Preparation method of product rich in high-activity lactoferrin

Abstract

The invention discloses a preparation method of a product rich in high-activity lactoferrin, and belongs to the technical field of food processing. According to the method, the lactoferrin is treated by adopting a high-voltage pulsed electric field technology instead of a traditional thermal sterilization technology, so that the damage of a thermal effect to the activity of the lactoferrin is avoided, and when the electric field intensity is below 30kV / cm, the retention rate of the lactoferrin can reach more than 95%; meanwhile, the method disclosed by the invention can improve the iron binding capacity, thermal stability, foamability and stability as well as emulsibility and stability of the lactoferrin. According to the invention, pathogenic microorganisms in the beverage can be effectively killed, the shelf life of the product is prolonged, and the safety of food microorganisms is ensured; and the combined treatment of lactoferrin and the pulsed electric field can enhance the sterilization effect, so that a small part of sub-lethal microorganisms generated by single pulsed electric field treatment further die. The application range of the lactoferrin is widened, and a new way is provided for processing of lactoferrin products.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and particularly relates to a method for preparing a product rich in highly active lactoferrin. Background Art

[0002] Lactoferrin (LF), the "gold of milk" in cow's milk, is an important iron-binding glycoprotein that is mainly found in various exosecretions of mammals, such as milk, tears and saliva. Lactoferrin has many powerful functional properties. First, it can act as an iron carrier to bind iron ions and promote the absorption and utilization of iron. This property makes lactoferrin have potential application value in the prevention and treatment of iron deficiency anemia. In addition, it can competitively deprive bacteria of the iron required for growth and directly bind to the microbial cell membrane to cause it to rupture. Therefore, lactoferrin also has antibacterial, antiviral, antifungal and antiparasitic effects. Another important function of lactoferrin is to regulate the immune system. It can bind to receptors on the surface of immune cells to exert immune regulation functions, while regulating the level of cytokines in the body, playing a role in anti-tumor, immune regulation and antioxidant activity.

[0003] The lactoferrin content in human milk is about 7 g / L, while the lactoferrin content in bovine milk is about 0.3-2.1 g / L. The amino acid sequence homology of bovine lactoferrin (bLF) and human lactoferrin (hLF) is 69%, and both have high similarity in biological activity and physicochemical properties. Therefore, the richness of bovine milk resources and related basic research on bovine lactoferrin have attracted much attention. The global bovine lactoferrin production has also increased rapidly from less than 80 tons per year in 2006 to more than 400 tons in 2023. Due to the huge demand for infant nutrition, immune health, sports nutrition, cosmetics, livestock and neonatal nutrition, the future bovine lactoferrin market is expected to grow at an annual rate of 10% to 12%.

[0004] However, lactoferrin is a heat-sensitive protein. Traditional pasteurization and ultra-high temperature instantaneous sterilization can cause different degrees of loss of lactoferrin. Traditional thermal sterilization technology has technical defects that are difficult to coordinate between long shelf life and lactoferrin retention rate; pasteurization faces challenges such as incomplete microbial inactivation, short shelf life (7-14 days), high storage conditions (2-6°C), and some loss of active proteins; although ultra-high temperature instantaneous sterilization can meet the requirements of commercial sterility, excessive heat treatment causes lactoferrin and other heat-sensitive active substances in cow's milk to be almost completely denatured and inactivated, and Maillard reaction is prone to occur, which seriously affects its nutrition and flavor substances. 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 technology.

[0005] At present, CN117918423A discloses a kind of milk with high active protein content and long shelf life and its production method. The invention uses raw milk as raw material, and can retain the flavor of milk and the content of its active substances after rapid preheating, high-voltage pulse electric field and aseptic homogenization. However, this method cannot solve the problem of maintaining the stability of lactoferrin activity and improving lactoferrin activity. Although this method uses pulse electric field to treat raw milk, it has a certain bactericidal effect, but it also needs to be preheated to (50-65℃) to ensure that the number of colonies is reduced (reaching 3 lg). Heating will inevitably lead to an increase in the conductivity of the solution, and the electric field will generate more Joule heat, thereby making the actual temperature of the material higher. It is known that the N-leaf denaturation temperature of lactoferrin is about 58℃, and the retention rate and activity of lactoferrin obtained by such 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 will detect inactivated lactoferrin. The new national standard method uses a selective heparin column to effectively intercept active lactoferrin, which is more targeted. Therefore, how to improve the retention rate and activity of lactoferrin while ensuring the bactericidal effect is a technical problem that needs to be solved in this field. Summary of the invention

[0006] To solve the above problems, the present invention provides a method for preparing a beverage rich in highly active lactoferrin, which can obtain lactoferrin with a high retention rate, has low energy consumption and high efficiency, and can ensure the microbial safety of the product, extend its shelf life, and improve the iron binding capacity, thermal stability, foaming performance and stability of lactoferrin, and emulsifying performance and stability of lactoferrin, so that the lactoferrin activity is higher and more stable. In addition, lactoferrin itself has antibacterial properties, and lactoferrin and pulsed electric field synergy have better bactericidal effects. This can not only greatly broaden the scope of application of lactoferrin, but also make useful explorations for the sustainable development of lactoferrin.

[0007] To achieve the above effects, the first object of the present invention is to provide a method for preparing a lactoferrin-rich product, the product including but not limited to a beverage, the method comprising the following steps: (1) Raw material processing adding lactoferrin powder to raw milk or sterile water to obtain a lactoferrin-rich solution; (2) Degassing treatment Degassing the lactoferrin-rich solution obtained in step (1); (3) Pulsed electric field sterilization treatment: The degassed solution obtained in step (2) is subjected to pulsed electric field sterilization treatment, wherein the electric field strength is set to 10-45 kV / 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 200-400 us; at the same time, the temperature in the pipeline of the pulsed electric field equipment is controlled not to be higher than 20°C; thus, a beverage rich in lactoferrin can be obtained.

[0008] Furthermore, the content of the lactoferrin powder in the fresh milk or sterile water in step (1) is 0.5-5%.

[0009] Furthermore, the electric field strength in step (3) is set to 10-30 kV / cm.

[0010] Furthermore, the method for controlling the temperature in the pipeline to be no higher than 20°C in step (3) is to place the pipeline in a water bath at 0-4°C.

[0011] Furthermore, when the electric field strength is 10-45 kV / cm, the lactoferrin retention rate in the lactoferrin product of the present invention can reach more than 90%; Preferably, when the electric field strength is below 30 kV / cm, the retention rates of the lactoferrin of the present invention in the four systems are all above 95%.

[0012] It was also found that the lactoferrin treated by the method of the present invention combined with the pulsed electric field treatment can produce a synergistic effect to enhance the bactericidal effect, further killing a small number of sublethal microorganisms produced by a single pulsed electric field treatment, which is of great value for ensuring the safety of food microorganisms.

[0013] The second object of the present invention is to provide a method for increasing the content of lactoferrin in a beverage, or the activity of lactoferrin, or the relative iron binding capacity of lactoferrin, or the thermal stability of lactoferrin, or improving the antibacterial ability of a beverage, the method comprising the following steps: (1) Raw material processing Add lactoferrin powder to raw milk or sterile water to obtain a lactoferrin-rich solution; (2) Degassing treatment Degassing the lactoferrin-rich solution obtained in step (1); (3) Pulsed electric field sterilization treatment: The degassed solution obtained in step (2) is subjected to pulsed electric field sterilization treatment, wherein the electric field strength is set to 10-45 kV / 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 200-400 us; at the same time, the temperature in the pipeline of the pulsed electric field equipment is controlled not to be higher than 20°C; thus, a beverage rich in lactoferrin can be obtained.

[0014] Furthermore, the content of the lactoferrin powder in the 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 in the pipeline to be no higher than 20°C in step (3) is to place the pipeline in a water bath at 0-4°C.

[0017] Furthermore, when the electric field strength is 10-45 kV / cm, the lactoferrin retention rate in the lactoferrin product of the present invention can reach more than 90%; Preferably, when the electric field strength is below 30 kV / cm, the retention rates of the lactoferrin of the present invention in the four systems are all above 95%.

[0018] It was also found that the lactoferrin treated by the method of the present invention combined with the pulsed electric field treatment can produce a synergistic effect to enhance the bactericidal effect, further killing a small number of sublethal microorganisms produced by a single pulsed electric field treatment, which is of great value for ensuring the safety of food microorganisms.

[0019] The third object of the present invention is to provide a product rich in highly active lactoferrin prepared by the preparation method of the present invention.

[0020] Furthermore, the product rich in highly active lactoferrin includes but is not limited to beverages.

[0021] Furthermore, the highly active lactoferrin-rich product 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%.

[0022] Beneficial Effects (1) The present invention uses a pulse electric field treatment method to treat a solution containing lactoferrin. By controlling the temperature during the pulse electric field treatment process to be below 20°C, the lactoferrin structure is not destroyed while the activity of lactoferrin is improved. The lactoferrin retention rate in the lactoferrin-rich raw milk and lactoferrin-rich aqueous solution, raw milk and bovine colostrum obtained after the treatment is high.

[0023] (2) Enhanced lactoferrin activity Protein denaturation, inactivation and aggregation induced by heat treatment often lead to reduced performance of lactoferrin. The present invention controls the temperature during the pulse electric field treatment to be below 20°C. On the one hand, after the pulse electric field treatment, the lactoferrin structure can be properly unfolded, and its iron binding ability, thermal stability, foaming property and foaming stability, emulsification property and emulsification stability can be improved, thereby further improving the quality of the beverage prepared by the present invention. On the other hand, lowering the temperature of the system during the pulse electric field treatment can reduce the conductivity of the material, which is not only conducive to avoiding the generation of electric sparks or arcs in the pulse electric field equipment and even emergency shutdown of the equipment, but also maintaining the stable operation of the equipment and extending the service life of the equipment.

[0024] (3) High retention rate of lactoferrin After being treated with different pasteurization intensities, the retention rate of lactoferrin is usually between 50% and 70%, and after being sterilized at ultra-high temperature, the lactoferrin is almost completely inactivated; however, after being treated with a pulsed electric field (electric field strength of 10-45 kV / cm) below 20°C according to the present invention, the retention rate of lactoferrin can reach more than 90%; when the electric field strength is below 30 kV / cm, the retention rates of lactoferrin in the four systems are all above 95%.

[0025] (4) Simple process, high efficiency, low energy consumption, good sterilization effect and long shelf life The present invention adopts pulse electric field technology, which can instantly cause irreversible "electroporation" to the cell membrane and cell wall of miscellaneous bacterial cells in the solution within microseconds, causing their metabolism to be disordered and the intracellular solution to flow out, thereby killing the microorganisms; and the pulse electric field can act on a wide range of microorganisms, and has a good killing effect on both Gram-negative bacteria and Gram-positive bacteria. The present invention can reduce the total colony count of raw milk rich in lactoferrin, aqueous solution rich in lactoferrin, raw milk and bovine colostrum by 3-5 logarithms, thereby greatly extending their shelf life.

[0026] (5) Lactoferrin and pulsed electric field synergistically improve the bactericidal effect Lactoferrin itself has good antibacterial properties. The pulsed electric field applied to a system rich in lactoferrin can enhance the bactericidal effect and further kill the small amount of sublethal microbial cells produced by the pulsed electric field. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The bactericidal effect of pulsed electric field with electric field strength of 10-45 kV / cm on raw milk, 0.5% lactoferrin raw milk, 5% lactoferrin aqueous solution and bovine colostrum.

[0028] Figure 2 The effect of pulse electric field treatment with an electric field strength of 10-45 kV / cm on the relative iron binding capacity of lactoferrin.

[0029] Figure 3 The effect of pulse electric field treatment with an electric field strength of 10-45 kV / cm on the foaming property and foaming stability of lactoferrin.

[0030] Figure 4 The effect of pulse electric field treatment with an electric field strength of 10-45 kV / cm on the emulsification and emulsification stability of lactoferrin.

[0031] Figure 5 The effect of pulse electric field treatment with an electric field strength of 15-45 kV / cm on the thermal stability of lactoferrin (N leaf).

[0032] Figure 6 The effect of pulse electric field treatment with an electric field strength of 15-45 kV / cm on the thermal stability of lactoferrin (C leaf).

[0033] Figure 7 The figure shows the changes in the secondary structure of lactoferrin under different electric field strength treatments of the present invention.

[0034] Figure 8 The figure shows the changes in the tertiary structure of lactoferrin under treatment with different electric field strengths according to the present invention. DETAILED DESCRIPTION

[0035] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0036] The test methods involved in the following embodiments are as follows: 1. Method for determination of colony count: The national standard "GB 4789.2-2022 National Food Safety Standard Food Microbiology Examination Determination of Total Colony Count" is adopted.

[0037] 2. Determination method of lactoferrin content: The national standard "GB 5009.299-2024 National Food Safety Standard Determination of Lactoferrin in Food" is adopted.

[0038] 3. Determination of relative iron binding capacity: (1) Dissolve lactoferrin in pH 7.55 mmol / L Tris-HCl buffer (containing 0.15 mol / L NaCl) to prepare a lactoferrin solution with a mass concentration of 2 mg / mL; (2) Take 0.9 mL of this solution and mix it with 0.1 mL of freshly prepared 0.1 mL NaHCO3 solution. After thorough mixing, react with 1.0 mL of 0.6 mmol / L Fe(NH4)2(SO4)2·6H2O solution (containing 0.01 mol / L HCl) at 25°C for 10 min. Then measure the absorbance at 465 nm. The absorbance of 10 mg / mL iron-saturated lactoferrin (Fe-LF) at 465 nm is 0.57. (3) The calculation equation for relative iron binding capacity is as follows:

[0039]

[0040] 4. Determination method of foaming property and foam stability: Homogenize the lactoferrin solution at 10,000 rpm for 2 min until it becomes foamy. Quickly transfer it to a 50 mL measuring cylinder. The volume of the foam after standing for 2 min is marked as V2, and the volume of the foam after standing for 30 min is marked as V. 30 ; Foaming ability (FA) and foaming stability (FS) are calculated according to the following formula:

[0041]

[0042] Where Vs is the initial liquid volume.

[0043] 5. Determination method of emulsification and emulsion stability: Take 10 mL of lactoferrin aqueous solution (5%) and 3 mL of soybean oil, mix them at 10000 rpm for 2 min, take 3 μL of the emulsion from the bottom of the beaker, mix it thoroughly with 3.0 mL of 0.1% (m / v) SDS solution, and use a UV spectrophotometer to measure the concentration of the emulsion at 0 min (A0) and 10 min (A10) after preparation. 10 ) when measuring the absorbance at 500 nm; The calculation equations for EAI and ESI are as follows:

[0044]

[0045] Where c is the protein concentration (0.05 g / mL), D is the dilution factor (1000), and α is the volume fraction of the oil phase (20%).

[0046] 6. Determination method of thermal stability: The thermal stability of lactoferrin was analyzed using differential scanning microcalorimetry (Nano-DSC): a certain amount of lactoferrin solution was taken, ultrapure water was used as the baseline, the temperature scanning range was 40~100 ℃, the heating rate was 1 ℃ / min, and the data was read using the Nano-DSC differential scanning microcalorimeter of TAInstruments-Waters LLC to study the thermal stability of lactoferrin.

[0047] Raw materials and equipment involved in the embodiments: The lactoferrin powder, raw milk and bovine colostrum involved in the following embodiments are all from Yili Dairy Research Institute, wherein the purity of the lactoferrin powder is ≥95%, and the iron content of lactoferrin is 5.4 mg / kg; the lactoferrin content in the raw milk is about 120 mg / kg, and the lactoferrin content in the bovine colostrum is about 5180 mg / kg.

[0048] The pulse electric field device involved in the following embodiments is the OSU-4L model of Ohio State University, USA, the pulse polarity is a unipolar or bipolar pulse, the waveform is a rectangular, sawtooth or exponential decay type, and the processing chamber is a continuous processing system; The sterile canning clean bench involved in the following embodiments was purchased from Shenzhen Sannuo Instrument Equipment Factory, model SW-CJ-1D; The 4°C refrigerator involved in the following embodiments was purchased from Haier Group, model number BCD-478WGHTD5DDYU1; The tube-sheet combined ultra-high temperature sterilizer involved in the following embodiments is purchased from Powerpoint International Company of Japan, model PT-20C-R.

[0049] The culture medium components involved in the following examples are as follows: The non-selective culture medium was BHI brain heart infusion broth agar.

[0050] The selective culture medium is VRBA crystal violet neutral red bile salt agar medium.

[0051] Embodiment 1: The pulse electric field processing method of the present invention comprises the following specific steps: (1) Raw material processing 1 g of lactoferrin powder was dissolved in 200 ml of raw milk, and magnetic stirring was performed at 500 rpm until the mixture was uniform and fully hydrated, thereby obtaining raw milk rich in lactoferrin; 10 g of lactoferrin powder was dissolved in 200 mL of sterile pure water and magnetically stirred at 500 rpm until it was uniform and fully hydrated to obtain an aqueous solution rich in lactoferrin; (2) Degassing treatment Degassing the raw milk, bovine colostrum, the lactoferrin-rich raw milk obtained in step (1), and the lactoferrin-rich aqueous solution under a pressure of 600 mm Hg; (3) Equipment disinfection: The pulse electric field equipment pipeline was fully disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence; (4) Pulsed electric field sterilization treatment: The degassed solution obtained in step (2) is placed in the pulsed electric field equipment pipeline after disinfection and cleaning in step (3) for pulsed electric field sterilization treatment, with the electric field intensity set to 10 kV / cm, the pulse width set to 2 us, the pulse frequency set to 100 Hz, and the total treatment time set to 300 us; at the same time, the temperature in the pipeline is controlled to be no higher than 20°C by a 4°C water bath condition; (5) Canned storage: The solutions obtained by the pulse electric field treatment in step (4) were aseptically bottled and stored in a refrigerator at 4°C.

[0052] Embodiment 2: The pulse electric field processing method of the present invention comprises the following specific steps: (1) Raw material processing 1 g of lactoferrin powder was dissolved in 200 ml of raw milk, and magnetic stirring was performed at 500 rpm until the mixture was uniform and fully hydrated, thereby obtaining raw milk rich in lactoferrin; 10 g of lactoferrin powder was dissolved in 200 mL of sterile pure water and magnetically stirred at 500 rpm until it was uniform and fully hydrated to obtain an aqueous solution rich in lactoferrin; (2) Degassing treatment Degassing the raw milk, bovine colostrum, the lactoferrin-rich raw milk obtained in step (1), and the lactoferrin-rich aqueous solution under a pressure of 600 mm Hg; (3) Equipment disinfection: The pulse electric field equipment pipeline was fully disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence; (4) Pulsed electric field sterilization treatment: The degassed solution obtained in step (2) is placed in the pulsed electric field equipment pipeline after disinfection and cleaning in step (3) for pulsed electric field sterilization treatment, with the electric field intensity set to 15 kV / cm, the pulse width set to 2 us, the pulse frequency set to 100 Hz, and the total treatment time set to 300 us; at the same time, the temperature in the pipeline is controlled to be no higher than 20°C by a 4°C water bath condition; (5) Canned storage: The solutions obtained by the pulse electric field treatment in step (4) were aseptically bottled and stored in a refrigerator at 4°C.

[0053] Embodiment 3: The pulse electric field processing method of the present invention comprises the following specific steps: (1) Raw material processing 1 g of lactoferrin powder was dissolved in 200 ml of raw milk, and magnetic stirring was performed at 500 rpm until the mixture was uniform and fully hydrated, thereby obtaining raw milk rich in lactoferrin; 10 g of lactoferrin powder was dissolved in 200 mL of sterile pure water and magnetically stirred at 500 rpm until it was uniform and fully hydrated to obtain an aqueous solution rich in lactoferrin; (2) Degassing treatment Degassing the raw milk, bovine colostrum, the lactoferrin-rich raw milk obtained in step (1), and the lactoferrin-rich aqueous solution under a pressure of 600 mm Hg; (3) Equipment disinfection: The pulse electric field equipment pipeline was fully disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence; (4) Pulsed electric field sterilization treatment: The degassed solution obtained in step (2) is placed in the pulsed electric field equipment pipeline after disinfection and cleaning in step (3) for pulsed electric field sterilization treatment, with the electric field intensity set to 20 kV / cm, the pulse width set to 2 us, the pulse frequency set to 100 Hz, and the total treatment time set to 300 us; at the same time, the temperature in the pipeline is controlled to be no higher than 20°C by a 4°C water bath condition; (5) Canned storage: The solutions obtained by the pulse electric field treatment in step (4) were aseptically bottled and stored in a refrigerator at 4°C.

[0054] Embodiment 4: The pulse electric field processing method of the present invention comprises the following specific steps: (1) Raw material processing 1 g of lactoferrin powder was dissolved in 200 ml of raw milk, and magnetic stirring was performed at 500 rpm until the mixture was uniform and fully hydrated, thereby obtaining raw milk rich in lactoferrin; 10 g of lactoferrin powder was dissolved in 200 mL of sterile pure water and magnetically stirred at 500 rpm until it was uniform and fully hydrated to obtain an aqueous solution rich in lactoferrin; (2) Degassing treatment Degassing the raw milk, bovine colostrum, the lactoferrin-rich raw milk obtained in step (1), and the lactoferrin-rich aqueous solution under a pressure of 600 mm Hg; (3) Equipment disinfection: The pulse electric field equipment pipeline was fully disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence; (4) Pulsed electric field sterilization treatment: The degassed solution obtained in step (2) is placed in the pulsed electric field equipment pipeline after disinfection and cleaning in step (3) for pulsed electric field sterilization treatment, with the electric field intensity set to 25 kV / cm, the pulse width set to 2 us, the pulse frequency set to 100 Hz, and the total treatment time set to 300 us; at the same time, the temperature in the pipeline is controlled to be no higher than 20°C by a 4°C water bath condition; (5) Canned storage: The solutions obtained by the pulse electric field treatment in step (4) were aseptically bottled and stored in a refrigerator at 4°C.

[0055] Embodiment 5: The pulse electric field processing method of the present invention comprises the following specific steps: (1) Raw material processing 1 g of lactoferrin powder was dissolved in 200 ml of raw milk, and magnetic stirring was performed at 500 rpm until the mixture was uniform and fully hydrated, thereby obtaining raw milk rich in lactoferrin; 10 g of lactoferrin powder was dissolved in 200 mL of sterile pure water and magnetically stirred at 500 rpm until it was uniform and fully hydrated to obtain an aqueous solution rich in lactoferrin; (2) Degassing treatment Degassing the raw milk, bovine colostrum, the lactoferrin-rich raw milk obtained in step (1), and the lactoferrin-rich aqueous solution under a pressure of 600 mm Hg; (3) Equipment disinfection: The pulse electric field equipment pipeline was fully disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence; (4) Pulsed electric field sterilization treatment: The degassed solution obtained in step (2) is placed in the pulsed electric field equipment pipeline after disinfection and cleaning in step (3) for pulsed electric field sterilization treatment, with the electric field intensity set to 30 kV / cm, the pulse width set to 2 us, the pulse frequency set to 100 Hz, and the total treatment time set to 300 us; at the same time, the temperature in the pipeline is controlled to be no higher than 20°C by a 4°C water bath condition; (5) Canned storage: The solutions obtained by the pulse electric field treatment in step (4) were aseptically bottled and stored in a refrigerator at 4°C.

[0056] Embodiment 6: The pulse electric field processing method of the present invention comprises the following specific steps: (1) Raw material processing 1 g of lactoferrin powder was dissolved in 200 ml of raw milk, and magnetic stirring was performed at 500 rpm until the mixture was uniform and fully hydrated, thereby obtaining raw milk rich in lactoferrin; 10 g of lactoferrin powder was dissolved in 200 mL of sterile pure water and magnetically stirred at 500 rpm until it was uniform and fully hydrated to obtain an aqueous solution rich in lactoferrin; (2) Degassing treatment Degassing the raw milk, bovine colostrum, the lactoferrin-rich raw milk obtained in step (1), and the lactoferrin-rich aqueous solution under a pressure of 600 mm Hg; (3) Equipment disinfection: The pulse electric field equipment pipeline was fully disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence; (4) Pulsed electric field sterilization treatment: The degassed solution obtained in step (2) is placed in the pulsed electric field equipment pipeline after disinfection and cleaning in step (3) for pulsed electric field sterilization treatment, with the electric field intensity set to 35 kV / cm, the pulse width set to 2 us, the pulse frequency set to 100 Hz, and the total treatment time set to 300 us; at the same time, the temperature in the pipeline is controlled to be no higher than 20°C by a 4°C water bath condition; (5) Canned storage: The solutions obtained by the pulse electric field treatment in step (4) were aseptically bottled and stored in a refrigerator at 4°C.

[0057] Embodiment 7: The pulse electric field processing method of the present invention comprises the following specific steps: (1) Raw material processing 1 g of lactoferrin powder was dissolved in 200 ml of raw milk, and magnetic stirring was performed at 500 rpm until the mixture was uniform and fully hydrated, thereby obtaining raw milk rich in lactoferrin; 10 g of lactoferrin powder was dissolved in 200 mL of sterile pure water and magnetically stirred at 500 rpm until it was uniform and fully hydrated to obtain an aqueous solution rich in lactoferrin; (2) Degassing treatment Degassing the raw milk, bovine colostrum, the lactoferrin-rich raw milk obtained in step (1), and the lactoferrin-rich aqueous solution under a pressure of 600 mm Hg; (3) Equipment disinfection: The pulse electric field equipment pipeline was fully disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence; (4) Pulsed electric field sterilization treatment: The degassed solution obtained in step (2) is placed in the pulsed electric field equipment pipeline after disinfection and cleaning in step (3) for pulsed electric field sterilization treatment, with the electric field intensity set to 40 kV / cm, the pulse width set to 2 us, the pulse frequency set to 100 Hz, and the total treatment time set to 300 us; at the same time, the temperature in the pipeline is controlled to be no higher than 20°C by a 4°C water bath condition; (5) Canned storage: The solutions obtained by the pulse electric field treatment in step (4) were aseptically bottled and stored in a refrigerator at 4°C.

[0058] Embodiment 8: The pulse electric field processing method of the present invention comprises the following specific steps: (1) Raw material processing 1 g of lactoferrin powder was dissolved in 200 ml of raw milk, and magnetic stirring was performed at 500 rpm until the mixture was uniform and fully hydrated, thereby obtaining raw milk rich in lactoferrin; 10 g of lactoferrin powder was dissolved in 200 mL of sterile pure water and magnetically stirred at 500 rpm until it was uniform and fully hydrated to obtain an aqueous solution rich in lactoferrin; (2) Degassing treatment Degassing the raw milk, bovine colostrum, the lactoferrin-rich raw milk obtained in step (1), and the lactoferrin-rich aqueous solution under a pressure of 600 mm Hg; (3) Equipment disinfection: The pulse electric field equipment pipeline was fully disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence; (4) Pulsed electric field sterilization treatment: The degassed solution obtained in step (2) is placed in the pulsed electric field equipment pipeline after disinfection and cleaning in step (3) for pulsed electric field sterilization treatment, with the electric field intensity set to 45 kV / cm, the pulse width set to 2 us, the pulse frequency set to 100 Hz, and the total treatment time set to 300 us; at the same time, the temperature in the pipeline is controlled to be no higher than 20°C by a 4°C water bath condition; (5) Canned storage: The solutions obtained by the pulse electric field treatment in step (4) were aseptically bottled and stored in a refrigerator at 4°C.

[0059] Example 9 The experimental method of treating bacteria with lactoferrin and electric field is as follows: 1. Escherichia coli ( E. coli ) Preparation of suspension: Transfer E. coli from nutrient agar slant medium to nutrient broth medium and culture at 37°C and 200 rpm in a shaker for 12 hours until the logarithmic growth phase. Centrifuge the E. coli culture at 4°C and 6000 rpm for 5 minutes, wash twice with sterile PBS buffer, and resuspend the cells in PBS buffer; 2. Contains E. coli ( E. coli ) Preparation of lactoferrin aqueous solution: The pulsed electric field processing method of this embodiment is the same as that of embodiment 1, except that step (1) is adjusted as follows: dissolving lactoferrin powder in sterile pure water, and magnetically stirring at 500 rpm until the lactoferrin powder is uniform and fully hydrated, to obtain aqueous solutions of lactoferrin at concentrations of 5 mg / mL, 10 mg / mL, and 20 mg / mL, respectively; The above-mentioned Escherichia coli ( E. coli ) suspension was mixed with 5 mg / mL, 10 mg / mL and 20 mg / mL aqueous solutions of lactoferrin at a ratio of 1:10 to obtain a bacterial concentration of 10 6 -10 8 CFU / mL of lactoferrin aqueous solution. Before treatment, the lactoferrin aqueous solution was stored in a refrigerator at 4°C.

[0060] 3. Lactoferrin and pulsed electric field combined to treat Escherichia coli E. coli : Escherichia coli ( E. coli ) 5 mg / mL, 10 mg / mL and 20 mg / mL lactoferrin aqueous solutions were passed into the pulsed electric field equipment pipeline after disinfection and cleaning in step (3) for pulsed electric field sterilization treatment, the electric field intensity was set to 20, 30 or 40 kV / cm, the pulse width was set to 2 us, the pulse frequency was set to 100 Hz, and the total treatment time was set to 300 us; at the same time, the temperature in the pipeline was controlled not to be higher than 20 ℃ under 4 ℃ water bath conditions; finally, the colony count was counted using selective and non-selective culture media (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 that can screen out completely healthy E. coli cell) The results of this embodiment are as follows: In the group without lactoferrin, as the electric field strength increased, the pulsed electric field had a negative effect on Escherichia coli ( E. coli ) The killing logarithm of the suspension increased gradually, and there was a difference of 0.3-0.6 lg between the selective culture medium and the non-selective culture medium, indicating that a single pulse electric field failed to completely kill a small number of microorganisms and might produce some sublethal microbial cells (i.e., in a damaged state that can be restored under suitable conditions).

[0061] In the group without pulsed electric field, with the increase of lactoferrin concentration, E. coli The killing logarithm of the bacteria increased, but the bactericidal effect was limited (less than 1 lg). E. coli The higher logarithm of killing may be due to the increase in lactoferrin concentration leading to the death of some sublethal microorganisms.

[0062] In the group where pulsed electric field and lactoferrin were applied simultaneously, the effect of electric field intensity and lactoferrin concentration on E. coli The killing logarithm increased accordingly, and the difference between the selective culture medium and the non-selective culture medium gradually decreased, indicating that the combined treatment of lactoferrin and pulsed electric field can cause further death of sublethal microbial cells produced by single pulsed electric field treatment, which is of great value for ensuring food microbial safety.

[0063] Table 1 Synergistic effect of lactoferrin and pulsed electric field treatment on E. coli Effect of kill log

[0064] Note: A is non-selective culture medium, B is selective culture medium.

[0065] Comparative Example 1: This comparative example is a method in which no electric field treatment is applied, and the specific steps are as follows: (1) Raw material processing 1 g of lactoferrin powder was dissolved in 200 ml of raw milk, and magnetic stirring was performed at 500 rpm until the mixture was uniform and fully hydrated, thereby obtaining raw milk rich in lactoferrin; 10 g of lactoferrin powder was dissolved in 200 mL of sterile pure water and magnetically stirred at 500 rpm until it was uniform and fully hydrated to obtain an aqueous solution rich in lactoferrin; (2) Degassing treatment Degassing the raw milk, bovine colostrum, the lactoferrin-rich raw milk obtained in step (1), and the lactoferrin-rich aqueous solution under a pressure of 600 mm Hg; (3) Equipment disinfection: The pulse electric field equipment pipelines were fully disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence; (4) No pulse electric field treatment: The degassed solutions were passed into the cleaned and sterilized pulse electric field equipment pipelines, but no electric field was applied. At the same time, the temperature in the pipelines was controlled to be no higher than 20°C under 4°C water bath conditions. (5) Canned storage: The solutions obtained by the pulse electric field treatment in step (4) were aseptically bottled and stored in a refrigerator at 4°C.

[0066] Comparative Example 2: This comparative example is a pasteurization method, and the specific steps are as follows: (1) Raw material processing 1 g of lactoferrin powder was dissolved in 200 ml of raw milk, and magnetic stirring was performed at 500 rpm until the mixture was uniform and fully hydrated, thereby obtaining raw milk rich in lactoferrin; 10 g of lactoferrin powder was dissolved in 200 mL of sterile pure water and magnetically stirred at 500 rpm until it was uniform and fully hydrated to obtain an aqueous solution rich in lactoferrin; (2) Degassing treatment Degassing the raw milk, bovine colostrum, the lactoferrin-rich raw milk obtained in step (1), and the lactoferrin-rich aqueous solution under a pressure of 600 mm Hg; (3) Pasteurization The degassed solutions were respectively passed into the sterilized tube-sheet combined ultra-high temperature sterilizer (72 °C, 15 s) for pasteurization; (4) The solutions obtained after pasteurization were aseptically packaged and stored in a refrigerator at 4°C.

[0067] Comparative Example 3 The pulsed electric field processing method of this comparative example is the same as that of Example 5, except that the 4°C water bath in step (4) is not used to control the temperature in the pipeline. The specific steps are as follows: (1) Raw material processing 1 g of lactoferrin powder was dissolved in 200 ml of raw milk, and magnetic stirring was performed at 500 rpm until the mixture was uniform and fully hydrated, thereby obtaining raw milk rich in lactoferrin; 10 g of lactoferrin powder was dissolved in 200 mL of sterile pure water and magnetically stirred at 500 rpm until it was uniform and fully hydrated to obtain an aqueous solution rich in lactoferrin; (2) Degassing treatment Degassing the raw milk, bovine colostrum, the lactoferrin-rich raw milk obtained in step (1), and the lactoferrin-rich aqueous solution under a pressure of 600 mm Hg; (3) Equipment disinfection: The pulse electric field equipment pipelines were fully disinfected and cleaned at room temperature using 4% NaOH aqueous solution, 10% hypochlorous acid disinfectant, and sterile pure water in sequence; (4) Pulsed electric field sterilization treatment: The degassed solutions obtained in step (2) are respectively placed in the pulsed electric field equipment pipelines disinfected and cleaned in step (3) for pulsed electric field sterilization treatment, with the electric field intensity set to 30 kV / cm, the pulse width set to 2 us, the pulse frequency set to 100 Hz, and the total treatment time set to 300 us; (5) Canned storage: The solutions obtained by the pulse electric field treatment in step (4) were aseptically bottled and stored in a refrigerator at 4°C.

[0068] Examples 1-8 and Comparative Examples 1-3: Experimental Results 1. Results of lactoferrin retention rate The results are shown in Table 2: Table 2 Retention rate of lactoferrin in different systems under different treatment conditions

[0069] (1) By Figure 1 It can be seen that Figure 1 The figure shows the effect of pulsed electric field treatment on the total number of colonies in raw milk, 0.5% lactoferrin raw milk, 5% lactoferrin aqueous solution and bovine colostrum when the electric field strength is 10-45 kV / cm, the treatment time is 300 us, and the water bath temperature is 4℃. It can be seen from the figure that with the increase of electric field strength, the total number of colonies decreases, and the sterilization effect is better; when the electric field strength is increased to 45 kV / cm, the total number of colonies in raw milk, 0.5% lactoferrin raw milk, 5% lactoferrin aqueous solution and bovine colostrum decreases by 5.54, 5.70, 5.95 and 5.15 logarithms, respectively. It can be seen that pulsed electric field treatment (PEF) has a good killing effect on microorganisms.

[0070] (2) As can be seen from Table 2, with the increase of electric field strength, the retention rate of lactoferrin slightly decreased. When the electric field strength was 30 kV / cm (the sterilization effect reached 3-5), the retention rate of lactoferrin in the four systems was above 95%. If the electric field strength was further increased to 45 kV / cm, the loss rate of lactoferrin still did not exceed 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 were 75.7%, 68.6%, 74.9% and 70.3%, respectively, which were significantly lower than the retention rates of lactoferrin after pulse electric field treatment. This shows that pulse electric field treatment has little damage to lactoferrin and can better maintain the content of lactoferrin.

[0071] (3) If, as shown in Comparative Example 3, a 4°C water bath is not used to control the temperature in the pulse electric field pipeline, the pipeline temperature cannot be controlled below 20°C during the pulse electric field treatment, and the Joule heat generated in the pipeline will continue to accumulate, causing the pipeline to heat up rapidly, and ultimately causing the lactoferrin retention rate after the electric field treatment to drop significantly (as shown in Table 2); at the same time, if a 4°C water bath is not used to control the temperature in the pulse electric field pipeline, the conductivity of the solution in the pipeline will increase, and the current in the pipeline will continue to increase. When it reaches a certain level, the pulse electric field pipeline may generate electric sparks and arcs, and even cause the equipment to be forced to shut down and stop, and cannot work normally; it can be seen that if a 4°C water bath is not used to control the temperature in the pulse electric field pipeline, it will cause great damage to the lactoferrin content and lactoferrin activity after the pulse electric field treatment, and it is not conducive to extending the service life of the equipment.

[0072] 2. Improve the relative iron binding capacity of lactoferrin The results are as follows Figure 2 , Figure 7 , Figure 8 As shown in Table 3: Table 3 Secondary structure of lactoferrin under different electric field strengths

[0073] Table 4 Relative iron binding capacity of lactoferrin under different electric field strengths

[0074] Depend on Figure 2 , Figure 7 , Figure 8As shown in Table 3, after the pulse electric field treatment, the spatial structure of lactoferrin was properly unfolded, the α-helix structure increased slightly, the β-folded structure decreased slightly, and more iron binding sites were exposed; the activity of lactoferrin, i.e., the iron binding capacity, was enhanced, and when treated with an electric field strength of 15-30 kV / cm, the relative iron binding capacity of lactoferrin exceeded 140%; while the relative iron binding capacity of the pasteurized group (72 ℃, 15 s) was only 87.7%, indicating that the heat treatment not only reduced 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, a 4 ℃ water bath is not used to control the temperature in the pulse electric field pipeline, the temperature of the pulse electric field pipeline cannot be controlled below 20 ℃ at this time, and the temperature rise of the pipeline will cause the relative iron binding capacity of lactoferrin to be greatly damaged, which is only 61.8%.

[0075] Under moderate field intensity treatment, lactoferrin can expose more iron binding sites, which enhances the iron binding reaction performance of lactoferrin. If the field intensity is further increased, the relative iron binding capacity of lactoferrin decreases, but it is still higher than that of the untreated group, the pasteurized group (72 ℃, 15 s) and the group without 4 ℃ water bath to control the temperature in the pulsed electric field pipe; when the pulsed electric field intensity reaches a certain value, the two-leaf ring structure of lactoferrin tends to loosen, causing the iron binding capacity of lactoferrin to decrease, but in general, the pulsed electric field treatment has a positive effect on the iron binding capacity of lactoferrin.

[0076] 3. Improve the foaming property and foaming stability of lactoferrin Foaming property refers to the ability of protein to form foam under stirring or mechanical action, while foaming stability refers to the ability of foam to maintain its structure and volume over a certain period of time. Protein molecules are amphiphilic, so they can reduce the surface tension of water and show good foaming properties when stirred vigorously; the determination of lactoferrin's foaming property and foaming stability is of great significance to the food industry. These characteristics determine the application effect of lactoferrin in food processing, especially in products that need to form foam structure (such as ice cream, cake, fermented dairy products, etc.), which can provide a scientific basis for food formulation and process optimization.

[0077] The results are as follows Figure 3 As shown: (1) By Figure 3 The results show that the foaming property of lactoferrin first increases with the increase of electric field strength, and then gradually decreases when the electric field strength reaches 25 kV / cm. When the electric field strength is 25 kV / cm, the foaming property of lactoferrin is the best, which is 133.6% of the pre-treatment property. At 45 kV / cm, its foaming property drops to 92.3% of the pre-treatment property.

[0078] (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, the foaming stability is 89.4% of that before treatment.

[0079] The improvement in lactoferrin's foaming performance is mainly the result of the improvement in protein structure under the action of a pulsed electric field. Under the action of the electric field, the protein unfolds, which is beneficial to the exposure of its internal groups, allowing it to fully exert its foaming properties. However, when the electric field is too strong and the structural changes are too drastic, the unfolded protein molecules interact with each other to form aggregates, forming molecular aggregates, reducing the protein solubility and the foaming performance of lactoferrin.

[0080] 4. Improve the emulsification and emulsion stability of lactoferrin Since lactoferrin has an amphiphilic structure, i.e., hydrophilic and lipophilic groups, in an oil-water mixture, the protein molecules tend to diffuse to the oil-water interface, with the hydrophobic part facing the lipid and the hydrophilic part facing the water phase, reducing the oil-water interfacial tension and film formation, thus having emulsifying properties. Because lactoferrin has unique functionality, such as antibacterial and immunomodulatory effects, it is often added to various foods to enhance their nutritional value and functionality. Therefore, the emulsification and emulsification stability determination of lactoferrin is of great significance to the food industry. The determination of emulsification stability is an important part of food quality control, which can help ensure that dairy products maintain their sensory and nutritional value during storage and shelf life. By determining the emulsification stability of lactoferrin, the stability of dairy products during food processing and storage, as well as the quality of the final product, can be evaluated.

[0081] The results are as follows Figure 4 As shown: Depend on Figure 4 It can be seen that when the electric field strength is 25 kV / cm, the emulsification of lactoferrin is the highest, which is 173.3% of that before treatment. When the electric field strength is 45 kV / cm, its emulsification drops to 95.3% of that before treatment. Pulsed electric field treatment also has a similar effect on the emulsification stability of lactoferrin. After pulsed electric field treatment, the spatial structure of lactoferrin unfolds, which not only increases the surface area of ​​lactoferrin in contact with the interface, but also exposes the internal groups of lactoferrin, enhances the surface hydrophobicity, and improves the emulsification performance; however, when the pulsed electric field exceeds a certain threshold, hydrophobic collapse and protein aggregation may occur, which reduces the emulsification performance of lactoferrin; in general, pulsed electric field treatment has a positive effect on the emulsification performance of lactoferrin under appropriate conditions. 5. Improve the thermal stability of lactoferrin The results are as follows Figure 5 and Figure 6As shown in the figure: after pulsed electric field treatment, the thermal stability of lactoferrin is improved, and the denaturation temperatures represented by its N leaf and C leaf are increased by 1-2 ℃ respectively; after treatment at 72 ℃ (15 s), the denaturation temperatures represented by its N leaf and C leaf are decreased by 0.18 ℃ and 1.90 ℃ respectively; the improvement of thermal stability under the action of pulsed electric field may be related to the change of its secondary structure under pulsed electric field treatment. After physical modification of lactoferrin structure under moderate conditions of pulsed electric field, its structure is more stable, so that lactoferrin can be more heat-resistant; while lactoferrin itself is sensitive to heat, and after treatment at 72 ℃ (15 s), the structure of lactoferrin becomes looser, which has caused certain damage to the structure of lactoferrin, thereby reducing its thermal stability.

[0082] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing a product rich in highly active lactoferrin, characterized in that: The product includes but is not limited to a drink, and the method includes the following steps: (1) Raw material processing adding lactoferrin powder to raw milk or sterile water to obtain a lactoferrin-rich solution; (2) Degassing treatment Degassing the lactoferrin-rich solution obtained in step (1); (3) Pulsed electric field sterilization treatment: The degassed solution obtained in step (2) is subjected to pulsed electric field sterilization treatment, wherein the electric field strength is set to 10-45 kV / 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 200-400 us; at the same time, the temperature in the pipeline of the pulsed electric field equipment is controlled not to be higher than 20°C; thus, a product rich in highly active lactoferrin can be obtained.

2. The preparation method according to claim 1, characterized in that The content of the lactoferrin powder in the fresh milk or sterile water in step (1) is 0.5-5%.

3. The preparation method according to claim 1, characterized in that: The electric field strength in step (3) is set to 10-30 kV / cm.

4. The preparation method according to claim 1, characterized in that: The method for controlling the temperature in the pipeline to be no higher than 20°C in step (3) is to place the pipeline in a water bath at 0-4°C.

5. A method for increasing the content of lactoferrin in a beverage, or the activity of lactoferrin, or the relative iron binding capacity of lactoferrin, or the thermal stability of lactoferrin, or improving the antibacterial ability of a beverage, characterized in that: The method comprises the following steps: (1) Raw material processing adding lactoferrin powder to raw milk or sterile water to obtain a lactoferrin-rich solution; (2) Degassing treatment Degassing the lactoferrin-rich solution obtained in step (1); (3) Pulsed electric field sterilization treatment: The degassed solution obtained in step (2) is subjected to pulsed electric field sterilization treatment, with the electric field intensity set to 10-45 kV / cm, the pulse width set to 1-3 us, the pulse frequency set to 50-150 Hz, and the total treatment time set to 200-400 us; at the same time, the temperature in the pipeline of the pulsed electric field equipment is controlled to be no higher than 20°C.

6. The method according to claim 5, characterized in that The content of the lactoferrin powder in the fresh milk or sterile water in step (1) is 0.5-5%.

7. The method according to claim 5, characterized in that The electric field strength in step (3) is set to 10-30 kV / cm.

8. The method according to claim 5, characterized in that The method for controlling the temperature in the pipeline to be no higher than 20°C in step (3) is to place the pipeline in a water bath at 0-4°C.

9. A product rich in highly active lactoferrin prepared by the preparation method according to any one of claims 1 to 4.

10. The product rich in highly active lactoferrin according to claim 9, characterized in that: The product rich in highly active lactoferrin refers to a lactoferrin product in which the lactoferrin retention rate can reach more than 90%.

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