Fresh milk sterilization method based on multi-stage high-voltage pulse electric field and fresh milk product

Abstract

The application belongs to the technical field of dairy product processing, and specifically discloses a fresh milk sterilization method based on multi-stage high-voltage pulse electric field and a fresh milk product. The multi-stage high-voltage pulse electric field is used for step-by-step temperature rise, the temperature of the milk is increased by 8-10 DEG C after each treatment chamber, the temperature after the last treatment chamber is increased to 60-70 DEG C, and the microorganisms in the milk are efficiently killed. The application uses the ohmic heat of the high-voltage pulse electric field to perform gradient temperature rise on the milk in the high-voltage pulse electric field treatment chamber, reduces the additional energy input, reduces the substrate protection effect of the milk system on the microorganisms, increases the fluidity of the cell membrane, increases the death efficiency of the microorganisms, reduces the heat treatment temperature of the milk, shortens the treatment time, and maximally retains the flavor and active substances of the milk. The shelf life of the milk product is prolonged, and the storage time at a temperature of 2-6 DEG C is greater than or equal to 20 days.

Description

Technical Field

[0001] This invention relates to the field of dairy processing technology, and in particular to a method for sterilizing fresh milk based on a multi-stage high-voltage pulsed electric field, and fresh milk products thereof. Background Technology

[0002] Among the many bioactive components of milk, lactoferrin, a natural glycoprotein with multiple physiological functions such as broad-spectrum antibacterial, antiviral, immunomodulatory, and promotion of intestinal iron absorption, has seen its activity retention level become a key indicator for measuring the nutritional quality of high-end dairy products. However, lactoferrin is highly heat-sensitive, and its molecular conformation is prone to irreversible denaturation during heat treatment, leading to loss of biological activity. Currently, the sterilization technology widely used in the dairy industry is still dominated by heat sterilization: pasteurization (72-85°C, 15-20 s) can kill pathogenic bacteria, but it will cause a loss of 40%-80% of lactoferrin. However, heat-resistant Bacillus in milk, such as Bacillus stearothermophilus, cannot be completely killed at 72-85°C. Pasteurized milk can only kill the vegetative cells, and the spores remain. If left at room temperature for a long time, they will multiply and spoil. Ultra-high temperature (UHT) sterilization (135-150°C, 2-5 s) can achieve long-term storage of products at room temperature, but it almost completely denatures and inactivates lactoferrin.

[0003] High-voltage pulsed electric field technology, as a highly promising non-thermal processing method, works by inducing transmembrane potentials on microbial cell membranes using high-voltage pulses. When the potential exceeds a critical value, irreversible electroporation occurs in the cell membrane, leading to microbial death. Theoretically, this technology can effectively inactivate microorganisms under ambient or low-temperature conditions. However, its effectiveness is significantly limited when directly applied to whole-component milk systems. Milk is not a simple suspension but a complex emulsion system composed of milk fat globules, casein micelles, and soluble components. In this system, large fat globules and protein aggregates create a physical "protective matrix effect" on the microorganisms dispersed within (especially bacteria encapsulated by protein networks or attached to fat globules). This protective effect weakens the effective electric field strength acting on the microbial cell membrane. To overcome this obstacle and achieve commercial sterility requirements, extremely high electric field strengths (>50 kV / cm) or extremely long processing times are typically required. This not only leads to increased electrode load and a surge in energy consumption, but also causes negative issues such as localized overheating, electrochemical modification of proteins, and rupture of milk fat globules due to excessive electrical treatment, severely impacting product flavor and stability. Therefore, relying solely on high-voltage pulsed electric field technology makes it difficult to achieve an ideal balance between energy consumption, equipment lifespan, sterilization effectiveness, and activity retention.

[0004] For example, Chinese patent application CN202410265304.4 discloses a method for rapidly preheating raw milk to 20℃-65℃ and then sterilizing it with a high-voltage pulsed electric field. Although this method can retain some active proteins, it has significant limitations: First, raw milk requires an additional heat source for external preheating, increasing energy loss and equipment complexity; second, this rapid external preheating causes thermal shock to heat-sensitive proteins (such as lactoferrin) in milk, leading to irreversible aggregation of protein structures and making it difficult to achieve the upper limit of extremely high retention rates; in addition, the sterilization effect is limited and cannot kill heat-resistant Bacillus and spores. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for sterilizing fresh milk and fresh milk products based on a multi-level high-voltage pulsed electric field. By leveraging the deep synergy between the physical perforation effect of the high-voltage pulsed electric field (PEF) and the thermobiological effect of the multi-level temperature rise generated by the high-voltage pulsed electric field itself, the technical bottleneck of single non-thermal or thermal processing is overcome.

[0006] To achieve the above objectives, the present invention is implemented according to the following technical solution: The first technical solution provided by this invention is a milk sterilization method based on multi-stage high-voltage pulsed electric field synergistic gradient temperature rise, comprising the following steps: S1. Fresh milk at a temperature of 4℃-10℃ is subjected to multi-stage high-voltage pulse electric field treatment. During the multi-stage high-voltage pulse electric field treatment, the ohmic heat generated by the high-voltage pulse electric field itself causes the fresh milk to heat up step by step. Each stage of treatment increases the temperature of the fresh milk by 8℃-10℃ until the temperature of the fresh milk reaches 60℃-70℃ after the last stage of treatment. S2. The fresh milk that has been treated by the multi-stage high-voltage pulse electric field is rapidly cooled to 2℃-10℃ and then aseptically filled.

[0007] Furthermore, the operating conditions for the multi-stage high-voltage pulse electric field treatment are: electric field strength 12kV / cm-35 kV / cm, pulse frequency 1Hz-300Hz, and pulse width 1μs-10μs.

[0008] Furthermore, the multi-stage high-voltage pulse electric field treatment is carried out by a number of high-voltage pulse electric field treatment chambers connected in series along the flow direction of the fresh milk, with the same number of treatment stages as the number of treatment stages. Each treatment chamber is equipped with an independent pulse power supply to achieve independent adjustment of the treatment parameters at each stage.

[0009] Preferably, the distance between the positive and negative electrode plates in the high-voltage pulse electric field processing chamber is 0.5cm-3.0cm, and the electrode plates are made of one of titanium, platinum or stainless steel.

[0010] Preferably, the multi-stage high-voltage pulse electric field processing has 6 stages: The electric field strength of the first and second stages is 12kV / cm-15kV / cm, the pulse frequency is 200 Hz-300Hz, and the pulse width is 6μs-10μs. These stages are used to gently heat fresh milk with a lower field strength and a wider pulse, inducing the transformation of microbial cell membrane lipids from the gel phase to the liquid crystal phase, reducing membrane rigidity, and simultaneously promoting increased fluidity of milk fat globule membranes and swelling of casein micelles. This weakens the matrix protection effect on microorganisms and slightly unfolds the structure of active proteins in the fresh milk to improve temperature tolerance. The electric field strength of the third and fourth stages is 15kV / cm-20kV / cm, the pulse frequency is 100Hz-200Hz, and the pulse width is 3μs-6μs. These stages are used to apply an enhanced electric field to induce irreversible electroporation of microbial cell membranes based on the reduction of the cell membrane electroporation threshold and the weakening of the matrix protection effect. The electric field strength of levels 5 and 6 is 20kV / cm-35kV / cm, the pulse frequency is 1Hz-100Hz, and the pulse width is 1μs-3μs. These are used to kill residual sublethal microorganisms and spores with high field strength and narrow pulses, while allowing the pulse to act on active proteins for a short time to reduce thermal damage.

[0011] Furthermore, the fresh milk is homogenized before being subjected to multi-stage high-voltage pulse electric field treatment.

[0012] Preferably, the homogenization process is carried out in a high-pressure homogenizer with a homogenization pressure of 150-250 bar and a processing time of 1-3 minutes.

[0013] The second technical solution provided by the present invention is a fresh dairy product, which is prepared by the sterilization method described above.

[0014] Furthermore, in the fresh dairy products, the retention rate of lactoperoxidase is ≥85%, the retention rates of α-lactalbumin and β-lactoglobulin are ≥90%, and the retention rate of lactoferrin is ≥80%.

[0015] The technical principle of the method of this invention is as follows: 1. A weakening mechanism based on the "protective matrix effect" of interface polarization As a typical emulsion-colloid complex system, the microstructure of milk significantly influences the electric field distribution. In this system, milk fat globules (0.1 μm-15 μm in diameter) consist of a low-conductivity fat core and a phospholipid-protein membrane, while casein micelles (50 nm-600 nm in diameter) are porous protein aggregates cross-linked with calcium phosphate. Under a high-voltage pulsed electric field, the presence of these non-conductive or weakly conductive phases induces interfacial polarization (Maxwell-Wagner polarization), causing the electric field lines to distort and redistribute around the microorganisms, forming a localized electric field shielding region, known as the "protective matrix effect."

[0016] This invention employs a multi-stage, stepped temperature increase (6 stages, each increasing by 10°C), which initially affects the physical structure of milk fat globules and casein micelles. As the temperature gradually increases (from an initial temperature of 4°C to 10°C and finally rising to below 70°C), the liquid crystal phase of the milk fat globule membrane transforms from a gel phase to a fluid phase, increasing membrane fluidity and reducing fat globule rigidity. Simultaneously, the hydrophobic interactions within the casein micelles are weakened, and the micelles undergo a certain degree of swelling or depolymerization. These changes reduce the microstructural density of the emulsion system, thereby significantly weakening its physical shielding effect on microorganisms. This exposes the previously encapsulated or attached microorganisms to a stronger local electric field, creating the prerequisites for electroporation.

[0017] 2. Electrosensitization-based synergistic mechanism of membrane lipid phase transition Microbial cell membranes are dynamic structures composed of phospholipid bilayers, and their fluidity directly determines their sensitivity to electric fields. At low temperatures (4℃-10℃), cell membrane lipids are in a highly ordered gel phase, resulting in strong membrane rigidity and high resistance to electroporation.

[0018] This process applies mild thermal stress to microorganisms through stepwise temperature increases (8℃-10℃ per increment). During this gradual temperature rise, the cell membrane lipids undergo a phase transition, gradually shifting from a gel phase to a more fluid liquid crystal phase. This moderate increase in membrane fluidity slightly improves membrane permeability and lowers the membrane breakdown threshold. When a subsequent pulsed electric field is applied to the cell membrane in the liquid crystal phase, irreversible electroporation is more easily induced at the same field strength. In short, the stepwise temperature increase acts as a "preheating and softening" process for the cell membrane, making subsequent electric field killing more efficient and thorough.

[0019] 3. Protein stability protection mechanism based on conformational dynamics Unlike conventional high-temperature sterilization (instantaneous high temperature), this invention avoids severe thermal shock. Gradual temperature increase provides a gradual adaptation process for the molecular conformation of whey proteins (especially α-lactalbumin, β-lactoglobulin, and lactoferrin). Studies have found that protein denaturation often begins with a co-operative transformation of local structures; slow temperature increase allows protein molecules to undergo conformational rearrangement through side-chain adjustments, thereby delaying irreversible aggregation caused by the sudden exposure of hydrophobic groups.

[0020] More importantly, the maximum temperature of this invention is strictly controlled below the thermal denaturation threshold window of 60℃-70℃. Within this temperature range, combined with the natural antibacterial activity of the lactoperoxidase system in milk, a triple barrier of "electric field-thermal-natural immunity" is formed. This mechanism effectively kills microorganisms while avoiding the drastic denaturation of whey proteins (especially the destruction of the helical twist structure of lactoferrin) and the aggregation and cross-linking of milk fat globule membrane proteins caused by temperatures exceeding 70℃, thereby ensuring the physical stability and nutritional activity of the product during a refrigerated storage period of up to 20 days.

[0021] Compared with existing technologies, this invention utilizes a multi-stage high-voltage pulsed electric field to gradually increase the temperature of the milk. The temperature of the milk increases by 8℃-10℃ in each stage of treatment, ensuring that the temperature reaches 60℃-70℃ after the final stage, thus achieving efficient sterilization of microorganisms in the milk. By subjecting the milk to gradient heating in the high-voltage pulsed electric field treatment chamber, additional energy input is reduced, while the protective matrix effect of the milk system on the microbial matrix is ​​decreased, cell membrane fluidity is increased, and the lethality of microorganisms is enhanced. It also has a good inactivation effect on thermoresistant Bacillus subtilis in milk, such as Bacillus stearothermophilus. Simultaneously, the lower heat treatment temperature and shorter treatment time of the milk maximize the preservation of its flavor and active substances. In the milk products of this invention, the retention rate of lactoperoxidase is ≥85%, the retention rates of α-lactalbumin and β-lactoglobulin are ≥90%, and the retention rate of lactoferrin is ≥80%. Through the implementation of this method, the shelf life of milk products can be extended, with a storage time of ≥20 days at 2℃-6℃. Attached Figure Description

[0022] Figure 1 Scanning electron microscope (SEM) images of *Bacillus stearothermophilus* after different treatments: a) untreated group (CK); b) Comparative Example 5 treatment group, i.e., heat treatment at 67°C for 15 s; c) Example 3 treatment group, i.e., after treatment with a six-stage high-voltage pulsed electric field synergistic gradient temperature rise, with a final outlet temperature of 67°C. Magnification is 100,000x, and the scale bar is 1 μm. Detailed Implementation

[0023] The present invention will now be described in detail, with reference to specific embodiments and experimental data, a method for sterilizing fresh milk based on a multi-stage high-voltage pulsed electric field and fresh milk products provided by the present invention.

[0024] In a first aspect, the present invention provides a method for sterilizing fresh milk based on a multi-stage high-voltage pulsed electric field, comprising the following steps: S1, subjecting fresh milk at a temperature of 4℃-10℃ to a multi-stage high-voltage pulsed electric field treatment, wherein the fresh milk is gradually heated by the ohmic heat generated by the high-voltage pulsed electric field itself during the multi-stage high-voltage pulsed electric field treatment, and each stage of treatment increases the temperature of the fresh milk by 8℃-10℃, until the temperature of the fresh milk reaches 60℃-70℃ after the last stage of treatment; S2, rapidly cooling the fresh milk after the multi-stage high-voltage pulsed electric field treatment to 2℃-10℃, and then aseptically filling it.

[0025] Traditional high-voltage pulsed electric field cooling or single-temperature-range treatments struggle to simultaneously achieve efficient sterilization and retention of active proteins. This invention utilizes the ohmic heat generated by the high-voltage pulsed electric field to construct a multi-stage, stepped temperature rise process (8℃-10℃ per stage), gradually increasing the material's temperature from an initial 4℃-10℃ to 60℃-70℃. This organically combines the "temperature effect" and the "electric field effect," ensuring effective sterilization of milk while protecting its active proteins, thereby improving the quality of the resulting sterilized fresh milk products. Specifically, this includes increasing the retention rates of lactoperoxidase, α-lactalbumin, β-lactoglobulin, and lactoferrin in milk.

[0026] In this invention, efficient sterilization is achieved through a gradual weakening of the matrix protection effect and the electrosensitization of the microbial cell membrane. Specifically, milk fat globules and casein micelles in cow's milk have a dense structure at low temperatures, which induces interfacial polarization and forms an electric field shielding region. As the temperature gradually increases, the fat globule membrane transforms from a gel phase to a fluid phase, and the casein micelles swell and depolymerize. This structural change is gradual, gradually removing the "physical shielding" of microorganisms and exposing them to the effective electric field, thus improving the sterilization effect. In addition, at low temperatures, cell membrane lipids are in a gel phase with a high electroporation threshold. Mild thermal stress of approximately 8°C-10°C per stage can drive the membrane lipids to gradually transform into a liquid crystal phase, reducing membrane rigidity and breakdown threshold. The pre-treatment achieves "preheating and softening," while the subsequent treatment implements efficient electroporation inactivation on the sensitized cell membrane, maximizing the killing effect.

[0027] Furthermore, high retention rates of active proteins in milk are achieved through conformational adaptation protection of active proteins. Specifically, protein thermal denaturation typically involves a process of "local delamination stacking → exposure of hydrophobic cores → irreversible intermolecular aggregation." Conventional instantaneous high-temperature sterilization, due to its excessively rapid heating rate, leaves proteins with insufficient time for conformational adjustment, leading to severe hydrophobic aggregation caused by simultaneous delamination stacking of multiple domains. The step-by-step heating method of this invention achieves protein activity protection through the following three points: First, kinetic protection is achieved by providing a time window for conformational rearrangement. Each stage only increases the temperature by 8°C-10°C, allowing fresh milk a certain residence time in each processing chamber. Protein molecules undergo a gradual process of "small heating → brief adaptation → further small heating," providing sufficient time for side chain fine-tuning to adapt to the current temperature and preventing simultaneous collapse of multiple domains. Second, thermodynamic protection is achieved by avoiding local overheating through ohmic heat step-by-step heating. Ohmic heat is generated uniformly within the fresh milk, eliminating temperature gradients caused by heat transfer surfaces. The protein experiences uniform temperature throughout, preventing preferential denaturation caused by local overheating. Third, structural protection is achieved through conformational pre-adaptation induced by pre-denaturation temperature. The first two stages of treatment raise the temperature of fresh milk to approximately 25℃-30℃, which is within the sub-denaturation temperature range of whey proteins. This induces slight conformational unfolding and increased flexibility of the protein, providing "thermal adaptation training" in advance for conformational adjustments at higher temperatures.

[0028] Under the aforementioned triple protection, the final temperature is strictly controlled at 60℃-70℃. This temperature is the optimal window verified by experiments: lactoferrin can maintain its native state after pre-conformation adaptation, with an extremely low denaturation rate; at the same time, it is sufficient to activate the natural antibacterial activity of the lactoperoxidase system, forming a synergistic bactericidal effect with the pulsed electric field.

[0029] Furthermore, the temperature of fresh milk treated with a multi-stage high-voltage pulsed electric field reaches 60℃-70℃. If the temperature remains at this level for too long, the active proteins may still gradually denature due to prolonged heat exposure, and any remaining sub-lethal microorganisms may reactivate and repair themselves. Therefore, step S2 rapidly cools the treated fresh milk to 2℃-10℃. On the one hand, rapid cooling "locks" the protein molecules in their conformationally adapted natural state, preventing hydrophobic aggregation during slow cooling in the intermediate temperature range. On the other hand, 2℃-10℃ is the standard refrigeration temperature for dairy products, which can effectively inhibit the growth of remaining microorganisms and spore germination. In addition, aseptic filling immediately after rapid cooling ensures that the product maintains physical stability and microbiological safety throughout the cold chain.

[0030] Among them, common methods in the field can be used to rapidly cool fresh milk that has been treated with multi-stage high-voltage pulse electric fields, including but not limited to rapid cooling through plate heat exchangers or shell-and-tube coolers.

[0031] This invention gradually weakens the matrix protection effect and achieves microbial electrosensitization through a stepped temperature rise, significantly improving the sterilization efficiency of the high-voltage pulsed electric field. It effectively inactivates not only common pathogenic and spoilage bacteria in milk, but also exhibits good inactivation effects on thermoresistant Bacillus species (such as *Bacillus stearothermophilus*) and their spores. Simultaneously, the entire process utilizes ohmic heat for stepped temperature rise, avoiding localized overheating and thermal shock caused by external heating, thus ensuring the activity and structural stability of active proteins. The sterilized fresh milk products show a lactoperoxidase retention rate ≥85%, α-lactalbumin and β-lactoglobulin retention rates ≥90%, and lactoferrin retention rate ≥80%, significantly superior to traditional pasteurization and UHT sterilization, maintaining a natural flavor and nutritional quality close to that of raw milk. Furthermore, the method of this invention does not introduce an external heat source, relying solely on the ohmic heat of the high-voltage pulsed electric field itself for temperature rise, reducing additional energy input and resulting in lower energy consumption than traditional heat sterilization methods.

[0032] It should be understood that the multi-stage high-voltage pulse electric field treatment described in this invention has at least 5 treatment stages. In some embodiments, the number of treatment stages is 5 to 8, preferably 6. The specific number of treatment stages can be determined based on factors such as the initial temperature of the fresh milk, the target temperature rise for each stage, and the final target temperature to be achieved. The process parameters for each stage of the high-voltage pulse electric field treatment can be the same, or they can be set independently according to the different treatment purposes of each stage. The multi-stage treatment is carried out sequentially along the flow direction of the fresh milk, that is, the fresh milk first flows through the first stage of treatment, and then enters the subsequent stages of treatment in sequence.

[0033] Furthermore, in some embodiments of the present invention, the operating conditions of the multi-stage high-voltage pulse electric field treatment are: electric field strength 12kV / cm-35 kV / cm, pulse frequency 1Hz-300Hz, and pulse width 1μs-10μs.

[0034] The above parameter range was set based on the technical concept of "using ohmic thermal gradient heating, synergistic electric field sterilization and protein protection" of this invention, and was determined through experimental optimization.

[0035] The electric field strength of 12 kV / cm-35 kV / cm provides sufficient driving force for the transmembrane potential to break through the critical breakdown threshold of microbial cell membranes, which is a necessary condition for achieving irreversible electroporation. The lower limit of this range is applicable to the initial mild treatment stage, where the milk temperature is low and the cell membrane is not yet sensitized. A lower field strength combined with a wider pulse width can induce the initiation of membrane lipid phase transition. The upper limit is applicable to the subsequent enhanced inactivation stage. Applying a high field strength under conditions of increased temperature and sensitized cell membranes can efficiently inactivate spores and sublethal microorganisms.

[0036] The pulse frequency of 1Hz-300Hz determines the number of pulse injections per unit time, directly affecting the heat generation rate of ohmic heat. Combined with independently adjustable processing chambers, the frequency can be flexibly selected for different processing stages: the high-frequency range is suitable for processing stages requiring rapid heating to drive matrix demasking and membrane lipid phase transitions, while the low-frequency range is suitable for processing stages requiring controlled energy input rates and precise final temperature limits, thus reliably achieving a stepped temperature rise of 8-10℃ per stage.

[0037] The pulse width range of 1μs-10μs covers the process requirements from wide-pulse gentle treatment to narrow-pulse enhanced inactivation. Wider pulses have higher single-pulse energy and are suitable for the front-end stage to drive the loosening of the matrix structure and the membrane lipid phase transition with ohmic heat; narrower pulses have higher peak power and lower thermal effect and are suitable for the back-end stage to reduce thermal damage to active proteins while ensuring sterilization effect.

[0038] In some embodiments of this invention, the specific energy value of the multi-stage high-voltage pulsed electric field treatment is 110 kJ / L-150 kJ / L, reflecting the total energy input accumulated across multiple stages. This energy level has been experimentally verified to raise the temperature of milk from 4°C-10°C to 60°C-70°C, precisely within the temperature window required to kill spores, while not exceeding the critical threshold for thermal denaturation of active ingredients such as lactoferrin. Combined with the step-by-step distribution of heat across each stage and inter-stage cooling control, precise heat delivery can be ensured, avoiding localized or overall overheating.

[0039] Furthermore, in some embodiments of the present invention, the multi-stage high-voltage pulse electric field treatment is performed using a number of high-voltage pulse electric field treatment chambers arranged in series along the flow direction of fresh milk, with the same number of treatment stages as the number of stages. Each treatment chamber is equipped with an independent pulse power supply to achieve independent adjustment of the treatment parameters at each stage. This allows for differentiated control of parameters while ensuring processing efficiency.

[0040] Furthermore, in some embodiments of the present invention, the distance between the positive and negative electrode plates of the high-voltage pulse electric field treatment chamber is 0.5cm-3.0cm, and the electrode plates are made of titanium, platinum, or stainless steel. The electrode spacing of 0.5cm-3.0cm is a result of comprehensively considering the electric field strength requirements, processing capacity, and ohmic heat control. A smaller spacing is beneficial for obtaining a higher field strength under the same power supply voltage, and a shorter residence time of fresh milk, facilitating precise control of the temperature rise at each stage; however, too small a spacing can easily lead to excessive local current density. The 0.5cm-3.0cm range provides adjustment space to adapt to the needs of different processing stages. The electrode plates are made of titanium, platinum, or stainless steel. These materials are all food-grade inert conductive materials, exhibiting good electrochemical stability under high-voltage pulse and salt-containing emulsion conditions. They can inhibit electrode corrosion and metal ion dissolution, and their excellent conductivity can reduce interfacial energy loss, effectively applying more electrical energy to the fresh milk to generate ohmic heat and electric field effects, ensuring long-term process stability.

[0041] Furthermore, in some embodiments of the present invention, the number of stages of the multi-stage high-voltage pulse electric field processing is 6 stages: The electric field strength of the first and second stages is 12kV / cm-15kV / cm, the pulse frequency is 200 Hz-300Hz, and the pulse width is 6μs-10μs. These stages are used to gently heat fresh milk with a lower field strength and a wider pulse, inducing the transformation of microbial cell membrane lipids from the gel phase to the liquid crystal phase, reducing membrane rigidity, and simultaneously promoting increased fluidity of milk fat globule membranes and swelling of casein micelles. This weakens the matrix protection effect on microorganisms and slightly unfolds the structure of active proteins in the fresh milk to improve temperature tolerance. The electric field strength of the third and fourth stages is 15kV / cm-20kV / cm, the pulse frequency is 100Hz-200Hz, and the pulse width is 3μs-6μs. These stages are used to apply an enhanced electric field to induce irreversible electroporation of microbial cell membranes based on the reduction of the cell membrane electroporation threshold and the weakening of the matrix protection effect. The electric field strength of levels 5 and 6 is 20kV / cm-35kV / cm, the pulse frequency is 1Hz-100Hz, and the pulse width is 1μs-3μs. These are used to kill residual sublethal microorganisms and spores with high field strength and narrow pulses, while allowing the pulse to act on active proteins for a short time to reduce thermal damage.

[0042] Specifically, the first and second stages use a low field strength combined with high frequency and wide pulses. The high frequency pulses rapidly inject ohmic heat to gently raise the temperature of fresh milk from 4℃-10℃ to about 25℃-30℃, driving the lipid transformation of microbial cell membranes from the gel phase to the liquid crystal phase to reduce membrane rigidity. At the same time, it promotes the increase of milk fat globule membrane fluidity and casein micelle swelling, weakens the physical protective effect of the matrix on microorganisms, and allows whey proteins to undergo conformational pre-adaptation at sub-denaturation temperature.

[0043] Based on the weakening of the preceding membrane lipid phase transition and matrix protection effect, the third and fourth stages moderately reduce the frequency to control the temperature rise rate and gradually increase the electric field strength to induce irreversible electroporation of the sensitized microbial cell membranes, thereby implementing the main body inactivation.

[0044] Levels 5 and 6 combine high field strength with low frequency and narrow pulses. Low frequency controls energy input to slow down the rate of temperature rise and ensure that the final temperature does not exceed 70°C. High field strength and narrow pulses effectively kill residual sublethal microorganisms and spores. At the same time, the short pulse duration reduces thermal damage to active ingredients such as lactoferrin.

[0045] Furthermore, the fresh milk is homogenized before being subjected to multi-stage high-voltage pulse electric field treatment.

[0046] On the one hand, high-pressure homogenization breaks down milk fat globules to the micrometer scale and disperses them uniformly through instantaneous pressure drop, shearing, and cavitation effects. The reduction and homogenization of fat globule size lowers the scale of the non-conductive phase in the emulsion system, reduces local electric field distortion caused by large fat globules during high-pressure pulsed electric field treatment, and makes the electric field distribution in milk more uniform. This increases the probability and intensity of microorganisms being exposed to the effective electric field, thereby enhancing the consistency and efficiency of subsequent multi-stage pulsed electric field sterilization and improving sterilization efficiency. On the other hand, the whey protein interface rearrangement induced by homogenization gives the proteins a certain degree of conformational flexibility, which works synergistically with the subsequent gradual thermal process of stepwise heating, further reducing the risk of irreversible denaturation of active proteins.

[0047] Preferably, the homogenization process is carried out in a high-pressure homogenizer at a pressure of 150-250 bar for 1-3 minutes. During homogenization, a cooling device is used to control the temperature of the milk mixture, ensuring that the fresh milk remains at a low temperature before entering the multi-stage high-pressure pulsed electric field treatment, thus maintaining the initial temperature baseline for the stepped heating process. More preferably, the milk is rapidly cooled to 4-10°C after homogenization before entering the multi-stage high-pressure pulsed electric field treatment.

[0048] The second technical solution provided by the present invention is a fresh dairy product, which is prepared by the sterilization method described above.

[0049] Furthermore, in the fresh dairy products, the retention rate of lactoperoxidase is ≥85%, the retention rates of α-lactalbumin and β-lactoglobulin are ≥90%, and the retention rate of lactoferrin is ≥80%.

[0050] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0051] Example 1: (1) High-pressure homogenization pretreatment: The initial bacterial count was selected to be approximately 8.7 × 10⁻⁶. 3 CFU / mL (inoculated with thermostable Bacillus-thermophilic Lipid-Bacillus 5.0 × 10⁻⁶) 2 White spot cow milk (with lactoperoxidase concentration of 11800 U / L, α-lactalbumin concentration of 1200 mg / L, β-lactoglobulin concentration of 3600 mg / L, and lactoferrin concentration of 1600 mg / L) was subjected to high-pressure homogenization at 10 ℃ for 3 min to break down milk fat globules and obtain homogenized milk.

[0052] (2) Multi-stage high-voltage pulsed electric field synergistic gradient temperature rise sterilization: Homogenized milk is pumped into a high-voltage pulsed electric field device containing six processing chambers for sterilization. The electrodes are made of stainless steel, and the distance between the positive and negative electrode plates in each processing chamber is 3 cm. The number of stages in the multi-stage high-voltage pulsed electric field treatment is 6. The electric field strength of the first and second stages is 12 kV / cm, the pulse frequency is 300 Hz, and the pulse width is 10 μs. These stages are used to gently heat fresh milk with a lower field strength and a wider pulse, inducing the transformation of microbial cell membrane lipids from the gel phase to the liquid crystal phase, reducing membrane rigidity, and simultaneously promoting increased fluidity of milk fat globule membranes and swelling of casein micelles. This weakens the matrix protection effect on microorganisms and slightly unfolds the structure of active proteins in the fresh milk to improve temperature tolerance. The electric field strength of the third and fourth stages is 15 kV / cm, the pulse frequency is 200 Hz, and the pulse width is 6 μs. These stages are used to apply an enhanced electric field to induce irreversible electroporation of microbial cell membranes based on the reduction of the cell membrane electroporation threshold and the weakening of the matrix protection effect. The electric field strength of levels 5 and 6 is 20 kV / cm, the pulse frequency is 100 Hz, and the pulse width is 3 μs. These are used to kill residual sublethal microorganisms and spores with high field strength and narrow pulses, while allowing the pulse to act on active proteins for a short time to reduce thermal damage.

[0053] After six stages of cumulative temperature rise, the milk temperature at the outlet of the final processing chamber reaches 62 ℃.

[0054] (3) Rapid cooling and aseptic filling: The milk processed in step (2) is rapidly cooled to 10 ℃, pumped into an aseptic tank, and filled in an aseptic filling machine to obtain the final fresh milk product, which is stored at 4 ℃.

[0055] Example 2: (1) High-pressure homogenization pretreatment: The initial bacterial count was selected to be approximately 9.8 × 10⁻⁶. 3 CFU / mL (inoculated with thermostable Bacillus-thermophilic Lipid-Bacillus 5.0 × 10⁻⁶) 2 Buffalo milk (CFU / mL) containing lactoperoxidase at a concentration of 12200 U / L, α-lactalbumin at a concentration of 1280 mg / L, β-lactoglobulin at a concentration of 3800 mg / L, and lactoferrin at a concentration of 1750 mg / L was homogenized under high pressure at 10 °C for 2 min to break down milk fat globules, resulting in homogenized buffalo milk.

[0056] (2) Multi-stage high-voltage pulsed electric field synergistic gradient temperature rise sterilization: Homogenized buffalo milk is pumped into a high-voltage pulsed electric field device containing six treatment chambers for sterilization. The electrode material is titanium, and the distance between the positive and negative electrode plates in each treatment chamber is 1 cm. The number of stages in the multi-stage high-voltage pulsed electric field treatment is 6. The electric field strength of the first and second stages is 13 kV / cm, the pulse frequency is 300 Hz, and the pulse width is 10 μs. These stages are used to gently heat fresh milk with a lower field strength and a wider pulse, inducing the transformation of microbial cell membrane lipids from the gel phase to the liquid crystal phase, reducing membrane rigidity, and simultaneously promoting increased fluidity of milk fat globule membranes and swelling of casein micelles. This weakens the matrix protection effect on microorganisms and slightly unfolds the structure of active proteins in the fresh milk to improve temperature tolerance. The electric field strength of the third and fourth stages is 16 kV / cm, the pulse frequency is 200 Hz, and the pulse width is 6 μs. These stages are used to apply an enhanced electric field to induce irreversible electroporation of microbial cell membranes based on the reduction of the cell membrane electroporation threshold and the weakening of the matrix protection effect. The electric field strength of levels 5 and 6 is 23 kV / cm, the pulse frequency is 100 Hz, and the pulse width is 3 μs. These are used to kill residual sublethal microorganisms and spores with high field strength and narrow pulses, while allowing the pulse to act on active proteins for a short time to reduce thermal damage.

[0057] After six stages of cumulative temperature rise, the milk temperature at the outlet of the final processing chamber reaches 62 ℃.

[0058] (3) Rapid cooling and aseptic filling: The buffalo milk processed in step (2) is rapidly cooled to 10 ℃, pumped into an aseptic tank, and filled in an aseptic filling machine to obtain the final fresh milk product, which is stored at 4 ℃.

[0059] Example 3: (1) High-pressure homogenization pretreatment: The initial bacterial count was selected to be approximately 5.3 × 10⁻⁶. 4 CFU / mL (inoculated with thermostable Bacillus-thermophilic Lipid-Bacillus 5.0 × 10⁻⁶) 2 White spot cow milk (with lactoperoxidase concentration of 10500 U / L, α-lactalbumin concentration of 1000 mg / L, β-lactoglobulin concentration of 3400 mg / L, and lactoferrin concentration of 1250 mg / L) was subjected to high-pressure homogenization at 10 ℃, a homogenization pressure of 250 bar, and a processing time of 1 min to break down milk fat globules, resulting in homogenized milk.

[0060] (2) Multi-stage high-voltage pulsed electric field synergistic gradient temperature rise sterilization: Homogenized milk is pumped into a high-voltage pulsed electric field device containing six processing chambers for sterilization. The electrode material is titanium, and the distance between the positive and negative electrode plates in each processing chamber is 2 cm. The number of stages in the multi-stage high-voltage pulsed electric field treatment is 6. The electric field strength of the first and second stages is 13 kV / cm, the pulse frequency is 250 Hz, and the pulse width is 7 μs. These stages are used to gently heat fresh milk with a lower field strength and a wider pulse, inducing the transformation of microbial cell membrane lipids from the gel phase to the liquid crystal phase, reducing membrane rigidity, and simultaneously promoting increased fluidity of milk fat globule membranes and swelling of casein micelles. This weakens the matrix protection effect on microorganisms and slightly unfolds the structure of active proteins in the fresh milk to improve temperature tolerance. The electric field strength of the third and fourth stages is 17 kV / cm, the pulse frequency is 150 Hz, and the pulse width is 5 μs. These stages are used to apply an enhanced electric field to induce irreversible electroporation of microbial cell membranes based on the reduction of the cell membrane electroporation threshold and the weakening of the matrix protection effect. The electric field strength of levels 5 and 6 is 27 kV / cm, the pulse frequency is 50 Hz, and the pulse width is 2 μs. These are used to kill residual sublethal microorganisms and spores with high field strength and narrow pulses, while allowing the pulse to act on active proteins for a short time to reduce thermal damage.

[0061] After six stages of cumulative temperature rise, the milk temperature at the outlet of the final processing chamber reaches 67 ℃.

[0062] (3) Rapid cooling and aseptic filling: The milk processed in step (2) is rapidly cooled to 10 ℃, pumped into an aseptic tank, and filled in an aseptic filling machine to obtain the final fresh milk product, which is stored at 4 ℃.

[0063] Example 4: (1) High-pressure homogenization pretreatment: The initial bacterial count was selected to be approximately 6.2 × 10⁻⁶. 4 CFU / mL (inoculated with thermostable Bacillus-thermophilic Lipid-Bacillus 5.0 × 10⁻⁶) 2 Buffalo milk (CFU / mL) containing 11000 U / L lactoperoxidase, 1100 mg / L α-lactalbumin, 3500 mg / L β-lactoglobulin, and 1400 mg / L lactoferrin was homogenized under high pressure at 10 °C for 2 min to break down milk fat globules, resulting in homogenized buffalo milk.

[0064] (2) Multi-stage high-voltage pulsed electric field synergistic gradient temperature rise sterilization: Homogenized buffalo milk is pumped into a high-voltage pulsed electric field device containing six treatment chambers for sterilization. The electrode material is titanium, and the distance between the positive and negative electrode plates in each treatment chamber is 3 cm. The number of stages in the multi-stage high-voltage pulsed electric field treatment is 6. The electric field strength of the first and second stages is 15 kV / cm, the pulse frequency is 275 Hz, and the pulse width is 8 μs. These stages are used to gently heat fresh milk with a lower field strength and a wider pulse, inducing the transformation of microbial cell membrane lipids from the gel phase to the liquid crystal phase, reducing membrane rigidity, and simultaneously promoting increased fluidity of milk fat globule membranes and swelling of casein micelles. This weakens the matrix protection effect on microorganisms and slightly unfolds the structure of active proteins in the fresh milk to improve temperature tolerance. The electric field strength of the third and fourth stages is 18 kV / cm, the pulse frequency is 180 Hz, and the pulse width is 5 μs. These stages are used to apply an enhanced electric field to induce irreversible electroporation of microbial cell membranes, based on the reduction of the cell membrane electroporation threshold and the weakening of the matrix protection effect. The electric field strength of levels 5 and 6 is 29 kV / cm, the pulse frequency is 70 Hz, and the pulse width is 2.5 μs. These are used to kill residual sublethal microorganisms and spores with high field strength and narrow pulses, while allowing the pulse to act on active proteins for a short time to reduce thermal damage.

[0065] After six stages of cumulative temperature rise, the milk temperature at the outlet of the final processing chamber reaches 67 ℃.

[0066] (3) Rapid cooling and aseptic filling: The buffalo milk processed in step (2) is rapidly cooled to 10 ℃, pumped into an aseptic tank, and filled in an aseptic filling machine to obtain the final fresh milk product, which is stored at 4 ℃.

[0067] Example 5: (1) High-pressure homogenization pretreatment: The initial bacterial count was selected to be approximately 4.9 × 10⁻⁶. 4 CFU / mL (inoculated with thermostable Bacillus-thermophilic Lipid-Bacillus 5.0 × 10⁻⁶) 2 White spot cow milk (with lactoperoxidase concentration of 9200 U / L, α-lactalbumin concentration of 800 mg / L, β-lactoglobulin concentration of 3200 mg / L, and lactoferrin concentration of 900 mg / L) was subjected to high-pressure homogenization at 10 ℃ for 3 min to break down milk fat globules and obtain homogenized milk.

[0068] (2) Multi-stage high-voltage pulsed electric field synergistic gradient temperature rise sterilization: Homogenized milk is pumped into a high-voltage pulsed electric field device containing six processing chambers for sterilization. The electrode material is titanium, and the distance between the positive and negative electrode plates in each processing chamber is 3 cm. The number of stages in the multi-stage high-voltage pulsed electric field treatment is 6. The electric field strength of the first and second stages is 14 kV / cm, the pulse frequency is 280 Hz, and the pulse width is 6 μs. These stages are used to gently heat fresh milk with a lower field strength and a wider pulse, inducing the transformation of microbial cell membrane lipids from the gel phase to the liquid crystal phase, reducing membrane rigidity, and simultaneously promoting increased fluidity of milk fat globule membranes and swelling of casein micelles. This weakens the matrix protection effect on microorganisms and slightly unfolds the structure of active proteins in the fresh milk to improve temperature tolerance. The electric field strength of the third and fourth stages is 19 kV / cm, the pulse frequency is 100 Hz, and the pulse width is 3 μs. These stages are used to apply an enhanced electric field to induce irreversible electroporation of microbial cell membranes based on the reduction of the cell membrane electroporation threshold and the weakening of the matrix protection effect. The electric field strength of levels 5 and 6 is 35 kV / cm, the pulse frequency is 50 Hz, and the pulse width is 1 μs. These are used to kill residual sublethal microorganisms and spores with high field strength and narrow pulses, while allowing the pulse to act on active proteins for a short time to reduce thermal damage.

[0069] After six stages of cumulative temperature rise, the milk temperature at the outlet of the final processing chamber reaches 70 ℃.

[0070] (3) Rapid cooling and aseptic filling: The milk processed in step (2) is rapidly cooled to 10°C, pumped into an aseptic tank, and filled in an aseptic filling machine to obtain the final fresh milk product, which is stored at 4°C.

[0071] Example 6: (1) High-pressure homogenization pretreatment: The initial bacterial count was selected to be approximately 7.2 × 10⁻⁶. 4 CFU / mL buffalo milk (containing lactoperoxidase at a concentration of 9800 U / L, α-lactalbumin at a concentration of 900 mg / L, β-lactoglobulin at a concentration of 3300 mg / L, and lactoferrin at a concentration of 1050 mg / L) was homogenized under high pressure at 10 ℃ for 3 min to break down milk fat globules, resulting in homogenized buffalo milk.

[0072] (2) Multi-stage high-voltage pulsed electric field synergistic gradient temperature rise sterilization: Homogenized buffalo milk is pumped into a high-voltage pulsed electric field device containing six treatment chambers for sterilization. The electrode material is titanium, and the distance between the positive and negative electrode plates in each treatment chamber is 3 cm. The number of stages in the multi-stage high-voltage pulsed electric field treatment is 6. The electric field strength of the first and second stages is 15 kV / cm, the pulse frequency is 200 Hz, and the pulse width is 9 μs. These stages are used to gently heat fresh milk with a lower field strength and a wider pulse, inducing the transformation of microbial cell membrane lipids from the gel phase to the liquid crystal phase, reducing membrane rigidity, and simultaneously promoting increased fluidity of milk fat globule membranes and swelling of casein micelles. This weakens the matrix protection effect on microorganisms and slightly unfolds the structure of active proteins in the fresh milk to improve temperature tolerance. The electric field strength of the third and fourth stages is 20 kV / cm, the pulse frequency is 130 Hz, and the pulse width is 4 μs. These stages are used to apply an enhanced electric field to induce irreversible electroporation of microbial cell membranes based on the reduction of the cell membrane electroporation threshold and the weakening of the matrix protection effect. The electric field strength of levels 5 and 6 is 35 kV / cm, the pulse frequency is 45 Hz, and the pulse width is 1.5 μs. These are used to kill residual sublethal microorganisms and spores with high field strength and narrow pulses, while allowing the pulse to act on active proteins for a short time to reduce thermal damage.

[0073] After six stages of cumulative temperature rise, the milk temperature at the outlet of the final processing chamber reaches 70 ℃.

[0074] (3) Rapid cooling and aseptic filling: The buffalo milk processed in step (2) is rapidly cooled to 10 ℃, pumped into an aseptic tank, and filled in an aseptic filling machine to obtain the final fresh milk product, which is stored at 4 ℃.

[0075] Comparative Example 1: (1) High-pressure homogenization pretreatment: Select the same batch of white flower milk as in Example 1, and perform high-pressure homogenization treatment at a temperature of 10 ℃ to obtain homogenized milk.

[0076] (2) Simple thermal sterilization: Homogenized milk is pumped into a tubular heat exchanger for conventional thermal sterilization without applying any pulsed electric field. The heating temperature is controlled to reach 62 ℃, and the milk is kept at 62 ℃ for 15 s by controlling the length and flow rate of the heat-insulating pipeline.

[0077] (3) Rapid cooling and aseptic filling: After the heat preservation in step (2), the milk is rapidly cooled to 10 ℃, pumped into an aseptic tank, and filled in an aseptic filling machine to obtain the final fresh milk product, which is stored at 4 ℃.

[0078] Comparative Example 2: (1) High-pressure homogenization pretreatment: Select the same batch of white flower milk as in Example 1, and perform high-pressure homogenization treatment at a temperature of 10 ℃ to obtain homogenized milk.

[0079] (2) High-voltage pulsed electric field sterilization with cooling: The homogenized milk is pumped into a high-voltage pulsed electric field device containing six processing chambers. The same multi-stage high-voltage pulsed electric field processing conditions as in Example 1 are set. However, during the processing, the external forced water cooling jacket of the device is activated to intervene in the cooling process, eliminate the heat effect generated by the electric field, and ensure that the temperature of the milk flowing through each processing chamber and at the final outlet is strictly controlled below 30 °C.

[0080] (3) Rapid cooling and aseptic filling: The milk processed in step (2) is cooled to 10 ℃, pumped into an aseptic tank, and filled in an aseptic filling machine to obtain the final fresh milk product, which is stored at 4 ℃.

[0081] Comparative Example 3: (1) High-pressure homogenization pretreatment: The same batch of buffalo milk as in Example 2 was selected and subjected to high-pressure homogenization at a temperature of 10 °C to obtain homogenized buffalo milk.

[0082] (2) Simple thermal sterilization: The homogenized buffalo milk is pumped into a tubular heat exchanger for conventional thermal sterilization without applying any pulsed electric field. The heating temperature is controlled to reach 62 ℃, and the buffalo milk is kept at 62 ℃ for 15 s by controlling the length and flow rate of the heat-insulating pipeline.

[0083] (3) Rapid cooling and aseptic filling: After the buffalo milk in step (2) is kept warm, it is rapidly cooled to 10 ℃, pumped into an aseptic tank, and filled in an aseptic filling machine to obtain the final fresh milk product, which is stored at 4 ℃.

[0084] Comparative Example 4: (1) High-pressure homogenization pretreatment: Select the same batch of buffalo milk as in Example 2 and perform high-pressure homogenization treatment at a temperature of 10 °C to obtain homogenized buffalo milk.

[0085] (2) High-voltage pulsed electric field sterilization with cooling: The homogenized buffalo milk is pumped into a high-voltage pulsed electric field device containing six processing chambers. The same multi-stage high-voltage pulsed electric field treatment as in Example 2 is set. However, during the treatment process, the external forced water cooling jacket of the device is activated to intervene in cooling, eliminate the heat effect generated by the electric field, and ensure that the temperature of the buffalo milk flowing through each processing chamber and at the final outlet is strictly controlled below 30 ℃.

[0086] (3) Rapid cooling and aseptic filling: The buffalo milk processed in step (2) is rapidly cooled to 10 ℃, pumped into an aseptic tank, and filled in an aseptic filling machine to obtain the final fresh milk product, which is stored at 4 ℃.

[0087] Comparative Example 5: (1) High-pressure homogenization pretreatment: Select the same batch of white flower milk as in Example 3, and perform high-pressure homogenization treatment at a temperature of 10 ℃ to obtain homogenized milk.

[0088] (2) Simple thermal sterilization: Homogenized milk is pumped into a tubular heat exchanger for conventional thermal sterilization without applying any pulsed electric field. The heating temperature is controlled to reach 67 ℃, and the milk is kept at 67 ℃ for 15 s by controlling the length and flow rate of the heat-insulating pipeline.

[0089] (3) Rapid cooling and aseptic filling: After the heat preservation in step (2), the milk is rapidly cooled to 10 ℃, pumped into an aseptic tank, and filled in an aseptic filling machine to obtain the final fresh milk product, which is stored at 4 ℃.

[0090] Comparative Example 6: (1) High-pressure homogenization pretreatment: Select the same batch of white flower milk as in Example 3, and perform high-pressure homogenization treatment at a temperature of 10 ℃ to obtain homogenized milk.

[0091] (2) Cooling high-voltage pulsed electric field sterilization: The homogenized milk is pumped into a high-voltage pulsed electric field device containing six processing chambers. The same multi-stage high-voltage pulsed electric field treatment as in Example 3 is set. However, during the treatment process, the external forced water cooling jacket of the device is turned on to intervene in the cooling, eliminate the heat effect generated by the electric field, and ensure that the temperature of the milk flowing through each processing chamber and at the final outlet is strictly controlled below 30 °C.

[0092] (3) Rapid cooling and aseptic filling: The milk processed in step (2) is cooled to 10 ℃, pumped into an aseptic tank, and filled in an aseptic filling machine to obtain the final fresh milk product, which is stored at 4 ℃.

[0093] Comparative Example 7: (1) High-pressure homogenization pretreatment: The same batch of buffalo milk as in Example 4 was selected and subjected to high-pressure homogenization at a temperature of 10 °C to obtain homogenized buffalo milk.

[0094] (2) Simple thermal sterilization: The homogenized buffalo milk is pumped into a tubular heat exchanger for conventional thermal sterilization without applying any pulsed electric field. The heating temperature is controlled to reach 67 ℃, and the buffalo milk is kept at 67 ℃ for 15 s by controlling the length and flow rate of the heat-insulating pipeline.

[0095] (3) Rapid cooling and aseptic filling: After the buffalo milk in step (2) is kept warm, it is rapidly cooled to 10 ℃, pumped into an aseptic tank, and filled in an aseptic filling machine to obtain the final fresh milk product, which is stored at 4 ℃.

[0096] Comparative Example 8: (1) High-pressure homogenization pretreatment: The same batch of buffalo milk as in Example 4 was selected and subjected to high-pressure homogenization at a temperature of 10 °C to obtain homogenized buffalo milk.

[0097] (2) High-voltage pulsed electric field sterilization with cooling: The homogenized buffalo milk is pumped into a high-voltage pulsed electric field device containing six processing chambers. The same multi-stage high-voltage pulsed electric field treatment as in Example 4 is set. However, during the treatment process, the external forced water cooling jacket of the device is activated to intervene in the cooling process, eliminate the heat effect generated by the electric field, and ensure that the temperature of the buffalo milk flowing through each processing chamber and at the final outlet is strictly controlled below 30 °C.

[0098] (3) Rapid cooling and aseptic filling: The buffalo milk processed in step (2) is rapidly cooled to 10 ℃, pumped into an aseptic tank, and filled in an aseptic filling machine to obtain the final fresh milk product, which is stored at 4 ℃.

[0099] Comparative Example 9: (1) High-pressure homogenization pretreatment: Select the same batch of white flower milk as in Example 5, and perform high-pressure homogenization treatment at a temperature of 10 ℃ to obtain homogenized milk.

[0100] (2) Simple thermal sterilization: Homogenized milk is pumped into a tubular heat exchanger for conventional thermal sterilization without applying any pulsed electric field. The heating temperature is controlled to reach 70 ℃, and the milk is kept at 70 ℃ for 15 s by controlling the length and flow rate of the heat-insulating pipeline.

[0101] (3) Rapid cooling and aseptic filling: After the heat preservation in step (2), the milk is rapidly cooled to 10 ℃, pumped into an aseptic tank, and filled in an aseptic filling machine to obtain the final fresh milk product, which is stored at 4 ℃.

[0102] Comparative Example 10: (1) High-pressure homogenization pretreatment: Select the same batch of white flower milk as in Example 5, and perform high-pressure homogenization treatment at a temperature of 10 ℃ to obtain homogenized milk.

[0103] (2) High-voltage pulsed electric field sterilization with cooling: The homogenized milk is pumped into a high-voltage pulsed electric field device containing six processing chambers. The same multi-stage high-voltage pulsed electric field treatment as in Example 5 is set. However, during the treatment process, the external forced water cooling jacket of the device is turned on to intervene in the cooling, eliminate the heat effect generated by the electric field, and ensure that the temperature of the milk flowing through each processing chamber and at the final outlet is strictly controlled below 30 °C.

[0104] (3) Rapid cooling and aseptic filling: The milk processed in step (2) is cooled to 10 ℃, pumped into an aseptic tank, and filled in an aseptic filling machine to obtain the final fresh milk product, which is stored at 4 ℃.

[0105] Comparative Example 11: (1) High-pressure homogenization pretreatment: Select the same batch of buffalo milk as in Example 6 and perform high-pressure homogenization treatment at a temperature of 10°C to obtain homogenized buffalo milk.

[0106] (2) Simple thermal sterilization: The homogenized buffalo milk is pumped into a tubular heat exchanger for conventional thermal sterilization without applying any pulsed electric field. The heating temperature is controlled to reach 70 ℃, and the buffalo milk is kept at 70 ℃ for 15 s by controlling the length and flow rate of the heat-insulating pipeline.

[0107] (3) Rapid cooling and aseptic filling: After the buffalo milk in step (2) is kept warm, it is rapidly cooled to 10 ℃, pumped into an aseptic tank, and filled in an aseptic filling machine to obtain the final fresh milk product, which is stored at 4 ℃.

[0108] Comparative Example 12: (1) High-pressure homogenization pretreatment: Select the same batch of buffalo milk as in Example 6 and perform high-pressure homogenization treatment at a temperature of 10°C to obtain homogenized buffalo milk.

[0109] (2) High-voltage pulsed electric field sterilization with cooling: The homogenized buffalo milk is pumped into a high-voltage pulsed electric field device containing six processing chambers. The same multi-stage high-voltage pulsed electric field treatment as in Example 6 is set. However, during the treatment process, the external forced water cooling jacket of the device is activated to intervene in the cooling process, eliminate the heat effect generated by the electric field, and ensure that the temperature of the buffalo milk flowing through each processing chamber and at the final outlet is strictly controlled below 30 °C.

[0110] (3) Rapid cooling and aseptic filling: The buffalo milk processed in step (2) is rapidly cooled to 10 ℃, pumped into an aseptic tank, and filled in an aseptic filling machine to obtain the final fresh milk product, which is stored at 4 ℃.

[0111] Comparative Example 13: (1) High-pressure homogenization pretreatment: Select the same batch of white flower milk as in Example 5, and perform high-pressure homogenization treatment at a temperature of 10 ℃ to obtain homogenized milk.

[0112] (2) Single-stage high-voltage pulsed electric field sterilization: Homogenized milk is pumped into a high-voltage pulsed electric field device containing only a single-stage processing chamber for sterilization. The electrode material is titanium, and the distance between the positive and negative electrode plates is 3 cm. The electric field strength is set to 35 kV / cm, the pulse frequency is set to 300 Hz, the pulse width is set to 10 μs, and the feed flow rate is reduced to 0.20 L / min to prolong the residence time of the milk in the single-stage processing chamber. The ohmic heat generated by the high-voltage pulsed electric field is used to make the outlet temperature of the milk reach 70 ℃ after single-stage processing. No inter-stage segmented adjustment or stepped temperature rise control is set throughout the process.

[0113] (3) Rapid cooling and aseptic filling: The milk processed in step (2) is rapidly cooled to 10 ℃, pumped into an aseptic tank, and filled in an aseptic filling machine to obtain the final fresh milk product, which is stored at 4 ℃.

[0114] Fresh milk products from Examples 1-6 and Comparative Examples 1-13 were stored at 4 °C. The shelf life of the prepared fresh milk products was then tested according to GB 19645 Pasteurized Milk and T / DAIM 003 Dairy Products Shelf Life Guidelines. The core causes of fresh milk spoilage are: germination of heat-resistant spores (waxy, coagulated, and Bacillus subtilis) + proliferation of psychrophilic bacteria residues. The test results are shown in Table 1.

[0115] Table 1 ; As shown in Table 1, the method of the present invention can extend the shelf life of milk products, with a storage time of ≥20 days at a temperature of 2℃-6℃.

[0116] Fresh milk products from Examples 1-6 and Comparative Examples 1-12 were stored at 4 °C for 15 days, and their retention rates of lactoperoxidase, α-lactalbumin, β-lactoglobulin, and lactoferrin were tested. The results are shown in Table 2.

[0117] Table 2 ; Table 2 shows that the synergistic process of this invention, at the same or lower maximum temperature, has a significantly better sterilization effect than pure PEF treatment, and is comparable to or even better than pure heat sterilization (especially at higher field strengths). This indicates that the stepped temperature rise significantly enhances the microbial inactivation efficiency of PEF; the retention rate of α-lactalbumin and β-lactoglobulin is much higher than that of pure heat sterilization, although slightly lower than that of pure PEF, but the sterilization of pure PEF is seriously insufficient; lactoferrin is the most heat-sensitive indicator, and this invention can still retain more than 80% of lactoferrin while achieving highly efficient sterilization (inactivation log number above 4.0), while traditional heat sterilization only retains 10-40%.

[0118] To further investigate the effects of different treatments on the cell morphology of *Bacillus stearothermophilus*, scanning electron microscopy was used to observe the untreated group, the control group (Example 5), and the treatment group (Example 3). The results are as follows: Figure 1 As shown.

[0119] Untreated groups (e.g.) Figure 1 In example a), *Bacillus stearothermophilus* was generally a relatively regular short rod-shaped or elliptical cell, with a relatively intact and dense cell surface, clear edge contours, and no obvious collapse, rupture, or leakage of contents. After being heat-treated at 67 ℃ for 15 s as in Comparative Example 5 (e.g., ...), ... Figure 1 (b) Some bacterial cells showed a certain degree of roughness and slight shrinkage on their surface, but most cells still maintained a relatively intact external morphology, indicating that simple heat treatment had a limited effect on the structural destruction of Bacillus stearothermophilus.

[0120] After undergoing the six-stage high-voltage pulsed electric field synergistic gradient temperature rise treatment in Example 3 (e.g.) Figure 1 In step c), the bacterial cells exhibited significant shrinkage, collapse, and irregular deformation; some cell surface structures were damaged and accompanied by fragmented material, resulting in a significant reduction in cell integrity. These results indicate that, compared to simple heat treatment at the same final temperature, the multi-stage high-voltage pulsed electric field synergistic gradient temperature rise treatment of this invention can cause more significant structural damage to *Bacillus stearothermophilus*, thus improving its inactivation effect.

[0121] In summary, this invention is equivalent to or even better than pure heat sterilization, and far superior to pure PEF; the retention rate of key heat-sensitive proteins such as lactoferrin is 30-50 percentage points higher than that of pure heat sterilization; the stepped temperature rise weakens the "protective matrix effect" of the milk system and "electrosensitizes" the microbial cell membrane, thereby significantly improving the sterilization efficiency of PEF without significantly increasing heat damage.

[0122] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for sterilizing fresh milk based on a multi-stage high-voltage pulsed electric field, characterized in that, Includes the following steps: S1. Fresh milk at a temperature of 4℃-10℃ is subjected to multi-stage high-voltage pulse electric field treatment. During the multi-stage high-voltage pulse electric field treatment, the ohmic heat generated by the high-voltage pulse electric field itself causes the fresh milk to heat up step by step. Each stage of treatment increases the temperature of the fresh milk by 8℃-10℃ until the temperature of the fresh milk reaches 60℃-70℃ after the last stage of treatment. S2. The fresh milk that has been treated by the multi-stage high-voltage pulse electric field is rapidly cooled to 2℃-10℃ and then aseptically filled.

2. The method for sterilizing fresh milk based on a multi-stage high-voltage pulsed electric field according to claim 1, characterized in that, The operating conditions for the multi-stage high-voltage pulse electric field treatment are: electric field strength 12kV / cm-35 kV / cm, pulse frequency 1Hz-300Hz, and pulse width 1μs-10μs.

3. The method for sterilizing fresh milk based on a multi-stage high-voltage pulsed electric field according to claim 1, characterized in that, The multi-stage high-voltage pulse electric field treatment is completed by using a number of high-voltage pulse electric field treatment chambers connected in series along the flow direction of the fresh milk, with the same number of treatment stages as the number of treatment stages. Each treatment chamber is equipped with an independent pulse power supply to achieve independent adjustment of the treatment parameters at each stage.

4. The method for sterilizing fresh milk based on a multi-stage high-voltage pulsed electric field according to claim 3, characterized in that, The positive and negative electrode plates of the high-voltage pulse electric field processing chamber are spaced 0.5cm-3.0cm apart, and the electrode plates are made of one of titanium, platinum or stainless steel.

5. The method for sterilizing fresh milk based on a multi-stage high-voltage pulsed electric field according to claim 1, characterized in that, The multi-stage high-voltage pulse electric field processing has 6 stages: The electric field strength of the first and second stages is 12kV / cm-15kV / cm, the pulse frequency is 200 Hz-300Hz, and the pulse width is 6μs-10μs. These stages are used to gently heat fresh milk with a lower field strength and a wider pulse, inducing the transformation of microbial cell membrane lipids from the gel phase to the liquid crystal phase, reducing membrane rigidity, and simultaneously promoting increased fluidity of milk fat globule membranes and swelling of casein micelles. This weakens the matrix protection effect on microorganisms and slightly unfolds the structure of active proteins in the fresh milk to improve temperature tolerance. The electric field strength of the third and fourth stages is 15kV / cm-20kV / cm, the pulse frequency is 100Hz-200Hz, and the pulse width is 3μs-6μs. These stages are used to apply an enhanced electric field to induce irreversible electroporation of microbial cell membranes based on the reduction of the cell membrane electroporation threshold and the weakening of the matrix protection effect. The electric field strength of levels 5 and 6 is 20kV / cm-35kV / cm, the pulse frequency is 1Hz-100Hz, and the pulse width is 1μs-3μs. These are used to kill residual sublethal microorganisms and spores with high field strength and narrow pulses, while allowing the pulse to act on active proteins for a short time to reduce thermal damage.

6. The method for sterilizing fresh milk based on a multi-stage high-voltage pulsed electric field according to claim 1, characterized in that, Before subjecting the fresh milk to multi-stage high-voltage pulse electric field treatment, the fresh milk is homogenized.

7. The method for sterilizing fresh milk based on a multi-stage high-voltage pulsed electric field according to claim 6, characterized in that, The homogenization process is carried out in a high-pressure homogenizer with a homogenization pressure of 150-250 bar and a processing time of 1-3 minutes.

8. A fresh dairy product, characterized in that, The fresh dairy product is prepared using the sterilization method described in any one of claims 1-7.

9. The fresh dairy product according to claim 8, characterized in that, In the fresh milk products, the retention rate of lactoperoxidase is ≥85%, the retention rate of α-lactalbumin and β-lactoglobulin is ≥90%, and the retention rate of lactoferrin is ≥80%.

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