Cold sterilization and freeze drying method for breast milk subjected to full-closed treatment and application of cold sterilization and freeze drying method

Abstract

The invention relates to a breast milk cold sterilization and freeze-drying method for full-closed treatment and application thereof, and is characterized in that the method comprises the following steps: injecting breast milk into a collection bag, carrying out vacuum sealing treatment, carrying out sterilization treatment by adopting cold sterilization, and carrying out freeze-drying to obtain freeze-dried breast milk; the cold sterilization comprises the steps of putting the collection bag into a closed pressure treatment cabin, and carrying out pressure treatment twice. According to the method disclosed by the invention, cold sterilization is adopted to uniformly transfer pressure to microbial cells in breast milk by utilizing the incompressible characteristic of a food-grade hydraulic medium on the premise of not depending on thermal action and avoiding damage of heat to nutritional ingredients, and efficient sterilization is realized by destroying cell membrane structures, enzyme active sites and genetic materials of microorganisms.

Description

Technical Field

[0001] This invention relates to the field of food sterilization and freeze-drying technology, and in particular to a fully enclosed method for cold sterilization and freeze-drying of breast milk and its application. Background Technology

[0002] Breast milk provides nutrition and various immune factors, enhancing the newborn's immune defense, neurological development, and gastrointestinal function. The World Health Organization recommends exclusive breastfeeding for the first six months of life, recognizing breast milk as the optimal nutritional choice for newborns. However, lactation is finite, and the rational utilization of excess breast milk during lactation is crucial to ensure the baby's growth and development after weaning. Excess breast milk requires appropriate processing methods and storage conditions. Currently, freeze-drying of breast milk can reduce its water content to 1-3% at freezing temperatures, helping to extend its shelf life. Furthermore, freeze-dried powder reduces the requirements for storage and transportation.

[0003] In the sterilization process, the European Milk Bank Association recommends low-temperature, long-duration pasteurization as the most common method for sterilizing surplus breast milk. This method, performed at 62.5°C for 30 minutes, can inactivate some bacteria, fungi, and viruses. However, low-temperature, long-duration pasteurization cannot kill heat-resistant spores and can damage the structure of nutrients in breast milk, such as vitamins, cytokines, immunoglobulins, lysozyme, and lactoferrin. The main goal of freeze-drying breast milk is long-term storage without loss of biological activity and with low microbial content, which low-temperature, long-duration pasteurization clearly cannot achieve. Common alternatives to low-temperature, long-duration pasteurization include high-temperature, short-duration pasteurization (75°C, 15-20 s) and ultra-high-temperature (UHT) sterilization (135°C, 2-5 s). Because of the higher temperatures, these methods are more effective at sterilization than low-temperature, long-duration pasteurization. However, they are essentially heat sterilization methods and still cause significant damage to heat-sensitive components in breast milk. Taking lysozyme in breast milk as an example, due to its heat intolerance, its activity decreases by 45% after ultra-high temperature instantaneous sterilization (and by 60% after low-temperature long-term pasteurization). Therefore, heat sterilization cannot meet the nutritional requirements of freeze-dried breast milk powder.

[0004] Furthermore, the existing preparation process for freeze-dried breast milk powder carries the risk of microbial contamination. Firstly, breast milk is collected from the breast using a breast pump and stored in a collection bag. It then needs to be transferred to a freeze-drying container, during which the breast milk comes into contact with the external environment. Studies show that approximately 30% of breast milk contamination occurs during this transfer step. Secondly, current collection bags use a single channel for both filling and retrieval. This means that the sample comes into contact with residual breast milk left at the filling point, posing a high risk of spoilage and bacterial growth. During freeze-drying, the freeze-drying container is open to allow ice crystals in the breast milk to sublimate into water vapor and escape. This means that the breast milk and the freeze-drying environment cannot be completely isolated, increasing the risk of contamination. Since freeze-dried breast milk powder is used directly for infant feeding, extremely high hygiene standards are required, and current processing methods cannot meet these requirements.

[0005] Therefore, developing a novel, fully enclosed freeze-drying method for sterilizing breast milk that can retain nutrients and ensure hygiene and safety is an urgent need to promote the effective utilization of surplus breast milk and ensure the healthy growth and development of newborns. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a fully enclosed method for cold sterilization and freeze-drying of breast milk, and its application. By combining cold sterilization with an integrated non-contact, anti-contamination freeze-drying bag for collecting breast milk, this invention achieves efficient sterilization and ensures the safety of breast milk microorganisms while maximizing the retention of heat-sensitive nutrients and bioactive substances in breast milk.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for cold sterilization and freeze-drying of breast milk in a fully enclosed manner. The method includes injecting breast milk into a collection bag, performing vacuum sealing treatment, sterilizing treatment by cold sterilization, and then obtaining freeze-dried breast milk by freeze-drying.

[0009] The cold sterilization process involves placing the collection bag into a sealed pressure chamber and subjecting it to two pressure treatments.

[0010] This invention utilizes a cold sterilization method combined with a fully enclosed process of an integrated non-contact, anti-contamination freeze-drying bag for collecting breast milk. This achieves efficient sterilization and ensures the safety of breast milk microorganisms while maximizing the retention of heat-sensitive nutrients and bioactive substances such as vitamins, immunoglobulins (especially stress-sensitive IgA and IgG), lysozyme, lactoferrin, and interleukins. Furthermore, the entire process from collection to powder production is sealed within the inner bag, with no contact with the external environment. This meets extremely high hygiene standards for infant feeding, promotes the effective utilization of excess breast milk, and provides a guarantee for the healthy growth of newborns.

[0011] Preferably, the cold sterilization includes placing the collection bag into a sealed pressure treatment chamber and injecting hydraulic medium for a first pressurization and a first pressure holding, followed by a pressure release and pause, then a second pressurization and a second pressure holding, and finally a pressure release and removal.

[0012] This invention employs cold sterilization, which, without relying on heat and avoiding the destruction of nutrients by heat, utilizes the incompressible properties of food-grade hydraulic media to uniformly transfer pressure to the microbial cells in breast milk. This achieves highly efficient sterilization by destroying the cell membrane structure, enzyme active sites, and genetic material of the microorganisms.

[0013] Preferably, the hydraulic medium includes any one or a combination of at least two of water, vegetable-based hydraulic oil, food-grade glycerin, or food-grade propylene glycol.

[0014] Preferably, the temperature of the hydraulic medium is 0℃ to 10℃. For example, it can be 0℃, 1℃, 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, 9℃, or 10℃, etc.

[0015] Preferably, the rate of the first pressurization is 10~20 MPa / min. For example, it can be 10 MPa / min, 12 MPa / min, 14 MPa / min, 16 MPa / min, 18 MPa / min, or 20 MPa / min, etc.

[0016] Preferably, the pressure of the first pressure holding is 100-800 MPa. The 100-800 MPa can be, for example, 100 MPa, 200 MPa, 300 MPa, 400 MPa, 500 MPa, 600 MPa, 700 MPa, or 800 MPa. Preferably, the pressure relief rate is 5-10 MPa / min, and the pressure after relief is 0-0.5 MPa. The 5-20 MPa / min can be, for example, 5 MPa / min, 6 MPa / min, 7 MPa / min, 8 MPa / min, 9 MPa / min, or 10 MPa / min. The 0-0.5 MPa can be, for example, 0 MPa, 0.1 MPa, 0.2 MPa, 0.3 MPa, 0.4 MPa, or 0.5 MPa.

[0017] Preferably, the pause time is 10 to 20 minutes. For example, it can be 10 minutes, 12 minutes, 14 minutes, 16 minutes, 18 minutes, or 20 minutes.

[0018] Preferably, the rate of the second pressurization is 10~20 MPa / min. For example, it can be 10 MPa / min, 12 MPa / min, 14 MPa / min, 16 MPa / min, 18 MPa / min or 20 MPa / min, etc.

[0019] Preferably, the pressure of the second pressure holding is 100~800 MPa, and the time is 3~20 min. The 100~800 MPa can be, for example, 100 MPa, 200 MPa, 300 MPa, 400 MPa, 500 MPa, 600 MPa, 700 MPa, or 800 MPa.

[0020] Preferably, the total time for the first and second pressure holding periods is 3 to 20 minutes. The 3 to 20 minutes can be, for example, 3 minutes, 5 minutes, 10 minutes, 15 minutes, or 20 minutes.

[0021] Preferably, the rate of re-depressurization is 5~20 MPa / min, and the pressure after depressurization is 0~0.5 MPa. The 5~20 MPa / min can be, for example, 5 MPa / min, 7 MPa / min, 9 MPa / min, 11 MPa / min, 13 MPa / min, 15 MPa / min, 17 MPa / min, 19 MPa / min, or 20 MPa / min. The 0~0.5 MPa can be, for example, 0 MPa, 0.1 MPa, 0.2 MPa, 0.3 MPa, 0.4 MPa, or 0.5 MPa.

[0022] Preferably, the collection bag includes a bag body and a threaded bag head, with the threaded bag head located at the top of the bag body.

[0023] In use, the collection bag of this invention connects the output end of the breast pump to the filling cap of the inner freeze-drying bag. Breast milk is then directly injected into the inner bag using the breast pump. After injection, the cap is closed to prevent leakage of breast milk or the entry of outside air. After collection, there is no need to transfer the breast milk; the double-layered collection bag containing the breast milk can be directly stored in a refrigerator or transported via a transport line. The outer antibacterial protective film isolates external contamination and inhibits microbial growth during storage and transport.

[0024] Preferably, the bag body includes an inner bag and an outer bag arranged facing inwards, with the outer edge of the inner bag and the inner edge of the outer bag fitted together.

[0025] Preferably, the outer bag is made of an antibacterial polyethylene terephthalate film.

[0026] The outer membrane of this invention is made of plastic film containing food-grade antibacterial ingredients, which has antibacterial and anti-collision functions and is suitable for refrigerator storage and transportation line transfer needs.

[0027] Preferably, the inner bag comprises an upper film and a lower film.

[0028] In this invention, the inner bag is composed of an upper film and a lower film, that is, the two sides of the inner bag are made of different materials. During freeze drying, the lower film faces down and is bonded to the plate of the freeze dryer to conduct temperature, while the upper film faces up and does not come into contact with the machine, and is responsible for air permeability.

[0029] Preferably, the material of the upper membrane includes expanded polytetrafluoroethylene membrane.

[0030] Preferably, the material of the lower membrane includes a low-density polyethylene film.

[0031] In this invention, the inner bag serves as a material storage bag. The upper membrane is a hydrophobic and breathable membrane, allowing only water vapor to pass through while blocking microorganisms and liquids. The lower membrane is a flexible heat transfer membrane, ensuring uniform heat transfer during freezing and drying, resulting in more uniform sample freezing.

[0032] Preferably, the vacuum sealing process includes vacuum sealing after tearing off the outer bag of the collection bag.

[0033] In this invention, the outer antibacterial protective film is removed during vacuum sealing, leaving only the inner freeze-dried functional bag containing breast milk. The inner bag is then placed in a vacuum packaging machine for vacuum sealing, further isolating it from the external environment and preparing it for subsequent sterilization.

[0034] Preferably, the collection bag is pre-frozen, heated for sublimation drying, and then the biological material is dried a second time to obtain the product.

[0035] Preferably, the pre-freezing temperature is -40℃ to -50℃, and the time is 2 to 4 hours. The -40℃ to -50℃ range can be, for example, -40℃, -42℃, -44℃, -46℃, -48℃, or -50℃. The 2 to 4 hours can be, for example, 2 hours, 2.2 hours, 2.4 hours, 2.6 hours, 2.8 hours, 3.0 hours, 3.2 hours, 3.4 hours, 3.6 hours, 3.8 hours, or 4.0 hours.

[0036] The flexible heat transfer properties of the inner bag lower membrane in this invention can ensure that breast milk freezes evenly and that water is completely converted into ice crystals.

[0037] Preferably, the sublimation drying process involves a vacuum degree of 10-30 MPa, a temperature of -10℃ to 0℃, and a time of 8-12 hours. The 10-30 MPa can be, for example, 10 MPa, 15 MPa, 20 MPa, 25 MPa, or 30 MPa. The -10℃ to 0℃ can be, for example, 0℃, 1℃, 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, 9℃, or 10℃. The 8-12 hours can be, for example, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours.

[0038] At this time, the water vapor generated by the sublimation of ice crystals in breast milk can escape directly to the condensation system of the freeze dryer through the hydrophobic and breathable membrane on the inner bag, thus removing moisture. The inner bag is always sealed, and the breast milk does not come into contact with the outside world.

[0039] Preferably, the secondary drying temperature is 20℃~30℃, and the time is 2~4 hours. The 20℃~30℃ can be, for example, 20℃, 22℃, 24℃, 26℃, 28℃, or 30℃. The 2~4 hours can be, for example, 2 hours, 2.5 hours, 3 hours, 3.5 hours, or 4 hours.

[0040] In this invention, secondary drying is used to further remove residual bound water from breast milk, reducing the water content of breast milk to 1%-3%, thus forming lyophilized breast milk powder.

[0041] In a second aspect, the present invention provides an application of the fully enclosed method for cold sterilization and freeze-drying of breast milk according to the first aspect in the preparation of freeze-dried breast milk.

[0042] The method of this invention can be applied to scenarios such as home breast milk storage, professional breast milk bank operation, and the infant food industry.

[0043] Home breast milk storage: Breastfeeding mothers can collect excess breast milk using a double-layered collection bag. The outer antibacterial film can protect the milk for short-term refrigeration. For long-term storage, the collection bag can be transferred to a processing station, where it will be sterilized and freeze-dried. The freeze-dried powder will then be sealed in an inner bag and stored at room temperature.

[0044] Professional breast milk bank operation: After receiving donated double-layer collection bags, the breast milk bank removes the outer film, vacuum seals, sterilizes and freezes the bags according to the procedure. The final freeze-dried powder can be stored at room temperature for 24 months, which greatly extends the storage period and there is no risk of contamination throughout the process. It can be safely supplied to premature infants and sick infants.

[0045] Infant food industry: As a large-scale production process for high-end freeze-dried breast milk powder, standardized double-layer collection bags and intermittent cold sterilization parameters ensure the stability of nutrient retention and microbiological indicators for each batch of products, meeting the needs of industrial production.

[0046] Compared with the prior art, the present invention has at least the following beneficial effects:

[0047] 1. The present invention employs cold sterilization, which, without relying on heat and avoiding the destruction of nutrients by heat, utilizes the incompressible properties of food-grade hydraulic media to uniformly transfer pressure to the microbial cells in breast milk, thereby achieving highly efficient sterilization by destroying the cell membrane structure, enzyme active sites, and genetic material of the microorganisms.

[0048] 2. This invention employs cold sterilization implemented within experimentally verified parameter ranges, which specifically protects stress-sensitive components such as IgA and IgG, with a total immunoglobulin retention rate of ≥98% and an interleukin retention rate of ≥99%. Compared with traditional heat sterilization methods, the nutrient retention rate is increased by 30-50%, maximizing the preservation of the natural nutrition and biological activity of breast milk.

[0049] 3. In this invention, breast milk is sealed within the inner freeze-drying bag from collection to production as freeze-dried powder, without any transfer operations. The inner bag is hydrophobic, breathable, and provides flexible heat transfer, balancing freeze-drying requirements with airtight protection, completely eliminating the risk of external contamination. Microbiological indicators are far superior to standards. The inner bag serves as both the collection bag and the freeze-drying bag, simplifying the process; the outer antibacterial film is suitable for refrigerated storage and transportation, eliminating the need for a special cold chain; and the final freeze-dried powder can be stored in a sealed container at room temperature for 24 months, significantly reducing energy consumption and costs associated with storage and transportation.

[0050] 4. The double-layer structure design of the collection bag of the present invention takes into account home collection, batch processing and industrial production. The intermittent sterilization parameters have been verified by actual testing and have strong stability, which can meet the standardization requirements of different scenarios. Attached Figure Description

[0051] Figure 1 A schematic diagram of the front structure of a freeze-drying bag for collecting breast milk.

[0052] Figure 2 A schematic diagram showing the structure of a freeze-dried bag for collecting breast milk, with one corner of the outer bag torn open.

[0053] Figure 3 A schematic diagram showing the placement of freeze-dried bags for collecting breast milk on the freeze dryer's trays.

[0054] Among them, 1-outer bag; 2-inner bag; 3-threaded bag head; 4-gradient mark; 5-sealed outlet; 6-upper film; 7-lower film. Detailed Implementation

[0055] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0056] Example 1

[0057] This embodiment involves cold sterilization and freeze-drying of breast milk.

[0058] (1) Structure of collection bag and pretreatment

[0059] Connect the breast pump to the threaded end of the collection bag, and collect breast milk into the bag using the pump. Close the cap to prevent leakage of breast milk or entry of outside air. The upper film is an expanded polytetrafluoroethylene (ePTFE) membrane (0.1 μm pore size, 0.1 mm thickness), and the lower film is a low-density polyethylene (LDPE) membrane (0.15 mm thickness). The outer film is an antibacterial polyethylene terephthalate (PET) membrane, 0.1 mm thick, which has good impact resistance and antibacterial properties. The specific structure is as follows... Figure 1 , Figure 2 and Figure 3 As shown.

[0060] The outer antibacterial protective film of the collection bag is removed, leaving only the inner freeze-drying functional bag containing breast milk. The inner bag is then placed in a vacuum packaging machine for vacuum sealing to further isolate it from the external environment and prepare it for subsequent sterilization.

[0061] (2) Cold sterilization

[0062] The system employs a closed pressure processing chamber, allowing the vacuum-sealed inner freeze-dried functional inner bag (containing breast milk) to be placed directly into the chamber without removing the breast milk, thus achieving a completely closed sterilization process.

[0063] Pretreatment: Pure water is injected into the sealed pressure processing chamber (Beijing Suyuan Zhongtian Technology Co., Ltd. CQC30L-600 high-pressure food processing device) to stabilize the medium temperature at 5°C, ensuring that the hydraulic medium completely covers the inner freeze-drying inner bag, and removing air from the chamber to avoid uneven local pressure during sterilization.

[0064] Intermittent pressurization operation: Activate the pressurization system to increase the chamber pressure to 400 MPa at a rate of 15 MPa / min, and maintain the pressure for the first time for 10 minutes. After the pressure maintenance, depressurize to 0 MPa at a rate of 7 MPa / min and pause for 15 minutes. Then, pressurize again to 400 MPa at a rate of 15 MPa / min for the second pressure maintenance, lasting 10 minutes. After the second pressure maintenance, slowly depressurize to 0 MPa at a rate of 5 MPa / min, remove the sterilized inner freeze-drying bag (the breast milk remains sealed inside), and proceed to the subsequent freeze-drying stage.

[0065] (3) Fully enclosed freeze drying

[0066] Pre-freezing treatment: Place the inner freeze-drying inner bag into the pre-freezing chamber of the freeze dryer and freeze at -45℃ for 3 hours. The flexible heat transfer characteristics of the lower membrane of the inner bag can ensure that the breast milk is frozen evenly and the water is completely converted into ice crystals.

[0067] Vacuum sublimation drying: After pre-freezing, turn on the vacuum system of the freeze dryer and control the vacuum degree in the freeze drying chamber at 20Pa. At the same time, slowly raise the temperature of the heating plate to -5℃ and maintain it for 10 hours.

[0068] Secondary drying: After sublimation drying is completed, the temperature of the heating plate is raised to 25°C and maintained for 3 hours to form lyophilized breast milk powder.

[0069] Example 2

[0070] This embodiment involves cold sterilization and freeze-drying of breast milk.

[0071] (1) Structure of collection bag and pretreatment

[0072] Connect the breast pump to the threaded end of the collection bag, and collect breast milk into the bag using the pump. Close the cap to prevent leakage of breast milk or entry of outside air. The upper film is an expanded polytetrafluoroethylene (ePTFE) membrane (0.1 μm pore size, 0.1 mm thickness), and the lower film is a low-density polyethylene (LDPE) membrane (0.15 mm thickness). The outer film is an antibacterial polyethylene terephthalate (PET) membrane, 0.1 mm thick, which has good impact resistance and antibacterial properties. The specific structure is as follows... Figure 1 , Figure 2 and Figure 3 As shown.

[0073] The outer antibacterial protective film of the collection bag is removed, leaving only the inner freeze-drying functional bag containing breast milk. The inner bag is then placed in a vacuum packaging machine for vacuum sealing to further isolate it from the external environment and prepare it for subsequent sterilization.

[0074] (2) Cold sterilization

[0075] The system employs a closed pressure processing chamber, allowing the vacuum-sealed inner freeze-dried functional inner bag (containing breast milk) to be placed directly into the chamber without removing the breast milk, thus achieving a completely closed sterilization process.

[0076] Pretreatment: Inject plant-based hydraulic oil into the sealed pressure treatment chamber, stabilize the medium temperature at 0℃, ensure that the hydraulic medium completely covers the inner freeze-drying functional bag, remove air from the chamber, and avoid uneven local pressure during sterilization.

[0077] Intermittent pressurization operation: Activate the pressurization system to increase the chamber pressure to 800 MPa at a rate of 20 MPa / min, and maintain the pressure for the first time for 1 minute. After the pressure maintenance, depressurize to 0 MPa at a rate of 5 MPa / min and pause for 10 minutes. Then, pressurize again to 100 MPa at a rate of 10 MPa / min and maintain the pressure for the second time for 2 minutes. After the second pressure maintenance, slowly depressurize to 0 MPa at a rate of 10 MPa / min, remove the sterilized inner freeze-drying bag (breast milk remains sealed inside the bag), and proceed to the subsequent freeze-drying stage.

[0078] (3) Fully enclosed freeze drying

[0079] Pre-freezing treatment: Place the inner freeze-drying inner bag into the pre-freezing chamber of the freeze dryer and freeze it at -40℃ for 4 hours. The flexible heat transfer characteristics of the lower membrane of the inner bag can ensure that the breast milk is frozen evenly and the water is completely converted into ice crystals.

[0080] Vacuum sublimation drying: After pre-freezing, turn on the vacuum system of the freeze dryer and control the vacuum degree in the freeze drying chamber at 10Pa. At the same time, slowly raise the temperature of the heating plate to -10℃ and maintain it for 8 hours.

[0081] Secondary drying: After sublimation drying, the temperature of the heating plate is raised to 30°C and maintained for 2 hours to form lyophilized breast milk powder.

[0082] Example 3

[0083] This embodiment involves cold sterilization and freeze-drying of breast milk.

[0084] (1) Structure of collection bag and pretreatment

[0085] Connect the breast pump to the threaded end of the collection bag, and collect breast milk into the bag using the pump. Close the cap to prevent leakage of breast milk or entry of outside air. The upper film is an expanded polytetrafluoroethylene (ePTFE) membrane (0.1 μm pore size, 0.1 mm thickness), and the lower film is a low-density polyethylene (LDPE) membrane (0.15 mm thickness). The outer film is an antibacterial polyethylene terephthalate (PET) membrane, 0.1 mm thick, which has good impact resistance and antibacterial properties. The specific structure is as follows... Figure 1 , Figure 2 and Figure 3 As shown.

[0086] The outer antibacterial protective film of the collection bag is removed, leaving only the inner freeze-drying functional bag containing breast milk. The inner bag is then placed in a vacuum packaging machine for vacuum sealing to further isolate it from the external environment and prepare it for subsequent sterilization.

[0087] (2) Cold sterilization

[0088] The system employs a closed pressure processing chamber, allowing the vacuum-sealed inner freeze-dried functional inner bag (containing breast milk) to be placed directly into the chamber without removing the breast milk, thus achieving a completely closed sterilization process.

[0089] Pretreatment: Inject food-grade propylene glycol into the sealed pressure treatment chamber, stabilize the medium temperature at 10°C, ensure that the hydraulic medium completely covers the inner freeze-drying functional bag, remove air from the chamber, and avoid uneven local pressure during sterilization.

[0090] Intermittent pressurization operation: Activate the pressurization system to increase the chamber pressure to 100 MPa at a rate of 10 MPa / min, and maintain the pressure for the first time for 12 minutes. After the pressure maintenance, depressurize to 0 MPa at a rate of 10 MPa / min and pause for 20 minutes. Then, pressurize again to 800 MPa at a rate of 20 MPa / min and maintain the pressure for the second time for 3 minutes. After the second pressure maintenance, slowly depressurize to 0 MPa at a rate of 20 MPa / min, remove the sterilized inner freeze-drying bag (breast milk remains sealed inside), and proceed to the subsequent freeze-drying stage.

[0091] (3) Fully enclosed freeze drying

[0092] Pre-freezing treatment: Place the inner freeze-drying inner bag into the pre-freezing chamber of the freeze dryer and freeze at -50℃ for 2 hours. The flexible heat transfer characteristics of the lower membrane of the inner bag can ensure that the breast milk is frozen evenly and that the water is completely converted into ice crystals.

[0093] Vacuum sublimation drying: After pre-freezing, turn on the vacuum system of the freeze dryer and control the vacuum degree in the freeze drying chamber at 30Pa. At the same time, slowly raise the temperature of the heating plate to 0℃ and maintain it for 12 hours.

[0094] Secondary drying: After sublimation drying, the temperature of the heating plate is raised to 20°C and maintained for 4 hours to form lyophilized breast milk powder.

[0095] Test Example 1

[0096] This test case explores the effects of the above embodiments.

[0097] This test case investigates and compares the nutritional components and microbial contamination rate of the freeze-dried breast milk powder from the above-described embodiments. Immunoglobulin G and immunoglobulin A were detected using enzyme-linked immunosorbent assay (ELISA), and the total bacterial count was determined according to the method in GB4789.2-2016.

[0098] The specific results are shown in Table 1.

[0099] Table 1

[0100]

[0101] The results above demonstrate that the method of this invention, which combines cold sterilization with an integrated non-contact, anti-contamination freeze-drying bag for collecting breast milk, achieves efficient sterilization and ensures the safety of breast milk microorganisms while maximizing the preservation of heat-sensitive nutrients and bioactive substances in breast milk.

[0102] Test Example 2

[0103] This test case explores the impact of different sterilization methods on the effectiveness.

[0104] To investigate the effects of different sterilization methods, this test case used fresh breast milk as a sample and divided it into three groups: cold sterilization, low-temperature long-time pasteurization, and ultra-high temperature instantaneous sterilization. The experimental procedures for each group are shown below:

[0105] Cold sterilization method group: Sterilization is performed using the cold sterilization method in Example 1 of this invention. A closed pressure processing chamber is used, and the vacuum-sealed inner freeze-dried functional inner bag (containing breast milk) is directly placed into the processing chamber without removing the breast milk, thus achieving a completely closed sterilization process.

[0106] Pretreatment: Pure water is injected into the sealed pressure processing chamber (Beijing Suyuan Zhongtian Technology Co., Ltd. CQC30L-600 high-pressure food processing device) to stabilize the medium temperature at 5°C, ensuring that the hydraulic medium completely covers the inner freeze-drying inner bag, and removing air from the chamber to avoid uneven local pressure during sterilization.

[0107] Intermittent pressurization operation: Activate the pressurization system to increase the chamber pressure to 400 MPa at a rate of 15 MPa / min, and maintain the pressure for the first time for 10 minutes. After the pressure maintenance, depressurize to 0 MPa at a rate of 7 MPa / min and pause for 15 minutes. Then, pressurize again to 400 MPa at a rate of 15 MPa / min and maintain the pressure for the second time for 10 minutes. After the second pressure maintenance, slowly depressurize to 0 MPa at a rate of 5 MPa / min and remove the sterilized inner freeze-dried functional bag.

[0108] Low-temperature, long-term pasteurization group: Fresh breast milk (using steam in a sterilization tank) is heated to 62.5°C and maintained at this temperature for 30 minutes. After sterilization, the breast milk needs to be cooled to about 4°C immediately to inhibit the growth and development of residual bacteria and extend the shelf life of the product.

[0109] Ultra-high temperature instantaneous sterilization: Fresh breast milk is heated to 135°C (using high-pressure steam at 205.8 kPa in an autoclave) and held for 5 seconds. After sterilization, the breast milk needs to be cooled to about 4°C immediately to inhibit the growth and development of residual bacteria and extend the product's shelf life.

[0110] After lyophilizing the samples from each group according to the method in Example 1, the retention rate of nutrients and the rate of microbial contamination were determined. Immunoglobulin G, immunoglobulin A, and total immunoglobulin were detected using enzyme-linked immunosorbent assay (ELISA). Lysozyme was detected according to GB / T 25879–2010. Lactoferrin was detected according to GB 5009.299-2024 using heparin affinity column combined with high-performance liquid chromatography (HPLC). Interleukin-2, interleukin-4, and interleukin-6 were detected using ELISA kits. Total protein was detected using the Kjeldahl method. Albumin was detected using HPLC. DHA and EP were detected using gas chromatography. Human milk oligosaccharides were detected according to T / CIFST025-2024 using ion chromatography-pulse amperometric detection. Total bacterial count was detected according to GB 4789.2-2016. Escherichia coli was detected according to GB... GB 4789.3-2016 specifies the plate count method for detection. Staphylococcus aureus was detected according to the method in GB 4789.10-2016, and Salmonella was detected according to the method in GB 4789.4-2016. Specific results are shown in Tables 2 and 3.

[0111] Table 2

[0112]

[0113] Table 3

[0114]

[0115] The experimental results above show that the intermittent cold sterilization method of the present invention has a much higher retention rate of stress-sensitive components such as IgA and IgG, as well as nutrients such as interleukin and total protein in breast milk than pasteurization and traditional heat sterilization methods, and can maintain the nutritional value of breast milk to the greatest extent.

[0116] This invention completely avoids the risk of microbial contamination in the transfer and open freeze-drying stages of traditional methods by sealing breast milk in the inner bag throughout the process. Furthermore, cold sterilization can effectively kill pathogenic bacteria. The microbial indicators of the final product not only meet the standards but are also far superior to those of traditional methods, thus satisfying the extremely high hygiene requirements for infant feeding.

[0117] Test Example 3

[0118] This test case explores the effect of different sterilization parameters on the sterilization effect.

[0119] This invention employs a process of pressurization, pressure holding, and pressure release to achieve a sterilization rate of ≥99.99% within a certain parameter range while maximizing the protection of pressure-sensitive components. Pressure value, medium temperature, and pressure holding time are the core parameters affecting sterilization effectiveness and nutrient retention. To verify the superiority of this invention, the following experiments were conducted.

[0120] (1) Effect of pressure value

[0121] This test case investigates the effect of pressure value on the effect based on Example 1. The pressure value of the first pressure holding and the pressure value of the second pressure holding are kept the same, namely 50 MPa, 100 MPa, 800 MPa and 1000 MPa respectively. The specific results are shown in Table 4.

[0122] Table 4

[0123]

[0124] The results show that the sterilization rate at 50 MPa pressure does not meet the sterilization requirements for infant food; sterilization needs can be met at pressures above 600 MPa. As the pressure increases, the retention rates of IgA and IgG gradually decrease. Lactoferrin has strong pressure tolerance, and its retention rate remains stable at over 96% at all pressures. Therefore, the pressure range of 100 MPa to 800 MPa is the optimal choice to balance sterilization effect and nutrient retention.

[0125] (2) Influence of medium temperature

[0126] This test case investigates the effects of medium temperatures of 0℃, 10℃, and 15℃ on the effect, based on Example 1. The specific results are shown in Table 5.

[0127] Table 5

[0128]

[0129] The results above show that: increasing the medium temperature accelerates the thermal motion of protein molecules. Although it does not affect the sterilization rate, it leads to a decrease in the conformational stability of proteins such as IgA and IgG. The retention rate of IgA decreases significantly when the temperature is above 10℃. At the same time, DHA, as an unsaturated fatty acid, is easily oxidized, resulting in a decrease in retention rate. Therefore, a medium temperature range of 0℃-10℃ can simultaneously ensure sterilization stability and nutritional protection.

[0130] (3) Effect of holding time

[0131] Based on Example 1, this test case investigated the total holding time for the first and second holding pressures when it was 2 min, 3 min, 20 min, or 25 min, and the specific results are shown in Table 6.

[0132] Table 6

[0133]

[0134] The results above show that when the total holding time is less than 3 minutes, the sterilization rate does not meet the standard; more than 15 minutes can meet the sterilization requirements, but when the time exceeds 20 minutes, the retention rates of IgA and IgG decrease with the extension of pressure exposure time. Therefore, 3-20 minutes is the total holding time range that ensures thorough sterilization and the highest nutrient retention rate.

[0135] Test Example 4

[0136] This test case explores the effects of different freeze-drying methods on the results.

[0137] In existing technologies, breast milk freeze-drying mainly employs an open-type freeze-drying method, where the breast milk directly contacts the freeze-drying chamber environment. This invention utilizes a fully enclosed freeze-drying technology, relying on the hydrophobic and breathable membrane of the breast milk freeze-drying bag to achieve water sublimation. Meanwhile, the pre-freezing temperature, sublimation vacuum degree, and secondary drying temperature are key parameters in the freeze-drying process. To verify the superiority of this invention, the following experiments were conducted.

[0138] (1) The effect of freeze-drying method

[0139] To investigate the effects of different freeze-drying methods on the results, this test case used fresh breast milk as the sample, and was divided into a fully enclosed group and an open group. The experimental procedures for each group are shown below:

[0140] Fully enclosed group: the method of Example 1.

[0141] Open-top group: Based on Example 1, the freeze-drying method is adjusted by replacing the breast milk freeze-drying bag with an ordinary tin foil box (open-top container), while other conditions and parameters remain unchanged.

[0142] The retention rate of nutrients, the rate of microbial contamination, and the water content of the samples after treatment in each group were tested. The specific results are shown in Table 7.

[0143] Table 7

[0144]

[0145] The results above show that the nutrient retention rates are similar between the two modes, indicating that the impact of freeze-drying on breast milk nutrition mainly depends on parameters such as temperature and vacuum level, and is unrelated to the freeze-drying method. In the traditional open freeze-drying method, breast milk is in direct contact with the air inside the freeze-drying chamber. Even if the chamber is sterile, there is still a risk of backflow of outside air when the pressure is released. The total number of microorganisms is more than 10 times higher than that in the fully enclosed mode, and Staphylococcus aureus was detected in some samples. In contrast, the fully enclosed mode completely isolates external contamination, and the total number of microorganisms meets food standards.

[0146] (2) Effect of pre-freezing temperature

[0147] This test case investigates the effects of -30℃, -40℃ and -50℃ on the effect based on Example 1. The specific results are shown in Table 8.

[0148] Table 8

[0149]

[0150] The results above show that: excessively high pre-freezing temperatures cause large ice crystals to form in breast milk, damaging the structure of mammary gland cells, making it prone to clumping and exceeding the water content standard after freeze-drying; while excessively low pre-freezing temperatures can form uniform fine ice crystals, they will prolong the freeze-drying time, increase energy consumption, and the DHA retention rate is not significantly different from that at -40℃; therefore, -40℃ is the optimal choice, which can achieve uniform freezing within 3 hours and results in the best product condition after freeze-drying.

[0151] (3) The effect of sublimation vacuum

[0152] This test case investigates the effects of 10 Pa, 20 Pa, or 30 Pa on the effect based on Example 1. The specific results are shown in Table 9.

[0153] Table 9

[0154]

[0155] The results above show that: the higher the vacuum level, the faster the sublimation rate, the shorter the freeze-drying time, and the lower the water content, resulting in stronger product stability; when the vacuum level decreases, the sublimation rate decreases, the freeze-drying time increases, and due to the untimely sublimation of water, vapor condenses on the surface, causing local collapse, but there is no significant difference in IgG retention rate.

[0156] (4) Effect of secondary drying temperature

[0157] This test case investigates the effects of 15℃, 25℃, or 35℃ on the effect, based on Example 1. The specific results are shown in Table 10.

[0158] Table 10

[0159]

[0160] The results above show that the core objective of secondary drying is to remove bound water from breast milk. If the temperature is too low, the drying time needs to be extended, and it is difficult to reduce the water content to below 2%. Within the scope of this invention, the water content can be reduced to 1.5% within 3 hours, while maximizing the protection of lactoferrin activity.

[0161] In summary, this invention combines cold sterilization with an integrated non-contact, anti-contamination freeze-drying bag for collecting breast milk, achieving efficient sterilization and ensuring the safety of breast milk microorganisms while maximizing the preservation of heat-sensitive nutrients and bioactive substances in breast milk.

[0162] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for cold sterilization and freeze-drying of breast milk in a fully enclosed environment, characterized in that, The method includes injecting breast milk into a collection bag, vacuum sealing it, sterilizing it with cold sterilization, and then freeze-drying it to obtain freeze-dried breast milk. The cold sterilization process involves placing the collection bag into a sealed pressure chamber and subjecting it to two pressure treatments.

2. The method for cold sterilization and freeze-drying of breast milk in a fully enclosed environment according to claim 1, characterized in that, The cold sterilization process involves placing the collection bag into a sealed pressure treatment chamber and injecting hydraulic medium for the first pressurization and the first pressure holding, followed by depressurization and a pause, then a second pressurization and a second pressure holding, and finally depressurization and removal.

3. The method for cold sterilization and freeze-drying of breast milk in a fully enclosed environment according to claim 2, characterized in that, The hydraulic medium includes any one or a combination of at least two of the following: water, vegetable-based hydraulic oil, food-grade glycerin, or food-grade propylene glycol. Preferably, the temperature of the hydraulic medium is 0℃~10℃.

4. The method for cold sterilization and freeze-drying of breast milk in a fully enclosed environment according to claim 2 or 3, characterized in that, The rate of the first pressurization is 10~20 MPa / min; Preferably, the pressure of the first pressure holding is 100~800 MPa; Preferably, the depressurization rate is 5~10 MPa / min, and the pressure after depressurization is 0~0.5 MPa; Preferably, the pause time is 10 to 20 minutes.

5. The method for cold sterilization and freeze-drying of breast milk using a fully enclosed process according to any one of claims 2-4, characterized in that, The rate of the second pressurization is 10~20 MPa / min; Preferably, the pressure of the second pressure holding is 100~800 MPa; Preferably, the total time for the first and second pressure holding periods is 3 to 20 minutes; Preferably, the rate of re-depressurization is 5~20 MPa / min, and the pressure after depressurization is 0~0.5 MPa.

6. The method for cold sterilization and freeze-drying of breast milk using a fully enclosed process according to any one of claims 1-5, characterized in that, The collection bag includes a bag body and a threaded bag head, with the threaded bag head located at the top of the bag body. Preferably, the bag body includes an inner bag and an outer bag that are arranged facing inwards, with the outer edge of the inner bag and the inner edge of the outer bag fitting together. Preferably, the outer bag is made of an antibacterial polyethylene terephthalate film; Preferably, the inner bag comprises an upper film and a lower film; Preferably, the material of the upper membrane includes expanded polytetrafluoroethylene membrane; Preferably, the material of the lower membrane includes a low-density polyethylene film.

7. The method for cold sterilization and freeze-drying of breast milk using a fully enclosed process according to any one of claims 1-6, characterized in that, The vacuum sealing process includes vacuum sealing after tearing off the outer bag of the collection bag.

8. The method for cold sterilization and freeze-drying of breast milk using a fully enclosed process according to any one of claims 1-7, characterized in that, The collection bag is pre-frozen, heated for sublimation drying, and then dried again by biological treatment to obtain the product. Preferably, the pre-freezing temperature is -40℃ to -50℃, and the time is 2 to 4 hours.

9. The method for cold sterilization and freeze-drying of breast milk in a fully enclosed manner according to claim 8, characterized in that, The sublimation drying process involves a vacuum of 10-30 MPa, a temperature of -10℃ to 0℃, and a time of 8-12 h. Preferably, the secondary drying temperature is 20℃~30℃ and the time is 2~4 hours.

10. The application of a fully enclosed method for cold sterilization and freeze-drying of breast milk according to any one of claims 1-9 in the preparation of freeze-dried breast milk.

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