Preservation system and method for anaerobic microorganisms

Abstract

The present invention relates to a method for maintaining anaerobic microorganism viability in a food product, such as milk, comprising storing the food product in a sealable storage container comprising an oxygen scavenging system. The present invention also relates to an oxygen scavenging system comprising a body (522) (the body (522) comprising a first end, a second end (526) and a first internal compartment), a lid (the lid being adapted to close the first end of the body), a gas permeable liquid impermeable membrane (527) (the membrane being adapted to close the second end of the body) and an oxygen scavenging material (OSM) (the OSM being adapted to be placed within the first internal compartment).

Description

Technical Field

[0001] This invention relates to an oxygen removal system and method for maintaining the survival rate of anaerobic microorganisms, particularly in food. Background Technology

[0002] Breast milk is the best source of nutrition for infants. In addition to essential nutrients (including proteins, fats, carbohydrates, vitamins, and minerals) and bioactive components, breast milk contains a wide variety of bacteria, fungi, and other microorganisms that constitute the milk microbiome. Anaerobic microorganisms present in human breast milk refer to microbial communities that thrive under anaerobic conditions. The greatest contribution of the maternal microbiome to the infant comes from the microorganisms in breast milk, accounting for more than 30% of the first infant's microbiome (Bogart et al., 2023, Cell and Host Microbiology, Vol. 31, No. 3, March 8, 2023, pp. 447-460). More than 200 species of bacteria are known to exist in human breast milk, including both aerobic and anaerobic bacteria belonging to different taxa, including Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. Some common genera include Bifidobacterium, Lactobacillus, Clostridium, Staphylococcus, Streptococcus, Veillonella, and Prevotella.

[0003] While aerobic (aerobic) bacteria also exist, anaerobic microbes play a crucial role in shaping the composition and function of the breast milk microbiome. These diverse microbes colonize the infant's gut and are essential for the development of the infant's immune system and resistance to disease. Some facultative or obligate anaerobic species belonging to the families Lactobacillus and Bifidobacteria are being explored and utilized for their health benefits in preventing and treating many pathological conditions and improving overall health. These anaerobic microbes help establish and maintain a healthy infant gut microbiome. They aid in the digestion and metabolism of complex carbohydrates in breast milk, producing short-chain fatty acids (SCFAs) as metabolic byproducts and supporting the development of the infant's immune system. Therefore, these microbes are often referred to as probiotics; they are live microbes that, when administered in adequate amounts, provide health benefits to the host.

[0004] Anaerobic bacteria in breast milk have also been found to produce enzymes that break down human milk oligosaccharides (HMOs), complex sugars abundant in breast milk but indigestible by infants. By metabolizing these HMOs, anaerobic microorganisms provide themselves with nutrients and produce metabolites that help regulate the developing immune system and promote the growth of other beneficial bacteria in the infant's gut, such as Bifidobacteria, Lactobacillus, and Lactobacillus mucosa. Furthermore, anaerobic microorganisms in breast milk may contribute to immune regulation and defense against pathogens. Some bacterial strains also produce antimicrobial compounds, such as bacteriocins, that inhibit the growth of harmful bacteria. In addition, the presence of specific anaerobic bacteria in breast milk is associated with a reduced risk of allergic diseases and improved gut health in infants.

[0005] Other components in breast milk that support a healthy gut microbiome and infant nutrition include antioxidants such as vitamin C. Antioxidants are crucial for protecting molecules involved in metabolism from losing electrons, thus preserving their function. They work by giving some of their electrons to reactive oxygen species (ROS), neutralizing the damaging effects of ROS. When breast milk is exposed to ambient oxygen, these antioxidants immediately begin to react with ROS, thus reducing the antioxidant capacity of breast milk. With this reduced antioxidant capacity, the environment becomes less favorable for the survival of probiotics, decreasing their availability to support the infant's metabolism after consuming breast milk.

[0006] The composition of anaerobic microbes in breast milk varies from person to person and may be influenced by factors such as maternal health, diet, and environmental exposure. Therefore, it is understandable that there are many complex interactions between the breast milk microbiome, maternal factors, and infant health outcomes.

[0007] Literature suggests that the method of breastfeeding—whether the mother uses a pump—is a key determinant of the composition of the milk microbiome. More specifically, a study by Moossavi et al. (Cell and Host Microbe, Vol. 25, No. 2, February 13, 2019, pp. 324-335) showed that pumping milk resulted in reduced microbiome abundance compared to direct breastfeeding, and was associated with the accumulation of potential pathogens and depletion of Bifidobacteria. Other studies have shown that when infants receive pumped breast milk, the levels of important Bifidobacteria co-occurring in breast milk and infant feces are reduced. It remains unclear why pumped breast milk contains a different microbiome. One possibility is the lack of retrograde inoculation, where the infant's oral microbiome is transferred to the breast milk. Another possibility is that certain bacteria are oxygen-sensitive and are depleted during pumping and subsequent storage.

[0008] While oxygen removal systems are known in the art, no oxygen removal system is disclosed for preserving naturally occurring anaerobic microorganisms in breast milk in a home environment. US 2014 / 0312000 discusses an oxygen-absorbing cap for preserving liquids such as wine. JP H04367472 discusses an oxygen absorber attached to the container opening. US 4287995 A discusses a container sealing member for preserving food products such as soy sauce, sake, sauces, wine, beer, juice, and vinegar. WO2014 / 041391 discusses a canister for containing active materials such as desiccants. US 5092914 A discusses an oxygen-absorbing cylinder that floats in a liquid. Schwab et al. (Front. Microbiol., November 20, 2019) discussed culturing the microbiome in human breast milk using an anaerobic container in a laboratory setting. The protocol discussed by Schwab et al. requires the use of the Anaerocultur A system placed within an anaerobic chamber.

[0009] Therefore, it is understandable that there is a need to improve the preservation of anaerobic microorganisms in freshly expressed breast milk. In particular, due to the differences in microbiome composition between individuals and its profound impact on infant health, there is a need to provide improved devices and methods for preserving expressed breast milk during use. Summary of the Invention

[0010] The inventors have developed a novel system and method for maintaining the survival rate of beneficial anaerobic microorganisms in culture media. Furthermore, this system has demonstrated its practicality in preserving the antioxidant content of foods and beverages beyond their typically expected shelf life.

[0011] In a first aspect, the present invention provides a method for maintaining the survival rate of anaerobic microorganisms in breast milk, the method comprising storing the breast milk in a sealable storage container, wherein the sealable storage container includes an oxygen removal system (OSS).

[0012] A sealable storage container can be selected from: a vessel, a bottle, a sealable bag, or a pouch. Appropriately, a sealable storage container is a baby bottle.

[0013] This milk includes mammalian milk. Specifically, it refers to human breast milk. More precisely, it refers to freshly expressed breast milk.

[0014] Anaerobic microorganisms include species selected from anaerobic bacteria, optionally from the Bifidobacteriaceae and / or Lactobacilliceae families, wherein the Bifidobacteriaceae family is suitably selected from Bifidobacterium infantis (Bifidobacterium infantis). Bifidobacterium infantis ), Bifidobacterium breve Bifidobacterium breve Bifidobacterium longum ( Bifidobacterium longum ), Bifidobacterium lactis ( Bifidobacterium lactis Bifidobacterium animalis ( Bifidobacterium animaliBifidobacterium densiflorum ( Bifidobacterium dentium ) and / or Bifidobacterium bifidum ( Bifidobacterium bifidum ).

[0015] In a second aspect, the present invention provides an oxygen removal system (OSS) for use with a sealable storage container to maintain the survival rate of anaerobic microorganisms in the culture medium contained therein, said OSS comprising:

[0016] a) A body including a first end and a second end, wherein the first end and the second end are open; and wherein the body defines a first internal compartment;

[0017] b) A lid adapted to seal the first end of the body;

[0018] c) An air-permeable but liquid-impermeable membrane, said membrane being adapted to seal the second end of the body; and

[0019] d) Oxygen removal material (OSM), said OSM being adapted to be placed in the first internal compartment.

[0020] In one embodiment, the OSS further includes a gasket adapted to provide a seal between an opening of the storage container and a lid adapted to close the opening of the storage container. In one embodiment, the gasket surrounds the body and extends radially outward therefrom. In another embodiment, the gasket comprises an elastic material, optionally including silicone rubber. In yet another embodiment, the gasket extends radially outward from the body for a distance of at least 1 cm, optionally at least 2 cm, and suitably at least 3 cm.

[0021] In one embodiment, the breathable but liquid-impermeable membrane is made of a hydrophobic material or includes a hydrophobic coating applied thereon. In another embodiment, the breathable but liquid-impermeable membrane further includes a hydrophobic coating.

[0022] In one embodiment, the oxygen removal material (OSM) is selected from one or more of the following: ferrous powder containing sodium chloride, optionally activated carbon, ferrous carbonate combined with a metal halide catalyst, ferrous oxide, unsaturated fatty acids, sodium bicarbonate, citric acid, and ascorbic acid.

[0023] In one embodiment, the amount of OSM used to place in the first internal compartment is at least 1g, at least 2g, suitably at least 5g, optionally at least 10g, at most 30g, suitably at most 40g, optionally at most 50g.

[0024] In one embodiment, the OSS also includes a sleeve. In one embodiment, the sleeve is truncated conical.

[0025] In one embodiment, the OSM is placed in a first internal compartment, with a first end sealed by a cap and a second end sealed by a breathable but liquid-impermeable membrane. In another embodiment, the second end is sealed by two breathable but liquid-impermeable membranes separated by a space.

[0026] In one embodiment, the breathable but liquid-impermeable membrane is covered by a liquid-impermeable cover. In one embodiment, the liquid-impermeable cover can be peeled off by a user.

[0027] In one embodiment, the storage container is selected from: a dish, a bottle, a sealable bag, or a pouch. In a preferred embodiment, the storage container is a baby bottle.

[0028] In one embodiment, the anaerobic microorganisms include species selected from anaerobic bacteria, optionally selected from the Bifidobacteriaceae and / or Lactobacilliceae families, wherein the Bifidobacteriaceae family is suitably selected from Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium lactis, Bifidobacterium dentalis, and / or Bifidobacterium bifidum.

[0029] In one embodiment, the OSS also includes an oxygen indicator. In one embodiment, the oxygen indicator is a dye.

[0030] In one embodiment, OSS selectively promotes the growth of probiotic anaerobic microorganisms and inhibits the growth of pathogenic microorganisms in a culture medium, wherein the culture medium is optionally mammalian milk, optionally human breast milk.

[0031] In one implementation, OSS retains the antioxidant content of the culture medium (such as milk).

[0032] In a third aspect, the present invention provides a method for maintaining the shelf life of freshly expressed breast milk, the method comprising storing the breast milk in a storage container, wherein the storage container includes an oxygen removal system (OSS). In one embodiment, the OSS is as described in the second aspect.

[0033] In a fourth aspect, the present invention provides a baby bottle that includes an oxygen removal system (OSS). In one embodiment, the OSS is as described in the second aspect of the invention.

[0034] In a fifth embodiment, the present invention provides a sealable container for maintaining the viability of anaerobic microorganisms therein, the container comprising the OSS of the second aspect of the present invention.

[0035] In a sixth embodiment, the present invention provides a lid for sealing a sealable container to maintain the survival rate of anaerobic microorganisms contained therein. The lid is adapted to contain OSS of the second aspect of the present invention.

[0036] In a seventh aspect, the present invention provides a method for selectively promoting the growth of probiotic anaerobic microorganisms and inhibiting the growth of pathogenic microorganisms in freshly expressed human breast milk, the method comprising storing breast milk in an infant bottle comprising the second aspect of the present invention, OSS.

[0037] Within the scope of this application, it is expressly intended that the various aspects, embodiments, examples, and alternatives listed in the foregoing paragraphs, claims, and / or the following description and drawings, particularly their various features, may be used independently or in any combination. That is, all embodiments and / or features of any embodiment may be combined in any way and / or in any combination, unless these features are incompatible. Attached Figure Description

[0038] One or more embodiments of the invention will now be described by way of example only with reference to the accompanying drawings, wherein:

[0039] Figure 1 illustrates one implementation of an oxygen removal system.

[0040] Figure 2 A storage container with a cap is shown, which includes one embodiment of an oxygen removal system according to the present invention.

[0041] Figure 3 It shows Figure 2 Bottle caps for storage containers

[0042] Figure 4 An alternative implementation of an oxygen removal system placed within a sealable container is shown.

[0043] Figure 5 An alternative implementation scheme for the oxygen removal system is shown.

[0044] Figure 6 A bottle cap including an oxygen removal system according to the present invention is shown.

[0045] Figure 7 An embodiment of an oxygen removal system with an integrated cover is shown.

[0046] Figure 8 Another embodiment of an oxygen removal system placed on the side wall of a storage container is shown.

[0047] Figure 9 An alternative embodiment of the oxygen removal system in a sealable container is shown.

[0048] Figure 10 The CFU counts of a probiotic *Bifidobacterium breve* strain isolated from human milk and exposed to oxygen at room temperature over time are shown. The mean with standard deviation is plotted. The CFU count of this strain decreases with increasing exposure time to oxygen.

[0049] Figure 11 The OD readings of the probiotic oxygen-sensitive Bifidobacterium infantis strain grown in MRS broth at 37°C are shown over time with and without this device. Mean values ​​with standard deviation are plotted. Statistical significance was assessed using two-way ANOVA, with p-values ​​indicated by asterisks (p-values ​​are indicated by asterisks). p<0.05, p<0.01, p<0.001, (p<0.0001). Growth was enhanced when stored in the device compared to when not stored.

[0050] Figure 12 The OD readings of a second probiotic, oxygen-sensitive Bifidobacterium infantis strain, grown in MRS broth at 37°C with and without this device are shown over time. Mean values ​​with standard deviations are plotted. Statistical significance was assessed using two-way ANOVA, with p-values ​​indicated by asterisks (p-values ​​are indicated by asterisks). p<0.05, p<0.01, p<0.001, (p<0.0001). Growth was enhanced when stored in the device compared to when not stored.

[0051] Figure 13 The OD readings of the probiotic *Lactobacillus mucilaginosus* strain grown in an MRS at 37°C with and without this device are shown over time. The mean with standard deviation is plotted. Statistical significance was assessed using two-way ANOVA, with p-values ​​indicated by asterisks (p-values ​​are indicated by asterisks). p<0.05, p<0.01, p<0.001, (p<0.0001). Growth was unaffected when stored in the device compared to when not stored.

[0052] Figure 14The OD readings of opportunistic bacteria *Staphylococcus aureus* grown in an MRS at 37°C are shown over time, with and without the device. The mean with standard deviation is plotted. Statistical significance was assessed using two-way ANOVA, with p-values ​​indicated by asterisks (p-values ​​are indicated by asterisks). p<0.05, p<0.01, p<0.001, (p<0.0001). Growth was inhibited when stored in the device compared to when not stored.

[0053] Figure 15 The CFU counts of the probiotic oxygen-sensitive Bifidobacterium breve strain inoculated into mixed pasteurized breast milk and stored at 4°C are shown with and without this device. Mean values ​​with standard deviation are plotted. Statistical significance was assessed using two-way ANOVA, with p-values ​​indicated by asterisks (p-values ​​are indicated by asterisks). p<0.05, p<0.01, p<0.001, (p<0.0001). The survival rate of the strain was enhanced when stored in the device compared with when not stored in the device.

[0054] Figure 16 The CFU counts of the probiotic oxygen-sensitive Bifidobacterium longum strain inoculated into mixed pasteurized breast milk and stored at 4°C are shown with and without this device. Mean values ​​with standard deviation are plotted. Statistical significance was assessed using two-way ANOVA, with p-values ​​indicated by asterisks (p-values ​​are indicated by asterisks). p<0.05, p<0.01, p<0.001, (p<0.0001). The survival rate of the strain was enhanced when stored in the device compared with when not stored in the device.

[0055] Figure 17The CFU counts of oxygen-sensitive Bifidobacterium bifidum strains inoculated into mixed pasteurized breast milk and stored at 4°C are shown with and without this device. Mean values ​​with standard deviation are plotted. Statistical significance was assessed using two-way ANOVA, with p-values ​​indicated by asterisks (p-values ​​are indicated by asterisks). p<0.05, p<0.01, p<0.001, (p<0.0001). Storing the strain in the device maintained its viability compared to storing it outside the device.

[0056] Figure 18 The CFU counts of the probiotic *Lactobacillus aeruginosa*, inoculated into mixed pasteurized breast milk and stored at 4°C, are shown with and without this device. The mean with standard deviation is plotted. Statistical significance was assessed using two-way ANOVA, with p-values ​​indicated by asterisks (*). p<0.05, p<0.01, p<0.001, (p<0.0001). Storing the strain in the device maintained its viability compared to storing it outside the device.

[0057] Figure 19 The CFU counts of the probiotic *Bifidobacterium infantis* (strain 1) inoculated into mixed pasteurized breast milk and stored at 4°C are shown with and without this device. Mean values ​​with standard deviation are plotted. Statistical significance was assessed using two-way ANOVA, with p-values ​​indicated by asterisks (p-values ​​are indicated by asterisks). p<0.05, p<0.01, p<0.001, (p<0.0001). The survival rate of strains stored in the device was higher than that when not stored in the device.

[0058] Figure 20The CFU counts of oxygen-sensitive infantile Bifidobacterium strains added to fresh mixed breast milk and stored at -20°C are shown, with and without this device. The mean with standard deviation is plotted. Statistical significance was assessed using two-way ANOVA, with p-values ​​indicated by asterisks (p-values ​​are indicated by asterisks). p<0.05, p<0.01, p<0.001, (p<0.0001). Storing the strain in the device maintained its viability compared to storing it outside the device.

[0059] Figure 21 Bar graphs are shown, illustrating the normalized abundance of (A) Bifidobacterium bifidum and (B) Bifidobacterium zoonotum species in breast milk at baseline (0 hours) and after 72 hours of storage at 4°C, with and without the device. Mean values ​​with standard errors are plotted. Statistical significance was assessed using a two-tailed t-test, with p-values ​​indicated by asterisks. (p<0.01). When the milk was stored with the device, the abundance of Bifidobacterium bifidum and Bifidobacterium animalis decreased significantly, while no significant changes were observed when it was stored with the device, indicating that it was preserved.

[0060] Figure 22 The CFU counts of pathogenic Klebsiella pneumoniae strains inoculated into pasteurized mixed breast milk and stored at room temperature over time are shown, with and without the device. Mean values ​​with standard deviation are plotted. Statistical significance was assessed using two-way ANOVA, with p-values ​​indicated by asterisks (p-values ​​are indicated by asterisks). p<0.05, p<0.01, p<0.001, (p<0.0001). The survival rate of the strain was reduced when stored in the device compared with when not stored in the device.

[0061] Figure 23 The CFU counts of pathogenic Staphylococcus aureus strains inoculated into pasteurized mixed breast milk and stored at room temperature over time are shown, with and without the device. Mean values ​​with standard deviation are plotted. Statistical significance was assessed using two-way ANOVA, with p-values ​​indicated by asterisks (p-values ​​are indicated by asterisks). p<0.05, p<0.01, p<0.001, (p<0.0001). The survival rate of the strain was reduced when stored in the device compared with when not stored in the device.

[0062] Figure 24 Antioxidant concentrations in breast milk samples stored at 4°C for three days (72 hours) with and without the device are shown. Mean values ​​with standard deviation are plotted. Statistical significance was assessed using two-way ANOVA, with p-values ​​indicated by asterisks (p-values ​​are indicated by asterisks). p<0.05, p<0.01, p<0.001, (p<0.0001). The antioxidant concentration was maintained when stored in the device compared to when not stored.

[0063] Figure 25 The diagram shows the pH values ​​of breast milk under refrigerator incubation (25A) and room temperature (25B) with and without the device.

[0064] Figure 26 A pie chart illustrates how participants identified spoilage in breast milk samples using a olfactory test. Of the 65 tests conducted, 71% (n=46) of participants perceived greater signs of spoilage in breast milk stored without the device, while 29% (n=19) selected breast milk stored with the device. This demonstrates the device's effectiveness in reducing perceived spoilage during storage.

[0065] Figure 27 A graph is shown depicting the changes in the red component of the RGB color of the oxygen-sensitive sticker (Emco) when placed in oxygen environments of (A) 5 cc and (B) 25 cc, with the required protective device present. The oxygen removal capability of the device is maintained for at least ten days compared to without the device.

[0066] Figure 28Photographs of methylene blue oxygen indicator solution filled to 15% capacity under three conditions are shown: in a vacuum (Tommee Tippee Colicsoothe Milk Air Remover), photographs of sterilized bottles with and without the device were captured at intervals of 0, 30, and 60 minutes. Figure 28 B shows the corresponding graph of pixel intensity processed in Fiji (ImageJ). The device successfully decolorized the methylene blue solution from blue to transparent within 60 minutes (indicating oxygen removal), with most of the oxygen removal occurring within 30 minutes. Small changes were observed under no-device and vacuum conditions.

[0067] Figure 29 Photographs show methylene blue oxygen indicator solutions filled in Sterifeed vials at full (FC) and half (HC) volumes using devices containing different scavengers (IMPAK, MIDKUIT, OXOID) at 0, 30, 60, and 90 minutes. All scavengers passed the test within 90 minutes, except for the IMPAK HC state. The photographs illustrate the flexibility and effectiveness of the cartridge design in scavenging oxygen with various absorbents. "Pass" indicates that the methylene blue has faded from blue to clear.

[0068] Figure 30 Photographs are shown of bottles of different sizes equipped with the device, filled to 15% capacity with methylene blue oxygen indicator solution. The photographs were taken at 0 minutes and 60 minutes. The device successfully decolorized the methylene blue solution from blue to clear within 60 minutes, and was suitable for all bottles.

[0069] Figure 31 A photograph is shown of methylene blue indicator solutions contained in Medela bottles equipped with devices of varying Tyvek membrane surface areas: small (S = 1.95 cm diameter), medium (M = 2.2 cm diameter), and large (L = 2.35 cm diameter). This trend indicates that a larger surface area results in faster deoxygenation, with complete decolorization (from blue to clear) observed within 60 minutes under all conditions.

[0070] Figure 32 Another embodiment of the oxygen removal system is shown, which includes a cover for connecting the oxygen removal system to an opening in a storage container. Detailed Implementation

[0071] The systems and apparatus described herein are particularly useful for preserving key ingredients, such as anaerobic microorganisms and antioxidant content in live foods, beverages, and biological samples, including but not limited to human breast milk.

[0072] Figure 1 illustrates an oxygen removal system (OSS) 100 suitable for storage containers to maintain the survival rate of anaerobic microorganisms in the culture medium within the storage container. The OSS includes a shell, an air-permeable but liquid-impermeable membrane, an oxygen removal material (OSM), and an oxygen indicator.

[0073] The housing 104 includes a first end 106 and a second end 108; and a first wall 110 and a second wall 114 surrounding a first internal space 112 extending between the first end 106 and the second end 108. In an alternative embodiment (not shown), the housing 104 further includes a second internal space defined between the first wall 110 and the second wall 114. In this embodiment, the second internal space extends at least to the second end 108 of the housing, optionally extending from the first end 106 of the housing to the second end 108.

[0074] In the embodiment of FIG1, the first end 106 and the second end 108 of the housing 104 are open. The second end 108 of the housing 104 is adapted to receive a gas-permeable but liquid-impermeable membrane 118 such that when a suitably sized membrane is introduced into the housing, the membrane can close the second end of the housing. In the embodiment shown in FIG1, the second end of the housing includes a lip 120 extending into the first internal space 112, such that the opening of the second end is smaller than the opening of the first end of the housing. This allows the gas-permeable but liquid-impermeable membrane to be securely placed within the first internal space, adjacent to the second end of the housing, thereby closing the second end. As used herein, “closed” means sealing an opening with a material that selectively restricts the entry or exit of gases and liquids over time. The membrane of the present invention selectively allows gases such as oxygen to pass through while substantially restricting the passage of liquids.

[0075] The first internal space of the housing is adapted to accommodate the main body 122. The main body includes a first end 124 and a second end 126, wherein the first end and the second end are open. The main body also defines a first internal compartment 128.

[0076] The first internal compartment 128 extends from a first end of the body to a second end of the body. In an alternative embodiment, the first internal compartment 128 extends from a point between a first end 124 and a second end 126 of the body 122 to the second end 126.

[0077] In the embodiment shown in Figure 1, the body further includes a second internal compartment 130. The second internal compartment extends at least to a second end of the body, optionally extending from a first end of the body to the second end. The second internal compartment is located near the first internal compartment. In an alternative embodiment, the second internal compartment at least partially surrounds the first internal compartment. Optionally, the second internal compartment completely surrounds the first internal compartment.

[0078] The cap 132 is adapted to seal the first end of the housing and / or body. As used herein, “seal” means to close an opening with a material such that the material substantially restricts the entry or exit of gases and liquids over time.

[0079] In one embodiment, the sealing ring 134 is used to seal a first end of the body and / or housing. Examples of sealing rings are known in the art, including mechanical gaskets for, for example, O-rings.

[0080] Oxygen removal material (OSM) is placed in the first internal compartment 128.

[0081] In the embodiment shown in Figure 1, the oxygen indicator may be placed in the second internal compartment 130. In an alternative embodiment, the oxygen indicator may include all or part of a printed image. Such an image may be attached to a transparent portion of the oxygen purging system so that it is visible from the outside of the system or container.

[0082] In another implementation of OSS, the body is a component of the shell.

[0083] Figure 5 Another implementation is illustrated. In this embodiment, body 522 includes a first end 524 and a second end 526. Body 522 defines a first internal compartment 528. The first internal compartment 528 extends from the first end of the body to the second end of the body. The second end 526 is sealed with a breathable but liquid-impermeable membrane 527. In this embodiment, a breathable but liquid-impermeable seal 529 is applied to the breathable but liquid-impermeable membrane 527. Oxygen scavenging material 521 is placed in the first internal compartment 528. The first end 524 is closed with a cap (not shown). Any suitable device can be used to close the first end. In this embodiment, the cap is heat-sealed to the first end 524.

[0084] In one embodiment, the oxygen removal system also includes a gasket 525. In another embodiment, the gasket is adapted to provide a seal between the opening of the storage container and a cap adapted to close the opening of the storage container.

[0085] The gasket 525 is basically circular, usually in the form of a ring. The gasket extends outward, allowing OSS to completely seal the opening of the storage container.

[0086] In the implementation scheme, the gasket is elastically stretchable and / or deformable.

[0087] In the implementation, the gasket comprises an elastic material. Suitably, the gasket is made of silicone or any other suitable elastomeric material, such as polyurethane rubber, thermoplastic polyurethane rubber, polytetrafluoroethylene, latex, polyisoprene, EPDM, SBR, etc.

[0088] In some embodiments, the gasket surrounds the body 522. In these embodiments, the gasket includes a central opening into which the OSS can be placed. In use, the gasket forms a liquid seal and a gas seal around the body.

[0089] In this implementation, the gasket is a skirted gasket. When the OSS is placed within the central opening of the gasket, the portion of the gasket near the central opening forms a skirt around the OSS.

[0090] In one embodiment, the body 522 includes a lip region 523 at its first end, such that the diameter of the body 522 in the lip region 523 is larger than the diameter of the central opening of the gasket. This allows the gasket to hold the OSS in place. Alternatively, the lip region is present within a cap of the body 522. In another embodiment, both the first end of the body and the cap include lip regions when the cap is placed to seal the first end of the body.

[0091] In the implementation scheme, the gasket extends radially outward from the body to form a flange, with an extension distance of at least 0.5 cm, optionally at least 1 cm, at least 1.5 cm, at least 2 cm, at least 2.5 cm, at least 3 cm, at least 3.5 cm, at least 4 cm, at least 4.5 cm, at least 5 cm, at least 5.5 cm, or at least 6 cm.

[0092] In the implementation, the gasket can be manually trimmed as needed to fit the opening of the storage container. This allows OSS to adapt to any suitable storage container, such as baby bottles from different manufacturers.

[0093] Figure 5 B illustrates one embodiment of the OSS (without gaskets or sleeves) of the present invention. Body 522 defines an internal compartment 528 including an OSM 521. A second end 526 of body 522 includes a breathable but liquid-impermeable membrane 527.

[0094] exist Figure 5 In C, the OSS also includes an airtight and liquid-impermeable membrane 529. This membrane prevents the airtight and liquid-impermeable membrane from contacting air before use. The user activates the OSS by removing or peeling off the airtight and liquid-impermeable membrane 529.

[0095] exist Figure 7 In the alternative embodiment shown, the cover 723 is attached to the body 722. In one embodiment, attachment is achieved via a hinge or locking mechanism.

[0096] Figure 9 Another embodiment of the OSS of the present invention is shown. The body 922 is placed inside the sleeve 927, which is placed in the central opening of the gasket 925. Figure 9B shows the OSS being placed in the opening of the baby bottle 910.

[0097] In the implementation scheme, the lid is an integral part of the main body.

[0098] exist Figure 32 In the illustrated embodiment, the OSS includes a top cover. The top cover 3220 is adapted to receive the body 3222 and attach to the opening of the storage container 3210. The top cover is made of silicone rubber. Any suitable elastomeric material can be used for the top cover (e.g., polyurethane rubber, thermoplastic polyurethane rubber, polytetrafluoroethylene, latex, polyisoprene, EPDM, SBR, etc.) such that the top cover can receive the body opposite the container opening. To use the OSS, the body is placed inside the top cover (after removing any airtight and liquid-impermeable membranes / coatings, if any), and then the top cover is inverted and attached to the opening of the storage container.

[0099] In one embodiment, the OSS includes a separate sleeve. The sleeve has a first end and a second end, both of which are open. The sleeve is shaped to accommodate a housing. In one embodiment, the sleeve is a truncated conical shape.

[0100] The sleeve completely or partially covers the outer casing. In one embodiment, the diameter of the sleeve is smaller than the cover of the outer casing, but larger than the body of the outer casing.

[0101] In one implementation, the sleeve has a lip such that the diameter of the lip is larger than the diameter of the gasket opening.

[0102] In one embodiment, the gasket can be stretched to cover the opening of the storage container. In this embodiment, the gasket is secured to the container opening by the elasticity of the material. Optionally, an O-ring can be used to hold the gasket in place. In another embodiment, the OSS can be placed on the side wall of the container. Examples are shown below. Figure 8 As shown in Figure A, container 800 includes an integrated oxygen removal system 805 in its sidewalls. This oxygen removal system includes an oxygen-permeable membrane 801, an oxygen removal material, and a body 822 surrounding the oxygen removal material 802. Container 800 also includes an airtight cover 803. Figure 8 In the embodiment shown in B, the container 800 further includes an additional layer 804 surrounding the oxygen removal system 805.

[0103] In another aspect, the present invention provides a cap for sealing a sealable container to maintain the survival rate of anaerobic microorganisms contained in a culture medium stored in the container. Figure 2 In the illustrated embodiment, the bottle cap 240 includes an opening 242 adapted to receive the oxygen purging system 204 of the second aspect of the invention. The OSS engages with the bottle cap 240 via a suitable mechanism (e.g., a push-fit mechanism). Figure 2In the middle, the O-ring 234 secures the cover 232 to the first end of the oxygen removal system 204. Figure 3 Showing more details Figure 2 The bottle cap is 240. Figure 3 In the middle, the bottle cap 340 includes a hole 342.

[0104] In another embodiment, the oxygen removal system is an integral part of the bottle cap. For example... Figure 4 As shown, the bottle cap 440 includes a first end 406 and a second end 408. A first internal space 428 is defined between the first end 406 and the second end 408. The first internal space 428 is adapted to contain an oxygen removal material 444. The oxygen removal material 444 can be placed directly within the first internal space 428. In another embodiment, the oxygen removal material can be placed in a film, a pouch, or another covering and then placed within the first internal space 428. An air-permeable but liquid-impermeable membrane 418 is located at the second end 408 of the bottle cap 440. The first end 406 of the bottle cap 440 is adapted to be sealed with a cap 432. The second end 408 of the bottle cap 440 is adapted to seal the storage container 402 to maintain the survival rate of anaerobic microorganisms or other components in the medium stored in the storage container. The sealing engagement of the cap 432 with the first end 406 can be achieved by a push-in fit or by means of... Figure 4 The threaded connection shown.

[0105] Oxygen indicators used in OSS (Optical Stimulus Oxygen Indicators) indicate the presence of oxygen (oxidized form or positive result) through color changes, which differs from indicating hypoxia (reduced form or negative result). Examples of dyes suitable for use as oxygen indicators include azure, indigo carmine, methylene blue, diphenylamine, fluorescent pink, or combinations thereof.

[0106] A permeable membrane is a membrane that selectively allows gases to pass through but not non-gaseous materials. The terms "permeable" or "breathable" refer to a material that allows gases, particularly oxygen (O2), to move from one side of the material to the other in either direction. As used herein, the related terms "breathable" and "semi-permeable" are synonymous with "permeable" and are used interchangeably herein. Typically, the material is contained within planar layers, sheets, films, or membranes. Permeable or breathable materials can be microporous or contained in composite materials, such as composite membranes. Therefore, permeable or breathable materials can be contained in layers (e.g., selective layers), coatings, or surface treatments of support material or gutter layers applied to a composite material. Thus, this type of membrane composite material has a selective layer that facilitates gas permeation, disposed on a support layer, for example, comprising multiple hollow fibers, providing mechanical strength to the entire material. Permeability is directly related to the concentration gradient of the permeate (such as a gas), the inherent permeability of the material, and the diffusion rate of the permeate within the membrane material. Examples of breathable membranes suitable for the systems and methods of the present invention include organic polymers, including low-density polyethylene, high-density polyethylene (e.g., Tyvek®), silanized alumina, and siloxane polymers, including polydimethylsiloxane (PDMS), etc.

[0107] In one embodiment, the breathable but liquid-impermeable membrane includes a hydrophobic coating. The term "hydrophobic material" refers to a material that repels or resists water. Exemplary hydrophobic materials include polyvinylidene fluoride (PVDF), octadecyltrichlorosilane (OTS), and hydrophobic polymers, including PLGA, etc.

[0108] The outer casing and body can be made of any suitable material, including lightweight plastics such as high-density polyethylene, polypropylene, polycarbonate or polyethyl sulfone, Tritan or polyamide, borosilicate glass, tinplate, foil or ceramic.

[0109] In use, an oxygen removal system is integrated into the storage container, exposing an air-permeable but liquid-impermeable membrane to the interior. The interior of the container typically includes a portion of the atmosphere that may contain oxygen. Oxygen permeates into the oxygen removal system, where it is absorbed by the oxygen removal material or otherwise isolated. Therefore, the oxygen removal system depletes the oxygen concentration within the storage container, making the environment more suitable for the survival of any anaerobic organisms that may be present in the culture medium. Depending on the selected OSM, even after short-term exposure to atmospheric conditions by opening and closing the bottle, the OSS can deplete the oxygen concentration within the storage container.

[0110] In a specific embodiment of the invention, the oxygen removal system can be used to pretreat the storage container before introducing a culture medium containing one or more anaerobic organisms. Therefore, introducing the culture medium may include rinsing with an oxygen-free gas (e.g., nitrogen rinsing) upon introduction of the culture medium to ensure that oxygen is not reintroduced into the pretreated storage container.

[0111] In one embodiment, OSS is produced and packaged under aseptic conditions to prevent microbial contamination. Alternatively or supplementally, OSS may be sterilized, for example, by exposure to high temperatures or chemicals. In another embodiment, OSS is wrapped in a covering such as a film to maintain its sterility.

[0112] The covering can also be used to protect the oxygen absorber from air exposure. It can be removed before use to minimize oxygen exposure prior to use.

[0113] In one embodiment, the sealable container is made of a material suitable for cryopreservation. "Cryopreservation" refers to a process that includes at least one step of lowering the temperature of biological material from a freezing temperature above that of the biological material (or a mixture of the biological material and the preservation composition) to a temperature below that freezing temperature. Cryopreservation includes freezing, vitrification, and lyophilization. Materials suitable for manufacturing the sealable container and / or shell of system 200 may include stainless steel, aluminum, glass, lightweight plastics such as high-density polyethylene, polypropylene, polycarbonate, or polyethersulfone, or polyamide.

[0114] In one embodiment, the sealable container is a bottle. Suitablely, the bottle is a baby bottle. Baby bottles are known in the art. A baby bottle may have a two-part design, comprising a body and a cap. The substantially hollow body is configured to hold liquid baby food, such as milk, preferably breast milk, and may include external threads on its upper outer wall that engage with internal threads provided on the inner wall of the cap, allowing the cap to be attached to the upper part of the body. The cap may be a nipple to facilitate infant feeding. The term "baby bottle" is interchangeable with "baby bottle" and "feeding bottle." Typically, a baby bottle can hold up to 330 ml of liquid.

[0115] In an alternative embodiment of the invention, the sealable container may be in the form of a pouch or bag, such as a milk collection and / or storage bag or a fecal collection bag for FMT, a lid compatible with a specific breast milk bottle, a box that can be inserted / removed, or a stopper for filling a hungate / conical flask, and the box may be installed inside the stopper to cultivate anaerobic bacteria. In the case of a pouch or bag, the sealable container is primarily defined by non-rigid or semi-rigid materials. These materials may include low-density polyethylene, polypropylene, and various plastic films. However, the sealing element may still include rigid materials, such as a cap or lid that can be combined with or otherwise secured to an oxygen removal system.

[0116] In one embodiment, the oxygen removal system may include a coupling mechanism for engaging the oxygen removal system with a container. The coupling member may be in the form of at least one circumferential rib that engages or connects with a corresponding portion on the container. Alternatively, the coupling mechanism may be a groove, clip, hook, or notch to allow the oxygen removal system to be attached to the container. In one embodiment, the coupling mechanism may be in the form of an adhesive.

[0117] In the implementation scheme, the OSS includes a coupling mechanism for engaging the OSS with the bottle cap. Any suitable coupling mechanism can be used, such as... Figure 6 The protrusion and groove mechanism shown in Figure A. The protrusion on the body 622 engages with the groove 623 in the cover 625.

[0118] exist Figure 6 In the alternative embodiment shown in B, the cover includes a fastening groove 627 that engages with the body 629.

[0119] In some embodiments, the oxygen removal system is disposable. In other embodiments, the oxygen removal system is reusable. In reusable embodiments, the oxygen removal system may be sterilized or otherwise cleaned. Oxygen removal material can be replenished with new material, and used oxygen removal material may be restored to its active state for repackaging in a new container or sent for recycling. Efforts should be made to promote the development of a sustainable circular economy to facilitate the reuse and recycling of materials used in the systems and methods of this invention.

[0120] In some implementations, the amount of OSM used to place in the first internal compartment is at least 1g, at least 2g, suitably at least 5g, at least 6g, at least 7g, at least 8g, at least 9g, at least 10g, at least 12g, at least 15g, at least 20g, and at most 30g, suitably at most 40g, and optionally at most 50g.

[0121] In use, the systems, apparatus, and methods of the present invention are suitable for preserving or improving the survival rate of anaerobic microorganisms. In one embodiment, the anaerobic microorganisms are anaerobic bacteria. Exemplary anaerobic bacteria include species of the genera *Bifidobacterium*, *Bacteroides*, and *Femtobacter*.

[0122] Bifidobacteria, Bacteroides, and Faecalibacterium are obligate anaerobes with numerous health benefits for humans. These genera are known to support digestive health by promoting the breakdown and absorption of nutrients, improving intestinal motility, and preventing gastrointestinal problems such as diarrhea and constipation. They also help maintain a balanced gut microbiome, which is crucial for overall digestive function. In particular, probiotic Bifidobacterium strains have been shown to modulate the immune system, stimulate antibody production, enhance immune responses, and reduce the risk of certain infections. They can help regulate the inflammatory response of the immune system and have been shown to be beneficial in treating conditions such as allergies and inflammatory bowel disease. Antibiotics disrupt the natural balance of gut bacteria, leading to diarrhea. Bifidobacterium probiotics have been used to prevent or alleviate antibiotic-associated diarrhea by restoring the balance of microbes in the gut. In infant health, Bifidobacterium species are crucial for establishing a healthy gut microbiome in infants. They are often added to infant formula or taken as supplements to promote the colonization of beneficial bacteria in the infant's gut and may reduce the risk of conditions such as colic, eczema, and allergies. However, the health benefits of Bifidobacterium can vary depending on the specific strain, dosage, and individual factors.

[0123] The systems, apparatus, and methods described herein allow for the periodic or continuous removal of oxygen from the atmospheric environment surrounding the culture medium. Therefore, the systems, apparatus, and methods described herein allow for the preservation of compositions and components that are easily degraded by oxygen.

[0124] Suitable, the culture medium may include consumable food, including milk, pharmaceutical or nutritional pharmaceutical compositions, or biological samples comprising live microbial components. Live microbial components refer to live microorganisms, such as bacteria, yeast, fungi, or combinations thereof.

[0125] In one specific embodiment of the invention, the culture medium comprises human breast milk, typically freshly expressed human breast milk.

[0126] In other embodiments, the food may include live fermented foods, such as foods that have been or are being fermented by live microorganisms, such as bacteria, yeast, or a combination of both. For example, such foods may include kombucha or pickles, or similar fermented products. The live microorganisms present in these types of foods may have probiotic properties, supporting a balance of beneficial bacteria in the gut and promoting digestive health. Pharmaceutical or nutritional compositions containing live microbial components may include probiotic supplements for the treatment of conditions including gastrointestinal infections or inflammatory diseases.

[0127] In other embodiments, the culture medium contains antioxidants, including vitamin E, vitamin C, vitamin A, polyphenols, metals (such as copper, zinc, and selenium). Other examples of antioxidants include coenzyme Q10, idebenone, lycopene, green tea polyphenols, cirebin, resveratrol, grape seed extract, barberry root (Mahonia aquifolium) extract, pomegranate extract, genistein, pycnogenol, curcumin, curcumin derivatives, tocopherols, Dunaliella salina extract, or combinations thereof. The systems, apparatus, and methods described herein are suitable for preserving the antioxidant content of media such as food, health products, and pharmaceuticals. The systems, apparatus, and methods described herein are particularly suitable for preserving the antioxidant content of milk, especially expressed breast milk.

[0128] In one embodiment of the invention, the total antioxidant capacity (TAC) of a portion of human breast milk is extended during storage when using the system, container, or apparatus of the invention, compared to when not in use. TAC describes the ability of antioxidants in various foods or bio-derived products to scavenge harmful free radicals, such as reactive oxygen species that affect shelf life, reduce nutritional potential, and promote spoilage. Antioxidants can neutralize these free radicals and prevent oxidation. TAC can be measured by various methods that use different chemical reactions to generate free radicals and monitor the reduction of free radicals by antioxidants, including TRAP (Total Radical Scavenging Parameter) assays (Young IS, (2001) J. Clin. Path 54:339). TRAP uses 2,2'-azobis(2-amidinylpropane) dihydrochloride (AAPH) to generate peroxy radicals, which are then captured by antioxidants present in the test sample. The oxidation reaction is monitored by measuring oxygen consumption using an oxygen electrode; the lag time before oxygen consumption is proportional to the TAC of the test sample. Therefore, TAC can comprehensively assess the antioxidant status of a food sample, but it cannot reflect the individual contributions of different antioxidants in the sample or their interactions. In specific embodiments of the invention, the TAC of samples (including food or beverages) is extended by at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, and optionally at least 6 months, compared to storage in a container excluding the claimed system or apparatus.

[0129] A biological sample is any sample collected from a subject (such as a human), including feces. In one embodiment, the system, apparatus, and method are used to maintain the viability of microorganisms in a fecal sample.

[0130] The method according to a first aspect of the invention maintains the viability of anaerobic microorganisms in milk, the method comprising storing milk in a sealable storage container, wherein the sealable storage container includes an oxygen removal system (OSS). In an embodiment, the OSS is as described in a second aspect of the invention.

[0131] The method disclosed in this paper also selectively promotes the growth of probiotic anaerobic microorganisms in freshly expressed human breast milk and inhibits the growth of pathogenic microorganisms.

[0132] The method disclosed in this paper can also be used to improve the survival rate of anaerobic microorganisms in probiotics.

[0133] definition

[0134] As used herein, the term "comprising" means that any of the elements listed must be included, and other elements may optionally be included. "Substantially constitutes" means that any referenced element must be included, excluding elements that would materially affect the essential and novel features of the listed elements, and other elements may optionally be included. "Includes" means that all elements other than those listed are excluded. Embodiments defined by each of these terms are within the scope of this invention.

[0135] The term "hydrophobic material" refers to materials that repel or resist water.

[0136] The terms “premature” and “low birth weight (LBW)” are used interchangeably to refer to newborns with a gestational age of less than 37 weeks and / or a birth weight of less than 2,500 grams.

[0137] As used herein, the term "oxygen scavenging material" refers to any material capable of collecting or removing oxygen from its environment. Suitable oxygen scavengers are, for example, oxidizable organic compounds and transition metal catalysts; olefinic unsaturated hydrocarbons and transition metal catalysts; quinones, reduced forms of photoreducing dyes, carbonyl compounds with absorbance in the ultraviolet spectrum; polymers having a polymer backbone, cyclic olefin side groups, and linking groups connecting the olefin side groups to the polymer backbone; copolymers of ethylene and strained cyclic alkylene groups; ethylene / vinylaralkyl copolymers; ascorbic acid; isoascorbic acid; sulfites; ascorbic acid and transition metal catalysts comprising simple metals or salts, or compounds, complexes, or chelates of transition metals; transition metal complexes or chelates of polycarboxylic acids, salicylic acid, or polyamines; tannins; or reducing metals such as iron. Other examples include ferrous iron powder containing sodium chloride, optionally containing activated carbon; ferrous carbonate combined with a metal halide catalyst; ferrous oxides; unsaturated fatty acids; sodium bicarbonate; citric acid; and ascorbic acid.

[0138] An "oxygen indicator" is a component that indicates the presence or absence of oxygen. Oxygen indicators include dyes that change color according to the presence or absence of oxygen. Exemplary dyes include azurlan, indigo carmine, methylene blue, diphenylamine, fluorescent pink, or combinations thereof.

[0139] "Milk" refers to milk obtained from mammals, formula milk, or a combination of both. Mammalian milk can refer to, for example, cow's milk, goat's milk, or human milk. Formula milk, often used interchangeably with infant formula, refers to artificial milk fed to infants as a substitute for breast milk. Formula milk can be derived from, for example, milk or plant-based sources such as soy and oats.

[0140] As used in this article, the term "anaerobic microorganisms" refers to microorganisms that can survive and grow in an atmosphere with less free oxygen than tropospheric air (i.e., less than about 10% free oxygen on a molar basis). Anaerobic microorganisms include organisms that can function in gases with free oxygen concentrations of less than about 10 mol%, or less than about 5 mol%, or less than about 2 mol%, or less than about 0.5 mol%.

[0141] "Bifidobacteria" and its synonyms refer to a genus of anaerobic bacteria beneficial to humans. Bifidobacteria are one of the major bacterial taxa constituting the gut microbiota of infants. They are beneficial commensal bacteria present in the gastrointestinal tract, contributing to the host's health. Different species of Bifidobacteria include *Bifidobacterium longum* subsp. infantis, *Bifidobacterium longum*, *Bifidobacterium animalis* subsp. lactis, *Bifidobacterium breve*, and *Bifidobacterium bifidum*. Each of these species can break down certain types of human milk oligosaccharides in breast milk and has been shown to be effective probiotic strains that can prevent the risk of diseases such as necrotizing enterocolitis in premature infants, and more broadly, improve the gut health of full-term infants.

[0142] The family Lactobacillus is a family that includes genera such as Lactobacillus, Lactobacillus mucosae, Lactobacillus casei, and Lactobacillus lactis, as well as other related genera. These genera are part of a diverse group of lactic acid bacteria known for their fermentation and probiotic functions.

[0143] The term "pathogen" is defined as including microorganisms, bacteria, viruses, and fungi, including but not limited to psychrophilic bacteria; lipophilic psychrophilic bacteria; proteophilic psychrophilic bacteria; mesophilic bacteria; Bacillus species, including Bacillus cereus; Clostridium species, including Clostridium perfringens and Clostridium botulinum; Cryptosporidium species; Campylobacter species, including Campylobacter jejuni; Listeria species, including Listeria monocytogenes; Escherichia species, including Escherichia coli and pathogenic Escherichia coli strains; and Mycobacterium species. Species, including Mycobacterium paratuberculosis; Pseudomonas species, including Pseudomonas fluorescens; Helicobacter species; Yersinia species, including Yersinia enterica; Toxoplasma species; Aeromonas species; Toxoplasma species, including Toxoplasma hominis; Streptococcus species; Staphylococcus species, including Staphylococcus aureus; Shigella species; Salmonella species, including Salmonella enteritidis, Salmonella Montegate, and Salmonella typhimurium; Cyclospora species, including Cyclospora cayeta. Cignatera Species of the genus Vibrio; species of the genus Orthomonas; species of the genus Entamoeba, including Entamoeba histolytica; species of the genus Candida, including Candida albicans; hepatitis viruses; astroviruses; caliciviruses; enteric adenoviruses; parvoviruses; and rotaviruses.

[0144] All references cited herein are incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0145] The present invention is further illustrated by the following non-limiting examples.

[0146] Example

[0147] Example 1: A probiotic strain of Bifidobacterium breve isolated from human milk is sensitive to oxygen.

[0148] Methods: Cultures of *Bifidobacterium breve* isolated from breast milk were inoculated onto BSM agar plates at different dilutions, in triplicate. These plates were exposed to aerobic conditions at staggered time points, with permissible oxygen exposure times ranging from 10 minutes to 4 hours. Subsequently, the plates were anaerobically incubated at 37°C for 48 hours, and colony counts were quantified. The mean of three replicates (n=3) was plotted for each time point, and error bars represent the standard deviation. Note that some error bars are too small to be displayed.

[0149] Results: The CFU count of the Bifidobacterium breve strain decreased with increasing exposure to oxygen, indicating a decreased survival rate of the strain under aerobic conditions. Figure 10 ).

[0150] Example 2: Growth of bacteria isolated from breast milk when using the device under optimal conditions

[0151] This device can promote the growth of probiotic oxygen-sensitive and oxygen-tolerant breast milk isolates, but under optimal growth conditions, it can inhibit the growth of pathogenic bacteria.

[0152] Methods: Bacterial strains isolated from breast milk, including *Bifidobacterium infantis* (strain 1), *Bifidobacterium infantis* (strain 2), *Bifidobacterium animalis*, *Lactobacillus fermentum*, and *Staphylococcus aureus*, were cultured in 5 mL MRS broth within 50 mL Falcon tubes under two conditions (with and without the apparatus). Triple cultures (n=3) were prepared under each condition and incubated at 37 °C. The optical density (OD) at 600 nm was measured over time by taking 100 μL aliquots from each Falcon tube.

[0153] Growth curves were compared under two conditions, with a focus on the time to reach stationarity and maximum OD. Repeated averages were plotted for each time point, with error bars representing standard deviations. Note that some error bars were too small to be displayed. Statistical significance was assessed using two-way ANOVA, with p-values ​​indicated by asterisks. p<0.05, p<0.01, p<0.001, p<0.0001).

[0154] Results: Under optimal growth conditions, the device significantly promoted the growth of oxygen-sensitive Bifidobacterium infantis strains (strain 1 and strain 2). Notably, without the device, these strains did not grow. Figure 11 and Figure 12 It is known that aerobic fermenting *Lactobacillus mucilaginosus* grows without significant difference in the presence or absence of this device, indicating that the device does not affect aerobic strains. Figure 13 Conversely, Staphylococcus aureus is an opportunistic pathogen that exhibits growth inhibition in the presence of the device. Figure 14 ).

[0155] Example 3: Using this device to preserve probiotics in breast milk

[0156] A: This device can preserve probiotic oxygen-sensitive breast milk isolates inoculated into mixed pasteurized breast milk. Survival rate

[0157] Methods: Bacterial strains isolated from breast milk, including *Bifidobacterium breve*, *Bifidobacterium longum*, *Bifidobacterium bifidum*, and *Lactobacillus fermentum*, were inoculated into mixed and pasteurized breast milk with or without the apparatus and stored in a refrigerator (4°C) for up to 6 days. Triple cultures (n=3) were prepared under each condition. Milk samples inoculated with bacteria were inoculated onto BSM agar at 0, 48, and 144 hours (6 days), anaerobically cultured at 37°C for 48 hours, and then quantitatively counted for colonies. *Bifidobacterium infantis* strains were also studied using the same method, but samples were taken at 0, 48, 72, and 96 hours. The replicates were plotted as mean at each time point, and error bars represent the standard deviation. Note that some error bars are too small to be displayed.

[0158] Statistical significance was assessed using a two-way ANOVA, with p-values ​​indicated by an asterisk (). p<0.05, p<0.01, p<0.001, p<0.0001).

[0159] Results: When stored with the device, all tested strains showed better survival rates during storage periods of 6 days or 4 days. At each time point (48 hours and 144 hours), the CFU counts of all strains in samples stored using the device were significantly higher than those in samples not using the device. This was also observed at time points for Bifidobacterium infantis (48 hours, 72 hours, and 96 hours). Statistical analysis demonstrated that the increased survival rate of probiotic strains in breast milk stored using the device indicates a significant preservation effect. This effect is achieved through… p A p-value <0.05 provides evidence that several strains showed more robust significance levels (e.g., p<0.01, p<0.001, (p<0.0001). These results highlight the device's ability to maintain the survival of probiotic strains in breast milk during extended refrigeration. These results are as follows: Figures 15 to 19 As shown.

[0160] B: This device can maintain the survival rate of oxygen-sensitive breast milk bacteria in fresh mixed breast milk stored in the refrigerator.

[0161] Methods: With and without the apparatus, a strain of *Bifidobacterium infantis* isolated from breast milk was inoculated into mixed and pasteurized breast milk and stored in a refrigerator (-20°C) for 7 and 21 days, respectively. Samples at each time point were stored separately to avoid the need for repeated freeze-thaw cycles. Triple cultures (n=3) were prepared under each condition. At each time point, the inoculated milk sample was inoculated onto BSM agar and anaerobically incubated at 37°C for 48 hours, followed by quantitative colony counting. The replicate mean for each time point was plotted, with error bars representing the standard deviation. Note that some error bars were too small to be displayed. Statistical significance was assessed using two-way ANOVA, with p-values ​​indicated by an asterisk (*). p<0.05, p<0.01, p<0.001, p<0.0001).

[0162] Results: Over time, the survival rate of Bifidobacterium infantis in the refrigerator (-20℃) decreased significantly. Figure 20 In samples without the device, a sharp decline in CFU counts was observed as early as 7 days. However, in samples stored with the device, the reduction in survival was significantly mitigated. (p<0.01), demonstrating the protective effect of the device. By day 21, the survival rate of Bifidobacterium infantis further decreased in samples stored without the device, while the CFU count remained stable in samples protected by the device. The preservation effect of the device was significantly better than that without the device. p<0.0001.

[0163] C: This device can preserve Bifidobacterium species present in breast milk microbiome samples.

[0164] Methods: Human breast milk was subjected to 16S amplicon sequencing using the CosmosID Kepler pipeline to obtain high-quality taxonomic assignments. Raw read counts assigned to species were filtered using the filterByExpr method in the edgeR package to remove species with sparse data (i.e., species with many zeros). Raw counts were normalized using counts per million (CPM) and log-transformed with a pseudo-count of 0.25. To test for preservation or reduction of bacterial abundance, normalized counts were compared between samples collected at 0 and 72 hours with and without the apparatus, focusing on the presence of Bifidobacterium species in the samples.

[0165] Results: Between the 0-hour and 72-hour time points when breast milk was not stored in the device, the abundance of Bifidobacterium species, Bifidobacterium animalis, and Bifidobacterium bifidum was significantly reduced (p=0.003 and p=0.005, respectively). Figure 21 However, the abundance of these same species did not decrease significantly when breast milk was stored in the device, indicating that Bifidobacteria in the milk were preserved. Interestingly, these two species were undetectable in milk samples stored for 72 hours without the device, suggesting that they were either completely absent or present at low abundances undetectable by sequencing, which explains the zero standard deviation in these samples.

[0166] Notably, with the exception of one species, the mean abundance of all other assessed Bifidobacterium species was reduced between 0 and 72 hours of milk storage without the device. However, these reductions did not reach statistical significance, likely due to the low sample size and inherent high variability between individuals. Nevertheless, the significant results and trends observed for Bifidobacterium species suggest that abundance was preserved in milk stored using the device, while abundance was reduced in samples not stored in the device.

[0167] Example 4: Using this device to inhibit opportunistic pathogens in breast milk

[0168] This device inhibits the survival rate of opportunistic pathogenic bacteria strains in pasteurized mixed breast milk.

[0169] Methods: Opportunistic pathogenic strains such as Klebsiella pneumoniae and Staphylococcus aureus were cultured and inoculated into mixed and pasteurized breast milk with and without the apparatus, and stored at room temperature. Triple cultures (n=3) were prepared under each condition. Samples were collected at specific time points and plated on LB agar: Klebsiella pneumoniae at 0, 4, 8, and 24 hours; Staphylococcus aureus at 0, 4, and 12 hours, aerobically incubated at 37°C for 24 hours, followed by colony counting and quantification. Repeated mean values ​​were plotted at each time point, with error bars representing standard deviations. If error bars were not visible, they were too small to be displayed. Statistical significance was assessed using two-way ANOVA, with p-values ​​indicated by an asterisk (p-value). p<0.05, p<0.01, p<0.001, p<0.0001).

[0170] Results: At the 4-hour time point, in accordance with the recommended guidelines for safe room temperature storage of breast milk, Klebsiella pneumoniae (Klebsiella pneumoniae) was observed under both with and without storage facilities. Figure 22 Staphylococcus aureus ( Figure 23 There was no statistically significant difference in bacterial survival rates between the two groups. This indicates that the presence of the device does not negatively impact the safety of breast milk in the early stages of storage, as the device does not lead to increased bacterial growth within the first 4 hours.

[0171] However, after the 4-hour time point, statistically significant differences were observed in the bacterial survival rates of all tested pathogens. The device effectively reduced the survival rates of Klebsiella pneumoniae and Staphylococcus aureus, thereby significantly inhibiting bacterial growth. These reductions were expressed as p-values. p<0.01 p<0.001 and Quantification was performed with p < 0.0001. This data indicates that the device significantly inhibited pathogen proliferation after 4 hours, supporting its practicality in protecting breast milk microbiome safety over extended room temperature storage periods.

[0172] Example 5: Using this device can extend the shelf life of breast milk.

[0173] A. Retention of antioxidants in breast milk

[0174] Methods: Freshly expressed breast milk was collected from donors and transported to the laboratory in a refrigerator with a maximum transport time of 1.5 hours. Upon arrival, samples were collected at 0 hours and stored at 4°C with and without a device. Samples were collected at 0 hours (n=30 donors), 72 hours with a device (n=27 donors), and 72 hours without a device (n=26 donors). The antioxidant molar concentration of each donor's sample was tested three times using an iron antioxidant status assay kit (Invitrogen™, ThermoFisher Scientific). Antioxidant capacity was calculated based on a standard curve provided by the kit manufacturer. Duplicate samples with differences exceeding 5% were excluded as outliers. The replicate mean was plotted for each time point, with error bars representing the standard deviation. Statistical significance was assessed using two-way ANOVA, with p-values ​​indicated by an asterisk (p-value). p<0.05, p<0.01, p<0.001, p<0.0001).

[0175] Results: At 4°C, the antioxidant concentration retained by breast milk stored using this device was significantly higher after 72 hours than that of breast milk not stored using this device. (p<0.0001). Furthermore, there was no significant difference in antioxidant concentration between milk stored using the device for 72 hours and freshly collected milk (0 hours) without using the device, demonstrating the device's effectiveness in long-term antioxidant preservation. In contrast, the antioxidant concentration decreased significantly from 0 hours to milk stored without using the device. p<0.0001) Figure 24 ).

[0176] B. The pH value of breast milk did not change after using the device.

[0177] Methods: Breast milk was collected as described in Example 2A, but this time with and without the equipment, the breast milk was stored at room temperature (four conditions in total: two with / without the equipment at 4°C, and two with / without the equipment at 22°C). Using a pH meter (HANNA HI-2210-02), the pH values ​​of the breast milk were measured at refrigerated conditions on day 0 (N=10), day 1 (N=6), day 2 (N=8), and day 3 (N=4), and at room temperature conditions on day 0 (N=10), day 1 (N=10), and day 2 (N=8). One-way ANOVA was used to compare the mean pH values.

[0178] Results: There were no significant differences in pH values ​​at each time point between the two temperatures, and the pH value gradually decreased with increasing storage time. This indicates that the device does not actively alter the pH value of breast milk compared to standard storage. Although there were no significant differences, a trend was observed where the pH value of breast milk stored at room temperature decreased faster than that stored under refrigeration conditions. Figure 25 This may be related to the growth of anaerobic bacteria, which produce lactic acid as a byproduct, leading to a decrease in pH.

[0179] C. Reducing breast milk spoilage through olfactory testing

[0180] Methods: Freshly expressed breast milk was collected from the donor and transported to the laboratory in a refrigerator, with a maximum transport time of 1.5 hours. The milk was then stored at 4°C for seven days, with or without the device. After the storage period, participants were given two samples of the same donor breast milk for olfactory testing: one sample stored with the device and the other not. This was a single-blind experiment; participants were not informed which sample was stored with the device. Participants were asked to evaluate the samples solely based on olfactory cues and answer, “Which sample shows signs of spoilage?”

[0181] A total of 13 donors' breast milk was tested, with 3-6 participants smelling each donor's milk on day 7. This involved a total of 14 participants, and 65 comparisons were made of the stored breast milk from the two treatment groups.

[0182] Results: Among the participants, 71% (n=46) perceived that breast milk stored without using the device showed greater signs of spoilage, compared to only 29% (n=19) who reported that breast milk stored with the device showed greater signs of spoilage. This indicates that the device effectively reduced the perceived spoilage during storage. Figure 26 ).

[0183] Example 6: Characterization of oxygen scavengers

[0184] A. This device reliably removes oxygen at different storage volumes.

[0185] Methods: ATCO oxygen indicator stickers (provided by Emco) were placed in 50 mL Ffalcon tubes containing 25 mL of distilled water (oxygen content: 5 cc) and a 100 mL empty Duran bottle (oxygen content: 25 cc) containing the device. Photographs were taken over ten days to observe the sticker color; the sticker remained bright yellow in the absence of oxygen and deep blue in the presence of oxygen. The device was then removed from both containers, and the stickers were photographed two days later. To quantify the color change, the RGB values ​​of the stickers were estimated (ImageJ). According to the ATCO oxygen indicator color scale, the red component of the RGB value decreased to 50 in the presence of oxygen and remained at 255 in the absence of oxygen. This information was used as a standard for evaluating the stickers in the experiment.

[0186] Results: The results are as follows Figure 27 As shown. The device was kept in an anaerobic environment for 10 days in 50 ml Falcon tubes and 100 ml Duran bottles, maintaining a yellow color and an RGB value of 255. In contrast, when the device was removed from both storage containers, the sticker turned a deep blue, indicating oxygen, with an RGB value of 50. This indicates that the device successfully removed all oxygen from the storage containers, and that standard storage does not actively remove oxygen.

[0187] B. Methylene blue oxygen indicator confirms that this device is significantly more effective than standard storage and vacuum storage in removing oxygen. live

[0188] Method: Sterifeed bottles were filled to 15% of their volume capacity with a methylene blue indicator solution (475 mmol potassium hydroxide, 185 mmol glucose, 17.4 µmol methylene blue), which is blue in the presence of oxygen and clear in the absence of oxygen. 15% volume was chosen to simulate the minimum volume of breast milk typically filled into the bottles, thus reflecting the maximum oxygen exposure of the milk. All bottles were vortexed at maximum speed for 15 seconds, and then photographed to capture the baseline blue color produced by the oxidation reaction. The Sterifeed bottles were then sealed with or without the apparatus (n=3). For apparatus conditions, the kit contained 9 g of OS (Midukit). The bottles were then vortexed again, and photographs were taken at 30 and 60 minutes.

[0189] Meanwhile, fill the Tommee Tippee Colicsoothe Milk Air Remover (TCMAR) to 15% of its capacity with methylene blue indicator solution. Then, apply a vacuum, and once complete, vortex the TCMAR again for 10 seconds and take a picture. Vortex the solution and take a picture every 30 minutes. Repeat this process three times (n=3).

[0190] To quantitatively analyze the data, the photographs were processed using ImageJ. These images were first converted to 32-bit grayscale, which assigned pixel intensity values ​​from 0 (black) to 255 (white). A region of interest (ROI) was selected for each image, and the average pixel intensity of the solution was measured to assess changes in color intensity. These average intensity values ​​were then plotted over time, allowing for quantitative comparisons of oxidation reactions under different conditions. Repeated averages were plotted at each time point, with error bars representing the standard deviation. Statistical significance was assessed using a two-way ANOVA, with p-values ​​indicated by asterisks. p<0.05, p<0.01, p<0.001, p<0.0001).

[0191] Result: Photo ( Figure 28 A) and the corresponding diagram ( Figure 28 (B) indicates that the device successfully decolorized the methylene blue oxygen indicator solution from blue to clear within 60 minutes, with most oxygen removal occurring at the 30-minute mark. In contrast, the color intensity changes observed under no-device and vacuum conditions were minimal, indicating almost no oxygen removal under these conditions. Figure 28 ).

[0192] C. Compare the device with different commercially available oxygen scavengers of varying weights.

[0193] Methods: Three oxygen absorbers were tested: IMPAK iron-free oxygen absorber, Midukit iron-oxygen absorber, and Oxoid™ AnaeroGen™ absorber, mounted on a Sterifeed bottle. Each absorber was used to fill a full box (FC) or half box (HC) at the following weights: IMPAK, 4.6 g and 2.3 g respectively; AnaeroGen, 3 g and 1.5 g respectively; Midukit, 9 g and 4.5 g respectively. A total of 25 mL of methylene blue indicator solution was added to the Sterifeed bottle, and the box and device were sealed. Another 25 mL of methylene blue indicator solution was added to a standard Sterifeed bottle and sealed as a control. Each bottle was vortexed at maximum speed for 15 seconds, and photographs were taken at 0, 30, 60, and 90 minutes after 10 seconds. Each condition was repeated three times. Once a sample passed the test, no further photographs were taken for that sample at subsequent time points.

[0194] Results: Photographs of the methylene blue indicator under different conditions showed that by 30 minutes, a full box of Oxoid, as well as a full and half-full box of Midukit, successfully removed all oxygen. By 60 minutes, a half-full box of Oxoid also removed all oxygen. At 90 minutes, a full box of IMPAK remover had removed all oxygen, while a half-full box of IMPAK did not completely remove oxygen within the specified time. These results indicate that Midukit is the most effective remover. Overall, the results suggest that the box design is flexible and effective, suitable for oxygen removal from various absorbers. The results of repeated experiments were consistent (n=3). Figure 29 ).

[0195] D. The methylene blue oxygen indicator confirms that the device can effectively remove oxygen from bottles of different sizes.

[0196] Method: Five bottles of different sizes (sterilized total volume = 168 mL, Avent total volume = 195 mL, Tommee Tippee total volume = 320 mL, Dr. Brown total volume = 300 mL, Medela total volume = 330 mL) were filled to 15% of their capacity with methylene blue oxygen indicator solution. Each bottle was vortexed at maximum speed for 10 seconds, and a photograph was taken to record the baseline blue color produced by the oxidation reaction. A total of 7.9 g OS was weighed and placed into the apparatus, which was then sealed inside each bottle. After 60 minutes, each bottle was vortexed for 10 seconds and photographed. Each condition was repeated three times.

[0197] Results: The device could remove oxygen from bottles of different sizes within 60 minutes, and the color of the methylene blue oxygen indicator changed from blue to transparent. The results of repeated experiments were consistent (n=3). Figure 30 ).

[0198] E. Devices for comparing different membrane surface areas

[0199] Methods: To determine the appropriate surface area of ​​the Tyvek membrane required to remove oxygen over one hour, different box sizes with varying Tyvek diameters were tested using Medela bottles (total volume = 330 mL). The diameters of the Tyvek membranes in each box were as follows: small box (S = 1.95 cm), medium box (M = 2.2 cm), and large box (L = 2.35 cm). Medela bottles were filled to 15% capacity with methylene blue oxygen indicator solution. Each bottle was vortexed for 15 seconds, and photographs were taken at 0, 30, and 60 minutes. Each condition was repeated three times.

[0200] Results: A clear trend was observed that a larger Tyvek membrane surface area resulted in faster oxygen removal, as indicated by the color change of the methylene blue solution from blue to clear. The largest container (L) exhibited the fastest oxygen removal rate, followed by the medium container (M) and the smallest container (S). Despite the differences in rate, oxygen was completely removed within 60 minutes under all conditions. The results of repeated experiments were consistent (n=3). Figure 31 ).

[0201] Although specific embodiments of the invention have been disclosed in detail herein, they are merely examples for illustrative purposes. The above embodiments are not intended to limit the scope of the appended claims. The inventors anticipate that various substitutions, alterations, and modifications can be made to the invention without departing from the spirit and scope of the invention as defined in the claims.

Claims

1. A method for maintaining the survival rate of anaerobic microorganisms in breast milk, the method comprising storing the breast milk in a sealable storage container, wherein the sealable storage container includes an oxygen removal system (OSS).

2. The method according to claim 1, wherein the storage container is selected from: a vessel, a bottle, a sealable bag, or a pouch.

3. The method according to claim 1 or 2, wherein the sealable storage container is a baby bottle.

4. The method according to any one of the preceding claims, wherein the milk comprises mammalian milk.

5. The method of claim 4, wherein the mammalian milk comprises human breast milk, suitably wherein the human breast milk is freshly expressed human breast milk.

6. The method according to any of the preceding claims, wherein the OSS comprises: a. A body comprising a first end and a second end, wherein the first end and the second end are open; and wherein the body defines a first internal compartment; b. A lid adapted to seal a first end of the body; c. A membrane that is breathable but impermeable to liquid, said membrane being adapted to seal a second end of the body; and d. Oxygen removal material (OSM), said OSM being adapted to be placed in a first internal compartment.

7. The method according to any one of the preceding claims, wherein the OSS further comprises a gasket adapted to provide a seal between the opening of the sealable storage container and a cap adapted to seal the opening of the sealable storage container.

8. The method of claim 7, wherein the gasket surrounds the body and extends radially outward therefrom.

9. The method of claim 7 or 8, wherein the gasket comprises an elastic material, optionally wherein the elastic material comprises silicone rubber.

10. The method according to any one of claims 7 to 9, wherein the gasket extends radially outward from the body by at least 1 cm, optionally at least 2 cm, suitably at least 3 cm.

11. The method according to any of the preceding claims, wherein the air-permeable but liquid-impermeable membrane is made of a hydrophobic material or includes a hydrophobic coating applied thereon.

12. The method according to any of the preceding claims, wherein the air-permeable but liquid-impermeable membrane further comprises an air-impermeable and liquid-impermeable covering.

13. The method according to any of the preceding claims, wherein the OSM is selected from one or more of the following: ferrous powder containing sodium chloride, optionally activated carbon, ferrous carbonate combined with a metal halide catalyst, ferrous oxide, unsaturated fatty acids, sodium bicarbonate, citric acid, and ascorbic acid.

14. The method according to any of the preceding claims, wherein, The amount of OSM used to place in the first internal compartment is at least 1g, at least 2g, suitably at least 5g, optionally at least 10g; at most 30g, suitably at most 40g; optionally at most 50g.

15. The method according to any one of the preceding claims, wherein the OSS further comprises a sleeve surrounding the body of the OSS, and optionally, wherein the gasket (when present) surrounds the sleeve.

16. The method according to any one of the preceding claims, wherein the anaerobic microbiome comprises microorganisms selected from anaerobic bacteria, optionally selected from Bifidobacteriaceae and / or Lactobacilliceae, wherein suitably the Bifidobacteria are selected from Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium animalis and / or Bifidobacterium bifidum.

17. An oxygen removal system (OSS) for use with a sealable storage container to maintain the survival rate of an anaerobic microbiome in a culture medium within the sealable storage container, said OSS comprising: a) A body including a first end and a second end, wherein the first end and the second end are open; and wherein the body defines a first internal compartment; b) A cover adapted to seal a first end of the body; c) A membrane that is permeable to air but impermeable to liquid, said membrane being adapted to seal a second end of the body; and d) Oxygen removal material (OSM), said OSM being adapted to be placed in the first internal compartment.

18. The OSS of claim 17 further includes a gasket adapted to provide a seal between the opening of the storage container and a cap adapted to close the opening of the storage container.

19. The OSS of claim 18, wherein the gasket surrounds the body and extends radially outward therefrom.

20. The OSS according to claim 18 or 19, wherein the gasket comprises an elastic material, optionally, the elastic material comprises silicone rubber.

21. The OSS according to any one of claims 17 to 20, wherein the gasket extends radially outward from the body by a distance of at least 0.5 cm, optionally at least 1 cm, at least 1.5 cm, at least 2 cm, at least 2.5 cm, at least 3 cm, at least 3.5 cm, at least 4 cm, at least 4.5 cm, at least 5 cm, at least 5.5 cm, or at least 6 cm.

22. The OSS according to any one of claims 17 to 21, wherein the breathable but liquid-impermeable membrane is made of a hydrophobic material or includes a hydrophobic coating applied thereon.

23. The OSS according to any one of claims 17 to 22, wherein the breathable but liquid-impermeable membrane further comprises a hydrophobic coating.

24. The OSS according to any one of claims 17 to 23, wherein the OSM is selected from one or more of the following: ferrous powder containing sodium chloride, optionally activated carbon, ferrous carbonate combined with a metal halide catalyst, ferrous oxide, unsaturated fatty acids, sodium bicarbonate, citric acid, and ascorbic acid.

25. The OSS according to any one of claims 17 to 24, wherein, The amount of OSM used to place in the first internal compartment is at least 2g, suitably at least 5g, optionally at least 10g; at most 30g, suitably at most 40g; optionally at most 50g.

26. The OSS according to any one of claims 17 to 25 further includes a sleeve.

27. The OSS of claim 26, wherein the sleeve surrounds the body, and optionally wherein the gasket (when present) surrounds the sleeve.

28. The OSS according to claim 26 or 27, wherein the sleeve is truncated conical.

29. The OSS according to any one of claims 17 to 28, wherein the OSM is placed in the first internal compartment, the first end is sealed by the cover, and the second end is sealed by the air-permeable but liquid-impermeable membrane.

30. The OSS according to any one of claims 17 to 28, wherein the breathable but liquid-impermeable membrane is covered by a breathable and liquid-impermeable covering.

31. The OSS of claim 30, wherein the airtight and liquid-impermeable cover is peelable by a user.

32. The OSS according to any one of claims 17 to 31, wherein the storage container is selected from: a vessel, a bottle, a sealable bag or a pouch.

33. The OSS according to claim 32, wherein the bottle is a baby bottle.

34. The OSS according to any one of claims 17 to 33, wherein the milk comprises mammalian milk, suitably wherein the mammalian milk is unsterilized.

35. The OSS according to any one of claims 17 to 34, wherein the anaerobic microbiome comprises microorganisms selected from anaerobic bacteria, the anaerobic bacteria optionally selected from Bifidobacteriaceae and / or Lactobacilliceae, wherein suitably the Bifidobacteria are selected from Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium animalis and / or Bifidobacterium bifidum.

36. A method for maintaining the shelf life of freshly expressed milk, the method comprising storing the milk in a storage container, wherein the storage container includes an oxygen removal system (OSS).

37. The method of claim 36, wherein the OSS is the OSS of any one of claims 17 to 35.

38. The method according to claim 36 or 37, wherein the milk is mammalian milk, optionally human breast milk.

39. A baby bottle comprising an oxygen removal system (OSS).

40. The baby bottle of claim 39, wherein the OSS comprises: a. A body comprising a first end and a second end, wherein the first end and the second end are open; and wherein the body defines a first internal compartment; b. A lid adapted to seal a first end of the body; c. A membrane that is breathable but impermeable to liquid, said membrane being adapted to seal a second end of the body; and d. Oxygen removal material (OSM), said OSM being adapted to be placed in a first internal compartment.

41. The baby bottle according to claim 40, wherein, The amount of OSM used to place in the first internal compartment is at least 1g, suitably at least 2g, suitably at least 5g, optionally at least 10g; at most 30g, suitably at most 40g; optionally at most 50g.

42. The baby bottle according to claim 40 or 41, wherein the OSM is selected from one or more of the following: ferrous iron powder containing sodium chloride, optionally activated carbon, ferrous carbonate combined with a metal halide catalyst, ferrous oxide, unsaturated fatty acids, sodium bicarbonate, citric acid, and ascorbic acid.

43. The baby bottle according to any one of claims 39 to 42, wherein the pad extends radially outward from the body by a distance of at least 0.5 cm, optionally at least 1 cm, at least 1.5 cm, at least 2 cm, at least 2.5 cm, at least 3 cm, at least 3.5 cm, at least 4 cm, at least 4.5 cm, at least 5 cm, at least 5.5 cm, or at least 6 cm.

44. The baby bottle according to any one of claims 39 to 43, wherein the OSS selectively promotes the growth of probiotic anaerobic microorganisms and inhibits the growth of pathogenic microorganisms in a culture medium, said culture medium optionally being mammalian milk, optionally being human breast milk.

45. A sealable container for maintaining the survival rate of anaerobic microorganisms contained therein, said container comprising any one of claims 17 to 35.

46. ​​A cap for sealing a sealable container for maintaining the survival rate of anaerobic microorganisms contained therein, wherein the cap is adapted to receive the OSS of any one of claims 17 to 35.

47. The OSS according to any one of claims 17 to 35, for maintaining the antioxidant content of the culture medium, for example, the culture medium being an emulsion.

48. The OSS according to any one of claims 17 to 35, wherein the OSS selectively promotes the growth of probiotic anaerobic microorganisms and inhibits the growth of pathogenic microorganisms in a culture medium, said culture medium optionally being mammalian milk, optionally being human breast milk.

49. The OSS according to any one of claims 17 to 35 further includes an oxygen indicator.

50. The OSS of claim 49, wherein the oxygen indicator is a dye.

51. A method for improving the growth of probiotic microorganisms or selectively promoting the growth of probiotic anaerobic microorganisms and inhibiting the growth of pathogenic microorganisms in freshly expressed human breast milk, the method comprising storing the breast milk in an infant bottle, the infant bottle comprising the OSS of any one of claims 17 to 35.

52. The method of claim 51, wherein the milk comprises Bifidobacteriaceae and / or Lactobacilliceae, optionally wherein the Bifidobacteria are selected from Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium animalis, and / or Bifidobacterium bifidum.

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