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Title:
PROCESS FOR PRODUCING A POWDERED COMPOSITION COMPRISING HUMAN BREAST MILK
Document Type and Number:
WIPO Patent Application WO/2020/243791
Kind Code:
A1
Abstract:
The invention provides a process for producing a powdered composition comprising human breast milk, the process comprising: enclosing an aqueous composition comprising human breast milk within a container, wherein a vapour-permeable barrier film forms at least a portion of container walls of the container; and freeze-drying the aqueous composition to produce the powdered composition, wherein water vapour sublimated during the freeze-drying egresses through the vapour-permeable barrier film.

Inventors:
CONDON KEVIN (AU)
Application Number:
AU2020/050577
Publication Date:
December 10, 2020
Filing Date:
June 05, 2020
Export Citation:
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Assignee:
AUSTRALIAN BREAST MILK BANK PTY LTD (AU)
International Classes:
A23C1/08; A23C3/00; A23C3/05; A23C9/20; F26B5/06; F26B25/12
Other References:
SALCEDO, JAIME ET AL.: "Human milk bactericidal properties: Effect of lyophilization and relation to maternal factors and milk components", JOURNAL OF PEDIATRIC GASTROENTEROLOGY AND NUTRITION, vol. 60, no. 4, 2015, pages 527 - 532, XP055767067, DOI: 10.1097/MPG.0000000000000641
FRANZ KOETTNITZ: "Freeze-Drying of Breast-Milk", LACTATION & BREASTFEEDING 2, 2018, XP055767081, Retrieved from the Internet
MANIN, LUCIANA P. ET AL.: "Evaluation of the Lipid Quality of Lyophilized Pasteurized Human Milk for Six Months by GC-FID and ESI-MS", JOURNAL OF THE BRAZILIAN CHEMICAL SOCIETY, vol. 30, no. 8, 2019, pages 1579 - 1586, XP055767146
OLIVEIRA, MARIANA M. ET AL.: "Development of a human milk concentrate with human milk lyophilizate for feeding very low birth weight preterm infants: A preclinical experimental study", PLOS ONE, vol. 14, no. 2, 2019, XP055733869
DILL, C. W. ET AL.: "Lipolytic activity during storage of human milk: accumulation of free fatty acids", JOURNAL OF FOOD PROTECTION, vol. 47, no. 9, 1984, pages 690 - 693, XP055767181
SOUSA, SÍLVIA G. ET AL.: "Human milk composition and preservation: evaluation of high-pressure processing as a nonthermal pasteurization technology", CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION, vol. 56, no. 6, 2016, pages 1043 - 1060, XP055696594, DOI: 10.1080/10408398.2012.753402
WESOLOWSKA, ALEKSANDRA ET AL.: "New achievements in high-pressure processing to preserve human milk bioactivity", FRONTIERS IN PEDIATRICS, vol. 6, 2018, pages 323
Attorney, Agent or Firm:
PHILLIPS ORMONDE FITZPATRICK (AU)
Download PDF:
Claims:
Claims

1. A process for producing a powdered composition comprising human breast milk, the process comprising:

enclosing an aqueous composition comprising human breast milk within a container, wherein a vapour-permeable barrier film forms at least a portion of container walls of the container; and

freeze-drying the aqueous composition to produce the powdered composition,

wherein water vapour sublimated during the freeze-drying egresses through the vapour-permeable barrier film.

2. A process according to claim 1 , wherein the temperature does not exceed 30°C during the freeze-drying.

3. A process according to claim 1 or claim 2, wherein the vapour-permeable barrier film is a porous polymeric film.

4. A process according to any one of claims 1 to 3, wherein the vapour-permeable barrier film comprises fibrous polyolefin, and preferably comprises bonded microfibers of HDPE.

5. A process according to any one of claims 1 to 4, wherein the container walls

comprise an impermeable receptacle portion for retaining the aqueous

composition and the vapour-permeable barrier film encloses the aqueous composition in the receptacle portion.

6. A process according to any one of claims 1 to 5, wherein the aqueous composition is contained within a sealable vessel located inside the container during the freeze-drying, wherein the sealable vessel is open during the freeze-drying and wherein the sealable vessel is sealed after the freeze-drying to enclose the powdered composition therein without first opening the container.

7. A process according to claim 6, further comprising sterilising the aqueous composition in the sealable vessel before the freeze-drying.

8. A process according to claim 7, further comprising enclosing the sealable vessel comprising the sterilised aqueous composition within the container and then opening the sealable vessel inside the container before the freeze-drying.

9. A process according to claim 7 or claim 8, wherein sterilising the aqueous

composition comprises pascalizing the aqueous composition in the sealable vessel.

10. A process according to any one of claims 1 to 6, further comprising sterilising the aqueous composition in the container before the freeze-drying.

11. A process according to claim 10, wherein sterilising the aqueous composition comprises pascalizing the aqueous composition in the container.

12. A process according to claim 11 , wherein the temperature of the aqueous

composition does not exceed 30°C during the pascalizing.

13. A process according to claim 11 or claim 12, wherein the aqueous composition is pascalized at a pressure of about 4000 bar (400 MPa) or greater.

14. A process according to any one of claims 11 to 13, wherein the aqueous

composition is contained within a sealed vessel located inside the container during the pascalizing, and wherein the sealed vessel is opened inside the container before the freeze-drying.

15. A process according to any one of claims 11 to 13, wherein:

i) the container comprises a selectively breakable seal which obstructs penetration of the aqueous composition through the vapour-permeable barrier film during the pascalizing; and ii) the selectively breakable seal is broken after the pascalizing, thereby allowing the water vapour to egress through the vapour-permeable barrier film during the freeze-drying.

16. A process according to claim 15, wherein the aqueous composition is retained in an impermeable compartment of the container during the pascalizing, the impermeable compartment isolated from the vapour-permeable barrier film by the selectively breakable seal.

17. A process according to claim 16, further comprising transferring the pascalized composition from the impermeable compartment to a permeable chamber of the container after breaking the selectively breakable seal, wherein the permeable chamber is enclosed by chamber walls comprising at least a portion of the vapour- permeable barrier film.

18. A process according to any one of claims 15 to 17, wherein the selectively

breakable seal comprises at least one selected from a zip seal and a clippable stick seal.

19. A process according to claim 15, wherein the selectively breakable seal comprises an impervious sealing layer laminated to at least the vapour-permeable barrier film, and wherein the selectively breakable seal is broken by at least partially removing the impervious sealing layer from the vapour-permeable barrier film.

20. A process according to any one of claims 11 to 19, wherein the entire container is enclosed within a removable outer impervious wrapping during the pascalizing.

21. A process according to claim 10, wherein sterilising the aqueous composition

comprises pasteurising the aqueous composition in the container.

22. A process according to any one of claims 1 to 21 , wherein the volume of the

aqueous composition enclosed within the container is no more than 100 ml.

23. A process according to any one of claims 1 to 22, wherein the aqueous

composition is frozen for freeze-drying within less than 10 seconds, preferably less than 5 seconds, of commencing the freezing.

24. A process according to any one of claims 1 to 23, wherein the freeze-drying

comprises heating the frozen aqueous composition to above -10°C within 48 hours of commencing the freeze-drying, and preferably comprises heating the frozen aqueous composition to above 0°C within 24 hours of commencing the freeze drying.

25. A process according to any one of claims 1 to 24, further comprising irradiating the powdered composition after the freeze-drying.

26. A process according to any one of claims 1 to 25, further comprising sealing the container in impermeable packaging after the freeze-drying.

27. A process according to any one of claims 1 to 26, wherein the aqueous

composition is human breast milk.

28. A system for producing a powdered composition comprising human breast milk, the system comprising:

a container configured to enclose an aqueous composition comprising human breast milk within container walls, wherein a vapour-permeable barrier film forms at least a portion of the container walls and wherein a removable impervious cover covers at least the vapour-permeable barrier film; and

a freeze-drier operable to freeze-dry the aqueous composition enclosed in the container when the removable impervious cover is at least partially removed from the vapour-permeable barrier film, thereby producing the powdered composition,

wherein water vapour sublimated during the freeze-drying egresses through the vapour-permeable barrier film.

29. A system according to claim 28, wherein the removable impervious cover is a polymeric film adhered to the vapour-permeable barrier film and removable therefrom by pealing.

30. A system for producing a powdered composition comprising human breast milk, the system comprising:

a sealable vessel configured to contain an aqueous composition comprising human breast milk;

a container configured to enclose the sealable vessel within container walls such that the sealable vessel can be opened and re-sealed when enclosed in the container, wherein a vapour-permeable barrier film forms at least a portion of the container walls; and

a freeze-drier operable to freeze-dry the aqueous composition contained in the sealable vessel when enclosed in the container, thereby producing the powdered composition,

wherein water vapour sublimated during the freeze-drying egresses through the vapour-permeable barrier film.

Description:
Process for producing a powdered composition comprising human breast milk Technical Field

[1 ] The invention relates to a process for producing a powdered composition comprising human breast milk. The process includes enclosing and freeze-drying an aqueous composition comprising human breast milk within a container. The container walls include a vapour-permeable barrier film, and water vapour sublimated during the freeze-drying egresses through the vapour-permeable barrier film.

Background of the Invention

[2] It is widely recognised that breast milk feeding provides optimum nutrition and other health benefits to infants. However, for a variety of reasons, a mother may be unable to supply sufficient, or any, breast milk via direct breast-feeding of her child. In such cases, donor human breast milk is a preferred alternative to formula feeding, particularly for pre-term or low birthweight infants. In other scenarios, mothers may desire to safely store their own breast milk for extended periods. For example, they may wish to retain a store of their breast milk to nourish further children, particularly where the mother is older or where there are medical reasons that render her capacity for future breast-feeding uncertain.

[3] It is critically important that donor milk or stored milk is safe when used. Expressed breast milk for donation is thus usually pasteurised to inactivate harmful pathogens. Pasteurisation with the Holder method, i.e. thermal treatment of bottled human breast milk in circulating water at 62.5°C for 30 minutes, is accepted by most human breast milk banks as a necessary compromise between microbial safety and immunologic retention. However, any thermal treatment inevitably degrades the complex natural balance of immune cells and bioactive proteins that is believed to safeguard a newborn infant’s optimum growth, immunological disease resistance, future development and long-term health. Moreover, there is a risk that poorly controlled temperature in the bath, uneven heat transfer through the bottle walls and into the entire volume of contained milk, or contamination of milk by the bath water, affects the reliability and safety of Holder pasteurisation. [4] A further issue with Holder pasteurised donor human milk (PDHM) and expressed breast milk for self-storage is the short usable lifetime. PDHM is typically frozen after pasteurisation to mitigate this. However, storage at between -20°C and -80°C for significant time periods has itself been shown to affect the desirable bioactivity of human breast milk. Guidelines from competent Health Authorities (e.g. New South Wales Health 2018) advise that PDHM should be stored frozen for no longer than 3 months after the date of pasteurisation, be used only within 24 hours of thawing in a refrigerator, and never be refrozen. The cold chain logistics and limited shelf life of frozen or unfrozen PDHM complicates the logistics of supplying donor breast milk to the point of need, and presents a heightened risk of infection or contamination after the product is transferred to the mother or nurse for use. In many remote areas or poor countries where breast milk banks are not established, cold chain facilities are not available or are inadequate, for example lacking data logging systems necessary to verify that sterilized breast milk has remained frozen until use. Such facilities add significant costs to the physical delivery and monitoring of PDHM when available.

[5] Dehydration of dairy products to produce a powdered product is one way to provide a longer usable lifetime and to reduce the risk of pathogen proliferation during storage and transfer. However, some powdering techniques such as spray-drying require the use of elevated temperatures which can also degrade thermally sensitive bioactive components in human breast milk, or are unsuited to preserve small amounts (such as single serve portions) of milk. Moreover, donated human breast milk would typically still require sterilization before powdering to ensure safety, and there is a further risk that human breast milk is contaminated with pathogens such as bacteria between sterilisation and powdering, during the powdering process itself or during subsequent processing and packaging of the powdered product.

[6] There is therefore an ongoing need for new processes for producing a powdered composition comprising human breast milk, which at least partially addresses one or more of the above-mentioned short-comings, or provide a useful alternative.

[7] A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that the document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

Summary of Invention

[8] The present invention relates to a process for producing a powdered composition comprising human breast milk. An aqueous composition comprising human breast milk is enclosed within a container, the container including a vapour- permeable barrier film which forms at least a portion of the container walls. The aqueous composition is freeze-dried in the container to produce the powdered composition, with the water vapour sublimated during the freeze-drying egressing through the vapour-permeable barrier film.

[9] An important role of the vapour-permeable barrier film is to maintain sterility in the container while permitting the necessary egress of gases, including sublimated water vapour, during freeze-drying of the enclosed aqueous composition. In particular, the vapour-permeable barrier film minimises or avoids ingress of micro particulate contaminants such as microbes during freeze-drying. It will thus be appreciated that enclosing the aqueous composition in the container means to seal it inside the container such that the only pathway for transport of gas / vapour between the inside and outside is through the vapour-permeable film. Thus, the powdered human breast milk produced according to the invention may have improved safety and/or longer usable life-time than powdered human breast milk produced by conventional dehydration techniques, including open-tray freeze-drying.

[10] It has been found that freeze-drying is itself capable of very significantly reducing the microbial load in human breast milk. By conducting the freeze-drying inside an enclosed container as disclosed herein, the full advantage of this inherent sterilisation can be realised since the ingress of microbes and other contaminants during or after the freeze-drying process can be avoided or minimised.

[11 ] In some embodiments, the human breast milk is initially sterilised while sealed inside a separate sealable vessel, which is then placed inside the container, opened before freeze-drying and re-sealed after freeze-drying while remaining enclosed within the container throughout. In other embodiments, the enclosed human breast milk is sterilised inside the container before freeze-drying and/or is retained in the container after freeze-drying for packaging and storage until use. The vapour- permeable barrier film may advantageously maintain sterility inside the container during such process steps also, thus improving safety, increasing usable lifetime and/or reducing costs when compared with processes in which the human breast milk is transferred between vessels during sterilisation, dehydration and packaging.

[12] In some embodiments, the enclosed human breast milk is sterilised by pascalization inside the separate sealed vessel or within the container before freeze drying. In such embodiments, a particularly high level of the desirable bioactivity of the human breast milk may be retained in the powdered composition, while still providing acceptable safety and ensuring sterility throughout the process.

[13] In accordance with a first aspect the invention provides a process for producing a powdered composition comprising human breast milk, the process comprising: enclosing an aqueous composition comprising human breast milk within a container, wherein a vapour-permeable barrier film forms at least a portion of container walls of the container; and freeze-drying the aqueous composition to produce the powdered composition, wherein water vapour sublimated during the freeze-drying egresses through the vapour-permeable barrier film.

[14] In some embodiments, the temperature does not exceed 37°C, or 30°C, or 25°C, during the freeze-drying.

[15] In some embodiments, the vapour-permeable barrier film is a porous polymeric film. In some embodiments, the vapour-permeable barrier film comprises fibrous polyolefin, such as bonded microfibers of HDPE.

[16] In some embodiments, the container walls comprise an impermeable receptacle portion for retaining the aqueous composition and the vapour-permeable barrier film encloses the aqueous composition in the receptacle portion.

[17] In some embodiments, the aqueous composition is contained within a sealable vessel located inside the container during the freeze-drying, wherein the sealable vessel is open during the freeze-drying and wherein the sealable vessel is sealed after the freeze-drying to enclose the powdered composition therein without first opening the container. The sealable vessel may comprise a flexible pouch with a removable screw-cap.

[18] In some such embodiments, the aqueous composition is sterilized in the sealable vessel before the freeze-drying. The process may further comprise enclosing the sealable vessel comprising the sterilised aqueous composition within the container and then opening the sealable vessel inside the container before the freeze-drying.

[19] In some embodiments, sterilising the aqueous composition comprises pascalizing the aqueous composition in the sealable vessel.

[20] In some embodiments, the process further comprises sterilising the aqueous composition in the container before the freeze-drying.

[21 ] In some such embodiments, sterilising the aqueous composition comprises pascalizing the aqueous composition in the container.

[22] In some embodiments the temperature of the aqueous composition does not exceed 30°C, or does not exceed 25°C, during the pascalizing. In some embodiments, the aqueous composition is pascalized at a pressure of about 4000 bar (400 MPa) or greater. In some embodiments, the aqueous composition is pascalized at a pressure of between about 4000 bar (400 MPa) and about 6000 bar (600 MPa) and a temperature of between about 20°C and about 30°C for between 1 and 5 minutes. In some embodiments, the aqueous composition is pascalized via a plurality of pressurisation steps comprising first and second pressurization steps at a pressure of about 3000 bar (300 MPa) or greater, wherein the pressure is at least partially released between the first and second pressurisation steps.

[23] In some embodiments, the aqueous composition is contained within a sealed vessel located inside the container during the pascalizing, and the sealed vessel is opened inside the container before the freeze-drying.

[24] In some embodiments wherein the aqueous composition is pascalized in the container before the freeze-drying: i) the container comprises a selectively breakable seal which obstructs penetration of the aqueous composition through the vapour-permeable barrier film during the pascalizing; and ii) the selectively breakable seal is broken after the pascalizing, thereby allowing the water vapour to egress through the vapour-permeable barrier film during the freeze-drying.

[25] In some embodiments, the aqueous composition is retained in an impermeable compartment of the container during the pascalizing, the impermeable compartment isolated from the vapour-permeable barrier film by the selectively breakable seal. In some embodiments, the pascalized composition is at least partially frozen before the selectively breakable seal is broken. In some embodiments, the process further comprises transferring the pascalized composition from the impermeable compartment to a permeable chamber of the container after breaking the selectively breakable seal, wherein the permeable chamber is enclosed by chamber walls comprising at least a portion of the vapour-permeable barrier film. The process may further comprise isolating and optionally detaching the permeable chamber from the impermeable compartment after transferring the pascalized composition.

[26] In some embodiments, the selectively breakable seal comprises at least one selected from a zip seal and a clippable stick seal. For example, it may be a double zip seal.

[27] In some embodiments, the selectively breakable seal comprises an impervious sealing layer laminated to at least the vapour-permeable barrier film. The selectively breakable seal may be broken by at least partially removing the impervious sealing layer from the vapour-permeable barrier film. The impervious sealing layer may be an adhesive polymeric film, for example removable from the vapour- permeable barrier film by pealing.

[28] In some embodiments, the container is sealed with a removable impervious cover during the pascalizing, the removable impervious cover covering at least the vapour-permeable barrier film, and the removable impervious cover is at least partially removed to allow the sublimated water vapour to egress through the vapour-permeable barrier film during the freeze-drying. The removable impervious cover may be a polymeric film. The entire container may be enclosed within a removable outer impervious wrapping during the pascalizing. [29] In some embodiments, sterilising the aqueous composition comprises pasteurising the aqueous composition in the container.

[30] In some embodiments, the entire container is sealed within a removable outer impervious cover layer during the pasteurising, and the pasteurising comprises immersing the container in a heated bath.

[31 ] In some embodiments, the aqueous composition is flash pasteurised.

[32] In some embodiments, the volume of the aqueous composition enclosed within the container is no more than 100 ml, for example between about 30 ml and 60 ml.

[33] In some embodiments, the aqueous composition is frozen for the freeze drying within less than 10 seconds, such as less than 5 seconds, of commencing the freezing.

[34] In some embodiments, the freeze-drying comprises heating the frozen aqueous composition to above -10°C within 48 hours of commencing the freeze drying. In some embodiments, the freeze-drying comprises heating the frozen aqueous composition to above 0°C within 48 hours, or within 24 hours, of commencing the freeze-drying.

[35] In some embodiments, the process further comprises irradiating the powdered composition after the freeze-drying, optionally within the sealed vessel or in the container.

[36] In some embodiments, the container walls further comprise an impermeable film. The container walls may be formed by heat sealing the impermeable film and the vapour-permeable barrier film together.

[37] In some embodiments, the process further comprises sealing the container in impermeable packaging after the freeze-drying.

[38] In some embodiments, the aqueous composition is human breast milk. [39] In accordance with a second aspect the invention provides a powdered composition comprising human breast milk, produced by a process according to any embodiment of the first aspect disclosed herein.

[40] In accordance with a third aspect the invention provides a system for producing a powdered composition comprising human breast milk, the system comprising: a container configured to enclose an aqueous composition comprising human breast milk within container walls, wherein a vapour-permeable barrier film forms at least a portion of the container walls and wherein a removable impervious cover covers at least the vapour-permeable barrier film; and a freeze-drier operable to freeze-dry the aqueous composition enclosed in the container when the removable impervious cover is at least partially removed from the vapour-permeable barrier film, thereby producing the powdered composition, wherein water vapour sublimated during the freeze-drying egresses through the vapour-permeable barrier film.

[41 ] In some embodiments, the container is a flexible polymeric pouch.

[42] In some embodiments, the removable impervious cover is a polymeric film adhered to the vapour-permeable barrier film and removable therefrom by pealing.

[43] In accordance with a fourth aspect the invention provides a system for producing a powdered composition comprising human breast milk, the system comprising: a sealable vessel configured to contain an aqueous composition comprising human breast milk; a container configured to enclose the sealable vessel within container walls such that the sealable vessel can be opened and re-sealed when enclosed in the container, wherein a vapour-permeable barrier film forms at least a portion of the container walls; and a freeze-drier operable to freeze-dry the aqueous composition contained in the sealable vessel when enclosed in the container, thereby producing the powdered composition, wherein water vapour sublimated during the freeze-drying egresses through the vapour-permeable barrier film.

[44] In some embodiments, the sealable vessel is adapted for pascalizing a liquid composition contained therein. [45] In accordance with a fifth aspect the invention provides a container for producing a powdered composition from an aqueous composition comprising human breast milk, the container comprising: container walls, wherein at least a portion of the container walls are flexible and wherein a vapour-permeable barrier film forms at least a portion of the container walls; and a selectively breakable seal which obstructs penetration through the vapour-permeable barrier film of an aqueous composition comprising human breast milk enclosed in the container during pascalizing of the aqueous composition to produce a pascalized composition, wherein breaking of the selectively breakable seal allows sublimated water vapour to egress through the vapour-permeable barrier film during freeze-drying of the pascalized composition to produce the powdered composition.

[46] In some embodiments, the container comprises an impermeable compartment for retaining the aqueous composition during the pascalizing, the impermeable compartment isolated from the vapour-permeable barrier film by the selectively breakable seal.

[47] In some embodiments, the container further comprises a permeable chamber enclosed by chamber walls comprising at least a portion of the vapour- permeable barrier film, wherein the permeable chamber is isolated from the impermeable compartment by the selectively breakable seal.

[48] In some embodiments, the pascalized composition is transferable from the impermeable compartment to the permeable chamber when the selectively breakable seal is broken. The pascalized composition may be transferable to the permeable chamber when frozen.

[49] In some embodiments, the selectively breakable seal comprises at least one selected from a zip seal and a clippable stick seal, and is preferably a double zip seal.

[50] In some embodiments, the container further comprises a removable impervious cover which covers at least the vapour-permeable barrier film, thereby sealing the container during the pascalizing, wherein the removable impervious cover is at least partially removable to allow the sublimated water vapour to egress through the vapour-permeable barrier film during the freeze-drying. The removable impervious cover nay be a polymeric film.

[51 ] In some embodiments, the selectively breakable seal comprises an impervious sealing layer laminated to at least the vapour-permeable barrier film, wherein the selectively breakable seal is breakable by at least partially removing the impervious sealing layer from the vapour-permeable barrier film.

[52] In some embodiments, the impervious sealing layer is an adhesive polymeric film, for example removable from the vapour-permeable barrier film by pealing.

[53] In some embodiments, the vapour-permeable barrier film is a porous polymeric film. The vapour-permeable barrier film may comprise fibrous polyolefin, such as bonded microfibers of HDPE.

[54] In some embodiments, the container further comprises a sealable mouth for transferring the aqueous composition into the container. The sealable mouth may comprise at least one zip seal, and preferably a double zip seal.

[55] In some embodiments, the container walls further comprise an impermeable film. The container walls may be formed by heat sealing the impermeable film and the vapour-permeable barrier film together.

[56] Where the terms“comprise”,“comprises” and“comprising” are used in the specification (including the claims) they are to be interpreted as specifying the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components, or group thereof.

[57] As used herein, the terms“first”,“second”,“third” etc in relation to various features of the disclosed devices are arbitrarily assigned and are merely intended to differentiate between two or more such features that the device may incorporate in various embodiments. The terms do not of themselves indicate any particular orientation or sequence. Moreover, it is to be understood that the presence of a“first” feature does not imply that a“second” feature is present, the presence of a“second” feature does not imply that a“first” feature is present, etc. [58] Further aspects of the invention appear below in the detailed description of the invention.

Brief Description of Drawings

[59] Embodiments of the invention will herein be illustrated by way of example only with reference to the accompanying drawings in which:

[60] Figure 1 schematically depicts a process for producing human breast milk powder in a tray container, according to an embodiment of the invention.

[61 ] Figure 2 schematically depicts a process for pasteurising human breast milk and producing human breast milk powder therefrom in a tray container, according to an embodiment of the invention.

[62] Figure 3 schematically depicts a process for producing human breast milk powder in a single pouch container, according to an embodiment of the invention.

[63] Figure 4 schematically depicts a process for producing human breast milk powder in a single pouch container, according to another embodiment of the invention.

[64] Figure 5 schematically depicts a process for pascalizing human breast milk and producing human breast milk powder therefrom in a single pouch container with external selectively breakable seal, according to an embodiment of the invention.

[65] Figure 6 schematically depicts a process for pascalizing human breast milk and producing human breast milk powder therefrom in a double pouch container with internal selectively breakable seal, according to an embodiment of the invention.

[66] Figure 7 schematically depicts a method for manufacturing the double pouch container depicted in Figure 6.

[67] Figure 8 schematically depicts the internal selectively breakable seal of the double pouch container depicted in Figure 6, according to an embodiment of the invention. [68] Figures 9 and 9A schematically depict the internal selectively breakable seal of the double pouch container depicted in Figure 6, according to another embodiment of the invention.

[69] Figures 10, 10A and 10B schematically depict a foldable double pouch container with internal double zip seal, for sequentially pascalizing and freeze-drying human breast milk according to an embodiment of the invention.

[70] Figure 11 is a photograph of tube containers for producing powder from enclosed human breast milk, according to an embodiment of the invention.

[71 ] Figure 12 schematically depicts a process for producing human breast milk powder in a pouch vessel enclosed within a permeable-walled container, according to an embodiment of the invention.

[72] Figure 13 schematically depicts a process for sequentially pascalizing human breast milk and then freeze-drying it to produce human breast milk powder in a pouch vessel, according to an embodiment of the invention.

[73] Figure 14 is a photograph of human breast milk sealed in an impermeable pouch for pascalizing, as performed in Example 2.

[74] Figure 15 is a photograph of frozen breast milk being transferred from an impermeable pouch (bag A), in which it was pascalized, to a complementary Tyvek- walled pouch (bag B) for freeze-drying, as performed in Example 3.

[75] Figure 16 is a photograph of the powdered breast milk, still contained in the Tyvek-walled pouch, as produced in Example 3.

[76] Figure 17 is a photograph of the powdered breast milk produced in Example 3. Detailed Description

Aqueous composition comprising human breast milk

[77] Provided herein is a process for producing a powdered composition from an aqueous composition comprising human breast milk. As used herein, human breast milk includes whole (i.e. unseparated) human breast milk and fractions thereof. In some embodiments, the aqueous composition consists of human breast milk, such as whole human breast milk, for example commingled donor breast milk collected by a breast milk bank or non-commingled expressed breast milk for self-storage by a mother. In other embodiments, the aqueous composition comprises one or more human breast milk fractions, such as a whey fraction or a fat fraction.

[78] The aqueous composition is typically liquid when enclosed in the container, but it is not excluded that it may be frozen when transferred into the container. The aqueous composition may optionally be sterilised before enclosure in the container, for example by conventional Holder pasteurisation, flash pasteurisation (High Temperature Short Time), high pressure processing, freeze pressure, irradiation, etc. Alternatively, it may be unsterilized when transferred, with sterilisation optionally effected inside the container as will be described hereafter.

[79] The aqueous composition may comprise, in addition to human breast milk, other components such as water soluble vitamins, and vitamin C in particular, that are generally reported as significantly decreased in thermal pasteurisation as well as Ca, Phos, fat. Such components may be added to the human breast milk before freeze drying as a convenient way to supplement the human breast milk powder produced in the process.

Container

[80] The aqueous composition is enclosed within a container during the freeze drying. The container includes container walls which enclose the aqueous composition inside the container. As used herein, container walls refer to all portions of a container which separate the interior volume of the container from the outside of the container, including base, side and upper portions, caps, lids and the like. As will be described in greater detail hereafter, a vapour-permeable barrier film forms at least a portion of the container walls.

[81 ] The size and geometry of the container is not considered to be particularly limiting on the scope of the invention. In some embodiments, the container is sized to enclose a single serve portion of human breast milk, for example no more than 100 ml, or between about 30 ml and 60 ml, such as about 50 ml. Thus, the powdered composition when diluted with water provides a single serve portion of reconstituted human breast milk. However, it is envisaged that larger containers, for example sized to enclose up to 1 litre of aqueous composition or even more, may also be used. This may be preferred when a large quantity of commingled donor milk must be powdered, for example for use in emergency relief for natural disasters.

[82] In some embodiments, the container is configured to retain the aqueous composition in a shallow pool, with a high surface to volume ratio. For example, the depth of the pool may be less than 20 mm, or less than 15 mm. Such a configuration may facilitate the speed and effectiveness of dehydration, as water vapour is more readily sublimed from the frozen breast milk during freeze-drying.

[83] In some embodiments, the container is configured such that at least a portion of the vapour-permeable barrier film forms an upper wall of the container, preferably situated above the liquid level of the aqueous composition enclosed in the container. Such configurations advantageously minimise contact between the human breast milk and the vapour-permeable barrier film, for example when transferring and enclosing the milk in the container, or when transferring the container into a freeze- drier. The aqueous composition may be retained in an impermeable receptacle portion of the container walls at the base of the container, such as a foil or thermoformed polymeric tray comprising one or more wells. The vapour-permeable barrier film may then be adhered to the receptacle portion to enclose the breast milk, for example by heat sealing or using an adhesive. Known methods of adhering or heat-sealing porous polymeric films such as Tyvek to freezer-grade polymeric trays may be used to produce such containers. For example, a porous polymeric film spot- coated with hot-melt adhesive may be heat-sealed to the tray rim to create an air-tight seal around the tray edges. Alternatively, an uncoated porous polymeric film may be heat sealed to a tray with hot-melt laminate structure, or adhered to the tray with a suitable curable adhesive.

[84] Alternatively, the vapour-permeable barrier film may be present as a window integrated into impermeable walls of the container, or part of a lid, for example a threaded lid which may be screwed onto an otherwise impermeable container.

[85] In some embodiments, the aqueous composition comprising human breast milk is contained inside a separate vessel which is then enclosed within the container having the permeable barrier film wall for freeze-drying. The outer, permeable-walled container need not directly contact the human breast milk and may thus be configured with dimensions suitable to enclose one, or more than one, of the vessels for freeze drying.

[86] In some embodiments, the enclosed vessel is an open vessel such as a tray, e.g. a foil or thermoformed polymeric tray comprising one or more wells configured to hold the milk. The enclosed vessel may be configured to retain the aqueous composition in a shallow pool, with a high surface to volume ratio. For example, the depth of the pool may be less than 20 mm, or less than 15 mm.

[87] In other embodiments, the separate vessel enclosed within the outer container is a sealable vessel, such as a bottle or a flexible pouch equipped with a screw-cap lid. The flexible pouch may have a threaded spout having an inner diameter in the range of 15 to 50 mm, to which a screw-cap can be fitted to seal the pouch. It has been found that human breast milk may be freeze-dried to powder while contained in a pouch vessel with open spout, which is itself enclosed within a larger container having vapour-permeable walls. As will be described in greater detail hereafter, this may advantageously allow the human breast milk to be sterilised inside the separate vessel, enclosed within the larger container while still sealed (before or after the sterilization), opened within the confines of the container, freeze-dried therein to produce the powder inside the sterile vessel, and optionally resealed in the vessel before opening the outer container.

[88] In some embodiments, the container is a flexible container, such as a bag or pouch formed of flexible film. In addition to the vapour-permeable barrier film portion, the pouch may include an impermeable film as part of, or the remainder of, the container walls. Suitable impermeable films may include polymeric films, for example a polyolefin film such as extruded polyethylene (e.g. high density polyethylene) film, metallic foils or foil-plastic laminates. Both the vapour permeable barrier film and the impermeable film are preferably approved by competent regulatory bodies for direct contact with food, and are preferably tough materials with resistance to damage during the intended use.

[89] Pouch-type flexible containers may conveniently be formed by heat-sealing or otherwise adhering complementary sheets of vapour-permeable barrier film and impermeable film together. For example, a single-pouch container may be manufactured by placing a rectangular sheet of polymeric vapour-permeable barrier film on top of a similarly sized rectangular sheet of impermeable polymeric film, and heat sealing the sheets together along three of the four edges.

[90] In some embodiments, the container walls of a pouch container may be formed substantially entirely of vapour-permeable barrier film. Commercially available header bags made of porous polymeric film (such as Tyvek) made be suitable for this purpose. In another example, a single sheet of vapour-permeable barrier film may be folded in half and heat-sealed along two of the three unjoined sides.

[91 ] When using a flexible container, the human breast milk may be retained in an impermeable receptacle such as a tray, or in a sealable vessel as a flexible pouch with spout and cap, placed inside the container to minimise contact between the human breast milk and the vapour-permeable barrier film. The flexible container may be spacious compared to the milk-holding tray or vessel inside, to allow a separation of the permeable walls from the milk and to provide a large permeable surface area through which the water can egress.

[92] The container may include an opening for transferring the aqueous composition into the container, the opening sealable to enclose the aqueous composition in the container for freeze-drying. In the case of a pouch-type container, the opening may be sealed by heat sealing the polymeric film walls adjacent to the opening, or with a sealing device such as one or more plastic zip seals (such as single, double or triple Ziplock seals) or a clippable stick seal (also known as a plastic bag sealer clip stick).

Vapour-permeable barrier film

[93] At least a portion of the container walls is formed by a vapour-permeable barrier film. As used herein, a vapour-permeable barrier film is a film with sufficient permeability to permit transmission of gases through the film, thus allowing the freeze-drying to take place, but which at least inhibits transmission of micro particulate materials, such as bacteria, through the film.

[94] In some embodiments, the vapour-permeable barrier film is a porous polymeric film. The porous polymeric film may comprise a polyolefin, for example high density polyethylene (HDPE) or poly(tetrafluoroethylene) (PTFE). The porous polymeric film may comprise a bonded web of polymeric fibres, in particular polymeric microfibers (i.e. having a diameter of less than 10 microns). Suitable microfibres may be formed by flash-spinning a fibre-forming composition containing the polymer, and the microfibers may then be laid in a web and thermally bonded to form the film. The partially bonded microfibers in the film are thus closely packed in a random distribution, providing a tortuous internal porosity in the film. This tortuous porosity permits transmission of gases, but blocks micro-particulate materials from penetrating through the film. The susceptibility of a porous material to micro-particulate penetration can be quantified by the % pMax value, as measured by ASTM F2638. In some embodiments, the vapour-permeable barrier film has a % pMax of less than 10%, or less than 1 %.

[95] The porous polymeric film may also resist infiltration by liquid aqueous compositions when a hydrophobic polymer is used, and diffusion of air through the film in the absence of a pressure gradient may be retarded by the tortuous porosity.

[96] Suitable vapour-permeable barrier films include porous polymeric films comprising FIDPE microfibers. The FIDPE microfibers may have a diameter of between 1 and 10 microns, such as about 4 microns. An example of such a film is Tyvek® film available from DuPont, for example Tyvek 1073B, 1059B or 2FS films. Tyvek film has been used as packaging for sterilized goods such as medical devices, where both breathability and exclusion of bacteria are important considerations. [97] In some embodiments, the vapour-permeable barrier film portion of the container walls is covered with a removable impervious cover prior to freeze-drying. The cover, typically present on the outer surface of the container, is then at least partially removed before freeze-drying to allow egress of the sublimated water vapour. The impervious cover may preserve the sterility and integrity of the vapour-permeable barrier film during manufacture of the container, or during one or more process steps before freeze-drying such as filling the container and sterilizing the aqueous composition in the container.

[98] The removable impervious cover may be an impermeable polymeric film. The entire container may be enveloped in an impermeable polymeric film wrapping, for example by vacuum sealing or shrink sealing. Alternatively, the removable impervious cover may cover only the vapour-permeable barrier film portion of the container walls. For example, the vapour-permeable barrier film may be a first layer of a multi-layered laminate structure, a second layer being an adhesive polymeric film or foil sealingly adhered to the vapour-permeable barrier film. The second layer may then be removed when desired by pealing it from the vapour-permeable barrier film.

Freeze-drying

[99] Freeze-drying, also known as lyophilisation, involves dehydrating an aqueous composition, frozen to a temperature below its triple point, at reduced pressure by sublimation of the water. Since the process requires exposure of the aqueous composition to low pressure (i.e. vacuum), freeze-drying of food products is conventionally performed in open trays in the vacuum chamber of a freeze-drier. While this is acceptable for many food products, contamination of human breast milk, for example by pathogens such as bacteria, during uncontained processing presents a more serious safety risk given the greater vulnerability of the infant consumers.

[100] In the process of the invention, by contrast, the aqueous composition comprising human breast milk is freeze-dried while enclosed in a container. The aqueous composition may be frozen for freeze-drying within the container, or it may be frozen before transfer into and enclosure within the container.

[101] The aqueous composition may be frozen initially to a temperature of below -10°C, such as between -10°C and -40°C, for example about -21 °C. Freezing may take place in the vacuum chamber of a freeze-drier, or in a separate freezer or freezing process.

[102] In some embodiments, the aqueous composition is subjected to accelerated freezing, by which it is meant that the aqueous composition is substantially frozen within less than 10 seconds, or less than 5 seconds, from commencing the freezing process. Accelerated freezing reduces the size of the crystals in the frozen composition compared to slow freezing processes, thus improving the uniformity and reproducibility of the subsequent dehydration. A powdered composition with good morphological properties may thus be produced. Moreover, rapid freezing is particularly important for breast milk which is prone to separation when standing in the liquid state. However, a balance should be struck between attaining these goals and the risk of freezing too quickly which may damage the lipid globules and thus compromise the bioactive properties of the milk. In some embodiments, the aqueous composition is thus frozen within 2 to 10 seconds from commencing the freezing process, such as from 3 to 5 seconds. Accelerated freezing may be accomplished by known methods such as using dry ice (solid CO2) or non CFC ozone safe propellant as the cooling medium. Prior to accelerated freezing, the milk may be agitated in the container to prevent separation either manually or using a machine including 360 degree over-turn rotation or a shaker machine.

[103] During the low-pressure stage of the freeze-drying, air or other gas inside the container is initially extracted through the vapour-permeable barrier film portion of the container walls, thus evacuating the container. Once the pressure is sufficiently reduced, water sublimes from the frozen aqueous composition and egresses through the vapour-permeable barrier film. As a result, the aqueous composition is dehydrated to form a powdered composition.

[104] The container enclosing the frozen human breast milk may be placed in the freeze-drier vacuum chamber, which is then evacuated to a set pressure of less than about 7 mbar, such as about 5 mbar or less. The vacuum chamber may be maintained at a set temperature below the freezing temperature during the initial stages of evacuation, such as between about -10°C and -40°C. Once the pressure in the container and the vacuum chamber is fully equilibrated, the frozen composition may be subjected to a temperature programme. [105] For example, the temperature may be slowly increased, for example at a rate of 5°C per hour, to a final target temperature of between 10°C and 30°C, for example 25°C. Alternatively, it may be more rapidly heated, such to heating to the final target temperature within 2 hours.

[106] In one exemplary embodiment, a 50 ml sample of human breast milk is enclosed in the container, with a liquid depth of 20 mm, and frozen to -38°C. The container is placed in the vacuum chamber of a freeze-drier. The vacuum chamber is initially set to -38°C and maintained at this temperature for 2 hours under vacuum. The temperature is then increased gradually to 25°C over two hours, then further increased to 37°C over 2 hours and held at this temperature for 18 hours before cooling and releasing the vacuum.

[107] As heat is added to the system, the water in the frozen human breast milk composition sublimates, and is extracted through the vapour-permeable barrier film. In some embodiments, the temperature does not exceed 37°C (i.e. human body temperature), or 30°C, or 25°C, or 22°C, during the freeze-drying. Maintaining a low temperature throughout, for example below human body temperature, protects the desirable bioactivity of the human breast milk from thermally induced degradation.

[108] The rate of heat input during freeze-drying may be optimised to achieve a desired balance between a beneficial sterilising effect of the freeze-drying itself, i.e. reducing the microbial load, and retention of the desirable bioactive properties of the milk. Without wishing to be bound by any theory, it is believed that a faster rate of heating, with higher temperatures reached at higher residual water content, may contribute to significant inactivation of pathogens potentially present in the milk. This may advantageously improve the safety and storage life of the powder milk composition compared with freeze-drying techniques where the aim is maximum retention of biological components. In some embodiments, the frozen composition may be heated to above -10°C within less 48 hours, or heated to above 0°C in less than 48 hours, such as less than 24 hours, during the freeze-drying.

[109] When sufficiently dehydrated by the freeze-drying, the aqueous composition is transformed into a powdered composition comprising human breast milk. The powdered composition may have a residual moisture content of below 5 wt%, or less than 3 wt%, such as between about 1 and 3 wt%. Such dehydration inactivates certain pathogens potentially present in milk, and is believed to render other pathogens dormant, thus preventing their proliferation during storage. By conducting the freeze-drying inside an enclosed container as disclosed herein, the sterility of the human breast milk is both enhanced by the freeze-drying process itself and protected by avoiding or minimising the ingress of microbes and other contaminants.

[110] When freeze-drying is complete, the vacuum in the vacuum chamber may be released. It will be appreciated that the powdered breast milk may become susceptible to reabsorption of water through the permeable film after freeze-drying, and steps may be adopted to minimise or avoid this. Optionally, the vacuum may be released with an inert gas such as nitrogen, so that the container enclosing the powdered composition is filled with the inert gas. Alternatively, the freeze-drying may be conducted in a controlled atmosphere environment, such as a dry room. The container may then be packaged in impermeable packaging, for example foil or polymeric packaging, optionally without first opening the container. The impermeable packaging assists to maintain sterility, exclude air and protect against light (e.g. UV) induced degradation during subsequent storage and transportation of the powdered breast milk composition.

[111] In some embodiments, the aqueous composition is contained inside a separate vessel which is itself enclosed within the container having the permeable barrier film wall during freeze-drying. The separate vessel may a sealable vessel, such as a bottle or a flexible pouch equipped with a screw-cap lid. As will be described in greater detail hereafter, the sealable vessel may be configured to allow the milk to be sterilised therein before freeze-drying, for example by pascalization.

[112] In some embodiments, the aqueous composition is sealed inside the separate vessel, optionally sterilised therein, and the vessel is then placed into and enclosed within the outer permeable-walled container. Once sealed in the container, the vessel may be opened, optionally after first freezing the aqueous composition. The aqueous composition is then subjected to freeze-drying such that a powder composition is produced inside the vessel within the outer container. Once freeze- drying is satisfactorily complete, the vessel may be resealed inside the container, and then if desired it may be removed from the container.

[113] One exemplary approach is to seal a single serve portion of human breast milk in a flexible polymeric pouch with a spout and air-tight screw-cap. Such pouches are commercially available, one example being the Tommee Tippee Express and Go pouch. Alternatively, a bespoke pouch designed to withstand sterilisation conditions, such as pascalization, when sealed may be used. The human breast milk therein may then optionally be sterilised, for example by pascalization, optionally frozen, and then enclosed within a flexible bag formed of porous polymeric film. If desired, the bag may be flushed with an inert gas to replace the air inside the bag. The breast milk is then frozen (if not already) and the flexible polymeric pouch is opened within the enclosing flexible bag. The breast milk is then freeze-dried to powder through the permeable walls of the flexible bag. Once a desired level of dehydration is obtained, the screw-cap is replaced on the spout of the flexible polymeric pouch, thus sealing the sterile milk powder in the pouch. The pouch may then be removed from the bag. When required for consumption, the pouch can be opened and the milk reconstituted with sterile water.

[114] The powdered composition comprising human breast milk is suitable to be reconstituted with sterile water when required for consumption. It may suitably be reconstituted to its original volume, or partially reconstituted, e.g. to c.a. 70 to 90 % of the original volume, to enrich the levels of protein, energy, calcium, phosphorus and other nutrients, producing a diet more suited to the nutritional needs of for example low-birth premature infants to accelerate weigh gain and body growth.

Sterilising

[115] In some embodiments, the aqueous composition comprising human breast milk is sterilised before the freeze-drying. The sterilization may be accomplished by methods including pasteurisation, pascalization, irradiation and ultrafiltration.

[116] In some embodiments, the aqueous composition is sterilized in a sealed vessel capable of being enclosed, and then opened, inside the container with a vapour-permeable barrier film wall. As disclosed herein, the sealable vessel may be a flexible polymeric pouch with a spout and air-tight screw-cap, but other sealable containers such as bottles and the like are envisaged.

[117] In some embodiments, the aqueous composition comprising human breast milk is sterilised inside the permeable-walled container before freeze-drying. Advantageously, the risk of contamination between sterilisation and freeze-drying is thus reduced.

[118] In some embodiments, the aqueous composition is sterilised in the separate sealed vessel and/or inside the permeable-walled container by pasteurisation, i.e. a thermal treatment to inactivate pathogens. The aqueous composition may be pasteurised by flash pasteurisation, also known as high- temperature short-time (HTST) pasteurisation. Thus, the aqueous composition may be rapidly heated to between about 70°C and 74°C, such as about 72°C, maintained at this temperature for a time of less than about 1 minute, such as 10 to 20 seconds, and then rapidly cooled to below 20°C. Flash pasteurisation is capable of inactivating many pathogens potentially present in human breast milk, while limiting the degradation of desirable bioactivity.

[119] In some embodiments, the aqueous composition is pasteurised by low- temperature long-time (LTLT) pasteurisation, an example of which is Holder pasteurisation. Thus, the aqueous composition may be heated to a temperature between 60°C and 64°C, for example 62.5°C for a time of between 10 minutes and one hour, such as about 30 minutes.

[120] Pasteurisation may be achieved by immersing the vessel / container in a heated water bath for a suitable contact time to achieve the required temperature. In embodiments where the human breast milk is already inside the container, the container is preferably covered with a removable impervious cover, for example a water-tight polymeric film, during the immersion to protect the vapour-permeable barrier film from contamination.

[121] In some embodiments, the aqueous composition comprising human breast milk is sterilised in the sealed vessel and/or inside the permeable-walled container by pascalization, also known as high pressure processing (HPP). Pascalization is a non- thermal food processing method, whereby microorganisms are inactivated by exposure to high pressure without relying on the application of heat. The process is often called “cold pasteurisation” and, in many applications, is uniquely able to preserve organoleptic properties, quality and micronutrients within the food, that would not be preserved through a heat/thermal process. Pascalization is able to neutralise many food pathogens and food spoilage microbes, thus achieving food safety outcomes and food preservation or shelf-life outcomes.

[122] Pascalization involves subjecting a liquid composition to extremely high hydraulic pressures, generally in excess of 3000 bar (300 mPa), such as between 3000 and 7000 bar, for example between 5500 and 6000 bar. Such high pressures, which are transmitted instantaneously and uniformly through the entire liquid volume, inactivate a wide range of potentially harmful pathogens, including bacteria and some viruses. However, since pascalization does not disrupt covalent bonds, some thermally sensitive bioactive components such as proteins and other nutritional and flavour components are not substantially degraded, or at least degraded less than in a pasteurisation process.

[123] The separate sealed vessel or permeable-walled container within which the human breast milk is pascalized must thus be sufficiently robust to withstand the high pressures. Moreover, since pascalization is generally conducted in a liquid (water) filled metal pressure vessel, equipped with high pressure pumps to provide the head, the vessel/container should itself be waterproof, or covered with a waterproof wrapping, when pascalized. Thus, as will be described in greater detail hereafter, the container may be protected with a robust impervious cover to prevent contamination of the vapour-permeable barrier film during the pressurisation.

[124] In some embodiments, the temperature of the aqueous composition does not exceed 30°C, or 25°C, or 20°C during the pascalizing. At lower temperatures, desirable bioactivity of the human breast milk may be preserved during the sterilisation. While a greater degree of pathogen inactivation may be achieved when pascalizing at higher temperatures, a desirable balance may be achieved between 3000 bar and 6000 bar at a temperature of 20°C to ensure safety while minimising degradation of desirable bioactivity. It will be appreciated that the temperature of the liquid aqueous composition will rise adiabatically during pressurisation (about 3 to 6°C per 1000 bar), so that the initial temperature should be set correspondingly below the maximum allowable temperature.

[125] In some embodiments, the aqueous composition is pascalized at a pressure of about 3000 bar or greater, or about 4000 bar (400 MPa) or greater, such as between about 4000 bar (400 MPa) and about 6000 bar (600 MPa). The aqueous composition may be subjected to high pressures for a time of 30 seconds or longer, for example between 1 and 5 minutes, such as about 3 minutes.

[126] In some embodiments, the aqueous composition is pascalized via a plurality of pressurisation steps, with at least partial release of the pressure between the steps. For example, the pascalization may involve at least two, or at least three or four, pressurisation steps at about 3000 bar (300 MPa) or greater, with the pressure released between each pressurisation for a time exceeding 30 seconds. Multi-step pascalization may, for example, improve the inactivation of bacterial spores.

[127] The parameters of the pascalization process may be selected to inactivate known pathogens which are potentially present in human breast milk. Such pathogens include viruses such as HIV 1 or 2, hepatitis B or C, human T-lymphotropic virus (HTLV) type I or II, cytomegalovirus (CMV), and non-spore forming bacteria. As a result of the pascalization, the total viable microbial count may be reduced to less than 10 CFU/ml, as determined by Standard Plate Count.

[128] Certain pathogens have been reported to be in deactivated (or inactivated) by pascalization, including but not limited to CMV, HIV, Hepatitis A. Other pathogens, for example syphilis, are less susceptible to deactivation by pascalization but survive poorly when dehydrated. Accordingly, the combination of pascalization followed by freeze-drying is believed to produce a significantly improved safety profile as different sets of pathogens are neutralised under each condition.

[129] In some embodiments, the powdered composition may be sterilized in the separate vessel or the permeable-walled container after the freeze-drying. For example, the powdered composition may be irradiated. The irradiation may be thermal, non-thermal, ionising or non-ionising radiation. [130] The sealable vessels / containers disclosed herein for processing human breast milk should generally be made from sterile materials, for example to the conditions required of medical grade packs for sterilised instruments, or irradiated to sterilise them prior to use.

Containers for pascalizing and freeze-drying

[131] Suitable containers for enclosing the liquid composition during sequential pascalizing and freeze-drying are flexible containers, i.e. where at least a portion of the container walls are flexible, for example polymeric pouches. Flexibility is required to withstand and transmit the pressure into the enclosed liquid. Furthermore, it is necessary that the milk-containing portion of the container is substantially liquid-full (i.e. with substantially no enclosed air pockets) when pascalized.

[132] If human breast milk is pascalized in the container, there is a risk that the milk may infiltrate the vapour-permeable barrier film portion of the container walls, causing clogging of its internal porosity or potentially even penetrating through the film to the outside of the container. Accordingly, the inventors have developed containers suitable to mitigate or avoid this risk. The containers include a selectively breakable seal which obstructs penetration of the aqueous composition through the vapour- permeable barrier film during the pascalizing.

[133] In some embodiments, the selectively breakable seal is located within the container, acting to prevent contact between the aqueous composition and the vapour-permeable barrier film during pascalization. Thus, the container may include within its interior an impermeable compartment for retaining the aqueous composition during pascalization. The selectively breakable seal isolates the impermeable compartment, and the aqueous composition contained therein, from the vapour- permeable barrier film. After pascalizing the aqueous composition, the selectively breakable seal is broken, without opening the container itself, thus allowing sublimated water vapour to egress from the aqueous composition during freeze drying. Selectively breakable seals capable of reversibly isolating parts of the interior of a flexible pouch-type container may include a plastic double zip seal, such as Ziplock seals, clippable stick seals, pressure-resistant stopcocks and screw-caps. Suitable plastic zip seals may be formed integrally in the polymeric material of pouch type containers during manufacture.

[134] In some embodiments, the selectively breakable seal divides the container into two portions, the impermeable compartment on one side of the seal and a permeable chamber, comprising the vapour-permeable barrier film portion of the container walls, on the other side of the seal. After pascalizing the aqueous composition in the impermeable compartment and then breaking the seal, the aqueous composition may be transferred into the permeable chamber. Optionally, the aqueous composition may be frozen before the transfer to avoid contacting the liquid with the vapour-permeable barrier film. Once transferred, the seal may be re formed between the impermeable compartment and permeable chamber, and the impermeable compartment may optionally be detached. The aqueous composition may then be freeze-dried in the residual container, with the sublimated water vapour egressing through the vapour-permeable barrier film as disclosed herein.

[135] Containers with internal seals (which may be termed “double-pouch containers” when formed of polymeric film and including both impermeable compartment and permeable chamber) may be protected during the pascalization by an impervious cover, as disclosed herein. The impervious cover covers at least the vapour-permeable barrier film, and preferably the entire container, thus preventing infiltration or contamination of the vapour-permeable barrier film by water in the pascalization pressure vessel.

[136] In some embodiments, the selectively breakable seal is located on the outside of the container walls. In such embodiments, the selectively breakable seal may comprise an impervious sealing layer which is laminated to the vapour- permeable barrier film. For example, the impervious sealing layer may include an adhesive polymeric film, removable from the vapour-permeable barrier film by pealing. In addition, a further impervious wrapping around the entire container may further assist to prevent ingress of fluids from the pressure vessel. While an external seal does not prevent contact between the aqueous composition and the vapour- permeable barrier film during pascalization, it ensures containment by preventing penetration of the aqueous composition through the walls. Moreover, it is considered that air trapped within the internal porosity of the vapour-permeable barrier film by the external seal may limit infiltration of the porosity by the aqueous composition. After the pascalization, the impervious sealing layer is delaminated from the vapour- permeable barrier film, so that sublimated water vapour can egress from the container during the subsequent freeze-drying.

System

[137] The invention also relates to a system for producing a powdered composition comprising human breast milk. The system comprises a container configured to enclose an aqueous composition comprising human breast milk within the container walls, and a freeze-drier operable to freeze-dry the aqueous composition enclosed in the container.

[138] The container, which may be a flexible polymeric pouch or bag as disclosed herein, includes a vapour-permeable barrier film as at least a portion of the container walls.

[139] In some embodiments, the vapour-permeable barrier film of the container is covered by a removable impervious cover on the outside of the container walls, which may protect the barrier film and the contents of the container from contamination prior to the freeze-drying step. The removable impervious cover may be an impermeable polymeric film sealing layer, adhered to the vapour-permeable barrier film as disclosed herein. The polymeric film may then be pealed away from the vapour-permeable barrier film.

[140] In use, the container is filled with the aqueous composition and sealed. For example, a single portion of expressed human breast milk may be transferred into the container by a mother desiring to preserve the milk for later use. The milk in the container may then optionally be sterilized and/or frozen and the container may optionally be transported to a remote freeze-drying facility. Immediately prior to freeze-drying, the impervious cover is removed and the container is placed in the freeze-drier to produce a powdered composition as disclosed herein. After freeze drying, the container may be further packaged and stored until required.

[141] In other embodiments, the system comprises a sealable vessel configured to contain the aqueous composition comprising human breast milk. The container is configured to enclose the sealable vessel within its walls such that the sealable vessel can be opened and re-sealed while enclosed in the container. For example, the vessel may be a flexible pouch with a spout and removable screw-cap which may be removed and replaced by manipulation through the flexible walls of the container.

[142] In use, the sealable vessel containing the aqueous composition is enclosed within the container, optionally sterilizing the milk therein, and the vessel is only opened when enclosed inside the container. The container is placed in the freeze- drier to produce a powdered composition as disclosed herein. After freeze-drying, the sealable vessel may be re-sealed while still inside the container, before removing it for further packaging and storage until required.

Embodiments

[143] An embodiment of the invention will now be described with reference to Figure 1. A single serve portion of liquid human breast milk 101 , for example commingled donated breast milk or expressed breast milk for self-storage by a mother, is transferred into the well of thermoformed polymeric tray 103, which may be sterilised before filling. Breast milk 101 may optionally be sterilised before transfer to the tray, for example by pasteurisation.

[144] A lid 105 of vapour-permeable barrier film (e.g. a porous polymeric film such as Tyvek) is then fitted by heat sealing or adhering the film to rim 107 of the tray. Breast milk 101 is thus enclosed in container 108 with the vapour-permeable film forming a part of the container walls. As depicted in Figure 1 , tray 103 includes a single well for receiving breast milk. Alternatively, the tray may include an array of multiple wells formed in an upper tray surface, each well capable of receiving a discrete portion of breast milk. The breast milk in each well may then be enclosed in its own container by heat-sealing a sheet of vapour-permeable film to the upper tray surface around the rim of each well, or the portions of breast milk may be enclosed by heat-sealing a sheet of vapour-permeable film to the edge of the upper tray surface surrounding all the wells.

[145] Closed container 108 is then transferred to a freeze-drier (not shown), where breast milk 101 is frozen to form frozen breast milk 109. Container 108 is then subjected to vacuum. Due to the vapour permeability of vapour-permeable film 105, air in the container is initially extracted to produce a vacuum inside the container. Thereafter, water vapour sublimates from frozen breast milk 109 and egresses through vapour-permeable film 105, as depicted by arrows 199. The sublimation dehydrates the human breast milk, resulting in the formation of human breast milk powder 113. The freeze-drying inactivates a range of pathogens potentially present in the breast milk. Once a satisfactory degree of dehydration is achieved, the vacuum is released, optionally with an inert gas such as nitrogen to avoid or minimise air ingress into container 108.

[146] Container 108 containing powdered breast milk may optionally be irradiated, and is then sealed in impermeable packaging 115, for example a foil pouch. The optional irradiation and packaging further protects human breast milk powder 113 from spoiling due to pathogen proliferation, air ingress or light exposure during storage and transport.

[147] Due to the enclosure of breast milk 101 in closed container 108 during freeze-drying, the ingress of contaminants, including microbes, is inhibited or avoided. The tortuous internal porosity of the vapour-permeable film permits egress of the water vapour sublimated during freeze-drying, but acts as a barrier to the entry of microbes. Accordingly, powdered composition 113 may have a longer usable life, or a lower risk of harmful microbial contamination and proliferation, than human breast milk which is freeze-dried in open trays.

[148] Another embodiment of the invention will now be described with reference to Figure 2, where features with similar numbering are as described for Figure 1. Container 108 thus includes tray 103 fitted with lid 105 of vapour-permeable barrier film, and breast milk 101 is enclosed in the container. Container 108 is sealed within waterproof (i.e. impermeable) polymeric film 223, for example by vacuum sealing, immersed in a temperature controlled water bath 224 and thus heated rapidly. Once the internal temperature of heated breast milk 101a reaches 72°C it is held at about that temperature for 10 to 20 seconds. The container is then withdrawn from the water bath and the breast milk is rapidly cooled to below 25°C. Polymeric film 223 is then removed. [149] The container is then placed in vacuum chamber 225 of a freeze-drier, further cooled until frozen to form frozen breast milk 109a, and subjected to vacuum. Due to the vapour permeability of vapour-permeable film 105, air in the container is initially extracted to produce a vacuum inside the container. Thereafter, water vapour sublimates from frozen breast milk 109a and egresses through vapour-permeable film 105, as depicted by arrows 199. The sublimation dehydrates the human breast milk, resulting in the formation of human breast milk powder 113a. After releasing the vacuum, the container may then optionally be irradiated and sealed in impermeable packaging.

[150] Rapid heating to 72°C, held for 10 to 20 seconds, followed by immediate cooling sterilises breast milk 101 by flash pasteurisation within container 108. Rapid heating and cooling in the container is readily achievable when using a relatively small volume of breast milk, such as a single serve portion, and limits the degradation of desirable bioactivity in the milk while still inactivating a variety of harmful pathogens that may be present. Moreover, due to the continuous enclosure of breast milk 101 in closed container 108 during both pasteurisation and freeze-drying, the ingress of contaminants, including microbes, is inhibited or prevented. The tortuous internal porosity of the vapour-permeable film permits egress of the water sublimated during freeze-drying, but acts as a barrier to the entry of microbes. Accordingly, powdered composition 113a may have a longer usable life, or a lower risk of harmful microbial contamination or proliferation, than pasteurised human breast milk which is freeze- dried in open trays or otherwise powdered.

[151] Another embodiment of the invention will now be described with reference to Figure 3, where features with similar numbering are as described for Figure 1. Fluman breast milk 101 is transferred into the well of polymeric tray 103, and the tray is then placed in pouch 308 through opening 310. Pouch 308 includes upper pouch wall 305 of vapour-permeable barrier film and impermeable lower pouch wall 307. The pouch may be formed by heat sealing a rectangular sheet of porous polymeric film (e.g. Tyvek) to a complementary rectangular sheet of impermeable polymeric film (such as extruded polyethylene film) along three edges (heat seal 31 1 depicted), leaving one edge unsealed to provide opening 310. [152] In a variation, pouch 308 may be formed of a single sheet of vapour- permeable porous polymeric film, folded in half and heat sealed around two edges, so that both pouch walls 305 and 307 are vapour permeable.

[153] With tray 103 placed inside the pouch, pouch opening 310 is sealed, for example with heat seal 313. Breast milk 101 is thus enclosed in closed pouch container 308a, and may be freeze dried as disclosed herein with reference to Figure 1. Water vapour sublimated during freeze-drying of breast milk 101 egresses through the porous polymeric film of upper pouch wall 305, while ingress of contaminants such as microbes is prevented or inhibited.

[154] Another embodiment of the invention will now be described with reference to Figure 4. Polymeric pouch 408 is provided, its walls including impermeable polymeric film 407 and vapour-permeable barrier film section 405. A plastic zip seal 417, such as a Ziplock seal, is provided adjacent to opening 410, to seal the pouch. In some embodiments, vapour-permeable barrier film section 405 is provided by heat sealing a suitably sized sheet of porous polymeric (e.g. Tyvek) film around the rim of a cut-out aperture in a wall of an otherwise impermeable polymeric pouch, via heat seal 411. Other means of producing a sealable polymeric pouch with a vapour- permeable wall section will be apparent to the skilled person. Optionally, at least the vapour-permeable barrier film section 405 is covered with a removable impervious cover (not shown), such as a pealable adhesive layer, which protects the film from contamination until its removal immediately prior to freeze-drying. Pouch 408 is preferably sterile prior to use.

[155] Fluman breast milk 401 , optionally already sterilised e.g. via pasteurisation, is then transferred into pouch 408 through opening 410, and the opening is sealed using plastic zip seal 417. Breast milk 401 is thus enclosed in closed pouch container 408a, with the vapour-permeable porous polymeric film 405 forming a part of the container walls. The porous polymeric film is resistant to penetration by the breast milk due to the hydrophobicity of its porous polymer and internal cohesive forces of the breast milk, and thus pouch 408 is able to satisfactorily retain the breast milk even if it comes into contact with the film. In a variation, a block of frozen breast milk is transferred into pouch 408 to minimise contamination of the porous polymeric film. [156] Closed container 408a is then placed in a freeze-drier (not shown), preferably with the porous polymeric film positioned on top, and breast milk 401 is frozen to form frozen breast milk 409. Container 408a is then subjected to vacuum. Due to the vapour permeability of porous polymeric film 405, air in the container is initially extracted to produce a vacuum inside the container. Thereafter, water vapour sublimates from frozen breast milk 409 and egresses through porous polymeric film 405, as depicted by arrows 499. The sublimation dehydrates the human breast milk, resulting in the formation of human breast milk powder 413. Once a satisfactory degree of dehydration is achieved, the vacuum is released, and the powdered breast milk in the pouch may then be irradiated and/or packaged in impermeable packaging for storage and transport.

[157] In a variation, closed container 408a, containing unsterilized human breast milk 408, is subjected to rapid heating and cooling, as described herein with reference to Figure 2, before the freeze-drying. The milk in the closed container is thus pasteurised to inactivate pathogens before freeze-drying.

[158] Another embodiment of the invention will now be described with reference to Figure 5. Polymeric pouch 508, with opening 510, is provided, its walls including impermeable polymeric film 507, for example extruded polyethylene film, and bi- layered laminate section 519. Laminate section 519 comprises an inner layer of vapour-permeable porous polymeric (e.g. Tyvek) film 505, and an impervious sealing layer 521 of adhesive impermeable polymeric film sealingly adhered to the porous polymeric film.

[159] Pouch 508 may be provided by heat-sealing a suitably sized sheet of bi- layered laminate film around the rim of a cut-out aperture in a wall of an otherwise impermeable polymeric pouch, as disclosed herein and schematically depicted in Figure 5. Alternatively, pouch 508 may be provided by heat sealing a rectangular sheet of bi-layered laminate film to a complementary rectangular sheet of impermeable polymeric film along three edges, leaving one edge unsealed to provide opening 510. Pouch 508 is preferably sterile prior to use. [160] Human breast milk 501 , for example a single serve portion of expressed milk for self-storage by a mother, is then transferred into pouch 508 through opening 510. Air inside the container is expelled and the opening is then sealed, either by heat-sealing with heat seal 511 as depicted or via another sealing device capable of withstanding pressure, such as a robust plastic double zip seal or a clippable stick seal. Breast milk 501 is thus enclosed in closed, liquid-full pouch container 508a. Vapour-permeable porous polymeric film 505 forms a part of the container walls, but the container is initially sealed by impervious sealing layer 521 which substantially blocks permeation of vapour into or out of the container through the porous polymeric film. The inward-facing porous polymeric film 505 is resistant to penetration by the breast milk due to the hydrophobicity of its porous polymer, the internal cohesive forces of the breast milk and the vapour-tight seal provided by impervious sealing layer 521 (as will be described in greater detail hereafter).

[161] Closed container 508a is then enveloped in a removable impermeable wrapping (not shown), for example by vacuum sealing it with polymeric film, to further assist to resist ingress of external fluids into the container. The container is then placed in pressure vessel 523, which is filled with water 527, and subjected to high pressure processing (i.e. pascalizing; represented by arrows 528) at 20°C. The pressure in the vessel may be maintained at a constant pressure of between 3000 and 6000 bar for between 1 and 5 minutes before release. Other pressure profiles, including multi-step pressurisation with intermediate pressure release, may be applied as described herein. Due to the flexibility of the container walls, the hydraulic pressure in the vessel is transmitted instantaneously and uniformly into breast milk 501 , without breaking the container through pressure, thus inactivating potentially harmful pathogens contained therein. However, impervious sealing layer 521 and the removable impermeable wrapping prevent ingress of water 527 into the container, and escape of breast milk 501 from the container by penetration through porous polymeric film 505. Indeed, without wishing to be bound by any theory, the inventors consider that penetration of the porous polymeric film’s tortuous internal porosity by breast milk 501 is limited and resisted by the gas trapped therein by the laminated impervious sealing layer 521. [162] After the high pressure treatment, container 508a now containing pascalized breast milk 501a is retrieved from the pressure vessel, and the removable impermeable wrapping is removed. Impervious sealing layer 521 is then delaminated from porous polymeric film 505 by pealing it away, without opening the container. Container 508a is then transferred to vacuum chamber 525 of a freeze-drier, preferably with the porous polymeric film positioned on top, and pascalized breast milk 501 a is frozen to form frozen breast milk 509. Alternatively, the milk may be frozen before delaminating sealing layer 521 from porous polymeric film 505. Container 508a is then subjected to vacuum. Due to the vapour permeability of porous polymeric film 505, water vapour sublimates from frozen breast milk 509 and egresses through porous polymeric film 505, as depicted by arrows 599. The sublimation dehydrates the human breast milk, resulting in the formation of human breast milk powder 513. Once a satisfactory degree of dehydration is achieved, the vacuum is released, and the powdered breast milk in the pouch may then be irradiated and/or packaged in impermeable packaging for storage and transport.

[163] Advantageously, pascalizing of breast milk 501 in the container at low temperature inactivates potentially harmful pathogens with only minimal impact on the milk’s desirable bioactivity, and ensures sterility inside container 508a before freeze drying. Moreover, due to the continuous enclosure of pascalized breast milk 501a in closed container 508a during freeze-drying and subsequent packaging, this sterility is maintained, since the ingress of contaminants, including microbes, is inhibited or avoided. Accordingly, powdered composition 513 may have a longer usable life, or a lower risk of harmful microbial contamination and proliferation, than human breast milk which is sterilised, freeze-dried, packaged, or transferred between vessels for any of these operations, without containment.

[164] Another embodiment of the invention will now be described with reference to Figure 6. Polymeric double pouch container 608, with an interior including compartment 612 and chamber 614, is provided. The walls of the double pouch, including upper wall 620 and lower wall 622, are formed from impermeable polymeric film 607, for example extruded polyethylene film, and vapour-permeable porous polymeric (e.g. Tyvek) film 605. The porous polymeric film forms part of the walls of chamber 614, but the walls of compartment 612 are formed only of the impermeable polymeric film. Compartment 612 has opening 610, and is separated from chamber 614 by selectively breakable seal 616, as will be described in greater detail hereafter. Pouch container 608 is preferably sterile prior to use.

[165] A method for manufacturing pouch 608 is schematically depicted in Figure 7. Upper wall 620 is produced by placing rectangular porous polymeric film 605 in partially overlapping arrangement with rectangular sheet 607a of impermeable polymeric film. Film 605 and sheet 607a are then heat sealed together, via heat seal 611 a, to form the rectangular upper wall 620. Upper wall 620 is then overlaid on lower wall 622, formed from rectangular impermeable polymeric sheet 607b. The upper and lower walls are then heat sealed together along three edges, via heat seals 611 b, 611 c and 611 d. The fourth edge 618 is left unsealed, thus providing mouth 610. Clippable stick seal 616a is then used to provide a pressure-resistant, selectively breakable seal between compartment 612 and chamber 614, as will be described in greater detail hereafter.

[166] Figure 8 schematically depicts a portion (denoted“A” in Figure 6) of double pouch container 408, including selectively breakable seal 616 according to an embodiment of the invention. Seal 616 is a robust plastic double zip seal, comprising first and second plastic zip seals 616a and 616b, such as Ziplock seals. When fastened, such a mechanism provides an airtight and pressure-resistant seal across the width of container 408 which prevents transfer of liquids between compartment 612 and chamber 614 when container 608 is subjected to high pressure processing conditions. Flowever, seal 616 may be reversibly broken when desired (denoted by arrow X) by mechanically unzipping first and second plastic zip seals 616a and 616b, thus providing channel 624 between compartment 612 and chamber 614.

[167] Figure 9 schematically depicts a portion (denoted“A” in Figure 6 and B in Figure 7) of double pouch container 408, including selectively breakable seal 616 according to another embodiment of the invention. Seal 616 in this embodiment is clippable stick seal 616c, comprising an elongated cylindrical stick 630 and a complementary elongated clip 632. Clip 632 has a C-shaped cross-sectional profile, capable of resilient outward deformation as elongated cylindrical stick 630 is clipped into place inside the C-shape. As depicted in Figures 9 and 9A, clippable stick seal 616c can be sealed and unsealed as desired by positioning stick 630 and clip 632 on opposite sides of the container polymeric film walls 620 and 622, and clipping and unclipping stick 630 into clip 632. When clipped in place, polymeric film walls 620 and 622 are tightly gripped together by stick seal 616c, thus providing an airtight and pressure-resistant seal across the width of container 408 which prevents transfer of liquids between compartment 612 and chamber 614 when container 608 is subjected to high pressure processing conditions.

[168] With continued reference to Figure 6, human breast milk 601 , for example commingled donor breast milk collected at a breast milk bank, is transferred through opening 610 into compartment 612 of pouch container 608. Air inside the container is expelled and the opening is then sealed, either by heat-sealing with heat seal 61 1d as depicted or via a sealing device capable of withstanding pressure, such as a robust plastic double zip seal. Breast milk 601 is thus enclosed in impermeable compartment 612 within closed pouch container 608a. Vapour-permeable porous polymeric film 605 forms a part of the container walls, but is isolated from the breast milk in compartment 612 by seal 616.

[169] Closed container 608a is then enveloped in a removable impermeable wrapping (not shown), for example by vacuum sealing it with polymeric film, to prevent contamination of chamber 614 and porous polymeric film 605 during pascalization. Porous polymeric film 605 may in addition be protected by an adhesive but pealably removable impervious sealing layer, as described herein with reference to Figure 5.

[170] Container 608a is then placed in pressure vessel 623 and subjected to high pressure processing (i.e. pascalizing) at 20°C as described herein. Due to the flexibility of the container walls 620 and 622, the hydraulic pressure in the vessel is transmitted instantaneously and uniformly into breast milk 601 , thus inactivating potentially harmful microbes contained therein. Flowever, the walls of compartment 612, formed of impermeable polymeric film 607, prevent contamination of the breast milk by external fluids and escape of breast milk 501 from the container, while the removable impermeable wrapping and the optional impervious sealing layer prevent contamination of the porous polymeric film and ingress of external fluids into chamber 614. Within the container, selectively breakable seal 616 retains breast milk 601 in compartment 612, thereby avoiding contact between the breast milk and porous polymeric film 605. Advantageously, the internal porosity of the vapour-permeable porous polymeric film thus remains pristine and in particular the risk of pore clogging by fats in the milk is avoided.

[171] After the high pressure treatment, pouch container 608a now containing pascalized breast milk is retrieved from the pressure vessel, and the breast milk is frozen to form frozen breast milk 609. Once frozen, selectively breakable seal 616 is broken, thus opening channel 624 between compartment 612 and chamber 614. The block of frozen breast milk 609 is then transferred from compartment 612 to chamber 614 through channel 624, for example by squeezing the container. Advantageously, transfer of the milk in an at least partially frozen state avoids or minimises contact between liquid breast milk and the porous polymeric film, thus further reducing the risk of pore clogging prior to sublimation. However, it is not excluded that liquid breast milk 601 is transferred to chamber 614 for freeze-drying therein.

[172] Once the breast milk has been transferred, chamber 614 may be isolated from compartment 612 by re-sealing seal 616 as depicted or by heat-sealing upper walls 620 and 622 together. Optionally, compartment 612 is then detached and discarded, for example by cutting it away.

[173] Residual container 608b is then transferred to the vacuum chamber of a freeze-drier (not shown), preferably with the porous polymeric film positioned on top, and the breast milk is freeze-dried as described herein. It will be appreciated that the impermeable wrapping and optional impervious sealing layer which protect the container during pascalizing should be removed, without opening the container, to allow the sublimation to proceed. This may be done at any time between the pascalizing step and evacuation during freeze-drying, but preferably as late as possible to best maintain sterility and avoid air ingress into the container. During freeze-drying, water vapour sublimates from frozen breast milk 609 and egresses through porous polymeric film 605, as depicted by arrows 699. The sublimation dehydrates the human breast milk, resulting in the formation of human breast milk powder 613. Once a satisfactory degree of dehydration is achieved, the vacuum is released, and the powdered breast milk in the pouch container may then be irradiated and/or packaged in impermeable packaging for storage and transport.

[174] Advantageously, pascalizing of breast milk 601 in the container at low temperature inactivates potentially harmful pathogens with only minimal impact on the milk’s desirable bioactivity, and ensures sterility inside container 608a before freeze drying. Moreover, due to the continuous enclosure of pascalized breast milk 601a in closed container 608b during freeze-drying and subsequent packaging, this sterility is maintained, since the ingress of contaminants, including microbes, is inhibited or avoided. Accordingly, powdered composition 613 may have a longer usable life, or a lower risk of harmful microbial contamination and proliferation, than human breast milk which is sterilised, freeze-dried, packaged, or transferred between vessels for any of these operations, without containment.

[175] Another embodiment of the invention will now be described with reference to Figures 10, 10A and 10B. Polymeric double pouch container 1008, seen in plan view in Figure 10 and side view in Figures 10A and 10B, has an interior including compartment 1012 and chamber 1014. The walls of the double pouch are formed from impermeable polymeric film 1007, for example extruded polyethylene film, and vapour-permeable porous polymeric (e.g. Tyvek) film 1005. The pouch may be fabricated by heat sealing porous polymeric and impermeable films as described herein; heat seals 1011 a-1011 d are depicted in Figures 10 and 10A. The porous polymeric film forms part of the walls of chamber 1014, but the walls of compartment 1012 are formed only of the impermeable polymeric film. Compartment 1012 is separated from chamber 1014 by plastic double zip seal 1016, and has opening 1010 which is reversibly sealable with double zip seal 1018.

[176] As seen in Figure 10B, container 1008 is folded (along axis A seen in Figure 10) during manufacture or immediately prior to use, such that porous polymeric film 1005 is sandwiched between layers of the impermeable film 1007. The edges of the folded pouch (adjacent heat seals 1011 b, 1011 c and 1011 d) are preferably then sealed, for example with adhesive tape (tape 1088 seen in Figure 10B), to hold the fold in place and to aseptically enclose the porous polymeric film within the fold and thus protect it from contamination before the freeze-drying step. Moreover, fold 1036 immediately adjacent double zip seal 1016 and the retention of chamber 1014 against compartment 1012 help to maintain the integrity of double zip seal 1016, thus preventing ingress of milk from compartment 1012 into chamber 1014 during pascalization.

[177] In use, human breast milk is transferred through opening 1010 into compartment 1012 of folded and sealed double pouch container 1008a. Air inside the container is expelled and opening 1010 is then sealed with double zip seal 1018. The breast milk is thus enclosed in impermeable compartment 1012 within closed pouch container 1008a, and isolated from vapour-permeable porous polymeric film 1005 by plastic double zip seal 1016.

[178] Container 1008a is then enveloped in removable impermeable wrapping, for example by vacuum sealing it with waterproof polymeric film. The container thus protected against contamination is placed in a pressure vessel and subjected to high pressure processing as described herein. Within the container, plastic double zip seals 1016 and 1018 retain the breast milk in compartment 1012, thereby avoiding leakage from the container and contact between the breast milk and porous polymeric film 1005.

[179] After the high pressure treatment, pouch container 1008a now containing pascalized breast milk is retrieved from the pressure vessel, and the breast milk is frozen. Once frozen, the impermeable wrapping is removed, the container is unfolded (e.g. after removing the adhesive tape holding the fold in place), and plastic double zip seal 1016 is broken, thus opening a channel between compartment 1012 and chamber 1014. Optionally, the block of frozen breast milk is transferred to chamber 1014, plastic double zip seal 1016 is resealed to enclose the milk in chamber 1014 and compartment 1012 is cut away. The container is then transferred to a freeze- drier and the breast milk is freeze-dried as described herein, with the sublimated water egressing through vapour-permeable barrier film 1005. In a further variation, the milk is not frozen at all before unfolding the container, unsealing double zip seal 1016 and transferring the container to the freeze-drier. [180] Another embodiment of the invention is described with reference to Figure 11 , which shows a photograph of Tyvek-walled tubes 1108 and 1108a. The tubes include an impermeable HDPE film forming a lower wall, a vapour-permeable film 1105 (e.g. made of Tyvek 2FS film) forming an upper wall and threaded spout 1110. The impermeable and vapour permeable films are heat sealed together via heat seal 1111 , with spout 1110 sealed between the films to form a tube opening which is sealable with cap 1110a.

[181] In use, raw human breast milk is transferred into tube 1108 through spout 1110, and cap 1110a is fitted to seal the tube. Closed tube 1108a is then placed inside water-proof plastic bag 1123 (e.g. a Ziplock bag) and immersed in a water bath controlled to 62°C for 30 minutes. The container is then withdrawn from the water bath, the breast milk is rapidly cooled to below 25°C, and the tube is removed from bag 1123. Tube 1108a, now containing Flolder-pasteurised human breast milk, is then placed in a freeze-drier and freeze-dried as described herein to form powdered human breast milk.

[182] In a variation, the filled tube is placed in a dry heater, such as in the well of a heated tray, so that pasteurisation can be effected without immersing the tube in a liquid.

[183] The freeze-dried human breast milk may be reconstituted in tube 1108a by adding a reconstitution liquid, e.g. water, into the tube through spout 1110. Optionally, the reconstitution liquid may be provided in a second container, e.g. an impermeable film tube, including a spout which is configured to mate with spout 1110 (e.g. via a complementary threaded mouth). The reconstitution fluid may be water, optionally fortified with minerals, vitamins etc. It may optionally include liquid human breast milk. The reconstitution fluid in the second tube is preferably sterile, and may be sealed until immediately before reconstitution. When reconstitution of the powder is tube 1108 is required, the two tubes are opened, connected via their complementary spouts, and the reconstitution liquid is forced into tube 1108 to mix with the human breast milk powder therein. Advantageously, this may ensure that the risk of contamination during reconstitution is reduced, for example in environments where the quality of available water is uncertain. [184] Another embodiment of the invention will now be described with reference to Figure 12. Flexible polymeric pouch vessel 1203 with threaded spout 1204 is filled with human breast milk 1201 , and the pouch vessel is then sealed with removable screw-cap 1206. As one example, pouch vessel 1203 may be a pre-sterilised, commercially available breast milk storage pouch, configured to fit to a breast pump, which may thus be filled with a single serve portion of breast milk by a donor.

[185] Breast milk 1201 , sealed inside pouch vessel 1203, may optionally then be sterilised, for example by Flolder pasteurisation as described herein. Alternatively, it may be further processed without a prior sterilisation step.

[186] Sealed pouch vessel 1203 containing the breast milk (preferably with a depth of less than 20 mm) is then rapidly frozen to form frozen breast milk 1209, taking care that the frozen milk does not block the spout. The sealed pouch vessel 1203 is then enclosed inside bag 1208 made of vapour permeable porous polymeric (e.g. Tyvek) film 1205, optionally flushing with nitrogen gas to replace the air inside the bag. Flexible bag 1208 is significantly larger than pouch vessel 1203. Screw-cap 1206 is then manually twisted off spout 1204 by manipulating it through the loosely fitting bag walls, and pouch vessel 1203 is pressed open to expand the pouch walls away from the frozen block of breast milk 1209.

[187] Sealed bag 1208 is then transferred to vacuum chamber 1225 of a freeze- drier, and the vacuum chamber is evacuated. Due to the vapour permeability of the porous polymeric film walls, a vacuum develops inside pouch vessel 1203 and water vapour sublimates from frozen breast milk 1209, passes through spout 1204 and egresses through porous polymeric film 1205, as depicted by arrows 1299. The sublimation dehydrates the human breast milk, resulting in the formation of human breast milk powder 1213. Once a satisfactory degree of dehydration is achieved, the vacuum is released, optionally with an inert gas. Screw-cap 1206 is then immediately screwed onto spout 1204 to seal the powdered breast milk 1213 in pouch vessel 1203. Once the pouch vessel is re-sealed, bag 1208 may be opened and pouch vessel 1203 removed. The powdered breast milk in the pouch vessel may then optionally be irradiated and/or packaged in impermeable packaging for storage and transport. When required for consumption, pouch vessel 1203 can be opened and rehydrated using purified water.

[188] Another embodiment of the invention will now be described with reference to Figure 13. Flexible polymeric pouch vessel 1303 with threaded spout 1304 is filled with human breast milk 1301 and the pouch vessel is sealed with removable screw- cap 1306 if not immediately further processed. Pouch vessel 1303 may also be a pre-sterilised, commercially available breast milk storage pouch, or a bespoke pouch configured for pascalizing when sealed.

[189] Immediately prior to the pascalization step, screw-cap 1306 is removed and clamp 1309 (or similar, such as a clippable stick seal) may be used to seal breast milk 1301 in the lower half of the pouch vessel, taking care to exclude air bubbles. Screw-cap 1306 may then be replaced (as shown) if capable of withstanding the high pressure conditions. Clamped and sealed pouch vessel 1303 containing breast milk 1301 is then placed in pressure vessel 1323, which is filled with water 1327, and subjected to high pressure processing (i.e. pascalizing; represented by arrows 1328) at 20°C. The pressure in the pressure vessel may be maintained at a constant pressure of between 3000 and 6000 bar for between 1 and 5 minutes before release. Other pressure profiles, including multi-step pressurisation with intermediate pressure release, may be applied as described herein. Due to the flexibility of the pouch vessel walls, the hydraulic pressure in the pressure vessel is transmitted instantaneously and uniformly into breast milk 1301 , thus inactivating potentially harmful pathogens contained therein. The clamp retains the milk in the lower portion of the pouch, thus preventing the spout and screw-cap from being directly exposed to the milk at high pressure.

[190] After the pascalizing step, sealed pouch vessel 1303 containing pascalized milk is retrieved from the pressure vessel, and rapidly frozen to form frozen breast milk 1309. Without unsealing it, pouch vessel 1303 is then enclosed inside flexible bag 1308 made of vapour-permeable porous polymeric (e.g. Tyvek) film 1305, optionally flushing with nitrogen gas to replace the air inside the bag. Clamp 1309 and screw-cap 1306 are then manually removed by manipulating them through the bag walls, and pouch vessel 1303 is pressed open to expand the walls away from the frozen block of breast milk 1309.

[191] Sealed bag 1308 is then transferred to vacuum chamber 1325 of a freeze- drier, and the vacuum chamber is evacuated. Due to the vapour permeability of the porous polymeric film walls, a vacuum develops inside pouch vessel 1303 and water vapour sublimates from frozen breast milk 1309, passes through spout 1304 and egresses through porous polymeric film 1305, as depicted by arrows 1399. The sublimation dehydrates the human breast milk, resulting in the formation of human breast milk powder 1313. Once a satisfactory degree of dehydration is achieved, the vacuum is released, optionally with an inert gas. Screw-cap 1306 is then immediately screwed onto spout 1304 to seal the powdered breast milk 1313 in pouch vessel 1303. Once the pouch vessel is sealed, bag 1308 may be opened and the pouch vessel removed. The powdered breast milk in the pouch vessel may then optionally be irradiated and/or packaged in impermeable packaging for storage and transport. When required for consumption, pouch vessel 1303 can be opened and rehydrated using purified water.

[192] In a variation of this method, clamped and sealed pouch vessel 1303 containing breast milk 1301 is enclosed within flexible bag 1308 before the pascalizing step. Flexible bag 1308 is then enveloped in a removable impermeable wrapping, for example by vacuum sealing it with polymeric film or in a polymeric pouch, to remove air and to prevent contamination of flexible bag 1308 during the pascalizing. Pouch vessel 1303 is thus tightly wrapped with a first layer comprising the porous polymeric film 1305 from the flexible bag walls and a second, water-proof layer comprising the impermeable wrapping. The entire assembly is then placed in pressure vessel 1323 and subjected to high pressure processing to sterilise the enclosed breast milk. Afterwards, the impermeable wrapping is removed and breast milk 1301 is frozen and subjected to freeze-drying through the porous polymeric film walls of flexible bag 1308 as described above. Advantageously, the entire process of sequential pascalizing and freeze-drying thus takes place while enclosed in flexible bag 1308, so that any risk of bacterial or other microparticulate contamination is minimised. EXAMPLES

[193] The present invention is described with reference to the following examples. It is to be understood that the examples are illustrative of and not limiting to the invention described herein.

Example 1.

[194] Freeze-drying of enclosed full cream cow’s milk was investigated as a model for human breast milk. Approximately 200 g of milk was enclosed in heat- sealed polymeric film pouches having one impermeable polymeric film wall and one Tyvek film wall. The Tyvek film wall surface area was c.a. 400 cm 2 , and the depth of the milk was about 8 mm when the pouch was laid flat. The pouches were then placed in a commercial freeze-drier with the Tyvek film facing up, and the enclosed milk was freeze-dried at a pressure of below 5 mbar, as shown in Table 1. After 16 hours, the milk was dehydrated to form an acceptably dry powder (c.a. 16 wt% residual mass).

Table 1.

Example 2.

[195] Test work was conducted using 180 litres of unpasteurised human breast milk. The milk was collected from hospitals and individual donors using normal breast milk banking collection procedures, frozen and stored in -20°C freezers and domestic refrigerator freezers. The milk was thawed prior to the experiments. [196] The liquid milk was packaged in impermeable high density polyethylene (HDPE) film pouches, with a liquid capacity of between 1.5 and 2 litres. The pouches, which had dimensions of approximately 500m long by 220 mm wide, were fabricated by heat-sealing two rectangular sheets of robust but flexible clear HDPE film together along three of the four edges. After transfer of milk into the pouch, as much air as possible was expelled and the fourth edge of the pouch was heat sealed to enclose the milk in the liquid-full pouch. An example of the sealed pouch, containing milk, is shown in Figure 14. Multiple filled pouches (total milk volume of at least 30 litres) were then placed in the pressure vessel of a commercial-scale high pressure processing machine. The milk was then pascalized at 6000 bar for 3 minutes at 20°C, without any ingress of water or leakage of milk from the bag.

[197] Microbiological analysis, in a NATA accredited food testing laboratory, showed that the HPP treatment significantly reduced the microbial load in the milk. Samples of the unprocessed milk were found to have total bacterial counts, measured by the Standard Plate Count (in colony forming units per millilitre (cfu/g)) ranging from 120 cfu/g to 48,000 cfu/g (mean of 19,278 cfu/g). The HPP processed milk was similarly tested and the total bacterial counts ranged from 0 cfu/g to 2 cfu/g (mean of 0.4 cfu/g).

[198] Unprocessed and HPP processed breast milk samples were also analysed for a range of pathogenic and indicator organisms, including Cronobacter species, Salmonella, Listeria monocytogenes, Staphylococcus aureus, Enterobacteriaceae and E. coli. Low numbers of Enterobacteriaceae (0 cfu/g to 390 cfu/g, mean of 146 cfu/g) were detected in the unprocessed donor breast milk and these were reduced to 0 cfu/g after HPP. Low numbers of Staphylococcus aureus (0 cfu/g to 3 cfu/g, mean of 0.8 cfu/g) were detected in the unprocessed donor breast milk and these were reduced to 0 cfu/g (0 cfu/g - 1 cfu/g, mean 0.2 cfu/g) after HPP. No Cronobacter species, Salmonella, Listeria monocytogenes or E. coli were detected in the unprocessed donor breast milk and none were detected in the HPP processed donor breast milk.

[199] Analysis of the microbiology results show that the HPP process applied to naturally contaminated human donor breast milk achieved a 4.6 log reduction in the bacterial load. The actual log reduction may be greater as this only assessed the natural level of contamination and may actually be equally effective upon higher levels of bacteria.

[200] Sealed samples of the HPP donor breast milk were stored at 0°C - 5°C and microbiologically analysed at intervals to assess the shelf-life. The HPP processed breast milk samples were analysed for Standard Plate Count, Spore forming bacteria, Yeasts and Moulds. The HPP donor breast milk remained microbiologically stable for 64 days (9.1 weeks) at which time the test sample was fully consumed. It is likely that the actual microbiological shelf-life is well beyond this time.

Example 3.

[201] A pouch containing 1411 g of HPP sterilized milk (as prepared in Example 2) was frozen at -21 °C, and opened on one side. The open mouth of the pouch (bag A) was placed into the open mouth of a complementary Tyvek-walled pouch (bag B) as seen in Figure 15, and the frozen block of milk was slid into the Tyvek-walled pouch. The Tyvek-walled pouch was formed from impermeable HDPE film with an integrated Ziplock seal at the base and an open mouth, and included a sheet of vapour-permeable Tyvek film 2FS film, heat sealed to the impermeable HDPE film, as part of the container walls on one side of the pouch. After transfer of the frozen milk, the Tyvek-walled pouch was heat-sealed closed at the mouth.

[202] The sealed pouch was transferred to a commercial freeze-drier and freeze- dried from an initial temperature of -21 °C at a pressure of below 5 mbar, heating at 5°C per hour to a final temperature of 25°C. Water sublimated during the freeze drying escaped through the permeable Tyvek film, leaving a fine breast milk powder product contained in the sealed pouch. The recovered mass was 240 g, corresponding to 17 wt% of the precursor milk. The residual water content of the breast milk powder was determined to be about 2 wt%. A photograph of the breast milk powder in the pouch is shown in Figure 16, while Figure 17 shows a sample of the powdered milk product with excellent powder properties.

[203] The breast milk powder was microbiologically analysed and showed insignificant change in microbial load compared to the starting HPP sterilized milk. If retained in the pouch and sealed in impermeable packaging, an extended usable shelf life of the breast milk powder can be expected.

Example 4.

[204] Donated human breast milk, 20 litres, was divided into 100 impermeable polymeric film pouches having double Ziplock seals. The pouches were sealed, taking care to exclude air, the sealed pouches were enclosed in a polymer overbag (20 pouches per overbag) and subjected to HPP at 600 bar for three minutes at 20°C. Although 15 of the 100 bags pouches leaked due to a failure of the pouch heat welding (i.e. on the sides of the pouches), the double Ziplock seals all retained the milk without leakage.

Example 5.

[205] A single serve portion of donated human breast milk (c.a. 50 ml) was placed in a commercially available 180 ml breast milk pouch (Tommee Tippee Express and Go Pouch), sealed with its screw-cap and frozen. The pouch (dimensions c.a. 130 mm by 100 mm) was then placed inside a Tyvek bag (Steripack Headerbag pouch made from coated 1073B Dupont coated Tyvek, dimensions c.a. 200 mm by 150 mm) and the bag was heat sealed to enclose the pouch. The pouch was then opened by manually removing the cap inside the bag, i.e. by gripping the pouch and lid through the Tyvek bag walls and twisting to unscrew.

[206] The Tyvek bag with enclosed pouch was then transferred to a commercial freeze-drier and freeze-dried from an initial temperature of -21 °C at a pressure of below 5 mbar, heating at 5°C per hour to a final temperature of 25°C. Water sublimated during the freeze-drying escaped through the open pouch spout (inner diameter 25.5 mm) and then through the permeable Tyvek film, leaving a fine breast milk powder product contained in the pouch.

[207] Once freeze-drying was complete, the screw-cap was screwed onto the pouch, thus sealing the freeze-dried breast milk powder in the pouch before opening the Tyvek bag. The residual water content of the breast milk powder was less than 3 wt%. Example 6.

[208] Human breast milk was inoculated with 10 7 cfu/ml of deliberately added pathogens S. aureus, E.coli, S.typhimurium and a mixture of all three. The inoculated milk was freeze-dried in a scientific freeze-drier as follows: the liquid milk with a depth of 10-12 mm was frozen to -15°C, held at -15°C for 38 hours under high vacuum, then warmed slowly to -5°C over 10 hours under vacuum. A fine breast milk powder with a water content of 2.2 wt% was thus obtained. The milk was then reconstituted to the same initial volume with sterile water.

[209] Samples of the untreated milk and the reconstituted milk were both cultured for 24 hours on agar. Microbiological analysis of the cultured milk samples showed that the freeze-drying significantly reduced the microbial load in the milk. The results are shown in Table 2 below.

Table 2.

[210] The results demonstrate a substantial reduction (log reduction) in the loading of pathogenic bacteria caused by freeze-drying alone.

Example 7.

[211] The bacteriostatic properties of raw (untreated) human breast milk, Holder pasteurised human breast and reconstituted freeze-dried human breast milk (produced using the process of Example 6) was investigated. The milk samples were inoculated with 10 5 cfu/ml various pathogens (E.coli, S. typhimurium) and the reproduction of the bacteria after 6 and 24 hours was evaluated. The results are shown in Table 3. Table 3.

[212] The results demonstrate that freeze-dried breast milk retains bacteriostatic properties, significantly greater than Holder pasteurisation.

[213] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is understood that the invention includes all such variations and modifications which fall within the spirit and scope of the present invention.