Method and system for producing sterile human dairy products

Abstract

To provide a human milk sterilization process that reduces or eliminates harmful pathogens such as Bacillus cereus and Clostridium botulinum while also maintaining the nutritional quality of human milk. [Solution] The present disclosure provides a method for sterilization and production of a sterile human dairy product. Raw human milk is provided from at least one donor. The raw human milk may contain both Bacillus cereus and Clostridium botulinum. The raw human milk is provided to a closed in-line sterilization system to produce the sterile human dairy product. The raw human milk passes through at least one bacterial clarifier and is further separated into a cream fraction and a skim fraction. The skim fraction can then be further concentrated to produce a retentate. A standardized dairy product is engineered by combining a portion of the cream fraction and a portion of the retentate to achieve a desired concentration of ingredients and / or nutrients.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 404,097, filed October 4, 2016, U.S. Provisional Application No. 62 / 439,408, filed December 27, 2016, and U.S. Provisional Application No. 62 / 486,884, filed April 18, 2017. The contents of each of the above-referenced applications are incorporated by reference in their entirety.

[0002] 2. Field of the invention The present invention relates to a method for continuous flow sterilization of human milk to produce a sterile human milk product. More specifically, the present invention relates to a method for the reduction of Bacillus cereus (Bacillus cereus) and Clostridium botulinum (Clostridium botulinum) in human milk by sterilization under conditions selected to provide a sterile human milk product suitable for remote consumption by infants, particularly premature infants. [Background technology]

[0003] 3.Background The provision of human breast milk for remote consumption by individuals, such as adults, adolescents, or infants, requires the reduction of the content of pathogens, such as bacteria, viruses, molds, spores, and yeasts, to acceptable levels, along with the simultaneous retention of the nutritional and biologically active substances in the milk that are beneficial to the individual when ingested.

[0004] The primary risk in the human breast milk supply for consumption is the presence of one or both of Bacillus cereus and Clostridium botulinum in the supply. Bacillus cereus originates from human milk donors. While not all donors will be carriers, pooling of donor milk during the production of human dairy products can result in more widespread contamination. Bacillus cereus is a heat-resistant, spore-forming, gram-positive bacterium that, if ingested, is harmful and causes infections of the bloodstream, lungs, and central nervous system, requiring treatment with antibiotics such as vancomycin, tobramycin, meropenem, and clindamycin. Clostridium botulinum causes botulism. The toxins produced by Clostridium botulinum can block nerve function, resulting in various forms of paralysis and therefore can be a serious and potentially fatal disease.

[0005] Many milk banks use pasteurization during human milk processing. When applied to human milk, pasteurization typically involves heating the milk to 62.5°C for 30 minutes ("holder pasteurization"). See Guidelines of the Establishment and Operation of a Donor Human Milk Bank, Human Milk Banking Association of North America (2015). Methods to eliminate Bacillus cereus and Clostridium botulinum employ more extreme processing conditions. These extreme processing conditions, as well as the conditions employed for holder pasteurization, often adversely affect the quality of sterilized human milk. That is, the process irreversibly damages nutritional proteins, lipids, and carbohydrates (e.g., oligosaccharides). Thus, while sterilized human milk is pathogen-free and safe for consumption, the nutritional content is also substantially altered such that the benefits provided by consuming sterilized human milk are lost. Furthermore, some processes used to reduce Bacillus cereus and Clostridium botulinum content involve the direct injection of heated streams into the milk, which risks the introduction of contaminants and requires downstream separation of water from the milk.

[0006] Therefore, there is a need for a human milk sterilization process that reduces or eliminates harmful pathogens such as Bacillus cereus and Clostridium botulinum while also maintaining the nutritional quality of human milk. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Guidelines of the Establishment and Operation of a Donor Human Milk Bank, Human Milk Banking Association of North America (2015) Summary of the Invention [Means for solving the problem]

[0008] 4. Abstract The present disclosure provides a method for sterilizing and producing a sterile human dairy product. Raw human milk is provided from at least one donor. The raw human milk may contain both Bacillus cereus and Clostridium botulinum. The raw human milk is provided to a closed in-line sterilization system to produce the sterile human dairy product. The raw human milk passes through at least one bacterial clarifier and is further separated into a cream fraction and a skim fraction. The skim fraction can then be further concentrated to produce a retentate. A standardized dairy product is engineered by combining a portion of the cream fraction and a portion of the retentate to achieve a desired concentration of ingredients and / or nutrients. In some embodiments, the standardized dairy product is fortified with additional ingredients and / or nutrients. The standardized dairy product is further sterilized through a countercurrent heat exchange process. Rapidly raising the temperature of the standardized dairy product to a target temperature and holding it at the target temperature for a duration eliminates pathogen content while also maintaining the nutritional quality of the sterilized dairy product. Importantly, the standardized dairy products of the present disclosure exhibit at least a 12 Log reduction in Clostridium botulinum content and greater than a 1,000 Log reduction in Bacillus cereus, making the standardized dairy products safe for consumption by adults, adolescents, and especially premature infants.

[0009] Thus, disclosed herein is a method for sterilizing human milk to produce a sterile human milk product, the method comprising the steps of receiving a human milk sample as an input by a sterilizer located in a closed in-line system, flowing the human milk sample in a first direction at a first flow rate through a tube of the sterilizer, heating the human milk sample in the tube by flowing a heating fluid in a second direction at a second flow rate, the heating fluid contacting an exterior surface of the tube, and cooling the heated human milk sample by the sterilizer.

[0010] In some embodiments, heating the human milk sample in the tube comprises increasing the temperature of the human milk sample to a temperature range of 130°C to 150°C and holding the temperature of the human milk sample within the temperature range for a duration of 3 to 15 seconds. In some embodiments, heating the human milk sample in the tube comprises increasing the temperature of the human milk sample to a temperature range of 135°C to 145°C and holding the temperature of the human milk sample within the temperature range for a duration of 6 to 14 seconds. In some embodiments, heating the human milk sample in the tube comprises increasing the temperature of the human milk sample to a temperature range of 138°C to 142°C and holding the temperature of the human milk sample within the temperature range for a duration of 8 to 13 seconds. In some embodiments, heating the human milk sample in the tube comprises increasing the temperature of the human milk sample to a temperature range of 140°C to 141°C and holding the temperature of the human milk sample within the temperature range for a duration of 12 to 13 seconds.

[0011] In various embodiments, the length of the tubing portion and the first flow rate of the fortified human milk sample are each predetermined to maintain the temperature of the fortified human milk within the temperature range for that duration.

[0012] In some embodiments, the first tube of the pharmaceutical-grade sterilizer has a diameter of between 0.25 inches and 10 inches. In some embodiments, a first flow rate of the fortified human milk sample through the tube of the pharmaceutical-grade sterilizer is between 0.25 gallons per minute and 25 gallons per minute. In some embodiments, the step of heating the human milk sample in the tube comprises preheating the human milk sample in a first section of the tube to a first temperature of between 80°C and 100°C and heating the human milk sample in a second section of the tube to a second temperature of between 130°C and 150°C.

[0013] In some embodiments, the human milk sample received by the pharmaceutical-grade sterilizer is not homogenized, hi some embodiments, the human milk sample is homogenized prior to being received by the sterilizer or is homogenized after being cooled by the sterilizer.

[0014] In various embodiments, a method for sterilizing human milk to produce a sterile human milk product includes eliminating pathogens from a raw human breast milk sample through one or more purification processes prior to receiving the human milk sample in a sterilizer. In some embodiments, the method further includes separating the clarified human milk sample into a cream sample and a skim sample, performing a concentration process on the skim sample to produce a retentate, and combining a portion of the cream sample with a portion of the obtained retentate to design a standardized human milk sample and produce a fortified human milk product. In some embodiments, the method of combining a portion of the cream sample with a portion of the obtained retentate includes detecting initial properties of the cream sample, comparing the detected initial properties of the cream sample to target properties for the fortified human milk product, and determining a portion of the cream sample and a portion of the obtained retentate based on the comparison.

[0015] In various embodiments, the method further includes the step of cooling the heated fortified human milk through an aseptic process followed by packaging the sterilized human milk product. In various embodiments, the sterilizer is one of a commercial-grade sterilizer or a pharmaceutical-grade sterilizer.

[0016] In some embodiments, the sterilized human dairy product has at least 63 calories per 100 milliliters of sterilized human dairy product. In some embodiments, the sterilized human dairy product has a total fat content of at least 3.0 g / 100 mL (3% by weight).

[0017] In various embodiments, the sterilized human milk products retain greater than 88% of the immunoglobulin A (IgA) compared to the raw human breast milk sample from which they were derived. In various embodiments, the sterilized human milk products retain greater than 77% of the immunoglobulin M (IgM) compared to the raw human breast milk sample from which they were derived. In various embodiments, the sterilized human milk products retain greater than 93% of the immunoglobulin G (IgG) compared to the raw human breast milk sample from which they were derived. In various embodiments, the sterilized human milk products retain greater than 64% of the antitrypsin compared to the raw human breast milk sample from which they were derived. In various embodiments, the sterilized human milk products retain greater than 81% of the lactoferrin compared to the raw human breast milk sample from which they were derived. In various embodiments, the sterilized human milk products retain greater than 78% of the lysozyme compared to the raw human breast milk sample from which they were derived. In various embodiments, the sterilized human milk products retain greater than 72% of the lactalbumin compared to the raw human breast milk sample from which they were derived. In various embodiments, the sterilized human milk products retain greater than 92% of the alpha casein compared to the raw human breast milk sample from which they were derived. In various embodiments, the sterilized human milk products retain greater than 92% of the beta casein compared to the raw human breast milk sample from which they were derived. In various embodiments, the sterilized human milk products retain greater than 92% of the kappa casein compared to the raw human breast milk sample from which they were derived. In other embodiments, the sterilized human milk products retain greater than 87% of the osteopontin compared to the raw human breast milk sample from which they were derived.

[0018] In various embodiments, the sterilized human milk products retain greater than 92% of total human milk oligosaccharides compared to the raw human breast milk sample from which the sterilized human milk products were derived. In various embodiments, the sterilized human milk products retain greater than 90% of fucosylated human milk oligosaccharides compared to the raw human breast milk sample from which the sterilized human milk products were derived. In various embodiments, the sterilized human milk products retain greater than 90% of 2'-fucosyllactose compared to the raw human breast milk sample from which the sterilized human milk products were derived. In various embodiments, the sterilized human milk products retain greater than 90% of 3'-fucosyllactose compared to the raw human breast milk sample from which the sterilized human milk products were derived. In various embodiments, the sterilized human milk products retain greater than 90% of sialylated human milk oligosaccharides compared to the raw human breast milk sample from which the sterilized human milk products were derived. In various embodiments, the sterilized human milk products retain greater than 90% of non-fucosylated human milk oligosaccharides compared to the raw human breast milk sample from which the sterilized human milk products were derived.

[0019] Thus, also disclosed herein is a closed in-line processing system comprising a sterilizer including a tube configured to cause a human milk sample to flow in a first direction at a first flow rate, and a container with a portion of the tube residing therein, the container configured to cause a heated fluid in contact with an exterior surface of the portion of the tube to flow in a second direction at a second flow rate to heat the human milk sample flowing through the first portion of the tube to a predetermined temperature for a duration.

[0020] In some embodiments, the predetermined temperature is within a temperature range of 130°C to 150°C, and the predetermined duration is between 3 and 15 seconds. In some embodiments, the predetermined temperature is within a temperature range of 135°C to 145°C, and the predetermined duration is between 6 and 14 seconds. In some embodiments, the predetermined temperature is within a temperature range of 138°C to 142°C, and the predetermined duration is between 8 and 13 seconds. In some embodiments, the predetermined temperature is within a temperature range of 140°C to 141°C, and the predetermined duration is between 12 and 13 seconds. In various embodiments, the sterilizer tubing is a spiral tubing, and the duration for which the human milk sample is held at the predetermined temperature depends on the number of spirals in the spiral tubing. In some embodiments, the first flow rate of the human milk sample through the pharmaceutical-grade sterilizer tubing is between 0.25 gallons per minute and 25 gallons per minute.

[0021] In various embodiments, the system further comprises one or more bacterial clarifiers, each bacterial clarifier configured to eliminate pathogens from the raw human breast milk sample; a milk separator in fluid communication with the last of the series of at least one bacterial clarifier, the milk separator configured to separate the clarified human milk sample into a cream sample and a skim sample; a milk concentrator in fluid communication with the milk separator, the milk concentrator further configured to produce a retentate by performing a reverse osmosis process on the skim sample received from the milk separator; and a milk combiner configured to combine a portion of the cream sample from the milk separator and a portion of the retentate from the milk concentrator.

[0022] In some embodiments, the milk standardizer is in fluid communication with the sterilizer and the homogenizer is not located in the closed in-line processing system prior to the sterilizer. In some embodiments, the system is capable of undergoing clean-in-place (CIP) methods of cleaning. In some embodiments, the sterilizer is a pharmaceutical-grade sterilizer. In some embodiments, the sterilizer is a commercial-grade sterilizer.

[0023] Thus, also disclosed herein are sterilized human dairy products comprising a bacterial aerobic plate count of less than 10 CFU per gram of sterilized human dairy product and lactoferrin at a concentration of 0.81 grams per liter to 13.28 grams per liter of sterilized human dairy product. In some embodiments, the sterilized human dairy product further comprises lysozyme at a concentration of 0.012 grams per liter to 0.105 grams per liter of sterilized human dairy product. In some embodiments, the sterilized human dairy product further comprises lactalbumin at a concentration of 1.87 grams per liter to 2.68 grams per liter of sterilized human dairy product. In some embodiments, the sterilized human dairy product further comprises antitrypsin at a concentration of 0.057 grams per liter to 0.40 grams per liter of sterilized human dairy product. In some embodiments, the sterilized human dairy product further comprises human milk oligosaccharides at a concentration of 8.8 grams per liter to 20.0 grams per liter of sterilized human dairy product. In some embodiments, the sterilized human dairy product further comprises 2'-fucosyllactose at a concentration of from 0.72 grams per liter to 4.3 grams per liter of sterilized human dairy product.

[0024] In some embodiments, the sterilized human dairy product has, per 100 milliliters, (a) 63-67 calories, (b) 30-34 calories from fat, (c) 1.2-1.8 grams saturated fat, (d) 10-16 mg cholesterol, (e) 10-20 mg sodium, (f) 4-10 grams total carbohydrate, (g) 4-7 grams sugars, (h) 1.2-1.6 grams protein, (i) 160-200 international units of vitamin A, and (j) 27-35 mg calcium. In some embodiments, the sterilized human dairy product has more than 63 calories per 100 milliliters. For example, sterilized human dairy products can have 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 calories per 100 milliliters of sterilized human dairy product.

[0025] In some embodiments, the sterilized human dairy product has a reduced amount of Bacillus cereus compared to the amount of Bacillus cereus in the raw human breast milk sample from which the sterilized human dairy product was sterilized, the reduced amount being at least a 500 log reduction of Bacillus cereus. In some embodiments, the sterilized human dairy product has a reduced amount of Clostridium botulinum compared to the amount of Clostridium botulinum in the raw human breast milk sample from which the sterilized human dairy product was sterilized, the reduced amount being at least a 12 log reduction of Clostridium botulinum.

[0026] In some embodiments, the sterilized human dairy product is contained within a sterile packaged product, hi some embodiments, the sterile packaged product is packaged in one of a bottle, a booster cup, a paper box, a paper brick, or a pouch.

[0027] In some embodiments, the sterilized human dairy product is contained within an extended shelf life packaged product, hi some embodiments, the extended shelf life packaged product is packaged in one of a bottle, a booster cup, a carton, a paper brick, or a pouch.

[0028] Various objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of embodiments of the invention, taken in conjunction with the accompanying drawings, in which like numerals represent like elements and in which: In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A method of sterilizing human milk to produce a sterile human milk product, comprising: receiving a human milk sample as an input by a sterilizer located in a closed in-line system; flowing the human milk sample in a first direction at a first flow rate through a tube of the sterilizer; heating the human milk sample within the tube by flowing a heating fluid in a second direction at a second flow rate, the heating fluid contacting an exterior surface of the tube; cooling the heated human milk sample through the sterilizer; A method comprising: (Item 2) The step of heating the human milk sample in the tube comprises: Raising the temperature of the human milk sample to a temperature range of 130°C to 150°C; maintaining the temperature of the human milk sample within the temperature range for a duration of 3 to 15 seconds; Item 1. The method according to item 1, comprising: (Item 3) The step of heating the human milk sample in the tube comprises: Raising the temperature of the human milk sample to a temperature range of 135°C to 145°C; maintaining the temperature of the human milk sample within said temperature range for a duration of 6 to 14 seconds; Item 1. The method according to item 1, comprising: (Item 4) The step of heating the human milk sample in the tube comprises: Raising the temperature of the human milk sample to a temperature range of 138°C to 142°C; maintaining the temperature of the human milk sample within the temperature range for a duration of 8 to 13 seconds; Item 1. The method according to item 1, comprising: (Item 5) The step of heating the human milk sample in the tube comprises: Raising the temperature of the human milk sample to a temperature range of 140°C to 141°C; maintaining the temperature of the human milk sample within the temperature range for a duration of 12 to 13 seconds; Item 1. The method according to item 1, comprising: (Item 6) 6. The method of any one of items 1-5, wherein the length of the portion of the tubing and the first flow rate of the human milk sample are each predetermined to maintain the temperature of the human milk sample within the temperature range for the duration. (Item 7) 7. The method of any one of items 1-6, wherein the first tube of the sterilizer has a diameter of 0.25 to 10 inches. (Item 8) 8. The method of any one of items 1-7, wherein the first flow rate of the human milk sample through the tubing of the sterilizer is between 0.25 gallons per minute and 25 gallons per minute. (Item 9) 9. The method of any one of items 1-8, wherein the human milk sample received by the sterilizer is not homogenized. (Item 10) 9. The method of any one of items 1-8, wherein the human milk sample is homogenized prior to receipt by the sterilizer or is homogenized after cooling by the sterilizer. (Item 11) 11. The method of any one of items 1-10, further comprising eliminating pathogens from the raw human breast milk sample through one or more purification processes prior to the sterilizer receiving the human milk sample. (Item 12) After the one or more purification processes and prior to the sterilizer receiving the human milk sample, separating the clarified human milk sample into a cream sample and a skim sample; performing a concentration process on the skim sample to produce a retentate; combining a portion of the cream sample with a portion of the obtained retentate to design a standardized human milk sample and produce a fortified human milk product; Item 12. The method of item 11, further comprising: (Item 13) combining a portion of the cream sample with a portion of the obtained retentate, detecting an initial characteristic of the cream sample; comparing the detected initial properties of the cream sample with target properties for the fortified human dairy product; determining a portion of the creep sample and a portion of the obtained retentate based on the comparison; Item 13. The method according to item 12, comprising: (Item 14) 14. The method of any one of items 1-13, further comprising the step of packaging the sterilized human dairy product following cooling the sterilized human dairy product through an aseptic process. (Item 15) 15. The method of any one of items 1-14, wherein the sterilizer is a pharmaceutical-grade sterilizer. (Item 16) 16. The method of any one of items 1-15, wherein the sterilized human dairy product has at least 63 calories per 100 milliliters of the sterilized human dairy product. (Item 17) 17. The method according to any one of items 1-16, wherein the sterilized human dairy product has a total fat content of at least 3.0 g / 100 mL (3% by weight). (Item 18) 18. The method of any one of items 1-17, wherein the sterilized human milk product retains greater than 88% of immunoglobulin A compared to the raw human breast milk sample from which the sterilized human milk product is derived. (Item 19) 19. The method of any one of items 1-18, wherein the sterilized human milk product retains greater than 77% of the immunoglobulin M compared to the raw human breast milk sample from which the sterilized human milk product is derived. (Item 20) 20. The method of any one of items 1-19, wherein the sterilized human milk product retains greater than 93% of immunoglobulin G compared to the raw human breast milk sample from which the sterilized human milk product is derived. (Item 21) 21. The method of any one of items 1-20, wherein the sterilized human milk product retains greater than 64% of the antitrypsin compared to the raw human breast milk sample from which the sterilized human milk product is derived. (Item 22) 22. The method of any one of items 1-21, wherein the sterilized human milk product retains greater than 81% of the lactoferrin compared to the raw human breast milk sample from which the sterilized human milk product is derived. (Item 23) 23. The method of any one of items 1-22, wherein the sterilized human milk product retains greater than 78% of lysozyme compared to the raw human breast milk sample from which the sterilized human milk product is derived. (Item 24) 24. The method of any one of items 1-23, wherein the sterilized human milk product retains greater than 72% of the lactalbumin compared to the raw human breast milk sample from which the sterilized human milk product is derived. (Item 25) 25. The method of any one of items 1-24, wherein the sterilized human milk product retains greater than 92% alpha-casein compared to the raw human breast milk sample from which the sterilized human milk product is derived. (Item 26) 26. The method of any one of items 1-25, wherein the sterilized human dairy product retains greater than 92% beta-casein compared to the raw human breast milk sample from which the sterilized human dairy product is derived. (Item 27) 27. The method of any one of items 1-26, wherein the sterilized human milk product retains greater than 92% of the kappa-casein compared to the raw human breast milk sample from which the sterilized human milk product is derived. (Item 28) 28. The method of any one of items 1-27, wherein the sterilized human milk product retains greater than 87% of osteopontin compared to the raw human breast milk sample from which the sterilized human milk product is derived. (Item 29) 29. The method of any one of items 1-28, wherein the sterilized human milk product retains greater than 92% of the total human milk oligosaccharides compared to the raw human breast milk sample from which the sterilized human milk product is derived. (Item 30) 30. The method of any one of items 1-29, wherein the sterilized human milk product retains greater than 90% of the fucosylated human milk oligosaccharides compared to the raw human breast milk sample from which the sterilized human milk product is derived. (Item 31) 31. The method of any one of items 1-30, wherein the sterilized human milk product retains greater than 90% of the 2'-fucosyllactose compared to the raw human breast milk sample from which the sterilized human milk product is derived. (Item 32) 32. The method of any one of items 1-31, wherein the sterilized human milk product retains greater than 90% of the 3'-fucosyllactose compared to the raw human breast milk sample from which the sterilized human milk product is derived. (Item 33) 33. The method of any one of items 1-32, wherein the sterilized human milk product retains greater than 90% of sialylated human milk oligosaccharides compared to the raw human breast milk sample from which the sterilized human milk product is derived. (Item 34) 34. The method of any one of paragraphs 1-33, wherein the sterilized human milk product retains greater than 90% of the non-fucosylated human milk oligosaccharides compared to the raw human breast milk sample from which the sterilized human milk product is derived. (Item 35) 1. A closed in-line processing system comprising: 1. A sterilizer comprising: a tube configured to cause the human milk sample to flow in a first direction at a first flow rate; a container within which the tubing section resides, the container configured to flow a heating fluid in contact with an exterior surface of the tubing section in a second direction at a second flow rate to heat the human milk sample flowing through the first tubing section to a predetermined temperature for a predetermined duration; The system includes a sterilizer. (Item 36) Item 36. The system according to item 35, wherein the predetermined temperature is within a temperature range of 130°C to 150°C, and the predetermined duration is 3 to 15 seconds. (Item 37) Item 36. The system of item 35, wherein the predetermined temperature is within a temperature range of 135°C to 145°C, and the predetermined duration is 6 to 14 seconds. (Item 38) Item 36. The system of item 35, wherein the predetermined temperature is within a temperature range of 138°C to 142°C, and the predetermined duration is 8 to 13 seconds. (Item 39) Item 36. The system according to item 35, wherein the predetermined temperature is within a temperature range of 140°C to 141°C, and the predetermined duration is 12 to 13 seconds. (Item 40) 40. The system of any one of items 35-39, wherein the first flow rate of the human milk sample through the tubing of the sterilizer is between 0.25 gallons per minute and 25 gallons per minute. (Item 41) one or more bacterial clarifiers, each bacterial clarifier configured to eliminate pathogens from the raw human breast milk sample; a milk separator in fluid communication with the last of the series of at least one bacterial clarifier, the milk separator configured to separate the clarified human milk sample into a cream sample and a skim sample; a milk concentrator in fluid communication with the milk separator, the milk concentrator being further configured to perform a reverse osmosis process on the skim sample received from the milk separator to produce a retentate; a milk combiner configured to combine a portion of the cream sample from the milk separator and a portion of the retentate from the milk concentrator; 41. The system of any one of items 35-40, further comprising: (Item 42) 42. The system of claim 41, wherein the milk combiner is in fluid communication with the sterilizer and a homogenizer is not located in the closed in-line processing system prior to the sterilizer. (Item 43) 43. The system of any one of items 35-42, wherein the system is capable of undergoing a clean-in-place (CIP) method of cleaning. (Item 44) 44. The system of any one of items 35-43, wherein the sterilizer is a pharmaceutical-grade sterilizer. (Item 45) 44. The system of any one of items 35-43, wherein the sterilizer is a commercial-grade sterilizer. (Item 46) 1. A sterilized human dairy product comprising: a bacterial aerobic plate count of less than 10 CFU per gram of said sterilized human dairy product; lactoferrin in a concentration of from 0.81 grams per liter to 13.28 grams per liter of said sterilized human dairy product; Contains sterile human dairy products. (Item 47) 47. The sterilized human milk product of item 46, comprising lysozyme in a concentration of from 0.012 grams per liter to 0.105 grams per liter of the sterilized human milk product. (Item 48) 48. The sterilized human dairy product of item 46 or 47, further comprising lactalbumin in a concentration of 1.87 grams per liter to 2.68 grams per liter of the sterilized human dairy product. (Item 49) 49. The sterilized human dairy product of any one of items 46-48, further comprising antitrypsin at a concentration of 0.057 grams per liter to 0.40 grams per liter of the sterilized human dairy product. (Item 50) 50. The sterilized human dairy product of any one of items 46-49, further comprising human milk oligosaccharides at a concentration of 8.8 grams per liter to 20.0 grams per liter of the sterilized human dairy product. (Item 51) 51. The sterilized human dairy product of any one of items 46-50, further comprising 2'-fucosyllactose in a concentration of from 0.72 grams per liter to 4.3 grams per liter of the sterilized human dairy product. (Item 52) 52. The sterilized human dairy product of any one of items 46-51, wherein the product has, per 100 milliliters, (a) 63 to 67 calories, (b) 30 to 34 calories from fat, (c) 1.2 to 1.8 grams of saturated fat, (d) 10 to 16 mg of cholesterol, (e) 10 to 20 mg of sodium, (f) 4 to 10 grams of total carbohydrates, (g) 4 to 7 grams of sugars, (h) 1.2 to 1.6 grams of protein, (i) 160 to 200 international units of vitamin A, and (j) 27 to 35 mg of calcium. (Item 53) 53. The sterilized human dairy product of any one of items 46-52, wherein the product has a reduced amount of Bacillus cereus compared to the amount of Bacillus cereus in a raw human breast milk sample from which the sterilized human dairy product was sterilized, wherein the reduced amount is at least a 500 log reduction of Bacillus cereus. (Item 54) 54. The sterilized human dairy product of any one of items 46-53, wherein the product has a reduced amount of Clostridium botulinum compared to the amount of Clostridium botulinum in the raw human breast milk sample from which the sterilized human dairy product was sterilized, wherein the reduced amount is at least a 12 log reduction of Clostridium botulinum. (Item 55) 55. A sterile packaged product comprising a sterilized human dairy product according to any one of items 46-54. (Item 56) 56. The sterile packaged product of item 55, wherein the sterile packaged product is packaged in one of a bottle, a booster cup, a paper box, a paper brick, or a pouch. (Item 57) 55. A shelf-life extended packaged product comprising a sterilized human dairy product according to any one of items 46-54. (Item 58) Item 58. The shelf life extended packaged product of item 57, wherein the shelf life extended packaged product is packaged in one of a bottle, a booster cup, a paper box, a paper brick, or a pouch. [Brief explanation of the drawings]

[0029] 5. Brief description of the drawings The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0030] [Figure 1A] FIG. 1A depicts a flow process within a system for processing raw human breast milk to produce a sterile human milk product, according to an embodiment of the present invention. [Figure 1B] FIG. 1B depicts an optional bacterial clarifier (synonymously, milk clarifier) device of the system, according to an embodiment of the present invention. [Figure 1C] FIG. 1C depicts an optional milk standardizer device of the system, according to an embodiment of the present invention. [Figure 2] FIG. 2 depicts a system diagram of a milk sterilizer in a closed in-line sterilization system according to an embodiment of the present invention. [Figure 3] FIG. 3 depicts a graph of dairy product temperature while flowing through a milk sterilizer, according to an embodiment of the present invention. [Figure 4] FIG. 4 depicts a flow chart for producing a sterilized human dairy product according to an embodiment of the present invention. [Figure 5] FIG. 5 depicts a flow chart for sterilizing dairy products according to an embodiment of the present invention. [Figure 6A] FIG. 6A tabulates parameters from several pilot-scale steps of a sterilization process according to an embodiment of the present invention, as further described in Example 7.1. [Figure 6B] FIG. 6B presents comparative nutritional data for raw human milk samples and sterilized human milk products prepared according to embodiments of the present invention. [Figure 6C] FIG. 6C presents the compositional profile of the first batch of raw milk samples and the sterilized human dairy product resulting from sterilization steps 4 and 11 as defined in FIG. 6A, where the retention of each compositional profile is calculated for the first batch of raw dairy product. [Figure 6D]FIG. 6D presents the compositional profile of the second batch of raw milk samples and the sterilized human dairy product resulting from sterilization steps 7 and 14 as defined in FIG. 6A, where the retention of each compositional profile is calculated for the second batch of raw milk product. [Figure 6E] FIG. 6E presents the quantified bacterial, mold, and yeast content for raw human milk and sterilized human milk products. [Figure 7A] 7A and 7B present the amounts of various supplements added per liter of milk sample to obtain a fortified milk sample. [Figure 7B] 7A and 7B present the amounts of various supplements added per liter of milk sample to obtain a fortified milk sample. [Figure 7C] FIG. 7C depicts the processing parameters used to sterilize each fortified milk sample and the Bacillus cereus log reduction of each fortified milk sample after sterilization. [Figure 8A] FIG. 8A depicts analytical data for the composition of the sterilized human dairy product processed in Example 7.3. [Figure 8B] FIG. 8B depicts the microbial counts from bottles 158, 1888, and 3151 compared to raw human milk. [Figure 9A] FIG. 9A presents analytical data and identifies the method criteria used to determine the various corresponding compositional components of the commercial-scale sterilized and fortified human dairy products produced in Example 7.4. [Figure 9B] FIG. 9B depicts the microbial counts of aseptically bottled sterilized fortified human milk products compared to raw fortified human milk. DETAILED DESCRIPTION OF THE INVENTION

[0031] The figures use like reference numbers to identify like elements. Letters following a reference number, such as "135A," indicate that the text specifically refers to the element with that particular reference number. A reference number in the text without a following letter, such as "135," refers to any or all of the elements in the figure bearing that reference number (e.g., "bacteria purifier 135" in the text refers to the reference numbers "bacteria purifier 135A" and / or "bacteria purifier 135B" in the figures).

[0032] 6. Detailed Description (6.1.Definition) It is to be understood that the invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0033] The detailed description of the present invention is divided into various sections for the convenience of the reader only, and disclosure found in any section may be combined with that in another section. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0034] Throughout this disclosure, various aspects of the invention may be presented in a range format. Ranges include the recited endpoints. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be construed as including all specifically disclosed subranges and individual numerical values within that range. For example, a description of a range such as 1 to 6 should be construed as including specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0035] Unless specifically stated otherwise or apparent from the context, as used herein, the term "or" is understood to be inclusive.

[0036] Unless specifically stated otherwise or clear from context, as used herein, the terms "a," "an," and "the" are understood to be singular or plural. That is, the articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0037] In this disclosure, the words "comprises," "comprising," "containing," "having," "includes," "including," and variations thereof have the meaning ascribed to them in United States patent law and allow for the presence of additional elements other than those expressly recited.

[0038] Unless specifically stated or otherwise clear from the context, as used herein, the term "about" or "approximately" is understood as within the general tolerances in the art, e.g., within two standard deviations of the mean, and is intended to encompass a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the stated value.

[0039] The term "raw human milk" or "raw human breast milk" composition is understood to mean human milk obtained from one or more human women without any form of pasteurization, sterilization, or decontamination. The raw composition is obtained from one or more donors, optionally has been pooled, and optionally may be contaminated with Bacillus cereus and other bacterial, viral, mold, spore, or yeast pathogens.

[0040] The term "sterilized human dairy" composition is understood to mean a dairy composition obtained after application of a method of sterilization according to various embodiments of the present invention.

[0041] 6.2. Systems and Methods for Producing Sterile Human Dairy Products (6.2.1. Sterilization System Devices) FIG. 1A depicts a flow process for processing raw human breast milk to produce a sterile human milk product, according to an embodiment of the present invention.

[0042] 1A, raw human breast milk 110 is provided to a closed in-line sterilization system 100 that produces a sterile human milk product 175. In various embodiments, once the raw human breast milk 110 enters the closed in-line sterilization system 100, the milk is not again exposed to the outside environment until it is collected as a sterile human milk product 175.

[0043] The system comprises a milk sterilizer 130 .

[0044] In various embodiments, the closed in-line sterilization system 100 further includes one or more additional devices, such as those shown in FIG. 1A , such as a bacterial clarifier (synonymously, a milk clarifier) 115, a milk standardizer 120, an enricher 125, or additional devices not shown in FIG. 1A , such as a milk homogenizer that homogenizes the milk sample or a deaerator that removes oxygen from the milk sample.

[0045] By way of example, in some embodiments, such as those preferred for producing fortified dairy products suitable for remote consumption by premature infants, system 100 includes a bacterial clarifier 115 and a milk standardizer 120. In some of these embodiments, system 100 further includes an fortifier 125 and / or a milk homogenizer. In other embodiments, such as those preferred for producing fortified dairy products designed for adult consumption, system 100 includes fortifier 125 but does not include milk standardizer 120. In some of these latter embodiments, system 100 further includes a bacterial clarifier (milk clarifier) 115. In some embodiments, the devices in closed in-line sterilization system 100 may be ordered differently. For example, the positions of bacterial clarifier (milk clarifier) 115, milk standardizer 120, and fortifier 125 in system 100 may be swapped.

[0046] In various embodiments, the devices of the closed in-line sterilization system 100 are in fluid communication with one another so that continuous flow processing of raw human breast milk 110 can occur as it is processed through the system 100. As depicted in FIG. 1A , for example, a bacterial clarifier (milk clarifier) 115 may be in fluid communication with a milk standardizer 120, which is further in fluid communication with an enricher 125, which is further in fluid communication with a milk standardizer 130. Tubes, pipes, or the like may be connected to the individual devices of the system 100 through welding, threads, or other suitable connections. In various embodiments, the closed in-line sterilization system 100 includes one inlet (e.g., through which raw human breast milk 110 is input) and one outlet (e.g., through which sterilized human dairy product 175 is output).

[0047] (6.2.1.1. Unprocessed human breast milk) The system 100 receives a batch of raw human breast milk 110 .

[0048] In typical embodiments, a batch of raw human breast milk 110 is prepared by pooling multiple individual milk donations. In some embodiments, the multiple milk donations are from a single donor. In typical embodiments, the multiple milk donations include milk from multiple donors.

[0049] In a typical embodiment, each donor collects multiple raw human milk samples and freezes each sample as soon as it is fully expressed. The multiple frozen raw human milk samples are then shipped frozen, pooled, and provided to system 100 for processing.

[0050] After receipt, the raw human breast milk from each donor is verified, i.e., determined to meet certain requirements. In a typical embodiment, one or more of an organoleptic test, a boiling mass test, an alcohol test, an acidity test, a resazurin test, a drug test, and a milk inhibitor test are performed. Typically, raw milk donations that do not pass the verification tests are discarded to prevent the donation from contaminating or adulterating the pooled raw human breast milk 110 provided to the system 100.

[0051] In various embodiments, prior to being provided to the closed in-line system 100, the raw human breast milk 110 is sampled to determine the characteristics or concentrations of components of the raw human breast milk 110. As used hereinafter, characteristics of the raw human breast milk 110 (and other dairy products described below) include the density of the milk and the total calories of the milk. In addition, the components of the raw human breast milk 110 (and other dairy products described below) include lipids / fats, proteins (whey, casein, albumin, etc.), vitamins, cholesterol, ionic salts (e.g., sodium, calcium, iron, etc.), carbohydrates, immunoglobulins, and oligosaccharides. These initial characteristics and / or concentrations of the components of the raw human breast milk 110 may be provided to the milk combiner 150 to design a standardized human dairy product. The process of designing a standardized human dairy product is described in further detail below.

[0052] In one embodiment, the total volume of the batch of raw human breast milk 110 is 1,200 liters. In other embodiments, the total volume of the batch of raw human breast milk 110 is more or less than 1,200 liters.

[0053] (6.2.1.2. Bacterial clarifier (milk clarifier)) Bacterial clarifier (and, synonymously, milk clarifier) 115 is a device or apparatus capable of removing some or all of the pathogens and / or pathogens (e.g., bacteria, mold, spore, and / or yeast contaminants) in raw human breast milk 110. In one embodiment of the system, bacterial clarifier 115 is the first device in system 100 that receives raw human breast milk 110 and outputs clarified milk to the next device, which in some embodiments is a milk standardizer 120. In other embodiments, bacterial clarifier 115 outputs clarified milk to an fortifier 125. In other embodiments, bacterial clarifier 115 outputs clarified milk directly to a milk sterilizer 130.

[0054] In some embodiments, the bacterial clarifier 115 comprises a single bacterial clarifier device 135 that outputs clarified milk 122 for input into the next device in the system 100. In some embodiments, the single bacterial clarifier removes up to 90% of pathogens, such as Bacillus cereus and / or Clostridium botulinum spores, from the milk.

[0055] In some embodiments, bacterial purifier 115 comprises multiple bacterial purifier devices. Reference is now made to FIG. 1B, which depicts two individual bacterial purifiers 135A and 135B connected in series, according to an embodiment of the present invention. In this embodiment, first bacterial purifier 135A removes a first portion of pathogens and / or pathogenic substances. The milk is then flowed to second bacterial purifier 135B, which further removes a second portion of pathogens and / or pathogenic substances. In various embodiments, flowing raw human breast milk 110 through first bacterial purifier 135A and second bacterial purifier 135B results in a dual bacterial purification process that removes up to 90% of pathogens from the milk. In some embodiments, the dual bacterial purification process removes up to 90% of Bacillus cereus spores from the milk. In some embodiments, the dual bacterial purification process removes up to 90% of Clostridium botulinum spores from the milk. The result of the dual bacterial purification process in these embodiments is purified milk 122 , which is provided to the next device in the system 100 .

[0056] In other embodiments, the bacterial purifier 115 employs more than two bacterial purifiers 135 in series to further increase the amount of pathogen clearance.

[0057] In various embodiments, each bacterial clarifier 135 is a centrifugal filtration device. An example of a suitable bacterial clarifier 135 is a GEA bacterial separator, such as the GEA Pathfinder. In such embodiments, human breast milk provided to the bacterial clarifier 135 is centrifuged at a predetermined speed for a duration. As the human breast milk 110 is centrifuged, solids above a certain density are collected according to the centrifugation speed and duration of centrifugation. These solids include larger pathogenic microorganisms (e.g., bacteria, viruses) as well as donor cells and other cellular material. The solids can then be further discharged from the bacterial clarifier 135 at periodic intervals. The purified human breast milk can then be provided to the next device in the system 100.

[0058] (6.2.1.3. Milk standardizer) 1A, a milk standardizer 120 designs a standardized human dairy product to possess target amounts of components (e.g., specific nutrients) and / or target characteristics. The milk standardizer 120 therefore ensures that milk provided for optional fortification and for sterilization is standardized so that each sterilized human dairy product 175 batch produced has reduced variability compared to another sterilized human dairy product 175 batch. Use of a milk standardizer 120 is preferred for aseptic dairy products designed for remote consumption by infants, particularly premature infants.

[0059] 1C depicts a milk standardizer 120 device of system 100 according to an embodiment of the present invention. For example, milk standardizer 120 includes a milk separator 140, a concentrator 145, and a milk combiner 150 that produces a standardized milk product 170 that is provided to the next device in system 100. Milk separator 140 separates a clarified milk sample into a cream fraction 155 and a skim fraction 160. Cream fraction 155 refers to the portion of the milk sample that contains a mixture of lipids (e.g., fat), while skim fraction 160 contains components such as ionic salts, proteins (e.g., lactose, whey, casein micelles, immunoglobulins, albumin, and the like), water-soluble vitamins, and human milk oligosaccharides (HMOs). In various embodiments, milk separator 140 is capable of maintaining the temperatures of the clarified milk, cream fraction 155, and skim fraction 160 at specific temperatures. For example, the milk separator 140 may apply refrigeration to maintain the temperature of the clarified milk and each fraction between approximately 4°C and 20°C. In other embodiments, the milk separator 140 may apply heating to maintain the temperature of the clarified milk and each fraction between approximately 45°C and 65°C. Depending on the temperature at which the milk is separated, the milk separator 140 may be designed accordingly, taking into account that the viscosity of the clarified milk may vary substantially at different temperatures. For example, the milk separator 125 may include disks responsible for separating the cream fraction 155 from the skim fraction 160. Thus, a cold milk separator may be designed with fewer disks (more space between disks) compared to a hot milk separator to ensure that the higher viscosity of milk at cold temperatures does not clog the milk separator 140.

[0060] In various embodiments, the milk separator 140 is a high-speed centrifuge (otherwise known as an ultracentrifuge) capable of applying centrifugal speeds of 50,000×g and above. An example of a milk separator 140 is a Tetra Pak Separator. Given that the cream fraction 155 possesses a lower density than that of the skim fraction 160, application of a predetermined centrifugal speed for a certain duration effectively separates the skim fraction 160 from the cream fraction 155. Thus, each individual fraction can be collected separately and subsequently processed. As depicted in FIG. 1C , the skim fraction 160 is provided to the concentrator 145, while the cream fraction 155 is provided directly to the milk combiner 150. In some embodiments, the cream fraction 155 is temporarily stored or held while the skim fraction 160 undergoes further processing.

[0061] Concentrator 145 further concentrates skim fraction 160 into a retentate. In one embodiment, concentrator 145 is a membrane filtration device that performs reverse osmosis on skim fraction 160 to concentrate components present in skim fraction 160 (e.g., salts, proteins, HMOs).

[0062] In some embodiments, concentrator 145 concentrates skim fraction 160 to obtain a retentate having target concentrations of components. For example, concentrator 145 may detect the initial concentrations of components in skim fraction 160 through a sensor or similar device. Exemplary component concentrations can be protein concentration, individual amino acid concentration, or vitamin concentration. The retentate with the target concentrations of components is then provided to milk combiner 150.

[0063] Milk combiner 150 designs the standardized human dairy product by combining cream fraction 155 and a portion of the retentate (e.g., concentrated skim fraction 160). In various embodiments, milk combiner 150 may further supplement the water initially removed by concentrator 145 when concentrating skim sample 160 to achieve a desired concentration in the standardized human dairy product.

[0064] In some embodiments, milk combiner 150 is configured with one or more sensors for detecting properties of cream fraction 155, the retentate, or both. For example, the detectable property may be the solution density or total calories of the cream fraction 155 or the retentate. Alternatively, or in addition, the sensors may be configured to detect the concentration of ingredients in the cream fraction 155 or the retentate. More specifically, the sensors of milk combiner 150 may detect the concentration of proteins in the retentate and the concentration of lipids (e.g., fats) in the cream fraction 155. Other ingredients detectable by the sensors include specific amino acids, vitamins, carbohydrates, oligosaccharides, and immunoglobulins.

[0065] In various embodiments, milk combiner 150 may be further configured to perform calculations or to communicate with a computing system. In some embodiments, milk combiner 150 includes a computing system. Such a computing system is hereinafter referred to as the computing system of milk combiner 150. The computing system may calculate desired portions of cream fraction 155 and retentate to be combined according to detected properties or concentrations of the components of cream fraction 155 and retentate detected by sensors of milk combiner 150. Additionally, the computing system, in some embodiments, may be configured with a memory that stores target properties or concentrations of the components of the standardized human dairy product.

[0066] As explained above, one example of a stored target characteristic or concentration of a component is the initial characteristic or concentration of the component in a previously sampled raw human breast milk 110. As another example, the stored target characteristic or concentration of a component may be a predetermined fixed number. Thus, the milk combiner 150 attempts to design a standardized dairy product that achieves the target characteristic or concentration of the component. In various embodiments, achieving the target characteristic or concentration of a component refers to achieving a component characteristic or concentration that is less than a certain percentage difference compared to the initial characteristic or concentration of the component in the raw human breast milk 110. In other embodiments, achieving the target characteristic or concentration of a component refers to achieving a component characteristic or concentration that is less than a certain percentage difference compared to a fixed, predetermined number.

[0067] As a more specific example, achieving a target density means that the standardized human dairy product has a density that is less than 10% different from a stored target density. As another example, achieving a target protein concentration means that the standardized human dairy product has a protein concentration that is less than 5% different from a stored target protein concentration. As a third example, achieving a target lipid concentration means that the standardized human dairy product has a lipid concentration that is less than 5% different from a stored target lipid concentration.

[0068] In an exemplary embodiment, milk combiner 150 receives cream fraction 155 and retentate and uses one or more sensors to detect the properties and / or concentrations of ingredients in cream fraction 155 and the retentate. A computing system of milk combiner 150 compares the detected properties and / or concentrations of the ingredients to stored target properties and / or concentrations of the ingredients. In one embodiment, the computing system identifies a single property or ingredient as a standard and determines the portions of cream fraction 155 and retentate to be combined so that a standardized human dairy product achieves the desired standard property or ingredient concentration. For example, a standardized human dairy product can be designed by combining cream fraction 155 and a portion of the retentate to have a target concentration of protein in the standardized human dairy product.

[0069] In various embodiments, to design the standardized human dairy product, the computing system prioritizes the properties and / or components of the standardized human dairy product to be achieved. In one embodiment, the property priorities are as follows: 1) density, 2) protein concentration, and 3) lipid concentration. In other embodiments, other priorities are established. As an example of this prioritization, the computing system may first determine which first portion of the cream fraction 155 and which first portion of the retentate to combine to achieve the desired density (e.g., within a pre-specified percentage difference). Next, the computing system determines whether combining the first portion of the cream fraction 155 and the first portion of the retentate also achieves the desired protein concentration (e.g., within that percentage difference). If not, the computing system may adjust the volumes of the first portion of the cream fraction 155 and the first portion of the retentate to meet the desired protein concentration while also maintaining the desired density. Similarly, the computing system may perform the same check with respect to lipid concentration. Thus, in some embodiments, the computing system may calculate a standardized human dairy product that achieves all properties included in the property priorities. In other embodiments, the computing system may generate standardized human dairy products that meet a subset of the characteristics according to the priority of the characteristics.

[0070] The milk combiner 150 combines the cream fraction 155 and a portion of the retentate as calculated by a computing system of the milk combiner 150. In various embodiments, the milk combiner 150 further hydrates the combined portion of the cream fraction 155 and the retentate. Thus, the milk combiner 150 designs a standardized human dairy product 170. The standardized human dairy product 170 is provided to the next device in the system 100.

[0071] In some embodiments, the milk standardizer 120 outputs the standardized human dairy product to the fortifier 125, as shown in Figure 1A. The fortifier 125 can provide supplemental nutrients to the standardized human dairy product, as described below.

[0072] In some embodiments, milk standardizer 120 provides the standardized dairy product to a milk homogenizer (not shown in FIG. 1A) located in-line between milk combiner 150 and milk sterilizer 130.

[0073] In other embodiments, the milk combiner 150 provides the standardized dairy product directly to the milk sterilizer 130 for further sterilization. Specifically, in these embodiments, the standardized dairy product provided to the milk sterilizer 130 is not pre-homogenized by any prior device within the closed in-line sterilization system 100. The lack of a homogenization step can help preserve the integrity of components in the standardized dairy product (e.g., proteins, HMOs, lipids, and the like) that may otherwise be destroyed or damaged by homogenization.

[0074] (6.2.1.4. Milk Homogenizer) When included within the system 100, the milk homogenizer homogenizes the human dairy product input thereto.

[0075] For example, a milk homogenizer uses a pressure-driven flow to force the standardized human dairy product through small physical passages at high velocities, thereby breaking down the fat globules in the standardized dairy product. Homogenization of the standardized human dairy product may improve the long-term stability of the dairy product, improve the flavor of the dairy product, and / or improve the appearance of the dairy product.

[0076] Depending on the intended use of the sterilized human milk product, the loss of nutritional value may outweigh these benefits. For sterilized human milk products 175 intended for remote consumption by premature infants, homogenization with a milk homogenizer is currently not preferred.

[0077] (6.2.1.5. Reinforcer) In various embodiments, the fortifier 125 provides the supplemental nutrients to achieve a target concentration of the added supplemental nutrients.

[0078] In various embodiments, the fortifier 125 provides one or more of fats, antibodies (e.g., IgA, IgG, IgE, and the like), colostrum such as bovine colostrum, proteins (amino acids, whey, casein, albumin, etc.), vitamins, cholesterol, ionic salts (e.g., sodium, calcium, iron, etc.), carbohydrates, and human milk oligosaccharides to the standardized human dairy product as supplemental nutrients. In some embodiments, the fortifier 125 adds a high pure nitrogen utilization (NNU) protein composition as described in U.S. Provisional Application No. 62 / 439,408, filed December 27, 2016, which is incorporated herein by reference in its entirety.

[0079] (6.2.1.6. Milk sterilizer) The milk sterilizer 130 eradicates pathogens present in the milk input thereto to ensure that the sterilized human dairy product 175 is suitable for remote consumption by individuals such as adults, adolescents, infants, and especially premature babies.

[0080] As discussed above, in some embodiments, milk sterilizer 130 directly receives raw human breast milk 110 as an input. In other embodiments, milk sterilizer 130 receives clarified human breast milk 122 as an input. In other embodiments, milk sterilizer 130 receives standardized dairy product 170 as an input, either before or after clarification. In some embodiments, the milk input into milk sterilizer 130 is fortified, and in other embodiments, the milk input into milk sterilizer 130 is not fortified. In some embodiments, the input milk is not homogenized. In other embodiments, the input milk is homogenized.

[0081] In a preferred embodiment, the milk sterilizer 130 performs an ultra-high temperature sterilization process to produce a sterilized human dairy product 175 .

[0082] For example, the milk sterilizer 130 rapidly heats the human dairy product to a target temperature. In addition, the human dairy product is held at the target temperature for a specified amount of time, hereafter referred to as the hold time. The target temperature, heating rate, and hold time applied to the human dairy product are selected to achieve a reduction in pathogen content while maximizing the integrity of the nutritional components within the human dairy product.

[0083] Reference is now made to Figure 2, which depicts a system diagram of a milk sterilizer 130 within a closed in-line sterilization system 100, in accordance with an embodiment of the present invention. In the depicted embodiment, the milk input into the sterilizer 130 is a standardized dairy product output by a milk standardizer 120 and, optionally, fortified by an fortifier 125.

[0084] The milk sterilizer 130 may include a preheater 205, a final heater 210, a holding tube 215, and a cooler 220. Additionally, the milk sterilizer 130 may include thermocouples between each device within the milk sterilizer 130 so that the temperature of the milk flowing through the milk sterilizer 130 may be determined and / or monitored at each step of the sterilization process. As shown in FIG. 2, the milk sterilizer 130 may further include a positive displacement pump 225 that drives the input dairy product through subsequent devices in the milk sterilizer 130. Various embodiments of the milk sterilizer 130 may include fewer devices than those disclosed herein in FIG. 2. As an example, the milk sterilizer 130 may include a single heater that performs a single heating process to heat the input dairy product to a target temperature, as opposed to both the preheater 205 and the final heater 210. Various embodiments of the milk sterilizer 130 may also include devices in addition to those disclosed herein in FIG. 2. For example, the milk sterilizer 130 may include a homogenizer located between any two of the devices depicted in FIG.

[0085] In various embodiments, the milk sterilizer 130 meets Good Manufacturing Practice (GMP) standards. For example, the tubing and connectors that come into contact with the input dairy product meet GMP standards. In other embodiments, the milk sterilizer 130 is a pharmaceutical-grade device that meets pharmaceutical-grade standards, such that the sterilization process performed by the milk sterilizer 130 is a pharmaceutical-grade sterilization process.

[0086] In a preferred embodiment, the milk sterilizer 130 is part of a closed-loop process that accepts dairy product as an input and provides sterilized human dairy product as an output. The sterilization process performed by the milk sterilizer 130 can be a continuous flow process. More specifically, the milk flowing through the milk sterilizer 130 can flow at a single, constant flow rate. In one set of embodiments, the milk flow rate is between 0.25 and 25 gallons per minute. In various embodiments, the milk flow rate is between 0.25 and 15 gallons per minute. In various embodiments, the milk flow rate is between 0.25 and 5 gallons per minute. In some embodiments, the milk flow rate is between 5 and 100 gallons per minute.

[0087] The preheater 205 preheats the input dairy product to a first target temperature. Referring now to Figure 3, a graph of the temperature of the dairy product during flow through the milk sterilizer 130 is shown, in accordance with an embodiment of the present invention. Specifically, the preheater 205 may preheat the input dairy product from an initial temperature (e.g., 0-25°C) to a first target temperature of 90°C. In other examples, the target temperature may be between 80°C and 100°C.

[0088] The preheater 205 may be a vessel, such as a vat, configured to receive an input dairy product through a milk inlet. The preheater 205 outputs a preheated dairy product through a milk outlet. Additionally, the preheater 205 can be configured to receive a preheating medium 230 through a heating inlet and output the preheating medium 230 through a heating outlet. In various embodiments, the milk inlet, milk outlet, heating inlet, and heating outlet are each located on the preheater 205 such that countercurrent heat exchange can occur between the input dairy product flowing through the milk inlet and milk outlet and the preheating medium flowing through the heating inlet and heating outlet. For example, the milk inlet and heating outlet are on one side of the preheater 205. The milk inlet and heating outlet can be adjacent to one another. Additionally, the milk outlet and heating inlet are on opposite sides of the preheater 205. Here, the milk outlet and heating inlet can also be adjacent to one another. Thus, the flow of input dairy product and the flow of preheating medium 230 within preheater 205 may be directional opposite to each other, thereby enabling efficient countercurrent heat exchange between the input dairy product and preheating medium 230 within preheater 205.

[0089] In various embodiments, the preheating medium 230 fed into the preheater 205 through the heating inlet is at or above the first target temperature. That is, the preheating medium 230 may be heated to or above the first target temperature in a separate chamber of the milk sterilizer 130 using electrical heating means. In other embodiments, the preheating medium 230 can be heated to or above the first target temperature in a separate chamber present within the preheater 205. Once the preheating medium 230 is at or above the first target temperature, it is then flowed through the preheater 205 to heat the input dairy product through countercurrent heat exchange.

[0090] The preheating medium 230 may be any type of heating fluid with a high specific heat capacity, such as water, mineral oil, and synthetic or organic-based solutions. In some embodiments, the preheating medium 230 may include heated steam. In other embodiments, the preheating medium 230 may be held at a target pressure that ensures that the preheating medium 230 is in liquid form (e.g., liquid water) as opposed to being in gas form (e.g., steam). This ensures that countercurrent heat exchange occurs between the liquid preheating medium 230 and the liquid milk in the preheater 205.

[0091] The milk inlet and milk outlet are connected in the preheater 205 by a tube through which the standardized milk flows. In some embodiments, the tube is a linear tube. In various embodiments, the tube may be a helical tube. The helical tube in the preheater 205 increases the surface area of the tube available for heat exchange between the preheating medium 230 in the helical tube and the input dairy product.

[0092] The tube is further configured to ensure that the input dairy product is heated to the first target temperature. For example, the length of the tube may be selected to allow the input dairy product to be preheated to the first target temperature and equilibrate within a threshold range of the first target temperature while still in the helical tube. As another example, the number of helices in the helical tube is selected to allow the input dairy product to be preheated to the first target temperature and equilibrate within a threshold range of the first target temperature while still in the helical tube. Additionally, the diameter of the tube may be between 0.25 inches and 10 inches. In some embodiments, the diameter of the tube is between 0.25 inches and 5 inches. In some embodiments, the diameter of the tube is between 0.25 inches and 1 inch. In some embodiments, the diameter of the tube is between 0.25 inches and 0.5 inches. In some embodiments, the diameter of the tube is 0.25 inches, 0.5 inches, 0.75 inches, 1 inch, 2 inches, 3 inches, 4 inches, 5 inches, 6 inches, 7 inches, 8 inches, 9 inches, or 10 inches.

[0093] Referring now to the final heater 210 of the milk sterilizer 130, in various embodiments, the final heater 210 may be similarly configured compared to the preheater 205. Generally, the above description regarding the preheater 205 may also apply to the final heater 210, unless expressly stated otherwise herein.

[0094] For example, the final heater 210 can include a milk inlet that receives the preheated dairy product from the preheater 205 and outputs the final heated dairy product through a milk outlet. Similarly, the final heater 210 includes a heating inlet that receives the final heating medium 235 and a heating outlet that outputs the final heating medium 235. The milk inlet, milk outlet, heating inlet, and heating outlet are each located on the final heater 210 such that countercurrent heat exchange can occur between the preheated dairy product flowing through the milk inlet and milk outlet and the final heating medium 235 flowing through the heating inlet and heating outlet. The milk inlet and heating outlet can be on one side of the final heater 210, while the milk outlet and heating inlet can be on the opposite side of the final heater 210. Thus, countercurrent heat exchange can occur between the final heating medium 235 and the preheated dairy product within the final heater 210.

[0095] Final heater 210 differs from preheater 205 in that final heater 210 raises the temperature of the preheated dairy product (e.g., at a first target temperature of 80°C to 100°C) to a second target temperature. Referring again to FIG. 3 , the second target temperature may be 130°C to 150°C. In one embodiment, the second target temperature may be 138°C to 142°C. In one particular embodiment, the second target temperature is 140°C to 141°C. To heat the preheated dairy product to the second target temperature, final heater 210 receives a final heating medium 235 that has been preheated to or above the second target temperature in a separate chamber. In various embodiments, final heating medium 235 may be held under a target pressure greater than atmospheric pressure to ensure that final heating medium 235 is in liquid form when countercurrent heat exchange occurs between the preheated dairy product and final heating medium 235. The pressure under which the final heating medium 235 is maintained may be higher than the pressure under which the preheating medium 230 is maintained, given the higher temperature of the final heating medium 235 .

[0096] The holding tube 215 is an insulated tube that maintains the temperature of the heated dairy product at or near the second target temperature for a holding time (e.g., a predetermined amount of time). The predetermined amount of time is selected so that bacterial (e.g., Bacillus cereus and Clostridium botulinum) levels are reduced while maintaining the integrity of the nutritional components (e.g., proteins, fats, immunoglobulins, oligosaccharides) in the sterilized dairy product. In one embodiment, the holding time is up to 50 seconds. In another embodiment, the holding time is between 2 and 20 seconds. In some embodiments, the holding time is between 3 and 15 seconds. In some embodiments, the holding time is between 6 and 14 seconds. In some embodiments, the holding time is between 8 and 13 seconds. In one embodiment, the holding time is between 12 and 13 seconds.

[0097] To achieve the desired retention time, the holding tube 215 can be specifically configured. As depicted in FIG. 2, the holding tube 215 may be a spiral tube. Given a constant flow rate of milk through the tube, the spiral tube can be designed with additional or fewer spirals (e.g., additional or shortened length) to increase or decrease the retention time. Alternatively, for a constant length of tube, the flow rate of milk through the tube can be adjusted by adjusting the diameter of the holding tube 215. Thus, increasing or decreasing the tube diameter results in a corresponding increase or decrease in retention time. The milk sterilizer 130 can have one or more interchangeable holding tubes 215 that can be installed between the final heater 210 and the cooler 220 to achieve a variety of different retention times.

[0098] Cooler 220 receives the heated dairy product from holding tube 215 and cools it to a target temperature. In some embodiments, the target temperature after cooling is between 15°C and 25°C. In other embodiments, the target temperature is room temperature (e.g., 23°C). In other embodiments, the target temperature is between 1°C and 8°C (e.g., 4°C), such as a refrigerated temperature.

[0099] In various embodiments, the cooler 220 can employ countercurrent heat exchange, similar to that of the preheater 205 and the final heater 210, to cool the heated dairy product. Generally, the above description regarding the configuration of the preheater 205 may also apply to the cooler 220, unless expressly stated otherwise herein. That is, the cooler 220 may have a milk inlet, a milk outlet, a cooling medium inlet, and a cooling medium outlet. The inlets / outlets may be positioned such that countercurrent heat exchange occurs between the heated dairy product and the cooling medium 240. Similar to the preheater 205 and the final heater 210, the milk inlet and cooling medium outlet may be on one side of the cooler 220, while the milk outlet and cooling medium inlet are on the opposite side, allowing for countercurrent heat exchange. In other embodiments, the cooler 220 can employ other cooling methods to cool the heated dairy product to a target temperature. The cooled dairy product output by the cooling medium 240 is hereinafter referred to as the sterilized human dairy product 175.

[0100] (6.2.1.7. Further processing and packaging) The sterilized human dairy product 175 obtained from the closed in-line sterilization system 100 can be further processed and / or packaged.

[0101] By way of example, the sterilized human dairy product 175 may further undergo a packaging step. In various embodiments, this packaging step is an aseptic packaging process that ensures that the sterilized human dairy product 175 remains unadulterated and safe for consumption. Such an aseptic packaging process may include the steps of package sorting, aseptic filling of the package with the sterilized human dairy product 175, heat sealing of the package, labeling and coding of the package, tamper-evident sealing of the package, and container tying. Aseptic packaging is currently preferred for sterilized human dairy product 175 intended for consumption by infants, such as premature infants. In other embodiments, the packaging process is an extended shelf life (ESL) packaging process. In various embodiments, the sterilized human dairy product 175 is packaged in a bottle or booster cup that facilitates feeding of the sterilized human dairy product 175 to infants, such as premature infants. In some embodiments, the packaging may be a paper box, a paper brick (e.g., a TETRA PAK aseptic brick), or a pouch.

[0102] In some embodiments, including those preferred for sterilized human dairy product 175 suitable for remote consumption by infants, particularly premature infants, the sterilized human dairy product 175 is aseptically packaged and meets pharmaceutical sterilization standards. More specifically, even if the sterilized human dairy product 175 is exposed to oxygen (e.g., the package is opened), the sterilized human dairy product 175 remains sterile and safe for consumption because pathogenic microorganisms have been sufficiently eradicated during the sterilization process. In other embodiments, the sterilized human dairy product 175 is ESL packaged and therefore can remain safe for consumption for an extended period of time after packaging.

[0103] (6.2.1.8. Device sterilization standards) Overall, each device in system 100 is designed to meet certain criteria.

[0104] For example, in one embodiment, bacterial clarifiers 135A and 135B, milk separator 140, concentrator 145, and milk combiner 150 each meet Grade "A" Pasteurized Milk Ordinance (PMO) standards. Milk sterilizer 130 can be designed to meet PMO standards. In some embodiments, milk sterilizer 130 is designed to meet even higher standards, i.e., sterilizer 130 can be designed to meet GMP or pharmaceutical-grade standards. As an example, to meet GMP or pharmaceutical-grade standards, sanitary welding is employed, while threaded fittings are avoided in milk sterilizer 130. In various embodiments, closed in-line sterilization system 100 is a clean-in-place (CIP) system, meaning that closed in-line system 100 does not need to be disassembled to be cleaned.

[0105] 6.2.2. Methods for Producing Sterile Human Dairy Products Reference is now made to FIG. 4, which depicts a flow chart for producing a sterilized human dairy product 175, according to an embodiment of the present invention.

[0106] Raw human breast milk 110 is first obtained. In a typical embodiment, for example, individual raw human breast milk donations (samples) are received from multiple human donors (405). Each raw human breast milk donation is verified (410) to determine if any donation fails to meet quality control standards. If the donation does not pass the quality control standards, it is then discarded. The verified samples are pooled (415). The pooled verified samples represent the raw human breast milk 110 that is then provided as input into the system 100. In various embodiments, the system is a closed in-line sterilization system 100.

[0107] 6.2.2.1. Methods for Producing Sterile Human Dairy Products for Remote Consumption by Infants For sterile human milk products intended for remote consumption by young children, such as premature babies, (i) maximum sterilization and pathogen removal, (ii) standardization, and (iii) nutrient retention are preferred.

[0108] 4, in a preferred embodiment, the system 100 eliminates (420) a portion (e.g., up to 90%) of pathogens from the raw human breast milk 110 through a purification process. In various embodiments, the process is a single bacterial purification process performed by a single bacterial clarifier 135 of the system 100. In various embodiments, the process is a dual bacterial purification process performed by a bacterial clarifier 135 of the system 100.

[0109] In an exemplary embodiment intended for remote consumption by an infant, such as a premature baby, the clarified milk 122 is then input into a milk standardizer 120 .

[0110] 1C, milk standardizer 120 then separates the clarified human milk into a cream fraction 155 and a skim fraction 160. In various embodiments, this separation process is performed by milk separator 140 of system 100.

[0111] System 100 further produces a retentate by concentrating skim fraction 160. In various embodiments, concentrator 145 of system 100 is a membrane filtration device that performs a reverse osmosis process on skim fraction 160 to produce a retentate. System 100 designs (435) a standardized human dairy product by combining a portion of cream fraction 155 with a portion of the retentate. In various embodiments, the step of combining cream fraction 155 and a portion of the retentate is performed by milk combiner 150 of system 100. To determine the cream fraction 155 and the portion of the retentate to be combined, milk combiner 150 detects the properties or concentrations of ingredients in each of cream fraction 155 and the retentate. Thus, certain ratios of cream fraction 155 and retentate can be combined to achieve target properties or concentrations of ingredients in the standardized human dairy product.

[0112] With regard to retention of nutritional value, it is currently preferred to omit homogenization.

[0113] The system 100 sterilizes (440) the standardized human dairy product. In various embodiments, the sterilization process is a countercurrent heat exchange process performed by the milk sterilizer 130 of the system 100. Reference is now made to FIG. 5, which depicts a flow chart for sterilizing a dairy product, according to an embodiment of the present invention. In particular, the flow process of FIG. 5 depicts the sterilization step 440 of FIG. 4 in greater detail.

[0114] The milk sterilizer 130 of the system 100 may be a pharmaceutical-grade device. In some embodiments, the milk sterilizer 130 receives a non-homogenized standardized human dairy product (505). The milk sterilizer 130 performs sterilization through a countercurrent heat exchange sterilization process. That is, the standardized human dairy product is flowed in a first direction at a first flow rate through tubing in the milk sterilizer 130 (510). In various embodiments, the tubing has a diameter between 0.25 and 10 inches. The standardized human dairy product is flowed through the tubing at a rate between 0.25 gallons per minute and 25 gallons per minute.

[0115] The milk sterilizer 130 heats the standardized human dairy product located within the tube by flowing a heating fluid in a second direction at a second flow rate (515). The heating fluid contacts the exterior surface of the tube. In various embodiments, the heating fluid is heated water, and the second direction in which the heated water flows is opposite the first direction in which the standardized human dairy product flows. Thus, as the standardized human dairy product flows through the tube, heat from the heated water flowing through the second tube can be easily transferred through the surface of the tube to heat the standardized human dairy product.

[0116] In various embodiments, the milk sterilizer 130 preheats the standardized human dairy product, then further heats the preheated human dairy product to a target temperature and holds the dairy product at the target temperature for a duration. The target temperature may be between 130°C and 150°C, and the standardized human dairy product may be held at the target temperature for 6 to 14 seconds. This process is refined to further eliminate pathogens that may be present in the standardized human dairy product, while also maintaining the ingredients and nutrients in the standardized human dairy product.

[0117] The milk sterilizer 130 cools 520 the heated human dairy product below the heated temperature to obtain a sterilized human dairy product 175. In various embodiments, the human dairy product is cooled to room temperature, between 15° C. and 25° C. In other embodiments, the human dairy product is cooled to a refrigeration temperature, between 1° C. and 8° C.

[0118] A sterilized human dairy product 175 is provided (525) by the milk sterilizer 130. For example, referring back to the flow process of Figure 4, the sterilized human dairy product 175 is obtained from the milk sterilizer 130 and packaged (445). In various embodiments, this may be an aseptic packaging process to ensure that the sterilized human dairy product is not contaminated prior to being provided for feeding to an infant. In such embodiments, the aseptically packaged sterilized human dairy product may be dispensed without refrigeration.

[0119] In some embodiments, the method further comprises fortifying the standardized dairy product before sterilization.

[0120] In specific embodiments, the method further comprises fortifying the standardized dairy product with colostrum. In typical embodiments, the colostrum is bovine colostrum. In some embodiments, the colostrum is ovine or caprine colostrum. In some embodiments, the method comprises fortifying the standardized dairy product with proteins or high pure nitrogen utilization (NNU) protein compositions as described in U.S. Provisional Application No. 62 / 439,408, filed December 27, 2016, which is incorporated herein by reference in its entirety. In some embodiments, the method comprises fortifying the standardized dairy product with antibodies, vitamins, ionic salts (e.g., sodium, calcium, iron, etc.), fats such as cholesterol, and / or carbohydrates such as human milk oligosaccharides.

[0121] 6.2.2.2. Methods for Producing Sterile Human Dairy Products for Remote Consumption by Adults In certain embodiments of a method for producing a sterilized human milk product for remote consumption by adults, the system 100 eliminates (420) a portion (e.g., up to 90%) of pathogens from raw human breast milk 110 through a purification process. In various embodiments, the process is a single or dual bacterial purification process performed by one or more bacterial clarifiers 135 of the system 100. However, sterilized human milk products intended for remote consumption by adults do not require as stringent sterility as sterilized human milk products intended for remote consumption by infants, particularly premature infants. Thus, in some embodiments, the method omits bacterial purification.

[0122] In some embodiments intended for remote consumption by adults, human breast milk, with or without pre-cleaning, is then input into milk standardizer 120 and standardized as described above with respect to sterilized human milk products for remote consumption by infants. However, sterilized human milk products intended for remote consumption by adults do not require as strict standardization as sterilized human milk products intended for remote consumption by infants, particularly premature infants. Thus, in some embodiments, the method omits standardization.

[0123] Sterilized human milk products intended for remote consumption by adults can be usefully fortified. In various embodiments, the method therefore includes a step of fortifying the milk prior to sterilization by the milk sterilizer 130.

[0124] In specific embodiments, the method further comprises fortifying the dairy product with colostrum. In typical embodiments, the colostrum is bovine colostrum. In some embodiments, the colostrum is ovine or caprine colostrum.

[0125] In some embodiments, the method includes fortifying a dairy product with a protein. In particular embodiments, the method includes fortifying a dairy product with a high pure nitrogen utilization (NNU) protein composition, as described in U.S. Provisional Application No. 62 / 439,408, filed December 27, 2016, which is incorporated herein by reference in its entirety. In the description set forth herein, a high pure nitrogen utilization (NNU) protein composition consists of free amino acids, i.e., amino acids that are not bound to any other amino acids by peptide bonds.

[0126] In a typical NNU-enriched embodiment, at least 95% by weight of the amino acids in the high NNU composition are free amino acids. In some embodiments, at least 96%, 97%, 98%, or even at least 99% of the amino acids in the high NNU composition are free amino acids. In a typical embodiment, less than 5% by weight of the amino acids in the high NNU composition are incorporated into peptides. In some embodiments, less than 4%, 3%, 2%, or even less than 1% by weight of the amino acids are incorporated into peptides. In certain embodiments, the composition does not contain any detectable peptides.

[0127] In typical embodiments, the high NNU composition comprises isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine, each as its L-isomer. In some embodiments, the high NNU composition further comprises L-histidine. In some embodiments, the high NNU composition further comprises one or more non-essential amino acids.

[0128] In typical embodiments, the amino acids are present in the ratios set forth in U.S. Patent No. 5,132,113, which is incorporated herein by reference in its entirety. In certain of these embodiments, the amino acids are present in the following proportions, in grams per 10 grams of composition: (a) Isoleucine from 1.217 to 1.647 (b) leucine from 1.827 to 2.735 (c) lysine 1.260-2.359 (d) 0.232 to 0.778 methionine (e) phenylalanine 0.843-1.314 (f) 0.970-1.287 threonine (g) 0.208-0.467 tryptophan (h) 1.260-1.900 valine

[0129] In certain embodiments, the amino acids are present in the proportions set forth in one of the eight compositions (I-VIII) of Table 1, in grams per 10 grams of composition. [Table 1]

[0130] In typical embodiments, the high NNU composition is added in an amount sufficient to ensure a protein concentration (F=6.38) of the final processed composition of at least 1.0 g / 100 g (1.0 wt%), 1.1 wt%, 1.2 wt%, 1.3 wt%, or at least 1.4 wt%. In certain embodiments, the high NNU composition is added in an amount sufficient to ensure a protein concentration of the final processed composition of at least 1.30 wt%, 1.31 wt%, 1.32 wt%, 1.33 wt%, 1.34 wt%, 1.35 wt%, 1.36 wt%, 1.37 wt%, 1.38 wt%, 1.39 wt%, or at least 1.40 wt%.

[0131] In some embodiments, the high NNU composition is added in an amount sufficient to ensure a protein concentration of the final processed composition greater than 1.4% by weight. In some of these high protein embodiments, the high NNU composition is added in an amount sufficient to ensure a protein concentration of the final processed composition greater than 1.40%, 1.41%, 1.42%, 1.43%, 1.44%, 1.45%, 1.46%, 1.47%, 1.48%, 1.49%, or even greater than 1.50% by weight. In some of these high protein embodiments, the high NNU composition is added in an amount sufficient to ensure a protein concentration of the final processed composition greater than 1.6%, 1.7%, 1.8%, 1.9%, or even greater than 2.0% by weight.

[0132] In various embodiments, the high NNU composition is added in an amount that provides at least 5% of the protein content of the final processed composition. In some embodiments, the high NNU composition is added in an amount that provides at least 6%, 7%, 8%, 9%, or at least 10% of the protein content of the final processed composition. In certain embodiments, the high NNU composition is added in an amount that provides at least 15%, 20%, or even at least 25% of the protein content of the final processed composition. In some embodiments, the high NNU composition is added in an amount that provides at least 30%, 35%, 40%, 45%, or even at least 50% of the protein content of the final processed composition.

[0133] In various embodiments, the high NNU composition is added in an amount that provides less than 50% of the protein content of the final processed composition. In specific embodiments, the high NNU composition is added in an amount that provides less than 45%, 40%, 35%, 30%, or 25% of the protein content of the final processed composition.

[0134] In various embodiments, the high NNU composition is added in an amount that provides more than 50% of the protein content of the final dairy product. These latter embodiments may be used alone or, in some embodiments, will be mixed with unfortified milk before administration. In some of these embodiments, the high NNU composition is added in an amount that provides at least 50%, 55%, 60%, 65%, 70%, 75%, or even at least 80%, 85%, 90%, or 95% of the protein content of the final processed dairy product.

[0135] In some embodiments, the dairy product is enriched with flavorings, hi some embodiments, the dairy product is enriched with natural colors.

[0136] In some embodiments, the dairy product is fortified with vitamins. In a specific embodiment, the dairy product is fortified with vitamin C. In a specific embodiment, the dairy product is fortified with vitamin D.

[0137] In some embodiments, the dairy product is fortified with minerals. In particular embodiments, the dairy product is fortified with calcium. In certain embodiments, the dairy product is fortified with magnesium.

[0138] In some embodiments, the dairy product may be degassed to remove gas from the dairy product. In various embodiments, the method therefore includes degassing the dairy product prior to sterilization by the dairy sterilizer 130. In some embodiments, the method includes degassing the dairy product after fortifying the dairy product.

[0139] In various embodiments of the method for producing a sterilized human dairy product intended for remote consumption by an adult, homogenization is omitted. In other embodiments, the dairy product is homogenized. In some embodiments, the dairy product is homogenized before sterilization. In other embodiments, the dairy product is homogenized after sterilization.

[0140] 6.3. Composition of Sterilized Human Dairy Products In various embodiments, the sterilized human dairy product 175 has 40-80 calories / 100 mL, 50-70 calories / 100 mL, or 60-70 calories / 100 mL. In some embodiments, the sterilized human dairy product 175 has 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 calories / 100 mL. In certain embodiments, the sterilized human dairy product 175 has 63.0-67.0 calories / 100 mL. In some embodiments, the sterilized human dairy product 175 has 65.0-66.0 calories / 100 mL.

[0141] In various embodiments, the sterilized human dairy product 175 has 24-40 calories from fat per 100 milliliters (mL). In some embodiments, the sterilized human dairy product 175 has 24-28 calories / 100 mL from fat, 28-32 calories / 100 mL from fat, 32-36 calories / 100 mL from fat, or 36-40 calories / 100 mL from fat. In some embodiments, the sterilized human dairy product 175 has 28-36 calories / 100 mL from fat. In some embodiments, the sterilized human dairy product 175 has 28 calories / 100 mL from fat, 29 calories / 100 mL from fat, 30 calories / 100 mL from fat, 31 calories / 100 mL from fat, 32 calories / 100 mL from fat, 33 calories / 100 mL from fat, 34 calories / 100 mL from fat, 35 calories / 100 mL from fat, or 36 calories / 100 mL from fat.

[0142] In various embodiments, the sterilized human dairy product 175 has a total fat content of at least 3.0 g / 100 mL (3% by weight), 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, or 4.0% by weight. In some embodiments, the sterilized human dairy product 175 has a total fat content of greater than 4.0% by weight.

[0143] In various embodiments, the sterilized human dairy product 175 includes 1.2 milligrams (mg), 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, or 1.8 mg of saturated fat per 100 mL of the sterilized human dairy product 175. In various embodiments, the sterilized human dairy product 175 includes 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, or 16 mg of cholesterol per 100 mL of the sterilized human dairy product 175. In various embodiments, the sterilized human dairy product 175 includes 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg of sodium per 100 mL of the sterilized human dairy product 175. In various embodiments, the sterilized human dairy product 175 includes 4g, 5g, 6g, 7g, 8g, 9g, or 10g of carbohydrates per 100mL of the sterilized human dairy product 175. In various embodiments, the sterilized human dairy product 175 includes 4g, 5g, 6g, or 7g of sugars per 100mL of the sterilized human dairy product 175. In various embodiments, the sterilized human dairy product 175 includes 1.2g, 1.3g, 1.4g, 1.5g, or 1.6g of protein per 100mL of the sterilized human dairy product 175. In various embodiments, the sterilized human dairy product 175 includes 160 international units (IU), 170 IU, 180 IU, 190 IU, or 200 IU of vitamin A per 100mL of the sterilized human dairy product 175. In various embodiments, the sterilized human dairy product 175 includes 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, or 35 mg per 100 mL of the sterilized human dairy product 175.

[0144] In various embodiments, the sterilized human dairy product 175 has a reduced amount of Clostridium botulinum (Clostridium botulinum) compared to the amount of Clostridium botulinum in raw human breast milk 110 obtained from one or more human donors. In some embodiments, the reduction in C. botulinum is a 12-50 log reduction, a 20-45 log reduction, or a 30-42 log reduction. In some embodiments, the sterilized human dairy product 175 has a 30 log, 31 log, 32 log, 33 log, 34 log, 35 log, 36 log, 37 log, 38 log, 39 log, 40 log, 41 log, or 42 log reduction in the level of C. botulinum compared to the level of C. botulinum in raw human breast milk 110.

[0145] In various embodiments, the sterilized human dairy product 175 has a reduced amount of Bacillus cereus compared to the amount of Bacillus cereus in raw human breast milk 110 obtained from one or more human donors. In various embodiments, the reduction of Bacillus cereus is greater than a 1,000 log reduction in the level of Bacillus cereus compared to the level in raw human breast milk 110.

[0146] In various embodiments, the sterilized human dairy product 175 has a bacterial aerobic plate count of less than 10 colony forming units (CFU) per gram of the sterilized human dairy product 175. In some embodiments, the sterilized human dairy product 175 has a yeast and mold count below 10 CFU per gram of the sterilized human dairy product 175.

[0147] In various embodiments, the sterilized human dairy product 175 retains greater than a certain percentage of components compared to raw human breast milk 110. In some embodiments, the retention of immunoglobulin A (IgA) is greater than 73%. In some embodiments, the retention of immunoglobulin M (IgM) is greater than 74%. In some embodiments, the retention of immunoglobulin G (IgG) is greater than 93%. In some embodiments, the retention of antitrypsin is greater than 55%. In some embodiments, the retention of lactoferrin is greater than 74%. In some embodiments, the retention of lysozyme is greater than 64%. In some embodiments, the retention of lactalbumin is greater than 65%. In some embodiments, the retention of alpha casein is greater than 86%. In some embodiments, the retention of beta casein is greater than 89%. In some embodiments, the retention of kappa casein is greater than 81%. In some embodiments, the retention of osteopontin is greater than 80%. In some embodiments, the retention of total HMOs is greater than 79%. In some embodiments, retention of fucosylated HMO is greater than 90%. Additionally, in some embodiments, retention of 2'-fucosyllactose is greater than 90%. In some embodiments, retention of 3'-fucosyllactose is greater than 90%. In some embodiments, retention of sialylated HMO is greater than 90%. In some embodiments, retention of non-fucosylated HMO is greater than 89%.

[0148] Referring to the absolute concentrations of components in sterilized human dairy product 175, in some embodiments, the concentration of lactoferrin in sterilized human dairy product 175 is between 0.81 g / L and 13.28 g / L. In other embodiments, the concentration of lactoferrin in sterilized human dairy product 175 is between 1 g / L and 12 g / L. In some embodiments, the concentration of lactoferrin in sterilized human dairy product 175 is 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, or 11 g / L.

[0149] In some embodiments, the concentration of lysozyme in sterilized human dairy product 175 is between 0.012 g / L and 0.105 g / L. In some embodiments, the concentration of lysozyme in sterilized human dairy product 175 is between 0.015 g / L and 0.105 g / L. In some embodiments, the concentration of lysozyme in the sterilized human dairy product 175 is 0.021 g / L, 0.022 g / L, 0.023 g / L, 0.024 g / L, 0.025 g / L, 0.026 g / L, 0.027 g / L, 0.028 g / L, 0.029 g / L, 0.030 g / L, 0.031 g / L, 0.032 g / L, 0.033 g / L, 0.034 g / L, 0.035 g / L, 0.036 g / L, 0.037 g / L, 0.038 g / L, 0.039 g / L, / L, 0.040g / L, 0.041g / L, 0.042g / L, 0.043g / L, 0.044g / L, 0.045g / L, 0.046g / L, 0.047g / L, 0.048g / L, 0.049g / L, 0.050g / L, 0.051g / L, 0.052g / L, 0.053g / L, 0.054g / L, 0.055g / L, 0.056g / L, 0.057g / L, 0.058g / L, 0.059g / L, 0.060g / L, 0.061g / L, 0.062g / L, 0.063g / L, 0.064g / L, 0.065g / L, 0.066g / L, 0.067g / L, 0.068g / L, 0.069g / L, 0.070g / L, 0.071g / L, 0.072g / L, 0 .073g / L, 0.074g / L, 0.075g / L, 0.076g / L, 0.077g / L, 0.078g / L, 0.079g / L, 0.080g / L, 0.081g / L, 0.082g / L, 0.083g / L, 0.0 84g / L, 0.085g / L, 0.086g / L, 0.087g / L, 0.088g / L, 0.089g / L, 0.090g / L, 0.091g / L, 0.092g / L, 0.093g / L, 0.094g / L, 0.09 5g / L, 0.096g / L, 0.097g / L, 0.098g / L, 0.099g / L, 0.100g / L, 0.101g / L, 0.102g / L, 0.103g / L, 0.104g / L, or 0.105g / L.

[0150] In some embodiments, the concentration of lactalbumin in sterilized human dairy product 175 is between 1.87 g / L and 2.68 g / L. In some embodiments, the concentration of lactalbumin in sterilized human dairy product 175 is between 2.0 g / L and 2.6 g / L. In some embodiments, the concentration of lactalbumin in sterilized human dairy product 175 is between 2.2 g / L and 2.4 g / L. In some embodiments, the concentration of lactalbumin in the sterilized human milk product 175 is 2.21 g / L, 2.22 g / L, 2.23 g / L, 2.24 g / L, 2.25 g / L, 2.26 g / L, 2.27 g / L, 2.28 g / L, 2.29 g / L, 2.30 g / L, 2.31 g / L, 2.32 g / L, 2.33 g / L, 2.34 g / L, 2.35 g / L, 2.36 g / L, 2.37 g / L, 2.38 g / L, or 2.39 g / L.

[0151] In some embodiments, the concentration of antitrypsin in sterilized human dairy product 175 is between 0.057 g / L and 0.40 g / L. In some embodiments, the concentration of antitrypsin in sterilized human dairy product 175 is between 0.10 g / L and 0.30 g / L. In some embodiments, the concentration of antitrypsin in the sterilized human dairy product 175 is 0.11 g / L, 0.12 g / L, 0.13 g / L, 0.14 g / L, 0.15 g / L, 0.16 g / L, 0.17 g / L, 0.18 g / L, 0.19 g / L, 0.20 g / L, 0.21 g / L, 0.22 g / L, 0.23 g / L, 0.214 g / L, 0.25 g / L, 0.26 g / L, 0.27 g / L, 0.28 g / L, or 0.29 g / L.

[0152] In some embodiments, the concentration of HMOs in sterilized human dairy product 175 is between 8.8 g / L and 20.0 g / L. In some embodiments, the concentration of HMOs in sterilized human dairy product 175 is between 10 g / L and 18 g / L. In some embodiments, the concentration of HMOs in sterilized human dairy product 175 is between 14 g / L and 16 g / L. In some embodiments, the concentration of HMOs in sterilized human dairy product 175 is 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, or 18 g / L.

[0153] In some embodiments, the concentration of fucosylated HMO in sterilized human dairy product 175 is between 5.46 g / L and 5.6 g / mL. In some embodiments, the concentration of fucosylated HMO in sterilized human dairy product 175 is 5.47 g / L, 5.48 g / L, 5.49 g / L, 5.50 g / L, 5.51 g / L, 5.52 g / L, 5.53 g / L, 5.54 g / L, 5.55 g / L, 5.56 g / L, 5.57 g / L, 5.58 g / L, or 5.59 g / L.

[0154] In some embodiments, the concentration of 2'-fucosyllactose in sterilized human dairy product 175 is between 0.72 g / L and 4.3 g / L. In some embodiments, the concentration of 2'-fucosyllactose in sterilized human dairy product 175 is between 1 g / L and 4 g / L. In some embodiments, the concentration of 2'-fucosyllactose in the sterilized human dairy product 175 is 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, 2.0 g / L, 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L, 2.5 g / L, 2.6 g / L, 2.7 g / L, 2.8 g / L, 2.9 g / L, 3.0 g / L, 3.1 g / L, 3.2 g / L, 3.3 g / L, 3.4 g / L, 3.5 g / L, 3.6 g / L, 3.7 g / L, 3.8 g / L, 3.9 g / L, or 4.0 g / L.

[0155] In some embodiments, the concentration of 3'-fucosyllactose in sterilized human dairy product 175 is 0.92 g / L to 0.93 g / L. In some embodiments, the concentration of sialylated HMO in sterilized human dairy product 175 is 1.08 g / L to 1.11 g / L. In some embodiments, the concentration of sialylated HMO in sterilized human dairy product 175 is 1.09 g / L to 1.10 g / L. In some embodiments, the concentration of nonfucosylated HMO in sterilized human dairy product 175 is 2.46 to 2.56 g / L. In some embodiments, the concentration of nonfucosylated HMO in sterilized human dairy product 175 is 2.47 g / L, 2.48 g / L, 2.49 g / L, 2.50 g / L, 2.51 g / L, 2.52 g / L, 2.53 g / L, or 2.54 g / L. [Example]

[0156] 7. Working Example The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, concentrations, etc.), but some experimental error and deviation should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0157] 7.1. Example 1: Pilot-Scale Ultra-High Temperature Sterilization of Human Milk Fourteen different sterilization processes were performed on human milk obtained from human donors using an Ultra-High Temperature Lab-25 Electric High Viscosity Hybrid Unit (MicroThermics, Raleigh, NC) to determine whether ultra-high temperature sterilization could be applied to human milk to achieve a high level of sterilization with the simultaneous preservation of the desired components of human milk.

[0158] The human milk was verified and pooled prior to sterilization.

[0159] Human milk was heated to a target temperature while undergoing countercurrent heat exchange and held at the target temperature for a duration. Figure 6A depicts parameters (e.g., temperature and hold time) from various sterilization processes. Generally, hold times, listed in terms of fluid element residence time (FERT), ranged from 6.6 seconds to a maximum of 12.6 seconds at temperatures ranging from 135°C to 145°C. FERT refers to the residence time of the element (e.g., milk) flowing through the center of the holding tube. A flow rate of 0.25 gallons / minute of human milk product was held constant across all processes. Additionally, the log reductions of Clostridium botulinum and Bacillus cereus per process are depicted in Figure 6A. Generally, the data confirm a 12-50 log reduction in Clostridium botulinum content and a greater than 1,000 log reduction in Bacillus cereus. This meets and exceeds the standard 12-log reduction (12D concept) for sterilization of a given food product.

[0160] Of note, additional human milk samples were processed using the processing parameters shown for step 8 (e.g., hold time = 8.1 seconds, temperature = 286°C). o F / 141.1°C). After sterilization, the additional sterilized human dairy products also exhibited a greater than 1,000 log reduction of Bacillus cereus.

[0161] Reference is now made to Figure 6B, which depicts comparative nutritional data for the raw human milk sample and the sterilized human milk product from process 11, which included a hold time of 8.1 seconds and a target temperature of 144.1°C. As shown in Figure 6B, for the 100g sample, minimal differences were observed in key nutritional components and properties, including total calories, total fat, cholesterol, sodium, carbohydrates, protein, vitamin A, vitamin C, calcium, iron, amino acids, and vitamins, compared to the raw human milk. That is, most of the components (except sodium) and properties of the sterilized human milk product exhibited 6% or less difference compared to the components and properties of the raw human milk sample.

[0162] Processing parameters for step 8 (e.g., holding time = 8.1 seconds, temperature = 286 oAn additional sterilized human dairy product that underwent ultra-high temperature sterilization using a temperature of 141.1°C (F / 141.1°C) was further analyzed for protein quality using standardized Food and Agriculture Organization (FAO) methods (e.g., FAO Document 51 (1991)). The Protein Digestibility Corrected Amino Acid Score (PDCAAS) of this sterilized human dairy product was 73.3 (% by weight).

[0163] Reference is now made to Figure 6C, which depicts the comparative concentrations and abundances of components measured by mass spectrometry in the first batch of raw human breast milk compared to the sterilized human milk products for two of the experimental steps, i.e., steps 4 and 11. The sterilized human milk products obtained from steps 4 and 11 were each derived from the first batch of raw human breast milk. The components quantified by mass spectrometry include immunoglobulins (IgA, IgM, and IgG), proteins (antitrypsin, lactoferrin, lysozyme, lactalbumin, alpha-casein, beta-casein, kappa-casein, and osteopontin), and human milk oligosaccharides (HMOs). The retention of each of the components in the standardized human milk products for each individual sterilization step is further depicted in Figure 6C.

[0164] Specifically, the Step 4 sterilization process resulted in greater than 80% retention of the majority of components, with the exception of IgM (78% retention) and antitrypsin (68% retention). Importantly, Figure 6C includes the concentration and retention of total HMOs, a component of human milk that is often overlooked in sterilized human milk products. Here, sterilized human milk products produced using the Step 4 sterilization process retained 92% of the total HMOs. The Step 11 sterilization process retained 93% of the total HMOs.

[0165] Reference is now made to Figure 6D, which further depicts the comparative concentrations and abundances of components measured by mass spectrometry in sterilized human milk products compared to a second batch of raw milk. Specifically, Figure 6D depicts the sterilized milk products obtained as a result of steps 7 and 14, as shown in Figure 6A. The sterilized human milk products obtained from steps 7 and 14 were each derived from a second batch of raw human breast milk. Here, the measured components of the sterilized milk products include immunoglobulins (IgA, IgM, and IgG), proteins (antitrypsin, lactoferrin, lysozyme, lactalbumin, alpha-casein, beta-casein, kappa-casein, and osteopontin), and human milk oligosaccharides (HMOs). Furthermore, the abundances of individual HMO components were further characterized, including fucosylated HMOs, sialylated HMOs, and non-fucosylated HMOs. Additionally, the abundance of individual components of fucosylated HMOs, including 2'-fucosyllactose (2-FL) and 3'-fucosyllactose (3-FL), was further quantified. Given that 2-FL and 3-FL are involved in defense against infectious diseases and reducing inflammation, the retention of 2-FL and 3-FL through the sterilization process is of interest.

[0166] Here, the Step 7 and Step 14 sterilization processes each resulted in greater than 80% component retention, with the majority of the components experiencing greater than 90% retention. It is noted that some component retention exceeded 100%, but these results may be due to measurement error. Notably, the retention of total HMOs for both Step 7 and Step 14 was nearly 100%. Similarly, the retention of 2-FL and 3-FL for Step 7 and Step 14 was also nearly 100%, respectively. This demonstrates that the sterilization process does not damage HMOs and can therefore ensure the bioavailability of important HMOs such as 2-FL and 3-FL, which may have therapeutic effects when consumed.

[0167] As shown in both Figures 6C and 6D, the sterilization process achieves high retention of components in the sterilized human milk product. Considering that Figures 6C and 6D, respectively, list retention rates by component, the absolute concentration of each component in the sterilized human milk product will depend on the concentration of each component in the raw human breast milk when obtained from the donor. As shown by the first and second batches of raw human milk samples (e.g., Figures 6C and 6D) and further confirmed by prior literature, the concentrations of various components in raw human breast milk (e.g., the components depicted in Figures 6C and 6D) vary widely. For example, prior literature has shown that lactoferrin levels in breast milk vary as a function of time postpartum (e.g., 1 day, 14 weeks, and 6 months). See Shashiraj, FM et al., European Journal of Clinical Nutrition 60:903-908 (2006). Additionally, levels of components in breast milk differ based on whether the birth was preterm or full-term. See Mehta, R. et al., Journal of Perinatology 31:58-62 (2011).

[0168] Specifically, the concentration of lactoferrin in raw human breast milk has been shown to range up to 14.92 g / L. See Turn, C.G. et al., Journal of Perinatology 37(5):507-512 (2017). Additionally, the concentration of lactoferrin in raw human breast milk has been shown to be as low as 1 g / L. See Montagne, P. et al., Advances in Experimental Medicine and Biology, vol. 501 (Springer, Boston, MA). Thus, considering the 81% retention achieved in step 11 of the sterilization process (as depicted in Figure 6C), the concentration of lactoferrin in sterilized dairy products can range from 0.81 g / L up to 13.28 g / L. Additionally, the concentration of lysozyme in raw human breast milk can be 0.015 g / L. See Hsu, Y. et al., Pediatrics and Neonatology 55:449-454 (2014). Thus, considering the 91% retention achieved in step 7 of the sterilization process, the concentration of lysozyme in sterilized dairy products can range from 0.012 g / L to 0.105 g / L (step 7 in Figure 6D). Additionally, the concentration of lactalbumin in raw human breast milk can range from 2.28 g / L to a maximum of 3.27 g / L. See Affolter, M. et al., Nutrients 8(8):504 (2016). Thus, considering the 82% retention achieved in step 4 of the sterilization process, the concentration of lactalbumin in sterilized dairy products can range from 1.87 g / L to 2.68 g / L. Additionally, the concentration of antitrypsin in raw human breast milk can range from 0.1 g / L to 0.4 g / L. See Chowanadisai, W. et al., Am. J. Clin. Nutr. 76(4):828-833 (2002). Thus, taking into account the near 100% retention of antitrypsin achieved in steps 7 and 11, the concentration of antitrypsin in sterilized dairy products can range from 0.057 (step 7) up to 0.4 g / L.

[0169] In addition, human milk oligosaccharide concentrations in human breast milk can range up to 20 g / L. See Gabrielli, O. et al., Pediatrics, 128(6):e1520-31 (2011). Figure 6C depicts a raw milk sample with 12 g / L of human milk oligosaccharides, while Figure 6D depicts a raw milk sample with 8.8 g / L of human milk oligosaccharides. Thus, considering the near-100% retention observed in steps 7 and 14, the concentration of HMOs in sterilized dairy products can range from 8.8 g / L (see step 14) up to 20 g / L. In addition, 2'-fucosyllactose concentrations in human breast milk can range from 1.1 to 4.3 g / L. See Puccio, J. Pediatr. Gastroenterol. Nutr. 64(4): 624-631 (2017). Therefore, taking into account the near 100% retention achieved in steps 7 and 14, the 2′-fucosyllactose concentration in the sterilized dairy product can range from 0.72 g / L (step 7) to 4.3 g / L.

[0170] Figure 6E depicts the quantified bacterial, mold, and yeast content for raw human milk and sterilized human milk products. Here, sterilized human milk products correspond to step 4 of the sterilization process as described above. Raw human milk corresponds to the same sample prior to sterilization. Standard aerobic plate counts of each sample were performed to determine the level of microorganisms in the sample. Serial dilutions of each sample were plated onto agar Petri dishes and incubated (37°C for 48 hours) to allow for microbial colony formation. The colony-forming units (CFU) of each serial dilution were visualized and quantified to determine the aerobic plate count of each Petri dish sample. Specifically, sterilized human milk products demonstrated significantly lower bacterial counts (<10 CFU per gram) compared to raw human milk (>250,000 CFU per gram).

[0171] The systems and methods as disclosed herein provide sterilized human dairy products with low bioburden of Bacillus cereus and Clostridium botulinum. Therefore, the sterilized human dairy products are safe for consumption by premature infants. Additionally, the sterilized human dairy products retain nutritional components at levels comparable to raw human milk compositions. Therefore, the sterilized human dairy products are suitable for consumption by premature infants.

[0172] 7.2. Example 2: Pilot-Scale Ultra-High Temperature Sterilization of Fortified Human Milk Human milk was obtained from human donors, validated, and pooled. The pooled human milk was divided into 13 batches, and each batch was fortified with one or more supplements. The supplements included amino acids, bovine colostrum, vitamin plain, vitamin C, vitamin D, magnesium bisglycinate, magnesium glycinate, calcium lactate, L-theanine, stevia, lactoenzymedica, vitamin cherry, flavoring amino acids, and methylsulfonylmethane. Reference is made to Figures 7A and 7B, which present the formulations of each fortified milk sample. Specifically, Figures 7A and 7B present the amount, in grams, of each supplement added to each liter of human milk to obtain each fortified milk sample.

[0173] Each fortified milk sample underwent ultra-high temperature sterilization using an Ultra-High Temperature Lab-25 Electric High Viscosity Hybrid Unit (MicroThermics, Raleigh, NC). See Figure 7C, which presents the processing parameters used to sterilize each fortified milk sample. Specifically, each fortified human milk sample was heated at 286°C while undergoing countercurrent heat exchange. o The mixture was heated to a target temperature of 141.1°F and held at the target temperature for 8.1 seconds. The flow rate of 0.25 gallons / minute for each fortified human dairy product was held constant across all runs.

[0174] Additionally, the log reduction of Bacillus cereus per process is depicted in Figure 7C. A greater than 1,000 log reduction of Bacillus cereus was observed in each sterilized fortified milk sample compared to the corresponding raw fortified milk sample counterpart.

[0175] 7.3. Example 3: Commercial-Scale Ultra-High Temperature Sterilization of Human Milk Ultra-high temperature sterilization of a commercial-scale batch of human milk was performed. A total of 50,000 fluid ounces (approximately 1,480 liters) of human milk from various human donors was sampled and pooled prior to sterilization. The pooled 50,000 fluid ounces of human milk were sterilized at the Tetra Pak Pilot Plant (Denton, TX), an FDA-registered food production facility, using a Tetra Therm Aseptic Flex to provide the ultra-high temperature treatment.

[0176] 50,000 fluid ounces of human milk undergoes countercurrent heat exchange and reaches a temperature of 289.4°C. o The milk was heated to a target temperature of 143°F / °C and held at the target temperature for 10.3 seconds. The human milk was flowed through the sterilization system at a flow rate of 8 gallons per minute. After sterilization of the human milk, the sterilized milk product underwent in-line homogenization at 1,800-2,500 psi. The homogenized sterilized milk product was then aseptically bottled into three 330 mL bottles.

[0177] A series of bottles were provided to Merieux Nutrisciences (Crete, IL) for determination of the composition of the sterilized human dairy products. Specifically, the sampled sterilized human dairy products were tested for the following properties and components: density (g / mL), calories (g), total fat (g), monounsaturated fat (g), polyunsaturated fat (g), saturated fat (g), trans fat (g), cholesterol (mg), sodium (g), potassium (mg), total carbohydrates (g), sugars (g), fructose (g), glucose (g), lactose (g), maltose (g), sucrose (g), galactose (g), protein (g), calcium (mg), iron (mg), moisture (g), ash (g), vitamin D2 (mcg), and vitamin D3 (mcg). In addition, the sterilized human dairy products were further tested for protein quality, as quantified using the Protein Digestibility Corrected Amino Acid Score (PDCAAS). Figure 8A presents analytical data. In addition, Figure 8A identifies the method criteria used to determine the various corresponding analytical components. Components were detected using official Association of Official Agricultural Chemists (AOAC) methods, Food and Agriculture Organization (FAO) methods, internal high performance liquid chromatography, or database calculations. The density of the sterilized human dairy sample was 1.016 g / mL.

[0178] Notably, the protein digestibility corrected amino acid score (PDCAAS) of the sterilized dairy product was 72.4 (% by weight). In comparison, the PDCAAS was 145 over 30 minutes. o A pasteurized human milk sample obtained from Mother's Milk Bank (San Jose, CA), which had previously undergone conventional pasteurization at F / 62.8°C, had a PDCAAS of 64.9 (wt%). Overall, the sterilized milk product possesses a 10% improvement in protein quality compared to the conventional pasteurized human milk sample.

[0179] Selected bottles were also submitted to Merieux NutriSciences (Salida, CA) for determination of Bacillus cereus, aerobic plate counts, total microbial counts, yeast, and mold in aseptically bottled sterilized human dairy products. Bottles were selected early during the sterilization process (bottle #158), late during the sterilization process (bottle #1888), and even later during the sterilization process (bottle #3151).

[0180] Reference is now made to Figure 8B, which depicts the microbial counts from bottles 158, 1888, and 3151 compared to raw human milk. Bacillus cereus counts were determined using the standardized AOAC 980.31 method for determining Bacillus cereus in foods. Aerobic plate counts for each sample were determined using the standardized AOAC 966.23 method. Total microbial counts were determined using the standardized USP method for total aerobic microbial counts. <61> Yeast and mold were determined using the standardized USP Microbiological Test for Non-Sterile Products. <61> was determined using the test.

[0181] Each of the three aseptically bottled, sterile, homogenized human milk products exhibited low levels (<100 CFU / g) of presumptive Bacillus cereus compared with the raw human milk sample, which exhibited significantly higher levels (300 CFU / g) of presumptive Bacillus cereus. Furthermore, each of the three aseptically bottled, sterile, homogenized human milk products exhibited low (<10 CFU / g) aerobic plate counts and low total microbial counts (<10 CFU / g). In contrast, the raw human milk sample exhibited a significantly higher aerobic plate count (780,000 CFU / g). Each of the three aseptically bottled, sterile, and homogenized human milk products also exhibited lower levels (<10 CFU / g) of yeast compared with the raw human milk sample, which exhibited significantly higher levels (4,200 CFU / g) of yeast. The aseptically bottled, sterilized and homogenized human dairy products contained within bottles 158, 1888, and 3151 pass industry standard quality control requirements and are considered commercially sterile.

[0182] 7.4. Example 4: Commercial-Scale Ultra-High Temperature Sterilization of Fortified Human Milk A total of 27,000 fluid ounces (800 liters) of human milk from various human donors was verified, pooled, and fortified prior to ultra-high temperature sterilization. Specifically, the milk was fortified with bovine colostrum (50.7 g per liter of milk), amino acids (70.5 g per liter of milk), ascorbic acid (3.1 g per liter of milk), and vitamin D (4.0 g per liter of milk). Organic stevia extract was also added. The 27,000 fluid ounces of fortified human milk was sterilized at the Tetra Pak Pilot Plant (Denton, TX), an FDA-registered food production facility, using a Tetra Therm Aseptic Flex to provide ultra-high temperature processing.

[0183] 27,000 fluid ounces of fortified human milk undergoes countercurrent heat exchange to produce 289.4 o The fortified human milk was heated to a target temperature of 143°F / °C and held at the target temperature for 10.3 seconds. The fortified human milk was flowed through the sterilization system at a flow rate of 8 gallons per minute. After sterilization of the fortified human milk, the sterilized fortified milk product underwent in-line homogenization at 1,800-2,500 psi. The sterilized fortified milk product was then obtained and aseptically bottled into 330 mL bottles.

[0184] Bottles were provided to Merieux Nutrisciences, located in Crete, IL, for determination of the composition of the sterilized fortified human dairy products. Specifically, the sterilized fortified human dairy products were tested for the following properties and components: density (g / mL), calories (g), total fat (g), monounsaturated fat (g), polyunsaturated fat (g), saturated fat (g), trans fat (g), cholesterol (mg), sodium (g), potassium (mg), total carbohydrates (g), sugars (g), fructose (g), glucose (g), lactose (g), maltose (g), sucrose (g), galactose (g), protein (g), calcium (mg), iron (mg), moisture (g), ash (g), vitamin D2 (mcg), and vitamin D3 (mcg). In addition, the sterilized fortified human dairy products were further tested for protein quality using PDCAAS. Figure 9A presents the analytical data and identifies the method criteria used to determine the various corresponding analytical components. Components were detected using official AOAC methods, internal high performance liquid chromatography, or database calculations. The density of the sterilized human dairy sample was 1.052 g / mL. Notably, the PDCAAS of the sterilized fortified dairy product was 100.0 (wt%), indicating a high protein quality of the sterilized fortified dairy product likely resulting from the fortification process.

[0185] Bottles of sterilized fortified human milk products were provided to Merieux NutriSciences, located in Salida, CA, to determine the presence of Bacillus cereus, yeast, and mold. Reference is now made to Figure 9B, which depicts the microbial counts of aseptically bottled sterilized fortified human milk products compared to raw fortified human milk. Bacillus cereus counts were determined using the standardized AOAC 980.31 method for determining Bacillus cereus in foods. Yeast and mold were determined using the standardized USP method for microbial testing of non-sterile products. <61> was determined using the test.

[0186] Aseptically bottled, sterilized, fortified human milk products exhibited low levels (<100 CFU / g) of presumptive Bacillus cereus, low levels (<10 CFU / g) of yeast, and low levels (<10 CFU / g) of mold. Notably, the levels of yeast in aseptically bottled, sterilized, fortified human milk products (<10 CFU / g) were significantly lower compared to their raw, fortified human milk counterparts (10,000 CFU / g).

Claims

1. A method for sterilizing raw human milk to produce a commercially available sterilized human milk product, comprising: removing a first fraction of pathogenic materials from the raw human milk using one or more clarifiers to form a human milk sample, the pathogenic materials comprising Bacillus cereus spores and Clostridium botulinum spores; receiving the human milk sample as an input by a sterilizer located in a closed in-line system; flowing the human milk sample in a first direction at a first flow rate through a tube of the sterilizer; heating the human milk sample within the tube through a countercurrent heat exchange process by flowing a heating fluid in a second direction at a second flow rate, the heating fluid contacting an exterior surface of the tube; cooling the heated human milk sample by the sterilizer to produce the commercially available sterilized human milk product; Including, heating the human milk sample in the tube, raising the temperature of the human milk sample to a temperature within the range of 130°C to 150°C; maintaining the temperature of the human milk sample within said temperature range for a duration of 3 to 15 seconds; wherein the commercially available sterilized human dairy product comprises: (i) a Clostridium botulinum content that is reduced by at least 12 logs relative to the Clostridium botulinum content present in the raw human milk from which the commercially available sterilized human milk product was derived; (ii) a Bacillus cereus content that is at least 500 log lower than the Bacillus cereus content present in the raw human milk from which the commercially available sterilized human milk product was derived; and (iii) human lactoferrin at a concentration of 81 w / v % or more of the concentration of human lactoferrin present in said raw human milk A method comprising:

2. 2. The method of claim 1, wherein the length of the tubing and the first flow rate of the human milk sample are each predetermined to maintain the temperature of the human milk sample within the temperature range for the duration.

3. The method of claim 1 or claim 2, wherein the tube has a diameter of between 0.64 cm and 25.4 cm (between 0.25 inches and 10 inches).

4. The method of any one of claims 1 to 3, wherein the human milk sample received by the sterilizer is not homogenized.

5. 4. The method of any one of claims 1 to 3, wherein the human milk sample is homogenized prior to receipt by the sterilizer or is homogenized after cooling by the sterilizer.

6. The method described in claim 1, wherein the one or more purifiers include a centrifugal filtration process.

7. Prior to the step of the sterilizer receiving the human milk sample, separating the clarified human milk sample into a cream sample and a skim sample; performing a concentration process on the skim sample to produce a retentate; combining a portion of the cream sample with a portion of the obtained retentate to design a standardized human milk sample and produce a fortified human milk product; further comprising Optionally, combining a portion of the cream sample with a portion of the obtained retentate comprises: detecting an initial characteristic of the cream sample; comparing the detected initial properties of the cream sample with target properties for a standardized human dairy product; determining a portion of the cream sample and a portion of the obtained retentate based on the comparison; The method of claim 1 , comprising:

8. 10. The method of claim 7, further comprising the step of packaging the commercially available sterilized human dairy product through an aseptic process following production of the commercially available sterilized human dairy product.

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