Methods, compositions, and devices for meeting dietary fatty acid needs

Abstract

Nutritional formulations containing long chain polyunsaturated fatty acids (LC-PUFAs) are provided, along with methods and devices for preparing the nutritional formulations and / or for administering the nutritional formulations. In some embodiments, a proportion of the LC-PUFAs in the nutritional formulation are in the form of monoglycerides and / or free fatty acids. In some embodiments, the nutritional formulation does not contain added lipase. Also provided are methods for providing nutrition to a subject, methods for improving fat absorption, methods for improving cognitive performance, methods for preventing chronic lung disease, and methods for reducing the length of time a patient requires total parenteral nutrition.

Description

[Technical field]

[0001] This application is related to U.S. Provisional Patent Application No. 61 / 600,207, filed February 17, 2012, and U.S. Provisional Patent Application No. 61 / 719,173, filed October 26, 2012, and claims priority under 35 U.S.C. § 119 to those provisional applications. [Background technology]

[0002] Long-chain fatty acids are important for human health and development. Long-chain fatty acids consumed in the diet are primarily in the form of triglycerides (TGs), in which three long-chain fatty acids are attached to a glycerol molecule via ester bonds. Absorption of long-chain triglycerides first requires the enzymatic action of lipases (e.g., pancreatic lipase), which digest the triglycerides by hydrolysis and break them down into monoglycerides and then into free fatty acids. Once available, these monoglycerides and free fatty acids are absorbed by endothelial cells in the small intestine, where they undergo re-esterification and are then transported to the liver and ultimately to tissues in the body for various physiological purposes (see D. Kasper et al., Harrison's Principles of Internal Medicine, 16th Edition, 2004). Medium-chain triglycerides can be absorbed through the intestinal lumen, whereas long-chain triglycerides cannot, and therefore pancreatic lipase is essential for proper long-chain fatty acid hydrolysis and absorption (see C. Jensen et al., Am. J. Clin. Nutr. 43:745-751 (1986)). However, some individuals, e.g., patients suffering from defective pancreatic productive activity, malabsorption, or pancreatic insufficiency, may not be able to properly break down long-chain triglycerides and may, as a result, suffer from inadequate absorption of fatty acids to maintain health. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Harrison's Principles of Internal Medicine, 16th ed. (2004) by D. Kasper et al. [Non-Patent Document 2] C. Jensen et al., Am. J. Clin. Nutr. 43:745-751 (1986) Summary of the Invention [Problem to be solved by the invention]

[0004] To improve the hydrolysis of long-chain triglycerides, commercially available lipase supplements may be added to the diet. However, for various reasons, lipase supplements do not necessarily solve the problem of low fatty acid absorption in all people who have a reduced capacity to break down long-chain triglycerides or who need to receive essential fatty acids for other reasons. For example, most commercially available lipase supplements are produced from animal pancreatic lipase, which is known to be extremely unstable at pH 7 or below. See, for example, U.S. Patent Application No. 2010 / 0239559; Harrison's Principles of Internal Medicine, 16th Edition, D. Kasper et al. (2004). By the time such lipase passes through the stomach, it is likely that a significant amount of it has been inactivated. Furthermore, not all lipases work to the same extent on hydrolysis of a given long-chain fatty acid, suggesting that lipase specificity is an important consideration (see R. Jensen et al., Lipids 18(3):239-252 (1983)). Moreover, in some populations with pancreatic insufficiency, such as preterm infants or patients in intensive care units, nutritional formulas are tightly regulated. In such controlled populations, supplementing approved formulas with additional ingredients may not be desirable or feasible. Furthermore, many fatty acid-supplemented formulas may contain medium-chain triglycerides, although there are clear medical benefits to ingesting long-chain fatty acids from the diet. Thus, there is a need for improved methods of enhancing hydrolysis of long-chain triglycerides.

[0005] Proper hydrolysis of long-chain polyunsaturated triglycerides (TG-LCPUFA) is particularly important for a variety of reasons. Long-chain polyunsaturated fatty acids (LC-PUFA) are crucial for neural and retinal development. Furthermore, some are designated "essential fatty acids" as humans cannot synthesize them and must obtain them from dietary sources. The major dietary source for the n-3 LC-PUFA docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) is their precursor, the essential fatty acid alpha-linolenic acid (ALA). However, endogenous enzymes are very inefficient in converting ALA to DHA and EPA. According to an official statement by the International Society for the Study of Fatty Acids and Lipids (ISSFAL), the conversion of ALA to DHA is about 1% in infants and much lower in adults (see Brenna et al., Prostaglandins Leukot Essent Fatty Acids, 80(2-3):85-91 (2009)). Thus, while DHA and EPA are not essential fatty acids in themselves, dietary sources of DHA and EPA are important. The major dietary source of the n-6 LC-PUFA arachidonic acid (ARA or AA) is linoleic acid (LA), an essential fatty acid. [Means for solving the problem]

[0006] Embodiments of the present invention solve these various problems by: (i) providing lipases that are significantly more efficient than others at hydrolyzing certain long chain triglycerides and esters, such as, for example, long chain polyunsaturated triglycerides and esters; (ii) providing nutritional formulations, such as, for example, medical nutritional formulations or infant formulas, that include pre-hydrolyzed components (i.e., monoglycerides and / or free fatty acids) of LC-PUFA triglycerides, LC-PUFA fatty acid esters, and / or other long chain triglycerides or long chain fatty acid esters; (iii) providing methods of producing such nutritional formulations, that include methods in which a formulation containing LC-PUFA triglycerides, LC-PUFA fatty acid esters, and / or other long chain triglycerides or long chain fatty acid esters is temporarily exposed to a lipase; and (iv) providing an apparatus designed to provide nutritional formulations that include monoglycerides and / or free fatty acids, such as, for example, LC-PUFA triglycerides and / or LC-PUFA fatty acid esters. In embodiments where the formulation is exposed to lipase briefly and the lipase is removed or separated from the formulation prior to ingestion, the present invention provides the advantage of ensuring the breakdown of LC-PUFA triglycerides, LC-PUFA fatty acid esters, and / or other long chain triglycerides or long chain fatty acid esters without the need for ingestion of exogenous lipase.

[0007] Therefore, some embodiments of the present invention provide a nutritional formulation. In some embodiments, the nutritional formulation comprises LC-PUFA. In some embodiments, more than 2% of the total LC-PUFA is in the form of monoglycerides and free fatty acids, i.e., less than 98% of the total LC-PUFA is in the form of triglycerides or esters. In some embodiments, the LC-PUFA monoglycerides and free fatty acids comprise more than 2.5%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 10%, more than 12%, more than 15%, more than 20%, more than 25%, more than 30%, more than 40%, more than 50%, or more than 75% of the total LC-PUFA in the nutritional formulation. In certain embodiments, the ratio of LC-PUFA monoglycerides and free fatty acids to triglycerides and esters is at least 0.08:1, at least 0.09:1, at least 0.1:1, at least 0.25:1, at least 0.5:1, at least 1:1, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 8:1, at least 10:1, or at least 20:1.

[0008] In certain embodiments, the nutritional formulation is formulated for administration to a premature infant. Other nutritional formulations encompassed by this specification are formulated for infants, toddlers, children, or adults who have a reduced ability to hydrolyze LC-PUFA triglycerides, LC-PUFA fatty acid esters, and / or other long chain triglycerides or long chain fatty acid esters, or who simply require additional essential dietary LC-PUFAs and / or other long chain fatty acids. In some embodiments, the nutritional formulations of the present invention are for subjects who are under 1 year old. In some embodiments, the subject is 1-4 years old. In some embodiments, the subject is 1-6 years old.

[0009] In certain embodiments, the nutritional formulations of the present invention are medical nutritional formulations, i.e., formulated to be consumed or administered orally or enterally under medical supervision, such as those dispensed through a hospital or pharmacy under prescription. Typically, medical nutritional formulations are formulated for the dietary management of a specific medical disorder, disease, or abnormal condition where special nutritional requirements exist. Medical nutritional formulations have a "Generally Recognized As Safe" status and must comply with FDA regulations regarding labeling, product requirements, and manufacturing.

[0010] In some embodiments, the nutritional formulation does not contain an additional lipase. In other embodiments, the nutritional formulation contains a lipase. In some embodiments, the lipase is selected from Chromobacterium viscosum, Pseudomonas fluorescens, Burcholderia cepacia, and Rhizopus oryzae lipases.

[0011] In some embodiments, the nutritional formulation comprises EPA, DHA, ARA, LA, and / or ALA.

[0012] Because free polyunsaturated fatty acids are unstable and rapidly degraded, the present invention also provides a convenient and effective method for preparing the nutritional formulation of the present invention immediately prior to ingestion by a subject. In certain embodiments, the method includes exposing a liquid nutritional composition comprising LC-PUFA triglycerides, LC-PUFA fatty acid esters, and / or other long-chain triglycerides and / or esters of long-chain fatty acids to lipase prior to ingestion by an individual in need of additional dietary LC-PUFAs and / or other long-chain fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 5 minutes, at least 8 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 45 minutes, or at least 60 minutes prior to ingestion. In some embodiments, the liquid nutritional composition is exposed to lipase for 1 minute or less, 2 minutes or less, 3 minutes or less, 5 minutes or less, 8 minutes or less, 10 minutes or less, 15 minutes or less, 30 minutes or less, 45 minutes or less, 60 minutes or less. In some embodiments, the liquid nutritional composition is exposed to the lipase for 24 hours or less. In certain embodiments, the lipase is selected from Chromobacterium viscosum, Pseudomonas fluorescens, Burcholderia cepacia, and Rhizopus oryzae lipases. In certain embodiments, the lipase may be removed from the nutritional formulation prior to ingestion. In other embodiments, the liquid nutritional composition comprising LC-PUFA triglycerides, LC-PUFA fatty acid esters, and / or other long chain triglycerides and / or esters is exposed to a lipase immobilized on a solid support prior to ingestion. In some embodiments, the lipase is immobilized on a solid support by covalent, ionic, or cross-linking bonds. In certain embodiments, the immobilized lipase is encapsulated within or attached to a permeable membrane.

[0013] Another aspect of the invention is a method of providing nutrition to a subject in need of dietary LC-PUFAs and / or other long-chain fatty acids by administering the formulation of the invention, such as a person with a reduced ability to break down long-chain triglycerides or long-chain fatty acid esters in the intestinal tract, a person suffering from pancreatic insufficiency, a person suffering from malnutrition, and a person receiving total parenteral nutrition. In some embodiments, the subject is a premature infant. In other embodiments, the subject is a full-term infant or a toddler. In certain embodiments, the subject is over 50 years of age, over 60 years of age, or over 70 years of age. In some embodiments, the subject is suffering from pancreatic insufficiency. In other embodiments, the formulation is administered through a feeding tube. In some embodiments, the nutritional formulation of the invention is administered to improve cognitive performance in people of all ages, to prevent chronic lung disease in preterm infants, to enhance neurological development in preterm infants, or to treat or prevent a number of other conditions associated with improvement through increased intake of long-chain fatty acids, such as EPA, DHA, ARA, LA, and ALA. Such conditions include, but are not limited to, Alzheimer's disease, bipolar disorder, depression, sepsis, acute respiratory stress, wound healing, cancer, cardiovascular disease, stroke, Parkinson's disease, schizophrenia, diabetes, multiple sclerosis, malnutrition, GI dysfunction, and chronic inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease.

[0014] Another embodiment of the present invention provides a method for reducing the time a patient requires total parenteral nutrition by administering the nutritional formulation of the present invention. This results in a reduced risk of intestinal atrophy and other complications associated with prolonged (more than 24 hours) total parenteral nutrition in such patients. Such methods can be used to reduce the recovery time of patients suffering from GI dysfunction, such as malabsorption, short bowel syndrome, IBD, pancreatic insufficiency, malnutrition before and after surgery, chemotherapy or radiation therapy, or other causes of malnutrition, cancer, wounds, and pressure ulcers. Such patients can receive the nutritional formulation of the present invention via a nasogastric tube. This method of delivery can be advantageous in situations where the patient suffers from altered gut motility, insufficient pancreatic enzyme secretion due to systemic inflammatory response syndrome, or other conditions that cause insufficient cleavage or absorption of LC-PUFA triglycerides, LC-PUFA fatty acid esters, and / or other long-chain triglycerides or esters of long-chain fatty acids. In an alternative embodiment where bypassing the stomach is advantageous, the nutritional formulation of the present invention can be administered via a nasojejunal tube. Other types of delivery devices may also be used to deliver the formulations of the present invention.

[0015] Healthy subjects can also benefit from increased absorption of LC-PUFAs, for example by reducing the risk of cardiovascular disease. Thus, in some embodiments, the present invention provides a method of improving fat absorption in a healthy subject, the method comprising feeding the subject a nutritional formulation of the present invention.

[0016] The present invention further provides an apparatus for preparing the nutritional formulation of the present invention. In some embodiments, the apparatus comprises a chamber containing at least one lipase, the chamber being capable of holding the liquid nutritional formulation so as to expose the liquid nutritional composition to the lipase. In some embodiments, the lipase in the container is immobilized on the inner surface of the container. In other embodiments, the lipase is immobilized on a support in the chamber. In some embodiments, the apparatus comprises a chamber made of a permeable membrane and containing the immobilized lipase in the chamber, such that the liquid nutritional composition passes through the permeable membrane to contact the lipase, but the lipase cannot pass through the permeable membrane. In some embodiments, the lipase contained in the chamber of the apparatus of the present invention is a microbial lipase. In some embodiments, the lipase is selected from bacterial lipases. In some embodiments, the lipase is selected from Chromobacterium viscosum lipase, Pseudomonas Fluorescens lipase, Burcholderia cepacia lipase, and Rhizopus oryzae lipase. In some embodiments, the lipase is selected from Chromobacterium viscosum lipase, Pseudomonas Fluorescens lipase, and Rhizopus oryzae lipase. In some embodiments, the lipase is Rhizopus oryzae lipase. In certain embodiments, for example, the following are provided: (Item 1) A nutritional formulation comprising long chain polyunsaturated fatty acids (LC-PUFAs), wherein greater than 5% of the LC-PUFAs are in the form of monoglycerides and / or free fatty acids. (Item 2) 2. The nutritional formulation of claim 1, wherein the LC-PUFA comprises one or more LC-PUFAs selected from the group consisting of DHA, ARA, and EPA. (Item 3) 3. The nutritional formulation of claim 2, wherein the LC-PUFA comprises DHA. (Item 4) 3. The nutritional formulation of item 2, wherein the LC-PUFA comprises EPA. (Item 5) 3. The nutritional formulation of claim 2, wherein the LC-PUFA comprises ARA. (Item 6) 3. The nutritional formulation according to item 2, wherein the LC-PUFA comprises DHA and EPA. (Item 7) 3. The nutritional formulation of claim 2, wherein the LC-PUFA comprises DHA and ARA. (Item 8) 3. The nutritional formulation of claim 2, wherein the LC-PUFA comprises DHA, EPA, and ARA. (Item 9) 9. The nutritional formulation according to any one of items 1 to 8, wherein the nutritional formulation is an infant formula. (Item 10) 10. The nutritional formulation according to any one of the preceding claims, further comprising a lipase selected from Chromobacterium viscosum lipase, Pseudomonas fluorescens lipase, and Rhizopus oryzae lipase. (Item 11) 11. The nutritional formulation of item 10, comprising no more than 0.1 milligrams of lipase per milligram of LC-PUFA. (Item 12) 10. The nutritional formulation according to any one of the preceding claims, wherein the nutritional formulation does not contain an added lipase. (Item 13) A nutritional formulation comprising a lipase selected from Chromobacterium viscosum lipase, Pseudomonas fluorescens lipase, and Rhizopus oryzae lipase. (Item 14) 14. The nutritional formulation according to item 13, further comprising DHA, wherein more than 5% of said DHA is in the form of monoglycerides and / or free fatty acids. (Item 15) 15. The nutritional formulation according to item 13 or 14, further comprising ARA, wherein more than 5% of said ARA is in the form of monoglycerides and / or free fatty acids. (Item 16) 16. The nutritional formulation according to any one of items 13 to 15, further comprising EPA, wherein more than 5% of said EPA is in the form of monoglycerides and / or free fatty acids. (Item 17) 17. The nutritional formulation according to any one of items 13 to 16, comprising no more than 0.1 milligrams of lipase per milligram of LC-PUFA. (Item 18) A method of preparing a nutritional formulation prior to ingestion by a subject, the method comprising exposing a liquid nutritional composition comprising LC-PUFA triglycerides and / or LC-PUFA esters to a lipase. (Item 19) The lipase is selected from the group consisting of Chromobacterium viscosum viscosum lipase, Pseudomonas fluorescens lipase, and Rhizopus oryzae lipase oryzae lipase. (Item 20) 20. The method according to claim 18 or 19, wherein the liquid nutritional composition is exposed to lipase for at least 1 minute prior to ingestion. (Item 21) 21. The method according to any one of items 18 to 20, wherein the liquid nutritional composition is exposed to lipase for 60 minutes or less prior to ingestion. (Item 22) 22. The method according to any one of items 18 to 21, comprising a step of preparing a liquid composition by adding a potable liquid to a solid or powder composition comprising LC-PUFA triglycerides and / or LC-PUFA esters and lipase, thereby exposing the liquid composition comprising LC-PUFA triglycerides and / or LC-PUFA esters to lipase. (Item 23) (a) preparing a liquid composition by adding a potable liquid to a solid or powder composition comprising LC-PUFA triglycerides and / or LC-PUFA esters; (b) exposing a lipase to the liquid composition, thereby exposing the LC-PUFA triglycerides and / or LC-PUFA esters to the lipase. (Item 24) 24. The method according to any one of items 18 to 23, further comprising removing the lipase from the liquid composition after exposing the LC-PUFA triglycerides and / or LC-PUFA esters to the lipase. (Item 25) The lipase is (a) exposing the lipase in the liquid composition to a molecule that binds the lipase, the molecule being immobilized on a solid support, thereby binding the lipase to the solid support; (b) separating the liquid composition from the solid support. (Item 26) Item 25. The method according to item 24, wherein the LC-PUFA triglycerides and / or LC-PUFA esters in the liquid composition are exposed to a lipase immobilized on a solid support, and the lipase is removed by separating the liquid composition from the solid support. (Item 27) 25. The method of claim 24, wherein the LC-PUFA triglycerides and / or LC-PUFA esters in the liquid composition are exposed to the lipase and then separated from the lipase by passing the liquid composition through a chamber, wherein the lipase is immobilized on at least a portion of the interior surface of the chamber. (Item 28) 25. The method of claim 24, wherein the LC-PUFA triglycerides and / or LC-PUFA esters in the liquid composition are exposed to the lipase and then separated from the lipase by a process comprising passing the liquid composition through a chamber, wherein the lipase is immobilized on a solid substrate contained within the chamber. (Item 29) 29. The method of claim 28, wherein the chamber is a column. (Item 30) 27. The method of claim 26, wherein the liquid composition comprising LC-PUFA triglycerides and / or LC-PUFA esters is exposed to the lipase and then separated from the lipase by a process comprising the step of exposing the liquid composition comprising LC-PUFA triglycerides and / or LC-PUFA esters to a vessel containing lipase immobilized on a solid support, and at least a portion of the inner surface of the vessel is permeable to LC-PUFA triglycerides and LC-PUFA esters but impermeable to the solid support. (Item 31) 31. The method according to any one of items 18 to 30, wherein more than 5% of the LC-PUFAs in the prepared nutritional formulation are in the form of monoglycerides and / or free fatty acids. (Item 32) 32. The method according to any one of items 18 to 31, wherein the LC-PUFA comprises one or more selected from the group consisting of DHA, ARA, and EPA. (Item 33) 33. The method according to any one of items 18 to 20 and items 22 to 32, wherein the liquid nutritional composition is exposed to lipase for 60 minutes or less prior to ingestion. (Item 34) 34. The method according to any one of items 18 to 33, wherein the liquid nutritional composition is exposed to no more than 0.1 milligrams of lipase per milligram of LC-PUFA in the liquid nutritional composition. (Item 35) 35. The method according to any one of items 18 to 34, wherein the liquid nutritional composition is exposed to lipase for 30 minutes or less prior to ingestion. (Item 36) A nutritional formulation prepared by the method according to any one of items 18 to 35. (Item 37) 13. A method of providing nutrition to a subject, comprising the step of supplying to said subject the nutritional formulation according to any one of items 1 to 17 and 36. (Item 38) 38. The method of claim 37, wherein the nutritional formulation is an infant formula. (Item 39) 37. A method for improving fat absorption in a subject, comprising the step of providing the nutritional formulation according to any one of items 1 to 17 and item 36 to the subject. (Item 40) 40. The method of claim 39, wherein the method reduces the total fat level in the subject's stool by at least 50%. (Item 41) 41. The method of claim 39 or 40, wherein the method reduces the level of at least one LC-PUFA selected from the group consisting of DHA, ARA, and EPA in the subject's stool by at least 50%. (Item 42) 42. The method according to any one of items 39 to 41, wherein the method increases the level of DHA, ARA, or both in the plasma of the subject. (Item 43) 43. The method according to any one of items 39 to 42, wherein the method increases the level of DHA, ARA, or both in the retina of the subject. (Item 44) 44. The method according to any one of items 39 to 43, wherein the method increases the level of DHA, ARA, or both in the heart of the subject. (Item 45) 45. The method according to any one of items 39 to 44, wherein the subject has pancreatic insufficiency, a defect in the productive activity of the pancreas, a reduced ability to hydrolyze LC-PUFA triglycerides or LC-PUFA esters, or a reduced ability to absorb LC-PUFA triglycerides or LC-PUFA esters. (Item 46) 46. ​​The method according to any one of items 39 to 45, wherein the nutritional formulation is provided to the subject via a feeding tube. (Item 47) 47. The method according to any one of items 39 to 46, wherein the subject is a preterm infant. (Item 48) The method according to any one of items 39 to 46, wherein the subject is at least 50 years old. (Item 49) 47. The method according to any one of items 39 to 46, wherein the subject is 6 years old or younger. (Item 50) 37. A method for improving cognitive performance in a subject over 50 years of age, comprising a step of feeding said subject the nutritional formulation according to any one of items 1 to 17 and item 36. (Item 51) 13. A method for preventing chronic lung disease in an infant, comprising feeding said infant the nutritional formulation according to any one of items 1 to 17 and item 36. (Item 52) A container comprising at least a first and a second compartment, said first compartment containing a nutritional formulation and said second compartment containing a lipase. (Item 53) The lipase is selected from the group consisting of Chromobacterium viscosum viscosum lipase, Pseudomonas fluorescens lipase, and Rhizopus oryzae lipase oryzae lipase. (Item 54) 54. The container according to claim 52 or 53, wherein the nutritional formulation is in powder form. (Item 55) 54. The container according to item 52 or item 53, wherein the nutritional formulation is in liquid form. (Item 56) 56. The container of any one of items 52 to 55, wherein the second compartment contains no more than 0.1 milligrams of lipase per milligram of LC-PUFA in the nutritional formulation contained within the first compartment. (Item 57) 1. An apparatus for preparing a formula comprising: a body wall for containing a liquid; and at least one lipase contained within the container. (Item 58) 58. The apparatus of claim 57, wherein the at least one lipase is immobilized within the container. (Item 59) 59. The device of claim 58, wherein the at least one lipase is attached to a structure in fluid communication with the interior of the vessel. (Item 60) 60. The device of claim 59, wherein the at least one lipase is attached to the structure by at least one of a covalent bond, an ionic bond, or a cross-linking bond within the structure. (Item 61) 61. The apparatus of claim 59 or 60, wherein the structure comprises an inner surface of the container. (Item 62) 61. The apparatus of claim 59 or 60, wherein the structure comprises a cap for the container. (Item 63) 61. The apparatus of claim 59 or 60, wherein the structure comprises a surface protrusion within the container. (Item 64) 61. The apparatus of claim 59 or 60, wherein the structure comprises a particle within the container. (Item 65) Item 65. The apparatus of item 64, wherein the particles comprise balls or beads. (Item 66) 66. The device according to any one of items 61 to 65, wherein the at least one lipase is encapsulated within a material that is permeable to fatty acids but impermeable to the lipase. (Item 67) 67. The apparatus of any one of items 57 to 66, wherein the container further comprises at least one opening in the container. (Item 68) Item 68. The device of item 67, wherein the opening is configured to connect to a feeding tube. (Item 69) 69. The device of claim 67 or 68, further comprising a valve. (Item 70) 1. An apparatus for preparing formula comprising a cap for a closure of a bottle, the cap comprising at least one lipase immobilized on or within a surface positioned for fluid contact with the interior of the bottle. (Item 71) 71. The device of claim 70, wherein the at least one lipase is immobilized on or within the surface by at least one of covalent bonding, ionic bonding, or inclusion within the structure. (Item 72) 72. The device of claim 70 or 71, wherein the surface comprises an inner surface of the cap. (Item 73) 73. The apparatus of any one of claims 70 to 72, wherein the surface comprises a protrusion configured to extend into the container. (Item 74) 74. The device according to any one of items 70 to 73, wherein the at least one lipase is encapsulated within a material that is permeable to fatty acids but impermeable to the lipase. (Item 75) An apparatus for hydrolyzing triglycerides and / or fatty acid esters in a nutritional formulation, the apparatus comprising a lipase attached to a solid support. (Item 76) The lipase is selected from the group consisting of Chromobacterium viscosum viscosum lipase, Pseudomonas fluorescens lipase, and Rhizopus oryzae lipase oryzae) lipase. (Item 77) 76. The apparatus of claim 75, comprising a vessel, wherein the lipase is immobilized on at least a portion of an inner surface of the vessel. (Item 78) 76. The apparatus of claim 75, comprising a vessel, wherein the lipase is immobilized on a solid substrate contained within the vessel. (Item 79) 79. The device according to claim 77 or 78, wherein the lipase is immobilized by covalent or cross-linking bonds. (Item 80) 80. The apparatus of any one of items 75 to 79, further comprising a feeding tube attached to the container. (Item 81) 13. A method for reducing the length of time a patient requires total parenteral nutrition, the method comprising enterally administering the nutritional formulation according to any one of items 1 to 17 and item 36. (Item 82) 50. The method according to any one of items 39 to 49, wherein the method increases the level of DHA, ARA, or both in red blood cells of the subject. (Item 83) 52. The method according to any one of items 39 to 51, wherein the method increases the level of one or more plasma components selected from the group consisting of triglycerides, cholesterol, HDL, and LDL. (Item 84) 52. The method according to any one of items 39 to 51, wherein said method does not significantly increase said accumulation of fat in the liver. (Item 85) 18. The nutritional formulation according to any one of items 1 to 8 or items 10 to 17, wherein the formulation is a nutritional formulation for adults. (Item 86) 50. The method of claim 49, wherein the subject is under 1 year old. (Item 87) 50. The method according to item 49, wherein the subject is 1 to 6 years old. (Item 88) 50. The method according to any one of items 39 to 49, wherein the method increases the level of vitamin A, vitamin E, or both in the plasma. (Item 89) 37. A method for increasing the plasma level of at least one vitamin selected from the group consisting of vitamin A and vitamin E, comprising the step of supplying to the subject the nutritional formulation according to any one of items 1 to 17 and 36. [Brief description of the drawings]

[0017] [Figure 1] 1 illustrates an apparatus and method for providing nutrition to an infant, according to certain embodiments. [Figure 2A] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 2B] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 2C] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 3A] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 3B] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 3C] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 4A] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 4B] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 5A] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 5B] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 6A] Photograph of a vial of Rhizopus oryzae lipase immobilized on polymer beads. [Figure 6B] 1 illustrates an apparatus for processing and administering a nutritional formulation, according to certain embodiments. [Figure 6C] 1 illustrates an apparatus for processing and administering a nutritional formulation, according to certain embodiments. [Figure 6D] A close-up of the device illustrated in FIG. 6C is shown. [Figure 7] 1 shows hydrolysis of DHA triglyceride by Rhizopus oryzae (RO) lipase. [Figure 8] Hydrolysis of ARA triglyceride by Rhizopus oryzae lipase. [Figure 9A] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 9B] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 9C] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 10A] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 10B] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 10C] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 11A] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 11B] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 11C] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 12] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 13A] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 13B] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 14] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 15] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 16] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 17A] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 17B] FIG. 1 illustrates a partial cross-sectional view of an apparatus for processing a formulation, according to certain embodiments. [Figure 18] 1 illustrates an apparatus for processing a formulation, according to certain embodiments. [Figure 19] FIG. 1 illustrates a partial cross-sectional view of an apparatus for processing a formulation, according to certain embodiments. [Figure 20] FIG. 1 illustrates a partial cross-sectional view of an apparatus for processing a formulation, according to certain embodiments. [Figure 21] FIG. 1 illustrates a partial cross-sectional view of an apparatus for processing a formulation, according to certain embodiments. [Figure 22] (Figure 22A) Fecal weights in EPI pigs during the control week ("Cont week", during which pigs were fed TG-LCPUFA-enriched infant formula) versus the treatment week ("Treat week", during which pigs were fed the same fortified formula that was either non-hydrolyzed ("CONT"), pre-hydrolyzed with Chromobacterium viscosum lipase ("CV"), or pre-hydrolyzed with Rhizopus oryzae lipase ("RO"). (Figure 22B) Total fecal fat content over the last 3 days of the treatment week in the same 3 groups of pigs. (Figure 22C) Coefficient of fat absorption (%CFA) is shown. [Figure 23]Figure 1 shows levels of ARA (A), EPA (B), and DHA (C) in feces from EPI pigs fed non-hydrolyzed formula ("CONT"), formula pre-hydrolyzed with CV lipase ("CV"), or formula pre-hydrolyzed with RO lipase. Asterisks indicate p<0.001. [Figure 24] Figure 1 shows levels of ARA (A) and DHA (B) in plasma of EPI pigs after 7 days of feeding non-hydrolyzed formula ("CONT"), formula pre-hydrolyzed with CV lipase ("CV"), or formula pre-hydrolyzed with RO lipase. Asterisks indicate p<0.05. [Diagram 25] 4 shows levels of ARA and DHA in retina (A) and adipose tissue (B) of EPI pigs after 7 days of feeding non-hydrolyzed formula ("CONT"), formula pre-hydrolyzed with CV lipase ("CV"), or formula pre-hydrolyzed with RO lipase ("RO"). Asterisks indicate p<0.05. [Figure 26] Shown are levels of ARA and DHA in heart (A) and kidney tissue (B) of EPI pigs after 7 days of feeding non-hydrolyzed formula ("CONT"), formula pre-hydrolyzed with CV lipase ("CV"), or formula pre-hydrolyzed with RO lipase ("RO"). Asterisks indicate p<0.05. [Figure 27] 1 shows the percent hydrolysis of DHA and ARA in Enfalac formulas with 100 mg (A), 500 mg (B), 1000 mg (C), or 2000 mg (D) of immobilized RO lipase. [Figure 28] The percent hydrolysis of DHA (A) and ARA (B) by RO lipase or pancreatin is shown. [Figure 29] 1 shows the study design for the 6-week porcine study described in Example 10. [Diagram 30]1 shows levels of ARA and DHA in red blood cells collected from healthy pigs fed TG-LCPUFA-enriched infant formula ("Healthy"), pigs with surgically induced exocrine pancreatic insufficiency fed TG-LCPUFA-enriched infant formula ("EPI"), and pigs with surgically induced exocrine pancreatic insufficiency fed TG-LCPUFA-enriched infant formula prehydrolyzed with immobilized RO lipase ("EPI+iRO"). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Long-chain polyunsaturated fatty acids Long-chain polyunsaturated fatty acids (LC-PUFAs) are hydrocarbon chains with two or more double bonds. Depending on the position of the first double bond relative to the methyl end, LC-PUFAs can be classified as omega-3 (n-3) or omega-6 (n-6) fatty acids. ALA and LA are the parent fatty acids of the n-3 and n-6 ​​PUFA families, respectively. These are considered "essential fatty acids" and humans cannot synthesize them, but rather must obtain them through the diet. This is because mammals cannot introduce double bonds beyond carbons 9 and 10 into fatty acids (see Blosover et al., Cell Biology: A Short Course, John Wiley & Sons, Inc. 39 (2011)). However, humans can produce additional long-chain PUFAs starting with ALA and LA.

[0019] Both ALA and LA are metabolized by a series of desaturation and elongation steps to produce other long-chain PUFAs. For example, ALA is metabolized to EPA and ultimately to DHA. LA is metabolized to ARA, an n6-fatty acid. However, the conversion of ALA to DHA and EPA and LA to ARA is not very efficient (see L. Arterburn et al., Am. J. Clin. Nutr. 83(Suppl):1467S-1476S (2006)). Studies have estimated that the conversion of ALA to DHA in humans is less than 5% (see B. Anderson and D. Ma, Lipids Health Dis. 8:33 (2009)). The liver contains the most active tissue for converting ALA to DHA and LA to ARA, and therefore plays an important role in providing DHA and ARA to less active tissues or organs such as the brain (see M. Martinez et al., J. Pediatr. 120:S129-S138 (1992)). Alternatively, these LC-PUFAs can be consumed directly from the diet. DHA and EPA are found in fish, walnuts, and flaxseed oil, while ARA can be obtained from animal fat sources, corn oil, soybean oil, and sunflower seed oil.

[0020] n-3 fatty acids The n-3 fatty acid DHA is important for neural and retinal development and function. It is the major long-chain PUFA in neural membranes and is essential for brain function, the establishment of brain circuits, and the transmission of nerve impulses. As a bound membrane component, DHA contributes to membrane fluidity, which is important for maintaining synaptic structure, neurotransmission, and synaptic plasticity (see G. Jicha et al., Clin. Interv. Aging 5:45-61 (2010)). DHA also influences signaling events that are essential for neuronal differentiation and survival, and has effects on neurotransmitter and eicosanoid levels and metabolism. The majority of DHA accumulation in the brain occurs during the first two years of life, at the beginning of the third trimester. In rodents and primates, inadequate supply of n-3 PUFA during this period has been shown to cause impairments in learning ability and neurotransmission (see M. Martinez et al., J. Pediatr. 120:S129-S138 (1992)). Supplementation with DHA in rats previously restricted to a DHA-deficient diet rescues performance in memory and learning tasks (see W. Chung et al., J. Nutr. 138(6):1165-1171 (2008)). In a study of healthy adolescent boys, 8 weeks of DHA supplementation significantly increased functional activation in the dorsolateral prefrontal cortex during an activation task compared to placebo (see R. McNamara et al., Am. J. Nutr. 91:1060-7 (2010)). Thus, DHA appears to be important not only for development but also for the maintenance of neuronal function.

[0021] DHA is also highly concentrated in the retina and has important effects on photoreceptor differentiation and activation of the visual pigment rhodopsin (H. Lauritzen et al., Prog. Lipid. Res. 40:1-94 (2001); M. Clandinin et al., J. Pediatr. 125:S25-32 (1994)). Inadequate supply of DHA during early development in primates and rodents leads to abnormal retinal physiology and reduced visual acuity (M. Reisbick et al., Dev. Psychol. 33:387-395 (1997); J. McCann et al., Am. J. Clin. Nutr. 82:281-295 (2005)). Similarly, in humans, infants fed a DHA-free formula for the first 12 months of life have been shown to have poorer visual acuity than infants fed a DHA-supplemented formula (E. Birch et al., Am. J. Clin. Nutr. 91(4):848-859 (2010)). DHA deficiency has also been associated with fetal alcohol syndrome, attention deficit hyperactivity disorder, cystic fibrosis, phenylketonuria, unipolar depression, aggressive hostility, and adrenoleukodystrophy (see A. Horrocks et al., Pharmacological Res. 40(3):211-225 (1999)).

[0022] The benefits of increasing the intake of DHA and other n-3 fatty acids have been described for a variety of diseases including, for example, Alzheimer's disease (AD), bipolar disorder (BP), depression including major depressive disorder (MDD) and postpartum depression, sepsis, acute respiratory stress, wound healing, cancer, cardiovascular disease, stroke, Parkinson's disease, schizophrenia, diabetes, multiple sclerosis, and chronic inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease.

[0023] For example, clinical trials in AD patients have shown that DHA provides therapeutic benefits. For a review of trials evaluating the effects of DHA in AD, see G. Jicha and W. Markesbery, Clin. Interv. Aging 5:45-61 (2010). Data from in vitro assays, cell culture systems, and murine models of AD support a direct role for n-3 PUFAs in amyloid processing in the brain. Moreover, in transgenic models of AD that produce amyloid, DHA supplementation reduces αβ levels (see M. Oksman et al., Neurobiol. Dis. 23(3):563-572 (2006)). In addition to available clinical trial data in patients with AD, a large study in healthy elderly people with mild memory complaints showed that subjects administered DHA performed better on learning and memory tests after 6 months compared to subjects taking a placebo. (Martek Press Release, May 4, 2010). Therefore, DHA may also play a beneficial role in preventing AD.

[0024] The therapeutic use of DHA has also been explored in patients with BP and MDD. See V. Balencia-Martinez et al., Expert. Rev. Neurother. 11(7):1029-1047 (2011) for a review of the effects of DHA in BP. Due to the difficulty of assessing DHA levels in brain tissue from human patients, the fatty acid composition in red blood cell membranes from blood samples was evaluated and found to contain much less DHA in patients with BP and MDD than in healthy controls (see R. McNamara et al., J. Affect. Disord. 126(1-2):303-311 (2010)). In autopsy studies, the fatty acid composition of the orbitofrontal cortex showed much lower levels of DHA in patients with BP compared to normal controls (see R. McNamara et al., Psychiatry Res. 160(3):285-299 (2008)). Moreover, in a 4-month, double-blind, placebo-controlled study, BP patients who took n-3 fatty acids had a much longer period of remission than those in the placebo group (Stoll et al., Arch. Gen. Psychiatry 56(5):407-412 (1999)). These studies suggest that DHA may be therapeutically beneficial in BD and MDD, particularly due to its mood-stabilizing effects.

[0025] DHA has also been shown to be beneficial for patients suffering from other forms of depression. For a review, see A. Logan et al., Lipids Health Dis. 3:25-32 (2004). Many studies have found that depressed patients have lower levels of n-3 in their blood. Similarly, increased plasma DHA has been associated with a decrease in the number of women reporting symptoms of postpartum depression. Several placebo-controlled studies have found that n-3 treatment improves the depressive system. For a review of the relationship between n-3 levels and depression, see A. Logan et al., Lipids Health Dis. 3:25-32 (2004).

[0026] In sepsis, an enteral diet enriched with EPA, gamma-linolenic acid, and antioxidants improved in-hospital outcomes and reduced mortality in patients with severe sepsis or septic shock requiring mechanical ventilation (see A. Pontes-Arruda et al., Crit. Care Med. 34(9):2325-2333 (2006)). Similar benefits in ventilator-free days, ICU-free days, reduced new organ damage, and reduced mortality have been reported in patients with acute respiratory stress fed a diet enriched with long-chain PUFAs and antioxidants (see J. Gadek et al., Crit. Care Med. 27(8):1409-1420 (1999)).

[0027] It has been reported that n-3 fatty acids also have beneficial effects on wound healing: through modifying the lipid microenvironment, n-3 fatty acids can also help to enhance epithelial cell remodeling and reduce inflammation (see D. Ruthig and K. Meckling-Gill, J. Nutr. 129:1791-1798 (1999); J. McDaniel et al., Wound Repair Regen. 19(2):189-200 (2011)).

[0028] EPA and DHA have shown protective effects in cancers such as prostate and breast cancer. This beneficial effect may be due to anti-inflammatory properties and mechanisms that reduce proliferation and promote apoptosis, such as downregulation of NF-κB. For a discussion of n-3 fatty acids in cancer, see B. Anderson and D. Ma, Lipids Health Dis. 8:33 (2009).

[0029] n-3 fatty acids have also been associated with beneficial effects in reducing the risk of cardiovascular disease in patients with cardiovascular disease and in healthy individuals. Similar beneficial effects have also been reported in stroke. Accordingly, the American Heart Association, as well as other health-related organizations, have issued recommendations regarding increased dietary intake of n-3 fatty acids (see P. Kris-Etherton et al., Circulation 106:2747-2757 (2002)). Possible mechanisms for the observed effects of n-3 fatty acids on cardiovascular health include triglyceride-lowering effects, blood pressure-lowering effects, reduced platelet aggregation, and stabilizing effects on the myocardium itself.

[0030] The benefits of n-3 fatty acids in some pathologies may be due to their broad anti-inflammatory effects. EPA and DHA generate resolvins, which are anti-inflammatory regulators with inflammation-resolving and immunomodulatory functions. For example, EPA and DHA exert an inhibitory effect on leukocyte chemotaxis and alter the production of inflammatory cytokines through reducing the activation of NF-κB in immune cells (see P. Calder, Int. Rev. Immunol. 28:506-534 (2009)). In general, n-3 PUFAs are associated with reduced proinflammatory T cell responses. When n-3 fatty acids are increased in animal diets, they alter the composition of T cell membrane microdomains in lipid rafts, reducing NF-κB activation, IL-2 production, and cell proliferation. Specifically, n-3 PUFAs affect the distribution and partitioning of the first signaling regulators of T cell activation, such as protein kinase C (see Y. Fan et al., J. Immunol. 173:6151-6160 (2004)). Also, n-3 fatty acids have been shown to reduce MHC class II expression on dendritic cells, effectively reducing antigen presentation to T cells, while n-6 fatty acids are associated with increased antigen presentation activity (see Sanderson et al., J. Leukoc. Biol. 62:771-777 (1997)). In mononuclear cell lines and peritoneal macrophages, DHA and EPA have anti-inflammatory properties mediated by G protein-coupled receptor 120 (GPR120). As a result, these fatty acids exhibit antidiabetic effects in vivo through suppressing macrophage-induced tissue inflammation (D. Oh et al., Cell 142(5):687-698 (2010)). The various immunomodulatory functions of n-3 PUFAs suggest that they may have an impact in many human diseases.

[0031] n-6 fatty acids Similar to n-3 fatty acids, n-6 fatty acids such as ARA play a vital role in neurodevelopment and brain function, with ARA accumulation in the brain during pre- and postnatal development (see B. Koletzo et al., J. Perinat. Med. 36(1):5-14 (2008)). n-6 fatty acids are important for normal development and immunity in general, and also stimulate skin and hair growth, maintain bone health, regulate metabolism, and maintain the reproductive system.

[0032] Long Chain PUFA Supplement For the last decade, health organizations have recommended the consumption of n-3 fatty acids in the diet due to their health benefits. DHA and EPA are commercially available as triglycerides or esterified forms in dietary supplements or pharmaceuticals (e.g., LOVAZA®, OMACOR®, and Vascepa™). DHA supplements can be derived from fish oil or from vegetarian sources such as flaxseed oil or algae. Supplements can be powders, liquid drinks, or tube-feeding formulations.

[0033] Infant formula is subject to the Federal Food, Drug, and Cosmetic Act and is defined as "a food that is claimed or represented for use solely as a food for infants in the special diet because it mimics breast milk or is a suitable total or partial substitute for breast milk." The FDA defines an infant as an individual 12 months of age or younger (21 CFR 105.3(e)). The predominant n-3 fatty acid in breast milk is DHA, which contains, on average, 7-8 mg / dL (ranging from 0.17% to 1.0% of total fatty acids) (see R. Yuhas et al., Lipids 41(9):851-858 (2006)). The amount of DHA in breast milk roughly reflects the maternal intake of DHA.

[0034] Commercially available TG-LCPUFA supplemented infant formulas include Enfamil formulas such as Enfamil LIPIL® and Enfamil PREMIUM®, Nestle formulas such as Baboo, Earth's Best Organic, Nestle Gerber GOOD START® and Nestle NAN®, Nutricia formulas such as NEOCATE® and APTAMIL®, Parent's Choice Organic, Similac formulas such as Pfizer's SMA GOLD®, Similac ADVANCE®, Similac EARLY SHIELD®, and ISOMIL®, and Ultra Bright Beginnings. Other infant formulas can also be supplemented with TG-LCPUFA. TG-LCPUFA supplemented formulas can be cow's milk or soy based and can be organic. In the United States, TG-LCPUFA supplemented infant formulas account for approximately 90% of product sales (Mead Johnson Nutrition).

[0035] TG-LCPUFA may also be added to follow-on formulas and beverages for infants, the elderly, and others who require nutritional or dietary supplementation with long-chain fatty acids. Such products include ENSURE®, PEDIASURE®, CARNATION®, BOOST®, CERELAC®, and SOUVENAID®. In addition, specialized formulations supplemented with esters of TG-LCPUFA or LC-PUFA can be used in conjunction with the methods and devices of the present invention in patients who require tube feeding. For example, enteral formulations can be commonly used in patients with preterm infants, renal failure, gastrointestinal disease or conditions resulting in GI dysfunction, intestinal resection, fat malabsorption, malnutrition, pancreatitis, hyperglycemia / diabetes, liver failure, acute and chronic pulmonary disease, or immunocompromised conditions. For a review of commercially available enteral formulations, see A. Malone, Pract. Gastr. 29(6):44-74 (2005). Nutritional formulas may be standard, basic, or specialized based on the patient's disease or condition. Commonly used standard formulas include, for example, ISOCAL®, NUTREN 1.0®, NUTREN 1.5®, NUTREN 2.0®, OSMOLITE 1.0®, OSMOLITE 1.2®, FIBERSOURCE 1.2®, JEVITY 1.2®, JEVITY 1.5®, PROBALANCE®, ISOSOURCE 1.5®, DELIVER 2.0®, NOVOSOURCE 2.0®, and TWOCAL HN®. Basic formulas may contain macronutrients, including polymeric and hydrolyzed formulas, and may be fiber-fortified. Disease-specific formulations include renal preparations such as, for example, MAGNACAL RENAL®, NEPRO®, NOVASOURCE RENAL®, SUPLENA® and NUTRI-RENAL®.

[0036] Gastrointestinal (GI) formulations can be used for nutritional management of patients with GI dysfunction, including those with severe protein or fat malabsorption, extensive intestinal resection, cystic fibrosis, cerebral palsy, short bowel syndrome, IBD, pancreatitis, Crohn's disease, diarrhea, gastrointestinal fistula, celiac disease, malabsorption syndrome, trauma / surgery, radiation enteritis, intestinal failure, and chylothorax. These formulations are also used for early postoperative nutritional support, nutritional supply, total parenteral nutrition (TPN) replacement, and dual nutritional support with TPN. GI formulations include, for example, PEPTAMEN® (which consists of 70% medium chain triglycerides and 30% long chain triglycerides to reduce the possibility of fat malabsorption), VIVONEX PLUS®, and VIVONEX PEDIATRIC®.

[0037] However, in individuals who suffer from impaired ability to hydrolyze long-chain triglycerides or esters of long-chain fatty acids, such as individuals with defective pancreatic production or pancreatic insufficiency, supplementing such formulations with DHA, EPA, and other n-3 fatty acids may not be sufficient to realize the benefits associated with these compounds. Long-chain triglycerides or fatty acid esters must be metabolized to monoglycerides and / or free fatty acids in order to be properly absorbed in the intestinal tract. The present invention provides a method to utilize existing commercially available long-chain PUFA supplements or newly designed formulations supplemented with long-chain PUFAs to provide ready-to-use formulations containing significantly higher concentrations of long-chain monoglycerides and / or free fatty acids. In some embodiments, the method is particularly effective in providing DHA, EPA, and ARA triglycerides or long chain monoglycerides and / or free fatty acids produced from esterified DHA, EPA, and ARA, so that the formulation will provide the maximum benefits associated with these important fatty acids to individuals who are otherwise unable to hydrolyze and absorb these important fatty acids.

[0038] Reduced capacity to hydrolyze long-chain triglycerides and fatty acid esters Pancreatic insufficiency is one of the conditions that leads to a decreased capacity to hydrolyze long-chain triglycerides. Pancreatic insufficiency is characterized by insufficient production of pancreatic exocrine enzymes, including pancreatic lipase. Pancreatic insufficiency can occur naturally at various stages of human life. For example, pancreatic lipase secretion starts at low levels around 30 weeks of gestation and remains at low levels for the first year of life. Thus, infants, especially preterm infants, can experience pancreatic insufficiency. As a result, if they are not breastfed, these infants are susceptible to poor fatty acid hydrolysis and absorption and do not receive the benefits associated with the intake of DHA, EPA, and other LC-PUFAs.

[0039] At the other end of the spectrum, otherwise healthy elderly people also experience pancreatic insufficiency or other declines in the ability to hydrolyze LC-PUFA triglycerides or esterified LC-PUFAs due to changes in the pancreas that occur as part of the natural aging process. These changes can include pancreatic atrophy, fibrosis, sclerosis, or lipomas. As a result, elderly people may experience symptoms of indigestion, including malnutrition, steatorrhea, diarrhea, abdominal pain, and weight loss, due to a decline in pancreatic exocrine enzymes (see K. Herzig et al., BMC Geriatrics 11:4-8 (2011)).

[0040] Pancreatic insufficiency or other reduction in the ability to hydrolyze LC-PUFA triglycerides or esterified LC-PUFAs may also result from disease or trauma. For example, pancreatitis is a state of inflammation in the pancreas, which causes pancreatic insufficiency. Pancreatitis can be either acute or chronic, including pancreatitis caused by alcoholism, idiopathic chronic pancreatitis, hereditary pancreatitis, traumatic pancreatitis, acute necrotizing pancreatitis, and autoimmune pancreatitis. Cystic fibrosis is also a cause of pancreatic insufficiency, especially in children and adolescents. Disorders that lower the pH in the duodenum, such as gastrinoma (Zollinger-Ellison syndrome), can inactivate lipase and cause pancreatic insufficiency. Pancreatic insufficiency can also be caused by gastrointestinal surgery in which parts of the stomach or pancreas are removed, gastrointestinal disorders such as pancreatic cancer, gastric ulcers, celiac disease, or Crohn's disease, or autoimmune disorders such as systemic lupus erythematosus (SLE) or inflammatory bowel disease (IBD).

[0041] Other causes of decreased ability to digest TG-LCPUFA, esterified LC-PUFA, and / or other long chain triglycerides and fatty acid esters include, for example, irritable bowel syndrome, hypertriglyceridemia, malnutrition including protein-calorie malnutrition, pancreatic and duodenal neoplasms, abdominal radiation therapy, hemochromatosis, primary sclerosing cholangitis, primary biliary cirrhosis, Schwachmann's syndrome, trypsinogen deficiency, enterokinase deficiency, or isolated lipase deficiency (see D. Kasper et al., Harrison's Principles of Internal Medicine, 16th Edition, 2004). A reduced ability to digest long-chain triglycerides or esterified long-chain PUFAs may also result from intestinal resection, cystic fibrosis, cerebral palsy, short bowel syndrome, IBD, pancreatitis, Crohn's disease, diarrhea, gastrointestinal fistula, celiac disease, malabsorption syndromes, trauma / surgery (particularly GI trauma or surgery), radiation enteritis, intestinal failure, chylothorax, cancer (particularly pancreatic or GI cancer), and / or wound healing. Although the exact cause is unknown, children with attention deficit hyperactivity disorder (ADHD) also have reduced levels of LC-PUFAs (see Burgress et al., Am. J. Clin. Nutri. 71(Suppl):327S-30S (2000)).

[0042] For example, cystic fibrosis (CF) patients have been shown to have reduced levels of LC-PUFAs (see Peretti et al., Nutrition & Metabolism 2:11-28 (2005)). CF patients undergoing pancreatic enzyme replacement therapy often suffer from ongoing fat malabsorption (see Kalivianakis, American Journal of Clinical Nutrition 69:127-134 (1999)). In some embodiments, the present invention provides compositions and methods for improving the absorption of fats, such as LC-PUFAs, in CF patients. In some embodiments, the present invention provides compositions for inducing weight gain in CF patients.

[0043] Cachexia and weight loss due to tissue catabolic state, nutrient re-routing, and malabsorption in advanced stages are common to advanced stages of many cancers, but pancreatic cancer (PC) is an exception in that weight loss and malabsorption are present in 80%-90% of patients at the time of diagnosis. Malabsorption resulting from exocrine defects, which are a major contributor to weight loss, is due to loss of pancreatic parenchyma, blockage of the pancreatic duct that inhibits enzymes from reaching the intestinal tract, and surgical procedures. The common end result of all of these mechanisms is steatorrhea and weight loss (see Damerla et al., J of Support Oncology 6:393-396 (2008)). Weight stabilization in PC is associated with improved survival and quality of life (see Davidson et al., Clinical Nutrition 23, 239-247 (2004)). In some embodiments, the present invention provides formulations and methods for improving fat absorption, such as LC-PUFAs, in PC patients. In some embodiments, the present invention provides compositions and methods for inducing weight gain in PC patients.

[0044] Some embodiments of the present invention improve upon current treatment options for pancreatic insufficiency and other conditions that reduce the ability to hydrolyze TG-LCPUFAs, esterified LC-PUFAs, and / or other long-chain triglycerides and fatty acid esters. In patients with reduced ability to hydrolyze TG-LCPUFAs, esterified LC-PUFAs, and / or other long-chain triglycerides and fatty acid esters, simply increasing the consumption of these nutrients without improving hydrolysis can lead to steatorrhea, abdominal pain, cramps, diarrhea, and other gastrointestinal complications. Pancreatic enzyme replacement therapy can also lead to complications. It has been observed that large amounts of pancreatic digestive enzymes can damage the large intestine and cause fibrotic colon disease (see D. Bansi et al., Gut 46:283-285 (2000); D. Borowitz et al., J. Pediatr. 127:681-684 (1995)). Another significant risk posed by lipase supplements is allergic reactions, since many commercially available lipase supplements are derived from animal sources. Thus, embodiments of the present invention providing prehydrolyzed long-chain triglycerides or long-chain PUFA esters, with or without the addition of lipase, will provide a better and safer method for treating pancreatic insufficiency or other reduced ability to digest long-chain triglycerides or esterified long-chain PUFAs.

[0045] Both n-3 and n-6 ​​fatty acids are important during development, but n-3 fatty acids are thought to be more important than n-6 fatty acids later in life. In some subjects, particularly some adults, it may be desirable to increase the ratio of (DHA and EPA):ARA. In particular, cystic fibrosis patients may benefit from increasing the ratio of (DHA and EPA):ARA in their plasma. However, currently available adult formulations generally have a low ratio of n-3:n-6 fatty acids. Furthermore, in subjects with impaired hydrolysis of TG-LCPUFA, simply increasing the consumption of n-3 TG-LCPUFA is unlikely to significantly improve the subject's ratio of (DHA and EPA):ARA, and the resulting increase in undigested TG-LCPUFA may cause gastrointestinal disorders.

[0046] Thus, some embodiments of the present invention provide formulations and methods for increasing the ratio of (DHA and EPA):ARA in a subject, particularly an adult subject. For example, some embodiments provide a method for preparing a formulation for adults, in which a formulation containing n-3 triglycerides and / or esters is exposed to a lipase that hydrolyzes the n-3 triglycerides and / or esters. In some embodiments, the prepared formulation contains a higher ratio of n-3:n-6 monoglycerides and / or free fatty acids, e.g., a higher ratio of free DHA and EPA to free ARA, than a corresponding formulation without lipase treatment. In some embodiments, the formulation contains more n-3 monoglycerides and / or free fatty acids than n-6 monoglycerides and / or free fatty acids (e.g., more free DHA and EPA than free ARA). In some embodiments, the formulation is prepared by exposing it to a lipase that has a higher activity on n-3 triglycerides and / or esters than on n-6 triglycerides and / or esters. In some embodiments, the enzyme is an RO enzyme.The present invention also provides a formulation in which the ratio of n-3:n-6 free fatty acid and / or monoglyceride is higher than the ratio of n-3:n-6 fatty acid found in the plasma of a subject, for example, the ratio of free DHA and EPA to free ARA is higher than that in the plasma of a subject.The present invention also provides a method in which such a formulation is administered to an adult subject.In some embodiments, the subject has cystic fibrosis.

[0047] Reduced capacity to hydrolyze long-chain fatty acids in preterm infants Long-chain PUFAs are important in infants for normal nervous system and retinal development and are highly accumulated in brain and retinal cell membranes, beginning at 30 weeks of gestation (see C. Martin et al., J. Pediatr. 159(5):743-749 (2011); A. Lapillone et al., Leukotrines Ess. Fatty Acids 81:143-150 (2009); J. McCann et al., Am. J. Clin. Nutr. 82:281-295 (2005); M. Martinez et al., J. Pediatr. 120:S129-S138 (1992)). Common fatty acids, including DHA, EPA, and ARA, and the lipases required to break down these fatty acids into monoglycerides and free fatty acids, are provided to the fetus through the placenta and then to the infant through breast milk. Preterm infants are at significantly higher risk of inadequate supply of fatty acids due to shortened gestational age, followed by reliance on external sources of their fatty acids after birth (see C. Martin et al., J. Pediatr. 159(5):743-749 (2011)). Furthermore, preterm infants do not produce sufficient levels of pancreatic lipase, and therefore they have difficulty hydrolyzing any long-chain fatty acids provided in their formula.

[0048] Preterm infants have been shown to have less DHA and a lower DHA / ARA ratio in both the brain and retina compared to full-term infants (see M. Martinez et al., J. Pediatr. 120:S129-S138 (1992)). In addition, in a retrospective study of fatty acid profiles in preterm infants, inadequate levels of long-chain PUFAs were associated with increased chronic lung disease and sepsis, possibly due to a dysregulated immune response (see C. Martin et al., J. Pediatr. 159(5):743-749 (2011)). These studies and others suggest that establishing adequate levels of long-chain PUFAs in preterm infants is a significant and potentially unmet problem, even with formulas supplemented with DHA and other long-chain triglycerides or long-chain fatty acid esters. The formulations, methods, and devices of the present invention will enable preterm infants to receive sufficient amounts of long-chain fatty acids and realize the associated medical benefits.

[0049] Decreased capacity to hydrolyze long-chain fatty acids in formula-fed infants. Formula-fed infants without supplemented fatty acids may also be deficient in long-chain PUFAs. It has been found that the levels of long-chain PUFAs are decreased in non-supplemented formula-fed infants compared to breast-fed infants (see B. Koletzo et al., J. Perinat. Med. 36(1):5-14 (2008)). Breast-fed infants may also be deficient in n-3 fatty acids due to the variable amount of DHA in breast milk and its relationship to the maternal dietary intake. A positive correlation between the amount of DHA in breast milk and visual and language development in breast-fed infants has been described (see S. Innis, J. Pediatr. 143:S1-S8 (2003)). Therefore, a diet containing DHA is recommended for lactating women. For formula-fed infants, all major formula manufacturers have introduced special infant formulas with fats containing DHA and ARA. However, reports on the benefits of these DHA- and ARA-enriched formulas are mixed. Some studies have shown significant benefits in cognitive development when infants receive long-chain PUFA-containing formulas, while others have not (see B. Koletzo et al., J. Perinat. Med. 36(1):5-14 (2008); E. Sarkadi-Nagy et al., J. Lipid Res. 45:71-80 (2004)). Recently, it has been shown that infants fed Enfamil LIPIL® containing DHA and ARA during the first year of life experienced improved immune outcomes, including improved respiratory health, compared to infants fed the same formula without lipids (see E. Birch et al., J. Pediatr. 156(6):902-906 (2010)). Overall, however, preclinical data do not show consistent benefits for infant development with current long-chain PUFA-supplemented formulas.

[0050] One explanation for the conflicting results of these studies is that some infants are unable to absorb the necessary amount of important fatty acids from the intestine when they consume formula supplemented with long-chain triglycerides or long-chain fatty acid esters. This inability to absorb fatty acids may be due to low levels of endogenous pancreatic lipase in infants. Because lipase is generally delivered to infants through breast milk, formula-fed infants cannot have sufficient levels of lipase to break down long-chain PUFAs or PUFA esters to monoglycerides and / or free fatty acids for absorption by the intestine. As a result, infants fed LC-PUFA-supplemented formulas still have a lower absorption of LC-PUFAs compared to breast-fed infants. Again, there is a clear need to allow for the hydrolysis and absorption of these fatty acids, rather than simply providing a fatty acid supplement.

[0051] Adding lipase to a statutory infant formula (or, for example, a medical nutritional formula) may require significant development research to screen, stabilize, and formulate a suitable lipase supplement. In untested, unformulated formulas, issues with lipase stability, lack of specificity, purity, and / or interference with other substances may result in excessive or potentially harmful levels of the enzyme. The bulk addition of a new substance beyond regulatory hurdles further introduces another variable of how well the formula is tolerated by individuals, particularly infants, who have a low capacity to hydrolyze long-chain triglycerides. This problem is present, for example, in the formulas described in U.S. Pat. No. 5,902,617 (Pabst) and U.S. Pat. No. 4,944,944 (Tang).

[0052] The present invention solves these various problems by providing an as-fed nutritional formulation that provides increased amounts of essential monoglycerides and free fatty acids that can be easily absorbed through the infant's intestinal tract. As a result, the formula-fed subject can be provided with the benefits of DHA, EPA, and ARA. In some embodiments, the nutritional formulation does not introduce any new ingredients beyond the pre-hydrolyzed fats present in the existing formula. In certain embodiments, formula-fed infants are provided with the benefits of fatty acids that breast-fed infants obtain without exposure to lipase supplements. In other embodiments, the nutritional formulation of the present invention contains a highly specific lipase that allows for the use of minimal amounts of lipase added to the infant formula to provide increased amounts of long chain monoglycerides and free fatty acids, particularly DHA, EPA, and ARA.

[0053] In some embodiments, the nutritional formulation improves fatty acid absorption. In some embodiments, the subject consumes the nutritional formulation for 3 days, 5 days, 7 days, 10 days, 14 days, 30 days, 60 days, or more. In some embodiments, such consumption of the nutritional formulation of the present invention can reduce total fat in stool, specifically, reduce the level of DHA, ARA, and / or EPA in stool. In some embodiments, the reduction is measured relative to the stool composition of the subject before starting to consume the nutritional formulation. In some embodiments, the reduction is measured relative to the stool composition of the subject that was fed a nutritional formulation that was not exposed to lipase, such as a currently available nutritional formulation, prior to consumption. The total fat, DHA, ARA, and / or EPA levels in stool can be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more. In certain embodiments, the levels of total fat, DHA, ARA, and / or EPA in the stool are reduced by between 50% and 80%. In some embodiments, the levels of total fat in the stool are reduced by at least 50%. In some embodiments, the levels of at least one LC-PUFA (such as DHA, ARA, or EPA) in the stool are reduced by at least 50%. In some embodiments, the levels of at least one LC-PUFA (such as DHA, ARA, or EPA) in the stool are reduced by at least 60%. In some embodiments, the levels of DHA, ARA, or EPA in the stool are each reduced by at least 50%. In some embodiments, the levels of DHA, ARA, or EPA in the stool are each reduced by at least 60%. In some embodiments, ingestion of the nutritional formulation improves plasma, red blood cell, and tissue accumulation of fat levels, including levels of DHA and ARA. Tissues can include retinal, cardiac, adipose, and renal tissue. In some embodiments, consumption of the nutritional formulation increases levels of DHA, ARA, or both in plasma, red blood cells, or both.In some embodiments, ingestion of the nutritional formulation increases the levels of DHA, ARA, or both in the retina. In some embodiments, ingestion of the nutritional formulation increases the levels of DHA, ARA, or both in the heart. In some embodiments, ingestion of the nutritional formulation increases the plasma levels of triglycerides, cholesterol, HDL, and / or LDL. In some embodiments, ingestion of the nutritional formulation increases the ratio of HDL to LDL in the plasma of a subject.

[0054] In some embodiments, ingestion of the nutritional formulation increases plasma levels of vitamin A and / or vitamin E. Without intending to be bound by theory, it is believed that this increase is due to vitamins A and E being typically provided as esters that must be hydrolyzed. Exposure to lipase in the various methods and compositions of the present invention enhances the hydrolysis of these vitamin esters, which leads to greater accumulation of vitamins A and E in the plasma.

[0055] In some embodiments, the intake of this nutritional formulation has a beneficial effect without significantly increasing the accumulation of fat in the liver. Fatty liver disease (FLD) is characterized by an increase in the accumulation of fat, especially triglycerides, in liver cells. This condition is also associated with other diseases that affect fat metabolism. It is normal for the liver to contain some fat by itself, and this does not cause symptoms. In some patients, fatty liver may be accompanied by liver inflammation and liver cell death (steatohepatitis). There is also an association with liver cancer (hepatocellular carcinoma). Insulin resistance, as well as increased consumption of carbohydrates and saturated fatty acids, and low intake of fiber and omega-3 fatty acids are all clearly related to the pathogenesis of FLD.

[0056] The causes of FLD include diet, medication, disease, and medical conditions. Excessive calorie consumption induces FLD, and excess calorie intake overwhelms the liver's ability to metabolize fat in a normal manner, which leads to fat accumulation in the liver. Many medications, including tamoxifen, amiodarone injection, amiodarone oral administration, and methotrexate, are associated with FLD. Fatty liver is also associated with type II diabetes, obesity, and high triglyceride levels in the blood, celiac disease, and Wilson's disease (a disorder of copper metabolism), rapid weight loss, and malnutrition.

[0057] Lipase Pancreatic insufficiency and other conditions associated with reduced capacity to hydrolyze long-chain triglycerides or long-chain fatty acid esters are currently treated with supplemental digestive enzymes, including pancreatic lipase. However, pancreatic enzymes, especially the pancreatic lipase present in these supplements, are often susceptible to the decomposition action of gastric acid and pepsin, so that only a small portion of the ingested enzyme reaches the duodenum in an active form (see E. Ville et al., Digestion 65:73-81 (2001)). However, many of the acid-protecting coatings have potential safety issues for infant populations or immunocompromised patients, since a significant portion of the delivered weight is plastic coating. Furthermore, even if acid-protecting coatings are helpful, some degree of malabsorption persists, requiring patients with pancreatic insufficiency to increase the dosage of enzyme supplements. This presence of fatty acid malabsorption of enterally coated enzymes may be due to the fact that the duodenum and upper jejunum of patients with pancreatic insufficiency are often acidic environments, so that the expected increase in pH is not achieved and the protective coating is not adequately dissolved to release the enzymes (D. Graham, New (See England J. Med. 296(23):1314-1317 (1977)). Both of these problems have been solved by increasing the dose of lipase administered. However, as previously mentioned, high doses of pancreatic enzyme supplements have been found to be associated with fibrotic colonic disease. Thus, some embodiments of the present invention provide nutritional formulations that include a higher content of long chain monoglycerides and / or free fatty acids without added lipase. Some embodiments provide nutritional formulations that include an optimal dose of lipase as described herein.

[0058] Lipases can be obtained from animals, plants, and many natural or genetically engineered microorganisms. Many, if not most, commercially available dietary lipase supplements are derived from animals and are particularly susceptible to degradation by digestive enzymes. A less frequently used alternative is microbial lipase, i.e., lipases produced in bacteria or fungi, such as yeast. Microbial lipases retain activity over a wider pH range than animal or plant lipases, thus eliminating the need for enteric tablets. However, microbial enzymes are susceptible to degradation by trypsin in the small intestine, thereby reducing their availability for degrading triglycerides and esters in the intestinal tract. In certain embodiments, the lipase used in the formulation, method, or device of the present invention is bacterial lipase, fungal lipase, or both.

[0059] The specificity and kinetics of individual lipases can vary widely. Lipase specificity is controlled by factors that affect the molecular properties of the enzyme, the structure of the substrate, and the binding of the enzyme to the substrate. Types of specificity include substrate specificity (i.e., a given lipase may be more active in degrading one type of fatty acid than another), and positional specificity (which involves preferential hydrolysis of ester bonds in the 1- and / or 3-positions of the glycerol backbone of triglycerides).

[0060] Currently, lipases produced by Chromobacterium viscosum, Pseudomonas fluorescens, Burcholderia cepacia, and Rhizopus oryzae have been reported to be effective in treating malaria, as well as ... Lipases produced by TG-DHA, TG-EPA, and TG-ARA have been shown to have greater specificity for DHA, EPA, and ARA than other lipases, such as those produced by Chromobacterium viscosum, Pseudomonas fluorescens, Burcholderia cepacia, and / or Rhizopus oryzae. As a result, lipase supplements or lipase-supplemented nutritional products containing TG-DHA, TG-EPA, and / or TG-ARA will provide increased hydrolysis of TG-DHA, TG-EPA, and / or TG-ARA. Thus, one aspect of the present invention provides a lipase dietary supplement or lipase supplemented nutritional product comprising Chromobacterium viscosum lipase, Pseudomonas Fluorescens lipase, Burcholderia cepacia lipase, and / or Rhizopus oryzae lipase. In some embodiments, the lipase is Chromobacterium viscosum lipase, Pseudomonas Fluorescens lipase, or Rhizopus oryzae lipase. In certain embodiments, the lipase is Rhizopus oryzae lipase.

[0061] Reference to a particular species of lipase, such as Chromobacterium viscosum lipase, Pseudomonas fluorescens lipase, Burcholderia cepacia lipase, and Rhizopus oryzae lipase, does not necessarily require that the lipase was prepared directly from a wild-type host cell. For example, the same lipase can be recombinantly produced in another host cell.

[0062] Another aspect of the present invention is the use of Chromobacterium viscosum, Pseudomonas fluorescens, In some embodiments, the lipase is selected from the group consisting of Chromobacterium viscosum, Chromobacterium fluorescens, Burkholderia cepacia, and Rhizopus oryzae lipases. viscosum lipase, Pseudomonas Fluorescens lipase, or Rhizopus oryzae lipase oryzae lipase. Additional aspects of the invention provide lipases having specific activity for DHA, EPA, and / or ARA comparable to the specific activity of one or more of Chromobacterium viscosum, Pseudomonas Fluorescens, Burcholderia cepacia, and Rhizopus oryzae as determined by reversed-phase high performance liquid chromatography (RP-HPLC) and described in Example 1. In some embodiments, the lipase has specific activity for DHA, EPA, and / or ARA comparable to the specific activity of one or more of Chromobacterium viscosum lipase, Pseudomonas Fluorescens lipase, or Rhizopus oryzae lipase. One embodiment of the present invention is a nutritional formulation containing less than 5,000 units of lipase (using units as assessed in a standard olive oil assay such as that described in Pharmaceutical Enzymes: Properties and Assay Methods, R. Ruyssen and A. Lauwers (Eds.), Scientific Publishing Company, Ghent, Germany (1978)). In other embodiments, the nutritional formulation contains less than 3,000 units of lipase. In some embodiments, the nutritional formulation contains less than 1,000 units. In a particular embodiment, the formulation containing less than 5,000, less than 3,000, or less than 1,000 units of lipase is an infant formula or a medical nutritional formula.

[0063] Immobilized Lipase The process of immobilizing enzymes and other proteins on insoluble supports is well known and described in the literature. Immobilization of lipase improves the stability of the enzyme, making it reusable and allowing products to be easily separated from the enzyme without being contaminated by lipase. In some embodiments, the lipase is covalently bound to the solid support, although non-covalent binding may also be used. Suitable methods of immobilizing lipase include, for example, adsorption, ionic bonding, covalent bonding, cross-linking, encapsulation, and entrapment in hydrophobic or hydrophilic polymeric and inorganic matrices. See Y. Ren et al., BMC Biotechnol. 11:63 (2011); V.Rurty et al., Biotechnol. Bioprocess Eng. 7:57-66 (2002). Lipase may be immobilized by direct binding to the support material or by binding through a linker. See, e.g., Stark and Holmberg, Biotechnol. and Bioeng. 34(7):942-950 (1989).

[0064] Immobilization by adsorption is reversible and typically involves hydrophobic interactions. It is simple and inexpensive but suffers from incomplete immobilization or leakage of the enzyme from the insoluble support. Examples of immobilized lipases using this method can be found in E. Lie et al., Chem. Technol. and Biotechnol. 50:549-553 (1991) (Candida cylindracea lipase, zeolite support); M. Basri et al., J. Chem. Technol. and Biotechnol. 59:37-44 (1994) (Candida rugosa lipase, polymeric support); H. Gunnlaughsdottir et al., Enzyme and Microbiol. Tech. 22:360-367 (1998) (Humicola lanuginose lipase, glass bead support). Suitable supports for immobilization by adsorption include ceramic beads, such as Toyonite (Toyo Denka Kogyo Co., Ltd.).

[0065] Ionic binding is based on electrostatic interactions between lipase and ionic groups of different charges on a matrix such as DEAE-cellulose or DEAE-Sephadex on a solid support. Ionic binding causes minimal changes in the shape of lipase, and often results in immobilized lipase with high activity. However, it should be understood that the binding force between enzyme and support is stronger than when using adsorption, but not as strong as covalent binding, and therefore leakage of lipase from support may occur.

[0066] Covalent binding is based on a covalent bond between the support material and the functional groups on the amino acids on the surface of the lipase. Possible functional groups in this binding of the enzyme to the support can be amino, carboxyl, sulfhydryl, hydroxyl, imidazole, or phenol groups that are not essential for the catalytic activity of the lipase. To protect the active sites, the immobilization can be carried out in the presence of a substrate or a competitive inhibitor. The great advantage of using covalent binding of the lipase to the support material is the strength of the bond, i.e., the stability of the immobilization. For an example of lipase immobilization by covalent binding, see S. Emi et al., European Polymer Journal 30(5):589-595 (1994). Suitable supports for covalent binding include, for example, Immobead™ (ChiralVision).

[0067] Cross-linking involves binding the lipase to itself to form a three-dimensional structure or to a solid structure using a cross-linking agent. For example, lipase can be cross-linked to chitosan beads. See S.H. Chiou et al., Prep. Biochem. Biotechnol. 37(3):265-275 (2007). Immobilization of lipase by encapsulation usually involves the formation of a porous coating or semi-permeable membrane around the lipase, which contains the lipase inside the porous material while allowing triglycerides and esters to pass freely. Immobilization of lipase by entrapment involves restricting the movement of the enzyme by trapping it within a lattice structure. Alginate beads may be used for this type of immobilization (see I. Bushan et al., J. Bioactive and Compatible Polymers 23(6):552-562 (2008)). Synthetic and natural polymers may be used. G. Fernandez-Lorente et al., J. Am. Oil Chem. Soc. (published online December 14, 2010) and G. Fernandez-Lorente et al., J. Am. Oil See also Chem. Soc. 88:1173-1178 (2011).

[0068] In certain embodiments, the formulations, methods, and devices of the present invention may utilize crystallized and cross-linked lipase as described in U.S. Pat. No. 6,541,606 (Margolin) for improved stability, with or without another form of immobilization, such as encapsulation.

[0069] In some embodiments, lipase is immobilized on magnetic nanoparticles (MNPs). These MNPs can be coated with a linker or polymer containing amino or epoxy functional groups to which lipase reacts. One suitable coating for MNPs is, for example, polydopamine. See, for example, Y. Ren et al., BMC Biotechnology 11:63 (2011). The use of MNPs for lipase immobilization has advantages such as biocompatibility, supermagnetism, small size, and low toxicity. The magnetic properties of the nanoparticles facilitate the removal of lipase from solution and also provide another means for attaching MNP-lipase to a solid support.

[0070] In some embodiments, the immobilized lipase is a microbial lipase. In some embodiments, the immobilized lipase is selected from bacterial lipases. In some embodiments, the immobilized lipase is selected from Chromobacterium viscosum. viscosum, Pseudomonas fluorescens, Burcholderia cepacia, and Rhizopus oryzae.

[0071] In certain embodiments, lipase (immobilized or not) is added to the formulation for 1, 2, 3, 4, 5, 10, 20, 30 minutes or more. Hydrolysis of LC-PUFA triglycerides and esters is measured by RP-HPLC. In certain embodiments, the percent hydrolysis of LC-PUFA triglycerides and esters is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% by 30 minutes. In embodiments, the percent hydrolysis of LC-PUFA triglycerides and esters is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% by 20 minutes. In embodiments, the percent hydrolysis of LC-PUFA triglycerides and esters is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% by 10 minutes. In certain embodiments, the lipase is a Rhizopus oryzae lipase.

[0072] Apparatus containing immobilized lipase According to various embodiments, the present disclosure provides an apparatus and method for preparing a nutritional product. The apparatus and method can be used to expose an infant formula or other nutritional product to lipase prior to consumption. Thus, the lipase breaks down fats and oils with the subsequent release of free fatty acids and monoglycerides. The apparatus and method would allow a convenient means for preparing a formula or other nutritional product. In some embodiments, the apparatus and method allows the infant or others consuming the product to avoid consuming exogenous lipase. In some embodiments, the apparatus and method allows the production of a formulation that includes monoglycerides and / or free fatty acids but does not contain any significant amount of lipase (as determined by ELISA).

[0073] 1, 2A-2C, 3A-3C, 4A-4B, 5A-4B, 9A-9C, 10A-10C, 11A-11C, 12, 13A-13B, 14, 15, 16, 17A-17B, 18, 19, 20, and 21 illustrate devices according to various embodiments of the present disclosure. As shown in FIG. 1, the device 100 of the present disclosure can include a container 110 configured to hold an infant formula 120 or other liquid nutritional product. As described in more detail below, the container 110 can include a lipase that is immobilized such that the formula 120 delivered to the infant through a nasogastric tube 114 or other feeding mechanism (e.g., a baby bottle) does not contain any appreciable amount of lipase. For example, the lipase can be immobilized on or in structures found along the walls or otherwise immobilized within the container such that the lipase is in fluid contact with the formula 120 within the container. Furthermore, as described with reference to various embodiments below, the formula can be added to the container 110 in a variety of ways that allow for lipase enzymatic processing within the container 110. For example, the fluid can be delivered through the tube 112 or poured into the container and then passed through a nasogastric tube or other device for delivery.

[0074] Throughout this disclosure, the devices and methods will refer to their use in processing or preparing nutritional formulations, such as, for example, infant formulas and medical nutritional formulations. It should be understood that the devices and methods can be used to process or prepare any type of nutritional formulation that may benefit from providing lipase treatment prior to consumption. Such products can include any nutritional formulation consumed by individuals with pancreatic insufficiency or by others with reduced capacity to hydrolyze long-chain triglycerides or esterified long-chain PUFAs.

[0075] 2A-2C and 3A-3C illustrate devices in more detail, according to various embodiments. As shown, devices 200-202, 300-302 can include a container 210, 310 for holding a liquid formulation. The container 210, 310 can include a variety of different types and shapes. For example, the container 210, 310 can include a glass or plastic jar or vial, a bag (e.g., silicone or other flexible material similar to an IV saline bag), a cylindrical container such as a syringe outer tube, or other container sized and shaped to hold a desired amount of formula or other product.

[0076] As previously discussed, the disclosed device allows for the formulation to be exposed to lipase to obtain the desired enzymatic action, while allowing the formulation to be conveniently consumed without subsequently consuming the lipase. Thus, in various embodiments, the lipase is immobilized within the container 210, 310 such that it remains within the container 210, 310 when the formulation is removed (e.g., through a nasogastric tube, a bottle nipple, or by transferring the formulation to another container) or can be removed from the formulation prior to consumption. In other embodiments, the lipase is immobilized within the container 210, 310, for example, on a removable solid support, allowing the lipase to be easily removed from the container while leaving the formulation intact within the container for later consumption.

[0077] 2A-2C show one configuration of the container 210, along with a particular embodiment for immobilizing lipase within the container 210. As previously discussed, the container 210 may have a variety of different materials, sizes, and shapes. Additionally, the container 210 may include one or more access ports 220, 230 to control the flow of the formula 260 into and out of the container.

[0078] The lipase may be immobilized within the container 210 in a variety of ways. For example, the lipase may be immobilized or contained within structures 250, 252 located on the inside of the container 210 (FIGS. 2A and 2C). Additionally or alternatively, the lipase 251 may be immobilized or contained within the walls of the container 210 (FIG. 2B). Thus, as soon as the formula 260 is placed inside the container, the formula 260 comes into contact with the lipase and produces the desired enzymatic action.

[0079] As previously discussed, lipase may be immobilized within the container by binding the lipase to structures 250, 252 within the container and / or to walls 251 of the container. The structures within the container may have a variety of shapes. For example, in certain embodiments, the structures may comprise beads, balls, or any other structures that may themselves be mobile within the container such that the structures flow within the formula. For example, as shown in FIG. 4A, the structures 250 may include beads or balls having surface walls 256 to which lipase 257 may be bound. Additionally, it should be understood that the structures 250, 252 may have a variety of different shapes or configurations (e.g., cube-like, ovoid, rod-like).

[0080] The configuration of the structures 250, 252 and / or the walls of the container 210 can be configured to provide a desired surface area over which the formula can be exposed to a sufficient amount of lipase for an acceptable period of time. For example, the structures 250, 252 can include multiple beads 250 (FIG. 2A) or rod-like structures 252 (FIG. 2C) to provide a high surface area for binding a sufficient amount of lipase. Alternatively, a lower amount of lipase may be preferred if a longer time is available to incubate the formulation with the lipase and / or if a lipase with a high enzymatic activity is used.

[0081] In various embodiments, the structures 250, 252 and / or the container are configured such that lipase is not retained within the formula 260 as soon as the formula 260 is removed from the container for consumption or storage. For example, the beads 250 or rod-like structures can be sized so that they do not pass through the relatively small access port 230. Alternatively or additionally, the structures can be attached to the walls of the container and / or the container can include a screen or filter sized to prevent the structures containing the formula 260 from moving. Additionally, the structures 250, 252 can have other properties that facilitate their separation from the formula. For example, the structures 250 can be formed from magnetic beads that can be removed by binding to a magnetic filter.

[0082] In some embodiments, rather than immobilizing the lipase by attachment to the structures within the container 210 and / or the walls of the container, the lipase 257' is contained within the structures 250, 252 and / or the walls 251 of the container. FIG. 4B illustrates one such embodiment. As shown, the structure 250' can include a bead or other shape having a wall 256'. This wall can be formed from a semi-permeable material that allows the inflow and outflow of the formula 260 but not the inflow and outflow of the lipase 257'. Such encapsulation may be used for other structures (e.g., 252) and / or walls of the container as well, such that the surface of the wall has a semi-permeable material within which the lipase may be contained.

[0083] The container may also have surface features that provide increased amounts of lipase and / or increased contact of the lipase with the formula 260. For example, the walls of the container may have ridges or other surface modifications to increase the surface area. Additionally, rather than containing a single open space, the container may contain variations in the flow path (e.g., a winding path that allows for extended or prolonged exposure to the lipase, and / or a collection of channels or tubes in which the lipase is immobilized and through which the formula can flow). See, for example, element 150 in FIG. 1 and element 2101 in FIG. 21.

[0084] In certain embodiments, the container may be manufactured and pre-packaged with lipase in any of the embodiments described herein. At the time of use, the container may be opened and the formula may be placed in the container and contacted with the lipase for a sufficient period of time to produce the desired enzymatic action. In other embodiments, structures such as beads 250 or rod-like structures having lipase immobilized on the surface of the structures or housed / encapsulated therein may be packaged and distributed, and these structures may be placed in a separate container containing the formula. In some embodiments, it may be effective to shake or stir the container containing the immobilized lipase and formula for a period of time.

[0085] As mentioned, the formula 260 can be placed into the container 210 via various access ports. For example, the container can include a top access port 220 and / or a bottom access port 230. The ports 220, 230 can be used for the inflow and outflow of the formula, respectively. Additionally, a single port can be used or multiple ports can be used. The ports can include structure configured to engage other devices that can be used for the delivery or transfer of fluid. For example, the ports can include connectors such as luer lock connections, threads, and / or conduits or tubes that can be engaged with nasogastric tubes. Additionally, the ports can be configured to engage with a bottle, a bottle nipple, or any other structure to facilitate the transfer of fluid to another container in delivery or to aid in delivery. Additionally, one or both of the ports 220, 230 can include a valve 140 (FIG. 1), 240 (FIG. 2A), or other fluid flow control mechanism.

[0086] 3A-3C illustrate a device of the present disclosure, according to certain embodiments. As shown, devices 300-302 include a container 310 for receiving formula 260. Additionally, container 310 may include a cap 322 or other closure, such as a threaded top for a jar or bottle. Similar to the embodiment shown in FIGS. 2A-2C, the device may include structures 350, 351, 351', and 353 that contain lipase immobilized on their surface and / or encapsulated therein.

[0087] The embodiments of Figures 3A-3C can provide for a more rapid separation of the formula from the structures containing lipase. For example, as shown in Figure 3A, the rod-like structures 350 can contain lipase, and after enzymatic treatment of the formula, the cap 322 can be removed, simultaneously removing the structures 350 and the lipase. Furthermore, the cap 322 can be replaced with another cap, a baby bottle nipple, or other fluid connection. Similarly, structures having other configurations similar to balls or beads 351, 351' (Figure 3B) can be sized for easy removal from the formula 260. For example, as shown, the beads 351, 351' are sized so that they can be easily removed manually or by filtration. Furthermore, the container 310 can provide lipase immobilized or contained on its inner surface 353, and after enzymatic treatment, the formula 260 can be transferred to another container or consumed by replacing the cap 322 with a connection to a baby bottle nipple or other delivery system.

[0088] Alternatively or additionally, the structures 350, 351, 351' can have a permeable exterior wall with additional components that provide for immobilized lipase housed within the structure. For example, structure 351' (FIG. 3B) illustrates one embodiment in which the structure 351' has a permeable exterior wall surrounding a number of beads 250. The exterior wall may comprise a mesh or other configuration that allows for easy movement of the formulation towards and away from the structure 351' to provide contact with the beads 250. Additionally, as described in various embodiments above, the beads can provide for lipase that can be immobilized on the surface of the beads or encapsulated within them.

[0089] As discussed above, the lipase-containing structure can be manufactured and distributed as a prepackaged component with the container 310. Alternatively, or in addition, the structure may be packaged and distributed separately from the container. For example, a cap 322 may be manufactured and distributed containing a rod-like structure 350 or beads 351, 351', or otherwise, having lipase housed therein or immobilized thereon. The cap may be configured for connection to a standard baby bottle, water bottle, or other container or device that may contain formula.

[0090] In other embodiments, the lipase may be provided such that the lipase contacts the formula as soon as it is placed in the container and / or during feeding or upon removal from the container. For example, FIG. 5A illustrates one device 500 according to an exemplary embodiment. The device 500 may include a standard baby bottle nipple, and the lipase may be immobilized on the periphery of the nipple or on the inner surface 510 of the nipple itself. In this way, during normal use, the formula will come into contact with the lipase. Similarly, the lipase may be contained within or on other structures that may be used for feeding, such as the fluid tube of a nasogastric feeding device.

[0091] Alternatively, the lipase can be provided in a separate element configured to allow contact of the formula with the lipase during normal fluid flow. For example, in one embodiment, the lipase can be contained within a housing 520 configured for engagement with a bottle closure, such as a nipple (FIG. 5A) or a bottle cap / top (FIG. 5B). The housing 520 can include a permeable wall that allows the formula to pass through the wall volume and contact the lipase provided therein.

[0092] The lipase contained within the housing 520 may be provided in a variety of forms. For example, in some embodiments, the lipase is immobilized on beads 550 within the housing 520 by binding or encapsulation, as described above. Additionally, the housing 520 may include an open mesh or other configuration that allows the formula to pass through it. For example, using the baby bottle configuration shown in FIG. 5A, an open mesh or flow path through the housing 520 would expose the formula to the lipase as it exits the bottle during feeding. Alternatively, as shown in FIG. 5B, the formula may be poured into or out of the top 530 of the housing, allowing the formula to be exposed to the lipase during filling or emptying of the container 310. The top 530 and / or any other portion of the housing 520 may be formed from a variety of materials. For example, the housing 520 may be formed from a membrane that allows for controlled fluid flow. Additionally, top portion 530 may be formed from a semi-permeable membrane that allows fluid (formula) to flow therethrough but does not allow lipase to pass through. Thus, the membrane forming top portion 530 may serve to immobilize lipase within container 310 without otherwise binding or immobilizing the lipase within container 310.

[0093] In various embodiments, the above described devices may include modifications to improve or otherwise control lipase activity. For example, the vessels 110, 210, 310 may include an agitation system that allows for continuous movement of the formula during incubation, thereby allowing the lipase to contact fatty acids found throughout the fluid volume. Additionally, the devices may include a system for controlling temperature to improve or control lipase activity.

[0094] Certain embodiments of the present invention provide a container containing a nutritional formulation and a lipase. In some embodiments, the lipase is in contact with the nutritional formulation in the container. In other embodiments, the lipase and the nutritional formulation are not in contact with each other in the container. In some embodiments, the nutritional formulation and the lipase are contained in separate compartments in the container. In some embodiments, the nutritional formulation is in dry form. In some embodiments, the nutritional formulation is in liquid form. In some embodiments, the lipase is contacted with the nutritional formulation by releasing the lipase into the compartment containing the nutritional formulation. In some embodiments, the lipase is contacted with the nutritional formulation by transferring the lipase and nutritional formulation to another container (e.g., by transferring to another container and emptying the lipase and nutritional formulation compartments). In some embodiments, liquid is added to the other container before or after transferring the lipase and nutritional formulation to the other container.

[0095] Devices according to the present disclosure can have many different shapes and / or configurations. For example, Figures 9A-9C, 10A-10C, 11A-11C, 12, 13A-13B, 14, 15, 16, 17A-17B, 18, 19, 20, 21 illustrate various additional shapes and / or configurations. In each of the configurations described in these figures, lipase can be immobilized using any of the methods described above (e.g., by immobilizing the lipase on a structure such as a bead within the device and / or by immobilizing the lipase in or on a wall or other surface of the device). Furthermore, particular configurations may be selected to provide a wide variety of characteristics, such as surface area, volume, amount of lipase, and / or exposure time of the material to the enzyme.

[0096] The devices illustrated in Figures 9A-9C, 10A-10C, 11A-11C, 12, 13A-13B, 14, 15, 16, 17A-17B, 18, 19, 20, and 21 can be configured to allow lipase contact in a variety of ways. For example, in various embodiments, the device can be partially or completely inserted into a container containing the formula to allow contact between the formula and the lipase. In other embodiments, the device is configured for in-line processing of the formula.

[0097] 9A-9C illustrate configurations for devices that may be placed within a container to process formula. As shown, devices 900, 920 (FIGS. 9A and 9C) can have a variety of shapes formed from an exterior wall 901, 901″ that surrounds a lipase. As shown above, the lipase 902 can be immobilized in a variety of ways, including attachment to beads. Additionally, device 910 (FIG. 9B) can include multiple pockets or openings 903 formed within one or more walls 901′. The particular configurations, number of pockets or openings, as well as the amount of lipase and / or volume of the device can be varied depending on the intended use and / or to control the lipase reaction rate.

[0098] In certain embodiments, the device may be configured to allow for changes in its size or shape. For example, Figures 10A-10C illustrate a device 1000 that can be compressed for storage, e.g., in a container 1001 prior to use. When needed, the container 1001 can be opened and the walls 1003 of the device can expand to produce a desired ratio of lipase volume 1002 to container volume. In some embodiments, the device 1000 includes a coil or spring 1004 that provides structural support and / or helps the device maintain a desired shape and / or volume.

[0099] In some embodiments, the device can include a wand-like extension to facilitate placement and removal of the lipase within the volume of formula. For example, Figures 11A-11C, 12, 13A-13B, 14 and 15 illustrate various exemplary configurations for devices including wand-like extensions. As shown, the device 1100, 1100', 1100", 1200, 1300, 1400, 1500 can include any of one or more pockets or openings 1101, 1201, 1301, 1401, 1501 arranged in various configurations near the distal region of the wand-like extension 1102, 1202, 1302, 1402, 1502. In some embodiments, the orientation of the pocket or opening 1301 can be adjusted, for example, to allow insertion into a narrow opening during use and / or to minimize storage space, as shown, for example, in Figures 13A-13B.

[0100] In various embodiments, the lipase may be attached to a portion of a cap or closure of a bottle or jar such that when the cap or closure is placed on the bottle or jar, the lipase may contact a fluid contained within the bottle or jar. For example, any of the devices shown herein may be attached to a surface of a cap or closure for contacting a formula container within a bottle or jar. Various configurations of devices 1600, 1700, 1800, 1900, 2000 including lipase attached to a cap or closure 1602, 1702, 1802, 1902, 2002 are illustrated in Figures 16, 17A-17B, 18, 19, and 20. As shown, the lipase may be contained within a pocket or opening 1601, 1705, 1805, 1901, 2001 having various shapes or configurations. Further, in some embodiments, the cap or closure member 1902, 2002 can include an opening 1910, 2010 for inserting or removing fluid from the container, and such opening 1910, 2010 can include a connector for a fluid tube, such as a Luer type connector.

[0101] In some embodiments, it may be desirable to treat the formula as it passes through the tubing (e.g., during formula feeding as shown in FIG. 1, or during transfer of the formula from one container to another). FIG. 21 illustrates another device 2100 for in-line treatment of lipase. The device 2100 may include a pocket or opening 2101 containing lipase, which may be immobilized as described above. Additionally, the pocket or opening 2101 may have a tortuous or curved flow path to allow for longer contact time between the lipase and the formula. In addition to this, the device 2100 may include openings 2110 at both ends to allow connection to tubes or conduits for inflow or outflow of the formula.

[0102] In various embodiments, the device may include a material that acts as a screen or mesh to prevent lipase from passing into the formula being ingested by the patient. For example, the devices shown in Figures 19 and 21 can include one or more meshes or screens 1906, 2106 to prevent lipase immobilized on beads or other structures from migrating into the formula being ingested.

[0103] In some embodiments, the lipase may be immobilized in or on a component of the container such that the lipase does not come into contact with the formula until further steps are initiated. For example, in one embodiment, the lipase may be contained within or on a portion of a cap or closure member that may include a mechanism for releasing the immobilized lipase into the container. For example, the lipase may be contained on or in a bead or other structure (see, e.g., element 1805 in FIG. 18 ) that is attached to or contained within the cap such that the lipase can be dripped into the container when desired (e.g., by twisting the cap or removing a barrier / attachment mechanism). Similarly, the lipase may be attached to or contained within a wall or other structure of the container and immobilized on a bead or other material, allowing the lipase to come into contact with the formula only when desired (e.g., by releasing the lipase into the container or removing a barrier that covers the lipase).

[0104] FIG. 6A is a photograph of a vial containing Rhizopus oryzae lipase immobilized on polymer beads. The immobilized lipase is in a dry granular form that can be added to the container or chamber of a device according to the invention, such as the device shown in FIGS. 6B-D. The immobilized lipase may be trapped within the chamber of the device by simply providing a filter at the outlet end of the chamber that contains pores large enough to allow the formula to pass through, but that retains the immobilized lipase within the chamber, while allowing the formula to pass through and exit the chamber. Alternatively, the lipase may be immobilized by coating an internal channel or chamber of the device such that the formula is exposed to the lipase as it passes through the chamber. Lipase in such a device can be used for continuous supply over an extended period of time due to increased stability and reusability of the lipase.

[0105] Nutritional Formulas Certain embodiments of the present invention provide a nutritional formulation. In some embodiments, the nutritional formulation is an infant formula. In some embodiments, the nutritional formulation is a medical nutritional formulation. In some embodiments, the nutritional formulation is exposed to lipase prior to ingestion. In some embodiments, this exposure allows for pre-hydrolysis of at least some of the lipids in the nutritional formulation. Thus, in some embodiments, the nutritional formulation is an "as-fed" formulation, i.e., a liquid formulation as constituted immediately prior to ingestion by the subject, and differs in composition from the formulation sold by the manufacturer. The term "nutritional formulation" does not encompass the composition in the subject's body after ingestion.

[0106] In some embodiments, the nutritional formulation comprises long chain fatty acids. In some embodiments, the nutritional formulation comprises one or more LC-PUFAs, such as DHA, ARA, and EPA. In some embodiments, the nutritional formulation comprises DHA. In some embodiments, the nutritional formulation comprises ARA. In some embodiments, the nutritional formulation comprises DHA and ARA. In some embodiments, the nutritional formulation comprises DHA, ARA, and EPA.

[0107] In some embodiments, more than 5% of the total long chain fatty acids in the nutritional formulation are in the form of monoglycerides and / or free fatty acids. In some embodiments, more than 5% of the total LC-PUFAs in the nutritional formulation are in the form of monoglycerides and / or free fatty acids. In some embodiments, more than 5% of the DHA are in the form of monoglycerides and / or free fatty acids. In some embodiments, more than 5% of the ARA are in the form of monoglycerides and / or free fatty acids. In some embodiments, more than 5% of the EPA are in the form of monoglycerides and / or free fatty acids.

[0108] In some embodiments, more than 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the total long chain fatty acids in the nutritional formulation are in the form of monoglycerides and / or free fatty acids. In some embodiments, more than 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the total LC-PUFAs in the nutritional formulation are in the form of monoglycerides and / or free fatty acids. In some embodiments, more than 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the DHA is in the form of monoglycerides and / or free fatty acids. In some embodiments, more than 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the ARA is in the form of monoglycerides and / or free fatty acids. In some embodiments, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 85%, more than 90%, more than 95%, or 100% of the EPA is in the form of monoglycerides and / or free fatty acids. In some embodiments, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 85%, more than 90%, more than 95%, or 100% of both the DHA and ARA are in the form of monoglycerides and / or free fatty acids. In certain embodiments, more than 90% of both the DHA and ARA are in the form of monoglycerides and / or free fatty acids. In certain embodiments, more than 95% of both the DHA and ARA are in the form of monoglycerides and / or free fatty acids.

[0109] In some embodiments, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the total long chain fatty acids in the nutritional formulation are in the form of monoglycerides and / or free fatty acids. In some embodiments, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the total LC-PUFAs in the nutritional formulation are in the form of monoglycerides and / or free fatty acids. In some embodiments, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the DHA is in the form of monoglycerides and / or free fatty acids.In some embodiments, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the ARA is in the form of monoglycerides and / or free fatty acids. In some embodiments, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the EPA is in the form of monoglycerides and / or free fatty acids. In some embodiments, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of both the DHA and ARA are in the form of monoglycerides and / or free fatty acids.In certain embodiments, at least 90% of both the DHA and ARA is in the form of monoglycerides and / or free fatty acids, hi certain embodiments, at least 95% of both the DHA and ARA is in the form of monoglycerides and / or free fatty acids.

[0110] In some embodiments of the invention, the approximate serving sizes of the nutritional formulas of the invention are about 100-110 mL for premature infant formulas, 90-150 mL (e.g., 148 mL) for term infant formulas, 230-500 mL (e.g., 235-250 mL) for enteral feeding, and 230-250 mL for pediatric and adult formulas. In some embodiments, each serving contains about 10-35 mg of ARA free fatty acids and monoglycerides (as obtained from the complete hydrolysis of TG-ARA in currently available preterm and term infant formulas) or about 40-50 mg of ARA free fatty acids or monoglycerides (as obtained from the complete hydrolysis of TG-ARA in currently available adult formulas). In some embodiments, each serving contains about 7-20 mg of DHA free fatty acids and monoglycerides (as obtained from the complete hydrolysis of TG-DHA in currently available preterm and term infant formulas) or about 10-40 mg of DHA free fatty acids or monoglycerides (as obtained from the complete hydrolysis of TG-DHA in currently available pediatric and adult formulas). An adult serving of 230-250 mL contains about 1,100 mg of EPA free fatty acids and monoglycerides and about 240 mg of DHA free fatty acids and monoglycerides (as obtained from the complete hydrolysis of TG-EPA and TG-DHA in some currently available adult formulas such as ProSure®). However, in some embodiments of the present invention, the ability to prehydrolyze TG-LCPUFA prior to ingestion allows for formulations to be produced with higher levels of LC-PUFA than in currently available formulas. Thus, in some embodiments, the amount of ARA and / or DHA free fatty acids and / or monoglycerides exceeds the amount that can be obtained from complete hydrolysis of TG-LCPUFA in currently available formulations. In some embodiments, a serving of the nutritional formulation of the present invention contains 50-100 mg of LC-PUFA free fatty acids and / or monoglycerides. In some embodiments, a serving of the nutritional formulation of the present invention contains 100-200 mg of LC-PUFA free fatty acids and / or monoglycerides.In some embodiments, an intake of the nutritional formulation of the present invention contains 200-300 mg of LC-PUFA free fatty acids and / or monoglycerides. In some embodiments, an intake of the nutritional formulation of the present invention contains 250-500 mg of LC-PUFA free fatty acids and / or monoglycerides. In some embodiments, an intake of the nutritional formulation of the present invention contains 500-1000 mg of LC-PUFA free fatty acids and / or monoglycerides. In some embodiments, an intake of the nutritional formulation of the present invention contains 1-2 g of LC-PUFA free fatty acids and / or monoglycerides. In some embodiments, an intake of the nutritional formulation of the present invention contains 2-3 g of LC-PUFA free fatty acids and / or monoglycerides.

[0111] In some embodiments, the nutritional formulation comprises fat, carbohydrate, and protein (or amino acids). In some embodiments, the infant formula of the present invention comprises one, more, or all of skim milk, lactose, vegetable oil (e.g., palm olein oil, coconut oil, soybean oil, and high oleic sunflower oil), whey protein concentrate, sugars, LC-PUFA, vitamins, and minerals. In some embodiments, the nutritional formulation comprises fats composed of medium chain fatty acids and fats composed of long chain fatty acids. In some embodiments, the nutritional formulation comprises fats composed of n-6 fatty acids and fats composed of n-3 fatty acids. In some embodiments, the nutritional formulation comprises LA and ALA.

[0112] In some embodiments, the nutritional formulation of the present invention does not contain added lipase. In some embodiments, the device and / or method of the present invention is used to expose the nutritional formulation to lipase, but the nutritional formulation is separated from the lipase before feeding, so that the "as-fed" nutritional formulation does not contain added lipase. A nutritional formulation without added lipase refers to a formulation in which lipase is undetectable or present at very low levels, for example due to leaching of immobilized lipase from the solid support into the formulation. In some embodiments, the nutritional formulation comprises no more than 0.02% (w / w) lipase, no more than 0.01% (w / w) lipase, no more than 0.005% (w / w) lipase, no more than 0.002% (w / w) lipase, no more than 0.001% (w / w) lipase, no more than 0.0005% (w / w) lipase, no more than 0.0002% (w / w) lipase, or no more than 0.0001% (w / w) lipase. In some embodiments, the nutritional formulation comprises less than 0.02% (w / w) lipase, less than 0.01% (w / w) lipase, less than 0.005% (w / w) lipase, less than 0.002% (w / w) lipase, less than 0.001% (w / w) lipase, less than 0.0005% (w / w) lipase, less than 0.0002% (w / w) lipase, or less than 0.0001% (w / w) lipase.

[0113] In some embodiments, the nutritional formulation comprises a lipase, such as Chromobacterium viscosum lipase, Pseudomonas fluorescens lipase, Burcholderia cepacia lipase, and Rhizopus oryzae lipase. oryzae lipase. In some embodiments, the lipase is selected from Chromobacterium viscosum lipase, Pseudomonas Fluorescens lipase, and Rhizopus oryzae lipase. In some embodiments, the lipase is a Chromobacterium viscosum lipase. In some embodiments, the lipase is a Pseudomonas Fluorescens lipase. In some embodiments, the lipase is a Rhizopus oryzae lipase.

[0114] In some embodiments, a serving of the nutritional formulation contains less than 5,000 units of lipase (using units assessed in a standard olive assay such as those in Pharmaceutical Enzymes: Properties and Assay Methods, R. Ruyssen and A. Lauwers (eds.), Scientific Publishing Company, Ghent, Belgium (1978)). In other embodiments, a serving of the nutritional formulation contains less than 3,000 units of lipase. In some embodiments, a serving of the nutritional formulation contains less than 1,000 units of lipase. In certain embodiments, the formulation containing less than 5,000 units, less than 3,000 units, or less than 1,000 units of lipase per serving is an infant formula or a medical nutritional formula.

[0115] In some embodiments, the nutritional formulation contains between 0.01 mg and 1 g of lipase per gram of total fat in the nutritional formulation (whether in free fatty acid, monoglyceride, ester, or triglyceride form). In some embodiments, the nutritional formulation contains between 0.1 and 500 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the nutritional formulation contains between 0.1 and 250 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the nutritional formulation contains between 0.1 and 200 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the nutritional formulation contains between 0.1 and 150 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the nutritional formulation contains between 0.1 and 100 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.1-50 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the nutritional formulation contains 1-50 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the nutritional formulation contains 25-75 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the nutritional formulation contains 1-100 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the nutritional formulation contains 50 mg or less of lipase per gram of total fat in the nutritional formulation.

[0116] In some embodiments, the nutritional formulation contains 0.001-10 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation (whether in free fatty acid, monoglyceride, ester, or triglyceride form). In some embodiments, the nutritional formulation contains 0.001-5 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-3 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-1 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-0.5 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-0.1 mg of lipase per milligram of total LC-PUFA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-0.05 mg of lipase per milligram of total LC-PUFA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.01-0.1 mg of lipase per milligram of total LC-PUFA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.02-0.08 mg of lipase per milligram of total LC-PUFA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.04-0.06 mg of lipase per milligram of total LC-PUFA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.1 mg or less of lipase per milligram of total LC-PUFA in the nutritional formulation.

[0117] In some embodiments, the nutritional formulation contains 0.001-10 mg of lipase per milligram of total DHA (whether in free fatty acid, monoglyceride, ester, or triglyceride form) in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-5 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-3 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-1 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-0.5 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-0.1 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-0.05 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.01-0.1 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.02-0.08 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.04-0.06 mg of lipase per milligram of total DHA in the nutritional formulation.

[0118] In some embodiments, the nutritional formulation contains 0.001-10 mg of lipase per milligram of total ARA (whether in free fatty acid, monoglyceride, ester, or triglyceride form) in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-5 mg of lipase per milligram of total ARA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-3 mg of lipase per milligram of total ARA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-1 mg of lipase per milligram of total ARA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-0.5 mg of lipase per milligram of total ARA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-0.1 mg of lipase per milligram of total ARA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-0.05 mg of lipase per milligram of total ARA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.01-0.1 mg of lipase per milligram of total ARA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.02-0.08 mg of lipase per milligram of total ARA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.04-0.06 mg of lipase per milligram of total ARA in the nutritional formulation.

[0119] In some embodiments, the nutritional formulation contains 0.001-10 mg of lipase per milligram of total EPA in the nutritional formulation (whether in free fatty acid, monoglyceride, ester, or triglyceride form). In some embodiments, the nutritional formulation contains 0.001-5 mg of lipase per milligram of total EPA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-3 mg of lipase per milligram of total EPA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-1 mg of lipase per milligram of total EPA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-0.5 mg of lipase per milligram of total EPA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-0.1 mg of lipase per milligram of total EPA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-0.05 mg of lipase per milligram of total EPA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.01-0.1 mg of lipase per milligram of total EPA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.02-0.08 mg of lipase per milligram of total EPA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.04-0.06 mg of lipase per milligram of total EPA in the nutritional formulation.

[0120] In some embodiments, the nutritional formulation is prepared by a method disclosed herein. In some embodiments, the nutritional formulation is prepared using an apparatus disclosed herein.

[0121] Method for preparing a nutritional formulation According to various embodiments, the present disclosure also provides a method of preparing a nutritional formulation. In some embodiments, the nutritional formulation is an infant formula. In some embodiments, the nutritional formulation is a medical nutritional formulation. In some embodiments, the nutritional formulation is a nutritional drink for adults (a complete nutritional drink, e.g., ENSURE, PEDIASURE, etc.).

[0122] In some embodiments, the method of preparing a nutritional formulation comprises exposing a liquid nutritional composition to a lipase. In some embodiments, the liquid nutritional composition comprises an LC-PIFA triglyceride or an LC-PUFA ester. In some embodiments, the liquid nutritional composition comprises a triglyceride or an ester of one or more LC-PUFAs selected from the group consisting of DHA, ARA, and EPA.

[0123] In some embodiments, the liquid nutritional composition is exposed to a lipase selected from Chromobacterium viscosum lipase, Pseudomonas Fluorescens lipase, Burcholderia cepacia lipase, and Rhizopus oryzae lipase. In some embodiments, the lipase is selected from Chromobacterium viscosum lipase, Pseudomonas Fluorescens lipase, and Rhizopus oryzae lipase. In some embodiments, the lipase is Chromobacterium viscosum lipase. In some embodiments, the lipase is a Pseudomonas Fluorescens lipase. In some embodiments, the lipase is a Rhizopus oryzae lipase.

[0124] The ingredients involved in these methods may be mixed in various orders. In some embodiments, the lipase is added to the liquid nutritional composition, thereby exposing the lipids in the liquid nutritional composition to the lipase. In some embodiments, the liquid nutritional composition is prepared by adding a potable liquid to a solid or powder form of the nutritional formulation. In some embodiments, the lipase is present in the solid or powder form of the nutritional composition prior to the addition of the potable liquid. In other embodiments, the lipase is added after the liquid nutritional composition is prepared. In some embodiments, the lipase and the solid or powder form of the nutritional composition are added to the potable liquid simultaneously.

[0125] In some embodiments, the liquid nutritional composition is exposed to lipase for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 5 minutes, at least 8 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 45 minutes, or at least 60 minutes prior to ingestion. In some embodiments, the liquid nutritional composition is exposed to lipase for 30 seconds or less, 1 minute or less, 2 minutes or less, 3 minutes or less, 5 minutes or less, 8 minutes or less, 10 minutes or less, 15 minutes or less, 30 minutes or less, 45 minutes or less, 60 minutes or less, 2 hours or less, 4 hours or less, 6 hours or less, 12 hours or less, or 24 hours or less.

[0126] In some embodiments, this method results in a nutritional formulation in which at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the total LC-PUFA in the nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the DHA is in the form of monoglycerides and / or free fatty acids. In some embodiments, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the ARA is in the form of monoglycerides and / or free fatty acids. In some embodiments, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the EPA is in the form of monoglycerides and / or free fatty acids.

[0127] For the purposes of this use, the exposure of a nutritional composition or formulation to lipase refers to the period of time that the liquid nutritional composition or liquid formulation is in contact with lipase, which may be in solution or immobilized.For the purposes of this use, the exposure to lipase ends when the formulation is ingested by a subject or when lipase is removed by separating the liquid formulation from the solid support on which the lipase is immobilized.In some embodiments, the liquid nutritional composition is exposed to lipase for 10 minutes or less, and at least 20% of the DHA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids.In some embodiments, the liquid nutritional composition is exposed to lipase for 10 minutes or less, and at least 20% of the ARA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids.In some embodiments, the liquid nutritional composition is exposed to lipase for 10 minutes or less, and at least 20% of the total LC-PUFA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids.

[0128] In some embodiments, the liquid nutritional composition is exposed to lipase for 10 minutes or less, and at least 40% of the DHA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 10 minutes or less, and at least 40% of the ARA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 10 minutes or less, and at least 40% of the total LC-PUFA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids.

[0129] In some embodiments, the liquid nutritional composition is exposed to lipase for 10 minutes or less, and at least 50% of the DHA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 10 minutes or less, and at least 50% of the ARA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 10 minutes or less, and at least 50% of the total LC-PUFA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids.

[0130] In some embodiments, the liquid nutritional composition is exposed to lipase for 10 minutes or less, and at least 60% of the DHA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 10 minutes or less, and at least 60% of the ARA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 10 minutes or less, and at least 60% of the total LC-PUFA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids.

[0131] In some embodiments, the liquid nutritional composition is exposed to lipase for 10 minutes or less, and at least 70% of the DHA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 10 minutes or less, and at least 70% of the ARA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 10 minutes or less, and at least 70% of the total LC-PUFA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids.

[0132] In some embodiments, the liquid nutritional composition is exposed to lipase for 20 minutes or less, and at least 20% of the DHA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 20 minutes or less, and at least 20% of the ARA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 20 minutes or less, and at least 20% of the total LC-PUFA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids.

[0133] In some embodiments, the liquid nutritional composition is exposed to lipase for 20 minutes or less, and at least 40% of the DHA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 20 minutes or less, and at least 40% of the ARA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 20 minutes or less, and at least 40% of the total LC-PUFA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids.

[0134] In some embodiments, the liquid nutritional composition is exposed to lipase for 20 minutes or less, and at least 50% of the DHA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 20 minutes or less, and at least 50% of the ARA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 20 minutes or less, and at least 50% of the total LC-PUFA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids.

[0135] In some embodiments, the liquid nutritional composition is exposed to lipase for 20 minutes or less, and at least 80% of the DHA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 20 minutes or less, and at least 80% of the ARA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 20 minutes or less, and at least 80% of the total LC-PUFA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids.

[0136] In some embodiments, the liquid nutritional composition is exposed to lipase for 20 minutes or less, and at least 90% of the DHA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 20 minutes or less, and at least 90% of the ARA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 20 minutes or less, and at least 90% of the total LC-PUFA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids.

[0137] In some embodiments, the liquid nutritional composition is exposed to lipase for 30 minutes or less, and at least 20% of the DHA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 30 minutes or less, and at least 20% of the ARA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 30 minutes or less, and at least 20% of the total LC-PUFA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids.

[0138] In some embodiments, the liquid nutritional composition is exposed to lipase for 30 minutes or less, and at least 40% of the DHA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 30 minutes or less, and at least 40% of the ARA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 30 minutes or less, and at least 40% of the total LC-PUFA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids.

[0139] In some embodiments, the liquid nutritional composition is exposed to lipase for 30 minutes or less, and at least 60% of the DHA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 30 minutes or less, and at least 60% of the ARA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 30 minutes or less, and at least 60% of the total LC-PUFA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids.

[0140] In some embodiments, the liquid nutritional composition is exposed to lipase for 30 minutes or less, and at least 70% of the DHA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 30 minutes or less, and at least 70% of the ARA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 30 minutes or less, and at least 70% of the total LC-PUFA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids.

[0141] In some embodiments, the liquid nutritional composition is exposed to lipase for 30 minutes or less, and at least 80% of the DHA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 30 minutes or less, and at least 80% of the ARA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the liquid nutritional composition is exposed to lipase for 30 minutes or less, and at least 80% of the total LC-PUFA in the resulting nutritional formulation is in the form of monoglycerides and / or free fatty acids.

[0142] In some embodiments, the lipase remains in the nutritional formulation when the nutritional formulation is provided to a subject. In other embodiments, the lipase is removed from the liquid nutritional composition before the liquid nutritional composition is provided to a subject. In some embodiments, the lipase is removed by exposing the liquid nutritional composition containing the lipase to a solid support immobilized with a molecule that binds the lipase, thereby binding the lipase to the solid support, and separating the liquid nutritional composition from the solid support. Because the lipase is immobilized on the solid support, separating the liquid nutritional composition from the solid support has the effect of removing the lipase from the liquid nutritional composition. In some embodiments, the lipase is immobilized on a solid support before the lipase is exposed to the liquid nutritional composition, and the lipase is removed by separating the liquid nutritional composition from the solid support. In some embodiments, the lipase is immobilized on at least a portion of the inner surface of the chamber or on a solid support contained within the chamber, and the liquid nutritional composition is temporarily exposed to the lipase by passing through the chamber. In some embodiments, the chamber is a column. In some embodiments, the liquid nutritional composition is exposed to a container containing lipase immobilized on a solid support, at least a portion of the interior surface of the container being made of a material that is permeable to triglycerides and esters but impermeable to the solid support.

[0143] In some embodiments, the method produces a nutritional formulation that does not contain added lipase. In some embodiments, the nutritional formulation is exposed to lipase, but the nutritional formulation is separated from the lipase prior to feeding, such that the as-fed nutritional formulation does not contain added lipase. A nutritional formulation that does not contain (or does not contain) added lipase refers to a formulation in which lipase is undetectable or present at only very low levels, for example due to leaching of immobilized lipase from the solid support into the formulation. In some embodiments, the nutritional formulation comprises no more than 0.02% (w / w) lipase, no more than 0.01% (w / w) lipase, no more than 0.005% (w / w) lipase, no more than 0.002% (w / w) lipase, no more than 0.001% (w / w) lipase, no more than 0.0005% (w / w) lipase, no more than 0.0002% (w / w) lipase, or no more than 0.0001% (w / w) lipase. In some embodiments, the nutritional formulation comprises less than 0.02% (w / w) lipase, less than 0.01% (w / w) lipase, less than 0.005% (w / w) lipase, less than 0.002% (w / w) lipase, less than 0.001% (w / w) lipase, less than 0.0005% (w / w) lipase, less than 0.0002% (w / w) lipase, or less than 0.0001% (w / w) lipase.

[0144] In some embodiments, the method includes exposing the nutritional formulation to less than 5,000 units of lipase per serving (as assessed by a standard olive assay, such as that described in Pharmaceutical Enzymes: Properties and Assay Methods, R. Ruyssen and A. Lauwers (Eds.), Scientific Publishing Company, Ghent, Belgium (1978)). In other embodiments, the nutritional formulation is exposed to less than 3,000 units of lipase per serving. In some embodiments, the nutritional formulation is exposed to less than 1,000 units of lipase per serving. In certain embodiments, the formulation exposed to less than 5,000 units, less than 3,000 units, or less than 1,000 units of lipase per serving is an infant formula or a medical nutritional formula.

[0145] In some embodiments, the method of the present invention exposes the nutritional formulation to between 0.01 mg and 1 g of lipase per gram of total fat in the nutritional formulation (whether in free fatty acid, monoglyceride, ester, or triglyceride form). In some embodiments, the method of the present invention exposes the nutritional formulation to between 0.1 and 500 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the method of the present invention exposes the nutritional formulation to between 0.1 and 250 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the method of the present invention exposes the nutritional formulation to between 0.1 and 200 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the method of the present invention exposes the nutritional formulation to between 0.1 and 150 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the method of the present invention exposes the nutritional formulation to between 0.1 and 100 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the method of the present invention exposes the nutritional formulation to 0.1-50 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the method of the present invention exposes the nutritional formulation to 1-50 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the method of the present invention exposes the nutritional formulation to 25-75 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the method of the present invention exposes the nutritional formulation to 1-100 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the method of the present invention exposes the nutritional formulation to 50 mg or less of lipase per gram of total fat in the nutritional formulation.

[0146] In some embodiments, the method exposes the nutritional formulation to 0.001-10 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation (whether in free fatty acid, monoglyceride, ester, or triglyceride form). In some embodiments, the method exposes the nutritional formulation to 0.001-5 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.001-3 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.001-1 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.001-0.5 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.001-0.1 mg of lipase per milligram of total LC-PUFA in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.001-0.05 mg of lipase per milligram of total LC-PUFA in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.01-0.1 mg of lipase per milligram of total LC-PUFA in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.02-0.08 mg of lipase per milligram of total LC-PUFA in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.04-0.06 mg of lipase per milligram of total LC-PUFA in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to no more than 0.1 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation.

[0147] In some embodiments, the method exposes the nutritional formulation to 0.001-10 mg of lipase per milligram of total DHA (whether in free fatty acid, monoglyceride, ester, or triglyceride form) in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.001-5 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.001-3 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.001-1 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.001-0.5 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.001-0.1 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.001-0.05 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.01-0.1 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.02-0.08 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.04-0.06 mg of lipase per milligram of total DHA in the nutritional formulation.

[0148] In some embodiments, a method of preparing a nutritional formulation comprises exposing a liquid nutritional composition to a device described herein. EXAMPLES

[0149] Example 1: Specific activity of lipase towards DHA and ARA To evaluate the enzymatic activity of various lipases on DHA and / or ARA triglycerides, experiments were performed in 2 mL glass vials (equipped with a magnetic stir bar) containing 0.1 M Tris buffer, pH 7.7, and the substrate DHA or ARA triglyceride. The reaction was started by adding the lipase solution. Lipases were obtained from the following commercial sources: Rhizopus oryzae (Amano DF-15, Amano Enzyme Inc., Nagoya, Japan), Chromobacterium viscosum (EMD CalBiochem, EMD Biosciences, Billerica, MA), and Pseudomonas Fluorescens (Amano AK, Amano Enzyme Inc., Nagoya, Japan). Other lipases are also available from commercial sources, such as Candida rugosa (Amano AY 30 or Amano 30, Amano Enzyme Co., Ltd., Nagoya, Japan), Aspergillus niger (Amano DS, Amano Enzyme Co., Ltd., Nagoya, Japan), Penicillium camembertii (Amano 50, Amano Enzyme Co., Ltd., Nagoya, Japan), Rhizomucor miehei (L4277, Sigma-Aldrich), Aspergillus oryzae (62285, Sigma-Aldrich), and Burcholderia cepacia (534641, Sigma-Aldrich). Lipase solutions were prepared from these commercial lipases without further purification except that the B. cepacia lipase was purified to homogeneity.

[0150] The vials were transferred to a 37°C water bath placed on a magnetic stirrer. 50 μl samples were taken at different time intervals, 0, 15, 30, 45, 60, 90, and 120 min, and added to HPLC vials containing 950 μl of transfer buffer (30% 10 mM ammonium phosphate buffer, pH 3.0 and 70% acetonitrile). The samples were then analyzed for either DHA free acid or ARA free acid by reversed-phase high performance liquid chromatography (RP-HPLC) using an Agilent HPLC 1100 series and a C8 RP column, monitoring at 215 and 220 nm. Commercially available standards were used to identify the free acid peaks by retention time: DHA triglyceride (Nu-check Prep, Inc. Lot No. T-310-D7-V), ARA triglyceride (Nu-check Prep, Inc. Lot No. T-295-JY14-V), DHA free acid form (Nu-check Prep, Inc. Lot No. U-84A-AU20-U), and ARA free acid form (Nu-check Prep, Inc. Lot No. U-71A-N11-U). The specific activities of the panel of lipases towards DHA and ARA in this assay are outlined in Table 1. In the opinion of the inventors, Chromobacterium viscosum (CV), Burcholderia cepacia (BC), Pseudomonas fluorescens (PF), and Rhizopus oryzae (RO) had substantially higher specific activity towards DHA and / or ARA than the other lipases tested, including Candida rugosa (CR). [Table 1]

[0151] Example 2: Enzymatic activity of Chromobacterium viscosum and Rhizopus oryzae lipases against DHA, ARA, and EPA in infant formulas To evaluate the enzymatic activity of CV and RO lipases on DHA, ARA, and EPA when supplemented into infant formula, cow's milk-based infant formula was prepared by dissolving 10 g of ENFAMIL® powder in 35 mL of water. Infant formula containing the substrate EPA, 0.1 M Tris buffer, pH 7.7, and 2.7 mg of substrates DHA (Nu-check Prep, Inc. Lot No. T-310-D7-V) and 5.4 mg of ARA (Nu-check Prep, Inc. Lot No. T-295) were added to a 1 mL glass vial (containing a magnetic stir bar). The reaction was initiated by the addition of the enzyme (i.e., lipase), and four concentrations of each enzyme were tested. The vial was transferred to a 37° C. water bath placed on a magnetic stirrer. 50 μl of each sample was taken at different time points, 0, 10, 20, 30, 45, and 60 min, and added to HPLC vials containing 950 μl of HPLC transfer buffer (30% 10 mM ammonium phosphate buffer, pH 3.0 and 70% acetonitrile). Samples were then analyzed for either DHA, ARA, or EPA acids by HPLC as described above.

[0152] The percent of total triglycosides decreased over time as the amount of free acid and monoglycerides increased. For example, the amount of DHA free acid increased over time when hydrolyzed with RO (Figure 7). Similarly, the amount of ARA free acid increased over time when hydrolyzed with RO (Figure 8).

[0153] The specific activity of each lipase in this assay was calculated based on the amount of free DHA, ARA, or EPA acids released in the infant formula and is shown in Table 2. [Table 2]

[0154] Example 3: Hydrolysis of DHA triglyceride and ARA triglyceride scale-up Lipases were evaluated for their ability to hydrolyze TG-DHA and TG-ARA when scaled up to amounts that could be used to supplement infant formula. Infant formula (milk) was prepared by dissolving 162 g Enfamil powder in 648 mL tap water (hot water, temperature was 37° C.). DHA triglyceride (442 mg, final concentration of DHA 0.54 g, 1.2% of total fat) and ARA triglyceride (885 mg, final concentration of ARA 1.08 g, 2.4% of total fat) were accurately weighed from the same source as in Example 2 and mixed with the infant formula powder before adding water. The reaction was carried out in a water bath under constant stirring. Fat hydrolysis was initiated by the addition of either CV or RO lipase. Formula samples were removed at 0, 15, and 30 minutes and analyzed for DHA and ARA hydrolysis by RP-HPLC as described above. The results are shown in Table 3 below. [Table 3]

[0155] Example 4: Enzymatic activity of immobilized Rhizopus oryzae lipase on TG-DHA or TG-ARA in infant formula and buffer To evaluate the enzymatic activity of immobilized RO lipase on TG-DHA or TG-ARA when supplemented in infant formula, cow's milk-based infant formula was prepared by dissolving 10 g of ENFAMIL® powder in 35 mL of water. The reaction was carried out as follows: 0.1 M Tris buffer, pH 7.7, and substrate (TG-DHA or TG-ARA) were added to a 1 mL glass vial (equipped with a magnetic stir bar). The reaction was started by adding lipase. The vial was transferred to a 37° C. water bath placed on a magnetic stirrer. 50 μl of each sample was taken at different time points at 0, 10, 20, and 30 min and added to an HPLC vial containing 900 μl of HPLC transfer buffer (30% 10 mM ammonium phosphate buffer, pH 3.0 and 70% acetonitrile). The samples were then analyzed for either DHA acid or ARA acid by RP-HPLC as described above.

[0156] The specific activity of lipase for hydrolysis of TG-DHA and TG-ARA was calculated based on the amount of free DHA acid or ARA acid released in the infant formula and the results are shown in Table 4. [Table 4]

[0157] Example 5: Animals and Surgical Procedures 5.1 Animals Experiments were performed on 12 pigs (9+3) obtained from university-bred pigs at the Swedish Agricultural University, Department of Agricultural Biosystems and Technology, Odarslov, weighing approximately 10±2 kg each. The animals were kept on a 12-h day-night cycle with light between 06.00 and 18.00 (6am-6pm) and darkness between 18.00 and 06.00 (6pm-6am). The pigs were individually housed in metabolic cages or separate pens equipped with dry sample troughs, drinking nipples and constant heat lamps (150 W). The pigs were allowed to move freely within the pens and were able to see each other.

[0158] 5.2 Feed During the post-surgery and pre-treatment periods, the pigs were fed a standard pig chow ("53908 Vaxtill 320 P BK", Lantmannen, Sweden) containing 17.5% crude protein, 3.9% crude fiber, 3.5% crude fat, and 5.2% ash, with 5000 IE / kg vitamin A, 500 IE / kg vitamin D, and 85 mg / kg vitamin E. The pigs were fed twice a day (2.0% of body weight per meal) between the hours of 09:00 and 10:00 (9am to 10am) and 17:00 and 18:00 (5pm to 6pm). During the adaptation period, a few days before the start of the experiment, the pigs were trained to consume infant formula (NAN Pro 1 Gold Infant Formula, Nestle). As pigs do not like to drink large amounts of liquid, the formula was prepared as a 1:4 dilution with tap water instead of a 1:7 dilution as recommended by the manufacturer to allow adequate consumption. The daily nutritional requirement was 400 kL / kg body weight, which corresponds to 40 g of formula powder / kg body weight. The daily ration was divided into four portions, with the first meal starting at 9 am and then every 3 hours, and the last meal being fed at 6 am. 100 g of NAN formula contains approximately 27.7% fat, 9.6% protein, and 57.8% carbohydrates.

[0159] 5.3 Infant Formulas Fortified with DHA and ARA Triglycerides According to the manufacturer, NAN Pro 1 Gold (Nestle) is a premium whey-dominant breastfeeding infant formula that is nutritionally complete and specially formulated for healthy infants from birth. It also contains fish oil to support brain and vision development. (http: / / www.nestlebaby.com / au / baby_nutrition / products / infant_formula / ) NAN Pro 1 Gold Ingredients: Milk Solids, Vegetable Oil (contains soy), Minerals (Calcium Citrate, Potassium Citrate, Potassium Chloride, Magnesium Chloride, Sodium Chloride, Sodium Sulfate, Ferrous Sulfate, Zinc Sulfate, Calcium Phosphate, Copper Sulfate, Manganese Sulfate, Potassium Iodide, Sodium Selenite), Omega LCPUFA (DHA, AA from Fish Oil), Emulsifier (Soy Lecithin), Vitamins [Sodium Ascorbate (vit C), dl-Alpha Tocopheryl Acetate (vit E), Niacinamide (niacin), Calcium Pantothenate, Retinyl Acetate (vit A), Thiamine Mononitrate (vit B1), Pyridoxine Hydrochloride (vit B6), Riboflavin (vit B2), Folic Acid, Phylloquinone (vit K1), Biotin, Cholecalciferol (vit D3)], cyanocobalamin (B12), L-histidine, taurine, inositol, nucleotides (cytidine 5'-monophosphate, uridine 5'-monophosphate, adenosine 5'-monophosphate, guanosine 5'-monophosphate), L-carnitine, cultures (bifidus). Table 5 below lists the breast milk and infant formulas, along with the infant and pig formulas for use in these studies. * The lipid composition of is outlined below. [Table 5] The total concentration of TG-DHA and TG-AA in the NAN formula was 0.22%, which is lower than the recommended level of 1%. Therefore, the NAN formula was fortified with TG-DHA and TG-AA derived from fish oil (NuCheck (http: / / www.nu-chekprep.com, ~40% TG-DHA and TG-ARA)) to reach final concentrations of 1% TG-DHA and 2% TG-DHA, respectively.

[0160] 5.4 Pancreatic duct ligation for induction of exocrine pancreatic insufficiency (EPI) Surgery to induce EPI was performed in 12+2 juvenile pigs aged 6-8 weeks. EPI generally develops fully 3-4 weeks after surgery. The onset of complete pancreatic insufficiency was confirmed by growth arrest (minimal or no weight gain) and / or the development of steatorrhea.

[0161] Example 6: Experimental Design, Procedures 6.1 Test Plan The study included three periods: adaptation, control, and test. During the 7-day adaptation period, pigs were trained to drink infant formula fortified with TG-DHA and TG-ARA. During the 7-day control period, pigs continued to receive infant formula fortified with TG-DHA and TG-ARA. During the 7-day test period, pigs received infant formula fortified with TG-DHA and TG-ARA that was either (a) unhydrolyzed, (b) prehydrolyzed with CV lipase, or (c) prehydrolyzed with RO lipase. Formula consumption was measured daily, fecal samples were collected for the last 3 days of each test period (72-hour collection), and blood samples were collected on day 7 of the control and test periods.

[0162] 6.2 Lipase Dosage The dosage of lipase and prehydrolysis time were determined based on the in vitro results (Example 3) and the daily nutritional requirements of pigs. NAN formula mixed with lipase RO or CV (~1300 U / g total fat) was incubated at 37°C for 15 min with shaking. Proposed formulation and lipase mixture: - Pig weight in the range of 11-14kg -Feed requirement: 40g of formula powder / kg body weight -Daily requirement: 500g powder / pig -Four meals per day -125g powder / pig / feed Diet preparation: 500 g powder + 1.5 L water (1:4 dilution) -Add the dry powder first -Add TG-PUFA oil (7.5 mL DHA and 15 mL ARA), mix thoroughly, add tap water from a 37° C. water bath and mix thoroughly. -For lipase treated formulas, mix in CV or RO lipase -Add water to final volume - Mix everything in a 37°C water bath for 15 minutes - Divide into 4 buckets and give each pig approximately 600mL of formula.

[0163] 6.2.1 Adaptation period (7-10 days) Approximately 7–10 days prior to the adaptation period, 12 pigs were placed in metabolic cages and trained to drink the TG-PUFA-enriched formula. On the first morning of the adaptation period, body weights were recorded before the morning meal.

[0164] 6.2.2 Control period (7 days) All selected pigs were fed infant formula as the sole dietary source, 4 times per day. Total daily formula consumption was measured during the entire experiment. Body weight was recorded on the morning of the first day of the control period, before the morning meal. 3 x 24 hour fecal samples were collected on days 5-7. Blood samples were collected on the last day of this period, 1 hour before the meal, and 1, 2, and 3 hours after the meal.

[0165] 6.2.3 Test period (7 days) All selected pigs were fed TG-PUFA enriched infant formula as the sole dietary source, 4 times per day. Total daily formula consumption was measured during the entire experiment. Body weight was recorded on the morning of the first day of the study period, before the morning meal. 3 x 24 hour fecal samples were collected on days 5-7. Blood samples were collected on the last day of the period, 1 hour before the meal, and 1, 2, and 3 hours after the meal.

[0166] Prior to the start of this period, pigs were randomly assigned to three groups based on body weight and willingness to drink formula: 1) One third of the EPI pigs (n=4) were fed formula prehydrolyzed with RO lipase. 2) One third of the EPI pigs (n=4) were fed formula prehydrolyzed with CV lipase. 3) One third of the EPI pigs (n=4) were fed formula alone The preparation of the formula and the lipase mixture is described above in paragraphs

[0201] (

[0157] ).

[0167] 6.3 Criteria for a positive response When compared to EPI pigs fed only formula supplemented with 2% TG-ARA and 1% TG-AA, there was a significant decrease in fecal LCPUFA, an increase in the coefficient of fat absorption (%CFA), and an increase in plasma LCPUFA concentrations. 6.4 Data analysis

[0168] Individual data were recorded at the time they occurred. Statistical analysis was performed using the Student's t-test. Differences were considered significant when p<0.05.

[0169] Example 7: RO and CV lipase improve fatty acid absorption in EPI pigs A well-established surgical model of exocrine pancreatic insufficiency (EPI) pigs was used to mimic preterm or term human infants with defective exocrine pancreatic function. The EPI surgical pig model was used essentially as described in Examples 5 and 6 to evaluate the effect of CV lipase or RO lipase prehydrolyzed infant formula on fatty acid absorption compared to non-hydrolyzed infant formula. The EPI pigs were 10 weeks old (+ / - 2 weeks), which corresponds to approximately 6 months of age for a human infant. Pigs were fed Nestle (NAN Pro 1 Gold) formula enriched with 2% ARA triglycerides (TG-ARA) and 1% DHA triglycerides (TG-DHA) derived from fish oil (NuCheck (http: / / www.nu-chekprep.com, ~40% TG-DHA and TG-ARA). Feeding was performed 4 times per day, every 3 hours. In the groups of pigs receiving prehydrolyzed formula, the formula was prehydrolyzed 15 min before feeding by mixing with CV lipase or RO lipase at 37°C. The duration of the experiment was 1 week, after which the fecal LC-PUFA concentration, the absorption of LC-PUFA in plasma, and the accumulation of LC-PUFA in tissues (retina, heart, liver, kidney, red blood cells, brain, and fat) were analyzed.

[0170] As shown in Figure 22A, EPI pigs fed formula prehydrolyzed with CV or RO lipase had significantly reduced stool weight (CV: >60% reduction, p<0.001; RO: ~30% reduction, p<0.05). Fat prehydrolysis with CV or RO lipase also significantly reduced total fat content in feces compared to control EPI pigs (Figure 22B) and significantly increased coefficient of fat absorption (%CFA) compared to controls (Figure 22C), where %CFA=(fat intake (g / 24 hr)-faecal fat (g / 24 hr)) / (fat intake (g / 24 hr)), n=3 / group, p=0.002 (CV vs. control), and p=0.003 (RO vs. control). When compared to controls, prehydrolysis with either CV or RO lipase caused a significant reduction in fecal ARA (36% and 65% reduction, respectively), EPA (78% reduction with both enzymes), and DHA (68% and 60% reduction, respectively) (Figure 23). These data indicate that prehydrolysis of formula with CV or RO lipase reduced fecal levels of total fat, ARA, DHA, and EPA, suggesting an improvement in the absorption of omega-3, omega-6, and total fatty acids.

[0171] In addition, pigs fed prehydrolyzed formulas had significantly increased plasma and tissue levels of ARA and DHA after 7 days of feeding compared to control pigs. For these studies, the control and CV lipase groups consisted of 4 pigs and the RO lipase group consisted of 3 pigs. Pre-meal plasma feeds were collected after 7 days of treatment and after an overnight fast. Plasma levels of ARA and DHA were significantly higher in pigs fed formulas prehydrolyzed with RO lipase compared to pigs fed nonhydrolyzed formulas (60% and 30%, respectively, p<0.05) (Figure 24). Plasma levels of ARA were also significantly higher in pigs fed formulas prehydrolyzed with CV lipase compared to pigs fed nonhydrolyzed formulas (40%, p<0.05) (Figure 24). ARA and DHA levels were also significantly increased (p<0.05) in the retina (Figure 25A) and adipose tissue (Figure 25B) of pigs fed formula prehydrolyzed with RO lipase compared to pigs fed nonhydrolyzed formula. Retinal levels of ARA were also significantly higher (p<0.05) in pigs fed formula prehydrolyzed with CV lipase compared to pigs fed nonhydrolyzed formula (Figure 25A). In pigs fed formula prehydrolyzed with CV or RO lipase, ARA levels were also significantly increased (p<0.05) in pig heart (Figure 26A, 60% and 20% increase, respectively) and kidney (Figure 26B) compared to pigs fed nonhydrolyzed formula. DHA levels were significantly increased (60%, p<0.05) in the heart of pigs fed formula prehydrolyzed with CV lipase compared to pigs fed skin hydrolyzed formula (Figure 26A). There were minimal or no changes in the liver, red blood cells, and brain, which may be explained by the relatively short duration of treatment in this study (7 days).

[0172] Example 8: Hydrolysis of TG-DHA and TG-ARA in infant formulas by immobilized lipase in "tea bags" Rhizopus oryzae (RO) lipase was covalently attached to acrylic beads and contained within a device resembling a teabag. Enfalac infant formula (25 g) was combined with tap water (88 mL) at 37°C. Reactions were carried out in glass bottles containing 100 mL of infant formula and teabags containing 100, 500, 1000, or 2000 mg of immobilized RO lipase. Each reaction was incubated at 37°C for 30 minutes with the bottle inverted. Samples were taken at 0, 1, 2, 3, 4, 5, 10, 20, and 30 minutes. Samples were analyzed for DHA and ARA by reversed-phase high-performance liquid chromatography (RP-HPLC).

[0173] At each concentration of immobilized RO lipase, the hydrolysis rates of DHA and ARA increased with increasing amounts of immobilized RO lipase (Figure 27). These data support the feasibility of the tea bag device for lipase prehydrolysis of milk formulas.

[0174] Example 9: Hydrolysis of TG-DHA and TG-ARA in infant formulas by immobilized lipase in cartridges Rhizopus oryzae (RO) lipase and Chromobacterium viscosum (CV) lipase were immobilized on macroporous acrylic polymer beads (Immobeads™; ChiralVision). Approximately 200 mg of RO lipase was used per gram of beads. Samples of the CV lipase-coated beads were irradiated (CVI) to determine the effect of irradiation on the efficacy of the immobilized lipase. Approximately 1.7 g of each bead preparation (RO, CV, and CVI) was loaded into a column with a bed volume of approximately 5 mL. Infant formula containing DHA and ARA triglycerides was passed over the column at a flow rate of 75 mL / hr. The column eluate was analyzed for hydrolysis of DHA and ARA by HPLC. The % hydrolysis of DHA and ARA triglycerides by CV, CVI, and RO lipases is shown in Table 6. [Table 6]

[0175] Example 10: RO Lipase vs. Pancreatin Rhizopus oryzae (RO) lipase showed greater activity towards DHA and ARA triglycerides than porcine pancreatin (Zenpep®), which contains a mixture of pancreatic lipase, protease, and amylase. 1.4 mL of infant formula was mixed with 100 uL of lipase (either pancreatin or RO lipase) and 100 uL of each of DHA and ARA triglycerides. The reactions were incubated at 37°C for 15 minutes. Samples were taken at 0, 1, 2, 4, 6, 8, 10, and 15 minutes and analyzed by RP-HPLC for DHA and ARA. DHA (Figure 28A) and ARA (Figure 28B) triglycerides were hydrolyzed over time by RO lipase but not by pancreatin.

[0176] Example 11: Six-week study: Long-term nutrition of pigs with formula prehydrolyzed with immobilized lipase Six healthy pigs and 20 pigs with surgically induced exocrine pancreatic insufficiency (EPI) (see Example 5) were subjected to a 2-week adaptation / control period followed by a 6-week test period (Figure 29). During the adaptation period, all pigs were fed NAN Pro 1 Gold infant formula (Nestle) ("IF"). During the test period, six of the healthy pigs ("Healty") and pigs with EPI ("EPI") were fed infant formula enriched with TG-LCPUFA (see Examples 5 and 6), and seven of the pigs with EPI were fed infant formula enriched with TG-LCPUFA that had been prehydrolyzed with immobilized RO enzyme using a "tea bag" device ("EPI+iRO"). The remaining pigs were removed from the study for various reasons, such as failure to surgically induce complete EPI.

[0177] For prehydrolysis, 2 liters of NAN Pro 1 Gold (Nestle) infant formula was prepared by mixing 1.5 liters of water at 37° C. with 500 g of powdered formula enriched with 50 mg / kg TG-DHA and 100 mg / kg TG-ARA (see Example 5, section 5.3). Five teabag-like devices containing RO lipase immobilized on beads (containing 1 gram of immobilized lipase in each "teabag") were added to the 2 liters of formula and mixed for 15 minutes at room temperature using a magnetic stirrer at constant mixing speed. This corresponds to 9,000 units (measured relative to olive oil) of immobilized RO lipase per 150 grams of total fat (60 U / g total fat) in the fortified formula. Before hydrolysis, the fortified formula contained 17.4 mmol / liter of non-esterified fatty acids. After hydrolysis, the formula contained 107.6 mmol / liter of non-esterified fatty acids.

[0178] Food consumption was measured daily. Blood and fecal samples were collected at the end of the adaptation period ("basal"), and after 1, 4, and 6 weeks of the treatment period. For the basal sample, feces were collected over a 48-hour period (2 x 24 hours). For the 1, 4, and 6 week samples, feces were collected over a 72-hour period (3 x 24 hours). At the completion of the treatment period, organs and tissues were collected for absorption and safety studies.

[0179] The prehydrolyzed formula was well tolerated with no treatment-related changes in feed intake, growth, organs (by gross examination), or general health. When the pigs were sacrificed at the end of the 6-week study, no evidence of fatty liver was seen by gross liver examination.

[0180] After 6 weeks, there was a statistically significant increase (20% and 36%, respectively) in red blood cell ARA (Figure 30A) and DHA (Figure 30B) levels in EPI pigs fed prehydrolyzed formula (EPI+iRO) when compared to EPI pigs fed non-prehydrolyzed TG-LCPUFA enriched formula (EPI). Red blood cell levels of ARA and DHA were not significantly different between healthy pigs and EPI pigs fed prehydrolyzed formula for 6 weeks.

[0181] There was a statistically significant increase in plasma levels of triglycerides, cholesterol, HDL, and LDL in EPI pigs fed prehydrolyzed formula for 6 weeks, as shown in Table 7. Plasma levels of triglycerides, cholesterol, HDL, and LDL were not significantly different between healthy pigs and EPI pigs fed prehydrolyzed formula for 6 weeks. [Table 7]

[0182] As shown in Table 8, pigs fed the prehydrolyzed formula for 6 weeks had increased plasma levels of vitamin A and vitamin E, but no significant differences were observed for vitamin D. There was a statistically significant difference in plasma levels of vitamin E between the EPI and EPI+iRO groups (p<0.05). There was a statistically significant difference in vitamin A between the EPI and healthy groups (p<0.05), but not between the EPI+iRO and healthy groups. [Table 8]

Claims

1. 1. A medical device for hydrolyzing a nutritional composition for consumption by a subject, the device comprising: one or more exterior walls forming an outer boundary of the device, the one or more exterior walls forming an enclosure and formed of a semi-permeable material; and an immobilized lipase contained within said enclosure; Including, the immobilized lipase is configured to hydrolyze fatty acid triglycerides within the nutritional composition; The medical device, wherein the semi-permeable material surrounds the immobilized lipase such that the immobilized lipase is contained inside the enclosure, and the nutritional composition can pass through the semi-permeable material.

2. The medical device of claim 1 , wherein the enclosure comprises a teabag-like structure.

3. The medical device of claim 1 , wherein the enclosure comprises a pocket.

4. The medical device of claim 1 , wherein the immobilized lipase is immobilized on at least one interior surface of the one or more exterior walls.

5. The medical device of claim 1 , wherein the immobilized lipase is immobilized in a structure contained within the enclosure.

6. The medical device of claim 5 , wherein the structures include at least one of particles, balls, or beads.

7. 10. The medical device of claim 1, wherein the immobilized lipase is immobilized by one or more of adsorption, ionic bonding, covalent bonding, cross-linking, encapsulation, or entrapment.

8. 2. The medical device of claim 1, wherein the immobilized lipase is selected from Chromobacterium viscosum, Pseudomonas Fluorescens, Burcholderia cepacia, or Rhizopus oryzae.

9. The medical device of claim 1 , comprising a plurality of enclosures.

10. The medical device of claim 1 , further comprising a rod-shaped extension having a distal end and a proximal end, the enclosure being connected to a distal region of the rod-shaped extension.

11. The medical device of claim 1 , wherein the one or more exterior walls form a plurality of pockets.

12. 1. A medical device for hydrolyzing a nutritional composition for consumption by a subject, the device comprising: a rod-shaped extension having a proximal end and a distal end; one or more walls forming one or more pockets at a distal region of the rod-shaped extension, the one or more walls being formed of a semi-permeable material; and a lipase immobilized within said one or more pockets; wherein said semi-permeable material surrounds said lipase such that said lipase is contained inside said one or more pockets, and wherein said nutritional composition can pass through said semi-permeable material.

13. The medical device of claim 12 , wherein the immobilized lipase is immobilized in a structure within the one or more pockets.

14. The medical device of claim 13 , wherein the structures include at least one of particles, balls, or beads.

15. The medical device of claim 12 , wherein a plurality of pockets are coupled to the distal region of the rod-shaped extension.

16. The medical device of claim 12 , wherein the immobilized lipase is configured to hydrolyze fatty acid triglycerides.

17. The medical device of claim 12 , wherein the one or more walls include a mesh that allows liquid to pass into and out of the one or more pockets.

18. 1. A method of preparing a nutritional composition for consumption by a subject, the method comprising:

2. Exposing a nutritional composition comprising triglycerides to the medical device of claim 1 to at least partially hydrolyze at least a portion of said triglycerides. A method comprising:

19. 20. The method of claim 18, wherein the nutritional composition is exposed to the medical device of claim 1 for 40 minutes or less.

20. The nutritional composition is intended to treat a range of conditions including Alzheimer's disease, bipolar disorder, depression, sepsis, acute respiratory stress, cancer, cardiovascular disease, stroke, Parkinson's disease, schizophrenia, diabetes, multiple sclerosis, malnutrition, GI dysfunction, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, short bowel syndrome, irritable bowel syndrome, systemic inflammatory response syndrome, celiac disease, Crohn's disease, cystic fibrosis, Zollinger-Ellison syndrome, hypertriglyceridemia, neoplasms, hemoglobinuria, and the like.

20. The method of claim 18, for provision to a subject having at least one of the following: gliomatosis, primary sclerosing cholangitis, primary biliary cirrhosis, diarrhea, Schwachmann's syndrome, trypsinogen deficiency, enterokinase deficiency, chylothorax, isolated lipase deficiency, premature birth, pancreatic insufficiency, pancreatitis, malabsorption, defective productive activity of the pancreas, reduced ability to hydrolyze triglycerides or esters, or reduced ability to absorb triglycerides or esters.

21. 1. A method of preparing a nutritional composition for consumption by a subject, the method comprising: inserting a medical device into a container containing a nutritional composition, the nutritional composition comprising a triglyceride; Including, The method, wherein the medical device comprises one or more walls forming an outer enclosure of the device, the one or more walls being formed of a semi-permeable material that is permeable to the nutritional composition, the device containing an immobilized lipase within the enclosure, the semi-permeable material surrounding the immobilized lipase such that the immobilized lipase is contained inside the outer enclosure.

22. agitating the nutritional composition.

22. The method of claim 21 further comprising:

23. 22. The method of claim 21, further comprising contacting said medical device with said nutritional composition for a period of time to allow said immobilized lipase to at least partially hydrolyze said triglycerides.

24. Removing the medical device from the nutritional composition.

22. The method of claim 21 further comprising:

25. 1. A medical device for hydrolyzing a nutritional composition for consumption by a subject, the device comprising: one or more exterior walls forming an outer boundary of the device, the one or more exterior walls forming a tea bag-like structure; and Lipase immobilized in the structure contained within the teabag-like structure wherein the immobilized lipase is configured to hydrolyze fatty acid triglycerides; The medical device, wherein the immobilized lipase is contained inside the teabag-like structure, and the nutritional composition can pass through the teabag-like structure.

26. 26. The medical device of claim 25, wherein the one or more exterior walls are semi-permeable.

27. 26. The medical device of claim 25, wherein the structures include at least one of particles, balls, or beads.

28. 26. The medical device of claim 25, wherein the immobilized lipase is immobilized by one or more of adsorption, ionic bonding, covalent bonding, cross-linking, encapsulation, or entrapment.

29. 26. The medical device of claim 25, wherein the immobilized lipase is selected from Chromobacterium viscosum, Pseudomonas Fluorescens, Burcholderia cepacia, or Rhizopus oryzae.

30. 26. The medical device of claim 25, wherein the one or more exterior walls comprise a mesh that allows liquid to pass into and out of the tea bag-like structure.

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