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

Abstract

To provide nutritional formulations comprising long-chain polyunsaturated fatty acids (LC-PUFAs), along with methods and devices for preparing and / or administering the nutritional formulations.SOLUTION: In some embodiments, a percentage of the LC-PUFAs in the nutritional formulation is in the form of monoglycerides and / or free fatty acids. In some embodiments, the nutritional formulations do not comprise added lipase. Also provided are methods for providing nutrition to a subject, methods for improving fat absorption, methods for improving cognitive ability, methods for preventing chronic lung disease, and methods for reducing the length of time a patient requires total parenteral nutrition.SELECTED DRAWING: Figure 7

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 hydrolyze the triglycerides, breaking 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 throughout the body for various physiological purposes (see D. Kasper et al., Harrison's Principles of Internal Medicine, 16th ed. (2004)). While medium-chain triglycerides can be absorbed through the intestinal lumen, 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, such as those suffering from defective pancreatic production, malabsorption, or pancreatic insufficiency, may be unable to properly break down long-chain triglycerides and may consequently suffer from inadequate fatty acid absorption to maintain health. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] D. Kasper et al., Harrison's Principles of Internal Medicine, 16th Edition (2004) [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 ability to break down long-chain triglycerides or who otherwise need to receive essential fatty acids. For example, most commercially available lipase supplements are derived 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; D. Kasper et al., Harrison's Principles of Internal Medicine, 16th Edition (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 are effective at hydrolyzing a given long-chain fatty acid to the same extent, 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 components may not be desirable or feasible. Furthermore, while many fatty acid-supplemented formulas may contain medium-chain triglycerides, there are clear medical benefits to consuming long-chain fatty acids from the diet. Therefore, improved methods for enhancing the hydrolysis of long-chain triglycerides are needed.

[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 considered "essential fatty acids," meaning humans cannot synthesize them and must obtain them from dietary sources. The primary dietary source of the n-3 LC-PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) is their precursor, the essential fatty acid alpha-linolenic acid (ALA). However, endogenous enzymes are highly inefficient at converting ALA to DHA and EPA. According to official publications by the International Society for the Study of Fatty Acids and Lipids (ISSFAL), the conversion of ALA to DHA is approximately 1% in infants and significantly 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 per se, dietary sources of DHA and EPA are important. The major dietary source of the n-6 LC-PUFA arachidonic acid (ARA or AA) is the essential fatty acid linoleic acid (LA). [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 formulations containing LC-PUFA triglycerides, LC-PUFA fatty acid esters, and / or other long-chain triglycerides or long-chain fatty acid esters are transiently 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 requiring 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 formulations are formulated for administration to premature infants. Other nutritional formulations encompassed herein 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 under 1 year of age. In some embodiments, the subject is 1 to 4 years old. In some embodiments, the subject is 1 to 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 a 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 "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 added 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 formulations of the present invention immediately prior to ingestion by a subject. In certain embodiments, the method comprises 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 needing 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, or 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 the solid support by covalent, ionic, or cross-linking bonding. In certain embodiments, the immobilized lipase is encapsulated within or attached to a permeable membrane.

[0013] Another aspect of the present 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 a formulation of the present invention to a subject in need of dietary LC-PUFAs and / or other long-chain fatty acids, such as a subject with a reduced ability to break down long-chain triglycerides or long-chain fatty acid esters in the intestinal tract, a subject suffering from pancreatic insufficiency, a subject suffering from malnutrition, and a subject receiving total parenteral nutrition. In some embodiments, the subject is a premature infant. In other embodiments, the subject is a full-term infant or 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 suffers from pancreatic insufficiency. In other embodiments, the formulation is administered via a feeding tube. In some embodiments, the nutritional formulation of the present invention is administered to improve cognitive performance in individuals of any age, 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 reduces the risk of intestinal atrophy and other complications associated with prolonged (more than 24 hours) total parenteral nutrition in such patients. Such a method can be used, for example, to shorten recovery times in 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 delivery method can be advantageous in situations where the patient suffers from altered intestinal 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 providing the subject with 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 includes a chamber containing at least one lipase, and the chamber can hold a liquid nutritional formulation so that the liquid nutritional composition is exposed 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 includes a chamber consisting of a permeable membrane containing the immobilized lipase, 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 more than 5% of the LC-PUFAs are in the form of monoglycerides and / or free fatty acids. (Item 2) 2. The nutritional formulation of item 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 item 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 item 2, wherein the LC-PUFA comprises ARA. (Item 6) 3. The nutritional formulation of item 2, wherein the LC-PUFA comprises DHA and EPA. (Item 7) 3. The nutritional formulation of item 2, wherein the LC-PUFA comprises DHA and ARA. (Item 8) 3. The nutritional formulation of item 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 items 1 to 9, 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 items 1 to 9, wherein the nutritional formulation does not comprise 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 the 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 the EPA is in the form of monoglycerides and / or free fatty acids. (Item 17) 17. The nutritional formulation of 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 derived from Chromobacterium viscosum viscosum lipase, Pseudomonas fluorescens lipase, and Rhizopus oryzae lipase oryzae lipase. (Item 20) 20. The method of 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 no more than 60 minutes before 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 a lipase, thereby exposing the liquid composition comprising LC-PUFA triglycerides and / or LC-PUFA esters to the 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 the step of removing the lipase from the liquid composition after the step of exposing the LC-PUFA triglycerides and / or LC-PUFA esters to the lipase. (Item 25) The lipase (a) exposing the lipase in the liquid composition to a molecule that binds to the lipase, wherein the molecule is 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) Item 25. The method of item 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 inner 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) Item 27. The method of item 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, wherein 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 before 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 before ingestion. (Item 36) A nutritional formulation prepared by the method according to any one of items 18 to 35. (Item 37) 10. A method of providing nutrition to a subject, comprising the step of supplying to the 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 subject with the nutritional formulation according to any one of items 1 to 17 and 36. (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 of any one of Items 39 to 44, wherein the subject has pancreatic insufficiency, a defect in the production 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 of 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) 10. A method for improving cognitive performance in a subject over 50 years of age, comprising feeding said subject the nutritional formulation according to any one of items 1 to 17 and item 36. (Item 51) 10. 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 first and second compartments, said first compartment containing a nutritional formulation and said second compartment containing a lipase. (Item 53) The lipase is derived from Chromobacterium viscosum viscosum lipase, Pseudomonas fluorescens lipase, and Rhizopus oryzae lipase oryzae lipase. (Item 54) 54. The container according to item 52 or item 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 container. (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 device 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 particles 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 in 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 a covalent bond, an ionic bond, or encapsulation 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 device of any one of items 70 to 72, wherein the surface comprises protrusions 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 in a material that is permeable to fatty acids but impermeable to the lipase. (Item 75) 1. 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 derived from Chromobacterium viscosum viscosum lipase, Pseudomonas fluorescens lipase, and Rhizopus oryzae lipase oryzae lipase. (Item 77) 76. The apparatus of claim 75, comprising a container, wherein the lipase is immobilized on at least a portion of an inner surface of the container. (Item 78) 76. The apparatus of claim 75, comprising a container, wherein the lipase is immobilized on a solid substrate contained within the container. (Item 79) 79. The device according to claim 77 or 78, wherein the lipase is immobilized by covalent or cross-linking bonding. (Item 80) 80. The device of any one of items 75 to 79, further comprising a feeding tube attached to the container. (Item 81) 37. A method for reducing the length of time a patient requires total parenteral nutrition, the method comprising enterally administering the nutritional formulation of any one of items 1 to 17 and 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 the 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 the method does not significantly increase the 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 item 49, wherein the subject is under 1 year old. (Item 87) Item 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 providing the subject with the nutritional formulation of any one of items 1 to 17 and 36. [Brief explanation 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 nutritional formulations, according to certain embodiments. [Figure 6C] 1 illustrates an apparatus for processing and administering nutritional formulations, according to certain embodiments. [Figure 6D] A close-up of the device illustrated in Figure 6C is shown. [Figure 7] 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] 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] 1 illustrates a partial cross-sectional view of an apparatus for processing a formulation, according to certain embodiments. [Figure 20] 1 illustrates a partial cross-sectional view of an apparatus for processing a formulation, according to certain embodiments. [Figure 21] 1 illustrates a partial cross-sectional view of an apparatus for processing a formulation, according to certain embodiments. [Figure 22] (Figure 22A) Fecal weight in EPI pigs during the control week ("Cont week," during which pigs were fed TG-LCPUFA-enriched infant formula) compared to 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 three days of the treatment week in the same three groups of pigs. (Figure 22C) Coefficient of fat absorption (%CFA) is shown. [Figure 23]Figure 1 shows the 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 the levels of ARA (A) and DHA (B) in the 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. [Figure 25] 1 shows the levels of ARA and DHA in the 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] 1 shows the 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] Figure 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] 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 pig study described in Example 10. [Figure 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 that had been prehydrolyzed with immobilized RO lipase ("EPI+iRO"). DETAILED DESCRIPTION OF THE INVENTION

[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" because humans cannot synthesize them but must obtain them through 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 through 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 predominant 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 component of junctional membranes, 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 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 early third trimester, during the first two years of life. In rodents and primates, inadequate supply of n-3 PUFAs 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 with 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 maintaining 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 increased 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. Furthermore, in a transgenic model of AD that produces 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-scale study in healthy elderly individuals with mild memory complaints showed that subjects administered DHA performed better on learning and memory tests after 6 months than subjects receiving 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 investigated in patients with BP and MDD. For a review of the effects of DHA in BP, see V. Balencia-Martinez et al., Expert. Rev. Neurother. 11(7):1029-1047 (2011). Due to the difficulty of assessing DHA levels in brain tissue from human patients, the fatty acid composition of erythrocyte membranes obtained 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 with normal controls (see R. McNamara et al., Psychiatry Res. 160(3):285-299 (2008)). Furthermore, in a 4-month, double-blind, placebo-controlled study, BP patients who took n-3 fatty acids had a significantly 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 patients with depression have reduced n-3 levels in their blood. Similarly, increased plasma DHA has been associated with a reduction in the number of women reporting symptoms of postpartum depression. Several placebo-controlled trials have found that n-3 treatment improves depressive systems. 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 decreased 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] n-3 fatty acids have also been reported to have beneficial effects on wound healing: through altering the lipid microenvironment, n-3 fatty acids can also help 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 downregulating 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 organizations, have issued recommendations for increasing 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 a stabilizing effect on the myocardium itself.

[0030] The benefits of n-3 fatty acids in several pathological conditions may be due to their broad anti-inflammatory effects. EPA and DHA generate resolvins, anti-inflammatory regulators with inflammation-resolving and immunomodulatory functions. For example, EPA and DHA exert inhibitory effects on leukocyte chemotaxis and alter the production of inflammatory cytokines through reducing NF-κB activation in immune cells (see P. Calder, Int. Rev. Immunol. 28:506-534 (2009)). In general, n-3 PUFAs have been associated with reduced proinflammatory T cell responses. Increased n-3 fatty acids in animal diets 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 distribution 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)). Furthermore, 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 (see 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 crucial role in neurodevelopment and brain function, with ARA accumulation in the brain during prenatal and postnatal development (see B. Koletzo et al., J. Perinat. Med. 36(1):5-14 (2008)). n-6 fatty acids are generally important for normal development and immunity, and also stimulate skin and hair growth, maintain bone health, regulate metabolism, and maintain the reproductive system.

[0032] Long-chain PUFA nutritional supplement For the past 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. Dietary 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 an infant food in special diets because it simulates breast milk or is suitable as a 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 an average of 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 closely reflects maternal DHA intake.

[0034] Commercially available TG-LCPUFA supplemented infant formulas include Enfamil formulas such as Enfamil LIPIL® and Enfamil PREMIUM®, Baboo, Earth's Best Organic, and Nestlé Gerber These include Nestle formulas such as 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 may be cow's milk or soy-based and may 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 requiring nutritional or dietary supplementation with long-chain fatty acids. Such products include ENSURE®, PEDIASURE®, CARNATION®, BOOST®, CERELAC®, and SOUVENAID®. Additionally, 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 requiring tube feeding. For example, small-enteral formulas can be commonly used in patients with preterm infants, renal failure, gastrointestinal disorders or conditions resulting in GI dysfunction, intestinal resection, fat malabsorption, malnutrition, pancreatitis, hyperglycemia / diabetes, liver failure, acute and chronic pulmonary disease, or immunocompromised states. For a review of commercially available small-enteral formulas, 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 1.5®, and others. 2.0®, and TWOCAL HN®. Basic formulations may contain macronutrients, including polymeric and hydrolyzed formulations, and may be fiber-fortified. Disease-specific formulations include, for example, renal preparations such as MAGNACAL RENAL®, NEPRO®, NOVASOURCE RENAL®, SUPLENA®, and NUTRI-RENAL®.

[0036] Gastrointestinal (GI) formulas can be used for the 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 formulas are also used for early postoperative nutritional support, nutritional delivery, total parenteral nutrition (TPN) replacement, and dual nutritional support with TPN. Examples of GI formulas include PEPTAMEN® (which consists of 70% medium-chain triglycerides and 30% long-chain triglycerides to reduce the potential for fat malabsorption), VIVONEX PLUS®, and VIVONEX PEDIATRIC®.

[0037] However, in individuals with impaired ability to hydrolyze long-chain triglycerides or long-chain fatty acid esters, such as those 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 for utilizing 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, this method is particularly effective in providing long chain monoglycerides and / or free fatty acids produced from DHA, EPA, and ARA triglycerides or 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 a condition that leads to a decreased ability 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 begins at low levels around 30 weeks of gestation and remains low throughout the first year of life. Therefore, infants, especially preterm infants, may experience pancreatic insufficiency. Consequently, if they are not breastfed, these infants are prone to poor fatty acid hydrolysis and absorption and miss out on 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 reductions in the ability to hydrolyze LC-PUFA triglycerides or esterified LC-PUFAs can also result from disease or trauma. For example, pancreatitis is an inflammatory condition in the pancreas that 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, particularly 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 part of the stomach or pancreas is 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 syndrome, trypsinogen deficiency, enterokinase deficiency, or isolated lipase deficiency (see D. Kasper et al., Harrison's Principles of Internal Medicine, 16th ed. (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 (especially GI trauma or surgery), radiation enteritis, intestinal failure, chylothorax, cancer (especially 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 persistent fat malabsorption (see Kalivianakis, American Journal of Clinical Nutrition 69:127-134 (1999)). In some embodiments, the present invention provides formulations and methods for improving the absorption of fats, such as LC-PUFAs, in CF patients. In some embodiments, the present invention provides formulations for inducing weight gain in CF patients.

[0043] While cachexia and weight loss due to tissue catabolic state, nutrient redirection, and malabsorption are common in advanced stages of many cancers, pancreatic cancer (PC) is exceptional, with weight loss and malabsorption occurring in 80% to 90% of patients at the time of diagnosis. Malabsorption, resulting from exocrine defects that contribute significantly to weight loss, is due to loss of pancreatic parenchyma, blockage of the pancreatic duct that prevents 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 patients with PC.

[0044] Some embodiments of the present invention improve upon current treatment options for pancreatic insufficiency and other conditions that result in a reduced ability to hydrolyze TG-LCPUFAs, esterified LC-PUFAs, and / or other long-chain triglycerides and fatty acid esters. In patients with a reduced ability to hydrolyze TG-LCPUFAs, esterified LC-PUFAs, and / or other long-chain triglycerides and fatty acid esters, simply increasing 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, as 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 added lipase, may 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 (DHA and EPA):ARA ratio. In particular, cystic fibrosis patients may benefit from increasing the (DHA and EPA):ARA ratio in their plasma. However, currently available adult formulas generally have a low ratio of n-3:n-6 fatty acids. Furthermore, in subjects with impaired TG-LCPUFA hydrolysis, simply increasing the consumption of n-3 TG-LCPUFA is unlikely to significantly improve the subject's (DHA and EPA):ARA ratio, 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 (DHA and EPA):ARA ratio in subjects, particularly adult subjects. For example, some embodiments provide methods 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 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 acids and / or monoglycerides is higher than the ratio of n-3:n-6 fatty acids found in the plasma of a subject, for example, a formulation in which the ratio of free DHA and EPA to free ARA is higher than that found 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] Decreased 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 the cell membranes of the brain and retina, 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, as well as 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 an inadequate supply of fatty acids due to shortened gestational age, followed by reliance on external sources of 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 as a result, 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)). Additionally, 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 remains a significant and potentially unmet need, even with formula 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 who are not supplemented with fatty acids may also be deficient in long-chain PUFAs. Long-chain PUFA levels have been shown to be lower in unsupplemented formula-fed infants compared with breast-fed infants (see B. Koletzo et al., J. Perinat. Med. 36(1):5-14 (2008)). Even breast-fed infants may be deficient in n-3 fatty acids because the amount of DHA in breast milk varies and is related to 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 specialized infant formulas containing fats containing DHA and ARA. However, reports regarding 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 with infants fed the same formula without lipids (see E. Birch et al., J. Pediatr. 156(6):902-906 (2010)). However, overall, preclinical data do not demonstrate consistent benefits for infant development from current long-chain PUFA-supplemented formulas.

[0050] One explanation for the contradictory results of these studies is that some infants are unable to absorb the necessary amounts of important fatty acids from the intestine when fed 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 do not have sufficient levels of lipase to break down long-chain PUFAs or PUFA esters into monoglycerides and / or free fatty acids for absorption by the intestine. As a result, infants fed LC-PUFA-supplemented formula still absorb less LC-PUFA than breast-fed infants. Again, there is a clear need to enable the hydrolysis and absorption of these fatty acids rather than simply providing a fatty acid supplement.

[0051] Adding lipase to a regulated infant formula (or, for example, a medical nutritional formula) can require significant development research to screen, stabilize, and formulate a suitable lipase supplement. In untested, unadjusted formulas, issues related to lipase stability, lack of specificity, purity, and / or interference with other substances can result in excessive or potentially harmful levels of the enzyme. The massive addition of a new substance that overcomes regulatory hurdles further introduces another variable: how well the formula will be tolerated by individuals, particularly infants, who have a low capacity to hydrolyze long-chain triglycerides. This problem exists, 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] Embodiments of the present invention solve these various problems by providing an as-fed nutritional formula 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, formula-fed recipients can receive the benefits of DHA, EPA, and ARA. In some embodiments, the nutritional formula does not introduce any new ingredients beyond the pre-hydrolyzed fats present in the existing formula. In certain embodiments, formula-fed infants receive the benefits of the fatty acids that breast-fed infants obtain without being exposed to lipase supplements. In other embodiments, the nutritional formulas of the present invention contain a highly specific lipase that allows for the use of minimal amounts of lipase added to 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 ingests the nutritional formulation for 3 days, 5 days, 7 days, 10 days, 14 days, 30 days, 60 days, or more. In some embodiments, such ingestion of the nutritional formulation of the present invention can reduce total fecal fat, specifically reducing the levels of DHA, ARA, and / or EPA in the feces. In some embodiments, this reduction is measured relative to the fecal composition of the subject before ingestion of the nutritional formulation. In some embodiments, this reduction is measured relative to the fecal composition of a subject fed a nutritional formulation that is not exposed to lipase, such as a currently available nutritional formulation, before ingestion. The levels of total fecal fat, DHA, ARA, and / or EPA 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 level of total fat in the stool is reduced by at least 50%. In some embodiments, the level of at least one LC-PUFA (such as DHA, ARA, or EPA) in the stool is reduced by at least 50%. In some embodiments, the level of at least one LC-PUFA (such as DHA, ARA, or EPA) in the stool is 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 this nutritional formulation improves plasma, red blood cell, and tissue accumulation of fat levels, including DHA and ARA levels. Tissues can include retinal, cardiac, adipose, and kidney tissue. In some embodiments, ingestion of the nutritional formula increases the 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 this nutritional formulation increases plasma levels of vitamin A and / or vitamin E. Without wishing 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 improves 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 formula has beneficial effects without significantly increasing the accumulation of fat in the liver. Fatty liver disease (FLD) is characterized by increased accumulation of fat, especially triglycerides, in hepatocytes. This condition is also associated with other diseases that affect fat metabolism. It is normal for the liver to contain some fat 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, 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; excessive calorie intake overwhelms the liver's ability to metabolize fat normally, leading to fat accumulation in the liver. Many medications, including tamoxifen, amiodarone injection, oral amiodarone, and methotrexate, are associated with FLD. Fatty liver is also associated with type II diabetes, obesity, high blood triglyceride levels, celiac disease, Wilson's disease (a disorder of copper metabolism), rapid weight loss, and malnutrition.

[0057] Lipase Pancreatic insufficiency and other conditions associated with a reduced ability to hydrolyze long-chain triglycerides or long-chain fatty acid esters are currently treated with supplemental digestive enzymes, including pancreatic lipase. However, pancreatic enzymes, particularly those present in these supplements, are often susceptible to the degradative effects of gastric acid and pepsin, so only a small fraction of the ingested enzyme reaches the duodenum in an active form (see E. Ville et al., Digestion 65:73-81 (2001)). However, many acid-protective coatings pose potential safety concerns for infants or immunocompromised patients, as a significant portion of the delivered weight is plastic coating. Furthermore, even when acid-protective coatings are effective, 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 the expected increase in pH is not achieved and the protective coating does not dissolve properly to release the enzyme (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 fibrosing colon disease. Accordingly, some embodiments of the present invention provide nutritional formulations that contain a higher content of long-chain monoglycerides and / or free fatty acids without added lipase. Some embodiments provide nutritional formulations that contain 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., lipase produced by 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-coated tablets. However, microbial enzymes are susceptible to degradation by trypsin in the small intestine, thereby reducing their availability for breaking down triglycerides and esters in the intestinal tract. In certain embodiments, the lipase used in the formulations, methods, or devices of the present invention is bacterial lipase, fungal lipase, or both.

[0059] The specificity and kinetics of individual lipases can vary greatly. 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 against Candida rugosa, Rhizomucor miehei, Penicillium camemberti, Aspergillus niger, and others. Lipases 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. Consequently, lipase supplements or lipase-supplemented nutritional products containing Chromobacterium viscosum, Pseudomonas fluorescens, Burcholderia cepacia, and / or Rhizopus oryzae may provide increased hydrolysis of TG-DHA, TG-EPA, and / or TG-ARA. Thus, one aspect of the present invention provides a lipase nutritional supplement or lipase-supplemented nutritional product containing 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 be prepared directly from the wild-type host cell; for example, the same lipase can be produced recombinantly 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 a Chromobacterium viscosum lipase. viscosum lipase, Pseudomonas fluorescens lipase, or Rhizopus oryzae lipase oryzae lipase. An additional aspect of the present invention provides a lipase having a 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 lipase, as determined by reverse-phase high-performance liquid chromatography (RP-HPLC), as described in Example 1. In some embodiments, the lipase has a 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 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 particular embodiments, 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 the lipase. In some embodiments, the lipase is covalently bound to the solid support, although non-covalent binding may also be used. Suitable methods for 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. Murty et al., Biotechnol. Bioprocess Eng. 7:57-66 (2002). The lipase may be immobilized by directly binding to the support material or by binding through a linker. See, for example, Stark and Holmberg, Biotechnol. and Bioeng. 34(7):942-950 (1989).

[0064] Adsorption immobilization is reversible and typically involves hydrophobic interactions. It is simple and inexpensive, but suffers from the drawbacks of 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, polymer support); and H. Gunnlaughsdottir et al., Enzyme and Microbiol. Tech. 22:360-367 (1998) (Humicola lanuginosa 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 the lipase and often results in immobilized lipase with high activity. However, it should be understood that the binding force between the enzyme and the support, although stronger than when using adsorption, is not as strong as covalent binding, and therefore leakage of lipase from the support may occur.

[0066] Covalent binding is based on a covalent bond between the support material and 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, which are not essential for the catalytic activity of the lipase. To protect the active site, immobilization can be performed in the presence of a substrate or competitive inhibitor. A major advantage of using covalent binding of lipase to the support material is the strength of the bond, i.e., the stability of the immobilization. For an example of covalent lipase immobilization, 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 using a cross-linking agent to bind the lipase to a solid structure. 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 typically involves forming a porous coating or semipermeable 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 also 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 lipases 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 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 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 ( The lipase is one or more selected from the group consisting of lipases from Pseudomonas viscosum, Pseudomonas fluorescens, Burcholderia cepacia, and Rhizopus oryzae.

[0071] In certain embodiments, lipase (whether 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] Device containing immobilized lipase According to various embodiments, the present disclosure provides devices and methods for preparing nutritional products. The devices and methods can be used to expose infant formula or other nutritional products to lipase prior to consumption. Thus, lipase breaks down fats and oils with the subsequent release of free fatty acids and monoglycerides. The devices and methods may allow for a convenient means for preparing formula or other nutritional products. In some embodiments, the devices and methods allow infants or others consuming the product to avoid consuming exogenous lipase. In some embodiments, the devices and methods allow for the production of formulations that contain monoglycerides and / or free fatty acids but do not contain any significant amounts 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, device 100 of the present disclosure can include a container 110 configured to hold infant formula 120 or other liquid nutritional product. As described in detail below, container 110 can include immobilized lipase such that formula 120 delivered to an 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, lipase can be immobilized on or within structures found along the wall or otherwise immobilized within the container such that the lipase is in fluid contact with 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 treatment 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 formula 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 others with a 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 devices allow for the exposure of formula to lipase for desired enzymatic action while allowing the formula to be conveniently consumed without subsequently consuming the lipase. Thus, in various embodiments, the lipase is immobilized within the container 210, 310 so that it remains within the container 210, 310 when the formula is removed (e.g., through a nasogastric tube, a bottle nipple, or by transferring the formula to another container), or so that it can be removed from the formula 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 the formula remains intact within the container for later consumption.

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

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

[0079] As previously described, lipase can 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 can have a variety of shapes. For example, in certain embodiments, the structures may comprise beads, balls, or any other structure that can be mobile within the container such that the structure flows within the formula. For example, as shown in FIG. 4A, structure 250 can include a bead or ball having surface walls 256 to which lipase 257 can be bound. Furthermore, it should be understood that structures 250, 252 can have a variety of different shapes or configurations (e.g., cube-like, ovoid, rod-like).

[0080] The configuration of structures 250, 252 and / or the walls of 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, 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, if a longer time is available to incubate the formulation with lipase and / or if a lipase with a high enzymatic activity is used, a lower amount of lipase may be preferred.

[0081] In various embodiments, the structures 250, 252 and / or the container are configured so that lipase is not retained within the formula 260 upon removal 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 in addition, 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. Furthermore, 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 structures within container 210 and / or the container walls, lipase 257' is contained within structures 250, 252 and / or container walls 251. Figure 4B illustrates one such embodiment. As shown, structure 250' may comprise a bead or other shape having wall 256'. This wall may be formed from a semi-permeable material that permits the inflow and outflow of formula 260 but not the inflow and outflow of lipase 257'. Such encapsulation may similarly be used for other structures (e.g., 252) and / or container walls, such that the surface of the wall comprises a semi-permeable material within which 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. Furthermore, 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, e.g., element 150 in FIG. 1 and element 2101 in FIG. 21.

[0084] In certain embodiments, a container may be manufactured and pre-packaged with a lipase according to any of the embodiments described herein. At the time of use, the container can be opened, and the formula placed in the container and allowed to contact 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 their surface or housed / encapsulated therein can be packaged and distributed, and these structures can be placed in a separate container containing the formula. In some embodiments, it may be effective to shake or agitate the container containing the immobilized lipase and formula for a period of time.

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

[0086] 3A-3C illustrate devices 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 screw-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 containing lipase immobilized on its surface and / or encapsulated therein.

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

[0088] Alternatively, or in addition, 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 structure 351' has a permeable exterior wall surrounding a number of beads 250. The exterior wall may include a mesh or other configuration that allows for easy movement of the formulation toward and away from structure 351' to provide contact with beads 250. Furthermore, as described in various embodiments above, the beads can provide for lipase, which can be immobilized on the surface of the beads or encapsulated within them.

[0089] As discussed above, this lipase-containing structure can be manufactured and distributed as a pre-packaged 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 containing a rod-like structure 350 or beads 351, 351′, or other material having lipase housed therein or immobilized thereon, may be manufactured and distributed. 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 can be provided so that it 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 can include a standard baby bottle nipple, and the lipase can be immobilized around the rim of the nipple or on the inner surface 510 of the nipple itself. In this way, the formula will come into contact with the lipase during normal use. Similarly, the lipase can be contained within or on other structures that may be used for delivery, such as the fluid tubing 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 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 can 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 can 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 can be injected into or poured from the top 530 of the housing, allowing the formula to contact the lipase while filling or emptying the container 310. The top 530 and / or any other portion of the housing 520 can be formed from a variety of materials. For example, the housing 520 can be formed from a membrane that allows 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, and 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 the nutritional formulation to another container (e.g., by transferring to another container and emptying the lipase compartment and the nutritional formulation compartment). In some embodiments, a liquid is added to the other container before or after transferring the lipase and the 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, and 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 can 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 exterior walls 901, 901″ surrounding a lipase. As noted above, 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 configuration, number of pockets or openings, and amount of lipase and / or volume of the device can be varied depending on the intended use and / or to control lipase reaction rate.

[0098] In certain embodiments, the device can 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 device walls 1003 can expand to create 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 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, devices 1100, 1100', 1100", 1200, 1300, 1400, and 1500 can include one or more pockets or openings 1101, 1201, 1301, 1401, and 1501 arranged in various configurations near the distal regions of the wand-like extensions 1102, 1202, 1302, 1402, and 1502. In some embodiments, the orientation of the pocket or opening 1301 can be adjusted, for example, to allow insertion into narrow openings and / or minimize storage space during use, as shown in Figures 13A-13B.

[0100] In various embodiments, the lipase can be attached to a portion of a cap or closure of a bottle or jar so that when the cap or closure is placed on the bottle or jar, the lipase can contact a fluid contained within the bottle or jar. For example, any of the devices shown herein can be attached to a surface of a cap or closure for contacting a formula container within the 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 can be contained within a pocket or opening 1601, 1705, 1805, 1901, 2001 having various shapes or configurations. Additionally, in some embodiments, the cap or closure member 1902, 2002 may include an opening 1910, 2010 for inserting or removing fluid from the container, and such opening 1910, 2010 may 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. Device 2100 can include a pocket or opening 2101 containing lipase, which can be immobilized as described above. Furthermore, pocket or opening 2101 can have a tortuous or curved flow path to allow for longer contact time between the lipase and the formula. Additionally, device 2100 can include openings 2110 at both ends to allow for connection to tubing or conduits for formula inflow or outflow.

[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 so 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, which may include a mechanism for releasing the immobilized lipase into the container. For example, the lipase may be contained on or within beads or other structures (see, e.g., element 1805 in FIG. 18 ) that are attached to or contained within the cap, allowing the lipase to be dripped into the container as needed (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, immobilized on beads or other material, allowing the lipase to contact the formula only when desired (e.g., by releasing the lipase into the container or removing a barrier covering the lipase).

[0104] Figure 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 present invention, such as the devices shown in Figures 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 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 the internal channel or chamber of the device so that the formula is exposed to the lipase as it passes through the chamber. Lipase in such devices can be used for continuous delivery over long periods of time due to the increased stability and reusability of the lipase.

[0105] Nutritional Formulas Certain embodiments of the present invention provide nutritional formulations. 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 a subject, and differs in composition from formulations sold by manufacturers. The term "nutritional formulation" does not encompass the composition in a subject's body after ingestion.

[0106] In some embodiments, the nutritional formulation includes a long-chain fatty acid. In some embodiments, the nutritional formulation includes one or more LC-PUFAs, such as DHA, ARA, and EPA. In some embodiments, the nutritional formulation includes DHA. In some embodiments, the nutritional formulation includes ARA. In some embodiments, the nutritional formulation includes DHA and ARA. In some embodiments, the nutritional formulation includes 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, greater 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, greater 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 this EPA is in the form of monoglyceride and / or free fatty acid.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 this DHA and ARA are in the form of monoglyceride and / or free fatty acid.In certain embodiments, more than 90% of this DHA and ARA are in the form of monoglyceride and / or free fatty acid.In certain embodiments, more than 95% of this DHA and ARA are in the form of monoglyceride and / or free fatty acid.

[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 are in the form of monoglycerides and / or free fatty acids, and in certain embodiments, at least 95% of both the DHA and ARA are in the form of monoglycerides and / or free fatty acids.

[0110] In some embodiments of the present invention, approximate serving sizes of the nutritional formulas of the present 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 the production of formulas 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 obtainable 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, a dose of the nutritional formulation of the present invention contains 200-300 mg of LC-PUFA free fatty acids and / or monoglycerides. In some embodiments, a dose of the nutritional formulation of the present invention contains 250-500 mg of LC-PUFA free fatty acids and / or monoglycerides. In some embodiments, a dose of the nutritional formulation of the present invention contains 500-1000 mg of LC-PUFA free fatty acids and / or monoglycerides. In some embodiments, a dose of the nutritional formulation of the present invention contains 1-2 g of LC-PUFA free fatty acids and / or monoglycerides. In some embodiments, a dose 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, carbohydrates, 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-PUFAs, 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 formulations of the present invention do not contain added lipase. In some embodiments, the devices and / or methods of the present invention are 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 Chromobacterium viscosum lipase. In some embodiments, the lipase is Pseudomonas fluorescens lipase. In some embodiments, the lipase is Rhizopus oryzae lipase.

[0114] In some embodiments, a serving of nutritional formula 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 nutritional formula contains less than 3,000 units of lipase. In some embodiments, a serving of nutritional formula contains less than 1,000 units of lipase. In specific embodiments, the formula 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 0.01 mg to 1 g of lipase per gram of total fat (whether in the form of free fatty acids, monoglycerides, esters, or triglycerides) in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.1 to 500 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.1 to 250 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.1 to 200 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.1 to 150 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.1 to 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 to 10 mg of lipase per milligram of total LC-PUFAs (whether in free fatty acid, monoglyceride, ester, or triglyceride form) in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001 to 5 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001 to 3 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001 to 1 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001 to 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-PUFAs in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001-0.05 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.01-0.1 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.02-0.08 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.04-0.06 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.1 mg or less of lipase per milligram of total LC-PUFAs 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 to 10 mg of lipase per milligram of total EPA (whether in free fatty acid, monoglyceride, ester, or triglyceride form) in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001 to 5 mg of lipase per milligram of total EPA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001 to 3 mg of lipase per milligram of total EPA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001 to 1 mg of lipase per milligram of total EPA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001 to 0.5 mg of lipase per milligram of total EPA in the nutritional formulation. In some embodiments, the nutritional formulation contains 0.001 to 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 for 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 beverage for adults (a complete nutritional beverage, e.g., ENSURE, PEDIASURE, etc.).

[0122] In some embodiments, the method for preparing a nutritional formulation comprises exposing a liquid nutritional composition to a lipase. In some embodiments, the liquid nutritional composition comprises an LC-PUFA triglyceride or an LC-PUFA ester. In some embodiments, the liquid nutritional composition comprises a triglyceride or 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 components involved in these methods may be mixed in various orders. In some embodiments, lipase is added to a liquid nutritional composition, thereby exposing 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, lipase is present in the solid or powder form of the nutritional composition prior to the addition of the potable liquid. In other embodiments, 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-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, hi 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 purposes of this use, exposing 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 purposes of this use, exposure to lipase ends when the formulation is ingested by a subject or when the 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-PUFAs in the resulting nutritional formulation are 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 having a lipase-binding molecule immobilized thereon, 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 a chamber or 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 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 comprises 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 specific 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 methods of the present invention expose the nutritional formulation to 0.01 mg to 1 g of lipase per gram of total fat (whether in the form of free fatty acids, monoglycerides, esters, or triglycerides) in the nutritional formulation. In some embodiments, the methods of the present invention expose the nutritional formulation to 0.1 to 500 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the methods of the present invention expose the nutritional formulation to 0.1 to 250 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the methods of the present invention expose the nutritional formulation to 0.1 to 200 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the methods of the present invention expose the nutritional formulation to 0.1 to 150 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the methods of the present invention expose the nutritional formulation to 0.1 to 100 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the methods of the present invention expose the nutritional formulation to 0.1 to 50 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the methods of the present invention expose the nutritional formulation to 1 to 50 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the methods of the present invention expose the nutritional formulation to 25 to 75 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the methods of the present invention expose the nutritional formulation to 1 to 100 mg of lipase per gram of total fat in the nutritional formulation. In some embodiments, the methods of the present invention expose 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 to 10 mg of lipase per milligram of total LC-PUFAs (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 to 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 to 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 to 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 to 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 to 0.1 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 to 0.05 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.01 to 0.1 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.02 to 0.08 mg of lipase per milligram of total LC-PUFAs in the nutritional formulation. In some embodiments, the method exposes the nutritional formulation to 0.04 to 0.06 mg of lipase per milligram of total LC-PUFAs 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 to 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 to 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 to 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 to 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 to 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. [Example]

[0149] Example 1: Specific activity of lipase on 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 initiated 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 reverse-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 number T-310-D7-V), ARA triglyceride (Nu-check Prep, Inc., lot number T-295-JY14-V), DHA free acid form (Nu-check Prep, Inc., lot number U-84A-AU20-U), and ARA free acid form (Nu-check Prep, Inc., lot number U-71A-N11-U). The specific activities of the panel of lipases toward DHA and ARA in this assay are summarized in Table 1. In the inventors' opinion, 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 formula 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 the 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 collected 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). The samples were then analyzed for either DHA, ARA, or EPA acids by RP-HPLC as described above.

[0152] The percent of total triglycosides decreased over time as the amount of free acid and monoglycerides increased. For example, when hydrolyzed with RO, the amount of DHA free acid increased over time (Figure 7). Similarly, when hydrolyzed with RO, the amount of ARA free acid increased over time (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 triglycerides and ARA triglycerides 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 of Enfamil powder in 648 mL of hot tap water (temperature was 37°C). DHA triglyceride (442 mg, final DHA concentration 0.54 g, 1.2% of total fat) and ARA triglyceride (885 mg, final ARA concentration 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 performed 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 initiated by adding lipase. The vial was transferred to a 37°C water bath placed on a magnetic stirrer. 50 μl of each sample was collected at different time points: 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 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. Animals were maintained on a 12-hour day / night cycle with light on from 06:00 to 18:00 (6 AM to 6 PM) and darkness from 18:00 to 06:00 (6 PM to 6 AM). Pigs were individually housed in metabolic cages or individual pens equipped with dry sample troughs, drinking teats, and constant heat lamps (150 W). Pigs were allowed to move freely within the pens and were visible to each other.

[0158] 5.2 Feed During the postoperative and pre-treatment periods, pigs were fed a standard pig diet (53908 Vaxtill 320 P BK, Lantmannen, Sweden) containing 17.5% crude protein, 3.9% crude fiber, 3.5% crude fat, and 5.2% ash, along with 5000 IE / kg vitamin A, 500 IE / kg vitamin D, and 85 mg / kg vitamin E. Pigs were fed twice daily (2.0% of body weight per meal) between 09:00 and 10:00 (9 AM to 10 AM) and 17:00 and 18:00 (5 PM to 6 PM). During the adaptation period, pigs were trained to consume infant formula (NAN Pro 1 Gold Infant Formula, Nestlé) for several days prior to the start of the experiment. Because 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 for 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 a.m. and then fed every three hours, with the final meal being fed at 6 a.m. 100 g of NAN formula contains approximately 27.7% fat, 9.6% protein, and 57.8% carbohydrates.

[0159] 5.3 Infant formula fortified with DHA and ARA triglycerides According to the manufacturer, NAN Pro 1 Gold (Nestlé) is a premium whey-based breastfeeding infant formula that is nutritionally complete and specially formulated for healthy infants from birth. It also contains fish oil to support brain and visual 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 selenate), 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), thiamin 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, culture (bifidus). Table 5 below lists the breast milk and infant formulas, along with the infant and pig formulas for use in these experiments. * The lipid composition of [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) EPI induction surgery was performed in 12+2 young pigs aged 6-8 weeks. EPI generally fully developed 3-4 weeks after surgery. The onset of complete pancreatic insufficiency was confirmed by growth arrest (minimal or no weight gain) and / or the onset of steatorrhea.

[0161] Example 6: Experimental Design and Procedure 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 lipase dosage 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 minutes with shaking. Proposed formulation and lipase mixture: -Pig weight in the range of 11-14 kg -Feed requirement: 40g of formula powder / kg body weight -Daily requirement: 500g of 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) Twelve pigs were placed in metabolic cages and trained to drink the TG-PUFA-enriched formula for approximately 7–10 days prior to the adaptation period. 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 four times per day as the sole food source. Total daily formula consumption was measured throughout the entire experiment. Body weight was recorded on the morning of the first day of the control period, before the morning meal. Three 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 four times per day as the sole dietary source. Total daily formula consumption was measured throughout the entire experiment. Body weight was recorded on the morning of the first day of the study period, before the morning meal. Three 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.

[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 (n=4) of the EPI pigs were fed formula prehydrolyzed with RO lipase. 2) One-third (n=4) of the EPI pigs were fed formula prehydrolyzed with CV lipase. 3) One-third of the EPI pigs (n=4) were fed formula only. The preparation of the formula and lipase mixture is described above in paragraphs

[0201] (

[0157] ).

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

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

[0169] Example 7: RO and CV lipases improve fatty acid absorption in EPI pigs A well-established surgical model of exocrine pancreatic insufficiency (EPI) in 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 infant formula prehydrolyzed with CV lipase or RO lipase on fatty acid absorption compared to non-hydrolyzed infant formula. The EPI pigs were 10 weeks old (+ / - 2 weeks), which corresponds to a human infant's age of approximately 6 months. Pigs were fed Nestlé (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 occurred four times per day, every 3 hours. In groups of pigs receiving prehydrolyzed formula, the formula was prehydrolyzed 15 minutes before feeding by mixing with CV or RO lipase at 37°C. The duration of the experiment was 1 week, after which fecal LC-PUFA concentrations, plasma LC-PUFA absorption, and LC-PUFA accumulation 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 the coefficient of fat absorption (%CFA) compared to controls (Figure 22C), where %CFA = (fat intake (g / 24 hr) - fecal 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 resulted in significant reductions in fecal ARA (36% and 65% reductions, respectively), EPA (78% reduction with both enzymes), and DHA (68% and 60% reductions, 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] Additionally, pigs fed the prehydrolyzed formula 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 four pigs, and the RO lipase group consisted of three pigs. Pre-meal plasma feed was collected after 7 days of treatment and an overnight fast. Plasma levels of ARA and DHA were significantly higher in pigs fed formula prehydrolyzed with RO lipase compared to pigs fed non-hydrolyzed formula (60% and 30%, respectively, p<0.05) (Figure 24). Plasma levels of ARA were also significantly higher in pigs fed formula prehydrolyzed with CV lipase compared to pigs fed non-hydrolyzed formula (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 non-hydrolyzed formula. Retinal ARA levels were also significantly higher (p<0.05) in pigs fed formula prehydrolyzed with CV lipase compared to pigs fed non-hydrolyzed formula (Figure 25A). In pigs fed formula prehydrolyzed with CV or RO lipase, ARA levels were also significantly increased (p<0.05) in the heart (Figure 26A, 60% and 20% increase, respectively) and kidney (Figure 26B) compared to pigs fed non-hydrolyzed 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 formula by immobilized lipase in "tea bags" Rhizopus oryzae (RO) lipase was covalently attached to acrylic beads and contained within a device resembling a tea bag. 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 tea bags 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 reverse-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 formula.

[0174] Example 9: Hydrolysis of TG-DHA and TG-ARA in infant formula 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 packed 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 / h. The column eluate was analyzed for DHA and ARA hydrolysis by HPLC. The percent 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 toward 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 μL of lipase (either pancreatin or RO lipase) and 100 μL of each of DHA and ARA triglycerides. The reaction was 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 feeding of pigs with formula prehydrolyzed with immobilized lipase Six healthy pigs and 20 pigs with surgically induced exocrine pancreatic insufficiency (EPI) (see Example 5) underwent a two-week adaptation / control period followed by a six-week test period (FIG. 29). During the adaptation period, all pigs were fed NAN Pro 1 Gold infant formula (Nestlé) ("IF"). During the test period, six of the healthy pigs ("Healthy") 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 TG-LCPUFA-enriched infant formula 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, including failure to surgically induce complete EPI.

[0177] For prehydrolysis, 2 liters of NAN Pro 1 Gold (Nestlé) infant formula was prepared by mixing 1.5 liters of water at 37°C with 500 g of powdered formula fortified 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 (each "teabag" contains 1 gram of immobilized lipase) were added to the 2 liters of formula and mixed for 15 minutes at room temperature using a magnetic stirrer at a 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 / L of non-esterified fatty acids. After hydrolysis, the formula contained 107.6 mmol / L of non-esterified fatty acids.

[0178] Food consumption was measured daily. Blood and fecal samples were collected at the end of the adaptation period ("baseline"), and after 1, 4, and 6 weeks of the treatment period. For the baseline 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 testing.

[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, gross liver examination showed no development of fatty liver.

[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) 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 difference was 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. An apparatus, the apparatus comprising: a container for receiving a nutritional composition comprising triglycerides and / or fatty acid esters; a first opening in the container, the first opening configured to connect the container to a feeding tube; a second opening in the container; and a lipase immobilized within the container such that the nutritional composition is exposed to the lipase when received within the container; wherein the lipase is configured to hydrolyze the triglycerides and / or fatty acid esters in the nutritional composition to form a nutritional formulation, the nutritional formulation comprising monoglycerides and free fatty acids, and wherein immobilization of the lipase limits the amount of lipase entering the nutritional composition to 0.1 milligrams of lipase or less per milligram of triglycerides and / or fatty acid esters exposed to the lipase.

2. 10. The device of claim 1, wherein the lipase is immobilized such that when the nutritional formula passes through the container, the immobilized lipase remains in the container.

3. The device of claim 1 , wherein the lipase is immobilized on the interior surface of the container.

4. The device of claim 1 , wherein the lipase is immobilized on a structure within the container.

5. The device of claim 4 , wherein the structure comprises a ball or bead within the container.

6. The device of claim 1 , wherein the first opening includes a connector for connecting to the feeding tube.

7. 10. The device of claim 1, wherein the container is a first container and the second opening is configured to facilitate transferring the nutritional composition to a second container.

8. 10. The device of claim 1, wherein the container is a first container and the second opening includes a connector for connection to a second container.

9. 10. The device of claim 1, wherein the feeding tube is a nasogastric tube or a nasojejunal tube.

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

11. 10. The device of claim 1, wherein the lipase is selected from the group consisting of Chromobacterium viscosum lipase, Pseudomonas fluorescens lipase, and Rhizopus oryzae lipase.

12. The device of claim 1 , further comprising a filter located at the first opening, the second opening, or both the first opening and the second opening of the container.

13. 1. A method for preparing a nutritional formulation, said method comprising: passing a nutritional composition comprising triglycerides through the device of claim 1 to expose the nutritional composition to the immobilized lipase to at least partially hydrolyze at least a portion of the triglycerides to form the nutritional formulation; The method, wherein after exposure to the lipase, the nutritional formulation contains fewer triglycerides and more monoglycerides and free fatty acids than before exposure to the lipase.

14. The nutritional formula is for use in a premature infant or a Gastrointestinal dysfunction, Fat malabsorption, short bowel syndrome, or Cystic fibrosis 14. The method of claim 13, wherein the method is provided to a subject having at least one of:

15. 14. The method of claim 13, wherein after the nutritional composition is exposed to the lipase, the nutritional formulation comprises more free fatty acids than monoglycerides.

16. 14. The method of claim 13, wherein the nutritional formulation is provided to a subject and the nutritional composition is exposed to the lipase for at least 1 minute prior to ingestion by the subject.

17. 14. The method of claim 13, wherein the nutritional formulation is provided to a subject and the nutritional composition is exposed to the lipase for 30 seconds or less prior to ingestion by the subject.

18. 14. The method of claim 13, wherein the nutritional formulation is provided to a subject and the nutritional composition is exposed to the lipase for 1 minute or less prior to ingestion by the subject.

19. 14. The method of claim 13, wherein the nutritional formulation is provided to a subject and the nutritional composition is exposed to the lipase for 5 minutes or less prior to ingestion by the subject.

20. 14. The method of claim 13, wherein the nutritional formulation is provided to a subject and the nutritional composition is exposed to the lipase for 60 minutes or less prior to ingestion by the subject.

21. 1. A method of providing a nutritional formulation, said method comprising: flowing at least one of cow's milk, infant formula, or enteral formula, comprising triglycerides and / or fatty acid esters, into a container of the device; hydrolyzing the triglycerides and / or fatty acid esters in at least one of the milk, infant formula, or enteral formula by exposing the milk, infant formula, or enteral formula to an immobilized lipase contained within the container of the device to form the nutritional formula; outputting the nutritional formula from the device to a feeding tube for ingestion by a subject; wherein the nutritional formulation comprises monoglycerides and free fatty acids, and wherein immobilization of the lipase limits the amount of lipase entering the nutritional composition to 0.1 milligrams of lipase or less per milligram of triglyceride and / or fatty acid ester exposed to the lipase.

22. 22. The method of claim 21, wherein the feeding tube is a nasogastric tube or a nasojejunal tube.

23. 22. The method of claim 21, wherein the lipase is immobilized on the interior surface of the container.

24. 22. The method of claim 21, wherein the lipase is immobilized on a structure within the container.

25. 25. The method of claim 24, wherein the structures comprise balls or beads within the container.

26. 22. The method of claim 21, wherein the lipase is immobilized on particles contained within the container.

27. 22. The method of claim 21, wherein the nutritional formulation output from the device comprises more free fatty acids than monoglycerides.

28. The nutritional formula is for use in a premature infant or a Gastrointestinal dysfunction, Fat malabsorption, short bowel syndrome, or Cystic fibrosis 22. The method of claim 21, wherein the method is provided to a subject having at least one of:

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