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

Abstract

To provide nutritional formulas comprising long-chain polyunsaturated fatty acids (LC-PUFAs), along with methods and devices for preparing and / or administering the nutritional formulas.SOLUTION: In some embodiments, a percentage of the LC- PUFAs in the nutritional formula are in the form of monogiycerides and / or free fatty acids. In some embodiments, the nutritional formulas 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 a related application to U.S. Provisional Patent Application No. 61 / 600,207, filed on February 17, 2012, and U.S. Provisional Patent Application No. 61 / 719,173, filed on October 26, 2012, and claims the priority based on these provisional applications under 35 U.S.C. § 119.

Background Art

[0002] Long-chain fatty acids are important for human health and development. Long-chain fatty acids consumed in the diet are mainly in the form of triglycerides (TG), where three long-chain fatty acids are bound to a glycerol molecule via ester bonds. The absorption of long-chain triglycerides first requires the enzymatic action of lipase (e.g., pancreatic lipase), which digests the triglycerides by hydrolysis, breaking them down into monoglycerides and further 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 the body's tissues for various physiological purposes (see D. Kasper et al., Harrison’s Principles of Internal Medicine, 16th Edition (2004)). Medium-chain triglycerides can be absorbed through the intestinal lumen, but long-chain triglycerides cannot, and thus 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 patients suffering from, for example, defects in pancreatic production activity, malabsorption, or pancreatic insufficiency, are unable to properly break down long-chain triglycerides and, as a result, may suffer from inappropriate fatty acid absorption, which is inadequate for maintaining health.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] In order to improve the hydrolysis of long-chain triglycerides, commercially available lipase nutritional supplements may be added to the diet. However, for various reasons, lipase nutritional supplements may have a reduced ability to break down long-chain triglycerides or may not necessarily solve the problem of low fatty acid absorption in all individuals who need to receive essential fatty acids for other reasons. For example, most commercially available lipase nutritional supplements are produced from animal pancreatic lipase, which is known to be significantly unstable at pH 7 or lower. See, for example, U.S. Patent Application No. 2010 / 0239559; By D. Kasper et al., Harrison’s Principles of Internal Medicine, 16th Edition (2004). A significant amount is likely to be inactivated by the time such lipase passes through the stomach. Furthermore, not all lipases act to the same extent on the hydrolysis of a given long-chain fatty acid, suggesting that lipase specificity is an important consideration (see By R. Jensen et al., Lipids 18(3): 239-252 (1983)). Moreover, in some populations with pancreatic insufficiency, such as premature infants or patients in the intensive care unit, the nutritional formulation is tightly regulated. It may not be desirable or possible to supplement an approved formulation in such controlled populations with additional ingredients. Furthermore, formulations supplemented with many fatty acids may contain medium-chain triglycerides, but there are obvious medical benefits to ingesting long-chain fatty acids from the diet. Therefore, an improved method for enhancing the hydrolysis of long-chain triglycerides is needed.

[0005] The appropriate hydrolysis of long-chain polyunsaturated triglycerides (TG-LCPUFA) is particularly important for various reasons. Long-chain polyunsaturated fatty acids (LC-PUFA) are extremely important for the development of the nervous system and retina. Furthermore, some of them are regarded as "essential fatty acids", which humans cannot synthesize and need to obtain from dietary sources. The main dietary sources of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) of n-3 LC-PUFA are the essential fatty acid, α-linolenic acid (ALA), which is their precursor. However, endogenous enzymes are very inefficient in the conversion of ALA to DHA and EPA. According to an official statement by the "International Society for the Study of Fatty Acids and Lipids" (ISSFAL), the conversion of ALA to DHA is approximately 1% in infants and quite low in adults (see Brenna et al., Prostaglandins Leukot Essent Fatty Acids, 80(2-3):85-91 (2009)). Therefore, although DHA and EPA are not essential fatty acids themselves, dietary sources of DHA and EPA are important. The main dietary source of arachidonic acid (ARA or AA) of n-6 LC-PUFA is linoleic acid (LA), which is an essential fatty acid.

Means for Solving the Problems

[0006] Embodiments of the present invention provide a lipase that is significantly more efficient than others in hydrolyzing, for example, certain long-chain triglycerides and esters such as long-chain polyunsaturated triglycerides and esters (i); provide a nutritional preparation such as a medical nutritional preparation or an infant formula containing pre-hydrolysis components (i.e., monoglycerides and / or free fatty acids) of, for example, LC-PUFA triglycerides, LC-PUFA fatty acid esters, and / or other long-chain triglycerides or long-chain fatty acid esters (ii); provide a method for producing such a nutritional preparation, including a method in which a preparation containing LC-PUFA triglycerides, LC-PUFA fatty acid esters, and / or other long-chain triglycerides or long-chain fatty acid esters is temporarily exposed to lipase (iii); and provide an apparatus designed to provide a nutritional preparation containing monoglycerides and / or free fatty acids, for example, LC-PUFA triglycerides and / or LC-PUFA fatty acid esters, thereby solving these various problems. In embodiments where the preparation is temporarily exposed to lipase and the lipase is removed or separated from the preparation before ingestion, the present invention provides the advantage of ensuring the breakdown of LC-PUFA triglycerides, LC-PUFA fatty acid esters, and / or other long-chain triglycerides or long-chain fatty acid esters without the need for ingestion of exogenous lipase.

[0007] Accordingly, some embodiments of the present invention provide a nutritional composition. In some embodiments, the nutritional composition 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 composition. 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 composition is formulated for administration to premature infants. Other nutritional compositions encompassed herein are formulated for infants, toddlers, children, or adults having 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 simply in need of additional essential dietary LC-PUFA and / or other long-chain fatty acids. In some embodiments, the nutritional composition of the present invention is for a subject less than 1 year old. 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 compositions of the present invention are formulated to be consumed or administered orally or enterally under medical supervision, such as medical nutritional compositions, i.e., those distributed through hospitals or pharmacies under prescription. Typically, medical nutritional compositions are formulated for the dietary management of specific medical disorders, diseases, or conditions where special nutritional requirements exist. Medical nutritional compositions have a "Generally Recognized As Safe" status and are required to comply with FDA regulations regarding labeling, product requirements, and manufacturing.

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

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

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

[0013] Another aspect of the invention is a method of providing nutrition to a subject in need of dietary LC-PUFAs and / or other long-chain fatty acids, such as a person with a reduced ability to break down long-chain triglycerides or long-chain fatty acid esters in the intestinal tract, a person suffering from pancreatic insufficiency, a person suffering from malnutrition, and a person receiving total parenteral nutrition, by administering a composition of the invention. In some embodiments, the subject is a premature infant. In other embodiments, the subject is a full-term infant or a young child. In certain embodiments, the subject is over 50 years old, over 60 years old, or over 70 years old. In some embodiments, the subject is suffering from pancreatic insufficiency. In other embodiments, the composition is administered through a feeding tube. In some embodiments, the nutritional composition of the invention is administered to improve cognitive ability in people of all ages, to prevent chronic lung disease in premature infants, to enhance neurological development in premature infants, or to treat or prevent many other conditions associated with improvements due to increased intake of long-chain fatty acids such as, for example, 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, eating disorders, 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 composition of the present invention. As a result, in such patients, the risk of intestinal atrophy and other complications associated with long-term (more than 24 hours) total parenteral nutrition is reduced. Such methods can be used, for example, to shorten the recovery time of patients suffering from malnutrition before and after malabsorption, short bowel syndrome, IBD, pancreatic insufficiency, surgery, chemotherapy or radiotherapy, or other causes of malnutrition, cancer, trauma, and GI dysfunction such as pressure ulcers. Such patients can receive the nutritional composition of the present invention via a nasogastric tube. This delivery method may be advantageous in situations where the patient is at risk of developing changes in intestinal motility, pancreatic enzyme secretion failure due to systemic inflammatory response syndrome, or other conditions that cause cleavage or absorption failure 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 it is advantageous to bypass the stomach, the nutritional composition of the present invention can be administered via a nasojejunal tube. Other types of delivery devices may also be used to deliver the composition of the present invention.

[0015] Also, healthy subjects can benefit from increased absorption of LC-PUFA, for example, by reducing the risk of cardiovascular disease. Thus, in some embodiments, the present invention provides a method for improving fat absorption in a healthy subject, the method comprising the step of supplying the nutritional composition of the present invention to the subject.

[0016] The present invention further provides an apparatus for preparing the nutritional composition of the present invention. In some embodiments, the apparatus comprises a chamber containing at least one lipase, and the chamber can hold a liquid nutritional composition so as to expose the liquid nutritional composition to the lipase. In some embodiments, the lipase in the container is immobilized on the inner surface of the container. In other embodiments, the lipase is immobilized on a support in the chamber. In some embodiments, the apparatus comprises a chamber made of a permeable membrane and containing immobilized lipase in the chamber, so that the liquid nutritional composition passes through the permeable membrane and contacts 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 composition 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) The nutritional composition according to item 1, wherein the LC-PUFA contains one or more LC-PUFAs selected from the group consisting of DHA, ARA, and EPA. (Item 3) The nutritional composition according to item 2, wherein the LC-PUFA contains DHA. (Item 4) The nutritional composition according to item 2, wherein the LC-PUFA contains EPA. (Item 5) The nutritional composition according to item 2, wherein the LC-PUFA contains ARA. (Item 6) The nutritional composition according to item 2, wherein the LC-PUFA contains DHA and EPA. (Item 7) The nutritional composition according to item 2, wherein the LC-PUFA contains DHA and ARA. (Item 8) The nutritional composition according to item 2, wherein the LC-PUFA contains DHA, EPA, and ARA. (Item 9) The nutritional composition according to any one of items 1 to 8, wherein the nutritional composition is milk powder for infants. (Item 10) The nutritional composition 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) The nutritional composition according to item 10, containing 0.1 mg or less of lipase per mg of LC-PUFA. (Item 12) The nutritional composition according to any one of items 1 to 9, wherein the nutritional composition does not contain additional lipase. (Item 13) A nutritional preparation comprising a lipase selected from Chromobacterium viscosum lipase, Pseudomonas Fluorescens lipase, and Rhizopus oryzae lipase. (Item 14) The nutritional preparation according to item 13, further comprising DHA, wherein more than 5% of the DHA is in the form of monoglyceride and / or free fatty acid. (Item 15) The nutritional preparation according to item 13 or 14, further comprising ARA, wherein more than 5% of the ARA is in the form of monoglyceride and / or free fatty acid. (Item 16) The nutritional preparation according to any one of items 13 to 15, further comprising EPA, wherein more than 5% of the EPA is in the form of monoglyceride and / or free fatty acid. (Item 17) The nutritional preparation according to any one of items 13 to 16, comprising lipase at 0.1 milligram or less per milligram of LC-PUFA. (Item 18) A method for preparing a nutritional preparation before ingestion by a subject, the method comprising exposing a liquid nutritional composition comprising LC-PUFA triglyceride and / or LC-PUFA ester to a lipase. (Item 19) The lipase is selected from the group consisting of Chromobacterium viscosum lipase, Pseudomonas Fluorescens lipase, and Rhizopus oryzae lipase, and the method according to item 18. viscosum) lipase, Pseudomonas Fluorescens lipase, and Rhizopus oryzae) lipase, and the method according to item 18. (Item 20) The method according to item 18 or item 19, wherein the liquid nutritional composition is exposed to the lipase for at least 1 minute before ingestion. (Item 21) The method according to any one of items 18 to 20, wherein the liquid nutrient composition is exposed to lipase for 60 minutes or less before ingestion. (Item 22) A method according to any one of items 18 to 21, comprising preparing a liquid composition by adding a drinking liquid to a solid or powder composition containing LC-PUFA triglyceride and / or LC-PUFA ester and lipase, thereby exposing the liquid composition containing LC-PUFA triglyceride and / or LC-PUFA ester to lipase. (Item 23) (a) A step of preparing a liquid composition by adding a drinking liquid to a solid or powder composition containing LC-PUFA triglyceride and / or LC-PUFA ester; (b) A method according to any one of items 18 to 21, comprising exposing lipase to the liquid composition, thereby exposing the LC-PUFA triglyceride and / or LC-PUFA ester to the lipase. (Item 24) A method according to any one of items 18 to 23, further comprising a step of removing the lipase from the liquid composition after the step of exposing the LC-PUFA triglyceride and / or LC-PUFA ester to the lipase. (Item 25) The lipase is (a) A step of exposing the lipase in the liquid composition to a molecule that binds to the lipase, and binding the lipase to the solid support by immobilizing the molecule on the solid support; (b) A method according to item 24, wherein the lipase is removed by a process comprising separating the liquid composition from the solid support. (Item 26) A method according to item 24, wherein the LC-PUFA triglyceride and / or LC-PUFA ester in the liquid composition is exposed to lipase immobilized on a solid support, and the lipase is removed by separating the liquid composition from the solid support. (Item 27) The LC-PUFA triglyceride and / or LC-PUFA ester in the liquid composition is exposed to the lipase, and then separated from the lipase by passing the liquid composition through a chamber, wherein the lipase is fixed to at least a part of the inner surface of the chamber, according to the method of Item 24. (Item 28) The LC-PUFA triglyceride and / or LC-PUFA ester in the liquid composition is exposed to the lipase, and then separated from the lipase by a process including the step of passing the liquid composition through a chamber, wherein the lipase is fixed to a solid substrate accommodated in the chamber, according to the method of Item 24. (Item 29) The method according to Item 28, wherein the chamber is a column. (Item 30) The liquid composition containing LC-PUFA triglyceride and / or LC-PUFA ester is exposed to the lipase, and then separated from the lipase by a process including the step of exposing the liquid composition containing LC-PUFA triglyceride and / or LC-PUFA ester to a container containing lipase immobilized on a solid support, wherein at least a part of the inner surface of the container is permeable to LC-PUFA triglyceride and LC-PUFA ester but impermeable to the solid support, according to the method of Item 26. (Item 31) More than 5% of the LC-PUFA in the prepared nutritional preparation is in the form of monoglyceride and / or free fatty acid, according to the method of any one of Items 18 to 30. (Item 32) The method according to any one of Items 18 to 31, wherein the LC-PUFA includes one or more selected from the group consisting of DHA, ARA, and EPA. (Item 33) The liquid nutritional composition is exposed to the lipase in 60 minutes or less before ingestion, according to the method of any one of Items 18 to 20 and Items 22 to 32. (Item 34) The method according to any one of items 18 to 33, wherein the liquid nutritional composition is exposed to lipase at 0.1 milligrams or less per milligram of LC-PUFA in the liquid nutritional composition. (Item 35) The method according to any one of items 18 to 34, wherein the liquid nutritional composition is exposed to lipase in 30 minutes or less before ingestion. (Item 36) A nutritional preparation prepared by the method according to any one of items 18 to 35. (Item 37) A method for providing nutrition to a subject, comprising the step of supplying the subject with the nutritional preparation according to any one of items 1 to 17 and item 36. (Item 38) The method according to item 37, wherein the nutritional preparation is formula milk for infants. (Item 39) A method for improving fat absorption in a subject, comprising the step of supplying the subject with the nutritional preparation according to any one of items 1 to 17 and item 36. (Item 40) The method according to item 39, wherein the method reduces the total fat level in the feces of the subject by at least 50%. (Item 41) The method according to item 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 feces of the subject by at least 50%. (Item 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) 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) 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) The method according to any one of items 39 to 44, wherein the subject has pancreatic insufficiency, a defect in pancreatic production activity, 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) The method according to any one of items 39 to 45, wherein the nutritional composition is supplied to the subject via a nutritional supply tube. (Item 47) The method according to any one of items 39 to 46, wherein the subject is a premature 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) The method according to any one of items 39 to 46, wherein the subject is 6 years old or younger. (Item 50) A method for improving cognitive ability in a subject over 50 years old, the method comprising the step of supplying the nutritional composition according to any one of items 1 to 17 and item 36 to the subject. (Item 51) A method for preventing chronic lung disease in an infant, the method comprising the step of supplying the nutritional composition according to any one of items 1 to 17 and item 36 to the infant. (Item 52) A container comprising at least first and second compartments, wherein the first compartment contains a nutritional composition and the second compartment contains lipase. (Item 53) The lipase is selected from the group consisting of Chromobacterium viscosum lipase, Pseudomonas Fluorescens lipase, and Rhizopus oryzae lipase, and the container according to item 52. viscosum) lipase, Pseudomonas Fluorescens lipase, and Rhizopus oryzae) lipase, and the container according to item 52. (Item 54) The container according to item 52 or item 53, wherein the nutritional preparation is in powder form. (Item 55) The container according to item 52 or item 53, wherein the nutritional preparation is in liquid form. (Item 56) The container according to any one of items 52 to 55, wherein the second compartment contains lipase at 0.1 milligrams or less per milligram of LC-PUFA in the nutritional preparation contained within the first compartment. (Item 57) An apparatus for preparing milk formula, a main body wall for containing a liquid, and at least one lipase contained within the container. (Item 58) The apparatus according to item 57, wherein the at least one lipase is immobilized within the container. (Item 59) The apparatus according to item 58, wherein the at least one lipase is attached to a structure in fluid communication with the interior of the container. (Item 60) The apparatus according to item 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) The apparatus according to item 59 or item 60, wherein the structure includes the inner surface of the container. (Item 62) The apparatus according to item 59 or item 60, wherein the structure includes the cap of the container. (Item 63) The apparatus according to item 59 or item 60, wherein the structure includes surface protrusions within the container. (Item 64) The apparatus according to item 59 or item 60, wherein the structure includes particles within the container. (Item 65) The apparatus according to item 64, wherein the particles include balls or beads. (Item 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) The device according to any one of items 57 to 66, wherein the container further includes at least one opening in the container. (Item 68) The device according to item 67, wherein the opening is configured to be connected to a nutritional supply tube. (Item 69) The device according to item 67 or item 68, further including a valve. (Item 70) An apparatus for preparing formula milk, including a cap for a closure member of a bottle, the cap including at least one lipase immobilized on or within a surface disposed for fluid contact with the interior of the bottle. (Item 71) The device according to item 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) The device according to item 70 or item 71, wherein the surface includes the inner surface of the cap. (Item 73) The device according to any one of items 70 to 72, wherein the surface includes a protrusion configured to extend into the container. (Item 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) An apparatus for hydrolyzing triglycerides and / or fatty acid esters in a nutritional formulation, the apparatus including a lipase attached to a solid support. (Item 76) The lipase is Chromobacterium viscosum lipase, Pseudomonas Fluorescens lipase, and Rhizopus oryzae lipase, and the device according to any one of items 57 to 75 selected from the group consisting of oryzae) lipase. (Item 77) The device according to item 75, comprising a container, wherein the lipase is immobilized on at least a part of the inner surface of the container. (Item 78) The device according to item 75, comprising a container, wherein the lipase is immobilized on a solid substrate accommodated in the container. (Item 79) The device according to item 77 or item 78, wherein the lipase is immobilized by a covalent bond or a crosslinking bond. (Item 80) The device according to any one of items 75 to 79, further comprising a nutrient supply tube attached to the container. (Item 81) A method for shortening the length of time a patient requires total parenteral nutrition, the method comprising the step of enterally administering the nutritional preparation according to any one of items 1 to 17 and item 36. (Item 82) 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) 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) 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) The nutritional preparation according to any one of items 1 to 8 or items 10 to 17, wherein the preparation is an adult nutritional preparation. (Item 86) The method according to item 49, wherein the subject is less than 1 year old. (Item 87) The method according to item 49, wherein the subject is 1 to 6 years old. (Item 88) 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) A method for increasing the plasma level of at least one vitamin selected from the group consisting of vitamin A and vitamin E, the method comprising supplying the nutritional composition according to any one of items 1 to 17 and item 36 to the subject. Brief Description of the Drawings

[0017]

Figure 1

Figure 2A

Figure 2B

Figure 2C

Figure 3A

Figure 3B

Figure 3C

Figure 4A

Figure 4B

Figure 5A

Figure 5B

Figure 6A

Figure 6B

Figure 6C

Figure 6D

Figure 7

Figure 8

Figure 9A

Figure 9B

Figure 9C

Figure 10A

Figure 10B

Figure 10C

Figure 11A

Figure 11B

Figure 11C

Figure 12

Figure 13A

Figure 13B

Figure 14

Figure 15

Figure 16

Figure 17A

Figure 17B

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Figure 28

Figure 29

Figure 30

DETAILED DESCRIPTION OF THE INVENTION

[0018] Long-chain polyunsaturated fatty acid Long-chain polyunsaturated fatty acids (LC-PUFA) are hydrocarbon chains having two or more double bonds. Depending on the position of the first double bond relative to the methyl terminus, LC-PUFA 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 referred to as "essential fatty acids" because humans cannot synthesize them and rather need to obtain them through diet. This is because mammals cannot introduce double bonds beyond carbons 9 and 10 in fatty acids (see Blosover et al., Cell Biology: A Short Course, John Wiley & Sons, Inc. 39 (2011)). However, humans can generate additional long-chain PUFAs starting from ALA and LA.

[0019] Both ALA and LA are metabolized by a series of desaturation and elongation steps to produce other long-chain PUFAs. For example, ALA is metabolized to EPA and ultimately to DHA. LA is metabolized to the n6-fatty acid ARA. However, the conversion of ALA to DHA and EPA and the conversion of LA to ARA are 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 tissues for converting ALA to DHA and LA to ARA, and thus the liver 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 is obtainable 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 the development and function of the nerves and retina. It is the major long-chain PUFA in the nerve membrane and is essential for brain function, the construction of brain circuits, and the transmission of nerve impulses. As a component of the conjunctival membrane, DHA contributes to the membrane fluidity that is important for maintaining synaptic structure, neurotransmission, and synaptic plasticity (see G. Jicha et al., Clin. Interv. Aging 5:45-61 (2010)). DHA also affects signaling events that are essential for neuron differentiation and survival and has effects on the levels and metabolism of neurotransmitters and eicosanoids. Most of the DHA accumulation in the brain occurs at the beginning of the third trimester during the first two years after birth. In rodents and primates, it has been shown that an inappropriate supply of n-3 PUFAs during this period causes learning ability and neurotransmission deficiencies (see M. Martinez et al., J. Pediatr. 120:S129-S138 (1992)). Supplementation of DHA in rats previously restricted to a DHA-deficient diet rescues the 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 the functional activation in the dorsolateral prefrontal cortex during the performance of an activation task compared with placebo (see R. McNamara et al., Am. J. Nutr. 91:1060-7 (2010)). Therefore, DHA is considered to be important not only for development but also for the maintenance of neuron function.

[0021] In addition, DHA is highly concentrated in the retina and has important effects on photoreceptor differentiation and the activation of the visual pigment rhodopsin (see H. Lauritzen et al., Prog. Lipid (See Res. 40:1-94 (2001); M. Clandinin et al., J. Pediatr. 125:S25-32 (1994)). Inadequate supply of DHA in the early stages of development of primates and rodents reduces abnormal retinal physiology and vision (see 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 formula without DHA for 12 months after birth have been shown to have lower vision than infants fed DHA-supplemented formula (see E. Birch et al., Am. J. Clin. Nutr. 91(4):848-859 (2010)). DHA deficiency is also 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 various diseases including, for example, Alzheimer's disease (AD), bipolar disorder (BP), 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 on AD patients have shown that DHA has a therapeutic effect. For a review of studies evaluating the effect of DHA in AD, see G. Jicha and W. Markesbery, Clin. Interv. Aging 5:45-61 (2010). Data from in vitro assays, cell culture systems, and murine models of AD support a direct role for n-3 PUFAs in amyloid processing in the brain. Moreover, in 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 valid clinical trial data in patients with AD, large-scale trials in healthy elderly subjects with mild memory complaints have shown that subjects administered DHA are superior in learning and memory tests after 6 months compared to placebo-treated subjects. (Martek Press Release, May 4, 2010). Thus, DHA can also play a beneficial role in preventing AD.

[0024] The therapeutic uses of DHA have also been investigated in patients with BP and MDD. For an examination 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 obtained from human patients, the fatty acid composition in erythrocyte membranes obtained from blood samples was evaluated and found to contain far 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 prefrontal cortex of the orbit was found to have far lower levels of DHA in BP patients than in normal controls (see R. McNamara et al., Psychiatry Res 160(3):285-299 (2008)). Moreover, in a four-month, double-blind, placebo-controlled trial, BP patients taking n-3 fatty acids had a much longer remission period than the placebo group (see Stoll et al., Arch Gen Psychiatry 56(5):407-412 (1999)). These studies suggest that DHA is therapeutically beneficial in BD and MDD, particularly due to its mood-stabilizing effects.

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

[0026] In sepsis, enteral diets enriched with EPA, gamma-linolenic acid, and antioxidants have been shown to improve in-hospital outcomes and reduce 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 terms of ventilator-free days and ICU-free days, reducing new organ failure and mortality, have been reported in patients with acute respiratory stress fed diets 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. By altering the lipid microenvironment, n-3 fatty acids can enhance epithelial cell reorganization and help 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 been shown to have 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 the down-regulation of NF-κB. For a review of the role of n-3 fatty acids in cancer, see B. Anderson and D. Ma, Lipids Health Dis. 8:33 (2009).

[0029] n-3 fatty acids are also 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. Therefore, the American Heart Association, as well as other health-related organizations, have issued recommendations regarding increased intake of n-3 fatty acids in the diet (see P. Kris-Etherton et al., Circulation 106:2747-2757 (2002)). Possible mechanisms for the observed effects of n-3 fatty acids on cardiovascular health include triglyceride-lowering effects, blood pressure-lowering effects, reduced platelet aggregation, and stabilizing effects on the myocardium itself.

[0030] The beneficial effects of n-3 fatty acids in several pathological conditions may be due to their broad anti-inflammatory actions. EPA and DHA generate resolvins, anti-inflammatory mediators with pro-resolving and immunomodulatory functions. For example, EPA and DHA alter the production of inflammatory cytokines by exhibiting an inhibitory effect on leukocyte chemotaxis and reducing the activation of NF-κB in immune cells (see P. Calder, Int. Rev. Immunol. 28:506-534 (2009)). In general, n-3 PUFAs are associated with a decrease in inflammatory T cell responses. When the n-3 fatty acids in animal feed are increased, the composition of the T cell membrane microdomains in lipid rafts changes, reducing NF-κB activation, IL-2 production, and cell proliferation. Specifically, n-3 PUFAs affect the distribution and partitioning of early signaling regulators of T cell activation, such as protein kinase C (see Y. Fan et al., J. Immunol. 173:6151-6160 (2004)). Also, n-3 fatty acids have been shown to reduce MHC class II expression in dendritic cells, effectively reducing antigen presentation to T cells, while n-6 fatty acids are associated with an increase in antigen-presenting 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 anti-diabetic effects in vivo by 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 are associated with ARA accumulation in the brain during pre- and postnatal development and play a very important role in neurodevelopment and brain function (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, also stimulate skin and hair growth, maintain bone health, regulate metabolism, and maintain the reproductive system.

[0032] Long-chain PUFA nutritional supplement For 10 years, health-related agencies have recommended dietary consumption of n-3 fatty acids for their health benefits. DHA and EPA are commercially available as triglycerides or esterified forms in nutritional supplements or pharmaceuticals (e.g., LOVAZA®, OMACOR®, and Vascepa™). DHA nutritional supplements can be derived from fish oil or from vegetarian sources such as flaxseed oil or algae. The nutritional supplement can be a powder, a liquid beverage, or a tube feeding formulation.

[0033] Infant formula is subject to the Federal Food, Drug, and Cosmetic Act and is defined as a "food that is represented for or described as being for use solely as a special dietary food for infants because it purports to be a substitute for breast milk or is suitable as a complete 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 major n-3 fatty acid in breast milk is DHA, which on average contains 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 almost reflects DHA intake in the mother.

[0034] Examples of commercially available TG-LCPUFA-supplemented infant formulas include Enfamil formulas such as Enfamil LIPIL® and Enfamil PREMIUM®, Baboo, Earth’s Best Organic, Nestle Gerber Examples include Nestle infant formulas such as GOOD START (registered trademark) and Nestle NAN (registered trademark), Nutricia infant formulas such as NEOCATE (registered trademark) and APTAMIL (registered trademark), Similac infant formulas such as Parent’s Choice Organic, Pfizer’s SMA GOLD (registered trademark), Similac ADVANCE (registered trademark), Similac EARLY SHIELD (registered trademark), and ISOMIL (registered trademark), and Ultra Bright Beginnings. Other infant formulas may also be supplemented with TG-LCPUFA. TG-LCPUFA-supplemented infant formulas may be based on cow milk or soy 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 milks and beverages for infants, the elderly, and other individuals who require nutritional supplements or dietary nutritional supplements containing long-chain fatty acids. Such products include ENSURE®, PEDIASURE®, CARNATION®, BOOST®, CERELAC®, and SOUVENAID®. In addition, specialized formulations supplemented with esters of TG-LCPUFA or LC-PUFA can be used in combination with the methods and devices of the present invention in patients who require tube feeding. For example, enteral formulations are commonly used in patients with premature infants, renal failure, gastrointestinal diseases or conditions causing GI dysfunction, bowel resection, fat malabsorption, malnutrition, pancreatitis, hyperglycemia / diabetes, liver failure, acute and chronic pulmonary diseases, or immunodeficiency states. For a review of commercially available enteral formulations, see A. Malone, Pract. Gastr. 29(6):44-74 (2005). The nutritional formulations may be standard, basic, or specialized based on the patient's disease or condition. Standard formulations commonly used include, for example, ISOCAL®, NUTREN 1.0®, NUTREN 1.5®, NUTREN 2.0®, OSMOLITE 1.0®, OSMOLITE 1.2®, FIBERSOURCE 1.2®, JEVITY 1.2®, JEVITY 1.5®, PROBALANCE®, ISOSOURCE 1.5®, DELIVER 2.0®, NOVOSOURCE 2.0®, and TWOCAL HN®. Basic formulations may contain macronutrients including polymeric and hydrolyzed formulations and may be fiber-enriched. Disease-specific formulations include, for example, renal formulations such as MAGNACAL RENAL®, NEPRO®, NOVASOURCE RENAL®, SUPLENA®, and NUTRI-RENAL®.

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

[0037] However, in people with a defect in pancreatic production activity or those suffering from pancreatic insufficiency, for example, or those suffering from an impairment in the ability to hydrolyze long-chain triglycerides or esters of long-chain fatty acids, even when such formulations are supplemented with DHA, EPA, and other n-3 fatty acids, it may not be sufficient to realize the beneficial effects 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 of providing an immediately available formulation containing significantly high concentrations of long-chain monoglycerides and / or free fatty acids using existing commercially available long-chain PUFA supplements or newly designed formulations supplemented with long-chain PUFA. In some embodiments, this method is particularly effective in providing long-chain monoglycerides and / or free fatty acids generated from DHA, EPA, and ARA triglycerides or esterified DHA, EPA, and ARA, so that this formulation will provide the maximum benefits associated with these important fatty acids to people who cannot hydrolyze and absorb these important fatty acids by other means.

[0038] Reduction in the ability to hydrolyze long-chain triglycerides and fatty acid esters Pancreatic insufficiency is one of the conditions that lead to a reduced 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 a human's life. For example, the secretion of pancreatic lipase starts at a low level around the 30th week of pregnancy and remains at a low level for the first year after birth. Therefore, infants, especially premature infants, may experience pancreatic insufficiency. As a result, if they are not breastfed, these infants are prone to poor fatty acid hydrolysis and absorption and do not receive the benefits associated with the intake of DHA, EPA, and other LC-PUFAs.

[0039] As a counterpoint, otherwise healthy elderly individuals also experience pancreatic insufficiency or other reductions 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 lipomatosis. As a result, the elderly may experience symptoms of indigestion, including malnutrition, steatorrhea, diarrhea, abdominal pain, and weight loss, due to a decrease in pancreatic exocrine enzymes (see K. Herzig et al., BMC Geriatrics 11:4-8 (2011)).

[0040] In addition, pancreatic insufficiency or other decreases in the ability to hydrolyze LC-PUFA triglycerides or esterified LC-PUFA may also result from disease or trauma. For example, pancreatitis is a state of inflammation in the pancreas, which causes pancreatic insufficiency. Pancreatitis can be either acute or chronic and includes pancreatitis caused by alcohol dependence, 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 young adults. Disorders that lower the pH in the duodenum, such as gastrinoma (Zollinger-Ellison syndrome), can inactivate lipase and cause pancreatic insufficiency. Pancreatic insufficiency may also be caused by gastrointestinal surgeries 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 reduced ability to digest TG-LCPUFAs, esterified LC-PUFAs, and / or other long-chain triglycerides and fatty acid esters include, for example, irritable bowel syndrome, hypertriglyceridemia, nutritional disorders including protein-calorie malnutrition, pancreatic and duodenal neoplasms, abdominal radiation therapy, hemochromatosis, primary sclerosing cholangitis, primary biliary cirrhosis, Shwachman syndrome, trypsinogen deficiency, enterokinase deficiency, or lipase alone deficiency (see D. Kasper et al., Harrison’s Principles of Internal Medicine, 16th Edition (2004)). Also, reduced ability to digest long-chain triglycerides or esterified long-chain PUFAs can occur due to bowel resection, cystic fibrosis, cerebral palsy, short bowel syndrome, IBD, pancreatitis, Crohn's disease, diarrhea, gastrointestinal fistula, celiac disease, malabsorption syndrome, trauma / surgery (especially GI trauma or surgery), radiation enteritis, bowel insufficiency, chylothorax, cancer (especially pancreatic cancer or GI cancer), and / or wound healing. The exact cause is unknown, but levels of LC-PUFAs are also reduced in children with attention deficit hyperactivity disorder (ADHD) (see Burgress et al., Am. J. Clin. Nutri. 71(Suppl):327S-30S (2000)).

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

[0043] Cachexia and weight loss due to catabolic state of tissues, altered nutrient pathways, and malabsorption in the advanced stage are common in the advanced stage of many cancers, but pancreatic cancer (PC) is exceptional in having weight loss and malabsorption, which are present in 80% - 90% of patients at the time of diagnosis. Malabsorption resulting from exocrine deficiency, a major factor in weight loss, is due to loss of pancreatic soft tissue, blockage of pancreatic ducts that inhibits enzymes from reaching the intestine, and surgical techniques. The common final result of all these mechanisms is steatorrhea and weight loss (see Damerla et al., J of Support Oncology 6:393 - 396 (2008)). Stabilization of weight 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 compositions and methods for improving the absorption of fats, such as LC - PUFAs, in PC patients. In some embodiments, the present invention provides compositions and methods for inducing weight gain in PC patients.

[0044] Some embodiments of the present invention improve current treatment options for pancreatic insufficiency and other conditions that reduce the ability to hydrolyze TG-LCPUFAs, esterified LC-PUFAs, and / or other long-chain triglycerides and fatty acid esters. In patients with a reduced ability to hydrolyze TG-LCPUFAs, esterified LC-PUFAs, and / or other long-chain triglycerides and fatty acid esters, simply increasing the consumption of these nutrients without improving hydrolysis can cause steatorrhea, abdominal pain, muscle spasms, 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, which are due to the fact that many commercially available lipase supplements are derived from animal sources. Thus, embodiments of the present invention that provide pre-hydrolyzed long-chain triglycerides or long-chain PUFA esters will provide a better and safer method for treating pancreatic insufficiency or other reduced ability to digest long-chain triglycerides or esterified long-chain PUFAs, with or without the addition of lipase.

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

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

[0047] Reduced ability to hydrolyze long-chain fatty acids in premature 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, which begins from 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)). Normal fatty acids including DHA, EPA, and ARA, as well as the lipases necessary 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 a significantly higher risk of inadequate fatty acid supply due to their shortened gestational period after their dependence 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)). In addition to this, in retrospective studies of the fatty acid profiles of preterm infants, inappropriate levels of long-chain PUFAs have been associated with an increase in chronic lung disease and sepsis, presumably due to dysregulation of the immune response (see C. Martin et al., J. Pediatr. 159(5):743-749 (2011)). Studies such as these suggest that establishing appropriate levels of long-chain PUFAs in preterm infants, even using formula supplemented with DHA and other long-chain triglycerides or long-chain fatty acid esters, is an important and potentially unmet problem. The compositions, methods, and devices of the present invention will enable preterm infants to receive sufficient amounts of long-chain fatty acids and recognize the associated medical benefits.

[0049] Reduced ability to hydrolyze long-chain fatty acids in artificially fed infants In addition, artificially fed infants who are not supplemented with fatty acids may also be deficient in long-chain PUFAs. The levels of long-chain PUFAs have been found to be lower in unsupplemented artificially fed infants compared to breastfed infants (see B. Koletzo et al., J. Perinat. Med. 36(1):5-14 (2008)). Even in breastfed infants, the amount of DHA in breast milk varies and is related to maternal diet intake, so there is a possibility of deficiency in n-3 fatty acids. A positive correlation has been demonstrated between the amount of DHA in breast milk and visual and language development in breastfed infants (see S. Innis, J. Pediatr. 143:S1-S8 (2003)). Therefore, a diet containing DHA is recommended for lactating women. For artificially fed infants, all major formula manufacturers have introduced special infant formulas containing fats with DHA and ARA. However, reports on the benefits of these DHA- and ARA-enriched formulas are inconsistent. Some studies have shown a significant effect on cognitive development when infants receive formula containing long-chain PUFAs, 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 for the first year of life experienced improved immune outcomes, including improved respiratory health, compared to infants fed the same lipid-free formula (see E. Birch et al., J. Pediatr. 156(6):902-906 (2010)). However, overall, the preclinical data do not show a consistent benefit for infant development with current long-chain PUFA-supplemented formulas.

[0050] One explanation for these conflicting results of the studies is that some infants are unable to absorb the required amount of essential fatty acids from the intestine when fed formula supplemented with long-chain triglycerides or long-chain fatty acid esters. The inability to absorb this fatty acid may be due to the low level of endogenous pancreatic lipase in the infant. Since lipase is generally carried to the infant through breast milk, formula-fed infants may not have sufficient levels of lipase to break down long-chain PUFAs or PUFA esters to monoglycerides and / or free fatty acids for intestinal absorption. As a result, infants fed LC-PUFA-supplemented formula still have lower absorption of LC-PUFA compared to breastfed infants. Again, there is a clear need not only to provide fatty acid supplements, but also to enable the hydrolysis and absorption of these fatty acids.

[0051] Adding lipase to legally defined infant formula (or, for example, medical nutrition formulations) may require significant development research to screen for suitable lipase supplements and to stabilize and formulate them. In unregulated formula where sufficient testing has not been done, problems related to lipase stability, lack of specificity, purity, and / or interference with other substances may result in the enzyme being at excessive or potentially harmful levels. The addition of large amounts of new substances that cross regulatory hurdles further introduces another variable of how well individuals, particularly infants, with low ability to hydrolyze long-chain triglycerides tolerate the formulation. This problem exists, for example, in the formulations described in U.S. Patent No. 5,902,617 (Pabst) and U.S. Patent No. 4,944,944 (Tang).

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

[0053] In some embodiments, this nutritional composition improves fatty acid absorption. In some embodiments, the subject ingests this nutritional composition over 3 days, 5 days, 7 days, 10 days, 14 days, 30 days, 60 days, or longer. In some embodiments, such ingestion of the nutritional composition of the present invention can reduce total fat in the feces, specifically, reduce 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 starting ingestion of this nutritional composition. In some embodiments, this reduction is measured relative to the fecal composition of a subject supplied with a nutritional composition not exposed to lipase, such as currently available nutritional compositions, prior to ingestion. The levels of total fat, DHA, ARA, and / or EPA in the feces 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 feces are reduced to between 50% and 80%. In some embodiments, the level of total fat in the feces 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 feces 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 feces is reduced by at least 60%. In some embodiments, the levels of DHA, ARA, or EPA in the feces are each reduced by at least 50%. In some embodiments, the levels of DHA, ARA, or EPA in the feces are each reduced by at least 60%. In some embodiments, ingestion of this nutritional composition improves plasma, erythrocyte, and tissue accumulation of fat levels including levels of DHA and ARA. Tissues can include retinal, cardiac, adipose, and renal tissues. In some embodiments, ingestion of this nutritional composition increases the levels of DHA, ARA, or both in plasma, erythrocytes, or both.In some embodiments, ingestion of this nutritional composition increases the levels of DHA, ARA, or both in the retina. In some embodiments, ingestion of this nutritional composition increases the levels of DHA, ARA, or both in the heart. In some embodiments, ingestion of this nutritional composition increases the plasma levels of triglycerides, cholesterol, HDL, and / or LDL. In some embodiments, ingestion of this nutritional composition increases the ratio of HDL to LDL in the plasma of the subject.

[0054] In some embodiments, ingestion of this nutritional composition increases the plasma levels of vitamin A and / or vitamin E. Without being bound by theory, this increase is thought to be due to the fact that vitamins A and E are typically provided as esters that are to be hydrolyzed. Exposure to lipase in the various methods and compositions of the present invention improves the hydrolysis of these vitamin esters, which further accumulates vitamins A and E in the plasma.

[0055] In some embodiments, ingestion of this nutritional composition has beneficial effects without significantly increasing the accumulation of fat in the liver. Fatty liver disease (FLD) is characterized by an increase in the accumulation of fat, particularly 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 by itself, which does not induce symptoms. In some patients, fatty liver may be accompanied by liver inflammation and hepatocyte death (steatohepatitis). There is also an association with liver cancer (hepatocellular carcinoma). Insulin resistance, as well as increased consumption of carbohydrates and saturated fatty acids, and low intake of fiber and omega-3 fatty acids are all clearly associated with the etiology of FLD.

[0056] Causes of FLD include diet, drug therapy, diseases, and medical conditions. Excessive calorie consumption induces FLD, as excessive calorie intake overwhelms the liver's ability to metabolize fat in the normal manner, leading to fat accumulation in the liver. Many drug therapies, including tamoxifen, amiodarone injection, oral amiodarone, and methotrexate, are associated with FLD. Fatty liver is also associated with type II diabetes, obesity, and high blood triglyceride levels, celiac disease, and Wilson's disease (abnormal 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 supplementary digestive enzymes including pancreatic lipase. However, pancreatic enzymes, particularly pancreatic lipase present in these nutritional supplements, are often sensitive to degradation by gastric acid and pepsin, so only a very small portion of the ingested enzyme reaches the duodenum in an active form (see E. Ville et al., Digestion 65:73-81 (2001)). However, many acid protection coatings have potential safety issues for the infant population or immunocompromised patients because a significant portion of the delivered weight is a plastic coating. Furthermore, even when the acid protection coating is helpful, some degree of malabsorption persists, necessitating an increased dosage of enzyme nutritional supplements in patients with pancreatic insufficiency. This presence of fatty acid malabsorption with enteric-coated enzymes may be due to the fact that the duodenum and upper jejunum of patients with pancreatic insufficiency are often in an acidic environment, so the expected increase in pH is not achieved and the protective coating does not dissolve properly to release the enzyme (see 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 mentioned above, high doses of pancreatic enzyme supplements have been found to be associated with fibrotic colon disease. Accordingly, some embodiments of the present invention provide nutritional formulations that contain higher levels of long - chain monoglycerides and / or free fatty acids without additional lipase. Some embodiments provide nutritional formulations that contain the optimal dose of lipase described herein.

[0058] Lipase can be obtained from animals, plants, and many natural or genetically engineered microorganisms. Although not most, many commercially available dietary lipase supplements are of animal origin and are particularly susceptible to degradation by digestive enzymes. An alternative with lower frequency of use is microbial lipase, i.e., lipase produced by bacteria or fungi such as yeast. Microbial lipase retains activity over a wider pH range than animal or plant lipase and thus eliminates the need for enteric - coated tablets. However, microbial enzymes are easily degraded by trypsin in the small intestine, thereby reducing their availability for decomposing 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 specificities and kinetics of individual lipases can vary widely. The specificity of lipase 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., the ability of a given lipase to be active in decomposing one type of fatty acid more than another lipase) and positional specificity (which involves the 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 are Candida rugosa, Rhizomucor miehei, Penicilium camemberti, Aspergilus niger It has been shown to have greater specificity for DHA, EPA, and ARA than other lipases such as lipases produced by Chromobacterium viscosum, Aspergillus niger, and Aspergillus oryzae. As a result, lipase nutritional supplements or lipase replenishment nutritional products containing Chromobacterium viscosum, Pseudomonas Fluorescens, Burcholderia cepacia, and / or Rhizopus oryzae will provide an increase in the hydrolysis of TG-DHA, TG-EPA, and / or TG-ARA. Accordingly, one aspect of the present invention provides a lipase nutritional supplement or lipase replenishment nutritional product comprising Chromobacterium viscosum lipase, Pseudomonas Fluorescens lipase, Burcholderia cepacia lipase, and / or Rhizopus oryzae lipase. In some embodiments, the lipase is Chromobacterium viscosum lipase, Pseudomonas Fluorescens lipase, or Rhizopus oryzae lipase. In certain embodiments, the lipase is Rhizopus oryzae lipase.

[0061] References to lipases of certain species, such as Chromobacterium viscosum lipase, Pseudomonas Fluorescens lipase, Burcholderia cepacia lipase, and Rhizopus oryzae lipase, do not necessarily require that the lipase be prepared directly from a wild host tumor. For example, the same lipase can be recombinantly produced in another host cell.

[0062] Another aspect of the present invention is to increase the absorption of DHA, EPA, and / or ARA by administering one or more of Chromobacterium viscosum, Pseudomonas Fluorescens, Burcholderia cepacia, and Rhizopus oryzae lipases as dietary supplements, or by pre-hydrolyzing a formulation containing DHA, EPA, and / or ARA with one or more of these enzymes. In some embodiments, the lipase is Chromobacterium viscosum lipase, Pseudomonas Fluorescens lipase, or Rhizopus is a lipase of Rhizopus oryzae. An additional aspect of the invention is a lipase having specific activity against DHA, EPA, and / or ARA that is determined by reverse-phase high-performance liquid chromatography (RP-HPLC) and is comparable to the specific activity of one or more of Chromobacterium viscosum, Pseudomonas Fluorescens, Burcholderia cepacia, and Rhizopus oryzae as described in Example 1. In some embodiments, this lipase has specific activity against DHA, EPA, and / or ARA that is comparable to the specific activity of one or more of Chromobacterium viscosum lipase, Pseudomonas Fluorescens lipase, or Rhizopus oryzae lipase. One embodiment of the invention is a nutritional formulation containing less than 5,000 units of lipase (using units as evaluated by a standard olive oil assay, such as those described in Pharmaceutical Enzymes: Properties and Assay Methods, R. Ruyssen and A. Lauwers (editors), Scientific Publishing Company, Ghent, Belgium (1978)). In other embodiments, this nutritional formulation contains less than 3,000 units of lipase. In some embodiments, this nutritional formulation contains less than 1,000 units. In certain forms, formulations containing less than 5,000, less than 3,000, or less than 1,000 units of lipase are infant formula or medical nutritional formulations.

[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, makes it reusable, and allows the product 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 bonds may also be used. Suitable methods for immobilization of lipase include, for example, adsorption, ionic binding, covalent binding, cross-linking, encapsulation, and entrapment in hydrophobic or hydrophilic polymer matrices and inorganic matrices. See Y. Ren et al., BMC Biotechnol. 11:63 (2011); V.R. Murty et al., Biotechnol. Bioprocess Eng. 7:57-66 (2002). The lipase may be immobilized by binding directly to the support material or through a linker. See, for example, Stark and Holmberg, Biotechnol. and Bioeng. 34(7):942-950 (1989).

[0064] Immobilization by adsorption is reversible and typically involves hydrophobic interactions. This is simple and inexpensive but has 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); H. Gunnlaughsdottir et al., Enzyme and Microbiol. Tech. 22:360 - 367 (1998) (Humicola lanuginose lipase; glass beads support). Suitable supports for immobilization by adsorption include, for example, ceramic beads such as Toyonite (Toyo Denko Kogyo Co., Ltd.).

[0065] Ionic binding is based on the electrostatic interaction between the 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 change in the form of the lipase and often results in an immobilized lipase with high activity. However, it should be understood that the binding force between the enzyme and the support, while stronger than when using adsorption, is not as strong as covalent binding, and thus leakage of the lipase from the support may occur.

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

[0067] Cross-linking involves binding lipase to itself to form a three-dimensional structure or using a cross-linking agent to bind 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 usually involves the formation of a porous coating or a semi-permeable membrane around the lipase, whereby the lipase is contained inside the porous material, but triglycerides and esters can pass through freely. Immobilization of lipase by confinement involves restricting the movement of the enzyme by confining the enzyme within a lattice structure. Alginate beads may be used for this type of immobilization (see I. Bushan et al., J. Bioactive and Compatible Polymers 23(6):552-562 (2008)). Synthetic and natural polymers may be used. See also G. Fernandez-Lorente et al., J. Am. Oil Chem. Soc. (published online on December 14, 2010) and G. Fernandez-Lorente et al., J. Am. Oil Chem. Soc. 88:1173-1178 (2011).

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

[0069] In some embodiments, the 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 the lipase reacts. One suitable coating for MNPs is, for example, polydopamine. See, for example, Y. Ren et al., BMC Biotechnology 11:63 (2011). Using MNPs for lipase immobilization has advantages such as biocompatibility, superparamagnetism, small size, and low toxicity. The magnetic properties of the nanoparticles facilitate removal of the lipase from solution and also provide another means for attaching the 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 one or more lipases selected from Chromobacterium viscosum, Pseudomonas Fluorescens, Burcholderia cepacia, and Rhizopus oryzae. viscosum), Pseudomonas Fluorescens, Burcholderia cepacia, and Rhizopus oryzae.

[0071] In certain embodiments, with or without immobilization, lipase 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, this lipase is Rhizopus oryzae lipase.

[0072] Device containing immobilized lipase According to various embodiments, the present disclosure provides an apparatus and method for preparing a nutritional product. This apparatus and method can be used to expose infant formula or other nutritional products to lipase prior to consumption. Thus, lipase decomposes fats and oils with subsequent release of free fatty acids and monoglycerides. This apparatus and method will enable a convenient means for preparing infant formula or other nutritional products. In some embodiments, this apparatus and method avoid exposing an infant or other person consuming the product to exogenous lipase. In some embodiments, this apparatus and method enable the production of a formulation that contains monoglycerides and / or free fatty acids but does not contain any significant amount of lipase (as determined by ELISA).

[0073] Figures 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 apparatuses according to various embodiments of the present disclosure. As shown in Figure 1, the apparatus 100 of the present disclosure can include a container 110 configured to hold formula 120 for infants or other liquid nutritional products. As will be described in detail below, the container 110 can include immobilized lipase such that the formula 120 supplied to an infant through a nasogastric tube 114 or other feeding mechanism (e.g., a feeding bottle) does not contain any recognizable amount of lipase. For example, the lipase can be immobilized in a structure seen along the walls or within the structure such that the lipase is in fluid contact with the formula 120 within the container, or can be immobilized within the container in other ways. Further, as will be described with reference to the various embodiments below, the formula can be added to the container 110 in various ways that enable enzymatic treatment of the lipase within the container 110. For example, fluid can be supplied through tube 112 or poured into the container, and then can pass through a nasogastric tube or other device for feeding.

[0074] Throughout the present disclosure, the apparatuses and methods will refer to use in processing or preparing nutritional formulations such as, for example, formula for infants and medical nutritional compositions. It should be understood that this apparatus and method can be used to process or prepare any type of nutritional formulation for which it may be beneficial to provide lipase treatment prior to consumption. Such products can include any nutritional formulation consumed by a person with pancreatic insufficiency or by others with a reduced ability to hydrolyze long - chain triglycerides or esterified long - chain PUFAs.

[0075] Figures 2A - 2C and 3A - 3C illustrate more detailed apparatuses according to various embodiments. As shown, apparatuses 200 - 202, 300 - 302 can include containers 210, 310 for holding a liquid formulation. Containers 210, 310 can include a variety of different types and shapes. For example, containers 210, 310 can include glass or plastic jars or vials, bags (e.g., silicone or other flexible materials similar to IV saline bags), cylindrical containers such as syringe outer tubes, or other containers sized and shaped to hold a desired amount of formula or other products.

[0076] As previously described, the apparatus of the present disclosure is capable of exposing formula to lipase to obtain a desired enzymatic effect, while at the same time allowing the formula to be conveniently consumed without consuming the lipase thereafter. Thus, in various embodiments, when the formula is removed (e.g., through a nasogastric tube, through the nipple of a feeding bottle, or by transferring the formula to another container), the lipase is immobilized within containers 210, 310 such that it remains within the containers 210, 310 or can be removed from the formula prior to consumption. In other embodiments, the lipase is immobilized within containers 210, 310, e.g., on a removable solid support, such that the formula can be left within the container for later consumption while at the same time the lipase can be easily removed from the container.

[0077] Figures 2A - 2C show one configuration of container 210, along with a particular embodiment for immobilizing lipase within container 210. As previously mentioned, container 210 may have a variety of different materials, dimensions, and shapes. In addition to this, 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 in the container 210 in various ways. For example, the lipase can be immobilized or contained within structures 250, 252 located inside the container 210 (Figs. 2A and 2C). In addition to, or instead of, this, the lipase 251 can also be immobilized or contained within the interior of the wall of the container 210 (Fig. 2B). Thus, when the formula 260 is placed inside the container, the formula 260 contacts the lipase, resulting in the desired enzymatic action.

[0079] As described above, the lipase can be immobilized in the container by binding the lipase to structures 250, 252 within the container and / or the wall 251 of the container. The structures within the container can have various shapes. For example, in certain embodiments, the structure can comprise beads, balls, or any other structure that can be mobile within the container such that the structure can flow within the formula. For example, as shown in Fig. 4A, the structure 250 can include beads or balls having a surface wall 256 to which the lipase 257 can bind. Further, it should be understood that the structures 250, 252 can have various different shapes or configurations (e.g., cube-like, ovaloid, rod-shaped).

[0080] The configuration of the structures 250, 252 and / or the wall of the container 210 can be configured to provide a desired surface area such that the formula can contact a sufficient amount of lipase for an acceptable period of time. For example, the structures 250, 252 can include a number of beads 250 (Fig. 2A) or rod-shaped structures 252 (Fig. 2C) to provide a high surface area for binding a sufficient amount of lipase. Alternatively, if more time is available for incubating the formula with the lipase and / or if a lipase with high enzymatic activity is used, a smaller amount of lipase may be suitable.

[0081] In various embodiments, the structures 250, 252 and / or the container are configured such that lipase is not retained within the formula 260 at the same time the formula 260 is removed from the container for consumption or storage. For example, the beads 250 or rod-like structures can be sized such that they do not pass through the relatively small access ports 230. Alternatively or in addition, the structure can be attachable to the wall of the container and / or include a screen or filter sized to prevent movement of structures containing the formulation 260. Further, the structures 250, 252 can have other properties that facilitate their separation from the formula. For example, the structure 250 can be formed from magnetic beads that can be removed by binding to a magnetic filter.

[0082] In some embodiments, rather than fixing the lipase by attachment of the structure within the container 210 and / or to the wall of the container, the lipase 257' is housed within the structures 250, 252 and / or the wall 251 of the container. FIG. 4B illustrates one such embodiment. As shown, the structure 250' can include beads or other shapes having a wall 256'. This wall can be formed from a semi-permeable material that permits the inflow and outflow of the formula 260 but does not permit the inflow and outflow of the lipase 257'. Such encapsulation can similarly be used for other structures (e.g., 252) and / or the wall of the container such that the surface of the wall has the semi-permeable material and lipase can be housed therein.

[0083] The container can similarly have an increased amount of lipase and / or a surface configuration that provides an increased contact of the lipase with the formula 260. For example, the wall of the container can have ridges or other surface modifications to increase the surface area. Further, rather than containing a single open space, the container can contain a modification of the flow path (e.g., a winding path that permits long-term or extended exposure to the lipase and / or an assembly of channels or tubes through which the lipase is immobilized and through which the formula can flow). See, for example, element 150 of FIG. 1 and element 2101 of FIG. 21.

[0084] In certain embodiments, the container may be manufactured and pre-packaged with lipase in any of the embodiments described herein. At the time of use, the container can be opened, the formula placed therein, and brought into contact with lipase for a sufficient time to effect the desired enzymatic action. In other embodiments, a structure such as bead 250 having lipase immobilized on the surface of or contained / enclosed within the structure or a rod-shaped structure is packaged and dispensed, and these structures can be placed within a separate container containing the formula. In some embodiments, it may be effective to shake or agitate the container containing the immobilized lipase and the formula over a period of time.

[0085] As described, the formula 260 can be placed within the container 210 via various access ports. For example, the container can include a top access port 220 and / or a bottom access port 230. Ports 220, 230 can be used for the inflow and outflow of the formula, respectively. In addition to this, it is possible to use a single port or multiple ports may be used. These ports can include structures configured to engage with other devices that can be used for the supply or transfer of fluid. For example, these ports can include connectors such as luer lock connections, threaded portions, and / or conduits or tubes that can engage with a nasogastric tube. In addition to this, these ports can be configured to engage with any other structure that facilitates the transfer of fluid to another container in the supply or aids in the supply, such as a feeding bottle, a feeding bottle nipple. Further, one or both of ports 220, 230 may be provided with a valve 140 (FIG. 1), 240 (FIG. 2A) or other fluid flow control mechanism.

[0086] Figures 3A - 3C illustrate the apparatus of the present disclosure according to a particular embodiment. As shown, apparatuses 300 - 302 include a container 310 for receiving the formula 260. In addition to this, the container 310 can include a cap 322 such as a threaded top for a jar or bottle or other closure means. Similar to the embodiment shown in Figures 2A - 2C, this apparatus may comprise structures 350, 351, 351' and 353 containing lipase immobilized on and / or encapsulated within its surface.

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

[0088] Alternatively, or in addition, the structures 350, 351, 351’ can have a permeable outer wall with additional components that provide immobilized lipase contained within the structure. For example, structure 351’ (FIG. 3B) illustrates one embodiment where structure 351’ has a permeable outer wall that surrounds a number of beads 250. The outer wall may comprise a mesh or other configuration that provides contact with the beads 250 and allows for easy movement towards and from the structure 351’ of the formulation. Further, as described in the various embodiments above, the beads can provide lipase that can be immobilized on the surface of the beads or encapsulated within them.

[0089] As described above, this structure containing lipase can be manufactured and distributed as a pre-packaged component with container 310. Alternatively, or in addition, the structure may be packaged and distributed separately from the container. For example, a rod-like structure 350 or beads 351, 351’ having lipase contained within or immobilized thereon, or a cap 322 containing other things, may be manufactured and distributed. This cap may be configured for connection with a standard feeding bottle, water cylinder, or other container or instrument that may contain the formula.

[0090] In other embodiments, lipase can be provided such that it contacts the formula simultaneously when the formula is placed within a container and / or during feeding or when removed from the container. For example, FIG. 5A illustrates one device 500 according to an exemplary embodiment. Device 500 can include a standard feeding bottle nipple, and the lipase can be immobilized on the peripheral edge of the nipple or the inner surface 510 of the nipple itself. In this way, during normal use, the formula will contact the lipase. Similarly, the lipase can be contained within or on the interior of other structures that can be used for feeding, such as the fluid tube of a nasogastric feeding device.

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

[0092] The lipase housed within the housing 520 can be provided in various forms. For example, in some embodiments, the lipase is immobilized on beads 550 within the housing 520 by bonding or encapsulation as described above. Further, the housing 520 can include an open mesh or other configuration that allows the formula to pass through it. For example, using the bottle configuration shown in FIG. 5A, the open mesh or flow path through the housing 520 will contact the formula with the lipase as the formula exits the bottle during feeding. Alternatively, as shown in FIG. 5B, the formula can be injected into or dispensed from the top 530 of the housing and the formula can be contacted with the lipase while filling or emptying the container 310. The top 530 of the housing 520 and / or any other portion can be formed from various materials. For example, the housing 520 can be formed from a membrane that allows a controlled fluid flow. Further, the top 530 can be formed from a semi-permeable membrane that allows the fluid (formula) to flow through it but does not allow the lipase to pass through. Thus, the membrane forming the top 530 can serve to immobilize the lipase within the container 310 without binding or immobilizing the lipase within the container 310 in another way.

[0093] In various embodiments, the apparatus described above may comprise modifications for improving or otherwise controlling lipase activity. For example, vessels 110, 210, 310 can include a stirring system that allows for continuous movement of the formula during incubation, thereby allowing lipase to contact the fatty acids found throughout the fluid volume. Further, the apparatus can 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 lipase. In some embodiments, the lipase is in contact with the nutritional formulation within the container. In other embodiments, the lipase and the nutritional formulation are not in contact within the container. In some embodiments, the nutritional formulation and the lipase are contained within separate compartments within the container. In some embodiments, the nutritional formulation is in a dry form. In some embodiments, the nutritional formulation is in a 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 and the nutritional formulation are contacted by transferring them to another container (e.g., by transferring them 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] The devices according to the present disclosure can have many different shapes and / or configurations. For example, FIGS. 9A - 9C, 10A - 10C, 11A - 11C, 12, 13A - 13B, 14, 15, 16, 17A - 17B, 18, 19, 20, 21 illustrate various additional shapes and / or configurations. In each of the respective configurations described with respect to these drawings, lipase can be immobilized using any of the methods described above (e.g., by immobilizing lipase on a structure such as beads within the device and / or by immobilizing lipase inside or on the walls or other surfaces of the device). Further, a particular configuration may be selected to provide a wide variety of characteristics such as surface area, volume, amount of lipase, and / or exposure time of the substance to the enzyme.

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

[0097] FIGS. 9A - 9C illustrate a configuration for a device that can be disposed within a container for processing formula. As shown, the devices 900, 920 (FIGS. 9A and 9C) can have various shapes formed from outer walls 901, 901” that surround the lipase. As shown above, lipase 902 can be immobilized in various ways including attachment to beads. Further, the device 910 (FIG. 9B) can include a plurality of pockets or openings 903 formed within one or more walls 901’. The particular configuration, the number of pockets or openings, and the amount of lipase and / or the volume of the device can vary depending on the intended use and / or to control the lipase reaction rate.

[0098] In certain embodiments, the device can be configured to allow for changes in its dimensions or shape. For example, FIGS. 10A - 10C illustrate a device 1000, which can be compressed, for example, for storage within a container 1001 before use. Optionally, the container 1001 can be opened, and the walls 1003 of the device can expand to produce a desired ratio of the lipase volume 1002 to the container volume. In some embodiments, the device 1000 includes coils or springs 1004 that provide structural support and / or assist the device in maintaining its desired shape and / or volume.

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

[0100] In various embodiments, the lipase can be attached to a part of the cap or closure member of a bottle or jar such that when the cap or closure member is disposed on the bottle or jar, the lipase can contact the fluid contained within the bottle or jar. For example, any of the devices shown herein may be attached to the surface of the cap or closure member for contacting the milk preparation container within the bottle or jar. Various configurations of devices 1600, 1700, 1800, 1900, 2000 including lipase attached to caps or closure members 1602, 1702, 1802, 1902, 2002 are illustrated in FIGS. 16, 17A - 17B, 18, 19, and 20. As shown, the lipase can be housed within pockets or openings 1601, 1705, 1805, 1901, 2001 having various shapes or configurations. Further, in some embodiments, caps or closure members 1902, 2002 can include openings 1910, 2010 for inserting or removing fluid from the container, and such openings 1910, 2010 can include connectors for fluid tubes, such as luer - type connectors.

[0101] In some embodiments, it may be desirable to process the milk preparation when it is passing through a tube (e.g., during milk preparation supply as shown in FIG. 1 or during transferring milk preparation from one container to another). FIG. 21 illustrates another device 2100 for in - line processing of lipase. The device 2100 can include a pocket or opening 2101 containing lipase that can be immobilized as described above. Further, the pocket or opening 2101 may have a tortuous or curved flow path to allow for a longer contact time between the lipase and the milk preparation. In addition to this, the device 2100 may include openings 2110 at both ends to enable connection to a tube or conduit for the inflow or outflow of the milk preparation.

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

[0103] In some embodiments, the lipase can be immobilized within or on a component of the container such that the lipase does not contact the formula until a further step is initiated. For example, in one embodiment, the lipase may be housed within or on a portion of a cap or closure member that can include a mechanism for releasing the immobilized lipase into the container. For example, the lipase can be housed on or within beads or other structures (see, e.g., element 1805 of FIG. 18), and the beads or other structures can be attached to or housed within the cap such that, if desired, the lipase can be dripped into the container (e.g., by twisting the cap or removing a barrier / attachment mechanism). Similarly, the lipase can be attached to or housed within the walls of the container or other structures and immobilized on beads or other materials such that the lipase can be contacted with 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 lipase from Rhizopus oryzae immobilized on polymer beads. The immobilized lipase is present in dry granular form that can be added to the vessel or chamber of an apparatus according to the present invention, such as the apparatus shown in FIGS. 6B - D. At the outflow end of the chamber, a filter is simply provided that contains holes large enough to allow the formula to pass through, while retaining the immobilized lipase within the chamber, permitting the formula to pass through and exit the chamber, while the immobilized lipase may be trapped within the chamber of the apparatus. Alternatively, the lipase may be immobilized by coating the internal channels or chambers of the apparatus such that the formula is exposed to the lipase as it passes through the chamber. The lipase in such an apparatus can be used for continuous supply over a long period of time for increased stability and reusability of the lipase.

[0105] Nutritional preparation Certain embodiments of the present invention provide a nutritional formulation. In some embodiments, the nutritional formulation is an infant formula. In some embodiments, the nutritional formulation is a medical nutritional formulation. In some embodiments, the nutritional formulation is exposed to lipase prior to ingestion. In some embodiments, this exposure enables 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 that is constituted immediately prior to ingestion by the subject and has a composition different from that of the formulation sold by the manufacturer. The term “nutritional formulation” does not include the composition within the body of the subject after ingestion.

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

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

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

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

[0110] In some embodiments of the present invention, the approximate serving size of the nutritional composition of the present invention is about 100-110 mL for preterm infant formula, 90-150 mL (e.g., 148 mL) for full-term infant formula, 230-500 mL (e.g., 235-250 mL) for enteral nutrition, and 230-250 mL for pediatric and adult formulations. In some embodiments, each intake contains about 10-35 mg of ARA free fatty acid and monoglyceride (as obtained from complete hydrolysis of TG-ARA in currently available preterm and full-term infant formulas) or about 40-50 mg of ARA free fatty acid or monoglyceride (as obtained from complete hydrolysis of TG-ARA in currently available adult formulations). In some embodiments, each intake contains about 7-20 mg of DHA free fatty acid and monoglyceride (as obtained from complete hydrolysis of TG-DHA in currently available preterm and full-term infant formulas) or about 10-40 mg of DHA free fatty acid or monoglyceride (as obtained from complete hydrolysis of TG-DHA in currently available pediatric and adult formulations). The 230-250 mL adult intake contains about 1,100 mg of EPA free fatty acid and monoglyceride and about 240 mg of DHA free fatty acid and monoglyceride (as obtained from complete hydrolysis of TG-EPA and TG-DHA in some currently available adult formulations such as ProSure®). However, in some embodiments of the present invention, the ability to pre-hydrolyze TG-LCPUFA prior to ingestion allows for the production of formulations having higher levels of LC-PUFA than currently available formulations. Thus, in some embodiments, the amount of free fatty acid and / or monoglyceride of ARA and / or DHA exceeds the amount obtainable from complete hydrolysis of TG-LCPUFA in currently available formulations. In some embodiments, the intake of the nutritional composition of the present invention contains 50-100 mg of LC-PUFA free fatty acid and / or monoglyceride. In some embodiments, the intake of the nutritional composition of the present invention contains 100-200 mg of LC-PUFA free fatty acid and / or monoglyceride.In some embodiments, the intake amount of the nutritional composition of the present invention contains 200-300 mg of LC-PUFA free fatty acids and / or monoglycerides. In some embodiments, the intake amount of the nutritional composition of the present invention contains 250-500 mg of LC-PUFA free fatty acids and / or monoglycerides. In some embodiments, the intake amount of the nutritional composition of the present invention contains 500-1000 mg of LC-PUFA free fatty acids and / or monoglycerides. In some embodiments, the intake amount of the nutritional composition of the present invention contains 1-2 g of LC-PUFA free fatty acids and / or monoglycerides. In some embodiments, the intake amount of the nutritional composition of the present invention contains 2-3 g of LC-PUFA free fatty acids and / or monoglycerides.

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

[0112] In some embodiments, the nutritional composition of the present invention does not contain additional lipase. In some embodiments, the apparatus and / or method of the present invention are used to expose the nutritional composition to lipase, but since the nutritional composition is separated from the lipase before supply, the "as-fed" nutritional composition does not contain additional lipase. A nutritional composition that does not contain additional lipase refers to, for example, a composition in which the lipase is undetectable or present at a very low level because the immobilized lipase leaches from the solid support into the composition. In some embodiments, the nutritional composition contains lipase at 0.02% (w / w) or less, 0.01% (w / w) or less, 0.005% (w / w) or less, 0.002% (w / w) or less, 0.001% (w / w) or less, 0.0005% (w / w) or less, 0.0002% (w / w) or less, or 0.0001% (w / w) or less. In some embodiments, the nutritional composition contains lipase less than 0.02% (w / w), less than 0.01% (w / w), less than 0.005% (w / w), less than 0.002% (w / w), less than 0.001% (w / w), less than 0.0005% (w / w), less than 0.0002% (w / w), or less than 0.0001% (w / w).

[0113] In some embodiments, this nutritional composition contains lipase. In some embodiments, this lipase is Chromobacterium viscosum lipase, Pseudomonas Fluorescens lipase, Burcholderia cepacia lipase, and Rhizopus It is selected from lipases of Chromobacterium viscosum, Pseudomonas Fluorescens, and Rhizopus oryzae. In some embodiments, the lipase is selected from the lipases of Chromobacterium viscosum, Pseudomonas Fluorescens, and Rhizopus oryzae. In some embodiments, the lipase is the lipase of Chromobacterium viscosum. In some embodiments, the lipase is the lipase of Pseudomonas Fluorescens. In some embodiments, the lipase is the lipase of Rhizopus oryzae.

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

[0115] In some embodiments, the nutritional composition 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 composition. In some embodiments, the nutritional composition contains 0.1 to 500 mg of lipase per gram of total fat in the nutritional composition. In some embodiments, the nutritional composition contains 0.1 to 250 mg of lipase per gram of total fat in the nutritional composition. In some embodiments, the nutritional composition contains 0.1 to 200 mg of lipase per gram of total fat in the nutritional composition. In some embodiments, the nutritional composition contains 0.1 to 150 mg of lipase per gram of total fat in the nutritional composition. In some embodiments, the nutritional composition contains 0.1 to 100 mg of lipase per gram of total fat in the nutritional composition. In some embodiments, the nutritional composition contains 0.1 to 50 mg of lipase per gram of total fat in the nutritional composition. In some embodiments, the nutritional composition contains 1 to 50 mg of lipase per gram of total fat in the nutritional composition. In some embodiments, the nutritional composition contains 25 to 75 mg of lipase per gram of total fat in the nutritional composition. In some embodiments, the nutritional composition contains 1 to 100 mg of lipase per gram of total fat in the nutritional composition. In some embodiments, the nutritional composition contains 50 mg or less of lipase per gram of total fat in the nutritional composition.

[0116] In some embodiments, this nutritional composition contains 0.001 to 10 mg of lipase per milligram of total LC-PUFA (whether in the form of free fatty acids, monoglycerides, esters, or triglycerides) in the nutritional composition. In some embodiments, this nutritional composition contains 0.001 to 5 mg of lipase per milligram of total LC-PUFA in the nutritional composition. In some embodiments, this nutritional composition contains 0.001 to 3 mg of lipase per milligram of total LC-PUFA in the nutritional composition. In some embodiments, this nutritional composition contains 0.001 to 1 mg of lipase per milligram of total LC-PUFA in the nutritional composition. In some embodiments, this nutritional composition contains 0.001 to 0.5 mg of lipase per milligram of total LC-PUFA in the nutritional composition. In some embodiments, this nutritional composition contains 0.001 to 0.1 mg of lipase per milligram of total LC-PUFA in the nutritional composition. In some embodiments, this nutritional composition contains 0.001 to 0.05 mg of lipase per milligram of total LC-PUFA in the nutritional composition. In some embodiments, this nutritional composition contains 0.01 to 0.1 mg of lipase per milligram of total LC-PUFA in the nutritional composition. In some embodiments, this nutritional composition contains 0.02 to 0.08 mg of lipase per milligram of total LC-PUFA in the nutritional composition. In some embodiments, this nutritional composition contains 0.04 to 0.06 mg of lipase per milligram of total LC-PUFA in the nutritional composition. In some embodiments, this nutritional composition contains 0.1 mg or less of lipase per milligram of total LC-PUFA in the nutritional composition.

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

[0118] In some embodiments, this nutritional composition contains 0.001 to 10 mg of lipase per milligram of total ARA (whether in the form of free fatty acids, monoglycerides, esters, or triglycerides) in the nutritional composition. In some embodiments, this nutritional composition contains 0.001 to 5 mg of lipase per milligram of total ARA in the nutritional composition. In some embodiments, this nutritional composition contains 0.001 to 3 mg of lipase per milligram of total ARA in the nutritional composition. In some embodiments, this nutritional composition contains 0.001 to 1 mg of lipase per milligram of total ARA in the nutritional composition. In some embodiments, this nutritional composition contains 0.001 to 0.5 mg of lipase per milligram of total ARA in the nutritional composition. In some embodiments, this nutritional composition contains 0.001 to 0.1 mg of lipase per milligram of total ARA in the nutritional composition. In some embodiments, this nutritional composition contains 0.001 to 0.05 mg of lipase per milligram of total ARA in the nutritional composition. In some embodiments, this nutritional composition contains 0.01 to 0.1 mg of lipase per milligram of total ARA in the nutritional composition. In some embodiments, this nutritional composition contains 0.02 to 0.08 mg of lipase per milligram of total ARA in the nutritional composition. In some embodiments, this nutritional composition contains 0.04 to 0.06 mg of lipase per milligram of total ARA in the nutritional composition.

[0119] In some embodiments, this nutritional composition contains 0.001 to 10 mg of lipase per milligram of total EPA (whether in the form of free fatty acids, monoglycerides, esters, or triglycerides) in the nutritional composition. In some embodiments, this nutritional composition contains 0.001 to 5 mg of lipase per milligram of total EPA in the nutritional composition. In some embodiments, this nutritional composition contains 0.001 to 3 mg of lipase per milligram of total EPA in the nutritional composition. In some embodiments, this nutritional composition contains 0.001 to 1 mg of lipase per milligram of total EPA in the nutritional composition. In some embodiments, this nutritional composition contains 0.001 to 0.5 mg of lipase per milligram of total EPA in the nutritional composition. In some embodiments, this nutritional composition contains 0.001 to 0.1 mg of lipase per milligram of total EPA in the nutritional composition. In some embodiments, this nutritional composition contains 0.001 to 0.05 mg of lipase per milligram of total EPA in the nutritional composition. In some embodiments, this nutritional composition contains 0.01 to 0.1 mg of lipase per milligram of total EPA in the nutritional composition. In some embodiments, this nutritional composition contains 0.02 to 0.08 mg of lipase per milligram of total EPA in the nutritional composition. In some embodiments, this nutritional composition contains 0.04 to 0.06 mg of lipase per milligram of total EPA in the nutritional composition.

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

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

[0122] In some embodiments, the method for preparing a nutritional composition includes exposing a liquid nutritional composition to lipase. In some embodiments, the liquid nutritional composition includes LC-PIFA triglycerides or LC-PUFA esters. In some embodiments, the liquid nutritional composition includes triglycerides or esters 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 Pseudomonas Fluorescens lipase. In some embodiments, the lipase is Rhizopus oryzae lipase.

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

[0125] In some embodiments, this 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, this 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, the method results in a nutritional composition in which at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the total LC-PUFA in the nutritional composition 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 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 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 EPA is in the form of monoglycerides and / or free fatty acids.

[0127] For the purposes of this use, exposure of a nutritional composition or formulation to lipase refers to a period of time during which a liquid nutritional composition or liquid formulation is in contact with lipase that can be in solution or immobilized. For the purposes of this use, exposure to lipase ends when the formulation is ingested by the 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, this 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, this 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, this 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, this 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, this 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, this 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, this liquid nutritional composition is exposed to lipase for 10 minutes or less, and at least 50% of the DHA in the resulting nutritional preparation is in the form of monoglycerides and / or free fatty acids. In some embodiments, this liquid nutritional composition is exposed to lipase for 10 minutes or less, and at least 50% of the ARA in the resulting nutritional preparation is in the form of monoglycerides and / or free fatty acids. In some embodiments, this 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 preparation is in the form of monoglycerides and / or free fatty acids.

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

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

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

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

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

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

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

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

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

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

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

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

[0142] In some embodiments, when the nutritional preparation is supplied to a subject, this lipase remains in the nutritional preparation as such. In other embodiments, this lipase is removed from the liquid nutritional composition before the liquid nutritional composition is supplied to a subject. In some embodiments, this lipase is exposed to a solid support immobilized with a molecule that binds to lipase in the liquid nutritional composition containing lipase, thereby binding the lipase to the solid support and removing the liquid nutritional composition from the solid support. Since 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, before the lipase is exposed to the liquid nutritional composition, the lipase is immobilized on a solid support, and this lipase is removed by separating the liquid nutritional composition from the solid support. In some embodiments, this lipase is immobilized on at least a part of the inner surface of the chamber or on a solid support housed in the chamber, and the liquid nutritional composition is temporarily exposed to 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, and at least a part of the inner surface of this container is 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 composition that does not contain additional lipase. In some embodiments, the nutritional composition is exposed to lipase, but the nutritional composition is separated from the lipase prior to feeding so that the as-fed nutritional composition does not contain additional lipase. A nutritional composition that does not contain (or is free of) additional lipase refers to a composition in which the lipase is undetectable or present only at very low levels, for example, because the immobilized lipase leaches from the solid support into the composition. In some embodiments, the nutritional composition contains lipase at 0.02% (w / w) or less, 0.01% (w / w) or less, 0.005% (w / w) or less, 0.002% (w / w) or less, 0.001% (w / w) or less, 0.0005% (w / w) or less, 0.0002% (w / w) or less, or 0.0001% (w / w) or less. In some embodiments, the nutritional composition contains lipase less than 0.02% (w / w), less than 0.01% (w / w), less than 0.005% (w / w), less than 0.002% (w / w), less than 0.001% (w / w), less than 0.0005% (w / w), less than 0.0002% (w / w), or less than 0.0001% (w / w).

[0144] In some embodiments, the method comprises exposing a nutritional composition to less than 5,000 units of lipase per serving, where the units are as assayed in a standard olive assay such as Pharmaceutical Enzymes: Properties and Assay Methods, R. Ruyssen and A. Lauwers (editors), Scientific Publishing Company, Ghent, Belgium (1978). In other embodiments, the nutritional composition is exposed to less than 3,000 units of lipase per serving. In some embodiments, the nutritional composition is exposed to less than 1,000 units of lipase per serving. In certain embodiments, the composition 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 composition.

[0145] In some embodiments, the method of the present invention exposes a nutritional composition to lipase at 0.01 mg to 1 g of lipase per gram of total fat (which may be in the form of free fatty acids, monoglycerides, esters, or triglycerides) in the nutritional composition. In some embodiments, the method of the present invention exposes a nutritional composition to lipase at 0.1 to 500 mg of lipase per gram of total fat in the nutritional composition. In some embodiments, the method of the present invention exposes a nutritional composition to lipase at 0.1 to 250 mg of lipase per gram of total fat in the nutritional composition. In some embodiments, the method of the present invention exposes a nutritional composition to lipase at 0.1 to 200 mg of lipase per gram of total fat in the nutritional composition. In some embodiments, the method of the present invention exposes a nutritional composition to lipase at 0.1 to 150 mg of lipase per gram of total fat in the nutritional composition. In some embodiments, the method of the present invention exposes a nutritional composition to lipase at 0.1 to 100 mg of lipase per gram of total fat in the nutritional composition. In some embodiments, the method of the present invention exposes a nutritional composition to lipase at 0.1 to 50 mg of lipase per gram of total fat in the nutritional composition. In some embodiments, the method of the present invention exposes a nutritional composition to lipase at 1 to 50 mg of lipase per gram of total fat in the nutritional composition. In some embodiments, the method of the present invention exposes a nutritional composition to lipase at 25 to 75 mg of lipase per gram of total fat in the nutritional composition. In some embodiments, the method of the present invention exposes a nutritional composition to lipase at 1 to 100 mg of lipase per gram of total fat in the nutritional composition. In some embodiments, the method of the present invention exposes a nutritional composition to lipase at 50 mg or less of lipase per gram of total fat in the nutritional composition.

[0146] In some embodiments, the method exposes the nutritional composition to lipase at 0.001 to 10 mg of lipase per milligram of total LC-PUFA (whether in free fatty acid, monoglyceride, ester, or triglyceride form) in the nutritional composition. In some embodiments, the method exposes the nutritional composition to lipase at 0.001 to 5 mg of lipase per milligram of total LC-PUFA in the nutritional composition. In some embodiments, the method exposes the nutritional composition to lipase at 0.001 to 3 mg of lipase per milligram of total LC-PUFA in the nutritional composition. In some embodiments, the method exposes the nutritional composition to lipase at 0.001 to 1 mg of lipase per milligram of total LC-PUFA in the nutritional composition. In some embodiments, the method exposes the nutritional composition to lipase at 0.001 to 0.5 mg of lipase per milligram of total LC-PUFA in the nutritional composition. In some embodiments, the method exposes the nutritional composition to lipase at 0.001 to 0.1 mg of lipase per milligram of total LC-PUFA in the nutritional composition. In some embodiments, the method exposes the nutritional composition to lipase at 0.001 to 0.05 mg of lipase per milligram of total LC-PUFA in the nutritional composition. In some embodiments, the method exposes the nutritional composition to lipase at 0.01 to 0.1 mg of lipase per milligram of total LC-PUFA in the nutritional composition. In some embodiments, the method exposes the nutritional composition to lipase at 0.02 to 0.08 mg of lipase per milligram of total LC-PUFA in the nutritional composition. In some embodiments, the method exposes the nutritional composition to lipase at 0.04 to 0.06 mg of lipase per milligram of total LC-PUFA in the nutritional composition. In some embodiments, the method exposes the nutritional composition to lipase at 0.1 mg or less of lipase per milligram of total LC-PUFA in the nutritional composition.

[0147] In some embodiments, the method exposes a nutritional formulation to lipase at 0.001 to 10 mg of lipase per milligram of total DHA (whether in the form of free fatty acids, monoglycerides, esters, or triglycerides) in the nutritional formulation. In some embodiments, the method exposes a nutritional formulation to lipase at 0.001 to 5 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the method exposes a nutritional formulation to lipase at 0.001 to 3 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the method exposes a nutritional formulation to lipase at 0.001 to 1 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the method exposes a nutritional formulation to lipase at 0.001 to 0.5 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the method exposes a nutritional formulation to lipase at 0.001 to 0.1 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the method exposes a nutritional formulation to lipase at 0.001 to 0.05 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the method exposes a nutritional formulation to lipase at 0.01 to 0.1 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the method exposes a nutritional formulation to lipase at 0.02 to 0.08 mg of lipase per milligram of total DHA in the nutritional formulation. In some embodiments, the method exposes a nutritional formulation to lipase at 0.04 to 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 includes exposing a liquid nutritional composition to an apparatus described herein.

Examples

[0149] Example 1: Specific Activity of Lipase Against DHA and ARA To evaluate the enzymatic activity of various lipases on DHA and / or ARA triglycerides, experiments were conducted 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. The 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 were also available from commercial sources such as Candida rugosa (Amano AY 30 or Amano 30, Amano Enzyme Inc., Nagoya, Japan), Aspergilus niger (Amano DS, Amano Enzyme Inc., Nagoya, Japan), Penicillium camembertii (Amano 50, Amano Enzyme Inc., Nagoya, Japan), Rhizomucor miehei (L4277, Sigma-Aldrich), Aspergilis oryzae (62285, Sigma-Aldrich), and Burcholderia cepacia (534641, Sigma-Aldrich). The lipase solutions were prepared from these commercial lipases without further purification, except that the B. cepacia lipase was purified to homogeneity.

[0150] The vial was transferred to a 37°C water bath placed on a magnetic stirrer. 50 μl of the sample was taken at different time intervals: 0, 15, 30, 45, 60, 90, and 120 minutes, and added to an HPLC vial containing 950 μl of mobile buffer (30% 10 mM ammonium phosphate buffer, pH 3.0 and 70% acetonitrile). The sample was then analyzed 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, for either DHA free acid or ARA free acid. Commercial standards: 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) were used to identify the free acid peaks by retention time. The specific activities of the lipase panel in this assay against DHA and ARA are summarized in Table 1. In the opinion of the present inventors, Chromobacterium viscosum (CV), Burcholderia cepacia (BC), Pseudomonas Fluorescens (PF), and Rhizopus oryzae (RO) had substantially higher specific activities against DHA and / or ARA compared to other lipases tested, including Candida rugosa (CR).

Table 1

[0151] Example 2: Enzymatic Activities of Lipases from Chromobacterium viscosum and Rhizopus oryzae against DHA, ARA, and EPA in Infant Formula To evaluate the enzymatic activities of CV and RO lipases on DHA, ARA, and EPA when supplemented in infant formula, an infant formula based on cow milk 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 substrate DHA (Nu-check Prep, Inc. lot number T-310-D7-V) and 5.4 mg of ARA (Nu-check Prep, Inc. lot number T-295) were added to a 1 mL glass vial (containing a magnetic stir bar). The reaction was initiated by adding the enzyme (i.e., lipase), and four concentrations of each enzyme were tested. The vial was transferred to a 37 °C water bath placed on a magnetic stirrer. 50 μl of each sample was taken at different time points, 0, 10, 20, 30, 45, and 60 minutes, and added to an HPLC vial containing 950 μl of HPCL mobile buffer (30% 10 mM ammonium phosphate buffer, pH 3.0 and 70% acetonitrile). The samples were then analyzed for either DHA acid, ARA acid, or EPA acid by PR-HPLC as described above.

[0152] The percentage of total triglycerides decreased over time as the amount of free acid and monoglyceride increased. For example, when hydrolyzed with RO, the amount of free DHA acid increased over time (Figure 7). Similarly, when hydrolyzed with RO, the amount of free ARA 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 acid, ARA acid, or EPA acid released in the infant formula and is shown in Table 2.

Table 2

[0154] Example 3: Hydrolysis of DHA Triglyceride and ARA Triglyceride Scale-up When scaled up to the amount that can be used to supplement infant formula, lipase was evaluated for its ability to hydrolyze TG-DHA and TG-ARA. Infant formula (milk) was prepared by dissolving 162 g of Enfamil powder in 648 mL of tap water (hot water, temperature was 37 °C). DHA triglyceride (442 mg, final concentration of DHA was 0.54 g, 1.2% of total fat) and ARA triglyceride (885 mg, final concentration of ARA was 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 a constant stirring state. Fat hydrolysis was initiated by adding either CV or RO lipase. Formula samples were taken out at 0, 15, and 30 minutes and analyzed for the hydrolysis of DHA and ARA 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 When supplemented in infant formula, to evaluate the enzymatic activity of immobilized RO lipase on TG-DHA or TG-ARA, infant formula mainly composed of milk was prepared by dissolving 10 g of ENFAMIL® powder in 35 mL of water. The reaction was carried out as follows. 0.1 M Tris buffer, pH 7.7, and substrate (TG-DHA or TG-ARA) were added to a 1 mL glass vial (equipped with a magnetic stir bar). The reaction was 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 taken at different time points of 0, 10, 20, and 30 minutes and added to an HPLC vial containing 900 μl of HPLC mobile buffer (30% 10 mM ammonium phosphate buffer, pH 3.0 and 70% acetonitrile). The sample was then analyzed for either DHA acid or ARA acid by RP-HPLC as described above.

[0156] The specific activity of lipase for the 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 The experiment was conducted on 12 pigs (9 + 3) obtained from pigs bred by the university at the Swedish Agricultural University, Department of Agricultural Biosystems and Technology in Odarslov, with each pig weighing approximately 10 ± 2 kg. The animals were maintained on a 12-hour light-dark cycle, with the light on from 06.00 to 18.00 (6 am to 6 pm) and the dark on from 18.00 to 06.00 (6 pm to 6 am). The pigs were individually housed in metabolic cages or individual barns equipped with a dry sample trough, drinking nipples, and a constant heating lamp (150 W). The pigs were allowed to move freely within the barn and were able to see each other.

[0158] 5.2 Feed During the post-operative and pre-treatment periods, the pigs were given a standard pig feed (「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 IU / kg of vitamin A, 500 IU / kg of vitamin D, and 85 mg / kg of vitamin E. The pigs were fed twice a day, at 09:00 - 10:00 (9 am - 10 am) and 17:00 - 18:00 (5 pm - 6 pm) (2.0% of body weight per meal). During the few days before the start of the experiment, i.e., the adaptation period, the pigs were trained to consume infant formula (NAN Pro 1 Gold Infant Formula, Nestle). Since pigs do not prefer to drink large amounts of liquid, the formula was prepared as a 1:4 dilution with tap water instead of the 1:7 dilution recommended by the manufacturer to allow for proper consumption. The daily nutritional requirement is 400 kL / kg body weight, which corresponds to 40 g of formula powder / kg body weight. The daily feed was divided into four portions, with the first meal starting at 9 am and given every 3 hours thereafter, and the last meal given at 6 am. 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 According to the manufacturer, NAN Pro 1 Gold (Nestle) is a high-quality whey-predominant starter 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 / ) Ingredients of NAN Pro 1 Gold: milk solids, vegetable oils (including 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 obtained from fish oil), emulsifier (soy lecithin), vitamins [sodium ascorbate (vit C), d-l alpha tocopheryl acetate (vit E), niacinamide (niacin), calcium pantothenate, retinyl acetate (vit A), thiamine nitrate (vit B1), pyridoxine hydrochloride (vit B6), riboflavin (vit B2), folic acid, phylloquinone (vit K1), biotin, cholecalciferol (vit D3)], cyanocobalamin (B12), L-histidine, taurine, inositol, nucleotides (cytidine 5'-monophosphate, uridine 5'-monophosphate, adenosine 5'-monophosphate, guanosine 5'-monophosphate), L-carnitine, cultures (Bifidus). Table 5 below outlines the lipid compositions of breast milk and infant formula, along with infant formula and piglet formula for use in these experiments. * [Table 5] ​The total concentration of TG-DHA and TG-AA in NAN formula milk is 0.22%, which is lower than the recommended level of 1%. Therefore, NAN formula milk was fortified with TG-DHA and TG-AA obtained from fish oil (NuCheck (http: / / www.nu-chekprep.com, ~40% TG-DHA and TG-ARA)) to reach the 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 on 12 + 2 young pigs at 6 - 8 weeks of age. EPI generally develops sufficiently 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, procedure 6.1 Test design The test included three periods: adaptation, control, and test. During the 7-day adaptation period, the pigs were trained to drink infant formula fortified with TG-DHA and TG-ARA. During the 7-day control period, the pigs were continuously given infant formula fortified with TG-DHA and TG-ARA. During the 7-day test period, the pigs were given infant formula fortified with TG-DHA and TG-ARA that was either (a) non-hydrolyzed, (b) pre-hydrolyzed with CV lipase, or (c) pre-hydrolyzed with RO lipase. Formula milk consumption was measured daily, fecal samples were collected on the last 3 days of each test period (72-hour collection), and blood samples were collected on the 7th day of the control and test periods.

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

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

[0164] 6.2.2 Control period (7 days) All selected pigs were given infant formula as the sole source of food four times a day. The total daily formula consumption was measured throughout the experiment. On the first morning of the control period, body weight was recorded before the morning feeding. Three 24-hour stool samples were collected on days 5 - 7. Blood samples were collected on the last day of this period at 1 hour before feeding and 1, 2, and 3 hours after feeding.

[0165] 6.2.3 Test period (7 days) All selected pigs were given TG-PUFA-enriched infant formula as the sole source of food four times a day. The total daily formula consumption was measured throughout the experiment. On the first morning of the test period, body weight was recorded before the morning feeding. Three 24-hour stool samples were collected on days 5 - 7. Blood samples were collected on the last day of this period at 1 hour before feeding and 1, 2, and 3 hours after feeding.

[0166] Before the start of this period, pigs were randomly assigned to the following three groups based on body weight and the willingness to drink formula milk. 1) Formula milk pre-hydrolyzed with RO lipase was given to one-third of the EPI pigs (n = 4). 2) Formula milk pre-hydrolyzed with CV lipase was given to one-third of the EPI pigs (n = 4). 3) Only formula milk was given to one-third of the EPI pigs (n = 4). The preparation of the formula milk and lipase mixture was described in the above paragraph

[0201] (

[0157] ).

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

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

[0169] Example 7: RO and CV lipases improve fatty acid absorption in EPI pigs Pigs with well - established exocrine pancreatic insufficiency (EPI), a surgical model, were used as a model mimicking premature or full - 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 pre - hydrolyzed infant formula with CV lipase or RO lipase on fatty acid absorption when compared to non - hydrolyzed infant formula. The EPI pigs were 10 weeks old (+ / - 2 weeks), which corresponds to approximately 6 months after birth in human infants. The pigs were given Nestle (NAN Pro 1 Gold) formula enriched with 2% arachidonic acid triglyceride (TG - ARA) and 1% docosahexaenoic acid triglyceride (TG - DHA) obtained from fish oil (NuCheck (http: / / www.nu - chekprep.com, ~40% TG - DHA and TG - ARA)). Diet supply was carried out 4 times a day, every 3 hours. In the group of pigs receiving pre - hydrolyzed formula, the formula was pre - hydrolyzed 15 minutes before supply by mixing it with CV lipase or RO lipase at 37°C. The duration of the experiment was 1 week, after which the LC - PUFA concentration in feces, plasma LC - PUFA absorption, and LC - PUFA accumulation in tissues (retina, heart, liver, kidney, erythrocytes, brain, and adipose) were analyzed.

[0170] As shown in Figure 22A, EPI pigs fed formula pre-hydrolyzed with CV lipase or RO lipase had significantly reduced fecal weights (CV: >60% decrease, p < 0.001; RO: ~30% decrease, p < 0.05). Pre-hydrolysis of fat by CV lipase or RO lipase also significantly decreased the total fat content in feces compared to control EPI pigs (Figure 22B) and significantly increased the fat absorption coefficient (%CFA) compared to controls (Figure 22C), where %CFA = (fat intake (g / 24 h) - fecal fat (g / 24 h)) / (fat intake (g / 24 h)), n = 3 / group, p = 0.002 (CV vs. control), and p = 0.003 (RO vs. control). When compared to controls, pre-hydrolysis with either CV lipase or RO lipase caused significant decreases in ARA (36% and 65% decreases, respectively), EPA (78% decrease with either enzyme), and DHA (68% and 60% decreases, respectively) in feces (Figure 23). These data indicate that pre-hydrolysis of formula with CV or RO lipase decreases the levels of total fat, ARA, DHA, and EPA in feces, suggesting an improvement in the absorption of ω-3, ω-6, and total fatty acids.

[0171] In addition, pigs fed pre-hydrolyzed formula had significant increases in plasma and tissue levels of ARA and DHA after 7 days of supply compared to control pigs. For these tests, the control and CV lipase groups consisted of 4 pigs each, and the RO lipase group consisted of 3 pigs. Plasma feed before the diet was collected after 7 days from the treatment and after an overnight fast. Plasma levels of ARA and DHA were significantly higher in pigs fed formula pre-hydrolyzed 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 pre-hydrolyzed with CV lipase compared to pigs fed non-hydrolyzed formula (40%, p<0.05) (Figure 24). ARA and DHA levels were also significantly increased in the retina (Figure 25A) and adipose tissue (Figure 25B) of pigs fed formula pre-hydrolyzed with RO lipase compared to pigs fed non-hydrolyzed formula (p<0.05). Retinal levels of ARA were also significantly higher in pigs fed formula pre-hydrolyzed with CV lipase compared to pigs fed non-hydrolyzed formula (p<0.05) (Figure 25A). In pigs fed formula pre-hydrolyzed with CV or RO lipase, ARA levels were also significantly increased in the heart (Figure 26A, 60% and 20% increases respectively) and kidneys (Figure 26B) of pigs compared to pigs fed non-hydrolyzed formula (p<0.05). DHA levels were significantly increased in the heart of pigs fed formula pre-hydrolyzed with CV lipase compared to pigs fed skin-hydrolyzed formula (60%, p<0.05) (Figure 26A). There were minimal or no changes in the liver, red blood cells, and brain, which can be explained by the relatively short duration (7 days) of the treatment in this test.

[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 placed in an apparatus similar to a tea bag. Enfalac infant formula (25 g) was combined with tap water (88 mL) at 37°C. Reactions were conducted 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 mixture 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 as the amount of immobilized RO lipase increased (Figure 27). These data support the practicality of the tea bag apparatus for pre-hydrolyzing formula with lipase.

[0174] Example 9: Hydrolysis of TG-DHA and TG-ARA in Infant Formula by Immobilized Lipase in a Cartridge Rhizopus oryzae (RO) lipase and Chromobacterium viscosum (CV) lipase were immobilized on macroporous acrylic polymer beads (Immobeads™; ChiralVision). Approximately 200 mg of RO lipase per gram of beads was used. Samples of CV lipase-coated beads were irradiated (CVI) to determine the effect of irradiation on the potency of the immobilized lipase. Approximately 1.7 g of each bead preparation (RO, CV, and CVI) was packed into columns with an approximate bed volume of 5 mL. Infant formula containing DHA and ARA triglycerides was passed over the columns at a flow rate of 75 mL / hour. Column eluents were analyzed by HPLC for hydrolysis of DHA and ARA. 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 against 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 the triglycerides of DHA and ARA. The reaction mixture was incubated at 37 °C for 15 minutes. Samples were taken at 0, 1, 2, 4, 6, 8, 10, and 15 minutes and analyzed for DHA and ARA by RP-HPLC. DHA (Figure 28A) and ARA (Figure 28B) triglycerides were hydrolyzed over time by RO lipase but not by pancreatin.

[0176] Example 11: 6-Week Study: Long-Term Nutritional Supply to Pigs with Infant Formula Pre-Hydrolyzed by Immobilized Lipase Six healthy pigs and 20 pigs with surgically induced exocrine pancreatic insufficiency (EPI) (see Example 5) were subjected to a 2-week adaptation / control period followed by a 6-week test period (Figure 29). During the adaptation period, all pigs were fed NAN Pro 1 Gold infant formula (Nestle) ("IF"). During the test period, six of the healthy pigs ("Healty") and pigs with EPI ("EPI") were fed infant formula fortified with TG-LCPUFA (see Examples 5 and 6), and seven of the pigs with EPI were fed TG-LCPUFA fortified infant formula pre-hydrolyzed with immobilized RO enzyme using a "tea bag" device ("EPI+iRO"). The remaining pigs were excluded from the study for various reasons, such as failure to surgically induce complete EPI.

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

[0178] Dietary 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 baseline samples, feces were collected over 48 hours (2 x 24 hours). For 1, 4, and 6-week samples, feces were collected over 72 hours (3 x 24 hours). At the end of the treatment period, organs and tissues were collected for absorption and safety testing.

[0179] The pre-hydrolyzed formula milk was durable with no change due to administration in dietary intake, growth, organs (by gross inspection), or general health status. When the pigs were sacrificed at the end of the 6-week test, no development of fatty liver was observed by gross inspection of the liver.

[0180] After 6 weeks, when compared to EPI pigs (EPI) given non-pre-hydrolyzed TG-LCPUFA fortified formula milk, in EPI pigs given pre-hydrolyzed formula milk (EPI+iRO), there were statistically significant increases in ARA (Figure 30A) and DHA levels (Figure 30B) in red blood cells (20% and 36% respectively). There was no significant difference in the red blood cell levels of ARA and DHA between healthy pigs and EPI pigs given pre-hydrolyzed formula milk for 6 weeks.

[0181] As shown in Table 7, in EPI pigs fed pre-hydrolyzed formula milk for 6 weeks, there were statistically significant increases in plasma levels of triglyceride, cholesterol, HDL, and LDL. There was no significant difference in plasma levels of triglyceride, cholesterol, HDL, and LDL between healthy pigs and EPI pigs fed pre-hydrolyzed formula milk over 6 weeks. [Table 7]

[0182] As shown in Table 8, pigs fed pre-hydrolyzed formula milk 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). For vitamin A, there was a statistically significant difference between the EPI and healthy groups (p<0.05), but not between the EPI+iRO and healthy groups. [Table 8]

Claims

【Claim 1】 The apparatus described in the specification.

Images