WO2013101516A1 - Methods for decreasing the incidence of necrotizing enterocolitis in infants, toddlers, or children using extracted genomic dna - Google Patents

Abstract

Disclosed are methods of reducing the incidence and/or risk of necrotizing enterocolitis in an infant, toddler, or child using nutritional compositions including extracted genomic bacterial DNA. The nutritional compositions including the extracted genomic DNA are further effective in reducing inflammation.

Description

METHODS FOR DECREASING THE INCIDENCE OF NECROTIZING ENTEROCOLITIS IN INFANTS. TODDLERS. OR CHILDREN USING

EXTRACTED GENOMIC DNA

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention hereby claims the benefit of the provisional patent application Serial No. 61/581,472, filed December 29, 2011, the disclosure of which is hereby incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

[0001 ] The present disclosure relates to extracted genomic DNA for reducing the incidence and or risk of necrotizing enterocolitis (NEC) and/or preventing and/or treating NEC in an infant, toddler, or child. More particularly, the present disclosure relates to human milk fortifiers, preterm and term infant formulas and pediatric formulas comprising extracted genomic DNA from

Lactobacillus' rhamnosus HNO01 that can reduce the risk of an infant, toddler, or child developing NEC.

BACKGROUND OF THE DISCLOSURE

[0002] The inflammatory response is an attempt by the body to restore and maintain homeostasis after invasion by an infectious agent, antigen challenge, or physical, chemical or traumatic damage. While the inflammatory response is generally considered a healthy response to injury, the immune system can present an undesirable physiological response if it is not appropriately regulated. Specifically, unregulated oxidation and associated inflammation are major causes of tissue damage and clinically significant disease in preterm and term infants. This is due in large part to the immaturity in function of the natural immune system of infants, and especially preterm infants.

[0003] Uncontrolled inflammatory responses in the immature gut may play a role in the pathogenesis of many intestinal inflammatory syndromes that present in newborns and children such as NEC. NEC is among the most common and devastating diseases in newborns. The mean prevalence of the disorder is about 7% among infants with a birth weight between 500 and 1500 g. The estimated rate of death associated with NEC ranges between 20 and 30%, with the highest rate among infants requiring surgery. Further, the excessive inflammatory process initiated in the highly immunoreactive intestine in NEC extends the effects of the disease systemically, affecting distant organs such as the brain and placing affected infants at a substantially increased risk for neurodevelopmental delays.

[0004] Breastfeeding has been associated with enhanced development and balanced growth and maturation of the infant's respiratory, gastrointestinal and immune systems, thereby providing protection of the infant to infection and inflammatory diseases. Breast milk appears to contain endogenous antioxidants, such as superoxide dismutase, glutathione peroxidase and catalase, or other non-enzymatic antioxidants such as glutathione, lactoferrin and polyphenols, in addition to exogenous antioxidants, such as vitamins A, C, E and selenium. Further, breast milk includes human milk oligosaccharides that not only act as pathogen receptor analogues, but activate immune factors by infant intestinal epithelial cells and/or associated immune cell populations. The function of these breast milk components, functioning as antioxidants and as immune modulators, includes not only the protection of breast milk lipids by peroxidation, but may also assist in the regulation of inflammatory responses to infection or other injury.

[0005] Not all infants receive human breast milk. Further, no prophylactic treatment(s) are currently available for the prevention of inflammatory diseases.

Therefore, development of safe and efficacious preventative or therapeutic methods would be beneficial, especially for preterm infants.

[0006] It would therefore be desirable to provide nutritional compositions that can modulate inflammation and enhance immunity such to reduce the incidences of NEC. It would further be advantageous if the nutritional compositions could support gastrointestinal maturity, gastrointestinal function, and mucosal immune development.

SUMMARY OF THE DISCLOSURE

[0007] The present disclosure is directed to nutritional compositions, including infant formulas, pediatric formulas, and child formulas, including genomic deoxyribonucleic acid (DNA) extracted from bacteria, including Lactobacillus rhamnosus HNOOl , as well as methods of using the compositions to reduce the incidence/risk of, treating and/or preventing NEC in an infant, toddler, or child. In some embodiments, the nutritional compositions further include one or more of prebiotics, human milk oligosaccharides (HMOs), long chain polyunsaturated fatty acids (LCPUFAs), and carotenoids in combination with the extracted genomic DNA.

[0008] One embodiment is a method of reducing the incidence of

necrotizing enterocolitis in an infant, toddler or child. The method comprises administering to an infant, toddler, or child at risk for necrotizing enterocolitis a nutritional composition comprising extracted genomic DNA from Lactobacillus rhamnosus HNOOl.

[0009] Another embodiment is a method of treating and/or preventing necrotizing enterocolitis in an infant, toddler, or child. The method comprises administering to an infant, toddler, or child at risk for necrotizing enterocolitis a nutritional composition comprising whole, extracted genomic bacterial DNA. The nutritional composition is selected from the group consisting of human milk fortifier, preterm infant formula, infant formula, pediatric formula, toddler formula, and follow- on formula.

[0010] Another embodiment is a nutritional composition comprising whole, extracted genomic DNA from Lactobacillus rhamnosus HNOOl.

[0011] Another embodiment is a nutritional composition comprising extracted genomic DNA from Lactobacillus rhamnosus HNOOl for use in reducing the incidence/risk of, treating, or preventing necrotizing enterocolitis.

[0012] It has been discovered that extracted genomic DNA, such as whole and/or fragmented extracted genomic DNA from Lactobacillus rhamnosus HNOOl, is highly effective in preventing and/or treating and/or managing and/or reducing the incidence of NEC in preterm infants, infants, toddlers, and children. It was previously demonstrated that toll-like receptor (TLR)-4 expression is increased in immature gut epithelial cells compared to mature epithelial cells. Further, activation of TLR-4 leads to translocation of NF-xl3, which is a transcription factor that regulates the expression of many genes involved in inflammatory responses, from cell cytoplasm to the nucleus, resulting in inflammatory responses. Lipopolysaccharide (LPS), a cell wall component of gram negative commensal and pathogenic microbes, is the agonist for TLR-4. Accordingly, as TLR-4 expression is increased in immature gastrointestinal epithelial cells, microbial colonization of the immature gastrointestinal tract can lead to increased inflammatory responses resulting in NEC. On the other hand, activation of TLR-9 in gastrointestinal epithelial cells attenuates TLR-4 signaling in the intestinal mucosa, thus attenuating inflammation (referred to as reciprocal signaling). Accordingly, previous attempts to reduce the incidence and severity of NEC include activating this reciprocal signaling process such as through administration of the TLR-9 agonist, CpG oligonucleotide (ODN), which attenuates TLR-4 signaling and expression.

[0013] Surprisingly, however, it has now been found that through

administration of extracted genomic DNA from Lactobacillus rhamnosus HNOOl, the incidence of NEC can be reduced without attenuating TLR-4. More particularly, it was found that administration of the extracted genomic DNA to mice increased TLR4 expression, as well as interleukin-10 (IL-10) expression, to levels similar to that of breast fed mice, and ameliorated clinical signs of NEC (e.g., diarrhea, decreases in body weight, and gastrointestinal histology). Further, while TLR-4 expression was increased, increased inflammation was not found. Particularly, it was unexpectedly found that the administration of extracted genomic DNA from Lactobacillus rhamnosus HNOOl modulated associated inflammatory biomarkers. For example, there was a decrease in inducible NO synthase (iNOS) and IL-6 expression compared to formula fed mice; that is, the expression levels were comparable to breast fed mice.

[0014] It has further been found that administration of extracted genomic DNA from Lactobacillus rhamnosus HNOOl supports gastrointestinal maturation, gastrointestinal function and mucosal immune development. Also, the nutritional compositions including extracted genomic DNA can be used to modulate allergy, respiratory infections, and chronic inflammation (e.g., irritable bowel disorder (IBD), Crohn's, ulcerative colitis).

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 depicts H&E staining of an intestinal section of a breast fed mouse as analyzed in Example 1. [0016] Figure 2 depicts H&E staining of an intestinal section of a breast fed mouse supplemented with DNA extracted from L. rhamnosus HNOOl as analyzed in Example 1.

[0017] Figure 3 depicts H&E staining of an intestinal section of a formula fed mouse as analyzed in Example 1.

[0018] Figure 4 depicts H&E staining of an intestinal section of a formula fed mouse supplemented with DNA extracted from L. rhamnosus HNOOl as analyzed in Example 1,

[0019] Figure 5 is a graph depicting intestinal iNOS mRNA expression in mice as analyzed in Example 1.

[0020] Figure 6 is a graph depicting intestinal IL-6 mRNA expression in mice as analyzed in Example 1.

[0021] Figure 7 is a graph depicting intestinal TLR-4 mRNA expression in mice as analyzed in Example 1.

[0022] Figure 8 is a graph depicting intestinal TLR-9 mRNA expression in mice as analyzed in Example 1.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0023] The nutritional compositions and methods described herein utilize genomic DNA that has been extracted from Lactobacillus rhamnosus HNOOl for controlling, preventing, treating and/or reducing NEC and/or the risk of NEC. In some embodiments, the extracted genomic DNA is included in the nutritional compositions in combination with prebiotics, anti-inflammatories (including long chain polyunsaturated fatty acids), and/or antioxidants.

[0024] These and other features of the nutritional compositions and methods, as well as some of the many optional variations and additions, are described in detail hereafter. [0025] The term "extracted genomic DNA" means DNA, either whole or fragmented, that has been purposely removed/isolated from a cell, including from Lactobacillus rhamnosus HNO01.

[0026] The terms "retort packaging" and "retort sterilizing" are used interchangeably herein, and unless otherwise specified, refer to the common practice of filling a container, most typically a metal can or other similar package, with a nutritional liquid and then subjecting the liquid-filled package to the necessary heat sterilization step, to form a sterilized, retort packaged, nutritional liquid product.

[0027] The term "aseptic packaging" as used herein, unless otherwise specified, refers to the manufacture of a packaged product without reliance upon the above-described retort packaging step, wherein the nutritional liquid and package are sterilized separately prior to filling, and then are combined under sterilized or aseptic processing conditions to form a sterilized, aseptically packaged, nutritional liquid product.

[0028] The terms "fat" and "oil" as used herein, unless otherwise specified, are used interchangeably to refer to lipid materials derived or processed from plants or animals. These terms also include synthetic lipid materials so long as such synthetic materials are suitable for oral administration to humans.

[0029] The term "probiotic" as used herein, unless otherwise specified, refers to a live or dead microorganism which, when administered in adequate amounts, confers a health benefit on the host. An example of a probiotic used herein is Lactobacillus rhamnosus HNO01.

[0030] The term "human milk oligosaccharide" or "HMO", as used herein, unless otherwise specified, refers generally to a number of complex carbohydrates found in human breast milk that can be in acidic or neutral form, and to precursors thereof. Exemplary non-limiting human milk oligosaccharides include 3'- sialyllactose, 6'-sialyllactose, 3'-fucosyllactose, 2'-fucosyllactose, and lacto-N-neo- tetraose. An exemplary human milk oligosaccharide precursor includes sialic acid. [0031] The terms "treat" or "treating" should not be taken to imply that an individual is treated until total recovery. Accordingly, these terms broadly include amelioration and/or prevention of the onset of the symptoms or severity of a particular condition (e.g., NEC).

[0032] The term "shelf stable" as used herein, unless otherwise specified, refers to a nutritional product that remains commercially stable after being packaged and then stored at 18-24°C for at least 3 months, including from about 6 months to about 24 months, and also including from about 12 months to about 18 months.

[0033] The terms "nutritional formulation" or "nutritional composition" as used herein, are used interchangeably and, unless otherwise specified, refer to nutritional liquids, nutritional powders, nutritional supplements, and any other nutritional food product as known in the art. The nutritional powders may be reconstituted to form a nutritional liquid, all of which comprise one or more of fat, protein and carbohydrate and are suitable for oral consumption by a human.

[0034] The term "nutritional liquid" as used herein, unless otherwise specified, refers to nutritional products in ready-to-drink liquid form, concentrated form, and nutritional liquids made by reconstituting the nutritional powders described herein prior to use.

[0035] The term "nutritional powder" as used herein, unless otherwise specified, refers to nutritional products in flowable or scoopable form that can be reconstituted with water or another aqueous liquid prior to consumption and includes both spraydried and drymixedldryblended powders.

[0036] The term "infant" as used herein, unless otherwise specified, refers to a person 12 months or younger. The term "preterm infant" as used herein, refers to a person bom prior to 36 weeks of gestation.

[0037] The term "toddler" as used herein, unless otherwise specified, refers to a person greater than one year of age up to three years of age.

[0038] The term "child" as used herein, unless otherwise specified, refers to a person greater than three years of age up to twelve years of age. [0039] The term "newborn" as used herein, unless otherwise specified, refers to a person from birth up to four weeks of age.

[0040] The term "infant formula" as used herein, unless otherwise specified, refers to liquid and solid human milk replacements or substitutes that are suitable for consumption by an infant.

[0041] The term "pediatric formula" as used herein, unless otherwise specified, refers to liquid and solid human milk replacements or substitutes that are suitable for consumption by an infant or toddler up to the age of 36 months (3 years).

[0042] The term "child formula" as used herein, unless otherwise specified, refers to liquid and solid human milk replacements or substitutes that are suitable for consumption by a child up to the age of 12 years.

[0043] The term "preterm infant formula" as used herein, unless otherwise specified, refers to liquid and solid nutritional products suitable for consumption by a preterm infant.

[0044] The term "human milk fortifier" as used herein, unless otherwise specified, refers to liquid and solid nutritional products suitable for mixing with breast milk or preterm infant formula or infant fonnula for consumption by a preterm or term infant.

[0045] The terms "inflammatory disease" or "inflammatory condition" as used herein, unless otherwise specified, refer to any disease, disorder, or condition characterized by inflammation. The term "infection-mediated inflammatory disease" as used herein, unless otherwise specified, refers to an inflammatory disease associated or induced by microbial infection, including viral and bacterial infection.

[0046] The terms "susceptible" and "at risk" as used herein, unless otherwise specified, mean having little resistance to a certain condition or disease, including being genetically predisposed, having a family history of, and/or having symptoms of the condition or disease. The nutritional compositions of the present disclosure are intended for use in the subset of individuals, including preterm and term infants, in need of assistance with respect to NEC as defined herein and not all individuals meet this requirement.

[0047] The terms "modulating" or "modulation" or "modulate" as used herein, unless otherwise specified, refer to the targeted movement of a selected characteristic.

[0048] All percentages, parts and ratios as used herein, are by weight of the total composition, unless otherwise specified. All such weights, as they pertain to listed ingredients, are based on the active level and, therefore, do not include solvents or by-products that may be included in commercially available materials, unless otherwise specified.

[0049] Numerical ranges as used herein are intended to include every number and subset of numbers within that range, whether specifically disclosed or not.

Further, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 2 to 8, from 3 to 7, from 5 to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

[0050] All references to singular characteristics or limitations of the present disclosure shall include the corresponding plural characteristic or limitation, and vice versa, unless otherwise specified or clearly implied to the contrary by the context in which the reference is made.

[0051] All combinations of method or process steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.

[0052] The nutritional compositions and methods may comprise, consist of, or consist essentially of the essential elements of the compositions and methods as described herein, as well as any additional or optional element described herein or otherwise useful in nutritional product applications. Product Form

[0053] The nutritional compositions of the present disclosure may be formulated and administered in any known or otherwise suitable oral product form. Any solid, liquid, or powder product form, including combinations or variations thereof, are suitable for use herein, provided that such forms allow for safe and effective oral delivery to the individual of the essential ingredients as also defined herein.

[0054] The nutritional compositions of the present disclosure include extracted genomic DNA, and in particularly suitable embodiments, whole, extracted genomic DNA from Lactobacillus rhamnosus HNOOl, or other bacteria, as described herein. The compositions may include the extracted genomic DNA alone or in combination with other immune enhancing factors including, but not limited to, prebiotics (and particularly, human milk oligosaccharides, GOS and/or FOS), antiinflammatories (and particularly, long chain polyunsaturated fatty acids (LCPUFAs)), and antioxidants, such as carotenoids and vitamins, as discussed below. Additional optional components including, for example, fats, carbohydrates, proteins, vitamins, and minerals may also be included in some of the nutritional composition embodiments of the present disclosure.

[0055] The nutritional compositions may be in any product form comprising the ingredients described herein, and which is safe and effective for oral

administration. The nutritional compositions may be formulated to include only some of the ingredients generally described herein, or may be modified with optional ingredients to foim a number of different product forms.

[0056] The nutritional compositions of the present disclosure are preferably formulated as dietary product forms, which are defined herein as those embodiments comprising the ingredients of the present disclosure in a product form that then contains at least one of fat, protein, and carbohydrate, and preferably also contains vitamins, minerals, or combinations thereof. The nutritional compositions will comprise extracted genomic bacterial DNA such as from Lactobacillus' rhamnosus HNO01, desirably in combination with at least one of protein, fat, vitamins, and minerals, to produce a nutritional composition.

[0057] The nutritional compositions may be formulated with sufficient kinds and amounts of nutrients to provide a sole, primary, or supplemental source of nutrition, or to provide a specialized nutritional product for use in individuals afflicted with specific diseases or conditions (e.g., NEC) or with a targeted nutritional benefit as described below.

[0058] Specific non-limiting examples of product forms suitable for use with the extracted genomic DNA-containing compositions as disclosed herein include, for example, liquid and powdered dietary supplements, liquid and powdered human milk fortifiers, liquid and powdered preterm infant formulas, liquid and powdered infant formulas, liquid and powdered elemental and semi-elemental formulas, liquid and powdered pediatric formulas, liquid and powdered toddler formulas, and liquid and powdered follow-on formulas suitable for use with infants and children.

Nutritional Liquids

[0059] Nutritional liquids include both concentrated and ready-to-feed nutritional liquids. These nutritional liquids are most typically formulated as suspensions or emulsions, although other liquid forms are within the scope of the present disclosure.

[0060] Nutritional emulsions suitable for use may be aqueous emulsions comprising proteins, fats, and carbohydrates. These emulsions are generally flowable or drinkable liquids at from about 1°C to about 25°C and are typically in the form of oil- in-water, water-in-oil, or complex aqueous emulsions, although such emulsions are most typically in the form of oil-in-water emulsions having a continuous aqueous phase and a discontinuous oil phase.

[0061] The nutritional emulsions may be and typically are shelf stable. The nutritional emulsions typically contain up to about 95% by weight of water, including from about 50% to about 95%, also including from about 60% to about 90%, and also including from about 70% to about 85%, of water by weight of the nutritional emulsions. The nutritional emulsions may have a variety of product densities, but most typically have a density greater than about 1.03 g/mL, including greater than about 1.04 g/mL, including greater than about 1.055 g/mL, including from about 1.06 g/mL to about 1.12 g/mL, and also including from about 1.085 g/mL to about 1.10 g/mL.

[0062] The nutritional emulsions may have a caloric density tailored to the nutritional needs of the ultimate user, although in most instances the emulsions comprise generally at least 19 kcal/fl oz (660 kcal/liter), more typically from about 20 kcal/fl oz (675-680 kcal/liter) to about 25 kcal/fl oz (820 kcal/liter), even more typically from about 20 kcal/fl oz (675-680 kcal/liter) to about 24 kcal/fl oz (800-810 kcal/liter). Generally, the 22-24 kcal/fl oz formulas are more commonly used in preterm or low birth weight infants, and the 20-21 kcal/fl oz (675-680 to 700 kcal/liter) formulas are more often used in term infants. In some embodiments, the emulsion may have a caloric density of from about 50-100 kcal/liter to about 660 kcal/liter, including from about 150 kcal/liter to about 500 kcal/liter. In some specific embodiments, the emulsion may have a caloric density of 25, or 50, or 75, or 100 kcal/liter.

[0063] The nutritional emulsion may have a pH ranging from about 3.5 to about 8, but are most advantageously in a range of from about 4.5 to about 7.5, including from about 5.5 to about 7.3, including from about 6.2 to about 7.2.

[0064] Although the serving size for the nutritional emulsion can vary depending upon a number of variables, a typical serving size is generally at least 1 mL, or even at least 2 mL, or even at least 5 niL, or even at least 10 mL, or even at least 25 mL, including ranges from about 1 mL to about 300 mL, including from about 4 mL to about 250 rnL, and including from about 10 mL to about 240 rnL.

Nutritional Solids

[0065] The nutritional solids may be in any solid form but are typically in the form of flowable or substantially flowable particulate compositions, or at least particulate compositions, that may optionally be compressed into tablets. Particularly suitable nutritional solid product forms include spray dried, agglomerated and/or dryblended powder compositions. The compositions can easily be scooped and measured with a spoon or similar other device, and can easily be reconstituted by the intended user with a suitable aqueous liquid, typically water, to form a nutritional composition for immediate oral or enteral use. In this context, "immediate" use generally means within about 48 hours, most typically within about 24 hours, preferably right after reconstitution.

[0066] The nutritional powders may be reconstituted with water prior to use to a caloric density tailored to the nutritional needs of the ultimate user, although in most instances the powders are reconstituted with water to fonti compositions comprising at least 19 kcal/fl oz (660 kcal/liter), more typically from about 20 kcal/fl oz (675-680 kcal/liter) to about 25 kcal/fl oz (820 kcal/liter), even more typically from about 20 kcal/fl oz (675-680 kcal/liter) to about 24 kcal/fl oz (800-810 kcal/liter). Generally, the 22-24 kcal/fl oz formulas are more commonly used in preterm or low birth weight infants, and the 20-21 kcal/fl oz (675-680 to 700 kcal/liter) formulas are more often used in term infants. In some embodiments, the reconstituted powder may have a caloric density of from about 50-100 kcal/liter to about 660 kcal/liter,

including from about 150 kcal/liter to about 500 kcal/liter. In some specific embodiments, the reconstituted powder may have a caloric density of 25, or 50, or 75, or 100 kcal/liter.

Extracted Genomic DNA

[0067] The nutritional compositions of the present disclosure include an extracted genomic DNA from probiotic bacteria. In addition to the extracted genomic DNA, a number of other components, including protein, fat, carbohydrate, prebiotics, anti-inflammatories/antioxidants, vitamins and minerals, for example, may also be optionally included in one or more of the nutritional compositions described herein and are further discussed below.

[0068] Extracted genomic DNA, such as whole and/or fragmented extracted genomic DNA from Lactobacillus rhamnosus HNO01, has been found to be highly effective in preventing and/or treating and/or managing and/or reducing the incidence of NEC in preterm infants, infants, toddlers, and children as described herein. Through administration of extracted genomic DNA from Lactobacillus rhamnosus HNO01, the incidence of NEC can be reduced without attenuating TLR-4, as described above. More particularly, it was found that administration of the extracted genomic DNA to mice increased TLR-4 expression, as well as interleukin-10 (IL-10) expression, to levels similar to that of breast fed mice, and ameliorated clinical signs of NEC (e.g., diarrhea, decreases in body weight, and gastrointestinal histology). Further, while TLR-4 expression was increased, increased inflammation was not found. Particularly, it was unexpectedly found that the administration of extracted genomic DNA from Lactobacillus rhamnosus HNO01 modulated associated inflammatory biomarkers. For example, there was a decrease in inducible NO synthase (iNOS) and IL-6 expression compared to formula fed mice; that is, the expression levels were comparable to breast fed mice.

[0069] It has further been found that administration of extracted genomic DNA from Lactobacillus rhamnosus HNO01 supports gastrointestinal maturation, gastrointestinal function and mucosal immune development. Also, the nutritional compositions including extracted genomic DNA can be used to modulate allergy, respiratory infections, and chronic inflammation (e.g., irritable bowel disorder (IBD), Crohn's, ulcerative colitis).

[0070] In some exemplary embodiments, the extracted genomic bacterial DNA may be from bacteria such as, but not limited to, Escherichia coli (Gram negative bacteria) or Lactobacillus or Bilidobacteria (Gram positive bacteria). In one specific embodiment, the extracted genomic bacterial DNA is derived from the probiotic Lactobacillus rhamnosus strain HNO01 (L. rhamnosus HNO01), also referred to as Lactobacillus rhamnosus DR20. It has surprisingly been discovered that extracted genomic DNA, and in some embodiments, whole, extracted genomic DNA from the probiotic L. rhamnosus HN001, is effective at treating and/or preventing NEC in an infant, toddler, or child when enterally administered to the infant, toddler, or child as described herein. It is believed that the extracted genomic DNA from L. rhamnosus HNO01 includes tolerogenic nucleic acids that have immunoregulatory properties that reduce inflammation causing NEC. Particularly, administration of the extracted genomic DNA activates inflammasomes, which can increase the ability of immune cells to rid the intestine of potentially pathogenic bacteria, thereby decreasing inflammation. Additionally, regulatory macrophages are stimulated, which further dampen inflammation.

[0071] L. rhamnosus HNO01 are heterofermentative bacteria that are Gram positive, non-motile, non-spore forming, catalase negative, facultative anaerobic rods exhibiting an optimal growth temperature of 37°C and an optimum pH of 6.0-6.5. See WO 2004/031389, herein incorporated by reference in its entirety. L. rhamnosus HNO01 are described in more detail in U.S. Patent No. 6,379,663, herein incorporated by reference in its entirety. L. rhamnosus HNOOl has previously been deposited with the Australian Government Analytical Laboratories (AGAL), deposit number

NM97/09514, on August 18, 1997 by the New Zealand Dairy Board, now known as Fonterra (Auckland, New Zealand). L. rhamnosus HNOOl is also commercially available from Danisco USA, Inc. (Madison, Wisconsin).

[0072] The genomic DNA may be extracted from the probiotic bacteria, and specifically L. rhamnosus HNOOl , using any methods known in the art that are sufficiently safe. Generally, gram-positive bacterial DNA extractions, such as extractions from L. rhamnosus HNOOl, are primarily completed with detergents, highly concentrated salt solutions, and organic solvents. Reagents such as sodium dodecyl sulfate (SDS) and ethylenediamine tetraacetic acid (EDTA) are used to destabilize microbial cell walls and cell membranes by disrupting polar interactions or by binding the divalent cations needed for membrane stability. Alternatively, bacterial cells can also be disrupted by use of a French press or microwave technique. Once cells are lysed, cetyltrimethylammonium bromide (CTAB), along with highly concentrated NaCl, is used to help purify bacterial DNA by combining with and dissociating polysaccharides and complex proteins. Phenol:chloroform:isoamyl alcohol (25:24:1) is the most potent reagent used in purifying DNA. Proteins are dissociated from DNA by phenol, whereas the chloroform denatures proteins and lipids; using these different organic solvents makes the deproteinization more efficient. The isoamyl alcohol is added to reduce foaming, which occurs during the extraction procedure. All three of these solutions together provide a sharp interface between an upper aqueous DNA containing phase and a lower organic phase containing protein and other cellular contaminants. [0073] DNA from the aqueous solution is treated with enzymes RNAse A and Proteinase K to remove RNA and further disassociate proteins and other cell wall components from DNA thereby aiding its isolation. Sodium acetate, as well as sodium chloride (NaCl), provide the positive charge, which neutralizes and stabilizes the DNA strands in order to enhance precipitation. Finally, ethanol is added, which decreases the dielectric effects of water, thereby allowing DNA precipitate to form.

[0074] Typically, about 1 mg extracted genomic DNA is extracted from 1 gram of bacterial paste, prepared as conventionally known in the art by growing bacterial cells on agarose plates supplemented with the appropriate nutrients for growth. It should be understood by one skilled in the art, however, that size of bacterial genome and loss of DNA due to the extraction procedure may influence the amount of genomic DNA recovered from the bacteria. It should further be recognized that recovery of genomic DNA may typically vary between about 10% and about 40% based on the extraction method used. Without being limiting, by way of example, based on the genomic size of Lactobacillus rhamnosus HNO01 and 100 percent recovery, extraction of 3.3 X 10¹⁰ bacteria will result in approximately 100 lag extracted genomic DNA.

[0075] Further, while discussed primarily herein as extracting whole, extracted genomic DNA, it should be recognized by one skilled in the art that the whole, extracted genomic DNA may further be fragmented using any DNA extraction methods known in the art. The nutritional compositions of the present disclosure may include whole, extracted genomic DNA, fragmented, extracted genomic DNA, and mixtures thereof.

[0076] Typically, the nutritional compositions will comprise extracted genomic DNA in sufficient amounts to provide a daily dose of at least 5 mg extracted genomic DNA per Kg body weight, including at least 10 [ig extracted genomic DNA per Kg body weight, including from 5 m extracted genomic DNA per Kg body weight to about 20 mg extracted genomic DNA per Kg body weight, including from 10 [ig extracted genomic DNA per Kg body weight to about 20 mg extracted genomic DNA per Kg body weight, including from about 100 [ig extracted, genomic DNA per Kg body weight to about 20 mg extracted genomic DNA per Kg body weight, and including from about 0.1 mg extracted genomic DNA per Kg body weight to about 10 mg extracted genomic DNA per Kg body weight.

[0077] Typically, the nutritional compositions of the present disclosure will include from about 0.00003% to about 3.5% by weight per volume of whole, extracted genomic DNA, including from about 0.00003%) to about 0.3%> by weight per volume, including from about 0.0001%> to about 0.25% by weight per volume, also including from about 0.001% to about 0.2% by weight per volume. By way of example, premature nutritional compositions for institutional and home use, typically providing approximately 24 kcal per fluid ounce, will include between about 0.0006% to about 1.3% by weight per volume genomic DNA. Human milk fortifiers may include from about 0.017% to about 3.4% by weight per weight genomic DNA. Accordingly, in one exemplary embodiment, a premature infant weighing approximately 1000 g and ingesting approximately 120 kcal per day of a nutritional composition comprising 0.07%) by weight per volume genomic DNA will receive about 1 mg of extracted genomic DNA on a daily basis.

[0078] In another embodiment, a nutritional composition for term infants may comprise from about 0.00012%) to about 0.23%> by weight per volume genomic DNA. Therefore, a term infant weighing approximately 3.5 kg and ingesting approximately 575 kcal from a composition including 0.0012% by weight per volume genomic DNA will receive about 1 Itg of extracted genomic DNA on a daily basis.

Prebiotics

[0079] The nutritional compositions of the present disclosure may optionally include a least one carbohydrate-based prebiotic in combination with the extracted genomic DNA. This carbohydrate-based prebiotic may be in addition to, or in place of, any other optional carbohydrate component as described herein. The prebiotics of the present disclosure are indigestible oligosaccharides that selectively stimulate the growth and/or activity of beneficial commensal or probiotic bacteria in the digestive system, and in particular L. rhamnosus HNO01, as well as other bacteria described herein. [0080] The carbohydrate-based prebiotic of the present disclosure may be selected from the group consisting of long chain fructooligosaccharides, short chain fructooligosaccharides (generally referred to as fructooligosaccharides), galactose- containing oligosaccharides (generally referred to as galactooligosaccharides), mammalian oligosaccharides (including oligosaccharides derived from human, bovine, ovine, caprine, and the like), milk oligosaccharides, and combinations thereof. Human milk oligosaccharides (HMO) are particularly desirable prebiotics.

[0081] Oligosaccharides are one of the main components of milk such as from mammals including humans, bovine, goats, sheep and the like. Human breast milk is particularly suitably as a source of oligosaccharides for use with the extracted genomic DNA of the present nutritional compositions. Human breast milk contains, on average, 10 grams per liter of neutral oligosaccharides and 1 gram per liter of acidic oligosaccharides. The composition of HMOs is very complex and more than 200 different oligosaccharide-like structures are known.

[0082] Suitable HMOs for use in the DNA-containing nutritional compositions may include acidic oligosaccharides, neutral oligosaccharides, nacetylglucosylated oligosaccharides, and HMO precursors. Specific non-limiting examples of HMOs that may be included individually or in combination in the compositions of the present disclosure include: sialic acid (i.e., free sialic acid, lipid-bound sialic acid, protein- bound sialic acid); D-glucose (Glc); D-galactose (Gal); Nacetylglucosamine

(GlcNAc); L-fucose (L-Fuc); D-fucose (D-fuc); fucosyl oligosaccharides (i.e., Lacto- N-fucopentaose I; Lacto-N-fucopentaose II; 2'- Fucosyllactose; 3'-Fucosyllactose; Lacto-N-fucopentaose III; Lacto-N-difucohexaose I; and Lactodifucotetraose); non- fucosylated, non-sialylated oligosaccharides (i.e., Lacto-N-tetraose and Lacto-N- neotetraose); sialyl oligosaccharides (i.e., 3'-Sialyl-3- fucosyllactose;

Disialomonofucosyllacto-N-neohexaose; Monofucosylmonosialyllacto-N-octaose (sialyl Lea); Sialyllacto-N-fucohexaose II; Disialyllacto-N-fiicopentaose II;

Monofucosyldisialyllacto-N-tetraose); and sialyl fucosyl oligosaccharides (i.e., 2'- Sialyllactose; 2-Sialyllactosamine; 3'-Sialyllactose; 3'-Sialyllactosamine; 6'- Sialyllactose; 6'-Sialyllactosamine; Sialyllacto-Nneotetraose c; Monosialyllacto-N- hexaose; Disialyllacto-N-hexaose I; Monosialyllacto-N-neohexaose I; Monosialyllacto-N-neohexaose II; Disialyllacto-N-neohexaose; Disialyllacto-N- tetraose; Disialyllacto-N-hexaose II; Sialyllacto-Ntetraose a; Disialyllacto-N- hexaose I; and Sialyllacto-N-tetraose b). Also useful are variants in which the glucose (Glc) at the reducing end is replaced by Nacetyl glucosamine (e.g., 2'- fucosyl-N-acetylglucosamine (2'-FLNac) is such a variant to 2'-fucosyllactose). These HMOs are described more fully in U.S. Patent Application No.

2009/0098240, which is herein incorporated by reference in its entirety. Other suitable examples of HMOs that may be included in the compositions of the present disclosure include lacto-N-fucopentaose V, lacto-N-hexaose, paralacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-neohexaose, monofucosyllactoN-hexaose II, isomeric fucosylated lacto-N-hexaose (1), isomeric fucosylated lacto-Nhexaose (3), isomeric fucosylated lacto-N-hexaose (2), difucosyl-para-lacto-Nneohexaose, difucosyl-para-lacto-N-hexaose, difucosyllacto-N-hexaose, lacto-Nneoocataose, para-lacto-N-octanose, iso-lacto-N-octaose, lacto-N-octaose, monofucosyllacto- neoocataose, monofucosyllacto-N-ocataose, difucosyllacto-Noctaose I, difucosyllacto-N-octaose II, difucosyllacto-N-neoocataose II, difucosyllacto-N- neoocataose I, lacto-N-decaose, trifucosyllacto-N-neooctaose, trifucosyllacto-N- octaose, trifucosyl-iso-lacto-N-octaose, lacto-N-difuco-hexaose II, sialyl-lacto-N- tetraose a, sialyl-lacto-N-tetraose b, sialyl-lacto-N-tetraose c, sialylfucosyl-lacto-N- tetraose I, sialyl-fucosyl-lacto-N-tetraose II, and disialyl-lacto-Ntetraose, and combinations thereof. Particularly suitable nutritional compositions include at least one of the following HMOs or HMO precursors: sialic acid (SA); 3'- Sialyllactose (3'SL); 6'-Sialyllactose (6'SL); 2'-Fucosyllactose (2 L); 3'- Fucosyllactose (3'FL); Lacto-N-tetraose and Lacto-N-neotetraose (LNnT), and in particular, combinations of 6'SL and 3'SL; combinations of 3'FL and. SA; combinations of 2'FL and 3'FL; combinations of 2'FL, 3'SL, and 6'SL; combinations of 3'SL, 3'FL, and LNnT; and combinations of 6'SL, 2'FL, and LNnT.

[0083] Other exemplary combinations include: SA, 3'SL, 6'SL, 3'FL, 2'FL, and LNnT; 3'SL, 6'SL, 3'FL, 2'FL, and LNnT; SA, 6'SL, 3'FL, 2'FL, and LNnT; SA, 3'SL, 3'FL, 2'FL, and LNnT; SA, 3'SL, 6'SL, 2'FL, and LNnT; SA, 3'SL, 6'SL, 3'FL, and LNnT; SA, 3'SL, 6'SL, 3'FL, and 2'FL; SA and 3'SL; SA and 6'SL; SA and 2'FL; SA and LNnT; SA, 3'SL, and 6'SL; SA, 3'SL and 3'FL; SA, 3'SL and 2'FL; SA, 3'SL and LNnT; SA, 6'SL and 3'FL; SA, 6'SL, and 2'FL; SA, 6'SL, and LNnT; SA, 3'FL, and 2*FL; SA, 3'FL, and LNnT; SA, 2'FL, and LNnT; SA, 3'SL, 6'SL, and 3'FL; SA, 3'SL, 6'SL and 2'FL; SA, 3'SL, 6'SL, and LNnT; SA, 3'SL, 3'FL, and 2'FL; SA, 3'SL, 3'FL, and LNnT; SA, 3'SL, 2'FL, and LNnT; SA, 6'SL, 3'FL, and 2'FL; SA, 6'SL, 2'FL, and LNnT; SA, 6'SL, 3'FL, and LNnT; SA, 3'FL, 2'FL, and LNnT; SA, 6'SL, 2'FL, and LNnT; SA, 3'SL, 3'FL, 2'FL, and LNnT; SA, 6'SL, 3'FL, 2'FL, and LNnT; SA, 3'SL, 6'SL, 3'FL, and LNnT; SA, 3'SL, 3'FL, 2'FL, and LNnT; SA, 3'SL, 6'SL, 2'FL, and LNnT; 3'SL, 6'SL, 3'FL, and 2'FL; 3'SL, 6'SL, 2'FL, and. LNnT; 3'SL, 3'FL, 2'FL, and LNnT; 3'SL, 6'SL, 3'FL, and LNnT; 3'SL, 6'SL, and 3'FL; 3'SL, 3'FL, and 2'FL; 3'SL, 2'FL, and LNnT; 3'SL, 6'SL, and 2'FL; 3'SL, 6'SL, and LNnT; 3'SL and 3'FL; 3'SL and 2'FL; 3'SL and LNnT; 6'SL and 3'FL; 6'SL and 2'FL; 6'SL and. LNnT; 6'SL, 3'FL, and LNnT; 6'SL, 3'FL, 2'FL, and LNnT; 3'FL, 2'FL, and LNnT; 3'FL and LNnT; and 2'FL and LNnT.

[0084] The HMOs may be present in the nutritional compositions in total amounts of HMO in the composition (mg of HMO per mL of composition) of at least 0.001 mg/mL, including from about 0.001 mg/mL to about 20 mg/mL, including from about 0.01 mg/mL to about 10 mg/mL, including from about 0.01 mg/mL to about 5 mg/mL, including from about 0.01 mg/mL to about 0.23 mg/mL of total HMO in the nutritional composition. Typically, the amount of HMO in the nutritional

composition may depend on the specific HMO or HMOs present and the amounts of other components, particularly the amount of extracted genomic DNA, in the nutritional compositions.

[0085] In one specific embodiment when the nutritional product is a nutritional powder, the total concentration of HMOs in the nutritional powder is from about 0.001% to about 5%, including from about 0.01% to about 1% (by weight of the nutritional powder).

[0086] In another specific embodiment, when the nutritional product is a ready- to-feed nutritional liquid, the total concentration of HMOs in the ready-to-feed nutritional liquid is from about 0.001% to about 0.50%, including from about 0.001% to about 0.15%), including from about 0.01% to about 0.10%, and further including from about 0.01%) to about 0.03% (by weight of the ready-to-feed nutritional liquid). [0087] In another specific embodiment when the nutritional product is a concentrated nutritional liquid, the total concentration of HMOs in the concentrated nutritional liquid is from about 0.002% to about 0.60%, including from about 0.002%) to about 0.30%), including from about 0.02%> to about 0.20%>, and further including from about 0.02%> to about 0.06%> (by weight of the concentrated nutritional liquid).

Anti-inflammatories/Antioxidants

[0088] In addition to the extracted genomic DNA described above, the nutritional products of the present disclosure may further optionally include antiinflammatories such as long-chain polyunsaturated fatty acids (LCPUFAs) and/or antioxidants such as carotenoids. LCPUFAs may be included in the nutritional compositions to provide nutritional support and to enhance growth and functional development of the intestinal epithelium and associated immune cell populations.

[0089] Exemplary LCPUFAs for use in the nutritional compositions include, for example, w-3 LCPUFAs and w-6 LCPUFAs. Specific LCPUFAs include docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (ARA), linoleic acid, linolenic acid (alpha linolenic acid) and gamma-linolenic acid derived from oil sources such as plant oils, marine plankton, fungal oils, and fish oils. In one particular embodiment, the LCPUFAs are derived from fish oils such as menhaden, salmon, anchovy, cod, halibut, tuna, or herring oil. Particularly preferred LCPUFAs for use in the nutritional compositions with the extracted genomic DNA include DHA, ARA, EPA, and combinations thereof.

[0090] In order to reduce any potential side effects of high dosages of LCPUFAs in the nutritional compositions, the content of LCPUFAs preferably does not exceed 3% by weight of the total fat content, including below 2% by weight of the total fat content, and including below 1 %> by weight of the total fat content in the nutritional composition.

[0091] The LCPUFA may be provided as free fatty acids, in triglyceride form, in diglyceride form, in monoglyceride form, in phospholipid form, or as a mixture of one or more of the above, preferably in triglyceride form. [0092] The nutritional compositions as described herein will typically comprise total concentrations of LCPUFA of from about 0.01 mM to about 10 mM and including from about 0.01 mM to about 1 mM. Alternatively, the nutritional compositions comprise total concentrations of LCPUFA of from about 0.001 g/L to about 1 g L.

[0093] Additionally, antioxidants such as carotenoids, and particularly, combinations of the carotenoids, lutein, lycopene, zeaxanthin and/or beta-carotene may further be included in the nutritional compositions of the present disclosure. Nutritional compositions containing these combinations, as selected and defined herein, can be used to modulate inflammation and/or levels of C-reactive protein in preterm and term infants.

[0094] It is generally preferable that the nutritional compositions comprise at least one of lutein, lycopene, zeaxanthin, beta-carotene to provide a total amount of carotenoid of from about 0.001 ug/mL to about 10 ug/mL. More particularly, the nutritional compositions comprise lutein in an amount of from about 0.001 p.g/mL to about 5 gg/mL, including from about 0.001 p.g/mL to about 0.0190 pg/mL, including from about 0.001 p.g/mL to about 0.0140 pg/mL, and also including from about 0.044 ug/mL to about 5 p.g/mL of lutein. It is also generally preferable that the nutritional compositions comprise from about 0.001 ug/mL to about 10 pg/mL, including from about 0.001 p.g/mL to about 0.0130 ps/mL, including from about 0.001 lig/mL to about 0.0075 ug/mL of lycopene, and also including from about 0.0185 ps/mL to about 5 ps/mL of lycopene. It is also generally preferable that the nutritional compositions comprise from about 1 ug/mL to about 10 tig/mL, including from about 0.001 tig/mL to about 0.025 lig/mL of beta-carotene, including from about 0.001 ug/mL to about 0.01 1 pg/mL of beta-carotene, and also including from about 0.034 p.g/mL to about 5 ttg/mL of beta-carotene. It should be understood that any combination of these amounts of beta-carotene, lutein, zeaxanthin, and lycopene can be included in the nutritional compositions of the present disclosure. Other carotenoids may optionally be included in the nutritional compositions as described herein. Any one or all of the carotenoids included in the nutritional compositions described herein may be from a natural source or artificially synthesized. [0095] Each of the carotenoids in the selected combinations can be obtained from any known or otherwise suitable material source for use in nutritional compositions, and each can be provided individually, or all together, or in any combination and from any number of sources, including sources such as multivitamin premixes containing other vitamins or minerals in combination with one or more of the carotenoids as described herein. Non-limiting examples of some suitable sources of lutein, lycopene, beta-carotene, or combinations thereof include LycoVit® lycopene (available from BASF, Mount Olive, NJ), Lyc-O-Mato® tomato extract in oil, powder, or bead form (available from LycoRed Corp., Orange, NJ), beta-carotene, lutein, or lycopene (available from DSM Nutritional Products, Parsippany, NJ), FloraGLO® lutein (available from Kemin Health, Des Moines, IA), Xangold® Natural Lutein Esters (available from Cognis, Cincinnati, OH), and Lucarotin® beta- carotene (available from BASF, Mount Olive, N.J.).

[0096] Any other antioxidants suitable for oral administration may be included for use in the nutritional compositions of the present disclosure, including, for example, vitamin A, vitamin E, vitamin C, retinol, and tocopherol, and

combinations thereof.

Macronutrients

[0097] The nutritional compositions including the extracted genomic DNA may be formulated to include at least one of protein, fat, and carbohydrate. In many embodiments, the nutritional compositions will include the extracted genomic DNA with protein, carbohydrate and fat.

[0098] Although total concentrations or amounts of the fat, protein, and carbohydrates may vary depending upon the product type (i.e., human milk fortifier, preterm infant formula, infant formula, etc.), product form (i.e., nutritional solid, powder, ready-to-feed liquid, or concentrated liquid) and targeted dietary needs of the intended user, such concentrations or amounts most typically fall within one of the following embodied ranges, inclusive of any other essential fat, protein, and/or carbohydrate ingredients as described herein. [0099] For the liquid preterm and term infant formulas, carbohydrate concentrations (including any carbohydrate-based prebiotic) most typically range from about 5% to about 40%, including from about 7% to about 30%, including from about 10% to about 25%, by weight of the preteint or term infant formula; fat concentrations most typically range from about 1% to about 30%, including from about 2% to about 15%, and also including from about 3% to about 10%, by weight of the preterm or term infant formula; and protein concentrations most typically range from about 0.5% to about 30%, including from about 1% to about 15%, and also including from about 2% to about 10%, by weight of the preterm or term infant formula.

[0100] For the liquid human milk fortifiers, carbohydrate concentrations (including any carbohydrate-based prebiotic) most typically range from about 10% to about 75%, including from about 10% to about 50%, including from about 20% to about 40%, by weight of the human milk fortifier; fat concentrations most typically range from about 10% to about 40%, including from about 15% to about 37%, and also including from about 18%) to about 30%), by weight of the human milk fortifier; and protein concentrations most typically range from about 5% to about 40%, including from about 10% to about 30%, and also including from about 15% to about 25%, by weight of the human milk fortifier.

[0101] The amount of carbohydrates, fats, and/or proteins in any of the liquid nutritional compositions described herein may also be characterized in addition to, or in the alternative, as a percentage of total calories in the liquid nutritional composition as set forth in the following table. These macronutrients for liquid nutritional compositions of the present disclosure are most typically formulated within any of the caloric ranges (embodiments A-F) described in the following table (each numerical value is preceded by the term "about"). Nutrient % Total Cal. Embodiment A Embodiment B Embodiment C

Carbohydrate 0-98 2-96 10-75

Protein 0-98 2-96 5-70

Fat 0-98 2-96 20-85

Embodiment D Embodiment E Embodiment F

Carbohydrate 30-50 25-50 25-50

Protein 15-35 10-30 5-30

Fat 35-55 1-20 2-20

[0102] In one specific example, liquid infant formulas (both ready-to-feed and concentrated liquids) include those embodiments in which the protein component may comprise from about 7.5% to about 25% of the caloric content of the formula; the carbohydrate component may comprise from about 35% to about 50% of the total caloric content of the infant formula; and the fat component may comprise from about 30% to about 60% of the total caloric content of the infant formula. These ranges are provided as examples only, and are not intended to be limiting. Additional suitable ranges are noted in the following table (each numerical value is preceded by the term "about").

[0103] When the nutritional product is a powdered preterm or term infant formula, the protein component is present in an amount of from about 5% to about 35%, including from about 8% to about 12%, and including from about 10% to about 12% by weight of the preterm or term infant formula; the fat component is present in an amount of from about 10% to about 35%, including from about 25% to about 30%, and including from about 26% to about 28% by weight of the preterm or term infant formula; and the carbohydrate component (including any carbohydrate-based prebiotic) is present in an amount of from about 30% to about 85%, including from about 45% to about 60%, and including from about 50% to about 55% by weight of the preteim or term infant formula.

[0104] For powdered human milk fortifiers the protein component is present in an amount of from about 1% to about 55%, including from about 10% to about 50%, and including from about 10% to about 30% by weight of the human milk fortifier; the fat component is present in an amount of from about 1% to about 30%, including from about 1% to about 25%, and including from about 1% to about 20% by weight of the human milk fortifier; and the carbohydrate component (including any carbohydrate-based prebiotic) is present in an amount of from about 15% to about 75%, including from about 15% to about 60%, and including from about 20% to about 50% by weight of the human milk fortifier.

[0105] The total amount or concentration of fat, carbohydrate, and protein, in the powdered nutritional compositions of the present disclosure can vary

considerably depending upon the selected composition and dietary or medical needs of the intended user. Additional suitable examples of macronutrient concentrations are set forth below. In this context, the total amount or concentration refers to all fat, carbohydrate, and protein sources in the powdered product. For powdered nutritional compositions, such total amounts or concentrations are most typically and preferably formulated within any of the embodied ranges described in the following table (each numerical value is preceded by the term "about").

F at

[0106] The nutritional compositions of the present disclosure may, in addition to the LCPUFAs described above, comprise an additional source or sources of fat. Suitable additional sources of fat for use herein include any fat or fat source that is suitable for use in an oral nutritional product and is compatible with the essential elements and features of such products. For example, in one specific embodiment, the additional fat is derived from short chain fatty acids.

[0107] Additional non-limiting examples of suitable fats or sources thereof for use in the nutritional products described herein include coconut oil, fractionated coconut oil, soybean oil, corn oil, olive oil, safflower oil, high oleic safflower oil, oleic acids (EMERSOL 6313 OLEIC ACID, Cognis Oleochemicals, Malaysia), MCT oil (medium chain triglycerides), sunflower oil, high oleic sunflower oil, palm and palm kernel oils, palm olein, canola oil, marine oils, fish oils, fungal oils, algae oils, cottonseed oils, and combinations thereof. Particularly desirable fats include MCT oil, soy oil, coconut oil, and high oleic safflower oil, all of which can be used alone or in combination. In specific embodiment, the fat system includes MCT oil, soy oil, and coconut oil alone, or in combination with DHA oil and/or ARA oil. In another specific embodiment, the fat system includes soy oil, high oleic safflower oil, MCT oil, and coconut oil alone, or in combination with DHA oil and/or ARA oil.

Protein

[0108] The nutritional compositions of the present disclosure may optionally further comprise protein. Any protein source that is suitable for use in oral nutritional compositions and is compatible with the essential elements and features of such products is suitable for use in the nutritional compositions.

[0109] Non-limiting examples of suitable proteins or sources thereof for use in the nutritional products include hydrolyzed, partially hydrolyzed or non-hydrolyzed proteins or protein sources, which may be derived from any known or otherwise suitable source such as milk (e.g., casein, whey), animal (e.g., meat, fish), cereal (e.g., rice, corn), vegetable (e.g., soy) or combinations thereof. Non-limiting examples of such proteins include milk protein isolates, milk protein concentrates as described herein, casein protein isolates, extensively hydrolyzed casein, whey protein, sodium or calcium casemates, whole cow milk, partially or completely defatted milk, soy protein isolates, soy protein concentrates, and so forth. In one specific embodiment, the nutritional compositions include a protein source derived from milk proteins of human and/or bovine origin. Particularly desirable proteins or sources thereof include non-fat milk, whey protein concentrate, and condensed skim milk, all of which can be used alone or in any combination. In one specific embodiment, the protein system includes a combination of non-fat milk and whey protein concentrate. In another specific embodiment, the protein system includes a combination of condensed skim milk and whey protein concentrate.

Carbohydrate

[0110] In addition to the carbohydrate-based prebiotics described above, the nutritional products of the present disclosure may further optionally comprise any carbohydrates that are suitable for use in an oral nutritional product and are compatible with the essential elements and features of such products.

[011 1] Non-limiting examples of suitable carbohydrates or sources thereof for use in the nutritional products described herein may include maltodextrin, hydrolyzed or modified starch or cornstarch, glucose polymers, corn syrup, corn syrup solids, rice-derived carbohydrates, pea-derived carbohydrates, potato-derived carbohydrates, tapioca, sucrose, glucose, fructose, lactose, high fructose corn syrup, honey, sugar alcohols (e.g., maltitol, erythritol, sorbitol), artificial sweeteners (e.g., sucralose, acesulfame potassium, stevia), and combinations thereof. A particularly desirable carbohydrate is a low dextrose equivalent (DE) maltodextrin. Particularly desirable carbohydrates (in addition to the carbohydrate-based prebiotics described above) include corn syrup, corn syrup solids, lactose, and maltodextrin. In one specific embodiment, the carbohydrate system is a combination of corn syrup and lactose. In another specific embodiment, the carbohydrate system is a combination of corn syrup solids and lactose. In another specific embodiment, the carbohydrate system is a combination of maltodextrin and lactose.

Other Optional Ingredients

[0112] The nutritional compositions of the present disclosure may further comprise other optional components that may modify the physical, chemical, aesthetic or processing characteristics of the products or serve as pharmaceutical or additional nutritional components when used in the targeted population. Many such optional ingredients are known or otherwise suitable for use in medical food or other nutritional products or pharmaceutical dosage forms and may also be used in the compositions herein, provided that such optional ingredients are safe for oral administration and are compatible with the essential and other ingredients in the selected product form.

[0113] Non-limiting examples of such optional ingredients include

preservatives, emulsifying agents, buffers, pharmaceutical actives, anti-inflammatory agents, additional nutrients as described herein, colorants, flavors, thickening agents and stabilizers, emulsifying agents, lubricants, and so forth.

[0114] The nutritional compositions may further comprise a sweetening agent, preferably including at least one sugar alcohol such as maltitol, erythritol, sorbitol, xylitol, mannitol, isolmalt, and lactitol, and also preferably including at least one artificial or high potency sweetener such as acesulfame K, aspartame, sucralose, saccharin, stevia, and tagatose. These sweetening agents, especially as a combination of a sugar alcohol and an artificial sweetener, are especially useful in formulating liquid beverage embodiments of the present disclosure having a desirable flavor profile. These sweetener combinations are especially effective in masking undesirable flavors sometimes associated with the addition of vegetable proteins to a liquid beverage. Optional sugar alcohol concentrations in the nutritional product may range from at least 0.01%, including from about 0.1% to about 10%, and also including from about 1 % to about 6%, by weight of the nutritional product. Optional artificial sweetener concentrations may range from at least 0.01%, including from about 0.05% to about 5%, also including from about 0.1% to about 1.0%, by weight of the nutritional product.

[0115] A flowing agent or anti-caking agent may be included in the nutritional compositions as described herein to retard clumping or caking of the powder over time and to make a powder embodiment flow easily from its container. Any known flowing or anti-caking agents that are known or otherwise suitable for use in a nutritional powder or product form are suitable for use herein, non-limiting examples of which include tricalcium phosphate, silicates, and combinations thereof. The concentration of the flowing agent or anti-caking agent in the nutritional composition varies depending upon the product form, the other selected ingredients, the desired flow properties, and so forth, but most typically range from about 0.1% to about 4%, including from about 0.5% to about 2%, by weight of the nutritional composition.

[0116] A stabilizer may also be included in the nutritional compositions. Any stabilizer that is known or otherwise suitable for use in a nutritional composition is also suitable for use herein, some non-limiting examples of which include gums such as xanthan gum. The stabilizer may represent from about 0.1% to about 5.0%, including from about 0.5% to about 3%, including from about 0.7% to about 1.5%, by weight of the nutritional composition.

[0117] The nutritional compositions may further comprise any of a variety of other vitamins or related nutrients, non-limiting examples of which include vitamin A, vitamin D, vitamin E, vitamin K, thiamine, riboflavin, pyridoxine, vitamin Bi₂, carotenoids (e.g., beta-carotene, zeaxanthin, lutein, lycopene), niacin, folic acid, pantothenic acid, biotin, vitamin C, choline, inositol, salts and derivatives thereof, and combinations thereof.

[0118] The nutritional compositions may further comprise any of a variety of other additional minerals, non-limiting examples of which include calcium, phosphorus, magnesium, iron, zinc, manganese, copper, sodium, potassium, molybdenum, chromium, chloride, and combinations thereof.

Methods of Manufacture

[0119] The nutritional compositions of the present disclosure may be prepared by any known or otherwise effective manufacturing technique for preparing the selected product solid or liquid form. Many such techniques are known for any given product form such as nutritional liquids or powders and can easily be applied by one of ordinary skill in the art to the nutritional compositions described herein.

[0120] The nutritional compositions of the present disclosure can therefore be prepared by any of a variety of known or otherwise effective formulation or manufacturing methods. In one suitable manufacturing process, for example, at least three separate slurries are prepared, including a protein-in-fat (PIF) slurry, a carbohydrate-mineral (CHO-MIN) slurry, a protein-in- water (PIW) slurry. The PIF slurry is formed by heating and mixing the oil (e.g., canola oil, corn oil, etc.) and then adding an emulsifier (e.g., lecithin), fat soluble vitamins, and a portion of the total protein (e.g., milk protein concentrate, etc.) with continued heat and agitation. The CHO-MIN slurry is formed by adding with heated agitation to water: minerals (e.g., potassium citrate, dipotassium phosphate, sodium citrate, etc.), trace and ultra trace minerals (TM/UTM premix), thickening or suspending agents (e.g. gellan gum, carrageenan). The resulting CHO-MIN slurry is held for 10 minutes with continued heat and agitation before adding additional minerals (e.g., potassium chloride, magnesium carbonate, potassium iodide, etc.), and/or carbohydrates (e.g., HMOs, fructooligosaccharide, sucrose, corn syrup, etc.). The PIW slurry is then formed by mixing with heat and agitation the remaining protein, if any. As DNA is water soluble, the extracted genomic DNA may be included in either the PIW or CHO-MIN slurries during manufacturing.

[0121] The resulting slurries are then blended together with heated agitation and the pH adjusted to 6.6-7.0, after which the composition is subjected to high- temperature short-time (HTST) processing during which the composition is heat treated, emulsified and homogenized, and then allowed to cool. Water soluble vitamins and ascorbic acid are added, the pH is adjusted to the desired range if necessary, flavors are added, and water is added to achieve the desired total solid level. The composition is then aseptically packaged to form an aseptically packaged nutritional emulsion. This emulsion can then be further diluted, heat-treated, and packaged to form a ready-to-feed or concentrated liquid, or it can be heat-treated and subsequently processed and packaged as a reconstitutable powder, e.g., spray dried, drymixed, agglomerated.

[0122] The nutritional solid, such as a spray dried nutritional powder or drymixed nutritional powder, may be prepared by any collection of known or otherwise effective techniques, suitable for making and formulating a nutritional powder. [0123] For example, when the nutritional powder is a spray dried nutritional powder, the spray drying step may likewise include any spray drying technique that is known for or otherwise suitable for use in the production of nutritional powders. Many different spray drying methods and techniques are known for use in the nutrition field, all of which are suitable for use in the manufacture of the spray dried nutritional powders herein.

[0124] One method of preparing the spray dried nutritional powder comprises forming and homogenizing an aqueous slurry or liquid comprising the extracted genomic DNA, and optionally protein, carbohydrate, and fat, and then spray drying the slurry or liquid to produce a spray dried nutritional powder. The method may further comprise the step of spray drying, drymixing, or otherwise adding additional nutritional ingredients, including any one or more of the ingredients described herein, to the spray dried nutritional powder.

[0125] Other suitable methods for making nutritional products are described, for example, in U.S. Pat. No. 6,365,218 (Borschel, et al.), U.S. Pat. No. 6,589,576

(Borschel, et al.), U.S. Pat. No. 6,306,908 (Carlson, et al.), U.S. Pat. Pub. No.

20030118703 Al (Nguyen, et al.), which descriptions are incorporated herein by reference to the extent that they are consistent herewith.

Methods of Use

[0126] The nutritional compositions as described herein can be used to reduce the incidence and/or risk of NEC in preterm infants, infants, toddlers, and children. The nutritional compositions as described herein can also be used to treat and/or prevent and/or reduce and/or control and/or minimize NEC, or the incidence of NEC.

Particularly, the nutritional compositions as described herein comprise extracted genomic DNA, such as from L. rhamnosus HNOOl, alone or in combination with one or more additional components, to provide a nutritional source for reducing inflammation, such as enteric inflammation, thereby reducing damage to the tissue lining and reducing cell death that can lead to NEC. Although it is within the scope of the present disclosure to treat any preterm infant, infant, toddler or child that actually has NEC, any of the preterm infants, infants, toddlers, and children may actually have NEC or may be at risk of getting NEC, may be susceptible to NEC, or may be in need of treatment for NEC. Because not all preterm infants, infants, toddlers, and children may fit into any of these categories, not all can benefit from each embodiment of the present disclosure as some of the embodiments disclosed herein are directed to a specific subclass or subgroup of the general population of preterm infants, infants, toddlers, and children.

[0127] Additionally, extracted genomic DNA when used in combination with LCPUFAs and/or carotenoids, can reduce oxidative stress, which is a metabolic condition in which there is an increased production and accumulation of oxidized biomolecules such as lipid peroxides and their catabolites, protein carbonyls, and oxidatively damaged DNA. The outcomes of oxidative stress range from unwanted changes in metabolism to inflammation and cell and tissue death.

[0128] The nutritional compositions of the present disclosure comprising the extracted genomic DNA may also provide optimal development and balanced growth and maturation of the infant's gastrointestinal and immune systems, thereby enhancing the infant's ability to resist systemic microbial infection (sepsis) and modulate inflammatory responses to infection.

[0129] Along with improved growth and maturation of the infant's immune system as described above, the use of the nutritional compositions of the present disclosure also functions as an immune modulator, thereby reducing infection- mediated inflammatory diseases in infants, toddlers, and children.

[0130] The nutritional compositions may also be used to modulate allergies and treat anchor prevent chronic inflammatory diseases including irritable bowel disorder, Crohn's and ulcerative colitis.

EXAMPLES

[0131] The following examples illustrate specific embodiments anchor features of the nutritional compositions of the present disclosure. The examples are given solely for the purpose of illustration and are not to be construed as limitations of the present disclosure, as many variations thereof are possible without departing from the spirit and scope of the disclosure. All exemplified amounts are weight

percentages based upon the total weight of the composition, unless otherwise specified.

[0132] The exemplified compositions are shelf stable nutritional compositions prepared in accordance with the manufacturing methods described herein, such that each exemplified composition, unless otherwise specified, includes an aseptically processed embodiment and a retort packaged embodiment.

EXAMPLE 1

[0133] In this Example, the effect of orally administered extracted genomic DNA from L. rhamnosus HNO01 (DR20) on NEC in a mouse model was evaluated. A mouse model was employed as the molecular signatures in mouse and human infant NEC intestine are comparable. Particularly, TLR-4/TLR-9 reciprocal expression is similar, as are the increased markers of inflammation (e.g., iNOS, IL-6, IL-1 13, and TNF-a).

[0134] Extracted genomic DNA from L. rhamnosus HNO01 (commercially available from Danisco USA, Inc., Madison, Wisconsin) was isolated using the phenol: chloroform extraction methodology described herein. The absence of demonstrable levels of cell wall components (e.g., lipoteichoic acid) or contaminating endotoxin levels was demonstrated by low levels of TNF-a producing monocytes as determined by a flow cytometric evaluation.

[0135] Swiss Webster mice, 7 to 10 days of age, were breast fed and treated with or without the extracted genomic DNA, 5 mg/g body weight, once daily by oral gavage, for 4 days prior to NEC induction and throughout the remainder of the study. NEC was induced as described by Gribar et al. (Gribar SC, Sodhi CP, Richardson WM, Anand RJ, Gittes GK, Branca MF, Jakub A, Shi XH, Shah S, Ozolek JA, Hackam DJ. Reciprocal expression and signaling of TLR4 and TLR9 in the pathogenesis and treatment of NEC. J Immunol. 2009 Jan l; 182(l):636-46). On study day 5, mice were gavage fed 250 pi, of formula (infant formula anine milk replacer at a ratio of 2:1 ) per 5 g body weight five times daily. A subset of mice continued to breast feed. All mice received 10-minute intervals of hypoxia (5% 02, 95% N₂) using a modular hypoxic chamber, twice daily. Mice were weighed daily, and activity level, coat appearance, and fecal consistency were noted. Mice were observed for 4 days then sacrificed. Upon sacrifice, 2 sections of the small intestine were removed for histological evaluation by Hematoxylin and Eosin (H&E) staining and for qRT PCT evaluation of TLR-4 and TLR-9 receptor expression and inflammatory markers. One section was taken 1 cm proximal to the ileocecal valve, and the second section was taken from the most diseased looking site. H&E sections were graded by a pathologist blinded to the study groups using a scoring system of: 0 - no inflammation; 1— mild inflammation; 2— moderate inflammation; 3— severe inflammation.

[0136] Mice in the breast fed group were the comparator normal control for this model. See Figures 1 and 2 for representative histology sections of ileum from breast fed mice and breast fed mice supplemented with extracted genomic DNA from L. rhainnosus HNOOl (DR20 DNA) (histology scores of 0 and 0.5, breast fed and breast fed supplemented with extracted genomic DNA, respectively). Formula fed mice developed severe NEC. Particularly, histology sections of the ileum of formula fed mice revealed prominence of inflammation in the lamina propria, shortened villi, and areas of necrosis and apoptosis (histology score 2.8 + 0.3 [mean + SEM, n = 6]). See Figure 3. To the contrary, formula fed mice supplemented with DR20 DNA developed less severe or no NEC compared to those without DNA supplementation. Mice in the extracted genomic DR20 DNA group retained normal ileal histological features including villi of normal length and mild inflammation (histology score 1.5 + 0.5 [mean + SEM, n = 8]). See Figure 4. Supportive of the histologic findings, markers of inflammation, iNOS (Figure 5) and IL-6 (Figure 6) were elevated in formula fed mice compared to breast fed mice. In formula fed mice supplemented with extracted genomic DNA, iNOS and. IL-6 were decreased compared to formula fed mice without DNA supplementation. Interestingly, TLR-4 expression was greater in formula fed mice supplemented with DR20 DNA compared to formula fed mice without DNA supplementation (Figure 7), but similar to breast fed mice while TLR-9 expression was decreased in formula fed DR20 DNA supplemented mice compared to formula fed mice without supplementation (Figure 8). These results indicate that the extracted genomic DNA is capable of reducing the incidence of NEC. EXAMPLES 2-4

[0137] Examples 2-4 illustrate ready-to-feed nutritional emulsions of the present disclosure for institutional administration to preterm infants, the ingredients of which are listed in the table below. All ingredient amounts are listed as kilogram per 1000 kilogram batch of product, unless otherwise specified.

EXAMPLE 5

[0138] Example 5 illustrates a human milk fortifier of the present disclosure, the ingredients of which are listed in the table below. All ingredient amounts are listed as kilogram per 10000 pound batch of product, unless otherwise specified.

EXAMPLE 6

[0139] Example 6 illustrates a ready-to-feed nutritional emulsion of the present disclosure for administration to preterm infants, the ingredients of which are listed in the table below. All ingredient amounts are listed as kilogram per 1000 kilogram batch of product, unless otherwise specified.

Genomic DNA 6.25 g

EXAMPLE 7

[0140] Example 7 illustrates a ready-to-feed nutritional emulsion of the present disclosure for administration to preterm infants, the ingredients of which are listed in the table below. All ingredient amounts are listed as kilogram per 50000 pound batch of product, unless otherwise specified. Ingredient Name Amount per 50000 lbs

Condensed Skim Milk 19656

Maltodextrin 5647

Lactose 2657

Soybean Oil 1786

High Oleic Safflower Oil 1722

Whey Protein Concentrate 1635

MCT Oil 1595

Coconut Oil 1186

Potassium Citrate 144.9

ARA Oil 65.8

Calcium Carbonate 51.3

Ascorbic Acid 86.1

Micronized Tricalcium Phosphate 98.0

Nucleotide-Choline Premix 53.3

Water-Soluble Vitamin Premix 41.3

DHA Oil 24.6

Magnesium Chloride 36.2

m-Inositol 36.7

Choline Chloride 9.15

Potassium Chloride 19.4

Vitamin ADEK Premix 9.60

Ferrous Sulfate 8.63

L-Carnitine 6.80

Beta-carotene (30% in vegetable oil) 250.00 g

Vitamin A Palmitate 116.63 g

Sodium Chloride 0 - 101.0

Potassium Phosphate Dibasic 0 - 101.0

Ascorbyl Palmitate 7.87

Mixed Tocopherols 3.75

Sodium Citrate 0 - 101.0

Potassium Hydroxide (processing aide) as needed

Genomic DNA 1.013

EXAMPLE 8

[0141] Example 8 illustrates a ready-to-feed nutritional emulsion of the present disclosure for administration to term infants, the ingredients of which are listed in the table below. All ingredient amounts are listed as kilogram per 1000 kilogram batch of product, unless otherwise specified.

Genomic DNA 0.12 g

EXAMPLE 9

[0142] Example 9 illustrates a ready-to-feed nutritional emulsion of the present disclosure for administration to term infants, the ingredients of which are listed in the table below. All ingredient amounts are listed as kilogram per 1000 kilogram batch of product, unless otherwise specified.

Claims

Claims:

1. A method of treating, preventing or reducing the incidence of necrotizing enterocolitis in an infant, toddler or child, the method comprising administering to an infant, toddler, or child at risk for necrotizing enterocolitis a nutritional composition comprising extracted genomic bacterial DNA wherein the nutritional composition is selected from the group consisting of human milk fortifier, preterm infant formula, infant formula, pediatric formula, toddler formula, and follow-on formula.

2. A method of claim 1, wherein the extracted genomic DNA is from

Lactobacillus rhainnosus HNO01.

3. A method of either one of claims 1 and 2, wherein the extracted DNA is selected from: whole, extracted genomic DNA; fragmented, extracted genomic DNA.

4. A method of any one of the preceding claims, wherein the infant, toddler, or child is administered at least 5 pg, preferably from 5μg to 20 mg of extracted genomic DNA/ g body weight per day.

5. A method of any one of the preceding claims, wherein the nutritional composition comprises at least one of a prebiotic, a long chain polyunsaturated fatty acid, or a carotenoid.

6. A method of any one of the preceding claims, wherein the prebiotic is preferably a human milk oligosaccharide, the long chain polyunsaturated fatty acid is selected from the group of docosahexanoic acid, arachidonic acid, eicosapentaenoic acid and combinations thereof, and the carotenoid is selected from the group of lycopene, lutein, beta-carotene, and combinations thereof.

7. A method of any one of the preceding claims, the nutritional composition comprising extracted whole, extracted genomic DNA from Lactobacillus rhamnosus HNO01, at 0.00003% to 3.5% by weight per volume of the nutritional composition.

8. A nutritional composition comprising whole, extracted genomic DNA from Lactobacillus rhamnosus HNO01.

9. A nutritional composition of claim 8, comprising from 0.00003% to 3.5% by weight per volume of whole, extracted genomic DNA.

10. A nutritional composition of either one of claims 8 and 9, further comprising at least one of a prebiotic, an anti-inflammatory, or an antioxidant.

1 1. A nutritional composition of claim 10, wherein the prebiotic is preferably a human milk oligosaccharide, the long chain polyunsaturated fatty acid is selected from the group of docosahexanoic acid, arachidonic acid, eicosapentaenoic acid and combinations thereof, and the carotenoid is selected from the group of lycopene, lutein, beta-carotene, and combinations thereof.

12. The use of a nutritional composition of any one of claims 8-11, for use in reducing the incidence of, treating, or preventing necrotizing enterocolitis.