HK1189456B - Method for inhibiting pathogens using a nutritional composition - Google Patents

Description

Methods of inhibiting pathogens using nutritional compositions

Technical Field

The present disclosure relates generally to the field of nutritional compositions, such as infant formula (infant formula), human milk fortifiers, pediatric dietary supplements, and the like, having a lactoferrin composition therein. More specifically, the present disclosure relates to methods of inhibiting the invasion mechanism of harmful bacterial pathogens and/or inhibiting the adhesion of at least one pathogen in the human gastrointestinal tract by administering to a human a nutritional composition comprising lactoferrin produced by a non-human source.

Background

There are various dietary compositions for humans, especially young people, that provide supplemental or essential nutrition during certain life stages. Generally, commercial dietary compositions for infants seek to mimic the composition and related functions of human milk as much as possible. By combining some of the physiologically active proteins with blended fat ingredients, the dietary compositions are formulated such that they mimic human milk, for use as a complete or partial replacement. Other ingredients often used in dietary compositions for infants may include carbohydrate sources such as lactose, as well as other vitamins, minerals and elements, which are believed to be present in human milk for absorption by the infant.

Some of the proteins present in human milk provide a defense against pathogens to prevent and suppress infection, while additionally promoting an immune response in infants. Proteins, including corrin-and lactoferrin, are thought to help infants protect against various bacterial pathogens through bacteriostatic and bactericidal activity.

Lactoferrin is one of the basic proteins in human milk, and is considered to be a glycoprotein having an average molecular weight of approximately 80 kilodaltons. It is an iron binding protein, has the ability to bind two molecules of iron in a reversible manner, and can promote the absorption of iron in the human intestinal tract. Functionally, lactoferrin regulates iron absorption and, as such, is able to bind iron-based free radicals and provide iron for an immune response.

Another effect of lactoferrin is its antimicrobial activity in defense against intestinal infections in humans in general, but in infants in particular. Lactoferrin is known to be both bacteriostatic and bactericidal, killing certain bacteria while inhibiting their growth and before they successfully invade intestinal cells.

In obtaining commercially viable (viable) dietary compositions, the addition of lactoferrin is often limited due to the expected loss of activity during processing. For example, in general, the temperature and pH requirements in processing infant formula and other products such as human milk fortifiers and various pediatric products reduce the specific functions of lactoferrin, resulting in lactoferrin not being included in the final formulation. In addition, lactoferrin is generally considered only for its iron-binding properties, and therefore may generally be excluded from formulation where this property is considered to be diminished by processing conditions.

Further, as is known in the art, human breast milk has relatively low iron, containing about 0.3 milligrams of iron per liter of breast milk. Although this amount is low, human infants have a high absorption rate, absorbing about half of the iron from breast milk. However, when a human infant is given a prior art formula with a high level of iron fortification, for example from about 10mg to about 12 mg per liter, the infant absorbs less than about 5% of the total iron. With such increased levels of iron in the prior art formulations, virtually all iron binding sites would be expected to be occupied, since lactoferrin is a known iron transporter.

Other problems with the prior art formulations include the inability to provide bacteriostatic effects. This is due in part to the use of lactoferrin with blocked or damaged binding sites, as the bacteriostatic effect is at least partially related to the extent to which lactoferrin present in the formulation binds iron.

Accordingly, it would be beneficial to provide nutritional compositions such as infant formulas, human milk fortifiers, pediatric dietary supplements, and the like, containing lactoferrin, particularly produced by non-human sources. Preferably, lactoferrin contained in the composition has a bacteriostatic effect even after processing under high temperature and low pH conditions. The combination of properties including the maintenance of the anti-invasive or anti-adhesion mechanisms of lactoferrin via high or low pH or high temperature conditions, such as during pasteurization, provides a dietary composition that can be at least partially protected against harmful bacterial pathogens.

Disclosure of Invention

Briefly, in one embodiment, the present disclosure relates to a method of inhibiting adhesion of one or more of the invasion mechanisms of bacterial pathogens and/or at least one pathogen using a nutritional composition comprising a lipid or fat, a protein source, a prebiotic composition, and lactoferrin from a non-human source, wherein the composition provides an active anti-invasion and anti-adhesion mechanism to undesirable bacterial strains found in the human gastrointestinal tract, even after processing including exposure to harsh environmental conditions.

In one embodiment, the present disclosure relates to a nutritional product comprising:

a. up to about 7g/100kcal of fat or lipid, more preferably from about 3g/kcal to about 7g/100kcal of fat or lipid;

b. a protein source of up to about 5g/100kcal, more preferably from about 1g/kcal to about 5g/100 kcal;

c. at least about 10mg/100kcal of lactoferrin, more preferably from about 70 mg/100kcal to about 220mg/100kcal of lactoferrin produced by a non-human source, most preferably from about 90mg/100kcal to about 190mg/100kcal of lactoferrin produced by a non-human source; and

d. about 0.1g/100kcal to about 1g/100kcal of a prebiotic composition comprising polydextrose (polydextrose) and/or galactooligosaccharide (galactooligosaccharide). More preferably, the nutritional composition comprises about 0.3g/100kcal to about 0.7g/100kcal of a prebiotic composition comprising a combination of polydextrose and galactooligosaccharide.

Preferably, the lactoferrin is non-human lactoferrin and/or human lactoferrin produced by a genetically modified organism. The term "organism" as used herein refers to any continuously living system, such as an animal, plant, fungus or microorganism. In a particularly preferred embodiment, the lactoferrin is used such that an effective amount of the lactoferrin containing nutritional composition can be administered to inhibit at least one invasion mechanism of at least one pathogen in the human gastrointestinal tract, even if during processing the nutritional composition has been exposed to certain processing conditions such as pH and temperature fluctuations typical of pasteurization. Lactoferrin contains an anti-invasion mechanism that can destroy adhesion factors and needles used by certain bacteria on human cells. In another particularly preferred embodiment, the lactoferrin is used such that an effective amount of the lactoferrin containing nutritional composition can be administered to inhibit the adhesion of at least one pathogen in the human gastrointestinal tract even if during processing the nutritional composition has been exposed to certain processing conditions such as pH and temperature fluctuations typical of pasteurization. Examples of such pathogens include enterotoxigenic escherichia coli (ETEC), enteropathogenic escherichia coli (EPEC), Haemophilus influenzae (haemaphilus influenza), shiga toxin-producing escherichia coli (STEC), enteroaggregative escherichia coli (EAEC), Salmonella Typhimurium serotype (Salmonella ser. Typhimurium), Shigella flexneri (Shigella flexneri), rotaviruses, noroviruses, Respiratory Syncytial Virus (RSV), adenoviruses, and combinations thereof.

Detailed description of the preferred embodiments

The present disclosure provides novel nutritional products that are readily digestible, provide physicochemical benefits, and/or provide physiological benefits. In one embodiment of the present disclosure, a nutritional composition comprises: a lipid or fat, a protein source, a prebiotic composition having at least 20% oligosaccharides, especially oligosaccharides comprising galacto-oligosaccharides (GOS), and lactoferrin that provides activity against invasive or anti-adhesion mechanisms to undesirable bacterial strains found in the human gastrointestinal tract. Lactoferrin comprised in the composition is produced by a non-human source. In certain embodiments, the prebiotic comprises a combination of galactooligosaccharide and polydextrose. More particularly, the compositions disclosed herein comprise:

a. up to about 7g/100kcal of fat or lipid, more preferably from about 3 to about 7g/100kcal of fat or lipid;

b. up to about 5g/100kcal of a protein source, more preferably from about 1 to about 5g/100kcal of a protein source;

c. d.about 0.1g/100kcal to about 1g/100kcal of a prebiotic composition comprising polydextrose and/or galactooligosaccharides. More preferably, the nutritional composition comprises about 0.3g/100kcal to about 0.7g/100kcal of a prebiotic composition comprising a combination of polydextrose and galactooligosaccharide; and

d. at least about 10mg/100kCal of lactoferrin, more preferably from about 70mg to about 220mg/100kCal of lactoferrin, and most preferably from about 90mg to about 190mg/100kCal of lactoferrin.

Definition of

As used herein, the term "prebiotic" refers to a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon that improve the health of the host.

The term "probiotic" refers to a microorganism with low or no pathogenicity that exerts beneficial effects on the health of the host.

As used herein, the term "infant" is generally defined as a human from about birth to 12 months of age.

A "preterm infant" is an infant born less than 37 weeks after gestation.

As used herein, "term infant" refers to an infant born at least 37 weeks after gestation.

"Children" are defined as persons from 12 months to about 12 years of age.

An "antibacterial effective amount" as used herein is generally defined as the amount of lactoferrin that provides at least one anti-invasion mechanism to the bacterial strain.

"lactoferrin produced by a non-human source" refers to lactoferrin produced or obtained by a source other than human breast milk. For example, lactoferrin as used in the present disclosure includes human lactoferrin and non-human lactoferrin produced by genetically modified organisms.

"biologically active lactoferrin" refers to lactoferrin having at least one anti-invasion or anti-adhesion mechanism to pathogens.

The term "non-human lactoferrin" as used herein refers to lactoferrin having an amino acid sequence different from that of human lactoferrin.

The term "organism" as used herein refers to any continuously living system, such as an animal, plant, fungus or microorganism.

The term "mimic" as used herein refers to the same or similar having or taking the form or appearance thereof, or having or producing symptoms.

Disclosed is a

In some embodiments, the nutritional product may be an infant formula. The term "infant formula" applies to compositions in liquid or powder form that meet the nutritional needs of infants by acting as a substitute for human milk. In the united states, the levels of infant formula are regulated by federal regulations set forth under 21 c.f.r. § 100, 106 and 107. These regulations define macronutrient, vitamin, mineral and other ingredient levels in an effort to mimic the nutritional and other properties of human breast milk. In a separate embodiment, the nutritional product may be a human milk fortifier, meaning that it is a composition that is added to human milk to increase the nutritional value of human milk. As a human milk fortifier, the disclosed compositions may be in powder or liquid form. In another embodiment, the disclosed nutritional products may be a children's nutritional composition.

The nutritional products of the present disclosure may provide minimal, partial, or complete nutritional support. The composition may be a nutritional supplement or a meal replacement. In some embodiments, the composition may be administered in conjunction with a food or nutritional composition. In this embodiment, the composition may be intermixed with the food or other nutritional composition prior to ingestion by the subject, or may be administered to the subject either before or after ingestion of the food or nutritional composition. The composition may be administered to a preterm infant receiving infant formula, human milk, a human milk fortifier, or a combination thereof.

The composition may, but need not, be nutritionally complete. The skilled artisan will recognize that "nutritionally complete" varies depending on a number of factors, including but not limited to age, clinical condition, and dietary intake of the subject to whom the term applies. Generally, "nutritionally complete" means that the nutritional compositions of the present disclosure provide sufficient amounts of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy for normal growth. As used for nutrients, the term "essential" refers to any nutrient that cannot be synthesized by the body in sufficient quantities for normal growth and to maintain health and thus must be supplemented by the diet. As used in nutrition, the term "conditionally essential" refers to a nutrient that must be supplemented by a diet under conditions in which the body does not have sufficient precursor compounds for endogenous synthesis to occur.

By definition, a composition that is "nutritionally complete" for a preterm infant will provide qualitatively and quantitatively sufficient amounts of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals and energy required for the growth of the preterm infant. By definition, a composition that is "nutritionally complete" for a full term infant will provide qualitatively and quantitatively adequate amounts of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals and energy required for growth of the full term infant. By definition, a composition that is "nutritionally complete" for a child will provide qualitatively and quantitatively adequate amounts of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy required for growth of the child.

The nutritional composition may be provided in any form known in the art, including a powder, gel, suspension, paste (paste), solid, liquid concentrate, or ready-to-use product. In a preferred embodiment, the nutritional composition is an infant formula, in particular an infant formula suitable for use as the sole source of nutrition for an infant.

In a preferred embodiment, the nutritional products disclosed herein may be administered enterally. As used herein, "enteral" refers to through or within the gastrointestinal or digestive tract, and "enteral administration" includes oral feeding, intragastric feeding, transpyloric administration, or any other introduction into the digestive tract.

Lactoferrin is an approximately 80kD, single chain polypeptide containing 1-4 glycans, depending on the species. The 3D structures of different species of lactoferrin are very similar, but not identical. Each lactoferrin contains two homologous leaves, termed N-and C-leaves, which refer to the N-terminal and C-terminal portions of the molecule, respectively. Each leaf is further composed of two sub-leaves or domains, which form a cleft where iron ions (Fe) are present³⁺) And (bi) carbonate anion in a synergistic manner. These domains are referred to as N1, N2, C1 and C2, respectively. The N-terminus of lactoferrin has a strong cationic peptide region that is responsible for many important binding properties. Lactoferrin has a very high isoelectric point (-pI 9) and its cationic nature plays a major role in its ability to defend against bacterial, viral and fungal pathogens. There are several clusters of cationic amino acid residues in the N-terminal region of lactoferrin that mediate the biological activity of lactoferrin against a wide range of microorganisms. For example, the N-terminal residues 1-47 of human lactoferrin (1-48 of bovine lactoferrin) are critical for the iron-independent biological activity of lactoferrin. In human lactoferrin, residues 2 to 5 (RRRR) and 28 to 31 (RKVR) are arginine-rich cationic domains in the N-terminus, particularly critical for the antimicrobial activity of lactoferrin. A similar region in the N-terminus was found in bovine lactoferrin (residues 17 to 42, FKCRRWQWRMKKLGAPSITCVRRAFA).

Lactoferrin from different host species may differ in its amino acid sequence despite having a positively charged amino acid in the terminal region of the inner leaf and sharing a relatively high isoelectric point, as described in "Perspectives on Interactions Between Lactoferrin and bacilli" appearing in the publication Biochemistry and Cell Biology, pp 275-281 (2006). Suitable lactoferrin for use in the present disclosure includes those having at least 48% homology to the amino acid sequence avgeqelrckcnwqwsgl at the HLf (349-364) fragment. In some embodiments, lactoferrin has at least 65% homology, and in some embodiments, at least 75% homology, to the amino acid sequence avgeqelcrcnqwsgl at the HLf (349-364) fragment. For example, non-human lactoferrin for use in the present disclosure includes, but is not limited to, bovine lactoferrin, porcine lactoferrin, equine lactoferrin, buffalo lactoferrin, goat lactoferrin, murine lactoferrin, and camel lactoferrin.

Surprisingly, the forms of lactoferrin included herein retain relevant activity even when exposed to conditions, i.e., low pH (i.e., below 7, and even as low as about 4.6 or less) and/or high temperature (i.e., above about 65 ℃, and as high as about 120 ℃) that would be expected to destroy or severely limit the stability or activity of human lactoferrin or recombinant human lactoferrin. These low pH and/or high temperature conditions may be expected during certain processing regimes, such as pasteurization, for nutritional compositions of the type described herein. For example, while bovine lactoferrin has an amino acid composition with only about 70% sequence homology to human lactoferrin, and is stable and remains active under conditions in which human or recombinant human lactoferrin becomes unstable or inactive, bovine lactoferrin has bactericidal activity against undesirable bacterial pathogens found in the human gut.

In U.S. patent No. 4,791,193, which is incorporated herein by reference in its entirety, okinogi et al disclose a method for producing high purity bovine lactoferrin. Generally, the disclosed method comprises 3 steps. The raw milk is first contacted with a weakly acidic cation exchanger to adsorb lactoferrin, followed by a second step in which washing is carried out to remove non-adsorbed substances. Followed by a desorption step in which lactoferrin is removed to produce purified bovine lactoferrin. Other methods may include steps as described in U.S. Pat. nos. 7,368,141, 5,849,885, 5,919,913, and 5,861,491, the disclosures of which are all incorporated by reference in their entireties.

In one embodiment, lactoferrin is present in the nutritional composition in an amount of at least about 10mg/100kCal, particularly when the nutritional composition is intended for use by children. In certain embodiments, the upper limit of lactoferrin is about 240mg/100 kCal. In another embodiment, when the nutritional composition is an infant formula, the lactoferrin is present in the nutritional composition in an amount of from about 70mg to about 220mg/100 kCal; in another embodiment, lactoferrin is present in an amount of about 90mg to about 190mg/100 kCal. The nutritional composition for infants may comprise lactoferrin in an amount of from about 0.5mg to about 1.5mg per ml of formula. In nutritional compositions replacing human milk, lactoferrin may be present in an amount of from about 0.6mg to about 1.3 mg per milliliter of formula.

Suitable fat or lipid sources for practicing the present disclosure may be any known or used in the art, including, but not limited to, animal sources, such as milk fat, butter, cream, yolk lipid; marine sources, such as fish oil, marine oil (marine oils), single cell oil; vegetable and vegetable oils, such as corn oil, canola oil, sunflower oil, soybean oil, palm olein, coconut oil, high oleic sunflower oil, evening primrose oil, rapeseed oil, olive oil, linseed (flaxseed) oil, cottonseed oil, high oleic safflower oil, palm stearin (palm stearin), palm kernel oil, wheat germ oil; medium chain triglyceride oils and emulsions and esters of fatty acids, and any combination thereof.

Milk protein sources that may be used in the practice of the present disclosure include, but are not limited to, milk protein powder, milk protein concentrate, milk protein isolate, skim milk solids, skim milk powder, whey protein isolate, whey protein concentrate, sweet whey, acid whey, casein, acid casein, caseinates (e.g., sodium caseinate, sodium calcium caseinate, calcium caseinate), and any combination thereof.

In one embodiment, the protein is provided as an intact protein. In other embodiments, the protein is provided as a combination of intact protein and partially hydrolyzed protein having a degree of hydrolysis between about 4% and 10%. In yet another embodiment, the protein source may be supplemented with glutamine-containing peptides.

In a particular embodiment of the present disclosure, the whey from which the protein is derived: the proportion of casein is similar to that found in human breast milk. In one embodiment, the protein source comprises from about 20% to about 85% whey protein. In one embodiment, the protein source may comprise from about 20% to about 80% casein. In yet another embodiment of the present disclosure, the protein source comprises from about 60% to about 85% whey and from about 15% to about 40% casein.

In one embodiment of the present disclosure, the nutritional composition may further comprise one or more probiotics. Any probiotic known in the art may be acceptable in this embodiment so long as it achieves the intended result. In a particular embodiment, the probiotic may be selected from Lactobacillus species, Lactobacillus rhamnosus gg (Lactobacillus rhamnosus gg), Bifidobacterium species, Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium breve (Bifidobacterium breve), and Bifidobacterium animalis subsp.

If included in the composition, the amount of probiotic may range from about 10 per day per kg body weight⁴To about 10¹⁰Individual colony forming units (cfu) change. In another embodiment, the amount of probiotic may be from about 10 per day per kg body weight⁶To about 10⁹Individual colony forming units varied. In yet another embodiment, the amount of probiotic may be at least about 10 per day per kg body weight⁶Individual colony forming units. Also, the disclosed compositions may also include probiotic conditioned media components.

In one embodiment, the probiotic may be viable or non-viable. As used herein, the term "viable" refers to living microorganisms. The term "non-viable" or "non-viable probiotic" refers to non-live probiotic microorganisms, their cellular components and metabolites. These non-viable probiotics may have been heat killed or otherwise inactivated, but retain the ability to beneficially affect the health of the host. The probiotics for use in the present disclosure may be naturally occurring, synthetic or developed biologically by genetic manipulation, whether such new sources are currently known or later developed.

The nutritional composition comprises one or more prebiotics. These prebiotics may be naturally occurring, synthetic or developed by genetically manipulating organisms and/or plants, whether these new sources are now known or later developed. In certain embodiments, the prebiotics included in the compositions of the present disclosure include those taught in U.S. patent No. 7,572,474, the disclosure of which is incorporated herein by reference.

Prebiotics for use in the present disclosure may include oligosaccharides, polysaccharides, and other prebiotics comprising fructose, xylose, soy, galactose, glucose, and mannose. More specifically, prebiotics for use in the present disclosure may include lactulose, lactosucrose, raffinose, glucooligosaccharides, inulin, polydextrose powder, galactooligosaccharides, fructooligosaccharides, isomaltooligosaccharides, soy oligosaccharides, lactosucrose, xylooligosaccharides, chitooligosaccharides, oligomannose, arabinooligosaccharides, sialyloligosaccharides, fucooligosaccharides, and gentiooligosaccharides.

In one embodiment, the total amount of prebiotics present in the nutritional composition may be from about 1.0 g/L to about 10.0 g/L of composition. Alternatively, the total amount of prebiotics present in the nutritional composition may be from about 2.0 g/L to about 8.0g/L of the composition. At least 20% of the prebiotics should comprise Galactooligosaccharides (GOS), polydextrose, or a mixture thereof. Preferably, the nutritional composition comprises polydextrose and galactooligosaccharides. Optionally, the nutritional composition comprises one or more additional prebiotics in addition to the polydextrose and/or galactooligosaccharides. In one embodiment, the amount of each of the galactooligosaccharides and/or polydextrose in the nutritional composition may range from about 1.0 g/L to about 4.0 g/L.

In one embodiment, the total amount of prebiotics present in the nutritional composition may be from about 0.1g/100kcal to about 1g/100 kcal. More preferably, the total amount of prebiotics present in the nutritional composition may be from about 0.3g/100kcal to about 0.7g/100 kcal. At least 20% of the prebiotics should comprise Galactooligosaccharides (GOS) and/or Polydextrose (PDX).

In one embodiment, the amount of galactooligosaccharides in the nutritional composition may be from about 0.2 g/100Kcal to about 1.0 g/100 Kcal. In another embodiment, the amount of galactooligosaccharides within the nutritional composition may be from about 0.1g/100Kcal to about 0.5 g/100 Kcal. In another embodiment, the amount of galactooligosaccharide is in the range of from about 0.2 g/100kcal to about 0.6 g/100 kcal. If polydextrose is used as the prebiotic, in one embodiment, the amount of polydextrose in the nutritional composition may be in the range of about 0.1g/100kcal to about 1g/100 kcal. In another embodiment, the amount of polydextrose ranges from about 0.2 g/100kcal to about 0.6 g/100 kcal. In yet another embodiment, if polydextrose is used in the prebiotic composition, in one embodiment, the amount of polydextrose in the nutritional composition may be in the range of about 0.1g/100Kcal to about 0.5 g/100 Kcal. In certain embodiments, the ratio of polydextrose to galactooligosaccharides in the prebiotic composition is about 9: 1 to about 1: 9.

preferably, the prebiotic composition, in combination with lactoferrin, inhibits the adhesion of one or more pathogens in the gastrointestinal tract when the nutritional composition is provided to a human. An example of one such pathogen is enterobacter sakazakii (enterobacter sakazakii, also known as Cronobacter sakazakii). Another pathogen whose adhesion is inhibited by the combination of lactoferrin and prebiotic compositions is escherichia coli.

The nutritional compositions of the present disclosure may also include a source of long chain polyunsaturated fatty acids (LCPUFAs) including docosahexaenoic acid (DHA). Other suitable LCPUFAs include, but are not limited to, alpha-linoleic acid, gamma-linoleic acid, linolenic acid, eicosapentaenoic acid (EPA), and arachidonic acid (ARA).

In one embodiment, the nutritional composition is supplemented with both DHA and ARA. In this embodiment, the weight ratio of ARA to DHA may be from about 1: 3 to about 9: 1. in one embodiment of the present disclosure, the ratio is about 1: 2 to about 4: 1.

the amount of long chain polyunsaturated fatty acids in the nutritional composition may vary from about 5mg/100kcal to about 100 mg/100kcal, more preferably from about 10mg/100kcal to about 50 mg/100 kcal.

The nutritional composition may be supplemented with oils containing DHA and ARA using standard techniques known in the art. For example, DHA and ARA can be added to a formulation by replacing an equivalent amount of oil normally present in the formulation, such as high oleic sunflower oil. As another example, oils containing DHA and ARA may be added to a formula by replacing the equivalent remainder (rest) of the total fat blend normally present in a formula without DHA and ARA.

If used, the source of DHA and ARA may be any source known in the art, such as marine oil, fish oil, single cell oil, yolk lipids and brain lipids. In some embodiments, the DHA and ARA are derived from single cell Martek oil, DHASCO and ARASCO ® or variants thereof, respectively. DHA and ARA may be in natural form, provided that the remainder of the LCPUFA source does not cause any substantial deleterious effect on the infant. Alternatively, DHA and ARA may be used in refined form.

In one embodiment of the present disclosure, the source of DHA and ARA is single cell oil as taught in U.S. patent nos. 5,374,567, 5,550,156, and 5,397,591, the disclosures of which are incorporated herein by reference in their entirety. However, the present disclosure is not limited to these oils.

In certain embodiments, the nutritional composition comprises from about 0.5mg/100kcal to about 5mg/100kcal of iron, including iron bound to lactoferrin.

One benefit of lactoferrin as used in embodiments of the present disclosure is its anti-invasive and anti-adhesion mechanisms in the human gastrointestinal tract. In particular, lactoferrin can disrupt certain bacterial invasion and cause the injection needles used for morbidity. Similarly, lactoferrin inhibits the adhesion of pathogens in the human gastrointestinal tract. One such example of a bacterium known to cause morbidity is escherichia coli, which can cause diarrhea in infants, children, and adults, and is considered an motivator for infantile diarrhea. Coli produces and transports bacterial proteins via needle-like complexes via a type III secretion system.

The secretion system of many gram-negative pathogenic bacteria is type III secretion, including the following bacteria: shigella, Salmonella, Pseudomonas and E.coli. The type III secretion system functions by using a needle to transport toxic proteins from the bacterial cytoplasm through the needle directly into the cytoplasm of the host cell. The use of needles provides for crossing multiple membranes, including the bilayer membrane of gram-negative bacteria and the eukaryotic cell membrane of human cells. Specifically, in the escherichia coli strain, the needle-shaped complex is composed of an escherichia coli secretory component f (escf), and escherichia coli secretory protein a (espa) is attached to a needle tip to form a substantially hollow structure, so that the component is passed from the bacteria to the host human cell. In this regard, bacterial proteins such as EspB can be introduced into host cells through this channel. Although The physiology of EspB is not fully understood in The paper "The Enteropothogenic E. coli effector EspB surface influencing and anti-Phagocytosis by Inhibiting myostatin Function", CellHosts and Microbe, pp 383-392 (2007), EspB is described as binding to Myosin, ultimately Inhibiting phagocytosis as a human immune response. Generally, myosin interacts with actin filaments to participate in cellular processes, such as eliminating phagocytosis by potential bacterial pathogens. When EspB released by e.coli in inhibiting phagocytosis inhibits the interaction between various myosin and actin filaments, adverse symptoms occur, resulting in diarrhea or other gastric distress in infants, children and adults (gastrostre).

One of the anti-invasion mechanisms of lactoferrin is to inhibit the transport of EspB into human cells. In particular, one mechanism may include inhibiting the formation of secretory structures necessary for the transport of EspB from bacteria. Lactoferrin is capable of degrading EspA, a protein responsible for transporting EspB into the tube-like structure of the host cell. Because EspA can be degraded by lactoferrin, it is not generated through the entrance of human cell membrane, thereby alleviating the morbidity caused by the entry of EspB into the cytoplasm of human cells. Also, lactoferrin may also have proteolytic activity, leading to degradation of EspB. Finally, lactoferrin effectively destroys the needle-like complexes associated with the pathogen secretion system, while degrading proteins responsible for symptoms including gastrointestinal distress and diarrhea.

Examples

The following examples are provided to illustrate embodiments of the nutritional compositions of the present disclosure, but should not be construed as limiting the disclosure in any way. Other embodiments within the scope of the claims herein will be apparent to those skilled in the art from consideration of the specification or practice of the nutritional compositions or methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a scope and spirit of the disclosure being indicated by the claims which follow the examples.

Example 1

This example illustrates the inhibition of pathogens, enterobacter sakazakii 4603 and 29004, and escherichia coli E2348/69, by lactoferrin alone and in combination with polydextrose and galactooligosaccharides.

Based on preliminary experiments, it was determined that the adhesion rate of the cultures of enterobacter sakazakii 4603 and 29004 to HEp-2 cells was the highest. After 6 hours of incubation, cultures of these Enterobacter sakazakii strains, as well as cultures of E.coli E2348/69 were harvested by centrifugation, washed with phosphate buffer, and resuspended in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum. HEp-2 cells (obtained from ATTC) in 75cm MEM containing 25ml of 10% FBS²In tissue culture flasks in CO₂The growth was in an incubator under tissue culture conditions. Confluent HEp-2 cells were harvested by adding 0.5ml of 0.25% trypsin-EDTA solution (Sigma) and incubating for 15 min under tissue culture conditions.Trypsin was inactivated with 0.5ml FBS, cells at approximately 3.6X 10 per well⁵Individual viable cells were seeded onto 12-mm diameter glass coverslips in 24-well tissue culture plates. The plates were incubated under tissue culture conditions for two days, or until confluence was reached, before starting each experiment.

Immediately before the start of the analysis, lactoferrin at final concentrations of 0.1 mg/ml, 0.6 mg/ml and 1 mg/ml was used alone and in combination with galacto-oligosaccharides (obtained from DOMO) and polydextrose (obtained from DMV) at final concentrations of 4 mg/ml and 16 mg/ml in a ratio of 1: 1 blend combination, and HEp-2 cells were added. Control wells without lactoferrin were also prepared. Then 900: 1 of a culture of Escherichia coli or Enterobacter sakazakii (containing about 10)⁷Cells) were added to each well (in triplicate). Tissue culture plates were then incubated at 37 ℃ with CO₂Incubate in incubator for 3 hours. The wells were then washed 5 times, non-adherent cells were removed, and adherent bacteria were counted by microscopic counting and quantitative real-time PCR.

For microscopic counting, coverslips were fixed with 100% methanol, stained with 10% giemsa for 15 minutes, washed with distilled water, and dried overnight. The coverslip was mounted on a microscope slide and viewed under a phase contrast microscope with a 100 x objective. Each coverslip was taken with 15 micrographs using the Motic Image software, according to the geometric pattern established throughout the coverslip. The number of cells and bacteria in each Image was counted using Image J Image analysis software. Adhesion was calculated as the number of bacteria adhered per HEp-2 cell. Adhesion inhibition was calculated as the number of bacteria adhered to each cell in the control minus the number of bacteria adhered to each cell in the treatment divided by the number of bacteria adhered to each cell in the control. For experiments using E.coli cultures, cells with E.coli microcolonies were counted manually. Cells with 4 or more bacteria were considered positive because of the typical local adhesion phenotype. The number of HEp-2 cells with adherent microcolonies was determined, and adhesion inhibition were calculated as described above. The experiments were performed in triplicate and repeated 3 times (n-9).

In addition to microscopic counting, adherent cells were also counted by quantitative real-time PCR (qRT-PCR), as described in Humphries et al, Interactions of organic Escherichia coli with a peptide and with an endogenous biopsys specificity, infection. Immun., 77, 4463-loop 4468 (2009). Briefly, genomic DNA was extracted from infected HEp-2 cells and quantified by qRT-PCR using oligonucleotide primers that amplified the enterobacter sakazakii 4603 and 29004 or the e.coli 2348/6916 s rRNA region. Properly diluted whole genome DNA was used as an internal control and a standard curve was prepared that correlates qPCR endpoints with cell concentration. The PCR mixture consisted of 11.25 μ l SYBR solution, 2.5 MasterMix, 1 μ l of each primer, and 5 μ l DNA template. The PCR reaction was performed using an Eppendorf Mastercycler Realplex 2.

Example 2

This example illustrates one embodiment of a nutritional product according to the present disclosure.

Describe kg/100kg

Carbohydrate, total amount 38.9

Protein, total 28.8

Fat, total 25.6

Prebiotics 4.5

Soybean lecithin 0.8

Lactoferrin 0.3

Calcium carbonate 0.5

Potassium citrate 0.2

Ferrous sulfate 0.05

Potassium chloride 0.048

Magnesium oxide 0.023

Sodium chloride 0.025

0.015 percent zinc sulfate

Copper sulfate 0.002

Magnesium sulfate 0.0003

Sodium selenite 0.00003

Choline chloride 0.144

Ascorbic acid 0.093

Nicotinamide 0.006

Calcium pantothenate 0.003

Palmitic acid vitamin A0.007

Vitamin B120.002

Vitamin D30.000001

Riboflavin 0.0008

Thiamine 0.0006

Vitamin B60.0004

Folic acid 0.0001

Vitamin K10.006

Biotin 0.00002

Inositol 0.03

Vitamin E acetate 0.01

Taurine 0.05

L-carnitine 0.001.

Example 3

This example illustrates another embodiment of a nutritional product according to the present disclosure.

Describe kg/100kg

Carbohydrate, total amount 24.7

Protein, total amount 31.9

Fat, total amount 39.3

Prebiotics 3.6

Lactoferrin 0.1

Calcium carbonate 0.15

Ferrous sulfate 0.03

Zinc sulfate 0.01

Copper sulfate 0.00025

Magnesium sulfate 0.0002

Sodium selenite 0.00001

Choline bitartrate 0.05

Ascorbic acid 0.004

Ascorbic acid sodium salt 0.04

Nicotinamide 0.007

Calcium pantothenate 0.0005

Palmitic acid vitamin A0.0005

Vitamin D30.0002

Riboflavin 0.0001

Thiamine 0.00005

Vitamin B60.00005

Folic acid 0.000067

Vitamin K10.00002

Vitamin E acetate 0.008

Taurine 0.02

0.2 part of fish oil

B-glucan 0.03.

Example 4

This example illustrates one embodiment of ingredients that can be used to prepare the nutritional products of the present disclosure.

872 ml of water

Lactose 65.6 mg

Vegetable oil blend 353.0 mg

Evaporated skimmed milk 34.0 mg

Whey protein concentrate 8.5 mg

Galacto-oligosaccharide 4.7 mg

Casein 3.5 mg

Polydextrose 2.4 mg

Lactoferrin solution (10%) 1.0 mg

Single cell DHA and ARA oil blend 0.94 mg

Mono-and diglycerides 0.7 mg

Calcium carbonate 0.44 mg

Calcium phosphate 0.4 mg

Potassium citrate 0.4 mg

Potassium chloride 0.4 mg

Soybean lecithin 0.4 mg

Sodium chloride 0.3 mg

Potassium phosphate 0.3 mg

Choline chloride 0.2 mg

Magnesium oxide 0.08 mg

0.08 mg of calcium hydroxide

Ferrous sulfate 0.07 mg.

Example 5

This example illustrates another embodiment of ingredients that can be used to prepare nutritional products according to the present disclosure.

686 ml of water

Reduced minerals whey 215 mg

Evaporated skim milk 67 mg

Vegetable oil blend 33 mg

Lactose 17 mg

Galacto-oligosaccharide 4.7 mg

Polydextrose 2.4 mg

Lactoferrin solution (10%) 1.0 mg

Single cell DHA and ARA oil blend 0.9 mg

Mono-and diglycerides 0.7 mg

Calcium carbonate 0.44 mg

Calcium phosphate 0.4 mg

Potassium citrate 0.4 mg

Potassium chloride 0.4 mg

Soybean lecithin 0.4 mg

Potassium phosphate 0.3 mg

Carrageenan 0.3 mg

Sodium citrate 0.2 mg

Choline chloride 0.2 mg

Magnesium oxide 0.08 mg

Calcium chloride 0.08 mg

Ferrous sulfate 0.07 mg.

All references cited in this specification, including but not limited to all papers, publications, patents, patent applications, lectures, texts, reports, manuscripts, manuals, books, web publications, journal articles, periodicals, and the like, are hereby incorporated by reference in their entirety. The discussion of the references herein is intended merely to summarize the assertions made by their authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinency of the cited references.

Although the preferred embodiments of the present disclosure have been described using specific terms, tools, and methods, such description is for illustrative purposes only. The words used are words of description rather than limitation. It is to be understood that changes and variations may be made by those skilled in the art without departing from the spirit or scope of the present disclosure, which is set forth in the following claims. Additionally, it should be understood that aspects of the various embodiments may be interchanged both in whole or in part. For example, while methods of producing commercial sterilized liquid nutritional supplements prepared according to those methods are exemplified, other uses are contemplated. Therefore, the spirit and scope of the appended claims should not be limited to the description of the preferred versions contained herein.

Claims (13)

1. Use of lactoferrin produced by a non-human source in the manufacture of a nutritional composition for inhibiting adhesion of bacterial pathogens in a human, comprising administering to the human the nutritional composition comprising:

a. a lipid;

b. a source of protein;

c. 0.1 to 1g/100kcal of prebiotic composition, wherein the prebiotic composition comprises at least 20% oligosaccharides; and

d. a source of lactoferrin produced from a non-human source isolated from a protein source, wherein the source of lactoferrin produced from a non-human source is present in an amount of at least 10mg/100kCal, wherein the lactoferrin retains its anti-adhesion capacity even after exposure to pH and temperature fluctuations or pasteurization.

2. Use of lactoferrin produced by a non-human source in the manufacture of a nutritional composition for inhibiting adhesion of bacterial pathogens in a human, comprising administering to the human the nutritional composition comprising:

a. fat;

b. a source of protein;

c. 0.1 to 1g/100kcal of prebiotic composition, wherein the prebiotic composition comprises at least 20% oligosaccharides; and

d. a source of lactoferrin produced from a non-human source isolated from a protein source, wherein the source of lactoferrin produced from a non-human source is present in an amount of at least 10mg/100kCal, wherein the lactoferrin retains its anti-adhesion capacity even after exposure to pH and temperature fluctuations or pasteurization.

3. Use according to claim 1 or 2, wherein the nutritional composition further comprises 5 to 100 mg/100kcal of a source of long chain polyunsaturated fatty acids comprising docosahexaenoic acid.

4. Use according to claim 3, wherein the source of long chain polyunsaturated fatty acids further comprises arachidonic acid.

5. Use according to claim 1, wherein the lipid is present at a level of up to 7g/100 kcal.

6. Use according to claim 2, wherein the fat is present at a level of at most 7g/100 kcal.

7. Use according to claim 1 or 2, wherein the protein source is present at a level of at most 5g/100 kcal.

8. Use according to claim 1 or 2, wherein the oligosaccharide comprises a galactooligosaccharide.

9. Use according to claim 8, wherein the oligosaccharide further comprises polydextrose.

10. Use according to claim 1 or 2, wherein the lactoferrin is present at a level of 70mg to 220mg/100 kCal.

11. Use according to claim 5, wherein the lipid source is present at a level of from 3g/100kcal to 7g/100 kcal.

12. Use according to claim 6, wherein the fat source is present at a level of from 3g/100kcal to 7g/100 kcal.

13. Use according to claim 7, wherein the protein source is present at a level of 1g/100kcal to 5g/100 kcal.