TWI626893B - Method of using a nutritional composition to inhibit pathogens - Google Patents

Abstract

A method for inhibiting the mechanism of bacterial invasion or attachment, which involves administering to a human a nutritional composition including lipids or fats; a source of protein; a probiotic composition; and at least about 10 mg / 100kCal Lactoferrin made from non-human sources.

Description

Method for inhibiting pathogens using nutritional composition

This document relates generally to the field of nutritional compositions such as infant formula, human milk fortified supplements, children's dietary supplements, etc., which have lactoferrin in the nutritional composition. More specifically, this article relates to a method for inhibiting the invasion of harmful bacterial pathogens and / or inhibiting at least one pathogen in the human gastrointestinal tract by administering to a human a nutritional composition including lactoferrin produced by non-human sources Method of attachment.

There are various dietary compositions for humans, especially young people, to provide supplemental or primary nutrition during certain life stages. In general, commercially available dietary compositions for infants mimic the composition of human milk and related functionalities as much as possible. By combining some of these proteins with physiological activity and blended fat ingredients, the dietary composition is modulated to make them mimic human milk for use as complete or partial substitutes. Other ingredients commonly used in infant dietary compositions may include, for example, carbohydrate sources of lactose and vitamins, minerals and elements that are present in human milk for absorption by infants.

Some of the proteins found in human milk provide defense against pathogens to prevent and suppress infections, while also boosting the immune response in infants. Proteins including haptocorrin (vitamin B12 binding protein) and lactoferrin are believed to help infants defend against various bacterial pathogens through bacteriostatic and bactericidal activities.

Lactoferrin is one of the main proteins in human milk and is considered to have There are glycoproteins with an average molecular weight of about 80 kilodaltons. It is an iron-binding protein that has the ability to bind two iron molecules in a reversible form and can promote iron uptake in the human small intestine. Functionally, lactoferrin regulates iron absorption and can thus bind iron-based free radicals and supply iron for immune responses.

Often, the additional role of lactoferrin is in its antimicrobial activity in defending human intestinal infections, especially in infants. Lactoferrin is known to inhibit the bacteriostatic and bactericidal properties of microorganisms before successfully invading small intestinal cells while inhibiting the growth of specific bacteria.

In order to obtain a commercially viable dietary composition, the addition of lactoferrin is often limited due to the expected loss of activity during processing. For example, the temperature and pH requirements of infant formulas and other fortified human milk supplements and various children's products often reduce the specific functions of lactoferrin, resulting in the absence of lactoferrin in the final preparation. In addition, lactoferrin is often considered only for its iron-binding properties; therefore, when this property is considered to be reduced by processing conditions, the modulator may generally not have lactoferrin.

Also, as is known in the art, human milk has a relatively low iron content (containing about 0.3 mg of iron per liter of human milk). Although low in content, human infants have high absorption rates, absorbing about half of the iron in human milk. However, when human infants are fed with prior art formulations with high levels of iron nutrition fortification, such as about 10 mg to about 12 mg per liter, the infant absorbs less than about 5% of the total iron content. With the high iron content in this prior art formulation, although lactoferrin is a known iron transporter, basically all lactoferrin iron binding sites are expected to be occupied.

Other difficulties with this prior art formulation include the inability to provide bacteriostatic effects . This is partly due to the use of lactoferrin with a blocked or damaged binding site, but the bacteriostatic effect is at least partially related to the degree of iron binding of the lactoferrin present in the formulation.

Therefore, it would be beneficial to provide nutritional compositions containing lactoferrin, especially lactoferrin made from non-human sources, such as infant formula, human milk nutrition supplements, dietary supplements for children, and the like. Preferably, the lactoferrin included in the composition has a bacteriostatic effect even after processing under high temperature and low pH conditions. The combination includes maintaining lactoferrin's resistance to invasion or resistance to mechanical attachment through high or low pH or high temperature conditions, such as during pasteurization, to provide a dietary composition that can at least partially protect against harmful bacterial pathogens.

Summary of invention

Briefly, in one embodiment, the present invention relates to the use of a nutritional composition comprising lipid or fat, a protein source, a probiotic composition, and lactoferrin from a non-human source for inhibiting the invasion of one or more bacterial pathogens. A method of mechanical transformation and / or attachment of at least one pathogen, wherein the composition provides an active anti-invasive mechanism against undesired bacterial strains in the human gastrointestinal tract even after processing including exposure to harsh environmental conditions.

In one embodiment, this document relates to a nutritional product comprising: a. Up to about 7 g / 100 kcal of fat or lipid, more preferably about 3 g / 100 kcal to about 7 g / 100 kcal of fat or Lipids; b. Protein sources up to about 5 g / 100 kcal, more preferably about 1 g / 100 kcal to about 5 g / 100 kcal of protein source; c. lactoferrin of at least about 10 mg / 100 kcal, more preferably about 70 mg / 100 kcal to about 220 mg / 100 kcal by non-human sources The lactoferrin produced is preferably about 90 mg / 100 kcal to about 190 mg / 100 kcal, lactoferrin manufactured from non-human sources; and d. About 0.1 g / 100 kcal to about 1 g / 100 A probiotic composition of kcal, which comprises polydextrose and / or galactooligosaccharide. More preferably, the nutritional composition comprises a probiotic composition of about 0.3 g / 100 kcal to about 0.7 g / 100 kcal, which comprises a combination of polydextrose and galactooligosaccharide.

Preferably, the lactoferrin is a non-human lactoferrin and / or a human lactoferrin produced by a genetically modified organism. The term "organism" as used herein refers to any continuous living system, such as an animal, plant, fungus, or microorganism. In a particularly preferred embodiment, the lactoferrin is used so that an effective amount of a lactoferrin-containing nutritional composition can be administered to inhibit at least one of the invasion mechanisms of at least one pathogen in the human gastrointestinal tract, even if During processing, the nutritional composition has been exposed to pH and temperature fluctuations typical of some pasteurized processing conditions. The lactoferrin includes an anti-invasion mechanism that can destroy the adhesion factors and injection needles used by certain bacteria on human cells. In another particularly preferred embodiment, the lactoferrin is used so that an effective amount of a lactoferrin-containing nutritional composition can be administered to inhibit the adhesion of at least one pathogen in the human gastrointestinal tract, even during processing. The same applies to the pH and temperature fluctuations of nutritional compositions that have been exposed to typical pasteurization-like processing conditions. Examples of such pathogens include enterotoxic E. coli (ETEC), enteropathogenic E. coli (EPEC), Haemophilus influenza , Shiga toxin-producing E. coli (STEC), enteroaggregative E. coli ( EAEC), Salmonella ser. Typhimurium, Shigella flexneri , Rotavirus, Norovirus, Respiratory Fusion Virus (RSV), Adenovirus and combinations thereof.

Provided herein are novel nutritional products that are easy to digest, provide physiochemical benefits, and / or provide physiological benefits. In one embodiment herein, the nutritional composition comprises a lipid or fat, a protein source, a probiotic composition with at least 20% oligosaccharides, particularly a galactose oligosaccharide (GOS), and lactoferrin, Provides active anti-invasion or anti-adhesion machinery against unwanted bacterial strains present in the human gastrointestinal tract. The lactoferrin contained in the composition is manufactured from a non-human source. In certain embodiments, the probiotic contains a combination of galactooligosaccharide and polydextrose. More specifically, the composition disclosed herein comprises: a. Up to about 7 g / 100 kcal of fat or lipid, more preferably about 3 to about 7 g / 100 kcal of fat or lipid; b. Up to A protein source of about 5 g / 100 kcal, more preferably a protein source of about 1 to about 5 g / 100 kcal; c. A probiotic composition of about 0.1 g / 100 kcal to about 1 g / 100 kcal, comprising Polydextrose and / or galactooligosaccharide. More preferably, the nutritional composition comprises a probiotic composition of about 0.3 g / 100 kcal to about 0.7 g / 100 kcal, which comprises a combination of polydextrose and galactooligosaccharides; and d. Lactoferrin of at least about 10 mg / 100 kCal, more preferably about 70 mg to about 220 mg / 100 kCal of lactoferrin, and most preferably about 90 mg to about 190 mg / 100 kCal of lactoferrin.

definition

As used herein, the term "probiotics" means non-digestible food ingredients that positively affect a host by selectively stimulating the growth and / or activity of one or a few bacteria in the colon that can improve host health.

The term "probiotic" means a low-pathogenic or non-pathogenic microorganism that has a positive effect on the health of the host.

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

"Premature babies" are babies born after about 37 weeks of pregnancy.

As used herein, "term infant" means an infant born after at least 37 weeks of pregnancy.

"Child" is defined as someone from 12 months to about 12 years old.

As used herein, an "antibacterially effective amount" is generally defined as the amount of lactoferrin that provides at least one anti-invasion mechanism against a bacterial strain.

As used herein, "lactoferrin produced from non-human sources" means lactoferrin manufactured or obtained from sources other than human milk. For example, lactoferrin as used herein includes human lactoferrin and non-human lactoferrin produced by genetically modified organisms.

"Bioactive lactoferrin" means lactoferrin with at least one anti-invasion or anti-adhesion mechanism against pathogenic bacteria.

As used herein, the term "non-human lactoferrin" refers to lactoferrin having an amino acid sequence that is different from the human lactoferrin amino acid sequence.

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

As used herein, the term "mimic" means having or taking some form or appearance, or having or producing similar symptoms.

Disclosure of invention

In some embodiments, the nutritional product may be an infant formula. The term "infant formula" refers to a composition that is a substitute for human milk that is in liquid or powder form and meets the nutritional needs of infants. In the United States, infant formula levels are specified in Title 21, Sections 100, 106, and 107 of the Code of Federal Regulations. These regulations define large amounts of nutrients, vitamins, minerals and other ingredients to mimic the nutritional and other properties of human milk. In another embodiment, the nutritional product may be a human milk fortified product, which means that it is a composition added to human milk to enhance the nutritional value of human milk. As a human milk fortified nutritional product, the disclosed composition may be in powder form or liquid form. In yet another embodiment, the nutritional product herein may be a nutritional composition for children.

The nutritional products in this article can provide minimal, partial, or full nutritional support. The composition may be a nutritional supplement or a meal replacement. In some embodiments, the composition may be administered along with a food or nutritional composition. In this embodiment, the composition may be intermixed with food or other nutritional composition prior to ingestion by the individual, or may be administered to the individual before or after ingestion of the food or nutritional composition by the individual. This composition can be administered to infant formula, breast milk, and human milk fortification camps. Premature babies with nutrition or their combination.

The composition may be (but not necessarily) nutritionally complete. Those skilled in the art will understand that "nutrition integrity" varies depending on many factors, including but not limited to the age of the individual to which the term applies, clinical conditions, and dietary intake. In general, "nutritive integrity" means that the nutritional composition herein provides all the appropriate amounts of carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditional essential amino acids, vitamins, minerals required for normal growth. And energy. When referring to nutrients, the term "essential" refers to any nutrient that the body cannot synthesize enough for normal growth and maintenance of health and therefore must be supplied through the diet. The term "conditionally necessary" when referring to a nutrient means that the nutrient must be supplied through the diet under conditions where the body cannot obtain a suitable amount of precursor compounds for endogenous synthesis.

For premature babies, the "nutritional integrity" composition is defined as providing a suitable amount of carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditional essential amino acids, Vitamins, minerals and energy. For term infants, the "nutritional integrity" composition is defined as providing the appropriate amount of carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, and conditional essential amino groups required for all growth of the term infant Acids, vitamins, minerals and energy. For children, the "nutritive integrity" composition is defined as providing the child with all the quality carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditional essential amino acids, vitamins, Minerals and energy.

The nutritional composition may be provided in any form known in the art, These include powders, gels, suspensions, pastes, solids, liquids, liquid concentrates or ready-made products. In a preferred embodiment, the nutritional composition is an infant formula, which is particularly suitable for use as an infant formula as the sole source of nutrition for infants.

In a preferred embodiment, the nutritional products disclosed herein can be administered enterally. As used herein, "intestinal tract" means through or within the gastrointestinal or digestive tract, and "parenteral administration" includes oral feeding, intragastric feeding, transpyloric administration, or Any other lead into the digestive tract.

Lactoferrin is a single-chain polypeptide of about 80 kD and contains 1-4 glycans depending on the species. The 3-D structures of lactoferrin in different species are very similar, but not exactly the same. Each lactoferrin contains two homotypic leaf-like structures, called N-lobe and C-lobe, which refer to the N- and C-terminus of the molecule, respectively. Each leaf-like structure is further composed of two secondary leaves or domains, which form a fissure, in which the iron ion (Fe ³⁺ ) and the (di) carbonate anion cooperate in close cooperation. These domain systems are called N1, N2, C1, and C2, respectively. The N-terminus of lactoferrin has a strong cationic peptide region, which 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 groups of cationic amino acid residues in the N-terminal region of lactoferrin to mediate the biological activity of lactoferrin against various microorganisms. For example, N-terminal residues 1-47 of human lactoferrin (1-48 for bovine lactoferrin) are critical to the iron-independent biological activity of lactoferrin. In human lactoferrin, residues 2 to 5 (RRRR) and 28 to 31 (RKVR) at the N-terminus are rich in arginine cation domains, which are particularly important for the antimicrobial activity of lactoferrin. A similar N-terminal region (residues 17 to 42; FKCRRWQWRMKKLGAPSITCVRRAFA) was also found in bovine lactoferrin.

As described in Perspectives on Interactions Between Lactoferrin and Bacteria (this article appears in BIOCHEMISTRY AND CELL BIOLOGY, pp. 275-281 (2006)), lactoferrin from different host species although usually has a relatively high isoelectric point , With positively charged amino acids in the inner lobe terminal region, but the amino acid sequence is different. Suitable lactoferrin for use herein includes having at least 48% homology to the amino acid sequence AVGEQELRKCNQWSGL of the HLf (349-364) fragment. In some embodiments, the lactoferrin has at least 65% homology to the amino acid sequence AVGEQELRKCNQWSGL of the HLf (349-364) fragment, and in embodiments, at least 75% homology. For example, non-human lactoferrin as used herein includes, but is not limited to, bovine lactoferrin, porcine lactoferrin, horse lactoferrin, buffalo lactoferrin, goat lactoferrin, murine lactoferrin, and camel lactoferrin protein.

Surprisingly, the lactoferrin forms contained herein are even exposed to low pH (ie, less than 7, and even as low as about 4.6 or less) and / or high temperatures (ie, more than about 65 ° C, and as high as about 120 ° C), which is expected to disrupt or severely limit the stability or activity of human lactoferrin or recombinant human lactoferrin, and still maintain the relevant activity. These low pH and / or high temperature conditions may be expected during certain processing methods for the types of nutritional compositions described herein, such as pasteurization. For example, the amino acid composition of bovine lactoferrin has only about 70% sequence homology with human lactoferrin, and is stable under conditions where human or recombinant human lactoferrin becomes unstable or inactive, and Still with At the same time, bovine lactoferrin has a fungicidal activity against undesired bacterial pathogens found in the human intestine.

In U.S. Patent No. 4,791,193, which is incorporated by reference in its entirety, Okonogi et al. Disclose a method for manufacturing high-purity bovine lactoferrin. Generally, the method, as disclosed, involves three steps. The raw milk raw material is first contacted with a weakly acidic cation exchanger to adsorb lactoferrin, followed by a second step, in which a rinse is performed to remove unadsorbed material. This is followed by a desorption step, at which time the lactoferrin is removed to produce purified bovine lactoferrin. Other methods may include steps as described in US Patent Nos. 7,368,141, 5,849,885, 5,919,913, and 5,861,491, all of which are incorporated herein by reference in their entirety.

In one embodiment, lactoferrin is present in the nutritional composition in an amount of at least about 10 mg / 100 kCal, especially when the nutritional composition is desired for children. In some embodiments, the upper limit for lactoferrin is about 240 mg / 100 kCal. In another embodiment, wherein the nutritional composition is an infant formula, lactoferrin is present in the nutritional composition in an amount of about 70 mg to about 220 mg / 100 kCal; in still another embodiment, lactoferrin The protein is present in an amount of about 90 mg to about 190 mg / 100 kCal. Nutritional compositions for infants may include lactoferrin in an amount of about 0.5 mg to about 1.5 mg per liter of formula. In replacing the nutritional composition of human milk, the content of lactoferrin may be from about 0.6 mg to about 1.3 mg per liter of the formula.

A suitable source of fat or lipid for practicing the present invention may be any known or user in the art, including but not limited to animal sources, such as: milk fat, butter, butter fat, Egg yolk lipids; aquatic sources such as: fish oil, aquatic fats, single-cell oils; vegetable and vegetable fats such as corn oil, rapeseed oil, sunflower oil, soybean oil, palmolein, coconut oil, high oleic acid Sunflower oil, evening primrose oil, canola oil, olive oil, linseed oil, cottonseed oil, high oleic safflower oil, palm stearin, palm kernel oil, wheat germ oil; medium chain triglycerides and Emulsifiers and esters of fatty acids; and any combination thereof.

Sources of cow's milk proteins useful in the practice herein include, but are not limited to, milk protein powder, milk protein concentrate, milk protein isolate, skim milk solids, skim milk, skim milk powder, whey protein, whey protein isolate, whey protein Concentrates, sweet whey, sour whey, casein, sour casein, casein salts (e.g. sodium caseinate, sodium caseinate calcium, caseinate calcium) and any combination thereof.

In one embodiment, the protein is provided as a whole protein. In other embodiments, the protein is provided as both a whole protein and a partially hydrolyzed protein, with a degree of hydrolysis ranging from about 4% to 10%. In yet another embodiment, the protein source may be supplemented with a glutamate-containing peptide.

In a specific embodiment herein, the protein-derived whey: casein ratio is similar to that in human milk. In one embodiment, the protein source comprises about 20% to about 85% whey protein. In another embodiment, the protein source may include about 20% to about 80% casein. In yet another embodiment herein, the protein source includes about 60% to about 85% whey and about 15% to about 40% casein.

In one embodiment herein, the nutritional composition may also contain one or more probiotics. As long as the desired results are achieved, any probiotics known in the art are acceptable in this example. In a specific embodiment, the probiotic bacteria may be selected from Lactobacillus strains, Lactobacillus rhamnosus GG, Bifidobacterium strains, Bifidobacterium longum strains. , Bifidobacterium brevis and Bifidobacterium animalis subsp . Lactis BB-12.

If included in the composition, the amount of the probiotic may be formed units (cfu) per day is from about ¹⁰⁴ to about ¹⁰¹⁰ colonies per kg body weight. In another embodiment, the amount of probiotics may be about 10 ⁶ to about 10 ⁹ cfu per kg of body weight per day. In yet another embodiment, the amount of probiotics may be at least about 10 ⁶ cfu per kg of body weight per day. In addition, the disclosed composition may also include a probiotic-conditioned medium component.

In one embodiment, the probiotic bacteria may be live bacteria or non-live bacteria. As used herein, the term "live bacteria" refers to living microorganisms. The term "non-living bacteria" or "non-living probiotics" means non-living probiotic microorganisms, their cellular composition and their metabolites. These non-living probiotic bacteria can be heat-killed or otherwise inactivated, but still retain the ability to have a beneficial effect on host health. In this context, the probiotics available may be developed by naturally occurring, synthetic or genetically manipulated organisms, whether such new sources are now known or later developed.

The nutritional composition contains one or more probiotics. Such probiotics can be developed by naturally occurring, synthetic or genetically manipulated organisms and / or plants, regardless of whether the new source is now known or later developed. In some real In the examples, the probiotics of the compositions included herein include the teachings of US Patent No. 7,572,474, the disclosure of which is incorporated herein by reference.

Probiotics useful herein may include oligosaccharides, polysaccharides, and other probiotics containing fructose, xylose, soy, galactose, glucose, and mannose. More specifically, the probiotics useful herein may include lactulose, lactosucrose, raffinose, gluco-oligosaccharides, inulin, polydextrose, polydextrose powder, galactooligosaccharides, and fructo-oligosaccharides. Sugar, isomalt oligosaccharide, soy oligosaccharide, lactulose oligosaccharide, xylose oligosaccharide, chito-oligosaccharide, mannose oligosaccharide, arabinose oligosaccharide, sialic acid oligosaccharide, trehalose oligosaccharide , And gentio-oligosaccharide.

In one embodiment, the total amount of probiotics present in the nutritional composition may be about 1.0 g to about 10.0 g per liter of the composition. Alternatively, the total amount of probiotics present in the nutritional composition may be about 2.0 g to about 8.0 g per liter of the composition. At least 20% of the probiotics should contain galactose oligosaccharides (GOS), polydextrose, or mixtures thereof. Preferably, the nutritional composition comprises polydextrose and galactooligosaccharide. Optionally, in addition to polydextrose and / or galactooligosaccharide, the nutritional composition comprises one or more additional probiotics. In one embodiment, each content of galactooligosaccharide and / or polyglucose 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 probiotics present in the nutritional composition may be about 0.1 g / 100 kcal to about 1 g / 100 kcal. More preferably, the total amount of probiotics stored in the nutritional composition may be about 0.3 g / 100 kcal to about 0.7 g / 100 kcal. At least 20% of this probiotic should contain galactose oligosaccharides (GOS) and / or polydextrose (PDX).

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

Preferably, when the nutritional composition is provided to a human, the probiotic composition combined with lactoferrin inhibits the adhesion of one or more pathogens in the gastrointestinal tract. One example of such a pathogen is Enterobacter sakazakii (or aka Cronobacter sakazakii ). Another pathogen is E. coli, whose attachment is inhibited by combining lactoferrin with the probiotic composition.

The nutritional formulas herein may also contain a long-chain polyunsaturated fatty acid (LCPUFA) source, which contains docosahexaenoic acid (DHA). Other suitable LCPUFAs include, but are not limited to, alpha-linolenic acid, gamma-linolenic acid, Linoleic acid, hypolinoleic 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 ARA: DHA weight ratio may be from about 1: 3 to about 9: 1. In an embodiment herein, the ratio is about 1: 2 to about 4: 1.

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

The nutritional composition can be supplemented with fats containing DHA and ARA using standard techniques known in the art. For example, DHA and ARA can be added to the formula by replacing equivalent amounts of fats and oils typically present in the formula (such as high oleic sunflower oil). As another example, fats and oils containing DHA and ARA can be added to the formulation by replacing equivalent amounts of the remaining whole fat blends that are normally present in formulations without DHA and ARA.

If used, DHA and ARA sources can be any known source in the art, such as aquatic oils, fish oils, single-cell oils, egg 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. The DHA and ARA can be in natural form, but the residue of the LCPUFA source will not cause any substantial harmful effects on the baby. Alternatively, the DHA and ARA can be used in a refined form.

In an embodiment herein, the sources of DHA and ARA are single-cell lipids as taught in U.S. Pat. Nos. 5,374,567, 5,550,156, and 5,397,591. The disclosures of these patents are incorporated herein by reference in their entirety. . However, this article is not limited to these greases.

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

The benefits of lactoferrin as used in the examples herein are its resistance to invasion and resistance to attachment in the human gastrointestinal tract. In particular, lactoferrin can destroy certain bacterial invasions and cause injection needles used for pathogenesis. Similarly, lactoferrin inhibits the adhesion of pathogens in the human gastrointestinal tract. One example of such bacteria known to cause disease is E. coli, which can cause diarrhea in infants, children, and adults, and is considered to be the cause of diarrhea in children. E. coli makes bacterial proteins and translocates them through a needle complex via a type III secretion system.

The secretion system of many Gram-negative pathogens is a type 3 secretion system, which includes the following bacteria: Shigella, Salmonella, Pseudomonas, and E. coli. The third type of secretion system uses the needle of the transported toxic protein to transport the toxic protein from the bacterial cytoplasm through the needle to the cytoplasm of the host cell. The needle is used to provide access through a multi-layered membrane including a two-layered membrane of gram-negative bacteria and human cell eukaryotic cell membranes. In particular, in E. coli strains, the acicular complex consists of E. coli secreted component F (EscF) and E. coli secreted protein A (EspA) attached to the tip of the needle, forming a generally hollow structure for the components The use of pathways from bacteria to host human cells. At this time, a bacterial protein such as EspB can be introduced into the host cell through this tube. Although the physiology of EspB may not be fully understood, in the article " The Enteropathogenic E. coli effector EspB facilitates microvillus effacing and Antiphagocytosis by Inhibiting Myosin Function " (in CELL HOSTS AND MICROBE, pp 383-392 (2007)) Describes that EspB binds to myosin and ultimately inhibits the phagocytosis of the human immune response. Generally, myosin proteins interact with actin filaments to participate in cellular processes, such as phagocytosis that clears possible bacterial pathogens. When EspB released by E. coli inhibits the interaction between various myosin proteins and actin filaments and inhibits phagocytosis, it can cause diarrhea or other stomach pains in infants, children, and adults, causing harmful symptoms.

Lactoferrin, one of the anti-invasion transgenic lines, inhibits the translocation of EspB into human cells. In particular, a mechanism may include inhibiting the formation of necessary secretory structures for the translocation of EspB from bacteria. Lactoferrin degrades EspA, a protein responsible for the tubular-like structure that is used to displace Esp B into host cells. When EspA can be degraded by lactoferrin, the passage through the human cell membrane will not be generated, thus reducing the incidence of EspB entering the cytoplasm of human cells. In addition, lactoferrin can also have proteolytic activity, causing the degradation of EspB. Finally, lactoferrin effectively interferes with the connection of the acicular complex to the pathogen's secretory system, while degrading proteins that cause symptoms including gastrointestinal pain and diarrhea.

Examples

The following examples are provided to illustrate the nutritional composition examples herein, but should not be construed as any limitation to this document. Those familiar with the technical field should consider this The description or implementation of the nutritional composition or method disclosed herein will clearly understand other embodiments within the scope of the patent application herein. This specification, together with this example, is only to be regarded as an example, and the scope and spirit of this document are specified by the scope of patent application following the example.

Example 1

This example exemplifies the inhibition of lactoferrin and lactoferrin combined with polydextrose and galactooligosaccharide pathogens, namely E. sakazakii 4603 and 29004, and E. coli E2348 / 69.

According to preliminary experiments, it was determined that E. sakazakii 4603 and 29004 cultured bacteria had the highest adhesion rate to HEp-2 cells. After 6 hours of incubation, these E. sakazakii strains and E. coli E2348 / 69 cultures were collected by centrifugation, rinsed with phosphate buffer, and resuspended in 10% fetal bovine serum-required minimal medium (MEM). HEp-2 cells (obtained from ATTC) were grown in 75 cm ² tissue culture flasks containing 25 ml of MEM supplemented with 10% FBS in tissue culture conditions in a CO ₂ incubator. Confluent HEp-2 cells were collected by adding 0.5 ml of a 0.25% trypsin-EDTA solution (Sigma) and culturing under tissue culture conditions for a total of 15 minutes. Trypsin was deactivated with 0.5 ml of FBS and cells were seeded on 12-mm diameter coverslips in a 24-well tissue culture plate with approximately 3.6 x 10 ⁵ viable cells per well. Plates were placed under tissue culture conditions for two days before starting each experiment or until reaching fullness.

Immediately before the test, lactoferrin and lactoferrin with final concentrations of 0.1 mg / ml, 0.6 mg / ml, and 1 mg / ml combined with 1: 1 galactooligosaccharide (obtained from DOMO) and polydextrose (from DMV (Obtained) was added to HEp-2 cells to a final concentration of 4 mg / ml and 16 mg / ml. Control wells without lactoferrin were also prepared. Subsequently, 900: 1, or E. sakazakii bacteria culture (number of cells containing about ¹⁰⁷⁾ were added to each well (repeated three groups). Thereafter, the tissue culture plate was placed in a CO ₂ incubator at 37 ° C. for a total of three hours. The wells were then washed five times to remove unattached cells, and the number of attached bacteria was counted by microscope counting and quantitative real-time PCR.

For microscope counting, coverslips were fixed with 100% methanol, stained with 10% Geimsa for 15 minutes, rinsed with distilled water, and dried overnight. The coverslip was fixed on a microscope slide and observed under a phase-contrast microscope with a 100x objective lens. Fifteen photomicrographs were taken of each cover glass using Motic Image software, and then a geometric pattern was created for the entire cover glass. Image J software was used to count the number of cells and bacteria in each image. Adhesion was calculated as the number of attached bacteria per HEp-2 cell. Adhesion inhibition was calculated by subtracting the number of bacteria attached to each cell in the control group from the number of bacteria attached to each cell in the control group and then dividing by the number of bacteria attached to each cell in the control group. For experiments using E. coli cultures, cells with E. coli microcolonies were manually counted. Cells with four or more bacteria were considered positive (with typical local attachment traits). The number of HEp-2 cells with attached microcolonies was measured, and the inhibition of adhesion was calculated as described above. The experiment was performed in a triplicate group and repeated three times (n = 9).

In addition to microscopic counting, attached cells are also counted by quantitative real-time PCR (qRT-PCR) (such as Humphries et al., Interactions of enteropathogenic Escherichia coli with pediatric and adult intestinal biopsy specimens during early adherence, Infect. Immun., 77, 4463-4468 (2009)). Briefly, genomic DNA was extracted from infected HEp-2 cells and quantified by qRT-PCR using oligonucleotide primers containing 16s rRNA regions of E. sakazakii 4603 and 29004 or E. coli 2348/69. An appropriately diluted whole genomic DNA was used as the internal control group, and a standard curve for qPCR endpoints as a function of cell concentration was prepared. The PCR mixture consisted of 11.25 μl SYBR solution, 2.5 MasterMix, 1 μl of each primer, and 5 μl of DNA template. This PCR reaction was performed using Eppendorf Mastercycler Realplex2.

Example 2

This example illustrates an example of a nutritional product according to this document.

Example 3

This example illustrates another embodiment of a nutritional product according to this document.

Example 4

This example illustrates one embodiment of the ingredients that can be used to prepare a nutritional product according to this document.

Example 5

This example illustrates another example of ingredients that can be used to prepare a nutritional product according to this document.

All references cited in this specification, including but not limited to all papers, journals, patents, patent applications, publications, texts, reports, manuscripts, brochures, books, web articles, journal articles, periodic publications, etc., All of them are incorporated herein by reference. The discussion of references in this article is intended only to summarize the author's claims and is not an admission that any reference constitutes prior art. The applicant reserves the right to challenge the accuracy and relevance of the cited references.

Although specific terms, devices, and methods have been used to describe the preferred embodiments herein, this description is for illustrative purposes only. The text used is descriptive rather than restrictive. It should be understood that those with ordinary knowledge in the art Changes and modifications may be made without departing from the spirit and scope of this document as claimed in the following patent application scope. In addition, it should be understood that various embodiments may be substituted in whole or in part for each other. For example, although methods for making commercially available sterile liquid nutritional supplements made according to these methods have been exemplified, other uses are also covered. Therefore, the spirit and scope of the appended patent application scope should not be limited to the preferred aspects contained in this description.

Claims (9)

A use of lactoferrin for manufacturing a nutritional composition for inhibiting the attachment of Cronobacter sakazakii in human body, the nutritional composition comprises: a) a lipid source; b) a protein source; c) isolated from A lactoferrin source produced from a non-human source from a protein source, wherein the lactoferrin source produced from a non-human source is present in an amount of at least 10 mg / 100 kCal, wherein the lactoferrin is exposed even to pH and temperature fluctuations Or it maintains its anti-adhesion ability after pasteurization; and d) a prebiotic composition of 0.1 to 1 g / 100 kcal, wherein the probiotic composition contains at least 20% of oligosaccharides. For example, the application in the scope of patent application, wherein the nutritional composition further comprises a long-chain polyunsaturated fatty acid source of about 5 to about 100 mg / 100 kcal, and the long-chain polyunsaturated fatty acid source includes docosahexaenoic acid. For example, the application in the second scope of the patent application, wherein the long-chain polyunsaturated fatty acid source further comprises arachidonic acid. For example, the application in the scope of claim 1, wherein the fat or lipid source is present in an amount of about 3 g / 100 kcal to about 7 g / 100 kcal. For example, the application in the scope of the patent application, wherein the protein source is present in an amount of about 1 g / 100 kcal to about 5 g / 100 kcal. For example, the use of item 1 of the patent scope, wherein the lactoferrin is present in an amount of about 70 mg / 100 kcal to about 220 mg / 100 kcal. For example, the use of item 1 of the patent scope, wherein the probiotic composition contains at least 20% of galactooligosaccharide, polydextrose, or a mixture thereof. For example, the use of item 7 of the patent application scope, wherein the probiotic composition contains at least 20% of galactose oligosaccharide. For example, the use of item 7 of the patent application scope, wherein the probiotic composition contains at least 20% of polydextrose.