Adherence Inhibition Of Pathogens By Prebiotic Oligosaccharides Patent Application (2025)

U.S. patent application number 13/854370 was filed with the patent office on 2013-10-31 for adherence inhibition of pathogens by prebiotic oligosaccharides. This patent application is currently assigned to Mead Johnson Nutrition Company. The applicant listed for this patent is Mead Johnson Nutrition Company. Invention is credited to Anja Wittke.

ADHERENCE INHIBITION OF PATHOGENS BY PREBIOTIC OLIGOSACCHARIDES

Abstract

The present disclosure relates to nutritional compositionscomprising a prebiotic component for inhibiting adherence ofpathogens in the gastrointestinal tract of a subject and to theprebiotic component, which comprises galactooligosaccharide,polydextrose or any mixture thereof. The nutritional compositionsof the present disclosure comprise a fat or lipid source, a proteinsource, and a carbohydrate source comprising an effective amount ofthe prebiotic component.

Related U.S. Patent Documents

Claims

1. A nutritional composition, wherein the nutritional compositionis effective at inhibiting the adhesion of pathogens in thegastrointestinal tract when fed to an infant or child, comprising:a fat or lipid source; a protein source; a carbohydrate sourcecomprising a prebiotic component comprising agalactooligosaccharide and polydextrose, wherein the combination ofa galactooligosaccharide and polydextrose is present in an amountof between about 1.0 g/L and about 10.0 g/L of the nutritionalcomposition; and at least one long chain polyunsaturated fatty acidin an amount of at least about 5 mg/100 kcal of the nutritionalcomposition.

2. The nutritional composition according to claim 1, wherein thenutritional composition is an infant formula.

3. The nutritional composition according to claim 1, wherein thenutritional composition is nutritionally complete.

4. The nutritional composition according to claim 1, wherein thenutritional composition is in a powdered form.

5. The nutritional composition according to claim 1, furthercomprising at least one probiotic.

6. The nutritional composition according to claim 5, wherein theprobiotic is selected from the group consisting of Bifidobacteriaspp., Lactobacillus spp. and combinations thereof.

7. (canceled)

8. The nutritional composition according to claim 1, wherein thelong chain polyunsaturated fatty acid is selected from the groupconsisting of docosahexaenoic acid, arachidonic acid, orcombinations thereof.

9. The nutritional composition according to claim 1, wherein thefat source is present at a level of about 3 to about 7 g/100kcal.

10. The nutritional composition according to claim 1, wherein theprotein source is present at a level of about 1 to about 5 g/100kcal.

11. (canceled)

12. The nutritional composition according to claim 1, wherein theratio of polydextrose to galactooligosaccharide is between about9:1 and 1:9.

13. The nutritional composition according to claim 1, wherein theprotein source is selected from the group consisting of wheyprotein, casein, casein protein, nonfat milk, hydrolyzed proteinand combinations thereof.

14-20. (canceled)

Description

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of commonlyassigned U.S. patent application Ser. No. 12/370,141, entitled"Method for Simulating the Functional Attributes of Human MilkOligosaccharides in Formula-fed Infants," which was filled on Feb.12, 2009 in the name of Bryon W. Petschow, which, in turn, claimspriority to U.S. patent application Ser. No. 11/172,123, filed Jun.30, 2005, which issued as U.S. Pat. No. 7,572,474, the disclosuresof each are incorporated by reference herein in their entirety.

BACKGROUND

[0002] 1. Technical Field

[0003] The present disclosure relates to enteral nutritionalcompositions comprising prebiotic component. More specifically, thenutritional compositions of the present disclosure contain aprebiotic component comprising galactooligosaccharide (GOS). Insome embodiments, the prebiotic component comprisesgalactooligosaccharide, polydextrose (PDX), a mixture ofgalactooligosaccharide and polydextrose, or a mixture ofgalactooligosaccharide, polydextrose and at least one otherprebiotic. Moreover, the disclosure is further directed to a methodof inhibiting adhesion of pathogens in the gastrointestinal tractof a human subject. The method comprises administering an effectiveamount of the prebiotic component.

[0004] 2. Background Art

[0005] Infants and children are exposed to a variety of pathogens,and consequently, the incidence of infectious diseases peaks in thefirst 4 years of life. Infants do not have mature immune systemsand are often incapable of mounting an effective immune response.Indeed, on a cellular level, infants have a decreased ability toconcentrate leukocytes where necessary. (Maternity and GynecologicCare, Bobak, Jensen, Zalar, Fourth Edition, p. 470) Consequently,infants are not able to limit invading pathogens due to ageneralized hypofunction of their inflammatory and immunemechanisms. As such, improving the immune response in infants andchildren would provide an opportunity to reduce the incidence ofinfections and to maintain or improve the overall health ofpediatric subjects.

[0006] Cronobacter sakazakii (also called Enterobacter sakazakii)is a motile, petrichious, non-sporeforming, Gram negativefacultative anaerobe. It is an opportunistic pathogen that has beenimplicated in severe forms of meningitis, necrotizingenterocolitis, and sepsis in neonates. Although the environmentalsource of the microorganism is not known, powdered infant formulahas been implicated in several outbreaks as well as other sporadiccases of infection. Of particular concern are reports of theorganism being isolated from powdered milk processing plants andhospital utensils such as spoons and other implements used toprepare infant formula. (Mullane, N. R., D, Drudy, P. Whyte, M.O'Mahony, A. G. M. Scanell, P. G. Wall, S. Fanning. 2006.Enterobacter sakazakii: properties and significance in dried infantmilk formula (IMF) powder. International Journal of DairyTechnology 59: 102-111.) Because colonization and subsequentinfection, and invasion of C. sakazakii require that the organismadhere to host cell surfaces, agents that inhibit or blockattachment of the pathogen to epithelial cells could be useful inreducing infections.

[0007] The infant gut microflora is rapidly established in thefirst few weeks following birth, and it has a great impact on aninfant's immune system. The nature of this intestinal colonizationis initially determined by the host's early exposure toenvironmental sources of microbes as well as to the health of theinfant. Whether the infant is breast-fed or formula-fed also has astrong influence on the intestinal bacterial population.

[0008] Human milk contains a number of factors that may contributeto the growth and population of the gut microflora of infants.Among these factors is a complex mixture of more than 130 differentoligosaccharides that reach levels as high as 8-12 g/L intransitional and mature milk. (Kunz, et al., Oligosaccharides inHuman Milk: Structure, Functional, and Metabolic Aspects, Ann. Rev.Nutr. 20: 699-722 (2000).) These oligosaccharides are resistant toenzymatic digestion in the upper gastrointestinal tract, and theyreach the colon intact, where they then serve as substrates forcolonic fermentation.

[0009] Human milk oligosaccharides are believed to elicit anincrease in the number of Bifidobacteria in the colonic microflora,along with a reduction in the number of potentially pathogenicbacteria. (Kunz, et al., Oligosaccharides in Human Milk: Structure,Functional, and Metabolic Aspects, Ann. Rev. Nutr. 20: 699-722(2000); Newburg, Do the Binding Properties of Oligosaccharides inMilk Protect Human Infants from Gastrointestinal Bacteria?, J.Nutr. 217:S980-S984 (1997).) One way that human milkoligosaccharides may increase the numbers of Bifidobacterium spp.and reduce the number of potentially pathogenic bacteria is byacting as competitive receptors and inhibiting the binding ofpathogens to the cell surface, Rivero-Urgell, et al.,Oligosaccharides: Application in Infant Food, Early Hum. Dev.65(S):43-52 (2001).

[0010] Because cow's milk and many commercially available infantformulas that are based on cow's milk provide only trace amounts ofoligosaccharides, prebiotics can be used to supplement the diet offormula-fed infants. Prebiotics have been defined as non-digestiblefood ingredients that beneficially affect the host by selectivelystimulating the growth and/or activity of one or a limited numberof cells in the colon that can improve the health of the host.

[0011] The incorporation of various prebiotic ingredients intoinfant formulas has been disclosed. For example, U.S. Patent App.No. 20030072865 to Bindels, et al. discloses an infant formula withimproved protein content and at least one prebiotic. The prebioticcomponent can be lacto-N-tetaose, lacto-N-fuco-pentaose, lactulose(LOS), lactosucrose, raffinose, galactooligosaccharide,fructo-oligosaccharide (FOS), oligosaccharides derived from soybeanpolysaccharides, mannose-based oligosaccharides,arabino-oligosaccharides, xylo-oligosaccharides,isomalto-oligo-saccharides, glucans, sialyl oligosaccharides, andfuco-oligosaccharides.

[0012] Similarly, U.S. Patent App. No. 2004/0191234 to Haschkediscloses a method for enhancing the immune response whichcomprises administering at least one prebiotic. The prebiotic canbe an oligosaccharide produced from glucose, galactose, xylose,maltose, sucrose, lactose, starch, xylan, hemicellulose, inulin, ora mixture thereof. The prebiotic can be present in an infantcereal.

[0013] Unfortunately, however, there are some disadvantages in theadministration of the above to formula fed infants. While they maybeneficially affect the population of probiotics in the gut, thefermentation of many of these prebiotic substances occurs at a veryrapid rate, which may produce excess gas, abdominal distension,bloating, and diarrhea. Therefore, the choice of prebioticsubstances in nutritional compositions and infant formulas shouldbe made with the goal of maximizing potential benefits, such asinhibiting the adhesion of pathogens, and minimizing unwantedside-effects.

BRIEF SUMMARY

[0014] Briefly, therefore, the present disclosure is directed, inan embodiment, to a nutritional composition comprising a prebioticcomponent that inhibits the adhesion of pathogens in thegastrointestinal tract and that is well-tolerated and does notproduce or cause excess gas, abdominal distension, bloating ordiarrhea. In some embodiments, the nutritional compositioncomprises a lipid or fat source, a protein source and an effectiveamount of the prebiotic component. In some embodiments, thenutritional composition also contains a source of long chainpolyunsaturated fatty acid (LCPUFA). In some embodiments, thenutritional composition is nutritionally complete, and in someembodiments, the nutritional composition includes a probiotic.

[0015] Accordingly, the prebiotic component containsgalactooligosaccharide, and in some embodiments, the prebioticcomponent additionally contains polydextrose, fructooligosaccharide(FOS), lactulose (LOS) or any combination or mixture thereof.

[0016] The prebiotic component contains an effective amount ofoligosaccharides such that adhesion of harmful bacteria isinhibited in the gastrointestinal tract when the prebioticcomponent is administered to a subject. The prebiotic compositioncan include a plurality of oligosaccharides, whereby thecombination of the oligosaccharides inhibits adherence of pathogensin the gastrointestinal tract. In certain embodiments, theprebiotic component comprises or consists of a combination ofgalactooligosaccharide and polydextrose. Moreover in someembodiments, the nutritional composition further includes a sourceof long chain polyunsaturated fatty acids.

[0017] The disclosure is also directed to a nutritional compositionthat inhibits the adhesion of pathogens in the gastrointestinaltract of a subject comprising, in some embodiments:

[0018] a. up to about 7 g/100 kcal of a fat or lipid, morepreferably about 3 to about 7 g/100 kcal of a fat or lipid;

[0019] b. up to about 5 g/100 kcal of a protein source, morepreferably about 1 to about 5 g/100 kcal of a protein source;

[0020] c. a prebiotic component comprising galactooligosaccharide;and

[0021] d. about 5 to about 100 mg/100 kcal of a source of longchain polyunsaturated fatty acids which include DHA, morepreferably about 10 to about 50 mg/100 kcal of a source of longchain polyunsaturated fatty acids which includes DHA.

[0022] In yet another embodiment, the disclosure is directed to anutritional composition having improved digestibility, thecomposition comprising a lipid or fat, a protein source, a sourceof long chain polyunsaturated fatty acids that includesdocosahexaenoic acid (DHA), and a prebiotic component thatcomprises at least 20% of an oligosaccharide mixture comprisinggalactooligosaccharide and/or a combination ofgalactooligosaccharide and polydextrose.

[0023] Additionally, the present disclosure relates to a method forinhibiting adhesion of pathogens in the gastrointestinal tract of ahuman subject.

[0024] In yet another embodiment, the present disclosure teaches amethod for reducing the incidence of diarrhea due to adhesion ofbacterial pathogens in the gastrointestinal tract.

[0025] It is to be understood that both the foregoing generaldescription and the following detailed description presentembodiments of the disclosure and are intended to provide anoverview or framework for understanding the nature and character ofthe disclosure as it is claimed. The description serves to explainthe principles and operations of the claimed subject matter. Otherand further features and advantages of the present disclosure willbe readily apparent to those skilled in the art upon a reading ofthe following disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 illustrates the adherence of C. sakazakii 4603 toHEp-2 cells in the presence of prebiotics (16 mg/ml). Adherence wasmeasured by microscopic counting (A) or by cultural enumeration(B). Statistically significant effects (p<0.05) are indicated bythe asterisk.

[0027] FIG. 2 illustrates adherence of C. sakazakii 29004 to HEp-2cells in the presence of prebiotics (16 mg/ml). Adherence wasmeasured by microscopic counting (A) or by cultural enumeration(B).

[0028] FIG. 3 shows the adherence of C. sakazakii 4603 to HEp-2cells in the absence (A) and presence of GOS (B), GOS-PDX (C); andPDX (D).

[0029] FIG. 4 illustrates the adherence of enteropathogenicEscherichia coli (E. coli E2348/69) to HEp-2 cells in the presenceof prebiotics (16 mg/ml). Adherence was measured by microscopiccounting. Significance (versus the control) is indicated byasterisks.

[0030] FIG. 5 illustrates the adherence of C. sakazakii 29004 toCaco-2 cells in the presence of prebiotics. Adherence was measuredby microscopic counting.

DETAILED DESCRIPTION

[0031] Reference now will be made in detail to the embodiments ofthe present disclosure, one or more examples of which are set forthherein below. Each example is provided by way of explanation of thenutritional composition of the present disclosure and is not alimitation. In fact, it will be apparent to those skilled in theart that various modifications and variations can be made to theteachings of the present disclosure without departing from thescope or spirit of the disclosure. For instance, featuresillustrated or described as part of one embodiment, can be usedwith another embodiment to yield a still further embodiment.

[0032] Thus, it is intended that the present disclosure covers suchmodifications and variations as come within the scope of theappended claims and their equivalents. Other objects, features andaspects of the present disclosure are disclosed in or are obviousfrom the following detailed description. It is to be understood byone of ordinary skill in the art that the present discussion is adescription of exemplary embodiments only and is not intended aslimiting the broader aspects of the present disclosure.

[0033] "Nutritional composition" means a substance or formulationthat satisfies at least a portion of a subject's nutrientrequirements.

[0034] "Pediatric subject" means a human that is less than 13 yearsof age. In some embodiments, a pediatric subject refers to a humansubject that is less than 8 years old.

[0035] "Infant" means a subject ranging in age from birth to notmore than about one year and includes infants from 0 to about 12months corrected age. The term infant includes low birth weightinfants, very low birth weight infants, and preterm infants. Thephrase "corrected age" means an infant's chronological age minusthe amount of time that the infant was born premature. Therefore,the corrected age is the age of the infant if it had been carriedto full term.

[0036] "Child" means a subject ranging in age from about 12 monthsto about 13 years. In some embodiments, a child is a subjectbetween the ages of one and twelve years old. In other embodiments,the terms "children" or "child" refer to subjects that are two,three, four, five or six years old. In other embodiments, the terms"children" or "child" refer to any range of ages between about 12months and about 13 years.

[0037] "Children's nutritional product" refers to a compositionthat satisfies at least a portion of the nutrient requirements of achild.

[0038] "Infant formula" means a composition that satisfies at leasta portion of the nutrient requirements of an infant. In the UnitedStates, the content of an infant formula is dictated by the federalregulations set forth at 21 C.F.R. Sections 100, 106, and 107.These regulations define macronutrient, vitamin, mineral, and otheringredient levels in an effort to stimulate the nutritional andother properties of human breast milk.

[0039] "Nutritionally complete" means a composition that may beused as the sole source of nutrition, which would supplyessentially all of the required daily amounts of vitamins,minerals, and/or trace elements in combination with proteins,carbohydrates, and lipids.

[0040] "Probiotic" means a microorganism with low or nopathogenicity that exerts beneficial effects on the health of thehost.

[0041] "Prebiotic" means a non-digestible food ingredient thatbeneficially affects the host by selectively stimulating the growthand/or activity of one or a limited number of bacteria in thedigestive tract that can improve the health of the host.

[0042] "Effective amount" means an amount that inhibits theadhesion of pathogens in the gastrointestinal tract of asubject.

[0043] "Prebiotic component" or "oligosaccharide mixture" means acombination of at least one prebiotic with at least one otherprebiotic. In a preferred embodiment, the prebiotic componentcomprises galactooligosaccharide and polydextrose.

[0044] The phrase "functional attributes" mean any inherent qualityor characteristic that causes something to occur. Examples offunctional attributes of human milk oligosaccharides in the presentdisclosure can include the increase of the population and speciesof beneficial bacteria, production of a short chain fatty acid(SCFA) profile that is high in acetic acid and low in butyric acid,production of a slow rate and low extent of fermentation ofprebiotics in the gut, and/or inhibition of adherence of pathogens,such as harmful bacteria, in the gastrointestinal tract of asubject.

[0045] The present disclosure describes a nutritional compositionfor a subject comprising a lipid source, a protein source and aprebiotic component, wherein the prebiotic component contains atleast one carbohydrate that inhibits the adhesion of pathogens inthe gastrointestinal tract. In other embodiments, the prebioticcomponent reduces the incidence of diarrhea and/or modulates stoolcharacteristics in a positive way.

[0046] The present disclosure also describes a method forinhibiting adherence of pathogens in the gastrointestinal tract ofa subject by administering an effective amount of a nutritionalcomposition comprising a fat or lipid source, a protein source anda source of galactooligosaccharide. In some embodiments, thegalactooligosaccharide may be administered as part of a prebioticcomponent. In some embodiments, the prebiotic may consist ofgalactooligosaccharide. In other embodiments, the prebioticcomponent may comprise the galactooligosaccharide administeredtogether with another carbohydrate, such as polydextrose. Theadministration of galactooligosaccharide, of polydextrose or of amixture thereof is physically well-tolerated by human subjects.

[0047] Suitable fat or lipid sources for practicing the presentdisclosure may be comprise any lipid source known in the art,including but not limited to, animal sources, e.g., milk fat,butter, butter fat, egg yolk lipid; marine sources, such as fishoils, marine oils, single-cell oils; vegetable and plant oils, suchas corn oil, canola oil, sunflower oil, soybean oil, palmolein,coconut oil, high oleic sunflower oil, evening primrose oil,rapeseed oil, olive oil, flaxseed (linseed) oil, cottonseed oil,high oleic safflower oil, palm stearin, soy lecithin, palm kerneloil, wheat germ oil, medium chain triglyceride

[0048] Bovine milk protein sources useful in practicing the presentdisclosure include, but are not limited to, milk protein powders,milk protein concentrates, milk protein isolates, nonfat milksolids, nonfat milk, nonfat dry milk, whey protein, whey proteinisolates, whey protein concentrates, sweet whey, acid whey, casein,acid casein, caseinate (e.g. sodium caseinate, sodium calciumcaseinate, calcium caseinate) and any combinations thereof.

[0049] In one embodiment, the proteins are provided as intactproteins. In other embodiments, the proteins are provided as acombination of both intact proteins and partially hydrolyzedproteins, with a degree of hydrolysis of between about 4% and 10%.In certain other embodiments, the proteins are more completelyhydrolyzed. In yet another embodiment, the protein source may besupplemented with glutamine-containing peptides.

[0050] In a particular embodiment of the disclosure, thewhey:casein ratio of the protein source is similar to that found inhuman breast milk. In an embodiment, the protein source comprisesfrom about 40% to about 80% whey protein and from about 20% toabout 60% casein.

[0051] The nutritional composition contains a prebiotic component.In some embodiments, the nutritional composition comprisesgalactooligosaccharide in addition to one or more other prebioticsas part of a prebiotic component. The term "prebiotic" as usedherein refers to indigestible food ingredients which exert healthbenefits upon the host. Such health benefits may include, but arenot limited to, selective stimulation of the growth and/or activityof one or a limited number of beneficial gut bacteria, stimulationof the growth and/or activity of ingested probiotic microorganisms,selective reduction in gut pathogens, inhibition of adherence ofgut pathogens and favorable influence on gut short chain fatty acidprofile. Such prebiotics may be naturally-occurring, synthetic, ordeveloped through the genetic manipulation of organisms and/orplants, whether such new source is now known or developedlater.

[0052] Prebiotics useful in the present disclosure may includeoligosaccharides, polysaccharides, and other prebiotics thatcontain fructose, xylose, soya, galactose, glucose and mannose.Prebiotic oligosaccharides are non-digestible food ingredients thatcan act as anti-adhesives against bacterial adherence by mimickingthe host cell receptor sites that intestinal pathogens recognize.Specifically, prebiotics useful in the present disclosure mayinclude polydextrose, polydextrose powder, lactulose, lactosucrose,raffinose, gluco-oligosaccharide, inulin, fructo-oligosaccharide,isomalto-oligosaccharide, soybean oligosaccharides, lactosucrose,xylo-oligosaccharide, chito-oligosaccharide, manno-oligosaccharide,aribino-oligosaccharide, siallyl-oligosaccharide,fuco-oligosaccharide, galactooligosaccharide, andgentio-oligosaccharides.

[0053] The galactooligosaccharide that is utilized in thenutritional composition(s) of the present disclosure is comprisedof several galactose-containing species of varying lengths. GOS isa mixture of oligosaccharides consisting of D-glucose andD-galactose. It is sometimes referred to astrans-galactooligosaccharide. It is produced from .beta.-lactose by.beta.-galactosidase, which can be obtained from Aspergillusoryzae. GOS has been suggested to increase calcium absorption andprevention of bone loss in adults. GOS has been identified as aprebiotic that is useful for administration to infants.

[0054] The amount of galactooligosaccharide in the nutritionalcomposition may, in an embodiment, be from about 0.1 mg/100 Kcal toabout 1.0 mg/100 Kcal. In another embodiment, the amount ofgalactooligosaccharide in the nutritional composition may be fromabout 0.1 mg/100 Kcal to about 0.5 mg/100 Kcal.

[0055] Meanwhile, the polydextrose of the present disclosure iscomposed of randomly cross-linked glucose units with several typesof glycosidic bonds, and its anti-adhesive activity has not beenpreviously studied. In some embodiments, PDX is synthesized from amixture of dextrose, about 10% sorbitol and about 1% citric acid.PDX is a non-digestible carbohydrate that is synthesized fromrandomly cross-linked glucose and sorbitol. It is not digested inthe upper GI tract and is only partially fermented in the lower GItract, making it a beneficial ingredient for digestive health. Thephysiological benefits of PDX include increased fecal bulk, reducedtransit time, lower fecal pH and reduced concentration ofputrefactive substances in the colon. In adults, PDX ingestion hasalso been shown to aid in the promotion and growth of beneficialbacteria in the intestine and production of SCFAs.

[0056] PDX has been identified as a prebiotic substance for adultsbased on its functions in the GI tract. PDX has not, however, beenidentified as a prebiotic that provides the benefits of the presentdisclosure and that can be administered to pediatric subjects, suchas infants or children. The gut microflora of infants is well knownto be less developed than that of an adult. While the microflora ofthe adult human consists of more than 10.sup.13 microorganisms andnearly 500 species, the gut microflora of an infant contains only afraction of those microorganisms, both in absolute number andspecies diversity. Because the bacterial populations and speciesvary immensely between the gut of an infant and an adult, it cannotbe assumed that a prebiotic substance that has a beneficial effecton adults would also have a beneficial effect on infants.

[0057] Polydextrose is commercially available from a variety ofsources. For example, STA-LITE.RTM. Polydextrose is available in 5lb bags from Honeyville Grain, Inc., located in Salt Lake City,Utah. Polydextrose is commercially available from DaniscoSweeteners, Ltd., located in the United Kingdom.

[0058] PDX is well-suited for incorporation into a nutritionalcomposition, such as an infant formula, as it contains only 1Cal/g, as compared to 4 Cal/g for typical prebiotics. It is alsohighly soluble and neutral tasting. Therefore, its addition to anutritional composition would not change the physical or tastecharacteristics of the composition.

[0059] The form of administration of PDX in the method of thedisclosure is not critical, as long as an effective amount isadministered. Most conveniently, the PDX is supplemented intonutritional composition, in some embodiments an infant formula,which is then fed to a subject.

[0060] The amount of polydextrose in the nutritional compositionmay, in an embodiment, be within the range of from about 0.1 mg/100Kcal to about 0.5 mg/100 Kcal. In another embodiment, the amount ofpolydextrose may be about 0.3 mg/100 Kcal. In a particularembodiment, galactooligosaccharide and polydextrose aresupplemented into the nutritional composition in a total amount ofabout at least about 0.2 mg/100 Kcal and can be about 0.2 mg/100Kcal to about 1.5 mg/100 Kcal.

[0061] In a particular embodiment of the present disclosure, PDX isadministered in combination with GOS. In this embodiment. PDX andGOS can be administered in a ratio of PDX:GOS of between about 9:1and 1:9. In another embodiment, the ratio of PDX:GOS can be betweenabout 5:1 and 1:5. In yet another embodiment, the ratio of PDX:GOScan be between about 1:3 and 3:1. In a particular embodiment, theratio of PDX to GOS can be about 1:1. In another particularembodiment, the ratio of PDX to GOS can be about 8:2.

[0062] An effective amount of the PDX:GOS combination may bebetween about 1.0 g/L and 10.0 g/L, administered daily. In anotherembodiment, an effective amount of the PDX:GOS combination may bebetween about 2.0 g/L and 8.0 g/L, administered daily. In aparticular embodiment, an effective amount of the PDX:GOScombination may be about 2 g/L of PDX and 2 g/L of GOS,administered daily.

[0063] In another specific embodiment of the present disclosure,galactooligosaccharide is administered in combination with FOS,which is also sometimes called oligofructose. FOS is acarbohydrate, specifically a chain polymer of the sugar fructose.FOS has been identified as a prebiotic that is useful foradministration to infants.

[0064] In another specific embodiment of the present disclosure,PDX is administered in combination with LOS. LOS is a semisyntheticdisaccharide formed from D-galactose and D-fructose and joined by a.beta.-glucosidic linkage. It is resistant to hydrolysis by humandigestive enzymes, but is fermented in the small intestine. It ishighly soluble and has a sweet taste. LOS has been identified as aprebiotic that is useful for administration to infants.

[0065] In this embodiment, PDX and LOS can be administered in aratio of between about 9:1 and 1:9, In another embodiment, theratio of PDX to LOS can be between about 5:1 and 1:5. In yetanother embodiment, the ratio of PDX to LOS can be between about3:1 and 1:3.

[0066] In a particular embodiment, the ratio of PDX to LOS canabout 5:5. In another particular embodiment, the ratio of PDX toLOS can be about 8:2.

[0067] An effective amount of the PDX:LOS combination may bebetween about 1.0 g/L and 10.0 g/L, administered daily. In anotherembodiment, an effective amount of the PDX:LOS combination may bebetween about 2.0 g/L and 8.0 g/L, administered daily. In aparticular embodiment, an effective amount of the PDX:LOScombination may be about 2 g/L of PDX and 2 g/L of LOS,administered daily.

[0068] In yet another embodiment of the present disclosure, PDX isadministered in combination with both GOS and LOS. In thisembodiment, the PDX:GOS:LOS combination can be administered in aratio of about 50:33:17. Alternatively, the ratio of thePDX:GOS:LOS combination can be about 1:1:1. In a particularembodiment, the ratio of PDX:GOS:LOS can be about 1:1.5:1.

[0069] An effective amount of the PDX:GOS:LOS combination may bebetween about 1.0 g/L and 10.0 g/L, administered daily. In anotherembodiment, an effective amount of the PDX:GOS:LOS combination maybe between about 2.0 g/L and 8.0 g/L, administered daily. In anembodiment, an effective amount of the PDX:GOS:LOS combination maybe about 2 g/L PDX, 2 g/L GOS and 2 g/L LOS, administered daily. Ina particular embodiment, an effective amount of the PDX:GOS:LOScombination may be about 2 g/L PDX, 1.32 g/L GOS and 2.6 g/L LOS,administered daily. In another embodiment, an effective amount ofthe PDX:GOS:LOS combination may be about 4 g/L PDX, 2.64 g/L GOSand 3.6 g/L LOS, administered daily.

[0070] The nutritional composition of the present application, insome embodiments, comprises a prebiotic component comprisinggalactooligosaccharide and polydextrose. The nutritionalcomposition of the present disclosure inhibits adhesion ofpathogens in the gastrointestinal tract of a subject. In someembodiments, the nutritional composition specifically inhibits theadherence of C. sakazakii 4603. In some embodiments, thenutritional composition inhibits the adherence of C. sakazakii29004.

[0071] The initial adherence of pathogens to host cell surfaces isone of the first steps prior to colonization and infection. Mosthuman pathogens that are able to adhere to host tissues have anadvantage over those that do not, since they are more likely toevade and resist the defense systems of the host. (Shoaf, K., G. L.Mulvey, G. D. Armstrong, R. W. Hutkins. 2006. Prebioticgalactooligosaccharides reduce adherence of enteropathogenicEscherichia coli to tissue culture cells, Infection and Immunity74:6920-6928.) Adherence between the pathogen and the host cells ismost commonly mediated via lectin-like adhesins expressed by thebacteria and ligand-like carbohydrates located on the surface ofthe host epithelial cells. Indeed, most intestinal pathogensrecognize specific monosaccharide residues or short oligosaccharidesequences as binding receptors. Bacterial variants that are unableto express functional adhesins are unable to adhere and initiateinfections, indicating that for these pathogens, adherence isrequired to cause disease (Isaacson, R. E. 1983, Bacterialadherence to mucosal surfaces: an attribute of virulence. BulletinEuropeen de Physiopathologie Respiratoire 19:75-80.). This findinghas led to the suggestion that agents that inhibit adherence canreduce incidences of infection, (Zorf, D. and Roth, S. 1996.Oligosaccharides anti-infection agents. Lancet 347:1017-1021)

[0072] The nutritional composition of the present disclosure uses aprebiotic component of oligosaccharide decoys that mimic thecarbohydrate binding sites ordinarily recognized by pathogens asbinding receptors on the surface of host epithelial cells. Theprebiotic component of oligosaccharides competes with the cognatesugars for binding to the adhesion and thereby inhibits bacterialadherence in the gastrointestinal tract.

[0073] Accordingly, to arrive at the nutritional composition of thepresent disclosure, the individual carbohydrates GOS and PDX, aswell as a component of GOS and PDX, were studied in an in vitromodel to assess their respective abilities to inhibit adherence ofpathogens to human cells lines.

[0074] Galactooligosaccharide and polydextrose were used foradherence inhibition assays performed with human cell lines (HEp-2and Caco-2 cells) infected with enteric pathogens Escherichia coli(EPEC strain E2348/69) or C. sakazakii strains (ATCC 4603, 29004,4593, 4583, ES415). The prebiotics GOS, PDX, or a prebioticcomponent comprising GOS and PDX were administered at aconcentration of 16 mg/ml in the assay and analyzed for theiradherence inhibition of the single pathogens to the epithelial celllines.

[0075] For adherence assays, confluent tissue culture monolayerswere prepared, and the prebiotics were mixed with C. sakazakii 4603or ATCC 29004 (approximately 10.sup.7 cells) prior to adding themto the HEp-2 cells. Following an incubation of 3 hours, which hadbeen determined in preliminary experiments to be the optimalincubation time for adherence, at 37.degree. C. in the CO.sub.2incubator, the wells were washed 5 times with PBS. Cells were thenfixed for microscopic enumeration or were instead detached andserial dilution plated on agar for culture enumeration.

[0076] For the adherence assays with EPEC, each prebiotic was addedat a final concentration of about 16 mg/ml and incubated with HEp-2cells for 30 minutes. Cells with microcolonies of EPEC or thatconsisted of four or more bacteria were considered positive forhaving a typical localized adherence phenotype. For C. sakazakiiadherence occurs via cluster formation and diffuse adherence.Adherence inhibition was calculated as the number of adheredbacteria per cell in the control minus the number of adheredbacteria per cell in the treatment divided by the number of adheredbacteria per cell in the control.

[0077] When measured by the microscopic method, a significantreduction (56%) in adherence of C. sakazakii 4603 was observed inthe presence of GOS (16 mg/ml). Adherence inhibition also occurred(48%) when a GOS-PDX component (8 mg/ml each) was tested, althoughPDX by itself had less effect. A similar pattern for this strain onHEp-2 cells was observed using cultural enumeration. Similarresults were also observed for Caco-2 cells and also for anotherstrain of C. sakazakii (ATCC 29004). Thus, the results suggest thatGOS and the prebiotic component of PDX and GOS have anti-adhesiveeffects on pathogens, such as C. sakazakii, and that the prebioticsdirectly inhibit the adherence of pathogenic organisms togastrointestinal epithelial cells.

[0078] In the compositions and methods of the present disclosure,an effective amount of a prebiotic component comprisinggalactooligosaccharide and polydextrose is administered to asubject for the purpose of simulating the functional attributes ofhuman milk oligosaccharides. In an embodiment, the total amount ofprebiotics present in the nutritional composition may be from about1.0 g/L to about 10.0 g/L of the composition. More preferably, thetotal amount of prebiotics present in the nutritional compositionmay be from about 2.0 g/L and about 8.0 g/L of the composition. Atleast 20% of the prebiotics may comprise a mixture ofgalactooligosaccharide and polydextrose. The amount of each ofgalactooligosaccharide and polydextrose in the nutritionalcomposition may, in an embodiment, be within the range of fromabout 1.0 g/L to about 4.0 g/L.

[0079] In some embodiments, the nutritional composition of thedisclosure contains a source of long chain polyunsaturated fattyacids (LCPUFAs) that comprises docosahexaenoic acid (DHA). Othersuitable LCPUFAs include, but are not limited to, .alpha.-linoleicacid, .gamma.-linoleic acid, linoleic acid, linolenic acid,eicosapentaenoic acid (EPA) and arachidonic acid (ARA).

[0080] In one embodiment, the nutritional composition issupplemented with both DHA and ARA. In this embodiment, the weightratio of ARA:DHA may be from about 1:3 to about 9:1. In oneembodiment of the present disclosure, this ratio is from about 1:2to about 4:1. In one embodiment, both DHA and ARA are administeredin combination with PDX.

[0081] The amount of long chain polyunsaturated fatty acids in thenutritional composition is advantageously at least about 5 mg/100Kcal, and may vary from about 5 mg/100 kcal to about 100 mg/100kcal, more preferably from about 10 mg/100 kcal to about 50 mg/100kcal.

[0082] The nutritional composition may be supplemented with oilscontaining DHA and ARA using standard techniques known in the art.For example, DHA and ARA may be added to the composition byreplacing an equivalent amount of an oil, such as high oleicsunflower oil, normally present in the composition. As anotherexample, the oils containing DHA and ARA may be added to thecomposition by replacing an equivalent amount of the rest of theoverall fat component normally present in the composition withoutDHA and ARA.

[0083] If utilized, the source of DHA and ARA may be any sourceknown in the art such as marine oil, fish oil, single cell oil, eggyolk lipid, and brain lipid. In some embodiments, the DHA and ARAare sourced from the single cell Martek oil, DHASCO.RTM., orvariations thereof. The DHA and ARA can be in natural form,provided that the remainder of the LCPUFA source does not result inany substantial deleterious effect on the infant. Alternatively,the DHA and ARA can be used in refined form.

[0084] In an embodiment of the present disclosure, sources of DHAand ARA are single cell oils as taught in U.S. Pat. Nos. 5,374,567;5,550,156; and 5,397,591, the disclosures of which are incorporatedherein in their entirety by reference. However, the presentdisclosure is not limited to only such oils.

[0085] In certain embodiments of the disclosure, the nutritionalcomposition may contain one or more probiotics. Any probiotic knownin the art may be acceptable in this embodiment provided itachieves the intended result. In a particular embodiment, theprobiotic may be selected from any Lactobacillus species,Lactobacillus rhamnosus GG, Bifidobacterium species,Bifidobacterium longum, and Bifidobacterium animalis subsp. lactisBB-12 (DSM No. 10140) or combinations thereof.

[0086] If included in the composition, the amount of the probioticmay vary from about 10.sup.4 to about 10.sup.10 colony formingunits (cfu) per kg body weight per day. An average infant weighsbetween about 2 kg and about 15 kg. Thus, in some embodiments, thenutritional composition of the present disclosure will comprisebetween about 2.times.10.sup.4 and 15.times.10.sup.10 cellequivalents of probiotic per daily serving to an infant. In anotherembodiment, the amount of the probiotic may vary from about10.sup.6 to about 10.sup.9 cfu per kg body weight per day; therebydelivering between about 2.times.10.sup.6 and 15.times.10.sup.9cell equivalents of probiotic per daily serving to an infant. Inyet another embodiment, the amount of the probiotic may be at leastabout 10.sup.6 cfu per kg body weight per day; thereby deliveringat least about 2.times.10.sup.6 cfu per day to an infant.

[0087] In an embodiment, the probiotic(s) may be viable ornon-viable. As used herein, the term "viable", refers to livemicroorganisms. The term "non-viable" or "non-viable probiotic"means non-living probiotic microorganisms, their cellularcomponents and/or metabolites thereof. Such non-viable probioticsmay have been heat-killed or otherwise inactivated, but they retainthe ability to favorably influence the health of the host. Theprobiotics useful in the present disclosure may benaturally-occurring, synthetic or developed through the geneticmanipulation of organisms, whether such new source is now known orlater developed.

[0088] In an embodiment, the nutritional composition comprising theprebiotic component is provided as a nutritionally complete infantformula, which contains suitable types and amounts of lipid,carbohydrate, protein, vitamins and minerals. In this embodiment,the amount of carbohydrate may vary from about 8 to about 12 g/100kcal, with protein from about 1 to about 5 g/100 kcal, lipid or fatfrom about 3 to about 7 g/100 kcal and may be supplemented with aneffective amount of the prebiotic component.

[0089] Nutritional compositions of the present disclosure may bemilk-based, in the form of liquid, evaporated, condensed or drymilk. In some embodiments, the nutritional composition may alsoinclude non-dairy liquid or solid food products, protein, flavorsor flavor masking agents, sweeteners and vitamins or dietarysupplements.

[0090] In some embodiments, the nutritional composition may benutritionally complete, containing suitable types and amounts oflipids, carbohydrates, proteins, vitamins and minerals to be asubject's sole source of nutrition. In one embodiment, thenutritional composition is a children's nutritional product. Inanother embodiment, the nutritional composition comprises an infantformula. In yet another embodiment, the nutritional compositioncomprises a nutritionally complete infant formula. In still anotherembodiment, the nutritional composition comprises a nutritionallycomplete children's nutritional product.

[0091] The disclosed nutritional composition may be provided in anyform known in the art, such as a powder, a gel, a suspension, apaste, a solid, a liquid, a liquid concentrate, reconstituteablepowdered milk substitute, or a ready-to-use product. Thenutritional composition may, in certain embodiments, comprise anutritional supplement, children's nutritional product, infantformula, human milk fortifier, growing up milk or any othernutritional composition designed for a pediatric subject.Nutritional compositions of the present disclosure include, forexample, orally-ingestible, health-promoting substances including,for example, chewable foods, beverages, tablets, capsules andpowders. The nutritional composition of the present disclosure maybe standardized to a specific caloric content, it may be providedas a ready-to-use product, or it may be provided in a concentratedform.

[0092] The nutritional composition of the present disclosure can beprepared using any method known in the art. In one embodiment, PDXis provided in powder form. It can be mixed with water and otheringredients in a mixing tank. If GOS and/or LOS are included in thecomposition, they can be provided in powdered or liquid form. Themixture can then be pasteurized, homogenized and spray-dried tomake a finished powder or canned and retorted to make a liquidproduct.

[0093] Furthermore, the prebiotic component can be administered asa nutritional supplement. For example, GOS can be ingested in theform of a pill, tablet, capsule, caplet, powder, liquid or gel. Inthis embodiment, the GOS can be ingested in combination with othernutrient supplements, such as vitamins, or in combination with aLCPUFA supplement, such as DHA or ARA.

[0094] In another embodiment, GOS can be provided in a formsuitable for infants selected from the group consisting offollow-on formula, beverage, milk, yogurt, fruit juice, fruit-baseddrink, chewable tablet, cookie, cracker, or a combinationthereof.

[0095] In a method of the present disclosure, the subject is aformula-fed infant. In one embodiment the subject is formula-fedfrom birth. In another embodiment, the subject is breast-fed frombirth until an age which is less than one year, and is formula-fedthereafter, at which time PDX supplementation begins.

[0096] In embodiments providing a children's nutritional product,one or more vitamins and/or minerals may be added in amountssufficient to supply the daily nutritional requirements of childrenbetween one and thirteen years old. It is to be understood by oneof ordinary skill in the art that vitamin and mineral requirementswill vary for children between the ages of one and thirteen years.Thus, the embodiments are not intended to limit the nutritionalcomposition to a particular age group but, rather, to provide arange applicable to children between the one and thirteen yearsold.

[0097] In embodiments providing a nutritional composition for achild, the composition may optionally include, but is not limitedto, one or more of the following vitamins or derivations thereof:vitamin B.sub.1 (thiamin, thiamin pyrophosphate, TPP, thiamintriphosphate, TTP, thiamin hydrochloride, thiamin mononitrate),vitamin B.sub.2 (riboflavin, flavin mononucleotide, FMN, flavinadenine dinucleotide, FAD, lactoflavin, ovoflavin), vitamin B.sub.3(niacin, nicotinic acid, nicotinamide, niacinamide, nicotinamideadenine dinucleotide, NAD, nicotinic acid mononucleotide, NicMN,pyridine-3-carboxylic acid), vitamin B.sub.3-precursor tryptophan,vitamin B.sub.6 (pyridoxine, pyridoxal, pyridoxamine, pyridoxinehydrochloride), pantothenic acid (pantothenate, panthenol), folate(folic acid, folacin, pteroylglutamic acid), vitamin B.sub.12(cobalamin, methylcobalamin, deoxyadenosylcobalamin,cyanocobalamin, hydroxycobalamin, adenosylobalamin), biotin,vitamin C (ascorbic acid), vitamin A (retinol, retinyl acetate,retinyl palmitate, retinyl esters with other long-chain fattyacids, retinal, retinoic acid, retinol esters), vitamin D(calciferol, cholecalciferol, vitamin D.sub.3,1,25,-dihydroxyvitamin D), vitamin E (.alpha.-tocopherol,.alpha.-tocopherol acetate, .alpha.-tocopherol succinate,.alpha.-tocopherol nicotinate, .gamma.-tocopherol), vitamin K(vitamin K.sub.1, phylloquinone, naphthoquinone, vitamin K.sub.2,menaquinone-7, vitamin K.sub.3, menaquinone-4, mnenadione,menaquinone-8, menaquinone-8H, menaquinone-9, menaquinone-9H,menaquinone-10, menaquinone-11, menaquinone-12, menaquinone-13),choline, inositol, .beta.-carotene and any combinationsthereof.

[0098] In embodiments providing a children's nutritional product,the composition may optionally include, but is not limited to, oneor more of the following minerals or derivations thereof: boron,calcium, calcium acetate, calcium gluconate, calcium chloride,calcium lactate, calcium phosphate, calcium sulfate, chloride,chromium, chromium chloride, chromium picolonate, copper, coppersulfate, copper gluconate, cupric sulfate, fluoride, iron, carbonyliron, ferric iron, ferrous fumarate, ferric orthophosphate, irontrituration, polysaccharide iron, iodide, iodine, magnesium,magnesium carbonate, magnesium hydroxide, magnesium oxide,magnesium stearate, magnesium sulfate, manganese, molybdenum,phosphorus, potassium, potassium phosphate, potassium iodide,potassium chloride, potassium acetate, selenium, sulfur, sodium,docusate sodium, sodium chloride, sodium selenate, sodiummolybdate, zinc, zinc oxide, zinc sulfate and mixtures thereof.Non-limiting exemplary derivatives of mineral compounds includesalts, alkaline salts, esters and chelates of any mineralcompound.

[0099] The minerals can be added to children's nutritionalcompositions in the form of salts such as calcium phosphate,calcium glycerol phosphate, sodium citrate, potassium chloride,potassium phosphate, magnesium phosphate, ferrous sulfate, zincsulfate, cupric sulfate, manganese sulfate, and sodium selenite.Additional vitamins and minerals can be added as known within theart.

[0100] In an embodiment, the children's nutritional composition maycontain between about 10 and about 50% of the maximum dietaryrecommendation for any given country, or between about 10 and about50% of the average dietary recommendation for a group of countries,per serving of vitamins A, C, and E, zinc, iron, iodine, selenium,and choline. In another embodiment, the children's nutritionalcomposition may supply about 10-30% of the maximum dietaryrecommendation for any given country, or about 10-30% of theaverage dietary recommendation for a group of countries, perserving of B-vitamins. In yet another embodiment, the levels ofvitamin D, calcium, magnesium, phosphorus, and potassium in thechildren's nutritional product may correspond with the averagelevels found in milk. In other embodiments, other nutrients in thechildren's nutritional composition may be present at about 20% ofthe maximum dietary recommendation for any given country, or about20% of the average dietary recommendation for a group of countries,per serving.

[0101] The children's nutritional composition of the presentdisclosure may optionally include one or more of the followingflavoring agents, including, but not limited to, flavored extracts,volatile oils, cocoa or chocolate flavorings, peanut butterflavoring, cookie crumbs, vanilla or any commercially availableflavoring. Examples of useful flavorings include, but are notlimited to, pure anise extract, imitation banana extract, imitationcherry extract, chocolate extract, pure lemon extract, pure orangeextract, pure peppermint extract, honey, imitation pineappleextract, imitation rum extract, imitation strawberry extract, orvanilla extract; or volatile oils, such as balm oil, bay oil,bergamot oil, cedarwood oil, cherry oil, cinnamon oil, clove oil,or peppermint oil; peanut butter, chocolate flavoring, vanillacookie crumb, butterscotch, toffee, and mixtures thereof. Theamounts of flavoring agent can vary greatly depending upon theflavoring agent used. The type and amount of flavoring agent can beselected as is known in the art.

[0102] The nutritional compositions of the present disclosure mayoptionally include one or more emulsifiers that may be added forstability of the final product. Examples of suitable emulsifiersinclude, but are not limited to, lecithin (e.g., from egg or soy),alpha lactalbumin and/or mono- and di-glycerides, and mixturesthereof. Other emulsifiers are readily apparent to the skilledartisan and selection of suitable emulsifier(s) will depend, inpart, upon the formulation and final product.

[0103] The nutritional compositions of the present disclosure mayoptionally include one or more preservatives that may also be addedto extend product shelf life. Suitable preservatives include, butare not limited to, potassium sorbate, sodium sorbate, potassiumbenzoate, sodium benzoate, calcium disodium EDTA, and mixturesthereof.

[0104] The nutritional compositions of the present disclosure mayoptionally include one or more stabilizers. Suitable stabilizersfor use in practicing the nutritional composition of the presentdisclosure include, but are not limited to, gum arabic, gum ghatti,gum karaya, gum tragacanth, agar, furcellaran, guar gum, gellangum, locust bean gum, pectin, low methoxyl pectin, gelatin,microcrystalline cellulose, CMC (sodium carboxymethylcellulose),methylcellulose hydroxypropyl methyl cellulose, hydroxypropylcellulose, DATEM (diacetyl tartaric acid esters of mono- anddiglycerides), dextran, carrageenans, and mixtures thereof.

[0105] All percentages, parts and ratios as used herein are byweight of the total formulation, unless otherwise specified.

[0106] The nutritional composition of the present disclosure may besubstantially free of any optional or selected ingredientsdescribed herein, provided that the remaining nutritionalcomposition still contains all of the required ingredients orfeatures described herein In this context, and unless otherwisespecified, the term "substantially free" means that the selectedcomposition may contain less than a functional amount of theoptional ingredient, typically less than 0.1% by weight, and also,including zero percent by weight of such optional or selectedingredient.

[0107] All references to singular characteristics or limitations ofthe present disclosure shall include the corresponding pluralcharacteristic or limitation, and vice versa, unless otherwisespecified or clearly implied to the contrary by the context inwhich the reference is made.

[0108] All combinations of method or process steps as used hereincan be performed in any order, unless otherwise specified orclearly implied to the contrary by the context in which thereferenced combination is made.

[0109] The methods and compositions of the present disclosure,including components thereof, can comprise, consist of, or consistessentially of the essential elements and limitations of theembodiments described herein, as well as any additional or optionalingredients, components or limitations described herein orotherwise useful in nutritional compositions.

[0110] As used herein, the term "about" should be construed torefer to both of the numbers specified in any range. Any referenceto a range should be considered as providing support for any subsetwithin that range.

EXAMPLES

[0111] The following examples are provided to illustrate someembodiments of the nutritional composition of the presentdisclosure but should not be interpreted as any limitation thereon.Other embodiments within the scope of the claims herein will beapparent to one skilled in the art from the consideration of thespecification or practice of the nutritional composition or methodsdisclosed herein. It is intended that the specification, togetherwith the example, be considered to be exemplary only, with thescope and spirit of the disclosure being indicated by the claimswhich follow the example.

Example 1

[0112] This example describes the experimental procedures andmethods used to assess the ability of galactooligosaccharide, ofpolydextrose and of a combination of galactooligosaccharide andpolydextrose to inhibit adherence of the pathogens to humancells.

[0113] Five C. sakazakii strains (Table 1) were obtained. In someexperiments, the enteropathogenic E. coli E2348/69 was also used.Prior to each experiment, freezer stocks of each organism wereplated onto Tryptic Soy Agar (TSA; Difco) and grown overnight at37.degree. C. A single colony was inoculated in 10 ml of TrypticSoy Broth (TSB; Difco) and incubated aerobically, without shaking.Preliminary experiments revealed that adherence rates were highestwhen cells were in late log phase. Therefore, after 6 hourincubation, cultures were harvested by centrifugation(3,184.times.g for 8 minutes). The cells were washed once withphosphate-buffered saline (PBS) and re-suspended in minimalessential medium (MEM; Hyclone, Logan, Utah) supplemented with 10%fetal bovine serum (FBS; Hyclone). Minimal essential medium waspre-equilibrated at tissue culture conditions (5% CO2, 95% relativehumidity, 37.degree. C.). Based on additional preliminaryexperiments, two strains (4603 and 29004) repeatedly gave thehighest adherence rates on HEp-2 cells and were used for allsubsequent experiments.

TABLE-US-00001 TABLE 1 Strains and sources Strains SourceEnterobacter sakazakii 4593 Milk powder isolate (Infant formula)Enterobacter sakazakii 4603 Milk powder isolate (Infant formula)Enterobacter sakazakii 29004 ATCC (deposited by CDC) Enterobactersakazakii 4583 Enterocolitis isolate Enterobacter sakazakii ES 415Meningitis isolate

[0114] HEp-2 cells were obtained from the American Type CultureCollection. Cells were grown in 75 cm.sup.2 tissue culture flaskscontaining 25 ml of MEM supplemented with 10% FBS in a CO.sub.2incubator at tissue culture conditions. Confluent HEp-2 cells wereharvested by adding 0.5 ml of 0.25% Trypsin-EDTA Solution andincubating for 15 minutes at tissue culture conditions. Trypsin wasinactivated with 0.5 ml of FBS. Cells were then seeded onto 12-mmdiameter glass cover slips in 24-well tissue culture plates atapproximately 3.6.times.10.sup.5 viable cells per well, and 500.mu.l of MEM supplemented with 10% FBS was added to each well.Plates were incubated under tissue culture conditions for two daysprior to the start of each experiment, or until confluence wasreached. Media was replaced one day before the experiment.

[0115] Galactooligosaccharide was obtained as a concentrated syrupand was lyophilized to give a final product of 95% total solids.The galactooligosaccharide and polydextrose were then prepared asconcentrated stock solutions with distilled sterile water at afinal concentration of 160 mg/ml. The solutions were filtersterilized by passage though 0.22 .mu.m filters. A component of PDXand GOS was prepared by mixing equal volumes to give 80 mg/ml ofPDX and 80 mg/ml of GOS in the component stock solution.Experiments using GOS, PDX, and a combination of GOS and PDX werealso performed with E. coli E2348/69.

[0116] Cell suspensions of C. sakazakii 4603 and ATCC 29004 wereprepared as described above. Prebiotics were mixed with bacterialcultures (in MEM supplemented with 10% FBS) prior to addition tothe HEp-2 and CaCo-2. The prebiotic solutions represented 10% ofthe final volume of the suspension, giving a final concentration of16 mg/ml of prebiotic in the assay mixtures. The plates wereincubated for 3 hours in the CO.sub.2 incubator at tissue cultureconditions. Preliminary experiments indicated that 3 hours ofincubation was optimal for adherence of the bacterial strains tothe HEp-2 tissue culture and to the CaCo-2 tissue culture. Thewells were then washed 5 times with PBS to remove non-adheredbacterial cells, All HEp-2 experiments were done in duplicate andreplicated 5 times. CaCo-2 experiments were done in duplicate andreplicated twice.

[0117] For microscopic enumeration, after washing five times withPBS, cells were fixed with 100% methanol for 20 minutes and stainedwith 10% Geimsa for 15 minutes. They were then washed once withsterile distilled water and dried overnight at room temperature.Cover slips were mounted on microscope slides and observed under aphase contrast microscope with the 100.times. objective. Fifteenpictures of each cover slip were taken using Motic Image softwarefollowing an established pattern throughout the entire cover slip.The number of cells and bacteria in each picture were counted usingImage J image analysis software. Adherence was calculated as thenumber of adhered bacteria per tissue culture. Adherence inhibitionwas calculated as the number of adhered bacteria per cell in thecontrol minus the number of adhered bacteria per cell in thetreatment divided by the number of adhered bacteria per cell in thecontrol.

[0118] For cultural enumeration, after washing 5 times with PBS,cells were detached by addition of 0.1% Triton X-100 for 30 minutesat room temperature. The cells were collected, and serial dilutionswere plated on agar. Plates were incubated aerobically at37.degree. C. for 24 hours. Adherence inhibition was calculated asthe number of adhered bacteria per ml in the control minus thenumber of adhered bacteria per ml in the treatment divided by thenumber of adhered bacteria per ml in the control.

Example 2

[0119] This example describes the experimental resultsdemonstrating the ability of galactooligosaccharide, polydextrose,and a component of galactooligosaccharide and polydextrose toinhibit adherence of pathogens to human cell lines.

[0120] Adherence of C. sakazakii 4603 to HEp-2 cells in thepresence and absence of prebiotics was measured by microscopic andculture methods. FIG. 1 illustrates the adherence of C. sakazakii4603 to HEp-2 cells in the presence of prebiotics (16 mg/ml).Adherence was measured by microscopic counting (A) or by culturalenumeration (B). Both methods revealed that C. sakazakii 4603adherence was lowest when GOS was present. By microscopicexamination, GOS addition led to a 56% reduction of binding by C.sakazakii 4603 to the HEp-2 cells (Table 2). Similarly, a 48%reduction occurred in the presence of the GOS-PDX component.However, PDX by itself did not significantly reduce adherence.

TABLE-US-00002 TABLE 2 Adherence inhibition of C. sakazakii 4603 asdetermined by microscopic counting Adherence Treatment.sup.1Inhibition (%) GOS 56 .+-. 8*.sup.2 PDX 33 .+-. 20 GOS-PDX 48 .+-.16* .sup.1Final concentration = 16 mg/ml .sup.2*= significant at p< 0.05 (n = 10)

[0121] Likewise, by cultural enumeration, GOS addition led to a 71%reduction of binding by C. sakazakii 4603 to the HEp-2 cells (Table3). Similarly, a 49% reduction occurred in the presence of theGOS-PDX component. PDX alone led to a 55% reduction inadherence.

TABLE-US-00003 TABLE 3 Adherence inhibition of C. sakazakii 4603 asdetermined by cultural enumeration Adherence Treatment Inhibition(%) GOS 71 .+-. 20*.sup.2 PDX 55 .+-. 26* GOS-PDX 49 .+-. 5*.sup.1Final concentration = 16 mg/ml .sup.2*= significant at p <0.05 (n = 10)

[0122] Strain ATCC 29004 gave similar results, with reductions of63% and 53% in the presence of GOS and the GOS-PDX component,respectively as determined by microscopic counting (Table 4).However, for this strain, even the PDX significantly inhibited(42%) adherence. Moreover, similar results were obtained whendetermined by cultural enumeration, as GOS led to an 83% adherenceinhibition, GOS-PDX led to an 80% inhibition and PDX led to a 58%adherence inhibition (fable 5). FIG. 2 illustrates adherence of C.sakazakii 29004 to HEp-2 cells in the presence of prebiotics (16mg/ml), showing the results for microscopic counting (FIG. 2(A))and cultural enumeration (FIG. 2 (B)) respectively.

TABLE-US-00004 TABLE 4 Adherence inhibition of C. sakazakii 29004as determined by microscopic counting Adherence Treatment.sup.1Inhibition (%) GOS 63 .+-. 8*.sup.2 PDX 42 .+-. 11* GOS-PDX 53 .+-.8* .sup.1Final concentration = 16 mg/ml .sup.2*= signfficant at p< 0.05 (n = 10)

TABLE-US-00005 TABLE 5 Adherence inhibition of C. sakazakii 29004as determined by cultural enumeration Adherence TreatmentInhibition (%) GOS 83 .+-. 6*.sup.2 PDX 58 .+-. 27* GOS-PDX 80 .+-.9* .sup.1Final concentration = 16 mg/ml .sup.2significant at p <0.05 (n = 10)

[0123] The cultural enumeration results confirmed those observed bymicroscopic examination for both strains (FIGS. 1 and 2 and Tables3 and 5). Yet, inhibition levels were somewhat higher when theculture data was used to calculate adherence inhibition. For strainC. sakazakii 4603, GOS and the GOS-PDX component reduced adherenceby 71% and 49%, respectively, with PDX by itself also inhibitingadherence by 55%. GOS, the GOS-PDX, and PDX alone treatmentsreduced adherence of strain C. sakazakii 29004 by 83%, 80%, and58%, respectively. Photomicrographs of the cover slips providedadditional confirmation that adherence of C. sakazakii 4603 toHEp-2 cells was reduced in the presence of GOS (FIG. 3). FIG. 3shows the adherence of C. sakazakii 4603 to HEp-2 cells in theabsence (FIG. 3(A)) and presence of GOS (FIG. 3(B)), GOS-PDX (FIG.3(C)); and PDX (FIG. 3(D)). These results suggest that GOS may havean anti-adhesive effect on C. sakazakii and directly inhibit theadherence of this organism to gastrointestinal epithelialcells.

[0124] To confirm that the GOS inhibited adhesion of pathogens,adherence inhibition experiments with E. coli E2348/69 and Hep-2tissue cultures were performed. The results (FIG. 4) showed thatGOS did indeed inhibit adherence of E. coli E2348/69 to Hep-2tissue cultures. FIG. 4 illustrates the adherence of E. coliE2348/69 to HEp-2 cells in the presence of prebiotics (16 mg/ml).Adherence was measured by microscopic counting. Significance(versus the control) is indicated by asterisks in FIG. 4.

[0125] Moreover, FIG. 5 shows that galactooligosaccharide andpolydextrose also inhibited the adherence of C. sakazakii (4603,29004) to Caco-2 cells. FIG. 5 illustrates the adherence of C.sakazakii 29004 to Caco-2 cells in the presence of prebiotics,where adherence was measured by microscopic counting.

Example 3

[0126] This example illustrates one embodiment of a nutritionalproduct according to the present disclosure.

TABLE-US-00006 TABLE 6 Nutritional Composition with PrebioticComponent Ingredient Per 10,000 L Demineralized Whey Solids 534.337kg Fat Component 339.695 kg Nonfat Milk Solids 191.234 kg Lactose136.321 kg Galactooligosaccharide Syrup Solid 35.096 kgPolydextrose 22.222 kg Potassium Citrate 7.797 kg Mono- andDiglycerides 7.233 kg Single Cell Arachidonic Acid Oil 6.486 kgCalcium Phosphate, Tribasic 4.185 kg Ascorbic Acid 1,403.323 gSodium Ascorbate 1,168.402 g Inositol 407.029 g Taurine 402.962 gCorn Syrup Solids 188.300 g Niacimarnide 89.857 g CalciumPantothenate 42.443 g Vitamin B12 23.613 g Biotin Trituration23.613 g Thiamin HCl 8.022 g Pyridoxine HCI 6.176 g Folic Acid2.260 g Lecithin Concentrate 3.694 kg Single Cell DocosahexaenoicAcid Oil 3.243 kg Carrageenan 2.826 kg Calcium Chloride 2.650 kgSodium Chloride 1.410 kg Maltodextrin 484.199 g CMP, free acid151.951 g AMP, free acid 33.944 g GMP, disodium salt 18.347 g UMP,disodium salt 7.559 g Ferrous Sulfate 0.620 kg Sodium Citrate 0.455kg Tocopheryl Acetate, DL-Alpha 160.882 g Soy Oil 139.612 g VitaminA Palmitate 17.253 g Cholecalciferol Concentrate 5.715 g Vitamin K,Liquid Phytonadione 0.538 g Zinc Sulfate 214.225 g Sodium Selenite51.112 g Cupric Sulfate 22.885 g Lactose 12.659 g Manganese Sulfate3.119 g Water, Deflouridated 10,311.900 kg

[0127] LOS is generated when lactose is heated at a hightemperature. Therefore, in this embodiment the product containsindigenous LOS. The level of indigenous LOS in the product isapproximately 2 g/L.

Example 4

[0128] This example illustrates one embodiment of a nutritionalproduct according to the present disclosure.

TABLE-US-00007 TABLE 7 Nutritional Composition with PrebioticComponent Ingredient Per 10,000 L Demineralized Whey Solids 534.337kg Fat Component 339.695 kg Nonfat Milk Solids 191.234 kg Lactose142.000 kg Galactooligosaccharide Syrup Solid 23.164 kgPolydextrose 22.222 kg Lactulose Syrup Solid 10.353 kg PotassiumCitrate 7.797 kg Mono- and Diglycerides 7.233 kg Single CellArachidonic Acid Oil 6.486 kg Calcium Phosphate, Tribasic 4.185 kgAscorbic Acid 1,403.323 g Sodium Ascorbate 1,168.402 g Inositol407.029 g Taurine 402.962 g Corn Syrup Solids 188.300 gNiacimarnide 89.857 g Calcium Pantothenate 42.443 g Vitamin B1223.613 g Biotin Trituration 23.613 g Thiamin HCI 8.022 g PyridoxineHCI 6.176 g Folic Acid 2.260 g Lecithin Concentrate 3.694 kg SingleCell Docosahexaenoic Acid Oil 3.243 kg Carrageenan 2.826 kg CalciumChloride 2.650 kg Sodium Chloride 1.410 kg Maltodextrin 484.199 gCMP, free acid 151.951 g AMP, free acid 33.944 g GMP, disodium salt18.347 g UMP, disodium salt 7.559 g Ferrous Sulfate 0.620 kg SodiumCitrate 0.455 kg Tocopheryl Acetate, DL-Alpha 160.882 g Soy Oil139.612 g Vitamin A Palmitate 17.253 g Cholecalciferol Concentrate5.715 g Vitamin K, Liquid Phytonadione 0.538 g Zinc Sulfate 214.225g Sodium Selenite 51.112 g Cupric Sulfate 22.885 g Lactose 12.659 gManganese Sulfate 3.119 g Water, Deflouridated 10,311.900 kg

[0129] LOS is generated when lactose is heated at a hightemperature. Therefore, in this embodiment the product containsindigenous LOS. The level of indigenous LOS in the product isapproximately 2.6 g/L.

Example 5

[0130] This example illustrates one embodiment of a nutritionalproduct according to the present disclosure.

TABLE-US-00008 TABLE 8 Nutritional Composition with PrebioticComponent Ingredient Per 10,000 L Demineralized Whey Solids 534.337kg Fat Component 339.695 kg Nonfat Milk Solids 191.234 kg Lactose119.321 kg Galactooligosaccharide Syrup Solid 46.327 kgPolydextrose 44.444 kg Lactulose Syrup Solid 20.706 kg PotassiumCitrate 7.797 kg Mono- and Diglycerides 7.233 kg Single CellArachidonic Acid Oil 6.486 kg Calcium Phosphate, Tribasic 4.185 kgAscorbic Acid 1,403.323 g Sodium Ascorbate 1,168.402 g Inositol407.029 g Taurine 402.962 g Corn Syrup Solids 188.300 gNiacimarnide 89.857 g Calcium Pantothenate 42.443 g Vitamin B1223.613 g Biotin Trituration 23.613 g Thiamin HCI 8.022 g PyridoxineHCI 6.176 g Folic Acid 2.260 g Lecithin Concentrate 3.694 kg SingleCell Docosahexaenoic Acid Oil 3.243 kg Carrageenan 2.826 kg CalciumChloride 2.650 kg Sodium Chloride 1.410 kg Maltodextrin 484.199 gCMP, free acid 151.951 g AMP, free acid 33.944 g GMP, disodium salt18.347 g UMP, disodium salt 7.559 g Ferrous Sulfate 0.620 kg SodiumCitrate 0.455 kg Tocopheryl Acetate, DL-Alpha 160.882 g Soy Oil139.612 g Vitamin A Palmitate 17.253 g Cholecalciferol Concentrate5.715 g Vitamin K, Liquid Phytonadione 0.538 g Zinc Sulfate 214.225g Sodium Selenite 51.112 g Cupric Sulfate 22.885 g Lactose 12.659 gManganese Sulfate 3.119 g Water, Deflouridated 10,325.600 kg

[0131] LOS is generated when lactose is heated at a hightemperature. Therefore, in this embodiment the product containsindigenous LOS. The level of indigenous LOS in the product isapproximately 3.6 g/L.

[0132] All references cited in this specification, includingwithout limitation, all papers, publications, patents, patentapplications, presentations, texts, reports, manuscripts,brochures, books, internet postings, journal articles, periodicals,and the like, are hereby incorporated by reference into thisspecification in their entireties. The discussion of the referencesherein is intended merely to summarize the assertions made by theirauthors and no admission is made that any reference constitutesprior art. Applicants reserve the right to challenge the accuracyand pertinence of the cited references.

[0133] Although embodiments of the disclosure have been describedusing specific terms, devices, and methods, such description is forillustrative purposes only. The words used are words of descriptionrather than of limitation. It is to be understood that changes andvariations may be made by those of ordinary skill in the artwithout departing from the spirit or the scope of the presentdisclosure, which is set forth in the following claims. Inaddition, it should be understood that aspects of the variousembodiments may be interchanged in whole or in part. For example,while methods for the production of a commercially sterile liquidnutritional supplement made according to those methods have beenexemplified, other uses are contemplated. Therefore, the appendedclaims should not be limited to the description of the versionscontained therein.

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