Patent Application
20130287895
1. Technical Field
The present disclosure relates to enteral nutritional compositions comprising prebiotic component. More specifically, the nutritional compositions of the present disclosure contain a prebiotic component comprising galactooligosaccharide (GOS). In some embodiments, the prebiotic component comprises galactooligosaccharide, polydextrose (PDX), a mixture of galactooligosaccharide and polydextrose, or a mixture of galactooligosaccharide, polydextrose and at least one other prebiotic. Moreover, the disclosure is further directed to a method of inhibiting adhesion of pathogens in the gastrointestinal tract of a human subject. The method comprises administering an effective amount of the prebiotic component.
2. Background Art
Infants and children are exposed to a variety of pathogens, and consequently, the incidence of infectious diseases peaks in the first 4 years of life. Infants do not have mature immune systems and are often incapable of mounting an effective immune response. Indeed, on a cellular level, infants have a decreased ability to concentrate leukocytes where necessary. (Maternity and Gynecologic Care, Bobak, Jensen, Zalar, Fourth Edition, p. 470) Consequently, infants are not able to limit invading pathogens due to a generalized hypofunction of their inflammatory and immune mechanisms. As such, improving the immune response in infants and children would provide an opportunity to reduce the incidence of infections and to maintain or improve the overall health of pediatric subjects.
Cronobacter sakazakii (also called Enterobacter sakazakii) is a motile, petrichious, non-sporeforming, Gram negative facultative anaerobe. It is an opportunistic pathogen that has been implicated in severe forms of meningitis, necrotizing enterocolitis, and sepsis in neonates. Although the environmental source of the microorganism is not known, powdered infant formula has been implicated in several outbreaks as well as other sporadic cases of infection. Of particular concern are reports of the organism being isolated from powdered milk processing plants and hospital utensils such as spoons and other implements used to prepare 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 infant milk formula (IMF) powder. International Journal of Dairy Technology 59: 102-111.) Because colonization and subsequent infection, and invasion of C. sakazakii require that the organism adhere to host cell surfaces, agents that inhibit or block attachment of the pathogen to epithelial cells could be useful in reducing infections.
The infant gut microflora is rapidly established in the first few weeks following birth, and it has a great impact on an infant's immune system. The nature of this intestinal colonization is initially determined by the host's early exposure to environmental sources of microbes as well as to the health of the infant. Whether the infant is breast-fed or formula-fed also has a strong influence on the intestinal bacterial population.
Human milk contains a number of factors that may contribute to the growth and population of the gut microflora of infants. Among these factors is a complex mixture of more than 130 different oligosaccharides that reach levels as high as 8-12 g/L in transitional and mature milk. (Kunz, et al., Oligosaccharides in Human Milk: Structure, Functional, and Metabolic Aspects, Ann. Rev. Nutr. 20: 699-722 (2000).) These oligosaccharides are resistant to enzymatic digestion in the upper gastrointestinal tract, and they reach the colon intact, where they then serve as substrates for colonic fermentation.
Human milk oligosaccharides are believed to elicit an increase in the number of Bifidobacteria in the colonic microflora, along with a reduction in the number of potentially pathogenic bacteria. (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 in Milk Protect Human Infants from Gastrointestinal Bacteria?, J. Nutr. 217:S980-S984 (1997).) One way that human milk oligosaccharides may increase the numbers of Bifidobacterium spp. and reduce the number of potentially pathogenic bacteria is by acting as competitive receptors and inhibiting the binding of pathogens to the cell surface, Rivero-Urgell, et al., Oligosaccharides: Application in Infant Food, Early Hum. Dev. 65(S):43-52 (2001).
Because cow's milk and many commercially available infant formulas that are based on cow's milk provide only trace amounts of oligosaccharides, prebiotics can be used to supplement the diet of formula-fed infants. Prebiotics have been defined as non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of cells in the colon that can improve the health of the host.
The incorporation of various prebiotic ingredients into infant formulas has been disclosed. For example, U.S. Patent App. No. 20030072865 to Bindels, et al. discloses an infant formula with improved protein content and at least one prebiotic. The prebiotic component can be lacto-N-tetaose, lacto-N-fuco-pentaose, lactulose (LOS), lactosucrose, raffinose, galactooligosaccharide, fructo-oligosaccharide (FOS), oligosaccharides derived from soybean polysaccharides, mannose-based oligosaccharides, arabino-oligosaccharides, xylo-oligosaccharides, isomalto-oligo-saccharides, glucans, sialyl oligosaccharides, and fuco-oligosaccharides.
Similarly, U.S. Patent App. No. 2004/0191234 to Haschke discloses a method for enhancing the immune response which comprises administering at least one prebiotic. The prebiotic can be an oligosaccharide produced from glucose, galactose, xylose, maltose, sucrose, lactose, starch, xylan, hemicellulose, inulin, or a mixture thereof. The prebiotic can be present in an infant cereal.
Unfortunately, however, there are some disadvantages in the administration of the above to formula fed infants. While they may beneficially affect the population of probiotics in the gut, the fermentation of many of these prebiotic substances occurs at a very rapid rate, which may produce excess gas, abdominal distension, bloating, and diarrhea. Therefore, the choice of prebiotic substances in nutritional compositions and infant formulas should be made with the goal of maximizing potential benefits, such as inhibiting the adhesion of pathogens, and minimizing unwanted side-effects.
Briefly, therefore, the present disclosure is directed, in an embodiment, to a nutritional composition comprising a prebiotic component that inhibits the adhesion of pathogens in the gastrointestinal tract and that is well-tolerated and does not produce or cause excess gas, abdominal distension, bloating or diarrhea. In some embodiments, the nutritional composition comprises a lipid or fat source, a protein source and an effective amount of the prebiotic component. In some embodiments, the nutritional composition also contains a source of long chain polyunsaturated fatty acid (LCPUFA). In some embodiments, the nutritional composition is nutritionally complete, and in some embodiments, the nutritional composition includes a probiotic.
Accordingly, the prebiotic component contains galactooligosaccharide, and in some embodiments, the prebiotic component additionally contains polydextrose, fructooligosaccharide (FOS), lactulose (LOS) or any combination or mixture thereof.
The prebiotic component contains an effective amount of oligosaccharides such that adhesion of harmful bacteria is inhibited in the gastrointestinal tract when the prebiotic component is administered to a subject. The prebiotic composition can include a plurality of oligosaccharides, whereby the combination of the oligosaccharides inhibits adherence of pathogens in the gastrointestinal tract. In certain embodiments, the prebiotic component comprises or consists of a combination of galactooligosaccharide and polydextrose. Moreover in some embodiments, the nutritional composition further includes a source of long chain polyunsaturated fatty acids.
The disclosure is also directed to a nutritional composition that inhibits the adhesion of pathogens in the gastrointestinal tract of a subject comprising, in some embodiments:
a. up to about 7 g/100 kcal of a fat or lipid, more preferably about 3 to about 7 g/100 kcal of a fat or lipid;
b. up to about 5 g/100 kcal of a protein source, more preferably about 1 to about 5 g/100 kcal of a protein source;
c. a prebiotic component comprising galactooligosaccharide; and
d. about 5 to about 100 mg/100 kcal of a source of long chain polyunsaturated fatty acids which include DHA, more preferably about 10 to about 50 mg/100 kcal of a source of long chain polyunsaturated fatty acids which includes DHA.
In yet another embodiment, the disclosure is directed to a nutritional composition having improved digestibility, the composition comprising a lipid or fat, a protein source, a source of long chain polyunsaturated fatty acids that includes docosahexaenoic acid (DHA), and a prebiotic component that comprises at least 20% of an oligosaccharide mixture comprising galactooligosaccharide and/or a combination of galactooligosaccharide and polydextrose.
Additionally, the present disclosure relates to a method for inhibiting adhesion of pathogens in the gastrointestinal tract of a human subject.
In yet another embodiment, the present disclosure teaches a method for reducing the incidence of diarrhea due to adhesion of bacterial pathogens in the gastrointestinal tract.
It is to be understood that both the foregoing general description and the following detailed description present embodiments of the disclosure and are intended to provide an overview or framework for understanding the nature and character of the disclosure as it is claimed. The description serves to explain the principles and operations of the claimed subject matter. Other and further features and advantages of the present disclosure will be readily apparent to those skilled in the art upon a reading of the following disclosure.
Reference now will be made in detail to the embodiments of the present disclosure, one or more examples of which are set forth herein below. Each example is provided by way of explanation of the nutritional composition of the present disclosure and is not a limitation. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the teachings of the present disclosure without departing from the scope or spirit of the disclosure. For instance, features illustrated or described as part of one embodiment, can be used with another embodiment to yield a still further embodiment.
Thus, it is intended that the present disclosure covers such modifications and variations as come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present disclosure are disclosed in or are obvious from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only and is not intended as limiting the broader aspects of the present disclosure.
“Nutritional composition” means a substance or formulation that satisfies at least a portion of a subject's nutrient requirements.
“Pediatric subject” means a human that is less than 13 years of age. In some embodiments, a pediatric subject refers to a human subject that is less than 8 years old.
“Infant” means a subject ranging in age from birth to not more than about one year and includes infants from 0 to about 12 months corrected age. The term infant includes low birth weight infants, very low birth weight infants, and preterm infants. The phrase “corrected age” means an infant's chronological age minus the amount of time that the infant was born premature. Therefore, the corrected age is the age of the infant if it had been carried to full term.
“Child” means a subject ranging in age from about 12 months to about 13 years. In some embodiments, a child is a subject between 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 12 months and about 13 years.
“Children's nutritional product” refers to a composition that satisfies at least a portion of the nutrient requirements of a child.
“Infant formula” means a composition that satisfies at least a portion of the nutrient requirements of an infant. In the United States, the content of an infant formula is dictated by the federal regulations set forth at 21 C.F.R. Sections 100, 106, and 107. These regulations define macronutrient, vitamin, mineral, and other ingredient levels in an effort to stimulate the nutritional and other properties of human breast milk.
“Nutritionally complete” means a composition that may be used as the sole source of nutrition, which would supply essentially all of the required daily amounts of vitamins, minerals, and/or trace elements in combination with proteins, carbohydrates, and lipids.
“Probiotic” means a microorganism with low or no pathogenicity that exerts beneficial effects on the health of the host.
“Prebiotic” means a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the digestive tract that can improve the health of the host.
“Effective amount” means an amount that inhibits the adhesion of pathogens in the gastrointestinal tract of a subject.
“Prebiotic component” or “oligosaccharide mixture” means a combination of at least one prebiotic with at least one other prebiotic. In a preferred embodiment, the prebiotic component comprises galactooligosaccharide and polydextrose.
The phrase “functional attributes” mean any inherent quality or characteristic that causes something to occur. Examples of functional attributes of human milk oligosaccharides in the present disclosure can include the increase of the population and species of 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 of prebiotics in the gut, and/or inhibition of adherence of pathogens, such as harmful bacteria, in the gastrointestinal tract of a subject.
The present disclosure describes a nutritional composition for a subject comprising a lipid source, a protein source and a prebiotic component, wherein the prebiotic component contains at least one carbohydrate that inhibits the adhesion of pathogens in the gastrointestinal tract. In other embodiments, the prebiotic component reduces the incidence of diarrhea and/or modulates stool characteristics in a positive way.
The present disclosure also describes a method for inhibiting adherence of pathogens in the gastrointestinal tract of a subject by administering an effective amount of a nutritional composition comprising a fat or lipid source, a protein source and a source of galactooligosaccharide. In some embodiments, the galactooligosaccharide may be administered as part of a prebiotic component. In some embodiments, the prebiotic may consist of galactooligosaccharide. In other embodiments, the prebiotic component may comprise the galactooligosaccharide administered together with another carbohydrate, such as polydextrose. The administration of galactooligosaccharide, of polydextrose or of a mixture thereof is physically well-tolerated by human subjects.
Suitable fat or lipid sources for practicing the present disclosure 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 fish oils, marine oils, single-cell oils; vegetable and plant oils, such as 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 kernel oil, wheat germ oil, medium chain triglyceride
Bovine milk protein sources useful in practicing the present disclosure include, but are not limited to, milk protein powders, milk protein concentrates, milk protein isolates, nonfat milk solids, nonfat milk, nonfat dry milk, whey protein, whey protein isolates, whey protein concentrates, sweet whey, acid whey, casein, acid casein, caseinate (e.g. sodium caseinate, sodium calcium caseinate, calcium caseinate) and any combinations thereof.
In one embodiment, the proteins are provided as intact proteins. In other embodiments, the proteins are provided as a combination of both intact proteins and partially hydrolyzed proteins, with a degree of hydrolysis of between about 4% and 10%. In certain other embodiments, the proteins are more completely hydrolyzed. In yet another embodiment, the protein source may be supplemented with glutamine-containing peptides.
In a particular embodiment of the disclosure, the whey:casein ratio of the protein source is similar to that found in human breast milk. In an embodiment, the protein source comprises from about 40% to about 80% whey protein and from about 20% to about 60% casein.
The nutritional composition contains a prebiotic component. In some embodiments, the nutritional composition comprises galactooligosaccharide in addition to one or more other prebiotics as part of a prebiotic component. The term “prebiotic” as used herein refers to indigestible food ingredients which exert health benefits upon the host. Such health benefits may include, but are not limited to, selective stimulation of the growth and/or activity of one or a limited number of beneficial gut bacteria, stimulation of the growth and/or activity of ingested probiotic microorganisms, selective reduction in gut pathogens, inhibition of adherence of gut pathogens and favorable influence on gut short chain fatty acid profile. Such prebiotics may be naturally-occurring, synthetic, or developed through the genetic manipulation of organisms and/or plants, whether such new source is now known or developed later.
Prebiotics useful in the present disclosure may include oligosaccharides, polysaccharides, and other prebiotics that contain fructose, xylose, soya, galactose, glucose and mannose. Prebiotic oligosaccharides are non-digestible food ingredients that can act as anti-adhesives against bacterial adherence by mimicking the host cell receptor sites that intestinal pathogens recognize. Specifically, prebiotics useful in the present disclosure may include 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, and gentio-oligosaccharides.
The galactooligosaccharide that is utilized in the nutritional composition(s) of the present disclosure is comprised of several galactose-containing species of varying lengths. GOS is a mixture of oligosaccharides consisting of D-glucose and D-galactose. It is sometimes referred to as trans-galactooligosaccharide. It is produced from β-lactose by β-galactosidase, which can be obtained from Aspergillus oryzae. GOS has been suggested to increase calcium absorption and prevention of bone loss in adults. GOS has been identified as a prebiotic that is useful for administration to infants.
The amount of galactooligosaccharide in the nutritional composition may, in an embodiment, be from about 0.1 mg/100 Kcal to about 1.0 mg/100 Kcal. In another embodiment, the amount of galactooligosaccharide in the nutritional composition may be from about 0.1 mg/100 Kcal to about 0.5 mg/100 Kcal.
Meanwhile, the polydextrose of the present disclosure is composed of randomly cross-linked glucose units with several types of glycosidic bonds, and its anti-adhesive activity has not been previously studied. In some embodiments, PDX is synthesized from a mixture of dextrose, about 10% sorbitol and about 1% citric acid. PDX is a non-digestible carbohydrate that is synthesized from randomly cross-linked glucose and sorbitol. It is not digested in the upper GI tract and is only partially fermented in the lower GI tract, making it a beneficial ingredient for digestive health. The physiological benefits of PDX include increased fecal bulk, reduced transit time, lower fecal pH and reduced concentration of putrefactive substances in the colon. In adults, PDX ingestion has also been shown to aid in the promotion and growth of beneficial bacteria in the intestine and production of SCFAs.
PDX has been identified as a prebiotic substance for adults based on its functions in the GI tract. PDX has not, however, been identified as a prebiotic that provides the benefits of the present disclosure and that can be administered to pediatric subjects, such as infants or children. The gut microflora of infants is well known to be less developed than that of an adult. While the microflora of the adult human consists of more than 1013 microorganisms and nearly 500 species, the gut microflora of an infant contains only a fraction of those microorganisms, both in absolute number and species diversity. Because the bacterial populations and species vary immensely between the gut of an infant and an adult, it cannot be assumed that a prebiotic substance that has a beneficial effect on adults would also have a beneficial effect on infants.
Polydextrose is commercially available from a variety of sources. For example, STA-LITE® Polydextrose is available in 5 lb bags from Honeyville Grain, Inc., located in Salt Lake City, Utah. Polydextrose is commercially available from Danisco Sweeteners, Ltd., located in the United Kingdom.
PDX is well-suited for incorporation into a nutritional composition, such as an infant formula, as it contains only 1 Cal/g, as compared to 4 Cal/g for typical prebiotics. It is also highly soluble and neutral tasting. Therefore, its addition to a nutritional composition would not change the physical or taste characteristics of the composition.
The form of administration of PDX in the method of the disclosure is not critical, as long as an effective amount is administered. Most conveniently, the PDX is supplemented into nutritional composition, in some embodiments an infant formula, which is then fed to a subject.
The amount of polydextrose in the nutritional composition may, in an embodiment, be within the range of from about 0.1 mg/100 Kcal to about 0.5 mg/100 Kcal. In another embodiment, the amount of polydextrose may be about 0.3 mg/100 Kcal. In a particular embodiment, galactooligosaccharide and polydextrose are supplemented into the nutritional composition in a total amount of about at least about 0.2 mg/100 Kcal and can be about 0.2 mg/100 Kcal to about 1.5 mg/100 Kcal.
In a particular embodiment of the present disclosure, PDX is administered in combination with GOS. In this embodiment. PDX and GOS can be administered in a ratio of PDX:GOS of between about 9:1 and 1:9. In another embodiment, the ratio of PDX:GOS can be between about 5:1 and 1:5. In yet another embodiment, the ratio of PDX:GOS can be between about 1:3 and 3:1. In a particular embodiment, the ratio of PDX to GOS can be about 1:1. In another particular embodiment, the ratio of PDX to GOS can be about 8:2.
An effective amount of the PDX:GOS combination may be between about 1.0 g/L and 10.0 g/L, administered daily. In another embodiment, an effective amount of the PDX:GOS combination may be between about 2.0 g/L and 8.0 g/L, administered daily. In a particular embodiment, an effective amount of the PDX:GOS combination may be about 2 g/L of PDX and 2 g/L of GOS, administered daily.
In another specific embodiment of the present disclosure, galactooligosaccharide is administered in combination with FOS, which is also sometimes called oligofructose. FOS is a carbohydrate, specifically a chain polymer of the sugar fructose. FOS has been identified as a prebiotic that is useful for administration to infants.
In another specific embodiment of the present disclosure, PDX is administered in combination with LOS. LOS is a semisynthetic disaccharide formed from D-galactose and D-fructose and joined by a β-glucosidic linkage. It is resistant to hydrolysis by human digestive enzymes, but is fermented in the small intestine. It is highly soluble and has a sweet taste. LOS has been identified as a prebiotic that is useful for administration to infants.
In this embodiment, PDX and LOS can be administered in a ratio of between about 9:1 and 1:9, In another embodiment, the ratio of PDX to LOS can be between about 5:1 and 1:5. In yet another embodiment, the ratio of PDX to LOS can be between about 3:1 and 1:3.
In a particular embodiment, the ratio of PDX to LOS can about 5:5. In another particular embodiment, the ratio of PDX to LOS can be about 8:2.
An effective amount of the PDX:LOS combination may be between about 1.0 g/L and 10.0 g/L, administered daily. In another embodiment, an effective amount of the PDX:LOS combination may be between about 2.0 g/L and 8.0 g/L, administered daily. In a particular embodiment, an effective amount of the PDX:LOS combination may be about 2 g/L of PDX and 2 g/L of LOS, administered daily.
In yet another embodiment of the present disclosure, PDX is administered in combination with both GOS and LOS. In this embodiment, the PDX:GOS:LOS combination can be administered in a ratio of about 50:33:17. Alternatively, the ratio of the PDX:GOS:LOS combination can be about 1:1:1. In a particular embodiment, the ratio of PDX:GOS:LOS can be about 1:1.5:1.
An effective amount of the PDX:GOS:LOS combination may be between about 1.0 g/L and 10.0 g/L, administered daily. In another embodiment, an effective amount of the PDX:GOS:LOS combination may be between about 2.0 g/L and 8.0 g/L, administered daily. In an embodiment, an effective amount of the PDX:GOS:LOS combination may be about 2 g/L PDX, 2 g/L GOS and 2 g/L LOS, administered daily. In a particular embodiment, an effective amount of the PDX:GOS:LOS combination 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 of the PDX:GOS:LOS combination may be about 4 g/L PDX, 2.64 g/L GOS and 3.6 g/L LOS, administered daily.
The nutritional composition of the present application, in some embodiments, comprises a prebiotic component comprising galactooligosaccharide and polydextrose. The nutritional composition of the present disclosure inhibits adhesion of pathogens in the gastrointestinal tract of a subject. In some embodiments, the nutritional composition specifically inhibits the adherence of C. sakazakii 4603. In some embodiments, the nutritional composition inhibits the adherence of C. sakazakii 29004.
The initial adherence of pathogens to host cell surfaces is one of the first steps prior to colonization and infection. Most human pathogens that are able to adhere to host tissues have an advantage over those that do not, since they are more likely to evade and resist the defense systems of the host. (Shoaf, K., G. L. Mulvey, G. D. Armstrong, R. W. Hutkins. 2006. Prebiotic galactooligosaccharides reduce adherence of enteropathogenic Escherichia coli to tissue culture cells, Infection and Immunity 74:6920-6928.) Adherence between the pathogen and the host cells is most commonly mediated via lectin-like adhesins expressed by the bacteria and ligand-like carbohydrates located on the surface of the host epithelial cells. Indeed, most intestinal pathogens recognize specific monosaccharide residues or short oligosaccharide sequences as binding receptors. Bacterial variants that are unable to express functional adhesins are unable to adhere and initiate infections, indicating that for these pathogens, adherence is required to cause disease (Isaacson, R. E. 1983, Bacterial adherence to mucosal surfaces: an attribute of virulence. Bulletin Européen de Physiopathologie Respiratoire 19:75-80.). This finding has led to the suggestion that agents that inhibit adherence can reduce incidences of infection, (Zorf, D. and Roth, S. 1996. Oligosaccharides anti-infection agents. Lancet 347:1017-1021)
The nutritional composition of the present disclosure uses a prebiotic component of oligosaccharide decoys that mimic the carbohydrate binding sites ordinarily recognized by pathogens as binding receptors on the surface of host epithelial cells. The prebiotic component of oligosaccharides competes with the cognate sugars for binding to the adhesion and thereby inhibits bacterial adherence in the gastrointestinal tract.
Accordingly, to arrive at the nutritional composition of the present disclosure, the individual carbohydrates GOS and PDX, as well as a component of GOS and PDX, were studied in an in vitro model to assess their respective abilities to inhibit adherence of pathogens to human cells lines.
Galactooligosaccharide and polydextrose were used for adherence inhibition assays performed with human cell lines (HEp-2 and 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 prebiotic component comprising GOS and PDX were administered at a concentration of 16 mg/ml in the assay and analyzed for their adherence inhibition of the single pathogens to the epithelial cell lines.
For adherence assays, confluent tissue culture monolayers were prepared, and the prebiotics were mixed with C. sakazakii 4603 or ATCC 29004 (approximately 107 cells) prior to adding them to the HEp-2 cells. Following an incubation of 3 hours, which had been determined in preliminary experiments to be the optimal incubation time for adherence, at 37° C. in the CO2 incubator, the wells were washed 5 times with PBS. Cells were then fixed for microscopic enumeration or were instead detached and serial dilution plated on agar for culture enumeration.
For the adherence assays with EPEC, each prebiotic was added at a final concentration of about 16 mg/ml and incubated with HEp-2 cells for 30 minutes. Cells with microcolonies of EPEC or that consisted of four or more bacteria were considered positive for having a typical localized adherence phenotype. For C. sakazakii adherence occurs via cluster formation and diffuse adherence. Adherence inhibition was calculated as the number of adhered bacteria per cell in the control minus the number of adhered bacteria per cell in the treatment divided by the number of adhered bacteria per cell in the control.
When measured by the microscopic method, a significant reduction (56%) in adherence of C. sakazakii 4603 was observed in the presence of GOS (16 mg/ml). Adherence inhibition also occurred (48%) when a GOS-PDX component (8 mg/ml each) was tested, although PDX by itself had less effect. A similar pattern for this strain on HEp-2 cells was observed using cultural enumeration. Similar results were also observed for Caco-2 cells and also for another strain of C. sakazakii (ATCC 29004). Thus, the results suggest that GOS and the prebiotic component of PDX and GOS have anti-adhesive effects on pathogens, such as C. sakazakii, and that the prebiotics directly inhibit the adherence of pathogenic organisms to gastrointestinal epithelial cells.
In the compositions and methods of the present disclosure, an effective amount of a prebiotic component comprising galactooligosaccharide and polydextrose is administered to a subject for the purpose of simulating the functional attributes of human milk oligosaccharides. In an embodiment, the total amount of prebiotics present in the nutritional composition may be from about 1.0 g/L to about 10.0 g/L of the composition. More preferably, the total amount of prebiotics present in the nutritional composition may be from about 2.0 g/L and about 8.0 g/L of the composition. At least 20% of the prebiotics may comprise a mixture of galactooligosaccharide and polydextrose. The amount of each of galactooligosaccharide and polydextrose in the nutritional composition may, in an embodiment, be within the range of from about 1.0 g/L to about 4.0 g/L.
In some embodiments, the nutritional composition of the disclosure contains a source of long chain polyunsaturated fatty acids (LCPUFAs) that comprises docosahexaenoic acid (DHA). Other suitable LCPUFAs include, but are not limited to, α-linoleic acid, γ-linoleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid (EPA) and arachidonic acid (ARA).
In one embodiment, the nutritional composition is supplemented with both DHA and ARA. In this embodiment, the weight ratio of ARA:DHA may be from about 1:3 to about 9:1. In one embodiment of the present disclosure, this ratio is from about 1:2 to about 4:1. In one embodiment, both DHA and ARA are administered in combination with PDX.
The amount of long chain polyunsaturated fatty acids in the nutritional composition is advantageously at least about 5 mg/100 Kcal, and may vary from about 5 mg/100 kcal to about 100 mg/100 kcal, more preferably from about 10 mg/100 kcal to about 50 mg/100 kcal.
The nutritional composition may be supplemented with oils containing DHA and ARA using standard techniques known in the art. For example, DHA and ARA may be added to the composition by replacing an equivalent amount of an oil, such as high oleic sunflower oil, normally present in the composition. As another example, the oils containing DHA and ARA may be added to the composition by replacing an equivalent amount of the rest of the overall fat component normally present in the composition without DHA and ARA.
If utilized, the source of DHA and ARA may be any source known in the art such as marine oil, fish oil, single cell oil, egg yolk lipid, and brain lipid. In some embodiments, the DHA and ARA are sourced from the single cell Martek oil, DHASCO®, or variations thereof. The DHA and ARA can be in natural form, provided that the remainder of the LCPUFA source does not result in any substantial deleterious effect on the infant. Alternatively, the DHA and ARA can be used in refined form.
In an embodiment of the present disclosure, sources of DHA and 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 incorporated herein in their entirety by reference. However, the present disclosure is not limited to only such oils.
In certain embodiments of the disclosure, the nutritional composition may contain one or more probiotics. Any probiotic known in the art may be acceptable in this embodiment provided it achieves the intended result. In a particular embodiment, the probiotic may be selected from any Lactobacillus species, Lactobacillus rhamnosus GG, Bifidobacterium species, Bifidobacterium longum, and Bifidobacterium animalis subsp. lactis BB-12 (DSM No. 10140) or combinations thereof.
If included in the composition, the amount of the probiotic may vary from about 104 to about 1010 colony forming units (cfu) per kg body weight per day. An average infant weighs between about 2 kg and about 15 kg. Thus, in some embodiments, the nutritional composition of the present disclosure will comprise between about 2×104 and 15×1010 cell equivalents of probiotic per daily serving to an infant. In another embodiment, the amount of the probiotic may vary from about 106 to about 109 cfu per kg body weight per day; thereby delivering between about 2×106 and 15×109 cell equivalents of probiotic per daily serving to an infant. In yet another embodiment, the amount of the probiotic may be at least about 106 cfu per kg body weight per day; thereby delivering at least about 2×106 cfu per day to an infant.
In an embodiment, the probiotic(s) may be viable or non-viable. As used herein, the term “viable”, refers to live microorganisms. The term “non-viable” or “non-viable probiotic” means non-living probiotic microorganisms, their cellular components and/or metabolites thereof. Such non-viable probiotics may have been heat-killed or otherwise inactivated, but they retain the ability to favorably influence the health of the host. The probiotics useful in the present disclosure may be naturally-occurring, synthetic or developed through the genetic manipulation of organisms, whether such new source is now known or later developed.
In an embodiment, the nutritional composition comprising the prebiotic component is provided as a nutritionally complete infant formula, 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/100 kcal, with protein from about 1 to about 5 g/100 kcal, lipid or fat from about 3 to about 7 g/100 kcal and may be supplemented with an effective amount of the prebiotic component.
Nutritional compositions of the present disclosure may be milk-based, in the form of liquid, evaporated, condensed or dry milk. In some embodiments, the nutritional composition may also include non-dairy liquid or solid food products, protein, flavors or flavor masking agents, sweeteners and vitamins or dietary supplements.
In some embodiments, the nutritional composition may be nutritionally complete, containing suitable types and amounts of lipids, carbohydrates, proteins, vitamins and minerals to be a subject's sole source of nutrition. In one embodiment, the nutritional composition is a children's nutritional product. In another embodiment, the nutritional composition comprises an infant formula. In yet another embodiment, the nutritional composition comprises a nutritionally complete infant formula. In still another embodiment, the nutritional composition comprises a nutritionally complete children's nutritional product.
The disclosed nutritional composition may be provided in any form known in the art, such as a powder, a gel, a suspension, a paste, a solid, a liquid, a liquid concentrate, reconstituteable powdered milk substitute, or a ready-to-use product. The nutritional composition may, in certain embodiments, comprise a nutritional supplement, children's nutritional product, infant formula, human milk fortifier, growing up milk or any other nutritional composition designed for a pediatric subject. Nutritional compositions of the present disclosure include, for example, orally-ingestible, health-promoting substances including, for example, chewable foods, beverages, tablets, capsules and powders. The nutritional composition of the present disclosure may be standardized to a specific caloric content, it may be provided as a ready-to-use product, or it may be provided in a concentrated form.
The nutritional composition of the present disclosure can be prepared using any method known in the art. In one embodiment, PDX is provided in powder form. It can be mixed with water and other ingredients in a mixing tank. If GOS and/or LOS are included in the composition, they can be provided in powdered or liquid form. The mixture can then be pasteurized, homogenized and spray-dried to make a finished powder or canned and retorted to make a liquid product.
Furthermore, the prebiotic component can be administered as a nutritional supplement. For example, GOS can be ingested in the form of a pill, tablet, capsule, caplet, powder, liquid or gel. In this embodiment, the GOS can be ingested in combination with other nutrient supplements, such as vitamins, or in combination with a LCPUFA supplement, such as DHA or ARA.
In another embodiment, GOS can be provided in a form suitable for infants selected from the group consisting of follow-on formula, beverage, milk, yogurt, fruit juice, fruit-based drink, chewable tablet, cookie, cracker, or a combination thereof.
In a method of the present disclosure, the subject is a formula-fed infant. In one embodiment the subject is formula-fed from birth. In another embodiment, the subject is breast-fed from birth until an age which is less than one year, and is formula-fed thereafter, at which time PDX supplementation begins.
In embodiments providing a children's nutritional product, one or more vitamins and/or minerals may be added in amounts sufficient to supply the daily nutritional requirements of children between one and thirteen years old. It is to be understood by one of ordinary skill in the art that vitamin and mineral requirements will vary for children between the ages of one and thirteen years. Thus, the embodiments are not intended to limit the nutritional composition to a particular age group but, rather, to provide a range applicable to children between the one and thirteen years old.
In embodiments providing a nutritional composition for a child, the composition may optionally include, but is not limited to, one or more of the following vitamins or derivations thereof: vitamin B1 (thiamin, thiamin pyrophosphate, TPP, thiamin triphosphate, TTP, thiamin hydrochloride, thiamin mononitrate), vitamin B2 (riboflavin, flavin mononucleotide, FMN, flavin adenine dinucleotide, FAD, lactoflavin, ovoflavin), vitamin B3 (niacin, nicotinic acid, nicotinamide, niacinamide, nicotinamide adenine dinucleotide, NAD, nicotinic acid mononucleotide, NicMN, pyridine-3-carboxylic acid), vitamin B3-precursor tryptophan, vitamin B6 (pyridoxine, pyridoxal, pyridoxamine, pyridoxine hydrochloride), pantothenic acid (pantothenate, panthenol), folate (folic acid, folacin, pteroylglutamic acid), vitamin B12 (cobalamin, methylcobalamin, deoxyadenosylcobalamin, cyanocobalamin, hydroxycobalamin, adenosylobalamin), biotin, vitamin C (ascorbic acid), vitamin A (retinol, retinyl acetate, retinyl palmitate, retinyl esters with other long-chain fatty acids, retinal, retinoic acid, retinol esters), vitamin D (calciferol, cholecalciferol, vitamin D3, 1,25,-dihydroxyvitamin D), vitamin E (α-tocopherol, α-tocopherol acetate, α-tocopherol succinate, α-tocopherol nicotinate, γ-tocopherol), vitamin K (vitamin K1, phylloquinone, naphthoquinone, vitamin K2, menaquinone-7, vitamin K3, menaquinone-4, mnenadione, menaquinone-8, menaquinone-8H, menaquinone-9, menaquinone-9H, menaquinone-10, menaquinone-11, menaquinone-12, menaquinone-13), choline, inositol, β-carotene and any combinations thereof.
In embodiments providing a children's nutritional product, the composition may optionally include, but is not limited to, one or 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, copper sulfate, copper gluconate, cupric sulfate, fluoride, iron, carbonyl iron, ferric iron, ferrous fumarate, ferric orthophosphate, iron trituration, 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, sodium molybdate, zinc, zinc oxide, zinc sulfate and mixtures thereof. Non-limiting exemplary derivatives of mineral compounds include salts, alkaline salts, esters and chelates of any mineral compound.
The minerals can be added to children's nutritional compositions in the form of salts such as calcium phosphate, calcium glycerol phosphate, sodium citrate, potassium chloride, potassium phosphate, magnesium phosphate, ferrous sulfate, zinc sulfate, cupric sulfate, manganese sulfate, and sodium selenite. Additional vitamins and minerals can be added as known within the art.
In an embodiment, the children's nutritional composition may contain between about 10 and about 50% of the maximum dietary recommendation for any given country, or between about 10 and about 50% 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 nutritional composition may supply about 10-30% of the maximum dietary recommendation for any given country, or about 10-30% of the average dietary recommendation for a group of countries, per serving of B-vitamins. In yet another embodiment, the levels of vitamin D, calcium, magnesium, phosphorus, and potassium in the children's nutritional product may correspond with the average levels found in milk. In other embodiments, other nutrients in the children's nutritional composition may be present at about 20% of the maximum dietary recommendation for any given country, or about 20% of the average dietary recommendation for a group of countries, per serving.
The children's nutritional composition of the present disclosure may optionally include one or more of the following flavoring agents, including, but not limited to, flavored extracts, volatile oils, cocoa or chocolate flavorings, peanut butter flavoring, cookie crumbs, vanilla or any commercially available flavoring. Examples of useful flavorings include, but are not limited to, pure anise extract, imitation banana extract, imitation cherry extract, chocolate extract, pure lemon extract, pure orange extract, pure peppermint extract, honey, imitation pineapple extract, imitation rum extract, imitation strawberry extract, or vanilla 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, vanilla cookie crumb, butterscotch, toffee, and mixtures thereof. The amounts of flavoring agent can vary greatly depending upon the flavoring agent used. The type and amount of flavoring agent can be selected as is known in the art.
The nutritional compositions of the present disclosure may optionally include one or more emulsifiers that may be added for stability of the final product. Examples of suitable emulsifiers include, but are not limited to, lecithin (e.g., from egg or soy), alpha lactalbumin and/or mono- and di-glycerides, and mixtures thereof. Other emulsifiers are readily apparent to the skilled artisan and selection of suitable emulsifier(s) will depend, in part, upon the formulation and final product.
The nutritional compositions of the present disclosure may optionally include one or more preservatives that may also be added to extend product shelf life. Suitable preservatives include, but are not limited to, potassium sorbate, sodium sorbate, potassium benzoate, sodium benzoate, calcium disodium EDTA, and mixtures thereof.
The nutritional compositions of the present disclosure may optionally include one or more stabilizers. Suitable stabilizers for use in practicing the nutritional composition of the present disclosure include, but are not limited to, gum arabic, gum ghatti, gum karaya, gum tragacanth, agar, furcellaran, guar gum, gellan gum, locust bean gum, pectin, low methoxyl pectin, gelatin, microcrystalline cellulose, CMC (sodium carboxymethylcellulose), methylcellulose hydroxypropyl methyl cellulose, hydroxypropyl cellulose, DATEM (diacetyl tartaric acid esters of mono- and diglycerides), dextran, carrageenans, and mixtures thereof.
All percentages, parts and ratios as used herein are by weight of the total formulation, unless otherwise specified.
The nutritional composition of the present disclosure may be substantially free of any optional or selected ingredients described herein, provided that the remaining nutritional composition still contains all of the required ingredients or features described herein In this context, and unless otherwise specified, the term “substantially free” means that the selected composition may contain less than a functional amount of the optional ingredient, typically less than 0.1% by weight, and also, including zero percent by weight of such optional or selected ingredient.
All references to singular characteristics or limitations of the present disclosure shall include the corresponding plural characteristic or limitation, and vice versa, unless otherwise specified or clearly implied to the contrary by the context in which the reference is made.
All combinations of method or process steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.
The methods and compositions of the present disclosure, including components thereof, can comprise, consist of, or consist essentially of the essential elements and limitations of the embodiments described herein, as well as any additional or optional ingredients, components or limitations described herein or otherwise useful in nutritional compositions.
As used herein, the term “about” should be construed to refer to both of the numbers specified in any range. Any reference to a range should be considered as providing support for any subset within that range.
The following examples are provided to illustrate some embodiments of the nutritional composition of the present disclosure but should not be interpreted as any limitation thereon. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from the consideration of the specification or practice of the nutritional composition or methods disclosed herein. It is intended that the specification, together with the example, be considered to be exemplary only, with the scope and spirit of the disclosure being indicated by the claims which follow the example.
This example describes the experimental procedures and methods used to assess the ability of galactooligosaccharide, of polydextrose and of a combination of galactooligosaccharide and polydextrose to inhibit adherence of the pathogens to human cells.
Five C. sakazakii strains (Table 1) were obtained. In some experiments, the enteropathogenic E. coli E2348/69 was also used. Prior to each experiment, freezer stocks of each organism were plated onto Tryptic Soy Agar (TSA; Difco) and grown overnight at 37° C. A single colony was inoculated in 10 ml of Tryptic Soy Broth (TSB; Difco) and incubated aerobically, without shaking. Preliminary experiments revealed that adherence rates were highest when cells were in late log phase. Therefore, after 6 hour incubation, cultures were harvested by centrifugation (3,184×g for 8 minutes). The cells were washed once with phosphate-buffered saline (PBS) and re-suspended in minimal essential medium (MEM; Hyclone, Logan, Utah) supplemented with 10% fetal bovine serum (FBS; Hyclone). Minimal essential medium was pre-equilibrated at tissue culture conditions (5% CO2, 95% relative humidity, 37° C.). Based on additional preliminary experiments, two strains (4603 and 29004) repeatedly gave the highest adherence rates on HEp-2 cells and were used for all subsequent experiments.
Enterobacter sakazakii 4593
Enterobacter sakazakii 4603
Enterobacter sakazakii 29004
Enterobacter sakazakii 4583
Enterobacter sakazakii ES 415
HEp-2 cells were obtained from the American Type Culture Collection. Cells were grown in 75 cm2 tissue culture flasks containing 25 ml of MEM supplemented with 10% FBS in a CO2 incubator at tissue culture conditions. Confluent HEp-2 cells were harvested by adding 0.5 ml of 0.25% Trypsin-EDTA Solution and incubating for 15 minutes at tissue culture conditions. Trypsin was inactivated with 0.5 ml of FBS. Cells were then seeded onto 12-mm diameter glass cover slips in 24-well tissue culture plates at approximately 3.6×105 viable cells per well, and 500 μl of MEM supplemented with 10% FBS was added to each well. Plates were incubated under tissue culture conditions for two days prior to the start of each experiment, or until confluence was reached. Media was replaced one day before the experiment.
Galactooligosaccharide was obtained as a concentrated syrup and was lyophilized to give a final product of 95% total solids. The galactooligosaccharide and polydextrose were then prepared as concentrated stock solutions with distilled sterile water at a final concentration of 160 mg/ml. The solutions were filter sterilized by passage though 0.22 μm filters. A component of PDX and GOS was prepared by mixing equal volumes to give 80 mg/ml of PDX and 80 mg/ml of GOS in the component stock solution. Experiments using GOS, PDX, and a combination of GOS and PDX were also performed with E. coli E2348/69.
Cell suspensions of C. sakazakii 4603 and ATCC 29004 were prepared as described above. Prebiotics were mixed with bacterial cultures (in MEM supplemented with 10% FBS) prior to addition to the HEp-2 and CaCo-2. The prebiotic solutions represented 10% of the final volume of the suspension, giving a final concentration of 16 mg/ml of prebiotic in the assay mixtures. The plates were incubated for 3 hours in the CO2 incubator at tissue culture conditions. Preliminary experiments indicated that 3 hours of incubation was optimal for adherence of the bacterial strains to the HEp-2 tissue culture and to the CaCo-2 tissue culture. The wells were then washed 5 times with PBS to remove non-adhered bacterial cells, All HEp-2 experiments were done in duplicate and replicated 5 times. CaCo-2 experiments were done in duplicate and replicated twice.
For microscopic enumeration, after washing five times with PBS, cells were fixed with 100% methanol for 20 minutes and stained with 10% Geimsa for 15 minutes. They were then washed once with sterile distilled water and dried overnight at room temperature. Cover slips were mounted on microscope slides and observed under a phase contrast microscope with the 100× objective. Fifteen pictures of each cover slip were taken using Motic Image software following an established pattern throughout the entire cover slip. The number of cells and bacteria in each picture were counted using Image J image analysis software. Adherence was calculated as the number of adhered bacteria per tissue culture. Adherence inhibition was calculated as the number of adhered bacteria per cell in the control minus the number of adhered bacteria per cell in the treatment divided by the number of adhered bacteria per cell in the control.
For cultural enumeration, after washing 5 times with PBS, cells were detached by addition of 0.1% Triton X-100 for 30 minutes at room temperature. The cells were collected, and serial dilutions were plated on agar. Plates were incubated aerobically at 37° C. for 24 hours. Adherence inhibition was calculated as the number of adhered bacteria per ml in the control minus the number of adhered bacteria per ml in the treatment divided by the number of adhered bacteria per ml in the control.
This example describes the experimental results demonstrating the ability of galactooligosaccharide, polydextrose, and a component of galactooligosaccharide and polydextrose to inhibit adherence of pathogens to human cell lines.
Adherence of C. sakazakii 4603 to HEp-2 cells in the presence and absence of prebiotics was measured by microscopic and culture methods.
1Final concentration = 16 mg/ml
2*= significant at p < 0.05 (n = 10)
Likewise, by cultural enumeration, GOS addition led to a 71% reduction of binding by C. sakazakii 4603 to the HEp-2 cells (Table 3). Similarly, a 49% reduction occurred in the presence of the GOS-PDX component. PDX alone led to a 55% reduction in adherence.
1Final concentration = 16 mg/ml
2*= significant at p < 0.05 (n = 10)
Strain ATCC 29004 gave similar results, with reductions of 63% 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 when determined by cultural enumeration, as GOS led to an 83% adherence inhibition, GOS-PDX led to an 80% inhibition and PDX led to a 58% adherence inhibition (fable 5).
1Final concentration = 16 mg/ml
2*= signfficant at p < 0.05 (n = 10)
1Final concentration = 16 mg/ml
2significant at p < 0.05 (n = 10)
The cultural enumeration results confirmed those observed by microscopic examination for both strains (
To confirm that the GOS inhibited adhesion of pathogens, adherence inhibition experiments with E. coli E2348/69 and Hep-2 tissue cultures were performed. The results (
Moreover,
This example illustrates one embodiment of a nutritional product according to the present disclosure.
LOS is generated when lactose is heated at a high temperature. Therefore, in this embodiment the product contains indigenous LOS. The level of indigenous LOS in the product is approximately 2 g/L.
This example illustrates one embodiment of a nutritional product according to the present disclosure.
LOS is generated when lactose is heated at a high temperature. Therefore, in this embodiment the product contains indigenous LOS. The level of indigenous LOS in the product is approximately 2.6 g/L.
This example illustrates one embodiment of a nutritional product according to the present disclosure.
LOS is generated when lactose is heated at a high temperature. Therefore, in this embodiment the product contains indigenous LOS. The level of indigenous LOS in the product is approximately 3.6 g/L.
All references cited in this specification, including without limitation, all papers, publications, patents, patent applications, presentations, texts, reports, manuscripts, brochures, books, internet postings, journal articles, periodicals, and the like, are hereby incorporated by reference into this specification in their entireties. The discussion of the references herein is intended merely to summarize the assertions made by their authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.
Although embodiments of the disclosure have been described using specific terms, devices, and methods, such description is for illustrative purposes only. The words used are words of description rather than of limitation. It is to be understood that changes and variations may be made by those of ordinary skill in the art without departing from the spirit or the scope of the present disclosure, which is set forth in the following claims. In addition, it should be understood that aspects of the various embodiments may be interchanged in whole or in part. For example, while methods for the production of a commercially sterile liquid nutritional supplement made according to those methods have been exemplified, other uses are contemplated. Therefore, the appended claims should not be limited to the description of the versions contained therein.
This application is a continuation-in-part of commonly assigned U.S. patent application Ser. No. 12/370,141, entitled “Method for Simulating the Functional Attributes of Human Milk Oligosaccharides in Formula-fed Infants,” which was filled on Feb. 12, 2009 in the name of Bryon W. Petschow, which, in turn, claims priority 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 disclosures of each are incorporated by reference herein in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12843610 | Jul 2010 | US |
| Child | 13854370 | US |
