Use of polydextrose for simulating the functional attributes of human milk oligosaccharides in formula-fed infants

[0001] The present invention relates to the use of polydextrose to simulate the functional properties of human milk oligosaccharides.

(2) Description of the Related Art [0002] The intestinal microflora of infants forms rapidly within the first few weeks after birth. The nature of this intestinal colonization is initially determined by early exposure to sources of microorganisms in the environment, as well as the health of the infant. A strong effect on the intestinal bacterial population is whether the infant is breastfed. breast-fed] or a mixture [ang. formula fed].

[0003] In breast-fed infants, for example, Bifidobacterium spp. Predominates among intestinal bacteria, and the share of Streptococcus spp. And Lactobacillus spp. Is lower. In contrast, the microflora of formula-fed infants is more diverse, contains Bifidobacterium spp. And Bacteroides spp., And more pathogenic species, such as Staphylococcus, Escherichia coli and Clostridia. The different species of Bifidobacterium in the faeces of breastfed and formula-fed infants are also different.

[0004] Bifidobacteria are generally considered to be "beneficial" bacteria and are known to protect against colonization by pathogenic bacteria. This is presumably by competing for cell surface receptors, competing for essential nutrients, producing antibacterial agents and producing inhibitory compounds such as short chain fatty acids (SCFAs) that can lower fecal pH and inhibit potentially pathogenic bacteria. Bifidobacteria are also associated with intestinal resistance to gastrointestinal (GI) and respiratory tract infections, and to increased immune function in children and infants. Therefore, promoting the intestinal environment in which Bifidobacteria dominate has become a goal in developing nutritional formulas for formula-fed infants.

[0005] Human milk (HM) contains a number of factors that may contribute to the growth and population of Bifidobacteria in the intestinal microflora of infants. Among these factors is a complex mixture of over 130 different oligosaccharides reaching levels as high as 8-12 g / l in transient 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 reach the colon intact, where they serve as substrates for fermentation in the colon.

[0006] Oligosaccharides from human milk are believed to cause an increase in the number of Bifidobacteria in the colonic flora 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: 699722 (2000); Newburg, Do the Binding Properties of Oligosaccharides in Milk Protect Human Infants from

Gastrointestinal Bacteria ?, J. Nutr. 217: S980-S984 (1997). The way in which HM oligosaccharides can increase the number of Bifidobacteria and reduce the number of potentially pathogenic bacteria is to act as competitive receptors and to inhibit 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). [0007] In addition to reducing the number of pathogenic bacteria and enhancing the Bifidobacteria population during the fermentation of oligosaccharides from human milk, they produce short chain fatty acids (SCFAs) such as acetic, propionic and butyric acids. It is believed that these short chain fatty acids contribute to caloric content, serve as the main source of energy for the intestinal epithelium, stimulate the absorption of sodium and water in the colon, and increase digestion and absorption in the small intestine. In addition, short chain fatty acids are thought to affect the overall health of the gastrointestinal tract by modulating gastrointestinal development and immune function.

[0008] Fermentation of human milk oligosaccharides also reduces fecal concentrations of ammonia, amine and phenol, which are considered to be the main components responsible for the smell of feces. Cummings & Macfarlane, The Control and Consequences of Bacterial Fermentation in the Human Colon, J. Appl. Bacteriol. 70: 443-459 (1991); Miner & Hazen, Ammonia and Amines: Components of Swine-Building Odor ASAE 12: 772-774 (1969); Spoelstra, Origin of Objectionable Components in Piggery Wastes and the Possibility of Applying Indicator Components for Studying Odour Development, Agric. Environ.

5: 241-260 (1980); O'Neill & Phillips, A Review of the Control of Odor Nuisance from Livestock Buildings: Part 3. Properties of the Odorous Substances which have been Identified in Livestock Wastes or in the Air Around them J. Agric. Eng. Res. 53: 23-50 (1992).

[0009] Due to the presence of oligosaccharides in human milk, the profile of short chain fatty acids of a breast-fed infant is very different from that of a formula-fed infant. For example, breast-fed infants produce virtually no butyrate, and acetate accounts for approximately 96% of total short chain fatty acid production. Lifschitz, et al., Characterization of Carbohydrate Fermentation in Feces of Formula-Fed and BreastFed Infants, Pediatr. Res. 27: 165-169 (1990); Siigur, et al., Faecal Short-Chain Fatty Acids in Breast-Fed and Bottle-Fed Infants. Acta. Paediatr. 82: 536-538 (1993); Edwards, et al., Faecal Short-Chain Fatty Acids in Breast-Fed and Formula-Fed Babies, Acta. Paediatr. 72: 459-462 (1994); Parrett & Edwards, In Vitro Fermentation of Carbohydrates by Breast Fed and Formula Fed Infants, Arch. Dis. Child 76: 249-253 (1997). However, when babies are fed the formula, they also have acetate (74%) as the main short-chain fatty acid in their faeces in addition to significant amounts of propionate (23%) and small amounts of butyrate (3%). These differences between the short-chain fatty acid profiles of breast-fed and formula-fed infants may affect the energy, digestion and overall health of the formula-fed infant.

bacteria in the colon, host ". Gibson, GR [0010] Because cow's milk and commercially available infant formula based on cow's milk only provide trace amounts of oligosaccharides, prebiotics are often used to supplement the diet of formula-fed infants. Prebiotics are defined as "non-digestible food ingredients that favorably affect the host by selectively stimulating the growth and / or activity of one or a limited number that can improve health & Roberfroid, MB, Dietary

Modulation of the Human Colonic Microbiota-Introducing the Concept of Probiotics, J. Nutr. 125: 1401-1412 (1995). Common prebiotics include fructooligosaccharides, glucooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, xylooligosaccharides and lactulose.

[0011] The incorporation of various prebiotic ingredients into infant formulas has been disclosed. Patent application Ser. No. 20030072865 to Bindels et al. Discloses, for example, an infant formula with a higher protein content and at least one prebiotic. The prebiotic component may be lacto-N-tetraose, lacto-N-fucopentose, lactulose (LOS), lactosaccharose, raffinose, galacto-oligosaccharide (GOS), fructo-oligosaccharide (FOS), oligosaccharides, polysaccharides, isosaccharides, soybean saccharides, , glucans, sialyl oligosaccharides and fucooligosaccharides.

[0012] Similarly, the patent application St. Ser. No. 20040191234 to Haschke discloses a method of increasing the immune response comprising administering at least one prebiotic. This prebiotic may be an oligosaccharide made from glucose, galactose, xylose, maltose, sucrose, lactose, starch, xylan, hemicellulose, inulin or mixtures thereof. The prebiotic may be present in infant cereal flakes.

[0013] Unfortunately, however, the administration of the above prebiotics to formula-fed infants has many disadvantages. Although they may favorably affect the probiotic population in the intestine, they do not produce a short chain fatty acid profile similar to that of breast-fed infants. In addition, the fermentation of many of these prebiotic substances is very fast, which often produces excess gas, enlargement of the abdomen, bloating and diarrhea. Therefore, the choice of prebiotic substances in infant formula should be aimed at maximizing potential benefits and minimizing such undesirable side effects.

[0014] Thus, it would be beneficial to administer a prebiotic substance simulating the functional properties of human milk oligosaccharides in infants, such as an increase in the population and species of beneficial bacteria in the infant's intestine, and the production of a short chain fatty acid profile similar to that of breast-fed infants. In addition, infants should be well tolerated of a prebiotic substance that should not produce or cause excess gas, abdominal enlargement, bloating or diarrhea.

SUMMARY OF THE INVENTION [0015] The present invention is defined by the appended claims.

[0016] Briefly, therefore, the present invention first indicates a new use of polydextrose (PDX) in the manufacture of an infant formula for simulating the functional properties of oligosaccharides from human milk by increasing acetate production and reducing butyrate production, to obtain a short chain fatty acid profile in formula-fed infants similar to that in breast-fed infants and by reducing the rate and extent of prebiotic fermentation in the intestine of formula-fed infants.

[0017] The present invention also indicates an infant formula containing PDX and GOS, wherein the total amount of PDX and GOS in the infant formula is between 1.0 g / L and 10.0 g / L, administered daily.

[0018] PDX can be used in the manufacture of infant formula for increasing the population and species of beneficial bacteria in formula-fed infants.

[0019] PDX can be used in the manufacture of an infant formula for producing a short chain fatty acid (SCFA) profile in formula-fed infants similar to that of breast-fed infants. In particular, PDX may result in increased acetate and reduced butyrate in the short chain fatty acid profile.

[0020] PDX can be used in the manufacture of infant formula to reduce the rate and degree of fermentation of prebiotics in the intestine of a formula-fed infant.

More specifically, the invention reduces total gas production and carbon dioxide production in the infant's intestine.

[0021] Some of the benefits found by the present invention include good infant tolerance and simulation of the functional properties of human milk oligosaccharides, such as the increased population and species of beneficial bacteria in the infant's intestine, optimization of faecal characteristics and production of a short chain acid profile fat profile similar to breast-fed infants.

BRIEF DESCRIPTION OF THE DRAWINGS [0022] For a more complete understanding of the present invention, reference is now made to the following descriptions, taken together with the accompanying drawings.

[0023] Figure 1 illustrates the production of total short chain fatty acids during fermentation of GOS, LOS, PDX2 and FOS.

[0024] Figure 2 illustrates changes in pH during fermentation of GOS, LOS, PDX2 and FOS.

[0025] Figure 3 illustrates the relative proportion of acetic acid production in the fermentation of GOS, LOS, PDX2 and FOS.

[0026] Figure 4 illustrates the relative proportion of propionic acid production in the fermentation of GOS, LOS, PDX2 and FOS.

[0027] Figure 5 illustrates the relative share of butyric acid production in the fermentation of GOS, LOS, PDX2 and FOS.

[0028] Figure 6 illustrates the relative proportions of acetic, propionic, butyric acids and the production of total short chain fatty acids in fermentation

GOS

LOS, PDX2 and FOS.

[0029] Figure 7 illustrates the production of total short chain fatty acids during the fermentation of various combinations of prebiotic carbohydrates.

[0030] Figure 8 illustrates the pH changes during fermentation of various combinations of prebiotic carbohydrates.

[0031] Figure 9 illustrates the production of total short chain fatty acids during the fermentation of various combinations of PDX and GOS.

[0032] Figure 10 illustrates the concentration of acetic acid produced during fermentation of various PDX and combinations

GOS.

[0033] Figure 11 produced during GOS.

[0034] Figure 12 produced during illustrates different fermentation concentrations illustrates the fermentation concentration of different propionic acid combinations of PDX and butyric acid combinations of PDX and

GOS.

[0035] Figure 13 illustrates gas production as total volume during fermentation of GOS, LOS, PDX2 and FOS.

[0036] Figure 14 illustrates gas production as a concentration of carbon dioxide during fermentation of GOS, LOS, PDX2 and FOS. [0037] Figure 15 illustrates the production of gases as the hydrogen concentration during the fermentation of GOS, LOS, PDX2 and FOS.

[0038] Figure 16 illustrates the production of gases as the concentration of hydrogen disulphide during fermentation of GOS, LOS, PDX2 and FOS.

[0039] Figure 17 is a summary of the prebiotic effects of human milk, FOS, LOS, GOS, PDX and various combinations thereof on fecal microflora.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0040] Embodiments of the invention will now be described in detail, one or several examples of which are given below. Each example is given as an explanation of the invention and not as a limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope of this invention. For example, features illustrated or described as part of an embodiment may be used in another embodiment to provide the next embodiment.

[0041] Thus, it is intended that the present invention include such modifications and changes as fall within the scope of the appended claims. Other objects, features and aspects of the present invention are disclosed or are apparent from the detailed description below. Those skilled in the art should be aware that this discussion is only a description of an exemplary embodiment and is not intended to limit the broader aspects of the present invention.

Definitions [0042] As used herein, the term "prebiotic" means an undigested nutrient that beneficially affects the host by selectively stimulating the growth and / or activity of one or a limited number of bacteria in the colon that may improve host health.

[0043] The term "probiotic" means a microorganism with low or no pathogenicity that has beneficial effects on host health.

[0044] The term "infant" as used herein means a human being less than about one year old. "Therapeutically effective amount", used in the amount providing application herein, means a prebiotic effect in a subject.

[0046] The term "simulating", as used herein, means having or taking the form or appearance of a symptomatic similarity or having or producing a symptomatic similarity.

[0047] The terms "functional properties" mean inherent property or characteristics that cause something to happen. Examples of the functional properties of human milk oligosaccharides in the present invention include an increase in the population and species of beneficial bacteria, the production of a short chain fatty acid profile with high acetic acid and low butyric acid content, and a low rate and low fermentation of prebiotics in the intestine.

[0048] The term "infant formula" as used herein means a composition that meets the nutritional requirements of an infant because it is a substitute for human milk. In the United States, the content of the infant formula is determined by federal regulations in chapters 100, 106 and 107 of document 21 of CRF These recipes define the amounts of macronutrient, vitamin, mineral and other ingredients to stimulate the nutritional and other properties of human breast milk.

Invention [0049] In accordance with the present invention, a new use of PDX in the production of infant formula simulating the functional properties of oligosaccharides from human milk in formula-fed infants has been discovered. The administration of PDX provides a beneficial effect on the population and species of probiotics, produces a short chain fatty acid profile that is similar to the profile of breast-fed infants and is physically well tolerated by infants.

[0050] PDX is an undigested carbohydrate that was synthesized from randomly cross-linked glucose and sorbitol. It is not digested in the upper gastrointestinal tract and is only partially fermented in the lower gastrointestinal tract, which makes it a beneficial ingredient for the health of the digestive system.

The physiological benefits associated with PDX include increased fecal mass, reduced transit time, lower fecal pH, and reduced concentration of putrefactive substances in the colon. Adults have also been shown to take PDX to help promote and grow beneficial bacteria in the gut and to produce short chain fatty acids.

[0051] PDX has been identified as a prebiotic substance for adults based on its activity in the gastrointestinal tract. Patent application Ser. No. 20040062758 to Mayr-Makinen et al. Relates, for example, to a composition that includes a probiotic and one or more prebiotics, wherein the prebiotic may be GOS, palatinozooligosaccharide, soybean oligosaccharide, geno-oligosaccharide, xylooligomers, non-degraded starch, lactosacrose, lactose lactose PDX. Similarly, the patent of Ser. No. 4,859,488 to Kana relates to liquid food containing PDX and oligosaccharides that is useful in the treatment of constipation.

[0052] PDX, however, has not been identified as a prebiotic that provides the benefits of the present invention and can be administered to infants. It is well known that the intestinal microflora of infants is far less developed than it is in the case of an adult. While the adult human microflora consists of more than 10¹³ of nearly 500 species of microorganisms, the infant's microflora contains only a fraction of these microorganisms relative to the unnamed number [ang. absolute number] as well as species diversity. Because bacterial populations and species differ so greatly between the infant gut and the adult intestine, it cannot be assumed that a prebiotic substance that has beneficial effects in adults will also have beneficial effects in infants.

[0053] In adults, PDX intake has been shown to increase acetate and butyrate production. Since butyrate is not present in significant amounts in breast-fed infants and has been associated with harmful effects if it was produced in significant quantities in the infant's intestine, PDX is not generally considered to be an appropriate ingredient in the infant's diet based on the observed gastrointestinal effects . So amazing and unexpected was that

PDX was actually metabolized mainly to acetate and propionate, with low butyrate formation. Thus, PDX not only showed a positive effect on the population and species of beneficial bacteria in the baby's digestive tract, but PDX also produced a short chain fatty acid profile that was very similar to the profile of breast-fed infants and would be extremely well tolerated by infants.

[0054] One particular reference regarding PDX in the context of administration to infants actually indicates the opposite of the present invention. In the patent application Ser. No. 20030157146 to Rautonen, it is claimed that PDX can stimulate the infant's immune system. In this application, however, the Applicant discloses that PDX actually reduces the Bifidobacteria population in the infant's intestine (Rautonen application, paragraph 0074). The applicant justifies this result by stating that "a large amount of Bifidobacteria may also cause less desirable physiological effects such as intestinal bacterial diseases and immunosuppression."

(Report by Rautonen, paragraph 0069).

[0055] Since the reference indicates that PDX actually reduces the Bifidobacteria population in the infant's intestine, this is in direct conflict with the teaching of the present application. In addition, the reference does not show that PDX increases acetate production, reduces butyrate production or fermentation of prebiotics in the intestine, and reduces the infant's speed.

[0056] According to the use of the present invention, the infant should be administered a therapeutically effective amount of PDX to simulate the functional properties of human milk oligosaccharides. A therapeutically effective amount of PDX may be between 1.0 g / L and 10.0 g / L, for daily administration. In another embodiment, the therapeutically effective amount of PDX may be between 2.0 g / L and 8.0 g / L, for daily administration.

[0057] PDX is commercially available from many sources. STA-LITE® PDX, for example, is available in 5-pound bags from Honeyville Grain, Inc., located in Salt Lake City, UT. Alternatively, Litesse® Ultra ™ PDX is commercially available from Danisco Sweeteners, Ltd., located in the United Kingdom.

[0058] PDX is well suited for inclusion in an infant formula as it contains only 1 inch / g, compared to 4 inch / g for typical prebiotics. It is also well soluble and neutral in taste. Therefore, its addition to an infant formula would not change the physical or taste characteristics of the composition.

[0059] In accordance with the use of the invention, PDX is added to the infant formula which is then to be fed to the infant.

[0060] The infant formula for use in the present invention is preferably nutritively complete and usually contains the appropriate types and amounts of lipids, carbohydrates, proteins, vitamins and minerals. The amount of lipids or fats can usually be from about 3 to about 7 g / 100 kcal. The amount of protein can usually be from about 1 to about 5 g / 100 kcal. The amount of carbohydrate usually can be from about 8 to about 12 g / 100 kcal. Protein sources may be any used in the art, for example, nonfat milk, whey protein, casein, casein protein, soy protein, hydrolyzed protein, amino acids and the like. Carbohydrate sources may be any used in the art, for example lactose, glucose, corn syrup powder, maltodextrin, sucrose, starch, rice syrup powder and the like. Lipid sources may be any used in the art, for example, vegetable oils such as palm oil, soybean oil, palm olein, coconut oil, medium chain triglyceride oil, highly oleic sunflower oil, highly oleic safflower oil and the like.

[0061] Conveniently, commercially available infant formulas may be used. Enfalac, Enfamil®, a mixture for premature babies, Enfamil®, Enfamil® with iron, Lactofree®, Nutramigen®, Pregestimil® or ProSobee® (available from Mead Johnson & Company, Evansville, IN, USA), for example, you can supplement with the right amounts of PDX and put this invention into practice.

[0062] In an embodiment of the invention, PDX should be administered with a prebiotic. The selected prebiotic is a prebiotic known in the art. Examples of prebiotics include, but are not limited to: FOS, inulin, glucooligosaccharide, GOS, isomalto-oligosaccharide, xylooligosaccharide, soybean oligosaccharides, chitooligosaccharide, gento-oligosaccharide, manno-oligosaccharide, glucosaccharosaccharosaccharosaccharose

the use of the present in combination with another can be any. [0063] In a particular embodiment of the use of the present invention, PDX should be administered in combination with GOS. GOS is a mixture of oligosaccharides consisting of Dglucose and D-galactose. Sometimes referred to as transgalacto-oligosaccharide. It is made from D-lactose by β-galactosidase, which can be obtained from Aspergillus oryzae. GOS has been suggested to increase calcium absorption and prevent bone atrophy in adults. GOS has been identified as a prebiotic that is useful for administration to infants in US Patent Application Ser. No. 20030072865 to Bindels and others.

[0064] In this embodiment of the use of the present invention, PDX and GOS should be administered in a PDX to GOS ratio between 9: 1 and 1: 9. In another embodiment, the PDX: GOS ratio can be between 5: 1 and 1: 5. In yet another embodiment, the PDX: GOS ratio may be between 1: 3 and 3: 1. In a particular embodiment, the PDX: GOS ratio can be about 5: 5. In another particular embodiment, the PDX: GOS ratio can be about 8: 2.

[0065] A therapeutically effective amount of the PDX: GOS combination may be between 1.0 g / L and 10.0 g / L for daily administration. In another embodiment, the therapeutically effective amount of the PDX: GOS combination may be between 2.0 g / L and 8.0 g / L for daily administration. In a particular embodiment, the therapeutically effective amount of the PDX: GOS combination may be between 2 g / L PDX and 2 g / L GOS for daily administration. [0066] In another specific embodiment of the use of the present invention, PDX should be administered in combination with LOS. LOS is a semisynthetic disaccharide formed from D19 galactose and D-fructose connected by a β-glucosidic bond. It is resistant to hydrolysis by human digestive enzymes but is fermented in the small intestine. It is well soluble and has a sweet taste. LOS has been identified as a prebiotic that is useful for administration to infants in US Patent Application Ser. No. 20030072865 to Bindels and others. LOS is commercially available from many sources.

[0067] In this embodiment of the use of the present invention, PDX and LOS should be administered in a ratio between about 9: 1 and 1: 9. In another embodiment, the PDX to LOS ratio may be between about 5: 1 and 1: 5. In yet another embodiment, the PDX to LOS ratio can be between about 3: 1 and 1: 3. In a particular embodiment, the PDX to LOS ratio can be about 5: 5. In another particular embodiment, the PDX to LOS ratio can be about 8: 2.

[0068] A therapeutically effective amount of the PDX: LOS combination may be between about 1.0 g / L and 10.0 g / L for daily administration. In another embodiment, the therapeutically effective amount of the PDX: LOS combination may be between about 2.0 g / L and 8.0 g / L for daily administration. In a particular embodiment, the therapeutically effective amount of the PDX: LOS combination can be about 2 g / L PDX and 2 g / L LOS for daily administration.

[0069] In yet another embodiment of the use of the present invention, PDX should be 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 may be about 1: 1: 1. In a particular embodiment, the PDX: GOS: LOS ratio can be about 1: 1.5: 1.

[0070] A therapeutically effective amount of the PDX: GOS: LOS combination may be between about 1.0 g / L and 10.0 g / L for daily administration. In another embodiment, the therapeutically effective amount of the PDX: GOS: LOS combination can be between about 2.0 g / L and 8.0 g / L for daily administration. In an embodiment, the therapeutically effective amount of the PDX: GOS: LOS combination may be about 2.0 g / L PDX, 2.0 g / L GOS and 2.0 g / L LOS for daily administration. In a particular embodiment, a therapeutically effective amount of the combination

PDX: GOS: LOS may be around 2 g / l PDX, 1.32 g / l GOS and 2.6 g / l LOS for daily administration. In another embodiment, the therapeutically effective amount of the PDX: GOS: LOS combination can be about 4 g / L PDX, 2.64 g / L GOS and 3.6 g / L LOS for daily administration.

[0071] In an embodiment of the invention, PDX may be combined with one or more probiotics for administration to an infant. Any probiotic known in this field is acceptable in this form of implementation. In a particular embodiment, the probiotic is selected from the group consisting of Bifidobacterium spp. Or Lactobacillus spp. In the embodiment, the probiotic is Lactobacillus rhamnosus GG (LGG). In another embodiment, the probiotic is Bifidobacterium lactis. In a specific embodiment, the probiotic is Bifidobacterium lactis Bb-12, available from Chr. Hansen Biosystems, located in Milwaukee, WI.

[0072] In other embodiments of the use of the present invention, the infant formula may contain other active agents such as long chain polyunsaturated fatty acids (LCPUFA). Suitable long chain polyunsaturated fatty acids include, but are not limited to, α-linoleic acid, γ-linoleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid (EPA), arachidonic acid (ARA) and docosahexaenoic acid (DHA). In an embodiment, PDX should be administered in combination with DHA. In another embodiment, PDX should be administered in combination with ARA. In yet another embodiment, PDX should be administered in combination with both DHA and ARA. A commercially available infant formula that contains DHA, ARA or a combination thereof can be supplemented with PDX and used in the present invention. Enfamil® LIPIL®, for example, which contains effective amounts of DHA and ARA, is commercially available and can be supplemented with LGG and used in the present invention.

[0073] In an embodiment, both DHA and ARA should be administered in combination with PDX. In this embodiment, the ARA: DHA weight ratio is usually from about 1: 3 to about 9: 1. Alternatively, the ratio may be from about 1: 2 to about 4: 1. In yet another alternative, the ratio can be from about 2: 3 to about 2: 1. In one particular embodiment, the ratio is about 2: 1.

[0074] An effective amount of DHA in an embodiment of the present invention is usually from about 3 mg per kg body weight per day to about 150 mg per kg body weight per day. In one embodiment of the invention, the amount is from about 6 mg per kg body weight per day to about 100 mg per kg body weight per day. In another embodiment, the amount is from about 10 mg per kg body weight per day to about 60 mg per kg body weight per day. In yet another embodiment, this amount is from about 15 mg per kg body weight per day to about 30 mg per kg body weight per day.

[0075] An effective amount of ARA in an embodiment of the present invention is usually from about 5 mg per kg body weight per day to about 150 mg per kg body weight per day. In one embodiment of the invention, the amount varies from about 10 mg per kg body weight per day to about 120 mg per kg body weight per day. In another embodiment, the amount varies from about 15 mg per kg body weight per day to about 90 mg per kg body weight per day. In yet another embodiment, the amount varies from about 20 mg per kg body weight per day to about 60 mg per kg body weight per day.

[0076] The amount of DHA in infant formulas for use in the present invention typically varies from about 5 mg / 100 kcal to about 80 mg / 100 kcal. In one embodiment of the present invention it ranges from about 10 mg / 100 kcal to about 50 mg / 100 kcal and in another embodiment from about 15 mg / 100 kcal to about 20 mg / 100 kcal. In a particular embodiment of the present invention, the amount of DHA is about 17 mg / 100 kcal.

[0077] The amount of ARA in infant formulas for use in the present invention typically varies from about 10 mg / 100 kcal to about 100 mg / 100 kcal. In an embodiment of the present invention, the amount of ARA varies from about 15 mg / 100 kcal to about 70 mg / 100 kcal. In another embodiment, the amount of ARA varies from about 20 mg / 100 kcal to about 40 mg / 100 kcal. In a particular embodiment of the present invention, the amount of ARA is about 34 mg / 100 kcal. [0078] The infant formula supplemented with oils containing DHA and ARA for use with the present invention may be made using standard techniques known in the art. They can be added to the blend, for example, by replacing an equivalent amount of oil, such as highly oily sunflower oil, usually present in the blend. As another example, oils containing DHA and ARA can be added to the blend by replacing an equivalent amount of the rest of the total fat blend normally present in the blend without DHA and ARA.

[0079] The source of DHA and ARA may be any source known in the art. In an embodiment of the present invention, the sources of DHA and ARA are oils obtained from unicellular organisms, referred to in patents of St. Ser. U.S. Patent No. 5,377,567; 5 550 156 and 5 397 591. The present invention is not limited to such oils, however. DHA and ARA can be in natural or refined form.

[0080] In an embodiment, the source of DHA and ARA is essentially free eicosapentaenoic acid (EPA). In one embodiment of the present invention, the infant formula contains, for example, less than about 16 mg EPA / 100 kcal; in another embodiment, less than about 10 mg EPA / 100 kcal, and in yet another embodiment, less than about 5 mg EPA / 100 kcal. One particular embodiment is essentially EPA-free. Another embodiment does not contain EPA, so that not even trace amounts of EPA are present in the mix.

[0081] The infant formula according to the present invention may be prepared using any method known in the art. In an embodiment, PDX is provided in the form of a powder. It can be mixed with water and other ingredients of baby formula in the mixing tank. If GOS and / or LOS are included in the infant formula, they can be provided in powder or liquid form. The mixture can then be pasteurized, homogenized and spray dried to produce a finished powder, or canned and transformed to produce a liquid product.

[0082] In the present invention, the infant is fed a formula. In one embodiment, the infant is fed the formula from birth. In another embodiment, the infant is breastfed from birth until less than one year, and then fed on the mixture, when PDX supplementation begins.

[0083] Human milk oligosaccharides can increase the population and species of beneficial bacteria in the gastrointestinal tract, have a short chain fatty acid profile with a high acetate content and a very low butyrate content and are slowly fermented avoiding the production of excess gas. As can be seen from the examples, administration of PDX, alone or in combination with other prebiotics, can be used to increase the population and species of beneficial bacteria in the gastrointestinal tract, can advantageously shift the production of short chain fatty acids towards greater acetate and propionate production, thereby limiting the production of butyrate and can slow down the rate of fermentation in the intestine, so that gas production is reduced while minimizing infant discomfort. Thus, administration of PDX, alone or in combination with other prebiotics, can simulate the functional properties of human milk oligosaccharides in formula-fed infants.

[0084] The following examples describe various embodiments of the present invention. Other embodiments falling within the scope of the claims contained in this application will be apparent to those skilled in the art after analyzing the description or practice of the invention disclosed in this application. It is intended that the description and examples be taken as examples only, the scope of the invention being defined by the claims following the examples. In the examples, all percentages are by weight unless otherwise indicated.

Example 1 [0085] This example illustrates the in vitro fecal fermentation model used in the present invention. The in vitro fecal fermentation model mimics the action of infant colon microbiota colon microbiota of infants]. During fermentation, carbohydrates are consumed and short chain fatty acids and gases are produced. After fermentation, the effect of prebiotics on the population and species of microorganisms present can be analyzed.

[0086] The individual carbohydrates tested were presented in Table 1.

Table 1: Individual carbohydrates

GOS: Vivinal GOS: Deb. No. 00026961 Ingredients of Borculo Domo;

received 09/17/02; purity 95.1%

LOS: Morinaga lactulose anhydride: MLC-A (F), series no

FRDL020926; Morinaga Milk Industry Co. Ltd; received 10/4/02; purity 97%

PDX: Sta-Lite III PDX: series no. DZ2K0351913; AE Staley

FOS: Raftilose P95 Fructo-oligosaccharides: series no

PCAB022B02; Raffinerie Notre-Dame / Orafti SA; received 9/6/02; purity 95.1%

PDX2: Litesse® Ultra ™ PDX: high molecular weight polymer, max. 22,000 MW; Danisco; lot no. V36020I INU: Raftiline® HP: long chain inulin DP> 23 (lot no: hptoh11oh1; Orafti BV; received October 2002;

DS 96.9%, Inulin 99.9%, Sucrose + Fructose + Glucose 0.1%).

[0087] Stool samples were taken from healthy infants at 2.513 months of age. Five experimental groups were run using different combinations of prebiotic carbohydrates in each fermentation group. The study involved twelve children in fermentation groups 1 and 2, 17 children in fermentation group 3, 19 children in fermentation group 4 and 23 children in fermentation group 5. In groups 1-3, only five children were able to give an acceptable sample. Children involved in the first fermentation were 4, 4, 4, 6, 6, 6, 8, 8, 9, 9, 9 and 10 months old, in the second fermentation at the age of 3, 4, 6, 6, 6, 7 , 8, 9, 10, 10, 12 and 13 months and in the third fermentation at the age of 2, 2.5, 3, 4, 4, 4, 4.5, 5, 5, 6, 6, 6, 9, 9, 10, 10

11 months. The children whose samples were used in fermentation were in the age group 1: 1, 6, 8, 9, 9, 9 months; Group 2: 4, 8, 10, 12, 13 months and Group 3: 2.5, 5, 6, 10, 11 months. In the 4 fermentation group, 10 children (of which one child twice) were able to give an acceptable sample. Donors to group 4 of fermentations were 2, 2.5, 4, 5, 7, 9, 9, 10, 11 and 15 months of age. In group 5 of fermentation, twelve children were able to give samples from which the four youngest donors were selected. Thus, donors were 5, 6, 6.5 and 6.5 months of age.

[0088] In vitro fecal fermentation was carried out according to the Karppinen method. Karppinen S., et al., In Vitro Fermentation of Polysaccharides of Rye, Wheat, and Oat Brans and Inulin by Human Faecal Bacteria, J. Sci. Food Agric. 80: 1469-76 (2000). [0089] In the present study, 100 mg carbohydrate samples were weighed into 50 ml bottles and hydrated using ml carbonate phosphate buffer pH 6.9. Samples were kept overnight in anaerobic conditions at 5 ° C until inoculum was formed. Faecal suspension (12.5%, weight / volume) was prepared under strict anaerobic conditions in the same buffer collecting fresh infant faeces. Eight mL of suspension was dosed into the substrate samples and the bottles were sealed in an anaerobic chamber to give a final 10% concentration of fecal suspension (weight / volume). Samples were incubated at 37 ° C for 1, 2, 4, 8 or 24 hours. Samples of 0 hours were prepared similarly in centrifuge tubes and quickly frozen using liquid nitrogen. All fermentation experiments contained fecal controls with no added carbohydrates.

[0090] Fermentation was terminated by removing the bottles from the water bath and placing them on ice; exceptionally, samples were kept at room temperature prior to gas measurement for direct sampling. The gas volume was measured and a gas sample (5 ml) was injected into the nitrogen-flushed bottle. The bottle was placed on ice after sampling. The fermentation sample was transferred to a centrifuge tube, the pH was measured and a sample (2 ml) was taken from the suspension for analysis of short chain fatty acids and quickly frozen in liquid nitrogen.

Example 2 [0091] This example illustrates the materials and methods necessary to determine the effectiveness of polydextrose as a prebiotic for formula-fed infants. In particular, this example illustrates the materials and methods necessary for the analysis of short chain fatty acids and gases.

[0092] Short chain fatty acids were extracted with (di) ethyl ether and analyzed by gas chromatography as described by Karppinen et al., Karppinen S., et al., In Vitro Fermentation of Polysaccharides of Rye, Wheat, and Oat Brans and Inulin by Human Faecal Bacteria, J. Sci. Food Agric. 80: 1469-76 (2000). Gases (hydrogen, carbon dioxide, methane, hydrogen disulfide and oxygen as quality control) were analyzed isothermally at 30 ° C by gas chromatography using a static headspace technique according to Karppinen and others.

Example 3 [0093] This example illustrates the effect of PDX on the in vitro short chain fatty acid profile produced by infant colon microbiota. Figures 1 and 2 show that the fermentation rate differs between different prebiotics. The production of total short chain fatty acids (the sum of acetic, propionic and butyric acids) is shown in Figure 1. The decrease in pH shown in Figure 2 also indicates the production of short chain fatty acids.

[0094] As can be seen from the drawings, PDX2 is a slowly fermentable carbohydrate, while FOS, GOS and LOS were rapidly and fully fermented. The fermentation rate of PDX2 was comparable to cereal dietary fibers. PDX2 fermented not only at the lowest initial rate, but also the degree of fermentation was only slightly above the value for the stool control. In contrast, the FOS fermentation rate was so high that it disappeared almost completely during the first sampling time points and produced the largest amount of short chain fatty acids among the prebiotics tested.

[0095] As shown in Figures 3-5, PDX2 fermentation leads to the highest propionate production and the lowest butyrate production after 24 hours. Acetate was still produced most of the short chain fatty acids during PDX fermentation, although the initial rate was much lower than for other substrates. The initial rate of propionate production from PDX2 was similar to that of other substrates, but higher levels were found at the end of fermentation. In contrast, fermentation of FOS, GOS and LOS showed increased concentrations of acetate and butyrate and reduced concentration of propionate. As a result, the combined relative proportion of acetate and propionate was much higher for PDX2 than for FOS, LOS or GOS. These results can also be seen in Figure 6. These results prove that PDX2 was the least butyrate producing substrate and the only substrate increasing the relative proportion of propionate.

[0096] These results are consistent with the in vitro study conducted by Wang, X. & Gibson, GR, Effects of the In Vitro Fermentation of Oligofructose and Inulin by Bacteria Growing in the Human Large Intestine, J. Appl. Bacteriol. 75: 373-380 (1993), in which faecal suspension from adult donors was used to ferment various carbohydrates. However, no increased production of propionate from PDX in vitro was demonstrated in an in vivo clinical trial with adult Chinese, Jie, Z. et al., Studies on the Effects of Polydextrose Intake on Physiological Functions in Chinese People, Am. J. Clin. Nutr. 72: 1503-09 (2000), in which three different PDX concentrations could increase butyrate and acetate levels, but not propionate. Higher butyrate production from GOS and FOS has also been shown in rats associated with human faecal flora (Djouzi, Z. et al., Compared Effects of Three Oligosacchardies on Metabolism of Intestinal Microflora in Rats Inoculated with a Human Faecal Flora, Br. J. Nutr. 78: 313-24 (1997).

Example 4 [0097] This example illustrates the effect of prebiotic combinations on the in vitro fermentation rate of infants using colon microbiota. Various combinations of prebiotic carbohydrates were selected to achieve the desired in vitro microbial fermentation rate. In this example, the combinations of substrates were compared for their fermentation rate (production of total short chain fatty acids) and pH changes as shown in Figures 7-8.

[0098] The addition of PDX to the GOS preparation slowed the fermentation rate of the combination, measured by the production of total short chain fatty acids (Fig. 7). Similarly, adding PDX to the LOS preparation slowed the fermentation rate of the combination. The addition of PDX to LOS or GOS also led to a more moderate decrease in pH as shown in Figure 8. This lower rate of acidification of the stool contents can lead to less irritation of the intestinal epithelium or anal area, increasing the infant's tolerance. The slower PDX pH lowering is consistent with the slower production of short chain fatty acids and the total in vitro fermentation rate compared to GOS and LOS. These results prove that PDX can be used to slow down the fermentation rate of PDX mixtures and traditional prebiotics such as GOS or LOS.

[0099] The effect of the PDX: GOS ratio on the production of total short chain fatty acids, acetate, propionate and butyrate was also investigated (Figures 9-12). Figure 9 shows that the PDX: GOS ratio of 8: 2 led to a lower production rate of total short chain fatty acids than the 5: 5 PDX: GOS ratio. Figure 9 confirms that a PDX: GOS ratio of 8: 2 produced less total short chain fatty acids than a 5: 5 ratio or

1: 9. Thus, these results show that a greater amount of PDX in the PDX: GOS mixture leads to a slower in vitro fermentation rate. The addition of PDX to GOS also tended to reduce the acetate and butyrate production rate, but had little effect on the overall rate and final propionate production.

Example 5 [0100] This example illustrates the effect of PDX on in vitro gas production by infant colon microbiota. The total gas production, shown in Figure 13 and measured as total volume per fermentation bottle, was approximately equal for GOS, LOS and FOS. In contrast, PDX leads to less total gas production during fermentation by the bacterial faecal microbiota of infants. The lower total gas production observed for PDX also indicates that it undergoes a slower fermentation than other prebiotics tested.

[0101] In addition to total gas production, carbon dioxide production is an important measure of infant tolerance to nutritional prebiotics. Carbon dioxide was the main gas product of all prebiotics tested. It was made in

3- and 44-76 times higher amounts than hydrogen or hydrogen disulphide, respectively.

[0102] Carbon dioxide production was generally the smallest for PDX compared to FOS, GOS and LOS (Fig. 14). Carbon dioxide was the main gas produced during FOS fermentation,

GOS and LOS, showing maximum levels between 320-380 pmol. In contrast, PDX showed much lower levels of carbon dioxide production (200 μmol). Hydrogen production from PDX by infant faecal microbiota was lower (about one third) than carbon dioxide production and significantly lower than the levels of hydrogen produced from FOS, GOS LOS (Figure 15). The formation of hydrogen disulphide from PDX was 1:44 compared to the formation of carbon dioxide and the maximum production of hydrogen disulphide was approximately the same concentration level for all prebiotics tested (Figure 16). Wang and Gibson also showed a larger share of carbon dioxide formation compared to hydrogen (1000 times) and methane (10 times). Wang, X. & Gibson,

GR, Effects of the In Vitro Fermentation of Oligofructose and Inulin by Bacteria Growing in the Human Large Intestine, J. Appl. Bacteriol. 75: 373-380 (1993). Since no methanogenesis was observed in this study, hydrogen disulfide was presumably formed from primary hydrogen. Levitt et al., Gas Metabolism in the Large Intestine, CRC Press, Boca Raton 131-154 (1995). It is possible that no hydrogen was detected due to its further metabolism to secondary gas, hydrogen disulphide, at late time points. Example 6 [0103] This example illustrates the materials and methods necessary to determine the effect of PDX on the population and species of infant colon microbiota. Briefly, the example uses the infant gut model to evaluate certain prebiotic compounds. The in vitro infant model used, based on the adult model, contained two 100 ml glass vessels connected in series, representing the proximal and distal areas of the infant's colon. The feed flow was regulated at a rate that took into account the shorter transit time in the infant's gut compared to the adult's gut. To model the in vivo pH differences within the colon, vessel 1 (V1) was controlled at pH 5.2 and vessel 2 (V2) was controlled at pH 6.7. The temperature was set to 37 ° C by means of a circulating water bath. Feed and culture vessels were magnetically mixed and maintained in an anaerobic atmosphere by the supply of oxygen-free nitrogen (15 ml / min).

[0104] After inoculating the system with an infant faecal suspension, two fermentation vats were left for up to 24 hours in batch mode. This allowed bacterial populations to achieve a balance in their new environment and increase in density. The feed flow was then turned on and the fermenter was in continuous culture mode for the rest of the experiment. The feed flow rate was set to 11.11 ml / h. In this study, the fermenters operated for 12 days, whereupon they were fed with the Enfalac baby formula (Mead Johnson Nutritionals, Evansville, IN) for 6 days and were fed with the Enfalac mixture and the added prebiotic or combination of prebiotics for a further 6 days.

[0105] Aseptically, 5 ml samples from V1 and V2 were then taken and prepared for culture-independent microbiological counting procedure for Fluorescent In Situ Hybridization (FISH) and microscopy to identify and count specific species of bacteria. The use of the FISH method allows accurate determination of the effect of prebiotics on specific bacterial populations in the proximal and distal areas of the infant's colon.

[0106] Prebiotics were added to the feeder individually or in combination at a total concentration of 7.5 g / L (0.75% w / v). The following oligosaccharides were used:

Table 2. Investigated prebiotics

<td>prebiotic</td><td>Type</td><td>Manufacturer</td>

<td>Lactulose (LOS)</td><td>Syrup</td><td>Morinaga Milk Indium. What. Ltd., Japan</td>

<td>galactooligosaccharide (GOS)</td><td>E0002 powder</td><td>delivered through Mead johnson</td>

<td>Polydextrose (PDX)</td><td>Powder 'Litesse Ultra'</td><td>Danisco</td>

<td>fructooligosaccharide</td><td>Powder Raftilose® P95</td><td>Orafti</td>

[0107] Donor infants were carefully selected, ideally at the age of 2-4 months, formula-fed (only where possible), healthy and recently not treated with antibiotics. A minimum age of 2 months was preferred as the microbiota of the infant's intestine was established.

Table 3. Donor information

<td>Code donor</td><td>Age</td><td>Feeding</td><td>Series fermentation</td>

<td>KB</td><td>16 weeks</td><td>SMA gold</td><td>F1</td>

<td>JS</td><td>13 weeks</td><td>Cow &amp; Gate</td><td>F2</td>

<td>F</td><td>19 weeks</td><td>SMA gold and feeding breast</td><td>F3</td>

<td>AE</td><td>9.5 the week</td><td>Breast-feeding</td><td>F4</td>

<td>AE</td><td>14 weeks</td><td>Breast-feeding</td><td>F5</td>

[0108] The microbiological flora of the infant intestine for fermentation studies was provided from freshly eliminated faeces of the infant. At least 3.5 g of stool sample was usually required. The stool sample was kept in a diaper, which immediately after taking away from the child, the guardian placed in an anaerobic jar with an open cartridge with anaerobic gas. It was taken and processed as quickly as possible (usually within an hour).

[0109] In the laboratory, feces were removed from the diaper and weighed.

fecal suspension by homogenization and pre-heated (overnight in 1 x PBS solution, using a stomacher homogenizer at an average speed of 120 seconds.

10% (w / v) samples were prepared in a deoxygenated anaerobic chamber. [0110] Each fermenter was inoculated with 5 ml of 10% w / v stool suspension. A sample of faecal suspension (sample S) was also taken for analysis.

[0111] The FISH method required 375 μl of faecal suspension sample (sample S) or sample from each fermenter to be counted with the duplicate bacteria. Each sample was bound by thorough stirring in 1.125 ml of a cold, filtered 4% (w / v) solution of paraformaldehyde in PBS pH 7.2 and stored overnight at 4 ° C (or at least for 4 hours).

[0112] The bound sample was centrifuged at 13,000 xg for 5 minutes and the supernatant discarded. The pellet was washed twice by resuspending in 1 ml of cold, filtered 1xPBS, each time settling the cells by centrifugation and discarding the supernatant. Finally, the pellet was completely suspended again in

150 μΐ filtered PBS; then mixed well with 150 μΐ 96% (v / v) ethanol. The cell preparation was stored at -20 ° C for at least 1 hour. before further processing.

[0113] In the hybridization step, 16 μΐ of the cell preparation (brought to room temperature) was mixed with 200 μΐ of filtered, pre-heated 2x hybridization buffer (30.3 mM Tris-HCl pH 7.2, 1.4 mM NaCl) containing 15, 1 ml / l 10% (w / v) SDS. This mixture was heated to the appropriate hybridization temperature and then mixed with the probe (50 ng / gl) in a ratio of 9: 1, respectively. The hybridization preparation was then returned to the hybridization oven for overnight incubation.

[0114] Finally, the hybridized cell preparation was collected on 0.2 gm filters for microscopic observations. Depending on the cell density, between 5 g and 100 g of the cell preparation was added to the filtered, pre-heated (to hybridization temperature) washing buffer (5-7 ml 20mM Tris-HCl pH 7.2, 0.9 M NaCl). Gl DAPI (4 ', 6-diamidino-2-phenylindole) was also added to the mixture to stain all cells and obtain full cell counts for each sample. It was then filtered under vacuum through

0.2m polycarbonate filter and placed on the microscope plate.

To minimize the loss of color in the fluorescent dye, a drop of SlowFade ™ (Molecular) was placed on the filter

Probes) and covered with a cover plate; the plates were then stored in the dark at 4 ° C until use. Bacteria labeled with a Cy3 fluorescent probe were counted using fluorescence microscopy (Leitz, Wetzlar, Germany) at 550 nm; UV light was used to count DAPI stained bacteria. Bacteria were counted in at least 15 fields selected at random and the average of them was used to estimate the number of cells per ml of the original sample.

[0115] Four comparisons, fermentation tests were carried out as given below.

Table 4. Fermentation series

<td>Fermentation Series</td><td colspan="2">Test substances</td>

<td>F1</td><td>FOS</td><td></td>

<td>F2</td><td>Human milk</td><td>PDX</td>

<td>F3</td><td>GOS</td><td></td>

<td>F4</td><td>1: 1 LOS: GOS</td><td>1: 1 PDX: LOS</td>

<td>F5</td><td>MOOSE</td><td>1: 1 PDX: GOS</td>

Example 7 [0116] This example illustrates the effect of PDX on the population and species of bacteria in the infant's intestine. In 1 fermentation series (F1), FOS was added to the food as a mix and used in the fermenter system. FOS, which was traditionally considered a good prebiotic ingredient, led to growth

Bifidobacteria and Clostridia and a decrease in Lactobacilli and

Bacteroides in V1. The addition of FOS to the food in the form of a blend did not lead to a change in Bifidobacteria and Lactobacilli levels and an increase in the level of Clostridia and Bacteroides in V2. [0117] In the F2 fermentation series, PDX and human milk were used in parallel fermenter systems. Human milk samples were delivered from the postpartum room and stored frozen. These were early milk samples of various volumes from several donors. Human milk food was used without diluting or adding lactose to maintain comparable levels of oligosaccharides and other nutrients. There was not enough human milk to use this fermenter for 12 days in parallel with the PDX fermenter. Therefore, more samples were taken on days 0, 4, 6 and 8. For comparative purposes, additional samples were collected from PDX fermenter on day 8 and also on day 11.

[0118] As might be expected, human milk favored the good growth of beneficial bacteria and Bifidobacteria and Lactobacilli and the reduction of Clostridia levels as shown in Figure 17. There was a clear increase in the population of Bifidobacteria and Lactobacilli in both vessels. Bacteroides numbers remained at a similar level throughout the fermentation.

[0119] The effects of adding PDX to the food in the form of a blend were also beneficial, with a significant increase in Lactobacilli and a decrease in both Clostridia and Bacteroides in both vessels (Figure 17).

[0120] In the F3 fermentation series, GOS was added to the food as a mix and used in a fermenter system. Adding GOS to food in the form of a blend had little effect on Lactobacilli in both vessels, but increased Bifidobacteria levels in V1 and V2 vessels and decreased Clostridia and Bacteroides levels in V1 vessel, but not V2.

[0121] The combination LOS: GOS (1: 1) was used compared to 1: 1 PDX: LOS in a parallel fermenter system during the F4 fermentation series. The LOS: GOS combination was effective in increasing the number of Lactobacilli in both vessels and Bifidobacteria in vessel V1 and in reducing the number of Bacteroides in vessel V1. The number of Clostridia decreased in V2, but increased in V1.

[0122] Supplementing food in the form of a 1: 1 PDX: LOS combination led to an increase in the number of Lactobacilli in V1, but to a slight decrease in the number of Bifidobacteria in each dish. Clostridia tended to decrease in both vessels, while the number of Bacteroides mainly decreased in V2.

[0123] In the F5 fermentation series, LOS was added to the food as a blend and used in a parallel fermenter system compared to a 1: 1 combination of PDX and GOS. Adding LOS to the food in the form of a blend increased the number of Lactobacilli in both vessels. However, the number of Clostridia also increased in V2, and the number of Bifidobacteria decreased in both vessels. Although the number of Bacteroides decreased in V1, this was not maintained in V2. Addition of PDX: GOS to food in the form of a blend increased Bifidobacteria and Lactobacilli levels in both vessels, but also increased Clostridia levels. Bacteroides levels only increased in V2.

[0124] In general, the number of Bifidobacteria increased in proportion to the total bacterial population in a V1 vessel with human milk, GOS, FOS, PDX and the PDX: GOS combination. In V2 GOS, the PDX: GOS combination and the LOS: GOS combination led to an increase in Bifidobacteria. Clostridia decreased in proportion to the total population in V1 with human milk, GOS and PDX and decreased in V2 with human milk, PDX and the LOS: GOS combination.

[0125] In a V1 vessel, Lactobacilli showed an increase after supplementation with LOS, PDX, human milk or PDX combinations, while increases in Lactobacilli in a V2 vessel with LOS, PDX, human milk and GOS combinations were observed. Increases in the percentage of Lactobacilli were particularly pronounced with PDX and the PDX: GOS combination and the LOS: GOS combination in V2.

[0126] Overall, PDX was effective in increasing levels of Lactobacilli and reducing levels of Clostridia and Bacteroides, with only a slight increase in Bifidobacteria in V1. The PDX: GOS combination also looked favorable for Bifidobacteria, which increased among the total number of bacteria (though not as a percentage of four groups) and increased the number of Lactobacilli at pH 5.2, but also had an adverse effect on increasing the number of Bacteroides.

[0127] When human milk was tested in a model system designed by the inventors, Bifidobacteria and Lactobacilli levels increased numerically, while Clostridia levels decreased numerically. This result was most consistently repeated with PDX and GOS, either alone or in combination with LOS or PDX. FOS, which is another carbohydrate currently used in various infant formulas, has been studied, but the same desired results were not obtained.

Example 8 [0128] This example illustrates one embodiment of an infant formula according to the present invention.

Table 5: Information on the nutrients of infant formulas

<td>Ingredient</td><td>For 10,000 liters</td>

<td>Demineralized whey powder</td><td>534.333 kg</td>

<td>Fat blend</td><td>339.695 kg</td>

<td>Skimmed milk powder</td><td>191,234 kg</td>

<td>Lactose</td><td>136.321 kg</td>

<td>Galacto-oligosaccharide syrup powder</td><td>35.096 kg</td>

<td>polydextrose</td><td>22,222 kg</td>

<td>Potassium Citrate</td><td>7,797 kg</td>

<td>Mono- and diglycerides</td><td>7.233 kg</td>

<td>Arachidonic acid oil obtained from</td><td>6,486 kg</td>

<td>unicellular organisms</td><td></td>

<td>Tribasic calcium phosphate</td><td>4.185 kg</td>

<td>Ascorbic acid</td><td>1403.323 g</td>

<td>Sodium ascorbate</td><td>1168.402 g</td>

<td>inositol</td><td>407.029 g</td>

<td>taurine</td><td>402.962 g</td>

<td>Powdered starch syrup</td><td>188,300 g</td>

<td>niacinamide</td><td>89.857 g</td>

<td>Calcium pantothenate</td><td>42.443 g</td>

<td>Vitamin B.₁₂</td><td>23.613 g</td>

<td>Biotin powder</td><td>23.613 g</td>

<td>Thiamine hydrochloride</td><td>8,022 g</td>

<td>Pyridoxine hydrochloride</td><td>6,176 g</td>

<td>Folic acid</td><td>2,260 g</td>

<td>Lecithin concentrate</td><td>3.694 kg</td>

<td>Docosahexaenoic acid oil obtained from unicellular organisms</td><td>3.243 kg</td>

<td>carrageenan</td><td>2.826 kg</td>

<td>Calcium chloride</td><td>2.650 kg</td>

<td>Sodium chloride</td><td>1.410 kg</td>

<td>maltodextrin</td><td>484.199 g</td>

<td>CMP, free acid</td><td>151.951 g</td>

<td>AMP, free acid</td><td>33.944 g</td>

<td>GMP, disodium salt</td><td>18.347 g</td>

<td>UMP, disodium salt</td><td>7.559 g</td>

<td>Iron (II) sulfate</td><td>0.620 g</td>

<td>Sodium Citrate</td><td>0.455 kg</td>

<td>DL-alpha-tocopheryl acetate</td><td>160.882 g</td>

<td>Soybean oil</td><td>139.612 g</td>

<td>Vitamin A Palmitate</td><td>17,253 g</td>

<td>Cholecalciferol concentrate</td><td>5.715 g</td>

<td>Vitamin K, liquid phytonadione</td><td>0.538 g</td>

<td>Zinc sulfate</td><td>214.225 g</td>

<td>Sodium Selenite</td><td>51.112 g</td>

<td>Copper (II) sulfate</td><td>22.885 g</td>

<td>Lactose</td><td>12.659 g</td>

<td>Manganese Sulphate</td><td>3.119 g</td>

<td>Defluorinated Water</td><td>10311,900 kg</td>

[0129] LOS is produced when lactose is heated at high temperature. Therefore, in this embodiment, the product contains a local LOS. The level of local LOS in the product is approximately 2 g / l.

Example 9 [0130] This example illustrates another embodiment of the infant formula according to the present invention.

Table 6. Information on the nutrients of the baby formula

<td>Ingredient</td><td>For 10,000 liters</td>

<td>Demineralized whey powder</td><td>534.333 kg</td>

<td>Fat blend</td><td>339.695 kg</td>

<td>Skimmed milk powder</td><td>191,234 kg</td>

<td>Lactose</td><td>142.000 kg</td>

<td>Galacto-oligosaccharide syrup powder</td><td>23.164 kg</td>

<td>polydextrose</td><td>22,222 kg</td>

<td>Lactulose Syrup Powder</td><td>10.353 kg</td>

<td>Potassium Citrate</td><td>7,797 kg</td>

<td>Mono- and diglycerides</td><td>7.233 kg</td>

<td>Arachidonic acid oil obtained from</td><td>6,486 kg</td>

<td>unicellular organisms</td><td></td>

<td>Tribasic calcium phosphate</td><td>4.185 kg</td>

<td>Ascorbic acid</td><td>1403.323 g</td>

<td>Sodium ascorbate</td><td>1168.402 g</td>

<td>inositol</td><td>407.029 g</td>

<td>taurine</td><td>402.962 g</td>

<td>Powdered starch syrup</td><td>188,300 g</td>

<td>niacinamide</td><td>89.857 g</td>

<td>Calcium pantothenate</td><td>42.443 g</td>

<td>Vitamin B.₁₂</td><td>23.613 g</td>

<td>Biotin powder</td><td>23.613 g</td>

<td>Thiamine hydrochloride</td><td>8,022 g</td>

<td>Pyridoxine hydrochloride</td><td>6,176 g</td>

<td>Folic acid</td><td>2,260 g</td>

<td>Lecithin concentrate</td><td>3.694 kg</td>

<td>Docosahexaenoic acid oil obtained from unicellular organisms</td><td>3.243 kg</td>

<td>carrageenan</td><td>2.826 kg</td>

<td>Calcium chloride</td><td>2.650 kg</td>

<td>Sodium chloride</td><td>1.410 kg</td>

<td>maltodextrin</td><td>484.199 g</td>

<td>CMP, free acid</td><td>151.951 g</td>

<td>AMP, free acid</td><td>33.944 g</td>

<td>GMP, disodium salt</td><td>18.347 g</td>

<td>UMP, disodium salt</td><td>7.559 g</td>

<td>Iron (II) sulfate</td><td>0.620 g</td>

<td>Sodium Citrate</td><td>0.455 kg</td>

<td>DL-alpha-tocopheryl acetate</td><td>160.882 g</td>

<td>Soybean oil</td><td>139.612 g</td>

<td>Vitamin A Palmitate</td><td>17,253 g</td>

<td>Cholecalciferol concentrate</td><td>5.715 g</td>

<td>Vitamin K, liquid phytonadione</td><td>0.538 g</td>

<td>Zinc sulfate</td><td>214.225 g</td>

<td>Sodium Selenite</td><td>51.112 g</td>

<td>Copper (II) sulfate</td><td>22.885 g</td>

<td>Lactose</td><td>12.659 g</td>

<td>Manganese Sulphate</td><td>3.119 g</td>

<td>Defluorinated Water</td><td>10311,900 kg</td>

[0131] LOS is produced when lactose is heated at high temperature. Therefore, in this form of implementation, the product contains both local and added LOS. The total LOS level in the product, including both added and local LOS, is approximately 2.6 g / L.

Example 10

This example illustrates yet another embodiment of the infant formula according to the present invention. Table 7. Information on the nutrients of the baby formula

<td>Ingredient</td><td>For 10,000 liters</td>

<td>Demineralized whey powder</td><td>534.333 kg</td>

<td>Fat blend</td><td>339.695 kg</td>

<td>Skimmed milk powder</td><td>191,234 kg</td>

<td>Lactose</td><td>119.321 kg</td>

<td>Syrup Powder galaktooligosacharydowego</td><td>46,327 kg</td>

<td>polydextrose</td><td>44.444 kg</td>

<td>Lactulose Syrup Powder</td><td>20.706 kg</td>

<td>Potassium Citrate</td><td>7,797 kg</td>

<td>Mono- and diglycerides</td><td>7.233 kg</td>

<td>Arachidonic acid oil obtained from organisms unicellular</td><td>6,486 kg</td>

<td>Tribasic calcium phosphate</td><td>4.185 kg</td>

<td>Ascorbic acid</td><td>1403.323 g</td>

<td>Sodium ascorbate</td><td>1168.402 g</td>

<td>inositol</td><td>407.029 g</td>

<td>taurine</td><td>402.962 g</td>

<td>Powdered starch syrup</td><td>188,300 g</td>

<td>niacinamide</td><td>89.857 g</td>

<td>Calcium pantothenate</td><td>42.443 g</td>

<td>Vitamin B.₁₂</td><td>23.613 g</td>

<td>Biotin powder</td><td>23.613 g</td>

<td>Thiamine hydrochloride</td><td>8,022 g</td>

<td>Pyridoxine hydrochloride</td><td>6,176 g</td>

<td>Folic acid</td><td>2,260 g</td>

<td>Lecithin concentrate</td><td>3.694 kg</td>

<td>Docosahexaenoic acid oil obtained from unicellular organisms</td><td>3.243 kg</td>

<td>carrageenan</td><td>2.826 kg</td>

<td>Calcium chloride</td><td>2.650 kg</td>

<td>Sodium chloride</td><td>1.410 kg</td>

<td>maltodextrin</td><td>484.199 g</td>

<td>CMP, free acid</td><td>151.951 g</td>

<td>AMP, free acid</td><td>33.944 g</td>

<td>GMP, disodium salt</td><td>18.347 g</td>

<td>UMP, disodium salt</td><td>7.559 g</td>

<td>Iron (II) sulfate</td><td>0.620 g</td>

<td>Potassium Citrate</td><td>0.455 kg</td>

<td>DL-alpha-tocopheryl acetate</td><td>160.882 g</td>

<td>Soybean oil</td><td>139.612 g</td>

<td>Vitamin A Palmitate</td><td>17,253 g</td>

<td>Cholecalciferol concentrate</td><td>5.715 g</td>

<td>Vitamin K, liquid phytonadione</td><td>0.538 g</td>

<td>Zinc sulfate</td><td>214.225 g</td>

<td>Sodium Selenite</td><td>51.112 g</td>

<td>Copper (II) sulfate</td><td>22.885 g</td>

<td>Lactose</td><td>12.659 g</td>

<td>Manganese Sulphate</td><td>3.119 g</td>

<td>Defluorinated Water</td><td>10325,600 kg</td>

[0133] LOS is produced when lactose is heated at high temperature. Therefore, in this form of implementation, the product contains both local and added LOS. The total LOS level in the product, including both added and local LOS, is approximately 3.6 g / L.