Probiotic Stabilization

Abstract

An ingestible composition including a probiotic contained in a mixture of hydrolyzed mammalian protein, one or more carbohydrates and a compound binder useful for nutrition of infants and children.

Claims

1. A liquid ingestible composition comprising a probiotic stabilized in a stabilization mixture, wherein the stabilization mixture comprises, on a dry weight basis, about 70% to about 85% of one or more carbohydrates; about 10% to about 20% of hydrolyzed mammalian protein, wherein at least 20% of the total hydrolyzed mammalian protein is comprised of peptides having a molecular weight of less than 2000 Daltons; and a compound binder comprising sodium alginate.

2. The composition of claim 1 wherein the probiotic comprises viable microbial cells.

3. The composition of claim 2 wherein the viable microbial cells comprise Lactobacillus rhamnosus.

4. The composition of claim 1 wherein the hydrolyzed mammalian protein comprises hydrolyzed casein.

5. The composition of claim 1 wherein at least 70% of the total hydrolyzed mammalian protein is comprised of peptides having a molecular weight of less than 2000 Daltons.

6. The composition of claim 1 wherein at least 90% of the total hydrolyzed mammalian protein is comprised of peptides having a molecular weight of less than 2000 Daltons.

7. The ingestible composition of claim 1, wherein the one or more carbohydrates comprises trehalose.

8. The composition of claim 1, wherein the composition is an infant formula.

9. The composition of claim 1, wherein the compound binder comprises about 4% to about 10% of the stabilization mixture.

10. A liquid nutritional composition comprising a lipid or fat; a protein source; about 5 to about 100 mg/100 kcal of a source of long chain polyunsaturated fatty acids which comprises docosahexaenoic acid; about 0.1 to about 1 mg/100 kcal of a prebiotic composition, wherein the prebiotic composition comprises at least 20% of an oligosaccharide; and a probiotic stabilized by a mixture comprising, on a dry weight basis, about 70% to about 85% of one or more carbohydrates; about 10% to about 20% hydrolyzed mammalian protein, wherein at least 20% of the total hydrolyzed mammalian protein is comprised of peptides having a molecular weight of less than 2000 Daltons; and a compound binder comprising sodium alginate.

11. The composition of claim 10 wherein the probiotic comprises non-viable microbial cells.

12. A liquid infant formula comprising: (a) a probiotic stabilized by a stabilization mixture comprising, on a dry weight basis, (i) about 70% to about 85% of one or more carbohydrates, wherein the one or more carbohydrates comprises trehalose; (ii) about 4% to about 10% compound binder, wherein the compound binder comprises sodium alginate; and (iii) about 10% to about 20% hydrolyzed mammalian protein, wherein at least 20% of the total hydrolyzed mammalian protein is comprised of peptides having a molecular weight of less than 2000 Daltons; (b) protein; and (c) at least one additional carbohydrate.

13. The infant formula of claim 12, wherein the probiotic comprises Lactobacillus rhamnosus GG.

14. The infant formula of claim 12, wherein the one additional carbohydrate comprises inulin.

15. The infant formula of claim 12, wherein the hydrolyzed mammalian protein comprises hydrolyzed casein.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present disclosure provides a novel stabilization formula and method, that provides stability and protection to biological materials for improving the viability of the target microorganism. The present disclosure includes a stabilization mixture comprising a hydrolyzed mammalian protein, which in certain embodiments is combined with one or more carbohydrates and a compound binder which together provide a protective matrix resulting in an increased shelf-life over other stabilization products utilizing non-hydrolyzed or hydrolyzed non-mammalian proteins.

DEFINITIONS

[0028] The term “probiotic” means a microorganism with low or no pathogenicity that exerts beneficial effects on the health of the host.

[0029] As used herein, the term “infant” is generally defined as a human from about birth to 12 months of age.

[0030] A “pre-term infant” is an infant born after less than about 37 weeks gestation.

[0031] A “full-term infant” as used herein means an infant born after at least about 37 weeks gestation.

[0032] “Children” are defined as humans over the age of about 12 months to about 12 years old.

[0033] An “effective amount” as used herein is generally defined as an amount of an agent that provides an observable result within the subject administered thereto.

[0034] The term “nutritional product” as used herein encompasses any consumable matter of either plant, animal or synthetic sources that may contain nutrients.

PREFERRED EMBODIMENTS

[0035] In the practice of the present disclosure, hydrolyzed mammalian protein is utilized as a component within a stabilization mixture for stabilizing biological material. As used herein, hydrolyzed protein is used to describe a degraded or decomposed protein molecule so that multiple peptide fragments are provided. Within the present disclosure, hydrolyzed protein and protein hydrolysate are used interchangeably to describe products of protein hydrolysis; extensively hydrolyzed protein is used to describe products of protein hydrolysis where at least 20%, more preferably at least 70%, and most preferably at least about 90%, of the hydrolyzed protein has a molecular weight of less than 2000 Daltons.

[0036] Hydrolyzed mammalian protein can be created from a variety of mammalian protein sources, including milk products and animal products. It may be created through a process of acid hydrolysis where mammalian protein is subjected to a strong acid and heated until the desired size ranges of amino acid fragments are created. Further types of protein hydrolysis include the use of enzymatic agents which digest protein molecules in creating shorter chains of amino acids. Common processes for hydrolyzing protein are known in the art and described in U.S. Pat. No. 4,377,601 issued to Conrad; U.S. Pat. No. 4,443,540 issued to Chervan et al.; U.S. Pat. No. 4,545,933 issued to Ernster; U.S. Pat. No. 4,757,007 issued to Satoh et al.; U.S. Pat. No. 4,873,108 issued to De Rooij et al.; U.S. Pat. No. 5,401,527 issued to Brown et al.; U.S. Pat. No. 6,214,585 issued to Kwong et al.; and U.S. Pat. No. 6,221,423 issued to Cho et al., the disclosures each of which are hereby incorporated by reference in their entirety.

[0037] Mammalian proteins that may be hydrolyzed for use in the stabilization mixture of the present disclosure include egg proteins, animal proteins, poultry, meat, serum albumen, glycol proteins, collagen, gelatin, milk proteins, casein, whey protein, albumen and others. In a preferred embodiment, the mammalian protein is a bovine protein. As previously defined, the hydrolyzed mammalian protein of any of the above types is, in a preferred embodiment, extensively hydrolyzed, meaning at least about 70% of the hydrolyzed protein yielding peptides having a molecular weight of less than about 2,000 Daltons.

[0038] In one embodiment, the hydrolyzed mammalian protein comprises hydrolyzed casein having over about 80%, advantageously over about 90%, of the peptides with a molecular weight of less than about 2,000 Daltons. Casein is understood to be a phospho-protein which comprises almost 80% of the total protein in bovine milk. The protein includes no disulfide bridges and as a result has little secondary or tertiary structure. Non-hydrolyzed casein includes casein variants having a molecular weight in the range of from about 19,000 Daltons to about 68,000 Daltons. One embodiment of a mammalian protein hydrolysate, such as casein hydrolysate, which may be used in practice of the present disclosure is one in which over 90% of the peptides have a molecular weight of less than 1,000 Daltons, with over 97% having a molecular weight of less than 2,000 Daltons. Less than 0.3% of the mammalian protein hydrolysate in certain embodiments is over 5,000 Daltons, illustrating that virtually all of the protein was hydrolyzed.

[0039] The use of the hydrolyzed mammalian proteins including the aforementioned hydrolyzed casein provides superior protection to probiotics, including Lactobacillus rhamnosus, beyond the protection observed through the use of larger peptide fragments or whole proteins. While not being bound by any theory, one possibility for the increased protection to the probiotics against both moisture and heat may involve the increase in the zeta potential of the surface resulting from the hydrolyzed mammalian protein. The zeta potential is a value which indicates the degree of repulsion between adjacent similarly charged particles within dispersion. Smaller compounds and molecules possess a high zeta potential which confers stability as the solution or dispersion will resist aggregation. Conversely, when the zeta potential is low attraction exceeds repulsion and the dispersion may break and flocculate. While hydrophobicity decreases, the magnitude of the zeta potential increases with an increasing degree of hydrolysis. The high degree of hydrolysis providing for many short peptide sequences may increase the zeta potential of the protein interface in contact with the probiotic and thus increase the stability of the biological agent to both heat and humidity. Of course, this does not explain the surprising differences between hydrolyzed mammalian and non-mammalian proteins.

[0040] In certain embodiments, the majority component of the stabilization mixture based on a dry weight basis is one or more carbohydrates, which may include polysaccharides, disaccharides and monosaccharides. Examples of monosaccharides may include glucose, fructose, ribose, galactose; disaccharides may include trehalose, sucrose, maltose, lactose, cellobiose; and polysaccharides may include glucose, fructose and galactose polymers, amylose, glycogen, cellulose, inulin, raffinose and stachyose, among other examples. In some embodiments, the carbohydrates may include disaccharides such as sucrose and trehalose, which are known to assist plant and microbial life during periods of low water availability. Trehalose, also known as mycose, is synthesized by fungi, plants and certain animals and functions as a key carbohydrate in anhydrobiosis where biological organisms withstand prolonged periods of desiccation. Physically, trehalose can form a gel phase and is useful in forming the protective matrix around the probiotic. A further embodiment provides that inulin may be included and provides a soluble fiber often considered as a prebiotic in nutritional applications. Advantageously, inulin passes through the stomach and duodenum undigested where it is available for digestion by the gut bacteria flora. The incorporation of inulin may help protect the probiotic from digestive enzymes and high acidity until the probiotic is properly located with the bacteria flora of the gut where inulin may be digested.

[0041] A further component of the stabilization mixture in some embodiments can be a compound binder (also termed a gelling agent) which may act as a thickener and produce a gel-like consistency. Examples of compound binders include alginates such as sodium alginate, chitosan, carboxymethylcellulose, among others. The incorporation of the compound binder provides for the formation of a viscous consistency providing for efficient matrix formation and a structural quality suitable for subsequent drying.

[0042] The stabilization mixture may also include additional ingredients that provide further benefits to either the probiotic or the individual ingesting the stabilized probiotic. These ingredients may comprise minerals, vitamins, antioxidants, trace elements, sterols, antioxidants, fatty acids, functional molecules, and combinations thereof. Other ingredients may include pectin, resistant starches, high amylose starches, guar, and locust bean gum, agar, xanthan, carrageenans, glucans, fructo-oligosaccharides, polyfructoses, maltodextrins, and combinations thereof.

[0043] Foaming of the mixture can be controlled by decreasing the amount of air present in the slurry. In certain embodiments, this can be achieved by mixing under vacuum so as to yield target slurry densities in the range of 1.0 to 1.2 g/cm.sup.3.

[0044] The stabilization mixture may be used to provide stability to a probiotic organism which may exert a beneficial effect on the health and welfare of individuals. Examples of suitable probiotics include but are not limited to yeasts such as Saccharomyces cereviseae, molds such as Aspergillus, Rhizopus, Mucor, and bacteria such as Lactobacillus. Specific examples of suitable probiotic micro-organisms are: Aspergillus niger, A. oryzae, Bacillus coagulans, B. lentus, B. licheniformis, B. mesentericus, B. pumilus, B. subtilis, B. natto, Bifidobacterium adolescentis, B. animalis, B. breve, B. bifidum, B. infantis, B. lactis, B. longum, B. pseudolongum, B. thermophilum, Candida pintolepesii, Clostridium butyricum, Enterococcus cremoris, E. diacetylactis, E. faecium, E. intermedius, E. lactis, E. muntdi, E. thermophilus, Lactobacillus acidophilus, L. alimentarius, L. amylovorus, L. crispatus, L. brevis, L. case, L. curvatus, L. cellobiosus, L. delbrueckii ss. bulgaricus, L. farciminis, L. fermentum, L. gasseri, L. helveticus, L. lactis, L. plantarum, L. johnsonii, L. reuteri, L. rhamnosus, L. sakei, and L. salivarius.

[0045] In an embodiment of the present disclosure Lactobacillus rhamnosus GG is utilized as a probiotic which may be stabilized by the present disclosure. Lactobacillus rhamnosus GG is described in U.S. Pat. No. 4,839,281, issued to Sharwood et al., which is hereby incorporated by reference in its entirety. Notably, Sharwood et al. describes Lactobacillus rhamnosus GG as being a species in which the bacteria have avid adherence to intestinal cells while being simultaneously able to survive at low pHs and produce large amounts of lactic acid.

[0046] The selected probiotic is preferably concentrated to a wet paste-like consistency prior to combining with the stabilization mixture of the present disclosure. Starting with probiotics in dry form is also an alternative. Concentration levels of selected probiotics include concentrations of from about 3× to about 20× though may include lesser or greater concentrations depending upon the specific probiotic biomass and subsequent processing steps. Generally, the preparation of a stabilized probiotic includes the steps of concentrating the selected probiotic or probiotics; providing components of the stabilization mixture in desired quantities; mixing the stabilization mixture with the concentrated probiotic; drying the stabilized probiotic and either packaging or combining the stabilized probiotic into a nutritional product which may include an infant formula.

[0047] In order to further illustrate the principles and operations of the present disclosure, the following example is provided. However, this example should not be taken as limiting in any regard.

EXAMPLE

[0048] Lactobacillus rhamnosus GG (LGG) is grown in a fermenter. The biomass is subsequently washed with buffer and centrifuged to obtain a LGG moist pellet. A stabilization mixture is pre-blended comprising on a dry weight basis approximately 75% trehalose, about 16.7% hydrolyzed casein, 5.2% inulin and approximately 3.1% sodium alginate. At a temperature of 37° C., the LGG moist pellet is mixed with the stabilization mixture and enough water to yield a total solids content of approximately 55%. The slurry is mixed under vacuum to yield a density of around 1.1 g/cm.sup.3.

[0049] The combination of stabilization mixture and LGG is then either vacuum-dried or freeze-dried to a final moisture content of approximately 3%. It is preferred for the mixture to be spread in a tray at a load ranging from 100 g/ft.sup.2 to 150 g/ft.sup.2. The mixture is dried ensuring product temperature is below 30° C. during removal of 80% of the total water. Temperature during removal of the remaining 17% moisture should not exceed 50° C. The dried, stabilized LGG may subsequently be ground and size selected through the use of sieves to obtain a product having a desirable size.

[0050] In optimizing the stabilization for probiotic, the multiple constituents may be varied in some embodiments. Carbohydrates may comprise from about 70% to about 85% of the stabilization mixture on a dry basis; the hydrolyzed mammalian protein may comprise from about 10% to about 20% of the stabilization mixture on a dry basis and the compound binder may comprise from about 0% to about 10% (i.e., up to about 10%) of the stabilization mixture on a dry basis.

[0051] The stabilized probiotic, with over 70% of the matrix protein having a molecular weight of less than 2,000 Daltons, may be packaged and sold commercially or may be instead combined with a variety of nutritional products. Such nutritional products may include both infant formulas and children's products useful for applications where one desires to incorporate a probiotic into a nutritional product that necessitates an improved shelf-life and stability.

[0052] One type of nutritional product for combination with a stabilized probiotic may be an infant formula. The term “infant formula” applies to a composition in liquid or powdered form that satisfies the nutrient requirements of an infant by being a substitute for human milk. In the United States, the content of an infant formula is dictated by the federal regulations set forth at 21 C.F.R. §§100, 106 and 107. These regulations define macronutrient, vitamin, mineral, and other ingredient levels in an effort to simulate the nutritional and other properties of human breast milk. Another nutritional product may be a human milk fortifier, meaning it is a composition which is added to human milk in order to enhance the nutritional value of human milk. As a human milk fortifier, the composition may be in powder or liquid form in combination with the stabilized probiotic.

[0053] The stabilized probiotic of the disclosure may be combined with a nutritional product that provides minimal, partial, or total nutritional support. The compositions may be nutritional supplements or meal replacements. The compositions may be administered in conjunction with a food or nutritional composition. The compositions containing the stabilized probiotic can either be intermixed with the food or other nutritional compositions prior to ingestion by the subject or can be administered to the subject either before or after ingestion of a food or nutritional composition. The compositions may be administered to preterm infants receiving infant formula, breast milk, a human milk fortifier, or combinations thereof.

[0054] The compositions for combination with the stabilized probiotic may, but need not, be nutritionally complete. The skilled artisan will recognize “nutritionally complete” to vary depending on a number of factors including, but not limited to, age, clinical condition, and dietary intake of the subject to whom the term is being applied. In general, “nutritionally complete” means that a nutritional composition provides adequate amounts of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy required for normal growth. As applied to nutrients, the term “essential” refers to any nutrient which cannot be synthesized by the body in amounts sufficient for normal growth and to maintain health and which therefore must be supplied by the diet. The term “conditionally essential” as applied to nutrients means that the nutrient must be supplied by the diet under conditions when adequate amounts of the precursor compound is unavailable to the body for endogenous synthesis to occur.

[0055] The composition which is “nutritionally complete” for the preterm infant will, by definition, provide qualitatively and quantitatively adequate amounts of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy required for growth of the preterm infant. The composition which is “nutritionally complete” for the term infant will, by definition, provide qualitatively and quantitatively adequate amounts of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy required for growth of the term infant. The composition which is “nutritionally complete” for a child will, by definition, provide qualitatively and quantitatively adequate amounts of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy required for growth of a child.

[0056] The stabilized probiotic created by the present disclosure may be combined with a nutritional composition provided in any form known in the art, including a powder, a gel, a suspension, a paste, a solid, a liquid, a liquid concentrate, or a ready-to-use product. In one combination, the nutritional composition is an infant formula, especially an infant formula adapted for use as sole source nutrition for an infant.

[0057] The nutritional products described for combining with the stabilized probiotic may be administered enterally. As used herein, “enteral” means through or within the gastrointestinal, or digestive, tract, and “enteral administration” includes oral feeding, intragastric feeding, transpyloric administration, or any other introduction into the digestive tract.

[0058] Suitable fat or lipid sources may be any known or used 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, palm kernel oil, wheat germ oil; medium chain triglyceride oils and emulsions and esters of fatty acids; and any combinations thereof.

[0059] Bovine milk protein of the nutritional product may 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.

[0060] The stabilized probiotic may be combined with a nutritional product having its proteins provided as intact proteins. In other embodiments, the proteins of the nutritional product are provided as a combination of both intact proteins and partially hydrolyzed proteins, with a degree of hydrolysis of between about 4% and 10%. Certain of these embodiments can be extremely hypoallergenic as both the stabilizer and the protein of the nutritional product contain only hydrolyzed protein. In yet another embodiment, the nutritional product may be supplemented with glutamine-containing peptides.

[0061] The whey:casein ratio of the protein source of the nutritional product may be similar to that found in human breast milk. In an embodiment, the protein source of the nutritional product comprises from about 40% to about 80% whey protein. In another embodiment, the protein source may comprise from about 20% to about 60% caseins.

[0062] When the stabilized probiotic is combined with the nutritional product, the composition may include an amount of probiotics from about 10.sup.4 to about 10.sup.10 colony forming units (cfu) per kg body weight per day. In other embodiments, the amount of the probiotic within the stabilized probiotic may vary from about 10.sup.6 to about 10.sup.9 cfu per kg body weight per day. In even further embodiments the composition of the nutritional product and stabilized probiotic may include an amount of probiotics from about of at least about 10.sup.6 cfu per kg body weight per day. In another embodiment, the concentration of the probiotic, for instance LGG, in the micromatrix is from about 10.sup.6 to about 10.sup.14 cfu per g, more preferably from about 10.sup.9 to about 10.sup.11 cfu per g.

[0063] In an embodiment, the stabilized probiotic(s) may be viable or non-viable, while viable are preferred. As used herein, the term “viable”, refers to live microorganisms, that is, comprising viable microbial cells. The term “non-viable” or “non-viable probiotic” means non-living probiotic microorganisms, their cellular components and metabolites thereof. Such non-viable probiotics may have been heat-killed or otherwise inactivated but may retain some ability to favorably influence the health of the host. The stabilized 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. Additionally, the nutritional product may include non-stabilized probiotics with the final composition including some stabilized probiotics and some non-stabilized probiotics.

[0064] The stabilized probiotics may also be combined with a nutritional composition containing one or more prebiotics. 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 (stabilized or not) microorganisms, selective reduction in 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 may include oligosaccharides, polysaccharides, and other prebiotics that contain fructose, xylose, soya, galactose, glucose and mannose. More specifically, prebiotics useful in the present disclosure may include lactulose, lactosucrose, raffinose, gluco-oligosaccharide, inulin, polydextrose, polydextrose powder, galacto-oligosaccharide, fructo-oligosaccharide, isomalto-oligosaccharide, soybean oligosaccharides, lactosucrose, xylo-oligosacchairde, chito-oligosaccharide, manno-oligosaccharide, aribino-oligosaccharide, siallyl-oligosaccharide, fuco-oligosaccharide, and gentio-oligosaccharides, and combinations thereof.

[0065] In some compositions of nutritional products including stabilized probiotics, 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 (in the liquid form). As noted, 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 should, in a preferred embodiment, comprise galacto-oligosaccharide (GOS).

[0066] In addition to galacto-oligosaccharide, the prebiotic composition can also comprise polydextrose (PDX). If polydextrose is used as a prebiotic, the amount of 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.

[0067] The amount of galacto-oligosaccharide in the nutritional composition may be from about 0.2 mg/100 Kcal to about 1.0 g/100 Kcal. In other compositions, the amount of galacto-oligosaccharide in the nutritional composition may be from about 0.1 g/100 Kcal to about 0.5 g/100 Kcal. If polydextrose is used as a prebiotic, the amount of polydextrose in the nutritional composition may, in an embodiment of the composition including stabilized probiotics, be within the range of from about 0.1 g/100 Kcal to about 0.5 g/100 Kcal. In another composition, the amount of polydextrose may be about 0.3 g/100 Kcal. Galacto-oligosaccharide and polydextrose may also be supplemented into the stabilized probiotic containing nutritional composition in a total amount of about 0.6 g/100 Kcal.

[0068] The nutritional formulation in which stabilized probiotics of the present disclosure may be combined there with, may contain a source of long chain polyunsaturated fatty acids (LCPUFAs) which comprise docosahexanoic acid (DHA). Other suitable LCPUFAs include, but are not limited to, α-linoleic acid, γ-linoleic acid, linoleic acid, linolenic acid, eicosapentanoic acid (EPA) and arachidonic acid (ARA).

[0069] In one embodiment, the final composition may be 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 this ratio is from about 1:2 to about 4:1.

[0070] The amount of long chain polyunsaturated fatty acids in the nutritional composition 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.

[0071] A 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 formula by replacing an equivalent amount of an oil, such as high oleic sunflower oil, normally present in the formula. As another example, the oils containing DHA and ARA may be added by replacing an equivalent amount of the rest of the overall fat blend normally present in the formula without DHA and ARA.

[0072] 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 compositions, 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.

[0073] In an embodiment of the nutritional composition combined with stabilized probiotics, 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.

[0074] In certain embodiments, the nutritional composition may be a milk-based nutritional composition which provides physiochemical and physiological benefits. As is known in the art, bovine milk protein comprises two major components: acid soluble whey protein and acid insoluble casein, with the latter representing about 80% of the total protein content of bovine milk. Upon entering the acidic environment of the stomach, casein precipitates and complexes with minerals forming semi-solid curds of varying size and firmness. Softer, smaller curds are easier for the body to digest than larger, harder curds. Curd formation may be an important consideration in the development of nutritional compositions, including, but not limited to infant formulas, medical foods, and premature infant formulas. As such, stabilized probiotics may be combined with compositions that include softer and smaller curds than standard infant formulas.

[0075] In other embodiments the stabilized probiotics of the present disclosure may be combined with nutritional compositions including bovine lactoferrin which retains its stability and activity in the human gut against certain undesirable bacterial pathogens. The nutritional composition of the disclosure may also contain TGF-β. In nutritional compositions, the level of TGF-β may be from about 0.0150 (pg/μg) ppm to about 0.1000 (pg/μg) ppm. In other nutritional compostions, the level of TGF-β in final composition including a stabilized probiotic is from about 0.0225 (pg/μg) ppm to about 0.0750 (pg/μg) ppm.

[0076] In some compositions, the level of TGF-β is from about 2500 pg/mL to about 10,000 pg/mL, more preferably from about 3000 pg/mL to about 8000 pg/mL.

[0077] In one embodiment, the ratio of TGF-fβ1: TGF-fβ2 is in the range of about 1:1 to about 1:20, or, more particularly, in the range of about 1:5 to about 1:15.

[0078] In some embodiments, the bioactivity of TGF-β in a nutritional composition is enhanced by the addition of a bioactive whey fraction. Any bioactive whey fraction known in the art may be used in this embodiment provided it achieves the intended result. In an embodiment, this bioactive whey fraction may be a whey protein concentrate. In a particular embodiment, the whey protein concentrate may be Salibra® 800, available from Glanbia Nutritionals. In a particular embodiment, the Salibra® 800 whey protein concentrate is 2.5% acidified. In another embodiment, the Salibra® 800 whey protein concentrate is 5% acidified. In yet another embodiment, the Salibra° 800 whey protein concentrate is 2% acidified. In a further embodiment, the Salibra° 800 whey protein concentrate is 3% acidified.

[0079] In another embodiment, the whey protein concentrate may be Nutri Whey 800, available from DMV International. In yet another embodiment, the whey protein concentrate may be Salibra-850, available from Glanbia Nutritionals. In still another embodiment, the whey protein concentrate may be Prolacta Lactalis WPI90, available from Lactilus Industrie U.S.A., Inc. In a further embodiment, the whey protein concentrate may be supplied by MG Nutritionals.

[0080] Accordingly, by the practice of the present disclosure, stabilized probiotics having heretofore unrecognized stability are prepared. The stabilized bacterial mixture exhibits exceptionally high stability through the use of hydrolyzed mammalian protein, especially hydrolyzed mammalian protein with over 70% of the peptides having a molecular weight of less than 2,000 Daltons. The stabilized probiotics are uniquely effective for nutritional applications with intermediate moisture levels (such as water activity as high as 0.4) where increased shelf life and stability in hot and humid environments are desired. The stabilized probiotics may be packed separately or be combined with any of the embodiments of nutritional compositions described herein.

[0081] 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.

[0082] Although preferred 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 both 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 spirit and scope of the appended claims should not be limited to the description of the preferred versions contained therein.