NZ718839B2 - Probiotic stabilization - Google Patents

Abstract

ingestible composition including a probiotic contained in a protective matrix of hydrolyzed protein, a first carbohydrate, a second carbohydrate, a lipid component and optionally a compound binder, useful for nutrition of infants and children.

Description

DESCRIPTION PROBIOTIC STABILIZATION TECHNICAL FIELD The present sure relates to the stabilization of biological material for ingestion by an individual. More particularly, the present disclosure s to a stabilization mixture comprising hydrolyzed protein, which provides improved stability to a probiotic organism when the probiotic is ed in a nutritional composition. The disclosure also es probiotic stabilization s.

BACKGROUND ART There are currently a variety of compositions for supplementing the nutrition of both humans and animals. These supplements may be provided to alter, reduce or increase the microflora within an individual’s gut so as to cause a desired effect on digestion. Ideally, mentation may cultivate an improved microflora for individuals, including humans, based upon the alteration of specific bacteria within the human’s gastrointestinal (GI) tract. This style of supplementation may be conducted through the use of probiotics, which are understood to be live microorganisms, that when administered in effective amounts confer a health or nutritional benefit to the host. One of the more common types of probiotics is a lactic acid bacterium which is able to convert sugars and other carbohydrates into lactic acid. This conversion lowers the pH within the gut of the host and provides fewer opportunities for l organisms to grow and cause problems through gastrointestinal infections.

A common technological challenge is introducing probiotics into the host in an appropriate manner, both for the maintenance of the probiotics as well as for the health of an individual. Current technologies include the utilization of encapsulation and stabilization ques for shielding the probiotics with a protective layer or matrix so that the protected microbe may be delivered to the riate location within the individual's GI tract. For example, in Batich et al.

(US. Patent No. 5,286,495), a process for microencapsulating cells is provided so that oxalate-degrading enzymes and bacteria may be encapsulated for both enteric and intraperitoneal stration. According to Batich et al., bacteria and enzymes can PCT/USZOl-UO6-l315 be successfully encapsulated in either alginate microcapsules or cellulose acetate phthalate microspheres. The model suggests that viability remains for the bacteria and enzymes so that the encapsulated cells reach the appropriate gastric region of the animal.

In U.S. Patent No. 5,733,568, issued to Ford, microencapsulated Lactobac/l/i bacteria are administered to the skin to treat or prevent recurrent skin infections. Lactobacil/us species are mixed with a glucose saline solution and gently stirred with a sodium alginate solution prior to being forced through a needle and dried to create gelled droplets. Other methods of encapsulation may include the addition of bacteria to a sion of polyvinyl povidone or ypropyl methylcellulose for encapsulating the bacteria.

In U.S. Patent No. 6,465,706, issued to Rogers et al., encapsulation of microbes is described for use in ontamination. Rogers et al. asserts that suitable encapsulation materials include natural or synthetic polymeric binders that encompasses both gels and foams as well as gelatin polymers. gh there have been developments concerning encapsulation and stabilization techniques for ning microorganisms for delivery into the digestive system of animals, there has been little development in encapsulation or stabilization techniques that protect the viability of probiotics during distribution and storage.

There is a need for a stabilization technique that is useful where circumstances preclude refrigeration, and further where such formulations may be exposed to s environments, especially those ated with tropical climates. In addition, the inherent moisture of the product poses a challenge in that tics generally are sensitive to water, especially in combination with high temperature. There is a need to deliver sufficient protection to probiotics under intermediate moisture ions (i.e. water activity of about 0.2 and higher, and up to about 0.4 or higher) and high temperatures during distribution and storage (i.e. temperatures of at least about 30°C, and up to and above 40°C) when incorporated into nutritional agents.

In particular, probiotics can provide a variety of benefits to a host, such as maintaining a healthy gastrointestinal flora, enhancing immunity, protecting against ea, atopic dermatitis and other diseases, etc. As such, there is a need for tics to be administered in various geographic locations, including tropical es, where the viability of the probiotic could be compromised. Conventional PCT/USZOl-UO6-I315 encapsulation and stabilization techniques are generally considered suitable only for non-humans and possess a chemical makeup that is ill-suited for infant formulas and/or for use by en, or known techniques have poor stability characteristics that significantly limit cial opportunities.

What is desired therefore, is a stabilization technique and a stabilized ial mixture using acceptable ients for either an infant formula or en’s ion, the stabilized mixture allowing for improved stability properties so that tics may be distributed in a wide variety of geographical locations and climates while ining a useful shelf-life. Further desired is a stabilization technology for the protection of probiotics, such as Lactobac/l/us rhamnosus, for use in nutritional compositions, such as infant formulas, supplements and children’s products. Indeed, a ation of characteristics, including improved stability combined with nutritional factors, provide an improved stabilization mixture applicable for prenatal, infant, and children's nutrition.

DISCLOSURE OF THE INVENTION In some embodiments, the present disclosure is directed to a nutritional composition comprising a lipid or fat source, a protein source, and a probiotic stabilized in a protective matrix, the protective matrix includes (i) a yzed protein, (ii) a first carbohydrate selected from the group consisting of sucrose, maltose, lactose, trehalose, maltotriose, maltodextrin having a se equivalent (”DE”) of about 2 to about 6, and any combination thereof, and (iii) a second carbohydrate selected from the group consisting of inulin, polydextrose, galactooligosaccharide, fructooligosaccharide, starch, maltodextrin having a dextrose equivalent of greater than about 8, and any combination thereof. In such embodiments, the nutritional composition comprises viable microbial cells, such as viable Lactobacfl/us rhamnosus cells. Also, the matrix may additionally se (i) sodium alginate and/or pectin and/or (ii) a lipid chosen from lecithin, monoglycerides, diglycerides, and any combination thereof. At least 20% of the yzed protein of the matrix may contain protein having a molecular weight of less than 2000 s.

Moreover, the hydrolyzed protein may comprise between about 10 and about 20% (w/w) of the protective matrix on a dry basis. Further, the hydrolyzed protein may include or consist solely of hydrolyzed casein. And the nutritional composition may be a powdered a, such as a powdered infant formula.

W0 2015/084531 PCT/U82014XO64315 In other embodiments, the present sure is directed to a nutritional composition sing a lipid or fat source, a protein , and a probiotic stabilized in a protective matrix, wherein the tive matrix includes (i) hydrolyzed protein, (ii) a first carbohydrate selected from the group consisting of e, maltose, lactose, trehalose, maltotriose, maltodextrin having a dextrose equivalent of about 2 to about 6, and any combination thereof, (iii) a second carbohydrate selected from the group consisting of inulin, polydextrose, galactooligosaccharide, fructooligosaccharide, starch, maltodextrin having a dextrose equivalent of greater than about 8, and any combination thereof, and (iv) a compound binder selected from the group consisting of sodium alginate, pectin, and any combination thereof.

In such embodiments, the nutritional composition may comprise viable microbial cells, such as viable LactobaC/Y/us rhamnosus cells. Also, the matrix may additionally comprise a lipid component, such as lecithin, a yceride and/or a diglyceride.

At least 20% ofthe hydrolyzed protein ofthe matrix may n protein having a molecular weight of less than 2000 Daltons. Moreover, the hydrolyzed protein may comprise between about 10 and about 20% (w/w) of the protective matrix on a dry basis. Further, the hydrolyzed protein may include or t solely of hydrolyzed casein. And the ional composition may be a powdered a, such as a powdered infant formula.

In another embodiment, the present disclosure is directed to a method for protecting a viable probiotic for use in a ed nutritional composition, the method includes the steps of (i) providing a viable probiotic, (ii) preparing a protective matrix for the tic by blending together (a) hydrolyzed casein, (b) a first carbohydrate selected from the group consisting of sucrose, maltose, e, trehalose, maltotriose, maltodextrin having a se equivalent of about 2 to about 6, and any combination thereof, (c) a second carbohydrate selected from the group consisting of inulin, polydextrose, galactooligosaccharide, fructooligosaccharide, starch, maltodextrin having a dextrose equivalent of greater than about 8, and any combination thereof, and (d) a lipid selected from the group consisting of lecithin, monoglycerides, diglycerides, and any combination thereof, (iii) combining the viable probiotic, the protective matrix and water to produce a mixture, (iv) drying the mixture to a final moisture content of about 4% or less, and (v) adding the dried PCT/USZOl-UO6-I315 mixture to a powdered nutritional product. In such an embodiment, the viable probiotic may be LactobaC/l/us rhamnosus.

These aspects and others that will become apparent to the skilled artisan upon review of the following description can be accomplished by providing a e including hydrolyzed mammalian protein for the stabilization of biological material, such as probiotics, to provide for increased stability of the biological material, resulting in the improved, long—term viability of the biological material. In an embodiment, the stabilization mixture advantageously provides for an ion of the shelf-life of probiotics such as Lactobac/l/us rhamnosus when compared to the use of non—hydrolyzed or yzed non—mammalian protein. The stabilization mixture may be combined with the probiotic in a variety of s including freeze drying, air , vacuum drying, spray drying and any combination f for preserving the tic.

It is to be understood that both the foregoing general ption and the following detailed description provide embodiments of the disclosure and are intended to provide an overview or framework of understanding to the nature and character of the disclosure as it is d.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. ’I is a graph that illustrates the stability of Lactobac/l/us rhamnosus (36 (L66) in a protective matrix (per the formulation presented in Table ’I hereinbelow) in a growing up milk having an available water (Aw) content of 0.28.

Fig. 2 is a graph that illustrates the stability of LGG without a protective matrix in a growing up milk having an available water (Aw) content of 0.28.

Fig. 3 presents scanning electron micrographs of stabilized LGG in a protective matrix (per the formulation presented in Table ’I hereinbelow).

BEST MODE FOR NG OUT THE INVENTION Reference now will be made in detail to the embodiments of the present disclosure, one or more examples of which are set forth hereinbelow. Each example is provided by way of explanation of the ional composition of the present disclosure and is not a limitation. In fact, it will be apparent to those d in the art that s modifications and variations can be made to the teachings of the t disclosure without departing from the scope of the disclosure. For instance, features PCT/USZOl-UO64315 illustrated or described as part of one embodiment can be used with r embodiment to yield a still further embodiment.

Thus, it is intended that the present disclosure covers such modifications and variations as come within the scope of the appended claims and their equivalents.

Other objects, features and aspects of the present disclosure are disclosed in or are s from the following detailed description. It is to be tood by one of ry skill in the art that the t discussion is a description of exemplary embodiments only and is not intended as limiting the broader aspects of the present disclosure.

The present disclosure provides a ization technique and a stabilized mixture (also referred to herein as a "stabilization mixture” or a "protective ”) that may be used for improving the ity of a biological material (also referred to herein as a "substrate”). In embodiments of the disclosure, the stabilized substrate may be a probiotic, wherein the various health benefits associated with the stabilized probiotic may be red to an individual upon ingesting a nutritional composition containing the stabilized probiotic.

While probiotics have been recognized as nutritionally beneficial, it is thought that the beneficial effects of probiotics are maximized if a probiotic microorganism is ingested by a subject when the microorganism is still alive. Thus, it is desirable for a viable probiotic to survive the ions of cturing and of ent into a consumable nutritional composition, such as a food or beverage, as well as to survive the subsequent shipping and storage time before the product is ingested and introduced to a subject's gastrointestinal tract. Many conventional probiotic compositions utilize an extremely high count of viable cells, with the understanding that a significant number of cells ultimately lose viability during the manufacturing process, transport, and e. Moreover, previously identified encapsulation and stabilization techniques provide some protection for probiotics, yet they do not provide a desired stability while aneously being functional for use in infant formula and/or children’s ional products.

By practice of the present disclosure, hydrolyzed mammalian protein is incorporated into a protective matrix. The hydrolyzed protein strengthens the protective matrix and increases the stability of probiotics that are protected by the matrix. As a result, - and children-compatible probiotics can be stabilized with PCT/USZOl-UO6-l315 a matrix ing hydrolyzed mammalian protein. The stabilized probiotics can be used in multiple nments, as the probiotics exhibit an ed viability.

Advantageously, probiotics stabilized by the protective matrix of the present disclosure can be incorporated into nutritional compositions and shipped over extended distances, as the probiotics will maintain viability even after extended ortation and storage time, due to the improved stability of the stabilized mixture.

The t method of providing stabilization to probiotics may e the use of a matrix for stabilizing a biological material, wherein the matrix includes a hydrolyzed mammalian protein, one or more carbohydrates and a compound .

While the protective matrix may be utilized for a variety of substances, in an embodiment, it is ed to protect probiotics, such as LactobaC/‘l/us rhamnosus. acfl/us rhamnosusis understood to possess relatively good bio- stability while having a high avidity for human intestinal mucosal cells. In use as a tic, Lactobac/l/us rhamnosusis thought to colonize the digestive tract and to balance intestinal microflora.

In creating the protective matrix for the probiotic, a hydrolyzed mammalian protein may be used to increase and strengthen the matrix around the probiotic. The hydrolyzed mammalian protein may include extensively hydrolyzed casein as well as other hydrolyzed mammalian proteins and is hypothesized to provide the increase in strength due to the characteristics associated with the short chain peptides comprising the hydrolyzed protein. In some embodiments, the hydrolyzed protein may be achieved by boiling mammalian protein in a strong acid or strong base or through an enzymatic degradation technique so as to break the protein down into shorter sequences of its component amino acids.

The protective matrix es for improved stability of the probiotic, meaning that a greater percentage of the probiotic cells are viable after processing, transportation and storage conditions. Specifically, the shelf life of viable probiotics is improved when compared to encapsulation and stabilization techniques using intact protein, hydrolyzed non-mammalian protein or n which is not as ively hydrolyzed as that specified herein.

The tive matrix of the present sure may be used in a multiplicity of processes for forming a stabilized probiotic product. These processes PCT/USZOl-If06-I315 include freezing, flash—freezing, freeze—drying, ambient air drying, vacuum drying, spray drying, low temperature drying, high temperature drying and any combination thereof. The resulting stabilized probiotic, r alone or integrated into a nutritional composition, possesses effective viability in a wide range of temperatures and conditions while ying improved shelf-life. Furthermore, the stabilized probiotic may be incorporated into a variety of prenatal, infant and children’s nutritional products for improving their gut microflora while simultaneously providing nutrition to the infant or child.

Accordingly, in one embodiment, the disclosure is directed to a method for stabilizing a biological material in a nutritional composition. Still r ment is a protective matrix for a probiotic. Yet another ment is a mixture for stabilizing a probiotic comprising one or more carbohydrates, a compound binder and hydrolyzed mammalian protein. A further ment is a method of increasing the shelf life of tics comprising stabilizing the probiotic with a stabilization mixture including hydrolyzed mammalian protein.

The present disclosure provides a novel stabilization mixture and method that provides stability and protection to biological materials, such as viable microorganisms. The present disclosure es a ization mixture comprising a hydrolyzed mammalian protein, which in certain embodiments is ed with one or more carbohydrates and a compound binder, which together provide a protective matrix resulting in an increased shelf-life over other encapsulation and ization products ing non-hydrolyzed or hydrolyzed non-mammalian ns.

DEFINITIONS The terms "protective matrix” and ”stabilization mixture" are used interchangeably hout the present disclosure.

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.

"Nutritional composition" means a substance or ation that satisfies at least a portion of a subject's nutrient requirements. The terms "nutritional(s)", "nutritional formula(s)", "enteral ional(s)", and "nutritional supplement(s)" are used as non-limiting examples of nutritional composition(s) throughout the present disclosure. Moreover, "nutritional composition(s)” may refer PCT/USZOl-UO6-I315 to liquids, powders, gels, , solids, concentrates, suspensions, or ready-to-use forms of l formulas, oral formulas, as for infants, formulas for ric subjects, formulas for children, growing-up milks and/or formulas for adults.

The term ”enteral” means deliverable through or within the gastrointestinal or digestive tract. "Enteral administration” includes oral feeding, intragastric feeding, transpyloric administration, or any other administration into the ive tract. "Administration" is broader than ”enteral administration” and includes parenteral administration or any other route of administration by which a substance is taken into a subject’s body.

"Pediatric subject" means a human less than 13 years of age. In some embodiments, a pediatric subject refers to a human subject that is less than 8 years old. In other embodiments, a pediatric t refers to a human subject between 1 and 6 years of age. In still further embodiments, a ric subject refers to a human subject between 6 and 12 years of age.

"Infant” means a human subject g in age from birth to not more than one year and es infants from O to 12 months corrected age. The phrase "corrected age" means an infant’s chronological age minus the amount of time that the infant was born premature. Therefore, the corrected age is the age of the infant if it had been carried to full term. The term infant includes low birth weight infants, very low birth weight s, and m infants. A "pre-term infant" is an infant born after less than about 37 weeks gestation. A "full-term infant" as used herein means an infant born after at least about 37 weeks gestation.

"Child” means a subject ranging in age from 12 months to about 12 years. In some embodiments, a child is a subject between the ages of 1 and 12 years old. In other embodiments, the terms "children" or "child" refer to subjects that are between one and about six years old, or between about seven and about 12 years old. In other embodiments, the terms ”children” or ”child” refer to any range of ages between 12 months and about 12 years.

”Children's nutritional product" refers to a composition that satisfies at least a portion of the nutrient requirements of a child. A growing-up milk (GUM) is an e of a children’s nutritional product.

As used herein, "hydrolyzed protein" means a product of protein hydrolysis. Within the present disclosure, hydrolyzed protein and protein hydrolysate WO 84531 ZOl-U064315 are used interchangeably to describe products of protein hydrolysis; extensively hydrolyzed protein is used to describe products of protein hydrolysis where at least 70%, more preferably at least about 90%, ofthe hydrolyzed protein has a molecular weight of less than 2000 Daltons.

The term "degree of hydrolysis" refers to the extent to which peptide bonds are broken by a ysis method.

The term "protein-free” means containing no measurable amount of protein, as measured by standard protein detection methods such as sodium dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis (SDS-PAG E) or size exclusion chromatography. In some embodiments, the nutritional composition is ntially free of protein, n antially free" is defined hereinbelow.

”Infant formula” means a composition that satisfies at least a portion of the nutrient requirements of an infant. In the United States, the content of an infant formula is dictated by the federal regulations set forth at 21 C.F.R. Sections 100, 106, and 107. These regulations define macronutrient, vitamin, mineral, and other ingredient levels in an effort to simulate the nutritional and other properties of human breast milk.

The term "growing-up milk" refers to a broad category of nutritional itions intended to be used as a part of a diverse diet in order to support the normal growth and pment of a child between the ages of about ’I and about 6 years of age.

”Milk-based” means sing at least one component that has been drawn or extracted from the mammary gland of a mammal. In some embodiments, a milk- based nutritional composition comprises components of milk that are derived from domesticated ungulates, ruminants or other s or any combination thereof.

Moreover, in some embodiments, milk-based means comprising bovine casein, whey, lactose, or any combination thereof. Further, "milk-based nutritional composition” may refer to any composition comprising any milk-derived or milk-based product known in the art.

"Nutritionally complete” means a composition that may be used as the sole source of nutrition, which would supply essentially all of the required daily amounts of vitamins, minerals, and/or trace elements in combination with ns, carbohydrates, and lipids. , ”nutritionally complete" describes a nutritional W0 2015/084531 PCT/U82014XO64315 composition that provides adequate amounts of carbohydrates, lipids, essential fatty acids, ns, essential amino acids, conditionally essential amino acids, vitamins, minerals and energy required to support normal growth and development of a subject.

The ition which is tionally complete” for the preterm infant will, by definition, provide qualitatively and quantitatively adequate amounts of all carbohydrates, lipids, essential fatty acids, ns, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy required for growth of the m infant. The composition which is "nutritionally complete” for the term infant will, by tion, provide qualitatively and quantitatively adequate amounts of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, ns, 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 s of all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, ionally essential amino acids, vitamins, minerals, and energy required for growth of a child.

As applied to nutrients, the term "essential" refers to any nutrient that cannot be synthesized by the body in amounts ient for normal growth and to maintain health and that, therefore, must be supplied by the diet. The term tionally essentia III 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 nous synthesis to occur.

The term "probiotic" means a microorganism with low or no pathogenicity that exerts beneficial effects on the health of the host. A "viable probiotic" means a live or active microorganism that exerts beneficial effects on the health of the host.

The term ”inactivated probiotic" or ”inactivated LGG" means a probiotic wherein the lic activity or reproductive ability of the referenced probiotic or Lactobacfflus rhamnosusGG (LGG) organism has been reduced or destroyed. The "inactivated probiotic" or "inactivated LGG" does, however, still retain, at the cellular level, at least a portion its biological glycol-protein and PCT/USZOl-U064315 DNA/RNA structure. As used herein, the term "inactivated" is synonymous with "non-viable”. In some embodiments, the vated LGG is heat-inactivated L66.

"Prebiotic" means a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the digestive tract that can improve the health of the host.

"Phytonutrient” means a chemical compound that occurs naturally in plants. Phytonutrients may be included in any derived substance or extract.

The term "phytonutrient(s)" encompasses several broad categories of compounds produced by plants, such as, for example, polyphenolic compounds, anthocyanins, proanthocyanidins, and flavanols (i.e. catechins, epicatechins), and may be derived from, for example, fruit, seed or tea extracts. r, the term phytonutrient includes all noids, phytosterols, thiols, and other plant-derived nds.

"B-glucan” means all B-glucan, including specific types of B-glucan, such as B-‘I,3-glucan or B-i,3;’|,o-glucan. Moreover, B-i,3;’|,o-glucan is a type of [34,3- glucan. Therefore, the term "B-’|,3-glucan" includes B-i,3;’|,6-glucan. n” means any naturally-occurring oligosaccharide or polysaccharide that comprises galacturonic acid that may be found in the cell wall of a plant. Different varieties and grades of pectin having varied physical and al properties are known in the art. Indeed, the structure of pectin can vary significantly n plants, n tissues, and even within a single cell wall. lly, pectin is made up of negatively charged acidic sugars (galacturonic acid), and some of the acidic groups are in the form of a methyl ester group. The degree of esterification of pectin is a measure of the percentage of the carboxyl groups attached to the galactopyranosyluronic acid units that are esterified with methanol.

Pectin having a degree of esterification of less than 50% (i.e., less than 50% of the carboxyl groups are methylated to form methyl ester groups) are classified as low-ester, low methoxyl, or low methylated (”LM”) pectins, while those having a degree of esterification of 50% or greater than 50%, (i.e., more than 50% of the carboxyl groups are methylated) are classified as high-ester, high methoxyl or high ated ("HM”) pectins. Very low ("VL”) pectins, a subset of low methylated pectins, have a degree of esterification that is less than approximately 15%.

PCT/USZOl-U06-l315 ’I 3 All percentages, parts and ratios as used herein are by weight of the total ional composition, including the stabilized probiotic, unless otherwise specified.

All amounts specified as stered ”per day” may be delivered in one unit dose, in a single serving or in two or more doses or servings administered over the course of a 24 hour period.

The ional ition of the present disclosure may be substantially free of any optional or selected ingredients described herein, provided that the remaining ional composition still contains all of the ed ingredients or features described herein. In this context, and unless otherwise specified, the term ”substantially free” means that the selected composition may contain less than a functional amount of the optional ingredient, typically less than 0.1% by weight, and also, including zero percent by weight of such optional or selected ingredient.

All references to singular characteristics or limitations of the t disclosure shall include the corresponding plural characteristic or limitation, and vice versa, unless otherwise specified or clearly d to the contrary by the context in which the reference is made.

All combinations of method or process steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced ation is made.

The methods and compositions of the present disclosure, including components thereof, can comprise, consist of, or consist essentially ofthe essential elements and limitations of the ments described herein, as well as any additional or optional ingredients, components or limitations described herein or ise useful in nutritional compositions.

As used herein, the term "about" should be ued to refer to both of the numbers specified as the endpoint(s) of any range. Any reference to a range should be considered as providing support for any subset within that range.

THE PROTECTIVE MATRIX In the practice of the present disclosure, hydrolyzed mammalian protein is utilized as a ent of a protective matrix for stabilizing biological material.

W0 2015/084531 PCT/US2014XO64315 Hydrolyzed mammalian protein can be created from a variety of ian 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 r chains of amino acids. Common processes for hydrolyzing protein are known in the art and described in US. Patent No. 601 issued to Conrad; U.S. Patent No. 4,443,540 issued to Chervan et al.; US. Patent No. 4,545,933 issued to Ernster; US. Patent No. 4,757,007 issued to Satoh et al.; US. Patent No. 4,873,108 issued to De Rooij et al.; US. Patent No. ,401,527 issued to Brown et al.; US. Patent No. 6,214,585 issued to Kwong et al.; and US. Patent No. 6,221,423 issued to Cho et al., the sures each of which are hereby incorporated by reference in their entirety.

Mammalian proteins that may be hydrolyzed for use in the stabilization e/protective matrix of the present disclosure include egg ns, animal ns, poultry, meat, serum albumen, glycol proteins, collagen, n, milk proteins, casein, whey protein, albumen and others. In an embodiment, the protective matrix es ian protein, such as 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 yzed protein yielding peptides having a molecular weight of less than about 2,000 Daltons.

In one embodiment, the hydrolyzed mammalian n 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 PCT/USZOl-UO6-l315 the mammalian protein hydrolysate in certain embodiments is over 5,000 Daltons, illustrating that virtually all of the protein was hydrolyzed.

The use of the hydrolyzed mammalian protein(s) in the protective matrix of the present disclosure, including the aforementioned yzed casein, provides superior protection to tics, including LactobaC/Y/us sus, beyond the protection observed through the use of larger e fragments or whole proteins.

While not being bound by any theory, one possibility for the increased protection to the probiotics t both moisture and heat may e the se in the zeta ial 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 on 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 ysis 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 ty. Of course, this does not explain the differences between hydrolyzed mammalian and non-mammalian proteins.

In some ments, the stabilization mixture may comprise between about 5 and about 25 grams of a hydrolyzed protein per 100 grams ofthe mixture on a dry basis. In certain embodiments, the stabilization mixture comprises between about 10 and about 20 grams of a hydrolyzed protein per 100 grams of the mixture on a dry basis. And in an embodiment, the stabilization mixture comprises about 15 grams of a hydrolyzed protein per 100 grams of the mixture on a dry basis. In some embodiments, the yzed n comprises hydrolyzed casein.

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. Indeed, the protective matrix may include lactulose, lactosucrose, raffinose, gluco-oligosaccharide, trehalose, inulin, polydextrose, galacto-oligosaccharide, fructo-oligosaccharide, isomaIto-oligosaccharide, soybean accharides, lactosucrose, xylo- PCT/USZOl-U064315 oligosaccharide, chito-oligosaccharide, oligosaccharide, aribinooligosaccharide , -oligosaccharide, fuco-oligosaccharide, gentio-oligosaccharides, and/or any combination thereof. In some embodiments, the protective matrix includes a first ydrate chosen from: sucrose, e, lactose, ose, maltotriose, maltodextrin having a dextrose equivalent of about 2 to about 6, and any combination thereof. In certain embodiments, the protective matrix includes a second carbohydrate chosen from: inulin, polydextrose, galactooligosaccharide, fructooligosaccharide, starch, maltodextrin having a dextrose equivalent of greater than about 8, and any combination thereof. |n some embodiments, the stabilization mixture may comprise between about 50 and about 80 grams of a first carbohydrate per 100 grams of the mixture on a dry basis; between about 60 and about 70 grams of a first carbohydrate per 100 grams of the mixture on a dry basis; or between about 65 and about 70 grams of a first ydrate per 100 grams of the mixture on a dry basis. The first carbohydrate may be chosen from: sucrose, maltose, e, trehalose, riose, maltodextrin having a dextrose equivalent of about 2 to about 6, and any combination thereof.

The stabilization mixture may also comprise between about ’I and about ‘IO grams of a second carbohydrate per 100 grams of the mixture on a dry basis; between about 4 and about 6 grams of a second carbohydrate per 100 grams of the mixture on a dry basis; or about 5 grams of a second carbohydrate per 100 grams of the mixture on a dry basis. In some embodiments, the second carbohydrate is chosen from: inulin, polydextrose, ooligosaccharide, oligosaccharide, starch, maltodextrin having a dextrose lent of greater than about 8, and any combination thereof.

A further component of the stabilization mixture can be a compound binder (also ed to as a gelling agent), which may act as a thickener and produce a gel-like consistency. Compound binders that may be included in the protective matrix of the present disclosure include alginates, such as sodium alginate, pectin, chitosan, carboxymethylcellulose, and mixtures thereof, among others. The incorporation of the compound binder provides for the ion of a viscous consistency providing for efficient matrix formation and a structural quality suitable for subsequent drying.

W0 2015/084531 PCT/USZOl-IXO64315 A compound binder can, in some embodiments, form a gum-like material and se the viscosity of mixtures to which it is added. Additionally, the compound binder may also provide for greater ease in mixing of the components together. For instance, sodium alginate may also possess emulsifier characteristics.

In some embodiments, the stabilization mixture may comprise LM pectin, HM pectin, VL pectin, or any mixture thereof. The included pectin may be soluble in water.

Pectins for use herein typically have a peak molecular weight of 8,000 Daltons or greater. The pectins of the present disclosure have a preferred peak molecular weight of between 8,000 and about 0, more preferred is between about 10,000 and about 200,000 and most preferred is between about 15,000 and about 100,000 Daltons. In some embodiments, the pectin of the present disclosure may be hydrolyzed pectin. In n embodiments, the protective matrix comprises yzed pectin having a molecular weight less than that of intact or unmodified pectin. The hydrolyzed pectin of the present sure can be prepared by any means known in the art to reduce molecular weight. Examples of said means are al hydrolysis, enzymatic hydrolysis and mechanical shear. A preferred means of reducing the molecular weight is by alkaline or neutral hydrolysis at ed temperature. In some embodiments, the protective matrix comprises partially hydrolyzed . In certain embodiments, the partially hydrolyzed pectin has a molecular weight that is less than that of intact or unmodified pectin but more than 3,300 Daltons.

The stabilization mixture may comprise between about 0.5 and about 5 grams of a compound binder, such as sodium alginate and/or pectin, per 100 grams of the mixture on a dry basis. In n embodiments, the stabilization mixture comprises between about 1 and about 3 grams of a nd binder per 100 grams of the mixture on a dry basis. And in an embodiment, the stabilization mixture comprises about 2 grams of a compound binder per 100 grams of the mixture on a dry basis. er, the ization mixture may also comprise at least one starch, source of starch and/or starch component. In some ments, the stabilization mixture may comprise native or modified starches, such as, for example, PCT/USZOl-U06-I315 ’I 8 waxy corn starch, waxy rice starch, waxy potato starch, waxy a starch, corn starch, rice starch, potato starch, tapioca starch, wheat starch or any mixture thereof.

Furthermore, the stabilization mixture may se a lipid component.

In some embodiments, the stabilization mixture comprises between about 0.5 and about 2 grams of a lipid component per 100 grams ofthe mixture on a dry basis. In certain embodiments, the stabilization mixture comprises between about 0.75 and about 1.25 grams of a lipid ent per 100 grams of the mixture on a dry basis.

And in an embodiment, the stabilization mixture comprises about 1 gram of a lipid component per 100 grams of the mixture on a dry basis. In some embodiments, the lipid component is chosen from: in, monoglycerides, diglycerides, and any combination thereof.

The stabilization mixture may also include additional ingredients that provide further benefits to either the probiotic or the individual ingesting the stabilized tic. These ingredients may comprise ls, vitamins, antioxidants, trace elements, sterols, antioxidants, fatty acids, functional molecules, and any combination thereof. Other ingredients may include resistant starches, high amylose starches, guar, and locust bean gum, agar, xanthan, carrageenans, s, and any combination thereof.

In some embodiments, the stabilization mixture may comprise between about 5 and about 20 grams of probiotic and/or other biological material per 100 grams of the e on a dry basis. In n embodiments, the stabilization mixture comprises between about 9 and about 12 grams of probiotic and/or other biological material per 100 grams of the mixture on a dry basis. And in an embodiment, the stabilization mixture comprises about 11 grams of probiotic and/or other biological material per ’IOO grams of the mixture on a dry basis. In another embodiment, the concentration of the probiotic, for instance LGG, in the protective matrix is from about 1 x 10" to about ’I x 1014 cfu per gram of the tive matrix, more preferably from about 1 x109 to about ’I x 10“ cfu per gram ofthe protective matrix.

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. es of suitable tics include but are not limited to yeasts such as Saccharomyces cereviseae, molds such as i/lus, Rhizopus, Mucor, and bacteria PCT/US201-U064315 such as Lactobac/l/us. Specific examples of suitable probiotic micro-organisms are: Aspergi/lus n/ger, A. oryzae, us coagulans, B. lentus, B. lichen/form/s, B. mesenz‘er/cus, B. pumf/us, B. subfi/l’s, B. natto, B/‘f/‘dobacz‘er/‘um ado/escenz‘is, B. an/h7a/fs, B. breve, B. b/‘fl'o’um, B. infant/'5, B. laCt/s, B. /0ngum, B. /0ngum B8536, B. /0ngum AH1206 (NCIMB: 41382), B. breve AH1205 (NCIMB: 41387), B. infant/5 35624 (NCIMB: 41003), B. /0ngum AH1714 (NCIMB 41676), B. anin7a/I'5 subsp. /aCt/'$ BB-12 (DSM No. 10140), B. pseudo/ongum, B. thermophi/um, Candida epes/I, C/osz‘ridium butyr/cum, Enterococcus cremor/s, E. d/lacez‘y/acz‘is, E faeC/um, E. edius, E. lad/'5, E. i, E. thermophi/us, Lactobac/l/us acidophI/us, L. a/I'menz‘ar/us, L. amy/o vorus, L. crispatus, L. brevis, L. case, L. curvatus, L. cellobiosus, L. o’e/brueC/(i/ ss. bu/gar/cus, L. farcim/n/s, L. fermentum, L. gassed L. he/vet/‘cus, L. 's, L. p/antarum, L. n/f, L. reuteri, L. rhamnosus, Lactobac/l/us rhamnosus GG (ATCC number 53103), L. sake/', L. sa/ivar/us and any combination thereof. In an embodiment, the stabilized probiotic(s) may be viable or non-viable. The stabilized probiotics useful in the present disclosure may be naturally-occurring, tic or developed through the c manipulation of organisms, r such new source is now known or later developed.

In an embodiment of the present disclosure Lactobac/l/us rhamnosus (36 is utilized as a probiotic that may be stabilized by the protective matrix of the present disclosure. LactobaC/Y/us rhamnosusGG is described in U.S. Patent Application 4,839,281, issued to od et al., which is hereby incorporated by reference in its entirety. Notably, Sharwood et al. describes Lactobacfl/us rhamnosus (36 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.

The selected probiotic is preferably concentrated to a wet paste-like tency 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 3X to about 20X though may include lesser or greater concentrations depending upon the specific probiotic biomass and uent processing steps. lly, the preparation of a stabilized probiotic includes the steps of concentrating the ed tic or probiotics; providing components of the 2014f06-l315 stabilization e in desired quantities; mixing the stabilization mixture with the concentrated probiotic; drying the stabilized probiotic and either packaging or combining the ized probiotic into a nutritional product, such as an infant formula.

In some embodiments, the present disclosure is directed to a method for protecting a viable probiotic for use in a ional composition, the method may include the steps of providing a viable tic, preparing a protective matrix for the probiotic by blending (i) hydrolyzed casein, (ii) a first carbohydrate selected from the group consisting of sucrose, e, lactose, trehalose, maltotriose, maltodextrin having a dextrose equivalent of about 2 to about 6, and any combination thereof, (iii) a second carbohydrate selected from the group ting of inulin, polydextrose, ooligosaccharide, fructooligosaccharide, starch, maltodextrin having a dextrose equivalent of greater than about 8, and any combination thereof, and (iv) a lipid selected from the group consisting of lecithin, monoglycerides, erides, and any combination thereof, then combining the viable probiotic, the protective matrix and water to produce a mixture and drying the mixture to a final moisture content of about 4% or less, and further adding the dried mixture to a powdered nutritional product.

In zing the stabilization for probiotic, the le constituents may be varied in some embodiments. In some embodiments, ydrates may comprise from about 70% to about 85% ofthe ization 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.

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.

Table 1 presents a sample embodiment of a stabilized probiotic e/protective matrix according to the present disclosure.

PCT/USZOl-U06-l315 TABLE 1: An embodiment of the tive matrix Ingredient Grams per 100g (dry basis) Probiotic (LGG, AH’I206 or 35624) 11 e, maltose, lactose, trehalose, riose, maltotriose enriched maltodextrin or low DE maltodextrin (preferably 2—6 DE) 69 Hydrolyzed casein 15 Inulin, PDX, GOS, FOS, starch (including modified versions) and/or maltodextrin (>8 DE) Fig. 1 illustrates the stability of LGG in a protective matrix prepared according to the embodiment presented in Table ’I, wherein the ized LGG is provided in a growing up milk having an available water (Aw) content of 0.28. Fig. 2 is a graph that illustrates the relative stability of LGG without a tive matrix in a growing up milk having an available water (Aw) content of 0.28. Fig. 3 presents scanning on micrographs of stabilized LGG in a protective matrix per the formulation presented in Table ’I.

Table 2 provides another example embodiment of a stabilized probiotic mixture/protective matrix according to the present disclosure.

W0 84531 PCT/US2014XO64315 TABLE 2: An embodiment of the protective matrix Ingredient Grams per 100g (dry basis) Probiotic (LGG, AH1206 or 35624) 11 Sucrose, maltose, lactose, trehalose, maltotriose, maltotriose enriched maltodextrin or low DE maltodextrin (preferably 2—6 DE) 67 Hydrolyzed casein 15 Inulin, PDX, GOS, FOS, starch (including modified versions) and/or maltodextrin (>8 DE) Sodium alginate or pectin Table 3 presents yet another example embodiment of a stabilized probiotic mixture/protective matrix according to the t disclosure.

TABLE 3: An embodiment of the protective matrix Ingredient Grams per 100g (dry basis) Probiotic (LGG, AH1206 or 35624) 11 Sucrose, maltose, e, trehalose, riose, maltotriose enriched maltodextrin or low DE maltodextrin (preferably 2-6 DE) 68 Hydrolyzed casein 15 Inulin, PDX, GOS, FOS, starch ding modified versions) and/or maltodextrin (>8 DE) 5 Lecithin, mono- or di-glyceride(s) 1 Table 4 also provides an example embodiment of a stabilized probiotic mixture/protective matrix according to the present disclosure.

ZOl-U06-l315 TABLE 4: An embodiment of the protective matrix Ingredient Grams per 100g (dry basis) Probiotic (LGG, AH1206 or 35624) 11 e, maltose, lactose, ose, maltotriose, maltotriose enriched maltodextrin or low DE maltodextrin (preferably 2—6 DE) 65 Hydrolyzed casein 15 Inulin, PDX, GOS, FOS, starch (including modified versions) and/or maltodextrin (>8 DE) 5 Sodium alginate or pectin 2 Lecithin, mono- or di-glyceride(s) 1 In order to further illustrate the principles and operations of the present disclosure, the following example is ed. However, this example should not be taken as limiting in any .

EXAMPLE Lactobac///us rhamnosus 66 (L66) 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 te. 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/cm3.

The ation 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 ’IOOg/ft2 to 1SOg/ft2.

The mixture is dried ensuring product temperature is below 30°C during l of 80% of the total water. Temperature during removal of the remaining 17% moisture PCT/USZOl-If06-I315 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.

IONAL TS FOR COMBINATION WITH A STABILIZED PROBIOTIC A stabilized probiotic prepared as described hereinabove may be combined with a nutritional product to form a novel nutritional composition.

For example, the stabilized tic may be combined with a nutritional product, such as an infant formula or children’s nutritional t, to form a stabilized nutritional composition. In another embodiment, the stabilized tic may be combined with a human milk ier, which is added to human milk in order to enhance the nutritional value of human milk. r, the stabilized probiotic of the disclosure may be combined with a nutritional product that provides minimal, partial, or total nutritional support. Such nutritional product(s) may be ional supplements or meal replacements. Indeed, the stabilized probiotic can be intermixed with food or other ional products prior to ingestion by a subject.

The nutritional product for combination with the stabilized probiotic may, but need not, be nutritionally complete. Likewise, the combination of the stabilized probiotic with a nutritional product may produce a nutritional composition that is nutritionally complete. In an ment, the ional composition of the disclosure is nutritionally complete and contains suitable types and amounts of lipid, ydrate, protein, vitamins and minerals.

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

The nutritional products described for combining with the stabilized probiotic may be administered enterally.

NUTRITIONAL COMPOSITIONS COMPRISING STABILIZED PROBIOTICS Again, a stabilized/protected probiotic prepared as described above may be combined with a nutritional product to form a novel nutritional composition.

W0 2015/084531 PCT/U82014XO64315 The nutritional composition may comprise any fat or lipid source that is 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; ble 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. The amount of lipid or fat in the ional composition typically varies from about ’I to about 7 9/1 00 kcal.

Further, the nutritional composition may comprise a source of bovine milk protein. The source of bovine milk protein may include, but is not limited to, milk n 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 (eg. sodium caseinate, sodium calcium caseinate, calcium caseinate) and any combination f.

In certain embodiments, the nutritional ition may comprise intact n. In other embodiments, the proteins ofthe nutritional ition are provided as a combination of both intact proteins and partially hydrolyzed proteins, with a degree of ysis of between about 4% and 10%. Certain of these embodiments can be extremely hypoallergenic, as both the stabilizer and the protein of the nutritional t contain only hydrolyzed protein. In yet another embodiment, the nutritional composition may be supplemented with glutamine- containing peptides.

The wheyzcasein ratio of the protein source of the ional composition may be similar to that found in human breast milk. In an embodiment, the protein source of the ional composition comprises from about 40% to about 80% whey protein. In another embodiment, the protein source may comprise from about 20% to about 60% caseins. The amount of protein in a nutritional composition typically varies from about ’I to about 7 9/100 kcal.

In other embodiments the nutritional composition comprises lactoferrin, which retains its stability and activity in the human gut t certain undesirable bacterial pathogens.

W0 2015/084531 PCT/USZOl-UO64315 The nutritional composition described herein can, in some embodiments, also se non-human lactoferrin, non-human lactoferrin produced by a genetically modified organism and/or human lactoferrin produced by a genetically modified organism. Lactoferrin is generally bed as an 80 kilodalton glycoprotein having a structure of two nearly identical lobes, both of which include iron binding sites. As described in " Perspectives on Interactions n Lactoferr/h andBacter/a" which ed in the publication BIOCHEMISTRY AND CELL Y, pp 275-281 (2006), lactoferrin from different host species may vary in an amino acid sequence though commonly possesses a relatively high ctric point with positively charged amino acids at the end terminal region of the internal lobe.

Lactoferrin has been recognized as having bactericidal and antimicrobial activities. In at least one embodiment, the lactoferrin is bovine lactoferrin.

Surprisingly, the forms of lactoferrin included herein maintain nt ty even if exposed to a low pH (i.e., below about 7, and even as low as about 4.6 or lower) and/or high temperatures (i.e., above about 65°C, and as high as about 120°C, conditions which would be expected to destroy or severely limit the ity or ty of human lactoferrin or recombinant human lactoferrin. These low pH and/or high temperature conditions can be expected during certain processing regimen for nutritional compositions of the types described herein, such as pasteurization.

In one embodiment, lactoferrin is present in the nutritional composition in an amount of from about 5 mg/’|00 kcal to about 16 mg/’|00 kcal. In another embodiment, lactoferrin is present in an amount of about 9 mg/’| 00 kcal to about 14 mg/’|00 kcal. In still further ments, the nutritional composition may comprise between about 75 mg and about 200 mg lactoferrin per 100 kcal. And in certain ments, the nutritional composition may comprise between about 90 mg and about 148 mg lactoferrin per 100 kcal.

The nutritional composition may also contain TG F-B. In some embodiments, the level of TG F-B may be from about 0.0150 (pg/pg) ppm to about 0.1000(pg/ug) ppm. In other embodiments, the level of TGF-B in final composition including a stabilized probiotic is from about 0.0225 (pg/pg) ppm to about 0.0750 (pg/pg) ppm.

PCT/USZOl-UO6-I315 In some embodiments of the nutritional composition, the level of TG F-B is from about 2500 pg/mL to about 10,000 pg/mL, more preferably from about 3000 pg/mL to about 8000 pg/mL. In an embodiment, the ratio of TGF-B1: TGF-BZ 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.

In some embodiments, the bioactivity of TG F-B 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 such embodiments provided it achieves the intended result. In an ment, 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.

The nutritional composition may comprise an amount of tic in addition to the stabilized probiotic. When the stabilized probiotic is combined with the nutritional product, the resulting nutritional ition may include a total amount of probiotics effective to provide from about 1 x 104 to about 1 x1010 colony forming units (cfu) per kg body weight per day to a subject. In other embodiments, the amount of the probiotic may vary from about 1 x106 to about 1 x109 cfu per kg body weight per day. In even further embodiments, the nutritional composition may include an amount of probiotics effective to e about 1 x 106 cfu per kg body weight per day.

In certain ments, the ional composition of the present disclosure ses n about 1 x106cfu probiotic and about 1 x101°cfu per 100 kcal of the composition. In some embodiments, the amount of probiotic may be in the range of about 1 x106cfu to about 1 x109cfu per 100 kcal of the composition.

Additionally, the ional composition may include abilized probiotics, with the final composition including some stabilized tics and some non-stabilized probiotics.

The nutritional composition may further comprise at least one prebiotic.

The term "prebiotic” as used herein refers to indigestible food ingredients that exert health benefits upon the host. Such health benefits may include, but are not d 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 PCT/USZOl-U06-I315 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 ped later.

Prebiotics may include oligosaccharides, polysaccharides, and other prebiotics that contain fructose, xylose, soya, ose, glucose and mannose. More specifically, prebiotics useful in the present sure may include lactulose, lactosucrose, raffinose, oligosaccharide, , polydextrose, xtrose powder, galacto- oligosaccharide, fructo-oligosaccharide, isomalto-oligosaccharide, soybean oligosaccharides, lactosucrose, xylo-oligosaccharide, chito-oligosaccharide, mannooligosaccharide , aribino-oligosaccharide, sialyl-oligosaccharide, ligosaccharide, and gentio-oligosaccharides, and combinations thereof.

In some embodiments, the total amount of prebiotics present in the nutritional composition may be from about 1.0 g/L to about 10.0 g/L ofthe composition (in the liquid form). In certain embodiments, the total amount of prebiotics t in the nutritional composition may be from about 2.0 g/L and about 8.0 g/L of the composition.

The nutritional composition may 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. If polydextrose is used as a prebiotic, the amount of polydextrose in the nutritional t may, in an embodiment ofthe composition including stabilized probiotics, be within the range of from about 0.1 mg/100 kcal to about 0.5 mg/100 kcal. In another composition, the amount of polydextrose may be about 0.3 mg/’|00 kcal. At least 20% of the prebiotics should, in a preferred embodiment, comprise polydextrose (PDX).

In certain embodiments, the nutritional composition comprises galacto- oligosaccharide. The amount of galacto-oligosaccharide in the ional composition may be from about 0.2 mg/100 kcal to about 1.0 mg/100 kcal. In other embodiments, the amount of galacto-oligosaccharide in the nutritional composition may be from about 0.1 mg/100 kcal to about 0.5 mg/100 kcal. Galacto— oligosaccharide and polydextrose may also be supplemented into the nutritional composition in a total amount of about 0.6 mg/100 kcal.

W0 2015/084531 PCT/Uszo14m64315 In some embodiments, the nutritional composition comprises an additional carbohydrate source, that is, a carbohydrate source provided in addition to the other ydrates described throughout the present disclosure. Suitable additional carbohydrate sources can be any used in the art, e.g., e, glucose, fructose, corn syrup solids, maltodextrins, sucrose, starch, rice syrup solids, and the like. The amount of additional carbohydrate in the nutritional composition lly can vary from between about 5 g and about 25 9/100 kcal. In some embodiments, the amount of carbohydrate is between about 6 g and about 22 g/1OO kcal. In other embodiments, the amount of carbohydrate is between about 12 g and about 14 g/1OO kcal.

The nutritional composition 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, o-linoleic acid, leic acid, linoleic acid, linolenic acid, eicosapentaenoic acid (EPA) and arachidonic acid (ARA).

In some embodiments, the nutritional composition may be supplemented with both DHA and ARA, and the weight ratio of ARAzDHA may be from about 1:3 to about 9:1. In certain ments the ARAzDHA ratio is from about 1:2 to about 4:1.

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/1OO kcal to about 50 mg/1OO kcal.

Moreover, a nutritional composition may be supplemented with oils containing DHA and ARA using standard techniques known in the art. As an example, the oils containing DHA and ARA may be added to a nutritional composition by ing an equivalent amount of the rest of the overall fat blend normally present in the nutritional ition.

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, ®, 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 . Alternatively, the DHA and ARA can be used in refined form.

W0 2015/084531 PCT/U82014XO64315 In an embodiment of the nutritional composition, sources of DHA and ARA are single cell oils as taught in U.S. Patent Nos. 5,374,567; 5,550,156; and ,397,591, the disclosures of which are orated herein in their entirety by reference.

In certain embodiments, the nutritional composition may be a milk- based nutritional composition that provides physiochemical and physiological ts. 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 tics may be combined with compositions that include softer and smaller curds than standard infant formulas.

One or more ns and/or minerals may also be added in to the nutritional composition in s sufficient to supply the daily nutritional requirements of a subject. It is to be understood by one of ordinary skill in the art that n and mineral requirements will vary, for example, based on the age of the child. For instance, an infant may have different vitamin and mineral requirements than a child between the ages of one and thirteen years. Thus, the embodiments are not intended to limit the nutritional composition to a particular age group but, rather, to e a range of acceptable vitamin and mineral components.

The ional ition may optionally include, but is not limited to, one or more of the following vitamins or derivations thereof: vitamin amin, thiamin osphate, TPP, thiamin triphosphate, TTP, thiamin hydrochloride, thiamin mononitrate), vitamin 82 (riboflavin, flavin mononucleotide, FMN, flavin adenine dinucleotide, FAD, lactoflavin, ovoflavin), vitamin 83 (niacin, nicotinic acid, nicotinamide, niacinamide, nicotinamide adenine eotide, NAD, nicotinic acid mononucleotide, NicMN, pyridinecarboxylic acid), vitamin 83-precursor phan, vitamin Bé (pyridoxine, pyridoxal, pyridoxamine, pyridoxine hydrochloride), pantothenic acid thenate, panthenol), folate (folic acid, folacin, PCT/USZOl-U064315 pteroylglutamic acid), vitamin 812(cobalamin, methylcobalamin, deoxyadenosylcobalamin, obalamin, hydroxycobalamin, adenosylcobalamin), biotin, vitamin C bic acid), vitamin A (retinol, retinyl acetate, l palmitate, retinyl esters with other hain fatty acids, retinal, retinoic acid, retinol ), vitamin D (calciferol, cholecalciferol, vitamin D3, -dihydroxyvitamin D), vitamin E (a-tocopherol, d-tocopherol acetate, d-tocopherol succinate, d-tocopherol nicotinate, G-tocopherol), vitamin K (vitamin K1, phylloquinone, naphthoquinone, vitamin K2, menaquinone-7, vitamin K3, menaquinone-4, menadione, menaquinone-8, inone—8H, menaquinone-9, menaquinone—9H, menaquinone—iO, menaquinone-i’l, menaquinone-’|2, menaquinone-‘I 3), choline, inositol, B-carotene and any combinations thereof.

Further, the nutritional composition may optionally include, but is not d to, one or more of the following minerals or derivations thereof: boron, calcium, m acetate, calcium gluconate, calcium chloride, calcium lactate, calcium phosphate, calcium e, chloride, chromium, chromium chloride, chromium picolonate, copper, copper e, copper gluconate, cupric sulfate, fluoride, iron, yl iron, ferric iron, ferrous fumarate, ferric hosphate, iron trituration, ccharide iron, iodide, iodine, magnesium, magnesium carbonate, ium hydroxide, magnesium oxide, magnesium stearate, magnesium sulfate, manganese, molybdenum, phosphorus, potassium, potassium phosphate, potassium iodide, potassium chloride, potassium acetate, selenium, sulfur, sodium, docusate sodium, sodium de, sodium selenate, sodium molybdate, zinc, zinc oxide, zinc sulfate and mixtures thereof. Non-limiting exemplary derivatives of mineral compounds include salts, alkaline salts, esters and chelates of any mineral compound.

The minerals can be added to nutritional compositions in the form of salts such as calcium phosphate, calcium glycerol phosphate, sodium citrate, potassium chloride, potassium phosphate, magnesium phosphate, ferrous sulfate, zinc sulfate, cupric sulfate, manganese sulfate, and sodium selenite. Additional vitamins and minerals can be added as known within the art.

The nutritional composition of the t disclosure may optionally include one or more of the following flavoring agents, including, but not limited to, ed extracts, volatile oils, cocoa or chocolate flavorings, peanut butter flavoring, cookie crumbs, a or any commercially available flavoring. Examples of useful PCT/USZOl-U06-l315 flavorings include, but are not limited to, pure anise extract, imitation banana extract, imitation cherry extract, chocolate extract, pure lemon extract, pure orange t, pure mint t, honey, imitation pineapple extract, imitation rum extract, imitation strawberry extract, or vanilla extract; or volatile oils, such as balm oil, bay oil, bergamot oil, cedarwood oil, cherry oil, cinnamon oil, clove oil, or peppermint oil; peanut butter, chocolate flavoring, a cookie crumb, butterscotch, toffee, and mixtures thereof. The amounts of flavoring agent can vary greatly depending upon the flavoring agent used. The type and amount of flavoring agent can be selected as is known in the art.

The nutritional composition of the present disclosure may optionally include one or more fiers that may be added for stability of the final product.

Examples of suitable emulsifiers include, but are not limited to, lecithin (9.9., from egg or soy), alpha lactalbumin and/or mono- and di-glycerides, and mixtures thereof.

Other emulsifiers are readily apparent to the skilled artisan and selection of suitable emulsifier(s) will depend, in part, upon the formulation and final product. In some embodiments, nutritional compositions of the present disclosure may comprise emulsifiers such as citric acid esters of mono- and/or diglycerides, diacetyl tartaric acid esters of mono- and/or diglycerides, and/or octenyl succinic anhydride modified starches.

The ional composition of the present disclosure may ally include one or more preservatives that may also be added to extend product shelf life. Suitable preservatives e, but are not limited to, ium sorbate, sodium sorbate, potassium benzoate, sodium benzoate, m disodium EDTA, and es f.

The nutritional composition of the present disclosure may optionally include one or more stabilizers. Suitable stabilizers for use in practicing the nutritional composition of the present disclosure include, but are not limited to, gum arabic, gum ghatti, gum karaya, gum anth, agar, furcellaran, guar gum, gellan gum, locust bean gum, pectin, low methoxyl pectin, gelatin, microcrystalline cellulose, CMC (sodium carboxymethylcellulose), methylcellulose hydroxypropyl methyl cellulose, hydroxypropyl cellulose, DATEM (diacetyl ic acid esters of mono- and diglycerides), dextran, carrageenans, and mixtures thereof.

W0 2015/084531 PCT/USZOl-H064315 The nutritional composition of the present disclosure may further include at least one additional phytonutrient, that is, another phytonutrient component in addition to the pectin, starch or other phytonutrient components described herein. Phytonutrients, or their derivatives, conjugated forms or sors, that are identified in human milk are preferred for inclusion in the nutritional composition. For example, in some embodiments, the nutritional composition of the present disclosure may comprise, in an 8 fl. oz. (236.6 mL) g, n about 80 and about 300 mg yanins, between about 100 and about 600 mg proanthocyanidins, between about 50 and about 500 mg ols, or any combination or mixture thereof. In other embodiments, the nutritional composition ses apple extract, grape seed extract, or a combination or mixture thereof.

Further, the at least one phytonutrient ofthe nutritional composition may be derived from any single or blend of fruit, grape seed and/or apple or tea t(s).

Examples of additional phytonutrients suitable for the nutritional composition include, but are not limited to, anthocyanins, proanthocyanidins, flavan- 3-ols (i.e.. catechins, epicatechins, etc.), flavanones, oids, isoflavonoids, stilbenoids (i.e. resveratrol, etc.) proanthocyanidins, anthocyanins, resveratrol, quercetin, curcumin, and/or any mixture f, as well as any possible combination of utrients in a purified or natural form. Certain components, especially plant- based ents of the nutritional compositions may provide a source of phytonutrients.

The phytonutrient component ofthe nutritional composition may also comprise naringenin, hesperetin, anthocyanins, quercetin, kaempferol, epicatechin, epigallocatechin, epicatechin-gallate, epigallocatechin-gallate or any combination thereof. In certain embodiments, the nutritional ition comprises between about 50 and about 2000 nmol/L epicatechin, between about 40 and about 2000 nmol/L epicatechin gallate, between about 100 and about 4000 nmol/L epigallocatechin gallate, between about 50 and about 2000 nmol/L naringenin, between about 5 and about 500 nmol/L kaempferol, between about 40 and about 4000 nmol/L hesperetin, between about 25 and about 2000 nmol/L anthocyanins, between about 25 and about 500 nmol/L quercetin, or a mixture thereof. rmore, the nutritional composition may comprise the metabolite(s) of a utrient or of its parent compound, or it may comprise other classes of dietary WO 84531 PCT/USZOl-U06-l315 phytonutrients, such as glucosinolate or sulforaphane. In certain embodiments, the nutritional composition comprises carotenoids, such as lutein, zeaxanthin, astaxanthin, lycopene, beta-carotene, alpha-carotene, gamma-carotene, and/or beta- cryptoxanthin.

The nutritional composition may also comprise isoflavonoids and/or isoflavones. Examples include, but are not limited to, genistein (genistin), daidzein in), glycitein, biochanin A, onetin, coumestrol, irilone, orobol, pseudobaptigenin, anagyroidisoflavone A and B, calycosin, glycitein, irigenin, 5-O- genistein, pratensein, prunetin, psi-tectorigenin, retusin, tectorigenin, iridin, ononin, puerarin, tectoridin, derrubone, e, wighteone, alpinumisoflavone, barbigerone, di-O—methylalpinumisoflavone, and 4'-methyl-alpinumisoflavone. Plant sources rich in vonoids, include, but are not limited to, soybeans, psoralea, kudzu, lupine, fava, chick pea, alfalfa, legumes and peanuts.

In an embodiment, the nutritional composition of the present disclosure comprises an effective amount of choline. An effective amount of choline is between about 20 mg choline per 8 fl. oz. (236.6 mL) serving to about 100 mg per 8 fl. oz. (236.6 mL) serving.

The disclosed nutritional composition may additionally comprise a source of B-glucan. Glucans are polysaccharides, specifically polymers of glucose, which are naturally occurring and may be found in cell walls of bacteria, yeast, fungi, and plants. Beta glucans (B-glucans) are themselves a diverse subset of glucose polymers, which are made up of chains of glucose monomers linked together via beta-type glycosidic bonds to form complex carbohydrates.

B-l,3-glucans are carbohydrate polymers purified from, for e, yeast, mushroom, bacteria, algae, or cereals. (Stone BA, Clarke AE. try and Biology of (1-3)-Beta-Glucans. LondonzPortland Press Ltd; 1993. ) The al structure of B-‘I,3-g|ucan depends on the source of the [5-1 ,3-glucan. er, various physiochemical parameters, such as solubility, primary structure, molecular weight, and ing, play a role in biological activities of [5-1 ,3-glucans. (Yadomae T., Structure and biological activities of fungal beta-1,3-glucans. ku . 2000;120:413—431.) B-l,3-glucans are naturally occurring polysaccharides, with or without B- lucose side chains that are found in the cell walls of a variety of plants, yeasts, PCT/USZOl-U064315 fungi and bacteria. ’| ,6-glucans are those containing glucose units with (1,3) links having side chains attached at the (1,6) on(s). B-i o s are a geneous group of glucose polymers that share structural commonalities, including a backbone of straight chain glucose units linked by a [54,3 bond with B- ’|,o-linked glucose es extending from this backbone. While this is the basic structure for the presently described class of B-glucans, some variations may exist.

For example, certain yeast B-glucans have additional regions of B(’|,3) branching extending from the B(’I ,6) branches, which add further complexity to their respective structures.

B-glucans derived from baker's yeast, Saccharomyces siae, are made up of chains of D-glucose molecules connected at the ’I and 3 positions, having side chains of glucose attached at the ’I and 6 positions. Yeast-derived B-glucan is an insoluble, fiber-like, complex sugar having the general structure of a linear chain of glucose units with a B-‘I,3 backbone interspersed with [5-1 ,6 side chains that are generally 6-8 e units in length. More specifically, B-glucan derived from baker’s yeast is poly-(’I ,o)-B-D-glucopyranosyl-(I ,3)-B-D-glucopyranose.

Furthermore, B-glucans are well tolerated and do not e or cause excess gas, abdominal distension, bloating or diarrhea in pediatric subjects. Addition of B-glucan to a nutritional composition for a pediatric subject, such as an infant formula, a growing-up milk or another children’s nutritional product, will improve the subject’s immune response by increasing resistance against invading pathogens and therefore maintaining or improving overall health.

The nutritional composition of the t disclosure may comprise B- . In some embodiments, the B-glucan is B-i,3;’|,o-glucan. In some embodiments, the B-I,3;’I,6-glucan is derived from baker’s yeast. The nutritional composition may comprise whole glucan particle B-glucan, particulate B-glucan, microparticulate B-glucan, PGG-glucan (poly-’I,6-B-D-glucopyranosyl-‘I,3—B-D- glucopyranose) or any mixture thereof. In some embodiments, microparticulate B- glucan comprises B-glucan particles having a er of less than 2 pm.

In some embodiments, the amount of an present in the composition is at between about 0.010 and about 0.080 g per ’IOOg of the nutritional composition. In other embodiments, the nutritional composition comprises between PCT/USZOl-U06-l315 about 10 and about 30 mg an per serving. In another embodiment, the nutritional ition comprises between about 5 and about 30 mg B-glucan per 8 fl. 02. (236.6 mL) serving. In other embodiments, the nutritional composition comprises an amount of B-glucan sufficient to provide between about 15 mg and about 90 mg B-glucan per day. In some embodiments, the nutritional composition may be red in multiple doses to reach a target amount of an delivered to the subject hout the day.

In some embodiments, the amount of B-glucan in the nutritional ition is n about 3 mg and about 17 mg per ’IOO kcal. In another embodiment the amount of B-glucan is between about 6 mg and about 17 mg per 100 kcal.

The nutritional composition may be expelled directly into a subject’s intestinal tract. In some embodiments, the nutritional composition is expelled ly into the gut. In some embodiments, the ition may be formulated to be consumed or administered enterally under the supervision of a physician and may be intended for the specific dietary management of a disease or condition, such as celiac disease and/or food allergy, for which ctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation.

The ional composition of the present disclosure is not limited to compositions comprising nutrients ically listed herein. Any nutrients may be delivered as part of the composition for the purpose of meeting nutritional needs and/or in order to optimize the nutritional status in a subject.

The nutritional composition of the present disclosure may be standardized to a specific caloric content, it may be provided as a ready-to-use t, or it may be provided in a concentrated form.

In some embodiments, the nutritional composition of the present disclosure is a growing-up milk. Growing-up milks are fortified milk-based beverages intended for children over ’| year of age (typically from 1-3 years of age, from 4-6 years of age or from 1-6 years of age). Growing-up milks are designed with the intent to serve as a complement to a diverse diet to provide additional insurance that a child achieves continual, daily intake of all ial vitamins and minerals, macronutrients plus additional functional dietary components, such as non-essential nutrients that have purported health-promoting properties.

PCT/USZOl-U06-I315 The exact composition of a nutritional ition according to the present disclosure can vary from market-to-market, depending on local regulations and dietary intake information of the population of interest. In some embodiments, nutritional compositions according to the disclosure include a milk protein source, such as whole or skim milk, plus added sugar and sweeteners to achieve desired sensory properties, and added vitamins and minerals. The fat composition is typically derived from the milk raw als. Total n can be targeted to match that of human milk, cow milk or a lower value. Total carbohydrate is usually targeted to provide as little added sugar, such as sucrose or fructose, as possible to achieve an able taste. Typically, Vitamin A, calcium and Vitamin D are added at levels to match the nutrient contribution of regional cow milk. Otherwise, in some embodiments, vitamins and minerals can be added at levels that provide approximately 20% ofthe dietary reference intake (DRI) or 20% of the Daily Value (DV) per serving. Moreover, nutrient values can vary between markets depending on the identified nutritional needs of the intended population, raw material butions and regional regulations.

In certain embodiments, the nutritional composition is hypoallergenic.

In other ments, the nutritional composition is kosher. In still further embodiments, the nutritional composition is a non-genetically modified product. In an embodiment, the nutritional formulation is sucrose-free. The nutritional composition may also be lactose-free. In other embodiments, the ional composition does not contain any medium-chain triglyceride oil. In some embodiments, no carrageenan is present in the composition. In other embodiments, the nutritional composition is free of all gums. ingly, by the practice of the present disclosure, stabilized probiotics having heretofore gnized stability are prepared. The stabilized ial mixture exhibits exceptionally high ity 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 s. The stabilized probiotics are ly effective for nutritional applications with intermediate moisture levels (such as water ty as high as 0.4) where increased shelf life and stability in hot and humid environments are desired. The stabilized probiotics may be W0 2015/084531 PCT/USZOl-U064315 packed separately or be combined with any of the embodiments of nutritional compositions described herein.

All references cited in this specification, including t limitation, all papers, ations, patents, patent applications, tations, 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 ofthe 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 cy and pertinence of the cited references.

Although preferred embodiments of the disclosure have been described using specific terms, devices, and methods, such description is for rative 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 t departing from the spirit or the scope ofthe t disclosure, which is set forth in the ing claims. In addition, it should be tood 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 ofthe preferred versions contained therein.

Claims (11)

1. A nutritional composition comprising a lipid or fat ; a protein source; and a probiotic stabilized in a protective matrix, the protective matrix comprising, a. a hydrolyzed protein; b. a first carbohydrate chosen from e, maltose, lactose, trehalose, maltotriose, maltodextrin having a dextrose equivalent of 2 to 6, and any combination f; c. a second carbohydrate chosen from , polydextrose, galactooligosaccharide, fructooligosaccharide, starch, maltodextrin having a dextrose equivalent of greater than 8, and any combination f; and d. a lipid chosen from lecithin, ycerides, diglycerides, and any combination f.

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 matrix further comprises a compound binder selected from sodium alginate, pectin, and any combination thereof.

5. The composition of claim 1, wherein the matrix further comprises .

6. The composition of claim 1, wherein at least 20% of the total hydrolyzed protein is comprised of protein having a molecular weight of less than 2000 s.

7. The composition of claim 1 wherein the hydrolyzed protein comprises from 10 percent to 20 percent (w/w) of the protective matrix on a dry basis.

8. The composition of claim 1, w herein the hydrolyzed protein comprises hydrolyzed casein.

9. The composition of claim 1, wherein the yzed protein consists of hydrolyzed casein.

10. The composition of claim 1, wherein the nutritional composition is a powdered infant formula.

11. A method for protecting a viable tic for use in a powdered ional composition, the method comprising: a. providing a viable probiotic; b. preparing a protective matrix for the probiotic by blending (i) hydrolyzed casein, (ii) a first carbohydrate chosen from sucrose, maltose, lactose, trehalose, maltotriose, maltodextrin having a dextrose equivalent of 2 to 6, and any combination thereof, (iii) a second ydrate chosen from inulin, polydextrose, galactooligosaccharide, fructooligosaccharide, starch, maltodextrin having a dextrose equivalent of greater than 8, and any combination thereof and (iv) a lipid chosen from lecithin, monoglycerides, diglycerides, and any combination thereof; c. combining the viable probiotic, the protective matrix and water to produce a mixture; d. drying the mixture of step (c) to a final moisture t of 4% or less; and e. adding the dried e of step (d) to a powdered nutritional product. 12 The method of claim 11, wherein the viable probiotic comprises Lactobacillus rhamnosus.