HK1242529A1 - Composition comprising heat labile milk proteins and method for preparing same - Google Patents

Description

Composition comprising heat-labile milk protein and method for preparing same

The application is a divisional application of an invention patent application with an international application number of 201180064301.5, namely, international application PCT/US2011/064054 which has an international application date of 2011, 12, month and 9 and enters China, and the invention is entitled "a composition containing heat-labile milk protein and a preparation method thereof".

Technical Field

The present disclosure relates generally to the field of nutritional compositions such as infant formulas, human milk fortifiers, pediatric dietary supplements, and the like comprising heat labile milk proteins and methods for preparing such compositions.

Background

Several proteins found naturally in milk have useful biological activities. These proteins can be found in whole milk as well as whey, casein or other milk protein fractions or isolates. For example, the protein lactoferrin found in human and non-human milk has a variety of different antibacterial and antiviral activities. Other milk proteins comprising lactoperoxidase and milk agglutinin (milk fat globule-EGF factor 8 protein) are also considered beneficial for reducing the risk of infection. Therefore, it is desirable to try to include these bioactive proteins in dairy dietary compositions for humans, such as infant formulas.

Unfortunately, attempts to include bioactive proteins in dairy based dietary compositions are often thwarted by the fact that the biological activity of certain dairy proteins can be lost or significantly reduced under the temperature conditions typically used to provide hygienic dairy compositions for human consumption. More particularly, many milk proteins are denatured or inactivated by the heat processing method. For example, lactoferrin and other bioactive milk proteins such as lactoperoxidase and milk agglutinin are somewhat unstable when subjected to pasteurization conditions such as 72 ℃ for 15 seconds. Other milk proteins that undergo denaturation or inactivation under high heat conditions are lactoferricin and transforming growth factor (TGF- β). Such proteins are particularly vulnerable to more severe processing conditions, such as processing at 130 ℃ to 145 ℃.

It would therefore be beneficial to provide a method for preparing a nutritional composition such as an infant formula, a human milk fortifier, a pediatric dietary supplement, etc., that has been subjected to high temperature processing conditions, but comprises a biologically active heat labile milk protein.

Disclosure of Invention

Briefly, the present disclosure, in one embodiment, relates to a method for preparing a composition. In one embodiment, the method comprises: a) providing a first composition comprising a fat or lipid source and a protein source and subjecting the first composition to a temperature of at least about 130 ℃; b) providing a second composition comprising heat-labile milk protein; and c) mixing the first composition with a second composition to form a third composition comprising a fat or lipid source, a protein source and a heat labile milk protein. In a preferred embodiment, the first and third compositions are nutritional compositions.

In some embodiments, the first composition comprises up to about 7 g/100kcal of a fat or lipid source, more preferably about 3 g/100kcal to about 7 g/100kcal of a fat or lipid source, and up to about 5 g/100kcal of a protein source, more preferably about 1g/100 kcal to about 5 g/100kcal of a protein source.

Preferably, the heat-labile milk protein in the second composition is lactoferrin, lactoperoxidase lactoferricin, TGF- β and/or milk agglutinin, more preferably the heat-labile milk protein is lactoferrin. Particularly preferably, the heat-labile milk protein is lactoferrin prepared from a non-human source.

In some embodiments, the second composition is a solution, preferably an aqueous solution comprising water, which has been used for food processing, for example, by reverse osmosis, Ultraviolet (UV) light treatment, irradiation, electrical pulses, hyperthermia, and the like. In one embodiment, the second composition has been filtered prior to mixing it with the first composition. In another embodiment, the heat labile milk protein has undergone sterilization upon mixing the second composition with the first composition. In another embodiment, the method further comprises aseptically packaging the third composition. Neither the second composition nor the third composition experiences a temperature greater than about 80 ℃.

In another embodiment, the present disclosure relates to a method for preparing a composition comprising the steps of: mixing a first composition comprising a fat or lipid source and a protein source that has been subjected to a temperature of at least about 130 ℃ with a second composition comprising heat labile milk protein that has not been subjected to a temperature of about 80 ℃ or greater to form a third composition comprising a fat or lipid source, a protein source, and heat labile milk protein; and aseptically packaging the third composition.

Many other objects, features and advantages of the present disclosure will become readily apparent to those skilled in the art upon a reading of the following description in conjunction with the accompanying drawings.

Drawings

The figure is a flow chart illustrating one embodiment of the disclosed method.

Detailed Description

In one embodiment, the present disclosure relates to a process for preparing a composition comprising a fat or lipid source, a protein source and a heat labile milk protein, comprising the steps of: a) subjecting a first composition comprising a fat or lipid source and a protein source to a temperature of at least about 130 ℃; b) providing a second composition comprising heat-labile milk protein; and c) mixing the first composition with the second composition to form a third composition comprising a fat or lipid source, a protein source and a heat labile milk protein. In preferred embodiments, the second composition is not subjected to a temperature of about 80 ℃ or greater.

Suitable fat or lipid sources for inclusion in the first composition may be any known or used in the art, including but not limited to animal sources such as milk fat, cream, butter fat, egg yolk lipids; marine sources such as fish oil, marine oil, single cell oil; vegetable and vegetable oils such as corn oil, canola oil, sunflower oil, soybean oil, palm olein, coconut oil, high oleic sunflower oil, evening primrose oil, rapeseed oil, olive oil, linseed (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 combination thereof.

In some embodiments, the protein source comprised in the first composition comprises milk protein. Milk protein sources for inclusion in the first composition include, but are not limited to, milk protein powder, milk protein concentrate, milk protein isolate, skim milk solids, skim milk powder, whey protein isolate, whey protein concentrate, sweet whey, acid whey, casein, acid casein, caseinate (e.g., sodium caseinate, sodium calcium caseinate, calcium caseinate), and any combination thereof.

In one embodiment, the protein is provided as an intact protein. In other embodiments, the protein is provided as a combination of both intact protein and partially hydrolyzed protein having a degree of hydrolysis of about 4% to 10%. In other embodiments, the protein is extensively hydrolyzed, with a degree of hydrolysis greater than 15%, even greater than 50% and even as high as 90% or higher. In another embodiment, the protein source may be supplemented with a glutamine-containing peptide.

In particular embodiments of the present disclosure, the protein source comprises whey protein and casein and the ratio of whey protein to casein is similar to that found in breast milk. For example, in some embodiments, the weight ratio of whey protein to casein is from about 20% whey to 80% casein to about 80% whey to 20% casein.

In some embodiments, the first composition and/or the third composition may be classified as an infant formula. The term "infant formula" applies to compositions in liquid or powder form that meet the nutritional needs of an infant by acting as a substitute for breast milk. In the united states, the contents of infant formulas are regulated by federal regulations set forth in 21 c.f.r. § 100, 106 and 107. These regulations define macronutrient, vitamin, mineral, and other ingredient levels in an attempt to mimic the nutritional and other properties of breast milk. In a separate embodiment, the first composition and/or the third composition may be a human milk fortifier, meaning that it is a composition that is added to human milk to increase the nutritional value of the human milk. As a human milk fortifier, the third composition may be in powder form or liquid form. In another embodiment, the disclosed first and/or third compositions may be a children's nutritional composition.

The first composition may be subjected to a temperature of 130 ℃ using equipment and methods familiar to the skilled person. Preferably, the first composition is subjected to a temperature of from about 130 ℃ to about 150 ℃ for a period of time of at least about 1 second. More preferably, the first composition is subjected to a temperature of from about 130 ℃ to about 150 ℃ for a time of from about 3 seconds to about 30 seconds.

A first composition comprising a fat or lipid source and a protein source is mixed with a second composition comprising a heat labile milk protein.

As used herein, a "heat labile milk protein" is protein 1) or is a protein that is found naturally in the milk of at least one type of mammal (i.e., a protein having substantially the same amino acid sequence as a protein found naturally in the milk of at least one type of mammal), or is an amino acid variant of a protein found naturally in the milk of at least one type of mammal; and 2) its biological activity is lost or diminished when subjected to elevated temperatures, such as temperatures greater than 80 ℃ or, in some cases, at least about 130 ℃. Preferably, the heat-labile milk protein is naturally found in the milk of at least one type of mammal. Such proteins may be isolated, for example, from the milk of at least one type of mammal or prepared by transgenic organisms. In another embodiment, the heat-labile milk protein is an amino acid variant of a protein found naturally in the milk of at least one type of mammal, prepared by, for example, removing, replacing, or adding one or more amino acids from or to the amino acid sequence of a protein found naturally in the milk of at least one type of mammal. Heat labile milk proteins useful in the present disclosure include, but are not limited to, lactoferrin, milk agglutinin, lactoperoxidase, lactoferricin, TGF- β, lysozyme, and immunoglobulins. Preferably, the heat-labile milk protein is lactoferrin, milk agglutinin and/or lactoperoxidase. Particularly preferably, the heat-labile milk protein is lactoferrin.

Lactoferrin is an approximately 80 kD single chain polypeptide containing 1-4 polysaccharides depending on the species. The 3-D structures of different classes of lactoferrin are very similar, but not identical. Each lactoferrin comprises two homologous leaves, called N-leaf and C-leaf, which refer to the N-terminal and C-terminal parts of the molecule, respectively. Each leaf also consists of two cotyledons or domains, which form a cleft in which the iron ion (Fe)³⁺) Is tightly bound in a coordinated fashion with the (bi) carbonate anion. These domains are referred to as N1, N2, C1, and C2, respectively. The N-terminus of lactoferrin has a strong cationic peptide region responsible for many important binding properties. Lactoferrin has a very high isoelectric point (about pI9) and its cationic nature plays a major role in its ability to resist bacterial, viral and fungal pathogens. N-terminal region of lactoferrin in modulating biological activity of lactoferrin against various microorganismsWithin the domain are several clusters of cationic amino acid residues. For example, the N-terminal residues 1-47 of human lactoferrin (1-48 of bovine lactoferrin) are critical for the iron-independent biological activity of lactoferrin. In human lactoferrin, residues 2 to 5(RRRR) and 28 to 31(RKVR) are arginine-rich cationic domains in the N-terminus, which is particularly critical for the antibacterial activity of lactoferrin. A similar region of the N-terminus is found in bovine lactoferrin (residues 17 to 42; FKCRRWQWRMKKLGAPSITCVRRAFA).

As published in publication Biochemistry and Cell Biology, pages 275-281 (2006) "Perspectives on Interactions Between Lactoferrin and Bacteria"lactoferrin derived from different host species may differ in their amino acid sequence, although it usually has a relatively high isoelectric point and positively charged amino acids in the terminal regions of the inner lobe. Suitable lactoferrin for use in the present disclosure includes those having at least 48% homology at the HLf (349-364) fragment to the amino acid sequence avgeqelrckcnwqwsgl. In some embodiments, lactoferrin has at least 65% homology at the HLf (349-364) fragment to the amino acid sequence avgeqelcrkcnwqwsgl, and in embodiments, at least 75% homology. For example, non-human lactoferrin for use in the present disclosure includes, without limitation, bovine lactoferrin, porcine lactoferrin, equine lactoferrin, buffalo lactoferrin, ovine lactoferrin, murine lactoferrin, and camel lactoferrin.

In a preferred embodiment, the lactoferrin is lactoferrin prepared by a non-human source. As used herein, "lactoferrin prepared by a non-human source" refers to lactoferrin prepared by or obtained from a source other than breast milk. For example, in some embodiments, the lactoferrin is human lactoferrin and/or non-human lactoferrin produced by a transgenic organism. As used herein, the term "organism" refers to any continuous living system such as an animal, a plant, a fungus, or a microorganism. As used herein, the term "non-human lactoferrin" refers to lactoferrin having an amino acid sequence that is different from the amino acid sequence of human lactoferrin. It is also preferred that lactoferrin is not hydrolysed. In one embodiment, the lactoferrin is bovine lactoferrin. In some embodiments, the lactoferrin is provided as an isolate and, among other things, as a component of the concentrated whey fraction.

A process for preparing bovine lactoferrin at high purity is disclosed in U.S. patent No. 4,791,193 to okinogi et al, which is incorporated herein by reference in its entirety. Generally, the disclosed method comprises three steps. The starting milk material is first contacted with a weakly acidic cation exchanger to absorb lactoferrin, followed by a second step in which washing takes place to remove unabsorbed material. A desorption step then occurs in which lactoferrin is removed to produce pure bovine lactoferrin. Other methods may include the steps described in U.S. Pat. nos. 7,368,141, 5,849,885, 5,919,913, and 5,861,491, the disclosures of which are all incorporated herein by reference in their entireties.

In some embodiments, the heat labile milk protein has been subjected to sterilization (no temperature above 80 ℃ applied) prior to mixing with the first composition. In one embodiment, the second composition is filtered through one or more filters (preferably the filter itself has been subjected to sterilization) prior to mixing with the first composition.

It is to be understood that in some embodiments, the first composition itself, which is subjected to a temperature of at least about 130 ℃, may comprise heat labile milk proteins. This may be the case if, for example, the biological activity of the heat labile milk protein is not critical. Thus, in some embodiments, a first composition comprising a fat or lipid source, a protein source, and a heat-labile milk protein is subjected to a temperature of at least about 130 ℃ prior to mixing with a second composition comprising a heat-labile milk protein (preferably a different heat-labile milk protein present in the first composition), wherein the second composition is not subjected to a temperature of greater than about 80 ℃.

The step of mixing the first and second compositions can be accomplished by methods familiar to the skilled artisan. More particularly, in some embodiments, the mixing of the first and second compositions may be accomplished by aseptic dosing and may be performed in a continuous or batch operation. For example, in one embodiment, the second composition is an aqueous solution, more preferably an aqueous solution comprising water, which has been used for food processing, for example by reverse osmosis treatment. The solution is then added to the first composition, which is preferably in liquid form. In a particularly preferred embodiment, the first composition is in the form of a liquid composition and the second composition is in the form of a solution that has been subjected to sterilization and a stream of the second composition is administered to the first composition.

In another preferred embodiment, the method of preparing a composition comprising heat-labile milk protein comprises: a) subjecting a liquid nutritional composition comprising a fat or lipid source and a protein source to a temperature of at least about 130 ℃; b) preparing a solution comprising heat-labile milk protein; c) subjecting the solution to disinfection; and d) mixing the liquid nutritional composition with the solution.

In another preferred embodiment, the third composition comprising lactoferrin is prepared by a process comprising: a) subjecting a liquid nutritional composition comprising a fat or lipid source and a protein source to a temperature of at least about 130 ℃ to form a first composition; b) preparing a solution comprising lactoferrin at a lactoferrin concentration of at least 1%; in some embodiments, the lactoferrin concentration is about 1% to about 30%, and in other embodiments, the lactoferrin concentration is about 1% to about 20% to form a second composition; c) subjecting the second composition to sterilization at a temperature of no greater than 80 ℃; and d) mixing the first composition with the second composition to form a third composition. In one embodiment, the process comprises preparing a solution comprising 1% to 20% lactoferrin and water that has been used for food processing, for example by reverse osmosis treatment. Also preferably, the step of subjecting the second composition to sterilization comprises filtering the second composition through one or more filters (preferably the filters themselves have been subjected to sterilization) at a temperature of less than about 60 ℃ and preferably from about 4 ℃ to about 60 ℃. In one embodiment, the pH of the second composition when in solution is about 2 to 7. In some embodiments, the step of mixing the liquid nutritional composition (i.e., the first composition) with the lactoferrin solution (i.e., the second composition) to form the third composition comprises administering a stream of the lactoferrin solution to the liquid nutritional composition.

In one embodiment, the amount of the second composition comprising lactoferrin mixed with the first composition is selected such that lactoferrin is present in the third composition in an amount of about 0.1 g/L to about 2 g/L. In another embodiment, the amount of the second composition comprising lactoferrin mixed with the first composition is selected such that lactoferrin is present in the third composition in an amount of at least about 10 mg/100kCal, particularly when the nutritional composition is intended for use by children. In some embodiments, the upper limit of lactoferrin in the third composition is about 300 mg/100 kCal. In another embodiment, where the third composition is an infant formula, the lactoferrin is present in the third composition in an amount of about 70 mg to about 220 mg/100 kCal; in another embodiment, lactoferrin is present in the third composition in an amount of about 90 mg to about 190 mg/100 kCal.

After mixing the first and second compositions, additional processing steps can be performed on the third composition, provided that any such additional processing steps do not result in inactivation or denaturation of any intact heat labile proteins in the third composition. For example, in a preferred embodiment, the third composition is aseptically packaged immediately after the step of mixing the first and second compositions, or after one or more additional steps. In another embodiment, immediately after the step of mixing the first and second compositions or after one or more additional steps, a third composition is combined with the already sterilized package and sealed under sterile conditions, for example at a temperature of about 4 ℃ to about 30 ℃. In another embodiment, after mixing the first and second compositions, the third composition is reduced to powder form by, for example, freeze-drying. The powder can then be reconstituted in liquid form by, for example, adding the powder to milk or water prior to administration to a human.

The third composition prepared by the methods disclosed herein may provide minimal, partial, or complete nutritional support. The composition may be a nutritional supplement or a meal replacement. In some embodiments, the composition may be administered in conjunction with a food or additional nutritional composition. In this embodiment, the composition can be mixed with a food or other nutritional composition prior to ingestion by the individual or can be administered to the individual prior to or after ingestion of the food or nutritional composition. In some embodiments, the third composition is administered to an infant or child. "Children (child)" and "children (children)" are defined as persons from 12 months to about 12 years of age. The term "infant" is generally defined as a person from birth to 12 months of age. In some embodiments, the composition may be administered to a preterm infant receiving infant formula, human milk, a human milk fortifier, or a combination thereof. "preterm infant" is an infant born after less than 37 weeks of gestation, whereas "term infant" refers to an infant born after at least 37 weeks of gestation.

The third composition may, but need not, be an infant formula and may be nutritionally complete. The skilled artisan will appreciate that "nutritionally complete" will vary depending on a variety of factors including, but not limited to, the age, clinical condition, and dietary intake of the individual to whom the term is applied. Generally, "nutritionally complete" means that the compositions of the present disclosure provide all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals, and energy in sufficient amounts for normal growth. The term "essential" when applied to nutrients means any nutrient that cannot be synthesized by the body in sufficient amounts for normal growth and to maintain health, and therefore must be provided by the diet. The term "conditionally essential" when applied to nutrients refers to nutrients that must be provided by the diet under conditions where the body cannot obtain sufficient amounts of precursor compounds for endogenous synthesis to occur.

A "nutritionally complete" composition for preterm infants will by definition provide all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals and energy in sufficient quantities in mass and quantities required for the growth of the preterm infant. A "nutritionally complete" composition for term infants will by definition provide all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals and energy in sufficient amounts, in mass and quantity, required for growth of the term infant. A "nutritionally complete" composition for children will, by definition, provide all carbohydrates, lipids, essential fatty acids, proteins, essential amino acids, conditionally essential amino acids, vitamins, minerals and energy in sufficient amounts, in mass and in amounts, required for growth of the child.

The third composition may be provided in any form known in the art, including a powder, gel, suspension, paste, solid, liquid concentrate, or ready-to-eat product. In a preferred embodiment, the third composition is an infant formula, in particular an infant formula suitable for use as the sole nutritional source for an infant.

In a preferred embodiment, the third composition may be administered enterally. As used herein, "enteral" means through or within the gastrointestinal tract or alimentary canal and "enteral administration" includes oral feeding, intragastric feeding, transpyloric administration, or any other means of introduction into the alimentary canal.

Preferably, the third composition prepared according to the present disclosure comprises one or more prebiotics, one or more probiotics, and/or one or more sources of long chain polyunsaturated fatty acids. As used herein, the term "probiotic" refers to microorganisms with low or no pathogenicity that exert beneficial effects on the health of the host and the term "prebiotic" refers to non-digestible food ingredients that beneficially affect the host by selectively stimulating host growth and/or activity or a limited number of bacteria in the colon that are capable of improving host health.

The inclusion of one or more prebiotics, one or more probiotics, and/or one or more sources of long chain polyunsaturated fatty acids (LCPUFAs) in the compositions of the present disclosure can be accomplished in a variety of ways. For example, in one embodiment, one or more of these components, such as prebiotics and/or long chain polyunsaturated fatty acids, are included in the first composition prior to subjecting the composition to a temperature of at least about 130 ℃. In another embodiment, one or more of these components, such as prebiotics and/or long chain polyunsaturated fatty acids, are present in the first composition after subjecting the composition to a temperature of at least about 130 ℃ but prior to mixing with the second composition. In other embodiments, any probiotic included is added to the third composition, for example during aseptic processing. Preferably, if it is desired to include a source of prebiotic, probiotic bacteria and/or long chain polyunsaturated fatty acids which lose their activity after being subjected to such temperature conditions, it is included in the third or second composition or added after the first composition has been subjected to a temperature of at least about 130 ℃.

As noted above, in one embodiment, one or more probiotics may be included in accordance with the present disclosure. Any probiotic known in the art may be acceptable in this embodiment as long as it achieves the desired effect. In a particular embodiment, the probiotic may be selected from lactobacillus (lactobacillus: (a)Lactobacillusspecies), Lactobacillus rhamnosus (Lactobacillus rhamnosus: (A)Lactobacillus rhamnosusGG), Bifidobacterium (Bifidobacteriumspecies), Bifidobacterium breve ((II)Bifidobacterium brevis) Bifidobacterium longum (b)Bifidobacterium longum) And Bifidobacterium animalis subsp lactis BB-12(Bifidobacterium animalis subsp. lactisBB-12)。

If included, the amount of probiotic in the third composition may be from about 10⁴To about 10¹⁰Colony forming units (cfu) per kg body weight per day. In another embodiment, the amount of probiotic may be from about 10⁶To about 10⁹cfu per kg body weight per day. In another embodiment, the amount of probiotic may be at least about 10⁶cfu per kg body weight per day. In addition, the disclosed compositions may also comprise probiotic conditioned media components.

In one embodiment, the one or more probiotics are viable. In another embodiment, the one or more probiotics are non-viable. As used herein, the term "viable" refers to living microorganisms. The term "non-viable" or "non-viable probiotic" refers to non-living probiotic microorganisms, their cellular components and their metabolites. Such non-viable probiotics may have been killed or otherwise inactivated by heating but retain the ability to beneficially affect the health of the host. The probiotics for use in the present disclosure may be naturally occurring, synthetic, or bred by genetic manipulation of organisms, whether such new sources are currently known or later developed. If a viable probiotic is used, preferably the probiotic is included in the second or third composition.

The compositions according to the present disclosure may also use one or more prebiotics. Such prebiotics may be naturally occurring, synthetic or bred by genetic manipulation of organisms and/or plants, whether such new sources are currently known or later developed. In some embodiments, prebiotics included in the compositions of the present disclosure include those taught by U.S. patent No. 7,572,474, the disclosure of which is incorporated herein by reference. Prebiotics for use in the present disclosure may include oligosaccharides, polysaccharides, and other prebiotics, which include fructose, xylose, soy, galactose, glucose, and mannose. More particularly, prebiotics for use in the present disclosure may include lactulose, lactosucrose, raffinose, oligo-glucose, inulin, polydextrose powder, galacto-oligosaccharide, fructo-oligosaccharide, isomalto-oligosaccharide, soy oligosaccharide, lactosucrose, xylo-oligosaccharide, chito-oligosaccharide (chito-oligosaccharide), oligomannose, arabino-oligosaccharide, sialyl-oligosaccharide, trehalose-oligosaccharide, and gentiooligosaccharide. Preferably, the prebiotic is polydextrose and/or galactooligosaccharide. Optionally, one or more additional prebiotics are used in accordance with the present disclosure in addition to the polydextrose and/or galactooligosaccharide.

In one embodiment, the prebiotic is included such that the total amount of prebiotic present in the third composition is from about 0.1g/100 kcal to about 1g/100 kcal. More preferably, the total amount of prebiotics present in the third composition is from about 0.3 g/100kcal to about 0.7 g/100 kcal. At least 20% of the prebiotics should comprise Galactooligosaccharides (GOS) and/or Polydextrose (PDX).

If polydextrose is used, the amount of polydextrose in the third composition may be in one embodiment from about 0.1g/100 kcal to about 1g/100 kcal. In another embodiment, the amount of polydextrose in the third composition is from about 0.2g/100 kcal to about 0.6 g/100 kcal.

If galactooligosaccharides are used, the amount of galactooligosaccharides in the third composition may be in one embodiment from about 0.1g/100 kcal to about 1g/100 kcal. In another embodiment, the amount of galactooligosaccharide in the third composition may be from about 0.2g/100 kcal to about 0.5 g/100 kcal. In some embodiments, the ratio of polydextrose to galactooligosaccharide in the third composition is from about 9:1 to about 1: 9.

One or more sources of long chain polyunsaturated fatty acids may also be used in accordance with the present disclosure. Preferably, the source of LCPUFAs comprises docosahexaenoic acid (DHA). Other suitable LCPUFAs include, but are not limited to, alpha-linoleic acid, gamma-linoleic acid, linolenic acid, eicosapentaenoic acid (EPA), and arachidonic acid (ARA).

In one embodiment, the first composition is supplemented with DHA and ARA. In this embodiment, the weight ratio of ARA to DHA may be from about 1:3 to about 9: 1. In one embodiment of the disclosure, the weight ratio of ARA to DHA is from about 1:2 to about 4: 1.

The amount of long chain polyunsaturated fatty acids in the third composition can vary from about 5 mg/100kcal to about 100 mg/100kcal, more preferably from about 10 mg/100kcal to about 50 mg/100 kcal.

The composition may be supplemented with oils containing DHA and ARA using standard techniques known in the art. For example, DHA and ARA may be added to the composition by replacing an equal amount of oil, such as high oleic sunflower oil, typically present in the composition. As another example, DHA and ARA containing oils may be added to the composition by replacing the remainder of the total fat mixture in an equal amount that is normally present in the composition (without DHA and ARA).

If used, the sources of DHA and ARA may be any known in the art, such as marine oils, fish oils, single cell oils, egg yolk lipids and brain lipids. In some embodiments, the DHA and ARA are derived from single cell Martek oil, DHASCO ® and ARASCO @ or variants thereof, respectively. DHA and ARA can be in native form, provided that the remainder of the LCPUFA source does not cause any significant detrimental effect on the individual. Alternatively, DHA and ARA can be used in a purified form.

In one embodiment of the disclosure, the sources of DHA and ARA are, for example, as described in No. 5,374,567; U.S. Pat. No. 5,550,156; and single cell oil as taught in U.S. patent No. 5,397,591, the disclosures of which are incorporated herein by reference in their entirety. However, the present disclosure is not limited to only such oils.

In particular embodiments, TGF- β is a heat-labile protein present in accordance with the present disclosure. TGF-. beta.may be present in its inactive form in the protein sources used herein. It is then activated in the human intestine by enzymes, extremes of pH and/or fission. In particular embodiments, the compositions of the present disclosure enhance the bioavailability or bioactivity of TGF- β in the human gut. This may include enhancing TGF- β signalling in humans. In one embodiment, the compositions of the present disclosure may enhance the biological activity of TGF- β in the human intestinal tract by at least about 5%, more advantageously at least about 15% or even at least about 25% or more, up to about 65%.

In particular embodiments of the present disclosure, the third composition comprises about 0.0150(pg/μ g) ppm to about 0.1000(pg/μ g) ppm of TGF- β. In another embodiment, the level of TGF- β in the third composition is about 0.0225(pg/μ g) ppm to about 0.0750(pg/μ g) ppm.

In particular embodiments of the present disclosure, the level of TGF- β in the third composition disclosed is from about 500 pg/mL to about 10,000 pg/mL of the composition, more preferably from about 3000 pg/mL to about 8000 pg/mL.

In one embodiment, the third composition is disclosed as having a ratio of TGF- β 1 to TGF- β 2 of about 1:1 to about 1:20 or more specifically about 1:5 to about 1: 15.

In some embodiments, the biological activity of TGF- β in the composition is enhanced by the addition of a biologically active whey fraction^®800 from Glanbia Nutritions. In a particular embodiment, Salibra^®800 whey protein concentrate is 2.5% acidified. In another embodiment, Salibra^®800 whey protein concentrate is 5% acidified. In another embodiment, Salibra^®800 whey protein concentrate is 2% acidified. In other embodiments, Salibra^®800 whey protein concentrate is 3% acidified.

In another embodiment, the Whey protein concentrate may be Nutri Whey 800, available from DMVInternational. In another embodiment, the whey protein concentrate may be Salibra-850, available from GlanbiaNutritions. In another embodiment, the whey protein concentrate may be Prolacta lactalis WPI90 available from lactlus industries u.s.a., Inc. In other embodiments, the whey protein concentrate may be provided by MGNutritionals.

Examples

The following examples are provided to illustrate embodiments of the nutritional compositions of the present disclosure and should not be construed as limiting in any way. Other embodiments within the scope of the claims herein will be apparent to those skilled in the art from consideration of the specification or practice of the nutritional compositions or methods disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the disclosure being indicated by the claims which follow the examples.

Example 1

This example illustrates one embodiment of ingredients that can be used to prepare the nutritional products of the present disclosure.

872 ml of water

Lactose 65.6 mg

Vegetable oil mixture 353.0 mg

Skimmed milk 34.0 mg

Whey protein 8.5 mg

Galacto-oligosaccharide 4.7 mg

Casein 3.5 mg

Polydextrose 2.4 mg

Lactoferrin solution (10%) 1.0 mg

DHA and ARA oil mixture 0.94 mg

Mono-and di-glycerides 0.7 mg

Calcium carbonate 0.44 mg

Calcium phosphate 0.4 mg

Potassium citrate 0.4 mg

Potassium chloride 0.4 mg

Lecithin 0.4 mg

Sodium chloride 0.3 mg

Potassium phosphate 0.3 mg

Choline chloride 0.2 mg

Magnesium oxide 0.08 mg

0.08 mg of calcium hydroxide

Ferrous sulfate 0.07 mg

Vitamin 0.03 mg

Mineral 0.03 mg.

Example 2

This example illustrates another embodiment of ingredients that can be used to prepare the nutritional products of the present disclosure.

686 ml of water

Whey 215 mg

Skim milk 67 mg

Vegetable oil mixture 33 mg

Lactose 17 mg

Galacto-oligosaccharide 4.7 mg

Polydextrose 2.4 mg

Lactoferrin solution (10%) 1.0 mg

DHA and ARA oil mixture 0.9 mg

Mono-and di-glycerides 0.7 mg

Calcium carbonate 0.44 mg

Calcium phosphate 0.4 mg

Potassium citrate 0.4 mg

Potassium chloride 0.4 mg

Lecithin 0.4 mg

Potassium phosphate 0.3 mg

Carrageenan 0.3 mg

Sodium citrate 0.2 mg

Choline chloride 0.2 mg

Magnesium oxide 0.08 mg

Calcium chloride 0.08 mg

Ferrous sulfate 0.07 mg

Vitamin 0.03 mg

Mineral 0.03 mg.

Nutritional compositions comprising the above components from examples 1 and 2, except lactoferrin, in liquid form were prepared and subjected to a temperature of about 135 ℃ to about 145 ℃ for about 3 seconds to about 30 seconds. A 1% -30% lactoferrin solution is prepared in reverse osmosis water and filtered using a sterilizing filter to produce a sterilized solution of lactoferrin. The liquid nutritional composition is mixed with the lactoferrin solution by administering a stream of the lactoferrin solution to the liquid nutritional composition. The resulting composition is aseptically packaged.

Referring now to the drawings, a flow chart of one embodiment of the disclosed method is indicated by reference numeral 10. In the method, a nutritional composition 100 is prepared. The nutritional composition 100 may include various heat stable ingredients as well as one or more heat labile proteins. In the processing step 20, the nutritional composition 100 is exposed to a temperature of at least 130 ℃ to form a first composition 120, which is sterile; in the first composition 120, any heat labile proteins in the nutritional composition 100 may be inactivated or denatured.

Continuing with the method shown in flow chart 10, a solution 200 comprising one or more heat labile proteins is prepared and subjected to a processing step 30, which may include filtering but does not include exposing the solution 200 to a temperature greater than 80 ℃ to form a second composition 220, which is sterile; the heat labile proteins in the second composition 220 are not denatured or inactivated.

First composition 120 and second composition 220 are then mixed in processing step 40 to form third composition 300 comprising intact heat labile proteins from second composition 220. The third composition 300 is then subjected to aseptic processing and packaging in a processing step 50 to provide an aseptically packaged composition 320.

All references cited in this specification, including but not limited to all documents, publications, patents, patent applications, lecture papers, articles, reports, manuscripts, profiles, books, web postings, journal articles, periodicals, and the like, are hereby incorporated by reference into this specification in their entirety. 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 pertinency of the cited references.

Although preferred embodiments of the present 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 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 scope of the present disclosure, which is set forth in the following claims. Further, it should be understood that aspects of the various embodiments may be interchanged both in whole or in part. For example, while methods for producing commercial sterilized liquid nutritional supplements prepared 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.

Claims (19)

1. A process for preparing a composition comprising a fat or lipid source, a protein source and a heat-labile milk protein, comprising the steps of:

a) providing a first composition comprising a fat or lipid source and a protein source and subjecting the first composition to a temperature of at least about 130 ℃;

b) providing a second composition comprising heat-labile milk protein; and

c) mixing the first composition with a second composition comprising heat labile milk protein to form a third composition comprising a fat or lipid source, a protein source, and heat labile milk protein.

2. The method of claim 1, wherein the second and third compositions do not experience a temperature greater than about 80 ℃.

3. The method of claim 1, wherein the second composition is an aqueous solution comprising water that has been treated for food processing.

4. The method of claim 2 wherein the water has been treated by reverse osmosis.

5. The method of claim 1, wherein the second composition has been filtered through one or more filters prior to mixing the second composition with the first composition.

6. The method of claim 1, further comprising the step of aseptically packaging the third composition.

7. The method of claim 1, wherein the heat-labile milk protein is selected from the group consisting of lactoferrin, lactoferricin, TGF- β, lactoperoxidase, milk lectin, and combinations thereof.

8. The method of claim 7, wherein lactoferrin is present in the third composition at a level of at least 10 mg/100 kCal.

9. The method of claim 5, wherein lactoferrin is present in the third composition at a level of 70 mg/100kCal to 220 mg/100 kCal.

10. The method of claim 1, wherein the fat or lipid source is present in the first composition at a level of about 3 g/100kcal to about 7 g/100 kcal.

11. The method of claim 1, wherein the protein source is present in the first composition at a level of about 1g/100 kcal to about 5 g/100 kcal.

12. A composition comprising a fat or lipid source, a protein source and a heat labile milk protein, the composition prepared by the process of:

a) subjecting a first composition comprising a fat or lipid source and a protein source to a temperature of at least about 130 ℃; and

b) mixing the first composition with a second composition comprising a heat-labile milk protein to form a third composition comprising a fat or lipid source, a protein source, and a heat-labile milk protein.

13. The composition of claim 12, wherein the heat labile milk protein has undergone sterilization when the second composition is mixed with the first composition.

14. The composition of claim 12, wherein the heat-labile milk protein is selected from the group consisting of lactoferrin, lactoferricin, TGF- β, lactoperoxidase, milk lectin, and combinations thereof.

15. The composition of claim 14, wherein the heat-labile milk protein is lactoferrin.

16. The composition of claim 15, wherein the lactoferrin is selected from the group consisting of non-human lactoferrin, human lactoferrin produced by a transgenic organism, and combinations thereof.

17. The composition of claim 12, wherein the third composition is an infant formula.

18. The composition of claim 12, wherein the fat or lipid source is present in the first composition at a level of about 3 g/100kcal to about 7 g/100 kcal.

19. The composition of claim 12, wherein the protein source is present in the first composition at a level of about 1g/100 kcal to about 5 g/100 kcal.