NZ626631B2

NZ626631B2 - Concentrated low water activity liquid human milk fortifier including extensively hydrolyzed protein

Info

Publication number: NZ626631B2
Application number: NZ626631A
Authority: NZ
Inventors: Jeffrey M Boff; Kevin B Mahan; Mustafa Vurma
Original assignee: Abbott Laboratories
Priority date: 2011-12-30
Filing date: 2012-11-21
Publication date: 2016-03-30

Abstract

Discloses a concentrated liquid nutritional composition comprising: from 5% to 50% by weight protein, on a dry weight basis, wherein at least a portion of the protein is hydrolysed casein having a degree of hydrolysis of from 20% to 80%; wherein the concentrated liquid nutritional composition has a solids content of greater than 50% by weight; and wherein the concentrated liquid nutritional composition has a water activity of less than 0.90. Further disclosed is a method of fortifying breast milk comprising mixing a liquid human milk fortifier with breast milk in a volume to volume ratio of less than or equal to 1:10. solids content of greater than 50% by weight; and wherein the concentrated liquid nutritional composition has a water activity of less than 0.90. Further disclosed is a method of fortifying breast milk comprising mixing a liquid human milk fortifier with breast milk in a volume to volume ratio of less than or equal to 1:10.

Description

CONCENTRATED LOW WATER ACTIVITY LI UID HUMAN MILK FORTIFIER INCLUDING EXTENSIVELY HYDROLYZED PROTEIN CROSS REFERENCE TO RELATED APPLICATIONS The present application hereby claims the beneﬁt of the provisional patent applications, Serial Nos. 61/581,636; and 61/581,637, ﬁled on December 30, 2011, the disclosures of which are incorporated by reference herein in their entirety.

FIELD OF THE DISCLOSURE {0002] The present disclosure relates to a stable, concentrated liquid human milk er. More ularly, the t disclosure relates to a long term , trated liquid human milk fortiﬁer having a low water activity, and in some embodiments, a low pH. The concentrated liquid human milk fortiﬁer may additionally includes extensively hydrolyzed casein, a high solids t, and a probiotic.

BACKGROUND Human milk is generally recognized as an ideal feeding for most infants due to its overall nutritional composition. Human milk provides infants with unique immunologic and developmental beneﬁts as compared generally to commercially available infant formulas. For some infants, however, especially m infants, human milk does not always meet the complete nutritional needs. Although these infants still generally beneﬁt from human milk, it is often desirable to supplement their human milk feedings with additional nutrients. Initially, these preterm infants may grow more rapidly than many of their term counterparts, and accelerated growth often requires additional nutrition, which is made possible by the use of a human milk fortiﬁer in combination with human milk.

Further, some infants, and ally preterm infants whose digestive tracts are immature, ence a delayed and abnormal pattern of gut colonization, particularly with regard to probiotics such as acteria and lactobacilli. This impaired intestinal colonization may predispose preterm infants to necrotizing enterocolitis (NEC) and increase the risk of bacterial translocation.

Most of the human milk ers described in the literature and cially available have been formulated as reconstitutable powders rather than liquids in order to minimize the volume displacement ofhuman milk by the fortiﬁer. The fortiﬁers may additional include probiotics. As tics are living organisms, addition of probiotics to a liquid formula risks inconsistent bacterial levels due to undesired proliferation followed by die-off over time. Undesired proliferation of probiotic microorganisms will negatively affect the nutritional proﬁle and physical stability of the ﬁnal product.

Recently, however, liquid human milk fortiﬁers, and speciﬁcally highly concentrated human milk fortiﬁer liquids, have received more attention as an ative to powders. Although these highly concentrated liquid human milk fortiﬁers do generally displace slightly more volume than the conventional powders, the s have the cant beneﬁt of being commercially sterile as they can be subjected to sufficient heat treatment during cturing, including optional c cturing for sterility.

Due to sensitive digestive systems and poor tolerance in many preterm infants, it may be ageous to utilize hydrolyzed proteins in human milk fortifiers. However, as ed to intact proteins, extensively hydrolyzed proteins (i.e., proteins having a degreeof hydrolysis of about 20% or more) tend to have poor ability to form long term stable emulsions. Additionally, the presence of high levels of insoluble minerals such as calcium salts may also cause a number of stability issues when used in combination with ively hydrolyzed proteins. As such, manufacturing long term stable concentrated liquid human milk fortiﬁers including extensively hydrolyzed proteins has proven difficult.

Many liquid human milk fortiﬁers have been manufactured with stabilizers, such as carrageenan. The stabilizers act to hold the nts and insolubles in solution over time and thus improve long term stability of the product. Although stabilizers such as carrageenan have generally proven to retard precipitation of many ingredients in the liquid nutritional formulations, this type of stabilizer is not ted in infant formulas and human milk fortifiers in many ies around the world. When stabilizers cannot be used in highly concentrated human milk fortifiers, it can be very difficult to e a long term stable highly concentrated human milk fortifier.

As such, there is a need for highly concentrated low water activity liquid human milk iers that are sufficiently long term stable that include hypoallergenic proteins, such as extensively yzed casein proteins, and optionally probiiotics. Additionally, it would be very beneficial if the highly concentrated low water activity human milk fortifier could be formulated to provide additional macro- and micro-nutrients without unwanted mineral fallout during storage.

BRIEF SUMMARY [00101 The present disclosure is directed to longterm stable high solids content and low water activity liquid human milk fortifiers including extensively yzed casein. In some embodiments, the extensively hydrolyzed casein is the sole source of n in the concentrated liquid human milk fortifier. The concentrated liquid human milk fortifiers may be, in some embodiments, hypoallergenic. e the liquid human milk fortifiers of the present disclosure have a high solids content and low water activity, their volume displacement when mixed with human breast milk and/or infant formula is minimized and their sterility is increased. [001 Oa] In an ment the present disclosure is also directed to a concentrated liquid nutritional composition comprising: from 5% to 50% by weight protein, on a dry weight basis, wherein at least a portion of the n is hydrolyzed casein having a degree of hydrolysis of from 20% to 80%; wherein the concentrated liquid nutritional composition has a solids content of greater than 50% by weight; and wherein the concentrated liquid nutritional composition has a water activity of less than 0.90. (10158294 1);GGG [001 Ob] In an embodiment the present disclosure is also directed to a method of fortifying breast milk, the method comprising: mixing a liquid human milk ier with breast milk in a volume to volume ratio of less than or equal to 1:10; wherein the liquid human milk fortifier comprises from 5% to 50% by weight protein, on a dry weight basis, and wherein at least a portion of the protein is hydrolyzed casein having a degree of hydrolysis of from 20% to 80%; wherein the liquid human milk fortifier has a solids content of greater than 50% by ; and wherein the liquid human milk ier has a water activity of less than 0.90.

The present disclosure is specifically directed to a concentrated liquid human milk fortifier sing from about 5% to about 50% by weight protein, on a dry weight basis, and ally a probiotic. At least a portion of the protein is extensively hydrolyzed casein. The concentrated liquid human milk fortifier has a solids content of greater than 50% by weight and has a water activity of less than 0.90. [00 12] The present disclosure is further specifically directed to an aseptically-sterilized concentrated liquid human milk fortifier sing from about 5% to about 50% by weight protein, on a dry weight basis, and optionally a probiotic. At least a portion of the protein is extensively hydrolyzed casein. The concentrated liquid human milk fortifier (10158294 1):GGG comprises a solids content of greater than 50% by weight, a pH of from 4.6 to 6.5, and a water activity of less than 0.90. [0013} The present disclosure is further speciﬁcally directed to a method of fortifying breast milk. The method comprises mixing a liquid human milk fortiﬁer with breast milk in a volume to volume ratio of 1:10 or less. The liquid human milk fortiﬁer comprises from about 5% to about 50% by weight protein, on a dry weight basis, and optionally a probiotic. At least a portion of the protein is extensively hydrolyzed casein. The liquid human milk fortiﬁer comprises a solids content of r than 50% by weight and a water activity of less than 0.90.

The t sure is further speciﬁcally directed to a concentrated liquid ional composition comprising from about 5% to about 50% by weight n, on a dry weight basis, wherein at least a portion of the n is extensively hydrolyzed casein, and optionally a probiotic. The liquid nutritional composition comprises a solids content of greater than 50% by weight and a water activity of less than 0.90. {0015} In some embodiments, the concentrated liquid human milk fortiﬁer r includes a stabilizer system. In one embodiment, the stabilizer system is comprised of an octenyl ic anhydride (OSA) modiﬁed starch (e.g., OSA modiﬁed corn starch) and a low acyl gellan gum. In another embodiment, the izer system is a single stabilizer such as OSA modiﬁed potato starch. The stabilizer system allows for the use of the extensively hydrolyzed casein protein without the ated ms of mineral fallout and poor emulsion stability, and allows for the fortiﬁer, in some embodiments, to be carrageenan-free.

It has been unexpectedly found that stable, concentrated liquid human milk fortiﬁers and concentrated liquid nutritional compositions can advantageously be prepared with a low water activity and/or low pH level. The low water activity, for example, a water activity of less than 0.90, and/or low pH, for example, a pH within the range of 4.6 to 6.5, of the liquid can reduce or even inhibit bacterial growth in the liquid such as from Cronobacter sakazakii, preventing subsequent food spoilage. Moreover, the low water activity and/or low pH provides an environment that hinders the growth of probiotic bacteria such as Biﬁdobacterz‘um and Laciobacillus species, while maintaining its viability. This improves product consistency and may prevent inconsistent ial levels in the gut upon product use due to uncontrolled proliferation of the probiotics followed by die-off over time.

A balanced colonization of tics, h both supplementing the trated liquids with one or more probiotics and providing the liquids with low water activity and/or low pH to reduce uncontrolled ial growth, can be provided so as to provide infants, and particularly, preterm infants, with a reduced bowel reservoir of pathogenic microorganisms, a reduced ence on broad-spectrum antibiotics, an increased gut mucosal barrier to pathogenic bacteria, and an up—regulation in protective ty. This is especially desirable for low birth weight and preterm infants delivered by caesarean section as these infants may be more at risk for slower pment of diverse and beneﬁcial intestinal ﬂora as compared to a lly born infant. The addition of probiotics further provides protection to the preterm infant against necrotizing enterocolitis and bacterial ocation.

Furthermore, by lowering one or both of water activity and pH of the concentrated liquids of the present disclosure, a sterile liquid may be produced having reduced microbial growth, and thus, r stability and longer shelf-life without extensive and severe heat treatment. By reducing the severity of heat treatment required to sterilize the concentrated liquids, the need to over-fortify ingredients (e.g., vitamins and minerals), which may be heat sensitive, may be reduced or eliminated. This r can reduce processing costs.

Supplementation with probiotics ﬁlrther provides for improved enteral nutrition, thereby improving weight gain typically desired of low birth weight and preterm infants.

Additionally, it has been unexpectedly discovered that concentrated liquids can be prepared to have higher solids ts; that is, solids contents of greater than 50% by weight, and more preferably, greater than 60% by weight. With a more concentrated liquid, and thus greater c density, the dose size required may also be reduced. This will allow for increased cost savings and t packaging size.

The stable concentrated liquids can further be prepared to include hypoallergenic extensively hydrolyzed casein proteins, which is beneﬁcial for use in low birth weight and preterm infants having ive digestive systems. These liquids including extensively yzed casein proteins can be prepared without disrupting the long term stability or emulsion properties of the liquid. By preparing a concentrated liquid by utilizing a stabilizer system including a octenyl succinic anhydride modiﬁed corn starch and low acyl gellan gum (or octenyl succinic anhydride modiﬁed potato starch as a sole stabilizer), the concentrated liquid may include up to 100% extensively hydrolyzed casein (by weight of the protein ent) while maintaining the desired emulsion and stability properties.

DETAILED DESCRIPTION The concentrated liquid compositions, and particularly concentrated liquid human milk fortiﬁers, of the present disclosure generally comprise extensively yzed casein, have a high solids content, and a low water activity, and optionally a probiotic. These concentrated liquid human milk fortiﬁers are long term stable and displace only a minimal amount of volume upon addition to human breast milk and/or infant formula.

The concentrated liquid human milk ers of the present disclosure address and provide a on to the longstanding problem ofproviding a sterile, long term stable, lergenic liquid human milk fortiﬁer, optionally including a probiotic, that can be used in combination with human breast milk or infant formula without signiﬁcant volume displacement. The concentrated liquid human milk fortiﬁers of the present disclosure not only provide the signiﬁcant beneﬁt of ed sterility as ed to powdered human milk fortiﬁers that may potentially be subject to microbial contamination, but also provide a stable human milk er that includes extensively hydrolyzed protein that can be more easily digested and absorbed into the gut of an infant, and ularly a preterm infant, as compared to intact proteins, and optinally includes a probiotic. The previous problems of providing a liquid human milk fortiﬁer with tic ty have been overcome in the human milk fortiﬁers sed herein.

By providing a long term stable trated liquid human milk fortiﬁer that can be based partly or solely on an ively hydrolyzed protein component, optionally in combination with a probiotic, the present disclosure now provides a sterile, concentrated liquid product that can be used in neonatal intensive care units in combination with human breast milk or infant formula for m and term infants to provide the infant with the additional nutrients, including both protein and minerals, required for growth and maturation. This can now be done with a highly sterile, stable product that is highly concentrated so as to be more like a powdered human milk fortiﬁer from a volume displacement perspective.

These and other elements or limitations of the concentrated liquids and methods of the present disclosure are described in detail hereinafter.

The terms "retort packaging" and "retort sterilizing" are used interchangeably herein, and unless otherwise speciﬁed, refer to the common practice of ﬁlling a container, most typically a metal can or other similar package, with a ional liquid and then subjecting the liquid-ﬁlled package to the necessary heat sterilization step, to form a sterilized, retort packaged, nutritional liquid product.

The term ”aseptic packaging" as used herein, unless otherwise speciﬁed, refers to the manufacture of a packaged product without reliance upon the above-described retort packaging step, wherein the nutritional liquid and e are sterilized separately prior to ﬁlling, and then are combined under sterilized or aseptic processing ions to form a ized, aseptically packaged, nutritional liquid product.

The term "infan " as used herein, refers generally to individuals less than about 1 year of age, actual or corrected.

The term ”preterm" are used herein refers to those infants born at less than 37 weeks gestation, having a birth weight of less than 2500 g, or both. {0029] The terms "fortiﬁer solids" or "total solids", unless otherwise speciﬁed, are used interchangeably herein and refer to all material components of the compositions of the present disclosure, less water.

The term “hypoallergenic” as used herein means that the concentrated liquid has a decreased tendency to provoke an allergic reaction in a user, such as a preterm or term infant, as compared to non-hypoallergenic s.

The term “extensively yzed’ as used herein refers to protein that has been enzymatically or acidically hydrolyzed to have a degree of hydrolysis of at least 20%.

Typically, extensively hydrolyzed proteins exist primarily as di- and tripeptides.

The terms “liquid nutritional composition” and “nutritional liquid” are used interchangeably herein, and unless otherwise ed, refer to nutritional products in concentrated form.

The term “adult ional t” as used herein includes formulas for generally maintaining or improving the health of an adult, and includes those formulas designed for adults who need to control their blood glucose. [00341 The terms “stable” and “shelf ” as used herein means that the concentrated liquid is resistant to separation of the liquid into two or more discernable layers (e.g., a top cream layer and a bottom serum layer) and precipitation/sediment formation on the bottom of a container for a time period after manufacture of at least three months, desirably at least six months, desirably at least twelve months, and more desirably at least en months.

All percentages, parts and ratios as used herein, are by weight of the total composition, unless otherwise speciﬁed. All such weights as they n to listed ingredients are based on the active level and, therefore, do not include ts or by- products that may be included in commercially available materials, unless otherwise speciﬁed. cal ranges as used herein are intended to include every number and subset of numbers within that range, whether speciﬁcally disclosed or not. Further, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 2 to 8, from 3 to 7, from 5 to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

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

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

The various ments of the concentrated liquids of the present disclosure may also be substantially free of any optional or selected ingredient or feature described herein, provided that the remaining concentrated liquid still contains all of the ed ingredients or features as described herein. In this context, and unless otherwise speciﬁed, the term antially free” means that the selected concentrated liquid contains 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.

The concentrated liquids and corresponding manufacturing methods of the present disclosure can comprise, consist of, or consist ially of the elements and limitations of the disclosure as described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise useful in the concentrated liquid.

Product Form While discussed primarily as a concentrated liquid human milk fortiﬁer, it should be recognized by one skilled in the art based on the sure herein that the concentrated compositions described herein may alternatively be used in liquid nutritional itions such as for use with suspensions, emulsions or clear or substantially clear liquids. The resulting trated liquid nutritional compositions may be used as adult liquid nutritional compositions and/or nal liquid nutritional compositions.

The concentrated liquid human milk fortiﬁers of the t disclosure have a solids content of at least 50%, or even at least 60%, including from about 50% to about 65%, and further ing from about 58% to about 62%. The concentrated liquid human milk fortiﬁers are liquids that are capable of being poured directly from a package containing them into human milk, formula, or mixtures thereof.

The concentrated liquid human milk fortiﬁers are generally formulated to have a caloric density of at least 1.25 kcal/ml (37 kcal/ﬂ oz), including from about 1.3 kcal/ml (38 kcal/ﬂ oz) to about 5 kcal/ml (149 kcal/ﬂ oz), and also including from about 1.4 kcal/ml (40 kcal/ﬂ oz) to about 2.7 kcal/ml (80 kcal/ﬂ oz), and also including from about 1.5 kcal/ml (44 kcal/ﬂ oz) to about 2.0 kcal/ml (59 kcal/ﬂ oz).

The concentrated liquid human milk fortiﬁers include packaged compositions further comprising a suitable unit dose package or container. The term "unit dose" as used herein refers to individual, single-use, packages of concentrated liquid human milk fortiﬁer containing an amount ofhuman milk fortiﬁer that can be used in a preparation of an infant g to provide sufﬁcient human milk er to supplement human milk for ate use, e.g., preferably within 8-24 hours, more preferably within 0-4 hours, of mixing with human milk.

The amount of fortiﬁed human milk prepared for a premature infant, for example, typically ranges from 25 ml to 150 ml a day. Consequently, a single unit dose is the riate amount of fortiﬁer solids to fortify a 25 ml preparation. Multiple packages can be used to prepare larger feeding volumes, especially for term infants.

The amount or volume of concentrated liquid human milk fortiﬁer in each unit dose package includes those embodiments in which the package contains an amount suitable to prepare an infant's next feeding. These unit dose packages typically contain ent fortiﬁer to provide from about 0.5 g to about 10 g of fortiﬁer solids, more typically from about 0.8 g to about 7.5 g of fortiﬁer solids, and even more typically from about 0.85 g to about 6.0 g of fortiﬁer solids.

The concentrated liquid human milk fortiﬁers of the present disclosure are preferably formulated so as to e fortiﬁed human milk having an osmolality of less than about 500 mOsm/kg water, preferably from about 300 mOsm/kg water to about 400 mOsm/kg water. Based on the disclosure herein, one skilled in the art can readily formulate the concentrated liquid human milk fortiﬁer with the appropriate carbohydrate sources and corresponding DE (dextrose equivalence) values to obtain or otherwise provide for the targeted osmolality of the human milk fortiﬁer when ed with human milk.

The concentrated liquid human milk fortiﬁers are r formulated to have a low water activity sufﬁcient to reduce or inhibit microbial growth, particularly growth of C. sakazakiz', Clostridium botulinum, Salmonella spp., Staphylococcus , and Escherichia coli, as well as many food ge microorganisms known in the art. ably, the liquid human milk fortiﬁers are prepared to have a water activity of less than 0.90, more preferably less than 0.875, and even more preferably less than 0.86.

The concentrated liquid human milk fortiﬁers may suitably have a pH ranging from about 3.5 to about 8.0, but are most advantageously in a pH range of from about 4.5 to about 7.5, including from about 4.5 to about 7.0, including from about 4.5 to about 6.7, including from about 4.5 to about 6.5, and including from about 4.5 to about 6.0. In some ments, the pH range is from about 5.5 to about 7.3, including from about 5.5 to about 7.0, ing from about 5.5 to about 6.5, and further including from about 5 .5 to about 6.0. In other embodiments, the pH range is from about 6.2 to about 7.2, including from about 6.2 to about 7.0, and including from about 6.2 to about 6.5.

Probiotics {0050] The concentrated liquid human milk fortiﬁers of the present sure comprise at least one probiotic. In some embodiments, the trated liquid human milk fortiﬁer es a combination of probiotics (at least two or more different probiotics) such that the fortiﬁer provides a gut colonization of various probiotics from various CCS.

Probiotics are live microorganisms thought to be healthy for the host organism.

Lactic acid bacteria (LAB) and bifidobacteria are the most common types of microbes used as probiotics. Probiotics maintain the microbial ecology of the gut and show physiological, immuno-modulatory and antimicrobial effects, such that the use of probiotics has been found to prevent and treat gastrointestinal diseases and/or disorders (e. g., NEC), pathogen-induced diarrhea and toxin-producing bacteria, urogenital infections, and atopic diseases. {0052] In order for microbes to exhibit ial probiotic s in vivo, the organisms should survive for extended time periods in the gastrointestinal tract. ore, it is desirable that tic strains be selected that possess qualities that prevent their rapid removal by gut contraction. Effective probiotic strains are able to survive gastric conditions and colonize the intestine, at least temporarily, by adhering to the intestinal lium.

Further, the probiotic selected should be capable of surviving manufacturing of the concentrated liquid human milk fortiﬁer as well as the extended periods of e of the fortiﬁer. ingly, in some embodiments, a heat resistant probiotic strain may be used to withstand the heat treatment(s) typically used in manufacturing of the concentrated liquid human milk fortiﬁer.

Additionally, as the concentrated liquid human milk fortiﬁer has low water activity, the fortiﬁer es a medium that supports dormancy of the added probiotic.

As used herein, “dormant” or “dormancy” refers to a state in which the bacterial cells have negligible activity, but are ultimately culturable; that is, the bacterial cells are in a ible state of metabolic shutdown. This dormancy state will further t the probiotic from various processing and storage conditions. [0055} In some embodiments, the probiotic may further be treated to provide the tic in a dormant state. By way of example, the probiotic may be in a form such as a freezedried form, an oil droplet, or a bacterial sion solution to further protect the probiotic. In some embodiments, the probiotic is encapsulated prior to addition to the concentrated liquid human milk fortiﬁer.

Suitable encapsulation technologies are known in the art. Encapsulation agents or als may include any encapsulation materials known in the art for use with probiotics, including protective hydrocolloids (e.g., pectins, alginates, gums), carbohydrates, starches, cellulose derivatives, proteins (e.g., dairy, egg, vegetable-based proteins), fat and waxes, fat derived compounds (e.g., lecithins, mono and diglycerides), s, ﬁlm forming agents, emulsiﬁers, surface active agents, carriers, dispersing agents, antioxidants and antimicrobials.

Non-limiting examples of tic strains for use in the concentrated liquid human milk fortiﬁers herein e the genus Lactobacillus ing L. acidophilus (e.g., L. acidophilus LA-S and L. acidophilus NCFM), L. amylovorus, L. brevis, L. bulgarz'cus, L. casez' spp. casei, L. casei spp. rhamnosus, L. crispatus, L. delbrueckii ssp. lactis, L. fermentum (e.g., L. fermentum CETC5716), L. helveticus, L. johnsom‘z’, L. paracasei, L. pentosus, L. rum, L. reuteri (e.g., L. reuterz' ATCC 55730, L. reuteri ATCC PTA-6475, and L. reuteri DSM 1793 8), L. sake, and L. rhamnosus (e.g., L. rhamnosus LGG and L. rhamnosus HNOOI); the genus Biﬁdobacterium including: B. animalis (e.g., B. animalis spp. lactis Bb-12), B. bifz‘dum, B. breve (e.g., B. breve M- 16V), B. infantis (e. g., B. infanris M-63 and B. infantis ATCC 15697), B. longum (e.g., B. longum BB536, B. longum AH1205, and B. Zongum AH1206), and B. Zactis (e.g., B. lactis HN019 and B. Zactis Bi07); the genus occus including: P. acidilactici; the genus Propionibacterium ing: P. acidzpropionicz’, P. freudenreichii, P. jensenii, and P. theom'z'; and the genus Streptococcus including: S. cremorz’s, S. Zactz‘s, and S. thermophilus. Particularly preferred probiotics e B. lactis and L. acidophilus.

The probiotics may be present in the concentrated liquid human milk fortiﬁer in a total amount of at least 103 CFU/mL composition, including from about 103 CFU/mL composition to about 1011 CFU/mL composition, and including from about l07 CFU/mL composition to about 1010 CFU/mL composition.

Extensively Hydrolyzed Casein Protein [0059} The concentrated liquid human milk fortiﬁers of the present disclosure may include hypoallergenic extensively hydrolyzed casein as a n . Generally, the concentrated liquid human milk fortiﬁers will e at least 35%, including at least 50%, including at least 60%, including at least 75%, including at least 90% and further ing about 100% extensively hydrolyzed casein, by total weight of total protein in the concentrated liquid human milk fortiﬁer. In one desirable embodiment of the present disclosure, the concentrated liquid human milk fortiﬁer includes 100% extensively hydrolyzed casein, by total weight of the protein in the concentrated liquid human milk fortiﬁer. In this desirable embodiment, the trated liquid human milk fortiﬁer is hypoallergenic. In some embodiments, the concentrated liquid human milk fortiﬁer will include from about 35% to 100%, ing from about 50% to 100%, further including from about 75% to 100% extensively hydrolyzed casein, by total weight of protein in the concentrated liquid human milk fortiﬁer. As discussed further below, in some embodiments of the present disclosure, the concentrated liquid human milk fortiﬁers of the present disclosure may optionally include other hypoallergenic or non-hypoallergenic proteins in addition to the extensively hydrolyzed casein protein.

Extensively yzed casein proteins suitable for use in the trated liquid human milk fortiﬁers of the present disclosure include those having a degree of hydrolysis of at least 10%, including from about 20% to about 80%, including from about % to about 60%, and further including from about 40% to about 60%. lly, the extensively hydrolyzed casein has a ratio of total amino nitrogen (AN) to total nitrogen (TN) of from about 0.2 AN to 1.0 TN to about 0.4 AN to about 0.8 TN. Suitable commercially available ively hydrolyzed caseins will generally have a protein level in the ingredient of from about 50% to about 95%, including from about 70% to about 90%. One suitable commercially available extensively hydrolyzed casein is Dellac CE90, which is a spray dried powder casein hydrolysate land Campina Domo, Amersfoort, The Netherlands).

Stabilizer System The concentrated liquid human milk fortiﬁers of the present disclosure optionally include a stabilizer . In one embodiment, the stabilizer system is a single stabilizer system including octenyl succinic anhydride (OSA) modiﬁed potato starch. In another embodiment, the stabilizer system is a synergistic two ent izer system. The ﬁrst component is an OSA modiﬁed starch, such as an OSA modiﬁed corn starch or an OSA modiﬁed potato starch. The second component is a low acyl gellan gum. These two components act in a synergistic manner to stabilize the concentrated liquid human milk er and retard the precipitation of nutrients therefrom.

The OSA modiﬁed starch, including the desirable OSA modiﬁed corn starch or OSA modiﬁed potato starch, is generally prepared by esterifying a dextrinized, ungelatinized waxy corn or potato starch with 1-octeny1 succinic anhydride. Methods of this type are well known in the art. One suitable commercially available OSA modiﬁed corn starch is N-CREAMERTM 46 (National Starch Food Innovation, Bridgewater, New Jersey). A suitable commercially available OSA modiﬁed potato starch is ELIANETM MC160 , The Netherlands).

The GSA-modiﬁed starch is present in the concentrated liquid human milk fortiﬁer in an amount of from about 0.1% to about 3.5%, including from about 0.6% to about 2.0%, ing from about 0.8% to about 1.5%, and further including about 1.2% by weight of the concentrated liquid human milk fortiﬁer.

The low acyl gellan gum (also known as and commonly referred to as deacylated gellan gum) may be a water-soluble polysaccharide produced by fermentation of a pure culture of Sphingomonas elodea. As used herein, “low acyl” means that the gellan gum has been d such that it forms ﬁrm, astic, brittle gels, that are heat stable, as compared to “high acyl” which forms soft, very elastic, non-brittle gels. One suitable commercially ble low acyl gellan gum is Kelcogel F (CP Kelco US. Inc., Atlanta Georgia).

The low acyl gellan gum is present in the concentrated liquid human milk fortiﬁer in an amount of from greater than 125 ppm to about 800 ppm, including from about 150 ppm to about 400 ppm, including from about 200 ppm to about 300 ppm and further including about 200 ppm. utrients The concentrated liquid human milk fortiﬁers and concentrated liquid nutritional compositions of the present disclosure generally se ydrate, fat, and protein utrients of sufﬁcient types and amounts that, when used alone or in combination with human milk or other feeding formula, help meet the nutritional needs of the user, especially the premature infant. The concentration of these macronutrients in the various embodiments of the present disclosure includes the ranges described hereinafter.

Protein {0067] The concentrated liquid human milk fortiﬁers of the present disclosure se a protein suitable for use in infants, especially preterm infants, at concentrations g from about 5% to about 50%, including from about 20% to about 40%, including from about 5% to about 30%, including from about 10% to about 25%, and also including from about 15% to about 25%, on a dry weight basis. In some embodiments, the protein may be at a concentration of less than 10%, on a dry weight basis. In some desirable embodiments, the n concentration may be from about 7 to about 15 grams, including from about 9 to about 12 grams of protein per 100 grams of final liquid product.

As noted above, the protein component of the concentrated liquid human milk fortiﬁers of the present disclosure is at least partially comprised of extensively hydrolyzed . In a particularly desirable embodiment of the present disclosure, the protein component of the concentrated liquid human milk fortiﬁer is entirely comprised of ively hydrolyzed casein. In embodiments wherein additional proteins sources (i.e., one or more protein sources in addition to the extensively hydrolyzed protein source) are to be used in the concentrated liquid human milk fortiﬁer in addition to the extensively hydrolyzed casein (i.e., the concentrated liquid human milk fortiﬁer protein component is not 100% ively hydrolyzed ), the fortiﬁer may still be made hypoallergenic by including additional hypoallergenic proteins such as soy n ysate, whey protein hydrolysate, rice protein hydrolysate, potato protein hydrolysate, ﬁsh protein hydrolysate, egg albumen hydrolysate, gelatin protein hydrolysate, pea n ysate, bean protein hydrolysate, combinations of animal and vegetable protein hydrolysates, and combinations thereof.

In this context, the terms in hydrolysates" or "hydrolyzed protein" are used interchangeably herein and include ively hydrolyzed proteins, wherein the degree of ysis is most often at least 10%, including from about 20% to about 80%, and also including from about 20% to about 60%, even more preferably from about 40% to about 60%. The degree of hydrolysis is the extent to which peptide bonds are broken by a hydrolysis method. The degree ofprotein hydrolysis for purposes of characterizing the extensively hydrolyzed protein component of these embodiments is easily determined by one of ordinary skill in the formulation arts by fying the amino nitrogen to total nitrogen ratio (AN/TN) of the n ent of the selected formulation. The amino nitrogen component is quantiﬁed by USP titration methods for determining amino nitrogen content, while the total nitrogen component is determined by the Tecator Kjeldahl method, all of which are well known methods to one of ordinary skill in the analytical chemistry art.

In other embodiments of the present disclosure, the concentrated liquid human milk fortiﬁer, in addition to the extensively hydrolyzed protein, may include an additional non-hypoallergenic protein source including for example, partially hydrolyzed or non- hydrolyzed (intact) protein, and can be derived from any known or otherwise suitable source such as milk (e.g., casein, whey, lactose-free milk protein es), animal (e.g., meat, ﬁsh), cereal (e. g., rice, corn), vegetable (e.g., soy, pea, bean), or combinations thereof. The protein can e, or be entirely or partially replaced by, free amino acids known or otherwise le for use in ional products, non-limiting examples of which include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-carnitine, L- cystine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L— lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-taurine, L-threonine, L- tryptophan, L-tyrosine, L-valine, and combinations thereof.

Carbohydrate The concentrated liquid human milk ers of the present sure comprise a carbohydrate suitable for use in infants, especially preterm infants, at concentrations most typically g up to about 75% by weight on a dry weight basis, including from about 5% to about 50%, and also including from about 20% to about 40%, by weight on a dry weight basis.

Carbohydrates le for use in the concentrated liquid human milk fortiﬁers may include hydrolyzed or intact, naturally and/0r ally modiﬁed, starches sourced from corn, tapioca, rice or , in waxy or non-waxy forms. Other non-limiting examples of suitable carbohydrate sources include hydrolyzed cornstarch, maltodextrin (i.e., non-sweet, nutritive polysaccharide having a DB value less than 20), com maltodextrin, glucose polymers, sucrose, corn syrup, corn syrup solids (i.e., polysaccharide having a DB value r than 20), e, rice syrup, se, high fructose corn syrup, indigestible oligosaccharides such as fructooligosaccharides (F08), and combinations thereof. The carbohydrates may comprise lactose or can be substantially free of lactose.

One embodiment of the present disclosure includes a non-reducing carbohydrate component, which may represent from about 10% to 100%, including from about 80% to 100%, and also including 100%, by weight of the total carbohydrate in the concentrated liquid human milk er. The selection of a non-reducing carbohydrate may enhance the product stability and is generally better tolerated by infants, especially premature infants. Non-limiting examples of non-reducing carbohydrates include sucrose or other carbohydrates that do not readily oxidize or react with Tollen's, ct's, or Fehling's reagents. The present disclosure therefore includes those embodiments comprising a ydrate component, wherein the carbohydrate component comprises a mono- and/or disaccharide such that at least 50%, including from about 80% to 100%, and also including 100%, of the mono- and/ or disaccharide is a non-reducing carbohydrate.

The concentrated liquid human milk fortiﬁers of the present disclosure also comprise a fat component suitable for use in infants, especially preterm infants, at concentrations most typically ranging up to about 40% by weight on a dry weight basis, including from about 10% to about 40%, and also including from about 15% to about 37%, and also including from about 18% to about 30%, by weight on a dry weight basis.

Fats suitable for use in the concentrated liquid human milk fortiﬁers of the present disclosure may include coconut oil, soy oil, corn oil, olive oil, er oil, high oleic safﬂower oil, MCT oil (medium chain triglycerides), sunﬂower oil, high oleic sunﬂower oil, structured triglycerides, palm and palm kernel oils, palm olein, canola oil, marine oils, cottonseed oils, and combinations thereof.

Suitable fats for use in the concentrated liquid human milk fortiﬁers include emulsiﬁers to help the various fortiﬁer components readily disperse when ed with human milk. Non-limiting examples of suitable emulsiﬁers include glyceryl monostearate, monoglycerides, diglycerides, distilled monoglycerides, soya bean lecithin, polyoxythylene stearate, polyoxyethylene sorbitan mono-oleate, polyoxyethylene sorbitan lmitate, polyoxyethylene sorbitan monostearate, ammonium phosphatides, polyoxyethylene an monolaurate, citric acid esters ofmono and diglycerides of fatty acids, tartaric acid esters of mono and diglycerides of fatty acids, and combinations thereof. Natural soy in is ally useful in this respect. 10077] The fat ent of the trated liquid human milk fortiﬁer may therefore optionally include any emulsiﬁer suitable for use in infant nutritional products.

Emulsiﬁer concentrations in these products may range up to about 10%, including from about 0.5% to about 10%, about 1% to about 10%, even more typically from about 1.5% to about 5%, by weight of the total fat component.

Another aspect of the t disclosure es those embodiments in which the weight ratio of fat to protein in the concentrated liquid human milk fortiﬁer is at least 0.3, including from about 0.4 to about 5, and also including from about 2 to about 4.

These ratios may be helpful in further stabilizing the trated liquid human milk fortiﬁer.

The concentrated liquid human milk fortiﬁers of the present disclosure also include those embodiments that se as part of the fat component one or more of arachidonic acid, docosahexaenoic acid, or combinations thereof, alone or in further combination with ic acid, linolenic acid, or both.

Vitamins and ls The trated liquid human milk fortiﬁers of the present disclosure may further comprise any of a variety of vitamins, non—limiting examples of which include vitamin A, vitamin D, vitamin E, vitamin K, thiamine, riboﬂavin, pyridoxine, vitamin B12, niacin, folic acid, pantothenic acid, biotin, vitamin C, choline, inositol, salts and derivatives thereof, and combinations f.

The concentrated liquid human milk fortiﬁers may also further comprise any of a variety of minerals known or otherwise suitable for use in infant or other nutritional formulas, non-limiting es of which include phosphorus, magnesium, m, zinc, manganese, copper, iodine, sodium, potassium, chloride, um, and combinations thereof.

The concentrated liquid human milk fortiﬁers of the present disclosure e those embodiments comprising per 100 kcal of fortiﬁer solids one or more of the following: vitamin A (from about 250 to about 6500 IU), vitamin D (from about 40 to about 1200 IU), n K, vitamin E (at least 0.3 IU), vitamin C (at least 8 mg), thiamine, vitamin B12, niacin, folic acid, pantothenic acid, biotin, choline (at least 7 mg), and inositol (at least 2 mg).

The concentrated liquid human milk fortiﬁers also include those embodiments comprising per 100 kcal of the fortiﬁer solids one or more of the following: calcium (at least 50 mg), phosphorus (at least 25 mg), magnesium (at least 6 mg), iodine, zinc (at least 0.5 mg), copper, manganese, sodium (from about 20 to about 60 mg), potassium (from about 80 to about 200 mg), chloride (from about 55 to about 150 mg) and um (at least 0.5 mcg).

Other Optional Ingredients The concentrated liquid human milk fortiﬁers of the present sure may further optionally comprise other ingredients that may modify the physical, chemical, aesthetic or processing characteristics of the compositions or serve as pharmaceutical or additional nutritional components when used in the targeted population. Many such optional ingredients are known for use in food and nutritional products, including infant formulas, and may also be used in the concentrated liquid human milk fortiﬁers of the present disclosure, provided that such optional materials are ible with the als described herein, are safe and effective for their intended use, and do not otherwise unduly impair product performance.

Non-limiting examples of such optional ingredients include preservatives, anti- oxidants, various pharmaceuticals, buffers, carotenoids, nts, , nucleotides and nucleosides, thickening agents, prebiotics, probiotics, sialic acid-containing materials, and other excipients or processing aids.

Aseptic Packaging In some embodiments, the concentrated liquid human milk fortiﬁers of the present disclosure may be sterilized and cally packaged. The aseptic packaging can be accomplished using any of a variety of techniques well known to those of ordinary skill in the formulation art, so long as the heat treatment is sufﬁcient to achieve long term shelf stability of the trated liquid. In one speciﬁc example, an c process is utilized that includes a high temperature short time (HTST) processing step (i.e., about 165°F (74°C) for about 16 seconds) or an ultra high temperature (UHT) processing step (i.e., about 292°F (144°C) for about 5 seconds).

A typical aseptic process in accordance with the present disclosure involves the preparation of a slurry from one or more ﬂuid combinations that may contain water and one or more of the following: carbohydrates, OSA modiﬁed starch, extensively yzed casein protein, fats, Vitamins and minerals. This slurry is lly emulsiﬁed, deaerated, homogenized and cooled to form a ized formula, and then aseptically packaged to form a sterilized, aseptically packaged concentrated liquid human milk fortiﬁer. Various other solutions may be added to the slurry at most any time before, during, or after processing.

Suitable aseptic packaging techniques include any of the well known aseptic packaging methods disclosed in the formulation arts for preparing ional formulation, all of which are lly directed to the sealing or ﬁlling of a sterilized liquid into a sterilized, air-tight container. Many variations on the basic method exist and are well known to those of ordinary skill in the formulation art, non-limiting examples of which are described in US. Pat. No. 6,096,358 (Murdick et al.); US. Pat. No. 6,227,261 (Das et al.); and US. Pat. No. 6,371,319 (Yeaton et al.), which descriptions are incorporated herein by reference.

The aseptically packaged embodiments ofthe present disclosure may include any container or package suitable for use with liquid human milk fortiﬁers and also capable of withstanding aseptic processing conditions (e.g., high temperature sterilization). Non- limiting examples of such containers include single or multi use bags, plastic bottles or containers, pouches, metal cans glass bottles, foil or other ﬂexible pouches, syringes, Vials, or any other container meeting the above-described criteria. {0090} The aseptically packaged ner for these ments is typically sterilized prior to being ﬁlled with its sterilized contents. The ner is most lly sterilized by the application of hydrogen peroxide or other suitable disinfectant to the inside e of the container. The hydrogen peroxide or other disinfectant is often applied in an atomized mist. After a disinfectant is d, the container may be transported along a conveyor system during which time the container may be subjected to one or more ngs of hot sterilized air, preferably hot, ized, dry air. The container is then preferably injected with nitrogen gas. The aseptically prepared container is then aseptically ﬁlled with sterilized product and sealed.

For c packaging, the concentrated liquid human milk fortiﬁer is lly heat treated with a high temperature short time (HTST) process or an ultra high temperature (UHT) process to sufﬁciently reduce the bioburden to allow the products to maintain commercially sterile over an extended shelf-life of the ﬁnished product exceeding about 12 months. The treated formula is then homogenized at 1000 psi or higher and aseptically packaged.

In an alternative embodiment, the trated liquid human milk fortiﬁers of the present disclosure may also be sterilized and retort packaged utilizing conventional means known in the art.

Methods of Use The concentrated liquid human milk er of the present disclosure is used in combination with human milk, other suitable infant formula, or combination of both, wherein the resulting fortiﬁed human milk or ed infant formula or combination of both has an osmolality le for oral administration to an infant. As noted, the lity will most typically be less than about 500 mOsm/kg water, more typically from about 300 mOsm/kg water to about 400 mOsm/kg water. {0094} As the solids content of the trated liquid human milk fortiﬁer has been increased in some embodiments as bed herein, the concentrated liquid human milk fortiﬁer of the present disclosure may be added directly to human milk (or infant formula or a combination of human milk and infant formula) in a volume to volume ratio of about 1:10 or less, including from about 1le to about 1:14, and also including from about 1:11 to about 1:12. The ratio is ultimately selected based primarily upon the ingredients and osmolality of the concentrated liquid human milk fortiﬁer and in View of the particular nutritional needs of the infant. The concentrated liquid human milk er may be added directly to every feeding or to a sufﬁcient number of feedings (e.g., once or twice daily) to provide optimal nutrition in view of the particular nutritional needs of the .

Human milk or other infant formula, after fortiﬁcation with the concentrated liquid human milk er will most typically have a calorie density ranging from about 40 kcal/ﬂ oz (1.4 kcal/ml) to about 80 kcal/ﬂ oz (2.7 kcal/ml), with the 56-80 kcal/ﬂ oz formulations (1.9-2.7 kcal/ml) being more useful in preterm infants, and the 40-55 koal/ﬂ oz (1 .4-1 .9 kcal/ml) formulations more useful for term infants.

The methods of the present disclosure include methods of providing nutrition to infants, especially preterm infants. As noted herein, preterm infants may especially beneﬁt from the use ofhuman milk fortiﬁers as the fortiﬁer can e onal nutrients to the preterm infant when combined with human breast milk and/or infant formula to foster quicker growth and development. In one particular ment, ion is ed to an infant by the addition of the concentrated liquid human milk fortiﬁer to human milk, infant formula, or combination thereof, followed by the administration of the fortiﬁed human milk, infant formula, or combination thereof, to the infant. 10097] Other alternative s of the present disclosure include using the human milk fortiﬁers as described herein to fortify human breast milk, infant formula, or a combination of human breast milk and infant formula to provide a fortiﬁed nutritional liquid for administration to an infant, and particularly a preterm infant. In one embodiment, human breast milk is fortiﬁed by mixing the concentrated human milk fortiﬁer with human breast milk, infant formula, or a combination thereof, in a volume to volume ratio of from about 1:3 to about 1:9.

The methods of the present disclosure also include a method of providing nutrition to users other than infants, such as adults and elderly. This method includes the addition of the concentrated ition to other liquid nutritionals such as suspensions, emulsions or clear or substantially clear liquids. The resulting concentrated liquid nutritional itions may be used as adult liquid nutritional compositions and medicinal liquid nutritional compositions.

Manufacture The concentrated liquid human milk fortiﬁers of the present sure may be prepared in accordance with the methods described hereinafte. [001001 In one embodiment, the concentrated liquid human milk fortiﬁer is prepared by lizing and combining/mixing ingredients into a homogeneous aqueous mixture which is subjected to a sufﬁcient l treatment and aseptic ﬁlling to achieve long term physical and microbial shelf stability. It should be recognized that as the probiotic used in the concentrated liquid human milk fortiﬁer cannot survive the aseptic heat treatment of the below described manufacturing process, the probiotic should be blended in after heat treatment, either by sterile stream or direct addition ately before ﬁlling of a sterilized, air-tight container. Alternatively, the probiotic may be treated for protection against the conditions of heat treatment by encapsulating a thermo-resistant tic as described . The ulated -resistant probiotic may then be included in the concentrated liquid human milk fortiﬁer prior to heat treatment. In yet other embodiments, the probiotic may be included in a separate sterile package from the concentrated liquid human milk fortiﬁer, and may be added to the concentrated liquid human milk fortiﬁer immediately prior to use.

To begin the manufacturing process, macronutrients hydrate, protein, fat, and minerals) are combined in several slurries er and with water. This blend is subjected to an initial heat treatment and then tested to verify proper nutrient .

Additional detail on this process is provided in the following paragraphs.

An intermediate aqueous carbohydrate—mineral (CHO—MIN) slurry is prepared by heating an appropriate amount of water. With agitation, the following soluble ingredients are added: maltodextrin, potassium citrate, ium chloride, potassium chloride, sodium chloride, and choline chloride. The carbohydrate-mineral slurry is held at elevated temperature under agitation until added to the blend.

An intermediate oil slurry is prepared by heating MCT oil and coconut oil to an elevated temperature and then adding distilled monoglycerides with agitation for a minimum of 10 minutes in order for the ingredient to dissolve. Soy oil, vitamin A palmitate, vitamin D3, alpha-tocopheryl-acetate, phylloquinone, ARA, DHA, and mixed carotenoids are then added with agitation to the oil blend. Insoluble mineral m source and ultra micronized tricalcium phosphate are added to the oil. Gellan gum and GSA-modiﬁed starch are then added to the oil blend with proper agitation. The oil blend slurry is maintained at an elevated ature under agitation until added to the blend.

The blend is prepared by combining the ingredient water, casein hydrolysate, all of the CHO—MlN slurry and whole oil blend slurry. The blend is maintained at 120°F for a period of time not to exceed two hours before further processing.

The blend is then homogenized using one or more in—line homogenizers at pressures from 1000-4000 psig with or without a second stage homogenization from 100- 500 psig followed by heat treatment using a UHTST (ultra-high temperature short time, 292-297°F for 1-30 seconds) process. After the appropriate heat treatment, the batch is cooled in a plate cooler to 33-45°F and then transferred to a refrigerated holding tank, Where it is subjected to analytical testing.

The next step in the manufacturing process involves optionally adding the probiotic along with any desired ns, trace minerals and water in order to reach the ﬁnal target total solids, probiotic, and vitamin/mineral contents. The ﬁnal batch is ﬁlled into a suitable container under aseptic ions or treated with a terminal sterilization s so the product will be stable at room temperature for an ed shelf—life.

Additional detail on this process is provided in the following paragraphs.

A trace l/vitamin/nutrient solution (STDl) is prepared by heating water to 80-100°F and adding the following ients with agitation: potassium citrate, ferrous sulfate, zinc sulfate, copper sulfate, manganese sulfate, sodium selenate, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, cyanocobalamin, folic acid, m pantothenate, niacinamide, biotin, m-inositol, nucleotide/choline premix, L-carnitine, L- leucine, L—tryptophan, and L-tyrosine.

] A vitamin C solution (STD2) is prepared by adding ic acid to water solution with agitation.

] All STDl and STD2 solutions are then added to the refrigerated batch, with agitation. The appropriate amount of ingredient on water is then added to the batch to achieve a target total solids level of 50-65%. The ﬁnal batch is then subjected to appropriate thermal treatment, the probiotic is optionally added, and ﬁlled into a le container under aseptic conditions and processes. (00110] The trated liquid human milk fortiﬁers of the t disclosure may, of course, be ctured by other known or ise suitable techniques not speciﬁcally described or shown herein without departing from the spirit and scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive and that all changes and equivalents also come within the description of the present disclosure. The following non-limiting examples will ﬁtrther illustrate the formulations and methods of the present disclosure.

EXAMPLES The following examples illustrate speciﬁc embodiments and/or features of the concentrated liquid human milk fortiﬁers of the present disclosure. The examples are given solely for the purpose of illustration and are not to be construed as limitations of the present disclosure, as many variations thereof are possible t departing from the spirit and scope of the disclosure. All iﬁed amounts are weight percentages based upon the total weight of the formulation, unless otherwise speciﬁed.

Examples [-4 In Examples 1—4, concentrated liquid human milk fortiﬁers are prepared in accordance with the present disclosure. The ingredients for the trated liquid human milk fortiﬁer are shown in the following table. All ingredient amounts are listed as kilogram per approximately 1000 kg batch of product, unless otherwise speciﬁed. _———-_ _—_—-_ OSA modiﬁed corn starch g—“u- OSA modiﬁed otato starch 0 __—— -——__—m- - 6-6 .1- -2 5.2 5-2 - 4.2 3,6 - 3.6 2.8 2.8 2.8 Mongglycerides 780.2 g 780.2 g 780.2 g 7802 g Sodium de 690.6 g 690.6 g 690.6 g 690.6 g M-Inositol 400.0 g 400.0 g 400.0 g 400.0 g Choline Chloride 320.0 g 320.0 g 320.0 g 320.0 g Tryptophan 300.0 g 300.0 g 300.0 g 300.0 g Zinc Sulfate 260.6 ; 260.6 g 260.6 g 260.6 g Niacinamide 254.6 g I 254.6 g 254.6 g 217-48 217.48 217-48 200-0 8 200-0 8 200.0 g Gellan Gum 199.6 ; 199.6 g 0 g 0 g Calcium Pantothenate 120.0 g_ 120.0 g 120.0 g 120.0 g Ferrous Sulfate 104.0 1 104.0 ; 104.0 ; 104.0 ; Vitamin A ate 51.5 g 51.5 g vin 30.0 g 30.0 g 22.0 : 22.0 g 17.8 g 12.0 1 Vitamin D3 Lutein 6.4 1 6.4 : Folic Acid 28 ; 2.8 ; 2.8 ; Beta Carotene 1880.0 ; 1880.0 1 1880.0 ; Biotin 1720.0 : 1720.0 : Man-anase Sulfate 1680.0 1 570.0 : 570-0 : 88.0 : 80.0 8 The concentrated liquid human milk fortiﬁer is prepared by solubilizing and combining/mixing ingredients into a homogeneous aqueous mixture which is subjected to a sufﬁcient thermal treatment and aseptic ﬁlling to achieve long term al and microbial shelf stability.

To begin the manufacturing process, utrients (carbohydrate, n, fat, and minerals) are combined in several slurries together and with water. This blend is subjected to an initial heat treatment and then tested to verify proper nt levels.

Additional detail on this process is provided in the following paragraphs.

An ediate s carbohydrate-mineral (CHO-MIN) slurry is prepared by heating an riate amount of water to 140-160°F. With agitation, the following soluble ingredients are added: sucrose, extrin, potassium citrate, magnesium chloride, potassium chloride, sodium chloride, and choline chloride. The carbohydrate- mineral slurry is held at 130-150°F under agitation until added to the blend.

An ediate oil slurry is prepared by heating MCT oil and coconut oil to 150 to 170°F and then adding distilled monoglycerides with agitation for a minimum of 10 minutes in order for the ingredient to dissolve. Soy oil, vitamin A palmitate, vitamin D3, Vitamin E, Vitamin K, ARA-containing oil, DHA-containing oil, lutein, and beta-carotene are then added with agitation to the oil blend. Insoluble mineral calcium source, and ultra micronized tricalcium phosphate is added to the oil. Gellan gum and GSA—modiﬁed starch are then added to the oil blend with proper agitation. The oil blend slurry is maintained at 130-150°F under agitation until added to the blend.

The blend is prepared by combining the ingredient water, casein hydrolysate, all of the N slurry and whole oil blend slurry. The blend is maintained at 120°F for a period oftime not to exceed two hours before further processing.

The blend is then homogenized using one or more in-line homogenizers at pressures from 1000-4000 psig with or without a second stage homogenization from 100- 500 psig ed by heat treatment using a HTST (high ature short time, 165- l85°F for 15-20 seconds) process. After the appropriate heat treatment, the batch is cooled in a plate cooler to 33-45°F and then transferred to a refrigerated holding tank, where it is subjected to analytical testing.

The next step in the manufacturing process involves adding vitamins, trace minerals, other ients, and water in order to reach the ﬁnal target total solids and vitamin/mineral contents. The ﬁnal pH of the product prior to thermal treatment is also adjusted. The ﬁnal batch is ﬁlled into a suitable container under aseptic conditions or treated with a terminal sterilization s so the product will be stable at room temperature for an extended shelf—life. onal detail on this process is provided in the following paragraphs.

A trace mineral/vitamin/nutrient solution (STDl) is prepared by heating water to 80-100°F and adding the following ingredients with agitation: potassium citrate, s sulfate, zinc sulfate, copper sulfate, manganese sulfate, sodium selenate, pyridoxine hydrochloride, riboﬂavin, thiamine hydrochloride, vitamin B12, folic acid, calcium pantothenate, niacinamide, , m-inositol, L—carnitine, L-leucine, L—tryptophan, and L-tyrosine.

] A vitamin C solution (STDZ) is prepared by adding ascorbic acid to a water solution with agitation.

All STDl and STDZ solutions are then added to the refrigerated batch, with agitation. The appropriate amount of ingredient dilution water is then added to the batch to achieve a target total solids level of SOD-60.0%. The ﬁnal pH of the product prior to thermal treatment is adjusted to >4.6—5.2 by addition of citric acid. The ﬁnal batch is then subjected to appropriate thermal treatment and ﬁlled into a suitable container under aseptic conditions and processes.

Example 5 In this Example, the concentrated liquid human milk fortiﬁer of e 1 was prepared and the overall water activity was ted. Four replicates of the concentrated liquid human milk fortiﬁer were evaluated using AquaLab CX-2 (Decagon Devices, Inc., Pullman, Washington).

Prior to evaluating the trated liquid human milk er, a standardization check was run on ted salt solutions of lithium chloride, magnesium chloride, and magnesium nitrate. The salt solutions were compared with distilled water. The results of the standardization check are shown in the table below.

Saturated Salt Solutions Target Standard Actual Reading Temperature (°C) Lithium Chloride 0.11 0.11 21.7 Magnesium Chloride 0.3 0.305 22.6 Magnesium Nitrate 0.5 0.526 21.8 DI Water 1.0 0.995 22.1 The water activity results of the concentrated liquid human milk fortiﬁer ates are shown in the table below. As the data indicate, the liquid human milk fortiﬁer has a very low water activity that is sufﬁcient to retard the growth of ed microbes during storage.

Replicate Reading ature (°C) 1 0.858 22.7 2 0.875 21.9 3 0.862 21.8 4 0.865 21.8 Average 0.865 22.1 Examples 6—9 Examples 6-9 were made in accordance with examples 1-4, tively, except Biﬁdobacterium Zactz's (probiotic) (1.0 kg) was added to each after the thermal treatment and before the batches are ﬁlled into a suitable container under aseptic ions and processes.

Claims

We claim:

1. A concentrated liquid nutritional composition comprising: from 5% to 50% by weight protein, on a dry weight basis, wherein at least a portion of the protein is hydrolyzed casein having a degree of ysis of from 20% to 80%; wherein the concentrated liquid nutritional composition has a solids content of greater than 50% by weight; and wherein the concentrated liquid nutritional composition has a water activity of less than 0.90.

2. The concentrated liquid nutritional composition of claim 1, r comprising a tic.

3. The concentrated liquid nutritional composition of claim 2, sing from 10 3 to 10" CFU/mL probiotic.

4. The concentrated liquid nutritional composition according to any one of claims 1-3, wherein the composition is an adult liquid nutritional composition.

5. The concentrated liquid nutritional composition according to any one of claims 1-3, wherein the ition is a liquid human milk fortifier.

6. The concentrated liquid nutritional composition according to any one of claims 1-3, wherein the composition has a pH of from 4.6 to 6.5.

7. The concentrated liquid nutritional composition according to any one of claims 1-3, further comprising carbohydrate, fat, vitamins, and ls.

8. The concentrated liquid nutritional composition according to any one of claims 1-3, wherein the concentrated liquid nutritional composition ses from 20% to 40% by weight protein, on a dry weight basis. (10115870 1):GGG

9. The concentrated liquid nutritional composition according to any one of claims 1-3, further comprising a izer system comprising an l succinic anhydride modified corn starch and a low acyl gellan gum, wherein the octenyl succinic anhydride modified corn starch is present in an amount of from 0.6% to 2.0% by weight of the composition, and wherein the low acyl gellan gum is present in an amount of from greater than 125 ppm to 800 ppm.

10. The concentrated liquid nutritional composition according to any one of claims 1-3, wherein the composition includes a stabilizer system sing an octenyl succinic anhydride ed potato starch.

11. The concentrated liquid ional composition according to any one of claims 1-3, wherein the protein is 100% by weight hydrolyzed casein having a degree of hydrolysis of from 20% to 80%.

12. The concentrated liquid nutritional composition ing to any one of claims 1-3, n the composition has a caloric density of from 1.25 kcal/mL to 5 kcal/mL.

13. A method of fortifying breast milk, the method comprising: mixing a liquid human milk fortifier with breast milk in a volume to volume ratio of less than or equal to 1:10; wherein the liquid human milk fortifier comprises from 5% to 50% by weight n, on a dry weight basis, and wherein at least a portion of the protein is hydrolyzed casein having a degree of hydrolysis of from 20% to 80%; wherein the liquid human milk fortifier has a solids content of greater than 50% by weight; and wherein the liquid human milk fortifier has a water activity of less than 0.90.

14. The method of claim 13, wherein the liquid human milk fortifier further comprises a probiotic. (101 15870_1):GGG

15. The method of claim 14, wherein the liquid human milk fortifier comprises from 103 to 1011 CFU/mL probiotic.

16. The method of either one of claims 14 or 15, n the probiotic is present in the liquid human milk fortifier in a form selected from the group ting of freezedried, an oil droplet, a bacterial suspension solution, and encapsulated.

17. The method according to any one of claims 13-15, wherein the liquid human milk fortifier is mixed with breast milk in a volume to volume ratio of from 1:14 to 1:11.

18. The method according to any one of claims 13-15, wherein the liquid human milk fortifier has a pH of from 4.6 to 5.2.

19. The method according to any one of claims 13-15, wherein the liquid human milk fortifier further comprises a stabilizer system comprising an octenyl succinic anhydride modified corn starch and a low acyl gellan gum.

20. The method according to any one of claims 13-15, wherein the fortified breast milk has a caloric density of from 1.4 kcal/mL to 2.7 kcal/mL.

21. A trated liquid nutritional composition ing to claim 1 and substantially as herein described with reference to the description and examples.

22. A method of ying breast milk, said method according to claim 13 and substantially as herein described with reference to the description and examples. Abbott Laboratories By the Attorneys for the Applicant SPRUSON & FERGUSON Per: (10774644_1):GGG