Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
PROBIOTIC STABILIZATION
TECHNICAL FIELD
[0001] The present disclosure relates to the stabilization of biological
material
for ingestion by an individual. More particularly, the present disclosure
relates to a
stabilization mixture comprising at least one phospholipid and at least one
glyceride,
which together provide improved stability to a probiotic organism when the
probiotic
is included in a nutritional composition. The disclosure also includes
probiotic
stabilization methods.
BACKGROUND ART
[0002] There are currently a variety of compositions for supplementing the
nutrition of both humans and animals. These supplements may be provided to
alter,
reduce or increase the microflora within an individual's gut so as to cause a
desired
effect on digestion. Ideally, supplementation may cultivate an improved
microflora
for individuals, including humans, based upon the alteration of specific
bacteria
within the human's gastrointestinal (GI) tract. This style of supplementation
may be
conducted through the use of probiotics, which are understood to be live
microorganisms, that when administered in effective amounts confer a health or
nutritional benefit to the host. One of the more common types of probiotics is
a
lactic acid bacterium which is able to convert sugars and other carbohydrates
into
lactic acid. This conversion lowers the pH within the gut of the host and
provides
fewer opportunities for harmful organisms to grow and cause problems through
gastrointestinal infections.
[0003] A common technological challenge is introducing probiotics into the
host in an appropriate manner, both for the maintenance of the probiotics as
well as
for the health and enjoyment of an individual. Current technologies include
the
utilization of encapsulation and stabilization techniques for shielding the
probiotics
with a protective layer or matrix so that the protected microbe may pass to
the
appropriate location within the individual's GI tract.
[0004] Although there have been developments concerning encapsulation and
stabilization techniques for containing microorganisms for delivery into the
digestive
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system of animals, there has been little development in encapsulation or
stabilization
techniques that protect the viability of probiotics during distribution and
storage.
There is a need for a stabilization technique that is useful where
circumstances
preclude refrigeration, and further where such formulations may be exposed to
various environments, especially those associated with tropical climates. In
addition,
the inherent moisture of the product poses a challenge in that probiotics
generally
are sensitive to water, especially in combination with high temperature. To
date, no
technology or technique has been identified to deliver sufficient protection
to
probiotics under intermediate moisture conditions (i.e. water activity of
about 0.2 and
higher, and up to about 0.4 or higher) and high temperatures during
distribution and
storage (i.e. temperatures of at least about 30 C, and up to and above 40 C)
when
incorporated into nutritional formulas.
[0005] In particular, probiotics can provide a variety of benefits to a
host, such
as maintaining a healthy gastrointestinal flora, enhancing immunity,
protecting
against diarrhea, atopic dermatitis and other diseases, etc. As such, there is
a need
for probiotics to be administered in various geographic locations, including
tropical
climates, where the viability of the probiotic could be compromised.
Conventional
encapsulation and stabilization techniques possess a chemical makeup that is
ill-
suited for infant formulas and/or for use by children, or known techniques
have poor
stability characteristics that significantly limit commercial opportunities.
[0006] What is desired therefore, is a stabilization technique and a
stabilized
bacterial mixture using acceptable ingredients for either an infant formula or
children's nutrition, the stabilized mixture allowing for improved stability
properties
so that probiotics may be distributed in a wide variety of geographical
locations and
climates while maintaining a useful shelf-life. Further desired is a
stabilization
technology for the protection of probiotics, such as Lactobacillus rhamnosus,
for use
in nutritional compositions, such as infant formulas, supplements and
children's
products. Indeed, a combination of characteristics, including improved
stability
combined with nutritional factors, provide an improved stabilization mixture
applicable for prenatal, infant, and children's nutrition.
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DISCLOSURE OF THE INVENTION
[0007] In some embodiments, the present disclosure is directed to a
nutritional
composition comprising a protein source and a probiotic, wherein the probiotic
is
stabilized in a protective matrix, the protective matrix includes at least one
phospholipid and at least one glyceride.
[0008] In such embodiments, the nutritional composition comprises viable
microbial cells, such as viable Lactobacillus rhamnosus cells. Also, the
matrix may
comprise a source of hydrolyzed protein, pectin, an additional lipid, or any
combination thereof. The nutritional composition may be a powdered formula,
such
as a powdered infant formula. Moreover, the at least one glyceride may include
a
monoglyceride, a diglyceride, or any combination thereof.
[0009] In another embodiment, the present disclosure is directed to a
method
for protecting a viable probiotic for use in a powdered nutritional
composition, the
method includes the steps of (i) providing a viable probiotic, (ii) preparing
a
protective matrix for the probiotic by blending together at least one
phospholipid
and at least one glyceride, and (iii) combining the viable probiotic, the
protective
matrix and water to produce a mixture; and (iv) drying the mixture of step
(iii) to a
final moisture content of about 4% or less. This method may comprise the
additional
step of a) adding the dried mixture to a powdered nutritional product or b)
enclosing
the dried mixture of step (iv) in a capsule. In such an embodiment, the viable
probiotic may be Lactobacillus rhamnosus, Bifidobacterium longum BB536,
Bifidobacterium longumsubsp. infantis 35624, Bifidobacterium animalissubsp.
lactis
BB-12, or any combination thereof.
[0010] These aspects and others that will become apparent to the skilled
artisan upon review of the following description can be accomplished by
providing a
mixture a phospholipid and a glyceride for the stabilization of biological
material,
such as probiotics, to provide for increased stability of the biological
material,
resulting in the improved, long-term viability of the biological material. In
an
embodiment, the stabilization mixture advantageously provides for an extension
of
the shelf-life of probiotics. The stabilization mixture may be combined with
the
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probiotic in a variety of methods including freeze-drying, air-drying, vacuum-
drying,
spray-drying and any combination thereof for preserving the probiotic.
[0011] It is to be understood that both the foregoing general description
and
the following detailed description provide embodiments of the disclosure and
are
intended to provide an overview or framework of understanding to the nature
and
character of the disclosure as it is claimed.
BEST MODE FOR CARRYING OUT THE INVENTION
[0012]Reference now will be made in detail to the embodiments of the present
disclosure, one or more examples of which are set forth hereinbelow. Each
example
is provided by way of explanation of the nutritional composition of the
present
disclosure and is not a limitation. In fact, it will be apparent to those
skilled in the art
that various modifications and variations can be made to the teachings of the
present
disclosure without departing from the scope of the disclosure. For instance,
features
illustrated or described as part of one embodiment can be used with another
embodiment to yield a still further embodiment.
[0013]Thus, it is intended that the present disclosure covers such
modifications and
variations as come within the scope of the appended claims and their
equivalents.
Other objects, features and aspects of the present disclosure are disclosed in
or are
obvious from the following detailed description. It is to be understood by one
of
ordinary skill in the art that the present discussion is a description of
exemplary
embodiments only and is not intended as limiting the broader aspects of the
present
disclosure.
[0014] The present disclosure provides a stabilization technique and a
stabilized mixture (also referred to herein as a "stabilization mixture" or a
"protective
matrix") that may be used for improving the stability of a biological material
(also
referred to herein as a "substrate"). In embodiments of the disclosure, the
stabilized
substrate may be a probiotic, wherein the various health benefits associated
with the
stabilized probiotic may be conferred to an individual upon ingesting a
nutritional
composition containing the stabilized probiotic.
[0015] While probiotics have been recognized as nutritionally beneficial,
it is
generally thought that the beneficial effects of probiotics are maximized if a
probiotic
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microorganism is ingested by a subject when the microorganism is still alive.
Thus, it
is desirable for a viable probiotic to survive the conditions of manufacturing
and of
placement into a consumable nutritional composition, such as a food or
beverage, as
well as to survive the subsequent shipping and storage time before the product
is
ingested and introduced to a subject's gastrointestinal tract. Many
conventional
probiotic compositions utilize an extremely high count of viable cells, with
the
understanding that a significant number of cells ultimately lose viability and
die
during the manufacturing process, transport, and storage.
[0016] By practice of the present disclosure, a lipid component comprising
at
least one phospholipid and/or at least one glyceride is incorporated into a
protective
matrix. The phospholipid and/or glyceride increase the stability of probiotics
that are
protected by the matrix. As a result, infant- and children-compatible
probiotics can
be stabilized with a matrix including at least one phospholipid and/or at
least one
glyceride. In some embodiments, the matrix further comprises at least one
additional lipid, that is, at least one lipid in addition to the phospholipid
and
glyceride. The stabilized probiotics can be used in multiple environments, as
the
probiotics exhibit an improved viability. Advantageously, probiotics
stabilized by the
protective matrix of the present disclosure can be incorporated into
nutritional
compositions and shipped over extended distances subjected to potentially
damaging temperature fluctuations, as the probiotics will maintain viability
even after
extended transportation and storage time, due to the improved stability of the
stabilized mixture.
[0017] The present method of providing stabilization to probiotics may
include
the use of a matrix for stabilizing a biological material, wherein the matrix
includes a
phospholipid, a glyceride, and additional components, such as one or more
additional lipids, one or more carbohydrates and/or a compound binder.
[0018] While the protective matrix may be utilized for a variety of
substances,
in an embodiment, it is utilized to protect at least one probiotic, such as
Lactobacillus
rhamnosus. Lactobacillus rhamnosus is understood to possess relatively good
bio-
stability while having a high avidity for human intestinal mucosal cells. In
use as a
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probiotic, Lactobacillus rhamnosus is thought to colonize the digestive tract
and to
balance intestinal microflora.
[0019] The protective matrix provides for improved stability of the
probiotic,
meaning that a greater percentage of the probiotic cells are viable after
processing,
transportation and storage conditions. Specifically, the shelf life of viable
probiotics
is improved when compared to other known stabilization techniques.
[0020] The protective matrix of the present disclosure may be used in a
multiplicity of processes for forming a stabilized probiotic product. These
processes
include freezing, flash-freezing, freeze-drying, ambient air drying, vacuum
drying,
spray drying, low temperature drying, and any combination thereof. The
resulting
stabilized probiotic, whether alone or integrated into a nutritional
composition,
possesses effective viability in a wide range of temperatures and conditions
while
displaying improved shelf-life. Furthermore, the stabilized probiotic may be
incorporated into a variety of prenatal, infant and children's nutritional
products for
improving their gut microflora while simultaneously providing nutrition to the
infant
or child.
[0021] Accordingly, in one embodiment, the disclosure is directed to a
method
for stabilizing a biological material in a nutritional composition. Still
another
embodiment is a protective matrix for a probiotic. A further embodiment is a
method of increasing the shelf life of probiotics comprising stabilizing the
probiotic
with a stabilization mixture including a phospholipid, a glyceride or any
combination
thereof.
[0022] The present disclosure provides a novel stabilization mixture and
method that provides stability and protection to biological materials, such as
viable
microorganisms. The present disclosure includes a stabilization mixture
comprising a
phospholipid, and a glyceride, which together provide a protective matrix
resulting in
an increased shelf-life over unprotected probiotics.
DEFINITIONS
[0023] The terms "protective matrix," "stabilization matrix," and
"stabilization
mixture" are used interchangeably throughout the present disclosure.
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[0024] An "effective amount" as used herein is generally defined as an
amount
of an agent that provides an observable result within the subject administered
thereto.
[0025] "Nutritional composition" means a substance or formulation that
satisfies at least a portion of a subject's nutrient requirements. The terms
"nutritional(s)", "nutritional formula(s)", "enteral nutritional(s)", and
"nutritional
supplement(s)" are used as non-limiting examples of nutritional composition(s)
throughout the present disclosure. Moreover, "nutritional composition(s)" may
refer
to liquids, powders, gels, pastes, solids, concentrates, suspensions, or ready-
to-use
forms of enteral formulas, oral formulas, formulas for infants, formulas for
pediatric
subjects, formulas for children, growing-up milks and/or formulas for adults.
[0026] The term "enteral" means deliverable through or within the
gastrointestinal or digestive tract. "Enteral administration" includes oral
feeding,
intragastric feeding, transpyloric administration, or any other administration
into the
digestive tract. "Administration" is broader than "enteral administration" and
includes parenteral administration or any other route of administration by
which a
substance is taken into a subject's body.
[0027] "Pediatric subject" means a human less than 13 years of age. In
some
embodiments, a pediatric subject refers to a human subject that is less than 8
years
old. In other embodiments, a pediatric subject refers to a human subject
between 1
and 6 years of age. In still further embodiments, a pediatric subject refers
to a
human subject between 6 and 12 years of age.
[0028] "Infant" means a human subject ranging in age from birth to not
more
than one year and includes infants from 0 to 12 months corrected age. The
phrase
"corrected age" means an infant's chronological age minus the amount of time
that
the infant was born premature. Therefore, the corrected age is the age of the
infant
if it had been carried to full term. The term infant includes low birth weight
infants,
very low birth weight infants, and preterm infants. A "pre-term infant" is an
infant
born after less than about 37 weeks gestation. A "full-term infant" as used
herein
means an infant born after at least about 37 weeks gestation.
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[0029] "Child" means a subject ranging in age from 12 months to about 12
years. In some embodiments, a child is a subject between the ages of 1 and 12
years
old. In other embodiments, the terms "children" or "child" refer to subjects
that are
between one and about six years old, or between about seven and about 12 years
old. In other embodiments, the terms "children" or "child" refer to any range
of
ages between 12 months and about 12 years.
[0030] "Children's nutritional product" refers to a composition that
satisfies at
least a portion of the nutrient requirements of a child. A growing-up milk
(GUM) is an
example of a children's nutritional product.
[0031] As used herein, "hydrolyzed protein" means a product of protein
hydrolysis. Within the present disclosure, hydrolyzed protein and protein
hydrolysate
are used interchangeably to describe products of protein hydrolysis;
extensively
hydrolyzed protein is used to describe products of protein hydrolysis where at
least
70%, more preferably at least about 90%, of the hydrolyzed protein has a
molecular
weight of less than 2000 Daltons.
[0032] The term "degree of hydrolysis" refers to the extent to which
peptide
bonds are broken by a hydrolysis method.
[0033] The term "protein-free" means containing no measurable amount of
protein, as measured by standard protein detection methods such as sodium
dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or size
exclusion chromatography. In some embodiments, the nutritional composition is
substantially free of protein, wherein "substantially free" is defined
hereinbelow.
[0034] "Infant formula" means a composition that satisfies at least a
portion of
the nutrient requirements of an infant. In the United States, the content of
an infant
formula is dictated by the federal regulations set forth at 21 C.F.R. Sections
100, 106,
and 107. These regulations define macronutrient, vitamin, mineral, and other
ingredient levels in an effort to simulate the nutritional and other
properties of
human breast milk.
[0035] The term "growing-up milk" refers to a broad category of
nutritional
compositions intended to be used as a part of a diverse diet in order to
support the
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normal growth and development of a child between the ages of about 1 and about
6
years of age.
[0036] "Milk-based" means comprising at least one component that has been
drawn or extracted from the mammary gland of a mammal. In some embodiments, a
milk-based nutritional composition comprises components of milk that are
derived
from domesticated ungulates, ruminants or other mammals or any combination
thereof. Moreover, in some embodiments, milk-based means comprising bovine
casein, whey, lactose, or any combination thereof. Further, "milk-based
nutritional
composition" may refer to any composition comprising any milk-derived or milk-
based product known in the art.
[0037] "Nutritionally complete" means a composition that may be used as
the
sole source of nutrition, which would supply essentially all of the required
daily
amounts of vitamins, minerals, and/or trace elements in combination with
proteins,
carbohydrates, and lipids. Indeed, "nutritionally complete" describes a
nutritional
composition that provides adequate amounts of carbohydrates, lipids, essential
fatty
acids, proteins, essential amino acids, conditionally essential amino acids,
vitamins,
minerals and energy required to support normal growth and development of a
subject.
[0038] The composition which is "nutritionally complete" for the preterm
infant
will, by definition, provide qualitatively and quantitatively adequate amounts
of all
carbohydrates, lipids, essential fatty acids, proteins, essential amino acids,
conditionally essential amino acids, vitamins, minerals, and energy required
for
growth of the preterm infant. The composition which is "nutritionally
complete" for
the term infant will, by definition, provide qualitatively and quantitatively
adequate
amounts of all carbohydrates, lipids, essential fatty acids, proteins,
essential amino
acids, conditionally essential amino acids, vitamins, minerals, and energy
required for
growth of the term infant. The composition which is "nutritionally complete"
for a
child will, by definition, provide qualitatively and quantitatively adequate
amounts of
all carbohydrates, lipids, essential fatty acids, proteins, essential amino
acids,
conditionally essential amino acids, vitamins, minerals, and energy required
for
growth of a child.
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[0039] As applied to nutrients, the term "essential" refers to any
nutrient that
cannot be synthesized by the body in amounts sufficient for normal growth and
to
maintain health and that, therefore, must be supplied by the diet. The term
"conditionally essential" as applied to nutrients means that the nutrient must
be
supplied by the diet under conditions when adequate amounts of the precursor
compound is unavailable to the body for endogenous synthesis to occur.
[0040] The term "probiotic" means a microorganism with low or no
pathogenicity that exerts beneficial effects on the health of the host. A
"viable
probiotic" means a live or active microorganism that exerts beneficial effects
on the
health of the host.
[0041] The term "inactivated probiotic" or "inactivated LGG" means a
probiotic wherein the metabolic activity or reproductive ability of the
referenced
probiotic or Lactobacillus rhamnosus GG (LGG) organism has been reduced or
destroyed. In an embodiment, the probiotic(s) may be viable or non-viable. As
used herein, the term "viable", refers to live microorganisms. The term "non-
viable"
or "non-viable probiotic" means non-living probiotic microorganisms, their
cellular
components and/or metabolites thereof. Such non-viable probiotics may have
been
heat-killed or otherwise inactivated, but they retain the ability to favorably
influence
the health of the host. The probiotics useful in the present disclosure may be
naturally-occurring, synthetic or developed through the genetic manipulation
of
organisms, whether such source is now known or later developed.
[0042] "Prebiotic" means a non-digestible food ingredient that
beneficially
affects the host by selectively stimulating the growth and/or activity of one
or a
limited number of bacteria in the digestive tract that can improve the health
of the
host.
[0043] "Phytonutrient" means a chemical compound that occurs naturally in
plants. Phytonutrients may be included in any plant-derived substance or
extract.
The term "phytonutrient(s)" encompasses several broad categories of compounds
produced by plants, such as, for example, polyphenolic compounds,
anthocyanins,
proanthocyanidins, and flavan-3-ols (i.e. catechins, epicatechins), and may be
derived
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from, for example, fruit, seed or tea extracts. Further, the term
phytonutrient
includes all carotenoids, phytosterols, thiols, and other plant-derived
compounds.
[0044] "13-9 lucan" means all P-glucan, including specific types of P-
glucan, such
as 3-1,3-glucan or 3-1,3;1,6-glucan. Moreover, 13-1,3;1,6-glucan is a type of
13-1,3-
glucan. Therefore, the term "3-1,3-glucan" includes 13-1,3;1,6-glucan.
[0045] "Pectin" means any naturally-occurring oligosaccharide or
polysaccharide that comprises galacturonic acid that may be found in the cell
wall of
a plant. Moreover, pectin may comprise molecules having a diverse chain length
formed by D-galacturonopyranosyl. Different varieties and grades of pectin
having
varied physical and chemical properties are known in the art. Indeed, the
structure of
pectin can vary significantly between plants, between tissues, and even within
a
single cell wall. Generally, pectin is made up of negatively charged acidic
sugars
(galacturonic acid), and some of the acidic groups are in the form of a methyl
ester
group. The degree of esterification of pectin is a measure of the percentage
of the
carboxyl groups attached to the galactopyranosyluronic acid units that are
esterified
with methanol.
[0046] Pectin having a degree of esterification of less than 50% (i.e.,
less than
50% of the carboxyl groups are methylated to form methyl ester groups) are
classified as low-ester, low methoxyl, or low methylated ("LM") pectins, while
those
having a degree of esterification of 50% or greater than 50%, (i.e., more than
50% of
the carboxyl groups are methylated) are classified as high-ester, high
methoxyl or
high methylated ("HM") pectins. Very low ("VL") pectins, a subset of low
methylated
pectins, have a degree of esterification that is less than approximately 15%.
Furthermore, in some embodiments, pectin molecules have the ability to
interact
with themselves and/or with divalent cations, such as calcium, through
electrostatic
interactions to form high molecular weight networks and gels. The degree of
esterification influences the extent of these intermolecular interactions.
[0047] "Lipid" refers to hydrophobic molecule(s) that may include free
fatty
acid(s) and/or molecules in which fatty acids are esterified to a hydroxyl
group of (i) a
saccharide, such as a mono- or di-saccharide, (ii) an alcohol, wherein the
alcohol may
comprise, for example, a long- or medium- carbon chain length (10 -20 units),
or (iii) a
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glycerol. These esterified or free fatty acids may be linear chains, saturated
or
unsaturated. Further, in some embodiments, the lipid component comprises
glycerides, phospholipids, sphingosine-derived molecules, such as
gangliosides,
cerebrosides, and sphingomyelins, or any combination thereof. And in certain
embodiments, the lipid component further includes molecules that are
hydrophobic
in nature, such as steroids, carotenoids, and/or fat-soluble vitamins.
[0048] "Glyceride" means any molecule derived from a glycerol or a sugar
backbone in which one or more of the hydroxyl groups is esterified with a
fatty acid.
In some embodiments, the glyceride component comprises mono-glycerides, di-
glycerides, mono-sugar esters, di-sugar esters, or a combination thereof.
[0049] "Phospholipid" means any molecule derived from a phosphatidic acid
in
which one or two carbons from the glycerol backbone are esterified to a fatty
acid,
and one of the terminal carbons is esterified to a phosphate group. In certain
embodiments, the phosphate group is a phosphodiester also esterified to
another
functional molecule, such as choline, serine, or inositol. In some
embodiments, the
phospholipid component comprises glycerophospholipid and/or
phosphoacylglycerols. Further, the phospholipid component may comprise lyso-
phospholipids wherein one of the carbons from the glycerol backbone remains as
a
non-esterified hydroxyl group. And in some embodiments, the phospholipid
component comprises lecithin, which may, for example, be extracted from
soybeans
or from egg yolk and may comprise a combination of phosphatidyl-choline,
phosphatidyl-inositol and phosphatidyl serine. In some embodiments, the
phospholipid may comprise soybean lecithin, egg lecithin, a milk fat derived
phospholipid or a combination thereof.
[0050] All percentages, parts and ratios as used herein are by weight of
the
total nutritional composition, including the stabilized probiotic, unless
otherwise
specified.
[0051] All amounts specified as administered "per day" may be delivered in
one unit dose, in a single serving or in two or more doses or servings
administered
over the course of a 24 hour period.
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[0052] The nutritional composition of the present disclosure may be
substantially free of any optional or selected ingredients described herein,
provided
that the remaining nutritional composition still contains all of the required
ingredients
or features described herein. In this context, and unless otherwise specified,
the
term "substantially free" means that the selected composition may contain less
than
a functional amount of the optional ingredient, typically less than 0.1% by
weight,
and also, including zero percent by weight of such optional or selected
ingredient.
[0053] All references to singular characteristics or limitations of the
present
disclosure shall include the corresponding plural characteristic or
limitation, and vice
versa, unless otherwise specified or clearly implied to the contrary by the
context in
which the reference is made.
[0054] All combinations of method or process steps as used herein can be
performed in any order, unless otherwise specified or clearly implied to the
contrary
by the context in which the referenced combination is made.
[0055] The methods and compositions of the present disclosure, including
components thereof, can comprise, consist of, or consist essentially of the
essential
elements and limitations of the embodiments described herein, as well as any
additional or optional ingredients, components or limitations described herein
or
otherwise useful in nutritional compositions.
[0056] As used herein, the term "about" should be construed to refer to
both
of the numbers specified as the endpoint(s) of any range. Any reference to a
range
should be considered as providing support for any subset within that range.
THE PROTECTIVE MATRIX
[0057] In the practice of the present disclosure, at least one
phospholipid and
at least one glyceride are utilized as components of a protective matrix for
stabilizing
biological material.
[0058] The use of the phospholipid and/or glyceride in the protective
matrix of
the present disclosure provides superior protection to probiotics, including
Lactobacillus rhamnosus beyond what was previously known in the art.
[0059] The lipid component of the protective matrix may comprise, but is
not
limited to animal sources, e.g., milk fat, butter, butter fat, milk
phospholipids, egg
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yolk lipid, egg yolk phospholipids; marine sources, such as fish oils, marine
oils, single
cell oils; vegetable and plant oils, such as corn oil, canola oil, sunflower
oil, soybean
oil, soybean phospholipids, palmolein, coconut oil, high oleic sunflower oil,
evening
primrose oil, rapeseed oil, olive oil, flaxseed (linseed) oil, cottonseed oil,
high oleic
safflower oil, palm stearin, palm kernel oil, wheat germ oil; medium chain
triglyceride
oils and emulsions and esters of fatty acids; and any combinations thereof.
[0060] In some embodiments, the stabilization mixture comprises between
about 0.5 and about 50 grams of a lipid component per 100 grams of the mixture
on
a dry basis. In certain embodiments, the stabilization mixture comprises
between
about 2.5 and about 30 grams of a lipid component per 100 grams of the mixture
on
a dry basis.
[0061] The stabilization mixture may comprise a phospholipid component. In
certain embodiments, the stabilization mixture includes between about 0.5 and
about
2 grams of a phospholipid component per 100 grams of the mixture on a dry
basis.
In some embodiments, the stabilization mixture comprises between about 0.75
and
about 1.25 grams of a phospholipid component per 100 grams of the mixture on a
dry basis.
[0062] Likewise, the stabilization mixture may include a glyceride
component.
In some embodiments, the stabilization mixture comprises between about 0.5 and
about 2 grams of a glyceride component per 100 grams of the mixture on a dry
basis.
In certain embodiments, the stabilization mixture comprises between about 0.75
and
about 1.25 grams of a glyceride component per 100 grams of the mixture on a
dry
basis. Moreover, the glyceride component may comprise monoglyceride(s),
diglyceride(s), or any combination thereof.
[0063] In certain embodiments, the majority component of the stabilization
mixture, based on a dry weight basis, is one or more carbohydrates, which may
include polysaccharides, disaccharides and monosaccharides. Indeed, the
protective
matrix may include lactulose, lactosucrose, raffinose, gluco-oligosaccharide,
trehalose, inulin, polydextrose, galacto-oligosaccharide, fructo-
oligosaccharide,
isomalto-oligosaccharide, soybean oligosaccharides, lactosucrose, xylo-
oligosaccharide, chito-oligosaccharide, manno-oligosaccharide, aribino-
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oligosaccharide, siallyl-oligosaccharide, fuco-oligosaccharide, gentio-
oligosaccharides, and/or any combination thereof. In some embodiments, the
protective matrix includes a first carbohydrate chosen from: sucrose, maltose,
lactose, trehalose, maltotriose, maltodextrin having a dextrose equivalent of
about 2
to about 6, and any combination thereof. In certain embodiments, the
protective
matrix includes a second carbohydrate chosen from: inulin, polydextrose,
galactooligosaccharide, fructooligosaccharide, starch, maltodextrin having a
dextrose
equivalent of greater than about 8, and any combination thereof.
[0064] In some embodiments, the stabilization mixture may comprise between
about 50 and about 80 grams of a first carbohydrate per 100 grams of the
mixture on
a dry basis; between about 60 and about 70 grams of a first carbohydrate per
100
grams of the mixture on a dry basis; or between about 65 and about 70 grams of
a
first carbohydrate per 100 grams of the mixture on a dry basis. The first
carbohydrate
may be chosen from: sucrose, maltose, lactose, trehalose, maltotriose,
maltodextrin
having a dextrose equivalent of about 2 to about 6, and any combination
thereof.
[0065] The stabilization mixture may also comprise between about 1 and
about
10 grams of a second carbohydrate per 100 grams of the mixture on a dry basis;
between about 4 and about 6 grams of a second carbohydrate per 100 grams of
the
mixture on a dry basis; or about 5 grams of a second carbohydrate per 100
grams of
the mixture on a dry basis. In some embodiments, the second carbohydrate is
chosen from: inulin, polydextrose, galactooligosaccharide,
fructooligosaccharide,
starch, maltodextrin having a dextrose equivalent of greater than about 8, and
any
combination thereof.
[0066] A further component of the stabilization mixture can be a compound
binder (also referred to as a gelling agent), which may act as a thickener and
produce
a gel-like consistency. Compound binders that may be included in the
protective
matrix of the present disclosure include alginates, such as sodium alginate,
pectin,
chitosan, carboxymethylcellulose, and mixtures thereof, among others. The
incorporation of the compound binder provides for the formation of a viscous
consistency providing for efficient matrix formation and a structural quality
suitable
for subsequent drying.
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[0067] A compound binder can, in some embodiments, form a gum-like
material and increase the viscosity of mixtures to which it is added.
Additionally, the
compound binder may also provide for greater ease in mixing of the components
together. For instance, sodium alginate may also possess emulsifier
characteristics.
[0068] In some embodiments, the stabilization mixture may comprise LM
pectin, HM pectin, VL pectin, or any mixture thereof. The included pectin may
be
soluble in water.
[0069] Pectins for use herein typically have a peak molecular weight of
8,000
Daltons or greater. The pectins of the present disclosure have a preferred
peak
molecular weight of between 8,000 and about 500,000, more preferred is between
about 101000 and about 200,000 and most preferred is between about 15,000 and
about 100,000 Daltons. In some embodiments, the pectin of the present
disclosure
may be hydrolyzed pectin. In certain embodiments, the protective matrix
comprises
hydrolyzed pectin having a molecular weight less than that of intact or
unmodified
pectin. The hydrolyzed pectin of the present disclosure can be prepared by any
means known in the art to reduce molecular weight. Examples of said means are
chemical hydrolysis, enzymatic hydrolysis and mechanical shear. A preferred
means
of reducing the molecular weight is by alkaline or neutral hydrolysis at
elevated
temperature. In some embodiments, the protective matrix comprises partially
hydrolyzed pectin. In certain embodiments, the partially hydrolyzed pectin has
a
molecular weight that is less than that of intact or unmodified pectin but
more than
3,300 Daltons.
[0070] The stabilization mixture may comprise between about 0.5 and about
5
grams of a compound binder, such as sodium alginate and/or pectin, per 100
grams
of the mixture on a dry basis. In certain embodiments, the stabilization
mixture
comprises between about 1 and about 3 grams of a compound binder per 100 grams
of the mixture on a dry basis. And in an embodiment, the stabilization mixture
comprises about 2 grams of a compound binder per 100 grams of the mixture on a
dry basis.
[0071] Moreover, the stabilization mixture may also comprise at least one
starch, source of starch and/or starch component. In some embodiments, the
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stabilization mixture may comprise native or modified starches, such as, for
example,
waxy corn starch, waxy rice starch, waxy potato starch, waxy tapioca starch,
corn
starch, rice starch, potato starch, tapioca starch, wheat starch or any
mixture thereof.
[0072] The stabilization mixture may also include additional ingredients
that
provide further benefits to either the probiotic or the individual ingesting
the
stabilized probiotic. These ingredients may comprise minerals, vitamins,
antioxidants, trace elements, sterols, antioxidants, fatty acids, functional
molecules,
and any combination thereof. Other ingredients may include resistant starches,
high
amylose starches, guar, and locust bean gum, agar, xanthan, carrageenans,
glucans,
and any combination thereof.
[0073] In some embodiments, the stabilization mixture may comprise between
about 5 and about 90 grams of probiotic and/or other biological material per
100
grams of the mixture on a dry basis. In some embodiments, the stabilization
mixture
comprises between about 50 and about 90 grams of probiotic and/or other
biological
material per 100 grams of the stabilization mixture. In certain embodiments,
the
stabilization mixture comprises between about 9 and about 12 grams of
probiotic
and/or other biological material per 100 grams of the mixture on a dry basis.
And in
an embodiment, the stabilization mixture comprises from about 10.2 to about
11.4
grams of probiotic and/or other biological material per 100 grams of the
mixture on a
dry basis. In another embodiment, the concentration of the probiotic, for
instance
LGG, in the protective matrix is from about 1 x 106 to about 1 x 1014 cfu per
gram of
the protective matrix, more preferably from about 1 x 109 to about 1 x 1011
cfu per
gram of the protective matrix.
[0074] The stabilization mixture may be used to provide stability to a
probiotic
organism which may exert a beneficial effect on the health and welfare of
individuals.
Examples of suitable probiotics include but are not limited to yeasts such as
Saccharomyces cereviseae, molds such as Aspergillus, Rhizopus, Mucor, and
bacteria
such as Lactobacillus. Specific examples of suitable probiotic micro-organisms
are:
Aspergillus niger, A. oryzae, Bacillus coagulans, B. lentus, B. licheniformis,
B.
mesentericus, B. pumilus, B. subtilis, B. natto, Bifidobacterium adolescentis,
B.
an/malls, B. breve, B. bifidum, B. infant/s, B. lactis, B. longum, B. longum
BB536, B.
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longum AH1206 (NCIMB: 41382), B. breve AH1205 (NCIMB: 41387), B. infant/s35624
(NCIMB: 41003), B. longum AH1714 (NCIMB 41676), B. animalissubsp. lactis BB-12
(DSM No. 10140), B. pseudolongum, B. thermophilum, Candida pintolepesii,
Clostridium butyricum, Enterococcus cremoris, E. diacetylactis, E. faecium, E.
intermedius, E. lactis, E. muntdi, E. thermophilus, Lactobacillus acidophilus,
L.
alimentarius, L. amylovorus, L. crispatus, L. brevis, L. case, L. curvatus, L.
cellobiosus,
L. delbruecla7 ss. bulgaricus, L. [arc/minis, L. fermentum, L. gasseri, L.
helveticus, L.
lactis, L. plan tarum, L. johnsonii, L. reuteri, L. rhamnosus, Lactobacillus
rhamnosus GG
(ATCC number 53103), L. sakei, L. salivarius and any combination thereof. In
an
embodiment, the stabilized probiotic(s) may be viable or non-viable. The
stabilized
probiotics useful in the present disclosure may be naturally-occurring,
synthetic or
developed through the genetic manipulation of organisms, whether such new
source
is now known or later developed.
[0075] In an embodiment of the present disclosure Lactobacillus rhamnosus
GG is utilized as a probiotic that may be stabilized by the protective matrix
of the
present disclosure. Lactobacillus rhamnosus GG is described in U.S. Patent
Application 4,839,281, issued to Sharwood et al., which is hereby incorporated
by
reference in its entirety. Notably, Sharwood et al. describes Lactobacillus
rhamnosus
GG as being a species in which the bacteria have avid adherence to intestinal
cells
while being simultaneously able to survive at low pHs and produce large
amounts of
lactic acid.
[0076] The selected probiotic is preferably concentrated to a wet paste-
like
consistency prior to combining with the stabilization mixture of the present
disclosure. Starting with probiotics in dry form is also an alternative.
Concentration
levels of selected probiotics include concentrations of from about 3X to about
20X
though may include lesser or greater concentrations depending upon the
specific
probiotic biomass and subsequent processing steps.
[0077] Generally, the preparation of a stabilized probiotic includes the
steps of
concentrating the selected probiotic or probiotics; providing components of
the
stabilization mixture in desired quantities; mixing the stabilization mixture
with the
concentrated probiotic; drying the stabilized probiotic and either packaging
or
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combining the stabilized probiotic into a nutritional product, such as an
infant
formula.
[0078] In some embodiments, the present disclosure is directed to a method
for protecting a viable probiotic for use in a nutritional composition, the
method may
include the steps of providing a viable probiotic, preparing a protective
matrix for
the probiotic by blending (i) a phospholipid component and (ii) a glyceride
component and any combination thereof, then combining the viable probiotic,
the
protective matrix and water to produce a mixture and drying the mixture to a
final
moisture content of about 4% or less, and further adding the dried mixture to
a
powdered nutritional product or to a capsule.
[0079] In optimizing the stabilization for probiotic, the multiple
constituents
may be varied in some embodiments as described herein and as shown in Tables 1-
4.
In some embodiments, the lipid may comprise from about 10% to 50% (w/w) of the
stabilization mixture. In some embodiments, the stabilization mixture may
comprise
about 5% to about 25% (w/w) of a phospholipid component, and from about 5% to
about 25% (w/w) of a glyceride component. In other embodiments, the
stabilization
mixture may comprise from about 5% to about 25% (w/w) of a phospholipid
component, from about 2.5 to about 12.5% (w/w) of a glyceride component, and
from about 2.5% to about 12.5% (w/w) of an additional/other lipid component.
In still
further embodiments, the total lipids of the stabilization mixture may
comprise from
about 3% to about 17% (w/w) of a phospholipid component, from about 3 to about
17% (w/w) of a glyceride component, and from about 3% to about 17% (w/w) of an
additional/other lipid component.
[0080] The stabilized probiotic may be packaged and sold commercially or
may
be instead combined with a variety of nutritional products or encapsulated
with other
functional components, such as docosahexaenoic acid. Such nutritional products
may
include both infant formulas and children's products useful for applications
where
one desires to incorporate a probiotic into a nutritional product that
necessitates an
improved shelf-life and stability.
[0081] Table 1 presents a sample embodiment of a stabilized probiotic
mixture/protective matrix according to the present disclosure.
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TABLE 1: An embodiment of the protective matrix
Ingredient Grams per 100g (wet basis)
Probiotic (LGG, AH1206 or 35624) 50-90
Phospholipid(s) 5-25
Glyceride(s) 5-25
[0082] Table 2 presents a sample embodiment of a stabilized probiotic
mixture/protective matrix according to the present disclosure.
TABLE 2: An embodiment of the protective matrix
Ingredient Grams per 100g (wet basis)
Probiotic (LGG, AH1206 or 35624) 50-90
Total Lipid(s) 10-50
Phospholipid(s) 5-25
Glyceride(s) optional
Other lipid (s) 5-25
[0083] Table 3 presents yet another example embodiment of a stabilized
probiotic mixture/protective matrix according to the present disclosure.
TABLE 3: An embodiment of the protective matrix
Ingredient Grams per 100g (wet basis)
Probiotic (LGG, AH1206 or 35624) 50-90
Total Lipid(s) 10-50
Phospholipid(s) 5-25
Glyceride(s) 2.5-12.5
Other lipid(s) 2.5-12.5
[0084] Table 4 also provides an example embodiment of a stabilized
probiotic
mixture/protective matrix according to the present disclosure.
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TABLE 4: An embodiment of the protective matrix
Ingredient Grams per 100g (dry basis)
Probiotic (LGG, AH1206 or 35624) 90-50
Total Lipid(s) 10-50
Phospholipid(s) 3.33-16.7
Glyceride(s) 3.33-16.7
Other Lipid(s) 3.33-16.7
NUTRITIONAL PRODUCTS FOR COMBINATION WITH A STABILIZED PROBIOTIC
[0085] A stabilized probiotic prepared as described hereinabove may be
combined with a nutritional product to form a novel nutritional composition.
[0086] For example, the stabilized probiotic may be combined with a
nutritional product, such as an infant formula or children's nutritional
product, to
form a stabilized nutritional composition. In another embodiment, the
stabilized
probiotic may be combined with a human milk fortifier, which is added to human
milk
in order to enhance the nutritional value of human milk.
[0087] Further, the stabilized probiotic of the disclosure may be combined
with
a nutritional product that provides minimal, partial, or total nutritional
support. Such
nutritional product(s) may be nutritional supplements or meal replacements.
Indeed,
the stabilized probiotic can be intermixed with food or other nutritional
products
prior to ingestion by a subject.
[0088] The nutritional product for combination with the stabilized
probiotic
may, but need not, be nutritionally complete. Likewise, the combination of the
stabilized probiotic with a nutritional product may produce a nutritional
composition
that is nutritionally complete. In an embodiment, the nutritional composition
of the
disclosure is nutritionally complete and contains suitable types and amounts
of lipid,
carbohydrate, protein, vitamins and minerals.
[0089] The stabilized probiotic created by the present disclosure may be
combined with a nutritional product provided in any form known in the art,
including
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a powder, a gel, a suspension, a paste, a solid, a liquid, a liquid
concentrate, or a
ready-to-use product. In one combination, the nutritional product is an infant
formula, especially an infant formula adapted for use as sole source nutrition
for an
infant.
[0090] The nutritional products described for combining with the
stabilized
probiotic may be administered enterally.
NUTRITIONAL COMPOSITIONS COMPRISING STABILIZED PROBIOTICS
[0091] Again, a stabilized/protected probiotic prepared as described above
may be combined with a nutritional product to form a novel nutritional
composition.
[0092] The nutritional composition may comprise any additional fat or
lipid
source that is known or used in the art, including but not limited to, animal
sources,
e.g., milk fat, butter, butter fat, egg yolk lipid; marine sources, such as
fish oils,
marine oils, single cell oils; vegetable and plant oils, such as corn oil,
canola oil,
sunflower oil, soybean oil, palmolein, coconut oil, high oleic sunflower oil,
evening
primrose oil, rapeseed oil, olive oil, flaxseed (linseed) oil, cottonseed oil,
high oleic
safflower oil, palm stearin, palm kernel oil, wheat germ oil; medium chain
triglyceride
oils and emulsions and esters of fatty acids; and any combinations thereof.
The
amount of lipid or fat in the nutritional composition typically varies from
about 1 to
about 7 g/100 kcal.
[0093] Further, the nutritional composition may comprise a source of
bovine
milk protein. The source of bovine milk protein may include, but is not
limited to,
milk protein powders, milk protein concentrates, milk protein isolates, nonfat
milk
solids, nonfat milk, nonfat dry milk, whey protein, whey protein isolates,
whey protein
concentrates, sweet whey, acid whey, casein, acid casein, caseinate (e.g.
sodium
caseinate, sodium calcium caseinate, calcium caseinate) and any combination
thereof.
[0094] In certain embodiments, the nutritional composition may comprise
intact protein. In other embodiments, the proteins of the nutritional
composition are
provided as a combination of both intact proteins and partially hydrolyzed
proteins,
with a degree of hydrolysis of from about 4% and 10%. Certain of these
embodiments can be extremely hypoallergenic, as both the stabilizer and the
protein
of the nutritional product contain only hydrolyzed protein. In yet another
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embodiment, the nutritional composition may be supplemented with glutamine-
containing peptides.
[0095] The whey:casein ratio of the protein source of the nutritional
composition may be similar to that found in human breast milk. In an
embodiment,
the protein source of the nutritional composition comprises from about 40% to
about
80% whey protein. In another embodiment, the protein source may comprise from
about 20% to about 60% caseins. The amount of protein in a nutritional
composition
typically varies from about 1 to about 7 g/100 kcal.
[0096] In other embodiments the nutritional composition comprises
lactoferrin,
which retains its stability and activity in the human gut against certain
undesirable
bacterial pathogens.
[0097] The nutritional composition described herein can, in some
embodiments, also comprise non-human lactoferrin, non-human lactoferrin
produced
by a genetically modified organism and/or human lactoferrin produced by a
genetically modified organism. Lactoferrin is generally described as an 80
kilodalton
glycoprotein having a structure of two nearly identical lobes, both of which
include
iron binding sites. As described in "Perspectives on Interactions Between
Lactoferrin
and Bacteria" which appeared in the publication BIOCHEMISTRY AND CELL BIOLOGY,
pp
275-281 (2006), lactoferrin from different host species may vary in an amino
acid
sequence though commonly possesses a relatively high isoelectric point with
positively charged amino acids at the end terminal region of the internal
lobe.
Lactoferrin has been recognized as having bactericidal and antimicrobial
activities. In
at least one embodiment, the lactoferrin is bovine lactoferrin.
[0098] Surprisingly, the forms of lactoferrin included herein maintain
relevant
activity even if exposed to a low pH (i.e., below about 7, and even as low as
about
4.6 or lower) and/or high temperatures (i.e., above about 65 C, and as high as
about
120 C, conditions which would be expected to destroy or severely limit the
stability
or activity of human lactoferrin or recombinant human lactoferrin. These low
pH
and/or high temperature conditions can be expected during certain processing
regimen for nutritional compositions of the types described herein, such as
pasteurization.
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[0099] In one embodiment, lactoferrin is present in the nutritional
composition
in an amount of from about 5 mg/100 kcal to about 16 mg/100 kcal. In another
embodiment, lactoferrin is present in an amount of about 9 mg/100 kcal to
about 14
mg/100 kcal. In still further embodiments, the nutritional composition may
comprise
from about 2 mg to about 200 mg lactoferrin per 100 kcal. And in certain
embodiments, the nutritional composition may comprise between about 90 mg and
about 148 mg lactoferrin per 100 kcal.
[0100] The nutritional composition may also contain TGF-13. In some
embodiments, the level of TGF-13 may be from about 0.0150 (pg/pg) ppm to about
0.1000 (pg/pg) ppm. In other embodiments, the level of TGF-13 in final
composition
including a stabilized probiotic is from about 0.0225 (pg/pg) ppm to about
0.0750
(pg/pg) ppm.
[0101] In some embodiments of the nutritional composition, the level of
TGF-13
is from about 2500 pg/mL to about 10,000 pg/mL, more preferably from about
3000
pg/mL to about 8000 pg/mL. In an embodiment, the ratio of TG F-131: TG F-132
is in
the range of about 1:1 to about 1:20, or, more particularly, in the range of
about 1:5
to about 1:15.
[0102] In some embodiments, the bioactivity of TGF-13 in a nutritional
composition is enhanced by the addition of a bioactive whey fraction. Any
bioactive
whey fraction known in the art may be used in such embodiments provided it
achieves the intended result. In an embodiment, this bioactive whey fraction
may be
a whey protein concentrate. In a particular embodiment, the whey protein
concentrate may be Salibra 800, available from Glanbia Nutritionals.
[0103] The nutritional composition may comprise an amount of probiotic in
addition to the stabilized probiotic. When the stabilized probiotic is
combined with
the nutritional product, the resulting nutritional composition may include a
total
amount of probiotics effective to provide from about 1 x 104 to about 1 x 1010
colony
forming units (cfu) per kg body weight per day to a subject. In other
embodiments,
the amount of the probiotic may vary from about 1 x 106 toabout 1 x 109 cfu
per kg
body weight per day. In even further embodiments, the nutritional composition
may
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include an amount of probiotics effective to provide about 1 x 106 du per kg
body
weight per day.
[0104] In certain embodiments, the nutritional composition of the present
disclosure comprises between about 1 x 106cfu probiotic and about 1 x 1010cfu
per
100 kcal of the composition. In some embodiments, the amount of probiotic may
be
in the range of about 1 x 106cfu to about 1 x 109cfu per 100 kcal of the
composition.
Additionally, the nutritional composition may include non-stabilized
probiotics, with
the final composition including some stabilized probiotics and some non-
stabilized
probiotics.
[0105] The nutritional composition may further comprise at least one
prebiotic.
The term "prebiotic" as used herein refers to indigestible food ingredients
that exert
health benefits upon the host. Such health benefits may include, but are not
limited
to, selective stimulation of the growth and/or activity of one or a limited
number of
beneficial gut bacteria, stimulation of the growth and/or activity of ingested
probiotic
(stabilized or not) microorganisms, selective reduction in gut pathogens, and
favorable influence on gut short chain fatty acid profile. Such prebiotics may
be
naturally-occurring, synthetic, or developed through the genetic manipulation
of
organisms and/or plants, whether such new source is now known or developed
later.
Prebiotics may include oligosaccharides, polysaccharides, and other prebiotics
that
contain fructose, xylose, soya, galactose, glucose and mannose. More
specifically,
prebiotics useful in the present disclosure may include lactulose,
lactosucrose,
raffinose, gluco-oligosaccharide, inulin, polydextrose, polydextrose powder,
galacto-
oligosaccharide, fructo-oligosaccharide, isomalto-oligosaccharide, soybean
oligosaccharides, lactosucrose, xylo-oligosaccharide, chito-oligosaccharide,
manno-
oligosaccharide, aribino-oligosaccharide, siallyl-oligosaccharide, fuco-
oligosaccharide,
and gentio-oligosaccharides, and combinations thereof.
[0106] In some embodiments, the total amount of prebiotics present in the
nutritional composition may be from about 1.0 g/L to about 10.0 g/L of the
composition (in the liquid form). In certain embodiments, the total amount of
prebiotics present in the nutritional composition may be from about 2.0 g/L
and
about 8.0 g/L of the composition.
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[0107] The nutritional composition may comprise polydextrose (PDX). If
polydextrose is used as a prebiotic, the amount of polydextrose in the
nutritional
composition may, in an embodiment, be within the range of from about 1.0 g/L
to
about 4.0 g/L. If polydextrose is used as a prebiotic, the amount of
polydextrose in
the nutritional product may, in an embodiment of the composition including
stabilized probiotics, be within the range of from about 0.1 mg/100 kcal to
about 0.5
mg/100 kcal. In another composition, the amount of polydextrose may be about
0.3
mg/100 kcal. At least 20% of the prebiotics should, in a preferred embodiment,
comprise polydextrose (PDX).
[0108] In certain embodiments, the nutritional composition comprises
galacto-
oligosaccharide. The amount of galacto-oligosaccharide in the nutritional
composition may be from about 0.2 mg/100 kcal to about 1.0 mg/100 kcal. In
other
embodiments, the amount of galacto-oligosaccharide in the nutritional
composition
may be from about 0.1 mg/100 kcal to about 0.5 mg/100 kcal. Galacto-
oligosaccharide and polydextrose may also be supplemented into the nutritional
composition in a total amount of about 0.6 mg/100 kcal.
[0109] In some embodiments, the nutritional composition comprises an
additional carbohydrate source, that is, a carbohydrate source provided in
addition
to the other carbohydrates described throughout the present disclosure.
Suitable
additional carbohydrate sources can be any used in the art, e.g., lactose,
glucose,
fructose, corn syrup solids, maltodextrins, sucrose, starch, rice syrup
solids, and the
like. The amount of additional carbohydrate in the nutritional composition
typically
can vary from about 5 g and about 25 g/100 kcal. In some embodiments, the
amount
of carbohydrate is between about 6 g and about 22 g/100 kcal. In other
embodiments, the amount of carbohydrate is between about 12 g and about 14
g/100 kcal.
[0110] The nutritional composition may contain a source of long chain
polyunsaturated fatty acids (LCPUFAs) which comprise docosahexaenoic acid
(DHA).
Other suitable LCPUFAs include, but are not limited to, a-linoleic acid, y-
linoleic acid,
linoleic acid, linolenic acid, eicosapentaenoic acid (EPA) and arachidonic
acid (ARA).
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[0111] In some embodiments, the nutritional composition may be
supplemented with both DHA and ARA, and the weight ratio of ARA:DHA may be
from about 1:3 to about 9:1. In certain embodiments the ARA:DHA ratio is from
about 1:2 to about 4:1.
[0112] The amount of long chain polyunsaturated fatty acids in the
nutritional
composition may vary from about 5 mg/100 kcal to about 100 mg/100 kcal, more
preferably from about 10 mg/100 kcal to about 50 mg/100 kcal.
[0113] Moreover, a nutritional composition may be supplemented with oils
containing DHA and ARA using standard techniques known in the art. As an
example, the oils containing DHA and ARA may be added to a nutritional
composition by replacing an equivalent amount of the rest of the overall fat
blend
normally present in the nutritional composition.
[0114] If utilized, the source of DHA and ARA may be any source known in
the
art such as marine oil, fish oil, single cell oil, egg yolk lipid, and brain
lipid. In some
compositions, the DHA and ARA are sourced from the single cell Martek oil,
DHASCO , or variations thereof. The DHA and ARA can be in natural form,
provided that the remainder of the LCPUFA source does not result in any
substantial
deleterious effect on the infant. Alternatively, the DHA and ARA can be used
in
refined form.
[0115] In an embodiment of the nutritional composition, sources of DHA and
ARA are single cell oils as taught in U.S. Patent Nos. 5,374,567; 5,550,156;
and
5,397,591, the disclosures of which are incorporated herein in their entirety
by
reference.
[0116] In certain embodiments, the nutritional composition may be a milk-
based nutritional composition that provides physiochemical and physiological
benefits. As is known in the art, bovine milk protein comprises two major
components: acid soluble whey protein and acid insoluble casein, with the
latter
representing about 80% of the total protein content of bovine milk. Upon
entering
the acidic environment of the stomach, casein precipitates and complexes with
minerals forming semi-solid curds of varying size and firmness. Softer,
smaller curds
are easier for the body to digest than larger, harder curds. Curd formation
may be
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an important consideration in the development of nutritional compositions,
including,
but not limited to infant formulas, medical foods, and premature infant
formulas. As
such, stabilized probiotics may be combined with compositions that include
softer
and smaller curds than standard infant formulas.
[0117] One or more vitamins and/or minerals may also be added in to the
nutritional composition in amounts sufficient to supply the daily nutritional
requirements of a subject. It is to be understood by one of ordinary skill in
the art
that vitamin and mineral requirements will vary, for example, based on the age
of the
child. For instance, an infant may have different vitamin and mineral
requirements
than a child between the ages of one and thirteen years. Thus, the embodiments
are
not intended to limit the nutritional composition to a particular age group
but, rather,
to provide a range of acceptable vitamin and mineral components.
[0118] The nutritional composition may optionally include, but is not
limited to,
one or more of the following vitamins or derivations thereof: vitamin B1
(thiamin,
thiamin pyrophosphate, TPP, thiamin triphosphate, TTP, thiamin hydrochloride,
thiamin mononitrate), vitamin B2 (riboflavin, flavin mononucleotide, FMN,
flavin
adenine dinucleotide, FAD, lactoflavin, ovoflavin), vitamin B3 (niacin,
nicotinic acid,
nicotinamide, niacinamide, nicotinamide adenine dinucleotide, NAD, nicotinic
acid
mononucleotide, NicMN, pyridine-3-carboxylic acid), vitamin B3-precursor
tryptophan, vitamin B6 (pyridoxine, pyridoxal, pyridoxamine, pyridoxine
hydrochloride), pantothenic acid (pantothenate, panthenol), folate (folic
acid, folacin,
pteroylglutamic acid), vitamin B12 (cobalamin, methylcobalamin,
deoxyadenosylcobalamin, cyanocobalamin, hydroxycobalamin, adenosylcobalamin),
biotin, vitamin C (ascorbic acid), vitamin A (retinol, retinyl acetate,
retinyl palmitate,
retinyl esters with other long-chain fatty acids, retinal, retinoic acid,
retinol esters),
vitamin D (calciferol, cholecalciferol, vitamin D3, 1,25,-dihydroxyvitamin D),
vitamin E
(a-tocopherol, a-tocopherol acetate, a-tocopherol succinate, a-tocopherol
nicotinate,
a-tocopherol), vitamin K (vitamin Kl, phylloquinone, naphthoquinone, vitamin
1(2,
menaquinone-7, vitamin K3, menaquinone-4, menadione, menaquinone-8,
menaquinone-8H, menaquinone-9, menaquinone-9H, menaquinone-10,
menaquinone-11, menaquinone-12, menaquinone-13), choline, inositol, 13-
carotene
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and any combinations thereof.
[0119] Further, the nutritional composition may optionally include, but is
not
limited to, one or more of the following minerals or derivations thereof:
boron,
calcium, calcium acetate, calcium gluconate, calcium chloride, calcium
lactate, calcium
phosphate, calcium sulfate, chloride, chromium, chromium chloride, chromium
picolonate, copper, copper sulfate, copper gluconate, cupric sulfate,
fluoride, iron,
carbonyl iron, ferric iron, ferrous fumarate, ferric orthophosphate, iron
trituration,
polysaccharide iron, iodide, iodine, magnesium, magnesium carbonate, magnesium
hydroxide, magnesium oxide, magnesium stearate, magnesium sulfate, manganese,
molybdenum, phosphorus, potassium, potassium phosphate, potassium iodide,
potassium chloride, potassium acetate, selenium, sulfur, sodium, docusate
sodium,
sodium chloride, sodium selenate, sodium molybdate, zinc, zinc oxide, zinc
sulfate
and mixtures thereof. Non-limiting exemplary derivatives of mineral compounds
include salts, alkaline salts, esters and chelates of any mineral compound.
[0120] The minerals can be added to nutritional compositions in the form
of
salts such as calcium phosphate, calcium glycerol phosphate, sodium citrate,
potassium chloride, potassium phosphate, magnesium phosphate, ferrous sulfate,
zinc sulfate, cupric sulfate, manganese sulfate, and sodium selenite.
Additional
vitamins and minerals can be added as known within the art.
[0121] The nutritional composition of the present disclosure may
optionally
include one or more of the following flavoring agents, including, but not
limited to,
flavored extracts, volatile oils, cocoa or chocolate flavorings, peanut butter
flavoring,
cookie crumbs, vanilla or any commercially available flavoring. Examples of
useful
flavorings include, but are not limited to, pure anise extract, imitation
banana extract,
imitation cherry extract, chocolate extract, pure lemon extract, pure orange
extract,
pure peppermint extract, honey, imitation pineapple extract, imitation rum
extract,
imitation strawberry extract, or vanilla extract; or volatile oils, such as
balm oil, bay
oil, bergamot oil, cedarwood oil, cherry oil, cinnamon oil, clove oil, or
peppermint oil;
peanut butter, chocolate flavoring, vanilla cookie crumb, butterscotch,
toffee, and
mixtures thereof. The amounts of flavoring agent can vary greatly depending
upon
the flavoring agent used. The type and amount of flavoring agent can be
selected as
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is known in the art.
[0122] The nutritional composition of the present disclosure may optionally
include one or more emulsifiers that may be added for stability of the final
product.
Examples of suitable emulsifiers include, but are not limited to, lecithin
(e.g., from
egg or soy), alpha lactalbumin and/or mono- and di-glycerides, and mixtures
thereof.
Other emulsifiers are readily apparent to the skilled artisan and selection of
suitable
emulsifier(s) will depend, in part, upon the formulation and final product. In
some
embodiments, nutritional compositions of the present disclosure may comprise
emulsifiers such as citric acid esters of mono- and/or diglycerides, diacetyl
tartaric
acid esters of mono- and/or diglycerides, and/or octenyl succinic anhydride
modified
starches.
[0123] The nutritional composition of the present disclosure may optionally
include one or more preservatives that may also be added to extend product
shelf
life. Suitable preservatives include, but are not limited to, potassium
sorbate, sodium
sorbate, potassium benzoate, sodium benzoate, calcium disodium EDTA, and
mixtures thereof.
[0124] The nutritional composition of the present disclosure may optionally
include one or more stabilizers. Suitable stabilizers for use in practicing
the
nutritional composition of the present disclosure include, but are not limited
to, gum
arabic, gum ghatti, gum karaya, gum tragacanth, agar, furcellaran, guar gum,
gellan
gum, locust bean gum, pectin, low methoxyl pectin, gelatin, microcrystalline
cellulose, CMC (sodium carboxymethylcellulose), methylcellulose hydroxypropyl
methyl cellulose, hydroxypropyl cellulose, DATEM (diacetyl tartaric acid
esters of
mono- and diglycerides), dextran, carrageenans, and mixtures thereof.
[0125] The nutritional composition of the present disclosure may further
include at least one additional phytonutrient, that is, another phytonutrient
component in addition to the pectin, starch or other phytonutrient components
described herein. Phytonutrients, or their derivatives, conjugated forms or
precursors, that are identified in human milk are preferred for inclusion in
the
nutritional composition. For example, in some embodiments, the nutritional
composition of the present disclosure may comprise, in an 8 fl. oz. (236.6 mL)
serving,
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between about 80 and about 300 mg anthocyanins, between about 100 and about
600 mg proanthocyanidins, between about 50 and about 500 mg flavan-3-ols, or
any
combination or mixture thereof. In other embodiments, the nutritional
composition
comprises apple extract, grape seed extract, or a combination or mixture
thereof.
Further, the at least one phytonutrient of the nutritional composition may be
derived
from any single or blend of fruit, grape seed and/or apple or tea extract(s).
[0126] Examples of additional phytonutrients suitable for the nutritional
composition include, but are not limited to, anthocyanins, proanthocyanidins,
flavan-
3-ols (i.e.. catechins, epicatechins, etc.), flavan ones, flavonoids,
isoflavonoids,
stilbenoids (i.e. resveratrol, etc.) proanthocyanidins, anthocyanins,
resveratrol,
quercetin, curcumin, and/or any mixture thereof, as well as any possible
combination
of phytonutrients in a purified or natural form. Certain components,
especially plant-
based components of the nutritional compositions may provide a source of
phytonutrients.
[0127] The phytonutrient component of the nutritional composition may also
comprise naringenin, hesperetin, anthocyanins, quercetin, kaempferol,
epicatechin,
epigallocatechin, epicatechin-gallate, epigallocatechin-gallate or any
combination
thereof. In certain embodiments, the nutritional composition comprises between
about 50 and about 2000 nmol/L epicatechin, between about 40 and about 2000
nmol/L epicatechin gallate, between about 100 and about 4000 nmol/L
epigallocatechin gallate, between about 50 and about 2000 nmol/L naringenin,
between about 5 and about 500 nmol/L kaempferol, between about 40 and about
4000 nmol/L hesperetin, between about 25 and about 2000 nmol/L anthocyanins,
between about 25 and about 500 nmol/L quercetin, or a mixture thereof.
Furthermore, the nutritional composition may comprise the metabolite(s) of a
phytonutrient or of its parent compound, or it may comprise other classes of
dietary
phytonutrients, such as glucosinolate or sulforaphane. In certain embodiments,
the
nutritional composition comprises carotenoids, such as lutein, zeaxanthin,
astaxanthin, lycopene, beta-carotene, alpha-carotene, gamma-carotene, and/or
beta-
cryptoxanthin.
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[0128] The nutritional composition may also comprise isoflavonoids and/or
isoflavones. Examples include, but are not limited to, genistein (genistin),
daidzein
(daidzin), glycitein, biochanin A, formononetin, coumestrol, irilone, orobol,
pseudobaptigenin, anagyroidisoflavone A and B, calycosin, glycitein, irigenin,
5-0-
methylgenistein, pratensein, prunetin, psi-tectorigenin, retusin,
tectorigenin, iridin,
ononin, puerarin, tectoridin, derrubone, luteone, wighteone,
alpinumisoflavone,
barbigerone, di-0-methylalpinumisoflavone, and 4'-methyl-alpinumisoflavone.
Plant
sources rich in isoflavonoids, include, but are not limited to, soybeans,
psoralea,
kudzu, lupine, fava, chick pea, alfalfa, legumes and peanuts.
[0129] In an embodiment, the nutritional composition of the present
disclosure
comprises an effective amount of choline. An effective amount of choline is
between
about 20 mg choline per 8 fl. oz. (236.6 mL) serving to about 100 mg per 8 fl.
oz.
(236.6 mL) serving.
[0130] The disclosed nutritional composition may additionally comprise a
source of B-glucan. Glucans are polysaccharides, specifically polymers of
glucose,
which are naturally occurring and may be found in cell walls of bacteria,
yeast, fungi,
and plants. Beta glucans (B-glucans) are themselves a diverse subset of
glucose
polymers, which are made up of chains of glucose monomers linked together via
beta-type glycosidic bonds to form complex carbohydrates.
[0131] [3-1,3-glucans are carbohydrate polymers purified from, for
example,
yeast, mushroom, bacteria, algae, or cereals. (Stone BA, Clarke AE. Chemistry
and
Biology of (1-3)-Beta-Glucans. London:Portland Press Ltd; 1993. ) The chemical
structure of P-1,3-glucan depends on the source of the P-1,3-glucan. Moreover,
various physiochemical parameters, such as solubility, primary structure,
molecular
weight, and branching, play a role in biological activities of [3-1,3-glucans.
(Yadomae
T., Structure and biological activities of fungal beta-1,3-glucans. Yakugaku
Zasshi.
2000;120:413-431.)
[0132] 3-1,3-glucans are naturally occurring polysaccharides, with or
without 3-
1,6-glucose or a1,4 glucose branched side chains, that are found in the cell
walls of a
variety of plants, yeasts, fungi and bacteria.
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[0133] 13-glucans derived from baker's yeast, Saccharomyces cerevisiae,
are
made up of chains of D-glucose molecules connected by
111 g lyc
and 3 positions, having side chains of glucose attached by
111 g lycc
1 and 6 positions. Yeast-derived 13-glucan is an insoluble, fiber-like,
complex sugar
having the general structure of a linear chain of glucose units with a 13-1,3
backbone
interspersed with r3-1,6 side chains that are generally 6-8 glucose units in
length.
More specifically, P-glucan derived from baker's yeast is poly-(1,6)-3-D-
glucopyranosyl-(1,3)-3-D-glucopyranose.
[0134] Furthermore,13-glucans are well tolerated and do not produce or
cause
excess gas, abdominal distension, bloating or diarrhea in pediatric subjects.
Addition
of 3-glucan to a nutritional composition for a pediatric subject, such as an
infant
formula, a growing-up milk or another children's nutritional product, will
improve the
subject's immune response by increasing resistance against invading pathogens
and
therefore maintaining or improving overall health.
[0135] The nutritional composition of the present disclosure may comprise
3-
glucan. In some embodiments, the 13-glucan is 3-1,3;1,6-glucan. In some
embodiments, the 13-1,3;1,6-glucan is derived from baker's yeast. The
nutritional
composition may comprise whole glucan particle 13-glucan, particulate 13-
glucan,
microparticulate 3-glucan, PGG-glucan (poly-1,643-D-glucopyranosy1-1,343-D-
glucopyranose) or any mixture thereof. In some embodiments, microparticulate
13-
glucan comprises 3-glucan particles having a diameter of less than 2 pm.
[0136] In some embodiments, the amount of P-glucan present in the
composition is at between about 0.010 and about 0.0809 per 100g of the
nutritional
composition. In other embodiments, the nutritional composition comprises
between
about 10 and about 30 mg 13-glucan per serving. In another embodiment, the
nutritional composition comprises between about 5 and about 30 mg P-glucan per
8
fl. oz. (236.6 mL) serving. In other embodiments, the nutritional composition
comprises an amount of P-glucan sufficient to provide between about 15 mg and
about 90 mg I3-glucan per day. In some embodiments, the nutritional
composition
may be delivered in multiple doses to reach a target amount of P-glucan
delivered to
the subject throughout the day.
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[0137] In some embodiments, the amount of P-glucan in the nutritional
composition is between about 3 mg and about 17 mg per 100 kcal. In another
embodiment the amount of 13-glucan is between about 6 mg and about 17 mg per
100 kcal.
[0138] The nutritional composition may be expelled directly into a
subject's
intestinal tract. In some embodiments, the nutritional composition is expelled
directly into the gut. In some embodiments, the composition may be formulated
to
be consumed or administered enterally under the supervision of a physician and
may
be intended for the specific dietary management of a disease or condition,
such as
celiac disease and/or food allergy, for which distinctive nutritional
requirements,
based on recognized scientific principles, are established by medical
evaluation.
[0139] The nutritional composition of the present disclosure is not
limited to
compositions comprising nutrients specifically listed herein. Any nutrients
may be
delivered as part of the composition for the purpose of meeting nutritional
needs
and/or in order to optimize the nutritional status in a subject.
[0140] The nutritional composition of the present disclosure may be
standardized to a specific caloric content, it may be provided as a ready-to-
use
product, or it may be provided in a concentrated form.
[0141] In some embodiments, the nutritional composition of the present
disclosure is a growing-up milk. Growing-up milks are fortified milk-based
beverages
intended for children over 1 year of age (typically from 1-3 years of age,
from 4-6
years of age or from 1-6 years of age). Growing-up milks are designed with the
intent
to serve as a complement to a diverse diet to provide additional insurance
that a
child achieves continual, daily intake of all essential vitamins and minerals,
macronutrients plus additional functional dietary components, such as non-
essential
nutrients that have purported health-promoting properties.
[0142] The exact composition of a nutritional composition according to the
present disclosure can vary from market-to-market, depending on local
regulations
and dietary intake information of the population of interest. In some
embodiments,
nutritional compositions according to the disclosure include a milk protein
source,
such as whole or skim milk, plus added sugar and sweeteners to achieve desired
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sensory properties, and added vitamins and minerals. The fat composition is
typically
derived from the milk raw materials. Total protein can be targeted to match
that of
human milk, cow milk or a lower value. Total carbohydrate is usually targeted
to
provide as little added sugar, such as sucrose or fructose, as possible to
achieve an
acceptable taste. Typically, Vitamin A, calcium and Vitamin D are added at
levels to
match the nutrient contribution of regional cow milk. Otherwise, in some
embodiments, vitamins and minerals can be added at levels that provide
approximately 20% of the dietary reference intake (DRI) or 20% of the Daily
Value
(DV) per serving. Moreover, nutrient values can vary between markets depending
on
the identified nutritional needs of the intended population, raw material
contributions and regional regulations.
[0143] In certain embodiments, the nutritional composition is
hypoallergenic.
In other embodiments, the nutritional composition is kosher. In still further
embodiments, the nutritional composition is a non-genetically modified
product. In
an embodiment, the nutritional formulation is sucrose-free. The nutritional
composition may also be lactose-free. In other embodiments, the nutritional
composition does not contain any medium-chain triglyceride oil. In some
embodiments, no carrageenan is present in the composition. In other
embodiments,
the nutritional composition is free of all gums.
[0144] Accordingly, by the practice of the present disclosure, stabilized
probiotics having heretofore unrecognized stability are prepared. The
stabilized
bacterial mixture exhibits exceptionally high stability through the use of
hydrolyzed
mammalian protein, especially hydrolyzed mammalian protein with over 70% of
the
peptides having a molecular weight of less than 2,000 Daltons. The stabilized
probiotics are uniquely effective for nutritional applications with
intermediate
moisture levels (such as water activity as high as 0.4) where increased shelf
life and
stability in hot and humid environments are desired. The stabilized probiotics
may be
packed separately or be combined with any of the embodiments of nutritional
compositions described herein.
[0145] All references cited in this specification, including without
limitation, all
papers, publications, patents, patent applications, presentations, texts,
reports,
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manuscripts, brochures, books, internet postings, journal articles,
periodicals, and the
like, are hereby incorporated by reference into this specification in their
entireties.
The discussion of the references herein is intended merely to summarize the
assertions made by their authors and no admission is made that any reference
constitutes prior art. Applicants reserve the right to challenge the accuracy
and
pertinence of the cited references.
[0146] Although preferred embodiments of the disclosure have been
described using specific terms, devices, and methods, such description is for
illustrative purposes only. The words used are words of description rather
than of
limitation. It is to be understood that changes and variations may be made by
those
of ordinary skill in the art without departing from the spirit or the scope of
the
present disclosure, which is set forth in the following claims. In addition,
it should be
understood that aspects of the various embodiments may be interchanged both in
whole or in part. For example, while methods for the production of a
commercially
sterile liquid nutritional supplement made according to those methods have
been
exemplified, other uses are contemplated. Therefore, the spirit and scope of
the
appended claims should not be limited to the description of the preferred
versions
contained therein.
