Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
USE OF NUTRITIONAL COMPOSITIONS INCLUDING LACTOFERRIN IN
SUPPORTING RESISTANCE TO DISEASES AND CONDITIONS
TECHNICAL FIELD
[0001] This disclosure relates generally to the field of nutritional
compositions, such as infant formulas, human milk fortifiers, children's
dietary
supplements, and the like, having lactoferrin, in particular, lactoferrin
produced by
a non-human source. More particularly, the disclosure relates to a method of
supporting resistance to a disease or condition caused by bacterial and viral
pathogens by administering to a human nutritional compositions including
lactoferrin.
BACKGROUND ART
[0002] There are currently a variety of dietary compositions for humans,
especially young humans, to provide supplemental or primary nutrition at
certain
stages in life. Generally, commercial dietary compositions for infants seek to
mimic
to the extent possible the composition and associated functionality of human
milk.
Through a combination of proteins, some of which have physiological activity,
and
blended fat ingredients, dietary compositions are formulated such that they
simulate human milk for use as a complete or partial substitute. Other
ingredients
often utilized in dietary compositions for infants may include a carbohydrate
source
such as lactose as well as other vitamins, minerals and elements believed to
be
present in human milk for the absorption by the infant.
[0003] Lactoferrin is one of the primary proteins in human milk and is
considered a glycoprotein having an average molecular weight of approximately
80
kilodaltons. It is an iron binding protein having the capacity to bind two
molecules
of iron in a reversible fashion and can facilitate the uptake of iron within
the
intestines for the human. Functionally, lactoferrin regulates iron absorption
and as
such can bind iron-based free radicals as well as donate iron for an
immunological
response.
[0004] In obtaining a commercially viable dietary composition, the addition
of
lactoferrin has generally been limited due to predicted losses of activity
during
processing. For example, generally, the temperature and pH requirements in
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processing infant formulas and other products such as human milk fortifiers
and
various children's products reduce specific functions of the lactoferrin,
causing
lactoferrin not to be included within a final formulation. In addition,
lactoferrin is
often considered only for its iron binding qualities; thus, lactoferrin may
generally
be excluded from a formulation where such properties are thought to be
diminished
by processing conditions.
[0005] Further, as known in the art, human breast milk is relatively low in
iron, containing about 0.3 milligrams of iron per liter of breast milk. While
this
quantity is low, human infants have high absorption rate, absorbing about half
of
the iron from the breast milk. However, when human infants are given prior art
formulas with high levels of iron fortification, for example, of from about 10
mg to
about 12 milligrams per liter, the infants absorb less than about 5% of the
total
iron. With such increased levels of iron within the prior art formulas,
virtually all
of the lactoferrin iron binding sites would be expected to be occupied, as
lactoferrin
is a known iron transport protein.
[0006] Additional complications of the prior art formulas include the
inability
of providing a bacterio static effect. This is partially due to the use of
lactoferrin
with blocked or damaged binding sites, as the bacteriostatic effect is at
least
partially related to the degree of binding to iron of the lactoferrin present
within
the formula.
[0007] Accordingly, it would be beneficial to provide a nutritional
composition, such as an infant formula, human milk fortifier, children's
dietary
supplement, and the like, which contains lactoferrin, in particular,
lactoferrin from
a non-human source. Given the prevalence of viral respiratory tract infections
in
humans, and infants and children in particular, it would be particularly
beneficial
to provide a nutritional composition that supports resistance to a viral
respiratory
tract infection. Preferably, the lactoferrin included in the compositions is
able to
support resistance to a disease or condition caused by bacterial and viral
pathogens
even after processing under conditions of high temperature and low pH.
DISCLOSURE OF THE INVENTION
[0008] Briefly, in an embodiment, the disclosure is directed to a method of
supporting resistance to a disease or condition, such as viral respiratory
tract
infection, in a human. In another embodiment, the disclosure is directed to a
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method of supporting resistance in a human to a disease or condition caused by
at
least one pathogen selected from the group consisting of Enterotoxigenic E.
coli
(ETEC), Enteropathogenic E. coli (EPEC), Haemophilus influenza, Shigatoxin
producing E. coli (STEC), Enteroaggregative E. coli (EAEC), Salmonella ser.
Typhimurium, Shigella flexneri, Rotavirus, Norovirus, Respiratory syncytial
virus
(RSV), Adenovirus, and combinations thereof. The method includes administering
to a human a nutritional composition comprising (1) a fat or lipid source, (2)
a
protein source, and (3) lactoferrin produced by a non-human source. In certain
embodiments, the disease or condition is a viral, lower respiratory tract
infection.
[0009] In an embodiment, the nutritional composition comprises:
a. up to about 7 g/100 kal of a fat or lipid source, more preferably about
3
g/100 kcal to about 7 g/100 kcal of a fat or lipid source;
b. up to about 5 g/100 kcal of a protein source, more preferably about 1
g/100 kcal to about 5 g/100 kcal of a protein source; and
c. at least about 10 mg/100 kCal of lactoferrin, more preferably about 70
mg to about 220 mg/100 kCal of lactoferrin, and most preferably about 90 mg to
about 190 mg/100 kCal of lactoferrin. Optionally, in certain embodiments, the
nutritional compositions may further comprise about 0.1 g/100 kcal to about 1
g/100
kcal of a prebiotic composition, such as a prebiotic composition comprising
polydextrose and/or galactooligosaccharide. More preferably, the nutritional
composition comprises about 0.3 g/100 kcal to about 0.7 g/100 kcal of a
prebiotic
composition which comprises a combination of polydextrose and
galactooligosaccharide. In certain embodiments, the disclosure is directed to
a
method of supporting resistance to a disease or condition in a human caused by
at
least one pathogen selected from the group consisting of ETEC, EPEC,
Shigatoxin
producing E. coli, EAEC, Salmonella ser. Typhimurium, Shigella flexneri or
combinations thereof.
[0010] Preferably, the lactoferrin is non-human lactoferrin and/or human
lactoferrin produced by a genetically modified organism. In one particularly
preferred embodiment, the lactoferrin used is such that an effective amount of
a
nutritional composition containing lactoferrin may be administered to the
individual to support resistance to a disease or condition caused by a viral
or
bacterial pathogen, even if, during processing, the nutritional composition
has been
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exposed to pH and temperature fluctuations typical of certain processing
conditions
like pasteurization.
BEST MODE FOR CARRYING OUT THE INVENTION
[0011] In an embodiment, the present disclosure provides a method of
supporting resistance to a disease or condition, such as viral respiratory
tract
infection, in a human caused by a bacterial or viral pathogen by administering
to
the human nutritional compositions that comprises a lipid or fat source, a
protein
source, and lactoferrin from a non-human source.
[0012] As used herein, each of "lactoferrin produced by a non-human source"
and "lactoferrin from a non-human source" means lactoferrin produced by or
obtained from a source other than human breast milk. For example, lactoferrin
for
use in the present disclosure includes human lactoferrin produced by a
genetically
modified organism as well as non-human lactoferrin. The term "organism", as
used
herein, refers to any contiguous living system, such as animal, plant, fungus
or
micro-organism. The term "non-human lactoferrin", as used herein, refers to
lactoferrin having an amino acid sequence that is different than the amino
acid
sequence of human lactoferrin.
[0013] Lactoferrins are single chain polypeptides of about 80 kD containing
1
¨ 4 glycans, depending on the species. The 3-D structures of lactoferrin of
different
species are very similar, but not identical. Each lactoferrin comprises two
homologous lobes, called the N- and C-lobes, referring to the N-terminal and C-
terminal part of the molecule, respectively. Each lobe further consists of two
sub-
lobes or domains, which form a cleft where the ferric ion (Fe3 ) is tightly
bound in
synergistic cooperation with a (bi)carbonate anion. These domains are called
Ni,
N2, Cl and C2, respectively. The N-terminus of lactoferrin has strong cationic
peptide regions that are responsible for a number of important binding
characteristics. Lactoferrin has a very high isoelectric point (¨pI 9) and its
cationic
nature plays a major role in its ability to defend against bacterial, viral,
and fungal
pathogens. There are several clusters of cationic amino acids residues within
the
N-terminal region of lactoferrin mediating the biological activities of
lactoferrin
against a wide range of microorganisms. For instance, the N-terminal residues
1-
47 of human lactoferrin (1-48 of bovine lactoferrin) are critical to the iron-
independent biological activities of lactoferrin. In human lactoferrin,
residues 2 to
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5 (RRRR) and 28 to 31 (RKVR) are arginine-rich cationic domains in the N-
terminus especially critical to the antimicrobial activities of lactoferrin. A
similar
region in the N-terminus is found in bovine lactoferrin (residues 17 to 42;
FKCRRWQWRMKKLGAPSITCVRRAFA).
[0014] As described in "Perspectives on Interactions Between Lactoferrin
and
Bacteria" which appeared in the publication BIOCHEMISTRY AND CELL BIOLOGY, pp
275-281 (2006), lactoferrins from different host species may vary in their
amino
acid sequences though commonly possess a relatively high isoelectric point
with
positively charged amino acids at the end terminal region of the internal
lobe.
Suitable lactoferrins for use in the present disclosure include those having
at least
48% homology with the amino acid sequence AVGEQELRKCNQWSGL at the HLf
(349-364) fragment. In some embodiments, the lactoferrin has at least 65%
homology with the amino acid sequence AVGEQELRKCNQWSGL at the HLf (349-
364) fragment, and, in embodiments, at least 75% homology. For example, non-
human lactoferrins acceptable for use in the present disclosure include,
without
limitation, bovine lactoferrin, porcine lactoferrin, equine lactoferrin,
buffalo
lactoferrin, goat lactoferrin, murine lactoferrin and camel lactoferrin.
[0015] Lactoferrin for use in the present disclosure may be, for example,
isolated from the milk of a non-human animal or produced by a genetically
modified
organism. For example, in U.S. Patent No. 4,791,193, incorporated by reference
herein in its entirety, Okonogi et al. discloses a process for producing
bovine
lactoferrin in high purity. Generally, the process as disclosed includes three
steps.
Raw milk material is first contacted with a weakly acidic cationic exchanger
to
absorb lactoferrin followed by the second step where washing takes place to
remove
nonabsorbed substances. A desorbing step follows where lactoferrin is removed
to
produce purified bovine lactoferrin. Other methods may include steps as
described
in U.S. Patent Nos. 7,368,141, 5,849,885, 5,919,913 and 5,861,491, the
disclosures
of which are all incorporated by reference in their entirety.
[0016] A benefit of lactoferrin, as used in certain embodiments of the
present
disclosure, is its ability to support resistance to a disease or condition
caused by
certain bacterial and viral pathogens, including ETEC, EPEC, Haemophilus
influenza, STEC, EAEC, Salmonella ser. Typhimurium, Shigella flexneri,
Rotavirus, Norovirus, RSV, Adenovirus, and combinations thereof.
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[0017] In one embodiment, lactoferrin is present in the nutritional
composition in an amount of at least about 10 mg/100 kCal, especially when the
nutritional composition is intended for use by children. In certain
embodiments,
the upper limit for lactoferrin is about 240 mg/100 kCal. In another
embodiment,
where the nutritional composition is an infant formula, lactoferrin is present
in the
nutritional composition in an amount of from about 70 mg to about 220 mg/100
kCal; in yet another embodiment, lactoferrin is present in an amount of about
90
mg to about 190 mg/100 kCal. Nutritional compositions for infants can include
lactoferrin in the quantities of from about 0.5 mg to about 1.5 mg per
milliliter of
formula. In nutritional compositions replacing human milk, lactoferrin may be
present in quantities of from about 0.6 mg to about 1.3 mg per milliliter of
formula.
[0018] As mentioned, in an embodiment, the nutritional compositions support
resistance to a viral respiratory tract infection. Humans, particularly
infants and
children, are vulnerable to viral respiratory tract infections. Viral, lower
respiratory tract infections, especially those caused by respiratory syncytial
virus,
can be particularly problematic. Indeed, respiratory syncytial virus is a
leading
cause of hospitalization for infants and children and can lead to, for
example,
bronchiolitis, pneumonia, and long-term complications such as wheezing. Thus,
in
a preferred embodiment, the disclosure is directed to a method of supporting
resistance to a viral, lower respiratory tract disclosure. In yet another
preferred
embodiment, the disclosure is directed to a method of supporting resistance to
a
respiratory syncytial virus infection. For example, the nutritional
compositions of
the disclosure may be used to support immune development to better enable the
body to fight viral respiratory tract infections, either preventing such
infections or
reducing their severity.
[0019] In certain embodiments, the nutritional composition is administered
prophylactically to a human who does not have a disease or condition such as
viral
respiratory tract infection and/or a disease or condition caused by at least
one
pathogen selected from the group consisting of ETEC, EPEC, Haemophilus
influenza, STEC, EAEC, Salmonella ser. Typhimurium, Shigella flexneri,
Rotavirus, Norovirus, RSV, Adenovirus, and combinations thereof. In other
embodiments, the human to whom the nutritional composition is administered has
viral respiratory tract infection or a disease or condition caused by the at
least one
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pathogen when the nutritional composition is administered.
[0020] Preferably, the human to whom the nutritional composition is
administered is an infant or a child. As used herein, the term "infant" is
generally
defined as a human from birth to 12 months of age. A "child" and "children"
are
defined as humans over the age of 12 months to about 12 years old.
[0021] Preferably, the lactoferrin in the nutritional compositions has the
same or similar activity against the disease or condition to that of
lactoferrin in free
form. In other words, preferably, the activity of the lactoferrin is not
significantly
diminished by inclusion in the nutritional composition. It is also preferred
the
lactoferrin included in the compositions is able to support resistance to a
disease or
condition caused by bacterial and viral pathogens even after processing under
conditions of high temperature and low pH. In one embodiment of the present
disclosure, the lactoferrin used is non-human lactoferrin.
[0022] For example, surprisingly, bovine lactoferrin maintains certain
bactericidal activity even if exposed to a low pH (i.e., below 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. 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. Yet, while
bovine lactoferrin has an the amino acid composition which has about a 70%
sequence homology to that of human lactoferrin, and is stable and remains
active
under conditions under which human lactoferrin becomes unstable or inactive,
bovine lactoferrin has bactericidal activity against undesirable bacterial
pathogens
found in the human gut.
[0023] In some embodiments, the nutritional composition may be an infant
formula. The term "infant formula" applies to a composition in liquid or
powdered
form that satisfies the nutrient requirements of an infant by being a
substitute for
human milk. In the United States, the content of an infant formula is dictated
by
the federal regulations set forth at 21 C.F.R. 100, 106 and 107. These
regulations
define macronutrient, vitamin, mineral, and other ingredient levels in an
effort to
simulate the nutritional and other properties of human breast milk. In a
separate
embodiment, the nutritional product may be a human milk fortifier, meaning it
is a
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composition which is added to human milk in order to enhance the nutritional
value of human milk. As a human milk fortifier, the disclosed composition may
be
in powder or liquid form. In yet another embodiment, the disclosed nutritional
product may be a children's nutritional composition.
[0024] The nutritional composition of the disclosure may provide minimal,
partial, or total nutritional support. The nutritional composition may be a
nutritional supplement or a meal replacement. In some embodiments, the
nutritional composition may be administered in conjunction with a food or
another
nutritional composition. In this embodiment, the nutritional composition can
either be intermixed with the food or other nutritional composition prior to
ingestion by the subject or can be administered to the subject either before
or after
ingestion of a food or nutritional composition. The nutritional composition
may be
administered to preterm infants receiving infant formula, breast milk, a human
milk fortifier, or combinations thereof. For purposes of the present
disclosure, a
"preterm infant" is an infant born after less than 37 weeks gestation, while a
"full
term infant" is an infant born after at least 37 weeks gestation.
[0025] The nutritional compositions may, but need not, be nutritionally
complete. The skilled artisan will recognize "nutritionally complete" to vary
depending on a number of factors including, but not limited to, age, clinical
condition, and dietary intake of the subject to whom the term is being
applied. In
general, "nutritionally complete" means that the nutritional composition of
the
present disclosure provides adequate amounts of all carbohydrates, lipids,
essential
fatty acids, proteins, essential amino acids, conditionally essential amino
acids,
vitamins, minerals, and energy required for normal growth. As applied to
nutrients, the term "essential" refers to any nutrient which cannot be
synthesized
by the body in amounts sufficient for normal growth and to maintain health and
which therefore must be supplied by the diet. The term "conditionally
essential" as
applied to nutrients means that the nutrient must be supplied by the diet
under
conditions when adequate amounts of the precursor compound is unavailable to
the
body for endogenous synthesis to occur.
[0026] 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
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acids, conditionally essential amino acids, vitamins, minerals, and energy
required
for growth of the preterm infant. The composition which is "nutritionally
complete"
for the full 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 full 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.
[0027] The nutritional composition may be provided in any form known in the
art, including a powder, a gel, a suspension, a paste, a solid, a liquid, a
liquid
concentrate, or a ready-to-use product. In one preferred embodiment, the
nutritional composition is an infant formula, especially an infant formula
adapted
for use as sole source nutrition for an infant.
[0028] In the preferred embodiments, the nutritional product disclosed
herein
may be administered enterally. As used herein, "enteral" means through or
within
the gastrointestinal, or digestive, tract, and "enteral administration"
includes oral
feeding, intragastric feeding, transpyloric administration, or any other
introduction
into the digestive tract.
[0029] Suitable fat or lipid sources for practicing the present disclosure
may
be any known or used in the art, including but not limited to, animal sources,
e.g.,
milk fat, butter, butter fat, egg yolk lipid; marine sources, such as fish
oils, marine
oils, single cell oils; vegetable and plant oils, such as corn oil, canola
oil, sunflower
oil, soybean oil, palmolein, coconut oil, high oleic sunflower oil, evening
primrose
oil, rapeseed oil, olive oil, flaxseed (linseed) oil, cottonseed oil, high
oleic safflower
oil, palm stearin, palm kernel oil, wheat germ oil; medium chain triglyceride
oils
and emulsions and esters of fatty acids; and any combinations thereof.
[0030] In certain embodiments, the protein source included in the
nutritional
composition comprises bovine milk proteins. Bovine milk protein sources useful
in
practicing the present disclosure include, but are not limited to, milk
protein
powders, milk protein concentrates, milk protein isolates, nonfat milk solids,
nonfat
milk, nonfat dry milk, whey protein, whey protein isolates, whey protein
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concentrates, sweet whey, acid whey, casein, acid casein, caseinate (e.g.
sodium
caseinate, sodium calcium caseinate, calcium caseinate) and any combinations
thereof.
[0031] In one embodiment, the proteins are provided as intact proteins. In
other embodiments, the proteins are provided as a combination of both intact
proteins and partially hydrolyzed proteins, with a degree of hydrolysis of
between
about 4% and 10%. In yet another embodiment, the protein source may be
supplemented with glutamine- containing peptides.
[0032] In a particular embodiment of the disclosure, the protein source
comprises whey and casein proteins and the ratio of whey to casein proteins
ratio is
similar to that found in human breast milk. For example, in certain
embodiments,
the weight ratio of whey to casein proteins is from about 20% whey:80% casein
to
about 80% whey: 20% casein
[0033] In certain embodiments of the disclosure, the nutritional
composition
may contain one or more probiotics. The term "probiotic" means a microorganism
with low or no pathogenicity that exerts beneficial effects on the health of
the host.
Any probiotic known in the art may be acceptable for use in the present
disclosure
provided it achieves the intended result. In a particular embodiment, the
probiotic
may be selected from Lactobacillus species, Lactobacillus rhamnosus GG,
Bifidobacterium species, Bifidobacterium longum, Bifidobacterium brevis, and
Bifidobacterium animalis subsp. lactis BB-12.
[0034] If included in the composition, the amount of the probiotic may vary
from about 104 to about 1010 colony forming units (cfu) per kg body weight per
day.
In another embodiment, the amount of the probiotic may vary from about 106 to
about 109 cfu per kg body weight per day. In yet another embodiment, the
amount
of the probiotic may be at least about 106 cfu per kg body weight per day.
Moreover,
the disclosed composition may also include probiotic-conditioned media
components.
[0035] In one embodiment, one or more of the probiotics is viable. In
another
embodiment, one or more of the probiotics is 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 metabolites thereof. Such non-viable probiotics may have been heat-killed
or
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otherwise inactivated but 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 new source is now known or later developed.
[0036] In one embodiment of the disclosure, the nutritional compositions
may
include a prebiotic composition comprising one or more prebiotics. As used
herein,
the term "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 colon that can improve the health of the host. A
"prebiotic composition" is a composition that comprises one or more
prebiotics.
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. In certain embodiments, the prebiotic included in
the
compositions of the present disclosure include those taught by U.S. Patent No.
7,572,474, the disclosure of which is incorporated herein by reference.
[0037] Prebiotics for use in the present disclosure 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
useful
in the present disclosure 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,
lactosucro se, raffinose, gluco-oligosaccharide, inulin, polydextrose,
polydextrose
powder, galactooligosaccharide, fructo-oligosaccharide, isomalto-
oligosaccharide,
soybean oligosaccharides, lacto sucrose, xylo-oligosacchairde, chito-
oligosaccharide,
manno-oligosaccharide, aribino-oligosaccharide, siallyl-oligosaccharide, fuco-
oligosaccharide, and gentio-oligosaccharides. Preferably, the nutritional
compositions comprise polydextrose (PDX) and/or galactooligosaccaharide (GOS).
Optionally, in addition to polydextrose and/or galactooligosaccaharide, the
nutritional compositions comprise one or more additional prebiotics.
[0038] If included in the nutritional compositions, the total amount of
prebiotics present in the nutritional composition may be from about 0.1 g/100
kcal
to about 1 g/100 kcal. More preferably, the total amount of prebiotics present
in
the nutritional composition may be from about 0.3 g/100 kcal to about 0.7
g/100
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kcal. At least 20% of the prebiotics should comprise galactooligosaccharide
and/or
polydextrose.
[0039] If polydextrose is used in the prebiotic composition, the amount of
polydextrose in the nutritional composition may, in an embodiment, be within
the
range of from about 0.1 g/100 kcal to about 1 g/100 kcal. In another
embodiment,
the amount of polydextrose in the nutritional compositions is within the range
of
from about 0.2 g/100 to about 0.6 g/100 kcal.
[0040] If galactooligosaccharide is used in the prebiotic composition, the
amount of galactooligosaccharide in the nutritional composition may, in an
embodiment, be from about 0.1 g/100 kcal to about 1 g/100 kcal. In another
embodiment, the amount of galactooligosaccharide in the nutritional
composition is
from about 0.2 g/100 kcal to about 0.5 g/100 kcal. In certain embodiments, the
ratio
of polydextrose to galactooligosaccharide in the prebiotic composition is
between
about 9:1 and about 1:9.
[0041] The nutritional formulation of the disclosure, in some embodiments,
may further contain a source of long chain polyunsaturated fatty acids
(LCPUFAs).
Preferably, the source of LCPUFAs comprise docosahexanoic acid (DHA). Other
suitable LCPUFAs include, but are not limited to, a-linoleic acid, y-linoleic
acid,
linoleic acid, linolenic acid, eicosapentanoic acid (EPA) and arachidonic acid
(ARA).
[0042] In one embodiment, the nutritional composition is supplemented with
both DHA and ARA. In this embodiment, the weight ratio of ARA:DHA may be
from about 1:3 to about 9:1. In one embodiment of the present disclosure, the
weight ratio of ARA:DHA is from about 1:2 to about 4:1.
[0043] 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.
[0044] The nutritional composition may be supplemented with oils containing
DHA and ARA using standard techniques known in the art. For example, DHA
and ARA may be added to the composition by replacing an equivalent amount of
an
oil, such as high oleic sunflower oil, normally present in the composition. As
another example, the oils containing DHA and ARA may be added to the
composition by replacing an equivalent amount of the rest of the overall fat
blend
normally present in the composition without DHA and ARA.
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[0045] 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 embodiments, the DHA and ARA are sourced from the single cell Martek
oils,
DHASCOO and ARASCOO respectively, 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.
[0046] In an embodiment of the present disclosure, sources of DHA and ARA
are single cell oils as taught in U.S. Pat. Nos. 5,374,567; 5,550,156; and
5,397,591,
the disclosures of which are incorporated herein in their entirety by
reference.
However, the present disclosure is not limited to only such oils.
[0047] In certain embodiments, the nutritional compositions comprise from
about 0.5 mg/100 kcal to about 5 mg/100 kcal of iron, including iron bound to
lactoferrin.
EXAMPLES
[0048] The following examples are provided to illustrate some embodiments
of the nutritional composition of the present disclosure but should not be
interpreted as any limitation thereon. Other embodiments within the scope of
the
claims herein will be apparent to one skilled in the art from the
consideration of the
specification or practice of the nutritional composition or methods disclosed
herein.
It is intended that the specification, together with the example, be
considered to be
exemplary only, with the scope and spirit of the disclosure being indicated by
the
claims which follow the example.
EXAMPLE 1
[0049] This Example exemplifies the effect of lactoferrin and infant
formula
on the growth of diarrengic E. coli strains in vitro.
[0050] Fifteen clinical diarrheageanic E. coli (DEC) strains from each DEC
group (Enterotoxigenic E. coli (ETEC), Enteropathogenic E. coli (EPEC),
Shigatoxin
producing E. coli (STEC), and Enteroaggregative E. coli (EAEC)) are obtained.
As
previously described, the strains are isolated from a Peruvian cohort study
from
children with diarrhea and identified by Real Time PCR for diaheagenic E. coli
groups. Additionally, fifteen clinical isolates of Salmonella ser. Typhimurium
and
fifteen isolates of Shigella flexneri are also obtained.
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[0051] All clinical strains and standard laboratory control strains are
grown
in McConkey agar medium over 24 hours at 37 C. For viability assays, the
strains
are grown in lysogeny broth at 37 C for 18 hours. The strains are then washed
twice in PBS and centrifugated at 4500 x g for 5 min. Only bacteria in mid-log
phase is used.
[0052] 0, 0.6, 1, 2, 4, 6, 8 and 10 mg/ml stock solutions of lactoferrin
and 0,
0.6, 1, 2, 4, 6, 8 and 10 mg/ml stock solutions of infant formula containing
2.1 g/100
kcal of milk proteins and 1.8 mg/100 kcal of iron are prepared in distilled
water.
[0053] Cultures of the clinical strains containing approximately, 2 x 108
logarithmic phase cells are inoculated in 96-well plates containing 1%
bactopeptone. Each plate is also inoculated, via microdilution, with the stock
solution of infant formula or lactoferrin such that each strain is tested
against 0,
0.6, 1, 2, 4, 6, 8 or 10 mg/ml of lactoferrin or infant formula. The plates
are
incubated at 37 C and monitored every 30 minutes for growth kinetics by serial
cultures of 10-fold dilutions. Growth is then monitored in a spectrophotometer
and/or ELISA reader. After incubation for 18-20 hours, the MIC for each strain
is
recorded as the lowest concentration of infant formula or lactoferrin that
caused
complete bacterial inhibition.
[0054] Synergistic assays testing the activity of both lactoferrin and
infant
formula against each strain are also performed, and the effect of the
lactoferrin/infant formula combination on bacterial growth is measured as
described above. For these synergistic assays, the concentrations of
lactoferrin and
infant formula are determined for each agent separately based on the results
of the
MIC study and growth kinetics.
EXAMPLE 2
[0055] This Example exemplifies the effect of lactoferrin and infant
formula
on the adherence of diarrengic E. coli strains to human intestinal epithelial
cells in
vitro.
[0056] A subconfluent layer of Hep2 cells (approximately 5 x 104 cells/well
in
a 24-well plate) is infected with the Enterotoxigenic E. coli,
Enteropathogenic E.
coli, Shigatoxin producing E. coli, Enteroaggregative E. coli, Salmonella ser.
Typhimurium or Shigella flexneri isolates described in Example 1. Infant
formula
with and without 10 mg/ml lactoferrin is then added thereto such that the
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concentration of bacterium to infant formula is in a ratio of 100:1. Then, the
infected layer of Hep2 cells is incubated at 37 C in 5% CO2 for 4 hours. The
Hep2
cells are then vigorously washed to remove non-adherent bacteria. The cells
are
fixed with 70% methanol, stained with 10% Giemsa solution and examined under a
microscope. Additionally, for Enteropathogenic E. coli, Shigatoxin producing
E.
coli, Shigella flexneri, and Salmonella ser. Typhimurium, fluorescent actin
staining
assay (FAS) assay is performed as previously reported.
EXAMPLE 3
[0057] This Example exemplifies the effect of lactoferrin and infant
formula
in supporting resistance to conditions or diseases caused by bacterial
pathogens in
vivo.
[0058] Healthy, female Balb/c strain mice between 6 and 8 weeks of age with
a weight between 20 and 24 g are obtained and separated into an experimental
group and control group. The experimental group is then fed 200 ill of infant
formula containing either 75 or 165 mg/ml of lactoferrin before infection and
the
control group is fed 200 ill of infant formula before infection. The amount of
infant
formula that is administered is adjusted so the amount the mice receive is
equivalent to 600 mg/kg and 1333 mg/kg per day of lactoferrin. The mice are
then
infected with 300 [1.1_, 108 colonyforming units (cfu) of Salmonella ser.
Typhimurium,
200 mt 108 cfuof Citrobacter rodentium (the murine model of EPEC) or 200 .1_,
108
cfu of Shigella flexneri. For infection and pre-treatment inoculations, a
gavage
needle is used. After infection, the mice receive infant formula containing
either 75
or 165 mg/ml of lactoferrin or infant formula alone ad libitum, respectively
for 7
days. Again, the amount of infant formula that is administered is adjusted so
the
amount the mice receive is equivalent to 600 mg/kg and 1333 mg/kg per day of
lactoferrin. The mortality, weight and clinical signs (piloerection, hunched
position,
and reduced movement) is monitored daily in all mice for 7 days after
infection.
The incidence of clinical signs is determined by comparing the behavior of
each
infected mouse for 15 minutes. At day 10 post-infection, the mice are
sacrificed and
cardiac puncture is performed for blood cultures. For histopathological
analysis,
organs (colon, liver and spleen) are removed. The degree of inflammation and
necrosis in the organs are studied with a pathologist blinded to group
assignment
to prevent bias.
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EXAMPLE 4
[0059] This Example exemplifies the effect of lactoferrin and infant
formula
in supporting resistance to conditions or diseases caused by viral pathogens
in vivo.
[0060] Example 3 is performed as described above, except that mice are
infected with strains of Rotavirus, Norovirus, Astrovirus, Adenovirus and
Calicivus
instead of the bacterial strains. The mice are monitored and studied as
described
above.
EXAMPLE 5
[0061] This example illustrates an embodiment of a nutritional product
according to the present disclosure.
Description kg per 100 kg
carbohydrate, total 38.9
protein, total 28.8
fat, total 25.6
prebiotics 4.5
soy lecithin 0.8
lactoferrin 0.3
calcium carbonate 0.5
potassium citrate 0.2
ferrous sulfate 0.05
potassium chloride 0.048
magnesium oxide 0.023
sodium chloride 0.025
zinc sulfate 0.015
cupric sulfate 0.002
manganese sulfate 0.0003
sodium selenite 0.00003
choline chloride 0.144
ascorbic acid 0.093
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Niacinamide 0.006
calcium pantothenate 0.003
vitamin A palmitate 0.007
vitamin B12 0.002
vitamin D3 0.000001
Riboflavin 0.0008
thiamin 0.0006
vitamin B6 0.0004
folic acid 0.0001
vitamin K1 0.006
biotin 0.00002
inositol 0.03
vitamin E acetate 0.01
taurine 0.05
L-carnitine 0.001
EXAMPLE 6
[0062] This
example illustrates another embodiment of a nutritional product
according to the present disclosure.
Description kg per 100 kg
carbohydrate, total 24.7
protein, total 31.9
fat, total 39.3
prebiotics 3.6
lactoferrin 0.1
calcium carbonate 0.15
ferrous sulfate 0.03
zinc sulfate 0.01
copper sulfate 0.00025
manganese sulfate 0.0002
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sodium selenite 0.00001
choline bitartrate 0.05
ascorbic acid 0.004
sodium ascorbate 0.04
niacinamide 0.007
calcium pantothenate 0.0005
vitamin A palmitate 0.0005
vitamin D3 0.0002
riboflavin 0.0001
thiamin 0.00005
vitamin B6 0.00005
folic acid 0.000067
vitamin K1 0.00002
vitamin E acetate 0.008
taurine 0.02
fish oil 0.2
B-glucan 0.03
EXAMPLE 7
[0063] This example illustrates one embodiment of ingredients that can be
used to prepare the nutritional product according to the present disclosure.
Water 872 ml
lactose 65.6 mg
vegetable oil blend 353.0 mg
nonfat milk evaporated 34.0 mg
whey protein concentrate 8.5 mg
galacto-oligosaccharide 4.7 mg
Casein 3.5 mg
polydextrose 2.4 mg
lactoferrin solution (10%) 1.0 mg
single cell DHA and ARA oil blend 0.94 mg
mono- and di-glycerides 0.7 mg
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calcium carbonate 0.44 mg
calcium phosphate 0.4 mg
potassium citrate 0.4 mg
potassium chloride 0.4 mg
soy lecithin 0.4 mg
sodium chloride 0.3 mg
potassium phosphate 0.3 mg
choline chloride 0.2 mg
magnesium oxide 0.08 mg
calcium hydroxide 0.08 mg
ferrous suflate 0.07 mg
EXAMPLE 8
[0064] This example illustrates another embodiment of ingredients that can
be used to prepare the nutritional product according to the present
disclosure.
Water 686 ml
reduced minerals whey 215 mg
nonfat milk evaporated 67 mg
vegetable oil blend 33 mg
lactose 17 mg
galacto-oligosaccharide 4.7 mg
polydextrose 2.4 mg
lactoferrin solution (10%) 1.0 mg
single cell DHA and ARA oil blend 0.9 mg
mono- and di-glycerides 0.7 mg
calcium carbonate 0.44 mg
calcium phosphate 0.4 mg
potassium citrate 0.4 mg
potassium chloride 0.4 mg
soy lecithin 0.4 mg
potassium phosphate 0.3 mg
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carrageenan 0.3 mg
sodium citrate 0.2 mg
choline chloride 0.2 mg
magnesium oxide 0.08 mg
calcium chloride 0.08 mg
ferrous suflate 0.07 mg
[0065] Preferably, the nutritional composition is administered to a human
and supports resistance to a disease or condition in the human caused by a
bacterial or viral pathogen, including viral respiratory tract infection.
[0066] All references cited in this specification, including without
limitation,
all papers, publications, patents, patent applications, presentations, texts,
reports,
manuscripts, brochures, books, internet postings, journal articles,
periodicals, and
the like, are hereby incorporated by reference into this specification in
their
entireties. The discussion of the references herein is intended merely to
summarize
the assertions made by their authors and no admission is made that any
reference
constitutes prior art. Applicants reserve the right to challenge the accuracy
and
pertinence of the cited references.
[0067] 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.
