Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
METHOD FOR INHIBITING PATHOGENS USING A NUTRITIONAL
COMPOSITION
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 compositions therein. More
particularly, the disclosure relates to a method for inhibiting the invasive
mechanisms of harmful bacterial pathogens and/or inhibiting the adhesion of at
least one pathogen in a human gastrointestinal tract by administering to the
human a nutritional composition including lactoferrin produced by a non-human
source.
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] Some of the proteins present within human milk provide for a defense
against pathogens to prevent and inhibit infection while additionally
promoting an
immunological response within the infant. Proteins including haptocorrin and
lactoferrin are understood to help infants defend against a variety of
bacterial
pathogens via bacteriostatic and bactericidal activity.
[0004] 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
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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.
[0005] An additional role of lactoferrin is its anti-microbial activity in
guarding against intestinal infections in humans generally, but especially in
infants. Lactoferrin has been known to be both bacteriostatic and bactericidal
in
inhibiting the growth of specific bacteria while also killing microbes prior
to a
successful invasion of intestinal cells.
[0006] 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
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.
[0007] 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 fed prior art
formulas with high levels of iron fortification, e.g. 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.
[0008] Additional complications of the prior art formulas include the
inability
of providing a bacteriostatic 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.
[0009] Accordingly, it would be beneficial to provide a nutritional
composition, such as an infant formula, human milk fortifier, children's
dietary
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supplement, and the like, which contains lactoferrin, in particular,
lactoferrin
produced by a non-human source. Preferably, the lactoferrin included in the
compositions has a bacteriostatic effect even after processing under
conditions of
high temperature and low pH. A combination of characteristics including the
maintenance of the anti-invasion or anti-adhesion mechanisms of lactoferrin
through either high or low pH or high temperature conditions, such as during
pasteurization, provide for a dietary composition that may at least partially
protect
against harmful bacterial pathogens.
DISCLOSURE OF THE INVENTION
[0010] Briefly, the present disclosure is directed, in an embodiment, to a
method for inhibiting one or more of the invasive mechanisms of a bacterial
pathogen and/or the adhesion of at least one pathogen using a nutritional
composition comprising a lipid or fat, a protein source, a prebiotic
composition, and
lactoferrin from a non-human source, where the composition provides active
anti-
invasion and anti-adhesion mechanisms against strains of undesirable bacteria
found in the human gastrointestinal tract, even after processing which
includes
exposure to harsh environmental conditions.
[0011] In an embodiment, the disclosure is directed to a nutritional
product
comprising:
a. up to about 7 g/100 kcal of a fat or lipid, more preferably about 3
g/kcal to
about 7 g/100 kcal of a fat or lipid;
b. up to about 5 g/100 kcal of a protein source, more preferably about 1
g/kcal to
about 5 g/100 kcal of a protein source;
c. at least about 10 mg/100 kcal of lactoferrin, more preferably from about
70
mg/100 kcal to about 220 mg/100 kcal of lactoferrin produced by a non-human
source, most preferably about 90 mg/100 kcal to about 190 mg/100 kcal of
lactoferrin produced by a non-human source; and
d. about 0.1 g/100 kcal to about 1 g/100 kcal of 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.
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[0012] Preferably, the lactoferrin is non-human lactoferrin and/or human
lactoferrin produced by a genetically modified organism. The term "organism",
as
used herein, refers to any contiguous living system, such as animal, plant,
fungus
or micro-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 inhibit at least one of the invasive mechanisms of at
least
one pathogen in the gastrointestinal tract of a human, even if, during
processing,
the nutritional composition has been exposed to pH and temperature
fluctuations
typical of certain processing conditions like pasteurization. The lactoferrin
includes anti-invasion mechanisms which may destruct the attachment factors
and
injection needle used by certain bacteria on human cells. In another
particularly
preferred embodiment, the lactoferrin used is such that an effective amount of
a
nutritional composition containing lactoferrin may be administered to inhibit
the
adhesion of at least one pathogen in the gastrointestinal tract of a human,
even if,
during processing, the nutritional composition has been exposed to pH and
temperature fluctuations typical of certain processing conditions like
pasteurization. Examples of such pathogens include 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.
BEST MODE FOR CARRYING OUT THE INVENTION
[0013] The present disclosure provides novel nutritional products that are
easily digested, provide physiochemical benefits, and/or provide physiological
benefits. In an embodiment of the present disclosure, a nutritional
composition
comprises a lipid or fat, a protein source, a prebiotic composition having at
least
20% of an oligosaccharide, especially one which comprises galacto-
oligosaccharide
(GOS), and lactoferrin which provides active anti-invasion or anti-adhesion
mechanisms against strains of undesirable bacteria found in the human
gastrointestinal tract. The lactoferrin included in the compositions is
produced by
a non-human source. In certain embodiments, the prebiotic comprises a
combination of galacto-oligosaccharide and polydextrose. More particularly,
the
composition disclosed herein comprises:
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a. up to about 7 g/100 kcal of a fat or lipid, more preferably about 3 to
about 7
g/100 kcal of a fat or lipid;
b. up to about 5 g/100 kcal of a protein source, more preferably about 1 to
about 5
g/100 kcal of a protein source;
c. d. about 0.1 g/100 kcal to about 1 g/100 kcal of 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; and
d. at least about 10 mg/100kCal of lactoferrin, more preferably about 70 mg
to
about 220 mg/100kCal of lactoferrin, most preferably about 90 mg to about 190
mg/100kCal of lactoferrin.
DEFINITIONS
[0014] 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.
[0015] The term "probiotic" means a microorganism with low or no
pathogenicity that exerts beneficial effects on the health of the host.
[0016] As used herein, the term "infant" is generally defined as a human
from about birth to 12 months of age.
[0017] A "preterm infant" is an infant born after less than 37 weeks
gestation.
[0018] A "full term infant" as used herein, means an infant born after at
least
37 weeks gestation.
[0019] "Children" are defined as humans from the age of 12 months to about
12 years old.
[0020] An "anti-bacterially effective amount," as used herein is generally
defined as an amount of lactoferrin that provides at least one anti-invasion
mechanism against strains of bacteria.
[0021] "Lactoferrin produced by a non-human source" means lactoferrin
which is produced by or obtained from a source other than human breast milk.
For
example, lactoferrin for use in the present disclosure includes human
lactoferrin
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produced by a genetically modified organism as well as non-human lactoferrin.
[0022] "Biologically active lactoferrin," means lactoferrin which possesses
at
least one anti-invasion or anti-adhesion mechanism against pathogens.
[0023] The term "non-human lactoferrin", as used herein, refers to
lactoferrin
having an amino acid sequence that is different from the amino acid sequence
of
human lactoferrin.
[0024] The term "organism", as used herein, refers to any contiguous living
system, such as animal, plant, fungus or micro-organism.
[0025] The term "simulating," as used herein means having or taking the
form or appearance of or having or producing a symptomatic resemblance to.
DISCLOSURE
[0026] In some embodiments, the nutritional product 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.11. 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 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.
[0027] The nutritional products of the disclosure may provide minimal,
partial, or total nutritional support. The compositions may be nutritional
supplements or meal replacements. In some embodiments, the compositions may
be administered in conjunction with a food or nutritional composition. In this
embodiment, the compositions can either be intermixed with the food or other
nutritional compositions 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
compositions may be administered to preterm infants receiving infant formula,
breast milk, a human milk fortifier, or combinations thereof.
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[0028] The 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.
[0029] 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.
[0030] 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.
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[0031] 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.
[0032] 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
(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).
[0033] 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%
9
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 for use in the present disclosure include, without
limitation,
bovine lactoferrin, porcine lactoferrin, equine lactoferrin, buffalo
lactoferrin, goat
lactoferrin, murine lactoferrin and camel lactoferrin.
[0034] Surprisingly, the forms of lactoferrin included herein maintain
relevant 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 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. For instance, while bovine lactoferrin has an the amino acid
composition which has only about a 70% sequence homology to that of human
lactoferrin, and is stable and remains active under conditions under which
human
or recombinant human lactoferrin become unstable or inactive, bovine
lactoferrin
has bactericidal activity against undesirable bacterial pathogens found in the
human gut.
[0035] In U.S. Patent No. 4,791,193,
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.
[0036] 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 of 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
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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.
[0037] 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.
[0038] 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 concentrates, sweet whey, acid
whey,
casein, acid casein, caseinate (e.g. sodium caseinate, sodium calcium
caseinate,
calcium caseinate) and any combinations thereof.
[0039] 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.
[0040] In a particular embodiment of the disclosure, the whey:casein ratio
of
the protein source is similar to that found in human breast milk. In an
embodiment, the protein source comprises from about 20% to about 85% whey
protein. In another embodiment, the protein source may comprise from about 20%
to about 80% casein. In yet another embodiment of the disclosure, the protein
source includes about 60% to about 85% whey and about 15% to about 40% casein.
[0041] In one embodiment of the disclosure, the nutritional composition may
also contain one or more probiotics. Any probiotic known in the art may be
acceptable in this embodiment provided it achieves the intended result. In a
11
particular embodiment, the probiotic may be selected from Lactobacillus
species,
Lactobacillus rharnnosus GG, Bifidobacterium species, Bifidobacteriurn longum,
Bifidobacterium breuis and Bifidobacterium animalis subsp. lactis BB-12.
[0042] If included in the composition, the amount of the probiotic may
vary
from about 104 to about 1049 colony forming units (du) 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 106cfu per kg body weight per day.
Moreover,
the disclosed composition may also include probiotic-conditioned media
components.
[0043] 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 metabolites thereof. Such non-viable probiotics may
have
been heat-killed or 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.
[00441 The nutritional composition contains 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.
[00451 Prebiotics useful in the present disclosure may include
oligosaccharides, polysaccharides, and other prebiotics that contain fructose,
xy]ose, 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-oligosacchairde, chito-oligosaccharide, manno-
oligosaccharide,
aribino-oligosaccharide, siallyl-oligosaccharide, fuco-oligosaccharide, and
gentio-
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oligosaccharides.
[0046] In an embodiment, 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. Alternatively, 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. At
least 20% of the prebiotics should comprise galacto-oligosaccharide (GOS),
polydextrose, or a mixture thereof. Preferably, the nutritional compositions
comprise polydextrose and galactooligosaccaharide. Optionally, in addition to
polydextrose and/or galactooligosaccaharide, the nutritional compositions
comprise
one or more additional prebiotics. The amount of each of galacto-
oligosaccharide
and/or 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.
[0047] In an embodiment, 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 kcal. At least
20%
of the prebiotics should comprise galactooligosaccharide (GOS) and/or
polydextrose
(PDX).
[0048] The amount of galacto-oligosaccharide in the nutritional composition
may, in an embodiment, be from about 0.2 g/100 Kcal to about 1.0 g/100 Kcal.
In
another embodiment, the amount of galacto-oligosaccharide in the nutritional
composition may be from about 0.1 g/100 Kcal to about 0.5 g/100 Kcal. In yet
another embodiment, the amount of galactooligsaccharide is within the range of
from about 0.2 g/100 kcal to about 0.6 g/100 kcal. 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 0.1 g/100 kcal to about 1 g/100
kcal.
In another embodiment, the amount of polydextrose is within the range of from
about 0.2 g/100 kcal to about 0.6 g/100 kcal. In yet another embodiment, 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 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.
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[0049] Preferably, the prebiotic composition, in combination with
lactoferrin,
inhibits the adhesion of one or more pathogens in the gastrointestinal tract
when
the nutritional compositions are provided to humans. An example of one such
pathogen is Enterobacter sakazakii (otherwise known as Cronobacter sakazakii).
Another pathogen for which adhesion is inhibited by the combination of
lactoferrin
with the prebiotic composition is E. coll.
[0050] The nutritional formulation of the disclosure may also contain a
source of long chain polyunsaturated fatty acids (LCPUFAs) which 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).
[0051] 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, this
ratio
is from about 1:2 to about 4:1.
[0052] 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.
[0053] 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 formula by replacing an equivalent amount of an
oil,
such as high oleic sunflower oil, normally present in the formula. As another
example, the oils containing DHA and ARA may be added to the formula by
replacing an equivalent amount of the rest of the overall fat blend normally
present
in the formula without DHA and ARA.
[0054] 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 oil,
DHASCO and ARAS CO 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.
[0055] In an embodiment of the present disclosure, sources of DHA and ARA
14
are single cell oils as taught in U.S. Pat. Nos. 5,374,567; 5,550.156; and
5,397,591.
However, the present disclosure is not limited to only such oils.
[0056] 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.
[0057] A benefit of lactoferrin as used in embodiments of the present
disclosure is its anti-invasion and anti-adhesion mechanism in the human
gastrointestinal tract. Specifically, lactoferrin may destroy the injection
needle
used by certain bacteria to invade and cause pathogenesis. Likewise,
lactoferrin
inhibits the adhesion of pathogens in the gastrointestinal tract of humans.
One
such example of a bacterium known to cause pathogenesis is Escherichia coli
which
may cause diarrhea in infants, children and adults and is realized as an agent
for
pediatric diarrhea. The K coli produces and translocates bacterial protein
through
a needle complex via a type III secretory system.
[0058] The secretory system of many gram-negative pathogenic bacteria is
a
type III secretion including the following bacteria: Shigella, Salmonella,
Pseudotnonas, and Escherichia coli. The type III secretory system functions
through use of a needle for the transport of virulent proteins from the
bacterial
cytoplasm through the needle directly into the host cell's cytoplasm. The use
of the
needle provides for a passage through the multiple membranes including the
double membranes of the gram-negative bacterium and the eukaryotic membrane
of the human cell. Specifically, in strains of E. coli the needle complex is
comprised
of E. coli secretion component F (EscF) with E. coli secreted protein A (EspA)
attaching to the tip of the needle, forming a generally hollow structure for
the
passage of components from the bacteria to the host human cell. At this point,
bacterial proteins such as EspB may be introduced into the host cell through
this
tube. While the physiology of EspB may not be fully understood in the article
"The
Enteropathogenic E. coli effector EspB facilitates micro villas effacing and
Ant iphagocytosis by Inhibiting Myosin Function" in CELL HOSTS AND MICROBE, pp
383-392 (2007), EspB is described as binding to myosins which ultimately
suppresses phagocytosis as a human immune response. Generally, myosin proteins
interact with actin filaments to participate in cellular processes such as
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phagocytosis in eliminating potential bacterial pathogens. Harmful symptoms
occur where EspB emitted by the E. coli inhibits the interaction between
various
myosin proteins and actin filaments in suppressing phagocytosis, leading to
diarrhea or other gastric distress in infants, children and adults.
[0059] One of the anti-invasion mechanisms of lactoferrin is in inhibiting
the
translocation of EspB into human cells. Specifically, one mechanism may
include
the inhibition of the formation of the necessary secretory structures for
translocating EspB from the bacteria. Lactoferrin is capable of degrading
EspA,
the protein responsible for the tube like structure for translocating Esp B
into the
host cell. As EspA may be degraded by lactoferrin, the portal through the
human
cell membrane would not be created thus alleviating pathogenesis created from
Esp
B entering into the human cell's cytoplasm. Furthermore, lactoferrin may also
possess proteolytic activity resulting in the degrading EspB. Ultimately,
lactoferrin effectively disrupts the needle complex associated with the
pathogen's
secretion system while simultaneously degrading proteins responsible for
symptoms including gastrointestinal distress and diarrhea.
EXAMPLES
[0060] The following examples are provided to illustrate 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 examples.
EXAMPLE 1
[0061] This Example exemplifies the inhibition of pathogens, namely E.
sakazakii 4603 and 29004 and E. coli E2348/69, by lactoferrin alone and in
combination with polydextrose and galactooligosaccharide.
[0062] Based on a preliminary experiment, it is determined that cultures of
E. sakazakii 4603 and 29004 give the highest adherence rates to HEp-2 cells.
After 6 hour incubation, cultures of these E. sakazakii strains as well as
cultures of
E. coli E2348/69 are harvested by centrifugation, washed with phosphate
buffered
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saline, and re-suspended in minimal essential medium (MEM) supplemented with
10% fetal bovine serum. HEp-2 cells (obtained from the ATTC) are grown in 75
cm2
tissue culture flasks containing 25 ml of MEM supplemented with 10% FBS in a
CO2 incubator at tissue culture conditions. Confluent HEp-2 cells are
harvested by
adding 0.5 ml of 0.25% Trypsin-EDTA Solution (Sigma) and incubating for 15
minutes at tissue culture conditions. Tryp sin is inactivated with 0.5 ml of
FBS,
and the cells are seeded onto 12-mm diameter glass coverslips in 24-well
tissue
culture plates at approximately 3.6 x 105 viable cells per well. Plates are
incubated
under tissue culture conditions for two days prior to the start of each
experiment,
or until confluency is reached.
[0063] Immediately before the start of the assay, lactoferrin at final
concentrations of 0.1 mg/ml, 0.6 mg/ml, and 1 mg/ml, alone and in combination
with a 1:1 blend of galactooligosaccharide (obtained from DOMO) and
polydextrose
(obtained from DMV) at final concentrations of 4 mg/ml and 16 mg/ml of, is
added
to the HEp-2 cells. Control wells containing no lactoferrin are also prepared.
Then, 900:1 of the E. coli or E. sakazakii culture (containing ca. 107 cells)
are added
to each well (in triplicate). Tissue culture plates are then incubated at 37 C
in a
CO2 incubator for three hours. The wells are then washed five times to remove
non-adherent cells, and adhered bacteria are enumerated by microscopic
enumeration and quantitative real-time PCR.
[0064] For microscopic enumeration, coverslips are fixed with 100%
methanol, stained with 10% Geimsa for 15 minutes, washed with distilled water,
and dried overnight. The coverslips are mounted on microscope slides and
observed under a phase contrast microscope with the 100x objective. Fifteen
photomicrographs of each coverslip are taken using Motic Image software
following
an established geometric pattern throughout the entire coverslip. The number
of
cells and bacteria in each image is counted using Image J image analysis
software.
Adherence is calculated as the number of adhered bacteria per HEp-2 cell.
Adherence inhibition is calculated as the number of adhered bacteria per cell
in the
control minus the number of adhered bacteria per cell in the treatment divided
by
the number of adhered bacteria per cell in the control. For experiments using
cultures of E. coli, cells with E. coli microcolonies are manually counted.
Cells with
four or more bacteria are considered positive for having a typical localized
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17
adherence phenotype. The number of HEp-2 cells with adhered microcolonies is
determined and adherence and adherence inhibition is calculated as described
above. Experiments are performed in triplicate and replicated three times (n =
9).
[0065] In addition to the microscopic enumeration, adhered cells are also
enumerated by quantitative real-time PCR (qRT-PCR), as described in Humphries
et al., Interactions of enteropathogenic Escherichia coli with pediatric and
adult
intestinal biopsy specimens during early adherence, Infect. Immun., 77, 4463-
4468
(2009). Briefly, genomic DNA are extracted from the infected HEp-2 cells and
quantified by qRT-PCR using oligonucleotide primers that amplify the 16s rRNA
region of E sakazakii 4603 and 29004 or E. coli 2348/69. Appropriately diluted
whole genomic DNA is used as internal controls and to prepare standard curves
relating qPCR endpoints to cell concentrations. The PCR mixture consists of
11.250 SYBR solution, 2.5 MasterMix, 1 pl of each primer, and 5 pd of DNA
template. The PCR reactions are performed using an Eppendorf Mastercycler
Realplex2.
EXAMPLE 2
[0066] 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
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cupric sulfate 0.002
manganese sulfate 0.0003
sodium selenite 0.00003
choline chloride 0.144
ascorbic acid 0.093
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 3
10067] 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
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zinc sulfate 0.01
copper sulfate 0.00025
manganese sulfate 0.0002
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 4
10068] 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
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lactoferrin solution (10%) 1.0 mg
single cell DHA and ARA oil blend 0.94 mg
mono- and di-glycerides 0.7 mg
calcium carbonate 0.44 mg
calcium phosphate 0.4 mg
potassium citrate 0.4 mg
potassium chloride 0.4 mg
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 5
10069] 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
21
potassium chloride 0.4 mg
soy lecithin 0.4 mg
potassium phosphate 0.3 mg
carrageenan 0.3 mg
sodium citrate 0.2 mg
choline chloride 0.2 mg
magnesium oxide 0.08 mg
calcium chloride 0.08 mg
ferrous suflate 0.07 mg
[0070] The discussion of the references cited 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.
[0071] 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.
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