U.S. patent application number 15/369741 was filed with the patent office on 2018-06-07 for methods for inducing adipocyte browning, improving metabolic flexibility, and reducing detrimental white adipocyte tissue deposition and dysfunction.
The applicant listed for this patent is Mead Johnson Nutrition Company. Invention is credited to Teartse Tim Lambers, Marieke H. Schoemaker, Eric A.F. van Tol, Yan Zhong.
Application Number | 20180153951 15/369741 |
Document ID | / |
Family ID | 61027636 |
Filed Date | 2018-06-07 |
United States Patent
Application |
20180153951 |
Kind Code |
A1 |
Zhong; Yan ; et al. |
June 7, 2018 |
Methods for Inducing Adipocyte Browning, Improving Metabolic
Flexibility, and Reducing Detrimental White Adipocyte Tissue
Deposition and Dysfunction
Abstract
This disclosure relates to methods of inducing adipocyte
browning, supporting metabolic flexibility, and/or reducing
detrimental WAT deposition or reducing WAT dysfunction in a subject
by administering extensively hydrolyzed casein and/or fractions
thereof ("eHC") to the subject, and/or by administering a long
chain polyunsaturated fatty acid (e.g., docosahexaenoic acid and/or
arachidonic acid) to the subject.
Inventors: |
Zhong; Yan; (Shanghai,
CN) ; Schoemaker; Marieke H.; (Rhenen, NL) ;
Lambers; Teartse Tim; (Nijmegen, NL) ; van Tol; Eric
A.F.; (Arnhem, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mead Johnson Nutrition Company |
Glenview |
IL |
US |
|
|
Family ID: |
61027636 |
Appl. No.: |
15/369741 |
Filed: |
December 5, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/716 20130101;
A61K 31/716 20130101; A61K 45/06 20130101; A23L 33/12 20160801;
A23V 2200/3262 20130101; A23V 2250/55 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A23V 2200/328 20130101; A23V
2250/1868 20130101; A23V 2250/1862 20130101; A23V 2250/28 20130101;
A61K 31/202 20130101; A23L 33/18 20160801; A23V 2002/00 20130101;
A61K 31/702 20130101; A61K 31/202 20130101; A61K 38/018 20130101;
A23L 33/125 20160801; A23L 33/19 20160801; A61K 35/741 20130101;
A23V 2002/00 20130101; A23V 2250/54246 20130101 |
International
Class: |
A61K 38/01 20060101
A61K038/01; A61K 31/202 20060101 A61K031/202; A61K 35/741 20060101
A61K035/741; A61K 31/716 20060101 A61K031/716; A61K 31/702 20060101
A61K031/702 |
Claims
1. A method for inducing adipose browning in a subject, the method
comprising administering to a subject a nutritional composition
comprising extensively hydrolyzed casein, extensively hydrolyzed
casein fractions, or combinations thereof, and/or a long chain
polyunsaturated fatty acid.
2. The method of claim 1, wherein the nutritional composition
comprises a protein source, wherein at least 1% of the protein
source comprises extensively hydrolyzed casein, extensively
hydrolyzed casein fractions, or combinations thereof, such that at
least 1% to 80% of the protein source comprises the following
individual peptides: SEQ ID NO:4, SEQ ID NO:13, SEQ ID NO:17, SEQ
ID NO:21, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,
SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:60, and SEQ ID NO:63.
3. The method of claim 2 wherein the protein source is present in
amount of from about 0.2 g/100 Kcals to about 5.6 g/100 Kcals of
the nutritional composition.
4. The method of claim 2, wherein the protein source further
comprises at least 10 individual peptides selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ
ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,
SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID
NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ
ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45,
SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID
NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ
ID NO:56, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62,
SEQ ID NO:64 and combinations thereof.
5. The method of claim 1, wherein the nutritional composition
comprises at least one long-chain polyunsaturated fatty acid.
6. The method of claim 5, wherein the at least one long-chain
polyunsaturated fatty acid is docosahexaenoic acid and arachidonic
acid.
7. The method of claim 6, wherein the docosahexaenoic acid is
present in an amount from about 5 mg/100 Kcal to about 75 mg/100
Kcal.
8. The method of claim 1, the nutritional composition further
comprising a culture supernatant from a late-exponential growth
phase of a probiotic batch-cultivation process.
9. The method of claim 1, the nutritional composition further
comprising a prebiotic comprising polydextrose and
galacto-oligosaccharide.
10. A method for increasing metabolic flexibility in a subject, the
method comprising administering to the subject a nutritional
composition comprising: extensively hydrolyzed casein, extensively
hydrolyzed casein fractions, or combinations thereof, and/or a long
chain polyunsaturated fatty acid.
11. The method of claim 10, wherein administration of the
nutritional composition to the subject when the subject is an
infant increases the metabolic flexibility of the subject in
adolescence and/or adulthood, and wherein metabolic flexibility is
measured by: a. reduced plasma levels of at least one of total
cholesterol, total triglycerides, free fatty acids, alanine
aminotransferase (ALT) and aspartate aminotransferase (AST) in the
subject in response to a high fat diet, as compared to a subject
who has not received the nutritional composition; or b. decreased
fasting insulin levels, improved glucose tolerance, enhanced
insulin sensitivity and/or increased plasma adiponectin levels, as
compared to a subject who has not received the nutritional
composition.
12. The method of claim 10, wherein the nutritional composition
comprises a protein source, wherein at least 1% of the protein
source comprises extensively hydrolyzed casein, extensively
hydrolyzed casein fractions, or combinations thereof, such that at
least 1% to 80% of the protein source comprises the following
individual peptides: SEQ ID NO:4, SEQ ID NO:13, SEQ ID NO:17, SEQ
ID NO:21, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,
SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:60, and SEQ ID NO:63.
13. The method of claim 12 wherein the protein source is present in
amount of from about 0.2 g/100 Kcals to about 5.6 g/100 Kcals of
the nutritional composition.
14. The method of claim 11, wherein the protein source further
comprises at least 10 individual peptides selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ
ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,
SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID
NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ
ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45,
SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID
NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ
ID NO:56, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62,
SEQ ID NO:64 and combinations thereof.
15. The method of claim 10, wherein the nutritional composition
comprises at least one long-chain polyunsaturated fatty acid.
16. The method of claim 10, the nutritional composition further
comprising a prebiotic which comprises polydextrose and
galacto-oligosaccharide.
17. A method for reducing detrimental WAT deposition or reducing
WAT dysfunction in a subject, the method comprising administering
to the subject a nutritional composition comprising: extensively
hydrolyzed casein, extensively hydrolyzed casein fractions, or
combinations thereof, and/or a long chain polyunsaturated fatty
acid.
18. The method of claim 17, wherein the nutritional composition
comprises a protein source, wherein at least 1% of the protein
source comprises extensively hydrolyzed casein, extensively
hydrolyzed casein fractions, or combinations thereof, such that at
least 1% to 80% of the protein source comprises the following
individual peptides: SEQ ID NO:4, SEQ ID NO:13, SEQ ID NO:17, SEQ
ID NO:21, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,
SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:60, and SEQ ID NO:63.
19. The method of claim 18 wherein the protein source is present in
amount of from about 0.2 g/100 Kcals to about 5.6 g/100 Kcals of
the nutritional composition.
20. The method of claim 18, wherein the protein source further
comprises at least 10 individual peptides selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ
ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,
SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID
NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ
ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45,
SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID
NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ
ID NO:56, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62,
SEQ ID NO:64 and combinations thereof.
Description
TECHNICAL FIELD
[0001] This disclosure relates to methods of inducing adipocyte
browning, supporting metabolic flexibility, and/or reducing
detrimental white adipose tissue (WAT) deposition or reducing WAT
dysfunction by administering extensively hydrolyzed casein and/or
fractions thereof ("eHC") to a subject, and/or by administering a
long chain polyunsaturated fatty acid (e.g., docosahexaenoic acid
and/or arachidonic acid).
BACKGROUND
[0002] Accumulation of excess white adipose tissue (WAT) adversely
affects metabolic health. In contrast, brown adipose tissue (BAT)
confers benefits on metabolic health, and is characterized by
increased energy-expenditure capacity in the form of heat
(thermogenesis). The negative effects of WAT may be reduced by
inducing the development of brown adipocytes or beige adipocytes
(also called `brite` (brown-in-white), induced BAT, recruitable BAT
and wBAT (white adipose BAT) in WAT, a process called "browning."
(See, Harms et al. (2013) NATURE MEDICINE 19:1252-1263.)
[0003] Like brown adipocytes, beige adipocytes express uncoupling
protein-1 (UCP1). Activated UCP-1 uncouples the respiratory chain
in mitochondria by reducing the proton gradient, thereby generating
heat from the combustion of substrates normally used to produce
ATP. Recent imaging studies suggest that obese adults have lower
mass and/or activity of UCP1-expressing adipocytes. (See, e.g.,
Cypess et al. (2009) N. ENGL. J. MED. 360:1509-1517.) Further, it
has been demonstrated by animal studies and in human adults that
browning activity can be induced in response to appropriate stimuli
such as cold exposure and nutrition stimulation.
[0004] According to the early programming concept, nutrition and
other environmental factors during sensitive time windows,
especially the first thousand days of an infant's life, can affect
the infant's metabolic flexibility later in life. Metabolic
flexibility relates to the ability to cope with dietary challenges
such as a high fat diet, or with inflammatory challenges due to
diet or other factors. Accordingly, there is a need in the art for
compositions and methods that can stimulate adipocyte browning and
the differentiation of WAT into BAT, especially in infants and
children, to improve metabolic outcomes. Further, there is a need
for compositions and methods to be provided to an infant or child
that can support the infant's metabolic flexibility later in
life.
BRIEF SUMMARY
[0005] In one aspect, the disclosure relates to a method for
inducing adipose browning in a subject, the method comprising
administering to a subject a nutritional composition comprising
extensively hydrolyzed casein, extensively hydrolyzed casein
fractions, or combinations thereof, and/or a long chain
polyunsaturated fatty acid.
[0006] In another aspect, the disclosure relates to a method for
improving metabolic flexibility in a subject. The method can
include administering to the subject a nutritional composition
comprising extensively hydrolyzed casein, extensively hydrolyzed
casein fractions, or combinations thereof, and/or a long chain
polyunsaturated fatty acid. In certain embodiments, administration
of the nutritional composition to the subject when the subject is
an infant improves the metabolic flexibility of the subject in
adolescence and/or adulthood, and wherein metabolic flexibility is
measured by (a) reduced plasma levels of at least one of total
cholesterol, total triglycerides, free fatty acids, alanine
aminotransferase (ALT) and aspartate aminotransferase (AST) in the
subject in response to a high fat diet, as compared to a subject
who has not received the nutritional composition; or (b) decreased
fasting insulin levels, improved glucose tolerance, enhanced
insulin sensitivity; and/or increased plasma adiponectin levels, as
compared to a subject who has not received the nutritional
composition.
[0007] In another aspect, the disclosure relates to reducing
detrimental WAT deposition or reducing WAT dysfunction in a
subject. The method can include administering to the subject a
nutritional composition comprising extensively hydrolyzed casein,
extensively hydrolyzed casein fractions, or combinations thereof,
and/or a long chain polyunsaturated fatty acid.
[0008] In certain embodiments, the extensively hydrolyzed casein
fraction has a molar mass distribution of greater than 500 Daltons.
In certain embodiments, the nutritional composition comprises a
protein which source, wherein at least 1% of the protein source is
a protein equivalent source which comprises extensively hydrolyzed
casein, extensively hydrolyzed casein fractions, or combinations
thereof, such that 1% to 80% of the protein source comprises the
following individual peptides: SEQ ID NO:4, SEQ ID NO:13, SEQ ID
NO:17, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:31, SEQ
ID NO:32, SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:60, and SEQ ID
NO:63. The protein source may further comprises at least 10
individual peptides selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ
ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25,
SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID
NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ
ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,
SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID
NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ
ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:58,
SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:64 and
combinations thereof.
[0009] In certain embodiments, the protein source is present in
amount of from about 0.2 g/100 Kcals to about 5.6 g/100 Kcals of
the nutritional composition.
[0010] In certain embodiments, the nutritional composition
comprises at least one long-chain polyunsaturated fatty acid. The
long-chain polyunsaturated fatty acid can be docosahexaenoic acid
and/or arachidonic acid. When docosahexaenoic acid is present it
can be present in an amount of at least about 5 mg/100 Kcal.
[0011] The nutritional composition in the disclosed method may be
an infant formula, and may, in some embodiments, further comprise
fat, carbohydrate, probiotic, prebiotic, or combinations thereof.
The prebiotic may include polydextrose and/or
galacto-oligosaccharide. The nutritional composition may also
comprise culture supernatant from a late-exponential growth phase
of a probiotic batch-cultivation process.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1A-1H shows luminescence images of Ucp1+/LUC mice in
response to ARA+DHA, hydrolyzed casein and a combination diet (CAD)
treatment before (A) and after (B) high fat diet feeding.
Quantification of luminescence for the dorsal (C), the chest and
neck (D) and abdomen (E) view images in response to ARA+DHA,
hydrolyzed casein, and the combination diet (CAD) before high fat
diet exposure (at 12 weeks of age) and similarly (F-H) after high
fat diet challenge measured (at 20 weeks of age). "HAR" is the
positive control for browning.
[0013] FIG. 2A-2E shows luciferase enzymatic activity in inguinal
BAT (iBAT; FIG. 2A), inguinal WAT (iWAT; FIG. 2B) in response to
ARA+DHA, hydrolyzed casein, and the combination diet (CAD) prior to
high fat diet feeding; *p<0.05, **p<0.01 vs. CON. Luciferase
enzymatic activity in iBAT (C), in iWAT (D) and epididymal WAT
(eWAT; E) in response to ARA+DHA, hydrolyzed casein, and the
combination diet (CAD) when exposed to high fat diet feeding as
measured at the end of the study. #p<0.01 vs. LFD-CON,
*p<0.05, **p<0.01 vs. HFD-CON.
[0014] FIG. 3A-F shows relative RNA expression of UCP1 in iBAT in
response to ARA+DHA, hydrolyzed casein, and the combination diet
(CAD) as measured at the end of the study (FIG. 3A); relative RNA
expression of UCP1 in iWAT in response to ARA+DHA, hydrolyzed
casein, and their combination diets (FIG. 3B); relative RNA
expression of UCP1 in eWAT in response to ARA+DHA, hydrolyzed
casein, and the combination diet (CAD) (FIG. 3C). Western blot
analysis of UCP1 in iBAT is shown in FIG. 3D. The ratio value of
UCP1/.beta.-actin, the density analysis of using NIH Image J
software for relative values is shown in (FIG. 3E). FIG. 3F shows
H&E staining and UCP1 immunohistochemistry in iBAT #p<0.01
vs. LFD-CON, *p<0.05, **p<0.01 vs. HFD-CON. *p<0.05,
**p<0.01 vs. HFD-CON.
[0015] FIG. 4A-C shows relative RNA expression of PRDM16 in iBAT,
iWAT and eWAT in response to ARA+DHA, hydrolyzed casein, and the
combination diet (CAD). FIG. 4D-F shows relative RNA expression of
PGC1.alpha. in iBAT, iWAT and eWAT in response to ARA+DHA,
hydrolyzed casein, and the combination diet (CAD). #p<0.01 vs.
Con-CON, *p<0.05, **p<0.01 vs. CON-HFD.
[0016] FIG. 5A-D shows that administration of ARA+DHA, hydrolyzed
casein (eCH), and their combination (CAD) improved glucose
tolerance and insulin sensitivity prior to high fat diet feeding
(FIG. 5A-B) or when exposed to a high fat diet feeding (FIG. 5C-D).
Glucose tolerance test was performed through intraperitoneal
injection of glucose into mice after 12 h fasting, and blood
glucose levels were measured at 0, 15, 30, 60, and 120 min later,
as shown in FIGS. 5A and C. Insulin tolerance test was performed
through intraperitoneal injection of insulin into mice after 6 h
fasting, and blood glucose levels were measured at 0, 15, 30, 60,
and 120 min later, as shown in FIG. 5B (*p<0.01, **p<0.05 vs.
CON) and FIG. 5D (*p<0.05, **p<0.01 vs. CON-HFD).
[0017] FIG. 6A-F shows the effects of administration of ARA+DHA,
hydrolyzed casein (eCH), and their combination (CAD) prior to and
during a high fat diet challenge in mice on TG (FIG. 6A); TC (FIG.
6B); ALT (FIG. 6C); AST (FIG. 6D); FFA (FIG. 6E); and insulin level
(FIG. 6F) as measured at the end of the study. #p<0.01 vs.
CON-CON, *p<0.05, **p<0.01 vs. CON-HFD.
[0018] FIG. 7A-D shows the effects of administration of ARA+DHA,
hydrolyzed casein (eCH), and their combination (eCH+ARA/DHA) prior
to and during a high fat diet challenge in mice on plasma
adipokines measurements as measured at the end of the study.
Adipokines measured are adiponectin (FIG. 7A), resistin (FIG. 7B),
leptin (FIG. 7C), and FGF21 (FIG. 7D). #p<0.01 vs. CON-CON,
*p<0.05 vs. CON-HFD.
[0019] FIG. 8A-F shows the anti-inflammatory effects of
administration of ARA+DHA, hydrolyzed casein (eCH), and their
combination (eCH+ARA/DHA) in mice as measured at the end of the
study. Interleukin 1 beta concentration expression is shown in FIG.
8A. Tumor necrosis factor-alpha concentration is shown in FIG. 8B.
Relative RNA expression of F4/80 in iBAT, iWAT and eWAT is shown in
FIG. 8C. Relative RNA expression of TNF.alpha. in iBAT, iWAT and
eWAT is shown in FIG. 8D. Relative RNA expression of IL1.beta. in
iBAT, iWAT and eWAT is shown in FIG. 8E. Relative RNA expression of
IL6 in iBAT, iWAT and eWAT is shown in FIG. 8F. #p<0.01 vs.
CON-CON, *p<0.05, **p<0.01 vs. CON-HFD.
DETAILED DESCRIPTION
[0020] 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.
[0021] 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.
[0022] "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
interchangeably 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, such as women who are lactating or pregnant.
In certain embodiments, the nutritional compositions are for
pediatric subjects, including infants and children.
[0023] The term "enteral" means 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.
[0024] "Pediatric subject" includes both infants and children and
refers herein to a human that is less than thirteen years of age.
In some embodiments, a pediatric subject refers to a human subject
that is less than eight years old. In other embodiments, a
pediatric subject refers to a human subject between about one and
about six years of age or about one and about three years of age.
In still further embodiments, a pediatric subject refers to a human
subject between about six and about twelve years of age.
[0025] "Infant" means a subject of not more than one year and
includes infants from zero to twelve 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, extremely low birth weight infants
and preterm infants. "Preterm" means an infant born before the end
of the 37.sup.th week of gestation. "Late preterm" means an infant
from between the 34.sup.th week and the 36.sup.th week of
gestation. "Full term" means an infant born after the end of the
37.sup.th week of gestation. "Low birth weight infant" means an
infant born weighing less than 2500 grams (approximately 5 lbs., 8
ounces). "Very low birth weight infant" means an infant born
weighing less than 1500 grams (approximately 3 lbs., 4 ounces).
"Extremely low birth weight infant" means an infant born weighing
less than 1000 grams (approximately 2 lbs., 3 ounces).
[0026] "Child" means a subject ranging in age from about twelve
months to about thirteen years. In some embodiments, a child is a
subject between about one and about twelve years old. In other
embodiments, the terms "children" or "child" refer to subjects that
are between one and about six years old, between about one and
about three years old, or between about seven and about twelve
years old. In other embodiments, the terms "children" or "child"
refer to any range of ages between about twelve months and about
thirteen years.
[0027] "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 is an example of a children's nutritional
product.
[0028] The term "degree of hydrolysis" refers to the extent to
which peptide bonds are broken by a hydrolysis method. The degree
of protein hydrolysis for purposes of characterizing the hydrolyzed
protein component of the nutritional composition is easily
determined by one of ordinary skill in the formulation arts by
quantifying the amino nitrogen to total nitrogen ratio (AN/TN) of
the protein component of the selected formulation. The amino
nitrogen component is quantified by USP titration methods for
determining amino nitrogen content, while the total nitrogen
component is determined by the Kjeldahl method, all of which are
well known methods to one of ordinary skill in the analytical
chemistry art.
[0029] When a peptide bond in a protein is broken by enzymatic
hydrolysis, one amino group is released for each peptide bond
broken, causing an increase in amino nitrogen. It should be noted
that even non-hydrolyzed protein would contain some exposed amino
groups. Hydrolyzed proteins will also have a different molecular
weight distribution than the non-hydrolyzed proteins from which
they were formed. The functional and nutritional properties of
hydrolyzed proteins can be affected by the different size peptides.
A molecular weight profile is usually given by listing the percent
by weight of particular ranges of molecular weight (in Daltons)
fractions (e.g., 2,000 to 5,000 Daltons, greater than 5,000
Daltons).
[0030] The term "molar mass distribution" when used in reference to
a hydrolyzed protein or protein hydrolysate pertains to the molar
mass of each peptide present in the protein hydrolysate. For
example, a protein hydrolysate having a molar mass distribution of
greater than 500 Daltons means that each peptide included in the
protein hydrolysate has a molar mass of at least 500 Daltons. To
produce a protein hydrolysate having a molar mass distribution of
greater than 500 Daltons, a protein hydrolysate may be subjected to
certain filtering procedures or any other procedure known in the
art for removing peptides, amino acids, and/or other proteinaceous
material having a molar mass of less than 500 Daltons. For the
purposes of this disclosure, any method known in the art may be
used to produce the protein hydrolysate having a molar mass
distribution of greater than 500 Dalton.
[0031] The term "protein source" includes any protein source or
protein equivalent source, such as soy, egg, whey, or casein, as
well as non-protein sources, such as peptides or amino acids.
Further, the protein source can be any used in the art, e.g.,
nonfat milk, whey protein, casein, soy protein, hydrolyzed protein,
peptides, amino acids, and the like. 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), soy bean
proteins, and any combinations thereof. The protein equivalent
source can, in some embodiments comprise hydrolyzed protein,
including partially hydrolyzed protein and extensively hydrolyzed
protein. The protein equivalent source may, in some embodiments,
include intact protein. More particularly, the protein source may
include a) about 20% to about 80% of the peptide component
described herein, and b) about 20% to about 80% of an intact
protein, a hydrolyzed protein, or a combination thereof.
[0032] The term "protein equivalent source" also encompasses free
amino acids. In some embodiments, the amino acids may comprise, but
are not limited to, histidine, isoleucine, leucine, lysine,
methionine, cysteine, phenylalanine, tyrosine, threonine,
tryptophan, valine, alanine, arginine, asparagine, aspartic acid,
glutamic acid, glutamine, glycine, proline, serine, carnitine,
taurine and mixtures thereof. In some embodiments, the amino acids
may be branched chain amino acids. In certain other embodiments,
small amino acid peptides may be included as the protein component
of the nutritional composition. Such small amino acid peptides may
be naturally occurring or synthesized.
[0033] The term "partially hydrolyzed" means having a degree of
hydrolysis which is greater than 0% but less than about 50%.
[0034] The term "extensively hydrolyzed" means having a degree of
hydrolysis which is greater than or equal to about 50%.
Accordingly, "extensively hydrolyzed casein fraction(s)" means
casein having a degree of hydrolysis which is greater than or equal
to about 50%. In some embodiments, extensively hydrolyzed may
include a degree of hydrolysis of greater than about 80%. In
further embodiments, extensively hydrolyzed may include a degree of
hydrolysis of greater than about 90%. "eHC" means extensively
hydrolyzed casein and/or fractions thereof.
[0035] The term "protein-free" means containing no measurable
amount of intact 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.
[0036] "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.
[0037] 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 normal growth and development of a
child between the ages of about 1 and about 6 years of age.
[0038] "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.
[0039] "Milk" means a component that has been drawn or extracted
from the mammary gland of a mammal. In some embodiments, the
nutritional composition comprises components of milk that are
derived from domesticated ungulates, ruminants or other mammals or
any combination thereof.
[0040] "Fractionation procedure" includes any process in which a
certain quantity of a mixture is divided up into a number of
smaller quantities known as fractions. The fractions may be
different in composition from both the mixture and other fractions.
Examples of fractionation procedures include but are not limited
to, melt fractionation, solvent fractionation, supercritical fluid
fractionation and/or combinations thereof.
[0041] "Fat globule" refers to a small mass of fat surrounded by
phospholipids and other membrane and/or serum proteins, where the
fat itself can be a combination of any vegetable or animal fat.
[0042] "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.
[0043] Therefore, a nutritional composition that is "nutritionally
complete" for a preterm infant will, by definition, provide
qualitatively and quantitatively adequate amounts of carbohydrates,
lipids, essential fatty acids, proteins, essential amino acids,
conditionally essential amino acids, vitamins, minerals, and energy
required for growth of the preterm infant.
[0044] A nutritional composition that is "nutritionally complete"
for a 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.
[0045] A nutritional composition that 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.
[0046] 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.
[0047] "Probiotic" means a microorganism with low or no
pathogenicity that exerts a beneficial effect on the health of the
host.
[0048] The term "non-viable probiotic" means a probiotic wherein
the metabolic activity or reproductive ability of the referenced
probiotic has been reduced or destroyed. More specifically,
"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. The "non-viable probiotic" does, however,
still retain, at the cellular level, its cell structure or other
structure associated with the cell, for example exopolysaccharide
and at least a portion its biological glycol-protein and DNA/RNA
structure and thus retains the ability to favorably influence the
health of the host. Contrariwise, the term "viable" refers to live
microorganisms. As used herein, the term "non-viable" is synonymous
with "inactivated".
[0049] "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 beneficial bacteria
in the digestive tract, selective reduction in gut pathogens, or
favorable influence on gut short chain fatty acid profile that can
improve the health of the host.
[0050] "Branched Chain Fatty Acid" ("BCFA") means a fatty acid
containing a carbon constituent branched off the carbon chain.
Typically the branch is an alkyl branch, especially a methyl group,
but ethyl and propyl branches are also known. The addition of the
methyl branch lowers the melting point compared with the equivalent
straight chain fatty acid. This includes branched chain fatty acids
with an even number of carbon atoms in the carbon chain. Examples
of these can be isomers of tetradecanoic acid, hexadecanoic
acid.
[0051] "Odd- and Branched-Chain Fatty Acid" ("OBCFA") is a subset
of BCFA that has an odd number of carbon atoms and have one or more
alkyl branches on the carbon chain. The main odd- and
branched-chain fatty acids found in bovine milk include, but are
not limited to, the isomers of tetradecanoic acid, pentadecanoic
acid, hexadecanoic acid, and heptadecanoic acid. For the purposes
of this disclosure, the term "BCFA" includes both branched-chain
fatty acids and odd-and-branched chain fatty acids.
[0052] "Trans-fatty acid" means an unsaturated fat with a
trans-isomer. Trans-fats may be monounsaturated or polyunsaturated.
Trans refers to the arrangement of the two hydrogen atoms bonded to
the carbon atoms involved in a double bond. In the trans
arrangement, the hydrogens are on opposite sides of the bond. Thus
a trans-fatty acid is a lipid molecule that contains one or more
double bonds in trans geometric configuration.
[0053] "Phospholipids" means an organic molecule that contains a
diglyceride, a phosphate group and a simple organic molecule.
Examples of phospholipids include but are not limited to,
phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine,
phosphatidylserine, phosphatidylinositol, phosphatidylinositol
phosphate, phosphatidylinositol biphosphate and
phosphatidylinositol triphosphate, ceramide phosphorylcholine,
ceramide phosphorylethanolamine and ceramide phosphorylglycerol.
This definition further includes sphingolipids, glycolipids, and
gangliosides.
[0054] "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 from, for example, fruit, seed or tea extracts.
Further, the term phytonutrient includes all carotenoids,
phytosterols, thiols, and other plant-derived compounds. Moreover,
as a skilled artisan will understand, plant extracts may include
phytonutrients, such as polyphenols, in addition to protein, fiber
or other plant-derived components. Thus, for example, apple or
grape seed extract(s) may include beneficial phytonutrient
components, such as polyphenols, in addition to other plant-derived
substances.
[0055] ".beta.-glucan" means all .beta.-glucan, including specific
types of .beta.-glucan, such as .beta.-1,3-glucan or
.beta.-1,3;1,6-glucan. Moreover, .beta.-1,3;1,6-glucan is a type of
.beta.-1,3-glucan. Therefore, the term ".beta.-1,3-glucan" includes
.beta.-1,3;1,6-glucan.
[0056] "Pectin" means any naturally-occurring oligosaccharide or
polysaccharide that comprises galacturonic acid that may be found
in the cell wall of a plant. 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.
[0057] 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 (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%.
[0058] As used herein, "lactoferrin from 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 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.
[0059] As used herein, "non-human lactoferrin" means lactoferrin
that has an amino acid sequence that is different than the amino
acid sequence of human lactoferrin.
[0060] "Pathogen" means an organism that causes a disease state or
pathological syndrome. Examples of pathogens may include bacteria,
viruses, parasites, fungi, microbes or combination (s) thereof.
[0061] "Modulate" or "modulating" means exerting a modifying,
controlling and/or regulating influence. In some embodiments, the
term "modulating" means exhibiting an increasing or stimulatory
effect on the level/amount of a particular component. In other
embodiments, "modulating" means exhibiting a decreasing or
inhibitory effect on the level/amount of a particular
component.
[0062] All percentages, parts and ratios as used herein are by
weight of the total formulation, unless otherwise specified.
[0063] 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.
[0064] 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. The compositions described herein may be free or
substantially free of any component described herein, included, for
example, any one or more of the following components: protein,
protein equivalent source, lipid, GOS, PDX, prebiotics, LGG,
probiotics, DHA, ARA, LCPUFAs, beta glucan (or any specific beta
glucan described herein), etc.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
Methods
[0069] The present disclosure relates to the discovery that
administering extensively hydrolyzed casein and/or fractions
thereof ("eHC") to a subject, and/or administering a long chain
polyunsaturated fatty acid (e.g., docosahexaenoic acid and/or
arachidonic acid) can induce adipocyte browning. Adipocyte browning
refers to the process of inducing the development of (1) brown
adipocytes and/or (2) beige adipocytes in white adipose tissue
(also called `brite` (brown-in-white), induced BAT, recruitable BAT
and wBAT (white adipose BAT). (See, Harms et al., supra.)
[0070] Adipocyte browning is beneficial to metabolic health. The
metabolic benefits of adipocyte browning may occur concurrently
with the process of adipocyte browning and/or may occur at a later
time. For example, if adipocyte browning is induced in an infant or
child, the infant or child may experience the metabolic benefits in
adolescence and/or adulthood in accordance with the early
programming concept.
[0071] In certain embodiments, induction of adipocyte browning is
determined by measuring increased expression of UCP1 in a subject.
In certain embodiments, increased UCP1 expression is measured by a
protein assay (e.g., an ELISA). In certain embodiments, a
nutritional composition is capable of inducing adipocyte browning
if administration of the nutritional composition to a Ucp-1
luciferase knock-in mouse causes increased luciferase activity.
[0072] Induction of adipocyte browning also may be determined by
detecting increased expression of other browning-relevant marker
genes, such as PGC1.alpha. (peroxisome proliferator-activated
receptor-.gamma. coactivator 1.alpha.) or PRDM16 (PR domain
containing 16) following administration of the nutritional
compositions described herein. Induction of adipocyte browning may
also be determined by assessing the function and amount of
mitochondria in adipocytes, using any method known in the art.
[0073] In further embodiments, the method relates to reducing
detrimental WAT deposition or reducing WAT dysfunction. In certain
embodiments, detrimental WAT deposition includes increased fat
mass, increased abdominal fat deposition, body weight gain,
increased weight of fat tissue deposits, and/or increased fatty
liver. In certain embodiments, WAT dysfunction includes impaired
fat tissue quality, increased hypertrophy of fat cells, increased
inflammation, altered adipokine profile, increased insulin
resistance and lipid overload to other organs (e.g., the
liver).
[0074] In certain embodiments, the compositions described herein
are capable of improving WAT function. For example, improved WAT
function is associated with improved insulin sensitivity and
smaller and more fat cells, and the presence of brown
adipocytes.
[0075] Administration of the compositions described herein to a
subject can increase metabolic flexibility in the subject. In
certain embodiments, administration of the compositions described
herein to a pediatric subject (e.g., an infant), can increase the
pediatric subject's metabolic flexibility later in life (e.g., in
adolescence and/or adulthood), in accordance with the early
programming concept. Metabolic flexibility refers to the ability to
cope with dietary challenges such as a high fat diet, or with
inflammatory challenges due to diet or other factors.
[0076] In certain embodiments, a subject exhibits an increase in
metabolic flexibility if the subject has reduced plasma levels of
at least one of total cholesterol, total triglycerides, free fatty
acids, alanine aminotransferase (ALT) and aspartate
aminotransferase (AST) in the subject in response to a high fat
diet, as compared to a subject who has not received (i.e., been
administered) the compositions described herein. In certain
embodiments, a subject exhibits an increase in metabolic
flexibility if the subject has decreased fasting insulin levels,
improved glucose tolerance, and enhanced insulin sensitivity, or
has an increased plasma adiponectin level, as compared to a subject
who has not received (i.e., been administered) a composition as
described herein.
[0077] In certain embodiments, administration of the compositions
described herein can also increase a subject's ability to cope with
inflammatory challenges. Increased ability to cope with
inflammatory challenges can be demonstrated, for example, by a
reduction in adipocyte inflammation in response to an inflammatory
stimulus or a high fat diet. A subject's level of inflammation can
be measured by measuring expression of pro-inflammatory cytokines
such as TNF-a, IL-1.beta., and IL-6 or by measuring the level of
macrophages in the subject. F4/80 is a macrophage-specific G
protein-coupled receptor, used as a macrophage marker in mice. In
certain embodiments, administration of the nutritional composition
is capable of increasing a subject's ability to maintain low
expression levels of at least one of TNF-a, IL-1.beta., IL-6 and
F4/80 in the subject in response to an inflammatory challenge or a
high fat diet. TNF-a and IL-1.beta. can be measured using ELISA and
RT-PCR assays. IL-6 and F4/80 can be measured using RT-PCR.
eHC Component
[0078] In certain embodiments, the eHC includes a peptide component
comprising SEQ ID NO:4, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21,
SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID
NO:51, SEQ ID NO:57, SEQ ID NO:60, and SEQ ID NO:63. In some
embodiments, the peptide component may comprise additional peptides
disclosed in Table 1. For example, the composition may include at
least 10 additional peptides disclosed in Table 1.
[0079] In another embodiment, the eHC further includes a peptide
component comprising at least 3 peptides selected from the group
consisting of SEQ ID NO:4, SEQ ID NO:13, SEQ ID NO:17, SEQ ID
NO:21, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ
ID NO:51, SEQ ID NO:57, SEQ ID NO:60, and SEQ ID NO:63, and at
least 5 additional peptides selected from Table 1.
[0080] Table 1 below identifies the specific amino acid sequences
that may be included in the eHC of the present disclosure.
TABLE-US-00001 TABLE 1 Seq ID Amino Acid Sequence (aa) 1 Ala Ile
Asn Pro Ser Lys Glu Asn 8 2 Ala Pro Phe Pro Glu 5 3 Asp Ile Gly Ser
Glu Ser 6 4 Asp Lys Thr Glu Ile Pro Thr 7 5 Asp Met Glu Ser Thr 5 6
Asp Met Pro Ile 4 7 Asp Val Pro Ser 4 n/a Glu Asp Ile 3 n/a Glu Leu
Phe 3 n/a Glu Met Pro 3 8 Glu Thr Ala Pro Val Pro Leu 7 9 Phe Pro
Gly Pro Ile Pro 6 10 Phe Pro Gly Pro Ile Pro Asn 7 11 Gly Pro Phe
Pro 4 12 Gly Pro Ile Val 4 13 Ile Gly Ser Glu Ser Thr Glu Asp Gln 9
14 Ile Gly Ser Ser Ser Glu Glu Ser 8 15 Ile Gly Ser Ser Ser Glu Glu
Ser Ala 9 16 Ile Asn Pro Ser Lys Glu 6 17 Ile Pro Asn Pro Ile 5 18
Ile Pro Asn Pro Ile Gly 6 19 Ile Pro Pro Leu Thr Gln Thr Pro Val 9
20 Ile Thr Ala Pro 4 21 Ile Val Pro Asn 4 22 Lys His Gln Gly Leu
Pro Gln 7 23 Leu Asp Val Thr Pro 5 24 Leu Glu Asp Ser Pro Glu 6 25
Leu Pro Leu Pro Leu 5 26 Met Glu Ser Thr Glu Val 6 27 Met His Gln
Pro His Gln Pro Leu Pro Pro Thr 11 28 Asn Ala Val Pro Ile 5 29 Asn
Glu Val Glu Ala 5 n/a Asn Leu Leu 3 30 Asn Gln Glu Gln Pro Ile 6 31
Asn Val Pro Gly Glu 5 32 Pro Phe Pro gly Pro Ile 6 33 Pro Gly Pro
Ile Pro Asn 6 34 Pro His Gln Pro Leu Pro Pro Thr 8 35 Pro Ile Thr
Pro Thr 5 36 Pro Asn Pro Ile 4 37 Pro Asn Ser Leu Pro Gln 6 38 Pro
Gln Leu Glu Ile Val Pro Asn 8 39 Pro Gln Asn Ile Pro Pro Leu 7 40
Pro Val Leu Gly Pro Val 6 41 Pro Val Pro Gln 4 42 Pro Val Val Val
Pro 5 43 Pro Val Val Val Pro Pro 6 44 Ser Ile Gly Ser Ser Ser Glu
Glu Ser Ala Glu 11 45 Ser Ile Ser Ser Ser Glu Glu 7 46 Ser Ile Ser
Ser Ser Glu Glu Ile Val Pro Asn 11 47 Ser Lys Asp Ile Gly Ser Glu 7
48 Ser Pro Pro Glu Ile Asn 6 49 Ser Pro Pro Glu Ile Asn Thr 7 50
Thr Asp Ala Pro Ser Phe Ser 7 51 Thr Glu Asp Glu Leu 5 52 Val Ala
Thr Glu Glu Val 6 53 Val Leu Pro Val Pro 5 54 Val Pro Gly Glu 4 55
Val Pro Gly Glu Ile Val 6 56 Val Pro Ile Thr Pro Thr 6 57 Val Pro
Ser Glu 4 58 Val Val Pro Pro Phe Leu Gln Pro Glu 9 59 Val Val Val
Pro Pro 5 60 Tyr Pro Phe Pro Gly Pro 6 61 Tyr Pro Phe Pro Gly Pro
Ile Pro 8 62 Tyr Pro Phe Pro Gly Pro Ile Pro Asn 9 63 Tyr Pro Ser
Gly Ala 5 64 Tyr Pro Val Glu Pro 5
[0081] Table 2 below further identifies a subset of amino acid
sequences from Table 1 that may be included and/or comprise the eHC
disclosed herein.
TABLE-US-00002 TABLE 2 Seq ID Amino Acid Sequence (aa) 4 Asp Lys
Thr Glu Ile Pro Thr 7 13 Ile Gly Ser Glu Ser Thr Glu Asp Gln 9 17
Ile Pro Asn Pro Ile Gly 6 21 Ile Val Pro Asn 4 24 Leu Glu Asp Ser
Pro Glu 6 30 Asn Gln Glu Gln Pro Ile 6 31 Asn Val Pro Gly Glu 5 32
Pro Phe Pro Gly Pro Ile 6 51 Thr Glu Asp Glu Leu 5 57 Val Pro Ser
Glu 4 60 Tyr Pro Phe Pro Gly Pro 6 63 Tyr Pro Ser Gly Ala 5
Nutritional Composition
[0082] In certain embodiments, the present disclosure relates
generally to nutritional compositions comprising a protein source,
wherein at least 1% of the protein source comprises the eHC and up
to 99% of the protein source comprises an intact protein, a
partially hydrolyzed protein, amino acids, or combinations thereof.
In embodiments, 1% to 80% of the protein source comprises the eHC
and 20% to 99% of the protein source comprises intact protein,
partially hydrolyzed protein, amino acids, or combinations thereof.
In still other embodiments, from 40% to 100% of the protein source
comprises the eHC and from 0 to 60% of the protein source comprises
an intact protein, a partially hydrolyzed protein, amino acids, or
combinations thereof. In yet other embodiments, from 40% to 70% of
the protein source comprises the eHC and from 30% to 60% of the
protein source comprises an intact protein, a partially hydrolyzed
protein, amino acids, or combinations thereof.
[0083] In another embodiment, 20% to 80% of the protein source
includes a peptide component comprising at least 3 peptides
selected from the group consisting of SEQ ID NO:4, SEQ ID NO:13,
SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:30, SEQ ID
NO:31, SEQ ID NO:32, SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:60, and
SEQ ID NO:63, and at least 5 additional peptides selected from
Table 1.
[0084] In some embodiments, the eHC may be present in the
nutritional composition in an amount from about 0.2 g/100 Kcal to
about 5.6 g/100 Kcal. In other embodiments the eHC may be present
in the nutritional composition in an amount from about 1 g/100 Kcal
to about 4 g/100 Kcal. In still other embodiments, the eHC may be
present in the nutritional composition in an amount from about 2
g/100 Kcal to about 3 g/100 Kcal.
[0085] The protein source disclosed herein may be formulated with
other ingredients in the nutritional composition to provide
appropriate nutrient levels for the target subject. In some
embodiments, the protein source is included in a nutritionally
complete formula that is suitable to support normal growth.
[0086] In other embodiments, the nutritional composition may
comprise a nutritional supplement or additive that may be added to
other nutritional formulations including, but not limited to,
foodstuffs and/or beverages. For the purposes of this disclosure,
"nutritional supplement" includes a concentrated source of
nutrient, for example the peptides identified herein, or
alternatively other substances with a nutritional or physiological
effective whose purpose is to supplement the normal diet
[0087] As discussed, the eHC may be provided as an element of a
protein source. In some embodiments, the peptides identified in
Tables 1 and 2, may be obtained by hydrolysis or they may be
synthesized in vitro by methods know to the skilled person. A
non-limiting example of a method of hydrolysis utilizing a
proteolytic enzyme is disclosed in U.S. Pat. No. 7,618,669 to
Rangavajla et al., which is hereby incorporated by reference in its
entirety however, other methods of hydrolysis may be used in
practice of the present disclosure.
[0088] In some embodiments, the protein source comprises a
hydrolyzed protein, such as casein, which includes partially
hydrolyzed protein and extensively hydrolyzed protein (i.e., the
eHC). In some embodiments, the eHC comprises an extensively
hydrolyzed casein and/or fractions thereof including peptides
having a molar mass distribution of greater than 500 Daltons. In
some embodiments, the eHC comprises peptides having a molar mass
distribution in the range of from about 500 Daltons to about 1,500
Daltons. Still, in some embodiments the eHC may comprise peptides
having a molar mass distribution range of from about 500 Daltons to
about 2,000 Daltons.
[0089] In some embodiments the protein source comprises partially
hydrolyzed protein having a degree of hydrolysis of less than 40%.
In still other embodiments, the protein source may comprise
partially hydrolyzed protein having a degree of hydrolysis of less
than 25%, or less than 15%.
[0090] In a particular embodiment, other than eHC, the nutritional
composition is protein-free and contains free amino acids as a
protein source. In this embodiment, the amino acids may comprise,
but are not limited to, histidine, isoleucine, leucine, lysine,
methionine, cysteine, phenylalanine, tyrosine, threonine,
tryptophan, valine, alanine, arginine, asparagine, aspartic acid,
glutamic acid, glutamine, glycine, proline, serine, carnitine,
taurine and mixtures thereof. In some embodiments, the amino acids
may be branched chain amino acids. In other embodiments, small
amino acid peptides may be included as the protein component of the
nutritional composition. Such small amino acid peptides may be
naturally occurring or synthesized. The amount of free amino acids
in the nutritional composition may vary from about 1 to about 5
g/100 Kcal. In an embodiment, 100% of the free amino acids have a
molecular weight of less than 500 Daltons. In this embodiment, the
nutritional composition may be hypoallergenic.
[0091] In an embodiment, where the protein source comprises intact
proteins, the intact proteins comprise from about 40% to about 85%
whey protein and from about 15% to about 60% casein.
[0092] In some embodiments, the nutritional composition comprises
between about 1 g and about 7 g of a protein source per 100 Kcal.
In other embodiments, the nutritional composition comprises between
about 3.5 g and about 4.5 g of protein source per 100 Kcal.
[0093] In certain embodiments, the nutritional composition of the
disclosure may contain a source of long chain polyunsaturated fatty
acid (LCPUFA), e.g., docosahexaenoic acid (DHA) and/or arachidonic
acid (ARA). Other suitable LCPUFAs include, but are not limited to,
linoleic (18:2 n-6), .gamma.-linolenic (18:3 n-6),
dihomo-.gamma.-linolenic (20:3 n-6) acids in the n-6 pathway,
a-linolenic (18:3 n-3), stearidonic (18:4 n-3), eicosatetraenoic
(20:4 n-3), eicosapentaenoic (20:5 n-3), and docosapentaenoic (22:6
n-3).
[0094] In certain embodiments the amount of LCPUFA in the
nutritional composition is at least about 5 mg/100 Kcal, and 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.
[0095] In certain embodiments, the amount of DHA in the nutritional
composition is at least about 17 mg/100 Kcal, and can vary from
about 5 mg/100 Kcal to about 75 mg/100 Kcal, or from about 10
mg/100 Kcal to about 50 mg/100 Kcal.
[0096] In an embodiment, especially if the nutritional composition
is an infant formula, the nutritional composition is supplemented
with both DHA and ARA. In this embodiment, the weight ratio of
ARA:DHA may be between about 1:3 and about 9:1. In a particular
embodiment, the ratio of ARA:DHA is from about 1:2 to about
4:1.
[0097] If included, the source of DHA and/or 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 single cell Martek oils, DHASCO.RTM. and
ARASCO.RTM., 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 subject.
Alternatively, the DHA and ARA can be used in refined form.
[0098] In an embodiment, sources of DHA and ARA are single cell
oils as taught in U.S. Pat. Nos. 5,374,657; 5,550,156; and
5,397,591, the disclosures of which are incorporated herein in
their entirety by reference. Nevertheless, the present disclosure
is not limited to only such oils.
[0099] In certain embodiments, the nutritional composition
comprises both eHC and a LCPUFA. In certain embodiments, the
nutritional composition comprises eHC, DHA and ARA. In certain
embodiments, administration of a nutritional supplement comprising
a combination of eHC and a LCPUFA (e.g., DHA and/or ARA) induces
adipocyte browning to a greater extent than either component alone,
reduces the risk of developing metabolic syndrome, and/or reduces
adipocyte inflammation.
[0100] The nutritional composition(s) of the present disclosure
including the eHC and/or LCPUFA may be administered in one or more
doses daily. Any orally acceptable dosage form is contemplated by
the present disclosure. Examples of such dosage forms include, but
are not limited to pills, tablets, capsules, soft-gels, liquids,
liquid concentrates, powders, elixirs, solutions, suspensions,
emulsions, lozenges, beads, cachets, and combinations thereof.
[0101] In some embodiments, the protein source comprising the eHC
and/or LCPUFA may be added to a more complete nutritional product.
In this embodiment, the nutritional composition may contain fats or
lipids and carbohydrate sources or components and may be used to
supplement the diet or may be used as the sole source of
nutrition.
[0102] In some embodiments, the nutritional composition comprises
at least one carbohydrate. The carbohydrate 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 the carbohydrate in the nutritional composition
typically can vary from between about 5 g/100 Kcal and about 25
g/100 Kcal. In some embodiments, the amount of carbohydrate is
between about 6 g/100 Kcal and about 22 g/100 Kcal. In other
embodiments, the amount of carbohydrate is between about 12 g/100
Kcal and about 14 g/100 Kcal. In some embodiments, the nutritional
composition comprises between about 3 g and about 8 g of a
carbohydrate. In some embodiments, corn syrup solids are preferred.
Moreover, hydrolyzed, partially hydrolyzed, and/or extensively
hydrolyzed carbohydrates may be desirable for inclusion in the
nutritional composition due to their easy digestibility.
Specifically, hydrolyzed carbohydrates are less likely to contain
allergenic epitopes
[0103] Non-limiting examples of carbohydrate materials suitable for
use herein include hydrolyzed or intact, naturally or chemically
modified, starches sourced from corn, tapioca, rice or potato, in
waxy or non-waxy forms. Non-limiting examples of suitable
carbohydrates include various hydrolyzed starches characterized as
hydrolyzed cornstarch, maltodextrin, maltose, corn syrup, dextrose,
corn syrup solids, glucose, and various other glucose polymers and
combinations thereof. Non-limiting examples of other suitable
carbohydrates include those often referred to as sucrose, lactose,
fructose, high fructose corn syrup, indigestible oligosaccharides
such as fructooligosaccharides and combinations thereof.
[0104] In one particular embodiment, the carbohydrate component of
the nutritional composition is comprised of 100% lactose. In
another embodiment, the additional carbohydrate component comprises
between about 0% and 60% lactose. In another embodiment, the
carbohydrate component comprises between about 15% and 55% lactose.
In yet another embodiment, the carbohydrate component comprises
between about 20% and 30% lactose. In these embodiments, the
remaining source of carbohydrates may be any carbohydrate known in
the art. In an embodiment, the carbohydrate component comprises
about 25% lactose and about 75% corn syrup solids.
[0105] In some embodiments, the carbohydrate may comprise at least
one starch or starch component. A starch is a carbohydrate composed
of two distinct polymer fractions: amylose and amylopectin. Amylose
is the linear fraction consisting of a-1,4 linked glucose units.
Amylopectin has the same structure as amylose, but some of the
glucose units are combined in an a-1,6 linkage, giving rise to a
branched structure. Starches generally contain 17-24% amylose and
from 76-83% amylopectin. Yet special genetic varieties of plants
have been developed that produce starch with unusual amylose to
amylopectin ratios. Some plants produce starch that is free of
amylose. These mutants produce starch granules in the endosperm and
pollen that stain red with iodine and that contain nearly 100%
amylopectin. Predominant among such amylopectin producing plants
are waxy corn, waxy sorghum and waxy rice starch.
[0106] The performance of starches under conditions of heat, shear
and acid may be modified or improved by chemical modifications.
Modifications are usually attained by introduction of substituent
chemical groups. For example, viscosity at high temperatures or
high shear can be increased or stabilized by cross-linking with di-
or polyfunctional reagents, such as phosphorus oxychloride.
[0107] In some instances, the nutritional compositions of the
present disclosure comprise at least one starch that is gelatinized
or pregelatinized. As is known in the art, gelatinization occurs
when polymer molecules interact over a portion of their length to
form a network that entraps solvent and/or solute molecules.
Moreover, gels form when pectin molecules lose some water of
hydration owing to competitive hydration of cosolute molecules.
Factors that influence the occurrence of gelation include pH,
concentration of cosolutes, concentration and type of cations,
temperature and pectin concentration. Notably, LM pectin will gel
only in the presence of divalent cations, such as calcium ions. And
among LM pectins, those with the lowest degree of esterification
have the highest gelling temperatures and the greatest need for
divalent cations for crossbridging.
[0108] Meanwhile, pregelatinization of starch is a process of
precooking starch to produce material that hydrates and swells in
cold water. The precooked starch is then dried, for example by drum
drying or spray drying. Moreover the starch of the present
disclosure can be chemically modified to further extend the range
of its finished properties. The nutritional compositions of the
present disclosure may comprise at least one pregelatinized
starch.
[0109] Native starch granules are insoluble in water, but, when
heated in water, native starch granules begin to swell when
sufficient heat energy is present to overcome the bonding forces of
the starch molecules. With continued heating, the granule swells to
many times its original volume. The friction between these swollen
granules is the major factor that contributes to starch paste
viscosity.
[0110] The nutritional composition of the present disclosure may
comprise native or modified starches, such as, for example, waxy
corn starch, waxy rice starch, corn starch, rice starch, potato
starch, tapioca starch, wheat starch or any mixture thereof.
Generally, common corn starch comprises about 25% amylose, while
waxy corn starch is almost totally made up of amylopectin.
Meanwhile, potato starch generally comprises about 20% amylose,
rice starch comprises an amylose:amylopectin ratio of about 20:80,
and waxy rice starch comprises only about 2% amylose. Further,
tapioca starch generally comprises about 15% to about 18% amylose,
and wheat starch has an amylose content of around 25%.
[0111] In some embodiments, the nutritional composition comprises
gelatinized and/or pre-gelatinized waxy corn starch. In other
embodiments, the nutritional composition comprises gelatinized
and/or pre-gelatinized tapioca starch. Other gelatinized or
pre-gelatinized starches, such as rice starch or potato starch may
also be used.
[0112] Suitable fats or lipids for use in the nutritional
composition of 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, palm
olein, 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.
[0113] The amount of lipids or fats is, in one embodiment, no
greater than about 7 g/100 Kcal; in certain embodiments, the lipid
or fat is present at a level of from about 2 to about 7 g/100 Kcal.
In certain embodiments, the nutritional composition comprises
between about 1 g and about 10 g per 100 Kcal of a lipid source. In
certain embodiments, the nutritional composition comprises between
about 2 g/100 Kcal to about 7 g/100 Kcal of a fat source. In
certain embodiments the fat source may be present in an amount from
about 2.5 g/100 Kcal to about 6 g/100 Kcal. In certain embodiments,
the fat source may be present in the nutritional composition in an
amount from about 3 g/100 Kcal to about 4 g/100 Kcal. In certain
embodiments, the nutritional composition comprises between about 3
g and about 8 g per 100 Kcal of a lipid source. In certain
embodiments, the nutritional composition comprises between about 5
and about 6 g per 100 Kcal of a lipid source.
[0114] In certain embodiments, the fat or lipid source comprises
from about 10% to about 35% palm oil per the total amount of fat or
lipid. In other embodiments, the fat or lipid source comprises from
about 15% to about 30% palm oil per the total amount of fat or
lipid. In yet other embodiments, the fat or lipid source may
comprise from about 18% to about 25% palm oil per the total amount
of fat or lipid.
[0115] In certain embodiments, the fat or lipid source may be
formulated to include from about 2% to about 16% soybean oil based
on the total amount of fat or lipid. In some embodiments, the fat
or lipid source may be formulated to include from about 4% to about
12% soybean oil based on the total amount of fat or lipid. In some
embodiments, the fat or lipid source may be formulated to include
from about 6% to about 10% soybean oil based on the total amount of
fat or lipid.
[0116] In certain embodiments, the fat or lipid source may be
formulated to include from about 2% to about 16% coconut oil based
on the total amount of fat or lipid. In some embodiments, the fat
or lipid source may be formulated to include from about 4% to about
12% coconut oil based on the total amount of fat or lipid. In some
embodiments, the fat or lipid source may be formulated to include
from about 6% to about 10% coconut oil based on the total amount of
fat or lipid.
[0117] In certain embodiments, the fat or lipid source may be
formulated to include from about 2% to about 16% sunflower oil
based on the total amount of fat or lipid. In some embodiments, the
fat or lipid source may be formulated to include from about 4% to
about 12% sunflower oil based on the total amount of fat or lipid.
In some embodiments, the fat or lipid source may be formulated to
include from about 6% to about 10% sunflower oil based on the total
amount of fat or lipid.
[0118] In some embodiments, the oils, i.e. sunflower oil, soybean
oil, sunflower oil, palm oil, etc. are meant to cover fortified
versions of such oils known in the art. For example, in certain
embodiments, the use of sunflower oil may include high oleic
sunflower oil. In other examples, the use of such oils may be
fortified with certain fatty acids, as known in the art, and may be
used in the fat or lipid source disclosed herein.
[0119] In some embodiments, the fat or lipid source includes an oil
blend including sunflower oil, medium chain triglyceride oil, and
soybean oil. In some embodiments, the fat or lipid source includes
a ratio of sunflower oil to medium chain triglyceride oil of about
1:1 to about 2:1. In certain other embodiments, the fat or lipid
source includes a ratio of sunflower oil to soybean oil of from
about 1:1 to about 2:1. In still other embodiments, the fat or
lipid source may include a ratio of medium chain triglyceride oil
to soybean oil of from about 1:1 to about 2:1.
[0120] In certain embodiments the fat or lipid source may comprise
from about 15% to about 50% w/w sunflower oil based on the total
fat or lipid content. In certain embodiments, the fat or lipid
source includes from about 25% to about 40% w/w sunflower oil based
on the total fat or lipid content. In some embodiments, the fat or
lipid source comprises from about 30% to about 35% w/w sunflower
oil based on the total fat or lipid content.
[0121] In certain embodiments the fat or lipid source may comprise
from about 15% to about 50% w/w medium chain triglyceride oil based
on the total fat or lipid content. In certain embodiments, the fat
or lipid source includes from about 25% to about 40% w/w medium
chain triglyceride oil based on the total fat or lipid content. In
some embodiments, the fat or lipid source comprises from about 30%
to about 35% w/w medium chain triglyceride oil based on the total
fat or lipid content.
[0122] In certain embodiments the fat or lipid source may comprise
from about 15% to about 50% w/w soybean oil based on the total fat
or lipid content. In certain embodiments, the fat or lipid source
includes from about 25% to about 40% w/w soybean oil based on the
total fat or lipid content. In some embodiments, the fat or lipid
source comprises from about 30% to about 35% w/w soybean oil based
on the total fat or lipid content.
[0123] In some embodiments, the nutritional composition comprises
from about 1 g/100 Kcal to about 3 g/100 Kcal of sunflower oil. In
some embodiments, the nutritional composition comprises from about
1.3 g/100 Kcal to about 2.5 g/100 Kcal of sunflower oil. In still
other embodiments, the nutritional composition comprises from about
1.7 g/100 Kcal to about 2.1 g/100 Kcal of sunflower oil. The
sunflower oil as described herein may, in some embodiments, include
high oleic sunflower oil.
[0124] In certain embodiments, the nutritional composition if
formulated to include from about 1 g/100 Kcal to about 2.5 g/100
Kcal of medium chain triglyceride oil. In other embodiments, the
nutritional composition includes from about 1.3 g/100 Kcal to about
2.1 g/100 Kcal of medium chain triglyceride oil. Still in further
embodiments, the nutritional composition includes from about 1.6
g/100 Kcal to about 1.9 g/100 Kcal of medium chain triglyceride
oil.
[0125] In some embodiments, the nutritional composition may be
formulated to include from about 1 g/100 Kcal to about 2.3 g/100
Kcal of soybean oil. In certain embodiments, the nutritional
composition may be formulated to include from about 1.2 g/100 Kcal
to about 2 g/100 Kcal of soybean oil. Still in certain embodiments,
the nutritional composition may be formulated to include from about
1.5 g/100 Kcal to about 1.8 g/100 Kcal of soybean oil.
[0126] In some embodiments, the term "sunflower oil", "medium chain
triglyceride oil", and "soybean oil" are meant to cover fortified
versions of such oils known in the art. For example, in certain
embodiments, the use of sunflower oil may include high oleic
sunflower oil. In other examples, the use of such oils may be
fortified with certain fatty acids, as known in the art, and may be
used in the fat or lipid source disclosed herein.
[0127] In some embodiments, the fat or lipid source provides from
about 35% to about 55% of the total calories of the nutritional
composition. In other embodiments, the fat or lipid source provides
from about 40% to about 47% of the total calories of the
nutritional composition.
[0128] In certain embodiments the nutritional composition may be
formulated such that from about 10% to about 23% of the total
calories of the nutritional composition are provided by sunflower
oil. In other embodiments, from about 13% to about 20% of the total
calories in the nutritional composition may be provided by
sunflower oil. Still, in other embodiments, from about 15% to about
18% of the total calories of the nutritional composition may be
provided by sunflower oil.
[0129] In some embodiments, the nutritional composition may be
formulated such that from about 10% to about 20% of the total
calories are provided by MCT oil. In certain embodiments, from
about 12% to about 18% of the total calories in the nutritional
composition may be provided by MCT oil. In certain embodiments,
from about 14% to about 17% of the calories of the nutritional
composition may be provided by MCT oil.
[0130] In some embodiments, the nutritional composition may be
formulated such that from about 10% to 20% of the total calories of
the nutritional composition are provided by soybean oil. In certain
embodiments, from about 12% to about 18% of the total calories of
the nutritional composition may be provided by soybean oil. In
certain embodiments, from about 13% to about 16% of the total
calories may be provided by soybean oil.
[0131] The nutritional composition may also contain one or more
prebiotics (also referred to as a prebiotic component) in certain
embodiments. Prebiotics exert health benefits, which 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
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 useful in the
present disclosure may include oligosaccharides, polysaccharides,
and other prebiotics that contain fructose, xylose, soya,
galactose, glucose and mannose
[0132] More specifically, prebiotics useful in the present
disclosure may include polydextrose (PDX), polydextrose powder,
lactulose, lactosucrose, raffinose, gluco-oligosaccharide, inulin,
fructo-oligosaccharide (FOS), isomalto-oligosaccharide, soybean
oligosaccharides, lactosucrose, xylo-oligosaccharide (XOS),
chito-oligosaccharide, manno-oligosaccharide,
aribino-oligosaccharide, siallyl-oligosaccharide,
fuco-oligosaccharide, galacto-oligosaccharides (GOS) and
gentio-oligosaccharides.
[0133] 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. More preferably, 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. In some embodiments,
the total amount of prebiotics present in the nutritional
composition may be from about 0.01 g/100 Kcal to about 1.5 g/100
Kcal. In certain embodiments, the total amount of prebiotics
present in the nutritional composition may be from about 0.15 g/100
Kcal to about 1.5 g/100 Kcal. Moreover, the nutritional composition
may comprise a prebiotic component comprising PDX. In some
embodiments, the prebiotic component comprises at least 20% w/w
PDX, GOS or a mixture thereof.
[0134] The amount of PDX in the nutritional composition may, in an
embodiment, be within the range of from about 0.015 g/100 Kcal to
about 1.5 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 some embodiments, PDX may be included in
the nutritional composition in an amount sufficient to provide
between about 1.0 g/L and 10.0 g/L. In another embodiment, the
nutritional composition contains an amount of PDX that is between
about 2.0 g/L and 8.0 g/L. And in still other embodiments, the
amount of PDX in the nutritional composition may be from about 0.05
g/100 Kcal to about 1.5 g/100 Kcal.
[0135] The prebiotic component also comprises GOS in some
embodiments. The amount of GOS in the nutritional composition may,
in an embodiment, be from about 0.015 g/100 Kcal to about 1.0 g/100
Kcal. In another embodiment, the amount of GOS in the nutritional
composition may be from about 0.2 g/100 Kcal to about 0.5 g/100
Kcal.
[0136] In a particular embodiment of the present disclosure, PDX is
administered in combination with GOS.
[0137] In a particular embodiment, GOS and PDX are supplemented
into the nutritional composition in a total amount of at least
about 0.015 g/100 Kcal or about 0.015 g/100 Kcal to about 1.5
mg/100 Kcal. In some embodiments, the nutritional composition may
comprise GOS and PDX in a total amount of from about 0.1 to about
1.0 mg/100 Kcal.
[0138] Lactoferrin can also be included in some embodiments of the
nutritional composition of the present disclosure. 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 (Fe.sup.3+) is
tightly bound in synergistic cooperation with a (bi)carbonate
anion. These domains are called N1, N2, C1 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
(.about.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 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).
[0139] 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.
[0140] 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. The oral electrolyte solutions
described herein can, in some embodiments comprise non-human
lactoferrin, non-human lactoferrin produced by a genetically
modified organism and/or human lactoferrin produced by a
genetically modified organism.
[0141] Suitable non-human lactoferrins for use in the present
disclosure include, but are not limited to, those having at least
48% homology with the amino acid sequence of human lactoferrin. For
instance, bovine lactoferrin ("bLF") has an amino acid composition
which has about 70% sequence homology to that of human lactoferrin.
In some embodiments, the non-human lactoferrin has at least 65%
homology with human lactoferrin and in some embodiments, at least
75% homology. Non-human lactoferrins acceptable for use in the
present disclosure include, without limitation, bLF, porcine
lactoferrin, equine lactoferrin, buffalo lactoferrin, goat
lactoferrin, murine lactoferrin and camel lactoferrin.
[0142] In one embodiment, lactoferrin is present in the nutritional
composition in an amount of at least about 15 mg/100 Kcal. In
certain embodiments, the nutritional composition may include
between about 15 and about 300 mg lactoferrin per 100 Kcal. In
another embodiment, where the nutritional composition is an infant
formula, the nutritional composition may comprise lactoferrin in an
amount of from about 60 mg to about 150 mg lactoferrin per 100
Kcal; in yet another embodiment, the nutritional composition may
comprise about 60 mg to about 100 mg lactoferrin per 100 Kcal.
[0143] In some embodiments, the nutritional composition 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. In certain
embodiments, the nutritional composition may comprise between about
0.1 and about 2 grams lactoferrin per liter. In some embodiments,
the nutritional composition includes between about 0.6 and about
1.5 grams lactoferrin per liter of formula.
[0144] In some embodiments, the nutritional composition of the
present disclosure comprises non-human lactoferrin, for example
bovine lactoferrin (bLF). bLF is a glycoprotein that belongs to the
iron transporter or transferrin family. It is isolated from bovine
milk, wherein it is found as a component of whey. The bLF that is
used in certain embodiments may be any bLF isolated from whole milk
and/or having a low somatic cell count, wherein "low somatic cell
count" refers to a somatic cell count less than 200,000 cells/mL.
By way of example, suitable bLF is available from Tatua
Co-operative Dairy Co. Ltd., in Morrinsville, New Zealand, from
FrieslandCampina Domo in Amersfoort, Netherlands or from Fonterra
Co-Operative Group Limited in Auckland, New Zealand.
[0145] There are known differences between the amino acid sequence,
glycosylation patterns and iron-binding capacity in human
lactoferrin and bLF. Additionally, there are multiple and
sequential processing steps involved in the isolation of bLF from
cow's milk that affect the physiochemical properties of the
resulting bLF preparation. Human lactoferrin and bLF are also
reported to have differences in their abilities to bind the
lactoferrin receptor found in the human intestine.
[0146] Though not wishing to be bound by this or any other theory,
it is believed that bLF isolated from whole milk has less
lipopolysaccharide (LPS) initially bound than does bLF that has
been isolated from milk powder. Additionally, it is believed that
bLF with a low somatic cell count has less initially-bound LPS. A
bLF with less initially-bound LPS has more binding sites available
on its surface. This is thought to aid bLF in binding to the
appropriate location and disrupting the infection process.
[0147] 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. Pat. 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. Pat. 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.
[0148] In certain embodiments, lactoferrin utilized in the present
disclosure may be provided by an expanded bed absorption ("EBA")
process for isolating proteins from milk sources. EBA, also
sometimes called stabilized fluid bed adsorption, is a process for
isolating a milk protein, such as lactoferrin, from a milk source
comprises establishing an expanded bed adsorption column comprising
a particulate matrix, applying a milk source to the matrix, and
eluting the lactoferrin from the matrix with an elution buffer
comprising about 0.3 to about 2.0 M sodium chloride. Any mammalian
milk source may be used in the present processes, although in
particular embodiments, the milk source is a bovine milk source.
The milk source comprises, in some embodiments, whole milk, reduced
fat milk, skim milk, whey, casein, or mixtures thereof.
[0149] In some embodiments, the process comprises the steps of
establishing an expanded bed adsorption column comprising a
particulate matrix, applying a milk source to the matrix, and
eluting the lactoferrin from the matrix with about 0.3 to about
2.0M sodium chloride. In other embodiments, the lactoferrin is
eluted with about 0.5 to about 1.0 M sodium chloride, while in
further embodiments, the lactoferrin is eluted with about 0.7 to
about 0.9 M sodium chloride.
[0150] The expanded bed adsorption column can be any known in the
art, such as those described in U.S. Pat. Nos. 7,812,138,
6,620,326, and 6,977,046, the disclosures of which are hereby
incorporated by reference herein. In some embodiments, a milk
source is applied to the column in an expanded mode, and the
elution is performed in either expanded or packed mode. In
particular embodiments, the elution is performed in an expanded
mode. For example, the expansion ratio in the expanded mode may be
about 1 to about 3, or about 1.3 to about 1.7. EBA technology is
further described in international published application nos. WO
92/00799, WO 02/18237, WO 97/17132, which are hereby incorporated
by reference in their entireties.
[0151] The isoelectric point of lactoferrin is approximately 8.9.
Prior EBA methods of isolating lactoferrin use 200 mM sodium
hydroxide as an elution buffer. Thus, the pH of the system rises to
over 12, and the structure and bioactivity of lactoferrin may be
comprised, by irreversible structural changes. It has now been
discovered that a sodium chloride solution can be used as an
elution buffer in the isolation of lactoferrin from the EBA matrix.
In certain embodiments, the sodium chloride has a concentration of
about 0.3 M to about 2.0 M. In other embodiments, the lactoferrin
elution buffer has a sodium chloride concentration of about 0.3 M
to about 1.5 M, or about 0.5 m to about 1.0 M.
[0152] In some embodiments the nutritional composition may include
an enriched lipid fraction derived from milk. The enriched lipid
fraction derived from milk may be produced by any number of
fractionation techniques. These techniques include but are not
limited to melting point fractionation, organic solvent
fractionation, super critical fluid fractionation, and any variants
and combinations thereof. In some embodiments the nutritional
composition may include an enriched lipid fraction derived from
milk that contains milk fat globules.
[0153] In certain embodiments, the addition of the enriched lipid
fraction or the enriched lipid fraction including milk fat globules
may provide a source of saturated fatty acids, trans-fatty acids,
monounsaturated fatty acids, polyunsaturated fatty acids, odd- and
branched-chain fatty acids (OBCFAs), branched-chain fatty acids
(BCFAs), (conjugated linoleic acid) CLA, cholesterol,
phospholipids, and/or milk fat globule membranes (MFGM) as well as
MFGM proteins to the nutritional composition
[0154] The milk fat globules may have an average diameter
(volume-surface area average diameter) of at least about 2 .mu.m.
In some embodiments, the average diameter is in the range of from
about 2 .mu.m to about 13 .mu.m. In other embodiments, the milk fat
globules may range from about 2.5 .mu.m to about 10 .mu.m. Still in
other embodiments, the milk fat globules may range in average
diameter from about 3 .mu.m to about 6 .mu.m. The specific surface
area of the globules is, in certain embodiments, less than 3.5
m.sup.2/g, and in other embodiments is between about 0.9 m.sup.2/g
to about 3 m.sup.2/g. Without being bound by any particular theory,
it is believed that milk fat globules of the aforementioned sizes
are more accessible to lipases therefore leading to better lipid
digestion.
[0155] In some embodiments the enriched lipid fraction and/or milk
fat globules contain saturated fatty acids. The saturated fatty
acids may be present in a concentration from about 0.1 g/100 Kcal
to about 8.0 g/100 Kcal. In certain embodiments the saturated fatty
acids may be present from about 0.5 g/100 Kcal to about 2.0 g/100
Kcal. In still other embodiments the saturated fatty acids may be
present from about 3.5 g/100 Kcal to about 6.9 g/100 Kcal.
[0156] Examples of saturated fatty acids suitable for inclusion
include, but are not limited to, butyric, valeric, caproic,
caprylic, decanoic, lauric, myristic, palmitic, stearic, arachidic,
behenic, lignoceric, tetradecanoic, hexadecanoic, palmitic, and
octadecanoic acid, and/or combinations and mixtures thereof.
[0157] Additionally, the enriched lipid fraction and/or milk fat
globules may comprise, in some embodiments, lauric acid. Lauric
acid, also known as dodecanoic acid, is a saturated fatty acid with
a 12-carbon atom chain and is believed to be one of the main
antiviral and antibacterial substances currently found in human
breast milk. The milk fat globules may be enriched with
triglycerides containing lauric acid at the Sn-1, Sn-2 and/or Sn-3
positions. Without being bound by any particular theory, it is
believed that when the enriched lipid fraction is ingested, the
mouth lingual lipase and pancreatic lipase will hydrolyze the
triglycerides to a mixture of glycerides including mono-lauric and
free lauric acid.
[0158] The concentration of lauric acid in the globules varies from
80 mg/100 ml to 800 mg/100 ml. The concentration of monolauryl in
the globules can be in the range of 20 mg/100 ml to 300 mg/100 ml
feed. In some embodiments, the range is 60 mg/100 ml to 130 mg/100
ml.
[0159] The enriched lipid fraction and/or milk fat globules may
contain trans-fatty acids in certain embodiments. The trans-fatty
acids included in the milk fat globules may be monounsaturated or
polyunsaturated trans-fatty acids. In some embodiments the
trans-fatty acids may be present in an amount from about 0.2 g/100
Kcal to about 7.0 g/100 Kcal. In other embodiments the trans-fatty
acids may be present in an amount from about 3.4 g/100 Kcal to
about 5.2 g/100 Kcal. In yet other embodiments the trans-fatty
acids may be present from about 1.2 g/100 Kcal to about 4.3 g/100
Kcal.
[0160] Examples of trans-fatty acids for inclusion include, but are
not limited to, vaccenic, or elaidic acid, and mixtures thereof.
Moreover, when consumed, mammals convert vaccenic acid into rumenic
acid, which is a conjugated linoleic acid that exhibits
anticarcinogenic properties. Additionally, a diet enriched with
vaccenic acid may help lower total cholesterol, LDL cholesterol and
triglyceride levels
[0161] In some embodiments, the enriched lipid fraction and/or milk
fat globules may contain OBCFAs. In certain embodiments, the OBCFAs
may be present in an amount from about 0.3 g/100 Kcal to about 6.1
g/100 Kcal. In other embodiments OBCFAs may be present in an amount
from about 2.2 g/100 Kcal to about 4.3 g/100 Kcal. In yet another
embodiment OBCFAs may be present in an amount from about 3.5 g/100
Kcal to about 5.7 g/100 Kcal. In still other embodiments, the milk
fat globules comprise at least one OBCFA.
[0162] Typically, an infant may absorb OBCFAs while in utero and
from the breast milk of a nursing mother. Therefore, OBCFAs that
are identified in human milk are preferred for inclusion in the
milk fat globules of the nutritional composition. Addition of
OBCFAs to infant or children's formulas allows such formulas to
mirror the composition and functionality of human milk and to
promote general health and well-being.
[0163] In some embodiments, the enriched lipid fraction and/or milk
fat globules may comprise BCFAs. In some embodiments the BCFAs are
present at a concentration from about 0.2 g/100 Kcal and about 5.82
g/100 Kcal. In another embodiment, the BCFAs are present in an
amount of from about 2.3 g/100 Kcal to about 4.2 g/100 Kcal. In yet
another embodiment the BCFAs are present from about 4.2 g/100 Kcal
to about 5.82 g/100 Kcal. In still other embodiments, the milk fat
globules comprise at least one BCFA.
[0164] BCFAs that are identified in human milk are preferred for
inclusion in the nutritional composition. Addition of BCFAs to
infant or children's formulas allows such formulas to mirror the
composition and functionality of human milk and to promote general
health and well-being.
[0165] In certain embodiments, the enriched lipid fraction and/or
milk fat globules may comprise CLA. In some embodiments, CLA may be
present in a concentration from about 0.4 g/100 Kcal to about 2.5
g/100 Kcal. In other embodiments CLA may be present from about 0.8
g/100 Kcal to about 1.2 g/100 Kcal. In yet other embodiments CLA
may be present from about 1.2 g/100 Kcal to about 2.3 g/100 Kcal.
In still other embodiments, the milk fat globules comprise at least
one CLA.
[0166] CLAs that are identified in human milk are preferred for
inclusion in the nutritional composition. Typically, CLAs are
absorbed by the infant from the human milk of a nursing mother.
Addition of CLAs to infant or children's formulas allows such
formulas to mirror the composition and functionality of human milk
and to promote general health and wellbeing.
[0167] Examples of CLAs found in the milk fat globules for the
nutritional composition include, but are not limited to, cis-9,
trans-11 CLA, trans-10, cis-12 CLA, cis-9, trans-12 octadecadienoic
acid, and mixtures thereof.
[0168] The enriched lipid fraction and/or milk fat globules of the
present disclosure comprise monounsaturated fatty acids in some
embodiments. The enriched lipid fraction and/or milk fat globules
may be formulated to include monounsaturated fatty acids from about
0.8 g/100 Kcal to about 2.5 g/100 Kcal. In other embodiments the
milk fat globules may include monounsaturated fatty acids from
about 1.2 g/100 Kcal to about 1.8 g/100 Kcal.
[0169] Examples of monounsaturated fatty acids suitable include,
but are not limited to, palmitoleic acid, cis-vaccenic acid, oleic
acid, and mixtures thereof.
[0170] In certain embodiments, the enriched lipid fraction and/or
milk fat globules of the present disclosure comprise
polyunsaturated fatty acids from about 2.3 g/100 Kcal to about 4.4
g/100 Kcal. In other embodiments, the polyunsaturated fatty acids
are present from about 2.7 g/100 Kcal to about 3.5 g/100 Kcal. In
yet another embodiment, the polyunsaturated fatty acids are present
from about 2.4 g/100 Kcal to about 3.3 g/100 Kcal.
[0171] In some embodiments, the enriched lipid fraction and/or milk
fat globules of the present disclosure comprise polyunsaturated
fatty acids, such as, for example linoleic acid, linolenic acid,
octadecatrienoic acid, arachidonic acid (ARA), eicosatetraenoic
acid, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and
docosahexaenoic acid (DHA). Polyunsaturated fatty acids are the
precursors for prostaglandins and eicosanoids, which are known to
provide numerous health benefits, including, anti-inflammatory
response, cholesterol absorption, and increased bronchial
function.
[0172] The enriched lipid fraction and/or milk fat globules of the
present disclosure can also comprise cholesterol in some
embodiments, at a level of from about 100 mg/100 Kcal to about 400
mg/100 Kcal. In another embodiment, cholesterol is present from
about 200 mg/100 Kcal to about 300 mg/100 Kcal. As is similar to
human milk and bovine milk, the cholesterol included in the milk
fat globules may be present in the outer bilayer membrane of the
milk fat globule to provide stability to the globular membrane.
[0173] In some embodiments, the enriched lipid fraction and/or milk
fat globules of the present disclosure comprise phospholipids from
about 50 mg/100 Kcal to about 200 mg/100 Kcal. In other
embodiments, the phospholipids are present from about 75 mg/100
Kcal to about 150 mg/100 Kcal. In yet other embodiments, the
phospholipids are present at a concentration of from about 100
mg/100 Kcal to about 250 mg/100 Kcal.
[0174] In certain embodiments, phospholipids may be incorporated
into the milk fat globules to stabilize the milk fat globule by
providing a phospholipid membrane or bilayer phospholipid membrane.
Therefore, in some embodiments the milk fat globules may be
formulated with higher amounts of phospholipids than those found in
human milk.
[0175] The phospholipid composition of human milk lipids, as the
weight percent of total phospholipids, has been measured as
phosphatidylcholine ("PC") 24.9%, phosphatidylethanolamine ("PE")
27.7%, phosphatidylserine ("PS") 9.3%, phosphatidylinositol ("PI")
5.4%, and sphingomyelin ("SM") 32.4%, (Harzer, G. et al., Am. J.
Clin. Nutr., Vol. 37, pp. 612-621 (1983)). Thus in one embodiment,
the milk fat globules comprise one or more of PC, PE, PS, PI, SM,
and mixtures thereof. Further, the phospholipid composition
included in the milk fat globules may be formulated to provide
certain health benefits by incorporating desired phospholipids.
[0176] In certain embodiments, the enriched lipid fraction and/or
milk fat globules of the present disclosure comprise milk fat
globule membrane protein. In some embodiments, the milk fat globule
membrane protein is present from about 50 mg/100 Kcal to about 500
mg/100 Kcal.
[0177] Galactolipids may be included, in some embodiments, in the
enriched lipid fraction and/or milk fat globules of the present
disclosure. For purposes of this disclosure "galactolipids" refer
to any glycolipid whose sugar group is galactose. More
specifically, galactolipids differ from glycosphingolipids in that
they do not have nitrogen in their composition. Galactolipids play
an important role in supporting brain development and overall
neuronal health. Additionally, the galactolipids,
galactocerebroside and sulfatides constitute about 23% and 4% of
total myelin lipid content respectively, and thus may be
incorporated into the milk fat globules in some embodiments.
[0178] Additionally, the nutritional compositions of the present
disclosure comprise at least one source of pectin. The source of
pectin may comprise any variety or grade of pectin known in the
art. In some embodiments, the pectin has a degree of esterification
of less than 50% and is classified as low methylated ("LM") pectin.
In some embodiments, the pectin has a degree of esterification of
greater than or equal to 50% and is classified as high-ester or
high methylated ("HM") pectin. In still other embodiments, the
pectin is very low ("VL") pectin, which has a degree of
esterification that is less than approximately 15%. Further, the
nutritional composition of the present disclosure may comprise LM
pectin, HM pectin, VL pectin, or any mixture thereof. The
nutritional composition may include pectin that is soluble in
water. And, as known in the art, the solubility and viscosity of a
pectin solution are related to the molecular weight, degree of
esterification, concentration of the pectin preparation and the pH
and presence of counterions.
[0179] Moreover, pectin has a unique ability to form gels.
Generally, under similar conditions, a pectin's degree of gelation,
the gelling temperature, and the gel strength are proportional to
one another, and each is generally proportional to the molecular
weight of the pectin and inversely proportional to the degree of
esterification. For example, as the pH of a pectin solution is
lowered, ionization of the carboxylate groups is repressed, and, as
a result of losing their charge, saccharide molecules do not repel
each other over their entire length. Accordingly, the
polysaccharide molecules can associate over a portion of their
length to form a gel. Yet pectins with increasing degrees of
methylation will gel at somewhat higher pH because they have fewer
carboxylate anions at any given pH. (J. N. Bemiller, An
Introduction to Pectins: Structure and Properties, Chemistry and
Function of Pectins; Chapter 1; 1986.)
[0180] The nutritional composition may comprise a gelatinized
and/or pregelatinized starch together with pectin and/or
gelatinized pectin. While not wishing to be bound by any theory, it
is believed that the use of pectin, such as LM pectin, which is a
hydrocolloid of large molecular weight, together with starch
granules, provides a synergistic effect that increases the
molecular internal friction within a fluid matrix. The carboxylic
groups of the pectin may also interact with calcium ions present in
the nutritional composition, thus leading to an increase in
viscosity, as the carboxylic groups of the pectin form a weak gel
structure with the calcium ion(s), and also with peptides present
in the nutritional composition. In some embodiments, the
nutritional composition comprises a ratio of starch to pectin that
is between about 12:1 and 20:1, respectively. In other embodiments,
the ratio of starch to pectin is about 17:1. In some embodiments,
the nutritional composition may comprise between about 0.05 and
about 2.0% w/w pectin. In a particular embodiment, the nutritional
composition may comprise about 0.5% w/w pectin.
[0181] 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 10,000 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 having a molecular weight less
than that of intact or unmodified pectin. The hydrolyzed pectin of
the present disclosure can be prepared by any process known in the
art to reduce molecular weight. Examples include chemical
hydrolysis, enzymatic hydrolysis and mechanical shear. A preferred
method of reducing the molecular weight is by alkaline or neutral
hydrolysis at elevated temperature. In some embodiments, the
nutritional composition 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.
[0182] The nutritional composition may contain at least one acidic
polysaccharide. An acidic polysaccharide, such as negatively
charged pectin, may induce an anti-adhesive effect on pathogens in
a subject's gastrointestinal tract. Indeed, nonhuman milk acidic
oligosaccharides derived from pectin are able to interact with the
epithelial surface and are known to inhibit the adhesion of
pathogens on the epithelial surface
[0183] In some embodiments, the nutritional composition comprises
at least one pectin-derived acidic oligosaccharide. Pectin-derived
acidic oligosaccharide(s) (pAOS) result from enzymatic
pectinolysis, and the size of a pAOS depends on the enzyme use and
on the duration of the reaction. In such embodiments, the pAOS may
beneficially affect a subject's stool viscosity, stool frequency,
stool pH and/or feeding tolerance. The nutritional composition of
the present disclosure may comprise between about 1 g pAOS per
liter of nutritional composition and about 6 g pAOS per liter of
nutritional composition.
[0184] In some embodiments, the nutritional composition comprises
up to about 20% w/w of a mixture of starch and pectin. In some
embodiments, the nutritional composition comprises up to about 19%
starch and up to about 1% pectin. In other embodiments, the
nutritional composition comprises about up to about 15% starch and
up to about 5% pectin. In still other embodiments, the nutritional
composition comprises up to about 18% starch and up to about 2%
pectin. In some embodiments the nutritional composition comprises
between about 0.05% w/w and about 20% w/w of a mixture of starch
and pectin. Other embodiments include between about 0.05% and about
19% w/w starch and between about 0.05% and about 1% w/w pectin.
Further, the nutritional composition may comprise between about
0.05% and about 15% w/w starch and between about 0.05% and about 5%
w/w pectin.
[0185] In some embodiments the nutritional composition comprises
sialic acid. Sialic acids are a family of over 50 members of
9-carbon sugars, all of which are derivatives of neuraminic acid.
The predominant sialic acid family found in humans is from the
N-acetylneuraminic acid sub-family. Sialic acids are found in milk,
such as bovine and caprine. In mammals, neuronal cell membranes
have the highest concentration of sialic acid compared to other
body cell membranes. Sialic acid residues are also components of
gangliosides.
[0186] If included in the nutritional composition, sialic acid may
be present in an amount from about 0.5 mg/100 Kcal to about 45
mg/100 Kcal. In some embodiments sialic acid may be present in an
amount from about 5 mg/100 Kcal to about 30 mg/100 Kcal. In still
other embodiments, sialic acid may be present in an amount from
about 10 mg/100 Kcal to about 25 mg/100 Kcal.
[0187] In one embodiment, the nutritional composition may contain
one or more probiotics. Any probiotic known in the art may be
acceptable in this embodiment. In a particular embodiment, the
probiotic may be selected from any Lactobacillus species, such as
Lactobacillus rhamnosus GG (LGG) (ATCC number 53103),
Bifidobacterium species, such as Bifidobacterium longum BB536
(BL999, ATCC: BAA-999), Bifidobacterium longum AH1206 (NCIMB:
41382), Bifidobacterium breve AH1205 (NCIMB: 41387),
Bifidobacterium infantis 35624 (NCIMB: 41003), and Bifidobacterium
animalis subsp. lactis BB-12 (DSM No. 10140), or any combination
thereof.
[0188] If included in the composition, the amount of the probiotic
may vary from about 1.times.10.sup.4 to about 1.5.times.10.sup.12
cfu of probiotic(s) per 100 Kcal. In some embodiments the amount of
probiotic may be from about 1.times.10.sup.6 to about
1.times.10.sup.9 cfu of probiotic(s) per 100 Kcal. In certain other
embodiments the amount of probiotic may vary from about
1.times.10.sup.7 cfu/100 Kcal to about 1.times.10.sup.8 cfu of
probiotic(s) per 100 Kcal.
[0189] 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.
[0190] In some embodiments, the nutritional composition may include
a source comprising probiotic cell equivalents, which refers to the
level of non-viable, non-replicating probiotics equivalent to an
equal number of viable cells. The term "non-replicating"is to be
understood as the amount of non-replicating microorganisms obtained
from the same amount of replicating bacteria (cfu/g), including
inactivated probiotics, fragments of DNA, cell wall or cytoplasmic
compounds. In other words, the quantity of non-living,
non-replicating organisms is expressed in terms of cfu as if all
the microorganisms were alive, regardless whether they are dead,
non-replicating, inactivated, fragmented etc. In non-viable
probiotics are included in the nutritional composition, the amount
of the probiotic cell equivalents may vary from about
1.times.10.sup.4 to about 1.5.times.10.sup.10 cell equivalents of
probiotic(s) per 100 Kcal. In some embodiments the amount of
probiotic cell equivalents may be from about 1.times.10.sup.6 to
about 1.times.10.sup.9 cell equivalents of probiotic(s) per 100
Kcal nutritional composition. In certain other embodiments the
amount of probiotic cell equivalents may vary from about
1.times.10.sup.7 to about 1.times.10.sup.8 cell equivalents of
probiotic(s) per 100 Kcal of nutritional composition.
[0191] In some embodiments, the probiotic source incorporated into
the nutritional composition may comprise both viable colony-forming
units, and non-viable cell-equivalents.
[0192] In some embodiments, the nutritional composition includes a
culture supernatant from a late-exponential growth phase of a
probiotic batch-cultivation process. Without wishing to be bound by
theory, it is believed that the activity of the culture supernatant
can be attributed to the mixture of components (including
proteinaceous materials, and possibly including (exo)polysaccharide
materials) as found released into the culture medium at a late
stage of the exponential (or "log") phase of batch cultivation of
the probiotic. The term "culture supernatant" as used herein,
includes the mixture of components found in the culture medium. The
stages recognized in batch cultivation of bacteria are known to the
skilled person. These are the "lag," the "log" ("logarithmic" or
"exponential"), the "stationary" and the "death" (or "logarithmic
decline") phases. In all phases during which live bacteria are
present, the bacteria metabolize nutrients from the media, and
secrete (exert, release) materials into the culture medium. The
composition of the secreted material at a given point in time of
the growth stages is not generally predictable.
[0193] In an embodiment, a culture supernatant is obtainable by a
process comprising the steps of (a) subjecting a probiotic such as
LGG to cultivation in a suitable culture medium using a batch
process; (b) harvesting the culture supernatant at a late
exponential growth phase of the cultivation step, which phase is
defined with reference to the second half of the time between the
lag phase and the stationary phase of the batch-cultivation
process; (c) optionally removing low molecular weight constituents
from the supernatant so as to retain molecular weight constituents
above 5-6 kiloDaltons (kDa); (d) removing liquid contents from the
culture supernatant so as to obtain the composition.
[0194] The culture supernatant may comprise secreted materials that
are harvested from a late exponential phase. The late exponential
phase occurs in time after the mid exponential phase (which is
halftime of the duration of the exponential phase, hence the
reference to the late exponential phase as being the second half of
the time between the lag phase and the stationary phase). In
particular, the term "late exponential phase" is used herein with
reference to the latter quarter portion of the time between the lag
phase and the stationary phase of the LGG batch-cultivation
process. In some embodiments, the culture supernatant is harvested
at a point in time of 75% to 85% of the duration of the exponential
phase, and may be harvested at about 5/6 of the time elapsed in the
exponential phase.
[0195] As noted, the disclosed nutritional composition may comprise
a source of .beta.-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 (.beta.-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.
[0196] .beta.-1,3-glucans are carbohydrate polymers purified from,
for example, yeast, mushroom, bacteria, algae, or cereals. The
chemical structure of .beta.-1,3-glucan depends on the source of
the .beta.-1,3-glucan. Moreover, various physiochemical parameters,
such as solubility, primary structure, molecular weight, and
branching, play a role in biological activities of
.beta.-1,3-glucans.
[0197] .beta.-1,3-glucans are naturally occurring polysaccharides,
with or without .beta.-1,6-glucose side chains that are found in
the cell walls of a variety of plants, yeasts, fungi and bacteria.
.beta.-1,3;1,6-glucans are those containing glucose units with
(1,3) links having side chains attached at the (1,6) position(s).
.beta.-1,3;1,6 glucans are a heterogeneous group of glucose
polymers that share structural commonalities, including a backbone
of straight chain glucose units linked by a .beta.-1,3 bond with
.beta.-1,6-linked glucose branches extending from this backbone.
While this is the basic structure for the presently described class
of .beta.-glucans, some variations may exist. For example, certain
yeast .beta.-glucans have additional regions of .beta.(1,3)
branching extending from the .beta.(1,6) branches, which add
further complexity to their respective structures.
[0198] .beta.-glucans derived from baker's yeast, Saccharomyces
cerevisiae, are made up of chains of D-glucose molecules connected
at the 1 and 3 positions, having side chains of glucose attached at
the 1 and 6 positions. Yeast-derived .beta.-glucan is an insoluble,
fiber-like, complex sugar having the general structure of a linear
chain of glucose units with a .beta.-1,3 backbone interspersed with
.beta.-1,6 side chains that are generally 6-8 glucose units in
length. More specifically, .beta.-glucan derived from baker's yeast
is
poly-(1,6)-.beta.-D-glucopyranosyl-(1,3)-.beta.-D-glucopyranose.
[0199] Furthermore, .beta.-glucans are well tolerated and do not
produce or cause excess gas, abdominal distension, bloating or
diarrhea in pediatric subjects. Addition of .beta.-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.
[0200] In some embodiments, the amount of .beta.-glucan present in
the composition is at between about 0.010 and about 0.080 g per 100
g of composition. In other embodiments, the nutritional composition
comprises between about 10 and about 30 mg .beta.-glucan per
serving. In another embodiment, the nutritional composition
comprises between about 5 and about 30 mg .beta.-glucan per 8 fl.
oz. (236.6 mL) serving. In other embodiments, the nutritional
composition comprises an amount of .beta.-glucan sufficient to
provide between about 15 mg and about 90 mg .beta.-glucan per day.
The nutritional composition may be delivered in multiple doses to
reach a target amount of .beta.-glucan delivered to the subject
throughout the day.
[0201] In some embodiments, the amount of .beta.-glucan in the
nutritional composition is between about 3 mg and about 17 mg per
100 Kcal. In another embodiment the amount of .beta.-glucan is
between about 6 mg and about 17 mg per 100 Kcal.
[0202] Addition of .beta.-glucan to a nutritional composition for a
pediatric subject, such as an infant formula, a growing-up milk or
another children's nutritional product, can improve the pediatric
subject's immune response by increasing resistance against invading
pathogens and therefore maintaining or improving overall
health.
[0203] 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.
[0204] The nutritional composition may optionally include, but is
not limited to, one or more of the following vitamins or
derivations thereof: vitamin B.sub.1 (thiamin, thiamin
pyrophosphate, TPP, thiamin triphosphate, TTP, thiamin
hydrochloride, thiamin mononitrate), vitamin B.sub.2 (riboflavin,
flavin mononucleotide, FMN, flavin adenine dinucleotide, FAD,
lactoflavin, ovoflavin), vitamin B.sub.3 (niacin, nicotinic acid,
nicotinamide, niacinamide, nicotinamide adenine dinucleotide, NAD,
nicotinic acid mononucleotide, NicMN, pyridine-3-carboxylic acid),
vitamin B.sub.3-precursor tryptophan, vitamin B.sub.6 (pyridoxine,
pyridoxal, pyridoxamine, pyridoxine hydrochloride), pantothenic
acid (pantothenate, panthenol), folate (folic acid, folacin,
pteroylglutamic acid), vitamin B.sub.12 (cobalamin,
methylcobalamin, deoxyadenosylcobalamin, cyanocobalamin,
hydroxocobalamin, 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 D.sub.3, 1,25,-dihydroxyvitamin D), vitamin E
(a-tocopherol, a-tocopherol acetate, a-tocopherol succinate,
a-tocopherol nicotinate, a-tocopherol), vitamin K (vitamin K.sub.1,
phylloquinone, naphthoquinone, vitamin K.sub.2, menaquinone-7,
vitamin K.sub.3, menaquinone-4, menadione, menaquinone-8,
menaquinone-8H, menaquinone-9, menaquinone-9H, menaquinone-10,
menaquinone-11, menaquinone-12, menaquinone-13), choline, inositol,
.beta.-carotene and any combinations thereof.
[0205] 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
picolinate, 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.
[0206] 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.
[0207] In an embodiment, the nutritional composition may contain
between about 10 and about 50% of the maximum dietary
recommendation for any given country, or between about 10 and about
50% of the average dietary recommendation for a group of countries,
per serving of vitamins A, C, and E, zinc, iron, iodine, selenium,
and choline. In another embodiment, the children's nutritional
composition may supply about 10-30% of the maximum dietary
recommendation for any given country, or about 10-30% of the
average dietary recommendation for a group of countries, per
serving of B-vitamins. In yet another embodiment, the levels of
vitamin D, calcium, magnesium, phosphorus, and potassium in the
children's nutritional product may correspond with the average
levels found in milk. In other embodiments, other nutrients in the
children's nutritional composition may be present at about 20% of
the maximum dietary recommendation for any given country, or about
20% of the average dietary recommendation for a group of countries,
per serving.
[0208] The nutritional compositions 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 is known in the
art.
[0209] The nutritional compositions 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.
[0210] The nutritional compositions 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, potassium citrate, calcium disodium
EDTA, and mixtures thereof. The incorporation of a preservative in
the nutritional composition including HMO ensures that the
nutritional composition has a suitable shelf-life such that, once
reconstituted for administration, the nutritional composition
delivers nutrients that are bioavailable and/or provide health and
nutrition benefits for the target subject.
[0211] In some embodiments the nutritional composition may be
formulated to include from about 0.1 wt % to about 1.0 wt % of a
preservative based on the total dry weight of the composition. In
other embodiments, the nutritional composition may be formulated to
include from about 0.4 wt % to about 0.7 wt % of a preservative
based on the total dry weight of the composition.
[0212] In some embodiments where the nutritional composition is a
ready-to-use liquid composition, the nutritional composition may be
formulated to include from about 0.5 g/L to about 5 g/L of
preservative. Still, in certain embodiments, the nutritional
composition may include from about 1 g/L to about 3 g/L of
preservative.
[0213] The nutritional compositions 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, carrageenan, and mixtures thereof.
[0214] The disclosed nutritional composition(s) may be provided in
any form known in the art, such as a powder, a gel, a suspension, a
paste, a solid, a liquid, a liquid concentrate, a reconstitutable
powdered milk substitute or a ready-to-use product. The nutritional
composition may, in certain embodiments, comprise a nutritional
supplement, children's nutritional product, infant formula, human
milk fortifier, growing-up milk or any other nutritional
composition designed for an infant or a pediatric subject.
Nutritional compositions of the present disclosure include, for
example, orally-ingestible, health-promoting substances including,
for example, foods, beverages, tablets, capsules and powders.
Moreover, 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. In some embodiments, the nutritional composition is in powder
form with a particle size in the range of 5 .mu.m to 1500 .mu.m,
more preferably in the range of 10 .mu.m to 300 .mu.m.
[0215] If the nutritional composition is in the form of a
ready-to-use product, the osmolality of the nutritional composition
may be between about 100 and about 1100 mOsm/kg water, more
typically about 200 to about 700 mOsm/kg water.
[0216] 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 and/or
starch components described hereinabove. Phytonutrients, or their
derivatives, conjugated forms or precursors, that are identified in
human milk are preferred for inclusion in the nutritional
composition. Typically, dietary sources of carotenoids and
polyphenols are absorbed by a nursing mother and retained in milk,
making them available to nursing infants. Addition of these
phytonutrients to infant or children's formulas allows such
formulas to mirror the composition and functionality of human milk
and to promote general health and well-being.
[0217] For example, in some embodiments, the nutritional
composition of the present disclosure may comprise, in an 8 fl. oz.
(236.6 mL) serving, 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).
[0218] For the purposes of this disclosure, additional
phytonutrients may be added to a nutritional composition in native,
purified, encapsulated and/or chemically or enzymatically-modified
form so as to deliver the desired sensory and stability properties.
In the case of encapsulation, it is desirable that the encapsulated
phytonutrients resist dissolution with water but are released upon
reaching the small intestine. This could be achieved by the
application of enteric coatings, such as cross-linked alginate and
others.
[0219] 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.), flavanones, 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.
[0220] Some amounts of phytonutrients may be inherently present in
known ingredients, such as natural oils, that are commonly used to
make nutritional compositions for pediatric subjects. These
inherent phytonutrient(s) may be but are not necessarily considered
part of the phytonutrient component described in the present
disclosure. In some embodiments, the phytonutrient concentrations
and ratios as described herein are calculated based upon added and
inherent phytonutrient sources. In other embodiments, the
phytonutrient concentrations and ratios as described herein are
calculated based only upon added phytonutrient sources.
[0221] In some embodiments, the nutritional composition comprises
anthocyanins, such as, for example, glucosides of aurantinidin,
cyanidin, delphinidin, europinidin, luteolinidin, pelargonidin,
malvidin, peonidin, petunidin, and rosinidin. These and other
anthocyanins suitable for use in the nutritional composition are
found in a variety of plant sources. Anthocyanins may be derived
from a single plant source or a combination of plant sources.
Non-limiting examples of plants rich in anthocyanins suitable for
use in the inventive composition include: berries (acai, grape,
bilberry, blueberry, lingonberry, black currant, chokeberry,
blackberry, raspberry, cherry, red currant, cranberry, crowberry,
cloudberry, whortleberry, rowanberry), purple corn, purple potato,
purple carrot, red sweet potato, red cabbage, eggplant.
[0222] In some embodiments, the nutritional composition of the
present disclosure comprises proanthocyanidins, which include but
are not limited to flavan-3-ols and polymers of flavan-3-ols (e.g.,
catechins, epicatechins) with degrees of polymerization in the
range of 2 to 11. Such compounds may be derived from a single plant
source or a combination of plant sources. Non-limiting examples of
plant sources rich in proanthocyanidins suitable for use in the
disclosed nutritional composition include: grape, grape skin, grape
seed, green tea, black tea, apple, pine bark, cinnamon, cocoa,
bilberry, cranberry, black currant chokeberry.
[0223] Non-limiting examples of flavan-3-ols which are suitable for
use in the disclosed nutritional composition include catechin,
epicatechin, gallocatechin, epigallocatechin, epicatechin gallate,
epicatechin-3-gallate, epigallocatechin and gallate. Plants rich in
the suitable flavan-3-ols include, but are not limited to, teas,
red grapes, cocoa, green tea, apricot and apple.
[0224] Certain polyphenol compounds, in particular flavan-3-ols,
may improve learning and memory in a human subject by increasing
brain blood flow, which is associated with an increase and
sustained brain energy/nutrient delivery as well as formation of
new neurons. Polyphenols may also provide neuroprotective actions
and may increase both brain synaptogenesis and antioxidant
capability, thereby supporting optimal brain development in younger
children
[0225] Preferred sources of flavan-3-ols for the nutritional
composition include at least one apple extract, at least one grape
seed extract or a mixture thereof. For apple extracts, flavan-3-ols
are broken down into monomers occurring in the range 4% to 20% and
polymers in the range 80% to 96%. For grape seed extracts
flavan-3-ols are broken down into monomers (about 46%) and polymers
(about 54%) of the total favan-3-ols and total polyphenolic
content. Preferred degree of polymerization of polymeric
flavan-3-ols is in the range of between about 2 and 11.
Furthermore, apple and grape seed extracts may contain catechin,
epicatechin, epigallocatechin, epicatechin gallate,
epigallocatechin gallate, polymeric proanthocyanidins, stilbenoids
(i.e. resveratrol), flavonols (i.e. quercetin, myricetin), or any
mixture thereof. Plant sources rich in flavan-3-ols include, but
are not limited to apple, grape seed, grape, grape skin, tea (green
or black), pine bark, cinnamon, cocoa, bilberry, cranberry, black
currant, chokeberry.
[0226] If the nutritional composition is administered to a
pediatric subject, an amount of flavan-3-ols, including monomeric
flavan-3-ols, polymeric flavan-3-ols or a combination thereof,
ranging from between about 0.01 mg and about 450 mg per day may be
administered. In some cases, the amount of flavan-3-ols
administered to an infant or child may range from about 0.01 mg to
about 170 mg per day, from about 50 to about 450 mg per day, or
from about 100 mg to about 300 mg per day.
[0227] In an embodiment of the disclosure, flavan-3-ols are present
in the nutritional composition in an amount ranging from about 0.4
to about 3.8 mg/g nutritional composition (about 9 to about 90
mg/100 Kcal). In another embodiment, flavan-3-ols are present in an
amount ranging from about 0.8 to about 2.5 mg/g nutritional
composition (about 20 to about 60 mg/100 Kcal).
[0228] In some embodiments, the nutritional composition of the
present disclosure comprises flavanones. Non-limiting examples of
suitable flavanones include butin, eriodictyol, hesperetin,
hesperidin, homeriodictyol, isosakuranetin, naringenin, naringin,
pinocembrin, poncirin, sakuranetin, sakuranin, steurbin. Plant
sources rich in flavanones include, but are not limited to orange,
tangerine, grapefruit, lemon, lime. The nutritional composition may
be formulated to deliver between about 0.01 and about 150 mg
flavanones per day.
[0229] Moreover, the nutritional composition may also comprise
flavonols. Flavonols from plant or algae extracts may be used.
Flavonols, such as isorhamnetin, kaempferol, myricetin, quercetin,
may be included in the nutritional composition in amounts
sufficient to deliver between about 0.01 and 150 mg per day to a
subject.
[0230] The phytonutrient component of the nutritional composition
may also comprise phytonutrients that have been identified in human
milk, including but not limited to 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.
[0231] In certain embodiments, the nutritional composition
comprises carotenoids, such as lutein, zeaxanthin, astaxanthin,
lycopene, beta-carotene, alpha-carotene, gamma-carotene, and/or
beta-cryptoxanthin. Plant sources rich in carotenoids include, but
are not limited to kiwi, grapes, citrus, tomatoes, watermelons,
papayas and other red fruits, or dark greens, such as kale,
spinach, turnip greens, collard greens, romaine lettuce, broccoli,
zucchini, garden peas and Brussels sprouts, spinach, carrots.
[0232] Humans cannot synthesize carotenoids, but over 34
carotenoids have been identified in human breast milk, including
isomers and metabolites of certain carotenoids. In addition to
their presence in breast milk, dietary carotenoids, such as alpha
and beta-carotene, lycopene, lutein, zeaxanthin, astaxanthin, and
cryptoxanthin are present in serum of lactating women and breastfed
infants. Carotenoids in general have been reported to improve
cell-to-cell communication, promote immune function, support
healthy respiratory health, protect skin from UV light damage, and
have been linked to reduced risk of certain types of cancer, and
all-cause mortality. Furthermore, dietary sources of carotenoids
and/or polyphenols are absorbed by human subjects, accumulated and
retained in breast milk, making them available to nursing infants.
Thus, addition of phytonutrients to infant formulas or children's
products would bring the formulas closer in composition and
functionality to human milk.
[0233] Flavonoids, as a whole, may also be included in the
nutritional composition, as flavonoids cannot be synthesized by
humans. Moreover, flavonoids from plant or algae extracts may be
useful in the monomer, dimer and/or polymer forms. In some
embodiments, the nutritional composition comprises levels of the
monomeric forms of flavonoids similar to those in human milk during
the first three months of lactation. Although flavonoid aglycones
(monomers) have been identified in human milk samples, the
conjugated forms of flavonoids and/or their metabolites may also be
useful in the nutritional composition. The flavonoids could be
added in the following forms: free, glucuronides, methyl
glucuronides, sulphates, and methyl sulphates.
[0234] 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-O-methylgenistein, pratensein, prunetin, psi-tectorigenin,
retusin, tectorigenin, iridin, ononin, puerarin, tectoridin,
derrubone, luteone, wighteone, alpinumisoflavone, barbigerone,
di-O-methylalpinumisoflavone, and 4'-methyl-alpinumisoflavone.
Plant sources rich in isoflavonoids, include, but are not limited
to, soybeans, psoralea, kudzu, lupine, fava, chickpea, alfalfa,
legumes and peanuts. The nutritional composition may be formulated
to deliver between about 0.01 and about 150 mg isoflavones and/or
isoflavonoids per day.
[0235] In an embodiment, the nutritional composition(s) of the
present disclosure comprises an effective amount of choline.
Choline is a nutrient that is essential for normal function of
cells. It is a precursor for membrane phospholipids, and it
accelerates the synthesis and release of acetylcholine, a
neurotransmitter involved in memory storage. Moreover, though not
wishing to be bound by this or any other theory, it is believed
that dietary choline and docosahexaenoic acid (DHA) act
synergistically to promote the biosynthesis of phosphatidylcholine
and thus help promote synaptogenesis in human subjects.
Additionally, choline and DHA may exhibit the synergistic effect of
promoting dendritic spine formation, which is important in the
maintenance of established synaptic connections. In some
embodiments, the nutritional composition(s) of the present
disclosure includes an effective amount of choline, which is about
20 mg choline per 8 fl. oz. (236.6 mL) serving to about 100 mg per
8 fl. oz. (236.6 mL) serving.
[0236] Moreover, in some embodiments, the nutritional composition
is nutritionally complete, containing suitable types and amounts of
lipids, carbohydrates, proteins, vitamins and minerals to be a
subject's sole source of nutrition. Indeed, the nutritional
composition may optionally include any number of proteins,
peptides, amino acids, fatty acids, probiotics and/or their
metabolic by-products, prebiotics, carbohydrates and any other
nutrient or other compound that may provide many nutritional and
physiological benefits to a subject. Further, the nutritional
composition of the present disclosure may comprise flavors, flavor
enhancers, sweeteners, pigments, vitamins, minerals, therapeutic
ingredients, functional food ingredients, food ingredients,
processing ingredients or combinations thereof.
[0237] The following examples describe embodiments of the present
disclosure. Other embodiments within the scope of the claims herein
will be apparent to one skilled in the art from consideration of
the specification or practice of the disclosed methods as disclosed
herein. It is intended that the specification, together with the
examples, be considered to be exemplary only, with the scope and
spirit of the disclosure being indicated by the claims which follow
the examples. In the examples, all percentages are given on a
weight basis unless otherwise indicated.
Example 1
[0238] The following example demonstrates that eHC, ARA/DHA and
their combination is capable of inducing adipocyte browning and
reducing metabolic disturbances later in life. eHC is denoted as
"casein" in the figures.
Ucp-1 Luciferase Knock-in Mouse Model
[0239] UCP1 is a key molecule in browning, and increased expression
is a measure for browning induction. In Ucp-1 luciferase knock-in
reporter mice, in vivo imaging of luciferase activity directly
reflects the UCP1 protein level in vivo. In this study, mice were
fed normal chow, and supplementation with ARA/DHA, eHC and their
combination began post-weaning at 4 weeks of age and continued for
8 weeks. At 12 weeks of age, animals were switched to a high fat
diet (HFD, including test ingredients ARA/DHA and/or eHC), and
feeding was continued for another 12 weeks. Browning activity (as
illustrated by luciferase signal) at different adipose tissue
depots and browning relevant marker genes were analyzed at
different time points. Other parameters included blood
biochemistry, glucose tolerance, inflammatory cytokines, as well as
adipocyte morphology and adipose tissue inflammation.
Results
Prior to HFD Feeding
[0240] Even in the absence of a high-fat diet, markers of browning
were upregulated by administration of ARA/DHA, eHC ("casein") and
the combination ("CAD"), compared to animals fed normal control
diet. As shown in FIG. 1A and FIG. 1C-E, animals fed ARA/DHA, eHC
("casein") and the combination (CAD) showed upregulation of UCP1 in
the dorsum, chest and neck and abdomen, as shown by increased
luciferase levels, compared to the animals fed normal control diet
(CON). Harmine (HAR) is an alkaloid natural product having browning
activity and is provided as a positive control. As shown in FIG.
2A-B animals fed ARA/DHA, eHC and the combination (CAD) showed
upregulation of UCP1 in both iBAT and iWAT, as measured by
luciferase expression, compared to the animals fed normal control
diet.
[0241] Further, administration of ARA/DHA, eHC and the combination
(CAD) resulted in reduced body weight gains and decreased tissue
weight of epididymal white adipose tissue (eWAT) and liver (data
not shown), better glucose tolerance and enhanced insulin
sensitivity compared to the animals fed normal control diet (see,
FIG. 6A-B).
[0242] These results suggest that supplementing ARA/DHA and/or eHC
led to significant browning induction, even in the absence of a
high-fat diet.
After HFD Feeding
[0243] After administration of a high-fat diet, markers of browning
were upregulated by administration of ARA/DHA, eHC and the
combination (CAD), compared to animals fed normal control diet. As
shown in FIG. 1B and FIG. 1F-H, animals fed ARA/DHA, eHC and the
combination (CAD) showed upregulation of UCP1 in the dorsum, chest
and neck and abdomen, as shown by increased luciferase levels,
compared to the animals fed normal control diet (CON). As shown in
FIG. 2C-E, animals fed ARA/DHA, eHC and the combination (CAD)
showed upregulation of UCP1 in both iBAT, iWAT and eWAT (with the
exception of eHC (casein) in eWAT), as measured by luciferase
expression, compared to the animals fed normal control diet. RNA
(FIG. 3A-C) quantitation, and Western blot (FIG. 3D, E) and tissue
staining (FIG. 3F) for UCP1 protein was consistent with the results
using the luciferase marker.
[0244] Further, as shown in FIG. 3A-C, UCP1 mRNA was upregulated in
iBAT, iWAT, and eWAT in animals fed ARA/DHA, eHC and the
combination (CAD).
[0245] In addition, following administration of ARA/DHA, eHC and
the combination (CAD), expression of other browning markers, PRDM16
and PGC1a, was increased in BAT and WAT depots, as shown in FIG.
4.
[0246] Administration of ARA/DHA, eHC and the combination (CAD)
also decreased fasting insulin level and improved glucose tolerance
and enhanced insulin sensitivity significantly. A glucose tolerance
test was performed through intraperitoneal injection of 10% glucose
at a dose of 1 g/kg body weight into mice after 12 h fasting. Blood
glucose was monitored from the tail vein blood using a glucometer
(ACCU-CHEK Advantage; Roche Diagnostics China, Shanghai, China) at
0, 15, 30, 60, and 120 min time points. As shown in FIG. 5A and
FIG. 5C, administration of ARA/DHA, eHC and the combination (CAD)
resulted in increased glucose tolerance both before and after a
high fat diet was administered.
[0247] An insulin tolerance test was performed through
intraperitoneal injection of 0.5 U/kg body weight recombinant human
insulin (Sigma) into mice after 6 h fasting, and blood glucose
levels were measured at 0, 15, 30, 60, and 120 min later. As shown
in FIG. 5B and FIG. 5D, administration of ARA/DHA, eHC and the
combination (CAD) resulted in increased insulin sensitivity both
before and after a high fat diet was administered.
[0248] Moreover, other plasma levels of risk factors for metabolic
syndrome including total plasma cholesterol, total triglycerides,
free fatty acids, ALT and AST were assessed. Serum concentrations
of total cholesterol (TC), triglycerides (TG), alanine
aminotransferase (ALT), aspartate aminotransferase (AST), free
fatty acid (FFA) were measured using COD-PAP and GPO-PAP methods
with automatic analyzer BAYER ADVIA-2400. As shown in FIG. 6, the
results revealed decreased levels in intervention groups compared
with HFD control group.
[0249] Plasma insulin, IL1.beta. and TNF-a concentrations were
determined by ELISA (R&D). Adiponectin, resistin, leptin, FGF21
levels were determined using ELISA. Plasma adiponectin level was
significantly increased in the intervention groups compared with
HFD group, and resistin and FGF21 levels were decreased, consistent
with the effects seen on insulin sensitivity. (FIG. 7.)
[0250] Furthermore, the intervention with ARA/DHA, eHC and the
combination showed strong inflammatory inhibiting effects. Plasma
IL-1.beta. and TNF-a and the relative expression of F4/80 (showing
macrophage infiltration), TNF-a, IL-1.beta. and IL-6 in fat tissues
were significantly decreased in the intervention groups compared
with the HFD group (FIG. 8).
[0251] In addition, body weight gain, white adipose tissue and
liver weight in the three invention groups was significantly lower
than in the HFD control group (data not shown).
[0252] Taken together, these results demonstrate that ARA/DHA, eHC
and the combination thereof induces adipose tissue browning,
increases metabolic flexibility, including improved glucose
tolerance and enhanced insulin sensitivity, and reduces detrimental
WAT deposition and WAT dysfunction.
Example 2
[0253] Table 3 provides an exemplary embodiment of a nutritional
composition according to the present disclosure and describes the
amount of each ingredient to be included per 100 Kcal serving.
TABLE-US-00003 TABLE 3 Nutrition profile of an example nutritional
composition per 100 Kcal Nutrient Minimum Maximum eHC (g) 1.0 7.0
Carbohydrates (g) 6 22 Fat (g) 1.3 7.2 Prebiotic (g) 0.3 1.2 DHA
(g) 4 22 Beta glucan (mg) 2.9 17 Probiotics (cfu) 0.5 5.0 Vitamin A
(IU) 9.60 .times. 10.sup.5 3.80 .times. 10.sup.8 Vitamin D (IU) 134
921 Vitamin E (IU) 22 126 Vitamin K (mcg) 0.8 5.4 Thiamin (mcg) 2.9
18 Riboflavin (mcg) 63 328 Vitamin B6 (mcg) 68 420 Vitamin B12
(mcg) 52 397 Niacin (mcg) 0.2 0.9 Folic acid (mcg) 690 5881
Panthothenic acid (mcg) 8 66 Biotin (mcg) 232 1211 Vitamin C (mg)
1.4 5.5 Choline (mg) 4.9 24 Calcium (mg) 4.9 43 Phosphorus (mg) 68
297 Magnesium (mg) 54 210 Sodium (mg) 4.9 34 Potassium (mg) 24 88
Chloride (mg) 82 346 Iodine (mcg) 53 237 Iron (mg) 8.9 79 Zinc (mg)
0.7 2.8 Manganese (mcg) 0.7 2.4 Copper (mcg) 7.2 41
[0254] 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 in whole or in part.
Therefore, the spirit and scope of the appended claims should not
be limited to the description of the preferred versions contained
therein.
[0255] 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.
Sequence CWU 1
1
6418PRTBovine 1Ala Ile Asn Pro Ser Lys Glu Asn 1 5 25PRTBOVINE 2Ala
Pro Phe Pro Glu 1 5 36PRTBOVINE 3Asp Ile Gly Ser Glu Ser 1 5
47PRTBOVINE 4Asp Lys Thr Glu Ile Pro Thr 1 5 55PRTBOVINE 5Asp Met
Glu Ser Thr 1 5 64PRTBOVINE 6Asp Met Pro Ile 1 74PRTBOVINE 7Asp Val
Pro Ser 1 87PRTBOVINE 8Glu Thr Ala Pro Val Pro Leu 1 5 96PRTBOVINE
9Phe Pro Gly Pro Ile Pro 1 5 107PRTBOVINE 10Phe Pro Gly Pro Ile Pro
Asn 1 5 114PRTBOVINE 11Gly Pro Phe Pro 1 124PRTBOVINE 12Gly Pro Ile
Val 1 139PRTBOVINE 13Ile Gly Ser Glu Ser Thr Glu Asp Gln 1 5
148PRTBOVINE 14Ile Gly Ser Ser Ser Glu Glu Ser 1 5 159PRTBOVINE
15Ile Gly Ser Ser Ser Glu Glu Ser Ala 1 5 166PRTBOVINE 16Ile Asn
Pro Ser Lys Glu 1 5 175PRTBOVINE 17Ile Pro Asn Pro Ile 1 5
186PRTBOVINE 18Ile Pro Asn Pro Ile Gly 1 5 199PRTBOVINE 19Ile Pro
Pro Leu Thr Gln Thr Pro Val 1 5 204PRTBOVINE 20Ile Thr Ala Pro 1
214PRTBOVINE 21Ile Val Pro Asn 1 227PRTBOVINE 22Lys His Gln Gly Leu
Pro Gln 1 5 235PRTBOVINE 23Leu Asp Val Thr Pro 1 5 246PRTBOVINE
24Leu Glu Asp Ser Pro Glu 1 5 255PRTBOVINE 25Leu Pro Leu Pro Leu 1
5 266PRTBOVINE 26Met Glu Ser Thr Glu Val 1 5 2711PRTBOVINE 27Met
His Gln Pro His Gln Pro Leu Pro Pro Thr 1 5 10 285PRTBOVINE 28Asn
Ala Val Pro Ile 1 5 295PRTBOVINE 29Asn Glu Val Glu Ala 1 5
306PRTBOVINE 30Asn Gln Glu Gln Pro Ile 1 5 315PRTBOVINE 31Asn Val
Pro Gly Glu 1 5 326PRTBOVINE 32Pro Phe Pro Gly Pro Ile 1 5
336PRTBOVINE 33Pro Gly Pro Ile Pro Asn 1 5 348PRTBOVINE 34Pro His
Gln Pro Leu Pro Pro Thr 1 5 355PRTBOVINE 35Pro Ile Thr Pro Thr 1 5
364PRTBOVINE 36Pro Asn Pro Ile 1 376PRTBOVINE 37Pro Asn Ser Leu Pro
Gln 1 5 388PRTBOVINE 38Pro Gln Leu Glu Ile Val Pro Asn 1 5
397PRTBOVINE 39Pro Gln Asn Ile Pro Pro Leu 1 5 406PRTBOVINE 40Pro
Val Leu Gly Pro Val 1 5 414PRTBOVINE 41Pro Val Pro Gln 1
425PRTBOVINE 42Pro Val Val Val Pro 1 5 436PRTBOVINE 43Pro Val Val
Val Pro Pro 1 5 4411PRTBOVINE 44Ser Ile Gly Ser Ser Ser Glu Glu Ser
Ala Glu 1 5 10 457PRTBOVINE 45Ser Ile Ser Ser Ser Glu Glu 1 5
4611PRTBOVINE 46Ser Ile Ser Ser Ser Glu Glu Ile Val Pro Asn 1 5 10
477PRTBOVINE 47Ser Lys Asp Ile Gly Ser Glu 1 5 486PRTBOVINE 48Ser
Pro Pro Glu Ile Asn 1 5 497PRTBOVINE 49Ser Pro Pro Glu Ile Asn Thr
1 5 507PRTBOVINE 50Thr Asp Ala Pro Ser Phe Ser 1 5 515PRTBOVINE
51Thr Glu Asp Glu Leu 1 5 526PRTBOVINE 52Val Ala Thr Glu Glu Val 1
5 535PRTBOVINE 53Val Leu Pro Val Pro 1 5 544PRTBOVINE 54Val Pro Gly
Glu 1 556PRTBOVINE 55Val Pro Gly Glu Ile Val 1 5 566PRTBOVINE 56Val
Pro Ile Thr Pro Thr 1 5 574PRTBOVINE 57Val Pro Ser Glu 1
589PRTBOVINE 58Val Val Pro Pro Phe Leu Gln Pro Glu 1 5 595PRTBOVINE
59Val Val Val Pro Pro 1 5 606PRTBOVINE 60Tyr Pro Phe Pro Gly Pro 1
5 618PRTBOVINE 61Tyr Pro Phe Pro Gly Pro Ile Pro 1 5 629PRTBOVINE
62Tyr Pro Phe Pro Gly Pro Ile Pro Asn 1 5 635PRTBOVINE 63Tyr Pro
Ser Gly Ala 1 5 645PRTBOVINE 64Tyr Pro Val Glu Pro 1 5
* * * * *