U.S. patent application number 14/981337 was filed with the patent office on 2016-05-26 for leucine-rich peptide compositions and methods for isolation.
This patent application is currently assigned to Glanbia Nutritionals (Ireland) Ltd.. The applicant listed for this patent is Eric D. Bastian, Brent Petersen, Loren S. Ward, Stanley Wrobel. Invention is credited to Eric D. Bastian, Brent Petersen, Loren S. Ward, Stanley Wrobel.
Application Number | 20160143983 14/981337 |
Document ID | / |
Family ID | 41114702 |
Filed Date | 2016-05-26 |
United States Patent
Application |
20160143983 |
Kind Code |
A1 |
Petersen; Brent ; et
al. |
May 26, 2016 |
Leucine-Rich Peptide Compositions and Methods for Isolation
Abstract
Disclosed are compositions comprising isolated peptides having a
leucine content of from about 12 to about 40 weight percent. Also
disclosed is a method for isolating leucine-rich peptides from
protein sources such as bovine whey and methods of use for these
peptides to provide beneficial effects in a human and/or animal
such as increasing blood flow, decreasing blood pressure,
increasing muscle mass, improving cognitive function, improving
cardiovascular function, etc.
Inventors: |
Petersen; Brent; (Twin
Falls, ID) ; Ward; Loren S.; (Twin Falls, ID)
; Bastian; Eric D.; (Twin Falls, ID) ; Wrobel;
Stanley; (Simpsonville, SC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Petersen; Brent
Ward; Loren S.
Bastian; Eric D.
Wrobel; Stanley |
Twin Falls
Twin Falls
Twin Falls
Simpsonville |
ID
ID
ID
SC |
US
US
US
US |
|
|
Assignee: |
Glanbia Nutritionals (Ireland)
Ltd.
Kilkenny
IE
|
Family ID: |
41114702 |
Appl. No.: |
14/981337 |
Filed: |
December 28, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14188676 |
Feb 24, 2014 |
9222117 |
|
|
14981337 |
|
|
|
|
Current U.S.
Class: |
514/15.6 ;
514/16.3 |
Current CPC
Class: |
C12P 21/06 20130101;
A61P 7/00 20180101; A61P 9/00 20180101; A23L 33/18 20160801; A23V
2002/00 20130101; A61P 43/00 20180101; A61P 3/02 20180101; A23L
33/175 20160801; A61K 31/198 20130101; A61P 9/08 20180101; A61K
38/018 20130101; A61P 9/12 20180101; A61P 9/10 20180101 |
International
Class: |
A61K 38/01 20060101
A61K038/01; A61K 31/198 20060101 A61K031/198 |
Claims
1. A peptide composition comprising peptides having a leucine
content of from about 12 to about 40 weight percent and a molecular
weight from about 200 to about 4,000, the peptides having been
isolated from a protein source by a method comprising the steps of
a) hydrolyzing the protein in the presence of at least one leucyl
aminopeptidase without additional proteases that are not leucyl
aminopeptidases to form a protein hydrolysate; b) deactivating the
aminopeptidase(s); and c) filtering the protein hydrolysate to
produce a permeate containing leucine-rich peptides having a
leucine content of from about 12 to about 40 weight percent and a
molecular weight of from about 200 to about 4,000 in the
permeate.
2. A peptide composition as in claim 1, wherein the method
comprises the additional step of concentrating the peptides by
removing aqueous fluid from the permeate.
3. The peptide composition of claim 1 wherein the protein source is
chosen from among the group consisting of legumes, fish, meat,
milk, blood, egg, corn, wheat gluten, maize, and combinations
thereof.
4. The peptide composition of claim 1 wherein the protein source is
chosen from among the group consisting of bovine milk, bovine whey,
whey protein concentrates, whey protein isolates, and combinations
thereof.
5. The peptide composition of claim 1 wherein the peptides are
concentrated by removing aqueous fluid from the permeate.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application from U.S.
patent application Ser. No. 14/188,676, filed Feb. 24, 2014, which
is a divisional of, and claims the benefit of priority of, U.S.
patent application Ser. No. 12/411,772, filed Mar. 26, 2009, which
claimed the benefit of priority of earlier-filed U.S. provisional
patent application No. 61/039,426, filed Mar. 26, 2008; the
contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to dietary peptide compositions for
stimulating protein synthesis, decreasing protein degradation,
producing vasodilation, increasing nitric oxide production, and
decreasing blood flow. More specifically, the invention relates to
leucine-rich peptide compositions and methods for their isolation
from proteins.
BACKGROUND OF THE INVENTION
[0003] Loss of muscle tissue often occurs as a result of aging,
malnutrition, and catabolic diseases such as burns, sepsis, and
cancer. Dietary protein supplementation may be beneficial, but
supplementation with the essential amino acid leucine has been
shown to be especially beneficial. Dietary leucine has, for
example, recently been shown to suppress the rate of myofibrillar
protein degradation and muscle weight loss in rats. Leucine also
stimulates muscle protein synthesis and modulates the activity of
various proteins involved in the control of mRNA translation.
Leucine may stimulate protein synthesis directly or through its
metabolite, .alpha.-ketoisocaproic acid. Leucine may stimulate
translation either independently or by interaction with the
mammalian target of rapamycin (mTOR).
[0004] Leucine is one of the branched-chain amino acids and is an
essential amino acid. It is the only amino acid that is converted
to acetyl-coenzyme A and alpha-ketoacids and is an important source
of nitrogen for synthesis of glutamine. In addition to its effects
on protein synthesis and degradation, leucine also stimulates
glucose uptake by protein kinase C (PKC), while insulin modulates
glucose uptake via protein kinase B.
[0005] Milk-derived whey is one good source of muscle-building
proteins. Whey protein isolates and whey protein concentrates are
used in protein-building dietary supplements. Whey proteins are a
good source of leucine, but for many individuals who need the
muscle-building amino acids dietary proteins can provide, it is
difficult to digest and/or absorb proteins. It is therefore
important to find ways to provide leucine-containing amino acid
compositions that provide amino acids in a more bioavailable form
for improving muscle protein synthesis.
SUMMARY OF THE INVENTION
[0006] The present invention relates to peptides having a leucine
content of from about 12% to about 40% ("leucine-rich peptides"),
those peptides being isolated from a protein source by a method
comprising the steps of hydrolyzing the protein in the presence of
one or more leucyl aminopeptidases, optionally in combination with
additional proteases, to produce a protein hydrolysate,
deactivating the enzymes, and filtering the protein hydrolysate to
provide a permeate comprising peptides having a molecular weight of
from about 200 to about 4,000, including sub-ranges in-between,
such as, for example, a molecular weight of from about 200 to about
1,000 or a molecular weight of from about 400 to about 1,000.
Optionally, the step of concentrating the leucine-rich peptides by
removing the aqueous fluid from the permeate may be added. Also
disclosed are method of use of such peptides for a variety of
beneficial effects, such as producing vasodilation, increasing
nitric oxide production, decreasing blood pressure, increasing
blood flow, increasing muscle tissue, improving wound healing, and
improving cognitive function.
[0007] The invention also relates to a method for isolating
leucine-rich peptides from a protein source, the method comprising
the steps of hydrolyzing the protein in the presence of one or more
leucyl aminopeptidases, optionally in combination with additional
proteases, to produce a protein hydrolysate, deactivating the
enzymes, filtering the protein hydrolysate to provide a permeate,
and concentrating the permeate containing the leucine-rich
peptides.
[0008] In various embodiments, the protein may be derived from
animal or plant sources, including, for example, legumes such as
soy or pea, fish, meat, milk, blood, egg, corn, wheat gluten,
maize, or combinations thereof. In some embodiments, the protein
source may be bovine milk, bovine whey, whey protein concentrates,
and/or whey protein isolates.
[0009] In various embodiments, the leucine content of the
leucine-rich peptides may be within various subranges of the about
12 to about 40 percent range, such as, for example, from about 12
to about 30 weight percent leucine, from about 15 to about 20
weight percent leucine, from about 15 to about 25 weight percent
leucine, and from about 20 to about 25 weight percent leucine.
DETAILED DESCRIPTION
[0010] The inventors have discovered that peptides having a leucine
content of from between about 12% and about 40% ("leucine-rich
peptides") may be isolated from a protein source by a method
comprising the steps of hydrolyzing the protein in the presence of
one or more (i.e., at least one) leucyl aminopeptidases, optionally
in combination with additional proteases, to form a protein
hydrolysate, deactivating the enzymes, filtering the protein
hydrolysate to isolate leucine-rich peptides, and concentrating the
leucine-rich peptides. In one embodiment, the method comprises
hydrolyzing the protein in the presence of one or more
aminopeptidases which may act as leucyl aminopeptidases (leucine
aminopeptidases), optionally supplemented with additional
proteases, to form a protein hydrolysate, deactivating the enzymes,
filtering the protein hydrolysate using ultrafiltration,
nano-filtration, or another suitable filtration method to give a
permeate containing the leucine-rich peptides, and concentrating
the leucine-rich peptides in the permeate using reverse osmosis,
evaporation, or another suitable method for concentrating the
peptides by removing the aqueous portion of the permeate from the
filtration step. The invention also provides compositions
comprising leucine-rich peptides prepared by the method.
[0011] Dietary leucine appears to have a number of beneficial
effects, including stimulation of muscle protein synthesis and
suppression of the rate of myofibrillar protein degradation and
muscle weight loss. Leucine also increases hypothalamic mTOR
signaling and decreases food intake and body weight (Cota, D. et
al. Science (2006) 312: 927-930). Leucine administration increases
leptin levels, and leptin is known to promote lipolysis in adipose
tissue, but has no apparent effect on lean tissue. Increased leptin
levels result in decreased hunger, decreased food consumption, and
increased cellular energy expenditure.
[0012] Protein sources such as standard whey protein hydrolysates
may contain as much as 10% leucine, but given the variety of
beneficial effects of this amino acid and the fact that it is not
synthesized in the body from other amino acids, it has been a goal
of the food and nutritional supplement industry to develop ways to
increase leucine levels in order to promote its beneficial effects,
especially its effects on muscle building and inhibition of muscle
wasting. The present invention provides amino acid compositions
that are rich in leucine and such compositions may be isolated from
readily-available protein sources using the method of the
invention.
[0013] The inventors proposed to use leucine aminopeptidase to
process whey proteins to produce bioactive/bioavailable
leucine-containing peptides which they have demonstrated to have a
variety of desirable effects. Leucine residues appear to play an
important role in the distinction between the effects of remarkably
similar proteins. For example, Angiotensin I differs from
Angiotensin II by two additional carboxyl-terminal amino
acids--histidine and leucine. Met-enkephalin, a neuropeptide, has
many similar effects to those of Leu-enkephalin, which appear to be
mediated by the aromatic side-chains of the amino acids in the
peptides. Both peptides consist of 5 amino acid residues.
Met-enkephalin and Leu-enkephalin differ, however, in the
carboxyl-terminal fifth amino acid residue. In Met-enkephalin, it
is methionine. In Leu-enkephalin it is leucine. Both appear to have
effects on arterial blood pressure and vasodilation, but in this
area the amino acid difference appears to be more significant, with
Leu-enkephalin appearing to have a stronger effect (Moore, R. H.
and D. Dowling, "Effects on Intravenously Administered Leu- or
Met-Enkephalin on Arterial Blood Pressure," Regulatory Peptides
(1980) 1(2): 77-87). While others have provided hydrolysates
containing a significant percentage of leucine present as a free
amino acid (see, for example, U.S. Pat. No. 6,875,456 to Delest, V.
et al.), the present invention provides small peptides (e.g., a
hydrolysate containing a significant percentage of di- and
tri-peptides) to deliver leucine in a more bioavailable/bioactive
form. These peptides have demonstrated surprising beneficial
results, such as potent inhibition of angiotensin converting enzyme
(ACE) inhibition, vasodilation, and stimulation of increased nitric
oxide production. Experiments demonstrate that the peptides of the
invention provide bioavailable/bioactive leucine, supplementation
with the peptides demonstrating many of the same beneficial effects
as those provided by leucine supplementation, but with additional
benefits not previously described as the result of leucine
supplementation.
[0014] Leucine-rich peptide compositions of the present invention
provide leucine in combination with other amino acids, but provide
increased amounts of leucine in relation to the other amino acids.
These compositions may readily be added as a powder to a drink
formulation, to a food product, to a nutritional supplement
composition such as a tablet, capsule, or other formulation, or to
a pharmaceutical preparation, for example. Additional flavorings,
carbohydrates, fats, proteins, vitamins, minerals, and other
suitable food or supplement ingredients may be included in such
compositions.
[0015] Peptides of the invention may be used to provide
compositions that provide a benefit to a human or animal in areas
including, but not limited to, cardiac health, sarcopenia,
endothelial function, maintaining healthy blood pressure or
lowering blood pressure, vasodilation, muscle growth and
development, sports nutrition, infant nutrition, prevention or
treatment of metabolic syndrome, cognitive function, eye health,
diabetes, improvement in glycemic index, mTOR activation, wound
healing, and skin care/treatment of skin disorders.
[0016] Studies have shown that human athletes given branched-chain
amino acids (BCAA) before and during exercise performed better on
complex cognitive tests following exercise (Hassmen, P. et al.,
Nutrition (1994) 10:405-10). When tested in dogs, BCAA
supplementation appeared to be especially effective at improving
cognitive performance during exercise in older dogs (Fretwell, L.
et al. J. Nutr. (2006) 136: 2069S-2071S). Compositions of the
present invention provide increased levels of the branched-chain
amino acid (BCAA) leucine, in conjunction with other amino acids in
a form that may be readily incorporated into tablets, capsules,
food products such as, for example, nutrition bars, supplements in
the form of powders, drink mixes, and other compositions for human
and/or animal consumption. These compositions may provide a benefit
for those individuals who desire to improve their cognitive
performance. Studies performed with the peptides of the invention
have demonstrated that supplementation with leucine-rich peptides
promotes vasodilation and increases blood flow, as well as
increasing the rate at which glucose is delivered to the tissues.
Older individuals, students, and others may derive a particular
benefit from foods, drinks, and/or supplements comprising
leucine-rich peptides of the invention.
[0017] Metabolic syndrome (Syndrome X) is a chronic disease that
affects at least 1 in every 5 adults in the United States. It is
often associated with obesity, but the defining characteristic is
abnormal glycemic control (glucose intolerance, insulin
resistance). Protein-rich diets, especially those comprising
significant amounts of leucine, are beneficial for the treatment of
obesity and for the treatment of the metabolic syndrome (Layman, D.
K. and D. A. Walker, J. Nutr. (2006) 136: 319S-323S). Compositions
of the present invention provide an excellent means by which the
necessary amino acids and the higher amounts of leucine necessary
to affect weight management and the metabolic syndrome may be
provided to a human or animal. Products such as "protein water"
(water containing whey protein isolate in combination with
sweeteners and flavorings), for example, may provide an additional
benefit if peptides of the present invention are used as the
protein component. Snack foods, nutrition bars, drinks, drink
mixes, and other products may also be formulated with the
leucine-rich peptides to provide compositions for use in weight
management and loss of fat. Leucine-rich peptide compositions may
be used to promote weight loss by incorporating those compositions
into one or more food products, or may be similarly used by
incorporating them into a nutritional supplement or nutritional
drink for ingestion at mealtime. Between-meal-snacks may also be
formulated with leucine-rich peptide compositions to decrease
hunger and food consumption.
[0018] Leucine-rich peptides of the invention may be added to whey
compositions, whey protein isolates, whey protein concentrates, and
other protein sources to improve muscle synthesis, decrease muscle
breakdown, decrease hunger and food consumption, improve blood
flow, and increase nitric oxide production in the blood vessels,
for example. These effects may be beneficial for a large population
of individuals, including those who voluntarily wish to build
additional muscle mass, those who are losing muscle mass through
the process of aging and/or injury, disease (e.g., sepsis, cancer)
or malnutrition, and those who desire to decrease the percentage of
adipose tissue to muscle mass in the body.
[0019] Leucine-rich peptide compositions of the present invention
are especially beneficial for aging individuals or others with
sarcopenia. During the aging process and beginning at about age 45,
human muscle mass decreases. The frailty that is associated with
aged individuals may be attributed in large part to this loss of
muscle mass, and that predisposes those individuals to further
disease or injury. Some of the muscle tissue loss in older
individuals may also be the result of malnutrition--especially in
terms of protein ingestion, as protein-rich foods may tend to be
more expensive and less readily available to individuals on fixed
incomes. Compositions of the present invention provide nutritional
supplements with which to address the needs of individuals in whom
aging or malnutrition has induced muscle loss.
[0020] Body-building involves the combination of nutritional
supplementation and exercise to increase muscle mass. Compositions
of the present invention are ideally suited for body-building
nutritional supplements. Leucine-rich compositions stimulate muscle
synthesis while generally decreasing the desire to overeat and gain
fat tissue. Furthermore, dietary leucine supplementation has been
shown to significantly improve endurance performance (Crowe, M. J.
et al., Eur. J. Appl. Physiol. (2006) 97(6): 664-72).
[0021] Leucine has demonstrated cardioprotective effects, in
addition to its many other beneficial effects. For example, leucine
protects heart muscle against myocardial ischemia and some
researchers believe it is also an anti-arrhythmic factor (Gabrys,
J. et al. (2002) Pharmacology Reviews and Communications 12:
101-108). Peptides of the present invention have been shown to act
as potent angiotensin converting enzyme (ACE) inhibitors. For
example, European Patent Number 1794189B1 (De Slegte, J et al.)
proposes that ACE-inhibitory peptides demonstrating the greatest
efficacy to date are those containing a significant percentage of
proline residues. Those inventors discuss the products of companies
such as Calpis and Valio, which provide tripeptides having two
proline residues per tripeptide. Reporting the results of their own
peptide preparation in terms of ACE inhibition (which is expressed
as an IC50 value--the concentration of peptide or hydrolysate
needed to reduce ACE activity by 50%), they indicate that their
hydrolysate produces an IC50 (expressed as .mu.g/ml) of 7.2.
Leucine-enriched peptides of the present invention, however, have
demonstrated IC50 values of from about 4.0 .mu.g/ml to about 9.6
.mu.grams/ml, with an ACE-inhibitory value of from 4.0 to 4.7 being
the norm for large-scale production lots.
[0022] Peptides of the present invention have also demonstrated
effectiveness in producing vasodilation, increased blood flow, and
increased nitric oxide (NO) production in the blood vessels (see
Example 1). This combination of effects clearly demonstrates the
positive effects of peptides of the invention on the cardiovascular
system, and therefore illustrates the benefits of peptides of the
invention as cardio-protective compositions and compositions for
improving vascular function, increasing blood flow, decreasing
blood pressure, increasing endurance capacity, etc. Compositions of
the invention may be provided or consumed as dietary supplements
and/or pharmaceutical compositions for maintaining healthy blood
pressure levels and for decreasing blood pressure in individuals
who have or are predisposed to elevated blood pressure levels. Such
compositions may be especially beneficial for individuals who are
diabetic, for example, who may have or be predisposed to high blood
pressure and who may also need the increased blood flow to tissues
in order to decrease tissue damage, especially in the
extremities.
[0023] Leucine supplementation has been determined to be more
beneficial than is generic supplementation with BCAAs for burn,
trauma, and sepsis patients (DeBandt, J. and L Cynober, J. Nutr.
(2006) 136: 308S-313S). Furthermore, leucine has an anabolic effect
on proteins in skin wounds and muscle, provided that adequate
additional amino acids are available. When compared with leucine
supplementation alone, leucine supplementation in conjunction with
protein supplementation provided a greater benefit for wound
healing (Zhang, X. et al., J. Nutr. (2004) 134: 3313-8).
Compositions of the present invention provide peptides having
enhanced leucine content and are therefore excellent food,
pharmaceutical, or nutritional compositions for administration to
patients with burns or other skin wounds, muscle injury, trauma,
sepsis, and other conditions in which the beneficial effects of
leucine may be enhanced by providing leucine in combination with
peptides or proteins. These compositions may be provided in oral
form, via intravenous means, or by injection. Leucine-rich peptide
compositions may also be used either orally or topically to enhance
skin tone and suppleness, and to promote overall skin health.
[0024] Leucine-rich peptides may also provide a benefit for blood
pressure regulation and inhibition of angiotensin converting enzyme
(ACE). For individuals desiring to better manage blood pressure and
increase blood flow to tissues, leucine-rich peptides of the
invention may be consumed in food products, drinks and drink mixes,
as food supplements such as tablets, capsules, powders, and other
formulations that may be easily self-administered at or between
mealtimes. By increasing blood flow and promoting vasodilation and
nitric oxide production, compositions of the invention may provide
a benefit by increasing microcirculation, promoting endothelial
health, and improving nutrient delivery to the tissues. These
effects may be especially important in individuals suffering from
diabetes, peripheral artery disease, and other diseases affecting
circulation, as well as the elderly, athletes, individuals
recovering from surgery, traumatic injury, burns, chronic wounds,
and other conditions where increased blood flow and nutrient
delivery may promote improved health.
[0025] A variety of beneficial effects have been associated with
nitric oxide production. NO relaxes smooth muscle and inhibits
vascular inflammation, inhibiting endothelial cells lining the
blood vessels from releasing inflammatory substances. NO inhibits
smooth muscle cell migration and proliferation and decreases
platelet adherence and aggregation. These effects may be especially
beneficial in those predisposed to the development of
atherosclerosis and to stroke, autoimmune disease, etc. NO appears
to regulate vascular inflammation in part by inhibiting exocytosis
of Weibel-Palade bodies, which can be triggered by thrombin,
histamine, fibrin, complement, leukotrienes, and ATP, for example
(Matsushita, K. et al. Cell (2003) 115: 139-150). Leucine-rich
peptides of the present invention have been shown in vivo to
increase nitric oxide production and vasodilation. Compositions of
the invention may therefore provide a benefit by preventing the
development of atherosclerotic lesions, decreasing vascular
inflammation, decreasing platelet adherence and aggregation, etc.
Compositions of the invention may therefore provide supplements
that provide a significant benefit in strengthening the
cardiovascular system, decreasing inflammation in the circulatory
system, preventing the development of plaques in the blood vessels,
and providing a more "heart-healthy" environment within the blood
vessels in general.
[0026] The method may be used to isolate leucine-rich peptides from
any source comprising leucine-containing proteins. Such sources may
include, but not be limited to, wheat gluten, maize/corn protein
isolates, egg proteins, soy and other legume proteins (e.g., pea),
fish, meat, blood/blood proteins, and milk proteins. One especially
valuable source of leucine-rich peptides is milk-derived whey, such
as, for example, bovine milk-derived whey, whey protein isolates,
and/or whey protein concentrates. Whey protein isolates generally
have a protein content of at least about 90 weight percent, while
whey protein concentrates may have from at least about 35 weight
percent to about 80 weight percent protein.
[0027] Enzyme sources for the method of the invention may be
readily identified by those of skill in the art. Leucyl
aminopeptidases (leucine aminopeptidases), for example, are
exopeptidases which hydrolyze the peptide bond adjacent to a free
amino group and react most rapidly with leucine-containing peptides
and proteins.
[0028] Using filtration, molecules are selectively passed through
or transported across a membrane, and this process may be even more
effective when a pressure gradient is used. Filtration is
particularly effective at isolating the leucine-rich peptides.
Methods of filtering molecules based upon molecular weight and
other properties are known to those of skill in the art and include
membrane filtration generally, ultrafiltration, and
nano-filtration, for example. At this point, the isolated
leucine-rich peptides are generally contained within a permeate,
which is fairly low in solids. The percentage of solids in the
composition (permeate) may be increased by removal of liquid
(aqueous fluid). This may be done by means known to those of skill
in the art, including but not limited to processes such as
evaporation and reverse osmosis.
[0029] Throughout this disclosure, where the term "comprising" is
used, it is intended that this term may be substituted with
"consisting of" and/or "consisting essentially of," as well. The
invention may be further described by means of the following
non-limiting examples:
EXAMPLES
Example 1
Production of Leucine Peptides
[0030] A high protein, low fat, low lactose liquid whey protein
isolate product was used as the beginning substrate. This product
was pumped into reaction tanks at a solids level of 17%. The
temperature was raised to 45.degree. C. and pH adjusted to 7.3 with
sodium hydroxide. A blend of proteases and aminopeptidases (e.g.,
leucine aminopeptidase) was then added at a level of 0.35% of the
solids. Hydrolysis was allowed to proceed for a 6-hour period with
hourly pH adjustments to maintain the pH at 7.3. The enzymes were
then deactivated by heating the solution to 65.degree. C.
[0031] Fractionation was performed using filtration, the
hydrolysate being passed through the filter to retain molecules
having a molecular weight of greater than 20,000. The permeate
coming from filtration system contained the leucine peptides,
having approximately 52% of the peptides in the molecular weight
range of less than 1,000; 41% of the peptides in the molecular
weight range of 1,000 to 4,000; and 6% of the peptides in the
molecular weight range of 4,000 to 20,000. These peptides were
further concentrated using reverse osmosis. The final product was
then dried and packaged.
[0032] Analysis of six preparations (lots) of peptides of the
invention is shown in Table 1, where Lot 06-187, Lot 06-257, and
Lot 06-259 are small-scale lots and Lot 07-041, Lot 07-046, and
07-055 are large-scale production lots. The degree of hydrolysis of
each lot is shown on the bottom row, with a range of hydrolysis of
from about 30% to about 50% producing the desired peptides in these
experiments. ACE inhibition is shown as the amount of peptide
needed to produce 50% ACE inhibition.
TABLE-US-00001 TABLE 1 Lot 06- Lot 06- Lot 06- Lot 07- Lot 07- Lot
07- 187 257 259 041 046 055 Tryptophan 1.52 1.52 1.45 1.34 1.42
1.37 Cystine 0.90 1.48 1.25 0.63 0.51 0.54 Methionine 3.84 4.47
5.19 5.43 4.95 5.21 Aspartic Acid 8.23 8.52 7.76 5.92 5.49 5.63
Threonine 7.14 8.60 8.75 10.77 10.33 11.08 Serine 4.31 4.36 4.10
4.78 4.38 4.43 Glutamic 13.43 13.42 12.20 8.78 8.34 8.47 Acid
Proline 4.23 4.79 3.97 2.26 2.28 2.37 Glycine 1.70 1.49 1.50 1.32
1.19 1.22 Alanine 7.20 6.25 6.53 7.63 7.10 7.08 Valine 7.38 6.51
6.98 6.87 7.48 7.54 Isoleucine 6.23 6.19 6.38 5.24 6.10 5.87
Leucine 14.98 14.80 16.51 22.88 24.45 23.12 Tyrosine 3.44 2.64 2.79
2.55 2.60 2.38 Phenylalanine 3.61 3.66 3.96 4.11 4.50 4.55 Lvsine,
Total 7.98 7.53 7.12 6.25 5.83 5.98 Histidine 1.81 1.79 1.86 1.73
1.76 1.75 Arginine 2.08 1.97 1.72 1.50 1.29 1.39 BCAA 28.6% 27.5%
29.9% 35.0% 38.0% 36.5% EAA 58.8% 59.2% 62.2% 67.8% 69.9% 69.4%
Protein 79.0 68.7 69.2 73.1 72.3 72.8 (N = 6.4) Protein (N = 83.7
72.8 73.3 77.5 76.6 77.2 6.78) Calcium 228 306 260 358 392 411
(mg/100 g) Sodium 3374 3829 4475 4015 2905 2722 Potassium 652 940
949 793 795 622 ACE 6.9 9.1 8.6 4.2 4.4 4.0 Inhibition (N = 6.4)
ACE 7.3 9.6 9.1 4.5 4.7 4.2 Inhibition (N = 6) Degree of 32.8%
35.9% 39.7% 47.3% 47.7% 47.9% Hydrolysis
Example 2
Stimulation of Increased Blood Flow, Vasodilation, NO
Production
[0033] Individuals were provided with 2 weeks of daily
supplementation with either the peptide product of the invention,
or placebo. After completion of the first 2 week supplementation
period and first day of vascular testing there was an interval of
1-2 weeks, after which the individuals started the second 2 week
supplementation period, each individual consuming the alternative
supplement. Individuals were evaluated at four separate times
during the protocol, and asked to fast for 12 hours, as well as to
avoid alcohol, caffeine, and exercise for 24 hours, and to consume
36 ounces of water the night before each evaluation and an
additional 12 to 16 ounces of water the morning of the visit, to
ensure that each person was adequately hydrated.
[0034] The peptide product was produced by Glanbia Nutritionals,
Twin Falls, Id. A single dose of 5 g was premeasured and placed in
individual packets with artificial sweetener. Individuals
participating in the study were provided with a 2-week supply and
instructed to consume one packet per day mixed in 300 mL water.
[0035] On the morning of vascular testing, individuals consumed one
packet containing 5 g of peptide product or placebo mixed in water.
Fasting measurements of flow mediated dilation (FMD) and forearm
blood flow (FBF) were determined. Fifteen minutes of recovery were
allowed between FMD and FBF measurements prior to ingestion of the
test beverage. Following these baseline measurements, subjects
consumed a single 5 g dose of either peptide product or placebo
mixed in 300 mL of water and artificial sweetener.
[0036] Post-ingestion FMD and FBF measurements were made
intermittently. Blood samples were obtained at 15, 30, 45, 60, 90,
and 120 minutes post-ingestion. Subjects rested in a comfortable
position for the entire duration of the test. To ensure
standardization between testing trials subjects were asked to
maintain their current level of physical activity during the study
period and to repeat their dietary intake from previously recorded
diet records the day prior to each vascular testing visit. Flow
mediated dilation (FMD) was assessed using standardized procedures
for performing high-frequency ultrasonographic imaging before (PRE)
and at 30, 60, and 90 min after ingestion of the test beverage. The
technique provokes the release of nitric oxide, resulting in
vasodilation that can be quantitated as an index of vasomotor
function. All tests were performed in a quiet,
temperature-controlled room after a 10 minute period in a supine
position. A blood pressure cuff was placed on the upper right arm
for occlusion. ECG leads were attached to monitor heart rate
throughout the procedure. The brachial artery was imaged above the
antecubital crease, and the transducer was placed to image the
brachial artery in a longitudinal axis with clear visualization of
the anterior and posterior vessel walls. When a clear image of the
anterior and posterior walls of the artery was obtained, the
transducer was held by a stereotactic clamp and the position held
constant for the duration of the data collection. Baseline brachial
artery diameter was recorded for 30 heart beats. A mark was made on
the arm where the image was collected. The cuff was inflated to 200
mm Hg for 5 min using a rapid cuff inflator (Hokanson E20,
Bellevue, Wash., USA) to occlude the brachial artery, and then
released. Arterial diameter was then assessed continuously for 300
heart beats after occlusion. Images of the brachial artery were
obtained using an Acuson 13.0-MHz linear array transducer and an
Aspen cardiac ultrasound system (Acuson Corp, Elmwood Park,
N.J.).
[0037] Image analysis was performed using MIA software (Medical
Imaging Applications, Iowa City, Iowa, USA). For baseline, the
average diameter taken from 30 frames was used. Three hundred
frames were recorded for post-occlusion. Peak post-occlusion
diameter was calculated by averaging the vessel diameter 5 frames
immediately before the observed peak diameter and the 5 frames
immediately after the same mark. Brachial artery FMD was calculated
and expressed as a percentage of the baseline diameter.
[0038] Forearm blood flow was measured in the same right arm using
venous occlusion strain gauge plethysmography. A calibrated
indium/gallium-filled silastic strain gauge, encircled around the
largest diameter of the right forearm, was connected to a
plethysmograph (EC6, Hokanson, Inc., Bellevue, Wash., USA). The
increase in forearm volume was measured after blocking the venous
efflux by an upper arm cuff inflated to 50 mmHg by a rapid cuff
inflator (Hokanson E20, Bellevue, Wash., USA) for 7 seconds during
each 15-second cycle to determine resting forearm blood flow
(R-FBF). This measurement was performed at rest (PRE) and 20, 50,
80, and 110 minutes after ingestion of the test beverage. The hand
circulation was excluded by a wrist cuff inflated to 220 mmHg for 1
min before and during each flow evaluation. Forearm blood flow was
estimated using specialized software (Noninvasive Vascular Program
3 (NIVP3), Hokanson, Bellevue, Wash., USA) which determined the
slope of the change in forearm volume and recorded blood flow in
terms of percent volume change per minute (%/min). Four
plethysmographic measurements were averaged to obtain values for
R-FBF. To determine reactive hyperemia induced forearm blood flow
(RHFBF), a blood pressure cuff on the upper right arm was inflated
to a pressure of 200 mmHg for 5 minutes. Upon release of the
occlusion, FBF was determined as described above. This measurement
was performed at rest (PRE) and 120 min after ingestion of the test
beverage.
[0039] A blood sample was obtained from a left arm vein after
subjects rested quietly for 10 minutes in the supine position.
Serum glucose and insulin concentrations were analyzed in duplicate
using a YSI glucose/lactate analyzer (YSI 2300 STAT, Yellow
Springs, Ohio) and commercially available ELISA [Diagnostic Systems
Laboratory (DSL), Webster, Tex.] (CV=7.0%) respectively, and used
to calculate an index of insulin resistance.
[0040] Body mass remained stable over the course of the study, and
there were no significant differences in systolic/diastolic blood
pressure after 2 weeks of peptide supplementation. However, results
demonstrated that blood vessel diameter, blood flow, and nitric
oxide levels were increased as a result of ingestion of the peptide
product of the invention.
Example 3
ACE Inhibition Assay
[0041] The ACE inhibition assay has been previously described by
Cushman and Cheung (Cushman, D. W. and Cheung, H. S., Biochem.
Pharmacol. (1971) 20: 1637). Briefly, substrate was prepared by
dissolving 21.475 mg of Hip-His-Leu ("HHL," Sigma, St. Louis, Mo.)
in 8 ml of phosphate buffered saline, with volume adjusted to 10 ml
and final pH to 8.3. A 10% w/w solution of peptide composition was
prepared using buffer as diluent. Angiotensin-converting enzyme
stock solution was prepared by diluting 0.1 unit of ACE (rabbit
lung, Sigma Chemical, St. Louis, Mo.) with buffer. To perform the
assay, 10 microliters of sample were placed into a small glass tube
with 200 .mu.l of HHL and 70 .mu.l of buffer. Tubes were placed in
a 37.degree. C. water bath and held for 3 minutes. Tubes were
removed from the water bath and 20 .mu.l of angiotensin-converting
enzyme were added to each tube. Tubes were vortexed and returned to
the water bath for 30 minutes. Tubes were then removed from the
water bath and 250 .mu.l HCl were added to stop the reaction.
Acetic ether (1.5 ml) was added to each tube with a glass pipette,
and tubes were again vortexed. Small tubes were independently
placed into plastic 50 ml tubes to be centrifuged for 2 minutes at
2000 rpm. Tubes were removed from the centrifuge with care being
taken not to disturb the ether layer. The ether layer (1 ml) was
removed and placed in a small 10 ml beaker, then placed on a hot
place to evaporate at approximately 100.degree. C. To each
evaporated sample, 1 ml of nanopure water was added. Absorbance was
read at 228 nm with a reaction mixture containing no inhibitor as
control.
* * * * *