U.S. patent application number 17/406501 was filed with the patent office on 2021-12-09 for active component encapsulated, protected and stabilized within a protein shell.
The applicant listed for this patent is NUABIOME LIMITED. Invention is credited to Sinead Doherty.
Application Number | 20210378973 17/406501 |
Document ID | / |
Family ID | 1000005787223 |
Filed Date | 2021-12-09 |
United States Patent
Application |
20210378973 |
Kind Code |
A1 |
Doherty; Sinead |
December 9, 2021 |
ACTIVE COMPONENT ENCAPSULATED, PROTECTED AND STABILIZED WITHIN A
PROTEIN SHELL
Abstract
A microcapsule includes an active component encapsulated within
a polymerized hydrolyzed protein shell. The microcapsule has an
average diameter that is less than one hundred micrometers as
determined by a laser diffractometer.
Inventors: |
Doherty; Sinead; (Dublin,
IE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NUABIOME LIMITED |
Dublin |
|
IE |
|
|
Family ID: |
1000005787223 |
Appl. No.: |
17/406501 |
Filed: |
August 19, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16504467 |
Jul 8, 2019 |
11135175 |
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17406501 |
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14897901 |
Dec 11, 2015 |
10449157 |
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PCT/EP2014/062154 |
Jun 11, 2014 |
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16504467 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 36/67 20130101;
A23L 33/175 20160801; A23L 33/105 20160801; A23L 33/10 20160801;
A61K 9/5052 20130101; A61K 9/5089 20130101; A23P 10/30 20160801;
A61K 31/198 20130101 |
International
Class: |
A61K 9/50 20060101
A61K009/50; A61K 31/198 20060101 A61K031/198; A23P 10/30 20060101
A23P010/30; A61K 36/67 20060101 A61K036/67; A23L 33/105 20060101
A23L033/105; A23L 33/10 20060101 A23L033/10; A23L 33/175 20060101
A23L033/175 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 12, 2013 |
EP |
13171757.1 |
Claims
1. A manufacture comprising a microcapsule, wherein said
microcapsule comprises an active component encapsulated within a
polymerized hydrolyzed protein shell and wherein said microcapsule
is of average diameter as determined by a laser diffractometer,
wherein said average diameter is less than one hundred micrometers.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/504,467, filed on Jul. 8, 2019, which is a divisional
application of U.S. application Ser. No. 14/897,901, filed on Dec.
11, 2015, now U.S. Pat. No. 10,449,157, which is a U.S. National
Stage of International Application No. PCT/EP2014/062154, filed on
Jun. 11, 2014, which claims the benefit of the Jun. 12, 2013
priority date of European Application No. 13171757.1, the contents
of which are herein incorporated by reference.
FIELD OF DISCLOSURE
[0002] This disclosure relates to microcapsules comprising an
active component encapsulated within a protein shell, and
comestible products, especially sports nutritional products,
comprising such microcapsules.
BACKGROUND
[0003] Creatine is recognized as a dietary supplement capable of
increasing muscle mass and muscle performance. It is provided in a
number of different forms, the most common of which is a powder
that comprises creatine monohydrate.
[0004] The ergogenic effect of creatine has been a subject of
systematic investigation. Most of these studies have demonstrated
significant positive effects of creatine on muscle mass, muscle
power, lean body mass, and performance at maximum short-duration
muscle exertion in various types of sports. Today, creatine
monohydrate is the most significant nutrition supplement in
sports.
[0005] Known forms of creatine monohydrate lack long-term stability
in water. Such forms must be made up shortly before being ingested.
Such a product therefore cannot be stored during the day.
[0006] In addition to creatine itself, namely creatine monohydrate,
numerous creatine salts such as creatine ascorbate, citrate,
pyruvate and others have proven to be suitable nutritional
supplements.
[0007] Uptake of creatine from the intestine and transport into the
muscles is controlled by an NaCl-dependent creatine transporter.
This uptake can promoted by the simultaneous intake of
carbohydrates and proteins. It has therefore been found that in
comparison with sole intake of creatine, that the combination of
creatine and carbohydrates can lead to a 60% greater rise in
creatine content in the muscle.
[0008] One formulation for enhancing creatine transport includes an
IGF-1 modulating substance, such as particular proteins, colostrum,
and recombinant IGF-1.
[0009] Despite its undisputed ergogenic and physiological effects,
creatine monohydrate suffers from a number of limitations. These
include low solubility and/or hydration capacity and poor stability
in aqueous solutions. In addition, relatively large doses are
required to elicit an ergogenic effect in the body.
[0010] Since creatine lacks marked stability in water or
corresponding aqueous solutions, creatine cycling by elimination of
water will generate creatinine. The rate of cycling depends on the
solution's pH and its temperature. Intestinal concentration does
not play any role in this process. Conversion to creatinine
proceeds rapidly, in particular in the acidic pH range between
three and four. The rapid breakdown of creatine in this medium
virtually rules out the use of aqueous or moist formulations for
human and animal nutrition. For example, based on the stomach pH
alone, a significant breakdown of creatine to creatinine can occur
depending on the residence time.
SUMMARY
[0011] The disadvantages of the prior art with regard to creatine
solubility and stability in aqueous solution and subsequent uptake
from the intestine and transport into the target tissue give rise
to the object of the invention of providing encapsulated
preparations of creatine. Encapsulation provides better protection
for creatine than previously demonstrated. This, in turn, helps
avoid breakdown of creatine to creatinine and therefore improved
creatine uptake from the intestine.
[0012] An important factor is the optimum uptake and thus retention
of the creatine in the target tissue. A further object of the
invention is to ensure that the creatine absorbed from the
intestine is optimally taken up into the target tissue rather than
being excreted via the kidneys or being converted into creatinine,
which is useless to the body and must likewise be excreted from the
body via the kidneys. Therefore the encapsulated systems have
acceptable organoleptic properties with improved bioavailability
for food and beverage applications.
[0013] The majority of creatine in the human body is in two forms:
the phosphorylated form, which makes up 60% of the stores, and the
free form, which makes up 40% of the stores. The average
70-kilogram young male has a creatine pool of around 120-140 grams.
This varies between individuals depending on the skeletal muscle
fiber type and quantity of muscle mass. The endogenous production
and dietary intake matches the rate of creatinine production from
the degradation of phosphocreatine and creatine at 2.6% and 1.1%
per day respectively. In general, oral creatine supplementation
leads to increased creatine levels within the body. Creatine can be
cleared from the blood by saturation into various organs and cells
or by renal filtration.
[0014] Three amino acids (glycine, arginine, and methionine) and
three enzymes (L-arginine:glycine amidinotransferase,
guanidinoacetate methyltransferase, and methionine
adenosyltransferase) are involved in creatine synthesis. The impact
creatine synthesis has on glycine metabolism in adults is low.
However the demand is more appreciable on the metabolism of
arginine and methionine.
[0015] Creatine ingested through supplementation is transported
into the cells exclusively by CreaT1. However, there exists another
creatine transporter, namely CreaT2, that is primarily active and
present in the testes.
[0016] Creatine uptake is regulated by various mechanisms, namely
phosphorylation and glycosylation as well as extracellular and
intracellular levels of creatine. CreaT1 has been shown to be
highly sensitive to the extracellular and intracellular levels
being specifically activated when total creatine content inside the
cell decreases.
[0017] In addition to cytosolic creatine, there exists a
mitochondrial isoform of CreaT1 that allows creatine to be
transported into the mitochondria. This intra-mitochondrial pool of
creatine appears to play a role in the phosphate-transport system
from the mitochondria to the cytosol. Myopathy patients have
demonstrated reduced levels of total creatine and phosphocreatine
as well as lower levels of CreaT1 protein, which is thought to be a
major contributor to these decreased levels.
[0018] In a first aspect, the invention features a process for
producing microcapsules. Such microcapsules comprise an active
component encapsulated within a polymerized hydrolyzed protein
matrix. The method includes the steps of providing a suspension of
hydrolyzed protein and an active component in a liquid ester,
treating the suspension to generate droplets of the suspension, and
immediately curing the droplets by immersion in a basic curing
solution. The ester in the suspension reacts with the basic curing
solution to release a salt that polymerizes the hydrolyzed protein,
thus encapsulating the active component. As used herein, a "basic"
solution is one with a pH that exceeds seven.
[0019] Microcapsules formed according to the process described
herein are surprisingly stable upon prolonged storage in aqueous
solution. For example, after as much as twenty-eight days of
storage, creatine monohydrate encapsulated in hydrolyzed whey
protein showed almost no loss in creatine concentration.
Furthermore, creatinine, which is a breakdown product of creatine,
was not detected at any significant levels even after twenty-eight
days of storage under the same conditions. Hence, it has been
possible to show that the encapsulation system is capable of
stability in aqueous solutions.
[0020] As used herein, the term "active component" refers to an
agent that is suitable for delivery to the gastrointestinal tract
of a mammal, including pharmaceutically active agents and health
food supplements including vitamins, minerals, co-factors, amino
acids, and the like. Preferably, the active component is an active
agent that partially or fully degrades in water or aqueous solution
and that is typically at least partially insoluble in water.
[0021] Examples of such active agents include creatine moieties
(for example creatine and its esters), beta-alanine, and amino
acids, especially L-amino acids such as L-leucine, L-glutamine and
the branched-chain amino acids typically comprising of leucine,
isoleucine, and valine. The active agent described herein can
comprise of one or more amino acids, which the body metabolizes in
the stomach and intestines. The active agent can comprise one or
more of the following amino acids: isoleucine, alanine, leucine,
phenylalanine, threonine, tryptophan, glycine, valine, proline,
histidine, serine, tyrosine, glutamate, glutamic acid, and
glutamine, in any form for example as salts, esters, branched-chain
amino acids, complexes, precursors, or derivatives.
[0022] In some embodiments, the active component comprises a
creatine moiety, for example creatine, or a creatine precursor or
derivative, examples of which include creatine monohydrate,
guanidinoacetic acid (a direct precursor of creatine), creatine
esters, creatinol, creatinol O-phosphate, or a mixture of at least
two of these compounds. Other creatine precursors include
guanidinoacetic acid, creatine esters, creatinol and creatinol
O-phosphate, which are known to be converted into creatine in the
body, are suitable encapsulation candidates for this specific
delivery action.
[0023] The active component is preferably selected from a creatine
moiety, L-Glutamine, Beta-alanine, L-Leucine or a branched-chain
amino acid, or derivatives or pre-cursors thereof. These amino
acids are consumed by a large number of athletes and those
interested in sports nutrition for a variety of reasons, such as
muscle mass gain, muscle recovery and increased energy. The vast
majority of Glutamine, Leucine and Beta-Alanine in nutritional
products is in the L conformation. Industry experts recommend
consumption of glutamine as a powder or capsule due to instability
in water and losses of the amino acids during digestion
processes.
[0024] Leucine, along with isoleucine and valine, is one of the
branched-chain amino acids, which play an important role in sports
nutrition. Leucine is an essential amino acid and as such cannot be
synthesized in the body. It must therefore be derived from the
diet. Hence, leucine stability and bioavailability are vital.
Generally speaking, branched-chain amino acids make up 15% of a
consumed protein. Even in high protein diets, levels of
branched-chain amino acids consumed, including levels of leucine,
may be low. This compromises bioavailability during gastric
transit. The ingredient is stable under ambient conditions; however
the effect of gastric transit on free-form leucine results in poor
absorption in the mammalian system.
[0025] Glutamine is an amino acid and is not recognized as
essential due to the body's ability to synthesize it. As the most
abundant amino acid in the body, it constitutes a little more than
five percent of the amino acids found in animal-derived protein
sources, such as meats, dairy, and eggs. The amino acid is consumed
by a large number of athletes and those interested in sports
nutrition for a variety of reasons such as muscle mass gain, muscle
recovery, and increased energy.
[0026] Industry experts recommend consumption of glutamine as a
powder or capsule due to stability issues with the glutamine in
water. For this reason it is recommended that consumers avoid
products such as pre-made drinks and bars containing the free amino
acid. Regardless of the form, up to 90 percent of ingested
glutamine is eliminated during digestion due to lack of protection
against digestive conditions. A fraction of consumed glutamine will
survive beyond the liver due the action of digestive enterocytes
and immune cells within the gut.
[0027] Beta-alanine is a nutritional supplement widely used by
athletes and bodybuilders to improve performance. This nonessential
amino acid occurs naturally in the body and is also found in foods
such as chicken, beef, pork and fish. Beta-alanine is the
rate-limiting precursor of carnosine (EFSA), which potentially
generates intramuscular carnosine and improved muscular endurance.
Beta-alanine can be generally provided as either a powder or in
gelatin capsules and its presence in foodstuffs can be measured by
established methods (EFSAS). General dosage is 1.6-6.4 grams of
beta-alanine per day for 4 weeks. However, an issue for
beta-alanine is parenthesis, i.e., a sensation of pins and needles,
that (pins and needles feeling. that occurs when administered in a
large dose. An encapsulation-delayed release strategy could benefit
the consumer and manufacturers by alleviating this issue. Indeed,
one manufacturer recommends consuming 3.2 grams per day but
breaking this amount into two to three doses per day. A delayed
release formulation would add convenience to this product.
[0028] For this reason, these amino acids and branched-chain
structures are suitable candidates for this formulation and
delivery using the methods and products described herein. When the
aforementioned sources are added to ready-to-consume supplements it
is usually in a peptide-bound form, such as glycyl-L-glutamine
hydrate. Hence the amount of active component per serving is
ultimately limited and restricted. Regardless of the form, on
average, up to ninety-two percent of ingested active agents are
eliminated during digestion. A fraction of consumed active agents
survives beyond the liver due the action of digestive enterocytes
and immune cells within the gut. In this manner, glutamine,
beta-alanine, L-leucine and branched-chain amino acids structure
should be regarded as particularly preferred amino acid
sources.
[0029] In some embodiments, the suspension comprises an extract of
Piper nigrum L, i.e., black pepper, or Piper longum L, i.e., long
pepper. Among these embodiments are those in which the extract
comprises 95% piperine, hereafter referred to as "pepper extract."
In such embodiments, polymerization of the hydrolyzed whey protein
encapsulates the active component and the pepper extract. In some
embodiments, the extract is bioperine.
[0030] Incorporation of the pepper extract into the microcapsules
enhances the absorption efficiency of the active component within
the gastrointestinal tract, especially when the active component is
a creatine moiety, such as creatine monohydrate. The encapsulation
system protects creatine better from conversion to creatinine in
the stomach than known methods. The presence of pepper extract in
the encapsulation matrix leads to a surprising improvement in
uptake from the intestine. This, in turn, translates into
distinctly higher bioavailability and thus better uptake into the
target tissue.
[0031] In this structural format, creatine that has been
encapsulated and stabilized by whey protein and black-pepper
extract is further incorporated into the encapsulation matrix to
aid creatine bioavailability in the blood. In a preferred
embodiment, the combination of creatine monohydrate, whey protein,
and phosphate and black-pepper extract further enhances creatine
encapsulation efficiency to greater than 99.5%. This leads to
enhanced creatine bio accessibility for muscle contraction and
exercise.
[0032] Preferably, the encapsulation system, i.e., the suspension,
has a pH value between 3 and 6 and ideally between 3.5 and 4.8. The
preferred initial carrier system is a mixture of an alcohol, acetic
acid, and hydrolyzed protein, with a protein of dairy origin being
preferred. The amount of ester produced may be freely selected over
a wide pH range, the preferred ratio being set at the selected pH
value of the formulation, which is established at a pH of between
three and six and preferably with pH above four. A particularly
useful pH is 4.8.
[0033] If the mixture ratio is correctly selected, namely if the
ratios of alcohol, acid, and protein are correct, there is
virtually no restriction on the amount of salt that can be released
from the ester reaction for further encapsulation purposes. For
instance, when a 1:1 mixture is used, a pH value greater than four
is inevitably established, this being independent of the total
amount of alcohol introduced.
[0034] The foregoing technique provides a pH value acceptable from
an organoleptic aspect while providing surprisingly good protection
of creatine from the influence of acids, and in particular, from
gastric acid. This helps to avoid the conversion of creatine to
creatinine. The action of the encapsulation process could not have
been predicted in the claimed pH range.
[0035] An unexpected result of the encapsulation process described
herein is one that goes well beyond reduced breakdown of creatine
in the stomach. Surprisingly, the process also results in improved
uptake of creatine by the cells themselves. It has accordingly been
possible to demonstrate that the methods and compositions described
herein lead to a distinctly greater rise in creatine concentrations
in the target tissue when compared with creatine monohydrate that
has not been encapsulated.
[0036] The use of sodium acetate salt in connection with a pepper
extract, such as bioperine, has demonstrated a surprisingly
significant influence on the bioavailability and uptake of creatine
into cells.
[0037] Using the encapsulation process as described herein
stabilizes creatine against acids and thus reduces the breakdown of
creatine in the stomach. Moreover, the presence of pepper extract
improves the uptake of creatine into the cells. Sodium ions of the
acetate buffer further assist uptake.
[0038] Typically, the active component is a creatine moiety. This
means a molecule or complex comprising creatine, for example a
creatine complex such as creatine monohydrate, or a creatine
derivative or precursor such as guanidinoacetic acid, which is a
direct precursor of creatine), creatine esters, creatine salts,
creatinol, creatinol O-phosphate, or mixtures of at least two of
these compounds. Examples of creatine salts include creatine
hydrochloride and creatine nitrate. However, the process described
herein is also applicable to the encapsulation of other active
components, especially active components, for oral delivery.
Examples include glutamine, oil-soluble bioactive agents, such as
vitamins and minerals, fatty acids, and fat-soluble colors or
flavors.
[0039] In some practices, the process includes an initial step in
which the ester is formed in-situ, typically by reaction between an
alcohol, preferably a weak alcohol, and an organic acid, preferably
a weak organic acid, in the presence of the hydrolyzed protein
and/or the active component.
[0040] In one embodiment, the suspension comprises a phosphate
moiety suitable for crosslinking hydrolyzed-protein in the formed
microcapsules. This embodiment is particularly suitable when the
active agent is a creatine moiety. The phosphate moiety may be, for
example, a phosphate salt, such as disodium phosphate. This
structural addition of phosphate cross-linkers into the extrusion
matrix enhances immediate creatine absorption and metabolism and
potentially enables the accelerated generation of high-energy
molecules, such as ATP, during exercise and creatine loading. In
some embodiments, the phosphate is added to the suspension at a
concentration of 0.01M to 0.05M, typically about 0.01M to 0.03M,
and ideally about 0.02M.
[0041] In a preferred embodiment, the suspension is formed by
mixing the active component, acetic acid, a weak alcohol, and
hydrolyzed protein together to form a suspension of hydrolyzed
protein and active component in an acetate ester.
[0042] A preferred embodiment features forming the suspension by
mixing a creatine moiety, for example, creatine monohydrate, acetic
acid, a weak alcohol, and hydrolyzed protein together to form a
suspension of hydrolyzed protein and the creatine moiety in an
acetate ester.
[0043] As used herein, the term "hydrolyzed" means that the protein
has been treated with protease enzymes to at least partially digest
native protein.
[0044] In some embodiments, the hydrolyzed protein has a degree of
hydrolysis of 18-85%. The "degree of hydrolysis" is defined as the
proportion of cleaved peptide bonds in a protein hydrolysate. The
degree of hydrolosis is determined using the OPA spectrophotometric
assay, which involves using N-acetyl-L-Cysteine (NAC) as the thiol
reagent.
[0045] Preferably, the hydrolyzed protein is hydrolyzed whey
protein, ideally hydrolyzed whey protein obtained from milk,
especially bovine milk. However, other types of hydrolyzed protein
may be employed including, for example, bovine collagen, pea, rice
or non-whey milk proteins.
[0046] A hydrolyzed protein of choice would be from a dairy source
ingredient with 90-95% protein (w/w) protein content. The ideal
ratio for milk proteins .beta.-lactoglobulin and
.alpha.-lactalbumin would be 3:1 to 5:1. In some preferred
embodiments, the ratio is approximately 4:1. In yet other preferred
embodiments, the ratio is 85:15.
[0047] Flavourzyme represents an ideal enzyme for hydrolysis of
dairy proteins. Flavourzyme is a protease-peptidase complex
produced by submerged fermentation of a selected strain of
Aspergillus oryzae, as produced by Novo Nordisk A/S.
[0048] A preferred embodiment uses Flavourzyme as standardized in
terms of leucine amino peptidase units (LAPS) by the manufacturer.
Hydrolysates used herein are prepared using a measure amount of
Flavourzyme with 1000 LAPU with defined hydrolysis conditions.
Temperatures can be within the range of 35-65.degree. C.,
preferably, 40-60.degree. C., ideally 45-50.degree. C. pH values
can be within the range 4-9, preferably, 5-8, ideally 6.5-7.5.
Hydrolysis is performed with an enzyme/substrate ratio (E/S) of
1/200, preferably, 1/150, ideally 1/100, on the basis of total
protein content.
[0049] The step of treating the suspension to generate droplets may
be carried out by extrusion. Some embodiments combine extrusion
with a break-up technique, such as liquid-jet break up. Such
methods may be carried out using an encapsulator.
[0050] However, it is also possible to generate the droplets by
other techniques including spray drying and spray chilling. All of
these techniques generate droplets of suspension in a stable
format, i.e., with little or no polymerization of hydrolyzed
protein. These droplets are immediately immersed in the basic
curing bath.
[0051] A typical suspension comprises hydrolyzed protein, a
phosphate moiety, and pepper extract in a liquid ester base. A
suitable protein is whey protein.
[0052] In some embodiments, the basic curing solution for formation
of microcapsules contains glycerol, preferably in an amount of 0.01
to 0.10M, and ideally in an amount of 0.04 to 0.07M. This has been
found to reduce surface tension during formation of droplets and
capsules. The presence of glycerol in the basic curing solution
results in glycerol being contained in the microcapsule membrane,
typically in an amount of about 0.05% glycerol. This results in
microcapsules with a satisfactory spherical shape and a
satisfactory size of between twenty and one hundred fifty
microns.
[0053] In some embodiments, the presence of glycerol reduces
surface tension during capsule formation and further contributes to
enhanced creatine encapsulation efficiency, for example with an
approximately 14.5% enhanced creatine yield.
[0054] Furthermore, glycerol incorporation into creatine
micro-capsules has the potential to further enhance water-holding
capacity and fluid retention in the muscle during creatine
absorption phase.
[0055] In a preferred embodiment, the presence of glycerol within
the encapsulation matrix promotes hydration and regeneration of
muscle. The methods and compositions described herein highlight the
need to include glycerol within the encapsulation matrix in the
presence of whey protein to ensure optimum encapsulation efficiency
and to further aid muscle fluid retention during and after creatine
absorption.
[0056] In another aspect, the invention features a method for
producing microcapsules that comprise an active component
encapsulated within a polymerized hydrolyzed protein matrix. Such a
method includes providing a suspension of hydrolyzed protein and an
active component in a liquid ester, treating the suspension to
generate droplets of the suspension, and immediately curing the
droplets by immersion in a basic curing solution. The ester in the
suspension reacts with the basic curing solution to release a salt
that polymerizes the hydrolyzed protein, thereby encapsulating the
active component. The basic curing solution comprises glycerol in
an amount of 0.04%-0.07% (v/v).
[0057] In some embodiments, generating droplets includes generating
droplets having a core and a coating. The core comprises the
suspension and the coating comprises hydrolyzed protein in a liquid
ester.
[0058] One method of generating such droplets comprises using an
extruder having concentric nozzles in which the inner nozzle
extrudes a core-forming stream and the outer nozzle extrudes the
coating-forming stream. In this method, the active agent is
contained within the core. The hydrolyzed protein in the coating is
polymerized when the droplets are immersed in the basic curing
solution. This particular method is suitable for generating
microcapsules in which the suspension comprises a non-aqueous base
such as an emulsion of oil and water. An example of such a base is
one that comprises a lipid-soluble component. In such methods, the
suspension additionally comprises the fat-soluble component and a
suitable dispersing agent, such as a fatty acid.
[0059] In one embodiment, the suspension comprises astaxanthin,
which as used herein is identified by CAS Number 472-61-7. The
addition of astaxanthin into the suspension further enhances
capsule longevity and shelf-life and possibly enhances muscle total
creatine content as compared to the ingestion of creatine
monohydrate alone. In some embodiments, the presence of astaxanthin
in whey protein encapsulation matrices provides an additional
protective barrier against water. This further protects creatine by
retarding its degradation into creatinine.
[0060] In the presence of an astaxanthin-hydrolyzed protein
formulation/suspension, a dispersing agent (i.e., fatty acid) must
be added to assist the dissolution of astaxanthin with hydrolyzed
protein. This dispersing agent is preferably an oil-based agent,
for example a fatty acid, for example lipoic acid or palmitic
acid.
[0061] The addition of astaxanthin in an oil-based agent, such as
lipoic acid or palmitic acid, will optimize the homogenous
dispersion of astaxanthin throughout the encapsulation matrix. In
some embodiments, the addition of astaxanthin using an oil-based
agent, such as lipoic acid, to the encapsulation formulation
potentially maximizes creatine uptake by the human skeletal muscle
when creatine monohydrate is ingested in an encapsulated form, as
outlined above.
[0062] Thus, in one embodiment, the suspension comprises hydrolyzed
protein, ideally hydrolyzed whey protein, an active agent, pepper
extract, and astaxanthin dissolved in a dispersing agent.
[0063] In another embodiment, the suspension comprises hydrolyzed
protein, ideally hydrolyzed whey protein, an active agent, pepper
extract, a phosphate moiety, and astaxanthin dissolved in a
dispersing agent.
[0064] The subject matter described herein has the potential to
significantly improve creatine protection against stomach acid, due
to the presence of whey protein matrices, to augment creatine
absorption, due to the presence of black-pepper extract and
astaxanthin oil-based dispersions, and to enhance creatine uptake
and retention in the muscle, assisted by the presence of glycerol
for enhanced ergogenic performance, bioavailability and
bioaccessibility, possibly catalyzed by the presence of
phosphate.
[0065] Thus, in a preferred embodiment, the subject matter
described herein provides a way to produce microencapsulates
comprising an active component, preferably a creatine moiety, that
is encapsulated within a polymerized hydrolyzed protein shell,
which is preferably a polymerized hydrolyzed whey protein
shell.
[0066] Such a method includes mixing an organic acid, an alcohol,
hydrolyzed protein, and an active component to generate a
suspension of hydrolyzed whey protein and the active component in a
liquid ester carrier; treating the suspension to generate an
aqueous formulation with addition of a phosphate moiety, a pepper
extract, or both; and treating the aqueous formulation to generate
droplets and immediately immersing the droplets in a basic curing
solution. The ester reacts with the basic curing solution to
release a salt that polymerizes the hydrolyzed whey protein
encapsulating the active component in the presence of the pepper
extract, the phosphate moiety, or both.
[0067] In another aspect, the invention features a method for
producing microcapsules comprising an active component, preferably
a creatine moiety, encapsulated within a polymerized hydrolyzed
protein shell, preferably a polymerized hydrolyzed whey protein
shell. Such a method includes mixing an organic acid, an alcohol,
hydrolyzed protein, and an active component to generate a
suspension of hydrolyzed whey protein and the active component in a
liquid ester carrier; treating the hydrolyzed protein suspension to
generate an emulsion, with addition of astaxanthin, and optionally
one or more of a phosphate moiety, and pepper extract, and
emulsified in the presence of a dispersing agent; treating the
emulsion to generate droplets and immediately immersing the
droplets in a basic curing solution optionally containing
additional phosphate and glycerol. The ester reacts with the basic
curing solution to release a salt that polymerizes the hydrolyzed
protein, encapsulating the active component in the presence of
astaxanthin and lipoic acid, and optionally pepper extract, a
phosphate moiety or both.
[0068] In the above practice, the droplets that are generated
comprise a core and a coating. This can be achieved using
concentric nozzles in which the emulsion is extruded through an
inner nozzle and a coating formulation, preferably comprising
hydrolyzed protein in a liquid ester, and optionally a phosphate
moiety, is extruded through the outer nozzle. The coating
formulation may also comprise the emulsion. The coating formulation
must comprise hydrolyzed protein in a liquid ester, but preferably
does not comprise the active agent.
[0069] Preferably, the process has an encapsulation efficiency of
between 92% and 98% as determined using the following equation:
Encapsulation efficiency (%)=((total loading creatine-creatine
losses)/total loading creatine).times.100
[0070] In some practices, the process employs creatine monohydrate,
typically crystalline creatine monohydrate. Ideally, the
crystalline creatine monohydrate has a prismoidal topography. In
some of these practices, the creatine monohydrate is spray-dried
creatine monohydrate. Among these are practices that include spray
drying to form crystalline creatine monohydrate, typically at low
temperature, an aqueous suspension of creatine monohydrate,
include, as an example, a suspension of creatine monohydrate in
alcohol.
[0071] Microcapsules formed according to the above-mentioned
methods were preferably prepared using the co-extrusion laminar jet
break-up technique (Encapsulator 1, Inotech, Switzerland). The
device was fitted with an inner nozzle (ranging from 20-300
micrometers) and an outer nozzle (ranging from 300-500 micrometers.
In a first formulation, this liquid ester suspension was treated
with phosphate and pepper extract and supplied to the inner nozzle
via sterile filtration coupled to a peristaltic pump to assist the
formation of liquid-core capsules.
[0072] In an alternative practice, the liquid ester suspension is
emulsified with pepper extract and astaxanthin and an oil-based
agent such as alpha lipoic acid. This results in a second
formulation. The first or second formulation will flow through the
inner nozzle and create the capsule inner core. The outer capsule
membrane is formed using the creatine liquid ester in the presence
of additional phosphate, which is supplied to the outer nozzle
using an air pressure regulator that enables flow rates ranging
from five to ten liters per hour under no more than 0.8 bar of
pressure.
[0073] Either the first or second formulation is extruded through a
heated nozzle into a weak basic environment. A typical nozzle is
between twenty and four hundred micrometers. A typical temperature
is thirty five degrees Celsius.
[0074] At this point, the pH increases and the ester reacts with
the base to release an acetate salt that instantly polymerizes the
protein suspension with simultaneous encapsulation of bioperine,
glycerol, phosphate, and bioactive material, e.g., creatine. If oil
core capsules are produced, again, the pH will increase, thereby
releasing an acetate salt that instantly polymerizes the protein
suspension with simultaneous encapsulation of bioperine, glycerol,
phosphate, astaxanthin, and alpha lipoic acid within the core with
bioactive material, e.g., creatine.
[0075] Having chosen flow rates that enable a stable jet of
creatine droplets through the nozzles, frequency and electrostatic
charge were set to have a stable bead chain visible in the strobe
light and a circular dispersion of the drops during their fall into
a gelling bath placed fifteen centimeters under the nozzle. This
gelling bath comprised 500 milliliters of di-sodium phosphate
buffer in 10 mM MOPS (3-(N-morpholino)propanesulfonic acid) with
0.04-0.07% w/v glycerol, at a pH of 7.4. The bath was magnetically
stirred to form a visible vortex. Droplet immersion of creatine
into this curing solution causes the instantaneous release of the
acetate salt that polymerizes the hydrolyzed protein. This further
encapsulates the creatine moiety within the capsule core and outer
whey protein membrane.
[0076] As a result, the first formulation generates creatine
monohydrate encapsulated in the presence of phosphate, glycerol
and, black pepper, surrounded by an outer membrane of hydrolyzed
whey protein.
[0077] The second formulation generates creatine monohydrate
encapsulated within an alpha-lipoic acid oil core in the presence
of phosphate, glycerol, and black pepper, further surrounded by an
outer membrane of hydrolyzed whey protein. Creatine capsules are
further incubated for twenty minutes in the basic curing buffer and
washed twice with 10 mM MOPS, with a final wash performed with
deionized water for thirty minutes.
[0078] As used herein, the term "microcapsule" refers to a particle
comprising an active component encapsulated within a hydrolyzed
protein shell and having an average diameter of less than a hundred
micrometers, ninety micrometers, eighty micrometers, seventy
micrometers, sixty micrometers, and fifty micrometers. Preferably,
the microcapsule has an average diameter of less than fifty
micrometers, forty micrometers, thirty micrometers, or twenty
micrometers. The method of measuring average diameter and D (v,
0.9) (size at which the cumulative volume reaches 90% of the total
volume), of micro-capsules is determined using a laser
diffractometer, such as the Mastersizer 2000 manufactured by Stable
Micro Systems, Surrey, UK, with a range of 0.2-2000 micrometers.
For particle size analysis, micro-bead batches were resuspended in
an ultrapure water, such as MILLI-Q water, and size distribution
was calculated based on the light intensity distribution data of
scattered light.
[0079] The term "protein gel" as used herein should be understood
to mean a sol in which the solid particles are meshed such that a
rigid or semi-rigid mixture results. The rigidity of the gel
structure will be determined by a texture analyzer, such as the
TA.XT analyzer. A gel is placed under a probe and, by running a
test, is compressed at 0.3 millimeters per second until it
collapses. The force, in grams, and the distance, in millimeters,
are measured and give the mechanical strength of the gel. The
process is repeated four to six times to ensure accuracy. The
strength of one gel can be calculated by dividing the strength
measured by the calculating the surface area of the gel particle
under the probe.
[0080] In some embodiments, the suspension comprises 10-25% or
10-20% hydrolyzed protein (w/v).
[0081] In some embodiments, the suspension comprises 75-90% or
80-90% active component (w/v).
[0082] In other embodiments, the suspension further comprises
0.01-0.05% pepper extract (w/v); 0.02-0.5 M phosphate moiety;
0.03-0.08% astaxanthin (w/v); and 0.60.9% dispersing agent
(w/v).
[0083] In yet other embodiments, the suspension comprises 10-25%
hydrolyzed protein (w/v); 75-90% active component (w/v); 0.01-0.05%
pepper extract (w/v); 0.02-0.5 M phosphate moiety; 0.03-0.08%
astaxanthin (w/v); and 0.6-0.9% dispersing agent (w/v).
[0084] In still other embodiments, the suspension comprises: 10-20%
hydrolyzed protein (w/v); 80-90% active component (w/v); 0.01-0.05%
pepper extract (w/v); 0.02-0.5 M phosphate moiety; 0.03-0.08%
astaxanthin (w/v); and 0.6-0.9% dispersing agent (w/v).
[0085] In still other embodiments, the hydrolyzed protein is
hydrolyzed whey protein.
[0086] In some embodiments, the active agent is a creatine
moiety.
[0087] In some embodiments, the pepper extract is bioperine.
[0088] In some embodiments, the dispersing agent is a fatty
acid.
[0089] In some embodiments, the dispersing agent is alpha-lipoic
acid.
[0090] In some embodiments, the suspension comprises: 0-20%
hydrolyzed whey protein (w/v), 80-90% creatine moiety (w/v),
0.025-0.035% bioperine (w/v), 0.03-0.04 M phosphate moiety,
0.04-0.06% astaxanthin (w/v), and 0.7-0.85% alpha-lipoic acid.
[0091] The term "liquid ester" should be understood to mean an
ester of an organic acid in a liquid form.
[0092] In some embodiments, the process includes an initial step in
which the ester is formed in-situ. Among these are embodiments in
which it is formed by reaction between an alcohol and an organic
acid.
[0093] Practices include those in which the alcohol is a weak
alcohol and those in which the organic acid is a weak organic
acid.
[0094] Also included are practices in which the step is carried out
in the presence of the hydrolyzed protein and/or the active
component.
[0095] The term "weak alcohol" should be understood to mean any of
a large number of colorless, flammable organic compounds that
contain the hydroxyl group (OH) and that slowly form esters with
acids. Simple alcohols, such as methanol and ethanol, are
water-soluble liquids, while more complex ones, like acetyl
alcohol, are waxy solids. Names of alcohols usually end in "ol."
Typical alcohol concentrations range from 0.2M-0.4 M (98%
purity).
[0096] Examples of weak organic acids include lactic acid, acetic
acid, formic acid, citric acid, and oxalic acid.
[0097] In some embodiments, the acid is acetic acid.
[0098] An organic acid is an organic compound with acidic
properties. The most common organic acids are the carboxylic acids,
whose acidity is associated with their carboxyl group --COOH.
Sulfonic acids, containing the group --SO.sub.2OH, are relatively
stronger acids. Alcohols, with --OH, can act as acids but they are
usually very weak. The relative stability of the conjugate base of
the acid determines its acidity. Typically, the acid has a
concentration of 0.5-0.65M.
[0099] Typically, the suspension has a concentration of carboxylic
ester of 0.1-0.6M, preferably 0.2-0.4M, and ideally about 0.3M.
[0100] In another aspect, the invention features a microcapsule
formed according to the methods described herein.
[0101] In yet another aspect, the invention features a multiplicity
of microcapsules formed according to the methods described
herein.
[0102] In another aspect, the invention features a comestible item
that comprises a multiplicity of microcapsules formed according to
the methods described herein. Examples of such comestible items
include a food product or beverage for human consumption.
[0103] In another aspect, the invention features a microcapsule
comprising an active component encapsulated within a polymerized
hydrolyzed protein shell. Among these are microcapsules having a
diameter of less than 100 micrometers, 90 micrometers, 80
micrometers, 70 micrometers, 60 micrometers, 50 micrometers.
Preferably, the microcapsules have an average diameter of less than
50 micrometers, 40 micrometers, 30 micrometers, or 20
micrometers.
[0104] Suitably, the microcapsule comprise pepper extract, ideally
bioperine, encapsulated within the polymerized hydrolyzed protein
shell.
[0105] Typically, the active component is a creatine moiety. As
used herein, a "creating moiety" is a molecule or complex
comprising creatine. Examples include a creatine complex, such as
creatine monohydrate, a creatine derivative, such as creatine ethyl
ester, and a creatine salt. Examples of creatine salts include
creatine hydrochloride and creatine nitrate.
[0106] In some embodiments, the microcapsules comprise alternative
or additional active components. Examples of alternative or
additional active components include active components for oral
delivery, such as glutamine, oil soluble bioactive substances, such
as vitamins and minerals, fatty acids, or fat soluble colors or
flavors. In some embodiments, the creatine monohydrate is
crystalline creatine monohydrate. Ideally, the crystalline creatine
monohydrate has a prismoidal topography (see FIG. 2A). Typically,
the creatine monohydrate is spray dried creatine monohydrate,
ideally crystalline creatine monohydrate obtained by spray drying
(typically at low temperature) an aqueous suspension of creatine
monohydrate, ideally a suspension of creatine monohydrate in
alcohol.
[0107] In some embodiments, the microcapsule comprises a phosphate
crosslinker, that crosslinks amino acids in the polymerized
hydrolyzed protein chains.
[0108] In some embodiments, the polymerized hydrolyzed protein
comprises glycerol.
[0109] In some embodiments, the hydrolyzed protein is hydrolyzed
whey protein, ideally hydrolyzed whey protein obtained from milk,
especially bovine milk. However, other types of hydrolyzed protein
can be employed. Examples include bovine collagen, pea, rice, or
non-whey milk proteins for hydrolyzing proteins.
[0110] A hydrolyzed protein of choice would be from a dairy source
ingredient with 90-95% protein (w/w) protein content. The ideal
ratio for milk proteins .beta.-lactoglobulin and
.alpha.-lactalbumin would be approx. 4:1, more specifically,
85:15.
[0111] Flavourzyme is a particularly good enzyme for hydrolysis of
diary proteins. Flavourzyme is a protease-peptidase complex
produced by submerged fermentation of a selected strain of
Aspergillus oryzae, as produced by Novo Nordisk A/S. It is
preferable to use Flavourzyme standardized in terms of Leucine
Amino Peptidase Units (LAPS) by the manufacturer. Hydrolysates for
the present invention are prepared using a measured amount of
Flavourzyme with 1000 LAPU with defined hydrolysis conditions.
[0112] Preferred temperatures are within the range of 35-65.degree.
C., preferably, 40-60.degree. C., ideally 45-50.degree. C.
[0113] Preferred pH values are within the range 4-9, preferably,
5-8, ideally 6.5-7.5.
[0114] Hydrolysis is typically performed with an enzyme/substrate
ratio (E/S) of 1/200, preferably, 1/150, and ideally 1/100, on the
basis of total protein content.
[0115] In some embodiments, the microcapsules comprises 10-25%
hydrolyzed protein (w/v).
[0116] In some embodiments, the microcapsule comprises 75-90%
active component (w/v).
[0117] In some embodiments, the microcapsule comprises 1.0-0.5%
pepper extract (w/v).
[0118] In some embodiments, the microcapsule comprises: 10-20%
hydrolyzed protein (w/v), 80-90% active component (w/v), and
0.01-0.05% pepper extract (w/v). Among these are embodiments that
also include a phosphate moiety.
[0119] In some embodiments, the microcapsule comprises: 10-20%
hydrolyzed protein (w/v), 80-90% active component (w/v), and
0.01-0.05% bioperine (w/v). Among these are embodiments that also
include one or more of a phosphate moiety, 0.04-0.07% glycerol
(w/v), 0.03-0.08% astaxanthin (w/v), and 0.6-0.9% alpha-lipoic acid
(w/v).
[0120] In some embodiments, the microcapsule comprises: 10-20%
hydrolyzed protein (w/v), 80-90% active component (w/v), 0.01-0.05%
bioperine (w/v), a phosphate moiety, 0.04-0.07% glycerol (w/v),
0.03-0.08% astaxanthin (w/v), and 0.6-0.9% alpha-lipoic acid
(w/v).
[0121] Embodiments also include those in which the microcapsule is
provided in the forms of powders, granular products, pastilles,
capsules, and effervescent tablets, solutions and gel products have
shown to be particularly suitable administration forms.
[0122] Still other embodiments use the creatine preparation in
combination with other active ingredient having a physiological
effect.
[0123] In some embodiments, the microcapsules are stable in water
for a period of at least twenty days.
[0124] In some embodiments, the microcapsules are stable in water
for a period of at least twenty-five days.
[0125] In some embodiments, the microcapsules are stable in water
for a period of at least or preferably twenty-eight days.
[0126] As used herein, "stable" means that there is no detectable
loss of encapsulated active agent after the time period for a 6.25%
suspension of microcapsules in water, i.e., five grams of
microcapsules dry weight in eighty grams of water.
[0127] In some cases, the encapsulation described herein benefits
animals. Practices therefore include administration to animals. If
the described creatine formulations are used as a feedstuff
additive, administration should in particular be regarded as
preferred for breeding and fattening animal and animals in
competitive sport. The methods also include administration to
horses, pigs, poultry, and fish.
[0128] In another aspect, the invention features a manufacture
comprising multiplicity of microcapsules as described herein.
[0129] Embodiments include those in which the manufacture includes
a comestible product, those in which it includes a comestible
sports nutrition product, those in which it includes a food, those
in which it includes a beverage, and those in which it includes a
supplement.
[0130] Embodiments also include those in which the manufacture
includes a powder, a particulate material, a unit dose product, and
a tablet.
[0131] In some embodiments, the manufacture includes a beverage,
including a sports nutritional beverage, that has a multiplicity of
microencapsulates as described herein suspended in a liquid
carrier.
[0132] In some embodiments, the manufacture comprises a snack bar,
including a sports nutritional snack bar, that includes a
multiplicity of microencapsulates as described herein suspended in
an edible carrier.
[0133] In some embodiments in which the manufacture is a comestible
product, including those in which it is a beverage and those in
which it is a snack bar, the manufacture includes gelled hydrolyzed
protein that has a degree of hydrolysis of less than 50%, a degree
of hydrolysis of less than 40%, a degree of hydrolysis of less than
30%, and a degree of hydrolysis of less than 20%.
[0134] In some embodiments in which the manufacture is a comestible
product, including those in which it is a beverage and those in
which it is a snack bar, the gelled hydrolyzed protein has a degree
of hydrolysis of between 80%-85%.
[0135] In another aspect, the invention features a method for
making a crystalline creatine monohydrate in which crystals have a
low particle size distribution and a stable crystalline structure.
In some practices, the average particle size is less than 10
micrometers. This is advantageous for applications in which the
crystalline creatine monohydrate is to be encapsulated.
[0136] Among other practices are those in which the crystalline
creatine monohydrate has a substantially prismoidal topography.
This is shown in FIG. 2A. Typically, at least 50%, 60%, 70%, or 80%
(v/v) of the crystals have a particle size of 1 to 10
micrometers.
[0137] In another aspect, the invention features a method of
preparing crystalline creatine monohydrate having a narrow particle
size distribution. Such a method include preparing an aqueous
suspension of creatine monohydrate and spray-drying the aqueous
suspension to generate crystalline creatine monohydrate having a
narrow particle size distribution. Typically, the spray-drying step
is carried out at a low temperature range of 30-70 degrees. A
preferable range is 40-60 degrees and an optimal range is 50-55
degrees.
[0138] In another aspect, the invention features a crystalline
creatine monohydrate formed according to a method of the
invention.
[0139] In another aspect, the invention features a non-therapeutic
method of increasing athletic performance in an individual
comprising the steps of administering, to the individual, a
comestible product as described herein in which the active
component comprises a creatine moiety, preferably creatine
monohydrate, and the microencapsulates in the preparation are
broken down in the gastrointestinal tract of the individual to
release the active component.
[0140] These and other features of the invention will be apparent
from the following detailed description and the accompanying
figures, in which:
BRIEF DESCRIPTION OF THE FIGURES
[0141] FIG. 1, which is spread across two sheets and six panels,
shows scanning electron microscope images of raw creatine
monohydrate,
[0142] FIG. 2 shows creatine monohydrate after spray drying in the
presence of pharmaceutical grade ethanol,
[0143] FIG. 3 shows X-ray diffraction data for consecutive steps
within an encapsulation process,
[0144] FIG. 4 shows data from an atomic force microscope
illustrating the presence of a second form of the creatine crystal
within milk protein encapsulation matrices,
[0145] FIG. 5 shows thermal gravimetrical analysis data of free
creatine and encapsulated creatine,
[0146] FIG. 6 shows creatine and creatine detection by
high-performance liquid chromatography ("HPLC") whereby creatine
eluted after 2.25 minutes and creatine generated a narrow peak
after 6.1 minutes,
[0147] FIGS. 7A and 7B show commercial creatine monohydrate
degradation in the aqueous incubation medium compared to
encapsulated creatine with standard deviation being the average for
eleven independent studies,
[0148] FIG. 8A is a scanning electron microscope image of an
incomplete coating of creatine using native whey protein,
[0149] FIG. 8B is a scanning electron microscope image of a single
encapsulated particle,
[0150] FIG. 8C is a scanning electron microscope image of an
additional hydrolyzed protein coating
[0151] FIG. 8D is a scanning electron microscope image of a
microparticle after initial intestinal digestion,
[0152] FIG. 8E is a scanning electron microscope image of released
creatine for absorption into the bloodstream,
[0153] FIG. 9 shows concentration of creatine monohydrate during
28-day storage in aqueous solution at pH 4.0 at room temperature
with four different treatments, namely hydrolyzed milk protein
capsules, hydrolyzed milk protein capsules with bioperine, native
whey protein capsules, and creatine in denatured whey protein
capsules at 25.degree. C. for up to 28 days followed by three hours
of exposure to ex vivo stomach contents at a pH of 1.6 for three
hours,
[0154] FIG. 10 shows concentration of creatinine during formation
during 28-day storage in aqueous solution at pH 4.0 at room
temperature with the following treatments: hydrolyzed milk protein
capsules, hydrolyzed milk protein capsules with bioperine, native
whey protein capsules, and creatine in denatured whey protein
capsules at 25.degree. C. for up to 28 days followed by three hours
of exposure to ex vivo stomach contents at a pH of 1.6, and
[0155] FIG. 11 shows various levels of creatine absorption tested
using standard absorption tests involving Caco-2 monolayers,
including tests of apical to basolateral permeability of free and
encapsulated creatine to mimic in vivo conditions, with the apical
permeability at a pH of six and the basolateral permeability at a
pH of seven.
DETAILED DESCRIPTION
[0156] The subject matter described herein includes a method for
controlling the timing of acetate-mediated polymerization of milk
proteins for the encapsulation of bioactive materials, with
particular interest in creatine monohydrate in the presence of
black pepper extract and astaxanthin, and also for controlling the
release of the encapsulated bioactive material by controlling
phosphate cross-linking and digestive properties of the
encapsulation system in order to enhance the absorption, uptake,
and muscle utility of active creatine.
[0157] The subject matter provides a bioactive material, and in
particular, creatine monohydrate, with structural features for long
term stability via encapsulation of an aqueous formulation that
contains milk protein, black pepper extract, bioperine, i.e.,
CAS:94-62-2, astaxanthin, i.e., CAS AS 472-61-7, an alcohol, and an
organic acid. This combination of substrates will naturally produce
an ester, which subsequently produces a salt upon reaction with a
weak base. This produces a polymerized protein matrix stabilized by
intra-molecular disulphide bonds. Residual alcohol generated during
this reaction is subsequently removed during the drying
process.
[0158] The incorporation of bioperine enhances creatine stability
against stomach acid and enzymatic digestion on the luminal side of
the gastro-intestinal tract. As a result of the digestibility of
the hydrolyzed whey protein capsules, encapsulated creatine will be
released at the proximal ileum to enable absorption and uptake of
creatine into the blood stream from the luminal side. In this way,
encapsulation promotes the absorption efficiency and
bioavailability of creatine monohydrate.
[0159] The presence of cross-linked phosphate enables the
accelerated generation of ATP during creatine administration. The
presence of glycerol promotes fluid retention during exercise and
muscle contraction. The incorporation of black pepper extract
and/or astaxanthin in the presence of alpha lipoic acid further
promotes the bio accessibility of creatine for muscles.
Furthermore, the presence of hydrolyzed milk protein eliminates the
allergenic nature of the final product.
[0160] The method provides mild process conditions for the
production of functional and bioavailable creatine monohydrate for
incorporation into a beverage. Previous inventions failed to
adequately protect creatine monohydrate from heat and low pH during
storage and delivery in beverage formats with added functional
ingredients to enhance bioavailability in the blood and subsequent
bio accessibility in the muscle.
[0161] This process for stabilization of bioactive material has the
ability to combine, protect, and release functional ingredients at
site-specific absorption sites in the gastro-intestinal tract to
achieve synergistic ergogenic effects with enhanced hydration
capacity to assist long term muscle contraction. Creatine capsules
are small, i.e., less than 50 microns, mono-dispersed, homogenous,
and spherically-shaped stabilized particles, with a narrow size
distribution. They are produced quickly and under mild and simple
encapsulation conditions at low cost and with high encapsulation
efficiencies, measured as a percentage of product encapsulated, for
commercial production.
[0162] The subject matter described herein includes an
encapsulation process for bioactive components that uses creatine
monohydrate as the test material. Aqueous suspensions are prepared
for initial molecular crystallization in the presence of
crosslinking agents. This is followed by extrusion encapsulation.
The technology enables the production of aqueous-core capsules or
oil-core capsules through incorporation of astaxanthin using an
oil-based dispersing agent.
Step 1: Molecular Stabilization
[0163] Scanning electron microscopy provided a valuable tool for
the visualization and ultimate optimization of the best
encapsulation system for efficient delivery of bioactive materials
such as creatine monohydrate. FIG. 1 shows an image of free
creatine monohydrate. It is clear that the structure of raw
creatine monohydrate is highly unstable as a monohydrate
material.
[0164] Large particles shown in panels A-D of FIG. 1 illustrate a
potential to break down into smaller particles with a greater
hydration capacity. Panel D of FIG. 1 illustrates dehydration
layers that typically correspond to an unstable compound. These are
pointed out by the arrow. Panels E and F of FIG. 1 illustrate the
unfavorable broad size distribution of commercially available
creatine monohydrate.
[0165] Particle sizes ranged all the way from a few microns to over
six hundred microns. This was not acceptable for either stability
or for further encapsulation. Hence, before initiating
encapsulation, it was imperative to generate creatine with a more
even distribution of sizes and a stable crystal structure.
[0166] To achieve the foregoing, creatine monohydrate was
spray-dried using pharmaceutical-grade ethanol at lower
temperatures. This maintained the functional attributes of
creatine. Following spray-drying, the creatine produced was
assessed for suitable size distribution and for its crystal
structure.
[0167] Panel A in FIG. 2 shows prismoidal creatine. This prismoidal
form ultimately generates a crystal with a large surface area. A
large surface area promotes bonding with the encapsulation polymer.
The creatine shown is suitable for encapsulation because the
particle sizes are less than ten microns.
Step 2: Encapsulation
Production of Aqueous Encapsulation Systems:
[0168] Micro-dispersed whey protein microcapsules were prepared
based on laminar jet break-up for the generation of whey protein
micro-capsules loaded with creatine monohydrate and bioperine.
Liquid ester carrier was delivered to a nozzle via a feed line. The
nozzle had a diameter that was between twenty and one thousand
micrometers. A PTFE membrane connected the nozzle to a vibrating
device. The vibrating device was insulated from the surrounding
structures by rubber mounts to avoid the generation of resonance
frequencies in the system.
[0169] The method includes preparing an aqueous formulation. Such a
formulation includes the bioactive material (i.e., creatine
monohydrate), milk protein, a pharmacological agent (i.e., a weak
alcohol), and an organic acid (i.e., acetic acid). This combination
of substrates naturally produces an acetate ester that is stable at
room temperature. However, no salt is present to initiate protein
polymerization. Therefore, the suspension remains in a fluid
state.
[0170] In a first formulation, the creatine liquid ester is treated
with phosphate and black-pepper extract and fed to the nozzle via
sterile filtration coupled to a peristaltic pump to assist the
formation of aqueous capsules. The protein-creatine-ester blend is
aseptically extruded through the assigned nozzle to generate a
steady stream of droplets regulated by air pressure enabling flow
rates ranging from ten to fifteen liters per hour under a pressure
of no greater than 0.6 to 0.8 bar.
[0171] After having chosen flow rates to generate a stable jet of
droplets through the nozzles, the next step is to set frequency and
electrostatic charge to cause formation of a stable bead chain
visible in a strobe light and a circular dispersion of drops during
falling into a gelling bath. The gelling bath comprised an alkaline
phosphate buffer (0.4M) placed fifteen centimeters under the
nozzle. The basic gelling bath was continuously agitated to avoid
coalescence or flocculation of microcapsules during curing.
[0172] Some practices include inducing charge on the mononuclear
droplets to promote their dispersion and to prevent coalescence
from occurring in the air and/or upon impact. Such coalescence
would result in formation of droplets and/or larger gelled
particles. The charge is applied at values ranging between 0.8-1.1
millivolts.
[0173] Upon landing in the phosphate gelling bath, high surface
tension may retard droplet movement. This retardation can result in
gelled particles having irregular shapes. In some instances this
delay can cause the droplet to burst. This would release the
creatine liquid ester carrier before encapsulation takes place.
[0174] To avoid this difficulty, it was useful to reduce surface
tension by adding surfactant and/or by slightly heating the
phosphate solution, for example to a temperature that is between
fifty and sixty degrees Celsius. Doing so permitted a drop to enter
the solution more quickly, thereby reducing a risk of its
deformation and promoting immediate encapsulation.
[0175] For this reason, it was important to include glycerol in the
phosphate gelling bath with a temperature of thirty-five degrees
Celsius. The presence of glycerol in the gelling bath also resulted
in glycerol being incorporated into the final creatine capsule. Due
to the fact that glycerol has favorable hydration properties for
muscle function, the inclusion of glycerol in the encapsulation
system comes with its own functional and ergogenic benefit.
[0176] The gelling bath comprised five hundred milliliters of
di-sodium phosphate buffer in 10 mM MOPS with 0.04-0.07% w/v
glycerol, 0.6-0.9% (w/v) alpha lipoic acid at a pH of 7.4. The bath
was magnetically stirred to form a visible vortex. Droplet
immersion of creatine into this curing solution caused the
instantaneous release of acetate salt, which in turn polymerized
the hydrolyzed protein, which further encapsulated the creatine
moiety within the gelled structure in the presence of black pepper
extract phosphate and glycerol.
[0177] During jet break-up and/or when entering the gelling bath, a
high negative charge was induced onto the droplet's surface using
an electrical potential of 0-2.15 kV between the nozzle and an
electrode that was placed directly underneath the nozzle. As
creatine droplets fell through the electrode, they were deflected
from their vertical path. This promoted droplet impact over a
larger area in the gelation solution. This enabled mono-disperse
capsules with a standard size deviation of less than .+-.1.5% to be
generated.
[0178] Within the gelling bath, several instantaneous reactions
occurred. When the droplet entered the gelling bath, the pH
increased and the ester reacted with the base to release an acetate
salt that quickly polymerized the protein suspension with
simultaneous encapsulation of bioperine, glycerol, phosphate and
bioactive material, such as creatine. This reaction produced
residual amounts of alcohol. This alcohol was subsequently removed
during the final drying process to alleviate any difficulties
associated with alcohol in food.
[0179] Pliable micro-beads were cured and/or polymerized at room
temperature in the phosphate buffer, recovered, and then washed
twice in sterile water. Matrix characterization was then performed
as a function of cure time in buffer, the cure time being less than
three hours. The beads were then washed twice with ten millimolar
MOPS, with a final wash performed with deionized water for thirty
minutes. Optimum parameters for a given protein-creatine suspension
were logged and utilized without adjustment during further batch
production.
[0180] The production of less than fifty milliliters of micro-beads
was sufficient to meet the requirements of preliminary studies.
Hence, the encapsulator resembled a batch-reactor. Commercial
production of aqueous gel creatine particles has been optimized
based on the aforementioned principle. As a result, this aqueous
encapsulation methodology generates creatine monohydrate
encapsulated in the presence of phosphate, glycerol and, black
pepper, in a gelled hydrolyzed whey protein matrix.
Preparation of Oil-Core Encapsulation Systems:
[0181] A concentric system with two running liquids was essential
for the generation of microcapsules with addition oil cores. This
was achieved by simultaneously supplying two feed lines to a
specifically designed concentric nozzle unit. This generated a
co-extruded laminar liquid jet, which was subsequently broken-up
into mononuclear drops by the application of a vibrational
frequency. The creatine liquid ester carrier was then gelled into
the desired mononuclear microcapsules, each comprising an inner oil
core and a whey protein outer membrane.
[0182] The capsule diameter was mainly dependent on the diameter of
the outer nozzle. As was the case for the single nozzle system used
for aqueous systems, it was possible to vary the size within a
certain range by increasing/decreasing the applied flow rate and
vibrational frequency. The diameter of the internal nozzle and the
flow rate of the material also affected the final capsule size. In
particular, increasing diameters and volumes resulted in larger
core volumes, therefore, larger microcapsules.
[0183] Micro-dispersed whey protein microcapsules were prepared
based on laminar jet break-up for the generation of whey protein
micro-capsules loaded with creatine monohydrate and bioperine. The
liquid ester carrier was delivered to the nozzle via a feed line
using two nozzles with diameters in the range twenty to a thousand
micrometers. The nozzle was connected, via a PTFE membrane, to a
vibrating device, which was insulated from the surrounding
structures by rubber mounts to avoid the generation of resonance
frequencies in the system.
[0184] Oil-based formulations were prepared. These comprised the
bioactive material (e.g., creatine monohydrate), milk protein, a
pharmacological agent (e.g., weak alcohol) and an organic acid
(e.g., acetic acid). This combination of substrates naturally
produced an acetate ester that was stable at room temperature.
However, no salt was present to initiate protein polymerization.
Therefore the suspension remained in a fluid state.
[0185] In a second formulation, creatine liquid ester was
emulsified with black pepper extract, astaxanthin, and an oil-based
agent such as alpha lipoic acid. This formulation would flow
through the inner nozzle, which was heated to thirty-five degrees
Celsius, and create the capsule's inner core. The outer capsule
membrane was formed using the creatine liquid ester in the presence
of additional phosphate, which was supplied to the outer nozzle
using an air-pressure regulator that enabled flow rates ranging
from five to ten liters per hour at an air pressure no greater than
0.7-0.9 bar.
[0186] At this point, the pH rose and the ester reacted with the
base to release an acetate salt that instantly polymerized the
protein suspension with simultaneous encapsulation of bioperine,
glycerol, phosphate, astaxanthin, and alpha lipoic acid within the
core with bioactive material, e.g., creatine. This reaction
produced residual amounts of alcohol, which was subsequently
removed during the final drying process, to avoid the presence of
alcohol in food.
[0187] Having chosen flow rates that enabled a stable jet of
creatine droplets through the nozzles, frequency and electrostatic
charge were then set to promote a stable bead chain that would be
visible in the strobe light and to cause circular dispersion of the
drops as they fell into a gelling bath that was placed fifteen
centimeters under the nozzle.
[0188] The production of less than fifty milliliters of micro-beads
was sufficient to meet the requirements of preliminary studies.
Hence the encapsulator resembled a batch-reactor.
[0189] The protein-creatine-ester blend was aseptically extruded
through the assigned nozzle into 0.4M alkaline phosphate buffer
tempered to thirty-five degrees Celsius. The buffer was
continuously agitated agitation to avoid coalescence or
flocculation of microcapsules during the curing process. The
gelling bath comprised five hundred milliliters of di-sodium
phosphate buffer in 10 mM MOPS with 0.04-0.07% w/v glycerol. The
gelling bath was maintained at a pH of 7.4 and also stirred
magnetically to form a visible vortex. Droplet immersion of
creatine into this curing solution caused the instantaneous release
of the acetate salt that polymerized the hydrolyzed protein. This
further encapsulated the creatine moiety within the capsule core
and outer whey protein membrane.
[0190] Some practices feature inducing a charge to the mononuclear
droplets to promote their dispersion and also to prevent
coalescence from occurring either in the air and/or upon impact,
which could result in the formation of duplets and/or larger
microcapsules. This charge must be applied at higher values
compared to the mono-centric nozzle system to enable similar
droplet dispersion to be achieved. This is due to the smaller
percentage of polyelectrolyte present in the droplet because of the
core material.
[0191] Upon landing in the phosphate gelling bath, high
surface-tension momentarily retards droplet movement. This can lead
to formation of oval capsules. In some instances this delay can
cause the droplet to burst, thus releasing the creatine liquid
ester carrier before encapsulation takes place. It is hypothesized
that this bursting is caused by the movement of the core liquid out
through the pre-hardened membrane protein when capsules are been
held back briefly at the surface of the hardening solution and
hence results in release, by bursting, of the core creatine
liquid.
[0192] To avoid the deleterious effects of high surface tensions,
it is useful to reduce the surface tension by either adding a
surfactant or by slightly heating the phosphate solution, for
example to a temperature that is between fifteen and sixty degrees
Celsius. This promotes quicker entry of the drop into the solution
and thereby suppresses its tendency to deform otherwise. It also
results in immediate encapsulation and thus results in a more
efficient encapsulation procedure.
[0193] For this reason, it is particularly useful to include
glycerol in the phosphate gelling bath and to temper the bath to
about thirty-five degrees Celsius. As a side benefit, having
glycerol in the bath results in incorporation of the glycerol into
the final creatine capsule. This permits the consumer to enjoy the
functional and ergogenic benefits of glycerol's favorable hydration
properties for muscle function.
[0194] During jet break-up and/or when entering the gelling bath, a
high negative charge was induced onto their surface by exposing the
drop to an electrical potential of up to 2.15 kilovolts between the
nozzle and an electrode that is placed directly underneath the
nozzle. As creatine droplets fall through the resulting electric
field, they are deflected from their vertical path. This results in
the drops' impact on the gelling bath occurring over a larger area.
This enabled mono-dispersed microcapsules with a standard size
deviation of less than .+-.1.5% to be generated.
[0195] Within the gelling bath, several essentially instantaneous
reactions occur. When a droplet enters the gelling bath, the pH
increases and the ester reacts with the base to release an acetate
salt that instantly polymerizes the protein suspension with
simultaneous encapsulation of bioperine, glycerol, phosphate and
bioactive material, e.g., creatine. This reaction produces residual
amounts of alcohol, which is subsequently removed during the final
drying process, thereby avoiding the contamination of food with
alcohol.
[0196] Pliable micro-beads were cured and/or polymerized at room
temperature in the phosphate buffer, recovered, and washed twice in
sterile water. A matrix characterization was then performed as a
function of cure time in the buffer, with the cure time varying
between zero and three hours. The product was then washed twice
with 10 mM MOPS, with a final wash performed with deionized water
for thirty minutes. Optimum parameters for a given protein-creatine
suspension were logged and used without adjustment during further
batch production.
[0197] The production of fewer than fifty milliliters of
micro-beads was sufficient to meet the requirements of preliminary
studies. Hence the encapsulator resembled a batch-reactor.
Commercial production of aqueous gel creatine particles has been
optimized based on the aforementioned principle. As a result, this
aqueous encapsulation methodology generates creatine monohydrate
encapsulated in the presence of phosphate, glycerol and, black
pepper, in a gelled hydrolyzed whey protein matrix. This oil-based
encapsulation system generates creatine monohydrate encapsulated
within an alpha-lipoic acid oil core in the presence of phosphate,
glycerol, and black pepper, further surrounded by an outer membrane
of hydrolyzed whey protein.
[0198] The incorporation of bioperine to the formulation enhances
the absorption efficiency of the bioactive within the
gastro-intestinal tract. The presence of hydrolyzed milk protein
eliminates the allergenic nature of the final product. This
formulation has been optimized for the production of more than a
thousand kilograms of encapsulated bioactive in a single batch
under sterile conditions.
[0199] The proposed aqueous and oil-core microcapsules containing
encapsulated creatine can be manufactured using the aforementioned
techniques on large-sale, for example more than four hundred liters
per day, by using vibrating jet technology and subsequently drying
by either drum drying or fluidized-bed drying. The dried product
can then be stored for subsequent addition to a beverage to assist
creatine bioavailability in the blood and, more importantly bio
accessibility of the creating to the muscle during exercise.
[0200] X-ray diffraction (XRD) is a versatile, non-destructive
technique utilized to detail the chemical composition and
crystallographic structure of creatine monohydrate before and after
the encapsulation process. In order to better convey an
understanding of the fundamental principles of X-ray diffraction
instruments, the terms "amorphous" and "crystalline" are defined
below.
[0201] In the "amorphous" state, atoms are randomly arranged as
they would be in a liquid. Whey protein is amorphous.
[0202] In a "crystalline" state, there exists a lattice, which is
regular three-dimensional distribution of atoms in space. A variety
of lattices exist, among which are cubic and rhombic lattices.
These atoms are arranged so that they form a series of parallel
planes separated from one another by a distance, d, that varies
according to the nature of the material. For any crystal, planes
exist in a number of different orientations, each with its own
specific d-spacing.
[0203] Commercial creatine monohydrate, in its raw form, is in a
first crystalline form that reacts readily with water. This form is
somewhat unstable. It is therefore desirable to transform it into a
second crystalline form, which is more stable.
[0204] FIG. 3 shows X-ray diffraction data representative of
creatine stability. After molecular stabilization, creatine
monohydrate appears to be less amorphous. This makes it less
vulnerable to creatinine production.
[0205] X-ray diffraction analysis also serves as a successful
method to determine encapsulation efficiency of the system. This is
because creatine is crystalline and whey protein, which serves as
the encapsulation matrix, is amorphous. Hence, if creatine is
successfully encapsulated by whey protein, X-ray diffraction will
not show any crystalline structures. This is because all the
creatine would have been amorphous whey protein.
[0206] However, if creatine is only partially encapsulated by whey
protein, X-ray diffraction data would reveal the existence of some
crystalline material. This would suggest the existence of free
crystalline creatine that has not interacted with whey protein.
[0207] In FIG. 3, X-ray diffraction data illustrates commercial
(raw) creatine monohydrate on the baseline curve L1 to be clearly
crystalline. The intensity of the two peaks midway along the
profile reveals this first crystalline form.
[0208] Following molecular stabilization using low-temperature
spray-drying, the second crystalline form is generated. The
existence of this second crystalline form is manifested in lesser
intensity of crystalline peaks illustrated for the L2 curve. Whey
protein encapsulation material was also analyzed to confirm this
amorphous form of whey protein and this was validated in the L3
curve.
[0209] During the encapsulation process, specialized hydrolyzed
whey protein demonstrated successful encapsulation efficiency for
creatine monohydrate. Interestingly addition of bioperine did not
adversely affect the encapsulation efficiency and full
encapsulation capacity in the X-ray diffraction profile, as shown
in curve L5. However utility of native and denatured whey protein
failed to successfully encapsulate creatine.
[0210] Based on the results shown in FIG. 3, it is evident that
creatine is efficiently encapsulated using hydrolyzed whey protein
in the presence of bioperine for enhanced absorption capacity. It
is clear that the first step generated an appropriate molecular
structure for efficient creatine encapsulation with hydrolyzed whey
protein in the presence of bioperine.
[0211] FIG. 4 shows atomic force microscopy data illustrating the
existence of embedded creatine monohydrate crystals within milk
protein encapsulation systems. These crystals have taken the second
crystal form, thus promoting protection of creatine from water.
Individual creatine crystals having an approximate size of between
ten and twenty micrometers may solely occupy a whey protein
capsule. However the functionality remains the same per batch of
encapsulated creatine produced.
Thermal Stability
[0212] The thermal gravimetrical analysis data shown in FIG. 5
compares raw and encapsulated creatine. Based on this data, there
was no change in the thermal properties or compositional structure
of commercial creatine as a result of having been encapsulated. The
thermal gravimetrical analysis thus demonstrates that the
degradation temperature of creatine remained the same before and
after encapsulation. Hence, in the presence of encapsulation
structures, creatine does not undergo undesirable degradation.
Furthermore, weight fluctuations were unaffected by changes in
temperature. This illustrates that the creatine monohydrate
retained its compositional structure and reactive properties
following encapsulation.
[0213] FIG. 6 shows detection of creatine and creatinine
concentrations using standardized high-performance liquid
chromatography. Following validation of the high-performance liquid
chromatography technique, stability trials were performed for free
and encapsulated creatine in water held at twenty-five degrees
Celsius for ten hours.
[0214] The results demonstrated that degradation of free creatine
followed first-order kinetics. Based on the slope of the line, the
first-order degradation rate constant was calculated as 0.0263 per
day at twenty-five degrees Celsius for free creatine
monohydrate.
[0215] Substantial conversion of creatine into creatinine was
recognized in aqueous formulations. These demonstrated significant
differences from those identified for encapsulated creatine.
[0216] Encapsulated formats revealed that no creatine had been
converted into creatinine in the presence of water after twelve
hours. Even after forty-eight hours of continued storage, there was
no evidence of creatinine production. This validates the
encapsulation conditions used for the protection of creatine in
beverages, particularly beverages that are intended as supplements
used by athletes engaging in sports.
[0217] FIGS. 7A and 7B illustrate the creatine content reduction in
water.
[0218] FIG. 7A shows that the concentration of commercial creatine
monohydrate fell by 66% following only fifteen minutes of
incubation in water at room temperature. After an hour, only about
9%.+-.1.34% of the initial creatine concentration remained. This
reduction demonstrates a direct correlation with an increase in
creatinine formation after fifteen minutes.
[0219] In contrast, as shown in FIG. 7B, encapsulated creatine was
significantly more stable in water solution. Even after three hours
at room temperature, there was no significant detection of
creatinine. Hence, creatine encapsulation provides a useful
delivery vehicle for creatine monohydrate in an aqueous
beverage.
[0220] Following this, accelerated shelf-life tests were conducted
with final sports-drink samples that were formulated according to
industrial standards. Encapsulated creatine demonstrated more than
three years of shelf-life stability in such aqueous environments.
Furthermore, high-performance liquid chromatography analysis
confirmed the absence of creatinine after completion of shelf-life
testing. Hence, encapsulated creatine fulfilled the stability
criteria for storage of beverage formulations.
[0221] The methods and compositions described herein provide
milk-protein encapsulation vehicles with desired mechanical
rigidity, resistance to deformation, strength, and resistance to
fracture in order to structurally protect creatine monohydrate from
aqueous solutions during long storage times with concomitant
release at the required systemic target site. Microencapsulates as
described herein demonstrated acceptable long-term storage
stability, namely as much as three years, with further sustained
stability in simulated stomach conditions in the presence of
pepsin. Microscopy and chromatography further validated the
targeted disintegration of protein matrices in physiological
intestinal conditions after several minutes with bioperine
providing enhanced absorption capacity.
[0222] The microbead degradation is catalyzed by the synergistic
effect of a neutral pH and enzymatic action. This property is one
that can be exploited for manufacture of specialized creatine
sports supplements. For this reason, optimization of encapsulation
conditions represented the basis of creatine stabilization in the
presence of creatine protective chaperones, such as milk protein
and bioperine.
[0223] Because bioperine is highly lipophilic, the concentration of
bioperine potentially increases the lipophilicity of the creatine
compound. This, in turn, would improve its ability to diffuse
through biological membranes.
[0224] In contrast, creatine is lipophobic. As such, creatine
generally requires a transporter to cross the lipid-rich plasma
membrane of a typical cell. The methods disclosed herein result in
a stable creatine-milk protein-bioperine moiety that demonstrated
reduced creatine degradation and increased half-life in aqueous
solutions. Hence, encapsulation in hydrolyzed milk protein
represents an excellent matrix for site-specific controlled
delivery and release of creatine with subsequent promotion of its
absorption at their target site.
[0225] FIGS. 8A-8E shows images of the progression of creatine
encapsulation in real-time.
[0226] FIG. 8A shows partial encapsulation of creatine using native
whey protein. It is apparent that encapsulation is not quite
complete. This can be compared to FIG. 8B, in which the native whey
protein has been replaced by hydrolyzed protein as an encapsulation
matrix.
[0227] FIG. 8C shows creatine encapsulated in whey protein with
bioperine outer membrane layers.
[0228] FIG. 8D shows the microcapsule having been partially
digested as a result of intestinal incubation. In FIG. 8D, one can
see erosion of protein matrix material as a result of the enzymatic
action of intestinal contents. After about three minutes of
intestinal incubation, creatine monohydrate was fully released for
subsequent absorption.
Creatine Storage Stability and Ex Vivo Digestion
[0229] The ability to adhere to the intestinal epithelium is
important for rapid absorption of encapsulated material into the
blood stream. As such, an important factor for efficacious
encapsulation of creatine is the extent to which the microparticles
adhere to the intestinal epithelium after intestinal liberation of
the encapsulated creatine.
[0230] Whey protein micro-particles have been found to be suitable
ex vivo delivery vehicles for delivery of active creatine along a
porcine gastro-intestinal tract with ileal tissue adhesion
indicating rapid absorption into the blood stream. After
twenty-eight days of storage in an aqueous solution at a pH of
four, creatine encapsulated in hydrolyzed protein illustrated
almost no loss in creatine concentration. Furthermore, creatinine
was not detected at any significant levels after 28-day storage in
hydrolyzed milk protein encapsulation systems.
[0231] FIG. 9 shows that subsequent gastric incubation maintained
complete creatine concentration with no detection of creatinine.
Creatine encapsulated in various forms of milk protein failed to
express significant protective properties for creatine after 28-day
water storage as illustrated in FIG. 10. Therefore, native and
denatured milk protein matrices expressed weak protective
properties for creatine and resulted in significant increases in
creatinine concentrations. Hence, hydrolyzed whey protein
encapsulation systems represent the only treatment capable of
providing storage stability and acid tolerance to creatine
monohydrate during beverage storage and stomach incubation.
Hydrolyzed protein provides an encapsulation vehicle capable of
maintaining maximum creatine concentrations of about 8 milligrams
per milliliter.
Absorption Capacity
[0232] Various levels of creatine absorption were tested using
standard absorption tests involving Caco-2 monolayers. Apical to
basolateral permeability of free and encapsulated creatine were
tested and prepared to mimic in vivo conditions i.e., apical
pH=6.0/basolateral pH=7.0). FIG. 11 shows that creatine absorption
was significantly enhanced as a result of electrostatic interaction
with bioperine. Electrophoretic mobility data demonstrated that
free creatine had a zeta potential of -2.4 mV compared to -23.14 mV
for creatine in the presence of bioperine. Hence, creatine
absorption was significantly enhanced as a result of the
electrostatic interaction generated during the formation of the
creatine-bioperine complex during the encapsulation process.
[0233] As illustrated in FIG. 11, it is clear that absorption of
encapsulated creatine was highly dependent on molecular charge of
creatine i.e., encapsulated creatine generated the substantial
molecular charge in the presence of bioperine at the pH utilized
during encapsulation. Hence electrostatic potential of encapsulated
creatine provided sufficient aqueous solubility for creatine
solubility in fluids of the absorption site and lipid solubility in
the presence of bioperine to allow sufficient partitioning of
creatine into lipoidal membranes and systemic circulation.
[0234] The invention is not limited to the embodiments herein
before described which may be varied in construction and detail
without departing from the spirit of the invention.
* * * * *