Note: Descriptions are shown in the official language in which they were submitted.
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REDUCTION OF ASTRINGENCY IN POLYPHENOL COMPOSITIONS
RELATED APPLICATIONS
[0001] This application is a continuation of United States Patent Application
Serial No. 11/958,556 filed on December 18, 2007, which is based on and claims
benefit to U.S. Application No. 11/616,572, filed on December 27, 2006, both
of
which are hereby incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates to microencapsulated polyphenol
compositions suitable for use in food and beverage products and methods for
producing such microencapsulated polyphenol compositions. Microencapsulation
significantly reduces the astringency and/or bitterness of the polyphenol
compositions and protects the polyphenol compositions from oxidation,
ingredient
interactions, enzymatic degradation, and/or the like while maintaining
gastrointestinal bioavailability within the digestive system.
BACKGROUND OF THE INVENTION
[0003] Naturally-occurring polyphenols derived from plants or plant materials
(e.g, tea, cocoa beans, and the like) are known to have antioxidant properties
as
well as providing other potential health benefits. Thus, considerable research
has
been carried out in recent years with regard to methods for obtaining such
polyphenols as well as methods for using them.
[0004] Generally, however, such polyphenol compositions are very astringent
and/or bitter. Thus, it is difficult to incorporate them into foods or
beverages in
biologically significant amounts without adversely affecting the taste and/or
organoleptic profile of such polyphenol-enriched foods or beverages.
[0005] Encapsulation has been used to provide delayed release and/or
protection of sensitive materials. For example, U.S. Patent 6,190,591
(February 20,
2001) provides a continuous, and relatively complicated, method for producing
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controlled release, discrete, solid particles (i.e., matrix) containing an
encapsulated
and/or embedded component such as a heat sensitive or readily oxidizable
pharmaceutically, biologically, or nutritionally active component. A release-
rate
component is introduced into the matrix to control the release of the active
component.
U.S. Patent 6,835,397 (December 28, 2004) provides an encapsulated yeast
composite
comprising a core containing the yeast and a coating containing an emulsified
lipid.
[0006] Japanese Patent Publication No. 2005-124540A (published May 19,
2005) provides a method for masking or reducing the astringency and bitterness
of
polyphenol compositions in which casein, especially acid casein, is included
in the
polyphenol composition. In this method, the polyphenol composition is mixed
with a
solution or suspension of casein in water, preferably adjusted to a neutral
pH. Such a
casein-containing polyphenol composition can be used directly in beverages or
dried to
form a powder for use in foods or beverages. Generally, to obtain sufficient
"masking"
of the polyphenols, about 40 to about 350 parts (and preferably about 60 to
150 parts)
casein was combined with about 100 parts of the polyphenol composition in
water.
This method does not appear as successful as desired since astringency can
still be
detected in the mouth (see Example 4 below).
[0007] It is desired to provide other and improved methods for reducing the
astringency and bitterness of polyphenol compositions. The present invention
provides
such improved methods for reducing the astringency and bitterness of
polyphenol
compositions. The present invention also provides polyphenol compositions
having
significantly reduced astringency and bitterness levels. The present
polyphenol
compositions can be added at significant levels to food and/or beverage
products
without adversely affecting the flavor and/or organoleptic properties of the
food or
beverage products.
SUMMARY OF THE INVENTION
[0008] The present invention relates to microencapsulation of polyphenol
compositions in order to significantly reduce astringency and bitterness
levels
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associated with the polyphenol components. The present polyphenol compositions
can
be added at significant levels to food and/or beverage products without
adversely
affecting the flavor and/or organoleptic properties of the food or beverage
products.
Thus, this invention provides polyphenol compositions which retain the
original
biological activities of polyphenois but without the specific bitterness and
astringency
normally associated with polyphenols.
DETAILED DESCRIPTION
[0009] The present invention relates to encapsulation or microencapsulation of
polyphenol compositions in order to significantly reduce astringency and
bitterness
levels normally associated with the polyphenol components. The present
polyphenol
compositions can be added at significant levels to food and/or beverage
products
without adversely affecting the flavor and/or organoleptic properties of the
food or
beverage products.
[0010] The present invention provides polyphenol compositions which are
essentially protected as they pass through the mouth but then allows release
of the
polyphenol compound contained therein released in the remainder of the
digestive
system (i.e., stomach, small intestines especially). By preventing or reducing
the
amounts of the polyphenol compounds from contacting the taste buds in the oral
cavity,
the present invention significantly reduces astringency and bitterness levels
normally
associated with polyphenols. The polyphenol compounds are, however, more fully
released within the digestive system where they can provide their health
benefits.
Microencapsulation of polyphenol compositions significantly reduces the
astringency
and/or bitterness of the polyphenol compositions and protects the polyphenol
compositions from oxidation, ingredient interactions, enzymatic degradation,
and the
like while maintaining gastrointestinal bioavailability within the digestive
system.
[0011] The encapsulated polyphenol compositions of the present invention can
be prepared using conventional encapsulation procedures and edible
encapsulating or
coating materials so long as the encapsulation allows the polyphenol
materials,
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especially when incorporated into food products, to pass through the oral
cavity without
significant release of the polyphenols, allows release of the polyphenols as
the
materials pass through the digestive system after the oral cavity, and
maintains
bioavailability when released. For purposes of this invention, "without
significant
release" is intended to mean that the release of polyphenols within the oral
cavity is
such that the astringency and/or bitterness normally associated with the
polyphenois is
effectively eliminated or reduced to levels which are acceptable for the
particular
product in which the polyphenols are incorporated.
[0012] Although synthetic polyphenois can be used, the polyphenols used in the
present invention are preferably naturally-occurring polyphenols derived from
plants or
plant materials (e.g, berries, tea, cocoa beans, coffee, vegetables, fruits,
and the like as
well as combinations thereof) which are known to have antioxidant properties
as well as
providing other potential health benefits. Such polyphenol compounds normally
include
catechin, epicatechin, gallocatechin, catechin gallate, epicatechin gallate,
gallocatechin
gallate, epigallocatechin gallate, epigallocatechin, tannic acid, gallotannin,
ellagitannin,
caffeic acid, dihydrocaffeic acid, chlorogenic acid, isochlorogenic acid,
genitisic acid,
homogenitisic acid, gallic acid, ellagic acid, rosemary acid, rutin,
quercetin, quercetagin,
quercetagetin, gossypetin, anthocyanin, leucoanthocyanin, proanthocyanidin,
enocyanin, and the like as well as their derivatives, polymers, and
stereoisomers. The
polyphenols can be extracted from these plants using conventional techniques
(e.g.,
extraction using one or more solvents selected from water, ethyl acetate,
methanol,
ethanol, isopropanol, and the like or mixtures thereof).
[0013] Generally the encapsulated polyphenols used in the present invention
are
in powdered form. Although the physical properties can vary depending on the
method
of encapsulation used and the product in which the polyphenois are to be
included, the
encapsulated polyphenols, in most embodiments, are preferably roughly
spherical and
have mean particle size of about 50 to about 1700 microns, preferably about 50
to
about 500 microns, and more preferably about 70 to about 120 microns. The
reduction
in particles size, if necessary, can be made before or after the encapsulated
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polyphenois are incorporated into the food product. Of course, so long as the
desired
reduction in astringency and bitterness are obtained while maintaining the
desired
organoleptic properties of the food product, encapsulated polyphenols having
other
shapes and/or particle sizes can be used.
[0014] For some products (e.g., those having a very smooth texture such as
chocolate) the particle size may preferably be in the lower portions of, or
even less
than, the ranges listed above. Thus, for example, encapsulated polyphenols
used in
the manufacture of chocolate preferably have, in the final product, a d90 of
about 15 to
about 100 microns (i.e., 90 percent of particles have a particle size equal to
or less than
the specific d90 value) and even more preferably of about 20 to about 30
microns in the
final product; such a particle size may be achieved by reducing the particle
size of the
encapsulated polyphenols either before they are added to the product or by
reducing
the particle size (e.g., milling) the encapsulated polyphenois during or after
the
manufacturing process of the product in which the encapsulated polyphenois are
to be
included.
[0015] The encapsulating material or coating material must be edible and
provide
controlled release within the human body when consumed. For purposes of this
invention, "controlled release" is intended to mean non-release or
significantly reduced
release during normal mastication conditions but then increased release
(essentially
complete release) while passage through the remainder of the digestive system
(i.e.,
stomach and/or small intestines) where bio-absorption can occur. By avoiding
or
significantly reducing release in the mouth during normal ingestion of food
containing
such polyphenol compositions, the astringency and bitterness normally
associated with
polyphenols is avoided, thereby allowing the incorporation of the encapsulated
polyphenol compositions in a wider range of food products and/or incorporation
at
higher levels without adversely affecting organoleptic properties of the food
product in
which they are incorporated. For purposes of this invention, "normal
mastication
processes" are intended to include normal chewing activities in the mouth
during
consumption of food up to the time the masticated food is swallowed. For
purposes of
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this invention, "normal digestive processes" are intended to include normal
digestive
process occurring after the masticated food is swallowed; generally, such
processes will
include mixing and digestion of the food in the stomach as well as passage of
the mixed
and digested food through the small intestines.
[0016] The encapsulating or coating composition should remain intact (thereby
preventing and/or substantially reducing release of the polyphenols) for at
least about
30 seconds, and preferably at least about 60 seconds, in the oral cavity
(essentially for
a time sufficient to allow chewing and swallowing of the food product
containing the
encapsulated polyphenois) but then be broken down, to allow release of the
polyphenols in the stomach and/or small intestines. For purposes of this
invention, the
actual mechanism by which the encapsulated polyphenois remain essentially
unreleased or by which release is delayed in the oral cavity but then provides
for
release in the remainder of the digestive systems is not critical.
[0017] Examples of suitable encapsulating or coating materials for use in the
present invention include lipids, gelatin, shellac, gum arabic, waxes,
polymers, mixtures
of proteins and carbohydrates, and the like as well as combinations thereof.
The
amount of encapsulating or coating material relative to the polyphenois is
generally an
effective amount to reduce astringency and/or bitterness normally associated
with
polyphenols while maintaining the organoleptic properties in acceptable
ranges.
Generally, the prepared encapsulated polyphenois contains about 60 to about 95
percent polyphenois and about 5 to about 40 percent encapsulating material,
and
preferably about 70 to about 90 percent polyphenols and about 10 to about 30
percent
encapsulating material.
[0018] Especially preferred lipid coating compounds for use in fluidized bed
encapsulation systems include hydrogenated palm oil, acetylated
monoglycerides,
hydrogenated cottonseed oil, hydrogenated soybean oil, hydrogenated coconut
oil,
cocoa butter, and the like. Preferably the gelatin coating composition
contains about 2
to about 20 percent gelatin, about 1 to about 5 percent glycerol, and about 75
to about
97 percent water. Type A or B gelatin can be used.
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[0019] Especially preferred mixtures of proteins and carbohydrates for use in
spray drying encapsulation systems include proteins such as milk proteins
(e.g., milk
protein isolate, sodium caseinate, total milk protein, whey protein, and the
like) and soy
proteins (e.g., soy protein isolate and the like) and carbohydrates such as
maltodextrin,
trehalose, corn syrup solids, and the like. More preferably, the protein is a
milk protein
with milk protein isolate and sodium caseinate being most preferred and the
carbohydrate is maltodextrin or trehalose with maltodextrin being most
preferred.
Generally the carrier system used in spray drying is aqueous based and
contains about
30 to about 70 percent protein and about 30 to about 70 percent carbohydrates,
and
more preferably about 35 to about 45 percent protein and about 55 to about 65
percent
carbohydrates. Generally, the spray dried prepared encapsulated polyphenols
contains
about 20 to about 60 percent polyphenois and about 40 to about 80 percent
encapsulating material, and preferably about 30 to about 50 percent
polyphenols and
about 50 to about 70 percent encapsulating material. Although not wishing to
be limited
by theory, it appears that in the spray drying system, the encapsulation may
be
achieved by physically absorbing and/or adsorbing the polyphenois along the
individual
protein chains. In addition to this physically absorption and/or adsorption,
the
polyphenols can also be essentially contained within a matrix formed by the
carrier
materials. Regardless of the mechanism or mechanisms involved, the spray dried
compositions provide an effective system for reducing the effects of the
polyphenols
(e.g., reduced astringency and/or bitterness).
[0020] The thickness and nature of the coating composition around the
polyphenol particles should be effective to prevent and/or delay release in
the oral
cavity and then to provide release in the remainder of the digestive system
and thus
reduce astringency and bitterness in the oral cavity. Generally, the coating
composition
forms an encapsulating layer, coating, or matrix protecting the polyphenol
particles of
about 1 to about 100 microns (about 0.001 to about 0.1 mm) thick, and
preferably about
to about 40 microns (about 0.01 to about 0.04 mm) thick. The encapsulating
layer,
coating, or matrix protecting the polyphenol particles may have a uniform or
non-
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uniform thickness. Generally, the encapsulated polyphenol compositions
contains
about 60 to about 95 percent polyphenois and about 5 to about 40 percent
encapsulating material, preferably about 70 to about 90 percent polyphenols
and about
to about 30 percent encapsulating material. Of course, the relative amounts of
the
polyphenols and encapsulating material and the thickness of the encapsulating
layer,
coating, or matrix may vary so long as the astringency and bitterness of the
polyphenois
are effectively reduced in the oral cavity. In some cases (e.g., the matrix
formed with
the spray drying encapsulating technique), it may be preferred to further
grind the
encapsulated polyphenol compositions in order to obtain the desired particle
size
distribution. Such further grinding may be carried out on the encapsulated
polyphenols
before or after introduction into the desired food product. Generally, the
particle size is
preferably adjusted to reduce or avoid the perception of grittiness while
maintaining the
desired reduction in astringency and bitterness levels in the final polyphenol-
containing
food product.
[0021] Of course, the appropriate particle size can be determined on a case-by-
case basis and will likely depend on the type of food product desired. For
example, for
a food product normally having a crunchy texture, a higher particle size can
be used
whereas a smooth texture product will likely require a smaller particle size.
For
products having a very smooth texture (e.g., high quality chocolate), the
particle size will
generally be preferred to be lower. Thus, for example, the encapsulated
polyphenols
used in the manufacture of chocolate preferably has a d90 of about 15 to about
100
microns (i.e., 90 percent of particles have particles equal to or less than a
specific
value) and even more preferably of about 20 to about 30 microns in the final
product;
such a particle size may be achieved by reducing the particle size of the
encapsulated
polyphenols either before they are added to the product or by milling the
polyphenol-
enriched product during or after the manufacturing process.
[0022] Encapsulation can be carried out using any conventional technique.
Examples of such techniques include fluidized bed encapsulation, extrusion,
spray
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drying, prilling, spinning disk, and the like. Preferably fluidized bed or
spray drying
systems are used for encapsulation.
[0023] The encapsulated polyphenol compositions of this invention are
especially
designed to allow incorporation of significant level of polyphenois in food
products for
human or animal consumption without the astringency and bitterness levels
normally
associated with polyphenols. Generally, the encapsulated polyphenol of this
invention
are incorporated into the desired food product at a level of about 1 to about
20 percent,
preferably at about 1 to about 10 percent, using any suitable technique. Thus,
for
example, encapsulated polyphenol compositions - especially those prepared by
spray
drying polyphenols using mixtures of proteins and carbohydrates - may be
incorporated
into dark or milk chocolate to significantly increase the amount of
polyphenois without
adversely affecting the organoleptic properties of the chocolate. For
polyphenol-
containing chocolate, it has been found that spray dried polyphenol
encapsulated
material can be added at many stages of the chocolate manufacturing process.
Such
spray dried polyphenol encapsulated material is preferably incorporated into
the
chocolate at or near the end of the conching treatment in conventional methods
for
making chocolate. The conched material containing the polyphenol encapsulated
material is preferably then ball milled and further treated using conventional
chocolate
making technology. The ball milling step appears to reduce the particle size
of the
polyphenol encapsulated material to levels effective to reduce or avoid the
perception
of grittiness while maintaining the desired reduced astringency and bitterness
levels. Of
course, other methods to obtain the desired particle size of the encapsulated
polyphenois in the final product can be used. For example, the spray dried
polyphenol
encapsulated material could be ground to a desired particle size before being
added to
the chocolate. Dark chocolate containing levels of about 500 mg or more
polyphenois
per 60 g chocolate and milk chocolate containing levels of about 200 mg or
more
polyphenols per 60 g chocolate can be prepared having good organoleptic
properties.
These levels represent a significant increase in polyphenol content as
compared to
traditional chocolate as well as polyphenol-enriched chocolate currently
available (i.e.,
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about 200 mg polyphenol per 60 g dark chocolate or about 100 mg polyphenol per
60 g
milk chocolate). The polyphenol-enriched chocolate currently available
generally have
been obtained by either (1) selecting starting materials with relative high
levels of
polyphenols and/or adjusting manufacturing conditions to help maintain the
polyphenol
levels in the starting materials or (2) adding increased levels of
polyphenols. Using the
first technique, significantly high levels of polyphenols (i.e., as high as
obtained in the
present invention) can generally not be obtained without significantly
astringency and
bitterness; and using the second technique, although high levels of
polyphenols can be
obtained, the astringency and bitterness associated with polyphenois becomes
very
apparent. The present invention, however, allows significantly higher
polyphenol levels
to be obtained without the astringency and bitterness normally associated with
polyphenols.
[0024] The invention will now be illustrated by specific examples which
describe
preferred embodiments of the present invention. They are not intended to limit
the
scope of the invention. Unless otherwise indicated, all ratios and percentages
throughout this specification are by weight. All patents and other
publications
discussed in this specification are hereby incorporated by reference.
[0025] Examples 1-4 illustrate encapsulated polyphenol compositions prepared
in a fluidized bed system. Examples 5-8 illustrate encapsulated polyphenol
compositions prepared in a spray drying system; the resulting spray dried
polyphenol
composition is then incorporated into chocolate.
[0026] Example 1. This example illustrates the microencapsulation of a
polyphenol composition with a lipid (a hydrogenated palm fat from Humko Oil
Products,
Cordova, TN). VitaBerryTM (Van Drunen Farms, Momence, IL) was used as the
polyphenol composition; it was stored in a freeze prior to use. VitaBerry T"'
is a
powdered blend of concentrated fruit extracts and whole-fruit powders which
contains
natural antioxidants having high oxygen radical absorbent capacity (ORAC)
values and
phytochemicals; the polyphenol content is about 30 percent. The polyphenol
composition was sieved to a size of 0.089 to 0.122 mm (about 140-100 mesh).
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[0027] The coating of the polyphenol composition was carried out using a hot-
melt fluidized bed system (Uni-Glatt GmbH, Ramsey, NJ). The fluidized bed
system
was started up about one hour in advance to allow the system to obtain
operating
temperature. The hydrogenated palm fat (99 g; melting point about 57 C) was
melted
using a hot plate. Thirty minutes before the coating was applied, the
polyphenol
composition was removed from the freezer and allowed to warm to room
temperature.
The polyphenol composition (200 g) was then added to the fluidized bed system
and
fluidization bed was started. After about 1 to 2 minutes (to allow the
polyphenol
composition to reach the operating temperature of about 70 C), the flow of the
melted
lipid was begun and then maintained at a rate of about 4 to about 5 ml/min.
Throughout the run the fluidized bed chamber was tapped with a rubber mallet
and the
shaker function was used approximately every two to three minutes to maintain
a
uniformly fluidized bed. After all of the hydrogenated palm fat had been fed,
the
fluidized bed was stopped and the encapsulated polyphenol composition was
removed.
The encapsulated polyphenol composition was spread on parchment paper and
allowed to cool for about 30 minutes. The ratio of the polyphenol composition
to lipid
was about 70:30. The encapsulated polyphenol composition was sieved to a size
of
0.250 to 1.7 mm and stored in a glass jar covered with aluminum foil in a
freezer.
Evaluation of the lipid-coated polyphenol composition is provided in Example
4.
[0028] Example 2. This example illustrates the microencapsulation of a
polyphenol composition (i.e., the VitaBerryTM used in Example 1) with a
gelatin (100
bloom Type A; Great Lakes Gelatin, Grayslake, IL) using essentially the same
equipment and procedure (except as noted) as in Example 1. The gelatin coating
composition was prepared by heating water (about 150 g) to about 100 C).
Gelatin
(about 10 g) was then slowly added with stirring. Once all the gelatin was
dissolved,
glycerol (about 2 g; (Dow Chemical, Pevely, MO) was added and stirring
continued for
about 5 minutes to obtain an uniform mixture. The coating solution is kept at
about
70 C and covered until used. The hot-melt fluidized bed system was modified so
that
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the inlet line for the coating composition could be heated so as to maintain
the coating
composition at a temperature of about 85 C as it entered the fluidized bed
chamber.
[0029] After allowing the polyphenol composition (about 50 g) to obtain the
operating temperature in the fluidized bed chamber, the flow of the coating
composition
(about 170 g) was begun and maintained at about 4 to 5 mI/min. Throughout the
run
the chamber was tapped with a rubber mallet and the filter blow back function
used
about every five to ten minutes to keep the filters clean and reduce excessive
powder
loss. After all of the gelatin solution had been applied to the polyphenol
composition,
the encapsulated polyphenol composition was removed as a dry powder. After
cooling,
was sieved to a size of 0.250 to 1.7 mm and stored in a glass jar covered with
aluminum foil in a freezer. The ratio of the polyphenol composition to gelatin
coating
was about 70:30. Evaluation of the gelatin-coated polyphenol composition is
provided
in Example 4.
[0030] Comparative Example 3. This example illustrates, for comparative
purposes, polyphenol compositions prepared by methods described in Japanese
Patent
Publication No. 2005-124540A. The same starting polyphenol composition as used
in
Examples 1 and 2 was used. Evaluations of the three comparative samples
described
herein are also provided in Example 4.
[0031] Sample 1. Acid casein (7.5g; Dairygold Co-Operative Society Limited,
Ireland) was mixed with 7.5 ml of 0.1 N sodium hydroxide in 80 ml deionized
water for
one hour at room temperature. The polyphenol composition (1 2g) was then added
and
mixing continued for an additional hour at room temperature. The solution was
then
poured into a small bread pan and covered with aluminum foil and stored in a
freezer
overnight. The frozen solution was then placed in a freeze dryer (Virtis
Genesis 25XL)
and freeze dried for approximately 3.5 days. After freeze drying, a casein-
containing
polyphenol composition in the form of a powder was obtained and then stored in
glass
jars in a freezer.
[0032] Sample 2. A second comparative sample was prepared exactly as in
Sample 1 above except that the initial casein solution contained 7.5g acid
casein, 7.5
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ml 0.1 N sodium hydroxide, and 0.475g sodium tripolyphosphate in 80 ml
deionized
water. The same polyphenol composition (12g) was added and treated as for
Sample
1. After freeze drying, a casein-containing polyphenol composition in the form
of a
powder was obtained and then stored in glass jars in a freezer.
[0033] Sample 3. Acid casein (49.4g; same as used in previous samples) and
sodium carbonate (3.3g) were added to deionized water (1 769.3g) and mixed
until
dissolved. The same polyphenol composition (79g) as used in the previous
samples
was slowly added and the entire solution was mixed. The resulting solution was
then
spray dried using an APV Anhydro Laboratory Spray Dryer Type PSD 52 at a flow
rate
of about 10m1/min, an inlet temperature of about 170 C, outlet temperature of
about
72 C (initial) to about 87 C (final) to obtain a powdered casein-containing
polyphenol
mixture, which was stored in a freezer.
[0034] Example 4. The compositions prepared in Examples 1-2 and
Comparative Example 3 were evaluated.
[0035] Dissolution. In order to evaluate treatment methods and their effect on
astringency and bitterness levels associated with the polyphenol components,
the
various samples were tested in various solutions to simulate (1) saliva from
the mouth,
(2) gastric juices from the stomach, and (3) intestinal fluids from the small
intestines.
Simulated saliva was obtained from A.S. Pharma (East Sussex, UK). Simulated
gastric
juices and simulated intestinal fluids were prepared according to United
States
Pharmacopeia (Edition 29, p. 3171). To simulate gastric and intestinal
digestion, 25 mg
of sample was weighed into 15 ml polypropylene centrifuge tubes, 10 ml of
solution
(warmed to 37 C) was added and the tube capped. The tubes were rotated end-
over-
end at 25 rpm and 37 C for 1 hour, then immediately drained through a glass
microfiber filter (VWR grade 691) where the undissolved material was retained.
A
sample of the filtrate was collected and analyzed for total phenolics by the
Folin-
Ciocalteu assay (see, Singleton et al., Am. J. Enol. Vitic., 16:144-158
(1965)). A similar
procedure was used to simulate contact with saliva, except tubes were rotated
for only
30 seconds prior to draining.
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[0036] The powdered polyphenol compositions (generally about 25 mg) were
incubated in the various simulated solutions (generally about 10 ml) at 37 C
for 30
seconds for the simulated salvia solution and for 1 hour for the simulated
gastric and
intestinal fluids. After each incubation, the amount of polyphenols released
into the
respective solutions from the test samples was determined using the Folin-
Ciocalteu
assay (see, Singleton et al., Am. J. Enol. Vitic., 16:144-158 (1965)); the
percent
recovery of polyphenois was then calculated. The following results were
obtained.
Recovery (%) of Polyphenols
Simulated Simulated Gastric Simulated Intestinal
Saliva Juices Juices
Example 1 10.3 89.6 86.5
(Inventive)
Example 2 9.4 91.0 79.9
(Inventive)
Comparative 21.4 88.2 84.8
Sample 1
Comparative 47.6 88.2 88.7
Sample 2
Comparative 21.9 85.9 84.8
Sample 3
As can be seen from this data, the inventive samples show significantly less
release of
polyphenols in the stimulated salvia solution than comparative samples. Thus,
when
the inventive samples are consumed, considerably less polyphenois will be
released in
the mouth, thereby significantly reducing astringency and bitterness levels.
Data from
the simulated gastric and intestinal fluids show that the inventive samples
will release
their polyphenois during the digestive process. Thus, as compared to the
comparative
samples, the inventive samples will provide their polyphenols during the
digestive
process (i.e., within the stomach and small intestines) and not within the
mouth.
[0037] Degradation by Polyphenol Oxidate. The various examples were also
evaluated to determine their ability to avoid degradation by polyphenol
oxidate. This set
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of experiments models the degradation of polyphenois in a model food matrix
due to
the presence of polyphenol oxidate enzyme. To determine the extent of
protection
against polyphenol oxidate enzyme, Comparative samples (about 7 mg; casein
treated)
or inventive samples (10 mg; encapsulated) from Comparative Example 3 (Samples
1
and 2 only) and Examples 1-2, respectively, were placed in 15 ml polypropylene
centrifuge tubes. In addition, an unencapsulated polyphenol sample (about 7
mg) was
treated in the same manner. Test solutions (5 ml) of 80:20 glycerol:water with
or
without polyphenol oxidate (2 mg/mI; Sigma-Aldrich, St. Louis, MO) was added
and
each tube capped. The tubes were rotated end-over-end for one hour at 22 C and
then
sparged with air at 30 minutes. Any enzymatic reactions were then stopped by
heating
the tubes in boiling water for 10 minutes; the samples were then cooled on
ice. The
resulting solutions were centrifuged for 20 minutes at 10,000G. The
supernatant was
collected and then evaluated for total phenolics using the Folin-Ciocalteu
assay as
above. The following results were obtained:
Recovery (%) of Polyphenols
Example 1 (Inventive) 76.4
Example 2 (Inventive) 96.3
Comparative Sample 1 93.4
Comparative Sample 2 75.4
Unencapsulated 70
[0038] Example 5. This example illustrates the general spray drying procedures
used to encapsulate polyphenol compositions as well as methods to evaluate the
effect
of the encapsulated polyphenois in chocolate.
[0039] All of the powdered ingredients, including the polyphenols, were mixed
in
a Hobart mixer (Hobart -50, 5 quart, paddle attachment, max 1725 rpm): dry
powders
were mixed at low speed. Water was then added slowly to the Hobart mixer at a
low
speed to avoid lumps, foaming, and/or large increases in viscosity; sieving
was
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performed if necessary. The resulting slurries were generally prepared so as
to obtain
about 45 percent solids.
[0040] The slurries were fed into a Niro Mobile MinorTM spray dryer (L/W/H
1800/925/2200 mm) using a peristaltic pump (Cole Parmer Masterflex L/S Easy-
Load).
Feeds were atomized into a spray using a vaned wheel rotating at high speed
(generally about 27000 rpm). Hot air entered the chamber around the wheel,
drying the
spray to produce a powder, which is then separated from the air in a cyclone.
Compressed air (4-5 bar) was used to power the atomizer and the dryer roof
(pneumatically lifted). During operation, the air inlet temperature was around
155-
170 C and the outlet temperature was around 95-105 C. The spray dryer and
atomizer
were disassembled and washed with water once or twice per day as needed to
prevent
extensive powder build up as well as significant cross contamination between
samples.
The spray dried polyphenol powder was collected for evaluation.
[0041] The resulting encapsulated polyphenois were then incorporated into a
commercial liquid dark chocolate mass taken from an operating commercial
production
line (i.e., after the conching step). This commercial chocolate mass contained
about
300 mg polyphenois per 100 g (averaged over several months of normal
production
runs). The spray dried polyphenol compositions were then manually added and
mixed
with the chocolate mass, and then manually tempered and moulded to produce
chocolate tablets. The amount of encapsulated polyphenol composition added was
adjusted to achieve an overall polyphenol level of at least about 500 mg
polyphenol per
60 g (or about 830 mg polyphenol per 100 g). The polyphenol content of the
polyphenol extracts used to prepare the spray dried compositions was
determined
using HPLC; this method measures intact procyanidin molecules (e.g.,
epicatechin and
catechin) as well as dimers, trimers, tetramers, and the like forms. The
chocolate
tablets were then stored at about 16 C for about three weeks to allow for fat
or cocoa
butter crystallization before sensory evaluations were conducted using a
trained test
panel.
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[0042] Example 6. This example provides early experiments using spray drying
to encapsulate grapeseed polyphenol extracts obtained from Planteextrakt.
Generally
the same spray drying and evaluation procedures as described in Example 5 were
used. The compositions used for spray drying consisted of a aqueous slurry
containing
the extract and the tested carriers in a ratio of 30/70. The encapsulated
compositions
were added at a level of about 5.3 percent to a liquid chocolate sample taken
from a
commercial production line. The following control samples were also prepared:
Control
1- chocolate (no additives); Control 2 - grapeseed extract (1.6 percent) mixed
into
chocolate (no carrier or spray drying); and Controls 3, 5, and 7 - mixing
powdered
grapeseed extract and carrier in the same proportions (no spray drying) into
chocolate.
Sensory evaluations were carried out on the chocolate samples after about 3
weeks.
[0043] The following samples were prepared and evaluated. Except for Control
1, all samples contained about 1.6 percent grapeseed extract; none of the
control
samples involved encapsulation; Samples 4, 6, 8, and 10 contained encapsulated
polyphenois prepared with various carriers (ratio of grapeseed extract to
carrier was
30/70).
Carrier/ Polyphenol Evaluation
Treatment* (mg/100g)
Control 1 No additives / 311 less astringent than any other sample
No encapsulation
No carrier / added strongly astringent, very sour, very bitter, earthy,
Control 2 grapeseed extract / 450 burnt notes, cocoa
No encapsulation
Control 3 Non-fat Dry Milk/ 410 Similar to Control 2
No encapsulation
4 Non-fat Dry Milk/ 397 less burnt, more chocolaty, less astringent, sour,
Encapsulated slightly salty aftertaste
Control Maltodextrin/ 408 Similar to Control 2
No Encapsulation
6 Maltodextrin/ 399 Astringency between Control 1 and Control 5
Encapsulated
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Carrier/ Polyphenol
Treatment* (mg/100g) Evaluation
Control 7 Whey Powder/ 407 Similar to Control 2
No Encapsulation
Whey Powder/ Sour aftertaste, less bitter, slightly chocolaty, less
8 Encapsulated 407 overall taste than Sample 10, similar to Control 2
Trehalose &
Control 9 Sodium Caseinate 408 More bitter aftertaste, more astringent than
(30/70)/ Sample 10 but less than Control 2
No Encapsulation
Trehalose & More chocolaty than Sample 4, less astringent
Sodium Caseinate than Control 2, slightly sour, less burnt than
(30/70)/ 408 Control 1 and Sample 4 (but more than Control 1),
Encapsulated less bitter, best of encapsulated samples; closer
to control 1 than to control 2
* Except for Control 1, all samples contained grapeseed extract.
[0044] Example 7. Using the procedures in Example 5 and guided by the results
of Example 6, the following components were used to prepare encapsulated
polyphenols. Many of these encapsulated polyphenols were then incorporated
into
chocolate for evaluation as described in Example 5. The spray dried
compositions are
presented below.
Protein' Carbohydrate" Polyphenolt Other$
Amount (%) Amount (%) Amount (%) Amount (%)
Alanate 155 Trehalose CocoanOX 70 -
1
22.5 47.5 30
Alanate 180 Trehalose CocoanOX 70 -
2
22.5 47.5 30
Alanate 180 Maltodextrin (10DE) CocoanOX 70 -
3
22.5 47.5 30
Alanate 180 Maltodextrin (10DE) CocoanOX 70 -
4
22.5 32.5 45
Alanate 180 Maltodextrin (10DE) CocoanOX 45 -
5
22.5 47.5 30
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Protein* Carbohydrate" Polyphenolt Other4
Amount (%) Amount (%) Amount (%) Amount (%)
Alanate 180 Maltodextrin (10DE) CocoanOX 45 -
6
22.5 32.5 45
Alanate 180 Maltodextrin (10DE) CocoanOX 70 Cocoa Butter
7
22.5 42.5 30 5
Alanate 180 Maltodextrin (10DE) CocoanOX 70 MM-100
8
22.5 47.4.5 30 0.1
Alanate 180 Maltodextrin (10DE) CocoanOX 70 Cocoa Butter
9
22.5 37.5 45 10
Alanate 167 Maltodextrin (10DE) CocoanOX 45 -
22.5 32.5 45
TMP 1104 Maltodextrin (10DE) CocoanOX 70 -
11
22.5 47.5 30
TMP 1104 Maltodextrin (10DE) CocoanOX 45 -
12
22.5 32.5 45
Pro-Fam 873 Maltodextrin (10DE) CocoanOX 70 -
13
22.5 47.5 30
Pro-Fam 873 Maltodextrin (10DE) CocoanOX 45 -
14
22.5 32.5 45
Alanate 385 Maltodextrin (10DE) CocoanOX 45 -
22.5 32.5 45
Alanate 155 is sodium caseinate; Alanate 180 is sodium caseinate; Alanate 167
is partially
hydrolyzed sodium caseinate; Alanate 385 is calcium caseinate; TMP 1104 is
total milk protein (i.e., milk
protein isolate), all obtained from Fonterra (New Zealand). Pro-Fam 873 is
isolated soy protein from ADM.
The carbohydrates were obtained from Cargill.
t CocoanOX 45 and CocoanOX 70 are natural cocoa extracts reported to contain
about 45 and 70
percent polyphenols, respectively, from Natraceutical Group.
$ MM-100 is a masking agent based on mono-ammonium glycyrrhizinate from Mafco.
[0045] Chocolate tablets were prepared containing the spray dried compositions
described in the table above using the procedure as described above in Example
5.
Additionally, standard chocolate samples containing only CocoanOX 45 and
CocoanOX
70 (i.e., no carriers or spray drying; Controls 1 and 2, respectively) were
also prepared.
The amounts of spray dried polyphenol compositions and the CocoanOX standards
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added were adjusted to bring the polyphenol levels to about 850 mg per 100 g
chocolate. The sensory results are shown below.
Detailed Sensory Evaluation Overall
Evaluation*
1 cocoa, fruity, chocolaty, slight astringent aftertaste, similar to sample 3
Good
2 strong cocoa, astringent (slight dry mouth), bitter, slight fruity,
chocolaty, slightly Good
sweet
3 strong cocoa, chocolaty, fruity, bitter, slight astringent aftertaste,
sweeter than Excellent
sample 11, balanced, slightly earthy
4 cocoa, more fruity, mid bitter (less than sample 3), more astringent
aftertaste Poor
sweeter than sample 3, slightly less cocoa, chocolaty, slightly astringent,
slightly Good
bitter, milder than 3 but close, balanced
6 strong cocoa, fruity, chocolaty, mid bitter, more astringent than samples 3,
11, and Good
17
7 cocoa, less fruity, slightly chocolaty, mid bitter, mid astringent (similar
to sample 4) Poor
8 astringent aftertaste, more bitter, less cocoa, less fruity, flat in
chocolaty Poor
9 less cocoa, mid bitter, very fruity, mid astringent (similar to samples 4
and 7), less Poor
balanced than sample 3
astringent aftertaste, bitter, cocoa Poor
11 mid cocoa, chocolaty, slightly bitter, fruity, slightly astringent
aftertaste, more sweet Excellent
12 cocoa, more chocolaty, less fruity, slightly sour and bitter aftertaste,
more Good
astringent than samples 3, 11, and 17 (dry mouth)
13 mild cocoa, slightly astringent, slightly sour, chocolaty, slightly fruity
Good
14 mid cocoa, fruity, bitter, slightly less astringent, chocolaty, balanced
Good
mildest sample, chocolaty, flat cocoa, creamy, less bitter, least astringent
Good
aftertaste
Control 1 less cocoa, very astringent, very bitter, earthy, less sweet, not
fruity Poor
Control 2 less cocoa, very astringent, very bitter, earthy, less sweet, not
fruity Poor
" Samples labeled good or excellent were considered acceptable.
[0046] Based on the sensory evaluation, compositions containing CocoanOX 45
were rated better when compared similar compositions containing CocoanOX 70
even
though the amounts were adjusted so that all compositions contained similar
overall
levels of polyphenols. Of the proteins tested, sodium caseinate, milk protein
isolate,
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and soy protein isolate were preferred. Of the carbohydrates tested,
maltodextrin,
trehalose, and corn syrups solids (25DE; data not shown) were preferred with
maltodextrin being most preferred; although the data is not presented here,
high-
maltose corn syrup, modified starch, and fibers (e.g., oligofructose (<10DE)
and inulin)
were tested but did not perform satisfactorily. Carrier systems containing
masking
agents (e.g., MM-100 or cocoa butter) were generally rated inferior to similar
systems
without the masking agents. The system using calcium caseinate (sample 15) was
among the mildest and had a very low astringency; but it was also very flat in
cocoa and
chocolate notes.
[0047] Of course, as those skilled in the art will realize, systems which are
effective in reducing astringency but which reduce or otherwise negatively
effect the
desired chocolate flavor attributes might be used, and might even be
preferred, in other
food products with different flavor profiles. It is the overall evaluation
that is important
since the encapsulated polyphenols must have, in addition to reduced
astringency, no
significant negative effects on the organoleptic properties of the food
product in which it
is being used.
[0048] Example 8. This example illustrates the incorporation of spray dried
encapsulated polyphenols into chocolate at various stages of a commercial
chocolate
production line. Except for the timing of the addition of encapsulated
polyphenols into
the chocolate and the chocolate production line, the procedures of Example 5
were
essentially used.
[0049] A first spray dried polyphenol composition was prepared comprising 47.5
percent trehalose, 22.5 percent sodium caseinate, and 30 percent CocoanOX 70.
The
first spray dried polyphenol composition had a d90 of about 82 microns; the
amount
added to the chocolate was adjusted to yield a final product containing about
500 mg
polyphenols/60 g chocolate. The first spray dried polyphenol composition was
then
added to separate runs of a pilot plant production process at the following
points:
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Sample 1- spray dried composition added with dry ingredients that are
subsequently refined to produce flakes;
Sample 2 - spray dried composition added at the beginning of conching process
(along with ingredients normally added at this point, e.g., cocoa liquor); and
Sample 3 - spray dried composition added at the end of conching process
(along with ingredients normally added at this point, e.g., lecithin, aroma,
cocoa butter).
A control sample was also prepared by mixing corresponding amounts of the
trehalose
and sodium caseinate (without any polyphenols and no spray drying) into a
finished
chocolate sample from a standard production run.
[0050] After three weeks to allow for fat or cocoa butter crystallization, the
chocolate samples were evaluated and the following results were obtained:
Sample Evaluation
Control very cocoa, fruity, sour, astringent, bitter
Sample 1 cocoa, most bitter, slight astringent in aftertaste, non-gritty, less
sour
F Sample 2 gritty, cocoa, fruity, bitter, slight astringent
Sample 3 gritty, cocoa, fruity, bitter, slight astringent, most similar to
Control
Although Sample 3 was the best polyphenol-containing sample, it was very
gritty.
Sample 2 was similar to Sample 3. Sample 1 had a good texture was more
astringent
as compared to Sample 3.
[0051] A second spray dried polyphenol composition was prepared comprising
32.5 percent maltodextrin, 22.5 percent sodium caseinate, and 45 percent
CocoanOX
45. The second spray dried polyphenol composition was then added to separate
runs of
a commercial chocolate production line at the following points:
Sample 1- spray dried composition added with dry ingredients that are
subsequently refined to produce flakes;
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Sample 2 - spray dried composition added at the end of conching process (along
with ingredients normally added at this point, e.g., lecithin, aroma, cocoa
butter); the
chocolate containing the encapsulated polyphenols was then subjected to
grinding in a
ball mill to reduce particle size. The finished encapsulated polyphenol-
containing
chocolate had a d90 of about 20 microns.
Sample 3 - a composition including the spray dried composition and a coarse
milled cocoa liquor were ground in a ball mill to provide a fine cocoa liquor
containing
enough encapsulated polyphenois to deliver essentially the same amount of
polyphenols
as Samples 1 and 2 in the finished product. The encapsulated polyphenol-
containing
fine cocoa liquor had a d90 of about 26 microns and was added at the beginning
of
conching process. The finished product had a similar d90.
[0052] After three weeks storage at about 16 C to allow for fat or cocoa
butter
crystallization, the chocolate samples were evaluated and the following
results were
obtained:
Sample Evaluation
Sample 1 strong cocoa, gritty, chocolaty, less fruit than Sample 2, more
astringent in
aftertaste, bitter
Sample 2 strong cocoa, fruity, creamy, astringent, slightly bitter, slightly
gritty (smooth but
with a few particles), well balanced
Sample 3 strong cocoa, mid fruity, slightly gritty, chocolaty, most bitter,
astringent
Sample 2 was the best spray dried polyphenol-containing sample of this series;
it was
perceived as the best in texture and taste, with a stronger fruity note and
slightly lower
astringency than the other samples, and the best balanced in overall taste.
Thus,
preferably the encapsulated polyphenols are added at the end of the conching
process
and the resulting mixture is then subjected to a further milling step before
completing the
production run to provide the finished chocolate product.
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