Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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LOW-SEDIMENT ACIDIC PROTEIN BEVERAGES
Field of the Invention
The present invention relates generally to specific types of low pH protein-
based
beverages (such as soy- and/or dairy-based types) that are properly suspended
to prevent
undesirable sedimentation of such protein constituents during storage. Such
beverages
include a thickening system comprising bacterial cellulose (BC) coated with
different
water soluble co-agents such that the BC-based component provides a network
forming
structure that suspends the target proteins and prevents any appreciable
sedimentation of
such proteins. Additionally, this system is capable of improving the
suspension of acidic
protein beverages fortified with insoluble calcium. The beverages encompassed
within
this invention exhibit certain stability benefits under typical storage
conditions and may,
depending upon the pH of the overall system, include additives that coat the
proteins to
prevent, or at least retard, aggregation of such constituent proteins when the
pH level
approaches their pertinent isoelectric point.
Background of the Invention
Soy- and dairy-based protein beverages have increased in popularity as the
availability of such products increases and improvements in organoleptic
properties for
such beverages occur. Currently, however, there are certain limitations
present for
widespread acceptance to consumers, primarily in terms of flavor and other
aesthetic
characteristics. A consumer is generally very particular about the beverage he
or she
ingests. As the populace becomes more health-conscious, such protein-based
types have
grown in acceptance. However, with such increased utilization comes the desire
to
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increase options in terms of taste, scent, and appearance in order to provide
a more
attractive product. Such an ultimate goal has proven rather difficult to
attain, mainly due
to shelf-life stability problems associated with the nutrient base-product
proteins present
within such beverages.
Dairy milk has been consumed for a very long time and is a staple product
after
pasteurization. There is a continued desire, however, to provide different
flavorings
within such a product such that pH issues remain a recurring problem with the
all-
important proteins present therein. Soy milk has found a foothold within
certain markets
particularly due to the absence of lactose within such products. Such soy
products,
however, exhibit similar problems as with the dairy protein-based compositions
in terms
of long-term shelf stability.
With either dairy or soy milks that possess a neutral or close to neutral pH,
the
proteins within such a target beverage can be easily suspended with typical
thickening
agents (such as carboxymethylcellulose and other cellulose ethers, pectin,
starch, xanthan
gum, guar gum, locust bean gum, carrageenan and the like). At such neutral pH
levels,
soy or milk proteins have a net negative charge, thereby reliably keeping the
protein
particles from aggregating, clustering, or otherwise creating large particles.
These typical
thickening agents are believed to impart an increase to the viscosity of the
water phase of
the target beverage. This aiding in the retention of the water phase of such a
target
beverage thus potentially limits the formation of protein precipitate to the
extent that the
protein remains soluble therein. Thus, these typical thickening agents provide
a manner
of minimizing protein sedimentation at neutral pH levels.
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The main problem exists when the pH level is lowered to a pH value of between
about 3.6 and 4.5, in order to accommodate the addition of organoleptic
enhancers, such
as flavorings, colorants, and the like. Off-note, or beany flavors of soy milk
may be
masked, or flavor enhancements may be added to dairy milk, by changing the
flavor and
lowering the pH of these beverages, thus increasing the organoleptic and/or
aesthetic
characteristics of such a target beverage. This can cause the protein
particles to exhibit a
decrease in charge density (i.e., a pH at or near the isoelectric point for
the particular
proteins present therein). At such a specific pH level, such proteins are
prone to thermal
denaturation, leading to significant and highly undesirable aggregation or
clustering of
the protein molecules and resulting in the above-noted undesirable
sedimentation from
solution. Despite the efficacy that typical stabilizers, such as pectin,
exhibit to minimize
association of protein during acidification of low pH soy protein beverages,
over time,
sedimentation may still occur in the pH range of 3.6 to 4.5. Surface
modifications and/or
homogenization of the target proteins prior to pectin addition has been
hypothesized as
well in order to aid in permitting proper and sufficient coating by the pectin
in solution
and thus reduce the propensity of protein to protein interactions that cause
the above-
discussed sedimentation problems. Unfortunately, such a suggested improvement
is quite
expensive and difficult to practice, and thus is not likely to be readily
followed in the soy
beverage market.
There is thus a need to overcome this sedimentation problem within low pH
protein-based beverages with a suspending aid that can meet the requirements
of long-
term storage stability. Even with thickening agents present, it has been
realized that if the
degree of aggregation of such proteins is sufficiently high, a suspension
including such
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constituent nutrients is very difficult to retain. At acidic pH levels, in
particular, certain
proteins, particularly those within soy and/or dairy beverages, exhibit such
undesirable
aggregation and thus are highly susceptible to deleterious interactions
between charged
portions thereof. Certain typical thickening agents may be used as coating
additives for
the protein constituents in order to reduce or, at best, delay, such
aggregation and
ultimate sedimentation. For instance, pectin may be introduced within such a
beverage
composition which is then adjusted to an acidic pH levels (i.e., below 4.5).
The pectin
will become, in essence, activated at such an acidic level, such that it may
not only
properly coat such proteins, but will prevent, or, more appropriately, reduce
protein-
protein interactions near its isoelectric point. Importantly, though, is that
pectin will not
prevent such aggregation and ultimate sedimentation on a long-term basis; as
such
beverages generally require a very long shelf life, such a system of protein
sedimentation
reduction does not provide, by itself, effective results for the
implementation of a low pH
system to increase flavor levels (as one example) within soy protein
beverages.
Basically, and unfortunately, such sedimentation, as alluded to above, will
invariably
eventually aggregate over time even with pectin present as a coating additive.
And, as a
result, if sufficient sedimentation of protein particles does occur over time,
such resultant
sediment will pack or cement strongly and will not easily become released,
even upon
vigorous shaking. In such a scenario, the resultant sediment will not be
ingested by the
consumer, and thus the desired benefit from the desired protein will be lost.
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Such pectin additives, however, do not provide the same type of significant,
but
limited, benefit when the pH is at a higher level (i.e., 5.0 to 6.0). At such
a pH level, the
pectin will not interact with the protein to the extent that proper coating
and protection
from such deleterious charged portion interactions will occur. At such a
higher pH level,
the proteins will not exhibit denaturation as readily as at a lower pH. The
heat of
processing, however, can still induce association and coagulation of proteins
even though
the subject formulation is present within this higher pH range (pH 5-6). With
the pectin
providing a certain degree of protection at lower pH levels, in essence the
resultant
interaction degree for the low pH pectin-only protected beverages will be
quite similar to
that as the higher pH level (i.e., 5.0) types, regardless of the presence of
pectin. Thus,
pectin alone will not provide a sufficient system of protection and thus
protein
sedimentation prevention within such acidic beverages, regardless of the
actual pH level
exhibited therein. Thus, a proper manner of not only potentially delaying such
protein
aggregation, but also providing a reliable long-term suspension system for
such acidic
protein-based beverages is of great necessity, particularly to increase the
potential market
for such products from an aesthetic perspective. To date, the best the market
has been
provided is the utilization of pectin alone as a coating additive, as noted
above. An
improvement in suspending systems, particularly with a solution that is low in
cost and
complexity and easy to incorporate within the beverage production methods, is
thus
highly desirable.
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Summary of the Invention
Accordingly, this invention encompasses a liquid composition comprising at
least
one protein-based material and at least one bacterial cellulose-containing
formulation
comprising at least one bacterial cellulose material and at least one
polymeric thickener
selected from the group consisting of at least one charged cellulose ether, at
least one
precipitation agent selected from the group consisting of xanthan products,
pectin,
alginates, gellan gum, welan gum, diutan gum, rhamsan gum, carrageenan, guar
gum,
agar, gum arabic, gum ghatti, karaya gum, gum tragacanth, tamarind gum, locust
bean
gum, and the like, and any mixtures thereof, wherein said liquid composition
exhibits a
pH level of at most 5.5.
Furthermore, this invention also encompasses a liquid composition comprising
at
least one protein-based material in an amount of between 0.1 and 20% by weight
and
exhibiting a pH level of at most 5.5, wherein said liquid composition exhibits
a
sedimentation level of protein of at most 10% after 24 hours of storage at a
temperature
of 22 C. Additionally, this invention further encompasses liquid composition
comprising
at least one protein-based material in an amount of between 0.1 and 20% by
weight, and a
source of insoluble calcium in an amount of between 0.05 and 5% by weight,
said liquid
composition exhibiting a pH level of at most 5.5; wherein said liquid
composition
exhibits a sedimentation level of protein of at most 10% and a sedimentation
level of
insoluble calcium of at most 10% after 24 hours of storage at a temperature of
22 C.
The possible charged cellulose ether within the bacterial cellulose-containing
formulation is a compound utilized to disperse and stabilize the reticulated
network in the
final end-use compositions to which such a bacterial cellulose-containing
formulation is
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added. The charged compounds facilitate, as alluded to above, the ability to
form the
needed network of fibers through the repulsion of individual fibers. Such a
network
provides an excellent network within a target beverage that exhibits
sufficient strength
and stability upon long-term storage, as well as thixotropic characteristics,
such that any
aggregated proteins present within such a target beverage will not appreciably
sediment
over time. The possible precipitation agent within the bacterial cellulose-
containing
formulation is a compound utilized to preserve the functionality of the
reticulated
bacterial cellulose fiber during drying and milling. Examples of such charged
cellulose
ethers include such cellulose-based compounds that exhibit either an overall
positive or
negative and include, without limitation, any sodium carboxymethylcellulose
(CMC),
cationic hydroxyethylcellulose, and the like. The precipitation (drying) agent
is selected
from the group of natural and/or synthetic products including, without
limitation, xanthan
products, pectin, alginates, gellan gum, propylene glycol alginate, rhamsan
gum,
carrageenan, guar gum, agar, gum arabic, gum ghatti, karaya gum, gum
tragacanth,
tamarind gum, locust bean gum, and the like. Preferably, though not
necessarily, a
precipitation (drying) agent is included.
As one potentially preferred embodiment, the formulation of bacterial
cellulose
and pectin produced thereby has the distinct advantage of facilitating
activation without
any labor- or energy-intensive activation required. Another distinct advantage
of this
overall method is the ability to collect the resultant bacterial cellulose-
containing
forinulation through precipitation with isopropyl alcohol, whether with a
charged
cellulose ether or a precipitation (drying) agent present therein. Thus, since
the bacterial
cellulose is co-precipitated in the manner described above, the alcohol-
insoluble
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polymeric thickener (such as xanthan or sodium CMC) appears, without intending
on
being bound to any specific scientific theory, to provide protection to the
bacterial
cellulose by providing a coating over at least a portion of the resultant
formed fibers
thereof. In such a way, it appears that the polymeric thickener actually helps
associate
and dewater the cellulosic fibers upon the addition of a nonaqueous liquid
(such as
preferably a lower alkyl alcohol), thus resulting in the collection of
substantial amounts
of the low-yield polysaccharide during such a co-precipitation stage. The
avoidance of
substantial amounts of water during the purification and recovery steps thus
permits
larger amounts of the bacterial cellulose to be collected ultimately. With
this novel
process, the highest amount of fermented bacterial cellulose can be collected,
thus
providing the high efficiency in production desired, as well as the avoidance
of, as noted
above, wastewater and multiple passes of dewatering and re-slurrying typically
required
to obtain such a resultant product. Furthermore, as noted previously, the
presence of a
drying agent, in particular, as one non-limiting example, a pectin product, as
a coating
over at least a portion of the bacterial cellulose fiber bundles, appears to
provide the
improvement in activation requirements when introduced within a target end use
composition. Surprisingly, there is a noticeable reduction in the energy
necessary to
effectuate the desired rheological modification benefits accorded by this
inventive
bacterial cellulose-containing formulation as compared with the previously
practiced
products of similar types. As well, since bacterial cellulose (hereinafter
referred to as
"BC") provides unique functionality and rheology as compared to a soluble
polymeric
thickener alone, the resultant product made via this inventive method permits
a lower cost
alternative to typical processes with improvements in reactivation
requirements,
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resistance to viscosity changes during high temperature food processing, and
improved
suspension properties during long term shelf storage.
Such target beverage are preferably dairy-based or soy-based and thus include
protein substances associated directly with such materials. However, other
types of
beverages that include proteins that exhibit an aggregation capability may
also be utilized
within the scope of this invention. Such beverages include, without
limitation, fruit
flavored milk or soy milk drinks, nutritional beverages, and yogurt smoothie.
Of
particular interest are protein-including beverages that are desirous of
proper suspension
in order to provide nutrients in such a suspension form after long-term
storage.
Detailed Description of the Invention
For purposes of this invention, the term "bacterial cellulose-containing
formulation" is intended to encompass a bacterial cellulose product as
produced by the
inventive method and thus including a polymeric thickener coating at least a
portion of
the resultant bacterial cellulose fiber bundles. The term "formulation" thus
is intended to
convey that the product made therefrom is a combination of bacterial cellulose
and a
polymeric thickener produced in such a manner and exhibiting such a resultant
structure
and configuration. The term "bacterial cellulose" is intended to encompass any
type of
cellulose produced via fermentation of a bacteria of the genus Acetobactel-
and includes
materials referred popularly as microfibrillated cellulose, reticulated
bacterial cellulose,
and the like.
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Bacterial cellulose may be used as an effective rheological modifier in
various
compositions. Such materials, when dispersed in fluids, produce highly
viscous,
thixotropic mixtures possessing high yield stress. Yield stress is a measure
of the force
required to initiate flow in a gel-like system. It is indicative of the
suspension ability of a
fluid, as well as indicative of the ability of the fluid to remain in situ
after application to a
vertical surface.
Typically, such rheological modification behavior is provided through some
degree of processing of a mixture of the bacterial cellulose in a hydrophilic
solvent, such
as water, polyols (e.g., ethylene glycol, glycerin, polyethylene glycol,
etc.), or mixtures
thereof. This processing is called "activation" and comprises, generally, high
pressure
homogenization and/or high shear mixing. The inventive bacterial cellulose-
containing
formulations of the invention, however, have been found to activate at low
energy
mixing. Activation is a process in which the 3-dimensional structure of the
cellulose is
modified such that the cellulose imparts functionality to the base solvent or
solvent
mixture in which the activation occurs, or to a composition to which the
activated
cellulose is added. Functionality includes providing such properties as
thickening,
imparting yield stress, heat stability, suspension properties, freeze-thaw
stability, flow
control, foam stabilization, coating and film formation, and the like. The
processing that
is followed during the activation process does significantly more than to just
disperse the
cellulose in base solvent. Such processing "tears apart" the cellulose fibers
to expand the
cellulose fibers. The bacterial cellulose-containing formulation may be used
in the form
of a wet slurry (dispersion) or as a dried product, produced by drying the
dispersion using
well-known drying techniques, such as spray-drying or freeze-drying to impart
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desired rheological benefits to a target fluid composition. The activation of
the bacterial
cellulose BC expands the cellulose portion to create a reticulated network of
highly
intermeshed fibers with a very high surface area. The activated reticulated
bacterial
cellulose possesses an extremely high surface area that is thought to be at
least 200-fold
higher than conventional microcrystalline cellulose (i.e., cellulose provided
by plant
sources).
The bacterial cellulose utilized herein may be of any type associated with the
fermentation product of Acetobacter genus microorganisms, and was previously
available, as one example, from CPKelco U.S. under the tradename CELLULONO.
Such aerobic cultured products are characterized by a highly reticulated,
branching
interconnected network of fibers that are insoluble in water.
The preparations of such bacterial cellulose products are well known. For
example, U.S. Pat. No. 5,079,162 and U.S. Pat. No. 5,144,021, both of which
are
incorporated by reference herein, disclose a method and media for producing
reticulated
bacterial cellulose aerobically, under agitated culture conditions, using a
bacterial strain
of Acetobacter aceti var. xylinum. Use of agitated culture conditions results
in sustained
production, over an average of 70 hours, of at least 0.1 g/liter per hour of
the desired
cellulose. Wet cake reticulated cellulose, containing approximately 80-85%
water, can
be produced using the methods and conditions disclosed in the above-mentioned
patents.
Dry reticulated bacterial cellulose can be produced using drying techniques,
such as
spray-drying or freeze-drying, that are well known.
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Acetobacter is characteristically a gram-negative, rod shaped bacterium 0.6-
0.8
microns by 1.0-4 microns. It is a strictly aerobic organism; that is,
metabolism is
respiratory, not fermentative. This bacterium is further distinguished by the
ability to
produce multiple poly P-1,4-glucan chains, chemically identical to cellulose.
The
microcellulose chains, or microfibrils, of reticulated bacterial cellulose are
synthesized at
the bacterial surface, at sites external to the cell membrane. These
microfibrils generally
have cross sectional dimensions of about 1.6 nm by 5.8 nm. In contrast, under
static or
standing culture conditions, the microfibrils at the bacterial surface combine
to form a
fibril generally having cross sectional dimensions of about 3.2 nm by 133 nm.
The small
cross sectional size of these Acetobacter-produced fibrils, together with the
concomitantly large surface and the inherent hydrophilicity of cellulose,
provides a
cellulose product having an unusually high capacity for absorbing aqueous
solutions.
Additives have often been used in combination with the reticulated bacterial
cellulose to
aid in the formation of stable, viscous dispersions.
The aforementioned problems inherent with purifying and collecting such
bacterial cellulose have led to the determination that the method employed
herein
provides excellent results to the desired extent. The first step in the
overall process is
provid'uig any amount of the target bacterial cellulose in fermented form. The
production
method for this step is described above. The yield for such a product has
proven to be
very difficult to generate at consistently high levels, thus it is imperative
that retention of
the target product be accomplished in order to ultimately provide a collected
product at
lowest cost.
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Purification is well known for such materials. Lysing of the bacterial cells
from
the bacterial cellulose product is accomplished through the introduction of a
caustic, such
as sodium hydroxide, or any like high pH (above about 12.5 pH, preferably)
additive in
an amount to properly remove as many expired bacterial cells as possible from
the
cellulosic product. This may be followed in more than one step if desired.
Neutralizing
with an acid is then typically followed. Any suitable acid of sufficiently low
pH and
molarity to combat (and thus effectively neutralize or reduce the pH level of
the product
as close to 7.0 as possible) may be utilized. Sulfuric acid, hydrochloric, and
nitric acid
are all suitable examples for such a step. One of ordinary skill in the art
would easily
determine the proper selection and amount of such a reactant for such a
purpose.
Alternatively, the cells may be lysed and digested through enzymatic methods
(treatment
with lysozyme and protease at the appropriate pH).
The lysed product is then subjected to mixing with a polymeric thickener in
order
to effectively coat the target fibers and bundles of the bacterial cellulose.
The polymeric
thickener must be insoluble in alcohol (in particular, isopropyl alcohol).
Such a thickener
is either an aid for dispersion of the bacterial cellulose within a target
fluid composition,
or an aid in drying the bacterial cellulose to remove water therefrom more
easily, as well
as potentially aid in dispersing or suspending the fibers within a target
fluid composition.
Proper dispersing aids (agents) include, without limitation, CMC (of various
types),
cationic HEC, etc., in essence any compound that is polymeric in nature and
exhibits the
necessary dispersion capabilities for the bacterial cellulose fibers when
introduced within
a target liquid solution. Preferably such a dispersing aid is CMC, such as
CEKOLO
available from CP Kelco. Proper precipitation aids (agents), as noted above,
include any
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number of biogums, including xanthan products (such as KELTROL , KELTROL T ,
and the like from CP Kelco), gellan gum, welan gum, diutan gum, rhamsan gum,
guar,
locust bean gum, and the like, and other types of natural polymeric
thickeners, such as
pectin, as one non-limiting example. Basically, the commingling of the two
products in
broth, powder or rehydrated powder form, allows for the desired generation of
the
polymeric thickener coating on at least a portion of the fibers and/or bundles
of the
bacterial cellulose. In one embodiment, the broths of bacterial cellulose and
xanthan are
mixed subsequent to purification (lysing) of both in order to remove the
residual bacterial
cells. In another embodiment, the broths may be mixed together without lysing
initially,
but co-lysed during mixing for such purification to occur.
The amounts of each component within the method may vary greatly. For
example, the bacterial cellulose will typically be present in an amount from
about 0.1% to
about 5% by weight, preferably from about 0.5 to about 3.0%, whereas the
polymeric
thickener may be present in an amount form 10 to about 900% by weight of the
bacterial
cellulose.
After mixing and coating of the bacterial cellulose by the polymeric
thickener, the
resultant product is then collected through co-precipitation in a water-
miscible
nonaqueous liquid. Preferably, for toxicity, availability, and cost reasons,
such a liquid is
an alcohol, such as, as most preferred, isopropyl alcohol. Other types of
alcohols, such as
ethanol, methanol, butanol, and the like, may be utilized as well, not to
mention other
water-miscible nonaqeuous liquids, such as acetone, ethyl acetate, and any
mixtures
thereof. Any mixtures of such nonaqueous liquids may be utilized, too, for
such a co-
precipitation step. Generally, the co-precipitated product is processed
througli a solid-
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liquid separation apparatus, allowing for the alcohol-soluble components to be
removed,
leaving the desired bacterial cellulose-containing formulation thereon.
From there, a wetcake form product is collected and then transferred to a
drying
apparatus and subsequently milled for proper particle size production. Further
co-agents
may be added prior to precipitation or to the wetcake or to the dried
materials in order to
provide further properties and/or benefits. Such co-agents include plant,
algal and
bacterial polysaccharides and their derivatives along with lower molecular
weight
carbohydrates such as sucrose, glucose, maltodextrin, and the like. Other
additives that
may be present within the bacterial cellulose-containing formulation include,
without
limitation, a hydrocolloid, polyacrylamides (and homologues), polyacrylic
acids (and
homologues), polyethylene glycol, poly(ethylene oxide), polyvinyl alcohol,
polyvinylpyrrolidones, starch (and like sugar-based molecules), modified
starch, animal-
derived gelatin, dairy proteins, soy proteins, other animal or plant-derived
proteins and
non-charged cellulose ethers (such as carboxymethylcellulose,
hydroxyethylcellulose,
and the like).
The bacterial cellulose-containing formulations of this invention may then be
introduced into the target inventive sufficiently low pH protein-based
beverages. Such
beverage compositions may include such bacterial cellulose-containing
formulations in
an amount from about 0.01% to about 1% by weight, and preferably about 0.03%
to
about 0.5% by weight of the total weight of the beverage composition and a
protein-
based material (preferably, though not necessarily dairy and or soy in nature)
in an
amount of from 0.1 to 20% by total weight of the beverage composition. Such
protein-
based materials include, again, without limitation, cow's milk, goat's milk,
soy milk,
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milk solids, whey proteins, caseins, soy protein concentrate, soy protein
isolate, and any
mixtures thereof. Other possible additives that may be included within this
low pH
beverage include, particularly, flavorings, preservatives, colorants,
stabilizers, sweeteners
(such as sugar, saccharin, and the like), fruit pulps, dietary fibers,
vitamins and minerals.
Preferred Embodiments of the Invention
The following non-limiting examples provide teachings of various inventive
beverages that are encompassed within this invention as well as comparatives
examples.
Suspension Aid Production
Example 1
BC was produced in a 1200 gal fermentor with final yield of 1.93 wt%. The
broth
was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm
of
lysozyme and 194 ppm of protease. A portion of the treated BC broth was mixed
with a
given amount of xanthan gum broth and CMC solution (BC/XG/CMC=3/1/1, dry
basis)
and the resultant mixture was then precipitated with IPA (85%) to form a press
cake. The
press cake was then dried and milled as in Example 1. The powdered formulation
was
then introduced into a STW sample in an amount of about 0.36% by weight
thereof, and
the composition was then mixed with a Silverson mixer at 8000 rpm for 5 min.
The
product viscosity and yield stress were 1057 cP and 3.65 dynes/cm2,
respectively.
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Example 2
BC was produced in a 1200 gal fermentor with final yield of 1.93 wt%. The
broth
was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm
of
lysozyme and 194 ppm of protease. A portion of the treated BC broth was mixed
with a
given amount of pectin solution (BC/Pectin=6/1, dry basis) and the resultant
mixture was
then precipitated with IPA (85%) to form a press cake. The press cake was
dried and
milled as in Example 1. The powdered formulation was then introduced into a
STW
sample in an amount of about 0.36% by weight thereof, wilh 20% CMC added
simultaneously, and the composition was then mixed with a Silverson mixer at
8000 rpm
for 5 min. The product viscosity and yield stress were 377 cP and 1.06
dynes/cm2,
respectively.
Example 3
BC was produced in a 1200 gal fermentor with final yield of 1.93 wt%. The
broth
was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm
of
lysozyme and 194 ppm of protease. A portion of the treated BC broth was mixed
with a
given amount of CMC solution (BC/CMC=3/1, dry basis) and the resultant mixture
was
then precipitated with IPA (85%) to form a press cake. The press cake was
dried and
milled as in Example 1. The powdered formulation was then introduced into a
STW
sample in an amount of about 0.36% by weight thereof, and the composition was
then
mixed with a Silverson mixer at 8000 rpm for 5 min. The product viscosity and
yield
stress were 432 cP and 1.39 dynes/cmz, respectively.
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Example 4
BC was produced in a 1200 gal fermentor with final yield of 1.93 wt%. The
broth
was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm
of
lysozyme and 194 ppm of protease. A portion of the treated BC broth was mixed
with a
given amount of pectin and CMC solutions (BC/Pectin/CMC=6/1/2, dry basis) and
the
resultant mixture was then precipitated with IPA (85%) to form a press cake.
The press
cake was dried and milled as in Example 1. The powdered formulation was then
introduced into a STW sample in an ainount of about 0.36% by weight thereof,
and the
composition was then mixed with a Silverson mixer at 8000 rpm for 5 min. The
product
viscosity and yield stress were 552 cP and 1.74 dynes/cm2, respectively.
Example 5
BC was produced in a 1200 gal fermentor with final yield of 1.4 wt%. The broth
was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm
of
lysozyme and 350 ppm of protease followed with another 350 ppm of
hypochlorite. A
portion of the treated BC broth was mixed with a given amount of xanthan gum
broth and
pre-hydrated CMC solution (BC/XG/CMC = 6/3/1, dry basis), then precipitated
with IPA
(85%), and dried and milled as in Example 1. The powdered formulation was then
introduced into a STW solution and 0.25% CaC12 solution in an amount of about
0.2%
by weight thereof, respectively, and the composition was then activated with
an
extensional homogenizer at 1500 psi for 2 passes. The product viscosities at 6
rpm were
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343 cP and 334 cP in STW and 0.25% CaC12 solutions, respectively. About 20 3.2
mm
diameter nylon beads (1.14 g/mL) were dropped into each of the solutions (in
STW or
0.25% CaC12 solution) and the solutions were left at room temperature for 24
hrs. None
of the beads settled down to the bottom of the beakers after the 24-hour time
period.
Example 6
BC was produced in a 1200 gal fermentor with final yield of 1.6 wt%. The broth
was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm
of
lysozyme and 350 ppm of protease followed with another 350 ppm of
hypochlorite. A
portion of the treated BC broth was mixed with a given amount of pre-hydrated
pectin
and CMC solutions (BC/Pectin/CMC = 6/3/1, dry basis), then precipitated with
IPA
(85%), and dried and milled as in Example 1. The powdered formulation was then
introduced into a STW solution and 0.25% CaCl2 solution in an amount of about
0.2%
by weight thereof, respectively, and the composition was then activated with
an
extensional homogenizer at 1500 psi for 2 passes. The product viscosities at 6
rpm were
306 cP and 293cP in STW and 0.25% CaC12 solutions, respectively. About 20 3.2
mm
diameter nylon beads (1.14 g/mL) were dropped into each of the solutions (in
STW or
0.25% CaC12 solution) and the solutions were left at room temperature for 24
hours.
None of the beads settled down to the bottom of the beakers after the 24-hour
time
period.
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Example 7
BC was produced in a 1200 gal fermentor with final yield of 1.6 wt%. The broth
was treated with 350 ppm of hypochlorite and subsequently treated with 70 ppm
of
lysozyme and 350 ppm of protease followed with another 350 ppm of
hypochlorite. A
portion of the treated BC broth was mixed with a given amount of pre-hydrated
CMC
solution (BC/CMC = 3/1, dry basis), then precipitated with IPA (85%), and
dried and
milled as in Example 1. The powdered formulation was then introduced into a
STW
solution and 0.25% CaC12 solution in an amount of about 0.2% by weight
thereof,
respectively, and the composition was then activated with an extensional
homogenizer at
1500 psi for 2 passes. The product viscosities at 6 rpm were 206 cP and 202 cP
in STW
and 0.25% CaC12 solutions, respectively. About 20 3.2 mm diameter nylon beads
(1.14
g/mL) were dropped into each of the solutions (in STW or 0.25% CaC12 solution)
and the
solutions were left at room temperature for 24 hours. None of the beads
settled down to
the bottom of the beakers after the 24-hour time period.
Each sample exhibited excellent and highly desirable viscosity modification
and
yield stress results. In terms of bacterial cellulose products, such results
have been
heretofore unattainable with bacterial cellulose materials alone and/or with
the low
complexity methods followed herein.
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Low pH Level Protein-Based Beverage Production and Analysis
Some initial comparative examples of pectin-containing soy-based beverages
were produced initially in order to demonstrate the stability of acid soy
drinks using such
high methoxyl (HM) pectin alone. These formulations are presented in Table 1,
below,
with the processing conditions listed thereafter. The soy protein was an
isolate available
from Solae under the tradename XT34N IP.
Table 1
0.20% Pectin 0.35% Pectin 0.50% Pectin
Control (Comp. Ex. 1) Com . Ex. 2) (Comp. Ex. 3)
Percent Grams Percent Grams Percent Grams Percent Grams
1.5% HM ectin solution 0.00 0 13.33 666.7 23.33 1166.7 33.33 1666.7
Deionized water 65.39 3269.5 52.06 2602.8 42.06 2102.8 32.06 1602.8
Soy protein isolate 1.56 78 1.56 78.0 1.56 78.0 1.56 78.0
Sugar 8.00 400 8.00 400.0 8.00 400.0 8.00 400.0
Oran e'uice 25.00 1250 25.00 1250.0 25.00 1250.0 25.00 1250.0
Sodium Citrate 0.05 2.5 0.05 2.5 0.05 2.5 0.05 2.5
To pH 4.0 To pH 4.0 To pH 4.0 To pH 4.0
50% citric acid solution
The soy protein isolate was dispersed into 25 C deionized (DI) water within a
flask using a high speed mixer (Caframo Stirrer). The resultant mixture was
then heated
to 70 C, held for 5 min and then cooled to ambient temperature (about 20-25
C). In a
separate flask, the HM pectin was dispersed into 50 C DI water using the same
type of
high speed mixer for 5 minutes and allowed to cool to ambient temperature. The
HM
pectin solution was then added to the soy isolate solution and stirred by hand
for about 3
minutes until the temperature was about - 25 C. The orange juice (no pulp
MINTJTE
MAIDO brand from The Coca-Cola Company) was then added to the resultant
solution.
Separately prepared was a dry blend of sodium citrate in the ainounts noted in
Table 1,
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above, the result of which was then introduced within the protein/pectin/juice
solution.
The pH was then adjusted to 4.0 using a 50% (w/v) citric acid solution while
stirring. An
ultrahigh temperature (UHT) process was then undertaken at 140.5 C for a 4.5
second
hold time, with further homogenization at 2000 psi (1500 first stage, 500
second stage),
and ultimate cooling to 30 C. The samples were then aseptically introduced
into
polyethylene terephthalate copolyester Nalgene bottles at 30 C for analysis.
Such
samples were then stored at room temperature for seven days and evaluated for
stability
and sedimentation.
Inventive samples were then prepared including certain bacterial-cellulose
containing formulations, such as BC:Xanthan:CMC (stabilizer A from Example 1,
above)
and BC:Pectin:CMC (stabilizer B from Example 6, above). Table 2 shows the
compositions made therefrom. The processes of preparing these are the same as
outlined
above.
Table 2
0.1% Stabilizer A 0.2% Stabilizer A 0.1% Stabilizer B 0.2% Stabilizer B
(Inv. Ex. 1) (Inv. Ex. 2) (Inv. Ex. 3) (Inv. Ex. 4)
Percent Grams Percent Grams Percent Grams Percent Grams
Deionized Water 65.29 3264.5 65.19 3259.5 65.29 3264.5 65.19 3259.5
Soy protein isolate 1.56 78 1.56 78 1.56 78 1.56 78
Sugar 8.00 400 8.00 400 8.00 400 8.00 400
Oran e'uice 25.00 1250 25.00 1250 25.00 1250 25.00 1250
Sodium Citrate 0.05 2.5 0.05 2.5 0.05 2.5 0.05 2.5
Stabilizer 0.10 5 0.20 10 0.10 5 0.20 10
50% citric acid solution to pH 4.0 to pH 4.0 to pH 4.0 to pH 4.0
Each of the control, comparative examples, and inventive examples from Tables
1
and 2 were stored for seven days at room temperature and evaluated. The
negative
control completely phase separated with a 50% clear upper layer and a thick
lower layer
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of sediment at the bottom half of the container. Upon adding 0.20% pectin, the
beverage
still formed a dense sediment at the bottom, with a cloudy upper layer that
constituted
90% of the beverage, indicating there was an insufficient amount of pectin
coating the
protein during the acidification step. The 0.35% and 0.50% pectin samples were
also
unstable due to the development of a visible pellet at the bottom of the
container,
however the mouthfeel of these beverages were different. The control and 0.20%
pectin
sample had an objectionable grainy texture, while 0.35% and 0.50% pectin
samples were
smooth, despite their instability.
Beverages produced with the Inventive BC-based stabilizer 1-4 demonstrated
noticeable improvements in stability over the pectin stabilized acid soy
drinks. Stabilizer
A showed improved stability over the control, with only 35% phase separation
at 0.10%
use level, compared to 50% phase separation in the control. The phase
separation was
further reduced to only 20% by increasing the concentration of Stabilizer A to
0.20%.
Beverages stabilized with stabilizer B showed no signs of phase separation.
The sensory
attributes of these BC-based only beverages were grainy in texture. The
results for these
beverage examples are provided in the following Table 3:
Table 3
0.1% 0.2% 0.1% 0.2%
0.2% 0.35% 0.50% Stabilizer Stabilizer Stabilizer Stabilizer
Control Pectin Pectin Pectin A A B B
% Phase 50 90 90 90 35 20 0 0
Separation
Cloudy Cloudy Cloudy
Visual Clear upper upper layer, upper layer, upper layer, Clear upper Clear
upper
Observations layer dense dense dense layer layer Stable Stable
sediment sediment sediment
Mouthfeel Grainy Grainy Smooth Smooth Grainy Grainy Grainy Grainy
Texture
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Further formulations were then prepared including both the inventive
stabilizers
and HM pectin in order to both improve the stability of the pectin only
formulation and
overcome the adverse texture of the BC-based stabilizer only formulation.
These new
inventive formulations were processed as follows and in accordance with the
process
steps outlined above after Table 1.
Table 4
0.05% Stabilizer B + 0.075% Stabilizer B + 0.10% Stabilizer B +
0.20% Pectin Inv. Ex. 5 0.20% Pectin Inv. Ex. 6 0.20% Pectin Inv. Ex. 7)
Percent Grams Percent Grams Percent Grams
1.5% HM pectin solution 13.3333 666.667 13.3333 666.667 13.3333 666.667
Deionized water 52.0042 2600.208 51.9779 2598.896 51.9517 2597.583
Soy protein isolate 1.5600 78.000 1.5600 78.000 1.5600 78.000
Sugar 8.0000 400.000 8.0000 400.000 8.0000 400.000
Oran e'uice 25.0000 1250.000 25.0000 1250.000 25.0000 1250.000
Stabilizer B 0.0500 2.500 0.0750 3.750 0.1000 5.000
Sodium Citrate 0.0500 2.500 0.0500 2.500 0.0500 2.500
50% citric acid solution to pH 4.0 to pH 4.0 to pH 4.0
Table 5
0.05% Stabilizer B + 0.075% Stabilizer B + 0.10% Stabilizer B +
0.35% Pectin Inv. Ex. 8 0.35% Pectin Inv. Ex. 9 0.35% Pectin inv. Ex. 10
Percent Grams Percent Grams Percent Grams
1.5% HM pectin solution 23.3333 1166.667 23.3333 1166.667 23.3333 1166,667
Deionized water 42.0042 2100.208 41.9779 2098.896 41.9517 2097.583
Soy protein isolate 1.5600 78.000 1.5600 78.000 1.5600 78.000
Sugar 8.0000 400.000 8.0000 400.000 8.0000 400.000
Oran e'uice 25.0000 1250.000 25.0000 1250.000 25.0000 1250.000
Stabilizer B 0.0500 2.500 0.0750 3.750 0.1000 5.000
Sodium Citrate 0.0500 2.500 0.0500 2.500 0.0500 2.500
50% citric acid solution to pH 4.0 to pH 4.0 to pH 4.0
Visual inspection after seven days showed that each of these inventive
combinations of stabilizer B with 0.20% pectin greatly improved the stability
of the
0.20% pectin beverage without BC-based stabilizer, Upon combining 0.05%
stabilizer B/
0.20% pectin, phase separation decreased from 90% shown in the pectin oizly
beverage,
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to just 40%. This reduction was further reduced to 25% phase separation by
using
0.075% stabilizer B/0.20% pectin, while only 10% instability was observed in
the 0.10%
stabilizer B/0.20% pectin combined system. Mouthfeel of all samples was
smooth, due
to the presence of pectin.
Although not presented above in tabular form, combinations of 0.35% pectin
with
Stabilizer A also showed improvements in stability over the pectin only
beverage. After
seven days, the sainples demonstrated 10% phase separation in both the 0.05%
and
0.075% stabilizer B/ 0.35% pectin stabilized beverages. Complete stability was
achieved
with 0.10% stabilizer B/ 0.35% pectin. Additionally, sensory evaluation of
these stable
samples indicated a smooth mouthfeel that lacked graininess. These data
suggest that
0.10% stabilizer B in combination with 0.35% provides the optimum stability
and
mouthfeel for this application. These results are presented below in Table 6.
Table 6
Inv. Ex. 5 Inv. Ex. 6 Inv. Ex. 7 Inv. Ex. 8 Inv. Ex. 9 Inv. Ex. 10
% Phase 40 25 10 10 10 0
Separation
Visual Clear upper Clear upper Clear upper Clear upper Clear upper Stable
Observations layer layer layer layer layer
Mouthfeel Smooth Smooth Smooth Smooth Smooth Smooth
Texture
Of further interest with this inventive system is the ability to demonstrate
the
functionality of BC-based stabilizers in suspending insoluble calcium in an
acidified
protein-based (soy, in this example, as a non-limiting selection) beverage.
Stabilizers B
and C (BC:CMC) (Example 3, above) were added to suspend calcium when used in
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addition to 0.35% pectin. The formulations were prepared as follows and in
accordance
with the process set forth after Table 1, above.
Table 7
0.10% 0.10%
Stabilizer B Stabilizer C
{- 0.35% {- 0.35%
Pectin Pectin
Control Inv. Ex. 11) Inv. Ex. 12)
Percent Grams Percent Grams Percent Grams
1.5% HM pectin solution 23.33 1166.5 23.33 1166.50 23.33 1166.50
Deionized water 41.74 2087.0 41.64 2082.00 41.64 2082.00
Soy rotein isolate 1.56 78.0 1.56 78.00 1.56 78.00
Sugar 8.00 400.0 8.00 400.00 8.00 400.00
Oran e'uice 25.00 1250.0 25.00 1250.00 25.00 1250.00
Tricalcium Phosphate 0.32 16.0 0.32 16.00 0.32 16.00
Stabilizer 0.00 0.0 0.10 5.00 '0.10 5.00
Sodium Citrate 0.05 2.5 0.0500 2.500 0.0500 2.500
50% citric acid solution to pH 4.0 to H 4.0 to pH 4.0
After seven days of room temperature, the control sample had the worst
stability,
due to formation of large sediment. The composition of the middle and bottom
portion of
the beverage was analyzed for solids and calcium content, in the stable and
unstable
regions, respectively. There was a higher amount of solids at the bottom
compared to the
center of the sample (15.16% vs. 11.80%). These unstable solids contained
2.15%
unstable calcium compared to 0.68% in the stable portion of the beverage, of
which the
difference in calcium and solids in the sediment was composed of protein and
sugars.
Both types of BC based stabilizers improved calcium suspension over the
control.
The difference in solids between the center and bottom of the sample in
stabilizers B and
C was negligible, as was the difference in calcium concentration, suggesting
that both
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BC-based stabilizers were capable of suspending protein in the acidified soy
beverage.
The results are tabulated below in Table 8.
Table 8
Location of Sample % Total solids % Ca in solids % Ca in Bev. % RDA
Center 11.80% 0.677% 0.080% 19.00%
Control
Bottom 15.16% 2.155% 0.327% 77.76%
Inv. Ex. 11 Center 13.16% 1.221% 0.161% 38.24%
Bottom 13.19% 1.242% 0.164% 39.00%
Inv. Ex. 12 Center 13.13% 1.229% 0.161% 38.41%
Bottom 13.18% 1.302% 0.172% 40.85%
Thus, the inventive stabilized beveraged exhibited excellent calcium
stabilization
and suspension versus the control even when in solution with potentially
aggregating
protein solids.
Further work was then undertaken to investigate the functionality of inventive
BC-based stabilizers co-processed with pectin in stabilizing low acidity (pH
5) soy
protein juice drinks. The formulations testing are listed below in Table 9.
Table 9
0.075% 0.10% 0.15%
Stabilizer B Stabilizer B Stabilizer B
Control Inv. Ex. 13) Inv. Ex. 14) Inv. Ex. 15)
Percent Grams Percent Grams Percent Grams Percent Grams
Nater 32.60 1630.00 32.53 1626.25 32.50 1625.00 32.45 1622.50
3oy Milk 35.00 1750.0 35.00 1750.0 35.00 1750.0 35.00 1750.0
3u ar 8.00 400.0 8.00 400.0 8.00 400.0 8.00 400.0
Xan e Juice 24.00 1200.0 24.00 1200.0 24.00 1200.0 24.00 1200.0
3odium Citrate 0.20 10.0 0.20 10.0 0.20 10.0 0.20 10.0
5tabilizer 0.00 0.0 0.075 3.8 0.10 5.00 0.15 7.5
Vanilla Extract 0.20 10.0 0.20 10.0 0.20 10.0 0.20 10.0
Citric Acid Solution (50% w/v) to pH 5.0 to pH 5.0 to pH 5.0 to H 5.0 to pH
5.0 to pH 5.0 to pH 5.0 to pH 5.0
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These formulations were prepared as follows: The soy protein isolate was first
dispersed into 25 C DI-water using an high speed mixer in a flask. The
solution was then
heated to 70 C, held for 5 minutes at that temperature, and then cooled to
ambient
temperature. The juice was then added to the soy milk while stirring. Sodium
citrate,
sugar and the inventive stabilizer were then dry blended in the amounts as
listed above
and added to the already mixed soy solution. The pH was then adjusted to 5.0
using a
50% (w/v) citric acid solution while stirring. An UHT process was then
undertaken at
140.5 C for a 4.5 second hold time, with homogenization at 2000 psi (1500
first stage,
500 second stage) and subsequent.cooling to 30 C. Bottles of polyethylene
terephthalate
copolyester Nalgene were then filled aseptically (as above) bottles at 30 C
for storage
and evaluation.
After 7 days room temperature storage, the control sample formed a protein
sediment at the bottom of the container, and demonstrated 80% phase
separation. Upon
adding 0.075% stabilizer B, stability improved to just 35% phase separation.
Increasing
the concentration to 0.10% further improved the stability to 10% phase
separation.
Complete stability was observed in the sample stabilized with 0.15% stabilizer
B. In
addition, all samples were orally evaluated and there was no graininess noted
in any the
samples. These data demonstrated that the BC based stabilizer is capable of
suspending
soy protein in the pH range near 5Ø The results are tabulated below in Table
10.
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Table 10
Control Inv. Ex. 13 inv. Ex. 14 Inv. Ex. 15
% Phase 80 35 10 0
Separation
Visual Clear upper Clear upper Clear upper Stable
Observations layer layer layer
Mouthfeel Smooth Smooth Smooth Smooth
Texture
Furthermore, experiments were then undertaken to investigate the functionality
of
inventive BC-based stabilizers in suspending heat-denatured milk proteins
within a
lightly acidified dairy based juice beverage. Two concentrations of stabilizer
B
(BC:Pectin:CMC) (Example 6, above) were added to the beverage and compared to
the
control sample. The formulations processed for this analysis were prepared as
follows in
accordance with Table 11 and in the outline below.
Table I1
0.15% 0.20%
Stabilizer B Stabilizer B
Control Inv. Ex. 16) Inv. Ex. 17)
Percent Percent Percent
Deionized Water 37.85 37.70 37.65
2.0% Milk 30.00 30.00 30.00
Sugar 8.00 8.00 8.00
Orange Juice 24.00 24.00 24.00
Vanilla Flavor 0.15 0.15 0.15
Citric Acid Solution
(50% w/v to pH 5.0 to pH 5.0 to pH 5.0
To prepare these beverage, Dl'water, milk, sugar and vanilla and were inixed
together using a high speed mixer. To this resultant mixture was slowly added
orange
juice, and the pH of the resultant composition was then adjusted to 5.0 using
a 50% (w/v)
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citric acid solution while stirring. An UHT process at 140.5 C for 4.5 seconds
hold time
was then undertaken, with homogenization at 2000 psi (1500 first stage, 500
second
stage), and subsequent cooling to 30 C. As above, polyethylene terephthalate
copolyester Nalgene bottles were then filled aseptically at 30 C and stored at
room
temperature for evaluation.
After 7 days of such storage, the control sample had completely failed,
forming a
dense sediment at the bottom of the container. Both samples using stabilizer B
had
completely uniform suspension of proteins in the drink. Oral evaluation of the
sample
demonstrated a noticeable grainy texture in the control sample, while 0.15%
Stabilizer B
and 0.20% Stabilizer B were smoother. The inouthfeel increased in thickness as
the
concentration of stabilizer B increased from 0.15% to 0.20%.
r Thus, in all instances, the inclusion of the suspension aid with BC imparted
excellent low phase separation, stable visual appearance, and excellent
mouthfeel,
particularly as compared with the control and the other comparative suspension
aid
systems.
While the invention will be described and disclosed in connection with certain
preferred embodiments and practices, it is in no way intended to limit the
invention to
those specific embodiments, rather it is intended to cover equivalent
structures and all
alternative embodiments and modifications as may be defined by the scope of
the
appended claims and equivalence thereto.
