Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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MICRO-CLUSTER COMPOSITIONS OF DRUGS, BIO-AFFECTING
COMPOSITIONS, BODY-TREATING COMPOSITIONS, CULTURE
MEDIA, FOODS AND BEVERAGES
FIELD OF THE INVENTION
The invention relates generally to micro-cluster liquids and methods of making
and using
them. The present invention provides a process of making micro-cluster liquid
and
methods of use thereof.
CONTENTS OF APPLICATTON
I. Micro-cluster Liquids and Methods of Making and Using Them
IT. Culture Media and Methods of Making and Using Culture Media
ITI. Drugs, Bio-affecting and Body-Treating Compositions
IV. Food or Edible Material and Beverages; Processes, Compositions, and
Products
I. MICRO-CLUSTER LIQUIDS AND METHODS OF MAKING AND USING
THEM
BACKGROUND OF THE INVENTION
Water is composed of individual HZO molecules that may bond with each other
through
hydrogen bonding to form clusters that have been characterized as five
species: un-bonded
molecules, tetrahedral hydrogen bonded molecules comprised of five (5) H20
molecules in
a quasi-tetrahedral arrangement and surface connected molecules connected to
the clusters
by 1,2 or 3 hydrogen bonds, (U.S. Patent 5,711,950 Lorenzen; Lee H.). These
clusters can
then form larger arrays consisting of varying amounts of these micro-cluster
molecules
with weak long distance van der Waals attraction forces holding the arrays
together by one
or more of such forces as; (1) dipole-dipole interaction, i.e., electrostatic
attraction
between two molecules with permanent dipole moments; (2) dipole-induced dipole
interactions in which the dipole of one molecule polarizes a neighboring
molecule; and (3)
dispersion forces arising because of small instantaneous dipoles in atoms.
Under normal
conditions the tetrahedral micro-clusters are unstable and reform into larger
arrays from
agitation, which impart London Forces to overcome the van der Waals repulsion
forces.
Dispersive forces arise from the xelative position and motion of two water
molecules when
these molecules approach one another and results in a distortion of their
individual
envelopes of infra-atomic molecular orbital configurations. Each molecule
resists this
distortion resulting in an increased force opposing the continued distortion,
until a point of
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proximity is reached where London Inductive Forces come into effect. If the
velocities of
these molecules are sufficiently high enough to allow them to approach one
another at a
distance equal to van der Waals radii, the water molecules combine.
There is currently a need for a process whereby large molecular arrays of
liquids can be
advantageously fractionated. Furthermore, there is a desire for smaller
molecular (e.g.,
micro-clusters) of water for consumption, medicinal and chemical processes.
SUMMARY OF THE INVENTION
The inventors have discovered that liquids, which form large molecular arrays,
such as
through various electrostatic and van der Waal forces (e.g.~ water), can be
disrupted
through cavitation into fractionated or micro-cluster molecules (e.g.,
theoretical tetrahedral
micro-clusters of water). The inventors have further discovered a method for
stabilizing
newly created micro-clusters of water by utilizing van der Waals repulsion
forces. The
method involves cooling the micro-cluster water to a desired density, wherein
the micro-
cluster water may then be oxygenated. The micro-cluster water is bottled while
still cold.
In addition, by overfilling the bottle and capping while the micro-cluster
oxygenated water
is dense (i. e., cold), the London forces are slowed down by reducing the
agitation which
might occur in a partially filled bottle while providing a partial pressure to
the dissolved
gases (e.g., oxygen) in solution thereby stabilizing the micro-clusters for
about 6 to 9
months when stored at 40 to 70 degrees Fahrenheit.
The present invention provides a process for producing a micro-cluster liquid,
such as
water, comprising subjecting a liquid to cavitation such that dissolved
entrained gases in
the liquid form a plurality of cavitation bubbles; and subj ecting the liquid
containing the
plurality of cavitation bubbles to a reduced pressure, wherein the reduction
in pressure
causes breakage of large liquid molecule matrices into smaller liquid molecule
matrices. In
another embodiment the liquid is substantially free of minerals and can be
water which
may also be substantially free of minerals. The embodiment provides for a
process which
is repeated until the water reaches about 140°C (about 60°C).
The cavitation can be
provided by subjecting the liquid to a first pressure followed by a rapid
depressurization to
a second pressure to form cavitation bubbles. The pressurization can be
provided by a
pump. In one embodiment the first pressure is about 55 psig to more than 120
psig. In
another embodiment the second pressure is about atmospheric pressure. The
embodiment
can be carried out such that the pressure change caused the plurality of
cavitation bubbles
to implode or explode. The pressure change may be performed to create a plasma
which
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dissociates the local atoms and reforms the atom at a different bond angle and
strength. In
another embodiment the liquid is cooled to about 4°C to 15°C.
Further embodiment
comprises providing gas to the micro-cluster liquid, such as where the gas is
oxygen. In a
further embodiment the oxygen is provided for about 5 to about 15 minutes.
In a further embodiment the invention provides a process for producing a micro-
cluster
liquid, comprising subjecting a liquid to a pressure sufficient to pressurize
the liquid;
emitting the pressurized liquid such that a continuous stream of liquid is
created;
subjecting the continuous stream of liquid to a multiple rotational vortex
having a partial
vacuum pressure such that dissolved and entrained gases in the liquid form a
plurality of
cavitation bubbles; and subjecting the liquid containing the plurality of
cavitation bubbles
to a reduced pressure, wherein the plurality of cavitation bubbles implode or
explode
causing shockwaves that break large liquid molecule matrices into smaller
liquid molecule
matrices. In a further embodiment the liquid is substantially free of minerals
and in an
additional embodiment the liquid is water, preferably substantially free of
minerals. The
invention provides that the process can be repeated until the water reaches
about 140°F
(about 60°C). In another embodiment the cavitation is provided by
subjecting the liquid to
a first pressure followed by a rapid depressurization to a second pressure to
form
cavitation bubbles. Further the invention provides that the pressurization is
provided by a
pump. In a further embodiment the first pressure.is about 55 psig to more than
120 psig
and, in another embodiment the second pressure is about atmospheric pressure,
including
embodiments where the second pressure is less than S prig. The invention also
provides
for micro-cluster liquid where the pressure change causes the plurality of
cavitation
bubbles to implode or explode. In a fiuther embodiment, the pressure change
creates a
plasma which dissociates the local atoms and reforms the atoms at a different
bond angle
2S and strength. The invention also provides a process where the liquid is
cooled to about 4°C
to 15°C. In another embodiment, the invention provides subjecting a gas
to the micro-
cluster liquid. Preferably, the gas is oxygen, especially oxygen administered
for about 5 to
1 S minutes and more preferably at pressure from about 15 to 20 psig.
The present invention also provides for a composition comprising a micro-
cluster water
produced according to the procedures noted above.
Still another aspect of the invention is a micro-cluster water which has any
or all of the
properties of a conductivity of about 3.0 to 4.0 ~.mhos/cm, a FTIR
spectrophotometric
pattern with a major sharp feature at about 2650 wave numbers, a vapor
pressure between
about 40°C and 70°C as determined by thermogravimetxic analysis,
and an 1'O NMR peak
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shift of at least about +30 Hertz, preferably at least about +40 Hertz
relative to reverse
osmosis water.
The present invention further provides for the use of the micro-cluster water
of the
invention for such purposes as modulating cellular performance and lowering
free radical
levels in cells by contacting the cell with the micro-cluster water.
The present invention further provides a delivery system comprising a micro-
cluster water
(e.g., an oxygenated microcluster water) and an agent, such as a nutritional
agent, a
medication, and the like.
Further, the micro-cluster water of the invention can be used to remove stains
from fabrics
by contacting the fabric with the micro-cluster water.
The details of one or more embodiments of the invention are set forth in the
accompanying drawings and the description below. Other features, objects, and
advantages of the invention will be apparent from the description and
drawings, and from
the claims.
All publications, patents and patent applications cited herein are hereby
expressly
incorporated by reference for all purposes.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a water molecule and the resulting net dipole moment.
FIG. 2 shows a large array of water molecules.
FIG. 3 shows a micro-cluster of water having 5 water molecules forming a
tetrahedral '
shape.
FIG. 4 shows an example of a device useful in creating cavitation in a liquid.
The device
provides inlets for a liquid, wherein the liquid is then subjected to multiple
rotational
vortexes reaching partial vacuum pressures of about 27" Hg. The liquid then
exits the
device at point A through an acceleration tube into a chamber less than the
pressure within
the device (e.g., about atmospheric pressure).
FIG. 5 shows FTIR spectra for RO water (Figure 5(a)) and processed micro-
cluster water
(Fig. 5(b)).
FIG. 6 shows TGA plots for RO water and oxygenated micro-cluster water.
FIG. 7 shows NMR spectra for RO water (Fig. 7(a)), micro-cluster water without
oxygenation (Fig. 7(b)) and micro-cluster water with oxygenation (Fig. 7(c)).
Fig. 8 shows a schematic illustration of a device for Raman spectroscopy.
Fig. 9 shows the effects of micro-clustered cell culture medium on macrophage
plasma
membranes.
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Fig. 10 shows the effects of micro-clustered cell culture medium on
intracellular pH.
Fig. 11 shows the effects of micro-clustered cell culture medium on the
viability of 293T
cells.
Figs. 12a and 12b show the effects of micro-clustered water on growth and
transfection of
two types of human cells.
Fig. 13 shows the effects of micro-clustered water on the expression profiles
of dendritic
cell markers.
Fig. 14 shows the effects of micro-clustered water on the functional state of
brain tissue
perfused with micro-clustered medium.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Liquids, including for example, alcohols, water, fuels and combinations
thereof, are
comprised of atoms and molecules having complex molecular arrangements. Many
of
these arrangements result in the formation of large molecular arrays of
covalently bonded
atoms having non-covalent interactions with adjacent molecules, which in turn
interact via
additional non-covalent interactions with yet other molecules. These large
arrays, although
stable, are not ideal for many applications due to their size. Accordingly it
is desirable to
create and provide liquids having smaller arrays by reducing the number of
rion-covalent
interactions. These smaller molecules are better able to penetrate and react
in biological
and chemical systems. In addition, the smaller riiolecular arrays provide
novel
characteristics that are desirable.
As used herein, "covalent bonds" means bonds that result when atoms share
electrons. The
term "non-covalent bonds" or "non-covalent interactions" means bonds or
interactions
wherein electrons axe not shared between atoms. Such non-covalent interactions
include,
for example, ionic (or electrovalent) bonds, formed by the transfer of one or
more
electrons from one atom to another to create ions, interactions resulting from
dipole
moments, hydrogen bonding, and van der Waals forces. Van der Waals forces are
weak
forces that act between non-polar molecules or between parts of the same
molecule, thus
bringing two groups together due to a temporary unsymmetrical distribution of
electrons
in one group, which induces an opposite polarity in the other. When the groups
are
brought closer than their van der Waals radii, the force between them becomes
repulsive
because their electron clouds begin to interpenetrate each other.
Numerous liquids are applicable to the techniques described herein. Such
liquids include
water; alcohols, petroleum and fuels. Liquids, such as water, are molecules
comprising
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one or more basic elements or atoms (e.g., hydrogen and oxygen). The
interaction of the
atoms through covalent bonds and molecular charges form molecules. A molecule
of
water has an angular or bent geometry. The H-O-H bond angle in a molecule of
water is
about 104.5° to 105°. The net dipole moment of a molecule of
water is depicted in FIG. 1.
This dipole moment creates electrostatic forces that allow for the attraction
of other
molecules of water. Recent studies by Pugliano et al., (Science, 257:1937,
1992) have
suggested the relationship and complex interactions of water molecules. These
studies
have revealed that hydrogen bonding and oxygen-oxygen interactions play a
major role in
creating large clusters of water molecules. Substantially purified water forms
complex
structures comprising multiple water molecules each interacting with an
adjacent water
molecule (as depicted in FIG. 2) to form large arrays. These large arrays are
formed based
upon, for example, non-covalent interactions such as hydrogen bond formation
and,as a
result of the dipole moment of the molecule. Although highly stable, these
large molecules
have been suggested to be detrimental in various chemical and biological
reactions.
Accordingly, in one embodiment, the present invention provides a method of
forming
fractionized or micro-cluster water as depicted in FIG. 3 having as few as
about 5
molecules of water.
The present invention provides small micro-cluster liquids (e.g., micro-
cluster water
molecules) a method for manufacturing fractionized or micro-cluster water and
methods of
use in the treatment of various biological conditions.
Accordingly, the present invention provides a method for manufacturing
fractionized or
micro-cluster liquids (e.g., water) comprising pressurizing a starting liquid
to a first
pressure followed by rapid depressurization to a second pressure to create a
partial vacuum
pressure that results in release of entrained gases and the formation of
cavitation bubbles.
The thermo-physical reactions provided by the implosion and explosion of the
cavitation
bubbles results in an increase in heat and the breaking of non-covalent
interactions holding
large liquid arrays together. This process can be repeated until a desired
physical-chemical
trait of the fractionized liquid is obtained. Where the liquid is water, the
process is
repeated until the water temperature reaches about 140° F (about 6,0
°C). The resulting
smaller or fractionized liquid is cooled under conditions that prevent
reformation of the
large arrays. As used herein, "water" or "a starting water" includes tap
water, natural
mineral water, and processed water such as purified water.
Any number of techniques known to those of skill in the art can be used to
create
cavitation in a liquid so long as the cavitating source is suitable to
generate sufficient
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energy to break the large arrays. The acoustical energy produced by the
cavitation
provides energy to break the large liquid arrays into smaller liquid clusters.
For example,
the use of acoustical transducers may be utilized to provide the required
cavitation source.
In addition, cavitation can be induced by forcing the liquid through a tube
having a
constriction in its length to generate a high pressure before the
constriction, which is
rapidly depressurized following the constriction. Another example, includes
forcing a
liquid through a pump in reverse direction through a rotational volute.
In one embodiment, a liquid to be fractionized is pressurized into a
rotational volute to
create a vortex that reaches partial vacuum pressures releasing entrained
gases as
cavitation bubbles when the rotational vortex exits through a tapered nozzle
at or close to
atmospheric pressure. This sudden pressurization and decompression causes
implosion
and explosion of cavitation bubbles that create acoustical energy shockwaves.
These
shockwaves break the covalent and non-covalent bonds on the large liquid
arrays, break
the weak array bonds, and form micro-cluster or fractionized liquid consisting
of, for
example, about five (5) H20 molecules in a quasi tetrahedral arrangement (as
depicted in
FIG. 3), and impart an electron charge to the micro-cluster liquid thus
producing
electrolyte properties in the liquid. The micro-cluster liquid is recycled
until desired
number of micro-cluster liquid molecules are formed to reach a given surface
tension and
electron charge, as determined by the temperature rise of the liquid over time
as cavitation
bubbles impart kinetic heat to the processed liquid. Once the desired surface
tension and
electron charge are reached the micro-cluster liquid is cooled until liquid
density increases.
The desired surface tension and electron charge can be measured in any number
of ways,
but is preferably detected by temperature. Once the liquid reaches a desired
density,
typically at about 4 to 15 °C, a gas, such as, for example, molecular
oxygen, can be
introduced for a sufficient amount of time to attain the desired quantity of
oxygen in the
micro-cluster liquid. The micro-cluster liquid is then aliquoted into a
container or bottle,
preferably filled to maximum capacity, and capped while the gassed micro-
cluster liquid is
still cool, so as to provide a partial pressure to the gassed micro-cluster
liquid as the
temperature reaches room temperature. This enables larger quantities of
dissolved gas to
be maintained in solution due to increased partial pressure on the bottles
contents.
The present invention provides a method for making a micro-cluster or
fractionized water
or liquid, for ease of explanation water will be used as the liquid being
described, however
any type liquid may be substituted for water. A starting water such as, for a
example,
purified or distilled water is preferably used as a base material since it is
relatively free of
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mineral content. The water is then placed into a food grade stainless steel
tank for
processing. By subjecting the starting water to a pump capable of supplying a
continuous
pressure of between about 55 and 120 psig or higher a continuous stream of
water is
created. This stream of water is then applied to a suitable device (see for
example FIG. 4)
capable of establishing a multiple rotational vortex reaching partial vacuum
pressures of
about 27" Hg, thereby reaching the vapor pressure of dissolved entrained gases
in the
water. These gases form cavitation bubbles that travel down multiple
acceleration tubes
exiting into a common chamber at or close to atmospheric pressure. The
resultant shock
waves produced by the imploding and exploding cavitation bubbles breaks the
large water
arrays into smaller water molecules by repeated re-circulation of the water.
The recycling
of the water creates increases results in an increase in temperature of the
water. The heat
produced by the imploding and exploding cavitation bubbles release energy as
seen in
sonoluminescence, in which the temperature of sonoluminance bubbles are
estimated to
range from 10 to 100 eV or 2,042.033 degrees Fahrenheit at 19,743,336
atmospheres.
However the heat created is at a sub micron size and is rapidly absorbed by
the
surrounding water imparting its kinetic energy. The inventors have determined
that the
breaking of these large arrays into smaller water molecules can be manipulated
through a
sinusoidal wave utilizing cavitation, and by monitoring the rise in
temperature one can
adjust the osmotic pressure and surface tension of the water under treatment.
The
inventors have determined that the ideal temperature for oxygenated micro-
cluster water
(Penta-hydrateTM) is about 140 degrees F (about 60 °C). This can be
accomplished by
using four opposing vortex volutes with a 6-degree acceleration tube exiting
into a
common chamber at or close to atmospheric pressure, less than 5 pounds
backpressure.
As mentioned above, the inventors have also discovered that liquids undergo a
sinusoidal
fluctuation in heat/temperature under the process described herein. Depending
upon the
desired physical-chemical traits, the process is repeated until a desired
point in the
sinusoidal curve is established at which point the liquid is collected and
cooled under,
conditions to inhibit the formation of large molecular arrays. Fox example,
and not by way
of limitation, the inventors have discovered that water processed according to
the methods
described herein undergoes a sinusoidal heating process. During the production
of this
water a high negative chaxge is created and imparted to the water. Voltages of
-350 mV to-
I volt have been measured with a superimposed sinusoidal wave with a frequency
of 800
cycles or higher depending on operating pressures and subsequent water
velocities. The
inventors have found that the third sinusoidal peak in temperature provides an
optimal
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number of micro-cluster structures for water. Although the inventors are under
no duty to
provide the mechanism or theory of action, it is believed that the high
negative ion
production serves as a ready source of donor electrons to act as antioxidants
when
consumed and further act to stabilize the water micro-clusters and help
prevent
reformation of the large arrays by aligning the water molecules exposed to the
electrostatic
field of the negative charge. While not wanting to be bound to a particular
theory, it is
believed that the high temperatures achieved during cavitation may form a
plasma in the
water which dissociates the H20 atoms and which then reform at a different
bond
association, as evidenced by the FTIR and NMR test data, to generate a
different structure.
It will be recognized by those skilled in the art that the water of the
present invention can
be further modified in any number of ways. For example, following formation of
the
micro-cluster water, the water may be oxygenated as described herein, fiu-ther
purified,
flavored, distilled, irradiated, or any number of further modifications known
in the art and
which will become apparent depending on the final use of the water.
In another embodiment, the present invention provides methods of modulating
the cellular
performance of a tissue or subject. The micro-cluster water (e.g., oxygenated
microcluster
water) can be designed as a delivery system to deliver hydration, oxygenation,
nutrition,
medications and increasing overall cellular performance and exchanging liquids
in the cell
and removing edema. Tests accomplished utilizing an RJL Systems Bio-Electrical
Impedance Analyzer model BIA101 Q Body Composition Analysis SystemTM
demonstrated substantial intracellular and extracellular hydration, changes in
as little as 5
minutes. Tests were accomplished on a 5~-year-old male 71.5" in height 269
lbs, obese
body type. Baseline readings were taken with Bio-Electrical Impedance
AnalyzerTM as
listed below.
As described in the Examples below it is contemplated that the micro-cluster
water of the
present invention provides beneficial effects upon consumption by a subject.
The subject
can be any mammal (e.g, equine, bovine, porcine, marine, feline, canine) and
is preferably
human. The dosage of the micro-cluster water or oxygenated micro-cluster water
(Penta-
hydrateTM) will depend upon many factors recognized in the art, which are
commonly
modified and adjusted. Such factors include, age, weight, activity,
dehydration, body fat,
etc. Typically 0.5 liters of the oxygenated micro-cluster water of the
invention provide
beneficial results. In addition, it is contemplated that the micro-cluster
water of the
invention may be administered in any number of ways known in the art,
including, for
example, orally and intravenously alone or mixed with other agents, compounds
and
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chemicals. It is also contemplated that the water of the invention may be
useful to irrigate
wounds or at the site of a surgical incision. The water of the invention can
have use in the
treatment of infections, for example, infections by anaerobic organisms may be
beneficially treated with the micro-cluster water (e.g., oxygenated
microcluster water).
In another embodiment, the micro-cluster water of the invention can be used to
lower free
radical levels and, thereby, inhibit free radical damage in cells.
In still another embodiment the micro-cluster water of the invention can be
used to remove
stains from fabrics, such as cotton.
The following examples are meant to illustrate but no limit the present
invention.
Equivalents of the following examples will be recognized by those skilled in
the art and
are encompassed by the present disclosure.
EXAMPLE 1
How to Make Micro-Cluster Water
Described below is one example of a method for making micro-cluster liquids.
Those
skilled in the art will recognize alternative equivalents that are encompassed
by the present
invention. Accordingly, the following examples is not to be construed to limit
the present
invention but are provided as an exemplary method for better understanding of
the
invention.
325 gallons of steam distilled water from Culligan Water or purified in 5
gallon bottles at
a temperature about 29 degrees C. ambient temperature, was placed in a 316
stainless steel
non-pressurized tank with a removable top for treatment. The tank was
connected by
bottom feed 2 114" 316 stainless steel pipe that is reduced to 1" NPT into a
20" U.S. filter
housing containing a 5 micron fiber filter, the filter serves to remove any
contaminants
that may be in the water. Output of the 20" filter is connected to a Teel
model 1 V458 316
stainless steel Gear pump driven by a 3HP 1740 RPM 3 phase electric motor by
direct
drive. Output of the gear pump 1" NPT was directed to a cavitation device via
1" 316
stainless steel pipe fitted with a 1" stainless steel ball valve used for
isolation only and
pasta pressure gauge. Output of the pump delivers a continuous pressure of 65
psig to the
cavitation device.
The cavitation device was composed of four small inverted pump volutes made of
Teflon
without impellers, housed in a 316 stainless steel pipe housing that are
tangentially fed by
a common water source fed by the 1 V458 Gear pump at 65 psig, through a 1/4"
hole that
would normally be used as the discharge of a pump, but are utilized as the
input for the
purpose of establishing a rotational vortex. The water entering the four
volutes is directed
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in a circle 360 degrees and discharged through what would normally be the
suction side of
a pump by the means of an 1" long acceleration tube with a 3l8" discharge
hole,
comprising what would normally be the suction side of a pump volute but in
this case is
utilized as the discharge side of the device. The four reverse fed volutes
establish
rotational vortexes that spin the water one 360 degree rotation and then
discharge the
water down the 5 degree decreasing angle from center line, acceleration tubes
discharging
the water into a common chamber at or close to atmospheric pressure. The
common
chamber was connected to a 1" stainless steel discharge line that fed back
into the top of
the 325-gallon tank containing the distilled water. At this point the water
made one
treatment trip through the device.
The process listed above is repeated continuously until the energy created by
the
implosions and explosions ofthe cavitation (e.g., due to the acoustical
energy) have
imparted its kinetic heat into the water and the water is at about 60 degrees
Celsius.
Although the inventors are under no duty to explain the theory of the
invention, the
inventors provide the following theory in the way of explanation and are not
to be bound
by this theory. The inventors believe that the acoustical energy created by
the cavitation
brakes the static electric bonds holding a single tetrahedral Micro-Clusters
of five H20
molecules together in larger arrays, thus decreasing their size and/or create
a localized
plasma in the water restructuring the normal bond angles into a different
structure of
water.
The temperature was detected by a hand held infrared~thermal detector through
a stainless
steel thermo well. Other methods of assessing the temperature will be
recognized by those
of skill in the art. Once the temperature of 60 degrees C. has been reached
the pump motor
is secured and the water is left to cool. An 8 foot by 8 foot insulated room
fitted with a
5,000 Btu. air conditioner is used to expedite cooling, but this is not
required. It is
important that the processed water not be agitated for cooling it should be
moved as little
as possible.
A cooling temperature of 4 degrees C. can be used, however 15 degrees C. is
sufficient
and will vary depending upon the quantity of water being cooled. Once
sufficiently cooled
to about 4 to 15 degrees C the water can be oxygenated.
Once the water is cooled to desired temperature, the processed water is
removed from the
325 gallon stainless steel tank into 5-gallon polycarbonate bottles for
oxygenation.
Oxygenation is accomplished by applying gas 02 at a pressure of 20 psig fed
through a
1 /4" ID plastic Iine fitted with a plastic air diffuser utilized to make fine
air bubbles (e. g. ,
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Lee's Catalog number 12522). The plastic tube is run through a screw on lid of
the 5
gallon bottle until it reaches the bottom of the bottle. The line is fitted
with the air diffuser
at its discharge end. The Oxygen is applied at 20 psig flowing pressure to
insure a good
visual flow of oxygen bubbles. In one embodiment (Penta-hydrate~M) the water
is
oxygenated for about five minutes and in another embodiment (Penta-hydrate
ProTM) the
water is oxygenated for about ten minutes.
Immediately after oxygenation the water is bottled in SOOmI PET bottles,
filled to
overflowing and capped with a pressure seal type plastic cap with inserted
seal gasket. In
one embodiment, the 0.5 L bottle is over filled so when the temperature of the
water
increases to room temperature it will self pressurize the bottle retaining a
greater
concentration of dissolved oxygen at partial pressure. This step not only
keeps more
oxygen in a dissolved state but also for preventing excessive agitation of the
water during
shipping.
EXAMPLE 2
A novel water prepared by the method of the invention was characterized with
respect to
various parameters.
A. Conductivity
Conductivity was tested using the.USP 645 procedure that specifies
conductivity
measurements as criteria for characterizing water. In addition to defining the
test protocol,
USP 645 sets performance standards for the conductivity measurement system, as
well as
validation and calibration requirements for the meter and conductivity.
Conductivity
testing was performed by West Coast Analytical Service, Inc. in Santa Fe
Springs, CA.
Conductivity Test Results
w_ l0~ RO Water Micro-cluster Water Micro-cluster
Water
Conductivity at 25°C.*
(~,mhos/cm) 5.55 3.16 3.88
* Conductivity values are the average of two measurements.
The conductivity observed for the micro-cluster water is reduced by slightly
more than
half compared to the RO water. This is highly significant and indicates that
the micro-
cluster water exhibits significantly different behavior and is therefore
substantively
different, relative to RO unprocessed water.
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B. Fourier Transform Infra Red Spectroscopy (FTIR)
Water, a strong absorber in the IR spectral region, has been well-
characterized by FTIR
and shows a major spectral line at approximately 3000 wave numbers
corresponding to O-
H bond vibrations. This spectral line is chaxacteristic of the hydrogen
bonding structure in
the sample. An unprocessed RO water sample, Sample A, and a unoxygenated micro-
cluster water sample, Sample B, were each placed between silver chloride
plates, and the
film of each liquid analyzed by FTIR at 25° C. The FTIR tests were
performed by West
Coast Analytical Service, Inc. in Santa Fe Springs, CA using a Nicolet Impact
400DTM
benchtop FTIR. The FTIR spectra are shown in Figure 5.
In comparing the FTIR spectra for the unoxygenated micro-cluster and RO
waters, it is
cleax that the two samples have a number of features in common, but also
significant
differences. A major sharp feature at approximately 2650 wave numbers in the
FTIR
spectrum is observed for the micro-cluster water (Figure 5(b)). The RO water
has no such
feature (Figure 5(a)). This indicates that the bonds in the water sample are
behaving
differently and that their energetic interaction has changed. These results
suggest that the
unoxygenated micro-cluster water is physically and chemically different than
RO
unprocessed water.
C. Simulated Distillation
Simulated distillations were carried out on RO water and unoxygenated micro-
cluster
water without oxygenation by West Coast Analytical Service, Inc. in Santa Fe
Springs,
CA.
Simulated Distillation Test Results
RO Water UnoxXgenated Micro-cluster
Water
Boiling Point range
(deg. C.) . 98-100 93.2-100
* Corrected for barometric pressure.
These results show a significant lowering of the boiling temperature of the
lowest boiling
fraction in the unoxygenated micro-cluster water sample. The lowest boiling
fraction for
micro-cluster water is observed at 93.2° C. compared with a temperature
of 98° C. for the
lowest boiling fraction of RO water. This suggests that the process has
significantly
changed the compositional make-up of molecular species present in the sample.
Note that
lower boiling species are typically smaller, which is consistent with all
observed data and
the formation of micro-clusters.
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D. Thermogravimetric Analysis
In this test, one drop of water was placed in a dsc sample pan and sealed with
a cover in
which a pin-hole was precision laser-drilled. The sample was subject to a
temperature
ramp increase of 5 degrees every 5 minutes until the final temperature. TGA
profiles were
run on both unoxygenated micro-cluster water and RO water for comparison.
The TGA analysis was performed on a TA Instruments Model TFA2950TM by
Analytical
Products in La Canada, CA. The TGA test results are shown in Figure 6. Three
test runs
utilizing three different samples are shown. The RO water sample is
designated, "Purified
Water" on the TGA plot. The unoxygenated micro-cluster water was run in
duplicate,
I 0 designated Super Pro 1 st test and Super Pro 2°d Test. The
unoxygenated micro-cluster
water and the unprocessed RO water showed significantly greater weight loss
dynamics. It
is evident that the RO water began losing mass almost immediately, beginning
at about
40° C until the end temperature. The micro-cluster water did not begin
to lose mass until
about 70° C. This suggests that the processed water has a greater vapor
pressure between
40 and 70° C. compared to unprocessed RO water. The TGA results
demonstrated that the
vapor pressure of the unxoygenated micro-cluster water was lower when the
boiling
temperature was reached. These data once again show that the unoxygenated
micro-cluster
water is significantly changed compared to RO water. These data once again
show that the
unoxygenated micro-cluster water also shows more features between the
temperatures of
75 and 100 + deg. C. These features could account for the low boiling
fractions) observed
in the simulated distillation.
E. Nuclear Magnetic Resonance (NMR) Spectroscopy
NMR testing was performed by Expert Chemical Analysis; Inc. in San Diego, CA
utilizing
a 600 MHz Bruker AM500TM instrument. NMR studies were performed on micro-
cluster
water with and without oxygen and on RO water. The results of these studies
are shown in
Figure 7. In 1' O NMR testing a single expected peak was observed for RO water
(Figure
7 (a)). For micro-cluster water without oxygen (Figure 7(b)), the single peak
observed was
shifted +54.1 Hertz relative to the RO water, and for the micro-cluster water
with oxygen
(Figure 7(c)), the single peak was shifted + 49.~ Hertz relative to the RO
water. The shifts
of the observed NMR peaks for the micro-cluster water and RO water. Also of
significance in the NMR data is the broadening of the peak observed with the
micro-
cluster water sample compared to the narrower peak of the unprocessed sample.
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EXAMPLE 4 - RAMAN SPECTROSCOPY
Raman spectroscopy , which is highly sensitive to structural modification of
liquids, was
employed to characterize and differentiate micro-cluster structures and micro-
clustered
molecular structure liquids. This study was based on obtaining and processing
spontaneous Raman spectra and allowing a registration of types of phase
transition in
liquid water at 4, 19, 36 and 75 degrees Celsius. The hydrogen bond network
and the
average per unit volume hydrogen bond concentration were determined, which led
to
characterization of waters produced by different methods and in particular
differentiation
and definition of water composition produced by the methods described above
for making
micro-clusters.
Figure 8 schematically illustrates the device used in these studies. The
source of
illumination was a Q-switched solid state Nd:YAG laser (Spectra Physics Corp.,
Mountain
View, CA) with two harmonics output at 1064 nm and its doubled frequency to
produce a
wavelength of 532 nm. A second harmonic generator comprised a KTP crystal
available
from Kigre, Tuscon, AZ. The first harmonic was at 1064 nrn with a pulse energy
of 200
mJ, width of 10 ns, and repetition rate of 6Hz. The optical mirror and
translucent cell
were obtained from CVC Optics, Albuquerque, NM. The spectrometer was obtained
from
Hamamatsu (Japan), and its auto-collimation system from Newport Corporation,
Costa
Mesa, CA. The electro-optical converter was from Texas Instruments, Houston,
TX.
The cell was filled with water as a test subject. The following water samples
were
studied: oxygenated micro-cluster water, unoxygenated micro-cluster water,
Millipore
(tm) distilled water, distilled water prepared in the laboratory, medical-
grade double
distilled injection water, bottled commercial reverse osmosis water, and tap
water
(unprocessed).The test water was subjected to strong ultrasonic fields
produced by a pulse
generator and a sine wave generator and a focusing horn. A laser beam was
directed into a
cell. Signals scattered at 90 degrees entered the spectrometer, which
contained a grating
unit,providing a dispersion of 2 nm/mm. A Raman scattering spectrum was
measured by a
detector.
The results indicated the modifications in micro-cluster water of the local
structure of the
hydrogen-bond net in the acoustic field. In particular, the modification
corresponded to a
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local decrease of the average distance between oxygen atoms to 2.80 angstroms,
enhancing the ordering of the net structure of hydrogen-bonded water molecules
to nearly
that of hexagonal ice, where this distance is 2.76 angstroms.
The test samples which contained micro-cluster water were shown to have about
a ten
degree Celsius higher cluster temperature compared to the other water samples,
which
indicated that the average cluster size was smaller in the micro-cluster
waters than in the
other water samples. Further, the micro-cluster waters represented a more
homogeneous
composition of cluster sizes than the other waters, i.e. a more homogenous
molecular
cluster structure.
II. CULTURE MEDIA, METHODS OF MAKING AND USING
The present invention involves compositions of culture media for biological,
agricultural,
pharmaceutical, industrial, and medical uses. The compositions comprise micro-
cluster
water. Methods of making and using the culture media compositions are within
the scope
of the invention.
General Description and Definitions
The practice of the present invention will employ, unless otherwise indicated,
conventional techniques within the skill of the art in (1) culturing animal
cells, plant cells,
and tissues thereof; microorganisms, subcellular parts, viruses, and
bacteriophage; (2)
perfusion of differentiated tissues and organs; (3) biochemistry; (4)
molecular biology; (5)
microbiology; (6) genetics; (7) chemistry. Such techniques are explained fully
in the
literature. See, e.g. Culture of Animal Cells: A Manual of Basic Technique,
4th edition,
2000, R. Ian Freshney, Wiley Liss Publishing; Animal Cell Culture, eds. J. W.
Pollard and
John M. Walker; Plant tissue Culture: Theory and Practice, 1983, Elsevier
Press; Plant
Cell Culture Secondary Metabolism Toward Industrial Application, Frank DiCosmo
and
Masanaru Misawa, CRC Press; Plant Tissue Culture Concept and Laboratory
Exercises,
2nd edition, Robert N. Trigiano and Dennis Gray, 1999, CRC Press; Plant
Biochemistry
and Molecular Biology, 2nd ed., eds. Peter J. Lea and Richard C. Leegood,
1999, John
Wiley and Sons; Experiments in Plant Tissue Culture, Dodds & Roberts, 3rd
edition;
Neural Cell Culture: A Practical Approach, vol. 163, ed. James Cohen and
Graham
Wilkin; Maniatis et al., Molecular Cloning: A Laboratory Manual; Molecular
Biology of
The Cell, Bruce Alberts, et.al., 4th edition, 2002, Garland Science: Microbial
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Biotechnology, Fundamentals of Applied Microbiology, Alexander N. Glazer and
Hiroshi
Nikaido 1995, W.H. Freeman Co.; Pharmaceutical Biotechnology, eds. Daan J.A.
Crommelin and Robert D. Sindelar, 1997, Harwood Academic Publishers)..
Relevant
periodicals include Cell Tissue Research; Cell; Science; Nature; Journal of
Immunology;
Thymus; International Journal of Cell Cloning; Blood; Hybridoma.
The following terminology will be used in accordance with the definitions set
out below in
describing the present invention.
The term "micro-clustered culture medium" as used herein refers to a culture
medium
which comprises micro-cluster water. The adjective "micro-clustered " which
modifies
any of the aqueous compositions including medium, media, liquid, gel,
composition,
constituent or ingredient refers to micro-clustered water in that composition,
i.e. which is
dissolved in or mixed with micro-cluster water.
As defined in the Oxford Dictionary of Biochemistry and Molecular Biology
(Oxford
University Press, 1997), the term "culture" refers to 1 (a) a collection of
cells, tissue
fragments, or an organ that is growing or being kept alive in or on a nutrient
medium (i.e.
culture medium); (b) any culture medium to which such living material has been
added,
whether or not it is still alive. 2. the practice or process of making,
growing, or
maintaining such a culture. 3. to grow, maintain or produce a culture.
A "cell" is the basic structural unit of all living organisms, and comprises a
small, usually
microscopic, discrete mass of organelle-containing cytoplasm bounded
externally by a
membrane and/or cell wall. Eukaryotes are cells which contain a cell nucleus
enclosed in
a nuclear membrane. Prokaryotes are cells in which the genomic DNA is not
enclosed by
a nuclear membrane within the cells.
"Culture medium" refers to any nutrient medium that is designed to support the
growth or
maintenance of a culture. Culture media are typically prepared artificially
and designed
for a specific type of cell, tissue, or organ. They usually consist of a soft
gel (often
referred to as solid or semi-solid medium) or a liquid, but occasionally they
are rigid
solids.
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"Tissue culture" refers to 1. the technique ox process of growing or
maintaining tissue cells
(cell culture), whole organs (organ culture) or parts of an organ, from an
animal or plant,
in artificial conditions; 2. any living material grown or maintained by such a
technique.
S
"Tissue" refers to any collection of cells that is organized to perform one or
more specific
function.
"Organ" is any part of the body of a multicellular organism that is adapted
andlor
specialized fox the performance of one or more vital functions.
"Organ culture" refers to a category of tissue culture, in which an organ or
part of an
organ, or an organ primordium, after removal from an animal or plant, is
maintained in
vitro in a nutrient medium with retention of its structure and/or function.
"Organelle" is any discrete structure in a unicellular organism or in an
individual cell of a
multicellular organism, that is adapted and/or specialized for the performance
of one or
more vital functions.
"Microbial biotechnology" refers to the use of cells, prokaryotic or
eukaryotic, in
production of proteins, recombinant and synthetic vaccines, microbial
insecticides,
enzymes, polysaccharides and polyesters, ethanol, amino acids, antibiotics; in
organic
synthesis and degradation by microbes (and by enzymes); and to environmental
applications, including sewage and wastewater microbiology; microbial
degradation of
xenobiotics; use of microorganisms in mineral recovery, and in removal of
heavy metals
from aqueous effluents. The broad scope of microbial biotechnology is, in
part, disclosed
in Microbial Biotechnology, Fundamentals of Applied Microbiology, Alexander N.
Glazer
and Hiroshi Nikaido 1995, W.H. Freeman Co.; Pharmaceutical Biotechnology, eds.
Daan
J.A. Crommelin and Robert D. Sindelar, 1997, Harwood Academic Publishers.
tell Culture Media - Fundamentals
The basic ingredients (as set forth below) of cell culture media - as
individual components,
as premixed components, dry or formulated with water -- are commercially
available from
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many vendors (e.g. Sigma Chemical, Invitrogen, Biomark, Cambrex, Clonetics to
name
just a few). Methods of formulating culture media with water are well known in
the art
(Culture Media for Cells, Organs, and Embryos, CRC Press, 1977; Animal Cells:
Culture
and Media: Essential Data, John Wiley & Son, 1995; Methods for Preparation of
Media,
Supplements and Substrata for Serum Free Animal Cell Culture in Cell Culture
Methods
for Molecular and Cell Biology, Vol. 1, Wiley-Liss, 1984). The media
compositions of
the invention comprise micro-cluster water. For the sake of listing the
various ways of
cell culturing Methods of cell culturing and types of cell media are well
known in the art,
and are briefly set forth below.
Types of cell cultures:
Primary cultures are taken directly from excised, normal animal tissue. These
tissues are
cultured either as an explant culture or cultured after dissociation into a
single cell
suspension by enzyme digestion. At first heterogeneous, these cultures are
later dominated
by fibroblasts. Generally, primary cultures are maintained in vitro for
limited periods,
during which primary cells usually retain many of the differentiated
characteristics of the
cells seen in vivo.
Continuous Cultures are comprised of a single cell type. These cells may be
serially
propagated in culture either for a limited number of cell divisions
(approximately fifty) or
otherwise indefinitely. Some degree of differentiation is maintained. Cell
banks must be
set up to maintain these cultures over long periods.
Culture Morphologv_
Cell cultures either growing in suspension (as single cells or small free-
floating clumps) or
as a monolayer attached to the tissue culture flask. Sometimes cell cultures
may grow as
semi-adherent cells in which there is a mixed population of attached and
suspension cells.
Txpes of Culture Media
In general, cultured cells require a sterile environment, a supply of
nutrients for growth,
and a stable culture environment, e.g. pH and temperature. Various defined
basal media
types have been developed and are now available commercially. These have since
been
modified and enriched with amino acids, vitamins, fatty acids and lipids.
Consequently
media suitable for supporting the growth of a wide range of cell types are now
available.
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The precise media formulations have often been derived by optimizing the
concentrations
of every constituent.
Vendors of culture media distribute via catalogs or the vendors' web sites to
those skilled
in the art literature for making and using culture media. For example, the
Sigma-Aldrich
company's web site discloses a book entitled Fundamental Techniques in Cell
Culture, A
Laboratory Handbook Online (Sigma-Aldrich Company), examples of different
media and
their uses are given in the table below. One of skill in the art would
substitutes micro-
clustered water for all or part of the non-micro-clustered water in the
culture media recited
below.
Table 1. Different types of culture medium and their uses
Balanced salt solutions PBS, Hanks BSS, Earles salts
DPBS (Prod. No. D8537 / D8662)
HBSS (Prod. No. H9269 / H9394)
EBSS (Prod. No. E2888) Form the basis of many complex media
Basal media MEM (Prod. No. M2279) Primary and diploid cultures.
DMEM (Prod. No. D5671) Modification of MEM containing increased level of amino
acids and vitamins. Supports a wide range of cell types including hybridomas.
GMEM (Prod. No. 65154) Glasgows modified MEM was defined for BHK-21 cells
Complex media RPMI 1640
(Prod. No. 80883) Originally derived for human leukaernic cells. It supports a
wide range
of mammalian cells including hybridomas
Iscoves DMEM
(Prod. No. 13390) Further enriched modification of DMEM which supports high
density
growth
Leibovitz L-15
(Prod. No. L5520, liquid) Designed for C02 free environments
TC 100 (Prod. No. T3160)
Grace's Insect Medium
(Prod. No. 68142)
Schneider's Insect Medium (Prod. No. 50146) Designed for culturing insect
cells
Serum Free Media CHO (Prod. No. C5467)
HEK293 (Prod. No. 60791) For use in serum free applications.
Ham F 10 and derivatives
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Ham F12 (Prod. No. N4888)
DMEM/F12 (Prod. No. D8062) NOTE: These media must be supplemented with other
factors such as insulin, transferrin and epidermal growth factor. These media
are usually
HEPES buffered
Insect cells Sf 900 II SFM, SF Insect-Medium-2 (Prod. No. 53902) Specifically
designed
for use with S~ insect cells
Basic I~redients of Media
Solutions of basic ingredients of media which comprise micro-clustered water
are included
in the compositions of the invention.
Inorganic salts
Carbohydrates
Amino Acids
Vitamins
Fatty acids and lipids
Proteins and peptides
Serum
Each type of constituent performs a specific function as outlined below:
Inorganic salts help to retain the osmotic balance of the cells and help
regulate membrane
potential by provision of sodium, potassium and calcium ions. All of these are
required in
the cell matrix for cell attachment and as enzyme cofactors.
Buffering Systems. Most cells require pH conditions in the range 7.2 - 7.4 and
close
control of pH is essential for optimum culture conditions. There are major
variations to
this optimum. Fibroblasts prefer a higher pH (7.4 - 7.7) whereas, continuous
transformed
cell lines require more acid conditions pH (7.0 - 7.4). Regulation of pH is
particularly
important immediately following cell seeding when a new culture is
establishing and is
usually achieved by one of two buffering systems; (i) a "natural" buffering
system where
gaseous C02 balances with the C03 / HC03 content of the culture medium and
(ii)
chemical buffering using a zwitterion called HEPES (Prod. No. H4034).
Cultures using natural bicarbonate/C02 buffering systems need to be maintained
in an
atmosphere of 5-10% C02 in air usually supplied in a C02 incubator.
Bicarbonate/C02 is
low cost, non-toxic and also provides other chemical benefits to the cells.
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HEPES (Prod. No. H4034) has superior buffering capacity in the pH range 7.2 -
7.4 but is
relatively expensive and can be toxic to some cell types at higher
concentrations. HEPES
(Prod. No. H4034) buffered cultures do not require a controlled gaseous
atmosphere.
Most commercial culture media include phenol red (Prod. No. P3532 / P0290) as
a pH
indicator so that the pH status of the medium is constantly indicated by the
color. Usually
the culture medium should be changed / replenished if the color turns yellow
(acid) or
purple (allcali).
Carbohydrates. The main source of energy is derived from carbohydrates
generally in the
form of sugars. The major sugars used are glucose and galactose however some
media
contain maltose or fructose. The concentration of sugar varies from basal
media containing
1g/1 to 4.5g/1 in some more complex media. Media containing the higher
concentration of
sugars are able to support the growth of a wider range of cell types.
Vitamins. Serum is an important source of vitamins in cell culture. However,
many media
are also enriched with vitamins making them consistently more suitable for a
wider range
of cell lines. Vitamins are precursors for numerous co-factors. Many vitamins
especially B
group vitamins are necessary for cell growth and proliferation and for some
lines the
presence of B 12 is essential. Some media also have increased levels of
vitamins A and E.
The vitamins commonly used in media include riboflavin, thiamine and biotin.
Proteins and Peptides. These are particularly important in serum free media.
The most
common proteins and peptides include albumin, transferrin, fibronectin and
fetuin and are
used to replace those normally present through the addition of serum to the
medium.
Fatty Acids and Lipids. Like proteins and peptides these are important in
serum free
media since they are normally present in serum. e.g. cholesterol and steroids
essential for
specialized cells.
Trace Elements. These include trace elements such as zinc, copper, selenium
and
tricarboxylic acid intermediates. Selenium is a detoxifier and helps remove
oxygen free
radicals.
It is time consuming to make media from the basic ingredients, and there is a
risk of
contamination in the process. Conveniently, most media are available as ready
mixed
powders or as l Ox and 1x liquid media. The commonly used media are listed in
the
catalogs of media vendors (e.g. Sigma-Aldrich Life Science Catalogue).
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If one skilled in the art purchases media ingredients as powder or l Ox media,
it is essential
that the water used to reconstitute the powder or dilute the concentrated
liquid is free from
mineral, organic and microbial contaminants. It must also be pyrogen free
(Prod. No.
W3500, water, tissue culture grade, Sigma-Aldrich). In most cases water
prepared by
reverse osmosis and resin cartridge purification with a final resistance of 16-
l~Mx is
suitable. Once prepared the media should be filter sterilized before use.
Obviously
purchasing lx liquid media direct from a vendor eliminates the need for this.
In all
instances, media of the invention involve micro-clustered water, preferably
tissue culture
grade, as a constituent. Vendors of media (e.g. Sigma-Aldrich, Invitrogen,
Clonetics) and
vendors of cells and cell cultures commonly purvey one or more of their
products (media,
media ingredients, and cells) in the form of kits which have containers for
the products.
The invention includes kits which comprise micro-clustered in its own
container or as an
ingredient of another container in the kit.
Serum. Serum is a complex mix of ~albumins, growth factors and growth
inhibitors and is
probably one of the most important components of cell culture medium. The most
commonly used serum is fetal bovine serum. Other types of serum are available
including
newborn calf serum and horse serum. The quality, type and concentration of
serum can all
affect the growth of cells and it is therefore important to screen batches of
serum for their
ability to support the growth of cells. Serum is also able to increase the
buffering capacity
of cultuxes that can be important for slow growing cells or where the seeding
density is
low (e.g. cell cloning experiments).
The culture media of the invention, which comprise micro-clustered water, and
methods of
making and using them are arbitrarily classified for purposes of this
application into use
for the following categories of biological entities. It is understood that
this classification
does not preclude the compositions or their methods of use from application in
more than
one category.
ANIMAL CELL, PER SE (E.G., CELL LINES, ETC.I
Compositions of the invention include:
1. A composition comprising micro-clustered culture medium, in particular
medium
formulated for use with animal cells.
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2. A composition comprising micro-clustered culture medium formulated for use
with
animal cells, and animal cells.
3. Compositions comprising animal cells made from using micro-clustered animal
cell culture media in methods enumerated below.
The culture media of the invention formulated for use with animal cells are
used for:
1. Propagating, maintaining or preserving an animal cell or composition
thereof.
2. Isolating or separating an animal cell or composition thereof.
3. Preparing a composition containing an animal cell.
Also covered by the invention are processes for preparing micro-clustered
animal cell
culture media, and for preparing compositions which comprise micro-clustered
animal cell
culture medium and animal cells. Vaccines are examples of products derived
from such
animal cell cultures.
Stem Cells
The compositions and methods of the invention are adapted for use with stem
cells.
Embryonal stem cells and lineage- or tissue-specific stem cells are important
models in
biomedical studies, but the availability and accessibility of research
materials in this
rapidly advancing field often become limiting. The compounds and methods of
the
invention are intended for expanding, preserving embryonic stem cells, as well
as
postnatally derived stem cells from a variety of strains and species.
(National Center for
Research Resources; American Type Culture Collection, Manasas, VA). Stem cells
are
also retrieved from bone marrow, subcutaneous fat, and the reticular dermis
bulge area.
Products available from the National Stem Cell Resource include: (a) nonhuman
embryonic stem cells, and lineage- or tissue-specific neonatally derived stem
cells from a
variety of species; these are available as either frozen vials, shipped on dry
ice; (b)selected
reagents related to stem cell characterization and utilization are available;
these include
antibodies, nucleic acid probes, cDNAs, genomic libraries and plasmid vectors
for
targeted mutagenesis or other stem cell-related purposes; (c) standardized
media, as they
are developed. Reagents identifying common traits among stem cell strains and
species
also will be available as they are identified or developed. These include
reagents for RT-
PCR and immunologically based assays. The present invention includes use of
micro-
clustered media and reagents for use with stern cells, including stem cell
retrieval.
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Microorganisms
Microorganisms include actinomycetales, unicellular algae, bacteria, fungi
(yeast and
molds), and protozoa.
Compositions of the invention include
1. Culture media comprising micro-cluster water for use with microorganisms.
2. Culture media comprising micro-cluster water and microorganisms.
The culture media of the invention involved with microorganisms are used for:
1. Propagating, maintaining or preserving microorganisms, or compositions of
microorganisms.
2. Preparing or isolating a composition containing a microorganism, which
processes
involve the use of micro-cluster water or culture media comprising micro-
cluster
water.
3. Isolating microorganisms.
Also covered by the invention are processes for preparing culture media
comprising
micro-cluster water, and for preparing compositions which comprises culture
media and
microorganisms.
VECTOR, PER SE (E.G., PLASMID, HYBRID PLASMID, COSMID, VIRAL
VECTOR, BACTERIOPHAGE VECTOR, ETC.1
These biological entities include self replicating nucleic acid molecules
which may be
employed to introduce a nucleic acid sequence or gene into a cell; such
nucleic acid
molecules are designated as vectors and may be in the form of a plasmid,
hybrid plasmid,
cosmid, viral vector, bacteriophage vector, etc.
Vectors or vehicles may be used in the transformation or transfection of a
cell.
Transformation is the acquisition of new genetic material by incorporation of
exogenous
DNA. Transfection is the transfer of genetic information to a cell using
isolated DNA or
RNA.
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A plasmid is an autonomously replicating circular extrachromosomal DNA
element. A
hybrid plasmid is a plasmid which has been broken open, has had DNA from
another
organism spliced into it, and has been repealed. A cosmid is a plasmid into
which phage
lambda "cos" sites have been inserted.
A viral vector (e.g., SV40, etc.) is a plant or animal virus which is
specifically used to
introduce exogenous DNA into host cells. A bacteriophage vector (e.g., phage
lambda,
etc.) is a bacterial virus which is specifically used to introduce exogenous
DNA into host
cells.
VIRUS OR BACTERIOPHAGE
These biological entities include a virus or bacteriophage which is a
microorganism that
(a) consists of a protein shell around a nucleic acid core of either
ribonucleic acid or
deoxyribonucleic acid, and (b) is capable of independently entering a host
microorganism,
and (c) requires a host microorganism, having both ribonucleic acid and
deoxyribonucleic
acid to replicate.
Compositions of the invention include
1. A composition of micro-clustered medium formulated for use with virus or
bacteriophage.
2. A composition of micro-clustered medium formulated for use with virus or
bacteriophage, which composition~comprises virus or bacteriophage.
The culture media of the invention involved with virus or bacteriophage are
used for:
1. Preparing or propagating virus or bacteriophage.
2. Purifying virus or bacteriophage.
3. Producing viral subunits.
Propagation is limited to processes concerned with the multiplication of
viruses and not
with processes concerned with the artificial alteration of genetic material
involving
changes in the genotype of the virus. Such processes of artificial alteration
of genetic
material are intended for processes of mutation, cell fusion, or genetic
modification, and
include (1) producing a mutation in an animal cell, plant cell or
microorganism, (2) fusing
animal, plant, or microbial cells, (3) producing a stable and heritable change
in the
genotype of an animal cell, plant cell, or a microorganism by artificially
inducing a
structural change in a gene or by incorporation of genetic material from an
outside source,
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or (4) producing a transient change in the genotype of an animal cell, plant
cell, or
microorganism by the incorporation of genetic material from an outside source.
A mutation is a change produced in cellular DNA which can be either
spontaneous, caused
by an environmental factor or errors in DNA replication, or induced by
physical or
chemical conditions. The processes of mutation included are processes directed
to
production of either directed or essentially random changes to the DNA of an
animal cell,
plant cell, or a microorganism without incorporation of exogenous DNA. It
should be
noted that in the art that incorporation of exogenous genetic material into a
cell or
microorganism or rearrangement of genetic material within a cell or
microorganism is not
necessarily considered a mutation.
In vitro mutagenesis, which is a method where cloned DNA is modified outside
of the cell
or microorganism and then incorporated into a cell or microorganism is not
considered to
be a mutation. Genetic material from an outside source may include chemically
synthesized or modified genes. Transient changes effected by incorporation of
genetic
material from an outside source involve expression of one or more phenotypic
traits
encoded by said genetic material. A transient change is one which is passing
or of short
duration. Methods producing nongenetically encoded changes effected by a
nucleic acid
molecule, such as antisense nucleic acid are not considered mutations.
These compositions and processes involve use with viruses of all types, i.e.,
animal, plant,
etc.
PLANT CELL OR CELL LINE, PER SE IE.G., TRANSGENIC, MUTANT, ETC.1
These biological entities include plant cells or cell lines, per se which may
be transgenic,
mutant, or products of other processes fox obtaining plant cells.
The compositions of the invention include:
1. A composition comprising micro-clustered water and medium formulated for
plant
cells or cell lines.
2. A composition comprising micro-clustered water and medium formulated for
plant
cells or cell lines and plant cells.
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The culture media of the invention involved with plant cells or cell lines are
used for:
1. Tn-vitro propagating
2. Maintaining or preserving plant cells or cell lines.
3. Isolating or separating plant cells.
4. Regenerating plant cells into tissues, plant parts, or plants, per se, with
or without
genotypic change occurring. (Total Lab Systems, Ltd., New Zealand; e.g.
Commercial
Propagation of Orchids in Tissue Culture: Seed Flasking Methods. Orchid Manual
Basics,
Kay S. Greisen, 2000, American Orchid Society; Plant Tissue Culture Protocols
as
disclosed in Sigma-Aldrich Co. web site and catalogs)
Subcellular Parts
It is understood that the compositions of the invention include media
formulated for
subcellular parts of microorganisms, aamal cells and plants, such as
organelles, i.e.
mitochondria, microsomes, chloroplasts, etc.. These media are used for
isolating or
treating subcellular parts. Methods of making these media are included in the
invention.
Media for Use With Differentiated Tissues or Or~,~;ans
The invention includes micro-clustered media adapted for use with
differentiated tissues or
organs, including blood. These media are used for the maintenance of a
differentiated
tissue or organ, i.e. maintained in a viable state in a nutrient or life
sustaining media.
Maintenance includes keeping an organ under conditions in which it produces a
product
(e.g., hormone) which is later recovered, or exhibits an activity (e.g.
synthesis of a
hormone).
Accordingly, the invention includes perfusion media formulated with micro-
clustered
water, which are used in processes for the maintenance of differentiated
tissue or organs
by continuously perfusing with a fluid, or compositions of the invention. U.S.
Patents
4,879,283; 4,873,230; and 4,798,824 (herein incorporated by reference)
disclose solutions
for perfusing and maintaining organs. D'Alessandro AM, Kalayoglu M, Sollinger
HW,
Pirsch JD, Southard JH, Belzer FO. Current status of organ preservation with
University
of Wisconsin solution. Arch Pathol Lab Med. 1991;115(3):306-310; Viaspan (r),
an organ
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perfusion and maintenance solution, manufactured by Barr Laboratories, Inc.
and used for
transplantation and viability preservation of organs and tissues.
Compositions of the invention include those formulated for freezing of
differentiated
tissues or organs, and used in processes for maintaining differentiated
tissues or organs by
freezing.
Compositions of the invention include those formulated for maintaining blood
or sperm in
a physiologically active state, and those formulated for methods of in vitro
blood cell
separation or treatment. Also included are compositions for artificial
insemination.
It is understood that micro-clustered compositions of the invention include
physiological
solutions or aqueous media which may not contain nutrient ingredients yet
still formulated
having pH, buffer capacity, osmolarity, conductance, sterility and which
otherwise are
used alone or in combination with other physiological solutions to maintain
living cells,
tissues, organs, and organisms. Examples of physiological solutions include,
but are not
limited to, Ringer's solutions, saline solutions, buffer solutions. These
solutions are
commonly known and used in handling biological materials, and are apparent to
those of
ordinary skill in the art.
Stimulation of growth or activit<~ using micro-clustered medium
Ee ects ofMicro-cluster Water oh Cellular Viabilit,~
A study was performed to determine the influence of micro-cluster water on
cell viability
as measured by cell membrane integrity.
A population of macrophages was subjected to growth medium which was
formulated
with micro-cluster water, and growth medium formulated with double distillated
water
(DDV~.
Macrophages were obtained by mice. 2 ml of Hanks solution (10 mM HEPES, pH
7.2)
was injected into the peritoneum of sacrificed mice. The solution, containing
macrophages, was collected. The cell concentration was adjusted to 106
cells/ml with
Hanks balanced salt solution.
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Generally, 20 microliter aliquots of the cell suspension were placed on glass
cover slips,
incubated for 45 minutes in a wet chamber, and then washed with Hanks solution
to
remove the cells attached to the glass surface.
The integrity of the cell membranes was determined by double staining the
cells with
ethidium bromide (EthBr, Sigma) and fluoresceindiacetate (FDA, Sigma). A
staining
solution was used which contained 5 microgramslml of EthBr and 5 micrograms/ml
of
FDA. Cells with damaged cell membranes were counted. The method is based on
the
ability of EthSr to enter cells which have damaged membranes. The EthBr binds
to DNA.
EthBr has a bright red fluorescence when bound to DNA. FDA easily penetrates
cells
from the medium and is structurally transformed to fluorescein which has
bright green
fluorescence. Accordingly, cells with intact plasma membranes accumulate
fluorescein,
whereas cells with damaged cell membranes allows fluorescein to easily leave
the cells.
As a result of this double staining, after five minutes, one observed cells
with intact
plasma membranes which had green fluorescence. Cells which had damaged plasma
membranes had red fluorescence.
In a first series of experiments, macrophages were incubated for 15 minutes in
media
containing EthBr and FDA. They were then thoroughly washed to remove free dyes
in the
extracellular media. Growth media was then replaced with 199 medium (199
Powder
medium - Russia, Paneko) prepared with either DDW or with micro-cluster water.
Dead
cells were then counted.
In a second series of experiments, cells were incubated for 230 minutes in
either 199 cell
medium prepared with DDW or micro-cluster water. Cells were then appropriately
stained to determine how many cells had died.
Figure 9 is an assessment of the number of macrophages with damaged plasma
membranes after incubation in 199 cell medium prepared on DDW or on micro-
cluster
water. The data is presented as percentage of cells with damaged plasma
membranes - P%
- after 15 minutes and 240 minutes of incubation in different 199 cell media.
The results
indicate that the amount of cells with damaged cell membranes was 2.6 times
greater in
cell medium prepared with double distilled water compared to medium prepared
with
micro-cluster water. Accordingly, it appeared that cell culture medium
formulated with
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micro-cluster water prolonged or increased the life of cells compared with the
effects of
cell culture medium formulated with DDW. Alternatively, it appeared that cell
culture
medium prepared with micro-cluster water inhibited damage to cell plasma
membranes
compared to cell culture medium prepared with DDW.
E acts ofMicro-cluster Water oh Iht~acellular~H
A study was performed to determine the influence of micro-cluster water on
intracellular
pH. Mouse macrophages were obtained as described above. Intracellular pH of
these
cells was determined after 15 minutes and after 240 minutes of incubation in
199 medium
prepared either with DDW or micro-cluster water.
Macrophage intracellular pH was measured based on a microspectrophotometric
method
using a fluorescent microscope (LUMAM 13, LOMO, Russia), which as a modified
system of fluorescence excitation and emission.
Fluorescence excitation was performed using a blue (lambda max=435 nm
photodiode.
Fluorescence was measured simultaneously at two different wavelengths by a two-
channel
system, which has lambdal=520 mn, lambda2=567 nm interference filters
respectively.
Fluorescence excitation and synchronous emission measurement was achieved with
a
built-in microcontxoller (LA-70M4).
Macrophages were incubated with fluorescent FDA (5 micrograms/ml) , which is a
pH
indicator, for 15 minutes. After incubation with the dye, the cells were
washed free from
dye in the surrounding medium. The cells were then placed in the medium in a
small petri
dish, and observed using a water immersion objective (x40). A pH calibration
curve was
established for a range of ionic conditions.
Cells, which had been incubated with FDA dye for 15 minutes and washed free
from dye
in the surrounding medium, were then placed in either 199 medium prepared with
DDW
or with micro-cluster water. Kinetic measurements of intracellular pH were
made with no
less than 30 microscopic observations, and repeated three times. Cells were
incubated for
as long as 230 minutes. Figure 10 illustrates the kinetics of intracellular pH
change (delta
pHi) in macrophages after replacement of incubation medium with 199 medium
prepared
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either with DDW or with micro-cluster water. The x-axis is time in seconds
after change
of cell medium. The y-axis is changes in intracellular pH - delta pHi. It can
be seen that
the intracelluar pH in a standard incubation medium 199-DDW and in 199-micro-
cluster
water were both about pH 7.15. After 15 minutes of incubation in 199-micro-
cluster
water, the pH increased by 0.16 unites. No significant change was observed in
macrophages incubating in 199-DDW during the same 15 minutes. After 230
minutes, a
0.43 increase was observed in the intracellular pH of the cells incubating on
199-micro-
cluster wafer. There was a negligible increase in intracellular pH of the
cells incubating
on 199-DDW. It is concluded that contacting cells with culture medium prepared
with
micro-cluster water instead of "normal" water increased the intracellular pH
of the cells.
A separate series of experiments using pig embryo kidney cells cultured with
199
mediums and with 10% bovine serum demonstrated increases in intracellular pH
and
robust cell viability when the growth mediums were prepared with micro-
clustered water
compared to growth mediums prepared with normal water.
E~'ects ofmicro-cluster water on~rowth and traps action o two tx~es of human
cells
A series of experiments was»performed to determine the effects of micro-
cluster water on
the growth of cells and on the transfection of cells in medium prepared with
micro-cluster
water.
The effects were studied using human epithelial cells (293T) and human
dendritic cells.
DMEM medium (Life Technologies, Gaithersburg, MD) was prepared from a lOx
concentrate by dilution in micro-cluster water obtained from AquaPhotonics,
Inc., San
Diego, CA). The cells were supplemented with 10% fetal calf serum (FCS).
In a parallel experiment, the cells were cultured with standard DMEM medium,
i.e.
medium prepared without micro-clustered water.
At days 0, 3, 6, and 9 the cells were stained with 0.4% trypan blue (Life
Technologies) to
determine the viability of the culture.
On day 1 of culturing, the 293T cells were subjected to transfection with an
HIV
molecular clone (which encodes GFP) by a calcium phosphate precipitation
method
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(Invitrogen, Carlsbad, CA). As a control, 293T cells cultured in standard DMEM
medium
were transfected with the same HIV molecular clone. The following day,
supernatants
were harvested from both HIV transfected cultures and assayed for HIV Gag p24
content
by ELISA. To find optimal dilution in the range of sensitivity of the method,
supernatants
were titrated by a factor of 10.
The harvested viruses were then used to infect primary cultures of dendritic
cells (DC).
Two cultures of DC were maintained in the medium prepared from concentrated
DMEM
and diluted by a factor of 10, one culture (experimental) in DMEM diluted with
micro-
cluster water, the other culture (control) diluted with normal water.
Infection was
monitored at the single-cell level by scoring the GFP-positive DC at fifth day
after HIV
exposure.
Results:
A. Viability tests, as shown in Figure 11, demonstrated that the micro-cluster
water
used as a solvent for medium preparation, improved 293T cell viability by 70%
at the 9th
day of culture over the cells cultured in medium prepared with normal water.
B. Replication of HIV in transfected 293T cell-cultures three-fold higher in
the
experimental cultures compared with the control cultures when supernatants
from the
respective cultures were titrated at the point of 3 log (Figure 12a).
C. Culturing of DC in a DMEM medium prepared with micro-cluster water and
exposure of DC to HIV harvested from 293T cells cultured in DMEM prepared with
micro-cluster water greatly enhanced the "permissivity" (Figure 12b) of DC to
HIV (35%
DC were infected in the experimental culture compared with 3.7% in the
control.)
These experiments demonstrated in a transformed cell-line, in a virus, and in
primary
cells, biological effects on these biological entities when micro-clustered
water replaced
normal water in the culture medium. There was 2-3 fold enhancement of the
cells'
viability; and an augmentation of either or both HIV replication and
replication rate in
vitro in the cell line and in the primary cell culture.
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E ects OfMicro-Cluster Water On The ExP~ession Pro ales O,~Cha~acteristic
Dendritic
Cell Markers
This study's objective was to monitor difference between the expression
profiles of
characteristic DC markers in media prep~:~red with de-ionized water and media
prepared
with micro-clustered water.
Experimental design and results. DC were cultured in media prepared from l OX-
concentrate MEM (Life-Technologies, Gaitersburg, MD) diluted to a final
concentration
either by de-ionized water or micro-clustered water. Both media were
supplemented with
cytokines IL-4 and GM-CSF (20 ng/ml). DC were generated according to standard
protocols (Sallusto et al., 1994), phenotyped on day 6 of differentiation and
cultured. On
days 30 and 69, respectively, phenotyping was repeated with the same
monoclonal
antibodies. The level of surface-marker expression was assessed by flow
cytometry using
FACscan reader (Bekton-Dickenson, CA).
Description of cell surface markers:
1. DC-SIGN - M.W. ~ 44K, cell-specific ICAM-3 receptor
Paper was attached about a function of DC-SIGN in dendritic cells.
2. CD4 - main HIV gp120 receptor, MW ~SSK. CD4 is an anchor place for HIV
envelope
proteins
3. CD 1 a - is an analog of MHC complex in professional antigen-presenting
cells, which is
responsible for presentation and processing of lipid antigens (non canonical
antigen-
presentation system).
4. CD80 - co-stimulatory molecule which provides signal 2 from antigen
presenting cell
(such as DC) for induction of T-cell proliferation.
5. CD83- maturation marker of dendritic cells (DC)
6. CXCR4 and CCRS - inflammatory chemokine receptors
7. MHC-II - Major Histo-Compatibility complex type II. Presents epitopes of
exogeneous
processed proteins.
As shown in Figure 12, during long-term culturing in medium prepared with
micro-
clustered water as a solvent, it was observed that a substantial change
occurred in the
pattern of expression of the CD83 marker. CD83 is a main indicator of DC
maturation.
DCs that express a low level of CD83 on day 60 and show typical morphology
(grown in
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suspension) are immature and functionally ready to take up foreign antigens.
Typically,
DCs exhibit such a phenotype in vitro (in standard medium) during first two
weeks of
differentiation. Further culturing in standard medium leads to a spontaneous
maturation
and cell-death mediated, most likely, through apoptosis. In a pilot
phenotyping
experiment it was detected that micro-clustered water (i) preserved immature
DC
phenotype and (ii) mediated DC surviving longer than 2.5 months. Phenotype
preservation was shown by analysis of expression of other markers (most
important are
DC-SIGN and MHC II) on the surface of DC cells. This analysis indicates that
micro-
clustered medium provides a satisfactory maintenance of functions typical to
immature
DC as seen by similarity of markers expression between DC in standard and
micro-
clustered media. The survival of DC's for more than 2.5 months was never
observed
before with standard medium formulation. Preliminary results demonstrated that
micro-
clustered water exhibited a biological activity reflected in modulation of DC
cell surface
markers.
Summarv
1. Micro-clustered water was fully applicable as a solvent for fine tissue
culture
experiments.
2. Contacting the cells with micro-clustered water altered the cells'
biological activity,
which was reflected in modulation of CD83 marker and elongation of a lifetime
span of DCs in vitro.
In Figure 13, the horizontal axis reflects ~he type of different receptors on
the cell surface.
The vertical axis represents responses (per cent of fluorescent intensity of
labeled
monoclonal antibodies bound to a specific receptors). Cells were stained with
the
2S respective monoclonal antibodies and signal was compared to the isotype
control (per cent
of ISO ~ 1.1%).
In Figure 13, the gray columns represent measurements after 6 days in control
medium.
The black columns after 30 days in control medium. The white columns after 60
days in
micro-clustered medium. Data were not obtained for normal water in a day 60
since the
cell culture underwent apoptosis at early date. Contrary to almost complete
die-off of the
cell in a standard medium, a surprisingly large number of cells in micro-
structured
medium showed a morphology of immature DC and a corresponding pattern of cell
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surface markers at day 60. Cell life survivability appeared to be enhanced by
micro-
clustered medium.
E ect O~Mic~o-Clustered Water Oh The Fu~ctio~al State OdfBrairc Tissue Perused
In
Artificial Cerebrosniual Fluid Prepared With Double Dr.'stilled Avid Micro-
Clustered
Waters.
The purpose of the study was to measure the effect of various types of waters
on the
functional state of brain tissue. Recording of an induced electrical signal
from the brain
IO sections in perfused fluid due to activity of hippocampus nervous cells was
used as the
testing method. According to the literature, the technology of making rat
brain sections
with a hippocampus of 300-450 ~,m in perfusion with artificial cerebrospinal
fluid allows
the brain tissue to keep its functional status for about 6-8 hours.
The method employed involved testing of the functional status of brain tissue
by
recording electrical neuron responses to the applied pulses of electric
current. Neuronal
reaction is very sensitive to the characteristics of perfusion medium.
Stimulation of the
axon group reflects the, change in membrane potential of postsynaptic cells,
which are
located in a region of the measuring electrode. The amplitude of the signal
depends on
the efficacy of the synaptic connections between stimulating axons and
postsynaptic
neurons and it also depends on the excitability of the postsynaptic neurons
themselves.
Declining functional activity of brain tissue is a result of a reduction in
the neurons
which are responding to the applied pulses of electric current. This is
directly correlated
with a decrease in summary amplitude.
The main advantages of the method involved easy access to the extracellular
space of
brain tissue in the specimen which made it possible to use chemical substances
of required
concentration directly. Furthermoe, there was an absence of interference due
to
respiration, heart beating, and animal movement, which make prolonged
measurements
difficult; experimental condition were easy to control in the absence of
anesthesia,
humoral, and hormonal influences. Also, it was relatively simple to use the
tested tissue
for biochemical and morphological analysis quickly after the
electrophysiological part was
completed.
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Requirements for the survivability of brain tissue sections. To maintain
viability of
isolated brain sections, artificial fluids are used which are similar in salt
composition to the
intercellular medium of the brain. However, the composition of cerebrospinal
fluid may
vary depending on the specific task.
Glucose was used as an energetic substrate in fluid. The pH of the fluid was
controlled
with a bicarbonate buffer. Osmotic pressure was in the range of 294-311mosm/l.
The
solution was also oxygenated by carbogen gas (mixture consisting of 95% oxygen
and 5%
CO2). Temperature was maintained in the range of 22-33 Celsius. Since the
sections were
without normal capillary blood flow, the exchange of substances was sustained
due to the
diffusion of oxygen, substrates, and metabolites between the incubation medium
and the
whole tissue section. Therefore, the thickness of the section had to be small
enough to
allow complete diffusion through the specimen. According to the empirical
formula used
in calculating the section thickness, the maximum value is approximately 600
microns and
this depends on the intensity of the oxidation process. During isolation,
section cells in the
surface layers with a size of 100 microns are damaged. Pyramidal cells are
approximately
the same size, so the section depth should be at least 300 microns
Experiments were conducted on brain tissue sections of Wistar rats, 1 month of
age.
Anesthesia was performed using ether. Rat brain was isolated and placed into
cold
artificial cerebrospinal (AC) fluid prepared with double distilled water. AC
fluid
composition: (mM): NaCI-130, KCl-3.5, NaH2*PO4-1.2, MgCl2 - 1.3, CaCl2-2.0,
NaHC03-25.0, and glucose. A Carbogen gas mixture was continuously pumped
through
the solution. Hippocampus sections of 400 microns were obtained using a
vibratome.
The sections were then placed into an incubation chamber, which contained AC
fluid, and
maintained at 22-25 Celsius. After 1 hour in the incubation chamber, the
sections were
transferred separately to the testing chamber, with AC fluid flowing through
it at the rate
of 3-5 mllmin. Stimulating electrical pulses (100 msec, 100-400 mA) were been
delivered
through bipolar wolfram electrodes (200 mm), which were located on the
ShafFer's
collators (nerve fibers, ending exciting synapses in the CAl region of
hippocampus).
Induced potentials, which are an electrical response to the stimulation of
assembly/totality, were recorded in the CAl region of hippocampus by using a
glass
microelectrode filled with AC fluid (resistance 0.5- 1.0 mW).
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Two series of experiments were performed. Standard AC fluid (A) was used as
the initial
100% level of signal in both series. In the first series of experiments, AC
fluid was
replaced with the solution having the same salt composition and double
distilled water -
(solution B). In the second series of experiments, micro-clustered water
replaced double
distilled water in solution B (solution C).
The perfusion system utilized made it possible to continuously switch the
supply of the
solutions into the testing chamber. The complete substitution of one solution
by another in
the chamber with a volume of 2 ml occurred during 1 minute. The amplitude of
induced
response was the comparative characteristic. To measure the induced negative
monophase
response, which is 30-40% of the maximum amplitude for the parameters of
power,
duration and location of stimulation were selected. Testing was produced with
a series of
10 single pulses with intervals of 10 msec. A series of pulses were applied at
intervals of
2 to I O minutes. The recorded signal was digitized by an analog-digital
converter and was
saved for the following analysis. Final data processing was completed using
Excel and
Origin software. Statistical analysis was performed using paired t tests. The
value of
P<0.05 was accepted as being statistically significant..
Result. Shaffer's collators were stimulated in the CAl region and the induced
response
was recorded after 0.5-4 msec and from 4-6 msec.
Figure I4 shows the dependence of focal potential measured from the rat
hippocampus on
the type of perfusing fluid. The horizontal axis represents the time after the
beginning of
the experiment. The vertical axis is the uxnplitude of electric signal (%
relative to signal
measured in standard AC fluid). Brain sections were placed in flowing standard
AC fluid
(A), fluid prepared with distilled (B), or micro-clustered water (C). Arrow
indicates
replacement of standard AC fluid with the test medium prepared with micro-
clustered
water. Results axe averaged for 14 sections from seven rats.
In the first series of experiments the dynamics of induced response amplitude
was
recorded after replacing standard AC solution with the solution prepared with
double
distilled water. Immediately after changing the solution, an increase in the
induced
response amplitude was observed with a maximum at 5 min 128.2% (Fig.l4). A
steady
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decrease in the amplitude was observed, to the point at which after one hour
the amplitude
decreased to only 31.7% of the initial value.
In the second series of experiments, micro-clustered water was used instead of
double
distilled water. Immediately after replacing the standard solution with the
solution
prepared with micro-clustered water, the amplitude of induced response sharply
increased,
with a maximum of 135.2% reached after 1-3 minutes. Afterwards, the amplitude
decreased slightly and after 1 hour it was down to 102%; 2 hours down to
94.8%.
Thus, the results obtained show that replacement of standard AC solution with
the solution
prepared with micro-clustered water, within experimental error, did not affect
the initial
amplitude of induced response for 2 hours. Replacement of the standard AC
solution with
the solution prepared with double distilled water resulted in a decrease to
31.7% (P<0.005)
amplitude after 1 hour.
'The study was stopped after 2 hours on the micro-clustered solution, as the
test unit was
out of solution. How long the rat brain tissue would have continued to be
viable should be
the subject of future studies. At the time the study was stopped the tissues
in micro-
clustered water still had an average amplitude of 94.8%.
Accordingly, a method of the invention includes stimulating or modulating the
growth or
activity of cells by contacting the cells for a sufficient period of time with
either micro-
clustered water or the micro-clustered media compositions of the invention.
This method
finds utility in using micro-clustered media to enhance the synthesis of
compounds or
products derived from culture of either animal cells, plant cells, or
microorganisms, or
from culture of organelles. Typically, the synthesis of compounds or products
by these
methods involves the preparation of a composition or compound which did not
exist in the
starting material.
The above examples illustrate that the compositions of the invention are
useful in methods
of regulating cell metabolism or physiology. Examples of such activities
include but are
not limited to altering or regulating the differentiation state of said cells,
ability of cells to
metabolize nutrient materials, cell cycle synchronization or lack thereof,
resistance or
sensitivity to particular compounds, alteration of intracellular pH. Other
methods of using
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the compositions of the invention find use in mere culturing of cells in a
medium, which
promotes normal cell growth and division.
Bionrocess Technolog,Y~Industri~.l Product Formation Through
Microbial Processes
The micro-cluster water and micro-clustered compositions of the invention are
generally
useful in bioprocess technology in small, medium and large scale processes,
and in
methods of production and product recovery or isolation, inoculum and medium
preparation, cultivation and downstream processing.
Industrial/pharmaceutical microbiology/biotechnology rely on aqueous
compositions,
methods of preparing and using them, and the resultant products in the form of
small-,
medium-, large/macromolecules (Microbial Biotechnology, Fundamentals of
Applied
Microbiology, Alexander N. Glazer and Hiroshi Nikaido 1995, W.H. Freeman Co.;
1S Pharmaceutical Biotechnology, eds. Daan J.A. Crommelin and Robert D.
Sindelar, 1997,
Harwood Academic Publishers). The cultivation of cells takes place in vessels
containing
an appropriate liquid growth medium. Production-scale cultivation is commonly
performed in bioreactors which are devices adapted for the growth or
propagation of a
microorganism or enzyme, or for the synthesis of a composition or compound
using a
microorganism or enzyme (Ibid, Crommelin, Chapter 3; Glazer at p. 2S0).
Accordingly,
the present invention includes the use of micro-clustered compositions in
bioreactors in
bioprocess technology as described herein.
The compositions of the present invention involve partial or complete
substitution of
2S micro-clustered compositions for aqueous compositions heretofore in use by
those of skill
in the art. Included in the invention are novel intermediate or final
products, which are
produced with the micro-clustered compositions, as well as methods of using
them
Some of the major products dependent on microbial/anirnal cell/plant cell
biotechnology
include fermented juices and distilled Liquors, cheese, antibiotics,
industrial alcohol, high
fructose syrups and amino acids, baker's yeast, steroids, vitamins, citric
acid, enzymes,
hormones, growth factors, vaccines, polysaccharide gums.
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Accordingly, the present invention includes micro-clustered compositions and
their use in:
I . Production of proteins in bacteria.
2. Production of proteins in yeast.
3. Production of recombinant and synthetic vaccines.
4. Production of microbial insecticides.
5. Production of enzymes
6. Production of microbial polysaccharides and polyesters
7. Production of ethanol
8. Production of amino acids
9. Production of antibiotics
10. Organic synthesis and degradation by enzymes and microbes
I 1. Environmental applications, including sewage and wastewater microbiology;
microbial degradation of xenobiotics; use of microorganisms in mineral
recovery, and in
removal of heavy metals from aqueous effluents.
EFFECTS OF MICRO-CLiTSTERED WATER ON MiTTATION RATES
The study of the effects of micro-clustered water at the cytogenetic level was
performed
using the methods of counting chromosomal aberrations and sister chromatid
exchange
(SCE) in the lymphocytes of peripheral human blood. In addition, the analysis
was
performed during the entire cell cycle process of human lymphocytes in cell
culture using
the method of counting the number of cells after one, two, and three
replication cycles.
The analysis of the frequency of chromosomal aberrations in a culture of human
lymphocytes is one of the main tests applied in the study of mutagenic
activity of
environmental factors and is approved by the (WHO) World Health Organization
(Methods fox the analysis of human chromosome aberrations. Eds. Buckton K. E.
and
Evans H. J. WHO, Geneva, 1973, p. 66).
The determination of SCE frequency is also one of the standard tests used in
the
evaluation of mutagenicity. This method possesses specificity and high
sensitivity in the
evaluation of mutagenic properties of chemical compounds (Sister Chromatid
Exchanges
(Parts A and B). Eds. Tice R. R. and Hollander A. Plenum Press, N.Y., London,
1984).
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The procedure of determining the frequency of SCE in a culture of human
lymphocytes
makes it possible to specifically evaluate the number of emergent SCE during
cell
culturing (Bochkov R.P., Chebotarev A.N., Platonova V.L, Debova G.A. Invention
Certificate No. 1,175,165. Government Committee of the USSR on Inventions and
Discoveries, 1985).
Specimen analysis for SCE was accomplished in parallel with the assessment of
the
number of metaphases after one, two, and three cycles of replication. from
this, the
determination of the average number of cell divisions and the duration of the
cell cycle
until the moment of cell fixation was made possible (Vedenkov V.G., Bochkov N.
P.,
Volkov LK., Urubkov A. R., Chebotarev A. N., Mathematical model of
determination
number of cells passing different number of divisions in culture. Proceeding
of Academy
of Sciences of USSR, v. 274, Nol, p186-189, 1984).
The evaluation of mutagenicity was based on the comparison of the frequency of
sister
chromatid exchange and chromosomal aberrations in human lymphocytes cultured
in cell
medium prepared with micro-clustered and standard deionized water.
Materials and Methods
Experiments were performed using the blood of a 58-year-old male and blood
from two
females, ages 26 and 61. Dry RPMI 1640 (Gibco) cell medium was used to prepare
the
dividing lymphocytes of peripheral blood in culture. Dry cell medium powder
was mixed
with 25 mM/ml of sodium bicarbonate (Serva) and 24 mM/ml HEPES (Serva) and
then
dissolved in deionized water (18 Mohm/cm) (control) or in micro-clustered
water. These
cell culture media solutions were then sterilized by passing them through
membrane filters
with a pore diameter of 0.22 m .
Cell cultures were prepared as follows: 1 ml of heparinized venous blood was
placed in
sterile plastic test tubes, then 0.015 ml of phytohemagglutinin P (Beckon &
Dickinson), 8
ml of RPMI 1640 medium (control or micro-clustered water based), and 1 ml of
embryonic calf serum were added (Biowest). Test tubes were shaken and placed
in an
incubator set at 37°C. Colchicine (Calbiochem) was added 2 hours prior
to fixation, with a
final concentration of 0.5 ~,g/ml.
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Cells were fixed after 48 hours of culturing to count chromosomal aberrations.
5-
bromodeoxyuridine was added (up to a final concentration of 10 p,glml) after
48 hour of
culturing to determine SCE in the cells. Then, cells were fixed after 80
hours.
10 ml of 0.55% potassium chloride (37°C) solution was added to the
cells before fixation
after centrifuge spin (10 min at 1000 r/min.) and the supernatant was removed.
Then, cells
were resuspended and left in the incubator for 10 minutes. The incubated cells
were fixed
with a mixture of methanol and glacial acetic acid (3:1) and cooled to -
10°C. The cells
were placed onto cooled wet glass slides, warmed, and left for at least 24
hours at room
temperature before staining.
The specimens on glass slides were stained by azure-eosin to count chromosomal
aberrations. Specimens wexe stained to determine SCE frequency in accordance
with
Chebotaxev A.N., Selezneva T.G., Platonova V.I. Modified method of
differential staining
of sister chromatids. Bulletin of experimental biology and medicine. V85, No
2, p.242-
243, 1978.
Student's t-Test was used to determine the difference in the average number
SCE per
cell.To evaluate the difference in the frequency of aberrations, a 2 x 2 size
chi-square test
is applied during the analysis of coupling tables. The same criteria was used
for evaluating
the changes in mitoses after the different number of replications of DNA, but
only for the
tables of 3 x 2 sizes.
Results of the Experiment
Sister chromatid exchanges
Two series of measurements were performed for each individual. In each series,
two
specimens were prepared and 25 metaphases were analyzed. Analysis showed that
medium frequency of SCE was not different for both specimens. In addition, the
average
number of SCE fox the series was not significantly different. Table 1 shows
the results of
SCE measurements.
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Table 1. Average SCE number per cell
-44-
Donor gender,Average std. Statistics
age Deviation (cell
number)
Deionized Water Micro-Clustered Df, t, P
Water
Male, 58 3,250,189 (100) 2,870,183 (100) 198; 1,44; 0,151
Female, 26 4,460,272 (100) 3,470,190 (100) 198; 2,98; 0.0032
Female, 61 4,310,269 (100) 3,810,236 (100) 198; 1,40; 0,164
Combined ~ 4,010,145 (300)~ 3,380,120 (300)~ 598; 3,311; 0.000985
~ ~ ~ I'~
The data presented in Table 1 for all individuals shows the SCE average number
per cell
was lower when micro-clustered water was used as the solvent of dry medium
RPMI 1640
compared to standard deionized water. This difference was statistically
significant at the
level of P<0.01 fox the 2nd individual. For the whole group, this statistical
difference was
even higher, at the level of P<O.OOI . Thus, SCE analysis revealed that using
micro-
clustered water as a solvent inhibited the frequency of mutation in a culture
of cells,
resulting in a smaller amount of damage in cell culture compared to standard
deionized
water.
Average number of divisions
Metaphases with uniformly stained sister chromatids were associated with first
mitosis.
Metaphases with one dark and one bright (arlequin chromosome) chromatid were
associated with second mitosis. In these cells, half of the chromosomal
material was bright
and the other half was dark. Cells having only 1/4 of their chromosomal
material dark and
3/4 bright were associated with third mitosis.
The average mitosis number was calculated by the formula:
n,~i)/( ~ ni)
The average number of cell divisions, taking the doubling-of the number of
cells after each
division into account was calculated according to the formula:
(~ i~n,l2'-r)/(~ n;/2'-1)
In these formulas i is the mitosis number, and ni is the number of cells of
the i-th mitosis.
The results showing the proportion of different mitoses in cells are presented
in Table 2.
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Table 2. Number of the 1st, 2nd and 3rd mitoses
Donor Type of Water Cell Average Average Statistics,
Number
gender, number number df
of of x2
,
age Mitosis divisionsmitosis ,
p
1 2 3
Male, 58 Deionized 128 239 71 I,58 1,87
Micro-Clustered 2; 14,23;
Water 75 220 92 1,75 2,04 0.0008
Female, Deionized 145 270 38 1,53 1,76
26
Micro-Clustered 2; 2,18;
Water 155 .235 32 1,48 1,71 0,34
Female, Deionized 185 240 20 1,42 1,63
61
Micro-Clustered 2; 1,63;
Water 198 224 15 1,38 1,58 0,44
Combined Deionized 458 749 129 1,51 1,75 2; 1,69;
Micro-Clustered 1,51 1,77 0,43
Water 428 679 139
Table 2 shows that for the first individual only, the cells in the medium with
micro-
clustered water divided more rapidly than in a medium prepared with standard
water.
However this effect was insignificant on the investigated group as a whole. On
the basis of
time that 5- bromodeoxyuridine was present (32 hour), during which it could
have been
incorporated into DNA resulting in brighter staining of chromosomal material,
it was
possible to determine the average time for the complete cell cycle process. It
gave
32/1.51=21.2 hours, which corresponded to the data found in the literature.
Chromosomal aberrations
Analysis of chromosomal aberrations was performed in 2 series of experiments
for each
individual, similar to the SCE analysis. In each series, 300 metaphases were
analyzed for
deionized and for micro-clustered waters. Data was not obtained for one of the
individual
women, age 61 years old. Analysis shows that for both series and for both
individuals
analyzed, the frequency of chromosomal aberrations did not differ for each
type of water.
Therefore, data for both series were combined. Table 3 shows the data on the
frequency of
chromosomal aberrations.
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Table 3. Frequency of chromosomal aberrations
Donor gender,Type of Metaphase Number of Frequency Statistics
of
Age Water number aberrant aberrant df, x2, P
metaphases metaphases
(%)
Deionized 600 19 3,17
Male, Micro- 1; 6,9;
58 Clustered 600 6 1,00 0_,0.~ 086
Water
Deionized 600 11 1,83
Female, Micro- 1; 2,28;
~
26 Clustered 600 5 0,83 0,1310
Water
Deionized ND ND ND
Female, Micro-
61 Clustered ND ND ND
Water
Deionized 1200 30 2,50
Combined Micro- 1; 8,96;
Clustered 1200 11 0,92 0,0028
Water
Table 3 shows that the frequency of aberrant metaphases during the use of
micro-clustered
water was significantly inhibited or reduced in the 58 year old male and also
in the 26 year
old female. The frequency of aberrant metaphases was less statistically
significant for
micro-clustered water compared with standard deionized water for the
individuals
analyzed as a whole.
Accordingly, this study showed that (1) a difference in cell cycle duration
was not
observed for deionized and micro-clustered waters; (2) sister chromatid
exchange
frequency was statistically lower in micro-clustered water; and (3) frequency
of
chromosomal aberrations was also lower in micro-clustered water. The use of
micro-
clustered water resulted in less mutageni., effects in comparison with
standard deionized
water.
The micro-clustered water inhibited the frequency of mutation in a culture of
cells, and
had a stabilizing effect on genetic material as evidenced by a lower sister
chromatid
exchange frequency and lower chromosomal aberrations in comparison with
standard
deionized water. As used herein, the term "genetic material" refers to a gene,
a part of a
gene, a group of genes, or fragments of many genes, on a molecule of DNA, a
fragment of
DNA, a group of DNA molecules, or fragments of many DNA molecules. Genetic
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material could refer to anything from a small fragment of DNA to the entire
genome an
organism. Accordingly, a method of the invention is directed to inhibiting the
frequency
of mutation of genetic material, the method involving the step of culturing
cells for a
sufficient time in a culture medium which comprises a sufficient amount of
micro-
s clustered water to inhibit the frequency cf mutation. The frequency of
mutation is
referenced with respect to a biological entity which could be cells in cell
culture, cells in
tissue, cells in organ culture, or cells in vivo. As detailed above, cells
include animal
cells, microorganisms, and plant cells. Effective culturing of cells situated
in vivo or ih
situ, involves administering a sufficient quantity of micro-clustered water or
medium
comprising micro-clustered water to a subject animal or plant which is
otherwise a
multicellular organism. Genetic material in biological entities of vectors,
viruses or
bacteriophage, and subcellular parts is subject as well to mutation inhibiting
effects of
micro-clustered water. It should be understood that the mutation-inhibiting
effect of
micro-clustered water is achieved by culturing or cultivating any of said
biological entities
1 S in micro-clustered water.
The invention is further directed to inhibiting the frequency of mutation in
the presence of
a mutagenic substance. The frequency of mutation' is referenced with respect
to a
biological entity which includes cells in cell culture, cells in tissue, cells
in organ culture,
or cells in vivo. As detailed above, cells include animal cells,
microorganisms, and plant
cells. Genetic material in biological entities such vectors, viruses or
bacteriophage; and
subcellular parts is subject as well to mutation inhibiting effects of micro-
clustered water.
Inhibiting Induced Muta~enesis in vitro To determine the frequency of
chromosome
aberrations in human lymphocytes, mitomycin C (the mutagen) is added to the
cell culture
in three different doses 24 hours before fixation. Control cells do not have
mutagens.
There are 4 experimental settings. Mutagenesis occurs before DNA synthesis.
Dioxydine~
is added to the lymphocyte culture in three different concentrations in order
to determine
the chromosome aberrations after DNA synthesis. All together, there are 16
settings: the
control, with no mutagens + 3 different concentrations of mitomycin C, the
control + 3
different concentrations of dioxydine, micro-clustered water + 3 different
concentrations
of mitomycin C, and Penta water + 3 different concentrations of dioxydine. 100
metaphases are analyzed for each setting, or 1600 cells are used. Mitomycin C
is also
added in three different doses 24 hours before the fixation to determine the
frequency of
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-48
sister chromatid exchange (SCE). However, the concentration of mutagen is one
order less
than it is for chromosome aberrations, plus control without mutagen. There are
4 settings
for the standard and 4 settings for micro-clustered water, which make 8
different settings.
25 metaphases are analyzed in each case - a total of 200 cells. Findings from
these
studies indicate that micro-clustered water inhibited the frequency of
mutation in the
presence of a mutagen.
Inhibiting Induced Muta~nesis in vivo Chromosome aberrations are counted in
mouse
bone marrow, 100 cells for each setting. Mice drink standard (control) and
micro-
clustered water over a 1S-day period. Mitomycin C is injected (3 doses +
control without
mutagen) 24 hours before animals are to be sacrificed and before cell
fixation. Dioxydine
is injected 2 hours prior to sacrifice and cell fixation (3 doses + control).
6 mice are in
each group; all together a total of 96 mice or 9600 cells. Findings from these
studies
indicate that micro-clustered water inhibited the frequency of mutation i~z
vivo in the
1 S presence of a mutagen.
[399056- drugs]
III. DRUGS, BIO-AFFECTING AND BODY
TREATING COMPOSITIONS
General Description and Def nitions
The practice of the present invention will employ, unless otherwise indicated,
conventional techniques within the skill of the art in (1) organic and
physical chemistry;
(2) biochemistry; (3) molecular biology; (4) pharmacology; (S) pharmacological
therapeutics; (6) physiology; (7) toxicology; (8) microbiology, (9) internal
medicine and
2S diagnostics. Such techniques are explained fully in the literature. See,
e.g. Maniatis et al.,
Molecular Cloning: A Laboratory Manual; Pharmaceutical Biotechnology, eds.
Daan J.A.
Crommelin and Robert D. Sindelar, 1997, Harwood Academic Publishers; Goodman &
Gilman's The Pharmacological Basis of Therapeutics, eds. Joel G. Hardman, Lee
E.
Limbird, Tenth Edition, 2001, McGraw Hill; Basic & Clinical Pharmacology,
Bernard G.
Katzung, Eighth Edition, 2001, McGraw Hill; Pharmaceutical Dosage Forms and
Drug
Delivery Systems, Howard C. Ansel, Loyd V. Allen, Jr., Nicholas G. Popovich,
Seventh
Edition, 1999, Lippincott, William & Wilkins; Harrison's Principles of
Internal Medicine
by Eugene Braunwald M.D. (Editor), Anthony S. Fauci M.D. (Editor), Dennis L.
Kasper
M.D. (Editor), Stephen L. Hauser M.D. (Editor), Dan L. Longo M.D. (Editor), J.
Larry
3S Jameson M.D. (Editor).
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The following terminology will be used in accordance with the definitions set
out below in
describing the present invention.
The term "drug" refers to a chemical agent intended for use in the diagnosis,
mitigation,
treatment, cure, or prevention of disease in human or in other animals.
Synonymous with
the term "drug" are the terms "bio-affecting agents" and "body-treating
agents." In a broad
sense, drugs axe substances that interact with living systems through chemical
processes.
These substances may be chemicals administered to a living body to achieve a
beneficial
therapeutic effect on some process within the patient or for their toxic
effects on
regulatory processes in parasites infecting the patient. It is understood that
the biological
properties are expressed on cells, tissues, and organs of living bodies. These
agents,
substances, or drugs are subjects of the micro-clustered compositions of the
invention.
The terms "medicinal activity," and "medical properties," and "active
ingredient" also refer
to the action of drugs on living tissue or bodies.
The terms "bio-affecting" and "body-treating" include subject matter defined
generally,
and in particular, by the classification definitions and examples or
embodiments disclosed
in the United States Manual of Classification, U.S. Patent Classification from
the United
States Patent and Trademark Office, in particular, Class 424 (and related
lines of
classification as disclosed therein): Drug, Bio-Affecting, and Body Treating
Compositions, which is hereby incorporated by reference. Further defined and
embodied
by Class 424 (and as described herein) are the terms and phrases "adjuvant or
carrier
compositions," "fermentates," "plant and animal extracts or body fluids or
material
containing plant or animal cellular structure" intended for use as bio-
affecting or body
treating compositions. The compositions of the invention are further defined
and
classified according to specific structures (e.g. layered tablet, capsule).
Processes of using
the compositions of the invention are embodied in Class 424, as well as
processes of
preparing the compositions.
Drugs are derived from plant or animal sources, as byproducts of microbial
growth,
through chemical synthesis, molecular modification of existing chemical
agents.
Sources of drugs: New drugs may be discovered from a variety of natural
animal, plant, or
microbial sources, or created synthetically in the laboratory. Plant materials
have served
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as a reservoir of drugs. Animals are a source of drugs, that are derived from
their tissues
or through their biologic processes. By way of non-limiting examples, hormonal
substances, such as thyroid extract, insulin and pituitary hormone are
obtained from the
endocrine glands of cattle, sheep, and swine. The urine of pregnant mares is a
rich source
of estrogens. Fermentates, which are compositions of or derived from bacteria
or the
microorganisms occurring in unicellular plants such as yeast, molds or fungi,
are well
known in the art (Glazer and Nikaido, Microbial Biotechnology, Fundamentals of
Applied
Microbiology, 2001, W.H. Freeman and Company).
The rubric "medical pharmacology" refers to the science of substances used to
prevent,
diagnose and treat disease.
Products of biotechnology contribute as well to pharmaceutical and diagnostic
compositions of the invention (Pharmaceutical Dosage Forms and Drug Delivery
Systems,
Howard C. Ansel, Loyd V. Allen, Jr., Nicholas G. Popovich, Seventh Edition,
1999,
Lippincott, William ~ Wilkins, See Chapter 18, incorporated by reference).
The term "prodrug" describes a compound that requires metabolic
biotransformation after
administration to produce the desired pharmacologically active compound.
The term "micy~o-clustered composition" as used herein refers to a composition
which
comprises micro-cluster water. The adjective "micro-clustered " which modifies
any of the
compositions of bio-affecting agents, body-treating agents, adjuvant or
carriers, or
ingredients thereof refers to micro-clustered water in that composition, i.e.
which is
dissolved in, mixed with, or otherwise combined with micro-cluster water.
A "cell" is the basic structural unit of all living organisms, and comprises a
small, usually
microscopic, discrete mass of organelle-containing cytoplasm bounded
externally by a
membrane and/or cell wall. Eukaryotes are cells which contain a cell nucleus
enclosed in
a nuclear membrane. Prokaryotes are cells in which the genomic DNA is not
enclosed by
a nuclear membrane within the cells.
"Tissue" refers to any collection of cells that is organized to perform one or
more specific
function.
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"Organ" is any part of the body of a multicellular organism that is adapted
and/or
specialized for the performance of one or more vital functions.
COMPOSITIONS OF THE INVENTION
The compositions of the invention are micro-clustered water compositions.
These
compositions comprise micro-clustered water and one or more agents selected
from one or
more of the group consisting of bio-affecting agents, body-treating agents,
and adjuvant or
carrier compositions.
SUMMARY OF THE INVENTION
The micro-clustered water compositions disclosed herein comprise one or more
agents
selected from one or more of the group consisting of bio-affecting agents,
body-treating
agents, and adjuvant or carrier compositions. The biological properties of the
body
treating agents include (a) preventing, alleviating, treating or curing
abnormal and
pathological conditions of the living body; (b) maintaining, increasing;
decreasing,
limiting or destroying a physiologic body function; (c) diagnosing a
physiological
condition or state by an in vivo test; and (d) controlling or protecting an
environment or
living body by attracting, disabling, inhibiting, killing, modifying,
repelling or retarding an
animal or micro-organism. Body treating agents may be selected from the group
of
agents intended for deodorizing, protecting, adorning or grooming a body.
The compositions of the invention can take the form of liquid, ointments,
creams, gels,
dispersions, powders, granules, capsules, tablets, and transdermal drug
delivery devices.
In any case, the compositions can be pha~~maceutical compositions.
Disclosed herein are methods of using the compositions of the invention, the
methods
involving a step of administering said composition to a living body, or
administering the
compositions ex vivo to cells, tissues, and organs.
In another aspect, methods are provided for preparing the compositions, the
methods
involving a step of combining micro-clustered water with one or more agents
selected
from one or more of the group consisting of bio-affecting agents, body-
treating agents, and
adjuvant or caxrier compositions.
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Formulation Fundamentals
-52-
Each particular pharmaceutical product which contains a drug or a body-
treating agent is a
formulation unique unto itself. In addition to the active therapeutic
ingredients, a
pharmaceutical formulation also contains a number of nontherapeutic or
phaxmaceutic
ingredients. It is through their use that a formulation achieves its unique
composition and
characteristic physical appearance. Pharmaceutic ingredients include such
materials as
fillers, thickeners, solvents, suspending agents, tablet coating and
disintegrants, stabilizing
agents, antimicrobial preservatives, flavors, colorants and sweeteners.
The formulation must be such that all components are physically and chemically
compatible, including the active therapeutic agents, the phaxmaceutic
ingredients and the
packaging materials.
Pharmaceutic Ingredients: Definitions and Tines
To prepare a drug substance into a dosage form or pharmaceutical composition,
pharmaceutic ingredients, which the art also refers to as adjuvants or
carriers, are required.
For example, in the preparation of pharmaceutic solutions, one or more
solvents are used
to dissolve the drug substance, flavors and sweeteners are used to make the
product more
palatable, colorants are added to enhance product, preservatives may be added
to prevent
microbial growth and stabilizers, such as antioxidants and chelating agents,
rnay be used to
prevent drug decomposition. In the preparation of tablets, diluents or fillers
are commonly
added to increase the bulk of the formulation, binders to cause the adhesion
of the
powdered drug and phaxmaceutic substances, anti-adherents or lubricants to
assist the
smooth tableting process, disintegrating agents to promote tablet break-up
after
administration, and coatings to improve stability, control disintegration, or
to enhance
appearance. Ointments, creams, and suppositories achieve their characteristic
features due
to the pharmaceutic bases which are utilized. Thus for each dosage form, the
pharmaceutic ingredients establish the primary feah~res of the product, and
contribute to
the physical form, texture, stability, taste and overall appearance.
The principal categories of pharmaceutic ingredients are numerous and well
known to
those skilled in the art. For the sake of not reproducing herein a catalog of
categories and
examples within each, Applicant refers the reader to the treatise
Pharmaceutical Dosage
Forms and Drug Delivery Systems, Howard C. Ansel, Loyd V. Allen, Jr., Nicholas
G.
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-53-
Popovich, Seventh Edition, 1999, Lippincott, William & Wilkins, which is
hereby
incorporated by reference. In particular, attention is focused on Chapter 3 -
Dosage Form
Design: Pharmaceutic and Formulation Considerations. Table 3.3 in this
reference
provides non-limiting examples of pharmaceutic ingredients, and examples
thereof. It is
understood that the micro-clustered compositions of the invention include
aqueous
compositions of pharmaceutic ingredients and/or excipients.
The reader should also be aware of the Handbook of Pharmaceutical Excipients
which
presents monographs on over 200 excipients used in pharmaceutical dosge form
I O preparation. Included in each monograph is such information as:
nonproprietary,
chemical, and commercial names; emperical and chemical formulas and molecular
weight;
pharmaceutic specifications and chemical and physical properties;
incompatibles and
interactions with other excipients and drug substances; regulatory status; and
applications
in pharmaceutic formulation or technology.
DOSAGE FORMS
In addition to liquid dosage forms of micro-clustered compositions, the micro-
clustered
compositions of the invention are directed to non-liquid dosage forms (as set
forth below)
which comprise micro-clustered water. See Pharmaceutical Dosage Forms and Drug
Delivery Systems, Howard C. Ansel, Loyd V. Allen, Jr., Nicholas G. Popovich,
Seventh
Edition, 1999, Lippincott, William & Wilkins.
Solid Dosage Forms and Modified-Release Drug Delivery Systems
Powders and Granules
Capsules and Tabets
Modified-Release Dosage Forms and Drug Delivery Systems
Semi-Solid and TransdermalSystems
Ointments, Creams, and Gels
Transdermal Drug Delivery Systems
Pharmaceutical Inserts
Suppositories and Inserts
Liquid dosage forms commonly comprises solutions and disperse systems. Sterile
dosage
forms and delivery systems involve parenterals, biologicals, ophthalmic
solutions and
suspensions.
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Novel and advanced dosage forms, delivery systems, and devices include
radiopharmaceuticals for diagnosis and for therapeutics, and liposomes.
The invention fiurther covers micro-clustered compositions, as described
above, in
combination with drug delivery systems, generally for parenteral delivery,
which
incorporate mechanical, electronic, and computerized components. Methods for
administering micro-clustered compositions to a living body which involve a
step using a
mechanical, electronic, or computerized component or device are within the
scope of the
present invention. Examples of these medical device assisted compositions
involve drug
delivery systems wluch include iontophoresis, phonophoresis, dialysis,
implanted pumps,
fluorocarbon propellant pumps, intravenous controllers and infusion pumps (
Chapter 19,
Ansel, incorporated by reference). Commonplace drug delivery systems which for
access
and delivery to the vascular system include syringes, needles or devices for
injection,
catheters, liquid composition containers, lines or tubing for delivering
liquids between
devices and/or the body or tissues.
SOLVENTS AND VEHICLES FOR INJECTION WHICH COMPRISE MICRO-
CLUSTERED WATER
The most frequently used solvent in the large scale manufacturer of injections
is Water for
Injection, USP. An aqueous vehicle is generally preferred for an injection,
and water is
used in the manufacture of injectable products. Examples of micro-clustered
waters
include:
Purified Water, USP, Sterile Water for Injection, USP, Bacteriostatic Water
fog Injection,
USP.
Sodium Chloride Injection, US, Bacteriostatic Sodium Chloride Injection, USP,
Ringer's
Injection, USP, Lactated Ringey~'s Injection, USP.
Bio-Affecting Agents and Body Treating Agents
Micro-clustered compositions of the invention include compositions of bio-
afFecting
agents and body-treating agents. "Bio-affecting agents" and "body-treating
agents" are
substances which may possess biological or medical properties as set forth
below. These
agents, substances, or drugs are components of the micro-clustered
compositions of the
invention. It is understood that the biological properties are expressed on
cells, tissues,
and organs of living bodies. The terminology of these biological or medical
properties, as
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used herein, is consistent with their usage in standard medical dictionaries
(e.g. Dorland's
Medical Dictionary), and treatises (e.g. The Pharmacological Basis of
Therapeutics, eds.
Joel G. Hardman, Lee E. Limbird, Tenth Edition, 2001, McGraw Hill; Basic &
Clinical
Pharmacology, Bernard G. Katzung, Eighth Edition, 2001, McGraw Hill;
Pharmaceutical
S Dosage Forms and Drug Delivery Systems, Howard C. Ansel, Loyd V. Allen, Jr.,
Nicholas G. Popovich, Seventh Edition, 1999, Lippincott, William & Wilkins.)
While body-treating agents may have medicinal effects, the primary meaning of
"body-
treating agents" for purposes of this invention is directed to agents
administered topically
to a living body and which are intended for deodorzing, protecting, adorning
or grooming
the body.
In general terms, the biological properties of the bio-affecting agents and
body treating
agents include:
1 S a. preventing, alleviating, treating or curing abnormal and pathological
conditions of the living body;
b. maintaining, increasing, decreasing, limiting or destroying a physiologic
body function;
c. diagnosing a physiological condition or state by an in vivo test;
d. controlling or protecting an environment or living body by attracting,
disabling, inhibiting, killing, modifying, repelling or retarding an animal or
micro-
2S organism.
Body-treating agents include, but are not limited to, dentifrices; topical sun
or radiation
screening or tanning preparations; manicure or pedicure compositions, bleach
for live hair
or skin; live skin colorants (e.g. lipstick); anti-perspirants or perspiration
deodorants; live
hair or scalp treating compositions; topical body preparations containing
solid synthetic
organic polymers (e.g. skin cosmetic coating).
Therapeutic Classification of the Bio-Affecting Agents
The following classification of drugs, which is non-limiting, is derived from
Goodman &
3S Gilman's The Pharmacological Basis of Therapeutics, eds. Joel G. Hardman,
Lee E.
Limbird, Tenth Edition, 2001, McGraw Hill, herein incorporated by reference
for the
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subject matter disclosed herein. The micro-clustered compositions of the
invention
comprise drugs which have one or more of the following medicinal activities.
Drubs Acting-at Synaptic and Neuroeffector Junctional Sited
S These agents affect neurotransmission in the autonomic and somatic motor
nervous
systems. Included are muscarinic receptor agonists and antagonists:
anticholinesterase agents; agents acting at the neuromuscular junction and
autonomic ganglia; catecholamines, sympathomimetic drugs, and adrenergic
receptor antagonists; S-hydroxytryptamine (serotonin): receptor agonists and
antagonists.
Dru s Acting on The Central Nervous System
These include general anesthetics and local anesthetics; therapeutic gases
(oxygen,carbon dioxide, nitric oxide, and helium; hypnotics and sedatives;
ethanol;
1 S drugs for treating psychiatric disorders, such as depression, anxiety
disorders,
psychosis, mania; drugs for treating epilepsies; drugs for treating central
nervous
system degenerative disorder; opioid analgesics; drugs for treating drug
addiction
and drug abuse.
Autacoid~ Dru Therapy of Inflammation
These include histamine, bradykinin, and their antagonists; lipid derived
autocoids:
eicosainoids and platelet activating factor; analgesic-antipyretic and
antiinflammatory agents and drug employed in the treatment of gout; drugs used
in
the treatment of asthma.
2S
Dru~;s Affecting Renal and Cardiovascular Function
These include diuretics; vasopressin and other agents affecting renal
conservation
of water; renin and angiotensin; drugs for treating myocardial ischemia;
antihypertensive agents and drugs for treating hypertension; drugs for
treating
heart failure; antiarrhythmic drugs; drugs for treating hypercholesterolemia
and
dyslipidemia.
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Drugs Affecting Gastrointestinal Function
These include agents for control of gastric acidity and treatment of peptic
ulcers
and gastroesophageal reflux disease; prokinetic agents, antiemetics, and
agents
used in irritable bowel syndrome; agents used for diarrhea, constipation, and
inflammatory bowel disease; agents used for biliary and pancreatic disease.
Chemotherapy of Parasitic Infections
These include agents used in the chemotherapy of protozoal infections, for
example, malaria, amebiasis, giardiasis, trichomoniasis, trypanosomiasis,
leishmaniasis; and for treating helminthiasis;
Chemotherapy of Microbial Diseases
These include antimicrobial agents such as sulfonamides, trimethoprim-
sulfamethoxazole, quinolones and agents for urinary tract infections;
penicillins,
cephalosporins, andother beta-lactam antibiotics; aminoglycosides; protein
synthesis inhibitors; drugs used in chemotherapy of tuberculosis,
mycobacterium
avium complex disease, and leprosy. Further included are antifungal agents,
antiviral agents, and antiretroviral agents.
Chemotherapy of Neonlastic Diseases
These include alkylating agents, nitrogen mustards, ethylenimines and
methylmelamines; alkyl sulfonates; nitrosoureas; folic acid analogs;
pyrimidine
analogs; purine analogs; natural products such as vinca alkaloids, paclitaxel,
epipodophyllotoxins; camptothecin analogs; antibiotics such as dactinomycin,
daunorubicin, doxorubicin, idarubicin; bleomycin, mitomycin; platinum
coordination complexes; hydroxyurea; porocarbazine; adrenocorticosteroids;
aminoglutethimide and other aromatase inhibitors; antiestrogens (e.g.
tamoxifen);
gonadotropin-releasing hormone analogs; antiandrogens; biological response
modifiers such as interleukins, granulocyte colony stimulating factor,
granulocytelmacrophage colony-stimulating factor; monoclonal antibodies.
Dru~~ iJsed for Immunomodulation
These include immunosuppressive agents, tolerogens, and immunostimulants.
These drugs include vaccines based on compositions of antibodies ranging from
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immune globulin to purified antibody compositions to monoclonal antibody
compositions.
Dru s Actin;a on the Blood and the Blood-Forming Organs
These include' hematopoietic agents, such as growth factors, minerals and
vitamins;
and anticoagulant, thrombolytic, and antiplatelet drugs.
Hormones and Hormone Antagonists
These include pituitary hormones and their hypothalamic releasing factors;
thyroid
and antithyroid drugs; estrogens and progestins; androgens;
adrenocorticotropic
hormone; adrenocortical steroids and their synthetic analogs; inhibitors of
the
synthesis and actions of adrenocortical hormones; insulin, oral hypoglycemic
agents; agents affecting calcification and bone turnover: calcium, phosphate,
parathyroid hormone, vitamin D, calcitonin.
1S
The Vitamins
These include water-soluble vitamins: the vitamin B complex and ascorbic acid;
and fat-soluble vitamins: vitamins A, K, and E.
Agents for Treating Dermatological Disorders Agents for Onhthamological
Treatment
ROUTE OF ADMINISTERING THE COMPOSITIONS OF THE INVENTION
Of the micro-clustered compositions of the invention which are intended for
administration to a living body, a variety of routes are available and chosen
by those of
skill in the art with reference to whether the composition is intended for
local or systemic
effects. A method of the invention involves using a composition of the
invention for
therapeutic or diagnostic purposes according to the medicinal or therapeutic
activities
described above. The method includes a step of administering or delivering the
composition via a route which could be oral, sublingual, parenteral,
epicutaneous (topical),
transdermal, conjunctival, intraocular, intranasal, aural, intrarespiratory,
rectal, vagina,
urethral. Those of skill in the therapeutic and diagnostic arts will find
guidance for
administering the compositions of the invention according to methods and
protocols
described in standard textbooks of general and specialized medicine.
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Ex vivo administration. Alternatively, the biological properties of the micro-
clustered
compositions of the invention are administered to and expressed on cells,
tissues, and
organs ex vivo. Evaluation, screening, and treating of mammalian cells,
tissues and
organs in culture are common protocols in gene therapy, stem cell therapy
(e.g. cord blood
stem cell transplantation), grafting or transplanting cells/tissues (e.g.
hematopoetic tissue),
tumor medicine (e.g. host/graft/tumor interactions) and reproductive medicine
(e.g.
embryo culture) (Autologous Blood and Marrow Transplantation X: Proceedings of
the
Tenth International Symposium, edited by Karel A. Dicke and Armand Keating,
May
2001; Bloodline Reviews; Blood and Marrow Transplantation Reviews; Ex Vivo
Cell
Therapy by Klaus Schindhelm and Robert Nordon). The aim of ex vivo therapy is
to
replace, repair, or enhance the biological function of damaged tissue or
organs. An ex vivo
process involves gathering cells from patients or donors, in vitro
manitpulation of to
enhance the therapeutic potential of the cell harvest, and subsequent
intravenous
transfusion.
METHODS OF PREPARING THE COMPOSITIONS OF THE INVENTION
Methods of preparing the micro-clustered compositions of the invention involve
a step of
combining or formulating one or more of a bio-affecting agent, body-treating
agent, or an
adjuvant or carrier compositions with micro-clustered water. Standard
treatises of
chemistry, clinical chemistry, medicinal chemistry, pharmacological sciences,
formulation
science are available to those of skill in the art for guidance in preparing
the compositions
of the invention.
DIAGNOSTIC COMPOSITIONS
The compositions of the invention include diagnostic compositions ( Mosby's
Manual of
Diagnostic and Laboratory Tests by Kathleen Deska Pagana, Timothy James
Paga.na);
methods of the invention include the use of micro-clustered diagnostic
composition in
diagnostic techniques performed in a living body (i.e. in vivo diagnosis or in
vivo testing),
or performed in vitro or ex vivo. Micro-clustered compositions comprising
contrast
agents for use in diagnostic radiological methods are included in the
invention. Diagnostic
reagents and methods for making them (Sigma Aldrich Co.; Worthington
Biochemical
Corporation; Wako Chemicals USA) and using them are well known in the art. The
invention includes kits which comprise micro-clustered compositions.
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IV. FOOD OR EDIBLE MATERIAL AND BEVERAGES: PROCESSES,
COMPOSITIONS, AND PRODUCTS
General Description and Definitions
The practice of the present invention wilt employ, unless otherwise indicated,
conventional food technology, food chemistry, food processing, organic- and
biochemistry
within the skill of the art. Such techniques for foods and beverages are fully
explained in
the literature. See, e.g. Potter, N.N. and Hotchkiss, J.H., Food Science,
Fifth Edition,
1998, Aspen Publishers; Belitz, H.D. and Grosch, W. Food Chemistry, Second
Edition,
1999, Springer; T.P. Coultate, Food: The Chemistry of Its Components, Fourth
Edition,
2002, Royal Society of Chemistry; Owen R. Fennema, Food Chemistry, 3rd
Edition,
1996, Marcel Dekker, Inc.; The Properties of Water.in Foods ISOPOW 6, Edited
by David
S. Reid; 1998 Shafiur Rahman, Food Properties Handbook, 1995, Culinary and
Hospitality Industry Publications Services; Brennan, J.G., Butters, J.R. et
al., 1990, Food
Engineering Operations, Chapman and Hall; Heldman, D.R., and Hartel, R.W.,
1997,
Principles of Food Processing, Chapman and Hall; Encyclopedia of Agricultural,
Food,
and Biological Engineering, 2003, Edited by: Dennis R. Heldman, Marcel Dekker,
Inc.;
Food Structure - Creation and Evaluation, 1987, eds. J.R. Mitchell and J.M.V.
Blanshard,
Woodhead Publishing Ltd.; Amorphous Food and Pharmaceutical Systems, 2002, ed.
H.
Levine, RSC Publishers; Ruan, Roger and Chen, Paul L., Water in Foods and
Biological
Materials, A Nuclear Magnetic Resonance Approach, 1998, Culinary and
Hospitality
Industry Publ. Services; Jose M. Aguilera and Stanley, David W.
,Microstructural
Principles of Food Processing and Engineering, Second Ed., 2000, Culinary and
Hospitality Industry Publ. Services; Functional Properties of Food
Macromolecules, eds.
J.R. Mitchell and D.A. Ledwaxd, 1986, Elsevier Applied Science Publ.; Roos,
Y.H.,
Phase Transitions in Foods, 1995, Academic Press. An extensive catalog of food
science
and technology reference books is available from American Technical
Publishers, Ltd.,
Hitchin, Herts., SG4 OSX, England. Water structure and behavior, including
water's role in
the hydration of food molecules, is exhaustively set forth online at
http://www.sbu.ac.ulc/water/. The references or patents cited herein are
incorporated to
the extent possible for teachings which are relevant for supplementing the
present
disclosure.
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The subject invention is directed to foods or edible materials and beverages,
which have
been hydrated with structured water. In one aspect, the invention comprises
foods or
edible materials and beverages which comprise structured water, and to
structured
ingredients or additives that are involved in preparing a structured or non-
structured
edible.
Another aspect of the invention involves the use of structured water in food
or beverage
rocessin a rocess which involves a ste of h dratin a food rocessin s stem b
p g, p p Y g p g Y Y
contacting structured water with at least one of the ingredients or products
of the food
processing system.
The invention is directed to the use of structured water as well as structured
compositions
for treating or perfecting a food material. In particular, the invention
covers methods of
using structured water in the various roles played by water including but not
restricted to
those set forth in the following table:
TABLE - Roles of Water in Food and Beverag-eses
Role Moisture RangeMechanism of Effect Quality Attribute
Affected
Solvent All-excluding Solution All
bound water
Reaction MediumAll-excluding Facilitation of chemical All
change
bound water
Reactant All Hydrolyzing agent Flavor, texture
Antioxidant Low Hydration and precipitationFlavor, color,
of
metal catalysts, bonding texture, nutritive
to
peroxides and functional value
groups of
proteins and carbohydrates,
promotes free radical
recombination.
Prooxidant Medium Reduction in viscosity Flavor, color,
increases
mobility of reactants texture, nutritive
and catalysts.
Swelling of solid matricesvalue
exposing catalytic surfaces
and
oxidizable groups
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Role Moisture Range' Mechanism of Effect Quality Attribute
Affected I
Structural- All Maintains the integrity Texture and
of proteins
intramolecular molecules attributes
affected by
enzymes
Structural- Low Hydrogen bonding to surfaceViscosity
Intermolecular groups on macromolecules
Hydrogen bonding to cross-linkingTexture -
in
sites of macromolecules dehydrated
foods
Structural- Medium and Influence on structure Rheological
High of
intermolecular emulsions (i.e. binding properties
to surface of
lipids). Influence the emulsions
interactions and
and conformation of gel textural
forming
polysaccharides and proteins.properties
of
gels.
The solute hydration role of structured water in food processing is further
characterized by
the classifications of the types of water-solute interactions as set forth in
the following
table reproduced from Fennema, Food Science, 3rd Edition.
Table 3 Classifications of Types of Water-Solute Interactions
Strength of interaction
compared to water-water
Type Example hydrogen bond a
W ater-iiee
Water-charged group on
organic molecule
Dipole-dipole Water-protein NH Approx. equal
Water-protein CO
Water-side chain OH
Hydrophobic hydration Water + R° ~ R(hydrated) Much less (~G > 0)
Hydrophobic interaction R(hydrated) + R(hydrated) ~ R2 Not comparable d (>
hydro-
(hydrated) + HZO phobic interaction; (~G < 0)
About 12-25 kJ/mol.
b But much weaker than strength of single covalent bond.
° R is alkyl group.
d Hydrophobic interactions are entropy driven, whereas dipole-ion and dipole-
dipole interactions are
enthalpy driven.
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The invention provides in general for structured products and compositions in
any
physical form, which are intended to be consumed via in whole or part via the
oral cavity
by human beings or animals. Further, structured water is included in the
invention in any
of its physical forms.
The scope of the present invention finds utility in the fields of food
engineering, food
chemistry, and food biology.
Food engineering involves food manufacturing, processing, packaging and
preservation.
Analogous to the roles of water, structured water, compositions thereof, and
methods of
processing foods that involve the structured water hydration methods described
herein find
applicability in fluid mechanics and mixing during extrusion, dough rheology,
predicting
diffusion of flavor compounds, understanding mechanism of expansion during
extrusion,
micro and macro structures of foods, baking and microwave processing,
simultaneous heat
and mass transfer during hybrid baking, and membrane-based technologies, as
well as ice
crystal size control during freezing, hot air jet impingement baking, health
promotion
through processed foods, food waste and by-product utilization, modified
atmosphere
packaging and smart packaging for microbial safety.
Food Chemistry applies chemical techniques, concepts and laws to determine the
kinds
and amounts of molecules in foods, their physical properties, and their
chemical
transformations during manufacture and storage. Structured water, compositions
thereof,
and methods of processing foods that involve the hydration methods described
herein find
applicability in a broad range from the analysis of food components to
measurements of
the molecular mobility of amorphous solids; chemical transformations of
lipids,
carbohydrates, and proteins, processing techniques such as extrusion, control
of
antimicrobial or ice-nucleating proteins; spectroscopic, mechanical, and
thermal
techniques for characterizing how the physical properties of amorphous, non-
crystalline,
solids modulate their chemical and physical properties and thus their shelf
life and
stability.
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DEFINITIONS
The meaning to be given to the various "art" terms appearing in the classes of
patentable
subject matter set forth herein, but which have not been included in the
glossary below, is
the same as that generally accepted or in common usage.
The terms "water" and "structured water" and "microclustered water" are used
interchangeably. The subject matter and scope of the "structured" inventions
is informed
by and analogous to the meaning of the term "water" as derived from the
context of its use
herein (U.S. Patent No. 6,521,248).
The terms "food" and "edible" will be used synonymously and interchangeable
herein.
Each ingredient or additive used in a food processing system, whether
naturally occurring
as a product of nature or synthetically produced, that becomes a part of an
edible
composition, or treats an edible composition or is either disclosed or claimed
as being
edible, is to be regarded as being edible.
Food or edible material includes beverages, as defined broadly in Class 426.
By way of
example, but not limitation, subclass 590 involves liquid intended to be drunk
or a
concentrate upon which the addition of aqueous material forms a liquid
intended to be
drunk.. Subclasses 569 (for beverages which form a foam) and 580 (for lacteal
containing
beverages) are included. Subclass 592 covers subject matter wherein the
product contains
ethyl alcohol. A detailed list of beverages, their definitions, and
classifications which
refer to the scope of subject matter within each is found in the U.S. Manual
of Patent
Classification, which is obtained from the United States Patent and Trademark
Office.
Compositions which comprise structured water are referred to herein as "micro-
clustered
compositions." The adjective "micro-clustered" modifies nouns which denote
compositions of matter (e.g. substances, additives, ingredients) and indicates
that the
modified composition of matter comprises micro-clustered water as a result of
otherwise
being hydrated at least in part by structured water. The acronym MCW stands
for
structured water.
A food processing system, in one aspect, involves breaking down the inherent
structures
within food materials or ingredients to a varying extent, and is therefore
concerned with all
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aspects of food -- the chemical and physical properties of food and its
constituents, the
processing and production of food, and the packaging and marketing of food,
which
represent components of a food processing system. Food quality - texture,
flavor release,
nutrient availability, moisture migration, and microbial growth -- are
influenced and
determined by the formation, stability and breakdown of structures within
foods. Each
ingredient or additive used in a food proc~essirig system, whether naturally
occurring as a
product of nature or synthetically produced, that becomes a part of an edible
composition,
or treats an edible composition or is either disclosed or claimed as being
edible, is to be
regarded as being edible. Food processing involves conversion of raw materials
and
ingredients into a consumer food or edible product. Food processing includes
any action
that changes or converts raw plant or animal materials into safe, edible, and
more palatable
foodstuffs. Improvement of storage or shelf life is another goal of food
processing.
HYDRATION CHEMISTRY IN FOOD PROCESSING
The present invention is directed to the use of structured water in food
processing. Water
in combination with carbohydrates, lipids, and proteins, represents one of the
main
components of foods. Accordingly, the invention is directed to methods of
achieving
combinations of structured water with carbohydrates, lipids and proteins in
food
processing systems.
Water's hydration properties depend, in part, on its clustering (Water
structure and
behavior, including water's role in the hydration of food molecules, is
exhaustively set
forth online at http://www.sbu.ac.uk/water/; and The Properties of Water in
Foods
ISOPOW 6, Edited by David S. Reid). Structured water's hydration properties
toward
biological macromolecules (particularly proteins and nucleic acids) is a
determinant of
their three-dimensional structures, and hence their functions, in solution.
Structured water is used in processing of foods to improve texture, mixing,
and flowing
properties, and functionality. MCW is also involved in a number of
interactions with other
components of foods. These interactions may contribute to the molecularly
disordered,
amorphous state, e.g., in low moisture foods. In amorphous food systems, the
glass
transition is the characteristic temperature range over which a stiff maternal
softens and
begins to behave in a leathery manner. This change is a temperature-, time-
(or frequency)
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and composition-dependent, material specific change in physical state, from a
"glassy"
mechanical solid to a "rubbery" viscous fluid
MCW plasticizes amorphous materials and enhances crystallization. The
plasticizing
effect of MCW gives rise to an increase in the molecular mobility that
facilitates the
arrangement of molecules and possibly enhances enzymatic reactions. The
availability of
MCW is a factor affecting rates of enzymatic reactions in amorphous food
systems. Food
materials are significantly plasticized by MCW. At increasing MCW contents,
the
materials also have higher water activities. Plasticizers are used to improve
flexibility and
workability of polymers as well as reduce viscosity.
Enzymatic reactions are often responsible for deleterious changes in low
moisture foods.
The rates of these changes may be related to changes in the physical state
such as the
glass transition. Water, in its normal and in its structured form, is the most
important
plasticizer of food materials. Water and other plasticizers also affect rates
of enzymatic
reactions. Food systems including carbohydrates, such as sugars, are very
susceptible to
crystallization even at reduced moisture level. Upon crystallization, the
sorbed water may
be expelled to the food materials changing the moisture level of the food
systems and
possibly affect rate of enzymatic reactions. Water's effect as a plasticizer
and its effects on
the rate of enzymatic reactions as a function of the texture of foods are
important factors
on maintaining quality and shelf life of low moisture food systems.
The physical state of food systems depends on the amount of water and other
plasticizers,
and the types of molecular interactions that involve all the components.
"Water binding" and "hydration" refer to the tendency of water to associate
with various
degrees of tenacity to hydrophilic or hydrophobic substances. Hydrophilic
solutes (i.e.
solutes or structures possessing hydrophilicity) interact with water with
greater or
comparable strength to water-water interactions whereas hydrophobic solutes
(i.e. solutes
or structures possessing hydrophobicity) only weakly interact with water with
strength far
less than water-water interactions.
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Methods for determining the hydration of molecular species which comprise food
and the
effects of hydration on food qualities are well known in the art (Shafiur
Rahman, Food
Properties Handbook, 1995, Culinary and Hospitality Industry Publications
Services).
Water competes for hydrogen bonding sites with intramolecular and
intermolecular
hydrogen bonding and is a major determinant of the conformation of
carbohydrates,
proteins, and lipids.
The contribution of water to protein structure
Hydration is very important for the three-dimensional structure and activity
of proteins.
Indeed, enzymes lack activity in the absence of water. In solution they
possess a
conformational flexibility, which encompasses a wide range of hydration
states, not seen
in the crystal or in non-aqueous environments. Equilibrium between these
states will
depend on the activi of the water within its microenvironment; i.e. the
freedom that the
water has to hydrate the protein. Thus, protein conformations demanding
greater hydration
are favored by more reactive water (e.g. high density water containing many
weak bent
and/or broken hydrogen bonds) and'drier' conformations are relatively favored
by lower
activity water (e.g. low-density water containing many strong intra-molecular
aqueous
hydrogen bonds).
The folding of proteins depends on the same factors as control the junction
zone formation
in some volysaccharides; i.e. the incompatibility between the low-density
water (LDW)
and the hydrophobic surface that drives such groups to form the hydrophobic
core. Tn
addition, water acts as a lubricant, so easing the necessary hydrogen bonding
changes.
Water molecules can bridge between the carbonyl oxygen atoms and amide protons
of
different peptide Links to catalyze the formation, and its reversal, of
peptide hydrogen
bonding. The internal molecular motions in proteins, necessary for biological
activity, are
very dependent on the degree of plasticizing, which is determined by the level
of
hydration. Thus internal water enables the folding of proteins and is only
expelled from
the hydrophobic central core when finally squeezed out by cooperative protein
chain
interactions. The position of the equilibrium around enzymes has been shown to
be
important fox their activity with the enzyme balanced between flexibility and
rigidity.
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Protein folding is driven by hydrophobic interactions, due to the unfavorable
entropy
decrease forming a large surface area of non-polar groups with water. In
protein
denaturation, water is critical, not only for the correct folding of proteins
but also for the
maintenance of this structure. The free energy change on folding or unfolding
is due to
the combined effects of both protein folding/unfolding and hydration changes.
Peptides and proteins play roles in foam, gels, emulsifying, flavor
precursors, flavor
compounds, and as enzymes. These properties are derived from the physico-
chemical
properties of amino acids and proteins. As described above, hydration of
proteins plays an
I 0 important role in the functionality of proteins, including binding of food
components by
proteins, gelation, swelling, production of dough, emulsifying, and foaming.
The catalytic
activity of enzymes and the regulation of enzyme reactions requires a
knowledge of
protein hydration and the aqueous microenvironment. Enzyme classes important
to food
processing include oxidoreductases, transferases, hydrolases, lyases,
isomerases, and
ligases. Water activity plays a key role in the regulation of enzyme
reactions.
Even in low moisture foods, enzymatic changes can occur despite the low water
activity.
The occurrence of these reactions reduces the storage stability of products.
Water can play
several different roles in food systems: (1) Water may act as second
substrate. It is well
known that the spatial structure of protein, which governs their functional
properties, is
stabilized by several kinds of interactions that include hydrogen bonds,
between polar
groups or between polar groups and water, and hydrophobic bonds associated
with the
structure of water around the protein molecule; (2) As disrupter of hydrogen
bond and
consequently contributing to the alteration of protein structure; (3) As a
solving medium
facilitating the diffusion of reactants; (4) As a reagent in the case of
hydrolysis reaction.
As a summary, enzyme activity depends on water-enzyme, water-substrate and
water-
matrix interactions. Also, matrix-substrate and matrix-enzyme interactions may
be
involved.
Finally, the occurrence of enzyme-catalyzed reactions in low moisture systems
requires a
certain quantity of water in order to facilitate both mobility and diffusion
of reactants.
This quantity may change according to the characteristics of the enzyme and
the
solubility and molecular size of the substrate.
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Enzymatic reactions involve the interaction of an enzyme with a substrate
where often
water is associated either as a solvent or a second substrate. The hydrolysis
of sucrose
requires that invertase is in contact with the hydrolytic bond of sucrose. If
the system is
dehydrated, the addition of water is necessary to restore the activity of the
enzyme. There
is, therefore, a requirement of mobility of the components. Water has to
diffuse through
the system, the enzyme may exert a certain mobility to reach the hydrolytic
bond, or the
substrate needs to move toward the active site of the enzyme. The rate of
enzymatic
reactions has to be dependent on the rate at which those motions take place,
which
depends in turn on the structure of the matrix of the systems. The presence of
polysaccharides in viscoelastic liquid for example has been shown to cause
entanglement
of the polysaccharide chain and restrict diffusion of water molecules.
In water restricted systems, it could be assumed that mobility would be
limited. The
activity of the enzyme would be dependent on its closeness to the substrate.
The enzyme
should, therefore, be distributed in such a way that it is available in the
vicinity of the
substrate. Poor miscibility could also lead to reduced reaction rates since it
may reduce
interactions between molecules. Composition, structure, and environmental
conditions
including moisture content, temperature, and pH, determine the physical state
and the
dynamics of the systems.
Whitaker (Principles of Enzymology for the Food Sciences,1994, 2nd ed., Marcel
Dekker,
Inc.; and Chapter 7 in Fennema, O.R. Food Chemistry, 3rd ed., 1996, Marcel
Dekker, Inc.)
elucidated the role of water on enzyme activity. Water plays at least four
important
functions in alI enzyme-catalyzed reactions: (1) folding of the protein, (2)
acting as a
transport medium for the substrate and enzyme, (3) hydration of the protein,
and (4)
ionization of prototropic groups in the active sites of the enzyme.
Nucleic acid hydration
Hydration is very important for the conformation and utility of nucleic acids.
Hydration is
greater and more strongly held around the phosphate groups, due to their
rather diffuse
electron distribution, but more ordered and more persistent around the bases
with their
more directional hydrogen-bonding ability. Because of the regular structure of
DNA,
hydrating water is held in a cooperative manner along the double helix in both
the major
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and minor grooves. The cooperative nature of this hydration aids both the
zipping
(annealing) and unzipping (unwinding) of the double helix.
Nucleic acids have a number of groups that can hydrogen bond to water, with
RNA having
a greater extent of hydration than DNA due to its extra oxygen atoms (i.e.
ribose 02') and
unpaired base sites. In DNA, the bases axe involved in hydrogen-bonded
pairing.
However even these groups, except for the hydrogen-bonded ring nitrogen atoms
(pyrimidine N3 and purine N1) are capable of one further hydrogen-bonding link
to water
within the major or minor grooves. Such solvent interactions are key to the
hydration
environment, and hence its recognition, around the nucleic acids and directly
contributes
to the DNA conformation.
Water Activity
Water activity has been an extremely useful tool in food science and
technology. It is
useful in relating to dynamics of moisture transfer and mapping of regions of
microbial
growth, physical changes and chemical reactions. Controlling water activity in
a food
processing system is critical for achieving a desired food stability, and for
predicting a
product's shelf life.
Water activity, aW, is a property of water in a material. In the mid 1970s,
water activity
came to the forefront as a major factor in understanding the control of the
deterioration of
reduced moisture and dry foods, drugs and biological systems. It was found
that the
general modes of deterioration, namely physical and physicochemical
modifications,
microbiological growth, and both aqueous and lipid phase chemical reactions,
were all
influenced by the thermodynamic availability of water as well as the total
moisture content
of the system. It is the difference in the chemical potential of water between
two systems
that results in moisture exchange and above a certain chemical potential as
related to the
aw of a system there is enough water present to result in physical and
chemical reactions.
The physical structure of a food or biological product, important from both
functional and
sensory standpoints, is often altered by changes in water activity due to
moisture gain or
loss. For example, the caking of powders is attributed to the amorphous-
crystalline state
transfer of sugars and oligosaccharides that occurs as water activity
increases above the
glass transition point. This caking interferes with the powder's ability to
dissolve or be free
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flowing and phase transitions can lead to volatile loss or oxidation of
encapsulated lipids.
The desirable crispiness of crackers, dry snack products such as potato chips,
and
breakfast cereals is lost if a moisture gain results in a water activity
elevated above a
threshold, again above the glass transition. Conversely, raisins and other
dried fruits may
harden due to the loss of water associated with decreasing water activity.
Thus, raisins or
other fruits in breakfast cereals are sugar coated to reduce the moisture loss
rate or are
modified with glycerol to reduce the water activity thereby preventing
moisture loss.
These procedures inhibit the net moisture transfer rate from the raisins to
the cereal,
therefore maintaining the cereal's crisp nature and the softness of the fruit
pieces in the
presence of a chemical potential driving, force. Finally, as aW, increases,
the permeability
of packaging films to oxygen and water vapor increases, due to swelling in the
rubbery
state.
Like physicochemical phenomena, the growth and death of microorganisms are
also
influenced by water activity. It has been repeatedly shown that each
microorganism has a
critical water activity below which growth cannot occur. For example,
Aspergillus
parasiticus does not grow below a certain water activity while the production
of aflatoxin,
a potent toxin; from the same organism is inhibited below a slightly higher
water activity.
For growth or toxin production to cease, key enzymatic reactions in the
microbial cell
must cease. Thus, the lowering of water activity inhibits these biochemical
reactions,
which in turn restricts microbial functioning as a whole. With spores, the
lower the water
activity, the more resistant they are to heat kill.
Microbially stable dry foods generally are defined as those with a water
activity below a
defined level, below which no known microbe can grow.
Water activity has been shown to influence the kinetics of many chemical
reactions.
Except for lipid oxidation reactions where the rate increases as water
activity decreases at
very low water activities, the rates of chemical reactions generally increase
with increasing
3 0 water activity.
When water interacts with solutes and surfaces, it is unavailable for other
hydration
interactions. The term 'water activity' describes the equilibrium amount of
water available
for hydration of materials; a value of unity indicates pure water whereas zero
indicates the
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total absence of water molecules. It has particular relevance in food
chemistry and
preservation.
Changes in water activity may cause water migration between food components.
Foods
containing macroscopic or microstructural aqueous pools of differing water
activity will
be prone to time and temperature dependent water migration from areas with
high water
activity to those with low water activity. a useful property used in the
salting of fish and
cheese but in other cases may have disastrous organoleptic consequences. Such
changes in
water activity may cause water migration between food components. Foods with
lower
water activity will tend to gain water, those with higher water activity tend
to lose water.
Control of water activity (rather than Water content) is very important in the
food industry
as low water activity prevents microbial growth (increasing shelf life),
causes large
changes in textural characteristics such as crispness and changes the rate of
chemical
reactions (increasing hydrophobe lipophilic reactions but reducing hydrophile
aqueous-
diffusion-limited reactions).
Free moisture has been identified in food art by the term water activity.
Water activity is
defined as the ratio of the vapor pressure of water in an enclosed chamber
containing a
food to the saturation vapor pressure of water at the same temperature. Water
activity is an
indication of the degree to which unbound water is found and, consequently, is
available
to act as a solvent or to participate in destructive chemical and
microbiological reactions.
Highly perishable foodstuffs have aw > 0.95. Growth of most bacteria is
inhibited below
about aw = 0.91; similarly most yeasts cease growing below a,~ = 0.87, and
most molds
cease growing below a,v > 0.80. The absolute limit of microbial growth is
about aw = 0.6.
As the solute concentration required to produce a,v < 0.96 is high (typically
> 1 molal), the
solutes (and surface interactions at low water content) will control the
structuring of the
water within the range where a,~ knowledge is usefully applied.
Many food preservation processes attempt to eliminate spoilage by lowering the
availability of water to microorganisms. Reducing the amount of free moisture
or unbound
water also minimizes other undesirable chemical changes, which can occur in
foods during
storage. The processes used to reduce the amount of unbound water in foods
include
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techniques such as concentration, dehydration, and freeze-drying. These
processes often
require intensive expenditure of energy and are not cost e~cient.
Control of water activity can be used successfully in achieving stability of
foods,
in prediction of moisture transfer between regimes in a mufti-component food,
for the
prediction of water vapor transfer through food packaging and the prediction
of the
final water activity of a mixture of components including dissolved species.
Molecular Mobility The molecular mobility (Mm) approach is a recent
development in
food science designed to explain how freezing and drying change the storage
stability of
foods and is an alternative and complementary method to water activity (aW)
ideas.
Most food materials do not form crystalline structures. To join in a crystal,
the molecule in
solution must slot into an existing lattice, rather like a jigsaw piece, it
can only fit in at one
orientation. Molecules rotate and flex in solution but they must be able to do
so fast
enough to form crystals before all the water leaves and movement stops. In
relatively slow
drying operations of small molecules crystals may have a chance to form: table
sugar and
salt are largely crystalline. However, large slow moving molecules or fast
drying
operations do not provide time for the crystals to grow and practically, in
most cases
crystals do not form. Instead, the solution becomes very viscous and
eventually behaves
like a rubber. If more water is removed the rubber becomes more and more
viscous until at
a critical point mobility effectively stops and the material can be considered
a glass. Both
glassy and rubbery materials are described as amorphous solids. Freezing can
be
considered a very similar process to drying. Water crystallizes as a pure ice,
which takes
no part in the solvation of the food material. As a food is frozen ice
crystals form leaving
the food in an increasingly dehydrated environment.
In each case the key parameter is molecular mobility - the capacity of the
molecules
present to move. Molecular mobility increases with temperature (the more
thermal energy
the molecules have the faster they move) and the concentration of small
molecules (almost
always water which acts as a molecular level lubricant or plasticizer). Drying
lowers the
moisture content and hence the molecular mobility of the solute. Freezing also
lowers the
water content (ice crystals form) but additionally the cooling reduces the
thermal energy of
the food molecules and therefore their mobility.
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The molecular mobility of a material is inversely related to its viscosity (if
the molecules
don't move much the liquid is thicker) and viscosity affects the rate of
diffusion limited
reactions. For a reaction between two molecules to occur, the molecules must
first collide
S and then have enough thermal energy to overcome the activation energy
barrier to
reaction.
The two technological approaches to getting food into a glassy state are
freezing and
drying. The molecular mobility approach is a novel complement to the aW method
of
understanding the role of water in food spoilage. In general molecular
mobility analysis is
better for diffusion limited reactions, frozen foods and physical changes,
they are about
equal for understanding crispness and stickiness, and aW is preferred for
dried foods and
non-diffusion limited processes. Some properties and behavioral
characteristics of food
that are dependent on molecular mobility are shown in the following table:
Table 7 Some Properties and Behavioral Characteristics of Foods That Are
Governed by
Molecular Mobility (Diffusion-Limited Changes in Products Containing Amorphous
Regions)
Dry or Semidry Foods Frozen Foods
Flow properties and stickiness Moisture migration (ice crystallization,
Crystallization and recrystalization formation of in-package ice)
Sugar bloom in chocolate Lactose crystallization ("sandiness"
in frozen
Cracking of foods during drying desserts)
Texture of dry and intermediate moistureEnzymatic activity
foods
Collapse of structure during secondaryStructural collapse of amorphous
(desorption) phase Burin,
phase of freeze-drying sublimation (primary) phase
of freeze-
Escape of volatiles encapsulated in drying
a solid,
amorphous matrix Shrinkage (partial collapse
of foam-like froze
Escape of volatiles encapsulated in desserts)
a solid,
amorphous matrix
Enzymatic activity
Maillard reaction
Gelatinization of starch
Staling of bakery products caused
by retrogradation
of starch
Cracking of baked goods during cooling
Thermal inactivation of microbial spores
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Glass Transition and Water Activiy: Physical Properties of the rubbery and_gl,
asst/ state
and food stability
Phase and state transitions. Phase transitions are changes in the state of
materials
occurring at well-defined transition temperatures- melting (solid to liquid)-
crystallization
(liquid to solid)- vaporization (liquid to gas)- condensation (gas to liquid).
A number of
materials, including foods, are noncrystalline but may exhibit properties of
solids or
liquids. Noncrystalline materials are amorphous materials, i.e., their
molecules are
arranged randomly. Amorphous materials are often supercooled liquids or
solids.
Supercooled liquids are often called "rubbers" and the solids are "glasses."
Transformation
between the supercooled liquid and solid states occurs over a temperature
range ,and the
transition is known as the "glass transition."
Glass transition is typical of inorganic and organic amorphous materials,
including such
food components as sugars and proteins. A number of material properties change
over the
glass transition temperature range.
Water Plasticisation. Water is the most important solvent, dispersion medium,
and
plasticizes in biological and food systems. Plasticization and its modulating
effect on
temperature location of the glass transition is a key technological aspect of
synthetic
polymer technology where a plasticizes is defined as a material incorporated
in a polymer
to increase the material's workability, flexibility, or extensibility. The
plasticizing effect
is usually described by the dependence of the glass transition temperature on
either the
weight, the volume, or molar fraction of water. Water plasticization can be
observed from
the decrease in the glass transition temperature with increasing water content
which may
also improve the detectability of the transition. Both carbohydrates and
proteins are
significantly plasticised by water, i.e., water acts as a softener, depressing
the glass
transition temperature. The glass transition of water, i.e., solid
noncrystalline water, is at
about -135°C. At high water contents the glass transition approaches
that of water. The
detectability of the glass transition often increases with increasing water
content-
decreasing broadness of the transition- increasing change in heat capacity
over the
transition temperature range.
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lass trahsitiohs in foods.
Understanding the glass transition and its relationships with physicochemical
changes is
very important for predicting the state and the behavior of food during
processing,
distribution, and storage.
The glass transition curve is a critical factor needed to understand physical
changes of
food. By way of example, in a cereal food processing system, it is important
to recognize
that if textural changes in a cereal system can be correlated with a glass
transition, and the
state diagram for the cereal food is known, then the processing and
environmental
conditions can be controlled such that the desired state for the food is
achieved and is also
retained during distribution and storage.
The amorphous state of nonfat food solids is typical of low moisture and
frozen foods.
Typical amorphous, glassy or rubbery foods are- dried fruits and vegetables-
extruded
snacks and breakfast cereals- hard sugar candies- free flowing powders- freeze
concentrated solids in frozen foods. The glass transition of food materials
can be observed
from a change in heat capacity, from a change in mechanical properties, and
from a
change in dielectric properties. The temperature range of the glass transition
is dependent
on the food material- low molecular weight food components, e.g., sugars, show
a clear
glass transition occurring over a temperature range of about 20°C- high
molecular weight
food components, e.g., proteins and starch, show a wide glass transition.
The glass transition temperature range is a specific property of each
material.
Carboh, due. Sugars have clear glass transitions. The glass transition
temperatures of
sugars increase with increasing molecular weight.
Proteins. Amorphous proteins are important structural biopolymers. Amorphous
proteins
are important structural components of cereal foods, e.g., gluten in bread.
The glass
transitions of proteins axe often difficult to determine calorimetricaily due
to a small
change in heat capacity and broadness of the transition.
Frozen materials. Ice formation during freezing results in freeze-
concentration of solutes.
The extent of freeze-concentration is dependent on the solutes and
temperature. At low
temperatures the freeze-concentrated solutes with unfrozen water vitrify,
i.e., the materials
contain a crystalline ice phase and a noncrystalline, glassy solute phase.
Some solutes
may crystallize, e.g., NaCI solution- freeze-concentrated sugars and foods
often vitrify.
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Maximally freeze-concentrated solutions show glass transition at an initial
concentration
dependent temperature above which ice melting has an onset temperature.
In defining the relationship between moisture content and chemical reaction
rates, polymer
sciences provides theories of glass transition and water activity to explain
the textural
properties of food systems and the changes which occur during food processing
and
storage such as stickiness, caking, softening and hardening. Food may be a
complicated
mixture of lipids, polysaccharides, sugars, proteins, etc. existing in
different phases. There
may be local differences in water content affecting the glass transition.
By way of examples, if an amorphous material exists in the glassy state, it is
hard and
brittle, e.g. for cereals it would represent a crisp product. In the rubbery
state the rriaterial
is soft and elastic, fox a fried snack or cereal this would represent an
undesirable soggy
state.
Thus glass transition theory provides a clearer approach to understanding the
physical and
texture changes of crisp cereals or snacks as water content increases. Texture
is an
important sensory attribute for many cereal based foods and the loss of
desired texture
leads to a loss in product quality and a reduction in shelf life. Saltine
crackers, popcorn,
puffed corn curls, puffed rice cakes, and potato chips lost crispness if the
water activity
exceeded a threshold. Crispness is attributed to intermolecular bonding of
starch forming
small crystalline-like regions when little water was present. These regions
require force to
break apart which gives the food a crisp texture. Above a certain water
activity, the water
was presumed to disrupt these bonds allowing the starch molecules to slip past
each other
when chewed. The crisp perception of dry cereal snacks was the result of
sounds
generated when chewed which diminished as the water activity was increased.
Loss of
crispness is well explained by the transition from the glassy to the rubbery
state.
Caking is another property that can be related to the glass transition. When a
sugar is in
solution and is dried, it is in the amorphous glassy state and the powder is
free flowing.. At
a high enough moisture or temperature, the material can enter the rubbery
state. In the
rubbery state, dried amorphous sugars tend to crystallize rapidly because of
increased
diffusion rates above a certain temperature, a condition resulting in
undesirable caking,
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which inhibits free flow. Caking follows characteristic the steps for
particles that are
wetted by water vapor.
The choice of ingredients and level of plasticizers such as water and other
small molecular
weight components influences the glass transition temperature of a food
product. In
general, as the molecular weight of a polymer increases within a homologous
series, the
glass transition temperature increases. The addition of plasticizers decreases
the glass
transition temperature.
I0 Ef_fects of Water on Diffusion in Food SXstems
A number of operations in food processing, and the stability of stored foods,
are affected
by diffusional properties of food systems, which include the foods themselves,
their
immediate environment within a package, and any barriers (packaging or
coating) used
with the foods. Water content and "water activity" affect these diffusional
properties
dramatically, by plasticizing food and/or packaging polymers and affecting
glass transition
temperatures of components, and in some cases, water may serve as an internal
transport
medium.
The 'term "additive," as used herein refers to a substance or a mixture of
substances used
primarily for purposes other than ifs nutritive value and added to a food in
relatively small
amounts to (1) impart or improve desirable properties (2) or suppress
undesirable
properties, and (3) may become a part of the food or be transitory in nature.
(Compare
ingredient below which in some instance may be an additive).
The term "basic ingredient," as used herein means a principal constituent
(except added
water) of a composition considered to be the fundamental part and by which the
composition is usually identified. Usually the basic ingredient constitutes
the major
portion of the composition, e.g., chocolate milk-milk is the basic ingredient.
in those
instances wherein a plurality of percentages of the ingredients are given that
ingredient
which constitutes 50 of the total composition (excluding added water) is
considered to be
the basic ingredient. The 50% may be determined by summing like ingredients,
e.g.,
lactose, whey and butter fat are all lacteal derived.
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"Carbohydrate" refers to a compound, the monomeric units of which contain at
least five
carbon atoms, and their reaction products wherein the carbon skeleton of
carbohydrate unit
is not destroyed. Alcohols and acids corresponding to carbohydrates, such as,
sorbitol
ascorbic acid, or mannonic acid are not considered as being carbohydrates.
The term "dry" refers to products which are as a complete product free or
relatively free
from water and under normal ambient conditions involve such characteristics,
but not
necessarily each and every one, as free flowing, dry to the touch, nontacky or
sticky,
nonadhesive, granular, powder, tablet, flake, flour, meal, particulate,
pellet, finely divided,
etc.
The term "ferment" refers to any enzyme or any living organism that is capable
of causing
or modifying a fermentation.
The term "ingredient" refers to a component part (usually a major one) of
mixture that
goes to make a food.
Ingredient or additive does not include packaging materials, containers, paper
products,
etc. or any other material which would not reasonably be regarded as being
edible.
However, in some instances, additive may be an ingredient.
"Isolated triglyceridic fat or oil" refers to fat or oil (as defined below)
that is free of any of
the plant or animal tissue from which it is derived.
"Package" refers to a mercantile combination of an edible material fully
encased,
encompassed, or completely surrounded by a solid material.
"Tissue" means material containing a certain amount of the original animal or
plant as
against an extract, which is considered to be devoid of original cellular
structure. Included
within the term are materials, which are chopped, cut, comminuted, pulverized,
milled,
slice, etc.
"Triglyceridic fat or oil" refers to esters of glycerol and a higher fatty
acid (i.e., a
monocarboxylic acid containing an unbroken chain of at least 7 carbon atoms
bonded to a
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carbonyl group) wherein the three available hydroxyl functions of the glycerol
are
esterified by a same or different fatty monocarboxylic acid. Triglycerides are
the chief
constituents of the naturally occurring fats and oils.
Included in the invention are foods or edible products which , with a focus on
water, could
be classified as follows:
Table I - Classif cation of Food Products According to a Water Content and
TXpe of
Appropriate Ph, s~-Chemical Approach
Physical State Product Examples Physico-Chemical Treatment
Dilute solutions/dispersions Drinks, soups Equilibrium thermodynamics,
refer to Henry's law
Semi-dilute Purees, jellies Polymer chemistry, chain
solution/dispersion (high entanglement, sol-gel
moisture content) transformations
Solids (high moisture) Fish, vegetable, meat, ice Biophysical chemistry,
colloid
cream science
(intermediate moisture) Preserves, sausages Materials science
(low moisture) Dried products, cereals Materials science, glasslrubber
transitions
IO
The following discussion sets forth physical-chemical principles used by those
skilled in
the art of food science for formulating edibles and ingredients.
Colloids and Rheology
Colloids are dispersions of small particles of one phase (the disperse phase)
in a second,
continuous phase. Colloids occur widely in foods. The study of colloids is
essentially the
study of the physical interactions between the surface of the particles in the
disperse phase
and between the continuous phase and the disperse phase. Rheology is the study
of
materials when deformed.
Many foods are colloidal and complex in nature with the continuous phase being
in the
form of a true solution and there being more than one disperse phase. Milk has
a
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continuous phase comprising polysaccharides, electrolytes and proteins in
aqueous
solution and disperse phases comprising both liquid fats and solid protein.
Emulsions and surface activity.
Emulsions are colloids where both disperse and continuous phases are liquid
and are the
most common type of food colloid. In the case of foods, they usually involve
an oil phase
and an aqueous phase and may be of two types:
~ oil in water (o/w) emulsions where the disperse phase is the oil
~ water in oil (w/o) emulsions where the disperse phase is the oil.
The phases in a emulsion may be exchanged by a process known as phase
inversio~z. A
common example of phase inversion in foods is butter making where cream is
converted
to butter by a process involving concentration and agitation. Once a
sufficient oil
concentration has been achieved, the agitation brings about a conversion of
the o/w
emulsion of cream to the w/o emulsion of butter. In the process, the oil
concentration is
further increased by the elimination of more aqueous phase as buttermilk. In
general
terms, the more stable form is determined by concentration.
Emulsifiers and Stabilizers
The process of forming an emulsion usually involves vigorous agitation to
break up the oil
into small droplets. Emulsion formation is assisted by the addition of
emulsifiers, which
help the break up process by reducing interfacial tension, thus these are
usually
surfactants. Common emulsifiers include detergents, glycerol mono stearate and
lecithin.
Table - Types of colloid
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Once the emulsion is formed, then it must be maintained which is the role of
stabilizers.
Emulsifiers can perform a stabilization role due to the electrostatic
interactions between
the hydrophilic pori:ion of the molecule. However this may not be enough and
stabilizers
may also needed. Stabilization may be achieved by the addition or presence of
macromolecules in the system. These may have two effects.
They may form a layer on the surface of the oil droplets which prevents the
droplets
meeting as a result of stearic hindrance. Insoluble proteins, such as casein
in milk often
perform this function.
They may dissolve in the continuous phase and increase its viscosity. In
foods, for
example, polysaccharides are often used for this purpose. Polysaccharide gums
such as
xanthan and carrageenan gums can produce substantial increases in viscosity on
addition
of small quantities as a consequence.
The breakdown of colloids involves particles coming together under the
influence of the
attractive forces and forming larger particles. There are various terms for
this process
depending on the exact nature of the process.
~ Flocculation is a Loose association of particles wluch is relatively easily
broken up
and the phases redispersed
~ Coagulation is a more strongly bound collection of particles. A Coagulated
disperse phase is not readily redispersed as inter-particle attraction is much
stronger than in flocculation.
~ Coalescence is when particles merge to form a single larger particle.
The first two definitions are somewhat loose and the two terms are sometimes
used
interchangeably. In general, flocculation occurs if there is a lowering of the
total surface
free energy as a consequence.
Coalescence
Coalescence is the combining of two particles to form a single larger
particle. The key
distinction is that flocs and coagulated particles retain a distinct identity,
but this is not the
case with coalescence.
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Coalescence is possible with both liquid and solid particles but is most
common with
liquids. The process involves a thinning of the continuous phase film between
the particles
until all the continuous phase has been expelled and the two particles merge.
Ostwald Ripening
If the disperse phase has any significant solubility in the continuous phase,
the
phenomenon called Ostwald ripening may occur. Owing to surface tension
effects, small
particles are generally more soluble than large particles. As a consequence,
large particles
tend to grow at the expense of small ones. If the process is sufficiently
rapid, the colloid
will be unstable. On the other hand control of this process is useful in
production of
photographic emulsions. In frozen foods, it can lead to deterioration during
long term
storage as the Larger ice crystals will tend to grow at the expense of the
smaller ones
leading to tissue damage.
Gels
Gels are formed when the interactions between the particles in the disperse
phase are
strong enough to form a rigid network. In such a case, the colloid behaves as
a solid and
under moderate shear stresses behaves elastically. In effect, a gel comprises
a continuous
floc filling the whole system..
In the case of gels based on macromolecules, there are regions of the
molecules where
there is attraction to other molecules - often in the form of hydrogen
bonding, or via some
form of ionic stabilization. 'The result, as in gels based on flocs, is a
three dimensional
network which behaves as if it were a solid.
Swelling of gels
The formation of the 3-D network that comprises a gel results in continuous
phase being
trapped within the gel. In many cases, the continuous phase is a solution and
the floc
network acts as a semi-permeable membrane. As a result, osmosis takes place
and the gel
will swell. The swelling tendency can be counteracted by applying an external
pressure,
the pressure required being known as the swelling pressure. This can reach
quite high
values. For example, driving wooden wedges into rock and soaking the wood can
cause a
sufficient swelling pressure to break the stone.
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Hydrocolloids are hydrophilic polymers, of vegetable, animal, microbial or
synthetic
origin, that generally contain many hydroxyl groups and may be
polyelectrolytes. They are
naturally present or added to control the functional properties of aqueous
foodstuffs. Most
important among these properties are viscosity (including thickening and
gelling) and
water binding but also significant are many others including emulsion
stabilization,
prevention of ice recrystallization and organoleptic properties.
Foodstuffs are very complex materials and this together with the
multifactorial
functionality of the hydrocolloids have resulted in several different
hydrocolloids being
required, the most important of which are: alginate, arabinoxyolan,
carragenan,
carboxymethylcellulose, cellulose, gelatin, beta-glucan, guar gum, gum arabic,
locust bean
gum, pectin, starch, xanthan gum.
Each of these hydrocolloids consists of mixtures of similar, but not
identical, molecules
and different sources, methods of preparation, thermal processing and
foodstuff
environment (e.g. salt content, pH and temperature) all affect the physical
properties they
exhibit. Descriptions of hydrocolloids often present idealized structures but
it should be
remembered that they are natural products (or derivatives) with structures
determined by
stochastic enzymic action, not laid down exactly by the genetic code. They are
made up of
mixtures of molecules with different molecular weights and no one molecule is
likely to
be conformationally identical or even structurally identical (cellulose
excepted) to any
other.
Mixtures of hydrocolloids show such a complexity of non-additive properties
that it is
only recently that these can be interpreted as a science rather than an art.
There is
enormous potential in combining the structure-function knowledge of
polysaccharides
with that of the structuring of water. The particular parameters of each
application must be
examined carefully, noting the effects required (e.g. texture, flow, bite,
water content,
stability, stickiness, cohesiveness, resilience, springiness, extensibility,
processing time,
process tolerance) and taking due regard of the type, source, grade and
structural
heterogeneity of the hydrocolloid(s).
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All hvdrocolloids interact with water, reducing its diffusion and stabilizing
its presence.
Generally neutral hydrocolloids are less soluble whereas polyelectrolytes are
more soluble.
Such water may be held specifically through direct hydrogen-bonding or the
structuring of
water or within extensive but contained inter- and intra-molecular voids.
Interactions
between hydrocolloids and water depend on hydrogen-bonding and therefore on
temperature and pressure in the same way as water cluster formation.
Similarly, there is a
reversible balance between entropy loss and enthalpy gain but the process may
be
kinetically limited and optimum networks may never be achieved. Hydrocolloids
may
exhibit a wide range of conformations in solution as the links along the
polymeric chains
can rotate relatively freely within valleys in the potential energy
landscabes. Large,
conformationally stiff hydrocolloids present essentially static surfaces
encouraging
extensive structuring in the surrounding water. Water binding affects texture
and
processing characteristics, prevents ~vneresis and may have substantial
economical
benefit. In particular, hydrocolloids can provide water for increasing the
flexibility
(plasticizing) of other food components. They can also effect ice crystal
formation and
growth so exerting a particular influence on the texture of frozen foods. Some
hydrocolloids, such as locust bean g_um and xanthan g_um, may form stronger
gels on
freeze-thaw due to kinetically irreversible changes consequent upon forced
association as
water is removed (as ice) on freezing.
As hydrocolloids can dramatically affect the flow behavior of many times their
own
weight of water, most hydrocolloids are used to increase viscosity (see rheolo
, which
is used to stabilize foodstuffs by preventing settling, phase separation, foam
collapse and
crystallization. Viscosity generally changes with concentration, temperature
and shear
strain rate in a complex manner dependent on the hydrocolloid(s) and other
materials
present. Mixtures of hydrocolloids may act synergically to increase viscosity
or
antagonistically to reduce it.
Many hydrocolloids also gel, so controlling many textural properties. Gels are
liquid-
water-containing networks showing solid-like behavior with characteristic
strength,
dependent on their concentration, and hardness and brittleness dependent on
the structure
of the hydrocolloid(s) present. Hydrocolloids display both elastic and viscous
behavior
where the elasticity occurs when the entangled polymers are unable to
disentangle in time
to allow flow. Mixtures of hydrocolloids may act synergistically, associating
to
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precipitate, gel or form incompatible biphasic s, s~-terns; such phase
confinement affecting
both viscosity and elasticity. Hydrocolloids are extremely versatile and they
are used for
many other purposes including (a) production of ~seudoplasticitv (i.e.
fluidity under shear)
at high temperatures to ease mixing and processing followed by thickening on
cooling, (b)
liquefaction on heating followed by gelling on cooling, (c) gelling on heating
to hold the
structure together (thermogelling), (d) production and stabilization of
multiphase systems
including films.
These properties of hydrocolloids are due to their structural characteristics
and the way
they interact with water. For example:
~ Hydrocolloids gel when intra- or inter-molecular hydrogen-bonding (and
sometimes salt formation) is favored over hydrogen bonding (and sometimes
ionic
interactions) to water to a sufficient extent to overcome the entropic cost.
Often the
hydrocolloids exhibit a delicate balance between hydrophobicity and
hydrophilicity. Extended hydrocolloids tend to tangle at higher concentrations
and
similar molecules may be able to wrap around each (forming helical junction
zones) other without loss of hydrogen bonding but reducing conformational
heterogeneity and minimizing hydrophobic surface contact with water so
releasing
it for more energetically favorable use elsewhere. Under such circumstances a
minimum number of links may need to be formed (i.e. a junction zone which, if
helical, generally requires a complete helix) to overcome the entropy effect
and
form a stable link. Where junction zones grow slowly with time, the
interactions
eliminate water and syneresis may occur (as in some jam and jelly).
~ Polysaccharide hydrocolloids stabilize emulsions primarily by increasing the
viscosity but may also act as emulsifiers, where their emulsification ability
is
reported as mainly being due to accompanying (contaminating or intrinsic)
protein
moieties. In particular, electrostatic interaction between ionic hydrocolloids
and
proteins may give rise to marked emulsification ability with considerable
stability
so long as the appropriate pH and ionic strength regime is continued.
Denaturation
of the protein is likely to lead to improved emulsification ability and
stability.
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~ Mixtures of hydrocolloids may avoid self aggregation at high concentration
due to
structural heterogeneity, which discourages crystallization but encourages
solubility. Hydrocolloids may interact with other food components such as
aiding
the emulsification of fats, stabilizing milk protein micelles or affecting the
stickiness of gluten.
~ The particle size of hydrocolloids and its distribution are important
parameters
concerning the rate of hydration and emulsification ability.
~ Negatively charged hydrocolloids change their structural characteristics
with
counter-ion type and concentration (including pH and ionic strength effects);
e.g. at
high acidity the charges disappear and the molecules become less extended.
~ Physical characteristics may be controlled by thermodynamics or kinetics
(and
hence processing history and environment) dependent on concentration. In
particular these may change with time in an monotonic or oscillatory manner.
~ Different hydrocolloids prefer low-density or higher densi water and other
hydrocolloids show compatibility with both. As more intra-molecular hydrogen-
bonds form so the hydrocolloids become more hydrophobic and this may change
the local structuring of the water. Mixed hydrocolloids preferring different
environments produce 'excluded volume' effects on each others effective
concentration and hence rheolo~v.
~ In the glassy state, conformational changes are severely inhibited, but the
water
held by hydrocolloids may act as plasticizer (allowing molecular motion)
greatly
reducing the glass transition temperature by breaking inter-molecular hydrogen-
bonding.
Gums and Starches' Controllinø Moisture Behavior
Understanding the mechanics of water's interactions within foods and how to
apply
polysaccharides such as gums and starches to control these interactions allows
designers to
take steps to improve product quality and extend shelf life.
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A classic example of this is dough for baked products. Here, water not only is
the solvent
that activates chemical and/or yeast leaveners, but is a processing aid
allowing the gluten
development that leads to the formation of a mixable, cohesive mass (dough)
that
subsequently can be formed and baked. The starches and gums themselves are
polymeric
ingredients that require activation by water as a plasticizer.
Gums and starches are polysaccharides consisting of a straight molecular
chain. Gums
have a functional group on one end of this chain and starches have various
branches on the
chain. The exact configuration varies depending on the material's source
In unmodified forms, both absorb water, swell in solution and act as mild
viscosifiers.
When activated by heat and/or mechanical action, gum and starch particles both
reorganize. Here is where the two begin to behave differently. Hydrated gums
molecules
have an affinity for one another and will gel. Starches, on the other hand,
continue to act
as individual molecules with an increased thickening capability. Various gums
and
starches behave in different ways and modifications of the basic material make
even more
variations possible (i.e. pregelatinized starch and cold-swelling gums.)
Flavor Components.
Water activity represents an important variable that influences the rate of
many chemical
reactions of flavor compounds. In complex aqueous systems, the way a food
matrix is
structured is of great importance to flavor release and flavor perception.
In aqueous food systems polysaccharides and proteins are generally the major
components
determining the structure of food products. Hydration of these macromolecular
components is of primary importance in order to follow up the consequences
when other
smaller molecules, such as aroma compounds, are present. The way these
volatile
compounds are trapped in food systems will determine flavor release and thus,
flavor
perception and the appearance of a product to the consumer.
Physico-chemical reactions involving flavor components -- whether between
flavors, or
between flavors and nonflavor components of food and the environment -- are
loosely
termed "flavor interactions." These interactions influence the quality,
quantity, stability
and the ultimate perception of flavor in food. Flavor is primarily a
combination of taste
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and odor, and along with appearance and texture, comprises the criteria for
sensory
acceptance of foods.
The term "artificial flavors" refers to those flavors that are added to foods,
or consisting of
compounds not existing in nature. Naturally occurring flavors, or those formed
by heating,
aging or fermentation, are considered "natural flavors." Naturally occurring
flavors that
are synthesized for addition to foods take on the label "nature-identical"
flavors.
Fruit flavors are formulated and compounded for specific applications. The
goal of the
product designer is to select flavors that perform optimally~within the
context of a
chemically reactive food product. Successfully achieving this goal requires
knowledge of
flavor interactions.
Physical and chemical flavor interactions occur continuously during food
growing,
harvesting, processing, storage and consumption. Interactions can be
attributed to various
types of chemical bonding: covalent bonding, hydrogen bonding, hydrophobic
bonding,
and the formation of inclusion complexes. The most commonly measured physical
aspects
of flavor interactions are binding, partitioning and release. Binding refers
to the absorption
of volatile and nonvolatile components of flavor onto the constituents of the
food matrix.
Partitioning describes the distribution of flavors in the aqueous, lipid or
gas phases
associated with the foodstuff and the package. The point at which flavor is
made available
to human sensory receptors is termed "release." Optimizing the time for flavor
release is
product-dependent, since longer times are needed for foods that are well-
chewed than for
drinks that spend only a few seconds in the mouth.
Flavors partition themselves between the oil and water phases differentially,
based on the
chemical structure of the flavor and the chain length of the fatty acids
present. In foods in
which fat has been reduced, the flavor release is affected by this
partitioning, since
flavorants in aqueous systems possess a higher equilibrium vapor pressure than
lipid
systems. Volatiles release more quickly from aqueous systems, and dissipate,
resulting in
less of a flavor impression on the human sensory organs.
Proteins possess little flavor of their own, but they bind several volatile
flavor components
particularly well in the presence of heat denaturation. Binding, due to
hydrophobic
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interactions and hydrogen-bonding, is reversible, as in the case of ketones,
hydrocarbons
and alcohol-based flavors. Covalent binding, such as Schiff base formation
(aldehydes and
amino groups), often is irreversible. Some of the factors influencing protein
binding to
volatiles are: temperature, pH, concentration and water presence. Proteins may
bind more
~ or less of a flavor component, depending on length and extent of heat
treatment. In dairy
proteins, several flavor components, such as a vanillin, benzaldehyde and d-
limonene,
were reduced by as much as 50% in solutions containing whey proteins or sodium
caseinate. Protein-flavor binding can reduce the impact of desirable flavors
and carry
undesirable flavors to sensory receptors. The most widely studied, documented
protein-
flavor interaction is the binding of off flavors to soy proteins.
Carbohydrates serve several important flavor-enhancement functions. Ranging in
size
from small to large, they function as sweeteners; browning-reaction
participants; fat
replacers; viscosity builders; and flavor encapsulators. Sugars serve as
carriers for flavors
by physical interaction in aqueous systems, and by chemical-binding in dry
ingredients.
Structures of larger carbohydrate molecules, such as starch and cyclodextrins,
can form
hydrophobic regions that serve as inclusion mechanisms for flavor compounds of
a like,
hydrophobic chemistry: The flavor molecules that fit-into these hydrophobic
regions are
called "guest molecules." These interactions are highly reversible, since no
other chemical
reaction takes place between the starch and the guest, other than the
hydrophobic
attraction. This interaction forms the basis for the molecular encapsulation
of flavors.
Polysaccharides, particularly hydrocolloids and gelling agents, bind flavor
components to
varying degrees. When the concentration of flavors is held constant -- and the
level of
polysaccharides increases -- perception of aroma and taste decreases, as a
result of
viscosity. The sweetness of sucrose, for example, is decreased when the
viscosity of a
solution of guar gum or carboxymethylcellulose is increased.
Carbohydrates also alter the volatility of aroma compounds. When compared to
flavor
compounds in a wafer solution, the addition of mono- and disaccharides
increases
volatility, and the addition of polysaccharides decreases volatility. The
effect of
carbohydrates on volatility is particularly important in food systems that use
fat replacers,
since volatiles are released at a faster rate when lipid content is low, due
to the weaker
interactions of carbohydrates with hydrophobic flavor compounds.
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Food matrices often are composed of proteins, carbohydrates and lipids, so
interactions
with flavors often occur between two or more components. The Maillard reaction
(also
known as nonenzymatic browning), in which reducing sugars react with amino
acids to
produce aromatic volatiles and browning products, is responsible for the
flavors formed
during thermal treatment of foods, such as chocolate, coffee, roasted meats,
bakery items
and caramel. The number and type of flavors produced by these reactions
depends on the
quantity and type of amino acids available to participate in the reaction
mixture. In
combination with lipid oxidation reactions, the Maillard reaction generates
flavor
compounds when carbonyl compounds (from degradation of sugar or lipids) react
with
amines or thiols during heating. Flavor reactions within a complex food matrix
seldom
occur in isolation, and are affected by the reactants, the intermediates and
the products of
other reactions.
Flavors and packaging interact as a result of three factors: migration of
packaging or food
components; permeation of the package by gas, water and organic vapors; and
exposure to
Light.
Protecting flavors from interactions that diminish or degrade them involves
minimizing
processing influences (heat, pH); environmental factors (evaporation, oxygen);
and
chemical interactions with the food matrix.
Flavor perception is related to the way aroma is released (or inversely
retained) from food
systems. Flavor release depends on the nature and concentration of flavor
compounds
present in the food, as well as on their availability for perception as a
result of interactions
between the major components and the flavor compounds in the food. Food
compositional
and structural factors, e.g. as a result of the presence of macromolecules,
and eating
behaviour determine perception and the extent of flavor release. Knowledge of
binding
behaviour of flavor compounds in relation to the major food components, their
rates of
partitioning between different phases, and the structural organization of food
matrices is of
great practical importance for the flavoring of foods, in determining the
relative retention
of flavors during processing or the selective release of specific compounds
during
processing, storage and mastication.
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The major mechanisms likely to occur in flavor release, are (i) specific
binding of aroma
molecules and (ii) entrapment of these molecules within a matrix. Specific
binding can
occur for some aroma molecules with proteins or with amylose. Additionally,
proteins
and polysaccharides affect the kinetics of aroma release as they influence the
transport of
aroma through the food into the air phase. Therefore, in complex aqueous
systems, the
way a food matrix is structured is of great importance to flavor release and
flavor
perception.
Different mechanisms controlling flavor release are likely to occur in food
systems.
Diffusion phenomena influenced by the viscosity of the system, unspecific
binding or
specific bindings to one of the macromolecular components are possibilities
for the
interactions of flavor molecules within the food matrix.
9VERVIEW OF FOOD PROCESSING
Food processing is an umbrella term, which describes all the activities of
manufacturing
food and beverages for human consumption, as well as prepared feeds for
animals. The
industry is defined as food and kindred products by Standard Industrial
Classification
(SIC) 20.
Food processing tends to break down the inherent structures within food
materials or
ingredients to a varying extent, and is therefore concerned with all aspects
of food -- the
chemical and physical properties of food and its constituents, the processing
and
production of food, and the packaging and marketing of food, which represent
components
of a food processing system. Food quality - texture, flavor release, nutrient
availability,
moisture migration, and microbial growth -- are influenced and determined by
the
formation, stability and breakdown of structures within foods.
Food processing involves conversion of raw materials and ingredients into a
consumer
food or edible product. Food processing includes any action that changes or
converts raw
plant or animal materials into safe, edible, and more palatable foodstuffs.
Improvement of
storage or shelf life is another goal of food processing.
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The purpose of food processing is to produce foods that between them provide
constituents of a balanced diet, are free from contamination, are appealing in
color, taste
and texture.
Food processing also drives an array of flavor chemistry reactions and the
perception of
flavor also depends on how the flavorful compounds are released during eating.
The
relationships between the structural, mechanical and physicochemical
properties of the
food and the perception of flavor and the formation of flavor compounds during
processing is dependent in part upon water hydration.
Food processing operations involve one or more of ambient temperature
processing,
mechanical processing, high temperature processing, low temperature
processing,
fermentation processing, and various post processing steps.
Ambient temperature processes include cleaning and sorting, peeling;
shredding, chopping
and milling; mixing, blending and forming. These often are preparation for
subsequent
operations.
Physical Separations include filtration, centrifuging; expression and
extraction; membrane
separations. These often involve recovering a particular component from a raw
material.
High temperature processes have two major purposes: Safety through
pasteurization and
sterilization; cooking, which modifies flavor, texture, nutritional qualities.
A single
process may serve both functions simultaneously. High temperature processes
include
sterilization and pasteurization; blanching; baking and roasting; frying;
microwave and
infra-red heating.
The purpose of blanching is as a pretreatment for dehydration, sterilization,
freezing. Heat
is sufficient to inactivate enzymes but not to cook but under processing is as
bad as over
processing.
Baking and Roasting are essentially the same process involving dry heating in
hot air.
Baking usually refers to dough products. Roasting usually refers to meat, nuts
and
vegetables. The surface of the treated substance undergoes chemical changes
developing
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color and flavor. The heat has nutritional effects in that the food easier to
eat and digest,
but there may be a loss of vitamins.
Frying is cooking in hot oil. Its purpose is to improve eating quality of the
food (flavor,
texture). Effects of frying are similar to those of baking. Because of direct
contact
between hot oil and food, fxying is generally quicker than roasting or baking.
Microwave and infra red heating use electromagnetic radiation for heating.
Microwave
heating involves short wavelength radiation. The frequency of the waves
coincides with
the natural vibration frequency of water molecules. Infra red is radiation
just beyond the
visible light region of the spectrum. The energy is dependant on temperature,
surface
properties, shape of the bodies.
Processing at low temperatures involves slowing the rate of microbial growth,
but does not
kill microbes. LJp to a point, the lower the temperature, the longer the shelf
life. Below -
I O oC, all microbial growth stops, but some residual enzyme activity may
remain. The
main function of chilling and freezing, therefore, is for storage and
prolonged shelf life.
Fermentation serves a number of purposes, including preservation, improving
nutritional
quality, improving digestibility, health benefits. There is a wide variety of
fermented
foods including dairy products, fermented meat and vegetables, beverages,
bread, etc.
Post processing operations include packaging and storage. The purposes of
these
operations include protection, display, increase storage life. Increasingly
modified
atmospheres are being used to increase shelf life, often by reducing oxygen
and increasing
nitrogen content.
Packaaing_Materials
Main packaging materials include metals, paper and board, glass, and polymers.
The
metals most widely used with foods are steel (usually found in the form of
tinplate
involved in canning), and aluminum used for three major food applications,
e.g. beverage
cans, foil containers, aerosol cans.
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Can manufacture
Cans are produced in two major forms. Three piece with rolled and soldered
side seams
and two separate end enclosures. Two piece in which sides and one end are
formed from
flat sheet and are seamless. The ends are sealed by a double seal which is
purely
mechanical. The interior of cans is usually coated with a suitable "enamel" to
protect
against tainting the food.
Paper and board paper
Various grades of paper are used. Draft paper is a strong paper often used for
paper sacks.
Vegetable parchment is a paper specially treated with acid to give it a
closer, smoother
texture. Sulphite paper is a lighter, weaker paper than kraft paper - often
used as paper
bags and sweet wrappers. Greaseproof paper is produced from sulphite pulp
where the
paper fibers are more thoroughly beaten to give a closer texture. It is
resistant to oil and
grease. Tissue is a soft resilient paper used for protection.
Aseptic packaging
Aseptic packaging is a process where the food is sterilized then filled into
sterile
containers under sterilized conditions which will prevent recontamination. It
differs from
in-pack sterilization in that the containers and food are sterilized
separately.
Aseptic processing
The shorter processing times possible mean the food is less processed leading
to less
destruction of vitamins and loss processed of flavors. Because the packaging
does not
have to be heated, a wider range of packaging is available. However, care must
be taken
to ensure sterility during the packaging operation packaging. Aseptic
processing permits
longer shelf life at normal temperatures with higher quality products.
Polymers for food packaging
Polymers are macromolecules based on a repeating unit derived from a small
molecule.
They may be natural - e.g. polysaccharides or synthetic. They possess a
variety of
properties useful to food packaging. Examples of polymers include
polyethylene, LDPE,
HDPE, polypropylene, polystyrene, olyvinyl chloride (PVC), polyethylene,
terephthallate
(PET), polycarbonate, polyamide (nylon), cellulose (cellulose acetate,
cellophane).
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Polymers may be classified as thermoplastic, which melts on heating; or
thermosetting,
which decomposes on heating.
UNIT OPERATIONS 1N FOOD
Evaporation
Evaporation is a process of concentrating a liquid by heating to evaporate the
water.
Evaporation may be used in foods for a number of purposes:
~ To pre-concentrate the food prior to some other process, usually drying or
to
reduce
transport costs
~ To improve the preservation qualities by reducing water activity eg. j am-
making.
~ To produce a product in its own right e.g. evaporated milk, fruit drinks.
Heat for evaporation is usually provided by condensing steam. Hence the
process involves
transferring latent heat from the steam to the evaporated water. Tt is usual
in food
evaporation, to carry out the evaporation under vacuum. This reduces the
boiling
temperature of the liquid and hence reduces thermal damage to the food. For
this reason,
short residence times in the evaporator are desirable. The most common types
of
evaporator are the thin film type where the liquid is spread in a thin film
over the inner
surface of a set of tubes, the steam being supplied to the outside of the
tubes. There are
two types of thin film evaporator, climbing film and falling film. Where a
high degree of
concentration is required, then multiple effect evaporation is employed. This
involves
carrying out the evaporation in a series of stages with the vapor generated in
one stage
being used as the heating steam for the next stage. This results in a
considerable degree of
steam economy.
Drying
Drying or dehydration of foods involves removing the water from a food to
reduce the
moisture content to a very low level (usually below 5% wt). The purpose of
drying foods
is to extend the storage life by reducing the water activity to practically
zero, thus
inhibiting microbial growth and enzyme activity. The normal processes of
drying involve
applying heat to the food and the drying process often results in irreversible
changes to the
food, such as non-enzymic browning and, vitamin degradation protein
denaturation.
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Unless carried out under carefully controlled conditions, drying can have a
significant
negative impact on the nutritional value of the food.
The drying process
Drying is normally carried out by heating the solid in air so that the water
evaporates into
the air. The drying process may be followed via a graph of moisture content vs
time. The
moisture content will eventually fall to a constant value. This is known as
the equilibrium
moisture content.
Drying mechanisms
Constant rate drying occurs when the solid material is completely covered with
a layer of
water.
Drying occurs by evaporation from the surface of the water layer and the rate
is governed
purely by the temperature and moisture content of the drying air. When
sufficient water
has evaporated so that a layer of water no longer covers the surface of the
solid, water has
to migrate from the interior of the solid by diffusion before it can evaporate
from the
surface of the solid. Under these circumstances, as the water content of the
interior falls,
the rate of diffusion to the surface falls and, hence the rate of evaporation
falls.
Drying rates and times
In the constant rate period, the drying rate is governed by surface
evaporation which is
effectively a function of the rate of heat transfer to the surface of the wet
solid.
Extraction
Solid-liquid extraction or leaching is a process of separating two solids by
contacting the
solid mixture with a solvent in which one solid is soluble and the other is
insoluble. This
process is widely used for recovering vegetable oils and also for instant tea
and coffee and
decaffeination of coffee. Extraction may be carried out batchwise or
continuously. The
most common way is using continuous countercurrent extraction in a manner
similar to
solvent extraction and adsorption.
FOOD ADDITIVES AND FOOD STRUCTURE
Important in making the food palatable and even attractive, these "minor"
additive
constituents of food often have little nutritional value. While they may be
present
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naturally in food, they are often added to the food to ensure control and
consistency of
properties. Additives affect foods' rheology and texture, colloidal
properties, colors,
including browning of foods, and flavorings
Food additives are often considered to be any substance not normally consumed
as a food
by itself and not normally consumed as a typical ingredient of a food.
Additives are
incorporated into foods so as to modify the properties (including the
processing properties)
of the food in some way. A distinction should be made between food additives
and food
contaminants.
A contaminant is an undesirable substance present in the food, which it is not
feasible to
completely remove (either for technical or economic reasons). An additive, on
the other
hand, is a substance, which is added deliberately for some specific purpose.
Food additives serve the following purposes:
1. Maintenance of the nutritional quality of food.
2. Enhancement of the keeping quality or stability of foods leading in a
reduction of
losses.
3. Making foods attractive to the consumer in a way that does not lead to
deception.
4. Providing essential aids in food processing.
It is also known in the art to use additives unethically to deceive the
consumer and to
disguise the use of poor ingredients or faulty processing and handling
techniques.
The major categories of food additives include
E number ' = Type of additive
Elxx Colors
... .. ,~"~W- ........ ~;,~
........ ........ .
E2xx "
Preservatives
E3xx Antioxidants, Emulsif ers, Stabilizers
and Thickeners
E4xx Sweeteners
ESxx Mineral Salts
E6xx Flavor Enhancers
E9xx Waxes and glazing agents
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Natural and Synthetic Additives
An additive can be called natural if it is actually isolated from a plant or
animal source
(using those terms broadly) or occurs in a plant or animal extract. If an
additive is
identical chemically to a compound occurring in nature but has actually been
chemically
synthesized, it referred to as mature identical. A synthetic additive is one
which does not
occur in nature and must be produced synthetically, such as a fermentation
process or by
other biotechnological methods.
The invention includes the following subject matter, described in United
States Class 426
of the Manual of Patent Classification. The categories, definitions, and
examples set forth
therein are to be interpreted according to the class definitions (and lines
with related
compound, process, and product classes) and patentable subject matter
classified therein as
set forth in United States Class 426 of the Manual of Patent Classification,
which is
hereby incorporated by reference.
A. Structured lMicroclusteredl Edible Products Or Compositions
1. Products or compositions which historically have been considered to be a
food, and
products or compositions which contain a naturally occurring material (i.e.,
plant or
animal tissue) which has been historically regarded as a food; e.g., milk,
cheese, apples,
bread, dough, bacon, whiskey, etc.).
2. Products or compositions which are known to have or are disclosed as having
nutritional effect.
3. Products or compositions which are closed or claimed as being edible or
which; perfect,
modify, treat, or are used in conjunction with an edible such as (1) or (2)
above or with
another edible, so as to become part of the edible composition or product, or
which
converts a nonedible to an edible form.
4. Mixtures of enzymes which are edible, per se, or which are used in
preparing a product
or composition proper for food or edible material.
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5. Products or compositions involved in foods or in compositions for making
foods which
contain a live micro-organism which enhances or perfects the digestive action
of the
intestinal tract, e.g., Bacillus acidophilus milk, etc.
6. Edible products or compositions which have structural characteristics.
7. Plural inorganic elements or minerals for fortification.
8. Edible bait.
B. Edible Food Products In Combination With Nonfood Materials Which Are
Generallw
1. Products or compositions of A above in combination with a package
structure, inedible
casing, a liner or base, an infusion bag, etc.
2. Compounds which have the same function as in (A. 1-3) in combination with
an
inedible material.
3. Potable water in a package.
4. Chewing gum and chewing gum bases, per se.
C. Flavoring And Sweetening Compositions
1. Flavoring compositions wherein at least one of the ingredients is not a
carbohydrate
type material.
2. Sweetening compositions wherein at least one of the ingredients is a
noncarbohydrate
type material.
D. Processes Of Administering The Products Or Composition Of A-C Above To An
Animal Via The Oral Cavitv.
F. Processes Of Administering A Compound Having The Same Function As The
Compositions Or Products Of A-C Above To An Animal Via The Oral Cavity,
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G. Processes Of Treating,Live Animals With A Product Compound. Or Ferment That
Perfects The Food Made From Said Animal In Combination With A Butchering
OperationYOr Processes Of Removing A Food Product From A Live Animal Followed
Bv
A_ Treatment Of The Removed Food Or A Butchering Operation Followed Bv An
eration.
H. Processes Of Prenarin T~ reatin_g Or Perfecting The Products Or
Compositions Of A-C.
I. Single Use Infusion Containers Or Receptacles Which Are Specific For
Preparing A
Food And Which Are Devoid Of Structure Which Specifically Cooperates With A
Food
Apparatus.
J. Compositions And Methods Of Use For Treating~,~ Or Perfecting A Food
Material.
Readers of skill in the art to which this invention pertains will understand
that the
foregoing description of the details of preferred embodiments is not to be
construed in any
manner as to limit the invention. Such readers will understand that other
embodiments
may be made which fall within the scope of the invention, which is defined by
the
following claims and their legal equivalents.
