Note: Descriptions are shown in the official language in which they were submitted.
~ W094/02029 Z~8~ PCT/~S93/06500
GLUCOMANNAN SPONGEOUS MATRICES
This invention relates to articles of manufacture
comprising spongeous matrices formed from coprocessed
mixtures of a glucomannan, especially konjac derived
glucomannan, and at least one other hydrocolloid;
processes for fabrication of the spongeous matrices; and
uses for the spongeous matrices.
Gels prepared from aqueous sols of konjac
glucomannan are known in the art. Japanese published
patent application 59-146560 discloses a spongy, porous
konjac glucomannan as a food additive. Japanese
published patent application 60-83564 discloses a water-
resistant food wrap containing konjac which "becomes a
water-resistant gel upon freezing." The thawed gel is
then dried to produce the food wrap. Similarly,
Japanese published patent application 60-141250
discloses the production of konjac which is water
insoluble and which does not absorb water, by drying or
freezing an alkaline sol or paste of konjac. Japanese
published patent application 59-227267 relates to a
method of freezing konjac to produce a product stable at
a pH of 10.0 to 12.2. See also Japanese published
patent application 59-227267 which discloses a method of
freezing, drying or dehydrating konjac to produce a
water insoluble or non-plastic product.
Gels containing agar or agarose are also known to
the art. U.S. patent 4,755,377 discloses aerated agar
gels formed by mixing gaseous components into the sol
~ before gelation. Gels containing agarose are commercial
products and are the subject of various patents.
Also known in the art are multi-component gels
containing konjac and one or more other components such
as starch (Japanese published patent application 62-
259550), alginic acid or starch with xanthan or
W094/02029 t ' ' PCT/US93/06500
galactomannan (Japanese published patent application 01-
166378), locust bean gum, plant protein and millet jelly
(Japanese published patent application 63-79S72), a
milk-type material (Japanese published patent
application 62-107751), alginic acid and propylene
glycol, starch and sodium glycolate, and starch and a
sodium stearate ester (Japanese published patent
applications 62-272952 and 62-195264), frozen bean curd
(Japanese published patent application 62-118859), and
various carrageenans (U.S. patent 4,427,704).
In addition, gels made from certain derivatives of
konjac (with or without other components) are also known
(Japanese patents 01-160467, 01-160468 and 02-150402).
In a first article embodiment, this invention
comprises an article of manufacture which is a spongeous
matrix, whose pores may be controlled as to size and/or
distribu~ion, when desired. The article of manufacture
is formed from a coprocessed mixture of (a) konjac
20 glucomannan and (b) at least one other aqueous gel-
forming polysaccharide.
Component (a) can be any glucomannan, of which
glucomannan derived from konjac is particularly
preferred. The konjac glucomannan is not limited as to
25 source and may be obtained from crude konjac flour, or
from purified or clarified konjac flour, or from konjac
glucomannan which has been chemically or physically
derivatized or modified.
Component (b) comprises a polysaccharide other than
a glucomannan which is capable of forming an aqueous
gel. Component (b) preferably comprises: agar,
agaroids, agarose, algin, alginates, carrageenan,
curdlan, gellan, a gel-forming chemical or physical
derivative of any of the foregoing, or a mixture
thereof. Component (b) more preferably comprises agar,
agarose, ~appa-carrageenan, a gel-formimg chemical
W094/02029 ~ ~4~ PCT/US93/06500
derivative or physical modification of any of the
foregoing, or a mixture thereof. Most pre~erably,
component (b) is agar, agarose, kappa-carrageenan, or a
mixture thereof.
Components (a) and (b) are present in a ratio [a:b]
based on their respective dry weights, of 1:0.125-8.0,
preferably 1:0.25-4.0, more preferably 1:0.5-2.
In a second article embodiment the article of
manufacture of this invention may be formed including an
additional component (c) which comprises one or more
water soluble polysaccharides other than (a) or (b), and
which need not form an aqueous gel. Component (c)
preferably comprises: guar gum, gum arabic, karaya gum,
locust bean gum, starch, tragacanth, or a mixture
thereof; more preferably: guar gum, locust bean gum,
starch, or a mixture thereof; and most preferably
starch.
Component (c) may be present in up to 200.0 % by dry
weight, preferably 0.5 to 200.0 % by dry weight compared
to the combined dry weight of (a) and (b). When
component (c) is starch, it preferably is present in 5.0
to 100.0 %, more preferably 20.0 to 75.0 % all by dry
weight compared to the combined dry weight of (a) and
(b).
In a third article embodiment, the spongeous matrix
may have a coating. The coating may be comprised of one
or more of the materials suitable for forming the
spongeous matrix itself, in which instance the coating
is in a non-spongeous form. Alternatively, the coating
may comprise a material other than that used for forming
the spongeous matrix. The coating may be a dried water-
soluble substance that otherwise would be added to the
matrix during use, for example a nutrient medium, buffer
salt, catalyst, reagent, or the like; to which water
would be added to activate or reactivate. The coating
may be water insoluble or soluble and independently may
~4~4
W094/02029 PCT/US93/06500 ~
. . .
be water permeable or impermeable, all depending upon
the use for which the inventive article is intended and
the environment in which the inventive article is
placed. Virtually all known water insoluble and/or
5 impermeable compositions such as silicone, resins, oils,
varnishes, and the like can be employed, depending on
the compatibility of the coating composition with the
intended use of the spongeous matrix. The coatings
themselves do not constitute a part of this invention
lO except when combined with the inventive spongeous
matrix.
In a fourth article embodiment, the inventive
spongeous matrix can be at least partially saturated
with a carrier medium. The preferred carrier is water,
15 although other liquids that do not adversely affect the
spongeous matrix for its intended purpose also may be
used. The substance carried may be medium soluble or
dispersible or may be medium borne particulate matter.
Depending upon the intended use of the inventive
20 spongeous matrix, the carrier medium may remain therein,
J or may be partially or completely removed. Examples of
carried substances include, but are not limited to: cell
growth or sustenance nutrient mediums, reagents,
pharmaceuticals, flavorings, colors, scents, cosmetics,
25 air fresheners, deodorants, adjuvants to any of the
foregoing, affinity or ion-exchange particulate, living
or dead cells or cellular matter, activated charcoal, or
mixtures thereof. This invention includes all
combinations of the inventive spongeous matrices with
30 these carried substances, although the substances are
themselves known.
In a fifth article embodiment, the inventive article
comprises the spongeous matrix of the above embodiments
in substantially dry form. For the purposes of this
35 embodiment "substantially dry" is defined as having no
readily expressible water, or as generally 20 % or less
_ W094/02029 - PCT/US93/06500
-- 5
by weight of water based on the total weight of the
spongeous matrix. The percentage weight of water will
vary depending upon the ambient atmospheric humidity.
Another group of embodiments of this invention are
processes for fabricating the above articles of
manufacture.
The first embodiment for fabricating articles of
manufacture according to this invention comprises the
sequential steps of:
A forming an aqueous sol of above ingredients (a),
(b), [and optionally (c)] present in the above
part by dry weight ratios;
B forming a gel by treatment of the sol with a
base of a strength and in a quantity sufficient
to make the sol of basic pH; [in a known but
less preferred gel-forming variation, when the
sol is retorted the pH can be acid, as low as
6.0, for example it can be 6.66 using a
phosphate buffer]i
C freezing the formed gel; and
D thawing the frozen gel, resulting in the
inventive spongeous matrix.
In step A the respective concentrations in water of (a)
25 and (b), are independently of each other 0.25 to 2.0 wt
%, preferably 0.5 to 2.0 wt %, more preferably 0.5 to
l.O wt %, all based upon the total weight of the sol.
The formation of the sol in step A preferably may be
accompanied by heating, although this is not required.
It is important that during freezing the cooling
gradient of the gel is relatively uniform and in
particular that local areas of greater cooling intensity
are avoided. A local "cold spot" during freezing may
prevent the production of a satisfactory spongeous
matrix by producing a local zone of extremely small
pores whose appearance is similar to a gel.
W094/02029 ~4~ PCT/US93/06500
Another factor affecting the nature of the spongeous
matrices is their rate of freezing during formation. It
has been observed that for a given component mixture and
ratio, a faster freezing rate usually results in smaller
pores and conversely that a slower freezing rate usually
results in larger pores. This observation fits well
with the above cold spot observation, since such cold
spots would be expected to produce faster local freezing
rates.
The second fabrication embodiment according to this
invention comprises including in step A of the first
embodiment the water soluble polysaccharide identified
above as component (c), present in the above dry weight
percentages relative to (a) and (b) combined.
The third fabrication embodiment according to this
invention comprises including in any of the other
embodiments an optional step E of the process, in which
at least a portion, preferably substantially all, of the
water content of the spongeous matrix is removed.
The fourth fabrication embodiment according to this
invention comprises sterilizing the spongeous matrix of
any of the foregoing processes, by any known method such
as gamma radiation, microwave exposure, or preferably
heating.
The fifth fabrication embodiment according to this
invention comprises using potassium carbonate as the
base used to form the gel in step B.
The sixth fabrication embodiment according to this
invention comprises adding sufficient base in step B to
result in a sol pH of 9.0 to 12Ø
The seventh fabrication embodiment according to this
invention comprises coating the inventive spongeous
matrix to form the product described in the third
article embodiment, by applying a coating comprising a
substance other than that of the spongeous matrix
itself. The coating application can be in any known
~ WO 94/02029 Z~L~a@~ PCr/US93/06500
manner, including dipping, spraying, and painting.
- The eighth fabrication embodiment according to this
invention comprises coating the inventive spongeous
matrix to form the product described in the third
article embodiment, by applying a coating comprising a
substance disclosed above as comprising the spongeous
matrix itself, either in sol or in thin gel form, but
without forming a spongeous matrix of the coating by the
inventive steps of freezing and thawing. The coating
application can be in any known manner, including
dipping, spraying, and painting.
Another group of embodiments of this invention are
uses of the inventive articles of manufacture.
The first use embodiment for the inventive articles
of manufacture comprises employing the above-described
spongeous matrices for the growth or storage of a living
plant material, optionally at least partially saturated
with a suitable growth or storage medium. The living
plant material may be a seed, embryo, graft, cutting,
young plant, callus, or any viable grouping of cells.
The second use embodiment relates to plant culture
and comprises the sequential steps of:
A - adding an aqueous plant nutrient medium to a
spongeous matrix according to this invention;
[the matrix and other ingredients optionally may
be sterilized];
B - inserting a living plant material into the
spongeous matrix; and
C - growing or maintaining the plant material
under known suitable respective growth or
maintenance physical conditions, for example
temperature, humidity, and radiation including
actinic to encourage either dormancy or growth.
D - optionally, when growing plant material~
after a root system sufficient to maintain the
W094/02029 ~ PCT/US93/06500
- 8 -
plant is established, the entire spongeous matrix
and contained plant may be placed into a soil-
cont~i n; ng growth medium, hydroponic system, or
the like.
The third use embodiment comprises the continuous or
intermittent replenishment or addition of plant nutrient
medium when employing the second use embodiment.
The fourth method of use embodiment comprises
employing the spongeous matrix of this invention as a
support for tissue or cell culture in a manner similar
to its use for plant material.
The fifth use embodiment comprises employing the
spongeous matrices as surgical sponges, [optionally
utilizing an above-described carried substance therein].
This use takes advantage of the inventive matrices slow
dissolution in aqueous fluids including body fluids
and/or its biodegradability. Naturally, the spongeous
matrices used for ~his ~urpose are firs~ sterili~ed by
known means.
The sixth use embodiment comprises forming the
spongeous matrices into suitable spacer shapes and
employing them in substantially dry form as a packing or
packaging material, [taking advantage of their inherent
biodegradability for eventual disposal].
The seventh use embodiment comprises employing the
spongeous matrices of this invention as filters for
gases and/or fluids.
Other than in the operating examples, or where
otherwise indicated, all numbers expressing quantities
of ingredients or reaction conditions used herein are to
be understood as modified in all instances by the term
"about".
It should be understood that the components
comprising the inventive article of manufacture are
complex organic polymers of natural origin and therefore
W094/02029 ~ 4 PCT/US93/06500
cannot be defined by exact chemical formulae. Most of
- the hydrocolloids and/or polysaccharides useful as
components in the inventive articles of manufacture are
genera rather than species, and where a genus is
referred to all known species thereof are probably
useful and are included unless otherwise indicated.
Illustrative of the foregoing, the Webster's New World
Dictionary of the American Language, second edition
(World Pub. Co., New York, 1972) defines gum arabic as
being obtained from several African acacias and
traqacanth as coming from any of various, especially
Asiatic, plants (genus A. stragalus) of the legume
family.
Unless indicated otherwise, Components (a) and (b),
and optional Component (c), refer to not only purified
materials, but also crude or native materials in which
the pure materials are present in an operative amount
for the purposes of this invention.
As used herein, the terms "chemical derivative" or
"chemically modified" used in referring to a component
of the inventive spongeous matrix refer to that
component having a substituent moiety, examples of which
moieties include, but are not limited to, acetyl, C1_4
alkyl, Cl_4 alkoxyl, C2_4 hydroxyl, carboxy C1_4 alkyl,
or an alkali metal or alkaline earth metal salt thereof
where appropriate. The removal of a pre-existing moiety
such as by deacetylation is also a possible chemical
modification.
As used herein, the terms "physical derivative" or
"physically modified" used in referring to a component
of the inventive spongeous matrix refer to that
component having properties which have been modified by
a physical manipulation which may or may not result in a
change of the complex organic polymers. The product of
depolymerization (degradation) in varying degrees is
also considered a physical derivative.
W094/02029 ~l 40a6 4 PCT/US93/06500 ~
-- 10 --
Component (a) - Glucomannan
The type and physical form of glucomannan useful in
this invention theoretically is not limited and includes
glucomannans derived from trees. Because of commercial
availability, and because of its known favorable
properties, konjac-origin glucomannan is preferred, and
references to "konjac" or "glucomannan" in this
application should be understood as to konjac-derived
glucomannan unless otherwise indicated.
Konjac glucomannan is a hydrocolloidal poly-
saccharide obtained from the tubers of various species
of Amorphophallus. It is a high molecular weight, non-
ionic glucomannan primarily consisting of mannose and
glucose at a respective molar ratio of approximately
1.6:1Ø This slightly branched polysaccharide is
connected by ~-1,4 linkages and has an average molecular
weight of 200,000 to 2,000,000 daltons. Acetyl moieties
along the glucomannan backbone contribute to water
solubility properties and are located, on average, every
9 to 19 sugar units.
Konjac flour is obtained by slicing, drying and then
wet- or dry-milling the AmorPhoPhallus tuber. This
material is then pulverized, sifted and air-classified.
This crude konjac flour contains numerous impurities
including starches, cellulose and nitrogen-containing
materials including proteins. Konjac flour is
dispersible in hot or cold water and forms a highly
viscous sol with a pH between 4.0 and 7Ø Solubility
is increased by heat and mechanical agitation.
Although this crude konjac flour is suitable for
producing the spongeous matrices of the invention, it
may optionally be further refined by such methods as
alcohol washing or by dissolution followed by
filtration.
Konjac flour is available as a commercial product
W094/02029 ~ 8~4 PCT/US93/06500
from a number of sources. One source, and method for
- preparing konjac flour, is disclosed in Marine Colloids
Bulletin K-1, "Nutricol~ Konjac Flour" (1989) (product
- and bulletin of FMC Corporation, Food Ingredient
(formerly Marine Colloids) Division, Philadelphia, PA,
19103 U.S.A.).
While konjac flour is preferred for use herein in
either crude or refined form, konjac tubers and raw or
semirefined konjac can also be employed to form the
inventive spongeous matrices, since the glucomannan is
contained within tiny sacs or granules in the tubers
which rupture upon hydration.
For most spongeous matrices, a higher molecular
weight konjac glucomannan is preferable, such as that
afforded by a native or crude konjac flour. A more
purified or a clarified konjac glucomannan is preferable
for certain particular uses of the spongeous matrix (for
example in medical, pharmaceutical or biotechnical
utilization). Clarified (purified) konjac is an
experimental product of FMC Corporation, Food Ingredient
Division, Philadelphia, Pennsylvania, U.S.A. It forms a
clear sol, as compared to the cloudy sol formed from
more crude konjac glucomannan. Such konjac glucomannan
usually has a lower molecular weight as a result of the
purification or clarification process. In such
instance, a higher concentration aqueous gel can be
obtained, and is preferably used to provide better
structural integrity of the spongeous matrix.
"Cold-melt" konjac, which is distinguished by its
unexpected property of liquifying at lower (cold)
temperatures and solidifying at higher temperatures also
may be used as the konjac glucomannan component (a)~
Cold-melt konjac and clarified konjac and their
manufacture are disclosed in pending Unites States
patent application 07/742,136 and/or 07/742,260 and
their cor~esponding Patent Cooperation Treaty
6~
"
W094/02029 PCT/US93/06500
publications, the contents of which are incorporated
herein by reference.
Component (b) - Aqueous Gel-Forminq Polysaccharide
Among the component (b) substances "agar", sometimes
referred to as agar-agar, is a phycocolloid derived from
red algae, such as Gelidium and Gracilaria, and is
primarily a polysaccharide mixture of a variety of
galactan molecules containing varying amounts of ester
sulfate and/or pyruvate and/or methoxyl groups, the
least ionic being termed "agarose".
Methods for the isolation of agarose from agar are
well known to the art. The particular source of the
agar or agarose is not important to the practice of the
present invention, although some minor variations in the
properties of the spongeous matrices may exist depending
on the source of the agar and/or agarose, and whether it
is used unmodified, or as a chemical or physical
derivative.
Carrageenan for use in the spongeous matrices of the
invention can be a mixture of the known carrageenan
fractions, but is preferably predominantly, (and most
preferably entirely) the kappa fraction.
Component (c) - Optional Water-Soluble Polysaccharides
Not all polysaccharides are suitable as the optional
third component (c). As noted in the following
examples, it was determined that chitosan is unsuitable
because it requires acid conditions to be water soluble
and the spongeous matrix forming processes require
alkaline conditions. Exposing a chitosan aqueous sol to
the alkaline conditions used in forming the inventive
spongeous matrices results in the chitosan coming out of
the sol. Gum arabic, karaya gum, and tragacanth are
each useful as component (c) when present at 5.0 % dry
weight, but work poorly or not at all at 50.0 % dry
W094/02029 ~ PCT/US93/06500
- 13 -
weight. These polysaccharides therefore are less
desirable as component (c), but still form compositions
at lower concentrations of up to approximately 25.0 %
dry weight, or when combined with starch, and are useful
in forming inventive articles of manufacture that have
utility for certain applications.
Starches that can be employed herein can be obtained
from any source, e.g. from corn, potatoes, tapioca,
rice, and wheat, and can be high or low in amylose.
The term "spongeous matrix" used to characterize the
articles of manufacture of this invention refers to
matrices having a predominantly sponge-like character
upon formation. For most (but not all) utilities, it is
preferred that they possess excellent structural
stability and mechanical strength, and in addition have
a porous, mostly interconnecting pocket-like structure,
from which liquid present can be readily expressed. By
the term "mostly interconnecting" is meant that at least
60% and generally at least 75% of the pockets
interconnect. In addition, many of the preferred
spongeous matrices when compressed exhibit rapid return
to their precompressed size and shape, with rapid uptake
of aqueous solutions, for example where the pores of the
spongeous matrix are substantially filled with the
aqueous solution when compressed, immersed in the
aqueous solution, and then pressure released. Moreover,
most of the spongeous matrices of the invention exhibit
at least some stability to short immersion in boiling
water.
While spongeous matrices can sometimes be formed
from the individual components of the present spongeous
matrices, for example from konjac glucomannan alone or
agar or agarose alone, such spongeous matrices do not
possess all of the favorable characteristics of the
spongeous matrices of the invention. For example, these
single component spongeous matrices tend to be weaker
W094/02029 ~ ~- 2~ PCT/US93/06500
- 14 -
than those of the present invention, possessing little
structural integrity and contain cavities more like
channels or tunnels rather than the mostly
interconnecting pockets characteristic of the inventive
articles of manufacture. Single component spongeous
matrices specifically are excluded from this invention.
The spongeous matrices of the invention have many
uses. One important use is in agriculture. Solid
aqueous nutrient gel media have been used previously as
supports in the growth and storage of seeds, plant
cells, tissues, explants, embryos, grafts, young plants,
and callus cultures. However, lack of air space in such
solid supports inhibits growth and provides a barrier to
root development and growth for the plant cultures. In
addition, it is often desirable to change the nutrient
media at different stages of growth by adding or
deleting specific nutrients or plant hormones. Such
desired change in nutrient media is not feasible in
solid media supports, but is with the inventive
spongeous matrices. Furthermore, prior to transplanting
the young plants from solid media it has been necessary
to separate them from the solid supports, a labor
intensive procedure that generally damages the fragile
root systems.
The inventive spongeous matrices can be hydrated in
aqueous nutrient media to provide a beneficial
combination of aqueous nutrient media and air space,
which also permits rapid continuous or intermittent
nutrient media replacement and/or exchange. Moreover,
plant root systems readily penetrate the spongeous
matrix supports, which need not be removed prior to
transplanting since the spongeous matrices are
completely biodegradable. In addition to plugs,
spongeous matrices in strip form can be used as
biodegradable seed tapes.
Another important use of the present spongeous
W094/02029 -;';t ~ PCT/US93/06500
- 15 -
matrices in sterile form is as surgical sponges and in
- the external treatment or packing of surface wounds or
conditions, for example as ear packings or for wound
cleaning in which medicaments can also be present in or
on the matrices. Where surgical spongeous matrices of
this invention purposely or accidentally are left in a
wound, they may present little problem where they can be
absorbed by body fluids, depending upon their component
materials.
A particularly important use for the spongeous
matrices of the invention is as packing or packaging
filler and protective materials which after use can be
reused, discarded or buried in landfills without concern
since the spongeous matrices are biodegradable. For
this purpose, the spongeous matrices can be formed in
any shape including those conventionally used for
packaging filler.
The inventive spongeous matrices can also be used in
many other fields such as for reagent delivery
substrates, adsorbents, controlled release devices,
chromatography packing, perfusion/dialysis equipment,
diagnostic reagent carriers, immobilized enzyme
reactors, collagen substitutes, cosmetic and air
freshener substrates, scent and flavoring carriers such
as lobster or fish baits, filters, electrophoresis
wicks, microbial growth supports and sampling devices,
filtration media, controlled release of reactants,
substrates for the release of insect repellants, and the
like.
In the process for fabricating the inventive
articles of manufacture, bases for forming the gels are
well known and those that can be used include (but are
not limited to) ammonia, alkali metal hydroxides,
(especially sodium and potassium) and alkali metal
carbonates, (especially sodium and potassium), potassium
carbonate being most preferred. Sufficient base is
W094/02029 PCT/US93/06500 ~ =
- 16 -
added to the sol to create an alkaline pH, preferably
one having a pH of 9.0 to 12.0, and the resulting sol is
then heated until it forms a uniform gel.
The thawed spongeous matrix, in an optional step E,
may be compressed to remove most of the liquid content
of the spongeous matrix. The spongeous matrix can be
dried completely in known manner such as by drying at
ambient or at an elevated temperature, by freeze-drying,
and by drying under a vacuum.
Preferably, prior to heating, the spongeous matrix
is rinsed by squeeze/swell cycles in water, and then in
a C1_6 alkanol or alkanol-water mixture, for example 65%
2-propanol.
The dry spongeous matrices of the invention can be
sterilized as desired by using heat (e.g., by
autoclaving while being protected from direct contact
with steam).
It was found that when the gel that forms in step B
is partially dried before the gel is frozen in step C,
the resulting sponge has a tough surface layer or skin,
which may be advantageous for some applications.
Alternatively, the skin can be removed prior to use.
Alternative methods of forming a skin include:
immersion of the formed spongeous matrix in a
dessicating medium such as isopropyl alcohol or acetone,
or coating the matrix with other (preferably water-
insoluble) known agents.
EXAMPLES
The following examples are given to further
illustrate the invention.
ExamPle 1
Koniac/Aqarose Composite Sponqeous Matrices
A 1% w/v clarified konjac sol was divided into a
W094/02029 PCT/US93/06500
- 17 -
number of 50 ml aliquots. Various amounts of agarose
powders were added to the aliquots and dissolved by
heating and stirring. The agarose samples were;
Seakem~ LE low electroendoosmosis agarose (0.5% w/v, 1%,
and 2%), and Seakem Gold agarose (1% and 2%). [Seakem~
agarose products are manufactured by FMC BioProducts,
Rockland, Maine 04841 U.S.A.]. Each sample was mixed
with 2 ml 5M NH40H, heated for 20 minutes in a boiling
water bath and then frozen. The samples were slowly
thawed at room temperature. The samples containing
Seakem LE and Seakem Gold agaroses formed spongeous
matrices with large cavities, which easily shed their
liquid when squeezed. As agarose concentration
increased, cavity size decreased. When squeezed flat
and released, the matrices rebounded to full size
indicating a vast increase in structural integrity over
sponges formed from the individual components. When
placed in water, the spongeous matrices completely
filled by capillary action. All but the 0.5% agarose-
containing spongeous matrix were soaked in 83% 2-
propanol, drained and then dried for an hour at 80C.
When placed back in water, they quickly rehydrated and
filled with water.
Example 2
Koniac/Aqar ComPosite Sponqeous Matrices
Crude konjac flour was used to prepare two 100 ml
samples of a 1% aqueous sol. One gram of Gracilaria-
derived agar was added to one sample and dissolved by
heating to boiling in a microwave oven. The sample was
then stirred in a hot water bath until fully dissolved.
Two aliquots were removed; one of 25 g and one of 50 g.
The smaller aliquot was diluted with water 1:1 thus
giving a concentration of 0.5% w/v konjac and 0.5% agar.
The second 100 ml konjac sol was mixed with 2 g of agar
and treated in the same manner. The four 50 g samples
(konjac/agar concentrations of: O. 5/0. 5~, 1/1~, O. 5/1~
W094/02029 PCT/US93/06500
- 18 -
and 1/2%) were all mixed with 0.375 ml of a lM K2CO3
solution and heated for 25 minutes in a boiling water
bath. The resulting gels were covered and frozen
overnight. The resulting spongeous matrix samples were
thawed and e~m; ned. The spongeous matrices appeared
very similar to the konjac/agarose matrices of Example 1
with minor exceptions. Whereas the agarose-containing
matrices were covered with a strong skin and had to be
forcibly squeezed to remove liquid, the konjac/agar
matrices had a thin skin which allowed most of the
internal liquid to drain away by gravity. The spongeous
matrices were processed and dried as previously
described. The dried samples all rehydrated and filled
with water. The 0.5~ konjac/1% agar matrix was the
fastest to swell while the 0.5~/0.5~ matrix was the
slowest.
Example 3
Koniac/Agarose Composite Sponqeous
Matrices bY Freeze Drying
A sol of 1% konjac from crude konjac flour
containing 1% Seakem~ LE agarose was prepared. A
portion of this (150 ml) was cast into a crystallizing
dish and gelled by adding 1.125 ml of lM K2CO3 in a
boiling water bath. The gel was placed in a freezer at
-15C for 1.5 hours and then transferred to a freezer at
-80C for 1 hour. The sample was placed in a
lyophilizer on a glass plate and dried under vacuum
(- o.l Torr) with a shelf temperature of 100F (about
38C). After 3 days the sample was removed and weighed
(3.59 g). The resulting spongeous matrix readily
hydrated and filled when placed in water. It had
numerous small uniform ca~ities.
~ W094/02029 ~ 86~ PCT/US93/06500
-- 19 -- - i .,,
Example 4
- Process Variations in Koniac/Agarose Matrix Formation
The composite spongeous matrices of Examples 1-3
were formed by gelling the konjac component (a) through
the use of a base and heat. The other component (b)
solidified as the hot gel cooled. A 100 ml solution of
1% konjac/1% LE agarose was prepared and split into two
equal aliquots. One sample was gelled as described in
Example 2 by heating the alkaline solution and cooling.
The second sample was mixed with the same amount of
K2CO3 solution but was iced immediately afterward to
rapidly set the agarose matrix. once gelled, the sample
was heated in a hot water bath (63C - 70C), a
temperature below the melting point of the agarose for
60 minutes. Both gels were frozen and thawed as
described in Example 2. The spongeous matrix formed by
the first procedure had large irregular cavities whereas
the second gel produced a spongeous matrix with smaller,
more consistent-sized cavities.
Example 5
Effects of PH durinq Gel Formation on Matrix Properties
A sol of 1% konjac from crude flour and 1% low
electroendoosmosis agarose was prepared as in example 1
and divided into seven 50 ml aliquots. Different
amounts of 5M NH40H ranging from 0.1 to 4 ml was stirred
into five of these aliquots. The pH of each was quickly
measured and the aliquots were gelled for 20 minutes in
a boiling water bath. The last 2 aliquots were mixed
with 0.1 and 0.2 ml volumes of 5_ NaOH and gelled in the
same manner. Their pH was also measured with a pH meter
(model 155 pH/ion meter; Corning Science Products;
Halstead, Essex; England). The volumes and type of
alkali used, as well as the corresponding pH's, are
shown in the following table:
W094/02029 ~408~4 PCT/US93/06500 ~
- 20 -
Alkali Concentration Volume (mL) pH
NH40H 5M 0.1 9.31
" ~ "" 0.5 9.60
" " "" 1.0 10.15
" " "" 2.0 10.40
" " "" 4.0 10.47
NaOH 5M 0.1 11.40
" " "" 0.2 11.80
All gels were frozen and thawed as previously
described, producing spongeous matrices. Several
general trends were observed in the matrices. When the
pH increased the number of spongeous cavities increased,
the size of the cavities decreased, and the homogeneity
of the spongeous matrix appeared to increase; the
reverse would be expected to be obtained upon decreased
pH.
ExamPle 6
Konjac / Aqar Sponqeous Matrix Concentration Variations
Comparison of ProPerties
A series of nine konjac (from crude konjac flour) +
agar spongeous matrices were prepared according to the
procedure of Example 2. Concentrations of the
components were either 0.5%, 0.75% or 1%. The matrices
were compared based on: 1) strength of the matrix, 2)
whether the spongeous matrix retained its original size
or shrank (before and after drying), and 3) whether the
spongeous matrix would rebound after being squeezed and
released.
The most desirable spongeous matrices were full
sized, durable and fully rebounded after a
squeeze/release cycle resulted from the following konjac
/ agar concentrations: 0.75%/1%, 0.5%/1~ and 1%/0.75%.
Intermediate samples were 1%/1% and 0.75%/0.75%. The
remaining matrices were generally soft and shrunken,
remaining flat after squeezing, but swelling when placed
in water.
~ W094/02029 ~4 ~ . PCT/US93/06500
- 21 -
Example 7
A. Sponqeous Matrix Stability in Boiling Water
- Three 200 ml spongeous matrices containing 1% konjac
(from crude konjac flour) and 1% of either agarose or
agar were prepared as described in Examples 1 and 2
respectively. The spongeous matrices were dried and
accurately weighed. The matrices were placed into 500
ml of hot water for 20 minutes and then boiled for an
additional 60 minutes. The agarose and agar containing
matrices appeared little changed. The spongeous
matrices were squeezed to remove excess liquid, rinsed
and treated with 83% 2-propanol for 30 minutes and dried
in a hot air oven. The samples were reweighed to
determine weight loss. The results are given below.
ComPosition Initial Weiqht Final Weight ~ Loss
konjac/agarose 3.711 g 2.758 g 25.7
konjac/agar 3.530 g 2.597 g 26.4%
The dried spongeous matrices were placed in water
and observed. Although the spongeous matrices did fill
with water, they lacked their initial structural
integrity and were partially collapsed.
B. Coated SPonqeous Matrix Stability In Boilinq Water
Two 1% konjac (from crude konjac flour) / 1% agar
spongeous matrices were formed and dried as described
above. The dried matrices were accurately weighed. As
a coating material, a 50 ml volume of a 1% crude konjac
solution was diluted to 200 ml or 0.25%. To this 1.5
ml of lMK2CO3 was added and the sample gelled in a
boiling water bath for 35 minutes. The gel was placed
in an ice bath where it cold melted, forming a cold-
melt konjac. This cold-melt sol was filtered through a
cloth filter to remove debris such as the sacs which
are found in kon~ac flour. One of the dry spongeous
W094/02029 ~ PCT/US93/06500 ~
; .
- 22 -
matrices was placed in this filtered cold-melt sol
coating material and allowed to hydrate. The matrix
was massaged to remove air bubbles and to allow
thorough coating. The spongeous matrix was removed,
squeezed to remove excess li~uid and hardened briefly
in 99% 2-propanol. The matrix was transferred to a
92OC oven for 30 minutes to partially dry, removed, and
recoated as before. Excess cold-melt liquid was
removed by squeezing and the spongeous matrix redried
at 75C overnight (no alcohol used). The matrix was
reweighed. Each matrix was placed in 300 ml of boiling
water for 60 minutes. Excess liquid was squeezed from
each spongeous matrix and both were then dried
overnight at 75C. The dried matrices were reweighed
to calculate the % weight loss due to leaching.
Sam~le Initial Weight Final Weight Wei~ht Loss % Weight Loss
Uncoated 1.678 g 1.039 g 0.639 g 38~
20 Coated 1.849 g 1.481 g 0.369 g 1996
Example 8
Three Component Spongeous Matrices
Two 50 ml samples of 1% konjac sol (from crude
konjac flour) were taken from a larger volume. To the
first sample, 0.25 g of agar and 0.25 g of kappa
carrageenan were mixed in and dissolved. The final
composition was 1% konjac, 0.5% agar and 0.5%
carrageenan. The first sample was gelled by heating in
a boiling water bath for 20 minutes after the addition
of 0.5 g KCl and 0.375 ml lM K2C03.
To the second sample, 0.25 g agar and 0.25 g
soluble starch were mixed in and dissolved. This was
35 gelled by adding 0.375 ml of lM K2C03 and heating as
with the first sample. The final gel composition was
W094/02029 ~ ~ PCT/US93/06500
1~ konjac, 0.5% agar and 0.5% starch.
The gels were frozen and thawed as described in
Example 1, to form spongeous matrices. The matrix
containing agar and carrageenan was very firm and thus
hard to squeeze. When placed in water, it swelled
slowly. The starch/agar containing matrix was softer
and easier to squeeze. It was also quicker to swell in
water than the first matrix. The starch containing
spongeous matrix was covered with a thin but leathery
skin.
Example 9
Konjac/Kappa Carraqeenan Composite Sponqeous Matrices
A. An aqueous sol of 1% (w/v) konjac (from crude
konjac flour) containing 3% kappa carrageenan was
prepared and divided into three 200 ml aliquots. To
the first aliquot, 2 g (1%) KCl was added under heating
to initiate gelation of the carrageenan. The hot
aliquot was allowed to cool and then frozen. The
second aliquot was mixed with 1.5 ml lM K2CO3 (for
konjac gelation) and 0.89% KCl (for carrageenan
gelation). The aliquot was heated in a boiling water
bath for a period of 30 minutes. The gel was allowed
to cool and then frozen. The third aliquot was mixed
with 0.5 ml of l.O M NaOH, heat-set for 30 minutes and
frozen overnight.
After thawing, the first sample (KCl only) was
transparent, with a rubbery, thick-walled corrugated
skin. The sample was firm, containing numerous large
irregular cavities, and would rebound to full size
after squeezing and releasing. It did not absorb much
water through capillary action and would only fill if
squeezed and released under water. The sample was
found to be hot-water soluble.
The second sample (KCl and K2CO3) was white,
rubbery, contained few cavities and had a thin skin.
;4
W094/02029 ~ PCT/US93/06500
- 24 -
The sponge would rebound after a squeeze/release cycle.
The sample did break up somewhat when placed in boiling
water but did not dissolve. There was evidence that at
least a portion of the carrageenan did leach out into
the water.
The third sample (NaOH) was very rubbery and tough
with very few cavities and looked and felt more like a
slightly fractured gel.
B. A series of duplicate 1% kappa carrageenan a~ueous
gels containing konjac (from crude konjac flour), in
increments of 0.2% (0.2 -> 1.0~), were prepared as
described above. One set of gels was set with KCl only
while the second was gelled with KCl, K2CO3 and heat.
The gels were frozen and thawed as previously
described.
For the first series (with KCl only), all gels were
transparent, rubbery with thick walls, tough and would
rebound after a squeeze/release cycle. (Only the
sample with 0.8% konjac did not rebound after
squeezing.) The cavities were irregular and generally
large. For those gels set with both alkali and KCl,
the gels were white, soft, thin walled and full of
small cavities. They would remain flat after a
squeeze/release cycle (except the sample which
contained 0.6% konjac) but would swell to full size in
water. The sponge containing 0.2% konjac was weak and
partially broke up when s~ueezed. As the konjac
concentration increased, the matrices became somewhat
more durable.
The spongeous matrix containing 1% konjac and 1%
kappa carrageenan was dried and weighed. The matrix
was then placed into 500 ml of hot water for 20 minutes
and then boiled for an additional 60 minutes. The
sponge turned white in color and became slimy. The
sponge was redried and reweighed to determine weight
loss. The result was:
W094/02029 PCT/US93/06500
- 25 -
initial weight - 3.564 g
- final weight - 2.754 g
percentage loss - 22.7%.
The dried matrlx was then placed in water and observed.
The ,,matrix was slimy with a thick rubber feel and
possessed no sponge-like characteristics.
~xamPle 10
Koniac/Gellan Gum Composite SPongeous Matrix
On hundred ml of a 2% (w/v) hot solution of gellan
gum was admixed with 100 ml of a 2% (w/v) hot solution
of konjac, to prepare 200 ml of 1%konjac / 1% gellan.
The mixture was cooled to 60C and 1.5 ml of lM K2C03
and MgS04 7H20 were admixed therein. A 150 mg portion
of the sample was introduced into a 6.5 x 6.5 x 10 cm
plastic container. The sample was heated in an 85C
water bath for 60 minutes. Then it was removed and
cooled at room temperature overnight, after which it
was frozen for 24 hours at -15C. The sample was
thawed at room temperature and ~m; ned. It was fully
spongeous. Although the sample was only about 75% of
its initial size, when squeezed and thawed it rebounded
fully at a moderate rate.
Exam~le 11
Koniac/Sodium Alginate ComPosite Matrices
Two hundred ml of 1% konjac / 1 % sodium alginate
(w/v) mixture was prepared by admixing 100 ml of a 2 %
(w/v) hot solution of sodium alginate with 100 ml of a
2% (w/v) hot solution of konjac. The sol was cooled to
600C and 1.5 ml of lM K2C03 and 0.4 g CaCl2 were
admixed. The alginate component began precipitating
immediately and its gel structure was disrupted by
subsequent mixing. Despite this, a 150 g portion of
the sol was placed in a 6.5 x 6.5 x 10 cm plastic
container and covered. The sample was heat-set for 60
W094/02029 PCT/US93/06500 _
- 26 -
~,
minutes in an 85C water bath. The gel was removed and
cooled overnight at room temperature and then frozen
for 24 hours at -15C. The sample was thawed at room
temperature and ~ ;ned. The sample was completely
porous while the matrix was soft and filled with many
small, fine pores. When squeezed and released, the
sample did not rebound significantly. Additionally,
the matrix crumbled quite easily, especially along its
edges.
A duplicate 200 ml sample of 1% konjac / 1% sodium
alginate was prepared in the above manner. When cooled
to 60C, only the K2C03 was added. The sol was gelled
by heating in the above manner. The gel was then
removed from the container and placed in 750 ml of 2%
CaCl2 for 48 hours to set the alginate component. The
gel, which shrank during this, was frozen and thawed in
the above manner. The matrix was fully porous, yet was
filled by much larger cavities. It was also much
firmer and quickly rebounded after being compressed and
released.
Example 12
Use of Inventive SPongeous Matrices for plant culture
A. A 150 ml volume of an aqueous sol containing 1%
crude, alcohol-washed konjac (from crude konjac flour)
and 1% agar was prepared as described in Example 2.
The sample was mixed with 1.125 ml of 1_ K2CO3 and cast
into a 6.5 x 6.5 x 10 cm plastic plant embryo culture
container (type GA 7, Magenta Corp.; Chicago, IL.).
The sol was then gelled by heating in an 85C hot water
bath for 35 minutes. The sample was frozen at -15C
overnight and thawed slowly at room temperature. The
resulting matrix was squeezed flat, placed in clean tap
water and allowed to reswell. This was repeated six
times, changing the water each time. The matrix's
capacity of absorption by capillary action was tested
W094/02029 PCT/US93/06S00
- 27 -
by slowly adding measured volumes of water and pouring
off the excess. Capacity was determined to be 37 ml
although the maximum would be much higher if air
bubbles were squeezed out and the sponge completely
filled. The sample was rinsed briefly in 2-propanol
and dried in a forced air oven at 55C.
The spongeous matrix was hydrated with 25 ml of
Murashige and Skoog salt base buffer (Hazelton Research
Products; Denver, PA). Two small slits were cut into
the surface of the sponge and a Kentucky Wonder~ pole
bean seed was inserted into each. The seeds began
germinating after 2 days. After 10 days, a complex
root system had developed and leaves had appeared. In
some places where the thin skin covering the sponge was
ripped or torn, roots had penetrated through to the
exterior. Throughout this period, the sponge was kept
moist by additional buffer. Two weeks after planting
the seeds, the seedlings were transplanted outside by
placing the entire sponge into the ground and slightly
covering it with soil. After nine days, the plants
were carefully excavated to ~mi ne the root system and
sponge. Roots which had already penetrated the skin
had begun to develop smaller tap roots whereas those
still within the sponge had not yet exited the matrix
which was beginning to show signs of deterioration.
After 7 weeks the plant was again excavated. There was
very little left of the spongeous matrix. What little
remained was a soft gel-like material which readily
fell away from the extensive root system which had
developed.
B. Two 1% konjac (from crude konjac flour)/1% agar
spongeous matrices were prepared by freeze/thawing gel
which were prepared in 50 ml disposable centrifuge
tubes. The samples were processed and dried as
described above. The dried matrices (~ 5 cm long) were
hydrated in water and holes were cut through them along
their long axis. Cuttings from a Coleus sp. house
W094/02029 ~ ~ PCT/US93/06500 ~
" .. ~
- 28 -
plant (~ 13 cmiin length) were inserted 4 cm into the
matrices. The matrices/cuttings were placed back into
centrifuge tubes containing water. Within 2 weeks
numerous roots had sprouted from the stem and had grown
throughout the sponge matrix.
Example 13
Sponqeous Matrices prePared varYinq ComPonent (c)
A solution of 1% glucomannan as crude konjac
tComponent (a)] containing 0.75% agar [Component (b)]
was prepared by dissolving 15 g konjac and 12 g agar in
1.5 l distilled water over 60 minutes in a hot water
bath. The sample was split into 100 g aliquots.
Various Component (c) gums were incorporated as dry
powders either by adding 0.088 g (5% w/w based on
konjac and agar) or 0.875 g (50% w/w). Each aliquot
was stirred and heated to effect dissolution. Samples
were cooled slightly and 0.75 ml of lM K2CO3 was
admixed. Each sample was heat-set in a boiling water
bath for 30 minutes. Gels were removed, cooled to room
temperature and then frozen overnight. The frozen
materials were thawed at room temperature and examined.
The results are summarized below.
ComPonent (c) Conc. Observations
Guar gum 5% spongy; slow, incomplete
swelling when placed in water,
n o r e b o u n d a f t e r
squeeze/release
Guar gum 50% as above but with slimy skin on
top surface
CLBG* 5% seemed normal; full-sized after
squeeze/release
CLBG* 50% " " ll ll
4~ Arabic, gum 5% spongy; swelled quickly in
water; no rebound after
squeeze/release
~ W094/02029 PCT/US93tO6500
~4~
- 29 -
Arabic, gum 50% mush; did not work
Karaya gum 5% similar to that with 5% arabic
Karaya gum 50% similar to arabic at 50%, did
not work
* = clarified locust bean gum
An attempt to incorporate chitosan as Component (c)
was not successful. Chitosan is only soluble under
acidic conditions, and was dissolved in aliquots of the
konjac/agar by adding acetic acid. However, when
alkali was added to adjust the pH for heat-setting, the
chitosan precipitated. An attempt was then made to gel
the sample by cooling the sol and allowing the agar
component to gel. At this point, the gel was soaked in
alkali to raise the pH. While this was in process,
much of the chitosan leached out of the gel and
precipitated in the alkaline buffer. Further efforts
to incorporate chitosan into a konjac/agar sponge were
then abandoned.
ComParison Example A
Attempts At Matrix Formation Usinq OnlY Konjac
Three 90 ml aliquots of 1% (w/v) clarified konjac
sol were prepared and then gelled by adding 3.6 ml of
5M NH40H and heating for 20 minutes in a boiling water
bath. One gel was frozen directly. The other two
samples were cold melted in an ice bath and one such
cold-melt sol was frozen directly. The second cold-
melt sol was adjusted to pH 3.5 with 5M HCl and then
frozen. All samples were frozen overnight (18 hours)
at x -15C. The three samples were thawed slowly at
room temperature. All three produced copious amounts
of syneresate. The syneresate from two of the samples
(frozen gel and frozen alkaline cold-melt sol) produced
a slight coag when dropped in 99% 2-propanol. The
W094/02029 PCT/US93/06500
~ 30 ~
syneresate from the acidified sample produced no such
coag. All three samples formed fibrous, spongy
matrices which had little structural integrity and
which were quite similar in texture and appearance.
One exception was that the matrix formed from acidified
cold-melt sol reswelled in liquid more rapidly than the
others after squeezing.
Comparison Example B
10Effect of Concentration on Freeze/Thaw Behavior
in Matrices ComPrisinq OnlY Koniac
Three crude konjac sols were prepared at 1%, 2% and
3% concentrations. The 1% sample (150 ml volume) was
mixed with 1.5 ml 1_ K2CO3 while the other 2 samples
(200 ml each) were mixed with 2 ml of the alkaline
solution. The samples were gelled 30 minutes in a
boiling water bath, covered and frozen overnight. The
samples were thawed and ~m ined. The 1% sample was
soft and squeezable. When compressed, liquid slowly
oozed out leaving a soft fibrous mat which was quite
tough and resilient, indicating insufficient structural
integrity. The mat remained flat after squeezing but
did swell somewhat when placed in water. This matrix
was full of very tiny pores. The 2% and 3% samples
were ess`entially unaffected by the freeze/thaw
procedure and appeared to be entirely intact. The
samples were very tough and rubbery and if anything,
appeared to be stronger than the initial gels. When
squeezed, there was very little (2%) or no (3%) liquid
expressed. There were no cavities or pores visible.
ComParison ExamPle C
Dryinq and Rehydration of Matrices
comPrisinq OnlY Koniac
35Duplicate S0 ml samples of 2% and 4% clarified
konjac were gelled with NH40H and heat. The gels were
~ W094/02029 PCT/US93/06500
2~ 64
- 31 -
cooled and frozen overnight and then slowly thawed.
The samples, which were firm and covered with a tough
skin, had to be squeezed and massaged to remove the
liquid trapped within the numerous cavities of the
sponge. When squeezed flat, the sample was very tough
and rubbery and when placed in water would swell back
to its original size. The samples were rinsed by
repeated squeezes/swell cycles in water and then in 65%
2-propanol. The samples were then dried in a 55C
oven. The dried sponges were lightweight and durable.
When placed in water, the samples rehydrated slowly to
their original size of 2 cm x 5 cm. No structural
integrity was evidenced by these matrices.
15Comparison ExamPle D
Attempt At Matrix Formation Usinq Only Agarose
A solution of 1~ Seakem~ LE agarose was prepared
and used to fill two 150 ml crystallizing dishes. The
samples were allowed to gel and were then frozen
overnight. The frozen gels, which had constricted
somewhat, were thawed slowly at room temperature. The
samples were covered with a smooth skin, which burst
open when squeezed. The resulting matrix was fibrous
and weak and had insufficient structural integrity, but
did swell when placed in water. The sample was rinsed,
treated with propanol and dried as described in example
2. The dried sample did not rehydrate when placed in
ambient water, but dissolved in hot water.
30ComParison Example E
Attempts at Matrix Formation Usinq OnlY Aqarose
Gel Concentration Variations
100 Ml samples of 1%, 2% and 3% Seakem~ LE agarose
were prepared. The samples were allowed to gel, cooled
to room temperature and were then frozen overnight.
The samples were thawed and examined. All sampies were
W094/02029 2 ~ ~8~ PCT/US93/06500 ~
,~
- 32 -
full sized although the 1% sample's sides were slightly
constricted. The 1% sample had a corrugated surface, a
few large irregular cavities, had no structural
integrity, was soft and easily broke up. The sample
did swell when placed in water. The 2% and 3% samples
resembled fractured gels with no visible cavities. The
samples were not compressible (3~) or released very
little water. Both were soft, yet somewhat brittle and
thus broke up very easily.
Comparison Example F
AttemPt At Matrix Formation Usinq Only Aqar
Three agar gels were prepared at 1%, 2% and 3%
concentrations. The starting material was a
Gracilaria-derived agar (Algas Marinas; Santiago,
Chile). The three gels were cooled and then covered,
frozen overnight and then thawed at room temperature.
The 1% sample was much like the 1~ agarose sample in
Comparison Example E. It was however softer and
weaker. After squeezing, the sample did not rebound
and remained compressed but did swell when placed in
water. The matrix, which contained some large and
randomly distributed cavities, was weak and easily
broke apart if force was used. The 2% sample was fully
compressible and when released, partially rebounded.
This sample also swelled in water. The matrix was
still rather weak and contained some cavities which
were smaller, more numerous and evenly distributed.
The 3% sample was much like the 2~ agar matrix although
it fully rebounded after being squeezed. Although it
was slightly stronger, the matrix was still fragile.
Comparison ExamPle G
AttemPt At Matrix Formation Using OnlY
35Kappa-Carraqeenan
W094/02029 i z ~ PCT/US93/06500
- 33 -
Two samples of kappa-carrageenan were prepared by
dispersing 1 and 2 gram quantities into lO0 ml volumes
of deionized water. Both samples were heated to
boiling to dissolve the material. Once fully
dissolved, 1 g of potassium chloride was added to both
the 1% and 2% w/v kappa-carrageenan sols. The samples
were allowed to cool, were frozen overnight and were
then slowly thawed at room temperature. Both samples
were very soft and mushy when squeezed. The somewhat
spongeous matrix was transparent and possessed very
little mechanical strength. Pores/cavities were
generally small but were somewhat irregular in size and
placement. The samples broke up very easily and were
hot-water soluble.
Comparison Example H
Attempts at Sponqeous Matrix Formation Usinq
Other Materials As Component (b)
A number of other materials were tried as potential
component (b) when mixed with konjac glucomannan from
crude konjac flour in attempts to form spongeous
matrices of adequate structural integrity and/or
meeting other acceptable parameters of this invention.
Some did not work at all while others produced poor
results. In some cases, the matrices formed were
interesting but lacked properties seen in the examples
according to this invention. In each of the following
examples (except where indicated), three gels were
prepared with concentrations of (konjac/other material)
of 1%/0.5%, 1%/1% and 0.5%/1%. The samples were
prepared by dissolving the other material in a prepared
solution of konjac, adding alkali and heating for 20
minutes in a boiling water bath. The gels were then
frozen and thawed as in Example 1.
8~;~
W094/02029 PCT/US93/06500
Htl) Clarified Locust Bean Gum (CLBG)
The thawed matrix samples were very soft,
slimy and had very small, uniform pores. They were
also quite weak and thus broke up readily during
squeezing. Samples remained flat after squeezing and
did not swell in water, even after drying. The liquid
which was expressed formed a coagulum when dropped into
83~ 2-propanol.
H(2) Clarified Guar
These samples were very similar to those
formed with CLBG although the empty matrix was tougher,
more resilient.
H(3) Polyvinvl Alcohol rPVA)
These samples were prepared with PVA (88%
hydrolyzed) with an average molecular weight of 125,000
(Aldrich Chemical Co., Inc.; Milwaukee, Wisconsin).
The thawed material was very weak, soft and slimy but
did contain many small uniform pores. When gently
squeezed, liquid would seep out. The empty matrix was
flat and quite tough and remained flat after squeezing
and would not swell in water before or after drying.
H(4) Methyl Cellulose
These samples were prepared from material
rated at 400 centipoise viscosity (2% @ 25C) from
Sigma Chemical Co. (St. Louis, MO). During the heating
phase, the samples foamed significantly generating many
tiny air bubbles. These samples were much like those
prepared with CLBG; soft, slimy and weak. Only the
sample containing 1% konjac/0.5~ methyl cellulose was
squeezed flat. After drying, it hydrated but did not
swell in room temperature water. However, when the
water was heated for 45 minutes, the matrix did
partially swell.
W094/02029 PCT/US93/06500
- 35 -
- H(5) Gelatin
Knox~ gelatin was used to prepare the 3
composite samples which were gelled, frozen and thawed
as previously described. The thawed materials had
little structural integrity and were not squeezable.
H(6) PolyethYlene-PolYoxyProPYlene Block Copolymer
In this series of gels, Pluronic~ F-127
polyethylene-polyoxypropylene block co-polymer (BASF
Corp.; Parsippany, N.J.) was employed as an additive.
The thawed samples had a jelly-like texture and
squeezing them was difficult, especially the 1%/1%
sample which could not be compressed. The liquid
expressed from the other 2 samples formed a coagulum in
2-propanol. The flat matrices would not swell in
water, yet after being dried, they did swell
appreciably but only in hot water.
H(7) Xanthan
For this experiment, concentrations of
konjac/xanthan (Kelco Keltrol~ T) were 1%/1% and
0.5%/0.5%. A duplicate of the 1%/1% was prepared and
allowed to gel without added alkali and heat. The
first 2 samples were gelled as previously described.
All gels were frozen and thawed. None of the thawed
samples displayed any sponge-like characteristics and
were either mush or gel-like.
H(8) CarboxYmethYl Aqarose (CMA)
A sample of CMA was used to prepare 2 gels.
one was 1% konjac containing 1% CMA and the second
sample was 1% konjac/0.5% CMA/0.5% low eeo
(electroendoosmosis) value agarose. After
freeze/thawing the gels, the first sample (1%/1%) was
observed to be mushy, was not squeezable, and there
W094/02029 ~ 4 PCT/US93/06500 ~
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were no visible cavities. The sample began breaking up
after a very small amount of fluid was expressed. The
second sample had more structural integrity and did
contain some visible cavities. It could be squeezed
flat but would remain that way until placed in water
where it would swell.
H(9) PolYacrylamide (PAA)
A solution containing 0.5~ konjac/5% PAA (36:1
acrylamide:bis-acrylamide) was prepared by dissolving
0.25 g konjac, 2.432 g acrylamide (Sigma Chemical Co.;
St. Louis, MO.) and 0.068 g of bis-acrylamide (American
International Chemical; Natick, MA) in 50 ml water at
room temperature. Once dissolved, 0.375 ml of lM
K2CO3, 12.5 ~ N,N,N',N'-tetramethylethylenediamine
(TEMED) and 250 ~ lof 10% ammonium persulfate (both
from FMC Corporation, Philadelphia, PA) were added and
the sample gelled for 20 minutes in a hot water bath (~
80C). This sample was frozen and thawed as previously
described. The thawed sample was gel-like and very
rubbery. There were no cavities visible and no liquid
could be squeezed from the sample.
Com~arison Example I
Inhibition of Matrix formation in Composite Gels
I(1) Addition of 10% NaCl
A 1% konjac/1% Seakem LE agarose sol (200 ml) was
prepared as described in Example 1. To this, 20 g (10%
w/v) NaCl was added. The sample was gelled by adding
1.5 ml lM K2CO3 and heating for 30 minutes in a boiling
water bath. A control was also prepared without the
added salt. Both gels were covered, frozen overnight
and thawed at room temperature. The control had formed
a typical sponge as described in Example 1. The sample
which contained 10% NaCl was a firm strong gel with no
evidence of cavities or fracturing. The sample was
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frozen again at -80C overnight and thawed. There was
some very slight fracturing but no sponge-like
properties.
If2) Addition of 5% Glycerol
A 200 ml solution of 1% konjac/1% agar was prepared
to which 10 g (5% w/v) of glycerol (EM Science; Cherry
Eill, N.J.) was added. The sample was gelled by adding
1.5 ml of lM K2CO3 and heating in a boiling water bath
for 30 minutes. The gel was covered, frozen overnight
and thawed. The sample was largely intact, showing
only minimal fracturing. The sample was not
compressible and there was no release of liquid.
