AU673848B2 - Glucomannan spongeous matrices - Google Patents

Description

OPI DATE 14/02/94 APPLN1. 1D 46720/93 iti AOPDATE 12/05/94 PCT NUM~BER PCT/US93/06500 i ll l I~ ~11111111 AU9346720 INTERNATIONAL APPLICATION PUiILISIiI3I UNDI(R THE' PATl UNT (GOPERATION TRLATN (PCTr El I~ ha h i i at i t in II)Il~lftJoiIrii.ii. i~ ~~k A61F 13115, A61K 9/14 Al 1(43)110Interna3tional Publicati i Date,. 3 FbruarN. 1994 (01,0294) (21, International Application Numnber: PCT US91 06500 (81) Designated Slates: AT. AU. 11) 110. Bit. BY. CA. CHii CZ, DE1. DK. 11S. 1-1. 011. IlU, J11 KRI Kit, KZ. LK, (22) International Filing Dale; 9 July 1993 (09 0)7 93) LU, MG. SIN. %IW. NL. NO, NZ. PT, RO, RU. SO., St. SK. UA. VN. European paItent (AT, BF. CH, DE, i)K. I-S, IR. GR. I14 iT. LU. MC. N, SE).

Plriorit.) data: OA\PI patent (I1F. IIJ. CF. CO. (1L CM. GA, ON. ML.

918.992 23JuIy 199 203A77,92) us MRI. N, SN, Ti). TO) (71) Applicant: HMC CORPORATION (US US], 1735 MarketI Published Street, Philadelphia, P~A 19103 07th internationat srearch report.

Before the expiration of the time limit for amendine the (72) Iniorst SNOW. William, Cu-tis 6 Brewster Street., claims and to he republished in the event of the receipt of R')Lkand. ML 04841 RENN, D~onald. Walter amendments, Brewster Point, Glen Cow., ME 04846 (US) 0 7 4) Agens: Fr-LLOWS. Charles. C. et FMC Corporation.

173n,5 Market Street, Philadelphia, PA (US).

6i7384 8 (54)'Tile: 0 LUC OMANNAN SPONGL[OUS MIATRICES (57) Abstract 1Tie imsention relates to articles of manufacture comprising spongeoui mnatrices \%hich may hawe a controlled pore size and or distribution, formed of: Component (it) glucomiannan; and Component at least one other aqueous gel-forming pol %accharide; and optionally Component at least one water soluble hydrocolloid other than the foregoing. The insention al~so relawes to processes for fabricating the spongeous matrices and their use as plant culture mnedia, as surgical sponges, and as packaging material, WO 94/02029 PCTA'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 waterresistant 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 konja" which is water insoluble and which does not absox. 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 W O 94/2029 PCT/US93/0650( 2 galactomannan (Japanese published patent application 01- 166378), locust bean gum, plant protein and millet jelly (Japanese published patent application 63-79572), 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 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 distribution, when desired. The article of manufacture is formed from a coprocessed mixture of konjac glucomannan and at least one other aqueous gelforming polysaccharide.

Component can be any glucomannan, of which glucomannan derived from konjac is particularly preferred. The konjac glucomannan is not limited as to 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 comprises a polysaccharide other than a glucomannan which is capable of forming an aqueous gel. Component 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 more preferably comprises agar, agarose, kappa-carrageenan, a gel-formimg chemical W\O 94 /02029) PCT/US93/06500 3 derivative or physical modification of any of the foregoing, or a mixture thereof. Most preferably, component is agar, agarose, kappa-carrageenan, or a mixture thereof.

Components and 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 which comprises one or morwater oluble polysaccharides other than or and 4 ,4AeM >4.pr (A t O wXA ECVP !K ar Of 1"alj'Ow.e. o{e bie.o iloi whichh need not form an aqueous ge Comp nent (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 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 and When component is starch, it preferably is present in to 100.0 more preferably 20.0 to 75.0 all by dry weight compared to the combined dry weight of and 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 watersoluble 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 Ie WO 9 c4/02029 PCT/US93/06500 4 be water permeable or irpermeable, 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 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 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, 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 spongeous matrix, the carrier medium may remain therein, 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, 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 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 embodiment "substantially dry" is defined as having no readily expressible water, or as generally 20 or less O 94l/02029 IPCT/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 tne 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 [and optionally 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 for example it can be 6.66 using a phosphate buffer]; 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) and are independently of each other 0.25 to 2.0 wt preferably 0.5 to 2.0 wt more preferably 0.5 to 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.

WO 94/02029 /2CT/S93/06500 6 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 present in the above dry weight percentages relative to and 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 12.0.

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. mhe coating application can be in any known O 4/02029 PCT/CS93/06500 7 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 plan±t 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 WO 9)4/02029 PCT/US93/06500 8 plant is established, the entire spongeous matrix and contained plant may be placed into a soilcontaining 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 d 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 this purpose are first sterilized 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 WO 94/02029 PCr/ US93/06500 9 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 and and optional Component 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, C1- 4 alkoxyl, C 2 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.

WO 94/02029 PCT/US93/06500 10 Component 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 polysaccharide obtained from the tubers of various species of Amorphophallus. It is a high molecular weight, nonionic glucomannan primarily consisting of mannose and glucose at a respective molar ratio of a- roximat.e.y 1.6:1.0. This slightly branched polysaccharide is connected by p-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 7.0. 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 WO 94/02029 PCT/US93/06500 11 from a number of sources. One source, and method for preparing konjac flour, is disclosed in Marine Colloids Bulletin K-l, "Nutricol® Konjac Flour" (1989) (product and bulletin of FMC Corporation, Food Ingredient (formerly Marine Colloids) Division, Philadelphia, PA, 19103 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 pongeous 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 Cold-melt konjac and clarified konjac and their manufacture are disclosed in pending Unites States patent application IWo o,/oZ5 and/or W~0~05o0 i and their corresponding Patent Cooperation Treaty 94/02029 M942C/US93/06500 12 publications, the contents of which are incorporated herein by reference.

Component Aqueous Gel-Forminq Poysaccharide Among the component substances "agar", sometimes referred to as agar-agar, is a phycocolloid derived from red algae, such as GAlidi and aclara, 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 Arrageenan fractions, but is preferably predominantly, (and most preferably entirely) the kappa fraction.

CoaIponent Optional Water-Soluble Polysaccharides Not all polysaccharides are suitable as the optional third component As noted in the following examples, it was determined that czitosan 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 when present at 5.0 dry veight, but work poorly or not at all at 50.0 dry

~F

WO 94/0jl2029) I'C'r/cS93/0650 13 weight. These polysa.charides therefore are less desirable as component 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, 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 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 comnressed, 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 O 94/02029' PCT/US93/06500 14 the'n 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 riot 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 WVO 94/02029 PCT/US93/06500 15 matrices in sterile form is as surgical sponges and in the external treatment or packing of surface wound, or conditions, for example as ear packings or for ,ound 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 ifter 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 WO 94/02029) 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 freeae-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 2-propanol.

The dry spongeous matricec of the invention can be sterilized as desired by using heat 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 waterinsoluble) known agents.

EXAMPLES

The following examples are given to further illustrate the invention.

Example 1 Konjac/Aqarose Composite Spongeous Matrices A 1% w/v clarified konjac sol was divided into a _I WO 94/02029 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 w/v, 1%, and and Seakem Gold agarose and [Seakem® agarose products are manufactured by FMC BioProducts, Rockland, Maine 04841 Each sample was mixed with 2 ml 5M NH 4 0H, 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% agarosecontaining spongeous matrix were soaked in 83% 2propanol, drained and then dried for an hour at 80 0

C.

When placed back in water, they quickly rehydrated and filled with water.

Example 2 Koniac/Agar Composite Spongeous Matrices Crude konjac flour was used to prepare two 100 ml samples of a 1% aqueous sol. One gram of Gracilariaderived 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: 0.5/1% WO 94/02029 PCT/US93/06500 18 and were all mixed with 0.375 ml of a IM K 2

CO

3 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 examined. 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/l% agar matrix was the fastest to swell while the matrix was the slowest.

Example 3 Koniac/Aqarose Composite Spongeous 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 1M K 2

CO

3 in a boiling water bath. The gel was placed in a freezer at 0 C for 1.5 hours and then transferred to a freezer at 0 C for 1 hour. The sample was placed in a lyophilizer on a glass plate and dried under vacuum 0.1 Torr) with a shelf temperature of 100 0 F (about 38 0 After 3 days the sample was removed and weighed (3.59 The resulting spongeous matrix readily hydrated and filled when placed in water. It had numerous small uniform cavities.

WO 94/02029 PC '9)6500 19 Example 4 Process Variations in Koniac/Acarose Matrix Formation The composite spongeous matrices of Examples 1-3 were formed by gelling the konjac component 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/l% 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

K

2

CO

3 solution but was iced immediately afterward to rapidly set the agarose matrix. Once gelled, the sample was heated in a hot water bath (63 0 C 70 0 C) 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 Effects of pH during 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 NH 4 0H 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 5M 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: WO 94/02029 PCT/US93/06500 20 Alkali Concentration Volume (mL) pH

NH

4 OH 5M 0.1 9.31 I" 0.5 9.60 i" 1.0 10.15 i" nI 2.0 10.40 I" r 4.0 10.47 NaOH 5M 0.1 11.40 II" ""Il 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 Spongeous 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 0.75% or 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: and 1%/0.75%.

Intermediate samples were and 0.75%/0.75%. The remaining matrices were generally soft and shrunken, remaining flat after squeezing, but swelling when placed in water.

WO 94/02029 PCT/US93/06500 21 Example 7 A. Sponceous 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 accuratelyweighed. 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 Weight 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 Spongeous Matrix Stability In Bo ling 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 ml of 1MK 2

CO

3 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 coldmelt konjac. This cold-melt sol was filtered through a cloth filter to remove debris such as the sacs which are found in konjac flour. One of the dry spongeous WO 94/02029 PCT17S93/06500 22 matrices was placed in this filtered cold-melt sol coating material and allowed to hydrate. The matrix was massaged to remove 'r bubbles and to allow thorough coating. The spongeous matrix was removed, squeezed to remove excess liquid and hardened briefly in 99% 2-propanol. The matrix was transferred to a 92°C 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 75°C 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 75°C. The dried matrices were reweighed to calculate the weight loss due to leaching.

Sample Initial Weight Final Weight Weight Loss Weight Loss Uncoated 1.678 g 1.039 g 0.639 g 38% Coated 1.849 g 1.481 g 0,369 g 19% Example 8 Three Component Spongeous Matrices Two 50 ml samples of 1% konjac sol (from c. ide 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 carrageenan. The fir:t sample was gelled by heating in a boiling water bath for 20 minutes after the addition of 0.5 g KC1 and 0.375 ml 1M K 2

CO

3 To the second sample, 0.25 g agar and 0.25 g soluble starch were mixed in and dissolved. This was gelled by adding 0.375 ml of 1M K 2

CO

3 and heating as with the first sample. The final gel composition was WO 94/02029 PCT/US93/06500 23 1% konjac, 0.5% agar and 0.5% starch.

The gels were frozen and thawed as described in Exal.ple 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 Koniac/Kappa Carraqeenan Composite Sponqeous Matrices A. An aqueous sol of 1% konjac (from crude konjac flour) containing 3% kappa carrageenan was prepared and divided into three 200 ml aliquots. To the first aliquot, 2 g KC1 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 1M K 2

CO

3 (for konjac gelation) and 0.89% KC1 (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 1.0 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 (KC1 and K 2 CO3) was white, rubbery, contained few cavities and had a thin skin.

WO 94/02029 PL'.'393/06500 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 aqueous gels containing konjac (from crude konjac flour), in increments of 0 2% (0.2 were prepared as described above. One set of gels was set with KCl only while the second was gelled with KCl, K 2

CO

3 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 nct rebound after squeezing.) The cavities were ir':egular 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 .lat after a squeeze/release cycle (except the sample which contained 0.6% konja-) but would swell to full size in water. The sponge containing 0.2% konjac was weak and partially broke up when squeezed. 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: WO 94/02029 PCT/US93/06500 25 initial weight 3.064 g final weight 2.754 g percentage loss 22.7%.

The dried matrix was then placed in water and observed.

The matrix was slimy with a thick rubber feel and possessed no sponge-like char-cteristics.

Example Koniac/Gellan Gum Composite Sponqeous Matrix On hundred ml of a 2% hot solution of gellan gum was admixed with 100 ml of a 2% hot solution of konjac, to prepare 200 ml of l%konjac 1% gellan.

The mixture was cooled to 60 0 C and 1.5 ml of 1M K 2 C0 3 and MgSO 4 *7120 were admixed therein. A 150 mg portion of the sample was introduced into a 6.5 x 6.5 x 10 cm plastic contaj.ier. The sample was heated in an 85 0

C

water bath for 60 minutes. Then it was removed and cooled at room temperature overnight, after which it was frozen for 24 hours at -15 0 C. The sample was thawed at room temperature and examined. It was fully spongecus. Although the sample was onl' about 75% of its initial size, when squeezed and thawed it rebounded fully at a moderate rate.

Example 11 Koniac/Sodium Alqinate Composite Matrices Two hundred ml of 1% konjac 1 sodium alginate mixture was prepared by admixing 100 ml of a 2 hot solution of sodium alginate with 100 ml of a 2% hot solution of konjac. The sol was cooled to 0 C and 1.5 ml of 1M K 2 C0 3 and 0.4 g CaC1 2 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 WO 94/02029 2I'C US93/0650 26 minutes in an 85 0 C water bath. The gel was removed and cooled overnight at room temperature and then frozen for 24 hours at -15 0 C. The sample was thawed at room temperature and examined. 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 K 2 C0 3 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% CaCl 2 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 1M K 2 C0 3 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 85°C hot water bath for 35 minutes. The sample was frozen at -15 0

C

overnight and thawed slowly at room temperature. The resulting matrix was squeezed flat, place 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 WO 94/02029 CI'rUS93/06500 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 55 0

C.

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 examine the root system and sponge. Roots which had already penetrated the skin had begun to develop smaller tap roots whereas those still within tne 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)/l% 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 WO 94/02029 I'C MUS93/06500 28 plant 13 cm in 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 Spongeous Matrices prepared varying Component (c) A solution of 1% glucomannan as crude konja" [Component containing 0.75% agar [Component was prepared by dissolving 15 g konjac and 12 g agar in 1 distilled water over 60 minutes in a hot water bath. The sample was split into 100 g aliquots.

Various Component gums were incorporated as dry powders e:.ther by adding 0.088 g w/w based on konjac and agar) or 0.875 g (50% Each aliquot was stirred and heated to effect dissolution. Samples were cooled slightly and 0.75 ml of 1M K2CO 3 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 Cone. Observations Guar gum 5% spongy; slow, incomplete swelling when placed in water, no rebound after 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% I" Arabic, gum 5% spongy; swelled quickly in water; no rebound after squeeze/release WO 94/02029 PCT/US93/06500 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 Using Only Koniac Three 90 ml aliquots of 1% clarified konjac sol were prepared and then gelled by adding 3.6 ml of

NH

4 0H 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 coldmelt sol was adjusted to pH 3.5 with 5M HCl and then frozen. All samples were frozen overnight (18 hours) at x -15°C. 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 WO 94/02029 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 Effect of Concentration on Freeze/Thaw Behavior in Matrices Comprising Only Koniac Three crude konjac sols were prepared at 2% and 3% concentrations. The 1% sample (150 ml volume) was mixed with 1.5 ml 1M K 2

CO

3 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 examined. 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 essentially 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 or no liquid expressed. There were no cavities or pores visible.

Comparison Example C Drying and Rehydration of Matrices Comprising Only Koniac Duplicate 50 ml samples of 2% and 4% clarified konjac were gelled with NH 4 0H and heat. The gels were WO 94/02029 PCT/US13/06500 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 2-propanol. The samples were then dried in a 55 0

C

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.

Comparison Example D Attempt At Matrix Formation Using 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.

Comparison Example E Attempts at Matrix Formation Using Only Agarose Gel Concentration Variations 100 Ml samples of 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 samples were WO 94/02029 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 or released very little water. Both were soft, yet somewhat brittle and thus broke up very easily.

Comparison Example F Attempt At Matrix Formation Using Only Aqar Three agar gels were prepared at 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 KapDa-Carraaeenan WO 94/02029 PCT/US93/06500 33 Two samples of kappa-carrageenan were prepared by dispersing 1 and 2 gram quantities into 100 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 geous matrix was transparent and possessed very Le 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 Spongeous Matrix Formation Using Other Materials As Component (b) A number of other materials were tried as potential Component 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 and The samples were prepared by dissolving the other material in a prepared solution of konjac, adding alkali and heating for minutes in a boiling water bath. The gels were then frozen and thawed as in Example 1.

WO 94/02029 PCT/US93/06500 34 H(1) 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) Polyvinyl Alcohol (PVA) 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 25°C) 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.

WO 94/02029 PCT/US93/06500 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) Polvethylene-Polyoxypropylene Block Copolvmer In this series of gels, Pluronic® F-127 polyethylene-polyoxypropylene block co-polymer (BASF Corp.; Parsippany, 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 and A duplicate of the 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) Carboxymethvl 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 was observed to be mushy, was not squeezable, and there WO 94/02029 PCT/US93/06500 36 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) Polvacrylamide (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 1M

K

2

CO

3 12.5 p1 N,N,N',N'-tetramethylethylenediamine (TEMED) and 250 p 1 of 10% ammonium persulfate (both from FMC Corporation, Philadelphia, PA) were added and the samplu gelled for 20 minutes in a hot water bath 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.

Comparison Example I Inhibition of Matrix formation in Composite Gels I(1) Addition of 10% NaCI A 1% konjac/l% Seakem LE agarose sol (200 ml) was prepared as described in Example 1. To this, 20 g w/v) NaCl was added. The sample was gelled by adding 1.5 ml 1M K 2 C0 3 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 WO 94/02029 PCT/US93/06500 37 frozen again at -80 0 C overnight and thawed. There was some very slight fracturing but no sponge-like properties.

1(2) Addition of 5% Glycerol A 200 ml solution of 1% konjac/l% agar was prepared to which 10 g w/v) of glycerol (EM Science; Cherry Hill, was added. The sample was gelled by adding ml of 1M K 2 C0 3 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.

Claims (36)

1. An article of manufacture which is a spongy matrix formed from a coprocessed mixture comprising: deacetylated glucomannan present in one part by dry weigA', and at least one other aqueous gel-f( ning polysaccharide selected from agar, agaroid, agarose, alginate, carrageenan, curdlan, gellan, pectin, a gel-forming chemical derivative of the foregoing, or a mixture thereof, present in 0.125 to 8.0 parts by dry weight;

2. The article of claim 1 which further comprises one or more water soluule polysaccharides in non-gelling form, which may be present in up to 200% by dry weight compared to the dry weight of and

3. The article of claims 1 or 2 characterized in that is konjac-derived, deacetylated glucomannan.

4. The article of any one of claims 1 to 3 characterized in that is agar, agarose, carrageenan, or a mixture thereof.

5. The article of manufacture of any one of claims 1 to 4 characterized in that is present and is in at least 0.5% by dry weight comrared to the combined dry weight of and and is guar gum, gum arabia, karaya gum, locust bean gum, starch, tragacanth, or a mixture thereof.

6. The article of manufacture of any one of claims 1 to 5 characterized in that is gual gum, locust bean gum, starch, or a mixture thereof.

7. The article of manufacture of claim 6 characterized in that is starch. o 39

8. The article of manufacture of claim 6 characterized in that is starch and is present in 20.0 to 75.0 by dry weight compared to the combined dry weight of and

9. The ar. cle of manufacture of any one of claims 1 to 8 chare,, rized in that is a substantially undegraded konjac. The article of manufacture of any one of claims 1 to 9 charactenzed in that is present in 0.25 to 4.0 parts by dry weight.

11. The article of manufacture of claim 10 characterized in that is present in 0.5 to 2.0 parts by dry weight.

12. The article of manufacture of any one of claims 1 to 11 characterized in that said spongeous matrix is at least partially saturated with a carriet medium and a carried substance which is medium-soluble, medium-dispersible, or medium-borne particulate matter.

13. The article of manufacture of claim 12 characterized in that said carrier medium ic water.

14. The article of manufacture of claim 13 in which said carrier medium has been at least partially removed.

15. The article of manuft.ture of any one of claims 12 to 15 characterized in that the carried substance is a: nutrient medium, reagent, pharmaceutical, flavoring, color, scent, cosmetic, air freshener, deodorant, adjuvant to any of the foregoing, affinity or ion-exchange particulat.:, cell or cellular particulate, S activated charcoal, or mixture thereof.

16. The article of manufacture of any one of claims 1 to 15 comprising a biodegradable sponge.

17. The article of manufacture of any one of claims 1 to 16 in substantially dry form.

18. The article of manufacture of claim 1 or 12 coated with a material selected from: i' r. a'erial other than that comprising said spongeous matrix, or to same material that comprises said spongeous matrix but in a non-spongeous form,

19. The article of manufacture of claim 18 characterized in that said coating is water-insoluble. The article of manufacture of claim 18 or 19 characterized in that said coating is water-permeable.

21. A process for fabricating an article of manufacture which is a spongeous matrix formed from a coprocessed mixture of the components: deacetylated glucomannan present in one part by dry weight, and at least one other aqueous gel-forming polysaccharide selected S; from agar, agaroid, agarose, alginate, carrageenan, curdlan, Sgellan, pectin, a gel-forming chemical derivative of the foregoing, or a mixture thereof, present in 0.125 to 8.0 parts by dry weight; S. said process characterized by the sequential steps of: A forming an aqueous sol comprising components and (b) present in a ratio a:b of 1:0.125-8.0 by dry weight; B gelling said aqueous sol by addition of sufficient base to result in a sol pH above 7.0, and optionally a gelling agent for component (a) or component C freezing said gel; and D thawing said gel to form said spongeous matrix. 41

22. The process of claim 21 characterized in that component is present in said spongeous matrix.

23. A process according to claim 21 or 22 whrein the spongeous matrix includes a component being one or more water soluble polysaccharides being in non-gelling form, which may be present in up to 200 by dry weight compared to the combined dry weight of and and wherein in step A the aqueous sol is formed from components and

24. The process of any one of claims 20 to 23 characterized in that the respective concentrations in water of components and independently are 0.25 to 2.0 wt based upon the total weight of said sol. The process of any one of claims 21 to 23 characterized in that the respective concentrations in water of components and independently are to 2.0 wt based upon the total weight of said sol.

26. The process of any one of claims 21 to 23 characterized in that the respective concentrations in water of components and independently are 0.5 to 1.0 wt based upon the total weight of said sol.

27. The process of any one of claims 21 to 26 characterized in that component consists of: agar, agaroid, agarose, alginate, carrageenan, curdlan, gellan, pectin, gel-forming chemical derivatives of the foregoing, or a mixture thereof.

28. The process of claim 27 characterized in that component is agar, agarose, carrageenan, or a mixture thereof.

29. The process of any one of claims 21 to 28 characterized in that the pH of said sol is adjusted to 9.0 to 12.0 by the addition of said base. The process of claim 29 characterized in that said base is ammonium hydroxide, an alkali metal hydroxide or carbonate, or an alkaline earth metal hydroxide or carbonate.

31. The process of claim 29 characterized in that said base is ammonium hydroxide, potassium carbonate, potassium hydroxide, sodium carbonate, or sodium hydroxide.

32. The process of claim 29 characterized in that said base is potassium carbonate.

33. The process of any one of claims 21 to 32 characterized in that the spongeous matrix is dewatered after step D.

34. The process of claim 33 characterized in that the spongeous matrix is dewatered until substantially dry, as hereinbefore defined. The process of claim 34 characterized in that the substantially dry spongeous matrix is sterilized. S36. A method for the growth or maintenance of a plant material characterized by the steps of: A. adding an aqueous nutrient medium to a spongeous matrix according to any one of claims 1 to 3; B. inserting the plant material into the nutrient-containing spongeous matrix; and C. growing or maintaining in viable condition the plant material in the spongeous matrix.

37. The method of claim 36 characterized in that said plant material is grown and comprises a callus, seed, embryo, explant, graft, or young plant. I\1e 1( 4 I 43

38. The method of claim 37 characterized in that the plant-containing spongeous matrix is placed into a soil-containing growth medium following Step C.

39. The method of claim 37 characterized by the intermittent or continuous addition of nutrients to the aqueous nutrient medium in the spongeous matrix. A surgical sponge characterized in that it comprises a sterilized biodegradable spongeous matrix according to any one of claims 1 to

41. A surgical sponge according to claim 40 further characterized in that it is coated with or contains at least one physiologically active substance.

42. A method for the packaging of one or more articles for storage or shipment including the step of packing the articles in a biodegradable material comprising one or more spongeous matrices according to any one of claims 1 to DATED this 4th day of September, 1996. FMC CORPORATION WATERMARK PATENT TRADEMARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA RHB:MED:JZ (DOC.8) AU4672093.WPC i *ii INTERNATIONAL SEARCH REPORT International iiplicatin Not. PCT/US93/06500 A. CLASSIFICATION OF SUBJECT MATT'ER :Please See Extrz Sheet. US CL :424/488, 426; 604/364, 368; 47/58, 80; 426/573, 46; 252/315.3 According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) U.S. 424/488, 426; 604/364, 368; 47/58, 80; 426/573, 46; 2521315.3 Documentation searched other than minimum documcntation to the extent that such documents are included in the fields searched Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) APS/JPO FREEZ? AND (GLUCOMANNAN# or Konja?) C. DOCUMENTIS CONSIDERED TO BE RELEVANT Categorya' Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. X Patent Abstracts of Japan, Group No. 00874, Vol. 15, No, 1-3, 22, 398, 09 October 1991, J03-1 64149 34, 35, 43 Y 4-11, 16, 17, 22, 28 X Patent Abstracts of Japan, Group No. C635, Vol. 13, No. 1, 22, 43 414, -13 September 1989, J01 -1 y 2-11, 23-33 X Patent Abstracts of Japan, Group No. 0470 Vol. 1 2, No. 22, 1, 43 22 January 1988, J62-1 75149 Y 2-11, 22-33 [j Further documents are listed in the continuation of Box C. See patent family annex. Specia categories of cited docuzments: *r Later docu ment published after the international fitig date or priority date sanl not in conflict with the application but cited to understand the docizmcutdefininr the general state ofthe ant which is not considered principle or theory underlying the invention to be pan o particular relevance X. document of particular relevance', the claimed invention cannot be earlier document publishls on or after the international fiing date consiered novel or cannot be considered to involve an inventive step *L document which may throw doubts on priority claim(s) or which is when the document is taken alone cited to establish the publication dame ol another citation or other Y. d n fpriua eeacl h lie neto antb aiecil ~considered to involve an inventive step when the document is .0 do..'tmsenz referring to an oral disclosure, use. exhibito or other combined with one or mome other such documents, such combination tt1.010 baing obvioua to a person skilld in the aut .P document publiscd prior to the international filing date but later than document memzber ofthe samne patent family the priority date claimed Date of the actual completion of the international search Date of mailing of the international search report DECEMBER 1993 O5A N 9 Name and mailing address of the ISAJUS Authorized officer ~1 Comniasiotncr of Patents and Trademnarks Box PCT A IL EZ AE Washington, D.C. 20231DAILSM TZ AE Facsimile No. NOT APPLICABLE ITelephone No. (703) 308-0451 Form PCTIISAI2IO (second sheet)(July 1992)* INTERNATIONAL SEARCH REPORT S.manatonal application No PCT/US93/06500 C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. Patent Abstracts of Japan, Group No. C440, Vol. 11, No. 251, 14 August 1987, J62-55052 Patent Abstracts of Japan, Group No. C399, Vol. 11, No. 24, 23 January 1987, J61-199758 US, A, 4,623,394 (Nakamura et al.) 18 November 1986, See entire document US, A, 4,943,444 (Nozaki et al.) 24 July 1990, See entire document, claims in particular. Patent Abstracts Japan, Group No. C256, Vol. 8, No. 273, 13 December 1984, J59-146560 Derwent Abstract, AN 92-170036/21, 18 March 1992, JPO

4084868-A US, A, 4,769,945 (Motoyama et al.) 13 September 1988 (note col. 2, line 63 to col. 4, line 37) US, A, 3,653,383 (Wise) 04 April 1972, See entire document US, A, 4,676,976 (Toba et al.) 30 June 1987, See entire document FMC Marine Colloids Nutrocol Konjac Flour, (1989) FMC Corporation Philadelphia, PA, USA 1, 43 2-11, 16, 34-35, 22-33 1-3, 43 2-11, 22-35 12-21 22-36 37-40 41-42 1-43 1-43 Form PCT/ISA/210 (contmuation of second sheet)(July 1992) Form PCT/15A1210 (contmnuation of second shct)(Julv 1992)« INTERNATIONAL SEARCH REPORT Inter onal application No PCT/US93/06500 Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet) This international report has not been established in respect of cenain claims under Article 17(2)(a) for the following reasons: 1. FO Claims Nos.: Sbecause they relate to subject matter not required to be searched by this Authority, namely: 2. Claims Nos.: Sbecause they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically: 3. C Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a). Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet) This International Searching Authority found multiple inventions in this international application, as follows: Please See Extra Sheet. 1. O- As all required additional search fees were timel. paid by the applicant. this international search report covers all searchable claims. 2. As all searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment of any additional fee. 3. As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims for which fees were paid, specifically claims Nos.: 4. O No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claims Nos.: Remark on Protest S The additional search fees were accompanied by the applicant's protest. O No protest accompanied the payment of additional search fees. Form PCT/ISA/210 (continuation of first sheet(l))(July 1992)* f INTERNATIONAL SEARCH REPORT litrnational application No. PCT/US93/06500 A. CLASSIFICATION OF SUBJECT MATTER: IPC A61F 13/15; A61K 9/14; A01C A01G 31/00 BOX II. OBSERVATIONS WHERE UNITY OF INVENTION WAS LACKING This ISA found multiple inventions as follows: I. Claims 1-11, 16-17, 22-36, and 43 are drawn to a sponge matrix and a method of making said sponge matrix, classified in Class 424, subclass 488 II. Claims 12-15, 18-21, and 41-42 are drawn to a sponge matrix impregnated with a substance, a sponge matrix coated with a coating, a surgical sponge which is coated or impregnated, classified in Class 604, subclass 634. III. Claims 37-40 are drawn to a method of growth maintenance of the plant material, classified in Class 47, subclass 58. Unity exist between claims in group I involving a sponge matrix and methods of making said matrix. The claims of group II lack unity with the claims of group I which does not include the special technical features further included in the compositions of Group II. The claims of Group III lack unity as corresponding to one of multiple uses of the spongeous matrices. Form PCT/ISA/210 (extra shee)(July 1992)*