CA2115141A1 - Clarified and cold-melt konjac glucomannan - Google Patents

Abstract

Clarified konjac glucomannan compositions substantially free of insoluble impurities, having a nitrogen content of no greater than about 0.60 wt %, and a turbidity potential as a 1.0 % aqueous sol of no greater than about 100 turbidity units as measured by the Formazin Turbidity Standard, as well as aqueous gels and sols, cold melt gels and spongy forms thereof. The resulting sols and gels may be used in foodstuffs, industrial biotechnical applications.

Description

2 1 i ~

CI,ARIFIED AND COLD--MELT RONJAC GLUCOMANNAN
This invention relates to clarified konjac (that is, purified glucomannan derived from konjac) and methods for preparing the same. It includes clarified konjac powders as well aæ sols and gels prepared therefrom. The clarified konjac glucomannan has enhanced purity and a low nitrogen content, and aqueous sols and gels thcreof have low turbidity. This invention also relates to aspects of the clarified 10 ~ konjac including a cold-melt product and to methods for making the above~products as well as varying the clarified konjac viscosity.
Konjac (Amorphophallus konjac) is a plant, the tuber of which is~the source of a well-known foodstuff 15~ in China and Japan, namely konjac~flour. This flour, which contains a~variety of insoluble materials described below~as well as a major~amount of desirable water-soluble substances, comprises a highly viscous sol of glucomannan~and solu le starches when 20 ~ reconstitutet~in wàter. The principal sol~uble conætituent~is~glucomannan, a polysaccharide comprised of~D-glucose;~and D-mannose,~ which is useful as an ingrediènt~in various foodstuffs, as well as in industrial~applications such~as~films, oil drilling 25 ~ fluids, and~paints. ~
There~are~numerous impurities in crude (native, unclarified~)~konjac flour, principally insoluble starches,~cellulose, and nitrogen-containing materials, including proteins, many of which impurities are ~derived from ~sacs" which~encapsulate the konjac flour in the tuber.~ As a result, the sols and gels of crude konjac flour~haYe~a highly turbid~, milky-white or cloudy appearance (due to water-swollen particulate impurities). ~ ~
U.S. Patent 3,928,3~22 to~Sugiyama et al. ~and U.S.
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3,973,008, which is c~mulative thereto) disclose a method for producing konjac mannan polysaccharide, i.e.
glucomannan, which comprises the principal ingredient ~: : of konjac flour, from raw konjac flour by first - 5 removing insolubIe components from an aqueous konjac flour sol by filtration or other conventional means, thereafter dialyzing the sol and subjecting the : resulting liquid~to freeze-drying to obtain a turbid, : : : cotton-like,~ low density fibrous product which is hard . 10 ~ to grind:and~poorly soluble in water.
Japanese~Patent Disclosure 01-49657, filed March 1, 1989, discloses~a konjac mannan product which has a nitrogenous component of not more than 0.2%. However, the method of::achieving this reduced nitrogen content ; 15 is not disclosed ~but appears to be by s~.mple dilution.
. U.S. Patent:~2,144,522 teaches a method for ; decolorizing~:and;.clarifying galactomannan gum sols such as locust bean gum which comprises contacting the gum sol with~activated carbon~in~the presence of aluminum 20` ~sulfate, the latter being added in amounts~ sufficient :to:form a double Al-Na~salt with sodium sulfate which .:is~-intrinsically~present~in~the activated carbon U~.S. Patent~3,346,556 discloses a method for 25~ preventing~the;degradation of galactomannan gums such as locust~bean~gum~resulting from~heat or pH changes :. :which~comprises~adding to aqueous gum sols polar organic oxygen-containing hydrophilic stabilizers such as alcohols,~ glycols, ketones or the like. Incidental to this proàess there is disclosed in one example : : (Example 5) à means for~clarifying locust bean gum by the conventional~:use:of~a fi~lter aid such as diatomaceous earth.
Japanese Patent:Disclosures 59-227,267 (Dec. 20, 1984), and 58-165,758 (Sept. 30, 1983) disclose methods , 2 ~

for treating aqueous sols of crude konjac flour with certain salts at pH's of 10 or below to obtain an insoluble form of konjac, principally for use as insoluble food products.
Japanese Patent Disclosure 63-68054 (March 26, 1988), discloses a reversibly soluble konjac gel product, but not the removal of insolubles which remain :
present in the product.
Gels formed from combinations of glucomannan derived from~crude konjac with other hydrocolloids, particularly polysaccharides such as carrageenan or xanthan gu~s~,~ are already known in the art. See, for example U.S. Patent 4,427,704.
This inventi~on provides dry clarified konjac glucomannan of low~nitrogen content; aqueous sols and gels thereof;~and methods for preparing each of the above products. The invention further provides:
methods for varying the viscosity potential of the clarified~konjac; co-processed hydrocolloids for~ed by 2~0~ ~combining the~clarified konjac with selected hydrocolloid~gums~; a cold-melt clarified konjac gel;
and further method and product variations.
The~term~-clarified" kon~ac, as used herein, refers to a konjac~glucomannnan which~is substantially free of 25~ insoluble~impurities, which has~a }ower nitrogen content than unclarified konjac, and which exhibits a lower;turbidity~than unclarified konjac when in the form of an aqueous~sol or gel. The term "crude"
konjac, as used~herein, refers to an unclarified or native kon~ac flour in which the glucomannan is still contained ~in the~sacs in which it occurs in nature, and ; various other impurities may be present.
, ~ ~
~ Generally, this invention encompasses clarified ~, ~
konjac characterized in that it comprises glucomannan ~ 35 derived from~konjac which is substantially free of :: :

WO93/~2571 PCT/US92/0659l ,,~. ,r; ~ ,S ~ ~' insoluble impurities, and has a nitrogen content of o to 0.60 wt % accompanied by an aqueous sol turbidity potential of 20 to 70 Turbidity Units as well as a continuum of a nitrogen content of 0 to 0.25 %
accompanied by an aqueous sol turbidity potential of 70 to 100 Turbidity Units.
In a first group of embodiments this invention provides clarified Xonjac characterized in that it co~prises glucomannan derived from konjac which is substantially free of insoluble impurities; and tA~ has ~; a nitrogen content of from more than 0.25 up to about , : ~
0.60 wt % and~an aqueous sol turbidity potential of from 20 to 70 turbidiey units as measured at 1.0 w/v %
concentration`using the Formazin Turbidity Standard; as ; 15 well as the continuum of tB~ a nitrogen content of 0.25 wt % or less,~and an aqueous sol turbidity potentiar of 20 to lO0 turbidity units as measured at l.0 w/v %
concentration~using the Formaz:in Turbidity Standard, of which ~B] is~preferred. More preferably, the clarified ~20 ~ konjac is characterized by a nitrogen content of 0.175 - ~ ~wt % or }ess~: and ~an aqueous sol turbidity potential of 20 to 70 turbidity units. Most preferably, the clarified konjac is~ characterized by a nitrogen content ; of 0.15~`wt~%~or less and an aqueous sol turbidity potential~of 2~0;to~60 turbidity units. This first group of embodiments also provides sols and gels of clarified konjac, cold-melt and spongy products, and methods for manufacturing the same.
`In a second group of embodiments this invention provides a clarified konjac characterized in that it comprises konjac-derived glucomannan which is substantially~free of insoluble impurities, has a ~;~nitrogen content of about 0.60 wt % or less, and has an aqueous sol turbidity potential of less than 20 turbidity units as measured at 1.0 w/v % concentration using the Formazin Turbidity Standard; as well as a method for preparing the same. Preferably, the clarified konjac is characterized by a nitrogen content of no greater than about 0.25%. MQre preferably the clarified konjac is characterized by a nitrogen content of no greater than about 0.175%. This second group of embodiments also provides sols and gels of this clarified konjac and a method for its manufacture.
The clarified~konjac of this invention also is characterized by~an aqueous sol viscosity potential of about 50 to 25,000 cps at a l w/v ~ concentration as ~ measured using a~Brookfield Viscometer Model LVTDV-II
;~ at 25C and 20 rpm, preferably a viscosity of about 1,000 to 25~,000~cps.
; 15 Generally, the method for the production of clarified konjàc~of this invention is characterize* by the consecutive~steps of: ~a~ preparing an aqueoUs sol of crude konjac comprising insoluble impurities and glucomannan;~t~b] contacting the crude konjac sol with ;;20~ an extraction-effective amount of an agent- capable of extracting~the~insoluble~impurities; [c] precipitating and~remo~ing~the insoluble impurities; ~d] forming a glucomannan~coagulate by~treating the remaining aqueous sol with a coagulant present in an amount sufficient to 2~5~ ~coagulate~æu~stantialIy all glucQmannan therein; and e]~removing~and~drying~the gIucomannan coagulate to rocover the~ ~ , clarified qlucomannan.
In the first group of embodiments, the clarified konjac of this invention~may be prepared by dispersing the konjac flour in water, and treating the resulting glucomannan~dispersion with one or more reagents together or sequent~ially, to~extract by aggregation, precipitation, or absorption~of the impurities present.
~ Such impurities are principally naturally-occurring in ;~ 3s the konjac tuber and comprise nitrogenous materials ~: ~
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WO93/0~71 PCT/US92/0659}

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such as proteins, insoluble fibers, and starches.
Extraction is then followed by separating the impurities from the dispersion, coagulating the resulting glucomannan from the remaining dispersion by the addition of a water-miscible coagulant such as an alcohol, and drying and grind ing the resulting coagulate to form the clarified konjac of this ;~ invention in powder form. These methods are particularly advantageous in that they can be completed sign~ficantly faster than known prior methods.
~: : ; In the~second group of embodiments, the method for production of the~clarified konjac is characterized by the steps of:
~a~ preparing:an aqueous sol of crude konjac comprising insoluble impurities~and glucomannan;
tb] contacting the crude konjac sol with an extraction salt seiected from one or more of dicalcium phosphate:~:calcium phosphate, magnesium phosphate, and aluminum~sulfate~(preferably calcium sulfate and aluminum;sulfa~te,~more preferably aluminu~ suifate) in an~amount;ef~fective to:extract the insoluble impurities by~precipitation;~
c~ precipitating and~removing the insoluble purities~
;td];forming a~glucomannan coagulate by treating the remaining~aqueous~:sol with isopropyl alcohol present in an amount~sufficient to coagulate substantially all ~:~ glucomannan;thérein; and~
e] removing and drying the glucomannan coagulate to recover the:clarified glucomannan.
:: OptionallQ~, clarified konjac sols, alone or with other components, may be further converted into : correspondingIy pure gels by known methods, such as by : addition of an alkali. The resulting gels may then be : 35 used in or as foodstuffs or in industrial compositions WO93~02571 PCT/US92/06591 2 1 ~

such as paints and other coatings.
;The clarified konjac gels have the unusual property of liquifying within specific low temperature ranges.
This is quite the reverse of the normal behavior of most hydrocolloid gels. Moreover, when cooled still further ?nd~then brought back to ambient temperature, the clarified konjac forms fibrous, porous, spongy, yet gel-like structures which, when compressed, rebound to their original form, and thus can serve as sponges to take up liguids and transport them to desired sites, such as to cells,~seeds, calli, or plantlets placed within them.;
The inventive methods afford additional advantages over unclarified (crude) konjac flour, namely improved ~ , ~
i~ 15 odor, color, solubility, and grindability. Crude konjac has a known distinct odor, and a tan to dark~
brown color (as~a dry~powder). Furthermore, crude konjac particles are not uniform in size and cannot be ground at~nor~al milling temperatures. Milling or 20 ~ other such grinding of crude konjac produces high temperatures~which destroy its viscosity potential in much the same way as~dry~heat degradation, and which ~ contribute to~its dark color.~ By contrast, the `~ ; clarified konjac of this~invention is a white powder 25~ ~which forms~a~clear sol, is odor-free and can readily be ground to~a uniform size. ~Additionally, clarified konjac~is~more~unifor~ in glucomannan content, and thus avoids the wide,;~ uncontrolled variations in viscosity or gel strength~which-occur with crude konjac~
Another~desireable property of the clarified konjac powder of this invention~is that, unlike crude konjac powder, clarified konjac hydrates rapidly in room tempe~ature~water with little effort, thereby ~ ~ facilitating~the utiIization of konjac in various ; ~35 recipes as well as the r~apid preparation of sols of WOg3/02571 ~ i PCT/US92/06591 different viscosities.
Figure 1 compares the nitrogen and turbidity values of the clarified konjac of this invention obtained using various extraction agents, with those of prior art products, including crude konjac~
Figure 2 compares the W absorbance properties of clarified konjac with crude konjac.
Other than in the operating examples, or where othexwise indicated, all numbers expressing quantities ~ of ingredients, parameters, or reaction conditions.used herein are to be understood as modified in all instances by~the~term "about".
The clarification of konjac to obtain a more purified glucomannan than is available from crude ` 15 ~ konjac affords~;several benefits, the most important of ` ~ which ~is that clarified konjac sol is essentially clear, althou~ a clarified~konjac gel has some turbidity.~ Unexpectedly,~when the clarified konjac sol is mixed with selected other hydrocolloid sols, and the 20~ mixture;then;~gelled, there~is a synergistic reaction ;which prod w es~clear, thermally reversible gels. Such clear gels are particularly useful in forming desserts and for~biotechnical applications where a clear gel is àdvantageous~ Hydrocolloids~particularly useful for ;25~ synergi~stically combining with~clarified konjac include clarified~xanthan,~ locust bean gum, amy}ose and amylopectin~starches, and~carrageenan. Gel forming hydrocolloids`su~h as agarose are merely additive and not synergist;ic. It is notable that crude xanthan and 30~ AMF (seaweed~flour,~;sometimes sold as carrageenan) are not adequate for this purpose, because the combinations o not produced the desired clear gel. Clarified konjac gel alone is somewhat cloudy, although less cloudy than crude konjac gel.
~ 35 Another important benefit of clarified konjac over ; ~
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2 1 1 ;~
g crude konjac is that it is more stable as a dry powder.
For example, crude konjac stored for 4 weeks at above room temperature (50C) loses 80% of its aqueous sol viscosity potential. By contrast, clarified konjac stored for the same time and at the same temperature loses only about 20% of its viscosity potential. It is believed that the increased storage stability is the result of the denaturing of enzymes present in the crude material, both by the initial heating of the sol and by the subsequent alcohol wash during the clarification process. It is a further benefit of clarification;~that clarified konjac is more easily rehydratable than crude konjac.

15` Crude Koniac 8tart~ina Material Crude~Xonjac flour starting material is a commercia} product available from a number of sources.
One~source,~and~method for preparing konjac flour, is disclosed in~Marine Colloids Bulletin K-1, "NUTRICOL~
2~0 ; Konjac Flour"~ (1989) tproduct and bulletin-of FMC
Corporation,~Marine Colloids Division, Philadelphia, Pennsylvani~a,~19103 U.S.A.]. Basically, the prccess involves slicing,~drying and~then wet- or dry-milling the AmorphophalIus tuber, followed by pulverization of ;the resulting~ konjac~to a p:owder ("flourn) which is sifted~and~air~classified.~ The resulting flour, as described in~the~above publication, consists of fine, ~`~ ; oval, whitish~granules containing "flour sacs"~ that ~' is, the glucomannan is encapsulated in a protein/fiber coating. This flour, when~hydrated for some time with ` agitation releases the encapsulated glucomannan to form a sol which is characteri~ed principally by its high viscosity, even;at 1% concentrations, substantial turbidity, and high nitrogen content. Viscosities in the range of 8,000 cps at a~1% by weight sol up to ~ 3~ ~ i 130,000 cps at 3~ are typically obtained after a heat (85C) and cool cycle, as measured on a Brookfield~ RVT
~; Viscometer, and an appropriate spindle, at 20 rpm and 25C tthe viscometer is a product of Brookfield Engineering Laboratories, Inc. Stoughton, Mass., U.S.A.~. Conversion of Brookfield centipoise (cps) readings into viscosity functions are discussed by Mitschka, P. in;~ eoloaica Act~ 21:207-209 (1982). As used herein, centipoise (cps) is equivalent to milli-Pascals-second ~mP-s).
, The crude konjac turbidity may vary considerably, depending upon the~concentration of the sol, but in the above viscosity range of from 8,000 cps up to 130,000 cps and concentrations of 1~ to 3%, turbidities o~ 100 to 300 turbidity units are conventionally obtained at 0.5 wt. % concentration, based on the Formazin Turbidity Standard ~(FTS) - Method 180.1 in "Methods of Chemical Analysis of Water and~Wastes" by EPA
Environmental~Moni*oring and Support Lab; March, 1~79.
;20~ At these turbidities the sol is generally ~ery cloudy to milky in~appearance.
The high~nitrogen content of the initial crude konjac~flour~is essentially a function of the amount of impurities;~present,~principally the tuber's naturally-25~ occurring~protein~and~the sac fiber coating which ; encapsulates~the glucomannan. The nitrogen content of the dry crude~flour~is~typically in the range of 0.3 to 1.3 wt. % of~nitrogen, although higher percentages are possible depending upon the variety of tuber used.
Product Descri~tion As a measure~of the significantly reduced amount ofimpurities present, the clarified products of this invention are characterized principally by their low nitrogen content~ and low turbidity as an aqueous sol or -11- 211.

gel. The corresponding viscosity of the product, in sol form, is also characteristically at a high level, and it is not adversely affected by the majority of agents that may be employed in the extraction process.
When prepared by the various methods of this invention, the clarified konjac is substantial~y free of insoluble impurities~ having a nitrogen content and turbidity as low as possible. A 1.0% aqueous sol according to this invention should have no greater than 100 (preferably 70, more preferably 60), turbidity units, as measured by a Mac8eth Coloreye Computer, model 1500,~(Kollmorgen Corp., Newburgh, N.Y.), and a Formazin Standard; and has a nitrogen content, (based on the weight of the dry product used to prepare the 801), of generally no greater than 0.25 (preferably 0.175, more preferably 0.}5) wt %. Within these ra~ges the clarified konjac 801 iS substantially transparent in appearance~and may be us~ed in a number of applications,~ particularly in c}ear foodstuffs and 20~ biotechnical,~or biomedical / diagnostic applications, where a~clear,~particle-free gel is essential, or where a~hiqhly viscous material~-is desired.
The product~of~;this invention may further be desoribed as~having a very wide, non-critical range at 25~ 1.0 wt % aqueous~sol of viscos~ities of from 50 to 25,000 centipoises, (as ~easured on a Brookfield Model LVTDV~ vi~soometer at~60~rpm and 25C) depending upon how the product is prepared. In general, the clarified product inherently has high viscosities, i.e. from :
30 ~ 1,000 to 25,000 cps, which are particularly useful for food formulations~,~ but this viscosity can be reduced to as little as~50 cps~by methods disclosed herein.
While the turbidity~of flour and product samples is generally determined by using visible light, ultra~iolet (W) light may also be employed to :
:
:::
: ~ :
~ ' ' WO93/02571 PCT/US9~/06591 '~ 1'1 i''l '~ 1. -characterize the clarified product and gauge the effectiveness of clarification procedures. This may be achieved by preparing 0.5~ sols of product, placing them in cuvettes and measuring their W absorbance between 200 and 320 nanometers (nm). Impurities, including DNA and protein, absorb W light in the 260-280 nm region and peaks in this area indicate their presence and relative amounts. As can be seen in Figure 2 and Table I, crude konjac samples contain a broad peak in this region and, overall, have a higher baseline of absorbance than clarified konjac samples, whîch lack the 260-280 peak. This is especially important for a~biotechnology separation medium where the presence of DNA or protein might interfere with performance.

TABLE I

Absorbance 2d Kon~ac Sam~le l A320 17~0 ¦- A260 ¦ A220 Crude ~ ~ 0.51400.8781 0.9634 2.6115 Clarified ~ ~ 0.06470.0967 0.1222 0.3118 ;25~ Method Descriptions The clarified~Xonjac of this invention may be prepared by~an aqueous extraction method comprising ~5 heating an aqueous sol of crude konjac flour containing insoluble impurities and contacting the heated sol with an extracting-effective amount of one or more extraction agents. The heating of the sol acts to break the natural sacs surrounding the glucomannan present in the crude konjac, and the extraction agent assists in removing protein impurities as well as the sacs themselves.

L '~ ~

The term "extracting" or "extraction", as used herein, means the separation of insoluble impurities from the konjac by aggregation, adsorption, precipitation or other means for rendering konjac flour substantially free of insoluble impurities.
Following the extraction, the 501 iS filtered to ~; remove the insoluble impurities, and the filtrate coagulated with a;~water-miscible coagulating agent such as isopropyl alcohol to recover the glucomannan ` 10 present. The coagulate is then dried and ground to particulate form,~to produce a clarified konjac flour according to this;invention.
The extraction~step may be varied somewhat depending upon the nature of the extraction agent ~employed and the~viscosity of the fi~al product d-sired.~ ~For example,~where the agent is a solid, ~t may be~b~ended with~the crude konjac flour~starting material, optionally with a filter aid, and the dry mixture~dispersed~with agit~tion into a sufficient 20 ~ a~ount of water~to~obtain the desired concentration of thé~resulti * ~clarified konjac glucomannan, 0.1 to 10 preferably~O.~5 to~3) wt~%~depending on the viscosity potential. ~
Alternatively,~ the ex~raction~agent may be added to 25~ th-~water either~before~or after the aqueous dispersal of~ the~flour,~ particularly~if an~acid is emp}oyed to ad~ust the~viscosity~of~the resulting prod~ct. While this dispersion may be carried out in water at ambient temperatures, preferably the water should be heated to 30~ temperatures~of ~from 70~to 10~0C, ~preferably 85 to 90C) for 15~to~60~minutes~or longer in order to speed up the process. ~emperatures, times, mixing rates and ` concentration of reactants may be varied routinely by those skilled in the art in order to optimize these ~ 35 operating conditions.
: ~ ~: : :

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WO93~02571 PCT/US92/06591 2 ~

Thereafter, the sol is filtered to remove the insoluble impurities, with or without a filter aid present. Filters such as glass wool, paper, cloth and fibrous mats may be used for this purpose, although any S filter which will remove insoluble particles is generally satisfactory. Filter aids which may be employed include per}ite and diatomaceous earth. The amount of filter aid is not critical, but is desirably employed in amounts of 1 to 5 times the weight of the konjac flour. The~filter cake is preferably then ; ~ washed with hot~water until no further clarified konjac glucomannan is;recovered.
The filtrate is next treated with a water-miscible coagulating agent for the glucomannan, and the 15~ coagulate recovered and dried. Useful coagulating agents include lower alcohols~such as methanol, ethanol, or~isopropyl alcohol or polar organic solvents such as acetone,~methylethyl ketone, or mixtures thereof. The amount of coagulating~agent is not 2;0~critical,;but it~should be added in~amounts sufficient to~recover the~glucomannan~from the sol generally in a weight ratio of~ 4~ or a volume~ratio of 2-3:1, coagulant:glucomannan. Alternatively,~ in place of coagulating~agents the~dry product~mày-be recovered 25~ ~d~irectly from sol~by such methods as freeze drying or spray drying.
The coagulate should be dried until it is capable .
of being ground to a fine powder. This may be : ,, i~ ; achieved, for example in a forced hot air oven at 30 ambient temperatures,~or even higher if viscosity reduction is desired. The resulting dry product is then ground to~;form partioles of desired size, preferably capable of passing through a 100 mesh (149 micron) screen.
~ 35 While the clarified, low-nitrogen product may be : ~ .

used in its dry, particulate form, for example in absorbent or texturizing applications, preferably it is used in sol form by redispersing the particles in water. The resulting clear sol may then be gelled in known manner and/or as described herein. The desired percent concentration of the dry composition in a sol or gel will depend largely on its intended use and its viscosity. Generally, O.l to lO (preferably 0.5 to 3.0) wt: %, based on the total weight of the sol may be ~0 employed, although these amounts are not critical. In addition, unless the process includes deliberate steps to reduce the viscosity of the product, it will ~ normally maintain a very high viscosity despite the ;~ several treatment~steps described herein.
As a further advantage of this invention, this clarified konjac sol normally develops a high viscosity, generally in the range of from l,OOO to 25,000 cps,~ at 1.0 w/v % (l g/lOO ml water) concentration and~25C, as measured on a Brookfield Viscometer, Model LVT DV-II, with a suitable spindle at 2~rpm or on a~model RVT at 20 rpm. Additionally, because~of~the rapid hydration properties of the clarlfied dry powder, it develops this viscosity rapidly.~ For~example, when~unc1arified konjac flour is 2~5 ~ dispersed in water to form a 1 wt % sol, the hydration step normally~takes about two hours at room temperature to~;form~a so1~of~;desired viscosity~ By contrast, under substantially the same conditions, hydration of the product of this~in~ention takes about 30 minutes to achieve~the same YisCosity~ Optionally, when desired, the viscosity~may be reduced to as low as about 50 cps at l w/v % and at 25C~
The resulting sol may~then readily be converted to a gel by known~means, for example by addition of an alkali such as K2C03 followed by heating. Unless the ~:

WO93/02~71 PCT/US92/06591 3~ 4 1 degree of polymerization has been deliberately modified during the processing, as described below, these gels generally possess~a 1% gel strength at 85C of from 100 to 31Og/cm2, when measured by a Marine Colloids Gel S Tester GT-2. (FMC Corporation, Marine Colloids Division, Philadelphia, Pennsylvania).

Extraction Aaents and Means In one embodiment, useful extracting agents are:
one or more salts~selected from the group comprising dicalcium phosphate, calcium phosphate, magnesioum phosphate, or aluminum sulfate (which is pre~erred), used together~or sequentially.
Among suitable; extraction agents are those useful l5~ for changing the~pH o} the crude sol in order to refine the konjac, for~example organic or inorganic acids ~uch as HCl and bases-~such as NaOH. The amount of such agent employed should be extractive-effective, that is, sufficient~to vary the~pH from l to 8.5, preferably 3 Z0 ~to;~8.5, w~ithin~which~ranges~the benefits described in the product~àre~obtained. At highly alkaline pH's the glucomannan,~ in addition to~being extracted, may also begin~to~gel~ prematurely, while at highly acidic pH's the viscosity~of~the resulting product may be reduced.
25~ ~owever, if this latter viscosity reduction is desired, then beneficially both extraction and viscosity reduction can~be~àchieved~virtually simultaneously.
Aqueous extraction of the crude konjac may also be achieved by the use of chelating agents such as alkali ; 30~ metal hexametaphosphates, ethylenediamine tetraacetic acid (EDTA),~and~nitrilotriacetic acid (NTA). The amount of chelating agent which should be used~should be that which is chelating-effective, preferably 1 to 50 wt % based on the weight of the crude konjac.
Other useful extraction agents in the konjac , WO93/02571 PCT/VS92/06~91 21 ~ ;Jl~

clarification method are ion exchangers such as cation exchanging carboxymethyl cellulose (CMC), anion exchanging diethyl-[2-hydroxypropyl~aminoethyl cellulose ~QAE), or diethylaminoethyl cellulose ~DEAE), desirably in extraction-effective amounts, preferably 5 to 15 wt % based on the weight of the crude konjac.
Soluble salts which may also be used as extraction agents include neutral salts such as sodium chloride;
basic salts such as sodium acetate; or acidic salts such as calcium chloride, or combinations thereof.
Additionally, there may be used for this purpose a phosphate buffer, for example a 0.005 M buffer, pH 7.3, prepared by mixing monobasic sodium phosphate with dibasic sodium phosphate in suitable amounts. When }5 utilized, the soluble salt or buffer should be present in an extraction-effective amount, preferably 5 to ~0 wt % based on the weight of the crude konjac.
In another e~odiment, insoluble salts may be employed as extraction agents, for example dicalcium phosphate, aluminum sulfate, calcium phosphate, magnesium phosphate, of which aluminum sulfate (alum) is preferred. If~desired, these salts may be formed n during the extraction steps by known means. When utilized, the insoluble salts should be present in an extraction-effective amount, preferably 1 to 25 (more preferably 5 to~l5) wt % based on the weight of the crude konjac. ~
It also has been found that organic solvents including lower alcohols such as isopropyl alcohol may be used for this purpose, as demonstrated in Example 5. When utilized, the organic solvents should be present in an extraction-effective amount.
Hot water alone (i.e. at 65 to 100C) may be used as an extraction agent, although this is not particularly satisfactory because turbidity may .

2 1 ~

increase under some circumstances (see Ex. 47~.

Certain Uses of Clarified Kon~ac The amount of clarified konjac employed when incorporated in foodstuffs or industrial compositions will necessarily be varied, and can be determined without undue experimentation by those skilled in the art based on the known usage of crude konjac. For example, in foodstuffs, amounts of O.l wt % may be used in cake mix, while in industrial applications such as films, oil drilling fluids, and paints, amounts ranging from l to 2% and up~ard may be employed.
The use of the clarified glucomannan of this ; invention in foodstuffs such as baked goods, dessert gels, and meats, results in~improved food properties.
For~example~, addition of the refined material to ca~e dough results ~n~improved texture, moistness, and rise of the final product.
In its gel~form the product~is useful as a food or food compone~nt,~ film former, and in various biotechnical~applications.

- Viscosity Re~uction~
In accordance~with a further aspect of this 25~ invention the~viscosity of the konjac sol, which is normally high,~may~be~reduced before, during, or after the extraction step by treatment of the glucomannan with a variety of`~reagents or other means to obtain viscosities of whatever reduced values are desired.
30 ~ Such sols of~reduced;viscosity~are;particularly useful in biotechnology for preparing gels of high concentration, and~in cosmetics for~texture control.
Moreover, if the~viscosity is lowered be~ore or during the extraction step the subsequent filtration step is 3S naturally greatly facilitated, as is the general : ~ :

WO93/02571 PCT/~S9~J06591 21 L 51 ~ 3 handling of the final product in sol form.
Means for reducing the viscosity of a 1.0 % w/v aqueous sol of the crude or clarified konjac (normally typically 14,500 cps) to 50 cps to 3,500 cps by ; 5 depolymerization, for use in biochemical or : pharmaceutical applications as well as for ease in ~: handling, are known in the art and any suitable method may be used. Such means include: exposure to gamma radiation; exposure to radiation other than gamma such as actinic; acid hydrolysis, including "Smith Degradation" involving reduction of periodate-oxidized polysaccharide with borohydride, followed by mild hydrolysis with acid tsee "Advances in Carbohydrate Chemistry and Biochemistry", Academic Press, New York, :1975, volume~31 page 203 et seq.]; alkaline hydrolysis;
catalytic hydrolysis, for example by using iron EDT~
(ethylenediaminetetraacetic acid) or NTA
~:~ (nitrilotriacetic:acid) with or without a transition metal addition; enzyme hydrolysis; mechanical shearing;
~thermal depolymerization such as by extended heating at 80 - 120C in a~dry or wet (aqueous sol) state; or other Xnown means.~ Many known techniques and aspects of~poly~ degradation useful in this invention are : ~: described in "Elements of Poly~er Degradation", by 2:5:~ Reich and:Stivala~,:McGraw Hill Book Co., New York, :1971.
;Reduction of viscosity by irradiation can be achieved by contacting the crude or clarified konjac with ga~ma rays, such as generated fr~m cobalt60, at : : 30 dosages ranging from 50 to 1200 Krad or above, in which ~ :~ case a direct correlation between dosage and viscosity ;~ is obtained, as shown in the examples below.
Alternatively, heat degradation of the crude or clarified glucomannan may be employed. For example, : 35 heating the glucomannan for a requisite number of WOg3/025~1 PCT/US92/06591 hours, or even days, at temperatures of from 50 to 200C, depending upon the reduced level of viscosity desired, will produce satisfactory results.
Among the chemical means, acid hydrolysis, or contact with acid vapors of, for example, 5M HCl, during an acid extraction of crude konjac, with or without previous heating of the sol, produces a konjac sol of lowered viscosity which may be filtered more rapidly. It will be understood that this same method ~; ~ 10 also may be used~to reduce the viscosity of the ; recovered product~after it has been clarified.
Reduction of viscosity with a base, on the other hand, where the pH remains above 12.5, yields unsatisfactory results in that the resulting dry product is either discolored or insoluble in water, or both. Moreover, at a pH between~9~and 12.5,~depending upon the base used~, the sol~wil-l start to gel prematurely.

Cold Melt Gels~/ Sols It has been found that the heat-set gel formed from a sol of the~clarified konjac o~ this invention exhibits similar~cold-melt properties to the crude onjac as used~in~ehe mentioned Japanese Patent Disclosures. ~A~major difference is that, analogous to 25~ the clarified~konjao~sols,~ the cold-mel*ed clarified konjac gels~form~a clear~liquid similar to a sol.
Specifically,~as~a~clarified konjac gel is cooled from room temperaturé, there is an almost linear softening of the gel and reduction~in gel strength. At 10C the 3~0~ - gel exhibits~a~ visible softening and at 5C it clearly becomes a liquid~ whose nature~has not been determined.
The liquid st~te;~continùes until about 0C, below which point the clàrifi~ed konjac gradually freezes into a solid (but not a gel). The cold liquid (at 5C to 0C) will reform into a gel upon heating or warming. The ~: -WO 93fO2571 PCI'/US92/06~91 211 )143 re-formation of a cold-melt sol, upon re-chilling, has been observed on several occasions, and on one occasion a clarified konjac was reversed from gel to liquid to gel at least three times. When clarified konjac is S cooled to freezing or slightly below and then brought back to room~temperature, it forms a clear, water-insoluble, spongy, dimensionally stable mass. It is known that this phenomenon occurs with crude konjac, however the spo~ngy mass formed with clarified konjac is noticeably lighter in color and contains none of the protein or other impurities found in crude konjac ` itself, and does; t have the characteristic odor of crude konjac. Because of this, it is contemplated that the spongy mass prepared from clarified konjac is suitable for various;med_cal app}ications such as implants and carriers for medications and for biotechnological applications requiring the absence of such contamination In order~to~ensure that the clarified kon3ac gel 20 ~possesses this~cold-melt" property, it is important that it be formed~under certain controlled conditions, primarily~with~respect to pH, as well as to the time the gel~takes to~;form~at any~given temperature. Other factors which may~also affect~the ability of the gel to melt at 1GW temperatures, include ion content and type.
;For example,~it has~ been found that as the glucomannan concéntration ~increases, the geI melts more slowly.
However, the concentration of clarified glucomannan in the gel is not critical, and may vary from .01 to 10 (preferably 1~to~5)~ wt %.
; In order to form a gel having cold-melt properties, the pH of the~sol obtained from the clarified glucomannan~first must be adjusted, desirably by heating it with an alkali at a temperature of from 65 3S to 130C until the gel is formed. The pH should ; , , W O 93/02571 PC~r/US92/06591 ~ 1 1 S ,l C~ ~, desirably be 9.6 to 12.3, preferably 10 to 11.5, employing such bases as NH40~, NaOH, K2C03, or mixtures thereof, of which NH40H is preferred. It has been found, moreover, that gels formed at the lower pH
S values within this alkaline range subsequently melt to a sol more rapidly. In addition, the pH of already-formed gels which were prepared at high pH values, (see Example 11), can be lowered by treatment with a buffer :
solution, to a ~H of 8-9 or lower without adversely affecting the cold-melt property of the gel. It has aIso been found~that the cold-melt property is adversely affected~by an extended gellation period, so that gel formation at elevated temperatures for short periods~of time is preferable to lower temperatures for ` 15 long periods.
Alternatively~, it has been~found that gels may ~e prepared at an acid pN t~instead of alkaline pH) if the ` preparation is carried out under retort conditions, that ~is, at~high~temperatures~while under pressure.
20~ For~example,~gels may be formed from clar~fied kon~ac sols at a pH~of 6.7, a temperature of 130C, and a pressure of~30~psi (about~2 atmospheres or 2.11 kgs/cm2) One~convenient~method~for gelation is by adding 25~ NH40H ;to a 1%~sol of clarified glucomannan unti} the desired p~is~achieved, e.g. 11.2;~ heating the alkaline sol for~a~bout~5~to~60~minutes,;~depending upon the amount employed,~(preferably 20 to 30 minutes at a '~ ~ temperatures'of;~from 50 to 120C, more preferably 80 to ~90C), until a gel forms;~and~thereafter cooling the gel in an ice'~bath,~ until it liquefies. The melted material can then~be~reformed to a gel by heating it until the gel~starts~to redevelop, generally starting at temperatures of 6C and above.
3S ~n certain cases, notably when NH40H is used as the , :

~::~::: :: :
; ~ :

WO93/02571 PCT/US92/0659l J `'~ ~ 1 base, it has been found that as the gel melts, barely visible spherical particles may form in the liquid which, for purposes of any further clarification of the liquid, may be removed by filtration. As a theoretical explanation, it is believed that the outer surface of these coacervate-like particles contain water soluble ~; starch which is present in the konjac flour.
Subject to those exceptions noted herein, the gels formed from the clarified konjac of this invention consistently exhibit cold-melt properties, that is, the gels liquefy when~exposed to temperatures below 5C, , down to 0C, at ambient pressure. If it is desired to keep the clarified konjac gel at low temperature without liquefying, this cold-melt~property can be avoided by the admixture of non-cold-melt hy ~ ocolloids,~principally such gums as xanthan, -~carrageenans, and~agaroids (especially agarose) or mixtures thereof.~ In some cases the clarified konjac will cogel with the hydrocolloid without the addition 20 ~ of alkali. Other~hydrocoI}oids may require added alkali, heat, specific ions, or similar means to form the gel, as~is~kn wn in the art.
It has also been found~that at specific reduced - ~ :
temperatures,~the;presence of gums in the alkali-set gel results in the reversible transformation of a gel from a spongy texture to one which is a clear elastic.
In addition~to the hydrocolloids, it has been found that certain ionic compounds at or above certain concentrations,~for example salts such as NaCl, may also be used }or the purpose of preventing cold melting of gels. In either case it~will be seen that variant cold-melt properties can be achieved on a selective basis by the addition of these materials.
The amounts of hydrocolloid or ionic compounds 3S necessary for preventing cold-melting of gels may be ;
~:

WO93/02571 P~T/VS92/06591 2 1 ~ ~. 1 4 1 varied considerably. For example, the addition of 10%
NaCl, i.e. ionic compound, by volume, will prevent cold melting. Alternatively, when a hydrocolloid is employed, the weight ratio of glucomannan to hydrocolloid in the gel may vary from about lO:l to l:lO. For example, the addition of l part by weight carrageenan or xanthan to 3 parts of glucomannan, based on the weight of the konjac in the gel will likewise prevent cold melting. However, it will be understood that if it is desired to modify the properties of the gel in other respects as well, the amount of these additions employed~can be increased accordingly.
The melted clarified konjac may be recovered in its liquid state and~stored or handled that way, if desired, as long as it is maintained at temperatures generally below 5C (at ambient pressure). In its ~
; cold, melted state the soI is notably stable at those temperatures. Alternatively, and more preferably, storage~in the form of the gel at appropriate pH values until it is ready~to be used, facilitates ~ts handling.
The unique property of this cold-melt sol makes it highly useful in~many ways, for example in b~iotechnology as~an electrophoresis medium, or in ~édical technology as a drug delivery mediun, e.g. by incorporation of~;a drug into the liquefied sol which could then be~hardened by warming it for storage or administration purposes. Foodstuffs and~beverages normally served cold could have their texture and consistency enhanced by making and/or storing a gel-containing food~under cold conditions untîl ready to be served, e.g. frozen desserts or the like; or conversely,;by adding the cold-melted sol to food in easily handled~ uid form and;then allowing it to set as a gel at room temperature.
In a further~embodiment, the cold-melted sol may be - 25 - 2 1 . .~ 1 4 ~

used in cell encapsulation or to deliver drugs topically. That is to say, by incorporation of a water-soluble or suspended drug in the sol, as for example topical anaesthetics, antibiotics, antiseptics or the like, this sol, upon application to a cut or burn, dries to form a thin film that slowly releases effective amounts of the drug to the affected area.

EXANPLES 1-2l: EXTRACTION AGENTS
A series of experiments was carrîed out demonstrating the preparation of the clarified konjac product Or this ~invention by means ~f the aqueous extraction of crude konjac flour with a variety of extraction agents. ~In each case (Examples 2-21) the 15~ ~ procedures of Example~l were followed,~except for the use~of different~extraction agents,~as indicated. As also shown in Example 1, a sol of the dry, ground product was prepared after which a viscosity measurement~was~made.
20~ Aqueous~ext~ractions,~alone or incorporating various salts~both~soluble~and insoluble, different pH's, chelating agents,~ion exchangers,~;etc.~ were used.
Time,~temperature,~konjac concentration and volumes werè identical~in~these extractions~ Filter aid usage 25~ varied~some~hat;~(0;~to 100 g)~; where no filter aid was used the~samples~were filtered~through a "cuno"-type cloth~filter.~ Thi~s~was done to speed the~processing and was effective in removing the insoluble sacs;
` smaller microscopic particles remaining could readily be removed with~a-filter aid, if desired. Coagulation (with isopropyl~alcohol) washing and recovery were the same as in Example 1 in all examples except for routine modifications~to suit specific cases.
As described~in more~detail below, in Examples 22-42, alkali was~then added~to the corresponding sols of :
;
:
:

2~llv~

Examples 1-~1 to form a gel, which was placed in crystallizing dishes and heated in a hot water bath.
Gel strength was measured immediately and the gels placed directly in an ice bath to be observed for melting. The melted gel was then allowed to incubate at room temperature overnight and observed for its regelling ability. The results of each of these experiments are also summarized below in Table II.

EXAMPLE 1 - Hot Water Six hundred ml of distilled water was heated to ~75 to 78C in a hot water bath. Six grams of crude konjac was added and stirred for 60 minutes while maintaining this temperature range. A 1 liter pressure filter bomb was assembled using only a fitted piece of cuno filter cloth~and then filled with boiling water which was then allowed to drain. The sample was poured into the filter bomb~and 10~psi (0.7 ~g/cm2) applied for 10 minutes. The pressure was gradually increased to 15, 25, 40, 45 ps~ 05, 1.75, 2.8, and 3.15 kg/cm2) and held at each~level for 15 minutes. The total filtration time was 70 minutes and 430 ml filtrate was collected. The filtrate was coagulated in 2x volume of 99% isopropyl alcohol (IPA), based on the volume of the 2~5 filtrate, and allowed to sband for 60 minutes. The coagulate was collected by vacuum filtration, on polyester cloth,~;squeezed dry, and transferred to 2x volume of 60% IPA for 30 minutes. The coagulate was again recovered, again treated with 99% IPA, and then ; ~ 30 dried at 55C~overnight (14 hours) in a forced hot-air oven. The sample weighed 3.57 ~; (59.5% yield) and was ground through a 40 mesh-screen (U.S. Standard Sieve Series). This material was used to prepare 200 ml of a 1 wt ~ aqueous sol by suspending 2 g in 200 ml distilled water. This was placed in a hot water bath WOg3/02571 PCT/US92/06591 - 27 ~

(-80C) and stirred with an overhead mixer for 45-60 minutes. The ssmple was poured into a 250 ml tall-form beaker and allowed to cool to ~5C. The viscosity was determined with a Brookfield Digital Viscometer (model LVTDV-~I) and found to be 18,400 cps (spindle #2, 0.3 rpm, 25C, 91.7% of maximum). ~

EXAMPLE 2 (pH 2) The following experiment illustrates the combined viscosity reduction and extraction of crude konjac with acid at low pH. The effect was to reduce the viscosity `before the extraction was completed.
The procedure outlined in Example l was repeated with the following~changes. The water was adj~tsted to pH 2 with l~.OM HCl~before heating. Filtration uas very quick, with S50 ml filtrate passing throùgh the filter bomb in 8 m~inutes~without the;need to apply any pressure. The amount of timé the coagulate sat after the initial coagulation was shortened to 45 minutes and 20 ~;a~final~15 minute hardening step in 99% IPA was employed. This process yielded 4.22 g (70.3%) of c1ar~ified g1ucomannan~which had a~1% viscosity of 57.3 cps~(Brookfield~LVTDV-II spind1e~;t1, 60~rpm, 25C, 57%
of~maximum)~

EYamP1eS~ 3 and 4 illustrate the~aqueous extraction, as in Example 2~ except that in Example 4 a base was used. It will be noted that while this procedure was , fully effective~at pH 7, at pH lO the results were poorer~because of thé partial gelling of the product at the higher pH values, which interfered with the : ~ , filtration step.~

EXAMPLE 3 ~PH 71 The procedure described in;Example 2 was repeated W093/02571 PCT/US~2/06591 2 ~ 1 v ~

using pH 7. The pH was controlled, as needed, with small amounts of O.lN NaOH and l.ON HCl. 250 ml of filtrate was collected in 95 minutes and then processed. The dried sample, 1.81 g or 30.1~, had a viscosity of 14,200.

EXAMPLE 4 f~H 10) In a like manner, as in Example 2, an aqueous extraction was carried out using pH 10 adjusted (l.ON
NaOH) water. Filtration was slow and only 150 ml of filtrate was collected (see table below for filtration times and pressures). The small amount of filtrate was observed to be~partially gelled and was discarded.

15 ~ ~Examples 5~and~6 illustrate the extraction process using two different chelating agents.

LE~5 (Hexametaphosphate - HMP~
Sodium hexametaphosphate~(3 g, 0.5% w/v) was added to the~hot water~prior to the;addition of the konjac.
In~this extraction~, 50 g Celatom diatomite (Eagle-Picher; Cincinnati,~Ohio) filter aid was mixed into the sample~before fiItration. After 109 minutes, 400 ml filtrate was~collected and processed (see details 25~ below).~ Aft-r~drying, 3.62 g (60.3% yield) was ground and used to prepare a 1% sol. mis:~material had a YiSCosity of 3~,010~cps~and~a gél strength of 124 g/cm2.
It also "cold-melted"~and reqelled upon warming.

EXAMPLE 6 (Ethylenediamine Tetraacetic Acid-E~TA3 n a manner si~milar to Example 5, another 6 g crude ~;~ konjac was extracted substituting 0.6 g ~0.1% w/v) disodium EDTA~for~hexamet4phosphate. Only 300 ml filtrate was collected after 120 minutes. This yielded 1.91 g or 31.9% after coagulation and drying. A 1% sol :

W093/~2571 PCT/~S92/06591 had a viscosity of 19,700 cps.

Examples 7-lO demonstrate the use of various soluble salts, or mixtures thereof, in the aqueous extraction of crude konjac, again using the general procedures of Example 1.

EXAMP~E 7 rNeutral Salt) 3 g NaCl was added prior to the addition of the konjac. The amount of filter aid was reduc~d to 25 g.
500 ml of filtrate'was collected in 110 minutes and ;processed as~above. This extraction produced 2.39 g (39.9% yield) of clarified konjac ~lucomannan having a 1% viscosity of~2~l,gOO cps.
~ ~ ~
EXAMPL~ 8 (8asic Salt) 3 g ~O.5%~ w/v,) sodium acetate was added to the water prior to~the~addition of the Xonjac. The filtrate (350~ml~collected during 120 minutes? was 20 ~ processed,~dried~and,then ground. The 3.674 g (61.2%
yield)~ was used~to prepare a 1% sol. The viscosity was ,measùred at~;4,~660 cps.

E$AMP~ES 9 AND lO (Acidic Salt~
,25~ ;CaC12.2H20 was~used~in~2~other~eYtractions 3.97 g;
(0.~5% w/v CaCl2~ In the first case (Example 9), 15 g filter aid Celatom~diatomite (Eagle-Picher, Cincinnati, Ohio) was used but filtration was difficult. Only 50 ml filtrate was collected after 200 minutes and the experiment was subsequently abandoned.
A second~attempt (Example 10) eliminated all filter aid. After 43 minutes, 5~5 ml filtrate was collected and processed yielding 4.06 g of dried product (67.7%
yield). A 1% sol'had a viscosity of 16,200 cps.
::
~ 35 ::

WO93/02571 PCT/US92~06591 EXAMPLE ll (PhosRhate Buffer) A 0.005 M phosphate buffer, pH 7.3, was prepared by mixing 39 ml 0.2~ monobasic sodium phosphate with 61 ml 0.2~ dibasic sodium phosphate. An aliquot of this, 25 S ml, was diluted to l~liter giving a final concentration of 0.005~. Six grams of crude konjac was extracted in this solution as~previously described. The filtrate, 250 ml obtained~after 68 minutes, was processed and dried. The~samp1e (1.957 g; 32.9% yield) had a 1%
viscosity of 1,380 cps.

The fol1Owin~examples (12-14) demonstrate the aqueous extraction of crude konjac with ion exchangers and with a polar organic solvent, (Example lS). As ~noted~in Examples~;33-35 (below), the pro~ducts of Examples~ 12-14~were~found not to cold melt. This may be~the result of~ion binding andlor aggregation effects.

20 ~ EXAMPLE 12 ~Cation Exchanaer-Car~oxYmethY1~Cellulose) (A.)~To~600~ml~disti1led~water, 0.6 q of water-so}uble~carboxymethyl cellulose (CMC) (l0~ w/w with konjac)~ was~added~before the~addition of konjac. No filter aid was~used and 500 ml filtrate was colIected 25 ~: ~in ~10 -inutes at~s~psi (.35 kgjcm2). After processing and~drying,~4.45~g~or 74.1% was~obtained. It had a 1%
viscosity o~15,~700 cps.~
B.) To~600~ml~deionized water, 0.6 g of insoluble, microgranular CMC 32 (Whatman Labsales; Hillsboro, 30~ Oregon) was~added~before the konjac. No filter aid was used and 575~ml~fi1trate was colIected in 32 minutes at pressures up to 20; psi tl.4 kg/cm2). Ater processing and drying, 3.9~ g;or 65.~1% was obtained. It had a 1%
viscosity of~lS,900 cps and after gellation, did not 35 cold melt. ~

WOs3/02~7l PCT/US92/06591 ~ i J' v;.~
- 31 ~

(C.) To 600 ml deionized water, 0.6 g of insoluble, fibrous CMC 23 ~Whatman Labsales; Hillsboro, Oregon) was added before the konja~. No filter aid was used and 575 ml filtrate was collected in 23 minutes at pressures up to 10 psi (.7 kg/cm2). After processing and drying, 4.06 g or 67.6% was obtained. It had a 1%
viscosity of 17,900 and also did not cold melt once a gel had been formed.

EXAMPLE 13 ~Anion Exchanaer Diethvlaminoeth~l Cellulose - DEAE~
0.6 g of DEAE oellulose was employed in an ; extraction. 500 ml of filtrate was collected in 35 minutes and was subsequently processed. The dried sample, 3.79 a or~63.1%, had a 1% viscosity of 16,100 cps.

EXAMPr~ 14 ~Anion~;Exchanaer Diethvl-r2-hYdroxypro~vll-aminoethyl Cellulose - OAE) 0.6 g QAE-~cellulose was added before the extraction.`~Filtr~ation was not as good as that for DEAE;~ only 300~ml;~was collected in 74 minutes, which yielded 1.96~g~(32~.6%) after~processing. This material had a 1% viscosi~ of 14,000 cps.

EXAMPLE~15 (20% Isopropyl Alcohol~
; Six grams~ of~the crude konjac was extracted in a mixture of 148 ml 99% isopropanol in 452 ml distilled water (20~ wj~w) ~as previously described. After 90 30 ~ minutes of filtration, 275 ml of filtrate was collected and then processed. A total of only 1.25 g (20.9~
yield) was recovered. This material had a viscosity of 11,800 cps. ~ ~ ~

In Exa~ples~15-21 insoluble salts were used to W093/02~71 PCT/US92/~Sgl 21~5~

adsorb impurities from the konjac in which the salts were formed in situ in Examples 18-21. It will be noted that when a filter aid was introduced by way of a modification to Example 18, as in Example 19, a clearer product was obtained. In a similar modification of Example 20, as in Example 21, but using a filter aid, much less filtrate was obtained.

EXAMPLE 16 (Dicalcium Phosphate) Dicalcium phosphate was also used in an extraction by adding 0.6 g ~10% w/w~with konjac) to the water before the addition of the konjac. After the "cook", 15 g of filter aid was added and filtration was carried out for 10 minutes at 25 psi (1.75 kg/cm2~ and then 40 minutes at 4a psi (2.~8 kg/cm2). Only 50 ml of filtrate wao col}ected during this time so the sample was r m ved~from the ~ilter bomb,~pooled with the small amount~of filtrate~and an additional B5 g of filter aid ~ixed in.~ Filtration~proceeded~for 120 minutes during 20 ~ which time 250 m1 of~filtrate was collected and subsequently processed. This process yielded 1.65 g;
27.4% yield)~. This material had a 1% viscosity of ; 4,410 cps~on a~Brookfield Viscometer, Model LTVDV-II, No. ~1 spindle.
EXAMPLE 17 (Aluminum Sulfate~
0.6 g aluminum sulfate was added~prior to the konjac. After 3~5 minutes, 500 ml of filtrate was collected and then processed. The dried sample, 3.15 g or 52.6%, was ~round and used to prepare a 1% sol and gel. The viscosity was determined to be 2,170 cps.

EXAMPLE 18 (Aluminum Sulfate - In Situ) To 600 ml distilled water, O.747 g monobasic sodium sulfate and O.847 g aluminum chloride was added with .

W O 93/02571 P(~r/VS92/06591 2 ~ ~ ~`J~

stirring. No filter aid was used and 500 ml filtrate was collected after 11 minutes at 5 psi (0.35 kg/cm2~.
This material was processed, dried and ground producing 4.20 g; ~70% yield). This material had a 1% viscosity of 1,720 cps.

EXA~PL~ l9 rAluminum Sulfate - In Situ) The above extraction was repeated with a few ~; changes`. 0.S26 g of the aluminum chloride (0.291 g anhydrous AlCl3) and 0.310 g monobasic sodium sulfate were used in this case. Additionally, 25 g of filter aid was added before filtration. Over 98 minutes, 450 ml of clear filtrate was collected and then processed.
After drying, 3.65 g (60.9% yield) of material was ground and used to~prepare a 1% sol. The sol, which was very clear,~had a viscosity of liSO cps.
~ ~ .
~ EXAMPLE 20 f~Dicalcium Phos~hate - In_Situ~
.
0.694 g~calcium~chloride and 0.~567 g monobasic ~sodium phosphate~was used in the extraction. Again, no filter aid was us`ed and 340 ml filtrate was collected and~processed~producing 2-90 gf (48.3% yield). This sample had a 1% viscosity~of 17,000 cps.

25~ ~EXAMPLE 21~rDicalcium Phosphate - In Situ) The above~extra~ction was-repeated. The amount of CaCl2~.2N2O was reduced to 0.382 g (0.288 g anhydrous ; ~ CaCl2 or 2.6 x ~lo~3 mols) whereas the amount of '~ ` NaH2PO4 dropped to 0.312 g (2.6 x 10-3 mols). Filter 30 ~ aid~(25 g) was~added before fiItration, which proceeded slowly. Only 150~ml of filtrate was collected after 147 minutes~. This extraction attempt was abandoned at this point.

::
:
.

~ 1 A ~; 1 d ~

~XAMPLES 22-42: GEL AND COLD-MELT FORMATION
A series of runs was carried out wherein each of the clarified konjac products of Examples 1-21, respectively, was gelled and thereafter tested for gel strength and cold-meltability, as Examples 22-42.
Results for these~are all in below Table II.
It will be noted~that of the recovered sols tested, only three of them, Examples 33-35, did not cold melt.
In general, however, it will be seen that the vast majority of the gel products of this invention are cold-meltable.

To 200 ml of a 1% soI of the product of Example 1 was added, with~stirring, 8 ml of 5 NH40H (i.e., 1 ml base/25 ml solution) to provide a pH o f about 10.5.^
n The 801 was heated~in a hot water bath for 60 minutes at a temperature of 85C during which time a gel ;formed. The gel was immediately tested for gel 20~ ~strength, and;then~placed in an ice bath until a temperature;of 4C~was obtained. The gel melted, as indicated in Table`II, to form a substantially clear sol~. When reheated, the gel reformed satisfactorily and was heat stàble :~ 25 ~:
, ~

In accordance~with the foregoing procedures of ;~ Example 22, but~;substituting as the starting materials the respective~products of Examples 2-21 (where recovered) for the~starting material of Example 22, there were obtained the corresponding gels. The gel strength and cold-melt properties of these gels is also reported in Table~II.

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WO 93/02571 PCI`/US92/06591 2 ~ 1 3 ~ ! ~ " 4 o FOOTNOTE:S FOR TABLE II

.
a Dist~llod wator b p~ adgu~ted and~asured at room temperature S c ~lter aid: Colato~ diatomite ~Eagle-PicAor;
Cinclnn~ti, OhioJ
:
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WO 93/02571 PCI`/US92/06591 ~ i 151~1 EXAMPLES 4 3--4 7: AI,UMINUM SULFATE AS EXTRACTION ~GENT
An additional series of experiments (Examples 43-45) were carried out in accordance with the process of this invention to demonstrate the effect of aluminum sulfate as extraction agent on the clarity, nitrogen content and viscosity of konjac flour. The results of these examples are reported below in Table III, together with comparative examples 46 and 47.
In each of Examples 43-45, subject only to certain variations shown in the footnotes, 7 g of konjac flour - (NUTRICOL brand, FMC Corporation, Marine Colloids Division, Philadelphia, Pennsylvania), 22 g of the perlite filter aid (FW-40 from Chemrock Corp., Thomaston, Me.), aluminum sulfate (in varying amounts shown in the table), and 0.25 ml of 3M NaOH were added to 800 ml of H20 (0.84 wt % konjac). The mixture was heated with agitation for 20 minutes at 90C, filtered ~through a warm fiIter bomb under pressure ranging from 10 to 40 psi (.7 to 2.8 kg/cm2) and the filtrate precipitated into 3 volumes of 70% isopropyl alcohol to , , .
form a coagulate.
The coagulate was dried in a forced-draft oven overnight at 60C~to produce a~ hardened cake which was ground up through~a No. 40 screen ~U.S. Standard Sieve S ies)(420 ~icrous)~. The dry particulate product was then reconstituted~as a 1 wt % aqueous sol. The viscosity and~turbidity of this sol were then measured, the results of which are also reported in Table III.
In comparative Example 46, the viscosity and turbidity of a sample of commercial konjac flour :: ~
dissolved in water to which no rea~ents had been added, was measured. In comparative Example 47, the dissolved konjac flour was processed in accordance with the method of this invention except that no aluminum sulfate or NaOH was added.

a. 1 wt. ~ sol.
. ~ea~ured ~t 12~rpm o~ ~ Brookfield~ Yisco~eter, Model LV~DY-~I, (No. 4 ~pindle) a~ 1 wt ~ concentr~tion ~nd at 25C.
:
c. In rurbid~ty; Unit~ ~. U. J ~a d on tAe Formazin Turbidity Standard as ~easured by a ~acBoth Coloreye m~chine. ~Aese turbidity v-1uos~were ori~inally measured on a~Fisher SpsctropAotometer ~, using a Fuller's EartA Standard. ~hey werff tAen converted to the Fo~azin ~tandard by co~rela~ion tudie~ which~co~pared sa~ple~ of the t~o ~t~ndards, u~ing ~tAe Fisher~unit.~ A final conversion to the ~cBetA/Fo~azin standard o~ tA~s ta~le ~as then obtainad v}a ~ correlatio~
~coefficient, as~;~de-~cribed below ~n Table VI, footnote (b).
d. ~nprocessed,~untreated ko~jac flour.
. Proce~sed witA~hot ~ater (1~ min. at ~5C), but not with aluminum s~lfate or NaOH.

W O 93/02571 PC~r/U~92/06591 2 ~ 4 ~

From the foregoing it will be seen, as shown in Example 44, that the viscosity of the clarified konjac may be maintained at very high levels, particularly for food use, by optimizing the amount of aluminum sulfate used. Also, the turbidity of the reconstituted, clarified konjac of this invention is significantly lower in Examples 43-45 of this invention as compared ; to the crude~konjac sol of comparative Example 46. The ` processing theating, etc.) of konjac with hot water in ~ ~` 10 the absence of aluminum sulfate ~Example 47) somewhat ; increased the turbidity of the reconstituted sol.
Of greater significance as shown in Example 44, at optimu~ concentrations of aluminum sulfate the viscosity of the~reconstituted clarified konjac surprisingly, and desirably, increased when compared to that of Examples~46 and 47.

The following~example illustrates a scaled-up version-of~the preoeding aluminum sulfate clarification 20 ~ procedure.

To a 225 gallo~ (about 852 liter) stainless steel tank, 140 gallons~(530 leters) cold water, 214 g 25 ~ aluminum sulfate ~(~4.71%~ and 162 ml 3M NaOH was added and~heated with~direct steam to 70C. 10 lbs. (4536 g) konjac flour and~31 lbs. of FW 40 filter aid was mixed in. Total volume;~was 160 gallons (606 liters) which was 0.75~ konjac w/v. The samp}e was heated to 85C
and held 15 minutes. The sample was then filtered in a preheated 18 inch~stainless~steel filter press with recycling occurring during the first 5 minutes. Total filtration time,~including flushing the filter press with hot water, was 6~0 minutes. The filtrate was coagulated in 300 gallons (1,136 liters) 85~ isopropyl WO 93/02571 PCr/US92/0659l ;3 L 41 alcohol IPA. The coagulate was recovered by screening and by pumping it through bags which were subsequently squeezed in a small press. The coagulate was then washed/hardened in 75 gallons of 85% IPA for 2 hours S with air agitation. The coagulate was recovered by screening and then squeezed by hand to remove excess liquid and subseguently dried at 55C overnight. The sample, 6.4 lbs. ~2.9 kg) or 64% yield, was ground through a 0.039 inch (2.4~m) screen. It had a nitrogen content of 0 .15% and a 1% turbidity of 11 NTUs ; (Ne~helometric Turbidity Units).

ExAMpLEs 49-53: GEL A~D CO~D-MELT SOL FORMATION
In a further series of runs, and in accordance with the general procedures of Example 22, the product of :
Example 48 was tested~for gelation and cold-meltability, using various bases and reaction conditions. ~ ~

~ ` EXAMPLE~49 To l00 ml of a 1 wt % aqueous sol of the product of Examp~le 48 was~added, with stirring, 4 ml 5M NH40H to provide a~pH o~10.08. The sol was heated in a boiling water bath for~20 minutes, during which the gel began 25~ to~form after 7 minutes. The gel was then placed directly in an ice~bath until it melted to form a substantial1y~clear sol.~ When reheated, the gel reformed satisfactorily.

EXAMPLE 50 ~Base variation~
The same procedure was followed as in Example 49, substituting sols of different bases for the ammonium hydroxide. Basic~solutions used were 5M NaOH, SM KOH, and 10% K2C03 with adjustments made for minor concentrakion variations. The cold-melt phenomenon was ~: :
:

W093/02571 PCT/US92/065gl 2 ~

observed with each; however, the formation of spherical bodies containing starch was only observed with the ammonium hydroxide cold-melt sol.

S EXANPLE 51 (Heatinq time variation) Three 50 g samples of l wt % clarified konjac were prepared as in Example 49. To each was added 2 ml SM
NH40H wi~h stirring. These were p}aced in a boiling water bath. Gelation was apparent after 7 minutes. At 20 minute intervals (20, 40 and 60 minutes) one beaker ~`~ was removed.~ The gels were allowed to cool to room temperature and~were then placed in an ice bath. All three gels cold-melted.

~EXAXPLE 52 ~H variationL
A. Six SO-g ~aliquots of 1 wt % of a~ueous clarified konjac were prepared in accordance with the procedures of Example 48. Using l.ON NaOH and O.lN HCl (to back titrate);,~ each beaker was adjusted to one of 2Q ~ the following~pN vaIues: 8.5, 9.0, 9.S, 10.0, lO.S and 11Ø These~were~all~placed in a boiling water bath for 20 minutes. ~Those samples with initial pH values lO,; iO.S~and 1l gelled and were removed after 20 minutes to cool at room temperature. Those samples at 25 ~ lower~pH did not~gel after;l hour in the water bath~
The 3 gels were~`pla~ced in an ice bath. The gel made at pH~10 melted ful~ly~and quicXly. The gel made at pH
lO.S melted slowly and only partially. The last gel pH 11.0) did~not melt~but softened considerably.
30 ~ B. A series~of SO-g aliquots of 1 wt % of aqueous clarified konjac was prepared~as in Example 49. To the first four aliquots, all;~;contained in beakers, 25, SO, 75 and 100 microIiters, respectively, of 5M NH40K was added. The remaining aliquots received increments of lOO microIiters~(maximum 2.1 ml~. The pH was checked ' WO93/02571 PCT/US92/065gl 2 1 1 ~

by pH meter and visually, by universal indicator. The gels were then heat set for 20 minutes, cooled to room temperature, covered, and allowed to stand at room temperature overnight ~16 hours). The beakers were all placed in an ice bath and monitored. The pH of those that melted was rechecked. Selected results are listed in the following table.

~ TABLE IV(a) :~ lQ
Volume NH40H:(~l) Initial DH Gelled MLelted Final ~H
25 ~ : 9.5 no --- 9.2 : 50 9.8 no --- 8.8 : : 75 9.8 weak yes 8.9 :
15100 10.0 yes yes 9.0 500 10. 4 n ~ 9.8 1000 ~ 10.8 ~ b 10.1 500 11. 3 H n na 2000 : 11.4 * partial na ~ ~
H : : : A similar series was run using 5M NaOH instead of NH40H. The~results were as follows:

` TABLE IV (b~
25~
Volume NAOH~ Initial DH Gelled Melted Final ~H
`: ~
9.2 no 7.3 ' 175 l0.4 " 8.2 :: : 30200 ~ 10.6 weak yes 8.5 ~` : 225 :~ 10.8 yes " 8.6 : 250 11.0 " " 8.7 300 11.2 " " 8.8 ::: :

WO93/0~571 PCT/US92/06591 2~ ? t~13 The following example illustrates the preparation of a gel at retort conditions and at a low pH.

For this example, 800 mls of a 2%-sol of clarified konjac from Example 48 was prepared by dispersing and dissolving it in a pH 6.6 phosphate buffer. This material was used to fill an aluminum can to capacity .
which was subsequently sealed. The can was placed in a pressure cooker~and heated at 130C at 30 psi (2.1 kg/cm2) for~60~minutes. After cooling, the can was opened. A soft~gel;was revealed. Several pieces were removed, placed in~a separate small beaker and then iced~. The gel melted fully and when heated in a hot , ~ 5 ~water bath (~-90C)~for 25 minutes, a much firmer gel :
reformed. ~;~

EXAMPLE~54~(Ge~l~and cold-melt sol stabilitiesL
Eight lOO-g~ wt % clarified konjac sols were ~prepared in~acco~dance with the~procedures~of Example 48.~ The~following~volume of-SM NH40H was added, in dup1icate,~ to~thé~samples;(resulting pH value is in parentheses)~ ml~(pH 10~.39), 2 ml (pH 10.58~), 3 ml (pH~10~.78)~and~4~ml~pH 10.90). All~eight samples were 25~ ~heat~set~for;20~;minutes in a boiling water bath. Four of;these geis,~ one~at each leveI, were covered with plastic wrap and~allowed~to stand at~room temperature for 10 days.~ The~other four gels were placed in an i~e bath after cooling. Only~the gels ~ormed at pH 10.39 ; 30~ and 10.~58 melted.~ The other two gels at pH 10.78 and l0~.90, softened but did not~melt. All four were ; covered with plastic and stored in a 9C refrigerator at 9~C.
: : :
~ The samples stored in the refrigerator were :::: :

, W093/0~71 PCT/US9t~06591 211~

examined after 8 days. The two lower pH aliquots were still in molten form, while the two higher pH samples were unchanged (soft gels). Small samples of the two cold melts were placed in a test tube and placed in a hoiling water bath for 10 minutes. Both formed gels, but these gels did not remelt.
The gels stored at room temperature were placed in an ice bath to check for meltability. The two lower pH
gels melted completely. The remaining gels, at pH
values of 10~78 and 10.90, melted substantially but not fully.

The following examples illustrate additional methods for~reducing the viscosity of the clarified l5~ kon~ac of this invention by means of irradiation.

EXAMPLES 55-63 ~Visoosity Reduction by Irradiation) Six 50 g aliquots of clarified konjac obtained by the process~of Example 48, and;a 100 g portion of an 20~ alcohol-washed~crude~konjac sample were irradiated by gamma rays (cobalt 60).
Sols (200~ml,~lS w/v) of~each sample, as well as sa ple~s~of the~or~iginal nondegraded materials, were ~prepared by heating the sample in a water bath and 25~ stirring with~an~overhead~mixer~for 60 minutes. The samples were~poured into 250 ml tall-form beakers and allowed to cool to room temperature. The viscosities were determined with a Brookfield digital viscometer as -1 described above. ~An aliquot (50 ml) of each sample was mixed with 2 l of~SM NH40H~and placed in a ~oiling water bath for 20 minutes to check for gelling ability.
Following gelation, the gels were placed on ice to check for cold-meltability. The results of each of these tests are shown below in Tab}e V.

:~ :

49 ~

TABLE V
Irrad. Level Viscosity Gel Example Koniac ~Xrad) (cps) ~ormed Melted Clarified 0 3360 yes yes 56 Clarified 50 1800 yes yes 57 Clarified l00 1230 yes yes 58 Clarified 200 641 yes yes ' ~ 59 Clarified 300 357 yes yes Clarified ~ 600 129 yes yes :
61 Clarified 900 58 weak yes 62 Crude ~0 11200 yes yes 63 Crude~ 300 1630 yes yes EXANPLES 64~-82~;!Nitroaen and Iurbidity Content of~Clarified Kon~ac Selected~products obtained from previous examples 20~ were~measured;to~determine their nitrogen ~ontent and turbidity~leve~ (Ex. 64-74). These results were compared with the'nitrogen content and turbidity level of~both~;crude.kon3aà flour, (Ex.~76 and 78-82) and the products~of the-~process described in U.S. Patent 25~ 3,9~28,322, ~Ex'.~75~) as well;as~those of the product of Ogasawara~et~al.,~ described~in "Electrophoresis on Konjac Mannan Geln~, Seibutsu Butsuri, ~, pp. 155-158 l987), ~Ex. 77)~which représents a slight modification of the U.S. 3,~928,~322 process. The results of all of 30~ these tests are~;set forth below in Table VI, and in Figure l, wherein~all nitrogen values are based on the dry weight of the~product.
In Table~VI,~as~described in footnote (b), certain of the turbidity~values were first obtained on a Fisher Spectrophotometer, Model II (Fisher Scientific, ~:: ~ :, WOg3/02571 PCT/US92/06591 ~11314~
-- so --Pittsburgh, Pa.), using a Formazin Standard and then converted to MacBeth Coloreye values. This conversion was carried out thro~gh a correlation study, as follows: 11 Formazin standards, with turbidity values S ranging from S to 400 NTU's, were prepared and measured (% transmittance) on both the MacBeth and Fisher units.
Additionally, S konjac samples (3 crude and 2 clarified) were prepared at concentrations of 1%, 0.5%, 0.2S% and 0.125% and~also measured on both units. The data (% transmittance) from these measurements were plotted against their turbidity values, as determined on each machine, and a correlation coefficient determined.

The process of U.S. Patent 3,928,322 - Sugiyama (Example 7S) was carried out as follows: ~
1. 2.5~g konjac~flour (89-9607) was suspended in S00 m} (O.S% w/v) tap water and heated at -S5-60C for 2 hours.~ ~
~ . .
2. The sol~was passed through a 115 mesh (125 micron) and then a 270 mesh (53 micron) metal screen to remove gross~insolubles~
3. The~sol~would not filter through a medium porosity glass filter (Pyrex 150 ml,~;A5TM 10-15) or a 0.2 ~ micron filter~so instead was heated to 90C and twice passed through a 14 inch - 1 inch diameter (35.6 cm~- 3.54 in diameter) bed of tightly packed glass wool.~ The filtrate, 300 ml, was very clear and appeared to be particle-free.
4. The filtrate was placed in a piece of dialysis tubing (Spectra/Por, 47.;7 mm x 75 mm, molecular weight cut off of 12 -> 14,000 daltons). The sample was dialyzed against 4 liters of tap water for 48 hours ~(the water was changed after 24 hours).

: :~;:

:
~:

5. The sample was then poured into 2 large crystallizing dishes and frozen.

6. Each aliquot was lyophilized at 0.6 Torr with a shelf temperature of 100F (37.8C) for 12 hours.

7. The dried sample was very white and quite fluffy.
~; The yield was 1.137 or 45.5%. Due to excessive static, the sample could not be ground and was wetted with~a~small amount of 20% isopropyl alcohol and then drled at 55C for 3 hours. The sample was then ground through a 40 mesh screen.

8.~ The sample~had a~nitrogen content of 0.07% and a 1.0% turbidlty of 128 Turbidity Units.

9. The process took a total of 68 hours to run.

15 ~
The Ogasawara~process (Example 77) was carried ~ut as follows:~
1. 10 g crude~konjao was suspended in 100 ml 50%
; ethanol~and ;stirred for 1 week.
;20 ~2. This materlal~was centrifugsd and the pellets were transferred to 100 ml 80% ethanol for 3 days with stlrring~
3.~Thls~was~again;centrlfuged (4000 rpm, 10 minutes) and the~pel~lets~transferred to l00 ml 100%
~ ~ (absolute)~ethanol for~l hour.
4. The~sample~was~recovered on ~54 Whatman filter paper by vacuu~ ~filtration, and dried in a 60C
oven for 6 hours.
5. 8.992 g of~material was recovered and was used to prepare a~5%~so1 in 178 ml. This was too viscous to treat, and was diluted 10 fold to 1780 ml (0.5%
w/v) and~allowed to sit overnight at room temperature.
6. This material was centrif~ged for 75 minutes at 9500 rpm.~
:
:;:

7. The supernatant (1700 ml) was dialyzed in lo volumes of distilled water for 3 days at room temperature.
8. The sample was removed from the dialysis tubing and centrifuged at 7500 rpm for 10 minutes.
9. Half of the sùpernatant was coagulated while the ; other half was placed in dialysis tubing and covered with polyethylene glycol (PEG 20) to reduce the volume from 850 ml to 450 ml.

10. This materia~l was frozen at -75C for 45 minutes and then lyophilized at 0.1 Torr and 100F (37.8C) ~; $or 3 days. ~

11. 2.19 g of the lyophilized material was recovered and was very~white and fluffy in appearance.

12. A}l samples~were vacuum dried to remove any moisture before testing. The lyophilized materi~l foamed excessively when the sol was prepared for turbidity measure~ments.
; 13. This process~took~a total of -384 hours to run.
20~ TA~LE VI~ -Corresp. ~ ~ ~ fral~ 1.0X ~bcBeth~d) ~,, Extr~ct-on A~ent e~ X llitro~en~C) ~u biditv ~ ~C 120,09 4~b) ~ 2 ` ~lu~, pitot p~ nt U 0.07 7 66 ~ 3 ~ pH 2~ 2 0.0810 67 ~ 4 ~ ~lu~, pitot p~t a 0.07 18 6B S ~lu~,~pilot pl-nt ~ 0.15 11 69 6 Alu~, pilot pl-nt 48 0.13 14 7 Sod~ cet~te 8 0.0945 71 8 Hot~ater 1 0 0745~b) 72 9 HMP ; 5 O.Q645(b) :: :: :
73 10 Insoluble CMC 23 12 0.03 50 74 11 Insoluble Ct~C 32 12 0.04 50 ~ ; 75 t2 Sugiy~ P~ltent (~bove) 0.07 128(b) ; 76 13 Crude/H~dro ~shed 0.11 177 :' ' :~
~ ' : :

2~ ~51~~ ~
-- 53 ~

Corresp. Frall 1.0X ~acBeth(d) Ex~ro~e Fi~. Extr~cti~n Aaent Ex~role X llitro~ C) TurbiditY

77 14 Og--~r- Publ. ~ bove) 0.31 ~o(b) 78 15 Crude 0.32 92~-~
79 16 Cru~e 0 30 1o1~e) 17 CrlJk 0.29 120~e) 81 18 Crude 0.41 155~) 0 J2 19 Cru~e 0.59 142~e) a~pl- cQntrifuged ~t ~000 rp~ for S mfnut~J before an~lyJis b. the values~originally o~ta~ned on a ~isher Spectrophoto~eter, 15~ thon con~ rfod into Nac~eth~-guivalents as doter~ined by a r~grossion lin-~obtain~d by plotting ~alues obta~nQd from a corr~lation study of id~ntical sa~ples measurod on both instrument~
, c. baJed on;the dry;~eight of the product.
d. -~m~-~urQd~on~a~-cBQth Color~y~ Comput~r ~S-rie~ 1500J, u~ing a ~or ~zin`~standard. ~-;e.~ O.S~ solution;turb~dity.
From tho~foregoing results it will be seen that whereas~:the:~:nitrogen and~turbidity values of the :25~ products of~this~invention (Examples 64-75) were both low,~the~corresponding values~of the crude konjac, as well~as one:~or:both of:the~Sugiyama~ and Ogasawara products,were:s~ignificantly~higher by comparison.

The following~example illustrates the inhibiting ~: :effect of hydrocolloids on:the cold-melt properties of : the clarified~gels of this~invention.

: : ~ .

: : ~

W093/~2571 PCT/US92/065gl :2 1 ~

EXAMPLE 83 (Added H~drocolloids Gums) Xanthan: 100 g of a hot 1% clarified konjac sol was mixed with 33 g of a 1~ w/v sol of xanthan (Keltrol T, Kelco Co., San Diego, Ca.). The mixture, which began gelling almost immediateIy, was heated in a hot water bath to melt the gel. Once melted, two 50 g aliquots were poured into beakers. Two ml of 5M NH40H
:
was stirred into each hot liquid sample. One was placed in a boiling water bath for 20 minutes while the other was allowed to cool to room temperature. Both samples formed gels~although they differed in ; appearance and~texture. The heat set gel was opaque and somewhat spongy while the second aliquot (not heat set) was clear and very elastic. The heat set gel, when placed in~an ice bath, became clear and elastic but did not~liquefy. When this transformed gel was~
reheated, it took on its original properties, that is, opaque and spongy.~ When placed in an ice bath, it again reverted to~the clear elastic gel.
20 ~ Carrageenan~ 33~g of a 1%~w/v CIC carrageenan sol (sodium, reduced-viscosity~kappa-form, a product of FMC
Corporation,~Marine Colloids Division, Philadelphia, Pennsylvania) was~mixed~with 100 g of a 1% clarified konjac -ol. Five~ml~of~5M~NH40H was added with 25;~ stirring~and~the sample was~heat set for 20 minutes. A
soft opaque;gel formed which, when placed in an ice bath, was transformed into a clear very elastic g~l, ~; but did not liquefy.
Agarose: 5 x 67 g samples of a 1% 3:1 clarified 3~0 glucomannan/agarose~sol were prepared~by mixing 50 g of a 1% konjac sol with 16.7 g agarose sol (SeaKem~ LE
agarose, FMC Corporation, Marine Colloids Division, ; Bioproducts~Group, Philadelphia, Pennsylvania). Two ml of SM NH40H were~added to~four of the aliquots and two of these were heat set in a boiling water bath for 20 minutes. ~ ~
`: :

2 ~

All five samples formed gels. Those gels formed with base and heat were opaque and very æoft. The gels which were not heat set (two with base, one without) were clear and tough. When a heat set gel was placed in an ice bath, it did not melt but was transformed ; into a clear:tough gel, analogous to the non heat-set samples.

:
.
:

.

:: :

.
; 30 ~: .

Claims (37)

CLAIMS:

1. Clarified konjac characterized in that it comprises glucomannan derived from konjac which is substantially free of insoluble impurities; and [A] has a nitrogen content of from more than 0.25 up to about 0.60 wt % and an aqueous sol turbidity potential of from 20 to 70 turbidity units as measured at 1.0 w/v % concentration using the Formazin Turbidity Standard; as well as in a continuum [B] a nitrogen content of 0.25 wt % or less, and an aqueous sol turbidity potential of 20 to 100 turbidity units as measured at 1.0 w/v % concentration using the Formazin Turbidity Standard.

2. The clarified konjac of claim 1 characterized by a nitrogen content of 0.25 wt % or less, and an aqueous sol turbidity potential of 20 to 100 turbidity units.

3. The clarified konjac of claim 1 characterized by a nitrogen content of 0.175 wt % or less and an aqueous sol turbidity potential of 20 to 70 turbidity units.

4. The clarified konjac of claim 1 characterized by a nitrogen content of 0.15 wt % or less and an aqueous sol turbidity potential of 20 to 60 turbidity units.

5. The clarified konjac of claim 1 characterized by an aqueous sol viscosity potential of 50 to 25,000 cps at a 1 w/v % concentration as measured using a Brookfield Viscometer Model LVTDV-II at 25°C and 12 rpm.

6. The clarified konjac of claim 5 characterized by a viscosity of 1,000 to 25,000 cps.

7. The clarified konjac of claim 1, 2, 3, 4, 5, and 6, characterized in that it comprises an aqueous sol.

8. The clarified konjac of claim 1, 2, 3, 4, 5, and 6, characterized in that it comprises an aqueous gel.

9. The clarified konjac of claim 8, characterized in that it comprises a mixture with at least one additional hydrocolloid before said gel is formed.

10. The clarified konjac gel of claim 9, characterized in that the additional hydrocolloid is selected from among carrageenan, xanthan, and agarose.

11. The clarified konjac gel of claim 10 characterized in that the weight ratio of clarified konjac to hydrocolloid in the gel mixture is about .1-10:1.

12. A clarified konjac comprising glucomannan derived from konjac which is substantially free of insoluble impurities; has a nitrogen content of 0.60 wt % or less; and has an aqueous sol turbidity potential of less than 20 turbidity units as measured at 1.0 w/v % concentration using the Formazin Turbidity Standard;
characterized in that it comprises a mixture with at least one additional hydrocolloid before said gel is formed.

13. The clarified konjac gel of claim 12, characterized in that the additional hydrocolloid is selected from among carrageenan, xanthan, and agarose.

14. The clarified konjac gel of claim 12, characterized in that the weight ratio of clarified konjac to hydrocolloid in the gel mixture is about .1-10:1.

15. The clarified konjac of claim 1 characterized in that it is in the form of a clear, water-insoluble, spongy, dimensionally stable mass.

16. A clarified konjac comprising glucomannan derived from konjac which is substantially free of insoluble impurities; has a nitrogen content of about 0.60 wt % or less; and has an aqueous sol turbidity potential of less than 20 turbidity units as measured at 1.0 w/v % concentration using the Formazin Turbidity Standard; characterized in that it is in the form of a clear, water-insoluble, spongy, dimensionally stable mass.

17. The clarified konjac of claim 1 characterized in that it is an aqueous cold-melt gel at temperatures above about 5°C which reversibly liquifies to a clear sol at temperatures between 5°C to 0°C.

18. A clarified konjac comprising glucomannan derived from konjac which is substantially free of insoluble impurities; has a nitrogen content of 0.60 wt % or less; and has an aqueous sol turbidity potential of less than 20 turbidity units as measured at 1.0 w/v % concentration using the Formazin Turbidity Standard;
characterized in that it is an aqueous cold-melt gel at temperatures above 5°C which reversibly liquifies to a clear sol at temperatures between 5°C to 0°C.

19. A method for the production of clarified konjac characterized by the consecutive steps of:
[a] preparing an aqueous sol of crude konjac comprising insoluble impurities and glucomannan;
[b] contacting the crude konjac sol with an extraction-effective amount of an agent capable of extracting the insoluble impurities;
[c] precipitating and removing the insoluble impurities;
[d] forming a glucomannan coagulate by treating the remaining aqueous sol with a coagulant present in an amount sufficient to coagulate substantially all glucomannan therein; and [e] removing and drying the glucomannan coagulate to recover the dry, clarified glucomannan.

20. The method of claim 19 characterized by selecting the extraction agent from among: chelating agents; soluble salts; insoluble salts; ion-exchangers;
organic solvents; hot water; or means for adjusting the pH of the sol.

21. The method of claim 19 characterized in that the extraction agent is an insoluble salt.

22. The method of claim 19 characterized in that the coagulant is isopropyl alcohol.

23. The method of claim 19 characterized by treating the aqueous crude konjac flour with a viscosity-reducing agent prior to extraction.

24. The method of claim 19 characterized by treating the aqueous sol remaining after extraction with a viscosity-reducing agent prior to treatment with the coagulant.

25. The method of claim 19 characterized by treating dry clarified glucomannan or an aqueous sol thereof before, during or after clarification with a viscosity-reducing agent.

26. The method of claim 19 characterized in that the viscosity-reducing agent is an acid which is simultaneously used as an extraction agent.

27. The method of claim 19 characterized in that the viscosity-reducing agent is gamma ray irradiation.

28. A method for the production of the spongy, dimensionally-stable mass of claim 15 characterized in that clarified konjac aqueous sol is cooled to freezing temperature or slightly below and then brought back to room temperature.

29. A method for the production of the spongy, dimensionally-stable mass of claim 16 characterized in that clarified konjac aqueous sol is cooled to freezing temperature or slightly below and then brought back to room temperature.

30. A method for the production of the cold-melt gel of claim 17 characterized in that the pH of a clarified konjac aqueous sol is adjusted to between about 9.6 and 12.3 before gel formation.

31. The method of claim 30 characterized in that the pH is adjusted to between 10.0 and 11.5.

32. The method of claim 30 characterized in that said gel formation is effected while heating for 5 to 60 minutes at a temperature of 50 to 120°C.

33. The method of claim 31 characterized in that said gel formation is effected while heating for 20 to 30 minutes at a temperature of 80 to 90°C.

34. A method for the production of the cold-melt gel of claim 18 characterized in that the pH of a clarified konjac aqueous sol is adjusted to between 9.6 and 12.3 before gel formation.

35. The method of claim 34 characterized in that the pH is adjusted to between 10.0 and 11.5.

36. The method of claim 34 characterized in that said gel formation is effected while heating for 5 to 60 minutes at a temperature of 50 to 120°C.

37. The method of claim 35 characterized in that said gel formation is effected while heating for 20 to 30 minutes at a temperature of 80 to 90°C.