Note: Descriptions are shown in the official language in which they were submitted.
WO 93/02571 PCI /US92/06591
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.
, ~ .
' .
.
W093/02571 PCT/US92/06591
2 -
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
,
WO93/02571 PCT/US92/06591
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 ~ ~'
-- 4 --
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
W093/02571 PCT/US92/06591
- 5 -
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
~: ~
. ~
;
I
WO93/0~71 PCT/US92/0659}
2 ~
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
; ~
::~:
.~
WO93/02571 PCT/US92/06591
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
WO93/02571 PCT/US92/06591
~ 3~ ~ i
-- 10 --
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
WO93/02571 PCT/US92/06591
-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
:
:
:::
: ~ :
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WO93/02571 PCT/US9~/06591
'~ 1'1 i''l '~ 1. -
- 12 -
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.
WO93/02571 PCT/US92/06591
L '~ ~
- 13 -
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.
: ~ ~: : :
:: ~
WO93~02571 PCT/US92/06591
2 ~
- 14 -
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
: ~ .
W093/02571 PCT/US92/06591
- 15 -
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
- 16 -
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~
- 17 -
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
.
WO93/02571 PCT/US92/06591
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
-- 19 --
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
- 20 -
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
- 21 -
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
- 23 -
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
- 24 -
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
WO93/02571 PCT/US92/06591
- 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
:
;
:
:
WO93/02571 PCT/US92/06591
2~llv~
- 26 -
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 ~
- 28 -
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
2115141
- 29 -
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
- 30 -
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~
- 32 -
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~
- 33 -
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.
::
:
.
W093/02571 PCT/US92/06591
~ 1 A ~; 1 d ~
- 34 -
~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.
EXAMPLE 22
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 ~:
, ~
EXAMPLES 23-42
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.
` 35
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WO 93/02571 PCI`/US92/06591
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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
:
d Oried coagulate,~y weight, calculated as twt
final product/wt start~ng materialJ x 100
e I wt ~ agueoù- ~ol of dried coagulate
f S~o a~ Example 9 but without filter aid
g ~e-au~ed in gr ~ ;
:~ ~
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WO 93/02571 PCI`/US92/06591
~ i 151~1
-- 41 --
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.
EXANPLE 48
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
-- 44 --
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 ~
- 45 -
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 ~
- 46 -
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
- 47 -
The following example illustrates the preparation
of a gel at retort conditions and at a low pH.
EXAMPLE S3
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~
- 48 -
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.
:~ :
WO93/02571 PCT/US92/06591
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).
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WO93/02571 PCT/US92/06591
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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.~
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WO93/02571 PCT/US92/06591
- 52 -
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
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WO93/02571 PCT/US92/06591
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.
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W093/~2571 PCT/US92/065gl
:2 1 ~
- 54 -
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. ~ ~
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WO93/02571 PCT/US92/06591
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- 55 -
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.
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