Agricultural and Biological Chemistry

ISSN: 0002-1369 (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tbbb19

Molecular Weight and Intrinsic Viscosity of Konjac

Gluco-mannan

Noriko Kishida, Satoshi Okimasu & Toshio Kamata

To cite this article: Noriko Kishida, Satoshi Okimasu & Toshio Kamata (1978) Molecular Weight
and Intrinsic Viscosity of Konjac Gluco-mannan, Agricultural and Biological Chemistry, 42:9,
1645-1650, DOI: 10.1080/00021369.1978.10863226

To link to this article: https://doi.org/10.1080/00021369.1978.10863226

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 Agric. Bioi. Chem., 42 (9), 1645 ~ 1650, 1978

Molecular Weight and Intrinsic Viscosity of Konjac Gluco-mannan

Noriko KISHIDA, Satoshi OKIMASU and Toshio KAMATA*

Laboratory of Food and Nutrition, Hiroshima Women's University, Hiroshima 734, Japan
*National Chemical Laboratory for Industry, Tokyo 151, Japan
Received December 6, 1977

For the purpose of making clear the macromolecular chemical properties of konjac
gluco-mannan, the light scattering and viscosity measurements were carried out for the aqueous
solutions of the partially methylated derivatives, and following results were obtained: 1) The
weight-average molecular weight, Mw,and the root mean square of radius of gyration, (S2)1/2,
were 100xl04~120xl04 and 1100~1300 A, respectively. 2) The (S2) versus Mw re-
lationship was represented by the equation of (S')=4.20 x 10-1. MW1.0B, and this fact
suggested a random coil as a molecular form in the solution. 3) The intrinsic viscosity, [r;],
versus Mw relationship was represented by the equation of [1)]=6.37xl0- 4·Mwo. 74, which
was considered to be useful for the purpose of estimating the molecular weight of konjac
gluco-mannan by viscometric method.

Konjac gluco-mannan (KGM) is a main In a previous paper,4) we have reported the

component of the konjac flour produced from method for preparing a water-soluble methyl-
the tubers of Amorphophallus Konjac C. Koch, KGM by a moderate treatment which may not
and has the rheological and biochemical charac- develop the depolymerization of KGM mole-
teristics inherent in this polysaccharide. These cules. Thus, the present paper deals with the
characteristics vary considerably according to results of the light scattering and viscosity
the methods of preparation and the strains or measurements for various methyl-KGM solu-
producing areas of tubers. Accordingly, it is tions.
of great interest to study such macromolecular
MATERIALS AND METHODS
chemical properties as molecular weight, mole-
cular form, intrinsic viscosity and their mutual KGM materials for methylation. Four strains of
relationships etc. of KGM, but only a few konjac fluors and 7 kinds of the partial hydrolyzates
shown in Table I were prepared as the KGM materials
reports have been made.
for methylation.
Torigata et aU) have reported the results of The flours were prepared specially for the present
the light scattering and viscosity measurements study from the tubers cultivated in 1974 at the agricul-
for an isoamyl acetate solution of nitro-KGM,
TABLE I. KGM MATERIALS FOR METHYLATION
but it is probable that KGM molecules may
have suffered some degradation during nitra- No.a Strain Producing Conditions of
prefecture hydrolysis
tion procedure. On the other hand, Sugiyama
Nl Zairai Hiroshima
et al. have reported the results of the light
N2 Akagi6dama Gunma
scattering measurements 2 ) for the aqueous solu- N3 Zairai Gunma
tions of native KGM, which was isolated by N4 Shina Gunma
a method in which konjac flour washed with HI Zairai Hiroshima 60°C, 300 min
50% ethanol was dissolved in water, centri- H2 180
H
" /I ,

fuged, dialyzed, and then lyophilized:) In H3

" " " , 120
H4 /I /I H , 60
their report, however, the viscosity measure- H5 50°C, 120
ment has not been made, perhaps owing to the " "
H6
" " " , 60
fact that the stability of the solution was in- H7
"
H
" , 30
sufficient judging from the result of our close a Nl ~ N4
are konjac flours and Hl ~ H7 are the
reexamination of their experiment. hydrolyzates.
 1646 N. KrSHIDA, S. OKlMASU and T. KAMATA
tural experimental stations of Gunma and Hiroshima
prefectures. Namely, the raw tubers were sliced ca.
5 mm in thickness, and dried in a hot-air dryer at 100 e0
until the moisture decreased to ca. 10 %. The dried
materials were pulverized for 8 hr by a stamp mill,
and then elutriated and sieved from the pulverized
materials. The flours were further purified by the
following method reported previously by Sugiyama
et al. 3 ) : About 50 g of flour was stirred with 250 ml of
50% ethanol for 3 hr at room temperature, and filtered
through double sheets of cheesecloth. This treatment
was repeated 4 times. The final residue was successive-
ly washed with 80% and absolute ethanol, and dried
in vacuo at 70°e.
It was confirmed by the following experiments that
the main component of the purified flours thus obtained
was KGM: The flour was dissolved in water at ca.
0.5 % with stirring for 3 hr at room temperature, and
the solution was centrifuged for 30 min at 10,000 rpm
to remove a small amount of insoluble substance.
The supernatant fluid was dialyzed for 3 days at lODe
through cellulose membrane against running distilled
water. This solution did not show at all any reactions
of starch-iodine, ninhydrin, and reducing sugars. In
addition, a mixture of 40 ml of this solution and 10 ml
of 0.5 N H 2S0 4 was heated in a boiling water bath for
24 hr under reflux. The hydrolyzate gave only two
spots, corresponding to D-mannose and D-glucose, on
usual paper chromatography. The molar ratio of
D-mannose to D-glucose was found to be ca. 1.6: 1.0
by measuring the spots with a densitometer.
Thus in the present work, the solutions of purified
flours were used as the KGM materials for the prepa-
ration of hydrolyzates or methyl-derivatives after the
centrifugation and/or the dialysis were performed.
The partial hydrolyzates were prepared from the
purified flour of Hiroshima prefecture. About 0.5 %
solution of the flour was prepared and centrifuged in
the same manner as described above. The hydrolysis
was carried out by heating 400 ml of the supernatant
with 100 ml of 0.5 N H 2 S0 4 for a limited time at the
fixed temperature. The solution was then neutralized
with 1 N NaOH, and dialyzed for 2 days. The product
was separated into 3 fractions by the fractional precipi-
tation with methanol. The middle fraction was taken,
dehydrated with acetone, and then dried in vacuo at
50°e. Each reaction conditions of hydrolysis is shown
in Table I.
Preparation of methyl-KGM. The KGM materials
mentioned above were dissolved in water at 0.5 % with
stirring for 3 hr at room temperature. In case of
flour, however, the solution was centrifuged and dialy-
zed in the same manner as described above. The exact
concentration of KGM in the dialyzed solution was
estimated by evaporating 30 g of the solution to dryness
at 105°e and weighing.
These solutions were submitted to methylation by
our method 4 ) reported previously on purpose to prepare
the water-soluble methyl-KGM having a degree of sub-
stitution (DS) of ca. 0.45, where a DS of 1 was defined
as one methoxyl group per hexose residue of KGM.
To an appropriate amount of each solution was added
60% NaOHto a final concentration of 1 %, and half
the specified amount of dimethyl sulfate was added to
this little by little, followed by standing for 1 hr at
30°e. After the concentration of NaOH in the solu-
tion was adjusted again to 1 % with 60% NaOH, the
other half of dimethyl sulfate was added in the same
manner, and the solution was allowed to stand for 3 hr
at 30 e with ocassional shaking. The resulting solu-
0
tion was successively dialyzed for 3 and 2 days through
cellulose membrane against running tap and running
distilled water, respectively. The product was pre-
cipitated and dehydrated with acetone, and dried in
vacuo at 50°e. Each methyl-KGM thus obtained was
entirely free from ash and nitrogen, and very soluble
in water. The DS was estimated by the previous
method 4 ) as shown in Table II.
Light scattering measurements. When unpolarized
light is used as a light source at the light scattering
measurement of a dilute polymer solution, the reduced
intensity, Ro, of scattered light is given by the following
equation. 5 )
KC/Ro=I/Mw' p(e)+2A 2 C (1)
K=27r2n02(dn/dc)2/Na· A04 (2)
Where Mw is the weight-average molecular weight of
polymer, c concentration (g/cc), A2 second virial co-
efficient, 110 refractive index of solvent, dn/dc the specific
refractive index increment, Ao wavelength of incident
light in vacuum, and Na Avogadro number. pee) is
called the particle scattering function, and the relation-
ship between pee) and the mean square of radius of
gyration, <S2), is represented by the following
equation.
P(e)=I-(l/3)(47r/Ao)2<S2)Sin 2(e/2)+... (3)
On the above equations, K is the optical constant
decided according to the kinds of polymer and solvent
Therefore, Ro of various concentrations of polymer
solution is measured at various angles, and then Mw,
A2 and <S2) are generally found by the well-known
Zimm-plot method') (See Fig. 1). However, the aque-
ous solution of methyl-KGM exhibits very high vis-
cosity in ordinary concentration for light scattering
measurements, and the optical clarification is difficult.
Furthermore, such a Zimm-plot is usually time-con-
suming. Thus in the present work, all the light scatter-
•
mg measurements were carried out for extremely dilute
solution, and the calculation of these molecular con-
stants was treated with one concentration method") in
light scatterning. .
 Molecular Weight and Intrinsic Viscosity of Konjac Gluco-mannan 1647

was centrifuged for 1 hr at 10,000 rpm. The viscosity

of the supernatant was then measured in various con-
10 centrations at 30±0.02°C with Ubbelohde-type visco-
meter. The flow time for water was 93.0 sec. The
intrinsic viscosity, [1]], was found by plotting reduced
viscosity against concentration and extrapolating to
zero concentration.
"0
The concentration assay of sample solution. The
......•
~

a: concentration of methyl-KGM in all the sample solu-

<> tions was estimated by the phenol-sulfuric acid meth-
"
'" ""
5
od,n improved upon the Dubois's method. As the con-
centration of solution has great influence on the data
obtained in light scattering and viscosity measure-
ments, the concentration assay must be carried out
with great accuracy. Therefore, it was perfonned
O L
________~I--------*I--------~I
1 2 3
cautiously under the same conditions as employed ip.
5in2 (912) + 1000 c making the calibration curve for methyl-KGM.

FIG. 1. Zimm-plot for Methyl-KGM Solution

(Sample, No. MH2). RESULTS AND DISCUSSION
•

Sample methyl-KGM (10~25 mg) was dissolved in Molecular weight and mean square of radius of
ca. 40 ml of dust free water, and the resulting solution gyration
was centrifuged for 1 hr at 30,000 rpm (90,000 G) by 2
The KclR o versus sin ({)12) plots obtained
Spinco Model L 4 Ultracentrifuge. Mter moderately for 11 kinds of methyl-KGM are shown in
diluting the supernatant with clean water, the solution
Fig. 2, and the values of Mw and <S2)1/2
was filtered into the light scattering cell through a
Corning ultrafine sintered glass filter. The light scatter- found in this figure are summarized in Table II.
ing measurements were carried out by Shimadzu light As seen in Table II, the values of M wand
scattering photometer (Type PG-21) at room tempera- <S2)1/2 for methyl-derivatives of native KGM
ture using the wavelength of 4358 A, and the angular are high as 100x 104 ", 120x 104 and 1l00",
distributions of light scattering were observed. The 1300 A, respectively, and no significant differ-
dn/dc of methyl-KGM solution was measured by
Shimadzu differential refractometer, and was found
ence due to the strain of KGM is observed.
to be 0.1453 (cc/g) for 4358 A.
All results of light scattering measurements were 10
MH3
calculated with least-squares method by an electronic
computer. "0
~
MH6
MH7
On one concentration method of light scattering, it "a:'" 5 MN2
MNI

is desirable to measure the reduced intensity of scattered ~

light on the condition that the error brought about by '"
ignoring the concentration dependent terms, A z, is
less than 2 %. Consequently, one concentration method
must be carried out for the solution of such concen-
• MHI
tration as satisfies the following equation. 6) •
1 MH2
Ro-;;;'Kx 10- 2/A 2 (4)
MH·,
As the value of K is already known in the present ex- MHS
MN4
MN3
periment, if the order of A2 is known it is possible to
set such measuring conditions as satisfies Eq. (4). The
value of A2 was found to be 3.5 x 10- 4 from the Zimm-
plot (see Fig. 1) for sample MH2. Thus the condition
of the present experiment was set using this value, and
o
the correction by A2 was applied to all data obtained. Sin2 (e/2)

Viscosity measurements. Sample methyl-KGM was FIG.2. Kc/Ro versus Sin 2 (0/2) Plots for Various
dissolved in water at 0.04~0.12 g/dl, and the solution Methyl-KGM Solutions.
1648 N. KISHIDA, S. OKIMASU and T. KAMATA

TABLE II. ABSTRACT OF THE RESULTS OBTAINED FOR VARIOUS METHYL KGM SOLUTIONS

Light scattering data Intrinsic Expansion

Sample DS of viscosity coefficient
No.a sample Concentration Mwx10- 4 <S2)1/2 [1J] (dljg) a.
of soln.(gjdl)
MH1 0.44 0.00802 16.0 427 4.6 1.11
MH2 0.45 670 21.7 506 5.6 1.13
MH3 0.45 616 26.3 571 7.4 1.20
MH4 0.44 310 48.7 792 10.9 1.23
MH5 0.45 368 56.8 827 11.4 1.22
MH6 0.43 319 59.5 873 13.4 1.27
MH7 0.44 304 81.3 1017 15.0 1.25
MN1 0.46 368 116 1304 19.9 1.32
MN2 0.45 309 111 1233 19.9 1.29
MN3 0.44 342 101 1136 18.6 1.29
MN4 0.45 314 98.5 1145 18.8 1.29
a These samples are the methyl derivatives of each KGM material shown in Table I.

On the other hand, Sugiyama et a[.2) have
carried out the same one concentration method
of light scattering as our work for various
sources of native KGM, and reported the
4 4
values of Mw-25.5x10 "'190x10 and
<S2>1/2=630", 2330 A. However, these
values for KGM prepared from common
flours are of almost the same order as our
data, except that these values for Shina Shu
(Chinese species) are extremely high (Mw=
190 X 10\ <S2>1/2 2330 A).
Furthermore, they have reported the values
of Mw-27.0x 104 and <S2>1/2 570 A (cor-
respond to the root mean squares of end to
end distance, <r2>1/2, of 1396 A) for the
deacetylated KGM, which was prepared by
the deacetylation of acetyl-KGM with alkali.
On the other hand, Torigata et aU) have
4 This fact is naturally understood to suggest
reported the values of Mw=27.l x 10 and
2
<r >1 /2= 1380 A (correspond to <S2>1/2 of
563 A) for the isoamyl acetate solution of
nitro-KGM. Sugiyama et af.2) have explained
these results by the assumption the KGM
molecules are degraded to one-fourth through
acetylation and deacetylation as well as in
case of nitration. These values of Mw and
<S2>1/2 are nearly equal to those that we
have obtained for a methyl-derivative (MH3
in Table II) of hydrolyzed KGM. Accordingly,
it is considered that the degradation of KGM
molecules surely occurs during the preparation.
From the facts mentioned above, it is Con-
eluded that no degradation of KGM molecules
occurs through the methylation and such values
obtained for methyl-derivatives show those of
native KGM. Moreover, the partial methyl-
lation of KGM brings about a remarkable
merit of the stability of the solution.
In addition, it has been well-known8 ) that
the molecular form of polymer in the solution
is elucidated from the <S2> versus· Mw
relationship. Accordingly, this relationship
of methyl-KGM was examined, and a good
linear relationship was then observed between
log <S2>1/2 and log Mw as shown in Fig. 3.
This is represented by the following equation,
and <S2> may be said to be almost propor-
tional to Mw.
<S2> 4.20 X 10- 1 • M Wl. 08 (5)
3.0
2.0 ;:-_ _ _ _--:' ;:--.....,...._ _ _ _ ~-
5.0 5.5 6.0
l09Mw
FIG. 3. Log <S2)1I2 versus .Log Mw Plot for
Methyl-KGM Solution.
 Molecular Weight and Intrinsic Viscosity of Konjac Gluco-mannan 1649

a random coil as the molecular form of KGM

in the solution.

Intrinsic viscosity versus molecular weight

relationship. A good linear relationship was 2
•
observed between log [r;] and log Mw as shown ...
in Fig. 4. This means that the [r;] versus Mw • •
relationship is in good conformity with the ••
•
following Sakurada-Houwink's equation 9 ) for
linear polymer. • •
1
(6)
In this equation, the values of K and a were
4 o 10
found to be 6.37 X 10- and 0.74, respectively.
These values are compatible with the chemical
structure and the <S2) versus Mw relation- FIG. 5. [7]]/M"/2 versus M"/ 2 Plot for Methyl-KGM
ship of methyl-KGM. Solution.
As for the intrinsic viscosity versus molecular
eluded volume effect has been reported.
weight relationship of KGM, Torigata et aU)
have reported the equation of [r;]= 11.6 x 10- 4 • [r;]=KM"/2 as 3 (7)
p O.95 (P is degree of polymerization.) for the
Where as is the expansion coefficient, which is
isoamyl acetate solution of nitro-KGM. We
a parameter concerning the extension of poly-
have attempted to reexamine their results with
mer chain in the solution. The value of con-
great interest, but we could not obtain the
stant, K, can be found from the [r;]/M"/ 2 versus
nitro-derivative in spite of the repeated trial of
M"i2 plots based on the Stockmayer-Fixman's
preparation by the reported procedure or
equation. 11)
under various conditions. Therefore, the
From the above standpoint, the [r;]/M"/2
nitration itself seems to be questionable as
versus M"/2 plots for methyl-KGM solution
the method for the macromolecular chemical
were made as shown in Fig. 5, and the value
study of KGM.
of K was found to be 0.85 in this figure. The
On the other hand, as for the relationship
values of as calculated from Eq. (7) are sum-
between the intrinsic viscosity for a linear poly-
marized in Table II. The as of the native
mer solution and the molecular weight, follow-
KGM derivatives shows almost the same value
ing Flory-Fox's equation" O) reflected an ex-
of ca. 1.30, but that of the hydrolyzed KGM
derivatives becomes somewhat smaller than this
1.5 with the lowering of molecular weight. On the
other hand, it has been known that the a.
values of various polymers show the highest of
1.0 ca. 1.7 for good solvent and the lowest of
,-,
.~ ca. 0.88 for poor solvent. Thus, it is concluded
en that the methyl-KGM molecules in aqueous
o
-' 0.5
solution are solvated considerably in the form
of a stretched random coil, and this fact is
supported also by the stability of the solution.
o L.-_ _ _--;: L.-_ _ _---: '---
5.0 55
. 6.0 Thus in the present paper, such macromole-
LogMw cular chemical properties as molecular weight,
4. Log [7]] versus Log Mw Plot for Methyl-
FIG.
molecular form, intrinsic viscosity and their
KGM Solution. mutual relationships etc. of KGM have been
1650 N. KISHIDA, S. OKIMASU and T. KAMATA
clarified for methyl-KGM solution. The rela-
tionship shown in Eq. (6) is expected to be
conveniently used for the purpose of estimating
the molecular weight of KGM by viscometric
method. However, as for the connection of
these properties with the rheological and bio-
chemical characteristics, further investigation
will be necessary.
Acknowledgements. We are sincerely grateful to
Dr. R. Senju and Prof. I. Sakata, Kyushu University,
for their helpful advice and instruction.
REFERENCES
1) H. Torigata, H. Inagaki and H. Kitano, Nippon
Kagaku Zasshi, 73, 186 (1952).
2) N. Sugiyama, H. Shimahara, T. Andoh, M. Take-
moto and T. Kamata, Agric. BioI. Chern., 36, 1381
(1972).
3) N. Sugiyama, H. Shimahara and T. Andoh, Bull.
Chern. Soc. Jpn., 45, 561 (1972).
4) N. Kishida and S. Okimasu, Agric. BioI. Chern.,
42, 669 (1978).
5) B. H. Zimm, J. Chern. Phys., 16, 1093 (1948).
6) T. Kamata and H. Nakahara, J. Colloid Interface
Sci., 43, 89 (1973).
7) T., Fujikawa, M. Shiomi, S. Mukai and M. Wada,
Nippon Nogeikagaku Kaishi, 48, 483 (1974).
8) A. Nakashima and M. Hosono, "Molecular
Matter Nature of Polymer," Vol. 1, Kagaku
Dojin Press, Kyoto, 1969, pp. 248~249.
9) A. Nakashima and M. Hosono, ibid., Vol. 2,
pp. 314~ 315.
10) P. J. Flory and T. G. Fox, J. Arn. Chern. Soc.,
73, 1904 (1951).
11) A. Nakashima and M. Hosono, "Molecular
Matter Nature of Polymer," Vol. 1, Kagaku
Dojin Press, 1969, pp. 205 ~ 206.