Rheological Property of Hydrolyzed Konjac Glucomannan

R. Ojima, T. Makabe, P. Prawitwong, R. Takahashi, M. Takigami* and S. Takigami Gunma University, 1-5-1, Tenjin-cho, Kiryu, Gunma 376-8515

Fax: 81-277-30-1141, e-mail: takigami@chem-bio.gunma-u.ac.jp *Gunma Industry Support Organization, Maebashi, Japan

Konjac glucomannan (KM) is a water soluble glucomannan with high molar mass. KM aqueous solution shows extremely high viscosity. KM can be depolymerized by chemical hydrolysis. The molar mass of hydrolyzed KM decreased with increase of acid concentration and hydrolysis time. The acid hydrolysis led chain scission of KM, but introduced no significant new chemical groups into the structure. KM molecular chain was hydrolyzed randomly by acid hydrolysis. The viscosity of KM aqueous solutions decreased with decreasing molar mass. KM solution changed to Newtonian fluid with decreasing molar mass at low concentration. KM with molar mass less than 320 kDa was Newtonian fluid in semi-dilute region. KM aqueous solution showed two kinds of critical overlap concentration of C* and C**. C* is onset of shrinkage of the polymer coils and C** is the critical concentration to attain their limiting size. The overlapping and entanglement of KM chains with high molar mass occurred at lower concentration as compared to those of KM with low molar mass. Key words: Konjac mannan, low molar mass, viscosity, rheology, critical overlap concentration

1. INTRODUCTION Konjac glucomannan (KM) is a neutral glucomannan

derived from tubers of Amorphophallas Konjac C. Koch, having β-1,4 linked β-D-glucose (G) and β-D-mannose (M) backbone approximately in the ratio of 1:1.6. KM has side chains and the branching positions are considered to be the C3 of G and M. An acetyl group is attached to ca.13 sugar residues [1]. Deacetylation occurs in the presence of alkali and a non-thermoreversible elastic gel is formed [2]. KM aqueous solution shows extremely high viscosity. Polysaccharides undergo depolymerization by various methods: γ-rays irradiation [3, 4], ultrasonic irradiation [3], chemical [5] and enzymatic hydrolyses [6].

From our previous study, the γ-rays irradiation led to chain scission of KM, but introduced no significant new chemical groups into the structure, apart from a small increase in content of carbonyl groups [7]. The viscosity of irradiated KM aqueous solutions decreased with decreasing molar mass.

Here, KM with lower molar mass was prepared by acid hydrolysis applying pressure. Characterization of the hydrolyzed KM was carried out using FT-IR, GPC-MALS and viscometers.

2. MATERIALS AND METHODS 2.1. Materials

Commercial konjac flour (Akagi Ohdama species) was supplied by Ogino Shoten Co. Ltd.(Gunma, Japan). The flour was purified by washing with ethanol aqueous solution for several times before use. Citric acid and other chemicals were special reagent grade and used as received. 2.2. Preparation of low molar mass KM

The purified KM and citric acid aqueous solution containing 0.001% NaN3 were put in an autoclavable

plastic bag and 3% KM aqueous solution was prepared. The KM solution was pressurized up to 0.247 MPa at 125°C using a pressure cooker. The hydrolysis of KM was performed by two ways: hydrolysis time was changed from 0 to 30 min using 0.5 mM citric acid and the acid concentration was varied from 0 to 1 mM at constant hydrolysis time of 20 min. 2.3. FT-IR measurement The hydrolyzed KM aqueous solution was centrifuged to remove cell wall debris before lyophilization. FT-IR spectra of the original and hydrolyzed KM were measured by a Magna 560 FT-IR spectrometer equipped with a Continuµm infrared microscope (Nicolet). A ZnSe polarizer was used as a high refractive index material. 2.4. Molar mass measurement

Molar masses of KM samples were determined by a multiangle laser light-scattering (MALS) using a MALS detector from DAWN DSP (Wyatt Technology) equipped with a vertically polarized He-Ne laser operated at wavelength of 632.8 nm. The photometer was connected to a size exclusion chromatography (SEC) column of GMPWXL (Tosoh) and a differential refractive index detector RI-71S (Shodex). The temperatures of the MALS flow cell and the column were controlled at 40 °C.

The KM solution was filtered through a 0.45 μm cellulose acetate membrane filter (Sartorius). Scattered light intensities at scattering angle between 15° and 163° were measured. The angular dependence of the scattering intensity was analyzed using Berry’s square-root plot to determine the radius of gyration and molar mass at each position of the peak. 50 mM sodium nitrate aqueous solution was used as both solvent and eluant at 1 mL/min.

Transaction of the Materials Research Society of Japan 34[3] 477-480 (2009)

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2.5. Rheological measurements The hydrolyzed KM was diluted with 0.001% NaN3

aqueous solution and centrifuged to remove cell wall debris before measurements. A RV-II rotation viscometer (Brookfield) and an Ubbelohde viscometer (Kusano Science) were used to measure viscosities of KM solutions. 3. RESULT AND DISCUSSION 3.1. FT-IR spectroscopy.

KM was hydrolyzed in 0.5 mM citric acid solution for scheduled time. Fig. 1 shows typical FT-IR spectra of original KM and acid hydrolyzed KM. For original KM, absorption peaks assigned to C=O stretching vibration of acetyl groups and C-O-C anti-symmetry stretching vibration of pyranose ring were observed at 1730 cm-1 and 1154 cm-1, respectively. Similar IR patterns were observed for the hydrolyzed KM. Since the absorbance ratio of C-O-C to C=O was hardly changed even at 30 min treatment, no elimination of acetyl groups occurred by the acid hydrolysis. The same results were observed for hydrolyzed KM at different acid concentration systems. Accordingly, it is inferred that the chemical structure of KM scarcely changed by acid hydrolysis. 3.2 Hydrolytic degradation

SEC-MALS chromatograms of the hydrolyzed KM are shown in Fig. 2. The chromatogram shifted to lower molar mass and the width of peak enlarged with hydrolysis time. It is inferred that the decrease of molar mass is caused by the main chain scission of KM by the hydrolysis. The weight average molar mass (Mw) and the weight average radius of gyration (RG) were determined and plotted against hydrolysis time in Fig. 3. The Mw and RG of original KM were 1030 kDa and 98 nm, respectively. The Mw and RG decreased significantly with increase of hydrolysis time up to 20 min and then decreased gradually. The Mw and RG of hydrolyzed KM prepared at 30 min were 359 k Da and 53.1 nm, respectively.

When the hydrolysis occurs randomly and obeys the first-order reaction, the following equation should be approved [8]. Where Mn (t) is number average molar mass at time t, Mn(0) is number average molar mass at time 0, Mm is molar mass of monomeric unit of 168, k is rate constant of hydrolytic degradation, and t is hydrolysis time, respectively.

Fig. 4 shows the relationship between 1/Mn and hydrolysis time. Since the value of 1/Mn increased linearly with increase of hydrolysis time, the acid hydrolysis obeys the first-order reaction and KM molecular chains were degraded randomly.

800 1000 1200 1400 1600 1800

C=O

C-O-C

Abs

orba

nce (

A.U

.)

Wavenumbers (cm-1)

(a)

(b)

(c)

(c) 30 min (359 kDa)(b) 20 min (483 kDa)(a) Original (1030 kDa)

Fig. 1. FT-IR spectra of hydrolyzed KM.

0

0.1

0.2

0.3

0.4

0.5

1.00E+04 1.00E+05 1.00E+06 1.00E+07

Molar mass (kDa)

Diff

eren

tial w

eigh

t fra

ctio

n(b)

(f)

(e) (d)

(c)

(a)

10 100 1000 10000

0.1

0.4

0.3

0.2

0

0.5

Fig. 2. Molar mass distribution of hydrolyzed KM. (a) original (1030 kDa); (b) 10 min (850 kDa); (c) 15 min (597 kDa); (d) 20 min (483 kDa); (e) 25 min (428 kDa); (f) 30 min (359 kDa).

0

20

40

60

80

100

2.0E+05

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8.0E+05

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1.2E+06

0 10 20 30

Hydrolysis time(min)

200

400

600

800

1000

1200

Mol

ar m

ass (

kD

a)

0

20

40

60

80

100

Wei

ght a

vera

ge R

G

（nm

）

20 30100

Fig. 3. Effect of hydrolysis time on molar mass and RG of hydrolyzed KM.

1/Mn (t)= kt/Mm+1/Mn (0) (1)

Log Mw4.0 4.5 5.0 5.5 6.0 6.50.0

0.2

0.4

0.6

0.8

1.0

1.20 kGy

Inte

nsity

(a.u

.)

1 kGy5 kGy

10 kGy

Log Mw4.0 4.5 5.0 5.5 6.0 6.50.0

0.2

0.4

0.6

0.8

1.0

1.20 kGy

Inte

nsity

(a.u

.)

1 kGy5 kGy

10 kGy

Rheological Property of Hydrolyzed Konjac Glucomannan478

0.00E+00

1.00E-06

2.00E-06

3.00E-06

4.00E-06

5.00E-06

0 500 1000 1500 2000

Hydrolysis time (sec)

0

1.0

2.0

3.0

4.0

5.01/

Mn

(10-6

Da)

500 1000 1500 20000

Fig. 4. 1/Mn vs. hydrolysis time of hydrolyzed KM.

3.3 Rheological property 3.3.1 Viscosity

The apparent viscosity (ηa) of 0.3 % KM aqueous solution was measured at 25°C using the rotation viscometer. Fig. 5 shows the relationships between ηa and shear rate for 0.3% KM aqueous solutions with various Mw. The ηa of original KM decreased drastically with decrease of the shear rate, which is the feature of pseudo-plastic fluids. The ηa decreased with decreasing molar mass, but 786 kDa KM still showed pseudo-plastic fluids behavior. When the molar mass decreased further, the ηa showed a constant value regardless of the shear rate. The solution of KM with molar mass less than 320 kDa changed to Newtonian fluid. The zero shear viscosity (η0) of KM solution was determined by extrapolation of the ηa value to shear rate of zero.

3.3.2 Critical overlap concentration

The η0 of KM and low molar mass KM solutions were determined at various concentrations. The specific viscosity (ηsp) of KM solutions was determined by an Ubbelohde viscometer in dilute solution. The values of η0 and ηsp were used as zero shear specific viscosity (ηsp0 ) [11].

The ηsp0 of original KM aqueous solution in the form of a double logarithmic plot against concentration of solution (C) is shown in Fig. 6. Three lines with different slopes can be drawn for the plot. The polymer concentration at the intersections of three lines is defined as the critical overlap concentration (C* and C**) [11-13]. The C* is the critical concentration of start to overlap with each other and the C** is the critical concentration of attaining their limiting size [11]. The C*and C** of the original KM were 0.033 g/dL and 0.072 g/dL, respectively.

Fig. 7 shows double logarithmic plots of the ηsp0 against solution concentration of KM with various molar mass. Similar linear relationship between log ηsp0 and log C was observed for low molar mass KM. Both critical overlap concentrations were determined and were plotted against molar mass of KM (Fig. 8). C* and C** shifted to high concentration side with decreasing molar mass. This means that the overlapping and entanglement of molecular chains of KM with high molar mass occur at lower concentration as compared to

those of KM with low molar mass. Fig. 9 shows a schematic model of distribution of

KM molecular chains in aqueous solution. The KM chains are essentially free to move individually below C* and start to overlap each other at C*. Polymer chains form transient network structure above C* and then the chains shrink until at C** where the polymer coils attain their limiting size. At semi-dilute concentration (C > C**), overlapping and entanglement of polymer chains are enhanced remarkably.

ηa

(mP

a·s)

0

200

400

600

0 20 40 60

Shear rate (s-1)

400

40200

200

600

600

ηa

(mPa

·s)

Fig. 5. Effect of acid concentration on molar mass and RG of hydrolyzed KM. ● original (1030 kDa); ○ 0.1 mM (786 kDa); ▲ 0.25 mM (736 kDa); ■ 0.5 mM (538 kDa); △ 0.75 mM (320 kDa); ◆ 1 mM (162 k Da).

log

ηsp

0(m

Pa·

s)

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

-2 -1.5 -1 -0.5 0

C**=0.072 g/dL

C*=0.033 g/dL

●Rotation viscometer○Ubbelohde viscometer

at 25 ºC

log C (g/dL)

Fig. 6. Relationship between KM concentration and zero shear specific viscosity (ηsp0 ) of original KM solution.

Transaction of the Materials Research Society of JapanR. Ojima et al. 34[3] 477-480 (2009) 479

0.5

C*

C* C**

C**

-1.5

-0.5

0.5

1.5

2.5

3.5

4.5

-2.5 -1.5 -0.5 0.5

log

hsp

0 (m

Pa·s

)

log C (g/dL)-2.5 -1.5 -0.5

-1.5

-0.5

1.5

3.5

4.5

2.5

0.5log

ηsp

0(m

Pa·

s)

0.5

Fig. 7. Relationship between KM concentration and zero shear specific viscosity (ηsp0) of KM solution with low molar mass. ● original (1030 kDa); ■0.5mM (538 kDa); ◆ 1mM (162 kDa).

-2

-1.5

-1

-0.5

0

1.00E+02 1.00E+03100 1000

log

C*

and

C**

(g/d

L)

Molar mass (kDa) Fig. 8. Relationships between two types of critical overlap concentration and molar mass of KM solution.

C < C* C=C* C=C** C > C**

KM concentration

highlow

low molar mass

original

Fig. 9. Schematic model of distribution of KM molecular chains in aqueous solution with deferent concentration.

4. CONCLUSIONS The acid hydrolysis led chain scission of KM, but

introduced no significant new chemical groups into the structure. KM molecular chain was hydrolyzed randomly by acid hydrolysis. The molar mass and RG decreased with increase of hydrolysis time and acid concentration. The apparent viscosity of hydrolyzed KM aqueous solution decreased with decreasing molar mass. KM aqueous solution showed pseudo-plastic fluid behavior at semi-dilute concentration. The solution of KM with low molar mass changed to Newtonian fluid with decreasing molar mass. KM aqueous solution showed two kinds of critical overlap concentration, C* is onset of shrinkage of polymer coils which continues until at C** where the polymer coils attain their limiting size. C* and C** increased with decreasing molar mass of KM. REFERENCES [1] M. Maeda, H. Shimahara and N.Sugiyama,

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Rheological Property of Hydrolyzed Konjac Glucomannan480

(Received March 31, 2009; Accepted June 9, 2009)