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Characteristics of glucomannan isolated from fresh tuber of Porang

(Amorphophallus muelleri Blume)

Research · August 2016

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Accepted Manuscript

Title: Characteristics of glucomannan isolated from fresh

tuber of Porang (Amorphophallus muelleri Blume)

Author: Anny Yanuriati Djagal Wiseso Marseno Rochmadi

Eni Harmayani

PII: S0144-8617(16)31032-3
DOI: http://dx.doi.org/doi:10.1016/j.carbpol.2016.08.080
Reference: CARP 11505

To appear in:

Received date: 24-3-2016

Revised date: 15-8-2016
Accepted date: 25-8-2016

Please cite this article as: Yanuriati, Anny., Marseno, Djagal Wiseso., Rochmadi,
., & Harmayani, Eni., Characteristics of glucomannan isolated from fresh
tuber of Porang (Amorphophallus muelleri Blume).Carbohydrate Polymers
http://dx.doi.org/10.1016/j.carbpol.2016.08.080

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Characteristics of glucomannan isolated from fresh tuber of Porang (Amorphophallus muelleri
Blume)

Anny Yanuriatia,b,*, Djagal Wiseso Marsenob, Rochmadi,c Eni Harmayanib

a
Department of Agricultural Technology, Sriwijaya University, Jl. Palembang Prabumulih Km. 32,
Inderalaya, Ogan Ilir 30662, South Sumatra, Indonesia
b
Faculty of Agricultural Technology, Gadjah Mada University, Jl. Flora No.1, Yogyakarta 55281,
Indonesia
c
Chemical Engineering Department, Faculty of Engineering, Gadjah Mada University, Jl. Grafika No.
2,Yogyakarta 55281, Indonesia

*Corresponding author:
Anny Yanuriati, Department of Agricultural Technology, Sriwijaya University, Jl. Palembang-
Prabumulih Km. 32, Indralaya, Ogan Ilir 30662, South Sumatra, Indonesia. Tel. +6281367740860; Fax:
+62711580276.
E-mail address: annyyanuriati@fp.unsri.ac.id

1
Highlights

 Glucomannan could easily and quickly be isolated from fresh porang tubers.

 Isolation was performed by repeated fresh tuber milling in ethanol and filtration.

 Isolation of glucomannan from fresh porang tubers required no further purification.

 Impurities of glucomannan isolated from fresh tubers could be removed completely.

 Glucomannan isolated from fresh tuber had high purity, viscosity and transparency.

2
Abstract

Porang is a potential source of glucomannan. This research objective was to find a direct glucomannan

isolation method from fresh porang corm to produce high purity glucomannan. Two isolation methods

were performed. In first method, sample was water dissolved using Al2(SO4)3 as flocculant for 15 (AA15)

or 30 (AA30) minutes with purification. In second method, sample was repeatedly milled using ethanol

as solvent and filtered for 5 (EtOH5) or 7 (EtOH7) times without purification. The characteristics of

obtained glucomannan were compared to those of commercial porang flour (CPF) and purified konjac

glucomannan (PKG). High purity (90.98%), viscosity (27,940 cps) and transparency (57.74 %) of

amorphous glucomannan were isolated by EtOH7. Ash and protein level significantly reduced to 0.57%

and 0.31%, respectively, with no starch content. Water holding capacity (WHC) of EtOH7 glucomannan

significantly enhanced, whereas its solubility was lower than those of PKG due to its ungrounded native

granular form.

Keywords: porang, Amorphophallus muelleri Blume, glucomannan, characteristics, isolation, fresh tuber.

3
1. Introduction

Amorphophallus muelleri Blume, locally called porang or iles kuning, is one of glucomannan potential

source in Indonesia due to its high level of glucomannan content. The tubers are not consumed and

unpalatable as they contain high levels of calcium oxalate. As highly demanded export commodity, the

tubers are sliced, dried to chips, milled to flour and exported without further processed into glucomannan,

despite its wide application, due to the complicated long process. Glucomannan has diverse function in

food, pharmaceutical (Alonso-Sande, Teijeiro-Osorio, Remuñán-López, & Alonso, 2009; Tester & Al-

Ghazzewi, 2013; C. Zhang, Chen, & Yang, 2014), cosmetics and chemical industries (Zhang, Xie, & Gan,

2005).

Available commercial glucomannan are extracted from dried tuber, especially konjac. Konjac

tuber parenchyma cortex comprises ordinary cells and large cells (idioblasts). The glucomannan granules

are located in egg-shaped idioblasts within parenchyma as single cells (Chua, Hocking, Chan, & Baldwin,

2013), encapsulated by scale like cell walls and dispersed all over the tuber (Takigami, Takiguchi, &

Phillips, 1997). Starch, cellulose and nitrogen containing materials in ordinary cells around idioblasts are

small (approximately 0.004 mm), while idioblasts contain very hard glucomannan granules with oval or

round shape, translucent, large with diameter between 0.25 – 0.75 mm and sized more than 5 – 10 times

than those of ordinary cells. The starch as agglomerated granules is easily broken into very fine particles

and insoluble in cold water whereas glucomannan granules are very difficult to be disintegrated for its

very hard form. The particles are bigger than the impurities (Zhao, Zhang, Srzednicki, Kanlayanarat, &

Borompichaichartkul, 2010) and soluble in water (Enomoto-rogers, Ohmomo, Takemura, & Iwata, 2014;

Luo, He, & Lin, 2013), but insoluble in ethanol (Li et al., 2014). Based on these characteristics, it is

possible to isolate glucomannan granules directly from fresh tuber by water dissolving with flocculants to

remove impurities followed by purification or only by repeated milling using ethanol and filtration

without further purification.

4
 Konjac glucomannan, a non-ionic polysaccharide with molecular weight > 1 x 106 Da (Chao et

al., 2012; Lin et al., 2010), consists of mannose and glucose residues linked by β-(1-4) with ratio of 1.6:1

(Koroskenyi & Mccarthy, 2001; Ratcliffe, Williams, Viebke, & Meadows, 2005) and the acetylation

degree around 5-10% (Gao & Nishinari, 2004). The ratio of glucose and mannose, the acetylation degree,

and the molecular weight could be different depends on the source of glucomannan (Gao & Nishinari,

2004; Koroskenyi & Mccarthy, 2001).

Basically, previous commercial glucomannan granules are extracted by slicing, drying, milling to

remove impurities which adhere to the glucomannan granules, followed by pulverizing to flour, sifting

and air clarifying (Ohashi, Shelso, Moirano, & Drinkwater, 2000; Zhao et al., 2010). Some remaining

impurities attached on the surface of glucomannan granules reduce the glucomannan concentration,

viscosity and transparency of konjac flour sol. The impurities encapsulated on the surface of the

glucomannan granules are not easy to be removed (Zhao et al., 2010).

They can only be detached partially as drying makes them attach to the granule surfaces more

firmly. Purification process is required to remove them for they turn the gel or sol to have higher

turbidity, with milky-white or cloudy appearance (Ohashi et al., 2000). Some methods were developed to

remove them by stirring in ethanol 50% (Chua et al., 2012) and simple centrifugation after flocculation

using Al2(SO4)3 (Tatirat & Charoenrein, 2011). The 2 methods required further long time purification

using high amount of high concentrated ethanol, while the others were by one-step procedure of

azeotropy-assisted acidic ethanol using citric acid (W. Xu, Wang, Jin, et al., 2014), and dimethyl

sulfoxide addition (Ye et al., 2014). All the methods used konjac flour from chips as material and still

need further ethanol washing.

Up to now, reports on direct glucomannan isolation from fresh tuber without drying are very rare.

The impurity removal will be more easily if the granules are isolated directly from fresh tuber without

tuber slices drying. The impurities surrounding the granules in fresh tuber could be detached more easily

since more firmly attaching impurities on the surface of glucomannan granules from drying could be

hindered. As a result, the obtained glucomannan will have higher yield, purity, viscosity, and

5
transparency even without further purification. This research objective was to find a direct glucomannan

isolation method from fresh porang corm to produce high purity glucomannan.

Hypotheses

The purity and the yield of porang glucomannan could be enhanced significantly as well as

viscosity and transparency of the sol by a direct isolation from fresh tuber since more firmly attaching

impurities on the surface of glucomannan granules from drying could be avoided, which resulted in easier

removal of the impurities.

2. Materials and methods

2.1. Materials

The materials were 2 year old porang tubers with average weight about 2.250 ± 250 g obtained

from Klangon Village, Madiun, Jawa Timur, Indonesia, ethanol 96%, and deionized water for

glucomannan isolation. All other chemicals were analytical grade from Merck Co.

2.2. Isolation and purification of glucomannan from Amorphophallus muelleri fresh tubers

Glucomannan were directly isolated from fresh tubers of porang using 2 methods, by water

dissolving using Al2(SO4)3 as a flocculant, followed by purification using ethanol and by repeated milling

using ethanol and glucomannan granules filtration to separate them from impurities without further

purification. In the first method, peeled tubers were cut and shredded. The shredded tubers were put in

sodium metabisulphite solution 200 ppm (w/v) and agitated for 4 hours. The glucomannan extract

dissolved in water (sol) was filtered and separated from the solid. The sol then was heated up to 40oC

with agitation, added with 10% Al2(SO4)3 for 15 or 30 minutes. Then the sol was centrifuged at 4000 rpm

for 20 minutes. The supernatant was then centrifuged at 4000 rpm for another 20 minutes and

precipitated using ethanol 96%. The precipitate was filtered, the residue of ethanol on the flakes was

evaporated, and the flakes were subsequently dried, ground, and sifted through a 60 mesh sieve.

6
 In second method, the clean tubers were sliced and milled in 50% ethanol around 12.000 rpm for

5 minutes, then filtered and pressed to get crude glucomannan. These milling, filtering and pressing

processes were repeated for 5 or 7 times and the obtained granules were dried at 40oC. The obtained

glucomannan characteristics were compared to CPF and PKG.

2.3. Glucomannan content

Glucomannan content was analyzed using 3,5-DNS colorimetric assay (Chua et al., 2012).

Glucomannan (0.2 g) was added to formic acid-sodium hydroxide buffer (0.1 mol/L; 50 ml) and

magnetically stirred for 4 hours at room temperature. The mixture was then diluted with formic acid-

sodium hydroxide buffer to 100 ml in a volumetric flask, followed by centrifugation (4000 rpm, 40 min,

25oC) to get supernatant as the glucomannan extract.

The glucomannan extract (5 ml) was put into a 25 ml volumetric flask and added with 3 M

sulphuric acid (2.5 ml). The resultant solution was mixed using a vortex, subsequently hydrolyzed for 90

min in a boiling water bath and allowed to cool to room temperature before the addition of 6 M sodium

hydroxide (2.5 ml). The solution was then made up to 25 ml with deionized (DI) water to form the

glucomannan hydrolysate. Both the glucomannan extract and hydrolysate were subjected to colorimetric

reactions and deionized water was used as a blank.

D-glucose standard solution (1 mg/ml) was diluted to 0.20%, 0.40%, 0.80%, 1.20%, and 1.60%

using deionized water (DI). About 1.5 ml of 1% 3,5 DNS solution was added to 2 ml of the sugar

standards. Each mixture was heated for 5 min in a boiling water bath and cooled to room temperature

before being diluted to 25 ml with DI water in a volumetric flask. Absorbance was then measured at 550

nm and a plot of the measured absorbance against the glucose content (mg) was constructed. A D-

mannose standard curve was built up by the procedure as described for glucose.

The glucomannan content (db) was calculated by following equation:

GM content (%) =

7
Where f = correction factor, T = glucose content of glucomannan hydrolysate (mg), To = glucose content

of glucomannan extract (mg), m = mass of glucomannan (200 mg) and w = water content of glucomannan

2.4. Chemical Compositions

The content of moisture, protein and ash were determined according to AOAC methods (Tatirat

& Charoenrein, 2011). The starch were analyzed qualitatively on glucomannan granules by staining

using I2-KI (Zhao et al., 2010). The presence of dark blue colour after staining indicated high starch

content of glucomannan.

2.5. Yield

The glucomannan yield (db) was calculated by following formula:

Where m1 = mass of dried glucomannan, m2 = mass of wet peeled porang tuber, w1 = water content of

dried glucomannan, and w2 = water content of wet peeled porang tuber.

2.7. Colour

Lightness (L*) of glucomannan powder was analyzed with a Minolta spectrophotometer. Dried

samples were put in a quartz silica cylinder and the lightness values were measured (Tatirat &

Charoenrein, 2011).

2.8. Morphology

Morphology of glucomannan granules obtained from repeated milling using ethanol and filtration

for 5 or 7 times was analyzed using Scanning Electron Microscope (SEM) (Tatirat & Charoenrein, 2011).

Dried glucomannan was placed on a stub and coated with thin layer gold sputters at 55 nm thickness onto

8
the samples and observed with a SEM-EDS Merck FEI type S50, EDAX AMETEK. The magnification

and accelerating voltage were displayed on the micrograph.

2.9. Transparency

One gram of glucomannan and 99 g or deionized water was dissolved until completely hydrated.

Transparency of the glucomannan sol was measured by UV-VIS Spectrometer at 550 nm (Ye et al.,

2014).

2.11. Viscosity apparent

Sol glucomannan (1%) was agitated at 150 rpm until completely hydrated. The measurement was

taken using a Brookfield Viscometer Model LVTDV-II at 25oC with spindle no.64 at 20 rpm.

2.12. Solubility

Glucomannan (0.1 g) was dispersed to 24.9 g deionized water and stirred for 1 hour. The mixture

was centrifuged for 20 minutes at 4000 rpm. About 10 g of the supernatant was dried to constant weight

at 105oC. The solubility was calculated by following equation (Du, Li, Chen, & Li, 2012):

Where m is the weight of soluble component in 10 g upper solution, W is the total weight of

glucomannan.

2.13. Water Absorbency

Glucomannan (0.1 g) was placed in previously weighed falcon and added with 30 g of deionized

water. The mixture was kept stay for 1 hour. The mixture was then centrifuged at 4000 rpm for 30

minutes. The supernatant was removed and the remained substance was weighed. The weight difference

9
between the two measurements was taken as the weight of the absorbed water (Koroskenyi & Mccarthy,

2001).

2.14. X-Ray Diffraction (XRD)

XRD patterns were determined using a Lab X XRD-6000 Shimadzu (Japan) equipped with a Cu

Kα target 40kV and 30 mA with a scan rate of 4oC/min. The diffraction angle ranged from 2Ø = 5 to 2Ø

= 60o. Crystallinity percentage (%) = area under the peaks/total curve area x 100 (Wang, Zhou, Wang, &

Li, 2015).

3. Results and discussion

3.1. Isolation of glucomannan from fresh tubers

The purity and the yield of glucomannan isolated by EtOH7 were enhanced significantly to

90.98% with low percentage of ash (0.57%) and protein (0.61%) without starch content (Table 1).

Table 1
Ash, starch, protein and yield of porang glucomannan compared to those of PKG and CPF
Treatment Glucomannan Ash Starch Protein Yield
(%)z (%) (I2-KI test) (%) (%)z
a b bc
AA15 76.83 2.07 +++++ 3.39 59.02b
b a b
AA30 85.72 1.40 +++++ 2.52 50.02a
bc a a
EtOH5 88.67 0.65 - 0.99 65.23c
EtOH7 90.98c 0.57a - 0.61a 61.05b
d a a
PKG 94.42 0.60 + 0.31 Na
CPF 79.91a 5.51c +++ 1.86b Na
Different superscripts among mean values in each column indicated significant difference (p ≤ 0.05).
z
data were based on dry basis
na = not available
- = no color, + = very light blue, ++ = light blue, +++ = blue, ++++ = dark blue, +++++= very dark blue

Compared to PKG, EtOH7-isolated glucomannan had lower purity, however, remained considered as

purified glucomannan based on the standard by The Ministry of the People’s Republic of China (2002).

The level of ash and protein on EtOH7 isolated glucomannan by was not significantly different from that

on PKG.

10
 The glucomannan granules of porang were similar to those of konjac. During milling, the softer

starch around the glucomannan and fiber cells from peripheral cell layers of the corms were first

disintegrated to tiny ash particles (Zhao et al., 2010). However, the glucomannan granules could not be

broken to fine particles as they were very hard. In addition, the size of glucomannan granules was bigger

than that of the impurities. These differences resulted in an easy separation of starch particles and other

impurities from glucomannan granules during milling and further filtration. The impurities leached out by

ethanol during filtration. The higher purities of glucomannan granules could be produced by longer

milling time than those by EtOH7. However, the low yield probably due to the partial small granules

leaching during filtration resulted in the decreasing of the EtOH7 isolated granules.

During milling in ethanol, the impurities (starch, protein, and ash) previously present interstitially

among the glucomannan granules in fresh tuber could be more easily detached from glucomannan

granules than those in flour derived from chips. Drying process hardened porang slices and stuck

impurities covering glucomannan granules more firmly. Smirnova, Mestechkina & Shcerbukhin (2001)

removed pigments and low molecular substances from glucomannan by boiling in 70% aqueous ethanol

solution for 1 hour. Xu, Wang, Ye, et al. (2014) also found that konjac flour was effectively purified by

using ethanol with controlling its temperature at 68oC. In this research, the increase in ethanol solution

temperature to 45-50oC during milling resulted in significant higher removal of impurities.

Milling and polishing of chips or konjac flour were only able to remove partial impurities while

some others were still attached to the glucomannan granules. Consequently, the impurity level of the

glucomannan granules was still high. Further purification by water dissolving and precipitation using

higher volume of high concentrated ethanol was required, even after stirring the konjac flour in 50%

ethanol for 90 minutes (Chua et al., 2012) or after simple centrifugation of water dissolved glucomannan

with flocculating agent (Tatirat & Charoenrein, 2011). Other methods, after impurities removal using

68oC citric acid ethanol (W. Xu, Wang, Jin, et al., 2014) and DMSO (Ye et al., 2014), the obtained

glucomannan need to be washed using higher amount of high concentrated ethanol to get high purity

glucomannan. On the other hand, the impurities covering glucomannan in fresh corm could be removed

11
more easily during milling in ethanol than those in chips or konjac flour, thus the EtOH7 glucomannan

had high purity without purification. The EtOH7 isolated glucomannan already contained a low level of

ash although slightly higher than those obtained by Tatirat & Charoenrein (2011) and by Xu, Wang, Jin,

et al. (2014) methods.

The ash content including calcium oxalate could be totally removed at longer duration of tuber

milling. This method would be easier and faster than the previous methods as drying of tuber, or further

washing or purification using higher amount of high concentrated ethanol as well as a flocculating agent

were no longer required.

3.1. Characteristics of porang glucomannan

3.1.1. Lightness values

Glucomannan lightness value isolated by both EtOH7 and by AA15 was not significantly

different from PKG (Fig.1).

Fig 1. Lightness values of CPF, PKG and glucomannan isolated by AA15, AA30, EtOH5 or EtOH7.
Each lightness value is shown beside of each picture label. Different superscripts among mean values
indicate significant difference (p ≤ 0.05).

The impurities covering glucomannan granules were more easily broken up to very fine particles

while the glucomannan granules were very hard to be ground. During milling in ethanol, the impurities in

12
the fresh tuber of porang can be gradually removed from surface of glucomannan granule. Longer milling

caused significantly higher impurities detachment which could be separated and ditched from

glucomannan granules during filtration. The higher removal level of impurities resulted in whiter

glucomannan granules. In addition, the native carotenoid on the tuber (Wootton, Luker-Brown, Westcott,

& Cheetham, 1993) could be removed as the pigment could be dissolved by ethanol and discarded during

filtration as well as browning inactivation due to ethanol temperature increase to 45-50oC during milling .

3.1.2. Morphology of glucomannan

SEM images of the glucomannan morphology are shown in Fig. 2.

Fig 2. SEM photographs of PKG (A), glucomannan granules isolated by EtOH5 (B) or EtOH7 (C) with
70X (1). 200x (2) and 750X magnification

13
Shape of native porang glucomannan granules isolated by EtOH7 and EtOH5 was round or oval with

rough, scale like-pattern while PKG was irregular as the glucomannan had already been ground. Surface

of CPF was smooth with blur scale-like pattern inside as the surface of the glucomannan granules was

still covered by impurities (Yanuriati, Haryadi, Rochmadi & Harmayani, 2013). The granules were

dispersed in the corm as single cells covered with scale-like cell walls (Takigami et al., 1997) and the

scale like pattern became more clearly appear on the surface of purified glucomannan granules

(Takigami, 2000). Li et al. (2009) found that native structure of konjac glucomannan was primarily

composed of lamella structure units. It appeared that scale like pattern on first lamella structure units of

glucomannan granules isolated by EtOH7 thinned and began to crack (Fig 2.C.). The layer could be

released by longer milling duration.

3.1.3. X-ray diffraction

Spectrums of X-ray diffraction describe the correlation graph between the diffraction of intensity

and angle. The sharp diffraction peak with light baseline is illustrated at a crystalline state, whereas the

widened peak diffraction appears solid and amorphous state. The X-ray curves of porang glucomannan

were shown in Fig 3.

Fig 3. XRD pattern of PKG, glucomannan isolated by EtOH7, AA15, and CPF

14
 Patterns of all porang glucomannan at 2Ɵ = 5 - 50o exhibited a very broad band with high peaks

around 19o-20o and small peaks around 35o. Native glucomannan had low degree of crystallinity around

or less than 5.43%, which indicated that all of them almost fully amorphous (Table 3). The same

patterns were also found in native konjac glucomannan (S. Wang et al., 2015; Yao et al., 2011) and

Amorphophallus corrugates glucomannan (An, Thien, Dong, Dung, & Du, 2011). The degree of

crystallization of native konjac glucomannan was higher than that of native porang glucomannan, about

16% (Pan et al., 2003 in Li et al., 2009). Figure 3 showed that an increase of crystallinity levels occurred

during glucomannan isolation. The highest increase of crystallinity degree was detected on EtOH7

isolated glucomannan (5.43%), followed by CPF (2.78%), AA15 (0.033%), and PKG (0.026%),

respectively.

The higher level of crystallinity on EtOH7 isolated glucomannan was stimulated by the use of

50% ethanol and ethanol temperature increase during milling. The crystallinity of glucomannan was

enhanced by ethanol (Wang, Zhong, Chen, Li, & Lv, 2011) since dehydration of glucomannan granules

strengthened the intermolecular and intramolecular hydrogen bonds and increased the crystallinity. In

addition, the exothermic ethanol during milling between 45oC-50oC also contributed to the increase in

crystallinity of the glucomannan granules. The amorphous parts of glucomannan granules were removed

by milling in ethanol. Li et al. (2009) found that 50% ethanol containing water activity around 0.25 had

only few number of activated water molecules which broke the hydrogen bonds between lamellar

structure units of konjac glucomannan granules. Thus, the crystallinity was still strongly maintained by

the inter and intramolecular bonds.

On the other hand, AA15 isolated glucomannan had lower crystallinity than those isolated by

EtOH7 and CPF which probably caused by purification process. Purification was performed by dissolving

in water, clarification using centrifugation and precipitation using ethanol. Prawitwong, Takigami, &

Phillips (2007) described that water was initially sorbed in the amorphous regions with less hydrogen

bonds. From these points, it could be hypothesized that the glucomannan chain could expand and the

water molecule had capability to break down the inter and intramolecular hydrogen bonds of

15
glucomannan. The weakness of the hydrogen bonds allowed water to form hydrogen bonds with the

molecules and increase of amorphous space region.

3.1.4. Transparency of porang glucomannan sol

Transparency of porang glucomannan was shown on Table 2. The most transparent sol of

glucomannan was produced by EtOH7 followed by EtOH5, which appeared more transparent compared

to PKG. In contrast, the transparency of glucomannan sols isolated by AA15 or AA30 was far lower

(Table 3) than those by EtOH7 or EtOH5. The presence of yellowish color in AA15 and AA30 isolated

glucomannan sols indicated that glucomannan still contained high level of impurities, such as protein,

starch (Table 1) including the native carotenoid.

Table 2
Physical characteristics of porang glucomannan
Treatments Viscosity (cps) Solubility (%) WHC (g water/g GMP) Transparency
(%)
AA15 102a 83.16b 24.21a 0.44a
a a a
AA30 134 74.16 24.91 2.15b
b a c
EtOH5 24,650 71.38 50.91 51.81e
b a c
EtOH7 27,940 74.59 52.11 57.74f
PKG 27,300b 98.43c 58.12d 47.83d
a a b
CPF 1,700 73.74 36.99 19.47c
Different superscripts among mean values in each column indicated significant difference (p ≤ 0.05).

In addition to the presence of a low level of starch content (Table 1), the less transparent of PKG

sol compared to the glucomannan sol from EtOH7 and EtOH5 could also be affected by the milling

process. The PKG had more further processes, such as purification, precipitation, drying and milling to

powder. During purification and drying, the intermolecular hydrogen bonds of glucomannan molecules

became closer and stronger (Alonso-Sande et al., 2009) to produce the very hard glucomannan, thus the

dried glucomannan precipitate was not easy to be milled. The precipitate powdering required a high

speed grinder resulted in high temperature frictions. The high temperature oxidized the glucomannan

granules which brought about decolourization and reduction on transparency and viscosity (Ohashi et al.,

2000).

16
3.1.5. Viscosity

Viscosity of glucomannan sol can be observed in Table 2. The highest viscosity level was found

in EtOH7 isolated glucomannan which was not significantly different from PKG and EtOH5 isolated

glucomannan. However, the sol viscosity of CPF, AA15 and AA30 isolated glucomannan were

significantly very low.

Glucomannan has poor solubility despite its hydrophilic property (Pan et al., 2013). The low

viscosity of the 2 last glucomannan was accounted for the low solubility of glucomannan. Not all

glucomannan granules had been dissolved during 4 hours extraction using water. As a result, some high

molecular granules might lose during centrifugation which lowered their glucomannan content.

Viscosity level was affected by molecular weight and purity. It was proportional to the molecular

weight (Luo, Yao, Zhang, Lin, & Han, 2012). Glucomannan had high molecular weight and very high

viscosity (Ojima et al., 2009). The viscosity lowered as the molecular weight reduced (Ojima et al., 2009;

Tatirat, Charoenrein, & Kerr, 2012). In this study, the molecular weight analysis by gel permeation

chromatography was conducted on EtOH7 isolated glucomannan whose Mn (number average molecular

weight), Mw (weight average molecular weight) and Mw/Mn were 6.47 x 105, 1.27 x 106 and 1.96

respectively due to its highest purity, viscosity and transparency. The AA15 and AA30 isolated

glucomannan still had higher impurities (ash, protein and starch) than EtOH7 and EtOH5 isolated

glucomannan (Table 1). The impurities had lower viscosity than glucomannan, resulted in reduction of

viscosity.

3.1.6. Solubility

Solubility of EtOH5, EtOH7, or AA30 isolated glucomannan was not significantly different,

except AA15 isolated glucomannan had significant higher solubility than the others. However, its

solubility remained significantly lower than PKG.

Solubility of glucomannan was affected by molecular weight and by the material morphology.

Lower molecular weight was less compact and had more porous particle (Luo et al., 2012) leading to

higher solubility. The solubility of glucomannan was also related to the hydroxyl and O-acetyl group

17
(Luo et al., 2013). In addition to high purity, the highest level of solubility of the PKG could be the

results of purification and grinding. Purification was performed by dissolution of glucomannan in water

and precipitation of the glucomannan sol in ethanol. During dissolving in water, the molecular chain

expanded (Luo et al., 2013) and the acetyl groups became be more exposed. The acetyl groups enhanced

the solubility and dispersion of glucomannan as the groups prevented the formation of intra and

intermolecular hydrogen (Alonso-Sande et al., 2009; Chen, Li, & Li, 2011) which became closer and

tighter during drying (Xu, Willför, & Holmbom, 2008). The changes of the molecular orientation did not

revert totally after drying and enhanced the solubility.

EtOH5 and EtOH7 isolated glucomannan were still in native granule form. Fig. 2B and 2C

showed the glucomannan granules were covered by first lamella structure which might also hinder the

solubility. Li et al. (2009) found that the native konjac glucomannan consisted of lamella structure units

containing both crystalline and amorphous regions. The connection zones between lamellar structures

contained booth loose and tight aggregation regions. These lamella structure units contributed the slow

hydration due to the gradual solubility. The solubility was higher in the amorphous than in the crystalline

regions.

3.1.7. Water holding capacity (WHC)

WHC of EtOH5 and EtOH7 isolated glucomannan rose significantly. On the other hand, WHC

of AA15 and AA30 isolated glucomannan was reduced significantly. Nonetheless, WHC of EtOH5 or

EtOH7 isolated glucomannan remained significantly lower than the PKG (Table 2). Strong structural

hydrogen bonds stimulated the low aqueous solubility (Kohyama, Sano, & Nishinari, 1996). However,

the formation of strong hydrogen bond between hydroxyl groups and water stimulated the high water

absorbency. The high water absorbency of native konjac glucomannan could reach 105.4 g water/g KGM

(Koroskenyi & Mccarthy, 2001). The cracks on some parts of first lamella unit on the EtOH7 isolated

glucomannan (Fig. 2C) contributed to the rate of water dispersion through the amorphous regions in the

lamellar structural units. The existence of water molecules in sufficient amount weakened and broke the

18
hydrogen bonding which resulted in the occurrence of bonds between water molecules and hydroxyl

group. However, the presence of some impurities covering glucomannan granule restrained the adsorption

of water which resulted in the lower WHC in AA15 and AA30 isolated glucomannan.

Conclusions

Repeated milling of sliced fresh porang tuber in ethanol followed by filtration without further

purification could be developed as an easier and faster novel method to isolate glucomannan, with

significantly high purity (90.98%), viscosity (27,940 cps) and transparency (57.74 %), since the level of

ash and protein content was reduced significantly to 0.57%, 0.31% with no starch content. The yield of

glucomannan was also enhanced by EtOH7. Besides no corm drying process was required for this

method, some other common steps could be eliminated, such as addition of antibrowning as well as

further washing or purification process using flocculants and higher volume of high concentration of

ethanol. The native glucomannan granules were still amorphous. The WHC was enhanced significantly

while the solubility was significantly lower than commercial PKG. In addition to high molecular weight

of native granular form, the lower solubility of the glucomannan could be contributed the gradual

solubility due to the lamellar structure units of glucomannan.

Acknowledgements

This research was partly supported by Post Graduate Grant in 2013 no. LPPM-

UGM/1396/LIT/2013 and Doctoral Dissertation Grant in 2015 no. 115/UN9.3.1/LT/2015 from Ministry

of Research, Technology and Higher Education, Republic of Indonesia. The authors also expressed

greatly thank to Dr. Anni Faridah for commercial PKG support.

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