Journal of Engineering Science and Technology
Special Issue on SOMCHE 2014 & RSCE 2014 Conference, January (2015) 1 - 8
© School of Engineering, Taylor’s University
HYDROLYSIS OF STARCH IN PORANG FLOUR USING
ALPHA AMYLASE
FADILAH*1,2, ROCHMADI2, S. SYAMSIAH2, HARYADI3
1
Chemical Engineering Department Sebelas Maret University, Jl. Ir. Sutami 36 A
Surakarta 57126, Indonesia
Chemical Engineering Department Gadjah Mada University, Jl. Grafika 2 Yogyakarta
55281, Indonesia
3
Faculty of Agricultural Technology Gadjah Mada University, Jl. Flora No. 1 Bulaksumur
Yogyakarta 55281, Indonesia
*Corresponding Author: [email protected]
2
Abstract
To obtain high purity glucomannan extracted from porang corm
(Amorphophallus muelleri Blume), an attempt was made to enzymatically
hydrolyse the starch which is considered as impurity. Purity of glucomannan
has great influence to its physicochemical properties. Starch hydrolysis was
carried out simultaneously with extraction of glucomannan. The objective of the
work was to study the effect of various factors, namely stirring speed, solid to
liquid ratio and the amount of enzyme added on the hydrolysis process. The
experiment was started by heating phosphate buffer solution to the desired
temperature and then adding certain amount of amylase. The porang flour was
then added into the solution and this time was considered as the starting time of
hydrolysis. Samples was taken at different time during the hydrolysis and
analysed for their sugar content. The result showed that all variable studied give
remarkable effect on the reducing sugar liberated during the process. By
increasing the stirring speed, the solid to liquid ratio and the amount of enzyme
added, the amount of reducing sugar increased. Increasing solid to liquid ratio
by 3 fold (3.33 g/L to 10 g/L) has increased the sugar produced by 4.6, while
increasing the stirring speed from 400 rpm to 600 rpm only increased the
reducing sugar concentration by 1.52 times and increasing the amount of
enzymes added by 4 fold (5 mL to 20 mL) only resulted in 1.24 times higher of
sugar produced. This indicates that solid/liquid ratio give more significant effect
in enzymatic hydrolysis of starch in porang compared with stirring speed and
the amount of enzymes do.
Keywords: Glucomannan purification, Starch, Alpha amylase, Reducing sugar.
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Fadilah et al.
Abbreviations
EFSA
FDA
TCM
USDA
European Food Safety Authority
Food and Drug Administration
Traditional Chinese Medicine
US Department of Agriculture
1. Introduction
Among natural biopolymers, glucomannan has received a great interest for
various important purposes. It has been used as TCM for thousands of years for
treatment of asthma, cough, hernia, breast pain, burns and skin disorders [1].
Currently, clinical studies show that glucomannan has ability to lower blood
sugar, lower blood cholesterol, helping weight loss and promote healthy micro
flora in intestinal [2]. Glucomannan is also used as emulsifier and stabilizer for
foods, drinks, and cosmetics products for its unique rheological and gelling
properties [1]. The use of glucomannan as food additive had been approved in
USA by FDA in 1994 and by USDA in 1996. Glucomannan is also authorized in
Europe by given an E425 agreement number by EFSA [1]. In pharmaceutical
area, glucomannan is used in drug delivery system due to its biodegradability and
good gel-forming ability [3, 4].
Glucomannan is a neutral polysaccharides composed mainly of ß,1-4 linked
D-mannose and D-glucose with molar ratio of 1.6:1. The backbone is lightly
branched, which short side branches at the C-3 position of the mannoses. Some
acetyl groups randomly present at the C-6 position of the sugar units [5]. This
acetyl groups, which is believed to contribute to the solubility and gelling
properties are located every 9 to 19 sugar units. Glucomannan have unique
properties. Its 1% solution has high viscosity, i.e., 30,000 cP, which is the highest
among 12 polysaccharides tested [6]. This relates to its high water absorbance
(100 g of water per g of glucomannan), and its high molecular weight, 105 – 106.
Glucomannan can be extracted from many botanical sources, but in large
quantity it could be only from the tuber of Amorphophallus sp. This perennial
herb is native to East Asia and belongs to the family of Araceae. As source of
glucomannan, Porang (Amorphophallus muelleri Blume) is cultivated in
Indonesia, as secondary crops under the teak, mahogany or sonokeling plantation [7].
In recent days, glucomannan can be extracted from the corms of
Amorphophallus sp. either by dry or wet processing method. Dry processing
method involves mechanical means in which the dried slice corms is grounded
and then purified via wind shifting or sieving. For its low quality, the product of
dry processing method have used in food industry. Product with higher quality
can be obtained by wet processing method. This method includes ethanol
isolation [8], use of aluminum sulphate solution [9], water extraction [8] and
enzymatic processing [10, 11].
Chua et al. [12] and Sugiyama [8] have conducted glucomannan extraction at
room temperature in relatively long time (three hours) to avoid gelatinization of
starch, which may entrained to glucomannan extract. To enhance the
glucomannan yield and to shorten the extraction time, it is needed to carry out the
extraction at higher temperatures. The gelatinization of the starch could be
overcome by hydrolysing it with amylase so the hydrolysate can be easily
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Special Issue 6 1/2015
Hydrolysis of Starch in Porang Flour Using Alpha Amylase
3
separated from the extracted glucomannan. Fadilah et al. [13] reported that using
commercial amylase commonly used in glucose factory was not appropriate for
this purpose. Commercial amylse may contain mannanase that would cleavage the
glucomannan chain. The use of amylase that free from mannanase is then preferable.
This work deals with simultaneous glucomannan extraction and starch
hydrolysis by amylase, especially focusing on the influence of time, stirring
speed, solid to liquid ratio and the amount of enzyme added on the hydrolysis of
starch in porang flour.
2. Materials and Methods
2.1. Materials
Porang flour was prepared following the method used by Fadilah et al. [13]. Alpha
amylase from Sigma (A 3403 Bachillus licheniformis) was diluted to 500 U/mL.
Phosphate buffer solutions containing Na2HPO4 and NaH2PO4 at pH 6 used as
medium for carrying out the simultaneous glucomannan extraction and enzymatic
hydrolysis of starch. Other chemicals used were 96% ethanol and 0.1 N HCl solutions.
2.2. Hydrolysis process
The hydrolysis processes were carried out according to the procedure followed by
Fadilah et al. [14] except for the treatment of the samples. At a certain time
interval, 100 ml of sample was taken out. An amount of 12.5 mL of this sample
was then added with 12.5 mL of 0.1 N HCl to stop the hydrolysis. All samples
were then centrifuged at 4000 rpm for 5 minutes. The reducing sugar content was
analysed using Nelson-Somogyi method with maltose used as the standard.
To study the influence of the solid to liquid ratio (mass of porang per volume
of solution), the experiments were conducted by keeping the stirring speed at 500
rpm, with the amount of enzyme of 20 mL, at temperature of 70 °C and solid to
liquid ratio were 3.33, 6.67 and 10 g/L. The influence of stirring speed were
conducted by carrying out the experiments with solid to liquid ratio was 10 g/L at
the temperature of 70 °C with stirring speed varied at 400, 500 and 600 rpm.
Experiments was also conducted by varying amount of enzyme added by
conducting the experiments with solid to liquid ratio was 10 g/L by keeping the
stirring speed at 500 rpm, at the temperature of 70 °C and with the amount of
enzyme added were 5, 10 and 20 mL.
3. Results and Discussion
During the process, it was observed that the starch granules took up water and
swelled. The starch underwent gelatinization. It is expected that the shearing
effect of stirring will remove the gelatinized outer layer and these long chained
starch molecules dissolved into solution [15]. Thus, hydrolysis will occur. At this
stage, amylase cleaves randomly the inner part of the starch molecule as it is an
endo type enzyme. Dextrines would be formed in the beginning, and as the
process continues the maltoses will accumulate, in which one of the molecules of
glucose has a free glucoside group and hence has reducing properties [16].
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Fadilah et al.
The experimental results of reducing sugar liberated during starch hydrolysis
by amylase at various solid to liquid ratio are shown in Fig. 1. As seen, higher
solid to liquid ratio produces higher initial rate (indicated by steeper slop of the
profile) and higher reducing sugar concentration. This fact may be related to the
quantity of the starch available to be hydrolysed. As the reaction rate is
proportional to the concentration of substrates, the higher the concentration of
starch the faster the reaction.
0.9
0.8
3.33 g/L
6.67 g/L
20
40
10 g/L
reducing sugar, g/L
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
60
time, minutes
80
100
120
Fig. 1. Reducing sugar liberated during hydrolysis of starch in porang flour
at various solid to liquid ratio (70 °C, 500 rpm, 20 mL enzymes).
As noticed, reducing sugar for all solid to liquid ratios increased rapidly
during the first 30 minutes of hydrolysis then decreased drastically before it
slowly increased again. The profile tended to reach constant value after 80
minutes. This winding curve differs from the results obtained by Kolusheva and
Marinova [16] and Sunaryanto et al. [17] which the profile of reducing sugar
released formed curves that constantly increase before it reached a constant value.
This phenomenon is still under investigation.
The highest concentration of reducing sugar obtained from different solid to
liquid ratios was 0.606 g/L, 0.198 g/L and 0.131 g/L from the solid to liquid ratios
of 10 g/L, 6.67 g/L and 3.33 g/L respectively. It is indicated that increasing
production of reducing sugar at solid to liquid ratio 10 g/L was 3 times higher
compared with that at 6.67 g/L and 4.6 times higher compared with that at 3.33 g/L.
Figure 2 shows the influence of stirring speed on the reducing sugar liberated
during the hydrolysis. Increasing the stirring speed has increased the amount of
liberated reducing sugar. It means that the enzymatic reaction was faster in the
higher stirring speed. Highest concentration of reducing sugar obtained from
different stirring speed was 0.775 g/L, 0.645 g/L and 0.51 g/L from the stirring
speeds at 600 rpm, 500 rpm and 400 rpm. The increasing of reducing sugar
liberated at 600 rpm was 1.2 times higher compared to at 500 rpm and 1.52 times
higher compared to at 400 rpm. This phenomenon can be attributed to the higher
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Hydrolysis of Starch in Porang Flour Using Alpha Amylase
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turbulence when the stirring speed increases, in which dissolution of gelatinized
starch increase too. Higher turbulence also means better contact between the
enzymes and the substrates. This leads to the higher products of reaction.
0.9
0.8
reducing sugar, g/L
0.7
0.6
0.5
0.4
0.3
0.2
0.1
400 rpm
500 rpm
600 rpm
0
0
20
40
60
80
time, minutes
100
120
Fig. 2. Reducing sugar liberated during hydrolysis of starch in porang flour at
various stirring speed (Solid to liquid ratio 10 g/L, 20 mL enzyme , 70 OC).
The influence of amount of amylase added can be seen in Fig. 3. Again the
same phenomenon of winding curves was observed. As seen in Fig. 3, higher
reducing sugar released at higher enzyme concentration. Highest reducing sugar
liberated during hydrolysis obtained from different amount of enzyme added were
0.52 g/L, 0.55 g/L and 0.65 g/L from the amount of enzyme added were 5 mL, 10
mL, and 20 mL respectively. Increasing the amount of enzyme added from 5 mL
to10 mL only increased the reducing sugar 1.05 times, and increasing the amount
of enzyme from by 4 fold (5 mL to 20 mL) only increased the reducing sugar 1.24
times. More enzymes gave more site for substrate to form enzyme-substrate
complex, so enhanced the reaction.
From the three variables studied affecting the hydrolysis, solid to liquid ratio,
i.e., the concentration of substrates, was more pronounced than two others.
Ingesson et al. [18] in their study also found similar results that the conversion of
cellulose was more affected by the substrate rather than shaking regime
employed. Sunaryanto et al. [17] studied the effect of different sago starch
concentration on the liquefaction using alpha amylase and reported that increasing
substrate concentration produced more reducing sugar but at very high
concentration less reducing sugar produced.
The increasing of stirring speed and the amount of enzyme has less effect to
hydrolysis. This may be due to the high viscosity of the solution as the starch
hydrolysis conducted simultaneously with glucomannan extraction. The presence
of extracted glucomannan made the solution very viscous. This viscous media
likely restricted the mobility of the enzyme thus affect the interaction of substrateenzyme and hence reduced the product reaction.
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Fadilah et al.
0.8
0.7
reducing sugar, g/L
0.6
0.5
0.4
0.3
0.2
0.1
5 mL
10 mL
20 mL
0
0
20
40
60
time, minutes
80
100
120
Fig 3. Reducing sugar liberated during hydrolysis of starch in porang flour at
various amount of Amylase added (Solid to liquid ratio 10 g/L, 500 rpm, 70oC).
Starch content in the porang flour was 12.28% (w/w). With solid to liquid
ratio 10 g/L, the concentration of the starch was 1.228 g/L. The highest reducing
sugar concentration obtained with this solid to liquid ratio was 0.775 g/L. with
stirring speed at 600 rpm and the amount of enzyme added was 20 mL. This
amount of enzyme is equal to 10,000 U. This enzyme dosage seem higher than
used by Kolusheva et al. [16], which was used 1,800 U for concentration of starch
250 g/L. Higher enzyme dosage was needed to ensure the starch hydrolysis run
well as it carried out in viscous solution.
4. Conclusions
Starch in porang flour hydrolyzed using amylase and the hydrolysis rate and yield
were influenced by stirring speed, amount of enzymes and ratio of solid to liquid.
Increasing the stirring speed, amount of enzyme and ratio of solid to liquid
increased the production of reducing sugar. The conversion of starch was more
affected by the ratio of solid to liquid, i.e., the concentration of substrates, rather
than stirring speed and the amount of enzymes. Triplicating the ratio of solid to
liquid from 3.33 g/L to 10 g/L increased the reducing sugar 4.6 times higher, while
increasing the stirring speed from 400 rpm to 600 rpm only increased the reducing
sugar concentration by 1.5 times and increasing the amount of enzymes added by 4
fold times only gave 1.24 times higher.
Acknowledgement
The authors would like to acknowlegde The Directorate General of Higher
Education, Indonesia, for financial support of this work through scholarship of
doctorate program (BPPS) at Gadjah Mada University to Fadilah and the research
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Hydrolysis of Starch in Porang Flour Using Alpha Amylase
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grant Hibah Penelitian Disertasi Doktor atau Doktor Baru (PDDB) PNBP 2014
Sebelas Maret University.
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