CHAPTER III ANALYSIS OF MAIN FLOUR AND BINDING AGENTS 3.1
Objectives To determine the characteristic of different rice flours
based on its amylose content and the binding agent (tapioca, sago,
and waxy rice flour) by physical analysis such as swelling,
solubility, microstructure by using SEM, viscous properties, X-ray
diffraction of rice flours, gelatinization temperature, and
chemical analysis such as amylose content. To determine the
characteristics of burgo dough which was made from rice flours with
the combination of three types of binding agent (tapioca, sago, and
waxy rice flour) through physical and chemical analysis, such as
water, protein, and starch content. 3.2 Materials and Methods 3.2.1
Materials Three kinds of rice flours used were derived from Indica
rice, which was purchased from the local market in South Sumatera,
Indonesia. So did with the binding agents, which are waxy Indica
rice, tapioca, and sago. As additional starch sample, native and
three kinds of chemically modified tapioca were supplied from Japan
starch company. Fresh burgo was prepared from the 9 combinations of
rice flours and binding agents.Combination Combination
Waxy rice A1 A2 A3
Tapioca B1 B2 B3
Sago C1 C2 C3
Rice Flour 1 Rice Flour 2 Rice Flour 3
3.2.2 Methods Preparation of starch sample. Purification of
starch was conducted by removing its protein for analysis of
amylose content, swelling-solubility power, RVA profile, DSC, and
X-Ray diffraction pattern. Especially for Apparent Amylose Content
(AAC) and Gel Permeation Chromatography (GPC) analysis, defatted
starch was also required. The method for protein and fat removed as
described in Chapter 2.2. Chemical composition. The chemical
analysis for measuring amylose content and moisture content based
on standard method. The iodine affinity of defatted starch was
determined by amperometric method according to Takeda, Hizukuri,
and Juliano (1987) with modification as described in Chapter 2.3.3.
The data recorder resulted a titration curve (Fig.4) which derived
the volume of KIO3 used for producing amylose-iodine complex.350
300 250 200 150 100 50 0 0 2 4 6Time (min.)
A
B8 10 12 14
Fig.4. Typical titration curve of AAC analysis A = the
continuous complexation between amylose and released iodine lead to
constant detector response ; B = the excess iodine due to
saturation of amylose-iodine complex results in increased detector
response (higher slope)
The calculation based on following equation.
The weight of iodine was derived from volume of KIO3 .From this
equation, the iodine affinity was converted to apparent amylose
content (AAC)
Swelling power and solubility index measurement. Modification of
method Li and Yeh (2001) was applied for determination of swelling
properties. The equation used for determining the swelling power
was as follow
Previously, solubilized starch (SS) was calculated as :
*Total carbohydrate content of supernatant was determined by
phenol-sulphuric assay as described in Chapter 2.3.2.
Pasting profile for burgo dough. Starch-water suspension was
prepared for about 8% w/w (adjusted based on the moisture content
of sample) from 28.0 g total weight, dry starch basis. The RVA
profile of sample was observed from the resulted curve (Fig 5)
RVA 4500 4000 3500 3000 Viscosity (RVU) 2500 2000 1500 1000 500
0 -500
Temperature 120
C100 Temperature (C)
A B
80 60 40
Pasting temperature
20 0
Time (sec.)
Fig 5. Typical RVA curve. The principle of the method is to
gelatinize a given amount of starch under precisely controlled
conditions including a fixed starch : water ratio (8% starch in
this experiment), a standard temperature profile (heating from 50
to 95C, holding, and cooling back to 50C), and a constant shear
rate (160rpm was used). The viscosity of the solution is calculated
based on the force required to maintain the constant shear rate (to
stir the swollen mass gel particles), and expressed RVA viscosity
units (RVU) ((Ikegwu et al., 2009). Each peak indicates different
characteristic of starch. Peak A represents peak viscosity, which
is a maximum viscosity of starch by application of heating and
shear stress. Peak B, trough viscosity, is used to calculate the
breakdown and setback viscosity, whereas C is introduced as final
viscosity. Pasting temperature is revealed by the initial time of
viscosity increment.
The analysis of thermal profile. Differential scanning
calorimetry (DSC) was used for resulting the thermal profile of
starches. Fig.6 shows the typical thermal profile curve
resulted
from the instrument. it is based on the principle when thermal
transition occurs, the energy absorbed by the sample is replenished
by increased energy input to the sample to maintain the temperature
balance. The area under the peak is directly proportional to the
entalphic change (H) and its direction indicates whether the
thermal event is endothermic or exothermic. At temperature about
1000 C, a peak is sometimes exist as the presence of amylose-lipid
complex.
DSC Profile Curve0 0 -100 -200 -300H
20
40
60
80
100
120
-400 -500 -600
Fig. 6.Typical DSC curve
The crystallinity degree of starches. Relative crystallinity of
starches was calculated based on the curve resulted from X-ray
diffractometer instrument as shown in Fig. 7. The area above the
smooth curve was taken as the crystalline portion and the lower
area between smooth curve and the linear baseline which connected
the two points of the intensity 2 of 30 and 10 in the samples was
taken as the amorphous section. In the present research, the
calculation based on weight was used. Therefore, the ratio of upper
area weight to total diffraction area weight was taken as the
degree of crystallinity.
Fig. 7.Wide-angle X-ray powder diffraction spectra showing
crystalline (upper region) and noncrystalline regions (adapted from
Cheetham and Tao, 1998)
Starch granule observation. The observation of starch granule
was carried out using Scanning Electron Microscopy (SEM). Starch
granules were fixed onto a circular specimen stub with double-sided
tape, coated with gold using an E-1010 ion sputter (Hitachi Science
Systems, Ltd., Hitachinaka, Japan) then observed using a S-4000
scanning electron microscope (SEM) (Hitachi Science Systems, Ltd.,
Hitachinaka, Japan) with an accelerating voltage of 3 kV.
3.3 Results and discussion Chemical composition of rice starch
and binding agents The chemical composition of samples involved the
measurement of amylose and moisture content as the result shown in
Table 1.
Table 1. Chemical components of samples No. 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. Sample Rice Flour 1 Rice Flour 2 Rice Flour 3 Waxy Rice
Flour Tapioca Sago Japan Tapioca SF-1700 SF-1900 SF-2800 Moisture
content (%) 14.77 13.90 13.53 13.67 15.27 15.27 10.87 11.20 13.70
12.47 Amylose content (%) 23.83 17.87 15.64 2.80 20.34 30.21 20.19
13.32 14.54 12.68
The amylose content of the grains of a rice plant can be seen as
the result of interaction between environmental factors and genetic
properties (Gomez, 1979). Champagne et al. (1999) revealed that
short grain rice contained low amylose while long grain showed high
amylose content. It was true for the result for present research.
Rice flour 1, rice flour 2, and rice flour 3 were firstly selected
based on their visual appearances and classified as long grain,
medium grain, and short grain rice, respectively. Rice flour type 1
contained amylose higher (23.83%) than medium (17.87%) and short
grain (15.64%). Rice flour type 3, short grain, which was firstly
assumed having low amylose content, was higher much than the
literature (Chapter 2). Yu et al. (2009) reported that Thai Jasmine
categorized as very low amylose content with the amount of AAC for
5.96%, while rice flour type 3 had 15.64% amylose. Waxy rice flour,
on the other hand, contained only 2.8% amylose. It was in agreement
with Yu et al. (2009) resulted that AAC of waxy rice flour was less
than 2%. This variation on amylose content could be also due to the
botanical origin of the rice. Nevertheless, the iodine colorimetry
method used to determine amylose content in current research had a
possibility to be
overestimated because the long chains of amylopectin could also
form a helical complex with iodine. It is important to gain this
kind of information since amylose contents is known having the
effects on characteristic of rice, such as textural properties of
cooked rice (Ong and Blanshard, 1995; Singh et al., 2005),
gelatinization and retrogradation behaviour of rice flour
(Varavinit et al., 2003). Especially for rice starch gel, Hibi and
Hikone (1998), Lu et al. (2009) and Mariotti et al. (2009) reported
its effects on the hardness, dynamic viscoelasticity and
retrogradation rate, respectively. High amylose rice is suggested
to cause high retrogradation of rice product, which will fairly
discussed in the result of pasting profile of the present research.
Tapioca and sago, on the other hand, contain amylose for more than
20%. The activity of the enzymes involved in starch biosynthesis
may be responsible for the variation in amylose content among the
various starches (Kross-mann & Lloyd, 2000). The typical
proportions of amylose and amylopectin in cassava starch are 20 and
80% respectively, although variations occur depending on the
cultivar and growing conditions. In this study, tapioca starch
contains 20.34% amylose. The additional type of binding agents
which were Japan tapioca and three kinds of chemically modified
tapioca were also measured for their amylose content. It is
observed that between native tapioca from Indonesia and native
tapioca from Japan only had slight difference in amylose content.
Chemical modification brings the reduction of amylose content of
three modified tapioca used in present research. The reduction
counted for 27.9 37.2%. The combination of main flours and binding
agents which have differences in amylose content could possibly
giving the expected result for the development of new product.
Swelling power and solubility index of burgo ingredients
Swelling power could indicate the characteristic of starch while
heating or cooking occurs. When starch molecules are heated in
excess water, the disruption of crystalline structure happens and
water molecules together with hydrogen bonding linked to the
exposed hydroxyl groups of amylose and amylopectin, which causes an
increase in granule swelling and solubility. It has been suggested
that amylose plays a role in restricting initial swelling because
this form of swelling proceeds more rapidly after amylose has rst
been exuded. The increase in starch solubility, with the
concomitant increase in suspension clarity is seen mainly as the
result of granule swelling, permitting the exudation of the amylase
(Singh et al., 2003). From three kinds of main ingredients used in
this research, their swelling and solubility power were analyzed
for treatment temperature 20, 40, 60 and 70o C as shown in Table 2.
Table 2. Swelling and solubility result of rice flours and binding
agents Sample 20o C Rice Flour 1 Rice Flour 2 Rice Flour 3 Waxy
Rice Flour Tapioca Sago 0,367 0,327 0,388 0,544 0,604 0,037
Solubility (%) 40 o C 0,526 0,509 0,498 0,686 1,129 0,574 60 o C
1,008 0,704 1,330 1,528 1,209 0,734 70 o C 3,631 2,939 1,538 20 o C
2,767 2,756 2,499 Swelling (%) 40 o C 2,870 2,789 2,563 2,688 3,232
2,509 60 o C 2,711 2,789 3,239 2,609 3,756 2,362 70 o C 7,181 7,331
3,996 17,578 20,116 4,006
57,648 2,607 18,148 2,244 8,261 2,706
From Table 2, it can be seen that at 200 C, the solubility power
of rice flours was ranging between 0.3270.388%, without significant
differences observed. Swelling power result, on the other hand, did
not give significant difference among the rice flours. The same
trend was also found for the analysis in temperature 40o C. The
highest solubility power value
was shown by rice flour type 1 (3,631%) at 700C while the
swelling power value ranging between 2,68 2,87%. Short grain rice,
on the other hand, exhibited the lowest swelling and solubility
value even in temperature 70o C. It can be further correlated with
the result of pasting temperature of the starch. Rice flour type 3
started to swell or gelatinize at temperature 81.6 0 C so that
temperature treatment on swelling and solubility measurement was
not enough to leach the amylose from the inside of rice starch
granule.By increasing the temperature during heating, granules
swelling increases as well. Nevertheless, it occurs only until a
specific temperature (Mandala & Bayas, 2004). Binding agent in
burgo production acts as the minor ingredient expected to prevent
disassociation of product during gelatinization. Moreover, when the
instant product produced, it requires strong compactness during
rehydration process. Tapioca, waxy rice, and sago, with amylose
levels from 2.8% to 30.21% dry base, were analyzed to obtain the
proper combination which will result high quality of product. Their
swelling and solubility power were examined using the same method
as previous main ingredients. Among the binding agents, tapioca
gave significant result of swelling power (20.116 %) and waxy rice
flour displayed the highest solubility index (57.648%). The high
swelling power of waxy rice flour (17.578%) could be explained by
the fact that amylopectin is predominant in improvement of starch
swelling, while the presence of amylose-lipid complex has been
known as swelling inhibitor. The amylopectin matrix gave more
consistency to the starch solution and was recognized as the
increase in swelling power. However, the highest swelling exhibited
by tapioca was in contrary to the theory. Regardless to the amylose
content, tapioca starch was known to have large size granule (as
proven further by next result of SEM) and in agreement with the
theory that large granule would swell more freely than small
granule. The swelling power and solubility also provide evidence of
the magnitude of interaction between starch chains within the
amorphous and crystalline (Singh et al., 2003). Nevertheless, the
result is in contrary with research conducted
by Li & Yeh (2001) reported that tapioca experienced
decreasing swelling power when more solids leached out during
cooking at higher temperatures. Sago exhibits the lowest degree of
solubility at 200 C, whereas it gives result of swelling power as
the highest among other binding agents at mentioned temperature. It
can be assumed that the amylose leaching from sago granules occured
earlier than the other binding agents. However, the rate of amylose
leaching of sago could be relatively very low due to the slow
increment of solubility and swelling even after reaching
temperature 70o C. the high amylose content of sago could also help
explain the result. When starch granules swell, the amylose inside
the granules leaches out simultaneously. A three dimensional
network formed and embedded the swollen granules in such a
continuous matrix which in turn decreasing the swelling power.
Ikegwu et al. (2009) conducted the research using starches from 13
improved cassava cultivars. It is resulted that the solubility
values ranged from 4.25 to 5.96%.
Swelling Power25 20 15 10 5 0 0 20 40 Temperature (C) 60 80
Solubility (%) Swelling (%) 70 60 50 40 30 20 10 0 0
Solubility Power
20
40 Temperature (C)
60
80
Fig 8. The swelling power and solubility index result
Information on swelling and solubility power of starch will help
food producer to create food product with desired characteristics.
Starch swelling can also be related to food quality. It is known
that wheat starches with high swelling power at 75o C resulted
better eating quality of instant fried noodles (Kim & Seib,
1993). Swelling powers of 3 types of rice flours and binding agents
are also shown in Figure 8. At a low temperature, the swelling
power of all starches gave almost similar trend. However, increase
in the heating temperature to more than 60o C led to the fact that
the swelling power of rice flour type 2 (medium grain rice) was
higher than the other rice flours. Hence, it absorbed more water at
high temperature than other starches. This evidence was being
strengthened by the RVA profile obtained. Moreover, at temperature
70o C, swelling power of all rice flours was still less than 20%.
The differences in the swelling power are partly affected by
hydrocarbon chains of internal lipids, which suppress hydration of
amorphous regions in starch granules (Tester & Morrison, 1990).
Short grain rice, rice flour type 3, shows the lowest swelling and
solubility power at 600 C. As mentioned earlier, waxy rice flour
started to swell at about 600 C. This result is consistent with
other research (Tester & Morrison, 1990; Rani &
Bhattacharya, 1995); Chung et al., 2010) showed that the starch
granules of waxy rice were greatly swollen at temperature range
55-63o C. The high swelling of waxy rice at this temperature is in
agreement with statement of Rani & Bhattacharya (1995) which
said that waxy starch exhibited a greater susceptibility to
swelling and rupture compared with high amylose rice. Since waxy
rice flour swelled much more than other rice flours (Figure 8), it
would seem that swelling is primarily a property of amylopectin. It
is strengthen by the fact that it contains amylose in lowest value.
The resulting swelling power indicates that the starch isolates
obtained were highly restricted type. However, lower swelling power
value suggests a more highly ordered arrangement in starch
granules. Sanni et al. (2005) reported that the swelling index of
granules reflect the extent of associative forces within the
granules, therefore the higher the swelling
index, the lower the associative forces. In term of producing
burgo, high degree swelling starch has been known to be less
resistant to granule disintegration. It has a relationship with the
significant viscosity decrease, either. Hence, main ingredient is
expected to give minimum effect from the swelling power in order to
result the rehydrated instant burgo with less deformation and high
compactness.
Pasting profile of main flours, binding agents, and
corresponding combination for burgo dough The series of structural
changes that take place in starch granules upon heating in excess
water is called gelatinization. In food applications,
gelatinization takes place during cooking. During gelatinization:
Upon initial hydration, the amorphous parts of the granules swell,
which increases the friction between granules and the viscosity. As
temperature increases and water penetration progresses, the
crystalline parts of the granules melt, allowing amylopectin
molecules to hydrate further, causing more swelling and viscosity
increase. When the granules reach a critical size, they break up,
which reduces the friction between them and causes the viscosity to
decrease. During cooling, the formations of amylose double helices
(retrogradation) or of amylose-lipid complexes if lipids are
present (Morrison, 1995) results in a viscosity increase. It is
known that the associative bonding of the amylose fraction is
responsible for the structure and pasting behaviour of starch
granule. The result of pasting profile of main flours and binding
agents is shown in Table 3.
Table 3. Pasting profile of rice flours and binding agents
Samplea Peak Rice flour 1 Rice flour 2 Rice flour 3 Waxy rice
flour Tapioca Sago Japan tapioca SF-1700 SF-1900 SF-2800a
Viscosity (RVU)b Trough 1711 1758 1235 1859 1375 8 1338 3573
2204 3769 Breakdown 367 907 1681 852 2677 191 2371 1337 452 2179
Final 3741 3682 1812 4035 2903 138 2651 6932 3952 6862 Setback 2030
1924 577 2176 1528 130 1313 3359 1748 3093
T pasting (o C) 82,05 80 76,3 81,6 71,65 76,45 69,15 71,45 71,45
61,65
2078 2665 2916 2711 4052 199 3709 4910 2656 5948
Mixtures consisted of 8% (w/w, dsb) starch in water. Measured in
Rapid ViscoAnalyser units
b
Discussing about the peak viscosity, short grain of Indica rice
had the highest peak viscosity (2916 RVU) compared with other rice
flours. According to the work reviewed by Dengate (1984) and
Crosbie (1991), the primary cause of higher peak paste viscosity
observed with reduced amylose starch may relate to the greater
swelling, and therefore reduced quantity of free water. It can be
possibly taken as one of the reasons since amylose content of rice
flour type 3 was the lowest among others. It is also in agreement
with research conducted by Chung et al. (2010) stated that long
grain, high amylose rice had the highest pasting temperature,
setback and final viscosity. Short grain, on the other hand, showed
the highest peak and breakdown viscosity. Peak viscosity of binding
agents, on the other hand, was ranging between 199-5948 RVU. The
variation could also be due to the effect of amylose content, which
is in accordance
with research resulted by Ikegwu et al. (2009). It is also
worthy to be noted that sago almost had no peak viscosity regarding
its lowest value (