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 (