chapter 3.analysis of main flour and binding agents of burgo

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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