APPLICATION OF SCANNING ELECTRON MICROSCOPY SEM TO

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2.6 APPLICATION OF SCANNING ELECTRON MICROSCOPY SEM TO

STARCH Scanning electron microscope SEM has been widely used in the area of research which is focused on starches, especially in examining granular morphology of starches. It gives satisfying information of starches granule size and shape among botanical sources which are necessary to be understood. In the best conditions, and with the most advanced equipment, the resolving power of the scanning electron microscope can reach 5 nm for biological matter Blond and Simatos, 1996. The morphological changes of starch during heating in excess water is also can be studied by SEM Eliasson and Gudmundssond, 2006. Collado and Corke 2003 suggested that functional properties of the starches are related not only to their structure as polymers but also to the packing of polymers within the granules. Bao and Bergman 2004 also reported that the clarity of starch suspensions which is very important for many food applications varies among different rice cultivars and may be attributed to amylose content and granular size. The mechanism of SEM in generating image reveals quite a bit of complexity, but it can be simplified Hafner, 2007. Principally, the sample is bombarded by electrons energy. Under this bombardment, each point of the object spontaneously emits varied radiations, depending on its chemical nature, surface state and the conditions of the electron probe emission. This radiation is captured by appropriate detectors Blond and Simatos, 1996. This instrument is composed by several supporting equipments such as a column containing a thermoelectronic emission system electron gun, an electron probe focusing system condenser and objective, and a scanning system electromagnetic deflection controlling the scan amplitude Blond and Simatos, 1996. One thing that must be concerned in SEM is sample preparation. Sample preparation for scanning electron microscopy depends on their hydration. The samples must be dehydrated before being placed under vacuum in the microscope column. If the moisture content does not exceed 16 e.g., cereal grains or dry products they can be observed as they are. Contrarily, if their moisture content is higher, it is essential to dehydrate them either by lyophilization or critical point drying Blond and Simatos, 1996. If it is preferred to avoid the inconvenience of dehydration, it is possible to use a cryotransfer stage. This special device allows the sample to be observed directly, after very fast freezing on a liquid nitrogen cooled stage -170 o C. Since the sample is preliminary fractured and coated with metal under vacuum at -170 o C, the temperature is never interrupted. The results with this technique are outstanding Blond and Simatos, 1996. The remarkable advantages of SEM are apparently admitted. However, this technique also presents certain drawbacks, like the inability to study hydrated media without resorting to complex techniques, and multitude of problems related to the preparatory techniques, which do not occur without fairly significant disruption in the sample material or the production of foreign elements that must be eliminated. These factors must be taken into account when interpreting the photographic documentation Blond and Simatos, 1996. As stated above that samples must be observed in dehydrated state, biological samples exhibit at three major drawbacks: 1 they are poor conductor, and thus poor emitters, causing strong discharges during observation, which disrupts the electromagnetic detection and consequently, the image; 2 the low energy electrons penetrate easily, so that the image is formed from both true secondary electrons and second generation electrons, which weakens image clarity, but accentuates its relief; 3 some substances, such as starch, are very sensitive to the action of high energy electrons, and break up when the intensity is too high Blond and Simatos, 1996. 11

III. RESEARCH METHODOLOGY

3.1 MATERIALS INSTRUMENTS

3.1.1 Materials

Pea flour Yunnan Xing Yi Foodstuff Co. Ltd., China, rice flour Thai Better Foods Co. Ltd., Thailand, and sticky rice flour Thai Better Foods Co. Ltd., Thailand, were obtained from local market. Potato amylose standard Sigma Chemical Co., St. Louis was used for apparent amylose content analysis, while for resistant starch analysis, Resistant Starch Assay Kits Megazyme International Ireland, Ltd. pancreatic α-amylase, amyloglucosidase, GOPOD reagent buffer and enzymes, D-Glucose standard solution, resistant starch control were used.

3.1.2 Instruments

. The main instruments were a scanning electron microscope SEM, LeO 1450 VP, England, a differential scanning calorimeter DSC, Mettler Toledo, TGASDTA 851e, Switzerland a rapid visco analyzer RVA, Model 4D, Newport Scientific, Australia, a texture analyzer TA-XT2, Stable Micro System, Texture Technologies Corp., USA, a spectrophotometer UV Vis. BiochromLibra S22, England, and color analyzer ColorQuest XE HunterLab, Hunter Associates Laboratory Inc., Virginia-USA.

3.2. EXPERIMENTAL DESIGN

This research was divided into three parts. The preliminary research was investigation on the properties of raw materials rice flour, sticky rice flour, and pea flour involved chemical composition, apparent amylose content, granular morphology, thermal and pasting properties. In experiment I Figure 3, the effect of mixing rice flour with sticky rice flour at various ratios 100:0, 97.5:2.5, 95:5, 92.5:7.5, 90:10 on apparent amylose content, granular morphology, thermal and pasting properties was also investigated. In experiment II Figure 4, trial was conducted to determine the solid content, proper cooking time and temperature to obtain rice cake. Similar trial was conducted for pea cake. Cakes resulted from various ratios of rice flour and sticky rice flour were then analyzed in terms of starch digestibility, textural and color properties. Commercially prepared pea cake and rice cake were also analyzed for comparison. In experiment III Figure 5 various cold setting conditions were conducted on pea cake and the extreme ratios of rice cake 100:0 and 90:10. First condition was 6 hours at room temperature ± 25 o C which represented vendors that make cakes in the morning, place them in room temperature then sell them in the afternoon or evening. Second condition was 6 hours at refrigeration temperature 4 o C which represented some vendors that place the cakes in iced box during time between after cooking to selling approximately 6 hours. Third condition was 24 hours at 4 o C which came from theoretical point of view that RS content will increase after storage for 24 hours or longer at refrigeration temperature for some starches. Experiment for 12 or 24 hours at room temperature was impossible to be conducted as spoilage occurred on the cakes. Clearer experimental steps can be seen on the following figures.