Diversity of the Starch Granule Morphology of Several Types Cassava (Minihot esculenta Crantz)

. Cassava ( Manihot esculenta Crantz), which is commonly called singkong is multipurpose crop that can be processed as food, feed and other cassava-based bioproduct. Potential application of cassava starch is determined by the properties of starch as the main component of cassava tuber biomass. The size of the cassava starch granules is different for each variety due to genetic and the environment factors in which it grows. The size of the starch granules affects the application of the starch. This study aimed to determine the shape and diameter of the starch granule in several types of cassava through microscopic analysis. The shape and the size of the 62 starch samples tested was varied. The starch granules shape was dominated by spheres form. The diameter of cassava starch granules ranged from 2.016 ± 0.015 µm – 3.318 ± 0.045 µm. The highest diameter was Tidung1.2 and the lowest was Tidung 2.2. This is an open acces article under the CC-BY license.


Making Starch
The manufacture of cassava starch begins with separating the cassava tubers and their skins by peeling them. Wash the peeled tubers thoroughly with water to separate the dirt. Clean peeled tubers are weighed. Furthermore, the tubers are grated or mashed with a machine until smooth. The shredded tubers are added with water (3 liters of water for every kg of tubers) to form pulp and knead so that more starch is released. The tuber pulp is then filtered with a cloth so that the starch escapes from the filter as a suspension of starch and fibers are left on the filter cloth. The starch suspension obtained is then collected in a settling vessel and precipitated for 12 hours. The liquid above the sediment is removed and the sediment is dried under sunlight to dry and then used for histochemical test of starch granules.

Histochemical Test of Starch with Lugol to Determine the Shape and Diameter of Starch
Granules The starch histochemical test was carried out using cassava starch samples. Then the sample was dripped with Lugol's solution (1: 2) and observed using a digital light microscope with a magnification of 40X. Observations were made in three fields of view for each sample. Microscopic profiles of starch were documented for analysis of granule shape and diameter. Images were analyzed using the ZEN application with a 2.5X zoom to determine the size and diameter in µm units. Starch diameter was calculated by repeating 20 granules in each of the three fields of view. The color of the starch granules as a reaction between the starch molecules and the solution was visually observed.

Forms of Starch Granules
Starch is a complex carbohydrate that is insoluble in water, colored white like a paste or powder and has no smell. Starch obtained from the extraction of cassava roots is white. According to [13] starch grains can be observer through a microscope so that it can be seen from the shape, size and form of starch grains, which solitary or assembled (compound starch grains). Starch in tubers is generally oval, or round. The original shape of the starch grains and the size distribution of the starch grains play a role in identifying the source of these starch grains. Based on the results of microscopic observations, in general cassava starch granules are round. The color variations of starch granules are categorized as blackish blue, bluish purple, and light purple ( Based on Figure 1, the starch forms found in the sample are round, oval, polygonal and truncated oval. The calculation of the ratio in the form of the percentage of each granule shape in all tested samples showed that the starch granule shape observed was predominantly round and the low percentage shapes (rarely) were oval and polygonal ( Table 1). The starch of all the samples tested after being dripped with Lugol's solution has a blackish color as shown in Figure 1 and visually. The color change occurs because there is a reaction between starch and Lugol's solution which indicates the presence of carbohydrate compounds. This is in accordance with that stated by [14] starch is purplish blue or black with lugol or iodine.

Diameter of Starch Granules
Microscopic observation of starch granules showed that the cassava starch varied in size. The largest diameter of starch was found in Tidung 1,2, which was 3,318± 0.045 µm and the lowest was found in Tidung 2,2, namely 2,016 ± 0.015 µm ( Table 2). The difference in size is the character of each type of starch [15]. Wang et al. (2009) stated that the characteristics of starch are different for each variety [16]. In Figure 2, analysis of the dots plot distribution of samples based on the diameter size category, it can be seen that the observed starch granule diameter is dominated by the size with an interval of 2-3 µm, only two samples have a diameter of more than 3, namely Tidung 1,2 and Crystal White Rose.  In Figure 2 it can be seen that the observed starch granule diameter size is dominated by size with 2-3µm intervals. The largest diameter size of starch was found in the hood 1.2 variety, namely 3.318± 0.045 µm and the lowest was found in Tidung 2, 2 varieties, namely 2.016 ± 0.015 µm ( Table  2). The difference in size is the character of each type of starch [15]. Wang et al. (2009) stated that the characteristics of starch are different in each variety [16].
The diameter size of the starch observed in the cassava starch samples tested was categorized as very small or small, with a diameter of 2.02 -3.32 µm. Unlike the Royang type of cassava starch from Thailand, which is planted by irrigation and rainfed, it has a small to large diameter range of 7.21 ± 33.78 µm which is influenced by irrigation and rainfed irrigation systems [17] [18]. This measure is in accordance with previous research by Lindeboom et al. (2004) who reported that starch grain size was classified into four groups, namely, small (<5 µm), small (5-10 µm), medium (10-25 µm), and large (> 25 µm). Meanwhile, according to Jading et al. (2011) there are two types of starch grains, namely small (5-10 µm) and large starch grain sizes, namely (25-40 µm).
The size of the starch granules has a very important role in the application of the food industry. Granule size is one of the functional properties that determines starch utilization [14]. Starch with small granule size has the ability to absorb water better and is easily digested by enzymes. It is important in the manufacture of liquid sugar (glucose, fructose and dextrins) from starch. Likewise for the manufacture of vermicelli, small starch is preferred because it can increase water absorption and the strength of the vermicelli before cooking, so that they are not easily broken/brittle [19]. In addition, the small starch granule size (<10 mm) and relatively the same as the potential fat globule is used as a fat replacer in the food industry [20].
Large granules are more resistant to hydrolysis, and the enzyme will break down small granules compared to large ones, and the enzymatic digestion pattern of starch granules at small sizes will be different from larger granule sizes [21]. In addition, cassava starch with large granule size has a higher gelatinization temperature than starch with small granule size [22].

Conclusion
The starch samples that were dripped with Lugol's solution were black in color and had various forms of starch granules, namely round, oval, polygonal and cut oval, of the 62 starch samples consisting 51 different types of cassava showing different granule diameter sizes with a range of 2.016 ± 0.015 µm -3,318 ± 0.045 µm. Based on the analysis of the distribution of samples according to diameter, there are 2 types of starch that have a larger diameter than the other samples, namely Tidung 1,2 and Crystal White rose.

Acknowledgement
Cassava starch samples were obtained at the Biotechnologi Research Center, Indonesian Institute of Science (LIPI) Cibinong, Bogor.