## 3 Results and Discussion
### Birefringence of Cassava Starches in HPP-treated Cassava Flours  
Microscope images showed varied sizes of cassava starch granules, as they have been reported to have a diameter ranging from 2.4-35 \(\upmu\)m [30,31]. The cassava starch granule has a spherical morphology with a truncated side, similar to these untreated samples but with a few distorted and damaged granules probably sustained during flour processing. There was no visible difference between untreated cassava flours, 300-400 MPa combinations, and 500 MPa for 10 min holding time. The micrographs of 500-30 min and all 600 MPa combination treatments revealed enlarged and swelled starch granules as well as aggregation (Figure 2; solid yellow arrow), which explains the notable change in viscosity of the slurries and formation of gel-like clumps. Starch granule aggregation was also reported for cassava starch at 600 MPa [32], leading to gel formation [33, 34, 35]. Other flours also showed granule aggregation [11,36] and a more continuous protein distribution [37]. Despite the swelling of starch granules in the cassava flour, there was minimal granular disintegration, a characteristic of HPP-treated starches [6]. Figure 1: Visual changes of untreated and selected treated cassava flour slurries; where (**A1**) is 0.10 MPa, 10% flour concentration; (**A2**) is 0.10 MPa, 20% flour concentration; (**A3**) is 0.10 MPa, 30% flour concentration; (**B1**) is 600 MPa–10 min, 10% flour concentration; (**B2**) is 600 MPa–10 min, 20% flour concentration; (**B3**) is 600 MPa–10 min, 30% flour concentration; (**C1**) is 600 MPa–30 min, 10% flour concentration; (**C2**) is 600 MPa–30 min, 20% flour concentration; (**C3**) is 600 MPa–30 min, 30% flour concentration. Polarized images of the flours revealed a Maltese cross pattern due to the radial orientation of the crystallites [19], as shown in Figure 2. Similar to light micrographs, untreated cassava flours, all 300-400 MPa combinations, and 500-10 min did not exhibit loss of birefringence (photos not shown). As seen from the starch granules' swelling, the 500-30 min and all 600 MPa combination treatments caused a visible dissolution of the Maltese cross. This phenomenon is intensified by increasing pressure levels, prolonging holding time, and decreasing flour concentration. This birefringence loss indicates the disorganization of molecular crystalline order [38] that came with pressure-induced gelatinization. At 500-30 min, the number of swollen starch granules increased with a decrease in flour concentration. Some swollen starch granules showed a hollow and gel-like center that appeared dark in polarized light with birefringence in the periphery (Figure 2; dashed red arrow). This suggests a loss of radial orientation and organization, supported by the report of [PERSON] et al. [39], wherein two distinct zones were seen from sliced HPP-treated potato starch granules, with the outer zone corresponding to unchanged structures and a gel-like network in the inner zone. Highly enlarged starch granules that showed no cross pattern (Figure 2; solid red arrow), indicating a complete loss of birefringence, were abundant in 600-10% FC samples, followed by 20% and least at 30% FC. All 600-30 min treatments had visually more loss of birefringence than their 600-10 min counterparts. Complete loss of birefringence was reported for isolated cassava starch granules from 550 MPa at 30%-30 min [40] to 600 MPa at 20%-5 min [34] and 50%-30 min [32], indicating pressure-concentration-time dependence. A similar finding can be reported for cassava flour, with a gradual birefringence loss at 500-30 min and all of the 600 MPa combination treatments. However, even at the highest pressure-lowest FC-longest HT (600-10%-30 min), random starch granules showing partial gelatinization (Figure 2; dashed red arrow). The 600 MPa combination treatment was also reported for 600 MPa combination treatments.