## 3 Results and Discussion
### Birefringence of Cassava Starches in HPP-treated Cassava Flours
#### 3.3.2 Long-Range Order of Cassava Starches  
The long-range order of starch granules can only be determined by XRD [43], which demonstrates the packing of double helices into ordered arrays [42]. From the diffractogram, the crystallinity from the long-range order was determined from the percentage of area under each prominent peak to the total area under the curve [25]. Analysis revealed a significant three-way interaction between pressure, FC, and HT (Table 1). The simple two-way interaction of pressure and FC against all levels of HT (both \(p<\) 0.001) and succeeding simple simple main effect of pressure across flour concentrations at certain levels of HT (all \(p<\) 0.001) were statistically significant at a significance level of 0.025%. Simple simple comparisons between pressures across all FCs at 10 min HT revealed that 600 MPa had significantly lowered %RC relative to other pressures, while no difference was found between lower pressures. This interaction is best visualized in Figure 4. On the other hand, at 10%-30 min, 500 MPa was significantly higher than 600 MPa but they were both significantly lower than other pressures. Conversely, at 20% to 30%-30 min, only 600 MPa was significantly lower than other pressures. The interaction between the variables demonstrated graduation in loss of %RC, which conforms with the varying degree of birefringence loss of the said samples. Furthermore, the statistically significant mean difference of 600 MPa to other pressures decreases with increasing FC at both HTs, revealing an inverse relationship of the hydration level of flour with %RC. However, the mean difference increased with HT between similar concentrations except for 10%, signifying a higher impact with longer HT. These suggest that 600 MPa is sufficient to alter the crystallinity or the long-range order of cassava starch in flour into a more amorphous state, but the intensity of alteration is dependent on flour concentration and holding time.  
XRD graphs can also be utilized to compare changes in crystallinity visually. The cassava flour samples exhibited a type C polymorph with a stronger type A orientation. It was characterized by distinct single peaks at 2\(\theta\)-15 and 23\({}^{\circ}\), a double peak at 17.5\({}^{\circ}\), and weak peaks at 20, 27, and 30\({}^{\circ}\). No difference in spectra was observed from the control to all 400 MPa treatment combinations. However, at 500-10%-30, the intensity of the double  
Figure 4: The relative crystallinity percentage (%RC) of untreated (0.1 MPa) and HPP-treated cassava flour at 10 min (**A**) and 30 min holding time (**B**). Error bars represent \(\pm\) standard deviation (n = 3).  
peak at 2\(\theta\)-17.5\({}^{\circ}\) and single peaks at 15 and 23\({}^{\circ}\) was slightly reduced (Figure 5A), while it was maintained in other 500 MPa combinations. Figure 5B reveals that at 600 MPa, the peaks at 2\(\theta\)-15 and 23\({}^{\circ}\) were dissolved progressively with decreasing FC and increasing HT. The doublet peak at 2\(\theta\)-17.5\({}^{\circ}\) turned into a singlet with decreasing intensity as FC decreased and HT increased. A similar decrease in intensity was reported for buckwheat flour and ascribed to crystallinity loss during the HPP treatment [44]. Moreover, a more prominent 2\(\theta\)-20\({}^{\circ}\) was observed, indicating the formation of amylose-lipid complexes during the process [17; 44; 45].