*3.3. Crystalline Structure of TPCS, TPCS-C, and TPCS-HC Films Aged for 15, 30, 45, 60, and 90 Days by Using XRD Analysis*

Overall, the TPCS, TPCS-C, and TPCS-HC films' crystallinity increased with the storage time. The longer the storage time, the higher the crystalline percentage. However, incorporating a single or hybrid filler slowed down or inhibited the retrogradation rate. Table 3 and Figure 4 shows the changes in the crystalline percentage of the TPCS, TPCS-C, and TPCS-HC films after 15, 45, and 90 days of aging. The virgin TPCS film demonstrates the highest rate of the crystals growth as the crystalline percentage reaches 28.7% in the 3-month storage. Meanwhile, TPCS/5BT and TPCS/5NC exhibit a lower crystallization percentage compared to the TPCS films. For TPCS-HC films, TPCS/2BT3NC and TPCS/4BT1NC proved to effectively prevent the retrogradation in starch for the first 45 days as the crystalline percentage only showed a minor increase of 3.7% and 2.2%, respectively. The crystalline percentage of TPCS/4BT1NC experienced a slight increase in crystallinity structure (2.9%) even though the storage period increased to 3 months. However, for TPCS/2BT3NC, the efficacy to inhibit retrogradation was only seen to be dominant in the first 45 days. This is because the crystallinity of the TPCS/2BT3NC increased drastically to 20% after 3 months of storage, showing that the retrogradation process has taken place.

**Table 3.** The changes in the crystalline percentage of TPCS, TPCS-C, and TPCS-HC films after 15, 45, and 90 days of storing.


The XRD diffraction pattern of the TPCS films, TPCS-C films, and TPCS-HC films that were stored for 15, 45, and 90 days was recorded. Upon the film drying process, the amylose chain will undergo fast retrogradation to form a Va-type or Vh-type crystalline structure with the inclusion of plasticizers, which is typically detected in 2θ = 7.8◦ , 15.0◦ , 20.8◦ , and 2θ = 17.0◦ , 18.3◦ , 22.1◦ respectively. However, the Va-type crystals are relatively unstable compared to the Vh-type crystalline structure; they would absorb water molecules and transform to the Vh-type crystalline during storage in ambient humidity [38]. Therefore, a small amount of Va-type crystalline residual structure was detected in TPCS films' XRD diffraction pattern. The complete transformation of crystalline usually happens in the first 20 days [11,39,40]. The crystalline structure change by absorbing water molecules agreed with the water absorption result of the pure TPS films. As the storage time increased to 45 days, the residual for Va-type crystalline disappeared and completely transformed into Vh-type crystalline. Meanwhile, as the TPS films were scanned on day 45, the intensity of the Vh seemed to reduce. This phenomenon was explained by Schmitt et al. where the Vh-type crystalline structure was superimposition with B-type crystalline during the slow retrogradation process, causing the intensity of the Vh to be reduced in the XRD diffraction diagram [16].

**Figure 4.** XRD scan for virgin TPCS, TPCS-C, and TPCS-HC films aged for (**a**) 15, (**b**) 45, and (**c**) 90 days from 2θ= 5.0–40.0° and (**d**) 90 days from 2θ = 10.0–30.2°. **Figure 4.** XRD scan for virgin TPCS, TPCS-C, and TPCS-HC films aged for (**a**) 15, (**b**) 45, and (**c**) 90 days from 2θ= 5.0–40.0◦ and (**d**) 90 days from 2θ = 10.0–30.2◦ .

*3.4. Recrystallization of Short-Chain Amylopectin in the TPCS, TPCS-C, and TPCS-HC Films Aged for 15, 45, and 90 Days as Detected through FTIR Analysis*  The FTIR spectra of TPCS, TPCS-C, and TPCS-HC in the range of 900–1200 cm−1 are shown in Figure 5. Many studies proved that the alteration of short-range molecular order could be obtained from the bands at 1022 cm−1 and 1047 cm−1 [45–47]. The number of amorphous regions and crystalline regions was represented by the bands at 1022 cm−1 and 1047 cm−1, respectively. The intensity ratio between the 1047 cm−1: 1022 cm−1 (C values) can be used to index the starch crystalline structure region in the amorphous area. The higher the Based on the XRD curves, the TPCS15 and TPCS/5NC15 and TPCS/5BT15 exhibited a firm Vh-type retrogradation peak compared to TPCS/4BT1NC15 and TPCS/2TB3NC15 for the first 15 days. The high intensity of the Vh structure strongly suggests the plasticizer was not effectively dispersed in the TPCS matrix, resulting in the formation of the Vh-plasticizer crystalline structure. Meanwhile, only a small Vh peak was detected in both TPCS-HC films, indicating that the hybrid filler more effectively interacts with the plasticizer and restricts the mobility of plasticizer molecules to prevent the formation of Vh-plasticizer crystalline structure.

C values, the more organized the TPCS matrix structure. The infrared spectra from 900 to 1200 cm−1 were deconvoluted, and the 1047 cm−1: 1022 cm−1 values were calculated and presented in Table 4. In the case of TPCS/5NC and TPCS/5BT, the increased intensity of the peak at 2θ = 22.2◦ over time can be due to the nucleating effect of the fillers. Generally, slow retrogradation consists of three major steps (nucleation, propagation, and maturation) in recrystallization [8]. The nucleation of the crystalline structure started at the early stage of film-forming. It was propagated throughout the storing time. The crystalline structure was finally matured after three months of storage and increased the intensity of the peaks. The biocomposite incorporating the single filler, regardless of BT or NT, exhibited a lower crystalline percentage than the TPCS films after 3 months, indicating that a single filler can suppress the retrogradation in starch. However, Lendvai et al. showed a contrary result where the addition of filler increases the retrogradation rate in starch. The increase in crystalline percentage may be attributed to poor filler dispersion in the TPS matrix. The agglomerated filler served as the nucleating site to facilitate the recrystallization of the starch chain [30].

The crystalline structure of the TPCS/4BT1NC only experienced slight changes after 3 months of storage, evidently proved by the slight alteration of the diffraction peaks. This result was also in agreement with the stable mechanical performance of the films. All the films exhibited a more defined peak as the storage time increased. A pronounced increase in peak intensity at 2θ = 22.2◦ was observed in TPCS, TPCS/5NC, and TPCS/5BT after 45 days of storage. However, the increased intensity of this peak was not observed in the TPCS-HC films, suggesting that the hybrid fillers can effectively form stable TPCS structures and reduce the retrogradation. However, as the NC ratio in the hybrid filler increased to 3, the peak intensity at 2θ = 22.2◦ for TPS/2BT3NC has a noticeable increase after 3 months of storage. This increase in peak intensity indicates that the semi-crystalline structure of the TPCS/2BT3NC is not stable with a long storage time. The inhibition of the retrogradation process is only prominent during the early storage period. TPCS/2BT3NC is only effective in preventing the fast retrogradation happening in starch, and it was less effective in suppressing the slow retrogradation happening in the TPCS films compared to the TPCS/4BT1NC.

The effectiveness in inhibiting the retrogradation in TPCS/4BT1NC and TPCS/2BT3NC may be attributed to the different ratios of the hybrid filler component. The increase in NC ratio in the TPCS-HC films resulted in the low TPS film's stability in long-term storage. Previous studies showed that the NC has a higher plasticizer affinity than the starch molecules. Drakopoulos et al. have studied the interfacial effect of TPS, microcrystalline cellulose, and plasticizer by using broadband dielectric spectroscopy (BDS). They showed that even though the microcrystalline cellulose and starch chains have high similarities in chemical structure, they possess different dimensional structures that affect their polarization toward plasticizers. The difference in dimensional structure makes the microcrystalline cellulose more hydrophilic, thus water molecules can more easily enter and decrease the polymer chain's mobility [41]. As the storage time increases, the plasticizer in the TPCS/2BT3NC will slowly get away from the amylopectin-rich phases. The voids left behind by the plasticizer causes more water to be diffused into the amylopectin structure and facilitates the recrystallization. This result agrees with the water absorption data of the TPCS/2BT3NC as the water absorption of TPCS/2BT3NC is slightly decreased compared to the TPCS/4BT1NC after 3 months of storage. This explained the sudden increase in crystalline percentage and intensity peak of the TPCS/2BT3NC at 2θ = 22.2◦ after 45 days of storage. The unexpected crystalline alteration was also seen in the study of A. Ujcic et al. However, they showed the opposite trend where the crystallinity of the TPS composite films decreased in the middle of the storage period by incorporating metal oxide. They proposed that the metal oxide induced the early crystalline structure detected in the TPS films. However, the metal oxide caused an unstable crystalline structure due to low compatibility with the starch chains, and the plasticizer can quickly destroy it during the recrystallization process [41]. This result also showed that the migration of plasticizers toward the fillers highly affected the TPS's retrogradation process. Moreover, it also showed that the retrogradation process highly influences the relationship of plasticizers with the filler in the TPS structure.

The agglomeration of plasticizer at the interfacial interaction between the NC in TPCS/2BT3NC and amylopectin will induce the amylopectin chain to form a trans crystallization structure on the surface of the NC as reported in many studies [33,34,42,43]. According to their studies, the existence of trans crystallization can be detected through the appearance of a peak at 2θ = 21.5◦ . The peak magnitude of TPCS/2BT3NC at 2θ = 22.2◦ was observed to be shifted away from 2θ = 22.5◦ to 21.7◦ from day 45 to 90 in Figure 4d. This shifting of diffraction peak was only observed for TPCS/2BT3NC90 and TPCS/5NC90. However, the TPCS/5NC90 only shows a slight shifting of peak compared to TPCS/2BT3NC90. The shifting of the peak is only seen in the films that contain NC in the structure, therefore this shifting of the peak is most probably related to the existence of transcrystalline structure in TPCS/2BT3NC90. The transcrystalline structure formed as an oriented crystalline layer of amylopectin on the NC with the inclusion of a plasticizer at the interfacial surface [44]. The transcrystalline structure in TPCS/2BT3NC may contribute to low melting enthalpy, as detected through the DSC analysis.
