*3.5. Moisture Absorption of TPCS, TPCS-C, and TPCS-HC Films Aged for 90 Days*

The accelerated retrogradation can happen due to the high amount of water absorption during storage and will cause the alteration of films' crystalline structure. The changing of crystalline structure will affect the mechanical and biodegradation properties of the samples. Water can absorb into the TPCS films and accelerate the retrogradation rate. Absorbed water molecules can increase the plasticizer content in the films and may facilitate the retrogradation rate. This phenomenon has been also described by other researchers [26,27]. Figure 6 shows the moisture absorption of the TPCS, TPCS-C, and TPCS-HC films throughout the 3-month storage period. As shown in Figure 6, the moisture absorption rate of TPCS-C and TPCS-HC is lower compared to the virgin TPCS. This shows that filler could create a more tortuous path for water molecules into the films, which can reduce the TPCS films' hydrophilic properties. All the films experienced different amounts of water absorption from the beginning of storage and reached their equilibrium moisture absorption after 10 days. The virgin TPCS film demonstrates the highest water absorption rate for the first 10 days and reached its maximum water absorption at 18.86%.

The high water absorption may be attributed to the presence of a large amount of hydroxyl groups in the starch structure, which causes a high affinity of the biopolymer to absorb water into its structure. However, after being stored for 3 months, the water absorption of the TPCS films seems to experience a slight decrease to 16.87%. This may be due to the retrogradation in the TPCS film structure, as shown in the XRD analysis, causing the expulsion of water molecules, and reducing the water content in the films. This decreasing water absorption effect was somewhat challenging to observe in the other films. TPCS/5NC has the second-highest water content compared to TPCS/5BT, TPCS/4BT1NC and TPCS/2TB3NC. This may be attributed to the higher affinity of NC toward water

molecules compared to BT. However, the high crystallinity structure of nanocellulose can reduce the water sensitivity and reduce the water permeation into the TPCS films, as reported in our previous study and other studies [49,50]. Apparently, the TPCS/4BT1NC film demonstrates the lowest water absorption rate compared to other films. The low water absorption of the TPCS/4BT1NC film indicates that BT and NC, when added in the ratio of 4:1, can most efficiently repel the water inclusion as the well-dispersed particles can create a more tortuous path for the entrance of the water. throughout the 3-month storage period. As shown in Figure 6, the moisture absorption rate of TPCS-C and TPCS-HC is lower compared to the virgin TPCS. This shows that filler could create a more tortuous path for water molecules into the films, which can reduce the TPCS films' hydrophilic properties. All the films experienced different amounts of water absorption from the beginning of storage and reached their equilibrium moisture absorption after 10 days. The virgin TPCS film demonstrates the highest water absorption rate for the first 10 days and reached its maximum water absorption at 18.86%.

The accelerated retrogradation can happen due to the high amount of water absorption during storage and will cause the alteration of films' crystalline structure. The changing of crystalline structure will affect the mechanical and biodegradation properties of the samples. Water can absorb into the TPCS films and accelerate the retrogradation rate. Absorbed water molecules can increase the plasticizer content in the films and may facilitate the retrogradation rate. This phenomenon has been also described by other researchers [26,27]. Figure 6 shows the moisture absorption of the TPCS, TPCS-C, and TPCS-HC films

sensitive to detecting long-range molecular crystallized structures [48].

*3.5. Moisture Absorption of TPCS, TPCS-C, and TPCS-HC Films Aged for 90 Days* 

*Polymers* **2022**, *14*, x FOR PEER REVIEW 17 of 21

After reaching 90 days of storage, the virgin TPCS film exhibits the highest increase in the C values compared to other films, indicating that the recrystallization of the shortchain amylopectin occurred more significantly. However, by adding a single filler or hybrid fillers, the recrystallization of short-chain amylopectin could be slowed down. The C values of the TPCS/4BT1NC, TPCS/5BT, and TPCS/5NC films have been consistently increased with the increase of storage time, which is in line with the increase in crystallinity percentage measured through the XRD analysis. However, the increase of the C value for TPCS/2BT3NC from 15 to 90 days showed little difference compared to the results obtained through the XRD analysis. The crystalline percentage calculated from XRD suggested that the TPCS/2BT3NC experienced a slow rate of crystal growth from 15 to 45 days, and the growth of crystalline structure was only detected after 45 days. However, the steady increase in the C-value as detected from the FTIR analysis indicates that the TPCS/2BT3NC possessed a consistent growth of crystalline structure from 15 to 90 days. The contradiction between the FTIR and XRD results could be due to the formation of a transcrystalline structure in the TPCS/2BT3NC sample, where the recrystallization of short-range molecular was more sensitive to be detected by FTIR. The transcrystalline structure in TPCS/2BT3NC was only detected in XRD analysis by shifting the peak at 2θ. The transcrystalline does not reflect the intensity of the XRD peak because XRD is more

**Figure 6.** Moisture absorption percentage for virgin TPCS, TPCS-C, and TPCS-HC films aged for 90 days.

The significantly reduced water permeability of the TPCS/4BT1NC film, when benchmarked with the virgin TPCS, could also be due to its greater aging resistance and low retrogradation rate. Fewer chain breakage, voids, and surface embrittlement in this TPCS hybrid biocomposite film make it more resistant to water absorption as it can reduce the permeation and penetration of water molecules into the films.

#### **4. Conclusions**

The virgin thermoplastic corn starch (TPCS), thermoplastic corn starch composite (TPCS-C), and thermoplastic corn starch hybrid biocomposite (TPCS-HC) films were stored for 3 months to study the effect of hybrid filler on the starch retrogradation through the tensile test, DSC, XRD, FTIR, and water absorption analyses. Results indicate that the presence of single filler or hybrid fillers (nanocellulose + bentonite) can minimize the degree of starch retrogradation, thus inhibiting the aging of the TPS film. The interactions between the TPCS, plasticizer, and hybrid filler have reduced the mobility of the starch molecular chains, minimizing the rearrangement of the starch molecular chain into the crystal structure, and subsequently retarded the retrogradation process of the biopolymer. On the contrary, the absence of the hybrid filler caused the migration of the plasticizer to the film's surface, allowing more free movement and arrangement of molecules for the formation of a more dominant amylopectin crystalline structure. This mechanism led to the retrogradation of this starch-based film.

The TPCS-C films showed moderate efficacy in reducing the starch retrogradation compared to the TPCS-HC. The TPCS-HC film is proven to possess the most negligible starch retrogradation and can maintain its mechanical stability throughout the 3-month aging period. Apparently, the ratio of nanocellulose (NC):bentonite (BT) hybrid filler in the TPCS-HC system affects the retrogradation and aging of the film during a long storing period (3 months). The high composition of the NC compared to BT in the biocomposite films can attract the migration of plasticizer molecules that accumulate in the interface of the NC and the amylopectin. This encourages the formation of the oriented crystalline layer of the amylopectin chains in the interface of the NC/amylopectin (known as a transcrystalline structure). This unstable transcrystalline region in the starch structure is responsible for the retrogradation phenomenon.

Overall, results indicate that the TPCS film incorporating the BT:NC hybrid filler in the ratio of 4:1 is the best hybrid biocomposite sample as it has demonstrated the most stable microstructure and mechanical properties upon the 3-month storing period. This suggests that the use of a green hybrid filler of nanocellulose and bentonite with the appropriate ratio/composition can be an efficient and environmentally friendly approach to avoid early biodegradation or aging of the starch-based film in the development of a sustainable packaging material.

**Author Contributions:** Conceptualization, A.F.O. and S.A.A.; data curation, D.S.L. and A.A.A.; formal analysis, D.S.L. and A.A.A.; investigation, A.F.O. and D.S.L.; methodology, A.F.O., D.S.L. and I.I.; project administration, A.F.O., S.A.A., I.I. and M.N.A.S.; software, M.N.A.S.; supervision, A.F.O. and S.A.A.; writing—original draft, D.S.L.; writing—review and editing, A.F.O. and A.A.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors gratefully acknowledge the funding and the financial support from the Fundamental Research Grant Scheme (FRGS)—(FRGS/1/2019/TK10/UNIMAP/03/2) funded by the Ministry of Higher Education Malaysia.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable for studies not involving humans.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.
