**1. Introduction**

The world is emphasizing the consumption of green energy and material for sustainability, thus biopolymers such as starch, polylactic acid, chitosan, and others are intensively developed to become the next potential material to replace the conventional plastics [1]. Starch-based biopolymers are highly attractive due to their high abundance on the earth, being environmentally friendly, and having the same process-structure property as fossilfuel plastic, which makes them viable as packaging materials [2]. Starch is semi-crystalline and made up of two different macromolecules: amylose and amylopectin. The ratio of amylose to amylopectin varies according to the botanical origin of starch. The properties of the starch and starch-based plastic are strongly dependent on the ratio of these two macromolecules [3]. Typically, except for waxy starch and high amylose starch, starch will contain a higher amount of amylopectin (70–80%) than amylose (20–30%) [4]. Amylopectin is highly branched, while amylose possesses a linear molecular structure. The crystalline structure of starch is mainly contributed by double helix branched amylopectin. The neighboring branching of amylopectin chains with a degree of polymerization from

**Citation:** Lai, D.S.; Osman, A.F.; Adnan, S.A.; Ibrahim, I.; Ahmad Salimi, M.N.; Alrashdi, A.A. Effective Aging Inhibition of the Thermoplastic Corn Starch Films through the Use of Green Hybrid Filler. *Polymers* **2022**, *14*, 2567. https://doi.org/10.3390/ polym14132567

Academic Editor: Evgenia G. Korzhikova-Vlakh

Received: 28 May 2022 Accepted: 22 June 2022 Published: 24 June 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

10 to 20 forms a double helix structure, causing the formation of high-order allomorphs. The interactions between double helix amylopectin and long chain amylose give rise to the semi-crystalline structure in the starch biopolymer. The alternate arrangement of the double helix crystalline lamella and the amylose amorphous lamella is known as a "growth ring", which becomes the basic structure of the starch granules.

It is clearly understood that the pristine form of starch has a poor processing ability due to an intense and strong hydrogen bonding network structure. Commonly, starch will degrade before reaching its melting temperature; therefore, plasticizers such as water, glycerol, polyol, and urea are required to weaken and break down the hydrogen bonding by forming the new hydrogen bonding with the plasticizer. Under shearing force, high temperature, and incorporation of plasticizers, granule starch can be processed into an amorphous and homogenous material known as thermoplastic starch (TPS). Glycerol and water are the most common plasticizers as they are compatible with the starch structure and were proven in many studies [5,6]. Glycerol and water can effectively reduce the intermolecular force of granule starch and loosen up the dense packing structure. Full disruption of the starch structure can be achieved through the presence of plasticizers and a sufficient supply of thermomechanical energy [7].

Virgin TPS films are rarely utilized due to their low mechanical properties and high moisture sensitivity. The high moisture absorption of the virgin TPS is due to the existence of a large number of hydroxyl groups in the starch structure [8]. Besides that, the metastability of the TPS structure may lead to recrystallization or retrogradation process during storage over a period. Retrogradation can deteriorate the characteristics of the TPS films as it will alter the mechanical properties and affect the quality of the final product. Consequently, this will limit the usage of TPS in many applications.

Many factors can lead to the retrogradation of the TPS films, such as humidity, storage time, and glass-transition temperature, Tg [7]. Retrogradation is a process of reconstruction of the crystalline structure of starch chains. Despite the controversy about the accurate mechanisms of retrogradation that happen in TPS films, retrogradation was identified as the main cause of the aging and brittleness of this bioplastic. Generally, the retrogradation process can be divided into three stages, (i) nucleation, (ii) propagation (growth of crystalline structure), and (iii) maturation (perfection of crystals) [8]. Two retrogradation processes occur during the aging of the TPS films: linear amylose structure recrystallizes into a single helix structure (fast retrogradation rate) and branched amylopectin recrystallizes into a double helix structure (slow retrogradation rate). The slow retrogradation of amylopectin compared to amylose is due to its limited chain dimensional and lower crystalline structure stability [8]. The retrogradation rate was highly dependent on the mobility freedom of starch molecules. The restoration of crystallinity structure, loss of water holding capacity, and increased stiffness of the films are the most commonly used methods to determine the retrogradation of the TPS films [9]. A large amount of research was conducted to prevent starch's retrogradation by incorporating different types of plasticizers such as glycerol, polyol, urea citric acid, formamide, and so on to reduce the interaction between the starch chains [9,10]. A plasticizer can be defined as a substance (typically a solvent) added to synthetic or natural polymers (biopolymers) to reduce brittleness by promoting plasticity and flexibility. Several papers have discussed the role of mono-plasticizer and co-plasticizer in controlling the retrogradation of the TPS films. The co-plasticization method is preferred to produce the TPS films as it can compensate for the disadvantages of each plasticizer to make better properties films compared to mono-plasticizer films. For instance, Esmaeili et al. employed glycerol and sorbitol as co-plasticizers to form the TPS films. The low mechanical strength of glycerol/TPS films was successfully enhanced by incorporating sorbitol. However, the film's ductility was greatly reduced by 80% compared to the glycerol/TPS films [11]. In another work, Khan et al. have proposed the use of boric acid and glycerol as co-plasticizer to reduce the starch retrogradation. They showed that the boric acid can provide a better anti-retrogradation effect compared to the glycerol, whereas the TPS films that contain co-plasticizers have higher moisture sensitivity compared to

glycerol/TPS films [12]. Glycerol was also proposed to be a co-plasticizer with other components containing amide groups such as urea, formaldehyde, and formamide in order to reduce the retrogradation in the TPS by forming a stronger hydrogen bonding [13–15]. Nonetheless, this kind of plasticizer is rather toxic to human health; therefore it is not suitable to be applied in food packaging material. Several researchers have proposed the incorporation of a co-plasticizer that can form crosslinking with the TPS chains such as citric acid, malic acid, and choline salts–DES to reduce the retrogradation of the TPS [16,17]. However, the ductility of the co-plasticizer films was significantly reduced, making them brittle and unsuitable for film packaging applications. Therefore, researchers have investigated the incorporation of additives into the TPS structure, such as filler or nanofiller to restrict the mobility of the TPS chains. Nanofillers are those fillers that have one of their dimensions in the nano-size range (less than 100 nm). Nanocelluse and nanoclays such as bentonite and montmorillonite are examples of the nanofiller that can be used to reinforce and improve the properties of the TPS films. The solid and compact interfacial bonding between the filler/nanofiller and the TPS chains may form a highly stable thermodynamic 3D networking structure that can prevent retrogradation [18].

The crystalline structure of starch acts like a cement structure embedded in the amorphous regions, increasing the films' stiffness, and reducing the elongation at the break of the films. Starch's retrogradation happens during storage by forming hydrogen bonding with its adjacent starch chains. Therefore, incorporating nanocellulose and bentonite is expected to interfere with the alignment of the starch chains, thus preventing them from "self-interactions". Lendvoi et al. have successfully reduced the retrogradation of TPS starch through the incorporation of bentonite. Intercalation of bentonite in between the TPS molecular chains has successfully suppressed the retrogradation of starch, however, at the cost of the flexibility of the films [19]. Balakrishnan et al. studied the effect of nanocellulose on the properties of the TPS films. They have concluded that the nanocellulose confined the chain segment of the TPS chains, reducing the mobility of the starch macromolecules and suppressing the retrogradation of starch during aging [20]. Several review papers have included more detailed information on the effect of nanofillers on the aging of the TPS films [6,21,22]. Moreover, the size and number of the hydroxyl group of the filler were proposed to play a significant part in preventing the retrogradation of starch. Meanwhile, some researchers found out that the hydroxyl group's reactivity and bond-forming ability are more important in forming a stable hydrogen bond with a starch chain to prevent retrogradation. Nevertheless, most of the research concluded that the prevention of retrogradation depends on the hydrogen strength forming between the starch, fillers, and plasticizers. The stronger the hydrogen formed between the starch chain, fillers, and plasticizer, the slower the retrogradation rate [22].

Recently, hybrid fillers have been studied as potential alternative components to inhibit the retrogradation in TPS films. TPS films produced by hybrid fillers may combine the good properties of both fillers and optimize the best mechanical performance for the films. The hydrophilic type of fillers, such as nanocellulose and nano-bentonite, are compatible with the TPS, which is also hydrophilic. Hybrid fillers were studied extensively to enhance the mechanical properties of TPS films. It has the vast potential to develop into packaging material. Many research papers studied the effect of hybrid filler on the TPS films but mainly focused on the mechanical strength, structure, and humidity properties [1,23,24]. There is still a lack of studies and reports on the relationship between hybrid fillers on the retrogradation rate of TPS films due to time-consuming data collection. The effect of storage time on the properties of the TPS hybrid biocomposite is somewhat limited. Our previous study found that a hybrid of nanocellulose (NC) and nano-bentonite (BT) has a synergy effect in enhancing the toughness of the TPS films [25]. Thus, to close the research gap in this field, this current work aims to analyze the effect of green hybrid filler addition (NC + BT) on the TPS film's retrogradation during aging. In this study, corn starch powder was utilized as raw material. Corn starch is a type of starch that is derived from maize (corn) grain, which can be obtained from the kernel and endosperm part of the plant. In

this article, thermoplastic starch derived from corn is referred to as thermoplastic corn starch (TPCS). The impact of hybrid filler on the TPCS film's mechanical property was studied throughout the storing period. A tensile test was applied to observe the mechanical stability throughout the 3-month storage. DSC, XRD, and FTIR analyses were employed to study the crystalline structural change, enthalpy crystalline melting, and crystalline melting temperature of the TPS films. These three tests are highly sensitive and have been proven in many studies as valuable and reliable tools to quantify the retrogradation that happens in TPS films [15,16]. All the data were compiled, analyzed, and correlated with the retrogradation process of the starch chains, to investigate the effectiveness of the hybrid filler in inhibiting the aging of the TPCS matrix.

#### **2. Experimental**

#### *2.1. Materials*

Corn starch (72% amylopectin, 28% amylose) was purchased from Sigma Aldrich (St. Louise, MO, USA). It was employed as a matrix material after being plasticized into thermoplastic form. The plasticized corn starch is referred to as thermoplastic corn starch (TPCS). The nanocellulose and bentonite were combined to form a hybrid filler (NC + BT) of the TPCS biocomposite film. The nanocellulose was extracted from the oil palm empty fruit bunch (OPEFB) fiber purchased from United Oil Palm Industries Sdn Bhd (Nibong Tebal, Malaysia). Natural nano-bentonite clay was obtained from Sigma-Aldrich (St. Louise, MO, USA). Details on the hybrid filler preparation can be found in our previous paper [14]. Glycerol was purchased from HmbG Chemicals (Hamburg, Germany). It was used as a plasticizer with deionized water.

#### *2.2. Preparation of TPCS, TPCS-C, and TPCS-HC Films*

The virgin TPCS films, TPCS biocomposite films (TPCS-C), and hybrid TPS biocomposite films (TPCS-HC) were prepared through the casting method. A total of 5 g of corn starch was dispersed in 100 mL distilled water and a 2 g glycerol mixture to form thermoplastic corn starch films. The mixture was kept stirring at 300 rpm using a heated magnetic stirrer for 30 min at 80 ◦C to obtain homogenous TPS gel. For TPCS-C and TPCS-HC, 5 wt% of single filler or hybrid filler were prepared and then subjected to an ultrasonication process before being incorporated into the TPCS matrix. There were two ratios of hybrid fillers selected: (i) 4BT:1NC and (ii) 2BT:3NC for producing the TPCS biocomposite films. These two ratios were selected based on our previous study where 4BT:1NC was found to be the optimum ratio to produce the toughest films, while 2BT:3NC was observed to result in the lowest toughness value to the TPCS-HC film.

In the preparation of the biocomposite films, the mixture of TPCS/BT/NC was continuously stirred for 10 min to achieve homogenous dispersion of hybrid filler in the matrix. The homogenous TPCS suspension was poured into an 8-inch round Teflon casting plate and dried in the oven for 24 h at 45 ◦C. The composition and the abbreviation of the samples are presented in Table 1.


**Table 1.** The formulation of TPCS, TPCS-C, and TPCS-HC films.

#### Storage Procedures for Aging Analysis Storage Procedures for Aging Analysis

**Hybrid Thermoplastic Starch (TPCS-HC)** 

The TPCS, TPCS-C, and TPCS-HC films were kept in a chamber with a constant humidity of 53% at 25 ◦C and stored for 15, 30, 45, 60, and 90 days. The film samples were taken out for testing and analysis after the specified storage period. Figure 1 shows the films' appearance after being stored for 15, 45, and 90 days. The TPCS, TPCS-C, and TPCS-HC films were kept in a chamber with a constant humidity of 53% at 25 °C and stored for 15, 30, 45, 60, and 90 days. The film samples were taken out for testing and analysis after the specified storage period. Figure 1 shows the films' appearance after being stored for 15, 45, and 90 days.

TPCS/4BT1NC 95 4 1 TPCS/2BT3NC 95 2 3

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(**c**)

**Figure 1.** The physical appearance of virgin TPCS, TPCS-C, and TPCS-HC films aged for (**a**) 15, (**b**) 45, and (**c**) 90 days. **Figure 1.** The physical appearance of virgin TPCS, TPCS-C, and TPCS-HC films aged for (**a**) 15, (**b**) 45, and (**c**) 90 days.

#### *2.3. Testing and Characterization of Films 2.3. Testing and Characterization of Films*

#### 2.3.1. Tensile Test 2.3.1. Tensile Test

A tensile test was carried out according to ASTM-638 Type V to determine the tensile properties of films with different storage duration using the Universal Instron Machine model-5582 (Norwood, MA, USA). The tensile test was performed with a 5 kN load sensor and 10 mm/min crosshead speed. The testing was carried out at room temperature (23 °C) with 53% humidity. Seven replicates were tested and the average values of tensile properties (tensile strength, Young's modulus, and elongation at break) were acquired from the Instron Merlin software Version 5.41.00 (Instron®, Norwood, MA, USA) and then rec-A tensile test was carried out according to ASTM-638 Type V to determine the tensile properties of films with different storage duration using the Universal Instron Machine model-5582 (Norwood, MA, USA). The tensile test was performed with a 5 kN load sensor and 10 mm/min crosshead speed. The testing was carried out at room temperature (23 ◦C) with 53% humidity. Seven replicates were tested and the average values of tensile properties (tensile strength, Young's modulus, and elongation at break) were acquired from the Instron Merlin software Version 5.41.00 (Instron®, Norwood, MA, USA) and then recorded.

#### orded. 2.3.2. Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry (DSC) analyses were carried out by TA instrument Q-10 (Lukens Drive, New Castle, DE, USA) to determine the rate of retrogradation of the TPCS-C and TPCS-HC. A total of 30 mg of samples were tested and analyzed between 25 ◦C to 180 ◦C with a heating rate of 10 ◦C/min in a nitrogen atmosphere to determine the enthalpy of crystallization of each sample. The reference pans used were 40 µL aluminum pans. According to ASTM D3418-03, the enthalpy of the crystalline structure was determined by measuring the area of the main endothermic peak, while the melt temperature was determined as the midpoint of onset and end temperature of the endothermic peak. The enthalpy change in temperature was calculated and identified by using TA universal analysis 2000 software version 4.5A (TA-instruments-Water L.C.C, Lukens Drive, New Castle, DE, USA). All the films were stored under the same humidity (53%) for 15, 45, and 90 days.

#### 2.3.3. X-ray Diffraction (XRD)

The crystalline structure of all the films was determined using the Bruker D2 Phaser X-ray diffractometer (Billerica, MA, USA) using Cu Kα X-rays. The samples were tested by using a scan rate of 0.1 s per step from 2θ = 10 − 40◦ . The analysis and identification of the peak was analyzed by using X'Pert HighScore plus under PANalytical B.V. Almelo, The Netherlands. Meanwhile, the calculation for the degree of crystallinity was performed using Originpro 2019 (b) under OriginLab Corporation Northampton, Massachusetts, USA. The testing was carried out at room temperature, 25 ◦C with 53% humidity. The crystallinity of the films was determined by calculating the subtracting the amorphous halo from the XRD scan shown in Formulation (1)

$$\text{Degree of crystallineity } (\%) = \frac{\text{I}\_{\text{o}} - \text{I}\_{\text{a}}}{\text{I}\_{\text{o}}} \times 100\text{\%} \tag{1}$$

where I<sup>o</sup> = Total surface area of the peak from the XRD-based line, I<sup>a</sup> = Amorphous halo from the XRD scan.

#### 2.3.4. Fourier Transform Infrared (FTIR)

TPCS, TPCS-C, and TPCS-HC films were scanned by using a Perkin Elmer spectrum 65 (Waltham, MA, USA) FTIR spectrometer with a wavelength range of 650–4500 cm−<sup>1</sup> , 16 scans and resolution of 4 cm−<sup>1</sup> . All the films were stored at the humidity of 53%, 25 ◦C, and stored for 15, 45, and 90 days before conducting the FTIR analysis. The infrared spectra range of 900–1200 cm−<sup>1</sup> was focused to calculate the intensity ratio of the spectra at a specific wavenumber (1045 cm−<sup>1</sup> :1022 cm−<sup>1</sup> ). The purpose was to study the short recrystallize structure of the films. The infrared spectrum range 900–1200 cm−<sup>1</sup> was deconvoluted, whereas the intensity ratio for 1045 cm−<sup>1</sup> :1022 cm−<sup>1</sup> was calculated by using the Origin 2019 (B) software (OriginLab Corporation Northampton, MA, USA).

#### 2.3.5. Moisture Absorption

The moisture absorption test was determined by using the samples of 10 × 10 × 0.2 mm dimension to quantify the amount of moisture absorption during the 3-month storage duration. All the produced films were kept in the sealed humidity chamber with a control relative humidity (RH) of 53% at 25 ◦C. The film samples were immediately weighed after being taken out from the humidity chamber, and weighed in a specific period. The moisture content of the films was calculated by using Equation (2):

$$\text{Moisture content} \left(\% \right) = \frac{\text{W} - \text{W}\_{\text{o}}}{\text{W}\_{\text{o}}} \times 100\% \tag{2}$$

where W = the film's weights after taking out from the humidity chamber and W<sup>o</sup> are the original mass of the films right after the films form. The test was repeated 3 times to obtain the average value of the moisture content [15].

#### **3. Results and Discussion 3. Results and Discussion**  *3.1. Mechanical Analysis of TPCS, TPCS-C, and TPCS-HC Films Aged for 15, 30, 45, 60, and 90*

*3.1. Mechanical Analysis of TPCS, TPCS-C, and TPCS-HC Films Aged for 15, 30, 45, 60, and 90 Days Days*  3.1.1. Tensile Strength

*Polymers* **2022**, *14*, x FOR PEER REVIEW 7 of 21

#### 3.1.1. Tensile Strength

Measuring the change of mechanical properties across a period is one of the effective ways to postulate the aging properties. Figure 2 summarizes the mechanical properties of the TPCS, TPCS-C, and TPCS-HC biocomposite films for 15, 30, 45, 60, and 90 days of storage. After being stored for 3 months, the tensile strength of the virgin TPCS increased from 3.01 MPa to 5.3 MPa, almost 76% during the storage. The gradual increase of the tensile strength indicates the retrogradation of the TPCS occurred due to enhancement in crystallinity, as detected through XRD and DSC analyses. More crystalline structure in the starch chains requires greater force to break the molecular bonds; therefore the tensile strength value increases [26]. Meanwhile, the tensile strength of the TPCS/4BT1NC sample has been slightly increased (from 7.05 MPa (15 days) to 8.31 MPa (90 days)) throughout the storage period. Measuring the change of mechanical properties across a period is one of the effective ways to postulate the aging properties. Figure 2 summarizes the mechanical properties of the TPCS, TPCS-C, and TPCS-HC biocomposite films for 15, 30, 45, 60, and 90 days of storage. After being stored for 3 months, the tensile strength of the virgin TPCS increased from 3.01 MPa to 5.3 MPa, almost 76% during the storage. The gradual increase of the tensile strength indicates the retrogradation of the TPCS occurred due to enhancement in crystallinity, as detected through XRD and DSC analyses. More crystalline structure in the starch chains requires greater force to break the molecular bonds; therefore the tensile strength value increases [26]. Meanwhile, the tensile strength of the TPCS/4BT1NC sample has been slightly increased (from 7.05 MPa (15 days) to 8.31 MPa (90 days)) throughout the storage period.

**Figure 2.** (**a**) Tensile strength, (**b**) Young's modulus, and (**c**) Elongation at break of TPS, TPSC, and HTPS films aged for 15, 30, 45, 60, and 90 days. **Figure 2.** (**a**) Tensile strength, (**b**) Young's modulus, and (**c**) Elongation at break of TPS, TPSC, and HTPS films aged for 15, 30, 45, 60, and 90 days.

Generally, the trend shown in these tensile test data can be attributed to the structural evolution of the TPS, which is also influenced by the plasticizer and hybrid filler. Tensile strength for all the films increases in the first 15 days to 30 days, indicating migration of plasticizers and rearrangement of starch microstructure that occur within the first 30 days. This observation was in accordance with previous studies, and those results were proved Generally, the trend shown in these tensile test data can be attributed to the structural evolution of the TPS, which is also influenced by the plasticizer and hybrid filler. Tensile strength for all the films increases in the first 15 days to 30 days, indicating migration of plasticizers and rearrangement of starch microstructure that occur within the first 30 days. This observation was in accordance with previous studies, and those results were proved by the XRD analysis which indicated the increase in the crystalline structure of the host

TPS in the first month of storage. An increase in the crystallinity was attributed to the migration of plasticizers, resulting in an increase in tensile strength [27–29]. However, the tensile strength of the TPCS/4BT1NC was relatively stable after the first 30 days compared to other films. This shows that the interaction between TPCS, plasticizer, and hybrid filler could effectively minimize the rearrangement of the starch molecular chain. TPCS/5BT and TPCS/5NC showed a gradual and constant increase in strength with increased storage time. Meanwhile, the TPCS films show a noticeable increase in tensile strength after 60 days. The rise of the TPCS films' tensile strength was contributed by the increased crystallization of the amylopectin structure in the TPCS matrix, as shown in DSC and XRD analyses. The increase of the recrystallized amylopectin region indicated that the 3D starch network formed was less stable compared to other films under the normal storage condition. The absence of filler/hybrid filler caused the migration of the plasticizer to the film's surface. The void left behind by the plasticizer causes amylopectin to form bonding with the adjacent molecular chains and form another crystalline structure.

It is worth mentioning that the retrogradation rate of the TPCS films is highly dependent on the TPCS's chain mobility. Strong interfacial bonding within the TPCS chains can reduce the starch chain mobility and thus, effectively hinder the retrogradation. The stronger the interfacial bonding, the slower the retrogradation rate. Therefore, incorporating a hybrid filler is postulated to reduce the mobility and retrogradation process of the starch chains, but at the same time increase the glass-transition temperature, Tg, of the TPCS. Meanwhile, for the virgin TPCS films, the high moisture absorption capability caused the decrease of Tg across the storage time. As the Tg decreases, the degree of plasticizer migration increases with the storage time [30]. Therefore, we can observe a sudden increase in tensile strength from 3.69 MPa (60 days) to 5.30 MPa (90 days). By comparing the tensile strength of the TPCS, TPCS-C, and TPCS-HC films for 3 months, the TPCS-HC was seen to show the best retention in the tensile strength value, suggesting that the hybrid fillers can effectively slow down or limit the retrogradation through solid interaction between the plasticizer and TPCS chains.

#### 3.1.2. Young's Modulus

The Young's modulus trend is almost identical to tensile strength, where the TPCS films show the highest increase in Young's modulus after 90 days of storage, even though the value is still the lowest among all the samples. The virgin TPCS film exhibits a 100% increase in Young's modulus from 10.1 MPa to 20.3 MPa after 3 months of storage. Meanwhile, the TPCS/4BT1NC demonstrates the lowest increase of Young's modulus (20% increase of Young's modulus from 37.2 MPa to 44.6 MPa). By comparing the increment percentage, it can be postulated that the TPCS/4BT1NC has relatively higher structural stability than other films. The progressive increase of the TPCS film's Young's modulus can be attributed to the retrogradation. During the storage, the amylose and amylopectin recrystallize into a single helix and double helix crystal structure through the expulsion of plasticizers located within their adjacent chains. This enhances the crystallinity of the TPCS and leads to the stiffening and brittleness of the TPCS film [31,32]. Obviously, the modulus of the TPCS/4BT1NC was rather stable throughout the storing period, indicating there was no drastic alteration of the crystalline structure causing the fluctuation in Young's modulus value.

The Young's modulus of TPCS/2BT3NC has shown a different trend than its tensile strength. The tensile strength of the TPCS/2BT3NC showed a slight decrease from days 45 to 90. However, the Young's modulus value showed a slightly increased from 32.2 MPa to 38.3 MPa. The slightly increased Young's modulus indicating the less chain mobility of TPCS/2BT3NC can be associated with the increased crystalline structure as proved in XRD analysis. However, DSC showed that the crystalline structure in TPCS/2BT3NC is highly unstable, and low energy is required to break the crystalline structure. Therefore, the increase in crystalline structure is not reflected in the tensile strength.

TPCS/5BT and TPCS/5NC biocomposites demonstrate a gradual increase of Young's modulus through the storage time. The increase of the modulus can be due to the increase in the crystalline structure of the TPCS-C chain or can be related to the improved reinforcing effect of filler with increased storage time. As the migration of plasticizers happens during storage, it will trigger the recrystallization in TPCS-C and increase the film's stiffness. The migration of plasticizers will promote a "closer" interaction between the filler and starch chains, which can improve direct interfacial interaction and form the glassy structure in the TPCS matrix [15].

#### 3.1.3. Elongation at Break

The elongation at break of the TPCS films has significantly decreased from 76.1% to 38.4% and became highly brittle as the storage time increased. The embrittlement of the TPCS films was one of the undesired characteristics caused by the retrogradation. It has been well documented that the amorphous structure of amylose and amylopectin can recrystallize into the different crystalline structures (single-crystalline or double helix crystalline structure) during the storage [3,16]. The different crystalline structure formed in TPCS films will cause heterogeneous phases occurs as amylose and amylopectin recrystallize through intra- and intermolecular bonding. The different extent of chain recrystallization rate and crystalline structure induces the hard and soft segment order in the TPCS matrix. The disparity hardness between these segment orders could increase the internal stress of TPCS films at the junction of crystalline structure when the film is stretched. The internal stress evolves into the microvoids, which results in premature failure and reduction in the elongation at break [16].

The elongation at the break of the TPCS/4BT1NC was gradually increased from 85.5% (15 days) to 112.3 % (30 days) but then decreased slightly to 92.6% (90 days). Interestingly, the TPCS/4BT1NC does not show a significant reduction in the elongation at break value compared to other films, indicating the hybrid fillers could stimulate stress relaxation in the hard segment order and enhance the soft segment order which reduces the internal stress build-up in the films as mentioned in our previous study [24]. Thus, the TPCS/4BT1NC is physically stable across the storage and aging.

Apparently, the results suggest that the stability toward the aging of the TPCS/2BT3NC film was less prominent as compared to the TPCS/4BT1NC film. The TPCS/2BT3NC exhibits a stable microstructure for the first 60 days; however, as the storage increased to 90 days, the microstructure stability was reduced, and retrogradation was detected after reaching 60 days. This shows that the ratio of NC/BT filler in the hybrid system would affect the long-term retrogradation of the TPCS films. It can be said that the high composition of the NC compared to BT in the biocomposite films can attract the migration of plasticizers toward the NC. The long storage period has caused an accumulation of plasticizers in the interface of NC and the amylopectin. This encouraged the crystallization of the amylopectin chains, forming the unstable transcrystalline region in the starch structure [33,34]. At the same time, the number of the plasticizer molecules in the amylopectin-rich region decreased, leading to reduced chain mobility and decreased elongation at break. As a result, elongation at break for the TPCS/5BT and TPCS/5NC films decreased along with the increase in storage time. However, the elongation at break for both films is still better than the virgin TPCS films, indicating that the single filler (BT or NC) can prevent retrogradation of the TPCS matrix to some extent, although not as good as the hybrid NC/BT filler.

#### *3.2. Thermal Analysis for TPCS, TPCS-C, and TPCS-HC Films Aged for 15, 45, and 90 Days*

Figure 3 presents the DSC thermal analysis of the virgin TPCS, TPCS-C, and TPCS-HC biocomposite films, which were stored for 15, 45, and 90 days. This thermal analysis was performed to study the thermal transition throughout the 3-month storage time. The thermal transition was detected by measuring the heat flow differences between the TPCS films and the reference pans, which can usually be related to the melting of the crystalline structure in the films.

structure in the films.

**Figure 3.** DSC heating curves of virgin TPCS, TPCS-C ,and TPCS-HC films aged for (**a**) 15, (**b**) 45, and (**c**) 90 days. **Figure 3.** DSC heating curves of virgin TPCS, TPCS-C, and TPCS-HC films aged for (**a**) 15, (**b**) 45, and (**c**) 90 days.

films and the reference pans, which can usually be related to the melting of the crystalline

The thermal transitions of the virgin TPCS and associate biocomposites were analyzed by observing the changes in the enthalpy of fusion of the melting endotherms (ΔHf) and melting temperature (Tm). The recrystallized structure of the retrograded starch films can be realized through the observed enthalpy changes. The increase of enthalpy melting can be related to the extent of recrystallization happening in the TPCS structure. The homogeneity and the quality of the crystallized structure formed by starch retrogradation also can be studied by the broadness and the intensity of the melting peak. A more complex, broad, and weak melting peak can be associated with a high number of the different crystalline structures formed in the matrix with varying morphologies. By comparing the DSC heating curves between the TPCS, TPCS-C, and TPCS-HC films, we can reveal the effect of single filler and hybrid filler on the recrystallization structure of TPCS. The melting temperatures (Tm) and enthalpy of fusion (ΔHf) for TPCS, TPCS-C, and TPCS-HC films are summarized in Table 2, while the DSC heating curves are shown in Figure 3. The thermal transitions of the virgin TPCS and associate biocomposites were analyzed by observing the changes in the enthalpy of fusion of the melting endotherms (∆Hf) and melting temperature (Tm). The recrystallized structure of the retrograded starch films can be realized through the observed enthalpy changes. The increase of enthalpy melting can be related to the extent of recrystallization happening in the TPCS structure. The homogeneity and the quality of the crystallized structure formed by starch retrogradation also can be studied by the broadness and the intensity of the melting peak. A more complex, broad, and weak melting peak can be associated with a high number of the different crystalline structures formed in the matrix with varying morphologies. By comparing the DSC heating curves between the TPCS, TPCS-C, and TPCS-HC films, we can reveal the effect of single filler and hybrid filler on the recrystallization structure of TPCS. The melting temperatures (Tm) and enthalpy of fusion (∆H<sup>f</sup> ) for TPCS, TPCS-C, and TPCS-HC films are summarized in Table 2, while the DSC heating curves are shown in Figure 3.

During the first 15 days, all the samples exhibited a broad and weak endothermic melting peak. TPCS15 films showed the highest enthalpy of melting (194.3 J/g) followed by TPCS/2BT3CN15 (186.6 J/g), TPCS/4BT1NC15 (178.5 J/g), TPCS/5BT15 (142.6 J/g) and TPCS/5NC15 (135.4 J/g). Even though the TPCS films have the highest melting enthalpy, it possesses the lowest T<sup>m</sup> (99.3 ◦C) and a broad endothermic peak, suggesting that the crystalline structure in the starch is non-uniform and less stable compared to other films. During the retrogradation process, the TPCS chains may form different structures and morphologies and result in distinct retrograded crystallites [35]. The differences of distinct retrograded crystalline structures may cause the different extent of the crystalline structure

formed in the TPCS matrix and result in the large and broad endothermic peak being detected. Meanwhile, incorporating single or hybrid fillers into the TPCS has increased the T<sup>m</sup> of the crystalline structure. Even though incorporating the BT and NC can hinder the recrystallization in the TPS films, studies also showed that the BT and NC may provide the "thermal shielding effect" to the matrix phase, which causes a higher melting temperature needed to break the structure [24,36]. Moreover, the difference in morphology between the BT and NC may contribute to different ways of matrix-filler interactions, which causes a difference in T<sup>m</sup> between the TPCS/5BT15 and TPCS/5NC15.

**Table 2.** Melting temperatures (Tm) and enthalpy of fusion (∆Hf) for TPCS, TPCS-C, and TPCS-HC films.


As the storage time increased to 45 days, the T<sup>m</sup> of the TPCS, TPCS-C, and TPCS-HC films increased, and the endothermic peak became sharper and more intense. The T<sup>m</sup> of TPCS/4BT1NC45 only shows a small increase (+4 ◦C), but the melting enthalpy showed a tremendous increase from 178.5 to 412.4 J/g. The significant growth of enthalpy could be due to the enhancement effect of hybrid fillers toward the crystalline structure which required higher thermal energy to melt the crystalline structure [24]. The melting temperature and enthalpy of the TPCS films also significantly increased by as much as 24.3 ◦C and 241.3 J/g from day 15 to 45. The increase of both parameters indicated the crystalline structure continued to develop into a more well-defined and stable crystal structure. Interestingly, the T<sup>m</sup> of the TPCS/2BT3NC45 was only increased by 18.2 ◦C. However, it shows only a small increase of melting enthalpy (128.5 J/g) from day 15 to 45 days. The small increment and broad melting enthalpy peak of TPCS/2BT3NC45 indicate that the crystalline structure formed in TPCS/2BT3NC45 is highly unstable and non-uniform in size.

As the storage time increased to 90 days, the T<sup>m</sup> and melting enthalpy of the films increased and become more well defined. The melting peak of the TPCS90 becomes broader and more intense compared to other films. The high-intensity TPCS90 melting peak can be related to a high retrogradation rate due to the high growth rate of the crystal structure. However, the broader peak observed could be due to the high number of imperfect and non-uniform crystals [23,37]. The higher Tm and sharper melting enthalpy of the TPCS/5NC90 and TPCS/5BT90 compared to the TPCS90 indicate that a more uniform and stable crystalline structure can be induced by adding NC or BT. Even though NC and BT can induce crystalline structure forming in the TPCS matrix, the overall increase in the melting enthalpy of TPCS/5NC and TPCS/5BT is rather smaller than the TPCS films from days 45 to 90. This shows that BT and NC can reduce the retrogradation of starch for a prolonged storage period. The DSC heating curve of the TPCS/2BT3NC90 revealed the lowest melting enthalpy of this sample compared to other samples, after 3 months of storage. The low melting enthalpy indicates that there is less thermal energy required to destroy the crystalline structure of the TPCS/2BT3NC90. Furthermore, the fluctuation in the melting temperature of the film suggests that the retrogradation process occurred to some extent. Meanwhile, the T<sup>m</sup> and melting enthalpy of the TPCS/4BT1NC show relatively stable across the 3-month storage period. The sharp and low intensity of melting enthalpy from the beginning of storage indicates the hybrid filler with a 4:1 ratio is highly favored to form a stable crystalline structure at the early stage of film-forming and restrict the mobility of the amylopectin, preventing the retrogradation phenomenon.
