*3.4. Mechanical Properties*

The effect of T-HNTs nanoparticles loaded with CIN on the mechanical properties of SA composite film was studied. As presented in Table 3, it was evident that with the addition of T-HNTs-CIN nanoparticle, the tensile strength (TS) of the SA/T-HNTs-CIN nanocomposite film improved, and its TS value was increased by 20.8% compared to the SA film. This was similar to the effect of halloysite nanotubes on the mechanical properties of carrageenan/gelatin films previously published by Akrami-Hasan-Kohal et al. [31]. The potential strain-induced arrangemen<sup>t</sup> of the clay particle layer in the polymer matrix and the interaction between the polymer and the hydrogen bonds in the clay minerals might contribute to the improvement of the tensile properties of the film [32,33]. The elongation at break (*ε*) of the film was not changed significantly by the T-HNTs-CIN nanoparticle addition. However, the addition of CIN significantly increased the flexibility of the film. Ahmed et al. also reported that adding clove essential oil to the film matrix increased the flexibility of the film [34]. The presence of the CIN in the SA matrix might hinder the polymer–polymer intermolecular attraction [35]. However, CIN was added to the SA matrix after being loaded by T-HNTs, and most of the CIN was present in the T-HNTs, thereby reducing the plasticizing effect of CIN.

**Table 3.** Mechanical properties of SA nanocomposite film.


Data are presented as mean ± standard deviation and different letters (a–c) within the columns shows the significant differences (*p* < 0.05), where a is the lowest value.

#### *3.5. Light Transmittance and Opacity of Film*

The UV-visible spectrum for SA nanocomposite film is displayed in Figure 3. It can be seen that SA film exhibited high light transmittance in both UV and visible regions, especially in the 300–800 nm region with a high light transmittance of more than 80%. The light transmittance of SA/CIN film at 240–300 nm was significantly lower than that of SA film. The anti-UV effect of CIN might be its own aromatic compound, and its chemical bond could absorb UV light [26]. Ahmed et al. had previously found a similar phenomenon

in the study of polylactide/cinnamon oil composite films [36]. Moreover, as can be seen from curves c and d in Figure 2, the transmittance of the composite film at all wavelengths was greatly reduced after the addition of T-HNTs in SA film.

**Figure 3.** Light transmittance of the nanocomposite film: (**a**) SA film, (**b**) SA/CIN film, (**c**) SA/T-HNTs film, and (**d**) SA/T-HNTs-CIN film.

Table 4 shows the transmittance values of the composite films at 240 (UV-C), 300 (UV-B), 360 (UV-A), and 600 nm (visible light) as well as the opacity of different formulation film. The light transmittances of SA film at 240, 300, 360, and 600 nm are 69.5, 81.8, 85.0, and 87.4%, respectively, and the transmittance value after the addition of T-HNTs-CIN decreased to 22.8, 40.9, 50.1, and 66.1%, respectively. This might be due to the combined action of T-HNTs and CIN. The presence of T-HNTs in the film matrix might block or diffract the light, thus affecting the transmittance of light at all wavelengths [8]. Huang et al. also found a similar phenomenon when halloysite was added during the preparation of agar-based nanocomposite films [37]. The transparency of SA composite film decreased with the addition of HNTs. At the same time, the color of the film was whitened by the addition of the white powder HNTs. It is noteworthy that the addition of T-HNTs-CIN resulted in a greater reduction in the transmittance of UV light than that of visible light. This means that by preparing the composite film with T-HNTs-CIN, the UV barrier properties can be improved without sacrificing the transparency of SA film. The nanocomposite film with high ultraviolet shielding performance has high application potential as a transparent ultraviolet blocking packaging material.


**Table 4.** Transmittance (%) and opacity and values of nanocomposite film in the visible, UV-A, UV-B, and UV-C regions.

Data are presented as mean ± standard deviation and different letters (a–d) within the columns shows the significant differences (*p*< 0.05), where a is the lowest value.

#### *3.6. Slow-Release Behavior of the CIN in Food Simulants*

Isooctane was used as food simulant to simulate food with hydrophobic fats. The cumulative release of CIN by the SA film without T-HNTs and the SA film with T-HNTs in food simulant are shown in Figure 4.

**Figure 4.** The cumulative release of CIN in the nanocomposite film: SA/CIN film and SA/T-HNTs-CIN film.

The whole process of the release experiment was carried out under stable environmental conditions (temperature of 20 ◦C, relative humidity 75%). The SA/CIN and SA/T-HNTs-CIN film in food simulant released most of CIN in the first 24 h, and the cumulative release of SA/CIN film was higher (58.95%), which was significantly higher than SA/T-HNTs-CIN film (28.57%). Subsequently, the release rate of CIN in the two films slowed down significantly. Finally, the release of CIN from SA/CIN film reached a peak of 59.97% at 72 h, and the release amount of CIN was about 16.64 mg, while the release of SA/T-HNTs-CIN film reached a stable level at 216 h, and the cumulative release of film stabilized at 60.31% (The release of CIN was about 17.44 mg). The presence of T-HNTs in SA/T-HNTs-CIN film slowed down the release rate of CIN, which in turn delayed the time when CIN reached stability. Shen et al. prepared novel sodium alginate-based double network hydrogel spheres after loading urea on HNTs, which reduced the release rate of urea [38]. The release of CIN in the SA composite film matrix into the food simulant is affected by many factors. First, the liquid molecules in the solvent diffuse from the outer surface of the film into the matrix of the SA composite film. Then, the polymer matrix network relaxes due to the presence of the solvent in the film matrix. Lastly, CIN is released from the relaxed polymer

matrix into the food simulating liquid until the thermodynamic equilibrium between the SA composite film and the food simulating liquid is reached. Of course, the last step is influenced not only by mass transfer, but also by the interaction between the volatile compounds and the matrix [25,39].

According to the steps described above for the release of the active compound from the SA composite film matrix, and in combination with the release curves of the two film systems in food simulation solution, it can be seen that the release rate of the SA composite film supported by T-HNTs was significantly slower than that of the SA/CIN film. This observed behavior could be explained by the retarded release of CIN by T-HNTs. The loading of CIN by T-HNTs was mainly the adsorption of intracavity and external surface of T-HNTs to CIN [40]. This increased the mass transfer steps of CIN in the release process, thereby prolonging the release time of CIN. Compared with CIN directly added to SA matrix, CIN was added to SA matrix in a HNTs loaded manner, and CIN was better protected to maintain its activity through the action of HNTs carrier, and the release time of CIN from the controlled release system was prolonged, thus maximizing the function of CIN. Therefore, it can be concluded that the presence of HNTs in SA/T-HNTs-CIN film controlled release system could effectively alleviate the initial burst release of CIN and prolong the action time of CIN. The SA/T-HNTs-CIN film slow-release system might have a good application prospect in the packaging of fatty foods.

#### *3.7. In Vitro Antibacterial Activity of the Film in the Release Experiment*

The effects of the manufactured film on the antibacterial activity of typical foodborne pathogens (*S. aureus* and *E. coli*) were investigated, and the results are shown in Figure 5. As expected, the presence of T-HNTs in the film matrix has no antibacterial activity against *S. aureus* and *E. coli.* However, on day 0 of the experiment, SA/CIN and SA/T-HNTs-CIN film decreased by 1.34 and 1.35 Log10CFU/mL, respectively, compared to the control group. This might be attributed to the presence of CIN in the polymer matrix. A large number of previous studies have shown that CIN had inhibitory effects on the growth of a variety of bacteria, because CIN might damage the cell membrane of bacteria, leading to changes in cytoplasmic leakage and membrane permeability [41,42]. Similarly, for *E. coli*, the number of colonies in the film containing CIN was significantly reduced by 0.65 and 0.57 Log10CFU/mL, respectively, compared with the control group. Interestingly, we found that *S. aureus* was more susceptible to CIN inhibition than *E. coli.* This phenomenon could be attributed to the fact that the cell wall of *E. coli* (Gramnegative bacterium) has an extra layer of lipopolysaccharide outer membrane than that of *S. aureus* (gram-positive bacterium). The lipopolysaccharide outer membrane has a good barrier effect on hydrophobic substances (CIN). Although it cannot completely block hydrophobic compounds, it limits the penetration of CIN into microbial cells and reduces the inhibitory effect [43].

In addition, with the increase of release time, the bacteriostatic effect of the film containing CIN on the two kinds of bacteria became less and less. There was no significant difference between SA/CIN film and SA in the colony count of *S. aureus* on the second day. However, the SA/T-HNTs-CIN film showed no significant difference in the number of colonies from the SA/T-HNTs film on 7 days. The same trend was observed for *E. coli*, where CIN was fixed by T-HNTs and then added to SA matrix, and the bacteriostatic duration was extended by 4 days compared with SA/CIN films. These results confirmed that the SA/T-HNTs-CIN film had a controlled release effect on CIN, successfully alleviating the interaction between the active compound and the surrounding environment and making it release slowly. This packaging system can be used as a new and promising alternative to renewable food packaging. Of course, future research in this area should focus more on increasing the loading of active substances or using multiple loading systems to improve their applicability.

**Figure 5.** Antimicrobial activity of the composite film: (**a**) *Staphylococcus aureus* and (**b**) *Escherichia coli.* Values followed by different letters (a–f) were significantly different (*p* < 0.05), where a is the lowest value.
