*3.5. Effect of Variations in PLA–Chitosan–TEO Concentration on Mechanical Characteristics of Food Packaging*

Tensile strength testing of food packaging is an important factor that must be studied further to determine its application in the food industry, particularly for applications to vegetables and fruits. Mobility mechanical properties are critical because they are required to support in vitro culture and implantation processes [46]. Furthermore, testing the mechanical properties of packaging materials is critical for determining the homogeneity of a polymer mixture and the mixed materials used in the manufacturing process. In this study, the mechanical characteristics of the PLA–chitosan–TEO material were analyzed by testing the tensile strength (Tensile Strength) of the sample using the UTM (Universal Testing Machine) in the Tensile Test Laboratory of the Chemical Engineering Department, Lhokseumawe State Polytechnic. The dimensions of the specimens used follow the dimensions of the specimens in ASTM D 638–99. In this graph, the highest tensile strength value was obtained in sample biofilm 3. The composition of each biofilm 1 material is 13.03 Mpa. Meanwhile, biofilm 2 (3 g of chitosan, 0.3 mL TEO and 0.5 mL of glycerol) and 3 (4 g of chitosan, 0.3 of TEO and 0.5 mL of glycerol) have tensile strength values of 15.15 MPa and 17.67 Mpa, respectively.

Figure 6 based on the three samples, the third sample has a large tensile strength value, which is because the high concentration of chitosan causes the tensile strength of the analysis to increase. The increase in tensile strength was due to the reduced water content in the bioplastic because it had been absorbed by chitosan as an organic polysaccharide. Therefore, the molecular structure of bioplastics becomes denser and more homogeneous, which causes the tensile strength to increase [47,48]. This is also in line with conducted research which states that the addition of chitosan in PLA causes the tensile strength to increase based on research that has been carried out by [49] regarding the effect of the homogenization method and the content of carvacrol on the microstructure and physical properties of chitosan-based films. Film-forming emulsions were made with chitosan (1.5%), Tween 80 (0.5%), and carvacrol (0.25%, 0.5%, and 1.0%). The homogenization method used is the rotor-stator with high- and low-pressure homogenization. The results showed that the use of carvacrol had a significant impact on the mechanical properties of the emulsified films. The tensile strength produced by the film was significantly reduced. The results of adding chitosan to bioplastics showed that there was an interaction in the mixed film (starch–chitosan bioplastic). Chitosan has a good filming property and has extensive water absorption in the acidic medium due to the chain relaxation effect generated in the macromolecular networks by protonated amino groups. The addition of chitosan can increase the tensile strength of bioplastics [50]. However, if more chitosan is added, the more the tensile strength value of the bioplastics will decrease. In other words, the resulting bioplastic will have brittle properties [51]. The table below is in line with research conducted by [52]: the strength of the prepared samples was not affected by the type of natural extract, but only by the used basic matrix/polymer. The explanation of different textural properties can be affected by the different pH since the preparation of films included PLA.

**Figure 6.** Comparison of the mechanical properties of each biofilm produced based on variations in chitosan. **Figure 6.** Comparison of the mechanical properties of each biofilm produced based on variations in chitosan.

The concentration of chitosan also affected the % elongation in PLA–chitosan–TEO biofilms samples. Elongation is a value that states the ability of a sample to be able to extend from its original shape or size as mentioned in Table 8. Figure 7 shows the elongation (%) of the PLA–chitosan–TEO biofilms obtained ranged from 18% to 25%. At a sample concentration of 2 gr chitosan, the lowest % extension value is obtained at a concentration of 2 g is only 18%. The chitosan concentration of 3 g, namely 22.7%, was the middle elongation obtained. The highest elongation came from biofilm 3 with 25% elongation. This is in line with the study of [53], where the minimum amount of chitosan used was set at 2 (by wt). It should be noted that starch could not be formed into films by glycerol solution. However, chitosan has an exceptional capacity to create film. As a result, it is also mentioned that biodegradable films were created using starch, and chitosan will provide a much better film because the films exhibited high strength and optimal elongation values. The results show that adding extracts to TEO and chitosan matrices improves the elongation and tensile strength of the biofilms formed. To The concentration of chitosan also affected the % elongation in PLA–chitosan–TEO biofilms samples. Elongation is a value that states the ability of a sample to be able to extend from its original shape or size as mentioned in Table 8. Figure 7 shows the elongation (%) of the PLA–chitosan–TEO biofilms obtained ranged from 18% to 25%. At a sample concentration of 2 gr chitosan, the lowest % extension value is obtained at a concentration of 2 g is only 18%. The chitosan concentration of 3 g, namely 22.7%, was the middle elongation obtained. The highest elongation came from biofilm 3 with 25% elongation. This is in line with the study of [53], where the minimum amount of chitosan used was set at 2 (by wt). It should be noted that starch could not be formed into films by glycerol solution. However, chitosan has an exceptional capacity to create film. As a result, it is also mentioned that biodegradable films were created using starch, and chitosan will provide a much better filmbecause the films exhibited high strength and optimal elongation values. The results show that adding extracts to TEO and chitosan matrices improves the elongation and tensile strength of the biofilms formed. To decrease the resultant error value, each sample test was performed three times.

decrease the resultant error value, each sample test was performed three times. **Table 8.** Mean values (± standard deviation) of elongation (%) obtained from biofilm formations of: **Table 8.** Mean values (± standard deviation) of elongation (%) obtained from biofilm formations of: (B1) 2 g chitosan; (B2) 3 g chitosan; (B3) 4 g chitosan after the elongation test each biofilms.


B3 25.04 ± 0.05 4 g chitosan, 0.3 mL TEO, and 0.5 mL glycerol

**Figure 7.** Mean values (±standard deviation) of elongation (%) obtained from biofilm formations of: 2 gr chitosan; 3 gr chitosan; 4 gr chitosan after the elongation test each biofilms. A statistical difference from SPSS is indicated (*p* < 0.05). **Figure 7.** Mean values (±standard deviation) of elongation (%) obtained from biofilm formations of: 2 gr chitosan; 3 gr chitosan; 4 gr chitosan after the elongation test each biofilms. A statistical difference from SPSS is indicated (*p* < 0.05).

#### *3.6. Techno Economic Challenges of the Developed and Future Research Directions 3.6. Techno Economic Challenges of the Developed and Future Research Directions*

Biofilms are complex microbial communities composed of one or more species submerged in an extracellular matrix with varying compositions based on the kind of food-producing environment and the invading species. Bacteria and fungi are examples of microorganisms that may form these biofilms. The presence of many bacterial species in a biofilm has significant ecological benefits since it facilitates the biofilm's adhesion to a surface. This can also happen in the absence of specific fimbriae in some species. Disinfectants such as quaternary ammonium compounds and other biocides are more resistant to mixed biofilms. In the food business, biofilms may build swiftly. The first two phases condition the material's surface and the reversible cell binding to that surface. The binding then becomes permanent, and the formation of microcolonies begins. Finally, the tridimensional structure of the biofilm is established, giving rise to a sophisticated ecosystem suitable for dispersal. Some biofilm-forming bacteria in food factory settings cause human diseases, which is very important in the food business. These pathogens may form biofilm structures on a variety of artificial substrates used in the food business, including stainless steel, polyethylene, wood, glass, and polypropylene. Biofilms are complex microbial communities composed of one or more species submerged in an extracellular matrix with varying compositions based on the kind of foodproducing environment and the invading species. Bacteria and fungi are examples of microorganisms that may form these biofilms. The presence of many bacterial species in a biofilm has significant ecological benefits since it facilitates the biofilm's adhesion to a surface. This can also happen in the absence of specific fimbriae in some species. Disinfectants such as quaternary ammonium compounds and other biocides are moreresistant to mixed biofilms. In the food business, biofilms may build swiftly. The firsttwo phases condition the material's surface and the reversible cell binding to that surface. The binding then becomes permanent, and the formation of microcolonies begins. Finally, the tridimensional structure of the biofilm is established, giving rise to a sophisticated ecosystem suitable for dispersal. Some biofilm-forming bacteria in food factory settings cause human diseases, which is very important in the food business. These pathogens may form biofilm structures on a variety of artificial substrates used in the food business, including stainless steel, polyethylene, wood, glass, and polypropylene.

Prior to selection, the in situ relevance of the biofilm model must also be reviewed. For example, before employing a specific model, it should be evaluated if the inoculum, flow regime (if any), and surfaces/interfaces fit the environment in the system being represented. To achieve in situ relevance, the essential drivers and factors in the system and environment must be recognized. Tools for evaluating how the model and results may be translated to practice should be established, and practitioners (including doctors and engineers) should be included in model review to guarantee in situ relevance. With an ever-increasing number of biofilm-related articles being published and a wide range of various methodologies becoming accessible, it can be difficult for researchers new to the subject (or to a specific subdiscipline within the field) to select the best appropriate model system. In addition, some instruction on data interpretation may be required (e.g.**,** how much biofilm reduction is needed in a particular model before considering it biologically meaningful). Although biofilms have an influence on many elements of life and biological sciences, they are considered a specific discipline. Prior to selection, the in situ relevance of the biofilm model must also be reviewed. For example, before employing a specific model, it should be evaluated if the inoculum, flow regime (if any), and surfaces/interfaces fit the environment in the system being represented. To achieve in situ relevance, the essential drivers and factors in the system and environment must be recognized. Tools for evaluating how the model and results may be translated to practice should be established, and practitioners (including doctors and engineers) should be included in model review to guarantee in situ relevance. With an ever-increasing number of biofilm-related articles being published and a wide range of various methodologies becoming accessible, it can be difficult for researchers new to the subject (or to a specific subdiscipline within the field) to select the best appropriate model system. In addition, some instruction on data interpretation may be required (e.g., how much biofilm reduction is needed in a particular model before considering it biologically meaningful). Although biofilms have an influence on many elements of life and biological sciences, they are considered a specific discipline.

#### *3.7. Comparative Table of Materials Used for Food Packaging Applications*

Table 9 is represent the difference process of producing food packaging with essential oil fillers as anti-bacterial and anti-oxidant chemicals that can extend the shelf life of components and protect product quality from free radicals.

**Table 9.** Comparative Table of Materials Used for Food Packaging Applications.


#### *3.8. Comparison between Produced Materials and Commercials Product*

The FTIR study of chitosan yielded the absorption regions of functional groups, as shown in Figure 8. The the red line shows that stretching OH group absorption emerged at a wave number of 3189.37 cm−<sup>1</sup> with a deacetylation temperature treatment of 100 ◦C for two hours, and the hydroxyl group (-OH) appeared at a wave number of 2980.20 cm−<sup>1</sup> . The N-H stretching group may be found at wave number 3004.61 cm−<sup>1</sup> . The vibration of the C-H range on aliphatic CH<sup>2</sup> is shown by the absorption at a wave number of 2779.37 cm−<sup>1</sup> . The appearance of bending vibration absorption CH<sup>2</sup> at a wave number of 1365.75 cm−<sup>1</sup> supports this. The absorption at 1567.09 cm−<sup>1</sup> indicates the C=O group (amide peak) that remains because the chitosan generated has not been entirely deacetylated. In the isolated chitosan IR spectra, N-H bending vibration of NH<sup>2</sup> is represented by the 1376 cm−<sup>1</sup> . The bending absorption of -CH<sup>3</sup> is modest and visible at the wave number 1389.45 cm−<sup>1</sup> . At the wave number 1306.09 cm−<sup>1</sup> , C-N stretching vibrations with low intensity were detected. The C-O bond range was discovered at wave numbers 1155.36 cm−<sup>1</sup> and 1114.86 cm−<sup>1</sup> .

Chitosan at a deacetylation temperature of 100 ◦C from commercial chitosan for two hours (black line) showed absorption of the stretching OH group at a wave number of 2992.11 cm−<sup>1</sup> and a hydroxyl group (-OH) at a wave number of 3180.91 cm−<sup>1</sup> . The N-H stretching group emerges at the wave number 3003.43 cm−<sup>1</sup> . Absorption at wavelength numbers 2945.87 cm−<sup>1</sup> and 2790. cm−<sup>1</sup> demonstrates the C-H stretching vibration on aliphatic CH2, which is reinforced by the presence of bending vibration absorption CH2 at wave number 1398.44 cm−<sup>1</sup> . The absorption at the wave number 1590.34 cm−<sup>1</sup> indicates the C=O group (amide peak) that remains because the chitosan generated has not been entirely deacetylated.

The area for pure substances is greater than for commercial products. The Figure 9 shows that the yield of the resultant extract increases, as well as the number of extraction steps, with the extraction stage, yielding more extract than the 1-stage extraction. Extraction with fewer solvents will be more effective than extraction with all the solvents at once.

This is due to the fact that at each stage, there will be contact with a new solvent, which provides a driving force in the form of variations in concentration and solubility at each stage, ensuring that a solute is always transferred from solid to solvent. The extract yield will eventually fall like Figure 10. This is due to the fact that in crosscurrent, multistage extraction, the solids employed at each stage are the same solids; therefore, the longer the extract is extracted, the more saturated it becomes until the extract gain no longer grows and decreases. *Polymers* **2021**, *13*, x FOR PEER REVIEW 22 of 26 at wave number 1398.44 cm**−**1. The absorption at the wave number 1590.34 cm**−**1 indicates the C=O group (amide peak) that remains because the chitosan generated has not been entirely deacetylated.

**Figure 8.** Comparison between extracted chitosan and commercial chitosan using FT-IR tools. **Figure 8.** Comparison between extracted chitosan and commercial chitosan using FT-IR tools.


**Figure 9.** Purity of extracted essential oil tested using gas chromatography-mass spectroscopy. **Figure 9.** Purity of extracted essential oil tested using gas chromatography-mass spectroscopy.

**Figure 10.** Purity of comerrcially essential oil tested using gas chromatography-mass spectroscopy.

Based on the research results on PLA as a biofilm, it was concluded that the maximum decomposition temperature obtained was at the temperature of 315.74 °C with variations of 4 g chitosan, 0.3 mL TEO, and 0.5 mL glycerol (biofilm 3). Molecules produce solid particles and are evenly distributed around the surface, affecting anti-microbes' properties. Based on the anti-microbial test, chitosan and TEO showed an increase in anti-bacterial activity. Moreover, the biofilms produced were able to fight *S. aureus* and *E. coli* bacteria in 9 days of exposure to open spaces. The concentration of chitosan affects the tensile strength of the PLA–chitosan–TEO sample: a higher

**4. Conclusions** 

**Figure 9.** Purity of extracted essential oil tested using gas chromatography-mass spectroscopy.

**Figure 10.** Purity of comerrcially essential oil tested using gas chromatography-mass spectroscopy. **Figure 10.** Purity of comerrcially essential oil tested using gas chromatography-mass spectroscopy.

#### **4. Conclusions 4. Conclusions**

Based on the research results on PLA as a biofilm, it was concluded that the maximum decomposition temperature obtained was at the temperature of 315.74 °C with variations of 4 g chitosan, 0.3 mL TEO, and 0.5 mL glycerol (biofilm 3). Molecules produce solid particles and are evenly distributed around the surface, affecting anti-microbes' properties. Based on the anti-microbial test, chitosan and TEO showed an increase in anti-bacterial activity. Moreover, the biofilms produced were able to fight *S. aureus* and *E. coli* bacteria in 9 days of exposure to open spaces. The concentration of chitosan affects the tensile strength of the PLA–chitosan–TEO sample: a higher Based on the research results on PLA as a biofilm, it was concluded that the maximum decomposition temperature obtained was at the temperature of 315.74 ◦C with variations of 4 g chitosan, 0.3 mL TEO, and 0.5 mL glycerol (biofilm 3). Molecules produce solid particles and are evenly distributed around the surface, affecting anti-microbes' properties. Based on the anti-microbial test, chitosan and TEO showed an increase in anti-bacterial activity. Moreover, the biofilms produced were able to fight *S. aureus* and *E. coli* bacteria in 9 days of exposure to open spaces. The concentration of chitosan affects the tensile strength of the PLA–chitosan–TEO sample: a higher concentration value of chitosan will make the bioscaffold material have an increased tensile strength value. The highest tensile strength value was in biofilm 3, with a composition of 4 g of chitosan concentration. However, if the addition of chitosan is continuous, it will increase the brittleness through higher water absorption so that further studies on the maximum amounts of chitosan as a filler in the manufacture of food packaging need to be considered. Biofilms in the food business are a severe economic and health concern. On the one hand, the presence of biofilms of food processing surfaces might result in financial losses due to corrosion on metal surfaces caused by certain bacteria, necessitating the replacement of these parts. Furthermore, certain bacterial species, such as Pseudomonas spp. and Bacillus spp., express a variety of proteolytic and lipolytic enzymes that can produce unpleasant smells and tastes (rancid, bitter). The impacted production batches must be removed and destroyed in these cases. On the other hand, and more importantly, biofilm development in food manufacturing is a critical public health issue. These biofilms may comprise bacterial (and occasionally fungal) species known to be harmful in healthy people, or they may solely target the immunocompromised (such as organ transplant recipients, oncology or HIV patients, etc.). These bacteria can cause food poisoning *(S. aureus)* as well as gastroenteritis *(E. coli)* and systemic illnesses in some situations *(E. coli* O157:H7).

**Author Contributions:** This study was carried out with the help of numerous persons who had the following responsibilities: Conceptualization, T.R.; methodology, J.P.S.; software, T.C.; validation, N.A.; formal analysis, M.H.M.H.; investigation, D.F.F.; resources, A.S.; data curation, A.E.H.; writing—original draft preparation, A.P.I. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by The Ministry of Education, Culture, Research, and Technology of the Republic Indonesia, grant number: 852/E4.1/AK.04.PT/PNL/2021.

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

**Informed Consent Statement:** Not appicable.

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

**Acknowledgments:** We would like to thank A.E.H., N.A., A.S., J.P.S., T.C., A.P.I., M.H.M.H. and D.F.F. for providing support of experimental instruments.

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

#### **References**

