2.2.6. Thermogravimetric Analysis (TGA)

Thermogravimetric analysis (TGA) was used to determine the decomposition temperature of the sample, which is c using a Perkin–Elmer instrument from Chemical Engineering's Laboratory at State Polytechnic of Lhokseumawe (Lhokseumawe, Indonesia) at a frequency of 1 Hz from −98 ◦C to 140 ◦C and a heating rate of 5 ◦C/min. Samples were then cut into small sizes. Finally, the position of the maximum tan value was used to determine the decomposition temperature of the sample.

### 2.2.7. Anti-Microbial Test

A method was developed for determination of the antibiotic susceptibility of anaerobic bacteria by use of a single-disc diffusion technique and incorporation of the inoculum in pour plates from Chemical Engineering's Laboratory at State Polytechnic of Lhokseumawe (Lhokseumawe, Indonesia). The method was standardized by the correlation of zone diameters with minimal inhibitory concentrations determined in the samples [21]. The antibacterial activity of biofilms was investigated using the agar media method in Petri dishes. Bacterial cultures grown in the mid-logarithmic phase were placed on agar media. *Escherichia coli* and *Staphylococcus aureus* were injected into agar media. After solidification of the agar coating, perfume solutions (15 mm in diameter) with different concentrations (5% and 10% by weight) were placed on the surface of the agar. The layers were incubated at 25 ◦C for 0 days to 9 days.

#### 2.2.8. Tensile Strength

The tensile strength of PLA–Chitosan–TEO was tested using a tensile strength tool according to the ASTM D 638–99 procedure from Chemical Engineering's Laboratory at State Polytechnic of Lhokseumawe (Lhokseumawe, Indonesia).

#### 2.2.9. Statistical Analysis Using Statistical Statistical Package for Social Science (SPSS)

To find out the results of the analysis of whether the characteristics of the biofilms have a significant effect or not, further analysis was carried out using SPSS (Statistical Package for Social Science) version 22.0 using the one-way ANOVA method. If the data are normally distributed and homogeneous, a one-way ANOVA test was carried out with a 95% confidence level.

All data regarding the mechanical test of the sample are listed in Tables 1–4. Table 1 presents tensile strength data for all samples using ANOVA method, Table 2 presents tensile strength data for all samples using ANOVA method for difference samples), Table 3 presents data % elongation for all samples using ANOVA method and Table 4 presents the data for % elongation for all samples using ANOVA method for difference samples.


**Table 1.** Tensile strength for all samples using ANOVA method.


**Table 2.** Tensile strength for all samples using ANOVA method for difference samples.

**Table 3.** % elongation for all samples using ANOVA method.


**Table 4.** % elongation for all samples using ANOVA method for difference samples.


Scheme 1 depicts the procedures required to produce chitin powder from shrimp shell waste using a variety of ways of methods.

Scheme 2 describes the steps that need to be taken to produce chitosan, the process of converting the acetyl group (NHCOCH3) in chitin to an amine group (NH2) in chitosan with the addition of NaOH.

Scheme 3 is a soxhletation method carried out by installing a soxhletation device tools. A number of raw materials are put into the cladding and 96% ethanol is added as a solvent.

Scheme 4 shows the manufacture of the biofilms is done by applying the principle of solution thermodynamics where the initial state of the solution is stable and then undergoes instability (addition of filler) in the phase change process (demixing), solidification (solidification) and phase transition so that at the stage it becomes solid after the addition of a high concentration polymer.

**Scheme 1.** Chitin production scheme [22]. **Scheme 1.** Chitin production scheme [22].

with the addition of NaOH.

Scheme 2 describes the steps that need to be taken to produce chitosan, the process of converting the acetyl group (NHCOCH3) in chitin to an amine group (NH2) in chitosan

*Polymers* **2021**, *13*, x FOR PEER REVIEW 8 of 26

**Scheme 2.** Chitosan production [23]. **Scheme 2.** Chitosan production [23].

**Scheme 3.** Turmeric essential oil extraction [24]. **Scheme 3.** Turmeric essential oil extraction [24].

after the addition of a high concentration polymer.

Scheme 4 shows the manufacture of the biofilms is done by applying the principle of solution thermodynamics where the initial state of the solution is stable and then undergoes instability (addition of filler) in the phase change process (demixing), solidification (solidification) and phase transition so that at the stage it becomes solid

**Scheme 4.** Biofilm manufacture [25]. **Scheme 4.** Biofilm manufacture [25].

### **3. Results and Discussion**

**3. Results and Discussion**  *3.1. Scanning Electron Microscopic Analysis (SEM)*

*3.1. Scanning Electron Microscopic Analysis (SEM)*  The scanning process of electron microscopy is based on the principle that an electron pistol produces an anode electron beam. In the direction of the sample, the magnet lens focuses on electrons. The whole sample is scanned by focused electron shafts through the scanner coil. The sample emits new electrons that are then received and sent The scanning process of electron microscopy is based on the principle that an electron pistol produces an anode electron beam. In the direction of the sample, the magnet lens focuses on electrons. The whole sample is scanned by focused electron shafts through the scanner coil. The sample emits new electrons that are then received and sent back to the monitor where the particle is sampled.

back to the monitor where the particle is sampled. Based on Figure 1, there were no chitosan particles in the analyses of Biofilm 1. The morphology of biofilms on the surface had space as no addition of glycerol as a binder had been found. Chitosan spreads evenly with Biofilm 2 particles and has a tight structure by adding TEO and glycerol. The difference between images (a) and (b) is shown in the image taken at 50× magnification. From the picture, it can be seen that the surface of the sample is hollow and cracked. The (a) sample surface is quite dominant with white spots and cracks, and in Figure 1b, the white spots are more clearly visible than in the previous image. These white spots are chitosan that has not been spread evenly and the mixing has not been uniform. A larger hollow in biofilm 3's surface appears. This proves that the mixture of materials in this sample has a fairly good level of Based on Figure 1, there were no chitosan particles in the analyses of Biofilm 1. The morphology of biofilms on the surface had space as no addition of glycerol as a binder had been found. Chitosan spreads evenly with Biofilm 2 particles and has a tight structure by adding TEO and glycerol. The difference between images (a) and (b) is shown in the image taken at 50× magnification. From the picture, it can be seen that the surface of the sample is hollow and cracked. The (a) sample surface is quite dominant with white spots and cracks, and in Figure 1b, the white spots are more clearly visible than in the previous image. These white spots are chitosan that has not been spread evenly and the mixing has not been uniform. A larger hollow in biofilm 3's surface appears. This proves that the mixture of materials in this sample has a fairly good level of homogeneity. In the morphology of this second sample, there are also just small numbers of white spots, which are chitosan and

TEO that have been evenly distributed [26,27]. Biofilm 3's particles are evenly distributed on the surface but have a smaller film layer because the glycerol is lower than in biofilm 2. According to [28], insufficient compositions in chitosan could spread evenly on the PLA polymer matrix with plasticizers and oregano oil. They were able to form homogeneous bonds that affected the properties. According to the findings of this study, TEO has been shown to bind biopolymer elements in chitosan into good bonds and produce complex biofilm surfaces when added to biofilms. The interaction of the two biopolymer elements demonstrates an attraction capable of replacing inorganic filmmakers [29,30]. The pore structure is a site where cell growth or proliferation occurs. Among the three sample outcomes, the third sample (biofilm 3) with a total composition variation of 4 g chitosan, 0.3 mL TEO, and 0.5 mL glycerol is the best sample based on morphology with the greatest structure and pores with greater porosity. *Polymers* **2021**, *13*, x FOR PEER REVIEW 11 of 26

#### *3.2. Fourier Infrared Spectroscopy Analysis (FTIR)* homogeneity. In the morphology of this second sample, there are also just small numbers of white spots, which are chitosan and TEO that have been evenly distributed [26,27].

The results of the analysis of the characteristics of turmeric essential oil from the extraction method by spectroscopy to obtain the value of the absorption results are used in Table 5. Qualitative analysis of different organic compounds can be confirmed from the characteristics of the vibration band appearing in the infrared spectrum region at a certain frequency influenced by certain functional groups. The transmittance percentage corresponding to the wave number is summed up in the total attenuated in the reflected IR spectrum, as shown in the figure above. There is an intense broad peak in the range of 2500–3200 cm−<sup>1</sup> , specifically at 2414.88 cm−<sup>1</sup> , corresponding to the polymer hydroxyl (OH) group. Another intense and branching peak in the range of 2256.71–2086.96 cm−<sup>1</sup> corresponds to methyl OH stretching and C-H bending, i.e., most of the aromatic compound and alcohol components. Other studies have shown that there is a functional group of turmeric essential oil that will be produced will depend on the species of curcuma used [31]. Biofilm 3's particles are evenly distributed on the surface but have a smaller film layer because the glycerol is lower than in biofilm 2. According to [28], insufficient compositions in chitosan could spread evenly on the PLA polymer matrix with plasticizers and oregano oil. They were able to form homogeneous bonds that affected the properties. According to the findings of this study, TEO has been shown to bind biopolymer elements in chitosan into good bonds and produce complex biofilm surfaces when added to biofilms. The interaction of the two biopolymer elements demonstrates an attraction capable of replacing inorganic filmmakers [29,30]. The pore structure is a site where cell growth or proliferation occurs. Among the three sample outcomes, the third sample (biofilm 3) with a total composition variation of 4 g chitosan, 0.3 mL TEO, and 0.5 mL glycerol is the best sample based on morphology with the greatest structure and pores with greater porosity.

(**a**) **Figure 1.** *Cont*.

**Figure 1.** Analysis result on the PLA variations of (**a**) biofilm 1, (**b**) 3 biofilm 2, and (**c**) biofilm 3 to observe the blendable properties between matrix (PLA) and filler (chitosan–TEO)*.*  **Figure 1.** Analysis result on the PLA variations of (**a**) biofilm 1, (**b**) 3 biofilm 2, and (**c**) biofilm 3 to observe the blendable properties between matrix (PLA) and filler (chitosan–TEO).

*3.2. Fourier Infrared Spectroscopy Analysis (FTIR)*  The results of the analysis of the characteristics of turmeric essential oil from the extraction method by spectroscopy to obtain the value of the absorption results are used in Table 5. Qualitative analysis of different organic compounds can be confirmed from the characteristics of the vibration band appearing in the infrared spectrum region at a certain frequency influenced by certain functional groups. The transmittance percentage corresponding to the wave number is summed up in the total attenuated in the reflected IR spectrum, as shown in the figure above. There is an intense broad peak in the range of The figure below shows a graph of the analysis of the PLA functional group. The graph shows the results of the FTIR test that from the optimum sample tensile strength in PLA, there exists N-H, C-H, O-H and C=O mentioned in Table 6. Where the NH group is present at the wave number 3336.85 cm−<sup>1</sup> with a wavelength range of 3300–3400 cm−<sup>1</sup> , the OH group is found at the number 3630.03 cm−<sup>1</sup> in the wave range of 3584–3700 cm−<sup>1</sup> and the CH group at the number 3630.03 cm−<sup>1</sup> in the wave range 3584–3700 cm−<sup>1</sup> . The C=O group is discovered at 1793.80 cm−<sup>1</sup> with a wave range of 1540–1870 cm−<sup>1</sup> , while the following group is located at 3294.42 cm−<sup>1</sup> with a wave range of 3267–3333 cm−<sup>1</sup> . This indicates that

2500–3200 cm−1, specifically at 2414.88 cm−1, corresponding to the polymer hydroxyl

no new functional groupings, but only PLA pure characteristics, have been discovered in line with research by [32].


**Table 5.** Wave number for functional groups of turmeric essential oil.

**Table 6.** Wave number for functional groups of pure PLA.


Turmeric essential oil interacts with the polymer matrix by forming intermolecular hydrogen bonds between their terminal hydroxyl group and the carbonyl groups of the ester moieties of both PLA and chitosan as mentioned in Table 7., in line with research [33] C-O stretching of alcohols and carboxylic acids, and N-H wagging of primary and secondary amines. These functional groups were predicted because chitosan is made up mostly of them. As evidenced by the appearance of a broad band at 3503 cm−<sup>1</sup> corresponding to phenolic OH stretching vibrations and an increased intensity and shift of bands attributed to C–O bending vibrations as show in Figure 2. The bands ascribed to isoprenoids' out-of-plane C-H wagging vibrations emerged at 2918 cm−<sup>1</sup> and 2922 cm−<sup>1</sup> when turmeric oil was added and mixed at a high speed to bind the matrix and filler.

**Table 7.** Wave number for each functional group of chitosan.


#### *3.3. Thermogravimetric Analysis (TGA)*

We examined TGA as a tool to evaluate differences in mass loss patterns in order to quantify differences in biofilm development. To the best of our knowledge, this is the first time TGA has been used to characterize a biofilm in Indonesia. Biofilm thickness is generally measured using confocal microscopy, which is a very efficient approach but necessitates expensive equipment and extensive training to produce high-quality pictures. TGA is frequently used for material characterisation since it needs little training and sample preparation and produces findings quickly. This is the temperature range in which bacterial organic matter is most likely to breakdown and burn; therefore, the difference is ascribed to a bacterial biofilm [34]. The TGA results are consistent with our findings. An active biofilm in direct contact with the carbon foam surface should generate a current by creating a potential difference between the anode and the cathode, as seen.

The researchers discovered a mass drop in humidity from 50 ◦C to 150 ◦C and a mass decrease from 250 ◦C to 400 ◦C owing to thermal deterioration of the two components. This is the temperature range in which bacterial organic matter is most likely to break down and burn; therefore, the difference is ascribed to a bacterial biofilm. As a result, the temperature of the biofilm breakdown was about 285 ◦C. Figure 3 depicts biofilms

degraded at 285.55 ◦C with a mass removal of 1.313 mg at 2 g chitosan, 0 mL TEO, and 0 mL glycerol. Biofilm 2, on the other hand, disintegrated at 274.02 ◦C with a mass removal of 1.54 mg. Furthermore, at 315.74 ◦C, samples with a weight removal of 1.74 mg were degraded using biofilm 3. *Polymers* **2021**, *13*, x FOR PEER REVIEW 14 of 26

**Figure 2.** Spectrum of FT-IR for each sample*.*  **Figure 2.** Spectrum of FT-IR for each sample.

degraded using biofilm 3. **Figure 3.** TGA analysis on biofilms to investigate sample resistance at a high temperature range before being applied as a food packaging*.*  **Figure 3.** TGA analysis on biofilms to investigate sample resistance at a high temperature range before being applied as a food packaging.

This research is in line with [35], in which weight loss and DTG (a derivative of TGA curves) of chitosan and Mandarin Essensial Oil–chitosan nanoparticles are presented.

The comparison between samples (a) and (b) based on Figure 4 shows that the physical change in the biofilm tested may have been the most degraded in the biofilm. The initial golden color became black after being burned. The sample's weight was also lost throughout the breakdown process. Each sample was examined, and it is known that the sample was broken down, owing to the combustion process in the TGA test equipment, resulting in the loss of carbon, water, or volatile components throughout the analysis method. Other combinations, such as chitosan, TEO, and glycerol, also had an effect

to 400 °C with pink line for chitosan dehydration and breakdown. Material structural modifications, such as the addition of Mandarin Essensial Oil, Tween20, and chitosan cross-linked with sodium tripolyphosphate show by black line, resulted in a new stage blue line (150–300 °C) in the chitosan nanoparticles and Mandarin Essensial Oil–chitosan

nanoparticles.

on the sample's heat resistance.

This research is in line with [35], in which weight loss and DTG (a derivative of TGA curves) of chitosan and Mandarin Essensial Oil–chitosan nanoparticles are presented. Chitosan went through two distinct stages. The first stage with green line (50–150 ◦C) was associated with free water loss. The second step used temperatures ranging from 250 ◦C to 400 ◦C with pink line for chitosan dehydration and breakdown. Material structural modifications, such as the addition of Mandarin Essensial Oil, Tween20, and chitosan cross-linked with sodium tripolyphosphate show by black line, resulted in a new stage blue line (150–300 ◦C) in the chitosan nanoparticles and Mandarin Essensial Oil–chitosan nanoparticles.

The comparison between samples (a) and (b) based on Figure 4 shows that the physical change in the biofilm tested may have been the most degraded in the biofilm. The initial golden color became black after being burned. The sample's weight was also lost throughout the breakdown process. Each sample was examined, and it is known that the sample was broken down, owing to the combustion process in the TGA test equipment, resulting in the loss of carbon, water, or volatile components throughout the analysis method. Other combinations, such as chitosan, TEO, and glycerol, also had an effect on the sample's heat resistance. *Polymers* **2021**, *13*, x FOR PEER REVIEW 16 of 26

**Figure 4.** The result of biofilm samples (**a**) before the bioplastic thermal resistance test, (**b**) after the bioplastic thermal resistance test. **Figure 4.** The result of biofilm samples (**a**) before the bioplastic thermal resistance test, (**b**) after the bioplastic thermal resistance test.

#### *3.4. Anti-Microbial Test 3.4. Anti-Microbial Test*

TEO additives to chitosan biofilms have been studied to enhance the temperature of decomposition as one of the criteria for biological degradation. Furthermore, because of its antiseptic properties for food, biofilms with antimicrobial compounds have great food preservation abilities. As a result, in addition to preserving food, it also ensures the safety, freshness, and longer shelf life of these goods. This is due to the presence of antimicrobial action in natural substances and extracts utilized as additions in chitosan products. As a result, the usage of chitosan as a packaging material has several applications and is aimed at food safety. TEO additives to chitosan biofilms have been studied to enhance the temperature of decomposition as one of the criteria for biological degradation. Furthermore, because of its antiseptic properties for food, biofilms with antimicrobial compounds have great food preservation abilities. As a result, in addition to preserving food, it also ensures the safety, freshness, and longer shelf life of these goods. This is due to the presence of antimicrobial action in natural substances and extracts utilized as additions in chitosan products. As a result, the usage of chitosan as a packaging material has several applications and is aimed at food safety.

It is critical to determine the antimicrobial activity, composition, structure, and functional groups of such extracts. Clove oil, thyme, cinnamon, rose salad, sage, and vanillin are the most active substances in the battle against germs. Several investigations It is critical to determine the antimicrobial activity, composition, structure, and functional groups of such extracts. Clove oil, thyme, cinnamon, rose salad, sage, and vanillin are the most active substances in the battle against germs. Several investigations have

have shown that they have antimycotic, non-toxic, and anti-parasitic effects. They can

Figure 5 presents microbial development through the 12-day testing process that occurred in B1 (biofilm 1), B2 (biofilm 2), and B3 (biofilm 3). The results showed that the lowest colony growth rate of 61 colonies/g *S. aureus* was found in B1 with the composition of 2 g of chitosan, 0 mL of TEO, and 0 mL of glycerol, followed by B2 with 3 g of chitosan, 0.3 mL of TEO, and 0.5 mL of the glycerol growth rate of 77 colonies/g *E. coli*. B3 biofilm showed the best results, wherein 4 g of chitosan, 0.3 mL of TEO and 0.5 mL of glycerol have been mixed. The test results showed a microbial reduction with the addition of chitosan compounds, and the addition of essential oil from turmeric increased the anti-bacterial activity. *S. aureus* is a Gram-positive bacteria that can attach to glass, metal, and plastic as abiotic surfaces and host tissues as biotic surfaces. The attachment of *S. aureus* to surfaces depends on the surface components of the bacterial microbes recognizing the adhesive matrix molecules for host proteins. To prevent the attachment of *S. aureus* to a surface through the matrix, the surface must be coated with shown that they have antimycotic, non-toxic, and anti-parasitic effects. They can also be linked to the function of those chemicals in plants, as well as the antibacterial capabilities of essential oils and their constituents [16,36].

Figure 5 presents microbial development through the 12-day testing process that occurred in B1 (biofilm 1), B2 (biofilm 2), and B3 (biofilm 3). The results showed that the lowest colony growth rate of 61 colonies/g *S. aureus* was found in B1 with the composition of 2 g of chitosan, 0 mL of TEO, and 0 mL of glycerol, followed by B2 with 3 g of chitosan, 0.3 mL of TEO, and 0.5 mL of the glycerol growth rate of 77 colonies/g *E. coli*. B3 biofilm showed the best results, wherein 4 g of chitosan, 0.3 mL of TEO and 0.5 mL of glycerol have been mixed. The test results showed a microbial reduction with the addition of chitosan compounds, and the addition of essential oil from turmeric increased the antibacterial activity. *S. aureus* is a Gram-positive bacteria that can attach to glass, metal, and plastic as abiotic surfaces and host tissues as biotic surfaces. The attachment of *S. aureus* to surfaces depends on the surface components of the bacterial microbes recognizing the adhesive matrix molecules for host proteins. To prevent the attachment of *S. aureus* to a surface through the matrix, the surface must be coated with anti-adhesion agents such as arylrhodamins, calcium chelators, silver nanoparticles, and chitosan [37–39]. *Polymers* **2021**, *13*, x FOR PEER REVIEW 17 of 26 anti-adhesion agents such as arylrhodamins, calcium chelators, silver nanoparticles, and chitosan [37–39].

**Figure 5.** Anti-Microbial test result on PLA the variated of B1 (Biofilm 1), B2 (Biofilm 2) and (Biofilm 3) B3*.* **Figure 5.** Anti-Microbial test result on PLA the variated of B1 (Biofilm 1), B2 (Biofilm 2) and (Biofilm 3) B3.

Bacteria adhere to surfaces in the form of a biofilm; therefore, one of the procedures that can prevent *S. aureus* pathogenesis is to prevent it from adhering to biotic and abiotic surfaces. Antibiofilm vaccinations are increasingly being used to inhibit *S. aureus* biofilm development [40]. Extracellular polysaccharides or cell wall-associated proteins identified in the biofilm matrix have the ability to stimulate an immune response that protects against *S. aureus* infection. Resistance to *S. aureus* can be acquired by active or passive vaccination with surface polysaccharides [41]. In contrast to *S. aureus*, *E. coli* bacteria live in the digestive system as part of the microbiota and may be a dangerous adversary. The presence of this bacterium can cause intestinal and extraintestinal infections in people and animals [42]. Based on the information gathered, *E. coli* has been added to the list of microorganisms of worldwide concern that cause the most prevalent illnesses in a variety of contexts, including communities, hospitals, and foodborne diseases [43]. These studies stated that combining chitosan and oregano oil as an essential oil could decrease water vapor permeability, puncture, and tensile strength. Still, they increased anti-bacterial and microbial properties due to the curcumin content and its essential oils, which inhibited the growth of causative bacteria, such as Bacillus sp, Bacteria adhere to surfaces in the form of a biofilm; therefore, one of the procedures that can prevent *S. aureus* pathogenesis is to prevent it from adhering to biotic and abiotic surfaces. Antibiofilm vaccinations are increasingly being used to inhibit *S. aureus* biofilm development [40]. Extracellular polysaccharides or cell wall-associated proteins identified in the biofilm matrix have the ability to stimulate an immune response that protects against *S. aureus* infection. Resistance to *S. aureus* can be acquired by active or passive vaccination with surface polysaccharides [41]. In contrast to *S. aureus*, *E. coli* bacteria live in the digestive system as part of the microbiota and may be a dangerous adversary. The presence of this bacterium can cause intestinal and extraintestinal infections in people and animals [42]. Based on the information gathered, *E. coli* has been added to the list of microorganisms of worldwide concern that cause the most prevalent illnesses in a variety of contexts, including communities, hospitals, and foodborne diseases [43]. These studies stated that combining chitosan and oregano oil as an essential oil could decrease water vapor permeability, puncture, and tensile strength. Still, they increased anti-bacterial and microbial properties due to the curcumin content and its essential oils, which inhibited the growth of causative bacteria, such as Bacillus sp, Shigella dysmetria, *S. aureus*, and

Shigella dysmetria, *S. aureus*, and *E. coli* [44,45]**.** Compared to the previously explained study, it can be concluded that the addition of TEO had shown an increased anti-bacterial

composite material was directly contaminated with the air, containing various microbe

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

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

*Food Packaging* 

types that can affect both physical and chemical ingredients.

*E. coli* [44,45]. Compared to the previously explained study, it can be concluded that the addition of TEO had shown an increased anti-bacterial activity, which was to inhibit microbial growth in the sample. That was because the composite material was directly contaminated with the air, containing various microbe types that can affect both physical and chemical ingredients.
