2.6.1. In Vivo Wound Healing Experiments

Animals

In this study, a total number of 20 six-week-old female Sprague–Dawley rats with a range of body weight from 200–250 g were used. They were randomly divided into three experimental groups of five rats each. The animals were acclimatized to the laboratory conditions for one week before the onset of the experiment. All rats were individually caged with a 12-h light/dark cycle, given adequate commercial pellets and water ad libitum throughout the study. All animal experiments were carried out under protocols approved by the Animal Ethics Committee (AEC)—UMT/JKEPHMK/2020/48, Universiti Malaysia Terengganu.

### Establishment of Wound Skin

Rats were anaesthetized using an intraperitoneal (i.p.) injection of ketamine (90 mg/kg) and xylazine (10 mg/kg). The dorsal skin was prepared by removing the hair with a razor blade and the surgical area was disinfected with 70% ethanol. Since the shaving procedure produced marked edema of the skin, the prepared rats were left for twenty-four hours before the wound was inflicted. After the rats were anesthetized with a combination of ketamine and xylazine via i.p., a full-thickness wound was created by using an 8-mm sterile skin biopsy punch. Each rat received two full-thickness wounds at their back dorsal.

#### Treatment of Hydrogel

All wounds in the treatment groups were dressed with GG and GVCO80 hydrogels, and followed by Opsite post-op waterproof film dressing (Smith and Nephew, Hull, England) as the secondary dressing. The dressings were then held in place with gauze to give mechanical protection to the dressing. The changing of dressing was done every 3 days to minimize the infection to the wound site. The Opsite film dressing acted as a positive control for comparison to the other treatments. The treated wound with GG dressing was considered a negative control. The rat's wound was photographed using a 13.1-megapixel Sony camera for evaluation of wound closure with the actual measurement. Macroscopic Observation of Wound

The wound measurement of the size taken at the time of biopsy was used to calculate the percent of wound contraction using equation:

$$\% \text{ wound contraction} = \frac{\text{W}\_0 - \text{W}\_t}{\text{W}\_0} \times 100 \tag{3}$$

where W<sup>0</sup> is the original wound area and W<sup>t</sup> is the wound area on the selected day after the biopsy. The measurements of wound size were taken on days 2, 4, 7, 11, and 14 consecutively throughout the study. The wound area was measured by placing the 1 mm<sup>2</sup> graph over the wound pictures. The squares were counted, and the area was recorded. The wound area was accessed by the same blinded observer.

#### 2.6.2. Ultrasound Imaging

The wound area also was analyzed by using real-time high-resolution 20 MHz ultrasound imaging equipment (Dermalab Combo, Cortex, Denmark) skin analyzer to produce images representing the cross-section of the wound skin. A standard echographic gel was applied and used as a medium between the probe and wound skin surface. The images produced were recorded.

#### 2.6.3. Histological Examination

Rats were euthanized on day 14 and skin samples that contained the wound area were taken for histological study. The skin samples were fixed with 10% buffered formalin for 24 h. The samples were embedded in paraffin and cut into 6 mm-thick sections for the middle part of the wounds. The sections were subsequently stained with hematoxylin and eosin (H & E) staining procedure. The H & E slides were visualized using a light microscope at 20x magnification.

#### Statistical Analysis

All data are presented as the mean ± standard deviation (SD). The data were processed by two-way ANOVA using statistical software analysis SPSS (version 20). The *p*-value < 0.05 was considered statistically significant.

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

#### *3.1. Formulation of Stabilizing VCO Microemulsion*

In general, water and oil were separated due to their high interfacial tension. Water is immiscible with virgin coconut oil (VCO) due to its high interfacial tension, which is typical for oils with water. The interfacial tension was reduced by introducing surfactant, which allowed the microemulsion polymerization process to take place. Nonionic surfactants, Triton X-100 and Tween 80, were used in preparing a stabilized VCO microemulsion through a ternary phase diagram. The optimum ratio from the ternary phase diagram was chosen in preparing the gellan gum–virgin coconut oil hydrogels (GVCO). The microemulsion has a characteristic of a clear solution, stable at room temperature and forming a one layer solution. The pseudo ternary phase diagrams consisting of virgin coconut oil microemulsion, distilled water, and surfactant with different hydrophilic–lipophilic balance (HLB) values were constructed using the surfactant titration method. Nine ratios of VCO: Water were constructed, i.e., 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, and 10:90 (*w*/*w*). Two types of nonionic surfactants were used, which were Tween 80 and Triton X-100 (Table 1). The mixture was observed using visual inspection after each addition of surfactant to the VCO and water. The samples were identified as microemulsions when they appeared transparent, and the liquid easily flowed. The results were plotted on a triangular graph as a ternary phase diagram, as shown in Figure 1.

on a triangular graph as a ternary phase diagram, as shown in Figure 1.

Triton X-100 (Table 1). The mixture was observed using visual inspection after each addition of surfactant to the VCO and water. The samples were identified as microemulsions when they appeared transparent, and the liquid easily flowed. The results were plotted

**Figure 1.** The ternary phase diagram of VCO microemulsion by using Tween 80 and TritonX-100 surfactants. **Figure 1.** The ternary phase diagram of VCO microemulsion by using Tween 80 and TritonX-100 surfactants.

The ternary phase diagram shows that different amounts of VCO, water, and Tween 80 can produce a stable oil-in-water microemulsion. The ratio of VCO: Water required a different amount of Tween 80 in the range from 55.59% to 69.50% to achieve a stable phase of microemulsions (Table 1). The ratios of 80:20 needed 55.59% of Tween 80 compared to the ratio of 70:30 and 60:40, which only needed 65.73% and 64.37%, respectively (Table 1). The volume of Triton X-100 required to produce a stable formulation in VCO: Water was higher than in Tween 80. The Triton X-100 amount ranged from 78.09% to 86.07% to produce stable microemulsions (Table 1). The amount of Triton X-100 was 83.24%, 83.63%, and 79.62% for ratios 80:20, 70:30, and 60:40, respectively. The formulation of VCO:Water: Tween 80 microemulsion was adopted due to the large area of microemulsion stability in the ternary phase diagram and lower amount of surfactant needed to form a stable mixture, compared to Triton X-100. Moreover, Tweens were reported to have been used widely in food, cosmetics, and pharmaceutical application due to minimal toxicity and low cost [23,24]. The ternary phase diagram shows that different amounts of VCO, water, and Tween 80 can produce a stable oil-in-water microemulsion. The ratio of VCO: Water required a different amount of Tween 80 in the range from 55.59% to 69.50% to achieve a stable phase of microemulsions (Table 1). The ratios of 80:20 needed 55.59% of Tween 80 compared to the ratio of 70:30 and 60:40, which only needed 65.73% and 64.37%, respectively (Table 1). The volume of Triton X-100 required to produce a stable formulation in VCO: Water was higher than in Tween 80. The Triton X-100 amount ranged from 78.09% to 86.07% to produce stable microemulsions (Table 1). The amount of Triton X-100 was 83.24%, 83.63%, and 79.62% for ratios 80:20, 70:30, and 60:40, respectively. The formulation of VCO:Water: Tween 80 microemulsion was adopted due to the large area of microemulsion stability in the ternary phase diagram and lower amount of surfactant needed to form a stable mixture, compared to Triton X-100. Moreover, Tweens were reported to have been used widely in food, cosmetics, and pharmaceutical application due to minimal toxicity and low cost [23,24].

#### *3.2. Physical Properties of the Hydrogels. 3.2. Physical Properties of the Hydrogels*

process could be monitored with ease.

The addition of VCO microemulsion to the gellan gum does not change the physical appearance of transparent free-standing GG hydrogel (Figure 2). The GVCO hydrogels at every concentration were transparent but becoming cloudy due to the increased amount of VCO microemulsion and supported by the transmittance result (Figure 2e). The transmittance for pure GG hydrogels was ≈ 99% compared to GVCO60 ≈ 85%, GVCO70 ≈ 70%, and GVCO80 ≈ 65% at λ = 700 nm. The transparent behavior of the GVCO hydrogel gives an advantage for the materials to be used as wound dressing material, and the healing The addition of VCO microemulsion to the gellan gum does not change the physical appearance of transparent free-standing GG hydrogel (Figure 2). The GVCO hydrogels at every concentration were transparent but becoming cloudy due to the increased amount of VCO microemulsion and supported by the transmittance result (Figure 2e). The transmittance for pure GG hydrogels was ≈ 99% compared to GVCO60 ≈ 85%, GVCO70 ≈ 70%, and GVCO80 ≈ 65% at λ = 700 nm. The transparent behavior of the GVCO hydrogel gives an advantage for the materials to be used as wound dressing material, and the healing process could be monitored with ease.

#### *3.3. Fourier Transform Infrared Spectroscopy (ATR–FTIR)*

ATR–FTIR spectra of the GVCO hydrogels confirm the presence of characteristic peaks of GG and VCO (Figure 3). The peaks at 2941 and 2895 cm−<sup>1</sup> are related to the saturated alkyl and the carbonyl groups of the fatty acids in VCO, respectively [25]. Peaks appearing at 1758, 1470, and 1419 cm−<sup>1</sup> were from the stretching of carboxylic (C=O), bending of

methylene (CH2), and bending of methyl (CH3) of VCO. The 1182 and 1142 cm−<sup>1</sup> peaks resulted from the stretching of ester C-O [26]. *Polymers* **2021**, *13*, x FOR PEER REVIEW 8 of 19

**Figure 2.** The physical appearance of (**a**) GG hydrogel, (**b**) GVCO60 (**c**) GVCO70, (**d**) GVCO80 hydrogels, and (**e**) transmittance of GG and GG–VCO hydrogels. **Figure 2.** The physical appearance of (**a**) GG hydrogel, (**b**) GVCO60 (**c**) GVCO70, (**d**) GVCO80 hydrogels, and (**e**) transmittance of GG and GG–VCO hydrogels.

**Figure 3.** ATR spectra of pure GG hydrogels, VCO, and GVCO hydrogels at different concentra-GVCO80 11 ± 0.3 9.3 ± 0.4 259 ± 7 6 ± 1.5 332 ± 11 **Figure 3.** ATR spectra of pure GG hydrogels, VCO, and GVCO hydrogels at different concentrations.

on the stress-at-break (σ), strain-at-break (γ), and Young's modulus (YM). Free-standing gellan gum (GG) hydrogel was brittle and almost impossible for use in biomedical applications such as a wound dressing material. To overcome this problem, VCO microemulsions were incorporated into GG as a plasticizer and to improve the flexibility of the materials. The incorporation of VCO microemulsion into GG caused the hydrogels to become durable, flexible, and easy to handle due to an increase in stress-at-break (σ) and Young's modulus (YM), but decreased slightly for strain-at-break (γ) values (Figure 4a). The σ and YM values of GG hydrogel were 4 ± 0.2 and 85 ± 7 kPa, respectively, and the addition of VCO microemulsion (GVCO80) increased both values to 11 ± 0.3 and 259 ± 7 kPa, respectively (Table 2). Although the γ value of the GVCO60 decreased to 7.9 ± 0.2% from 10.7 ± 0.4% (GG hydrogel), the addition of higher content of VCO microemulsion resulted in the γ value increasing to 9.3 ± 0.4% compared to GVCO60 hydrogel films. Thus, the plasticizer

**Table 2.** Summary of the stress-at-break (σ), strain-at-break (γ), Young's modulus (YM), swelling degree, and water vapor transmission rate (WVTR) of gellan gum (GG) and GVCO hydrogels at

Control - - - - 1547 ± 32 GG 4 ± 0.2 10.7 ± 0.4 85 ± 7 3 ± 0.6 964 ± 47 GVCO60 6 ± 0.1 7.9 ± 0.2 134 ± 6 12 ± 4.0 391 ± 13 GVCO70 7 ± 0.4 8.5 ± 0.2 175 ± 8 9 ± 1.5 344 ± 23

**Modulus (YM) (kPa)**

**Swelling Degree (%)**

**WVTR (g m−2 d−1**

**)**

effect of the VCO microemulsion in GCVO hydrogel films was proven.

different ratios. Control for WVTR is a test without hydrogel film.

**Strain (γ) (%)**

**Stress (σ) (kPa)**

tions.

*3.4. Mechanical Performances*

GG has a broad band at 3476 cm−<sup>1</sup> , which is a typical region for stretching of the O-H group (3000–3500 cm−<sup>1</sup> ) [27]. A new distinguishing peak observed at 1638–1675 cm−<sup>1</sup> in GVCO hydrogels correlates to the shifting of -C-H (-CH2) bending scissoring of VCO and C=C due to the appearance of phenolic content and stretching of that aromatic ring [28]. The stretching alkyl signal in VCO microemulsion at 2943 cm−<sup>1</sup> shifted to 2930–2920 cm−<sup>1</sup> in GVCO hydrogels confirming the hydrogen bonding interaction in GVCO blends [28].

A significant change of enlargement peak of GVCO hydrogel can also be observed around 1149–1109 cm−<sup>1</sup> , due to an increased stretching vibration of esters group of gellan gum and VCO. The ATR–FTIR spectra of GVCO show all prominent peaks of VCO with significant changes in variation and shifting of transmittance intensities, indicating that GG and VCO microemulsion interact to produce a stable network hydrogel.

#### *3.4. Mechanical Performances*

Mechanical performance is crucial to evaluate and understand the strength of the materials. Table 2 shows the tensile properties of GVCO hydrogels at the different ratios on the stress-at-break (σ), strain-at-break (γ), and Young's modulus (YM). Free-standing gellan gum (GG) hydrogel was brittle and almost impossible for use in biomedical applications such as a wound dressing material. To overcome this problem, VCO microemulsions were incorporated into GG as a plasticizer and to improve the flexibility of the materials. The incorporation of VCO microemulsion into GG caused the hydrogels to become durable, flexible, and easy to handle due to an increase in stress-at-break (σ) and Young's modulus (YM), but decreased slightly for strain-at-break (γ) values (Figure 4a). The σ and YM values of GG hydrogel were 4 ± 0.2 and 85 ± 7 kPa, respectively, and the addition of VCO microemulsion (GVCO80) increased both values to 11 ± 0.3 and 259 ± 7 kPa, respectively (Table 2). Although the γ value of the GVCO60 decreased to 7.9 ± 0.2% from 10.7 ± 0.4% (GG hydrogel), the addition of higher content of VCO microemulsion resulted in the γ value increasing to 9.3 ± 0.4% compared to GVCO60 hydrogel films. Thus, the plasticizer effect of the VCO microemulsion in GCVO hydrogel films was proven.

**Table 2.** Summary of the stress-at-break (σ), strain-at-break (γ), Young's modulus (YM), swelling degree, and water vapor transmission rate (WVTR) of gellan gum (GG) and GVCO hydrogels at different ratios. Control for WVTR is a test without hydrogel film.


The mechanism of GG hydrogel formation is closely related to the conformational transition from the coil to helix structures [13]. Normally, GG hydrogels are produced by physical crosslinking methods induced by temperature variation or by the presence of divalent cations [29]. Normal hydrogels are usually spontaneously formed by weak secondary forces such as hydrogen bonding, van der Waals interactions, and ionic bonding [30]. In an aqueous solution at high temperatures (≈60 ◦C), the gellan gum chain is in a disordered single-coil state. Upon cooling from 70 to 30 ◦C, the gellan solution promotes the formation of double helices stabilized by internal hydrogen bonding [31]. This facilitates the tight packaging of gellan gum chains, resulting in fragile hydrogels. However, the addition of VCO microemulsion that contains lauric acid (C12 ≈ 78%) and a hydroxyl group is responsible for promoting the formation of hydrogen bonds between gellan gum and VCO microemulsion [28]. This bonding replaces the hydrogen bonds between gellan gum chains and thus decreases the intermolecular bonds along polymer chains and improves strain-at-break [6] of the hydrogels regarding the content of VCO microemulsion. The

bonding is expected to increase the stress-at-break values and give more strength to the hydrogels. Another factor that was contributing to improving flexibility of the GVCO hydrogels could be due the polymerization process. The packed and solid structure of GG and GVCO hydrogels was observed in the cross-section of hydrogels. This shows that VCO microemulsion and GG were homogenous, and no separation within the mixture occurred. *Polymers* **2021**, *13*, x FOR PEER REVIEW 10 of 19

**Figure 4.** (**a**) Typical stress–strain curves of GG and GVCO hydrogels, (**b**–**e**) scanning electron microscopy images of the cross-sectional area of (**b**) pure gellan gum (GG) hydrogels, (**c**) GVCO60, (**d**) GVCO70, and (**e**) GVCO80 hydrogels. **Figure 4.** (**a**) Typical stress–strain curves of GG and GVCO hydrogels, (**b**–**e**) scanning electron microscopy images of the cross-sectional area of (**b**) pure gellan gum (GG) hydrogels, (**c**) GVCO60, (**d**) GVCO70, and (**e**) GVCO80 hydrogels.

#### The mechanism of GG hydrogel formation is closely related to the conformational *3.5. Swelling Ratio, and Water Vapor Transmission Rate (WVTR)*

transition from the coil to helix structures [13]. Normally, GG hydrogels are produced by physical crosslinking methods induced by temperature variation or by the presence of divalent cations [29]. Normal hydrogels are usually spontaneously formed by weak secondary forces such as hydrogen bonding, van der Waals interactions, and ionic bonding [30]. In an aqueous solution at high temperatures (≈60 °C), the gellan gum chain is in a disordered single-coil state. Upon cooling from 70 to 30 °C, the gellan solution promotes the formation of double helices stabilized by internal hydrogen bonding [31]. This facili-The swelling degree of GVCO hydrogels is summarized in Table 2. The standard buffer solution with pH ≈ 7.2 was used to mimic human body fluid. At pH ≈ 7.2, the number of ionic groups is highest in the solution. The GVCO60 absorbed the maximum swelling rates ≈ 12 ± 4, which is an increment of four-fold more than GG hydrogel. The increase of swelling is expected due to the reaction of –OH groups of VCO with the –OH and –COOH of the polymeric chains [32]. These functional groups can relieve the entanglement of polymeric chains, thus weakening the hydrogen bonding among the

tates the tight packaging of gellan gum chains, resulting in fragile hydrogels. However, the addition of VCO microemulsion that contains lauric acid (C12 ≈ 78%) and a hydroxyl

and VCO microemulsion [28]. This bonding replaces the hydrogen bonds between gellan gum chains and thus decreases the intermolecular bonds along polymer chains and improves strain-at-break [6] of the hydrogels regarding the content of VCO microemulsion. The bonding is expected to increase the stress-at-break values and give more strength to the hydrogels. Another factor that was contributing to improving flexibility of the GVCO hydrogels could be due the polymerization process. The packed and solid structure of GG and GVCO hydrogels was observed in the cross-section of hydrogels. This shows that hydrophilic groups. This decreases the degree of physical crosslinking and therefore improves the water uptake of the hydrogels. However, the swelling degree for GVCO70 and GVCO80 hydrogels decreased slightly upon adding a higher concentration of VCO microemulsion, 9 ± 1.5% and 6 ± 1.5%, respectively. The other reason could be due to the hydrophobicity property of VCO itself; the greater the amount of VCO, the greater water resistance of the hydrogels expected.

The water vapor transmission rate (WVTR) of control and GVCO hydrogels are in the range of 332–1547 g m−<sup>2</sup> d −1 (Table 2). The value decreased upon the addition of VCO microemulsion that can be expected due to tight packaging of VCO and GG blends, which then interrupted the water vapor transmission rate. The tight packaging of GG and VCO microemulsion resulted in reducing the surface tension and increasing the stability of water retention in GVCO hydrogels. However, these values remain within the range of WVTR values (90–2893 g m−<sup>2</sup> d −1 ), as reported for eight commercially available synthetic wound dressings [33].

### *3.6. Thermal Analysis*

Thermogravimetric analysis (TGA) was conducted to study the thermal stability of GG and GVCO hydrogels. The thermogram and derivative thermogram of GG and GVCO hydrogels are presented in Figure 5. The degradation below T ≤ 100 ◦C, which occurred for GG hydrogels, is common due to evaporation of moisture in the hydrogels [6]. The degradation of GG and GVCO hydrogels shows a similar trend, in which the degradation occurred at the onset temperature ≈ 54 ◦C, and the offset temperature at ≈ 160 ◦C (Table 3). However, there are differences in the peak of GVCO hydrogels at ≈ 130 ◦C. The GVCO80 hydrogels (containing the highest concentration of VCO) show three small peaks at ≈ 130 ◦C and are assumed due to a few degradation processes of polymer materials. The GVCO60 and GVCO70 hydrogels have two peaks (at ≈ 130 ◦C), and a broader peak at the center (Figure 5). The appearance of these small peaks at ≈ 130 ◦C shows that the addition of VCO exhibits a better outcome in increasing the thermal stability of hydrogels due to a certain degree of interaction between GG and VCO. The weight loss of the GVCO hydrogels is further supported the thermal behavior of the hydrogels, in which the weight loss of GVCO60, GVCO70, and GVCO80 decreased to 86%, 89%, and 88%, respectively, compared to GG hydrogel at 95% (Table 3). Decreased weight loss reflects the increases in the thermal behavior of a material [34]. Similar phenomena have been observed for other oil-based materials [35,36].


**Table 3.** Thermal gravimetric properties of gellan gum (GG) and GVCO hydrogels.

**Figure 5.** (**a**) Thermogravimetric thermograms and (**b**) derivative thermograms of GG and GVCO hydrogels at different concentrations. **Figure 5.** (**a**) Thermogravimetric thermograms and (**b**) derivative thermograms of GG and GVCO hydrogels at different concentrations.

#### **Table 3.** Thermal gravimetric properties of gellan gum (GG) and GVCO hydrogels. *3.7. Antibacterial Studies*

**Sample <sup>T</sup><sup>o</sup> (°C) Temperature Offset (°C) Weight Loss (%)** GG 54 157 95 GVCO60 48 165 86 GVCO70 53 155 89 GVCO80 50 157 88 *3.7. Antibacterial Studies*  GG is well known for its behavior to promote cell growth. For that reason, the result confirming that the control (GG hydrogel) shows no antibacterial activities examined through a qualitative method (inhibition zones) after incubating for 24 h against Gramnegative (*Escherichia coli* and *Klebsiella pneumoniae*) and Gram-positive (*Staphylococcus*  GG is well known for its behavior to promote cell growth. For that reason, the result confirming that the control (GG hydrogel) shows no antibacterial activities examined through a qualitative method (inhibition zones) after incubating for 24 h against Gramnegative (*Escherichia coli* and *Klebsiella pneumoniae*) and Gram-positive (*Staphylococcus aureus* and *Bacillus subtilis*) bacteria (Figure 6a–d). The addition of VCO microemulsion (GVCO60, GVCO70, and GVCO80) in gellan gum hydrogels, however, results in no clear zone of inhibition around the samples against all bacteria (Figure 6). It has been elaborated that the VCO does not possess antibacterial activity on its own, but rather is induced by its free fatty acids, particularly lauric acid (C12), and to a small extent by capric acid (C10) and caprylic acid (C8) [37]. In other words, the VCO must be metabolized to release those components and exert its antimicrobial effects [38]. Another mechanism proposed for the antibacterial effect of VCO is the lipolyze process with lipase and water to form monoglyceride, for which the structure consists of glyceride molecules attached to either sn-1 or sn-2 position

of the glycerol [39]. The derivative of monoglycerides, known as monolaurin or monoester of the lauric acid is the most effective in inhibiting the microorganism by disrupting the cell membrane and the cytoplasm of cells [40]. or sn-2 position of the glycerol [39]. The derivative of monoglycerides, known as monolaurin or monoester of the lauric acid is the most effective in inhibiting the microorganism by disrupting the cell membrane and the cytoplasm of cells [40].

*aureus* and *Bacillus subtilis*) bacteria (Figure 6a–d). The addition of VCO microemulsion (GVCO60, GVCO70, and GVCO80) in gellan gum hydrogels, however, results in no clear zone of inhibition around the samples against all bacteria (Figure 6). It has been elaborated that the VCO does not possess antibacterial activity on its own, but rather is induced by its free fatty acids, particularly lauric acid (C12), and to a small extent by capric acid (C10) and caprylic acid (C8) [37]. In other words, the VCO must be metabolized to release those components and exert its antimicrobial effects [38]. Another mechanism proposed for the antibacterial effect of VCO is the lipolyze process with lipase and water to form monoglyceride, for which the structure consists of glyceride molecules attached to either sn-1

*Polymers* **2021**, *13*, x FOR PEER REVIEW 13 of 19

**Figure 6.** (**a**) *Escherichia coli* (*E. coli*), (**b**) *Klebsiella pneumoniae* (KP), (**c**) *Staphylococcus aureus* (S.A), and (**d**) *Bacillus subtilis* (BS) shown in the figure. Note: The number refers to samples: (1) penicillin (positive control), (2) GVCO80, (3) GVCO70, (4) GVCO60, and (5) gellan gum (negative control). **Figure 6.** (**a**) *Escherichia coli* (*E. coli*), (**b**) *Klebsiella pneumoniae* (KP), (**c**) *Staphylococcus aureus* (S.A), and (**d**) *Bacillus subtilis* (BS) shown in the figure. Note: The number refers to samples: (1) penicillin (positive control), (2) GVCO80, (3) GVCO70, (4) GVCO60, and (5) gellan gum (negative control).

In this particular study, the GVCO hydrogels do not show antibacterial properties against Gram-negative (*Escherichia coli* and *Klebsiella pneumoniae*) and Gram-positive (*Staphylococcus aureus* and *Bacillus subtilis*) bacteria, maybe due to lesser contact of VCO with the agar, which reduces the chances of the latter to diffuse into the agar. As discussed earlier, the antibacterial activities of VCO are triggered by the monolaurin of lactic acid, which depends on the concentration of VCO. Higher content of VCO may contain a higher content of monoglyceride to transform to monolaurin derivatives and thus exert the antimicrobial effects. However, our result is contrary to a few studies reported previously [36,41]. Although these studies examined the film on the agar spread with Gram-negative and Gram-positive bacteria, which has the lesser contact of essential oil to the agar, but their observation shows promising antibacterial activities. Lee and coworkers reported In this particular study, the GVCO hydrogels do not show antibacterial properties against Gram-negative (*Escherichia coli* and *Klebsiella pneumoniae*) and Gram-positive (*Staphylococcus aureus* and *Bacillus subtilis*) bacteria, maybe due to lesser contact of VCO with the agar, which reduces the chances of the latter to diffuse into the agar. As discussed earlier, the antibacterial activities of VCO are triggered by the monolaurin of lactic acid, which depends on the concentration of VCO. Higher content of VCO may contain a higher content of monoglyceride to transform to monolaurin derivatives and thus exert the antimicrobial effects. However, our result is contrary to a few studies reported previously [36,41]. Although these studies examined the film on the agar spread with Gram-negative and Gram-positive bacteria, which has the lesser contact of essential oil to the agar, but their observation shows promising antibacterial activities. Lee and coworkers reported the antibacterial activities of hydroxypropyl methylcellulose/ oregano essential oil nanoemulsion (HPMC/ORNE) composite films against Gram-positive (*S. aureus*, *B. cereus*, and *L. monocytogenes*) and Gram-negative bacteria (*E. coli*, *S. typhimurium*, *P. aeruginosa*, and *V. parahaemolyticus*) [41]. Their results show that the diameters of the inhibition zones increased significantly (*p* < 0.05) with increasing oregano essential oil concentration, with films containing over 5.0% (*v*/*v*) ORNE showing antibacterial activity against all tested strains, particularly against *S. typhimurium*. The authors claimed the antibacterial activity was attributed to the concentration of its most active compound, namely carvacrol, and other phenolics such as thymol and p-cymene, which can act synergistically, and later

disrupting the outer membrane of bacteria, resulting in increased permeability and loss of ions, ATP, and other cytoplasm contents, thus inhibiting bacterial growth [41]. Another study reported by Fangfang and coworkers also shows the antibacterial activity of virgin coconut oil (VCO) incorporated into potato starch-based biodegradable (PSBB) films [36]. According to the authors, the antibacterial effect of films with different VCO concentrations is due to metabolization of the lauric acid in VCO and produce mononucleotides, which have antibacterial properties against *E. coli* and *S. aureus* [36]. Further study should be carried out to understand the possible interaction of VCO with bacteria on the agar at higher concentrations.
