Flavor, antimicrobial activity, and physical properties of composite film prepared with different surfactants

Abstract Different surfactants (lecithin, Tween‐20, and Tween‐80) were added in composite film during the preparation. Flavor, antimicrobial activity, and physical properties of ginger essential oil ‐gelatin film were investigated, in order to study the effect of surfactants on the properties of film. The flavor of GEO was not detected in the film prepared with Tween‐20 and film prepared with Tween‐80, and these two films exhibited stronger antimicrobial activity; film prepared with lecithin possessed higher value in thickness, elongation at break, water solubility, ΔE and opacity, lower value in water vapor property, and tensile strength; attenuated total reflectance‐Fourier transform infrared spectrum results suggested, Tween‐20 and Tween‐80 enhanced the strength of covalent bond, and lecithin weakened the strength of hydrogen bond; and the result of scanning electron microscope showed that Tween‐20 and Tween‐80 improved the dispersion of oil droplets in film. Therefore, this study suggested that surfactants had an influence on the physical properties and molecular structure of a resulting film; in addition, Tween‐20 and Tween‐80 could reduce the flavor of GEO in film, improving the antimicrobial activity of film.

Surfactant had the function of provoking stable state between water phase and oil phase, forming homogeneity distribution of oil droplets (Tongnuanchan, Benjakul, & Prodpran, 2014). Essential oil should be mixed with surfactant to obtain a stable and uniform film solution due to the insolubility of oil in water (Peng, Yin, & Li, 2013;Song, Zuo, et al., 2018). Different types of surfactants were used in the preparation of composite film; Lee, Lee, Yang, and Song (2016) used Tween-80 as surfactant during the preparation of essential oil-protein film; Tongnuanchan, Benjakul, Prodpran, and Nilsuwan (2015) mixed palm oil and lecithin in advance, and then, the mixture was added into film solution; Prodpran, Benjakul, and Artharn (2007) selected Tween-20 as surfactant to emulsify palm oil; Tongnuanchan et al. (2014) studied the influence of different surfactants on thermal properties of essential oil film. However, the effect of different surfactants on the physical properties of essential oil films remained not clear, especially on the flavor and antimicrobial activity. Therefore, the aim of this work was to prepare GEO film with different surfactants (hydrophilic/hydrophobic); in order to figure out the effect of surfactants on flavor, antimicrobial properties, mechanical capacities, WVP, water solubility (WS), color, light transmittance, attenuated total reflectance-Fourier transform infrared spectrum (ATR-FTIR), and scanning electron microscope (SEM) were applied.

| Preparation of GEO films prepared with different surfactants
Ginger essential oil film mixed with different surfactants was prepared on the basis of Liu et al. (2017). Firstly, fish gelatin (8%, w/v) was dissolved in distilled water at 60°C for 90 min. Glycerol was added in gelatin solution (10%, w/w, based on the weight of gelatin), and this solution named mixture A. Then, GEO and surfactant (lecithin/ Tween-20/Tween-80) were mixed at the ratio of 1:1 (w/w), and this was mixture B. Mixture B was added into mixture A to obtain the GEO concentration of 0.5% (w/v, based on distilled water), and this was mixture C. Mixture C was stirred at 25 ± 2°C for 30 min. Then, mixture C (8 ml) was cast onto a rimmed plastic plate (90 × 90 mm 2 ) and dried at 25 ± 2°C and 50 ± 5% relative humidity (RH) for 48 hr.
Film without adding surfactant was used as control film. These film samples were prepared for analysis.

| The flavor of GEO and the flavor of GEO in films
The flavor of GEO: GEO was diluted to appropriate concentration with dichloromethane solution. The volatile flavor components of GEO were detected by gas chromatography-mass spectrum (GC-MS) (Trace1300/ISQ; Thermo Fisher) coupled with the column of HP-5 (30 m × 0.25 mm × 0.25 μm). The injection volume of GEO diluent was 1 μl, and the initial oven temperature was 40°C, then raised to 290°C at a rate of 6.5°C/min, keeping this temperature for 2 min.
The mass detector was carried out in an electron impact mode with ionization energy at 70 eV, and helium gas was selected as carrier gas with the speed of 1 ml/min (Mohamed, El-Emary, & Ali, 2010).
The flavor of GEO in films: Films were immersed into dichloromethane solution and stirred continuously for 30 min. 1 μl of immersed solution was injected into GC-MS following the method of GEO test.

| Antimicrobial activity
The antimicrobial activity of film was tested on Gram-negative E. coli and Gram-positive S. aureus with the method of Vilela et al. (2017).
All the reagents and vessels were sterilized at 121°C for 20 min. The method of plate serial dilution was applied to calculate the bacteria number, and the unit of bacteria number was measured in log cfu/ ml. LB medium was used as a bacterial culture medium. The initial bacterial concentration was adjusted to 5 log cfu/ml. 160 mg film was added to LB medium and cultured with bacterial; LB medium without film was used as blank sample. All samples were incubated at 37°C for 24 hr in static condition. The lower value of log cfu/ml demonstrated higher antimicrobial activity. All samples were measured three times. Mitutoyo) was used to measure films thickness. Ten random locations were selected as a test point. The values of thickness were used to calculate TS, water vapor permeability (WVP), and opacity.

| Water vapor permeability
Water vapor permeability was measured based on ASTM E96-95 method (ASTM, 1995). The dried CaCl 2 was placed in glass cups (40 mm wide, 25 mm depth) to keep the RH of inner space 0%. Films were used to seal the glass cup, and then, these cups were placed in desiccator that contains distilled water at 30°C. Weight changes were recorded hourly. WVP was calculated by the following formula: where w represented the grow weight (g) of the glass cup, l denoted the thickness (m) of the film, A was the sealed areas (m 2 ), t was the interval time (s), and (P 2 − P 1 ) was the difference vapor pressure aside the film (4,244.9 Pa at 30°C). All the films were tested three times to obtain the averaged value.

| Mechanical properties
The method of ASTM (2012) was used to examine TS and EAB by Texture Analyzer (Stable Micro System TA.TX-plus). The trigger force was 5 g, and the test speed was 1 mm/s. Films were cut into strips with 20 mm wide and 50 mm long for experiments. TS and EAB were calculated by the following formula: where F max was the maximum force (N) that need to apart the strips, A was the cross-sectional area of the strips (m 2 ), ΔL was the changed length of strips, and L 0 was the initial length of strips (L 0 = 30 mm). Each type of films was tested for 10 times.

| Water solubility
Films were dried at 105°C to obtain a constant weight M 1 , films were immersed into distilled water at 20°C and kept agitating for 24 hr, and then, remained film was dried again at 105°C to obtain a constant weight M 2 ; the following formula was used to calculate WS: where M 1 was the initial weight before immersion and M 2 was the weight of retain film after immersion. Each film was tested three times (Rubilar et al., 2013).

| Color and opacity
Film's color was tested by colorimeter (CR-10; Konica Minolta optics Inc). The data were obtained by covering film on the standard white plate. Film's color was represented by the value of L (lightness/ darkness), a (redness/greenness), b (yellowness/blueness), and ΔE; ΔE was the total color. The value of ΔE was calculated by the following formula: where the value of L*, a*, and b* were came from standard white plate (L* = 92.56, a* = −0.49, b* = −0.25). Each type of film samples was tested eight times (Moradi et al., 2012).
UV-visible spectrophotometer (Nanodrop200; Thermo Fisher) was used to test the opacity of film. The opacity value was calculated by the formula: where A 600 was the absorbance at 600 nm and x was the thickness of film (mm) (Wu et al., 2015).

| Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) analysis
The measurement was conducted by the method of Lin et al.

| Scanning electron microscope
The microstructure of film was analyzed by SEM (S-3400N; Hitachi). Surface and cross-section microstructure were obtained according to the method of Nilsuwan, Benjakul, and Prodpran (2018).

| Statistical analysis
Statistical tests were performed by Statistical Package for Social Science (SPSS). Duncan test and analysis of variance (ANOVA) were used for analysis, and the difference was considered to be statistically significant if p < .05.

| The flavor of GEO and the flavor of GEO in film
The flavor compounds of GEO are illustrated in Table 1. The flavor compounds of GEO were detected to determine whether the flavor of film was affected by GEO flavor. A total of 18 flavor compounds were detected through GC-MS, including 15 terpenes, 2 aldehydes, and 1 phenol, and these compounds consisted of the flavor of GEO.
The mixed flavor of GEO included spicy, woody, herbal, minty, citrus, and another plant flavor. Seven compounds (camphene, α-myrcene, α-pinene, α-phellandrene, copaene, bisabolene, gingerol) contributed to spicy flavor, which accounted for 88.37% of all flavor compounds, whereas only 11.63% of all flavor compounds came from other 11 compounds. It could be speculated that spicy was the main flavor of GEO. The compounds of gingerol had the flavor of spicy, which accounted for 18.42% of all GEO volatile compounds, and accounted for 43.68% of all flavor compounds. Therefore, gingerol might be a characteristic flavor compound in GEO (Karunakaran & Sadanandan, 2019).
Ginger essential oil flavor compounds in the film are shown in Table 2. It could be noted that two compounds (gingerol and hexanal) in the film were the same as GEO. As a characteristic compound of GEO, gingerol was detected in control film (2.64%) and film prepared with lecithin (0.46%), but it was not detected in the film prepared with Tween-20 and film prepared with Tween-80. The detection of gingerol in the film affected the sensory quality of the film. Hexanal had the flavor of grass, tallow, and fat, and the area% of hexanal in each film was close (1.48%-1.57%), while the area% of hexanal in GEO was 0.38%. Hexanal was speculated to be derived from fish gelatin rather than GEO for two reasons: (a) The area% of hexanal in GEO was lower than that of other compounds, and it was difficult to be detected preferentially compared with other compounds with high area%; (b) the resource of gelatin was fish gelatin, and hexanal was a characteristic fish flavor according to previous reports. Liu, Tao, Mccrummen, Hanson, and Wang (2016) found that hexanal was an off-flavor in catfish fillet; hexanal compounds were observed in fresh fish by Morsy et al. (2016) and Song, Dai, Shen, Peng, and Zhang (2018) considered that hexanal was one of the key volatile compounds of fish oil.
Ginger essential oil tended to be accumulated on the surface of control film without the effect of surfactants, and thus, GEO was easily eluted by solvent and detected by GC-MS. HLB value was the balance between the two-phase. The HLB value of lecithin, Tween-20, and Tween-80 was 4.0, 16.7, and 15.0, respectively; higher HLB value indicated better hydrophilic or polar properties; and lower HLB possessed the opposite characteristics (Schmidts, Dobler, Nissing, & Runkel, 2009

| Antimicrobial activity
The antimicrobial activity of film prepared with different surfactants is exhibited in Figure 1.

| Thickness
The thickness of films prepared with different surfactants is shown in Table 3
Surfactants could contribute to the uniform distribution of the lipid in the film matrix, which were beneficial to promote the moisture resistance of film (Dickinson, 2003;Xiao et al., 2016). Thus, film incorporated with surfactants possessed lower WVP than control film, regardless of the type of surfactants. Higher HLB value represented better hydrophilic or polar properties, and lower HLB possesses the opposite characteristics (Schmidts et al., 2009). Adding nonpolar and hydrophobic materials would reduce the absorptivity and diffusivity of water vapor, due to decreased WVP (Arfat et al., 2014;Xiao et al., 2016). Thus, film prepared with lecithin had lower WVP value than films prepared with Tween-20 and film prepared with Tween-80. In conclusion, film prepared with different surfactants had prominent difference in WVP, and the WVP values of film were ranked from high to low: control film, Tween-20 film, Tween-80 film, lecithin film.

| Mechanical properties
Tensile strength and EAB are shown in Table 3, and these two parameters were used to evaluate the mechanical properties of film.

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). There was no significant difference in mechanical properties between Tween-20 film and Tween-80 film, because the hydrophilicity of Tween-20 and Tween-80 was similar.

| Water solubility
Water solubility of films incorporated with different surfactants is illustrated in Table 3. Control film possessed the lowest WS (14.86%); WS increased with the addition of surfactants, and film prepared with lecithin had the highest WS (19.29%); the value of WS in Tween-20 film (16.69%); and Tween-80 film (17.15%) was close.
WS reflected the film's resistance to water, film with low WS helped to maintain the integrity of the food and extended the shelf life of the food. WS was affected by the internal structure of film. Film incorporated with surfactants formed coarseness surface and loose structure, which would increase the contact area between the film and water, enhancing the WS of film (Song, Zuo, et al., 2018). Film prepared with lecithin had looser structure and higher WS due to the bulky structure of lecithin.

| Color and opacity
Color of film is expressed through the value of ΔE, L, a, and b. As exhibited in Table 4 Opacity of films at 600 nm is shown in Table 4, and control film had the lowest opacity (1.84) among all films; opacity of film increased with the addition of surfactants, and film prepared with Tween-20 (7.27) and film prepared with Tween-80 (7.18) had lower opacity than film prepared with lecithin (8.96). The transparency and light scattering intensity of the films were related to the oil droplet size of the dispersed phase. As the size of oil droplets increased, the intensity of light scattering was enhanced due to lower transparency and higher opacity of the film (Monedero, Fabra, Talens, & Chiralt, 2009;Nur Hanani & Aelma Husna, 2018;Sánchez-González, Vargas, González-Martínez, Chiralt, & Cháfer, 2009). Uniform structure in protein solution was formed easily by hydrophilic surfactant compared with that by hydrophobic surfactant (Song, Zuo, et al., 2018;Sothornvit, Rhim, & Hong, 2009). Lecithin had weak dispersion effect than Tween-20 and Tween-80, and film prepared with lecithin likely formed nonuniform oil droplets in film due to the enhanced intensity of light-scattering by larger size of oil droplets; thus, opacity of film prepared with lecithin became higher. A similar result was obtained from Andreuccetti et al. (2011), and film prepared with lecithin had higher opacity than that of film prepared with yucca extract, which was a hydrophilic surfactant. No significant difference in opacity was achieved between film prepared with Tween-20 and film prepared with Tween-80 because of the similar hydrophilic property of Tween-20 and Tween-80.

| Scanning electron microscope
The surface morphologies and freeze-fractured cross-section morphologies of film incorporated with different surfactants are shown in Figure 3. In terms of surface morphologies, control film exhibited confirm and smooth surface with faint oil stain. After the addition of surfactants, oil droplets were appeared in film; oil droplets presented in Tween-20 film and Tween-80 film were uniform; and oil droplets presented in lecithin film were nonuniform. In cross-sectional morphologies, control film was compacted and drape, and loose structure of films was formed by the addition of surfactants.
The structure of film was related to the properties of film. The structure of control film was firm and smooth, and thus, control film possessed the lowest thickness, EAB and WS, and the highest thickness, TS. Essential oil in film hindered the interaction between proteins upon the addition of surfactants, composite film formed looss structure and flexible region, due to increased thickness, EAB and WS, decreased TS. were not detected, and the antimicrobial activity was higher.

| CON CLUS ION
The

CO N FLI C T O F I NTE R E S T
The authors declared that they have no conflicts of interest in this work.
F I G U R E 4 Simplified illustration of the change in the properties of film prepared with different surfactants

E TH I C A L A PPROVA L
The study did not involve human and animal testing.