Characterization of antimicrobial and antioxidant performance of dihydroeugenol and its glycoside incorporated into whey protein isolate films

1 Introduction

Due to increasing environmental awareness, the development of bio-based plastics is gaining more and more attention, as for many years, traditional commercial food packaging materials were generally petroleum-based plastics such as polyethylene (PE), polypropylene (PP) and polystyrene (PS) (Kunwar et al., 2016). For this reason, some degradable films composed of biocompatible macromolecules with good film-forming properties, such as polysaccharides and proteins, seem to be promising alternatives for plastic packaging (Zhang et al., 2020; Zhang and Jiang, 2020).

To ensure the effectiveness of biodegradable films, several criteria must be met. These include satisfactory mechanical properties, effective barriers against water vapor, oxygen, and ultraviolet light, biodegradability, thermal stability, resistance to food-borne bacteria, desirable sensory attributes, cost-effective production, and compatibility with the packaged product (Guimarães et al., 2018; Yadav et al., 2020). Proteins have been successfully used to form films and coatings. Well-studied proteins, such as whey protein, casein, wheat gluten, soy protein or zein, have been used to develop films obtained from renewable resources with faster degradability than other polymeric materials (Ramos Ó. L. et al., 2012; Cinelli et al., 2014; Mohammadi et al., 2020).

Whey proteins include different globular proteins such as β-lactoglobulin, α-lactalbumin, bovine serum albumin, immunoglobulins, and proteose-peptone polypeptides (Lent et al., 1998). β-lactoglobulin is the main whey protein and dominates gelling and aggregation behavior in whey protein formulations (Hammann and Schmid, 2014). The widespread availability and versatility of whey proteins make them highly suitable for the production of films and coatings. Whey proteins possess the unique ability to undergo conformational changes and interact with one another, forming modified three-dimensional networks. These properties make whey proteins an excellent choice for developing films and coatings with enhanced functionalities (Schmid and Müller, 2019; Alizadeh-Sani et al., 2020).

Essential oils, products of plants’ secondary metabolism, have been considered safe and effective alternatives for microbial control and can be added to films (Lang and Buchbauer, 2012; Wen et al., 2016). Active packaging refers to the interaction between the packaging material and the food it contains, where the packaging itself serves a functional role. One such function is antimicrobial activity, which can be achieved through the incorporation of essential oils into films. This active packaging approach offers various benefits for stored food, such as extending shelf life and reducing microbial growth. By leveraging the antimicrobial properties of essential oils, these films can contribute to preserving the quality and safety of packaged food products (Raut and Karuppayil, 2014; Catarino et al., 2017; Noori et al., 2018). Furthermore, glycosylation has been widely used as a structural modification method that optimizes the physicochemical and biological properties of the starting compound (Hiroyuki et al., 2002; Kurosu et al., 2002) and has been applied in other eugenol derivatives with the evaluation of antifungal activity where the results showed a significant increase in the activity after glycosylation (De Souza et al., 2014).

Based on this, dihydroeugenol and deacetylated dihydroeugenol glycoside compounds, previously synthesized in a previous work by our group (Resende et al., 2017), were used as antimicrobial additives in the production of whey protein isolate (WPI) films with cellulose nanofiber. Subsequently, the synthesized films were evaluated for its ability to inhibit the growth of Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Salmonella Enteritidis and its physical and optical properties were evaluated to verify if adding the compounds interferes with the film’s characteristics.

2 Materials and methods

2.1 Chemicals

The dihydroeugenol (PubChem CID: 17739) was provided by the Laboratory of Pharmaceutical Chemistry of the Federal University of Alfenas, and the synthesis and characterization of glycoside of dihydroeugenol were synthesized at the same laboratory, according to Resende et al. (2017). Whey protein isolate (WPI 9400) with 90% protein was purchased from Hilmar Ingredients (Hilmar CA, United States). The glycerol and sodium chloride were obtained from Merck (Darmstadt, Germany), bacteriological peptone and yeast extract were bought from Kasvi (Sao Paulo, Brazil). Brain Heart Infusion (BHI) broth/agar, TSA (Tryptic Soy Agar), 2,2-diphenyl-1-picrylhydrazyl (DPPH), cellulose nanofiber, and ethanol were purchased by Sigma Aldrich, (Darmstadt, Germany).

2.2 Microorganisms, standardization, and maintenance of the inoculum

The bacterial strains used were E. coli ATCC 25922; L. monocytogenes ATCC 19117; S. aureus ATCC 8702 and Salmonella Enteritidis S64. The stock cultures were stored in freezing medium (15 mL of glycerol; 0.5 g of bacteriological peptone; 0.3 g of yeast extract; 0.5 g of sodium chloride; 100 mL of distilled water, pH 7.0). The cultures were reactivated by inoculating 100 µL aliquots into tubes containing 10 mL of Brain Heart Infusion (BHI) broth and incubated at 37°C for 24 h. The cultures were standardized at 10⁸ CFU mL⁻¹ through a growth curve (OD 600 nm) with counting on BHI agar plates, incubated at 37°C, for 24 h.

2.3 Development of active packaging films incorporated with dihydroeugenol and its glycoside

The WPI films were prepared according to Zinoviadou et al. (2009) and Azevedo et al. (2015), with adaptations. The control film, labeled as “Control,” was produced by dissolving WPI and glycerol in distilled water. In a separate step, cellulose nanofiber was dispersed in the glycerol solution. All solutions were individually subjected to magnetic stirring for 30 min. After stirring, the solutions were poured into each other, and magnetic stirring was continued for 10 min at room temperature. At the end of this stirring, a film-forming solution of 12% (m/v) WPI, 30% (m/m) glycerol and 1% (m/m) cellulose nanofiber in relation to WPI was obtained. Then the pH was adjusted to 8 with 2N NaOH and the final solution was subjected to ultrasonic homogenizer (Sonifier Cell Disruptor Branson–Model 450D, Manchester, United Kingdom) for 10 min, at 80 W and 25°C. The solutions were then heated at 90°C for 30 min in a water bath for protein denaturation and then cooled to room temperature and poured onto plastic plates.

For active treatments, dihydroeugenol and its glycoside were added in a final concentration of 6% (v/v) relative to the WPI after the water bath to maintain the constituent’s preventing evaporation or thermal degradation. The solutions were then subjected to ultrasonic homogenizer again. The thickness control was performed by the volume applied to the support, corresponding to 16.36 mL in a 78.53 cm³ area. The films were dried at room temperature for 24 h ensuring the solvent’s slow evaporation and film formation.

2.4 Determination of the antimicrobial activity of packages

The evaluation of antibacterial potential was performed by the disc diffusion method, according to Bauer et al. (1966), with some modifications. After broth growth, aliquots of 200 μL of the standardized culture of E. coli; L. monocytogenes; S. aureus and Salmonella Enteritidis were plated on TSA (Tryptic Soy Agar) surface. On the already inoculated TSA, active film discs with a diameter of 15 mm were arranged. The disks were previously subjected to UV light for 30 min on each side and then applied to each quadrant of the plate containing the respective bacteria. The plates were incubated at 37°C for 48 h and after the incubation the halo size was measured using a vernier calipers. As a negative control, films without the addition of dihydroeugenol and its glycoside, were evaluated. The results were expressed in millimeters by averaging the values obtained in three repetitions.

2.5 Antioxidant activity of active films

The method applied, 2,2-diphenyl-1-picrylhydrazyl (DPPH), is based on the ability of the DPPH radical to be scavenged and discolored in the presence of an antioxidant compound, upon reduction, the purple color of the radical changes to yellow at the same time that the absorbance decreases at 515 nm resulting in the reduction of absorbance values (Foti, 2015).

The WPI films (0.1 g) were cut into small pieces and mixed with 2 mL of ethanol 80% for solubilization. The mixture was vigorously vortexed for 3 min and kept at room temperature for 3 h. Then it was stirred again. An aliquot of the extract was mixed with 3.9 mL of 2,2-diphenyl-1-picrylhydrazyl (DPPH) 0.1 mM in ethanol 80%. The mixture was vigorously vortexed for 1 min and kept in the dark for 30 min. The absorbance was measured at 515 nm using a spectrophotometer (SP 2000UV). Ethanol 80% was used as a negative control and was mixed with DPPH 0.1 mM. The radical scavenging activity (SA) was calculated according to equation (Byun et al., 2010):

$\left(\%\right)\mathrm{S}\mathrm{A}=\left(\mathrm{A}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{A}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\right)/\left(\mathrm{A}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)\mathrm{x}100(1)$

Wherein:A_control: negative control absorbance.A_sample: sample absorbance.

2.6 Water solubility

The water solubility of films was determined according to the method of Rhim et al. (2005). The samples (3 × 5 cm) were weighed (P1) and placed in beakers containing 30 mL of deionized water. The beakers were covered with parafilm and stored at 25.0°C ± 0.5°C for 24 h. After this period, pieces of undissolved films were recovered using filter paper. The filtrate was dried at 105°C for 24 h and then weighed (P2). The solubility in water was determined from the undissolved dry matter and calculated according to the following equation:

$\left.\left(\%\right)\mathrm{W}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{s}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}=\left(\left(\mathrm{P}1-\mathrm{P}2\right)\mathrm{x}100\right)/\mathrm{P}1\right)(2)$

2.7 Optical properties

2.7.1 Color

For determining the color of WPI films, colorimeter Color Quest XE, Hunter Lab (Reston, VA, United States) and Comission Internationale de L’Éclairage (CIE) system (L*, a*, b*) with a light source D65, an observer angle of 10° and mode reflectance were used. The parameters (L*, a*, b*), hue angle (h°) and chroma saturation index (C*) were analyzed. L* indicates brightness ranging from 0 (black) to 100 (white); a* indicates the color intensity ranging from green (−) to red (+); b* indicates the color intensity ranging from blue (−) to yellow (+) (Sarker and Oba, 2019).

2.7.2 Transparency

To determine the transparency of WPI films, transmittance percentage (% T) at 600 nm was measured using a GBC UV/VIS 918 spectrophotometer (Shimadzu, Tokyo, Japan) according to ASTM (2015) method. The transparency (T600) was calculated according to the following equation:

$\mathrm{T}600=\left(\left(\mathrm{Log}\%\mathrm{T}\right)\right)/\partial (3)$

Where ∂ is the film thickness (mm).

2.7.3 Statistical analysis

The results obtained for the WPI films incorporated with dihydroeugenol and glycoside of dihydroeugenol were submitted to analysis of variance (ANOVA), and the averages were compared through the Tukey test at a 5% significance level, using SISVAR 5.1 software (Ferreira, 2011). Results were expressed as mean values ± standard deviation (n = 3).

3 Results

3.1 Evaluation of antimicrobial activity of active films

The results of measuring the inhibition halos (mm) formed by the antibacterial activity of WPI films incorporated with dihydroeugenol or glycoside are shown in Table 1. The obtained results show that there was no inhibition of bacterial growth when control film was used without addition of synthesized compounds. The addition of compounds dihydroeugenol and its glycoside was effective against all bacteria tested (halos ranging from 7.5 to 23 mm). Significant difference (p < 0.05) in treatments were seen for all the evaluated microorganisms, and WPI film added with dihydroeugenol glycoside was able to form inhibition halos larger than WPI film added with dihydroeugenol.

TABLE 1

TABLE 1. Inhibition Halos (mm) formed by the Antibacterial Activity of WPI Films with cellulose nanofiber incorporated with dihydroeugenol or glycoside as antimicrobial agents.

3.2 Antioxidant activity of WPI active films

The results showed that there was not statistically significant (p > 0.05) difference between the antioxidant activity of the films made with the addition of dihydroeugenol (39.79% ± 8.05%) and to those made with glycoside (43.42% ± 6.82%) (Figure 1). The control sample showed that the antioxidant activity of the film prepared from WPI, without other additives, presents a value of 15.1% ± 1.63%. Based on this result, the compounds synthesized significantly (p < 0.05) increased the antioxidant activity.

FIGURE 1

FIGURE 1. Antioxidant assay in the evaluation of DPPH radical intercept of whey protein isolate (WPI). Control: WPI film without the addition of dihydroeugenol and its glycoside. WPID: WPI film with the addition of dihydroeugenol (6% v/v), WPIG: WPI film with the addition of dihydroeugenol glycoside (6% v/v). Each bar represents the means ± SD, considering the results obtained in at three independent experiments. Bars followed by different letters differ significantly (p < 0.05) by the Tukey test.

3.3 Films solubility

The results of the films solubility are showed in Figure 2, relatively a low solubility (p < 0.05) was observed of the produced films, with averages of 23% ± 2.87% and 24% ± 3.92% for the film with dihydroeugenol and the film with glycoside, respectively, with no significant difference (P ˃ 0.05) in solubility between them. The solubility of control film presented an average value of 68% ± 3.49%, indicating that the synthesized compounds added to the film decreased the solubility of films, due to their hydrophobic character.

FIGURE 2

FIGURE 2. Water solubility of whey protein isolate (WPI). Control: WPI film without the addition of dihydroeugenol and its glycoside. WPID: WPI film with the addition of dihydroeugenol (6% v/v), WPIG: WPI film with the addition of dihydroeugenol glycoside (6% v/v). Each bar represents the means ± SD, considering the results obtained in at three independent experiments. Bars followed by different letters differ significantly (p < 0.05) by the Tukey test.

3.4 Optical properties

3.4.1 Color

The attributes of color are important because they influence the acceptance of products by consumer, All parameters were compared according to Minolta (2007) and are presented in Table 2. The L * attribute (luminosity) had no significant (p < 0.05) variation between the two treatments with the addition of compounds. This parameter reflects how clear the sample is, the use of the dihydroeugenol (87.91 ± 0.59) and dihydroeugenol glycoside (89.43 ± 1.41) additives decreased the L* value in relation to the control film (97.20 ± 0.04), but visually are clear enough for food packaging.

TABLE 2

TABLE 2. Color parameters of WPI films produced with addition of dihydroeugenol and glycoside.

Values of a*, which may range from −90 to 70, were significantly higher for the glycoside containing film (−2.36 ± 0.24), showing its better coloring ability in relation to dihydroeugenol (−1.47 ± 0.04). However, the values of b*, may range from −80 to 100, did not vary significantly (p > 0.05) among the dihydroeugenol (28.17 ± 0.98) and its glycoside films (24.22 ± 4.90), but showed a tendency to yellow (highest value) when compared to the control film (9.49 ± 1.69).

The saturation index, chroma (C*), defines color intensity, and values close to zero are indicative of neutral colors (gray) and values around 60 indicate vivid and intense colors (Minolta, 2007), there is not vary significantly (p > 0.05) among the dihydroeugenol (28.20 ± 0.97) and its glycoside films (24.30 ± 4.96), both presented intermediate values at interval of 0–60, characterizing a medium intensity in the colors, however, the control film showed a lower (p < 0.05) value of c* (9.54 ± 0.31). The Hue angle (h°) represents the color tone, and, through it, the sample position can be estimated in color solid (Minolta, 2007). All films analyzed presented values close to 90°, with no significant difference (p > 0.05) between them, this value was presented in a shade of yellow color.

3.4.2 Transparency

The transparency of the WPI films studied in our work are shown in Figure 3. The control film had an average transparency of 11.63 ± 1.3 log (%T)/mm, while the film with the addition of the dihydroeugenol and glycoside compounds obtained an average transparency of 9.95 ± 0.76 and 8.92 ± 0.98 log (%T)/mm, respectively. It was observed that there was no difference (P ˃ 0.05) in transparency for the treatments performed, and compared to WPI films without additives, the transparency was reduced, probably due to the characteristic staining of the synthesized compounds.

FIGURE 3

FIGURE 3. Transparency of whey protein isolate (WPI). Control: WPI film without the addition of dihydroeugenol and its glycoside. WPID: WPI film with the addition of dihydroeugenol (6% v/v), WPIG: WPI film with the addition of dihydroeugenol glycoside (6% v/v). Each bar represents the means ± SD, considering the results obtained in at three independent experiments.

4 Discussion

To confirm any effect of the incorporation of dihydroeugenol and its glycoside components in whey protein films, the antimicrobial, antioxidant, and physicochemical properties were evaluated in our work. The efficacy of essential oils as antimicrobial agents when added to films has been reported (Gomes et al., 2017; Noori et al., 2018; Pavlátková et al., 2023).

The choice to work with dihydroeugenol and its synthesized glycoside was made according to the results previously found in the work of our research group Resende et al. (2017), in which dihydroeugenol showed to be more active than eugenol beyond the tested bacteria Escherichia coli, Listeria monocytogenes, Salmonella enteritidis, Staphylococcus aureus. The results are also in agreement with those found in our work, where the glycoside presented a lower Minimal Bactericidal Concentrations when compared to dihydroeugenol, showing a higher antimicrobial potency of glycoside.

To the best of our knowledge, this is the first study of the incorporation of eugenol or dihydroeugenol in WPI films for comparative purposes. However, a study evaluated the antimicrobial activity of polyhydroxybutyrate films incorporated with eugenol. In contrast to S. aureus, the zone of inhibition formed was 14 ± 1 mm and was compared to E. coli. This value was 19 ± 1 mm and the diameter of film discs was 6 mm (Narayanan et al., 2013). In the WPI films incorporated with dihydroeugenol, the observed effect on E. coli was lower compared to the findings reported in the referenced study. However, against S. aureus, the effect was higher than what was observed in the cited work. On the other hand, in the WPI films incorporated with the glycoside, the antimicrobial activity was higher against both bacteria compared to the film incorporated with eugenol.

Sanla-Ead et al. (2012) developed cellulose films and used them as additives with eugenol and cinnamaldehyde, both in a concentration of 1% v/v. Among other tests, the researchers evaluated the antibacterial activity of the films, by the same method used in the present study, except for the disc diameter of 5 mm. The results obtained by the researchers showed no activity on tested microorganisms, among them L. monocytogenes, S. aureus, E. coli and Salmonella Enteritidis. The researchers attributed the results to low concentration and solubility of compounds in films.

Thus, it can be verified that, based on the few works available in literature, it is possible to make a brief comparison of these results with eugenol analog incorporated into other types of films. In the evaluated cases, the WPI film incorporated with dihydroeugenol glycoside proved to be more efficient against the tested bacteria. Thus, the films produced can be evaluated for application in food, increasing their shelf life, and benefiting consumers in the search for natural preservatives and sustainable packaging.

Based on the results of the antimicrobial activity of the active film produced with glycoside, a comparison was made between the activity against the different microorganisms evaluated. It is possible to verify that the mean inhibition zones across the Gram-negative bacteria E. coli and S. Enteritidis were significantly similar. Likewise, it occurred to the middle front of the Gram-positive S. aureus and L. monocytogenes. In general, it is possible to realize a significantly higher activity against Gram-positive bacteria, suggesting a better membrane permeability of these cells by the active agent. According to Dorman and Deans (2000), Gram-negative bacteria have an outer membrane that is rich in polysaccharides that forms a hydrophilic surface. This hydrophilic character may present a barrier to the permeability of lipophilic substances, as derivatives of essential oils, thus explaining the greater resistance of Gram-negative bacteria to these compounds.

Studies have indicated that eugenol possesses inhibitory effects on the growth of E. coli, L. monocytogenes, and S. aureus (Sanla-Ead et al., 2012). It has been observed to disrupt their cellular membranes and induce leakage of potassium ions (Walsh et al., 2003; Gill and Holley, 2006). In the work by Devi et al. (2010), was demonstrated the significant potential of eugenol as an effective antibacterial compound against Salmonella typhi. At bactericidal concentrations, eugenol primarily acts by disrupting the cytoplasmic membrane, leading to increased permeability. This disruption causes the leakage of ions and loss of cellular contents, including intracellular proteins, ultimately resulting in cell death. Furthermore, the findings reveal that eugenol possesses chemo-attractant properties toward S. typhi.

Films composed of ethylene-vinyl acetate (EVA) blended with eugenol was studied in the work of Nostro et al. (2013), following a 24–48 h incubation period, the film with eugenol at a concentration of 7 wt% exhibited promising activity. The results showed that the combination of EVA with eugenol exhibited activity against S. aureus, S. epidermidis, and E. coli, with a decrease in optical density of 20%–30%. Additionally, the EVA + eugenol combination displayed weak activity against S. aureus and P. aeruginosa strains, decreasing optical density by 15%–20%.

The nanocomposite film with sodium alginate, containing 1.5% of clove essential oil, in which the major compound is eugenol, effectively inhibited the growth of L. monocytogenes and S. aureus during the first 7, 6 days of the storage period, respectively. For E. coli, the highest concentration completely inhibited its growth during the first 6 days of the study period, although the antimicrobial effectiveness decreased afterward (Alboofetileh et al., 2014).

In theory, hydrophilic glycosyl groups have the ability to interact with starch molecules and effectively permeate between polysaccharide chains. This interaction acts as a pseudo-plasticizer, leading to improved physical properties of biodegradable films. Consequently, glycosides have the potential to serve as a valuable tool for solubilizing hydrophobic bioactive compounds in aqueous solutions, while simultaneously enhancing the performance of polysaccharide-based films (Wang et al., 2023). Additionally, the formation of micelles by glycosides can further contribute to enhanced antimicrobial activity. On the other hand, the primary way in which eugenol exerts its antimicrobial effects is through its free hydroxyl group. This hydroxyl group has the ability to hinder certain enzymes by binding to proteins and it can also disrupt the cell membrane, thereby increasing its permeability (Souza et al., 2022).

Among the different approaches, the antioxidant effect can also be exploited by adding dihydroeugenol and its synthesized glycoside in biodegradable films. Radical species act as initiators in the lipid oxidation chain, causing undesirable effects on food (López-De-Dicastillo et al., 2012). Based on this result, the compounds synthesized significantly increased the antioxidant activity, when compared with the control sample, agreeing with Moradi et al. (2012), since they are derived from eugenol that has antioxidant activity already elucidated. Additionally, the addition of phenolic compounds, such as the products used, greatly increases antioxidant activity, as reported by Siripatrawan et al. (2013) and Chang-Bravo et al. (2014).

Therefore, the elimination of these radicals by the packaging that will wrap food, by the incorporation of antioxidant species, shows itself as an innovative and interesting alternative for food preservation. In addition to radical scavenging ability, it is important that the antioxidant agent does not affect the overall properties of the film, a fact observed in the present study, where the addition of compounds did not negatively affect the characteristics of the film.

Among the physicochemical properties of the films, despite the hydrophilic character of the glycosidic subunit, the dihydroeugenol glycoside did not increase the film solubility because, besides its aglycone subunit being hydrophobic, hydrogen bonds between the glycosidic subunit and the WPI matrix may occur, preventing the breakdown of the structure by water molecules. So, the solubility of control film presented a higher solubility value, indicating that the synthesized compounds added to the film decreased the solubility of films, due to their hydrophobic character, poor water-soluble biodegradable films are desirable for packaging and protecting food with medium/high humidity. Ramos et al. (2012) and Fairley et al. (1996) reported in their work the partial insolubility of WPI films and attributed this characteristic to the presence of strong intermolecular bonds (disulfide bonds) between the protein molecules inside the polymeric matrix of the WPI films.

The transparency of a film is important so that it allows the consumer to view the product stored there. In this way, it becomes an attribute that influences the purchase of the product. In the work of Al-Salem (2009), in which polyethylene films with UVA additives and light transformers (Tinuvin 494 FB, Tinuvin NOR 371, Chimasorb 81 and Irgastab) were evaluated, not essential oils, the values of transparency calculated by the equation used in this work, were around 19.5, being significantly more transparent than the films synthesized with the addition of dihydroeugenol and its glycoside. On the other hand, the transparency value of the control, only the WPI film was similar to that reported in other studies (Qazanfarzadeh and Kadivar, 2016; Sukyai et al., 2018). Sothornvit et al. (2010) found a transparency of 14.38 in WPI films, and the addition of other compounds was able to significantly reduce this transparency.

The visual appearance of films is often an important factor in consumer acceptance (Kurek et al., 2014). In general, the color of films with the addition of dihydroeugenol and its glycoside showed a tendency to yellow. The presence of fats and phospholipids is responsible for the tendency of these films to turn into a yellowish color (Lorenzen and Schrader, 2006; Ramos et al., 2013). In the work of Gohargani et al. (2020), the incorporation of Zataria multiflora essential oil in chitosan-whey protein-based film containing bionanocomposite tio2, caused a slight reduction in lightness (L*) and an increase in e yellowness, the same was observed in our work.

5 Conclusion

The present study showed that WPI film added with dihydroeugenol glycoside was able to form inhibition halos larger than WPI film added with dihydroeugenol, proving the efficacy of glycoside against B. cereus, E. coli, S. aureus and S. Enteritidis, with significantly higher activity against Gram-positive bacteria, suggesting a better membrane permeability of these cells by the active agent evaluated. Furthermore, the compounds synthesized significantly increased the antioxidant activity when compared to the control (without dihydroeugenol and glycoside). The results also showed a relatively a low solubility of the produced films (23% ± 2.87% and 24% ± 3.92%, dihydroeugenol and glycoside, respectively), presenting a possible applicability, since poor water-soluble biodegradable films are desirable for packaging and protecting food with medium/high humidity. Overall, this study demonstrated that Incorporation of dihydroeugenol and its glycoside in whey protein isolate film could effectively be considered promising candidates to be used as active packaging for food products as an antibacterial and antioxidant film for packaging food with medium/high humidity.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author.

Author contributions

DD, DC, MD, and RP contributed to the conception and design of the study, DC, MD, RP contributed to the methodology, DR and LL contributed to the investigation, DD and RS provided the resources, DR, LS, and NT contributed to the data curation, DR wrote the first draft of the manuscript. DD, RS, DC, MD, LS, NT, and DC wrote the review and editing, DR and LS contributed to the visualization, DD, DC, RP, and MD were the supervisors of the study, DD contributed to the project administration. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico do Brasil (CNPq) [grant numbers 312336/2014-4, 423095/2016-1], Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) [grant number CAG - APQ-03478-16], and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) [PNPD20131289].

Conflict of interest

The author(s) RS declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

References

Al-Salem, S. M. (2009). Influence of natural and accelerated weathering on various formulations of linear low density polyethylene (LLDPE) films. Mat. Des. 30, 1729–1736. doi:10.1016/j.matdes.2008.07.049

CrossRef Full Text | Google Scholar

Alboofetileh, M., Rezaei, M., Hosseini, H., and Abdollahi, M. (2014). Antimicrobial activity of alginate/clay nanocomposite films enriched with essential oils against three common foodborne pathogens. Food control. 36, 1–7. doi:10.1016/j.foodcont.2013.07.037

CrossRef Full Text | Google Scholar

Alizadeh-Sani, M., Mohammadian, E., and McClements, D. J. (2020). Eco-friendly active packaging consisting of nanostructured biopolymer matrix reinforced with TiO2 and essential oil: application for preservation of refrigerated meat. Food Chem. 322, 126782. doi:10.1016/J.FOODCHEM.2020.126782

PubMed Abstract | CrossRef Full Text | Google Scholar

ASTM (2015). “ASTM (2015) D1746–15: standard test method for transparency of plastic sheeting,” in Annual book of ASTM standards 2015 (United States: ASTM International).

Google Scholar

Azevedo, V. M., Dias, M. V., Borges, S. V., Costa, A. L. R., Silva, E. K., Medeiros, É. A. A., et al. (2015). Development of whey protein isolate bio-nanocomposites: effect of montmorillonite and citric acid on structural, thermal, morphological and mechanical properties. Food Hydrocoll. 48, 179–188. doi:10.1016/j.foodhyd.2015.02.014

CrossRef Full Text | Google Scholar

Bauer, A. W., Kirby, W. M., Sherris, J. C., and Turck, M. (1966). Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45, 493–496. doi:10.1093/ajcp/45.4_ts.493

PubMed Abstract | CrossRef Full Text | Google Scholar

Byun, Y., Kim, Y. T., and Whiteside, S. (2010). Characterization of an antioxidant polylactic acid (PLA) film prepared with α-tocopherol, BHT and polyethylene glycol using film cast extruder. J. Food Eng. 100, 239–244. doi:10.1016/j.jfoodeng.2010.04.005

CrossRef Full Text | Google Scholar

Catarino, M. D., Alves-Silva, J. M., Fernandes, R. P., Gonçalves, M. J., Salgueiro, L. R., Henriques, M. F., et al. (2017). Development and performance of whey protein active coatings with Origanum virens essential oils in the quality and shelf life improvement of processed meat products. Food control. 80, 273–280. doi:10.1016/j.foodcont.2017.03.054

CrossRef Full Text | Google Scholar

Chang-Bravo, L., López-Córdoba, A., and Martino, M. (2014). Biopolymeric matrices made of carrageenan and corn starch for the antioxidant extracts delivery of Cuban red propolis and yerba mate. React. Funct. Polym. 85, 11–19. doi:10.1016/j.reactfunctpolym.2014.09.025

CrossRef Full Text | Google Scholar

Cinelli, P., Schmid, M., Bugnicourt, E., Wildner, J., Bazzichi, A., Anguillesi, I., et al. (2014). Whey protein layer applied on biodegradable packaging film to improve barrier properties while maintaining biodegradability. Polym. Degrad. Stab. 108, 151–157. doi:10.1016/J.POLYMDEGRADSTAB.2014.07.007

CrossRef Full Text | Google Scholar

De Souza, T. B., Orlandi, M., Coelho, L. F. L., Malaquias, L. C. C., Dias, A. L. T., De Carvalho, R. R., et al. (2014). Synthesis and in vitro evaluation of antifungal and cytotoxic activities of eugenol glycosides. Med. Chem. Res. 23, 496–502. doi:10.1007/s00044-013-0669-2

CrossRef Full Text | Google Scholar

Devi, K. P., Nisha, S. A., Sakthivel, R., and Pandian, S. K. (2010). Eugenol (an essential oil of clove) acts as an antibacterial agent against Salmonella typhi by disrupting the cellular membrane. J. Ethnopharmacol. 130, 107–115. doi:10.1016/j.jep.2010.04.025

PubMed Abstract | CrossRef Full Text | Google Scholar

Dorman, H. J. D., and Deans, S. G. (2000). Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J. Appl. Microbiol. 88, 308–316. doi:10.1046/j.1365-2672.2000.00969.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Fairley, P., Monahan, F. J., Bruce German, J., and Krochta, J. M. (1996). Mechanical properties and water vapor permeability of edible films from whey protein isolate and sodium dodecyl sulfate. J. Agric. Food Chem. 44, 438–443. doi:10.1021/jf9505234

CrossRef Full Text | Google Scholar

Ferreira, D. F. (2011). Sisvar: a computer statistical analysis system. Ciência agrotecnologia 35, 1039–1042. doi:10.1590/s1413-70542011000600001

CrossRef Full Text | Google Scholar

Foti, M. C. (2015). Use and abuse of the DPPH• radical. J. Agric. Food Chem. 63, 8765–8776. doi:10.1021/acs.jafc.5b03839

PubMed Abstract | CrossRef Full Text | Google Scholar

Gill, A. O., and Holley, R. A. (2006). Disruption of Escherichia coli, Listeria monocytogenes and Lactobacillus sakei cellular membranes by plant oil aromatics. Int. J. Food Microbiol. 108, 1–9. doi:10.1016/j.ijfoodmicro.2005.10.009

PubMed Abstract | CrossRef Full Text | Google Scholar

Gohargani, M., Lashkari, H., and Shirazinejad, A. (2020). Study on biodegradable chitosan-whey protein-based film containing bionanocomposite TiO2and Zataria multiflora essential oil. J. Food Qual. 2020, 1–11. doi:10.1155/2020/8844167

CrossRef Full Text | Google Scholar

Gomes, M. de S., Cardoso, M., das, G., Guimarães, A. C. G., Guerreiro, A. C., Gago, C. M. L., et al. (2017). Effect of edible coatings with essential oils on the quality of red raspberries over shelf-life. J. Sci. Food Agric. 97, 929–938. doi:10.1002/jsfa.7817

PubMed Abstract | CrossRef Full Text | Google Scholar

Guimarães, A., Abrunhosa, L., Pastrana, L. M., and Cerqueira, M. A. (2018). Edible films and coatings as carriers of living microorganisms: a new strategy towards biopreservation and healthier foods. Compr. Rev. Food Sci. Food Saf. 17, 594–614. doi:10.1111/1541-4337.12345

PubMed Abstract | CrossRef Full Text | Google Scholar

Hammann, F., and Schmid, M. (2014). Determination and quantification of molecular interactions in protein films: a review. Mater. (Basel) 7, 7975–7996. doi:10.3390/ma7127975

PubMed Abstract | CrossRef Full Text | Google Scholar

Hiroyuki, D., Toshiyuki, S., Kohtaro, K., Kuniki, K., and Shoji, U. (2002). Enzymatic synthesis of l-menthyl alpha-maltoside and l-menthyl alpha-maltooligosides from l-menthyl alpha-glucoside by cyclodextrin glucanotransferase. J. Biosci. Bioeng. 94, 119–123. doi:10.1263/jbb.94.119

PubMed Abstract | CrossRef Full Text | Google Scholar

Kunwar, B., Cheng, H. N., Chandrashekaran, S. R., and Sharma, B. K. (2016). Plastics to fuel: a review. Renew. Sustain. Energy Rev. 54, 421–428. doi:10.1016/J.RSER.2015.10.015

CrossRef Full Text | Google Scholar

Kurek, M., Galus, S., and Debeaufort, F. (2014). Surface, mechanical and barrier properties of bio-based composite films based on chitosan and whey protein. Food packag. Shelf Life 1, 56–67. doi:10.1016/J.FPSL.2014.01.001

CrossRef Full Text | Google Scholar

Kurosu, J., Sato, T., Yoshida, K., Tsugane, T., Shimura, S., Kirimura, K., et al. (2002). Enzymatic synthesis of alpha-arbutin by alpha-anomer-selective glucosylation of hydroquinone using lyophilized cells of Xanthomonas campestris Wu-9701. J. Biosci. Bioeng. 93, 328–330. doi:10.1263/jbb.93.328

PubMed Abstract | CrossRef Full Text | Google Scholar

Lang, G., and Buchbauer, G. (2012). A review on recent research results (2008-2010) on essential oils as antimicrobials and antifungals. A review. Flavour Fragr. J. 27, 13–39. doi:10.1002/ffj.2082

CrossRef Full Text | Google Scholar

Lent, L. E., Vanasupa, L. S., and Tong, P. S. (1998). Whey protein edible film structures determined by atomic force microscope. J. Food Sci. 63, 824–827. doi:10.1111/j.1365-2621.1998.tb17908.x

CrossRef Full Text | Google Scholar

López-De-Dicastillo, C., Gómez-Estaca, J., Catalá, R., Gavara, R., and Hernández-Muñoz, P. (2012). Active antioxidant packaging films: development and effect on lipid stability of brined sardines. Food Chem. 131, 1376–1384. doi:10.1016/j.foodchem.2011.10.002

CrossRef Full Text | Google Scholar

Lorenzen, P. C., and Schrader, K. (2006). A comparative study of the gelation properties of whey protein concentrate and whey protein isolate. Lait 86, 259–271. doi:10.1051/lait:2006008

CrossRef Full Text | Google Scholar

Minolta, K. (2007). Precise color communication. Japan: Konica Minolta Photo Sensing Inc.

Google Scholar

Mohammadi, M., Mirabzadeh, S., Shahvalizadeh, R., and Hamishehkar, H. (2020). Development of novel active packaging films based on whey protein isolate incorporated with chitosan nanofiber and nano-formulated cinnamon oil. Int. J. Biol. Macromol. 149, 11–20. doi:10.1016/J.IJBIOMAC.2020.01.083

PubMed Abstract | CrossRef Full Text | Google Scholar

Moradi, M., Tajik, H., Razavi Rohani, S. M., Oromiehie, A. R., Malekinejad, H., Aliakbarlu, J., et al. (2012). Characterization of antioxidant chitosan film incorporated with Zataria multiflora Boiss essential oil and grape seed extract. LWT - Food Sci. Technol. 46, 477–484. doi:10.1016/J.LWT.2011.11.020

CrossRef Full Text | Google Scholar

Narayanan, A., Neera, M., and Ramana, K. V. (2013). Synergized antimicrobial activity of eugenol incorporated polyhydroxybutyrate films against food spoilage microorganisms in conjunction with pediocin. Appl. Biochem. Biotechnol. 170, 1379–1388. doi:10.1007/s12010-013-0267-2

PubMed Abstract | CrossRef Full Text | Google Scholar

Noori, S., Zeynali, F., and Almasi, H. (2018). Antimicrobial and antioxidant efficiency of nanoemulsion-based edible coating containing ginger (Zingiber officinale) essential oil and its effect on safety and quality attributes of chicken breast fillets. Food control. 84, 312–320. doi:10.1016/j.foodcont.2017.08.015

CrossRef Full Text | Google Scholar

Nostro, A., Scaffaro, R., D’Arrigo, M., Botta, L., Filocamo, A., Marino, A., et al. (2013). Development and characterization of essential oil component-based polymer films: a potential approach to reduce bacterial biofilm. Appl. Microbiol. Biotechnol. 97, 9515–9523. doi:10.1007/s00253-013-5196-z

PubMed Abstract | CrossRef Full Text | Google Scholar

Pavlátková, L., Sedlaříková, J., Pleva, P., Peer, P., Uysal-Unalan, I., and Janalíková, M. (2023). Bioactive zein/chitosan systems loaded with essential oils for food-packaging applications. J. Sci. Food Agric. 103, 1097–1104. doi:10.1002/jsfa.11978

PubMed Abstract | CrossRef Full Text | Google Scholar

Qazanfarzadeh, Z., and Kadivar, M. (2016). Properties of whey protein isolate nanocomposite films reinforced with nanocellulose isolated from oat husk. Int. J. Biol. Macromol. 91, 1134–1140. doi:10.1016/J.IJBIOMAC.2016.06.077

PubMed Abstract | CrossRef Full Text | Google Scholar

Ramos, Ó. L., Fernandes, J. C., Silva, S. I., Pintado, M. E., and Malcata, F. X. (2012a). Edible films and coatings from whey proteins: a review on formulation, and on mechanical and bioactive properties. Crit. Rev. Food Sci. Nutr. 52, 533–552. doi:10.1080/10408398.2010.500528

PubMed Abstract | CrossRef Full Text | Google Scholar

Ramos, Ó. L., Reinas, I., Silva, S. I., Fernandes, J. C., Cerqueira, M. A., Pereira, R. N., et al. (2013). Effect of whey protein purity and glycerol content upon physical properties of edible films manufactured therefrom. Food Hydrocoll. 30, 110–122. doi:10.1016/j.foodhyd.2012.05.001

CrossRef Full Text | Google Scholar

Ramos, O. L., Silva, S. I., Soares, J. C., Fernandes, J. C., Poças, M. F., Pintado, M. E., et al. (2012b). Features and performance of edible films, obtained from whey protein isolate formulated with antimicrobial compounds. Food Res. Int. 45, 351–361. doi:10.1016/j.foodres.2011.09.016

CrossRef Full Text | Google Scholar

Raut, J. S., and Karuppayil, S. M. (2014). A status review on the medicinal properties of essential oils. Ind. Crops Prod. 62, 250–264. doi:10.1016/j.indcrop.2014.05.055

CrossRef Full Text | Google Scholar

Resende, D. B., Martins, H. H. D. A., Souza, T. B. D., Carvalho, D. T., Piccoli, R. H., Schwan, R. F., et al. (2017). Synthesis and in vitro evaluation of peracetyl and deacetyl glycosides of eugenol, isoeugenol and dihydroeugenol acting against food-contaminating bacteria. Food Chem. 237, 1025–1029. doi:10.1016/j.foodchem.2017.06.056

PubMed Abstract | CrossRef Full Text | Google Scholar

Rhim, J. W., Lee, J. H., and Kwak, H. S. (2005). Mechanical and water barriers properties of soy protein and clay mineral composite films. Food Sci. Biotechnol. 14, 112–116.

Google Scholar

Sanla-Ead, N., Jangchud, A., Chonhenchob, V., and Suppakul, P. (2012). Antimicrobial activity of cinnamaldehyde and eugenol and their activity after incorporation into cellulose-based packaging films. Packag. Technol. Sci. 25, 7–17. doi:10.1002/pts.952

CrossRef Full Text | Google Scholar

Sarker, U., and Oba, S. (2019). Salinity stress enhances color parameters, bioactive leaf pigments, vitamins, polyphenols, flavonoids and antioxidant activity in selected Amaranthus leafy vegetables. J. Sci. Food Agric. 99, 2275–2284. doi:10.1002/jsfa.9423

PubMed Abstract | CrossRef Full Text | Google Scholar

Schmid, M., and Müller, K. (2019). Whey protein-based packaging films and coatings. Whey Proteins Milk Med 2019, 407–437. doi:10.1016/B978-0-12-812124-5.00012-6

CrossRef Full Text | Google Scholar

Siripatrawan, U., Vitchayakitti, W., and Sanguandeekul, R. (2013). Antioxidant and antimicrobial properties of Thai propolis extracted using ethanol aqueous solution. Int. J. Food Sci. Technol. 48, 22–27. doi:10.1111/j.1365-2621.2012.03152.x

CrossRef Full Text | Google Scholar

Sothornvit, R., Hong, S. I., An, D. J., and Rhim, J. W. (2010). Effect of clay content on the physical and antimicrobial properties of whey protein isolate/organo-clay composite films. LWT - Food Sci. Technol. 43, 279–284. doi:10.1016/J.LWT.2009.08.010

CrossRef Full Text | Google Scholar

Souza, V. V. M. A., Almeida, J. M., Barbosa, L. N., and Silva, N. C. C. (2022). Citral, carvacrol, eugenol and thymol: antimicrobial activity and its application in food. J. Essent. Oil Res. 34, 181–194. doi:10.1080/10412905.2022.2032422

CrossRef Full Text | Google Scholar

Sukyai, P., Anongjanya, P., Bunyahwuthakul, N., Kongsin, K., Harnkarnsujarit, N., Sukatta, U., et al. (2018). Effect of cellulose nanocrystals from sugarcane bagasse on whey protein isolate-based films. Food Res. Int. 107, 528–535. doi:10.1016/J.FOODRES.2018.02.052

PubMed Abstract | CrossRef Full Text | Google Scholar

Walsh, S. E., Maillard, J., Russell, A. D., Catrenich, C. E., Charbonneau, D. L., and Bartolo, R. G. (2003). Activity and mechanisms of action of selected biocidal agents on Gram-positive and-negative bacteria. J. Appl. Microbiol. 94, 240–247. doi:10.1046/j.1365-2672.2003.01825.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Wang, F., Zhan, J., Ma, R., and Tian, Y. (2023). Simultaneous improvement of the physical and biological properties of starch films by incorporating steviol glycoside-based solid dispersion. Carbohydr. Polym. 311, 120766. doi:10.1016/j.carbpol.2023.120766

PubMed Abstract | CrossRef Full Text | Google Scholar

Wen, P., Zhu, D. H., Wu, H., Zong, M. H., Jing, Y. R., and Han, S. Y. (2016). Encapsulation of cinnamon essential oil in electrospun nanofibrous film for active food packaging. Food control. 59, 366–376. doi:10.1016/J.FOODCONT.2015.06.005

CrossRef Full Text | Google Scholar

Yadav, S., Mehrotra, G. K., Bhartiya, P., Singh, A., and Dutta, P. K. (2020). Preparation, physicochemical and biological evaluation of quercetin based chitosan-gelatin film for food packaging. Carbohydr. Polym. 227, 115348. doi:10.1016/J.CARBPOL.2019.115348

PubMed Abstract | CrossRef Full Text | Google Scholar

Zhang, W., and Jiang, W. (2020). Antioxidant and antibacterial chitosan film with tea polyphenols-mediated green synthesis silver nanoparticle via a novel one-pot method. Int. J. Biol. Macromol. 155, 1252–1261. doi:10.1016/J.IJBIOMAC.2019.11.093

PubMed Abstract | CrossRef Full Text | Google Scholar

Zhang, W., Li, X., and Jiang, W. (2020). Development of antioxidant chitosan film with banana peels extract and its application as coating in maintaining the storage quality of apple. Int. J. Biol. Macromol. 154, 1205–1214. doi:10.1016/J.IJBIOMAC.2019.10.275

PubMed Abstract | CrossRef Full Text | Google Scholar

Zinoviadou, K. G., Koutsoumanis, K. P., and Biliaderis, C. G. (2009). Physico-chemical properties of whey protein isolate films containing oregano oil and their antimicrobial action against spoilage flora of fresh beef. Meat Sci. 82, 338–345. doi:10.1016/j.meatsci.2009.02.004

PubMed Abstract | CrossRef Full Text | Google Scholar