**Active Edible Films Based on Arrowroot Starch with Microparticles of Blackberry Pulp Obtained by Freeze-Drying for Food Packaging**

**Gislaine Ferreira Nogueira 1, Farayde Matta Fakhouri 2,3,\*, José Ignacio Velasco <sup>2</sup> and Rafael Augustus de Oliveira <sup>1</sup>**


Received: 18 July 2019; Accepted: 19 August 2019; Published: 23 August 2019

**Abstract:** This research work evaluated the influence of the type of incorporation and variation in the concentration of blackberry pulp (BL) and microencapsulated blackberry pulp (ML) powders by freeze-drying on the chemical and physical properties of arrowroot starch films. Blackberry powders were added to the film-forming suspension in different concentrations, 0%, 20%, 30% and 40% (mass/mass of dry starch) and through two different techniques, directly (D) and by sprinkling (S). Scanning electron microscopy (SEM) images revealed that the incorporation of blackberry powder has rendered the surface of the film rough and irregular. Films incorporated with BL and ML powders showed an increase in thickness and water solubility and a decrease in tensile strength in comparison with the film containing 0% powder. The incorporation of blackberry BL and ML powders into films transferred colour, anthocyanins and antioxidant capacity to the resulting films. Films added with blackberry powder by sprinkling were more soluble in water and presented higher antioxidant capacity than films incorporated directly, suggesting great potential as a vehicle for releasing bioactive compounds into food.

**Keywords:** blackberry; arrowroot starch; gum arabic; freeze-drying; water solubility; water vapor permeability; anthocyanins; antioxidant capacity; powder; food packaging

#### **1. Introduction**

The production of active edible films for active edible packaging is gaining interest from researchers and the industry due to their potential to control the quality and stability of many food products (dried fruits, meat and fish, among others) [1,2]. Current research on active packaging has been focusing on the encapsulation of natural bioactive compounds, antimicrobial and antioxidant agents, vitamins, aromas and dyes, within biodegradable packaging materials [1–12]. This approach can improve protection properties and also generate custom properties, such as antioxidants and antimicrobials, innovative flavours, aromas and colours [4–6].

Due to these aspects, countless research projects are being conducted in this direction. For instance, the incorporation of green tea extract into chitosan films [7]; red raspberry extract, rich in anthocyanins, into isolated soy protein films [8]; natural extract of beet and carrot into hydroxypropyl methylcellulose films [9]; oil resin, oregano, olive, pepper, garlic, onion and cranberry into chitosan films; and casein into carboxymethyl cellulose films [10], among many other studies.

Films containing blackberry pulp presented anti-inflammatory activity and increased cell viability [11]. The use of blackberry in films is promising and deserves further exploration, since this

fruit is an excellent agricultural resource for combining nutritional properties and biological activities in the same food, which can bring benefits both to the food in which it will be packed, due to its antioxidants, and to the consumer in terms of nutrition. Blackberry is a rich source of antioxidant compounds, such as phenolic acids, tannins and anthocyanins [13,14].

Several studies have reported higher antioxidant capacity in blackberries based on their oxygen radical absorbance capacity in comparison with other fruits, such as strawberries, red raspberries and red wine grapes [15–17]. In addition, the microencapsulation of blackberry pulp as a possibility to maintain the stability of its antioxidant compounds when exposed to unfavourable conditions (e.g., high temperature, light, oxygen) is a viable alternative and a promising technology to preserve its functionality [18].

Among the techniques of microencapsulation, freeze-drying is the most used in the food industry; low temperatures are applied, which favour the preservation of bioactive materials that are sensitive to high temperatures [19,20]. Freeze-drying is a technique based on dehydration by sublimation of a frozen product. During this procedure, blackberry pulp is homogenized with the encapsulating agent and then co-lyophilized, obtaining in the end a material with a dry aspect [21], which can be easily reduced to powder with microparticles with diameters of μm-mm [22].

The incorporation of microparticles of blackberry into the film can protect antioxidant compounds, and allow their release, in a controlled manner, into the food during storage. In addition, the way of incorporating these microparticles influences their location within the film matrix and this may influence this release, as well as the physico-chemical properties (microstructure, mechanical and barrier properties, colour, anthocyanins content and antioxidant capacity) presented by the film [23].

Recently, Nogueira, Fakhouri and Oliveira [24] reported methods used for the preparation of starch films incorporated, directly and by sprinkling, with dried blackberry microparticles. In the direct incorporation method, the blackberry powder was added directly into the filmogenic solution and later deposited onto the support plate for drying; whereas, in the incorporation method by sprinkling, the filmogenic solution was previously deposited onto the support plate and, subsequently, the blackberry powder was sprinkled with a stainless steel sieve onto its surface. According to the authors, the resulting films were homogeneous and the incorporation of blackberry powder into the films did not make them sticky; instead, they could be easily manipulated. Most of the blackberry particles remained intact after their direct incorporation, as well as by sprinkling, into the film-forming suspension, since it was possible for the naked eye to visualize particles in the resulting dry film. Moreover, using the sieve to sprinkle the powder onto the film-forming solution allowed the blackberry particles to fall evenly across each surface area of the film. Nogueira, Fakhouri and Oliveira [23] confirmed this behaviour through scanning electron microscope (SEM) images, in which particles added by sprinkling into the filmogenic solution tend to remain on the surface, differing from particles added directly into the filmogenic solution, which tend to be more integrated in the resulting film matrix. The fact that the particles are located on the surface of the film allows greater contact with the food or aqueous media, resulting in a faster release of the particles and solubilization of the film. When the film is consumed along with the food, it should be able to release encapsulated compounds to the food system or during passage into the gastrointestinal tract after consumption [23,25]. For active edible films, high solubility in aqueous media is a desirable feature [25].

The incorporation of encapsulated blackberry pulp into the film can promote a controlled release of the antioxidant compounds onto the food surface during storage, where it will act to prevent oxidation and the formation of undesirable food flavours and textures [26–28]. In the work carried out by Talón et al. [27], sunflower oil oxidation was prevented when using films incorporated with encapsulated eugenol. Pork belly packaged with Job's tears starch film containing 0.5% clove bud essential oil exhibited a lower degree of lipid oxidation determined by peroxide and thiobarbituric acid reactive substances than a non-packaged sample during storage [28].

Given the great potential as active packaging, further studies are still necessary for a better understanding of the influence of blackberry microparticles on properties of arrowroot starch films. This knowledge will be important in the future to more efficiently develop active films incorporated with blackberry microparticles and for their eventual application as active food packaging and partial substitutes for non-biodegradable plastic packaging used for specific food wrappers such as sushi, or to be consumed as fruit strips as a source of nutritional compounds.

Thus, the aim of this research work was to develop edible and active arrowroot starch films incorporated, directly (D) and by sprinkling (S), with blackberry pulp (BL) and microencapsulated blackberry pulp (ML) powders by freeze-drying. The influence of the method of incorporation and the variation in the concentration of blackberry powders in properties of starch films were investigated. The blackberry powders were characterized with regard to drying process, moisture content, water activity, hygroscopicity, solubility, microstructure, colour, anthocyanins content and antioxidant properties. The films were characterized regarding microstructure, colour, anthocyanins content, antioxidant properties, thickness, water activity, moisture content, water solubility, water vapor permeability and mechanical properties.

#### **2. Materials and Methods**

#### *2.1. Materials*

In this work, we used frozen blackberries (*Rubus fruticosus*), cv. Tupy, containing total solid content of 10.3 g/100 g of pulp (Agro Monte Verde Eirelli", MG, Brazil), gum arabic InstantgumVR (colloids Naturels, São Paulo, Brazil) containing 14.00% ± 0.10% of moisture content, 1.38% ± 0.16% of proteins, 0.37% ± 0.02% of lipids, 3.79% ± 0.10% of ash and 80.46% ± 0.10% of carbohydrates [29], arrowroot starch containing 15.24% ± 0.19% of water, 0.40% ± 0.03% of protein, 0.12% ± 0.01% of fat, 0.33% ± 0.01% of ash, 83.91% ± 0.10% of carbohydrates [29] and amylose content of 35.20% ± 1.63% [30] and glycerol P.A. (Reagen, Quimibrás Indústrias Químicas S.A.- Rio de Janeiro, Brazil) as plasticizing agent. All other reagents used for the analysis presented analytical grade.

#### *2.2. Production of Blackberry Pulp (BL) and Microencapsulated Blackberry Pulp (ML) Powders by Freeze-Drying*

The production of blackberry pulp (BL) and microencapsulated blackberry pulp (ML) powders by freeze-drying followed the methodology described by Nogueira, Fakhouri and Oliveira [23]. A portion of frozen blackberry pulp with and without encapsulating agent (encapsulating agent consisting of arrowroot starch and arabic gum mixture (1:1 mass/mass) in ratio of 1:1.78 (mass/mass, blackberry pulp solids for encapsulating agent) was homogenized in a homogenizer mixer at room temperature for 5 min, and freeze-dried (Mod. 501, Edwards Pirani, Crawley, West Sussex, UK), with initial temperature of −40 ◦C, pressure of 0.1 mmHg and final temperature of 25 ◦C per 2 h, with total cycle time of 48 h. The resulting dried product was ground in a hammer mill (MR Manesco and Ranieri Ltd.a, model MR020, Piracicaba, Brazil) and sieved (28 mesh). The powders were stocked in hermetic pots, coated with aluminium foil to protect against photodegradation and, subsequently, stored in desiccators containing dried silica gel at 25 ◦C.

#### Characterization of Blackberry Powder

The blackberry powders were characterized with regard to drying process as the ratio between powder solid mass and the mass of total solids in the feed solution, in triplicate. The moisture content of powders was gravimetrically determined, in triplicate, by drying the samples in a vacuum oven at 60 ◦C until constant weight [29]. For the determination of water activity, AquaLab Lite apparatus (Decagon Devices Inc., Pullman, WA, USA) was used, by direct reading of the samples, in triplicate, at 25 ◦C.

Hygroscopicity was determined following the methodology described by Cai and Corke [31], with modifications. Samples (about 1 g in Petri dishes) of each powder were placed, in triplicate in desiccators containing saturated sodium chloride (NaCl) solution (75.7% relative humidity at 25 ◦C). After one week, each sample was weighed and hygroscopicity was expressed as grams of water absorbed per 100 g of dry solids.

For the determination of solubility, the method proposed by Eastman and Moore (1984), cited by Cano-Chauca, Stringheta, Ramos and Cal-Vidal [32] was followed. One gram of each sample was added to 100 mL of distilled water and maintained at high stirring speed in the magnetic stirrer for 5 min. Then, the solution was centrifuged at 3000 G for 5 min. An aliquot of 25 mL of the supernatant was transferred to pre-weighed Petri dishes and submitted to drying in the oven at 105 ◦C until constant weight. Solubility (%) was calculated by weight difference.

#### *2.3. Incorporation of Blackberry Powders into Film-Forming Solution*

#### 2.3.1. Preparation of Film-Forming Solution

Film-forming solution was prepared by dispersing arrowroot starch in distilled water (4%, mass/mass), as optimized by Nogueira, Fakhouri and Oliveira [33], and heated at 85 ◦C in a thermostatic bath, under constant agitation, for about 5 min. Then, glycerol was added to the starch solution at a concentration of 17% (mass/mass of total solids, as optimized by Nogueira, Fakhouri and Oliveira [33]) and homogenized. Blackberry powders were added to the film-forming solution in concentrations of 0%, 20%, 30% and 40% (mass/mass of total solids). This incorporation of blackberry powder into the film-forming solution was performed in two ways, directly (D) and by sprinkling (S), following the methodology proposed by Nogueira, Fakhouri and Oliveira [24].

#### 2.3.2. Direct Incorporation of Blackberry Powders (D) into Film-Forming Solution

Blackberry pulp powders (BL) and microencapsulated blackberry pulp powder (ML) were added directly into the film-forming solution and homogenized manually with the aid of a drumstick. Aliquots of 25 mL of resulting solutions were dispensed onto Plexiglas plates (12 cm diameter). Films were dried for about 24 h, at room temperature (25 ± 5 ◦C). After being removed from the plates, the films were conditioned at 25 ◦C and 55% ± 3% of relative humidity for 48 h before their characterization.

#### 2.3.3. Incorporation of Blackberry Powder by Sprinkling (S) into Film-Forming Suspension

Aliquots of 25 mL of resulting film-forming solution (solution of arrowroot starch and glycerol obtained according to item 2.3.1) were initially deposited onto Plexiglas plates (12 cm diameter). Blackberry pulp powder (BL) and microencapsulated blackberry pulp powder (ML) were homogeneously sprinkled, through a stainless-steel sieve with 53 mesh, onto all the surface area of the film-forming solution already disposed on the plates [24]. Films were removed from the support plates after drying for 24 h at room temperature (25 ± 5 ◦C). Films were stored at 25 ◦C and 55 ± 3% relative humidity for 48 h before their characterization.

#### *2.4. Films Characterization*

#### 2.4.1. Visual Aspect

Visual and tactile analyses were performed in order to select the most homogeneous films that were flexible for handling when removed from plates. Films without these characteristics were rejected.

#### 2.4.2. Microstructure

The morphological characteristics of the surface and the cross section developed from the film samples were examined under a scanning electron microscope (Leo 440i, Electron Microscopy/Oxford, Cambridge, England). Film samples were placed on double-sided carbon adhesive tape adhered to a stub, submitted to the application of a gold layer (model K450, Sputter Coater EMITECH, Kent, UK and observed in a scanning electron microscope operated at 20 kV.

#### 2.4.3. Colour Determination

The colours of the films were measured using a Hunter Lab colorimeter (Color Quest XE 2819, USA). The equipment was set with D65 illuminant and calibrated with a standard white reflector plate. Three films of each treatment were evaluated. The CIELab, mainstream color space coordinate system defined by International Commission on Illumination (CIE) was used for determining a\*, b\* and L\* parameters, where L\* is luminosity (L\* = 0 black and L\* = 100 white), a\* is the greenness and redness of samples (+a\* = red and -a\* = green) and b\* represents the blueness and yellowness (+b\* = yellow and -b\* = blue). Colour difference (ΔE \*) between the films were calculated according to Nogueira, Fakhouri and de Oliveira [24].

#### 2.4.4. Anthocyanins Content

The anthocyanins content in the films and powders was determined by the method employed by Sims and Gamon [34], with adaptations. Film samples were previously macerated in liquid nitrogen, weighed in triplicate and homogenized with 3 mL of cold acetone/Tris-HCl solution (80:20, volume/volume, pH 7.8 0.2 M) for 1 min. The samples remained at rest for 1 h, protected from light and centrifuged for 15 min at 3500 rpm. The supernatants were immediately read in a spectrophotometer (B422 model, Micronal) in visible region at 537 nanometers (anthocyanins). The acetone/Tris-HCl solution was used as blank sample. Absorbance values were converted to mg/100 g of blackberry pulp solids.

#### 2.4.5. Antioxidant Capacity

Antioxidant capacity was determined by the ABTS (2.2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) method, which estimates the sample's capacity to isolate ABTS radicals. These ABTS radicals were formed by the reaction of 140 mM potassium persulfate with standard 7 mM ABTS solution, stored in the dark for 16 h at room temperature. Then, ABTS (P.A.) was diluted with ethanol to obtain the absorbance value of 0.70 nm ± 0.05 nm to 734 nm. Samples of the films and powders were extracted in methanol solution (50% methanol in distilled water, *v*/*v*) and then in acetone solution (70% acetone in distilled water, *v*/*v*) to determine their antioxidant capacity. Aliquots of 30 μL of extract were added to 3 mL of ABTS radical and kept in the dark for 6 min. Standard curve was fitted with Trolox [6-hydroxy-2.5.7.8-tetrametilchroman-2-carboxylic acid] at concentrations ranging from 100 to 2000 μM. Results were calculated according to an equation fitted by standard curve and expressed by μg.g−<sup>1</sup> solid of Trolox equivalent (TE). All of the analyses were performed in triplicate.

#### 2.4.6. Film Thickness, Water Activity and Moisture Content

Film thickness was measured using a micrometre (Mitutoyo brand, model MDC 25 M, MFG/Japan). For each film, thickness was determined by randomly measuring 10 different regions of the film. The moisture content of films was gravimetrically determined, in triplicate, by drying the samples in an air-forced oven 105 ºC for 24 h [29]. The water activity was determined using AquaLab Lite apparatus (Decagon Devices Inc., Pullman, WA, USA), by direct reading of the samples, circularly sized into 35 mm in diameter, in triplicate, at 25 ◦C.

#### 2.4.7. Solubility in Water

The method proposed by Gontard, Guilbert, and Cuq [35] was used for determining the water solubility of film samples, which were cut in circles (2 cm of diameter) and weighed (initial dry weight at 105 ◦C for 24 h). Then, they were placed individually into beakers containing 50 mL of distilled water, and stirred at 75 rpm for 24 h at 25 ± 2 ◦C. Finally, the samples were removed and dried at 105 ◦C until constant weight (final dry weight). The solubility of films (%) was calculated as the percentage of total soluble matter.

#### 2.4.8. Water Vapor Permeability

Water vapor permeability tests were conducted using the ASTM E96-80 method [36]. Film samples were placed, in triplicate, in acrylic permeation cells containing dried calcium chloride (0% relative humidity at 25 ◦C). These cells were weighed and placed in a desiccator maintained at 25 ◦C and 75% RH using saturated sodium chloride solution.

Water vapor transferred through the film was determined by the mass gain of calcium chloride. Cells were weighed daily for at least 7 days. For each film sample, the thickness was determined by random measurements of 5 different regions of the film. The water vapor permeation rate (WVP) was obtained following the equation described by Nogueira, Fakhouri and de Oliveira [24].

#### 2.4.9. Mechanical Properties

Tensile strength and elongation at break were obtained using a texturometer conducted according to the ASTM standard method D 882-83 [37], with modifications [38]. For each film, six samples were cut in rectangular strips (100 mm × 25 mm). Thickness was determined by randomly measuring 5 different regions of the sample, before analyses. For this test, films were fixed by two distal claws, initially 50 mm apart, which moved at the speed of 1 mm/s. Tensile strength (MPa) was calculated by dividing the maximum force at the moment of rupture (N) by cross-sectional area of the film (m2). Elongation at break (%) was calculated dividing the difference between the distance at the moment of rupture (cm) and the initial separation distance (cm) by the distance at the moment of rupture (cm), multiplied by 100.

#### *2.5. Statistical Analysis*

Significant differences between average results were evaluated by analysis of variance (ANOVA) and Tukey test at 5% level of significance, using SAS software (Cary, NC, USA).

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

#### *3.1. Characterization of Blackberry Powder*

Figure 1 shows microstructure of BL and ML powders observed through SEM images. External morphology of freeze-dried BL and ML resembled a broken glass structure of variable sizes, with typical folds, slight cracks and porosity on the surface due to the loss of water content during the freeze-drying process. Similar characteristics were observed by Yamashita [21] and Franceschinis, Salvatori, Sosa and Schebor [39] for freeze-dried blackberry (*Rubus* spp.) powder.

Furthermore, for ML powder, it was possible to observe spherical particles distributed throughout their vitreous structures. It is believed that these particles are the result of the microencapsulation of blackberry pulp by the mixture of arrowroot starch and arabic gum used as an encapsulating agent. Shi et al. [40] produced nanoparticles of starch by spray-drying and freeze-drying methods. Nanoparticles of starch produced by both methods were spherical and showed very similar morphology.

Table 1 shows the results of the characterization of blackberry pulp (BL) and microencapsulated blackberry pulp (ML) powders obtained by freeze-drying. The yield obtained from the blackberry pulp's drying process, with or without encapsulating agents, was high. BL and ML powders presented low water content and the water activity was less than 0.3, indicating that they are biochemically and microbiologically stable because below that value (Aw < 0.3) there are interruptions in non-enzymatic reactions and there is no growth of microorganisms [41].

**Figure 1.** Scanning electron micrographs of freeze-dried blackberry pulp (BL, images **A**, **B** and **C**) and freeze-dried microencapsulated blackberry pulp (ML, images **D**, **E** and **F**) powders: images (**A**,**D**) 250× magnification, (**B**,**E**) 1000× magnification, (**C**,**F**) 3000 × magnification.

ML powder was significantly (*p* < 0.05) less hygroscopic and soluble than BL powder. This fact happened because arrowroot starch and arabic gum are materials with low hygroscopicity; consequently, the microencapsulation of blackberry pulp tends to reduce the hygroscopicity of resulting powders. Besides, although arabic gum is highly soluble [42], arrowroot starch in its native form presents low solubility in water at room temperature. This fact probably contributed to the decrease in the solubility of the blackberry powder.


**Table 1.** Characterization of blackberry powder obtained by freeze-drying.

Same letters in the same line show no statistical difference (*p* > 0.05).

Although microencapsulation is a widely used method to protect bioactive compounds against adverse environmental conditions, such as pH, light and oxygen [18], no significant differences (*p* > 0.05) were observed between the values of anthocyanins and antioxidant properties for BL and ML powders. Since freeze-drying is a method that does not use high temperatures during the drying process and is based on dehydration by the sublimation of a frozen product, these factors probably contributed to the maintenance of bioactive compounds and antioxidant capacity [21].

The resulting powders presented a reddish colour, typical of blackberry pulp. ML powder showed significantly (*p* < 0.05) lighter coloration than BL powder, due to the presence of an encapsulating agent which has a lighter colour.

#### *3.2. Films Characterization*

#### 3.2.1. Visual Aspect

The incorporation of blackberry powders into the film-forming solution gave the arrowroot starch films a reddish colour, which can be observed in Figure 2. In larger concentrations, the colour was more visually remarkable. All films could be removed from the plates and, in general, had good appearance and transparency. Films with 0%, 20%, 30% and 40% of blackberry pulp powder (BLD) and microencapsulated blackberry pulp powder (MLD) directly incorporated were visually homogeneous, continuous and very flexible for handling. On the other hand, films with blackberry powders (BL and ML) incorporated by sprinkling (S) were brittle and sensitive to handling.

**Figure 2.** Photographic images of the films samples with 0%, 20%, 30% and 40% of freeze-dried blackberry pulp (BL) and freeze-dried microencapsulated blackberry pulp (ML) incorporated directly (D) and by sprinkling (S): (**A**) 0%; (**B**) 20% BLD; (**C**) 30% BLD; (**D**) 40% BLD; (**E**) 20% BLS; (**F**) 30% BLS; (**G**) 40% BLS; (**H**) 20% MLD; (**I**) 30% MLD; (**J**) 40% MLD; (**K**) 20% MLS; (**L**) 30% MLS; (**M**) 40% MLS.

#### 3.2.2. Microstructure

The morphological characteristics of the films can be observed in Figure 3. Differently from the film with 0%, which presented organized polymer matrix and regular surface (Figure 3A–C), films developed with 40% of blackberry powders presented rough surfaces and morphological characteristics directly associated to those observed in BL and ML powders. Shi et al. (2013) also observed that the incorporation of starch nanoparticles obtained by spray drying and freeze-drying made the surface of starch film rough. According to the authors, protuberances found on the film

surface resulted from the presence of starch nanoparticles. The structural characteristics exhibited by the resulting films also varied according to the type of technique, directly or by sprinkling, employed in the incorporation of the blackberry powders into the film-forming solution. The cross-section images of films incorporated directly with 40% BL and ML (Figure 3D–F,J–L, respectively) revealed the presence of dispersed and agglomerated blackberry particles within the starch network, which resulted in organised and disorganised regions.

**Figure 3.** Scanning electron microscope (SEM) images of the films with 0% and 40% of freeze-dried blackberry pulp (BL) and freeze-dried microencapsulated blackberry pulp (ML) incorporated directly (D) and by sprinkling (S): (**A**) surface of control film; (**B**,**C**) cross section of control film; (**D**) surface of 40% BLD film; (**E**,**F**) cross section of 40% BLD film; (**G**) surface of 40% BLS film; (**H**,**I**) cross section of 40% BLS film; (**J**) surface of 40% MLD film; (**K**,**L**) cross section of 40% MLD film; (**J**) surface of 40% MLS film; (**K**,**L**) cross section of 40% MLS film. Images (**A**), (**C**), (**F**), (**I**), (**L**) and (**O**) with 250× magnification and images (**B**), (**D**), (**E**), (**G**), (**H**), (**J**), (**K**), (**M**) and (**N**) with 1000× magnification.

This result may have been obtained due to the poor dispersion of powders in high concentrations in the film-forming solution, given their high viscosity. Similar characteristics were observed by Sartori and Menegalli [43] in film containing solid lipid microparticles. Castillo et al. [44] observed randomly dispersed nano-agglomerates and individual platelets of talc in nanocomposite films by transmission electron microscopy (TEM). Mukurumbira, Mellem and Amonsou [25] observed that the incorporation of nanocrystals into starch films made their surfaces irregular and rough. According to the authors, these changes could be attributed to the presence of aggregated nanocrystals and, possibly, the interactions between nanocrystals and amylose in the starch. Ortega, Giannuzzi, Arce and García [45] incorporated silver nanoparticles into starch films and also observed the presence of agglomerates of nanoparticles in the gelatinized starch suspension.

For films embedded with sprinkled blackberry powder it was observed that the use of a sieve to sprinkle BL and ML powders over the film-forming solution allowed them to become adhered to the film surface after drying. In addition, as can be seen in Figure 3G–I, the ML particles penetrated more into the starch matrix than the BL particles, which tended to remain mostly on the surface of the matrix. Probably, ML particles are denser than BL (Figure 3M–O).

#### 3.2.3. Colour Determination

Table 2 presents colour parameters of film samples with 0% [24], 20%, 30% and 40% of blackberry pulp (BL) and microencapsulated blackberry pulp (ML) incorporated directly (D) and by sprinkling (S). Films incorporated with blackberry exhibited colorimetric parameters correlated with those found for BL and ML powders (Table 1), differing significantly from the film with 0% [24] which was colourless.

**Table 2.** Colour parameters of film samples with 0%, 20%, 30% and 40% of freeze-dried blackberry pulp (BL) and freeze-dried microencapsulated blackberry pulp (ML) incorporated directly (D) and by sprinkling (S). Results of the control film characterization (0%) were published by Nogueira, Fakhouri and Oliveira [24].


Same letters in the same column show no statistical difference (*p* > 0.05).

The results indicate that the incorporation and concentration of BL and ML significantly (*p* < 0.05) affected chromaticity parameters (a\* and b\*) of films. With the incorporation of BL and ML into the film with 0% [24], values of b\* increased, varying from negative to positive, showing a propensity for yellow coloration. Similarly, Li et al. [46] observed that the addition of starch nanocrystals resulted in the yellowing of pea starch films.

A relevant increase in values of a\* was also observed, evidencing a tendency for red coloration and the presence of anthocyanins pigments. It was reported that a\* coordinate is attributed to the anthocyanins content in blackberry, which is responsible for the red colour of BL and ML powders [21,47]. Once again, low values indicate that films developed with BL and ML powders exhibited colour shades ranging from red to yellow-orange, correlating with film visual observations.

Ortega et al. [45] also observed that the incorporation and concentration of silver nanoparticles significantly affected chromaticity parameters (a\* and b\*); although, in both cases the corresponding values were very low and, visually, nanocomposite films remained colourless.

As expected, the incorporation of BL and ML powders and their concentration also significantly affected (*p* < 0.05) colour difference (DE). The increase from 20% to 40% in the concentration of BL and ML incorporated directly and by sprinkling into arrowroot starch film (0% [24]) caused a statistically significant increase (*p* < 0.05) in a\* and b\* values, leading to a decrease in luminosity L\* and increase in total colour difference ΔE\* (Table 2).

#### 3.2.4. Anthocyanins Content and Antioxidant Capacity

Table 3 shows anthocyanins content (mg/100 g of blackberry solids) and antioxidant capacity (μmol of Trolox/g of blackberry solids) of films after the drying process. In the absence of blackberry powder (BL and ML), arrowroot starch film showed an insignificant amount of anthocyanins content and antioxidant capacity. Thus, it is clear that the anthocyanins content and antioxidant capacity presented by arrowroot starch films incorporated with BL and ML are directly related to their content in blackberry powders (Table 1).

It is important to add that there was a decrease in the anthocyanins content of films with BL and ML powders when compared with the initial amount of the respective powders. This happened because anthocyanins present great susceptibility to degradation when exposed to environmental factors such as temperature, light, pH and oxygen [48] during film production, resulting in their decrease in dried films. Maniglia, Tessaro, Lucas and Tapia-Blácido [49] also reported losses of phenolic compounds due to a possible oxidative degradation of phenolic groups caused by heating applied during the preparation of film-forming solution and during the film-drying process. Nevertheless, in this research work, increasing the concentration of blackberry incorporated into the film led to a slight increase in anthocyanins content.

**Table 3.** Anthocyanins content and antioxidant capacity of films with 0%, 20%, 30% and 40% of freeze-dried blackberry pulp (BL) and freeze-dried microencapsulated blackberry pulp (ML) incorporated directly (D) and by sprinkling (S). Results of the control film characterization (0%) were published by Nogueira, Fakhouri and Oliveira [24].


Same letters in the same column show no statistical difference (*p* > 0.05). \* For 0% [24] film, anthocyanins content and antioxidant capacity is expressed by total solids, Total Anthocyanins (mg/100 g of total solids) and ABTS (μmol of Trolox/g of total solids).

In scientific literature there are several works reporting a high correlation between phenolic compound content and antioxidant capacity [50,51]. However, increasing the concentration from 20% to 40% BL and ML powders incorporated into the film-forming solution has shown a tendency to increase the anthocyanins content in the resulting films. This same trend was not observed for the antioxidant capacity. A similar behaviour was observed by Nogueira et al. [52] for arrowroot starch films incorporated with blackberry pulp, and by Chang-Bravo, López-Córdoba, and Martino [53] for extracts of yerba mate and propolis. According to Chang-Bravo, López-Córdoba, and Martino [53], for both

extracts, DPPH(2,2-Diphenyl-1-picryl-hidrazil) radical scavenging activity increased proportionally to their polyphenols content until reaching a plateau in which the antioxidant capacity became independent from the concentration.

As for the antioxidant capacity, the type of incorporation of BL and ML powders, either directly or by sprinkling, had more influence than the variation in their concentration in films. The films incorporated with 30% and 40% of BL and ML by sprinkling had the highest antioxidant capacity. The fact that the particles of BL and ML remained on the surface of the film probably facilitated the extraction of bioactive compounds, due to a greater number of particles in direct contact with the extraction solvent, which consequently generated greater antioxidant activity. However, in direct incorporation, the BL and ML powders integrated into the polymer matrix, which probably reduced their surface area when in direct contact with extraction solvents. This fact may have hindered the extraction of bioactive compounds and, consequently, generated lower antioxidant capacity.

Moreover, it is important to emphasize that there are great varieties of bioactive compounds with antioxidant capacity in fruit extracts [50]. In addition to anthocyanins, other bioactive phenolic acids, tannins and ascorbic acid [14] may be also present in films with blackberry, contributing to the total antioxidant capacity of BL and ML powders.

#### 3.2.5. Water Activity and Moisture Content

Water activity and moisture content values of films are shown in Table 4. The water activity obtained for the control film (0% [24]) and films containing BL and ML ranged from 0.37 to 0.55. Films can be considered stable against microbial proliferation. According to Quek, Chock and Swedlund [54], in general, food with aw < 0.6 is considered microbiologically stable and, in case of any spoilage, it is induced by chemical reactions rather than by microorganisms.

Moisture content in the control film (0% [24]) and in films containing BL and ML ranged from 7.88% to 13.65%. Similar results were found for films made of amadumbe and potato starch with amadumbe starch nanocrystals (0, 2.5, 5 and 10%), which presented moisture content ranging from 9.3% to 13.4% and 14.8% to 16.7%, respectively [25].

The incorporation of BL and ML powders by sprinkling (S) led to a significant decrease (*p* < 0.05) in the moisture content of films in comparison with those incorporated directly and the control films (0% [24]). Li et al. [46] observed a decrease in moisture content with the incorporation of starch nanocrystals into pea starch films. In this study, the impact of particles falling onto the film-forming solution by sprinkling may have triggered a discontinuity of polymer matrix on its surface, favouring water release from the starch structure. Low water content in BLS and MLS films may be one of the possible causes of brittleness and fragility of these films.

**Table 4.** Water activity and moisture content of films with 0%, 20%, 30% and 40% of freeze-dried blackberry pulp (BL) and freeze-dried microencapsulated blackberry pulp (ML) incorporated directly (D) and by sprinkling (S). Results of the control film characterization (0%) were published by Nogueira, Fakhouri and Oliveira [24].


Same letters in the same column show no statistical difference (*p* > 0.05).

#### 3.2.6. Film Thickness, Water Solubility, Water Vapor Permeability and Mechanical Properties

Table 5 shows thickness (mm), water solubility (%), water vapor permeability (g.mm/m2.day.kPa), tensile strength (MPa) and elongation at break (%) of films with 0% (0%, [24]), 20%, 30% and 40% of BL and ML powders incorporated directly (D) and by sprinkling (S).

**Table 5.** Thickness (mm), water solubility (%), water vapour permeability (g.mm/m2.day.kPa), tensile strength (MPa) and elongation at break (%) of films with 0%, 20%, 30% and 40% of freeze-dried blackberry pulp (BL) and the freeze-dried microencapsulated blackberry pulp (ML) incorporated directly (D) and by sprinkling (S). Results of the control film characterization (0%) were published by Nogueira, Fakhouri and Oliveira [24].


Same letters in the same column show no statistical difference (*p* > 0.05).

Water solubility, water vapor permeability and mechanical properties are directly influenced by film thickness, among other factors [45]. Thickness of films (0%, [24]) made from arrowroot starch and of films incorporated with BL and ML powders ranged from 0.065 mm (0% [24]) to 0.173 mm (40% MLS). The increase of 20% to 40% in the concentration of BL and ML, incorporated directly and by sprinkling into the arrowroot starch matrix (0% [24]), caused a statistically significant increase (*p* < 0.05) in the thickness of the control film (0%, [24]). This increase in thickness is due to the increase in solid content, since the same volume of the film-forming solution was used in the plate area. Besides, the agglomeration of blackberry particles in the polymer matrix, evidenced by prominences on the surface of films by SEM (Figure 3), may have contributed to the increase in film thickness. Ortega et al. [45] also observed that silver nanoparticles caused a slight increase in film thickness. The authors also attributed these results to the increased solid content and possible agglomeration of silver nanoparticles.

Water solubility, water vapor permeability and mechanical property are important parameters for choosing the application of biopolymer films [25,55–59]. Some applications, considering the potential use of these new polymer films instead of synthetic packaging, may require low water solubility, strength and flexibility to tolerate the typical effort made by packaging materials during handling and food transportation, maintaining the integrity of the product [25,55,56]. Some other applications, such as encapsulation, may require significantly higher solubility [25,55] in order to allow release of the encapsulated material into the surroundings.

The type of incorporation and the variation of BL and ML concentrations had significant effect (*p* < 0.05) on the solubility, tensile strength and elongation at break of starch films. In general, water solubility increased significantly (*p* < 0.05) while tensile strength decreased after increasing the concentrations of BL and ML powders (20%, 30% and 40%) in films, in comparison with the control film (0%, [24]), by direct incorporation. This behaviour could be attributed to a possible reduction of intermolecular attraction forces caused by agglomerations of BL and ML [25,44,45], which led to the disrupting and discontinuity of starch matrix [23] (Figure 3). Consequently, the polymer network

was less dense, facilitating water permeation in its structure and solubilization, and reducing its resistance and increasing flexibility. Films prepared with babassu mesocarp flour and starch isolated from babassu mesocarp by casting exhibited similar behaviour [49].

Films incorporated directly with BL and ML were less water soluble than films incorporated with blackberry powder by sprinkling. The location of blackberry particles probably influenced this behaviour. As showed in Figure 3, blackberry powder particles incorporated by sprinkling tended to remain on the surface of the film, unlike directly incorporated particles, which were introduced into the polymer starch network. The fact that the particles of blackberry powder stayed on the surface of the films is believed to have enabled a greater and direct contact of these particles with the water. Additionaly, the fact that the particles are porous and hydrophilic probably led to their solubilization and the formation of holes on the surface of the films, which allowed water molecules to go inside the starch matrix and then its solubilization [23]. The same phenomenon of increased water absorption was observed in the chitosan/starch films with halloysite nanotubes, which was attributed to the increased porosity and hydrophilicity of the nanocomposite films [60]. The addition of hydrophilic clay into kafirin films also affected the hydrophilicity of the film due to the presence of Si-OH groups [61].

As equally observed for water solubility, water vapor permeability presented by films was also significantly affected (*p* < 0.05) by the incorporation of blackberry powder, as well as by the variation in its concentration. According to Ludueña, Vázquez, and Alvarez [62], the passage of water molecules through a polymeric material is the balance between three principal mechanisms: film crystallinity, tortuous pathways through the polymeric matrix, and the presence of structural defects on the surface.

Films with only 20% of powder presented decreasing water vapor permeability in relation to the film with 0% (0%, [24]), as previously reported by Shi et al. [40] for starch films containing spray dried and vacuum freeze-dried starch nanoparticles. At low concentrations, BL and ML powders were easily dispersed into the film-forming solution, increasing compactness of the films, which may have hindered the passage of water molecules [40,46,63]. It is also possible that the presence of blackberry powder particles within the starch matrix, as well as on the surface, introduced a tortuous path for the passage of water molecules, which may have led to a decreasing behaviour in water vapor permeability. A similar result regarding a decrease in water vapor permeability was observed when chitin nano-whiskers (CNWs) was incorporated into the maize starch–based films [64].

In a single polymer film, the diffusible molecules migrate from a straight (middle) path that is perpendicular to the film surface. Whereas, in films with nanocomposites, the diffusion molecules must navigate through tortuous paths due to the presence of particles or platelets, as well as through interfacial zones of different permeability characteristics in comparison with those with pure polymer [64–69]. In theory, the longer the diffusive pathway of the penetrant, water molecules in this case, the lower the permeability [25,63].

However, when more than 20% of BL and ML powders were incorporated directly, the particles tended to form aggregations, as observed in the microstructure of 40% BLD and MLD films (Figure 3). The aggregation of BL and ML particles reduces the interaction between the active surface area and the polymer matrix. This fact tends to weaken their adhesion to the starch matrix interface, destroying the orderly structure of the film to 0% , increasing water vapor permeability [25,64], and following the same tendency observed for water solubility.

#### **4. Conclusions**

This study has developed a new understanding of the influence of the incorporation of blackberry particles obtained by freeze-drying into the properties of arrowroot starch-based films. The addition of blackberry pulp particles causes interactions with the film matrix, inducing changes in the properties of films. These interactions alter the microstructure, as well as the chemical, mechanical, and barrier properties of the films. It was found that the incorporation of blackberry pulp particles does not only make the surface of arrowroot starch rough and irregular, but also thicker, more flexible, soluble in

water, and less mechanically resistant. Additionally, blackberry pulp particles transferred colour, anthocyanins and antioxidant capacity to the arrowroot starch film.

Properties presented by the resulting films were also influenced by the concentration and the type of method used, direct or by sprinkling, for the incorporation of blackberry powders into the film-forming solution. Films with only 20% BL and ML presented lower water vapor permeability rates than the film with 0%. This behaviour was attributed to a better dispersion of blackberry powder at low concentrations in the film-forming solution, as well as to the introduction of tortuous paths in the starch matrix. At concentrations above 30%, there was an increase in water vapor permeability due to the presence of agglomerated blackberry powder particles. Films incorporated with blackberry powder by sprinkling had higher antioxidant capacity and were more soluble in water, showing great potential to be used as a vehicle for releasing bioactive compounds into the surroundings.

**Author Contributions:** Conceptualization, F.M.F.; Funding acquisition, J.I.V. and R.A.d.O.; Investigation, G.F.N. and F.M.F.; Supervision, J.I.V. and R.A.d.O.; Writing—original draft, G.F.N.; Writing—review and editing, F.M.F. and R.A.d.O.

**Funding:** The research leading to these results has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 712949 (TECNIOspring PLUS) and from the Agency for Business Competitiveness of the Government of Catalonia.

**Acknowledgments:** The authors thank the Coordination for the Improvement of Higher Education Personnel (CAPES, No 01-P-3712/2017) and the School of Agricultural Engineering – University of Campinas for their financial support.

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

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Effect of Cinnamon Extraction Oil (CEO) for Algae Biofilm Shelf-Life Prolongation**

#### **Maizatulnisa Othman 1,\*, Haziq Rashid 1, Nur Ayuni Jamal 1, Sharifah Imihezri Syed Shaharuddin 1, Sarina Sulaiman 1, H. Saffiyah Hairil 2, Khalisanni Khalid <sup>3</sup> and Mohd Nazarudin Zakaria <sup>4</sup>**


Received: 26 November 2018; Accepted: 17 December 2018; Published: 20 December 2018

**Abstract:** This study was conducted to improve the life-span of the biofilm produced from algae by evaluating the decomposition rate with the effect of cinnamon extraction oil (CEO). The biofilm was fabricated using the solution casting technique. The soil burying analysis demonstrated low moisture absorption of the biofilm, thus decelerating the degradation due to low swelling rate and micro-organism activity, prolonging the shelf-life of the biofilm. Hence, the addition of CEO also affects the strength properties of the biofilm. The maximum tensile strength was achieved with the addition of 5% CEO, which indicated a good intermolecular interaction between the biopolymer (algae) and cinnamon molecules. The tensile strength, which was measured at 4.80 MPa, correlated with the morphological structure. The latter was performed using SEM, where the surface showed the absence of a separating phase between the biofilm and cinnamon blend. This was evidenced by FTIR analysis, which confirmed the occurrence of no chemical reaction between the biofilm and CEO during processing. The prolongation shelf-life rate of biofilm with good tensile properties are achievable with the addition of 5% of CEO.

**Keywords:** cinnamon extraction oil; algae; biodegradation; shelf-life; food packaging

#### **1. Introduction**

Quotidian plastic materials for food packaging in the market is proffered using a synthetic polymer base like polyproline and polyethylene, which is strenuous to decompose. Based on every day activities of Malaysian households and industries, high amounts of solid waste materials seem to have polluted and harmed the landfill with poison. In the interest of conserving the landfill, while overcoming the solid waste pollution issues, a few groups of individuals. such as researchers, the government, and industrial players [1], came up with several solutions. Starting off, the government promotes the routine of recycling, reducing, and reusing on a daily basis to the community [2]. Industrial players and researchers further focus on recycling plastics to be used as secondary materials and attempts to recycle and convert those materials into synthetic fiber threads and yarns to produce jerseys, shoes, and other textile products [3]. Nonetheless, the main problems are nowhere near solved

at this point. The solid waste keeps on continuing its revolution in our daily life. The development of the first environmentally friendly materials consist partly of conventional plastic polyethylene (PE) or polypropylene (PP) and partly of nature's own material—chalk (40% by weight). Until early 2004, the research growth on producing the bio-based product was expanding throughout the entire globe [4]. PLA (polylactic acid) films, produced from lactic acid, have shown the highest commercial potential and are now produced on a comparatively large scale. Most bioplastics are produced based on natural biomaterials, such as corn, starch, and soybean [5]. Moreover, other food harvests, for example, cassava, wheat, potato, and sago, have also been transformed into plastic to supplant oil-based plastic [6]. Since those products are food assets for human beings, continuous transformation of those yields into plastic will soon interfere with human sustenance supply by reducing the world's sustenance assets. In an effort to prevent interference with food assets, other biomaterials should be assessed. Contemporaneous edible films have the potential to substantially reduce the environmental burden due to food packaging and limit moisturization, aroma, and lipid migration between food components [7]. Dealing with the biopolymer materials, it is difficult to avoid fugacious shelf-life issues. Biopolymer, which derives from natural sources, is commonly known for having transitory shelf-life as compared to synthetic biopolymer. Problems and errors may arise during the storage time either for logistic purposes or during transportation of the biopolymer to the industrial consumer. Humidity, temperature, micro-organism, and fungi attack may affect the quality and deteriorate the strength of the biopolymer. Accordingly, this study focuses on producing biofilm with sustainable shelf-life, high quality, low toxicity, and cheap costs, which could provide efficient food chain supply. Therefore, algae are chosen to be used in biopolymer film fabrication. Algae, or seaweed, is an environmental asset that exists in boundless amounts, which can be cultivated naturally. The Agarose chemical structure provides a good support for films. In addition, it is reported that the films, which are made of algae, are transparent, strong, and flexible [2]. In order to enhance the shelf-life and improve the strength of the algae film, cinnamon extract was used to act as a co-primer and anti-microbial in the film. Based on a previous study, anti-microbial polymer film was able to restrain microbial development, hence, broadening the time span of usability of sustenance [6]. Cinnamaldehyde is an organic compound with the formula of C6H5CH=CHCHO and occurs naturally as a predominant trans (E) isomer, giving cinnamon its flavor and odor [1]. It is a type of flavonoid that is naturally synthesized by the shikimate pathway [2]. This pale yellow, viscous liquid occurs in the bark of cinnamon trees and other species of the genus *Cinnamomum* sp. The essential oil of cinnamon bark contains about 50% cinnamaldehyde [3]. Cinnamaldehyde is also used as a fungicide [8]. Proven effective on over 40 different crops, cinnamaldehyde is typically applied to the root systems of plants [5]. Its low toxicity and well-known properties makes it ideal for agricultural activities. Thus, "cinnamaldehyde" is an effective insecticide, and its scent is also known to repel animals, such as cats and dogs [8]. It has also been tested as a safe and effective insecticide against mosquito larvae [9]. At a concentration of 29 ppm, cinnamaldehyde can kill half of Aedes aegypti mosquito larvae within 24 h [10]. The "trans-cinnamaldehyde" also works as a potent fumigant and practical repellent for adult mosquitos [11]. By adding cinnamon active chemicals into the packaging system [5], the growth rate of microorganisms in food can be inhibited or reduced. Among other antimicrobials, cinnamaldehyde, which is a major component of cinnamon, also possesses antimicrobial activity and has been utilized in the processing of milk, chicken, and meat [5,6]. The objectives of this study were (1) to assess the suitable percentage loading of cinnamon extract with algae film, and (2) to characterize the effect of cinnamon with the biofilm based on the soil bury test, tensile test, FTIR, and SEM.

#### **2. Materials and Methods**

Raw algae and cinnamon were purchased from a local store, located in Gombak, Malaysia, while glycerol and acetic acid were then purchased from Sigma Aldrich (Selangor, Malaysia). For the preparation of raw materials of the biofilm, algae were processed though cleaning, drying, and shredding into powder form (ranging from 50 μ–100 μ). The cinnamon oil was collected using the microwave essential oil extraction method. Temperature was set at 60 ◦C for 6 min as the cinnamon started to steam up and condensation took place for the production of the extraction oil. Next, we let the extraction oil cool down for another 20 min before collecting it. Using a separator, we collected the oil and applied low heat (33–35 ◦C) to separate the oil and water. After 20 min of heating process, the oil particle moved on top of the water surface. The oil was collected using the pipet and placed in the vial.

#### *2.1. Solution Casting Method*

The algae biofilm was prepared using the solution casting method. The ingredients to prepare biofilm with CEO is as follows: 2% algae powder, three different percentages of CEO at 1%, 3%, and 5%; 1.5 mL glycerol solution; 1%, 3%, 5%, 7%, and 9% acetic acid with 0.2% molarity and distilled water were weighed individually using an electronic mass balance. Acetic acid was obtained in liquid form. It was diluted to 0.2% (*w*/*v*) using distilled water. The algae, glycerol, acetic acid, and distilled water were mixed in a beaker, which was then heated up to 90 ◦C on a hot plate and held at that temperature for 25 min. The stirring speed of the magnetic stirrer was set at a constant speed of 250 rpm, to avoid the formation of bubbles and maintain the homogeneity of the solution. Then, the mixed solution was cooled down to 65 ◦C for 35 min. During cooling, stirring was continued to prevent the formation of bubbles and solidification of the solution. The second batch of the biofilm was repeated with the addition of the CEO. Pure biofilms (0% CEO) were set up as a control sample. The solution was cast into a square form (18 × 27 cm) of the acrylic plate. Upon casting, the drying process took place in an oven at a temperature of 50 ◦C for 24 h. The biofilm thickness was measured using an electronic gauge (Digitronic Caliper, Gombak, Selangor, Malaysia), with accuracy ranging between 0.1% and 1% as a function of thickness value (0–100 μm or 0–1000 μm). Seven replicates were made for each type of biofilm formulation.

#### *2.2. Soil Burial Test*

The compostability of the biofilms and CEO additions were performed according to soil bury test ISO/DIS 17088. The biofilm dimensions of 20 mm × 20 mm were cut and weighed and five replicates were made for each formulation. The bury test area was plotted at a cool and shaded corner of the garden. The soil temperature was based on the normal climate change, which is from 33 to 35 ◦C, while soil type was black garden soil. Each sample was buried in a convenient depth of 50 mm to allow for aerobic soil bury composting, as the compost has to be turned at regular intervals in this process. The area was plotted with granite or brick to prevent interruption or error during the investigation. Each time the specimen was retrieved from the ground, the plotted area was covered with layers of dried leaves or thin layers of soil to allow air to permeate the hole and accelerate the growth and expansion of fungi or bacteria.

#### *2.3. Tensile Test*

The Instron tensile test ASTM D882-02 machine (Gombak, Selangor, Malaysia) was used for this test. The load of the machine was set at 5 kN with the speed at 10 mm/min. Seven replicates of strips for each composition were cut at dimensions of 70 mm × 10 mm. The result of the tensile strength and elongation at break were assessed through the graph of the stress-strain curve.

#### *2.4. Fourier Transform Infrared Spectrometer (FTIR)*

An FTIR Spectrometer (Perkin Elmer System spectrum 100; PerkinElmer, Gombak, Selangor, Malaysia) is an analytical technique used to identify organic, polymeric, and, in some cases, inorganic materials. The FTIR analysis method uses infrared light to scan test samples and observe chemical properties. The resolution was set up at 4cm−<sup>1</sup> in a spectral range of 4000 to 600 cm−<sup>1</sup> and 32 scans per sample. Different peaks (various functional groups of chemical elements) of the IR spectrum were observed along the selected initial angle to the final angle.

#### *2.5. Scanning Electron Microscopy (SEM)*

The surface morphology of the films was studied using a Scanning Electron Microscope (SEM) JSM 5600 (Gombak, Selangor, Malaysia) with magnifications up to 1000×. Prior to carrying out the observation, the samples were subjected to sputter coating with a layer of carbon using a Polaron SC515 (Gombak, Selangor, Malaysia). This procedure was performed to ensure the sample morphology could be clearly observed under SEM and to prevent any electrostatic charging during observation.

#### **3. Results and Discussions**

#### *3.1. Biofilm Thickness*

In a polymer film packaging application, thickness is a crucial aspect, which requires specific attention from the material design. The thickness of the biofilm will highly influence other important properties, such as the strength, elasticity, and moisture content. Researchers [5] found that the main purpose of effective biofilm for food packaging is to secure the food from food pathogens, thus extending the shelf-life of the food, and will ensure the quality of the food and its nutrients to be intact. The general thickness of biofilms for packaging is ±0.3 mm [12]. Table 1 shows the thickness of the algae-based film by varying algae sample.


**Table 1.** Thickness of algae-based film with and without CEO.

The second batch formulation was focused on biofilm with the addition of several percentages of CEO. Based on this observation, it could be highlighted the importance of using CEO compared to the cinnamon powder. By using CEO, it was easier to control the thickness of the biofilm, as the resulting film thickness was not significantly different compared to the thickness of the control biofilm (0%, as depicted in Table 1). This is because the CEO used in the solution form mixed well within the blends. In comparison, using the cinnamon powder, the course cinnamon particle will not dissolve in the biofilm solution during processing, hence affecting the thickness of the bioplastic. In industry, this parameter is important for food packaging. This trend was similar to the previous study [13], where whey and pectin protein powder were incorporated into cinnamon and researchers had difficulty to control the thickness of the biofilm.

#### *3.2. Algae-Based Biofilm with Acetic Acid*

#### 3.2.1. Tensile Data

(A) Tensile strength, (B) modulus elasticity and (C) elongation at break of algae-based biofilms were affected by the different percentages of acetic acid, as demonstrated in Figure 1. The first attempt to produce algae-based biofilm failed because the sample was too fragile, as the algae is rich with starch content and becomes hydrophilic in nature. The glycerol was chosen to alter the delicateness of the biofilm. Once again, the biofilm produced was low in strength, as the particles of glycerol may have leaked during processing and the biofilm produced was found to tear easily. Next, the flexibility of the biofilm was improved with the help of the acetic acid content, to aid the glycerol to be fully efficient in the algae-based biofilm. Acetic acid is a weak acid that has one carboxylic acid group and is usually use in food additive [5]. Although there has been no specific research on the usage of acetic acid as

crosslinking agents, it has reported that the presence of acetic acid increased the interfacial interaction in the properties of coconut shells filled with low a polyethylene composite [14]. Researchers [15] also indicated that the combination of acetic acid with CO2 packaging can extend the shelf life from 12 to 20 days for chicken retail cuts without negatively affecting the quality and sensory properties of the broiler meat. The addition of acetic acid into the blend may help the infusion of glycerol into the algae molecular structure by accelerating the disintegration and suspension of algae sediment. Previous researchers also stated similar strength results in a PVA and chitosan blend with glycerol and acetic acid [16]. However, from the analysis done, the addition of 1%, 3%, 5%, and 7% acetic acid in the biofilm decreases the (A) tensile strength to half compared to the control biofilm, probably due to the different molecular structures of the acetic acid, even though it comes from the same carboxylic acid family [17]. Figure 1 also illustrated a reduction in (B) modulus of algae-based biofilms, similar to the tensile strength, with an increase in the concentration of acetic acid. Based on the figure, the lowest elastic property was recorded for 7% acetic acid, further than this percentage will continuously drop the modulus strength of the biofilm. This finding was similar to previous attempts using glycerol to increase the percentage of citric acid by up to 15% [3]. On the other hand, the (C) elongation at break (Eb) results were vice versa to the tensile and modulus strength results. Based on the Eb graph, it was found that the biofilms were capable of resisting changes in shape without crack formation with the addition of the acetic acid. Figure 1 demonstrated that the (C) elongation at break gradually increased with the increasing percentage of acetic acid by up to 7%. The highest elongation at break was measured with 5% acetic acid at 27.34% of Eb, while the lowest Eb was shown by 0% acetic acid at 20.14%. Physically, the biofilm with the addition of acetic acid was better in flexibility, less fragile, and good in modulus. The biofilm obtained was transparent and could not easily tear off when folded. However, the addition of acetic acid neither improves the tensile strength nor the elongation at break of the biofilm.

**Figure 1.** Effect of acetic acid on (**A**) tensile strength; (**B**) modulus strength; and (**C**) elongation at break of algae-based biofilm.

#### 3.2.2. Soil Burial Test

The algae used in this research to form a biofilm is a green algae species known as Neochloris Oleoabundans or Ettlia Oleoabundans [18]. These unicellular green algae are freshwater based and rich in starch content [18,19]. Starch is hydrophilic in nature and easy to degrade due to microbial and moisture contact. The degradation of algae-based bioplastic film via starch by micro-organisms in the soil produced carbon dioxide, biomass formed by extraction of algae carbon, and soluble CEO compound. From the soil bury test analysis, gradual biodegradation was observed in the biofilm surface degradation as shown in Figure 2. Figure 2 demonstrates the physical appearance of the algae biofilm after soil burial test for 28 weeks. The samples for soil bury test were exposed to the actual weather. Under rainy conditions, excess water permeated through the soil and diffused into the biofilm samples causing swelling and softening of the biofilm. Based on the physical observation, the biofilm started to deteriorate after 14 weeks and onwards, most likely due to hydrophilic nature of the algae. At the end of the 28 week period, the sample could barely retain the shape and began to wrinkle and tear apart. From the pictures, the sample showed high number of pores, and the number of pores continues to spread and increase in size as the length of soil bury test was prolonged. This confirmed that the biofilm sample had undergone biodegradation phases.

**Figure 2.** Physical appearance of algae film after soil burial at the **7th**, **14th**, **21th**, and **28th** weeks.

#### *3.3. Algae-Based Biofilm with CEO*

#### 3.3.1. Tensile Data

Algae-based biofilm with 3% acetic acid was found to yield good (A) tensile strength and (B) modulus properties, and, therefore, was selected to be used with CEO. This percentage was selected to be used for further investigation alongside the addition of different ranges of CEO (phase two). The percentages of CEO tested were 1%, 3%, 5%, 7%, and 9%. The control sample is label as 0 in the tabulated figures. Figure 3 demonstrates the (A) tensile strength of algae film, which increases with increasing percentages of CEO. The control sample without acetic acid possessed the least tensile strength. Meanwhile, the maximum tensile strength was achieved with 5% CEO due to the good intermolecular interaction between algae and starch and cinnamon molecules. This finding was also supported by a previous study [20], which also recorded a similar pattern where tensile strength increased with the addition of cinnamon bark oil into the alginate film. Based on Figure 3, the (B) modulus elasticity of the algae-based biofilm was found to display the same trend as the tensile strength results. The addition of 5% CEO was found to increase the stiffness of the algae-based biofilm up to 0.323 GPa, compared to algae-based biofilm with 1%, 3%, 7%, and 9% of CEO, which recorded lower modulus strength. However, in this study, the 5% CEO loading did affect the (C) elongation at break compared to the other percentages. Figure 3 indicated that the 5% CEO loading has low elongation at break compared to the 3% and 7% of CEO loading. Based on the current findings, 5% CEO with 3% acetic acid yielded a good and accepted elongation at break of the algae-based biofilm. This was made possible with the right amount of acetic acid in strengthening and adhering to the intermolecular bonds between the algae and cinnamon molecules. Hence, the addition of acetic acid into algae-based biofilm clearly indicated that the acetic acid molecules affect the adjacent molecules by increasing the distance and reducing the internal force, resulting in a more flexible film. The interference with adjacent molecules affects the intermolecular and intramolecular linkage of the polymer, thus strengthening the structure of the algae-based biofilm [21].

**Figure 3.** Effect of CEO on (**A**) tensile strength; (**B**) modulus strength; and (**C**) elongation at break of algae-based film.

#### 3.3.2. Soil Burial Test

Figure 4 depicted the physical appearance and Figure 5 shows the SEM analysis of biofilm with 5% CEO after soil burial at the 7th, 14th, 21th, and 28th weeks, respectively. The analysis is similar to control algae-based biofilm in Figure 2, it was noticed that the color of the biofilms with CEO turned darker and the darkening of the biofilms is a sign of biodegradation [22]. Changes in the appearance of the biofilms are explainable through the high-moisture absorption property and low intensity of cinnamaldehyde in the CEO percentages [23]. Based on the physical appearance of the biofilm in Figure 4, the sample in this research would have behaved similarly to the findings by Zhang et al. [20], where the alginate films incorporated with cinnamon bark oil showed less biodegradation potential compared to the alginate film without cinnamon bark oil. Therefore, it can be postulated that the addition of 3% acetic acid into the recipe assists in reducing the decomposition rate of the biofilm with CEO, compared to the sample of 5% CEO without acetic acid content. From the SEM analysis in Figure 5, the agglomeration of biofilm became more obvious as the biofilm began to swell, which in turn caused slow degradation. The addition of 5% of CEO into the biofilm exhibited physical changes. Besides which, different volumes of CEO used in this study resulted in varying biodegradation rates and behaviors. The antimicrobial (cinnamaldehyde functional group) and repellent properties of cinnamon may also decelerate the degradation rate of the film. These findings are in accordance with the previous research [23], where the higher the cinnamon percentage, the slower the composability rate. The film with 5% CEO demonstrated lesser pore percentages. A higher amount of CEO tends to reduce the degradation rate because of the hydrophobicity of the acetic acid, and the strong aroma of the cinnamon itself may repel insects and micro-organisms from attacking the biofilm [24].

**Figure 4.** Physical appearance of biofilm 5% of CEO at the **7th**, **14th**, **21th**, and **28th** weeks.

**Figure 5.** SEM analysis of algae film with 5% CEO after soil burial at the **7th**, **14th**, **21th**, and **28th** weeks.

#### *3.4. Fourier Transform Infra-Red (FTIR) Spectroscopy Data*

Figure 6A illustrated the FTIR spectra of biofilm with 5% CEO content and B biofilm with 3% of acetic acid, respectively, both displaying individual peaks within the range of 4000–500 cm−1. Peak A presented a broad absorption band at about 3310 cm−1, which represents the hydroxyl (OH) group. The peak at 2924 cm−<sup>1</sup> was recognized due to the C–H stretching of methane. Besides this, the peak at 1606 cm−<sup>1</sup> was formed due to the stretching vibration of the conjugated peptide bond formation by amine (NH2) and acetone groups in the algae. The peak at 1441 cm−<sup>1</sup> was due to an ester sulfate group. The characteristic peaks at 1013 cm−<sup>1</sup> and 931 cm−<sup>1</sup> indicated C–O stretching groups of 3,6-anhydrogalactose [25]. In addition, based on the FT-IR spectrum of CEO in A, the absorption band or frequency ranged from 3500 cm−<sup>1</sup> to 3200 cm−<sup>1</sup> broad, exhibiting the presence of O–H stretch. The specific absorbance band at 1635 cm−<sup>1</sup> revealed the stretching vibration of the C=O bond for cinnamaldehyde [25]. Due to the influence of conjugation and an aromatic ring, the peak is wider than usual for aldehyde compounds. A strong absorption band between 900 cm−<sup>1</sup> and 690 cm−<sup>1</sup> indicated the presence of aromatic C=C bonds [26]. Cinnamaldehyde is the main active component in cinnamon, which can be used as a natural antimicrobial in food preservation to retard or inhibit the bacterial growth of pathogenic and spoilage bacteria, which in turn extends the shelf life of the food products [20]. Since there was no peak observed at 1700–1720 cm−<sup>1</sup> in Figure 6B, which shows that there was no crosslinking between acetic acid and the algae-based blends due to the absence of chemical reaction. The sighting of a peak at wavenumber ranges between 1700–1720 cm−<sup>1</sup> indicates the presence of cellulose-fatty acids ν(C=O), a stretching vibration of the esters. The slope was transmitted obviously in (A), however, slowly lowering down with the addition of acetic acid as showed in sample (B). The combination of CEO and acetic acid was significantly reduced the cellulose fatty acid presence in the algae as shown in sample (C) which is possibly occurs due to the formation of a physical reaction between CEO, acetic acid and algae [26]. Figure 6C represents the FT-IR spectrum of 3% acetic acid blends with 5% CEO biofilms. A peak at 1716 cm−<sup>1</sup> was observed, indicating an association with C=O,

which is attributed to the carboxyl and ester carbonyl bands. This confirmed the existence of acetic acid in the specimen. However, the peak at 3328 cm−<sup>1</sup> became less intense when cinnamon was added into the formulation.

**Figure 6.** FTIR spectra of (**A**) Biofilm with 5% CEO; (**B**) biofilm with 3% acetic acid; and (**C**) biofilm with 5% CEO and 3% acetic acid.

#### **4. Conclusions**

The tensile test of biofilm demonstrated good enhancement upon the incorporation of 5% CEO. The biofilm achieved tensile strength at 4.8 MPa and elongation of 15%. Based on the SEM morphology, higher amounts of CEO in the presence of acidic acid leads to a reduction in the degradation rate of the biofilm. The biofilm demonstrated a continuous phase and exhibited a characteristic band at 1716 cm−<sup>1</sup> in the FTIR analysis. Hence, in conclusion, 5% CEO and 3% acetic acid are the suitable blend that could tremendously affect the tensile behavior and the biodegradation rate of the biofilm.

**Author Contributions:** Conceptualization, M.O. and K.K.; methodology, H.R.; validation, S.I.S.S., S.S., and H.S.H.; formal analysis, H.R.; investigation, M.N.Z.; resources, N.A.J.; writing—original draft preparation, M.O.; writing—review and editing, H.R. and H.S.H.; visualization, H.R. and H.S.H.; supervision, M.O.; project administration, K.K.

**Funding:** This research was funded by IIUM under publication fund and Malaysia Government.

**Acknowledgments:** Would like to acknowledge IIUM, MARDI, and UITM bio-composite department for support given for administrative and technical support.

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

#### **References**


© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Use of Orange Oil Loaded Pectin Films as Antibacterial Material for Food Packaging**

#### **Tanpong Chaiwarit 1, Warintorn Ruksiriwanich 1, Kittisak Jantanasakulwong <sup>2</sup> and Pensak Jantrawut 1,\***


Received: 24 September 2018; Accepted: 11 October 2018; Published: 14 October 2018

**Abstract:** This study aims to develop orange oil loaded in thin mango peel pectin films and evaluate their antibacterial activity against *Staphylococcus aureus.* The mango peel pectin was obtained from the extraction of ripe Nam Dokmai mango peel by the microwave-assisted method. The thin films were formulated using commercial low methoxy pectin (P) and mango pectin (M) at a ratio of 1:2 with and without glycerol as a plasticizer. Orange oil was loaded into the films at 3% *w/w*. The orange oil film containing P and M at ratio of 1:2 with 40% *w/w* of glycerol (P1M2GO) showed the highest percent elongation (12.93 ± 0.89%) and the lowest Young's modulus values (35.24 ± 3.43 MPa). For limonene loading content, it was found that the amount of limonene after the film drying step was directly related to the final physical structure of the film. Among the various tested films, P1M2GO film had the lowest limonene loading content (59.25 ± 2.09%), which may be because of the presence of numerous micropores in the P1M2GO film's matrix. The inhibitory effect against the growth of *S. aureus* was compared in normalized value of clear zone diameter using the normalization value of limonene content in each film. The P1M2GO film showed the highest inhibitory effect against *S. aureus* with the normalized clear zone of 11.75 mm but no statistically significant difference. This study indicated that the orange oil loaded in mango peel pectin film can be a valuable candidate as antibacterial material for food packaging.

**Keywords:** antibacterial activity; food packaging; orange oil; pectin film

#### **1. Introduction**

Currently, environmental problems and food safety have been of great public concern. Using packaging materials consisting of biopolymer is considered environmentally advantageous. Active packing has been defined as 'a type of packaging in which the package, the product, and the environment interact to extend shelf life or improve safety and convenience or sensory properties, while maintaining the quality and freshness of the product [1]. One of the major concerns of the food industry is the spoilage of foods and food poisoning caused by microbial contamination, which occurs mainly on the surface as a result of the post processing and food handling process [2]. Thus, packaging possessing an antimicrobial property might be novel and gain interest for active packaging to be used in the food industry.

Pectin, one of the main structural water-soluble polysaccharides derived from plant cell walls, has been utilized in several fields, such as the pharmaceutic, cosmetic, and food industries, because of its excellent gelling property. Moreover, pectin can be extracted by simple methods from various plant materials. Mango peel, which is an agro-waste substantially arisen from fruit processing products in Thailand, was found to be a rich source of pectin [3–5].

Natural aromatic compounds and flavors such as fruit and vegetable essential oils are also extensively used in the food industry. For one clear example, orange oil exhibiting a significant bacterial inhibitory effect is one of the most beneficial and frequently used essential oils. Limonene, a major constituent found in orange oil, has been an active compound implicated in an antimicrobial property [6]. Limonene is a clear liquid at 25 ◦C with a citrus-like taste and odor. It is slightly soluble in glycerol, soluble in ethanol and carbon tetrachloride, and miscible with fixed oil. Limonene has demonstrated antibacterial activity as has been shown to inhibit the growth of many bacterial species in vitro, for example, *Staphylococcus aureus*, *Salmonella enteritidis*, *Escherichia coli*, *Klebsiella pneumoniae,* and *Proteus vulgaris* [7–9]. Limonene, a lipophilic compound, is able to penetrate through lipids of a bacterial cell, distribute cell structure, and render them more permeable. Then, it causes cell death by extensive leakage of intracellular fluid and ions [10]. There were some previous studies of film loading bioactive compounds that showed antimicrobial activity for preservative packaging. For example, gelatin film containing bergamot and lemongrass essential oils against *Escherichia coli*, *Listeria monocytogenes*, *Staphylococcus aureus*, and *Salmonella typhimurium.* Hydroxy methylcellulose film incorporated with kiam wood extract against *E. coli*, *S. aureus* and *L. monocytogenes* and starch film containing saponin against *E. coli*, *S. typhi*, and *E. erogenous* [11–13]. In the current study, the bio-based packaging materials have been developed by incorporating selected components, that is, essential oil derived from oranges, into a mango pectin film packaging model. The developed orange oil loaded pectin films were characterized and evaluated for their antibacterial activity against *S. aureus.*

#### **2. Materials and Methods**

#### *2.1. Materials*

Commercial low methoxy pectin (LMP; Unipectine OF300C; DE = 30% and DA = 0%) was purchased from CargillTM , Saint Germain, France. Orange oil was purchased from Thai-China flavors and Fragrance Industry Co., Ltd., Nonthaburi, Thailand. Calcium chloride (CaCl2) was purchased from Merck, Damstadt, Germany. Mueller Hinton agar (MHA) was purchased from Becton Dickinson, Holdrege, NE, USA. Tryptic soy broth (TSB) was obtained from HiMedia Laboratories Pty. Ltd., Mumbai, India. *S. aureus* (ATCC 25923) was obtained from the BIOTEC, Manassas, VA, USA. Distilled water served as the solvent for preparing film solutions.

#### *2.2. Mango Pectin*

Mangoes (cv. Nam Dokmai) were collected from Chiang Mai province in Thailand, during June to August 2018. The mangoes were washed and the peels were stripped with a peeling knife, and then dried in a hot air oven at 50 ± 2 ◦C. The dried peel was ground to a fine powder using a high-speed food processor, and was then screened using a sieve (number 30; diameter 0.6 mm). Pectin from mango peel was extracted using the microwave-assisted method [14]. First of all, mango peel powder was mixed with acidified water pH 1.5. The mixture was extracted using microwave oven, irradiated at a frequency of 2450 MHZ at 500 W for 20 min. Then, it was centrifuged at 9000 rpm for 10 min. After that, supernatant was precipitated with ethanol and the precipitated pectin was washed by ethanol three times. The mango peel pectin was dried in hot air oven at 40 ◦C. The dried mango peel pectin was ground to a fine powder and kept in desiccator for further experiment.

#### *2.3. Identification of Major Compounds of Orange Oil*

The major compounds of orange oil used in this study were analyzed by gas chromatography mass spectrometer (GC-MS analyzer) from Agilent-Technologies (Santa Clara, CA, USA). A split ratio of 1:650 was used to inject the sample of 1.0 μL. The compounds of the sample were separated using an HP-5 MS capillary column (30 m × 0.25 mm, film thickness 0.25 μm). The carrier gas was helium and the flow rate was set as 1.5 mL/min. The data obtained from GC-MS analysis were compared with the National Institute of Standards and Technology mass spectral library for compound identification.

#### *2.4. Preparation of Films Loaded with Orange Oil*

The minimum inhibitory concentration (MIC) of orange oil against *S. aureus* was evaluated before loading orange oil into the film formulations. The MIC was evaluated using the broth dilution method [8]. Orange oil was prepared in various concentrations from 0.5 to 150 mg/mL by dilution with Tween® 80. The orange oil concentration above MIC was then selected and used for loading in the prepared film, using the ionotropic gelation with solvent casting techniques [15]. Briefly, film forming solution 3% *w*/*v* was prepared using commercial low methoxyl pectin (P) mixed with the extracted mango peel pectin (M) in various ratios with and without 40% glycerol (G), basic on dry pectin weight as the plasticizer. Orange oil (3% *w/w*) was added into pectin solution and then mixed until a homogeneous mixture was obtained. After that, the steps that were previously described were followed [15]. The film without mango pectin nor glycerol was used as a control film (P3M0O) in order to focus on the effect of mango pectin in thin film formulation. Furthermore, only mango pectin cannot form gel by this technique. Thus, the combinations of both pectin in the film formulation were prepared.

#### *2.5. Characterization of Films Loaded with Orange Oil*

The mechanical properties of the films loaded with orange oil were tested by a texture analyzer TX.TA plus (Stable Micro Systems, Surrey, UK). Each experiment was repeated six times. The tensile strength, percent elongation, and Young's modulus was calculated [15]. The film morphology was examined using scanning electron microscopy (SEM, JSM-5410LV, JEOL Ltd., Peabody, MA, USA). The SEM were performed at 15 kV under low vacuum mode. The morphology of film's matrix at magnifications of ×500 was evaluated.

#### *2.6. Limonene Loading Content*

The limonene loading content in the films was determined using GC-MS, which was previously described [16]. The limonene loading was calculated by the following equation: %Limonene loading = the actual quantity of limonene/the theoretical quantity of limonene × 100.

#### *2.7. Film Sterility Test*

Films containing orange oil were cut into a circle with a diameter of 4.0 mm, which is the same size as the disc that was used in the anti-bacterial activity test. The cut films were sterilized by ethylene oxide and tested for sterility by the direct inoculation in culture medium method [17,18]. Tryptic soy broth media and its containing *S. aureus* were used as negative and positive control, respectively. Film samples were placed in the media and evaluated the microbial growth sign by visual observation after incubation at 37 ± 2 ◦C for seven days.

#### *2.8. Anti-Bacterial Activity of Films Loaded with Orange Oil*

In vitro anti-bacterial activity of films was determined using the agar disc diffusion method [19]. *S. aureus* was activated in TSB and incubated for 6–10 h. Absorbance was then measured at 600 nm. The activated *S. aureus* turbidity had to equal 108 cfu/mL and was used and cultivated on MHA plates. The films loaded with orange oil were put onto the MHA plate. Ampicillin was also used as a positive control. After that, the MHA plates were incubated at 37 ◦C for 24 to 48 h, and the diameter of the clear zone was measured, including the diameter of the film or disc, using a Vernier caliper. Each experiment was performed in triplicate.

#### *2.9. Statistical Analysis*

An analysis of variance (ANOVA) was carried out with SPSS software version 16.0 (SPSS Inc., Chicago, IL, USA).

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

#### *3.1. Identification of Orange Oil*

GC-MS analysis of orange oil showed 19 known compounds including hydrocarbons, aldehydes, alcohol, and ketone (Table 1). The major compound of the oil was limonene (84.57%), which was exhibited at the retention time of 4.801 min. Other compounds were also found in the oil, such as *cis*-limonene oxide (1.86%), β-myrcene (1.06%) and *trans*-limonene oxide (0.88%). Thus, limonene was used as the marker for further experiments in this study. Previous studies have also identified limonene to be the major component (67.44–94.50%) in orange oil [20–22].


**Table 1.** Identified compounds of orange oil. GS-MC—gas chromatography mass spectrometer.

#### *3.2. Morphology and Mechanical Properties of Film Loaded with Orange Oil*

All film formulations' compositions are shown in Table 2. The film without orange oil (P1M2 film) exhibited a smooth surface with dense matrix, whereas orange oil films showed rough surface with the presence of micropores inside the film's matrix. When orange oil was loaded, we observed the increasing of the thickness of orange oil films, which was related to the size and amount of micropores inside the film's matrix (Table 2 and Figure 1). The more orange oil evaporated, the greater the number and size of micropores, which resulted in a loose structure of the matrix and increased film thickness. Our results are consistent with Jouki et al. 2014, which found that quince seed film showed the loose film's matrix when oregano oil was incorporated [23]. Furthermore, in a previous study, the effect of grape pomace extract in chitosan film was investigated. Film structure discontinuities were induced by incorporation of wax or oil into the polysaccharide matrix [24]. Moreover, this study found that the greater the mango peel pectin ratio, the greater the number of micropores as seen in P3M0O film dense film matrix with less micropores than P1M2O film. The mechanical properties of films loaded with orange oil are shown in Table 3. Young's modulus of P1M2GO film significantly decreased to 35.24 MPa compared with 145.34 MPa for this film without orange oil. These results may be because of the amount of micropores in the polymer matrix. A previous study showed that quine seed mucilage films incorporated with the highest amount of oregano essential oil, which had many micropores, exhibited the best mechanical properties with the lowest tensile strength and Young's modulus, as well as the highest percent elongation. The works of [23,25] also found that percent elongation of citrus pectin film incorporated with clove bud oil increased as a result of oils existing in the film matrix in the form of oil droplets, which can be easily deformed and improve the film's extensibility.

**Figure 1.** Scanning electron microscopy (SEM) of film's matrix of P3M0O (**a**), P1M2O (**b**), P1M2GO (**c**), and P1M2 (**d**) films.

**Table 2.** Film composition and thickness. LMP—low methoxy pectin.


**Table 3.** Orange oil loading content, tensile strength, elongation, and Young's modulus of films containing orange oil.


Note: For each test, the different letters are statistically different (*p* < 0.05).

#### *3.3. Limonene Oil Loading Content*

The limonene loading contents of films loaded with orange oil are shown in Table 3. The highest limonene loading content (86.17%) was obtained from P3M0O film, whereas P1M2GO film exhibited the lowest limonene loading content (59.25%). This result found that the texture of the orange oil film played an important role in limonene loading contents. Micropores indicated that orange oil had evaporated from the polymer matrix. Film with a lower amount and smaller size of micropores, like P3M0O, can encapsulate more limonene content. While the P1M2GO film with larger amount and size of micropores showed the lowest limonene content.

#### *3.4. Film Sterility and Anti-Bacterial Activity*

The product sterility test was used to confirm that the film samples were sterile, before testing the anti-bacterial activity. In sterility test, there was no microbial growth in any of the film samples. Growth of *S. aureus* was observed in the positive control, while no sign of *S. aureus* growth was observed in the negative control. The results of examination of the anti-microbial activity of films against *S. aureus*

using the agar disk diffusion method are shown in Table 4. All films loaded with limonene exhibited a clear zone. In the direct comparisons of the clear zone diameter of film formulations containing different limonene loading content, the P3M0O film, which had the highest limonene loading content (86.17%), showed the widest clear zone diameter (10.02 mm), whereas P1M2GO film, which exhibited the lowest limonene loading content (59.25%), showed the smallest clear zone diameter (8.08 mm). Thus, the anti-bacterial activity of the films in this study was dependent on the amount of limonene remaining in the film. Generally, in a hydrophilic polymer such as pectin, water molecules from agar penetrate into the polymer matrix resulting in swelling of film; thus gradually widening the meshes of the polymer network and leading to greater release of active compounds into the surroundings [22]. However, in order to normalize limonene content in each film, the normalized clear zone was calculated and presented in Table 4. Interestingly, the normalized clear zone dimeter of P1M2GO film (11.75 mm) was higher than the others, but no statistically significant difference existed between the films. It may be concluded that when we consider film composition in the same amount of limonene loading content, the P1M2GO film, which showed better mechanical properties, tended to show higher anti-bacterial activity. In addition, our recent study found that loading orange oil in the form of microemulsion was able to reduce micropores in the film's matrix and increase limonene loading capacity as well as anti-bacterial activity [16].


**Table 4.** Anti-bacterial activity of films against *S. aureus.*

Note: ND = not detected. Normalized clear zone (nm) = (limonene content of P3M0O/limonene content of each film) × clear zone diameter of each film.

#### **4. Conclusions**

Films loaded with orange oil were prepared and their morphology, tensile properties, loading content, and anti-bacterial activity were investigated. The addition of orange oil decreased the Young's modulus value, which appears to be related to the amount of micropores in the film's matrix. Interestingly, the P1M2GO film, which had the best mechanical properties, exhibited the lowest limonene loading content (59.25%). For the test of anti-microbial against *S. aureus*, the P1M2GO film showed the highest normalized clear zone (11.75 mm), but no significant difference existed. This study indicated that the orange oil loaded in mango peel pectin film can be a valuable candidate as an antibacterial material for food packaging. However, this orange oil film needs further development, especially the increasing of the limonene loading content. Other technologies such as preparation of orange oil in micro-emulsion or nano-emulsion could be used in order to enhance the limonene loading content.

**Author Contributions:** The study was designed by all of authors. The all of experiments were conducted by T.C., W.R., and K.J. under suggestions of P.J. The manuscript was written by T.C. and P.J.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors gratefully acknowledge the Franco-Thai Cooperation Programme in Higher Education and Research/Franco-Thai Mobility Programme/PHC SIAM 2018-2019, Graduate School Chiang Mai University (GSCMU) and Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand for the financial support. The authors would like to thank Assist. Sasithorn Sirilun for all of the anti-bacterial activity testing support.

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

#### **References**


© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
