chlorophyll in alginate-based edible film in inhibiting spoilage of fish snacks

Edible films are environmentally biodegradable materials used for food packaging. The edible green alga Caulerpa racemosa has antimicrobial properties; however, its chlorophyll-based bioactive compounds can be damaged when heated so it is prepared in microcapsules. Our research evaluated the effect of Caulerpa microcapsules on an alginate-based edible film on film properties and food spoilage. The microcapsules were used at concentrations of 0%, 0.5%, 1%, and 1.5%. The edible film was measured for film properties, total phenolic content (TPC), antioxidant activity (DPPH), functional group (FTIR) and microstructure (SEM). Food spoilage was evaluated on a popular fish-based product (fish bubble snacks). Bacterial strains of Escherichia coli and Staphylococcus aureus were used to evaluate antimicrobial effectiveness of the edible films. The addition of Caulerpa microcapsules had no significant negative effect on physical properties of the alginate-based film, while the smoother and more homogenous surface should enhance the barrier properties of the film. The slow and evenly distributed release of active compounds from the microcapsules increased resistance to Rhizopus sp. and significantly reduced the proliferation of E. coli but not S. aureus on coated fish snacks. Caulerpa racemosa can be used to enhance the effectiveness of alginate-based films in delaying spoilage and could extend product shelf-life.


Introduction
An edible film is a thin edible layer that is used to coat food and can be consumed together with this food. The role of the edible film is to prevent external contamination and retard the deterioration of food so that food quality and safety can be maintained (1). Alginates are complex polysaccharides containing mannuronic and guluronic acids (2). They can form stable gels at high and low temperatures and in low pH environments and can be used for numerous applications in food processing (3). Glycerol is one of the plasticizers that can be added to alginate-based edible films to improve the strength, solubility and elasticity (4).
Ideally, a high-quality edible film will also contain substances that can retard food spoilage, for example through enrichment with natural antimicrobial agents (5,6). Chlorophyll extracts from green algae have shown antibacterial activity (7). The bioactive compounds in crude phenolic extract of the green alga Caulerpa racemosa have antibacterial properties (8)(9)(10)(11). Furthermore, the bioactive compounds from the methanolic fraction have bactericidal and larvicidal properties (9,12). However, bioactive chlorophyll compounds are easily damaged when heated, for example during food processing or cooking (13)(14)(15).
Microencapsulation is a nanotechnology method which can be used to prevent damage to bioactive compounds by protecting the encapsulated compounds during edible film processing (16). This technology has applied in several industries, including the pharmaceutical industry, and in medical applications (17,18). Microencapsulation methods used to increase the stability of bioactive compounds include spray drying and freeze drying (16,19). While spray drying is arguably the most popular method (16), and has shown advantages for some compounds, in particular greater regularity in the size and shape of the nanoparticles produced (19,20), this method can damage compounds sensitive to elevated temperatures (21). Freeze drying, also known as lyophilisation, is relatively simple, carried out at low temperatures, and also avoids oxidation or chemical modification (21). Freeze dried microencapsulated bioactive compounds can also be more stable and less prone to damage in cold or hot temperatures (22,23).
The presence and growth of fungi is related to the moisture content of food and that many fresh food products are readily susceptible to microbial spoilage (24), while the range of water activity (aw) of foods with intermediate moisture content is conducive for fungal growth (25). According to Nagaraj et al., Caulerpa racemosa could be an antifungal and/or antibacterial agent, as it contains phenolic compounds (9). Freeze dried microencapsulated chlorophyll extracts from C. racemosa are stable and can release potentially antimicrobial compounds in a slow and controlled manner (26).
Based on these considerations, encapsulated chlorophyll from C. racemosa could be applied as a potential solution to prevent some types of microbial food spoilage by enhancing the antimicrobial activities of protective edible film. This study evaluated the physical properties of an alginate-based edible film enriched with microcapsules containing chlorophyll from C. racemosa and its effectiveness as a barrier for preventing microbial growth. The rationale was that the enriched edible film would contain antimicrobial agents that could be released gradually over time and thus extend the shelf-life of the product.

Collection of Materials and Preparation
Fresh Caulerpa racemosa was harvested from Karimunjawa Island, Central Java, Indonesia. The macroalgal sample was dried in a shady place for 4-5 days to reduce its water content to 80-20%. Alginate (Merck, Germany) was obtained from CV. Total Equipment Pharmacy in Semarang, Indonesia. Glycerol (Merck, Germany) and distilled water were purchased from Indrasari Chemical Agencies, Semarang, Indonesia. The ingredients for fish bubble snacks were purchased from the local market in Semarang, Indonesia.
The extraction of active compounds from fresh Caulerpa racemosa was based on (27) with modification. The C. racemosa was first dried and then a weighing 100 grams was chopped and macerated at room temperature through total immersion in 400 ml of industrial ethanol (Merck, Germany) as the solvent. Ethanol is safe for consumption so it is used as a solvent in this study. Ethanol is a solvent known to be able to extract bioactive components such as curcumin and anthocyanins with the highest total phenol content and antioxidant activity compared to methanol, water, and acetone (28)(29)(30). The sample was macerated for ± 48 h then filtered. Residual ethanol was removed from the C. racemosa extract through rotary evaporation (RE-2000E, China). The extract was then freeze-dried (Ningbo Yinzhou Sjia Lab Equipment Co, Ltd).
The microencapsulation process was performed using a solution of 10% Caulerpa extract, 1% Tween 80 (polyoxyethylene sorbitan monooleate, a non-ionic and non-toxic surfactant) (Merck, Germany), 10% coating materials, and distilled water to reach 100%. The coating materials used were fish gelatine and Arabic gum in the ratio 0.5:9.5 (0.5% and 9.5% of the mixture). The mixture was homogenized (Ultra Turrax Homogenizer WiseTis HG-15D, Germany) at 10,000 rpm for 3 minutes. The homogenized solution was then frozen at -35°C for 24 h (23). The frozen samples were then freeze dried at -100°C vacuum pressure for ± 48 h.
The edible film was prepared by adding 2% alginate to distilled water. The alginate was dissolved on a hot plate with a magnetic stirrer to prevent coagulation. Glycerol (2.5 %) was added when the temperature reached 50°C, and the mixture was then stirred at a moderate speed for 30 min. Caulerpa microcapsules were added in three different concentrations: 0.5%, 1%, and 1.5%, with 0% as a control. Edible film composition was prepared based on Table 1. The mixture was homogenized and then poured onto a glass plate and oven dried at 60°C for 4 h. A dough was made by combining the dough ingredients (mashed potatoes, wheat flour, eggs, garlic, pepper, and sugar) in a mixer. Small balls of dough (called bubbles) with a diameter of approximately 2 cm were formed and stuffed with raw barracuda fish filleted. These bubbles were then fried, coated in beaten egg and covered with breadcrumbs and kept at -18°C for 24 hours.

Edible Film Analysis
The analysis for edible film including several tests. The thickness of the edible film was measured at five points using a screw micrometre (Mitutoyo, Japan). Tensile strength and percent elongation were measured using a Universal Testing Machine (Texture Analyser CT3 Brookfield, USA). Small rectangular strips (5 mm wide x 50 mm long) were cut from the edible film. Tensile strength and the Young Modulus (MPa) were determined based on maximum force per unit area (Newton/m 2 ) exerted on the film before it failed (ripped). Percent elongation (in %) was obtained by dividing the difference between the maximum (just before failure) and initial length (50 mm) by the initial film length then multiplying the result by 10 following (31).
The gravimetric method was applied to measure Water Vapour Transmission Rate (WVTR). The edible film samples, together with 10 grams of silica gel (RH=0%), were placed in a jar containing NaCl 40% (w/v) (RH=75%) at 25°C. The silica gel absorbed the water vapour transmission escaping through the film (32).
Total phenolic content was measured based on (33). Folin-Ciocalteu reagent was applied to the sample and the results expressed in Gallic acid equivalent per gram of extract. Antioxidant activity (DPPH) was measured by adding 1 mL of 2.2-diphenyl-1-picrylhydrazil (DPPH) to a sample which was placed in methanol, then homogenized and stored in a dark room at room temperature for 25 minutes. The absorbance was then read using a UV-Vis spectrophotometer (Shimadzu, Japan) at 760 nm. The percentage of DPPH free radicals was calculated following (11). The functional group was analysed through Fourier Transform Infrared Spectroscopy (FTIR) at wavelengths of 400-4000 cm -1 following (11).
The edible film microstructure was subjected to scanning electron microscopy (SEM) according to (11). The samples were attached to a SEM unit (Jeol JSM I 00 651 OLA, Japan) using a two-sided adhesive tape and then coated. The SEM was operated at 20 kV with a magnification of 1000X and the surface morphology was recorded as a scaled digital image.
Bacterial strains of Escherichia coli and Staphylococcus aureus were used to evaluate the antimicrobial effectiveness of edible film in this study. Bacterial samples were prepared from beef (10 g) and placed in Stomacher bags with 90 ml of Mueller Hinton solution in a Colworth Stomacher 400 (Seward Ltd., England). These were then incubated for 18 h at 37°C before the test, following (34). Circles of edible film with a diameter of 5 mm were laid on top of agar medium (set in a petri dish) which had been spread with 0.1 ml of fungal culture. The petri dish then was incubated at a temperature of 25°C for 72 h. After the incubation period, the diameter of the inhibition zone and the diameter of the clear zone formed were measured following (35).

Statistical Analysis
All procedures were performed in triplicate (3 replicates) and results presented (tabulated) as mean ± standard deviation (SD). Data were analysed through analysis of variance (ANOVA). If there were any significant differences between treatments, a post-hoc Tukey test was conducted. Significance was evaluated at the 95% confidence level (α = 0.05). Statistical analyses were performed in SPSS Software version 21.

Edible Film Physical Characteristics
The addition of C. racemosa microcapsules increased the thickness of the edible film and affected several other properties ( Table 2). The microcapsules were in powder form and increased the total mass. The thickness of the edible film is influenced by the raw materials and the addition of active ingredients, as stated in (31). The addition of C. racemosa increased the soluble solids, and therefore thickness was also increased. A similar effect was described from the addition of oregano essential oil (36). It is likely that the increased thickness affected the tensile strength, elongation, young modulus, and WVTR.
Tensile strength, elongation, and WVTR decreased with increased microcapsule content. Furthermore, the glycerol used as a plasticizer also increased the film matrix in the edible film, as found by (37). The alginate in an edible film influence both the elongation and tensile strength (38). Savoury Essential Oil (SEO) added to nanocomposites can decrease tensile strength and Young modulus and increase percent elongation (17). The WVTR is influenced by the ratio between hydrophobic and hydrophilic materials, as well as the thickness: the thicker the edible film, the lower the WVTR, thought to be due to the increased time needed for the diffusion of water vapour (39).
The total phenolic content in edible film enriched with C. racemosa microcapsules ( Table 3) showed that the TPC and antioxidant activity also increased at higher enrichment rates. The control (unenriched) edible film had a TPC of 0.89±0.01 mg GAE/g. The TPC level increased in line with addition of chlorophyll microcapsules. This is similar to the results of (40) who found that both the total phenol content and the antioxidant content of edible film increased with the addition of oregano oil or black cumin oil. TPC likely affected the antioxidant activity, which also increased with higher microcapsule enrichment. Measuring the DPPH is a widely accepted quantitative test for antioxidant activity (41). The antioxidant activity reached an optimum DPPH scavenging activity level of around 62% with the highest C. racemosa microcapsule enrichment level (1.5%).

Antimicrobial Activity
An inhibition zone size of 1.30-2.95 mm was observed for the bacterium E. coli. However, no inhibition zone was observed for S. aureus (Table 4). The phenolic extract of Caulerpa showed some activity against E. coli. Gram-positive microorganisms (S. aureus and B. cereus) have been found more susceptible to the Articoat DLP 02 (AR) than the Gram-negative group (34). High antibacterial activity against Erwinia amylovora and Pseudomonas sp. is reported from films enriched with Gallic phenolic acids (42). The addition of savoury essential oil (SEO) to agar-cellulose nanocomposite films was less effective against Gram-negative bacteria (E. coli) than Gram-positive bacteria (Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus) (17). Alginate film enriched with essential oil of oregano has also been reported as significantly more effective against Grampositive than against Gram-negative bacteria (36). Alginate-based edible film combined with cinnamon oil obtained an inhibition zone of 36.00 mm against S. aureus (43), while edible alginate can prolong shelf-life of fresh fruit for up to 28 days (44).

Antifungal Activity
With respect to antifungal activity, Rhizopus type fungi were observed on the surface of fish bubble snacks after storage at room temperature (Table 5). Fungal growth appeared much more rapidly on the control (no microcapsule enrichment) than on the product treated with chlorophyll microcapsule enriched film, indicating an antifungal activity of the chlorophyll microcapsules at all levels (0.5%, 1%, 1.5%) tested in this study.

Edible Film Physical Characteristics
Fourier Transform Infrared Spectroscopy (FTIR) detected the presence of the C-H functional group in the three edible film formulations with chlorophyll microcapsules on the edible film (Figure 1 a,b,c), but not in the control (Figure 1d). Chlorophyll has a C-O aldehyde functional group at a wavelength of 1583-1709 cm -1 , C-H groups at 2809-3012 cm -1 , and O-H groups at 3029-3639 cm -1 (45). The C-H group is a determining group for chlorophyll compounds because it is located close to the tetrapyrrole ring bonded to metal ions (46). Based on the FTIR spectra, the edible film contained chlorophyll microcapsules, which were detected by the presence of a C-H group expressing chlorophyll and appeared at a wavelength around 2900 cm -1 . This group was not detected in control (d). The interaction of chlorophyll microcapsules with edible films is indicated by a shift in wavelength, especially for the C-O and O-H groups (47). The O-H group shifted from 1413 to 1414 cm -1 while the C-O shifted from 1590 to 1594 cm -1 . This indicates that there was an interaction between the chlorophyll microcapsules and the edible film.

Film Microstructure -Scanning Electron Microscopy (SEM)
The addition of microencapsulated chlorophyll affected the microscopic morphology of the edible film. The SEM images ( Figure 2) show a clearly visible difference between the surface texture of edible film with and without chlorophyll microcapsule enrichment. The control film (no enrichment) had a rough surface (Figure 2a), while the addition of microcapsules produced a much flatter, smoother and more homogenous surface (Figure 2b). This characteristic should enhance the barrier properties of the edible film (48). The surface of the film was homogeneous and smooth, indicating that there was no agglomeration and the microcapsules were evenly distributed on the surface of the film. The results of this study are in accordance with that shown by (49) who found that microcapsules with gelatine and gum Arabic coating materials can form cross-links with the film matrix so that they can be dispersed into the film. This causes the mechanical properties of the film to be better than the control.

Conclusions
The enrichment of alginate-based edible film with chlorophyll microcapsules enabled the chlorophyll microcapsules to blend into the alginate-based edible film where they migrated and slowly released compounds which were able to prevent or retard some microbial growth on the fish bubble snack product tested. The enrichment increased the film thickness, improved the surface texture of the film, increased the resistance of the coated food (fish bubble snacks) to the growth of the mould Rhizopus sp., and increased resistance to the proliferation of E. coli but not of S. aureus. At room temperature the antifungal effect was twice as strong as for the same product without enrichment of the edible film coating. The antifungal properties of the enriched edible film extended the shelf-life of the product tested.

Data Availability Statement
Not applicable.

Conflicts of Interest
Authors may declare no conflict of interest.