Physical and functional properties of fish gelatin-based film incorporated with mangrove extracts

Materials

Starry triggerfish (Abelistes stellaris) skins were provided from a small-scale fish industry of the District of Tuban, East Java, Indonesia, and delivered in iceboxes, with a fish/ice ratio of 1:3 (w/w), to the Faculty of Fisheries and Marine Science, Universitas Brawijaya. Upon arrival, the samples were cut into small pieces (0.5 × 0.5 cm²) using clean scissors. The samples were stored in a freezer (−20 °C) until use. Mangrove (B. gymnorhiza and S. alba) leaves were obtained from the Sendang Biru coastal area, Malang, East Java, Indonesia. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) was purchased from Sigma–Aldrich (Germany). Sodium hydroxide (NaOH), acetic acid (CH₃COOH), and methanol (CH₃OH) were procured from Merck (Germany). Mueller-Hilton agar was supplied by Sigma–Aldrich (Germany). All the solvents and chemicals used for this study were of analytical grade.

Fish gelatine extraction

The extraction of gelatine involved an acid-aided process following the method of Gudmunson & Hafsteinsson (1997). First, the frozen skins were thawed with running water. The prepared samples were then soaked in 0.1 M NaOH with a sample-to-solution ratio of 1:5 (w/v). The mixture was stirred for 2 h at 30 ± 2 °C. The alkaline solution was replaced hourly. After being washed with dH₂O (pH 7), the samples were soaked in a solution of 0.6 M acetic acid at a 1:5 (w/v) ratio for 2 h. Acid-soaked skin was then washed with dH₂O (until pH 7). Afterwards, the samples underwent an extraction process with dH₂O (pH 7) at a sample-to-solution ratio of 1:3 (w/v). The extraction process was carried out in a water bath for 4 h at 55 °C. The mixtures were filtered with filter paper. The filtrates were then dried in an oven for 48 h at a temperature of 55 °C. To obtain gelatine powder, the dried gelatine sheets were ground.

Preparation of mangrove leaf extracts

The leaves of B. gymnorhiza and S. alba were authenticated by the botanist from Clungup Mangrove Conservation Centre, Malang Indonesia prior used for experiment. The leaves were then air-dried for three weeks until the moisture content was less than 5% before they were pulverized and weighed for the experiment (Paputungan, Wonggo & Kaseger, 2017). The extraction was started by adding methanol (90% v/v) to 100 g of crushed leaves at a ratio of 1:3 (w/v). The samples were extracted using the maceration method for 72 h at room temperature. The solution was replaced daily with fresh methanol. The collected solutions were filtered using Whatman no. 40 filter paper. A vacuum rotary evaporator (set at 40 °C) was used to obtain crude extracts of mangrove leaves. The collected mangrove extracts were then kept at 4 °C in a chiller until further use.

Preparation of active films

The active film solutions were prepared by dissolving 5 g of fish gelatine with 100 mL of distilled water under magnetic stirring at 50 °C for 30 min. The mixtures were added to 0.5% (v/v) glycerol with heating at 45 °C for 15 min with continuous stirring. To find the best treatment of the active films, different mangrove leaf extract concentrations ranging from 0% to 0.35% (w/v) were added to the film solution. The mixtures were continuously stirred until the solution was thoroughly mixed. The mixtures were then poured onto a 20 × 20 cm² pan and oven-dried using an electric oven at 55 °C for 18–20 h.

Determination of amino acid composition

The amino acids were analysed using ultra-performance liquid chromatography (UPLC). The hydrolysis of the gelatine sample was carried out in an acidic environment using 6 N HCl heated at 110 °C for 22 h. As an internal standard, α-amino butyric acid (AABA) was used. The AccQ-Fluor reagent kit was used as a reagent for amino acid derivatization. The UPLC conditions were as follows: column: ACCQ-Tag Ultra C-18, flow rate: 0.5 mL per min with an injection volume of 1 µL. The elution system was carried out in a gradient with a specific composition (Waters, 2016).

Thickness

The thickness of the film was measured using a screw thread micrometer (Mitutoyo Corp., Japan). Five separate measurements were taken (including the centre spot as the reference point) on the 20 × 20 cm² edible film area. The thickness value of the edible film measured was equal to the average of the five measurements (Chae & Heo, 1997).

Mechanical properties

Tensile strength (TS) and elongation at break (EAB) percentages were measured using an SSB 0500 model Tensile Strength and Elongation from Tester Industries based on a previously reported method (ASTM, 2003). Prior to measurement, active films were conditioned in a desiccator with 75% relative humidity (RH) for 24 h. The maximum force value for cutting the measured film was observed on the tool display. The strength was determined based on the maximum load at the time the film broke, while the percentage of elongation was calculated based on the film’s length when the film broke. The active packaging surface area was measured according to a 3 × 7 cm² area. TS can be calculated using the following formula: ${\displaystyle \mathrm{TS}=\frac{F}{A}}$

where F is the tensile stress (N) and A is the cross-sectional area (mm²).

Water vapour transmission rate (WVTR)

The WVTR of each film was measured according to (ASTM, 1987). The active packaging was cut with a diameter of 3.5 cm and placed between the containers (glass drinks). The first container contained water, while the second container was given silica gel with a constant weight. The samples were set aside for approximately 24 h, and water vapour transmission was calculated according to the following formula: ${\displaystyle \text{WVTR}\left(\frac{g}{m2day}\right)=\frac{W}{A}}$

where W is the slope of weight gained against time plot (g), and A is the area of film (m²).

pH

The procedure for testing the degree of acidity (pH) was as follows: 0.2 grams of sample was dispersed in 20 mL of distilled water at a temperature of 80 °C. The sample was then homogenized using a magnetic stirrer. The measurement of the degree of acidity (pH) was carried out using a pH meter when the sample reached room temperature (BSI, 1975).

Moisture content

The moisture content was measured according to the method of Indonesian National Standard (SNI 06-3735-1995). Five grams of active packaging was weighed and placed in an empty cup which were previously dried in the oven and cooled in a desiccator. The cup containing the sample was then covered and put in an oven at 100−102 °C for 6 h.

The moisture content was expressed by the percentage of weight lost during drying according to the equation (W₀ − W ₁/ W₀) ×100%, where W₁ represents the weight of the film after drying (g) and W₀ represents the initial weight of the film (g).

Determination of antioxidant activity

The DPPH radical-scavenging activity was determined according to the method published by Hanani, Yee & Khaizura (2019). Approximately 25 mg of active packaging was weighed and dissolved in five mL ethanol to obtain a solution. 0.1 mL of the sample solution was then mixed with 3.9 mL of 0.01 mmol L⁻¹ DPPH. Each mixture was homogenized and incubated for 30 min in the dark at room temperature. The absorbance value was measured using a UV-Vis spectrophotometer at 517 nm. The percentage of radical scavenging was calculated by dividing the difference of absorbance values of control (A_control) and the sample (A_sample) by the absorbance of the control, using the equation (A _control- A_sample/ A_control) ×100%.

Determination of antibacterial activity

The antibacterial activity was determined by the method of Hanani, Yee & Khaizura (2019). The bacterial strains used in this study consisted of E. coli, B. subtilis, Salmonella sp., and S. aureus. Prior to inhibition testing, the active film solution was prepared from 5 g of dried fish gelatine with the addition of 0.5% (v/v) glycerol in 100 mL of distilled water. Different concentrations of mangrove extract (0, 0.05, 0.15, 0.25, and 0.35% (w/v)) were added to the mixed solution. Afterwards, blank discs (six mm in diameter) were immersed in the different treated solutions for 15 min. The treated discs were laid on the Petri discs inoculated by four strains in Muller-Hilton agar (3 × 10⁸ cfu/mL), and the treatments were then incubated at 37 °C for 16 h. The diameter of the inhibition zones was measured using the following formula:

Zone of inhibition (mm) = zones free of bacterial growth –diameter of the disc.

Statistical analysis

All experiments were subjected to analysis of variance (ANOVA) using a general linear model, and mean comparisons were applied using a Tukey test. Except for antibacterial experiments, data are presented as the mean from three independent experiments ± SD of the results. Statistical analysis was carried out using Minitab, Version 18, a statistical software program (Minitab Pty Ltd., Sydney, NSW, Australia). The significant level was set at p < 0.05.

Amino acid composition of fish gelatine

The amino acid composition of A. stellaris skin gelatine is tabulated in Table 1. The presence of L-glycine (29.60%) and L-proline (12.53%) was higher than that of other amino acids in A. stellaris gelatine.

Appearance of the films

The appearance of active packaging from A. stellaris gelatine film and different concentrations of mangrove extracts is shown in Fig. 1. A higher concentration of leaf extracts resulted in a darker colour of the active packaging.

Thickness

The thicknesses of fish gelatine films containing two species of mangrove extracts are shown in Table 2. The average thickness of all the films was in the range of 137–143 µm, and the highest film thickness was observed in the gelatine-based film containing 0.35% B. gymnorhiza extract.

Tensile strength (TS)

Table 2 presents the TS values of fish gelatine added mangrove extracts with different concentrations. Based on statistical analysis, the type of mangrove leaves did not significantly affect the TS of active packaging. In contrast, the extract concentration and interaction between mangrove leaf types and extract concentration gave significantly different results (p < 0.05).

Elongation at break (EAB)

Based on the results in Table 2, the type of mangrove leaves and the concentration of mangrove leaf extract significantly affected the EAB values of the active packaging (p < 0.05).

Water vapour transmission rate (WVTR)

The WVTR of packaging films incorporated with 0−0.35% mangrove extracts is shown in Table 2. The results showed that two species of mangroves (B. gymnorhiza and S. alba) and different concentrations of mangrove leaf extracts were significantly different compared to the WVTR values of the active packaging films (p < 0.05).

pH value

The average pH value of the active packaging ranged from 5.75 to 6.6. The concentration of mangrove leaf extracts significantly affected (p < 0.05) the pH. In contrast, there was no significant difference between the mangrove species.

Moisture content

Table 2 shows that the moisture content of the active packaging ranged from 6−6.50%. Incorporating a higher concentration of mangrove leaf extracts significantly increased the moisture content (p < 0.05).

Antioxidant activity

The antioxidant activity of the edible film in this study was measured using the DPPH radical-scavenging assay. Figure 2 shows the antioxidant activity of films incorporated with B. gymnorhiza and S. alba extracts. With the increasing concentration of mangroves extracts, the scavenging effect increased significantly (p < 0.05) from 12.39% to 57.32% and from 12.36% to 60.98% for B. gymnorhiza and S. alba extracts, respectively.

Antibacterial activity

Four bacterial strains were used to determine the antibacterial activities of fish gelatine-derived films incorporated with B. gymnorhiza and S. alba leaf extracts. The inhibition zones of the films are shown in Table 3. The films with higher concentration of mangroves extracts, showed significantly (p < 0.05) increased antibacterial activities.