A biodegradable film based on sodium alginate–gelatin involving a mulberry anthocyanin extract for fish oil packaging

Yawen Lv; Jingjing Yang; Ting Luo; Hongmei Liao; Yawen Lv; Jingjing Yang; Ting Luo; Hongmei Liao

doi:10.48130/fia-0026-0011

Driven by the 'dual carbon' strategy and the imperative of a circular economy, food packaging is undergoing a ternary upgrade. In this study, mulberry anthocyanin extract (MAE) was upcycled into a natural, dual-function active principle that concurrently delivers antioxidant capacity and ultraviolet (UV) barrier activity. It was incorporated with an edible sodium alginate–gelatin (SG) based film to engineer a next-generation biodegradable antioxidant film. The dose-dependent impacts of MAE on the film's mechanical signature, radical-scavenging efficacy, optical performance, and practical utility in fish oil packaging were systematically examined. MAE obtained from a residue of mulberry juice fermentation had 296.86 mg/g total polyphenols and 146.50 mg/g anthocyanins, primarily cyanidin-3-glucoside and cyanidin-3-rutinoside. When 0.2 mg/mL MAE (SG-2MAE) was added, the tensile strength of SG films increased from 48.33 to 50.98 MPa. When 0.4 mg/mL MAE was incorporated, the antioxidant activity of SG-4MAE film against 2,2'-biazido-bis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) increased by 158%, whereas transmittance (600 nm) decreased by 33.7%, indicating enhanced UV blocking. When applied for packaging fish oil, the SG-MAE film delayed oxidation better than the unpackaged sample after 4 weeks' storage by directly blocking oxygen, releasing antioxidant constituents, and adsorbing residual oxygen. In summary, SG-MAE films showed excellent comprehensive properties and high antioxidant properties, and could be used as active packaging materials for fish oil.

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Introduction

Traditional packaging materials, such as polyethylene, polypropylene, and metal-based packaging, have caused environmental pollution problems because of their nonbiodegradability and potential toxicity. As consumers become more aware of sustainability and environmental issues, it is urgent to develop biodegradable and edible films using natural biopolymers. Sodium alginate, a naturally occurring polysaccharide extracted from brown seaweed, is widely recognized for its excellent film-forming properties, biodegradability, and nontoxic nature^[1]. When gelatin is mixed with sodium alginate, the film's tensile strength, elongation at break, and physical qualities improve, allowing it to handle stringent food packaging standards. It has reported that sodium alginate and gelatin can be used as packaging materials for soybean. Gelatin, a protein derived from collagen, is commonly used in food packaging because of its ability to enhance biopolymeric films' mechanical properties, flexibility, and thermal stability^[2]. Biodegradable films composed of gelatin exhibit good mechanical properties; however, these films still have some limitations. Packaging films based on gelatin may dissolve, swell, or decompose upon contact with moisture^[3]. It has been shown that the hardness of gelatin increases linearly with an increase in gelatin concentration, and gelatin below its melting temperature (37–40 °C) exhibits cold solidification, which is unfavorable for consumption^[4]. However, compounding gelatin with other biodegradable packaging materials can significantly improve its moisture resistance, elasticity, flexibility, and stiffness^[5]. In addition, our previous study found that films prepared with a sodium alginate to gelatin ratio of 7:3 has excellent mechanical properties and good barrier properties, and the addition of rose polyphenols effectively delayed the oxidation of edible oils.

Natural antioxidants have become an important trend in the the future development of foods. Introducing natural antioxidants such as polyphenols (phenolic acids, flavonoids, anthocyanins, lignans, and stilbenes), carotenoids (lutein and carotene), and vitamins (vitamins E and C) into packaging films can further enhance their functional properties^[6]. Anthocyanins are water-soluble flavonoid pigments found readily available in various fruits and vegetables, with free radical scavenging and antioxidant activities^[7]. Anthocyanins shows strong antioxidant activity and act as antioxidants by interrupting the oxidation reaction chain. Films with anthocyanins inhibit photo-oxidation by improving the optical barrier property. One study has been carried out on a functional edible film of olive oil^[8]. Mulberry pomace, the residue product of processed materials, is rich in anthocyanins and can be used as an anthocyanin source^[9].

Fish oil is usually stored at low temperatures, away from light, and in airtight containers. It has abundant omega-3 polyunsaturated fatty acids (PUFAs), which are known to have positive health effects especially in preventing inflammatory, neurological, and cardiovascular diseases^[10]. However, PUFAs' susceptibility to oxidative degradation remains a significant challenge in developing and preserving fish oil-based products^[11]. The oxidation of unsaturated fatty acids leads to rancid odors, reduced nutritional value, and the generation of potentially harmful oxidation products such as aldehydes and hydroperoxides. Therefore, ensuring the oxidative stability of fish oils is essential to maintain their quality, functionality, and shelf life throughout storage and distribution. It has suggested that we could use natural antioxidants like gallic acid to prevent oxidation^[12] or to scavenge peroxide radicals formed at the start of the oxidation reaction before the chain reaction^[13]. A novel fish oil package may be a potential choice. However, few studies have used a biodegradable film for packaging fish oil. Therefore, we prepared a film with light barrier and antioxidant properties for packaging fish oil based on sodium alginate and gelatin with mulberry anthocyanins.

This study aimed to prepare a novel biodegradable packaging film with sodium alginate and gelatin (SG), incorporating a mulberry anthocyanin extract (MAE) to act as an antioxidant and provide light-blocking properties. The primary objectives were to evaluate the potential application of SG-MAE films in packaging fish oil, thus offering a sustainable and efficient alternative to conventional packaging materials. The incorporation of MAE into the SG film matrix not only imparts antioxidant ability but also contributes to the film's ability to act as a light barrier. This dual functionality is crucial for protecting fish oil, which is highly susceptible to oxidation when exposed to light. It is expected to provide valuable insights into the development of active packaging materials that can effectively protect light-sensitive foods such as fish oil. The findings will highlight the potential of using natural antioxidants and light-blocking agents in biodegradable films, thereby offering a more sustainable and functional packaging solution.

Materials and methods

Materials

Hydrochloric acid, methanol, ethanol, and gelatin were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Chemicals such as 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH), 2,2'-biazido-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), copper sulfate, sodium alginate, and fish oil (18% eicosapentaenoic acid, 20% omega-3 PUFAs) were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Mulberry pomace was obtained from a local fruit wine fermentation enterprise in Wuxi City.

Extraction and purification of anthocyanins from mulberry pomace
MAE was extracted and purified according to Ai et al.^[14] with slight modifications. Specifically, mulberry pomace was mixed with a 60% ethanol aqueous solution (v/v, pH = 4) at a solid–liquid ratio of 1:10, and the mixture was stirred at 40 °C for 1 h under dark conditions. Then the mixture was centrifuged at 7,552 × g for 10 min, and the suspension was concentrated using an evaporator (RV8, Eka, Staufen, Germany) at 40 °C under atmospheric pressure. The residue was the crude anthocyanin extract, which was stored at −20 °C for further purification over the next 7 d.

The crude extract was first passed through an AB-8 macroporous resins column (50 mm × 400 mm) at a flow rate of 1 mL/min. Then the anthocyanin extract was eluted from the AB-8 macroporous resin with 70% ethanol containing 0.1% HCl (v/v), at a flow rate of 1 mL/min. The eluant was collected and concentrated by evaporation. Finally, the eluant was freeze-dried with a lyophilizer (DC801, Yamato Scientific Co., Ltd., Japan) and stored at −20 °C for further application in the film-forming process.

Characterization of MAE

The total phenolics and anthocyanins

The total phenolic in MAE was determined using the Folin–Ciocalteu assay^[15]. In short, a 1 mg/mL MAE solution was prepared with deionized water, then each 1 mL of the MAE solution was mixed with 1 mL Folin–Ciocalteau reagent, 3 mL Na₂CO₃ (7.5%, w/v), and 5 mL distilled water. Then the mixture was incubated at 30 °C for 1 h away from the light, and its absorbance at 760 nm was measured using an ultraviolet (UV) mini-124 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Total phenolics of the samples are presented as mg of gallic acid equivalents per 1-g sample.

The anthocyanin content was determined using the differential pH method^[16]. Briefly, the absorbance at 510 and 700 nm of the MAE solution was measured. The content was expressed as mg cyanidin-3-O-glucoside equivalent/g extract, and was calculated by Eqs (1) and (2) as follows:

$ A={\left({A}_{510}-{A}_{700}\right)}_{{{pH}_{1.0}}}-{\left({A}_{510}-{A}_{700}\right)}_{{{pH}_{4.5}}} $

(1)

$ Total\;anthocyanins\;\left(mg/g\right)=\dfrac{A\times MW\times DF\times V\times 10}{\varepsilon \times L\times M} $

(2)

where, A is the absorbance, MW is the molecular weight of cyanidin-3-O-glucoside (449.2 g/mol), DF is the dilution concentration, V is the volume of extracted solvent (mL), ɛ is the extinction coefficient of cyanidin-3-O-glucoside (28,000 L/cm/mol), L is the radiant length (cm), and M is the mass of the anthocyanin extract (g).

Main anthocyanins in MAE determined by ultrahigh-performance liquid chromatography–tandem mass spectrometry

The anthocyanins in MAE were analyzed by ultrahigh-performance liquid chromatography–tandem mass spectrometry (UPLC-MS) according to Chen et al.^[17]. The MAE samples were dissolved in distilled water to prepare a 1 mg/mL MAE solution and then centrifuged at 5,180 × g for 10 min at 4 °C. The specific detection conditions of UPLC-MS were as follows: a bridged ethylene hybrid (BEH) C18 column (100 × 2.1 mm, 1.7 μm); a diode array detector (DAD) with a detection wavelength at 535 nm; an injection volume of 2 μL. The mobile phase consisted of water (Solvent A) and acetonitrile (Solvent B), each containing 0.1% formic acid. The flow rate was 0.3 mL/min. The column was maintained at 45 °C, and a gradient elution was performed over 10 min. The mass spectrometry (MS) detection parameters were optimized for electrospray Ionization positive mode (ESI+) scanning with a mass range of 20–1,000 m/z. Additional MS conditions included a gas temperature of 400 °C, a gas flow rate of 700 L/h, a collision energy of 25 eV, a cone voltage of 30 V, a nebulizer pressure of 50 psi, and a capillary voltage of 3.5 kV.

Antioxidant activity of MAE

The antioxidant activity of MAE was determined according to a previous study with slight modifications^[14]. To determine the DPPH scavenging activity, each 100-μL MAE solution (0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) was mixed with 4 mL of a DPPH solution (0.14 mmol/L) and reacted for 30 min in the dark. To determine the ABTS scavenging activity, each 100 μL MAE solution (0.01–1.0 mg/mL) was mixed with 3.9 mL of an ABTS solution (consisting of a 1:1 mixture of 7 mmol/L ABTS and 2.45 mmol/L K₂S₂O₈) and reacted for 30 min in the dark. Then the absorbance of the mixture at 517 and 734 nm was determined with a spectrophotometer (UV mini-124, Shimadzu Corporation, Kyoto, Japan) for calculating the free radical scavenging ability of DPPH and ABTS, respectively. Deionized water instead of the MAE solution was used as a control, and the free radical scavenging activity was calculated according to Eq. (3):

$ Free\; radical\; scavenging\; activity\; (\text{%})=\left(1-\dfrac{A_{\mathit{1}}}{A_{\mathit{0}}}\right)\times100 $

(3)

where, A₀ is the absorbance value of the control, and A₁ is the absorbance value of samples.

pH sensitivity of MAE

The pH sensitivity was determined by dissolving 2 mg of MAE in 20-mL buffer solutions with varying pH levels (pH 1.0–12.0) for 3 min. Subsequently, each 10-mL solution was transferred into a glass sample vial, and the color change was recorded with a smartphone. Additionally, the visible absorption spectra of the MAE solutions were scanned from 450 to 700 nm using a spectrophotometer (UV mini-124, Shimadzu Corporation, Kyoto, Japan).

Preparation of SG-MAE films
According to our previous study^[18], the SG-MAE films were prepared as following: 0.05 mg/mL sodium alginate and gelatin solutions were prepared separately and were mixed at a ratio of 7:3 to prepare SG solution. Then 2, 4, 6, 8, or 10 mg of MAE was added to the mixture, with 1 g glycerol as a plasticizer to obtain film-forming solutions. Then the mixture was treated with an ultrasonic cell grinder (Scientz-IID, SCIENTZ, Ningbo, China) at 500 W for 15 min at 25 °C. After defoaming with a vacuum (Sorvall LYNX 4000, Thermo Fisher Scientific, Waltham, America), each 20-mL film-forming solution was spread onto petri dishes (10 cm × 10 cm) and dried at 25 °C for 24 h to prepare SG-MAE films. These films were marked as SG-2MAE, SG-4MAE, SG-6MAE, SG-8MAE, and SG-10MAE, according to the amount of MAE. The SG film without MAE was used as a control, labeled CK. Before further tests, these films were peeled off and stored at 25 °C and 53% relative humidity for more than 48 h.

Rheological characterization of the film-forming solutions
According to a previous study by Ai et al.^[14], the rheological properties of the film-forming solutions were examined using a rheometer (DISCOVERY HR-3, TA Instruments, New Castle, Delaware, USA). The apparent viscosity of the film-forming solution was measured at 25 °C by using a 40-mm aluminum parallel plate with a plate spacing of 1.0 mm, and the shear rate was 0.1–100 s⁻¹. The viscosity (η, Pas) and shear rate (γ, s⁻¹) data were analyzed using a power law model, as shown in Eq. (4):

$ \eta =K{\gamma }^{n-1} $ (4)

where, K is the consistency index (Pa sⁿ), and n is the flowing behavior index.

Thickness, water solubility, moisture content, and water vapor permeability

The thickness, water solubility, moisture content, and water vapor transmission rate of the films were determined according to a previous study^[14]. The thickness of the films was determined at 10 randomly selected locations in each film using a digital micrometer (digital outside micrometer, GREENER, Yantai, China).

The moisture content (MC) of the film was determined by drying the film at 105 °C until a constant weight W₁ (g) was reaached^[16]. The MC was calculated by Eq. (5).

$ {MC}\;\left({\text{%}}\right)=\dfrac{{W}_{0}-{W}_{1}}{{W}_{0}}\times {100} $

(5)

where, W₀ is the initial weight of the film, and W₁ is the weight of the film after drying.

To detect the water solubility (WS), dried films were weighed (M₀) and put into 50 mL of deionized water under shaking at 25 °C for 24 h. Then the water-insoluble matter was removed and dried at 105 °C to a constant weight (M₁). WS was calculated by Eq. (6).

$ {WS}\;\left({\text{%}}\right)=\dfrac{{{M}}_{{0}}-{{M}}_{{1}}}{{{M}}_{{0}}}\times {100} $

(6)

The water vapor permeability (WVP) was determined by adding 20 mL of deionized water to a bottle, sealing the bottle with a film sample (4 cm × 4 cm), and placing the bottle in a desiccator for 7 d. We recorded the change in weight of the tubes daily. The procedure was repeated three times for each film. WVP was calculated by Eq. (7).

$ WVP=\dfrac{{\Delta m\times d}}{{S\times \Delta P \times T}} $

(7)

where, Δm is the mass of water transmitted (g), d is the film's thickness (mm), S is the active area of water vapor transmission (3.14 × 10⁻⁴ m²), T is the time (s), and ΔP is the difference in the water vapor between two sides of the film (3.167 kPa) (at 25 °C)^[16].

According to Wang et al.^[16], the oxygen permeability (OP) was measured by sealing a container containing linoleic acid (2 mL) with the film and then incubating it at room temperature for 7 d. The OP value was calculated using Eq. (8).

$ OP=\dfrac{{\Delta w}}{{a \times t}} $

(8)

where, Δw is the change in the weight of linoleic acid (g), a is the surface area of the film (m²), and t is the duration (days).

Structural characterization of SG-MAE films

The intermolecular interactions in the films were analyzed using the attenuated total reflection attachment of the infrared spectrometer (Nicolet iS10, Thermo Scientific™, Massachusetts, USA). The measurements were conducted with 32 scans at a resolution of 4 cm⁻¹, covering a wavelength range of 600–4,000 cm⁻¹. Additionally, the crystalline properties of the films were assessed using an X-ray diffractometer (D2 PHASER, Bruker, Karlsruhe, Germany). The X-ray analysis was performed with Cu-Kα radiation at 40 kV at a range of 2θ of 5° to 60° and a scan rate of 4° min⁻¹.

Mechanical properties

A texture analyzer (TA. XTPlus, Stable Micro System, Surrey, UK) equipped with a tensile grips (TG) probe was utilized to determine the mechanical properties, including tensile strength (TS) and elongation at break (EB). The films were cut into long strips with an initial gap of 40 mm and a test speed of 10 mm/s. Each film was tested three times. We calculated TS and EB according to Eqs (9) and (10), respectively.

$ TS\;\left({MPa}\right)=\dfrac{{F}}{{w \times d}} $

(9)

where, F is the maximum tensile force at break (N), w is the width of the film (mm), and d is the thickness of the film (mm).

$ EB\; \left(\text{%}\right)=\dfrac{L_{\mathit{\mathrm{1}}}-L_{\mathit{\mathrm{0}}}}{L_{\mathit{\mathrm{0}}}} $

(10)

where, L₁ is the maximum tensile length of the film reached at the time of fracture (mm), and L₀ is the initial length of the clamping distance (mm).

Scanning electron microscopy images of SG-MAE films

The surface and cross-sectional morphologies of the films were characterized using scanning electron microscopy (SEM). The sample was sputter-coated with a layer of gold to enhance conductivity and observed under an accelerating voltage of 3 kV for morphological characterization.

Water contact angle
The water contact angle (WCA) of the films was determined using a video-based optical contact angle-measuring instrument (OCA15EC, Dataphy Instruments Co., Ltd., Dusseldorf, Germany). The films were placed on the sample stage, and 8 μL of deionized water was slowly dispensed onto the film's surface using a 2-mL microsyringe. A digital image of the water droplet was captured using a camera equipped with a macrolens. The WCA (°) was then calculated and recorded using the instrument's software^[14].

Transmittance and color properties

The transparency of the films was measured as follows. The films were cut into rectangular strips (4 cm × 1 cm), and their light transmission was scanned at 200–800 nm with a spectrophotometer (UV mini-124, Shimadzu Corporation, Kyoto, Japan).

Their color properties were measured with a portable colorimeter (cs-820N, Hangzhou Caipu Technology Co., Ltd., Hangzhou, China). The following color parameters were measured at five randomly selected locations in each sample: L* (brightness), a* (red to green), b* (yellow to blue), and the total color difference (ΔE) calculated according to Eq. (11)^[14]:

$ \Delta E=\sqrt{{\left({{L}}^{{*}}{-L}_{{0}}^{{*}}\right)}^{{2}}+{\left({{a}}^{{*}}{-a}_{{0}}^{{*}}\right)}^{{2}}+{\left({{b}}^{{*}}{-b}_{{0}}^{{*}}\right)}^{{2}}} $

(11)

where, L₀*, a₀*, and b₀* are the initial values of the films, and L*, a*, and b* are the values of the films after storage.

Antioxidant activity of SG-MAE films

The antioxidant activity of the films was measured according to a previous study^[18]. Briefly, 0.2 g of each film was dissolved in 10 mL ethanol (50% dissolved in water), then the antioxidant activity was measured according to the method of examining the rheological characterization of film-forming solution.

Fish oil packaging
The SG and SG-4MAE films were cut into pouches (4 cm × 4 cm), heat-sealed using a heat-sealing machine (Dapai, Shenzhen Dingsheng Electric Appliance Co., LTD., Shenzhen, China), and sealed to pack 2 g of fish oil. The effect of top-cover packaging on the fish oil was also investigated: 10 mL of edible oil in a glass bottle was covered with a SG or SG-4MAE film (4 cm × 4 cm) and then fixed with a rubber band. Unpackaged fish oil and fish oil in ziplock bags were used as controls. All the samples were stored at 25 ± 3 °C and 53% ± 2% relative humidity for 4 weeks. To quantitatively assess the extent of oxidation, the peroxide value (POV), thiobarbituric acid value (TBA), and acid value (AV) of the fish oil were measured weekly.

Evaluation of fish oil's quality during storage

Peroxide value
According to Zhu et al.^[19], 1 g of fish oil, 30 mL of trichloromethane/acetic acid (3:2 volume ratio), and 1 mL of saturated potassium iodide were mixed in a conical flask. The mixture was homogenized and reacted in the dark for 3 min. Then 100 mL of distilled water was added, and the reaction mixture was titrated with a 0.01 mol/L sodium thiosulfate solution, using a 1 mL soluble starch solution (1%, w/w) as an indicator. A blank titration was conducted in the same way without the oil sample. POV (mmol/kg) was calculated using Eq. (12).

$ POV=\dfrac{\left(V-V_0\right)\times c}{2m}\times1,000 $ (12)

where, V and V₀ are the volume of sodium thiosulfate solution (mL) consumed by the sample and the blank solution, respectively; c is the concentration of the sodium thiosulfate standard solution (mol/L); and m is the weight of fish oil (g).

Thiobarbituric acid value
Samples of 50–250 mg of fish oil were mixed with 25 mL of 1-butanol, then the 5-mL mixture was reacted with 5 mL of TBA reagent at 95 °C for 2 h. After heating, these tubes were rapidly cooled in an ice-water bath to terminate the reaction and prevent further oxidation^[19]. The absorbance of the samples at 530 nm was tested, and the TBA value was calculated via Eq. (13).

$ {T}BA=\dfrac{50\times ( A-B) }{m} $ (13)

where, A is the sample absorbance, B is the blank absorbance, and m is the weight of fish oil (mg).

Acid value
According to the method of Wang et al.^[20], 0.5 g of fish oil was mixed with 50 mL of ether/isopropanol (1:1, volume ratio). The mix was then titrated using 0.001 mol/L NaOH solution with phenolphthalein as an indicator. A blank titration was treated in the same way without oil. AV (mg/g) was calculated by using Eq. (14).

$ AV=\dfrac{\left({V-}{{V}}_{{0}}\right)\times {c\times 40}}{{m}} $ (14)

where, V and V₀ are the volume of the NaOH solution (mL) consumed by the sample and blank solution, respectively; c is the concentration of the NaOH standard solution (mol/L); 40 is the molar mass of NaOH (g/mol); and m is the weight of fish oil (g).

Shelf life prediction
The patterns of change in the quality of food usually conform to zero- or one-level models, so the oxidation kinetic equations for fish oil during storage at 25 °C were established using Eq. (15)^[21]. The end of shelf life was predicted and calculated as the acid value of fish oil reaching 3 mg/g.

$ \ln\;\left({AV}\right)=kt+a $ (15)

where, AV is the acid value of edible oil at t days of storage (mg/g), k is the rate constant of oxidative rancidity (1/d), t is the number of days of storage (days), a is ln (AV₀), and AV₀ is the initial acid value.

Statistical analysis
The results are reported as the mean ± standard deviation. Data were subjected to one-way analysis of variance (ANOVA) by SPSS 24 software (IBM, New York, USA) according to Duncan's multiple range tests (p < 0.05). A significance level of p <0.05 was used to determine statistical significance.

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Films	Thickness (μm)	Water solubility (%)	Moisture content (%)	Water vapor permeability (× 10⁻¹¹ g/[m·s·Pa])	Oxygen permeability (g/dm²)
SG	80.12 ± 4.11^c	63.11 ± 0.77^a	19.58 ± 1.62^a	2.21 ± 0.01^a	3.16 ± 0.21^c
SG-2MAE	82.69 ± 3.29^c	62.89 ± 2.31^a	17.83 ± 1.68^b	1.50 ± 0.01^d	5.22 ± 0.54^b
SG-4MAE	80.07 ± 2.71^c	62.31 ± 1.23^a	16.11 ± 1.57^c	1.73 ± 0.03^c	5.43 ± 0.67^b
SG-6MAE	85.61 ± 4.21^b	60.03 ± 0.91^b	16.53 ± 2.36^c	1.85 ± 0.01^b	5.51 ± 0.61^b
SG-8MAE	83.09 ± 2.81^bc	58.21 ± 0.89^c	18.73 ± 0.82^ab	1.97 ± 0.17^ab	5.47 ± 0.35^b
SG-10MAE	89.93 ± 1.01^a	58.11 ± 0.77^c	17.42 ± 0.80^b	1.65 ± 0.01^bc	6.20 ± 0.23^a
SG-MAE, sodium alginate–gelatin film with 0.2–1.0 mg/mL mulberry pomace anthocyanin extract. Different letters in the same column represent significant differences at p < 0.05.

Films	L*	a*	b*	ΔE	T_{(600 nm)} (%)
SG	90.94 ± 0.26^a	−0.75 ± 0.30^f	15.14 ± 0.20^a	−	71.84 ± 0.98^a
SG-2MAE	76.14 ± 1.50^b	10.26 ± 0.33^e	13.87 ± 0.29^b	26.46 ± 1.46^e	58.77 ± 0.55^b
SG-4MAE	64.78 ± 0.61^c	20.23 ± 0.00^d	14.59 ± 0.38^a	40.2 ± 1.33^d	47.63 ± 1.85^c
SG-6MAE	56.28 ± 1.27^d	24.46 ± 0.78^c	15.06 ± 0.29^a	49.89 ± 1.79^c	40.34 ± 0.97^d
SG-8MAE	43.32 ± 3.03^e	30.19 ± 0.53^b	15.26 ± 0.58^a	63.35 ± 2.56^b	28.55 ± 1.60^e
SG-10MAE	36.92 ± 0.05^f	36.29 ± 0.80^a	16.13 ± 0.77^a	71.93 ± 0.57^a	22.3 ± 0.14^f
SG-MAE, sodium alginate–gelatin film with 0.2–1.0 mg/mL MAE. Different letters in the same column represent significant differences at p < 0.05.

Packaging	Kinetic equation	R²	Shelf life (days)
Control	ln(AV) = 0.053t − 0.173	0.97	24.6
Top-cover with SG film	ln(AV) = 0.048t − 0.170	0.97	26.5
Top-cover with SG-4MAE film	ln(AV) = 0.042t − 0.223	0.99	31.6
Ziplock bags	ln(AV) = 0.043t − 0.196	0.96	30.3
SG bag	ln(AV) = 0.033t − 0.315	0.95	42.8
SG-4MAE bag	ln(AV) = 0.022t − 0.282	0.95	62.6
Control: Unpackaged fish oil; ziplock bags: Fish oil packaged in plastic ziplock bags; SG bag and SG-4MAE bag: Fish oil packaged in bags made of sodium alginate–gelatin (SG) film and sodium alginate–gelatin film containing 0.4 mg/mL mulberry pomace anthocyanin extract; top-covering with SG film and SG-4MAE film: Fish oil packaged in glass bottles and covered with SG or SG-4MAE films.

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A biodegradable film based on sodium alginate–gelatin involving a mulberry anthocyanin extract for fish oil packaging

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