Article

Hake Fish Preservation Using Plant-Based Impregnated

Polylactic Acid Food Films as Active Packaging
Fini Sánchez-García 1 , Noelia D. Machado 2 , María Tirado-Fernández 1 , Cristina Cejudo-Bastante 2, * ,
Ana M. Roldán 1 , Casimiro Mantell-Serrano 2 and Lourdes Casas-Cardoso 2, *

1 Chemical Engineering and Food Technology Department, Faculty of Sciences, Marine Research
Institute (INMAR), University of Cadiz, P.O. Box 40, 11510 Puerto Real, Spain; fini.sanchez@uca.es (F.S.-G.);
maria.tirafer@alum.uca.es (M.T.-F.); ana.roldan@uca.es (A.M.R.)
2 Chemical Engineering and Food Technology Department, Faculty of Science, Wine and Agrifood Research
Institute (IVAGRO), University of Cadiz, Avda. República Saharaui, s/n, 11510 Puerto Real, Spain;
noelia.machado@uca.es (N.D.M.); casimiro.mantell@uca.es (C.M.-S.)
* Correspondence: cristina.cejudo@uca.es (C.C.-B.); lourdes.casas@uca.es (L.C.-C.)

Abstract: Global fish consumption has steadily increased; however, fishery products
are difficult to preserve. Active packaging has emerged as an alternative to improve its
conservation. In this work, fresh hake fillets were packaged in commercial polylactic
acid films impregnated with olive leaf extract using supercritical CO2 . The impregnation
was performed at 35 ◦ C and 400 bar for 1 h. The ABTS assay was used to determine the
antioxidant activity, and migration tests were performed using food simulants A and D2 for
10 days at 5 ◦ C. The fresh fillets were packaged in impregnated and control films and stored
for 12 days at 4 ◦ C. The microbiological, physical (drip loss, aw , pH, and color) and chemical
parameters (total volatile base and trimethylamine) were analyzed. The impregnated
films presented a 706 µg extract mg−1 polymer, showing a 2-fold extract release using
food simulant D2 than simulant A. After hake storage using impregnated films, reduced
microbial count, and drip loss, maintaining the pH stability was obtained. The color turned
Academic Editors: Igor Tomasevic,
yellowish and no detectable olfactory presence of the extract was noted. The chemical
Marco Iammarino, Annalisa Mentana
parameters were similar in both types of films. The proposed biodegradable packaging
and Rosalia Zianni
with olive by-products preserves moisture and controls microbial growth, representing an
Received: 14 December 2024
eco-friendly alternative.
Revised: 7 January 2025
Accepted: 8 January 2025
Published: 10 January 2025 Keywords: hake fish; polylactic acid; olive leaf extract; supercritical fluids
Citation: Sánchez-García, F.;
Machado, N.D.; Tirado-Fernández, M.;
Cejudo-Bastante, C.; Roldán, A.M.;
Mantell-Serrano, C.; Casas-Cardoso, L. 1. Introduction
Hake Fish Preservation Using Over the past decades, global fish consumption has steadily increased, driven by eco-
Plant-Based Impregnated Polylactic
nomic growth and improved living standards [1]. Reflecting this trend, the 2024 edition of
Acid Food Films as Active Packaging.
The State of World Fisheries and Aquaculture reported that the global fisheries and aquaculture
Appl. Sci. 2025, 15, 643.
https://doi.org/10.3390/ production reached 223.2 million tons in 2022, a 4.4% increase from 2020. This included
app15020643 185.4 million tons of aquatic animals, with a global per capita consumption of 20.7 kg, two
times higher over the last 60 years [2]. This rise underscores the growing demand for fish
Copyright: © 2025 by the authors.
Licensee MDPI, Basel, Switzerland. as a vital source of high-quality proteins and essential nutrients including omega-3 fatty
This article is an open access article acids, minerals, and vitamins [3].
distributed under the terms and However, preserving fishery products presents a significant challenge due to rapid
conditions of the Creative Commons microbial, enzymatic, and chemical reactions during storage, which degrade the quality,
Attribution (CC BY) license
flavor, texture, and nutritional value [4]. These challenges underscore the need for innova-
(https://creativecommons.org/
tive approaches, such as active packaging, to mitigate spoilage and enhance shelf life. As a
licenses/by/4.0/).

Appl. Sci. 2025, 15, 643 https://doi.org/10.3390/app15020643

Appl. Sci. 2025, 15, 643 2 of 22

consequence, advanced preservation techniques have been developed [5], among which
active packaging (AP) has emerged as a promising solution. According to the European
Union Guidance to Commission Regulation No. 450/2009, AP provides an extra function,
in addition to a protective barrier against external altering factors. This type of packaging
can absorb food-derived chemicals from the food or its environment (absorbers), or it
can release substances into the food or the environment surrounding the food such as
preservatives, antioxidants, and flavorings (emitters) [6]. In this sense, AP may not only
extend the shelf life of perishable foods more efficiently than traditional packaging, but can
also align with modern food safety standards by integrating active components that can
absorb or release substances to enhance food preservation [7,8].
In recent years, there has been growing interest in incorporating natural antioxidants,
such as polyphenols, tocopherols, essential oils, and plant extracts, to replace synthetic ones
like butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), which have
raised health concerns [9]. The addition of polyphosphates is another controversial point
in food safety [10]. Among these natural sources, plant extracts have gained attention for
their bioactive properties and their potential to valorize agro-industrial by-products [11].
Among the agri-food residues, those derived from the production of olive oil from Olea
europaea L. tree are of particular interest.
The Mediterranean region produces about 60% of the world’s olive output, generating
large amounts of by-products like olive pomace and olive leaves [12]. It is well-known
that olive leaves are rich in phenolic compounds including simple phenols, flavonoids,
and secoiridoids [13]. These bioactive compounds can be extracted using conventional
extraction techniques such as Soxhlet and heat-reflux extraction or by using alternative
techniques such as microwave, ultrasound, and high-pressure assisted extraction [14].
Olive leaf extracts have shown potential in active packaging applications such as loaded
polyethylene terephthalate/polypropylene films (PET/PP) for vegetables (e.g., cherry
tomatoes and sunflower seed preservation) [15,16], and in meat (e.g., the development of
biodegradable carrageenan films for lamb meat preservation) [17].
Parallel to these developments, the food packaging industry is increasingly adopting
biodegradable polymers to minimize the environmental impact. Polymers in food packag-
ing have distinct advantages such as their adequation to food properties and composition
to increase their preservation, which makes the commercialization of packaged food the
strategy most often used by supermarkets to provide a wide variety of fresh and frozen
products. Nowadays, the major focus on polymer-based packaging is to minimize the
environmental impact by utilizing biodegradable polymers as sustainable alternatives to
conventional materials derived from fossil fuels [18]. Polylactic acid (PLA), a biodegradable
polymer derived from renewable resources like corn has garnered significant attention
in the packaging industry. It offers several advantages including high transparency, low
permeability to water and gases, recyclability, and a short degradation period, making it a
viable alternative to conventional plastics like polystyrene (PS), low-density polyethylene
(LDPE), and PET [18–20]. Recent studies have highlighted promising results in food preser-
vation. For example, PLA films loaded with rice straw extracts demonstrated antioxidant
and antimicrobial properties, successfully preserving pork meat by inhibiting microbial
growth, oxidation, discoloration, and weight loss. The extracts were incorporated into
PLA using melt blending and compression molding [21]. Another study evaluated bilayer
films composed of a PLA-tilapia fish gelatin-sodium alginate loaded with β-cyclodextrin
inclusion complexes of cinnamaldehyde and thymol. These active bilayer films extended
the shelf life of Japanese sea bass (Lateolabrax japonicus) by at least 3 days. As can be noted,
these active films were prepared by the coating method [22].
Appl. Sci. 2025, 15, 643 3 of 22

One innovative method for incorporating active compounds into biodegradable poly-
mers is supercritical CO2 (scCO2 ) impregnation. The scCO2 is a state reached above the
critical temperature (TC = 31.1 ◦ C) and pressure (PC = 73.8 bar) where the characteristics of
the liquid and gas phases become nearly indistinguishable. The main advantage of this
solvent is the ability to adjust the diffusivity, viscosity, and density in front of small changes
in the pressure and temperature parameters [23]. During supercritical impregnation, CO2 is
pressurized and heated above its critical point, allowing it to dissolve the active compounds
and then impregnate them into the polymeric material. Once the pressure is released, the
CO2 evaporates, leaving the active substances embedded within the material. This method
is efficient, non-toxic, and environmentally friendly [24]. The scCO2 impregnation of PLA
films intended for food active packaging has been explored using carvone [25,26], and
thymol [27], and extracts such as thyme [28] and clove [29]. However, the performance of
these types of packaging in real food matrices has been scarcely evaluated.
Given this context, the main objective of this work was to produce active packaging
for hake fillets using a natural extract as a source of bioactive compounds impregnated
in a commercial biodegradable polymer employing supercritical technologies. For this
purpose, a commercial PLA-based film was impregnated with olive leaf extract using
the scCO2 technique. The resulting films were assessed for their bioactivity, and extract
migration properties. Their effectiveness in preserving minimally processed hake fillets
was evaluated over a 12-day storage period. The impact of the impregnated films on
the shelf life of the hake fillets was determined through comprehensive analyses of the
physicochemical, microbiological, and sensory parameters, offering insights into their
potential for enhancing food preservation.

2. Materials and Methods

2.1. Materials
The biodegradable PLA-based film (10 µm thickness) was provided by Eversia (Murcia,
Spain). The Olea europaea leaves (<3% moisture) were obtained from an olive producer (San
José de Lora de Estepa SCA Coop, Seville, Andalusia, Spain), dried at room temperature
(~27 ◦ C) for 48 h, and stored in the dark before use.
Carbon dioxide (CO2 , 99.99% purity) was purchased from S.E. Carburos Metálicos
S.A. (Barcelona, Spain). Ethanol (food grade, 96% v/v) was obtained from Cosela (Sevilla,
Spain).
The reactants 2,2′ -azino-bis-(3-ethylbenzothiazoline-6-sulfonic) (ABTS), potassium
peroxodisulfate (K2 S2 O8 , ≥99.0%), trichloroacetic acid ACS reagent (TCA, ≥99.0%), boric
acid ACS reagent (99.5%), potassium carbonate (K2 CO3 ), formalin solution neutral buffered
10%, chloride acid (HCl), sodium chloride ACS reagent (NaCl, 99.0%), glycerol formal, and
trypan blue were purchased from Sigma-Aldrich (Hamburg, Germany).
PCA agar and MRS agar were provided by Laboratorios Conda S.A. (Madrid, Spain),
and refined sunflower oil with a maximum acidity of 0.2◦ was obtained from a local market
(Cadiz, Spain).

2.2. Enhanced Solvent Extraction of Olive Leaves

High-pressure equipment from Thar Technologies (model SF1000, Pittsburgh, PA,
USA) was used for the extraction process. The procedure was conducted in batch mode,
with some modifications from a previously reported method [16]. The dried olive leaves
were ground until a size of 2–3 mm was reached by using a Phillips HR 3655/00 1400 W
blender (China). A paper cartridge containing 121 g of ground olive leaves and 650 mL of
ethanol was placed into a 1000 mL high-pressure vessel. The mixture of CO2 with ethanol
as a cosolvent allows for the extraction of more polar compounds [30]. CO2 was pumped
Appl. Sci. 2025, 15, 643 4 of 22

at a steady flow rate of 10 g min−1 until the pressure reached 120 bar. The system was held
at 80 ◦ C for 24 h. The working pressure and temperature were selected according to the
optimization performed in a previous study [16]. Afterward, it was depressurized at a rate
of 40 bar min−1 . The extract was then collected and stored at 4 ◦ C in the dark.
The concentration of the extract was determined by calculating the ratio of the dry
extract mass to the final volume of extract, expressed in g L−1 . Antioxidant activity was
assessed according to the method outlined in Section 2.4.

2.3. Supercritical Solvent Impregnation

The supercritical impregnation of olive leaf extract into the biodegradable PLA films
was performed using high-pressure equipment from Thar Technologies (model SF500, Pitts-
burgh, PA, USA). The procedure followed was similar to that previously documented [31].
A film measuring 23 × 38 cm was wrapped in a metal mesh and placed into a 500 mL
high-pressure vessel with 15 mL of olive leaf extract at the bottom of the vessel (Figure 1).
The pressurization was performed at a rate of 10 g min−1 . Once the pressure of 400 bar
was reached at 35 ◦ C, these conditions were sustained in batch mode for 1 h. After that,
the pressure was released at a rate of 0.66 bar s−1 . The impregnated film was then recov-
ered from the vessel and stored at 4 ◦ C in the dark before use. The films were green and
transparent with an average weight of 136 mg and a 10 µm thickness.

Figure 1. Schematic of the supercritical solvent impregnation process (P = pressure, T = temperature,

BPR = back pressure regulator).

2.4. ABTS Radical Scavenging

The antioxidant capacity was assessed using the ABTS radical cation decoloriza-
tion method (2,2′ -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)). The antioxidant com-
pounds led to the reduction of this radical, resulting in a color change from bluish-green to
complete colorlessness [32].
First, a stock solution was prepared by mixing 38.4 mg of ABTS with 6.6 mg of K2 S2 O8
in a final volume of 10 mL of water (Solution 1). This solution was left to stand in the
dark for at least 12 h. For use, Solution 1 was diluted to achieve an absorbance of 0.70 at a
wavelength of 750 nm (Solution 2) using a SynergyTM HTX Multi-Mode Microplate reader
provided by Biotek® Instruments, Inc. (Winooski, VT, USA).
The antioxidant activity of the olive leaf extract was determined as follows. An aliquot
of 3 µL of extract solution at different concentrations (1000–66,000 ppm) was added to
300 µL of Solution 2. After 10 min of reaction in the dark at room temperature, the
absorbance at 750 nm was measured. A control was prepared by adding 3 µL of ethanol
Appl. Sci. 2025, 15, 643 5 of 22

to 300 µL of Solution 2. The percentage of inhibition (% OI) was calculated according to

Equation (1):
% OI = (AC − A10min )/A0 × 100, (1)

where AC is the absorbance of the control and A10min is the absorbance of ABTS in the
presence of the olive leaf extract after 10 min of reaction. A calibration curve was obtained
from the graph % OI vs. extract concentration (ppm) (Equation (2)).

y = 1.8 × 10−3 x + 2.015x − 9.119, R2 = 0.991, (2)

From this curve, the concentration of the extract required to achieve a 50% reduction
in the initial ABTS concentration (EC50 ) was determined.
For the antioxidant activity of the impregnated film, pieces of plastic (~13 mg) were
cut and placed in 5 mL of ethanol. These were then subjected to ultrasound treatment
for 15 min to withdraw all the impregnated extract from the plastic. An aliquot of 3 µL
of this solution was added to 300 µL of Solution 2 and left to react for 10 min in the dark.
Afterward, the absorbance at 750 nm was measured, and the % OI was calculated from
Equation (1). All experiments were performed in duplicate.

2.5. Migration Test into Food Simulants

Migration tests were conducted using food simulants following Regulation (EU)
No. 10/2011 for plastic materials and articles intended to come into contact with food [33].
The food simulants chosen were ethanol 10% v/v (food simulant A) and sunflower oil as
the vegetable oil (food simulant D2).
Small portions (~0.35 g) of the impregnated and non-impregnated (control) films were
placed inside a tube in contact with 35 mL of food simulant. The tubes were stored at 5 ◦ C
for 10 days. Then, the absorbance at 650 nm (wavelength of extract maximal absorbance)
was measured using a SynergyTM HTX Multi-Mode Microplate reader provided by Biotek®
Instruments, Inc. (Winooski, VT, USA). The migration was calculated using a calibration
curve (Equation (3)) using extract solutions at different concentrations (3.19–31.88 ppm).

y = 0.087x − 0.0146, R2 = 0.992 (3)

2.6. Preparation of Fish Packages

In order to evaluate the efficiency of impregnated films in preserving fresh-cut seafood
products, hake fillets were selected. These fillets were obtained from the loins of fresh hake
purchased and filleted at a local market (Puerto Real, Cadiz).
The hake fillets were packaged in impregnated and non-impregnated (control) films
using a vacuum packing machine by Metro-Professional GVS1000 (Düsseldorf, Germany). All
samples were stored for 12 days at 4 ◦ C for subsequent monitoring and control of their shelf
life. Samples were analyzed at 0, 2, 5, 7, 9, and 12 days to establish significant control points for
monitoring and determining the shelf life of the fresh-cut, packaged, and stored fillets.
A fillet of ~116 g was introduced into each package. Samples were prepared in duplicate.
Each duplicate consisted of one sample from the loin and the tail (Figure 2a) to consider the
difference in fish musculature in these two areas, thus obtaining maximum representativeness.
Samples were analyzed using microbiological methods, and physical parameters were also
determined (aw , pH, and color). The rest were frozen at −20 ◦ C until the chemical parameter
assay was carried out (TVB, total volatile bases and TMA, trimethylamine).
Appl. Sci. 2025, 15, 643 6 of 22

Figure 2. Appearance of the hake fillets packaged into impregnated and non-impregnated films.
(a) Conformation of the package and (b) appearance along storage.

2.7. Monitoring and Control of the Shelf Life of the Packaged Product
2.7.1. Physical and Chemical Analysis of the Packaged Product
Water activity (aw ) was measured in triplicate using a hygrometer (Aqualab Serie 3,
Decagon Devices, Pullman, WA, USA) calibrated with standard salt solutions and an
internal temperature control to measure the aw at 25 ◦ C (±0.2 ◦ C). Samples of the fish fillets
Appl. Sci. 2025, 15, 643 7 of 22

(~1.0 g) were placed in measurement capsules (Decagon Devices, Pullman, WA, USA),
ensuring complete coverage of the capsule’s base.
The pH values of the fish fillets were determined in triplicate using a digital pH meter
(Micro pH 2001, Crison Instruments, penetration pH electrode for solids and semi-solids
SensIONTM , Barcelona, Spain). The pH meter was calibrated every 10 measurements using
buffer solutions at pH 4.0 and 7.0.
The rate of drip loss of the fish fillets during storage was calculated as percent water
lost compared to the initial sample weight according to Equation (4):

Drip loss (%) = (W 1 − W 2 )/W 1 × 100 (4)

where W 1 is the weight of the fish fillet at a predetermined time and W 2 is the initial weight
of the fish fillet [34].
The muscle and skin color of the fish fillets were measured through a Minolta colorime-
ter CM-2600d (Minolta Co., Osaka, Japan) equipped with a standard D65 illuminant. The
calibration was performed with both white and black standard plates. SpectraMagicTM
NX software (ve. 1.9, Pro USB) was used to record the measurements and analyze color.
The a*, b*, and L* parameters were determined corresponding to the red-green component
(red for a* > 0 and green for a* < 0), the yellow-blue component (yellow for b* > 0 and blue for
b* < 0), and lightness (black for L* = 0 and white for L* = 100), respectively. Color change (∆E)
was calculated on each sampling day in comparison to day 0, according to Equation (5) [35]:
√
∆E = [(∆L*)2 + (∆a*)2 + (∆b*)2 ] (5)

which indicated the overall color difference between the fish fillet after storage and the
original sample at day 0. The determination of ∆E allowed us to predict differences to the
naked eye by analyzing the variation in the CIELab parameters. According to Abadias
et al. [36], ∆E < 1.5 represents slight differences, 1.5 < ∆E <3.0 is considered different to a
trained eye, and when ∆E > 3.0, the samples are statistically different.
The contents of total volatile base nitrogen (TVB-N) and trimethylamine nitrogen
(TMA-N) in the fish fillet samples were measured by employing Conway Microdiffusion
Units Bel-Art™ Conway Diffusion Cells, SP Scienceware™ (Fisher Scientific S.L., Madrid,
Spain), according to a previously reported method by Sánchez-García et al. [37]. Each analysis
was repeated in duplicate, and the concentration was also expressed as mg 100 g−1 .

2.7.2. Microbial Analysis

Microbiological analysis was performed by the direct counting of microbial cells under an
optical microscope (Leica Microsystem GmbH, Wetzlar, Germany) using a Neubauer chamber
(Improved Neubauer Chamber, Brand GmbH & Co. KG, Wertheim, Germany). This method
was proposed by Sánchez-García et al. [37] as an easy and quick approximation to assess the
number of microorganisms involved in freshness loss in fish fillets. For this purpose, 1 g of
muscle from different points of each sample was poured into 9 mL of NaCl 0.9% w/v in an
AV-100 biological safety cabinet from Burdinola (Bizkaia, Spain). The mixture was then shaken
for 5 min using an ultrasonic device. After homogenization, samples (1 mL) were stained with
methylene blue (1% v/v) to increase the contrast of microbial cells for subsequent analysis in
the Neubauer chamber. Counts were assessed in duplicate, and the results were expressed as
the number of microbial cells per mL, using Equation (6).

No. germs/mL = (No. cells counted × 16)/(No. squares counted × volume of square × dilution × 2) (6)
Appl. Sci. 2025, 15, 643 8 of 22

2.7.3. Sensory Evaluation of the Packaged Product (QIM)

The methodology used to evaluate the quality index method (QIM) scheme was mod-
ified based on the method described earlier by Cardenas Bonilla et al. [38] for fresh cod
(Gadus morhua) fillets with skin, a white fish similar to the one in this study. Here, the QIM
scheme for the sensory evaluation of fresh fish fillets was performed by three evaluators who
observed and registered the changes occurring in the samples from day 0 until spoiled. Each
descriptor evaluated the received scores in which zero corresponded to very fresh fish. The
scores increased according to spoilage, up to three for each descriptor, following the evaluation
form shown in Table 1. In the sensory evaluation, a score was assigned to each descriptor
evaluated, and the arithmetic mean of the scores obtained at each stage of the analysis was
calculated, allowing for the determination of the freshness index (FI), based on the freshness
indices established for white fish by Council Regulation (EC) No. 2406/96 [39].

Table 1. The QIM scheme for fresh hake fillet with skin.

Parameter Character Descriptor Description Score

Skin Brightness Iridescent pigmentation 0
Pigmentation without brightness 1
Rather dull 2
Dull 3
Mucus Uniform, thin, transparent 0
Slightly cloudy, 1
Little thicker, milky, slimy 2
Clotted, thick, yellowish 3
Flesh Bright Transparent, bluish 0
Slightly opaque 1
Opaque 2
Milky 3
Texture Very firm, elastic 0
Firm, hard 1
Rather soft, flexible 2
Very soft, tend to crumble 3
Odor Fresh, neutral (algae, sea) 0
Seaweedy, marine, grass 1
Sour milk, slightly sweet 2
Acetic, ammonia, rancid 3
Color Bright white 0
White, greyish 1
Some yellowish, a little muted 2
Quality index (0–18) Yellow, over all pink, muted 3

3. Results and Discussion

The extraction of olive leaves was performed using enhanced solvent extraction,
as previously described. This method employs ethanol in combination with CO2 in a
supercritical state, allowing for a deeper penetration of the solid matrix to be extracted.
This leads to increased solubility of the analytes, reduced surface tension and viscosity of
the solvent, and an enhanced mass transfer rate. As a result, a rapid extraction process with
high yields and low solvent consumption is performed [40]. For example, in this study,
a yield of 27% was obtained using 650 mL of ethanol and 24 h, 1.5-fold higher than that
obtained after 24 h of maceration with 70% aqueous ethanol [41].
The antioxidant activity of the olive leaf extract is mainly attributed to its polyphenol pro-
file [42]. The composition of this extract was previously analyzed by Cejudo-Bastante et al. [16],
Appl. Sci. 2025, 15, 643 9 of 22

who identified oleuropein from the secoiridoid family as the major phenolic compound,
luteolin-7-glucoside from the flavonol family, and hydroxytyrosol from the simple phenols.
Together, these compounds accounted for 90% of the total polyphenol content in the extract.
In this study, a final dried extract concentration of 66.15 ± 2.85 mg mL−1 with an EC50
of 28.53 ± 1.23 µg mL−1 was obtained, showing good antioxidant activity comparable to
our previous reports [15,16,31,43,44]. The comparison with our previous work indicates
that the extraction procedure is reproducible. Regarding other extraction methods, the
EC50 value obtained in this study using the ABTS assay was slightly superior to those
reported for other olive leaf varieties extracted through cold maceration with pure ethanol
and several steps (23.82–38.32 mg L−1 ) [45]. This result validates the potential of the olive
leaf extract for applications in food preservation due to its antioxidant activity. Based on
this, this extract was then used for the supercritical impregnation of the PLA-based film.

3.1. Characterization of Impregnated PLA Based-Film

The impregnation conditions were selected according to a previous report where
the highest antioxidant activity of the olive leaf extract impregnated PET/PP films was
achieved at 400 bar and 35 ◦ C [31]. In the present work, the impregnated PLA film presented
an oxidation inhibition of 28.13 ± 0.49%, indicating a concentration of 706 µg extract mg−1
PLA. These results were higher than those found for the same extract using PLA filaments
at 400 bar and 55 ◦ C with an antioxidant activity of ~15% and a loading of 0.5 mg g−1
polymer [43]. These differences can be attributed to several factors: the film or filament
form of the PLA, which affected the performance of supercritical impregnation, the higher
CO2 density employed in this work, which enhanced the solvent power (972.2 kg m−3 at
400 bar and 35 ◦ C compared to 906.85 kg m−3 at 400 bar and 55 ◦ C) as well as the slower
depressurization rate used in this study (0.66 vs. 2 bar s−1 ) that decreased the dragging
of the extract along with CO2 [24,46]. The morphology (form) of the polymer can affect
the diffusion kinetics of bioactive compounds within the polymeric matrix. Specifically,
in a film, a thinner structure allows for faster impregnation, while in a deeper matrix,
diffusion occurs at a slower rate. Furthermore, the larger surface area exposed in a film
compared to a filament provides more area for impregnation, resulting in potentially
higher impregnation yields in films than in filaments. Moreover, changes in CO2 density
significantly impact the efficiency of impregnation. As observed in a previous study, the
impregnation of bioactive compounds in olive leaf extract increases with CO2 density [16].
Thus, a higher density results in a greater concentration gradient between the fluid phase
and the polymer, which enhances the impregnation process. The depressurization rate also
affects the impregnation efficiency as a faster pressure drop rate leads to the simultaneous
release of soluble compounds along with CO2 or their desorption from the polymer matrix,
which can reduce the impregnation loading [46].
When the PET/PP films were impregnated with olive leaf extract, lower loadings were
found at the same impregnation conditions (<100 µg extract mg−1 PET/PP) [16,47]. The
differences in the PLA and PET/PP film loading could have resulted from the chemical
interactions between the bioactive components that were soluble in the supercritical fluid phase
and the polymers. The presence of hydroxyl moieties in the polyphenols can form strong
H-bonds with carbonyl and ether groups present in the PLA and PET polyesters. In addition,
weaker interactions, such as π-stacking and other Van der Waals forces, could occur between
the aromatic rings of the main olive extract compounds and the terephthalate unit of PET.
However, the presence of hydrophobic methyl groups in the PP layer of PET/PP films can
reduce the affinity of polyphenols for this matrix, leading to considerably lower loadings [44].
Migration testing is used to evaluate the quality and safety aspects of packaging materials
in food contact applications. This analysis helps to understand the interactions between ad-
Appl. Sci. 2025, 15, 643 10 of 22

ditives, packaging materials, and food products. Furthermore, migration is desirable when
the incorporated additives are intended to prolong the product’s shelf life. Since food prod-
ucts are complex, food simulants are used to mimic food systems. Ethanol 10% v/v (food
simulant A) was designed to represent hydrophilic foods, and vegetable oil (food simulant
D2) to simulate lipophilic foods. If the testing conditions are not technically feasible with
food simulant D2, migration tests should be conducted using ethanol 95% v/v and isooc-
tane [48]. In this study, the maximum amount of extract released from the PLA films into
food simulant A was 57.305 ± 2.3 mg dm−2 . For food simulant D2, the migration was
121.827 ± 11.9 mg dm−2 after 10 days at 5 ◦ C. Similar trends have previously been reported
by other authors using polymeric films impregnated with olive leaf extract. Fiorentini et al.
found migration values of 0.56 ± 0.48 mg dm−2 into simulant A and 2.01 ± 0.27 mg dm−2
into simulant D2 (olive oil) after 10 days at 40 ◦ C using PLA films [49]. Cejudo-Bastante et al.
also obtained higher migration values using ethanol 95% v/v (simulant C) in comparison
with distilled water (simulant A) after 10 days at 40 ◦ C using PET/PP films (0.15 ± 0.012 vs.
0.09 ± 0.012 mg dm−2 , respectively) [16].
According to current legislation, plastic materials and articles shall not transfer their
constituents into food in quantities exceeding the overall limit of 10 mg dm−2 [33]. The
values observed in the present study significantly exceeded this regulation, thus prompting
discussion on two critical aspects. Firstly, this legislation applies specifically to the migra-
tion of substances incorporated into plastics to produce a technical effect in the finished
product or those used to provide a suitable medium for polymerization. It does not, how-
ever, address bioactive compounds that are not detrimental to human health. Currently,
there is no specific legislation governing active packaging. Secondly, while food simulants
provide a controlled environment for studying the migration and interaction of compounds,
they have inherent limitations when extrapolating results to real food matrices. Real food
systems exhibit greater complexity, with variable compositions (e.g., fats, proteins, water
content), pH levels, and structural properties that can significantly influence the diffusion
and activity of bioactive compounds. These complexities highlight the need for further
research to validate findings in real matrices and inform the development of regulations
specific to active packaging.
These results suggest that films impregnated with olive extract may perform better
with lipophilic media, making the proposed packaging potentially more effective at preserv-
ing fatty foods. In this sense, hake fillets were selected as a model of fatty food considering
a total fatty acid content of 0.97–1.97% w/w [50]. Moreover, due to the rapid deterioration
of fatty foods, as previously mentioned, they represent an appropriate choice for evaluating
the effectiveness of the proposed active packaging.

3.2. Preservation of Hake Fillets

3.2.1. Monitoring of Freshness and Sensory Evaluation of the Hake Fillets
Considering the migratory nature of the active packaging, it is worth considering
that the inclusion of new compounds could eventually modify the sensory perception of
the food. This modification can be perceived not only by decreasing the appearance of
off-odors, but by also providing other new compounds from the active extracts. Therefore,
the evaluation of the effectiveness of these new packaging materials should involve a
sensory analysis. The prediction of the impact on the sensory properties, especially when
natural extracts are employed, is of primary importance as they can modify organoleptic
properties and lead consumers to reject them.
The appearance of the hake fillets analyzed during the trial is presented in Figure 2,
and the scores of the QIM are shown in Table 2.
Appl. Sci. 2025, 15, 643 11 of 22

Table 2. Changes in the skin and flesh parameters of the fish fillets were assessed during preservation
at 4 ◦ C at each sampling point.

Freshness Quality Parameters Change During Storage

Parameter Descriptor Description Score
Sample Day 0 Day 2 Day 5 Day 7 Day 9 Day 12
Skin Brightness Iridescent pigmentation 0 Control 0 - - - - -
Impregnated 0 0 - - - -
Pigmentation without brightness 1 Control - 1 - - - -
Impregnated - - 1 1 - -
Rather dull 2 Control - - 2 2 - -
Impregnated - - - - 2 -
Dull 3 Control - - - - 3 3
Impregnated - - - - - 3
Mucus Uniform, thin, transparent 0 Control 0 - - - - -
Impregnated 0 0 0 - - -
Slightly cloudy, 1 Control - 1 1 - - -
Impregnated - - - 1 1 -
Little thicker, milky, slimy 2 Control - - - 2 2 -
Impregnated - - - - - 2
Clotted, thick, yellowish 3 Control - - - - - 3
Impregnated - - - - - -
Flesh Bright Transparent, bluish 0 Control 0 0 - - - -
Impregnated 0 0 0 - - -
Slightly opaque 1 Control - - 1 - - -
Impregnated - - - 1 1 -
Opaque 2 Control - - - 2 2 2
Impregnated - - - - - 2
Milky 3 Control - - - - - -
Impregnated - - - - - -
Texture Very firm, elastic 0 Control 0 0 - - - -
Impregnated 0 0 0 - - -
Firm, hard 1 Control - - 1 - - -
Impregnated - - - 1 - -
Rather soft, flexible 2 Control - - - 2 - -
Impregnated - - - - 2 2
Very soft, tend to crumble 3 Control - - - - 3 3
Impregnated - - - - - -
Odor Fresh, neutral (algae, sea) 0 Control 0 0 - - - -
Impregnated 0 0 0 - - -
Seaweedy, marine, grass 1 Control - - 1 1 - -
Impregnated - - - 1 1 1
Sour milk, slightly sweet 2 Control - - - - 2 2
Impregnated - - - - - -
Acetic, ammonia, rancid 3 Control - - - - - -
Impregnated - - - - - -
Color Bright white 0 Control 0 0 - - - -
Impregnated 0 0 0 - - -
White, greyish 1 Control - - 1 1 - -
Impregnated - - - 1 - -
2 Control - - - - 2 -
Some yellowish, a little muted
Impregnated - - - - 2 -
3 Control - - - - - 3
Yellow, over all pink, muted
Impregnated - - - - - 3
Overall score 18 Control 0 2 7 10 14 16
Impregnated 0 0 1 6 9 14

Packages were opened on each sampling day, and the sensory analysis was conducted
before chemical analysis. The fillets packaged in both non-impregnated and impregnated
films exhibited similar trends in visual appearance. This was also corroborated by the
color analysis described in Section 3.2.2. A high correlation was found between the total
score (sum of all attributes) and storage time. This correlation indicates that the descriptors
gradually deteriorated over time (from days 2 to 12). The control samples stood out for their
faster and more pronounced evolution to deterioration. The attributes were independent
of each other but changed and received higher scores with storage time, being higher in the
control samples. In the impregnated samples, it remained at almost 0 until day 5, evolving
well below the control samples on days 7 and 9, and with a value of 14 on day 12 as opposed
to 16 in the control samples. During storage, the flesh color progressively turned yellowish,
and the firmness decreased in both samples without remarkable differences (Table 2). This
phenomenon could be explained as follows. Although the control sample deteriorated
slightly faster than the impregnated sample, the impregnated sample gradually acquired
Appl. Sci. 2025, 15, 643 12 of 22

a greenish hue from the impregnation process. As a result, the yellow coloration of both
samples appeared similar. Regarding texture, the skin became dry and wrinkled, partic-
ularly from day 5 onward. By day 7, a thicker mucus on the skin was observed in both
samples, though the hake packaged in the non-impregnated film showed a more advanced
deterioration. Regarding the odor properties, a sour aroma was detected in the control
samples from day 9 onward, whereas samples packaged in the impregnated film exhibited
a slight vegetal note that masked the sour perception related to loss of freshness. Up to
this point, no detectable olfactory presence of the extract was noted. The influence on the
visual or odor properties of food was previously reported when using active packaging
functionalized with natural extracts in meat and fish preservation. Jebely Jaban et al. [51]
evaluated the acceptance of chicken fillets in terms of the sensory properties of samples
packaged into a bioactive ZnO-Vicia villosa protein isolate-based edible coating and con-
cluded that the coated samples exhibited a slower rate of sensory loss when compared to
the control sample. Additionally, Khalid et al. [52] carried out a preservation study of beef and
chicken meat using carboxymethyl cellulose enriched with pomegranate extract nanoparticles.
In their study, 10 panelists evaluated the sensory texture, odor, and color in comparison to
the control samples. The panelists scored higher on the samples packaged with active films
because the microbial and chemical degradation was less perceived aromatically and improved
the color quality in both kinds of meat. Furthermore, Haridevamuthu et al. [53] evaluated the
storage of shrimp in chitosan-based films with 1, 2, and 4% of Terminalia catappa leaf extracts.
Quantitative descriptive analysis was carried out on samples that were unwrapped, wrapped
with non-functionalized films, and those functionalized at the three levels. Panelists scored the
samples better, as the % extract added to the films increased at all days of storage for the color,
aroma, texture, and overall acceptability descriptors, without reporting the influence of the
extract on sensory perception.
According to the microbial results further discussed in Section 3.2.3, from day 7 on-
ward, the microbiological count exceeded the marketability limit of the fish. Thus, the
impact of the extract on some of the visual and olfactory attributes was not considered to
exert any significant influence during the commercial shelf life of the fish. Nevertheless,
slight differences were observed in the other sensory attributes in the flesh and skin, denot-
ing a delay in the deterioration reactions. These results will be corroborated analytically in
the following sections.

3.2.2. Effects of Storage on the Physical and Chemical Properties

Color plays a key role in food quality control, as consumers often associate color with
the degree of freshness of the product [54]. The color determination was carried out from
the color measurements of the different parts of the fish (flesh, light skin, and dark skin)
using the time zero sample as the reference. Table 3 and Figure 3 show the evaluation of
the color parameters and the total color variation (∆E) of the packaged fillets considering
the values of the initial sample.
The ANOVA multifactorial analysis (p < 0.05) of all coordinates (L*, a* and b*) in the
three parts of the fillets revealed that the factor “days of storage” had more influence on
the variation of the parameters than the packaging material and showed a statistical effect
over the values obtained in all cases. Additionally, the packaging material influenced the
b* values of light skin and the a* and b* values of flesh, being the parts of the fillet with
more statistical differences from the initial point.
Appl. Sci. 2025, 15, 643 13 of 22

Table 3. CIELab parameters of the hake fillets along storage time.

Storage Time (Days) 0 2 5 7 9 12

Flesh
L* Control 42.06 ± 0.75 49.23 ± 0.95 1 A 43.24 ± 0.33 aB 40.85 ± 2.39 B 44.26 ± 0.92 bC 41.22 ± 0.21 bB
Impregnated film 47.88 ± 0.93 1 A 47.16 ± 1.68 1 bA 41.26 ± 0.20 B 37.87 ± 1.30 1 aC 34.70 ± 0.97 1 aD
a* Control −3.005 ± 0.37 −2.96 ± 0.23 bA −4.30 ± 0.34 1 B −4.14 ± 0.08 1 B −4.16 ± 0.21 1 bB −3.31 ± 0.18 bA
Impregnated film −3.82 ± 0.20 1 aA −4.99 ± 0.16 1 BC −4.50 ± 0.31 1 AC −5.36 ± 0.16 1 aBC −4.38 ± 0.31 1 aAC
b* Control −1.378 ± 0.64 1.42 ± 1.04 aAB 0.34 ± 3.01 AB −0.08 ± 3.00 aA 3.53 ± 0.28 1 aB 3.93 ± 0.21 1 B
Impregnated film 6.69 ± 0.39 1 bA 2.60 ± 1.26 1 B 6.04 ± 1.64 1 bA 6.05 ± 0.21 1 bA 3.19 ± 0.37 1 A
Light skin
L* Control 7.18 ± 0.02 73.06 ± 0.06 1 aA 66.53 ± 0.19 1 B 57.61 ± 1.07 1 Ca 59.49 ± 0.44 1 CDa 60.88 ± 0.77 1 bD
Impregnated film 69.80 ± 0.96 bA 66.53 ± 0.15 1 B 59.74 ± 0.85 1 Cb 61.35 ± 0.07 1 Cb 57.68 ± 0.89 1 aD
a* Control −1.58 ± 0.01 −2.07 ± 0.01 1 A −1.86 ± 0.08 A −1.96 ± 0.11 aA −0.79 ± 0.03 1 bB −3.86 ± 0.13 1 C
Impregnated film −1.81 ± 0.20 A −2.90 ± 0.01 1 B −0.46 ± 0.42 1 bC −2.33 ± 0.01 1 aD −4.07 ± 0.23 1 E
b* Control −0.22 ± 0.05 1.33 ± 0.06 1 aA 0.08 ± 0.58 aA 1.82 ± 0.94 1 aAB 4.44 ± 0.11 1 BC 6.74 ± 1.30 1 C
Impregnated film 11.29 ± 1.05 1 bA 5.80 ± 0.11 1 bB 10.61 ± 0.16 1 bA 3.62 ± 0.06 1 B 4.74 ± 2.88 1 B
Dark skin
L* Control 46.88 ± 0.68 42.15 ± 0.14 A 33.07 ± 0.40 1 aB 34.80 ± 0.67 1 B 32.56 ± 3.88 1 B 35.80 ± 3.72 1 B
Impregnated film 47.79 ± 1.30 A 40.12 ± 0.14 1 bB 35.63 ± 0.84 1 BC 38.04 ± 0.94 1 B 31.06 ± 0.31 1 C
a* Control −1.53 ± 0.05 2.92 ± 1.73 1 b 4.28 ± 0.12 1 b 1.50 ± 0.21 a 2.62 ± 0.70 1 2.10 ± 0.91 1
Impregnated film −1.77 ± 3.37 aA 1.57 ± 1.43 1 aB 3.26 ± 0.01 1 bB 1.11 ± 0.30 AB 1.88 ± 0.19 1 B
b* Control 3.68 ± 0.19 10.95 ± 1.51 1 A 10.82 ± 0.85 1 A 9.55 ± 0.10 1 aA 11.66 ± 1.00 1 bA 21.00 ± 1.32 1 bB
Impregnated film 10.01 ± 0.16 1 AB 12.15 ± 0.05 1 B 15.12 ± 1.41 1 bC 8.06 ± 0.76 1 aA 12.83 ± 1.47 1 aBC
1 Difference respecting the initial point. Lower case indicates differences among the treated and not treated samples on
the same day for each parameter measured. Capital letters indicate differences along days in the same row.

Figure 3. Color variation (∆E) of the (a) flesh, (b) light skin, and (c) dark skin. Dotted lines indicate
the tendency lines of the data along storage time. Lowercase letters indicate differences between the
impregnated and control samples at the same storage time. Capital letters indicate differences along
storage days in the same treatment (p < 0.05) (n = 4).
Appl. Sci. 2025, 15, 643 14 of 22

Analyzing the evolution of each part individually, the L* values of the flesh tended to
have lower values along storage, both when using the control and the impregnated films
with respect to the initial sample, with statistical differences from day 5. Samples packaged
into impregnated films showed more differences to the initial day than those packaged
under the control film. Regarding the a* parameter, the values decreased along storage in
all samples, showing differences among the treatments from day 9. Therefore, the influence
of the chlorophylls was not as determinant on the color variation as the evolution of the
fish itself. In this sense, despite the high green color of the impregnated films, the fish did
not show a green tonality. On the other hand, the color difference of the fillets was mostly
due to the significant increase in the b* values when packaged using the impregnated films.
This denotes an increase in the yellow hue promoted by the presence of the extract. When
analyzing the skin, in general, the color variations of both the light and dark parts were
mostly influenced by the storage time rather than the film treatment in all parameters,
showing the same trends as those previously mentioned on the flesh. The most evident
differences in the treatments were due to the b* values, where these values were statistically
higher in the impregnated films from day 7 in the case of the dark skin, and from day 2 in
the light skin, tending to yellow hues.
In a comparison with the initial sample, the differences were related to the higher
spoilage of the fillets (Section 3.2.3) in the control film, and to the possible cession of the
compound when the impregnated film was used. In all samples, differences were observed
with respect to day 0, which shows the fast spoilage of the fish. In general terms, fish
packaged with the impregnated film showed higher ∆E values, mainly due to a higher b*
contribution (yellow hue). However, it must be noted that the differences in ∆E values
among the impregnated and control films were generally less than 3 in most cases (Figure 3,
lowercase letters). This denotes only slight variations that are imperceptible to the naked
eye, as can also be observed in Figure 2.
Figure 4 shows the effect of the storage time on the drip loss, water activity and pH
values of the hake fillets packaged in the impregnated and non-impregnated films (control).
The maintenance of moisture in fresh-cut products with a high aw is a key point in the poly-
mer formulation. It should prolong the humidity as much as possible, avoiding condensation
inside the package, to prevent the proliferation of microorganisms. González-Buesa et al. [55]
pointed out that PLA material has a relatively high water vapor permeability coefficient.
As a result, when kept refrigerated, the raw material can gradually lose moisture. This phe-
nomenon was reflected in both the aw reduction and the drip loss observed in both cases,
and in the samples packaged in impregnated PLA to a lesser extent [55]. As can be seen in
Figure 4a, the behavior in this study was similar. Drip loss showed an increasing tendency
in both cases, being higher in the control film (24.69 ± 2.59%) compared to the impregnated
one (18.17 ± 1.48%) at the end of the experiment. This drip loss was clearly reflected in the
aw , whose values decreased for both types of samples. This decrease was more pronounced
in the non-impregnated samples at the end of the storage time (Figure 4b). Therefore,
impregnation appears to have preserved the moisture of the samples over long storage
periods. In the study carried out by Mezhoudi et al. [54], it was observed that the coating
of hound shark (Mustelus mustelus) fillets with edible gelatin enriched with Moringa oleifera
extract reduced syneresis and decreased the weight loss by 26% over the control (fillets
without coating). They also reported higher values than those observed in this work for
impregnated film. However, this result suggests that a PLA coating impregnated with
natural extracts may effectively control moisture loss in fish fillets during cold storage.
Appl. Sci. 2025, 15, 643 15 of 22

Figure 4. Evolution of (a) drip loss (%), (b) water activity (aw ), and (c) pH during preservation at
4 ◦ C at each sampling point. Data are expressed as the mean ± standard deviation (n = 3). Lowercase
letters indicate differences between the impregnated and control samples at the same storage time.
Capital letters indicate differences along storage days in the same treatment (p < 0.05).

Regarding the pH, Browmik et al. [56] identified pH values as a biomarker for fish
and meat freshness, noting that values over 7 suggest the onset of food deterioration.
Figure 4c shows a separation of the pH increase tendency among the impregnated and
non-impregnated films from day 2. On days 5 and 7, the hake fillets packaged in the
impregnated films exhibited steady pH values, remaining slightly above 7, indicating a
delay in spoilage. During the same period, the pH values of hake fillets packaged in the
control films were statistically higher, suggesting a faster rate of spoilage. When fish are
caught under stressful conditions, biochemical processes occur that affect the pH. Initially
(post-catch), a decrease in pH can be observed due to glycogenesis reactions that release
lactic acid, which accumulates in muscle tissue [57]. However, once the fish begins to
deteriorate, basic compounds are generated that can increase the pH such as volatile bases,
trimethylamine, and ammonia [58] as well as the formation of alkaline compounds due to
microbiological activity or endogenous enzymes present in the fish [59]. Therefore, these
changes in pH can be indicative of the degree of freshness of the fish and its quality.
On the other hand, Figure 5 shows the evolution in the chemical parameters of the
packaged hake fillets over the storage period.
Appl. Sci. 2025, 15, 643 16 of 22

Figure 5. Evolution of the (a) total volatile base (TVB-N) and (b) trimethylamine (TMA-N) during
preservation at 4 ◦ C at each sampling point. Data are expressed as the mean ± standard deviation
(n = 3). Lowercase letters indicate differences between the impregnated and control samples at the same
storage time. Capital letters indicate differences along storage days in the same treatment (p < 0.05).

The total volatile basic nitrogen (TVB-N) is one of the most widely used measurements
of seafood quality and is a common chemical indicator of marine fish spoilage [60,61]. Both
the bacterial activity and biochemical modifications resulting from the autolytic activity of
fish produce a series of basic nitrogenous compounds, such as ammonia, trimethylamine
(TMA-N), dimethylamine, and monoethylamine, known as the total volatile basic nitrogen
(TVB-N), which are considered representative of this alteration [62]. The concentration of
TVB-N in freshly caught fish usually varies between 5 and 20 mg 100 g−1 [63]. Based on
previous studies [64–67], Jinadasa B. proposed that the quality classification of fish and fish
products regarding TVB-N values would be “high quality” up to 25 mg 100 g−1 , “good quality”
up to 30 mg 100 g−1 , “limit of acceptability” up to 35 mg 100 g−1 , and “spoilt” above
35 mg 100 g−1 [59]. Feng et al. [34] determined that a gelatin/chitosan coating could
improve the quality of fresh fish fillets and prevent spoilage when refrigerated at 4 ◦ C.
They showed that the TVB-N for the control group increased dramatically from 3.59 mg
100 g−1 at day 0 to 93.52 mg 100 g−1 at day 17. This result suggests that the freshness of
the control fillet decreased quickly and became spoilt during cold storage. In this study,
the TVB-N (Figure 5a) showed the same trend. The samples exposed to the impregnated
and control films increased from 10.20 mg 100 g−1 at day 0 to values near 80 mg 100 g−1
at day 12. There comes a time of advanced deterioration in which not only volatile bases
are produced, but also acids, depending on the types of microorganisms developing in the
environment, which is why volatile bases remain or fall and the pH drops (Figure 4c).
TMA-N (Figure 5b) is the main component of the TVB-N fraction. This compound is
produced by the bacterial reduction of trimethylamine oxide (TMAO) and is considered
responsible for the characteristic “unpleasant smell” of fish [68]. The results showed that
TMA-N seemed to be the main component of TVB-N, with similar trends in both graphs.
In this case, the higher values of TBV-N to TMA indicated the hydrolysis of proteins and
the appearance of the other amines from TMAO, either due to microbial activity or the
endogenous enzymatic activity [69].

3.2.3. Effects of Storage on the Microbial Count

Fish is excellent for microbial growth due to its high-water content, relatively high
pH, and abundance of nutrients such as proteins and non-protein nitrogen compounds
Appl. Sci. 2025, 15, 643 17 of 22

typically found in fish tissues [70]. Microbial proliferation depends on the initial microflora,
conditioned largely by the aquatic environment from which it originates and the handling it
has undergone. This proliferation generates the metabolization of non-protein nitrogenous
compounds present in fish. These compounds contribute to the characteristic off-flavors
and odors of spoiled fish as well as biogenic amines, which are a health and food safety
concern [70–72].
Microbial spoilage and pathogenic bacteria will overgrow on chilled fish and cause
the final deterioration. After this, the fish is no longer organoleptically, visually, and
microbiologically acceptable for consumption. The recommended microbiological limit
for seafood, particularly for fresh fish, is 107 CFU g−1 , according to the International
Commission on Microbiological Specifications for Foods [73]. Figure 6 shows the microbial
count of the packaged hake fillets during the storage time.

Figure 6. Evolution of the microbial count (log UFC g−1 of sample) during preservation at 4 ◦ C at
each sampling point. Data are expressed as the mean ± standard deviation (n = 2). Lowercase letters
indicate differences between the impregnated and control samples at the same storage time. Capital
letters indicate differences along storage days in the same treatment (p < 0.05).

In this work, the initial microbial count values were very high, as were the pH values,
volatile bases, and TMA. As seen in previous studies of fresh hake [74,75], fresh samples
of excellent quality showed TMA-N levels below 1 mg 100 g−1 , maintained for 5–6 days.
However, in this study, the initial value was 3–4 mg 100 g−1 and immediately increased
exponentially. This phenomenon suggests that the hake may have been caught several
days before purchase. Nonetheless, the results showed an identical evolution in both cases,
obtaining statistically lower values for the hake packaged into impregnated films from
day 5. Between days 2 and 5, the impregnated films seemed to exert a bacteriostatic effect,
delaying the microbial growth by 3 days. In this sense, the microbial count remained
in the range of 107 CFU g−1 for three days longer, which could potentially increase the
commercial shelf life of the fish. The bacteriostatic effect of active packaging has previously
been reported in the application of fresh food. For instance, Amaro-Blanco et al. [76] re-
ported a delay in the growth of mesophilic, lactic acid, and psychrotrophic bacteria, molds,
and yeasts of dry-cured shoulder sliced from Iberian pigs when packaged into polyamide
polyethylene films enriched with olive leaf extract. The experiments were compared with
the application alone and in combination with high-pressure processing. The authors con-
cluded that the physical treatment exerted a bactericidal action instead of the bacteriostatic
Appl. Sci. 2025, 15, 643 18 of 22

action of the AP tested. Hong-Ting et al. [77] analyzed the application of carvacrol-loaded
curdlan hydrogels for the preservation of seabass. Although the antimicrobial effect of
carvacrol was demonstrated in vitro against Shewanella putrefaciens, Vibrio parahaemolyticus,
and Vibrio harveyi, the effectiveness in the inactivation of the pathogens when inoculated
over the fish was not that evident, showing, in the best cases, a bacteriostatic effect. The
authors suggested that the fish matrix was a medium enriched in nutrients that promoted
microbial growth and hindered the microbial inhibitory effect of the extracts. In agreement,
Yin et al. [78] incorporated oregano essential oils into glucomannan-based pads and tested
their preservative effect on yellow croaker. They observed that the essential oils exerted a
bacteriostatic effect on the total viable count of bacteria in the fish fillets, which improved
the shelf life of large yellow croaker fillets under refrigeration for an additional 4 days,
similar to the effect observed, according to the results shown in Figure 6.

4. Conclusions
Active packaging has been proposed as a non-thermal preservation strategy for fresh
foods, offering a less intrusive alternative to methods such as high hydrostatic pressure,
pulsed electric fields, or ultrasound, which act directly on the food. While active packaging
is unlikely to achieve the same drastic reductions in microbial populations, its bacterio-
static effect through the temporary release of active substances offers a subtler approach.
However, limitations exist, as the concentration of active compounds in the polymer must
be carefully balanced to maintain the sensory and safety attributes of the food. Thus, the
key challenge lies in optimizing the migration kinetics of bioactive compounds, preserving
the sensory qualities, assessing the gastrointestinal viability, and achieving a demonstrable
bacteriostatic effect to ensure the success of active packaging technologies.
This study demonstrated the practical potential of biodegradable polylactic acid films
impregnated with olive leaf extract as an eco-friendly alternative for fish preservation. The
supercritical fluid impregnation process provided excellent results in the functionalization
of films. This technology generated PLA-based films with antioxidant activity and demon-
strated differential extract release in food simulants, suggesting tailored applications for
various food types. The migration tests pointed to the aptitude of this film for their action
in food with a certain fatty content such as white fish. The proposed active packaging
effectively reduced the microbial growth and minimized drip loss while maintaining the
pH stability in fresh hake fillets over 12 days of storage at 4 ◦ C. The bactericidal effect of the
olive leaf extract was evident during days 2 and 5, maintaining the mesophilic bacteria con-
centration on the hake fillets, which could have an impact on the marketability of the fish.
In contrast, the control samples showed a more pronounced loss of freshness as evidenced
by both the sensory and analytical results. Although the packaging caused a yellowish
hue in the hake fillets, it did not alter the distinctive fish odor profile, demonstrating the
sensory neutrality of the olive leaf extract. Despite the impregnated films having a strong
green color, this did not result in any green coloration of the fish.
All in all, these findings highlight the potential of supercritical CO2 impregnation as
an efficient technology for developing bioactive films with natural extracts. This technology
shows great promise for application in highly perishable products, such as seafood, offering
an innovative and sustainable solution for food preservation.

Author Contributions: Conceptualization, F.S.-G., N.D.M., C.C.-B. and L.C.-C.; Methodology, F.S.-G.,
M.T.-F., L.C.-C. and C.C.-B.; Formal analysis, F.S.-G., N.D.M. and C.C.-B.; Investigation, F.S.-G., M.T.-F.
and L.C.-C.; Writing—original draft preparation, F.S.-G., N.D.M. and C.C.-B.; Writing—review and
editing, A.M.R. and L.C.-C.; Supervision, F.S.-G., C.C.-B., A.M.R. and L.C.-C.; Project administration,
C.M.-S., L.C.-C. and C.C.-B.; Funding acquisition, C.M.-S. and L.C.-C. All authors have read and
agreed to the published version of the manuscript.
Appl. Sci. 2025, 15, 643 19 of 22

Funding: This work was framed within Project “TED2021-131822B-I00”, financed by MCIN/AEI/
10.13039/501100011033 and the European Union NextGenerationEU/PRTR. These results are also
part of PROYEXCEL_00920 (2021 Call), supported by the Program of Aid For Research Projects
of Excellence, on a Competitive Concurrence Basis, aimed at Entities Qualified as Agents of The
Andalusian Knowledge System, within the scope of The Andalusian Plan for Research, Development,
and Innovation (PAIDI 2020), Department of University, Research, and Innovation of the Regional
Government of Andalusia.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: The original contributions presented in this study are included in the
article. Further inquiries can be directed to the corresponding author.

Acknowledgments: The results of this paper are framed in the iM-PACK project, which is supported
by the PRIMA Program. The PRIMA program is supported by the European Union.

Conflicts of Interest: The authors declare no conflicts of interest.

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