
==== Front
Food Chem X
Food Chem X
Food Chemistry: X
2590-1575
Elsevier

S2590-1575(24)00626-6
10.1016/j.fochx.2024.101738
101738
Research Article
Monitoring fish freshness with pH-sensitive hydrogel films containing quercetin or eucalyptol
Mirzaei Akbar a
Mirzaei Ghazaleh a
Nezafat Zahra a
Javanshir Shahrzad shjavan@iust.ac.ir
a⁎
Karimkhani Mohammad Mahdi b
Jamshidi Abdollah b
a Pharmaceutical and Heterocyclic Compounds Research Laboratory, Chemistry Department, Iran University of Science and Technology, Tehran, Iran
b Department of Food Hygiene and Aquaculture, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran
⁎ Corresponding author. shjavan@iust.ac.ir
22 8 2024
30 10 2024
22 8 2024
23 1017389 5 2024
11 8 2024
12 8 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
This research developed pH-sensitive smart films using carboxymethyl cellulose (CMC) and collagen (COL), combined with either quercetin (QCT) or eucalyptol (EUC), to prevent fish meat spoilage. COL, extracted from isinglass, was confirmed as type I through SDS-PAGE. The films were characterized using FESEM, FTIR, and TGA. The addition of QCT or EUC enhanced antioxidant levels to 60.16% and 70.83%, respectively, up from a baseline of 10.4%. It also increased tensile strength from 3.32 ± 0.22 to 11.8 ± 0.25 and 13.2 ± 0.27 MPa, and enhanced elongation at break from 5 ± 3.1% to 27.7 ± 1.1% and 30.15 ± 2.1%. Fish meat packaged with QCT showed a lower spoilage rate due to the antibacterial and antioxidant effects of EUC and QCT (TVBN = 7.37 ± 0.01), compared to CMC/COL film (TVBN = 10.11 ± 0.02) and non-packaged fish (TVBN = 11.23 ± 0.01). The films exhibit >80% transparency, highlighting their suitability for food packaging. CMC/COL/QCT is preferred for fish packaging because it offers better mechanical properties and lower TVB-N levels.

Highlights

• Developing films with collagen extracted from fish waste and containing QCT or EUC to prevent fish spoilage during packaging.

• Investigating the effectiveness of pure flavonoids versus flavonoid-containing extracts as pH indicators.

• Comparing the mechanical, antioxidant, and antibacterial properties of pure flavonoids and flavonoid extracts.

Keywords

Antibacterial
Carboxymethyl cellulose
Collagen
Quercetin
Eucalyptol
Intelligent packaging
Fish freshness
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pmc1 Introduction

Today, with the development of science and technology, human demand for fresh food has also increased. Integrating science and technology in agriculture, food distribution, and consumer behavior has significantly influenced the demand for fresh food (Bourgeois & Sette, 2017; Konfo et al., 2023). Packaging is increasingly critical in improving supply chain profitability and reducing environmental impact(de Castro Moura Duarte & Picanço Rodrigues, 2024; Konfo et al., 2023). The packaging is no longer exclusively the envelope inside which to store and protect the product, but a trend that is growing more and more in many application sectors and which makes it possible to make packaging “talking”, become a vector of information, an instrument of exchange between manufacturer and consumer (Halmans, 2024). It can be applied as a protective layer to avoid food contamination and preserve food quality (Verghese, Lewis, Lockrey, & Williams, 2013; Zambujal-Oliveira & Fernandes, 2024). The primary function of traditional food packaging is indeed to protect from contamination (dust, micro-organisms, chemical substances) and external agents (oxygen) responsible for the contamination, accelerated degradation of their quality and safety, transport and store this perishable food and cannot provide information about the freshness of the food to consumers (Shao et al., 2021). At the same time, increasing concerns associated with the protection of the environment have generated significant efforts in terms of recycling, besides the appearance of new packaging concepts such as bio-sourced, renewable, biodegradable, and/or compostable(Shafi & Bajpai, 2023). These requirements have prompted researchers to design and manufacture new intelligent packaging. In this type of food packaging, indicators are integrated to provide information on the freshness of food (Lu et al., 2020; Yue et al., 2024). Smart packaging has potential applications in the food industry, due to its ability to monitor changes in the condition and quality of packaged products such as freshness, spoilage, and bacterial contamination, during transport and storage (Hammond et al., 2015; Taghinia, Abdolshahi, Sedaghati, & Shokrollahi, 2021; Yue et al., 2024). There are different designs for the preparation of smart food packaging including biological and chemical indicators such as integrity, time, temperature, and freshness indicators (Otles & Sahyar, 2016). Research has concentrated on incorporating natural indicators into smart packaging to monitor gas and temperature changes in food (Mohammadian, Alizadeh‐Sani, & Jafari, 2020). One of the most attractive smart FIs is pH-sensitive colorimetric materials because of their real-time food quality and freshness monitoring feature (Ezati, Priyadarshi, Bang, & Rhim, 2021).

Freshness indicators normally use dyes sensitive to pH changes caused by product spoilage. The alteration of the pH measured on meat and fish during the experimental phase thus causes a change in the color of the packaging to alert consumers (Riahi, Sadrabad, Jebali, Moghaddam, & Mohajeri, 2019). In fact, during meat and fish spoilage due to the activity of microorganisms, volatile nitrogenous compounds are produced, which cause the pH of the environment to change. Therefore, the use of pH colorimetric indicators can be useful in detecting spoilage(X. Wang et al., 2019; X. Zhang et al., 2019).

In general, smart packaging consists of two components: polymer substrate and active pigment or dye that act as pH colorimetric indicators (Alizadeh-Sani, Mohammadian, Rhim, & Jafari, 2020; X. Wang, Yong, et al., 2019). Among different substrates, natural polymer resources including seaweeds (Akbar & Mustari, 2024), collagen and polysaccharides such as carboxymethyl cellulose, starch, carrageenan, chitosan(Tonekaboni, Mehdikhani, & Yazdani, 2024), carrageenan, etc. have been of interest due to their biocompatibility, availability, low price, and natural origin (Al-Tayyar, Youssef, & Al-Hindi, 2020; Yildirim-Yalcin, Tornuk, & Toker, 2022). In the meantime, carboxymethyl cellulose has a special place in food packaging due to its gelling properties, transparency, biocompatibility, and non-toxicity(Costa et al., 2023). In recent years, the use of natural pigments such as curcumin, quercetin (Mirzaei, Jorshari, Jananshir, Noori, & Mahdavi, 2024), anthocyanins (Chen, Zhang, Bhandari, & Yang, 2020), and plant extracts such as phenolic acid in oats (OPC) and ascorbate palmitoyl (AP) (Shi, Li, & Liu, 2021), mango peel extract, fenugreek extract, etc. have been reported as a pH indicator for detecting food spoilage food spoilage along with various substrates.

There are also reports of the use of cellulose/curcumin to preserve the freshness of fish with a visual monitor(W. Wang et al., 2024). In addition, special attention has been paid to using anthocyanins and their rich extracts in food packaging. In a recent study, litchi extract was utilized as an anthocyanin-rich substrate, and incorporated into a polymer blend of chitosan, polyvinyl alcohol (PVA), and gelatin to create composite films (Guo, Shao, Ma, Zhang, & Lu, 2023). Additionally, a colorimetric indicator for fish spoilage has been developed using anthocyanin extracted from red poppy, which has been incorporated into a protein isolate and chitin nanofiber matrix. This approach has demonstrated acceptable performance (Tavassoli et al., 2024). The fruitfulness of using anthocyanin-rich date extract in carboxymethyl cellulose substrate confirms the potential performance of anthocyanins in monitoring the freshness of food as an indicator of pH detection(Elhadef et al., 2024).

In addition to polysaccharides, the use of proteins such as collagen has a special place in food packaging(Tang et al., 2022; R. Zhang, Liu, Han, Ren, & Jiang, 2024). Collagen as a neutral polymer used for food, cosmetics, drug delivery, and tissue engineering applications, is mainly extracted from terrestrial animals by different methods (Gaikwad & Kim, 2024). However, extracting hydrolyzed collagen from pigs and cattle has certain limitations due to health issues, such as swine flu and bovine spongiform encephalopathy (BSE, “mad cow disease”) (Shui, Li, Chae, Xu, & Bressler, 2023). In addition, religious issues must be taken into account (Bhuimbar, Bhagwat, & Dandge, 2019). The demand for marine collagen has surged significantly in recent years due to its unique properties and health benefits. Sourced from the skin, scales, and bones of marine and freshwater fish, marine collagen offers advantages over traditional animal-derived collagen, primarily due to its low molecular weight, contributing to reduced inflammatory reactions and fewer contaminants (Rahman, Gogoi, Dubey, & Chowdhury, 2023). Alternative sources of collagen, including marine invertebrates such as jellyfish and sponges, have also gained attention. Despite these advantages, the standalone use of polysaccharides or collagen often presents limitations, particularly concerning mechanical strength and durability. To address these challenges, combining or cross-linking polysaccharides with collagen presents an effective strategy to enhance mechanical properties.

Recent studies have demonstrated that incorporating collagen or gelatin into carboxymethyl cellulose (CMC) -based films can significantly improve the mechanical properties vital for food packaging applications (Zhu et al., 2024)). However, the investigation of quercetin (QCT) in intelligent food packaging has been limited, particularly its effectiveness in monitoring the freshness of fish products when integrated into a CMC-collagen matrix. Furthermore, no published reports explore the potential of eucalyptus extract (EUC) as a rich source of QCT specifically for fish packaging contexts. This research aims to evaluate the efficacy of Quercetin (QCT) and Eugenol (EUC) as natural flavonoid preservatives for monitoring the freshness of fish. Both QCT and EUC are recognized for their significant antibacterial and antioxidant properties, which can potentially extend the shelf life of fish products. In pursuit of this objective, we are investigating the synergistic effects of combining these plant extracts with carboxymethyl cellulose (CMC) and collagen—specifically marine collagen sourced from isinglass, which is derived from the swim bladders of fish. To facilitate freshness monitoring, we are developing two types of biodegradable, pH-sensitive blended hydrogel films that incorporate QCT and EUC. These films are designed not only to enhance the preservation of fish but also to serve as indicators of spoilage, providing a dual function that is both sustainable and effective. This study will contribute to the field of food preservation by creating innovative packaging solutions that leverage natural compounds, thereby promoting food safety and reducing waste.

We also examined the films' antibacterial and antioxidant properties and measured total volatile basic nitrogen (TVBN) levels to assess their potential application in fish packaging. This research highlights the innovative use of bioactive compounds in developing intelligent packaging solutions that address both food safety and sustainability concerns.

2 Experimental

2.1 Chemicals and apparatuses

All materials were bought from Aldrich and Merck Co. CMC (Cass No. 9004-32-4) and QCT (Cass No. 6151–25-3) were purchased from Merck Co. Isinglass was bought from ANGEL brand. The prepared films were characterized using UV–vis and FT-IR with features 1700 spectrophotometer from Shimadzu and the NICOLET IR100 apparatus and carried out in the 400–4000 cm−1 region respectively. Field emission scanning electron microscopy (FESEM, ZEISS Sigma 300) was used to study the structure and morphology of synthesized films.

2.2 Preparation of EUC

Eucalyptus (E. camaldulensis Dehnh) leaves were collected from trees on the university campus and washed with tap water. In the next step, it was dried at room temperature and away from light, and after cutting into small pieces, they were soaked (10 g) in a mixture of ethanol:water (70:30) for 24 h. The mixture was then subjected to ultrasonication (E300H,30 KHz, Shiraz New Ultrasound Company)) at 50 °C for 2 h. Finally, the blend was filtered with filter paper(42 μm), and the solvent was evaporated by a rotary evaporator(Heidolph Hei-VAP Precision ML/G5 Rotary) and completely dried at 40 °C and pressure 175 mmHg.

2.3 Extraction of COL from IG

COL extraction from IG involves two steps: a) pretreatment of IG; and b) collagen extraction by Acid Soluble Collagen (ASC). The type of extracted COL was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

2.3.1 Pre-treatment

Non-collagenous material was removed by soaking IG in 1:10 w/v of 0.1 M sodium hydroxide for 6 h at room temperature. The tissue was washed with distilled water until a neutral pH was achieved. For the decalcification, a solution of 0.5 M EDTA-2Na (pH 7.5) was used under stirrer conditions (48 h 1:10(w/v) IG/EDTA solution) at room temperature. Finally, the fat was removed by soaking in butyl alcohol) 1:10 w/v of 10%) at 25 °C. After stirring for 24 h, these glasses were filtered and washed several times with distilled water to remove butyl alcohol and fat.

2.3.2 Acid soluble collagen (ASC) extraction

Considering that COL is more soluble in acid, acetic acid (100 mL, 0.5 M) was used for extraction of COL under stirrer conditions (24 h at 25 °C). In the next step, COL was precipitated using NaCl (2.5 M), and the precipitate was collected by centrifugation (universal 320, pol ideal tajhiz, Iranian company) at 12000 rpm for 10 min (Hettich(. The COL was dissolved in acetic acid (20 mL, 0.5 M) and for 20 h placed in dialysis tubing and dialyzed (12KD, sigma alderich) against distilled water at 25 °C.

2.3.3 Spectrophotometric determination of QCT

The results of total flavonoid content (TPC) were expressed as QCT milliequivalents (meq) per gram of dry EUC(Handayani, 2021; Mirzaei et al., 2024). AlCl3 colorimetric method was used for this aim. The EUC was mixed with methanol (0.5 mL of 1:10 g/mL), 0.1 mL of 10% aluminum chloride, 0.1 mL of potassium acetate 1 M, and 2.8 mL of distilled water. The absorbance of the mixture was determined using a spectrophotometer UV–Vis at 415 mm to detect quercetin in triplicate. The same sample without AlCl3 was used as a blank solution. The calibration curve was prepared by preparing QCT solutions at concentrations of 10 to 100 μg/mL in methanol (Handayani, 2021; Mirzaei et al., 2024).

2.3.4 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was performed on 7.5% polyacrylamide gels containing 1% SDS and Coomassie brilliant blue R250 was used for staining. COL for electrophoresis was dissolved in acetic acid, 2% SDS, and 2% mercaptoethanol and was heated at 100 °C for 2 min.

2.4 Preparation of CMC/COL/QCT or CMC/COL/EUC blended hydrogel film

For this purpose, we first sonicated a solution of CMC in water (0.5 g of CMC in 40 mL of distilled water) using an ultrasonic probe at 90 W power for 30 mins (Topsonic-UHP-1200). A solution of 4 g of IG in 100 mL of acetic acid (0.5 M) was stirred at room temperature for 24 h and then filtered. In the next step, IG solution (20 mL) and glycerol (1.5 mL) were added to the sonicated solution of CMC. In the final step, 10 mL of a solution of QCT (0.024 g) in methanol (80%) was added under stirrer conditions for 30 min (RT-Heidolfh MR Hei-End 505–50,000-00). The blended film is prepared by drying the obtained hydrogel in an oven at 40 °C (Memmert UNB 300). The above steps were repeated to prepare a film containing EUC. EUC solution (0.012 g in 5 mL of solvent) was added so that the final film contains a similar amount of QCT. To be able to deduce the effect of the freshness indicators, a standard film was prepared without EUC or QCT.

2.4.1 Light-transmittance and UV–vis measurement

The transparency of the films is checked by UV–vis spectroscopy, for this purpose, pieces of film (4 cm × 1 cm) were placed directly in the cell of the spectrophotometer with air as a transparency reference. UV–vis spectra of CMC/COL, CMC/COL/EUC, and CMC/COL/QCT films, EUC, and QCT solution at different pH were measured by spectrophotometer. For this purpose, the pH of the eucalyptus extract solutions and the films were adjusted to the desired values (pH:1–12) using HCl or NH3 (0.1 mol/L) solutions.

2.5 Freshness indicator application of synthesized films for fish

To investigate pH sensing properties of films on fish (Trout), the films containing EUC or QCT were fixed on the headspace of a packing dish of fish (10 g). In the next step, the dishes were closed with Parafilm® and incubator (shimaz co) at 25 °C and 75% RH. The level of TVBN (total volatile base nitrogen) of fish was measured by the Kjeldahl method. First, fish (10 g) was homogenized in 100 mL of distilled water and then centrifuged (3000 rmp, 10 min). Then, 5 mL of the homogenized sample was filtered and transferred to the Kjeldahl distillation system, and 5 mL of MgO suspension (10 g/L) was added to it. Condensates were collected in a boric acid solution (20 g/L) and titrated with 0.1 mol/L hydrochloride. Also, the sample without coating film was used as a blank.

2.5.1 Film morphology and structure

Micrographs of the films were investigated using FESEM TESCAN MIRA3 and each specimen was sputtered with gold for 5 min with a thickness of 5 nm. Determination of interaction between CMC, COL, QCT, or EUC was performed using Fourier Transform Infrared Spectroscopy (FTIR) (See supporting information, Fig. S1).

2.6 Antioxidant performance of the synthesized films

2.6.1 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay

The antioxidant activity of CMC/COL films was performed according to previously reported methods (Kahya et al., 2022) with some slight modifications. Briefly, 200 mg of dry film sample was weighed and cut into small pieces. 10 mL of de-ionized water was added to the films. After 2 h incubation of the film pieces in water About 1 mL of the extracts was added to 3 mL methanol and 1 mL methanolic solution of (2,2-Diphenyl-1-picrylhydrazyl) DPPH radicals (0.012 g/100 mL). Then, the resulting mixture was stirred and incubated at ambient temperature (120 min). Finally, the absorbance was read at 517 nm against a blank. The scavenging ability was quantified as follows:Scavenging activity%=A517nmof control−A517nmof sampleA517nmof control×100.

2.6.2 Ferric reducing antioxidant power assay (FRAP)

The ferric-reducing antioxidant power (FRAP) assay was applied according to the described method by Kahya 2022(Kahya et al., 2022). To prepare the FRAP reagent, 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) pre-dissolved in 40 mM HCl solution, 20 mM FeCl3 solution and 0.3 mM acetate buffer (pH 3.6) was mixed in 1:1:10 ratio, respectively. Before use, the FRAP reagent was kept for 10 min at 37o C. Film extracts were prepared with 100 mg dry film and 5 mL of de-ionized water after 2 h of incubation. 1 mL FRAP reagent, 450 μL deionized water, and a volume of 50 μL film extract were mixed in a glass vial and then incubated for 10 min at 37o C. Absorbance of solutions was measured at 593 nm wavelength.

2.6.3 Determination of total phenolic content (TPC)

Briefly, 500 μL of diluted solution of Folin–Ciocalteau reagent (1:10 (v/v) ratio) was added to 100 μL of the film extract (100 mg dry film dissolved in 2.5 mL de-ionized water) After 1 min, 1.5 mL of 20%(w/v) sodium carbonate was added and stirred well. This mixture was kept at ambient temperature (2 h, dim light). Afterward, the absorption of the solution was determined at 760 nm using a Shimadzu UV–vis spectrophotometer. Distilled water was used as a control. The total amount of phenolic compounds was expressed as Gallic acid equivalent (GAE) defined as mg Gallic acid per g of dried film. All tests were carried out in triplicate.

2.7 Antimicrobial performance of the synthesized films

The antimicrobial activities of the films against meat spoilage bacteria including S.aureus ATCC 6538 and E. coli ATCC 8739 were investigated. The antibacterial activity of the films was determined using the total viable colony count method. The purchased bacteria were cultured in 50 mL of Luria-Bertani broth for 24 h at 37 °C. 6 mL of bacterial suspension was centrifuged at 5000 r /min for 5 min. The upper cleaning fluid was removed, and the precipitation was diluted with PBS buffer solution and cultured for another 24 h with the same steps. After that, the precipitation was diluted with PBS buffer to ∼107 CFU/ mL 200 μL of diluted bacterial suspension was dropped on the surface of the films (3 cm × 3 cm), and the plates were incubated at 37 °C for 18 h with mild shaking and samples rinsed by 2 mL PBS buffer. Then 20 μL of the above liquid was taken and coated evenly over the solid medium. The numbers of colonies were recorded after the coated petri dish was incubated in 3, 6, 9, 12,15, and 18 h in an oven at 37 °C. Film-free culture media and film A were tested as the negative and positive control, respectively (Zhang et al., 2022).

2.8 Mechanical properties

According to ISO 527, the TA-XTPlus Texture Analyzer (Stable Micro Systems, Co., UK) is used to obtain of the tensile strength (TS) and elongation at break (EB) with three repetitions, and the results were averaged. The TS and EB of the films (1.0 cm * 2.0 cm) were determined, from which the values of Young's modulus could be determined.

2.9 Thermogravimetric analysis (TGA)

The thermal stability was determined by thermogravimetric analysis (STA 504 Germany Bahr Company) at the temperature range of 25–800 °C under the oxygen atmosphere (Fig. S7). The interpretation is provided in the supporting information file

2.10 Spectrophotometric determination of the amount QCT

To conduct a comparison of the effect of EUC extract and QCT in the collagen film, it is imperative to determine the precise amount of quercetin present in the extract. For this purpose, a QCT calibration curve was drawn (Fig. S2, SI) and the TPC value was evaluated. This will enable us to accurately measure and analyze the effects of both EUC extract and QCT on the collagen film. The TPC result was calculated from the regression equation of the standard plot (y = 0.004× + 0.014, R2 = 0.9746), divulging that the concentration of the QCT was 125 μg/mL.

2.11 Statistical analysis

Statistical analysis was conducted using an unpaired Student's t-test for three independent experiments, with results presented as the mean ± standard deviation (SD). Significance was defined at p < 0.05(Wei et al., 2024).

3 Results and discussions

Previous literature has shown that the physical and chemical properties of packaging films based on polysaccharides and proteins are affected by the type and concentration of plant extracts (Shah et al., 2023). In fact, by the interaction between the phenolic groups of the extract with the structure of proteins or polysaccharides, properties such as mechanical, color change, antioxidant, and antibacterial (Al-Tayyar et al., 2020; Liu et al., 2018).

3.1 Characterization of ASC extracted

The purity, type, and molecular weight of the extracted collagen from IG were determined using SDS-PAGE. (Fig. S3, SI) displays two distinct bands at approximately 100 and 130 kDa, corresponding to the α1 and α2 chains(Wei et al., 2024). High molecular weight components, including β-chain (dimer) and γ-chain (trimer), were observed in both ASCs (Bhuimbar et al., 2019). These results indicate that the extracted collagen from fish is type I, as evidenced by previous studies. Additionally, the presence of low molecular weights in the SDS pattern suggests that acid treatment can result in the cleavage of protein chains and the formation of low molecular weight peptides.

3.2 Light-transmittance, UV–vis measurement, and field emission scanning electron microscopy (FESEM)

Fig. S4 (SI) displays the changes in absorption UV–vis spectra of Eucalyptus (EUC) or Quercetin (QCT) solutions at different pH values. The color of EUC solutions changes from bright yellow to reddish, while QCT solutions change from bright yellow to light yellow (Masek et al., 2018). The reason for this color change should be investigated in the molecular structure of QCT or EUC. According to Fig. S5 (SI), QCT has three important parts: (i) the catechol structure in the B-ring; (ii) the double bond that is conjugated with the 4-oxo function in the C-ring; and (iii) the -OH groups in the A-ring (Masek et al., 2018). The color change is not simply the result of the deprotonation of QCT hydroxyl groups. By changing the pH of the environment, ring C undergoes chemical changes, leading to the loss of resonance between rings A and B, which is observed as a color change(Jurasekova, Domingo, García-Ramos, & Sánchez-Cortés, 2014; Mirzaei et al., 2024). This color change leads to the shift of the peaks to a lower wavelength (Jurasekova et al., 2014). The π → π* transitions of rings A and B in the QCT structure appear at 250 nm (pH = 6), which are shifted to 325 nm with a greater intensity peak in alkaline (pH = 11 or 12) conditions. EUC contains different compounds such as flavonoids (e.g., QCT and rutin), and tannins (e.g. Ellagic acid). EUC is a natural dye that ranges in color from yellow to yellowish-brown. The major coloring component of Eucalyptus bark is quercetin. The presence of tannins and other flavonoids may cause a difference in color between the EUC and QCT solutions(Ali, Nisar, & Hussain, 2007; Cadahía, Conde, García-Vallejo, & Fernández de Simón, 1997; Mongkholrattanasit et al., 2013; Vázquez, Santos, Freire, Antorrena, & González-Álvarez, 2012). The comparison of pH changes in the prepared films shows that both films show excellent color changes.

Fig. 1 shows FESEM images, revealing cracks on the surface of the pure films resulting from the impact of an electron beam during magnified FESEM imaging. These cracks increase with magnification and electron beam approach, as seen in Fig. 1a-c. However, CMC/COL films containing QCT (Fig. 1d-f) and EUC (Fig. 1g-i) do not display these cracks. Red arrows confirm QCT particles or EUC, and red circles indicate small cracks in the CMC/COL/EUC film. EUC or QCT increases the films' strength, but agglomerated particles can reduce transparency(Liu et al., 2018). CMC/COL/EUC films are also slightly damaged. In general, the transparency of food packaging is one of the most effective factors in gaining consumer trust and allows for checking food quality and freshness(Kuang, Yang, & Zou, 2024). Various food crises such as corruption and pollution have led consumers to pay more attention to the healthfulness of food(Deng & Srinivasan, 2013; Ma, Zhuang, & Ma, 2020). The produced films showed >80% transparency, making them suitable for food packaging. CMC/COL/QCT has better light transmission than CMC/COL and CMC/COL/EUC. The incorporation of QCT and EUC significantly changes the film's light transmittance (Fig. 1j). The accepted threshold for transparency in materials is approximately 80% transmittance, though materials exceeding 90% transmittance are regarded as highly transparent. Notable examples include flat glass, which achieves a transmittance of 93% in the visible spectrum and commonly used food packaging polymers such as polycarbonate (PC), poly(methyl methacrylate) (PMMA), and polystyrene (PS), which can reach transmittance values of up to 92% (Lin, Bilotti, Bastiaansen, & Peijs, 2020).Fig. 1 FESEM images of a-c) pure film, d-f) CMC/COL/QCT, and g-i) CMC/COL/EUC, j) light transmittance of films.

Fig. 1

3.3 Freshness of fish using synthesized films

The health of food is directly related to the health of humans and the environment(Clark, Springmann, Hill, & Tilman, 2019). Proper food packaging prevents food spoilage to a great extent(Yudiastuti et al., 2023). Proteins in different types of meat are attacked by bacteria and various volatile nitrogenous compounds such as ammonia, dimethylammonium, and trimethylamine are produced, which leads to a change in the pH value (de Oliveira Filho et al., 2021). According to the film pH sensing characteristics, the CMC/COL/EUC and CMC/COL/QCT films were employed to monitor fish freshness. According to Fig. 2 (a, b), the color of the blended films changed from yellow to orange on the 3rd day.Fig. 2 Color response of a) CMC/COL/QCT, b) CMC/COL/EUC films at different pH values and applications of sensing films for monitoring fish freshness c) 0 days and d) after 3 days.

Fig. 2

To investigate the process of fish spoilage and the inhibiting effect of the prepared films on the speed of spoilage, TVB-N tests were performed and the obtained values are reported in Table 1. As we expected, the antibacterial and antioxidant properties of EUC or QCT reduced the spoilage rate of fish meat. After three days of fish meat storage, TVB-N value changed from 6.17 ± 0.02 to 7.37.3 ± 0.01 and 9.79.7 ± 0.01 for CMC/COL/QCT and CMC/COL/EUC films, respectively. In the case of fish meat packed with CMC/COL film, the TVBN value equal to 10.11 ± 0.02 was obtained. Upon comparing the outcomes of packaged fish meat that underwent QCT treatment (TVBN = 7.37.3 ± 0.01), CMC/COL film (TVBN = 10.11 ± 0.02), and non-packaged fish meat (TVBN = 11.23 ± 0.01), it is apparent that the film containing QCT exhibited a superior ability to preserve the fish meat. Despite the EUC extract being successful in mitigating fish meat spoilage (TVBN = 9.79 ± 0.01), the QCT-containing film proved to be more effective.Table 1 TVBN test for the freshness of the fish.

Table 1Samples	TVB-N (mg/100 g)	Storage time (h)	Storage temp. (°C)	Reference	
Initial	6.17 ± 0.02	0	25	This study	
CMC/COL	10.11 ± 0.02	72	25	idem	
CMC/COL/QCT	7.3 ± 0.01	72	25	idem	
CMC/COL/EUC	9.7 ± 0.01	72	25	idem	
No film	11.23 ± 0.01	72	25	idem	
Rose anthocyanins extract	13.43 ± 1.15	144	4	(Y. Yang et al., 2022)	
CMC/polyvinyl alcohol/anthocyanins	33.39	40	4	(Y. Wang et al., 2022)	
Potato starch/blueberry anthocyanins/ chondroitin sulfate	28.47	36	4	(Bao et al., 2022)	
CMC/date pits anthocyanins	21.6 ± 0.56	72	4	(Elhadef et al., 2024)	

In a recent study, Yang and colleagues explored the use of films made from sodium alginate/sodium carboxymethyl cellulose containing rose anthocyanins extract for preserving shrimp and pork meat. The findings showed that the films significantly reduced the amount of TVB-N in the meat compared to control samples. Shrimp meat packed with the rose anthocyanins extract film had a TVB-N level of 13.43 ± 1.15 mg/100 g after three days at 4 °C, while pork meat with the anthocyanins film had a TVB-N level of 33.39 mg/100 g after four days at 4 °C (Yun Wang, Zhang, & Zhang, 2022). For a detailed comparison of the results with previous literature, please refer to Table 1.

3.4 Antimicrobial performance of the prepared films

The ability to resist bacterial growth is of utmost importance for the efficacy of active film preparations (L. Zhang et al., 2022). However, the pure film sample exhibited suboptimal antibacterial properties, as demonstrated by the data presented in Fig.S6. This may be attributed to the inadequate concentration of antibacterial agents in CMC and Isinglass which are more effective on gram-positive bacteria. Quercetin and eucalyptus extract are present in samples CMC/COL/QCT and CMC/COL/EUC, and they gradually release antioxidant and antibacterial constituents over time, leading to a logarithmic drop in the bacterial count. At the end of 18 h, it was observed that S. aureus had a greater reduction in bacterial count when compared to E. coli. The reduction in bacteria for sample CMC/COL/QCT was 2 logs CFU/mL, while for sample CMC/COL/EUC, it was >2 logs CFU/mL. The potent antibacterial properties of samples CMC/COL/QCT and CMC/COL/EUC can be attributed to QCT and eucalyptus extract, respectively. These two compounds are responsible for the higher sensitivity of Gram-positive bacteria than Gram-negative bacteria, leading to a more pronounced reduction trend.

In the past literature, the sensitivity of gram-positive bacteria to quercetin and eucalyptus extract has been mentioned, for example: QCT, an essential plant flavonoid, possesses a variety of pharmacological activities. Extensive literature investigates its antimicrobial activity and possible mechanism of action. QCT has been shown to inhibit the growth of different Gram-positive and Gram-negative bacteria as well as fungi and viruses(Nguyen & Bhattacharya, 2022). The exact mechanism by which QCT exhibits antibacterial properties is not yet fully understood, but there are several hypotheses. One suggestion is that QCT accumulates in the cell wall of bacteria and interacts with peptidoglycan, leading to the destruction of the cell wall and ultimately causing bacterial inactivity (Li et al., 2022). So, peptidoglycan concentration in the bacterial cell wall is the main factor affected by QCT(L. Wang et al., 2019; D. Yang, Wang, Long, & Li, 2020).

Essential oil derived from fresh leaves of Eucalyptus globulus contains 11 components and exhibits greater activity against Gram-positive bacteria than against Gram-negative bacteria(Nadjib, Amine, Abdelkrim, Fairouz, & Maamar, 2014). The results of our study are consistent with previous research, as noted in the reports above.

4 Conclusion

Food packaging is a critical topic in the food industry, involving various methods and materials, each with its strengths and weaknesses. The primary objective of this study was to develop and produce packaging using natural materials that are safe for both the environment and human health post-use. The worldwide findings indicate that transitioning from plastic-based materials to those derived from natural polymers has the potential to lessen both environmental impacts and health risks associated with packaging. There is a notable cause for concern regarding the ingestion of food products contaminated by plastic throughout the food production chain. The degradation of plastic waste into microplastics poses a threat to natural resources, notably marine life. The potential consumption of microplastics by humans within the food chain raises serious health concerns, including the risk of developing severe illnesses such as cancer(Pourebrahimi & Pirooz, 2023). To this end, we prepared a pH-sensitive smart film using carboxymethyl cellulose (CMC) and collagen (COL) containing either eucalyptus (EUC) or quercetin (QCT). The results indicated no significant differences in the films' transparency, antioxidant activity, and antibacterial properties. However, the mechanical properties of the CMC/COL film were enhanced with the addition of QCT compared to EUC. Additionally, the total volatile basic nitrogen (TVB-N) level was lower after three days with QCT, making it the preferred option.

Despite these positive outcomes, challenges remain in implementing this technology on an industrial scale. The quality of EUC is influenced by factors such as plant species, growth period, harvesting time, and extraction method. Similarly, the quality of the polymer substrate and pigment concentration significantly impact the performance and quality of the final product. Therefore, further research is necessary to address these issues. In conclusion, this study is dedicated to elevating food quality while safeguarding human health and environmental safety.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Funding

No funding to declare.

CRediT authorship contribution statement

Akbar Mirzaei: Writing – original draft, Validation, Methodology, Investigation. Ghazaleh Mirzaei: Writing – original draft, Validation, Methodology, Investigation. Zahra Nezafat: Writing – original draft, Validation, Investigation. Shahrzad Javanshir: Writing – review & editing, Supervision, Conceptualization. Mohammad Mahdi Karimkhani: Writing – original draft, Investigation, Formal analysis. Abdollah Jamshidi: Writing – review & editing, Validation, Investigation, Formal analysis.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

Supplementary material Explore the multimedia component for additional insights and a deeper understanding of the study

Image 1

Data availability

“Data supporting this study are included within the article and/or supporting materials”

Acknowledgments

Not applicable.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.fochx.2024.101738.
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