Evaluation of edible coating on quality, biochemical components and microbial community under cooling and freezing conditions on fresh-cut artichoke

Plant material and experimental design

The artichoke experiment was conducted over two consecutive growing seasons (2024–2025). A French hybrid cultivar was used and obtained immediately after harvest from Rayyan Farm and Factory Fresh, Kafr El-Dawar, Beheira Governorate, Egypt. To minimize enzymatic browning and oxidative deterioration, the harvested artichokes were immersed in a 2% (w/v) ascorbic acid solution prior to further processing to preserve it until it is transported to the laboratory.

Sample preparation and packaging

Following pretreatment, artichoke samples were coated with edible coatings using a brush application technique and subsequently air-dried at ambient temperature to ensure uniform film formation. The coated samples were packaged in low-density polyethylene (LDPE) bags (47 × 33 cm; 0.03 mm thickness) and polypropylene (PP) bags (40 μm thickness), supplied by the Department of Manufacturing, Packaging, and Food Packaging Engineering Research, Food Technology Institute, Agricultural Research Center, Giza, Egypt. All samples were stored for further analyses.

Preparation of edible coating formulations

Whey protein coating

Whey protein was obtained from (≥ 95% purity, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s specifications. The whey protein used without additional flavoring agents, sweeteners, or other commercial additives. A solution was prepared by dissolving 100 g of whey protein in 1 L of distilled water. The solution was heated at 45 °C for 30 min under continuous stirring. A calcium chloride (CaCl₂) solution (20 g L⁻¹) was prepared separately and heated at 45 °C for 15 min under stirring. The two solutions were then combined under continuous stirring. Subsequently, 20 mL of lemon oil and 2 mL of Tween 20 (20%) were added as an emulsifier. The final coating was applied to the samples using a brush technique. All treatments were performed at room temperature (20–25 °C). After coating, samples were air-dried under ambient conditions. This treatment was applied to enhance tissue firmness and reduce enzymatic activity during storage¹¹.

Calcium chloride (CaCl₂) was incorporated into the coating formulations because calcium ions play an important role in maintaining cell wall integrity through the formation of calcium pectate cross-links within the middle lamella. This interaction enhances tissue firmness, delays softening, reduces membrane permeability, and contributes to the suppression of enzymatic browning and senescence-related metabolic activities during storage. In addition, calcium treatments have been widely reported to improve postharvest quality and extend the storage life of fresh fruits and vegetables^12,13.

Gum Arabic (GA) coating (Acacia seyal)

GA (Talha gum) derived from Acacia seyal was obtained from a certified local supplier. The material was cleaned, finely ground, and used without preservatives or additives.

A solution was prepared by dissolving 100 g L⁻¹ GA in distilled water, followed by heating at 60 °C for 30 min under continuous stirring until complete dissolution. The solution was cooled to room temperature before the addition of 20 mL peppermint oil and 2% Tween 20 as an emulsifier. The coating was applied using a brush technique. GA served as the sole film-forming biopolymer and was used to enhance moisture retention and postharvest quality¹⁴.

Carboxymethyl cellulose (CMC) coating

CMC were obtained from (Sigma-Aldrich, St. Louis, MO, USA) was used without further purification. A solution was prepared by dissolving 60 g of CMC in 750 mL of distilled water under stirring, followed by the addition of 250 mL ethanol. The mixture was heated to 80 °C until complete dissolution. Finally, 20 mL clove oil and 2% Tween 20 were added. The coating was applied using a brush technique. The formulation was prepared freshly prior to use¹⁵.

Control: Uncoated samples served as the control

The objective of this study was to evaluate the efficacy of complete edible coating formulations enriched with essential oils as practical postharvest preservation systems rather than to investigate the individual contribution of each coating component. Similar formulation-based approaches have been widely adopted in studies evaluating edible coatings for fresh produce preservation, where the performance of the integrated coating system represents the primary focus^16,17. Therefore, treatments containing the coating matrices alone, without essential oil incorporation, were not included in the experimental design.

The selected biopolymer–essential oil combinations were chosen based on previous studies demonstrating their suitability for edible coating applications and postharvest preservation. Whey protein has been widely reported as an effective film-forming matrix with excellent oxygen barrier and emulsifying properties, facilitating the incorporation and controlled release of essential oils such as lemon oil^18,19. GA possesses superior emulsification capacity and coating stability, making it an appropriate carrier for bioactive compounds including peppermint oil^20,21. Likewise, (CMC) was selected because of its excellent film-forming ability, transparency, flexibility, and moisture barrier properties, while clove oil was incorporated due to its strong antioxidant and antimicrobial activity^22,23. Therefore, these formulations were selected as representative functional coating systems to evaluate their effectiveness in preserving the quality and storage stability of fresh-cut artichoke.

Methods for extracting essential oils

In the laboratory of the Department of postharvest and handling Vegetables Research, Horticultural Research Institute, Agricultural Research Center, Giza, Egypt, 20 g of mint leaves, clove flower buds, and lemon fruit peel were taken after cleaning and drying. The plant material was procured from a certified local herbal supplier. The essential oil was extracted by steam distillation using a Clevenger-type apparatus for 3–4 h. The obtained oil was then dried over anhydrous sodium sulfate, filtered, and stored in amber glass bottles at 4 °C until further analysis to prevent oxidation and volatilization of its active compounds.

There were three methods of storage used:

1. 1.

   The refrigeration stage at 0 °C with 95% relative humidity,

2. 2.

   Individual Quick Freezing (IQF) at -30 °C.

3. 3.

   The two techniques are combined: IQF at -30 °C and thawing at 0 °C in the refrigeration stage with 95% relative humidity.

The qualities listed below were recorded after a two-week storage period for each treatment. Approximately 1000 kg of freshly harvested artichokes were used throughout the entire study. The material was subdivided among all coating, packaging, and storage treatments. Each treatment consisted of three independent biological replicates, with each replicate containing approximately 1 kg of fresh-cut artichoke packed separately in an individual package. The package was considered the experimental unit for statistical analysis.

The total carbohydrates

The total carbohydrate content of the sample is determined using the colorimetric phenol-sulfuric acid method, as outlined by²⁴.

Protein content

Protein determined using the Kjeldahl method outlined by²⁵.

Total sugars

The concentration of the total sugars in a sample can be determined using the colorimetric approach known as the Anthrone method. The anthrone reagent interacts with sugars in an acidic environment to produce a blue-green hue. The anthrone reagent and sulfuric acid are combined with the sample, which is then cooked until the reaction is finished. Next, the mixture is allowed to cool, and at 620 nm, its absorbance is determined. The amount of sugar in the original sample and the absorbance are linearly correlated²⁶.

Folic acid

Spectrophotometric technique was created to measure folic acid by²⁷.

Inulin content

The inulin was extracted from the artichoke tuber samples using an accelerated solvent extraction technique. After that, spectrophotometry was used to determine the amount of fructose in the hydrolysates. The spectrophotometric technique is based on measuring the absorbance at 350 nm of the triiodide complex produced by the periodate oxidation of fructose, upon the addition of potassium iodide²⁸.

Cynarin content

Cynarin determined according to²⁹.

Total phenols

The total Phenolic concentration was calculated using methodologies developed by^30,31.

Where: C= Concentration obtained from the calibration in µg/ml, DF= Total dilution factor; W= weight of the sample in grams, 1000 = conversion from µg/ml to mg/ml.

Ascorbic acid (vit. C)

The vitamin C content is determined using UV spectroscopy, which measures the amount of vitamin C in a sample. According to³².

Polyphenol oxidase (PPO) enzyme activity

Polyphenol oxidase (PPO) activity was determined according to the method of³³ with slight modifications. Fresh tissue (1 g) was homogenized in 10 mL of cold phosphate buffer (0.1 M, pH 6.8) and centrifuged at 10,000 ×g for 15 min at 4 °C. The reaction mixture contained 2.9 mL of catechol solution (0.1 M) prepared in phosphate buffer and 0.1 mL of enzyme extract. The increase in absorbance at 420 nm was recorded for 3 min using a UV–Vis spectrophotometer. One unit of PPO activity was defined as the change in absorbance of 0.001 per minute under the assay conditions and expressed as U g⁻¹ fresh weight.

Malondialdehyde (lipid oxidation)

The MDA concentration was determined using an extinction coefficient of 155 mM^− 1 cm^− 134 after deducting the absorbance at 600 nm from the absorbance at 532 nm.

Superoxide anion (O₂
⁻)

The superoxide anion (O₂⁻) Fifty milligrams of the leaf samples were incubated in an extraction medium made up of 100 µM ethylenediaminetetraacetic acid (EDTA) disodium salt, 20 µM nicotinamide adenine dinucleotide (NADH), and 20 mM sodium phosphate buffer at pH 7.8 to measure the superoxide anion (O₂⁻) concentration³⁵. Adding 100 µL of 25.2 mM epinephrine in 0.1 N HCl started the reaction. The absorbance was measured at 480 nm for 5 min after the samples had been incubated for 5 min at 28 °C with continuous mixing. The buildup of adrenochrome was determined using a molar absorption coefficient of 4.0 × 103 M^− 136 to calculate superoxide anion production.

Hydrogen peroxide content

Using 300 mg of samples that were homogenized in an extraction medium composed of 50 mM potassium phosphate buffer at pH 6.5 and 1 mM hydroxylamine, the hydrogen peroxide content (H₂O₂) concentration was ascertained. The mixture was then centrifuged for 15 min at 10,000×g at 4 °C. After that, 30 µL samples of the supernatant were added to a reaction medium containing 100 µM FeNH₄SO₄, 25 mM sulfuric acid, 250 µM xylenol orange, and 100 mM sorbitol³⁷. The absorbance of the samples was measured at 560 nm after they were kept in the dark for 30 min.

Determination of Potassium

It was estimated Flame photometrically using Ienway Flame photometer model Corning 400 according to³⁸.

Determination of Fe

Using the method described by³⁹, the heavy metal content was examined using the Perkin Elmer Model 5100 electrothermal atomic absorption spectrometer.

Texture

The texture of the sample was measured using a TA.XT2i Plus texture analyzer (Stable Micro Systems Ltd., Godalming, Surrey, UK). Fresh-cut artichoke was placed on the test platform with the skin facing upward. The probe (P/2, 2 mm cylindrical probe) was compressed to 50% of the total sample height at a pre-test speed of 2 mm/s, a test speed of 1 mm/s, and a post-test speed of 2 mm/s. The maximum force was recorded as the hardness of the sample, while the number of peaks represented the sample fragility (brittleness).

Weight loss percent

The formula for the percentage of weight reduction (WLP %) used by⁴⁰ [(initial – final weight)/initial weight] × 100 was used to calculate the WLP% following the storage period.

Ethylene

Ethylene gas determined according to⁴¹.

Respiration rate

Respiratory rate a gas-tight container of known volume is filled with product in the closed system, and the container, which initially contained ambient air as its atmosphere, is closed. The changes in CO₂ concentration over a specific period are measured and used to calculate respiration rates⁴².

Microorganism

Pathogenic strains including

Bacteria

The bacterial strains used in this investigation (Gram positive and Gram negative) were kindly supplied by the Microbiology Department, Faculty of Agricultural Cairo University. These strains were Staphylooccus aureus, ATTCC 25,923, E. coli, ATCC 25,922, Bacillus cereus ATCC 33,018 Cultures were maintained on nutrient agar slants (4 °C) and sub cultured at 37 °C in nutrient broth for 24 h prior to incubation.

Fungi

Isolates of fungi were Aspergillus niger and obtained from Microbiology Center (CATM), Ain-Shams Uni.

Media used

All media consist of (gL^− 1). Plate count agar medium, Potato dextrose agar medium and Nutrient Agar medium.

Microbiological methods

Total count bacteria were determined on plate count agar according to⁴³.

Yeast and mold were enumerated using Potato dextrose agar according to⁴⁴.

Determination of antibacterial and antifungal activity

For the antibacterial activity test, prepared coating solution was kept in a 100 mL beaker and sterile filter paper discs were immersed in the solution. Antibacterial and antifungal activities of the coating formulations were evaluated against the selected bacterial and fungal strains using the disc diffusion method according to⁴⁵. The plates were incubated at 35 °C overnight for bacteria and at 27 °C for 3 days for fungi. Inhibition zones were measured and expressed in (mm). Antimicrobial activity was evaluated using the edible coating formulations themselves rather than the dried films or coated artichoke samples. The disc diffusion assay was performed to assess the inhibitory potential of each coating solution containing the respective biopolymer matrix and essential oil components against selected bacterial and fungal strains. Therefore, the recorded inhibition zones represent the antimicrobial efficacy of the coating formulations under in vitro conditions. In contrast, microbial counts determined during storage were used to evaluate the effectiveness of coated and packaged artichoke samples in suppressing microbial proliferation under practical storage conditions. Two commercially available polymeric film bags were used as packaging materials. The investigated polymer materials were low-density polyethylene (LDPE) 20 × 30 cm of 40 μm thickness and polypropylene (BOPP Biaxial oriented) 20 × 30 cm of 24 μm thickness. The average oxygen transmission rates (OTR)of the PE and PP films are 4000 cc ∕ m² ∕ day and 1000 cc ∕ m² ∕ day ‚while the water vapor transmission rates (WVPR) of films is LDPE 20 gm ∕ m² ∕ day and 70 gm ∕ m² ∕ day respectively.

Characterization of edible films

Film thickness

Film thickness was measured using a Mitutoyo micrometer (Model MDC-25 M, MFG / Japan). The final value represented the average of 10 random measurements taken at different parts of the film as reported by (R)⁴⁶.

Water vapor transmission

The water vapor transmission rate [g/ (s.m²)] and water vapor permeability (WVP) through films were determined gravimetrically using the⁴⁷ Method E96-95. A circular test cup was used to determine the WVP of the film. The film was first cut into circular shape that was larger than the inner diameter of the cup, the cup was filled with 50% distilled water and the film was sealed at the top using paraffin oil, then the cups were placed in a desiccator containing anhydrous calcium chloride. The weights of the cups were recorded every hour during 10 h and to specimens of each film were tested. Linear regression was used to estimate the slope of this line in g/h. The water vapor transmission rate (WVPR) and water vapor permeability were determined using the following equation:

Where, ∆m/∆t is the moisture gain weight per time (g/s), A is the surface area of the film (m²) L is the film thickness (mm) and is the difference in relative humidity⁴⁸.

Transparency of edible films

Transparency acts as an additional factor to exploit the constituents of edible film⁴⁹. Transmittance to visible light was measured at wavelength 550 nm.

Water solubility of edible films

The solubility of different edible films was determined according to method given by⁵⁰. The film solubility expresses the percentage of the films dry matter solubilized after immersion of the per-weighted films (2 × 3 cm) in 80 ml of distilled water for 24 h at room temperature with constant agitation. The remained pieces of the film after immersion filtered using filter paper, were dried at 60 °C in an oven constant weight. Film solubility(S%) was calculated from the following equation:

Statistical analysis

The experiment was conducted as a factorial arrangement consisting of four coating treatments (control, whey protein + lemon oil, GA + Peppermint oil, and CMC + clove oil), three storage conditions (cooling, IQF, and cooling + IQF), and two packaging materials (polyethylene and polypropylene). Each treatment combination was replicated three times. One package containing 1 kg of fresh-cut artichoke represented one biological replicate and was considered the experimental unit for statistical analysis.

The experiment was arranged as a completely factorial design consisting of three factors: (i) edible coating treatment with four levels [control, whey protein + lemon oil, GA + Peppermint oil, and CMC + clove oil], (ii) storage condition with three levels [cooling at 0 °C, (IQF) at -30 °C, and cooling + IQF], and (iii) packaging material with two levels [polyethylene (PE) and polypropylene (PP)]. Therefore, a total of 24 treatment combinations (4 × 3 × 2) were evaluated. Each treatment combination was replicated three times, and each replicate package was considered an independent experimental unit.

Data were analyzed using a three-factor factorial ANOVA considering coating treatment, storage condition, and packaging material as fixed factors “COSTATE” program version (6.400) as reported by ⁵¹. Main effects and all possible interactions were evaluated. Mean separation was performed using Duncan’s multiple range test at P ≤ 0.05. Results are presented as mean ± standard deviation of three independent biological replicates.