Chitosan, extracted for potential upgrading, was characterized by Fourier transform infrared (FTIR) spectroscopy using KBr pellets in the scanning range of 400–4,000 cm⁻¹. The FTIR spectra of chitosan were measured (Bruker Vertex 70) with a resolution of 4 cm⁻¹.

The FTIR spectra of chitosan extracted by citric acid were obtained in a frequency range of 400–4,000 cm⁻¹. The degree of deacetylation (DD) of the extracted chitosan was calculated from the FTIR spectra using the method proposed by Khan et al. (2002).

The chitosan extracted with an organic acid was characterized by an X-ray diffractometer (X'Pert-Pro) with CuKa radiation (voltage = 40 kV, intensity = 30 mA). The X-ray diffractometer includes a 2θ goniometer.

The morphology of the extracted chitosan was investigated by a scanning electron microscope (SEM) (JEOL JSM-IT500HR) at a magnification range of × 600.000, an accelerating voltage of 0.5–30.0 kV, and a maximum sample size of 200 mm in diameter and 75 mm in height.

Experimental design matrix analysis (Table 2) leads to response functions presenting coded factors effects (X₁ and X₂) and their interaction which has the following formula:

Mathematical analysis interactions between factors (X₁ and X₂) present response surfaces and allows the identification of areas where there is a maximum or minimum response of studied response functions (Y_aa, Y_pig, and Y_fi) in the experimental domain.

The effect of treatment on prolonging the storage of post-harvest strawberries was studied. The percentages of contamination by Aspergillus niger were calculated using the following formula:

Pigmentation of treated and untreated strawberries was evaluated by measuring the concentration of anthocyanin pigments by using the spectroscopic method (Giusti and Wrolstad, 2001).

The firmness of processed strawberries was taken into account the hardness of fruits. This parameter was evaluated by making microscopic observations of strawberry tissue sections. Firmness is a variable that mainly affects the final fruit appearance. A quantitative assessment was developed by a jury based on a questionnaire.

The final extracted product was characterized by Fourier transform infrared (FTIR) spectroscopy. The later was used to identify functional groups in extracted chitosan. The assignment of absorption bands is based on the literature data (Shigemasa et al., 1996; Duarte et al., 2002; Kumirska et al., 2010).

The FTIR spectral data of chitosan extracted, by citric acid, revealed the following peaks (Figure 1): Peak at 3328.21 cm⁻¹ is attributed to OH stretching vibrations, while peaks at 2919.99 and 2870.06 cm⁻¹ are attributed to the CH-stretching aliphatic vibration in the -CH and -CH₂ groups. Amide frequencies consist of N-H bond stretching of amide I and N-H straining vibrations of -NH₂ groups are observed at 1649.55 and 1584.17 cm⁻¹, respectively. Peak at 1374.14 cm⁻¹ is attributed to the symmetric deformation of C-H in the -CH₃ group. The 1318.70 cm⁻¹ peak is attributed to the vibration modes of amide III. Absorption bands at 1149.69 cm⁻¹ correspond to anti-symmetric stretching of the C-O-C bridge. Stretching vibrations are also observed at 1060.58 and 1022.79 cm⁻¹ for the C-O stretching vibration of alcohol groups. Many authors have observed similar FTIR bands (Dimzon and Knepper, 2015; De Queiroz Antonino et al., 2017; Kumari et al., 2017).

Deacetylation degree (DD) of extracted chitosan was calculated from the FTIR spectra. In FTIR spectra, different absorption bands represent different functional groups present in chitosan. The determination of DD was done by two absorption bands, a characteristic band that represents the amide group of N-acetyl-D-glucosamine and a reference band that represents a group present in both N-acetyl-D-glucosamine and D-glucosamine (Dimzon and Knepper, 2015).

In the present work, the deacetylation degree of chitosan extracted by citric acid is 80.86%. This parameter is very important because it influences the physical, chemical, and biological properties of this biopolymer. Deacetylation degrees of the chitosan samples, obtained from the shells of white shrimp, were 94.23 and 92.45% when the chitin samples were converted into chitosan by the removal of the acetyl group with sodium hydroxide (NaOH) at 40% (w/v) and 60% (w/v), respectively (Kuyyogsuy, 2020).

Chitosan extracted from organic acid was characterized by the X-ray diffraction technique (Figure 2). This later made it possible to analyze the crystallinity of the extracted chitosan. X-ray diffraction pattern (XRD) showed two broad and characteristic diffraction peaks at 2θ around 20° and 30° which are typical fingerprints of semi-crystalline chitosan character. Kuyyogsuy (2020) reported that the XRD pattern of chitosan obtained, from white shrimp shells, revealed characteristic diffraction peaks at 2θ around 10° and 20° corresponding to the crystalline nature of chitosan. Samples of chitosan synthesized from different sources such as fish, shrimp, and crab shells were analyzed by X-ray diffraction (XRD) and diffraction peaks were observed at different values of 2θ such as 20°, 30°, 45°, and 65° (Kumari et al., 2017).

Morphological characteristics of extracted chitosan were studied by scanning electron microscopy (SEM). Figure 3 shows the surface morphology of chitosan extracted by citric acid at 100 and 5 μm. Chitosan micrographs showed a relatively smooth top surface and fibrous structures. The figure (5 μm) reveals the fibrous nature of extracted chitosan.

Natural variables (acetic acid and chitosan) coding into synthetic variables (X₁ and X₂) allowed us to develop an experimental design type 2² (4 experiments). Results of variance analysis of experimental design are shown in Table 4. Table 4 validates the mathematical model and Table 5 validates the coefficients of the model.

Obtained F (8.6) value is higher than the critical one F′ (4.07) illustrated in the Fisher Snedecor table, which confirms the mathematical model validity illustrated in Table 5.

Response function mathematical model (Equation 1) related to anti-fungal activity against Aspergillus niger obtained is as follows:

Student test performed on the coefficients (Table 5) shows that they have a significant effect and consequently we retained all of them. Coefficients analysis sign shows that studied factors and their interactions have a negative effect on the microbial load of strawberries (i.e., its reduction). This is consistent with results obtained from the study of the chitosan effect on Aspergillus niger, where the latter shows strong anti-fungal activity on Aspergillus niger mycelial growth. Effects of studied factors (X₁ and X₂) and their interaction (X1*X₂) are shown in Figure 6.

The interaction of the studied factors is shown in Figure 7 where we observe the response surface according to coded variables (acetic acid and chitosan), which delimits the experimental domain. This figure shows that anti-fungal activity against Aspergillus niger is maximal when acetic acid and chitosan are at the highest level (1.5 and 3%, respectively). Thus, the contamination of strawberries by Aspergillus niger is maximal when both factors X₁ and X₂ are at the lowest levels (0.5 and 1%, respectively).

Natural variables (acetic acid and chitosan) coding into synthetic variables (X₁ and X₂) allowed us to develop an experimental design type 2² (4 experiments) illustrated in the Materials and methods section. Variance analysis of experimental design results is shown in Table 6. Table 6 validates the mathematical model and Table 7 validates the coefficients of the model.

Obtained F (39.06) value is higher than the critical one F′ (4.07) illustrated in the Fisher Snedecor table, which confirms mathematical model validity as shown in Table 7.

Response function mathematical model (Equation 2) related to strawberries pigmentation stabilization obtained is as follows:

Student test performed on the coefficients (Table 7) shows that they have a significant effect and consequently we retained all of them. Coefficients analysis sign shows that studied factors and their interactions have a positive effect on the pigmentation stabilization of strawberries. Chitosan protected anthocyanin pigments from degradation during storage due to its antioxidant properties. Acetic acid contributed as a solvent in stabilizing the pigmentation of treated strawberries. Effects of studied factors (X₁ and X₂) and their interaction (X1*X₂) are shown in Figure 8.

The interaction of studied factors is shown in Figure 9 where we observe the response surface according to coded variables (acetic acid and chitosan), which delimits the experimental domain. This figure shows that pigmentation stabilization is maximal when acetic acid and chitosan are at the highest level (1.5 and 3%, respectively).

Natural variables (acetic acid and chitosan) coding into synthetic variables (X₁ and X₂) allowed us to develop an experimental design type 2² (4 experiments) illustrated in the Materials and methods section. Variance analysis of experimental design results is shown in Table 8. Table 8 validates the mathematical model and Table 9 validates the coefficients of the model.

Obtained F (422.55) value is higher than the critical one F′ (4.07) illustrated in the Fisher Snedecor table, which confirms mathematical model validity as shown in Table 9.

Response function mathematical model (Equation 3) related to strawberries firmness improvement obtained is as follows:

Student test performed on the coefficients (Table 9) shows that they have a significant effect and consequently we retained all of them. Coefficient analysis sign shows that the studied factors and their interactions have a positive effect on the firmness improvement of strawberries. Chitosan preserved the cellular structures of fruits as a post-harvest treatment due to its functional properties. This polymer regulates parameters responsible for the ripening of fruits during the storage period. Acetic acid contributes as a chitosan vector in the preservation of cellular structures of strawberries, by inhibiting oxidative enzymes such as polyphenol oxidases. Factors effects (X₁ and X₂) and their interaction (X1*X₂) are shown in Figure 10.

The interaction of studied factors is shown in Figure 11 where we observe the response surface according to the coded variables (acetic acid and chitosan), which delimits the experimental domain. This figure shows that firmness improvement is maximal when acetic acid and chitosan are at the highest level (1.5 and 3%, respectively).

According to obtained results, we find that edible coating made from chitosan extracted with organic acid (citric acid) significantly reduced microbial load of deterioration in strawberries. Treated strawberries retained their initial pigmentation for a longer duration when compared to untreated strawberries. Maturation of treated strawberries is thus delayed by a few days when compared to batch control and therefore treatment carried out has maintained cellular integrity in treated strawberries during the storage period. Acetic acid contributed as a solvent in stabilizing pigmentation and preserving cellular structures in treated strawberries. Soaking strawberries in an elaborated preservative solution extends the storage life of fruits and is considered to be very sensitive to post-harvest losses.

In this study, we used the strain Aspergillus niger as a model to develop a biological, less expensive, and effective anti-fungal treatment. The treatment elaborated by the biological chitosan and the acetic acid limits the proliferation of the molds which produce hydrolytic enzymes which degrade the cellular structures (firmness) and the pigments (anthocyanin). The stabilization of the cellular strucures limits or reduces cellular self-destruction. Strawberries attacked by molds ripen and spoil rapidly as fungal hydrolases degrade the polymers in strawberries into monomers that are assimilated by the bacteria that take hold afterward. The elaborate treatment ensures normal or slightly delayed ripening because the tissue structures are more stable. The prolongation of the conservation of strawberries lasts several days (7–8 days), a justified time sufficient to manage well the marketing of these very fragile fruits. This invention will give great competitiveness in the national and international market. The management of the sales of the strawberries treated by this process will be easier following prolonged conservation which allows the producers to manage time and space at the level of competitiveness.

The chitosan coating can be used to maintain the quality of strawberries by reducing growth and consequently enhancing the shelf life during cold storage compared to the uncoated treatment (Jiang et al., 2020). In addition, the chitosan (61 kDa) coating was more effective in delaying the pH increase and could better delay the reduction of strawberry flavonoids during storage. A study done by Abdipour et al. (2020) reported that the application of chitosan (1%) edible coating and UV irradiation maintained the post-harvest quality and reduced the weight loss during the storage of treated sweet cherry fruit. The chitosan coating on the strawberries' surface acted as a semi-permeable barrier to gases and water, thus reducing respiration and water loss and neutralizing dehydration and shrinkage of the fruit (Jongsri et al., 2016; Cazón and Vázquez, 2019). Chitosan coating has good mechanical strength on the surface of strawberries and could slow down texture degradation (Jiang et al., 2020). In addition, Petriccione et al. (2015) reported that chitosan treatment significantly reduced water loss and delayed the qualitative changes in the color of post-harvest strawberries. Furthermore, changes in the total polyphenol, anthocyanin, and flavonoid contents and the antioxidant capacity of chitosan-coated strawberries were delayed. In vivo bioassays showed that strawberries immersed in chitosan (7.5, 10, and 15 mg mL⁻¹) displayed significant inhibition of mycelial growth and spore germination of post-harvest fungal strawberry pathogens (Arceo-Martínez et al., 2019).

Strawberries are highly perishable fruits that degrade quickly post-harvest, which explains their short shelf life. Fungal deterioration is one of the main causes of post-harvest losses of these fruits considered to be fragile. Storing strawberries at low temperatures is the most commonly applied method to improve their shelf life (Saleem et al., 2021). However, this technique without the use of other combined durable treatments is not considered appropriate due to the highly perishable nature of strawberries once released to the market. Other post-harvest techniques have been reported to extend the shelf life of strawberries, namely, edible coatings (Qamar et al., 2018; Quintana et al., 2021). These improve the visual appearance and maintain the post-harvest quality of coated fruits and vegetables. Chitosan coating has been applied post-harvest to many fruits and vegetables such as strawberries (Ban et al., 2018; Trinetta et al., 2020).

In various studies, chitosan has been proven to possess broad-spectrum antimicrobial activity, which can inhibit a huge variety of fungi and bacteria growth. This biopolymer is a potential antimicrobial substance due to its good biocompatibility, biodegradability, non-toxicity, and availability. The electrostatic interaction between chitosan and amine groups on fungi cell surface is the key that gives it ability to bind, alter or destroy the microbial cell surface (Qin et al., 2020). This explains the anti-fungal activity of chitosan against Aspergillus niger in strawberries in our study. Chitosan exhibits key anti-fungal properties that inhibited the growth of the mycelial fungus recognized as a food pathogen and spoilage microorganism. Aspergillus genus strains often contaminate products in the food industry. This currently encourages manufacturers to overcome this problem by using natural ingredients that meet food safety standards. Aspergillus niger is by far the most common Aspergillus species responsible for the post-harvest decay of fresh fruits, namely, apples, pears, peaches, citrus, grapes, figs, strawberries, mangoes, and melons (Pitt and Hocking, 2009). Thus, chitosan is a marine biological polysaccharide that has attracted much interest for antimicrobial applications (Chuan et al., 2019; Shariatinia, 2019).

Chitosan is considered a biopolymer with excellent antioxidant properties due to the presence of the amine group and two hydroxyl groups that give the polymer the ability to scavenge free radicals (Anraku et al., 2018). The use of antioxidants as food additives protects foodstuffs against oxidation, which generally affects food quality. Food attractive color suggests freshness and quality to consumers and influences purchasing decisions. For this reason, food coloring is an important parameter to consider in the food industry. Anthocyanins are natural pigments sensitive to degradation by exogenous and endogenous factors (Ge et al., 2018). Coatings and films are alternative methods to extend and protect these pigments from degradation, due to their bioactive properties, so chitosan has been used in many of these applications (Harkin et al., 2019). In this study, chitosan treatment ensured anthocyanin pigments stability in strawberries during the storage period. Acetic acid contributed as a solvent and reagent against oxidation enzymes (polyphenol oxidase) in stabilizing the pigmentation of treated strawberries.

As an active food packaging, chitosan can be applied to food as an edible thin film or an edible coating. The coating is a thin layer formed as a covering on a food product, which is applied in liquid form, by dipping the product in a chitosan solution (or spraying the product with chitosan solution) (Muxika et al., 2017). Chitosan has a wide spectrum of biological actions such as antioxidant activity, antimicrobial function, anti-fungal properties, film-forming properties, and food additive properties, which could be used in the food industry to improve safety, quality, and food shelf life (Kulawik et al., 2019). In this study, a coating made with chitosan improved post-harvest stability of strawberries for a long time by keeping their initial pigmentation, reducing the microbial load of deterioration, and preserving cellular structures when compared to control strawberries, and as a result, the treatment realized extended shelf life of strawberries.

Applying chitosan film or coating can improve perishable fruits and shelf life of vegetables as a post-harvest treatment. Overall, chitosan regulates gas exchange (CO₂ and O₂), post-harvest fruit respiration rate, and ethylene production rate during the storage period (Cazón and Vázquez, 2019). Furthermore, studies show that applying chitosan has a beneficial effect on maintaining firmness, titratable acidity, moisture content, and color retention throughout storage in treated vegetables and fruits (Leceta et al., 2015). In our study, acetic acid contributed as a chitosan vector in preserving the cellular structures of strawberries.

The results of this work can help the decision-makers (legislation and law in force) to elaborate organic quality standards according to the official bulletin which forms a schedule of conditions to be respected and followed by the producers. This contribution is double-edged, elimination of toxic acids for public health and protection for the environment. Thus, this work is a milestone that contributes to sustainable development.

Today's consumers demand less use of chemicals to extend the shelf life of food products. For this reason, there has been a growing interest in recent times to develop materials with film-forming ability and antimicrobial and antioxidant properties, which help improve food safety and shelf life. Due to its functional properties, chitosan is considered a natural alternative to synthetic antimicrobials and antioxidants. This biodegradable polymer is gaining more attention in the development of new active food packaging materials.