The Individual and Combined Impacts of Ozonation and Chitosan Bio-coating on Health-Promoting Bioactive Substance and Storage Stability of Fresh Aronia

The Individual and Combined Impacts of Ozonation and Chitosan Bio-coating on Health-Promoting Bioactive Substance and Storage Stability of Fresh Aronia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Individual and Combined Impacts of Ozonation and Chitosan Bio-coating on Health-Promoting Bioactive Substance and Storage Stability of Fresh Aronia Nur Ebru Güleryüz Ok, Muhammed Yüceer, Çiğdem Uysal Pala, Cengiz Caner This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6862137/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The focus of the study is to analyze the individual and synergistic effects of ozone (8 ppm for 5 minutes) and 1% chitosan bio-coating application on enhancing the storability of fresh aronia with high antioxidant and anthocyanin content. The fresh aronia were ozonated (8 ppm 5 min), chitosan-coated, and after being coated after ozonation (8 ppm 5 min), placed in polypropylene (PP) trays and stored for 4 months (+ 4°C). Analyses were carried out on the 1st day, 1st, 2nd, 3rd, and 4th months. The pH, Brix, color values, gas concentrations, weight loss, texture profile analyses, fungal development, monomeric anthocyanin, and total phenolic content were analyzed during storage. Results At the end of storage, the weight loss was 0.81% in the ozone group, 0.72% in the coated groups, and 0.68% in chitosan and ozonated ones, while it was 1.2% in the control groups. While the Brix in the control group increased from 18.3 to 20.3; at the end of storage, it was 19.7 in the ozone group, 19.8 in the coated ones, and 19.55 in the chitosan and ozonated ones. While the pH in the control group increased from 3.56 to 3.93, it was 3.8 in the ozone group, 3.82 in the coated ones, and 3.81 in the ozonated and coated groups. The rate of respiration was minimized as the gas composition O 2 /CO 2 in the headspace was achieved with the synergistic impacts of ozonation and coating. At the end of the 4th month, while it decreased to 380 gf on average in the control samples, this value was determined as 424 gf in the ozonated ones, 453 gf in the chitosan-coated, and 470 gf in the chitosan-coated samples after ozonation. Ozonation highlighted efficacy in inhibiting mold growth. The extractability of anthocyanin (3314 mg cyn 3-glu/100 g) phenol (660 mg) was significantly enhanced with ozonation at the end of the storage. Conclusion Ozonation and ozonation after chitosan applications demonstrated a significant potential to maintain anthocyanins in fresh berries by improving their extractability and minimizing degradation during storage. Additionally, ozonation positively influenced anthocyanins and total phenolic content. Ozonation was used as an elicitor to improve the bioactive substances in the fresh aronia. It was observed that the combined use of ozonation and chitosan coating enhanced storage stability and bioactive compounds in the fresh aronia. Aronia Chitosan Ozone Equilibrium Modified Atmosphere Packaging (EMAP) Anthocyanins Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction The consumption of fresh produce, such as berries, has recently received remarkable attention due to people's awareness of its health properties in the past decade. Aronia melanocarpa berries (black chokeberry) have one of the highest contents of potential antioxidants, such as anthocyanins, flavonoids, and proanthocyanidin polyphenols, in the plant kingdom that may be helpful in the reduction of risk of diabetes, cancer, cardiovascular, and gastrointestinal disease 1 . Hjartaker, et al. 2 found that consuming more berries has been correlated with a lower risk of developing from cancer mortality (8–10%), stroke, and digestive system cancer. Aronia berries are significantly lesser-known when compared to other berries, but they have gained popularity over the last decade, mainly due to recognition of the health benefits of underutilized berries, especially aronia berry 1 . Fresh berries such as aronia have a limited storage stability with 30%−60% losses depending upon the supply chain stages 3 , mainly due to it continuing with metabolic processes after postharvest 4 . Berries are highly perishable horticultural produce. Also, postharvest diseases, such as gray mold during the storage period, lead to important economic losses 1 . The availability of fresh berries in retail stores throughout the year caused by their positive health-promoting properties is very important for increased consumption. Thus, helping in maintaining its highest qualities is necessary for extending storability for enhancing sustainability. Since fresh aronia are harvested fully ripened, they are susceptible to decay in their high quality due to moisture through transpiration, mechanical damage, softening, susceptibility toward postharvest diseases, and changes in phytochemicals during postharvest storage 5 . Therefore, it must be preserved in its superior quality to ensure its consumption throughout the year. The demand for safe, fresh produce with high nutritional value, high sensory properties, and extended storage stability using sustainable innovative emerging technologies has increased in recent years. Various environmentally friendly emerging post-harvest methods such as ultrasound, plasma, ozonation, electrolyzed water, high-pressure processing, edible coatings, and equilibrium-modified atmosphere packaging (EMAP) have become more important in recent years in the preservation of fresh produce and reduce their ecological footprint 6 . Today, the focus is on ozone, sustainability, cheapness, ease of application, the powerful antimicrobial agent characterized by easy decomposition without leaving any toxins/residues and oxidation, and approval by the FDA as an antibacterial agent for direct contact with food 78 . Several research studies have evaluated that microbial loads can be lowered by ozone treatmensts 8 . Horvitz, et al. 9 have revealed that the highest reduction in B. cinerea , Salmonella enterica , and E. coli in Andean blackberry after role of ozonations (0.7 ppm, for 3 min.) when compare the 0.4, 0.5 and 0.6 ppm (3 min). Similarly, Shah, et al. 5 reported that gray mold by B. cinerea was minimized by ozonation (2 to 3 ppm for 3 h) in red raspberry and maintaining higher contents of TA, SSC, and surface colors 6 . Ozone storage (0.1 and 0.3 ppm) suppressed fungal development for 12 days. Surface color and anthocyanin content were better retained in the stored berries 10 . Ozone application maintained firmness in kiwi (300 ppb) 11 , papaya (1.5–3.5 ppm) 12 , strawberries (2 min at 300 ppb) 13 . Ozone applications (40–200 ppb) slowed down the yellowing of the broccoli, but 700 ppb was reported to be undesirable tissue browning 8 . The application of the ozone in foods mostly concentrates on the microbiological control of fish, poultry, dairy, and meat products, while its applications in fruits and vegetables, especially fresh berries, are still limited 14 . The applications of the optimized ozone (2, 5, and 10 ppm) and contact time (3 and 9 min) in fresh “Angelino” plum preservation enhanced bioactive substances, including anthocyanins and organic acids, during storage but had minimal impact on the general quality 4 . Therefore, to enhance the storage stability of fresh berries, it is necessary to combine emerging nonthermal technologies, such as sustainable bio-coating. The sustainable coatings are considered a revolutionary strategy in post-harvest preservation, combining durability and ecological potential into a one-shot solution to preserve the quality of perishable products. Of all these biocoatings, chitosan has attracted attention in the recent past because of its inherent antibacterial properties and suitability with a broad range of bioactivities 15 . Chitosan biocoatings are to act a main role in the strawberries in lowering the permeability of the O 2 /CO 2 and moisture in trays 16 . Chitosan biocoatings act as barriers by producing tiny physical layers on the fruits that can significantly reduce postharvest losses by controlling the mass transfer of gases (O 2 /CO 2 ) leads to slowing down the metabolic activity and minimizing the respiration rate of the fresh fruits. Hence enhancing its storage stability and qualities of fresh produce. It is especially useful for fresh produce that contains a high level of water, like fresh berries 17 . Like biocoatings, an EMAP is to play a crucial role in regulating the mass transfer of gases (O 2 /CO 2 ), and moisture established inside the headspace using the film permeability (O 2 /CO 2 ) and the respiration rates of the packaged fruits in the trays. By using, EMAP improves the storage stability of fresh produce by suppressing respiration rates and ethylene production 18 . Therefore, EMAP must be combined to maintain the high quality of aronia during extended storage 17 . Because of the strategic nature of the market, researchers are using emerging techniques such as equilibrium modified atmosphere packaging (EMAP) and sustainable chitosan bio-coatings to preserve perishable berries for longer storage stability. There are a number of techniques that are used to preserve the post-harvest quality (phenol levels and antioxidant activities) of fresh berries including modified atmosphere packaging and coatings 19 , 20 . However, there are a few studies reported on deep research into the impacts of ozonation and chitosan biocoating on the storability of “fresh aronia’ during extended storability. Therefore, comprehensive detail research is essential to extend the postharvest storage stability of fresh berries such as fresh aronia. Based on our literature review, there is a lack of existing research papers on the combined ozone (8 ppm) and chitosan coatings for fresh aronia storage stability. For that reason, the main goal of this study was to provide novel insights into mitigation and improvement of storage stability of fresh aronia with high levels of the bioactive compounds such as total monomeric anthocyanin and phenolic. 2. Materials and Methods 2.1 Sample processing. Fresh aronia fruits ( Aronia melanocarpa ) used in this study were supplied from a local producer in Yalova province (Producer No: M-77-26) in the 2021 season. Fresh aronia berries were placed in a 5-L transparent glass chamber and treated with 8 ppm ozone gas with the help of a diffuser for 5 minutes using TKZ-6G ozone generator (Teknozone, Izmir). The solutions of the chitosan bio-coating were produced by dissolving 1% chitosan (w/w) in 1% acetic acid (0.5% glycerol as plasticizer) 21 . After the application, 100 g of aronia were placed in PP trays (190*144*H65 cm) and heat-sealed using the MAP-25 (MAP-25, Apack Ltd. Şti., Istanbul, Turkey) with normal air atmosphere. Packaged aronias were stored at + 4°C and monthly samplings were carried out. The analyses were carried out on the 1st, 1st, 2nd, 3rd, and 4th months of storage, randomly selecting packaged trays from each application, with 2 replications and 2 parallels. Application groups and application parameters: Control 90 µm microperforated films (EMAP) 1% Chitosan coating 8 ppm ozonation (5 min.) 8 ppm ozonation (5 min.) after 1% chitosan coating applications Weight loss, in-package gas concentration (O 2 /CO 2 ), water-soluble DM, pH, titratable acidity, color values ​​(L * , a * and b * ), texture profile analysis (TPA: firmness-hardness, external adhesiveness, internal adhesiveness, chewiness, gumminess and springness), mold status, monomeric anthocyanin amount, and total phenolics content of fresh aronia were determined on the 1st day and then 1st, 2nd, 3rd, and 4th months of storage. 2.2 Gas composition in trays The gas (O 2 /CO 2 ) levels in the headspace in the trays were measured by inserting the needle into the trays before opening with OxyBaby, the gas analyzer (HTK, Hamburg, Germany) 22 . 2.3 pH Measurement and Total Soluble Solid Content After opening the package, aronia berries were separated into three sets, and berries were placed in a blender, and then the obtained pulp was transferred into a beaker. Analyses were carried out using the pH meter (Atagon PAL-1 Osoka, Japan) 22 and Pal-1refractometer (Atago Co., Tokyo, Japan) and then shown as Brix 23 at room temperatures. 2.4 Surface Color The surface of the fresh aronia “L * ”, “a * ”, and “b * ” values was analyzed using a CR-400 ChromaMeter (Minolta Co. Ltd., Japan) 23 . 2.5 Texture Profile Analysis (TPA) The TPA properties were measured using the Texture Analyzer TA-XT Plus (Stable Micro Systems Co., Surrey, England) using P-2CYL cylindrical probe based on a trigger force of 1.0 N. Properties such as hardness (N), springiness (unitless), stickiness (N.s), chewiness (N) and gumminess were analyzed automatically using the software 17 2.6 Total Anthocyanin Analysis The total monomeric anthocyanin amount is measured by the pH differential method (colored at pH 1.0 and colorless at pH 4.5). The extracted aronia juice was filtered and then scanned using the spectrophotometer (7205 UV/Visible, Jenway Co., UK). The absorbance measurement was carried out at 520 nm and at 700 nm wavelength at pH 1.0 and pH 4.5 according to Caner, et al. 17 . Results are presented in mg cyanidin-3-glucoside equivalents/100 mg sample. 2.7 Total Phenolic Content (TPC) The Folin-Ciocalteu method was followed. A spectrophotometer (7205 UV/Visible, Jenway Co., UK) was used to analyze absorbance at 765 nm with a blank as the reference. TPC was quantified using a gallic acid calibration curve and written as mg of gallic acid equivalents (GAE)/100 g of sample. - CUPRAC The CUPRAC analysis, based on the reduction of Cu (II) to Cu (I) by aronia antioxidants, leading to the formation of a colored Cu (I)-neocuproine (Nc) complex with maximum absorbance at 450 nm, was measured. For the assay, ethanolic aronia extracts were sequentially mixed with 0.01 M CuCl 2 and 1 M ammonium acetate buffer (pH 7.0) and incubated at 25°C for 30 minutes to allow complete complex formation. A spectrophotometer (7205 UV/Visible, Jenway Co., UK) was used to read the absorbance of the resulting Cu (I)-Nc chelate at 450 nm). The antioxidant capacity was quantified by means of a Trolox standard curve, and the outcomes were expressed as mg Trolox equivalents (TE) per 100 g sample. - DPPH The scavenging of DPPH (2,2-diphenyl-1-picrylhydrazyl) free radicals assay was measured according to Benvenuti, et al. 24 . The extracts (10–40 µL) were mixed with 300 µL of 1 mM DPPH solution and adjusted to 3 mL with ethanol. After incubation for 15 minutes in the dark at room temperature, the spectrophotometer was carried out to measure the absorbance at 517 nm. DPPH scavenging activity (%) was evaluated based on the reduction in absorbance, and the half-maximal effective concentration (EC 50 ) was determined as mg of sample from inhibition percentage versus concentration curves. 2.7 Visual evaluation (fungal decay evaluation) Fungal growth on the fresh aronia was visually counted and recorded as the number of fruits with mold or visible fungal growth, decay, or lesions. 22 . 2.8 Statistical Analysis The MIXED procedure of SAS V-9 (Statistical Analysis Systems, Cary, NC, USA) was used for statistical evaluation of the results. The effects of ozonation, chitosan bio-coating, storage time, and their interactions were analyzed using the Tukey test based on p < 0.05. 3 Results and Discussion The weight loss The fresh, ripe Aronia melanocarpa contains high amounts of water (74 to 83%) 25 . Consequently, postharvest water loss, which happens naturally in fresh aronia, leads to cell shrinkage, withering, and shriveling through storage. Even at 3–10% water loss can adversely affect the texture of the fruits and make them unmarketable and consumer acceptance. This is mainly caused by the berry's susceptibility to water loss, which alters its physical characteristics that lead to wilting, shrinkage, and reduced firmness. Control of the moisture loss rate is necessary to preserve the desired texture and also the appearance of the fresh aronia berries. According to Konishi, et al. 26 the cuticle, calyx, and stomata are the pathways of water loss in fruit. A role in berry water loss is thought to be played by the cuticular membrane covering the fruit surface. All aronia groups showed weight loss during storage (Fig. 1 ). The weight loss in fresh fruits of 3–10% is the most important sign of loss of freshness. The weight losses were 1.2% in the control group, 0.8% in ozone-applied samples, 0.72% in chitosan-coated groups, and 0.67% in chitosan-coated groups after ozonation at the end of the storage. The highest significant weight loss was measured in the untreated ones (1.2%) at the end of the storage (p < 0.05). Implementing chitosan coatings can significantly reduce the weight losses of fresh aronia. Fruits such as the mango 27 , kiwi 28 , grape berry 29 , strawberry (0, 0.3, and 1.0 ppm for 24 h) 30 Pomegranate 31 , raspberry 32 , blackberry fruit 33 and physalis angulata L. berries 34 have shown that chitosan biocoatings lower the rate of stomata's transpiration, leading to lower weight loss, which is the main advantage of edible coatings. The lower reduction in the weight loss value of chitosan-coated aronia is most likely because of the impact of chitosan biocoating layers on the fruit acting as a semi-permeable barrier to gases and moisture, thus minimizing the water loss, respiration rate, and microbial infection 35 . The biocoatings act as a modified atmosphere, which delays the aging of aronia more effectively by controlling metabolic processes 36 . If coating is applied properly can help maintain optimum moisture levels around the fresh aronia fruits and prevent excessive water loss by creating a protective layer on the aronia surfaces and controlling the mass transfer of gases. Ozone-treated fruits had consistently lower respiration rates in comparison with control ones at the same storage time. The ozone on the postharvest life of fresh fruits includes its capability to minimize postharvest deterioration and oxidize ethylene, thereby minimizing the metabolic process and preventing deterioration of fresh produce quality during storage 37 , 38 . After chitosan coatings or ozone application, the water evaporation in fresh aronia could be remarkably prevented, as could the moisture loss. Research has published that ozonation effectively closes stomata, retards cell wall metabolism, and delays cell wall degradation that maintains the stability of fresh fruits during storage. Ozone impact on the control of the activities of the enzymes reduced the activities of peroxidase (POD) and polyphenol oxidase (PPO ) 39 . This impact can be connected to the fact that ozonation suppressed the fresh fruit transpiration, which led to a lower moisture loss. 30 . Appropriate amounts of ozonation efficaciously retard the minimizing of aronia water loss during storage. Similar publications have highlighted that ozonation for kiwi (45 mg/m 3 every 12 h) 39 , fresh garlic (5 ppm at 4°C) 40 , fresh-cut pitaya (2, 4, and 6 mg L – 1 20 min 7 , strawberry (0, 0.3, and 1.0 ppm) 30 for 24 h, table grapes [(0.8–1.2 µl/L continuous)] 41 , red peppers [0, 7 ppm ozonation 1, 3 and 5 min] 42 , Andean blackberries (0.4, 0.5, 0.6, and 0.7 ppm, for 3 min.) 9 , and blueberries (4 ppm or 2.5 ppm ozonataion) 43 . According to Contigiani, et al. 44 the weight loss of strawberries after ozonation (3.5 mg L − 1 ; 5–15 min) were dose-dependent. Ozonization (5 min) significantly minimized weight loss, but weight loss in ozonated strawberries (15 min.) was higher than in unozonated ones. An appropriate level of ozonation has the positive effect of suppressing weight losses 40 . The minimized weight loss in the ozonated fresh aronia could be connected to its proportionally thicker cuticle compared with other fresh fruits such as tomatoes, cranberries, and strawberries. In this study, with a weight loss of 1.2%, which is much lower than the 3–10%, all aronia sets would still have been marketable during the extended storage periods. Headspace Gases Fresh fruits respire to carry out their metabolic activities using O 2 producing CO 2 after harvest. Respiratory rate is the main index of the metabolic rate of fresh produce and therefore of their post-harvest shelf life 9 . In a trays packaging, the concentration of the gases (O 2 /CO 2 ) in the headspace changes to create an EMAP during storage (Fig. 2 a and 2 b). When the O 2 level in the trays decreased, rates of respiration slowed down. At the same time, as CO 2 level rise, they lead to minimizing not only the respiration rate and C₂H₂ production but also suppressed inhibition of mold/bacteria growth 17 , 45 It was found that while the O 2 level in the headspace of the trays increased, the CO 2 level also increased, and the equilibrium O 2 level stabilized between 15.5–16.7% for the ozone-treated groups. There were no significant differences between treatments (p > 0.05). Similar results were published for low-dose ozone-treated melons, mulberries 46 , and strawberries treated 47 . It was observed that O 2 levels reduced and that aronia maintained their stability for an extended period of storage (Fig. 2 a). All groups showed a similar pattern, i.e. a continuous and steady reduction in the O 2 concentration and an increase in the CO 2 concentration during storage (Fig. 2 b). It was clearly highlighted that the untreated trays maintained the highest respiration rate throughout the storage period. It has been analyzed that ozone is potential in lowering the rate of respiratory and helps to create EMAP in trays. It was noticed that the highest CO₂ concentration occurred in the untreated groups, while the least CO 2 level was achieved in the control ones (Fig. 2 b). This gas level is highly related to the rate of aronia respiration during storage. The trays with ozone-treated groups lower in the O 2 levels and increased in CO 2 levels during storage. EMAP for the aronia was more successful when ozone was used (Fig. 2 a and 2 b), the rates of respiration delay created extended storage stability. Decreased O 2 and increased CO₂ levels slow down the rate of aronia respiration more than control and also result in lower enzymatic activity, which affects less utilization of substrates and extends the storage periods 48 . In this research, the O 2 level was effectively lowered to 17.1% and CO 2 concentrations up to about 4.2% in the untreated one at the end of the storage. Ozone treatments also decreased the rates of respiration, causing lower O 2 utilization and CO 2 . The similar inhibitive impacts of ozonation on rates of respiration during storage of Andean blackberries, melons, and mulberries have been reported by Horvitz, et al. 9 , Chen, et al. 49 and Han, et al. 46 This can be the results of ozone effectively suppressing the microbial respiration process, thus minimizing the respiration rate of the fruit to a certain extent. This was agreed with the ozonation obstacle biosynthesis of ethylene by blocking Acetyl-CoA carboxylase ( ACC ) synthesis 50 . Brix The key parts of solids soluble in fruit juice are sugars that were shown on the °Bx scale. The effective retention of soluble solid content (SSC) is crucial for keeping fresh fruit well-stored and edible. The water-SSC of the aronia increased during storage in all groups due to continuous breathing. The SSC in the ozonated samples was relatively gentle, whereas the SSC in the ozonated group was better maintained than in the untreated one. The SSC of the aronia gradually increased for all treatments (18.3 to 19.5–20.3° Brix) at the end of storage (Fig. 3 ). The storage time has a significant effect on Brix values (p < 0.05) and with an increase in storage, it can be seen that the amount of soluble solids in the control is lower compared to the ozonated aronia. The untreated exhibited the highest in SSCx (18.3), while the 19.7 (O 3 ), 19.8 (coated), and 19.5 (ozonation with coated) were at the end of storage. It was similarly reported that ozonation postpones the reducing of SSC in fresh aronia. The increase in SSC of all samples during storage can be explained by the decrease in water level and the conversion of complex carbohydrates into monosaccharides 51 . It is related to the fact that ozonation slows down carbohydrate metabolism 52 Ozonization and storage time of fresh aronia caused significant changes in SSC. It is highlighted that the fresh aronia applied with ozone has a slower decrement in SSC. This can be as the chemical impact of ozone slows down enzyme activities related to carbohydrate metabolism, while maintaining a high level of soluble solute. Loss of acidity due to aging is another reason for the decline in SSC 53 . The results highlighted that ozone treatment was efficacious in delaying the increase in SSC content. Thus, the quality of the fresh berry was preserved at a later stage of storage. pH value Both the pH level of the control and the ozone-treated aronia are slightly increased over the storage period due to respiration metabolism that is related to the use of the organic acid through the TCA pathway. This increase is due to the degradation of organic acids during the storage periods 54 . Ozone and EMAP create a MAP that avoids the quick decline of organic acid by minimizing the respiration rate 51 . The influence of ozone and coating variety on pH over the storage period (p < 0.05). While the pH of fresh aronia on the initial day was 3.56 in the untreated one, it increased to 3.93 at the end of 4 months; in aronia with ozonation, it was noticed that these changes progressed much more slowly after 2 months and were limited to 3.81 at the end of 4 months. The coated ones slowed down these changes significantly in pH values compared with the control (3.83), ozonation after coatings (3.82) and untreated groups (3.93). Nevertheless, the highest pH during the entire storage period was found in the untreated samples. The higher respiration rates resulted in the higher utilization of organic acids and accumulation of higher CO₂ concentrations inside the trays 55 . Regarding ozonation or coatings applied significantly slow down the respiration and also the pH of aronia when compared to the control groups. By slowing down the rate of respiration, the modified atmosphere created by the coatings also prevents high levels of organic acid consumption. The ozonation suppressed the respiration and slowed down the rate of the respirations. The lower pH was observed in the ozonated samples, where O 2 /CO 2 is in equilibrium, due to the reduction in respiration rates compared to untreated samples, as a result of the slower increase in pH. During the 12-day study, the chitosan coating did not affect the pH of fresh strawberries. This is because chitosan coatings and ozonation create a modified atmosphere that suppresses organic acid decomposition by suppressing the respiration rate 16 , 56 – 58 . The higher respiration rates resulted in the higher utilization of organic acids and accumulation of higher CO₂ concentrations inside the trays 55 . The higher CO 2 percentage in the packaging may cause CO 2 to dissolve faster, which may increase the amount of HCO 3 56 . Regarding ozonation and chitosan coatings significantly slow down the respiration rates and also the pH of aronia (Fig. 4 ) when compared to the control. These results are in agreement with those published by Caner, et al. 17 , who published that strawberries’ pH rises and depends on different EMAPs used during storage. Color For the consumer, color is a significant criterion in determining acceptance or rejection of the fruit, as evidenced by the case of fresh berries 17 . The loss of fruit surface color during storage can be attributed to two factors: firstly, an increased respiration rate, and secondly, the processes of browning and the loss of labile anthocyanin pigment (red) 59 . The color of aronia depends on their content of natural predominant pigments, mainly anthocyanins, where cyanidin glycoside (98.4%) present in aronia 60 . Our results show that the L* value was slightly increased over the storage of the aronia berries, from initial days (23) to (24) at 4 months of storage; nevertheless, the increase was not significant in either the control or ozone-treated samples during the storage period (Fig. 5 a). The L* value of the ozonated aronia increased over the storage and was always higher than the unozonated aronia fruit. Aronia exhibited a slight increase in the L* values over storage. This could be caused by inactivating the enzymes involved in browning 61 . Notably, the L parameter of fresh produce can be affected by surface damage, the enzymatic action of polyphenoloxidase on phenolic substrates, and the structure of cell wall macromolecules 62 . This is agreed with Tiwari, et al. 63 report that ozonated tomatoes and ozone-treated strawberries (1.0 ppm and 1.5 ppm) exhibited higher L* values than the untreated groups over storage 61 . It has been established that storage periods do not exert a significant effect on color change (L* value). However, no significant difference (p > 0.05) was observed between the ozonated samples in terms of brightness (L* value). The red color intensities (a* values) in aronia were more intense when aronia were coated. The a* values in aronia indicating the red color intensities were more intense compared with control groups (Fig. 5 b). The a* values ​​of ozonated and chitosan coating groups to fresh aronia exhibited higher values ​​compared to the control throughout the storage (p < 0.05). It was determined that a* values ​​were significantly higher as a result of the combined application of ozone and chitosan coating (p < 0.05). In fact, at the end of storage, the a* value of ozonated after chitosan coatings groups had significantly the highest value (1.58) compared to chitosan coatings (1.26) groups, but not ozonated (1.41) and all control groups were found to be significantly lower compared to treated groups (p < 0.05) (Fig. 5 b and 5 c). These results are similar to the previous research conducted by Panou, et al. 61 . The coatings and ozonation delay the deterioration of anthocyanin pigments 61 . Ozonation played a significant role in the brightness of the appearance of aronia and reducing the change of bright appearance, highlighting that ozonation could retard and coatings enhance the surface of aronia and consequently maintain higher commercial appearance. Texture Profile Analysis Hardness; During storage periods, fresh aronia undergoes cell wall modification; furthermore, the number of cells between adhesions decreases, leading to increased softening, which involves extensive depolymerization of pectin and partial hemicellulose, dissolution of pectin, and loss of neutral sugars in the pectin side chain 58 . The fresh aronia undergoes a softening process in berries that is related to the following activities of fruit-softening enzymes, including pectin methyl esterase (PME), polygalacturonase (PG), and pectate lyase (PL); and the accumulation of reactive oxygen species (ROS) 5 . The process of pectate lyase (PG) activity involves the cleavage of pectin acid, which in turn results in the disassembly of the components of cell walls and a concomitant reduction in firmness. 64 . During the storage periods, all groups exhibited decreased firmness. While the chitosan-coated after ozonation groups had the greatest hardness value than the other groups, and all treated groups had significantly greater values than the control groups (p < 0.05). The application of edible coatings has been demonstrated to create a tiny surface barrier layer around the berries, thereby preventing moisture evaporation and delaying berry softening. The hardness decreased continuously as a function of storage periods, from 530 gf on the first day to 380–470 gf after 4 months of storage (Fig. 6 ). This value was observed to decrease the most in the control group at 380 gf. In the 4th month, the best values ​​were obtained in the chitosan coating and also in the combination of ozone and chitosan coatings with a decrease of 13.5–18.7% at 453–470 gf, while the highest percentage decrease of 28% was observed in the control group (380 gf) compared to initial values. The findings revealed that there existed a considerable discrepancy between the treatments and the control group following a period of four months of storage (p < 0.05). It was determined that the decrease in hardness was delayed in the products coated with chitosan and ozone (p < 0.05). Therefore, it was determined that the combination of ozonation and chitosan coatings optimum treatment for enhancing the firmness values of fresh aronia during storage. The combination of chitosan coating and ozonation has the capacity to diminish the activity of cell-wall decomposition enzymes. This process is conducive to safeguarding the integrity of the cell wall, impeding berry softening, and prolonging the storage stability of the berry. It was demonstrated that the application of a coating of the polysaccharide known as 'chitosan' resulted in a significant enhancement of the mechanical strength of the cell-wall structure and concomitantly delayed the process of loss of firmness. As a result, it was observed that the softening of aronia berries was slowed down and their storage stability was extended. The effectiveness of the process in maintaining the integrity of the material is partly related to the reduction of respiration rates caused by inhibition of mass transfer by the polysaccharide. It has been hypothesized that delays in softening may be attributable to ozonation reducing physiological degradation mechanisms. Furthermore, ozonation has been found to reduce water loss from berries 17 , 65 . Ozonation suppressed the decomposition of cell-wall polysaccharides by inhibiting the activity of enzymes (PG, PME) related to cell-wall modification. So ozonation significantly maintains the cell wall structure and ultimately maintains the firmness of aronia due to its maintaining both propectin and cellulose content compared to control groups. Therefore, ozonation is a powerful technique to regulate cell-wall metabolism, postphone cell-wall degradation, and extend the storage stability of aronia throughout storage 39 . It is evident that ozone has an effect on the breakdown of ethylene molecules, thus slowing the rate of pectin degradation and reducing the respiration rate of fresh aronia. These effects contribute to a delay in the softening of the fruit during storage 61 , 66 . In agreement with our results that the hardness was maintained after ozonation (0.075 ppm, 0.15 ppm, 0.25 ppm) compared to the control during storage 17 , 55 . Pectin and hemicellulose degradation of cell walls, changes in headspace composition, and more importantly, polygalacturonase (PG) and pectinmethylesterase (PME) activities may be limited by the CO 2 levels in the packaging 67 . No significant differences were observed on day one of the storage experiment between the treated subjects and the control sample. The higher hardness values were recorded for coated, after- ozonated aronia samples when compared to the control over the storage periods. On the second month of storage, significant differences were recorded between the treated with ozone or coated aronia and the hardness of the control ones. In control ones, a decrease in hardness was observed in the range of 28% (Fig. 6 ). In ozonated aronias, chitosan-coated or ozonation after chitosan, this rate decreased to around 18.7%, 15.5%, or 13.5% when compared to initial days of storage (p < 0.05). Consistently decreased in hardness as a function of storage times, from the initial day 530 gf to 380–469 gf at end of the storage. These values decreased the most to 380 gf in the control and 423 gf O₃, 453 gf coated samples, and 470 gf ozonation after coating at the end of the storage. The highest hardness values (470 g) were measured at 470 gf with a decrease of 13.5% in the coating after ozonation, the lowest percentage of reduction at initial hardness (P < 0.05). Therefore, it was determined that the combination of ozone and chitosan application in maintaining the hardness values of fresh aronia throughout storage. The maintaining of hardness can be related to the minimizing of physiological degradation mechanicms by ozonation and prevents water loss of fresh berries 17 , 23 . The outcomes highlighted a strong and negative correlation between water loss during storage and hardness levels 65 . The weakening of the cell wall, in conjunction with an increase in cell separation due to a reduction in turgor pressure within the cells during storage, has been demonstrated to result in economic loss 68 . It is evident that the presence of ozone and chitosan coatings contributes to the maintenance of cell wall strength, cell-to-cell adhesion, turgor, and decreased respiration rates in fresh Aronia, resulting in enhanced performance 16 . It is hypothesized that the levels of carbon dioxide achieved in the trays, as well as the headspace composition, could limit the degradation of cell walls by pectin and hemicellulose, and could inhibit pectin methylesterase (PME) and polygalacturonase (PG) activities 69 . In agreement with our results that Caner, et al. 17 . the hardness was maintained after ozonation (0.075 ppm, 0.15 ppm, 0.25 ppm) compared to control during storage 55 . As a result, berry softening is delayed and fresh aronia quality is maintained. This is associated, at least in part, with the use of a coating to create EMAP and ozonation, with a view to reducing respiration rates. The adhesiveness , no statistically different results were obtained between the groups during storage (P > 0.05). In contrast to the control group, the ozone treatment and the coated treatment exhibited adhesiveness maintenance. The springiness significantly decreased (0.097 to 0.07–0.081), especially the lowest in the untreated ones (0.07), while ozonated (0.0078), coated (0.008) and ozonation after coatings (0.081) at the end of storage. Nevertheless, no substantial disparities were observed between the ozonated and coated groups at the end of the storage. The results of the study demonstrated that the treatments had a significant impact on the cohesiveness. A steady decline in cohesion was measured during the storage period due to decomposition of the cell wall and middle lamella pectin 70 . The untreated group had the highest decrements (from 0.07 to 0.053), while ozone (0.07 to 0.057), chitosan (0.07 to 0.059), ozonation after coatings (0.07 to 0.06) had lower decrements. Among those treated, there were no significant changes in cohesion. The decrease in the cohesion value may have developed due to the tissue softening that developed due to the dissolution of the pectin-containing material in the middle lamellae of the adjacent cells of the aronia fruit 4 . A steady decline in cohesiveness was measured through the storage due to decomposition of the cell wall and middle lamella pectin 70 . There were no significant changes in the cohesiveness of the treatment group. In the case of gumminess (N) values ​​​​decreased in all groups during the storage. The least decrease was measured in the fruit group coated after ozone application (11.34% compared to the first day), and the most decrease was measured in the control group (26.68% compared to the first day). This decrease is more pronounced in the control group, decreasing from 33 to 24.1 in the control group, 27.8 in the ozonated group, 28.8 in the chitosan-coated group, and 29.4 in the ozonation after coatings at end of the storage due to the softening resulting in a softer and more elastic aronia texture. The hydrolysis of starch is the cause of lower gumminess 23 . Since gumminess is obtained by multiplying hardness and internal adhesiveness, gumminess values ​​​​were parallel to the change in the product of these two values 23 . In addition, it was seen that after 120 days in gumminess values, only ozone-applied samples, only chitosan-coated samples, and chitosan-coated samples after ozone application were not statistically different from each other (p > 0.05). The chewiness values decreased in all groups during storage. The decrease in chewiness is more pronounced than in cohesiveness. There was a reduction in chewiness as a result of the reduction in hardness. Ozonation lowers the decreasing rate that helps to limit the activity of cellulolytic and pectic enzymes. Chewiness decreased from 3.15 to 2.76 (control), 2.91 in ozonated ones, 2.96 in chitosan-coated ones, and 3.01 in ozonated ones after chitosan coatings, with 12,3%, 2,91%, 2,96% and also 3,10%, respectively, at the end of storage. In addition, while only ozone-treated samples and only chitosan-coated samples were not statistically different from each other after 120 days in terms of chewability, the chewiness value in chitosan-coated samples after ozonation was measured higher than these two groups. Therefore, it can be concluded that ozone and coating application slow down the enzymatic reactions in the fruit. The minimal decrease in TPA values in chitosan-coated samples after ozone enables the determination of the synergistic effect of ozone and coating application. Possibly, ozonation and chitosan coating helped maintain fresh aronia textural parameters, especially hardness, which highlights changes in the structure of the cell wall. There was a high correlation between the TPA parameters of fresh aronia and the changes in its weight loss, the Brix value, and the pH value. Total monomeric anthocyanin Aronia is the richest natural source of polyphenols, including anthocyanins, proanthocyanidins, flavonols, flavanols, and phenolic acids compared to other fruits. Aronia contains more anthocyanins than blueberries (about twice as many), açaí (about four times) and goji (350 times) 71 . There have been reports of high levels of loss and changes in chemical structure, as anthocyanins are known to be unstable compounds 72 . These substances are responsible for the high bioactivity of aronia, and the main polyphenolic substances of aronia have a wide range of health benefits. Approximately 25% of the total polyphenols in aronia are anthocyanins. They are mainly four different cyanidin glycosides, primarily cyanidin-3-galactoside (68.9%) and cyanidin-3-arabinoside (27.5%) 73 . When the initial day storage, it was observed that the total content of initial monomeric anthocyanins can vary between 2773 (in control), 2792 (coated), 2865 (ozonated), and 2885 (ozonation after coatings) mg/100 g in initial days (Fig. 7 ). These high values ​​were not significantly different from the others due to great varations (p < 0.05). The significant enhancements were measured in anthocyanin contentsat end of the storage (p < 0.05). It is clearly seen that the ozonation at the beginning of storage and also at the end of the storage, in terms of increasing anthocyanin extraction, has a positive impact towards extracting them at higher levels compared to the unozonated groups. At the end of storage, the amount of anthocyanin was 2780 in the control, while the significantly highest amounts (3315 in ozonated and 3326 in ozonation after coatings) were detected in the ozonated samples (p < 0.05). Ozonation and ozonation after coatings gave more successful extraction results than the other groups during the storage (p < 0.05). In fact, it has been published that ozonation stimulates the biosynthesis of phenolic substances such as anthocyanins in fresh fruits. Admane, et al. 74 . Ozonation after coatings exhibited the best extractability performance during storage based on the bioactive compounds, including anthocyanins 4 . Similarly, Pinto, et al. 75 published that the combination of ozonation and MAP significantly maintained anthocyanin contents in blueberries. In the study conducted by Piljac-Žegarac and Šamec 76 on the effect of post-harvest storage temperature on the content of the cherry, sour cherry, and strawberry fruit samples, an increase in the amount of anthocyanin was observed in those stored at + 4°C. Similarly, the anthocyanin content in the berry skin of O 3 -treated grapes (5, 10, 20 µL L-1 for 30 min) was significantly higher than in the control 74 . O 3 contact leads to changes in grape secondary metabolism, improving the synthesis of phenolics, including stilbenes and anthocyanins. Furthermore, ozonation enhanced the extractability in ozonated grapes 77 . It has been confirmed that the amount of bioactive compounds such as anthocyanins can be efficiently increased in a couple of days between the ages of the leaves, depending on the concentration of ozone (100 ppb; 16 h and 200 ppb; 24 h) 78 . O 3 applications are often reported to enhance bioactive substances, inluding polyphenols and antioxidant volatiles. The literature suggests that low concentrations and short treatments give the best results in most cases. Grapes treated with O 3 at the right dose and for the right time promote the biosynthesis of metabolites. On the other hand, it modifies the permeability of the skin, resulting in a greater extraction of polyphenols. The berries' response to moderate stress, which induces the biosynthesis of polyphenols, is probably responsible for the increase in anthocyanins 77 . Therefore, ozonation affects the structure of the cell wall and the composition of the cell membrane, in addition to inducing the biosynthesis of phenolic compounds, and facilitates the extraction of these substances. It has been shown that ozonation can cause increased anthocyanin amounts by enhancing the phenylpropanoid biosynthesis pathway in strawberries 79 . Total phenolics Phenolic compounds, or polyphenols are among the most common compounds of secondary metabolism in various plants (up to 200–300 mg per 100 g). Phenolic compounds affect odor and taste formation and can inhibit enzymes associated with the development of human diseases. An increase in total phenol content was observed on the 15th day and a decrease on the other days. The highest reduction of total phenol content was in the untreated groups, from 838 to 610 (∼28% loss), while the significantly highest amounts (660 for O 3 , 637 for chitosan, and 656 for coatings after ozonation) were measured (p < 0.05) (Fig. 8 ). These results highlighted that the ozonations maintained stability of the total phenol amount in comparison with the unozonated ones. In response to ozone treatment, flavonoid biosynthesis and phenylalanine metabolism were the main targets of the regulatory effect of ozone on phenolic compounds 7 . Oxidizing enzymes are inactivated by ozone. This slows or stops the decomposition of phenolic compounds 80 . This may be due to the oxidation-reduction potential of ozone application to fruits, which increases intracellular reactive oxygen species responsible for the harmful effect on nucleic acids, paving the way for damage to bacterial cell walls. In addition, another reason for the increase seen on the 15th day may be due to the phenylalanine ammonia-lyase enzyme, which is activated by cold heat stress experienced in the fruit at refrigerator temperature. Recent publications have underlined that appropriate ozonation can significantly enhance peroxidase activity, maintain high levels of total phenolics and flavonoids, and improve the antioxidant capacity of berry fruits. This result is similar to the study conducted by on aronia fruits stored at + 4°C and − 20°C and by Chen, et al. 81 on strawberry samples treated with ozone at different concentrations (1, 3, and 5 ppm for 10 hours). The increase in polyphenol content can be explained mainly by the release of polyphenols from the cell structure due to cellular degradation and increased extractability. The decrease observed on the following days indicates that polyphenols released outside the cell are subject to faster degradation. Among the different O 3 concentrations applied (1,5 and 9 µL/L and time intervals of 1, 12, 24, and 48 h), 5 µL/L applied for 48 h was found to be optimal for the induction of bioactive compounds such as total phenolics, flavonoids, water-soluble polysaccharides, and ganoderic acids. A twofold increase was observed 82 . Compared to uncoated fruits, coated fruits showed a slower rate of decline in phenolic content, most likely due to reduced oxygen permeability and hence reduced enzyme activity 83 . For apples coated with CH-OLE 2%, the lowest decrease in total phenolic content was 1.24 mg GAE g-1. Conversely, fruits that had not been coated exhibited the most substantial decreases in total phenolic content, reaching 0.28 mg GAE g-1 by the conclusion of the storage period. The decline in phenolics could be attributable to the reduction in sugar levels. This is because sugar is known to be a stimulant for the metabolic pathways involved in phenol production 84 . During storage, the aging phenomenon, degradation of pectic substances in the cell wall, and microbial infection lead to the breakdown of intracellular substances, oxygenation, and disruption of the integrity of the cell membrane. This raises the enzyme activity of polyphenol oxidase (PPO), which is in charge of the phenol oxidation. Earlier research has shown that chitosan decreases PPO activity and raises the levels of phytochemicals in strawberries. However, the decrease in phenolics during long-term storage can be explained by cell wall degradation and exposure of phenolics to enzymatic oxidation 85 . Visual evaluation. Fresh aronia is a perishable fruit, and its storage stability is often adversely affected by molds. Out of these, Penicillium sp., Alternaria sp. and Bothrytis sp. are the main spoilage-causing agents in fresh aronia fruits 86 . These pathogens can infect fresh berries after harvest, causing rot and spoilage, and can cause significant deterioration during cold storage 58 . When the total number of rotten and moldy fresh aronia berries in the trays is counted during storage. No mold growth was observed in the chitosan-coated after-ozonated berry groups. There was only 1/1 rotten/mold growth observed in the chitosan-coated and also ozonated groups at the end of the storage. The highest rotten/mold growth was observed in 2 months (1/1), (2/4) 3 months, and (4/5) at the end of the storage for the untreated groups (Table 1 ). It was clearly observed that with the combination of the chitosan and ozonation, any fungal and rotton decay were detected during storage. On fresh aronia, the combination of the chitosan and ozonation had a major effect on molds and decays, whereas the use of individual chitosan coatings or ozonation does significantly affect suppression on fresh aronia samples (Fig. 9 ). Table 1 Effect of ozonation and chitosan coatings on rotten/moldy percentage of aronia in package (4 months storage) Treatments * Day 1 Day 15 Month 1 Month 2 Month 3 Month 4 Control 0/0 0/0 0/0 1/1 2/4 4/5 Ozonation 0/0 0/0 0/0 0/0 0/0 1/1 Chitosan coating 0/0 0/0 0/0 0/0 0/0 1/1 Ozonation & chitosan coating 0/0 0/0 0/0 0/0 0/0 0/0 Similar results were published that Bessi et al. (2014) underlined that ozonation can effectively suppress the development of mold and bacteria on the surface of the citrus. Likewise, the number of viable yeast and molds in uncoated arils was more than in coated arils 31 . Since ozone and chitosan, due to cationic amino groups, have broad-spectrum antimicrobial properties 58 . The microbial load increased in coated strawberries when stored. However, the density of microbial cells was lower than that found in untreated strawberries 87 . In the study conducted by Pinto, et al. 75 it was reported that red raspberry fruits exposed to gaseous ozone (6 ppm) for 16 hours after packaging in PET (polyethylene terephthalate) trays reduced mold growth by 1.37 log CFU/g and provided better visual quality for 15 days. In addition, another study found that chitosan coating suppressed the growth of Rhizopus sp . and B. cinerea on fresh raspberry fruits during cold storage 5 . Lemic, et al. 88 published the inhibitory effect of ozonation on mold growth in citrus, while Gorzelany, et al. 80 underlined that the ozone applications had a suppressed impact in lowering the yeast, and molds on the saskatoon berries. Contigiani, et al. 44 highlighted that ozone treatments (3.5 mg/L for 5 − 15 min) fresh strawberries had a significant reduction in yeast or mold incidence compared to untreated berries. The most common moulds in fresh aronia berries that cause significant deterioration were Penicillium sp., Alternaria sp. and Bothrytis sp. during storage. Fresh aronia is a perishable fruit, and its storage stability is often adversely affected by molds . Out of these, Penicillium sp., Alternaria sp. and Bothrytis sp. are the main spoilage causing agents in fresh aronia fruits 86 . These pathogens can infect fresh berries after harvest, causing rot and spoilage, and can significant significant deterioration during cold storage 58 . When the total number of rotten and moldy fresh aronia berries in the trays is counted during storage. No mold growth was observed in the chitosan coated after ozonated berries groups. There were only 1/1 rotton/mold growth observed in the chitosan coated and also ozonated groups at end of the storage. The highest rotton/mold growth were observed in 2 mounts (1/1), (2/4) third mounts and (4/5) at end of the storage for the untreated groups (Table 1 ). It was clearly observed that the combination of the chitosan and ozonation, any fungal and rotton decay were detected during storage. On fresh aronia, when combination of the chitosan and ozonation was applied had a major effect on molds and decays, whereas the use of individual chitosan coatings or ozonation does affect significantly suppressed on fresh aronia samples. Similar results were published that Bessi et al. (2014) underlined that ozonation can efficently supresed the development of mold and bacteria on surface of the citrus. Similarly, the number of viable yeasts and moulds was higher in the uncoated arils than in the coated arils 31 . Since ozone and chitosan due to cationic amino groups have broad-spectrum antimicrobial properties 58 . The microbial populations grew during storage in coated strawberries. However, the microbial cell densities were lower than in untreated strawberries 87 . In the study conducted by Pinto, et al. 75 , it was reported that red raspberry fruits exposed to gaseous ozone (6 ppm) for 16 hours after packaging in PET (polyethylene terephthalate) trays reduced mold growth by 1.37 log CFU/g and provided better visual quality for 15 days. In addition, another study found that chitosan coating suppressed the growth of Rhizopus sp. and B. cinerea on fresh raspberry fruits during cold storage 89 . Lemic, et al. 88 published the inhibitory effect of ozonation on mold growth in citrus, while Gorzelany, et al. 80 underlined that the ozone applications had a suppresed impact in lowering the yeast, and molds on the saskatoon berries. Contigiani, et al. 44 higligted that ozona treatments (3.5 mg/L for 5 − 15min) fresh strawberries had a significant reductions in yeast or mold incidence compared to untreated berries. 4. Conclusion Our results underlined that, ozonation and chitosan coating treatments individually alone significantly improved the storability of fresh aronia. The combination of chitosan biocoating after ozonation has a significant effect on slowing down the softening and weight loss of aronia, and the effect improved its fresh aronia firmness properties during storage. This could be due to a semi-permeable barrier in the chitosan coating that prevents water vapor and gases from entering the coated berries, thereby reducing transpiration. The obtained allowed a proper mass transfer exchange suitable for fresh berries and normal physiological weight loss, thus delaying degradation and extending storage stability. Coated and ozonated aronia showed lower fungal development and less decay than untreated ones while retaining better color in the treated samples. Ozonation increased the bioactive compounds, mainly anthocyanins and polyphenols. O 3 application is very feasible. It is easy to incorporate into the chitosan coatings and can be used not only for extending storage stability, but also for promoting the health-related substance of fresh aronia, helping to improve their health-related quality of life. It was observed that the surface color of fresh aronia (L* and a) was improved. Furthermore, it has been demonstrated that post-harvest exposure to aronia can result in a substantial reduction in the utilization of ozonation and chitosan coatings, attributable to the latter's inherent antimicrobial characteristics. At the end of this study, it was found that the shelf life was one month longer with the synergistic effect of chitosan coatings after ozonation compared to the control groups. Hence, the notable synergistic effects carried out by the best performance in preserving fresh aronia by the combined applications of ozonation and chitosan coatings make it a versatile, sustainable secure substitute for artificial preservatives within the food industry. It is imperative that fresh fruits and vegetables be coated with a preservative solution following the ozonation process, prior to their being packaged, in order to prevent the growth of microbes. In light of the growing demand from consumers for safe, organic produce, the utilization of ozone and chitosan coatings has emerged as a sustainable alternative to artificially produced preservatives. These coatings have been demonstrated to provide a viable and sustainable approach to enhancing the storage stability of food products. In view of the fact that severe oxidative stress has been identified as a feature of metabolic syndrome, polyphenol-rich extracts have the potential to be of particular use to patients suffering from the condition. In this study, the authors posit that the polyphenolic extract of fresh berries could be of interest for use in the formulation of dietary supplements and advocate for further study in this area. Researchers have dealt with this drawback by increasing chitosan coatings using neutral lipids and/or nanoclays and blending with proteins and/or polysaccharides. We further investigated the impact on sensory qualities as consumer acceptance of ozone and chitosan coatings in commercial scenarios. Declarations Data Availability Data is available on request from the authors. Ethical Statement Ethics approval was not required for this research. This study does not involve any human or animal testing. Conflict of Interest The authors declare no conflict of interest. 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C. (1998). Antifungal effects of chitosan coating on fresh strawberries and raspberries during storage. The Journal of Horticultural Science and Biotechnology , 73 , 763–767. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6862137","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":473098209,"identity":"9a255297-f653-4b27-b2e4-7715584346a9","order_by":0,"name":"Nur Ebru Güleryüz Ok","email":"","orcid":"","institution":"Çanakkale Onsekiz Mart University","correspondingAuthor":false,"prefix":"","firstName":"Nur","middleName":"Ebru Güleryüz","lastName":"Ok","suffix":""},{"id":473098210,"identity":"0e479b27-bd43-4e94-b7dc-a89c721a49b1","order_by":1,"name":"Muhammed Yüceer","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYBADOSjNTLwWY4QWNiK1JDYQrUV3Ro7hhw8V99L727vTJBgqrBMb5Hsf4NVidiPHWHLGmeLcGWfObpNgOJOe2MDGbkBIixkzb1tC7gaJ3G0SjG2HgVoIuAys5e+/hHQDsJZ/xGphbEhIgGhpIEbLmWfFkj3HEgyBftlskXAs3biNLY2AluPJGz/8qEmQ52/v3XjjQ421bD/zMfxaGAQSkDggNuGY5D9AUMkoGAWjYBSMdAAA/JpCAkJ9rEsAAAAASUVORK5CYII=","orcid":"","institution":"Çanakkale Onsekiz Mart University","correspondingAuthor":true,"prefix":"","firstName":"Muhammed","middleName":"","lastName":"Yüceer","suffix":""},{"id":473098211,"identity":"ceb293f7-b51c-46c6-b5ca-be54028e570d","order_by":2,"name":"Çiğdem Uysal Pala","email":"","orcid":"","institution":"Çanakkale Onsekiz Mart University","correspondingAuthor":false,"prefix":"","firstName":"Çiğdem","middleName":"Uysal","lastName":"Pala","suffix":""},{"id":473098212,"identity":"08d26592-03fc-44d9-82e2-63a2077cdffe","order_by":3,"name":"Cengiz Caner","email":"","orcid":"","institution":"Çanakkale Onsekiz Mart University","correspondingAuthor":false,"prefix":"","firstName":"Cengiz","middleName":"","lastName":"Caner","suffix":""}],"badges":[],"createdAt":"2025-06-10 10:38:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6862137/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6862137/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85044117,"identity":"576840e5-43aa-4a8c-b51a-1735db123007","added_by":"auto","created_at":"2025-06-20 09:59:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":52184,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ozonation and chitosan coatings on weight loss values (%) of Aronia (4 months storage).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6862137/v1/3a6a6fc7bb5e9675b2eaac96.png"},{"id":85045471,"identity":"2a2fff35-7a0d-4d3f-8924-2940ece481b7","added_by":"auto","created_at":"2025-06-20 10:15:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":40311,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea.\u0026nbsp; \u003c/strong\u003eEffect of ozonation and chitosan coatings on O\u003csub\u003e2\u003c/sub\u003e gas concentration values (%) of Aronia (4 months storage).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u0026nbsp; \u003c/strong\u003eEffect of ozonation and chitosan coatings on CO\u003csub\u003e2\u003c/sub\u003e gas concentration values (%) of Aronia (4 months storage).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6862137/v1/6f0cc5f61a6f33775e875022.png"},{"id":85044118,"identity":"55a49828-b25b-4841-9b14-494a1f6ad48d","added_by":"auto","created_at":"2025-06-20 09:59:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":38818,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ozonation and chitosan coatings on total soluble solids values of Aronia (4 months storage).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6862137/v1/31b95d30325389e094fdaf81.png"},{"id":85044124,"identity":"d03e1694-c491-4f98-8e23-d3830825fc4c","added_by":"auto","created_at":"2025-06-20 09:59:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":34614,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ozonation and chitosan coatings on pH values of Aronia (4 months storage).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6862137/v1/f2dfa1ccdb1309e8471051a7.png"},{"id":85044623,"identity":"ac4ccb6a-453c-4920-bf07-12da14767827","added_by":"auto","created_at":"2025-06-20 10:07:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":39681,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eEffect of ozonation and chitosan coatings on L* color values of Aronia (4 months storage).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb. \u003c/strong\u003eEffect of ozonation and chitosan coatings on a* color values of Aronia (4 months storage).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec. \u003c/strong\u003eEffect of ozonation and chitosan coatings on b* color values of Aronia (4 months storage).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6862137/v1/b3b27f883361d3d33dc12a30.png"},{"id":85044122,"identity":"9c952906-3e1f-4c12-a7b3-da52167b0e9e","added_by":"auto","created_at":"2025-06-20 09:59:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":53715,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ozonation and chitosan coatings on texture profile analysis values of Aronia (4 months storage).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6862137/v1/7595b5f722331c0784c916b0.png"},{"id":85044622,"identity":"b0e7cce3-7841-4760-a812-1b43ebf1126e","added_by":"auto","created_at":"2025-06-20 10:07:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":37627,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ozonation and chitosan coatings on total anthocyanin content (ppm) values of Aronia (4 months storage).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6862137/v1/1d8a904cc6822214e21ba56e.png"},{"id":85044619,"identity":"5c6ee0b5-2c8a-4005-b4b4-8d9ceee6ed7a","added_by":"auto","created_at":"2025-06-20 10:07:18","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":41048,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ozonation and chitosan coatings on total phenol content (ppm gallic acid) values of Aronia (4 months storage).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6862137/v1/09660f6afe34c92052e4af62.png"},{"id":85044628,"identity":"91328dcb-9378-4fcf-93b0-ac16ffc77594","added_by":"auto","created_at":"2025-06-20 10:07:18","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":137709,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ozonation and chitosan coatings on visual evaluation of aronia at the of the storage period (2\u003csup\u003end\u003c/sup\u003e months) a) control group, b) ozonation, c) chitosan coating, d) ozonation \u0026amp; chitosan coating.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6862137/v1/53965e7b2865de771482e0e4.png"},{"id":87662160,"identity":"9d4e929d-70c2-4b2d-911c-f5319f10b231","added_by":"auto","created_at":"2025-07-27 10:31:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1469758,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6862137/v1/f669f3a3-4e55-41c9-8de0-41f3eecc17bf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Individual and Combined Impacts of Ozonation and Chitosan Bio-coating on Health-Promoting Bioactive Substance and Storage Stability of Fresh Aronia","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe consumption of fresh produce, such as berries, has recently received remarkable attention due to people's awareness of its health properties in the past decade. Aronia melanocarpa berries (black chokeberry) have one of the highest contents of potential antioxidants, such as anthocyanins, flavonoids, and proanthocyanidin polyphenols, in the plant kingdom that may be helpful in the reduction of risk of diabetes, cancer, cardiovascular, and gastrointestinal disease \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Hjartaker, et al. \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e found that consuming more berries has been correlated with a lower risk of developing from cancer mortality (8\u0026ndash;10%), stroke, and digestive system cancer.\u003c/p\u003e \u003cp\u003eAronia berries are significantly lesser-known when compared to other berries, but they have gained popularity over the last decade, mainly due to recognition of the health benefits of underutilized berries, especially aronia berry \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Fresh berries such as aronia have a limited storage stability with 30%\u0026minus;60% losses depending upon the supply chain stages \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, mainly due to it continuing with metabolic processes after postharvest \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Berries are highly perishable horticultural produce. Also, postharvest diseases, such as gray mold during the storage period, lead to important economic losses\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The availability of fresh berries in retail stores throughout the year caused by their positive health-promoting properties is very important for increased consumption. Thus, helping in maintaining its highest qualities is necessary for extending storability for enhancing sustainability. Since fresh aronia are harvested fully ripened, they are susceptible to decay in their high quality due to moisture through transpiration, mechanical damage, softening, susceptibility toward postharvest diseases, and changes in phytochemicals during postharvest storage \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Therefore, it must be preserved in its superior quality to ensure its consumption throughout the year.\u003c/p\u003e \u003cp\u003eThe demand for safe, fresh produce with high nutritional value, high sensory properties, and extended storage stability using sustainable innovative emerging technologies has increased in recent years. Various environmentally friendly emerging post-harvest methods such as ultrasound, plasma, ozonation, electrolyzed water, high-pressure processing, edible coatings, and equilibrium-modified atmosphere packaging (EMAP) have become more important in recent years in the preservation of fresh produce and reduce their ecological footprint \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eToday, the focus is on ozone, sustainability, cheapness, ease of application, the powerful antimicrobial agent characterized by easy decomposition without leaving any toxins/residues and oxidation, and approval by the FDA as an antibacterial agent for direct contact with food \u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e .\u003c/p\u003e \u003cp\u003eSeveral research studies have evaluated that microbial loads can be lowered by ozone treatmensts\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Horvitz, et al. \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e have revealed that the highest reduction in \u003cem\u003eB. cinerea\u003c/em\u003e, \u003cem\u003eSalmonella enterica\u003c/em\u003e, and \u003cem\u003eE. coli\u003c/em\u003e in Andean blackberry after role of ozonations (0.7 ppm, for 3 min.) when compare the 0.4, 0.5 and 0.6 ppm (3 min). Similarly, Shah, et al. \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e reported that gray mold by \u003cem\u003eB. cinerea\u003c/em\u003e was minimized by ozonation (2 to 3 ppm for 3 h) in red raspberry and maintaining higher contents of TA, SSC, and surface colors \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Ozone storage (0.1 and 0.3 ppm) suppressed fungal development for 12 days. Surface color and anthocyanin content were better retained in the stored berries \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Ozone application maintained firmness in kiwi (300 ppb) \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, papaya (1.5\u0026ndash;3.5 ppm) \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, strawberries (2 min at 300 ppb) \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Ozone applications (40\u0026ndash;200 ppb) slowed down the yellowing of the broccoli, but 700 ppb was reported to be undesirable tissue browning \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe application of the ozone in foods mostly concentrates on the microbiological control of fish, poultry, dairy, and meat products, while its applications in fruits and vegetables, especially fresh berries, are still limited \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe applications of the optimized ozone (2, 5, and 10 ppm) and contact time (3 and 9 min) in fresh \u0026ldquo;Angelino\u0026rdquo; plum preservation enhanced bioactive substances, including anthocyanins and organic acids, during storage but had minimal impact on the general quality\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Therefore, to enhance the storage stability of fresh berries, it is necessary to combine emerging nonthermal technologies, such as sustainable bio-coating. \u003c/p\u003e \u003cp\u003eThe sustainable coatings are considered a revolutionary strategy in post-harvest preservation, combining durability and ecological potential into a one-shot solution to preserve the quality of perishable products. Of all these biocoatings, chitosan has attracted attention in the recent past because of its inherent antibacterial properties and suitability with a broad range of bioactivities \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Chitosan biocoatings are to act a main role in the strawberries in lowering the permeability of the O\u003csub\u003e2\u003c/sub\u003e/CO\u003csub\u003e2\u003c/sub\u003e and moisture in trays \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Chitosan biocoatings act as barriers by producing tiny physical layers on the fruits that can significantly reduce postharvest losses by controlling the mass transfer of gases (O\u003csub\u003e2\u003c/sub\u003e/CO\u003csub\u003e2\u003c/sub\u003e) leads to slowing down the metabolic activity and minimizing the respiration rate of the fresh fruits. Hence enhancing its storage stability and qualities of fresh produce. It is especially useful for fresh produce that contains a high level of water, like fresh berries \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLike biocoatings, an EMAP is to play a crucial role in regulating the mass transfer of gases (O\u003csub\u003e2\u003c/sub\u003e/CO\u003csub\u003e2\u003c/sub\u003e), and moisture established inside the headspace using the film permeability (O\u003csub\u003e2\u003c/sub\u003e/CO\u003csub\u003e2\u003c/sub\u003e) and the respiration rates of the packaged fruits in the trays. By using, EMAP improves the storage stability of fresh produce by suppressing respiration rates and ethylene production \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Therefore, EMAP must be combined to maintain the high quality of aronia during extended storage \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Because of the strategic nature of the market, researchers are using emerging techniques such as equilibrium modified atmosphere packaging (EMAP) and sustainable chitosan bio-coatings to preserve perishable berries for longer storage stability.\u003c/p\u003e \u003cp\u003eThere are a number of techniques that are used to preserve the post-harvest quality (phenol levels and antioxidant activities) of fresh berries including modified atmosphere packaging and coatings \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, there are a few studies reported on deep research into the impacts of ozonation and chitosan biocoating on the storability of \u0026ldquo;fresh aronia\u0026rsquo; during extended storability. Therefore, comprehensive detail research is essential to extend the postharvest storage stability of fresh berries such as fresh aronia. Based on our literature review, there is a lack of existing research papers on the combined ozone (8 ppm) and chitosan coatings for fresh aronia storage stability. For that reason, the main goal of this study was to provide novel insights into mitigation and improvement of storage stability of fresh aronia with high levels of the bioactive compounds such as total monomeric anthocyanin and phenolic.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Sample processing.\u003c/h2\u003e \u003cp\u003eFresh aronia fruits (\u003cem\u003eAronia melanocarpa\u003c/em\u003e) used in this study were supplied from a local producer in Yalova province (Producer No: M-77-26) in the 2021 season. Fresh aronia berries were placed in a 5-L transparent glass chamber and treated with 8 ppm ozone gas with the help of a diffuser for 5 minutes using TKZ-6G ozone generator (Teknozone, Izmir).\u003c/p\u003e \u003cp\u003eThe solutions of the chitosan bio-coating were produced by dissolving 1% chitosan (w/w) in 1% acetic acid (0.5% glycerol as plasticizer) \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. After the application, 100 g of aronia were placed in PP trays (190*144*H65 cm) and heat-sealed using the MAP-25 (MAP-25, Apack Ltd. Şti., Istanbul, Turkey) with normal air atmosphere. Packaged aronias were stored at +\u0026thinsp;4\u0026deg;C and monthly samplings were carried out. The analyses were carried out on the 1st, 1st, 2nd, 3rd, and 4th months of storage, randomly selecting packaged trays from each application, with 2 replications and 2 parallels.\u003c/p\u003e \u003cp\u003eApplication groups and application parameters:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eControl 90 \u0026micro;m microperforated films (EMAP)\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e1% Chitosan coating\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e8 ppm ozonation (5 min.)\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e8 ppm ozonation (5 min.) after 1% chitosan coating applications\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eWeight loss, in-package gas concentration (O\u003csub\u003e2\u003c/sub\u003e/CO\u003csub\u003e2\u003c/sub\u003e), water-soluble DM, pH, titratable acidity, color values ​​(L\u003csup\u003e*\u003c/sup\u003e, a\u003csup\u003e*\u003c/sup\u003e and b\u003csup\u003e*\u003c/sup\u003e), texture profile analysis (TPA: firmness-hardness, external adhesiveness, internal adhesiveness, chewiness, gumminess and springness), mold status, monomeric anthocyanin amount, and total phenolics content of fresh aronia were determined on the 1st day and then 1st, 2nd, 3rd, and 4th months of storage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Gas composition in trays\u003c/h2\u003e \u003cp\u003eThe gas (O\u003csub\u003e2\u003c/sub\u003e/CO\u003csub\u003e2\u003c/sub\u003e) levels in the headspace in the trays were measured by inserting the needle into the trays before opening with OxyBaby, the gas analyzer (HTK, Hamburg, Germany) \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 pH Measurement and Total Soluble Solid Content\u003c/h2\u003e \u003cp\u003eAfter opening the package, aronia berries were separated into three sets, and berries were placed in a blender, and then the obtained pulp was transferred into a beaker. Analyses were carried out using the pH meter (Atagon PAL-1 Osoka, Japan) \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and Pal-1refractometer (Atago Co., Tokyo, Japan) and then shown as Brix \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e at room temperatures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Surface Color\u003c/h2\u003e \u003cp\u003eThe surface of the fresh aronia \u0026ldquo;L\u003csup\u003e*\u003c/sup\u003e\u0026rdquo;, \u0026ldquo;a\u003csup\u003e*\u003c/sup\u003e\u0026rdquo;, and \u0026ldquo;b\u003csup\u003e*\u003c/sup\u003e\u0026rdquo; values was analyzed using a CR-400 ChromaMeter (Minolta Co. Ltd., Japan) \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Texture Profile Analysis (TPA)\u003c/h2\u003e \u003cp\u003eThe TPA properties were measured using the Texture Analyzer TA-XT Plus (Stable Micro Systems Co., Surrey, England) using P-2CYL cylindrical probe based on a trigger force of 1.0 N. Properties such as hardness (N), springiness (unitless), stickiness (N.s), chewiness (N) and gumminess were analyzed automatically using the software \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Total Anthocyanin Analysis\u003c/h2\u003e \u003cp\u003eThe total monomeric anthocyanin amount is measured by the pH differential method (colored at pH 1.0 and colorless at pH 4.5). The extracted aronia juice was filtered and then scanned using the spectrophotometer (7205 UV/Visible, Jenway Co., UK). The absorbance measurement was carried out at 520 nm and at 700 nm wavelength at pH 1.0 and pH 4.5 according to Caner, et al. \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Results are presented in mg cyanidin-3-glucoside equivalents/100 mg sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Total Phenolic Content (TPC)\u003c/h2\u003e \u003cp\u003eThe Folin-Ciocalteu method was followed. A spectrophotometer (7205 UV/Visible, Jenway Co., UK) was used to analyze absorbance at 765 nm with a blank as the reference. TPC was quantified using a gallic acid calibration curve and written as mg of gallic acid equivalents (GAE)/100 g of sample.\u003c/p\u003e \u003cp\u003e \u003cb\u003e- CUPRAC\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe CUPRAC analysis, based on the reduction of Cu (II) to Cu (I) by aronia antioxidants, leading to the formation of a colored Cu (I)-neocuproine (Nc) complex with maximum absorbance at 450 nm, was measured. For the assay, ethanolic aronia extracts were sequentially mixed with 0.01 M CuCl\u003csub\u003e2\u003c/sub\u003e and 1 M ammonium acetate buffer (pH 7.0) and incubated at 25\u0026deg;C for 30 minutes to allow complete complex formation.\u003c/p\u003e \u003cp\u003eA spectrophotometer (7205 UV/Visible, Jenway Co., UK) was used to read the absorbance of the resulting Cu (I)-Nc chelate at 450 nm). The antioxidant capacity was quantified by means of a Trolox standard curve, and the outcomes were expressed as mg Trolox equivalents (TE) per 100 g sample.\u003c/p\u003e \u003cp\u003e \u003cb\u003e- DPPH\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe scavenging of DPPH (2,2-diphenyl-1-picrylhydrazyl) free radicals assay was measured according to Benvenuti, et al. \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The extracts (10\u0026ndash;40 \u0026micro;L) were mixed with 300 \u0026micro;L of 1 mM DPPH solution and adjusted to 3 mL with ethanol. After incubation for 15 minutes in the dark at room temperature, the spectrophotometer was carried out to measure the absorbance at 517 nm. DPPH scavenging activity (%) was evaluated based on the reduction in absorbance, and the half-maximal effective concentration (EC\u003csub\u003e50\u003c/sub\u003e) was determined as mg of sample from inhibition percentage versus concentration curves.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Visual evaluation (fungal decay evaluation)\u003c/h2\u003e \u003cp\u003eFungal growth on the fresh aronia was visually counted and recorded as the number of fruits with mold or visible fungal growth, decay, or lesions. \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e.\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical Analysis\u003c/h2\u003e \u003cp\u003eThe MIXED procedure of SAS V-9 (Statistical Analysis Systems, Cary, NC, USA) was used for statistical evaluation of the results. The effects of ozonation, chitosan bio-coating, storage time, and their interactions were analyzed using the Tukey test based on p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and Discussion","content":"\u003cp\u003e \u003cb\u003eThe weight loss\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe fresh, ripe \u003cem\u003eAronia melanocarpa\u003c/em\u003e contains high amounts of water (74 to 83%) \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Consequently, postharvest water loss, which happens naturally in fresh aronia, leads to cell shrinkage, withering, and shriveling through storage. Even at 3\u0026ndash;10% water loss can adversely affect the texture of the fruits and make them unmarketable and consumer acceptance. This is mainly caused by the berry's susceptibility to water loss, which alters its physical characteristics that lead to wilting, shrinkage, and reduced firmness. Control of the moisture loss rate is necessary to preserve the desired texture and also the appearance of the fresh aronia berries. According to Konishi, et al. \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e the cuticle, calyx, and stomata are the pathways of water loss in fruit. A role in berry water loss is thought to be played by the cuticular membrane covering the fruit surface.\u003c/p\u003e \u003cp\u003eAll aronia groups showed weight loss during storage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The weight loss in fresh fruits of 3\u0026ndash;10% is the most important sign of loss of freshness. The weight losses were 1.2% in the control group, 0.8% in ozone-applied samples, 0.72% in chitosan-coated groups, and 0.67% in chitosan-coated groups after ozonation at the end of the storage. The highest significant weight loss was measured in the untreated ones (1.2%) at the end of the storage (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eImplementing chitosan coatings can significantly reduce the weight losses of fresh aronia. Fruits such as the mango \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, kiwi \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, grape berry \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, strawberry (0, 0.3, and 1.0 ppm for 24 h) \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Pomegranate \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, raspberry \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, blackberry fruit \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e and physalis angulata L. berries \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e have shown that chitosan biocoatings lower the rate of stomata's transpiration, leading to lower weight loss, which is the main advantage of edible coatings.\u003c/p\u003e \u003cp\u003eThe lower reduction in the weight loss value of chitosan-coated aronia is most likely because of the impact of chitosan biocoating layers on the fruit acting as a semi-permeable barrier to gases and moisture, thus minimizing the water loss, respiration rate, and microbial infection\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. The biocoatings act as a modified atmosphere, which delays the aging of aronia more effectively by controlling metabolic processes \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. If coating is applied properly can help maintain optimum moisture levels around the fresh aronia fruits and prevent excessive water loss by creating a protective layer on the aronia surfaces and controlling the mass transfer of gases.\u003c/p\u003e \u003cp\u003eOzone-treated fruits had consistently lower respiration rates in comparison with control ones at the same storage time. The ozone on the postharvest life of fresh fruits includes its capability to minimize postharvest deterioration and oxidize ethylene, thereby minimizing the metabolic process and preventing deterioration of fresh produce quality during storage \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAfter chitosan coatings or ozone application, the water evaporation in fresh aronia could be remarkably prevented, as could the moisture loss. Research has published that ozonation effectively closes stomata, retards cell wall metabolism, and delays cell wall degradation that maintains the stability of fresh fruits during storage. Ozone impact on the control of the activities of the enzymes reduced the activities of peroxidase (POD) and polyphenol oxidase (PPO\u003cem\u003e)\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. This impact can be connected to the fact that ozonation suppressed the fresh fruit transpiration, which led to a lower moisture loss. \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAppropriate amounts of ozonation efficaciously retard the minimizing of aronia water loss during storage. Similar publications have highlighted that ozonation for kiwi (45 mg/m\u003csup\u003e3\u003c/sup\u003e every 12 h) \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, fresh garlic (5 ppm at 4\u0026deg;C) \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, fresh-cut pitaya (2, 4, and 6 mg L\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e 20 min \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, strawberry (0, 0.3, and 1.0 ppm)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e for 24 h, table grapes [(0.8\u0026ndash;1.2 \u0026micro;l/L continuous)] \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, red peppers [0, 7 ppm ozonation 1, 3 and 5 min] \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, Andean blackberries (0.4, 0.5, 0.6, and 0.7 ppm, for 3 min.) \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, and blueberries (4 ppm or 2.5 ppm ozonataion) \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAccording to Contigiani, et al. \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e the weight loss of strawberries after ozonation (3.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; 5\u0026ndash;15 min) were dose-dependent. Ozonization (5 min) significantly minimized weight loss, but weight loss in ozonated strawberries (15 min.) was higher than in unozonated ones. An appropriate level of ozonation has the positive effect of suppressing weight losses \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The minimized weight loss in the ozonated fresh aronia could be connected to its proportionally thicker cuticle compared with other fresh fruits such as tomatoes, cranberries, and strawberries.\u003c/p\u003e \u003cp\u003eIn this study, with a weight loss of 1.2%, which is much lower than the 3\u0026ndash;10%, all aronia sets would still have been marketable during the extended storage periods.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHeadspace Gases\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFresh fruits respire to carry out their metabolic activities using O\u003csub\u003e2\u003c/sub\u003e producing CO\u003csub\u003e2\u003c/sub\u003e after harvest. Respiratory rate is the main index of the metabolic rate of fresh produce and therefore of their post-harvest shelf life \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In a trays packaging, the concentration of the gases (O\u003csub\u003e2\u003c/sub\u003e/CO\u003csub\u003e2\u003c/sub\u003e) in the headspace changes to create an EMAP during storage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). When the O\u003csub\u003e2\u003c/sub\u003e level in the trays decreased, rates of respiration slowed down. At the same time, as CO\u003csub\u003e2\u003c/sub\u003e level rise, they lead to minimizing not only the respiration rate and C₂H₂ production but also suppressed inhibition of mold/bacteria growth \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIt was found that while the O\u003csub\u003e2\u003c/sub\u003e level in the headspace of the trays increased, the CO\u003csub\u003e2\u003c/sub\u003e level also increased, and the equilibrium O\u003csub\u003e2\u003c/sub\u003e level stabilized between 15.5\u0026ndash;16.7% for the ozone-treated groups. There were no significant differences between treatments (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Similar results were published for low-dose ozone-treated melons, mulberries \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, and strawberries treated \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt was observed that O\u003csub\u003e2\u003c/sub\u003e levels reduced and that aronia maintained their stability for an extended period of storage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eAll groups showed a similar pattern, i.e. a continuous and steady reduction in the O\u003csub\u003e2\u003c/sub\u003e concentration and an increase in the CO\u003csub\u003e2\u003c/sub\u003e concentration during storage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). It was clearly highlighted that the untreated trays maintained the highest respiration rate throughout the storage period. It has been analyzed that ozone is potential in lowering the rate of respiratory and helps to create EMAP in trays. It was noticed that the highest CO₂ concentration occurred in the untreated groups, while the least CO\u003csub\u003e2\u003c/sub\u003e level was achieved in the control ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). This gas level is highly related to the rate of aronia respiration during storage.\u003c/p\u003e \u003cp\u003eThe trays with ozone-treated groups lower in the O\u003csub\u003e2\u003c/sub\u003e levels and increased in CO\u003csub\u003e2\u003c/sub\u003e levels during storage. EMAP for the aronia was more successful when ozone was used (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), the rates of respiration delay created extended storage stability. Decreased O\u003csub\u003e2\u003c/sub\u003e and increased CO₂ levels slow down the rate of aronia respiration more than control and also result in lower enzymatic activity, which affects less utilization of substrates and extends the storage periods \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. In this research, the O\u003csub\u003e2\u003c/sub\u003e level was effectively lowered to 17.1% and CO\u003csub\u003e2\u003c/sub\u003e concentrations up to about 4.2% in the untreated one at the end of the storage.\u003c/p\u003e \u003cp\u003eOzone treatments also decreased the rates of respiration, causing lower O\u003csub\u003e2\u003c/sub\u003e utilization and CO\u003csub\u003e2\u003c/sub\u003e. The similar inhibitive impacts of ozonation on rates of respiration during storage of Andean blackberries, melons, and mulberries have been reported by Horvitz, et al. \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, Chen, et al. \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e and Han, et al. \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e This can be the results of ozone effectively suppressing the microbial respiration process, thus minimizing the respiration rate of the fruit to a certain extent. This was agreed with the ozonation obstacle biosynthesis of ethylene by blocking Acetyl-CoA carboxylase (\u003cem\u003eACC\u003c/em\u003e) synthesis \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBrix\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe key parts of solids soluble in fruit juice are sugars that were shown\u003c/p\u003e \u003cp\u003eon the \u0026deg;Bx scale. The effective retention of soluble solid content (SSC) is crucial for keeping fresh fruit well-stored and edible. The water-SSC of the aronia increased during storage in all groups due to continuous breathing. The SSC in the ozonated samples was relatively gentle, whereas the SSC in the ozonated group was better maintained than in the untreated one. The SSC of the aronia gradually increased for all treatments (18.3 to 19.5\u0026ndash;20.3\u0026deg; Brix) at the end of storage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The storage time has a significant effect on Brix values (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and with an increase in storage, it can be seen that the amount of soluble solids in the control is lower compared to the ozonated aronia. The untreated exhibited the highest in SSCx (18.3), while the 19.7 (O\u003csub\u003e3\u003c/sub\u003e), 19.8 (coated), and 19.5 (ozonation with coated) were at the end of storage. It was similarly reported that ozonation postpones the reducing of SSC in fresh aronia.\u003c/p\u003e \u003cp\u003eThe increase in SSC of all samples during storage can be explained by the decrease in water level and the conversion of complex carbohydrates into monosaccharides \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. It is related to the fact that ozonation slows down carbohydrate metabolism \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e Ozonization and storage time of fresh aronia caused significant changes in SSC. It is highlighted that the fresh aronia applied with ozone has a slower decrement in SSC. This can be as the chemical impact of ozone slows down enzyme activities related to carbohydrate metabolism, while maintaining a high level of soluble solute. Loss of acidity due to aging is another reason for the decline in SSC \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe results highlighted that ozone treatment was efficacious in delaying the increase in SSC content. Thus, the quality of the fresh berry was preserved at a later stage of storage.\u003c/p\u003e \u003cp\u003e \u003cb\u003epH value\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBoth the pH level of the control and the ozone-treated aronia are slightly increased over the storage period due to respiration metabolism that is related to the use of the organic acid through the TCA pathway. This increase is due to the degradation of organic acids during the storage periods \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Ozone and EMAP create a MAP that avoids the quick decline of organic acid by minimizing the respiration rate \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe influence of ozone and coating variety on pH over the storage period (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). While the pH of fresh aronia on the initial day was 3.56 in the untreated one, it increased to 3.93 at the end of 4 months; in aronia with ozonation, it was noticed that these changes progressed much more slowly after 2 months and were limited to 3.81 at the end of 4 months. The coated ones slowed down these changes significantly in pH values compared with the control (3.83), ozonation after coatings (3.82) and untreated groups (3.93). Nevertheless, the highest pH during the entire storage period was found in the untreated samples. The higher respiration rates resulted in the higher utilization of organic acids and accumulation of higher CO₂ concentrations inside the trays \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Regarding ozonation or coatings applied significantly slow down the respiration and also the pH of aronia when compared to the control groups. By slowing down the rate of respiration, the modified atmosphere created by the coatings also prevents high levels of organic acid consumption. The ozonation suppressed the respiration and slowed down the rate of the respirations. The lower pH was observed in the ozonated samples, where O\u003csub\u003e2\u003c/sub\u003e/CO\u003csub\u003e2\u003c/sub\u003e is in equilibrium, due to the reduction in respiration rates compared to untreated samples, as a result of the slower increase in pH. During the 12-day study, the chitosan coating did not affect the pH of fresh strawberries. This is because chitosan coatings and ozonation create a modified atmosphere that suppresses organic acid decomposition by suppressing the respiration rate \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe higher respiration rates resulted in the higher utilization of organic acids and accumulation of higher CO₂ concentrations inside the trays \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. The higher CO\u003csub\u003e2\u003c/sub\u003e percentage in the packaging may cause CO\u003csub\u003e2\u003c/sub\u003e to dissolve faster, which may increase the amount of HCO\u003csub\u003e3\u003c/sub\u003e \u003csup\u003e56\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRegarding ozonation and chitosan coatings significantly slow down the respiration rates and also the pH of aronia (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e) when compared to the control. These results are in agreement with those published by Caner, et al. \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, who published that strawberries\u0026rsquo; pH rises and depends on different EMAPs used during storage.\u003c/p\u003e \u003cp\u003e \u003cb\u003eColor\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor the consumer, color is a significant criterion in determining acceptance or rejection of the fruit, as evidenced by the case of fresh berries\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The loss of fruit surface color during storage can be attributed to two factors: firstly, an increased respiration rate, and secondly, the processes of browning and the loss of labile anthocyanin pigment (red) \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. The color of aronia depends on their content of natural predominant pigments, mainly anthocyanins, where cyanidin glycoside (98.4%) present in aronia \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur results show that the L* value was slightly increased over the storage of the aronia berries, from initial days (23) to (24) at 4 months of storage; nevertheless, the increase was not significant in either the control or ozone-treated samples during the storage period (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The L* value of the ozonated aronia increased over the storage and was always higher than the unozonated aronia fruit.\u003c/p\u003e \u003cp\u003eAronia exhibited a slight increase in the L* values over storage. This could be caused by inactivating the enzymes involved in browning \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Notably, the L parameter of fresh produce can be affected by surface damage, the enzymatic action of polyphenoloxidase on phenolic substrates, and the structure of cell wall macromolecules \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. This is agreed with Tiwari, et al. \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e report that ozonated tomatoes and ozone-treated strawberries (1.0 ppm and 1.5 ppm) exhibited higher L* values than the untreated groups over storage \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. It has been established that storage periods do not exert a significant effect on color change (L* value). However, no significant difference (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) was observed between the ozonated samples in terms of brightness (L* value).\u003c/p\u003e \u003cp\u003eThe red color intensities (a* values) in aronia were more intense when aronia were coated.\u003c/p\u003e \u003cp\u003eThe a* values in aronia indicating the red color intensities were more intense compared with control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The a* values ​​of ozonated and chitosan coating groups to fresh aronia exhibited higher values ​​compared to the control throughout the storage (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). It was determined that a* values ​​were significantly higher as a result of the combined application of ozone and chitosan coating (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In fact, at the end of storage, the a* value of ozonated after chitosan coatings groups had significantly the highest value (1.58) compared to chitosan coatings (1.26) groups, but not ozonated (1.41) and all control groups were found to be significantly lower compared to treated groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). These results are similar to the previous research conducted by Panou, et al. \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe coatings and ozonation delay the deterioration of anthocyanin pigments \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Ozonation played a significant role in the brightness of the appearance of aronia and reducing the change of bright appearance, highlighting that ozonation could retard and coatings enhance the surface of aronia and consequently maintain higher commercial appearance.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTexture Profile Analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHardness;\u003c/b\u003e During storage periods, fresh aronia undergoes cell wall modification; furthermore, the number of cells between adhesions decreases, leading to increased softening, which involves extensive depolymerization of pectin and partial hemicellulose, dissolution of pectin, and loss of neutral sugars in the pectin side chain \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. The fresh aronia undergoes a softening process in berries that is related to the following activities of fruit-softening enzymes, including pectin methyl esterase (PME), polygalacturonase (PG), and pectate lyase (PL); and the accumulation of reactive oxygen species (ROS) \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The process of pectate lyase (PG) activity involves the cleavage of pectin acid, which in turn results in the disassembly of the components of cell walls and a concomitant reduction in firmness. \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDuring the storage periods, all groups exhibited decreased firmness. While the chitosan-coated after ozonation groups had the greatest hardness value than the other groups, and all treated groups had significantly greater values than the control groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The application of edible coatings has been demonstrated to create a tiny surface barrier layer around the berries, thereby preventing moisture evaporation and delaying berry softening. The hardness decreased continuously as a function of storage periods, from 530 gf on the first day to 380\u0026ndash;470 gf after 4 months of storage (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This value was observed to decrease the most in the control group at 380 gf. In the 4th month, the best values ​​were obtained in the chitosan coating and also in the combination of ozone and chitosan coatings with a decrease of 13.5\u0026ndash;18.7% at 453\u0026ndash;470 gf, while the highest percentage decrease of 28% was observed in the control group (380 gf) compared to initial values. The findings revealed that there existed a considerable discrepancy between the treatments and the control group following a period of four months of storage (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). It was determined that the decrease in hardness was delayed in the products coated with chitosan and ozone (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Therefore, it was determined that the combination of ozonation and chitosan coatings optimum treatment for enhancing the firmness values of fresh aronia during storage. The combination of chitosan coating and ozonation has the capacity to diminish the activity of cell-wall decomposition enzymes. This process is conducive to safeguarding the integrity of the cell wall, impeding berry softening, and prolonging the storage stability of the berry. It was demonstrated that the application of a coating of the polysaccharide known as 'chitosan' resulted in a significant enhancement of the mechanical strength of the cell-wall structure and concomitantly delayed the process of loss of firmness. As a result, it was observed that the softening of aronia berries was slowed down and their storage stability was extended. The effectiveness of the process in maintaining the integrity of the material is partly related to the reduction of respiration rates caused by inhibition of mass transfer by the polysaccharide. It has been hypothesized that delays in softening may be attributable to ozonation reducing physiological degradation mechanisms. Furthermore, ozonation has been found to reduce water loss from berries \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Ozonation suppressed the decomposition of cell-wall polysaccharides by inhibiting the activity of enzymes (PG, PME) related to cell-wall modification. So ozonation significantly maintains the cell wall structure and ultimately maintains the firmness of aronia due to its maintaining both propectin and cellulose content compared to control groups.\u003c/p\u003e \u003cp\u003eTherefore, ozonation is a powerful technique to regulate cell-wall metabolism, postphone cell-wall degradation, and extend the storage stability of aronia throughout storage \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. It is evident that ozone has an effect on the breakdown of ethylene molecules, thus slowing the rate of pectin degradation and reducing the respiration rate of fresh aronia. These effects contribute to a delay in the softening of the fruit during storage \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn agreement with our results that the hardness was maintained after ozonation (0.075 ppm, 0.15 ppm, 0.25 ppm) compared to the control during storage \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Pectin and hemicellulose degradation of cell walls, changes in headspace composition, and more importantly, polygalacturonase (PG) and pectinmethylesterase (PME) activities may be limited by the CO\u003csub\u003e2\u003c/sub\u003e levels in the packaging \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNo significant differences were observed on day one of the storage experiment between the treated subjects and the control sample. The higher hardness values were recorded for coated, after- ozonated aronia samples when compared to the control over the storage periods. On the second month of storage, significant differences were recorded between the treated with ozone or coated aronia and the hardness of the control ones. In control ones, a decrease in hardness was observed in the range of 28% (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In ozonated aronias, chitosan-coated or ozonation after chitosan, this rate decreased to around 18.7%, 15.5%, or 13.5% when compared to initial days of storage (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Consistently decreased in hardness as a function of storage times, from the initial day 530 gf to 380\u0026ndash;469 gf at end of the storage. These values decreased the most to 380 gf in the control and 423 gf O₃, 453 gf coated samples, and 470 gf ozonation after coating at the end of the storage. The highest hardness values (470 g) were measured at 470 gf with a decrease of 13.5% in the coating after ozonation, the lowest percentage of reduction at initial hardness (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Therefore, it was determined that the combination of ozone and chitosan application in maintaining the hardness values of fresh aronia throughout storage.\u003c/p\u003e \u003cp\u003eThe maintaining of hardness can be related to the minimizing of physiological degradation mechanicms by ozonation and prevents water loss of fresh berries \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The outcomes highlighted a strong and negative correlation between water loss during storage and hardness levels \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. The weakening of the cell wall, in conjunction with an increase in cell separation due to a reduction in turgor pressure within the cells during storage, has been demonstrated to result in economic loss \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. It is evident that the presence of ozone and chitosan coatings contributes to the maintenance of cell wall strength, cell-to-cell adhesion, turgor, and decreased respiration rates in fresh Aronia, resulting in enhanced performance \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. It is hypothesized that the levels of carbon dioxide achieved in the trays, as well as the headspace composition, could limit the degradation of cell walls by pectin and hemicellulose, and could inhibit pectin methylesterase (PME) and polygalacturonase (PG) activities \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn agreement with our results that Caner, et al. \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. the hardness was maintained after ozonation (0.075 ppm, 0.15 ppm, 0.25 ppm) compared to control during storage \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. As a result, berry softening is delayed and fresh aronia quality is maintained. This is associated, at least in part, with the use of a coating to create EMAP and ozonation, with a view to reducing respiration rates.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe adhesiveness\u003c/b\u003e, no statistically different results were obtained between the groups during storage (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In contrast to the control group, the ozone treatment and the coated treatment exhibited adhesiveness maintenance.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe springiness\u003c/b\u003e significantly decreased (0.097 to 0.07\u0026ndash;0.081), especially the lowest in the untreated ones (0.07), while ozonated (0.0078), coated (0.008) and ozonation after coatings (0.081) at the end of storage. Nevertheless, no substantial disparities were observed between the ozonated and coated groups at the end of the storage.\u003c/p\u003e \u003cp\u003eThe results of the study demonstrated that the treatments had a significant impact on the cohesiveness. A steady decline in cohesion was measured during the storage period due to decomposition of the cell wall and middle lamella pectin\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. The untreated group had the highest decrements (from 0.07 to 0.053), while ozone (0.07 to 0.057), chitosan (0.07 to 0.059), ozonation after coatings (0.07 to 0.06) had lower decrements. Among those treated, there were no significant changes in cohesion. The decrease in the cohesion value may have developed due to the tissue softening that developed due to the dissolution of the pectin-containing material in the middle lamellae of the adjacent cells of the aronia fruit \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. A steady decline in cohesiveness was measured through the storage due to decomposition of the cell wall and middle lamella pectin \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. There were no significant changes in the cohesiveness of the treatment group.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn the case of gumminess (N)\u003c/b\u003e values ​​​​decreased in all groups during the storage. The least decrease was measured in the fruit group coated after ozone application (11.34% compared to the first day), and the most decrease was measured in the control group (26.68% compared to the first day). This decrease is more pronounced in the control group, decreasing from 33 to 24.1 in the control group, 27.8 in the ozonated group, 28.8 in the chitosan-coated group, and 29.4 in the ozonation after coatings at end of the storage due to the softening resulting in a softer and more elastic aronia texture. The hydrolysis of starch is the cause of lower gumminess \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Since gumminess is obtained by multiplying hardness and internal adhesiveness, gumminess values ​​​​were parallel to the change in the product of these two values \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In addition, it was seen that after 120 days in gumminess values, only ozone-applied samples, only chitosan-coated samples, and chitosan-coated samples after ozone application were not statistically different from each other (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe chewiness\u003c/b\u003e values decreased in all groups during storage. The decrease in chewiness is more pronounced than in cohesiveness. There was a reduction in chewiness as a result of the reduction in hardness. Ozonation lowers the decreasing rate that helps to limit the activity of cellulolytic and pectic enzymes. Chewiness decreased from 3.15 to 2.76 (control), 2.91 in ozonated ones, 2.96 in chitosan-coated ones, and 3.01 in ozonated ones after chitosan coatings, with 12,3%, 2,91%, 2,96% and also 3,10%, respectively, at the end of storage. In addition, while only ozone-treated samples and only chitosan-coated samples were not statistically different from each other after 120 days in terms of chewability, the chewiness value in chitosan-coated samples after ozonation was measured higher than these two groups.\u003c/p\u003e \u003cp\u003eTherefore, it can be concluded that ozone and coating application slow down the enzymatic reactions in the fruit. The minimal decrease in TPA values in chitosan-coated samples after ozone enables the determination of the synergistic effect of ozone and coating application. Possibly, ozonation and chitosan coating helped maintain fresh aronia textural parameters, especially hardness, which highlights changes in the structure of the cell wall. There was a high correlation between the TPA parameters of fresh aronia and the changes in its weight loss, the Brix value, and the pH value.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTotal monomeric anthocyanin\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAronia is the richest natural source of polyphenols, including anthocyanins, proanthocyanidins, flavonols, flavanols, and phenolic acids compared to other fruits. Aronia contains more anthocyanins than blueberries (about twice as many), a\u0026ccedil;a\u0026iacute; (about four times) and goji (350 times) \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThere have been reports of high levels of loss and changes in chemical structure, as anthocyanins are known to be unstable compounds \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. These substances are responsible for the high bioactivity of aronia, and the main polyphenolic substances of aronia have a wide range of health benefits. Approximately 25% of the total polyphenols in aronia are anthocyanins. They are mainly four different cyanidin glycosides, primarily cyanidin-3-galactoside (68.9%) and cyanidin-3-arabinoside (27.5%) \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhen the initial day storage, it was observed that the total content of initial monomeric anthocyanins can vary between 2773 (in control), 2792 (coated), 2865 (ozonated), and 2885 (ozonation after coatings) mg/100 g in initial days (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These high values ​​were not significantly different from the others due to great varations (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The significant enhancements were measured in anthocyanin contentsat end of the storage (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). It is clearly seen that the ozonation at the beginning of storage and also at the end of the storage, in terms of increasing anthocyanin extraction, has a positive impact towards extracting them at higher levels compared to the unozonated groups. At the end of storage, the amount of anthocyanin was 2780 in the control, while the significantly highest amounts (3315 in ozonated and 3326 in ozonation after coatings) were detected in the ozonated samples (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Ozonation and ozonation after coatings gave more successful extraction results than the other groups during the storage (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In fact, it has been published that ozonation stimulates the biosynthesis of phenolic substances such as anthocyanins in fresh fruits. Admane, et al. \u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. Ozonation after coatings exhibited the best extractability performance during storage based on the bioactive compounds, including anthocyanins \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Similarly, Pinto, et al. \u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e published that the combination of ozonation and MAP significantly maintained anthocyanin contents in blueberries. In the study conducted by Piljac-Žegarac and Šamec \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e on the effect of post-harvest storage temperature on the content of the cherry, sour cherry, and strawberry fruit samples, an increase in the amount of anthocyanin was observed in those stored at +\u0026thinsp;4\u0026deg;C. Similarly, the anthocyanin content in the berry skin of O\u003csub\u003e3\u003c/sub\u003e-treated grapes (5, 10, 20 \u0026micro;L L-1 for 30 min) was significantly higher than in the control \u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. O\u003csub\u003e3\u003c/sub\u003e contact leads to changes in grape secondary metabolism, improving the synthesis of phenolics, including stilbenes and anthocyanins. Furthermore, ozonation enhanced the extractability in ozonated grapes \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. It has been confirmed that the amount of bioactive compounds such as anthocyanins can be efficiently increased in a couple of days between the ages of the leaves, depending on the concentration of ozone (100 ppb; 16 h and 200 ppb; 24 h) \u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eO\u003csub\u003e3\u003c/sub\u003e applications are often reported to enhance bioactive substances, inluding polyphenols and antioxidant volatiles. The literature suggests that low concentrations and short treatments give the best results in most cases. Grapes treated with O\u003csub\u003e3\u003c/sub\u003e at the right dose and for the right time promote the biosynthesis of metabolites. On the other hand, it modifies the permeability of the skin, resulting in a greater extraction of polyphenols. The berries' response to moderate stress, which induces the biosynthesis of polyphenols, is probably responsible for the increase in anthocyanins \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTherefore, ozonation affects the structure of the cell wall and the composition of the cell membrane, in addition to inducing the biosynthesis of phenolic compounds, and facilitates the extraction of these substances.\u003c/p\u003e \u003cp\u003eIt has been shown that ozonation can cause increased anthocyanin amounts by enhancing the phenylpropanoid biosynthesis pathway in strawberries \u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTotal phenolics\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePhenolic compounds, or polyphenols are among the most common compounds of secondary metabolism in various plants (up to 200\u0026ndash;300 mg per 100 g). Phenolic compounds affect odor and taste formation and can inhibit enzymes associated with the development of human diseases.\u003c/p\u003e \u003cp\u003eAn increase in total phenol content was observed on the 15th day and a decrease on the other days. The highest reduction of total phenol content was in the untreated groups, from 838 to 610 (\u0026sim;28% loss), while the significantly highest amounts (660 for O\u003csub\u003e3\u003c/sub\u003e, 637 for chitosan, and 656 for coatings after ozonation) were measured (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These results highlighted that the ozonations maintained stability of the total phenol amount \u003cem\u003ein comparison with\u003c/em\u003e the unozonated ones. In response to ozone treatment, flavonoid biosynthesis and phenylalanine metabolism were the main targets of the regulatory effect of ozone on phenolic compounds\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Oxidizing enzymes are inactivated by ozone. This slows or stops the decomposition of phenolic compounds \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e. This may be due to the oxidation-reduction potential of ozone application to fruits, which increases intracellular reactive oxygen species responsible for the harmful effect on nucleic acids, paving the way for damage to bacterial cell walls. In addition, another reason for the increase seen on the 15th day may be due to the phenylalanine ammonia-lyase enzyme, which is activated by cold heat stress experienced in the fruit at refrigerator temperature.\u003c/p\u003e \u003cp\u003eRecent publications have underlined that appropriate ozonation can significantly enhance peroxidase activity, maintain high levels of total phenolics and flavonoids, and improve the antioxidant capacity of berry fruits. This result is similar to the study conducted by on aronia fruits stored at +\u0026thinsp;4\u0026deg;C and \u0026minus;\u0026thinsp;20\u0026deg;C and by Chen, et al. \u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e on strawberry samples treated with ozone at different concentrations (1, 3, and 5 ppm for 10 hours). The increase in polyphenol content can be explained mainly by the release of polyphenols from the cell structure due to cellular degradation and increased extractability. The decrease observed on the following days indicates that polyphenols released outside the cell are subject to faster degradation. Among the different O\u003csub\u003e3\u003c/sub\u003e concentrations applied (1,5 and 9 \u0026micro;L/L and time intervals of 1, 12, 24, and 48 h), 5 \u0026micro;L/L applied for 48 h was found to be optimal for the induction of bioactive compounds such as total phenolics, flavonoids, water-soluble polysaccharides, and ganoderic acids. A twofold increase was observed \u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. Compared to uncoated fruits, coated fruits showed a slower rate of decline in phenolic content, most likely due to reduced oxygen permeability and hence reduced enzyme activity \u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e. For apples coated with CH-OLE 2%, the lowest decrease in total phenolic content was 1.24 mg GAE g-1.\u003c/p\u003e \u003cp\u003eConversely, fruits that had not been coated exhibited the most substantial decreases in total phenolic content, reaching 0.28 mg GAE g-1 by the conclusion of the storage period.\u003c/p\u003e \u003cp\u003eThe decline in phenolics could be attributable to the reduction in sugar levels. This is because sugar is known to be a stimulant for the metabolic pathways involved in phenol production \u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. During storage, the aging phenomenon, degradation of pectic substances in the cell wall, and microbial infection lead to the breakdown of intracellular substances, oxygenation, and disruption of the integrity of the cell membrane. This raises the enzyme activity of polyphenol oxidase (PPO), which is in charge of the phenol oxidation. Earlier research has shown that chitosan decreases PPO activity and raises the levels of phytochemicals in strawberries. However, the decrease in phenolics during long-term storage can be explained by cell wall degradation and exposure of phenolics to enzymatic oxidation \u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eVisual evaluation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eFresh aronia\u003c/em\u003e is a perishable fruit, and its storage stability is often adversely affected by molds. Out of these, \u003cem\u003ePenicillium sp., Alternaria sp.\u003c/em\u003e and Bothrytis sp. are the main spoilage-causing agents in fresh aronia fruits \u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e. These pathogens can infect fresh berries after harvest, causing rot and spoilage, and can cause significant deterioration during cold storage \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. When the total number of rotten and moldy fresh aronia berries in the trays is counted during storage. No mold growth was observed in the chitosan-coated after-ozonated berry groups. There was only 1/1 rotten/mold growth observed in the chitosan-coated and also ozonated groups at the end of the storage. The highest rotten/mold growth was observed in 2 months (1/1), (2/4) 3 months, and (4/5) at the end of the storage for the untreated groups (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It was clearly observed that with the combination of the chitosan and ozonation, any fungal and rotton decay were detected during storage. On fresh aronia, the combination of the chitosan and ozonation had a major effect on molds and decays, whereas the use of individual chitosan coatings or ozonation does significantly affect suppression on fresh aronia samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of ozonation and chitosan coatings on rotten/moldy percentage of aronia in package (4 months storage)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatments\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eDay 15\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMonth 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMonth 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMonth 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMonth 4\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2/4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4/5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOzonation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1/1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChitosan coating\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1/1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOzonation \u0026amp; chitosan coating\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSimilar results were published that Bessi et al. (2014) underlined that ozonation can effectively suppress the development of mold and bacteria on the surface of the citrus. Likewise, the number of viable yeast and molds in uncoated arils was more than in coated arils \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Since ozone and chitosan, due to cationic amino groups, have broad-spectrum antimicrobial properties \u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. The microbial load increased in coated strawberries when stored. However, the density of microbial cells was lower than that found in untreated strawberries \u003csup\u003e\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the study conducted by Pinto, et al. \u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e it was reported that red raspberry fruits exposed to gaseous ozone (6 ppm) for 16 hours after packaging in PET (polyethylene terephthalate) trays reduced mold growth by 1.37 log CFU/g and provided better visual quality for 15 days. In addition, another study found that chitosan coating suppressed the growth of \u003cem\u003eRhizopus sp\u003c/em\u003e. and \u003cem\u003eB. cinerea\u003c/em\u003e on fresh raspberry fruits during cold storage \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLemic, et al. \u003csup\u003e\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e published the inhibitory effect of ozonation on mold growth in citrus, while Gorzelany, et al. \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e underlined that the ozone applications had a suppressed impact in lowering the yeast, and molds on the saskatoon berries. Contigiani, et al. \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e highlighted that ozone treatments (3.5 mg/L for 5\u0026thinsp;\u0026minus;\u0026thinsp;15 min) fresh strawberries had a significant reduction in \u003cem\u003eyeast\u003c/em\u003e or mold incidence compared to untreated berries.\u003c/p\u003e \u003cp\u003eThe most common moulds in fresh aronia berries that cause significant deterioration were Penicillium sp., Alternaria sp. and Bothrytis sp. during storage. Fresh aronia is a perishable fruit, and its storage stability is often adversely affected by \u003cem\u003emolds\u003c/em\u003e. Out of these, Penicillium sp., Alternaria sp. and Bothrytis sp. are the main spoilage causing agents in fresh aronia fruits \u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e. These pathogens can infect fresh berries after harvest, causing rot and spoilage, and can significant significant deterioration during cold storage \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. When the total number of rotten and moldy fresh aronia berries in the trays is counted during storage. No mold growth was observed in the chitosan coated after ozonated berries groups. There were only 1/1 rotton/mold growth observed in the chitosan coated and also ozonated groups at end of the storage. The highest rotton/mold growth were observed in 2 mounts (1/1), (2/4) third mounts and (4/5) at end of the storage for the untreated groups (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It was clearly observed that the combination of the chitosan and ozonation, any fungal and rotton decay were detected during storage. On fresh aronia, when combination of the chitosan and ozonation was applied had a major effect on molds and decays, whereas the use of individual chitosan coatings or ozonation does affect significantly suppressed on fresh aronia samples.\u003c/p\u003e \u003cp\u003eSimilar results were published that Bessi et al. (2014) underlined that ozonation can efficently supresed the development of mold and bacteria on surface of the citrus. Similarly, the number of viable yeasts and moulds was higher in the uncoated arils than in the coated arils \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Since ozone and chitosan due to cationic amino groups have broad-spectrum antimicrobial properties \u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. The microbial populations grew during storage in coated strawberries. However, the microbial cell densities were lower than in untreated strawberries \u003csup\u003e\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the study conducted by Pinto, et al. \u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e, it was reported that red raspberry fruits exposed to gaseous ozone (6 ppm) for 16 hours after packaging in PET (polyethylene terephthalate) trays reduced mold growth by 1.37 log CFU/g and provided better visual quality for 15 days. In addition, another study found that chitosan coating suppressed the growth of Rhizopus sp. and B. cinerea on fresh raspberry fruits during cold storage \u003csup\u003e\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLemic, et al. \u003csup\u003e\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e published the inhibitory effect of ozonation on mold growth in citrus, while Gorzelany, et al. \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e underlined that the ozone applications had a suppresed impact in lowering the yeast, and molds on the saskatoon berries. Contigiani, et al. \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e higligted that ozona treatments (3.5 mg/L for 5\u0026thinsp;\u0026minus;\u0026thinsp;15min) fresh strawberries had a significant reductions in \u003cem\u003eyeast\u003c/em\u003e or mold incidence compared to untreated berries.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eOur results underlined that, ozonation and chitosan coating treatments individually alone significantly improved the storability of fresh aronia. The combination of chitosan biocoating after ozonation has a significant effect on slowing down the softening and weight loss of aronia, and the effect improved its fresh aronia firmness properties during storage. This could be due to a semi-permeable barrier in the chitosan coating that prevents water vapor and gases from entering the coated berries, thereby reducing transpiration. The obtained allowed a proper mass transfer exchange suitable for fresh berries and normal physiological weight loss, thus delaying degradation and extending storage stability. Coated and ozonated aronia showed lower fungal development and less decay than untreated ones while retaining better color in the treated samples. Ozonation increased the bioactive compounds, mainly anthocyanins and polyphenols. O\u003csub\u003e3\u003c/sub\u003e application is very feasible. It is easy to incorporate into the chitosan coatings and can be used not only for extending storage stability, but also for promoting the health-related substance of fresh aronia, helping to improve their health-related quality of life. It was observed that the surface color of fresh aronia (L* and a) was improved. Furthermore, it has been demonstrated that post-harvest exposure to aronia can result in a substantial reduction in the utilization of ozonation and chitosan coatings, attributable to the latter's inherent antimicrobial characteristics.\u003c/p\u003e \u003cp\u003eAt the end of this study, it was found that the shelf life was one month longer with the synergistic effect of chitosan coatings after ozonation compared to the control groups.\u003c/p\u003e \u003cp\u003eHence, the notable synergistic effects carried out by the best performance in preserving fresh aronia by the combined applications of ozonation and chitosan coatings make it a versatile, sustainable secure substitute for artificial preservatives within the food industry. It is imperative that fresh fruits and vegetables be coated with a preservative solution following the ozonation process, prior to their being packaged, in order to prevent the growth of microbes. In light of the growing demand from consumers for safe, organic produce, the utilization of ozone and chitosan coatings has emerged as a sustainable alternative to artificially produced preservatives. These coatings have been demonstrated to provide a viable and sustainable approach to enhancing the storage stability of food products. In view of the fact that severe oxidative stress has been identified as a feature of metabolic syndrome, polyphenol-rich extracts have the potential to be of particular use to patients suffering from the condition. In this study, the authors posit that the polyphenolic extract of fresh berries could be of interest for use in the formulation of dietary supplements and advocate for further study in this area.\u003c/p\u003e \u003cp\u003eResearchers have dealt with this drawback by increasing chitosan coatings using neutral lipids and/or nanoclays and blending with proteins and/or polysaccharides. We further investigated the impact on sensory qualities as consumer acceptance of ozone and chitosan coatings in commercial scenarios.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is available on request from the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval was not required for this research. This study does not involve any human or animal testing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) received no nancial support for the research, authorship, and/or publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCC and CUP designed the study. NEGO, CC, MY and CUP made the sampling and analysis. CC, MY and CUP wrote the manuscript text and prepared the table and figures. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSharma, P., Bratley, K., Ford, T., Ristvey, A. G., \u0026amp; Volkis, V. V. (2025). 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Antifungal effects of chitosan coating on fresh strawberries and raspberries during storage. \u003cem\u003eThe Journal of Horticultural Science and Biotechnology\u003c/em\u003e, \u003cem\u003e73\u003c/em\u003e, 763\u0026ndash;767.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Aronia, Chitosan, Ozone, Equilibrium Modified Atmosphere Packaging (EMAP), Anthocyanins","lastPublishedDoi":"10.21203/rs.3.rs-6862137/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6862137/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe focus of the study is to analyze the individual and synergistic effects of ozone (8 ppm for 5 minutes) and 1% chitosan bio-coating application on enhancing the storability of fresh aronia with high antioxidant and anthocyanin content. The fresh aronia were ozonated (8 ppm 5 min), chitosan-coated, and after being coated after ozonation (8 ppm 5 min), placed in polypropylene (PP) trays and stored for 4 months (+\u0026thinsp;4\u0026deg;C). Analyses were carried out on the 1st day, 1st, 2nd, 3rd, and 4th months. The pH, Brix, color values, gas concentrations, weight loss, texture profile analyses, fungal development, monomeric anthocyanin, and total phenolic content were analyzed during storage.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAt the end of storage, the weight loss was 0.81% in the ozone group, 0.72% in the coated groups, and 0.68% in chitosan and ozonated ones, while it was 1.2% in the control groups. While the Brix in the control group increased from 18.3 to 20.3; at the end of storage, it was 19.7 in the ozone group, 19.8 in the coated ones, and 19.55 in the chitosan and ozonated ones. While the pH in the control group increased from 3.56 to 3.93, it was 3.8 in the ozone group, 3.82 in the coated ones, and 3.81 in the ozonated and coated groups. The rate of respiration was minimized as the gas composition O\u003csub\u003e2\u003c/sub\u003e/CO\u003csub\u003e2\u003c/sub\u003e in the headspace was achieved with the synergistic impacts of ozonation and coating. At the end of the 4th month, while it decreased to 380 gf on average in the control samples, this value was determined as 424 gf in the ozonated ones, 453 gf in the chitosan-coated, and 470 gf in the chitosan-coated samples after ozonation. Ozonation highlighted efficacy in inhibiting mold growth. The extractability of anthocyanin (3314 mg cyn 3-glu/100 g) phenol (660 mg) was significantly enhanced with ozonation at the end of the storage.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOzonation and ozonation after chitosan applications demonstrated a significant potential to maintain anthocyanins in fresh berries by improving their extractability and minimizing degradation during storage. Additionally, ozonation positively influenced anthocyanins and total phenolic content. Ozonation was used as an elicitor to improve the bioactive substances in the fresh aronia. It was observed that the combined use of ozonation and chitosan coating enhanced storage stability and bioactive compounds in the fresh aronia.\u003c/p\u003e","manuscriptTitle":"The Individual and Combined Impacts of Ozonation and Chitosan Bio-coating on Health-Promoting Bioactive Substance and Storage Stability of Fresh Aronia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-20 09:59:13","doi":"10.21203/rs.3.rs-6862137/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d7c96988-08b6-4a7d-80b8-304c0de68170","owner":[],"postedDate":"June 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-27T10:23:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-20 09:59:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6862137","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6862137","identity":"rs-6862137","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}