Synergistic Effects of Chitosan and Organic Acids on the Antimicrobial, Antifungal and Antioxidant Properties of Edible Coatings

Synergistic Effects of Chitosan and Organic Acids on the Antimicrobial, Antifungal and Antioxidant Properties of Edible Coatings | 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 Synergistic Effects of Chitosan and Organic Acids on the Antimicrobial, Antifungal and Antioxidant Properties of Edible Coatings Andrea Paola Guisolis, María de los Ángeles Dublan, Eliana Castañares, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6613506/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 This study examines the effectiveness of a chitosan-based edible coating, enhanced with citric, ascorbic, and acetic acids, in improving the microbiological quality and extending the shelf life of tomatoes ( Solanum lycopersicum ). Various formulations were tested for their antimicrobial and antifungal properties, targeting bacteria such as Escherichia coli and Staphylococcus aureus , and fungi like Alternaria arborescens and Alternaria tenuissima . The selected formulation—comprising 1% chitosan, 0.5% glycerol, and 1% each of citric and ascorbic acids—demonstrated significant antibacterial, antifungal, and antioxidant effects. Antibacterial tests revealed stronger inhibition against S. aureus than E. coli , while antifungal activity showed 100% growth inhibition in both Alternaria strains. Additionally, tomatoes treated with this coating displayed prolonged antioxidant activity and reduced microbial counts, emphasizing its potential in improving food safety and reducing spoilage. Citric Ascorbic Shelf life Solanum lycopersicum Tomato Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. INTRODUCTION Post-harvest losses in horticultural crops primarily stem from handling during the journey from harvest to consumer. Various factors contribute to their degradation, including mechanical damage, inadequate storage, and poor handling and transportation practices [ 1 ]. While tomatoes ( Solanum lycopersicum ) are among the most widely cultivated and consumed vegetables globally, their production results in significant losses and waste [ 2 ]. Changes in fruit quality can be mechanical, physiological, or pathological. Mechanical damage can lead to visual abnormalities, increased respiratory activity, and altered metabolism, significantly affecting the physicochemical composition of the pericarp and locular tissue, as well as their overall sensory characteristics. Additionally, water loss after harvesting adversely impacts the quality of these fruits, which is closely linked to temperature, relative humidity, cultivar, and storage duration [ 3 ]. Moreover, microbial attacks from organisms such as Alternaria alternata , Botrytis cinerea , Geotrichum , Rhizopus stolonifer , and bacteria like Erwinia spp. are responsible for the rapid deterioration of tomatoes, leading to economic losses [ 4 ]. In this context, exploring post-harvest management alternatives for fruits and vegetables is essential to extend their shelf life, ensuring a safe and high-quality product supply. While various environmentally friendly technologies have been tested, the ideal approach includes strategies that protect the environment and minimise waste production. Our research, alongside studies such as those by Fukuyama et al. (2024) [ 5 ], focuses on reducing food waste and post-harvest losses in alignment with UN Sustainable Development Goal 12 (Responsible Production and Consumption), which aims to halve global food waste per capita by 2030. This objective can be achieved by repurposing materials typically regarded as waste to produce novel compounds of interest or to extend the shelf life of perishable products. Edible coatings have emerged as a promising strategy for prolonging the shelf life of fresh and freshly cut fruits and vegetables [ 6 , 7 ]. These coatings can be made from proteins, carbohydrates, lipids, or a combination of these, often incorporating additional components such as antimicrobial and antioxidant agents. They can be applied through spraying or immersion. Functionally, edible coatings reduce water loss and browning, decrease ethylene production, enhance appearance, and extend commercial viability [ 6 , 8 ]. Furthermore, these coatings must exhibit mechanical properties that ensure proper adhesion to the food and withstand handling without damage [ 9 – 11 ]. The availability of environmentally compatible biopolymers for formulating edible coatings presents a significant challenge for the modern agri-food industry. In this regard, chitosan—a compound obtained through the deacetylation of chitin, primarily sourced from crustacean exoskeletons—represents a promising alternative due to its natural biodegradability, low toxicity, ease of availability, and ability to trigger defence responses in plant tissues. Additionally, it possesses strong antioxidant properties that can eliminate free radicals and serves as a matrix for incorporating other substances, enhancing antimicrobial and antioxidant capabilities [ 12 , 13 ]. While some researchers have explored the effects of various edible coatings on preserving the physicochemical attributes of tomatoes, further investigation is needed on the synergistic combination of chitosan with other components, such as organic acids, for these applications. Therefore, the objective of this study was to design a formulation of edible coating based on chitosan, citric acid, ascorbic acid, and acetic acid, aiming to exhibit in vitro antimicrobial activity and evaluate its effectiveness in preserving the microbiological quality of tomatoes during refrigerated post-harvest storage. 2. MATERIAL AND METHODS 2.1.1 Materials Chitosan : Sigma Aldrich Chemical Co., Germany. Glacial acetic acid : Biopack, Argentina. Glycerol : Anedra, Argentina. Citric Acid : Biopack, Argentina. Ascorbic acid : Biopack, Argentina. Escherichia coli ATCC35218 and Staphylococcus aureus ATCC 25923 : American Type Culture Collection, USA. Luria Bertani Broth (LB): Britania, Argentina. NaCl Biopack, Argentina. Müller Hinton agar : Oxoid LTD, England. Alternata arborescens (EGS39128) and Alternata tenuissima (EGS 34015) : Dr. Emmory G. Simmons, Indiana, USA. Modified Potato Dextrose Agar: MPDA : Britannia, Argentina. DPPH : Sigma Aldrich, USA. Methanol : Anedra, Argentina. Plate count agar- PCA : Britania, Argentina. Violet red bile agar-VRBA : Britania, Argentina. Glucose potato agar-GPA : Britania, Argentina. Chloramphenicol : Sigma Aldrich, USA. Eosin methylene blue agar-EMB : Britania, Argentina. Baird Parker medium-BP base : Britania, Argentina. Potassium tellurite : Britania, Argentina. Eeosin methylene blue agar-EMB : Britania, Argentina. 2.1.2 Formulation preparation Chitosan solutions at 1% and 2.5% w/v were prepared by weighing 10 g and 25 g of the compound (low molecular weight, 95000 Da, 92% DD-Deacetylation Degree), respectively. In each case, they were mixed with 10 mL of glacial acetic acid and 800 mL of distilled water, with magnetic stirring until complete dissolution. Various coating formulations containing glycerol (0.5% v/v) were prepared to enhance their plasticizing properties. The properties of different combinations of citric, ascorbic, and acetic acids at various concentrations were investigated. Before bringing to final volume, these agents were added to the chitosan and glycerol solution in adequate quantities to achieve the study concentrations. The systems were mixed and shaken until fully dissolved, reaching the specified final volume. 2.1.3 Sample Collection and Tomato Conditioning Round-type tomatoes ( Solanum lycopersicum ) grown in a non-climate-controlled greenhouse were harvested in a horticultural facility located in the city of Azul, Province of Buenos Aires (36°47′00″S 59°51′00″W). When they reached physiological ripeness, they were randomly selected with a grade 2 colour tone on the ripeness colour chart. Tomatoes of uniform size, with no physical damage and free of disease were chosen. The fruits were taken to the CAIVA Laboratory (Food Quality, Safety and Added Value), Faculty of Agronomy of Azul, UNCPBA, where they were washed by immersing them in tap water at 20 ± 1°C and then placed on a worktable on absorbent paper for air-drying at room temperature. In cases where bacteria were intentionally inoculated to evaluate behaviour, the tomatoes were disinfected with a 1% sodium hypochlorite solution and washed with sterile distilled water. A batch of 100 tomatoes was initially used for the different experimental determinations a posteriori (Fig. 1 ). A completely randomised design was used to select the samples, dividing the harvested tomatoes into two groups. The collection of plant material ( Solanum lycopersicum , tomato), complied with relevant institutional, national, and international guidelines and legislation. 2.2. Bacterial and fungal strains, and culture conditions Escherichia coli ATCC35218 and Staphylococcus aureus ATCC 25923 were the model strains used in this work as Gram negative and Gram positive, respectively. The strains were cultivated overnight in Luria Bertani Broth (LB) at 36 ± 1°C (Tecno Dalvo Cultivation Oven) For the experiments, both bacterial cells were collected by centrifugation (Presvac, DCS-16-RC) at 4000 rpm for 5 min, rinsed with sterile NaCl solution (0.85% m/V), and resuspended to obtain an equivalent to the 0.5 McFarland scale (Turbidity Standard). Inoculants prepared in this way were used in the subsequent assays. Single spore isolates of Alternaria arborescens EGS39128 and A. tenuissima EGS 34015 (Dr. Emmory G. Simmons, Indiana, USA) were cultivated on plates containing Potato Carrot Agar (PCA) according to Simmons (2007) [ 14 ] and incubated at 23°C under an alternating cycle of 8 hours of cool white fluorescent daylight and 16 hours of darkness for 7 days (Briket M4200 Growth chamber). From these plates, 4 mm diameter mycelial discs were obtained using a sterile punch for use in subsequent assays. 2.3 Antibacterial activity The antibacterial activity of the individual components of the formulation was determined by the well diffusion method on Müller Hinton agar [ 15 ] against E. coli ATCC 35218 and S. aureus ATCC25923. First, a 0.5 Mc Farland scale inoculum, as indicated in section 2.2 , was swabbed on the agar surface and then 50 µL of each solution was put on a well of 6 mm diameter. Then, agar plates were incubated at 36 ± 1°C (Tecno Dalvo Cultivation Oven) for 24 hours. After incubation, the inhibition was interpreted as the agar surface area without bacterial growth around the well. Two measurements were taken perpendicular to each other at the zone of inhibition of growth using a digital calliper (Hamilton), and the values were averaged. The solutions tested were chitosan at 1 and 2,5% m/V and citric and ascorbic acids at 0.1; 0.5; 0.75, 1 and 2% m/V, individually and combined (chitosan-citric acid; chitosan-acetic acid; chitosan- ascorbic acid; chitosan- ascorbic- acetic acid; chitosan- ascorbic- citric acid; chitosan- acetic- citric acid; chitosan- citric - ascorbic acid). This assay was employed as a screening method to define the solutions to be further evaluated, and the determinations were conducted in triplicate. 2.4. Determination of radial mycelial growth inhibition. The effect of chitosan on the mycelial growth of Alternata arborescens (EGS39128) and Alternata tenuissima (EGS 34015) was determined by inoculating a 4 mm diameter mycelial disc of both fungi on Modified Potato Dextrose Agar (MPDA) containing chitosan (1%), a combination of ascorbic and citric acids (0.1%, 0.5%, 0.75%, 1%, and 2% each), and chitosan (1%) combined with acids (0.1%, 0.5%, 0.75%, 1%, and 2%). This procedure was performed in triplicate, and the plates were incubated at 30 ± 1°C. After seven days, the mycelial diameter was measured on plates exposed to the evaluated agents and control plates. The variation in diameter was measured using a Truper vernier calliper. The activity of the agents against the fungal cultures was calculated using the following equation [ 13 ]: %Inhibition: [1 - (dt/dc)] × 100 Where dc is the average diameter of mycelial growth in the control, and dt is the average diameter in the treatment. 2.5. Assessment of free radical detoxifying effect against DPPH The DPPH free radical scavenging detoxifying effect was performed following the method described by Hsu et al. (2003) [ 16 ], with some modifications. For this purpose, a 0.5 mL aliquot of the methanolic extract of tomato was mixed with 2.5 mL of a cold methanolic solution containing 0.1 mM DPPH, and it was kept in the dark at 4 ± 1 ºC for 60 minutes. This procedure was carried out in triplicate. The absorbance of the reaction mixture was measured at a wavelength of 517 nm using an UV-visible spectrophotometer (Biochrom Libra S22). Methanol at 80% was used as a blank instead of the extract. The detoxifying free radical inhibition effect of DPPH was calculated using the following formula: DPPH detoxifying effect (%I) = [1 - (A 517 sample/A 517 blank)] x 100 The evaluated solutions were composed of chitosan at 1% and 2.5% w/v, glycerol at 0.5% w/v, and/or different combinations of citric acid (CitA) and ascorbic acid (AscA) at various concentrations (0.1%, 0.5%, 0.75%, 1%, and 2% w/v, respectively). 2.6. Bacterial growth curve Once the coating formulation that yielded the most desirable responses was selected, a growth curve was conducted for the bacteria E. coli ATCC35218 and S. aureus ATCC25923 in LB broth (control) and LB broth supplemented with chitosan and ascorbic and citric acids in the proportions that showed promising results in terms of their antimicrobial and antioxidant capacity. The optical density at 600 nm was recorded (using a UV-visible spectrophotometer Biochrom Libra S22) every 60 minutes during incubation with agitation at 36 ± 1°C (Tecno Dalvo Cultivation Oven). The determinations were conducted in triplicate. 2.7. Effect of the designed formulation on Solanum lycopersicum (tomato) 2.7.1. Application of the coating on tomato Round type Solanum lycopersicum samples, grown in an uncontrolled greenhouse environment, were sourced from a local producer (36°47′00″S 59°51′00″W) at the physiologically mature stage corresponding to a "pintón" colour tone (grade 2 on the harvest maturity colour chart). The tomatoes were harvested without physical damage, disease free, and with uniform sizes. They were prepared by immersing them in tap water at 20 ± 1°C and then air drying on absorbent paper at room temperature. Subsequently, the designed and selected edible coating was applied to the samples by spraying it evenly over the entire surface and allowed to dry at room temperature (20 ± 1°C). This procedure of spraying the coating on the tomatoes was performed in duplicate, with airing done before the second application, as mentioned. 2.7.2. DPPH free radical detoxifying effect (%I) in tomatoes The plant sample was ground using a grinder (Liliana AM680, 650 W), 1 g of the sample was weighed, and 2.5 mL of a methanol: acetic acid solution (40:1) was added. Extractions were carried out by ultrasonic bath agitation (Testlab TB04TA) for 15 minutes. Subsequently, the mixture was centrifuged (Presvac, DCS-16-RC) for 5 minutes at 4000 rpm at 4 ± 1°C, and the supernatant was reserved. This process was repeated twice, and the obtained extracts were combined. The volume of the centrifuged supernatant was measured. The DPPH free radical detoxifying effect was performed following the method described in section 2.5 . 2.7.3. Enumeration of indicator microorganisms in tomatoes Untreated and coated samples were separately ground under aseptic conditions to prepare a homogenate of 1 g of tomato in a 0.85% w/v NaCl solution. Subsequently, 10 fold dilutions were prepared from 10 − 1 to 10 − 5 . Aliquots were plated in triplicate on agar plates for plate count agar (PCA), violet red bile agar (VRBA), and glucose potato agar (GPA) supplemented with chloramphenicol (0.05% w/v) for the enumeration of total mesophilic aerobes, total coliforms, and yeasts and moulds, respectively. Plates were incubated at the specified temperature and time for each group of microorganisms: total mesophilic aerobes for 24 hours at 30 ± 1°C, total coliforms for 18–24 hours at 35 ± 1°C, and yeasts and moulds for five days at 25 ± 1°C. The enumeration results were reported as CFU/g PF or log CFU/g PF. 2.7.4. Inoculation of tomatoes with reference bacteria Inoculums of E. coli ATCC 35218 and S. aureus ATCC 25923 were prepared at 0.5 on the McFarland scale in 0.85% w/v NaCl solution, and the surface of both treated and untreated tomatoes was inoculated with 100 µL of each bacterial suspension. Bacterial counts were performed at zero, seven, fourteen, and twenty-one days. This inoculation was performed in triplicate for each bacterium and for each time point of analysis. In each case, the entire tomato was blended, and a homogenate was prepared from 1 g of the sample and sterile 0.85% w/v NaCl solution in a 1:10 ratio, with dilutions ranging from 10 − 1 to 10 − 5 . For E. coli enumeration, aliquots were plated in triplicate on eosin methylene blue agar (EMB), while Baird Parker medium (BP base) supplemented with egg yolk and 1% potassium tellurite solution was used for S. aureus enumeration. Plates were incubated for 22 ± 2 hours at 37 ± 1°C. Enumeration results were standardised to CFU/g of PF or log CFU/g of PF. 2.8. Statistical analysis All experiments were conducted in triplicate. The results were analysed using general linear models and mixed models with Infostat version 2020p. Adjusted means and standard errors (SE) were calculated for Treatment*Day. Fisher's LSD test (α = 0.05) was used for multiple comparisons. 3. RESULTS AND DISCUSSION 3.1 Antimicrobial activity of chitosan Chitosan solutions at 1% and 2.5% w/v exhibited antimicrobial activity against E. coli ATCC35218 and S. aureus ATCC25923 (Table 1 ). For E. coli , the inhibitory effect was dose dependent, with significant differences favouring higher concentrations of chitosan. Specifically, a concentration of 1% w/v resulted in an average inhibition zone diameter of 0.65 cm, whereas when chitosan at 2.5% w/v was evaluated, the mean diameter was 1 cm. In contrast, for S. aureus , there was no statistically significant variation in inhibition when the concentration of chitosan was increased, resulting in inhibition zones of 1 cm and 1.1 cm in diameter, respectively. These results are consistent with those reported by Uranga et al. (2018) and Wrońska et al . (2021) [ 17 , 18 ], who found that as the concentration of chitosan increased, the inhibition of E. coli DH5α and spoilage bacteria also increased in their respective studies. This antimicrobial effect of chitosan may be due to the interaction of the protonated free amino groups (-NH 3 + ) of chitosan (cationic nature due to its preparation in a weakly acidic medium) with the negative charges on the bacterial cell wall, which alters cell permeability and affects viability [ 19 ]. Furthermore, considering the structural differences in the cell walls of Gram positive and Gram negative bacteria, the response is expected to differ for the two strains tested. On the other hand, citric, ascorbic, and acetic acids, when evaluated individually or in combination at various concentrations, did not show antimicrobial activity, except for citric acid at concentrations greater than 0.75% w/v. The latter resulted in inhibition zones ranging from 0.6 to 0.8 cm in diameter for E. coli (Table 1 ). Concerning acids, their antimicrobial effectiveness is intricately linked to the pH of the solution and the presence of the acid in its undissociated form. The undissociated form exhibits enhanced permeability through the lipid membranes of bacterial cells, facilitating entry. Upon entry, it dissociates, resulting in cytoplasmic acidification and a subsequent reduction in intracellular pH. Hydrogen ions must be eliminated to counteract this effect, disrupting membrane function that gives rise to an inhibitory mechanism significantly impacting bacterial metabolism. This alteration modifies the hydrogen ion and electrical charge gradients with the exterior and interferes with amino acid and phosphate transport systems [ 20 , 21 ]. For this reason, when choosing an organic acid as an antimicrobial agent, pH and pKa (which provides an idea of the acid's strength and ability to dissociate) should be considered. Furthermore, there have been cases where Escherichia coli strains grew at pH levels close to 2, showing viability even after several hours of exposure [ 22 ]. In contrast, authors such as Gyawali et al. (2020) and Holloway et al. (2011) [ 23 , 24 ] have reported that using organic acids as sole components would not prevent the growth of Staphylococcus aureus . These results are also consistent with those found by Ibrahim et al. (2008) and Tajkarimi and Ibrahim (2011) [ 25 , 26 ], who observed insignificant effects on foodborne pathogens such as Salmonella and E. coli O157:H7 when using ascorbic or lactic acid at different concentrations [ 23 ]. Regarding the combination of acids and their effect as antimicrobials, the results obtained are consistent with those found by Al Rousan et al. (2018) [ 27 ], who studied combinations of citric and acetic acids at various concentrations and did not achieve complete inhibition of the growth of Escherichia coli O157:H7, Salmonella typhimurium , and Staphylococcus aureus after two days of incubation in tabbouleh salads. On the other hand, Tajkarim and Ibrahim (2011) [ 26 ] assessed the synergistic potential of the combination of ascorbic and lactic acids. While there was a significant reduction in the growth of E. coli O157:H7 in this study, they also found that the combination of these organic acids did not completely inhibit the development of this bacterium. Finally, glycerol did not affect the growth of the tested bacteria. These results were expected since the primary function of this compound is not to inhibit microbial growth but is often used to provide edible coatings with greater flexibility, resistance, and reduced brittleness [ 28 ]. When formulations that include the components evaluated individually were analysed, it was observed that the antimicrobial activity of chitosan was enhanced by the presence of acids in the formulation (Fig. 2 ), reflecting a synergistic effect between the components. In all cases, there was a greater inhibitory power on the growth of S. aureus , as the diameter of the inhibition zones was more significant than those formed in the presence of E. coli . To prevent the development of S. aureus , combinations of chitosan (1% w/v) with all three acids at 0.75% w/v (each) and with CitA and AscA at 0.1% w/v (each) yielded the best results, with average inhibition zones of 1.85 cm in diameter. Meanwhile, when chitosan and the acids were at 1% w/v in both formulations, comparable and statistically non-significant inhibition was observed between them. Regarding the inhibition of E. coli , the formulation containing CitA and AscA (each at 1% w/v) and the formulations with CitA, AscA, and AcA (each at 0.75% and 2% w/v) achieved inhibition zones of 0.9 cm in diameter. At the same time, the rest of the combinations ranged from 0.7 to 0.85 cm, respectively. These results align with other authors demonstrating the advantage of combining chitosan with various compounds to provide edible coatings with greater antimicrobial efficacy. In this regard, the combination of chitosan with cinnamon oil, lemon essential oil, and leaf extract of Sonneratia caseolaris is effective in microbiological control of strawberries, cherry tomatoes, and bananas [ 29 – 31 ]. Table 1 Antimicrobial effect of chitosan, ascorbic acid, citric acid, acetic acid, and glycerol. Component Concentration (% w/v) E. coli ATCC35218 (cm) S. aureus ATCC25923 (cm) Chitosan 1 0.60 ± 0,05 1 ± 0,1 2,5 1 ± 0,1 1.1 ± 0,1 AscA 0,1 NI NI 0,5 NI NI 0,75 NI NI 1 NI NI 2 NI NI CitA 0,1 NI NI 0,5 NI NI 0,75 0,7 ± 0,07 NI 1 0,65 ± 0,05 NI 2 0,8 ± 0,06 NI AcA 0,1 NI NI 0,5 NI NI 0,75 NI NI 1 NI NI 2 NI NI Glycerol 0,5 NI NI CitA + AscA + AcA 2 NI NI CitA + AscA 2 NI NI CitA + AcA 2 NI NI AscA + AcA 2 NI NI * AscA: ascorbic acid; CitA.: citric acid; AcA.: acetic acid; NI: no inhibition. **Data are presented as means ± standard deviation of three independent replicates. Means labelled with different letters differ (P < 0.05). 3.2. Antioxidant capacity of chitosan and formulations As mentioned earlier, the antioxidant activity of chitosan may be attributed to the ability of its free residual amino groups to react with the free radicals present in the environment. However, this activity is directly influenced by concentration, molecular weight, and degree of deacetylation. It increases when compounds with antioxidant properties are added, which can act through radical scavenging, hydrogen donation, metal chelation, and singlet oxygen quenching [ 32 , 33 ]. According to Fig. 3 , 1% chitosan solutions demonstrated an antioxidant capacity of approximately 30%. However, when combined with the evaluated acids, it tripled the percentage of inhibition, with an average of 92.34% I and values ranging from 89–96% I, confirming their significant synergy and the effectiveness of these combinations. These results are consistent with the literature. Akalin et al. (2021) [ 34 ] found that the combined use of chitosan, pectin, and silver nanoparticles inhibits the growth of Gram-negative bacteria such as E. coli . Debbabi et al. (2017) [ 35 ] demonstrated the positive effect of a coating formulated with 1% chitosan and 10% citric acid for controlling two Gram-positive bacteria ( S. aureus and S. epidermidis ) as well as two Gram-negative bacteria ( E. coli and P. aeruginosa ). 3.3. Evaluation of formulation behaviour on tomatoes Based on the results obtained regarding antimicrobial and antioxidant activity and considering the use of the minimum concentrations required to achieve a significant effect in these two studied capacities, the formulations of 1% w/v chitosan with the addition of 0.5% w/v glycerol and 1% w/v citric acid and ascorbic acid, and 1% w/v chitosan with the addition of 0.5% w/v glycerol and 0.75% w/v citric acid, ascorbic acid, and acetic acid were evaluated. The formulation containing acetic acid among its components was observed to have detrimental effects on the tomato epidermis after seven days of application (Fig. 4 ) and favoured the proliferation of fungi. Conversely, formulations without acetic acid did not show any harmful effects on the epidermis of the treated tomatoes. Consequently, it was decided to discard the formulations designed with acetic acid and continue the studies on the formulation containing 1% w/v chitosan with the addition of 0.5% w/v glycerol and 1% w/v citric acid and ascorbic acid. 3.4. Evaluation of the antifungal activity of the formulation Once the formulations containing acetic acid were discarded, the effect of using only 1% w/v chitosan and its combinations of citric and ascorbic acid was evaluated. Finally, formulations designed with 1% w/v chitosan, the addition of 0.5% v/v glycerol, and citric and ascorbic acids (ranging from 0.1 to 2% w/v) were also assessed for their impact on the growth of Alternaria tenuissima (EGS 34015) and Alternaria arborescens (EGS 39128). When analysing the combination of acids, complete inhibition of mycelial growth in A. tenuissima was observed at a concentration of 1% w/v. For the rest of the solutions evaluated, the growth of these fungi varied between 10% and 40% (Fig. 5 ). Regarding A. arborescens (EGS 39128), complete inhibition was achieved starting from a concentration of 0.75% w/v of acids, while with acid mixtures at 0.1% and 0.5% w/v, mycelial growth inhibition was 61% and 83%, respectively (Fig. 5 ). Regarding using chitosan (1% w/v) as the sole component and combined with the evaluated organic acids, mycelial growth inhibition was 100% for all proposed concentrations and tested strains (Fig. 5 ). On one hand, this effect could be explained by the fact that chitosan may act as a chelating agent for metals and essential nutrients, which weaken fungi and inhibit their growth [ 36 , 37 ]. On the other hand, chitosan in combination with acids may affect the permeability of the fungal cell membrane, leading to the breakdown of the cell structure. Furthermore, chitosan forms a protective layer on the surface of the tomatoes, acting as a barrier that limits pathogen access and enhances the overall effectiveness of the treatments. Other authors have observed damage and deformation of hyphae and spores of Alternaria when growing on the surface of fruits pre-treated with chitosan [ 38 ]. In this context, Guo et al. 2020 [ 39 ] proposed that the mechanism of action of submicron chitosan dispersions on Alternaria alternata involves an intracellular action mechanism that allows chitosan to accumulate within the cell or associate with the cell wall, cell membrane, or septum, causing cellular damage that affects hyphal development and spore germination. Furthermore, Das et al. (2023) [ 40 ] suggested that the mechanism of action of chitosan combined with essential oil from Angelica archangelica would primarily target the plasma membrane against Botrytis cinerea . 3.5 Bacterial growth curve of bacteria exposed to the formulation. While the antimicrobial activity against S. aureus and the Alternaria strains proved effective with the formulation of 1% chitosan, 0.5% glycerol, and 0.1% citric and ascorbic acids each, in the case of E. coli , a concentration of acids of at least 1% was required to achieve more significant inhibition. Therefore, the formulation of 1% chitosan, 0.5% glycerol, and 1% citric, and ascorbic acids (EC) was selected for evaluating its effect on the growth of S. aureus ATCC 25923 and E. coli ATCC35218 strains. When the strains were grown in LB broth supplemented with chitosan, glycerol, ascorbic and citric acids to reach the EC concentrations, a significant inhibition of microbial growth was observed, preventing them from reaching the exponential growth phase during the tested period. In contrast, in the control culture (unsupplemented LB), both bacteria multiplied until reaching the stationary phase (Fig. 6 ) within seven to eight hours of incubation. This assay confirmed the antimicrobial effectiveness of the selected formulation as an edible coating (EC). Furthermore, in this context, the addition of agents to chitosan-based edible coatings has shown antimicrobial effectiveness. Specifically, the introduction of clove and cinnamon essential oils significantly enhanced the antibacterial and antibiofilm properties of the material against Escherichia coli , Enterococcus hirae , Staphylococcus aureus , and Pseudomonas aeruginosa . Biofilm inhibition values ranged from 69.76–96.97%, depending on the bacterial species [ 41 ]. These findings highlight the potential of chitosan-based formulations, both in edible coatings and active films, to improve food safety and preservation. Moreover, several studies have investigated the mechanisms of action of organic acids on bacteria, including the alteration of intracellular pH, protein denaturation, and membrane damage, which contribute to bacterial death. However, bacteria employ detoxification mechanisms such as efflux pumps and metabolic adjustments to resist acid stress [ 42 ]. Additionally, the combination of organic acids with chitosan may enhance the antimicrobial efficacy by modifying the permeability of the bacterial cell membrane, thereby limiting these detoxification responses and improving the overall inhibitory effect. 3.6. Effectiveness of the applied coating on tomatoes 3.6.1. Effect of coating application on indicator microorganisms in tomatoes. One of the primary causes of food deterioration is microbiological activity, leading to a loss of food quality. As tomatoes ripen, there is a progressive increase in microorganisms, particularly moulds and yeasts. Therefore, using coatings composed of potential microbial inhibitors and controllers is of interest for food preservation. In this regard, when the coating was applied to freshly harvested tomatoes, it was observed that, although the coated fruits exhibited a lower count of total mesophilic aerobes and coliforms compared to the control, the differences were not statistically significant over the test period (Fig. 7 , a and b). As for the variable of moulds and yeasts, as the monitoring period progressed, the cfu/g (Colony Forming Units per gram) increased. Only on the seventh day post treatment were statistically significant differences observed with lower counts in the coated tomatoes (Fig. 7 c). Other authors have found similar behaviours in tomatoes coated with sapote gum with chitosan (1%) or with a solution of alginate, chitosan, and Flourensia cernua extract, including a delay in the appearance of microorganisms, as observed in this study for total coliforms [ 43 , 44 ]. On the other hand, pomegranates coated with chitosan and ascorbic acid [ 45 ] showed a significant reduction in populations of total mesophilic aerobes, yeasts, and moulds during the storage time (28 days). The combination of chitosan and organic acids, which are part of the formulated mixture in this study, results in the presence of positively charged free amino groups. This creates an environment that affects the integrity of the microbial cell wall, leading to changes in membrane permeability and disrupting normal cellular function. Consequently, the counts obtained from treated samples are lower. However, mechanisms of detoxification by bacteria in response to antimicrobial compounds have been identified, which could explain why the differences between treated and control groups may not be statistically significant [ 46 ]. 3.6.2. Effect of coating application on bacterial inoculants on tomatoes Additionally, the antimicrobial effect of EC application on tomatoes was evaluated when intentionally inoculated with S. aureus ATCC 25922 or E. coli ATCC 35218 on the fruit's surface. Concerning the Gram positive strain, a favourable effect was observed on day seven post inoculation, as 2 logs reduced the count in the treated samples compared to the initial load. In contrast, in the untreated tomatoes, the population increased by at least 1 log (Fig. 8 a). Regarding the inoculation with E. coli , the treated tomatoes maintained the microbial load from the beginning of the assay until day seven. At the same time, the controls showed a growth of 0.4 logs, resulting in a statistically significant difference in favour of the EC application (Fig. 8 b). On the day fourteen post inoculation, neither the control samples nor those inoculated with both bacteria exhibited microbial growth. This highlights the effectiveness of using the coating in controlling the proliferation of model microorganisms commonly found in food, as used in this assay. In this regard, Jin et al. (2022) [ 47 ] indicated that chitosan treatment on tomatoes inoculated with Salmonella enterica and Listeria monocytogenes demonstrates antimicrobial effectiveness, as it reduced the bacterial population to non-detectable levels by the second day following treatment. 3.7 Antioxidant capacity in coated tomatoes Regarding antioxidant activity, the coated tomatoes showed an increase in their ability to inhibit the DPPH radical, both when compared to untreated samples and in terms of their evolution throughout the storage period. In other words, the percentage of DPPH radical inhibition (%I) was significantly higher than the treated samples for all days evaluated (Table 2 ). This increase is likely due to the presence of ascorbic acid as part of the formulation, as this compound is a potent antioxidant, and as demonstrated in this study, the proposed formulation for the EC exhibits a high antioxidant capacity. Other researchers have shown similar trends in tomatoes when using chitosan-based coatings with other agents, such as aloe vera and mint essential oil [ 33 , 48 ]. Likewise, Zhou et al. (2023) [ 49 ] demonstrated that the catechol-functionalized chitosan coating effectively preserves fruit quality by maintaining firmness and colour, reducing weight loss, and providing antioxidant protection, thereby extending the shelf life of strawberries and bananas. Similar results were found by Umbayda et al. (2024) [ 50 ], who tested the efficacy of a chitosan and macadamia nut oil mixture to preserve the antioxidant properties and physicochemical characteristics of tomatoes. Additionally, when Das et al. (2023) [ 40 ] evaluated the use of chitosan combined with essential oil from Angelica archangelica on table grapes ( Vitis vinifera L.), they found that this coating was effective in preserving enzymatic antioxidants, reducing respiration rates, and enhancing sensory quality Overall, these studies underscore the significant role that chitosan-based coatings play in enhancing the antioxidant capacity and quality of fruits during storage, highlighting their potential as effective preservation strategies in the agricultural industry. Table 2 Antioxidant capacity was measured as % inhibition of DPPH (%I) for tomatoes coated with 1% chitosan, 0.5% glycerol, 1% citric acid, and 1% ascorbic acid, as well as the control, stored for 21 days. Time (days) Control EC 0 31,6 ± 1,8 b 43,2 ± 2,9 a 7 53,1 ± 0,9 a 50,1 ± 2,3 b 14 57,8 ± 0,8 b 61,1 ± 2,5 a 21 64,0 ± 0,7 b 68,9 ± 3,6 a * Data are presented as means ± standard deviation of three independent replicates. Means labelled with different letters differ (P < 0.05). 4. CONCLUSION The chitosan-based coating formulated with citric and ascorbic acids offers a promising approach to enhancing tomato shelf life and microbial safety. This study highlights the synergistic effect of chitosan and organic acids, which not only inhibits microbial growth but also maintains the antioxidant capacity of the tomatoes throughout storage. These findings suggest that this eco-friendly edible coating could serve as a sustainable solution to minimize food waste, improve the quality of fresh produce, and potentially benefit the fresh produce industry by meeting increasing demands for food safety and quality. Declarations Funding Declaration This research was funded by Faculty of Agronomy, National University of the Center of the Province of Buenos Aires. Data availability statement The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Author Contribution A.P.G Investigation (Equal) Methodology (Equal) Writing - original draft (Equal)M.A.D Formal analysis (Equal) Methodology (Equal) Visualization (Equal)K.E.D Formal analysis (Equal) Visualization (Equal)R.K.N Conceptualization (Lead) Formal analysis (Lead) Supervision (Lead) Writing - review & editing (Equal) References Janghu S, Kumar V, Yadav A (2024) Post-Harvest Management of Fruits and Vegetables. Perspectivas actuales en agricultura y ciencia de los alimentos, vol. 7, 125–148. Ed. BP International Publisher, Bengala Occidental, India 10.9734/bpi/cpafs/v7/7984E Trombino S, Cassano R, Procopio D, Di Gioia ML, Barone E (2021) Valorization of Tomato Waste as a Source of Carotenoids. 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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-6613506","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":458245152,"identity":"f07ba5be-38db-4c55-9069-7c7a16e2ab0c","order_by":0,"name":"Andrea Paola Guisolis","email":"","orcid":"","institution":"Universidad Nacional del Centro de la Provincia de Buenos Aires, Facultad de Agronomía, CAIVA","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"Paola","lastName":"Guisolis","suffix":""},{"id":458245153,"identity":"5040f552-89dc-4936-b8c5-29275ff9ff04","order_by":1,"name":"María de los Ángeles Dublan","email":"","orcid":"","institution":"Universidad Nacional del Centro de la Provincia de Buenos Aires, Facultad de Agronomía, CAIVA","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"de los Ángeles","lastName":"Dublan","suffix":""},{"id":458245154,"identity":"7dfad87d-6152-4868-89fc-eecd1ce48504","order_by":2,"name":"Eliana Castañares","email":"","orcid":"","institution":"Universidad Nacional del Centro de la Provincia de Buenos Aires, Facultad de Agronomía, BIOLAB","correspondingAuthor":false,"prefix":"","firstName":"Eliana","middleName":"","lastName":"Castañares","suffix":""},{"id":458245155,"identity":"b4c83f5b-bee2-41bc-8672-2b59e51d8e2c","order_by":3,"name":"Karina Elizabeth Díaz","email":"","orcid":"","institution":"Universidad Nacional del Centro de la Provincia de Buenos Aires, Facultad de Agronomia","correspondingAuthor":false,"prefix":"","firstName":"Karina","middleName":"Elizabeth","lastName":"Díaz","suffix":""},{"id":458245156,"identity":"c8b4bb1c-9e11-46dd-a69f-de56acc27986","order_by":4,"name":"Rosa Karina Nesprias","email":"data:image/png;base64,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","orcid":"","institution":"Universidad Nacional del Centro de la Provincia de Buenos Aires, Facultad de Agronomía, CAIVA","correspondingAuthor":true,"prefix":"","firstName":"Rosa","middleName":"Karina","lastName":"Nesprias","suffix":""}],"badges":[],"createdAt":"2025-05-07 15:38:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6613506/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6613506/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83038597,"identity":"4c653d24-1964-4243-ab70-d364e5399807","added_by":"auto","created_at":"2025-05-19 10:25:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":440370,"visible":true,"origin":"","legend":"\u003cp\u003eSample of tomatoes harvested and transported to the laboratory of the Faculty of Agronomy, UNCPBA.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6613506/v1/adc286db9aa183a972d16ebc.png"},{"id":83038601,"identity":"3eb4acbd-fec8-4313-9a7a-ee5b3e8f9efa","added_by":"auto","created_at":"2025-05-19 10:25:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":34485,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntimicrobial activity of the formulations\u003c/strong\u003e. Inhibitory effect on the growth of a) \u003cem\u003eE. coli\u003c/em\u003e ATCC 35218 and b) \u003cem\u003eS. aureus\u003c/em\u003e ATCC 25923 using formulations based on 1% w/v chitosan, 0.5% w/v glycerol, and the addition of different combinations of evaluated acids at concentrations ranging from 0.1 to 2% w/v (Citric: C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e, Ascorbic: C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e, and Acetic: C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e\n\u003cp\u003eData are presented as means ± standard deviation of three independent replicates. Means labelled with different letters differ (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6613506/v1/6914d4bf5bdefa52319feb92.png"},{"id":83040871,"identity":"a31f2a18-38a5-41ff-ad66-6da44cbd0b70","added_by":"auto","created_at":"2025-05-19 10:41:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":37680,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntioxidant capacity of chitosan and formulations\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e. \u003c/strong\u003e\u003c/em\u003eDPPH inhibition of chitosan 1% (w/v) and formulations based on 1% (w/v) chitosan, 0.5% (v/v) glycerol, and the addition of different combinations of evaluated acids at concentrations ranging from 0.1 to 2% w/v (Citric: C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e, Ascorbic: C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e, and Acetic: C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). Data are presented as means ± standard deviation of three independent replicates. Means labelled with different letters differ (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6613506/v1/b5b0f7733c9d3728676e2135.png"},{"id":83038604,"identity":"277a7466-b90f-4f24-8054-6150e8f27820","added_by":"auto","created_at":"2025-05-19 10:25:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":445028,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of coating on tomato characteristics.\u003c/strong\u003eRepresentative images depicting the effects of applying edible coatings on tomatoes are presented at 7 days. These coatings include: a) 1% w/v chitosan, 0.5% w/v glycerol, and a combination of acids (Citric Acid + Ascorbic Acid + Acetic Acid at 0.75%), b) 1% w/v chitosan, 0.5% w/v glycerol, and a combination of citric acid + ascorbic acid at 1%, and c) a control group without coating. The images were captured seven days after application.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6613506/v1/6a29437a322af13c4351190a.png"},{"id":83039178,"identity":"bbf74cb0-f106-4b9e-8c03-ff11a0fd7161","added_by":"auto","created_at":"2025-05-19 10:33:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":26853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFungal mycelium inhibition by the coated formulations. \u003c/strong\u003eInhibition of radial mycelial growth in \u003cem\u003eA. tenuissima\u003c/em\u003e (EGS 34015) and \u003cem\u003eA. arborescens (\u003c/em\u003eEGS 39128) exposed to mixtures of organic acids (citric and ascorbic). Data are presented as means ± standard deviation of three independent replicates. Means labelled with different letters differ (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6613506/v1/cfb0c6512e1b6501e004c4e9.png"},{"id":83039180,"identity":"7e4c555b-8732-45db-8e67-5f1ae0955c31","added_by":"auto","created_at":"2025-05-19 10:33:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":19546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of coating formulation on bacterial development. \u003c/strong\u003eGrowth curves of \u003cem\u003eS. aureus \u003c/em\u003eATCC25923 (a) and \u003cem\u003eE. coli\u003c/em\u003e ATCC35218 (b) in Luria Bertani broth without supplementation (squares) and supplemented (dots) with chitosan (1%), glycerol (0.5%), and citric and ascorbic acids (1% each). Data are presented as means ± standard deviation of three independent replicates. Means labelled with different letters differ (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6613506/v1/f4b5b0aaeecccdbdc0951a40.png"},{"id":83038602,"identity":"2f5a8491-ab75-45d0-9cc7-684510ee43f0","added_by":"auto","created_at":"2025-05-19 10:25:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":22698,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicrobiological quality of coated tomatoes.\u003c/strong\u003e Total mesophilic aerobic counts (a), total coliform counts (b), and yeast and mould counts (c) in tomato samples coated with chitosan (1%), glycerol (0.5%), citric acid (1%), and ascorbic acid (1%), as well as the control, during 21 days of storage. ND indicates that bacteria were not detected at the time of testing. Data are presented as means ± standard deviation of three independent replicates. Means labelled with different letters differ (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6613506/v1/fb6973c745e648700229cfc8.png"},{"id":83038605,"identity":"01444557-1bb8-4004-aed8-7d091cd2262c","added_by":"auto","created_at":"2025-05-19 10:25:35","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":24504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of edible coating on the proliferation of bacteria in tomatoes.\u003c/strong\u003e Counts of \u003cem\u003eS. aureus\u003c/em\u003e ATCC 25923 (a) and \u003cem\u003eE. coli \u003c/em\u003eATCC 35218 (b) inoculated in tomato samples coated with chitosan (1%), glycerol (0.5%), citric acid (1%), and ascorbic acid (1%), as well as the control, stored for 21 days. ND indicates that bacteria were not detected at the time of testing. Data are presented as means ± standard deviation of three independent replicates. Means labelled with different letters differ (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6613506/v1/c1a20c501da44bf595918754.png"},{"id":95000853,"identity":"83e2ffe3-bcf1-45ef-8d1b-607eb923c554","added_by":"auto","created_at":"2025-11-03 09:00:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2990159,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6613506/v1/fa62ad2e-a090-4eae-8ec2-a38586fb9f9e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synergistic Effects of Chitosan and Organic Acids on the Antimicrobial, Antifungal and Antioxidant Properties of Edible Coatings","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003ePost-harvest losses in horticultural crops primarily stem from handling during the journey from harvest to consumer. Various factors contribute to their degradation, including mechanical damage, inadequate storage, and poor handling and transportation practices [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. While tomatoes (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) are among the most widely cultivated and consumed vegetables globally, their production results in significant losses and waste [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Changes in fruit quality can be mechanical, physiological, or pathological. Mechanical damage can lead to visual abnormalities, increased respiratory activity, and altered metabolism, significantly affecting the physicochemical composition of the pericarp and locular tissue, as well as their overall sensory characteristics. Additionally, water loss after harvesting adversely impacts the quality of these fruits, which is closely linked to temperature, relative humidity, cultivar, and storage duration [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Moreover, microbial attacks from organisms such as \u003cem\u003eAlternaria alternata\u003c/em\u003e, \u003cem\u003eBotrytis cinerea\u003c/em\u003e, \u003cem\u003eGeotrichum\u003c/em\u003e, \u003cem\u003eRhizopus stolonifer\u003c/em\u003e, and bacteria like \u003cem\u003eErwinia spp.\u003c/em\u003e are responsible for the rapid deterioration of tomatoes, leading to economic losses [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this context, exploring post-harvest management alternatives for fruits and vegetables is essential to extend their shelf life, ensuring a safe and high-quality product supply. While various environmentally friendly technologies have been tested, the ideal approach includes strategies that protect the environment and minimise waste production. Our research, alongside studies such as those by Fukuyama \u003cem\u003eet al.\u003c/em\u003e (2024) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], focuses on reducing food waste and post-harvest losses in alignment with UN Sustainable Development Goal 12 (Responsible Production and Consumption), which aims to halve global food waste per capita by 2030. This objective can be achieved by repurposing materials typically regarded as waste to produce novel compounds of interest or to extend the shelf life of perishable products. Edible coatings have emerged as a promising strategy for prolonging the shelf life of fresh and freshly cut fruits and vegetables [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These coatings can be made from proteins, carbohydrates, lipids, or a combination of these, often incorporating additional components such as antimicrobial and antioxidant agents. They can be applied through spraying or immersion. Functionally, edible coatings reduce water loss and browning, decrease ethylene production, enhance appearance, and extend commercial viability [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, these coatings must exhibit mechanical properties that ensure proper adhesion to the food and withstand handling without damage [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The availability of environmentally compatible biopolymers for formulating edible coatings presents a significant challenge for the modern agri-food industry. In this regard, chitosan\u0026mdash;a compound obtained through the deacetylation of chitin, primarily sourced from crustacean exoskeletons\u0026mdash;represents a promising alternative due to its natural biodegradability, low toxicity, ease of availability, and ability to trigger defence responses in plant tissues. Additionally, it possesses strong antioxidant properties that can eliminate free radicals and serves as a matrix for incorporating other substances, enhancing antimicrobial and antioxidant capabilities [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile some researchers have explored the effects of various edible coatings on preserving the physicochemical attributes of tomatoes, further investigation is needed on the synergistic combination of chitosan with other components, such as organic acids, for these applications. Therefore, the objective of this study was to design a formulation of edible coating based on chitosan, citric acid, ascorbic acid, and acetic acid, aiming to exhibit in vitro antimicrobial activity and evaluate its effectiveness in preserving the microbiological quality of tomatoes during refrigerated post-harvest storage.\u003c/p\u003e"},{"header":"2. MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1.1 Materials\u003c/h2\u003e \u003cp\u003e \u003cb\u003eChitosan\u003c/b\u003e: Sigma Aldrich Chemical Co., Germany. \u003cb\u003eGlacial acetic acid\u003c/b\u003e: Biopack, Argentina. \u003cb\u003eGlycerol\u003c/b\u003e: Anedra, Argentina. \u003cb\u003eCitric Acid\u003c/b\u003e: Biopack, Argentina. \u003cb\u003eAscorbic acid\u003c/b\u003e: Biopack, Argentina. \u003cb\u003eEscherichia coli\u003c/b\u003e \u003cb\u003eATCC35218\u003c/b\u003e and \u003cb\u003eStaphylococcus aureus\u003c/b\u003e \u003cb\u003eATCC 25923\u003c/b\u003e: American Type Culture Collection, USA. \u003cb\u003eLuria Bertani Broth\u003c/b\u003e (LB): Britania, Argentina. \u003cb\u003eNaCl\u003c/b\u003e Biopack, Argentina. \u003cb\u003eM\u0026uuml;ller Hinton agar\u003c/b\u003e: Oxoid LTD, England. \u003cb\u003eAlternata arborescens\u003c/b\u003e \u003cb\u003e(EGS39128)\u003c/b\u003e and \u003cb\u003eAlternata tenuissima\u003c/b\u003e \u003cb\u003e(EGS 34015)\u003c/b\u003e: Dr. Emmory G. Simmons, Indiana, USA. \u003cb\u003eModified Potato Dextrose Agar: MPDA\u003c/b\u003e: Britannia, Argentina. \u003cb\u003eDPPH\u003c/b\u003e: Sigma Aldrich, USA. \u003cb\u003eMethanol\u003c/b\u003e: Anedra, Argentina. \u003cb\u003ePlate count agar- PCA\u003c/b\u003e: Britania, Argentina. \u003cb\u003eViolet red bile agar-VRBA\u003c/b\u003e: Britania, Argentina. \u003cb\u003eGlucose potato agar-GPA\u003c/b\u003e: Britania, Argentina. \u003cb\u003eChloramphenicol\u003c/b\u003e: Sigma Aldrich, USA. \u003cb\u003eEosin methylene blue agar-EMB\u003c/b\u003e: Britania, Argentina. \u003cb\u003eBaird Parker medium-BP base\u003c/b\u003e: Britania, Argentina. \u003cb\u003ePotassium tellurite\u003c/b\u003e: Britania, Argentina. \u003cb\u003eEeosin methylene blue agar-EMB\u003c/b\u003e: Britania, Argentina.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2 Formulation preparation\u003c/h2\u003e \u003cp\u003eChitosan solutions at 1% and 2.5% w/v were prepared by weighing 10 g and 25 g of the compound (low molecular weight, 95000 Da, 92% DD-Deacetylation Degree), respectively. In each case, they were mixed with 10 mL of glacial acetic acid and 800 mL of distilled water, with magnetic stirring until complete dissolution.\u003c/p\u003e \u003cp\u003eVarious coating formulations containing glycerol (0.5% v/v) were prepared to enhance their plasticizing properties. The properties of different combinations of citric, ascorbic, and acetic acids at various concentrations were investigated. Before bringing to final volume, these agents were added to the chitosan and glycerol solution in adequate quantities to achieve the study concentrations. The systems were mixed and shaken until fully dissolved, reaching the specified final volume.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.3 Sample Collection and Tomato Conditioning\u003c/h2\u003e \u003cp\u003eRound-type tomatoes (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) grown in a non-climate-controlled greenhouse were harvested in a horticultural facility located in the city of Azul, Province of Buenos Aires (36\u0026deg;47\u0026prime;00\u0026Prime;S 59\u0026deg;51\u0026prime;00\u0026Prime;W). When they reached physiological ripeness, they were randomly selected with a grade 2 colour tone on the ripeness colour chart. Tomatoes of uniform size, with no physical damage and free of disease were chosen.\u003c/p\u003e \u003cp\u003eThe fruits were taken to the CAIVA Laboratory (Food Quality, Safety and Added Value), Faculty of Agronomy of Azul, UNCPBA, where they were washed by immersing them in tap water at 20\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and then placed on a worktable on absorbent paper for air-drying at room temperature. In cases where bacteria were intentionally inoculated to evaluate behaviour, the tomatoes were disinfected with a 1% sodium hypochlorite solution and washed with sterile distilled water.\u003c/p\u003e \u003cp\u003eA batch of 100 tomatoes was initially used for the different experimental determinations \u003cem\u003ea posteriori\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A completely randomised design was used to select the samples, dividing the harvested tomatoes into two groups.\u003c/p\u003e \u003cp\u003eThe collection of plant material (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e, tomato), complied with relevant institutional, national, and international guidelines and legislation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Bacterial and fungal strains, and culture conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC35218 and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923 were the model strains used in this work as Gram negative and Gram positive, respectively. The strains were cultivated overnight in Luria Bertani Broth (LB) at 36\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C (Tecno Dalvo Cultivation Oven) For the experiments, both bacterial cells were collected by centrifugation (Presvac, DCS-16-RC) at 4000 rpm for 5 min, rinsed with sterile NaCl solution (0.85% m/V), and resuspended to obtain an equivalent to the 0.5 McFarland scale (Turbidity Standard). Inoculants prepared in this way were used in the subsequent assays.\u003c/p\u003e \u003cp\u003eSingle spore isolates of \u003cem\u003eAlternaria arborescens\u003c/em\u003e EGS39128 and \u003cem\u003eA. tenuissima\u003c/em\u003e EGS 34015 (Dr. Emmory G. Simmons, Indiana, USA) were cultivated on plates containing Potato Carrot Agar (PCA) according to Simmons (2007) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and incubated at 23\u0026deg;C under an alternating cycle of 8 hours of cool white fluorescent daylight and 16 hours of darkness for 7 days (Briket M4200 Growth chamber). From these plates, 4 mm diameter mycelial discs were obtained using a sterile punch for use in subsequent assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Antibacterial activity\u003c/h2\u003e \u003cp\u003eThe antibacterial activity of the individual components of the formulation was determined by the well diffusion method on M\u0026uuml;ller Hinton agar [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] against \u003cem\u003eE. coli\u003c/em\u003e ATCC 35218 and \u003cem\u003eS. aureus\u003c/em\u003e ATCC25923. First, a 0.5 Mc Farland scale inoculum, as indicated in section \u003cspan refid=\"Sec6\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e, was swabbed on the agar surface and then 50 \u0026micro;L of each solution was put on a well of 6 mm diameter. Then, agar plates were incubated at 36\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C (Tecno Dalvo Cultivation Oven) for 24 hours. After incubation, the inhibition was interpreted as the agar surface area without bacterial growth around the well. Two measurements were taken perpendicular to each other at the zone of inhibition of growth using a digital calliper (Hamilton), and the values were averaged. The solutions tested were chitosan at 1 and 2,5% m/V and citric and ascorbic acids at 0.1; 0.5; 0.75, 1 and 2% m/V, individually and combined (chitosan-citric acid; chitosan-acetic acid; chitosan- ascorbic acid; chitosan- ascorbic- acetic acid; chitosan- ascorbic- citric acid; chitosan- acetic- citric acid; chitosan- citric - ascorbic acid). This assay was employed as a screening method to define the solutions to be further evaluated, and the determinations were conducted in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Determination of radial mycelial growth inhibition.\u003c/h2\u003e \u003cp\u003eThe effect of chitosan on the mycelial growth of \u003cem\u003eAlternata arborescens\u003c/em\u003e (EGS39128) and \u003cem\u003eAlternata tenuissima\u003c/em\u003e (EGS 34015) was determined by inoculating a 4 mm diameter mycelial disc of both fungi on Modified Potato Dextrose Agar (MPDA) containing chitosan (1%), a combination of ascorbic and citric acids (0.1%, 0.5%, 0.75%, 1%, and 2% each), and chitosan (1%) combined with acids (0.1%, 0.5%, 0.75%, 1%, and 2%). This procedure was performed in triplicate, and the plates were incubated at 30\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. After seven days, the mycelial diameter was measured on plates exposed to the evaluated agents and control plates. The variation in diameter was measured using a Truper vernier calliper. The activity of the agents against the fungal cultures was calculated using the following equation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]:\u003c/p\u003e \u003cp\u003e \u003cb\u003e%Inhibition: [1 - (dt/dc)] \u0026times; 100\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWhere dc is the average diameter of mycelial growth in the control, and dt is the average diameter in the treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Assessment of free radical detoxifying effect against DPPH\u003c/h2\u003e \u003cp\u003eThe DPPH free radical scavenging detoxifying effect was performed following the method described by Hsu et al. (2003) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], with some modifications. For this purpose, a 0.5 mL aliquot of the methanolic extract of tomato was mixed with 2.5 mL of a cold methanolic solution containing 0.1 mM DPPH, and it was kept in the dark at 4\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u0026ordm;C for 60 minutes. This procedure was carried out in triplicate. The absorbance of the reaction mixture was measured at a wavelength of 517 nm using an UV-visible spectrophotometer (Biochrom Libra S22). Methanol at 80% was used as a blank instead of the extract. The detoxifying free radical inhibition effect of DPPH was calculated using the following formula:\u003c/p\u003e \u003cp\u003e \u003cb\u003eDPPH detoxifying effect (%I) = [1 - (A\u003c/b\u003e \u003csub\u003e \u003cb\u003e517\u003c/b\u003e \u003c/sub\u003e \u003cb\u003esample/A\u003c/b\u003e \u003csub\u003e \u003cb\u003e517\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eblank)] x 100\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe evaluated solutions were composed of chitosan at 1% and 2.5% w/v, glycerol at 0.5% w/v, and/or different combinations of citric acid (CitA) and ascorbic acid (AscA) at various concentrations (0.1%, 0.5%, 0.75%, 1%, and 2% w/v, respectively).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Bacterial growth curve\u003c/h2\u003e \u003cp\u003eOnce the coating formulation that yielded the most desirable responses was selected, a growth curve was conducted for the bacteria \u003cem\u003eE. coli\u003c/em\u003e ATCC35218 and \u003cem\u003eS. aureus\u003c/em\u003e ATCC25923 in LB broth (control) and LB broth supplemented with chitosan and ascorbic and citric acids in the proportions that showed promising results in terms of their antimicrobial and antioxidant capacity. The optical density at 600 nm was recorded (using a UV-visible spectrophotometer Biochrom Libra S22) every 60 minutes during incubation with agitation at 36\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C (Tecno Dalvo Cultivation Oven). The determinations were conducted in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Effect of the designed formulation on Solanum lycopersicum (tomato)\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1. Application of the coating on tomato\u003c/h2\u003e \u003cp\u003eRound type \u003cem\u003eSolanum lycopersicum\u003c/em\u003e samples, grown in an uncontrolled greenhouse environment, were sourced from a local producer (36\u0026deg;47\u0026prime;00\u0026Prime;S 59\u0026deg;51\u0026prime;00\u0026Prime;W) at the physiologically mature stage corresponding to a \"pint\u0026oacute;n\" colour tone (grade 2 on the harvest maturity colour chart). The tomatoes were harvested without physical damage, disease free, and with uniform sizes.\u003c/p\u003e \u003cp\u003eThey were prepared by immersing them in tap water at 20\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and then air drying on absorbent paper at room temperature. Subsequently, the designed and selected edible coating was applied to the samples by spraying it evenly over the entire surface and allowed to dry at room temperature (20\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C). This procedure of spraying the coating on the tomatoes was performed in duplicate, with airing done before the second application, as mentioned.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2. DPPH free radical detoxifying effect (%I) in tomatoes\u003c/h2\u003e \u003cp\u003eThe plant sample was ground using a grinder (Liliana AM680, 650 W), 1 g of the sample was weighed, and 2.5 mL of a methanol: acetic acid solution (40:1) was added. Extractions were carried out by ultrasonic bath agitation (Testlab TB04TA) for 15 minutes. Subsequently, the mixture was centrifuged (Presvac, DCS-16-RC) for 5 minutes at 4000 rpm at 4\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, and the supernatant was reserved. This process was repeated twice, and the obtained extracts were combined. The volume of the centrifuged supernatant was measured. The DPPH free radical detoxifying effect was performed following the method described in section \u003cspan refid=\"Sec9\" class=\"InternalRef\"\u003e2.5\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.7.3. Enumeration of indicator microorganisms in tomatoes\u003c/h2\u003e \u003cp\u003eUntreated and coated samples were separately ground under aseptic conditions to prepare a homogenate of 1 g of tomato in a 0.85% w/v NaCl solution. Subsequently, 10 fold dilutions were prepared from 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e. Aliquots were plated in triplicate on agar plates for plate count agar (PCA), violet red bile agar (VRBA), and glucose potato agar (GPA) supplemented with chloramphenicol (0.05% w/v) for the enumeration of total mesophilic aerobes, total coliforms, and yeasts and moulds, respectively. Plates were incubated at the specified temperature and time for each group of microorganisms: total mesophilic aerobes for 24 hours at 30\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, total coliforms for 18\u0026ndash;24 hours at 35\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, and yeasts and moulds for five days at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. The enumeration results were reported as CFU/g PF or log CFU/g PF.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.7.4. Inoculation of tomatoes with reference bacteria\u003c/h2\u003e \u003cp\u003eInoculums of \u003cem\u003eE. coli\u003c/em\u003e ATCC 35218 and \u003cem\u003eS. aureus\u003c/em\u003e ATCC 25923 were prepared at 0.5 on the McFarland scale in 0.85% w/v NaCl solution, and the surface of both treated and untreated tomatoes was inoculated with 100 \u0026micro;L of each bacterial suspension. Bacterial counts were performed at zero, seven, fourteen, and twenty-one days. This inoculation was performed in triplicate for each bacterium and for each time point of analysis. In each case, the entire tomato was blended, and a homogenate was prepared from 1 g of the sample and sterile 0.85% w/v NaCl solution in a 1:10 ratio, with dilutions ranging from 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e. For \u003cem\u003eE. coli\u003c/em\u003e enumeration, aliquots were plated in triplicate on eosin methylene blue agar (EMB), while Baird Parker medium (BP base) supplemented with egg yolk and 1% potassium tellurite solution was used for \u003cem\u003eS. aureus\u003c/em\u003e enumeration. Plates were incubated for 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2 hours at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. Enumeration results were standardised to CFU/g of PF or log CFU/g of PF.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were conducted in triplicate. The results were analysed using general linear models and mixed models with Infostat version 2020p. Adjusted means and standard errors (SE) were calculated for Treatment*Day. Fisher's LSD test (α\u0026thinsp;=\u0026thinsp;0.05) was used for multiple comparisons.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Antimicrobial activity of chitosan\u003c/h2\u003e \u003cp\u003eChitosan solutions at 1% and 2.5% w/v exhibited antimicrobial activity against \u003cem\u003eE. coli\u003c/em\u003e ATCC35218 and \u003cem\u003eS. aureus\u003c/em\u003e ATCC25923 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For \u003cem\u003eE. coli\u003c/em\u003e, the inhibitory effect was dose dependent, with significant differences favouring higher concentrations of chitosan. Specifically, a concentration of 1% w/v resulted in an average inhibition zone diameter of 0.65 cm, whereas when chitosan at 2.5% w/v was evaluated, the mean diameter was 1 cm. In contrast, for \u003cem\u003eS. aureus\u003c/em\u003e, there was no statistically significant variation in inhibition when the concentration of chitosan was increased, resulting in inhibition zones of 1 cm and 1.1 cm in diameter, respectively. These results are consistent with those reported by Uranga \u003cem\u003eet al.\u003c/em\u003e (2018) and Wrońska \u003cem\u003eet al\u003c/em\u003e. (2021) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], who found that as the concentration of chitosan increased, the inhibition of \u003cem\u003eE. coli\u003c/em\u003e DH5α and spoilage bacteria also increased in their respective studies. This antimicrobial effect of chitosan may be due to the interaction of the protonated free amino groups (-NH\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e) of chitosan (cationic nature due to its preparation in a weakly acidic medium) with the negative charges on the bacterial cell wall, which alters cell permeability and affects viability [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Furthermore, considering the structural differences in the cell walls of Gram positive and Gram negative bacteria, the response is expected to differ for the two strains tested.\u003c/p\u003e \u003cp\u003eOn the other hand, citric, ascorbic, and acetic acids, when evaluated individually or in combination at various concentrations, did not show antimicrobial activity, except for citric acid at concentrations greater than 0.75% w/v. The latter resulted in inhibition zones ranging from 0.6 to 0.8 cm in diameter for \u003cem\u003eE. coli\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConcerning acids, their antimicrobial effectiveness is intricately linked to the pH of the solution and the presence of the acid in its undissociated form. The undissociated form exhibits enhanced permeability through the lipid membranes of bacterial cells, facilitating entry. Upon entry, it dissociates, resulting in cytoplasmic acidification and a subsequent reduction in intracellular pH. Hydrogen ions must be eliminated to counteract this effect, disrupting membrane function that gives rise to an inhibitory mechanism significantly impacting bacterial metabolism. This alteration modifies the hydrogen ion and electrical charge gradients with the exterior and interferes with amino acid and phosphate transport systems [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor this reason, when choosing an organic acid as an antimicrobial agent, pH and pKa (which provides an idea of the acid's strength and ability to dissociate) should be considered.\u003c/p\u003e \u003cp\u003eFurthermore, there have been cases where \u003cem\u003eEscherichia coli\u003c/em\u003e strains grew at pH levels close to 2, showing viability even after several hours of exposure [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In contrast, authors such as Gyawali \u003cem\u003eet al.\u003c/em\u003e (2020) and Holloway \u003cem\u003eet al.\u003c/em\u003e (2011) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] have reported that using organic acids as sole components would not prevent the growth of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. These results are also consistent with those found by Ibrahim \u003cem\u003eet al.\u003c/em\u003e (2008) and Tajkarimi and Ibrahim (2011) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], who observed insignificant effects on foodborne pathogens such as \u003cem\u003eSalmonella\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e O157:H7 when using ascorbic or lactic acid at different concentrations [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegarding the combination of acids and their effect as antimicrobials, the results obtained are consistent with those found by Al Rousan \u003cem\u003eet al.\u003c/em\u003e (2018) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], who studied combinations of citric and acetic acids at various concentrations and did not achieve complete inhibition of the growth of \u003cem\u003eEscherichia coli\u003c/em\u003e O157:H7, \u003cem\u003eSalmonella typhimurium\u003c/em\u003e, and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e after two days of incubation in tabbouleh salads. On the other hand, Tajkarim and Ibrahim (2011) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] assessed the synergistic potential of the combination of ascorbic and lactic acids. While there was a significant reduction in the growth of E. coli O157:H7 in this study, they also found that the combination of these organic acids did not completely inhibit the development of this bacterium.\u003c/p\u003e \u003cp\u003eFinally, glycerol did not affect the growth of the tested bacteria. These results were expected since the primary function of this compound is not to inhibit microbial growth but is often used to provide edible coatings with greater flexibility, resistance, and reduced brittleness [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhen formulations that include the components evaluated individually were analysed, it was observed that the antimicrobial activity of chitosan was enhanced by the presence of acids in the formulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), reflecting a synergistic effect between the components. In all cases, there was a greater inhibitory power on the growth of \u003cem\u003eS. aureus\u003c/em\u003e, as the diameter of the inhibition zones was more significant than those formed in the presence of \u003cem\u003eE. coli\u003c/em\u003e. To prevent the development of \u003cem\u003eS. aureus\u003c/em\u003e, combinations of chitosan (1% w/v) with all three acids at 0.75% w/v (each) and with CitA and AscA at 0.1% w/v (each) yielded the best results, with average inhibition zones of 1.85 cm in diameter. Meanwhile, when chitosan and the acids were at 1% w/v in both formulations, comparable and statistically non-significant inhibition was observed between them. Regarding the inhibition of \u003cem\u003eE. coli\u003c/em\u003e, the formulation containing CitA and AscA (each at 1% w/v) and the formulations with CitA, AscA, and AcA (each at 0.75% and 2% w/v) achieved inhibition zones of 0.9 cm in diameter. At the same time, the rest of the combinations ranged from 0.7 to 0.85 cm, respectively.\u003c/p\u003e \u003cp\u003eThese results align with other authors demonstrating the advantage of combining chitosan with various compounds to provide edible coatings with greater antimicrobial efficacy. In this regard, the combination of chitosan with cinnamon oil, lemon essential oil, and leaf extract of \u003cem\u003eSonneratia caseolaris\u003c/em\u003e is effective in microbiological control of strawberries, cherry tomatoes, and bananas [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\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\u003eAntimicrobial effect of chitosan, ascorbic acid, citric acid, acetic acid, and glycerol.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eComponent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003cp\u003e(% w/v)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e \u003cp\u003eATCC35218\u003c/p\u003e \u003cp\u003e(cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e \u003cp\u003eATCC25923\u003c/p\u003e \u003cp\u003e(cm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eChitosan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0,05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0,1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0,1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0,1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eAscA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eCitA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,7\u0026thinsp;\u0026plusmn;\u0026thinsp;0,07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,65\u0026thinsp;\u0026plusmn;\u0026thinsp;0,05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,8\u0026thinsp;\u0026plusmn;\u0026thinsp;0,06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eAcA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlycerol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCitA\u0026thinsp;+\u0026thinsp;AscA\u0026thinsp;+\u0026thinsp;AcA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCitA\u0026thinsp;+\u0026thinsp;AscA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCitA\u0026thinsp;+\u0026thinsp;AcA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAscA\u0026thinsp;+\u0026thinsp;AcA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNI\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\u003e* AscA: ascorbic acid; CitA.: citric acid; AcA.: acetic acid; NI: no inhibition.\u003c/p\u003e \u003cp\u003e**Data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of three independent replicates. Means labelled with different letters differ (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Antioxidant capacity of chitosan and formulations\u003c/h2\u003e \u003cp\u003eAs mentioned earlier, the antioxidant activity of chitosan may be attributed to the ability of its free residual amino groups to react with the free radicals present in the environment. However, this activity is directly influenced by concentration, molecular weight, and degree of deacetylation. It increases when compounds with antioxidant properties are added, which can act through radical scavenging, hydrogen donation, metal chelation, and singlet oxygen quenching [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. According to Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, 1% chitosan solutions demonstrated an antioxidant capacity of approximately 30%. However, when combined with the evaluated acids, it tripled the percentage of inhibition, with an average of 92.34% I and values ranging from 89\u0026ndash;96% I, confirming their significant synergy and the effectiveness of these combinations. These results are consistent with the literature. Akalin \u003cem\u003eet al.\u003c/em\u003e (2021) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] found that the combined use of chitosan, pectin, and silver nanoparticles inhibits the growth of Gram-negative bacteria such as \u003cem\u003eE. coli\u003c/em\u003e. Debbabi \u003cem\u003eet al.\u003c/em\u003e (2017) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] demonstrated the positive effect of a coating formulated with 1% chitosan and 10% citric acid for controlling two Gram-positive bacteria (\u003cem\u003eS. aureus and S. epidermidis\u003c/em\u003e) as well as two Gram-negative bacteria (\u003cem\u003eE. coli and P. aeruginosa\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Evaluation of formulation behaviour on tomatoes\u003c/h2\u003e \u003cp\u003eBased on the results obtained regarding antimicrobial and antioxidant activity and considering the use of the minimum concentrations required to achieve a significant effect in these two studied capacities, the formulations of 1% w/v chitosan with the addition of 0.5% w/v glycerol and 1% w/v citric acid and ascorbic acid, and 1% w/v chitosan with the addition of 0.5% w/v glycerol and 0.75% w/v citric acid, ascorbic acid, and acetic acid were evaluated. The formulation containing acetic acid among its components was observed to have detrimental effects on the tomato epidermis after seven days of application (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and favoured the proliferation of fungi. Conversely, formulations without acetic acid did not show any harmful effects on the epidermis of the treated tomatoes. Consequently, it was decided to discard the formulations designed with acetic acid and continue the studies on the formulation containing 1% w/v chitosan with the addition of 0.5% w/v glycerol and 1% w/v citric acid and ascorbic acid.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Evaluation of the antifungal activity of the formulation\u003c/h2\u003e \u003cp\u003eOnce the formulations containing acetic acid were discarded, the effect of using only 1% w/v chitosan and its combinations of citric and ascorbic acid was evaluated. Finally, formulations designed with 1% w/v chitosan, the addition of 0.5% v/v glycerol, and citric and ascorbic acids (ranging from 0.1 to 2% w/v) were also assessed for their impact on the growth of \u003cem\u003eAlternaria tenuissima\u003c/em\u003e (EGS 34015) and \u003cem\u003eAlternaria arborescens\u003c/em\u003e (EGS 39128).\u003c/p\u003e \u003cp\u003eWhen analysing the combination of acids, complete inhibition of mycelial growth in \u003cem\u003eA. tenuissima\u003c/em\u003e was observed at a concentration of 1% w/v. For the rest of the solutions evaluated, the growth of these fungi varied between 10% and 40% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Regarding \u003cem\u003eA. arborescens\u003c/em\u003e (EGS 39128), complete inhibition was achieved starting from a concentration of 0.75% w/v of acids, while with acid mixtures at 0.1% and 0.5% w/v, mycelial growth inhibition was 61% and 83%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegarding using chitosan (1% w/v) as the sole component and combined with the evaluated organic acids, mycelial growth inhibition was 100% for all proposed concentrations and tested strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). On one hand, this effect could be explained by the fact that chitosan may act as a chelating agent for metals and essential nutrients, which weaken fungi and inhibit their growth [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. On the other hand, chitosan in combination with acids may affect the permeability of the fungal cell membrane, leading to the breakdown of the cell structure. Furthermore, chitosan forms a protective layer on the surface of the tomatoes, acting as a barrier that limits pathogen access and enhances the overall effectiveness of the treatments. Other authors have observed damage and deformation of hyphae and spores of \u003cem\u003eAlternaria\u003c/em\u003e when growing on the surface of fruits pre-treated with chitosan [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In this context, Guo \u003cem\u003eet al.\u003c/em\u003e 2020 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] proposed that the mechanism of action of submicron chitosan dispersions on \u003cem\u003eAlternaria alternata\u003c/em\u003e involves an intracellular action mechanism that allows chitosan to accumulate within the cell or associate with the cell wall, cell membrane, or septum, causing cellular damage that affects hyphal development and spore germination. Furthermore, Das et al. (2023) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] suggested that the mechanism of action of chitosan combined with essential oil from \u003cem\u003eAngelica archangelica\u003c/em\u003e would primarily target the plasma membrane against \u003cem\u003eBotrytis cinerea\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Bacterial growth curve of bacteria exposed to the formulation.\u003c/h2\u003e \u003cp\u003eWhile the antimicrobial activity against \u003cem\u003eS. aureus\u003c/em\u003e and the \u003cem\u003eAlternaria\u003c/em\u003e strains proved effective with the formulation of 1% chitosan, 0.5% glycerol, and 0.1% citric and ascorbic acids each, in the case of \u003cem\u003eE. coli\u003c/em\u003e, a concentration of acids of at least 1% was required to achieve more significant inhibition. Therefore, the formulation of 1% chitosan, 0.5% glycerol, and 1% citric, and ascorbic acids (EC) was selected for evaluating its effect on the growth of \u003cem\u003eS. aureus\u003c/em\u003e ATCC 25923 and \u003cem\u003eE. coli\u003c/em\u003e ATCC35218 strains. When the strains were grown in LB broth supplemented with chitosan, glycerol, ascorbic and citric acids to reach the EC concentrations, a significant inhibition of microbial growth was observed, preventing them from reaching the exponential growth phase during the tested period. In contrast, in the control culture (unsupplemented LB), both bacteria multiplied until reaching the stationary phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) within seven to eight hours of incubation. This assay confirmed the antimicrobial effectiveness of the selected formulation as an edible coating (EC).\u003c/p\u003e \u003cp\u003eFurthermore, in this context, the addition of agents to chitosan-based edible coatings has shown antimicrobial effectiveness. Specifically, the introduction of clove and cinnamon essential oils significantly enhanced the antibacterial and antibiofilm properties of the material against \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eEnterococcus hirae\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. Biofilm inhibition values ranged from 69.76\u0026ndash;96.97%, depending on the bacterial species [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. These findings highlight the potential of chitosan-based formulations, both in edible coatings and active films, to improve food safety and preservation.\u003c/p\u003e \u003cp\u003eMoreover, several studies have investigated the mechanisms of action of organic acids on bacteria, including the alteration of intracellular pH, protein denaturation, and membrane damage, which contribute to bacterial death. However, bacteria employ detoxification mechanisms such as efflux pumps and metabolic adjustments to resist acid stress [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Additionally, the combination of organic acids with chitosan may enhance the antimicrobial efficacy by modifying the permeability of the bacterial cell membrane, thereby limiting these detoxification responses and improving the overall inhibitory effect.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Effectiveness of the applied coating on tomatoes\u003c/h2\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.6.1. Effect of coating application on indicator microorganisms in tomatoes.\u003c/h2\u003e \u003cp\u003eOne of the primary causes of food deterioration is microbiological activity, leading to a loss of food quality. As tomatoes ripen, there is a progressive increase in microorganisms, particularly moulds and yeasts. Therefore, using coatings composed of potential microbial inhibitors and controllers is of interest for food preservation.\u003c/p\u003e \u003cp\u003eIn this regard, when the coating was applied to freshly harvested tomatoes, it was observed that, although the coated fruits exhibited a lower count of total mesophilic aerobes and coliforms compared to the control, the differences were not statistically significant over the test period (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, a and b). As for the variable of moulds and yeasts, as the monitoring period progressed, the cfu/g (Colony Forming Units per gram) increased. Only on the seventh day post treatment were statistically significant differences observed with lower counts in the coated tomatoes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). Other authors have found similar behaviours in tomatoes coated with sapote gum with chitosan (1%) or with a solution of alginate, chitosan, and \u003cem\u003eFlourensia cernua\u003c/em\u003e extract, including a delay in the appearance of microorganisms, as observed in this study for total coliforms [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. On the other hand, pomegranates coated with chitosan and ascorbic acid [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] showed a significant reduction in populations of total mesophilic aerobes, yeasts, and moulds during the storage time (28 days).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe combination of chitosan and organic acids, which are part of the formulated mixture in this study, results in the presence of positively charged free amino groups. This creates an environment that affects the integrity of the microbial cell wall, leading to changes in membrane permeability and disrupting normal cellular function. Consequently, the counts obtained from treated samples are lower. However, mechanisms of detoxification by bacteria in response to antimicrobial compounds have been identified, which could explain why the differences between treated and control groups may not be statistically significant [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.6.2. Effect of coating application on bacterial inoculants on tomatoes\u003c/h2\u003e \u003cp\u003eAdditionally, the antimicrobial effect of EC application on tomatoes was evaluated when intentionally inoculated with \u003cem\u003eS. aureus\u003c/em\u003e ATCC 25922 or \u003cem\u003eE. coli\u003c/em\u003e ATCC 35218 on the fruit's surface. Concerning the Gram positive strain, a favourable effect was observed on day seven post inoculation, as 2 logs reduced the count in the treated samples compared to the initial load. In contrast, in the untreated tomatoes, the population increased by at least 1 log (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Regarding the inoculation with \u003cem\u003eE. coli\u003c/em\u003e, the treated tomatoes maintained the microbial load from the beginning of the assay until day seven. At the same time, the controls showed a growth of 0.4 logs, resulting in a statistically significant difference in favour of the EC application (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). On the day fourteen post inoculation, neither the control samples nor those inoculated with both bacteria exhibited microbial growth. This highlights the effectiveness of using the coating in controlling the proliferation of model microorganisms commonly found in food, as used in this assay. In this regard, Jin \u003cem\u003eet al.\u003c/em\u003e (2022) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] indicated that chitosan treatment on tomatoes inoculated with \u003cem\u003eSalmonella enterica\u003c/em\u003e and \u003cem\u003eListeria monocytogenes\u003c/em\u003e demonstrates antimicrobial effectiveness, as it reduced the bacterial population to non-detectable levels by the second day following treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Antioxidant capacity in coated tomatoes\u003c/h2\u003e \u003cp\u003eRegarding antioxidant activity, the coated tomatoes showed an increase in their ability to inhibit the DPPH radical, both when compared to untreated samples and in terms of their evolution throughout the storage period. In other words, the percentage of DPPH radical inhibition (%I) was significantly higher than the treated samples for all days evaluated (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This increase is likely due to the presence of ascorbic acid as part of the formulation, as this compound is a potent antioxidant, and as demonstrated in this study, the proposed formulation for the EC exhibits a high antioxidant capacity. Other researchers have shown similar trends in tomatoes when using chitosan-based coatings with other agents, such as aloe vera and mint essential oil [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Likewise, Zhou \u003cem\u003eet al.\u003c/em\u003e (2023) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] demonstrated that the catechol-functionalized chitosan coating effectively preserves fruit quality by maintaining firmness and colour, reducing weight loss, and providing antioxidant protection, thereby extending the shelf life of strawberries and bananas. Similar results were found by Umbayda \u003cem\u003eet al.\u003c/em\u003e (2024) [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], who tested the efficacy of a chitosan and macadamia nut oil mixture to preserve the antioxidant properties and physicochemical characteristics of tomatoes.\u003c/p\u003e \u003cp\u003eAdditionally, when Das et al. (2023) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] evaluated the use of chitosan combined with essential oil from \u003cem\u003eAngelica archangelica\u003c/em\u003e on table grapes (\u003cem\u003eVitis vinifera\u003c/em\u003e L.), they found that this coating was effective in preserving enzymatic antioxidants, reducing respiration rates, and enhancing sensory quality\u003c/p\u003e \u003cp\u003eOverall, these studies underscore the significant role that chitosan-based coatings play in enhancing the antioxidant capacity and quality of fruits during storage, highlighting their potential as effective preservation strategies in the agricultural industry.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntioxidant capacity was measured as % inhibition of DPPH (%I) for tomatoes coated with 1% chitosan, 0.5% glycerol, 1% citric acid, and 1% ascorbic acid, as well as the control, stored for 21 days.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime (days)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEC\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e31,6\u0026thinsp;\u0026plusmn;\u0026thinsp;1,8\u003c/b\u003e \u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e43,2\u0026thinsp;\u0026plusmn;\u0026thinsp;2,9\u003c/b\u003e \u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e53,1\u0026thinsp;\u0026plusmn;\u0026thinsp;0,9\u003c/b\u003e \u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e50,1\u0026thinsp;\u0026plusmn;\u0026thinsp;2,3\u003c/b\u003e \u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e57,8\u0026thinsp;\u0026plusmn;\u0026thinsp;0,8\u003c/b\u003e \u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e61,1\u0026thinsp;\u0026plusmn;\u0026thinsp;2,5\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e64,0\u0026thinsp;\u0026plusmn;\u0026thinsp;0,7\u003c/b\u003e \u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e68,9\u0026thinsp;\u0026plusmn;\u0026thinsp;3,6\u003c/b\u003e \u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\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\u003e \u003cb\u003e*\u003c/b\u003e Data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of three independent replicates. Means labelled with different letters differ (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. CONCLUSION","content":"\u003cp\u003eThe chitosan-based coating formulated with citric and ascorbic acids offers a promising approach to enhancing tomato shelf life and microbial safety. This study highlights the synergistic effect of chitosan and organic acids, which not only inhibits microbial growth but also maintains the antioxidant capacity of the tomatoes throughout storage. These findings suggest that this eco-friendly edible coating could serve as a sustainable solution to minimize food waste, improve the quality of fresh produce, and potentially benefit the fresh produce industry by meeting increasing demands for food safety and quality.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eFunding Declaration\u003c/h2\u003e\n\u003cp\u003eThis research was funded by\u0026nbsp;Faculty of Agronomy, National University of the Center of the Province of Buenos Aires.\u003c/p\u003e\n\u003ch2\u003eData availability statement\u003c/h2\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.P.G Investigation (Equal) Methodology (Equal) Writing - original draft (Equal)M.A.D Formal analysis (Equal) Methodology (Equal) Visualization (Equal)K.E.D Formal analysis (Equal) Visualization (Equal)R.K.N Conceptualization (Lead) Formal analysis (Lead) Supervision (Lead) Writing - review \u0026amp; editing (Equal)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e \u003cli\u003e\u003cspan\u003eJanghu S, Kumar V, Yadav A (2024) Post-Harvest Management of Fruits and Vegetables. 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Appl Food Res 4(1):2772\u0026ndash;5022. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.afres.2024.100434\u003c/span\u003e\u003cspan address=\"10.1016/j.afres.2024.100434\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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":"Citric, Ascorbic, Shelf life, Solanum lycopersicum, Tomato","lastPublishedDoi":"10.21203/rs.3.rs-6613506/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6613506/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study examines the effectiveness of a chitosan-based edible coating, enhanced with citric, ascorbic, and acetic acids, in improving the microbiological quality and extending the shelf life of tomatoes (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e). Various formulations were tested for their antimicrobial and antifungal properties, targeting bacteria such as \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, and fungi like \u003cem\u003eAlternaria arborescens\u003c/em\u003e and \u003cem\u003eAlternaria tenuissima\u003c/em\u003e. The selected formulation\u0026mdash;comprising 1% chitosan, 0.5% glycerol, and 1% each of citric and ascorbic acids\u0026mdash;demonstrated significant antibacterial, antifungal, and antioxidant effects. Antibacterial tests revealed stronger inhibition against \u003cem\u003eS. aureus\u003c/em\u003e than \u003cem\u003eE. coli\u003c/em\u003e, while antifungal activity showed 100% growth inhibition in both \u003cem\u003eAlternaria\u003c/em\u003e strains. Additionally, tomatoes treated with this coating displayed prolonged antioxidant activity and reduced microbial counts, emphasizing its potential in improving food safety and reducing spoilage.\u003c/p\u003e","manuscriptTitle":"Synergistic Effects of Chitosan and Organic Acids on the Antimicrobial, Antifungal and Antioxidant Properties of Edible Coatings","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-19 10:25:31","doi":"10.21203/rs.3.rs-6613506/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":"ae5abb30-c7eb-4e8d-b561-5b33a2cf317c","owner":[],"postedDate":"May 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-03T08:24:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-19 10:25:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6613506","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6613506","identity":"rs-6613506","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}