Chitosan–putrescine nanoparticle coating attenuates postharvest decay and maintains ROS scavenging system activity of strawberry cv. ‘Camarosa’ during cold storage

The current research was conducted on strawberry cv. ‘Camarosa’ fruits in a greenhouse in Zanjan, Iran, in 2020. The fruits were harvested at the commercial maturity stage (>80% of the surface red colour) and were selected in terms of uniformity in shape and size and absence of visual damage. Then, they were immediately transferred to the postharvest physiology laboratory of the Department of Horticulture, University of Zanjan, to be applied with the treatments.

Chitosan-coated putrescine nanoparticles (CTS-PUT NPs) were synthesised at the Nanochemistry Laboratory of the University of Maragheh in Iran, following the method described by Mahmoudi et al. (2022) with some modifications. To obtain a clear solution of CTS, 0.1 g of low-molecular weight CTS powder was added to 25 mL of 1% acetic acid solution (w/v) and mixed at a rate of 300 rpm at 70°C for 2 hr. Subsequently, 0.1 g of PUT was dissolved in 15 mL of distilled water and slowly added to the CTS solution. The ratio of tripolyphosphate (TPP) to CTS was 1:2.5, so 0.4 g of TPP was dissolved in 5 mL of distilled water and gradually added to the CTS and PUT solution, resulting in the formation of CTS nanoparticles through crosslinking. Strawberries were then immersed in solutions containing 1 mM or 2 mM of PUT, 0.1% (w/v) CTS, 0.1% (w/v) CTS-PUT NPs or deionised water (control) for 5 min at 20°C. After dipping treatment, the strawberries were placed on a kraft paper and allowed to dry for 1 h at room temperature (20°C). Following drying, the fruits were sealed in polyethylene film bags of 0.03 mm in thickness with 4–5 holes (7–8 mm) to maintain the composition of air within the container and stored in a cold storage facility at 4°C with a relative humidity of 85%–90% for 12 days. They were randomly allocated into five groups of 240 for each treatment in three replications (80 per replication). Three boxes from each treatment served as three replications. The samples were taken out of storage on days 3, 6, 9 and 12, and their shelf life was evaluated after 24 hr at 20°C. After that, samples of fruit from each treatment/replicate were collected for quality measurements. Also, the additional samples of fruits were frozen in liquid nitrogen and stored at −80°C immediately. The frozen samples were used to determine the content of ascorbic acid, malondialdehyde (MDA), hydrogen peroxide (H₂O₂), flavonoids, total anthocyanin, and total phenolics; total antioxidant capacity; and the antioxidant activity of catalase (CAT) and superoxide dismutase (SOD).

The decay percentage was assessed by counting the number of rotten fruits at each sampling step and determining the difference between the number of initial healthy fruits and rotten fruits in each box. It was expressed in percent (Meng et al., 2008).

Tissue firmness was measured on both sides of fruits using a texture analyser (FT011, Facchini srl., Alfonsine [RA], Italy) of a probe diameter of 5 mm. It was expressed in newtons (N) (Meng et al., 2008).

The TSS content of the fruits was determined using a digital refractometer (PAL-1, Atago Co, Tokyo, Japan). To measure TA, 10 mL of fruit juice was mixed with 45 mL of distilled water and two drops of phenolphthalein as an indicator. Then, it was titrated with sodium hydroxide 0.1 N until its pH reached 8.1. The amount of NaOH consumed was used to calculate acidity in terms of the dominant acid (citric acid) (Hernández-Muñoz et al., 2006).

Ascorbic acid was measured by the method of titration with potassium iodide for which 10 mL of the fruit juice was poured into a beaker, and 2 mL of starch 1% was added was added to it. The resulting solution was titrated against the iodine solution and the titration process was continued until the formation of dark blue colour. The amount of ascorbic acid was expressed in mg · 100 g⁻¹ FW (Saini et al., 2006).

To measure total anthocyanin, phenol and flavonoid contents and antioxidant capacity, 1 g of the strawberry fruit tissue was mixed with methanol 80%. Then, its volume was adjusted to 10 mL, and the extract was centrifuged at 12000 × g for 10 min.

The total anthocyanin content (TAC) was measured by using the pH differential method. First, 200 μL of the fruit supernatant was added to 1800 μL of the potassium chloride buffer (pH = 1) and 1800 μL of the sodium acetate buffer (pH = 4.5). The diluted solutions were allowed to stand for 15 min to equilibrate. Finally, the absorbance was recorded at 510 nm and 700 nm, and the anthocyanin content was calculated in mg · 100 g⁻¹ FW (Pineli et al., 2011).

To measure the total flavonoid, 250 μL of the extract was added to 75 μL of sodium nitrite 5%, 150 μL of aluminium chloride 10% and 500 μL of sodium hydroxide 1 M, and its volume was adjusted to 2.5 mL by adding distilled water. After 5 min, the absorbance of the solution was read at 507 nm using a spectrophotometer (Specorp 250 Jena-History, Germany) (Pineli et al., 2011). The standard curve was drawn based on the absorbance at certain concentrations of quercetin.

The total phenol content was measured by using the Folin–Ciocalteau reagent for which 100 μL of the extract along with 2 mL of carbonate sodium 2% was poured into a test tube, and after 5 min, it was added to 100 μL of the diluted Folin–Ciocalteau (50%) reagent. The absorbance was read at 720 nm using a spectrophotometer (Specorp 250 Jena-History). To draw the standard curve, gallic acid was prepared at different rates, and their absorbance was recorded. The total phenol content was reported in mg · 100 g⁻¹ FW (Pineli et al., 2011).

The total antioxidant capacity was determined by using the DPPH method. First, a solution of 0.1 mM DPPH was prepared. Then, 50 μL of the fruit extract was added to 1950 μL of the DPPH solution (0.1 mM), and the volume was adjusted to 2 mL. The absorbance was read at 517 nm after 10 min using a spectrophotometer (Specorp 250 Jena-History, Germany). The absorbance of the samples was compared with the DPPH solution. The total antioxidant activity was calculated by the following equation in percent (Pineli et al., 2011).

where Ac is the absorbance of the control and As is the absorbance of the sample.

To measure MDA, 1 g of the fruit tissue was added to 5 mL of trichloroacetic acid 10% solution and centrifuged at 10000 × g for 5 min. Then, 2 mL of the supernatant was mixed with 2 mL of trichloroacetic acid 1% and 0.6 g of thiobarbituric acid. The mixture was kept in a hot water bath at 100°C for 20 min, immediately cooled down in ice, and re-centrifuged at 10000 × g for 5 min. Finally, MDA absorbance was read at 532 and 600 nm, and the result was expressed in nmol · g⁻¹ FW.

in which A532 is the absorbance at 532 nm, A600 is the absorbance at 600 nm, W is the sample weight and V is the TCA volume.

To measure H₂O₂, 1 g of the strawberry fruit tissue was ground in 5 mL of trichloroacetic acid 1%. The resulting extract was centrifuged at 10000 × g at 4°C for 5 min. In the next step, 750 μL of the resulting supernatant was mixed with 250 μL of 10 mM potassium phosphate buffer (pH = 7) and 1 mL of potassium iodide 1 M. Then, the absorbance was read at 390 nm using a spectrophotometer. The H₂O₂ content was then determined using a standard curve in nmol g⁻¹ FW of fruit fresh tissue (Ma et al., 2016).

To measure the activity of antioxidant enzymes including SOD and CAT, one gram of fruit tissue was ground in 3 mL of 50 mM potassium phosphate buffer (KH₂PO₄) (pH = 7.8) containing 0.2 mM Na₂EDTA and 2% (w/v) polyvinylpyrrolidone (PVP). Then, the homogenous compound was centrifuged at 12000 × g at 4°C for 20 min, and the supernatant was used as the enzymatic extract.

To measure SOD, 50 μL of the enzymatic extract was first mixed with 2 mL of 50 mM phosphate buffer, 100 mL of ethylenediaminetetraacetic acid (EDTA) and 200 μL of nitroblue tetrazolium (NBT). It was then exposed to 40-W fluorescent light for 10 min. In the next step, 50 μL of 0.15 mM riboflavin was added to the mixture and was exposed to the same light for 12 min. Finally, the absorbance was read at 560 nm. One unit of the SOD activity expressed as U · g⁻¹ FW is based on the amount of enzyme that can inhibit NBT oxidation by 50% (Chen et al., 2011).

To determine the CAT activity, 100 μL of the enzymatic extract was added to 50 μL of H₂O₂ and 200 μL of 50 mM phosphate buffer with an acidity of 7. The CAT activity was recorded with a spectrophotometer based on the decline in absorbance at 240 nm in 2 min. The results were expressed in U · g⁻¹ FW (Chen et al., 2011).

The study was conducted as a factorial experiment in a completely randomised design with three replications. The first factor was assigned to PUT treatment at two levels (1 mM and 2 mM), CTS (0.1% w/v) and CTS-PUT NPs (0.1% w/v), as well as a control. The second factor included the storage time (3 days, 6 days, 9 days and 12 days). Two-way analysis of variance (ANOVA) was performed using the SPSS statistical software package program version 18.0 (SPSS Inc., Chicago, IL, USA), and significant differences between mean values were compared using Duncan’s multiple range test at a level of 5%. Data were expressed as the mean ± standard error (SE).

According to Table 1, the treatments positively influenced the reduction of the fruit decay percentage over 12 days of storage. The decay percentage was zero in both control and treated fruits on the 3rd day of storage. PUT reduced the decay of strawberries during 9 days and 12 days of storage effectively. At the end of the storage period, the lowest decay was 16.66% related to the treatment of CTS-PUT NPs and 15.83% related to 0.1% CTS, and the highest was 57.5% observed in the control fruits (Table 1).

Strawberries contain a lot of water and have susceptible tissue, so they are easily exposed to fungal pathogens during the postharvest period. In this regard, grey mould (Botrytis cinerea), which can grow at low temperatures, inflicts significant losses (Harker et al., 2000). Presently, it is highly important to propose safe methods for extending the shelf life of strawberries. One method is to use edible coatings, e.g., CTS and nanocomposites, which can increase the longevity and preserve the quality of strawberries (Ribeiro et al., 2007). In this research, the fruits treated with CTS-PUT NPs were infected less than the control fruits, reflecting the effective role of these treatments in the control of fungal infections. Similar results were observed in a study by Bal and Ürün (2021) in which the decay incidence of strawberries coated with CTS and CTS + PUT was lower than that of control and PUT-treated fruits during storage. It has been stated that an increase in internal polyamines, such as PUT, can be effective in controlling fungal diseases including grey mould, and this shows the role of these compounds in defensive responses of plants to pathogens (Walters, 2003). By increasing the activity of biomolecules and strengthening the defensive system against pathogens, the PUT treatment reduced decay in peaches (Abbasi et al., 2019), mandarins (Ennab et al., 2020) and strawberries (Zokaei Khosroshahi et al., 2007). CTS has natural antimicrobial activity and elicits defensive responses (Terry and Joyce, 2004). This coating is a poly-cation that consumes negative charges on the cell surface and changes cell permeability, thereby causing the leakage of intracellular electrolytes and proteins (Avadi et al., 2004). It also penetrates fungal cells and suppresses and slows down the synthesis of mRNA and proteins (Li et al., 2015). CTS has been used to control postharvest diseases of many fruits, such as pears (Yu et al., 2008), jujubes (Xing et al., 2015) and grapes (Meng et al., 2008) with results similar to our findings. Nanoparticle coatings have tiny particles, so they reduce the diameter of respiratory pores on the fruit surface. Consequently, their permeability to oxygen and water decreases, and fruit decay is retarded (Xing et al., 2019).

According to Table 1, fruit tissue firmness was reduced in both control and treated fruits over the storage period. The highest firmness was observed in the treatments of CTS and CTS-PUT NPs. At the end of the storage period, the lowest firmness was 0.9 N observed in the control, and the highest was 2.9 N observed in fruits treated with CTS-PUT NPs (Table 1). The applications of CTS-PUT NPs and CTS effectively preserved the tissue firmness of strawberries.

The loss of firmness is accompanied by changes in the metabolism of cell wall carbohydrates and the structural components of cell walls. These changes are induced by the activity of cell wall-degrading enzymes (Valero et al., 2002). PUT binds to pectin of cell walls, thereby suppressing the cell wall-degrading enzymes, such as polygalacturonase and pectin methylesterase, which are responsible for postharvest softening of fruits (Martínez-Romero et al., 2002). The application of polyamines, such as PUT, to apricots (Martínez-Romero et al., 2002), peaches (Abbasi et al., 2019) and papayas (Hanif et al., 2020) contributed to preserving fruit firmness. Similar to our findings, CTS reduced the activity of cell wall-degrading enzymes, thereby leading to the preservation of fruit firmness of mangoes (Chien et al., 2007), tomatoes (Liu et al., 2007) and cornelian cherries (Duan et al., 2011).

Based on the results, the treatments inhibited the loss of TSS during storage effectively (Table 1). In the 6 days of storage, the highest TSS content was exhibited by the fruits treated with CTS-PUT NPs (7.3°Brix), those treated with CTS (7.5°Brix) and the control (7.2°Brix). At the end of storage, no significant differences were observed in TSS among the treatments. The lowest TSS content was 4.4°Brix related to the control (Table 1).

In the 6 days of storage, the highest TA was observed in the treatments of CTS-PUT NPs and CTS and the lowest in the treatments of 1 mM and 2 mM PUT. The differences among the fruits stored for 12 days were not significant. The lowest TA at the end of the storage period was observed in the control. All treatments were effective in preserving the TA of the strawberries at the end of the storage period (Table 1).

The concentration of TSS continuously increases over the process of fruit maturing due to the hydrolysis of polysaccharides and organic acids (Piero et al., 2005). Respiration contributed to the consumption of organic acids and the reduction of TA in fruits (Zokaei Khosroshahi et al., 2007). The effect of PUT on TSS and TA can be associated with the reduction of respiration and the retardation of the ripening process. Similar results have been reported for plums (Davarynejad et al., 2015), apricots (Martínez-Romero et al., 2002) and mangoes (Wannabussapawich and Seraypheap, 2018). CTS and its nanocomposite coatings create a semipermeable layer around fruits and a controlled internal atmosphere, thereby reducing respiration and ethylene synthesis, preventing the effect of ethylene and extending fruits’ storage life. Consequently, TSS consumption and reduction are hindered (Bautista-Banos et al., 2006; Xing et al., 2020). According to our results, the CTS treatment prevented the decline in TSS in papayas (Bautista-Banos et al., 2003), mangoes (Xing et al., 2020) and tomatoes (Meena et al., 2020).

The ascorbic acid content was decreased in both control and treated fruits during the storage. The comparison of the means revealed that no treatment had any effects on preventing the loss of ascorbic acid on the 3rd day of storage and the highest ascorbic acid content was observed in the control. On the 9th and 12th days of storage, the highest ascorbic acid content was associated with the treatments of CTS-PUT NPs and 2 mM PUT. At the end of the storage period, all treatments effectively prevented the excessive loss of ascorbic acid in the strawberries. The lowest ascorbic acid (18.16 mg · 100 g⁻¹ FW) was related to the control (Table 1).

Ascorbic acid is a major factor in strawberry quality and is very sensitive to oxidation. It rapidly oxidises during storage. PUT helps preserve ascorbic acid by reducing the activity of ascorbate peroxidase (APX) (Ennab et al., 2020). The application of PUT in mangoes (Razzaq et al., 2014), mandarins (Ennab et al., 2020) and apricots (Davarynejad et al., 2013) contributed to preserving the amount of ascorbic acid. CTS reduces the oxidation of ascorbic acid by reducing the amount of oxygen, thereby preventing the decline ascorbic acid (Wang and Gao, 2013). CTS and CTS nanocomposites have effectively been used to prevent the decline in ascorbic acid in kiwifruits (Vivek and Subbarao, 2016), litchis (Sun et al., 2010) and mangoes (Chien et al., 2007).

In the present study, the TAC of the strawberries first increased but then started to decrease during the storage period. The results showed no significant difference between the coating treatments and the control in total anthocyanin on the 3rd day of storage. The anthocyanin content was increased in all fruits on the 6th day. The highest anthocyanin content was related to the CTS-PUT NPs and 2 mM PUT treatments. At the end of the storage period, the lowest TAC was observed in the control fruits and the highest was related to the treatments of CTS-PUT NPs and CTS (Figure 1A).

Figure 1.

Strawberry is rich in anthocyanins. The dominant anthocyanin in this fruit is pelargonidin-3-glucoside (Petriccione et al., 2015). The amount of anthocyanin during the postharvest period increases due to its biosynthesis, but it decreases at the end of storage due to the senescence process (Hu et al., 2011). During stress, the activity of the PAL enzyme, which plays a key role in the biosynthesis pathway of phenol compounds, increases, resulting in an increase in anthocyanin synthesis (Keutgen and Pawelzik, 2007). PUT increases anthocyanin by inducing the enzymes involved in its synthesis, e.g., phenylalanine ammonia-lyase and chalcone isomerase. It also binds to anthocyanin and inhibits its oxidation and degradation (Barman et al., 2014). The application of PUT to pomegranates (Barman et al., 2014) and strawberries (Bal and Ürün, 2021) prevented the postharvest decline in anthocyanin.

CTS inhibits postharvest anthocyanin loss by controlling the ripening process, lowering oxygen levels, and suppressing the activity of peroxidase and polyphenol oxidase enzymes. CTS and nanocomposite coating effectively preserved anthocyanin in strawberries (Resende et al., 2018) and plums (Chang et al., 2019), which agrees with our findings.

It was found that increasing the storage time decreased the total phenol content in all treatments. On the 3rd day of storage, the highest amount of total phenols was related to the treatments of CTS and 2 mM PUT, showing significant differences from the other treatments (Figure 1B). Since the 6th day of storage, the total phenol content was decreased over time so that the lowest amount at the end of the storage period was related to the control and 1 mM PUT and the highest was related to the treatments of CTS-PUT NPs and 2 mM PUT. These treatments differed from the other treatments significantly.

The total flavonoid content was decreased with increasing the storage time. The fruits treated with CTS had the highest total flavonoid content over the whole storage period. On the 12th day of storage, the lowest amount of flavonoid was observed in the control and 1 mM PUT treatments and the highest in the treatments of CTS and CTS-PUT NPs, exhibiting significant differences from the other treatments (Figure 1C).

The results for the antioxidant capacity revealed a decrease in this capacity with the increase in the storage period, but there was not a significant difference between the coating treatments and the control until the 9th day (Figure 1D). The antioxidant capacity of the control was dropped from the 9th day to the end of the storage period and showed strongly significant differences with the treated fruits. However, the fruits treated with the edible coatings did not differ from one another in the total antioxidant activity significantly. In general, antioxidant contents increased and then decreased during storage. Determining antioxidant capacity is a method for expressing the biological properties of fruits, which play a key role in preserving fruit quality during the storage period (Wannabussapawich and Seraypheap, 2018). Wang and Gao (2013) and Bal and Ürün (2021) reported a positive effect of CTS- and CTS + PUT-based edible coatings on the antioxidant activity of strawberry. High values of the antioxidant content observed in berries treated with CTS, CTS-PUT NPs and 2 mM PUT could be attributed to the high level of phenolic compounds. Previous studies indicate a linear correlation between the phenolic content and antioxidant capacity of fruits (Razzaq et al., 2014; Bal and Ürün, 2021). The decline in phenolic content during extended storage periods may result from the breakdown of cell structure, leading to the release of phenolics that are subsequently exposed to enzymatic oxidation. Research on pomegranates (Mirdehghan and Rahemi, 2007), plums (Davarynejad et al., 2015), kiwifruits (Jhalegar et al., 2012) and zucchinis (Palma et al., 2015) showed that PUT can effectively inhibit the loss of phenol compounds at the postharvest period. PUT prevents the postharvest loss of phenol compounds by increasing the activity of PAL and preventing the oxidation of these compounds (Hosseini et al., 2018). CTS is used to increase the synthesis of most phenol compounds in tissues of plants. For example, it increases the total phenol compounds in strawberries (Resende et al., 2018). Furthermore, the plants treated with CTS and CTS-coated titanium dioxide preserved higher levels of phenols, flavonoids and flavonoids in mangoes (Xing et al., 2020). Previous studies have shown that the treatment of CTS and nanocomposite coating increased phenols in blueberries (Xing et al., 2021) and grapes (Gohari et al., 2021).

Based on the results, the MDA content increased during the storage period, but the treatments were effective in inhibiting the increase in the MDA content. The lowest MDA content on the 3rd day of storage was related to the treatment of CTS-PUT NPs, which differed from the other treatments and the control significantly. The highest MDA content at the end of the storage time was related to the control, differing from the other treatments significantly, whereas no significant difference was observed among the coating treatments (Figure 2A).

Figure 2.

As the storage period was extended, the H₂O₂ content was increased in both treated and control fruits. On the 6th day of storage, the lowest H₂O₂ content was observed in the fruits treated with CTS-PUT NPs, differing from the other treatments and the control significantly. At the end of the storage period, the control fruits exhibited the highest H₂O₂ content and differed from the other coating treatments significantly. But the coating treatments did not differ from one another significantly (Figure 2B). The CTS-PUT NPs, CTS and PUT treatments effectively inhibited the increase in H₂O₂ in the strawberries during the storage period.

The first cell structures in plants that are damaged by chilling stress are lipids and proteins of the plasma membrane (Liang et al., 2003). The increase in free oxygen radicals damages most cell structures and components, such as fats, proteins and carbohydrates. The treatment of PUT decreased H₂O₂, MDA and electrolyte leakage by increasing the activity of antioxidant enzymes (Palma et al., 2015). Separate studies have reported the effect of PUT on decreasing MDA and H₂O₂ in peaches (Abbasi et al., 2019) and kiwifruits (Yang et al., 2016). CTS and CTS nanocomposites effectively hindered the increase in MDA and H₂O₂ during storage (Xing et al., 2021), which can be ascribed to the CTS and CTS nanocomposites’ potential to inducing a defensive system (Badawy and Rabea, 2009; Vieira et al., 2016). The application of CTS and CTS nanocomposite coatings significantly reduces the MDA increase in peppers and cucumbers (Xing et al., 2011; Zhang et al., 2015).

According to the results, the CAT activity was the lowest in the CTS treatment on the 3rd day of storage, differing from the other treatments significantly. On days 6 and 9, the CAT activity increased so that its highest activity was related to the control and its lowest activity to the treatment of CTS (Figure 2C). At the end of the storage, the CAT activity was decreased so that the lowest was related to the control, which differed from the coating treatments significantly. But, no significant differences were observed among the coating treatments.

The comparison of the means revealed that the SOD activity was gradually increased until the 9th day of storage, but it, then, started to decrease. The highest SOD activity was related to the treatments of CTS and CTS-PUT NPs, which differed from the control and other coating treatments significantly (Figure 2D). At the end of the storage, the highest and the lowest activity of this enzyme were related to the treatment of CTS and the control, respectively. They differed from the other treatments significantly. In general, the treatment of CTS prevented the loss of SOD activity significantly.

During fruit ripening, the synthesis and accumulation of reactive oxygen species (ROS) such as H₂O₂ and superoxide anions increase continuously. So, the continuous increase in oxidation induces stress at the fruit ripening stage (Sudhakar et al., 2001). ROS induces oxidative stress on lipid membranes, nucleic acids and proteins. Plants are equipped with defensive mechanisms to eliminate ROS damage. ROS can be scavenged by the plants’ enzymatic defence including SOD, APX and CAT or by its nonenzymatic antioxidants such as ascorbic acid, phenols and glutathione (Gill and Tuteja, 2010). Polyamines are significantly effective in increasing plant tolerance to stress. They act as the deactivators of free oxygen radicals. The antioxidant nature of these compounds may be related to the suppression of NADPH oxidase and the inhibition of the accumulation of superoxide radicals (Martin-Tanguy, 2001).

Superoxide radicals are converted to H₂O₂ by SOD, which is then converted to water and superoxide anions by CAT and peroxidase (Wannabussapawich and Seraypheap, 2018). The strawberries treated with 2 mM PUT and CTS-PUT NPs showed higher CAT activity than the control and other treatments. CTS and nano-silica coatings hindered the reduction of the activity of antioxidant enzymes in loquat fruits at the postharvest stage by suppressing free radicals and preserving the integrity of cell membranes (Song et al., 2016). The CTS coating and cellulose nanocomposite films effectively prevented the reduction of the activity of CAT, SOD and APX in mangoes (Xiao et al., 2021) and eggplants (Attia et al., 2021).