Shelf‐life extension of Fragaria × ananassa Duch. using selenium nanoparticles synthesized from Cassia fistula Linn. leaves

Abstract Fragaria × ananassa Duch. (Strawberry) fruit is susceptible to postharvest diseases, thus decrease in quality attributes, such as physiological and biochemical properties leads to decrease in shelf life. The objective of the present study was to check the effect of Selenium NP's and packaging conditions on the shelf life of strawberry (Fragaria × ananassa Duch) fruits. The shelf life was observed with 4 days intervals and examined for characteristics such as physiological weight loss, moisture content, percentage decay loss, peroxidase, catalase, and DPPH radical scavenging. The quality change of postharvest Fragaria × ananassa Duch. was monitored by the application of selenium nanoparticles (T1 plant extract in 10 mM salt solution, T2 plant extract in 30 mM salt solution, T3 plant extract in 40 mM salt solution, T4 distilled water; control) in different packaging materials (plastic bags, cardboard, and brown paper) at different storage conditions (6°C and 25°C). 10 mM, 20 mM, and 30 mM solution of sodium selenite salt, prepared from 1 M stock solution. Selenium nanoparticles were synthesized using Cassia fistula L. extract and sodium selenite salt solution. Polyvinyl alcohol (PVA) was used as a stabilizer. The nanoparticles were characterized through UV–visible spectroscopy and X‐Ray diffractometer (XRD). It was observed that the strawberry Fragaria × ananassa Duch. Treated with T1 (CFE and 10 mM salt solution) stored in plastic packaging at ±6°C showed the best physiological parameters and hence the treatment is recommended for storage without affecting the quality of strawberry fruit up to 16 days.

(approximately 1-2 days at room temperature and 3-4 days in the refrigerator) because of mechanical damage, excessive softness, rot induced by microbial development, moisture loss, and physiological abnormalities, during growth, harvesting, and storage (Panou et al., 2019). Several studies have documented effective postharvest management of strawberry fruits. The quality of strawberry fruits was maintained during storage period through the application of hot air and Pichia guilliermondii (Zhao et al., 2023). Pichia guilliermondii is documented to cause Fingernail infection (Zhang et al., 2020). Strawberry fruits packed in corrugated fiberboard packaging box treated with chlorine dioxide along with ethylene and moisture absorber were documented to have shelf life of 8.66 days (Dutta et al., 2023). Nanotechnology provides many benefits to the food industry in maintaining food taste, texture, sensitivity, quality, shelf life, and food safety. Nanotechnology is also used in food industries for preparation, packaging, marketing, and better yield and standard (Nile et al., 2020). The addition of nanoparticles into packaging materials provides quality and extension in the shelf life of fruits.
The integration of NPs into polymers has empowered the development of more resistant packaging that is also cost-effective (He et al., 2018). Application of proline-coated chitosan nanoparticles on strawberry fruits increased the ascorbic acid content, phenolic content, total soluble sugars, enzymatic, and non-enzymatic antioxidant activity during cold storage period (Bahmani et al., 2022). Argininecoated chitosan nanoparticles application on plum fruits reduced the weight loss, chilling injury, maintains the texture, ascorbic acid content, and enhanced the antioxidant activities (POD, CAT, APX, and SOD) during storage period at 1°C . Postharvest application of glycine betainecoated chitosan nanoparticles enhanced the shelf life of plum fruits for up to 40 days during storage period at 1°C. Increase in DPPH activity, phenolic content, flavonoids, and anthocyanin was also observed in plum fruits during storage period .
During the past 20 years, there has been a resurgence in green technology to improve the yield, nutritional, and postharvest quality of fruits and vegetables. Green synthesis of nanoparticles is an advanced method over other methods because it is simple, cost-effective, eco-friendly, and reproducible, and provides more secure products (Emamifar & Mohammadizadeh, 2015). Selenium (Se) initiates the biosynthesis of hormone and plays a defensive role in both adults and children. Se is reported to play a role in the biosynthesis of thyroid hormone; moreover, it is also involved in muscle movement, triggering an immune system against microbial infections and reproductive functions. An optimum intake of Se can eliminate the risk of cancer cell formation in humans. Se plays a defensive role in plants, as it reduces the damage caused by extreme climatic changes. It is known as a beneficial element for higher plants as it increases the production of secondary metabolites, antioxidant mechanism, and improves photosynthesis (Lanza & Reis, 2021). Selenium nanoparticles are getting increased popularity as a micronutrient and a dietary supplement in nanomedicine (Panou et al., 2019). Se nanoparticles are reported to have high bioavailability, significant bioactivities, and low cytotoxicity (El-Ramady et al., 2016). Preharvest foliar application of selenium enhances the nutritional profile of kiwi fruits during storage period for 90 days at 1°C (Ghafouri et al., 2022).
The overall production cost in Pakistan is projected to be Rupees 100,000 per acre, whereas the income is rupees 200,000 per acre and the land area is 78 acres, with a yearly yield of 274 tons (Bangulzai et al., 2014). Therefore, improved infrastructure, postharvest production methods, and harvesting facilities can support it in domestic and foreign markets (Rajwana et al., 2016).
The present study was designed to observe the potential efficacy of SeNPs on physiochemical properties, antioxidant activities (enzymatic and non-enzymatic), and storability in different conditions (different temperatures and packaging materials), Selenium nanoparticles were synthesized, characterized, and applied to postharvest strawberry fruits. The objective of the present study was to determine the effect of selenium nanoparticle and storage conditions on the shelf life enhancement of strawberry (Fragaria × ananassa Duch).

| MATERIAL S AND ME THODS
The study was carried out in the Molecular Taxonomy Lab, Department of Botany, Lahore College for Women University, Lahore, from September to April 2022.

| Plant collection for nanoparticle synthesis
Fresh leaves of Cassia fistula L. were collected, cleaned, air-dried at room temperature, and then ground into fine powder with the help of a grinder. The powder was sieved and kept in an air-tight container for further use (Bhalodia & Shukla, 2011).

| Preparation of plant extract
About 10 g of plant powder was boiled in 100 mL of distilled water at 70°C for 20 min in a water bath. The supernatant was filtered using Whatmann filter paper No. 1 to remove the impurities. The supernatant was collected, re-filtered, and centrifuged at 0.02236 g for 10 min and stored in jars for further use (Mulla et al., 2020).

| Characterization of selenium nanoparticles
Surface plasmon resonance of selenium nanoparticles was monitored by UV-visible spectrophotometer (Metash -Model V-5800).
UV-vis spectrometric readings were recorded at a scanning speed of 200-800 nm wavelength range at the interval of 1 nm. To set the zero line, distilled water was used as a blank. The crystalline structure of selenium nanoparticles was confirmed by X-ray diffractometer (XRD). XRD was performed in the 2 θ range from 10° to 90°.

| Application of selenium nanoparticles on strawberry fruits
Strawberry fruits of uniform size, shape, and no physical or mechanical damage were purchased from the local market of Lahore, Pakistan and are transferred to the laboratory. The experimental design followed for the study was randomized complete split block design with four treatments, each treatment was run in triplicates.
Selenium nanoparticles, 0.1% solution was prepared and 10 mL of each conc. was sprayed on 10 randomly selected strawberry fruits. Two different temperatures, room (25°C) and cold storage (4°C), three different packaging materials (polythene, paper, and cardboard) were selected for the application of nanoparticles.
Treatments selected for the application of nanoparticles based on the production of nanoparticles included T 1 plant extract in 10 mM salt solution, T 2 plant extract in 20 mM salt solution, T 3 plant extract in 30 mM salt solution, and T 4 distilled water as control. Synthesized selenium nanoparticles were sprayed on the postharvested strawberry fruits bought from a local market in Lahore.
Sprayed fruits were stored in three different packaging materials, such as polythene bags, paper bags, and cardboard packaging.
Effect of SeNPs and storage conditions: temperature (6°C, 25°C) and packaging materials (plastic, cardboard, paper) on the shelf life of strawberry fruits were checked at 4 days intervals by observing the physiological weight loss, moisture content, decay percentage, enzymatic and non-enzymatic antioxidant activities (Li et al., 2011).

| Effect of SeNPs application on strawberry fruits
2.6.1 | Physiological weight loss, moisture content, and percentage decay loss

Physiological weight loss (PWL)
Physiological weight loss in strawberry fruits was calculated by comparing the original weight Wo (weight measured after applying treatment) and the weight (W) after 4 days interval (Hernández-Munoz et al., 2008). The percentage physiological weight loss was measured by the formula: where, PWL = Physiological weight loss, w 0 = Original weight, w = Weight measured after 4 days interval.

Moisture content
Empty petri dishes were weighed and 1.5 g of sample was added to each petri dish. These petri dishes were placed in an oven at 120°C for 2 h and then placed in a desiccator for cooling. Again, the weight of petri plates was noted and compared with the initial weight (Gharezi et al., 2012). The percentage of moisture content was calculated by: where, Loss of weight = weight of petri dish and sample -weight of dried sample; Weight of sample = weight of petri dish and sampleweight of empty petri dish.

Percentage decay loss
Percentage decay loss was measured according to Ali et al. (2010).
It was calculated by counting the number of fruits that decayed on 4 days' interval divided by the number of fruits included on the initial day of the experiment. The formula used to measure the percentage decay loss was (% decay loss = FD-FI × 100), where FD = no. of decayed fruits and FI = no. of initial fruits.

| Increase in shelf life
The length of the extended shelf life was calculated by counting the number of days extended as compared with the control treatment (Moneruzzaman et al., 2008).

| Microbial susceptibility analysis
The antimicrobial analysis was performed on nano-treated strawberry fruits by agar well diffusion method following the methodology of Daoud et al. (2015). Bacterial strains (Pseudomonas

Extraction of crude enzyme
A quantity of 0.2 g of strawberry fruit was ground under liquid nitrogen, then added 0.5 mL of 1 mM phosphate buffer saline (pH 7.0); 1 mL of 1 mM EDTA, 1 mL of 1 mM ascorbic acid, 1 mL riboflavin, 1 mL of 0.1% PVP. The samples were centrifuged at 0.0118 g for 15 min.
The supernatant was collected and used for the determination of peroxidase and catalase enzyme activity (Kamińska et al., 2022).

Determination of peroxidase activity
Peroxidase activity was carried out by taking 1 mL of sodium phosphate buffer (pH = 7.0), 1 mL of H 2 O 2 , 1 mL guaiacol, and 0.5 mL enzyme extract to make the final reaction mixture, and absorbance was observed with UV-vis spectrophotometer (Metash -Model V-5800) at 420 nm. Peroxidase activity was expressed in units of the enzyme that causes the absorbance increase of 0.1 per minute in U g −1 FW (unit per gram fresh weight).

Determination of catalase activity
A volume of 0.5 mL enzyme extract was taken and 1 mL of hydrogen peroxide was added and the activity was checked with UV-visible spectrophotometer (Metash -Model V-5800) at 240 nm resulting from the decomposition of hydrogen peroxide. One catalase unit was represented as the amount of H 2 O 2 (μmol) decomposed by 1 g of tissue within 1 min (Kamińska et al., 2022).

| Non-enzymatic antioxidant activity
Non-enzymatic antioxidant activity of the strawberry fruits during storage period was evaluated by three assays named Total Phenolic content assay, DPPH Assay, and Total antioxidant assay.

Total phenolic content (TPC) assay
Total phenolic content was evaluated by following the methodology of Dewanto et al., 2002. A volume of 0.1 mL of each extract was mixed with 2.8 mL of 10% Na 2 CO 3 and 0.1 mL of 2 N Folin-Ciocalteu (FC) reagent. The absorbance of the reaction mixture was measured at ƛ max 725 nm by UV-visible spectrophotometer (Metash -Model V-5800). The results were obtained from a calibration curve (y = 0.005x + 0.047, R 2 = 0.998) of gallic acid (12.5-100 μg/mL) and represented as gallic acid equivalent (GAE) per gram dry weight.

DPPH (diphenyl picryl Hydrazyl radical) assay
DPPH radical scavenging activity assay was done by following the methodology of Kahraman and Feng (2021). A volume of 0.2 mL of each sample was mixed with 3.9 mL of DPPH solution and vortex.
The samples were kept in dark for 15 min. UV-vis spectrophotometer (Metash -Model V-5800) was used for the measurement of absorbance values at ƛ max 517 nm using DPPH as negative control and buthylhydroxytoluene (BHT) as positive control. The antioxidant activity was calculated as the following: where A c is the absorbance of the control and A t is the absorbance of samples.

Total antioxidants assay
Total antioxidant potential of the strawberry extracts was performed according to the methodology of Aftab et al. (2019). A volume of 0.1 mL of each extract was mixed with 1.9 mL of the reagent solution (0.6 M sulfuric acid, 4 mM ammonium molybdate, and 28 mM sodium phosphate). The incubation of the reaction mixture was done at 95°C for 60 min and was allowed to cool at room temperature. The antioxidant activity was expressed as the sample absorption at ƛ max 695 nm using UV-vis spectrophotometer.

| Statistical analysis
Effects of different treatments, storage condition: temperature (6 and 25°C) of packaging materials on strawberry fruits' shelf life was subjected to two-way analysis of variance (ANOVA) using IBM SPSS statistics 20 software and OriginPro 2022. Duncan's multiple range test was used to evaluate the differences between mean values deemed significant at p < .05 by using IBM SPSS 20 software.

| Synthesis of selenium nanoparticles
The color change from pale to reddish brown among all reaction mixtures within 48 hours indicated the synthesis of selenium nanoparticles. Surface plasmon resonance of all three reaction mixtures was determined by UV-visible spectroscopy and crystalline structure by X-ray diffraction (XRD).

| UV-Visible analysis
The bioreduction of selenium ions in the solution was monitored by diluting a small amount of solution of 10 folds with distilled water

| XRD (X-Ray diffraction) analysis
The crystalline structure and the average size of green synthesized selenium nanoparticles were analyzed with X-ray diffractometer and are presented in Figure  The study was similar to the outcomes of Ghaderi et al. (2022), in which SeNPs were synthesized from the aqueous extract of Abelmoschus esculentus.

| Antimicrobial profile of SeNPs
The antimicrobial profile of green synthesized selenium nanopar-  Water content is directly associated with macronutrients (proteins).
Fruits and vegetables lose water through transpiration and respiration soon after harvest. And decrease in water content results in the reduction of weight and freshness (Singh et al., 2022). The reason behind this was that at cold storage, the process of ripening of fruit slowed and moisture loss was decreased. The findings were in correspondence with Saleh and Abu-Dieyeh (2022).
The study also documented the factors involved in the decay of strawberry fruits which include an increase in skin permeability for loss of moisture, an increase in respiration rate, and vulnerability to decaying organisms.
The senescence rate of postharvest fruits and vegetables is determined by respiration rate. Respiration involves a series of redox reactions that utilize carbohydrates and organic acids as substrates.
Increase in respiration rate causes an increase in the metabolic rate of fruits (Du et al., 2022). Sang et al. (2022)

| Increase in shelf life
The effect of different parameters on the shelf life of strawberry fruits was observed and depicted in Figure 7. The length of the shelf life was calculated by observing the strawberry fruits after

| Peroxidase (POD) activity
During storage period, peroxidase activity of strawberry fruits was observed. As given in Table 2, a decrease in POD activity was observed in control strawberry fruits. A significant increase in peroxidase activity was observed in strawberry fruits treated with SeNPs.
Strawberry fruits treated with SeNPs stored at 6°C showed an increase in POD activity for up to 16 days, as compared with fruits stored at 25°C. The T1 (10 mM SeNPs) application on strawberry fruits was observed to increase the POD activity for up to the 16th day. Peroxidase decreases the production of reactive oxygen species and thus enhances the shelf life of fruits and vegetables (Huang et al., 2023).

| Catalase (CAT) activity
Catalase (CAT) plays a vital role in neutralization by decomposition of H 2 O 2. During storage period, catalase activity was observed and it is presented in Table 3. The CAT activity of the treated strawberry fruit in plastic packaging was generally higher in room storage (25°C) than that in cold storage (6°C) on day 4. Because at high temperature, the metabolism of fruit is fast, and low temperature slows down the metabolic process of fruits. Maximum CAT activity of 0.25 ± 0.01 was recorded in T 1 stored in plastic packaging at 6°C on day 4 which increased to 0.45 ± 0.00 on day 12. An increasing trend in catalase activity during storage period was observed in SeNPs-treated strawberry fruits stored at 6°C than the control treatments and the fruits kept at 25°C.
In cold storage, the CAT activity was increased as the days of storage increased. Increase in the catalase activity of strawberry fruits up to 5 days during storage period was observed by Haider et al. (2022). The study checked the effect of application of Eucalyptus leaves extract on postharvest strawberry fruits.

| Total phenolic content and antioxidant activities
Tables 4-6 present the total phenolic content and antioxidant profiles of strawberry fruits packed in different packaging materials

| Relationship of strawberry fruit characteristics
Pearson's correlation coefficient test was applied to investigate the inter-correlation of strawberry fruits' quality attributes during storage period (Table 7). Both positive and negative significant correlations were found among fruit traits (physiological weight loss, moisture content, physiological decay loss, peroxidase, catalase, total phenolic content, DPPH, and Total antioxidant activity). Physiological weight loss is positively correlated with moisture content (r 2 = 0.753).
Maintenance of moisture content maintains the weight of the fruits.
A decrease in moisture content reduces the weight of the fruits.
Physiological decay loss was found to be negatively correlated with moisture content. A decrease in moisture content increases the decay percentage. Positive correlation (r 2 = 0.95) was observed between moisture content and peroxidase activity suggesting that the moisture content lowers the production of reactive oxygen species.
DPPH radical scavenging activity and total antioxidant activity are positively correlated with the total phenolic content of the fruits. The results are endorsed by the findings of Khedr (2022).
Pearson correlation coefficient test was also performed to check the effect of each treatment on strawberry fruits' quality attributes during storage period (Table 8a-d). Among all the treatments, control treatment showed non-significant relationship among quality attributes of strawberry fruits during storage period as compared with other treatments. However, T1 application was observed to have significant negative and positive correlations among quality attributes of strawberry fruits as compared with other treatments. The study confirmed the relationship of moisture content with physiological weight loss and physiological decay loss. Phenolic compounds scavenge reactive oxygen species; therefore, are known as antioxidants.
They play an important role in the defense mechanism of fruits and vegetables. Total phenolic content protects plants from pathogen TA B L E 4 Total phenolic contents among all treatments in different packaging at cold and room storage.

Zone of Inhibition (mm)
Conc.

Zone of Inhibition (mm)
Conc.

| CON CLUS ION
The results of the recent study indicate that the green synthesized SeNPs possess antioxidant and antimicrobial potential. Green synthesized SeNPs application on postharvest strawberry fruits increases the shelf life, keeps up the quality attributes, and enhances peroxidase activity, catalase activity, total phenolic content, and antioxidant capacity.

ACK N OWLED G EM ENT
In this research work special acknowledgement goes towards research center of the Future University in Egypt. Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2023R155), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors confirm that they have no conflicts of interest with respect to the work described in this manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.