Combination effects of calcium chloride and nano-chitosan on the postharvest quality of strawberry (Fragaria x ananassa Duch.)

Postharvest Biology and Technology 162 (2020) 111103

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Combination effects of calcium chloride and nano-chitosan on the postharvest quality of strawberry (Fragaria x ananassa Duch.)

T

Van T.B. Nguyen, Duyen H.H. Nguyen, Ha V.H. Nguyen* Department of Food Technology, School of Biotechnology, International University — Vietnam National University HCMC, Vietnam

A R T I C LE I N FO

A B S T R A C T

Keywords: Strawberry Volatile compounds SEM Nano-chitosan Postharvest quality

This study aimed to investigate influences of calcium chloride (CaCl2) concentrations combined with nanochitosan coating on the quality of strawberry during postharvest storage. The fruit were dipped in different concentrations of calcium chloride (1 %, 2 %, 3 %, 4 %) before being coated with 0.2 % nano-chitosan. Physicochemical analysis including the overall quality index, weight loss, firmness, titratable acidity, total soluble solid, L-ascorbic acid content, antioxidant capacity, total phenolic content, total anthocyanin content, and malondialdehyde content were performed in 3 d intervals until fruit became unmarketable. Among six examined treatments, a combination of 3 % CaCl2 and nano-chitosan (NCTS) was the most effective one as maintaining the highest score of overall quality index of strawberry stored at 4 °C up to 15 d. The treatment also significantly reduced weight loss, preserved L-ascorbic acid, total anthocyanin contents, antioxidant capacity, and retarded malondialdehyde production. The scanning electron microscope image showed a smooth surface of strawberries coated with 3 % CaCl2 combined 0.2 % nano-chitosan. There has no bitterness detected in the treated strawberries after being stored 15 d at 4 °C. The major volatile compounds determined in the initial day were remained until the 15th d of storage.

1. Introduction Strawberry (Fragaria × ananassa Duch.) is a non-climacteric fruit containing a great variety of bioactive compounds including phenolic constituents, anthocyanins, vitamins and minerals (Giampieri et al., 2015). However, postharvest handling and storage of fresh strawberries is difficult mostly due to their high susceptibility to mechanical injury, water loss, microbial decay, physiological deterioration and high respiration rate (Liu et al., 2018). There have been various techniques studied to extend the shelf life of fresh strawberries such as cold storage (Han et al., 2004), modified atmosphere packaging (Nielsen and Leufvén, 2008), heat treatments (Civello et al., 1997), bioactive compounds (Liu et al., 2018) and edible coating (Sogvar et al., 2016; Badawy et al., 2017). Storage life of strawberry was within 1 d at room temperature (Mercantila, 1989) or prolonged from 5 to 10 d at 0 °C (SeaLand, 1991). Calcium (Ca2+) is a crucial divalent cation plant nutrient required for building structure of cell wall and cellular membranes. It can form cross-linking with de-esterified pectic residues to create a structure known as pectin “egg-box” (White and Broadley, 2003). This model plays an important role in strengthening cell wall and enhancing tissue firmness (White and Broadley, 2003). As a result, fruit softening is ⁎

remarkably delayed. Moreover, calcium is able to delay ripening and senescence-related processes including nutritional loss and susceptibility to pathogens (Conway et al., 1992, 1994b). Calcium has also been suggested to improve tissue antioxidant capacity as well as inhibit physiological disorders (White and Broadley, 2003; de Freitas and Mitcham, 2012; Saure, 2014). Besides, calcium plays an important role in defending against plant pathogens. This could be due to the capability of calcium in inhibiting pathogen pectolytic enzyme activities (Conway et al., 1994a). Although postharvest calcium treatments express positive effects on fruit quality, excessive calcium concentration could lead to side effects on the fruit surface such as discoloration (Conway et al., 1994b) and undesirable sensory taste (Hanson et al., 1993). A number of earlier studies reported that the combination between calcium and other postharvest methods can provide considerable advances over the calcium treatment alone (Aguayo et al., 2008; Hussain et al., 2012). Chitosan and nano-chitosan coating for prolonging the storage-life of plant foods has been reported in several studies (Eshghi et al., 2014; Nguyen and Nguyen, 2020). However, the combining between calcium and nano-chitosan in strawberries is not found in the literature. From those reasons, the objectives of this study were to investigate

Corresponding author. E-mail address: [email protected] (H.V.H. Nguyen).

https://doi.org/10.1016/j.postharvbio.2019.111103 Received 4 July 2019; Received in revised form 17 December 2019; Accepted 20 December 2019 0925-5214/ © 2019 Elsevier B.V. All rights reserved.

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supernatant was collected for titratable acidity and total soluble solid analyses. The titratable acidity (TA) of strawberry fruit was measured using the pH titration method (Hernández-Muñoz et al., 2006) using 0.1 M NaOH until reach pH 8.1. pH values were assessed using pH meter (HI 9126, Hanna Instruments Inc., Romania). The data of TA was expressed as gram of citric acid per kg of fruit fresh weight basis.

the effects of calcium chloride treatment combined with nano-chitosan coating on the quality of strawberry during storage. 2. Materials and method 2.1. Materials 2.1.1. Sample preparation Strawberry fruit (Fragaria x ananassa Duch.) were harvested from the orchard located in Lam Dong province when the red color covered 75 % of the fruit surface. Fruit were selected for uniformity in maturity stage, size, shape and color as well as the absence of mechanical damage and disease. The fruit were rinsed in tap water before treatments.

TA (gkg−1)=

Along with TA determination, total soluble solid content (TSS) was analyzed using the digital refractometer (RX- 5000, Atago Co., LTD., Japan) at 25 °C. The results of TSS were expressed as percentage (%). 2.2.5. Determination of L-ascorbic acid content The L-ascorbic acid content (AAC) was estimated following by a method of Kapur et al. (2012) with slight modifications. Strawberry fruit (5 g) was homogenized with 50 mL metaphosphoric acid – acetic acid solution, followed by centrifugation at 1822 x g for 15 min (UNIVERSAL 320R, Andreas Hettich GmbH and Co. KG, Germany). After that, 4 mL of the supernatant was mixed with 0.23 mL of 3 % bromine water, 0.13 mL of 10 % thiourea, and 1 mL of 2,4-dinitrophenyl hydrazine. The mixture was incubated at 37 °C in 3 h before being cooled in 30 min and treated with 5 mL of chilled 85 % H2SO4. The absorbance of samples was taken at 521 nm (GENESYS 10 UV–vis, Thermo Fisher Scientific, Inc., USA). L-ascorbic acid was used as standard solution. The results were expressed in g kg−1 fresh weight.

2.1.2. Experimental design According to Nguyen and Nguyen (2020), 0.2 % nano-chitosan solution that was prepared and supplied by Dalat Nuclear Research Institute, Vietnam with particle size was 250 nm (Saharan et al., 2013), showed the most effective coating solution for maintaining the postharvest quality of strawberries. Therefore, in the current work, there were six treatments: Control (untreated fruit); 0 % CaCl2 + 0.2 % nanochitosan (NCTS); 1 % CaCl2 + 0.2 % NCTS; 2 % CaCl2 + 0.2 % NCTS; 3 % CaCl2 + 0.2 % NCTS and 4 % CaCl2 + 0.2 % NCTS. Briefly, strawberry fruit were immersed in CaCl2 (Merck, Darmstadt, Germany) solution with different concentrations: 1 %; 2 %; 3 %; and 4 % in 1 min and drained at room temperature in 1 h before dipping into 0.2 % nanochitosan (NCTS) solution in 1 min (Nguyen and Nguyen, 2020). The treated fruit then were stored at 4 °C. Physico-chemical analysis was performed in each 3 d interval. Each treatment was done in triplicate.

2.2.6. Determination of total anthocyanin content The total anthocyanin content (TAC) of samples was quantified by the pH-differential method, followed by Giusti and Wrolstad (2001) with slight modifications. Briefly, 2 g of strawberry was homogenized with 0.025 M potassium chloride buffer, pH 1.0, and heated at 50 °C for 3 h. The mixture was centrifuged at 1822 x g for 20 min (UNIVERSAL 320R, Andreas Hettich GmbH and Co. KG, Germany). Then, the supernatant was diluted with 0.025 M potassium chloride buffer, pH 1.0, and 0.4 M sodium acetate buffer, pH 4.5 with appropriate dilution factor. The dilution was stand for 15 min for the equilibration. The absorbances for each dilution were taken at 496 nm and 700 nm (GENESYS 10 UV–vis, Thermo Fisher Scientific, Inc., USA). TAC was calculated and expressed as g kg−1 fresh weight.

2.2. Analytical methods 2.2.1. Measurement of the overall quality index Decay rate and shriveling score was assessed using a 1–5 visual rating scale (Fig. 1). A score of 3 was minimum acceptable quality before strawberries become unmarketable. Thus, when the deterioration was visible or when the rating of at least one of the quality attributes was at 3 or lower, the treatments were terminated (do Nascimento Nunes, 2015). 2.2.2. Measurement of weight loss The weight loss of strawberry was expressed as differences between the original weight and the weight recorded after 3 d intervals (Hernández-Muñoz et al., 2008). The original weight was measured right after applying treatments using digital balance (TXB- 622 L, Shimadzu Co, LTD., Japan). The weight loss percentage was calculated by following formula:

Weight loss ( %) =

Volume of NaOH(mL) × 0.1 M × 0.064 ×100 10 g of sample×102

2.2.7. Determination of total phenolic content The total phenolic content of strawberry was determined using the Folin–Ciocalteu colorimetric method, as modified by Boeing et al. (2014). Strawberry fruit (1 g) was homogenized with 10 mL of acetone:water (7:3, v/v). The homogenates was centrifuged at 1822 x g for 10 min at 4 °C (UNIVERSAL 320R, Andreas Hettich GmbH and Co. KG, Germany). After that, 1 mL of the supernatant was reacted with 5 mL of 10 % Folin–Ciocalteu reagent and 4 mL of 7.5 % sodium carbonate. The mixture was stand at room temperature for 60 min and measured at the absorbance of 765 nm (GENESYS 10 UV–vis, Thermo Fisher Scientific, Inc., USA). Gallic acid was used as standard solution. The results were expressed as GA equivalents in a fresh weight basis (g kg−1).

m 0−m × 100 m0

Where m0: weight of sample before storage (g), and m: weight of sample after storage intervals (g) 2.2.3. Measurement of firmness Fruit for each treatment were sliced into halves and each half was measured in center zone. The firmness of samples was measured using a Digital Fruit Hardness Tester (FR- 5120, Lutron electronic enterprise Co., LTD., Taiwan) according to Hernández-Muñoz et al. (2006) and 2 mm diameter tip was used. The cross-head speed was 2 mm s−1 and the penetration depth was 2 mm. The firmness was assessed as the maximum penetration force (N) reached during tissue breakage.

2.2.8. Determination of antioxidant capacity The antioxidant capacity (AC) of strawberry extracts was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging assay as described by Hangun-Balkir and McKenney (2012) with minor modifications. To prepare the extract, 2 g of fresh sample was homogenized with 80 % ethanol and centrifuged at 1822 x g for 15 min at 4 °C (UNIVERSAL 320R, Andreas Hettich GmbH and Co. KG, Germany). Then, 2 mL of 0.1 mM DPPH reagent was added to 2 mL of the supernatant (equivalent 80 % ethanol volume as control). The solution was kept in dark condition at room temperature for 30 min. Finally, the absorbance at 517 nm was recorded (GENESYS 10 UV–vis, Thermo

2.2.4. Determination of total soluble solid and titratable acidity At first, 10 g of strawberry puree was homogenized using a blender with 0.1 L of distilled water. After centrifugation at 1822 x g for 10 min (UNIVERSAL 320R, Andreas Hettich GmbH and Co. KG, Germany), the 2

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Fig. 1. Visual quality scores and descriptors for strawberry (Nguyen and Nguyen, 2020).

Fisher Scientific, Inc., USA). Antioxidant capacity was expressed as the percentage of DPPH radical scavenging capacity following the below formula:

%DPPH scavenging=

(A control − A sample) A control

Where in ε (molar extinction coefficient): 155 mM−1 cm−1 for MDA DF: dilution factor

x100

2.2.10. Scanning electron microscopy (SEM) Fresh strawberries of control, 0 % CaCl2 + NCTS, and 3 % CaCl2 + NCTS treatment at the 6th d were frozen at -80 °C for 1 d, then freeze dried and cut into a piece of 2 mm × 2 mm × 1 mm (Fonseca et al., 2005). Samples were sent to the scanning electron microscope for observing SEM images.

Where in Acontrol: The absorbance of control Asample: The absorbance of sample 2.2.9. Determination of malondialdehyde content The malondialdehyde (MDA) content was assessed by the thiobarbituric acid (TBA) reaction (Liu et al., 2018). Briefly, 1 g of fresh strawberry was homogenized in 10 mL of ice-cold 0.1 % trichloroacetic acid (TCA), followed by centrifugation at 1822 x g for 10 min (UNIVERSAL 320R, Andreas Hettich GmbH and Co. KG, Germany). Then, 1 mL of sample aliquot taken from the supernatant was mixed with 4 mL of 0.25 % TBA in 10 % TCA. After incubation at 95 °C for 15 min, the mixture was quickly cooled in an ice bath and centrifuged at 1822×g for 10 min (UNIVERSAL 320R, Andreas Hettich GmbH and Co. KG, Germany). The absorbance was taken at 532 and 600 nm. The malondialdehyde content was calculated using the following formula and expressed as micromoles of MDA per kilogram of fruit fresh weight basis (μmol kg−1):

MDA content(μmolkg−1) =

2.2.11. Sensory evaluation Sensory quality of strawberries was evaluated by using a descriptive rating test (Lu et al., 2018). In brief, 30 semi-trained panelists (10 males and 20 females, ages 20–30 years) with no problem with taste or odor ability were chosen. Eight examined attributes for the descriptive rating test were color, firmness, sweetness, sourness, strawberry flavor, juiciness, bitterness, and off-flavor. The test was conducted at day 0 of untreated strawberries and day 15 of the 3 % CaCl2 + NCTS treated ones. Before conducting the sensory evaluation, strawberries were taken out of the refrigeration an hour to equilibrate to room temperature. Fruit were chosen with uniform in size, shape, and color to minimize any bias caused by presentation. The panelists were arranged to individual booth and the test was performed under white light. Each panelist was provided 5 strawberry fruit and ask to evaluate the score between 0 (absence of the sensation) and 5 (strong intensity). Between

(A532-A 600)×DF ε ×103 3

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became unmarketable after 12 d with the appearance of fruit surfacedamage symptoms. This finding agreed with the report of Manganaris et al. (2007), who found that high concentrations of calcium salts accelerated flesh softening processes in peach fruit. A plausible explanation for these adverse effects is the substrate delocalization and cell wall disintegration when immersing fruit into high salt concentrations (Souty et al., 1993; Manganaris et al., 2007).

evaluations, water was given to rinse the palate, and a break in 30 s was enforced. 2.2.12. GC–MS analysis At first, 5 g of fresh strawberries was homogenized (A11 Basic, IKAWerke GmbH & Co.KG, Germany) and mixed with 5 mL of acetone before being shaken well at 350 rpm for 2 h. The acetone extract was then filtered through a 0.45 μm membrane filter. The collected solution was used for GC–MS analysis in the day of extraction. GC–MS analysis was performed using Scion equipment (SCION 456GC, SCION SQ, USA). The system was controlled by MS Workstation 8 for Scion software. The extracted sample (1 μL) was injected into the gas chromatograph through a RESTEK Rxi-5 ms column (30 m × 0.25 mm (i.d.), 0.25 μm df). It was operated with the injector temperature at 250 °C. The initial column temperature was held at 50 °C for 1 min, increased to 80 °C at a rate of 30 °C min−1, increased to 180 °C at a rate of 5 °C min−1, increased to 300 °C at a rate of 60 °C min−1 and finally held isothermally for 3 min. Total analysis time was 27 min. Helium was used as the carrier gas with the flow rate at 1 mL min1. For the MS analysis, the ionization voltage was 70 eV. The samples were injected in split mode as 50:1. Mass spectral scan range was set at 50–500 amu at full scan mode. The ionization source temperature was 250 °C.

3.2. Weight loss The results recorded for the weight loss percentage of control and treated strawberries are presented in Fig. 2B. The weight loss increased significantly (p < 0.05) during the entire storage period for all treatments. Among the treatments, the combinations of CaCl2 and NCTS coating had better effects on delaying the weight loss of strawberry than the control and NCTS coating alone. At the end of the storage time, 3 % CaCl2 + NCTS was the best treatment in preventing weight loss of strawberries. It is proved that loss of weight in fruit is mainly due to water evaporation caused by transpiration and respiration processes (Sogvar et al., 2016). Chitosan coating is believed to act as a semipermeable barrier against oxygen, carbon dioxide, and moisture, thus reducing respiration, oxidation reaction, and water loss (Gol et al., 2013). In addition, it is claimed that postharvest calcium treatments might change gas diffusion rates in the tissue, resulting in the inhibition of respiratory metabolism (Hewett and Thompson, 1992; Saftner et al., 1999). Therefore, the combination of NCTS coating and CaCl2 treatment was more effective in reducing weight loss than the control or NCTS coating alone, as shown in Fig. 2B.

2.3. Statistical analysis All statistical analyses were performed using Minitab software (version 17). The data were analyzed by one-way ANOVA and presented as means ± standard deviation (SD) with p < 0.05 as significant levels.

3.3. Firmness 3. Results and discussion Changes in firmness of both control and treated strawberries during storage at 4 °C are shown in Fig. 2C. Firmness of the initial fruit was 3.72 N and decreased during storage, but this decrease was effectively delayed by the calcium treatment. After the first 3 d of storage, there was an increasing in firmness of all samples, and that of calcium treated samples (except 1 % CaCl2 + NCTS) was greater than non-treated one. This could be induced by the water loss in fruit cells during storage, leading to raising of force needed to break up the fruit flesh (Chen et al., 2011). In addition, Lara et al. (2004) has proved that the application of

3.1. The overall quality index The effect of different CaCl2 concentrations on the visual quality score of strawberries as compared to control sample is shown in Fig. 2A. It is noted that the combination of 3 % CaCl2 and NCTS coating maintained the overall quality of strawberries up to 15 d; meanwhile no differences (p > 0.05) were obtained among CaCl2 levels of 0 %, 1 %, and 2 %. It was noted that strawberries treated with 4 % CaCl2 + NCTS

Fig. 2. Effects of different calcium chloride concentrations combined with 0.2 % nano-chitosan on the overall quality index (A), weight loss (B), firmness (C), and titratable acidity (D) of strawberries stored at 4 ± 1 °C for 15 d. The error bars represent standard deviations of triplicate assays with the confidence interval of 95 %. 4

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Muñoz et al., 2008). TSS content of the control and treated strawberries then slowly declined until the end of the storage time. It is found that sugar is one of the main substrates for respiratory metabolism in fruit. Therefore, the use of sugar for respiration processes might cause the reduction in TSS level (Gol et al., 2013). At 6th and 9th d of storage, TSS values of calcium treated samples were higher than untreated one. It is reported that calcium treatment might delay cell wall degrading process by binding with pectic substances presented in cell wall and middle lamella (Lara et al., 2004). As a result, the amount of sugar used for respiration in calcium treated fruit might be lower than that of untreated one. In addition, at 12th and 15th d, fruit treated with 3 % CaCl2 + NCTS contained significantly higher amounts of TSS compared to that of 4 % CaCl2 + NCTS (p < 0.05).

exogenous calcium ions to strawberry fruit preserved cell wall and middle lamella structure, both of which contributed to fruit firmness. In other words, under the action of pectin methyl-esterase enzyme during ripening stage, more pectin carboxyl groups are generated, leading to the stimulation of calcium ion binding to pectin (Conway and Sams, 1983; Stanley et al., 1995). Calcium ions supplied from the treatment with 1 % and 2 % CaCl2 might be insufficient to form a cross-linking with carboxyl groups in pectin. When strawberries were immersed into the higher concentrations of CaCl2 (3 % and 4 %), fruit firmness retention was increased. 3.4. Titratable acidity Changes in titratable acidity (TA) of strawberry by storage time are shown in Fig. 2D. No significant difference (p > 0.05) in TA content was observed between treated samples at the initial and final d. However, there were marked increases (p < 0.05) in TA of both control and treated fruit within 6 d of storage. The increased TA values could be resulted from the increment of Cl− ion (Souty et al., 1993). Moreover, the rising of L-ascorbic acid content (Fig. 23B) might also contribute to the increasing of TA content (Chen et al., 2011). Reduction of TA levels after 6 d of storage might be due to metabolic changes in fruit resulted from the use of organic acids in the respiratory process (Sogvar et al., 2016).

3.6. L-Ascorbic acid content For the control as well as the treated strawberries, L-ascorbic acid content (AAC) showed an increasing trend in entire storage period (Fig. 3B). The increment in AAC might be due to the synthesis of ascorbic acid from D-galacturonic acid released from cell wall pectin (Liu et al., 2018) or from D-glucose found in strawberry fruit (Chen et al., 2011). Meanwhile, a possible explanation for AAC lost during the last 3 d of storage is the autoxidation occurred when the ascorbic acid combine with oxygen in the air (Sogvar et al., 2016). At the 15th d, using the 3 % CaCl2 + NCTS showed a better effect in maintaining AAC of fresh strawberries compared to the 4 % CaCl2 + NCTS treatment (Fig. 3B).

3.5. Total soluble solid Total soluble solid (TSS) of control and treated samples during storage are presented in Fig. 3A. TSS level at the beginning of storage was 6.93 % and then remarkable increased (p < 0.05) at the 3rd d in all treatments. A possible explanation for this phenomenon is the solubilization of polyuronides and hemicelluloses presented in cell wall (Hernández-Muñoz et al., 2008). Moreover, the water loss due to transpiration might also contribute to the increment in TSS (Hernández-

3.7. Antioxidant capacity During storage, antioxidant capacity (AC) was declined significantly (p < 0.05) either in control or treated strawberries with the lowest value obtained from the control fruit (Fig. 3C). The data demonstrated positive effects of the combination between calcium treatment and NCTS coating. It has been known that chitosan coating may form a Fig. 3. Effects of different calcium chloride concentrations combined with 0.2 % nanochitosan on the total soluble solid content (A), L-ascorbic acid content (B), antioxidant capacity (C), total phenolic content (D), total anthocyanin content (E), and malondialdehyde content (F) of strawberries stored at 4 ± 1 °C for 15 d. The error bars represent standard deviations of triplicate assays with the confidence interval of 95 %.

5

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3.8. Total phenolic content There were increases and then decreases of total phenolic content (TPC) over the entire storage period (Fig. 3D). The increment of TPC could be due to the phenolics and flavonoids production via the phenylpropanoid metabolism (Singh et al., 2010). It is claimed that the degradation of cell structure at the fruit senesce stage might be responsible to the decline of TPC (Gol et al., 2013). Furthermore, the application of NCTS coating may form a selective barrier reducing oxygen supply for enzymatic oxidation of phenolic compounds (Gol et al., 2013), thus; TPC of treated samples were maintained longer than uncoated fruit. 3.9. Total anthocyanin content Changes in the anthocyanins content (TAC) of control and coated strawberry fruit stored at 4 °C are shown in Fig. 3E. It can be seen that during 6 d of storage, TAC of the coated samples increased gradually, similar to reported studies (Gol et al., 2013; Sogvar et al., 2016). A possible explanation is the continued synthesis of anthocyanins through flavonoid and anthocyanin pathway after harvesting (Kalt et al., 1999; Ananga et al., 2013). However, afterward, TAC of all coated fruit was significantly decreased (p < 0.05) until the end of storage (Fig. 3E). This could be due to anthocyanins degradation, which was related to water loss during storage, induced physiological stress and promoted fruit senescence (Nunes et al., 2005). Loss of water caused membrane disintegration and cellular content leakage (Ben-Yehoshua and Rodov, 2003), both of which contributed to the reduction of anthocyanins concentration. In addition, the rising of enzyme activities including polyphenol oxidase might also led to the decrease in anthocyanin content of strawberries during storage (Eshghi et al., 2014). In general, 3 % CaCl2 + NCTS showed more positive effect (p < 0.05) in delaying the loss of TAC in comparison with 4 % CaCl2 + NCTS coated fruit during 15 d of storage (Fig. 3E). 3.10. Malondialdehyde content Malondialdehyde (MDA) is a secondary end product of membrane lipid peroxidation. It is usually considered as an indicator to assess the progress of cell oxidative damage (Wang et al., 2015). As shown in Fig. 3F, MDA content of all treatments increased during storage at 4 °C. MDA content in control fruit was significantly higher (p < 0.05) than that of treated fruit after 6 d of storage. At the 9th d, the production of MDA was significantly delayed (p < 0.05) in 3 % CaCl2 + NCTS and 4 % CaCl2 + NCTS treatments as compared with others. According to Pareek (2017), CaCl2 added may bind to phospholipids and proteins in the membranes to protect cellular membranes against reactive oxygen species (ROS). Furthermore, by accumulating antioxidant compounds such as ascorbic acid, postharvest calcium treatment can increase tissue antioxidant capacity (Pareek, 2017). In addition, it was also found that the increase of MDA level could be prevented by applying NCTS coating (Nguyen and Nguyen, 2020). The coating might act as a semi-permeable barrier against oxygen responsible for lipid peroxidation, thereby alleviating oxidative damage during cold storage (Petriccione et al., 2015). Therefore, the combination of 3 % or 4 % CaCl2 and NCTS should be considered as the appropriate treatments for reducing MDA content after 15 d of storage (Fig. 3F).

Fig. 4. Scanning electron microscopy (SEM) images of fruit surface of control (A), 0 % CaCl2 + NCTS (B), and 3 % CaCl2 + NCTS fruit (C), stored at 4 °C after 6 d.

selective barrier reducing oxygen supplying for oxidation reaction (Gol et al., 2013). Hence, the reduction of AC in treated fruit was delayed effectively. Moreover, calcium treatment has been proved to retain antioxidant activity as well as inhibit oxidative metabolism in fresh produce during ripening and senescence (Pareek, 2017). In this research, an incorporation of 3 % and 4 % CaCl2 with 0.2 % NCTS coating was suggested to maintain AC in the berries.

3.11. Scanning electron microscopy Scanning electron microscopic (SEM) image was carried out to investigate the changes in the fruit surface structure of control, non-calcium treated (0 % CaCl2 + NCTS) and calcium treated (3 % CaCl2 + NCTS) samples after 6 d of storage at 4 °C (Fig. 4). Evidently, several cracks were observed on the surface of the control (Fig. 4A). This might be due to the remarkable water loss (as shown in Fig. 2B), which were 6

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strawberry aroma and taste. Interestingly, bitterness attribute of 3 % CaCl2 + NCTS sample was not recognized at 15th d. Lawless et al. (2004) reported that the bitterness caused by calcium salts can be suppressed by sucrose and citric acid, which presented naturally in strawberries (Mahmood et al., 2012). In addition, the formation of cross-linking between calcium ion and carboxyl groups in pectin reduced the amount of free calcium ion responsible for the unpleasant taste (bitterness). 3.13. GC–MS analysis Table 1 illustrates volatile compounds detected in 3 % CaCl2 + NCTS treated strawberries in the initial and the 15 d of storage. The major ones were 3-methyl-2,5-furandione; 2,5-dimethyl-2,4-dihydroxy3(2 H)-furanone, and 5-hydroxymethylfufural of which 5-hydroxymethylfufural was more dominant. According to Poerwono et al. (2001), 3-methyl-2,5-furandione or citraconic anhydride was formed when citric acid was heated; thus, this compound might represent for the changes of citric acid in fruit. It is noticed that calcium treatment maintained a certain amount of 2,5-dimethyl-2,4-dihydroxy-3(2 H)furanone (DMHF) that was contributing significantly to the flavor of strawberry fruit (Wein et al., 2002). Although DMHF content was decreased slightly from 0.96 % to 0.92 %, the strawberry flavor might be retained by the compound 2,5-dimethyl-4-methoxy-3(2 H)-furanone (DMMF) found at 15th day (Table 1). Indeed, DMMF was an enzymatic methylation product of DMHF and it is also reported to be a key strawberry aroma (Kallio, 2018). Moreover, Fig. 5 points out that the intensity score for strawberry flavor in the sensory evaluation was still stand around 4 at the end of storage duration. Beside furan group, organic acids included butanoic acid and propanoic acid and fatty acids which were n-hexadecanoic acid and oleic acid were detected at the final day of storage.

Fig. 5. Descriptive rating scale of fresh strawberries at the 0th d and strawberries treated with 3 % CaCl2 + NCTS at the 15th d, stored at 4 °C. Five-point intensity scale: 0 = not at all; 1 = weak; 2 = fairly; 3 = moderate; 4 = quite; 5 = very. Data are expressed as means ± SD of scores given by thirty panelists. Different letters indicate significant differences with the confidence interval of 95 %.

Table 1 Volatile compounds found in strawberries treated with 3 % CaCl2 + NCTS stored at day 0 and day 15. Volatile compounds

3-methyl-2,5-furandione 2,5-dimethyl-2,4-dihydroxy-3(2 H)-furanone 5-hydroxymethylfufural 2,5-dimethyl-4-methoxy-3(2 H)-furanone Butanoic acid n-hexadecanoic acid Oleic acid Propanoic acid

Storage duration Day 0

Day 15

4.48 % 0.96 % 62.09 % – – – – –

3.88 % 0.92 % 60.58 % 0.51 % 1.91 % 0.64 % 0.68 %

4. Conclusions The results of this study indicate that the combination of 3 % CaCl2 and NCTS coating brought a significantly higher overall quality index and a better positive impact than other treatments on physical properties including weight loss, fruit firmness, and chemical properties regarding L-ascorbic acid, total anthocyanin contents and antioxidant capacity of strawberries stored at 4 °C within 15 d. The treatment created a smooth surface outside protecting strawberries. Meanwhile, coating the fruit with 4 % CaCl2 and NCTS had significantly preserved fruit firmness, antioxidant capacity and total phenolic content. The both treatments significantly delayed MDA accumulation. Sensory evaluation showed a weak off-flavor; however no bitterness at all for the 3 % CaCl2 + NCTS coated strawberries after the 15 d storage at 4 °C.

-: compound was not detected.

results of transpiration and respiration processes. On the contrary, only a few small cracks appeared as treated with NCTS coating alone (Fig. 4B). It is generally known that chitosan coating reduced respiration rate and water loss (Gol et al., 2013). In treatment of 3 % CaCl2 + NCTS, there was an absence of cracks on surface (Fig. 4C). In comparison with NCTS coating alone, using the mixture of CaCl2 and NCTS created a smooth surface for strawberries. This might be due to the cross-linking formation between de-esterified pectic residues and exogenous calcium ion during ripening period as discussed in section 3.3.

Acknowledgement The authors would like to thank the infrastructure support of International University-Vietnam National University Ho Chi Minh city, Vietnam.

3.12. Sensory evaluation Results of the descriptive rating test are shown in Fig. 5. In brief, the scores of color, firmness and juiciness of strawberries were reduced (p < 0.05) after 15 d of storage. The results obtained supported the data presented in section 3.1, 3.2, 3.3 and the reasons could be the loss of water during storage and activities of endogenous enzymes (Lara et al., 2004; Sogvar et al., 2016; Badawy et al., 2017).Moreover, Chisari et al. (2007) found that polyphenol oxidase enzymes created undesirable brown or black pigments, which negatively affecting the surface color of strawberries (Chisari et al., 2007). In terms of sweetness and sourness, there were insignificant differences (p > 0.05) in the evaluation of the first and the last day of storage. However, the detected intensity of strawberry flavor at 15th d was weaker than that of 0th d. This might be due to the loss and changes of volatile compounds contributing to the

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