Skip to main content
NCBI home page
Journal of Food Science and Technology logoLink to Journal of Food Science and Technology
. 2022 Mar 12;59(8):3296–3306. doi: 10.1007/s13197-022-05412-5

Aloe vera gel coating delays softening and maintains quality of stored persimmon (Diospyros kaki Thunb.) Fruits

Muhammad Shahzad Saleem 1, Shaghef Ejaz 1,✉, Muhammad Akbar Anjum 1, Sajid Ali 1, Sajjad Hussain 1, Aamir Nawaz 1, Safina Naz 1, Mehdi Maqbool 2, Abdul Manan Abbas 1
PMCID: PMC9304499  PMID: 35876768

Abstract

The effect of Aloe vera (AV) gel coating was studied on antioxidant enzymes activities, oxidative stress, softening and associated quality attributes of persimmon fruits. The fruits were coated with 0 and 50% AV-gel coating and stored for 20 days at 20 ± 1 ºC. AV-gel coated fruits exhibited considerably less weight loss, hydrogen peroxide level, electrolyte leakage and malondialdehyde content. AV-gel coated fruits had significantly higher ascorbate peroxidase, peroxidase, superoxide dismutase and catalase activities. In addition, AV-gel coating suppressed pectin methylesterase, polygalacturonase and cellulase activities and showed higher ascorbic acid, DPPH scavenging antioxidants and phenolics, and lower sugars and carotenoids. To the best of our knowledge, these results are the first evidence that AV-gel coating modulates the activities of cell wall degrading enzymes to delay ripening in climacteric fruits. So, AV-gel coating prohibited the onset of senescence by activating enzymatic antioxidant system, accumulating bioactive compounds and suppressing cell wall degradation.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13197-022-05412-5.

Keywords: Diospyros kaki, Fruit firmness, Oxidative stress, Ripening, Antioxidative enzymes, Softening enzymes

Introduction

Persimmon (Diospyros kaki Thunb.) is being intensively cultivated from Japan up to the regions near to Mediterranean Sea and distant Brazil (Senica et al. 2016). Persimmon fruit provides dietary fiber, minerals, vitamins, polyphenols, flavonoids, carotenoids, steroids and terpenoids to humans. These bioactive compounds regulate genes linked with cholesterol accumulation and, thus, reduce oxidative stress and cholesterol accumulation in humans (Pérez-Burillo et al. 2018).

Persimmons show climacteric behavior with a rapid rise in ethylene production and respiration rate, resulting in limited marketing time and increased fruit spoilage. Storing the persimmon fruits below 10 ºC compromises its quality; for example, at 5 ºC, mottling and translucence of peel, gelling and darkening of flesh, loss of flavor and juice and the production of off odors occur (Fahmy and Nakano 2016). The loss of firmness is largely due to solubilization and polymerization of pectin and decomposition of cellulose during ripening. Two major enzymes, namely pectin methylesterase (PME) and polygalacturonase (PG), are involved in the degradation of pectin, whereas cellulase (CEL) enzyme catalyzes cellulolysis reaction. Fruit softening is also linked with associated cellular oxidation and peroxidation processes. During postharvest phase, several internal metabolic processes stimulate the production of reactive oxygen species (ROS). Accumulation of ROS, such as superoxide (O2•−) and hydroxyl (OH•) ions and hydrogen peroxide (H2O2) increases during ripening that triggers fruit softening by loosening cell wall (Payasi et al. 2009). ROS promote cellular breakdown and senescence by destabilizing cellular redox homeostasis, regulating expression of mitochondrial proteins, modifying DNA base and peroxidizing membrane-lipids (Tian et al. 2013). In such scenario, a robust defense mechanism consisting of enzymatic antioxidants, such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX) etc., and non-enzymatic antioxidants, such as phenolics, carotenoids, ascorbic acid etc., can mitigate the oxidative stress by scavenging ROS (Huan et al. 2016). Therefore, the techniques that maintain cell wall structure and cellular redox homeostasis by regulating enzymatic activities and ROS production are required for extended postharvest storage of fruits (Saleem et al. 2020).

Edible coatings have been widely studied in extending the storage and shelf life of fresh fruits and vegetables. Edible coatings such as, chitosan, Aloe vera and gum arabic, are barrier to moisture, microbes, oxygen and carbon dioxide and thus modify the internal physiological functions of fruits and vegetables, resulting in the extension of storage or shelf life of the coated produce (Guillén et al. 2013; Khaliq et al. 2016; Saleem et al. 2020, 2021). This microenvironment limits the activities of cell wall-degrading enzymes and allows the fruit to retain firmness during storage (Maftoonazad et al. 2008). Various combinations of coatings, such as gum arabic with chitosan (Maqbool et al. 2010) or carboxymethyl cellulose (Shakir et al. 2022), or multi-layer coating (Ali et al. 2014) have been found to be highly efficient. Aloe vera gel (AV-gel) has shown the promise of a viable plant-based edible coating by stimulating enzymatic and non-enzymatic defense mechanisms (Mendy et al. 2019) and inhibiting surface browning (Ali et al. 2019a) and flesh softening (Khaliq et al. 2019). AV-gel based coating reduced POD and polyphenol oxidase activities and enhanced CEL, SOD and APX activities in harvested litchi fruits (Ali et al. 2019b). Similarly, extension in postharvest life of climacteric fruits, such as plum (Martínez-Romero et al. 2017) and papaya (Kuwar et al. 2015), has also been reported in response to AV-gel application.

To our knowledge, the role of AV-gel coating in delaying fruit softening and regulating ROS metabolism in persimmon fruits to extend its storage life is not studied yet. Therefore, in this study, modulation of stress indicators like lipid peroxidation, H2O2 accumulation and membrane damage and response of antioxidant enzymes, including CAT, SOD, POD and APX, and bioactive antioxidants have been investigated during ripening of AV-gel coated persimmon fruits at ambient storage. The activity of cell wall-degrading proteins, i.e. PME, PG and CEL, has also been investigated as a marker for fruit ripening and softening.

Materials and methods

Plant material and preparation of Aloe vera Gel Coating

Freshly harvested fruits of persimmon (cv. ‘Fuyu’) were sorted for uniformity in size and maturity (yellow-orange color stage) and freedom from any injury or decay. Fruits had total soluble solids (TSS) values from 10 to 12%. The fruits were disinfected with 0.01% NaOCl solution for two min and, after rinsing, were air dried. For preparing AV-gel coating, mature leaves from Aloe vera plants (≥ 3-year-old) were first disinfected with 2% NaOCl and then rinsed with distilled water. The rind of Aloe vera leaves (~ 1 kg) was peeled off to extract colorless mucilaginous gel. The gel matrix was blended, filtered through two layers of muslin cloth to remove the fibers and pasteurized at 70 °C for 45 min. For improving the strength and plasticity of AV-gel coating, 1.5% glycerol was added. The gel was cooled to room temperature before adjusting its pH to 3.7.

Application of AV-gel Coating

Based on preliminary trial on persimmon fruits, where the fruits were coated with 0% (control), 25%, 50% and 100% AV-gel, 50% AV-gel was selected for this research work. The experiment was laid out using completely randomized design replicated thrice. Before applying AV-gel coating on day 0, 15 fruits per replication were analyzed for quality and biochemical attributes. Thereafter, the fruits were distributed to two groups (control or non-coated and AV-gel coated group), each containing 225 fruits. Before application of coating, internal fruit tissue temperature was brought to 20 ± 2 ºC. The fruits of control group were immersed in deionized water, whereas the fruits of AV-gel coated group were immersed in AV-gel coating solution for three minutes. The coating solution temperature was maintained at 20 ± 2 ºC. Extra coating solution was drained by keeping the coated fruits on a stainless-steel mesh. The fruits were then dried in air for 90 min at 20 °C and packed in sterilized baskets. Finally, the fruits were stored at 20 ± 1 °C and 85 ± 2% relative humidity for 20 day. The fruits were sampled on the 4th, 8th, 12th, 16th and 20th day of application of the AV-gel coating for various analyses.

Fruit Weight Loss (%) and Electrolyte Leakage

Weight loss (%) of persimmon fruits was calculated from initial weight and final weight (weight on each sampling day) and expressed in percent.

For determining plasma membrane leakage, 0.3 cm thick fruit tissue-discs of 1 cm diameter were continuously shaken in 50 mL deionized water for 30 min (Yang et al. 2011) and the electrical conductivity (EC1) of the solution was recorded. Then this solution was heated to 100 °C for 30 min and the electrical conductivity (EC2) was recorded after bringing the solution to room temperature. These two electrical conductivities were used to calculate relative electrolyte leakage of cell membranes which was expressed in percentage as follows.

graphic file with name M1.gif

Malondialdehyde Content and Hydrogen Peroxide

For malondialdehyde (MDA) content determination, 1 g of sample was extracted in 10% trichloroacetic acid (15 mL) and centrifuged for 20 min at 10,000 g. The supernatant was reacted with 0.6% 2-thiobarbituric acid (2 mL). A UV-Vis spectrophotometer (UV-1602; BMS, Spain) was used for taking absorbance at 450, 532 and 600 nm (Ali et al. 2019b). MDA levels were presented as µmol kg− 1 fresh biomass.

For the measurement of H2O2 concentration, 1 g of fruit pulp was homogenized with 0.1% trichloroacetic acid (1 mL) and centrifuged for 15 min at 12,000 g (Velikova and Loreto 2005). 500 µL of supernatant was added to phosphate buffer (0.5 mL, pH 7.0) and 1 mol L− 1 potassium iodide (1 mL). A wavelength of 390 nm was set to measure the absorbance of the solution. H2O2 concentration was denoted as µmol kg− 1 fresh weight.

Activities of Antioxidant Enzymes

Crude Enzyme Extraction

Precooled phosphate buffer (4 °C, pH 7.2) was used to homogenize persimmon fruit tissues. The homogenate was then centrifuged for 10 min at 12,000 g. The supernatant thus obtained was designated as enzyme extract that was stored at -80 °C unless required for further analysis.

Ascorbate Peroxidase Activity

For determining the activity of APX assay, enzyme extract (100 µL), 0.5 mmol L− 1 L-ascorbic acid (100 µL) and 0.15 mmol L− 1 H2O2 (100 µL) were added to a cuvette containing phosphate buffer (700 µL, pH 5). Subsequently, by using the spectrophotometer, absorbance of the solution was observed at 290 nm at an interval of 30 s up to 90 s. To calculate APX activity, mean values were used, and the activity was expressed as U kg− 1 (Nakano and Asada 1987).

Catalase Activity

For CAT activity, enzyme extract (100 µL), 20 mmol L− 1 H2O2 (500 µL) and phosphate buffer (2.4 mL, pH 5) were added into a quartz cuvette. Further, the change in the absorbance of the solution was spectrophotometrically observed after 0, ½, 1, 1½ and 2 min at 240 nm and CAT activity was expressed as U kg− 1 (Ali et al. 2016).

Peroxidase Activity

The production of tetraguaiacol, in the presence of H2O2, from the dehydrogenation of guaiacol was measured at 470 nm to determine the activity of POD enzyme. For this, the reaction mixture consisting of enzyme extract (200 µL), 40 mmol L− 1 H2O2 (300 µL, as a substrate), phosphate buffer (1 mL, pH 5) and 20 mmol L− 1 guaiacol (100 µL) was taken in a cuvette and its absorbance was recorded at 0, ½, 1 and 1½ min. For the estimation of POD activity, mean values were derived, and the activity was expressed as U kg− 1 (Ali et al. 2016).

Superoxide Dismutase Activity

SOD inhibits photochemical reduction of nitro blue tetrazolium, which determines its enzymatic activity. In a cuvette, enzyme extract (100 µL) along with phosphate buffer (1 mL, pH 5), distilled water (1 mL), 22 µmol L− 1 methionine (300 µL) and 20 µmol L− 1 nitro blue tetrazolium (100 µL) were taken. The cuvette was exposed to UV-light for 15 min. Thereafter, 0.6 µmol L− 1 riboflavin (100 µL) was added to the reaction mixture as a substrate. The reaction mixture was observed for its absorbance at 560 nm after 0, ½, 1 and 1½ min. Using the mean values, SOD activity was calculated and expressed as U kg− 1 (Ali et al. 2016).

Determination of Activities of Cell Wall Degrading Enzymes

Pectin Methylesterase Activity

The PME activity was determined following a modification of the procedure of Hagerman and Austin (1986). Fruit tissue sample (2 g) was homogenized in 2 M NaCl solution (8 mL) at 4 °C. The homogenate was shaken for 4 h keeping the temperature at 4 °C before centrifuging at 20,000 g during 10 min at 4 °C. The pH of clear supernatant was adjusted to 7.5 with 0.1 N HCl or 0.1 N NaOH. 750 µL distilled water (pH 7.5) was added to a cuvette containing 2.0 mL pectin (0.5% w/v) esterified from citrus fruit and 150 µL bromothymol blue (0.01% w/v). pH of the reaction mixture was adjusted to 7.5 and 100 µL of the supernatant was introduced to initiate the reaction. Subsequently, absorbance of the solution was observed at 620 nm. A reduction of 0.1 S− 1 in absorbance was defined as the one unit of PME activity at 620 nm and presented as U kg− 1.

Extraction for Polygalacturonase and Cellulase Activity

To extract enzyme protein, 1 g homogenized sample was added to 8 mL cold sodium acetate (40 mM, pH 5.5) containing 2 mL NaCl (0.2 M) and insoluble polyvinylpolypyrrolidone (2% w/v). The mixture was incubated at 4 °C for 15 min, followed by centrifugation at 10,000 g for 20 min at 4 °C. Finally, the supernatant was collected as enzyme extract and used for analysis of activities of PG and CEL as suggested by Deng et al. (2005).

Polygalacturonase Activity

The enzyme extract (100 µL) was added to reaction mixture containing ice-cooled 500 µL 0.2 M sodium acetate (pH 4.5) and 400 µL 1% citrus pectin. This reaction mixture was then incubated for 1 h at 37 °C before adding 1000 µL of 1% dinitrosalicylic acids. The mixture was then heated to boiling temperature for 5 min before cooling it and adding the substrate. An increase of 0.1 min− 1 in absorbance was defined as the one unit of PG activity at 540 nm and presented as U kg− 1.

Cellulase Assay

For the determination of CEL activity, 100 µL extract was introduced to a reaction mixture containing 400 µL of carboxymethyl cellulose (1% w/v), 500 µL sodium acetate buffer (0.1 M, pH 5.0). Then the reaction mixture was incubated at 37 °C for 1 h before adding 1,000 µL of dinitrosalicylic acid (1%) in it and heating in boiling water for 5 min. An increase of 0.1 min− 1 in absorbance was defined as the one unit of CEL activity at 540 nm and presented as U kg− 1.

Protein Content

Bradford assay was used to determine total protein content (Bradford 1976). The absorbance of the samples and standard (bovine serum albumin) was observed at 595 nm.

Total Soluble Solids, Titratable Acidity and Reducing, Non-reducing and Total Sugars

TSS of persimmon juice were measured by refractometer. Titratable acidity (%) was determined by titrating diluted persimmon fruit juice (1: 4) with 0.1 N NaOH until titration solution reached at pH 8.2 (Ejaz et al. 2015). Reducing, non-reducing and total sugars were evaluated as suggested by Hussain et al. (2017).

Non-enzymatic Antioxidants

Total Phenolic Content

Total phenolics were estimated as described by Singleton and Rossi (1965). For this purpose, the sample was centrifuged at 12,000 g at 4 °C for 15 min. Then 200 µL supernatant was added to a mixture consisting of Folin-Ciocalteu’s reagent and 800 µL Na2CO3 (7.5%) solution. Further, the volume was made up to 10 mL by adding deionized water. Finally, absorbance of the solution was recorded at 765 nm and total phenolics were presented as g kg− 1 fresh weight.

Total Antioxidants

Total antioxidants in persimmon fruits were analyzed by their capability to scavenge DPPH radicals (Brand-Williams et al. 1995) and then compared with trolox (an antioxidant standard). Antioxidant compounds were extracted from homogenized 1 g sample of frozen persimmon pulp using 70% ethanol (10 mL). Afterwards, 0.1 mmol L− 1 DPPH (2 mL) was added to filtered extract (1 mL) before incubating it in dark for 30 min and recording its absorbance at 520 nm. Ethanol (70%) was used as blank and 2 mL DPPH/ mL 70% ethanol as control. The standard curve using trolox was produced and the results were presented as µmol trolox equivalent kg− 1 fresh weight.

Total Carotenoids

Using the equations given by Lichtenthaler (1987), total carotenoid content were estimated in persimmon fruit pulp. Carotenoids were extracted in the dark by homogenizing the persimmon pulp (1 g) in 80% acetone pre-chilled to 4 °C. To neutralize the organic acids, 0.5 g CaCO3 was also added during grinding. Then the homogenate was centrifuged for 5 min at 10,000 g at 4 °C. The resulting supernatant was collected, and 5 mL chilled acetone was again added to the residue to re-extract carotenoids in the same way. Both supernatants were mixed well, and the absorbance was noted at 646.8, 663.2 and 470 nm. For blank, 80% acetone was used, and total carotenoids were expressed as mg kg− 1 fresh weight.

Ascorbic Acid Content

Persimmon fruit juice (10 mL) was filtered through two layers of cheese cloth. Oxalic acid solution (0.4%) was added to the juice before filtering it and titrating against 2, 6-dichlorophenolindophenol. Ascorbic acid content was expressed as mg kg− 1 fresh weight (Ali et al. 2019b).

10. Statistical Analysis

The variances within the data were analyzed with ANOVA technique using significance level of 5%. The means of the treatments were compared by using least significance difference test (α = 0.05). The statistical analysis was processed with SPSS Statistics (version 19) software.

Results and Discussion

Effect of AV-gel Coating on Fruit Weight Loss

Water loss in fresh fruits and vegetables decreases their freshness, storage duration and economic value. Therefore, water loss is the most undesirable physiological process in harvested horticultural produces. In this study, weight loss increased in non-coated and AV-gel coated fruits from the 4th to 20th day of storage period (Fig. 1a). However, coated persimmon fruits showed significantly lower weight loss on each sampling day. On 20th day, AV-gel coated fruits had 1.67-fold less weight loss than non-coated control. Cellular respiration and desiccation of horticultural fresh produces, particularly fruit, result in the loss of their mass (Ali et al. 2019b). Pressure gradient between fruit surface and its surroundings determines the rate of water loss. Polysaccharide based coatings, such as AV-gel, act as a barrier and check metabolic activities, such as respiration and transpiration, which, in turn, reduce the loss of moisture from the fruit and, hence, prevent overall fruit weight loss (Guillén et al. 2013; Ali et al. 2018; Khaliq et al. 2019). Khoshgozaran-Abras et al. (2012) also reported that if AV-gel is incorporated in a coating, it significantly reduces its water vapor permeability and, hence, strengthens the barrier property of the coating. Therefore, AV-gel coating, in this study, decreased desiccation and prevented weight loss in persimmon fruits under ambient storage conditions.

Fig. 1.

Fig. 1

Open in a new tab

Effect of Aloe vera gel coating on fruit weight loss (a), electrolyte leakage (b), malondialdehyde content (c) and hydrogen peroxide (d) of persimmon fruits. Data are reported as mean ± SE (n = 3). Different letters indicate statistical differences by LSD test at p˂0.05

Electrolyte Leakage, MDA and H2O2 Contents

Generally, electrolyte leakage, MDA concentration and H2O2 production increased in control and AV-gel coated persimmons with the increase in storage time (Fig. 1b, c and d). The increase in electrolyte leakage was significantly higher in control compared to that in AV-gel coated persimmon fruits. On the 20th day of storage, AV-gel coated persimmon fruits showed markedly less (1.21-fold) electrolyte leakage compared to non-coated fruits (Fig. 1b). Similarly, AV-gel coated persimmons exhibited significantly lower MDA content than control fruits. On the 20th day of storage, MDA content was found to be significantly lower (1.12-fold) in AV-gel coated persimmon fruits than in control fruits (Fig. 1c). On the 4th day of storage, difference in H2O2 production between control and AV-gel coated persimmons was non-significant. However, as storage time passed, the rate of H2O2 production increased but this increase was markedly lower in AV-gel coated persimmon fruits than in control (Fig. 1d). Overall, the production of H2O2 content was markedly less (1.13-fold) in AV-gel coated fruits compared to control persimmons on the 20th day of storage period.

Electrolyte leakage and accumulation of H2O2 and MDA are markers of cellular damage. Their concentration generally increases during fruit ripening (Huan et al. 2016) because prolonged storage of fruits and vegetables stimulates production of ROS such as H2O2 that damages membrane fatty acids and, eventually, increases the production of MDA contents. However, AV-gel coating reduced all these stress markers in stored persimmon fruits. Impairment of membrane fatty acids, linoleic and linolenic fatty acids, leads to loss of osmotic balance, deactivation of membrane-bound enzymes, influx of fluids and ions and loss of proteins and RNA, leading to senescence (Qin et al. 2009). Maalekuu et al. (2006) also reported that oxidation of membrane lipids in pepper fruits increased membrane ion leakage that deteriorated pepper quality during storage. However, membrane integrity and electrolytes release can be reduced by applying an appropriate coating to a produce. Ali et al. (2019a) also found that AV-gel based coating protected membranes by inhibiting loss of structural integrity, resulting in lower electrolyte leakage in AV-gel coated lotus root slices during cold storage.

In this study, higher H2O2 and lipid peroxidation in non-coated fruits indicated that persimmon ripening was characterized by an oxidative stress, as reported in other climacteric fruits such as in papaya by Kuwar et al. (2015) and in mango by Rosalie et al. (2015). H2O2 stimulates ripening of a fruit through increased synthesis of ethylene, leading to early onset of senescence. Therefore, low intracellular H2O2 levels delay ripening, stabilize membrane structures and eventually increase storage life of fresh fruits. This can be achieved by applying an edible coating such as AV-gel (Rasouli et al. 2019; Saleem et al. 2020). An edible coating protects membrane fatty acids and reduces production of ROS, subsequently suppressing the production of MDA in stored fruits. Therefore, in the current work, MDA production was low in AV-gel coated persimmon fruits probably due to reduced oxidative damage as was also reported earlier in AV-gel coated lotus root slices by Ali et al. (2019a). So, AV-gel coating mitigated oxidative stress and maintained cell health, thus delaying senescence of persimmon fruit tissues.

Effect of AV-gel Coating on Antioxidant Enzymes Activities

APX enzyme activity continuously decreased in AV-gel coated and non-coated persimmon fruits from the 4th to 20th day of storage (Fig. 2a). However, a higher reduction in APX enzyme activity was noted in non-coated persimmon fruits than in AV-gel coated fruits. At the end of the experiment, APX enzyme activity was considerably higher (1.38-fold) in AV-gel coated fruits than in non-coated fruits. CAT and POD enzymes activities also uninterruptedly reduced from the 4th to 20th day of storage in both, non-coated and AV-gel coated persimmon fruits. After 20 days of storage time, CAT and POD activities were markedly higher (1.25 and 1.43-fold, respectively) in AV-gel coated persimmon fruits than in untreated control fruits (Fig. 2b and c). As far as SOD enzyme activity is concerned, it decreased by 8th day, then increased by 12th day and again decreased by 20th day in both treatments. However, AV-gel coated fruits always showed higher SOD activity, reaching up to 1.19 times higher than non-coated fruits after 20 days of storage period (Fig. 2d).

Fig. 2.

Fig. 2

Open in a new tab

Effect of Aloe vera gel coating on ascorbate peroxidase (a), catalase (b), peroxidase (c) and superoxide dismutase (d) enzymes activities of persimmon fruits. Data are reported as mean ± SE (n = 3). Different letters indicate statistical differences by LSD test at p˂0.05

Climacteric rise in the respiratory metabolism of a harvested fruit, such as persimmon, results in overproduction of ROS. During ripening and senescence, oxidative stress damages mitochondrial proteins (such as outer membrane transporter 'porin'), antioxidant proteins and Kreb’s cycle enzymes including malate dehydrogenase and aconitase (Qin et al. 2009). Therefore, storage of persimmon fruits after harvest leads to oxidative stress. SOD, POD, CAT and APX are antioxidative enzymes linked with reducing oxidative damage (Payasi et al. 2009). SOD is a metal containing protein that catalyzes the dismutation reaction of superoxide radical anions into H2O2 and molecular oxygen. Further, H2O2 is scavenged by CAT and APX in peroxisomes and other membranous organelles. APX reduces H2O2 to water via ascorbate–glutathione cycle while oxidizing ascorbate to monodehydroascorbate and dehydroascorbate molecules. Antioxidant enzymes scavenge different ROS and, hence, alleviate oxidative stress, consequently enhancing the storage potential of persimmon fruits in response to postharvest treatments. AV-gel coating maintains higher activities of these enzymes and reduces fruit tissue senescence which eventually results in extended storage life and acceptable quality of a produce (Ali et al. 2019a; Hassanpour 2015). This is the reason that AV-gel coated persimmons showing higher SOD, POD, CAT and APX activities mitigated oxidative stress-induced damages by scavenging ROS. This oxidative stress mitigation, in this study, was also evident from less electrolyte leakage and low accumulation of H2O2 and MDA in coated persimmons.

Effect of AV-gel Coating on Cell Wall-Degrading Enzymes

Activities of softening enzymes, PG, PME and CEL, progressively increased in both treatments with increased storage period. However, the increase in PME, PG and CEL enzymes activities was significantly higher in control compared to AV-gel coated persimmon fruits (Fig. 3a, b and c). Overall, the lowest activity of PME, PG and CEL was noted on 4th day and the highest was observed on 20th day of storage. On the 20th day of storage, AV-gel coated persimmon fruits showed markedly reduced PME (1.49-fold), PG (1.42-fold) and CEL (1.63-fold) enzymes activities as compared to control (Fig. 3a, b and c).

Fig. 3.

Fig. 3

Open in a new tab

Effect of Aloe vera gel coating on pectin methylesterase (a), polygalacturonase (b) and cellulase (c) enzymes activities of persimmon fruits. Data are reported as mean ± SE (n = 3). Different letters indicate statistical differences by LSD test at p˂0.05

Cell wall degrading enzymes like PG, PME and CEL modulate textural fruit softening. As ripening progresses, fruit softening also increases due to increase in the activities of PG and PME which depolymerize the chain length of pectin components of cell wall. CEL hydrolyzes cellulose during ripening contributing to cell wall weakening. However, softening of a fruit could be retarded by delaying the increase in the activities of CEL, PG and PME enzymes (Zhao et al. 2019). The edible coatings like AV-gel coating reduces the activities of these hydrolases by limiting the concentration of oxygen and carbon dioxide, thus preserving fruit firmness during storage (Maftoonazad et al. 2008). Shi et al. (2019) also argued that pre-storage coating application significantly delays the increase in PG, PME and CEL activities during postharvest storage. So, the activities of PG, PME and CEL were delayed in AV-gel coated group that in turn contributed to the extension of persimmon fruit storage life. To the best of our knowledge, this is the first report describing the role of AV-gel coating in modulation of the activities of cell wall degrading enzymes including PG, PME and CEL in any stored fruit or vegetable.

Effect of AV-gel Coating on Total Soluble Solids, Reducing and Non-reducing Sugars and Titratable Acidity

TSS increased at markedly reduced rate in AV-gel coated persimmons from the 4th to 20th day of storage compared to that in control fruits (Fig. 4a). After 20 days of storage, TSS were found to be 1.19-fold lower in AV-gel coated persimmon fruits than in control. The loss in titratable acidity was noticeably higher in non-coated fruits than in AV-gel coated persimmon fruits. At the end of the experiment, titratable acidity in AV-gel coated persimmon fruits was significantly higher (1.29-fold) than in non-coated fruits (Fig. 4b). A general increasing trend was observed in both treatments for reducing, non-reducing and total sugars with the increase in storage period (Fig. 5a, b and c). However, the increase in sugars was significantly higher in non-coated fruits than in AV-gel coated fruits, clearly indicating a fast ripening process occurring in non-coated fruits.

Fig. 4.

Fig. 4

Open in a new tab

Effect of Aloe vera gel coating on total soluble solids (a) and titratable acidity (b) of persimmon fruits. Data are reported as mean ± SE (n = 3). Different letters indicate statistical differences by LSD test at p˂0.05

Fig. 5.

Fig. 5

Open in a new tab

Effect of Aloe vera gel coating on reducing (a), non-reducing (b) and total sugars (c) of persimmon fruits. Data are reported as mean ± SE (n = 3). Different letters indicate statistical differences by LSD test at p˂0.05

A rapid increase in sugars is an indicator of fast ripening process in climacteric fruits that leads to shorter postharvest life. However, starch hydrolysis can be reduced with the application of a suitable edible coating. For example, AV-gel coated sapodilla fruits showed a less increase in sugars during storage at ambient temperature (Khaliq et al. 2019). A higher rate of metabolic activities, such as ethylene production and respiration rate, consumes various organic acids present in fruit cells (Khaliq et al. 2019) that leads to rapid senescence of a produce. Coatings based on AV-gel modify the internal microenvironment of a fruit resulting in the reduction of respiration process (Mendy et al. 2019). This could be the reason that, in this study, there was significantly less loss of titratable acidity and sugars in AV-gel coated persimmon fruits. Like our findings, reduced titratable acidity has been observed in AV-gel coated fruits of peach, plum (Guillén et al. 2013), sapodilla (Khaliq et al. 2019) and papaya (Mendy et al. 2019). Mustafa et al. (2014) also observed increase in total soluble solids and decrease in titratable acidity with storage duration in coated tomatoes.

Effect of AV-gel Coating on Non-Enzymatic Antioxidants

AV-gel coated persimmon fruits exhibited significantly less loss of total phenolics than control fruits (Fig. 6a). On average, total phenolics decreased at markedly slow rate in AV-gel coated persimmons from the 4th to 20th day of storage compared to those in control fruits. At the end of the experiment, total phenolics were found to be significantly higher (1.42-fold) in AV-gel coated persimmon fruits than those in non-coated fruits. By the 4th day of storage, the difference in total antioxidants between control and AV-gel coated persimmons was non-significant (Fig. 6b). However, as storage time progressed, total antioxidants were significantly less in non-coated fruits than in AV-gel coated persimmon fruits. Overall, total antioxidants were significantly higher (1.14-fold) in AV-gel coated fruits compared to control persimmons at the end of the experiment.

Fig. 6.

Fig. 6

Open in a new tab

Effect of Aloe vera gel coating on total phenolic content (a), total antioxidants (b), total carotenoids (c) and ascorbic acid content (d) of persimmon fruits. Data are reported as mean ± SE (n = 3). Different letters indicate statistical differences by LSD test at p˂0.05

Generally, total carotenoids increased at markedly slow rate in AV-gel coated persimmon fruits from the 4th to 20th day of storage compared to that in control fruits (Fig. 6c). On the 20th day of storage, total carotenoids were found to be substantially lower (1.18-fold) in AV-gel coated persimmon fruits than in non-coated fruits (Fig. 6c). Ascorbic acid content (AsAC) reduced with the increase in the time of storage (Fig. 6d). This rate of AsAC decrease was noticeably higher in non-coated fruits than in AV-gel coated fruits. Overall, AsAC content in AV-gel coated persimmon fruits was significantly higher (1.26-fold) than that of non-coated fruits on the 20th day of storage period (Fig. 6d).

Most insoluble-bound phenolics form covalent bond with cell wall components. Therefore, breakdown of cell structure with the onset of senescence oxidizes phenolics and, thus, decreases their concentration. However, edible coatings maintain cell wall structure and act as barrier to oxygen and thus inhibit enzymatic oxidation of phenolics (Ali et al. 2019b). This is the reason that 50% AV-gel in this study maintained higher phenolic content. Mendy et al. (2019) also observed that 50% AV-gel coating maintained higher phenolics content in papaya fruits than control (0% AV-gel).

Persimmon fruits have numerous antioxidants including ascorbic acid, phenols and carotenoids (Naser et al. 2018; Plaza et al. 2012) that contribute to their antioxidant profile. In general, antioxidants reduce in a stored fruit due to their oxidation by enzymes and ROS during senescence (Khaliq et al. 2019). However, use of edible coatings, such as AV-gel, reduces the oxidative stress and maintains the integrity of cellular compartments in which antioxidants are localized (Hassanpour 2015; Khaliq et al. 2019). Therefore, a higher total antioxidants concentration in AV-gel coated persimmon fruits might be due to higher concentrations of phenolics, carotenoids and ascorbic acid content in these fruits.

AsAC and carotenoids are molecular antioxidants that provide reducing power to cellular membrane structure to overcome the oxidation stress (Rosalie et al. 2015). AsAC directly scavenges few ROS, such as O2 •− and H2O2, or indirectly reduces H2O2 via APX (Ali et al. 2019a). The enzyme ascorbic acid-oxidase regulates the concentration of ascorbic acid by oxidizing it. Therefore, AsAC reduces sharply due to its oxidation under long term storage of fruit. However, earlier reports have suggested that edible coating based on AV-gel inhibited its oxidation and sustained its higher levels in contrast to no coating (Hassanpour 2015; Mendy et al. 2019); the same was also observed in this investigation.

Persimmon fruits are a rich source of carotenoids like β-carotene, lutin, zeaxanthin and β-crytoxanthin which accumulate during ripening stage (Naser et al. 2018; Plaza et al. 2012), transforming the color of a fruit from yellowish orange to reddish orange (Naser et al. 2018). So, AV-gel coating in this study suppressed the accumulation of carotenoids, probably by inhibiting the ripening related processes, and inhibited the change of color of persimmon fruits (Supplementary Fig. S1). Therefore, AV-gel coating in this study also showed higher carotenoid content than control at the end of the experiment.

Conclusions

AV-gel coating application reduced oxidative stress, as was obvious from lower H2O2 level, membrane leakage and MDA content. The lower oxidative stress was accompanied with significantly higher activities of APX, SOD, POD and CAT enzymes and higher levels of antioxidants including phenolics, DPPH scavenging antioxidants and ascorbic acid. AV-gel coating also delayed ripening and softening by suppressing activities of PME, PG and CEL enzymes and slowing the compositional changes in sugars, titratable acids and total carotenoids. So, AV-gel coating delayed senescence by activating antioxidant system and suppressing cell wall degrading mechanism. Therefore, AV-gel coating can be utilized to delay ripening and softening of persimmon fruits during ambient storage conditions.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (667.3KB, docx)
Supplementary Material 2 (15.5KB, docx)

Authors’ contributions

MSS and AMA carried out the experiment; SE conceived the idea and supervised the work; MAA and SA developed the methodology and wrote the MS; SH and AN analyzed the data and prepared the graphs; SN edited the MS; MM revised the MS and prepared response to the reviewers’ comments.

Funding

The authors received no specific grant or award for this research work.

Conflicts of interest/Competing interests

The authors declare that they have no competing interests.

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

All data generated or analysed during this study are included in this published article.

Code Availability

Not applicable.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. Ali A, Zahid N, Manickam S, Manickam S, Siddiqui Y, Alderson PG. Double layer coatings: A new technique for maintaining physico-chemical characteristics and antioxidant properties of dragon fruit during storage. Food Bioprocess Technol. 2014;7:2366–2374. doi: 10.1007/s11947-013-1224-3. [DOI] [Google Scholar]
  2. Ali S, Khan AS, Malik AU, Shahid M. Effect of controlled atmosphere storage on pericarp browning, bioactive compounds and antioxidant enzymes of litchi fruits. Food Chem. 2016;206:18–29. doi: 10.1016/j.foodchem.2016.03.021. [DOI] [PubMed] [Google Scholar]
  3. Ali A, Yeoh WK, Forney C, Siddiqui MW. Advances in postharvest technologies to extend the storage life of minimally processed fruits and vegetables. Crit Rev Food Sci Nutr. 2018;58:2632–2649. doi: 10.1080/10408398.2017.1339180. [DOI] [PubMed] [Google Scholar]
  4. Ali S, Khan AS, Anjum MA, Nawaz A, Naz S, Ejaz S, Hussain S. Aloe vera gel coating delays post-cut surface browning and maintains quality of cold stored lotus (Nelumbo nucifera Gaertn.) root slices. Sci Hortic. 2019;256:108612. doi: 10.1016/j.scienta.2019.108612. [DOI] [Google Scholar]
  5. Ali S, Khan AS, Nawaz A, Anjum MA, Naz S, Ejaz S, Hussain S. Aloe vera gel coating delays postharvest browning and maintains quality of harvested litchi fruit. Postharvest Biol Technol. 2019;157:110960. doi: 10.1016/j.postharvbio.2019.110960. [DOI] [Google Scholar]
  6. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  7. Brand-Williams W, Cuvelier ME, Berset C. Use of free radical method to evaluate antioxidant activity. LWT - Food Sci Technol. 1995;28:25–30. doi: 10.1016/S0023-6438(95)80008-5. [DOI] [Google Scholar]
  8. Deng Y, Wu Y, Li Y. Changes in firmness, cell wall composition and cell wall hydrolases of grapes stored in high oxygen atmospheres. Food Res Int. 2005;38:769–776. doi: 10.1016/j.foodres.2005.03.003. [DOI] [Google Scholar]
  9. Ejaz S, Jezik KM, Stumpf W, Gosch C, Halbwirth H, Stich K. Amelioration of an open soilless cultivation system for microgardening spinach (Spinacia oleracea L.) Zemdirbyste. 2015;102:201–208. doi: 10.13080/z-a.2015.102.026. [DOI] [Google Scholar]
  10. Fahmy K, Nakano K. Effective transport and storage condition for preserving the quality of `Jiro’ persimmon in export market. Agric Agric Sci Procedia. 2016;9:279–290. doi: 10.1016/j.aaspro.2016.02.115. [DOI] [Google Scholar]
  11. Guillén F, Díaz-Mula HM, Zapata PJ, Valero D, Serrano M, Castillo S, Martínez-Romero D. Aloe arborescens and Aloe vera gels as coatings in delaying postharvest ripening in peach and plum fruit. Postharvest Biol Technol. 2013;83:54–57. doi: 10.1016/j.postharvbio.2013.03.011. [DOI] [Google Scholar]
  12. Hagerman AE, Austin PJ. Continuous spectrophotometric assay for plant pectin methyl esterase. J Agric Food Chem. 1986;34:440–444. doi: 10.1021/jf00069a015. [DOI] [Google Scholar]
  13. Hassanpour H. Effect of Aloe vera gel coating on antioxidant capacity, antioxidant enzyme activities and decay in raspberry fruit. LWT - Food Sci Technol. 2015;60:495–501. doi: 10.1016/j.lwt.2014.07.049. [DOI] [Google Scholar]
  14. Huan C, Jiang L, An X, Yu M, Xu Y, Ma R, Yu Z. Potential role of reactive oxygen species and antioxidant genes in the regulation of peach fruit development and ripening. Plant Physiol Biochem. 2016;104:294–303. doi: 10.1016/j.plaphy.2016.05.013. [DOI] [PubMed] [Google Scholar]
  15. Hussain SB, Anjum MA, Hussain S, Ejaz S, Ahmed M. Physico-chemical profiling of promising sweet orange cultivars grown under different agro-climatic conditions of Pakistan. Erwerbs-Obstbau. 2017;59:315–324. doi: 10.1007/s10341-017-0327-9. [DOI] [Google Scholar]
  16. Khaliq G, Mohamed MTM, Ghazali HM, Ding P, Ali A. Influence of gum arabic coating enriched with calcium chloride on physiological, biochemical and quality responses of mango (Mangifera indica L.) fruit stored under low temperature stress. Postharvest Biol Technol. 2016;111:362–369. doi: 10.1016/j.postharvbio.2015.09.029. [DOI] [Google Scholar]
  17. Khaliq G, Ramzan M, Baloch AH. Effect of Aloe vera gel coating enriched with Fagonia indica plant extract on physicochemical and antioxidant activity of sapodilla fruit during postharvest storage. Food Chem. 2019;286:346–353. doi: 10.1016/j.foodchem.2019.01.135. [DOI] [PubMed] [Google Scholar]
  18. Khoshgozaran-Abras S, Azizi MH, Hamidy Z, Bagheripoor-Fallah N. Mechanical, physicochemical and color properties of chitosan based-films as a function of Aloe vera gel incorporation. Carbohydr Polym. 2012;87:2058–2062. doi: 10.1016/j.carbpol.2011.10.020. [DOI] [Google Scholar]
  19. Kuwar U, Sharma S, Tadapaneni VRR. Aloe vera gel and honey-based edible coatings combined with chemical dip as a safe means for quality maintenance and shelf life extension of fresh‐cut papaya. J Food Qual. 2015;38:347–358. doi: 10.1111/jfq.12150. [DOI] [Google Scholar]
  20. Lichtenthaler HK. Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. Methods Enzymol. 1987;148:350–382. doi: 10.1016/0076-6879(87)48036-1. [DOI] [Google Scholar]
  21. Maalekuu K, Elkind Y, Leikin-Frenkel A, Lurie S, Fallik E. The relationship between water loss, lipid content, membrane integrity and LOX activity in ripe pepper fruit after storage. Postharvest Biol Technol. 2006;42:248–255. doi: 10.1016/j.postharvbio.2006.06.012. [DOI] [Google Scholar]
  22. Maftoonazad N, Ramaswamy HS, Marcotte M. Shelf-life extension of peaches through sodium alginate and methyl cellulose edible coatings. Int J Food Sci Technol. 2008;43:951–957. doi: 10.1111/j.1365-2621.2006.01444.x. [DOI] [Google Scholar]
  23. Maqbool M, Ali A, Alderson P. A combination of gum arabic and chitosan can control anthracnose caused by Colletotrichum musae and enhance the shelf-life of banana fruit. J Hort Sci Biotechnol. 2010;85:432–436. doi: 10.1080/14620316.2010.11512693. [DOI] [Google Scholar]
  24. Martínez-Romero D, Zapata PJ, Guillén F, Paladines D, Castillo S, Valero D, Serrano M. The addition of rosehip oil to Aloe gels improves their properties as postharvest coatings for maintaining quality in plum. Food Chem. 2017;217:585–592. doi: 10.1016/j.foodchem.2016.09.035. [DOI] [PubMed] [Google Scholar]
  25. Mendy TK, Misran A, Mahmud TMM, Ismail SI. Application of Aloe vera coating delays ripening and extend the shelf life of papaya fruit. Sci Hortic. 2019;246:769–776. doi: 10.1016/j.scienta.2018.11.054. [DOI] [Google Scholar]
  26. Mustafa MA, Ali A, Manickam S, Siddiqui Y. Ultrasound-Assisted Chitosan-Surfactant Nanostructure Assemblies: Towards Maintaining Postharvest Quality of Tomatoes. Food Bioprocess Technol. 2014;7:2102–2111. doi: 10.1007/s11947-013-1173-x. [DOI] [Google Scholar]
  27. Nakano Y, Asada K. Purification of ascorbate peroxidase in spinach chloroplasts; its inactivation in ascorbate-depleted medium and reactivation by monodehydroascorbate radical. Plant Cell Physiol. 1987;28:131–140. doi: 10.1093/oxfordjournals.pcp.a077268. [DOI] [Google Scholar]
  28. Naser F, Rabiei V, Razavi F, Khademi O. Effect of calcium lactate in combination with hot water treatment on the nutritional quality of persimmon fruit during cold storage. Sci Hortic. 2018;233:114–123. doi: 10.1016/j.scienta.2018.01.036. [DOI] [Google Scholar]
  29. Payasi A, Mishra NN, Chaves ALS, Singh R. Biochemistry of fruit softening: an overview. Physiol Mol Biol Plants. 2009;15:103–113. doi: 10.1007/s12298-009-0012-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pérez-Burillo S, Oliveras MJ, Quesada J, Rufián-Henares JA, Pastoriza S. Relationship between composition and bioactivity of persimmon and kiwifruit. Food Res Int. 2018;105:461–472. doi: 10.1016/j.foodres.2017.11.022. [DOI] [PubMed] [Google Scholar]
  31. Plaza L, Colina C, Ancos B, De, Sánchez-Moreno C, Pilar Cano M. Influence of ripening and astringency on carotenoid content of high-pressure treated persimmon fruit (Diospyros kaki L.) Food Chem. 2012;130:591–597. doi: 10.1016/j.foodchem.2011.07.080. [DOI] [Google Scholar]
  32. Qin G, Wang Q, Liu J, Li B, Tian S. Proteomic analysis of changes in mitochondrial protein expression during fruit senescence. Proteomics. 2009;9:4241–4253. doi: 10.1002/pmic.200900133. [DOI] [PubMed] [Google Scholar]
  33. Rasouli M, Saba MK, Ramezanian A. Inhibitory effect of salicylic acid and Aloe vera gel edible coating on microbial load and chilling injury of orange fruit. Sci Hortic. 2019;247:27–34. doi: 10.1016/j.scienta.2018.12.004. [DOI] [Google Scholar]
  34. Rosalie R, Joas J, Deytieux-Belleau C, Vulcain E, Payet B, Dufossé L, Léchaudel M. Antioxidant and enzymatic responses to oxidative stress induced by pre-harvest water supply reduction and ripening on mango (Mangifera indica L. cv. `Cogshall’) in relation to carotenoid content. J Plant Physiol. 2015;184:68–78. doi: 10.1016/j.jplph.2015.05.019. [DOI] [PubMed] [Google Scholar]
  35. Saleem MS, Ejaz S, Anjum MA, Nawaz A, Naz S, Hussain S, Ali S, Canan I. Postharvest application of gum arabic edible coating delays ripening and maintains quality of persimmon fruits during storage. J Food Process Preserv. 2020;44:e14583. doi: 10.1111/jfpp.14583. [DOI] [Google Scholar]
  36. Saleem MS, Anjum MA, Naz S, Ali S, Hussain S, Azam M, Sardar H, Khaliq G, Canan İ, Ejaz S. Incorporation of ascorbic acid in chitosan-based edible coating improves postharvest quality and storability of strawberry fruits. Int J Biol Macromol. 2021;189:160–169. doi: 10.1016/j.ijbiomac.2021.08.051. [DOI] [PubMed] [Google Scholar]
  37. Senica M, Veberic R, Grabnar JJ, Stampar F, Jakopic J. Selected chemical compounds in firm and mellow persimmon fruit before and after the drying process. J Sci Food Agric. 2016;96:3140–3147. doi: 10.1002/jsfa.7492. [DOI] [PubMed] [Google Scholar]
  38. Shakir MS, Ejaz S, Hussain S, Ali S, Sardar H, Azam M, Ullah S, Khaliq G, Saleem MS, Nawaz A, Anjum MA (2022) Synergistic effect of gum Arabic and carboxymethyl cellulose as biocomposite coating delays senescence in stored tomatoes by regulating antioxidants and cell wall degradation. Int J Biol Macromol (In Press). doi: 10.1016/j.ijbiomac.2022.01.073 [DOI] [PubMed]
  39. Shi Z, Yang H, Jiao J, Wang F, Lu Y, Deng J. Effects of graft copolymer of chitosan and salicylic acid on reducing rot of postharvest fruit and retarding cell wall degradation in grapefruit during storage. Food Chem. 2019;283:92–100. doi: 10.1016/j.foodchem.2018.12.078. [DOI] [PubMed] [Google Scholar]
  40. Singleton VL, Rossi JA. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic. 1965;16:144–158. [Google Scholar]
  41. Tian S, Qin G, Li B. Reactive oxygen species involved in regulating fruit senescence and fungal pathogenicity. Plant Mol Biol. 2013;82:593–602. doi: 10.1007/s11103-013-0035-2. [DOI] [PubMed] [Google Scholar]
  42. Velikova V, Loreto F. On the relationship between isoprene emission and thermotolerance in Phragmites australis leaves exposed to high temperatures and during the recovery from a heat stress. Plant Cell Environ. 2005;28:318–327. doi: 10.1111/j.1365-3040.2004.01314.x. [DOI] [Google Scholar]
  43. Yang H, Wu F, Cheng J. Reduced chilling injury in cucumber by nitric oxide and the antioxidant response. Food Chem. 2011;127:1237–1242. doi: 10.1016/j.foodchem.2011.02.011. [DOI] [PubMed] [Google Scholar]
  44. Zhao Y, Zhu X, Hou Y, Wang X, Li X. Effects of nitric oxide fumigation treatment on retarding cell wall degradation and delaying softening of winter jujube (Ziziphus jujuba Mill. cv. Dongzao) fruit during storage. Postharvest Biol Technol. 2019;156:110954. doi: 10.1016/j.postharvbio.2019.110954. [DOI] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (667.3KB, docx)
Supplementary Material 2 (15.5KB, docx)

Data Availability Statement

All data generated or analysed during this study are included in this published article.

Not applicable.

Articles from Journal of Food Science and Technology are provided here courtesy of Springer

RESOURCES