Scholarly article on topic 'Effects of anti-coloring agents on blackening inhibition and maintaining physical and chemical quality of fresh-cut okra during storage'

Effects of anti-coloring agents on blackening inhibition and maintaining physical and chemical quality of fresh-cut okra during storage Academic research paper on "Agriculture, forestry, and fisheries"

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{"Okra pods" / "Phenolic content" / Texture / "Microbial load" / "Coloring process"}

Abstract of research paper on Agriculture, forestry, and fisheries, author of scientific article — M.A. Saleh, A.M. El-Gizawy, R.E.L. El-Bassiouny, H.M. Ali

Abstract Fresh-cut okra pods were stored in sealed polypropylene bags at 5°C and 95% RH for 8days. Pods were dipped in 0.5% solution of cysteine, ascorbic acid, CaCl2, or citric acid for 5min before storage. The main observed undesirable physiological and morphological alterations were weight loss, increasing microbial load, softening texture, and decreasing the phenolic content with blackening in color. CaCl2 was effective in increasing cell membrane integrity leading to improving texture, minimizing weight loss, decreasing microbial load, and preventing polyphenoloxidase (PPO) from contacting its phenolic substrates and thus reducing blackness. Ascorbic acid and cysteine were best anti-coloring agents since their strong ability to inhibit PPO and reacting with the resulted colored quinones to give colorless products. Reducing blackness was found parallel to decreasing phenolic content, indicating the role of the phenolic oxidation in the blackening process in okra pods during storage. Citric acid was less effective in enhancing the examined physical and chemical properties.

Academic research paper on topic "Effects of anti-coloring agents on blackening inhibition and maintaining physical and chemical quality of fresh-cut okra during storage"

Annals of Agricultural Science (2013) 58(2), 239-245

Faculty of Agriculture, Ain Shams University Annals of Agricultural Science

www.elsevier.com/locate/aoas

ORIGINAL ARTICLE

Effects of anti-coloring agents on blackening inhibition and maintaining physical and chemical quality of fresh-cut okra during storage

M.A. Saleh a *, A.M. El-Gizawy b, R.E.L. El-Bassiouny a, H.M. Ali

a Postharvest and Handling of Vegetable Crops Department, Horticulture Research Institute, Agriculture Research Center, Giza, Egypt

b Horticulture Department, Faculty of Agriculture, Ain Shams University, Egypt c Agricultural Biochemistry Department, Faculty of Agriculture, Ain Shams University, Egypt

Received 28 April 2013; accepted 9 May 2013 Available online 5 September 2013

KEYWORDS

Okra pods; Phenolic content; Texture; Microbial load; Coloring process

Abstract Fresh-cut okra pods were stored in sealed polypropylene bags at 5 °C and 95% RH for 8 days. Pods were dipped in 0.5% solution of cysteine, ascorbic acid, CaCl2, or citric acid for 5 min before storage. The main observed undesirable physiological and morphological alterations were weight loss, increasing microbial load, softening texture, and decreasing the phenolic content with blackening in color. CaCl2 was effective in increasing cell membrane integrity leading to improving texture, minimizing weight loss, decreasing microbial load, and preventing polyphenoloxidase (PPO) from contacting its phenolic substrates and thus reducing blackness. Ascorbic acid and cys-teine were best anti-coloring agents since their strong ability to inhibit PPO and reacting with the resulted colored quinones to give colorless products. Reducing blackness was found parallel to decreasing phenolic content, indicating the role of the phenolic oxidation in the blackening process in okra pods during storage. Citric acid was less effective in enhancing the examined physical and chemical properties.

© 2013 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams

University.

Introduction

* Corresponding author. Tel.: +20 (202) 44441172. E-mail address: Journalaaru@yahoo.com (M.A. Saleh). Peer review under responsibility of Faculty of Agriculture, Ain-Shams University.

Okra (Abelmoschus esculentus L), a tropical and African origin vegetable, is produced in many warm-weather countries e.g. India, Pakestan, Turky, Iran, Nigeria, Ghana, Greece, and southern USA and is considered one of the most important vegetables in Egypt (Arapitsas, 2008; Falade and Omojola, 2010) because of its nutritional content and medicinal potentials against inflammation, gastric irritation (Arapitsas, 2008), and colon cancer (Babarinde and Fabunmi, 2009). Okra is a

0570-1783 © 2013 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University. http://dx.doi.org/10.1016/j.aoas.2013.07.008

good source of viscous mucilage, proteins (Karakoltsidis and Constantinides, 1975), and dietary fibers (Adom et al., 1996) in addition to contents of vitamin C, polyphenols (Arapitsas, 2008), fats, carbohydrates (Baxter and Waters, 1990), and many minerals e.g. Na, K, Mg, Ca, Fe, Zn, and Mn (Al-Wandawi, 1983); Recently, there has been an increasing market demand for minimally processed fresh-cut fruits and vegetables. However, okra has a short shelf-life and extremely perishable because of the high water contents and respiration rate (Finger et al., 2008; Falade and Omojola, 2010); in addition, the damage in tissues caused by fresh-cut preparation is a major contributor to induce undesirable biochemical pathways, leading to browning or blackening of surface cutting, lowering nutative value (Brecht, 1995; Queiroz etal., 2008), providing sites for microbial infection, accelerating respiration rate, water loss, and ethylene production (Finger et al., 2008; Falade and Omojola, 2010). Phenolic compounds are well known to play an important role in the browning process since polyphenoloxidase (PPO) can catalyze their oxidation by molecular oxygen in two steps: first, hydroxylation in the ortho position to give a catecholic structure and second oxidation of catechols to o-quinones that can be self-condensed or polymerized with other biochemicals to produce brown pigments responsible for the undesirable color change. Therefore, damaged tissues in fresh-cut products expose phenolic compounds in vacuoles to the membrane-bound polyphenoloxidase resulting in color darkening (PiliZota and Subaric, 1998; Yoruk and Marshall, 2003; Gacche et al., 2006).

Various approaches to control the extent of browning or blackening have been investigated; in general, enzymatic browning can be avoided or minimized by thermal inactivation of PPO or by using chemical additives. Treatments with ascorbic acid, cysteine, citric acid, and oxalic acid can inhibit poly-phenoloxidase during storage; ascorbic acid can also reduce the resulted quinones back to the starting catechols before browning process takes place, in addition to its strong antiox-idant activity (Abo-Shady et al., 2007; Ali et al., 2013), while cysteine, under certain conditions, may react with quinones to give colorless products (Altunkaya and Gokmen, 2008; Queiroz et al., 2008; Chang, 2009). Other treatments such as calcium chloride (CaCl2) maintain visual quality by keeping the integrity of the cell wall and retarding vegetable flesh softening (Luna-Guzman and Barrett, 2000).

The objective of this study was to determine the effect of some anti-coloring agents on reducing browning or blackening, preserving phenolic and water contents, maintaining physical quality, and inhibiting pathogen infection of fresh-cut okra during storage.

Materials and methods

Freshly harvested okra (Zara cultivar) was obtained from a commercial farm in Esmailia Governorate, Egypt. Pods were harvested at immature stage in the first week of August in 2010 and 2011 seasons and transported immediately, under cooling condition, to the postharvest laboratory, Horticulture Research Institute, Giza. Samples were selected free of visual damage or defects and uniform in color and size (40-50 mm long and 12-15 mm diameter).

All cutting utensils (knife and cutting board) used in removing okra pod calyx were washed with soap and water then rinsed with 100 ppm sodium hypochlorite solution prior to

use. Fresh-cut okra was randomly divided into 5 groups for the following treatments:

(1) Dipping in 0.5% solution of cysteine for 5 min.

(2) Dipping in 0.5% solution of ascorbic acid (AA) for 5 min.

(3) Dipping in 0.5% solution of calcium chloride (CaCl2) for 5 min.

(4) Dipping in 0.5% solution of citric acid (CA) for 5 min.

(5) Dipping in distilled water (control).

All fresh-cut okra samples were packed in sealed polypropylene bags (15 x 15 cm and 20i thickness). Each bag contained 20 pods and stored at 5 0C and 95% RH; a complete randomized design was used. At each interval, three bags were used as replicates for the following measurements:

(1) Weight loss percentage (WL) was calculated by the following formula: WL = [(Wi - Wf)/Wi] x 100, where Wi is initial fruit weight (gm) and Wf is final fruit weight (gm) at a given time.

(2) General appearance (GA) was determined visually using a scale from 1 to 9; where 9 = excellent, 7 = good, 5 = fair, 3 = poor and 1 = unusable. Samples rating below 5 were considered unmarketable.

(3) Texture was measured by using TA-1000 texture analyzer instrument with a penetrating cylinder of 1 mm diameter. Penetration to a constant distance (3 and 5 mm) inside the pulps with a constant speed 2 mm/s. was performed and the peak of resistance was recorded (gm/cm3).

(4) Color change was determined by using a Minolta Chroma meter model CR-400 for measuring the L* and Hue angel (F = 180 + tan-1(b*/a*) where L*, a* and b* are Hunter parameters.

(5) Total microbial count was obtained by the pour plate method using nutrient agar as the growth medium. In sterile Petri dishes, 1 ml of the homogenized diluent sample was poured then 10 ml of nutrient agar was gently dispensed and swirled. The plates were inverted after solidification and incubated at 37 0C for 24 h. The colonies were counted and the number of colonies per plate was multiplied by the dilution factor to obtain the total viable counts per ml of the original sample. Microbial colonies were counted using a Protos Colony Counter (Model 50000, Synoptics Ltd., Cambridge, UK) and reported as log CFU/g of tissue.

(6) Total soluble phenols were determined spectrophoto-metrically by the method of Folin-Ciocalteu (Folin and Ciocalteu, 1927; Hyodo et al., 1978); results were reported as mg (gallic acid)/g fresh weight.

(7) Statistical analysis was performed where data were analyzes by two-way analysis of variance (ANOVA) method using SPSS version 16. Fischer's least significant difference (LSD) at 5% probability level was used to examine significant effects.

Results and discussion

To examine the effects of different treatments on preserving the physical and chemical conditions of fresh-cut okra during

storage, pods were stored, as recommended previously, in polypropylene pages (Babarinde and Fabunmi, 2009) at 95% RH and 5 0C (Finger et al., 2008).

Weight loss

Data in Table 1 showed a progressive increase in the weight loss percentage of fresh-cut okra during storage. The increasing weight loss is in consistence with the report of Gupta and Muk-herjee (1982) and could be attributed to the loss of moisture through transpiration and dry matter contents through respiration processes (Adetuyi et al., 2008). The rate of weight loss was least after 8 days when fresh-cut okra was treated with calcium chloride in the two seasons (0.92% and 0.87% for seasons 2010 and 2011, respectively) which could be due to strengthen the cell wall (Bolin and Huxsoll, 1989) and reducing the respiration rate in the stored vegetables (Fallik et al., 1999).

Texture

Data in Table 2 revealed that significant reduction in pod texture had occurred by prolongation of the storage period. These results agree with those obtained by Sargent et al. (1996). The decrease in pod texture was attributed to gradual breakdown of protopectin to water soluble lower molecular weight fractions leading to the increase in the rate of pod softening (Wills et al., 2007).

Among the examined compounds, CaCl2 was distinctive among other treatments and significantly reduced the texture loss of fresh-cut okra during storage relative to control group. The favorable effect of CaCl2 could be due to stabilization of membrane systems through the formation of Ca-pectates which increase the rigidity of the middle lamella and cell wall (Poovaiah, 1986). Generally, any treatment capable of delaying softening is potentially helpful in extending the postharvest shelf-life and maintaining the product quality. The interaction between treatments and storage periods indicates that CaCl2 treatment was superior in maintaining fresh-cut texture during the storage period.

General appearance (GA)

Data in Table 3 indicate that the general appearance of the control fresh-cut okra samples was deteriorated severely with

a poor grade (3 and 2.33 in both seasons, respectively) after 8 days. The decrease in GA during storage period resulted from shriveling, wilting, and color change in pods (Sargent et al., 1996). However, treatments by cysteine, ascorbic acid and CaCl2 significantly enhanced the general appearance relative to control after the storage period in both seasons. However, okra samples treated with cysteine showed the best general appearance where pods kept their good GA (7 and 7.67) till the end of the storage period in both seasons, respectively. Moreover, ascorbic acid or CaCl2 treated samples showed good appearance till 6 days of storage then dropped to a fair level at the end of the storage period in both seasons.

Total microbial count

Data in Table 4 showed that microbial growth was increased with increasing the storage period particularly in control samples; however, samples of all treatments had significant lower level of microbial load after 8 days relative to control samples. Ascorbic acid and CaCl2 were the most effective treatments for reducing the total microbial count in both seasons, while cys-teine and citric acid were less effective. These results are in agreement with those reported by Luna-Guzman and Barrett (2000); CaCl2 may have provided an inhibitory effect on micro-bial growth, while water alone (control) allowed for spore spreading and thus increasing microbial load; besides, calcium chloride and ascorbic acid can lower intracellular pH or reduce water activity (Shelef, 1994; Whitaker, 1994), which provides a protective antimicrobial barrier against food borne pathogens in products (Weaver and Shelef, 2007). In addition, microflora is usually restricted to fungal and lactic acid bacteria at low pH (Luna-Guzman and Barrett, 2000). Also, calcium could enhance tissue texture leading to providing protection from fungal or bacterial attack by stabilizing or strengthening cell walls (Bolin and Huxsoll, 1989).

Color and total phenolic content changes

The color was measured by recording the lightness/darkness parameter (L* value) and hue angel (ho). Darkness of control pods was observed clearly during storage period (Table 5) where there was not only significant but also remarkable decrease in the L* value from 58.29 and 55.64 at the zero time to 45.71 and 40.28 after 8 days of control samples in the two

Table 1 Effect of anti-coloring agents on weight loss percentage of fresh-cut okra during storage in 2010 and 2011 seasons.

Treatments 2010 Season Mean 2011 Season Mean

Storage period (days) Storage period (days)

2 4 6 8 2 4 6 8

Control 0.70 0.98 1.30 1.46 1.11 0.58 0.89 1.16 1.39 1.01

Cysteine 0.64 0.94 1.14 1.38 1.03 0.52 0.84 1.09 1.20 0.91

Ascorbic acid 0.67 0.90 1.20 1.42 1.05 0.55 0.90 1.13 1.22 0.95

Calcium chloride 0.42 0.63 0.79 0.92 0.69 0.32 0.54 0.68 0.87 0.60

Citric acid 0.66 0.92 1.26 1.41 1.06 0.57 0.86 1.11 1.30 0.96

Mean 0.62 0.87 1.14 1.32 0.51 63.8 1.03 1.19

LSD Season 2010 Season 2011

Treatment 0.04 Storage period 0.09 Treatment x storage period 0.11 0.05 0.07 0.09

Table 2 Effect of anti-coloring agent on texture of fresh-cut okra during storage in 2010 and 2011 seasons.

Treatments 2011 Season 2010 Season

Storage period (days) Mean Storage period (days) Mean

0 2 4 6 8 0 2 4 6 8

Control 13.21 12.58 12.64 11.82 11.03 12.31 15.34 14.97 14.40 13.74 12.98 14.29

Cysteine 13.21 12.96 12.52 11.94 11.12 12.35 15.34 15.06 14.41 13.91 13.15 14.37

Ascorbic acid 13.21 12.82 12.41 11.75 11.20 12.28 15.34 15.00 14.52 13.82 13.04 14.34

Calcium chloride 13.21 13.13 12.97 12.54 12.17 12.80 15.34 15.21 15.06 14.87 14.65 15.03

Citric acid 13.21 12.93 12.57 11.54 11.15 12.28 15.34 14.92 14.45 13.85 13.11 14.33

L.S.D Season 2010 Season 2011

Treatments 0.06 0.07

Storage period 0.12 0.14

Treatments x storage period 0.17 0.19

Table 3 Effect of anti-coloring agent on general appearance (score) of fresh-cut okra during storage in 2010 and 2011 seasons.

Treatments

2011 Season

2010 Season

Storage period (days)

Mean Storage period (days)

0 2 4 6 8 0 2 4 6 8

Control 9.00 8.33 7.00 5.00 3.00 6.46 9.00 8.33 6.33 4.33 2.33 6.19

Cysteine 9.00 9.00 9.00 8.33 7.00 8.46 9.00 9.00 9.00 9.00 7.67 8.73

Ascorbic acid 9.00 9.00 8.33 7.00 5.00 7.66 9.00 9.00 9.00 7.67 5.67 8.07

Calcium chloride 9.00 9.00 8.33 7.67 6.33 8.06 9.00 9.00 9.00 7.67 5.00 7.93

Citric acid 9.00 8.33 7.00 5.67 4.33 6.86 9.00 9.00 7.67 5.00 3.67 7.01

Mean 9.00 8.73 7.93 6.73 5.13 9.00 8.86 8.20 6.73 5.13

L.S.D Season 2010 Season 2011

Treatments Storage period Treatments x storage periods 0.21 0.25 0.31 0.26 0.31 0.36

Table 4 Effect of anti-coloring agent on total microbial count (log10 CFU) of fresh-cut okra during storage in 2010 and 2011 seasons.

Treatments 2011 Season 2010 Season

Storage period (days) Mean Storage period (days) Mean

0 2 4 6 8 0 2 4 6 8

Control 0.45 1.24 1.72 3.14 4.86 1.96 0.53 2.27 3.08 4.12 5.23 3.05

Cysteine 0.45 0.96 1.79 2.82 3.78 1.96 0.53 1.98 2.32 3.15 3.87 2.37

Ascorbic acid 0.45 0.83 1.02 1.91 2.52 1.35 0.53 1.36 1.96 2.24 2.79 1.82

Calcium chloride 0.45 0.72 1.10 1.82 2.64 1.35 0.53 1.54 1.84 2.31 2.68 1.78

Citric acid 0.45 1.10 1.66 2.91 3.56 1.94 0.53 1.84 2.45 3.12 3.92 2.37

Mean 0.45 0.97 1.45 2.52 3.47 0.53 1.79 2.33 3.03 3.69

L.S.D Season 2010 Season 2011

Treatment 0.18 0.12

Storage period 0.22 0.18

Treatment x storage period 0.26 0.20

seasons, respectively, while other treatments showed also decrease in the L* value but at lesser extent. The best treatments were cysteine, ascorbic acid, and CaCl2 where the L* value remained closer to initial value (~56.5 and 54.5 in the two seasons, respectively) at the end of the experimental period, while citric acid was less effective.

Hue angle (ho) is a useful parameter to show the color difference (Voss, 1992). In consistent with the L* value results, hue angle of control samples showed also much color

deterioration after 8 days (from 120.38 and 123.24 to 110.69 and 112.84 in the two seasons, respectively). Cysteine, ascorbic acid, and CaCl2 treatments gave again the best results where the hue angle dropped only to ~118 and 122 in both seasons, respectively, during the storage period while citric acid was also less effective in preventing color change (Table 6).

Total soluble phenolic content was decreased throughout the storage period (Table 7) in a parallel trend to that observed

Table 5 Effect of anti-coloring agent on color (L* value) of fresh-cut okra during storage in 2010 and 2011 seasons.

Treatments

2010 Season

2011 Season

Storage period (days)

Mean Storage period (days)

0 2 4 6 8 0 2 4 6 8

Control 58.29 53.17 50.61 48.24 45.71 51.2 55.64 53.39 50.17 45.22 40.28 48.94

Cysteine 58.29 57.62 57.21 57.06 56.94 57.42 55.64 55.23 55.04 54.90 54.72 55.11

Ascorbic acid 58.29 57.46 57.00 56.82 56.61 57.24 55.64 55.11 54.82 54.62 54.32 54.90

Calcium chloride 58.29 57.45 56.94 56.72 56.51 57.18 55.64 55.16 54.71 54.69 54.12 54.86

Citric acid 58.29 56.03 55.12 53.11 50.14 54.54 55.64 54.11 50.29 46.17 40.73 49.39

Mean 58.29 56.34 55.37 54.39 53.18 55.64 54.6 53.01 51.12 48.83

L.S.D Season 2010 Season 2011

Treatment Storage period Treatments x Storage period 0.28 0.31 0.34 0.26 0.30 0.34

Table 6 Effect of anti-coloring agent on hue angle (ho) of fresh-cut okra during storage in 2010 and 2011 seasons.

Treatments 2010 Season 2011 Season

Storage period (days) Mean Storage period (days) Mean

0 2 4 6 8 0 2 4 6 8

Control 120.38 117.92 115.23 114.71 110.69 115.79 123.24 120.27 117.14 114.39 112.84 117.58

Cysteine 120.38 120.10 119.94 119.61 119.23 119.85 123.24 122.92 122.75 122.41 122.16 122.70

Ascorbic acid 120.38 119.75 119.23 119.06 118.75 119.43 123.24 122.27 122.03 121.78 121.54 122.17

Calcium chloride 120.38 119.43 119.00 118.82 118.54 119.23 123.24 122.04 121.83 121.64 121.13 121.97

Citric acid 120.38 118.20 117.13 115.28 112.42 116.68 123.24 120.78 117.71 115.27 113.68 118.14

Mean 120.38 119.08 118.11 117.50 115.93 123.24 122.86 120.29 119.01 118.27

L.S.D Season 2010 Season 2011

Treatment 0.32 0.42

Storage period 0.37 0.46

Treatment x storage period 0.41 0.49

Table 7 Effect of anti-coloring agent on total phenolic content (mg (gallic acid)/g fresh weight) of fresh-cut okra during storage in 2010 and 2011 seasons.

Treatments 2010 Season 2011 Season

Storage period (days) Mean Storage period (days) Mean

0 2 4 6 8 0 2 4 6 8

Control 10.62 9.75 9.10 8.23 7.91 9.12 11.29 10.31 9.73 9.2 8.42 9.79

Cysteine 10.62 10.25 10.04 9.52 9.14 9.91 11.29 11.02 10.82 10.41 9.94 10.69

Ascorbic acid 10.62 10.12 9.91 9.35 9.07 9.81 11.29 10.95 10.71 10.35 9.62 10.58

Calcium chloride 10.62 10.17 9.87 9.39 9.0 9.81 11.29 10.87 10.52 10.17 9.57 10.48

Citric acid 10.62 10.02 9.17 8.45 8.03 9.26 11.29 10.54 10.12 9.64 8.73 10.06

Mean 10.62 10.06 9.62 8.99 8.63 11.29 10.74 10.38 9.95 9.26

L.S.D Season 2010 Season 2011

Treatment 0.18 0.28

Storage period 0.22 0.30

Treatment x storage period 0.26 0.32

of color change where the phenolic content of control samples respectively. In addition, cysteine, ascorbic acid, and CaCl2 was decreased remarkably during the experimental period treatments were still better in preserving the phenolic content from 10.62 and 11.29 to 7.91 and 8.42 in the two seasons, till the end of storage period (>9mg/g fresh weight in both

seasons) than that of citric acid (8.03 and 8.73 mg/g fresh weight) in the two seasons, respectively.

The decrease in phenolic content upon fresh-cut treatment is due to the oxidation by polyphenoloxidase to give the colored quinones (Chang, 2009). During storage, the enzymatic oxidation is continued, and the resulted quinones are polymerized non-enzymatically to give darker pigments, which explains the parallel consumption of phenols with the development of blackness throughout the storage period. The superiority of ascorbic acid and cysteine treatments is attributed to their dual roles in inhibiting color formation; while ascorbic acid reduces quinones back to the colorless cat-echols, cysteine reacts with quinones to give colorless products; besides, both of them are PPO inhibitors (PiliZota and Subaric, 1998; He and Luo, 2007; Chang, 2009). The main function of CaCl2 is strengthen the cell wall and stabilizing the cell membrane (Picchioni et al., 1995; Luna-Guzman and Barrett, 2000), thus keeps PPO, which is membrane-bound enzyme, away from its phenolic substrates present mainly in vacuoles leading to preserving phenolic content and inhibiting browning process (Jiang et al., 2004; Queiroz et al., 2008; Huanga et al., 2012); in addition, chloride ion is known PPO inhibitor (PiliZZota and Subaric, 1998). Citric acid acts mainly as enzyme inhibitor by chelating the copper from the enzyme active site; besides, any used acidulates may lower the suitable pH for maximum PPO activity (He and Luo, 2007).

In conclusion, the main undesirable changes occurred in stored okra pods were texture softening, blackening in color and increasing microbial load. Softening was relieved by treatment with CaCl2 while microbial load was best treated with ascorbic acid and CaCl2. In the meantime, cysteine, ascorbic acid, and CaCl2 could preserve color changes till the end of the experimental period. Therefore, deterioration in the general appearance, weight, texture, color and phenolic content could be mostly preserved in fresh-cut okra pods in polypropylene bags up to 8 days at 9 °C and 95% RH upon dipping in 0.5% solution of cysteine, ascorbic acid, or CaCl2 for 5 min.

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