Effect of Chlorine and Silver Grafted zeolite-LDPE Composite Bags Packaging on the Postharvest Quality of Sapota (Manilkara Achras)

ABSTRACT

In this study, zeolite-LDPE composite bags with or without an infusion of antimicrobial compound (chlorine or silver) were employed as novel packaging materials. The influence of packaging innovation on physico-chemical parameters and shelf life of sapota (Manilkara achras, Family: Sapotaceae) fruits under cold storage condition was investigated. Sapota fruits first packed in antimicrobial compound synergized zeolite-LDPE composite bags and then placed in corrugated fiber board (CFB) box showed significantly lower physiological loss in weight, respiration rate, total soluble solids (TSS), disease score and higher color (L*, chroma and hue-angle) values for pulp, titratable acidity, firmness and more shelf-life than the sapota fruits packed in corrugated fiber board (CFB) box alone. Our results suggest that zeolite-LDPE composite bags synergized with antimicrobial compounds could maintain the postharvest quality and increase the shelf-life of sapota fruits.

KEYWORDS:

• Zeolite
• Manilkara achras
• synergized
• LDPE (Low Density Poly Ethylene)
• Days after storage (DAS)

Introduction

Sapota fruits are classified under extremely high ethylene producers; they are highly perishable in nature, ripe quickly, lose moisture rapidly, spoiled faster and achieve senescence speedily after harvest (Singh et al., 2017). The level of ethylene production in sapota fruits increases slowly from the beginning of ripening and reaches its peak at 144 h, followed by a decline (Selvaraj and Pal, 1984).

Ethylene is a gaseous plant hormone that plays a major role in the regulation of the metabolism of harvested horticultural crops at very low concentrations (Zhang et al., 2012). The post-harvest life of both climacteric and non-climacteric fruits can be influenced by ethylene. This hormone affects their quality attributes, the development of physiological disorders and post-harvest diseases (Ernst, 2011). Effects of ethylene on quality attributes viz., external appearance, texture, flavor and nutritive value of fruits have been extensively reported (Saltveit, 1999; Ernst, 2011). Any closed environment such as truck trailer, shipping container, warehouses, cold rooms, and consumer size package results in an increase in the concentration of ethylene. Therefore, the need to control ethylene activity to extend the postharvest life of fruits through improvement in packaging, introducing anti-ethylene substances is greater than ever.

Zeolite is a large and diverse class of volcanic aluminosilicate crystalline material which has many useful applications (Khosravi et al., 2015). The use of zeolite as an adsorbent has started in the 1930s followed by Milton, who used zeolite for air purification (Kamarudin, 2006). Zeolite is a nanoporous crystalline alumina silicate having a trihedral and tetrahedral structure. It contains large vacant spaces or cages in its structure that provide space for adsorption of cations or large molecules such as water and ethylene (Khosravi et al., 2015). It has a rigid, three-dimensional crystalline structure consisting of a network of interconnected channels and cages. Water moves freely in and out of these pores, but the zeolite framework remains rigid (Kamarudin, 2006). Moreover, the incorporation of antimicrobial compounds into zeolite-LDPE composite bags can further improve the physical, mechanical and biological properties of the bag (Lee et al., 2017).

Among inorganic antimicrobial agents, chlorine and silver compounds could highly inhibit microbial growth and show strong biocidal effects on many species of bacteria including Escherichia coli (Kim et al., 2007; Lee et al., 2009; Yang et al., 2009). The interaction of chlorine and silver ions with microbial cytoplasmic components and nucleic acids can inhibit the respiratory chain enzymes and interferes with the membrane permeability, limiting the development of bacteria, fungi, and yeast (Russel and Hugo, 1994). In this study, the effect of antimicrobial compounds synergized zeolite-LDPE composite bags packaging on the postharvest quality of M. achras fruits was evaluated and recorded for the first time.

Materials and Methods

Materials and Treatments

The present investigation was undertaken in the Department of Post Harvest Technology, University of Horticultural Sciences, Bagalkot, Karnataka, India during the year 2018–19. Sapota fruits (cv. Kalipatti) of uniform size and shape, free from any visible damage, scratches, and decay were selectively harvested manually from a commercial orchard at the proper maturity stage. The maturity stage was judged as the right stage based on the skin color of the fruits which changed from light brown to dark brown (potato-like color) and brown scale-like structure on the surface of fruit was disappeared. The fruits were brought to the laboratory in plastic crates. Soon, the plastic crates containing fruits were placed in the cold room for pre-cooling by room cooling method at 13°C for 12 h. Then, fruits were packed in zeolite-LDPE composite bags with or without outer CFB box viz., Silver-zeolite-LDPE composite bag (T₁), Zeolite-LDPE composite bag (T₂), Chlorine-zeolite-LDPE composite bag (T₃), Silver-zeolite-LDPE composite bag + CFB box (T₄), Zeolite-LDPE composite bag + CFB box (T₅), Chlorine-zeolite-LDPE composite bag + CFB box (T₆), Commercially used CFB (T₇) and Control (without any package) (T₈) @ 6 fruits/treatment (per bag) and stored under refrigerated (13 ± 1°C, 85–90% RH) conditions. A thermostat of the walk-in cold room maintained the set temperature. Relative humidity in the storage chamber was maintained with the help of a humidifier.

Preparation of Antimicrobial Compounds Synergized zeolite-LDPE Composite Bags

In the production of the composite bags, finely ground natural zeolite was combined with commercial polyethylene. Polyethylene was purchased from Dhwani polytech industries, Mumbai, India as polyethylene beads. Zeolite was received from Asian Pacific zeolite molecular sieve market region in an unprocessed form. The zeolite mineral was in the form of large pieces about 20 × 18 x 8 cm in dimension as received from the region. These were crushed in a hammer mill (Laboratory Mill Model 4, Arthur H. Thomas Company, USA). Chlorine and silver powders were received from Kudaim Industries, Goa. Polyethylene beads were dissolved in a solvent and zeolite and antimicrobial compounds were added to this solution. The solvent used was xylene as it is the most suitable for polyethylene. Polyethylene in the form of beads was put into xylene in a 1:10 ratio and the mixture was kept at 75°C for 1 h while mixing the solution intermittently. Upon obtaining a homogeneous solution, the solution was divided into three groups. The first group solution was added with silver and zeolite powder @ 1 part of polyethylene, the second group was added with chlorine and zeolite powder @ 1 part of polyethylene and the third group was added with only zeolite powder @ 1 part of polyethylene. Then, the mixture formed was poured as a thin film in a petri dish and dried in a hot air oven at 80°C until all xylene evaporated leaving a thick and brittle film as a residue. Finally, the obtained film was cut into very small pieces, i.e., 0.02 m x 0.05 m rectangular strips, placed between heat stable and non-sticking papers and hot-pressed at 130°C for 5 min to obtain the sheet. Then, the sheets were carved into bags by sealing the ends.

Then, fruits were packed in those bags. Each pack of fruits was kept undisturbed until the scheduled date of observation. Thus, there were so many numbers of packs under each treatment as the number of times the fruits were observed at a scheduled interval. Sapota fruits were observed for 8 times for each treatment. There were eight packs each containing six fruits for each treatment. All the fruit-packs removed from storage condition on the scheduled day of observation were used to record different observations. Thus, each pack in the storage condition passed through the storage time undisturbed until it was finally taken out to observe for different parameters.

Physiological Loss in Weight (%)

The sapota fruits in each treatment were weighed at the beginning of storage and recorded as initial weight. On the subsequent dates of observation during storage, the fruits were weighed and the weight was recorded as the final weight on each date of observation. The cumulative loss in weight of fruits was calculated and expressed as percent physiological loss in weight using the formula given below.

$\mathrm{P}\mathrm{h}\mathrm{y}\mathrm{s}\mathrm{i}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{g}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{l}\mathrm{o}\mathrm{s}\mathrm{s}\mathrm{i}\mathrm{n}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\left(\mathrm{\%}\right)=\frac{\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}-\mathrm{F}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{l}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}}{\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}}\times 100$

Respiration Rate (Ml CO₂ Kg⁻¹h⁻¹)

The rate of respiration was measured by static headspace method using a gas analyzer (Make: PBI, DANSENSOR, and CHECKMATE 2) and expressed as ml CO₂kg⁻¹h⁻¹. To carry out this method, four sapota fruits were trapped in 3000 ml airtight containers having twist-top lid fitted with a silicone rubber septum at the center of the lid. The containers were kept for an hour for the accumulation of respiratory gases at the headspace. After the specified time, the headspace gas was sucked to the sensor of the analyzer through the hypodermic hollow needle and the displayed value of evolution of CO₂ concentration (%) was recorded. Further, the rate of respiration was calculated on the basis of the amount of CO₂ evolved from the fruit per unit weight per unit time using the following formula.

$\mathrm{R}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{i}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\mathrm{C}{\mathrm{O}}_2/\mathrm{k}\mathrm{g}/\mathrm{h}\right)=\frac{\mathrm{C}{\mathrm{O}}_2\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\mathrm{\%}\right)\times \mathrm{H}\mathrm{e}\mathrm{a}\mathrm{d}\mathrm{s}\mathrm{p}\mathrm{a}\mathrm{c}\mathrm{e}}{100\times \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{f}\mathrm{r}\mathrm{u}\mathrm{i}\mathrm{t}\left(\mathrm{k}\mathrm{g}\right)\times \mathrm{T}\mathrm{i}\mathrm{m}\mathrm{e}\left(\mathrm{h}\mathrm{r}\right)}$

Color Value of Pulp (L*, Chroma and Hue-angle)

Color of the fruit peel was measured using Color Flex EZ colorimeter (Make: CFEZ 1919, Hunter Associates Laboratory., Inc., Reston, Virginia, USA Model No. 45/0, Serial No. CFEZ1919) fitted with 45 mm diameter aperture. The instrument was calibrated using black and white tiles provided by the manufacturer. Color was expressed in terms of L* (lightness/darkness), chroma or saturation (C*) which is the relation of a color to a neutral gray of the same lightness (Kevin, 2002). Hue-angle is the index or purity of the color and it shows the brightness of fruits (Shafiee et al., 2010).

Total Soluble Solids (TSS) (°B)

The juice extracted by squeezing the homogenized fruit pulp through muslin cloth was used to measure the TSS. It was determined by using a digital refractometer (Make: Hanna Instruments, Romania) replicated three times and the mean was expressed in °Brix.

Titratable Acidity (%)

Five grams of homogenized pulp was made up to 100 ml and filtered through a muslin cloth. Then, 10 ml of the filtrate was taken for titration against 0.1 N NaOH solution using phenolphthalein as an indicator. The appearance of light pink color was considered as the endpoint. The acidity was calculated and expressed as percent malic acid.

$\mathrm{A}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{i}\mathrm{t}\mathrm{y}\left(\mathrm{\%}\right)=\frac{\mathrm{T}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e}\times \mathrm{N}\mathrm{o}\mathrm{f}\mathrm{N}\mathrm{a}\mathrm{O}\mathrm{H}\times \mathrm{V}\mathrm{o}\mathrm{l}.\mathrm{m}\mathrm{a}\mathrm{d}\mathrm{e}\mathrm{u}\mathrm{p}\times \mathrm{E}\mathrm{q}.\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{a}\mathrm{c}\mathrm{i}\mathrm{d}}{\mathrm{V}\mathrm{o}\mathrm{l}.\mathrm{o}\mathrm{f}\mathrm{a}\mathrm{l}\mathrm{i}\mathrm{q}\mathrm{u}\mathrm{o}\mathrm{t}\times \mathrm{V}\mathrm{o}\mathrm{l}.\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}\mathrm{n}\times 1000}\times 100$

Firmness (Newtons)

Fruit firmness was determined with TAXT Plus Texture Analyzer (Make: Stable Micro Systems, UK, Model: Texture Export Version 1.22). The force with the samples got pierced was recorded in the graph and the peak force value in the graph was taken as the firmness value in terms of Newton force (N). The following instrument settings were used during the experiment.

Type of probe used :Piercing probe

Test option :Return to start

Test speed :5.0 mm/s

Posttest speed :10.0 mm/s

Distance :50 mm

Load cell :5 kg

Disease Score (%)

Disease occurrence on fruits was scored by visual inspection of fruits during storage. For the disease sore, damage caused by fungi or bacteria was considered based on the scale mentioned below. Finally, disease scoring was calculated with the following formula.

$\mathrm{D}\mathrm{i}\mathrm{s}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{s}\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{e}\left(\mathrm{\%}\right)=\frac{\mathrm{S}\mathrm{u}\mathrm{m}\mathrm{o}\mathrm{f}\mathrm{a}\mathrm{l}\mathrm{l}\mathrm{d}\mathrm{i}\mathrm{s}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{g}}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{g}\times \mathrm{M}\mathrm{a}\mathrm{x}\mathrm{i}\mathrm{m}\mathrm{u}\mathrm{m}\mathrm{d}\mathrm{i}\mathrm{s}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{g}\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{e}}\times 100$

Disease scale:

(0) No lesions

1. 5% to <15% lesions

2. 15 to <25% lesions

3. 25 to <50% lesions

4. 50 to <75% lesions

5. 75 to 100% lesions

Shelf Life (Days)

The number of shelf-life days was decided based on physiological loss in weight (PLW). A PLW of 10% was considered as the upper limit for the determination of shelf life (Jaya, 2010).

Experimental Design and Data Analysis

The first experiment was carried out with three replicate fruits and the experiment was repeated three times and pooled data were subjected to statistical analysis. Fruits were arranged in Complete Randomized Design. The second experiment was carried out for three replicate fruits and repeated three times. Randomly selected eight fruits were taken to analyze physiological loss in weight, respiration rate, color value of pulp (L*, chroma, and hue-angle), Total Soluble Solids (TSS), titratable acidity, firmness, disease score and shelf life and all the experiments were repeated 3 times. The data of the experiment were analyzed as applicable to a completely randomized design (CRD). Statistical analyses of experiments were performed using Web Agri Stat Package (WASP) Version 2 (Jangam and Thali, 2010). The level of significance used in ‘F’ and ‘t’ was p = .01 and also p = .05 for some parameters. Critical difference values were calculated whenever F-test was found significant.

Results and Discussion

Physiological Loss in Weight (PLW) (%)

PLW (%) of sapota fruits in this study increased (1.46% at 3 DAS to 17.67% at 21 DAS) with the increase in the duration of storage (Table 1). Respiration and transpiration by which the fruit loses water and cause the weight loss are advanced by the gases like ethylene and oxygen that raise respiration (Raorane et al., 2012). The PLW among the treatments on each observed day were found to be statistically significant. Minimum PLW was noted in T₆ (Chlorine-zeolite-LDPE composite bag + CFB box) packaging treatment throughout the storage (3 DAS-0.98%; 6 DAS-1.23%; 9 DAS-2.81%; 12 DAS-5.30%; 15 DAS-7.03%; 18 DAS-10.42% and 21 DAS-12.05%). However, it was statistically similar to T₄ (Silver-zeolite-LDPE composite bag + CFB box) and T₅ (Zeolite-LDPE composite bag + CFB box). Zeolite has the property to adsorb gases like ethylene and oxygen from the storage environment (Khosravi et al., 2015). Fruit respiration and transpiration in this study was therefore probably retarded resulting in minimum weight loss. However, zeolite-LDPE variant packages combined with CFB boxes (T₄, T₅, T₆) exhibited better performance over their counterparts without the combination of CFB (T₁, T₂, T₃). The humid microclimate created within CFB in T₄ (Silver-zeolite-LDPE composite bag + CFB box), T₅ (Zeolite-LDPE composite bag + CFB box) and T₆ (Chlorine-zeolite-LDPE composite bag + CFB box) might have reduced the drive for removal of moisture from the fruits. Abhay et al. (2015) recorded minimum physiological loss in weight in Dwarf Cavendish fruits packed in a ventilated polyethylene bag along with KMnO₄ up to 18 days of storage. Sapota fruits maintained without any packaging (T₈) followed by T₇ (Commercially used CFB) recorded higher PLW percentage throughout the storage period.

Table 1. Effect of antimicrobial compounds synergized zeolite-LDPE composite bags on physiological loss in weight (PLW) (%) of sapota fruits under refrigerated condition (13°C).

	Physiological loss in weight (PLW) (%)
Treatments	3 DAS	6 DAS	9 DAS	12 DAS	15 DAS	18 DAS	21 DAS	Mean
T1	1.49^cd	3.87^ab	4.85^bc	8.98^bc	11.18^bc	14.68^bc	17.90^bcd	8.99
T2	1.66^bc	2.09^bc	6.07^b	10.21^ab	11.91^bc	16.11^b	19.58^bc	9.66
T3	1.41^cd	3.82^ab	4.64^bcd	8.86^bc	10.78^bc	14.34^bc	17.52^cd	8.76
T4	1.07^e	1.49^c	3.09^cd	6.49^cd	8.55^cd	12.10^de	13.25^e	6.57
T5	1.25^de	3.62^ab	4.38^bcd	8.52^bcd	10.15^cd	12.93^cd	15.47^de	8.04
T6	0.98^e	1.23^c	2.81^d	5.30^d	7.03^d	10.42^e	12.05^e	5.68
T7	1.83^ab	2.26^bc	6.38^b	11.37^ab	14.00^ab	16.28^b	21.64^ab	10.53
T8	2.04^a	4.47^a	8.59^a	13.03^a	16.12^a	18.41^a	23.96^a	12.37
Mean	1.46	2.85	5.10	9.09	11.21	14.40	17.67
S.Em±	0.08	0.61	0.67	1.11	1.15	0.67	1.30
CD@1%	0.39	2.52	2.79	4.61	4.76	2.79	5.39

Respiration Rate (Ml CO₂ Kg⁻¹h⁻¹)

Fresh sapota fruits used in the study had a respiration rate of 78.09 ml CO₂/Kg/hr. With storage, the mean of respiration rate increased in all the treatments up to 15 days and a decrease at 18 and 21 DAS (Table 2). The data revealed significant differences with respect to the respiration rate of sapota during 21 DAS. The rate was found to be minimum in the treatment T₆ (Chlorine-zeolite-LDPE composite bag + CFB box) throughout the storage (3 DAS-81.4; 6 DAS-92.2; 9 DAS-108.5; 12 DAS-128.9; 15 DAS- 195.6; 18 DAS-192.3; 21 DAS-164.1 ml CO₂/Kg/hr) in comparison to all other treatments. In the present study, hurdle to respiration process was created by low temperature as well as zeolite-composed package. Porous zeolite is effective in adsorbing gases such as oxygen, carbon dioxide and ethylene and water vapor, and thereby reducing respiration (Khosravi et al., 2015). This is possibly due to the biocidal effect of chlorine (Kim et al., 2007; Lee et al., 2009; Yang et al., 2009) leading to delay in fruit senescence. However, T₆ treatment had a non-significant difference with T₃ (Chlorine-zeolite-LDPE composite bag), T₄ (Silver-zeolite-LDPE composite bag + CFB box) and T₅ (Zeolite-LDPE composite bag + CFB box) throughout the storage except at 12 DAS when T₆ differed significantly over all other treatments. Reduced respiration of fruits in zeolite-LDPE variants (with or without chlorine or silver) in CFB boxes (T₄, T₅, T₆) than those without CFB (T₁, T₂, T₃) could be due to beneficial synergistic effect of combined packages. Sapota fruits kept open condition (T₈) had a significantly higher respiration rate followed closely by T₇ (Commercially used CFB) throughout the storage period. This defends the beneficial effect of zeolite-LDPE bags.

Table 2. Effect of antimicrobial compounds synergized zeolite-LDPE composite bags on respiration rate (ml 0f CO₂/Kg/hr) of sapota fruits under refrigerated condition (13°C).

	Respiration rate (ml 0f CO2/Kg/hr)
Treatments	3 DAS	6 DAS	9 DAS	12 DAS	15 DAS	18 DAS	21 DAS	Mean
Initial	78.09
T1	89.1^bcd	98.7^abc	122.2^cd	153.8^cd	208.9^cd	199.2^b	183.9^bc	150.82
T2	91.5^abc	100.8^ab	125.3^bc	160.6^bc	213.8^bc	201.5^ab	185.6^abc	154.15
T3	87.8^bcde	96.0^abc	118.6^de	149.0^de	206.8^cd	198.8^b	180.3^c	148.18
T4	83.4^de	92.4^c	111.8^fg	137.6^f	200.6^de	196.4^bc	168.3^de	141.50
T5	85.7^cde	94.3^bc	115.1^ef	144.5^ef	204.8^cde	196.6^bc	171.7^d	144.67
T6	81.4^e	92.2^c	108.5^g	128.9^g	195.6^e	192.3^c	164.1^e	137.57
T7	93.9^ab	102.7^a	128.4^ab	163.6^ab	220.4^ab	202.8^ab	189.8^ab	157.37
T8	96.2^a	104.1^a	131.0^a	169.6^a	224.7^a	206.0^a	192.7^a	160.61
Mean	88.62	97.65	120.11	150.95	209.45	199.20	179.55
S.Em±	2.33	2.74	1.53	2.77	3.45	2.14	2.47
CD@1%	9.65	8.23	6.36	11.46	14.26	6.41	10.22

*DAS – Days After Storage

Color Value of Pulp (L*, Chroma, and Hue-angle)

Instrumental L* values of sapota fruit pulp revealed significant differences at 3 DAS to 15 DAS, but not during later storage period of 18 and 21 DAS (Table 3). Significantly maximum L* value was observed in T₆ (Chlorine-zeolite-LDPE composite bag + CFB box) at 3, 6, 9, 12 and 15 days of storage (70.4, 69.9, 61.7, 57.8 and 53.9, respectively) and remained on par with all other treatments at 18 and 21 DAS (49.8 and 45.6). Flesh color of sapota fruits changes from pale yellow or pale pink to orange or dark brown color upon repining (Perez et al., 2000). Orange color is due to the synthesis of carotenoids and brown color is due to the synthesis of phenols during ripening (Casas Alencaster, 1977). Maximum L* value was observed in T₆ indicate a delay in ripening. Minimum L* value was seen in T₈ (Control) followed by T₇ (Commercially used CFB) almost throughout the storage. In this present study, instrumental color values (L*, Chroma, and Hue-angle) of sapota pulp gradually decreased during storage under both ambient and refrigerated (13°C) conditions.

Table 3. Effect of antimicrobial compounds synergized zeolite-LDPE composite bags on L* color value of pulp of sapota fruits under refrigerated condition (13°C).

	L* value
Treatments	3 DAS	6 DAS	9 DAS	12 DAS	15 DAS	18 DAS	21 DAS	Mean
Initial	70.4
T1	70.4^a	63.4^bc	55.0^bcd	51.5^bc	47.0^bcd	42.8	39.8	52.84
T2	68.7^ab	61.1^c	53.8^cde	49.3^cd	45.8^cd	40.5	38.9	51.15
T3	70.4^a	63.7^bc	55.3^a	51.7^bc	47.2^bcd	42.6	39.2	52.87
T4	70.4^a	67.6^ab	59.2^ab	55.2^ab	51.7^ab	46.2	43.3	56.22
T5	70.4^a	65.0^abc	57.8^abc	53.9^b	49.4^abc	44.9	41.5	54.70
T6	70.4^a	69.9^a	61.7^a	57.8^a	53.9^a	49.8	45.6	58.44
T7	66.3^bc	59.8^c	51.4^de	47.3^d	43.5^d	38.3	37.8	49.20
T8	64.5^c	59.4^c	49.1^e	47.0^d	43.3^d	38.0	37.4	48.38
Mean	68.93	63.73	55.41	51.71	47.72	42.88	40.43
S.Em±	1.03	1.98	1.60	1.25	1.80	2.28	1.25
CD@1%	4.29	5.96	6.63	5.19	7.44	NS	NS

Chroma values were maximum in T₆ (Chlorine-zeolite-LDPE composite bag + CFB box) at 3, 6, 9, 12, 15, 18, and 21 DAS (44.2, 43.9, 41.9, 38.6, 35.5, 32.1, and 27.8, respectively) (Table 4). Significantly minimum chroma value was seen in T₈ (Control) followed by T₇ (Commercially used CFB). Instrumental hue-angle values of sapota fruit pulp revealed significant at 3, 6, 12, 18 and 21 DAS, but non-significant differences among the treatments at 9 and 15 DAS (Table 5). Significantly maximum hue-angle value was observed in T₆ (Chlorine-zeolite-LDPE composite bag + CFB box) at 3, 6, 12, 18 and 21 days of storage (57.6, 53.9, 49.1, 42.5, and 39.1, respectively). However, the treatment T₆ was on par with T₄ (Silver-zeolite-LDPE composite bag + CFB box) and T₅ (Zeolite-LDPE composite bag + CFB box) for up to initial 12 days of storage. Minimum hue-angle value throughout the storage was seen in T₈ (Control) (3 DAS-47.0, 6 DAS-43.3, 9 DAS-38.0, 12 DAS-33.8, 15 DAS- 31.5, 18 DAS-28.3, 21 DAS-24.0).

Table 4. Effect of antimicrobial compounds synergized zeolite-LDPE composite bags on chroma color value of pulp of sapota fruits under refrigerated condition (13°C).

	Chroma
Treatments	3 DAS	6 DAS	9 DAS	12 DAS	15 DAS	18 DAS	21 DAS	Mean
Initial	44.2
T1	39.2^cd	39.4^c	37.1^cde	32.1^abcd	29.6^bcd	26.4	23.5	32.47
T2	38.9^cd	37.0^d	35.2^def	30.6^bcd	27.1^cde	24.5	21.9	30.74
T3	39.8^c	39.7^c	37.8^bcd	32.5^abcd	29.9^bcd	26.8	24.7	33.02
T4	43.3^a	43.1^a	41.6^ab	36.2^ab	33.4^ab	30.2	26.6	36.34
T5	41.5^b	41.2^b	39.4^abc	34.4^abc	31.8^abc	28.7	25.4	34.62
T6	44.2^a	43.9^a	41.9^a	38.6^a	35.5^a	32.1	27.8	37.71
T7	37.8^de	35.8^d	33.5^ef	28.9^cd	25.9^de	22.4	20.6	29.27
T8	37.4^e	33.5^e	31.8^f	26.3^d	23.5^e	20.6	16.1	27.02
Mean	40.26	39.20	37.28	32.45	29.58	26.46	23.32
S.Em±	0.55	0.40	1.32	2.31	1.76	3.06	2.80
CD@1%	2.28	1.68	5.46	6.95	7.30	NS	NS

*DAS – Days After Storage

Table 5. Effect of antimicrobial compounds synergized zeolite-LDPE composite bags on hue-angle color value of pulp of sapota fruits under refrigerated condition (13°C).

	Hue angle
Treatments	3 DAS	6 DAS	9 DAS	12 DAS	15 DAS	18 DAS	21 DAS	Mean
Initial	57.6
T1	51.5^bcd	47.0^bcd	44.8	42.0^bc	40.5	36.5^abcd	32.8^bcd	42.15
T2	49.3^cd	45.8^cd	42.5	39.5^cd	36.2	33.2^bcd	29.6^cde	39.44
T3	51.7^bcd	47.2^bcd	44.6	42.9^bc	40.9	36.7^abcd	33.7^abc	42.52
T4	55.2^ab	51.7^ab	48.2	47.2^a	45.6	40.7^ab	37.8^ab	46.62
T5	53.9^abc	49.4^abc	46.9	45.7^ab	43.8	38.8^abc	35.9^ab	44.91
T6	57.6^a	53.9^a	50.8	49.1^a	47.0	42.5^a	39.1^a	48.57
T7	47.3^d	43.5^d	40.3	36.2^de	33.9	30.1^cd	27.3^de	36.94
T8	47.0^d	43.3^d	38.0	33.8^e	31.5	28.3^d	24.0^e	35.12
Mean	51.68	47.72	44.51	42.05	39.92	35.85	32.52
S.Em±	1.93	1.59	3.37	1.35	3.51	2.97	2.07
CD@5%	5.81	4.77	NS	4.05	NS	8.93	6.21

Minimum chroma and hue values in T₈ and T₇ might be due to browning of pulp associated with the high concentrations of phenols in ripe fruits (Casas Alencaster, 1977). Similarly, higher L*, chroma and hue-angle color values in fruits packed in zeolite-LDPE bags with or without antimicrobials (T₁ to T₆) could be reasoned to ethylene adsorbing capacity of zeolite. Porous zeolite is effective in adsorbing ethylene thereby reducing carotenoids and phenols synthesis in fruits (Khosravi et al., 2015). Hence, the presence of higher ethylene around fruits in T₇ (Commercially used CFB) and T₈ (Control) and reduced presence of ethylene around fruits in antimicrobial compounds synergized zeolite-LDPE bags could be responsible for lower and higher L*, chroma and hue-angle value in those treatments, respectively. Additional modification of atmosphere caused by CFB and the antimicrobial effect of silver in T₄ (Silver-zeolite-LDPE composite bag + CFB box) and chlorine in T₆ (Chlorine-zeolite-LDPE composite bag + CFB box) might be the reason for still higher L*, chroma and hue-angle as a microbial contribution to enhancing ethylene in these packages is minimized.

Total Soluble Solids (TSS) (°B)

TSS was found to increase gradually along the 21-day period of storage (Table 6). But this parameter TSS did not present statistical difference, Minimum TSS was found in T₆ (Chlorine-zeolite-LDPE composite bag + CFB box) at all the days of observation (3 DAS-16.41 °B; 6 DAS-17.72°B; 9 DAS-18.90°B; 12 DAS-19.30°B; 15 DAS-20.26°B; 18 DAS-22.80°B and 21 DAS-24.71°B). Zomoroid (2005) recorded minimum TSS in cut apples stored along with a pouch of zeolite (1 gm) for a period of 6 days. Zeolite has adsorbing property of oxygen, carbon dioxide and ethylene gases from the storage environment, thus checking the biochemical differentiation and metabolic reorganization leading to a reduction in TSS (Khosravi et al., 2015). Maximum TSS was observed in T₈ (Control) at all the seven observations taken during the study (3 DAS-18.09 °B; 6 DAS-21.78°B; 9 DAS-22.97°B; 12 DAS-23.74°B; 15 DAS-24.92°B; 18 DAS-24.31°B and 21 DAS-28.82°B). However, a difference of 3–4°B between the treatments practically has a tremendous impact on taste, flavor, and ripening of fruit. In climacteric fruits like sapota, the conversion of starch to sugars in the fruit is an important component of the ripening process, giving the fruit its distinctive sweet flavor as well as a precursor for many of the aromatic flavor compounds (Kays, 1998).

Table 6. Effect of antimicrobial compounds synergized zeolite-LDPE composite bags on TSS (°B) of sapota fruits under refrigerated condition (13°C).

	TSS (°B)
Treatments	3 DAS	6 DAS	9 DAS	12 DAS	15 DAS	18 DAS	21 DAS	Mean
Initial	16.36
T1	17.21	19.62	20.93	22.32	22.84	23.83	25.91	21.80
T2	17.48	19.98	21.56	22.87	23.09	23.98	26.36	22.18
T3	17.02	19.07	20.22	21.98	22.61	23.55	25.74	21.45
T4	16.69	18.79	19.19	20.02	21.48	22.97	24.95	20.58
T5	16.85	18.91	19.85	20.88	21.84	23.16	25.30	20.97
T6	16.41	17.72	18.90	19.30	20.26	22.80	24.71	20.01
T7	17.82	20.02	22.19	23.07	24.26	24.00	27.28	22.66
T8	18.09	21.78	22.97	23.74	24.92	24.31	28.82	23.51
Mean	17.19	19.48	20.72	21.77	22.66	23.57	26.13
S.Em±	0.44	2.14	2.10	2.90	2.22	1.22	0.40
CD@5%	NS	NS	NS	NS	NS	NS	NS

*DAS – Days After Storage

Titratable Acidity (%)

Titratable acidity (%) was found to increase from the initial value (0.14%) up to 3 DAS and later declined in all the storage treatments of sapota (Table 7). At all the days of observation, maximum acidity was associated with T₆ (Chlorine-zeolite-LDPE composite bag + CFB box) (3 DAS-0.60%; 6 DAS-0.58%; 9 DAS-0.55%; 12 DAS-0.51%; 15 DAS-0.49%; 18 DAS-0.46% and 21 DAS-0.42%). The most important feature of zeolite is effective adsorption of gases such as oxygen, carbon dioxide, and ethylene and water vapor leading to reduced respiration and metabolism of fruits (Khosravi et al., 2015). Reduction in metabolism could reduce the use of organic acids as substrates in respiration (Islam et al., 1996), thus resulting in higher acidity in fruits. The T₆ (Chlorine-zeolite-LDPE composite bag + CFB box) recorded higher acidity than the fruits without any package (T₈). Further, higher titratable acidity in antimicrobial compounds synergized zeolite-LDPE variants in CFB boxes (T₄, T₅, T₆) than those without CFB (T₁, T₂, T₃) could be due to beneficial synergistic effect of combined packages. Lower titratable acidity observed in fruits kept in CFB alone (T₇) strongly justifies the effect of zeolite-LDPE bags with or without antimicrobial compounds.The treatment T₈ (Control) showed significantly minimum titratable acidity (3 DAS-0.28%; 6 DAS-0.26%; 9 DAS-0.23%; 12 DAS-0.20%; 15 DAS-0.18%; 18 DAS-0.18% and 21 DAS-0.13%) closely followed by T₇ (Commercially used CFB) throughout the study duration. Dhua et al. (2006) and Bhutia et al. (2011) revealed that sapota fruits packed along with ethylene absorbers showed marked retardation of ripening and also maintenance of higher acidity in comparison to control fruits.

Table 7. Effect of antimicrobial compounds synergized zeolite-LDPE composite bags on titratable acidity (%) of sapota fruits under refrigerated condition (13°C).

	Titratable acidity (%)
Treatments	3 DAS	6 DAS	9 DAS	12 DAS	15 DAS	18 DAS	21 DAS	Mean
Initial	0.14
T1	0.42^e	0.39^de	0.36^de	0.33^cde	0.30^d	0.28^c	0.26^bcd	0.33
T2	0.38^f	0.36^e	0.32^ef	0.30^def	0.28^de	0.26^cd	0.24^cde	0.30
T3	0.47^d	0.44^cd	0.41^cd	0.38^bcd	0.35^cd	0.33^bc	0.30^abc	0.38
T4	0.56^b	0.53^cd	0.51^ab	0.47^ab	0.44^ab	0.40^ab	0.38^ab	0.47
T5	0.51^c	0.49^ab	0.46^bc	0.43^abc	0.41^bc	0.38^ab	0.36^abc	0.43
T6	0.60^a	0.58^a	0.55^a	0.51^a	0.49^a	0.46^a	0.42^a	0.51
T7	0.31^g	0.28^f	0.26^fg	0.23^ef	0.21^ef	0.18^de	0.16^de	0.23
T8	0.28^g	0.26^f	0.23^g	0.20^f	0.18^f	0.15^e	0.13^e	0.20
Mean	0.44	0.41	0.38	0.35	0.33	0.30	0.28
S.Em±	0.02	0.04	0.05	0.03	0.05	0.05	0.03
CD@1%	0.03	0.09	0.10	0.17	0.10	0.12	0.17

Firmness (Newtons)

In this study, irrespective of the treatments, firmness (N) of fruits decreased along the storage duration (Table 8). Firmness (N) was found to decrease gradually along the period of storage of 21 days. This is witnessed by initial value (9.87 N) and means values recorded at 3, 6, 9, 12, 15, 18 and 21 DAS (8.99 N, 8.54 N, 8.35 N, 7.76 N, 7.19 N, 6.74 N, and 5.28 N, respectively). The steady reduction in fruit firmness during the storage period is due to the natural process of ripening in almost all fleshy fruits as a result of biochemical changes in the cellular structure (Dharmasenal and Kumari, 2005). The depletion of fruit firmness during ripening is apparently the result of the cell wall hydrolysis mechanism. This is related to increased activity of endogenous enzymes like pectinase and pectin methylesterase which break down pectin material, or cause reduction of turgor pressure, which diminishes with loss of water or dehydration caused by tissue perspiration during storage (Beaulieu and Gorny, 2001; Edelky et al., 2016).

Table 8. Effect of antimicrobial compounds synergized zeolite-LDPE composite bags on firmness (N) of sapota fruits under refrigerated condition (13°C).

	Firmness (N)
Treatments	3 DAS	6 DAS	9 DAS	12 DAS	15 DAS	18 DAS	21 DAS	Mean
Initial	9.87
T1	9.02^bcd	8.52^cd	8.38^bc	7.55^cd	7.21^ab	6.72^cd	5.27^cd	7.52
T2	8.69^cd	8.32^de	8.12^cd	7.33^cde	7.04^cd	6.55^cd	5.02^de	7.29
T3	9.01^bcd	8.59^bcd	8.37^bc	7.73^c	7.29^bc	6.71^cd	5.46^bc	7.59
T4	9.42^ab	8.92^ab	8.70^ab	8.43^ab	7.69^ab	7.29^ab	5.78^ab	8.03
T5	9.26^abc	8.78^abc	8.58^ab	8.22^b	7.45^abc	6.93^bc	5.59^abc	7.83
T6	9.50^a	9.14^a	8.93^a	8.69^a	7.82^a	7.42^a	5.91^a	8.20
T7	8.61^d	8.11^e	7.97^d	7.16^de	6.72^de	6.38^de	4.80^e	7.10
T8	8.45^d	7.95^e	7.77^d	6.97^e	6.30^e	5.97^e	4.42^f	6.83
Mean	8.99	8.54	8.35	7.76	7.19	6.74	5.28
S.Em±	0.20	0.12	0.12	0.14	0.14	0.14	0.12
CD@1%	0.84	0.51	0.52	0.59	0.60	0.58	0.49

*DAS – Days After Storage

The firmness (N) of sapota fruits showed significant differences among the treatments during the storage period (Table 8). Maximum firmness was observed in T₆ (Chlorine-zeolite-LDPE composite bag + CFB box) (3 DAS-9.50 N; 6 DAS-9.14 N; 9 DAS-8.93 N; 12 DAS-8.69 N; 15 DAS-7.82 N; 18 DAS-7.42 N and 21 DAS-5.91 N). However, the treatment T₆ (Chlorine-zeolite-LDPE composite bag + CFB box) was on par with T₄ (Silver-zeolite-LDPE composite bag + CFB box) and T₅ almost throughout the storage period. The treatment T₈ (Control) showed significantly minimum firmness throughout the storage (3 DAS-8.45 N; 6 DAS-7.95 N; 9 DAS-7.77 N; 12 DAS-6.97 N; 15 DAS-6.30 N; 18 DAS-5.97 N and 21 DAS-4.42 N) and this was non-significant with T₇ (Commercially used CFB) throughout the study period. These results are in conformity with the findings of Kaur and Kaur (2018) where they observed maximum firmness (7.48 kg/cm²) in the banana fruits packed in a polybag with KMnO₄ after 8 days of storage. Similarly, Elamin and Abu-Goukh (2010) reported that bananas packed along with KMnO₄ showed more delay in climacteric peak and more firmness (8.23 Kg/cm²) compared to the fruits packed without KMnO₄.

Significantly minimum firmness was observed in T₈ (Control) under refrigerated storage (4.42 N at 21 DAS) condition. Salunkhe and Desai (1984) reported that controlled atmosphere storage or modified atmosphere packaging inhibits the breakdown of pectin substances, which retains fruit texture and remains firmer for a longer period.

Disease Score (%)

Sapota fruits showed significant differences among the treatments for disease scoring (%) during storage of 21 days (Table 9). The visual score for diseases increased with the increase in storage period. Sapota with no disease at the beginning were healthy up to 15 days, but showed an increase in score for disease from 18 DAS (32.12%) to 21 DAS (47.83%). The treatment T₆ (Chlorine-zeolite-LDPE composite bag + CFB box) witnessed significantly minimum disease score (18 DAS-11.19% and 21 DAS-24.29%) over the rest of the treatments throughout the storage period. The significantly maximum score for diseases over all other treatments was noted in T₈ (Control) both at 18 DAS (57.36%) and 21 DAS (78.67%). Among inorganic antimicrobial agents, silver and chlorine could effectively inhibit microbial growth and show strong biocidal effects on many species of bacteria including Escherichia coli (Kim et al., 2007; Lee et al., 2009; Yang et al., 2009). The interaction of silver and chlorine ions with microbial cytoplasmic components and nucleic acids can inhibit the respiratory chain enzymes and interfere with the membrane permeability, limiting the development of bacteria, fungi, and yeast (Russel and Hugo, 1994). In general, all variants of polybags in this study performed better when combined with CFB boxes (T₄, T₅, T₆) than if they were employed alone (T₁, T₂, T₃). Additional modification of atmosphere caused by CFB in T₄ (Silver-zeolite-LDPE composite bag + CFB box), T₅ (Zeolite-LDPE composite bag + CFB box) and T₆ (Chlorine-zeolite-LDPE composite bag + CFB box) might be the reason for less proliferation of diseases. Between the two antimicrobial agents used in the package, chlorine appeared to be more effective than silver. Chlorine is the most commonly used sanitizer due to its efficacy, cost-effectiveness ratio and simple use (Petri et al., 2011). Washing raw produce with water-containing sodium hypochlorite (NaOCl) is the most commonly used method for removing pathogens from the surfaces of vegetables (Arah et al., 2016; El-Ramady et al., 2015; Povratanak et al., 2015).

Table 9. Effect of zeolite-LDPE composite bags on disease score (%) and shelf life (days) of sapota fruits under refrigerated condition (13°C).

	Disease score (%)
Treatments	18 DAS	21 DAS	Mean
Initial	NIL			Shelf-life (days)
T1	26.85^d	39.17^e	33.01	13.33^d
T2	39.37^c	55.91^c	47.64	11.66^e
T3	22.00^e	37.48^e	29.74	13.66^d
T4	14.70^f	31.25^f	22.97	16.00^b
T5	36.68^c	46.51^d	41.59	14.66^c
T6	11.19^f	24.29^g	17.74	17.66^a
T7	48.81^b	69.38^b	59.09	11.33^e
T8	57.36^a	78.67^a	68.01	9.66^f
Mean	32.12	47.83		13.49
S.Em±	1.56	1.33		0.30
CD@1%	6.47	5.52		1.28

*DAS –Days After Storage

Shelf-life (Days)

Sapota fruits recorded significantly maximum shelf life (days) in T₆ (Chlorine-zeolite-LDPE composite bag + CFB box) (17.66 days) (Table 9). It was however non-significant with T₁ (Silver-zeolite-LDPE composite bag), T₂ (Zeolite-LDPE composite bag), T₃ (Chlorine-zeolite-LDPE composite bag) and T₄ (Silver-zeolite-LDPE composite bag + CFB box). Significantly minimum shelf life (days) was recorded in T₈ (Control) (12.00 days) and this treatment was on par with T₇ (Commercially used CFB) (9.66 days).

The packaging yielded an extra shelf-life of 8 days over control (T₈) and 6.33 days over T₇ (Commercially used CFB). Slow rate of ripening of sapota fruits in T₆ (Chlorine-zeolite-LDPE composite bag + CFB box) is documented in terms of slower decrease in fruit firmness, reduced respiration rate, physiological loss in weight and disease score. The realization of more shelf life in T₆ (Chlorine-zeolite-LDPE composite bag + CFB box) was mainly due to retarding ethylene effect on the ripening process and due to the effect of chlorine on post-harvest decay. Altered micro-environment and cushioning benefit provided by CFB box in T₆ (Chlorine-zeolite-LDPE composite bag + CFB box) was supportive to the health and wholesomeness of sapota fruits.

Conclusion

Our results have shown that antimicrobial compounds synergized zeolite-LDPE composite bags packaging had a positive effect on the postharvest quality of M. achras fruits. The preservation effect of antimicrobial compounds synergized zeolite-LDPE composite bags could be attributed to its intrinsic ethylene adsorbing property as well as antimicrobial property. Thus, sapota fruits packaged with antimicrobial compounds synergized zeolite-LDPE composite bags showed a lower physiological loss in weight, respiration rate, total soluble solids (TSS), disease score and higher pulp color (L*, chroma and hue-angle) values, titratable acidity, firmness, and more shelf-life. These indicated that antimicrobial compounds synergized zeolite-LDPE composite bags could be explored as a novel active packaging material for the postharvest storage of sapota fruits.

Acknowledgments

We are thankful to the Department of Post Harvest Technology, College of Horticulture, University of Horticultural Sciences, Bagalkot, Karnataka, India for providing the laboratory facilities and technical support.