Influence of Types of Modified Atmospheric Packaging (MAP) Films on Cold-Storage Life and Fruit Quality of ‘Kinnow’ Mandarin (Citrus nobilis Lour X C. deliciosa Tenora)

ABSTRACT

An investigation was aimed to evaluate the efficacy of different MAP films viz., low-density polyethylene (LDPE)-film (25 μ), LDPE-film (38 μ), polypropylene (PP)-film (25 μ), PP-film (38 μ), LDPE-film (25 μ) with pinholes, LDPE-film (38 μ) with pinholes, PP-film (25 μ) with pinholes, and PP-film (38 μ) with pinholes on cold-storage life and fruit quality of ‘Kinnow’ (Citrus nobilis Lour x C. deliciosa Tenora) mandarin. The packed and control fruits were kept under cold-storage conditions (5–7 °C and 90–95 % RH) up to 75 days. The fruits were sampled following 0, 30, 45, 60, and 75 days cold storage and assessed for different quality parameters. The results indicate variability among the different MAP treatments for weight loss, spoilage loss, juice content, firmness, pectin content, activities of fruit softening enzymes, soluble solids concentration (SSC), titratable acidity (TA), ascorbic acid content, total phenol content, total antioxidant activity, total carotenoids content, and organoleptic sensory attributes. Among all MAP treatments, PP-film (25 μ) with pinholes was the most effective in maintaining various fruit quality parameters such as SSC, TA, ascorbic acid, sensory attributes, and retarding the activities of fruit softening enzymes such as pectin methylesterase (PME) and cellulase fruit up to 60 days in cold storage. Positive correlation was found among ascorbic acid content, total antioxidant activity, and total phenols and between PME and cellulase activity.

KEYWORDS:

• Postharvest
• map
• shelf-life
• pectin
• fruit softening enzymes
• mandarin

Introduction

‘Kinnow’ mandarin fruit is a bountiful source of bioactive compounds such as ascorbic acid, total carotenoids, hesperidins, naringin, hydrocinnamic acid, ferulic acid, and cyaniding glucoside (Sogi and Singh, 2001). These bioactive compounds boost the immune system against coronary-artery diseases, tumor, and diverse infections as well as abetting the absorption of Fe and Zn in humans (Kelebek et al., 2008). ‘Kinnow’ mandarin fruit is in huge demand in domestic as well as international markets due to its nutritional value, flavor, and enriching taste. ‘Kinnow’ mandarin is commercially grown in India and Pakistan as well as to a certain extent in California and Arizona (Khalid, 2013). India and Pakistan are prominent exporters of ‘Kinnow’ mandarin fruit to Russia, Iran, Sri Lanka, China, Thailand, and the middle-east (Dhatt and Mahajan, 2011; Khalid, 2013). Harvesting span of ‘Kinnow’ is very short and extends from December to February. Limited cold and chain, sporadic refrigeration facilities and a short harvesting period lead to an oversupply in the markets consequently contribute to 35–40 % postharvest losses and reduce profit to the growers (Mahajan et al., 2006). The extension of storage life of ‘Kinnow’ mandarin fruit offers fascinating panorama to elevate its export and minimize the glut in local markets as well as mitigate post-harvest losses.

Numerous approaches such as cold storage (Singh and Jain, 2004), post-harvest dip in sodium bicarbonate (Singla et al., 2018), salicylic acid (Haider et al., 2020) and coatings with carboxy methylcellulose, chitosan, and beeswax (Baswal et al., 2020a), dip application of methyl jasmonate or salicylic acid and fumigation of 1-methylcyclopropene (Baswal et al., 2020b) resulted in a modest success in extending cold storage life and maintaining quality of ‘Kinnow’ fruit. The nominal cost of MAP seems to be an ideal and attractive approach, particularly in the developing countries to extend the post-harvest life and maintain the quality of fresh horticultural produce (Mangaraj and Goswami, 2011). The effect of various polymeric films viz., MAP with and without perforation, cling film wrapping, shrink film, and vacuum packaging have been examined to extend the storage life of different types of fresh horticultural produce. MAP combined with cold storage reduces the rate of respiration of fresh horticultural produce in conjunction with restricted exchange of gases and moisture through a packaging material (Rai and Paul, 2007). Earlier, the combination of polymeric film packaging with low-temperature storage delayed mandarin (Citrus reticulata cv. Blanco) fruit softening (Bhattarai and Shah, 2017) accumulated higher aroma volatile compounds in raspberry (Rubus ideus L.) (Giuggioli et al., 2015) and maintained overall fruit quality in peach (Prunus persica (L.) (Mahajan et al., 2015). Recently, Singh et al. (2018) reported that ‘Kinnow’ fruit packed in shrink film, cling film, and low-density polyethylene (LDPE) film delayed the moisture loss, ripening, and senescence as well as maintained the fruit quality up to 25 days during cold storage. Miri et al. (2018) reported that combined influence of wax coating (180 mg 100 ml⁻¹ wax, 20 mg 100 ml⁻¹ imazalil, and 50 mg 100 ml⁻¹ thiabendazole) and polyethylene film (19 μ thickness) effectively reduced weight loss in cold-stored ‘Kinnow’ fruit up to 90 days. Mahajan and Singh (2014) also previously claimed that ‘Kinnow’ mandarin fruit wrapped in shrink film (15 μ) enhanced the storage life and maintained the overall quality up to 20 days under ambient super-market conditions (18–20^°C, 80–85 % RH). All these reports claim that the different types of polymeric films in combination with ambient or low temperature extend storage life and maintain quality in a range of fruit crops. Therefore, it is surmised that different types of MAP films may affect the levels of pectin, activities of fruit softening enzymes and various quality parameters during storage. However, no information is available on the effects of different types of polymeric packaging films on the dynamics of pectic substances, total carotenoids, total phenols, total antioxidant activity, and activities of fruit softening enzymes in cold-stored ‘Kinnow’ mandarin and warrants to be investigated. Therefore, the objective of the present investigation was to evaluate different types of polymeric packaging films on fruit weight loss, spoilage, firmness, level of pectin and activities of fruit softening enzymes such as pectin methylesterase (PME) and cellulase, SSC, TA, ascorbic acid content, total phenols content, total antioxidant activity, total carotenoids content, and sensory attributes of cold-stored ‘Kinnow’ mandarin fruit.

Materials and Methods

Fruit

‘Kinnow’ fruit were harvested at commercial maturity (12:1 sugar-acid ratio) in the third week of January from 15-year-old selected healthy trees at Regional Fruit Research Station, Abohar (30^°55′N, 54^°30′E). The fruits were randomly harvested around the tree canopy with the help of clipper by retaining a small button of the stalk. Following the harvest, the fruits were transported to the Postharvest laboratory of Department of Fruit Science, College of Horticulture, Punjab Agricultural University, Ludhiana. Uniform size and blemish-free fruit (250–300 g) were selected and washed with 0.01% chlorinated water (Sodium hypochlorite 4% @ 2.5 ml L⁻¹) and allowed to dry overnight to remove the excess surface moisture (Anonymous, 2018). The fruits were harvested from different trees but grown in the same block of orchard during two consecutive years of investigations.

Packaging and Storage Conditions

To conduct the experiment 12 fruits were packed in each MAP film. Miscellaneous MAP films were used viz., LDPE-film (25 μ), LDPE-film (38 μ), (PP)-film (25 μ), PP-film (38 μ), LDPE-film (25 μ) with pinholes, LDPE-film (38 μ) with pin holes, PP-film (25 μ) with pinholes and PP-film (38 μ) with pinholes (Sol Pack System, India). Unpacked fruits were treated as a control. To facilitate ventilation,16, 16, 18, and 13 pinholes of 0.5 mm diameter were made in each side of LDPE-film (25 μ), LDPE-film (38 μ), PP-film (25 μ), PP-film (38 μ). Packed and the control fruits were stored at 5–7^°C and 90–95% RH. Three packs each from LDPE-film (25 μ), LDPE-film (38 μ), PP-film (25 μ), PP-film (38 μ), LDPE-film (25 μ) with pinholes, LDPE-film (38 μ) with pinholes, PP-film (25 μ) with pinholes, PP-film (38 μ) with pinholes, respectively were taken out following 30, 45, 60 and 75 days of cold storage. Following each cold storage period, fruit weight loss, spoilage, juice content, firmness, levels of pectin and activities of PME and cellulase, soluble solids concentration (SSC), titratable acidity (TA), ascorbic acid, total phenols, total carotenoids, total antioxidant activity, and sensory evaluations were assessed. Whilst these quality parameters were also assessed from the freshly harvested fruit to represent zero-day of cold storage. The experiments during both consecutive years were laid out by following two factors (MAP treatments and cold storage periods) in factorial completely randomized design, included three replications. Each replication included 12 fruits.

Estimation of Fruit Weight Loss, Spoilage, Juice Contents and Firmness

The weight loss, spoilage, juice content, and firmness were determined by using the methods explained previously in detail by Baswal et al. (2020a, 2020b). Weight loss of both packed and unpacked fruit was determined at each storage interval. Spoilage loss was estimated on the basis of counting the number of spoiled fruit divided by the initial number of fruit multiplied by 100. The fruit juice was extracted with the help of screw-type extractor, weighed in grams, and estimated in percentage (W/W). The firmness of both packed and unpacked fruit was estimated with the help of a penetrometer (Model FT- 327, USA) using 8 mm stainless steel probe and expressed in terms of kg force (kg f) as explained previously (Baswal et al., 2020a).

Determination of Levels of Pectin and Activities of PME and Cellulase

Pectin content was estimated by following the method of Ruck (1961) with slight modifications as detailed previously by Baswal et al. (2020a) and expressed as mg Ca pectate 100 ml⁻¹ juice. The activity of PME was determined by estimating the increase in acidity after the hydrolysis of pectin by the enzyme with slight modifications as previously explained by Baswal et al. (2020a) and expressed in terms of mmol of pectin hydrolyzed min⁻¹ L⁻¹. Cellulase activity was estimated by using the method of Denison and Koehn (1977) with slight modifications described earlier by Baswal et al. (2020a) and expressed as g glucose min⁻¹ kg⁻¹ protein.

SSC, TA, Ascorbic Acid, Total Phenols, Total Antioxidant Activity, Total Carotenoids, and Sensory Evaluation

The SSC of the fruit juice was estimated using a hand refractometer (Atago Co., Tokyo, Japan) and expressed in percentage after making the temperature correction at 20 °C. The TA was determined as per standard procedure (AOAC, 2005) and expressed as percentage. Ascorbic acid content from the fresh fruit juice was determined by using Ranganna (1994) methods with slight modifications as previously described by Baswal et al. (2020a) and expressed as mg 100 ml⁻¹ juice. Total phenols content was determined by the following method described earlier by Bray and Thorpe (1954) with slight modifications as previously detailed by Baswal (2019) and expressed as mg 100 ml⁻¹ juice. Estimation of total antioxidant activity was done by adding 0.1 ml of ‘Kinnow’ juice extract in 3.9 ml aliquot of DPPH solution (0.0780 mM) made in 100 ml of 95 % methanol. The mixture was kept in dark for 30 min. The change in absorbance of the sample was recorded using spectrophotometer (Epoch Biotech, USA) at 517 nm wavelengths for 30 min against 95 % methanol as reagent blank (Brand-Williams et al., 1995). Total antioxidant activity was expressed using the following equation:

Total carotenoids content was estimated by following the method detailed by Gao and Wu (2005) with slight modifications explained recently by Baswal et al. (2020a) and expressed as mg 100 ml⁻¹ juice. Sensory attributes were evaluated by a panel of 10 trained judges based on taste, texture, flavor, and overall appearance of the fruit using a 9-point ‘Hedonic scale’ (Amerine et al., 1965). The panelists evaluated the differences between the samples where 0–6 displayed extremely unsuitable; 7 moderately suitable; 8 very much suitable and 9 extremely suitable for consumption as previously by Baswal et al. (2020a, 2020b).

Statistical Analysis

The pooled data obtained from two consecutive years of investigations (2016–17 and 2017–18) and subjected to two-way ANOVA using procedures of the Statistical Analysis System 9.3 (S.A.S. Institute Inc., Cary, NC, USA). Means were separated using the least significant difference (LSD) values at a significance level of P ≤ 0.05. The data from both years were pooled due to homogeneity of variance during both the years. Correlation among ascorbic acid content, total antioxidant activity, and total phenols and between PME and cellulase activities was performed taking into account all the sampling data.

Results

Weight Loss

Weight loss was significantly increased from 0.85 % to 2.75 % with extension in cold storage period from days 30 to 75. However, fruit packed in PP-film (25 μ) with pinholes resulted significantly lowest weight loss (0.47 %) as compared with the control (6.68 %) and all other MAP treatments. A significant interaction was detected between different MAP treatments and cold storage period for weight loss. The fruit packed in PP-film (25 μ) with pinholes exhibited significantly lower weight loss (0.28 %, 0.51 %, 0.53 %, and 1.03 %) as compared with the control fruit (4.36 %, 6.86 %, 9.49 %, and 12.66 %) and all other MAP treatments in 30, 45, 60, and 75 days cold-stored fruit respectively (Table 1).

Table 1. Physiological weight and spoilage loss of ‘Kinnow’ mandarin fruit influenced by different MAP treatment and cold storage periods during 2017 and 2018

Physiological weight loss (%)							Spoilage (%)
Storage time (Days)							Storage time (Days)
Treatment (T)	0	30	45	60	75	Mean (T)	0	30	45	60	75	Mean (T)
LDPE-film (25 μ)	0	0.45	0.74	0.95	1.57	0.74	0	8.33	12.50	33.33	83.33	27.50
LDPE-film (38 μ)	0	0.60	0.88	0.89	1.48	0.77	0	8.33	12.50	49.99	79.16	30.00
LDPE-film (25 μ) with pinholes	0	0.30	0.55	0.79	1.34	0.59	0	0	0	0	20.83	4.17
LDPE-film (38 μ) with pinholes	0	0.49	0.73	1.00	1.23	0.69	0	0	0	4.33	50.00	10.87
PP-film (25 μ)	0	0.25	0.91	1.91	2.45	1.10	0	16.66	20.83	33.33	91.66	32.50
PP-film (38 μ)	0	0.45	0.74	0.95	1.52	0.74	0	0	0	12.50	91.66	20.83
PP-film (25 μ) with pinholes	0	0.28	0.51	0.53	1.03	0.47	0	0	0	0	12.49	2.50
PP-film (38 μ) with pinholes	0	0.49	0.74	0.94	1.46	0.73	0	0	0	12.49	75.00	17.50
Control	0	4.36	6.86	9.49	12.66	6.68	0	0	0	20.83	33.33	10.83
Mean (SI)	0	0.85	1.41	1.94	2.75		0	3.70	5.09	18.53	59.72
LSD (p ≤ 0.05)	Treatment (T) = 0.078 Days (ST) = 0.058 ST x T = 0.17						Treatment (T) = 0.92 Days (ST) = 0.68 ST x T = 2.05

The data were transformed using square root transformation for weight and spoilage loss but only non-transformed values are given. Each value represents a pooled mean of 2 years data (2017 and 2018)

Spoilage

In spite of different MAP treatments, an elevation in spoilage (3.70 %–59.72 %) was noticed with extension in cold storage period from days 30 to 75. However, the fruit packed in PP-film (25 μ) with pinholes resulted significantly lowest mean spoilage loss (2.50 %) as compared with the control (10.83 %) and all other MAP treatments. A significant interaction was observed between different MAP treatments and storage period for spoilage loss. The fruit packed in PP-film (25 μ) with pinholes displayed significantly reduced spoilage loss (0 %, , and 12.49 %) as compared to PP-film (25 μ) without pinholes (20.83 %, 33.33 %, and 91.66 %) and all other MAP treatments in 45, 60, and 75 days cold-stored fruit respectively (Fig. 1, 2 and Table 1).

Figure 1. Fruit packed in PP-film (25 μ) with pinholes (A) 30 days after cold storage; (B) 45 days after cold storage; (C) 60 days after cold storage and (D) 75 days after cold storage

Figure 2. Fruit packed in PP-film (25 μ) (A) 30 days after cold storage; (B) 45 days after cold storage; (C) 60 days after cold storage and (D) 75 days after cold storage

Juice Content

The extension in cold storage period from days 30 to 75 resulted a significant decline in juice content (46.33 % to 40.90 %). Among all the MAP treatments, fruit packed in PP-film (25 μ) with pinholes recorded significantly highest mean juice content (47.10 %) as compared with the control (42.63 %) and all other MAP treatments. A significant interaction was observed between different MAP treatments and storage period for percent juice content. The fruit packed in PP-film (25 μ) with pinholes showed significantly higher juice content (45.51 % and 45.40 %) as compared with the control and all other MAP treatments during all the cold storage period except 30 and 45 days respectively (Table 2).

Table 2. Juice content and firmness of ‘Kinnow’ mandarin fruit influenced by different MAP treatment and cold storage periods during 2017 and 2018

Juice (%)							Firmness (kg force)
Storage time (Days)							Storage time (Days)
Treatment (T)	0	30	45	60	75	Mean (T)	0	30	45	60	75	Mean (T)
LDPE-film (25 μ)	49.00	45.19	44.71	42.96	41.52	44.67	6.19	5.19	4.84	4.19	3.25	4.73^e
LDPE-film (38 μ)	49.00	44.72	43.83	42.94	40.89	44.27	6.19	5.95	4.94	3.59	2.70	4.68^e
LDPE-film (25 μ) with pinholes	49.00	48.59	47.79	45.32	42.71	46.68	6.19	6.50	6.10	4.58	3.78	5.43^a
LDPE-film (38 μ) with pinholes	49.00	46.79	46.46	45.10	42.77	46.03	6.19	6.50	6.30	4.17	3.37	5.31^b
PP-film (25 μ)	49.00	44.75	41.93	41.46	38.05	43.04	6.19	4.83	4.57	4.49	2.80	4.58 ^f
PP-film (38 μ)	49.00	45.44	44.67	43.31	41.74	44.83	6.19	6.01	5.06	4.44	2.72	4.88^d
PP-film (25 μ) with pinholes	49.00	48.41	47.19	45.51	45.40	47.10	6.19	7.29	6.21	4.19	3.33	5.44^a
PP-film (38 μ) with pinholes	49.00	46.71	44.99	43.43	40.51	44.93	6.19	7.07	5.67	4.25	2.57	5.15 ^c
Control	49.00	46.37	45.02	38.26	34.48	42.63	6.19	4.54	3.93	3.39	3.01	4.21 ^g
Mean (SI)	49.00	46.33	45.18	43.14	40.90		6.19^a	5.99^b	5.29 ^c	4.14^d	3.06^e
LSD (p ≤ 0.05)	Treatment (T) = 0.65 Days (ST) = 0.48 ST x T = 1.45						Treatment (T) = 0.096 Days (ST) = 0.072 ST x T = 0.21

The data were transformed using square root transformation for juice content but only non-transformed values are given. Each value represents a pooled mean of 2 years data (2017 and 2018)

Firmness

A concomitant decline in firmness (5.99 kg f to 3.06 kg f) was noticed with extension in cold storage period from days 30 to 75. The fruit packed in PP-film (25 μ) with pinholes recorded significantly highest mean firmness (5.44 kg f) as compared with the control (4.21 kg f) and all other MAP treatments. There was a significant interaction observed between different MAP treatments and cold storage period for firmness. The fruit packed in PP-flim (25 μ) with pinholes resulted significantly higher firmness during all the cold storage periods (Table 2).

Pectin

The extension of cold storage period from days 30 to 75 manifested a significant decline in pectin content (0.51 mg Ca pectate 100 ml⁻¹ juice to 0.16 mg Ca pectate 100 ml⁻¹ juice). However, the fruit packed in PP-film (25 μ) with pinholes exhibited significantly highest mean pectin content (0.41 mg Ca pectate 100 ml⁻¹ juice) compared with the control (0.34 mg Ca pectate 100 ml⁻¹ juice) and all other MAP treatments. A significant interaction was noticed between different MAP treatments and cold storage period for pectin content. The fruit packed in PP-film (25 μ) with pinholes showed significantly higher content of pectin (0.36, 0.32, and 0.19 mg Ca pectate 100 ml⁻¹ juice) in 45, 60, and 75 days cold-stored fruit respectively as compared to all other MAP treatments and control fruit (Table 3). Whilst the fruit packed in LDPE-film (25 μ) with pinholes manifested significantly higher content of pectin (0.57 mg Ca pectate 100 ml⁻¹ juice) in 30 days cold-stored fruit as compared to all other MAP treatments and control (Table 3).

Table 3. Pectin content, PME, and cellulase activity of ‘Kinnow’ mandarin fruit influenced by different MAP treatments and cold storage periods during 2017 and 2018

Pectin (mg Ca pectate 100 ml^−1 juice)							Pectin methylesterase activity (mmol pectin hydrolyzed min^−1 L^−1)						Cellulase activity (g glucose min^−1 kg^−1 protein)
Storage time (Days)							Storage time (Days)						Storage time (Days)
Treatment (T)	0	30	45	60	75	Mean (T)	0	30	45	60	75	Mean (T)	0	30	45	60	75	Mean (T)
LDPE-film (25 μ)	0.65	0.49	0.35	0.21	0.16	0.37	0.59	2.63	3.73	2.45	1.81	2.24	1.26	3.43	3.93	3.24	2.57	2.89
LDPE-film (38 μ)	0.65	0.48	0.26	0.22	0.18	0.36	0.59	2.77	3.95	2.45	1.89	2.33	1.26	4.16	7.38	1.65	0.95	3.08
LDPE-film (25 μ) with pinholes	0.65	0.57	0.34	0.27	0.16	0.40	0.59	2.25	2.38	2.49	1.76	1.89	1.26	1.68	2.44	3.16	0.80	1.87
LDPE-film (38 μ) with pinholes	0.65	0.54	0.34	0.28	0.15	0.39	0.59	2.26	2.43	2.56	1.97	1.96	1.26	2.82	3.09	3.15	0.16	2.10
PP-film (25 μ)	0.65	0.46	0.30	0.23	0.12	0.35	0.59	2.91	3.52	2.70	2.18	2.38	1.26	3.01	5.89	4.92	1.20	3.26
PP-film (38 μ)	0.65	0.54	0.34	0.23	0.15	0.38	0.59	2.13	4.35	2.13	1.94	2.23	1.26	3.27	5.81	1.97	1.11	2.69
PP-film (25 μ) with pinholes	0.65	0.53	0.36	0.32	0.19	0.41	0.59	1.78	1.92	2.61	1.54	1.69	1.26	1.78	2.05	2.26	0.73	1.62
PP-film (38 μ) with pinholes	0.65	0.52	0.34	0.28	0.16	0.39	0.59	2.10	2.20	4.10	1.70	2.14	1.26	2.73	3.48	3.67	0.49	2.33
Control	0.65	0.44	0.25	0.21	0.13	0.34	0.59	3.50	3.98	2.24	1.71	2.41	1.26	6.81	8.30	1.63	0.35	3.67
Mean (SI)	0.65	0.51	0.32	0.25	0.16		0.59	2.48	3.16	2.64	1.83		1.26	3.30	4.71	2.85	0.93
LSD (p ≤ 0.05)	Treatment (T) = 0.00091 Days (ST) = 0.00068 SI x T = 0.020						Treatment (T) = 0.046 Days (ST) = 0.034 ST x T = 0.10						Treatment (T) = 0.064 Days (ST) = 0.048 ST x T = 0.14

Each value represents a pooled mean of 2 years data (2017 and 2018)

PME Activity

The extension of cold storage period from 30 to 75 days displayed a significant change in PME activity (2.48–1.83 mmol pectin hydrolyzed min⁻¹ L⁻¹). However, the fruit packed in PP-film (25 μ) with pinholes resulted significantly lowest mean PME activity (1.69 mmol pectin hydrolyzed min⁻¹ L⁻¹) as compared with the control (2.41 mmol pectin hydrolyzed min⁻¹ L⁻¹) and all other MAP treatments. There was an interaction observed between different MAP treatments and cold storage period for PME activity. The fruit packed in PP-film 25 μ with pinholes showed significantly reduced pectin methylesterase activity in 30 and 45 days cold-stored fruit as compared with the control and all other MAP treatments. However, the fruit packed in PP-film 25 μ with pinholes following 60 days cold storage showed significantly reduced activity of pectin methylesterase as compared to all other MAP treatments and control. Whilst the fruit packed in PP-film 25 μ with pinholes showed significantly reduced activity of pectin methylesterase in 75 days cold-stored fruit as compared with the control and all other MAP treatments (Table 3).

Cellulase Activity

Cellulase activity was significantly changed from 3.30 to 0.93 g glucose min⁻¹ kg⁻¹ protein with extension in cold storage period (days 30 to 75). The fruit packed in PP-film (25 μ) with pinholes resulted significantly lowest mean cellulase activity (1.62 g glucose min⁻¹ kg⁻¹ protein) as compared with the control (3.67 g glucose min⁻¹ kg⁻¹ protein). A significant interaction was detected between different MAP treatments and cold storage period for cellulase activity. The fruit packed in LDPE-film (25 μ) with pinholes preceding 30 days of cold storage registered lowest activity of cellulase as compared to all other MAP treatments and control. However, the fruit packed in PP-film (25 μ) with pinholes showed significantly reduced activity of cellulase in 45 days cold-stored fruit. Whilst the control fruit following 60 days of cold storage period exhibited significantly reduced activity of cellulase as compared to all the MAP treatments. The fruit packed in LDPE-film (38 μ) with pin holes recorded significantly reduced activity of cellulase in 75 days cold-stored fruit as compared to all the MAP treatments and control fruit (Table 3).

SSC

SSC exhibited an identical trend with a progress in cold storage period (days 30–75). A significant rise in SSC was observed up to 60 days of cold storage thereafter it declined significantly till end of the storage. However, fruit packed in PP-film (25 μ) with pinholes recorded significantly highest mean SSC (10.73 %) as compared with the control fruit (9.67 %) and all other MAP treatments. A significant interaction was found between different MAP treatments and cold storage period for SSC. The fruit packed in PP-film (25 μ) with pinholes showed significantly higher SSC (10.57 %, 11.10 % and 11.37% respectively) in 30, 45, and 75 days cold-stored fruit as compared to all other MAP treatments and control. While, the fruit packed in LDPE-film (25 μ) with pinholes following 60 days cold storage resulted significantly higher SSC (12.80 %) as compared to all other MAP treatments and control fruit (Table 4).

Table 4. Soluble solids concentration (SSC), titratable acidity (TA), and ascorbic acid content of ‘Kinnow’ mandarin fruit influenced by different MAP treatments and cold storage periods during 2017 and 2018

SSC (%)							TA (%)						Ascorbic acid (mg 100 ml^−1 juice)
Storage time (Days)							Storage time (Days)						Storage time (Days)
Treatment (T)	0	30	45	60	75	Mean (T)	0	30	45	60	75	Mean (T)	0	30	45	60	75	Mean (T)
LDPE-film (25 μ)	8.60	9.90	10.27	11.72	10.62	10.22	1.24	0.86	0.77	0.63	0.44	0.79	33.92	22.83	22.32	20.30	18.60	23.60^d
LDPE-film (38 μ)	8.60	9.20	10.40	10.77	10.67	9.93	1.24	0.84	0.68	0.62	0.50	0.78	33.92	22.55	21.81	20.13	18.39	23.36^de
LDPE-film (25 μ) with pinholes	8.60	10.27	10.67	12.80	10.82	10.63	1.24	0.96	0.89	0.76	0.39	0.85	33.92	24.71	24.09	20.47	19.76	24.59^ab
LDPE-film (38 μ) with pinholes	8.60	10.55	10.75	11.10	10.85	10.37	1.24	0.98	0.78	0.63	0.46	0.82	33.92	25.77	23.06	21.39	18.22	24.47^abc
PP-film (25 μ)	8.60	9.27	9.87	10.90	10.45	9.82	1.24	0.76	0.64	0.63	0.51	0.76	33.92	22.42	20.75	20.32	17.72	23.03^ef
PP-film (38 μ)	8.60	10.00	10.55	11.10	11.00	10.25	1.24	1.01	0.80	0.55	0.37	0.80	33.92	28.10	23.45	19.05	16.22	24.15 ^c
PP-film (25 μ) with pinholes	8.60	10.57	11.10	12.00	11.37	10.73	1.24	1.18	0.85	0.79	0.48	0.91	33.92	23.38	23.22	22.96	20.99	24.90^a
PP-film (38 μ) with pinholes	8.60	9.55	10.80	11.62	11.48	10.41	1.24	0.94	0.78	0.63	0.44	0.81	33.92	25.71	23.00	21.28	17.93	24.37^bc
Control	8.60	8.70	9.67	10.95	10.45	9.67	1.24	0.71	0.62	0.60	0.53	0.74	33.92	21.73	20.66	19.76	18.39	22.90 ^f
Mean (SI)	8.60	9.78	10.46	11.44	10.86		1.24	0.92	0.76	0.65	0.46		33.92^a	24.14^b	22.49 ^c	20.63^d	18.47^e
LSD (p ≤ 0.05)	Treatment (T) = 0.19 Days (ST) = 0.14 ST x T = 0.43						Treatment (T) = 0.017 Days (ST) = 0.013 ST x T = 0.038						Treatment (T) = 0.43 Days (ST) = 0.32 ST x T = 0.97

Each value represents a pooled mean of 2 years data (2017 and 2018)

TA

The extension in cold storage period from days 30 to 75 resulted a significant decline in TA (0.92 %–0.85 %). The fruit packed in PP-film (25 μ) with pinholes exhibited significantly highest mean TA (0.91 %) as compared with the control (0.74 %) and all other MAP treatments. A significant interaction was noticed between different MAP treatments and cold storage period for TA. The fruit packed in PP-film (25 μ) with pinholes following 30 and 60 days cold storage resulted significantly higher TA as compared to all other MAP treatments and control. Whilst the fruit packed in LDPE-film (25 μ) with pinholes following 45 days cold storage resulted significantly higher TA as compared to all other MAP treatments and control. However, the control fruit manifested significantly higher TA in 75 days cold-stored fruit as compared to all other MAP treatments (Table 4).

Ascorbic Acid

Ascorbic acid content significantly decreased (24.14 mg 100 ml⁻¹ juice to 0.184 mg 100 ml⁻¹ juice) with an extension in cold storage period from days 30 to 75. However, the fruit packed in PP-film (25 μ) with pinholes showed significantly highest mean ascorbic acid content (24.90 mg 100 ml⁻¹ juice) as compared with the control (22.90 mg 100 ml⁻¹ juice). There was a significant interaction noticed between different MAP treatments and cold storage period for ascorbic acid content. The fruit packed in PP-film (25 μ) with pinholes manifested significantly higher content of ascorbic acid in 60 and 75 days cold-stored fruit as compared to all other MAP treatments and control. Whilst, the fruit packed in LDPE-film (25 μ) with pinholes exhibited significantly higher of ascorbic acid content in 45 days cold-stored fruit as compared to all other MAP treatments and control. The fruit packed in PP-film (38 μ) following 30 days cold storage resulted significantly higher content of ascorbic acid as compared to all other MAP treatments and control (Table 4).

Total Phenols

Total phenols significantly declined (182.30 mg 100 ml⁻¹ juice to 30.77 mg 100 ml⁻¹ juice) with the extension in cold storage period from days 30 to 75. Among all the MAP treatments, the fruit packed in PP-film (25 μ) with pinholes recorded significantly highest mean total phenols content (126.39 mg 100 ml⁻¹ juice) contrasted to control (89.65 mg 100 ml⁻¹ juice). A significant interaction was found between different MAP treatments and cold storage period for total phenols content. The fruit packed in PP-film (25 μ) with pinholes exhibited significantly higher total phenols content in 45 and 60 days cold-stored fruit as compared to all other MAP treatments and control. While, the fruit packed in LDPE-film (25 μ) following 30 and 75 days cold storage resulted significantly higher content of total phenols as compared to all other MAP treatments and control (Table 5).

Table 5. Total phenols, total antioxidant activity, and total carotenoids content of ‘Kinnow’ mandarin fruit influenced by different MAP treatments and cold storage periods during 2017 and 2018

Total phenols (mg 100 ml^−1 juice)							Total antioxidant activity (%)						Total carotenoids (mg 100 ml^−1 juice)
Storage time (Days)							Storage time (Days)						Storage time (Days)
Treatment (T)	0	30	45	60	75	Mean (T)	0	30	45	60	75	Mean (T)	0	30	45	60	75	Mean (T)
LDPE-film (25 μ)	129.98	212.96	110.48	80.89	42.90	115.45^d	26.60	22.76	18.41	13.66	8.51	17.99^d	0.42	0.52	0.48	0.30	0.19	0.38
LDPE-film (38 μ)	129.98	165.32	153.80	90.77	20.56	112.09^e	26.60	23.52	18.58	12.13	8.22	17.81^de	0.42	0.57	0.36	0.26	0.24	0.37
LDPE-film (25 μ) with pinholes	129.98	185.05	180.41	89.04	25.08	121.91^b	26.60	25.03	18.51	14.80	11.03	19.20^b	0.42	0.64	0.61	0.31	0.23	0.45
LDPE-film (38 μ) with pinholes	129.98	196.03	146.56	89.75	38.25	120.12^bc	26.60	24.04	17.16	13.92	11.53	18.65 ^c	0.42	0.60	0.52	0.35	0.25	0.43
PP-film (25 μ)	129.98	158.77	94.72	71.56	18.68	94.74 ^f	26.60	23.37	17.51	12.07	8.63	17.64^e	0.42	0.53	0.35	0.27	0.26	0.37
PP-film (38 μ)	129.98	211.18	115.21	82.52	40.63	115.91^d	26.60	25.46	17.28	11.51	11.28	18.43 ^c	0.42	0.56	0.45	0.35	0.18	0.40
PP-film (25 μ) with pinholes	129.98	191.15	163.90	120.19	26.74	126.39^a	26.60	25.18	19.31	15.35	11.63	19.62^a	0.42	0.76	0.53	0.49	0.41	0.53
PP-film (38 μ) with pinholes	129.98	193.33	148.57	87.23	36.92	119.21 ^c	26.60	23.85	15.90	14.93	11.43	18.54 ^c	0.42	0.71	0.47	0.23	0.22	0.41
Control	129.98	126.93	93.94	70.27	27.11	89.65 ^g	26.60	19.59	13.81	9.90	6.50	15.28 ^f	0.42	0.59	0.35	0.23	0.15	0.35
Mean (SI)	129.98 ^c	182.30^a	134.18^b	86.91^d	30.77^e		26.60^a	23.65^b	17.39 ^c	13.14^d	9.86^e		0.42	0.61	0.46	0.31	0.24
LSD (p ≤ 0.05)	Treatment (T) = 2.53 Days (ST) = 1.88 ST x T = 5.66						Treatment (T) = 0.33 Days (ST) = 0.24 ST x T = 0.73						Treatment (T) = 0.00091 Days (ST) = 0.00068 ST x T = 0.020

Each value represents a pooled mean of 2 years data (2017 and 2018).

Total Antioxidant Activity

Total antioxidant activity significantly decreased (23.65 %–9.86 %) with prolongation in cold storage period from days 30 to 75. Fruit packed in PP-film (25 μ) with pinholes resulted significantly highest mean total antioxidant activity (19.62 %) as compared with the control (15.28 %) and all other MAP treatments. There was a significant interaction detected between different MAP treatments and cold storage period for total antioxidant activity. Following all the cold storage period (days 30 to 75) fruit packed in PP-film (25 μ) with pinholes maintained significantly higher total antioxidant activity as compared with the control and all other MAP treatments (Table 5).

Total Carotenoids

The total carotenoids content significantly declined (0.61 mg 100 ml⁻¹ juice to 0.24 mg 100 ml⁻¹ juice) with extension in cold storage period from days 30 to 75. However, the fruit packed in PP-film (25 μ) with pinholes exhibited significantly highest mean total carotenoids content (0.53 mg 100 ml⁻¹ juice) as compared with the control (0.35 mg 100 ml⁻¹ juice) and all other MAP treatments. There was a significant interaction observed between different MAP treatments and cold storage period for total carotenoids content. The fruit packed in PP-film (25 μ) with pinholes resulted significantly higher content of total carotenoids (0.76, 0.49, and 0.41 mg 100 ml⁻¹ juice) in 30, 60, and 75 days cold-stored fruit as compared with the control fruit and all other MAP treatments. Whilst the fruit packed in LDPE-film (25 μ) with pinholes preceding 45 days cold storage showed significantly higher content of total carotenoids (0.61 mg 100 ml⁻¹ juice) as compared to all other MAP treatments and control (Table 5).

Organoleptic Sensory Attributes (Hedonic Scale 1–9)

The extension in cold storage period from days 30 to 75 resulted a significant decline in the sensory attributes (8.38–4.0). Though the fruit packed in PP-film (25 μ) with pinholes recorded significantly highest mean sensory attributes (7.94) as compared with the LDPE-film (38 μ) without pinholes and control (6.07 and 6.65, respectively). A significant interaction was found between different MAP treatments and the cold storage period for the sensory attributes. The fruit packed in PP-film (25 μ) with pinholes exhibited significantly the higher score for sensory attributes as compared to all other MAP treatments and the control during all the cold storage periods except 75 days (Table 6).

Table 6. Organoleptic sensory attributes (Hedonic scale 1–9) of ‘Kinnow’ mandarin fruit influenced by different MAP treatments and cold storage periods during 2017 and 2018

Organoleptic sensory attributes (Hedonic scale 1–9)
Storage time (Days)
Treatment (T)	0	30	45	60	75	Mean (T)
LDPE-film (25 μ)	7.50	8.34	7.75	6.94	4.97	7.10
LDPE-film (38 μ)	7.50	8.32	7.70	6.83	0	6.07
LDPE-film (25 μ) with pinholes	7.50	8.57	8.25	7.95	6.68	7.79
LDPE-film (38 μ) with pinholes	7.50	8.72	8.07	7.74	6.04	7.62
PP-film (25 μ)	7.50	8.45	7.55	7.13	0	6.13
PP-film (38 μ)	7.50	8.40	7.61	7.04	0	6.11
PP-film (25 μ) with pinholes	7.50	8.72	8.50	8.35	6.63	7.94
PP-film (38 μ) with pinholes	7.50	8.65	7.87	7.17	5.85	7.41
Control	7.50	7.20	6.63	6.12	5.80	6.65
Mean (SI)	7.50	8.38	7.77	7.25	4.00
LSD (p ≤ 0.05)	Treatment (T) = 0.14 Days (ST) = 0.10 ST x T = 0.31

The data were transformed using square root transformation for sensory attributes but only non-transformed values are given. Each value represents a pooled mean of 2 years data (2017 and 2018)

Correlation Analysis

Ascorbic acid content, total antioxidant activity, total phenols, PME, and cellulase activity were found to be significantly correlated with each other. Total antioxidant activity was positively significantly correlated with ascorbic acid content and total phenols. While ascorbic acid content, total antioxidant activity, and total phenols were significantly but negatively correlated with PME and cellulase activities. However, PME activity was positively significantly correlated with cellulase activity (Table 7).

Table 7. Correlation among ascorbic acid content, total antioxidant activity, total phenols, PME, and cellulase activity of cold-stored ‘Kinnow’ fruit

	Ascorbic acid content	Total phenols	Total antioxidant activity	PME
Total phenols	0.916*
Total antioxidant activity	0.870*	0.904*
PME	−0.922*	−0.813*	−0.805*
Cellulase	−0.977*	−0.903*	−0.907*	0.963*

*Significant at p ≤ 0.05

Discussion

Elevated weight loss under different storage conditions may attributed to the increased rate of respiration and transpiration leading to moisture loss (Salisbury and Ross, 1992). The delayed weight loss in the fruit packed in PP-film (25 μ) with pinholes may be due to restricted respiratory process initiated by fruit inside the package (Ben Yehoshua, 1985). The increased relative humidity inside package around the fruit surface may also be ascribed to the reduced weight loss (Rai et al., 2011). Earlier, MAP in LDPE film and shrink film significantly delayed weight loss in ‘Kinnow’ mandarin and peaches (Mahajan et al., 2015; Mahajan and Singh, 2014; Mandal, 2015). The fruit packed in PP-film (25 μ) with pinholes delayed spoilage may be due to its lower water vapor transmission rate (WVTR) than other packaging films (Figure 1). While the fruit packed in non-ventilated packaging film exhibited significantly highest spoilage (Figure 2) even higher than that of control possibly due to accumulation of excessive moisture inside the package which consequently facilitates disease development on the fruit (Robertson, 1993). Previously, MAP in shrink film, HDPE film, LDPE film, and perforated PP film exhibited significantly reduced spoilage loss in ‘Kinnow’, peaches, and Japanese pear (Mahajan et al., 2015; Nath et al., 2012; Randhawa et al., 2009; Singh et al., 2018). The higher juice content in fruit packed in PP-film (25 μ) with pinholes possibly due to elevated relative humidity consequently reduced weight loss of the fruit (Bisen et al., 2012). Earlier, MAP in shrink film and HDPE film maintained significantly higher juice content in ‘Kinnow’ and sweet orange cv. Mosambi (Ladaniya, 2003; Ladaniya and Singh, 2001; Randhawa et al., 2009). Loss in firmness may ascribed to the breakdown of insoluble protopectins into soluble pectin as well as by hydrolysis of starch (Mattoo et al., 1975). Fruit packed in PP-film (25 μ) with pinholes exhibited significantly higher firmness may be due to a higher level of pectin and reduced activities of the fruit softening enzymes (Table 3). Previously, MAP in shrink film and PP film exhibited significantly highest firmness in ‘Kinnow’, peaches, and Japanese pear (Mahajan et al., 2015; Mahajan and Singh, 2014; Nath et al., 2012; Singh et al., 2018). Loss in pectin content may ascribed to higher activities of pectolytic enzymes on natural pectin content in fruit (Nara et al., 2001). The fruit packed with PP-film (25 μ) with pinholes resulted significantly highest pectin content possibly due to lower activities of fruit softening enzymes such as PME and cellulase (Table 3). Likewise, MAP in LDPE film, shrink film, and OPP film resulted significantly highest content of pectin in sapota, apple, and radish (Mohamed et al., 1996; Schreiner et al., 2003; Wijewardane and Guleria, 2013). Fruit softening is associated with higher activities of cell wall deteriorating enzymes (Marin-Rodriguez et al., 2002). The fruit packed with PP-film (25 μ) with pinholes significantly retarded the activities of fruit softening enzymes such as PME and cellulase may be due to build up of a higher concentration of CO₂ inside the bag as CO₂ has been reported to have an antagonistic effect on ethylene biosynthesis (Khan and Singh, 2008). Earlier, MAP resulted in reduced activities of fruit softening enzymes in cold-stored nectarine cv. Maria Aurelia and plum cv. Tegan Blue (Khan and Singh, 2008; Ozkayaa et al., 2016) .

The concurrent rise in SSC during cold storage attributed to degradation of starch into soluble sugars (Wills et al., 1980). The fruit packed with PP-film (25 μ) with pinholes delayed an initial increase in SSC may be due to retarded ripening and senescence processes (Mahajan et al., 2015). Earlier, MAP in shrink film recorded significantly highest SSC in ‘Kinnow’ and peaches (Mahajan et al., 2015; Mahajan and Singh, 2014; Singh et al., 2018). Polymeric packaging films delay ripening and senescence processes thereby maintain a higher level of acidity. The fruit packed in PP-film (25 μ) with pinholes maintained significantly highest TA possibly due to slow consumption of organic acid during mitochondrial respiration during ripening (Kaushal and Thakur, 1996). Likewise, MAP with shrink film exhibited significantly highest TA in ‘Kinnow’ and peaches (Mahajan et al., 2015; Mahajan and Singh, 2014; Singh et al., 2018). Loss in ascorbic acid content ascribed to the action of ascorbic acid oxidase catalyzes auto-oxidation of ascorbic acid to dehydroascorbic acid (Mapson, 1970). The fruit packed with PP-film (25 μ) with pinholes resulted significantly highest content of ascorbic acid possibly due to inhibition of auto-oxidation of ascorbic acid to dehydroascorbic acid during cold storage. Previously, MAP with shrink film and LDPE film exhibited significantly highest juice content in ‘Kinnow’ and papaya (Azene et al., 2014; Mahajan and Singh, 2014; Singh et al., 2018).

Post-harvest changes in phenolic compounds varies with the genetic potential and environmental factors of the crops (Harborne, 1998). The decline in total phenols during cold storage is ascribed to its breakdown due to stimulation of enzymatic activity (Fawole and Opara, 2013). The fruit packed with PP-film (25 μ) with pinholes manifested significantly highest content of total phenols may be due to delayed stimulation of the activity of enzymes involved in phenolic degradation during cold storage (Tomas-Barberan and Espin, 2001). Earlier, MAP with Xtend® film and shrink and cling films accumulated significantly highest content of total phenols in pomegranate cv. Bhagwa and guava (Kumar et al., 2017; Rana et al., 2015). However, contrarily, Mohebbi et al. (2015) reported that control (untreated) fruit exhibited significantly highest content of total phenol in cherry cv. Cornelia possibly due to faster post-harvest ripening inferred by elevated ethylene production, softening, and a rapid loss in TA. Fruit packed with PP-film (25 μ) with pinholes exhibited significantly highest total antioxidant activity possibly due to the reflection of higher content of total phenol (Khaliq et al., 2015). Earlier, MAP in PP film, Xtend® film and D-955 film maintained significantly highest total antioxidant activity in cherry, raspberry, and mango fruit (Khorshidi et al., 2011; Moor et al., 2014; Rao and Shivashankara, 2015).

Loss in carotenoids content may ascribed to its susceptibility to oxidation and geometric isomerization of its polyene chain (Sanchez-Moreno et al., 2003). The fruit packed in PP-film (25 μ) with pinholes exhibited significantly content of total carotenoids possibly due to packaging films resulted modified atmosphere around the fruit, in turn delay the respiration and senescence process simultaneously accumulate higher content of total carotenoids. Previously, MAP with shrink film resulted significantly highest content of total carotenoids in ‘Kinnow’ and mango (Mahajan and Singh, 2014; Rao and Shivashankara, 2015). The fruit packed in PP-film (25 μ) with pinholes resulted significantly highest score for sensory attributes may be due to delayed ripening and senescence process that led to the development of better flavor and aroma (Jawandha and Kirandeep, 2017). While the fruit packed with non-ventilated packaging films exhibited significantly lowest score for sensory attributes possibly due to initiation of anaerobic respiration inside package simultaneously develops off-flavors and volatile compounds (Robertson, 1993). Earlier, MAP with shrink film exhibited significantly the highest score for sensory attributes in ‘Kinnow’ and peaches (Mahajan et al., 2015; Mahajan and Singh, 2014). A positive correlation among ascorbic acid content, total antioxidant activity, and total phenols may be due to the fact that ascorbic acid is a powerful antioxidant in fruits and imparts the antioxidant potential of the juice (Reddy et al., 2010). Hence, the antioxidant activity in the sampled fruit cannot be ascribed solely to their phenolic content but also to the actions of other antioxidant compounds present in the fruits and their interactions. Previously, a positive correlation was also found between total phenols and other bioactive compounds in cold-stored plum and cherry fruit (Diaz-Mula et al., 2009; Serrano et al., 2009). A negative correlation of PME and cellulase activities with ascorbic acid content, total phenols, and total antioxidant activity possibly due to the fact that both PME and cellulase are the fruit softening enzymes and deteriorate the fruit quality during storage (Fisher and Bennett, 1991). Recently, Singh et al. (2020) also observed a positive correlation between PME and cellulase activity in pear cv. Punjab Beauty fruit.

Conclusion

MAP using PP film (25 μ) with pinholes significantly reduced weight loss, spoilage loss, firmness loss retarded the activities of fruit softening enzymes and maintained significantly highest content of juice, ascorbic acid, total phenols, total antioxidant activity, total carotenoids, and sensory attributes in cold-stored ‘Kinnow’ fruit up to 60 days.

Acknowledgments

The financial aid from the University Grants Commission, Ministry of Human Resources and Development, India to A K Baswal is gratefully acknowledged.