Bioactive chitosan based coatings: functional applications in shelf life extension of Alphonso mango – a sweet story

Introduction

Chitosan, a derivative of chitin which belongs to a family of polysaccharides obtained from crustacean wastes and consists of β (1–4) – linked-2-acetamide-2-deoxy-d-glucose units. It is a natural, biodegradable, biocompatible polysaccharide which can form transparent films [1]. Plasticity of chitosan and other barrier characteristics can be improved by blending either physically or chemically with desirable polymers/additives [2]. Packaging/coating is presently important in post-harvest preservation of fruits, vegetables, and processed foods for assured shelf life extension. The raw materials of food, as well as the innumerable number of processed food products made available in the market are highly perishable and need effective and efficient packaging systems for extending their shelf life and availability. Chitosan may be used for the development of an environment-friendly alternative for creating functional polymers with various applications including food packaging [3]. Fruits and vegetables are surface-coated with a thin layer of edible polymer film to inhibit transpiration and minimize adverse changes, otherwise lower their market value and taste properties [4]. Edible coatings comprise mainly natural substances, including wax, polysaccharides and proteins [5]. Chitosan has been proven one of the best edible and biologically safe preservative coatings for different types of foods because of its antimicrobial actions, lack of toxicity and biochemical properties. It has been proven that the chitosan can control numerous pre- and postharvest diseases of fresh fruits [6]. Previous studies from our lab regarding chitosan edible coatings has indicated, the extended shelf life of the fruits and vegetables by minimizing the rate of respiration and reducing the water loss [7].

According to FAOSTAT, it was estimated that annually more than 18 million tons (year 2013–1014) of mango will be produced in India and tops in the world [8]. Alphonso mango is known as the “King of mango” because of its delicious tropically fruity sweet taste and juicy saffron/golden colored non-fibrous tender texture of its flesh [9].

Fully ripened Alphonso mangoes also release an aromatic fruity fragrance in the air. These qualities have been attributed to a unique combination of geographical conditions which are mainly present in Western India, especially near coastlines of Maharashtra. As ground and weather profiles required to cultivate this fruit are scarce, Alphonso mangoes are produced in a very limited quantity each year. These mangoes are harvested only once every year in the months of April and May and most of the stock is sold and consumed locally within India. The rest is exported to countries like Middle east, USA, UK, Australia with large populations of Indian diaspora. Alphonso Mango is highly perishable hence shelf life is very limited, which leads to huge post-harvest loss every year. The current study was conducted to investigate the shelf life extension of mangoes using developed chitosan based formulation on quality parameters (biochemical and organoleptic). Since chitosan is known eliciting (defense mechanism) bio-polymer, upon coating it can interacts with fruit to modify the physiology of mango apart from protection through barrier.

Materials and methods

Methods

Alphonso mango fruits were harvested at physiologically matured stage. Fruits were desapped, air-dried, tray packed and then brought carefully to the Lab. Fruits were sorted for uniformity (size, color and weight). Then the mangoes were further washed with clean water and treated with fungicide (Benamyl, 500 ppm, optional depending upon orchard maintenance) for 5 min and kept at room temperature. Dried mangoes were dipped into the chitosan based coating formulation for about 2 min and fruits were individually air dried completely (as shown in Fig. 1). They were further stored at room temperature (30 ± 3°C) and 40–50% RH for 2 weeks (15 days). Mangoes were analyzed for different parameters at regular intervals for 2 weeks and the results were compared with control (untreated) fruits.

Fig. 1:

Flow chart for chitosan based formulation coating process. *Transferred technology.

Results and discussion

Initial experiments, in the process of developing chitosan based formulation for extending Alphonso mango had yielded very significant results with respect to extension of shelf life. Visually, the coated mangoes were green, hard and fresh even after 14 days, whereas the control fruits got fully ripened between 7 and 8 days’ time. But when we examined the inside of the coated fruits on 14^th day developed with off flavor and was still unripe [even Ethrel dip (ethylene induction) did not ripen the fruits], which was further confirmed with gas chromatography (detail not given) resulted in anaerobic fermentation (ethanol production) (Fig. 2). This may be mainly because of chitosan being a good barrier for oxygen had completely halted the respiration process of fruits. A number of chemical and biochemical processes that take place during the storage of fruits also contribute to the deterioration of their overall quality.

Fig. 2:

Initial experiments showing unsuccessful coating formulations (M1F1 and M1F2) for Alphonso mangoes resulted in extended shelf life but, lead to anaerobic fermentation of fruits due to very slow rate of respiration.

After several modifications in terms of concentration, viscosity and incorporation of food additives to improve formulation’s barrier properties and studies over the period of time, resulted in developing M4F2 formulation which gave better results in extending the shelf life of Alphonso mango. The rheological (Fig. 3) parameter showed sort of Bingham fluidic behavior for M4F2 unlike chitosan solution. Complete characterization for coatings is practically difficult when compare to thin casted dried films with respect to mechanical and barrier properties, hence was not attempted.

Fig. 3:

Rheological comparison (shear stress vs. shear rate) between chitosan solution and developed formulation (M4F2) at different concentration. The chitosan solution showed typical shear thinning non-Newtonian fluids whereas M4F2 showed Bingham plastic behavior.

Microbial studies for developed formulation

The data regarding contamination or microbial growth incidence for the formulation was recorded for 30 days storage period by conducting microbial experiments using PDA and EMB agar plate methods at room temperature. There were no microbial growth was found in formulation solution kept for 21 days for both bacterial and fungal growth as shown in Fig. 4. Chitosan is known for its number of health benefits as a coating or packaging materials as it is an effective antioxidant [12], capable of retaining vitamin C in fruit and vegetables, and it has bacteriostatic and bactericidal properties [13]. Hence, chitosan is a highly recommended polymer for the production of edible film coatings [6, 14].

Fig. 4:

Microbiological analysis for chitosan based formulation (M4F2).

Mango storage studies

Physiological loss in weight

Physiological weight loss (%) were observed for both control sample and coated samples, but coated samples were observed with lesser rate of weight loss between from day 4 to day 10 at room temperature stored mangoes. But a significant change was not observed in the overall storage period (Fig. 5).

Fig. 5:

Physiological loss in weight of both control and coated fruits till 12^th day of storage studies.

Along with PLW, managing temperature is the most critical factor in the process of ripening for mature-green mangoes. For mangoes when held at 27–30°C during ripening, the skin of the fruit becomes better colored and the fruit acquire a good flavor. Ripening is retarded when mangoes are held above 30°C and or subject to chilling injury if held below 13°C, as per our observation (data not shown) and as indicated by others [15]. Sometimes, coatings may help in withstanding sudden changes in temperatures during storage at room temperatures, but still lot of scientific evidence will be required for this statement.

Half-matured fruit and fruits from early season harvests underwent limited ripening and were less acceptable. For maximizing storage potential, suggestions were made by controlling harvest maturity and storage temperature for progressive harvests throughout the season [16].

Chemical characteristics

Total soluble solid (°Brix) content

Significant variation was observed in TSS content between control and coated fruits during storage. The results exhibited that TSS content of mango pulp developed in a continuous stream with the expansion of storage period. The developing trend gradually increased from initial (day 0) to 14^th day from ~10°Brix to ~20°Brix (Table 1).

Table 1:

Changes with respect to Alphonso mango storage parameters; brix, acidity and carotenoid content during 7 days interval between control and coated fruits.

Days/parameters	0 Day	7th Day		14th Day
	RT control/coated	RT control	RT coated	RT coated
TSS (°Brix)	9.11	17.79	12.01	19.79
Acidity (%)	2.58	1.39	1.17	0.35
Carotenoids (mg/100 g)	2.22	5.54	3.82	8.02

Average values of three replicates from five fruits.

But in control fruits, the total brix was achieved at maximum 18°Brix till 7–8 days of storage whereas coated fruits still showed around 12°Brix at the same time (7^th day). After 10 days of storage control fruits were not available for comparative study since fruits were over ripened and soft. The coated fruits showed slower or delayed process of ripening and achieved maximum brix of ~20°Brix at 14^th day of storage period.

Titratable acidity

Titratable acidity was perceived to be significant at different days after storage. At various days of storage, the control fruits kept at room temperature showed higher titratable acidity as compared to the coated fruits. Titratable acidity reduced with the advancement of storage period.

In all the storage period, lower titratable acidity (1.39%) was derived from control fruits at 7^th day, interestingly still lower values for the mangoes which were coated (averaged, 1.17%) and achieved lowest (0.35%) on 14^th day (Table 1). This phenomena happening might be possible due to coating (treatment) resulted in triggered biochemical postharvest changes and delayed ripening that caused higher diminishing trend of titratable acidity, while control fruits with normal ripening resulting in lower decreasing trend of titrable acid content.

Carotenoids

Carotenoids are synthesized in mango fruit during ripening. The edible part (pulp) turns from pale yellow to yellow and deep yellow to orange-yellow during ripening due to carotenoid content development. Carotenoid content in control and coated fruits showed significant changes at different storage intervals. The results exhibited that carotenoid content of mango pulp increased gradually from initial (2.22 mg/100 g) to 14^th day (8.02 mg/100 g) in coated fruits (Table 1).

Sensory and HPLC analysis

Panelists rated samples individually under sensory evaluation. Samples were served one at a time in random order. Using scoresheets provided, panelists were requested to rate samples relative to ratings of standards. Based on ratings, panelists then rated two mango samples, labeled Sample A and Sample B, respectively. During evaluation, panelists noted that flavor properties were better and also they experienced interestingly more sweetness for coated samples (15–16^th day) compared to control (9^th day). The influence of sugars and acids on the flavor notes intensity, especially the aromatics and chemical feeling factors, including sweetness is not new but noteworthy [17].

To study the sugar profile, especially sucrose, fructose and glucose, the pooled pulp (from five fruits) for each (control and coated) was subjected to HPLC for determination. Sugars, corresponding to the different peak retention time in the chromatograms were assessed. Only fructose and sucrose were observed in control samples whereas the presence of glucose along with sucrose and fructose was observed in certain ratio for coated samples (Fig. 6). Sweetness is the major determinant of quality and marketability of fruits. In most of the fruits, glucose and fructose form the major proportion of soluble sugars [18]. Even though, the fructose is couple of times sweeter to glucose, in our study increased glucose to fructose ratio may improve the overall fruit sweetness and flavor along with the sucrose in certain critical ratio. Sweetness of fruits depends mainly on the type and composition of sugars present, which is primarily a genotype dependent. Sugar content is also dependent on total solids, pH, titratable acidity and fruit size [19]. In addition, sugar content varies with plant nutrition, climate, soil and also fruits storage conditions [20]. Chitosan is well known eliciting compound to act as primary initiation molecule for signal transduction and play a key role in the regulation of both physiological and metabolic events in plants [6].

Fig. 6:

HPLC sugar profile for control and coated mangoes determined. Inset: standard fructose, glucose and sucrose HPLC chromatogram.

Further, the mangoes peel (exocarp) structure for control and coated fruits of equal maturity were directly studied using Olympus microscope with attached camera and computer. Surface structural features were photographed in particular magnification. Based on close observation, the coated and completely dried mangoes showed the red specked epidermal surface (oil filled pits) compared to control fruits. But, if the surface of the mango was exposed for longer duration with coating formulation before drying, this red speck intensifies as shown in the Fig. 7. This is just a preliminary observation made regarding possible elicitation of the formulation in bringing about internal biochemical changes of the fruits in the process of ripening in addition to partial barrier properties to slow down respiration. Detailed future understanding of molecular action of developed formulation and the additive factors controlling sweetness and the shelf life is important to design strategies for enhancing quality of fruits.

Fig. 7:

Picture showing drying (separately) of mangoes after coating and microscopic surface peel topology (exocarp) of control and coated fruits.

Further, this study was extended for scale-up experiments (> 500 kgs of Alphonso mango) by procuring from Devgad, Maharashtra region showed the average shelf life of 17 days for coated mangoes at room temperature (28±2°C) for M4F2 formulation. Since, few incidence of stem end rot disease (for both control and coated) and also persistence of green peel color for ripened coated mangoes was observed, future work to address the same is in progress.

Conclusions

Shelf life studies by applying chitosan based formulation (M4F2) for Alphonso mango revealed that the coated mangoes, loss of weight gradually increased with the storage time and was linear for both coated and control fruits. The M4F2 thus prepared may be stored for ~30 days at room temperature (30 ± 2°C) without microbial spoilage and without changing its rheological properties. M4F2 enhanced the TSS and carotenoid content progressively, and decreased the titratable acidity drastically compared to control fruits. Coating application also decreased the incidence of microbial spoilage of fruits and also the anthracnose incidence. The data revealed that, applying a chitosan based coating effectively prolongs the quality and improves the sensory attributes with the extension of the shelf life of mango at room temperature. In addition to these changes, it was also observed that standardized chitosan-based coating M4F2 also enhanced the sweetness in coated fruits compared to control. HPLC studies revealed that the presence of glucose along with sucrose and fructose in critical ratio enhanced the sweetness in the coated mango samples compared to control mangoes. But this study does not minimize the significance of volatiles and additional flavor compounds on perception of mango flavor, but does establish sugars and acids as key taste compounds in mango.