Enhancement of Shelf Life of Guava Fruits by Application of Chitosan based Nanoemulsion

BSTRACT Background: Edible coating is an effective and widely used approach for preservation of quality of fruits. The physiological changes remain intact with edible coating that leads to maintain the nutritional quality. The current study is an endeavor to develop an appropriate edible coating material that may be applied over fruits as freshness indicators. Methods: Chitosan nanoemulsion was prepared with ultrasonication and guava samples were dipped in prepared nanoemulsion. Quality evaluation of guava samples was evaluated during 15 day storage in triplicates. Result: Guava fruits were coated with chitosan based emulsion to preserve its nutritional qualities. Weight loss, TSS, acidity (%), ascorbic acid and 2,2 diphenyl-1-picrylhydrazyl (DPPH) inhibition activity were measured in coated and control guava samples. Samples coated with guava were found to preserve overall quality. TSS and acidity content, which is ripening indicator, was found to be not affected significantly in coated sample whereas in control samples TSS increased and acidity decreased during storage. The changes in weight loss were significantly more in control samples than coated samples. Ascorbic acid and antioxidant activity variation was observed more in control samples.


INTRODUCTION
Guava (Psidium guajava L.) belongs to family Myrtaceae and considered as the commercial fruit crops of India.The guava fruit has major portion of edible part and represents excellent source of vitamin C and rich source of minerals like calcium, phosphorus, iron as well as the source of vitamin A, pantothenic acid, riboflavin, thiamin and niacin.Further, the polyphenols and carotenoids content provide its significantly high nutritional value (Thaipong et al., 2006).However, the fruits tend to rapid perish ability due to high respiration rate and consequently high metabolic activities being a climacteric nature that ripens after harvesting (Singh and Pal, 2008).It is susceptible to chilling injury when stored below 10C, therefore require constant attention to prevent spoilage loss during transportation and marketing (Reyes and Paull, 1995).
Edible coatings have been conceptualized widely now days to enhance the shelf life of whole as well as fresh-cut fruits remarkably.In fact, the physiological processes such as respiration, degradation of cell wall, transpiration and deterioration by microbial action improves by the appropriate coating method, thereby maintaining the quality of the fruit and vegetable (Ali et al., 2011).They could be good alternative in place of synthetic preservatives.Moreover, edible coatings can be used as antimicrobial agents which helps in preventing the microbial spoilage and consequently extending the shelf life of the fruits (Raybaudi-Massilia et al., 2008).Generally, the materials polysaccharides, proteins and lipids for fabrication of edible coating can be used to extend the shelf life of various horticultural commodities (Chen et al., 2016;Gardesh et al., 2016).Pectin was used successfully as coating material to preserve colour and quality of dehydrated pineapple during storage (Saini and Sharma, 2016).Carboxy methyl cellulose was found suitable for edible coating due to its film solubity and elongation ability (Dipanwita and Kandeepan, 2017).W hey protein is extensively used for edible coating to enhance the shelf life of foods (Ranganathan et al., 2019).
Among all these coating materials chitosan (polysaccharide) based edible coatings are gaining popularity in recent times.Chitosan is obtained by N-deacetylation of chitin in an alkaline environment (Butola 2019).Chitosan is a non-toxic, non-allergenic, biodegradable, biocompatible and film-forming material with beneficial biological effects (antifungal, antibacterial, antitumour) (Chaudhary et al., 2020).Chitosan films are ecofriendly, biodegradable and having good mechanical properties that selectively allow gas permeability for CO 2 and O 2 .The respiration rate of fruits decreases as the edible chitosan coating applied to the surface (Haaji et al., 2018).However, chitosan coating Enhancement of Shelf Life of Guava Fruits by Application of Chitosan based Nanoemulsion possesses high water vapour permeability thereby its application gets restricted in humid environment (Candir et al., 2018).Water vapour permeability signifies the organoleptic characteristics of stored fruit for its ability to fight against dehydration or rehydration of the film; Chitosan as an edible coating has low water solubility therefore acquires rigid crystal structure and not used directly.Chitosan and apple peel extract were used as edible coating for preserving nutritional qualities of strawberries.Chitosan (1, 2, or 3%) were used as edible coating for mangoes to maintain firmness, reduce water loss by preventing starch degradation (Silva et al., 2017).In the present study, chitosan based coating was applied on freshly cut guava fruit samples and various observation were made as an indicator for its freshness during fifteen days storage time.

MATERIALS AND METHODS
Guava fruits of varieties (Allahabadi Surkha) were purchased from local market, Dehradun.The fruits were selected on the basis of their uniformity in size, color and absence of mechanical injury.Tween80, glycerol and chitosan were purchased from Himedia, Mumbai, India.All other chemicals used in the study were of analytical grade.The study was conducted at Department of Food Technology, Uttaranchal University.

Preparation of chitosan nanoemulsion for edible coating
Chitosan (1% w/v) was dissolved in glacial acetic acid (1% v/v) and stirring was done with blender at 800 rpm for 5 min.The pH was kept to 5.2.Glycerol (0.75%) was added as plasticizer followed by addition of few drops of Tween 80 (1 g/100 mL chitosan solution) into the emulsion.Homogenization was done further at 5000 rpm for 10 minutes.The solution was then ultrasonicated at frequency 40 kHz using sonicator (Labman, LMUE-6, India).

Particle size of nanoemulsion
The particle size and zeta-potential value of chitosan emulsions was estimated by using particle size analyzer (Zetasizer Nano-ZS 90, Malvern Instruments, UK).Samples were diluted (1:100) with distilled water to avoid multiple scattering.

Weight loss
Five guava fruits cut samples were used for weight loss (%) determination.Weight loss was calculated by weighing fruits at the 0 th day and after fifteen days storage duration.Results were indicated as a percent weight loss.

Titratable acidity and total soluble solids (TSS)
10 g guava cut samples was mixed properly with help of blender with 50 ml distilled water and then after properly filtered.(%) Titratable acidity was determined by titration of obtained filtrate sample with standard solution (0.1 M) of NaOH using phenolphthalein as an indicator.Titratable acidity was expressed as % citric acid.
Total soluble solids expressed as  Brix was assayed using portable refractometer (Erma, India).

Ascorbic acid
Ascorbic acid (mg ascorbic acid per 100 g fruit pulp) content in coated guava samples was estimated by procedure followed by AOAC (1990).Ten gram of guava sample was extracted with 90 mL of 3% metaphosphoric acid.Centrifugation of samples was carried out at 5000×g for 15 min in properly mixed samples.The supernatant (10 mL) was collected for titration against 2, 6 dichlorophenol indophenol dye till pink rose color persists for about 20 s.

Antioxidant activity
Antioxidant activity of guava samples was analyzed by DPPH radical scavenging method (Wani et al., 2018).Guava fruit extract (0.1 mL) was added with 3.9 mL DPPH.The reaction mixture containing sample with DPPH was kept for about 30 min. in dark and absorbance was taken at 517 nm using UV-Spectrophotometer (Systronic-AU2701).The results were expressed as (% inhibition) by the equation given below:

RESULTS AND DISCUSSION
Total soluble solids and titratable acidity (%) TSS and TA (%) of freshly cut samples coated with chitosan samples are shown in Table 1.These parameters are noteworthy for predicting for organoleptic quality of fruit as it helps in determination of soluble sugar and organic acid contents of fruits.The TSS of control fruit samples was observed to increase from 11.06±0.004 Brix to 13.33±0.004 Brix during storage (Fig 1).This increase could be featured due to the less respiration rate, deconstructed cell wall and the increase in dry matter due to water loss.Guava categorizes as climacteric fruit and having tendency to show rise in total soluble solids (TSS) content during storage (Singh and Pal, 2008).However, guava samples coated with chitosan were found to observe the gradual decrease in TSS (11.10±0.007 Brix to 10.45±0.004 Brix) during 15 day storage kept at 4  C (Fig 1 ) which was due to slow rate of metabolic activity by presence of functional edible coating (Radi et al., 2018).
The TA (%) of the uncoated fruits decreased from 0.92% to 0.70%, while in coated fruits this decrease was comparatively lower up to 0.88% at 15 day of storage.The titratable acidity (TA) was reported to decrease as well by earlier studies over storage since organic acids gets utilized during respiration process (Gol et al., 2015).The decrease in acidity was observed in uncoated fruits as the organic acids utilizes in form of substrates for respiratory metabolism as well as energy liberation during storage (Dubey et al., 2019).Earlier studies of Lim et al. (2011) also reported a decrease in titratable acidity of sweet cherries fruits.

Weight loss
The weight loss during storage in treated samples is presented in Fig 2 .In control guava fruit sample the weight loss was found to be 47% after 15 days of storage.However, coated sample had observed less weight loss i.e. 8.6% during storage.The less weight loss in chitosan nanoemulsion coated sample was due to barrier properties provided by chitosan network.Wang and Rhim, (2016) observed the same pattern of weight loss in apricots coated with chitosan biopolymer in combination with polyphenolic extract.Chen et al., (2016) and Synowiec et al., (2014)

Ascorbic acid degradation
Ascorbic acid significantly found in fruits and provides various health activities including immune system, antioxidative characteristics, healthy skins and gums.
The decrease in ascorbic acid in guava samples is shown in Table 2.The decrease in ascorbic acid content was found significantly from its original value 300 mg/100g to 100 mg/ 100 for uncoated samples during 15 day storage.However, the ascorbic acid decreased to 280 mg/100 g from its original value.The decrease in ascorbic acid was mainly due to presence of ascorbic oxidase that causes oxidative deterioration of ascorbic acid.Chitosan had ability to be effective in controlling the losses of ascorbic acid for coating on fruits such as guava (Hong et al., 2012) and papaya (Ali et al., 2011).

DPPH antioxidant activity
The antioxidant activity decreased to 30% from its original activity of 70% in control sample whereas in coated sample the activity decreased only to 62%.In control sample the phenolic compounds generally degrades due to senescence and high rate of respiration (Ghasemnezhad et al., 2010).In our study the antioxidant activity was found more in all  Enhancement of Shelf Life of Guava Fruits by Application of Chitosan based Nanoemulsion coated fruits than control Samples.In fact, the coating of fruits modifies the internal atmosphere thereby reducing the risk of oxidative destruction of antioxidant compounds.Wang and Gao (2013) reported higher levels of total phenolic content and antioxidant activity in chitosan coated strawberries during storage.Chiabrando and Giacalone, (2015) observed similar observation in case of Chitosan coated blueberries.

Particle size of chitosan nanoemulsion
The particle size and zeta potential of chitosan nanoemulsion were found to be 202 nm (Fig 3 ) and -23 mV.The particle size ranges between 78-342 nm.The zeta potential between -30 to +30 mV is considered for stable nanoemulsion (Heurtault, 2003).Zeta potential plays decisive role in providing stability of nanoemulsion.In general, sufficient electrical charge requires to prevent aggregation of droplets by producing the repulsive force between droplet.Ghaderi-Ghahfarokhi et al. (2017) obtained the particle size of 235.6 nm produced by chitosan emulsion containing cinnamon essential oil using an ultrasonic water bath for 1 hr.

CONCLUSION
Chitosan based nanoemulsion have small size and large surface thereby having ability to provide antimicrobial and barrier properties against oxygen and moisture thus helps in preventing from deteriorative action in guava fruits.The present study confirmed that the use of chitosan nanoemulsion could augment the quality of the guava fruit.Moreover, degradation of ascorbic acid and antioxidant activity was found to preserve with application of edible coating.Edible coating could be an alternative approach in food industries to reduce post harvest quality losses in fruits.

%
also demonstrated reduction in weight loss in orange and apple fruit by incorporation of fig fruit and sweet basil seed extract into coating respectively.

Table 2 :
Ascorbic acid and DPPH inhibition activity (%) content in guava samples during storage.