Influence of Chitosan-Based Edible Coating on the Shelf Life and Nutritional Quality of Guava (Psidium guajava L.) Fruit in Room and Refrigerated Temperatures

1 Plant Protection Division, Nuclear Institute for Agriculture and Biology, Jang Road Faisalabad, Pakistan 2 Department of Applied Chemistry, Government College University Faisalabad, Faisalabad 38000, Pakistan 3 Natural Medicines and Products Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 4 Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 5 Department of Biochemistry, Government College University Faisalabad, Faisalabad 38000, Pakistan*


Introduction
Guava (Psidium guajava L.) is an important fruit, and it belongs to the family of Myrtaceae. Guava fruit has great importance in Latin American countries like Colombia, Brazil, Mexico, Africa, and Asia [1-2]. It is cultivated on 62,300 with a production of 512,300 tons in Pakistan [3], and is ranked 4th in terms of total area production after citrus, mango, and apples [4]. Guava is composed entirely of minerals, vitamins. It contains 82% water, 0.7% protein, and 11% carbohydrates. It includes 2-6 % more vitamin C than citrus, 10-30% more than banana, and 10 times more than papaya. About 93% portion of guava fruit is edible [5]. Due to the high respiration rate, harvested guava attained a fast ripening stage and subsequently decayed incidence [6][7][8][9][10].
A passive barrier that delays the environmental effects on food products is known as adequate food packaging. Researchers are currently developing such a packaging material that positively impacts the environment and food, but it would also have good preservation properties [11]. The packaging with antimicrobial or antioxidant properties is proved to play an essential role in the stability of fresh food but extends its shelf life too [12]. The edible coating based on chitosan is derived from natural sources by deacetylation of chitin. Studies have shown that it is harmless to humans, wildlife, pets, and the environment.
Furthermore, it is effective for inhibiting the decay of fruits and extending shelf life [13][14]. Previous studies have documented that chitosan and its derivatives-based coatings are efficient in inhibiting a wide range of fungi (Aider, 2010;No et al., 2007). The pathogen's damage caused in fruits and plants is minimized as chitosan-based coating triggers the defensive mechanisms. The studies focused on postharvest properties have documented that chitosan is active in inhibiting fungal growth in strawberries [15][16]. The coating on strawberries has reported its low levels do not change the fruit's astringency, and its coating does not alter consumer acceptance of strawberries in storage [17]. In another research Devlieghere et al. [18] have observed that chitosan-based coating not only reduced the propagation of harmful germs and pathogens, but it effectively affected the decay of fruits during storage.
In our previous study [4], the use of aloe vera (AV) based coating has proved effective in maintaining nutrition and extends the shelf life of guava fruits. In the current study, chitosan with different levels (0.5, 1.0, 1.5, 2.0%) was applied on guava stored for 12 days at room and refrigerated temperatures. The results would be useful for further development and edible coating materials.

Sample collection
The commercial cultivar of guava 'Gola" was selected, and samples were collected from the orchard of Ayub Agriculture Research Institute Faisalabad, Pakistan (30º31′5″N, 73º74′0″E, 184m above sea level) having tropical weather conditions. The fruits of same cultivar, full green color, and firm maturity were selected. The selection criteria include fruits free from injury, damage, or infection with insects, and the guava samples were uniform in size, shape, and color.

Preparation of chitosan solutions
The stock solutions of chitosan were prepared following the method Chen et al. [19]. Chitosan (Sigma-ID 448869-250G; degree of deacetylation ≥75, MW: 50-190 kDa,) was used to prepare 500 ml solution, 0, 0.5, 1, 1.5, and 2% (w/v), taking the accurate weight of 2.5, 5.0, 7.5 and 10 g of chitosan in a volumetric flask and diluted up to 500 ml with distilled water and added glacial acetic acid (3 mL), placed in a water bath for 15 minutes until it was properly mixed and cooled down at room temperature.

Treatment of guava fruits
A total of five groups of guava fruits were distributed into five groups containing each with 20 fruits. Each group's fruits were dipped into chitosan solutions (control, 0.5%, 1.0%, 1.5%, and 2.0% for 2 minutes, respectively, and each treatment was performed in triplicate. Treated guavas fruits were dried for 30 min at room temperature and stored in sintered polyurethane plastic bags (33 cm x 25 cm, 10 cm) and stored up to 12 days at room temperature (25 °C and at refrigerated temperature 4 °C. The quality parameters and shelf-life attributes were calculated after 3 days intervals.

Determination of total soluble solids (TSS)
All treatments, including control of guava fruits, were converted into juices. The total soluble solids were calculated using a digital hand Refractometer (PAL-1, Atago, Tokyo, Japan) and expressed in terms of °Brix.

Total sugars, reducing sugar, and non-reducing sugar
The amount of total sugar, reducing sugar, and non-reducing sugar was determined using our previously validated method [4]. Briefly, a sample of 0.5 mL was mixed with 0.25 mL phenol and added 1.25 mL of H2SO4 (conc), and placed in a water bath at 30 °C for 25 mint. The absorbance of the sample was noted at 490 nm. The amount of reducing sugar in the juice was estimated by the method of 3,5 dinitrosalicylic acid (DNS) method. The Juice sample 200 µL was extracted with 1 mL of DNs and 1.8 mL of distilled water. Then the solution was kept in a water bath for 15 mint at 100 °C. Finally, the solution was allowed to cool, and added 9 mL of distilled water and absorbance was noted at 540 nm. However, the non-reducing sugar was calculated by subtracting the value of reducing sugar from total sugar.

Determination of Malondialdehyde (MDA)
The estimation of the lipid peroxidation level in the guava juices was determined using the thiobarbituric acid (TBA) method [4,20]. The absorbance was measured at 532 nm and 600 nm.

Detection of Vitamin C, total phenolic contents (TPC), and total flavonoids (TF)
The detection of vitamin C (ascorbic acid) was carried as described by Hameed et al. [21]. The juice sample of 100 µL was mixed with 900 µL of distilled water. Then 100 µL of metaphosphoric acid and 100 µL of dichloroindophenol (DCIP) were added to the solution and mixed properly. The ascorbic acid content in the supernatant was measured by the dinitrophenylhydrazine method.
The assessment of total phenolic content (TPC) was done by Folin-Ciocalteu (F-C) colorimetric method as described by Nair et al. [22]. The TPC concentrations were expressed as µM/g. The detection of total flavonoids (TF) in guava juices was done as described by Lin and Tang [23]. The guava juice samples were diluted with aluminum chloride (AlCl3) and potassium acetate (CH3CO2K). The standard linear curve of quercetin and rutin with a range of 0.005-0.1 mg/mL was used to detect flavonoids, and the amount was expressed as mg/g of fruit extract.
The sample of 10 mL of guava juice was extracted with 25 mL of methanol and sonicated for 20 min. After sonication, the sample was centrifuged at 10,000 rpm for 10 minutes at 25 °C [24]. Then, methanol 5 mL was added and again subjected to centrifugation at 10,000 rpm for 5 minutes. Then, the solution was filtered with Whatman (no 42) filter paper. Then 5g of sodium sulfate anhydrous was added, and finally, the solution was filtered with a microsyringe filter (0.45 µm, Merck) for further HPLC analysis.

Determination of crude fiber, potassium, and sodium levels
The crude fiber in guava juice was determined by AOAC method 962.09. Briefly, sample 3 g of juice was mixed in 5 mL of 1.25% sulfuric acid and heated at100 °C for 30 minutes. Then, the solution was cooled and filtered by Whatman filter paper No. 42. The 2 mL of 1.25% NaOH was added to the residue and mixed. Finally, the residue was ignited in the furnace at 600 °C. The crude fiber was calculated using the following formula Crude fiber = − The estimation of potassium and sodium was done as the method described [25]. A stock solution of potassium from potassium chloride 100 µg/mL was prepared, and similarly, 100 µg/mL stock solution of Na sodium chloride was prepared. The standard curves included a range of 0, 5, 10, 15, 20, and 25 µg/mL for further calculations.

Statistical analysis
The data results were subjected to statistical analysis using SPSS (version 26 for Windows, SPSS Inc., Chicago, USA). The normality of data was analyzed using a normal distribution (Shapiro-Wilks test). To investigate the difference in treatment means and different storage temperatures, ANOVA was applied. The least significant difference (LSD) was used to analyze the difference of treatment during the storage period. A probability value of 0.05 was used to determine the statistical significance.

Results and discussion
The influence of chitosan coating on guava fruit stored at room and refrigerated temperature is shown in Fig 1.  Fig 1: Photographs of control, 0.50% chitosan, 1% chitosan, 1.5% chitosan, and 2% of chitosan-coated fruits at the 0th, 3 rd , 6 th , 9 th and 12th day of storage at room and refrigerated temperature The amount of total soluble solids (TSS) in guava fruits with different chitosan treat-2 ments at room and refrigerated temperatures is shown in Fig 2. In control samples 3 stored at room temperature, TSS levels were gradually decreased except on the 6th day 4 (increased from 10 to 11 °Brix). The highest increase of TTS levels (16.02 to 17.2 and 16.5 5 to 17.2 °Brix) was observed at 12th-day storage (refrigerated temperature) at chitosan 6 treatments of 1.5% and 2.0%, respectively. In chitosan treatment, guava samples both 7 stored at room temperature and refrigerated temperature  13 The presence of sugars in guava fruit imparts sweetness which directly linked the flavor 14 and taste of fruit. The influence of chitosan treatments at room and refrigerated tempera-15 ture on total sugars (A), reducing sugars (B), and non-reducing sugars (C) is represented 16 in Fig 3. The levels of total sugars in control (room temperature) were increased from 210.5 17 to 295.5 mg/g, and in control (refrigerated temperature) increased from 211.5 to 270.5 mg/g 18 (Fig 3A). However, with chitosan treatment, levels insignificantly decrease the levels of 19 total sugars (room temperature storage period) or retain the levels of total sugars in guava 20 fruits (refrigerated temperature) samples. The total sugar levels were significantly differ- 21 [36]. In another study decrease in TPC 147 levels were reported in litchi fruit [37].   The levels of total flavonoids (Quercetin) µg/g and rutin (µg/g) after applying differ-175 ent levels of coating with chitosan in guava fruits at different stored temperatures 176 are shown in Fig 5C and 5D.  known due to their use as ingredients in various multivitamin supplements [38]. 186 The phenolics and flavonoids' mechanism both react with hydroxyl radicals and 187 superoxide anion radicals [39][40]. The levels of crude fiber (Fig 6A), potassium (Fig 6B), and sodium (6C) 198 were determined and observed the influence of chitosan coating in guava fruits