Molds and Yeasts

Molds and yeasts analysis was carried out according to Pérez-Grijalva et al. [30] with some modifications. Samples of 1 mL were poured into potato dextrose agar (pH = 3.5 ± 0.1) and incubated at 25 ± 1 ◦C; colonies (CFU/g) were evaluated after 3 days of incubation.

#### Mesophilic Aerobic Bacteria

Mesophilic aerobic bacteria count was performed as described by Alsharjabi et al. [29], using plate count agar, pouring 1 mL of sample and incubating for 48 h at 37 ◦C. Results were reported as Log10 CFU/g.

#### 2.2.7. Statistical Analysis

Samples were analyzed in triplicate. Data were evaluated by one-way analysis of variance (ANOVA) and significant differences were analyzed by the Tukey test (*p* < 0.05).

#### **3. Results and Discussion**

#### *3.1. Edible Coating Characterization*

#### 3.1.1. Texture and Weight Loss

Firmness and weight loss changes of papaya with and without coating at different storage times are shown in Figure 2. Coated fruits required a larger force to penetrate 5 mm of pulp than the uncoated ones, which after 10 days of storage showed firmness loss of 92.02%, whereas coated papaya showed firmness reduction of 47.36%. This behavior is similar to that reported by Cortez-Vega et al. [31], who applied a fish-protein-based coating on minimally processed papaya. According to firmness values, uncoated papaya achieved its final stage of ripening after 5 days of storage (close to 10 N), while coated fruit reached this stage after 15 days [4]. Figure 2b shows that uncoated papaya exhibited an increasing trend in weight loss with storage time, up to 33.3% after 15 days of storage, whereas the coated fruit did not display any significant change in weight during 15 days.

Similar results were obtained by Adetunji et al. [32], who applied a layer of chitosan on papaya surface, but did find weight losses even with this coating. Hazarika et al. [33] applied coatings of carboxymethyl cellulose, chitosan, and *Aloe vera* on papaya, which again decreased but not suppressed weight losses.

**Figure 2.** Physical changes during the ripening of papaya with and without coating during storage at room temperature: (**a**) papaya pulp firmness; (**b**) weight loss. a–f: used on top of reported values, indicate that if the same letter appears at different times, the values compared are not significantly different (*p* > 0.05).

#### 3.1.2. pH, Soluble Solids, Titratable Acidity, and Vitamin C

Along the storage period of papayas, the acidity showed a decreasing trend, but after 5 and 10 days, coated papaya exhibited significantly (*p* < 0.05) lower acidity than the uncoated fruits (Figure 3a). However, at the end of storage time, there was no significant difference in this parameter. A similar behavior was reported by Ali et al. [34], who applied different chitosan concentrations as coatings to papaya, and found that at least 2% chitosan (*w*/*v*) was required to create an internal atmosphere to reduce acidity changes. Thus, in the present study, the applied coating did not show this feature due to similar acidity after 15 days of storage for coated and uncoated papayas.

**Figure 3.** Chemical changes during papaya ripening, coated and uncoated, during storage time at room temperature: (**a**) titratable acidity (% cítric acid); (**b**) vitamin C (g/kg); (**c**) pH; (**d**) soluble solids. a–f: used on top of reported values, indicate that if the same letter appears at different times, the values compared are not significantly different (*p* > 0.05).

Vitamin C content is a characteristic factor in the papaya ripening process that is related to oxidative degradation during maturation stages [35]. Figure 3b reveals a decrease in vitamin C of coated papaya after 5 days of storage, whereas the opposite was shown by the uncoated fruit, which agrees with Yurena et al. [36], who reported that vitamin C increased with maturation stage. Similar behavior was described by Wall [37] for papaya from different cultivars. Vitamin C increase may be related to increased lipid peroxidation, since this oxidative phenomenon induces intensification of antioxidant compounds such as ascorbic acid [38].

Up to 10 days of storage for both coated and uncoated papayas showed a similar trend in pH values, and after 15 days both treatments showed a sharp pH reduction significantly different from the initial value, but similar between them (Figure 3c). Reduction of pH during storage time was attributed to organic acids production, which is related to the papaya ripening process [31]. Soluble solids of coated and uncoated papayas did not show any significant difference (*p <* 0.05) during the storage time, increasing significantly in relation to the initial value after 15 days (Figure 3d). Jayanthunge et al. [39] obtained similar results by storing papaya in microperforated polyvinyl chloride containers, and suggested that soluble solids increased as a result of the different metabolic activities in tissues leading to pectin decomposition and carbohydrates hydrolysis into simple sugars during storage.

#### 3.1.3. Color

Papaya samples color changed from mostly green to yellow-red over time (Figure 4), associated with chlorophyll degradation [5]. Uncoated papaya reached yellow-red coloration and did not reveal significant changes during the fifteen days of storage (Supplementary Figure S1), whereas coated papaya showed a gradual change from green to yellow-red as storage time increased (Figure S2). Figure 4b shows chromatic change in *a*\* parameter, where uncoated papaya reached a red color from day 5, without any more significant changes in the remaining storage time, while coated papaya gradually acquired this coloration.

Figure 4c shows significant changes for coated papaya in the *b*\* chromatic value from 5–10 days, while uncoated papaya does not show significant changes from 5 to 15 days. According to Barragán-Iglesias et al. [40], there are five papaya ripening levels based on color and days after anthesis (DAA). Physiological maturity is reached at 135 DAA and the surface is green; ripeness ready for consumption occurs at 156 DAA, and 100% of the surface is orange-red.

They defined three intermediate stages depending on the extent of orange-red color of the fruit surface: 143 DAA (25%), 149 DAA (50%), and 153 DAA (75%). From the evaluated color of papaya samples, it is concluded that the application of coating delayed papaya ripening reaching maturity for consumption after 15 days, whereas uncoated papaya reached this stage after 5 days.

#### 3.1.4. Volatile Compounds

The volatile compounds identified are listed in Table 1, and are in agreement with those obtained in other studies [31,41–43]. These results indicate that coating application affects the papaya ripening process. Fuggate et al. [41] and Almora et al. [44] reported papaya chemical changes during the intermediate stages of ready-for-consumption ripeness, where the amount of ethyl butanoate, ethyl hexanoate, and alcohols increased. These increments were also noted for coated and uncoated papayas, but the uncoated fruit produced ethyl butanoate after 5 days, whereas coated papaya produced it after 10 days, and its concentration after 15 days was higher in uncoated papaya. In relation to ethyl hexanoate, it was detected after 15 days of storage, with higher concentration in the uncoated papayas.

**Figure 4.** Papaya color changes in storage with and without edible coating: (**a**) *L*\*; (**b**) *a*\*; (**c**) *b*\*. a–d: used on top of reported values, indicate that if the same letter appears at different times, the values compared are not significantly different (*p* > 0.05).


**Table 1.** Volatile compounds generated during papaya ripening with and without edible coating.


**Table 1.** *Cont.*

Among alcohols related to papaya ripening [44] are 2-ethylhexanol and benzyl alcohol, which increased in similar quantities with storage time in coated and uncoated papayas. Ethanol was detected in both coated and uncoated papayas after 5 days, but concentration was higher in uncoated fruits at all storage times. According to Lee et al. [45], compounds such as 2-ethyl hexanol, acetic acid, butyric acid, ethyl acetate, ethyl butanoate, ethyl hexanoate, and methyl acetate are related to the papaya fermentation process. Uncoated papayas generated ethyl butanoate after 5 days, whereas coated fruits generated it after 10 days. Acetic acid appeared after 15 days in both treatments, whereas butyric acid generation was about 10 times higher in uncoated than in coated papayas (Table 1). In addition, higher amounts of these compounds were detected in uncoated papayas at all storage times. It must be pointed out that only uncoated papayas generated methyl acetate from day 5 until the end of storage time. Thus, these results indicate that coating application delays papaya fermentation.

### *3.2. Microbiological Analysis*

Microbial contamination is an important cause of papaya postharvest losses. Average initial values in papaya surface were 35.3 ± 5.5 colony-forming units (CFU)/g of total coliforms, 548.5 ± 97.3 CFU/g of mesophilic aerobic bacteria, 239.75 ± 28.5 CFU/g of yeasts, and 0 molds. Figure 5a shows total coliforms population, where a decrease is observed for the first 10 days in coated papaya, whereas population increased in uncoated papaya. A similar behavior is observed for mesophilic aerobic bacteria, where coating application allowed the reduction of 2 Log10 CFU g−<sup>1</sup> after 15 days of storage, while uncoated papaya increased their population by 2 Log10 CFU g−<sup>1</sup> (Figure 5b). Coated papaya showed a fungal population <10 UFC g<sup>−</sup>1, whereas the uncoated fruits at the end of storage exhibited a population of 206 CFU g−<sup>1</sup> (Figure 5c). Antimicrobial activity was most noticeable in yeasts (Figure 5d); coated papaya revealed a population decrease of 3 Log10 CFU g−<sup>1</sup> versus population increase to about 4.7 Log10 CFU g−<sup>1</sup> for uncoated papayas, showing a completely spoilt fruit (Figure S1d). Similar

results have been obtained using starch-based edible coatings added with nisin and LAE, achieving inhibition against bacteria and fungi [17,18].

**Figure 5.** Antimicrobial activity of papaya with and without edible coating: (**a**) total coliforms; (**b**) mesophilic aerobic bacterial; (**c**) fungi; (**d**) yeasts. a–e: used on top of reported values, indicate that if the same letter appears at different times, the values compared are not significantly different (*p* > 0.05).

#### *3.3. Image Analysis*

Images showing textural changes in coated and uncoated papayas are shown in Figure 6A. Entropy is an indicator of the complexity of the texture image, and measures the disorder or randomness [46]. Entropy values decreased gradually for coated and uncoated papayas during the first days, being significantly different (*p* < 0.05) until 10 days of storage. After this time, coating application permitted lower entropy values than those of uncoated papaya, indicating a surface with less damage. Fruit texture is an important factor that can be used to determine the stage of ripening, because during this process, a detachment of parenchyma cell wall takes place [47]. These results indicate that coated papaya displays a cell membrane system more ordered and with less mobility.

The angular second moment (ASM) is a parameter that measures the homogeneity of an image [48], and ASM showed a time-dependent increment (Figure 6B). Samples with edible coating showed higher values than those without it. Images with edible coating presented higher homogeneity values due to the stage of ripening, and in uncoated papayas due to fungi present on the papayas' surface.

**Figure 6.** Image analysis of papaya with and without edible coating: (**a**) entropy; (**b**) angular second moment; (**c**) fractal dimension.

The fractal dimension (FD) or fractal texture is a measure of the degree of roughness of the images; the higher the values of fractal dimension, the rougher or more complex the gray level images (Figure 7), while lower FD values can be associated with simpler or smoother images. After 20 days, the FD of uncoated papaya is smaller than that of the coated fruit, and roughness was attributed to fungal growth. Samples without edible coating showed more complex images than coated papayas (Figure 6C); this could be due to changes derived from the papayas' maturation.

**Figure 7.** Grayscale papaya crops (8 bits) after different days of storage. 0 days (initial time): with coating (**a**,**b**); without coating (**c**,**d**). 5 days: with coating (**e**,**f**); without coating (**g**,**i**). 10 days: with coating (**j**,**k**); without coating (**l**,**m**). 15 days: with coating (**n**,**o**); without coating (**p**,**q**).
