Effect of postharvest exogenous edible coating treatments on inhibitor browning and maintaining quality of fresh mushroom

The shelf-life of fresh mushrooms is limited to 1-3 days at ambient conditions and antioxidant characters are reduced and acquire a brown colour during post-harvest storage. Therefore, the present study was conducted to investigate the effect of some postharvest exogenous edible coating treatments of immersed in six different solutions for 5 min of calcium chloride (2%, 4%), chitosan (1%, 2%), CMC (1%, 2%) and control (distilled water) on reducing the browning, microbial load and maintaining quality during storage for 15 days at 4 ± 1 °C and 95% RH, during two successive seasons of 2021 and 2022 in 4th and 6th of January in the first and second seasons respectively. The obtained results revealed that dipping mushroom plants in a solution of chitosan 2% or calcium chloride 4% was the best treatment for reducing weight loss, while chitosan 2% for 5 min was the most effective treatment in reducing browning, PPO activity, total phenolic, flavonoids contents and inhibiting microbial counts for 12 days at 4 ± 1 °C and 95% RH.


Introduction Introduction Introduction Introduction
Button mushroom is one of the most used mushrooms in the world. It contains essential elements and phytochemicals which have antioxidant abilities and benefits to human health. The short shelf-life is mostly explained by the water loss and high respiration levels. (Zhang et al., 2016). Mushrooms are usually used as food and as functional food as well as for medical purpose due to their high concentrations of minerals, proteins, low cholesterol contents and presence of several bioactive compounds (Wani et al., 2010). Browning is a significant factor that limits consumer mushrooms acceptability. Browning occurs in much vegetables because of the increase in PPO activity, phenolic compounds, lipid peroxidation and membrane integrity (Saba and Sogvar, 2016).
2 Calcium chloride is an important treatment for extending the shelf life of fresh fruits is applying edible coating on the surface of the fruit (Luna-Guzman and Barrett, 2000), chitosan (Chong et al., 2015) and carboxymethylcellulose (Sangsuwan et al., 2008), in conjunction with low temperature. Edible coating potentially prolongs the shelf life of fruits due to the formation of a barrier layer blocking the exposure of gas and moisture. The barrier layer can delay the quality deterioration of fruits by reducing respiration, delaying volatile loss, decreasing the broke of moisture migration and thus extending the shelf life of fruits (Rojas -Grau et al., 2009). Treatment with calcium chloride increased the amount of internal calcium in the cell wall, which contributes to the retention of rigidity and inhibition of respiration, ethylene production and aging in mushrooms (Rageh, 2018). Applying CaCl2 treatment as calcium helps in maintaining cellular organization and regulating enzyme activities, thereby reducing moisture loss associated with senescence (Singh et al., 2016) on the mushroom. The potential of calcium in imparting stability to vacuole membranes and further slowing the enzymatic browning was studied by (Beelman et al., 1987). They also indicated that CaCl2 inhibited the browning of mushrooms. Calcium dips raise the possibility of producing fruit less susceptible to flesh browning symptoms.
Chitosan has been applied successfully as a coating on the food surface to extend the shelf life effectively without compromising the natural tastes of the product. The chitosan films are used as coating of fresh fruits and vegetables (mushroom, apples, oranges, tomatoes, pepper, cantaloupe …etc.) because they are flexible, offer valuable properties such as elasticity, selective permeability and act as an antimicrobial barrier against pathogens (Zhelyazkov et al., 2012;Hussein et al., 2015). An additional positive effect of chitosan coating is related to its ability to extend the storage life of fruits and vegetables. Chitosan forms a semipermeable film that regulates the gas exchange and reduces transpiration losses and fruit ripening is slowed down. Also, the respiration rate and hence water loss are reduced (Bautista-Banos et al., 2006). However, chitosan coating partially inhibited enzyme activity and delayed the increase in phenolic content and enzyme activities of fresh-cut mushroom. Thus, it seems that the effect of chitosan coating on anti-browning is not only due to the reduction in oxygen supply but also to the inhibitory effects of enzyme activities (Eissa, 2008) on the fresh-cut mushroom.
Carboxymethyl cellulose (CMC) is one of the polysaccharides used in an edible coating. Carboxymethyl cellulose is flexible and transparent and can act as a barrier to moisture and oxygen. Previous studies reported the application of carboxy methyl cellulose as a suitable coating material for several products. It is a linear, longchain, water-soluble, anionic polysaccharide that can be used as a fruit coating. Purified CMC is a white to cream-colored, tasteless, odourless, free-flowing powder. Edible coatings can also serve as carriers of food additives, anti-browning and antimicrobial agents, colorants, flavors, nutrients, and spices (Srivastava and Bala, 2016) on button mushroom. Moreover, Haffez (2016) reported that coating fresh-cut cantaloupe with methylcellulose extended the shelf life of the product, and also showed a beneficial effect on reducing decay, weight loss, colour change, loss of firmness and delayed the softening of fruit and texture change. The objective of this study was to investigate the potential of some edible coating such as calcium chloride, chitosan and carboxy methylcellulose individually in different concentrations to extend shelf life and maintain the quality of fresh mushroom during storage at 4 °C and 95% RH.

Materials and Methods Materials and Methods Materials and Methods Materials and Methods
Mushrooms (Agaricus bisporus) were obtained from the outlet of the Food Technology Research Institute -Agricultural Research Center in Giza, Egypt, during the two seasons 2021 and 2022 on January 4 in the first season and on January 6 in the second season. Then Button mushrooms were taken to the laboratory of Vegetable Crops Postharvest and Handling Dept., Giza Governorate, Egypt. Button mushrooms were selected with uniformity size, colour and free of blemishes, visual damage or defects for postharvest experiment.

Preparation of edible coating solution:
Chitosan is a commercial product; it includes chitosan in a proportion of 90-95% (2-Amino-2-deoxybeta-D-glucosamine) (EL-Badawy 2014). Chitosan was bought from El-Gomhouria Company, Egypt. Chitosan coating at (1 or 2%) was prepared by dissolving 10 g or 20 g chitosan powder in 1000 ml of distilled water, respectively and homogenized by a magnetic stirrer. Glycerol (1.5% W/V) was added to the mixture as a plasticizer.
Carboxymethyl cellulose (CMC) was bought from Technogene Company, Egypt. CMC coating at (1 or 2%) was prepared by dissolving 10 g or 20 g of CMC powder in a water ethyl alcohol mixture (2:1) at 75 °C under the high-speed mixer (900 rpm) for 15 min. then, glycerol (1.5% W/V) was added and the solution was stirred for another 10 min under the same conditions. Calcium chloride solution coating at (2 or 4%) was prepared by dissolving 20 g or 40 g CaCl2 powder in 1000 ml of distilled water respectively and homogenized by a magnetic stirrer. Button mushrooms were washed with tap water and then treated with the following, treatments: 1-Dipping in calcium chloride solution at 2% for 5 min. 2-Dipping in calcium chloride solution at 4% for 5 min. 3-Dipping in chitosan solution at 1% for 5 min. 4-Dipping in chitosan solution at 2% for 5 min. 5-Dipping in carboxy methyl cellulose solution at 1% for 5 min. 6-Dipping in carboxy methyl cellulose solution at 2% for 5 min. 7-Control (dipping in distilled water). All treatments were air-dried after being directly dipped in air and packed in polypropylene bags (25 x 30 cm) with a thickness of 20 mm. Each sachet contains 100 g mushrooms represented as an experimental unit (EU). 12 replicates were selected from each treatment and stored at 4 °C and 95% relative humidity for 12 days. Samples were randomly drawn in 3 replicates and samples were organised in a complete randomized design.

Quality attributes:
Studied characters were examined directly after treatment and at 3 days intervals to determine the following: 1-Browning Index (BI): The colour of the mushroom cap was measured using a Minolta Chroma Meter (Model CR-155, Minolta Camera Co., Osaka, Japan), using the Hunter Lab Color Scale. Mushroom colour has been commonly measured using the L* value. Also changes in other parameters of the hunter scale (a* and b*) related to browning (Aguirre et al., 2008). BI was determined by the method of Ruangchakpet and Sajjaanantakul (2007) using the following equation: Browning Index (BI) = [100 (x -0.31)] / 0.17, where x = (a* + 1.75L*) / (5.645L* + a* -0.3012b*), L* value indicates lightness of the colour, which range from 0 (dark) to 100 (white). The positive value of a* indicates the red colour, while the negative value indicates the green colour. The positive value of b* indicates the yellow colour, while the negative value indicates the blue colour.
2-Polyphenol oxidase (PPO) activity: the activity of PPO was determined in the extract during storage period by the method of Pizzocaro et al. (1993).
3-Total phenolic contents: Total phenolic contents were measured according to Singleton and Rossi, (1965). 4-Total bacteria counts (TBC) and total yeast and mold counts (TYMC): The population of total bacteria counts (TBC) and total yeast and mold counts (TYMC) were determined by the method of Gonulalan et al. (2003). 5-Weight loss (%) was measured. 6-Flavonoids contents were determined using the method of Barros et al. (2008).

Statistical analysis
The experimental design was factorial with two factors in a CRD with three replicates. Duncan's Multiple Range Test at 5% level of significance was used to compare between means according to Sendecor and Cochran (1982).

Results Results Results
Browning Index (BI) Data in Figure (1) showed a significant difference between edible coating treatments in the browning Index of fresh mushrooms during storage. The Browning Index of fresh mushrooms was increased with the prolongation of the storage period. These results were recorded with (Rageh 2018) on button mushrooms and (Eissa 2008) on fresh-cut mushrooms.

5
All treatments were better than the control however; fresh mushroom dipped in a solution of chitosan at 2% was the most effective treatment for reducing the browning Index during storage in two seasons. In another word, this treatment gave the lowest value of the browning Index in both seasons, while untreated (control) obtained the highest ones in this concern. Fresh mushroom treated with CMC at 1% was less effective in reducing the browning Index when compared with the other treatments (Table 1). These results were true in the two seasons and agree with Eissa (2008) for chitosan, Anshu and Anju (2018) for CaCl2, Srivastava and Bala (2016) for CMC and Ban et al. (2014) for chitosan and CaCl2. Concerning the interaction between edible coating treatments and storage period on browning Index, data recorded that fresh mushroom dipped in chitosan at 2% gave the lowest value in browning index till 12 days (end of storage period) at 4 °C, while, untreated control had the highest value in the browning index after 12 days of storage.

Polyphenol oxidase activity (PPO)
The presented data in Figure 3 indicated that PPO activity was increased with the prolongation of the storage period in the two tested seasons. The increase in PPO activity may be due to the activation from latent to fully active form. PPO enzyme activity increased in fruit after harvest and the activity of phenols was closely associated with the development of browning, these results are in an agreement with those reported by Mirshekari et al. ( Figure 3. Figure 3. Figure 3. Figure 3. Effect of storage periods on the activity of PPO during both seasons. Concerning the effect of CaCl2, chitosan and carboxy methyl cellulose (CMC), data in Figure 4 revealed that fresh mushroom dipped in chitosan 2% were the most effective treatments in reducing PPO activity with significant differences between this treatment and the others, in both seasons. The highest mean values of PPO activity were obtained from untreated control, in the two seasons. These results are in agreement with those obtained by Eissa (2008) for chitosan.
Regarding the interaction between CaCl2, chitosan and carboxy methyl cellulose (CMC) treatments and storage periods, data revealed that all treatments reduced PPO activity with significant differences among them compared with the untreated control after 12 days of storage, in the two seasons (Table 2).     Total phenolic contents: Data in Figure (5) indicated that the prolongation of the storage period led to decrease total phenolic contents. This decrease could be attributed to the oxidation of the PPO enzyme giving the coloured quinones and quercetin was oxidized directly by PPO. Similar results were obtained by Rageh (2018); Queiroz et al. (2008) on button mushroom. Also, the effect of edible coating treatments maintained the total phenolic loss as compared with control, fresh cut mushroom treated with chitosan 2% seems to be most effective in reducing total phenolic loss with significant differences between all treatments in the two seasons, followed by chitosan 1% (Figure 6).   8 Figure 6. Figure 6. Figure 6. Figure 6. Effect of edible coating treatments on total phenol (mg/g) during both seasons Regarding the interaction between CaCl2, chitosan and carboxy methyl cellulose (CMC) treatments and storage periods, data in Table 3 showed that, the interaction between edible coating treatments revealed that all treatments reduced total phenolic loss with significant differences among them compared with the untreated control after 12 days of storage and the lowest value was obtained from untreated control, in the two seasons. In general, the interaction between edible coating treatments and storage period was significant in the two seasons. After 9 days of storage, fresh-cut mushrooms treated with chitosan 2% gave the highest total phenolic content, while untreated control gave the lowest ones at the same period (Table 3). Total bacterial count (TBC) Data in Figure (7) indicated that bacterial growing in fresh mushrooms increased with increasing storage period, mainly in the control in both seasons. These results were similar to the results of Rageh (2018) on button mushroom and (Eissa 2008) on the fresh cut mushroom. The results in Figure (8) showed significant differences in bacterial growth between edible coating treatments and control. Fresh mushrooms treated with all studied treatments had lower levels of bacterial load in comparison to untreated control treatment, while, dipped in chitosan coating at 2% provided the lowest count in bacterial count followed by chitosan coating at 1% in the two seasons, while the other treatments gave less effective in reducing bacterial growth in both seasons and agree with Jiang et al. (2011). Data in Table 4 showed that the interaction between edible coating treatments and storage period was significant during storage periods. After 9 days of storage showed that fresh mushroom treated with chitosan at 2% and chitosan at 1% were effective in inhibition bacterial growth with significant differences between them ,these were true in two seasons and agree with (Rageh, 2018) on button mushroom and (Eissa, 2008) on the fresh cut mushroom. Fig  Fig Fig  Figure 8. ure 8. ure 8. ure 8. Effect of edible coating treatments on total bacterial count (cfu/g) of button mushroom during both seasons

Total yeast and mold counts (TYMC)
Data in Figure (9) indicated that TYMC mushrooms augmented with the extension of the storage period, mainly in the control in both seasons. The same trend was recorded with Rageh (2018).
Recording the effect of edible coating treatments, data in Figure (10) showed significant differences in TYMC between all treatments and the control. Fresh mushrooms dipped in chitosan at 2% and chitosan at 1% treatments gave the lowest significant number in TMYC treatment with no significant differences between them, followed by CaCl2 at 4%, however the other treatments were less effective in decreasing this character. The high levels of TYMC were recorded with untreated control during storage in both seasons. These results are in accordance with Eissa (2008) for chitosan and Chikthimmah et al. (2005) for CaCl2. Figure 9. Figure 9. Figure 9. Figure 9. Effect of storage periods on yeast and mold count (cfu/g) of button mushrooms during both seasons Data given in Table 5 indicated that TYMC of fresh cut mushroom were decreased with increasing chitosan concentration. The untreated fresh-cut mushroom was contaminated with yeasts and molds (TYMC) of untreated mushroom after 12 days stored at 4 •C was 3.13 and 3 log colony-forming units (cfu/g) in the tow season, whereas the TYMC reduced by 1 log value at mushroom coated with 2% chitosan, 1% chitosan and 4% CaCl2 respectively. This rate is accepted as a maximal count of decontaminated fruit desired by the fruit trade (Lee et al., 2004). Furthermore, no growth of yeasts, bacteria and molds was noticed after 6 days stored at 4 •C in mushroom coated with 1% and 2% chitosan. Our result showed that 2% chitosan was effective for decreasing the harmful microorganisms (TYMC) in mushroom. Concerning the interaction data in Table 6 exhibited that after 12 days of storage fresh mushrooms dipped in chitosan at %2 was the most effective treatment in reducing TYMC in both seasons. Figure 10. Figure 10. Figure 10. Figure 10. Effect of edible coating treatments on yeast and mold count (cfu/g) of button mushrooms during both seasons Weight loss percentage Data in Figure (11) show the impact of chitosan (CH), CaCl2 and carboxy methyl cellulose (CMC) treatments on the weight loss percentage of fresh mushroom during cold storage. Results indicated that there were significant increases in weight loss of fresh cut mushroom during the storage period in the two seasons. Figure 11. Figure 11. Figure 11. Figure 11. Effect of storage periods on weight loss percentage of fresh mushroom during both seasons Similar results were obtained by Rageh (2018) and Chong et al. (2015). Concerning the effect of postharvest treatments on weight loss percentage, data shown in Figure 12 revealed that there were significant differences between all treatments and untreated control in weight loss percentage during storage, however, all treatments retained their weight during storage as compared with control. Moreover, fresh-cut mushroom treated with CaCl2 at the two concentrations, resulted in a prominent reduction in weight loss percentage with significant differences between them in the two seasons, followed by chitosan at 1% and CMC at 2% with no significant differences between them in the two seasons. The highest value of weight loss percentage was recorded with untreated control. These results were in agreement with Attia (2014) for CaCl2 Chong et al. (2015) for CaCl2 or chitosan and Nadim et al. (2015) for methylcellulose. Concerning the interaction between postharvest treatments and storage period, data in Table 6 indicated that fresh-cut mushroom treated with CaCl2 at 4% was the most obvious in reducing the loss of weight loss percentage during all storage period in the two seasons while the highest value of weight loss percentage was recorded with untreated control in both seasons. Figure 12. Figure 12. Figure 12. Figure 12. Effect of edible coating treatments on weight loss percentage of fresh mushroom during both seasons 13 Total flavonoids contents Data in Figure 13 showed that total flavonoid contents decreased with the prolongation of the storage period. Concerning the effect of postharvest treatments, data in Figure 14 revealed that all treatments were effective in maintaining total flavonoid contents during storage compared with untreated control. Moreover, fresh mushrooms dipped in chitosan 2% were the most effective treatments in reducing the loss of total flavonoid contents in the two seasons, followed by chitosan 1% in the two seasons however, the other treatments were less effective in this concern. The lowest value was obtained from untreated control in the two seasons. These results in agreement with Rageh (2018) for calcium chloride and Jiang et al. (2012) for chitosan. Figure 13. Figure 13. Figure 13. Figure 13. Effect of storage periods on flavonoids (mg/g) of button mushroom during both seasons  Figure 14. Figure 14. Figure 14. Figure 14. Effect of edible coating treatments on flavonoids (mg/g) of button mushroom during both seasons For the interaction between postharvest treatments and storage period, after 12 days of storage, results in Table 7 indicated that fresh-cut mushrooms dipped in chitosan 2% had significantly higher values of total flavonoid content compared with other treatments, followed by chitosan 1%. These results were true in the two seasons, while the lowest total flavonoid content was obtained with control in both seasons.

Discussion Discussion Discussion
Edible coating Edible coating potentially prolongs the shelf life of fruits due to the formation of barrier layer blocking the exposure of gas and moisture. The barrier layer can delay the quality deterioration of fruits by reducing the respiration, delaying volatiles loss, decreasing the broke of moisture migration and thus extend the shelf life of fruits (Rojas -Grau et al., 2009).

Browning Index (BI)
The reduction of the browning Index using chitosan may be due to that chitosan coatings could delay postharvest senescence of fruits and vegetables associated with colour changes and dehydration (Ban et al., 2014).

Polyphenol oxidase activity (PPO)
The reduction of PPO activity under chitosan treatments may be due to that chitosan is a cationic polysaccharide recognized as an excellent protein binder, the decrease in enzyme activity was not simply the result of inadequate extraction procedure but a result of the binding of the enzymes to chitosan (Baldwin et al., 1995;Van Der Lubben et al., 2001). However, chitosan coating partially inhibited enzyme activity and delayed the increase in phenolic content and enzyme activities of fresh-cut mushrooms. Thus, it seems that the effect of chitosan coating on anti-browning is not only due to the reduction in oxygen supply but also to the inhibitory effects of enzyme activities (Li and Yu, 2000;Jiang and Li, 2001). The main function of calcium lactate is to strengthen the cell wall and stabilized the cell membrane (Luna-Guzman and Barrett, 2000), thus keeping PPO, which is a membrane-bound enzyme, away from its phenolic substrates present mainly in vacuoles leading to preserving phenolic content and inhibiting browning process.

Total phenolic contents
A lower level of total phenolic of the chitosan-treated slices may be due to the inhibition of PAL activity. However, the decrease in polyphenols content was due to the binding of the polyphenols to chitosan. Similar results were obtained by Eissa (2008). Rabea et al. (2003). Revealed that the antimicrobial of chitosan is probably caused by the interaction between chitosan and the microbial cell membranes, which leads to the leakage of proteinaceous and other intracellular constituents. Chitosan can also penetrate to the nuclei of fungi and interferes with RNA and protein synthesis. Krasaekoopt and Mabumrung (2008) found that the fresh-cut fruits coated with chitosan at 1% or 2% reduced the psychrotroph counts below the detectable level of lower than 100 cfug-1 throughout the storage period, also reduced the number of yeast and mold counts to lower than 200 log cfu g -1 . This was probably due to the fungicidal action of chitosan that caused alteration in the function of the cellular membrane (Fang et al., 1994). The favourable effect of CaCl2 Luna-Guzman and Barrett (2000) found that CaCl2 has provided an inhibitory effect on microbial growth while control treatment allowed for spore spreading and increased counts. The reduction of microorganisms in fresh cut mushroom treated with CaCl2 may be due to calcium salts can lower intracellular pH or reduce water activity (Shelef, 1994) which provides a protective antimicrobial barrier against food-borne pathogens in the product (Weaver and Shelef, 1993). Also, CaCl2 treatment may have provided an inhibitory effect on microbial growth (Weaver and Shelef, 1993).

Total yeast and mold counts (TYMC):
Calcium reduced water activity (Beelman et al., 1987) which provides a protective antimicrobial barrier against food-borne pathogens in products (Crowe et al., 2005) in addition, microflora is usually restricted to fungal and lactic acid bacteria at low pH (Chikthimmah et al., 2005). Also, calcium-enhanced tissue develops resistance to fungal attack by stabilizing or strengthening cell walls, thereby making them more resistant to harmful enzymes produced by bacteria and fungi, and it also delays the aging of fruits (Picchioni et al., 1995).

Weight loss percentage
Increases in weight loss of fresh cut mushroom during the storage period can be attributed to the loss of water which is relatively easy to evaporate (Vasey, 2006). Bolin and Huxsoll (1989) found that the effect of CaCl2 on reducing weight loss is likely due to strengthening the cell wall and reducing the respiration rate in the stored vegetable fruits. Chong et al. (2015) revealed that CaCl2 or chitosan treatments can effectively prevent moisture loss in fresh-cut honeydew melon by coating the fruit surface, the migration of moisture from inner fruits to the surface slowed down, leading to a reduced rate of moisture loss and possibly reduced respiration rate, resulting in lowering weight loss. The favourable effect of chitosan treatment in reducing weight loss may be due to the chitosan coating forms a layer of semi-transparent to smooth the pericarp surface (Dong et al., 2004) and can be used as a protective barrier to reduce respiration and transpiration rates through fruit surface (Kester and Fernema, 1986). Also, Shiri et al. (2013) found that chitosan coating reduced weight loss during storage as it enables epidermal tissues to control water loss and reduce respiratory activity, the barrier to water vapor, reducing moisture loss and delaying fruit dehydration (Baldwin et al., 1995). Carboxymethyl cellulose coatings are effective physical barriers to moisture loss and slower rates of weight loss in coated fruits because of the cover features for gas diffusion of stomata, the organelles that regulate the transpiration process and gas exchange between the fruit and the surroundings (Almenar et al., 2006). Thereby, weight loss reducing during storage. CMC edible coating provides a semi-permeable barrier to O2, CO2, H2O and moisture transfer slowing down fruit respiration (Baldwin et al., 1995).

Total flavonoids contents
The effect of chitosan in this concern could be due to the fast oxidation of flavonoids compound on the mushroom surface, directly in contact with the O2. Enzymatic oxidation of phenolics via polyphenol oxidase (PPO) has been associated with mushroom browning. Previous research shows a significant positive relationship between total phenolic and antioxidant activity in mushrooms, the higher radical scavenging activity in chitosan-coated mushrooms in the present study could be mainly attributed to its higher level of total phenolic compounds. The main function of CaCl2 is to strengthen the cell wall and stabilize the cell membrane (Jones and Jacobsen, 1983), thus keeping PPO, which is a membrane-bound enzyme, away from its phenolic substrates present mainly in vacuoles leading to preserving phenolic content and inhibiting browning process (Koushki et al., 2011).

Conclusions Conclusions Conclusions
It could be concluded that, dipping mushroom fresh-cut in chitosan at 2% could be considered as an effective method in increasing its shelf life and keeping it at high quality during shelf life. That was clear by reducing deterioration rate (browning -PPO activity -total phenolic loss -total bacterial, yeast and mold counts). Moreover, keeping its contents of total flavonoids for up to 12 days at 4 °C.