The combined effect of exposure time to sodium chlorite (NaClO2) solution and packaging on postharvest quality of white button mushroom (Agaricus bisporus) stored at 4 °C

Mushrooms have been consumed due to their nutritional and medicinal properties for centuries and are also one of the best sources of vitamins, especially vitamin B (Wani et al., 2010). More than hundreds of edible mushroom species exist in nature, but a few of those are cultivated commercially on industrial scale. Among those species, the button mushroom (Agaricus bisporus) is the most common edible mushroom all over the world (Doymaz, 2014a; Moradian et al., 2018). Button mushrooms have no cuticle to protect them from physical damage, microbial attack and water loss. They have very high respiration rate and high water content, making them prone to microbial spoilage and enzymatic browning (Brennan et al., 2000; Zalewska et al., 2018). Pseudomonas spp. have been isolated as the most abundant microorganism responsible for the spoilage of mushrooms (Simón & González-Fandos, 2010; Venturini et al., 2011). The shelf-life of mushrooms is very limited, and they lose their commercial value within a few days (Doymaz, 2014b; Gantner et al., 2017). This is a big problem for the distribution and marketing of the fresh mushrooms. Therefore, prolonging the shelf-life of mushrooms without any adverse effect on their quality would be important for the mushroom processors as well as the consumers. The most preferred way to extend the shelf-life is washing the mushrooms with solutions having antibrowning and/or antimicrobial properties to control respiration rate, and enzymatic and microbiological activity. Washing the mushrooms with antibrowning and antimicrobial agents including hydrogen peroxide (Brennan et al., 2000), sodium hypochlorite (Simón & González-Fandos, 2010), citric acid (Simón & González-Fandos, 2010), malic acid (Singla et al., 2012), acetic acid (Sedaghat & Zahedi, 2012) and ascorbic acid (Sedaghat & Zahedi, 2012) reduced the microbial activity to some extent, but the shelf-life of the mushrooms is still limited.


Introduction
Mushrooms have been consumed due to their nutritional and medicinal properties for centuries and are also one of the best sources of vitamins, especially vitamin B (Wani et al., 2010). More than hundreds of edible mushroom species exist in nature, but a few of those are cultivated commercially on industrial scale. Among those species, the button mushroom (Agaricus bisporus) is the most common edible mushroom all over the world (Doymaz, 2014a;Moradian et al., 2018). Button mushrooms have no cuticle to protect them from physical damage, microbial attack and water loss. They have very high respiration rate and high water content, making them prone to microbial spoilage and enzymatic browning (Brennan et al., 2000;Zalewska et al., 2018). Pseudomonas spp. have been isolated as the most abundant microorganism responsible for the spoilage of mushrooms (Simón & González-Fandos, 2010;Venturini et al., 2011). The shelf-life of mushrooms is very limited, and they lose their commercial value within a few days (Doymaz, 2014b;Gantner et al., 2017). This is a big problem for the distribution and marketing of the fresh mushrooms. Therefore, prolonging the shelf-life of mushrooms without any adverse effect on their quality would be important for the mushroom processors as well as the consumers. The most preferred way to extend the shelf-life is washing the mushrooms with solutions having antibrowning and/or antimicrobial properties to control respiration rate, and enzymatic and microbiological activity. Washing the mushrooms with antibrowning and antimicrobial agents including hydrogen peroxide (Brennan et al., 2000), sodium hypochlorite (Simón & González-Fandos, 2010), citric acid (Simón & González-Fandos, 2010), malic acid (Singla et al., 2012), acetic acid (Sedaghat & Zahedi, 2012) and ascorbic acid (Sedaghat & Zahedi, 2012) reduced the microbial activity to some extent, but the shelf-life of the mushrooms is still limited.
Sodium chlorite (NaClO 2 ) (SC) is an effective antibrowning and antimicrobial agent. The use of SC solution (0.5-1.2 g/L) has been approved by the US Food and Drug Administration (FDA) for sanitizing food products, including processed fruits and vegetables (U. S. Food and Drug Administration, 2019). Lu et al. (2007) used the SC solution ranging from 0.1 to 1 g/L to inhibit enzymatic browning in apple slices and found that the SC solution of 1 g/L was the most effective treatment in reducing the browning in sliced apples. Allende et al. (2009) used the SC solution in concentration of 1 g/L to reduce total aerobic mesophilic bacteria and Escherichia coli O157:H7 on the fresh-cut cilantro, and they observed about 3 log 10 CFU/g reductions in microbial counts. Wei et al. (2017) used the SC solution in concentration of 1 g/L to reduce aerobic bacteria, E. coli O157:H7, moulds, yeasts and Salmonella Typhimurium on fresh strawberry, cherry tomato and red bayberry. They reported that the SC solution resulted in considerable reductions in microbial populations. These studies revealed that the SC solution can be used to inhibit browning and limit the microbial growth in foods.
The effect of the SC solution on the postharvest quality of white button mushroom and Pseudomonas spp. counts during storage were not known and not investigated. Therefore, the main objective of this work was to extend the shelf-life of white button mushrooms by investigating the effect of washing time with the SC solution followed by packaging with a non-perforated polyvinyl chloride (PVC) film on the quality parameters including colour, firmness, weight loss, pH and gas composition, and Pseudomonas spp. counts in the white button mushrooms during storage.

Materials and methods
White button mushrooms (Agaricus bisporus) were obtained from MUPA Agriculture and Industry Inc. (Izmit, Kocaeli, Turkey). Mushrooms were picked at the closed cap stage (cap diameter 3.5-4.5 cm). They were immediately shipped to the laboratory at 4 °C which is the industrial practice for storage and delivery of the mushrooms. Damaged, bruised and shrivelled mushrooms were discarded. After the stipes were trimmed at 1 cm, the whole mushrooms were washed with 1 g/L SC (Merck KGaA, Darmstadt, Germany) solution for 30, 60 and 120 s. No abnormal odour formation was detected after the washing treatments. Approximately ten mushrooms were placed in polystyrene trays (∼125 g/tray) with the dimension of 22.5 × 13.5 × 3 cm. Some of the trays were overwrapped with a PVC film with a thickness of 9 µm and O 2 permeability of 33,000 mL/m 2 day atm under passive modified atmosphere packaging (MAP) conditions, and the others were not overwrapped with any kind of packaging film. Three different trays were used for each treatment in each sampling day. Every measurement was repeated at least three times. All the mushroom samples were stored at 4 °C ± 0.2 °C for 12 days in an incubator (Binder KB53, Binder GmbH, Tuttlingen, Germany). Mushrooms used in the experiments were categorized as follows: i) Control: Mushrooms not subjected to the SC solution and stored without any overwrapping material; ii) Control-P: Mushrooms not subjected to the SC solution and stored with a PVC overwrapping material; iii) SC-30: Mushrooms washed with the SC solution for 30 s and stored without any overwrapping material; iv) SCP-30: Mushrooms washed with the SC solution for 30 s and stored with a PVC overwrapping material; v) SC-60: Mushrooms washed with the SC solution for 60 s and stored without any overwrapping material; vi) SCP-60: Mushrooms washed with the SC solution for 60 s and stored with a PVC overwrapping material; vii) SC-120: Mushrooms washed with the SC solution for 120 s and stored without any overwrapping material and viii) SCP-120: Mushrooms washed with the SC solution for 120 s and stored with a PVC overwrapping material.

Colour analysis
The colour of the mushroom samples was measured in CIELab colour space where L* is the lightness component or luminance from black (0) to white (100), a* is a chromatic component from green (−120) to red (+120) and b* is a chromatic component from blue (−120) to yellow (+120) using a chroma meter (CR-400, Konica Minolta, Tokyo, Japan) equipped with a D 65 illuminant source. Prior to colour measurements, the chroma meter was calibrated with its white calibration tile (Y = 86.6, x = 0.3188 and y = 0.3364). Total colour difference (∆E*) values are calculated using Equation 1 (Borchert et al., 2014):

Microbiological analysis
Twenty-five grams of mushrooms were aseptically weighed and homogenized using a stomacher (Bag Mixer 400VW, Interscience, Woburn, MA, USA) at high speed for 2 minutes with 225 mL of sterile peptone (0.1%, w/v) water (Oxoid, Basingstoke, UK). Serial dilutions (10 -2 -10 -8 ) were made in serial dilution tubes by mixing 1 mL sample with 9 mL of 0.1% (w/v) sterile peptone water. Pseudomonas spp. were determined in King's B medium (Biolife Italiana, Milan, Italy) as given by King et al. (1954), following incubation at 25 °C for 48 h (Simón et al., 2005;Simón & González-Fandos, 2010). Pseudomonas spp. counts were determined by taking three separate trays for each treatment and each sampling point. Results were expressed as the average of three separate measurements in terms of log 10 CFU/g.

Texture measurements and weight loss
The firmness of the mushrooms was measured using a texture analyser (Lloyd TA1, Lloyd Instruments Ltd., West Sussex, UK) equipped with a load cell of 250 N. Cylindrical probe with a 12.5 mm diameter was used for the whole mushrooms. The probe speed was set to a constant speed of 2 mm/s, and the contact force was 0.5 N. Samples were penetrated to a depth of 5 mm. Force and time data were recorded with Llyod software, and the maximum force was expressed as the firmness (N) of the mushrooms. Results were reported as the average of nine measurements.
The weight loss of the mushroom samples was determined using a digital balance (Mettler Toledo, PB03-S, Columbus, OH, USA) with an accuracy of ±0.001 g. Triplicate measurements were done, and average value was reported as the weight loss. The weight loss (%) is given by Equation 2: where w L is the weight loss (%), w i is the initial weight (g) and w f is the final weight (g) of the mushrooms.

Headspace gas composition analysis and pH
Oxygen (O 2 ) and carbon dioxide (CO 2 ) levels inside the packages were measured using a gas analyser (Check Mate-II, PBI Dansensor, Ringsted, Denmark). Samples were taken with a syringe inserted through a gas tight septum placed on the exterior of the film. Measurements were performed in triplicate, and results were reported as the average of triplicate measurements.
Mushroom samples were homogenised using a kitchen type blender (MB450, Tefal, Cedex, France), and homogenised mushrooms were filtered through a muslin cloth. The pH of the filtrate was measured using a digital pH meter (SevenEasy S20-K, Mettler Toledo, Columbus, OH, USA). Measurements were done in triplicate, and average value was given.

Statistical analysis
Experiments were performed using a completely randomized design. One-way analysis of variance (ANOVA) was performed using Matlab 7.12.0 (R2011a) software (MathWorks Inc., Natick, MA, USA) in order to analyse the data. Statistical differences between the treatment means were determined by post hoc analysis using Tukey's multiple range test. The differences between the means are regarded as statistically significant if the p-value ≤ 0.05. Data were shown as mean ± standard deviation.

Effect of washing with SC solution and packaging on mushroom colour
Colour change in mushrooms is a crucial factor affecting the consumer's preference. The L* value of all the mushroom samples decreased while ∆E* values increased during storage at 4 °C (Table 1). Previously conducted studies on Agaricus bisporus showed that L* value could be used as a criterion to determine the mushroom quality and shelf-life (Aguirre et al., 2008;Taghizadeh et al., 2010).
Mushrooms with L* value of lower than 80 would not be acceptable at wholesale level (Gao et al., 2014;Lagnika et al., 2014;Lopez-Briones et al., 1993). This grading criterion is the most widely used indicator to determine the mushroom shelf-life both in the industry and research (Aguirre et al., 2008).
Washing the mushrooms with the SC solution and packaging with the PVC film slowed down the colour change of the mushrooms (Table 1). Similar results were also reported by Lu et al. (2007) who observed that the SC solution in concentration of 1 g/L effectively decreased the change in the L* value of apple slices. The initial L* value of the fresh mushrooms was 90.14. After 4 days of storage, the L* value of the control was significantly (p ≤ 0.05) lower than the L* value of the fresh mushrooms while there was no significant difference (p > 0.05) in the L* value of SCP-120 as compared with the L* value of the fresh mushrooms after 8 days of storage. The L* value of SCP-120 after 12 days of storage was significantly (p ≤ 0.05) higher than the L* value of the control on day 12th. This means that washing of the mushrooms with the SC solution and packaging with the PVC film retard browning and reduce the colour change of the mushrooms during 12 days of storage.
The browning and change in the colour of mushrooms is considered to occur due to polyphenol oxidase activity (Xu et al., 2016). Oxidation of phenolic compounds by polyphenol oxidase causes browning (Waliszewski et al., 2007). SC is a mixed type of inhibitor for polyphenol oxidase activity, the key enzyme responsible for browning reaction (Lu et al., 2006). SC inactivates mushroom tyrosinase, which is the major polyphenol oxidase responsible for browning in button mushrooms (Cliffe-Byrnes & O'Beirne, 2008). Therefore, the treatments with SC are responsible for slowing down the polyphenol oxidase activity of mushrooms. Lu et al. (2007) also found that SC was the effective inhibitor of polyphenol oxidase activity and enzymatic browning in apple slices.
After 12 days of storage, the highest L* and ∆E* values were obtained for the SCP-60 and SCP-120. These results confirm that subjecting the mushrooms to the SC solution longer than 60 s does not have a significant (p ≤ 0.05) effect on colour. Therefore, it was decided that SCP-60 was the most appropriate treatment to minimize the colour change in white button mushrooms.

Effect of washing with SC solution and packaging on Pseudomonas spp. counts on mushroom
Pseudomonas spp. play a key role on the postharvest quality of the mushrooms. The initial count of Pseudomonas spp. on fresh mushrooms in this study was found to be 7.59 log 10 CFU/g which is in good agreement with the Pseudomonas spp. counts on mushrooms found in previous studies which ranged from 6.9 to 8.1 log 10 CFU/g at harvest time (Simón et al., 2005;Venturini et al., 2011). Washing the mushrooms with SC solution and packaging with the PVC film were found to be effective in reducing the Pseudomonas spp. counts (Table 2).
Similar results related to the inhibitory effect of SC solution on microorganisms were also reported for different food products. Allende et al. (2009) found that SC solution in concentration of 1 g/L reduced total aerobic mesophilic bacteria on the fresh-cut cilantro by 3 log 10 CFU/g. Wei et al. (2017) also found that SC solution caused a decrease in the total bacteria in strawberry by 2.9 log 10 CFU/g. Pseudomonas spp. counts increased with the storage time. Washing the mushrooms with the SC solution resulted in the mushrooms with lower Pseudomonas spp. counts than the controls. After 8 days of storage, the number of Pseudomonas spp. on mushroom samples washed with the SC solution for 30, 60 and 120 s was lower than the initial number of Pseudomonas spp. on fresh mushroom. This indicates that SC is an effective antimicrobial agent and subjecting the mushrooms to the SC solution even for short times has a high inhibitory effect on the Pseudomonas spp.
The lowest Pseudomonas spp. counts on mushrooms were found for the SCP-120 at each sampling day. However, statistical analysis showed that there was no significant difference (p > 0.05) in the numbers of Pseudomonas spp. for the mushrooms subjected to the SC solution for 60 and 120 s regardless of overwrapped with the PVC film or not at any sampling day. In other words, treating the mushrooms with the SC solution for 60 or 120 s has the same inhibitory effect on the Pseudomonas spp. irrespective of packaging throughout 12 days of storage.

Effect of washing with SC solution and packaging on mushroom texture and weight loss
The firmness of the mushrooms is often one of the most important quality parameters considered by the consumers, and it is related to change in weight loss (Gao et al., 2014). The initial firmness of the fresh mushrooms was found to be 40 N, and the firmness decreased with the storage time (Figure 1). In other words, all the mushroom samples softened during 12 days of storage at 4 °C. One of the important reasons of the softening is the degradation of cell walls in mushrooms caused by bacterial enzymes and microorganisms especially Pseudomonas spp. that breaks down the intracellular matrix (Gao et al., 2014). The other important reason of the softening is shrinkage of the cell due to loss of water (dehydration). The firmness of the mushrooms washed with the SC solution was higher than the firmness of the control samples. The tendency in the bacteria-induced softening and the softening due to the loss of water could be slowed down with the aid of the SC washing treatments.
The firmness of all the mushroom samples treated with the SC solution and packed with the PVC film were significantly (p ≤ 0.05) higher than the firmness of the mushrooms solely treated with the SC solution and the controls after 8 days of storage ( Figure 1). This means that the SC washing of the mushrooms combined with the packaging retards softening of the mushrooms during storage. Similar results regarding the effect of packaging material on the firmness of mushrooms were also reported by González-Fandos et al. (2000) who found that the change in the firmness values of the mushrooms was delayed by packaging the mushrooms with the PVC film that increases the CO 2 level in the package and decreases the respiration rate. After 12 days of storage, the firmness value of the control decreased to 7 N while the firmness values of SCP-60 and SCP-120 were 27.4 N and 23.1 N, respectively. In other words, the decrease in the firmness of SCP-60 was lower than the decrease in the firmness of SCP-120. This indicated that subjecting the mushrooms to the SC solution longer than 60 s has an adverse effect on the firmness of the mushrooms. Therefore, SCP-60 was found to be the most appropriate treatment to minimize the change in the firmness of mushrooms.  Dehydration is important and directly affects the mushroom quality because mushrooms lose water from their epidermal layer during the postharvest storage (Khan et al., 2014). Weight loss increased with the storage time for all the mushroom samples ( Figure 2). The maximum weight loss occurred in the control and reached almost 40% after 12 days of storage. There was a significant difference (p ≤ 0.05) between the weight loss of control and SC-60 and SC-120 over the entire storage period. This means that subjecting the mushrooms to the SC solution longer than 30 s has reduced the weight loss. When the mushrooms were packed with the PVC film, weight loss was significantly (p ≤ 0.05) reduced as compared with the unpacked mushrooms treated with the SC solution over the entire storage period. Similar results were also reported by Oz et al. (2015) who found that packaging slowed down the dehydration of the mushrooms. After 12 days of   storage, the weight loss for the mushroom samples packed with the PVC film was less than 10%. This reveals that the PVC film acts as a good barrier against water loss from the mushrooms.

Effect of washing with SC solution and packaging on headspace gas composition and pH
O 2 and CO 2 concentrations inside the packages were measured immediately after the mushrooms were packed. O 2 levels in the packages decreased while CO 2 levels increased with the storage time because of the high respiration rate and the gas permeability properties of the PVC film (Gantner et al., 2017). There was no significant difference (p > 0.05) in gas composition values of the packaged mushroom samples. The rate of change in O 2 and CO 2 levels inside the packages was high during the first 4 days of storage, and then the rate of change of O 2 and CO 2 was diminished and levelled off. This means that the respiration rate of the mushrooms in the first 4 days was high and then the respiration rate decreased due to an increase in the CO 2 levels inside the packages. Similar results were also reported by Lagnika et al. (2011) who observed that an increase in CO 2 levels inside the packages caused an inhibitory effect on the respiration rate of mushrooms stored under MAP at 4 °C. The concentration of O 2 inside the packages decreased to around 15% while the CO 2 concentration increased to about 4% after 12 days of storage ( Figure 3). pH is important during storage because it affects stability and shelf-life of foods. The initial pH value of the fresh Agaricus bisporus was found to be 6.65 which is in good agreement with the pH values (6.50-6.92) reported for fresh Agaricus bisporus (Aday, 2016;Masson et al., 2002). The pH values of the mushroom samples varied between 6.58 and 6.85 during 12 days of storage at 4 °C ( Figure 4). There was no significant difference (p > 0.05) in pH values of the mushrooms stored at 4 °C for 12 days. This means that washing the mushrooms with the SC solution or packaging with the PVC film has not any effect on the pH values of the mushrooms stored at 4 °C for 12 days.

Conclusion
Treating the mushrooms with the SC solution had beneficial effects on maintaining the postharvest quality and reducing the Pseudomonas spp. counts. Washing the mushrooms with the SC solution and packaging with the PVC film slowed down the colour change of the mushrooms. The firmness of the mushrooms washed with the SC solution was higher than the firmness of the control, but treating the mushrooms with the SC solution longer than 60 s had an adverse effect on the mushroom firmness. Among different treatments used in our study, mushrooms washed with the SC solution for 60 s and stored at 4 °C with a PVC overwrapping material were found to be the most appropriate treatment to minimize the change on the postharvest quality of mushrooms. PVC film reduced the water loss and decreased the respiration rate of the mushrooms. Washing the mushrooms with the SC solution resulted in the mushrooms with lower Pseudomonas spp. counts than the controls. SC was an effective antimicrobial agent and subjecting the mushrooms to the SC solution for short period of times had a high inhibitory effect on the Pseudomonas spp. Washing the mushrooms with the SC solution could be used by the mushroom processors as an alternative to other postharvest treatments used for the mushrooms. The change on the postharvest quality of mushrooms could be minimized and mushroom shelf-life could be prolonged by the SC washing and packaging.