Comparative Evaluation of Storage Temperatures on Physiological and Microbiological Parameters of Chemically Treated Button Mushroom (Agaricus bisporus) Stored under Modified Atmospheric Packaging (MAP)

The growing demand for low-cholesterol, low-fat foods rich in nutrients, vitamins, and medicinal properties is expected to drive the global button mushroom market. However, button mushrooms have a short post-harvest lifespan, typically lasting only 1-4 days at ambient conditions and 4-6 days under refrigeration. This rapid deterioration is primarily attributed to high transpiration rates, metabolic activity, and moisture loss from their delicate epidermal structure (Ares et al., 2007). The perishable nature of button mushrooms, coupled with significant post-harvest losses (30–35%), poses challenges for long-distance transportation and international trade. Common post-harvest issues such as browning, veil-opening, weight loss, and microbial degradation often result in substantial economic losses.
       
Modified atmosphere packaging (MAP), involving the alteration of ambient air composition to create an optimal environment for decreasing the mushroom’s respiration rate and extending its shelf life, has shown promise in mitigating these challenges. Treatments such as chemical applications (e.g., calcium chloride, sorbitol and citric acid), MAP and low-temperature storage have been demonstrated to effectively prolong the shelf life of button mushrooms to 12-16 days (Koushki et al., 2011; Oliveiraa et al., 2012).
       
The primary objective of this study is to determine the storage duration of button mushrooms packed under MAP while maintaining acceptable microbiological counts. Assessment of total plate count and yeast mold estimation will provide insights into the suitability of button mushrooms for consumption purposes (Farzana et al., 2016).

Button mushrooms (Agaricus bisporus) were obtained from the Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, in the morning. Chemicals were supplied from Himedia (Mumbai, India). Button mushrooms were packed in polybags made of Polyamide 20µm-Polyethylene 70µm with EVA (Ethylene-vinyl acetate sealant layer). Oxygen permeability and water vapour transmission rate were 50 cm³/m².day.bar (both at 23°C and 0% RH) and 2.8g/m².day (at 23°C and 85% RH), respectively.
 
Processing and packaging procedures
 
Fresh button mushrooms, free from wounds and blemishes, were selected and cleaned. Fresh button mushrooms free from physical injuries were dipped in a solution of CaCl₂ (2.5%, w/v), citric acid (3%, w/v) and sorbitol (0.1 %, w/v) for 4 min and were air-dried for 12 hours (at 25±5°C and RH 85±5%). Test samples were prepared by placing (40±5 g) chemically treated button mushrooms in polyethylene packets of Polyamide 40 µm-Polyethylene 120 µm with EVA (Ethylene-vinyl acetate sealant layer). Packaging button mushrooms followed chemical treatment under different MAP treatments by modified atmospheric packaging unit (VAC-STAR* S 220 MP, Sugiez, Switzerland).
       
In case of MAP treatments we had taken three MAP treatments i.e.
I. MAP1 (12% CO₂; 6% O₂).
II. MAP2 (12% CO₂; 6% O₂).
III. MAP3 (Control).
       
The button mushrooms after chemical treatments and MAP packaging were stored at 8±1°C and 12±1°C for 16 days, and observations have recorded an interval of 4 days with the three replications.
       
After providing with different MAP treatments, the button mushroom air tight packets were stored at three different temperatures i.e.
I.  Low temperature (8±1°C).
II. Normal temperature (12±1°C).
 
 
Headspace gas analysis
 
Percentage CO₂ and O₂ concentrations were determined with the help of a gas analyzer (MAP Mix 9001 ME, PBI Dansensor, Ringsted, Denmark) using a needle were inserted in samples through the septum to assure the closure of the hole, the gas composition was recorded.
 
Total soluble solids
 
Button mushroom extract was prepared and was analyzed with the help of a refractometer (Model RFM 970, Bellingham and Stanley, UK) (Eissa, 2008) for finding total soluble solids.
 
Ascorbic acid content 
 
Ascorbic acid standard
 
100 mg of L-ascorbic acid was measured and volume was made up to 100 ml with 3% HPO₃ solution (Harris and Ray, 1935).
 
2, 6-di-chlorophenol indophenol dye solution
 
42 mg of sodium carbonate was dissolved in distilled water and 52 mg of 2, 6-di-chlorophenol indophenols was mixed into it followed by volume make up to 200 ml.
 
Standardization of dye
 
5 ml of the standard ascorbic acid solution was taken into a 100 ml flask, followed by 5 ml of the 3% HPO₃ solution. Microburette was filled with the dye solution. The ascorbic acid solution was titrated with the dye solution until the appearance of pink colour. Titrated value was recorded.
 
Dye factor: 0.5/Titrate value.
 
Oven-dried button mushrooms were ground into powder form in a mixer grinder and samples 2 g were taken and dissolved in 3 % HPO₃ and volume was made up to 100ml. The aliquot was centrifuged at 2000 g for 15 minutes. 5 ml of supernatant was taken into 10 ml of 3% of HPO₃ and titrated against the dye (V₂ in ml). The initial and final reading of the dye solution was recorded in the presence of pink colour. The concentration of ascorbic acid in mg/g sample was estimated by formula:                             
   
Where,
W = Weight of sample taken.
V₁ = Sample of extract taken.
V₂ = Required dye solution for titration.
 
Total polyphenol content (TPC)
 
Oven-dried button mushrooms at 65°C were taken and ground into fine powder. Dried mushroom powder (1 g) was mixed with 15 ml of distilled water. The aliquot was kept on a shaker incubator at 150 rpm for 24 hours at room temperature. Button mushroom samples were taken followed by filtration and were centrifuged at 2000 rpm for 15 minutes. Total polyphenol compounds of mushrooms extract were calculated with the help of the Folin-Ciocalteu reagent. An aliquot of 100µL (1:5 dilution) was mixed with 100 µl of Folin–Ciocalteu reagent (1:1 dilution with distilled water) and 2 ml of 10% sodium carbonate solution was added.The absorbance was recorded in a spectrophotometer  at 750 nm. The same extract of button mushroom prepared for total polyphenols was used for finding the radical scavenging activity. The absorbance of the samples was recorded at 515 nm using a spectrophotometer.
 
Total plate count (TPC)
 
One gram of Agaricus bisporus was homogenized in 9.0ml sterile 0.1% peptone water for 30 seconds to a homogenous suspension. 17.5 g of plate count agar was dissolved in 1000 ml distilled water followed by heating for complete dissolution. All the procedure was done in sterilized conditions in laminar flow (Omicron Sensing Inc. U.S.A, model: H2000-25mmWC). Plates were incubated at 37°C for 24 hours in an incubator (Metrex Scientific Instruments (P) Ltd., New Delhi). The colonies were counted with the help of a digital colony counting meter, and colonies were expressed in a log colony-forming unit per gram (log cfu/gm). (Harrigan, 1998).
 
Yeast and moulds estimation
 
Yeast and molds were determined by the pour plate method. In aseptic condition, 25 g of test pieces were kept in a conical flask with a glass stopper which contains 225 ml of phosphate-buffered saline. Then conical flask contents were transferred into a sterile blender jar and blended at low speed for 1 minute. The 10^-1 (1:10) dilution was obtained by adding 1 ml of aliquot to 99 ml of phosphate-buffered saline. Then again, we used a sterile pipette to transfer 1ml of 10^-1 dilution to 99 ml of phosphate-buffered saline followed by cap placing shaking for 3 minutes (Teramura et al., 2015) :
   
Statistical analysis
 
All the observations were recorded in triplicates, and data were processed to two-way analysis of variance (ANOVA) considering different chemical, MAP treatment and storage time. S.Em± (Standard error of the mean) and critical difference at 5% was calculated (Dean and Voss, 1999).

The blanched samples spoiled after 12 days, whereas chemically treated and MAP packed samples were intact even after 16 days. After 16 days of the storage period, CM1 had better retention in the quality parameter than other samples.
 
Headspace gas composition
 
A rapid decline in oxygen (O₂) [Fig 1(A) and Fig 1(B)] concentration and a simultaneous increase in carbon dioxide (CO₂) [Fig 2(A) and Fig 2(B)] concentration were observed at 8°C. Similar observations of decreased O₂ and increased CO₂ concentrations under modified atmosphere packaging (MAP) treatments during storage were documented in previous studies by researchers (Antmann et al., 2008; Jafri et al., 2013). The higher transpiration rates of button mushrooms at elevated temperatures, along with the permeability of polyethylene packaging bags, are likely contributing factors to the changes in gas concentration during storage.
 

 

 
Total soluble solids
 
An increase in TSS is possibly due to converting polysaccharides like cellulose, starch, pectin etc., into simple sugars like glucose and fructose. The rise in TSS of sample CM1 was slower at both 8°C and 12°C than other samples (Díaz-Mula et al., 2011). The TSS of CM1 after 16 days of storage at 8°C and 12°C was 6.09% and 7.10%, respectivelyas shown in [Fig 3(A) and Fig. 3(B)].  Higher temperature increases the tissue temperature of button mushrooms, which initiates the biochemical reactions responsible for the deterioration of mushroom quality (Gholamia et al., 2017).
 

 
Ascorbic acid content
 
A sharp decline in ascorbic acid content was recorded with the storage period. Ascorbic acid is thermolabile in nature, and blanching in hot water can lead to appreciable loss of its concentration. Low-temperature storage is possibly the most suitable method for controlling ascorbic acid loss (Ishaq and Obirinakem, 2015). The rate of decrease of ascorbic acid content was higher at 12°C as compared to 8°C as shown in in [Fig 4(A) and Fig 4(B)]. A minimum decrease in ascorbic acid content was recorded for CM1 at 8°C.
 

 
Total polyphenol content
 
Polyphenol compounds with anti-oxidant activity are present in methanolic extracts button mushrooms (Tsai et al., 2007). Total phenolic content in fresh button mushrooms was recorded as 3.50 mg GAE/g fresh weight, continuously falling off with storage time. Sample CM2 (chemically treated, 6% CO₂; 12% O₂) observed a minimum (2.75 mg GAE/g fresh weight) decrease in total phenolic content at 8°C after 16 days. A significant reduction in total phenolic content (P<0.05) was recorded in all the samples. At the end of the storage period of 16 days, reduced phenolic content was found to be maximum at 12°C. Blanched samples lost more than 50% of phenolic content after 12 days of storage at 12°C as depicted in in [Fig 5(A) and Fig 5(B)] (Jiang et al., 2012).
 

 
Free radical scavenging activity (RSA)
 
Radical scavenging activity of fresh button mushroom was found to be 10.25 mg AA/100 g fresh weight which follows the decreasing trend with the storage period reported by Alasalvar et al., (2005) for modified air packed purple carrots and Jiang et al., (2012) for chitosan-coated mushrooms. Studies showed fall-off in ascorbic acid content, total polyphenol content and free radical scavenging activity go along with the same pattern, indicating radical scavenging activity relies on both ascorbic acid content and phenol content (Utto et al., 2013; Jafri et al., 2013). An inference that can be drawn was that the combined effect of MAP, chemical and temperature treatments could effectively maintain the anti-oxidant activity of button mushroom more prominent than MAP treatment in maintaining anti-oxidant activity (Sami et al., 2021). The trends are shown in Fig 6(A) and Fig 6(B).
 

 
Total plate count
 
TPC shows a steep shoot up with both temperature and storage period. Considering the general trend of temperature effect on TPC, it was evident that an increase in temperature increases the exponential growth phase of bacterial count with a short duration (Gaglio et al., 2019).  This investigation observed that 44% of chemically and MAP treated button mushrooms were in the maximum desirable count of the total. After 12 days of the storage period, bacterial colonies were too numerous to count (TNTC), so recorded data was taken up to the 4^th dilution. Citric acid provides an acidic environment to the bacteria, which helps to reduce the growth of colonies (Rossouw and Korsten, 2016).
 
Yeast and molds count
 
Button mushroom surface content of yeast and molds increases sharply with the storage period (Eldin et al., 2016). Initially, yeast and molds count recorded for fresh button mushroom was 30.23 cfu g^-1 × 10^-2, which reached 229.67 cfu g^-1 × 10^-2 at the end of the storage period. Samples treated with CaCl₂, citric acid and sorbitol and packed in  12% CO₂, 6% O₂ stored at 8^oC were best in controlling yeast and mold counts for more extended periods.

All chemical, MAP, and temperature treatments exhibited a general decline in quality parameters, with particularly rapid degradation observed at 12^oC. However, button mushrooms packed under a modified atmosphere demonstrated superior performance even after extended storage periods of up to 12 days at 8°C (Singh et al., 2018). The accelerated degradation at higher temperatures may be attributed to tissue softening, possibly resulting from protein and polysaccharide degradation, as well as vacuole disruption. Chemical treatments involving calcium chloride, citric acid, and sorbitol yielded the most favorable outcomes compared to blanched samples. Given the highly perishable nature of button mushrooms, distinct preservation methods such as treatment with calcium chloride, citric acid, sorbitol, or overwrapping with polyethylene films containing modified atmospheres are essential. Various alternative techniques, including refrigeration, calcium chloride cultivation, ionizing radiation, and coating, can also be explored to enhance the post-harvest shelf life of button mushrooms.

The corresponding author is highly thankful to Banaras Hindu University for providing her UGC Fellowship during her doctoral programme.

The authors would hereby like to declare that there is no conflict of interests that could arise.