The standardization of the micro perforation was carried out by the adjustment of the point time (PT) which determines the focusing time of the beams and the optimal diameter and number of the holes. Several PT’s were tried in nine replicates and the optimum PT has been determined to obtain standard diameter and circular holes. The diameters of the holes were determined via a stereomicroscope (Leica CLS 150 XE) and their image was monitored by scanning electron microscopy (SEM Thermo Scientific Apreo II, USA). Minimum three replicates were performed for this process.

The number of holes micro perforated on the packaging film was calculated by considering the following parameters: The CO₂ transmission rate of the microhole, the headspace volume, the CO₂ volume in excess in the headspace. The CO₂ transmission rate was calculated by using the mean diameter of the measurements and equation (3) (González et al., 2008).

(3) T R = a 1 A h a 2

Where;

TR = CO₂ or O₂ transmission rate (mL day^-1); a₁, a₂ are the constants for O₂ or CO₂; A_h = area of each microhole (μm²).

The PBI Dansensor Checkmate (Ringsted, Denmark) digital analyzer was used to determine the O₂ and CO₂ concentrations in the headspace.

10 g of sample was homogenized with distilled water of 50 mL and then the measurement was performed with a pH meter.

It was determined gravimetrically and expressed in terms of percentage. The weight loss was calculated using the following equation:

(4) Δ M = M i − M t M t × 100

Where:

ΔM, M_i, and M_t denote the percentage change in the weight loss, initial weight, and weight at the instance of measurement respectively.

The color analysis was carried out by assessing L*, a*, and b* values. The instrument was calibrated with black and white standard plates before the analysis. The reported data are the mean of eight measurements made for each sample. Total color difference (ΔE) and browning index (BI) were calculated by using the equations below.

(5) Δ E   =   ΔL 2   +   Δa 2   +   Δb 2

(6) BI= 100 ( x-0.31 ) 0.172

(7) x =  a + 1.75L 5.645L + a - 3.012b

Five slices of each sample lot were analyzed in terms of hardness. The test velocity and the compression rate were adjusted as 1.5 mm.s^-1 and 75% respectively. The hardness of the sample was expressed in terms of the maximum force (g). The analysis was conducted in triplicate.

Briefly, 10 g of mushroom samples were added into the stomacher bags containing 90 mL 0.1% peptone solution. The samples were serially diluted in 0.1% peptone solution to the appropriate concentration after homogenization. The analysis was performed by the pour plate method using Plate Count Agar (PCA, Merck). The colonies were counted and the CFU g^-1 was calculated after incubation of the plates at 30 ±1 °C for 72 hours.

10 grams of the samples were weighed into Stomacher bags under aseptic conditions, and decimal serial dilutions were prepared until 10^-7. 0.1 mL of each decimal dilution were spread over the surface of Pseudomonas CFC Agar (Merck) plates and incubated at 25 °C ±1 °C for 48 h ± 2 h. After incubation, Petri dishes containing 30-300 colonies were counted.

The deterioration of the mushrooms might be caused by the impact of several anaerobic conditions, which also depend on the O₂ level in the headspace and accelerate the spoilage when the O₂ level becomes less than 1%, which may result in the tissue softening and undesired odors (Parentelli et al., 2007). Almenar et al. (2007) and De Reuck, Sivakumar and Korsten (2009) revealed that extremely low oxygen levels in the headspace might trigger anaerobic reactions which result in off-odors and offflavors. Thus, the O₂ level measured in the headspace might be a good indicator for microbial spoilage. On the other hand, CO₂ was claimed to have an important role in the retention of textural characteristics, when it is present at high concentrations in the headspace (Gonzalez-Fandos et al., 2001; Simón, Gonzáles-Fandos and Tobar, 2005). The quantitative changes in the headspace gas composition were presented in Figure 4.

As seen in Figure 4, the O₂ level in the headspace declined to 0.45% after 4 days of storage in the non-perforated samples, whereas it fell below 1% (0.4%) after 9 days of storage in the microperforated samples. Consequently, one might state that the microbiological spoilage might have started after 4 days in the non-perforated samples, and after 9 days of storage in the microperforated group.

The changes in the pH of the samples during the storage period were observed and the measurements were summarized in Table 1. No statistical difference could be detected among the samples in both groups concerning the storage period. The storage time and/or the microperforation process did not statistically affect the pH value. The pH values ranged from 6.69 to 6.92 and 6.86 in the nonperforated and microperforated samples respectively. These results are in agreement with those of several studies (Jaworska et al., 2010).

Over the 10 days of storage, the slight increase in the pH (1.9% and 3.2% for microperforated and non-perforated respectively) of the mushrooms was also reported by several studies (Tao et al., 2006). On the other hand, in another study (Oliveira et al., 2012b), it was reported a slight decrease in the pH from 6.74 to 6.5 after 7 days of storage. However, the pH values of the control samples were slightly higher than those of microperforated samples. This result was also stated by several scientists (Gholami, Ahmadi and Farris, 2017).

The weight loss during the storage period has been considered as one of the main factors influencing the microbial spoilage of the mushrooms. Regardless of the type of packaging material, the spoilage proceeds along the storage period. For most of the fruits and vegetables, a weight loss of 5 – 6% indicates a significant reduction in their commercial value due to the spoilage (Guillame et al., 2010). The changes in the weight loss of the mushrooms stored in non-perforated and microperforated packaged samples were presented in Table 2.

As presented in Table 2., the weight loss in the nonperforated and microperforated mushrooms was below 5% indicating that there was no decrease in the commercial value of the mushrooms during the storage period (Mahajan et al., 2007). Also, no significant difference was determined between the microperforated and nonperforated groups during each storage period. This result is in agreement with those of the previous research (El-Seifi et al., 2019). No statistical difference for the storage period could be detected among the non-perforated mushroom samples until the 9^th day of storage. On the other hand, a statistically significant difference was determined among the microperforated samples for each storage period (p <0.05). The weight loss on the 10^th day of the storage period was lower in the microperforated mushrooms (2.80%) than that of the non-perforated samples (3.59%). The scientists (El-Seifi et al., 2019) also reported a similar result for the dill in PP packages with 8 microholes of diameter (40 μ) after 7 days at 0 ºC plus 4 days at 10 ºC storage.

The Hunter Lab (L*, BI, and ΔE ) values of the nonperforated and microperforated fresh-sliced mushroom samples were shown in Table 3. As presented in Table 3., the L* values tended to decrease throughout the entire storage period in both groups of samples (non-perforated and microperforated). The results are consistent with the data of some other studies (Oliveira et al., 2012b; Gholami, Ahmadi and Farris, 2017). The L* values appeared to be higher in the microperforated samples than those of the non-perforated ones. This issue was evident for the undesired browning reactions that might have taken place in the non-microperforated samples. For all storage periods examined in this study, there was a statistical significance between the microperforated and nonperforated groups in the L*, BI, and ΔE values (p <0.05). The most important factor which might have an impact on the customer preferences for the mushrooms (Agaricus bisporus) is to be considered as the appearance and the browning in the tissue after being harvested. The enzymatic activities and microbial contamination might be responsible for the browning on the surface of the mushrooms (Nerya et al., 2006). Hence, the total color difference (ΔE), the lightness (L* ), and the browning index (BI) have been regarded as the most critical parameters during the storage and the changes in these values were presented in Table 3.

As presented in Table 3., the browning index and the total color difference values for both samples increased throughout the 10 day storage period. The L* values decreased considerably along the storage period in both groups. However, the L* values of non-perforated samples were lower compared to those of the microperforated ones during all storage periods. The difference in the L* values between microperforated and non-perforated samples was found statistically significant for each period (p <0.05). The BI values of both groups increased during the entire storage period. However, a statistically significant difference between the two groups was detected in all storage periods (p <0.05). BI values were determined as 70.96 and 80.79 and ΔE values were determined as 42.53 and 46.45 for microperforated and non-perforated samples respectively on the 10^th day of storage. BI values increased approximately by 257% and 307% since the beginning of the storage. In several studies (Oliveira et al., 2012b; Pogorzelska-Nowicka et al., 2020) such an increase in the BI values was observed along the storage period. The browning index and total color difference values of the microperforated samples were lower than compared to those of the non-perforated samples which might be associated with the microbial spoilage and enzymatic reactions that started earlier in the non-perforated samples. Several authors (Lin et al., 2017) reported that high CO₂ concentration might give rise to damage in the cap surface tissue of the mushrooms which also stimulates the browning and leads to higher BI values. On the 4^th day, the CO₂ concentrations in the package were close to 20% for microperforated and non-perforated packages. Hence, the increase in the BI values might be associated with the rise in the CO₂ concentration.

The textural characteristics of the mushrooms can be harshly influenced due to enzymatic activity and water loss during storage (Gholami, Ahmadi and Farris, 2017). Loss of turgor and softening of the tissues were reported as the main causes of the poor textural quality in the mushrooms by several authors (Guillame et al., 2010; Liu et al., 2010; Khan et al., 2014; Qin et al., 2015). The hardness is the most common feature used for the description of quality attributes for the button mushrooms (Agaricus bisporus). The higher the hardness, the better the sensory properties of the mushrooms (Gholami, Ahmadi and Farris, 2017). The changes in the hardness of the fresh-sliced mushrooms in the nonperforated and microperforated packaging films during the storage were presented in Table 4.

The aging process, the loss in the tissue hardness, and the overripening of the mushrooms lead to tissue softening characterized by a spongious texture (Villaescusa and Gil Muñoz, 2003). As expected, the hardness values of both groups of samples declined throughout the storage period. As shown in Table 4, no statistically significant changes in the hardness values of the microperforated samples could be observed during the first four days of storage. On the other hand, the decrease in the hardness values of non-perforated samples was higher than in those of the microperforated ones during each storage period. Therefore, one might state that the softening process occurred more obviously in the non-perforated samples. This result might be evident for the beneficiary effect of microperforation in terms of the retardation of tissue softening.

After 10 days of storage, the decline in the hardness was approximately 38% and 66% for the microperforated and non-perforated samples respectively. The loss in the hardness might be associated with the loss of cell urgency caused by the cell disruption, intercellular expansion of the pileal surface (Zivanovic, Buescher and Kim, 2000), the protein and polysaccharide degradation which also affect the cell permeability (Oliveira et al., 2012a). In the study (Pogorzelska-Nowicka et al., 2020), it was reported a higher decline (72.5%) in the firmness of the mushrooms in perforated packages after 7 days of storage at 15 °C. They used a macro-perforation design with 6 holes of a diameter of 330 microns which could allow more tissue softening due to the higher transmission rates of oxygen as a result of wider surface area.

Investigation of the pathogenic microorganisms was carried out at the beginning of the storage (day 0).

No pathogenic microorganisms were detected in the freshsliced mushroom samples.

The shelf life of the mushrooms was severely influenced by the growth of total aerobic mesophilic bacteria and Pseudomonas spp. Thus, analysis of the total aerobic mesophilic bacteria count and Pseudomonas spp. counts were performed along with the shelf life. The results were presented in Figure 5.

As shown in Figure 5, since the initial microbial load was over 5 log CFU.g^-1 the number of total microorganisms rapidly increased during the first days of storage. The total count of Pseudomonas spp. increased from 5.40 (day 0) to 8.88 log CFU.g^-1 in the microperforated packaged samples at the end of the storage period (day 10), while a quite rapid increase in the number of Pseudomonas spp. was observed in the non-perforated packaged samples on the 4^th day by reaching a value of 8.78 log CFU.g^-1. On the other hand, the TAMB count increased more slowly compared to the count of Pseudomonas spp. and reached in the microperforated and non-perforated packaged samples 8.79 log CFU.g^-1 and 8.92 log CFU.g^-1 respectively at the end of the storage. On the other hand, the TAMB count increased more slowly compared to the count of Pseudomonas spp. and reached in the microperforated and non-perforated packaged samples 8.79 log.CFU g-1 and 8.92 log CFU.g^-1 at the end of the storage. The studies concerning the determination of the shelf life of the fresh-sliced mushrooms reported similar results to our findings and revealed that Pseudomonas spp. was regarded as one of the main factors leading to microbial spoilage of mushrooms (Gonzalez-Fandos et al., 2000; Srivastava et al., 2010; Wang et al., 2017). Unfortunately, no legislative regulation specific to the microbial spoilage is available about the fresh-cut produce except for the pathogenic microorganisms. However, basically above the level of 8 log CFU.g^-1 can be considered as the inception of deterioration, even though no risks in terms of food safety might arise at this level and it is important for food quality because Pseudomonas spp. are saprophytic microorganisms that lead to deterioration in mushrooms. As a result of the microbial spoilage, several qualitative defects such as changes in color, undesired odor, and softening might be encountered which affect the consumer demand (Simón, González-Fandos and Tobar, 2005). Based on the counts of Pseudomonas spp. and TAMB, we can conclude that the microbial deterioration started on the 7^th day of storage in the microperforated packaged samples whereas it started on the 4^th day of storage in the non-perforated packaged samples. Tissue softening due to microbial spoilage and/or drip loss occurred in the microperforated packaged samples on the 7^th day according to the hardness values. On the other hand, there was a remarkable decrease in the hardness in the non-perforated samples on the 2^nd day.

The changes in the sensory characteristics of the samples during storage were presented in Table 5.

As summarized in Table 5, the scores of the odor, texture, appearance, and overall acceptability decreased with the storage period for each group of samples. However, the scores for the sensorial attributes of the microperforated packaged samples were higher than the non-perforated packaged samples. The sensorial attributes were maintained for microperforated packaged samples until the 9^th day of storage and on this period the sensorial scores were below 3 which is the limiting value to accept the food in 5 points hedonic scale for the sensorial attributes.

However, the sensorial properties of non-perforated packaged samples were acceptable until the 4^th day of storage. Since we couldn’t analyze on the 5^th or 6^th day of the storage, it is not clear if the non-perforated packaged samples were acceptable or not on these days. However, the non-perforated packaged samples weren’t accepted because of their poor sensorial attributes all including texture, odor, appearance, and overall acceptance on the 7^th day of storage. For this reason, it is not clear whether the non-perforated packaged samples were acceptable or not after the 4^th day of storage. Several scientists (Eastwood and Burton, 2002) reported that the consumers mostly prefer the mushrooms with fewer spots and better appearance even though the aged mushrooms have a better taste. Thus, the overall acceptability of the consumers depends much more on the color, texture, and appearance than the flavor of the mushrooms. Based on the sensorial properties, we can conclude that the shelf life of fresh-sliced mushrooms can be extended to 8 days with microperforated packaging. Whereas the shelf life of fresh-sliced mushrooms was less than 7 days (4, 5, or 6 days). It was shown that the microperforation process was effective to keep the sensorial properties and extending the shelf life of fresh-sliced mushrooms as stated by (Dhalsamant et al., 2015).