E ﬀ ect of Titanium Dioxide Nanocomposite Material and Antimicrobial Agents on Mushrooms Shelf-Life Preservation

: Mushrooms have limited shelf-life and it can be prolonged if suitable conditions and treatments are e ﬀ ectively applied. In this study, nanocomposite material and antimicrobial agents with a combination of chitosan were used as novel packaging material for mushroom preservation. The microbiological analysis, physicochemical properties, headspace gas analysis, and polyphenol oxidase activity (PPO) during cold storage were investigated. As compared with control, coated mushrooms with chitosan (CHS), and nano-titanium dioxide CHSTiO 2 thymol + tween-80 CHSTiO 2 / TT80 coating treatment showed signiﬁcantly ( p ≤ 0.05) lower respiration rate, microbial contaminations (4.27 log CFU / g), and (5.93 log CFU / g) for total yeast / mold and aerobic plate counts, respectively. The weight loss ratio was the lowest for CHSTiO 2 / TT80 (10.88% loss) followed by CHSTiO 2 (11.76% loss). CHSTiO 2 / TT80 recorded a higher electrolyte leakage rate (25.84%) and acidity. While the lowest PPO activity was established for CHSTiO 2 (17.09 U mg − 1 Protein), while the lowest values for total soluble solid concentrations were reported for CHSTiO 2 / TT80 mushrooms (4.91%). These results indicated that CHSTiO 2 / TT80 coating treatment might delay the aging degree of white button mushrooms and be investigated as a novel packaging material for other food products in the future.


Introduction
Mushrooms are highly perishable due to their high respiration, metabolic activities, and transpiration rates [1]. Themushroom's shelf-life can be prolonged if the suitable conditions and treatment are effectively applied, as chilling at 4 • C can prolong the shelf life up to 3-4 days compared to the ambient temperature (1-2 days) [2]. Singh et al. [3], reported that the properties,

Weight Loss Ratio and Texture Measurements
The weight loss ratio (%) for each mushroom treatments was evaluated by dividing the weight change by using a digital balance with an accuracy of 0.01 g and calculated by Equation (3): where, w i is the initial weight, while w f is the weight during the storage period. The firmness of mushroom samples was detected on the top side of nine mushroom pieces for each treatment by using an FHR-1 (1 kg) a texture analyzer with a speed of 2 mm/s, diameter 5 mm cylinder-type, 250 N load cell, and 0.5 Ncontact force (Nippon CO., Tokyo, Japan) [15].

Headspace Gas Analysis and pH
The gas compositions, carbon dioxide (CO 2 ) and oxygen (O 2 ), inside the headspace of packaged mushrooms were detected by using a gas chromatograph (GC) (Check Mate-II, Ringsted, Denmark) Propaq-Q and Molecular Sieve 5A columns (25 m × 0.5 mm i.d. × 1 pm)were used for CO 2 and O 2 determinations, respectively. Mushroom samples were detected by a gas-tight syringe septum and placed on the film exterior, while the carrier of the gas was the helium flow of (2.5 mL/min) and detection of FID (250 • C). The chromatography was applied in triplicate with a thermal conductivity detector [16]. The pH value of the mushroom juices was detected after homogenization, filtration, and the use of a digital pH meter (S20-K, Columbia, OH, USA).

Enzyme Activity Analysis and Total Soluble Solids
The polyphenol oxidase activity (PPO) was evaluated by using a kit (Solarbio, Beijing, China) according to the protocol method reported by the manufacturer's instructions by mixing 1 mL pyrocatechol (50 mM) and 1 mL of sodium phosphate buffer (100 mM, pH 7.0) with detection at 410 nm. (PPO) activity was expressed at U mg −1 Protein [17]. The total soluble solids (TSS) of mushroom samples were homogenized, filtered by a 40 µm filter pape, and evaluated by a hand-held refractometer with a resolution of 0.01in three replications (Krüss, Hamburg, Germany) [18].

Membrane Permeability Analysis and Open Cap Percents
Randomly selected mushroom bodies (5 g) were cut into several parts, leaving the pileus intact and suspended in a 50 mL beaker of deionized water. Electrical conductivity was detected and recorded (P0) then detected again after 10 min (P1) after the addition of deionized water several times and Processes 2020, 8, 1632 4 of 12 soaking for 1 h. Mushroom tissues were boiled for 10 min then (P2) was detected after cooling [19]. Electrolyte leakage rate (%) was described by Equation (4): The quality of white button mushrooms was detected according to the development of the opening cap and described by Equation (5) [20]: where, N t is the total mushroom numbers and N ac is the open cap numbers.

Statistical Analyses
The normal test (Kolmogorov_Smirnov) was done to check the normal distribution of the samples. Analysis of variance (ANOVA) was used to compare the storage period for each treatment. Duncan's tests as post hoc were performed to investigate the differences between days interval at p ≤ 0.05. The computer program SPSS software for windows version 22.0 was used for statistical analysis (Statistical Package for Social Science, Armonk, NY, USA: IBM Corp) at significant levels 0.05 (p-Value ≤ 0.5), while the charts were drawn by Origin 8 software.

Microbiological Analysis
The yeast and mold counts of CHSTiO 2 and CHSTiO 2 /TT80 coating treatments did not vary significantly for the first 9 days but were raised by prolonging the storage time, Table 1. The yeast and mold counts of CHS coating (6.17 log CFU/g) and CHSTiO 2 coating (6.13 log CFU/g) treatments were higher than (control) samples on days 12. The lowest count was established in CHSTiO 2 /TT80 coating treatment (4.27 log CFU/g). It was noticed that the addition of thymol and tween-80 as antimicrobial agents have suppressed the growth of total yeast and mold loads [12]. Rok [14] reported that the pH values and high sugar contents are the mean reasons for microbial growth enhancement.
According to Table 1, aerobic plate counts were efficiently obtained for CHSTiO 2 /TT80 coating treatments (5.93 log CFU/g). Furthermore, CHS and CHSTiO 2 coating treatments established parallel values (6.43 log CFU/g). The reduction of aerobic plate counts in CHSTiO 2 /TT80 mushroom samples can be due to the presence of thymol and tween-80 as antimicrobial agents. In agreement with the aerobic plate counts, Karimirad et al. [21] reported the strong effect of chitosan nanoparticles on the mushroom contaminations and shelf life extension. The combination of nano-films with the antimicrobial agents leads to control the electronegative and polycationic on the surface of the mushrooms for the modification of cell permeability [12].

Color Attribute Changes
Mushrooms have a very short shelf-life due to turning brown and losing quality within a few days as the majority significant parameter for customer approval is the color [4]. The L* value reduced and the browning index increased with the storage period onwards. The results for color attribute changes are shown in Figure 1. Although, on the sixth day, L* value values were in parallel in all coating treatments compared with (control), Figure 1a. CHS coating treatment preserved lightness (12.81% loss) as compared to other coatings treatments, whereas CHSTiO 2 /TT80 mushroom samples established the maximum (20.91% loss) on day 12. Weight loss and enzyme activities might be the major reasons for the optical reduction. Parameter L*, depending on the mushroom reflectivity surface that can show the luminosity [22]. Gholami et al. [2] reported that the lower lightness values can be due to the coating films that can cause some changes on the mushroom surfaces.
a* and b* values of all treatments were raised for the duration of the storage period, while the increase in control samples (9.12-24.56% loss) was greater compared with the coated mushrooms, respectively Figure 1b,c. The larger a* value linked to the enzymatic browning increase during the storage period [5].
The browning index can be influenced by L* value decrease, as it is one of the major quality features for white mushroom deterioration measurements and freshness. As shown in Figure 1d, the browning index values raised with the upwards of storage days. Attractively, at day 12, CHSTiO 2 /TT80 (0.71% loss) mushrooms significantly reduced the browning index compared with the control and other treatments. As a result, the presence of thymol and tween-80 could delay repining and preserve color. Lin et al. [23] established that modified atmosphere packages also can control the browning index, physiological injuries, and cell membrane damage.
Processes 2020, 8, x FOR PEER REVIEW 5 of 11 mushroom contaminations and shelf life extension. The combination of nano-films with the antimicrobial agents leads to control the electronegative and polycationic on the surface of the mushrooms for the modification of cell permeability [12].

Color Attribute Changes
Mushrooms have a very short shelf-life due to turning brown and losing quality within a few days as the majority significant parameter for customer approval is the color [4]. The L* value reduced and the browning index increased with the storage period onwards. The results for color attribute changes are shown in Figure 1. Although, on the sixth day, L* value values were in parallel in all coating treatments compared with (control), Figure 1a. CHS coating treatment preserved lightness (12.81% loss) as compared to other coatings treatments, whereas CHSTiO2/TT80 mushroom samples established the maximum (20.91% loss) on day 12. Weight loss and enzyme activities might be the major reasons for the optical reduction. Parameter L*, depending on the mushroom reflectivity surface that can show the luminosity [22]. Gholami et al. [2] reported that the lower lightness values can be due to the coating films that can cause some changes on the mushroom surfaces.
a* and b* values of all treatments were raised for the duration of the storage period, while the increase in control samples (9.12-24.56% loss) was greater compared with the coated mushrooms, respectively Figure 1b,c. The larger a* value linked to the enzymatic browning increase during the storage period [5].
The browning index can be influenced by L* value decrease, as it is one of the major quality features for white mushroom deterioration measurements and freshness. As shown in Figure 1d, the browning index values raised with the upwards of storage days. Attractively, at day 12, CHSTiO2/TT80 (0.71% loss) mushrooms significantly reduced the browning index compared with the control and other treatments. As a result, the presence of thymol and tween-80 could delay repining and preserve color. Lin et al. [23] established that modified atmosphere packages also can control the browning index, physiological injuries, and cell membrane damage.
After 12 days of the storage period, the largest ΔE* values were established for the CHS (48.39), Figure 1e. Walkowiak-Tomczak et al. [24] confirmed that when the total color variation values were more than five it observed two colors impression. The minimum color differences were observed for CHSTiO2/TT80 (ΔE* from 37.79 to 40.50), as a total color variation of more than two means a significant color difference. Tarlak et al. [15] confirmed that color can be influenced by several factors such as enzyme oxidations and microbial population. Rok et al. [25] confirmed that the reduction of the color attribute could be due to the polyphenolic compounds oxidation reactions.

Weight Loss Ratio and Texture Measurements
Weight loss can be occurred due to several factors such as respiration, microbial growth, and transpiration during the storage period. Figure 2a shows a significant (p ≤ 0.05) weight loss during storage for all samples. As expected, the weight losses were significantly the lowest for CHSTiO2/TT80 (10.88% loss) followed by CHSTiO2 (11.76% loss) compared with the control samples due to the presence of nanocomposite material and antimicrobial agents which retained the respiration, inhibited the microbial growth, and delayed the enzyme activities [25].
The mushroom texture is a vital item for overall acceptance, which is influenced by the quality during the marketing [26]. Control samples were reduced rapidly during the storage onwards by 3.91 N from the initial value, Figure 2b. It was also obvious that the firmness of CHS mushrooms was significantly (p ≤ 0.05) suppressed on day 12 and had 5.11 N. Firmness loss can be influenced by several factors such as the biochemical and microbial processes [27]. However, CHSTiO2 mushrooms established that the nanocomposite material might decrease cell-wall-degrading enzyme activities, due to its high anti-oxidation capacities during the storage time [14]. Our results were linked with the finding of Gholami et al. [2] who established that applying nanocomposite materials can enhance the firmness of mushroom samples. After 12 days of the storage period, the largest ∆E* values were established for the CHS (48.39), Figure 1e. Walkowiak-Tomczak et al. [24] confirmed that when the total color variation values were more than five it observed two colors impression. The minimum color differences were observed for CHSTiO 2 /TT80 (∆E* from 37.79 to 40.50), as a total color variation of more than two means a significant color difference. Tarlak et al. [15] confirmed that color can be influenced by several factors such as enzyme oxidations and microbial population. Rok et al. [25] confirmed that the reduction of the color attribute could be due to the polyphenolic compounds oxidation reactions.

Weight Loss Ratio and Texture Measurements
Weight loss can be occurred due to several factors such as respiration, microbial growth, and transpiration during the storage period. Figure 2a shows a significant (p ≤ 0.05) weight loss during storage for all samples. As expected, the weight losses were significantly the lowest for CHSTiO 2 /TT80 (10.88% loss) followed by CHSTiO 2 (11.76% loss) compared with the control samples due to the presence of nanocomposite material and antimicrobial agents which retained the respiration, inhibited the microbial growth, and delayed the enzyme activities [25].
The mushroom texture is a vital item for overall acceptance, which is influenced by the quality during the marketing [26]. Control samples were reduced rapidly during the storage onwards by 3.91 N from the initial value, Figure 2b. It was also obvious that the firmness of CHS mushrooms was significantly (p ≤ 0.05) suppressed on day 12 and had 5.11 N. Firmness loss can be influenced by several factors such as the biochemical and microbial processes [27]. However, CHSTiO 2 mushrooms established that the nanocomposite material might decrease cell-wall-degrading enzyme activities, due to its high anti-oxidation capacities during the storage time [14]. Our results were linked with the finding of Gholami et al. [2] who established that applying nanocomposite materials can enhance the firmness of mushroom samples.

Headspace Gas Analysis and pH
The changes in headspace gas concentrations control and coated mushroom samples are presented in Figure 3. However, after three days of the storage period, O2 concentration ratio was reduced in mushroom samples during the respiration process, Figure 3a. Decreases values differed according to the coating treatments. In detail, O2 concentration in control samples (19.25%) + CHS (16.57%) decreased slightly, while in both CHSTTiO2 (5.52%) and CHSTiO2/TT80 (2.17%), the O2 concentration consumptions were very low due to the effective oxygen barrier properties of nanocomposite material and antimicrobial agents. A subtle increase was detected from day 9 to day 12 for CHSTiO2 and CHSTiO2/TT80 mushrooms, which might be a sign of O2 permeation through the mushroom packages. In contrast to oxygen, carbon dioxide concentration was raised to reach (5.73%) in (control) samples on day 12 due to the respiration and permeation, while the deposition of other coating treatments did not, Figure 3b. Qin et al. [28], reported similar values for carbon dioxide concentration as it may influence by white button mushroom quality.
Mushroom samples CHSTiO2/TT80 followed by CHESTiO2 established the best results as there were slightly higher acidity values from the initial pH value, Figure 3c. The decrement in pH was recorded for CHS mushroom samples 6.40 compared with the inertial ph 6.56 on day 12. Higher O2 concentrations activate microorganisms on foods as visual bacteria growth was observed with the storage time on control and CHS samples. The pH of the coated mushroom with CHS decreased during storage can be due to the microbial growth population rate [29]. The evaluated pH values were in agreement with the results from the literature [3,30].

Headspace Gas Analysis and pH
The changes in headspace gas concentrations control and coated mushroom samples are presented in Figure 3. However, after three days of the storage period, O 2 concentration ratio was reduced in mushroom samples during the respiration process, Figure 3a. Decreases values differed according to the coating treatments. In detail, O 2 concentration in control samples (19.25%) + CHS (16.57%) decreased slightly, while in both CHSTTiO 2 (5.52%) and CHSTiO 2 /TT80 (2.17%), the O 2 concentration consumptions were very low due to the effective oxygen barrier properties of nanocomposite material and antimicrobial agents. A subtle increase was detected from day 9 to day 12 for CHSTiO 2 and CHSTiO 2 /TT80 mushrooms, which might be a sign of O 2 permeation through the mushroom packages. In contrast to oxygen, carbon dioxide concentration was raised to reach (5.73%) in (control) samples on day 12 due to the respiration and permeation, while the deposition of other coating treatments did not, Figure 3b. Qin et al. [28], reported similar values for carbon dioxide concentration as it may influence by white button mushroom quality.  Mushroom samples CHSTiO 2 /TT80 followed by CHESTiO 2 established the best results as there were slightly higher acidity values from the initial pH value, Figure 3c. The decrement in pH was recorded for CHS mushroom samples 6.40 compared with the inertial ph 6.56 on day 12. Higher O 2 concentrations activate microorganisms on foods as visual bacteria growth was observed with the storage time on control and CHS samples. The pH of the coated mushroom with CHS decreased during storage can be due to the microbial growth population rate [29]. The evaluated pH values were in agreement with the results from the literature [3,30].

PPO Activity and TSS Concentration
In all the coating treatments, the PPO activity (Figure 4a) was raised with the progress of the storage time, while the maximum enzyme value was detected in control (45.49 U mg −1 Protein) after 12 days. The lowest PPO activity was established forCHSTiO 2 (17.09 U mg −1 Protein), while its initial value was (12.62 U mg −1 Protein). Besides, CHS obtained lower PPO activity (32.29 U mg −1 Protein) compared with the control (45.49 U mg −1 Protein) could be due to the chitosan component. Karimirad et al. [21] explained that mushroom browning is a result of phenolic oxidation by polyphenol oxidase activity. Meanwhile, Wei et al. [6] reported that PPO enzyme is the main reason for browning in mushrooms as it catalyzes the polyphenolic matrix to create dyes, which reduces the marketability. Consequently, nanocomposite material inhibited color changes and the ability to retain antioxidant phenolics in mushroom bodies.
TSS concentration decreased with the storage period due to higher respiration and ripening rates [8]. CHSTiO 2 /TT80 mushrooms (4.91%) showed the best results as compared to the control (5.15%), Figure 4b. This study shows that the senescence rate was the lowest in the case of nanocomposite material with the addition of thymol and tween-80 as antimicrobial agents [6]. TSS concentration decreased with the storage period due to higher respiration and ripening rates [8]. CHSTiO2/TT80 mushrooms (4.91%) showed the best results as compared to the control (5.15%), Figure 4b. This study shows that the senescence rate was the lowest in the case of nanocomposite material with the addition of thymol and tween-80 as antimicrobial agents [6].

Membrane Permeability and Open Cap Percents
Membrane permeability percentage reflected frequently by the electrolyte leakage rate [31]. According to Figure 5a, the electrolyte leakage rate increased as long as the storage time in all coated samples, indicating a decrease in the mushroom membrane integrity. Furthermore, CHSTiO 2 /TT80 mushrooms (25.84%) exhibited a significantly (p ≤ 0.05) lower electrolyte leakage rate than the other samples, which might be attributed to the presence of thymol and tween-80. Other reports suggested the lower value of mushroom membrane integrity was directly related to the mushroom browning and lipid peroxidation [32][33][34].

Membrane Permeability and Open Cap Percents
Membrane permeability percentage reflected frequently by the electrolyte leakage rate [31]. According to Figure 5a, the electrolyte leakage rate increased as long as the storage time in all coated samples, indicating a decrease in the mushroom membrane integrity. Furthermore, CHSTiO2/TT80 mushrooms (25.84%) exhibited a significantly (p ≤ 0.05) lower electrolyte leakage rate than the other samples, which might be attributed to the presence of thymol and tween-80. Other reports suggested the lower value of mushroom membrane integrity was directly related to the mushroom browning and lipid peroxidation [32][33][34].
Cap opening was raised in all coating treatments with the duration of the storage period, and the highest value was detected for uncoated samples (76.49%) after 12 days, Figure 5b. At the end of the experiment, the value of cap opening was in-between 30.08 and 31.61% in CHSTiO2 + CHSTiO2/TT80 samples, respectively, which prevented the water vapor from affecting the packaged mushrooms. The cap opening percentile is regarded as the maturity indicator and refers to moisture content loss and mushroom aging [35,36]. In addition, according to our findings, high CO2 and low O2 concentrations have a positive effect on the cap opening reduction and preventing repining.

Conclusions
The results of this research work have established that CHSTiO2/TT80 coating treatment has a positive effect on button mushroom preservation. The preservation effect might be attributed to the combination of chitosan, nanocomposite material, and antimicrobial agents (thymol and tween-80) CHSTiO2/TT80. Thus, it showed lower respiration rate, weight loss, browning degree, and microbial contaminations, and higher electrolyte leakage rate and acidity than those coated with CHS and CHSTiO2. Meanwhile, CHSTiO2 coating treatment established the lowest polyphenol oxidase activity andCHS maintained lightness. These results indicated that CHSTiO2/TT80 coating treatment might be investigated as a novel packaging material for other consumable vegetables and fruit products in the future. Semi nano-films mainly with the addition of (thymol-tween) is suggested for nanotechnology application researches and preservation manufacturing.  Cap opening was raised in all coating treatments with the duration of the storage period, and the highest value was detected for uncoated samples (76.49%) after 12 days, Figure 5b. At the end of the experiment, the value of cap opening was in-between 30.08 and 31.61% in CHSTiO 2 + CHSTiO 2 /TT80 samples, respectively, which prevented the water vapor from affecting the packaged mushrooms. The cap opening percentile is regarded as the maturity indicator and refers to moisture content loss and mushroom aging [35,36]. In addition, according to our findings, high CO 2 and low O 2 concentrations have a positive effect on the cap opening reduction and preventing repining.

Conclusions
The results of this research work have established that CHSTiO 2 /TT80 coating treatment has a positive effect on button mushroom preservation. The preservation effect might be attributed to the combination of chitosan, nanocomposite material, and antimicrobial agents (thymol and tween-80) CHSTiO 2 /TT80. Thus, it showed lower respiration rate, weight loss, browning degree, and microbial contaminations, and higher electrolyte leakage rate and acidity than those coated with CHS and CHSTiO 2 . Meanwhile, CHSTiO 2 coating treatment established the lowest polyphenol oxidase activity andCHS maintained lightness. These results indicated that CHSTiO 2 /TT80 coating treatment might be investigated as a novel packaging material for other consumable vegetables and fruit products in the future. Semi nano-films mainly with the addition of (thymol-tween) is suggested for nanotechnology application researches and preservation manufacturing.