The effect of carbon dioxide on the quality of the mushrooms

Mushrooms' quality may be significantly changing depending on their type, strain, growing cycle, packing, cooling, postharvest handling (PHH), and conditions of storage. This work aimed to define the influence of the type and mushrooms' strain, the regime of the PHH by carbon dioxide on their preservation (marketability, loss of weight (LW)), changes in the chemical substances, and physiological activity (intensity respiration (IR) and heat release (HR)). Mushrooms Agaricus bisporus (AB) (strains ІБК-25 and ІБК-15) and Pleurotus ostreatus (PO) (strains НК-35 and Amycel 3000) were used for testing. Three regimes of treatment by CO2 with a concentration of 20% were applied: 2 h; 12 h and 22 h. The control was the mushrooms without treatment by CO2. Changes in the chemical substances such as dry matters (DM), protein nitrogen (PN), and ascorbic acid (AA) in the researched mushrooms were observed. The best result of mushroom preservation was provided by the regime of CO2 treatment during 12 h. The yield of marketable AB was 94.9% (IBK-25) and 94.2% (IBK-15) comparison to control 93.5%, and 92.5%, respectively. The regime of PHH 2 h almost has no influence but 22 h harmed this indicator. PHH of mushrooms by carbon dioxide was promoted to preserve the DM and increasing concentration of CO2 was supplied better results. Thus, DM at the end of storage in the AB of strain IBK-25 depend on the regime were 8.5, 8.6, and 8.4%, against – 8.3% in the control variant. Significant quantitative changes in the PN and AA as a result of treatment by CO2 were not established. PHH also affected the IR and HR. The increased duration of treatment by CO2 inhibited the intensity of physiological processes in the mushrooms. But, as in previous cases, the best result was provided PHH by 20% CO2 during 12 h. Similar trends of treatments effect by carbon dioxide were observed in the mushrooms of PO.


INTRODUCTION
In the case of CAS, the product is stored in cold storage chamber where created atmospheric composition with certain relative humidity (RH) and concentration of O2 and CO2 that is maintained constant throughout storage. CAS reduces IR of mushrooms, their texture changes, and brown discoloration (enzymatic browning) as a result shelf life is extended (Djekic et al., 2017;Park et al., 2020). A major disadvantage of CAS is the significant cost of equipment and its maintenance. MAP is a method of storage when fresh product is in a sealed package and is creating a modified atmosphere by respiratory gas exchange, namely oxygen intake and carbon dioxide evolution (Zhang, Pu, and Sun, 2018; Zalewska et al., 2018). Equilibrium concentrations of O2 and CO2 are consequently established in the case the rate of gas permeation through the packaging material equals respiratory gas exchange (Gantner et al., 2017; Gholami, Ahmadi and Farris, 2017). The balance of the gas exchange depends on product weight, temperature, the respiration rate of a certain product, permeability O2 and CO2 through packaging material, free volume in the package, and film area. MAP helps to extend the shelf life and keep quality as a result creation of a corresponding atmosphere around the products that are packaged in plastic films (Ozturk, Havsut and Yildiz, 2021; Vunduk et al., 2021). There are two methods of creating a modified atmosphere: active and passive modifications. The product is only sealed in a polymeric package at the passive modification. The atmosphere is modified as a result of the fresh product breathing and permeation of gases into the package. It takes a long time to reach the steady-state conditions within the package in passive modification. In the case of the active modification, air initially compulsorily is pushed into the package. Therefore, a stable state of the atmosphere is reached after packaging quickly (Li et al., 2014; Han Lyn et al., 2020). MAP of mushrooms has been shown successfully to delay senescence and keep quality after harvest.

Modified Humidity Packaging
The many polymeric films used for fresh product packaging have lower water vapor transmission rates compared to rates of breathing of this product. As a result, the packages creating saturated conditions of water vapor. The high relative humidity in the package (HRHP) can be cause condensation of water vapor within a package and promote microbial growth. It is may be increasing or decreasing the spoilage depending on the products, their breathing coefficients, and water potentials. There are two possible ways for reaching the desired HRHP: perforation of the package (Dhalsamant et al., 2015) and use of in-package water-absorbing compounds like calcium chloride that can be keeping the required RH (Villaescusa and Gil, 2003). Combination MAP with MHP promotes improving the shelf-life of fresh mushrooms. The best result for keeping mushrooms obtained at the HRHP of 87 -90% during the storage.
The disadvantages of MAP and MHP are the need for special packaging materials, water-absorbing compounds, and packaging equipment that increases the cost of products. Besides, as a result, MAP storage takes place excessive accumulation of CO2 that may be damage the cell membrane and physiological injuries to the mushrooms, such as severe enzymatic browning and loss of firmness (Varoquaux et al., 1999).
The perspective method of PHH of mushrooms and extended their shelf life is the treatment by carbon dioxide with high concentration during a short time.
The effect of this influence depends on the stage developmental of mushrooms, the concentration of CO2, and the time of exposure (Fonseca, Oliveira and Brecht, 2002; Li et al., 2013).
This work aimed to investigate the effect of the short-term treatment by high concentrations of carbon dioxide on the mushrooms' quality and their physiological activity.

Scientific hypothesis
Mushrooms are products with a high level of water in the fruiting bodies (more than 90%). That explains high IR and metabolic activity to comparison other horticultural crops and causer loss of the sensory quality of mushrooms (browning and texture changes). Mushrooms have a large loss of moisture as a result of evaporation, especially at the storage in the reduced RH. The shelf life of mushrooms depends on their type, packing, regimes, and ways of cooling, technologies of PHH, and conditions of transportation.

Samples
Materials of the study were mushrooms of AB (strains ІBK-25 and ІBK-15) and PO (strains НК-35 and Amycel 3000) from the collection of Institute of Botany after name M.G. Xolodnogo of National Academy Science of Ukraine. These strains are widespread, suitable to grow all year, and have universal purposes. Mushrooms AB and PO were supplied by the Trynchuk Mushroom Farm (Kyiv region, Fastiv district, Borova village) and transported to the National University of Life and Environmental Sciences of Ukraine (Kyiv, Ukraine) at 2 ±2 °С and 90% ±1 RH.

Animals and Biological Material:
No animals and biological materials were used for the studies Instruments The four cold chambers КH-6U with volume 6 m 3 were used for the mushrooms treatment by CO2 and their following storage. The required concentration of CO2 was created by the rotameter of the brand RM-2.5 GUZ (GK PriborMarket, Russia). The gaseous medium in the chamber was stirred by fans.
Concentration CO2 in the chambers was monitoring by a VTI-2 gas analyzer (Thermal Engineering Institute, Russia). The temperature and RH in the chamber were controlled daily. The air temperature was measured by alcohol thermometer TLC-5 (PJSC "Glass Device", Ukraine, I accuracy class) with the division price of 0.5 °C but RH -by August psychrometers (Lab Time, Ukraine, I accuracy class), daily.
Samples were weighted by laboratory scales ADG2200С (AXIS) from the company "Scales of AXIS Ukraine" with the 2 nd class of accuracy.

Laboratory Methods
For chemical analyses, only the first wave mushrooms were used. In mushrooms, before storage and after 6 days were determined DM, PN, and AA.
Content of AA -restoring the Tillman's reagent, by extraction acid solution of mushrooms sample followed by filtration of the resulting substrate by the titrimetric method according to the state standard of Ukraine DSTU 7803 (2015).

Description of the Experiment
The scheme of researches is presented in Figure 1. Sample preparation: Mushrooms that were used in the investigations were harvested at the peak of fruiting of the first, second, and third waves. Mushrooms were harvested manually, immediately placed in plastic boxes with the volume of 5 kg where they were stored. Every sample before storage was weighed, numbered, and added a label with indication weight, temperature, time of the start storage, and repeatability.

PHH of mushrooms by CO2
Regimes PHH of mushrooms of AB and PO: 20% СО2 during 2 h; 20% СО2 during 12 h and 20% СО2 during 22 h. The control was the mushrooms without treatment by carbon dioxide. The temperature of the product storage was 1 °C. Time after mushrooms' harvesting to the PHH by CO2 did not exceed 3 h.

Density of carpophore
The density of carpophore (DC) of fruiting bodies of AB mushrooms before and after storage was determined by the formula: (1) Where: DC -density of carpophore (g.cm -1 ); mc -weight of carpophore (g); dh -diameter of mushroom hat (cm).

Loss of weight
LW was determined by the method of fixed samples. Samples were weighed before storage and every day. The calculation LW was performed by the formula: (2) Where: LW -loss of weight (g); Wbs -the weight of the sample before storage (g); Weds -the weight of a sample of every day of storage (g).

Intensity of respiration
The IR of mushrooms was determined experimentally in desiccators every day during the storage. This method is based on the absorption of CO2 by solutions of alkalis (Ba(OH)2) with known concentration and followed by the determination of the amount of alkali that did not react with acid for titration (HCl). Simultaneously, the alkali was titrated from a desiccator without mushrooms (control). IR was determined by the formula: (3) Where: IR -the intensity of respiration (mL.kg -1 .h -1 for CO2); Qcquantity of acid which was used for control titration (mL); Qe -quantity of acid which was used for titration in the experiment (mL); wm -the weight of the mushrooms (g); t -time duration of the product in the desiccator (min); Tcorrection to a titter of the 0.1 N alkali; a -average between atmospheric pressure at the start and end of the experiment; b -average between the temperature at the start and end of the experiment.

Number of samples analyzed:
The weight of samples for investigations was up to 4 kg. The average sample for chemical analysis was 20 mushrooms fruit bodies with average weight. Number of repeated analyses: 3 Number of experiment replication: Every variant was replied three times. Mushrooms were stored for 6 days. The marketable quality, the chemical composition of mushrooms, the yield of marketable products, and natural LW were determined before and after the storage. The intensity of physiological processes was monitored daily.

Statistical Analysis
The experiment was established as completely randomized designs with three repetitions in 2017 -2019 years. The data are reported as mean values ± standard deviation (SD). As the statistical analysis software was used Microsoft Excel version 2016.

RESULTS AND DISCUSSION
Many publications report that reduced O2 and elevated CO2 concentration have beneficial effects on the shelf life of mushrooms ( The atmospheres with a high concentration of CO2 can potentially reduce respiration rate, production of ethylene, sensitivity, decay, and physiological changes in the mushrooms ( So, low concentrations of CO2 did not allow obtaining the desired effect but high has negatively affected the quality and shelf life of the mushrooms. To establish the optimal regime of AB and PO mushrooms treatment by carbon dioxide, we were kept them in sealed chambers with 20% CO2 in the environment for 2, 12, and 22 h, and then stored with temperature 1 °C for 6 days in the normal atmosphere. The influence of parameters and regimes of the PHH on the quality of mushrooms was determined by organoleptic and chemical indexes. Organoleptic evaluation of AB and PO showed that PHH by 20% CO2 for 2 h had almost no effect on changes in color, smell, and consistency of fruiting bodies. Prolonged treatment by carbon dioxide during 22 h significantly deteriorated the color of mushrooms ( Figure  2). In our opinion, prolonged contact of mushroom tissues with high concentrations of CO2 has caused respiratory disorders and inhibited the biochemical processes associated with oxygen deficiency. This caused the death of individual cells, enzymatic browning, and a decrease in the fruit density of mushrooms, which corresponding with the results obtained by other researchers (Briones et al., 1992). Besides, products had a slight characteristic smell of carbon dioxide.
A positive effect on the organoleptic characteristics of the mushrooms was established in the variant of treatment by 20% CO2 for 12 h. There was no foreign smell; the fruiting bodies remained whole and dense. As can be seen in Figure  2, the mushrooms of AB had advantages over control in appearance at this regime.
However, it should be noted that photos do not fully demonstrate changes in the appearance of mushrooms. The difference is more clearly visible in the organoleptic evaluation products in the boxes. The better appearance of mushrooms at this regime, compared with the control, can be explained by inhibition of the activity of the enzyme tyrosine as a result of CO2 treatment. As a consequence, the formation of melanins slows down, which causes enzymatic browning of AB. A similar result in terms of inhibition of the enzyme tyrosine activity as a result of treatment by high concentrations of CO2 was obtained in the investigations of straw mushrooms (Volvariella volvacea) (Jamjumroon et al., 2013; Jamjumroon et al., 2012). PO in contrast to the AB did not change in appearance at different regimes of CO2 treatment.
Preservation of AB and PO depending on the regime of PHH was determined by the results of studies of three cycles of cultivation (repetitions). The highest yield of marketable products was observed at the regime of treatment by 20% CO2 within 12 h (Table 1). For mushrooms AB of the IBK-25 strain, on average, was 94.9% that is 1.4% more than the control variant (93.5%). For strain IBK-15, this index was 94.2% (in the control variant -92.5%). The duration of treatment during 2 h did not significantly affect the output of marketable fruit bodies. Depending on the growing cycle, they were 93.3 -93.7%, while in the control variant -93.0 -93.9%.
Negatively affect had a variant of treatment by CO2 for 22 h on the yield of marketable products. On average, during three growing cycles, their quantity decreased relative to control by 1.8-1.9%, depending on the strain of the AB. The regime of treatment by 20% CO2 during 2 h had little effect on the natural LW ( Table 2) and quantity of non-marketable fruiting bodies of mushrooms. Such a shortterm treatment cannot significantly change the vital processes that take place in the mushrooms after harvest. The treatment by carbon dioxide also affected the  Note: * -The first growing cycle; ** -The second growing cycle; *** -The third growing cycle. Values are means ± standard deviation, p ≤0.05.
DC of mushrooms (Table 1). There best values were observed for treatment during 12 h (5.56 and 5.65 g.cm -1 ), and in the control variant -5.48 and 5.58 g.cm -1 , respectively.
The regime of the mushrooms treatment during 22 h had positive effects on the natural LW (Table 2). In this regime, a natural loss is slightly lower (4.0 -4.2% depending on the strain), compared with the control (4.8 -5.0%), but at the same time in the experimental version significantly increases the quantity of non-marketable fruiting bodies (from 1.8 -2.5 to 4.4 -5.1%, respectively).
The fruiting bodies of AB of the strain IBK-15 have larger fruiting bodies compared to the strain IBK-25, respectively, and a larger area of moisture evaporation. This, in turn, affected the natural LW during storage (4.2 -5.0% for strain IBK-15) against 4.0 -4.8% for strain IBK-25, depending on the regime of PHH.
The tendencies concerning the influence of PHH of 20% СО2 on the natural LW, quantity of a commodity, and noncommodity fruit bodies in the AB were characteristic and for PO (Table 3). There was a slight increase in the yield of marketable products -by 0.6 -0.9% relative to the control for the twelve-hour treatment by carbon dioxide. This is due to the breaking of vital processes in mushrooms as a result of CO2 treatment. The total LW during storage of PO mushrooms in these conditions was formed mainly due to natural losses. For strain NK-35 their value was 5.2%, for Amycel 3000 -4.7%. The quantity of non-commodity fruit bodies was 0.5 and 1.5%, respectively (Table 4). PO of strain Amycel 3000 had a higher number of non-marketable fruiting bodies that explain the botanical features of the formation of their clusters. The clusters of this strain had a large number of small fruit bodies, which after storage for 6 days dried up or became watery. The treatment OP by CO2 during 22 h harmed the quantity of non-marketable products.
Their numbers increased up to 3.4 and 3.6%, compared with controls -0.8 and 1.8%, depending on the strain. Some mushrooms had brown spots. In our opinion, the reason for this is the burns of the tissues of the mushrooms by carbonic acid that was formed by the interaction of condensate, which appeared as a result of respiration of products and carbon dioxide of high concentration.
Natural losses of mushrooms during storage were formed due to the evaporation of moisture and loss of chemical substances (Tables 5 and 6).
The postharvest handling by carbon dioxide during the storage of AB mushrooms contributes to the preservation of DM. It explains by the inhibition effects of CO2 on the respiration processes and development of the mushroom's fruiting body that decreased sugar consumption. Thus, at the end of storage for the mushroom of strain IBK-25, the amount of DM in the fruiting bodies of the experimental variants were 8.5, 8.6, and 8.4%, while in the control variant -8.3%.  13 Note: * -The first growing cycle; ** -The second growing cycle; *** -The third growing cycle. Values are means ± standard deviation, p ≤0.05.
Carbon dioxide in most cases did not significantly affect the changes in the amount of PN and AA compared to the control. Only after treatment duration by 22 hours, the amount of PN partially changed.
For strain IBK-25 its amount was 3.5% for the experimental variant and 3.7% for the control; for strain IBK-15 -3.4 and 3.5%, respectively. This may be due to oxygen deficiency and protein breakdown. Similar trends in the effect of postharvest handling by carbon dioxide on the content of chemical substances were observed for OP mushrooms. The amount of DM in mushrooms of strain NK-35 during the storage period decreased from 12.5 to 11.3 -11.7% depending on the exposure and in the control variant -up to 11.4%.
Most DM was preserved in fruiting bodies after twelve hours of treatment: in strain NK-35 -11.7% and in the strain Amycel 3000 -11.5%, that it is significantly more than the control variant.
There are similar to the AB in PO mushrooms wasn't observed significant changes in the quantity AA during the treatment by CO2. 2 Note: * -The first growing cycle; ** -The second growing cycle; *** -The third growing cycle. Values are means ± standard deviation, p ≤0.05.    Analysis of the IR of mushrooms indicated that after harvesting quantity of carbon dioxide released during respiration begins to increase sharply, and then decreases during storage due to low temperatures (Table 7 and 8).
Significantly affected the amount of CO2 released had postharvest treatment by carbon dioxide, especially in the first 3 days.
As can be seen from Table 7 and Table 8, there is a tendency that with increasing duration of treatment by CO2, the process of respiration is suppressed. It is the least intense respiration was as a result of treatment CO2 during 22 h. It was typical for both AB mushrooms and OP.
Thus, for AP of strain IBK-25 after a day of storage, the amount of CO2 released in the experimental variant was 7.5 but in the control -9.2 mL.kg -1 .h -1 for CO2; after two days -8.1 and 11.3 mL.kg -1 .h -1 for CO2; after three days -4.0 and 5.3 mL.kg -1 .h -1 for CO2, respectively. In the future, the IR of the control and experimental variants was gradually equalized and at the end of storage did not differ significantly (2.1 -2.3 mL.kg -1 .h -1 for CO2 in the experimental variants and 2.5 in the control) (Table 7 and  8).
Based on the data on the intensity of respiration, calculations of the HR of mushrooms were performed (Tables 9 and 10).
Postharvest short-term treatment from 2 till 22 h by carbon dioxide at a concentration of 20% affected the average HR of mushrooms during storage.
Since the intensity of HR directly depends on the IR, so the trends of its change are similar.

CONCLUSION
Postharvest short-term handling mushrooms of AB and PO by carbon dioxide at a concentration of 20% is an effective method that reduces the level of natural losses, increases the yield of marketable products, helps preserve their chemical substances, and inhibits the intensity of physiological processes in the product (IR and HR). According to all the obtained data, the most effective is the application of the regime of mushrooms treatment by CO2 during 12 h. Increasing the time of treatment by carbon dioxide up to 22 h most effectively inhibits the processes of respiration (on the 0.4 -3.2% for AB and the 0.6 -5.4% for PO) and HR (on the 0.1 -0.8% for AB and the 0.1 -1.4% for PO), compared with the control and depending on the mushrooms' strain and duration of their storage. However, this regime promoted sharply increases the quantity of nonmarketable products (on the 2.6% for AB and the 1.8 -2.6% for PO) compared to the control and depending on the strain and wave of fruiting.