Extremely low-oxygen storage: aerobic, anaerobic metabolism and overall quality of apples at two temperatures

: The objective of this work was to evaluate the effect of extremely low-oxygen storage on the aerobic, anaerobic metabolism and overall quality maintenance of ‘Royal Gala’ and ‘Galaxy’ apples stored at two temperatures. For ‘Royal Gala’ apples, four treatments were evaluated in a two-factor scheme (2 × 2), with two storage conditions: [1] 1.2 kPa O 2 + 2 kPa CO 2 ; [2] 0.8 kPa O 2 + 1.6 kPa CO 2 ; and two temperatures (1.0 and 1.5 °C). For ‘Galaxy’ apples, the experiment was conducted in a two-factor (3 × 2) scheme with three storage conditions: [1] 1.2 kPa O 2 + 2 kPa CO 2 ; [2] 0.8 kPa O 2 + 1.6 kPa CO 2 ; [3] 0.4 kPa O 2 + 1.2 kPa CO 2 , and two temperatures (1.0 and 1.5 °C). The ‘Royal Gala’ apples stored under 0.8 kPa O 2 + 1.6 kPa CO 2 at 1.5 °C had higher flesh firmness, healthy fruit and soluble solids along with lower incidence of flesh breakdown and mealiness. The ‘Galaxy’ apples stored under 0.4 kPa O 2 + 1.2 kPa CO 2 , at temperature of 1.5 °C had lower 1-carboxylic acid-1-aminocyclopropane (ACC) oxidase enzyme activity, ethylene production, occurrence of mealiness, when compared to 1.0 °C. The storage of these two cultivars at 1.5 °C with extremely low O 2 partial pressures is possible. The possibility to increase the storage temperature from 1 °C, which is traditionally used for these mutants, to 1.5 °C allows to keep better quality with the advantage of energy saving.


INTRODUCTION
Most apples produced in Brazil belong to the 'Gala' cultivar and its spontaneous mutants, including 'Galaxy' and 'Royal Gala' , totalizing a production of 326,658 t in 2017/2018 crop, representing 66% of the national apple production (Agapomi 2018). These mutants stand out for presenting a good red coloration of epidermis (Brackmann et al. 2009(Brackmann et al. , 2012Weber et al. 2013), high juiciness and better balance between acidity and soluble solids (Thewes et al. 2015).
Temperature is the most important factor for the maintenance of fruit quality during storage (Both et al. 2018;Brackmann et al. 2008). The reduction in temperature suppresses the respiratory pathway, ethylene production and, consequently, the reduction of biochemical changes in the fruit (Kruse et al. 2011;Saltveit 1999;Steffens et al. 2007). The ideal temperature for controlled atmosphere storage of 'Gala' apples and their mutants ranges from 0.0 to 1.0 °C (Both et al. 2017;Brackmann et al. 2009;Thewes et al. 2017a). However, recent studies reported the possibility of increasing the storage temperature of some types of apples without quality losses (Anese et al. 2019;Weber et al. 2019). The application of 1-methylcyclopropene (1-MCP) to apples stored under controlled atmosphere (CA) allowed an increase of the storage temperature to 2.5 °C, obtaining an electrical energy saving of 26 and 35% for the 'Jonagold' and 'Gala' apples, respectively (Kittemann et al. 2015). The use of dynamic controlled atmosphere by chlorophyll fluorescence (DCA-CF) allowed 20% of energy savings in apples stored for seven months (Kittemann et al. 2015). In both studies, the increase in temperature did not result in Extremely low-oxygen storage fruit quality losses; however, quality maintenance varied among cultivars and some were more sensitive to the increase in temperature. Apple cultivars respond differently to the increase in temperature and the auxiliary technique adopted, such as the application of 1-MCP, DCA-CF and ultra-low oxygen (ULO) (Köpcke 2015). The increase of O 2 partial pressure in the storage room cause an increase in the fruit metabolism, resulting in a lower quality maintenance (Steffens et al. 2007). Therefore, it is important to conduct studies to verify the feasibility to increase the storage temperature and its interaction with extremely low O 2 partial pressures in maintaining fruit quality.
The most widely used method for the storage of apples in Brazil is CA (Brackmann et al. 2012;Weber et al. 2013). Thus, O 2 partial pressure are reduced, CO 2 partial pressure increased, storage temperature reduced and relative humidity increased to levels close to 100% inside the rooms, in order to reduce respiration and ethylene production (Gorny and Kader 1996;Ke et al. 1991;Lumpkin et al. 2015). The fruit tolerance to CA depends on several factors such as cultivar, maturation stage, harvest year, O 2 and CO 2 partial pressure and duration of storage (Bulens et al. 2012;Watkins 2008). Regarding the storage in CA, O 2 partial pressure is maintained above the anaerobic compensation point, which consists of the O 2 concentration that causes lower production of CO 2 in the storage chamber (Boersig et al. 1988), consequently the fruit are not at the point of minimum respiration. This causes quality losses, such as low flesh firmness, decrease of aroma (Thewes et al. 2015) and increase in decay incidence after storage under refrigerated atmosphere (RA) and CA (Antoniolli et al. 2011).
The ULO storage consists in maintaining O 2 partial pressure below 1.0 kPa, thus keeping the metabolism of the fruit closer to the O 2 minimum limit, which favors a greater maintenance of the postharvest quality of apples. The storage of apples in low O 2 reduces the oxidative metabolism, the 1-carboxylic acid-1-aminocyclopropane (ACC) oxidase enzyme activity and ethylene production (Bekele et al. 2016;Imahori et al. 2013;Mditshwa et al. 2017;Thewes et al. 2015). 'Fuji' apples stored at 2.5 °C under 0.7 to 1.0 kPa of O 2 had lower flesh firmness loss and lower physiological disorders incidence when compared to the O 2 partial pressures of 2.0 and 3.0 kPa (Argenta and Brackmann 1994). 'Royal Gala' apples stored in DCA-RQ with O 2 partial pressure between 0.13 and 0.41 kPa presented higher flesh firmness, higher percentage of healthy fruit and lower incidence of physiological disorders as compared to CA with 1.2 kPa O 2 + 2.0 kPa CO 2 (Both et al. 2017).
The objective of this work was to evaluate the effect of extremely low-oxygen storage on the aerobic, anaerobic metabolism and overall quality maintenance of 'Royal Gala' and 'Galaxy' apples stored at two temperatures.

Experimental material, establishment and monitoring of CA conditions
After harvest, the apples were selected by discarding those with decay, inadequate size or mechanical damage. Each treatment was composed of four replicates of 25 fruit and the design was completely randomized. For 'Royal Gala' apples, 4 treatments were carried out in a factorial scheme (2 × 2), with two atmosphere conditions: [1] 1.2 kPa O 2 + 2.0 kPa CO 2 ; [2] 0.8 kPa O 2 + 1.6 kPa CO 2 ; and two temperatures (1.0 and 1.5 °C) (Both et al. 2017;Brackmann et al. 2009 In order to establish the atmosphere conditions, the fruit were stored in hermetically sealed chambers, with a volume of 0.233 m 3 , which were allocated inside cold rooms, and the storage temperature was set according to each treatment. The O 2 partial pressure was reduced by using N 2 from a nitrogen generator that uses the pressure swing adsorption principle (PSA) to sweep and dilute the O 2 inside the chambers. The O 2 was reduced to 5.0 kPa on the first day of storage and 1.0 kPa·day -1 on the following five days through fruit respiration and N 2 flushing, until the preestablished partial pressure for each treatment was reached. The partial pressures of CO 2 in the chambers were obtained by their accumulation from fruit respiration. The replacement of O 2 consumed by the fruit was carried out by injecting cold atmospheric air with an automatic control system. The excess CO 2 produced by fruit respiration was absorbed by the circulation of the gas in the chamber through a CO 2 absorber with calcium hydroxide. The CA conditions were measured daily and corrected by an automatic control system (Valis, Lajeado, RS, Brazil) connected to an O 2 and CO 2 analyzer (Siemens, model Ultramat 23, Germany).
V. Ludwig et al.

Analysis of fruit quality and metabolism
The laboratory analyses were performed after nine months of storage plus seven days of shelf life at 20 °C (± 1.0). The following parameters were analyzed:

Ethylene production
Determined by stowing of 1.5 kg sample in a 5 L container, which was hermetically closed for about 1 h. After, two samples of 1 mL of the headspace were taken from the container and injected into a gas chromatograph, with a Poropak N80100 column and flame ionization detector (FID), with column, injector and detector temperature of 90, 140 and 200 °C, respectively. The results were expressed in ng C 2 H 4 ·kg -1 ·s -1 .

Respiration rate
Determined by stowing of 1.5 kg sample in a 5 L container, which was hermetically closed for about 1 h, headspace air was flushed through an Isocell Oxicarb CO 2 analyzer, to determine the CO 2 concentration. The results were expressed in µg CO 2 ·kg -1 ·s -1 .

Internal ethylene and internal CO 2
Determined according to methodology proposed by Thewes et al. (2015). Results were expressed in µg·L -1 and mg·100 mL -1 , respectively.

ACC oxidase enzyme activity
Determined from samples of the epidermis (3 g) collected from the equatorial region of the fruit. These samples were immersed in a solution containing 0.1 mmol·L -1 ACC and in a 2-(N-morpholino) ethanesulfonic acid (MES) buffer solution with a 6.0 pH. After 30 min, the samples were withdrawn and placed in 50 mL hermetically sealed syringes, to which 1 mL of CO 2 was added. After 30 min, the concentration of ethylene contained in the syringes was determined by the same chromatograph used to determine ethylene production. Data was expressed in ng C 2 H 4 ·kg -1 ·s -1 , according to Bufler (1986).

Flesh firmness
Determined with a penetrometer equipped with a 11 mm tip inserted on two opposite sides in the equatorial region of the fruit, from where the epidermis have been previously removed. The data were expressed in Newton (N).

Soluble solids and titratable acidity
Soluble solids (SS) were determined with the aid of a refractometer, from the juice extracted from the samples. The results were expressed in percentage. Titratable acidity was obtained by titration with 0.1 N NaOH, of a solution containing 10 mL of juice diluted in 100 mL of distilled water, until the pH of 8.1. Results were expressed as g malic acid·100 g -1 .

Physiological disorders, decay incidence and healthy fruit amount
Determined by counting fruit that presented external and/or internal symptoms such as flesh breakdown, mealiness, decay and pulp cracking, according to Thewes et al. (2015). The health fruit was quantified by the difference among the Extremely low-oxygen storage total number of fruit and fruit with any symptom of decay, mealiness and incidence of flesh breakdown. The results were expressed in percentage of fruit affected.

Acetaldehyde, ethanol and ethyl acetate
Slices of equatorial region of the fruit were taken and centrifuged with a juicer Philips Walita centrifuge to extract the juice. Subsequently, 10 mL of the juice was packed in 20 mL vials. The juice sample was kept exposed in a water bath at 70 °C for 30 min, so that the anaerobic metabolism compounds could volatilize into the headspace of the vial. Afterwards, a 100 μL sample of headspace air was injected into a gas chromatograph equipped with a 60 m long capillary column (DN-WAX Dani). The injector, column and detector temperatures were 180, 60 and 250 °C, respectively. The results were expressed in µL·L -1 .

Statistical analysis
Analysis of variance (ANOVA) was performed for each variable. The means were compared by Tukey's test with a 5% probability of error, using the statistical software SISVAR version 5.6. The data that presented significant difference by ANOVA were submitted to partial least square (PLS) analysis, before the data were auto-scaled in order to obtain the same weights for all variables (mean = 0 and variance = 1). The Unscrambler X software (version 9.7, CAMO A/S, Trondheim, Norway) was used.

Partial least square (PLS)
The PLS analysis allowed an overview of the experiment and correlated the influence of the storage conditions to the analyzed parameters. For 'Royal Gala' apples, the increase in storage temperature was found to be correlated to a higher concentration of soluble solids, greater percentage of healthy fruit, higher concentration of ethanol and ethyl acetate (Fig. 1a). On the other hand, the higher activity of the ACC oxidase enzyme, ethylene production at chamber opening and at 7 days of shelf life, as well as the internal ethylene concentration and respiration rate at 7 days, presented a correlation with the storage condition of 1.2 kPa O 2 + 2.0 kPa CO 2 . For 'Royal Gala' apples the respiration rate was correlated to 1.0 kPa O 2 + 2.0 kPa CO 2 (Both et al. 2014a). The storage under 0.8 kPa O 2 + 1.6 kPa CO 2 was correlated with the internal concentration of CO 2 and respiration rate at chambers opening. For 'Galaxy' apples, the concentration of ethanol and internal CO 2 presented an association to the storage under higher temperature (Fig. 1b). According to Both et al. (2018), storage of 'Galaxy' apples at 2.0 °C resulted in higher ethanol concentration than at 1.0 °C. The storage with 1.2 kPa O 2 + 2.0 kPa CO 2 was correlated to the higher production of ethylene at chambers opening and at 7 days of shelf life, internal ethylene concentration, ACC oxidase enzyme activity and mealiness. In contrast, fruit stored under 0.4 kPa O 2 + 1.2 kPa CO 2 and 0.8 kPa O 2 + 1.6 kPa CO 2 had a correlation to flesh firmness, healthy fruit, titratable acidity, acetaldehyde concentration, decay, pulp cracking and flesh breakdown, fact that did not lead to these treatments having a lower healthy fruit percentage (Fig. 2).

Ethylene production, respiration rate, ACC oxidase activity and internal ethylene concentration
'Royal Gala' apples stored under 1.2 kPa O 2 + 2.0 kPa CO 2 presented a higher ethylene production, after shelf life, when stored at 1.0 °C when compared to 1.5 °C (Fig. 3a). 'Galaxy' apples stored at 2.0 °C had a higher ethylene production at seven days of shelf life compared to apples stored at temperatures of 1.0 and 1.5 °C. Storage under 0.8 kPa O 2 + 1.6 kPa CO 2 condition produced less ethylene immediately after chambers opening and at seven days shelf life when compared to 1.2 kPa O 2 + 1.2 kPa CO 2 (Fig. 3a). 'Galaxy' apples submitted to 0.4 kPa O 2 + 1.2 kPa CO 2, regardless of temperature, had V. Ludwig et al. lower ethylene production compared to the 1.2 kPa O 2 + 2.0 kPa CO 2 and 0.8 kPa O 2 + 1.6 kPa CO 2 conditions, which may be explained by the lower ACC oxidase enzyme activity (Fig. 3a). For 'Galaxy' apples at chamber opening, fruit stored under 0.8 kPa O 2 + 1.6 kPa CO 2 at 1.5 °C had a lower ethylene production compared to 1.0 °C. Blueberries stored at 2 °C had higher ethylene production than storage at 10 °C, probably the low temperature caused an abiotic stress increasing the ethylene production (Álvarez-Hernández et al. 2019). The ethylene production by fruit stored under 0.4 kPa O 2 + 1.2 kPa CO 2 was not influenced by the storage temperature. Fruit stored under 1.2 kPa O 2 + 1.2 kPa CO 2 showed higher concentration of ethylene compared to 0.4 kPa O 2 + 1.2 kPa CO 2 and 0.8 kPa O 2 + 1.6 kPa CO 2 (Fig. 3a). Fruit stored under 0.4 kPa O 2 + 1.2 kPa CO 2 , after seven days of shelf life, had a lower ethylene production than the fruit kept under 0.8 kPa O 2 + 1.6 kPa CO 2 , this may be explained by the lower activity of the ACC oxidase enzyme of fruit stored under 0.4 kPa O 2 + 1.2 kPa CO 2 at 1.5 °C (Fig. 3a), similar results were found by Both et al. (2014b), Thewes et al. (2015), and Weber et al. (2013). The increased concentration of ethanol in the fruit can inhibit the ACC oxidase enzyme activity and the ethylene production (Thewes et al. 2015). It was observed that ethanol has a suppressive effect in the transcription genes   Figure 1. (a) Scores of treatments (bold words) and correlations, the partial last square of 'Royal Gala' apples stored during nine months plus seven days at 20 °C; (b) scores of treatments (bold words) and correlations, the partial last square of 'Galaxy' apples stored during nine months plus seven days at 20 °C.
Extremely low-oxygen storage of the ACC oxidase enzyme (BO-ACO1 e BO-ACO2) and, consequently, the production of ethylene in broccoli (Asoda et al. 2009;Podd and Van Staden 1998). The respiration rate in 'Royal Gala' apples stored under 1.2 kPa O 2 + 2.0 kPa CO 2 was lower than fruit stored under 0.8 kPa O 2 + 1.6 kPa CO 2 at 1.0 °C (Fig. 3b). Fruit stored under 0.8 kPa O 2 + 2.0 kPa CO 2 must have suffered chilling injury, which increased their respiration rate, because when kept at 1.5 °C, no difference between 1.2 kPa O 2 + 2.0 kPa CO 2 and 0.8 kPa O 2 + 1.6 kPa CO 2 was found. The oxygen lowering below 1.0 kPa causes an increase in the respiration rate, if they are submitted to low temperatures (Brackmann et al. 2008). After 7 days of shelf life, no difference in the respiration rate was found between the fruit stored under 1.2 kPa O 2 + 2.0 kPa CO 2 and 0.8 kPa O 2 + 1.2 kPa CO 2 , at 1.0 °C. When the   temperature was raised to 1.5 °C, the fruit stored under 0.8 kPa O 2 + 1.2 kPa CO 2 maintained lower respiration rate as compared to 1.2 kPa O 2 + 2.0 kPa CO 2 (Fig. 3b). For 'Galaxy' , at 7 days of shelf life, the condition 1.5 °C and 1.2 kPa O 2 + 2.0 kPa CO 2 resulted in higher fruit respiration rate than the ones stored under 0.4 kPa O 2 + 1.2 kPa CO 2 . However, the temperature elevation did not increase the respiration rate of fruit stored under 0.4 kPa O 2 + 1.2 kPa CO 2 , evidencing the effect of the ethanol production increase (Fig. 4b) in the reduction of ethylene production and, consequently, a reduction in the respiration rate. When the fruit were stored at 1.0 °C, the respiration rate was greater in the fruit stored under 0.8 kPa O 2 + 1.6 kPa CO 2 when compared to all other conditions (Fig. 3b). The internal ethylene concentration (IEC) in 'Royal Gala' , at chamber opening, was lower under 0.8 kPa O 2 + 1.6 kPa CO 2 when compared to 1.2 kPa O 2 + 2.0 kPa CO 2 at 1.0 °C (Fig. 3b). This finding may be related to the lower respiratory   Extremely low-oxygen storage metabolism and ethylene production of the fruit stored under 0.8 kPa O 2 + 1.6 kPa CO 2 (Fig. 3a, b). The elevation of storage temperature caused a higher IEC in the fruit stored under 1.2 kPa O 2 + 2.0 kPa CO 2 . The high IEC is related to the postclimacteric state (Bender et al. 2000). Apples stored under 1.2 kPa O 2 + 2.0 kPa CO 2 at 1.5 °C had lower IEC than to 1.0 °C, evidencing that they could already have passed by climacteric stage. On the other hand, fruit maintained under Means followed by equals letters, lowercase in the atmosphere condition and uppercase in the temperature storage, do not differ by Tukey's test at 5% of error probability (p < 0.05). *ns: not significant difference.   Ludwig et al. 0.8 kPa O 2 + 1.6 kPa CO 2 had the lowest IEC and ethylene production at 1.0 °C (Fig. 3a). For 'Galaxy' apples, the storage under 1.2 kPa O 2 + 2.0 kPa CO 2 resulted in higher IEC compared to a 0.8 kPa O 2 + 1.6 kPa CO 2 and 0.4 kPa O 2 + 1.2 kPa CO 2 at 1.0 °C. After the period of shelf life, the conditions of 0.4 kPa + 1.2 kPa CO 2 and 0.8 kPa O 2 + 1.6 kPa CO 2 presented a lower IEC than 1.2 kPa O 2 + 2.0 kPa CO 2 at 1.0 °C (Fig. 3b). 'Royal Gala' apples stored under 0.8 kPa O 2 + 1.6 kPa CO 2 at 1.0 °C had lower ACC oxidase activity in relation to 1.2 kPa O 2 + 2.0 kPa CO 2 (Fig. 3a). At 1.5 °C, the low oxygen did not reduce the activity of the ACC oxidase. When comparing the storage temperatures, the lowest ACC oxidase activity was observed at 1.5 °C when fruit were submitted to 1.2 kPa O 2 + 2.0 kPa CO 2 . For 'Galaxy' apples, the lowest activity of ACC oxidase was observed in the fruit under 0.4 kPa O 2 + 1.2 kPa CO 2 condition (Fig. 3b). This lower activity led to a lower ethylene production and lower respiration rate (Fig. 3a).

Flesh firmness, physiological disorders and healthy fruit
Flesh firmness in 'Royal Gala' apples was higher at 1.5 °C than at 1.0 °C (Fig. 2a). Among the storage conditions, the highest flesh firmness was observed under 0.8 kPa O 2 + 1.6 kPa CO 2 , which may be due to the lower ACC oxidase enzyme activity and lower ethylene production (Fig. 2a, 3a). Ethylene activates enzymes that degrade the cell wall (Payasi et al. 2009;Prasanna et al. 2007), causing higher loss of flesh firmness during storage, such as β-galactosidase, α-1-arabinofuranosidase, pectin methyl esterase and polygalacturonase (Ortiz et al. 2011;Wei et al. 2010). For 'Galaxy' apples, the higher flesh firmness maintenance was found under 0.4 kPa O 2 + 1.2 kPa CO 2 when compared to all the other CA conditions at 1.0 °C. Similar results were found in 'Royal Gala' apples stored under extremely low oxygen partial pressure, where the low oxygen (0.4 kPa O 2 + 1.2 kPa CO 2 ) maintained higher flesh firmness as compared to 1.2 kPa O 2 + 2.0 kPa CO 2 storage (Berghetti et al. 2020). This may be related to the higher production of ethanol (Fig. 4a), which reduces the ACC oxidase enzyme activity and the ethylene production. However, when the fruit were stored at 1.5 °C, no difference between the storage conditions was found in ACC oxidase activity. For 'Galaxy' apples, the highest maintenance of flesh firmness was found in the fruit stored under 0.8 kPa O 2 at 1.5 °C (Fig. 2a). The increase of the temperature resulted in a greater flesh firmness maintenance of 'Galaxy' apples stored under DCA-QR 1.3 (Both et al. 2018), and in 'Gala' and 'Imperial Gala' stored under CA (1.8 kPa of O 2 + 2.0 kPa of CO 2 ) (Mazzurana et al. 2016).
The incidence of mealiness is one of the main troubles of the 'Gala' apples group and it is responsible for causing commercial depreciation. This is correlated to the advancement of ripening and degradation of the cell's lamella. The CA condition did not influence the incidence of mealiness for 'Royal Gala' apples ( Fig. 2a); however, a lower percentage of mealiness was found at temperature of 1.5 °C than at 1.0 °C (Fig. 2a). On the other hand, 'Galaxy' apple submitted to 0.4 kPa O 2 + 1.2 kPa CO 2 presented lower incidence of mealiness than in any of the other CA conditions, due to a lower activity of the ACC oxidase enzyme, internal ethylene concentration and lower ethylene production at chamber opening and at 7 days of shelf life (Fig. 3a, b).
Flesh breakdown is also correlated to the advancement of ripening and is caused by the leakage of the vacuole content rich in polyphenols that encounters the polyphenol oxidase enzyme of the cell cytosol causing dark color in the pulp. Flesh breakdown may be induced by several factors, such as high concentration of anaerobic metabolism compounds, CO 2 concentration above 1 % and cell freezing (Fan et al. 2011;James and Jobling 2009). The incidence of flesh breakdown in 'Royal Gala' apples was lower at 1.5 °C than at 1.0 °C. Increase in the temperature from 0 to 3 °C reduced the occurrence of flesh breakdown in 'Pink Lady' (James et al. 2005) and 'Nicoter' apples (Weber et al. 2019). The greater incidence of flesh breakdown may be correlated to the production of ethylene under low-temperature storage (Fig. 1). No significant difference was found in the incidence of flesh breakdown between the different temperatures and CA conditions for 'Galaxy' apples.
The percentage of healthy fruit in 'Royal Gala' was higher at 1.5 °C than at 1.0 °C (Fig. 2b), due to the lower incidence of flesh breakdown and mealiness in comparison to fruit stored at 1.0 °C (Fig. 2a). No difference was found regarding the percentage of healthy fruit, pulp cracking and decay incidence for 'Galaxy' apples. This result suggests that the temperature of 1.5 °C can be used in apple fruit industry.
Extremely low-oxygen storage

Soluble solids and titratable acidity
Soluble solids (SS) are a feature that directly influences the consumers acceptance of apples. For 'Royal Gala' apples, the amount of SS after the shelf life period was greater at 1.5 °C than at 1.0 °C (Fig. 4a). For 'Galaxy' apples, there was no difference in amount of SS, regardless of the condition and temperature of storage.

Anaerobic metabolism
Fruit anaerobic metabolism may occur during the storage of apples in ULO, along with the production of anaerobic metabolic compounds such as ethanol, acetaldehyde and ethyl acetate.
For 'Royal Gala' apples, the concentration of ethanol was greater under 0.8 kPa O 2 + 1.6 kPa CO 2 than in 1.2 kPa O 2 + 2.0 kPa CO 2 at 1.5 °C (Fig. 4b), probably because the fruit stored under lower oxygen partial pressure activated more anaerobic metabolism, producing more ethanol. The acetaldehyde concentration was lower under 0.8 kPa O 2 + 1.6 kPa CO 2 when compared to 1.2 kPa O 2 + 2.0 kPa CO 2 at 1.0 °C (Fig. 4b). The accumulation of acetaldehyde in the fruit tissue can be a response to fruit senescence (Ke et al. 1994;Wright et al. 2015). A greater incidence of mealiness occurred under 1.2 kPa O 2 + 2.0 kPa CO 2 condition, which reduces the diffusion of gases ) and consequently induces the production of acetaldehyde (Thewes et al. 2017b). Additionally, fruit kept at 0.8 kPa O 2 + 1.6 kPa CO 2 probably had a lower pyruvate decarboxylase enzyme (PDC) expression and activity, which is induced by the ethylene (Harb et al. 2011). Therefore, the lower ethylene production was observed in the fruit stored under 0.8 kPa O 2 + 1.6 kPa CO 2 (Fig. 3a).
Nevertheless, for 'Royal Gala' apples, an inverse response was noticed at 1.5 °C since the fruit stored under 1.2 kPa O 2 + 2.0 kPa CO 2 presented a lower concentration of acetaldehyde in comparison to 0.8 kPa O 2 + 1.6 kPa CO 2 . The storage under extremely low oxygen partial pressure results in an increased production of anaerobic metabolism compounds, including an elevated acetaldehyde concentration (Pesis 2005); however, a lower concentration of acetaldehyde was found under 0.8 kPa O 2 + 1.6 kPa CO 2 at 1.0 °C in comparison to 1.5 °C. The use of low partial pressure of O 2 associated with low temperature probably caused damage to the tissue and, consequently, increased the occurrence of physiological disorders such as mealiness and flesh breakdown, besides reducing the flesh firmness (Fig. 2a).
The concentration of ethyl acetate has an important role in the formation of the aroma of apples; however, when present in high concentrations, it can cause off-flavors (Wright et al. 2015). The concentration of ethyl acetate was greater under 1.2 kPa O 2 + 2.0 kPa CO 2 than under 0.8 kPa O 2 + 1.6 kPa CO 2 for 'Royal Gala' , when stored at 1.0 °C (Fig. 4a). This is due to the fact that there is a higher production of acetaldehyde and ethanol under 1.2 kPa O 2 + 2.0 kPa CO 2 (Fig. 4a), since the acetaldehyde is converted into ethanol by the ADH and, from an esterification reaction with acetic acid, forms ethyl acetate (Ke et al. 1994;Lee et al. 2014). However, at 1.5 °C, the condition of 0.8 kPa O 2 + 1.6 kPa CO 2 provided a greater concentration of ethyl acetate than 1.2 kPa O 2 + 2.0 kPa CO 2 (Fig. 4a). When stored under 0.8 kPa O 2 + 1.6 kPa CO 2 , a greater concentration of ethyl acetate occurred at 1.5 °C in relation to 1.0 °C.
The lowest concentration of ethanol for 'Galaxy' apples was found under 0.4 kPa O 2 + 1.2 kPa CO 2, at 1.0 °C, in comparison to 1.2 kPa O 2 + 2.0 kPa CO 2 , without differing from 0.8 kPa O 2 + 1.6 kPa CO 2 , probably due to the lower conversion of acetaldehyde into ethanol under 0.4 kPa O 2 + 1.2 kPa CO 2 (Fig. 4a). However, when 'Galaxy' were stored at 1.5 °C, an inverse response was observed in the conditions of 0.4 kPa O 2 + 1.2 kPa CO 2 and 0.8 kPa O 2 + 1.6 kPa CO 2, the fruit presented greater concentration of ethanol than in 1.2 kPa O 2 + 2.0 kPa CO 2 , due to a larger production of acetaldehyde, that was converted into ethanol by the action of the ADH enzyme. However, the elevated production of acetaldehyde is due to the anaerobic metabolism and not due to the senescence of the fruit, since these had a lower respiration rate and a lower percentage of mealiness (Fig. 2a, 3a).
A lower concentration of ethanol was noticed when the fruit were stored under 0.8 kPa O 2 + 1.6 kPa CO 2 at 1.0 °C than when submitted to 1.5 °C. A similar response was found when the fruit were stored under 0.4 kPa O 2 + 1.2 kPa CO 2 at 1.0 °C. The temperature increase from 1.0 °C to 18 °C resulted in a more than 10-fold increase in the anaerobic metabolism in apples fruit (Boeckx et al. 2018). Another related factor is the chilling injury combined with low oxygen partial pressure (Brackmann et al. 2012). Fruit stored under 1.2 kPa O 2 + 2.0 kPa CO 2 presented the lowest concentration of acetaldehyde in comparison to all the other storage conditions. V. Ludwig et al.

CONCLUSION
The best storage condition for 'Royal Gala' apples is 0.8 kPa O 2 + 1.6 kPa CO 2 at 1.5 °C, which maintains a higher percentage of healthy fruit, soluble solids, flesh firmness, lower flesh breakdown and mealiness. However, lower O 2 partial pressures, such as 0.4 kPa, should be studied for this cultivar in the future.
For 'Galaxy' apples the storage under 0.4 kPa O 2 + 1.2 kPa CO 2 at 1.5 °C was the most efficient, because it reduces the ACC oxidase enzyme activity, along with the ethylene production and decreases the incidence of mealiness. The temperature can be increased from 1 °C, which is traditionally used for these mutants, to the temperature of 1.5 °C, which can allow saving electrical energy.
There was an increase in the ethanol concentration by increasing the temperature from 1.0 to 1.5 °C for 'Galaxy' and 'Royal Gala' apples stored under 0.4 kPa O 2 + 1.2 kPa CO 2 conditions. 'Galaxy' apples stored under 0.4 kPa O 2 + 1.2 kPa CO 2 and 0.8 kPa O 2 + 1.6 kPa CO 2 results in higher concentration of ethanol compared to 1.2 kPa O 2 + 2.0 kPa CO 2 .