Introduction

In Mexico, approximately 360 thousand t of fresh strawberries (Fragaria ananassa) are produced each year, out of which 77 thousand (21.3 %) are exported, with a value of 142.1 million dollars (^{FAOSTAT, 2012}). The main cultivated varieties are: Festival, Sweet Charlie, Galaxy, Camino Real, Albion, Camarosa, and Diamante. All these varieties come from mother plants imported from the USA to Mexico, where they are multiplied in nurseries, then transplanted, and grown in field or protected systems. In the Colegio de Postgraduados, Mexico, studies were carried out in order to generate Mexican varieties, which are under technological evaluation for production, pests and diseases resistance, harvest season, and fruit quality. The varieties that have stood out are: CP-LE7, Zamorana, and Jacona; the last two are awaiting their acceptance by the Registro Nacional Agropecuario loaf the Secretaría de Agricultura, Ganadería, Pesca y Alimentación of Mexico.

Strawberry fruits are very likely to decay or go bad quickly, due to their high metabolic activity, extreme susceptibility to mechanical damage, water loss, and rot development, caused mainly by Botrytis cinerea (^{Anderson et al., 2004}). Temperature is one of the main factors that accelerate the deterioration of strawberry fruits; therefore, designing an adequate cold chain is important to achieve a shelf life that fulfills established commercialization requirements. Strawberries are harvested and packaged in the field, and then precooled up to storage or transport temperature (or both). According to ^{Nunes et al. (1995)}, a 6 h delay in cooling fruits harvested at 30 °C significantly diminishes firmness, and affects its losses. Recommended refrigeration temperatures for strawberry fruits range from 0 to 0.5 °C (Anderson et al., 2004). Atmospheres with high CO₂ concentrations (10 to 20 %) are used during transport and refrigerated storage of strawberry fruits, in order to delay the damage caused by tissue softening, accelerated water loss, and rot development (^{Tudela et al., 2003}). However, depending on the cultivar, production technology, and concentration of CO₂, the anaerobic metabolism is stimulated, which -depending on the exposure time- favors the accumulation of ethanol and acetaldehydes, with the consequent perception of unpleasant flavors and aromas (Deell, 2006). The strawberry fruits response to refrigeration treatment -with or without atmospheres enriched with CO₂- varies with the cultivar, and ^{Pelayo et al. (2003)} reported differences in postharvest life from 2 to 4 d among the strawberry cultivars Aromas, Selva, and Diamante.

The biochemical and physiological responses of the fruits in the Mexican strawberry cultivars to the use of controlled atmospheres and refrigeration temperatures are unknown. Therefore, the objective of this research was to evaluate the effect of two high CO₂ concentrations for a short period in the changes associated with quality, rot control, and shelf life of three Mexican strawberry cultivars. The hypothesis was that the strawberry varieties have similar maturation; therefore, fruit quality will have the same behavior and response to the treatments.

Materials and Methods

For this research, fruits from three Mexican strawberry cultivars CP-LE7, Zamorana, and Jacona, as well as from the introduced cultivar Festival (control) were harvested. The plants were grown in a commercial plot from the production area of Tangancicuaro (19° N, 102° W), State of Michoacan. Local climate is temperate tropical. From each cultivar, a random sample of red fruits with – ripeness was harvested. Defective fruits -with physical or pathogen damage- were discarded; then, two treatments were formed with healthy fruits, according to the storage conditions: fruits stored at 3 °C with 5 % CO₂ atmosphere (3 °C+5 % CO₂), and at 3 °C with 10 % CO₂ atmosphere (3 °C+10 % CO₂). The fruits were stored for 3 and 5 d in the treatment conditions, plus, in each case, 2 d in environment conditions (20±2 °C, 50-60 % RH). For each CO₂ treatment, four 4-L containers were used per cultivar; 800 g of fruit were placed in each one. In order to establish the desired atmospheres, a gas mixer was used (Postharvest Research, UC, Davis, CA, USA, Series 916-753-4587 with 6 mixing stations). In order to multiply and distribute the gas mixtures generated, boards were used with calibrated capillaries, with the following flows -for 5 % and 10 % CO₂, respectively-: CP-LE7 (1.78 and 1.93 L min^-1), Zamorana (2.00 and 2.13 L min^-1), Jacona (1.99 and 2.12 L min^-1), and Festival (2.00 and 2.14 L min^-1). In order to monitor the gases, a Telaire 7001 CO₂ Monitor with ± 0.5 % effectiveness was used. In addition, gas flow was humidified prior to the entrance of the distribution boards to maintain a 90 % RH in the containers.

In order to evaluate the rot development caused by Botritys cinerea, an incidence analysis was carried out with 50 fruits from each cultivar. Fruits were harvested and exposed to the established CO₂ concentrations. The number of healthy and rotting fruits was quantified after 5 d stored under CO₂ and refrigeration treatment. Data were reported as percentage of healthy and rotting fruits.

The evaluations were carried out at the time of harvest at 3 and 5 d with treatments and after the fruits were 2 d under environmental conditions. The recorded variables were: acetaldehyde concentration in pulp, using the method described by Davis and Chase (1969). In order to do so, 5 g of pulp per cultivar were weighed and placed in one 25-mL vial, which was hermetically sealed. Each vial was one replicate, and there were five replicates per cultivar. These vials were incubated 10 min in a bain-marie at 32 °C; then, they were shaken for 5 s; finally, a 1 mL sample was taken from the headspace and injected in a Hewlett Packard gas chromatograph, model 5900 II (CA, USA). The operating temperatures were: 170 °C in the column (injector inlet), 180 °C in the Detector A or TCD (Thermal Conductivity Detector), 180 °C in the FID detector (Flame Ionization Detector), and 150 °C in the oven. Previously, an equal number of vials were prepared with a 5 mL solution of ethanol and acetaldehyde with known concentration (standard), following the same procedure. The data for both metabolites were reported in mg 100 g^-1 fruit. The weight losses were evaluated in a sample of 10 fruits per treatment, at the end of the environment exposure; based on that data, the weight losses were calculated with the following formula:

Fruit color was measured in 10 fruits per treatment and cultivar, around their horizontal diameter, with a Hunter Lab tristimulus colorimeter model D25A (Virginia, USA). The CIELAB L*a* and b* parameters were recorded, which were used to calculate the hue angle (°h=atan(b*/a*)), and the saturation index (Chroma (C)=(a²+b²)^1/2). Pulp firmness was measured individually in a sample of 5 fruits per treatment and cultivar, on the opposite sides of the average diameter, using a digital force gage model FVD-30 with a conic probe (Wagner Instruments, CT, USA), and data were recorded in Newtons (N). Titratable acidity (AT) was evaluated based on the determination of citric acid content (%), according to the ^{AOAC method (1990)}: in 5 fruits per treatment and cultivar, a 5-g sample per fruit was individually weighed; then, each sample was homogenized with 50 mL of distilled water; subsequently, a 5-mL aliquot was taken from the filtrate, and it was titrated with NaOH 0.1 N, using phenolphthalein in alcohol solution (2.5 %) as indicator. The pH of the same filtrate was determined using a potentiometer (Corning model 12, NY, USA). In the juice of the same number of fruits per treatment and cultivar, the total soluble solids content was individually measured according to the ^{AOAC method (1990)}, using a digital refractometer (Atago model Pr-100, Guangzhou, China), and the values were recorded in °Brix. In 5 fruits per treatment and cultivar, the total sugar content was determined individually using the Anthrone colorimetric method: 1.0 g of pulp per fruit was weighed, then mixed with 60 mL of 80 % alcohol in an Erlenmeyer flask. This mixture was boiled for 10 min, and cooled down to room temperature. Then, it was filtered, and adjusted to a volume of 25 mL, out of which a 1.0-mL aliquot was taken, dehydrated in a bain-marie at 55 °C, and 60 mL of distilled water were added. A1.0 mL aliquot of this solution, 3 mL of distilled water plus 6 mL of Anthrone reagent were added to a test tube placed in an ice bath. The test tubes were then placed in a bath with boiling water for 3 min, before cooling them rapidly with ice water. Finally, the absorbance was read at 600 nm. For calculation purposes, a standard glucose curve was obtained, and the data were reported as g sugars 100 g^-1 fruit.

Results and Discussion

The analysis of the fruits of each variety at harvest time did not show any significant differences with regard to the variables evaluated. After analyzing the data at the end of the treatments, the analysis of variance showed that regarding the internal quality, Zamorana fruits had higher acidity (1.13 % citric acid), as compared to the others cultivars. It is important to point out that the SST, the SST/AT ratio, pH, total sugars, and acetaldehyde kept similar concentrations in the four cultivars, after the controlled atmosphere treatments, and their values were close to the initial ones. After the fruits were kept 2 d under environment conditions of temperature and atmosphere, the total sugar content increased significantly: CP-LE7 showed the highest content (9.02 g 100 g^-1 fruit), in addition to a greater accumulation of acetaldehyde (26.02 mg 100 g^-1 fruit). The results of the volatile compounds concentration -as a response to the fruits’ exposure to high CO₂ concentrations- presented changes only with regard to acetaldehyde concentration, but no ethanol was detected (Table 1). According to ^{Ke et al. (1991)}, in horticultural products exposed to stress condition -due to low O₂ concentrations, high CO₂ concentrations, or both- anaerobic respiration is stimulated, which favors the accumulation of acetaldehyde and ethanol. ^{Pelayo et al. (2007)} report that the exposure of ‘Camarosa’ strawberries to a 19.7 % CO₂ atmosphere at 5 °C favored the accumulation of fermentative metabolites, which caused changes to the aroma profile and undesirable changes to the fruit taste. The acetaldehyde concentration results show that the exposure to high CO₂ levels stimulates anaerobic metabolism, because, in all treatments, the acetaldehyde concentration increased, compared to the concentration at harvest time. ^{Pelayo et al. (2003)} documented differences in the accumulation of total fermentative metabolites among Selva, Diamante, and Aromas strawberry cultivars, after they were stored in a 19.7 % CO₂ atmosphere at 5 °C for 7 d. This suggests that the acetaldehyde accumulation results show differences in the sensitivity to the accumulation of anaerobic metabolites among the Mexican strawberry cultivars studied.

The fruit external quality variables showed that Jacona had brighter fruits (L=34.54), while Festival produced fruits with a higher saturation index (C=32.33). The Mexican varieties had firmer fruits than the commercial variety did, and their values were higher than 20 N; CP-LE7 stood out with 22.35 N. The firmness in the four varieties decreased after 2 d under environment conditions, but the fruit of the Mexican varieties remained firmer than the Festival fruits did. The weight loss during the high CO₂ concentration and refrigeration treatments was low: CP-LE7 presented a greater loss (1.78 %) and Zamorana showed better resistance to fruit dehydration (0.88 %). This implies that -under the said storage conditions- Mexican Zamorana strawberry cultivar is less sensitive to weight loss, compared to other cultivars. Regarding the storage time, the greatest weight losses occurred when the fruits were transferred to room temperature and environment atmosphere conditions, and Festival lost up to 19.84 %. Color, luminosity, hue angle, and fruit saturation index of the fruits decreased under these conditions, and the commercial variety had the highest values (L=32.75, °h=34.13, C=26.63); Jacona was the only variety whose values were close to the values of the commercial variety (Table 1).

The evaluation time had no significant effect on the variables (p˃0.05). The fruits of the four cultivars were taken out the controlled atmospheres and, 2 d after being at room temperature and under normal atmosphere conditions, significant statistical differences were found. Therefore, the AT (0.87 % citric acid) was higher in fruits with 5 d of treatment, the SST/AT ratio was higher at 3 d (10.41), as well as the pH of the fruits (4.03). Strawberries subjected to 5 d exposure time showed greater firmness (19.39 N) and reduced weight loss up to 43 %; they had a better and brighter color (L=33.33); and their saturation index was higher (C=26.72) (Table 2). These results revealed that the color of the strawberry cultivars studied changed as a result of the effect of CO₂ concentrations, as well as exposure times. Such changes are attributed to the differences in the synthesis and degradation of anthocyanins, the modification of the type of anthocyanins -as a result of changes in the fruit’s pH-, and the effect of treatments with high CO₂ concentrations of that induce changes in the vacuolar pH (^{Halcroft and Kader, 1999}; ^{Pelayo et al., 2003}; ^{Boledon et al., 2010}).

The statistical analysis of the CO₂ concentration showed significant changes in the fruits of the four cultivars. There was a higher accumulation of total sugars (8.58 g 100 g^-1 fruit), and pH (4.20) with the 5 % concentration of CO₂, but fruits lost more weight (1.4 %). After the fruits were moved normal conditions of temperature and atmosphere, the lowest CO₂ concentration gave the best results: less sour fruits (0.77 % citric acid), higher total sugar content (8.49 g 100 g^-1 fruit), fruits with greater firmness (19.47 N), and brighter fruits (L=32.1), deeper (°h=34.1), and less opaque (C=26.1) reds; nevertheless, fruit weight loss was lower under the highest concentration (10 % CO₂) (Table 3). ^{Holcroft et al. (1999)}) and Vicente et al. (2003) point out that CO₂ enriched atmospheres delay softening during storage conditions, while firmness decreases rapidly when fruits are placed under room temperature and normal atmosphere conditions. Likewise, ^{Harker et al. (2000)} reported that high CO₂ concentrations have a positive effect on the reduction of firmness loss in strawberry fruits associated with changes in the apoplast’s pH. This promotes the precipitation of soluble pectins and improves the cell-to-cell adhesion at the middle-layer level.

The time*concentration interaction showed that the combination of 5 d+10 % CO₂ at the end of the treatment, and 5 d+5 % CO₂ plus 2 d at room temperature caused greater acetaldehyde content in the fruits (26.29 and 24.39 mg 100 g^-1 fruit). In each cultivar, the total sugar content was lower in the treatments with 10 % CO₂ (Table 4). Depending on how long the fruit was stored at 3 °C, the total sugar content was significantly lower in the treatments with 10 % CO₂ in 3 and 5 d. In this respect, the level of glucose, fructose, and total sugars in strawberry fruits decreases because of the 19.7 % of CO₂ treatment (^{Pelayo et al., 2003} and ²⁰⁰⁷); meanwhile, fruit sugar content is lower when stored under a higher CO₂ concentration. Therefore, there is an effect on the accumulation. However, the metabolism related to this decrease in the sugar content of strawberries is still unknown. Nevertheless, ^{Watkins (2000)} has set forth the hypothesis that the stimulation of the respiratory activity (at glycolysis level) responds to a tissue stress condition imposed by the high CO₂ concentration.

Depending on the storage time, the greater weight losses occurred after the refrigeration and CO₂ treatment when the fruits were stored at room temperature and normal atmosphere conditions. The greatest weight loss occurred in the fruits under 3 d+5 % of CO₂ treatment stored at 3 °C (23.81 %), even though, there were already significant losses (1.46 %) at the end of the treatment (Table 4). The time*concentration of CO₂ interaction revealed that the fruit firmness in all the treatments decreased significantly when the fruits were placed under room temperature and atmosphere conditions; the treatment that kept the highest fruit firmness was 5 d+5 % CO₂, at the end of the treatment (23.24 N), as well as 2 d later (20.81 N).

According to the time*concentration of CO₂ interaction, the color of the fruits stored under the 5 d+10 % CO₂ treatment at 3 °C had the highest parameters at the end of treatment (L=33.9, °h=35.33, C=31.97). When fruits were put under room temperature and normal atmosphere conditions resulted in significant losses of L (darker), C (less brightness) and °h values, indicating a deeper red shade in the strawberry fruits (Table 4).

In a 10 % CO₂+3 °C controlled atmosphere, the rot index decreased considerably, because all the varieties had levels lower than 4 %. The 5 % CO₂+3 °C treatment also decreased the presence of the pathogen in the fruits, but it was not that significant (Figure 1). According to ^{Wszelaki et al. (2003)}, a high CO₂ concentration effectively suppresses mycelial growth, spore germination, and elongation of the Botrytis cinerea germ tube; it is also fungistatic to most fungi. However, once the fruit is removed from the controlled atmosphere, it is no longer protected, and the fungus can start to grow again. In addition, they concluded that the continuous application of controlled atmospheres with 15 kPa CO₂ (14.8 % CO₂) during 14 d was the method that best controlled the development of Botrytis cinerea in the fruits. In addition, ^{Piña-Dumoulín et al. (2001)} also found that controlled atmospheres with 15 % O₂+20 % CO₂ significantly retarded fungus growth in blackberries stored at 2 °C.

Figure 1 Rot percentage by Botrytis cinerea in three Mexican strawberry varieties, and one commercial variety, subject to two controlled atmosphere treatments with 3 °C+5 % CO₂, and 3 °C+10 % CO₂ for 5 days. 

Conclusions

The Mexican cultivars (CP-LE7, Zamorana, and Jacona) had firmer fruits compared to the commercial cultivar, which provides them with greater resistance during the post-harvest manipulation. Each cultivar presented at least one outstanding attribute: CP-LE7, total sugar content; Zamorana, less sensitivity to weight loss; and Jacona, bright red fruits. The rot percentage was controlled by the two controlled atmospheres applied in the four cultivars. A controlled atmosphere with 10 % CO₂ had greater control over Botrytis cinerea, while an atmosphere with 5 % CO₂ had best results in keeping fruit quality.