Introduction

Strawberry production in México is estimated at 360 426 tonnes, which represents l7.9% of world production, placing it as the second largest producer after the United States of America (^{FAOSTAT, 2012}), commercially, the exports level reaches 76 890 tonnes with a value of 142 053 million dollars (^{FAOSTAT, 2011}), mainly as a fresh product.

Currently strawberry production in México is based on the varieties Festival, Sweet Charlie and Galaxia, among others, developed in the Universities of California and Florida, USA, all of them with specific characteristics in terms of yield, resistance to pests and diseases, production season and fruit quality. New varieties have been generated in México, some of which are under evaluation, both in protected and field conditions, with the objective of defining the characteristics of plant development and the requirements of production technology; it is also necessary to know the physiological and biochemical changes related to plant maturation and post-harvest senescence, in order to define crop indexes, quality patterns and response to technologies aiming to prolong shelf life.

Several factors, such as ripeness maturity, temperature, relative humidity, rot and physical damage incidence, favor the loss of commercial, nutritional and sensorial quality of strawberry fruits in an accelerated way (^{Pineli et al., 2011}). The most commonly used strawberry harvest index is when the fruit takes a pink (– ripening) or a red (ripe-firm) color. ^{Mitcham and Mitchell (2002)} mention that the best state for the strawberry harvest is when the fruits have reached at least – red color in order to achieve a longer shelf life.

Among the technologies used to extend shelf life of this product are refrigeration in normal atmosphere (air) or with high concentrations of CO₂. Regarding to this, it has been reported (^{Ayala-Zavala, 2004}) that the conservation at 0 °C maintains the quality of strawberry fruits for 7-9 days, being a limitation the decay development by Botrytis cinerea and loss of antioxidant capacity (^{Peng et al., 2011}). The use of CO₂ enriched atmospheres (10-15% CO₂) prolonged shelf life to 9-13 days (^{Pelayo et al., 2003}) and preserved firmness, total soluble solids, pH and titratable acidity (^{Pelayo-Zaldívar et al., 2007}). In contrast, ^{Holcroft and Kader (1999a)} report that high concentrations of CO₂ negatively affect fruit color, anthocyanin concentration and the formation of fermentative metabolites leading to a change in taste (^{Pelayo-Zaldívar et al., 2007}). The objective of this paper was to evaluate the effect of two concentrations of CO₂ on the quality and shelf life of the fruit of two Mexican varieties of strawberry stored under refrigeration.

Materials and methods

For the experiment, plants of two varieties of Mexican strawberry denominated as Jacona and Zamorana obtained by the Program of Genetic Improvement of Fruit of the Colegio de Postgraduados in Agricultural Sciences, Campus Montecillo, México, as well as the Festival variety generated in the University of Florida, USA, taking it as a reference, and were produced under greenhouse conditions. In order to harvest the fruits, the – red state (^{standard: NMX-FF-062-SCFI-2002}) was used as criterion, establishing six treatments in total with 40 fruits each, resulting from the combination of variety factor (Festival, Jacona and Zamorana) and CO₂ concentration (0.03 and 15%) in the atmosphere, all stored for 8 days at 2 ±1 °C and 85-90% of relative humidity.

In order to establish the atmosphere with 15% of CO₂ the Mixing Board UC-Davis gas mixing system and panels were used to maintain a flow of 184 mL min^-1. At constant pressure; the gases for the mixture were air (0.03% CO₂) and CO₂ from a pressurized cylinder with 100% concentration. The gas entrainment system was complemented by placing the fruits in a total of 24 bottles (4/treatment), which were sealed and a continuous system was established. CO₂ measurements were carried out using a temperature and CO₂ recording equipment (TELAIRE brand, model 7001, manufactured in Goleta, California, USA) and data logger (HOBO brand, model U-12-012, manufactured in Bourne, Massachusetts, USA). The evaluated variables were: accumulated weight loss calculated based on the weight difference between the initial value and the corresponding value at the end of the evaluation period, expressing the data as percentage (%),pulp firmness using a Wagner Texturometer model FDV -30 with a metal base Chatillon, adapted with a 5 mm conical strut, reporting the data in Newtons (N); external color (measured at the maximum equatorial part of the fruit) was determined using a Hunter Lab colorimeter (Reston, Virginia, USA, model D-25), obtaining with the Lab-Hunter values the luminosity, pitch angle or °h (180 + tan^-1 b/a) and saturation index or chroma (a² + b² )^½ (Arias et al., 2000), total soluble solids (% SST).

Determined by a Atago digital refractometer model PR-100, with a scale of 0-32% according to the ^{AOAC (1990)} method, titratable acidity (% citric acid) and pH of the pulp were measured according to the methodologies described By ^{AOAC (1990)}, ascorbic acid (vitamin C) determined according to the 2,6-dichlorophenol indophenol method described by ^{AOAC (2000)} and reported in mg 100 g^-1, ethanol and acetaldehyde production (mg 100 g^-1) by the method proposed by Davis and Chace (1969) using a Hewlett Packard gas chromatograph model 5890 II which operated at a 180 °C temperature in the flame ionization detector, furnace temperature of 150 °C isothermal and column Paraplot Q (25 m/0.22 nm internal diameter).

For the statistical analysis, a completely randomized design with factorial arrangement was applied and a means comparison with Tukey̕ s tests (α= 0.05) was used in order to detect significant differences between treatments. The measures of firmness, color and weight loss were performed in a sample composed of 10 replicates per treatment, being the experimental unit one fruit; for the rest of the variables the sample consisted of five replicates and the experimental sample of two fruits. Data were analyzed using the SAS^® 9.0 statistical package (^{SAS, 2000}).

Results and discussion

According to the results obtained (Table 1), after 8 days of storage at 2 ±1 °C, it was observed that both the variety factor and the CO₂ concentration did not show significant differences, which indicated that, in terms of weight loss, the strawberry fruits of the three cultivars showed similar behavior when exposed to an atmosphere with 0.03% or 15% of CO₂. It should be noted that the weight loss after the established storage period comprised from 1.49 to 2.49%, which did not provoke wilting symptoms, since this range was lower than the critical level of water loss for wilt damages of 6% reported by ^{Robinson et al. (1975)}, obtained by the daily cumulative evaluation of these losses.

Table 1 Analysis of variance (p- value) in the main interaction effects of the variety and CO₂ concentration, in the response variables studied. 

Valores en la misma columna con letras iguales no son estadísticamente diferentes (α= 0.01).

Regarding to the pulp firmness, no differences were observed due to the effect of the cultivar factors and CO₂ concentration (Table 1), the determined values were between 0.675 to 0.918 N. However, in relation to the initial firmness value, Jacona and Zamorana showed significantly firmness loss over ʻFestivalʼ during storage (Figure 1), suggesting differences in cell wall metabolism resulting from the genetic improvement of this variety (^{Perkins, 1995}; ^{Jiménez-Bermúdez, 2002}). It should be noted that Mexican varieties showed greater firmness (Figure 1) at the beginning of storage (Day 1) than the ʻFestivalʼ variety which, according to ^{Gooding (1976)}, is indicative of greater resistance to both mechanical and by pathogens damages.

Figure 1 Results of the analysis of variance (p- value) for firmness variable, in fruits evaluated at the time of harvest and after the treatments. Means with the same letter are statistically the same (Tukey, p≤ 0.05).  

As for the fruit color, after eight days of refrigeration at 2 ±1 °C, there were significant differences in the luminosity value due to the variety factor, being higher in ʻFestivalʼ (31.58), not showing differences by effect of the CO₂ concentration within each variety (Table 1). Regarding to storage time inrelation to the initial value, the luminosity decreased significantly only in the Mexican varieties, independently of the CO₂ concentration, which showed a slight darkening of the fruits (Figure 2). A similar response has been reported by ^{Holcroft and Kader (1999a)}, who found that the luminosity of fruits of the Selva variety stored for 5 and 10 days at 5 °C decreased as the storage time advanced and were not affected by the concentration of CO₂ (air and 20 kPa) in the surrounding atmosphere.

Figure 2 Results of the analysis of variance (p-value) for the color variable (luminosity), in fruits evaluated at the time of harvest and after treatments. Means with the same letter are statistically equal (Tukey, p≤ 0.05).  

Regarding to the pitch angle color attribute (°h), it was observed that at the end of storage there were no significant differences due to the effect of the variety as well as the concentration of CO₂; in addition, no significant differences were found in all 6 treatments (Table 1) resulting from combinations of the variety factors and CO₂ concentration. Regarding the time of harvest, in the fruits of the three studied varieties, the value of the pitch angle (°h) decreased significantly after the storage period established (Figure 3), which was indicative of changes in the red tonality of The fruits, mainly due to the anthocyanins oxidation effect due to the senescence advance (^{Bodelon et al., 2010}). As for the saturation index (Chroma), significant differences were also observed due to the variety factor, being higher in ʻFestivalʼ (Table 1).

Figure 3 Results of the analysis of variance (p- value) for the color variable (pitch angle, °h), in fruits evaluated at harvest time and after treatments. Means with the same letter are statistically equal (Tukey, p≤ 0.05).  

The results of the six treatments also showed that the fruits of the Festival variety exposed under normal atmosphere (0.03% of CO₂) showed the highest chroma value (30.33), indicating an inferior progression of senescence. As regards storage time, there were no differences in the saturation index (Chroma) within each variety (Figure 4), a response that coincides with that reported by ^{Holcroft and Kader (1999b)} where there were no significant differences in the chroma value in fruits stored for 0, 5 and 10 days in a controlled atmosphere (20 kPa CO₂) at 5 °C.

Figure 4 Results of the analysis of variance (p-value) for the color variable (saturation index or chroma), in fruits evaluated at the time of harvest and after treatments. Means with the same letter are statistically equal (Tukey, p≤ 0.05).  

After 8 days of storage at 2 ±1 °C, the total soluble solids content (SST) showed significant differences due to the variety factor, being ʻFestivalʼ higher (7.9 °Brix) than ʻZamoranaʼ (6 °Brix) (Table 1). ^{Gil et al. (1977)} also observed that exposure in normal atmosphere (air) or under enriched atmospheres with concentrations of 10, 20 and 40% CO₂ in strawberry fruits (Fragaria x ananassa cv. Selva) did not affect the content of SST. On the other hand, in relation to the storage time, within each variety, only a significant decrease of SST in ʻZamoranaʼ (Figure 5) was shown, suggesting a greater metabolic activity in this variety.

Figure 5 Results of the analysis of variance (p- value) for the total soluble solids (°Brix), in fruits evaluated at the time of harvest and after treatments. Means with the same letter are statistically equal (Tukey, p≤ 0.05).  

The pH of the pulp after 8 days of storage at 2 ±1 °C, was significantly different for both the variety effect and CO₂ concentration, being higher in ʻFestivalʼ, compared to ʻJaconaʼ, which coincided with a lower (0.73%) and higher (0.88%) citric acid content, respectively, also with significant differences. As for the treatment effect, in those fruits exposed to the atmosphere of 0.03% the pH was significantly higher than the treatment of 15% of CO₂; however, the citric acid content was similar (Table 1), suggesting differences in the metabolism of organic acids when used as a respiratory substrate. ^{Holcroft and Kader (1999a)} reported that storage at 5 °C and CO₂ concentration greater than 20 kPa increased the pH and decreased the titratable acidity in strawberry fruits of Selva variety while, the fruits exposed to a concentration of 10 kPa, showed increase in pH but the titratable acidity remained similar to the control (air), coinciding this behavior with the results obtained in the study for the three varieties studied. Based on the storage time, in the three varieties the pH tended to increase with respect to the initial value and only in the Zamorana variety, the citric acid content decreased significantly, which showed a different postharvest behavior of this variety in relation to ʻJaconaʼ and ʻFestivalʼ.

According to the results obtained in the concentration of ascorbic acid (vitamin C), there were significant differences due to the effect of the variety (Table 1), showing ʻFestivalʼthe highest concentration (45.79 mg 100 g^-1), then Jacona (43.772 mg 100 g^-1) and finally Zamorana (36.812 mg 100 g^-1). On the other hand, CO₂ treatment (15%) significantly maintained a higher content of this compound. No significant differences were observed in the interaction of the CO₂ concentration and concentration factors; however, the fruits of the three varieties treated with 15% of CO₂ compared to the fruits stored in normal atmosphere maintained a higher concentration of this vitamin, respectively, being 41.750 versus 39.503 mg 100 g^-1 in ‘Festival’, 39.953 versus to 33.67 mg 100 g^-1 in ‘Zamorana’ and 48.937 versus 38.607 mg 100 g^-1 in ‘Jacona’ (Table 1).

Analysis by variety of ascorbic acid content (Figure 6) showed that ‘Jacona’ and ‘Zamorana’ did not show significant changes, relative to the initial value, due to storage conditions; but not in Festival which tended to decrease significantly, which supposes a tendency to lose ascorbic acid when senescence advanced by an effect of oxidation until dehydroascorbic acid. According to ^{Lee and Kader (2000)}, vitamin C loss is influenced by factors such as variety and storage conditions, and this post-harvest vitamin C loss is reduced in fruits stored in atmospheres with 10% CO₂.

Figure 6 Results of the analysis of variance (p- value) for the variable ascorbic acid concentration, in fruits evaluated at the time of harvest and after treatments. Means with the same letter are statistically equal (Tukey, p≤ 0.05).  

Regarding the ethanol and acetaldehyde contents, the results obtained after 8 days at 2 ±1 °C the fruits treated with 15% CO₂ significantly increased the content of ethanol and acetaldehydes in the pulp (Table 1), being the Festival variety the most likely to accumulate these metabolites. The fruits with the highest content of ethanol and acetaldehydes were those of the Festival variety stored in controlled atmosphere (15% of CO₂) with values of 33.95 and 12.06 mg 100 g^-1, respectively (Table 1). The increase of fermentative metabolites has been reported in fruits stored at 5 °C and an atmosphere composed by air + 20 kPa CO₂ in contrast to fruits stored at 5 °C and normal atmosphere (air), which do not present an increase (^{Pelayo et al., 2003}). ^{Larsen and Watkins (1995)} reported values of 36.6 mg 100 g^-1 of ethanol and 1.38 mg 100 g^-1 of acetaldehyde in fruits treated with 20% CO₂ for 12 days at 0 °C, these compounds showed a slight bad taste. Likewise, the fruits of Festival variety showed a slight aroma and characteristic flavor of fermentation after storage, which evidenced the induction of anaerobic respiration, a response that did not occur in fruits of the Jacona and Zamorana varieties.

Conclusions

The strawberry fruits of the three cultivars, after being exposed to an atmosphere with 0.03% or 15% CO₂ for 8 days at 2 ±1 °C, showed similar behavior in terms of weight loss, firmness, luminosity, tone angle, saturation index and total soluble solids.

During storage, the fruits of the Mexican varieties Jacona and Zamorana showed greater firmness at the moment of harvest, but during the storage it shows more accelerated firmness losses with respect to ‘Festival’. On the other hand, the value of the tone angle of the three varieties decreases significantly, independently of the atmosphere type, which results in a different red tone than the initial one.

Storage at 0.03% or 15% CO₂ for 8 days at 2 ± 1 °C increases the pH of the pulp, mainly in the ‘Festival’ variety, but there are no changes in citric acid content; regardless of the variety, fruits stored at a concentration of 15% of CO₂ maintain a higher content of vitamin C than fruits stored with 0.03% (normal atmosphere).

After storage for 8 days at 2 ±1 °C, the concentration of ethanol and acetaldehyde increased significantly in those in fruits exposed 15% of CO₂ compared to those stored in air (0.03% of CO₂), the variety with the highest accumulation and perception of fermentative metabolites is Festival.

It is necessary to test other concentrations and periods of storage that allow to define the best option to maintain the quality of the Mexican varieties.