Fresh strawberries (Fragaria × ananassa Duch. cv. Goha) with red color on 70% - 80% of the fruit surface were hand-harvested from a commercial firm in Muju, Republic of Korea. Strawberry fruits were freshly harvested in the early morning and immediately transported to the postharvest laboratory of National Institute of Horticultural and Herbal Science, Suwon by a refrigerated truck maintaining a temperature of 4˚C. Fruits were graded for uniformity of color and size, and damaged fruits were removed. About 300 g fruits were then placed in transparent plastic box (16 × 12 × 8 cm) with several holes on the top and bottom portion of the box. One box of strawberry was considered as one replicate and four replicates were used in each treatment. Strawberry boxes were subjected to different concentrations and time of CO₂ treatment using acrylic chamber (60 × 30 × 30 cm) and CO₂ cylinder attached to a pressure gauge and a gun-like injector. Strawberries were treated with air (control), 15% CO₂ gas for 1 or 3 hours (15% CO₂-1 h or 15% CO₂-3 h), 30% CO₂ gas for 1 or 3 hours (30% CO₂-1 h or 30% CO₂-3 h), 50% CO₂ gas for 1 or 3 hours (50% CO₂-1 h or 50%CO₂-3 h) and stored at 4˚C for up to 13

days. Strawberry boxes were placed in the chamber, tightened the chamber opening and CO₂ was supplied to the chamber using small holes of the chamber where CO₂ concentration was monitored with a gas analyzer. The treatments were conducted at room temperature and when CO₂ concentration reached at expected level in different treatments, the chamber was completely closed and transferred to 4˚C storage room. After stipulated treatment duration, the chambers were taken out from the storage room, opened and only strawberry boxes were immediately transferred to 4˚C storage room. Evaluations of sensory, physical and biochemical qualities were carried out on 0, 3, 6, 9 and 13 days of storage.

Surface color of randomly selected seven fruit from each replicate was measured using a colorimeter (CR-400 Minolta, Osaka, Japan) which provided CIE L*, a* and b* values. Measurements were taken from opposite sides around the equatorial zone of the fruit. The meter was calibrated using the manufacturer’s standard white plate (Y 93.5, x 0.3155, y 0.3320). L* refers to the lightness and ranges from black = 0 to white = 100. A negative value of a* indicates green, while a higher positive number indicates red color. Positive and negative b* indicate yellow and blue color, respectively. These values were then converted into chroma {}, which indicates the intensity or color saturation.

The firmness was measured using a texture analyzer (TA Plus, Lloyd Instruments, Ametek Inc., UK) fitted with a flat probe. Each fruit was placed horizontally on the stationary platform and compressed 8 mm at a rate of 5, 2 and 10 mm∙s⁻¹ as the pre-test, test and post-test speed, respectively. With a running load cell of 20 N, the probe was attached to a creep meter equipped with the software (NEXYGEN™MT v 4.5, Lloyd Instruments, Ametek Inc., UK) for automatic analysis using a computer. The maximum force (N) developed during the test was recorded. Eight strawberry fruit from each replicate were randomly selected and a total of 32 data from four replicates were averaged from each treatment. All measurements were taken at room temperature (20˚C ± 2˚C).

Total soluble solid (TSS), pH and titratable acidity (TA) were measured according to the AOAC [15] procedures. On each evaluation day, 3 - 5 fruit from each replicate was wrapped with 2 layers of cotton cloth and squeezed with a hand pressed juice maker. TSS of the resultant juice was measured in terms of brix using a refractometer (PAL-1, Atago Co. Ltd, Tokyo, Japan). The pH was determined using a pH meter (D-55122, Schott Instruments GmbH, Germany) with a glass electrode. Titratable acidity was measured by diluting each 5 mL aliquot of strawberry juice in 20 mL distilled water and then titrating to pH 8.2 using 0.1 N NaOH and finally the results were expressed as percentage of citric acid.

The sensory analysis of strawberry sample was carried out by an 8-member (aged 24 - 48) expert panel. The members of the panel were trained to recognize and score overall visual quality, appearance, visual texture, of strawberry sample prior to the test. These sensory qualities were evaluated by using 9-point scale (9 = excellent, 7 = good, 5 = fair, 3 = poor and 1 = unusable). A score of 6 was considered as the limit of marketability. Softening score of individual fruit in a box was carried out on a 0 - 4 scale, where 0 = normal (0% surface softened), 1 = trace (up to 10% surface softened), 2 = slight (10 to 25% surface softened), 3 = moderate (25% - 50% surface softened), and 4 = severe (>50% surface softened). Softening index was then calculated from the following formula

The experiment was conducted with four replications per treatment. Statistical analyses of the data were carried out using SAS software (SAS Institute, Cary, NC, USA). The level of significance was calculated from the F value of ANOVA. Mean comparison was achieved by Duncan’s multiple range test. Prior to the final experiment, two preliminary experiments were conducted with limited replications that resulted similar trend.

After treated with CO₂ gas, strawberry fruit firmness increased in all longer duration treatments and both of 50% CO₂ treatment (Figure 1). Sharp increases in firmness were observed in 50% CO₂ treated strawberry, whereas slight increase were found in strawberry samples treated with 15% and 30% CO₂ for 3 h. At the beginning of storage, fruit firmness increased in all samples except 50% CO₂ treatment. Firmness of 15% CO₂-3 h increased gradually and both of 30% CO₂ treated samples increased until 6 days of storage and then declined slightly or just maintained. Although increases in fruit firmness were reported in few studies by the application of CO₂ [14] [16] , our results revealed that time of treatment and concentration greatly affect the firmness of studied straw- berry variety. Matsumoto et al. [14] also reported that the magnitudes of the increases in fruit firmness were different among cultivars and principally due to a shift toward significantly greater abundance of chelator-asso- ciated pectins at the expense of water- and alkali-soluble pectins. Furthermore, significant increase in chelator- soluble polymers and a concomitant decrease in water-soluble polymers were reported in skin and flesh tissues of strawberries exposed to high CO₂ environment [8] [16] . The increasing pattern of firmness at the end of storage could be a consequence of water loss (data not shown) from the surface of fruit that made it farmer. However, we recorded significant decline in other parameters like sensory qualities (discussed later) on 13 days and hence, firmness measured instrumentally was less evident on this day.

Total soluble solid of control sample declined throughout the storage whereas it slightly increased or maintained in CO₂ treated samples (Figure 2). However, all samples treated with CO₂ for longer duration showed a sudden decline on 3 days of storage and the TSS were recovered thereafter. Overall, CO₂ treated samples exhibited higher TSS compared to untreated control sample. Among the CO₂ treated samples, TSS of 15% CO₂-3 h treat- ment increased insignificantly until the end of storage while TSS of 30% CO₂-1 h treatment declined gradually though higher values were recorded at the beginning of storage. Although Pelayo-Zaldivar et al. [17] found re- duced TSS in strawberry stored in air or air + 20 kPa CO₂ environment for 3 and 6 days, Blanch et al. [18] re- ported the highest accumulation of total sugar was occurred when strawberries were exposed to a continuous gas mixture containing 20% CO₂ + 20% O₂ + 60% N₂. Gil et al. [1] found slight decline in TSS after 5 days in strawberries stored in air and after 10 days in different concentration of CO₂ enriched environment. Unlike TSS, pH was lightly affected by CO₂ treatment and we found little decline in pH after CO₂ treatment (Figure 2).

However, sample treated with 30% and 50% CO₂ for 3 h showed significant decline (p < 0.05) in pH compared to control sample on treatment day. Overall, pH of CO₂ treated samples did not show significant variation (p > 0.05) either among them or in between storage days which is in consistent with the results of Gil et al. [1] on strawberries stored in different concentration of CO₂ enriched environments. Similarly, Pelayo-Zaldivar et al. [17] observed no specific trend in pH of strawberry during storage at elevated CO₂.

Titratable acidity, on the other hand, slightly increased by CO₂ treatment and 30% CO₂-3 h and both of 50% CO₂ treatments showed greater increase (p < 0.05) in TA compared to control or other treatments (Figure 2). However, TA values of shorter duration CO₂ treated samples declined slightly or maintained until the end of storage whereas these values were slightly increased at the end of storage in samples treated with CO₂ for longer duration. Unlikely, Gil et al. [1] found insignificant decline in TA in “Selva” strawberries stored at 40% CO₂. It was reported that the changing pattern of TSS, pH and TA are variable depending on the many factors such as cultivars, growing seasons, cultivation methods and nutrient supply as well as postharvest handling including storage condition [1] [17] - [19] . “Goha” strawberry is a new cultivar in Korea and usually grown in summer season which might have unique physio-biochemical characteristics and probably thereby response in a differently way to that of other cultivars in respect to CO₂ treatments reported by various researchers. The initial TA of this strawberry was 0.87% whereas TA of 15%, 30% and 50% CO₂ for 3 h treated samples were 0.93, 1.13 and 1.10%, respectively. In an earlier study, Shin et al. [19] reported 0.79% TA of “Goha” strawberry which was nearly similar to our result. The slight decline in TA of during storage could be due to the further ripening of the fruit and in consistent with those reported by Han et al. [20] and Shin et al. [19] .

Since color is the most important factor affecting the overall quality and appearance of strawberry, we measured surface color using Hunter color spaces. It revealed that color parameters were less affected by CO₂ application (Figure 3). L* value, which indicates the lightness, gradually declined (p < 0.05) in control sample when storage time elapsed and similar trend was found in 15% CO₂-1 h treated sample but a sudden increase was occurred at the end of the storage . However, L* values were almost maintained in other CO₂ treated samples although insignificant inclining or declining fluctuations were observed. Similarly, Zheng et al. [21] also found no significant variation of L* value during storage of strawberry. No remarkable change was observed in a* and chroma values among the treatments and insignificant increasing, decreasing or maintaining patterns were recorded (Figure 3). Sample treated with 15% CO₂-3 h condition showed nearly constant a* and chroma values throughout the storage. Increases in redness (higher a* value) is a general pattern observed during storage of mature strawberry owed to the ripening process which fall after certain period [22] . Since we used about 3/4 portions matured (red color) strawberry, these results may imply that color of strawberry was maintained in the selected storage condition. Our results also suggest that postharvest application of CO₂ did not affect the color of strawberry. In agreement with our result, Shin et al. [19] found no variation in Hunter L*, a* and b* values in ‘Goha’ strawberry during storage. It was also reported that skin color of strawberry was not markedly affected by treatments with different concentration of CO₂ [1] .

Using a nine-point scoring method, overall quality, appearance and visual texture were evaluated on each evaluation day. Sensory scores of these three parameters decreased gradually with the increases in storage time in all treatments (Table 1). Although samples of different treatments received marketable scores until 6 days of storage, interestingly only sample treated with 15% CO₂ for 3 h yielded slightly higher value to the marketable limit set at 6 in a 9-point scale on 9 days of storage. Among other treatments, 15% CO₂-3 h showed the highest visual and appearance quality on 6 days of storage which were not maintained in later stages. Sample treated with 15% CO₂-3 h also showed comparatively higher values of appearance and visual texture until 9 days of storage. Consequently, we recorded an insignificant (p > 0.05) lower value of softening index in this sample among the treatments on 9 days of storage (Table 1). This result suggests that there is a correlation between softening index and overall quality of strawberry during storage. In many cases, softening is the first visual indication of the deterioration of strawberry quality rather than decay or calyx discoloration or changes in surface color. However, there was no difference among the treatments in either of the sensory parameters on 13 days of storage regardless of treatment condition. It seems that shelf-life of “Goha” strawberry ends near about 9 days. Although we observed greater increase in firmness (measured instrumentally) of 50% CO₂ treated strawberry throughout the storage, sensory quality of those samples were not consistent with firmness values. This result indicates that CO₂ not only affect the firmness properties of strawberry, but also affect other biochemical properties that substantially influence sensory qualities. Suppakul et al. [23] reported that high levels of carbon dioxide can induce changes in taste of strawberry and develop undesirable anaerobic glycosis in fruit. In consistence with our sensory results, Shin et al. [19] also found 9 days marketable limit of “Goha” strawberry with different treatments over its untreated control.