Changes in Volatile Compounds in Short-Term High CO2-Treated ‘Seolhyang’ Strawberry (Fragaria × ananassa) Fruit during Cold Storage

‘Seolhyang’ strawberry is harvested before it is fully ripened and treated with CO2 to extend the shelf-life. However, the volatile changes in the ‘Seolhyang’ strawberry after short-term CO2 treatment have not been investigated, although the volatile profile is an important quality attribute. Herein, we investigated the effect of short-term high CO2 treatment on the changes in the composition of volatile compounds in ‘Seolhyang’ strawberries at two ripening stages (i.e., half-red and bright-red) during cold storage using headspace solid-phase microextraction and gas chromatography-mass spectrometry. Furthermore, the effect of CO2 treatment on fruit quality with respect to the aroma was investigated. A total of 30 volatile compounds were identified. Storage increased the volatile compound concentrations, and the total concentration of volatiles in the CO2-treated strawberries was lower than that of the untreated strawberries during storage. The production of some characteristic strawberry volatiles (e.g., 4-methoxy-2,5-dimethyl-3(2H)-furanone) was inhibited in CO2-treated strawberries. However, CO2 treatment helped maintain the concentrations of hexanal and 2-hexenal, which are responsible for the fresh odor in strawberries. Interestingly, CO2 treatment suppressed the production of off-odor volatiles, acetaldehyde, and hexanoic acid during strawberry storage. Thus, short-term CO2 treatment may help maintain the fresh aroma of strawberries during cold storage.


Introduction
'Seolhyang' (Fragaria × ananassa Duch) strawberry was developed in 2005 by crossbreeding the 'Akihime' and 'Red Pearl' cultivars in Korea [1]. This cultivar, which has a sweet taste and a characteristic sweet aroma, accounted for 87.5% of the Korean strawberry production in 2017 [2]. The strawberry cultivar is exported from Korea to various Asian countries. In 2020, approximately 1602 and 1208 tons of strawberries were exported from Korea to Hong Kong and Singapore, respectively [3]. However, the 'Seolhyang' cultivar, owing to its soft texture, has a short shelf-life compared with other traditional strawberry cultivars, such as 'Maehyang' and 'Keumhyang' [4]. Thus, harvesting unripened strawberries and postharvest treatments may prolong the shelf-life of 'Seolhyang' strawberry to enable shipping over long distances [4].
. Figure 1. A representative GC chromatogram of CO2-treated bright-red strawberries after 9 d storage at 10 °C. Volatile compound codes are provided in Table 1.
At day 0, some volatiles occurred or were present in higher concentrations only the bright-red strawberries. For example, (2E)-hexenyl acetate was not present in half-r strawberries but was present in bright-red strawberries at day 0. (2E)-Hexenyl acetate w also reported in seven Fragaria × ananassa cultivars by Dong et al. [20]; however, it was n present in the 'Camarosa' and 'Chandler' strawberries harvested in India [22]. Thus, t volatile composition of strawberries may be affected by the cultivar type, maturity, and agricultural practices, such as the growing region. [23].
During cold storage, (2E)-hexenyl acetate and ethyl octanoate were newly produc in half-red and bright-red strawberries, respectively. The volatile composition of stra berries during cold storage was affected by the ripening stage when harvested. For exa ple, ethyl octanoate was not found in fresh strawberries (day 0) during the two ripeni stages; however, it was produced only in bright-red strawberries after cold storage, r gardless of CO2 treatment. Previous studies found that ethyl octanoate was not present strawberries of the 'Cigaline', 'Chandler', 'Albion', and 'Festival' cultivars [24][25][26]. Wa et al. reported that ethyl octanoate is formed after 5 d of storage in 'Hong Yan' strawb ries [27]. The concentrations of some volatiles decreased or were absent after CO2 tre ment. For example, the acetaldehyde content decreased, and 2-butanone was absent bright-red strawberries after CO2 treatment.  Table 1.
At day 0, some volatiles occurred or were present in higher concentrations only in the bright-red strawberries. For example, (2E)-hexenyl acetate was not present in half-red strawberries but was present in bright-red strawberries at day 0. (2E)-Hexenyl acetate was also reported in seven Fragaria × ananassa cultivars by Dong et al. [20]; however, it was not present in the 'Camarosa' and 'Chandler' strawberries harvested in India [22]. Thus, the volatile composition of strawberries may be affected by the cultivar type, maturity, and/or agricultural practices, such as the growing region. [23].
During cold storage, (2E)-hexenyl acetate and ethyl octanoate were newly produced in half-red and bright-red strawberries, respectively. The volatile composition of strawberries during cold storage was affected by the ripening stage when harvested. For example, ethyl octanoate was not found in fresh strawberries (day 0) during the two ripening stages; however, it was produced only in bright-red strawberries after cold storage, regardless of CO 2 treatment. Previous studies found that ethyl octanoate was not present in strawberries of the 'Cigaline', 'Chandler', 'Albion', and 'Festival' cultivars [24][25][26]. Wang et al. reported that ethyl octanoate is formed after 5 d of storage in 'Hong Yan' strawberries [27]. The concentrations of some volatiles decreased or were absent after CO 2 treatment. For example, the acetaldehyde content decreased, and 2-butanone was absent in bright-red strawberries after CO 2 treatment.  Figure 2 shows the sum of the volatile compounds of the strawberries. Before storage (0 d), the sum of the volatiles in bright-red strawberries (B0h, 3787 µg/kg) was 1.24-fold higher than that of the half-red strawberries (H0h, 3044 µg/kg). These findings agree with those of previous studies, showing that the sum of the volatiles in 'Cigaline' strawberries increased during maturity [25]. After 9 d of storage, the volatile concentrations of strawberries used as control increased to a greater extent (B9D, 8186 µg/kg; H9D, 10,198 µg/kg) than those of CO 2 -treated strawberries (BT9D, 5130 µg/kg; HT9D, 5480 µg/kg). Thus, short-term CO 2 treatment of strawberries at two ripening stages may inhibit the production of volatiles during 9 d of storage. The MANOVA results showed that CO 2 treatment and storage time affected the concentrations of the total volatiles (i.e., sum of volatiles) (p < 0.001), as shown in Supplementary Materials, Table S1.  Figure 2 shows the sum of the volatile compounds of the strawberries. Before storage (0 d), the sum of the volatiles in bright-red strawberries (B0h, 3,787 µ g/kg) was 1.24-fold higher than that of the half-red strawberries (H0h, 3,044 µ g/kg). These findings agree with those of previous studies, showing that the sum of the volatiles in 'Cigaline' strawberries increased during maturity [25]. After 9 d of storage, the volatile concentrations of strawberries used as control increased to a greater extent (B9D, 8,186 µ g/kg; H9D, 10,198 µ g/kg) than those of CO2-treated strawberries (BT9D, 5,130 µ g/kg; HT9D, 5,480 µ g/kg). Thus, short-term CO2 treatment of strawberries at two ripening stages may inhibit the production of volatiles during 9 d of storage. The MANOVA results showed that CO2 treatment and storage time affected the concentrations of the total volatiles (i.e., sum of volatiles) (p < 0.001), as shown in Table S1.

Changes in the Composition of Volatile Compounds in Strawberries
The volatile composition of strawberries varied with maturity. For example, brightred strawberries showed a higher ester/aldehyde ratio compared to that of half-red strawberries at day 0. After 9 d of storage, the aldehyde contents of half-red strawberries (H9D) and bright-red strawberries (B9D) decreased to 78% and 71%, respectively, compared to that at day 0. However, the ester contents of H9D and B9D increased 3.0-fold and 1.8-fold, respectively, compared to that at day 0. The ester content of CO2-treated half-red strawberries (HT9D) was 68.5% that of the total volatiles. This value was similar to that of the half-red  The volatile composition of strawberries varied with maturity. For example, bright-red strawberries showed a higher ester/aldehyde ratio compared to that of half-red strawberries at day 0. After 9 d of storage, the aldehyde contents of half-red strawberries (H9D) and bright-red strawberries (B9D) decreased to 78% and 71%, respectively, compared to that at day 0. However, the ester contents of H9D and B9D increased 3.0-fold and 1.8-fold, respectively, compared to that at day 0. The ester content of CO 2 -treated half-red strawberries (HT9D) was 68.5% that of the total volatiles. This value was similar to that of the half-red. The relative concentrations of individual volatile compounds in the control and CO 2treated strawberries at two ripening stages-(A) half-red and (B) bright-red-during storage are shown in Tables 2 and 3. The relative concentrations of characteristic volatile compounds of the strawberries are shown in Figure 3. The concentration of each volatile compound was described as the relative concentration of the appropriate internal standard. In half-red strawberries, the hexanal concentration significantly decreased from 217 µg/kg (H0h) to 41 µg/kg (HT9D) and 56 µg/kg (H9D) after 9 d in storage (p < 0.05). Furthermore, the 2-hexenal concentrations significantly decreased from 975 µg/kg (H0h) to 413 µg/kg (H9D) and 333 µg/kg (HT9D). No significant difference was observed in the hexanal and 2-hexenal concentrations between H9D and HT9D (p > 0.05). The hexanal concentrations of stored bright-red strawberries (B9D, 25 µg/kg; BT9D, 48 µg/kg) decreased compared to those in fresh bright-red strawberries (B0h, 160 µg/kg). Furthermore, the 2-hexenal concentrations of stored bright-red strawberries (B9D, 164 µg/kg; BT9D, 308 µg/kg) decreased compared to those of fresh half-red strawberries (B0h, 871 µg/kg). The 2-hexenal concentration of BT9D was significantly higher than that of B9D (p < 0.05). A previous study reported that the concentrations of hexanal and 2-hexenal, the characteristic volatile compounds responsible for the fresh and fruity odor of fresh strawberries, decreased during storage [28]. Hexanal may help delay the ripening process through the inactivation of phospholipase D, membrane lipid-degrading enzymes, and downstream oxidative signaling, and enhance shelf-life, as shown in mango fruits [29]. Moreover, hexanal may help reduce postharvest deterioration by preventing the growth of microorganism and fungi [30]. Interestingly, postharvest hexanal treatment on 'Rubygem' strawberries increased the alcohol percentage of the fruit at the end of their shelf-life [15]. However, in this study, short-term CO 2 treatment on half-red strawberries did not increase the alcohol content at the end of the storage period. In this study, short-term CO 2 treatment did not affect the hexanal and 2-hexenal concentrations in half-red strawberries but maintained their concentrations in bright-red strawberries. Thus, CO 2 treatment on bright-red strawberries may help maintain fruit quality during storage.
Characteristic strawberry volatile compounds include esters, such as ethyl butanoate, ethyl hexanoate, methyl butanoate, and methyl hexanoate [5]. Among them, methyl hexanoate showed a higher concentration than that of other esters. It has been previously suggested as a maturity marker for other strawberry cultivars (e.g., 'Albion', 'Malling Pearl', 'Florin', 'Charlotte', 'Anabelle', 'Florin 2', 'Montery', 'San Andreas', and 'Portola') [31]. The methyl hexanoate concentration of B0h (330 µg/kg) was 5.9-folds higher than that of H0h (56 µg/kg) before storage (day 0). The methyl hexanoate concentration of control half-red control strawberries increased and then decreased during storage. Ayala-Zavala et al. also showed that the methyl hexanoate concentration of commercially matured 'Chandler' strawberries increased 1.8-fold when stored at 5 • C for 5 d, but it decreased to the concentrations lower than the original concentration when harvested [32]. However, the methyl hexanoate concentration continued to increase at a slower rate in CO 2 -treated half-red (HT) strawberries but not in the in control half-red strawberries. The methyl hexanoate concentrations decreased during early storage (≈2 d) and then increased both in the control and CO 2 -treated bright-red strawberries. The concentrations decreased to a greater extent in CO 2 -treated bright-red strawberries until 2 d of storage and increased at a slower rate in CO 2 -treated bright-red strawberries compared to the control bright-red strawberries stored under the same conditions. Thus, CO 2 treatment may induce a decline in the methyl hexanoate concentration during the early storage period and then inhibit methyl hexanoate formation during storage, regardless of the ripening stage. Interestingly, the production of methyl butanoate, ethyl butanoate, and ethyl hexanoate was inhibited in CO 2 -treated half-red strawberries during storage; however, the effect was not observed or was less frequently observed in bright-red strawberries. The concentration of esters in CO 2 -treated bright-red strawberries was 81-98% that of the control at 9 d of storage.
Acetaldehyde is responsible for the pungent aroma, which is the off-odor in strawberries [38]. The acetaldehyde concentration of half-red strawberries (H0h, 899 µg/kg) was similar to that of bright-red strawberries (B0h, 881 µg/kg). The acetaldehyde concentration of half-red strawberries (H0h, 899 µg/kg) increased after 9 d of storage (H9D, 986 µg/kg). In the case of bright-red strawberries, the acetaldehyde concentration (B0h, 881 µg/kg) increased after 9 d of storage (B9D, 988 µg/kg). The acetaldehyde concentration of H9D (986 µg/kg) was 2.0-fold higher than that of HT9D (505 µg/kg). The acetaldehyde concentration of B9D (988 µg/kg) was 2.2-fold higher than that of BT9D (440 µg/kg). Thus, CO 2 treatment may inhibit acetaldehyde formation and help maintain the aroma quality of strawberries during storage, regardless of maturity. A previous study reported that the acetaldehyde content was lower in strawberries (Fragaria vesca L.) stored in a controlled atmosphere with a higher CO 2 concentration compared to that in the untreated control [38]. High CO 2 treatment inhibited pyruvate decarboxylase activity that decarboxylates pyruvate to yield acetaldehyde during storage [39].
2,3-Butanedione is responsible for the pungent off-odor of strawberries [40]. The 2,3butanedione concentration of bright-red strawberries (B0h, 92 µg/kg) was 2.9-fold higher than that of half-red strawberries (H0h, 32 µg/kg). The 2,3-butanedione concentration was lower in CO 2 -treated strawberries during storage compared to untreated strawberries at the same storage time, regardless of the ripening stage. Thus, CO 2 treatment may improve the aroma quality of strawberries during storage regardless of the maturity based on lower concentrations of 2,3-butanedione compared to the control during storage.
Hexanoic acid is also related to a rancid off-odor in strawberries [40]. The concentration of hexanoic acid of half-red strawberries increased from 26 µg/kg (H0h) to 370 µg/kg (H9D) and 162 µg/kg (HT9D). In bright-red strawberries, the concentration of hexanoic acid increased from 87 µg/kg (B0h) to 664 µg/kg (B9D) and 502 µg/kg (BT9D). Thus, hexanoic acid concentrations increased during storage regardless of the ripening stage; however, CO 2 treatment may inhibit hexanoic acid production during storage. Similar to our findings, the hexanoic acid concentrations of (untreated) 'Sweet Charlie' strawberries increased during 9 d storage at 4 • C [10]. However, the hexanoic acid concentrations of 'Pajaro' strawberries decreased during 12 d storage in 20% CO 2 at 0 • C [41]. In addition to hexanoic acid, various fatty acids, including linoleic acid and linolenic acid, were identified in nine strawberry cultivars in a previous study [42]. Some ester compounds, such as methyl butanoate, ethyl butanoate, and methyl hexanoate, are formed by the β-oxidation of fatty acids in strawberries [18]. In this study, the ester compound production may have been inhibited during storage by CO 2 treatment possibly owing to inhibited fatty acid oxidation during storage.

Principal Component Analysis (PCA) and the Heatmap of Volatiles
A PCA was performed using the volatile compounds data from all samples ( Figure 4A and B). The first two principal components (PC1 and PC2) accounted for 61.03% of the total variation. PC1 and PC2 contributed to 45.47% and 15.56% of the total variation, respectively. The control strawberries in mostly late storage periods (H9D, B6D, and B9D) were separately grouped from other samples. As storage progressed, the strawberries were spread on the positive side of PC1. They were positively correlated with characteristic strawberry volatiles, such as 4-methoxy-2,5-dimethyl-3(2H)-furanone, methyl butanoate, ethyl butanoate, methyl hexanoate, and ethyl hexanoate. Acetaldehyde and 2,3-butanedione, and hexanoic acid, that are responsible for the off-odor of strawberries, were spread on the positive side of PC1. These volatiles correlated with H9D, B6D, and B9D. Hexanal and 2-hexenal, that are responsible for the fruity odor of strawberries, were spread on the negative side of PC1. These volatiles correlated with the half-red strawberries during the early storage period (e. g., H0h, H6h, H1D, HT6h, and HT1D). Additionally, CO 2 -treated strawberries were spread on the negative side of PC2. Especially, CO 2 -treated bright-red strawberries were spread toward the negative side of PC2 compared to CO 2 -treated half-red strawberries. (2E)-2-hexenyl acetate and 3,7-dimethyl-1,6-octadien-3-ol concentrations positively correlated with CO 2 -treated strawberry groups. Thus, these may be used as potential markers of short-term CO 2 treatment in strawberries. Previously, 3,7-dimethyl-1,6-octadien-3-ol was reported as a key component of the muscat table grape [43]. The concentration of 3,7-dimethyl-1,6-octadien-3-ol was higher in CO 2 -treated strawberry groups compared to the control groups, regardless of the ripening stage. Ten volatiles (4-methoxy-2,5-dimethyl-3(2H)-furanone, hexanoic acid, methyl octanoate, methyl butanoate, 2-heptanone, ethyl butanoate, butyric acid, hexyl acetate, ethyl octanoate, and hexyl butanoate) were selected from the loading plot based on their high variables importance in projection (VIP) values over 1, as they contributed to the discrimination of the strawberries samples.  , and the "non-specific filtering" option in the XLSTAT program was not used. Missing data were estimated using the nearestneighbor algorithm. Hierarchical clustering was conducted based on Euclidian distances.

Strawberry Samples
'Seolhyang' strawberries were harvested at two different ripening stages from a strawberry farm located in Jinju, Gyeongsangnam-do, Korea (35.1800 • N, 128.1076 • E) in March 2019. The 'Seolhyang' strawberries were grown in a commercial greenhouse using conventional soil cultivation practices. The samples were transported to the Korea Food Research Institute (KFRI; Wanju, Korea), which was located at a distance of 175 km from the farm, using a refrigerated truck. Undamaged strawberries of uniform size and shape were selected for the study. The ripening stages were grouped into half-red (80% maturity) or bright-red (100% maturity) based on surface color development. Half of the selected fruits under each ripening group were treated with CO 2 by exposing the samples to air saturated with 30% CO 2 and 70% N 2 for 3 h at 25 • C. Thus, the samples were divided into four subgroups (i.e., half-red control, half-red CO 2 -treated, bright-red control, and bright-red CO 2 -treated). Approximately 15-20 fruits from each subgroup were placed in a plastic bin and covered with a lid. The two bins were removed periodically at 0, 6, 24, and 48 h, and 3, 6, and 9 d to sample the strawberries stored at 10 • C. To compare the changes in the volatile profiles of strawberries following CO 2 treatment, the volatile composition of strawberries after 6 h was investigated. Thus, there were 8 (time point) × 2 (half-red and bright-red) × 2 (control and CO 2 treated) × 2 (duplicate) = 64 samples for strawberries stored at 10 • C. Each of the samples at the time points were immediately frozen in liquid nitrogen and stored at −80 • C until analysis.

Determination of Strawberry Volatiles using HS-SPME-GC/MS
The sample preparation method was adopted from our previous study with modifications [44]. Frozen strawberries (300 g) were ground using a blender (BL682 Auto-IQ, Nutri Ninja, Boston, MA, USA) for 1 min. Then, 5 g of the ground frozen strawberries was transferred to a glass headspace vial (Gerstel, Baltimore, MD, USA), and 5 mL of saturated sodium chloride solution was added. An internal standard mixture (octanal-d16, ethyl butanoate-d3, and n-hexyl-d13 alcohol) was added to the glass headspace vial with a final concentration of 10 µg/kg and was sealed immediately using a magnetic crimp cap (Gerstel).
After 10 min of equilibration, a 2 cm solid phase microextraction (SPME) fiber coated with divinylbenzene/carboxen/ polydimethylsiloxane (DVB/CAR/PDMS) was used to extract the volatiles. The volatiles were absorbed for 30 min at 40 • C and desorbed for 30 s at 250 • C. Volatiles in strawberry were determined using gas chromatography (7890B, Agilent technologies, Santa Clara, CA, USA) coupled with a mass selective detector (5977B, Agilent technologies) and separated on a DB-WAX capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness; Agilent technologies). The oven temperature was set to 40 • C for 2 min; the temperature was increased by 4 • C/min to 120 • C and held at 120 • C for 5 min. Then, the temperature was increased to 250 • C at a rate of 20 • C/min and held at 250 • C for 5 min. The solvent delay was set to 3 min. Helium (99.999%) was used as a carrier gas at a flow rate of 1 mL/min. The inlet temperature was 250 • C, and an inlet liner (0.75 mm i.d., Agilent technologies) was used for the splitless mode. The temperatures of the transfer line, ion source, and quadrupole were 240 • C, 230 • C, and 150 • C, respectively. The electron energy was 70 eV. Mass spectra were obtained in the range of m/z 30-350 at a rate of 3.06 scans/s.

Identification and Relative Quantification of Volatiles in Strawberries
Volatiles were identified by comparing their mass spectra and the retention time of authentic standards if available or tentatively by searching libraries (Willey and NIST 08 mass spectral libraries) for compounds with no authentic standard available. Eighty percent of the match score was used as a cut-off, and Kovats' retention index was used to confirm the identification of volatiles. Kovats' retention index was calculated using the retention time of each volatile compound and an authentic alkane standard mixture.
Relative volatile concentrations were calculated according to formula (1) presented below [45]. Briefly, the extracted ion peak area of each volatile was divided by the extracted ion peak area of the respective internal standard (octanal-d16 for aldehydes, ketones, and furanones; n-hexyl-d13 alcohol for alcohols and acids; ethyl butanoate-d3 for esters). The relative concentration of each volatile was converted by multiplying the area ratio with 10 µg internal standard/kg strawberry. The results were expressed on a fresh weight basis.
Concentration µg kg = extracted ion peak area extracted ion peak area o f internal standard internal standard 10 µg kg (1)

Statistical Analyses
Statistical analyses were conducted using SPSS statistics 23 software (SPSS, Inc., Chicago, IL, USA). Significant differences in the volatile composition among the strawberry fruits were confirmed by an analysis of variance at p < 0.05. Duncan's test was used as a post hoc analysis. A multivariate analysis of variance (MANOVA) was conducted to evaluate the effects of maturity, storage time, CO 2 treatment, and their interactions on the volatile composition of strawberries. PCA, heatmap, and hierarchical cluster analyses were performed using XLSTAT (XLSTAT, ver. 2017.03, Microsoft Excel Add-in software, New York, NY, USA). The data format for PCA was an observation/variable table, and Pearson's correlation type was used as the PCA type. Based on the volatile profile of strawberries, heatmap and hierarchical cluster analyses were conducted to demonstrate similarities and differences among the strawberries depending on maturity, storage period, and short-term CO 2 treatment. The total number of features was the same as the number of determined volatiles in the strawberries (n = 30), and the "non-specific filtering" option in the XLSTAT program was not used.

Conclusions
To the best of our knowledge, this is the first study investigating the effect of short-term CO 2 treatment on the volatile composition of 'Seolhyang' strawberries. A total of thirty volatiles were identified using HS-SPME with GC-MS in 'Seolhyang' strawberries stored at 10 • C for 9 d. The volatile composition of the strawberries changed depending on the ripening stages, storage time, and short-term CO 2 treatment. Ripening and storage increased the total volatile concentrations and CO 2 treatment inhibited the production of volatiles. The production of some characteristic strawberry volatiles, such as methyl hexanoate and 4-methoxy-2,5-dimethyl-3(2H)-furanone, was inhibited in CO 2 -treated strawberries, especially in half-red strawberries, during cold storage. However, CO 2 treatment helped maintain the hexanal and 2-hexenal concentrations, which are responsible for the fresh odor in strawberries. Interestingly, CO 2 treatment decreased the formation of rancid offodor volatiles, such as acetaldehyde, 2,3-butanedione, and hexanoic acid during storage, regardless of the ripening state. Thus, short-term CO 2 treatment may help maintain a fresh odor and reduce the off-odor, improving strawberry aroma characteristics during storage. Short-term CO 2 treatment may be applied to 'Seolhyang' strawberries for long-distance shipping to improve the strawberry aroma characteristics during shipping. In future research, the role of short-term CO 2 treatment in improving the aroma quality of 'Seolhyang' strawberries can be confirmed using descriptive sensory analysis.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules27196599/s1, Table S1: Multivariate analysis of variance F-values and p-values for the volatile contents of all strawberries.