These raspberry drupelets were held in a –80℃ freezer until use. Raspberry volatiles were analyzed using a method modified from Forney et al. . Frozen raspberry drupelets were removed from the -80℃ freezer and 5 g were added to 100 g NaCl saturated H2O, and homogenized in a blender for 1 min. Five mL of the homogenate was transferred to a 20 mL headspace vial . Headspace volatiles were then analyzed by solid phase micro extraction gas chromatography mass spectroscopy . Vials were incubated at 50℃ for 10 min, and then the headspace was exposed to a gray SPME fiber for 10 min with agitation. The fiber was desorbed at250℃ for 15 min onto a BD-WAX UI column held at 35℃ for 5 min, then ramped to 240℃ at 0.167℃/s and held at 240℃ for 4.5 minutes. Helium was the carrier gas at a flow of 16.7 ml/s. Peaks were initially identified through comparison with NIST Mass spectral library . Retention indices of these compounds were used to further verify identity by comparison against standard compounds and relatively quantified with GC using a BD-FATWAX UI column. The same method was followed except the sample amount was 1 mL and nitrogen was used as the carrier gas. The samples’ peak area was multiplied by the reference standard concentration, drainage collection pot and the result was divided by the peak area of the reference standard.A descriptive sensory analysis was performed with 12 panelists who were trained ahead of the sensory evaluations to align their sensory perception. There were four one-hour training sessions over two weeks. During the training, panelists were provided with references for each attribute to compare against the training samples.

The sensory evaluations took place in the UC Davis Department of Plant Sciences Sensory Lab, equipped with five separate evaluation booths with individual computers with sensory analysis software . Samples were prepared the morning of evaluation and stored at 5℃. The samples were brought to room temperature before being tasted by the panel. One sample included 3-4 raspberries and was provided to the panelists in sealed sensory tasting cups. Each sample was blinded with random 3-digit codes generated by the software .Panelists tasted three replications of raspberries at harvest , and again for each treatment after 5, 10, and 13-days in atmosphere storage and evaluated their taste, texture, and flavor. The panelists were instructed to cleanse their palates with crackers and water in between samples. On day 10, there was only one replication of the air treatment and two replications each of 15 kPa and 5 kPa atmospheres appropriate for sensory evaluation due to decay growth. On day 13, there were no samples of the air treatment, 5 kPa treatment had two replications, 8 kPa treatment had thee replications and 15 kPa treatment had two replications. The panelists measured the intensity of sensory attributes of the raspberry samples and marked their score for each given attribute on a 10 cm straight line anchored with less and more using sensory evaluation software . This software transmuted the markings for each attribute into a numerical value ranging from 1 to 10 units, where 1 was less and 10 was more intensity. The tasted attributes were sweetness, acidity/tartness, firmness , juiciness, raspberry flavor, and off-flavor. The tasting lexicons were decided and agreed upon during the training.Data were analyzed using R statistical program . A total of 4 treatments and 3 replications across the four evaluation dates were analyzed for instrumental and sensory qualities of the raspberries. Data were assessed through ANOVA followed by Fishers Least Significance Difference test to reveal significant differences among treatments and evaluation times.

A correlation analysis was also conducted to investigate the relationship between volatile compounds and sensory attributes. The sensory data was analyzed using principal component analysis using R and R Studio software andPCA plots are presented for 5- and 10-day evaluations. The sensory data on day 13 was insufficient for analysis due to decay.Storing raspberries under high CO2 atmospheres reduced fruit softening in a concentration dependent manner . Raspberries stored in 15 kPa atmosphere did not soften until day 13, and only slightly. Raspberries stored in 8 and 5 kPa CO2 had intermediate firmness throughout storage, and softened gradually, while raspberries stored in 0.03 kPa atmosphere lost firmness quickly during storage and had the lowest firmness among all the atmosphere treatments at each evaluation. Across all evaluation’s times, raspberries stored in 15 kPa atmosphere were most firm, raspberries stored in air were least firm, and raspberries stored in 5 or 8 kPa atmosphere were intermediate and not different from each other .A total of 14 volatile compounds were detected in the raspberry fruit . There were five terpenes , three alcohols , two aldehydes and one each of ester , ketone , alkyne and carboxylic acid . The aromatic volatiles limonene, linalool, hexanoic acid and α-terpineol increased in concentration over time, particularly in 0.03 kPa atmosphere stored raspberries . However, storage in elevated CO2 atmospheres resulted in significantly lower concentrations of these volatiles as well as α-ionone . Raspberries held in 15 kPa atmosphere exhibited a sharp and significant increase in the fermentative volatiles, acetaldehyde and ethanol, on day 5 and had higher concentrations than raspberries stored in other atmosphere treatments . Acetaldehyde, ethyl acetate, and ethanol all increased in concentration overtime, particularly on the last day of storage .

Raspberry fruit mouthfeel firmness, raspberry flavor and TSS decreased over time and juiciness and off-flavor increased over time . Firmness scores, both hand and mouthfeel, were significantly higher in raspberries stored in 15 kPa atmosphere followed by fruit held in 8 kPa atmosphere. The trend was opposite for juiciness and sweetness, where raspberries held in 0.03 kPa or 5 kPa atmosphere had the highest juiciness scores, and fruit held in 15 kPa atmosphere had a lower sweetness score than fruit held in air atmosphere. Tartness score was higher in fruit held in 8 kPa or 15 kPa atmosphere than fruit held in 0.03 kPa atmosphere. Raspberry flavor was significantly higher in fruit held in 8 kPa atmosphere than in 5 kPa atmosphere . After five days of storage, the PCA biplot showed that raspberry firmness was strongly associated with the 15 kPa atmosphere treatment, and less so with the 8 kPa atmosphere treatment . Fermentative volatiles: acetaldehyde, ethyl acetate, and ethanol were also clustered with the 15 kPa atmosphere treatment, and less so with the 8 kPa atmosphere treatment. Raspberry flavor was most closely associated with sweetness andtetradecane at the bottom of the biplot. No treatments were closely associated. Tartness, TA, TSS, heptanol, α-terpineol and limonene were associated with each other and the 5 kPa atmosphere treatment at the top of the bi-plot. Sweetness, off-flavor, and juiciness were clustered with each other and the 0.03 kPa atmosphere treatment. Most of the aromatic volatiles were associated closely with the 0.03 kPa and 5 kPa atmosphere treatments, and on the opposite side of the bi-plot from firmness and 8 kPa and 15 kPa atmosphere treatments. After ten days, firmness remained associated with treatments with high CO2 concentrations . Raspberry flavor, TSS, TA, and tartness were associated with each other and the 8 kPa atmosphere treatment, and raspberry flavor shifted to the top of the bi-plot. Juiciness, sweetness, tetradecane and heptanone were clustered on the top left with the 5 kPa atmosphere treatment. At the bottom of the bi-plot, ethanol and ethyl acetate were associated with the 15 kPa and 0.03 kPa atmosphere treatments, respectively, and acetaldehyde was in between 15 kPa and 0.03 kPa atmosphere. Aromatic volatiles maintained their association with lower CO2 atmosphere treatments, and were also associated with off-flavor as at ten days .Across evaluation days, total soluble solids and juiciness were positively correlated . Sweetness was negatively correlated with hand firmness and tartness, and mouthfeel firmness and juiciness were negatively correlated. Acetaldehyde and ethanol were negatively correlated and tetradecane was positively correlated with juiciness and TSS . 2-Heptanol was positively correlated with juiciness. Hexanal, hexanoic acid, α-ionone, linalool, and α-terpineolwere negatively correlated with hand firmness, and all but hexanal were positively correlated with sweetness. Limonene was the only volatile significantly correlated with off-flavor , and α-terpineol was the only volatile correlated with tartness .Raspberry firmness remained stable or decreased more slowly with increasing CO2 concentration in storage, with the highest firmness in 15 kPa CO2. In agreement with our results, Haffner et al. , found that an atmosphere of 15% CO2, 10% O2 maintained the firmness of five raspberry cultivars stored for seven days at 1℃.Strawberries exposed to high CO2 exhibited changes in apoplastic pH, square plastic pot which may have induced cell to cell adhesion by precipitation of soluble pectin . This may explain why high CO2 stored raspberries were perceived as firmer by our sensory panelists. However, firmer fruit tasted less sweet to the sensory panelists. Stec et al. reported that firmer kiwifruit tasted less sweet than softer ones. This finding aligned with the general notion that softer fruit have more ripe fruit characteristics such as sweetness, juiciness and higher aroma intensity .

This can explain the negative correlation of juiciness with firmness in our experiment. In addition, storage under high CO2 atmospheres also might have inhibited further ripening of the fruit which would inhibit fruit softening. High CO2 atmospheres also reduced development of leakiness and color darkening. Exposure to CO2 atmospheres can induce fermentative metabolism , likely due to its capacity to disrupt enzyme systems . Elevated CO2 has been reported to induce development of alcoholic flavors in fruit if the concentration is too high for longer times . High CO2 enhances the activity of pyruvate decarboxylase and alcohol dehydrogenase, but reduces activity of alcohol acetyltransferase. As a result, acetaldehyde and ethanol accumulate and this trigger further production of ethyl esters and reduction of other esters, thus, enhancing alcoholic flavor . Larsen suggested that accumulation of ethyl acetate was linked to development of off-flavor in raspberries in some cultivars. High CO2 atmospheres can impact the lipoxygenase pathway which is involved in the formation of aromatic volatile compounds through effects on enzymes or by limiting substrates due to production of fermentative volatiles . In our study, fermentative volatiles were strongly associated with storage in the higher CO2 atmospheres early in storage, but after 10 days of storage, raspberries stored in air also accumulated fermentative volatiles, likely due to over-ripening. In addition, very low O2 atmospheres can contribute to off-flavors. Joles et al. reported that raspberries stored in 3% O2 developed off-flavor because fermentative respiration occurs when O2 levels drop below this critical level . However, this might not be the case for our experiment, because our lowest O2 level was ≥ 6 kPa. The one exception might be the 15 kPa atmosphere. The combination of 15 kPa CO2 with 6 kPa O2 could have resulted in additional impacts on fruit metabolism given the relatively low O2 concentration, resulting in a stronger impact of the 15 kPa atmosphere on fruit quality. The concentration of individual fermentative volatiles was as much as 1000-fold higher than the aromatic volatiles, and the fermentative volatiles were 4 to 300-fold higher in raspberries stored under 15 kPa CO2 compared to fruit stored in air. Accumulation of acetaldehyde, ethanol and ethyl acetate can contribute to objectionable changes in taste . However, in our experiment, the off flavor sensory score was lower in fruit stored in 15 or 8 kPa atmosphere than in 5 kPa atmosphere, and was moreclosely clustered on the bi-plot with aromatic volatiles than fermentative volatiles. Limonene was most highly correlated with off-flavor. The concentrations of ethanol detected in our raspberry samples appear to be below the corresponding odor threshold of 990 µl /L, . This may explain why our sensory panelists did not sense any off-flavor in raspberries stored in 15 kPa atmosphere, even when fermentative volatile concentrations were significantly higher in those fruit than in fruit stored in lower CO2 atmospheres. The odor threshold is the lowest concentration of a volatile that can be smelled and can vary as much as 106 to 108 among volatiles in fruit . Therefore, the most abundant volatile is not necessarily always the dominant fruit aroma. Alcohols usually have considerably higher threshold values, near 990 µl /L , and therefore contribute less to aroma building than their corresponding aldehydes .