Carbon Dioxide–induced Changes in Color and Anthocyanin Synthesis of Stored Strawberry Fruit

Anthocyanin concentrations increased in both external and internal tissues of ‘Selva’ strawberries (Fragaria ×ananassa Duch.) stored in air at 5 °C for 10 days, but the increase was lower in fruit stored in air enriched with 10 or 20 kPa CO2. Flesh red color was less intense in CO2 storage than in air storage. Activities of phenylalanine ammonia lyase (PAL) and UDP glucose : flavonoid glucosyltransferase (GT) decreased during storage, with decreases being greater in both external and internal tissues of strawberry fruit stored in air + 20 kPa CO2 than in those kept in air. Activities of both PAL and GT in external tissues of strawberries stored in air + 10 kPa CO2 were similar to those in fruit stored in air, while enzyme activities in internal tissues more closely resembled those from fruit stored in air + 20 kPa CO2. Phenolic compounds increased during storage but were not affected by the storage atmosphere. The pH increased and titratable acidity decreased during storage; these effects were enhanced in internal tissues by the CO2 treatments, and may in turn have influenced anthocyanin expression. Anthocyanin synthesis continues in harvested fruit, particularly those stored in air, even at low storage temperatures. Increases have been observed in lowbush blueberry fruit (Vaccinium angustifolium Ait.) (Kalt and McDonald, 1996), where anthocyanin concentration increased by 18% after storage for 2 weeks at 1 °C; in pomegranates (Punica granatum L.), where an increase of 71% was observed after 6 weeks of storage at 10 °C (Holcroft et al., 1998); and in ‘Blomidon’ (Kalt et al., 1993) and ‘Selva’ strawberry fruit (19% increase in whole fruit or 31% in external tissues after storage for 10 d at 5 °C) (Gil et al., 1997). Treatment with CO2 inhibits this postharvest increase in anthocyanin concentration (Gil et al., 1997; Holcroft et al., 1998) by affecting anthocyanin biosynthesis, degradation, or both. The biosynthesis of anthocyanins has been well studied (Holton and Cornish, 1995; Stafford, 1990). The first step of the phenylpropanoid pathway is the production of cinnamic acid from phenylalanine, produced via the shikimic acid pathway, by the action of phenylalanine ammonia lyase (PAL; EC 4.3.1.5). Cinnamic acid is converted into coumaric acid, which is modified to the CoA form. Three molecules of malonyl CoA combine with ρ-coumaroyl-CoA and form required concentration (e.g., 10 ± 1 kPa). The composition of the gases was checked regularly with a gas chromatograph equipped with a thermal conductivity detector (model 211; Carle Instruments, Anaheim, Calif.) or an infrared gas analyzer (model 2000R; Horiba Instruments, Irvine, Calif.). Color. All the fruit were analyzed initially and after 5 and 10 d of storage at 5 °C. At each evaluation time 12 fruit per replicate were returned to the jars and held in air at 5 °C for a further 3 d. External color was measured on opposite sides of the fruit using a Minolta chromameter (model CR-200; Minolta, Ramsey, N.J.), which provided CIE L*, a*, and b* values. These values were used to calculate chroma (C* = [a* + b*]), which indicates the intensity or color saturation, and hue angle (h° = arctangent [b*/a*]) where 0° = red-purple; 90° = yellow; 180° = bluish-green and 270° = blue (McGuire, 1992). The fruit were sliced in half and internal flesh color was measured on both halves. External and internal color was also measured on the fruit that had been returned to air for 3 d. pH, TA, and TSS. Fruit halves were separated into external tissues (epidermis and about one-third of the cortex), internal (pith, bundle zone, and the remainder of the cortex), and the juice was analyzed separately for total soluble solids (TSS), pH, and titratable acidity (TA). Soluble solids content was measured using an Abbe refractometer (model 10450; American Optical, Buffalo, N.Y.). A 4-g sample of juice was diluted with 20 mL of distilled water, and pH and TA were measured using an automatic titrator fitted with pH meter and an autoburette (PHM85 Precision, ABU80; Radiometer, Copenhagen, Denmark). TA was expressed as percentage of citric acid. Anthocyanin extraction and analysis. Twenty halves per replicate were frozen in liquid N2 and kept at –80 °C until analyzed for anthocyanin and enzyme activities. The frozen tissue was removed from the freezer, allowed to thaw slightly, and separated into external and internal tissues as described previously. The separated tissue was immediately refrozen in liquid N2 before being ground in an Oster blender. Anthocyanins were extracted, separated, and quantified as described by Gil et al. (1997). Enzyme extraction and analysis. The method of Lister et al. (1996) was used to extract and analyze PAL and GT, with the only difference being in the GT assay. UDP-glucose replaced UDP-galactose, and the product, quercetin 3-glucose, was quantified by the high-performance liquid chromatography (HPLC) method described in Holcroft et al. (1998). Total and individual phenolics. Total soluble phenolic concentration was measured with a commercially available Folin & Ciocalteau phenol reagent (Sigma Chemical Co., St. Louis) using ρ-coumaric acid as a standard (Singleton and Rossi, 1965). The extracts were prepared for HPLC analysis by dilution with 80% ethanol prior to analysis. All samples and standards were run in duplicate and absorbance was measured at 660 nm. naringenin chalcone, which is then converted into the flavanone, naringenin. The next step is the formation of dihydroflavonol, which is reduced to flavan-3,4-diol, or leucoanthocyanin, and then is converted to anthocyanidin. Finally, the glucose molecule is attached by the action of UDP glucose : flavonoid glucosyltransferase (GT; EC 2.4.1.91), resulting in the anthocyanin. Our hypothesis is that elevated CO2 atmospheres during storage adversely affect anthocyanin biosynthesis in strawberry fruit, and that this effect is greater in the internal tissues of the fruit. To test this hypothesis we measured anthocyanin concentration and the specific activity of PAL and GT, two important enzymes involved in biosynthesis, in external and internal tissues of strawberry fruit. Other phenolic compounds were also analyzed in an attempt to understand whether the storage atmosphere could affect the partitioning of the precursors into various phenolic compounds. Since color expression and stability of anthocyanins is affected by pH, we report CO2induced pH changes in fruit tissues. Materials and Methods Experimental setup. ‘Selva’ strawberries were harvested in Watsonville, Calif., and transported to the Univ. of California, Davis, in an air-conditioned vehicle and stored overnight at 5 °C. Damaged berries were discarded and remaining berries were sorted for uniform size and color. Sixty fruit were placed in each 9.5-L jar and three jars (replicates) were used per treatment. The jars were ventilated with a continuous flow of humidified air, air + 10 or air + 20 kPa CO2 at a flow rate of 170 mL·min using needle valve flow boards. The gases were maintained within 10% of the