Plant material: A minimum of 500 g berries was collected for each selection in New York and Washington. For some cultivars, such as Heritage, a number of samples were harvested over the season and harvest dates were sampled separately (a total of 2000 to 5000 g fruit). All berries were frozen at -20 C immediately after harvest and held at -20 C until shipped. Frozen fruit were shipped to Lane, OK in December and held at -20 C until analyzed. Analyses were done within 4 months of receipt.
Preparation of samples. Two 50 g samples were weighed for each selection, then pureed using a blendor followed by one minute with a Polytron homogenizer. Samples were filtered through cheesecloth to remove seeds. A 5 g aliquot of each sample was then added to 25 ml of water:methanol:acetone:formic acid (400 ml:400 ml: 200 ml:0.6 ml), polytronned briefly, and used for total phenolics, FRAP, and total anthocyanin.
SSC, pH, titratable acidity.A 0.5 g sample of the puree was placed on a digital refractometer (Atago, Model PR 100) to measure soluble solids content. A 10 g sample of puree was added to 90 ml double distilled water (wt:wt) and a stir bar added for pH and titratable acidity measurements. The pH was measured using an Orion pH meter and electrodes designed for purees. Titratable acidity was measured using a digital buret ( ) and 0.1 N NaOH, where mls to an endpoint of 8.1 were quantified, and acidity based as percent citric acid (0.064 meq).
Total anthocyanin. Total anthocyanin was determined using the pH differential method of Giusti and Wrolstad (2001)(3). A subsample of the solvent-extracted puree (0.1, 0.5 or 0.8 ml) was added to pH 1.0 and pH 4.5 buffers and absorbance at 510 and 700 nm measured using a Shimazdu UV160 spectrophotometer. Each selection had duplicate runs made for each sample (a total of 4 to 16 reps per selection).
Total Phenolics. Total phenolics were determined with the Folin-Ciocalteau reagent, following the method of Singleton and Rossi (1965)(4). One ml of the solvent-extracted puree was added to one ml of F-C reagent (0.25N), followed by one ml of sodium carbonate (1N) and 7 ml water.
FRAP. Ferric reducing absorbance power was determined following a modified method of Benzie and Strain (1996)(1) and Deighton et al. (2000)(2). The method was modified for a non-automated spectrophotometer (Shimazdu UV 160) using a wavelength of 593 nm. FRAP reagent was pipetted into multiple cuvettes, then addition of samples to the reagent was staggered to every 20 seconds. Mixing of samples and reagents was done by rapid aspiration and expulsion of fluid with the pipette tip held just under the surface of the mixture.
ORAC. Oxygen radical absorbing capacity (ORAC) was determined on subsamples from some selections using a microplate reader and fluorescein as the fluourescent probe (Wada and Ou, 2002).(5)
Reference List
1. Benzie,I.F.F.; Strain,J.J. The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power"; the FRAP assay. Anal.Biochem. 1996, 239, 70-76.
2. Deighton,N.; Brennan,R.; Finn,C.; Davies,H.V. Antioxidant properties of domesticated and wild Rubus species. J.Sci.Food Agric. 2000, 80, 1307-1313.
3. Giusti,M.M.; Wrolstad,R.E. Anthocyanins. Characterization and measurement with UV-visible spectroscopy. Current Protocols in Food Analytical Chemistry, F 1.2-F 1.13. 2001. New York, John Wiley & Sons.
4. Singleton,V.L.; Rossi,J.A. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Amer. J. Enology Viticulture 1965, 16, 144-158.
5. Wada,L.; Ou,B. Antioxidant activity and phenolic content of Oregon caneberries. J Agric Food Chem. 2002, 50, 3495-3500.