Materials and Methods. An initial group of thirteen genotypes cloned from selected native stock and commercial varieties were planted in replicated blocks with a minimum of four plants in each block in 2018. An additional fifty-four genotypes were planted in four expansion plots totaling two acres of cultivation area between 2019 and 2021. Not all genotypes displayed productivity over the course of the study, with less than half of genotypes producing sufficient materials for testing. Elderberry fruit was harvested during the 2019 through 2021 summer season (July-August). Certain genotypes were evaluated on berries collected in winter months as available. Elderflower samples were collected in summer of 2020. All material analyzed in the study was grown and collected by Hyldemœr + Co., then vacuum-sealed and frozen at -20 °C until being transported to the University of Florida Postharvest Lab and kept frozen (-30 °C) for later analysis. Berries were analyzed for soluble solids content (SSC), total titratable acidity (TTA), pH, juice content, total anthocyanin content and total antioxidant capacity (FRAP); while flowers were measured for phenolic content.

Quality analyses. All analyses were performed under yellow light (UV filtering) to prevent degradation of nutritional compounds. Fruit samples were ground by hand using a small metal spatula then centrifuged at 12,000 rpm for 20 minutes at 4 °C. The blended berry samples (10 to 20 g) resulted in 5 to 10 ml of juice. The juice was filtered through four layers of cheesecloth and was used to assess SSC, TTA, and FRAP.

A digital Refractometer (model r2i300, Reichert Analytical Instruments) was used to determine soluble solids content (SSC, °Brix); pH and total titratable acidity (TTA, % citric acid basis) were measured using an automatic potentiometer (model 719 S Titrino, Metrohm). TTA was determined by diluting 6 ml of juice with 50 ml deionized water, and then titrating with 0.1 N sodium hydroxide (NaOH) to an endpoint of pH 8.2 and expressed as percent citric acid.                                            

Antioxidant capacity was measured using the FRAP (ferric reducing antioxidant power) assay (Benzine and Strain, 1996; Pulido et al., 2000). Clarified juice was evaluated against a Trolox standard, mixed with a working solution of 1:1:10 FeII TPTZ to FeIII TPTZ to a sodium acetate buffer and read at an absorbance of 593 nm. Results were expressed as Trolox equivalents (TE µmol/g f.w.).

Total anthocyanin content was extracted using acidified methanol outlined in Lee et al. (2005). Elderberry tissue (0.3 g) was homogenized with 10 mL of 0.5% formic acid in methanol (v/v) then centrifuged at 12,000 rpm at 4 °C for 20min. The supernatant was retained, and the sample was re-extracted with 5 ml acidified methanol, vortexed 1 min then centrifuged again. The supernatant was combined with the previously retained supernatant and completed to 15 ml with acidified methanol. The extract (0.6 ml) was mixed with 2.4 ml potassium chloride pH 1 buffer and pH 4.5 buffer and measured using a spectrophotometer (Powerwave XS2, BioTek, Winooski, VT, USA) at 510 and 700 nm. Pigment concentration was calculated using the following formula: Abs520 × dilution factor × (molecular weight (MW) of cyanidin-3-glucoside equivalents (CGE)/molar extinction coefficient) where MW of CGE = 449.2 and the molar extinction coefficient = 26,900. Results were expressed as CGE (mg/g f.w.).  Total phenolic content for flowers were analyzed with the Folin-Ciocalteu method reported by Singleton and Rossi (1965) using gallic acid as the standard. Once the flowers were removed from the vacuum sealed bag, they immediately turned brown (Fig. 4). Elderflower (2 g) was homogenized with 2 mL of calcium chloride then centrifuge at 4,500 rpm at 4 °C for 20 minutes. The sample (0.1 mL) was diluted with 5.9ml water then added to 2.5 mL of 0.2 N Folin-Ciocalteu reagent and 2.0 mL of 7.5 % sodium carbonate then incubated in a water bath at 45 °C for 15 min. Aliquots of 250 μL were pipetted into a 96-well plate and absorbance read at 765 nm in a microplate reader (PowerWave XS2, Biotek). The results were expressed as gallic acid equivalents (GAE mg/100 g) of flower fresh weight.

 Literature Cited

Benzie, I.F. and Strain, J.J.. 1996. The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant

power": the FRAP assay. Analytical Biochem. 239(1):70-76.

Cata, A., Stefanut, M.N., Pop, R., Tanasie, C., Mosoarca, C., and Zamfir, A.D. 2016. Evaluation of

antioxidant activities of some small fruits containing anthocyanins using electrochemical and chemical

methods. Croatica Chemica Acta 89(1): 1-12. http://dx.doi.org/10.5562/cca2656

Kaack, K.V. and Knudsen, B.F. 2015. Horticultural production of flowers and berries of elder

(Sambucus nigra) as raw material for processing of foods and pharmaceuticals. Acta Hortic. 1061: 201-

208. https://doi.org/10.17660/ActaHortic.2015.1061.21

Lee, J., R.W. Durst, R.E. Wrolstad. 2005. Determination of total monomeric anthocyanin pigment

content of fruit juices, beverages, natural colorants, and wines by the pH differential method:

Collaborative study. J. AOAC Int. 88:1269–1278.

Mikulic-Petkovsek M., Samoticha J., Eler K., Stampar F., Veberic R. 2015. Traditional elderflower

beverages: a rich source of phenolic compounds with high antioxidant activity. J Agric Food Chem.

63(5):1477-87. doi: 10.1021/jf506005b.

Ozgen, M., Scheerens, J.C., Reese, R.N., and Miller R.A. 2010. Total phenolic, anthocyanin contents

and antioxidant capacity of selected elderberry (Sambucus canadensis L.) accessions. Pharmacognosy

Magazine 23(6): 198-203.

Perkins-Veazie, P., Thomas A.L., Byers P.L., Finn C.E. 2015. Fruit Composition of Elderberry

(Sambucus spp.) Genotypes Grown in Oregon and Missouri, USA. Acta Hortic. 1061:219-224. doi:

10.17660/ActaHortic.2015.1061.24.

Pulido, R., Bravo, L. and Saura-Calixto, F. 2000. Antioxidant Activity of Dietary Polyphenols as

Determined by a Modified Ferric Reducing/Antioxidant Power Assay. Journal of agricultural and food

chemistry. 48:3396-3402.

Singleton, V.L. and Rossi J.A. 1965. Colorimetry of Total Phenolics with Phosphomolybdic-

Phosphotungstic Acid Reagents. Am J Enol Vitic.16:144-158.