Analysis of the Antioxidant Qualities of Flowers and Fruits of Several Commercial Varieties of Sambucus nigra ssp. Canadensis (The North American Black Elderberry) in Florida

Final report for FS19-317

Project Type: Farmer/Rancher
Funds awarded in 2019: $9,971.00
Projected End Date: 03/14/2022
Grant Recipient: Farmer
Region: Southern
State: Florida
Principal Investigator:
Heather Martin
Hyldemoer & Co., LLC
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Project Information

Abstract:

With the demand for Elderberry continuing to rise, our farm seeks to determine which of nearly 60 varieties of Sambucus nigra ssp. Canadensis and Sambucus nigra grown on our farm show promise for commercial production within Florida. Commercial production success hinges upon two main points. The first of which is finding those varieties that are best suited for sustainable cultivation in Florida's unique environment and the second is  conducting compositional quality analysis to determine which varieties are the most marketable based upon their individual compositions. With increased  knowledge on these factors, it is possible to educate Florida growers on the viability of Sambucus as a commercial crop.

Project Objectives:

From the analysis of both the flower and fruit of Sambucus nigra ssp. Canadensis and Sambucus nigra, our farm will determine which of the nearly 60 varieties we planted have the highest value. These analyses will be shared with potential growers to educate them on the feasibility of utilizing specific varieties of Sambucus as a commercial crop throughout Florida.

Cooperators

Click linked name(s) to expand/collapse or show everyone's info
  • Dr. Juanita Popenoe (Educator)
  • Dr. Kevin Athearn (Educator)
  • Dr. Steve Sargent (Educator)
  • Dr. Ali Sarkhosh (Educator)
  • David Jarnagin (Researcher)

Research

Materials and methods:

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. 88:12691278.


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.

 

 

 

 

 

Research results and discussion:

Results – 2019 season. For the elderberry genotypes tested, the SSC ranged from 6.95 to 13.65 °Brix, TTA from 0.27% to 0.59% and the SSC/TTA ratio from 17.12 to 33.93. The pH was similar for all genotypes and averaged 5.2. The antioxidant activity (FRAP) ranged from 1.25 to 18.73 (µmol/g). Genotypes 34 and 38 showed promise in the field so they were analyzed for total anthocyanins. Genotype 34 (8.67 CGE mg/g) had much higher average anthocyanin content than the average of five samples of genotype 38 taken in the main harvest season during July (1.12 CGE mg/g) (Table 1). The phenolic content for elderflower ranged from 4.7 to 8.85 (GAE mg/g) (Table 2).

Results – 2020 season. The values for most parameters measured were higher for the 2020 harvest season. It was also observed that the fruit were larger and contained more seeds. For the genotypes tested in 2020, the SSC ranged from 7.37 to 15.93 °Brix, TTA from 0.38% to 0.58%, and SSC/TTA ratio from 20.85 to 37.32. The pH was similar for all genotypes and averaged 5.0. The antioxidant activity (FRAP) ranged from 6.7 to 37.9 (µmol/g) and total anthocyanins from 1.49 to 12.92 (CGE mg/g) (Table 3).

Results – 2021 season. Genotype 38, Sample 49 (38-00N-Z1.5) had the lowest FRAP (1.17 µmol/g) and anthocyanin (0.65 mg/g) compared to other samples. Genotype JLAV-1, Sample 54 (JLAV 1-03-Z0.5) had both high FRAP (32.59 µmol/g) and anthocyanin (13.24 mg/g) results compared to other samples. The image shows the color difference between blended samples 49 (38-00N-Z1.5) on the left and 54 (JLAV 1-03-Z0.5) on the right. According to the field notes, sample 49 was picked underripe. Sample 54 was taken from an establishment year plant at only 10 months in-ground in the same plot and on the same harvest date as sample 49. Table 4 (page 7) shows the average analysis per genotype. Full data for all individual samples in the study is available from Hyldemœr + Co. upon request. For the genotypes tested in 2021, the average SSC ranged from 8.02 to 13.83 °Brix, TTA from 0.30% to 0.67%, and SSC/TTA ratio from 13.39 to 27.28. The pH was similar for all genotypes and averaged 4.8. The antioxidant activity (FRAP) ranged from 6.27 to 32.57 (µmol/g) and total anthocyanins from 3.6 to 26.57 (CGE mg/g) (Table 4).

Summary. The cumulative results from three harvest seasons (2019, 2020 and 2021) were similar or slightly higher than the values found in the literature, though several samples tested at significantly higher ranges than found in the literature in terms of antioxidant capacity and anthocyanin content. In 2020 when the flower sample bags were opened they immediately oxidized (turned brown); however, the data was similar to other reported results. The reported values for elderberry (Sambucus canadensis L.) were 8 to 12 °Brix, 0.75 to 1.4% TTA, 4.6 pH (Kaack et al., 2015; Ozgen et al., 2010; Perkins-Veazie et al., 2015), 2 to 6 Cy-3GE mg/g Anthocyanin content, and 20 to 24 TE µmol/g Antioxidant capacity (Ozgen et al., 2010; Perkins-Veazie et al., 2015). Elderflower phenolic content was reported as 10 GAE mg/g (Mikulic-Petkovsek et al., 2015).

Table 1. Season 2019: Elderberry fruit antioxidant activity, total anthocyanins, SSC, TTA, SSC/TTA ratio, pH and juice content - postharvest.

 

Sample from Genotype Specimen

 

 

Harvest date

Antioxidant activity - FRAP

 

Anthocyanin

 

SSC

 

TTA

 

SSC/TTA Ratio

 

pH

 

Juice content (ml/g)

TE

(µmol/g)

±

s.d.

Cy- 3GE

(mg/g)

±

s.d.

 

°Brix

±

s.d.

Citric acid

%

±

s.d.

 

SSC/TTA

±

s.d.

 

pH

±

s.d.

27-01-400

7/25/2019

9.77

1.58

 

 

10.17

0.45

0.59

0.05

17.12

0.02

4.58

0.02

0.45

27-02-400

7/6/2019

5.38

0.41

 

 

9.07

0.15

0.32

0.03

28.66

0.02

5.39

0.02

0.44

29-01-100

7/11/2019

13.13

0.80

 

 

10.87

0.45

0.45

0.05

24.09

0.06

4.98

0.03

0.52

29-01-100

7/25/2019

16.26

3.93

 

 

10.73

0.21

0.39

0.01

27.47

0.03

5.31

0.06

0.53

29-03-100

7/11/2019

15.92

0.50

 

 

10.60

0.00

0.50

0.02

21.22

0.01

4.92

0.01

0.62

30-02-200

8/30/2019

8.39

0.31

 

 

12.80

0.42

0.38

0.00

33.68

0.01

5.37

0.01

0.38

31-01-100

7/25/2019

5.54

0.57

 

 

8.60

0.36

0.37

0.02

22.99

0.06

5.21

0.06

0.41

31-03-400

7/25/2019

4.16

0.21

 

 

9.13

0.47

0.38

0.01

23.81

0.03

5.45

0.03

0.37

34-01-200

7/11/2019

9.15

1.01

7.20

0.76

10.47

0.47

0.52

0.05

20.13

0.01

4.75

0.01

0.31

34-02-200

7/15/2019

6.15

1.54

10.72

2.23

12.73

0.15

0.49

0.03

26.04

0.04

4.93

0.04

0.22

34-15-400

7/11/2019

5.76

0.39

8.09

0.11

11.70

0.87

0.45

0.01

25.92

0.05

5.26

0.05

0.30

38-02-200

7/9/2019

2.92

0.13

2.24

0.03

8.55

0.07

0.33

0.03

25.79

0.04

5.00

0.04

0.21

38-02-400

11/6/2019

15.88

0.19

4.30

1.00

10.15

0.07

0.45

0.00

22.66

0.04

4.87

0.04

0.43

38-04-400

11/6/2019

18.73

2.55

5.73

0.04

9.70

0.42

0.47

0.01

20.70

0.01

4.70

0.01

0.43

38-09-400

7/18/2019

2.67

0.47

1.39

0.16

9.45

0.35

0.28

0.02

33.93

0.02

5.71

0.02

0.22

38-14-400

7/18/2019

1.49

0.20

0.33

0.03

7.80

0.00

0.32

0.00

24.15

0.06

5.38

0.06

0.20

38-15-400

7/18/2019

2.32

0.07

0.98

0.18

9.15

0.07

0.30

0.01

30.55

0.02

5.75

0.02

0.22

38-19-400

7/11/2019

1.25

0.20

0.66

0.07

6.95

0.35

0.27

0.00

25.60

0.01

5.36

0.01

0.22

50-01-400

7/25/2019

8.88

0.76

 

 

12.80

0.42

0.49

0.01

26.10

0.01

5.08

0.01

0.31

51-01-200

8/30/2019

13.56

0.13

 

 

13.65

0.64

0.48

0.06

28.59

0.00

5.36

0.00

0.28

53-01-100

7/11/2019

8.95

0.25

 

 

9.60

0.14

0.36

0.00

26.34

0.01

5.29

0.01

0.30

TE= trolox equivalents; Cy-3GE= cyanidin 3-glucoside equivalents. Data based on fresh weight and represents the mean (n=3).

 

Table 2. Season 2019: Elderflower total phenolics - postharvest.

Sample from genotype specimen

 

Harvest date

Total phenolics (mg/g GAE)

 

±

s.d.

27-01-400

6/6/2019

7.53

0.06

27-03-100

5/30/2019

6.73

0.02

29-03-100

5/30/2019

6.59

0.04

29-03-100

6/6/2019

6.16

0.02

30-02-200

7/19/2019

4.66

0.04

31-04-400

6/4/2019

6.22

0.06

34-19-400

6/14/2019

5.57

0.01

38-02-200

5/30/2019

8.85

1.26

50-F1-400

5/30/2019

6.63

0.02

51-01-200

8/30/2019

7.76

0.02

GAE = gallic acid equivalents. Data based on fresh weight and represents the mean (n=3).

Table 3. Season 2020: Elderberry fruit antioxidant activity, total anthocyanins, SSC, TTA, SSC/TTA ratio, pH and juice content - postharvest.

 

Sample from genotype specimen

 

 

Harvest date

Antioxidant activity - FRAP

 

Anthocyanin

 

SSC

 

TTA

SSC/TTA

Ratio

 

pH

 

Juice content (ml/g)

TE

(µmol/g)

±

s.d.

Cy-3GE

(mg/g)

±

s.d.

 

°Brix

±

s.d.

Citric acid %

±

s.d.

SSC/ TTA

±

s.d.

 

pH

±

s.d.

27-08-400

8/1/2020

37.88

0.18

12.92

0.80

15.93

0.59

0.58

0.06

27.93

3.97

4.63

0.06

0.6

28-01-200

7/23/2020

16.72

0.18

8.97

1.72

13.90

0.20

0.39

0.10

37.32

10.13

5.20

0.01

0.6

29-03-800

8/1/2020

21.61

0.15

10.78

0.97

10.37

0.31

0.47

0.13

23.18

7.14

5.11

0.01

0.5

31-08-400

8/1/2020

21.24

0.02

7.52

0.09

11.33

0.25

0.54

0.07

20.85

2.54

4.88

0.04

0.6

32-03-400

5/25/2020

18.07

0.11

2.63

0.23

11.83

0.12

0.49

0.11

24.58

5.30

4.85

0.03

0.6

33-08-400

7/23/2020

17.05

0.07

6.19

1.05

11.60

0.44

0.38

0.09

31.40

6.13

5.29

0.05

0.6

34-19-400

8/1/2020

21.76

0.04

10.89

0.56

10.83

0.12

0.53

0.12

21.12

4.77

4.88

0.01

0.6

38-03-400

8/1/2020

6.68

0.01

1.49

1.49

7.37

0.23

0.38

0.19

22.24

10.98

4.88

0.03

0.6

TE= trolox equivalents; Cy-3GE= cyanidin 3-glucoside equivalents. Data based on fresh weight and represents the mean (n=3).

Table 4. Season 2021: Elderberry fruit antioxidant activity, total anthocyanins, SSC, TTA, SSC/TTA ratio, pH and juice content - postharvest.

 

Sample average of genotype

Antioxidant activity - FRAP

 

Anthocyanin

 

SSC

 

TTA

 

SSC/TTA Ratio

 

pH

 

Juice content (ml/g)

TE

(µmol/g)

±

s.d.

Cy-3GE

(mg/g)

±

s.d.

 

°Brix

±

s.d.

Citric acid %

±

s.d.

 

SSC/TTA

±

s.d.

 

pH

±

s.d.

27

9.36

na

14.34

na

11.45

na

0.58

na

19.83

na

4.65

na

0.45

28

22.41

4.03

14.51

4.41

11.23

2.93

0.51

0.03

22.09

4.51

4.79

0.21

0.59

29

14.21

1.28

17.91

5.29

10.82

1.96

0.51

0.09

21.57

5.78

4.83

0.16

0.51

31

15.55

4.34

12.04

4.11

12.01

1.58

0.57

0.07

21.19

2.63

4.81

0.18

0.53

32

9.79

na

13.61

na

10.70

na

0.48

na

22.18

na

4.87

na

0.65

33

12.97

0.34

10.35

1.78

11.95

0.67

0.54

0.01

21.99

1.47

4.81

0.13

0.58

34

12.00

0.40

7.21

2.51

10.06

0.23

0.38

0.06

26.72

4.08

5.04

0.27

0.51

36

15.28

1.47

23.30

6.33

13.83

4.00

1.03

0.03

13.39

3.50

4.26

0.14

0.44

38

6.27

1.01

3.60

2.50

8.21

1.15

0.30

0.03

27.28

2.90

4.93

0.10

0.58

DNS-01

18.51

na

26.57

na

10.15

na

0.61

na

16.69

na

4.41

na

0.54

DNS-07

18.19

na

17.21

na

8.05

na

0.43

na

18.94

na

4.74

na

0.49

DNS-10

18.11

na

13.89

na

10.50

na

0.51

na

20.58

na

4.68

na

0.48

DNS-11

28.63

na

22.91

na

10.15

na

0.66

na

15.47

na

4.50

na

0.60

DNS-15

26.39

na

17.04

na

11.85

na

0.52

na

22.77

na

4.68

na

0.51

DNS-16

23.31

3.24

13.63

0.34

10.05

0.78

0.39

0.05

25.81

5.32

5.00

0.23

0.53

DNS-19

24.51

na

23.79

na

12.20

na

0.57

na

21.25

na

4.86

na

0.53

DNS-23

26.65

na

22.56

na

11.40

na

0.67

na

17.09

na

4.66

na

0.48

DNS-26

32.57

na

25.39

na

11.60

na

0.48

na

23.95

na

4.99

na

0.51

DNS-30

9.11

2.30

6.79

4.92

8.07

0.88

0.44

0.05

18.52

3.68

4.84

0.19

0.44

FGWR

20.68

1.91

22.85

6.98

12.30

2.12

0.63

0.01

19.71

3.68

4.49

0.26

0.56

JLAV1

20.71

7.70

12.21

4.01

8.02

0.75

0.35

0.02

23.12

3.01

5.13

0.14

0.54

JLAV5

30.15

3.38

17.95

1.50

9.81

0.88

0.37

0.01

26.88

2.67

5.15

0.20

0.59

TE= trolox equivalents; Cy-3GE= cyanidin 3-glucoside equivalents. Data based on fresh weight and represents the mean (n=3).

 

 

Discussion. Though approximately 40% of genotypes present in the trial plots proved sufficiently productive to analyze for compositional quality, relatively few genotypes displayed meaningful, consistent productivity. Of the most consistently productive genotypes, a relatively large group were demonstrated to be capable of producing fruit with high concentrations of antioxidants and anthocyanins by previously reported standards. Some potentially productive genotypes were eliminated from further trialing expansion. For example, genotype 38 reliably produced harvest volumes in excess of nearly any other genotype. Because of its high productivity and large cymes, this genotype needs to be trellised to keep the fruit off the ground effectively. This additional management, labor and material cost was not demonstrated to be worthwhile long-term given the relatively poor compositional quality analysis for genotype 38 over the 3-year study period. Conversely, the JLAV and DNS genotypes series grown in the same trial plots, but with only one year of analysis performed, demonstrated early precocity and very high levels of antioxidant capacity and anthocyanins. Ongoing trialing of genotypes selected through field and compositional quality analysis will focus on a limited number of genotypes, only 4 of which were present in the original 13 genotypes at the beginning of the study period: genotypes 29, 31, 33, and 34. Additional genotypes added over the 2019-2021 establishment period which warrant further study for cultivation in the Southeast include: the FGW, DNS and JLAV series of genotypes.

 

Participation Summary
1 Farmer participating in research

Educational & Outreach Activities

1 Curricula, factsheets or educational tools
2 Journal articles
1 Tours

Participation Summary:

2 Farmers
10 Ag professionals participated
Education/outreach description:

Our farm partnered with Dr. Juanita Popenoe, Dr. Kevin Athearn, Dr. Steve Sargent and Dr. Ali Sarkhosh on EDIS publications on elderberry cultivation and economics in Florida.

https://edis.ifas.ufl.edu/pdf/HS/HS139000.pdf

https://edis.ifas.ufl.edu/publication/FE1093

The final compositional quality analysis report is available on our website at, https://hyldemoerfarms.com/comp-qual-2021. It will also be presented at the Florida Wine and Grape Growers Association (FWGGA) annual meeting in June 2022 and mailed to interested parties.

Learning Outcomes

2 Farmers reported changes in knowledge, attitudes, skills and/or awareness as a result of their participation

Project Outcomes

2 Farmers changed or adopted a practice
1 Grant received that built upon this project
3 New working collaborations
Project outcomes:

This project demonstrates that Sambucus (elderberry) is a viable alternative crop in Florida. The trialing of nearly 60 varieties has eliminated those varieties that cannot be grown sustainably in Florida, providing a headstart for other potential Sambucus growers in Florida.

Information Products

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.