This document summarizes research projects focused on phenolic compounds, proteins, and protein hydrolysates found in cereal grains like sorghum. The projects involve extracting and purifying phenolic compounds using alkaline extractions and separating proteins using techniques like HPLC, electrophoresis, and chromatography. The extracts and fractions are then analyzed to study their antioxidant activities, enzymatic functions, and bioactivities. Publications and presentations are also referenced reporting on the isolation of protein fractions from crops, separation of sorghum proteins, and production of sorghum protein hydrolysates.

1. Research Projects
 Phenolic / Antioxidant Compounds
– Alkaline Extraction of Phenolics from Intact Cereal Grain2
– Separation of Anthocyanins from Black Sorghum Bran
 Proteins
– Separation of Kafirins on Surface Porous RP-HPLC Columns5
– IE-HPLC Method to Characterize Alcohol Soluble Proteins3
– Isolation and Characterization of Sorghum Protease Inhibitor
 Protein Hydrolysates
– Functional and Bioactivity of Sorghum Protein Hydrolysates6

2. Phenolics
 Samples
– Sorghum
• Flour
• Bran
• Grain
– Oats, Wheat, Corn Grain
 Purification
– NaOH and Alcohol Extraction
 Analysis
– RP-HPLC
– Antioxidant Activity (AOC)
• Fluorescence detection
Ref. 2: AOC of NaOH Sorghum bran extracts
(treated & untreated) over time at ambient
conditions.

3. Protein Samples
 Solubility
– Water
– Salt
– Alcohol
– Detergent
– Reducing and Alkylating agents
 Sorghum, Corn, Wheat, Rice, Oats, Millet
 Cereal grain, bran, flour, leaf tissue, DDGS
 Camelina, Algae isolate

4. Protein Purification
 Selective Extractions
– Targeting protein subclasses
 Precipitation
– TCA
– Acetone
– Ethanol
– pH
– Boiling
 Dialysis
 Spin Columns
 Fractionation
– Collecting fractions from LC runs
Ac / 3.6 mM NH
OH
0 4 8 12 16 20 24 28 32
0.10
0.08
0.06
0.04
0.02
0.00
Time, min
Absorbance, 254 nm
A: 10 mM NH
4
4
100
80
60
40
20
0
% B, 1 M NH
4
Ac / 25 mM NH
4
OH
Orange area represents the location of
active fraction eluted by Low-Pressure LC.

5. Protein Analysis - Separations
 Chromatography
– HPLC (Agilent/ChemStation)
• RP, SEC, Ion-exchange
– Low-pressure LC (Bio-Rad/BioLogic)
• SEC, Ion-exchange, Hydrophobic Interaction, Chromatofocusing
– 2D Separations
 Electrophoresis
– SDS-PAGE
• Coomassie Blue
• Silver Stain
• Reverse Zymography
– Capillary Electrophoresis
Ref. 3

6. Protein Analysis - Assays
 In-vitro Digestibility
– Pepsin
– Trypsin
– Chymotrypsin
– Alcalase
– Papain
– Bromelain
– Neutrase
 Colorimetric
– Enzymatic Inhibitory Activity
– BCA
– Degree of Hydrolysis (OPA)
 Functionality
– Emulsion
– Foaming
– Solubility
– Antioxidant Activity
Ref. 6: Emulsion Activity Index (bars) &
Stability (line)

7. Publications
1) Li, N., Qi, G., Sun, X.S., Wang, D., Bean, S., Blackwell, D., “Isolation and Characterization of
Protein Fractions Isolated from Camelina Meal.” Transactions of the American Society of
Agricultural and Biological Engineers. 2014, 57, 169.
2) Blackwell, D.L., Herald, T.J., Bean, S.R., Gadgil, P. “Alkaline extraction of phenolic compounds
from intact sorghum kernels.” International Journal of Food Science & Technology. 2012, 47,
2671.
3) Blackwell, D.L. and Bean, S.R. “Separation of alcohol soluble sorghum proteins using non-porous
cation-exchange columns.” Journal of Chromatography A. 2012, 1230, 48.
4) Bean, S.R., Ioerger, B.P., Smith, B.M., Blackwell, D.L. Sorghum protein structure and chemistry:
Implications for nutrition and functionality. In: Advances in Cereal Science: Implications to food
processing and health promotion. Awika, J.A., Piironen, V., Bean, S.R., Eds.; American Chemical
Society: Washington DC, 2011; pp 131-147.
5) Bean, S.R., Ioerger, B.P., Blackwell, D.L. “Separation of kafirins on surface porous reversed
phase–high-performance liquid chromatography columns.” Journal of Agricultural and Food
Chemistry. 2011, 59, 85.

8. Presentations
6) Blackwell, D.L., B.M. Smith, S.R. Bean, M. Tilley, “Production of sorghum
protein hydrolysates”, IFT Annual Meeting, Las Vegas, Nevada, June 2012.
(Poster)
7) Blackwell, D.L. and S.R. Bean, “RP-HPLC Analysis of Sorghum Protein
Digestibility”, ACS National Meeting, Anaheim, California, March 2011.
(Poster)
8) Blackwell, D.L., B.P. Ioerger, S.R. Bean, “Improved RP-HPLC Separations
on Sorghum Proteins: Methods and Applications”, SICNA Meeting, Mead,
Nebraska, August 2010. (Poster)
9) Blackwell, D.L., B.P. Ioerger, S.R. Bean, “Improved RP-HPLC Separations
on Sorghum Proteins: Methods and Application”, HPLC Meeting, Boston,
Massachusetts, June 2010. (Poster)