Project Title:
Optimizing extraction methods to improve
utilization of wheat gluten in
bio-product manufacturing
By:
Shannon Williamson
Mentors:
Dr. Andrew Ross
Dr. Kaichang Li
Crop and Soil Science
Wood Science
Oregon State University
Oregon State University
Temporary wet strength resins (TWSR)
• When paper/paper products become wet their tensile strength
decreases dramatically. Therefore, resins are added to these
products to increase wet strength for a duration of time.
• Two types of resins: Temporary and permanent that can provide
either short or long lasting wet strength.
• Ren and Li (2005) developed a temporary wet-strength additive from
wheat gluten for use in the paper and paperboard industry.
Why wheat gluten?
• Wheat gluten is widely available, inexpensive and biodegradable.
• Currently the paper industry uses glyoxalpolyacrylamide (GPA) which is manufactured from
petro-chemicals.
• Wheat gluten is a renewable resource
• The new temporary wet-strength additive could be used
in the industry if the wheat gluten extraction process
was refined.
Wheat endosperm protein composition
• Albumins and Globulins~15-20% (Not useful for TWSR)
• Gliadins and Glutenins~75-80%
• This new additive has been made with only the gliadins
– only using about half of the available protein.
Gliadins vs. Glutenins
• Gliadins are soluble in 70% ethanol
• Glutenin
– is the residue leftover from the extraction of gliadin.
• Glutenin subunits are not soluble...
– due to the large size of the protein subunits.
– due to inter-subunit disulfide bonds
Approach
Chemical Modification of wheat gluten
I.
Gluten becomes soluble in a variety of chemicals: soaps, detergents,
hydrochloric acid, sodium hydroxide, 70% ethanol are just a few
possibilities.
II.
Solubilization by deamidation is also used; this can be in acidic or
basic solutions. Removal of the amide group of glutamine results in a
change in the potential ionic charge on the protein.
III.
Enzymatic modification is a process that hydrolyzes peptide bonds and
increases the solubility of gluten.
Outline of reaction scheme
•
Measure protein content of gluten with LECO and perform HPLC
analysis of existing proteins
•
8% (w/v) ratio of gluten in aqueous solution
•
Addition of enzyme to modify gluten protein
•
The fractions will be separated by centrifugation
•
Remeasure protein content and examine HPLC
•
Sonication of existing residues (insoluble fraction)
Enzymes
•
Two enzymes utilized
1. Bacterial protease from Bacillus licheniformis( Subtilisin A)
2. Fungal protease from Aspergillus oryzae
•
•
Both are endoproteases with broad specificity toward native and denatured
proteins
Both perform best in an environment pH= 6.0-9.0
Chromatogram for untreated gluten
•
•
•
Gluten – sonicated 1 min (black)
Gluten- sonicated 3 min (green)
Gluten – sonicated 5 min (blue)
Glutenin Peak
Gliadin
Peak
0.06
AU
0.04
0.02
0.00
12.00
13.00
14.00
15.00
16.00
17.00
18.00
19.00
Minutes
20.00
21.00
22.00
23.00
24.00
25.00
SE-HPLC Data for Run 1-1 and 1-2
•
•
•
•
Red- 1 minute sonication of gluten
Blue- No sonication of gluten
Black-Run - Fungal protease - 1 hr. treatment
Green-Run - Fungal protease - 30 minute treatment
0.12
0.10
0.08
0.06
AU
0.04
0.02
0.00
-0.02
-0.04
-0.06
12.00
14.00
16.00
18.00
20.00
Minutes
22.00
24.00
26.00
28.00
Results of Enzymatic Treatment
•
~100% of protein was soluble in water
•
Solubilized protein has a considerably reduced mass due to hydrolysis
•
With the protein in solution the TWSR was then made according the Ren
and Li (2005)
Resin From Hydrolyzed Gluten Protein
•
Hydrolyzed protein was modified with glycidyltrimethylammonium chloride
(GTA)
•
GTA modified protein underwent another reaction with glyoxal to create a
GTA-gluten protein-glyoxal resin.
•
When this last reaction was performed between GTA-gliadin and glyoxal, a
significant change in viscosity was observed.
•
In the case of my enzymatic treated protein, no change in viscosity was
observed.
Conclusions and Future goals
• Total solubilization of gluten protein is possible with enzymatic
treatment.
• Resin made from low molecular weight gluten proteins failed to yield
a usable product.
• Future approaches
– Hydrolyze less to keep MW closer to the MW of gliadin
– Separate gliadin from glutenin, followed by hydrolysis of glutenin and
then recombining with gliadin
Acknowledgments
•
Ernest and Pauline Jaworski Fund for Summer Research Experiences for
Underserved Undergraduates in Plant Science
•
Howard Hughes Medical Institute
•
Dr. Andrew Ross and Dr. Jae Ohm
•
Dr. Kaichang Li
•
Crop and Soil & Wood Science Department at Oregon State University