1. Impact of various extrusion processes on Zein Gordon W. Selling 2015 Biopolymers and Bioplastics Meeting

2. Zein Composition Zein is the predominant corn protein Storage protein inside protein bodies of seed endosperm M.W. ~ 10,000 – 28,000 (+ dimers…) Four types , ,  low  Different solubility  -Zein (20 and 22,000 Da) is the main type Can be extracted from co-products of bioethanol production using aqueous alcohols Other zein types protein body wall Historically used in fiber and coating applications

3. Zein Melt Processing Historically used solution routes to process Melt processes explored relatively recently Improved understanding of impact of extrusion needed Zein as supplied begins to degrade (w/o shear) ~ 220 °C by DSC and TGA

4. Zein Degradation During Melt Processing Single screw extruder – single pass Varied extrusion temperature Varied throughput* Examined two plasticizers TEG / water* Twin screw extruder – single pass Varied extrusion temperature Varied screw rpm & throughput* Single screw extruder – multiple passes * - Insufficient time to present here

5. Zein Melt Processing – Single Screw Extruder - Single Pass Extrudate appearance changed with temp 100 °C160 °C260 °C

6. Extrusion Affects on Color With increased extrusion temperature extrudate color* becomes darker (L), greener (a) and bluer (b) Large change >160 °C * Compression molded samples

7. Extrusion Affects on Molecular Weight - Temp Changes in m.w. (SDS-PAGE) observed Cross-linking occur ~ 140 °C; low m.w. produced ~ 180 °C; hydrolysis ~ 240 °C SDS-PAGE w/o DDT * DDT - dithiothreitol SDS-PAGE w/ DDT

8. NMR & IR Spectroscopy 300 °C 100 °C Differences observed in three regions of C 13 spectra Addn peaks @ 170-180 ppm – COOH, COOR due to peptide hydrolysis and/or ester formation Loss of signal 50-65 ppm & growth 20-35 ppm suggest loss of C- OH & gain C-C=O – ROH oxidized to carboxyls IR - amide I & II changes above ~240 °C Change in Amide 1 & Amide 2 w/ Temp (Height 1658 vs. 1533) IR Spectra (select samples)

9. Extrusion Affects on Protein Structure - CD Near & far UV CD spectroscopy indicate changes in 3 and 2° structure Rapid structural changes occur in 3° above 220 °C Rapid structural changes occur in 2° above 240 °C @ 262 nm – 220 C Near-UV CD Spectra @ 222 nm – 240 C Far-UV CD Spectra

10. Extrusion Affects on Tensile Properties Produced compression molded samples Formulations above 220 °C too brittle to test With increasing extrusion temp. different molding conditions required Properties begin to decline above 140 °C

11. Zein Melt Processing – Twin Screw Extruder - Single Pass Lower residence time process Changes observed in extrudate with temperature 180 C 220 C 300 C

12. Twin Screw - Color Development Color change* occurs at higher temp versus single screw process * Compression molded samples

13. Twin Screw Extrusion – Molecular Weight M.W. changes occur Smaller in magnitude Different temperatures vs. single screw process ~200 °C observe cross- linking 140 °C for single screw Low m.w. materials produced ~ 220 °C 180 °C for single screw ~300 °C observe large degradation 260 °C for single screw w/ DTT* * DTT-dithiothreitol

14. Extrusion Affects on Tensile Properties Produced compression molded samples Formulations above 260 °C too brittle to test Increasing temp. required different molding conditions Properties begin to decline above 220 °C (140 °C for single screw)

15. Effect of Multiple Passes – Single Screw Experiments carried out at 145 °C (w/ 10% TEG) M.W. and color changes beginning ~145 °C With each pass torque / pressure increased Indicative of increased m.w.

16. Zein Extrudate Appearance After 1 st pass little change in appearance First Second Third

17. Extrusion Affects on Molecular Weight - Passes SDS-PAGE w/ and w/o DDT* illustrate that much of the m.w. growth is due to disulfide bonds Some permanent branches present * DTT-dithiothreitol

18. Extrusion Affects on Protein Structure Near-UV CD spectroscopy - changes in 3° structure observed after 1 st -2 nd pass Far-UV CD spectroscopy - changes in 2° structure observed after 3rd pass IR & NMR spectroscopy display few changes Far-UV CD Spectra (90% EtOH/water) Far-UV CD Molar Ellipticity @ 222 nm (90% EtOH/water) Near-UV CD Spectra (90% EtOH/water) Near-UV CD Molar Ellipticity @ 262 nm (90% EtOH/water)

19. Recycling Affects on Tensile Properties Compression molded samples displayed few differences

20. Conclusions Significant as-is protein thermal degradation begins ~ 220 °C Extrusion processing can have a large impact on zein properties – Single Screw Extrudate appearance changes across entire spectrum of processing conditions Processing can be well behaved at one set of conditions Color begins to rapidly change above 160 °C NMR suggests general hydrolysis, oxidation, ester formation Protein cross-linking occurring ~ 140 °C Protein cleavage dominates above ~ 180 °C Protein 3° structure rapidly changes at 220 °C followed by 2° structure at 240 °C Tensile strength and elongation begin to decline above 140 °C Compression molding conditions change

21. Conclusions Extrusion processing can have a large impact on zein properties – Twin Screw Color, m.w. and tensile property changes with temp in similar trend to single screw process High color and crosslinking occurs ~60 °C higher Massive hydrolysis not observed Tensile strength loss starts ~ 220 °C Temperature impact on properties observed, less significant than for single screw Residence time Ultimate processing temp of 200 °C possible

22. Conclusions Zein recycling Processing changes required with each pass Minimal color change with each pass M.W. changes with each pass Disulfide bond formation dominant driver Little hydrolysis or permanent crosslinks Protein 2° and 3° structural changes occur Little changes in physical properties Extrusion window for zein relatively large 80-140 °C and even up to 200 °C possible Zein may be recycled

23. Acknowledgments Kelly Utt & Ashley Maness for performing analyses & running extruder. Kathy Hornback for running the extruder Art Thompson, Karl Vermillion, Jon Friesen (ISU) – SEM, NMR and CD analysis