Impact of various extrusion processes on Zein

Gordon W. Selling2015 Biopolymers and Bioplastics Meeting

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 (~70%)a , (~10%)b , (~20%), (g d low) Different solubility

a-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

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

-2.8

-2.1

-1.4

-0.7

0.0

20 70 120 170 220

Temperature (oC)

Hea

t F

low

(W

/gra

m)

a

1

100 200 300 400 5000

20

40

60

80

100

Rem

aini

ng M

ass

(%)

Temperature (oC)

0.0

2.5

5.0

7.5

10.0

Derivative M

ass (%/m

in)

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

Zein Melt Processing – Single Screw Extruder - Single Pass

Extrudate appearance changed with temp

100 °C

160 °C

260 °C

FormulationFeed

throat 1 2 & 3 rpm

Ave. Throughput

(gpm)

Molding Temp

(oC)**

Zein/ 5% H2O 65 90 100 30 8

" " 65 110 120 45 12" " 65 130 140 40 9" " 65 150 160 40 10" " 65 170 180 40 10" " 55* 190 200 40 11" " 55* 210 220 40 10" " 55* 230 240 40 12" " 55* 250 260 40 10" " 55* 270 280 120 9" " 55* 290 300 40 7

Zein/ 10% TEG 65 90 100 80 10 82" " 65 110 120 70 10" " 65 130 140 60 9 93" " 65 150 160 65 13 104" " 65 170 180 60 12 93" " 55* 190 200 65 12 113" " 55 210 220 70 11 88" " 55 230 240 60 10" " 55 250 260 60 10" " 55 270 280 90 10" " 55 290 300 100 6

** Molding time and pressure were 20 min and 3250 pounds per sample respectively

Zone Temp (oC)

* Feed problems required reduction in throat temperature, surging led to difficulties on controlling throughput which became worse as temperature increased

FormulationFeed

throat 1 2 & 3 rpm

Ave. Throughput

(gpm)

Molding Temp

(oC)**

Zein/ 5% H2O 65 90 100 30 8

" " 65 110 120 45 12" " 65 130 140 40 9" " 65 150 160 40 10" " 65 170 180 40 10" " 55* 190 200 40 11" " 55* 210 220 40 10" " 55* 230 240 40 12" " 55* 250 260 40 10" " 55* 270 280 120 9" " 55* 290 300 40 7

Zein/ 10% TEG 65 90 100 80 10 82" " 65 110 120 70 10" " 65 130 140 60 9 93" " 65 150 160 65 13 104" " 65 170 180 60 12 93" " 55* 190 200 65 12 113" " 55 210 220 70 11 88" " 55 230 240 60 10" " 55 250 260 60 10" " 55 270 280 90 10" " 55 290 300 100 6

** Molding time and pressure were 20 min and 3250 pounds per sample respectively

Zone Temp (oC)

* Feed problems required reduction in throat temperature, surging led to difficulties on controlling throughput which became worse as temperature increased

Extrusion Affects on Color

With increased extrusion temperature extrudate color* becomes darker (L), greener (a) and bluer (b) Large change >160 °C

Extrusion Temp L a b as-is 100 C 240 Cas-is 24.2 10.1 21.5100 19.0 9.7 17.7140 17.1 9.5 17.2160 16.9 9.8 17.2180 15.0 3.9 10.7200 15.1 2.4 9.9220 14.8 0.5 9.1240 13.5 -0.3 10.4

* Compression molded samples

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

100 125 150 175 200 225 250

Del

ta E

Extrusion temp (C)

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

Mar

ker

As-

is10

0

120

140

160

180

200

220

240

260

280

300

225 kDa150 kDa100 kDa75 kDa

35 kDa

25 kDa

50 kDa

5 kDa

10 kDa

15 kDa

* DDT - dithiothreitol

SDS-PAGE w/ DDT

Ma

rke

rA

s-is

100

120

140

160

180

200

220

240

260

280

300

As-

isM

ark

er

225 kDa150 kDa

75 kDa

50 kDa

10 kDa

15 kDa

35 kDa

25 kDa

5 kDa

100 kDa

NMR & IR Spectroscopy

300 °C

100 °C

Differences observed in three regions of C13 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)

y = 0.0015x + 1.1704R² = 0.8352

y = 0.0135x - 1.5557R² = 0.9822

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

100 140 180 220 260 300

Rat

io A

bs 1

658/

Abs

153

3

Temperature (C)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1500160017001800

Ab

sorb

anc

e

Wavenumber (cm-1)

As-is

100

240

300

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

y = 0.28x - 316R² = 0.99

y = 5.7x - 1452R² = 0.94

-300

-250

-200

-150

-100

-50

0

50100 150 200 250 300

[Q] l

x 10

2d

eg c

m2 /

dm

olExtrusion Temperature (C)

-300

-250

-200

-150

-100

-50

0

50

260 270 280 290

[q] l

x 10

2,de

g cm

2 /dm

ol

nm

As-is

220

240

300

@ 222 nm – 240 C

Far-UV CD Spectra

-120

-100

-80

-60

-40

-20

0

200 210 220 230 240nm

[q] l

x 1

05 , deg

cm

2 /dm

ol

100

As-Is

160

220

260

280

300

y = 0.295x - 140.1R2 = 0.77

y = 0.925x - 290.9R2 = 0.97

-120

-100

-80

-60

-40

-20

090 140 190 240 290

[q] l

x 10

5 , d

eg c

m2 /

dmol

Extrusion Temperature (oC)

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

Extrusion Temp (ºC)

Molding Temp (ºC)

Tensile Str. (MPa)

Elong. (%)

Modulus (MPa)

100 82 33 11 383140 93 32 11 429160 104 24 7 436180 104 16 4 488200 113 14 4 426220 88 19 5 476

Zein Melt Processing – Twin Screw Extruder - Single Pass

Lower residence time process

Changes observed in extrudate with temperature

180 C

220 C 300 C

Zones->

1 2 3 4 5 6 7 8* rpm Throughput

21 80 90 115 120 175 180 180 150 67

21 85 95 125 130 195 200 200 150 67

21 85 100 135 140 210 220 220 150 67

21 85 105 145 150 225 240 240 150 67

21 85 110 155 160 245 260 260 150 67

21 85 115 165 170 265 280 280 150 67

21 85 120 175 180 275 300 300 150 67

Twin Screw - Color Development

Color change* occurs at higher temp versus single screw process

* Compression molded samples

0

2

4

6

8

10

12

14

100 150 200 250 300

Del

ta E

Extrusion temp (C)

Single Screw

Twin Screw

Temp L* a* b* L* a* b*100 19.4 10.2 19.1 22.1 6.2 14.8120 22.0 6.9 15.1140 17.6 9.6 17.7 22.4 6.7 14.8160 17.5 10.5 17.9 19.5 7.4 16.6180 16.2 4.1 10.5 17.9 7.3 15.9200 15.6 2.4 9.6 23.2 8.2 19.0220 15.1 0.8 9.1 22.3 7.2 19.5240 14.4 -0.6 9.5 17.8 2.1 10.4260 19.8 2.1 10.4280 20.7 1.0 8.4300 18.6 0.9 7.4

Single Screw Twin Screw

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

Protein Markers

(kDa)

Temp - >

140 160 160 180 200 220 240 260 280 300

22515010075503525

1510

5

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)

Extrusion Temp

Compression Mold temp

TS (Mpa) TS s

Elo (%) Elo s

YM (Mpa) YM s

80 93 40 1 13 1 355 23100 93 39 2 13 1 332 31120 93 40 2 13 1 359 17140 93 39 1 11 1 388 9160 104 39 0 12 1 372 12180 104 39 1 13 1 348 24200 116 31 3 9 1 396 35220 116 27 1 8 1 389 32240 116 17 2 5 1 375 15260 116 2 0 3 3 159 76

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.

y = 2.6171x + 20.623R² = 0.9324

0

10

20

30

40

0 2 4 6 8

Tor

qu

e (M

Pa)

Number of Passes

PassScrew RPM

Average Through-Put

(gr/min)

Average Torque (MPa)

Average Pressure (MPa)

1 115 14.5 7.3 0.2

2 15 14.2 26.2 1.1

3 12 15.6 29.5 2.4

4 13 16.0 28.5 2.8

5 12 15.2 34.4 3.9

6 12 14.1 36.9 5.1

7 12 14.9 38.9 5.9

y = 0.95x - 0.7429R² = 0.9926

0

2

4

6

0 2 4 6 8

Pre

ssu

re (

MP

a)

Number of Passes

Zein Extrudate Appearance After 1st pass little change in appearance

First Second Third

# Passes L* a* b* dE

1 20.0 8.2 17.7

2 24.9 7.8 19.7 5.3

3 25.7 7.2 19.1 5.9

4 26.1 6.3 16.7 6.5

5 27.2 5.4 14.3 8.5

6 31.3 5.3 15.2 11.9

7 28.9 4.1 11.4 11.7

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

1 A - Without DTT B - With DTT 2

225150100

7550

35

25

15

10

5

Mar

ker

1 2 3 4 5 6 7 Mar

ker

1 2 3 4 5 6 7

Extrusion Affects on Protein Structure

Near-UV CD spectroscopy - changes in 3° structure observed after 1st-2nd 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)

-200

-150

-100

-50

0200 220 240 260

[q] l

x 1

05m

deg c

m2 /d

mol

Wavelength (nm)

As-is1

23

5

4

7 6

Far-UV CD Molar Ellipticity @ 222 nm (90% EtOH/water)

-200

-175

-150

-125

-100

-75

-1 0 1 2 3 4 5 6 7

[q] l

x 10

5m

deg

cm

2 /d

mol

Number of Passes

sssssssss

Near-UV CD Spectra (90% EtOH/water)

-700

-600

-500

-400

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-200

-100

0

100260 270 280 290

[q] l

mde

g cm

2 /dm

ol

Wavelength (nm)

1

2

0

3 - 7

Near-UV CD Molar Ellipticity @ 262 nm (90% EtOH/water)

-450

-375

-300

-225

-150

-75

0

75-1 0 1 2 3 4 5 6 7 8

[q] l

md

eg c

m2 /

dm

ol

Number of Passes

Recycling Affects on Tensile Properties

Compression molded samples displayed few differences

# Passes

TS(MPa) TS s

Elo (%) Elo s

YM (MPa) YM s

1 30 2 9 1 416 132 29 3 7 1 449 303 26 4 7 1 440 444 27 1 7 1 421 175 26 2 7 1 430 456 28 4 7 1 450 417 31 5 7 1 462 12

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

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

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

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