Structuration and mechanical properties of gels made from gluten proteins

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Structuration and mechanical properties of gels made

from gluten proteins

Mohsen Dahesh, Amélie Banc, Agnès Duri-Bechemilh, Marie Helene Morel,

Laurence Ramos

To cite this version:

Mohsen Dahesh, Amélie Banc, Agnès Duri-Bechemilh, Marie Helene Morel, Laurence Ramos.

Struc-turation and mechanical properties of gels made from gluten proteins. International soft matter

conference (ISMC), Sep 2013, Rome, Italy. 2013. �hal-01601702�

Structuration and Mechanical Properties of Protein Gels

Made From Gluten Proteins

Mohsen Dahesh

1,2

_{, Amélie Banc}

1

_{, Agnès Duri}

2

_{, Marie-Hélène Morel}

2

,

_{Laurence Ramos}

1

1

_{Laboratoire Charles Coulomb UMR5221, CNRS and Université of Montpellier 2, 34095 Montpellier}

France

2

_{Ingénierie des Agropolymères et Technologies Emergentes UMR 1208, CIRAD, INRA, Montpellier}

SupAgro, 34000 Montpellier, France

Wheat gluten proteins are among the most complex protein networks in nature, due in particular to their poor solubility in water and to their viscoelastic behavior. Gluten networks are often considered as transient networks comprising extensible biopolymer segments of flexible or semiflexible chains between junction points1_{. However, the exact} structure of the network, the nature of the junction points and the way it get structured under shear remain to be clarified. Here we report the visco-elastic behavior of model systems composed of gluten proteins near gelation. We build model systems by dispersing in ethanol-water mixtures two major protein groups, gliadins and glutenins, that we have purified from gluten. Rheological properties show a slow evolution over time scales of the order of days of the linear frequency dependence complex modulus of the samples, with a concentration-dependent liquid to solid transition. Interestingly, we find that all data acquired at different protein concentrations and different times after sample preparation can be scaled onto a master curve showing a cross-over from a soft solid behavior at low frequency to a visco-elastic fluid at high frequency. Rheological data are completed by scattering experiments in order to elucidate the complex structure of the materials. For gel samples, the scattering profiles display at small length scales features typical of polymer and evidences at larger length scale a fractal structure that we interpret as being due to the highly disordered state of the junction points. Biochemical assays are also performed to elucidate the origin of the sample aging.

Background

•Gluten can be defined as the rubbery mass proteins that remain when wheat dough is washed by water to discard starch granules and water-soluble constituents.

•Gluten proteins play a key role in baking quality of wheat product by conferring water absorption capacity, cohesivity, visco-elasticity on wheat dough.

•Gluten contains hundreds of protein components which are present either as monomers or polymers. These proteins can be classified mainly by two broad groups: gliadin and glutenin

•Gluten is like a ‘two component glue’, in which gliadins can be understood as a ‘plasticizer’ or ‘solvent’ for glutenins. A proper mixture of both fractions is essential to impart the viscoelastic properties of wheat dough and the quality of the end product2_.

What is gluten?

Abstract

Viscosityof wheat dough?

2. Polymeric glutenins

(150,000<Mw<8,000,000 g/mol)

Elasticity of wheat dough?

Motivation

Wheat is the third most-produced cereal in the world after maize and rice. Wheat is mostly used in food industry to make product like bread which is prepared by wheat dough baking.

Gluten plays a key role in the bread making properties of wheat dough and gives the unique visco-elastic properties of wheat dough. Many efforts has been done to reveal structuration and mechanical properties of wheat dough but there is not a firm answer for the moment.

Because of complexity of gluten composition we decided to study the structure and visco-elasticity of purified fractions.

Model systems made from gluten proteins

References

1. Trevor S. K. Ng et al, J. Rheol. 2008, 52(2), p417-449 2. H. Wieser et , Food Microbiology 2007, 24, p115–119 3. S.Uthayakumaran, et al , Rheol Acta , 2002, 41: p162–172 4. A. N. Falcao, Macromolecules, 1993, 26, p5350-5364

5. Neutron, X-rays and Light: Scattering Methods Applied to Soft Condensed Matter Edited by P.Linder & Th. Zemb, O Glatter - 2002 - Amsterdam: North-Holland.

6. V.Trappe, et al, Macromolecules 1997,30,p2365-2372.

Gluten dispersed In Ethanol/water (50% v/v) C≈ 0.1wt%

Centrifugation _overnightCooling at 4 C

Non-soluble proteins

Model system A

Model system A+B

107 106 105 104 103 MW (g/mol) Model system A Gliadin 107 106 105 104 103 MW (g/mol) Model system A+B Glutenin Gliadin

To study gluten complex system, a purification protocol was developed from gluten. Two model systems were obtained.

Rheology of model system A

100 101 102 10-3 10-2 10-1 100 101 102 103 G' G'' 23% G' G'' 28% G' G'' 35% G' G'' 43% G' , G' ' (P a ) frequency (rad/s) 1 0,8

Rheology of model system A+B

10-2 10-1 100 101 102 103 101 102 103 G' G'' G', G '' ( Pa) Strain % 10-1 100 101 102 101 102 G' G'' Day3 G' G'' Day7 G' G'' Day25 G' , G' ' (P a ) frequency (rad/s) 10-2 10-1 100 101 102 103 10-1 100 101 102 100 101 10-1 100 G' G'' Day3 G' G'' Day7 G' G'' Day25 b* [G ', G '' ] a*[frequency] b a 10-1 100 101 102 100 101 102 G' G'' 18,6% G' G'' 20% G' G'' 22% G' G'' 24% G' , G' ' (Pa) frequency (rad/s) DAY7 10-1 100 101 102 103 104 105 100 101 102 103 104 100101102103104 10-1 100 101 102 G' G'' 18,6% G' G'' 20% G' G'' 22% G' G'' 24% * [G', G'' ] *[frequency] 10-4 10-2 100 102 104 106 10-1 101 103 105 10-1101103105 10-1 101 103 or b* [ G' , G'' ] a or *[Frequency] G'0 or b a or 0 10 20 30 40 50 60 10-3 10-1 101 103 18,6% 20% 22% 24% 24% 26% 28% 30% 36% 40% G'0 (Pa)

Aging time (day)

01020304050607080 1E-3 0,01 0,1 1 G'0 /G' Aging time /t* 18 20 22 24 26 28 30 32 34 36 38 40 42 0 1 2 3 4 t* (day) % 18 20 22 24 26 28 30 32 34 36 38 40 42 -4 0 4 8 Ln (G' Pa ) 10-3 10-1 101 103 G' (Pa) G' =(3.5x10-12) x exp(- x ) Structural Study

Small angle X-ray scattering Model system A+B

10-1 100 10-1 100 101 102 6% 28% I (q) Arbit rary unit q (nm-1) q-5/3 q-2 q-5/3 10-1 ₁₀0 10-1 100 101 102 28% I( q) arbit rary unit q (nm-1) q-2 q-5/3 10-1 2x10-1 3x10-1 4x10-1 5x10-1 1 1,5 2 2,5 3 3,5 4 S [nm ] C (g/ml) C-0,8

Protein Purification Protcol

Model system Protein fractions [%] Glutenins Gliadins

A 6.5% 93.5%

A+B 55.5% 44.5%

Rheological properties of Model system A was done by measuring complex moduli G΄( storage ) and G˝ (loss) in the linear regime. It shows viscous behavior (Newtonian) untill certain concentration. After Ф=43% viscolelastic fluid behavior can be observed.

Increasing concentration

Pure gliadin (A) dispersion in EtoH/water 40%v/v

Increasing concentration

(Gliadin+Glutenin) dispersion in EtoH/water 50% v/v

Turbid Opaque Transparent

Transparent Transparent Transparent

Very large range linear regime compared with wheat dough (from 15343 to 10-3_{) and gluten (3x10}-2_).3

Scaling

Collapsed master curve showing scaled G΄(ω,t) (closed symbol) and G˝ (ω,t) (open symbols) as functions of the scaled frequency. Inset shows the linear relationship between scaling factors: (a and b)

=>Self-similarity in the structure of the network at

different agingtimes

Scaling

Measuring G΄(ω,Ф),G˝(ω,Ф) shows liquid-solid transition (gel point) increasing concentration at given aging day.

Collapsed master curve showing scaled G΄(ω,Ф) (closed symbol) and G˝(ω,Ф) (open symbols) as functions of the scaled frequency..Inset shows the linear relationship between scaling factors: (α and β)

Collapsed master curve showing scaled G΄(ω,t,Ф) (closed symbol) and G˝(ω,t,Ф) (open symbols) as functions of the scaled frequency.Inset shows the linear relationship between scaling factors: ( a , α and b, β)

Low-frequency elastic modulus (G0΄) versus aging time

t* charateristic time of gelation Exponential behavior of G΄∞ Scaling

for dilute regime I(q)≈q-5/3

Self-avoiding walk behavior For semidilute regime I(q)= ξS mesh size of entanglement network D fractal dimension at large scale

Increasing concentration

SAXS data can be fitted with a semidilute polymer solution model (blob model4,5₎

ξS

•Length scales ≤ ξS= dilute solution behavior for the individual chains.

•Length scales ≥ ξS Semidilute polymer behavior .

1800 1700 16001500 1400 1300 1200 0,0 0,5 1,0 1,5 2,0 168016401600 0 1 2 DAY1 DAY3 DAY8 DAY11 No rmalise d ab so rban ce % wavenumber (cm-1) Amide l Amide l

Aging effect study (model system A+B )

Infrared spectroscopy

No change in the secondary structure of the model systemA+B with time

Small angle X-ray scattering

10-2 ₁₀-1 ₁₀0 10-1 100 101 102 Day 2 Day 20

I(q) arbitrary unit

q (nm-1) 22%

No evolution of SAXS spectra in the range studied. Measurement at q<(0.037nm-1)is required.

Conclusion & outlooks

10-5 10-4 10-3 10-2 0,0 0,2 0,4 0,6 45 C=6mg/ml G 2( (s) Fit by single exponential

0,01 0,02 0,03 e-5 e-4 e-3 e-2 e-1 45 C=6mg/ml G2 ( ) (s) Slope Dq2 Cumulant 0,00000 0,0002 0,00040,0006 0,0008 200 400 600 800 1000 1200 6mg/ml 1/s ) q2(nm-2) Diffusion Constant 0,01 0,02 106 107 108 DAY 2 DAY16 DAY57 Re x/KC (g/mol) q(nm-1)

Very dilute dispersion of Model system A+B

Dynamic light scattering

1 2 3 4 5 6 7 8 120 140 160 180 200 220 DAY2 DAY16 DAY57 Rg (nm) C (mg/ml) Measuring G΄(ω,t), G˝(ω,t) Shows liquid-solid transition (gel point) with time

•Monodisperse (γ=0.015 Polydispersity) •RH=100 5nm

•Not compact object •Large q behavior: internal mode of polymer chains6

Static light scattering

There is aggregation of the very dilute dispersion with time. It finally leads to macroscopic phase separation.

Two model system developed from gluten proteins Model systemA :

Mechanical properties shows Newtonian behavior. Model systemA+B:

• Mechanical properties shows a concentration and time dependent liquid-solid transition (gelation). Moreover frequency-dependent complex moduli (G΄,G˝) can be scaled onto a master curve.

=> Self-similarity of the model system with different concentrations and aging times.

• G΄ has exponential dependence on Ф untill certain concentration

• Dilute regime structure:

• monodisperse objects with internal dynamic mode (self avoiding walk in good solvent)

Semidilute regime structure

The model system can be described by a semidilute polymer model at small scale and fractal structure at large scale

Outlooks

1. What is respective role of the gliadin and glutenin in the Model systemA+Bgelation?

2. Is it helpful to study mechanical properties of mixture of model systemAandA+B?

3. What is the structural origin of aging in model systemA+B? 4. Shear rheology of model systemA+Bversus concentration

and time is also helpful for structuration study.

Self-similarity in the structure of the network at different concentrations Time evolution Titl Titl compar ANALY SIS

Cooking OR Washing with

water