“During two weeks of vacation you will lose 50% of your non-life supporting IQ

(basically everything above IQ 100)”.

Altai Mountains, Western Mongolia, 2011

Surface rheology of protein - polysaccharide stabilized interfaces

Leiden, 27.9.2011

Varvara Mitropoulos, Patrick Rühs, Peter Fischer

Institute of Food, Nutrition and Health, ETH Zurich, Switzerland

But there is always hope: D.T. Wasan once in Zurich

“All my Ph.D. students working on interfacial

rheology eventually became monks”

Surface coverage: Isotherm controlled (Soluble surfactant)

dA1 G1 g1

dA2 << dA1 G1 = G2 g1 = g2

Surface area A not relevant

Surface coverage, i.e. interfacial tension, is controlled by isotherm

No significant shear rheological responses

Surface coverage: Area controlled (Insoluble surfactant)

dA1 G1 g1

dA2 << dA1 G1 << G2 g1 >> g2

Surface coverage G and Surface pressure p depend on accessible surface area A

Interfacial rheological response in shear and dilatation

Surface coverage: Time controlled (Proteins)

dA1 G1 = 0 g1

After realistic time: dA1 G1 < G2 g1 >> g2

Surface coverage G and surface pressure p depend on adsorption time (non-existing isotherm)

Protein denaturation leads to inhomogeneous morphology of interfacial film

Adsorption overlay dilatational properties (shear less affected)

After infinite time: dA1 G1 < G2 < G3 g1 >> g2 >> g3

Protein interfaces: Effect of bulk concentration

Insufficient bulk concentration C1

Insufficient bulk concentrations (indeed very small concentrations) lead to weak interfacial films if any

Sufficient bulk concentration C1

DARPin (Designed ankyrin repeat proteins) Ni1C (Mw = 10.8 kDa)*

* V. Mitropoulos et al.: Soft Matter 7 (2011) 7612

Air or oil phase

Aqueous phase

Diffusion

Adsorption/ Desorption

Conformation change (denaturation)

Network formation (2D Gels*)

J. Benjamins: Dissertation, Wageningen University (2002) E. Dickinson: J Chem Soc, Faraday Trans 94 (1998) 1657

<<0

Protein interfaces: (Very simple) adsorption model

Surface-active proteins are polyampholytes

and decrease the interfacial tension as a function of

adsorption time

Consequences for (mainly) dilatational interfacial rheology

g(t)

A(t)

Conformational adaptation of protein during interfacial adsorption

Area change influences protein concentration and protein structure at the investigated interface

Miyano K, Abraham BM, Ting L, Wasan DT (1983) J Colloid Interface Sci, 92(2):297

Dilatational interfacial rheology: Adsorption vs. Rheology I

Diffusion-controlled surfactants: e.g. Lucassen-VdT, Loglio, Petrov/Joos

Most protein systems are not purely diffusion controlled: Globular proteins undergo conformation changes at interfaces

POE-20-cetyl ether

Elastic parameter E0= 28.2 mN/m Diffusion relaxation time t0 = 625 s

Ovalbumin (globular protein, pI 4.5-4.75 Mr - 4300, MW - 45000)

Viscoelastic response is corrupted by adsorption kinetics

P. Ruehs et al.: Society of Rheology Meeting, Cleveland (2011)

Fibrils made out of b-lactoglobulin

(for general cooking recipe ask Leonard; our fibers were made according to Jung JM, Gunes DZ, Mezzenga R: Langmuir 26 (2010) 15366-15375)

Dilatational interfacial rheology: Adsorption vs. Rheology II

Steady long-tern adsorption

Oscillation: Interfacial tension is ahead of area change!

B. Warburton: Proceedings of the Conference on Theoretical Rheology, Aberystwyth/UK (1971)

The growth of linear viscoelasticity: Anisotropochrony

During experimental work on the formation of interfacial molecular multilayers it is necessary to consider the concept of linear viscoelastic materials which have properties varying with respect to time. There are, indeed, many other practical examples of such temporal changes of properties, e.g. the setting of concrete, the curing of rubbers and the gelling of starch based table sweets, to mention but a few. Also, in the field of biochemistry, the enzymolysis of proteins and polysaccharides involves changes of viscoelastic parameters where the growth constants are negative. It is suggested that this phenomenon could be called anisotropochrony. The phenomenon is quite distinct from thixotrophy, rheopexy, or dilatancy and it is assumed that the rheological investigation is of such low amplitude that no interference with the intrinsic growth process occurs. The behaviour of an elementary viscoelastic solid, having properties which vary linearly and slowly with time, has already been published for creep conditions. The work has been extended to cover oscillatory conditions, where the rate of growth of the complex modulus is small compared with the period of oscillation.

Protein interface: Summary Surface concentration is controlled by adsorption time (no isotherm)

Protein adsorption and denaturation interferes with rheological response (mainly dilatation)

Denaturation depends on available space (first come – fully denaturation, last to come – squeeze in)

Morphology of adsorption layer is unknown at any time

Mixed interfaces from proteins and biopolymers

Bulk aggregation

Pure interfacial aggregation of proteins

Interfacial aggregation of mixture

Protein – Biopolymer material investigated I

Erni P: Soft Matter 7 (2011) 7586

Protein – Biopolymer material investigated II

Sagis L: Rev. Mod. Phys. (2011) accepted

Dickinson E: FHC 17 (2011) 25

Biopolymers at interfaces: “His Master’s Voice”

Biopolymers stabilized emulsions and foams I

•  Partly used as indirect prove that BP-P mixture aggregate at interfaces

•  Claimed emulsion or foam stabilization could be bulk complexation of BP-P *

•  Droplet and bubble size normally increase due to bulky interfacial aggregates

Emulsion: B-LG – Gum Arabic: Complexation through strong attractive electrostatic interaction (Bouyer E et al. JCIS 354 (2011) 467) Pectin – Gum Arabic: (Nakauma M et al.: FHC 22 (2008)1254) * Pectin – B-LG: Interface structure controlled by bulk properties (Ganzevles RA et al.: Langmuir 22 (2006) 10089) Chitosan – Whey protein: Higher stability by higher steric stabilization (Laplante S et al.: CHP 65 (2006) 479) * i-carrageenan – B-LG: BP-P complexes no not stabilize due to bridging fluculation (Gu YS et al.: JAFC 52 (2004) 3626) * Amylopectin – Casein: Bulk phase separation (de Bont PW et al.: FHC 16 (2002) 127) * Methylcellulose – BSA: Resistance to coalescence related to adsorption layer (Sarker DK et al.: Coll Surf B 12 (1999) 147) *

Biopolymers stabilized emulsions and foams II

•  Partly used as indirect prove that BP-P mixture aggregate at interfaces

•  Claimed emulsion or foam stabilization could be bulk complexation of BP-P*

•  Droplet and bubble size normally increase due to bulky interfacial aggregates

Foam: Xanthan, Guar, k-carrageenan – Egg Albumin: BP-P coacervation (Miquelim JN et al.: FHC 24 (2010) 398) * Alginate, l-carrageenan – Whey protein: Anionic non-surface active BP (Perez AA, AIChE J 56 (2010) 1107) * B-LG – Gum Arabic: Foams are stabilized by electrostatic complexes or coacervates (Schmitt C et al.: Langmuir 21 (2005) 7786) *

Phase separation in biopolymer mixtures

Food term: Biopolymer mixtures Physical term: Colloid - semi-flexible polymer mixtures

Original work: [1] H.G Bungenberg de Jong: Colloid Science, Vol 2, Elsevier, 1949

[2] S. Asakura et al.: J. Chem. Phys. 22 (1954) 1255

Grinberg et al.: FHC: 11 (1997) 145 A. Syrbe et al.: Int. Dairy J. 8 (1998) 179 C.G. de Kruif, R. Tuinier: FHC 15 (2001) 555 V. Tolstoguzov: FHC 17 (2003) 1 S.L. Turgeon et al.: COCIS 8 (2003) 401

Phase separation: Example and basic phase diagrams

gelatine-rich

dextran-rich

Biopolymers are limited co-soluble: At low c due to no entropical penalty At high c in form of phase-separated saturated blends

Segregation Complexation

Binodal

Tie line

Region of compatibility Mixture made from: Polymer (Protein) P1 Biopolymer P2 Solvent S1

Dickinson E: Food Hydrocolloids 17 (2003) 25 A. Syrbe et al.: Int. Dairy J. 8 (1998) 179

Phase separation: Example in 3D

Locus Bean Gum LBG (Carob) & Skimmed Milk Powder (micellar casein)

Schorsch, Jones and Norton: FHC (1999)

Associative phase separation: Charge systems

•  Most frequently (though not only) observed when the two biopolymers have opposite net charge

•  Inducing charges by pH at IP of protein (+ ue charges at protein surfaces) •  Inducing charges by increasing salt concentration (salting in, salting out)

pH

Char

ge

0

+ ue Region where charge on protein and anionic polysaccharide is opposite

Protein isoelectric pH

Protein Anionic polysaccharide

~ pH ~ 5

- ue

Cartoon by Bettina Wolf

Charged systems: Example in 3D

•  Total polymer concentration very low

•  Critical gelling region reached by slow reduction in pH with glucono-δ-lactone.

•  Gel strength maximum where charges on the two polymers were equal and opposite.

•  Some structure formation above the protein isolectric point.

Laneuville SI et al: Langmuir 22 (2006) 7351-7357

pH induced gelation of 2:1 mixture of b-lactoglobulin and xanthan (0.1% total polymer concentration)

B-LG – biopolymer composites: The Patino collection I*

*Any paper by J.M. Rodriguez Patino et al. (e.g. AIChE J. 52 (2006) 2627)

B-LG: isoelectric point 5.1 Progylene Glycol Alginates: Different degree of esterfication Surface active trizma buffer: pH 7 – 7.1, T – 20°C

B-LG – biopolymer composites: The Patino collection II

R. Baeza et al.: AIChE J. 52 (2006) 2627

B-LG: isoelectric point 5.1 Xanthan: Not surface active trizma buffer: pH 7 – 7.1, T – 20°C

B-LG: isoelectric point 5.1 Lamdba-carrageenan: Surface active trizma buffer: pH 7 – 7.1, T – 20°C

B-LG – Biopolymer composites: Dilatational moduli

R. Baeza et al.: AIChE J. 52 (2006) 2627

Cartoon for surface active and non-surface active BP

R. Baeza et al.: AIChE J. 52 (2006) 2627

Competitive adsorption

Complexation

Imcompatibility

B-LG – low methoxyl pectin composites

B-LG: isoelectric point 5.1 Low methoxyl pectin: Mw =15000, Mw/Mn = 2.4 Not surface active Acetate buffer: pH 4.5, ionic strength 9mM

Pure pectin: High negative charged sysytem

B-LG – pectin complexation

B-LG – Pectin composite: Adsorption from bulk

High pectin concentration (0.2 – 6): Negative charge prevents film formation At neutral charges b-LG film properties dominates

B-LG – Pectin composite: Sequential adsorption

Sequential adsorption of pectin to existing PAL forms different film.

B-LG – Pectin composite: Adsorption cartoon

Bulk adsorption at high pectin concentration: Negative charge prevents film formation

Pre-adsorbed b-LG film: PAL dominates the film properties

Adsorption cartoon revisited (with help of some neutrons)

Neutron reflectivity measurements can provide (i) layer thickness, (ii) multi-layer structure, and (iii) layer roughness.

R.A.Ganzevles et al.: JCIS 317 (2008) 137

B-LG – pullulan composite: Role of charge density

B-LG: isoelectric point 5.1 Pullulan (glucose trimer unit): Mw = 150000 g/mol Not surface active Solution: pH 4.5

Pure pectin: High negative charged sysytem

Increased Charge density: Bulk complexation promoted

Increasing concentration, i.e., charge density of pullulan promotes bulk complexation: Weak interfacial film.

Low charge ratio (high pullulan concentration, i.e. large negative charge versus low b-LG concentration) features weak interfacial film.

At neutral conditions (charge ratio 1) a moderate film formation.

Summary: See pectin cartoon!

Sequential adsorption

At least one of the systems (sim-system) is thermodynamical blocked.

Gum Acacia: A protein – biopolymer composite by nature

Gum Arabic and modified starch stabilized interfaces and emulsions

Question: Interfacial properties of beverage emulsions containing gum arabic or modified starches as emulsifier

• Gum arabic is widely used as emulsifier and stabilizer in beverage emulsions.

• Not as bad as diamonds, but shortage through drought & civil war lead to efforts to replace it by mesquite gum or hydrophobically modified starch (octenyl succinate substituted starch).

P. Erni et al.: BM 8 (2007) 3458

Plant exudates

Exudate gum plants • Acacia Senegal or Acacia Seyal • Tragacanth • Karaya (Sterculia tree) • Mastic Gum (Pistacia lentiscus) • …

Production • Acacia Senegal or Acacia Seyal trees are cut • Exudate picked, kibbled, sieved • Dissolved, decanted, centrifuged or filtered • Pasteurization • Spray- or roller-dried

Verbeken D et al.: Appl. Microbiol. Biotechnol. 63 (2003) 10 Islam AM et al. Food Hydrocolloids 11 91997) 493

OIL

WATER

Gum Arabic - a protein/polysaccharide hybrid

• Wattle: High molecular arabinogalactan-protein complex (AGP, 1 - 10%), Responsible for emulsion stabilizing properties • Blossoms: Low molecular mass arabinogalactan polysaccharide fraction, protein deficient (AG, 89 - 98%) • Glycoprotein fraction containing the rest of the protein (GP, < 1%)

*Renard et al.: Biomacromolecules 7 (2006) 2637 Gaspar Y et al.: Plant Molecular Biology 47 (2001)161

Working principle and deficiencies in beverage emulsions • Emulsify flavor oils (orange) under acid conditions • Surface activity is low in comparison to other food proteins • 30% of dissolved GA associates with interface

Wattle Blossom Model:

Composition of Gum Arabic

Acacia Gums consist of a continuum of molecular species with varying protein:polysaccharide ratio, MW, and charge

Accessible by: Hydrophobic interaction chromatography (HIC), size exclusion chromatography, Multi-angle LS, UV, IR spectroscopy, CD and titration Renard et al.: Biomacromolecules 7 (2006) 2637

AG AGP GP

CH2 Amids

Gum Arabic and modified starch: Structure build-up I

General: • MS achieves equilibrum condition faster • Equilibrium interfacial tension is lower for MS • Absolute decrease is larger for GA

= 12.2 mN/m

= 8.9 mN/m

Both not ideal: • High Mw • Conformational change • Irreversible adsorption

P. Erni et al.: BM 8 (2007) 3458

Gum Arabic and modified starch: Structure build-up II

• The interfaces are strongly cross-linked (G‘>>G‘‘)

• Interfaces (a/w and o/w) show a viscoelastic solid like behavior

• GA could be used at a/w-interface

• Film formation time is important.

Rheological response during film formation:

P. Erni et al.: BM 8 (2007) 3458

Gum Arabic and modified starch: Viscoelasticity

= 1%

GA G’ > G’’ Scale-free (rubber-like, soft glass)

MS G’’ >> G’

GA G(t) = St-n (S ≈ 0.1, n ≈ 0.2, i.e. close to bulk gel values, relaxation time > 100 s

MS very week relaxation

P. Erni et al.: BM 8 (2007) 3458

Gum Arabic and modified starch: Shear deformation

GA: LVE ≈ 1 - 2%, indicates a rigid, inflexible structure, breakdown MS: viscous film

Creep flow

Fracture

Shear-thinning of ruptures layer

P. Erni et al.: BM 8 (2007) 3458

Gum Arabic and modified starch: Dilatational moduli

IE*I = d / (dA/A0)

• Interfacial dilatation response for MS is weak but still considerable!*

• Stronger film build by GA

t = 2 h

*Contrast to very weak shear response: - shear probes intermolecular interaction - dilatation show ability to change in concentration (ad-, desorption)

P. Erni et al.: BM 8 (2007) 3458

Thank you

Richard Nixon (quoted by Thomas Pynchon in “Gravities Rainbow”,

performed by Ernest in Sherman’s Lagoon)