Protein Interactions / assemblies and subsequent properties. STLOpenday

Croguennec, Thomas

Famelart, Marie-Hélène

English

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Protein Interactions / assemblies and subsequent
properties
Thomas Croguennec, Marie-Hélène Famelart
To cite this version:
Thomas Croguennec, Marie-Hélène Famelart. Protein Interactions / assemblies and subsequent prop-
erties. STLOpendays, Institut National de Recherche Agronomique (INRA). UMR UMR INRA /
AgroCampus Rennes : Science et Technologie du Lait et de l’?uf (1253)., Mar 2019, Rennes, France.
￿hal-02097220￿
Marie-Hélène FAMELART / Thomas CROGUENNEC 
Interactions - Structures - Functionalities
STLOpen Days
19-21 March 2019
Protein Interactions / assemblies 
and subsequent properties 
STLOpen Days
19-21 March 2019
2
• New trends in Europe driven by consumer’s expectations
• More natural and healthier food products
• 100% milk products 
(without non-dairy additives)
• Innovative products
(new uses and consumption habits)
General introduction
• Additives are currently used to control dairy product quality 
(texture, heat stability, phase separation,…) 
• Milk proteins are customizable into different assemblies 
that exhibit different properties (water holding capacity, heat 
stability,…) than native proteins → could be used to replace 
partly or totality food additives in dairy products
PROFIL  
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3
Dairy manufacturers:
• add value to dairy compounds 
(e.g. aggregated whey proteins)
MILK
CHEESE
(20 millions tonnes)
WHEY
♦ 10% of the milk volume
♦ >80% of the milk proteins 
(caseins)
♦ 90% of the milk volume
♦ <20% of the milk proteins 
(whey proteins)
Whey proteins : Large amount available around the world 
General Introduction
From waste stream to value-added,
« gutter-to-gold » Smithers, 2008
- Exceptional biological value 
(amino acid composition)
- Ligand carriers (vitamins, 
minerals, fatty acids)
- Bioactive proteins and 
peptides
Excellent for 
human nutrition
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1. Whey protein aggregates for controlling emulsion 
heat stability and texture
2. Whey protein aggregates as texturizing ingredients
Topics of this presentation
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Introduction: Whey protein aggregates for controlling emulsion 
heat-stability and texture
• Whey proteins : - natural emulsifiers (adsorb to oil/water interface)
- heat-sensitive proteins (heat treatments are required to 
extent food product shelf life) 
→ heat sensitive emulsions 
→ texturized (gelled) emulsions
Heat stability of whey protein emulsions (pH 7)
Stable/fluid
Fluid emulsions: Protein 
amount too low to build 
a continuous network
Oil droplet
Denatured 
whey proteins 
Unstable/Gelled
Oil droplet
Non-adsorbed 
whey proteins 
Adsorbed 
whey proteins 
Gelled emulsions : Network of 
proteins entrapping oil droplets
Gelled emulsions obtained by 
using non-dairy gelling agents
fluid emulsions obtained by using
non-dairy stabilizing agentsDairy industries
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• To design whey protein emulsions at high protein concentration 
(>4%) that are fluid after heating?
• To design texturized (gelled) emulsions at low whey protein 
concentrations (<3%)? 
Research Questions
How to control the texture of whey protein emulsions in a large 
range of protein concentrations without non-dairy additives?
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How to design whey protein emulsions at high protein concentrations 
that are fluid after heating in the absence of non-dairy additives?
Strategy
Stability of
the continuous phase
(replace native whey
proteins by aggregated
whey proteins)
1
Stability of the oil 
droplets (replace native 
whey proteins by caseins)
2
Competition for the 
oil droplet surface
3
Adsorbed proteins
(caseins)
Protein in the continuous phase
(dense and large whey protein
aggregates)
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Thermal Stability of the emulsions
Caseins (%)
W
h
e
y
p
ro
te
in
a
g
g
re
g
a
te
s
(%
)
Caseins
Whey
proteins
8
How to design whey protein emulsions at high protein concentrations 
that are fluid after heating in the absence of non-dairy additives?
Results
Critical casein
concentration
Observation at:
Oil droplet scale
Interface scale
(after 1 min of 
heating at 120°C)
(after 30 min of 
heating at 120°C)
3
4
5
6
7
8
9
0.0 0.2 0.4 0.6 0.8 1.0
Marie Chevallier, PhD thesis
STLOpen Days
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Casein Oil droplet
How to design whey protein emulsions at high protein concentrations 
that are fluid after heating in the absence of non-dairy additives?
- by adding sufficient amount of caseins to cover oil droplet surface (control of 
the size and stability of the oil droplets)
→ estimated value: mass of oil (g) × specific surface (m2/g of oil) × protein interfacial load (g/m2) 
(protein interfacial load ∼ 2mg/m2 for caseins) 
Main findings
Casein
Whey proteins aggregates in the 
continuous phase of the emulsions
Oil droplet
- By selecting large and dense whey protein aggregates (small number, low
interfacial adsorption rate and low reactivity on heating)
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How to obtain texturized (gelled) emulsions at low whey protein 
concentrations without non-dairy additives ? 
Strategy
Connect oil droplets 
with whey proteins
aggregates (low density)
1
Control inter-droplet 
distance (oil content/ 
homogenization pressure)
2

H
o
m
o
g
e
n
iza
tio
n
 
p
re
ssu
re
Caseins for stabilizing oil droplets interface
0.01
0.10
1.00
10.00
100.00
0 10 20 30 40 50
In
te
r-
d
ro
p
le
t
d
is
ta
n
ce
 (
µ
m
)
Oil content (%)
Whey protein aggregates used as connectors
(low density/elongated aggregates → factal aggregates)
Size of the whey protein aggregates
≈
inter-droplet distance 
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Results
How to obtain texturized (gelled) emulsions at low whey protein 
concentrations without non-dairy additives ? 
0
0.5
1
1.5
2
0 1 2 3
Casein (%)
W
h
e
y
p
ro
te
in
a
g
g
re
g
a
te
s
(%
)
Unstable
FluidGel Viscous
Emulsion texture changes with the oil 
droplet surface composition
Observations at:
Emulsion scale
Interface scale
Thibault Loiseleux, PhD thesis
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12
How to obtain texturized (gelled) emulsions at low whey protein 
concentrations without non-dairy additives ? 
- Select aggregates of low density (fractal aggregates of whey proteins)
- Use whey protein aggregates as « connector » at the surface of the oil droplets (size of 
the aggregates ∼ distance between oil droplets)
- In combination with the homogenization pressure, use caseins as emulsifiers to control 
the size of the oil droplets (distance between oil droplets)
« connected » oil droplets
Whey protein aggregates
(= connecting agent)
Caseins (with the homogenizing
pressure, they control the oil droplet size 
and the distance between oil droplets)
The number of « connectors » defines the texture of the 
emulsions (gel, viscous, fluid)
Main findings
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Introduction: whey proteins as texturizing ingredients
Whey proteins: • Heat-induced aggregates produced by heating 
treatments in solution for increased functional 
properties
• Dry heating or heating a protein powder: process used 
for white egg powders for increasing foaming, 
emulsifying and gelling properties (Kato et al. 1989), 
• Dry heating  of dairy powders: less studied and not 
used 
• Dry heating: a way to produce other changes of whey 
proteins 
 Limited diffusion of solutes = less aggregation
 Traces or addition of lactose, degradation products of 
Maillard intermediates such as dicarbonyl 
 crosslinks
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How to custom protein assemblies with targeted sizes?
How to custom their functional properties such as their ability 
to entrap large amounts of water and deliver high viscosities?
• to replace food texturizing additives
Research Questions
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15
How to custom protein assemblies with targeted sizes?
pH 9.5
Drying Dry heating Suspension
100 °C
200 µm30 µm
Powder Dry-heated
powder in 
suspension
Whey 
proteins
Elise Schong, PhD thesis
STLOpen Days
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16
pH 9.5
Drying
100 µm
50 µm
Whey 
proteins
Powder
• Dry heating  powder no longer soluble
• Crosslinks between proteins
• Microparticles have a size and shape related to 
the size of the powder before dry heating 
Dry heating Suspension
100 °C
Dry-heated
powder in 
suspension
How to custom protein assemblies with targeted sizes? 
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17
How to custom their functional properties such as their ability to entrap 
large amounts of water and deliver high viscosities? 
• Yield of transfer of whey proteins in microparticles with:
 time of dry heating
 addition of lactose (0.2, 0.3 and 0.5 g/10 g protein)
• 1 g of powder can lead to 0.9 g of microparticles
• 1 g of powder leads to around 15 g of wet particles
• The water content is around 95% of the wet weigh of microparticles
• Due to the porous structure of powders
0.0
0.2
0.4
0.6
0.8
1.0
0 5 10
D
ry
 y
ie
ld
 (
g
 d
ry
 
m
ic
ro
p
a
rt
ic
le
s/
g
 p
o
w
d
e
r)
Time of dry heating (h)
Lac 0.2
Lac 0.3
Lac 0.5
0
5
10
15
20
25
0 5 10
W
e
t 
y
ie
ld
 (
g
 w
e
t 
m
ic
ro
p
a
rt
ic
le
s/
g
 p
o
w
d
e
r)
Time of dry heating (h)
Lac=0.2
Lac=0.3
Lac=0.5
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18
How to custom their functional properties such as their ability to entrap 
large amounts of water and deliver high viscosities? 
• Porous structure of powders  Microparticles entrap a large amount of water
• Ability of microparticles to swell in the water phase: by a factor around 4
Time after water addition
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19
How to custom their functional properties such as their ability to entrap 
large amounts of water and deliver high viscosities? 
D
ry heating
=
 cross-links
DH powder
Proteins
+ aqueous phase
swelling
Repulsions between 
proteins in the microparticle
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20
How to custom their functional properties such as their ability to entrap 
large amounts of water and deliver high viscosities? 
Spray dried powder: 23 g macroparticles/kg suspension
0.0001
0.001
0.01
0.1
1
10
100
1000
0.01 0.1 1 10 100 1000
V
is
co
si
ty
 (
P
a
.s
)
Shear rate (s-1)
Simplesse100
24h DH
72h DH
48h DH
36h DH
• Very high viscosity as compared to micro-particulated whey proteins (d[4,3]~10 µm)
• Viscosity decreases with increased dry heating duration
 reduced d[4,3]  
 reduced water content
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• Mechanism of formation and nature of the protein crosslinks is
still speculative
• We can form microparticles only at pH > 8.0
• The longer the whey protein solution is stored at pH
9.5, the higher the amount of microparticles
• Role of:
 alkaline pH  protein denaturation? Increased exposure
of lysine residues?
 lactose  Degradation via Maillard reactions? or
degradation products of lactose at high temperature?
Is still under study.
Conclusions
pH 9.5
Whey proteins
STLOpen Days
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22
• Whey protein aggregates: high diversity of structures  High diversity
of functionalities
 Large and dense aggregates (microgels) → heat-stability
(solutions/emulsions)
 Low density aggregates (fractal) → functionality dependent on the size
o Small fractal aggregates : cold-gelation (CaCl2 or acidic pH), limited
syneresis
o Medium fractal aggregates : connectivity between droplets, texture
o Large fractal aggregates : increase viscosity
 Very large porous aggregates (microparticles) → water entrapment,
texture
• Whey protein aggregates can be produced at the industrial scale
(poster N°4 - Domitille de Guibert: “Designing innovative technological
routes for the production and stabilization by drying of protein fractal
assemblies”).
General conclusions
STLOpen Days
19-21 March 2019
23
• Chevallier, M., Riaublanc, A., Cauty, C., Hamon, F., Rousseau, F., Thevenot, J., Lopez, C., Croguennec, T., 2019, 
The repartition of whey protein microgels and caseins between fat droplet surface and the continuous phase 
governs the heat stability of emulsions. Colloids and surface A, 563, 217-225.
• Schong, E., Famelart M.H., 2019, Influence of lactose on the formation of whey protein microparticles obtained 
by dry heating at alkaline pH. Food Hydrocolloids, 87, 477-486.
• Chevallier, M., Riaublanc, A., Lopez, C., Hamon, P., Rousseau, F., Thevenot, J., Croguennec, T., 2018, Increasing 
the heat-stability of whey protein-rich emulsions by combining the functional role of WPM and caseins. Food 
hydrocolloids, 76, 164-172.
• Kharlamova, A., 2017, texture des matrices laitières avec des agrégats de proteins laitières. PhD thesis, 
Université du Maine, France.
• Loiseleux, T., Rolland-Sabate, A., Garnier, C., Guilois, S., Croguennec, T., Anton, M., Riaublanc, A., 2018, 
Determination of hydro-colloidal characteristics of milk protein aggregates using Asymmetrical Flow Field-Flow 
Fractionation coupled with Multiangle Laser Light Scattering and Differential Refractometer (AF4-MALLS-DRi). 
Food hydrocolloids, 74, 197-206
• Schong, E., Famelart M.H., 2018, Dry heating of whey proteins leads to formation of microspheres with useful 
functional properties. Food Research International, 113, 210-220.
• Schong, E., Famelart, M.H., 2017, Dry heating of whey proteins. Food Research International, 100, 31-44.
• Famelart, M.H., Schong, E., Croguennec T., 2018, Dry heating a freeze-dried whey protein powder: Formation of 
microparticles at pH 9.5. J. Food Eng., 224, 112-120.
• Chevallier, M., Riaublanc, A., Lopez, C., Hamon, P., Rousseau, F., Croguennec, T., 2016, Aggregated whey 
proteins and trace of caseins synergistically improve the heat stability of whey protein-rich emulsions. Food 
hydrocolloids, 61, 487-495.
• Patent WO2018185192 (A1) - 2018-10-11 - « Stabilized protein aggregate particules and process for the 
preparation of said particles », M. H. Famelart, T. Croguennec, P. Schuck, E. Schong, T. Sevrin.
Publications in relation to the study
THANK YOU FOR
YOUR ATTENTION
MERCI
Please visit http://www.rennes.inra.fr/stlo_engSTLOpen Days
19-21 March 2019


Protein Interactions / assemblies and subsequent properties

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Protein Interactions / assemblies and subsequent properties

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