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Yves LE LOIR - STLO
STLOpen Days19-21 March 2019
Welcome
Kinetics of heat-induced denaturation of whey proteins and characterization of protein aggregates in
model infant formulas
Amira HALABI Amélie DEGLAIRE Marie HENNETIER Frédéric VIOLLEAU Said BOUHALLAB Didier DUPONT Thomas CROGUENNEC
8th ISFRS 18th June 2019
ETH Zurich
France
Science &
technology of milk &
eggs
Brittany
STLOpen Days19-21 March 2019
1
Infant milk formulas (IMFs)
Goal for the IMF formulation: to mimic the human milk composition, in particular the protein profile
Way of feeding of world infants0-5 months of age in 2018
41%
59% UNICEF global databases, 2018
Benefits of breastfeeding
WHO UNICEF, 2018
STLOpen Days19-21 March 2019
2
Milk proteins
The CN:WP ratio and the WP composition have to be modified for the IMF formulation
Two categories of milk proteins
Calcium phosphate
κ-casein
αS1-, αS2-, β-, κ- caseins50-600 nm
Glantz et al., 2010
Tolkach et al., 2007; Kulozik et al., 2011; Vogel, 2012
Caseins (CN)
Whey proteins (WP)
60%
C40%
20%
80%
40%
60%
Milk protein composition
Chatterton et al., 2013
0
5
10
15
20
25
30
35
40
g o
f p
rote
ins
/ L
of
liqu
id m
ilkCaseins
α-LA β-LG LF Others
WP
CN
Others (Immunoglobulins Igs, Serum albumin, … )
α-lactalbumin(α-LA)
β-lactoglobulin(β-LG)
Lactoferrin(LF)
STLOpen Days19-21 March 2019
3
Consequences
o Protein digestibility?
o Protein structural modifications
Needs
o Extend shelf life
o Microbiological safety
Heat treatments of IMFsWhy heat treatment?
Lack of knowledge of the heat treatment impact on the IMF proteins
Liquid preparation
Adapted from Human Milk Biochemistry and Infant Formula Manufacturing Technology, 2014
Skimmed bovine milk
Processing manufacture of IMFs
Liquid IMFs(1.3% of proteins)
DilutionSterilization
Dry ingredients(Lactose, WP isolates, minerals)
PasteurizationHomogenizationConcentration by evaporation
Pasteurized concentrate
Fat
STLOpen Days19-21 March 2019
4
Objectives and strategy
Heat treatment impact on the protein fraction of IMFs mimicking
the human milk protein profile
Heat treatments
Prediction of the WP denaturation rate and the protein structure in heated IMFs, based on their WP composition and the heat treatment parameters
Part 1
Kinetics of heat-induced
denaturation of WP
Part 2
Characterization of heat-
induced protein aggregates
Control IMF CN:WP 40:60
Without modification of the proportion of the bovine milk
whey proteins
LF+ IMF CN:WP 40:60
LF quantity closes to that of human milk
LF+ ALA+ IMF CN:WP 40:60
LF and α-LA quantities close to those of human milk
Model IMFs
STLOpen Days19-21 March 2019
5
Heating temperatures 807572.5 77.57067.5 °C
Part 1: Kinetics of heat-induced denaturation of WP
Skimmedbovine milk
Caseins
α-LA β-LG LF
14 10
39
46
32
618 15
40 40 40
0
20
40
60
80
100
Control LF+ LF+ ALA+ IMFs
% o
f to
tal p
rote
ins
60% WP
40% CN
CN
WP
IMFs
1.3% of proteins
IMFs
Lactose Minerals ProteinsWP isolate
α-LALF
STLOpen Days19-21 March 2019
6
Strategy
Part 1: Kinetics of heat-induced denaturation of WP
Glass tube
Water bath
IMF
Heat treatment
Acetic acid / Sodium acetate Buffer
Precipitation of the denatured WP at pH 4.6
40°C – 2 min14,000xg – 20 min – 25°C
Supernatant Native WP
Quantification of native WP Reverse phase - high performance liquid
chromatography
STLOpen Days19-21 March 2019
7
Frac
tio
n o
f n
ativ
e α
-LA
Holding time (min)
α-LA
Frac
tio
n o
f n
ativ
e LF
Holding time (min)
LF
Frac
tio
n o
f n
ativ
e β
-LG
Holding time (min)
β-LG
No modification of the heat denaturation rate of LF by the presence of the other WP
LF+ ALA+ IMF
Control IMF
LF+ IMF
Part 1: Kinetics of heat-induced denaturation of WPKinetics of thermal denaturation of WP in IMFs at 75°C
Decrease of the heat denaturation rate of α-LA with the β-LG content reduction
Increase of the heat denaturation rate of β-LG in the presence of LF
LF+ ALA+ Control LF+ Caseins
α-LA β-LG LF
CN
WP
Control IMF
LF+ IMF
LF+ ALA+ IMF
LF+ IMF
STLOpen Days19-21 March 2019
8
Part 2: Characterization of heat-induced protein aggregates
WP denaturation rate = 65%67.5°C 80°C
Heating temperatures or
Skimmedbovine milk
Caseins
α-LA β-LG LF
14 10
39
46
32
618 15
40 40 40
0
20
40
60
80
100
Control LF+ LF+ ALA+ IMFs
% o
f to
tal p
rote
ins
60% WP
40% CN
CN
WP
IMFs
1.3% of proteins
IMFs
ProteinsWP isolate
α-LALF
Lactose Minerals
STLOpen Days19-21 March 2019
9
Protein separation and characterization Asymmetric Flow Field Flow Fractionation -
Multi angle laser light scattering (AF4 - MALLS)
Protein concentration at 3 kDa
Protein aggregate morphologyTransmission Electron Microscopy
(TEM)
Strategy
Part 2: Characterization of heat-induced protein aggregates
Denatured WP precipitation
Supernatant Native WP
Quantification of native WP Reverse phase - high performance
liquid chromatography
Glass tube
Water bath
IMF
Heat treatmentWP denaturation rate = 65%
STLOpen Days19-21 March 2019
10
AF4 – MALLS fractograms of the unheated and heated IMFs at 67.5°C and 80°C
LF+ ALA+ Control LF+ Caseins
α-LA β-LG LF
CN
WP
Part 2: Characterization of heat-induced protein aggregates
Native WP
Denatured WP aggregate
CN micelle
Pro
tein
co
nte
nt
(g.L
-1)
Elution time (min)
Control IMF
Unheated 67.5°C
Control IMF
Unheated
Elution time (min)
Pro
tein
co
nte
nt
(g.L
-1)
Control IMF
Pro
tein
co
nte
nt
(g.L
-1)
Elution time (min)
Unheated 67.5°C 80°C Oldfield et al., 1998b; Anema et al., 2003
STLOpen Days19-21 March 2019
11
AF4 – MALLS fractograms of the unheated and heated IMFs at 67.5°C and 80°C
LF+ ALA+ Control LF+ Caseins
α-LA β-LG LF
CN
WP
Part 2: Characterization of heat-induced protein aggregates
Native WP
Denatured WP aggregate
CN micelle
Pro
tein
co
nte
nt
(g.L
-1)
Elution time (min)
Unheated
LF+ IMF
Elution time (min)
Pro
tein
co
nte
nt
(g.L
-1)
LF+ IMF
67.5°CUnheated
Elution time (min)
Pro
tein
co
nte
nt
(g.L
-1)
LF+ IMF
67.5°CUnheated 80°C
STLOpen Days19-21 March 2019
12
AF4 – MALLS fractograms of the unheated and heated IMFs at 67.5°C and 80°C
LF+ ALA+ Control LF+ Caseins
α-LA β-LG LF
CN
WP
Part 2: Characterization of heat-induced protein aggregates
Native WP
Denatured WP aggregate
CN micelle
Elution time (min)
Pro
tein
co
nte
nt
(g.L
-1)
Unheated
LF+ ALA+ IMF
Unheated 67.5°C
LF+ ALA+ IMF
Elution time (min)
Pro
tein
co
nte
nt
(g.L
-1)
Unheated 67.5°C
LF+ ALA+ IMF
Elution time (min)
Pro
tein
co
nte
nt
(g.L
-1)
80°C
STLOpen Days19-21 March 2019
13
Variation of the nature and morphology of protein aggregates according to the IMF and the heat treatment parameters
LF+ ALA+ Control LF+ Caseins
α-LA β-LG LF
CN
WP
Part 2: Characterization of heat-induced protein aggregates
Native WP
Denatured WP aggregate
CN micelle
Protein aggregate morphology of the IMFs heated at 80°C by transmission electron microscopy
200 nm 200 nm200 nm
80°CTEM
Control IMF 67.5°C
LF+ IMF LF+ ALA+ IMF
80°C
Unheated IMFs AF4 - MALLS
AF4 - MALLS
STLOpen Days19-21 March 2019
15
Take home messages
o Characterization of heat-induced protein aggregates
• Composition and morphology of protein aggregates depend on the WP composition of
IMFs and the heat treatment parameters
Objectives
Study of the heat treatment impact on the protein fraction of IMFs mimicking the human milk
protein profile
Strategy
o Kinetics of heat-induced denaturation of WP
o Characterization of heat-induced protein aggregates
Resultso Kinetics of heat-induced denaturation of WP
• No modification of the heat denaturation rate of LF by the presence of the other WP
• Decrease of the heat denaturation rate of α-LA with the β-LG content reduction
• Increase of the heat denaturation rate of β-LG in the presence of LF
STLOpen Days19-21 March 2019
16
Acknowledgements
PhD funding Research supports
Marie HennetierFrédéric Violleau
Agnès BurelAurélien Dupont
Pascaline HamonArlette LeducMarie-Bernadette Maillard
THANK YOU FORYOUR ATTENTION
MERCI
Please visit http://www.rennes.inra.fr/stlo_engSTLOpen Days19-21 March 2019
Thank you for your attention
STLOpen Days19-21 March 2019
Supplemented data
17
STLOpen Days19-21 March 2019
18
Frac
tio
n o
f n
ativ
e W
P
Unheated 62.5°C30 min
75°C15 sec
90°C15 sec
75°C10 min
Low pasteurization treatment: native WP for IMFs without LF
Fraction of residual native WP in IMFs after pasteurization treatments
Part 1: Kinetics of heat-induced denaturation of WP
α-LA
β-LG
LF
Control LF+
LF+ ALA+
IMF
WP
LF+ ALA+ Control LF+ Caseins
α-LA β-LG LF
CN
WP
High pasteurization treatment: native WP for IMFs without β-LG
STLOpen Days19-21 March 2019
19
Nat
ive
WP
co
nte
nt
(g.L
-1)
Variation of the residual native WP contents according to the IMF and the heat treatment parameters
Contents of residual native WP in IMFs at 65% of WP denaturation rate
LF+ ALA+ Control LF+ Caseins
α-LA β-LG LF
CN
WP
α-LA
β-LG
LF
Control LF+
LF+ ALA+
IMF
WP
Part 2: Characterization of heat-induced protein aggregates
STLOpen Days19-21 March 2019
20
Oldfield et al., 1998b; Anema et al., 2003
AF4 – MALLS fractograms of the unheated and heated IMFs at 67.5°C and 80°C
Pro
tein
co
nte
nt
(g.L
-1)
Elution time (min)
67.5°C 80°CUnheated
Control IMF
Elution time (min)
67.5°C 80°C
Pro
tein
co
nte
nt
(g.L
-1)
Unheated
LF+ IMF
Elution time (min)
67.5°C 80°C
Pro
tein
co
nte
nt
(g.L
-1)
Unheated
LF+ ALA+ IMF
LF+ ALA+ Control LF+ Caseins
α-LA β-LG LF
CN
WP
Part 2: Characterization of heat-induced protein aggregates
Native WP
Denatured WP aggregate
CN micelle
Variation of the nature and shape of protein aggregates according to the IMF and the heat treatment parameters
STLOpen Days19-21 March 2019
21
AF4 – MALLS data for the CN micelle population of the unheated and heated IMFsat 67.5°C and 80°C
LF+ ALA+ Control LF+ Caseins
α-LA β-LG LF
CN
WP
Part 2: Characterization of heat-induced protein aggregates