kinetics of heat-induced denaturation of whey proteins and ...©sentation - isfrs - amira -...

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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

[email protected]


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


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)


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


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


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


6

Strategy


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


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


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


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


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%


10

AF4 – MALLS fractograms of the unheated and heated IMFs at 67.5°C and 80°C


α-LA β-LG LF

CN

WP


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


11



α-LA β-LG LF

CN

WP


Native WP


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


12



α-LA β-LG LF

CN

WP


Native WP


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


13

Variation of the nature and morphology of protein aggregates according to the IMF and the heat treatment parameters


α-LA β-LG LF

CN

WP


Native WP


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


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


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

[email protected]


Supplemented data

17


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


α-LA

β-LG

LF

Control LF+

LF+ ALA+

IMF

WP


α-LA β-LG LF

CN

WP

High pasteurization treatment: native WP for IMFs without β-LG


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


α-LA β-LG LF

CN

WP

α-LA

β-LG

LF

Control LF+

LF+ ALA+

IMF

WP



20

Oldfield et al., 1998b; Anema et al., 2003


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


α-LA β-LG LF

CN

WP


Native WP


CN micelle

Variation of the nature and shape of protein aggregates according to the IMF and the heat treatment parameters


21

AF4 – MALLS data for the CN micelle population of the unheated and heated IMFsat 67.5°C and 80°C


α-LA β-LG LF

CN

WP


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