FIE FIELD-FLOW FR FRACTIONATION TECHNIQUES FOR MIL ILK PROTEIN - - PowerPoint PPT Presentation

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FIE FIELD-FLOW FR FRACTIONATION TECHNIQUES FOR MIL ILK PROTEIN - - PowerPoint PPT Presentation

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FIE FIELD-FLOW FR FRACTIONATION TECHNIQUES FOR MIL ILK PROTEIN CHARACTERIZATION MARIE HENNETIER 1 , AMIRA HALABI 3 , AGNS BUREL 5 , AMLIE DEGLAIRE 3 , THOMAS CROGUENNEC 3 , AUDREY ROMELARD 4 , ALAIN BANIEL 4 FRDRIC VIOLLEAU 1,2 1

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20 20th Int International Sy Symposium on n Fie Field- an and Flo Flow-Based Se Separations 23–27th February 2020 University of Vienna 25-02-20

FIE FIELD-FLOW FR FRACTIONATION TECHNIQUES FOR MIL ILK PROTEIN CHARACTERIZATION

MARIE HENNETIER1, AMIRA HALABI3, AGNÈS BUREL 5, AMÉLIE DEGLAIRE3, THOMAS CROGUENNEC3, AUDREY ROMELARD4, ALAIN BANIEL4 FRÉDÉRIC VIOLLEAU1,2

1TOULOUSE FFF CENTER, UNIVERSITÉ DE TOULOUSE, INP-PURPAN, TOULOUSE, FRANCE 2LABORATOIRE DE CHIMIE AGRO-INDUSTRIELLE, LCA, UNIVERSITÉ DE TOULOUSE, INRA, TOULOUSE 3 AGROCAMPUS OUEST-INRA SCIENCE & TECHNOLOGIE DU LAIT ET DE L'OEUF (STLO), RENNES, FRANCE 4 INGREDIA, ARRAS, FRANCE 5 PLATEFORME MRIC, MICROSCOPY - RENNES IMAGING CENTER, RENNES, FRANCE

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I. Introduction : Milk proteins

  • II. AsFlFFF analysis of Infant Milk Formulation (IMF)
  • III. Study of Whey proteins by 2D AsFlFFF-RPLC

Conclusion

  • IV. Denatured whey proteins studied by Centrifugal-FFF
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I

Introduction

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SLIDE 5

5

1

2 categories of milk proteins

Tolkach et al., 2007; Kulozik et al., 2011; Vogel, 2012

Whey Proteins (WP)

Autres (Immunoglobulin, Serum Albumin… ) α-lactalbumin (α-LA) β-lactoglobulin (β-LG) Lactoferrin (LF) Calcium Phosphate κ-casein αS1-, αS2-, β-, κ- caseins

Glantz et al., 2010

Caseins (CN)

50-600 nm

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Milk Milk activities activities in T TFFFC platform and his his partners partners

6

2

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Milk Milk activities activities in T TFFFC platform and his his partners partners

7

2

Casein micelles Whey Proteins Ultrafiltration Denaturation Functional protein ingredients

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Milk Milk activities activities in T TFFFC platform and his his partners partners

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2

Casein micelles Whey Proteins Ultrafiltration Denaturation Functional protein ingredients Formulation Infant full-cream milk formulation Infant skim milk formulation

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SLIDE 9

Milk Milk activities activities in T TFFFC platform and his his partners partners

9

2

Casein micelles Whey Proteins Ultrafiltration Denaturation Functional protein ingredients Formulation Infant full-cream milk formulation Infant skim milk formulation Cheese production Whey proteins

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Milk Milk activities activities in T TFFFC platform and his his partners partners

10 10

2

Casein micelles Whey Proteins Ultrafiltration Denaturation Functional protein ingredients Formulation Infant full-cream milk formulation Infant skim milk formulation Cheese production Whey proteins Drink Formulation

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SLIDE 11

Milk Milk activities activities in T TFFFC platform and his his partners partners

11 11

2

Casein micelles Whey Proteins Ultrafiltration Denaturation Functional protein ingredients Formulation Infant full-cream milk formulation Infant skim milk formulation Cheese production Whey proteins Drink Formulation Analysis by FFF techniques

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Milk Milk activities activities in T TFFFC platform and his his partners partners

12 12

2

Casein micelles Whey Proteins Ultrafiltration Denaturation Functional protein ingredients Formulation Infant full-cream milk formulation Infant skim milk formulation Cheese production Whey proteins Drink Formulation Analysis by FFF techniques

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II II

As As-FlFFF FlFFF analysis lysis of Infant ant Milk k Formulat mulation ion (IMF) F)

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SLIDE 14

As As-FlFFF FlFFF analysis analysis of

  • f Infant

Infant Milk Milk Formulatio Formulation n (IMF) (IMF)

14 14 Casein micelles Whey Proteins Ultrafiltration Denaturation Functional protein ingredients Formulation Infant full-cream milk formulation Infant skim milk formulation Cheese production Whey proteins Drink Formulation Analysis by FFF techniques

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Milk composition

  • n

15 15 The CN:WP ratio and the WP composition have to be modified for the Infant milk formulation (IMF) to mimic the human milk composition

1

Chatterton et al., 2013

5 10 15 20 25 30 35 40

g of proteins /L

LF

Caseins β-lactoglobulin α-lactalbumin Lactoferrin Others Whey proteins Caseins

60% 40% 60% 40% 20% 80%

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Infant milk f formulatio ions ns IMFs, their heat treatment treatment and and their their character characteriz izati ation

  • n

16 16 Control IMF

CN:WP 40:60 Without modification

  • f the proportion of

the bovine milk whey proteins

LF+ IMF

CN:WP 40:60 LF quantity closes to that of human milk

3 Infant Milk Formulation IMFs

LF+ ALA+ IMF

CN:WP 40:60 LF and α-LA quantities close to those of human milk

2

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Infant milk f formulatio ions ns IMFs, their heat treatment treatment and and their their character characteriz izati ation

  • n

17 17 Control IMF

CN:WP 40:60 Without modification

  • f the proportion of

the bovine milk whey proteins

LF+ IMF

CN:WP 40:60 LF quantity closes to that of human milk

3 Infant Milk Formulation IMFs

LF+ ALA+ IMF

CN:WP 40:60 LF and α-LA quantities close to those of human milk

2

Heat Treatment Model IMFs Transmission Electronic Microscopy AsFlFFF-MALS-QELS-RI 67.5°C 80°C

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AF4 AF4-MALS MALS-QE QELS-RI RI fractogram ams of t the u unheated and heated IMFs at 6 67.5°C and 8 80°C : C Control IMF IMF

18 18

Concentration (g.L-1) Unheated Time (min)

3

Control IMF : RI Signal Mw/Rgw = 3 Sphere

Native WP WP aggregate Casein Micelle Partially desintegrated Casein Micelle

Eluent : 50mM NaCl + 5mM CaCl2

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AF4 AF4-MALS MALS-QE QELS-RI RI fractogram ams of t the u unheated and heated IMFs at 6 67.5°C and 8 80°C : C Control IMF IMF

19 19

3

67.5°C Unheated Concentration (g.L-1)

Control IMF : RI Signal

Non aggregated WP WP aggregate Casein Micelle Partially desintegrated Casein Micelle

Eluent : 50mM NaCl + 5mM CaCl2

Mean Mw ;Rgw ;Rhw : waste of k-cas

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SLIDE 20

AF4 AF4-MALS MALS-QE QELS-RI RI fractogram ams of t the u unheated and heated IMFs at 6 67.5°C and 8 80°C : C Control IMF IMF

20 20

3

Concentration (g.L-1) Time (min) Unheated 67.5°C 80°C

Mw Control IMF : RI Signal

Non aggregated WP WP aggregate Casein Micelle Partially desintegrated Casein Micelle

Eluent : 50mM NaCl + 5mM CaCl2

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AF4 AF4-MALS MALS-QE QELS-RI RI fractogram ams of t the u unheated and heated IMFs at 6 67.5°C and 8 80°C : L LF+ IMF IMF 21

21

4

Concentration (g.L-1) Time (min) Unheated

LF LF+

+ IM

IMF : : RI RI Si Signal

Mw and Rgw Mw/Rgw = 2,6 not spherical

Native WP WP aggregate Casein Micelle Partially desintegrated Casein Micelle

Eluent : 50mM NaCl + 5mM CaCl2

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SLIDE 22

AF4 AF4-MALS MALS-QE QELS-RI RI fractogram ams of t the u unheated and heated IMFs at 6 67.5°C and 8 80°C : L LF+ IMF IMF 22

22

3

Temps (min) Concentration (g.L-1)

LF LF+

+ IM

IMF : : RI RI Si Signal

67.5°C Unheated

Non aggregated WP WP aggregate Casein Micelle Partially desintegrated Casein Micelle

Mw Mw/Rgw = 2,8

Eluent : 50mM NaCl + 5mM CaCl2

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SLIDE 23

AF4 AF4-MALS MALS-QE QELS-RI RI fractogram ams of t the u unheated and heated IMFs at 6 67.5°C and 8 80°C : L LF+ IMF IMF 23

23

3

Time (min) Concentration (g.L-1)

LF LF+

+ IM

IMF : : RI RI Si Signal

67.5°C Unheated 80°C

Rgw/Rhw=1.8 Mw/Rgw=2.3

Non aggregated WP WP aggregate Casein Micelle Partially desintegrated Casein Micelle

Eluent : 50mM NaCl + 5mM CaCl2

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SLIDE 24

AF4 AF4-MALS MALS-QE QELS-RI RI fractogram ams of t the u unheated and heated IMFs at 6 67.5°C and 8 80°C : L LF+ ALA ALA+ IMF IMF

24 24

4

Time (min) Concentration (g.L-1) Unheated

LF LF+

+ ALA+ IM

IMF

Native WP WP aggregate Casein Micelle Partially desintegrated Casein Micelle

Eluent : 50mM NaCl + 5mM CaCl2

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SLIDE 25

AF4 AF4-MALS MALS-QE QELS-RI RI fractogram ams of t the u unheated and heated IMFs at 6 67.5°C and 8 80°C : L LF+ ALA ALA+ IMF IMF

25 25

4

Unheated 67.5°C

LF LF+

+ ALA+ IM

IMF

Temps (min) Concentration (g.L-1)

Non aggregated WP WP aggregate Casein Micelle Partially desintegrated Casein Micelle

Eluent : 50mM NaCl + 5mM CaCl2

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SLIDE 26

AF4 AF4-MALS MALS-QE QELS-RI RI fractogram ams of t the u unheated and heated IMFs at 6 67.5°C and 8 80°C : L LF+ ALA ALA+ IMF IMF

26 26

4

Unheated 67.5°C

LF LF+

+ AL

ALA+ IM IMF

Temps (min) Concentration (g.L-1) 80°C

Rgw Rhw

Non aggregated WP WP aggregate Casein Micelle Partially desintegrated Casein Micelle

Mw/Rgw=2.9

Eluent : 50mM NaCl + 5mM CaCl2

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Transmis nsmission sion Electron ctronic ic Microsco roscopy py

27 27

200 00 nm nm

80 80°C

TEM

Con Control l IM IMF

80 80°C

AF4

5

Non aggregated WP WP aggregate Casein Micelle Partially desintegrated Casein Micelle

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Transmis nsmission sion Electron ctronic ic Microsco roscopy py

28 28

200 00 nm nm

80 80°C

TEM

Con Control l IM IMF

80 80°C

AF4

5

200 00 nm nm

LF LF+

+ IM

IMF

Non aggregated WP WP aggregate Casein Micelle Partially desintegrated Casein Micelle

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Transmis nsmission sion Electron ctronic ic Microsco roscopy py

29 29

200 00 nm nm

80 80°C

TEM

Con Control l IM IMF

80 80°C

AF4

5

200 00 nm nm

LF LF+

+ ALA

LA+ IM IMF LF LF+

+ IM

IMF

Non aggregated WP WP aggregate Casein Micelle Partially desintegrated Casein Micelle

200 00 nm nm

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III III

Study dy of

  • f Whey

Whey proteins teins by 2D AsFlFFF lFFF-RPLC RPLC

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Study of W Whey proteins by 2 2D-AsFlFFF AsFlFFF- RPHPLC RPHPLC

31 31 Casein micelles Whey Proteins Ultrafiltration Denaturation Functional protein ingredients Formulation Infant full-cream milk formulation Infant skim milk formulation Cheese production Whey proteins Drink Formulation Analysis by FFF techniques

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Whey y Proteins teins Analysis lysis by As As-FlFFF FlFFF (1D) (1D)

32 32

1

Industrial whey protein isolate : they’re few percents of caseins inside Objective : separation of all proteins

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Whey y Proteins teins Analysis lysis by As As-FlFFF FlFFF (1D) (1D)

33 33

1

0.00 0.20 0.40 0.60 0.80 1.00 1.20 10 11 12 13 14 15 16 17 18 19 20 Normalized Concentration Time (min)

Whey proteins and standards RI signal

Whey proteins a-lactalbumin b-lactoglobulin BSA Immunoglobulin a/b/k casein mix

  • The different whey proteins are identified but not very good separated by AsFlFFF
  • By HPLC it’s not possible to separate caseins from whey proteins

Industrial whey protein isolate : they’re few percents of caseins inside Objective : separation of all proteins

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Whey y Proteins teins Analysis lysis by 2D AsFlFFF lFFF-RPLC RPLC

34 34 1rst dimension : As-FlFFF and 2nd dimension : RPLC HPLC : Agilent and Thermo system ; As-FlFFF : Wyatt The sample is collected at the end of As-FlFFF in a column-guard and injected again online in the column every 80 seconds

2

2nd Dimension 1rst Dimension

1D Pump 1D Sampler 1D As-FlFFF 1D UV Detector 2D Pump 2D UV Detector 2D Column

2D LC Valve

+ Column guards = Collector + Injector

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Whey Proteins Analysis by 2 2D AsFlFFF AsFlFFF-RPL PLC : UV signal mapping

35 35

3

2D RPLC (sec) 1D As-FlFFF (min) Standards of Whey Proteins

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Whey Proteins Analysis by 2 2D-AsFlFFF AsFlFFF-RPH PHPLC LC : UV s signal mapping

36 36

3

β-lac A β-lac B 2D RPHPLC (sec) 1D As-FlFFF (min) Standards of Whey Proteins

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37 37 β-lac A β-lac B BSA monomer 2D RPHPLC (sec) 1D As-FlFFF (min)

Whey Proteins Analysis by 2 2D-AsFlFFF AsFlFFF-RPH PHPLC LC : UV s signal mapping 3

Standards of Whey Proteins

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38 38 β-lac A β-lac B BSA monomer α-lac 2D RPHPLC (sec) 1D As-FlFFF (min)

Whey Proteins Analysis by 2 2D-AsFlFFF AsFlFFF-RPH PHPLC LC : UV s signal mapping 3

Standards of Whey Proteins

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39 39 IgG β-lac A β-lac B BSA monomer α-lac 2D RPHPLC (sec) 1D As-FlFFF (min)

Whey Proteins Analysis by 2 2D-AsFlFFF AsFlFFF-RPH PHPLC LC : UV s signal mapping 3

Standards of Whey Proteins

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40 40 IgG β-lac A β-lac B BSA monomer α-lac α-casein (aggregated and non-aggregated in 1D) 2D RPHPLC (sec) 1D As-FlFFF (min)

Whey Proteins Analysis by 2 2D-AsFlFFF AsFlFFF-RPH PHPLC LC : UV s signal mapping 3

Standards of Whey Proteins

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41 41 IgG β-lac A β-lac B BSA monomer α-lac α-casein (aggregated and non-aggregated in 1D) β-casein 2D RPHPLC (sec) 1D As-FlFFF (min)

Whey Proteins Analysis by 2 2D-AsFlFFF AsFlFFF-RPH PHPLC LC : UV s signal mapping 3

Standards of Whey Proteins

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42 42 IgG β-lac A β-lac B BSA monomer α-lac α-casein (aggregated and non-aggregated in 1D) β-casein k-casein 2D RPHPLC (sec) 1D As-FlFFF (min)

Whey Proteins Analysis by 2 2D-AsFlFFF AsFlFFF-RPH PHPLC LC : UV s signal mapping 3

Standards of Whey Proteins

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IV IV

Denature atured whey whey proteins teins studied died by by Centrifugal trifugal-FFF FFF

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Denatured Denatured whey whey proteins proteins studied studied by by Centrifuga Centrifugal-FF FFF

44 44 Casein micelles Whey Proteins Ultrafiltration Denaturation Functional protein ingredients Formulation Infant full-cream milk formulation Infant skim milk formulation Cheese production Whey proteins Drink Formulation Analysis by FFF techniques

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Denatured Denatured whey whey proteins proteins studied studied by by As As- FlFFF FlFFF

45 45

1 10 100 1000 0.00 0.20 0.40 0.60 0.80 1.00 20 40 60 80 100 rg (nm) Relative scale Time (min)

RI/MALS/rg

LS RI rg

1

There are no big aggregate in concentration Mass recovery : 44% Whey proteins heated at high temperature Eluent : 50mM NaCl

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Denatured Denatured whey whey proteins proteins studied studied by by Centrifuga Centrifugal-FF FFF

46 46

1 10 100 1000 0.00 0.20 0.40 0.60 0.80 1.00 1.20 10 20 30 40 50 60 Rg (nm) Relative scale Time (min)

RI/MALS/ Rg LS RI Rg

2

Mass recovery of 92% : No membrane : less interaction Detergent in the eluent Eluent : 50mM NaCl + 0.1% Novachem (detergent) (Postnova system)

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Denatured Denatured whey whey proteins proteins studied studied by by Centrifuga Centrifugal-FF FFF

47 47

1 10 100 1000 0.00 0.20 0.40 0.60 0.80 1.00 1.20 10 20 30 40 50 60 Rg (nm) Relative scale Time (min)

RI/MALS/ Rg 1: Objets less dense and big in the void peak : whey proteins non aggregated

51% Mass fractions 1

LS RI Rg

2

Eluent : 50mM NaCl + 0.1% Novachem (detergent) Mass recovery of 92% : No membrane : less interaction Detergent in the eluent

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SLIDE 48

48 48

1 10 100 1000 0.00 0.20 0.40 0.60 0.80 1.00 1.20 10 20 30 40 50 60 Rg (nm) Relative scale Time (min)

RI/MALS/ Rg 1: Objets less dense and big in the void peak 2: Rg from 25 to 850nm

51% 23% 1 2

LS RI Rg

2 Denatured Denatured whey whey proteins proteins studied studied by by Centrifuga Centrifugal-FF FFF

Mass fractions Eluent : 50mM NaCl + 0.1% Novachem (detergent) Mass recovery of 92% : No membrane : less interaction Detergent in the eluent

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49 49

1 10 100 1000 0.00 0.20 0.40 0.60 0.80 1.00 1.20 10 20 30 40 50 60 Rg (nm) Relative scale Time (min)

RI/MALS/ Rg 1: Objets less dense and big in the void peak 2: Rg from 25 to 850nm 3: More than 1µm : MALS can’t calculate the size Fraction collection to do batch DLS

51% 23% 26% 1 2 3

LS RI Rg

2 Denatured Denatured whey whey proteins proteins studied studied by by Centrifuga Centrifugal-FF FFF

Mass fractions Eluent : 50mM NaCl + 0.1% Novachem (detergent) Mass recovery of 92% : No membrane : less interaction Detergent in the eluent

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Batch DLS of t the 3 3 f fractions

50 50 1 Mean Rh: 68 nm Between 10 nm to 200 nm 2 Mean Rh : 219 nm Between 45 nm to 1,2µm 3 Mean Rh : 692 nm Between 100 nm to 4,5µm 1 2 3

3

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Co Conc nclus lusion ion an and d ou

  • utl

tloo

  • ok
  • 1rst

study : casein-WP pasteurized structures are different in function

  • f

the WP composition in IMF and heat temperature :

  • Addition of lactoferrin in IMF destabilize the casein micelles
  • Addition
  • f

Lactoferrin form sturctures with ams around the micelle after heat treatment at 80°C

  • Addition
  • f

Lactoferrin + α-lac form smaller structures with WP aggregates inside the micelles at 80°C Digestibility

  • f

this structures? Scale-up for formulation and pasteurization : Stuctural difference?

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SLIDE 52

Co Conc nclus lusion ion an and d ou

  • utl

tloo

  • ok
  • 1rst study : casein-WP pasteurized structures are different in function of

the WP composition in IMF and heat temperature :

  • Addition of lactoferrin in IMF destabilize the casein micelles
  • Addition of Lactoferrin form sturctures with ams around the micelle

after heat treatment at 80°C

  • Addition
  • f

Lactoferin + α-lac form smaller structures with WP aggregates inside the micelles at 80°C Digestibility

  • f

this structures? Scale-up for formulation and pasteurization: Stuctural difference?

  • 2nd

study : 2D-AsFlFFF-RPHPLC can separate all proteins

  • f

WP and casein mix Quantitative experiment with calibration curve and industrial mix study

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SLIDE 53

Co Conc nclus lusion ion an and d ou

  • utl

tloo

  • ok
  • 1rst study : casein-WP pasteurized structures are different in function of

the WP composition in IMF and heat temperature :

  • Addition of lactoferrin in IMF destabilize the casein micelles
  • Addition of Lactoferrin form sturctures with ams around the micelle

after heat treatment at 80°C

  • Addition
  • f

Lactoferin + α-lac form smaller structures with WP aggregates inside the micelles at 80°C Digestibility

  • f

this structures? Scale-up for formulation and pasteurization: Stuctural difference?

  • 2nd

study : 2D-AsFlFFF-RPHPLC can separate all proteins

  • f

WP and casein mix Quantitative experiment with calibration curve and industrial mix study

  • 3rd

study : Centrifugal-FFF allow to have a better mass recovery for denatured WP

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SLIDE 54

Thank you for your attention!

Amira Halabi, Amélie Deglaire, Thomas Croguennec, Audrey Romelard, Alain Baniel, Frédéric Violleau

The TFFFC team: Estelle N’Tsiba, Audrey Ric, Mireille Gaucher, Kévin Pascotto

Acknowledgmen t:

https://www.linkedin.com  TFFFC

To follow us:

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Annexes