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

FIE FIELD-FLOW FR FRACTIONATION TECHNIQUES FOR MIL ILK PROTEIN CHARACTERIZATION MARIE HENNETIER 1 , AMIRA HALABI 3 , AGNÈS BUREL 5 , AMÉLIE DEGLAIRE 3 , THOMAS CROGUENNEC 3 , AUDREY ROMELARD 4 , ALAIN BANIEL 4 FRÉDÉRIC VIOLLEAU 1,2 1 TOULOUSE FFF CENTER, UNIVERSITÉ DE TOULOUSE, INP-PURPAN, TOULOUSE, FRANCE 2 LABORATOIRE 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 20th Int 20 International Sy Symposium on n Fie Field- an and Flo Flow-Based Se Separations 23 – 27th February 2020 University of Vienna 25-02-20

I. Introduction : Milk proteins II. AsFlFFF analysis of Infant Milk Formulation (IMF) III. Study of Whey proteins by 2D AsFlFFF-RPLC IV. Denatured whey proteins studied by Centrifugal-FFF Conclusion

I Introduction

1 5 2 categories of milk proteins Caseins (CN) Whey Proteins (WP) κ -casein α S1 -, α S2 -, β -, κ - caseins 50-600 nm Calcium Phosphate α -lactalbumin β -lactoglobulin Lactoferrin Glantz et al., 2010 (α -LA) (β -LG) (LF) Autres (Immunoglobulin, Serum Albumin… ) Tolkach et al., 2007; Kulozik et al., 2011; Vogel, 2012

Milk Milk activities activities in T TFFFC platform and 2 his his partners partners 6

Milk Milk activities activities in T TFFFC platform and 2 his his partners partners 7 Ultrafiltration Whey Proteins Casein micelles Denaturation Functional protein ingredients

Milk Milk activities activities in T TFFFC platform and 2 his his partners partners 8 Ultrafiltration Whey Proteins Casein micelles Infant full-cream milk Denaturation formulation Formulation Functional protein ingredients Infant skim milk formulation

Milk activities Milk activities in T TFFFC platform and 2 his his partners partners 9 Ultrafiltration Cheese production Whey proteins Whey Proteins Casein micelles Infant full-cream milk Denaturation formulation Formulation Functional protein ingredients Infant skim milk formulation

Milk Milk activities activities in T TFFFC platform and 2 his his partners partners 10 10 Ultrafiltration Cheese production Whey proteins Whey Proteins Casein micelles Infant full-cream milk Formulation Denaturation formulation Formulation Functional protein ingredients Infant skim milk formulation Drink

Milk Milk activities activities in T TFFFC platform and 2 his his partners partners 11 11 Analysis by FFF techniques Ultrafiltration Cheese production Whey proteins Whey Proteins Casein micelles Infant full-cream milk Formulation Denaturation formulation Formulation Functional protein ingredients Infant skim milk formulation Drink

Milk Milk activities activities in T TFFFC platform and 2 his his partners partners 12 12 Analysis by FFF techniques Ultrafiltration Cheese production Whey proteins Whey Proteins Casein micelles Infant full-cream milk Formulation Denaturation formulation Formulation Functional protein ingredients Infant skim milk formulation Drink

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

As As-FlFFF FlFFF analysis analysis of of Infant Infant Milk Milk Formulatio Formulation n (IMF) (IMF) 14 14 Analysis by FFF techniques Ultrafiltration Cheese production Whey proteins Whey Proteins Casein micelles Infant full-cream milk Formulation Denaturation formulation Formulation Functional protein ingredients Infant skim milk formulation Drink

1 Milk composition on 15 15 40 Caseins Caseins 35 Whey proteins 30 Others g of proteins /L 25 Lactoferrin 80% β -lactoglobulin 20 α -lactalbumin 15 40% 10 40% 5 60% 20% 60% 0 Chatterton et al., 2013 LF The CN:WP ratio and the WP composition have to be modified for the Infant milk formulation (IMF) to mimic the human milk composition

Infant milk f formulatio ions ns IMFs, their heat 2 treatment and treatment and their their character characteriz izati ation on 16 16 3 Infant Milk Formulation IMFs Control IMF LF + ALA + IMF LF + IMF CN:WP 40:60 CN:WP 40:60 CN:WP 40:60 Without modification LF quantity closes LF and α -LA of the proportion of to that of human quantities close to the bovine milk whey milk those of human milk proteins

Infant milk f formulatio ions ns IMFs, their heat 2 treatment treatment and and their their character characteriz izati ation on 17 17 3 Infant Milk Formulation IMFs Control IMF LF + ALA + IMF LF + IMF CN:WP 40:60 CN:WP 40:60 CN:WP 40:60 Without modification LF quantity closes LF and α -LA of the proportion of to that of human quantities close to the bovine milk whey milk those of human milk proteins Transmission Electronic Microscopy 67.5°C Model IMFs Heat Treatment 80°C AsFlFFF-MALS-QELS-RI

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 3 IMF IMF 18 18 Eluent : Control IMF : RI Signal 50mM NaCl + 5mM CaCl 2 M w /Rg w = 3 Sphere Native WP Concentration (g.L -1 ) WP aggregate Casein Micelle Partially desintegrated Casein Micelle Time (min) Unheated

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 3 IMF IMF 19 19 Eluent : Control IMF : RI Signal 50mM NaCl + 5mM CaCl 2 : waste of k-cas Mean M w ;Rg w ;Rh w Non aggregated WP Concentration (g.L -1 ) WP aggregate Casein Micelle Partially desintegrated Casein Micelle Unheated 67.5°C

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 3 IMF IMF 20 20 Eluent : Control IMF : RI Signal 50mM NaCl + 5mM CaCl 2 M w Non aggregated WP Concentration (g.L -1 ) WP aggregate Casein Micelle Partially desintegrated Casein Micelle Time (min) Unheated 67.5°C 80°C

AF4 AF4-MALS MALS-QE QELS-RI RI fractogram ams of t the u unheated LF + IMF 4 and heated IMFs at 6 67.5°C and 8 80°C : L IMF 21 21 Eluent : LF + + IM LF IMF : : RI RI Si Signal 50mM NaCl + 5mM CaCl 2 M w /Rg w = 2,6 not spherical Native WP Concentration (g.L -1 ) M w and Rg w WP aggregate Casein Micelle Partially desintegrated Casein Micelle Time (min) Unheated

AF4 AF4-MALS MALS-QE QELS-RI RI fractogram ams of t the u unheated LF + IMF 3 and heated IMFs at 6 67.5°C and 8 80°C : L IMF 22 22 Eluent : LF + + IM LF IMF : : RI RI Si Signal 50mM NaCl + 5mM CaCl 2 M w Non aggregated WP Concentration (g.L -1 ) M w /Rg w = 2,8 WP aggregate Casein Micelle Partially desintegrated Casein Micelle Temps (min) Unheated 67.5°C

AF4 AF4-MALS MALS-QE QELS-RI RI fractogram ams of t the u unheated LF + IMF 3 and heated IMFs at 6 67.5°C and 8 80°C : L IMF 23 23 Eluent : LF + + IM LF IMF : : RI RI Si Signal 50mM NaCl + 5mM CaCl 2 Non aggregated WP M w /Rg w =2.3 Concentration (g.L -1 ) Rg w /Rh w =1.8 WP aggregate Casein Micelle Partially desintegrated Casein Micelle Time (min) Unheated 80°C 67.5°C

AF4 AF4-MALS MALS-QE QELS-RI RI fractogram ams of t the u unheated LF + ALA ALA + and heated IMFs at 6 67.5°C and 8 80°C : L 4 IMF IMF 24 24 Eluent : + ALA + IM LF + LF IMF 50mM NaCl + 5mM CaCl 2 Native WP Concentration (g.L -1 ) WP aggregate Casein Micelle Partially desintegrated Casein Micelle Time (min) Unheated

AF4 AF4-MALS MALS-QE QELS-RI RI fractogram ams of t the u unheated LF + ALA ALA + and heated IMFs at 6 67.5°C and 8 80°C : L 4 IMF IMF 25 25 Eluent : + ALA + IM LF + 50mM NaCl + 5mM CaCl 2 LF IMF Non aggregated WP Concentration (g.L -1 ) WP aggregate Casein Micelle Partially desintegrated Casein Micelle Temps (min) 67.5°C Unheated

AF4 AF4-MALS MALS-QE QELS-RI RI fractogram ams of t the u unheated LF + ALA ALA + and heated IMFs at 6 67.5°C and 8 80°C : L 4 IMF IMF 26 26 Eluent : ALA + IM 50mM NaCl + 5mM CaCl 2 LF + + AL LF IMF M w /Rg w =2.9 Non aggregated WP Concentration (g.L -1 ) Rg w WP aggregate Rh w Casein Micelle Partially desintegrated Casein Micelle Temps (min) 67.5°C Unheated 80°C

Transmis nsmission sion Electron ctronic ic Microsco roscopy py 5 27 27 Non aggregated WP Control Con l IM IMF WP aggregate 80 80°C Casein Micelle AF 4 Partially desintegrated Casein Micelle 80°C 80 TEM 200 00 nm nm

Transmis nsmission sion Electron ctronic ic Microsco roscopy py 5 28 28 Non aggregated WP Con Control l IM IMF LF + + IM LF IMF WP aggregate 80 80°C Casein Micelle AF 4 Partially desintegrated Casein Micelle 80 80°C TEM 200 00 nm nm 200 00 nm nm

Transmis nsmission sion Electron ctronic ic Microsco roscopy py 5 29 29 Non aggregated WP LA + IM Con Control l IM IMF LF + + IM LF IMF LF LF + + ALA IMF WP aggregate 80 80°C Casein Micelle AF 4 Partially desintegrated Casein Micelle 80°C 80 TEM 200 00 nm nm 200 00 nm nm 200 00 nm nm

III III Study dy of of Whey Whey proteins teins by 2D AsFlFFF lFFF-RPLC RPLC