1. food and drink
2. desserts and baking

DEVELOPMENT OF A NEW DAIRY
INGREDIENT FOR THE UTILIZATION OF
BUTTERMILK CONSTITUENTS
Thèse
Maxime Saffon
Doctorat en Sciences et Technologie des Aliments
Philosophiæ Doctor (Ph.D)
Québec, Canada
© Maxime Saffon, 2013
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RESUME
L’utilisation du babeurre pour la formulation alimentaire est limitée à cause de la capacité
de rétention d’eau importante de ses phospholipides. L’objectif de ce projet était de
développer une nouvelle approche de valorisation des constituants du babeurre. Ce sousproduit de l’industrie laitière est riche en composants d’intérêts qui ont des propriétés
nutritionnelles, santés, et fonctionnelles prometteuses comme les constituants de la
membrane de globule de matière grasse (MFGM) et les phospholipides. Les deux
principaux procédés de production d’agrégats protéiques laitiers ont été combinés donnant
un traitement thermique intensif à pH acide des protéines du lactosérum en présence des
constituants du babeurre. Les résultats ont d’abord montré qu’il était possible de substituer
différentes proportions de protéines du lactosérum par des protéines du babeurre et que la
présence des constituants du babeurre entrainait la formation d’agrégats protéiques variés
avec une capacité de rétention d’eau plus faible. Les résultats ont révélé que des agrégats
protéiques étaient préformés lors de la préparation des babeurres incluant les protéines du
lactosérum, les caséines et les protéines de la MFGM. Les phospholipides sont intégrés aux
agrégats par l’intermédiaire de la MFGM à des températures faibles (65°C) alors qu’ils
semblent s’associer directement avec les protéines à des températures plus élevées (
80°C). Par la suite, les agrégats du babeurre agissent à titre de noyau d’agrégation pour les
protéines issues du lactosérum. Le type d’interactions formées entre les protéines a un
impact significatif sur les propriétés physiques et fonctionnelles des agrégats. En dernier
lieu, il a été possible d’utiliser ces agrégats variés pour la production de yaourt ferme. Les
agrégats lactosérum:babeurre ont agi à titre d’agent passif plutôt que d’agent actif mais des
interactions entre les agrégats et les protéines du lait écrémé ont été observées. Ces
associations seraient initiées par les groupements thiols libres des agrégats présents avant le
chauffage. Cependant, la mise en solution de la poudre d’agrégats doit être strictement
contrôlée. Ce projet propose une nouvelle approche pour l’utilisation du babeurre ainsi
qu’une meilleure compréhension du comportement à la chaleur de ses constituants.
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ABSTRACT
The use of buttermilk in food formulation is limited due to the extensive water-holding
capacity of its phospholipids. The goal of this project was to develop a new approach for
the valorization of buttermilk’s constituents. This by-product is rich in valuable
components with promising nutritional, healthy, and functional properties such as the milk
fat globule membrane (MFGM) constituents, the phospholipids. The two main processes of
production of dairy aggregates have been combined resulting of the intensive heatdenaturation of whey proteins at low pH (4.6) in presence of proteins from buttermilk.
First, results showed that it was possible to substitute whey proteins by different levels of
buttermilk proteins in the process and that the presence of buttermilk constituents led to the
formation of mixed aggregates with new functional properties such as a low water-holding
capacity. Results revealed that aggregates are pre-formed during the preparation of the
buttermilk concentrates involving whey proteins, casein, and MFGM proteins.
Phospholipids are integrated to the aggregates through the MFGM at low temperature
(65°C), but seem to directly interact with the proteins at higher temperatures ( 80°C).
These pre-formed aggregates from buttermilk can act as aggregation nucleus for the
proteins from whey. The types of interactions that occur between the proteins significantly
affected the properties of the aggregates such as their water-holding capacity, their size, and
the solubility of the powder. Finally, it was possible to use the mixed aggregates in the
production of set-type yogurt. Whey:buttermilk aggregates were acting more like a passive
than a reactive filler, but some possible interactions with the proteins from the skim milk
were observed due to the high concentration of thiol groups of the aggregates before
heating. However, the dispersibility of the powder must be strictly controlled. Overall, this
project proposed a new approach for the use of buttermilk and allowed a better
understanding of the thermal behavior of its constituents.
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TABLE OF CONTENTS
RESUME ........................................................................................................................................................ III
ABSTRACT ..................................................................................................................................................... V
TABLE OF CONTENTS.............................................................................................................................. VII
TABLE LIST ................................................................................................................................................... XI
FIGURE LIST .............................................................................................................................................. XIII
EQUATION LIST.......................................................................................................................................XVII
ABREVIATIONS ........................................................................................................................................ XIX
ACKNOWLEDGMENTS............................................................................................................................ XXI
FOREWORD .............................................................................................................................................. XXV
CHAPTER 1
INTRODUCTION............................................................................................................... 1
CHAPTER 2 LITERATURE REVIEW ................................................................................................... 5
2.1
BUTTERMILK AND WHEY............................................................................................................................. 5
2.1.1 Buttermilk ...................................................................................................................................................... 5
2.1.2 Whey ................................................................................................................................................................. 7
2.2
CONSTITUENTS FROM BUTTERMILK AND WHEY ..................................................................................... 8
2.2.1 Caseins ............................................................................................................................................................. 8
2.2.2 Whey proteins ........................................................................................................................................... 10
2.2.3 Milk fat globule membrane proteins .............................................................................................. 13
2.2.4 Phospholipids from buttermilk ......................................................................................................... 17
2.3
PRINCIPLES OF PROTEIN AGGREGATION ................................................................................................ 19
2.3.1 Definitions of native state, denaturation, and reversibility ................................................. 19
2.3.2 Nucleation................................................................................................................................................... 20
2.3.3 Description of the different pathways .......................................................................................... 20
2.3.4 Bonds, interactions, and exchanges in milk protein aggregation .................................... 22
2.3.5 Effects of experimental conditions .................................................................................................. 24
2.4
HEAT-INDUCED AGGREGATION OF MILK PROTEINS ............................................................................. 26
2.4.1 Aggregation mechanisms of -lactoglobulin and -lactalbumin ................................... 26
2.4.2 Heat-induced interactions with minor whey proteins ........................................................... 31
2.4.3 Heat-induced interactions involving casein micelles.............................................................. 33
2.4.4 Heat-induced aggregation mechanisms of MFGM constituents ........................................ 36
2.5
APPLICATIONS FOR DAIRY PROTEIN AGGREGATES IN FOOD FORMULATION .................................... 42
2.5.1 Role of aggregates as fat mimetic ................................................................................................... 42
2.5.2 Role of aggregates as water holder ................................................................................................ 45
CHAPTER 3
HYPOTHESIS, GOAL, AND OBJECTIVES ................................................................. 47
CHAPTER 4 THERMAL AGGREGATION OF WHEY PROTEINS IN THE PRESENCE OF
BUTTERMILK CONCENTRATE .............................................................................................................. 49
4.1
RÉSUMÉ ....................................................................................................................................................... 50
4.2
ABSTRACT ................................................................................................................................................... 51
4.3
INTRODUCTION .......................................................................................................................................... 51
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4.4
MATERIALS AND METHODS ...................................................................................................................... 53
4.4.1 Materials...................................................................................................................................................... 53
4.4.2 Preparation of whey and buttermilk concentrates ................................................................. 53
4.4.3 Heating experiments .............................................................................................................................. 54
4.4.4 Treatments with N-ethylmaleimide (NEM) and power ultrasound ................................ 56
4.4.5 Analytical methods ................................................................................................................................. 56
4.4.6 Particle size distribution and surface area ................................................................................. 58
4.4.7 Determination of rheological properties...................................................................................... 58
4.4.8 Statistical analysis .................................................................................................................................. 59
4.5
RESULTS ...................................................................................................................................................... 59
4.5.1 Aggregation yield .................................................................................................................................... 59
4.5.2 Water-holding capacity ........................................................................................................................ 61
4.5.3 Consistency coefficient (k) ................................................................................................................... 62
4.5.4 Flow behavior index (n)........................................................................................................................ 63
4.5.5 Particle size distribution ...................................................................................................................... 64
4.6
DISCUSSION ................................................................................................................................................. 66
4.7
CONCLUSIONS ............................................................................................................................................. 68
4.8
ACKNOWLEDGMENTS ................................................................................................................................ 69
CHAPTER 5 EFFECT OF BUTTERMILK COMPONENTS ON THE HEAT-INDUCED
DENATURATION OF WHEY PROTEINS ...............................................................................................71
5.1
RÉSUMÉ ....................................................................................................................................................... 72
5.2
ABSTRACT ................................................................................................................................................... 73
5.3
INTRODUCTION .......................................................................................................................................... 73
5.4
MATERIALS AND METHODS ...................................................................................................................... 76
5.4.1 Materials...................................................................................................................................................... 76
5.4.2 Preparation of whey protein concentrates.................................................................................. 76
5.4.3 Preparation of spray dried buttermilk concentrates .............................................................. 77
5.4.4 Heat-induced aggregation .................................................................................................................. 78
5.4.5 Treatment with N-ethylmaleimide (NEM) .................................................................................. 78
5.4.6 Analytical method ................................................................................................................................... 78
5.4.7 Gel electrophoresis .................................................................................................................................. 79
5.4.8 Confocal laser scanning microscopy............................................................................................... 80
5.4.9 Statistical analysis .................................................................................................................................. 80
5.5
RESULTS AND DISCUSSION........................................................................................................................ 82
5.5.1 Composition ............................................................................................................................................... 82
5.5.2 Free thiol groups concentration ....................................................................................................... 83
5.5.3 Gel electrophoresis .................................................................................................................................. 85
5.5.4 Confocal laser scanning microscopy images............................................................................... 89
5.5.5 Proposed mechanism for association of whey proteins with buttermilk constituents
93
5.6
CONCLUSIONS ............................................................................................................................................. 94
5.7
ACKNOWLEDGEMENTS.............................................................................................................................. 95
CHAPTER 6 EFFECT OF HEATING OF WHEY PROTEINS IN THE PRESENCE OF MILK FAT
GLOBULE MEMBRANE EXTRACT OR PHOSPHOLIPIDS FROM BUTTERMILK .......................97
6.1
RÉSUMÉ ....................................................................................................................................................... 98
6.2
ABSTRACT ................................................................................................................................................... 99
6.3
INTRODUCTION .......................................................................................................................................... 99
6.4
MATERIALS AND METHODS ................................................................................................................... 101
6.4.1 Materials....................................................................................................................................................101
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6.4.2 Isolation of the milk fat globule membrane .............................................................................. 101
6.4.3 Heat treatment ....................................................................................................................................... 102
6.4.4 Analytical method ................................................................................................................................. 102
6.4.5 Confocal laser-scanning microscopy ............................................................................................ 103
6.4.6 Statistical analysis ................................................................................................................................ 104
6.5
RESULTS AND DISCUSSION ....................................................................................................................104
6.5.1 Free thiol group concentration ....................................................................................................... 104
6.5.2 Gel electrophoresis ................................................................................................................................ 108
6.5.3 Confocal laser-scanning microscopy images ............................................................................ 109
6.5.4 Thin layer chromatography ............................................................................................................. 113
6.6
CONCLUSIONS...........................................................................................................................................115
6.7
ACKNOWLEDGEMENTS ...........................................................................................................................116
CHAPTER 7 EFFECT OF FREE THIOL GROUP REACTIVITY DURING THE FORMATION
OF HEAT-INDUCED AGGREGATES FROM WHEY AND BUTTERMILK ON THEIR
PROPERTIES 117
7.1
RÉSUMÉ .....................................................................................................................................................118
7.2
ABSTRACT .................................................................................................................................................119
7.3
INTRODUCTION ........................................................................................................................................119
7.4
MATERIALS AND METHODS ...................................................................................................................121
7.4.1 Materials.................................................................................................................................................... 121
7.4.2 Preparation of the whey and buttermilk concentrates ....................................................... 122
7.4.3 Preparation of protein aggregates powders ............................................................................ 123
7.4.4 Particle size distribution in heated liquid mixtures .............................................................. 124
7.4.5 Protein solubility index at pH 6.8 ................................................................................................... 125
7.4.6 Exposition of free thiol groups of the mixed aggregates .................................................... 125
7.4.5 Statistical analysis ................................................................................................................................ 125
7.5
RESULTS ....................................................................................................................................................126
7.5.1 Heat-induced aggregation characterization ........................................................................... 126
7.5.2 Properties of the mixed aggregates .............................................................................................. 128
7.6
DISCUSSION ..............................................................................................................................................130
7.7
CONCLUSION.............................................................................................................................................132
7.8
ACKNOWLEDGMENTS .............................................................................................................................133
CHAPTER 8 EFFECT OF SUBSTITUTION OF SKIM MILK POWDER BY
WHEY:BUTTERMILK HEAT-DENATURED PROTEIN AGGREGATES IN MODEL SET-TYPE
YOGURT
135
8.1
RÉSUMÉ .....................................................................................................................................................136
8.2
ABSTRACT .................................................................................................................................................137
8.3
INTRODUCTION ........................................................................................................................................137
8.4
MATERIALS AND METHODS ...................................................................................................................138
8.4.1 Materials.................................................................................................................................................... 138
8.4.2 Preparation of whey:buttermilk heat-denatured aggregates.......................................... 139
8.4.3 Yogurt production................................................................................................................................. 139
8.4.4 Analytical methods ............................................................................................................................... 140
8.4.5 Confocal laser scanning microscopy............................................................................................. 142
8.4.6 Statistical analysis ................................................................................................................................ 143
8.5
RESULTS AND DISCUSSION .....................................................................................................................144
8.5.1 Whey:buttermilk protein aggregates composition and properties ............................... 144
8.5.2 Particle size in skim milk ................................................................................................................... 145
8.5.3 Exposure of thiol groups .................................................................................................................... 146
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8.5.4 Distribution of particles in enriched skim milk before acidification .............................148
8.5.5 Texture of yogurts .................................................................................................................................148
8.5.6 Water-holding capacity of yogurt gels ........................................................................................151
8.5.7 Simulation of the gel formation ......................................................................................................153
8.7
CONCLUSIONS .......................................................................................................................................... 156
8.8
ACKNOWLEDGEMENTS........................................................................................................................... 156
CHAPTER 9 GENERAL CONCLUSIONS.......................................................................................... 157
9.1
ACHIEVEMENTS AND ORIGINAL CONTRIBUTIONS .............................................................................. 157
9.2
SIGNIFICANCE OF THE RESULTS ............................................................................................................ 163
9.3
QUESTION YET TO BE ANSWERED AND PERSPECTIVES ..................................................................... 164
CHAPTER 10
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LITERATURE CITED.............................................................................................. 167
TABLE LIST
Table 1.1: Proposed applications for the use of buttermilk in food formulation
(Vanderghem et al., 2010). ............................................................................................. 2
Table 2.1: Comparison between the gross composition of buttermilk and skim milk
(Ramachandra Rao et al., 1995; Walstra et al., 2006). ................................................... 5
Table 2.2: Comparison between the gross composition of milk, acida, and sweetb cheese
whey (Britten et al., 2002; Smithers, 2008). ................................................................... 8
Table 2.3: Summary of physical and chemical properties of whey proteins adapted from
Cayot and Lorient (1998), Kinsella and Whitehead (1989a), Morr and Ha (1993), and
Rüegg et al. (1977)........................................................................................................ 11
Table 2.4: Summary of physical and chemical properties of bovine milk fat globule
membrane proteins adapted from Cheng et al. (1988), Dewettinck et al. (2008), Heid
et al. (1996), Hvarregaard et al. (1996), Pallesen et al. (2001), Singh (2006), and
Stammers et al. (2000). ................................................................................................. 15
Table 2.5: Lipid composition of buttermilk (adapted from Keenan and Dylewski (1995) and
Walstra et al. (2006)...................................................................................................... 18
Table 2.6: Possible positive and negative effects of fat replacers (adapted from Senanayake
and Shahidi (2005). ....................................................................................................... 43
Table 4.1: Composition (% DM) of the various whey-buttermilk mixtures concentrated at
9.5% protein (w/v) used for the heating experiments ................................................... 57
Table 4.2: Summary of the significance (P values) calculated by the analysis of variance of
data of each contrasts for each variables (n = 3)........................................................... 59
Table 4.3: Average particle size and span of untreated and treated whey-buttermilk
mixtures acidified at pH 4.6, denatured at 90°C and homogenized at 65.50 MPa. ...... 65
Table 5.1: Summary of the significance (P value) by the analysis of variance of data of
each contrast for each variable (n = 3). ......................................................................... 81
Table 5.2: Composition (% DM) of whey protein concentrate (WPC) and the different
buttermilk concentrates (BC) powders used for the heating experiment with rBC:
regular buttermilk concentrate, rBCSFE: regular buttermilk concentrate after
supercritical fluid extraction, wBC: whey buttermilk concentrate, wBCSFE: whey
buttermilk concentrate after supercritical fluid extraction. ........................................... 82
Table 6.1: Summary of the significance calculated by the analysis of variance of free thiol
group concentration (n = 3). ....................................................................................... 105
Table 7.1: Summary of the significance (P value) calculated by the analysis of variance of
data of each contrasts for each variables (n = 3)......................................................... 126
Table 8.1: Summary of the significance of the effects of WBAP and time on the texture and
water-holding capacity of yogurt gels, as calculated by analysis of variance (n = 3).143
Table 8.2: Summary of the significance of the effects of WBAP and time on the response of
milk to heating, as calculated by analysis of variance (n = 3). ................................... 144
Table 8.3: Composition of heat-denatured whey:buttermilk aggregates (WBAP), whey
permeate (WP), and powdered skim milk. ................................................................. 145
Table 8.4: Summary of the mean rupture force, adhesiveness and relaxation of yogurt gels,
as a function of percent substitution of powdered skim milk with whey:buttermilk
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aggregates as enrichment of the starting milk. A Duncan post-test was applied to
compare the means to the control. .............................................................................. 150
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FIGURE LIST
Figure 2.1: Schematic representation of the submicelle model of the casein micelle
(represented by Horne (2006). ........................................................................................ 9
Figure 2.2: Schematic representation of bovine milk fat globule membrane (from JiménezFlores’ group). .............................................................................................................. 14
Figure 2.3: Schematic representation of major protein aggregation pathways..................... 20
Figure 2.4: Schematic representation of the currently proposed pathways of formation of
the heat-induced whey proteins/-casein complexes in heated skim milk from Donato
et al. (2009). .................................................................................................................. 34
Figure 4.1: Experimental procedure used to prepare aggregated buttermilk and whey
proteins mixtures........................................................................................................... 55
Figure 4.2: Effect of protein composition, NEM and ultrasound on aggregation yield of
different mixtures of whey and buttermilk concentrates (9.5 % protein), acidified at
pH 4.6 and co-denatured at 90°C. ................................................................................. 60
Figure 4.3: Effect of protein composition, application of ultrasound and addition of NEM
on water-holding capacity (WHC) of mixed buttermilk-whey protein mixture (9.5 %
protein) heated at pH 4.6 (90°C). .................................................................................. 61
Figure 4.4: Effect of protein composition, application of ultrasound and addition of NEM
on consistency index (k) of mixed buttermilk-whey protein mixture (9.5 % protein)
heated at pH 4.6 (90°C). ............................................................................................... 62
Figure 4.5: Effect of protein composition, NEM and ultrasound on flow behavior indices of
different mixtures of cheese whey and buttermilk concentrates (9.5 % protein),
acidified at pH 4.6 and co-denatured at 90°C. .............................................................. 63
Figure 5.1: 15% Non-reducing SDS gels of the mixtures. 1: protein standard, 2: WPC, 3:
WPC + rBC, 4: WPC + rBCSFE, 5: WPC + wBC, 6: WPC + wBCSFE. ........................ 83
Figure 5.2: Effects of time (a) and composition of the mixture (b) on the accessibility of
free thiol groups during heating with constant stirring at temperature up to 90°C at pH
4.6. ................................................................................................................................ 84
Figure 5.3: Evolution of the protein profile of WPC as a function of heating time (min)
under non-reducing (a) and reducing (b) conditions. ................................................... 86
Figure 5.4: Evolution of the protein profile of the WPC + rBC mixture as a function of
heating time (min) under non-reducing (a) and reducing (b) conditions. ..................... 86
Figure 5.5: Evolution of the protein profile of the WPC + wBC mixture as a function of
heating time under non-reducing (a) and reducing (b) conditions. ............................... 86
Figure 5.6: Evolution of the protein profile of the WPC (a), WPC + rBCSFE mixture (b),
and WPC + wBC (c) in presence of NEM as a function of heating time under nonreducing conditions. ...................................................................................................... 88
Figure 5.7: Confocal laser scanning miscroscope pictures taken at 100X of WPC at 0, 15,
and 25 minutes of heating before (top) and after (bottom) analysis with ImageJ. ....... 89
Figure 5.8: Confocal laser scanning microscope pictures taken at 100X of WPC + rBC
mixture at 0, 15, and 25 minutes of heating before (top) and after (bottom) analysis
with ImageJ. .................................................................................................................. 90
Figure 5.9: Evolution of the average surface area observed with a confocal laser scanning
microscope at 100X as a function of interactions time*composition (a) and
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time*condition. In a), mixtures were presented as follows:  = WPC;  = WPC +
rBC;  = WPC + rBCSFE;  = WPC + wBC  = WPC + wBCSFE. In b), conditions
were presented as follows:  = absence of NEM;  = presence of NEM. ................ 91
Figure 5.10: Three-dimensional confocal laser scanning microscope pictures of WPC +
rBC mixture before heating. ......................................................................................... 92
Figure 6.1: Effect of interactions composition*temperature (a) and temperature*pH (b) on
the exposition of free thiol groups during heating of WPI (W.), WPI+MFGM extract
(W.+M.), and WPI+phospholipid powder (W.+P.) under constant stirring. ............. 105
Figure 6.2: Pictures of heated WPI (a), WPI+MFGM (b), and WPI+PL (c) at pH 4.6 at
80°C and after 60 minutes of sedimentation in ice-melted water bath. ..................... 107
Figure 6.3: Evolution of the protein profiles in the different mixtures at 0 (a), 10 (b), 15 (c),
20 (d) minutes of heating at 80°C using SDS-PAGE under non-reducing conditions.
Letters a to d correspond to samples at pH 6.8 and letters a' to d' to pH 4.6. ............ 108
Figure 6.4: Three-dimensional confocal laser-scanning microscope pictures of WPI heated
for 25 minutes at 80°C at pH 4.6. ............................................................................... 110
Figure 6.5: Three-dimensional confocal laser-scanning microscope pictures of the
WPI+MFGM extract heated for 15 minutes at 80°C at pH 4.6. ................................ 111
Figure 6.6: Three-dimensional confocal laser-scanning microscope pictures of WPI+PL
heated for 15 minutes at 65°C at pH 4.6 .................................................................... 112
Figure 6.7: Three-dimensional confocal laser-scanning microscope pictures of WPI heated
for 15 minutes at 80°C at pH 6.8. ............................................................................... 113
Figure 6.8: Evolution of the phospholipids profiles in WPI+PL at 0 (a), 10 (b), 15 (c), 20
(d), and 25 (e) minutes of heating time at 80°C. Picture was converted in negative
mode in order to make the spot easier to see.............................................................. 114
Figure 7.1: Effect of the presence of NEM during formation of mixed aggregates from
heating (90°C – 5min) at low pH (4.6) mixtures containing 50% of the protein from
WPC and 50% from buttermilk concentrates on the number-weighted average particle
size distribution in the mixtures. ................................................................................ 127
Figure 7.2: Effect of the presence of NEM during formation of mixed aggregates from
heating (90°C – 5min) at low pH (4.6) mixtures containing 50% of the protein from
WPC and 50% from buttermilk concentrates on the aggregation yield. .................... 128
Figure 7.3: Effect of the presence of NEM during formation of mixed aggregates from
heating (90°C – 5min) at low pH (4.6) mixtures containing 50% of the protein from
WPC and 50% from buttermilk concentrates on the water-holding capacity of the
aggregates. .................................................................................................................. 128
Figure 7.4: Effect of the composition and the presence of NEM during formation of mixed
aggregates from heating (90°C – 5min) at low pH (4.6) mixtures containing 50% of
the protein from WPC and 50% from buttermilk concentrates on nitrogen solubility
index of the powders. Aggregates formed in the absence of NEM are represented in
black and aggregates formed in the presence of NEM are represented in grey. ........ 129
Figure 7.5: Effect of the composition and the presence of NEM during formation of mixed
aggregates from heating (90°C – 5min) at low pH (4.6) mixtures containing 50% of
the protein from WPC and 50% from buttermilk concentrates on the accessibility of
free thiol groups of the mixed aggregates. ................................................................. 130
Figure 8.1: Particle size distribution profile in skim milk after 5 min at 85°C, with added
powdered skim milk substituted 0% () 20% (), 40% (), 60% (), 80% () and
100% () with WBAP. .............................................................................................. 146
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Figure 8.2: Effect of milk composition on thiol group exposure following heating at 85°C
for 25 minutes (including ramp time). A Duncan post-test was applied to compare the
means to the control. ................................................................................................... 147
Figure 8.3: Images of control (enriched with powdered skim milk) skim milk (a) and skim
milk + WBAP aggregates (0.6 g/100 mL; b) at pH 6.8, using the Nikon C1 confocal
laser scanning microscope at 60X. Proteins were dye in green and phospholipids in
red. .............................................................................................................................. 148
Figure 8.1: Firmness of control set-type yogurt, and yogurts enriched with whey:buttermilk
protein aggregates. A Duncan post-test was applied to compare the means to the
control. ........................................................................................................................ 149
Figure 8.5: Water-holding capacity of set-type yogurt, and yogurts made from milk
enriched with whey:buttermilk protein aggregates. .................................................... 152
Figure 8.6: Confocal laser scanning microscope (Nikon C1) images taken of gels of control
skim milk (a) and skim milk + WBAP (0.6 g/100 mL; b) at 60X with a zoom of
2.31X; proteins were dyed green and phospholipids are red. Images a’ and b’ were
processed with ImageJ to color the cluster in red. ...................................................... 153
Figure 8.7: General trends observed in the videos of gel formation as a function of the pH
and time....................................................................................................................... 155
Figure 9.1: Schematic representation of the proposed mechanism of aggregation of proteins
from WPC and the properties of the heat-induced protein aggregates. ...................... 161
Figure 9.2: Schematic representation of the proposed mechanism of aggregation of
constituents from WPC + BC mixtures and the properties of the heat-induced protein
aggregates. .................................................................................................................. 162
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EQUATION LIST
Equation 4.1: Calculation of the aggregation yield .............................................................. 57
Equation 4.2: Calculation of the water-holding capacity of protein aggregates ................... 57
Equation 4.3: Formula of the Power Law Model ................................................................. 58
Equation 7.1: Calculation of the aggregation yield ............................................................ 124
Equation 7.2: Calculation of the water-holding capacity of the aggregates ....................... 124
Equation 7.3: Calculation of the protein solubility index at pH = 6.8 ................................ 125
Equation 8.1: Calculation of the relaxation of acid set-type yogurt ................................... 141
Equation 8.2: Calculation of the water-holding capacity of the gel ................................... 141
Equation 8.3: Calculation of the theoretical concentration of thiol group.......................... 142
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ABREVIATIONS
-LA: alpha-lactalbumin
-LG: beta-lactoglobulin
-CN: -casein
ADPH: adipophilin
BC: buttermilk concentrate
BSA: bovine serum albumin
BTN: butyrophilin
CD36: cluster of differentiation 36
CLSM: confocal laser scanning microscope
FABP: fatty-acid binding protein
k: Consistency coefficient
n: Flow behavior index
MFGM: milk fat globule membrane
MUC1: Mucin1
MWP: microparticulated whey proteins
NEM: N-ethylmaleimide
PAS 6/7: periodic acid Shiff 6/7
PAS III: periodic acid Schiff III
PC: phosphatidyl choline
PE: phosphatidyl ethanolamine
PI: phosphatidyl inositol
PL: phospholipids
xix
PP: permeate powder
PS: phosphatidyl serine
SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis
SM: sphingomyelin
SMP: skim milk powder
SH/SS: Thiol/Disulfide
TLC: thin layer chromatography
WHC: water-holding capacity
WPC: whey protein concentrate
WPI: whey protein isolate
XDH/XO: xanthine dehydrogenase/oxidase
xx
ACKNOWLEDGMENTS
This work represents an achievement of four years of research during which I crossed the
path of many important people that I shall therefore acknowledge.
First of all, I would like to express my sincere gratitude to my advisor Dr. Yves Pouliot.
When I met Dr. Pouliot in 2007, I was a kid with a head full of dreams. With his trust,
advice, continual support, confidence, and his guidance I grew up quickly in my thinking,
the planning of my experiments, and the discussion of my results. Most of all, Dr. Pouliot
always had a challenge waiting for me, and at the end the kid was able to realize all of his
dreams. I feel very fortunate and I am very thankful for all the opportunities he gave me. As
the song goes “Je ne peux pas te dire ce que je ne peux pas écrire. Il faudrait que j’invente
des mots qui n’existent pas dans le dico” but one day I will find a way to express all of my
thankfulness towards you.
I would also like to express my gratitude to my co-advisor Dr. Michel Britten. Dr Britten
has always been very generous in his advice, explanations, comments, corrections, and
support. His powerful brain has been the cause of few sleepless nights, but I believe that
working along Dr. Britten made me stronger in my thinking, the expression of my ideas,
and definitely opened my eyes. I hope that our paths will cross again in the future and I will
be always ready to discuss with you the different possibilities to explain one result.
I would also like to express my gratitude to my co-advisor Dr. Rafael Jiménez-Flores. We
often joke by saying that working along Dr. Jiménez-Flores would be the biggest challenge
I faced during this four years of grad school. I found this to be true but it was also the most
enjoyable challenge of all. He welcomed me in his arms (no second degree here) in 2010
and later in his scientific team in 2011. Dr. Jiménez-Flores has been a source of inspiration
for me and definitely a source of joy. I am thankful for his guidance, his support, and the
trust he put in me. I am also thankful for the opportunity to manage different projects,
xxi
advise different candidates in their studies, meet people from industries, and present my
results all over California.
I am also thankful toward Dr. Jean-Christophe Vuillemard for accepting to do the prereading of this thesis. Dr. Vuillemard has always been very supportive toward this project
since day one. He has been generous many times with his comments and his suggestions
for the advancement of my experiments. Many thanks to Dr. Fanny Guyomarc’h for joining
the committee of my thesis. Dr Guyomarc’h’s work has been a source of inspiration during
the planning of many parts of this project.
I am thankful toward Drs. Laurent Bazinet, Sylvie Gauthier, and Sylvie Turgeon. They
have been very supportive during the last four years regarding my advancements in my
scientific and personal life.
Of course, nothing would have been done without the help of some person. Many many
many thanks to the extraordinary Diane Gagnon form the STELA Research Group for
teaching me all the lab techniques that I know, for her patience, her availability, her
support, and of course her candies … Many thanks to the people from the DPTC to
welcome me, for their patience, their understanding, their help, their support, and for never
have laughing at my French accent. Many thanks to the interns that have worked at my
side: Justine, Eva, Véronique, Sylvain, Camille et Mélanie.
I would like to express all of my gratitude to Frederic Lehance. Frederic was the first
person to make me appreciate Quebec City and the Quebec experience in general. He
supported from day one (back in 2007) in my studies and in my personal life. He has
always been here for me the last six years, listening to my problems, giving me support and
advice. He is the belgio-punko big brother that I always wanted.
I also must acknowledge the incredible support I had from my friends at home (particularly
Dr. Alexandre). I established very strong relationships since I moved in Canada, and I
xxii
would like to express my gratitude to my very best friends Dr. Jeremie and Dr. Romain
who have always been here for me. The expression “away from the eyes, away from the
heart” is not in their vocabulary, so I never felt alone and lost during my Californian
experience. Many thanks to the very good friends that I made during this journey:
Benjamin, Laure, Bertrand, Cyril, Ryan, Mason, BJ, Anthony, and David.
I am very grateful to have met my lovely girlfriend Melissa. Her support,
comprehensiveness, understanding, encouragement gave me the strength to pursue and
complete my degree. Thanks my love for being at my side, even in this scary cold French
speaking part of the world. Many thanks to her family for being so supportive and for
letting her come in Quebec City.
I am very fortunate to have a wonderful family. I have an enormous respect for them
because letting go of their little boy was probably not easy, but they always expressed their
understanding and their gigantic support. I am very proud of you and would like to express
all of my love to you Danièle & Lucien, Céline & Julien & Justine & Nathan, Marthe &
Dominique, Marie-Rose & Hubert.
Last but not least, I am thankful towards Le Fond Québécois de la Recherche sur la Nature
et les Technologies (FQRNT) – Novalait Inc. – Ministère de l’Agriculture et de
l’Alimentation du Québec (MAPAQ) for their financial support.
xxiii
xxiv
FOREWORD
The work carried out in this thesis was aimed to develop a new approach to use whey and
buttermilk, and to understand the interactions that occur between components of both
liquids. This thesis contains all the results obtained during the course of realization of the
project. Results are presented in the form of five published, submitted, or in preparation
research articles.
The first article «Thermal aggregation of whey proteins in the presence of buttermilk
concentrate» has been submitted and published in «Journal of Food Engineering N°103» in
2011. Authors are Maxime Saffon, Michel Britten, and Yves Pouliot.
The second article «Effect of buttermilk components on the heat-induced denaturation of
whey proteins» is in preparation for a submission in «Journal of Agriculture and Food
Chemistry». Authors are Maxime Saffon, Rafael Jiménez-Flores, Michel Britten, and Yves
Pouliot.
The third article «Effect of heating of whey proteins in the presence of milk fat globule
membrane extract or phospholipids from buttermilk» is pending for submission. Authors
are Maxime Saffon, Rafael Jiménez-Flores, Michel Britten, and Yves Pouliot.
The fourth article «Effect of buttermilk constituents on the properties of heat-induced
protein aggregates» is pending for submission. Authors are Maxime Saffon, Rafael
Jiménez-Flores, Michel Britten, and Yves Pouliot.
The fifth article «Effect of substitution of skim milk powder by whey:buttermilk heatdenatured aggregates in model set-type yogurt» is in preparation for a submission in the
Special Issue «Structure of Flavor of Dairy Products» of Foods. Authors are Maxime
Saffon, Rafael Jiménez-Flores, Michel Britten, and Yves Pouliot.
xxv
For each article, Maxime Saffona planned and achieved the experiments, presented the
results, and wrote the article. In consequence, he is the first author of all of them. Dr.
Jiménez-Floresb, co-supervisor of this project, participated to the planning, the discussion
of the results, and the revision of the writing from the second to the fifth articles. He is
second author on these papers. Dr. Michel Brittenc, co-supervisor of this project,
participated to the planning, the discussion of the results, and the revision of the writing of
all articles. He is third author on all of them. Dr. Yves Pouliota, supervisor of this project,
participated to the planning, the discussion of the results, and the revision of the writing of
all articles. He is the last author on all of them.
a
STELA Dairy Research Center, Institute of Nutrition and Functional Foods (INAF), Laval University,
Quebec City, QC, Canada, G1V 0A6.
b
Dairy Products Technology Center (DPTC), California Polytechnic State University, San Luis Obispo, CA,
USA, 93405.
c
Food Research and Development Center (FRDC), Agriculture and Agri-Food Canada, St-Hyacinthe, QC,
Canada, J2S 8E3.
xxvi
A Danièle, Lucien, Céline
Et mes grands-parents
«Les portes de l’avenir s’ouvrent
à ceux qui savent les pousser»
-Michel Colucci
1
CHAPTER 1
INTRODUCTION
The worldwide consumption of milk or dairy products is constantly increasing. It is
estimated that the production of milk increases about 1.5% every year in order to satisfy the
needs of consumers. The increase of consumption of dairy products such as cheese or butter
also increases the production of dairy by-products such as whey or buttermilk. As an
example, the production of whey and buttermilk in Canada in 2008 has been calculated
around 3,535,000 tons and 82,600 tons, respectively (Statistics Canada, 2009). Over the
years, the opinion about these by-products has changed. It is now recognized that whey and
buttermilk constituents have interesting biologic, nutritional properties, and health-benefits.
Many applications have been developed to use whey in food formulation, such as in cheese,
bakery, pastry, and delicatessen productions. Unfortunately, buttermilk has not found as
many industrial applications as whey yet. Recently, Vanderghem et al. (2010) summarized
ten potential applications for buttermilk or buttermilk constituents that have been tested in
research labs over the last 20 years (Table 1.1).
Buttermilk is a unique product due to its concentration of milk fat globule membrane
(MFGM) and associated material (proteins, phospholipids, and sphingolipids) that have
been associated with very promising health properties ranging from anti-viral to anticancer. Buttermilk contains other constituents with potential food applications such as
caseins (75% of the total proteins), whey proteins (8 to 15% of the total proteins), and polar
lipids. However, it has been demonstrated that the exploitation of the buttermilk
constituents is complicated due to some irreversible changes that appear along the process.
Previous work of Morin (2006) showed that the pasteurization of the cream is critical
because it affects the solubility at pH 4.6 of MFGM proteins, modifies the surface of the fat
membrane, and increases the accessibility of phospholipids. Phospholipids have an
important capacity to hold water, so the increase of their exposure limits the use of
buttermilk in dairy products such as cheese or yogurt. Above all, pasteurization of cream
1
has been correlated to the poor coagulation properties of buttermilk. Upon the treatment,
MFGM proteins initiate heat-induced interactions with other proteins.
Table 1.1: Proposed applications for the use of buttermilk in food formulation (Vanderghem et al.,
2010).
Sources
Desired Properties
Fresh buttermilk
milk
sweet Viscosity agent
Cheddar cheese
buttermilk
Condensed
Effects
Agent for the heat Recombined evaporated Stability increased
stability
Ultrafiltered
Applications
Higher
apparent
viscosity
sweet Agent for the yield, Pizza cheese
cream buttermilk
Higher
cheese
texture,
meltability,
moisture
and
coagulation
Decreased
Moisture retention
yield,
content.
chewy,
Reduced meltability and
gel strength
Buttermilk
Moisture retention
Bread
Higher water absorption,
Agent for the dough
Increased resistance to
and
extension,
sensory
properties
Better
sensory score
Whey proteins are also very sensitive to heating. A process has been developed to use whey
protein aggregates in food formulation. Some of their industrial applications are, however,
limited due to the water-holding capacity of denatured whey proteins. Combination of
buttermilk and whey could be the key to better exploit their constituents in food
formulation by reducing their individual negative effects such as extensive water retention.
It is now clear that caseins, whey proteins, and MFGM proteins interact together under
heating. It can be thought that it is possible to control the composition and properties of
aggregates by using different ratio and/or experimental conditions.
2
The goal of this work was to develop a new approach to use buttermilk constituents based
on the knowledge of the thermal behavior of its constituents in order to obtain new
potential dairy ingredients with texture modification applications for food formulation.
3
4
CHAPTER 2
LITERATURE REVIEW
2.1 Buttermilk and whey
2.1.1 Buttermilk
Buttermilk is the aqueous phase expulsed after the formation of large butter grains during
churning of cream (Boudreau & St-Amant, 1984). As air is incorporated during the buttermaking process, proteins from the cream unfold and form unstable foam with the air
bubbles. Upon mechanical stress, the foam is destabilized resulting of fat clumping and
phase inversion. Butter becomes solid while the water and soluble particles form the
buttermilk. Most of the protein, minerals, lactose, and water from the cream are recovered
in the buttermilk (Table 2.1). A high portion of the MFGM is also present in the buttermilk.
Table 2.1: Comparison between the gross composition of buttermilk and skim milk (Ramachandra Rao
et al., 1995; Walstra et al., 2006).
Constituents
Buttermilk
Total Solids (%)
9.50 – 10.50
Skim Milk
9.40
Lactose (%)
3.60 – 4.30
4.80
Proteins (%)
3.30 – 3.90
3.36
Fat (%)
0.30 - 0.70
0.07
Phospholipids (%)
Ash (%)
0.07 – 0.18
0.55 – 0.90
0.015
0.88
Churning 1 kilogram of cream will typically yield 0.5 kilogram of butter and 0.5 kilogram
of buttermilk. Liquid buttermilk is prone to oxidation, so this product is concentrated by
evaporation and then sprays dried in order to extend the shelf life. Currently, dried
buttermilk is mainly used for animal feeding (swine, bovine), but also used in the food
industry for its emulsion properties and its positive impacts on the texture and taste of
products.
5
Buttermilk is the richest dairy product in phospholipids. In consequence, addition of
phospholipids (through substitution of milk by buttermilk) has been tested for the
production of low-fat cheese (Turcot et al., 2002; Turcot et al., 2001). Addition of
buttermilk increased the cheese yield, but notable changes in the texture and taste of cheese
have been recorded. These results have been correlated to the high water-holding capacity
of phospholipids. Authors concluded that the amount of phospholipids should be carefully
calculated before addition in cheese. They suggested enriching the milk for the cheesemaking process (40 gram of proteins per kilogram of milk) with less than one-gram of
phospholipids per kilogram of milk to make sure that the texture or taste will not be
affected. Earlier, Mistry et al. (1996) have reported that it is possible to substitute milk with
liquid buttermilk prior to the cheese-making process without changing the properties of the
cheese if the level of substitution is under five percent. Later, Raval and Mistry (1999)
observed that cheeses had a lower fat content (lower free oil) and harder bodies if the milk
was supplemented with buttermilk (5%). However, cheese had a lower meltability, and a
higher apparent viscosity comparatively to the control cheese.
2.1.1.1
Specialized «biorefinery» approach
As described above, buttermilk is rich in minor components associated with the MFGM
fragments (proteins, phospholipids). These components have demonstrated promising
functional properties and health benefits. Morin (2006) focused on the development of an
approach for the separation of MFGM material using microfiltration. Their results showed
that the similitarity between the size of the casein micelle and the MFGM fragments made
the separation very challenging. Later, they demonstrated that separation can be improved
by addition of a cream washing step prior to the microfiltration in order to remove the
casein micelles.
However, Bédard ST-Amand (2009) have shown that the industrial HTST pasteurization of
the cream is critical for the MFGM fragments. Their results showed that several proteins
(probably located at the surface of the bilayer) have broken off the fragments. In
6
opposition, some of the available Bovine Serum Albumin and β-lactoglobulin proteins are
associated with the MFGM fragments.
Regarding the difficulties to separate buttermilk constituents and the modification that
occur during the butter-making process (solubilization of some MFGM proteins), a global
approach for the utilization of buttermilk seems more appropriate.
2.2.1.2
Global approach
As described in Table 1.1, over ten applications have been proposed for the utilization of
buttermilk in food formulation. The proposed applications are ranged from an agent for
moisture retention in bread to a viscosity agent for cheddar cheese.
It is also possible to vary the composition of buttermilk for the valorization of a specific
constituent. For example, Costa et al. (2010) have proposed an approach to produce a whey
buttermilk powder enriched in milk fat globule membrane phospholipids. Whey buttermilk
is a very unique product that is richer in MFGM material comparatively to regular
buttermilk. By combining ultrafiltration (10X) and CO2-supercritical fluid extraction (350
bars; 50°C) they obtained a powder containing 73% of whey proteins and 21% of lipids of
which 61% were phospholipids.
2.1.2 Whey
Whey is the aqueous phase separated from casein network formed through chymosin or
mineral/organic acid during cheese-making or casein manufacture process. Depending on
the cheese production, whey components represent approximately 50% of the initial milk
solids with 20% of the proteins and 100% of the lactose (Table 2.2). Whey has been
considered as a waste product during many years and as one of the most polluting food byproducts due to its high content in lactose (> 75% of the dry basis).
7
Table 2.2: Comparison between the gross composition of milk, acida, and sweetb cheese whey (Britten et
al., 2002; Smithers, 2008).
Total solids (%)
Milk
Acid whey
Sweet whey
12.80
6.60
6.80
Lactose (%)
4.90
4.40
4.90
Total proteins (%)
3.50
0.70
0.70
Caseins (%)
2.80
< 0.1
< 0.1
Whey proteins (%)
0.70
0.70
0.80
Fat (%)
3.70
0.06
0.15
Ash (%)
0.70
0.37
0.31
However, whey represents a rich and heterogeneous content of protein with nutritional,
biological and food attributes that give to it potential applications in food processing.
Different technologies have been developed to concentrate (35 to 80% of protein) or isolate
(> 90% of protein) whey proteins such as ultrafiltration or ultrafiltration/diafiltration. Whey
protein concentrates (WPC) or isolates (WPI) have a relatively low value, and are widely
produced in dairy industry. These powders have different applications in food processing
industries where water binding and texturization are required, such as gelation, thermal
stability, foam formation, and emulsification. Their uses vary from manufactured meats to
formulated foods or reformed food products (Chatterton et al., 2006; Foegeding et al.,
2002).
2.2 Constituents from buttermilk and whey
2.2.1 Caseins
Caseins are the predominant proteins in buttermilk with around 75% of the total protein
content (Walstra et al., 2006). Caseins are very different comparatively to other proteins.
They have little secondary and tertiary structures, and are hydrophobic with a high charge.
The exposed hydrophobic groups give the protein a strong ability for self-interactions or
association with other proteins (Walstra et al., 2006).
8
In a milk system, caseins are present as colloidal particles named casein micelles. Caseins
micelles are composed of four major proteins: S1 (33%),  (33%), S2 (11%),  (11%),
plus calcium phosphate (8 g.100g-1 of casein), a small portion of proteose peptone and
some enzymes (Brulé et al., 1997; Walstra et al., 2006). Casein micelles have an average
diameter ranged from 40 to 300 nanometers and have a negative charge. The presence of
casein micelles in milk is very important because it determines the physical stability of
milk products during process and storage and the viscosity of products concentrated in
proteins (Walstra et al., 2006).
Over the years, various structural models have been proposed for the casein micelles. The
most enduring model has been initiated by Schmidt (1980), and consists of submicelles
connected together via hydrophobic bonds and calcium phosphate (Figure 2.1).
Figure 2.1: Schematic representation of the submicelle model of the casein micelle (represented by
Horne (2006).
Each submicelle would be composed of 20 to 25 casein molecules, and would have an
average diameter ranges from 12 to 15 nanometers. The submicelles located at the outside
9
of the micelles would contain one or two -caseins. The «hairy layer» of the casein
micelles would be formed by the C-terminal end of (~ 75 amino acid residues). The hairy
layer would be hydrophilic, negatively charged and would provide colloidal stability to the
casein micelle (Walstra et al., 2006).
-casein molecules are mostly present as oligomer of about 120 kDa. Each oligomer
contains from 5 to 11 monomers. -casein differs from the other caseins, due to the
presence of two cysteine residues that form an intermolecular disulfide bond (Cys11-Cys88),
and because are involved in SH/SS exchanges with whey proteins.
In buttermilk, caseins are also mostly found in micellar form. However, some variation in
the size of the casein micelle and its properties due to the processing can be expected.
Morin et al. (2008) found that the casein micelles from buttermilk have poor coagulation
properties due to the cream pasteurization. As describe later, heat-induced interactions
occur between -lactoglobulin and -casein located at the surface of the micelles.
2.2.2 Whey proteins
Whey proteins represent between 8 to 15% of the total proteins in buttermilk (depending of
the churning process) (Sodini et al., 2006), while they represent almost 100% of the
proteins in whey.
The two major whey proteins are -lactoglobulin (-LG) and -lactalbumin (-LA), and
they represent from 70 to 80% of the total protein content. The minor proteins are serum
albumin (BSA), immunoglobulin (Ig), proteose peptone (PP), lactoferrin (LF) and
lactoperoxydase (LP), and they represent about 20 % of the total proteins (Table 2.3).
10
Table 2.3: Summary of physical and chemical properties of whey proteins adapted from Cayot and
Lorient (1998), Kinsella and Whitehead (1989a), Morr and Ha (1993), and Rüegg et al. (1977).
Proteins Concentration (g.L-1) Mol. Weight (kDa)
pI
S-S (SH) TU (°C)
2.0 – 4.0
18.4
5.3
2 (1)
72.8
-LG
1.0 – 1.5
14.2
4.8
4
65.2
-LA
Ig
0.6 – 1.0
0.015 to1.0
5.5 – 6.8
BSA
0.1 – 0.4
66.0
5.1
17 (1)
62.2
PP
0.3 – 0.8
4.0 – 40.0
4.1 – 5.6
0
ND
LF
0.1
80.0
8.4 – 9.0
17
65 / 90
LP
0.01 – 0.03
78.0
9.5
6 (3)
ND
pI = isoelectric point; TU = unfolding temperature; ND = not determined.
Bovine -lactoglobulin represents about 50 to 55% of total whey proteins with a
concentration of 2.0 to 4.0 g.L-1 of milk (Morr et al., 1993). The protein is present as a
dimer of 36,400 Da at a neutral pH (Sawyer & Kontopidis, 2000), and starts to dissociate
into two identical monomers at a pH above 6.5 or below 3.5 (Hambling et al., 1992;
Sawyer et al., 2000; Townend et al., 1969). Monomeric -lactoglobulin is a small acidic
globular protein of 162 amino acids with a molecular weight around 18,300 Da (slightly
different with genetic variants). The secondary structure of the protein consists of three
short helices, an -helix, and nine strands of anti- parallel -sheets (Kinsella et al., 1989a).
Hydrophobic, ionic, and hydrogen-bond interactions between the peptide chains stabilize
the secondary and tertiary structure of the protein. In the native form, the disulphide bridges
Cys66-Cys160 and Cys106-Cys119 also stabilize its structure, and a free thiol group (free –SH)
is available in position Cys121 (Kinsella et al., 1989b). In the native form of the protein, the
free thiol group and the bond Cys106-Cys119 are in the hydrophobic cleft (Sawyer, 2003).
The free thiol group is mainly responsible for the thermal irreversible unfolding and
aggregation of the protein. -lactoglobulin is a resistant carrier of retinol, palminate, fatty
acids, vitamin D and cholesterol as review by de Wit (1998) and Madureira et al. (2007).
Foegeding et al. (2006) have demonstrated that -lactoglobulin has a comparable foam
overrun capacity to egg white (albumin) for the formation of meringues, angel food cake or
similar products. Their results brought the idea that WPC, WPI or -lactoglobulin isolate
could provide a cost-effective alternative to egg albumin in food formulation. The high
nutritional value and health benefits (anti-hypertensive, anti-cancer, hypocholesterolemic,
opiodergic, and anti-microbial), and the variety of its functional properties has already
11
made -lactoglobulin a primary choice for modern food and beverage formulation
(Chatterton et al., 2006).
Bovine -lactalbumin represents about 22% of total whey proteins, and it is the second
major protein with a concentration of 1.0 to 1.5 g.L-1 of milk (Morr et al., 1993). lactalbumin is also a small globular protein of 123 amino acids with a 14,200 Da molecular
weight. Four disulphide bridges stabilize its structure in position Cys6-Cys120, Cys28-Cys111;
Cys61-Cys77, and Cys73-Cys91 (Morr et al., 1993). -lactalbumin does not have a free thiol
group. The biological function of -lactalbumin is to support the biosynthesis of lactose (de
Wit, 1998). Bovine -lactalbumin is an excellent source of the essential amino acids
tryptophan and cysteine, and its high homology to human -lactalbumin made it useful for
the development of infant formulas.
Bovine serum albumin is present at about 0.1 to 0.4 g.L-1 in cow milk (Morr et al., 1993).
Bovine serum albumin is one of the biggest whey protein with 582 amino acid residues,
and a molecular weight around 66,000 Da (66,267). This protein possesses seventeen
intermolecular disulphide bonds, and a free thiol group at the residue Cys34. The unfolded
protein (rupture of intramolecular disulphide bonds) exposes hydrophobic side groups
initially buried to the aqueous phase. Bovine serum albumin binds free fatty acid for
transportation in the blood, and is an important source for the production of glutathione in
the liver (de Wit, 1998; Morr et al., 1993).
The immunoglobulins (Ig) are an heterogenous family of glycoproteins from 15,000 to
1,000,000 Da, and are present at the concentration of 0.6 to 1.0 g.L-1 in cow milk (Morr et
al., 1993). Korhonen et al. (2000) have reported that bovine serums contain three major
classes of immunoglobulins that are IgG, IgM, and IgA with a similar structure: two
identical light chains (23 kDa) and two identical heavy chains (53 kDa) connected together
through disulphide bonds. Approximately 80% of the bovine immunoglobulins are IgG (1
or 2). Immunoglobulins are antibodies that act as a carrier of passive immunity to the
newborn, and as an antimicrobial agent (de Wit, 1998; Madureira et al., 2007).
12
The proteose peptone (PP) fraction of milk is an heterogeneous group of
phosphoglycoproteins formed following proteolysis of the N-terminal region in the
sequence of -casein by plasmin, and possibly other milk proteins, lipoproteins, and
proteolipids. These molecules are amphiphilic because of their charged phosphate groups
and sequences of hydrophobic amino acid residues (Kinsella et al., 1989a).
Lactoferrin (LF) is present at 0.02 to 0.35 g.L-1 in bovine milk and 2.0 to 5.0 g.L-1 in human
milk. Lactoferrin is a monomeric glycoprotein of 689 amino acids residues and has a
molecular weight of around 75,000 Da (76,110). The protein possesses seventeen
intermolecular disulphide bonds, but no free thiol group (Kinsella et al., 1989a). Lactoferrin
is a transport agent for ferric iron (Fe3+), so it makes iron more available for absorption in
the gut. Lactoferrin also has antibacterial activity (de Wit, 1998).
Lactoperoxidase (LP) is present at the concentration of 0.01 to 0.03 g.L-1 of milk. It is one
of the most abundant enzyme in whey (0.25 to 0.5 % of the total whey proteins).
Lactoperoxidase is a single peptide chain containing 612 amino acid residues.
Lactoperoxidase has 4 to 5 potential sites for N-glycosylation. The protein also possesses
15 half cystines (Cals et al., 1991), and a ferric iron (Fe3+). Lactoperoxidase is a main
defensive system agent in mammals because of its antimocrobial and antiviral properties.
The protein provide a strong protection against invading micro-organisms (Madureira et al.,
2007).
2.2.3 Milk fat globule membrane proteins
The bovine milk fat globule membrane (MFGM) represents between 2% and 6% of the
total mass of the fat globule and is composed of a complex mix of proteins, glycoproteins,
phospholipids, triglycerides, cholesterol, enzymes, and minor constituents. The
composition of the MFGM varies as a function of the size of the fat globule, the breed, the
health status of the cow, or the stage of lactation (Keenan & Mather, 2006). The protein
13
content of the MFGM varies from 25% to 60% depending on the MFGM extraction
techniques and the analytical methods. Over 40 proteins have been identified. For a long
time, MFGM proteins have been classified according to their relative migration during
polyacrylamide gel electrophoresis in presence of SDS. The nomenclature of the MFGM
has been clarified by the Milk Protein Nomenclature Committee of the American Dairy
Science Association and reported by Mather (2000). The main proteins of bovine MFGM
are: Mucin 1 (MUC1), Xanthine dehydrogenase/oxidase (XDH/XO), Periodic acid Schiff
III (PAS III), Cluster of Differentiation (CD36), Butyrophilin (BTN), Adipophilin (ADPH),
Periodic acid Schiff 6/7 (PAS 6/7), and Fatty-acid binding protein (FABP) (Singh, 2006)
(Table 2.4). All these proteins are located through the membrane in specific positions
depending on their physical and physiological properties (Figure 2.2). The overall
isoelectric point of the MFGM was determined around 4.8 (Kanno & Kim, 1990). At higher
pH, the MFGM will be charged negatively.
Figure 2.2: Schematic representation of bovine milk fat globule membrane (from Jiménez-Flores’
group).
14
Table 2.4: Summary of physical and chemical properties of bovine milk fat globule membrane proteins
adapted from Cheng et al. (1988), Dewettinck et al. (2008), Heid et al. (1996), Hvarregaard et al. (1996),
Pallesen et al. (2001), Singh (2006), and Stammers et al. (2000).
Proteins
Percent of the
Molecular Weight
MFGM proteins
(kDa)
pI
-SS- (SH) Td (°C)
(%)
MUC1
n.f.
160 to 200
< 4.5
0 (0)
n.f.
XDH/XO
20
150
7.7
11 (38)
< 60
PAS III
5
95 to 100
n.f.
n.f.
n.f.
CD36
5
76 to 78
n.f.
3 (0)
n.f.
BTN
20 to 43
67
5.0 to 5.4
1 (0)
58
ADPH
n.f.
52
7.5 to 7.8
n.f.
n.f.
PAS 6/7
n.f.
47 - 52
5.6 to 7.6
9 (0)
> 80
FABP
n.f.
13
n.f.
n.f.
n.f.
pI = isoelectric point; Td= temperature of denaturation; n.f. = not found
Mucin 1 (MUC1) is a mucin-type glycoprotein that is associated with the cream fraction
and is found at a concentration of 40 mg.L-1 of milk. It is a polymorphic protein of 160,000
Da to 200,000 Da. Mucin 1 is strongly bound to the serum-exposed side of the MFGM, but
can also become soluble after a short heat-shock. In this case, the protein is soluble in the
serum. Bovine MUC1 possesses one cysteine residue in the membrane-spanning region and
three cysteine residues in the cytoplasmatic tail (Pallesen et al., 2001). Snow et al. (1977)
reported that the protein contains 50% of carbohydrate (w/w) including sialic acid (30.5%),
N-acetylglucosamine (22.3%), galactose (15.9%), N-acetylgalactosamine (14.0%), mannose
(11.1%), and fucose (5.8%). The protein is acidic with an isoelectric point under 4.5, due to
its high content in sialic acid. The protein also possesses a highly glycosylated filamentous
portion located outside the MFGM. The role of Mucin 1 has been uncertain for many years,
however Kvistgaard et al. (2004) and Mather (2000) reported that the protein has a
protective effect against physical damages and rotavirus. These effects are attributed to the
filamentous portion of the protein.
15
Xanthine dehydrogenase/oxidase (XDH/XO) is the most abundant enzyme of the MFGM.
The protein is present in the form of a homodimer of 300,000 Da, where each monomer is
estimated to be around 148,000 Da. Twenty-two disulphide bonds reinforce the structure of
the protein. XDH/XO also contain thirty eight thiol residues, four of them are available and
thirty four are buried in the structure (Cheng et al., 1988). XDH/XO is a peripheral-attached
protein that is generally considered as weakly bound to the membrane, because it is
possible to remove approximately 60% of the total protein using appropriate buffers. In
opposition, the remaining 40% are firmly attached to the membrane (Mather, 2000). The
role of XDH/XO has been uncertain for many years, but it is believed that the protein could
have an antimicrobial function in the gut due to production of hydrogen peroxide.
Periodic acid Schiff III (PAS III) is a glycoprotein of 95,000 Da to 100,000 Da that has
been poorly characterized. However, it is known that PAS III is present in the serumexposed portion of the membrane (Mather, 2000).
Cluster of Differentiation (CD36) is an integral protein of the MFGM of 76,000 Da to
78,000 Da. The protein possesses ten cysteines residues (Berglund et al., 1996). Six of the
ten form the disulfide bonds Cys242-Cys310, Cys271-Cys332, and Cys312-Cys321, and the other
four are acylated near the intracellular side of the membrane (Rasmussen et al., 1998). The
protein does not have a free thiol group. CD36 is strongly attached to the membrane; even a
centrifugation treatment does not permit to remove the protein from the membrane. CD36
could act as a scavanger receptor by binding to apoptotic cells and cell fragments and
precipitating their elimination by phagocytosis (Mather, 2000).
Butyrophilin (BTN) is the most abundant protein in bovine MFGM and is a protein of
67,000 Da. The protein is strongly attached to the membrane and can resist to a
centrifugation treatment or extraction with chaotropic agents and detergents (Mather et al.,
1977). The structure of the protein is stabilized by a disulphide bond between the two antiparallel -strands (Stammers et al., 2000). BTN denatures at a temperature of 58°C and
16
forms aggregates through non-native disulfide cross-bridges at the same temperature
(Appel et al., 1982).
Adipophilin (ADPH) is a protein of 52,000 Da strongly attached to the membrane even
after extraction with salts and non-ionic detergents (Heid et al., 1996). ADPH has not been
widely studied, however it is known that the protein could conserve a hydrophobic binding
region that could mediate associations with lipid droplets or eventually proteins. ADPH
binds five to six molecules of fatty acids per molecule of proteins (Heid et al., 1996).
Periodic acid Schiff 6/7 (PAS 6/7) are glycoproteins of 52,000 Da and 47,000 Da
respectively (Hvarregaard et al., 1996). Nine disulphide bridges stabilized the structure of
the proteins (Cys6-Cys17; Cys11-Cys29; Cys31-Cys40; Cys48-Cys59; Cys53-Cys76; Cys78-Cys87;
Cys234-Cys238; Cys252-Cys409; Cys91-Cys247), but the proteins have no free thiol group. PAS
6/7 are poorly attached to the membrane because the proteins can be present as soluble
form in skim milk or buttermilk (Mather, 2000). PAS 6/7 is probably the most heat-stable
MFGM because the proteins are stable until 80°C (Ye et al., 2002).
Fatty-acid binding protein (FABP) is the smallest protein of the MFGM with a molecular
weight of 13,000 Da that is mainly poorly attached to the membrane (Brandt et al., 1988).
2.2.4 Phospholipids from buttermilk
The lipid content of buttermilk usually ranges from 0.3% to 0.7%. Compositions of lipids
in buttermilk and in milk fat globule membrane have been well characterized as shown in
Table 2.5. Buttermilk is richer in polar lipids such as phospholipids, sphingolipids, and
gangliosides with a content ranging from 0.07% to 0.18% of the total lipids comparatively
to whole milk (0.035% of the total lipids), skim milk (0.015% of the total lipids) or whey
(0.02% of the total lipids). In bovine milk, about 50% to 60% of the phospholipids are
integrated to the milk fat globule membrane (fragmented or not), and represent between
26% to 31% of the total lipids of the membrane (McPherson & Kitchen, 1983; Singh,
17
2006). The most common classes of phospholipids found in the membrane are
Sphingomyelin (SM), Phosphatidyl choline (PC), Phosphatidyl ethanolamine (PE),
Phosphatidyl inositol (PI), Phosphatidyl serine (PS), Lysophosphatidyl choline and
represent 22%, 36%, 27%, 11%, 4%, and 2% of the total phospholipids from the MFGM,
respectively. These classes generally contain high levels of esterified long-chain fatty acid
(C16; C18:1), but low levels of short-chain fatty acid (C4 to C10) (McPherson et al., 1983).
The MFGM components and particularly the phospholipids have been extensively
investigated over the last twenty years due to their possible roles in nutrition or human
health. Agents against colon cancer, gastrointestinal pathogens, Alzheimer’s disease,
depression, stress, support for the recovery of the liver from toxic chemical attack or
chronic viral are different examples of health-benefits of the phospholipids from the
MFGM (Dewettinck et al., 2008; Spitsberg, 2005). It has also been reported that SM and
PC are important sources of choline that is important for the synthesis and transmission of
important neurotransmitters and even maybe for the brain development.
Table 2.5: Lipid composition of buttermilk (adapted from Keenan and Dylewski (1995) and Walstra et
al. (2006).
Constituents
In Buttermilk
Constituents
(% of dry basis)
In MFGM
(% of total lipids)
Total lipids
2.9 to 7.4
Triglycerides
62
Phospholipids (PL)
0.6 to 1.9
Diglycerides
9
18
PE
42.9% of PL
Sterols
0.2 to 2.0
PI
8.9% of PL
Free fatty acids
0.6 to 6.0
PS
8.6% of PL
Phospholipids
26 to 31
PC
19.1% of PL
SM
17.9% of PL
CER
7.7% of PL
2.3 Principles of protein aggregation
Wang et al. (2010) defined protein aggregates as a protein in a non-native state whose size
is at least twice as that of the native protein. In chemistry, biochemistry, and pharmacology
the term oligomers or critical oligomers is preferred (Modler et al., 2003).
Individual protein molecules have the capacity to fold or (partially) unfold many times.
These changes depend on environmental conditions such as intrinsic (structure of protein)
or extrinsic factors (pH, temperature, pressure, etc) and lead to an energetically unfavorable
unfolded state. This situation is due to the exposition of hydrophobic regions. To regain
stability, protein molecules refold again or self-associate to form aggregates.
Aggregation results in the absence of normal functionality of the protein. Protein
aggregation has been well-studied because a large number of human disorders, ranging
from type II diabetes to Parkinson’s and Alzheimer’s diseases, are associated with protein
aggregation (Dobson, 2001; Mattson et al., 1999). Aggregates can also induce a cellular
toxicity when they are present in organs such as the liver, heart, and brain (Varadarajan et
al., 2000).
2.3.1 Definitions of native state, denaturation, and reversibility
The «native state» of a protein corresponds to its operative or functional form. In their
native states, most of the proteins are folded into rigid and well-defined three-dimensional
structures. In protein aggregation, the term “native” is used to define the initial state of the
protein before denaturation.
“Denaturation” refers to a major change from the original native structure that occurs
without severance of any of the primary chemical bonds (Tanford, 1968). However, this
definition is subject to controversy, such as the meaning of a major change.
19
Tanford (1968) simply defined the “reversibility” as the ability of the protein to return
(reversible) or to not return (irreversible) to the native conformation. It means that the
reaction is reversible only if the native structure can be recovered. In most cases, the
process becomes irreversible due to a secondary reaction that follows a major
conformational change (i.e unfolding). These reactions can be induced by the use of
temperature, acid or basic pH, etc.
2.3.2 Nucleation
Most of the protein aggregation mechanisms are nucleation-dependent and, in consequence,
are initiated by the formation of an aggregation nucleus (Wang et al., 2010). There is no
definition of an aggregation nucleus in the literature, however, Krishnan (2003) defined a
nucleus as small as the size of a dimer. In opposition, Baynes et al. (2005) observed a small
multimer as a nucleus. In the case of -lactoglobulin, Schokker et al. (1999) observed the
presence of non-native dimers and oligomers in the early stages of the heat-induced
denaturation. Later, non-native monomers, denatured proteins, and/or small aggregates are
incorporated at the surface of the nucleus to form large aggregates. This second step is
named as aggregation, elongation, fibrillation, or polymerization in the literature (Wang et
al., 2010).
2.3.3 Description of the different pathways
Wang et al. (2010) summarized the major protein aggregation pathways as presented in
Figure 2.3.
Figure 2.3: Schematic representation of major protein aggregation pathways
20
Aggregation through unfolding intermediates and unfolded states is the first pathway and
named (1) in Figure 2.3. Under normal conditions, a solution of proteins is a balance
between proteins in a native state and a small amount of unfolding intermediate proteins (I
in Figure 2.3). The latter of the two is divided into native-like intermediate or unfolded-like
intermediate depending on the degree of unfolding. These intermediate proteins are
considered as precursors of the physical aggregation process because they expose more
hydrophobic areas and are more flexible than native proteins. Interactions of these
intermediates lead to the formation of aggregates (A in Figure 2.3). Later in the process,
aggregates interact with each other. As soon as aggregates reach a certain size or solubility
limit, they become insoluble (P in Figure 2.3).
Many proteins do not go through the intermediate state. These proteins can directly form
aggregates physically from the native state through self-association (pathways 2a in Figure
2.3). These associations are due to electrostatic, hydrophobic, or van der Waals
interactions. Self-association is defined as the formation of reversible aggregates and is
considered as the precursor of irreversible aggregates. In fact, the distinction of the
formation of intermediates and the self-association can be difficult to demonstrate. So,
Wang et al. (2010) reminded that formation of intermediates is related to conformational
stability and self-association is related to colloidal stability.
Aggregation can also be due to chemical denaturation and chemical linkage between
protein chains (pathways 2b in Figure 2.3). Formaldehyde-mediated cross-linking,
dityrosine formation, oxidation, and Maillard reactions have been reported by Wang et al.
(2010) as possible cross-linking pathways. Nevertheless, the most common chemical
linkage is the intermolecular disulfide bond exchange. Surface-located and internally
located cysteines are both involved in the formation of disulfide bonds.
Protein aggregation through chemical degradation is the last major pathway (pathways 3 in
Figure 2.3). The most known examples are auto-oxidation, oxidation (Rosenfeld et al.,
21
2009), dimerization (Roostaee et al., 2009), hydrolysis (Van Buren et al., 2009), and
glycation (Wei et al., 2009). These reactions can change protein hydrophobicity,
secondary/tertiary structures, or the kinetics of protein unfolding.
Theoretically, other pathways can lead to formation of protein aggregates. Proteins that are
largely unfolded in natural conditions or proteins that have only two states (N or D) can
directly form aggregates from their denatured state (pathways 2a’ in Figure 2.3). The
denatured state can also undergo chemical degradation and form aggregates either directly
(pathway 2b’ in Figure 2.3) or indirectly (pathway 3’ in Figure 2.3).
Of course, the same protein can aggregate by different pathways, but one pathway often
dominates and should be controlled first. Protein aggregation can be reversible or
irreversible. The reversibility of aggregation is due to its ability to disaggregate and mainly
depends on the stage of the aggregation process (early stage = reversible; late-stage =
irreversible), environmental conditions (heat often induces irreversible aggregation), and
the size of the aggregates.
2.3.4 Bonds, interactions, and exchanges in milk protein
aggregation
Both covalent and non-covalent interactions are involved in the heat-induced protein
aggregation reactions in milk. The types of covalent bonding that occur during the heatinduced aggregation of milk proteins are thiol-thiol oxidation (also called formation of a
disulfide or disulfide bonds), and mainly thiol/disulfide exchange (SH/SS). The first one
appears in aerobic biological systems, and is characterized by a formal two-electron
oxidation of two thiols (-SH) to a disulfide (-SS-) with the reduction of oxygen (Gilbert,
1990):
2 R-SH  R-SS-R + 2e- + 2 H+
(1)
Later, Gilbert (1993) defined the thiol/disulfide exchange reaction by the nucleophilic
attack of a thiol on one of the two sulfurs of a disulfide bond to oxidize one thiol while
22
reducing another disulfide. This reaction occurs with an intra-molecular –SH group or an
intermolecular protein free –SH group.
RSH + R’SSR’  RSSR’ + R’SH
(2)
RSH + RSSR’  RSSR + R’SH
(3)
This reaction depends on the differences in stability between the two disulfides and the two
thiols, and is generally reversible. The exchange will become irreversible in some processes
such as strong heat treatments where the reversible oxidation of one or more -SH groups
precedes the irreversible oxidation of one or more additional -SH groups (Cappel &
Gilbert, 1986). It is generally accepted that the SH/SS exchanges occur during the heatinduced denaturation of -lactoglobulin. The free thiol group of the protein (Cys121) attacks
one of the two-disulfide bonds of another protein (Cys106-Cys119 or Cys66-Cys160). A free
thiol groups is now available in the second protein. Following this mechanism, Livney and
Dalgleish (2004) have found SH/SS exchanges between the residues Cys66 and Cys160 from
-lactoglobulin with residues Cys11 and Cys88 from -casein, respectively.
Non-covalent bonds are critical in maintaining the three-dimensional structure of large
molecules such as proteins and are also involved in the intermolecular binding of large
molecules. There are four types of non-covalent bonds: hydrogen bond, ionic bond, van der
Waals forces, and hydrophobic interactions. The role that plays these interactions is still
uncertain in the heat-induced aggregation of milk proteins. Hydrophobic interactions seem
to play an important role for whey protein aggregation, but less comparatively to SH/SS
exchanges. The unfolded proteins expose both thiol and hydrophobic groups that were
initially buried in the three-dimensional structure of the -lactoglobulin. Results of Oldfield
et al. (2000) showed that 10 to 15% of the -lactoglobulin available in their system
aggregated through hydrophobic interactions during the first step of their heat-induced
denaturation at 85°C, then followed by SH/SS exchanges. Controversially, Galani and
Apenten (1999) concluded that the hydrophobic interaction become important only at
temperatures between 90°C and 110°C.
23
2.3.5 Effects of experimental conditions
Temperature is often judged as the most critical factor that affects protein aggregation.
Changing the environmental temperature (increasing or decreasing) will destabilize the
proteins and will eventually promote protein aggregation. Aggregation can occur even at
room temperature. In the case of milk protein aggregation, temperature and holding time
will determine the level and the rate of interactions. At lower temperatures (75°C to 85°C),
the rate of aggregation between whey proteins and casein micelles increases slowly. At
higher temperatures (90 to 100°C) the rate increases rapidly at first and then reaches a
plateau (Anema & Li, 2003a). Reducing activation energy, enhancing hydrophobic
exchanges, increasing diffusion of proteins, and increasing the frequency of the molecular
collisions are other examples of the effects of variation of the environmental temperatures
(Wang et al., 2010).
Physico-chemical environment has a strong effect on protein aggregation and particularly
the ionic strength and the protein concentration. However pH is estimated the most critical
one. Variations of pH will change the distribution of surface charges on proteins, and
directly affect
intramolecular
folding
interactions,
intermolecular
protein-protein
interactions, and the rate of protein aggregation (Wang et al., 2010). At high basic or acid
pH, proteins are heavily charged so the repulsion forces are important. Controlling the pH
of the buffer (to reach the pI of the proteins) will reduce the repulsion forces, increase the
attraction forces, or positively affect the intramolecular and intermolecular interactions
between proteins. Anema and Li (2003b) showed that the interaction between whey
proteins and casein micelles were more important at pH 6.5 comparatively to pH 6.7.
Donato and Guyomarc’h (2009) reported later in their review that ~ 100% of the whey
proteins associate with the casein micelle at pH 6.2, while (60 to 85%) associate at pH 6.4
and only (10 to 15%) at pH 6.9 during heating of skim milk for several minutes at
temperatures ranging from 80 to 120°C.
Wang et al. (2010) also explained that proteins can complex with both positive and
negative ions present in the solution through electrostatics interactions. These associations
can change the surface charge, and consequently affect the charge-charge interactions
24
during protein aggregation. Livney et al. (2003) found that a high ionic strength promotes
heat-induced disulfide bonding between the two major whey proteins.
The composition of the protein solution also affects the denaturation of proteins. It has been
demonstrated that lactose and some other soluble non-protein components present in milk
have a protective role against unfolding of -lactoglobulin. Spiegel (1999) demonstrated
that the rate of thermal unfolding of -lactoglobulin (< 85°C) slows down by increasing the
content of lactose. Their results showed that a denaturation degree of 0.8 was obtained after
40 seconds at 80°C at a lactose concentration of 1.5%, while 35 minutes were needed at the
lactose concentration of 13.5%. Arakawa and Timasheff (1982) explained that sugars
change the surface tension of water. Consequently, proteins stabilize their structure by
preferentially interacting with water or soluble components. Later, Spiegel and Huss (2002)
obtained the same results by varying the amount of calcium in their system. Once again,
they referred the increase of the heat stability in the presence of calcium to the theory of the
preferential interaction of proteins with water. Similar results and explanations were given
by Anema et al. (2006). These authors found that, in general, soluble non-protein
components and lactose delay the irreversible denaturation of -lactoglobulin and lactalbumin. On the other hand, they reported that increasing the protein concentration
increases the rate of denaturation. However, impact of the soluble non-protein components
is stronger comparatively to the concentration of proteins, but only for -lactoglobulin. This
result means that in concentrated milk the denaturation rate of -lactoglobulin is still
delayed. Wang et al. (2010) explained that increasing the protein concentration has three
possible consequences: a decrease of aggregation phenomena due to crowding effect; an
increase of aggregation phenomena due to better chance of association; or a precipitation
due to solubility limit. But in most cases, increasing protein content promotes proteinprotein association as reported by Anema et al. (2006).
25
2.4 Heat-induced aggregation of milk proteins
2.4.1 Aggregation mechanisms of -lactoglobulin and lactalbumin
2.4.1.1
Thermal behavior of whey proteins
The thermal behavior of whey proteins, especially -lactoglobulin, is a complex
phenomenon that depends on the experimental conditions (temperature, pH, time, ionic
strength). To illustrate the complexity, the following paragraph only present the result from
heating experiment up to 150°C at pH > 6.8 (De Wit, 2009).
At 20°C and at pH near to 6.0, -lactoglobulin is present as a dimer. The dimer starts to
dissociate into two identical monomers at pH above 7.5 or below 3.5 (Townend et al.,
1969), and/or at temperature up to 55°C. At pH ranging from 6.0 to 8.5, -lactoglobulin
undergoes a reversible change of conformation that corresponds to a discrete change in the
specific optical rotation of the protein (Tanford et al., 1959). This conformational
modification of the tertiary structure appears from the native to the reversible conformation
of the protein, and results in exposure of the thiol group (Cys121) (Dunnil & Green, 1965).
The transition from native form to unfolded form is negligible at pH < 6.8 and at 50°C, but
represents 18 % at pH 7.0 (Pantaloni, 1964).
At temperatures between 60 to 70°C, more reversible conformational changes appear.
Pantaloni (1964) found that irreversible modifications characterized by the exposition of
the free thiol group (Cys121) appear at 70°C, but at low amount (6 %). Iametti et al. (1996)
concluded that the monomer form of -lactoglobulin has a poorly defined tertiary structure
at 70°C. Following this irreversible structure modification, monomers expose large
hydrophobic surfaces and a reactive thiol group. They defined this conformation as “molten
globule state”. Prabakaran and Damodaran (1997) reported the exposure of the buried
26
Cys121 and the presence of the disulphide bond Cys106-Cys119 at temperature from 63°C to
65°C due to a melting of the E-H -strand region of the molecule.
The irreversible unfolding step of -lactoglobulin appears at temperature range from 70°C
to 80°C depending of the pH value, and more specifically close to 78.5°C (Schokker et al.,
1999). Results also showed that a reactive monomer (unfolded) forms a reactive dimer with
another reactive monomer via thiol-disulphide exchange, thiol-thiol oxidation, and noncovalent interactions (Prabakaran et al., 1997; Schokker et al., 1999). When the
concentration of reactive dimer becomes important a polymerization reaction appears.
Results of Schokker et al. (1999) showed that the reactive -lactoglobulin monomers form
non-native dimers and oligomers in the first step of the aggregation. Then, the formation of
dimers and trimers occurs as a function of the heating time. The size of aggregates
increased supposedly by addition of reactive monomers or dimers to small aggregates.
Their results also showed the presence of pentamers consisting of 100 -lactoglobulin
units.
The accessibility of the free thiol groups increases by increasing the temperature (> 80°C).
At these temperatures, protein aggregation occurs via both thiol/disulphide exchange and
hydrophobic interactions (Sawyer, 1968). Residual native or reformed secondary structures
of -lactoglobulin unfold completely at temperature above 125°C may be due the
breakdown of disulphide bonds (Watanabe & Klostermeyer, 1976).
2.4.1.2
Mechanisms of aggregation
According to their model, Roefs and de Kruif (1994) proposed that the heat-induced
denaturation of β-lactoglobulin processes via a three-step mechanism: initiation,
propagation, and termination. The model is based on a thiol-catalyzed aggregation
mechanism where the free and active thiol groups (-SH) acts as a radical, and quickly and
continuously polymerize -lactoglobulin monomers. The initiation step starts by reversible
reactions, and then by a (pseudo) irreversible reaction. The reversible reactions consist of
27
the separation of dimers (B2) in monomers (B) at temperature below 70°C. They defined
the irreversible reaction by the exposition of the free thiol group of the unfolded βlactoglobulin (B*):
B2  …  B => B*
(4)
A thiol/disulfide exchange is formed between a disulfide bond of a “native” monomer and
the free thiol group of an unfolded β-lactoglobulin as described in 1.4. The monomer then
exposes a free thiol group and can initiate a SH/SS exchange with another protein, etc.
Results of Creamer et al. (2004) demonstrated that the disulfide bond Cys66-Cys160 has been
broken during the heat treatment, and that Cys160 became free. In consequence, the group
Cys160 probably plays an important role for the polymerization process. The propagation
step is linear because it involves only one free thiol and one disulfide bond per monomer.
However, the mechanism can be repeated many times until two reactive multimers (Bi* and
Bj*) form a disulfide bond by thiol-thiol oxidation:
B + Bi* => Bi+1*
i1
(5)
Bi* + Bj* => Bi+j
i,j 1
(6)
As recalled by Oldfield et al. (1998), the unfolding of β-lactoglobulin is also likely to
expose some hydrophobic residues ([B]). The following reactions could also occur under
specific environmental conditions:
B2  …  B => [B]
(7)
[B] + [B] => [Bi+1]
i 1
(8)
[Bi+1] => Bi+1*
i 1
(9)
The results of their study did not permit the confirmation of this phenomenon, but later
Oldfield et al. (2000) reported that 15% to 20% of the total β-lactoglobulin were present as
hydrophobic formed aggregates at 85°C. However, the portion decreased to 0% with the
increase of the temperature and heating time. According to their results, they concluded that
28
hydrophobic interactions between proteins could occur during the initial step of the
aggregation of β-lactoglobulin, and that aggregates became stronger by internal disulfide
bonds. Hoffmann and Van Mill (1997), Sawyer (1968), and Xiong et al. (1993) have also
demonstrated that it is possible to form whey protein aggregates through hydrophobic
interactions if the free thiol groups are blocked by a chemical reactant such as Nethylmaleimide (NEM).
Oldfield et al. (1998) also proposed a heat-induced aggregation for -lactalbumin (A). The
unfolded protein does not expose free thiol groups, so aggregates are formed mainly
through hydrophobic interactions. However, reactions are reversible because no aggregates
are found in solution at room temperature after heating (Gezimati et al., 1997). The possible
reaction involved the presence of hydrophobic sites of the unfolded protein ([A]) at
temperatures between 75°C to 85°C:
A  [A]
(10)
[A] + [A]  [Ai+1]
(11)
[Ai+1] + [A]  …  [An]
(12)
In a complex solution such as milk, -lactalbumin can form a heat-induced protein
complex with β-lactoglobulin by both hydrophobic interactions ([AB]) and SH/SS
exchanges (AB*). Gezimati et al. (1997) concluded that the free thiol group of βlactoglobulin can initiate SH/SS exchange reactions that incorporate both proteins in the
final complex. Once again, the process could involve a propagation, and termination step:
A + B2  A + B => [A] + [B] + B*
(13)
[A] + B* or [B] => [AB] => (AB)i*  (AB)i+j* (14)
Over the years, other mechanisms have been proposed. The summary of the different heatinduced aggregation mechanisms of whey proteins involves a general trend corresponding
to the dissociation of dimers into monomers, exposition of a free thiol group by the
unfolded β-lactoglobulin, and formation of aggregates through both SH/SS exchange
reactions and non-covalent bonds. It is also important to take into consideration the
29
experimental conditions. Prior, it has been discussed that factors such as temperature, pH,
ionic strength, composition, etc., strongly affect the denaturation behavior of proteins. For
example, in their study Roefs and de Kruif (1994) used a buffer containing 87% of βlactoglobulin with a few percent of -lactalbumin, less that 1% of salt (< 0.1% Ca2+), 2% of
lactose, 2% of soluble non-protein nitrogen compounds, and around 3% of water. Their
buffers were ranged from 2 to 95 g of β-lactoglobulin per liter of double-distilled water at
pH comprised between 6.75 and 6.95. They heated the buffers up to 65°C for different
times.
De Wit (2009) has recently reported that all the “new” proposed mechanisms are
contradictory with older studies, and particularly with the «well-known chemistry of the
SH/SS interchange in proteins in this pH range» explained by Cecil (1963). The author
concluded that the SH/SS interchanges are induced by the presence of thiolate ions (RS-),
and not based on a polymerization induced by a free thiol group (-SH) as proposed in Roefs
and de Kruif’s model. The following equations represent the basis of the reduction of
protein disulfides by thiolate ions and exchange reactions between disulfides. The reactions
stop when the thiolates are oxidized in disulfide bonds:
RS- + R’SSR’  RSSR’ + R’S-
(15)
RS- + R’SSR  RSSR + R’S
(16)
De Wit (2009) has also explained that a continuous polymerization is impossible according
to the two previous reactions (15 and 16). According to him, heat-induced aggregation can
occur only by the oxidation of the thiolate groups of the monomers, dimers, and tetramers.
Bauer et al. (2000) have proposed a model slightly deviated from Roef and de Kruif’s.
Their observations indicate that the mechanism is divided into four stages such as: 1)
Equilibrium between monomers and dimers; 2) «Activation» of monomers; 3) SH/SS
exchanges to form dimers, trimers, and tetramers; 4) Formation of large aggregates. Their
30
results also suggest the importance of an aggregation nucleus, formed from four oligomers,
as a prerequisite for the heat-induced aggregation of β-lactoglobulin.
Schokker et al. (1999) have also demonstrated that non-native dimers and oligomers were
formed in the early stages of the denaturation of β-lactoglobulin, presumably through
SH/SS exchanges, thiol-thiol oxidation and non-covalent interactions at 78.5°C. Later,
large aggregates ranging from 105 to 2x 106 Da were formed continuously by incorporation
of monomers and/or small aggregates, mainly through SH/SS exchanges. Unlike other
studies, their model proposes non-linear aggregates. They found that the formation of
aggregates involves more than one free thiol group.
According to more recent studies and reviews, models proposed by Schokker et al. (1999)
and Bauer et al. (2000) seem to be the most coherent. They defined the heat-induced
aggregation of β-lactoglobulin as nucleus-dependent reactions that occur really fast. Their
models are also closer to the general trend reported by Wang et al. (2010) on the
aggregation pathways of proteins aggregation. However, they are mainly based on
thiol/disulfide exchanges or thiol-thiol oxidation. The role of non-covalent interactions is
still uncertain. Galani et al. (1999) concluded that the importance of non-covalent
interactions is more important at high temperature of heating ranging from 90°C to 110°C.
Using a free thiol-blocking agent (N-ethylmaleimide), Kitabatake et al. (2001)
demonstrated that it was possible to form heat-induced aggregates of β-lactoglobulin.
2.4.2 Heat-induced interactions with minor whey proteins
It is generally accepted that -lactoglobulin dominates the formation of whey protein
aggregates when all whey proteins are present. The aggregation mechanism of this protein
has been studied extensively in different buffers, at a wide range of temperatures, pH, or
ionic strength. The aggregation mechanisms of other whey proteins, especially minor whey
proteins, are less studied. However it appears that minor whey proteins can affect the
aggregation process of the major whey proteins.
31
Using one and two-dimension SDS-PAGE, Havea et al. (1998) showed that protein
aggregates were formed involving both hydrophobic interactions and SH/SS exchanges
during the heating of whey protein concentrate at 75°C at pH 6.9. Aggregates were
composed of -lactoglobulin, -lactalbumin, bovin serum albumin (BSA), and
Imunnoglobulin H and L.
Gezimati et al. (1996) reported that bovine serum albumin in a solution responds in a
similar way to the heat treatment than -lactoglobulin. Havea et al. (2000) also defined the
aggregation mechanism of the protein as thiol catalyzed polymerization close to lactoglobulin’s, but with the exceptions that BSA begins to aggregate at a lower
temperature, and without the initial separation of the dimer into two monomers. During its
unfolding, BSA loses its native form for a transformed monomer form with an exposed
thiol group (Cys34) that can promote inter SH/SS exchanges. Gezimati et al. (1996) also
reported that BSA can be found as hydrophobic aggregates. Later, Gezimati et al. (1997)
confirmed the presence of hydrophobicaly formed BSA aggregates at 70°C. They
suggested that this form could be an intermediate in the formation of a gel network, and
that SH/SS exchange will stabilize the aggregates. Authors have also found that lactoglobulin and BSA can interact together during heating, however, direct evidence of
SH/SS inter-exchanges were found only in the second study. Havea et al. (2000) proposed a
possible mechanism for the aggregation of -lactalbumin in the presence of BSA where
reactive polymers of BSA would interact with unfolded -lactalbumin through
hydrophobic interactions. The complexes are then stabilized by SH/SS exchanges between
the free thiol group of the BSA and presumably the disulfide bond Cys6-Cys120 of lactalbumin. Then, the complex could interact with another unfolded -lactalbumin to form
SH/SS exchanges with the newly exposed free thiol group of the attached -lactalbumin.
However, they concluded that the SH/SS exchange process was reversible, resulting in the
possible liberation of oligomers of -lactalbumin.
32
2.4.3 Heat-induced interactions involving casein micelles
Heat-induced interactions of casein micelles with whey proteins have been extensively
studied since the first hypothesis of Sawyer et al. (1963). It is known that whey proteins,
particularly -lactoglobulin, can associate with the casein micelles through SH/SS
exchanges with the -caseins (Donato et al., 2007; Morr & Josephson, 1968; Sawyer, 1968;
Vasbinder et al., 2003a). Anema et al. (2003a) have observed an increase in size of the
casein micelle ranging from 30 to 35 nm during the early stages of heating of skim milk at
pH 6.5, while only an increase of around 10 nm was observed at pH 6.7. Earlier, Smits and
van Brouwershaven (1980) revealed that 83% of the total amount of -lactoglobulin was
associated with the casein micelle during heating of skim milk at 90°C at pH 5.8, while
76%, 44%, and 24% of the total -lactoglobulin was associated at the same temperature but
at pH 6.6, 6.8, and 7.3, respectively. Vasbinder et al. (2003a) have reported that 65% of the
total amount of -lactoglobulin and 50% of the total amount of -lactalbumin were
associated with the casein micelles during heating of skim milk at 80°C at pH 6.9.
However, -lactalbumin could associate with the casein micelles only in the presence of lactoglobulin. Later, Livney et al. (2004) reported evidence (but low confidence) of
formation of disulfide bonds between residues Cys66 of -lactoglobulin and Cys11 of casein and between residues Cys160 of -lactoglobulin and Cys88 of -casein.
Despite the new evidences concerning the heat-induced interactions of whey proteins and
casein micelles, the mechanism is still unclear. Donato et al. (2009) summarized the latest
and more coherent assumptions corresponding in four possible mechanisms trying to
answer two major questions. The first question is to determine if whey proteins aggregate
prior to the association with the casein micelles. The second question is to determine if casein dissociates from the micelle before or after association with whey proteins (Figure
2.4).
33
Figure 2.4: Schematic representation of the currently proposed pathways of formation of the heatinduced whey proteins/-casein complexes in heated skim milk from Donato et al. (2009).
Corredig and Dalgleish (1999), Dalgleish et al. (1997), Guyomarc’h et al. (2003a), and
Vasbinder et al. (2003a) have reported that -lactoglobulin and -lactalbumin could
interact together in the serum phase during heating of milk, before interacting with the
casein micelles (I.A in Figure 2.4). They defined whey protein aggregates as intermediates
34
in the heat-induced association of whey proteins with the casein micelles. Elfagm and
Wheelock (1977) have already observed that two steps were involved in the complex
formation between whey protein and -casein: 1) Changes in the whey protein structures
that do not involve -casein: 2) Interactions between whey proteins and -casein. Later,
Elfagm and Wheelock (1978) have confirmed these observations and observed that the
presence of casein micelles facilitated interactions between -lactoglobulin and lactalbumin. Using a stronger heat-treatment, Guyomarc’h et al. (2003a) have isolated
soluble aggregates and micelle-bound aggregates. The first type was composed of lactoglobulin and -lactalbumin. However, their results did not indicate that whey protein
aggregates are present at the end of heating. In fact, their results showed that -caseins
were present in all of the type of aggregates, and few s1-casein. Guyomarc’h et al. (2003b)
concluded that soluble aggregates initiated the destabilization of the casein micelles
because of their interactions with the micelle surface. They suggested that hydrophobic
interactions might be involved if the pH of the buffer led to decrease the repulsive charge
interactions.
Nevertheless, Eubert and Brunner (1982) have observed in their model system that
aggregation of -lactoglobulin does not seem necessary for the association with the casein located at the surface of the casein micelles and that a partial denaturation of lactoglobulin could be sufficient for the formation of micelle-bound aggregates (I.B in
Figure 2.4). Guyomarc’h et al. (2009) have also observed that whey proteins integrated casein to the formation of protein aggregates with similar structure comparatively to whey
protein aggregates and without a previous step. They suggested that hydrophobic bonding
and/or SH/SS exchanges were involved.
On the other hand, most of the studies agreed that the dissociation of -casein from the
casein micelles is a major point for the understanding of the association with whey proteins.
Anema and Klostermeyer (1997) have observed that at pH ranged from 6.3 to 7.1, a small
proportion of casein micelles was dissociated at 20°C, but the quantity of the soluble
caseins increased with the increase of the temperature. The maximum dissociation was
35
observed at 70°C, before the irreversible denaturation of whey proteins. Later, results of
Anema (2008) confirmed that the dissociation of -casein from the micelles can occur
before the association with whey proteins. Moreover, their results strongly indicated that
heat-denatured whey proteins preferentially associate with soluble -casein (present in the
serum-phase) than -casein located at the surface of the casein micelle (II.C in Figure 2.4).
Oppositely, Parker et al. (2005) have reported that the addition of caseinates in skim milk
hardly affected the formation of heat-induced aggregates in the serum. Later, Donato et al.
(2007) have also observed that addition of -casein had little or no effect on the formation
of protein aggregates during heating of milk. These results suggest that denatured whey
proteins preferentially associate with the -casein located at the surface of the casein
micelle rather than soluble -casein or caseinates (II.D in Figure 2.4). Aggregates may
dissociate from the casein micelle in the latest stages of heating.
Proposed mechanisms are difficult to compare because most of the studies have been
conducted at different pH, different temperatures, different heating systems, and with
different kind of proteins (purified, isolated, in milk, etc). Donato et al. (2007), for
example, suggested that -casein in solution is highly charged and has less hydrophobic
sites than -casein located at the surface of the casein micelles. It is most likely that the
presented mechanisms co-exist more than compete with each other.
2.4.4 Heat-induced aggregation mechanisms of MFGM
constituents
2.4.4.1
MFGM proteins
Little information is available on the denaturation (including aggregation) mechanisms of
MFGM proteins. Ye et al. (2002) were able to extract the MFGM from whole milk and to
remove whey proteins. Their results showed that protein aggregates were formed during
heating through intermolecular disulfide bonds between XO, BTN, and a small proportion
36
of PAS 6. Their study revealed that XO and BTN were denatured at low temperatures
(60°C, 10 min), while PAS 6/7 were stable until 80°C. Appel et al. (1982) already reported
that BTN forms heat-induced aggregates through SH/SS exchanges at 58°C.
Heat-induced aggregates between MFGM and other dairy proteins have been found in
different dairy products. Although, the exact mechanism of association remains uncertain,
and different pathways could occur. Corredig and Dalgleish (1998) found protein
aggregates including whey proteins and MFGM proteins in buttermilk. These interactions
were induced by the pasteurization of cream even at a temperature as low as 65°C. They
also observed that association between MFGM proteins and -lactoglobulin was
predominant at higher temperatures (> 65°C), but not for -lactalbumin. Authors suggested
that both non-covalent interactions and SH/SS exchanges may be involved, but their results
did not permit to confirm their statement.
It has been hypothesized that the association between whey proteins or -casein through
SH/SS exchanges is probably initiated by the MFGM proteins. For example, XO has 22
disulfide bonds, 38 free thiol groups with 4 available in the native form and 34 buried in the
structure (Cheng et al., 1988). Furthermore, they demonstrated that all the free thiol groups
become available after the unfolding of the protein. Due to the unfolding of the MFGM
proteins at a lower temperature comparatively to whey proteins, free thiol groups of the
MFGM proteins are exposed during the first step of the whey protein denaturation, even
before the exposition of the free thiol group of -lactoglobulin, but after the dissociation of
-lactoglobulin into monomers (Ye et al., 2004b).
Ye et al. (2004b) confirmed that the major whey proteins interact with MFGM proteins via
SH/SS exchanges during heating of whole milk starting at 60 to 65°C for 10 min. The
amount of -lactoglobulin and -lactalbumin linked to the MFGM increases with an
increase of the temperature up to 80°C, and then stays constant. They determined that only
1% of the total amount of -lactoglobulin and 0.8% of the total amount of -lactalbumin
were associated with the MFGM material during heating of whole cream. It appeared that
37
XO and BTN were not affected by the heat treatment, while both PAS 6 and PAS 7 were
affected. They also found that a small proportion of -casein associated with the MFGM
proteins via SH/SS exchanges.
In another study, Ye et al. (2004a) tried to better understand the aggregation mechanisms
that occur between MFGM proteins, -lactoglobulin, and -lactalbumin. The authors
summarized the overall process in accordance with the literature as follows: 1) Unfolding
of MFGM, -lactoglobulin, -lactalbumin; 2) Association of polypeptides of MFGM and
self-association of whey proteins; 3) Interactions between unfolded proteins or small
aggregates of MFGM, -lactoglobulin, -lactalbumin. Their results permitted to propose
two mechanisms for the heat-induced association between MFGM proteins and lactoglobulin.
At temperatures above the -lactoglobulin (B2) irreversible unfolding (60°C to 70°C), they
proposed a mechanism in two steps. Under the effect of the temperature, -lactoglobulin
partially unfolds (B), but does not expose its free thiol group located in the residue Cys121.
In opposition, MFGM proteins (MFGM*) become irreversibly unfolded (MFGM**) and
expose more free thiol groups (*). At this temperature, MFGM proteins self-associate
through SH/SS exchange ((MFGM)). The MFGM protein complexes still possess available
thiol groups (*):
B2  …  B
(17)
MFGM* => MFGM**
(17’)
MFGM* + MFGM** => (MFGMi+1)*
(18)
Then, partially unfolded -lactoglobulin associate with the MFGM proteins or with MFGM
protein aggregates. The formation of the complex ((B*MFGM)) involves SH/SS exchanges
and permits the release of one thiol group of the -lactoglobulin priorly involved in a
intramolecular disulfide bond:
B + MFGM** or (MFGMi)* => (B*MFGM)*
38
(19)
At higher temperatures (75°C to 95°C), the mechanism is slightly different because the lactoglobulin completely unfolds and exposes its free thiol groups (B*):
B2  …  B => B*
(20)
MFGM* => MFGM**
MFGM* + MFGM** => (MFGMi+1)*
(20’)
i1
(21)
The formation of the complex between -lactoglobulin and MFGM proteins ((BMFGM))
involves SH/SS exchanges initiated by the free thiol groups of the MFGM proteins and the
free thiol group of -lactoglobulin. Authors did not suggest that this association release a
thiol group of the -lactoglobulin. However, it is easy to think that the complex remains
reactive due to the large amount of free thiol groups of the MFGM proteins:
(MFGMi)* + B* => (BMFGM)*
(22)
It has not been explained yet why the proportion of whey proteins that associate with the
proteins of the membrane is limited and relatively low (~1.0 and ~ 0.25 mg of protein.g-1
fat for -lactoglobulin and -lactalbumin, respectively) (Ye et al., 2004a). A possible
explanation is that whey proteins preferably interact with each other or with -casein
during heating and that the aggregates could not interact with the MFGM proteins.
Corredig and Dalgleish (1996) reported that casein micelles offer a better surface area for
heat-induced interactions with whey proteins based on their diameters (50 to 300 nm)
comparatively to fat globules (0.5 to 10 m). Another possible explanation is that the
membrane offers a limited number of sites for interactions with other proteins upon
heating. Once all the available sites have been taken, any interaction can occur between
MFGM and other proteins (Ye et al., 2004a).
2.4.4.2
Involvement of minor lipids in the heat-induced
aggregation of MFGM
Houlihan et al. (1992) concluded that heat-induced aggregation of whey proteins with the
milk fat globule membrane in whole milk is mostly due to protein-protein interactions,
however they suggested that membrane lipids could also be involved. Their idea is based
39
on the fact that membrane proteins are lost during the heat treatment while the hydrophobic
regions of phospholipids become accessible.
As a general trend, Dufourcq and Faucon (1977) explained that the interaction between
proteins and phospholipids is possible due to a ionic interaction between the lysine or
arginine residues of the protein and the negative groups of the phospholipids such as
phosphate or carboxylic. In a second step, hydrophobic residues (tryptophan) interact with
the bilayer.
Brown et al. (1983) studied the complex formation between solvent-treated -lactoglobulin
and phosphatidylcholine in an aqueous solution. They concluded that proteins in their
native conformation (secretory) are unable, in general, to form a complex with lipids. A
certain degree of unfolding is necessary to expose the initially buried helical regions and
hydrophobic groups. The complex formation can start as soon as these regions and/or
groups are exposed. According to their model, lipid-protein interactions are initiated by an
ionic attraction between charged amino acid residues in the protein and the polar head
group of the lipid in order to position the lipid. The complex is then stabilized by
hydrophobic interactions between the hydrophobic side of the helix and the hydrocarbon
chain of the lipid. A similar binding mechanism has been observed with milittin to
phospholipids (Dufourcq et al., 1977), and glycophorin (Ong et al., 1981). Cornell and
Patterson (1989) have reported interactions between -lactoglobulin and phospholipids at
pH 4.4. Their results are consistent with the idea that positively charged groups of the
protein interact with negatively charged lipids. At pH 4.6, -lactoglobulin has a positive net
charge and PC is isoelectric (other phospholipids are ionized at pH > 4.0) (Hauser &
Phillips, 1979). Cornell et al. (1989) supposed that an electrostatic attraction between lactoglobulin and PC could be expected around this pH. Between five to seven positive
charges of the protein (depending on the conformation) could welcome the negative
charges of PC. Earlier, Brown et al. (1983) have also reported bondings between lactoglobulin and PC after a treatment with a helix-forming solvent. They observed that an
ionic interaction permitted the position of the lipid and protein molecule, and then
40
hydrophobic interactions stabilize the complex. Later, Lefèvre and Subirade (1999; 2000)
have observed using Fourier transform infrared spectrophotoscopy hydrophobic
interactions between -lactoglobulin and SM and electrostatic interactions between the
protein and PS. Their study also suggested that -lactoglobulin does not bond with PC.
However, Spector and Fletcher (1970) concluded that -lactoglobulin offers only one highenergy binding site for long chain free fatty acids (FFA), and one weak energy-bonding
site. In opposition, serum albumin offers six high-energy binding sites.
Barratt et al. (1974) studied interactions between casein (-, S1-, -) fractions and
synthetic lecithins (C10, C12, C14), or synthetic lysolecithins (C10-C20). Their results showed
that only S1- was able to complex with dicapryl (C10) and dilauroyl (C12) lecithin, and with
lysolecithins. It appeared that complexes formed with short-chain lysolecithins (C10, C12)
were not stable. Conversely, long-chain lysolecithins (C14, C16, and C18) formed stable
lipid-protein complexes with S1-casein. Their results showed that additional molecules of
lysolecithin could interact with the formed lipoprotein (lysolecithins-S1-casein). They also
found that about 30 to 35 molecules of lysolecithin (C14-C18) were bonded to one S1casein. Previously, Patrick et al. (1972) found that the fatty acid chains of the lysolecithin
molecules were bounded hydrophobically to the proteins whilst the polar groups of the
lysolecithin molecules remain free. It is known that the protein hydrophobicity is important
in lipid binding, however, Barrat et al. (1974) judged their results surprising because - and
-casein are more hydrophobic than S1-casein. Their tendency to interact with
phospholipids should be consequently greater.
Recently, Gallier et al. (2012) used a model system to understand the structure of the
MFGM and understand lipid-protein interactions at the surface of the globule. Their atomic
force microscopy images clearly demonstrated interactions between -casein and
phospholipids from the bovine MFGM. They concluded that both electrostatic interactions
due to the negative net charge of the protein at pH = 7 and hydrophobic interactions due to
its high hydrophobicity were involved. Their study did not permit to identify which
41
phospholipids were involved in the complex formation, however based on their previous
work they supposed that PE may be involved.
2.5 Applications for dairy protein aggregates in food formulation
Many dairy products like cheese or yogurt are classified as protein-rich food products. In
these products, proteins serve as sources of essential amino acids and as a texture agent.
Coombe and Kett (2005) and Zemser et al. (1998) have shown that the structure of protein
is closely correlated to its functionality. Primary structure properties have been defined as
hydrophobic/hydrophilic residues, the number of disulfide bonds and free sulfhydryl
groups, and the net charge of the protein (Chiti et al., 2003; Chiti et al., 2002). This
information is sufficient to control texture of food during well-known process, but is
insufficient to innovate with new textures, new properties, and new sensory or nutritional
properties. In fact, industries use only a few different types of protein because of the poor
literature about the folding of proteins, and their denaturation behavior. Moreover, thermal
processes change the physical and functional properties of proteins. Some changes are
desirable because they improve the texture (yogurt), but in most cases are undesirable
(specially in UHT treatments). In consequence, the use of dairy protein aggregates is
limited to increase the cheese yield or to substitute fat in low-fat or fat-free products.
2.5.1 Role of aggregates as fat mimetic
A portion of fat can be removed in some food systems without a noticeable change in the
texture of the product or the taste, but total removal is rare. Fat replacers have been
developed to replace the fat in food systems without any changes in the final physical
properties of the matrix and hypothetically with any changes in the flavor or savor. The
definition of fat replacers and fat mimetics have been given by the American Dietetic
Association (2005), the Calorie Control (2004), Glueck et al. (1994), and Omayma and
Youssef (2007) as follows:
42
«The ideal fat replacer(s) recreates some of all the functional properties of fat, while also
significantly reducing fat and calorie content, and without any of the undesirable properties.
A fat mimetic is an ingredient that mimics one or more of the sensory and physical
functions of fat in the food by holding water. They provide from 0 to 9 kcal g-1».
The protein-based mimetics are made through physical aggregation of proteins from milk,
whey, corn, and egg white. The aggregates from whey and corn proteins are more suitable
for food preparation including heating treatments, but not for extreme heating or frying.
Protein-based substitutes can replace 75 to 100% of the fat in food systems (Glueck et al.,
1994) and they are partially or fully digested and absorbed, with a lower calorie level due to
their low energy density. Regarding food regulation, fat mimetics are food additives or
generally recognized as safe (GRAS) substances. Their use is approved only if it has been
demonstrated that they are safe through long-term ingestion (American-DieteticAssociation, 2005). The common and usual name of the fat replacer must be labeled on the
package in conformation of the Nutrition Labeling and Education Act of 1994. Possible
positive and negative effects of fat replacers are summarized in Table 2.6.
Table 2.6: Possible positive and negative effects of fat replacers (adapted from Senanayake and Shahidi
(2005).
Benefits
Reduction of total fat in the diet
Reduction of energy intake in the diet
Replacement of saturated fat in the diet
Reduction of intake cholesterol
Reduction of absorption of cholesterol
Reduction of triacylglycerol levels
Reduction of coronary heart disease risk
factors
Increase the intake of complex carbohydrates
No flavor compromise
No trans-fatty acids
Maintain mouth-feel, texture, and moisture
Inconvenients
Reduction of intake of fat-soluble
vitamins
Reduction of essential fatty acids intake
Compensatory energy intake
Increase of the cost
Alteration of the function of the digestive
tract
Possible gastrointestinal tract disturbance
The process of preparation of denatured proteins has been introduced for the first time by
Walker (1970) as the Alfa-Laval Centriwhey process. Initially, the whey was taken after the
43
cheese-making process, and cooled down to 8°C to adjust its pH to 6.25. Whey was heated
at 95°C for twenty minutes, and then pH was decreased to 4.6 during the heating. The
denatured proteins were separated at 40°C by sedimentation or centrifugation, and the
denatured whey protein concentrate was finally cooled down to 13°C. This process has
been widely used in France to incorporate whey proteins in Camembert, Coulommier,
Demi-Carré, Port Salut, and Bel Pease cheeses since the seventies (Lawrence, 1993). With
years, the process has been modified or improved. First, Lelièvre (1990) concluded that the
size of the heat-denatured whey protein is critical, and related to their incorporation in a
food system. He suggested adding an homogenization treatment as the last step of the
process in order to reduce the average particle size distribution between 0.1 and 10 m.
This range has been judged optimal to trap the denatured whey proteins in the casein
matrix. The pH can also be decreased to 4.6 at the beginning of the denaturation instead of
just at the end (Lebeuf et al., 1998; Punidadas et al., 1999). Following these two major
changes, the name of this ingredient changed for microparticulated and denatured whey
proteins (MWP).
CPKelco US Inc., and Parmalat-Ingredients have not revealed their industrial processes for
the production of Simplesse and Dairy-Lo products, but were probably inspired by the
Alfa-Laval Centri-whey process. Simplesse products are made through a simultaneous
heating and shearing of whey proteins or a mix of milk and egg proteins. Dairy-Lo is
manufactured by ultrafiltering sweet whey followed by a partial thermal denaturation of
proteins by heating.
Several studies have been developed to investigate the impact of the use of protein-based
fat mimetics into cheese and yogurt processes. Simplesse and Dairy-Lo products are low
calorie protein-based fat mimetics. Glueck et al. (1994) calculated for example that using
one gram of Simpless in a food formulation really decreases the calorie content of the food
product. In fact, three grams of fat (9 kcal.g-1) can be replaced by one gram of Simpless®
(4 kcal.g-1), so a decrease of 5 kcal per gram of fat replaced can be expected. However, the
use of this type of ingredient in dairy products is often limited due to the high water-
44
binding capacity of denatured proteins. Depending on the products, it is generally advisable
to add them in proportions of 0.5% to 1.5% of the total weight.
Seydim et al. (2005) have shown that Dairy Lo has very promising texture and sensory
properties for the production of acid set-type yogurt. Addition of Dairy Lo at 1.7% of the
final weight (w/w) did not affect the whey separation, but increased the gel strength. The
firmness of yogurts supplemented with Dairy-Lo was comparable to full-fat yogurts after
one and seven days of storage but significantly higher comparatively to low-fat yogurts. In
opposition, Sandoval-Castilla et al. (2004) have reported that yogurts supplemented with
Dairy Lo (1.05% of the final weight (w/w)) were lower in tension and firmness but higher
in cohesiveness and adhesiveness comparatively to their control full fat yogurts. They have
also observed that the use of protein-based fat mimetics yielded yogurt with a more open
and less dense protein network. Tamine et al. (1995) have reported that a low proportion of
Simplesse® 100 (1.5% of the final weight (w/w)) interacted with the milk protein during
preparation of the base, but most of the interactions took place during gelation. Yogurts
supplemented with Simplesse® 100 were the lowest in firmness and the most susceptible to
serum separation.
2.5.2 Role of aggregates as water holder
Lawrence (1993) summarized an alternative method to use proteins aggregates for the
production of cheese by heating the milk at higher temperatures comparably to
pasteurization in order to denature the whey proteins and form aggregates with the casein
micelles. During the coagulation, the denatured whey proteins remain attached to the
micelle and become a part of the gel matrix. This method has been extensively used for the
yogurt-making process because it leads to the formation of gels with higher viscosity and
firmness (Dannenberg & Kessler, 1988; Lucey et al., 1998b; Lucey et al., 1997; ParnellClunies et al., 1986), with more homogeneous microstructure with lower porosity of the
network (Kalab et al., 1976; Parnell-Clunies et al., 1987), and with higher whey retention
(Dannenberg et al., 1988; Puvanenthiran et al., 2002). The method has also been
extensively investigated for the cheese-making process because it increases the production
yield. However, it is often judged controversial for the cheese production because the
45
formation of aggregates limits the primary phase of the enzymatic reaction and prevents
fusion of the casein micelles. Furthermore, cheeses have excessive moisture and texture
defects (Guyomarc'h, 2006). It is believed that some improvements such as alkaline heattreatment can be added in order to improve this method. Later, Donato and Guyomarc’h
(2009) concluded that the use of whey protein/-casein or whey proteins/casein micelle is
limited due to little information available on the formation of theses complexes, their
properties, the conditions to control their properties, and their nutritional properties.
Buttermilk seems to be a promising candidate for the formation of whey protein//-casein
aggregates. The use of buttermilk is however very challenging because this by-product is
complex and is still not fully understood. Moreover, its constituents have been subjected to
different rough processing steps. There is a lot of work to be done to understand the
structure and properties of the proteins from buttermilk. There is even more work to be
done to fully understand the microstructure and composition of dairy protein-based
aggregates. Finally, it is important to determinate if the protein-based aggregates become a
part of the end products or not. Tamine et al. (1995) and more recently Sandoval-Castilla et
al. (2004) have concluded that the added denatured proteins were involved in the structure
of the yogurt by means interactions with the casein micelles. This information is capital
because it would increase the use of protein aggregates.
46
CHAPTER 3
HYPOTHESIS,
GOAL,
AND
OBJECTIVES
It is generally accepted that skim milk and buttermilk are similar in composition.
Considering the literature presented, it seems obvious that caseins, whey proteins and
MFGM constituents from buttermilk interact with whey proteins upon heating. However,
buttermilk has been subject to intensive treatments such as skimming, pasteurization,
churning. Previous studies from this research group have clearly demonstrated that buttermaking process affects the constituents of buttermilk. These modifications probably affect
the sensitivity of buttermilk to heat treatment and particularly the formation of heat-induced
aggregates. Examples of modification could be different pathways for aggregation or
implication of new constituents.
The general hypothesis of this doctoral thesis is that a better understanding of buttermilk
constituents and particularly their reaction to heat treatment in presence of whey proteins
can control the properties of the resulting aggregates. While using whey proteins aggregates
during formulation may not be able to fulfill the texture requirements of the final product,
addition of buttermilk during the heat-induced aggregation can modify the physical and
functional properties of aggregates.
Objectives for this work were:
1. To develop heat-induced protein aggregates by combination of whey and buttermilk
concentrates.
2. To understand the effect of buttermilk constituents on the heat-induced formation of
whey protein aggregates.
47
3. To characterize the contribution of phospholipids from buttermilk to the formation
of heat-induced whey protein-buttermilk aggregates.
4. To understand the effect of the type of interactions that occurred during the
association of proteins on the properties of the aggregates.
5. To evaluate the potential of whey:buttermilk heat-induced aggregates as
replacement of skim milk powder for the production of acid-set type yogurts.
48
CHAPTER 4
THERMAL AGGREGATION OF WHEY
PROTEINS IN THE PRESENCE OF BUTTERMILK
CONCENTRATE
A preliminary experience have been designed in order to determine the optimal processing
parameters (pH, temperature, and holding time) in order to produce aggregates with lower
water-holding capacity comparatively to whey protein aggregates. Statistical analysis of the
data revealed that the temperature of 90°C led to a more efficient production of
sedimentable protein aggregates (higher aggregation yield) as a function of the buttermilk
proportion in the mixture than the temperature of 80°C and that a lower pH (4.6) led to the
formation of less hydrated protein aggregates as a function of the buttermilk proportion in
the mixture comparatively to higher pH (5.4 or 6.2). Statistical analysis revealed that the
holding time had no significant difference on the water-holding capacity of aggregates or
on the aggregation yield. The following parameters were selected: heating at pH 4.6 at
90°C and holding time of 5 minutes,
The work presented in this chapter aimed at characterizing the impact of ratio whey to
buttermilk on the denaturation process of cheese whey on the functional properties of
denaturated protein aggregates. We also investigated in this project the impact of applying
power ultrasound on the functional properties of aggregates.
Results of this part of the project were published essentially in this form in Journal of Food
Engineering (2011), 103:244-250. Results of this part of the project were also presented in
two posters at 2010 ADSA Joint Annual Meeting in Denver (CO, USA), one poster at the
Journée Scientifique GP3A in Quebec City (QC, Canada), and one poster at 2010 Journée
Technologique Novalait in Drummondville (QC, Canada).
49
4.1 Résumé
L’objectif de cette étude était d’évaluer l’impact de l’addition d’un concentré de protéines
du babeurre sur le procédé d’agrégation des protéines du lactosérum. Des concentrés
protéiques de lactosérum et de babeurre ont été mélangés puis chauffés (pH 4,6; 90°C).
L’ajout de babeurre a significativement augmenté le rendement d’agrégation et diminué la
capacité de rétention d’eau des agrégats. La modification des propriétés rhéologiques
suggère que les protéines du lactosérum dénaturées ont interagi avec les protéines du
babeurre. L’ajout d’un agent bloqueur de groupements thiols a permis de montrer que les
interactions covalentes et non covalentes étaient impliquées dans la formation d’agrégats.
La formation d’agrégats fut également influencée par un traitement aux ultrasons pendant le
chauffage. Il apparait que sous l’influence de la cavitation et de la turbulence générée par
les ultrasons, le rendement d’agrégation est augmenté et que la capacité de rétention d’eau
des agrégats est diminuée.
50
4.2 Abstract
The objective of this study was to assess the impact of adding buttermilk concentrate to the
denaturation and microparticulation process of cheese whey protein concentrate. For this
purpose, the two concentrates were mixed and co-denatured (pH 4.6; 90 °C) and
homogenized. The presence of buttermilk significantly increased aggregation yield and
decreased water-holding capacity of aggregates up to volumetric buttermilk - whey protein
ratio of 75:25. Modifications of rheological properties suggest that denatured whey proteins
interacted with caseins. A thiol blocker, N-ethylmaleimide, was added before heating to
measure the role of disulphide bond formation in the aggregation process. Results showed
that both covalent and non-covalent interactions were involved in the formation of
aggregates. Ultrasound treatment was applied during denaturation process and was shown
to influence aggregate formation. It appeared that under turbulence and cavitation
conditions, aggregation yield was increased and water- holding capacity decreased.
4.3 Introduction
A number of technological approaches have been developed to increase the retention of
whey proteins into cheese (Lawrence, 1989; Lawrence, 1993; Lelièvre, 1990). A widely
used approach consists in the addition of denatured and aggregated whey proteins to cheese
milk. However, one of the most serious challenges is to optimize the size and water-holding
capacity of the aggregated whey proteins. Indeed, larger aggregates readily interfere with
the casein network and they are poorly retained in cheese curd (Lelièvre, 1990). Such a
problem can, however, be circumvented by homogenization of the aggregates, thus
facilitating retention (Mignot & Tracard, 1976; Punidadas et al., 1999). The addition of
whey proteins into cheese has proved to increase yield but to reduce quality. Another
problem associated with the incorporation of aggregated proteins is related to their effect on
cheese moisture. Excessive cheese moisture is a common defect that limits the amount of
whey proteins that can be added to cheese milk.
51
Substituting buttermilk to cheese milk at a concentration over 5–10% to milk has been
shown to generate texture defects and high moisture (Joshi et al., 1994). However, other
studies have shown that buttermilk could be a valuable product for increasing the moisture
content and improve the texture of low fat cheese, essentially because it contains fragments
of milk fat globule membrane (MFGM) including phospholipids, and whey proteins
(Mistry et al., 1996; Raval et al., 1999; Turcot et al., 2002; Turcot et al., 2001).
It was shown by Morin et al. (2008) that the poor coagulation properties of buttermilk were
related to cream pasteurization that probably induced important modifications of the
MFGM surface during the heating process. Many authors have reported that whey proteins
had the potential to interact with -casein (-CN) (Donato et al., 2007; Morr et al., 1968;
Sawyer, 1969; Vasbinder & de Kruif, 2003b) and particularly with serum-phase -casein
after the dissociation of -CN from the micelle (Anema, 2008). In addition, Ye et al.
(2004a,b) demonstrated that -lactoglobulin (-lg) and -lactalbumin (-la) associated
with the MFGM by thiol–disulphide bonds in heated milk.
In line with these observations, we hypothesized that co-denaturation of cheese whey and
buttermilk proteins would induce the formation of aggregates between whey proteins–-CN
– MFGM by means of SH/SS interchanges as well as by other non-covalent interactions
such as ionic, hydrophobic or van der Waals. Also, the aggregation yield and water-holding
capacity of the aggregates would be dependent to the ratio between whey and buttermilk, as
well as on the availability of free-SH groups and on the possibilities of rearrangements
during aggregation process.
N-ethylmaleimide (NEM), a thiol blocker, can be used to prevent or slow down the
thiol/disulphide interchange reactions between denatured milk proteins (Alting et al., 2000;
Hoffmann et al., 1997). Havea et al. (2004) have shown that gels formed by non-covalent
interactions were more rigid than those formed by disulphide bonds. We also hypothesized
that applying physical treatment that would promote rearrangement of the aggregates
during heating and would increase the compactness and reduce the hydration of the
52
aggregates. Ultrasound treatments are known to generate cavitation, turbulence and heat
(Patist & Bates, 2008). High intensity ultrasound (20 kHz) generates shear forces that were
effective in reducing viscosity of dairy ingredients (Zisu et al., 2009) and in homogenizing
fat globule (Ashokkumar et al., 2009; Jambrak et al., 2008).
The use of ultrasound treatments in food processing is considered as an emergent potential
alternative to heat or to homogenization treatment (Patist et al., 2008). Moreover, under
turbulent conditions due to ultrasound treatment, particle mobility is increased, which
promotes the formation of aggregates (Walstra, 1983). The challenge remains, however, to
control yield.
The objective of the present work was to characterize the heat-induced aggregation process
of whey protein–buttermilk concentrate mixtures with different buttermilk protein content
(0%, 25%, 50%, 75% and 100%) on the aggregation yield and water-holding capacity of
the aggregates. The effects of blocking free- SH by addition of NEM and applying power
ultrasound (20 kHz) during aggregation process were also assessed.
4.4 Materials and methods
4.4.1 Materials
Fresh Mozzarella cheese whey and buttermilk were obtained in a local cheese factory
(L’Ancêtre, Bécancour, QC, Canada). N-ethylmaleimide (NEM) was obtained from Alfa
Aestar (Ward Hill, MA, USA). All other reagents were from Fisher Scientific (Ottawa, ON,
Canada).
4.4.2 Preparation of whey and buttermilk concentrates
Fresh Mozzarella cheese whey and buttermilk were skimmed by using a pilot scale milk
separator (Alfa Laval, Uppsala, Sweden). Bacterial contamination of cheese whey was
53
reduced by microfiltration (TetraPak MSF1, Lund, Sweden) through a 1.4 m membrane
(Membralox®, Bazet, France). Microfiltered whey and buttermilk were concentrated by
ultrafiltration (UF) through a 5 kDa membrane (Romicon, Koch Membrane Systems,
Wilmington, MA, USA) to a final protein concentration of 9.5% (w/v) in the UF-retentate.
The concentrates were frozen to -28 °C until further analysis.
4.4.3 Heating experiments
Concentrates were mixed at the following buttermilk:whey protein ratios: 0:100; 25:75;
50:50; 75:25 and 100:0. Mixtures (200 mL) were adjusted to pH 4.6 by the slow addition of
HCl 1 N and heated from 4 °C to 90 °C in a thermostatically-controlled water bath, under
constant stirring for 25 min (including come-up time) at 168 rpm agitation rate. After
thermal treatment, mixtures were cooled to 30 °C in an ice bath and homogenized for five
passes at 65.5 MPa using an Emulsiflex-C5 (Avestin Canada, ON, Canada). The overall
process used for the preparation of aggregates is summarized in Figure 4.1.
54
Figure 4.1: Experimental procedure used to prepare aggregated buttermilk and whey proteins
mixtures.
55
4.4.4 Treatments with N-ethylmaleimide (NEM) and power
ultrasound
A concentration of 9.5 mM N-ethylmaleimide (NEM) was used in accordance with the
experiments of Alting et al. (2000) in order to completely block the free thiol groups. NEM
(9.5 mM) was added in whey–buttermilk concentrate (9.5% protein concentration) mixtures
(200 mL) before heat treatment.
Power ultrasounds were applied using a constant frequency 20 kHz probe (Virsonic-550,
The Virtis Company, Gardiner, NY, USA). The power level was fixed to 275W. The
titanium tip was immersed 2 cm underneath the surface during the treatment. The
treatments were applied to the mixtures after 10 min of thermal denaturation, until the end
of heating (25 min).
4.4.5 Analytical methods
4.4.5.1
Composition analyses
Total nitrogen content was determined by the Dumas combustion method (IDF, 2002)
using a LECO equipment (Protein Analyzer model FP-528 Leco Instruments Ltd.,
Mississauga, ON, Canada). Nitrogen values were converted into protein values using 6.38
as the nitrogen conversion factor. Fat was determined using the Mojonnier extraction
method (IDF, 2008) and lactose by enzymatic method (IDF, 2001). Total solids were
obtained by microwave drying (Smart System 5, CEM Corp., Matthews, NC, USA) and ash
was measured by incineration in a muffle furnace at 550 °C for 20 h (AOAC, 1990). The
overall composition of each mixture as determined experimentally is summarized in Table
4.1.
56
Table 4.1: Composition (% DM) of the various whey-buttermilk mixtures concentrated at 9.5% protein
(w/v) used for the heating experiments
% Dry Matter
Proteins
Lactose
Fat
Ashes
a
100:0
73.0 ± 0.2
10.0 ± 0.2
7.7 ± 0.1
1.7 ± 0.0
75:25
64.0 ± 1.8
14.6 ± 0.5
10.6 ± 0.1
3.0 ± 0.0
50:50
59.8 ± 3.0
17.6 ± 0.6
11.7 ± 0.2
4.3 ± 0.2
25:75
54.1 ± 1.8
20.8 ± 0.4
12.6 ± 0.4
5.2 ± 0.0
0:100
52.1 ± 0.3
26.9 ± 0.3
14.3 ± 0.3
6.0 ± 0.0
a
refer to the relative proportion of total proteins of cheese whey and buttermilk in the
mixtures.
4.4.5.2
Determination of aggregation yield and water-holding
capacity
The aggregated material was separated by centrifugation (SORVALL-RC-5B, Du Pont
Company, Newtown, CT, USA) at 15,000g for 20 min at 20 °C. Aggregation yield
(Equation 4.1) was calculated from protein content in the supernatant:
Equation 4.1: Calculation of the aggregation yield
Aggregation Yield (%) =
[ P ]M - [ P ]S ´100
[ P]M
where [P]M was the protein concentration in mixture (%)
and [P]S was the protein concentration in supernatant (%).
Water-holding capacity (Equation 4.2) was estimated from the moisture of the
centrifugation pellet and reported on protein content.
Equation 4.2: Calculation of the water-holding capacity of protein aggregates
WHC (gwater /g protein ) =
[ M] P
[ P]P
where [M]P was the moisture content in pellet (g)
and [P]P was the protein content in pellet (g).
57
4.4.6 Particle size distribution and surface area
The particle size distribution of homogenized mixtures was measured using a Mastersizer
2000 laser diffraction system (Malvern Instruments, Worcestershire, UK). Samples were
shaken by inversion for 1 min and dispersed directly into the recirculating cell until a 12–
19% obscuration value was obtained. Agitation rate of dispersant (deionised water) was set
to 320 rpm and measurements were started after 5 min of agitation. Data was obtained from
the average of five measurements by samples and expressed as the volume-weighted
average particle size, also known as the D[4,3] value.
4.4.7 Determination of rheological properties
Flow curves were obtained using a controlled strain rheometer (ARES 100 FR, TA
Instrument, New Castle, Delaware, USA). A coaxial geometry with a cup (35 mm
diameter) and a bob (35 mm length, 33 mm diameter) was used. Samples were poured
directly (13 mL) into the measuring system of the rheometer at 25 °C. The shear rate and
the shear stress were interpreted using the rheometric computer program. The values of the
flow behavior index (n) and the consistency coefficient (k) were obtained from plots of log
shear stress versus log shear rate between 5 and 500 s-1, according to the power law model
(Equation 4.3).
Equation 4.3: Formula of the Power Law Model
log t = logk + n logg
where  is the shear stress (Pa),
 is the shear rate (s-1),
n is the flow behavior index,
and  is the consistency index (Pa.sn)
58
4.4.8 Statistical analysis
All experiments were performed in triplicate, so each value represents the mean of three
measurements. The effect of ratio, addition of a thiol blocker and application of ultrasound
on aggregation yield, WHC, rheological properties and particle size distribution were tested
according to a factorial design. Statistical analyses were carried out using SAS software
(SAS Institute Inc., Cary, NC). Table 4.2 summarizes significance for each treatment on
each variable. Results were considered significantly different when P < 0.05.
Table 4.2: Summary of the significance (P values) calculated by the analysis of variance of data of each
contrasts for each variables (n = 3).
Significance (P values)
Contrast
Aggregation yield
WHC
k
n
Model
< 0.0001
0.0311
< 0.0001 < 0.0001
Ratio (R)
< 0.0001
0.0009
< 0.0001 < 0.0001
NEM (N)
0.0045
0.0470
0.0011 < 0.0001
Ultrasound (U)
< 0.0001
0.0002
0.0433
0.0017
R*N
0.0557
0.9627
0.3598
0.0019
R*U
0.2054
0.4440
0.0001
0.0009
N*U
0.6547
0.4843
0.3214
0.0007
R*N*U
0.0989
0.5816
0.5104
0.9726
a
Logarithmical transformations were applied
Particle sizea
0.0009
0.0040
0.0010
0.0874
0.0036
0.3023
0.9403
0.3069
4.5 Results
4.5.1 Aggregation yield
The effects of buttermilk to whey protein ratio, ultrasound and NEM treatments on
aggregation yield are shown in Figure 4.2.
59
Aggregation Yield (%)
100
100:0
75:25
50:50
25:75
0:100
80
60
40
Control (R)
R*U
R*N
Figure 4.2: Effect of protein composition, NEM and ultrasound on aggregation yield of different
mixtures of whey and buttermilk concentrates (9.5 % protein), acidified at pH 4.6 and co-denatured at
90°C.
Aggregation yields for pure cheese whey and buttermilk concentrates were respectively
63.3 ± 1.9% and 86.1 ± 5.5%. In concentrate mixtures, the aggregation yields significantly
increased (P < 0.0001) according to the proportion of buttermilk. There seems to be no
deviation to the activity rule, suggesting that the components in the mixture might
aggregate independently of each other.
Ultrasound treatment significantly increased (P = 0.0001) aggregation yields and this effect
was dependent on the proportion of buttermilk in the mixtures. Aggregation yield increased
by 33% for pure whey protein concentrates, but the increase was only 3% for pure
buttermilk concentrates. The aggregation yields in mixed concentrates treated with
ultrasound were higher than for control concentrates, with a minimum aggregation yields
observed at 75% cheese whey protein ratio (83.7 ± 1.8%) and a maximum at pure
buttermilk concentrates (89.5 ± 4.0).
Adding thiol blocker (NEM) significantly increased (P = 0.0045) the aggregation yields of
mixed protein concentrates. On average, aggregation yields increased by 6%. Figure 4.2
60
suggests that NEM was more efficient to increase the aggregation yields in mixtures with
high proportion of cheese whey protein concentrates. However statistical analysis indicates
non-significant interaction with protein ratio (P = 0.0557).
For protein mixtures treated with ultrasound, NEM had a significant effect (P = 0.0007) on
aggregation yields (results not shown).
4.5.2 Water-holding capacity
The effects of treatments on WHC are shown in Figure 4.3. Only the principal effects were
presented since the second and third level statistical interactions between the factors were
WHC (gwater/gprotein)
not significant (Table 4.2).
3.0
3.0
3.0
2.5
2.5
2.5
2.0
2.0
2.0
1.5
1.5
1.5
1.0
0
25 50 75 100
Buttermilk protein fraction
1.0
1.0
no
yes
Ultrasound
0
9.5
[NEM] mM
Figure 4.3: Effect of protein composition, application of ultrasound and addition of NEM on waterholding capacity (WHC) of mixed buttermilk-whey protein mixture (9.5 % protein) heated at pH 4.6
(90°C).
WHC of aggregates formed in pure cheese whey and buttermilk concentrates were
respectively 2.75 ± 0.08 gwater/gprotein and 2.21 ± 0.14 gwater/gprotein. In concentrate mixtures, WHC
of aggregates significantly decreased (P = 0.0009) according to the proportion of buttermilk
to reach minimum values at 75% buttermilk protein fraction (2.06 ± 0.16 gwater/gprotein).
61
Ultrasound treatment significantly increased (P = 0.0433) the WHC of aggregates. On
average, WHC increased by 0.21 gwater/gprotein comparatively to control mixtures.
Adding NEM significantly increased (P = 0.0470) the WHC of aggregates. On average,
WHC increased by 0.18 gwater/gprotein comparatively to control mixtures.
4.5.3 Consistency coefficient (k)
The effects of buttermilk to whey protein ratio, ultrasound and NEM on consistency
Consistency Coefficients (MPa.s)
coefficient are shown in Figure 4.4.
1500
1500
1500
1000
1000
1000
500
500
500
0
0
0
0
25 50 75 100
Buttermilk protein fraction
no
yes
Ultrasound
0
9.5
[NEM] mM
Figure 4.4: Effect of protein composition, application of ultrasound and addition of NEM on
consistency index (k) of mixed buttermilk-whey protein mixture (9.5 % protein) heated at pH 4.6
(90°C).
Consistency coefficients of pure cheese whey and buttermilk concentrates were
respectively 253.2 ± 86.8 MPa.s and 705.6 ±141.6 MPa.s. In concentrate mixtures, k
significantly increased (P < 0.0001) according to the proportion of buttermilk.
62
Ultrasound treatment significantly increased (P = 0.0017) k comparatively to control
mixtures. On average, k increased by 100 MPa.s. The maximum values were obtained with
75% buttermilk protein ratio (988.4 ± 174.2 MPa.s).
Adding NEM significantly increased (P = 0.0011) k comparatively to control mixtures. On
average, k increased by 85 MPa s. The maximum values were obtained with pure
buttermilk protein ratio (983.8 ± 116.2 MPa s).
4.5.4 Flow behavior index (n)
The effects of buttermilk to whey protein ratio, ultrasound and NEM on flow behavior
index are illustrated in Figure 4.5. A decrease of viscosity with increasing shear rate is
indicative of a pseudoplastic behavior (n < 1). Flow behavior index (n) of control and
treated mixtures indicated a pseudoplastic behavior.
1.0
100:0
75:25
50:50
25:75
0:100
Flow Behavior Index
0.8
0.6
0.4
0.2
0.0
Control
Ultrasound
NEM
Figure 4.5: Effect of protein composition, NEM and ultrasound on flow behavior indices of different
mixtures of cheese whey and buttermilk concentrates (9.5 % protein), acidified at pH 4.6 and codenatured at 90°C.
Flow behavior indexes of pure cheese whey and buttermilk concentrates were respectively
0.751 ± 0.121 and 0.423 ± 0.072. In concentrate mixtures, n significantly decreased (P <
0.0001) according to the proportion of buttermilk in the mixture.
63
Ultrasound treatment significantly decreased (P = 0.0009) n and this effect was dependent
to the proportion of buttermilk in the mixtures. Flow behavior indexes decreased by 0.124
with pure cheese whey concentrates, and, surprisingly, only by 0.008 with pure buttermilk
concentrates. Flow behavior indexes of mixtures treated with ultrasound were lower than
for control concentrates, with a minimum n observed at 75% buttermilk protein ratio (0.300
± 0.0047) and a maximum at pure cheese whey concentrates (0.627 ± 0.0019).
Adding NEM significantly decreased (P < 0.0001) n and this effect was dependent to the
proportion of buttermilk in the mixtures. Flow behavior indexes decreased by 0.135 and
0.130 respectively for pure cheese whey and buttermilk concentrates. Flow behavior
indexes of mixtures treated with NEM were lower than for control concentrates, with a
minimum n observed at pure buttermilk concentrates (0.0293 ± 0.031) and maximum at
pure cheese whey concentrates (0.616 ± 0.073).
4.5.5 Particle size distribution
The effect of control and treatments on particle size distribution of homogenized mixtures
is reported in Table 4.3.
The particle size distributions of mixtures was significantly affected (P = 0.0040) by the
proportion of buttermilk in the mixtures. It appeared that the size increased when whey
proteins were in higher or equal proportion compared to buttermilk proteins (4.4 ± 0.1 µm
with pure whey to 20.9 ± 10.2 µm with 50:50 ratios) and decreased with higher
concentration of buttermilk (7.3 ± 3.9 µm with 25:75 to 5.7 ± 0.80 µm with pure
buttermilk).
64
Table 4.3: Average particle size and span of untreated and treated whey-buttermilk mixtures acidified
at pH 4.6, denatured at 90°C and homogenized at 65.50 MPa.
Mixtures
D[4,3] (µm)
Span
100:0a
4.4 ± 0.1
1.6 ± 0.8
75:25
12.7 ± 1.8
3.4 ± 1.1
50:50
20.9 ± 10.2
2.2 ± 1.2
25:75
7.3 ± 3.9
2.0 ± 1.0
0:100
5.7 ± 0.8
1.2 ± 1.1
100:0
6.4 ± 4.4
6.1 ± 0.8
75:25
58.9 ± 32.1
17.4 ± 1.3
50:50
4.8 ± 2.2
2.0 ± 0.8
25:75
6.6 ± 4.4
1.5 ± 1.5
0:100
10.1 ± 8.8
1.6 ± 0.3
Absence of NEM
Presence of NEM
a
refer to the relative proportion of total proteins form cheese whey and buttermilk in the
mixtures.
The aggregate size in co-denatured cheese whey–buttermilk protein mixtures was affected
(P = 0.036) by both protein composition and NEM addition. Particle size decreased when
mixture contained high proportion of whey proteins (6.4 ± 4.4 µm with pure whey to 4.8 ±
2.2 µm with 50:50 ratios) and decreased with higher concentration of buttermilk solids (6.6
± 4.4 µm to 10.1 ± 8.8 µm with pure buttermilk). As a general trend, aggregates formed in
presence of NEM were larger in mixture containing higher proportion of cheese whey
proteins. However, a different behavior was observed for mixture with 50% buttermilk
protein, where NEM had no effect on particle size.
Ultrasound treatment had no significant effect (P = 0.0874) on particle size distributions of
aggregates.
65
4.6 Discussion
Our data show that it is possible to integrate buttermilk concentrate in a
denaturation/microparticulation process of whey proteins in order to increase aggregation
yields and decrease WHC of aggregates.
Increasing the buttermilk protein fraction up to 75% led to higher aggregation yields and
lower WHC combination. These results suggest that the presence of casein and eventually
the dissociation of -casein from the micelle at low pH (4.6) before heating is more
favorable to increase aggregation yield. However, it is not clear if aggregation results only
from association of denatured whey proteins with each other or with -casein or both.
Blond and Montupet (1989) had observed that protein WHC decreases in function of
denaturated protein percentage. In line with this observation and knowing that caseins
interact with denaturated whey proteins during heat treatment, the present results show that
the co-denaturation of a low proportion of whey protein with a higher proportion of caseins
results from the formation of condensed and low hydrated aggregates.
Modifications of rheological properties and particle size distribution of aggregates caused
by substitution of whey by buttermilk suggest that casein micelles participate in the
aggregation process. Vasbinder et al. (2003b) showed that at low pH (<6.6) most of the
whey protein aggregates are located at the surface of the casein micelles. Whey protein
aggregates are larger in size than native protein and the association with -casein increases
the size of the casein aggregates and led to increase viscosity of mixtures (higher k and
lower n). However, an increase of viscosity could be due to some physicochemical changes
in the mixture, such as the volume fraction occupied by the aggregates, a change in their
size, shape or hydration, or also to an increase of attachment between particles. Considering
these different possibilities, it can be thought that increasing proportion of casein in the
mixture increased the number of binding sites and favored interactions between aggregated
particles.
66
Hoffmann et al. (1997) have shown that the reactivity and the accessibility of -lg free
thiols were higher when the pH was close to that of their pK value (8.2). In our pH
condition (4.6), it is not clear if aggregation results only by thiol/disulphide bonds or noncovalent exchanges such as ionic, hydrophobic, van der Waals or both.
Kitabatake et al. (2001) concluded that -lg linked with NEM interact with other -lg–
NEM complexes mainly by hydrophobic interactions. Our results show that the origin of
interactions between denatured proteins (disulphide bonds or non-covalent or both) does
not affect aggregation yields. In accordance with Xiong et al. (1993), we hypothesized that
reduced SH/SS interchange reactions would increase the molecular flexibility and that this
modification could favor dehydration of aggregates by homogenization. However, it
appeared that WHC of aggregates increased in presence of NEM. Havea et al. (2004) have
reported that heat-induced whey protein concentrate gels formed by covalent interactions
were rubbery and gels formed by non-covalent interactions were rigid. It is possible that the
rigidity of these aggregates prevent their dehydration. Our observations on the changes in
rheological properties and particle size distribution suggest that aggregates formed in the
presence of NEM were larger (higher k, lower n) than those formed in untreated mixtures.
Walstra (1983) suggested that turbulent conditions (homogenization) and heat generated by
ultrasound treatment cause an acceleration of heat-induced aggregation/cross-linking and
prevent the formation of disulphide bonds between denatured whey proteins. Our results
show that the use of ultrasound increases protein aggregation in mixtures with high
proportion of whey proteins. These results are in agreement with Villamiel and Jong (2000)
who have shown that the denaturation of whey proteins (-lg and -la) was higher when
the ultrasonic treatment was performed during heating. Moreover, the denatured whey
proteins preferentially interact with dissociated -casein (Anema, 2008), and Taylor and
Richardson (1980) had shown that sonication can generate free caseins by the disruption of
casein micelle in milk treated with ultrasound treatment. Ertugay et al. (2004) demonstrated
that cavitation and turbulence effects of ultrasounds are similar to conventional
homogenization at a low power level (<180 W) and better at higher power levels (>180 W),
67
and that ultrasonic homogenization could reduce the size of milk fat globule (Ertugay et al.,
2004; Villamiel et al., 2000). Zisu et al. (2009) and Daubert et al. (2006) have found a
correlation between the decrease of whey protein size treated with power ultrasound and
the decrease in viscosity. Jambrak et al. (2009) and Kresic et al. (2008) observed an
increase in the apparent viscosity caused by a particle size decrease. Our results show that
ultrasound treatment did not affect directly the particle size but promoted the formation of
aggregates (highest yields), and especially for pure cheese whey proteins and 75% cheese
whey protein ratio. In fact, the homogenization and shear stress caused by cavitation during
ultrasound treatment probably modified the aggregation/cross-linking by aggregates
remodeling or rearrangement during the aggregation process but it did not affect WHC of
aggregates.
Applying ultrasound on mixtures treated with NEM showed a significant impact for
aggregation yields. We hypothesize that cavitations and turbulence could affect the
interaction whey protein– NEM because yields were similar to results obtained in presence
of ultrasound.
4.7 Conclusions
This present study shows that co-denaturation of cheese whey and buttermilk concentrates
leads to the formation of protein aggregates. Higher proportions of buttermilk protein in the
mixtures increased yields and decreased water-holding capacity of aggregates. Preventing
the formation of disulphide bonds between denatured whey proteins increased WHC of
aggregates. This approach could be used to control WHC of aggregates. Rearrangement
caused by ultrasound treatment during the denaturation increased yields and decreased
WHC of aggregates. More work should be done in order to evaluate the potential use of the
lowest hydrated aggregates in cheese making process and of the highest hydrated
aggregates in yogurt manufacture.
68
4.8 Acknowledgments
This work was funded by the Le Fonds Québécois de la Recherche sur la Nature et les
Technologies (FQRNT, Quebec city, Quebec, Canada) – Novalait Inc. (Sainte-Foy,
Quebec, Canada) – Ministère de l’Agriculture et de l’Alimentation du Québec (MAPAQ,
Quebec city, Quebec, Canada).
69
CHAPTER 5
EFFECT
COMPONENTS
ON
OF
BUTTERMILK
THE
HEAT-INDUCED
DENATURATION OF WHEY PROTEINS
The work presented in the previous chapter demonstrated that 1) it is possible to integrate
buttermilk constituents into the process of denaturation of whey proteins; 2) addition of
buttermilk constituents led to the formation of mixed aggregates with different properties;
3) higher proportions of buttermilk proteins in the mixtures increased yields and decreased
water-holding capacity of aggregates.
The work presented in this chapter aimed at better understanding the heat-induced
aggregation mechanisms of whey proteins that occur in presence of buttermilk constituents.
Experiments of this chapter were conducted during an internship at Dairy Product
Technology Center (DPTC) at California Polytechnic State University in San Luis Obispo
under the supervision of Dr Rafael Jiménez-Flores.
Results of this part of the project will be submitted essentially in this form in Journal of
Agriculture and Food Chemistry. Results of this paper were also presented in one poster at
2012 Dairy Ingredients Symposium in San Francisco (CA, USA), one poster at 2012
Journée Technologique Novalait in Drummondville (QC, Canada), and in one oral
presentation at 2012 ADSA Joint Annual Meeting in Phoenix (AZ, USA).
71
5.1 Résumé
L’objectif de cette étude était d’obtenir une meilleure compréhension du phénomène
d’agrégation induit par la chaleur entre les protéines du lactosérum et les constituants du
babeurre. Des poudres de concentrés de babeurre ont été préparées à partir de babeurre
régulier et de babeurre de lactosérum puis mises en solution dans un concentré de protéines
du lactosérum avant d’être chauffées à 90°C (pH 4,6) en présence ou non d’un agent
bloqueur de groupements thiols. Les gels SDS-PAGE en conditions réductrices ont montré
que la majorité de protéines n’était pas présente sous formes natives avant le chauffage. Les
images en trois dimensions montrent des interactions entre les protéines, la matière grasse
et les phospholipides. Les résultats suggèrent que des particules réactives sont formées lors
de la préparation du lactosérum et du babeurre. Ces agrégats “préformés” du babeurre
agiraient à titre de noyaux d’agrégation pour les protéines du lactosérum.
72
5.2 Abstract
The objective of this study was to better understand the heat-induced aggregation
occurring between whey proteins and buttermilk constituents as affected by free thiol
groups reactivity. Two different spray dried buttermilk concentrates were prepared from
regular and whey buttermilks. A portion of these powders was treated by supercritical fluid
extraction (CO2) to reduce the amount of non-polar lipids. Powders were hydrated in liquid
whey protein concentrate, and mixtures were adjusted to pH 4.6 and heated up to 90°C. The
dispersions were heated with or without thiol blocking N-ethylmaleimide (5 mM). The
presence of buttermilk constituents decreased the accessibility of free thiol groups during
heating. SDS-PAGE gels under non-reducing conditions showed a decreased in native
whey and MFGM proteins during heating and the presence of large aggregates at the
surface of the stacking and the resolving gels, even in presence of N-ethylmaleimide. SDSPAGE gels under reducing conditions showed that a large amount of proteins were not
present in their native forms before heating. Confocal laser-scanning microscope pictures
confirmed the presence of non-soluble particles corresponding to denatured proteins, fat
globules, milk fat globule membrane fragments and proteins aggregates before heating.
Three-dimensional images suggested interactions between proteins and fat globules or
phospholipid. Overall, results suggest that reactive particles were formed during processing
of whey or buttermilk powders involving proteins and fat through both thiol/disulfide
exchanges and hydrophobic interactions. It appeared that these “pre-formed” particles
would act as a nucleus during heat-induced denaturation of whey proteins and would
control the size and number of protein aggregates.
5.3 Introduction
Denatured and aggregated whey proteins have been widely used as fat mimetic in low fat
cheese (Lawrence, 1989; Lawrence, 1993; Lebeuf et al., 1998; Punidadas et al., 1999), or in
yogurt as a texture modifier (Sandoval-Castilla et al., 2004; Tamine et al., 1995). Authors
reported an increase of the production yield in both processes. However, it is generally
73
accepted that the use of denatured whey proteins is limited due to their excessive waterholding capacity.
Our previous work showed that it was possible to reduce the water-holding capacity of the
whey protein aggregates by substituting whey with buttermilk before the heat treatment
(Saffon et al., 2011). The lower water-holding capacities were obtained with 50% and 75%
of buttermilk protein in the mixtures. The increase of the proportion of buttermilk proteins
also significantly changed the physical and functional properties of the heat-induced
aggregates as follows: an increase of the aggregation yield and consistency coefficient of
the mixtures and a decrease of the flow behavior index of the mixtures as function of the
buttermilk protein fraction. As a general trend, the average diameters of particles were
higher in presence of buttermilk.
The unfolding and aggregation mechanisms of whey proteins under heating conditions have
been studied but are still unclear. Roefs and de Kruif (1994) proposed a mechanism for lactoglobulin denaturation following three steps (initiation, propagation, termination) based
on a polymerization by the exposed free thiol groups of the unfolded -lactoglobulin.
Schokker et al. (1999) observed the formation of oligomers of -lactoglobulin during the
first steps of the denaturation by thiol/disulfide (SH/SS) interchange reactions, thiol/thiol
oxidation, and non-covalent associations. All of the studies agreed on the importance of the
SH/SS interactions between a heat-induced unfolded protein and a monomer. Oldfield et al.
(1998) suggested that the -lactoglobulin also exposes hydrophobic groups during the
initiation step that could participate in the mechanism. Later, the same group found that 15
to 20% of the -lactoglobulin were present as aggregates induced by hydrophobic
interactions at 85°C (Oldfield et al., 2000). Several research groups have confirmed the role
of the hydrophobic interactions during the heat-induced aggregation of -lactoglobulin
proteins in experiments involving a thiol-blocking agent (Hoffmann et al., 1997; Sawyer,
1968; Xiong et al., 1993). The aggregation mechanism of pure -lactalbumin only involves
hydrophobic interactions due to the absence of a free thiol group, but thiol/disulfide
interactions are possible between -lactoglobulin and -lactalbumin when they are heated
74
together. Bauer et al. (2000) concluded that the initial presence of small oligomers such as
monomers, dimers and tetramers are important for the heat-induced aggregation of lactoglobulin. In their review on protein aggregation, Wang et al. (2010) explained that the
majority of protein aggregation phenomena are nucleation-dependent and that an
aggregation nucleus initiates the inter-molecular interactions.
In line with these observations, we hypothesized that the -casein (-CN) and the
constituents of the milk fat globule membrane (MFGM) from the buttermilk act as nucleus
for the aggregation of whey proteins.
It has been shown that MFGM proteins denature at lower temperature (~ 60°C)
comparatively to whey proteins (Ye et al., 2002). Later, the same group demonstrated that
the maximal amounts of whey proteins associated with the MFGM are only 1 % and 0.8 %
of the total -lactoglobulin and -lactalbumin respectively during heating of whole milk at
temperatures ranging from 60 to 95°C during heat treatment of whole milk (Ye et al.
2004a,b). Several other research groups have found the same proportion during the heat
treatment of skim milk or cream (Corredig et al., 1998; Kim & Jiménez-Flores, 1995).
Buttermilk protein composition is globally similar to skim milk with a majority of caseins
(75 % of total proteins), whey proteins (8 to 15% of total proteins) and MFGM proteins
(Sodini et al., 2006; Walstra et al., 2006). Processing steps such as pasteurization of the
cream and/or churning are likely to induce modifications of the buttermilk constituents. For
example, Morin et al. (2008) showed that the solubility at pH 4.6 of MFGM proteins is
affected by the heat treatment of the cream. They also demonstrated the poor coagulation
properties of buttermilk are correlated to the pasteurization of cream. Several changes are
initiated by this treatment such as the modification of the surface of the membrane and the
increase of the accessibility of phospholipids.
The objective of the present work was to better understand the aggregation mechanisms of
whey proteins in presence of buttermilk constituents as affected by free thiol group
75
reactivity during heating. Different buttermilk powders were produced from buttermilk and
whey buttermilk to understand the role of each buttermilk constituent. The importance of
hydrophobic interactions during the heat-induced formation of aggregates was investigated
by addition of a thiol-blocking agent (N-etylmaleimide) prior the heat treatments. The
aggregate composition was characterized by confocal laser-scanning microscopy and gel
electrophoresis.
5.4 Materials and methods
5.4.1 Materials
Fresh whey (unpasteurized; milk pasteurized at 74.4°C for 16.1 seconds) from TILL and
Gouda cheese productions and milk cream (unpasteurized; milk pasteurized at 74.4°C for
16.1 seconds) were obtained in California Polytechnic State University’s processing plant
(San Luis Obispo, CA, USA). Hilmar Ingredients (Hilmar, CA, USA) donated the whey
cream from cheddar cheese production (pasteurized at 82.2°C for 35 seconds; milk
pasteurized at 73.3°C for 16 seconds). Lissamine Rhodamine B was obtained from Avanti
Polar Lipids Inc. (Alabaster, AL, USA) and Fast green FCF from Sigma (ST Louis, MO,
USA). Elman’s reagent was from Thermo Scientific (Rockford, IL, USA). Nethylmaleimide and all other reagents were from Fisher Scientific (Fair Lawn, NJ, USA).
5.4.2 Preparation of whey protein concentrates
Fresh cheese whey was skimmed by using a pilot scale milk separator (Alfa Laval,
Uppsala, Sweden). Bacterial contamination of whey was reduced by microfiltration using a
pilot plant scale system (T-12 model, GEA-Niro Filtration, Hudson, WI, USA) through a
1.4 m membrane (Koch Membrane Systems, Wilmington, MA, USA). Microfiltered whey
was concentrated using two 10 kDa spiral polymeric ultrafiltration membranes (Koch
Membrane Systems, Wilmington, MA, USA) fitted in parallel up to a protein concentration
of 4.0 % (w/v). Whey permeate was collected and frozen until used for protein blend
formulations. The retentate was diluted sequentially in the feed tank with 3 diavolumes of
76
DI water. The diafiltration was stopped when a final concentration of 5.2% of protein (w/v)
was obtained. The whey protein concentrate (WPC) was then frozen to – 35°C until further
analysis. The concentration trial run was performed with whey from three different
productions.
5.4.3 Preparation of spray dried buttermilk concentrates
Regular and whey cream were processed at 12°C using a continuous butter-churn for pilot
plant (Egli, Butschwil, Switzerland). Immediately after churning, regular and whey
buttermilks were filtrated through cheesecloth, and skimmed, using a pilot scale milk
separator (Alfa Laval, Uppsala, Sweden). Skim buttermilks were concentrated by UF (GEA
R-12 model, Milwaukee WI) using two 10 kDa spiral polymeric membranes fitted parallel
on the module, and then the retentate was diluted sequentially in the feed tank with 3
diavolumes of DI water. The DF was conducted until a concentration of 5% of protein
(w/v) was reached. Buttermilk concentrates (BC) were spray-dried using a Niro Filterlab
Spray-drier (Hudson, WI, USA) at 35 bars, and with inlet and outlet air temperatures of
185°C and 95°C, respectively. A portion of each powder was submitted to supercritical
fluid extraction (SFE) (Thar Designs, Inc., Pittsburgh, PA, USA) to remove the non polar
lipids according to Costa et al. (2010). Circulated deionized water (+ coolant) at 3°C was
used for cooling different zones in the SFE apparatus. Carbon dioxide tanks were filled and
inspected by A & R Welding Supply (San Luis Obispo, CA, USA). The system conditions
were controlled manually by Windows 2000-based software. Approximately 125 g of each
sample were submitted to an extraction cycle for maximum non-polar lipid removal using
the following conditions: 7200 g of CO2 at a flow of 20 g.min-1, extraction pressure of 350
bar, and temperatures of 35°C. Powders were stored at 10°C under the following codes:
regular buttermilk concentrate (rBC), whey buttermilk concentrate (wBC), regular
buttermilk concentrate treated by SFE (rBCSFE), and whey buttermilk concentrate treated by
SFE (wBCSFE). Gross composition of powders is presented in Table 5.2.
77
5.4.4 Heat-induced aggregation
WPC was diluted with UF permeate to decrease the protein concentration from 5.2% to
2.6% (w/v). BC powder was then dispersed in diluted WPC to increase the total protein
concentration to 5.2% (w/v) again. Whey-buttermilk mixtures were stirred overnight at
4°C. Denaturation process followed the previous proposed design (Chapter 4). Briefly, 250
mL blends were adjusted to pH 4.6 with 1N HCL and heated from 8°C to 90°C in a
thermostatically controlled water-bath maintained at 97°C for a total time of 25 minutes.
Mixtures were constantly stirred using a caframo lab mixer during heating (speed 5). Nondiluted WPC (5.2% protein) was used as a control. All heating experiments were repeated
three times.
5.4.5 Treatment with N-ethylmaleimide (NEM)
NEM (5 mM) was used to completely block the exposed free thiol group during heating
(Alting et al., 2000). NEM was added to mixtures before overnight stirring.
5.4.6 Analytical method
5.4.6.1
Composition
Gross composition was determined using recommended methods (AOAC, 2003). Nitrogen
content was determined using Kjeldahl method, and converted to protein using a
conversion factor of 6.38. Lipid content was determined using the Mojonnier method, total
solids content by forced air oven drying, and ash content by gravimetric method of
incineration in a furnace at 550°C. Lactose content was determined by enzymatic method
(IDF, 2001).
5.4.6.2
Free thiol groups concentration
Accessible thiol groups concentration was measured according to Ellman (1959). 250 L of
mixtures were taken every five minutes during heating and mixed with 2.5 ml of 0.1M
78
sodium phosphate buffer (containing 1mM EDTA; pH 8.0), and 0.1 mL of DTNB reagent
solution (4 mg in 1 mL of sodium phosphate buffer). The tubes were mixed by hand and
incubated at room temperature for 30 min. Absorbance was measured at 412 nm using a
Spectra Max Plus spectrophotometer (Sunnyvale, CA, USA) after setting a blank.
Absorbance of every sample was also measured without the addition of DTNB. The free
thiol concentration was expressed as mole per gram of proteins.
5.4.7 Gel electrophoresis
Protein profiles were determined using the sodium dodecyl sulfate polyacrylamide gel
electrophoresis technique (SDS-PAGE) under reducing and non-reducing conditions
according to Laemmli (1970). Samples were taken every five minutes during heating and
diluted 1:10 in deionized water, and then with alkaline buffer (non-reducting condition).
Diluted samples were also diluted 1:2 in Laemmli buffer (95% Laemmli solution + 5% Mercaptoéthanol (v/v)) and heated for five minutes at 95°C (reducting condition). After a
quick spin, supernatants were loaded in 15% acrylamide SDS gels. Analyses were
conducted in a mini-Protean II system (Bio-Rad Laboratories Ltd., Hercules, Ca, USA) at
90 V. Gels were stained with Coomassie Brilliant Blue (Sigma Chemical Compagny, ST
Louis, MO, USA). Protein classes were determined according to their molecular weight by
comparison with a molecular weight standard (Precision Plus All Blue Protein Standard,
Bio-Rad laboratories Ltd.).
The aggregated material of each mixture was separated at the end of heating (25 minutes)
by centrifugation (Beckman L7-35, Brea, CA, USA ) at 15,000g for 20 minutes at 20°C.
Samples were taken and diluted 1:2 in deionized water, and then 1:2 in the proper buffer as
described above. Samples corresponded to the soluble material were labelled “sup” in the
legend of the gels.
79
5.4.8 Confocal laser scanning microscopy
Samples of the mixtures were taken at 0, 15, and 25 minutes of heating and diluted 1:2 in
deionized water. 50 L of the diluted samples were dyed with 2 L of Lissamine
Rhodamine B and 2 L of Fast Green FCF. Samples were mixed, and then stored 20
minutes in the dark. 25 L were deposited on a slide, and fixed to the slide with agarose (50
L; 0.5% w/v in deionized water). Observations were performed using an Olympus
Fluoview FV1000 inverted confocal laser scanning microscope (Olympus America Inc.,
Center Valleyr, PA, USA) equipped with four lasers. A 100x oil-immersion objective
(UPLSAPO 100X) was used for all images. Four pictures per samples (800 x 800 pixels;
63.16 x 63.16 µm) were taken and analyzed by using ImageJ (U.S. National Institutes of
Health, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/). Briefly, pictures were transformed
in 8-bit, and then made binary. The software counted particles, and the average particle
surface area was expressed in m2.
Three-dimensional images were obtained by scanning the sample across a defined section
along the z-axis.
5.4.9 Statistical analysis
All experiments were performed in triplicate, so each value represents the mean of three
measurements. Three different and independent variables were selected and named as
follows: Composition that expressed the effect of addition of different buttermilk
concentrates, Time that expressed the effect of the holding time in the water bath, NEM that
expressed the effect of blocking the free thiol groups. Data was tested according to a
factorial design 5*2*2 for the number of particles, a factorial design 5*3*2 for the surface
area of particle, and a factorial design 5*6 only for the concentration of free thiol groups
(any results with NEM). Statistical analysis was carried out using SAS software (SAS
Institute Inc., Cary, NC, USA), using the PROC GLM procedure. Mean comparisons were
performed using a Duncan post-test if appropriate. Table 5.1 summarizes significance for
each treatment on each variable. Results were considered significantly different when P <
0.05.
80
Table 5.1: Summary of the significance (P value) by the analysis of variance of data of each contrast for
each variable (n = 3).
Significance (P values)
Contrast
[free –SH]*
Number of particles Surface area**
Model
< 0.0001
0.7211
< 0.0001
Composition
0.0002
0.3469
< 0.0001
Time (B)
< 0.0001
0.6660
< 0.0001
NEM (C)
n.a.
0.7058
0.0230
A*B
0.9670
0.4033
0.0150
A*C
n.a.
0.9336
0.6037
B*C
n.a.
0.6749
0.0471
A*B*C
n.a.
0.3138
0.8118
(A)
* The effect “blocs” was evaluated significant, so the software automatically re-adjusted
the model. This result means that three whey were different. The first whey was a mix from
TILL and Gouda cheeses production, while the second and third whey were from TILL
cheese production only.
** The effect “blocs” was evaluated significant, so the software automatically re-adjust the
model. This result means that there is a variability regarding the analysis of the pictures.
Pictures were taken in order to illustrate samples in their entirety (small particles, large
particles, etc) and corresponded to a mean of four pictures per samples.
81
5.5 Results and discussion
5.5.1 Composition
The composition of the WPC and BC powders is shown in the Table 5.2, and the protein
profile of the mixture in Figure 5.1.
Table 5.2: Composition (% DM) of whey protein concentrate (WPC) and the different buttermilk
concentrates (BC) powders used for the heating experiment with rBC: regular buttermilk concentrate,
rBCSFE: regular buttermilk concentrate after supercritical fluid extraction, wBC: whey buttermilk
concentrate, wBCSFE: whey buttermilk concentrate after supercritical fluid extraction.
Liquid
WPC
Protein
Fat
Lactose
Ash
5.2  0.2
0.8  0.7
4.4  4.4
0.2  0.4
% of Dry Matter
Powder
rBC
69.2  1.7
22.4  1.4
4.8  1.4
4.5  0.0
rBCSFE
79.1  0.8
5.7  0.4
7.5  2.7
6.4  0.0
wBC
72.1  5.1
20.3  0.8
4.8  1.4
2.7  0.2
wBCSFE
70.8  2.2
13.8  1.5
12.9  5.2
2.9  0.0
Protein content of regular buttermilk concentrate (rBC) and whey buttermilk concentrate
(wBC) was similar and averaged 70.6%. The fat content decreased from 22.4  1.4% to 5.7
 0.4% and from 20.3  0.8% to 13.8  1.5% after CO2-SFE treatment respectively on
regular and whey buttermilk concentrates due to the removal of the non-polar lipids.
The protein profile of the mixtures showed that -lactoglobulin was the main protein in the
mixtures (Figure 5.1). Some proteins from the MFGM membranes were found, and are
identified according to their molecular weight reported by Singh (2006) such as Periodic
acid Shiff 6/7 (PAS 6/7: 48-54 kDa), Adipophilin (ADPH; 52 kDa), Butyrophiline (BTN:
82
67 kDa), Cluster of Differenciation or Periodic acid Shiff IV (CD36 or PAS IV: 76-78
kDa), Periodic acid Shiff III (PAS III: 95-100 kDa) Xanthine dehydrogenase/oxydase
(XDH/XO: 150 kDa), Mucine 1 (MUC1: 160-200 kDa), and BRCA 1 (210 kDa). Fatty acid
binding protein (FABP; 13 kDa) was not found. Casein was found in the mixtures WPC +
rBC and WPC + rBCSFE, and was present as trace level in the WPC, WPC + wBC and
WPC + wBCSFE mixtures.
Figure 5.1: 15% Non-reducing SDS gels of the mixtures. 1: protein standard, 2: WPC, 3: WPC + rBC,
4: WPC + rBCSFE, 5: WPC + wBC, 6: WPC + wBCSFE.
5.5.2 Free thiol groups concentration
Heating time and mixture composition had significant effect on free thiol groups
concentration, but the interaction between these two variables was not significant (Table
5.1). As presented in Figure 5.2, the concentration of free thiol groups significantly
increased from 0.29 ± 0.27 µmol/gprot to 8.16 ± 3.19 µmol/gprot after heating 10 minutes, and
then remained constant (Figure 5.2a). It is known that whey proteins, especially lactoglobulin and Bovin Serum Albumin, are subjected to irreversible conformational
changes upon heating leading to the exposition of free thiol groups (Pantaloni, 1964;
Havea, 200). These changes are negligible at low temperatures (< 55°C), but increase with
the temperature (~ 80°C).
83
Figure 5.2: Effects of time (a) and composition of the mixture (b) on the accessibility of free thiol
groups during heating with constant stirring at temperature up to 90°C at pH 4.6.
As expected, the concentration of free thiol groups was higher in the WPC comparatively to
other mixtures (Figure 5.2b). In presence of caseins from rBC or rBCSFE, the exposure of
free thiol groups was reduced but not significantly. The low levels detected were due to a
lower proportion of -lactoglobulin in rBC, but were surprisingly higher comparatively to
our expectations. Caseins do not expose free thiol groups during heating. It was expected
that the 1:1 ratio between whey and buttermilk material would reduce the concentration of
free thiol groups by 50% in presence of caseins.
Surprisingly, the concentration of free thiol groups was lower in the WPC + wBC mixture
(not significant) and WPC + wBCSFE mixture (significant) comparatively to in WPC. The
reactivity of the proteins and constituents from whey buttermilk seems, in consequence, to
be more affected by the butter-making (pasteurization, churning) that the constituents from
regular buttermilk. Morin et al. (2008) have already reported some effects of the cream
pasteurization such as the poor coagulation properties of regular buttermilk, but to our
84
knowledge no study gives examples of the impact of the processing of whey buttermilk on
its constituents. Knowing that the constituents of whey buttermilk have been subjected to
two pasteurizations (73.3°C for 16 sec and 82.2°C for 35 sec), the lower concentration of
free thiol groups in WPC + wBC and WPC + wBCSFE mixtures could be explained by a
loss of reactivity of whey buttermilk constituents during processes including oxidation of
free thiol group or SH/SS interchange reactions between the different proteins.
The concentration of thiol groups was equal to 0 mole/gprot in presence of N-ethylmaleimide
(NEM) regardless to heating time or composition (data not shown).
5.5.3 Gel electrophoresis
Figures 5.3 to 5.5 present the evolution of native proteins in WPC and mixtures as a
function of heating time.
85
Figure 5.3: Evolution of the protein profile of WPC as a function of heating time (min) under nonreducing (a) and reducing (b) conditions.
Figure 5.4: Evolution of the protein profile of the WPC + rBC mixture as a function of heating time
(min) under non-reducing (a) and reducing (b) conditions.
Figure 5.5: Evolution of the protein profile of the WPC + wBC mixture as a function of heating time
under non-reducing (a) and reducing (b) conditions.
86
Content of native -lactoglobulin (-LG) and -lactalbumin (-LA) decreased after 10
minutes of heating in WPC (Figure 5.3a), but after 15 minutes in presence of caseins (WPC
+ rBC) (Figure 5.4a) but only after 5 minutes in WPC + wBC (Figure 5.5a). In all the
mixtures the amount of native BTN decreased as a function of the heating time. The
amounts of CD 36 or PAS IV, MUC 1 or BRCA 1, and PAS 6/7 remained constant in
WPC, but were decreasing after 10 minutes of heating in the mixtures. Native XO was
present only before heating in all the blends. In all mixtures, the amount of residual proteins
in the wells seemed to increase from 0 to 15 minutes of heating and then slightly decreased
until the end of heating (25 minutes). As a general trend, electrophoresis separation of
proteins conducted under non-reducing conditions showed that native forms of XO and
BTN were lost in the first 5 to 15 minutes of heating following by the decrease of the native
forms of -LG and -LA. Ye et al. (2002) already reported that the MFGM proteins are
sensitive at lower temperatures comparatively to the other dairy proteins. Later, they have
proposed mechanisms of heat-induced aggregation between MFGM proteins, lactoglobulin, and -lactalbumin (Ye et al., 2004a; Ye et al., 2004b). These heat-induced
associations appear after dissociation of -LG into monomers and before exposition of its
free thiol groups. Free thiol groups of MFGM proteins initiate SH/SS interchange reactions.
After reduction, residual proteins were observed in the wells for the mixture, but not for the
WPC, and the MFGM proteins PAS III and FABP. The bands of -lactoglobulin, lactalbumin and MFGM proteins were more abundant after reduction and even for the
sample before heating. This result clearly demonstrates that protein aggregates were formed
previously to the heat-induced denaturation during the preparation of the WPC or powders.
Gels under reducing conditions show that whey proteins, caseins, MFGM proteins were
involved in the formation of aggregates during the process of preparation through SH/SS
interchange reactions (mainly) and non-covalent exchanges. Residual proteins remained in
the well after reduction indicating that some high molecular weight particles were formed
through other types of interactions during heating such as hydrophobic interactions and van
der Waals forces.
87
Figure 5.6: Evolution of the protein profile of the WPC (a), WPC + rBCSFE mixture (b), and WPC +
wBC (c) in presence of NEM as a function of heating time under non-reducing conditions.
Figure 5.6 shows the evolution of native proteins during heating in presence of NEM. In all
mixtures, the amount of XO was the only band to visually decrease in intensity after 5 or 10
minutes. The amount of residual proteins in the wells was constant for the WPC except at 5
minutes were the amount was higher. Some particles remained in the wells after reduction
(result not shown). In WPC + rBC, WPC + rBCSFE, and WPC + wBC, the amount of
residual proteins in the wells stayed constant, even after reduction. For WPC + wBCSFE
mixture, the amount of residual proteins in the wells was higher at the end (after 10
minutes) than at the beginning of heating. In presence of the thiol-blocking agent, high
molecular weight particles were present in the well or at the beginning of the resolving
gels. Sawyer (1968), Xiong et al. (1993), Hoffmann and Van Mil (1997) have observed the
formation of whey protein aggregates during a heat treatment in the presence of Nethylmaleimide (NEM) via non-covalent interactions, especially hydrophobic interactions.
Gels under reducing conditions (results not shown) confirmed that non-covalent
interactions also contributed to the formation of protein aggregates during the preparation
and the heating between whey proteins and buttermilk constituents. The role of non-
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covalent interactions in dairy protein aggregation is, however, still not well defined.
Oldfield et al. (2000) have demonstrated that 15 to 20 % of the available -lactoglobulin
aggregated via hydrophobic interactions during the first steps of heat-induced denaturation
followed by covalent interactions at 85°C.
5.5.4 Confocal laser scanning microscopy images
Figures 5.7 and 5.8 present the average distribution of proteins (green) and phospholipids
(red) at 0, 10 and 25 minutes of heating in WPC and WPC + wBC mixture respectively.
Figure 5.7: Confocal laser scanning miscroscope pictures taken at 100X of WPC at 0, 15, and 25
minutes of heating before (top) and after (bottom) analysis with ImageJ.
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Figure 5.8: Confocal laser scanning microscope pictures taken at 100X of WPC + rBC mixture at 0, 15,
and 25 minutes of heating before (top) and after (bottom) analysis with ImageJ.
At t0, more non-soluble proteins and phospholipids were found in the WPC + wBC mixture
than in WPC. Phospholipids were not visible at t0 in whey. Phospholipids remained close to
protein particles in WPC, but did not interact with them. In WPC + wBC mixture,
phospholipids seemed to be part of the protein aggregates, as evidenced by close contacts
between fat globules or fat globules with proteins or both. The binary transformation
confirmed that interactions between phospholipids and proteins were hardly found in whey,
but phospholipids were involved in the structure of the protein aggregates in the presence
of buttermilk constituents and even since the beginning of heating. Statistical analysis
(Table 5.1) showed no significant difference between samples for the number of particles
per images observed at 100X, but the interactions composition*heating time (P = 0.0150)
and heating time*NEM (P = 0.0471) had significant effect on the average surface area.
Figure 5.9a represents the evolution of the surface area of particles observed at 100X as a
function of composition and heating time.
90
size time*condi
4
a)
b)
Average area observed at 100X
(µm2)
Average area observed at 100X
(µm2)
4
3
3
2
2
1
0
normal
NEM
1
0
10
20
Time (min)
30
0
0
15
Time (min)
25
Figure 5.9: Evolution of the average surface area observed with a confocal laser scanning microscope at
100X as a function of interactions time*composition (a) and time*condition. In a), mixtures were
presented as follows:  = WPC;  = WPC + rBC;  = WPC + rBCSFE;  = WPC + wBC  = WPC +
wBCSFE. In b), conditions were presented as follows:  = absence of NEM;  = presence of NEM.
At every time, the average surface area of particles in WPC was higher comparatively to
the blends. Figure 5.9b represents the evolution of the surface area of particles observed at
100X as a function of time and in presence or absence of NEM. As a general trend, the
presence of NEM significantly reduced the average surface area of particles in the mixtures.
Confocal laser-scanning microscope pictures analysis revealed that WPC and mixtures
contained non-native proteins and non-soluble MFGM material before heating, especially
in whey buttermilks. Small protein aggregates and protein – phospholipid/MFGM
aggregates were also present. The loss of solubility and the formation of small aggregates
were probably due to the pasteurization of the milk/cream or the mechanical forces
occurring during the cheese-making process, butter-making process, filtration, and spray
drying. Increasing the time of heating and the temperature did not increase the number of
non-soluble particles in WPC or in the mixtures, while the particle size stayed constant in
mixtures. Non-soluble particles already present in the mixtures became bigger by
association with denatured proteins or small aggregates in mixtures.
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Three-dimensional images were taken to visualize interactions between proteins and
phospholipids/MFGM. Pictures confirmed that some non-native proteins or protein
aggregates were found even at t0 in whey and mixtures. Figure 5.10 shows that some
interactions between proteins and phospholipids/MFGM were also present at t0, but only in
WPC + buttermilk mixtures. The presence of phospholipids (red) or interactions between
proteins and phospholipids/MFGM were more important after treatment of the buttermilk
powders by C02-supercritical fluid extraction (not presented). After heating, it appeared that
phospholipids were mainly present at the surface of the proteins aggregates, but some small
red dots were also located inside the structure of aggregates at t15 and t25. This phenomenon
was observed in every mixtures, but mainly in WPC + wBC.
Figure 5.10: Three-dimensional confocal laser scanning microscope pictures of WPC + rBC mixture
before heating.
Three-dimensional pictures confirm that the interaction between phospholipids/MFGM and
proteins were present in the mixtures, including in samples treated by NEM, but were
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limited in WPC. Surprisingly, these non-soluble particles were almost non-detected in
WPC. Similar trend was observed with liquid whey buttermilk after UF/DF (results not
shown), meaning that the presence of a large amount of non-soluble particles (denatured
proteins, fat, and protein aggregates) in the mixture before heating is not to be attributed to
poor dispersibility of buttermilk powder ingredients. Three-dimensional pictures confirmed
that some proteins covered fat globules or MFGM fragments. More surprisingly,
phospholipids, viewed as little red dots were present on the side of the aggregates or even
buried in the structure of the protein aggregates. This phenomenon was more frequent after
C02-SFE treatment of the buttermilk. Unfortunately, it is impossible to identify the nature
of the small aggregates and their mechanism of interaction. They could be a small fragment
of MFGM (proteins + phospholipids) or free phospholipids. Interactions between
phospholipids (lecithin, lysolecithin, phosphatidylcholine) with s1-casein or lactoglobulin have been demonstrated (Barratt et al., 1974; Brown et al., 1983; Spector et
al., 1970). In all cases, the complexation of phospholipids and proteins involves first an
ionic attraction between the charged amino-acid residues (protein) and the polar head group
(lipid), and then hydrophobic interactions to stabilize the complex. In the case of lactoglobulin, its helix formation is determinant (Brown et al., 1983). Interactions between
phospholipids and proteins could also explain the presence of particles in the wells of gel
after reduction.
5.5.5 Proposed mechanism for association of whey proteins with
buttermilk constituents
The results of this study suggest that the heat-induced association between whey proteins
from WPC and buttermilk constituents involves the following steps:
Preliminary reactions: Formation of small aggregates during the preparation of buttermilk
powders. Regarding the temperatures involved in the process of preparation of the BC
powders it appears evident that proteins from buttermilks are pre aggregated, including
whey proteins, caseins, and MFGM proteins. Even if the mechanisms of heat-induced
aggregation of dairy proteins are still uncertain, it has been well demonstrated that whey
93
proteins interact with -casein at the surface of the casein micelles (Livney et al., 2004;
Morr et al., 1968; Sawyer, 1968; Vasbinder et al., 2003a) or with soluble -casein (Alting
et al., 2003; Anema, 2008; Anema et al., 2003a) or with MFGM proteins (Ye et al., 2004a;
Ye et al., 2004b) during heating. Formation of aggregates may also integrate free
phospholipids.
Step 1: «Activation» of the pre formed aggregates from BC. Wang et al. (2010) reminded
us that most of the protein aggregation mechanisms are nucleation-dependent, and are
initiated by formation of an aggregation nucleus. Krishnan et al. (2003) defined the
aggregation nucleus as a small particle with a size close to a -LG dimer, while Baynes et
al. (2005) observed small -LG multimers as nuclei. Schokker et al. (1999) observed the
presence of non-native dimers and oligomers in the early stages of -LG denaturation. In
presence of buttermilk, it appears that aggregation nucleus could be non-native whey
proteins or small protein aggregates or MFGM fragments or -caseins or a combination.
Step 2: Aggregation of denatured proteins from WPC with aggregated buttermilk
constituents. Based on studies from Schokker et al. (1999) and Baynes et al. (2005),
monomers or small aggregates of whey proteins were probably rapidly incorporated into
the early-formed particles to form large aggregates through mainly SH/SS interchange
reactions and non-covalent interactions.
Confocal pictures revealed a higher accessibility of MFGM fragments in whey buttermilk
but not in regular buttermilk, presumably due to the more complex interactions of caseins
that are present in regular buttermilk.
5.6 Conclusions
The present study shows that the heat-induced denaturation mechanism of whey proteins
changes in the presence of buttermilk constituents. The presence of caseins, whey proteins,
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and MFGM components from buttermilk reduced the concentration of free thiol groups,
and delayed the loss of native -lactoglobulin and -lactalbumin. In presence of whey
buttermilk, the concentration of free thiol groups was also reduced, but the loss of native
whey proteins was accelerated. SDS PAGE confirmed that MFGM proteins seemed
involved in the aggregation mechanism of whey proteins, however interaction of
MFGM/phospholipids were poorly represented in WPC compared to mixtures. Buttermilk
contained a small but perhaps important amount of non-soluble proteins. Small aggregates
were also present, and involved both thiol/disulfide and hydrophobic bonds between whey
protein, casein and MFGM components. Confocal pictures revealed a higher accessibility
of MFGM fragments in whey buttermilk concentrate but not in regular buttermilk
concentrate, presumably due to the more complex interactions of caseins that are present in
regular buttermilk. The presence of phospholipids buried in the structure of the aggregates
suggests that they could participate to the formation of the aggregates in the early stages of
heating.
5.7 Acknowledgements
This work was funded by the Le Fond Québécois de la Recherche sur la Nature et les
Technologies (FQRNT, Quebec City, Quebec, Canada) – Novalait Inc. (Sainte-Foy,
Quebec, Canada) - Ministère de l’Agriculture et de l’Alimentation du Québec (MAPAQ,
Quebec City, Quebec, Canada).
95
96
CHAPTER 6
EFFECT OF HEATING OF WHEY
PROTEINS IN THE PRESENCE OF MILK FAT
GLOBULE
MEMBRANE
EXTRACT
OR
PHOSPHOLIPIDS FROM BUTTERMILK
The work presented in the previous chapter demonstrated that 1) protein aggregates are preformed during the preparation of the concentrates; 2) pre-formed aggregates act as
aggregation nucleuses during the heat treatment of the mixtures. Results also suggested that
phospholipids from regular or whey buttermilk are integrated to the formation of the
protein aggregates.
The work presented in this chapter aimed at proving that phospholipids from buttermilk can
act as a nucleus during aggregation of whey proteins. Experiments of this chapter were
conducted during an internship at Dairy Product Technology Center (DPTC) at California
Polytechnic State University in San Luis Obispo under the supervision of Dr Rafael
Jiménez-Flores.
Results of this part of the project will be submitted after the publication of Chapter 5.
Results of this paper will be presented in a oral presentation at 2013 ADSA Joint Annual
Meeting in Indianapolis (IN, USA), and in a poster form at 2013 STELA Colloque in
Montreal (QC, Canada).
97
6.1 Résumé
L’objectif de cette étude était de déterminer si les phospholipides interviennent lors de
l’agrégation des protéines du lactosérum. Les phospholipides ont été obtenus à partir d’un
isolat de membranes de globule de matière grasse (MFGM) ou d’une poudre commerciale
de phospholipides laitiers (PL). Les PL ont été mis en solution (1 % p/v) avec un isolat de
protéines du lactosérum (IPL) (5 % p/v) et chauffés à 65 ou 80°C (pH 4,6 ou 6,8).
L’addition de phospholipides n’a pas affecté significativement la libération de groupements
thiols mais un délai de 5 min a été observé pour la diminution des formes natives des
protéines à pH 6,8. Les profils de chromatographie ont révélé une absence de
phospholipides libres après 20 min de chauffage à 80°C à pH 4,6 dans le mélange IPL-PL.
Les images en trois dimensions ont confirmé la présence d’interactions entre les protéines
et les PL dans les mélanges.
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6.2 Abstract
The objective of this study was to understand if the phospholipids from buttermilk can act
as heat-induced aggregation nucleus for whey proteins. For this purpose, phospholipids
were obtained from a milk fat globule membrane (MFGM) extract from whey buttermilk or
from a commercial dairy phospholipids powder (PL). Whey proteins isolate (WPI) was
dispersed in water to a concentration of 5% protein (w/v), and the pH was adjusted to 4.6 or
6.8. Solutions were heated to 65°C or 80°C for 25 min under constant stirring. MFGM
extract or PL powder were dispersed in the WPI solutions at 1.0% (w/v) before heating.
The aggregate composition was characterized with the Ellman’s reagent (free thiols), onedimensional gel electrophoresis technique, thin layer chromatography (TLC), and three
dimensional confocal laser-scanning microscopy. All experiments were performed in
triplicate and the concentration of free thiol groups was tested according to a factorial
design. Addition of phospholipids or MFGM extract did not significantly affect the
liberation of free thiol group of whey proteins but delayed the loss of native form of major
whey proteins by 5 min at pH 6.8. TLC profiles showed no traces of free phospholipids
after 20 min of heating in WPI-PL mixtures at 80°C at pH 4.6 when phospholipids were
added to the mixtures. 3D images confirmed that only few interactions occurred between
whey proteins and MFGM proteins/phospholipids in the whey solution, while the
interactions were frequent in presence of MFGM extract or phospholipids. Overall, our
results suggest that phospholipids from buttermilk are involved in the formation of protein
aggregates at pH 4.6 through the MFGM fragments at 65°C while they seem to directly
interact with the proteins at 80°C.
6.3 Introduction
Microparticulated whey proteins (MWP) have been developed to mimic fat globules and to
increase retention of whey protein in cheese (Lawrence, 1993; Punidadas et al., 1999) or to
modify the texture of yogurts (Sandoval-Castilla et al., 2004; Tamine et al., 1995). Whey
proteins are concentrated by ultrafiltration, and then heat-denatured at 90-95°C for several
99
minutes at acid (4.2 to 4.6) or neutral (6.2 to 6.7) pHs (Lebeuf et al., 1998; Punidadas et al.,
1999). The addition of MWP has to be controlled due to the excessive water-holding
capacity of denatured whey proteins. In consequence, MWP are used in very low amounts
during formulation (~ 1.0 to 1.5 % of total weight).
Our previous work showed that it was possible to control the water-holding capacity of
heat-denatured whey proteins by substituting whey with buttermilk before the heat
treatment (Saffon et al., 2011). Our latest results demonstrated that protein aggregates were
formed during butter-making process involving whey proteins, caseins, and milk fat
globule membrane (MFGM) proteins. These preformed aggregates acted as an aggregation
nucleus during whey protein denaturation (Chapter 5). Results also suggested that
phospholipids could act as an aggregation nucleus too.
Brown et al. (1983) concluded that -lactoglobulin in its native form is generally unable to
form a complex with phosphatidylcholine or lipids. A certain degree of unfolding is
necessary to expose initially buried helical regions and hydrophobic groups. According to
their model, lipid-protein interactions are initiated by a ionic attraction between charged
amino acid residues in the protein and the polar head group of the lipid in order to position
the lipid. Then the complex is stabilized by hydrophobic interactions between the
hydrophobic side of the helix and the hydrocarbon chain of the lipid. Similar observations
have been reported by Bos and Nylander (1996) and Ong et al. (1981). Dufourcq et al.
(1977) and Ong et al. (1981) observed that the charged residues involved are lysine or
arginine for the protein and phosphate or carboxylic groups (negative) for
the
phospholipids. In their review on the binding properties of -lactoglobulin, Kontopidis et
al. (2004) proposed that the monomeric form of the protein has one ligand-binding site, and
potentientially another weaker site (not yet located) that can bind linear molecules such as
the fatty acids hydrocarbon chains of the hydrophobic tails of the phospholipids or retinol.
Phospholipids can also be attached to whey proteins through the MFGM proteins. Fat
globules are disrupted during churning, so the membrane is segmented and contains a
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complex mix of proteins, phospholipids, glycoproteins, triglycerides, cholesterol, enzymes,
and different minor components. Ye et al. (2004a,b) demonstrated that whey proteins and
MFGM proteins interact during heating of milk.
The objective of the present work was to determine if phospholipids from buttermilk can
interact with whey proteins during a heating treatment. Whey proteins were heated in
solution in presence of MFGM extract from whey buttermilk or in presence of
phospholipids from regular buttermilk. The aggregate composition was characterized by gel
electrophoresis, thin layer chromatography, and three-dimensional confocal laser-scanning
microscope images.
6.4 Materials and methods
6.4.1 Materials
Hilmar Ingredients (Hilmar, CA, USA) donated the whey cream (pasteurized at 82.2°C for
35 seconds; milk pasteurized at 73.3°C for 16 seconds) from cheddar cheese. BiPro whey
protein isolate (WPI) was obtained from Davisco Foods Ingredient, Inc. (Eden Prairie, MN,
USA), and phospholipids concentrate 700 (the following components have been detected
by HPLC: neutral lipids, lactosylceramide, sphingomyeline (SM), phosphatidylcholine
(PC), phosphatidyléthanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS))
was manufactured at Fonterra Ltd (Edgecumbe, New Zealand). Lissamine Rhodamine B
was obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA), and Fast Green FCF
from Sigma (St-Louis, MO, USA). Ellman’s reagent was from Thermo Scientifics
(Rockford, IL, USA). All other reagents were from Fisher Scientific (Fair Lawn, NJ, USA).
6.4.2 Isolation of the milk fat globule membrane
Whey cream was processed at 12°C using a continuous butter churn for pilot plant (Egli,
Butschwil, Switzerland). Immediately after churning, the whey buttermilk was filtrated
101
through cheesecloth. Filtrated buttermilk was centrifuged at 25,000 rpm for 120 minutes at
4°C (Beckman L7-35 Ultracentrifuge, Beckman Coulter, Inc., USA). The supernatant was
removed, and the pellet was dissolved in PBS buffer in presence of Triton X 100. The
hydrated pellet was centrifuged at 25,000 rpm for 120 minutes at 4°C. The supernatant was
removed, and the pellet hydrated in water to a 10% (w/v) solution. The solution was mixed
in a blender, and sonicated for 1 minute, and finally stored at -20°C until further analysis.
6.4.3 Heat treatment
WPI powder was dispersed in deionized water to a concentration of 5.0% protein (w/v),
and the pH was adjusted to 4.6 with 1N HCl or 6.8 with 1N NaCl. The solution was then
dispersed overnight at 4°C. WPI solutions were heated from 8°C to 65°C or 80°C in a
thermostatically controlled water-bath maintained at 72°C or 87°C for a total time of 25
minutes. Solutions were constantly stirred using a caframo lab mixer during heating (speed
5). Heated solutions were cooled down at room temperature. One gram of MFGM extract
or phospholipids powder per 100 mL of WPI solution were also added before stirring. All
heating experiments were repeated three times.
6.4.4 Analytical method
6.4.4.1
Concentration of free thiol groups
Accessibility of free thiol groups was measured according to Ellman (1959). 250 L of
mixtures were taken every five minutes of the heating time, and mixed with 2.5 mL of
0.1M sodium phosphate buffer (containing 1mM EDTA; pH 8.0), and 0.1 mL of DTNB
reagent solution (4 mg in 1 mL of sodium phosphate buffer). The tubes were mixed by
hand, and incubated at room temperature for 30 minutes. Absorbance was measured at 412
nm using Spectra Max Plus spectrophotometer (Sunnyvale, CA, USA) after setting a blank.
Absorbances of each sample were also measured without the addition of DTNB. The free
thiol concentration was determined in mole/gprot.
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6.4.4.2
Gel electrophoresis
Protein profiles were determined using the sodium dodecyl sulfate polyacrylamide gel
electrophoresis technique (SDS-PAGE) under non-reducing conditions according to
Laemmli (1970). Samples were taken at 0, 10, 15, and 20 minutes of the heating time, and
diluted 1:10 in deionized water, and then with alkaline buffer (non-reducing conditions). 25
L of samples were loaded in 15% acrylamide SDS-PAGE gels. Analyses were conducted
in a mini-Protean II system (Bio-Rad Laboratories Ltd., Hercules, CA, USA) at 90 V. Gels
were stained with Coomassie Brilliant Blue (Sigma Chemical Compagny, St Louis, MO,
USA). Protein classes were determined according to their molecular weight by comparison
with a molecular weight standard (Precision Plus All Blue Protein Standard, Bio-Rad
laboratories Ltd.).
6.4.4.3
Thin layer chromatography
Samples from the solution WPI+PL were taken at 0, 10, 15, 20, and 20 minutes of the
heating time, and 5 L were loaded directly on a glass backed silica TLC plate using a
glass capillary. Plates were deposed in a chamber filled with a polar solvent mixture
containing 65 mL of chloroform, 25 mL of methanol, and 4 mL of deionised water. Plates
were maintained in chamber for 60 to 90 minutes, and then dry into vacuum oven for 15
minutes. Dried plates were placed overnight in iodine chamber with iodine pellet.
6.4.5 Confocal laser-scanning microscopy
Sample of the mixtures were taken at 0, 15, and 25 minutes of heating and diluted 1:2 in
deionized water. 50 L of the diluted samples were dyed with 2 L of Lissamine
Rhodamine B and 2 L of Fast Green FCF. Samples were mixed and stored 20 minutes in
the dark. 25 L of the dyed solution were deposited onto a slide, and fixed with agarose (50
L; 0.5% w/v in deionized water). Three-dimensional images were taken by scanning the
sample across a defined section along the z-axis using an Olympus Fluoview FV1000
inverted confocal laser-scanning microscope (Olympus America Inc., Center Valley, PA,
USA).
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6.4.6 Statistical analysis
All heating treatments and experiments were performed in triplicate. Data from the
concentration of free thiol groups data was tested according to a factorial design
composition*time*temperature*pH (3*2*2*2). Statistical analysis was carried out using
SAS software (SAS Institute Inc., Cary, NC, USA) using the PROC GLM procedure.
Results were considered significantly different when P < 0.05.
6.5
Results and discussion
6.5.1 Free thiol group concentration
The statistical analysis of data presented in Table 6.1 shows that only interactions
composition*temperature (P = 0.0094) and temperature*pH (P = 0.0049) were significant
on the concentration of free thiol groups. Figure 6.1 presents the effects of these
interactions on the accessibility of thiol groups during heating.
104
Table 6.1: Summary of the significance calculated by the analysis of variance of free thiol group
concentration (n = 3).
Significance (P value)
Contrast
[Free –SH] Significance Contrast [Free –SH] Significance
Composition (A) 0.5958
n.s
B*C
0.1335
n.s
Time (B)
0.0141
n.s
B*D
0.0924
n.s
Temperature (C)
0.0280
n.s
C*D
0.0049
**
pH (D)
0.0626
n.s
A*B*C
0.4071
n.s
A*B
0.7979
n.s
A*B*D
0.8675
n.s
A*C
0.0094**
**
A*C*D
0.6712
n.s
A*D
0.4444
n.s
B*C*D
0.9643
n.s
a)
2.0
b)
2.5
2.0
[-SH] free (10-5 mole.gprot-1)
[-SH] free (10-5 mole.gprot-1)
1.5
1.5
1.0
1.0
0.5
0.5
0.0
C
pH
4.
6_
80
°
C
pH
6.
8_
80
°
C
pH
6.
8_
65
°
C
65
°
4.
6_
pH
0°
C
0°
C
W
.+
P.
8
.8
0°
C
W
.+
M
W
.8
5°
C
W
.+
P.
6
5°
C
.6
W
.+
M
W
.6
5°
C
0.0
Figure 6.1: Effect of interactions composition*temperature (a) and temperature*pH (b) on the
exposition of free thiol groups during heating of WPI (W.), WPI+MFGM extract (W.+M.), and
WPI+phospholipid powder (W.+P.) under constant stirring.
As shown in Figure 6.1a, the average concentration of free thiol groups exposed during
heating significantly increased in the mixtures between 65°C and 80°C.
105
As shown in Figure 6.1b, the average concentration of free thiol groups exposed during
heating at pH 4.6 or 6.8 significantly increased between 65°C and 80°C.
It has been known since Pantaloni (1964) that the exposition of the free thiol group of the
-lactoglobulin (Cys121) is initiated at 70°C, but in a low proportion (6%), and defined the
irreversible conformation change of the protein. However, -lactoglobulin is irreversibly
unfolded at a temperature close to 78.5°C, making the free thiol groups accessible for
thiol/disulfide exchanges (Schokker et al., 1999). Increasing the temperature above 80°C
led to increase the liberation of free thiol groups. Hoffmann et al. (1997) demonstrated that
aggregation of -lactoglobulin is related to the pH dependent reactivity of the free thiol
group. Their results showed that the accessibility of the free thiol group of the protein is
better at pH = 8.0 comparatively to pH < 7.0 due to the pK value of thiol group around 8.2.
To summarize, the presence of milk fat globule membrane extracts (MFGM) or
phospholipids from buttermilk did not affect the accessibility of thiol groups of lactoglobulin.
However, the presence of MFGM extract or phospholipids affected the formation of the
aggregates. Figure 6.2 illustrates differences between pellets from the sedimentation of
mixtures following the heat treatment.
106
Figure 6.2: Pictures of heated WPI (a), WPI+MFGM (b), and WPI+PL (c) at pH 4.6 at 80°C and after
60 minutes of sedimentation in ice-melted water bath.
At pH 4.6, the height of pellets was smaller and the texture was harder in WPI
comparatively to the other mixtures. In the mixtures WPI+MFGM extract and WPI+PL the
texture of the pellet was softer and easy to disturb, while in WPI it was comparable to a gel.
Increasing the temperature from 65°C to 80°C, only changed the height of pellets. No
pellets were observed at pH = 6.8 for both temperatures meaning that the heat treatment
was not strong enough at this pH to form non-soluble aggregates. Our previous results have
demonstrated that the presence of buttermilk constituents during the denaturation of whey
proteins controls the size and the number of particles (Chapter 5 and 6). In general, addition
of buttermilk constituents decreased the number and the size of heat-induced aggregates.
The present results seem to confirm that whey proteins form a strong network of aggregates
while heating alone, and that the presence of buttermilk constituents changes the physical
properties (such as size) of heat-induced aggregates.
107
6.5.2 Gel electrophoresis
Evolution of native proteins as a function of heating time (80°C) is presented in Figure 6.3.
Figure 6.3: Evolution of the protein profiles in the different mixtures at 0 (a), 10 (b), 15 (c), 20 (d)
minutes of heating at 80°C using SDS-PAGE under non-reducing conditions. Letters a to d correspond
to samples at pH 6.8 and letters a' to d' to pH 4.6.
At pH 6.8, content of native -lactoglobulin and -lactalbumin decrease after 15 minutes of
heating time (until the end) in WPI+MFGM and WPI+PL, but only after 10 minutes in
WPI. We have already reported that the presence of buttermilk constituents delays the loss
of native -lactoglobulin and -lactalbumin. It is known since Sawyer et al. (1963) that the
main whey proteins interact which each other during a heat treatment. The aggregation
process involves mainly thiol/disulfide interchange reactions, but also thiol/thiol oxidation,
and hydrophobic interactions (Oldfield et al., 1998; Schokker et al., 1999). The decrease of
the amount of whey proteins at each heating time is correlated to the process of
aggregation.
108
Under non-reducing conditions, only three MFGM proteins were present in the mixtures
before heating such as Butyrophiline (BTN: 67 kDa) and Cluster of Differenciation or
Periodic Acid Shiff IV (CD36 or PAS IV: 76-78 kDa). All the other MFGM proteins were
not detected by the migration. Ye et al. (2002) have reported that MFGM proteins are
sensitive at lower temperatures comparatively to whey proteins. The pasteurization
treatment of the whey or whey cream used for the present work could have been sufficient
to denature most of the MFGM proteins. Their results also suggested that xanthine
dehydrogenase/oxydase (XDH/XO: 150 kDa) and butyrophiline form a protein complex
with a higher molecular weight during heating above 60°C. This complex could be the band
located around 250 kDa in our gels. However, Butyrophyline was still present after 10
minutes of heating at pH 6.8, but only in a low amount, while the two other MFGM
proteins were absent. Ye et al. (2004a,b) have demonstrated that MFGM protein can
interact with -lactoglobulin and -lactalbumin during heating of milk, but only in a very
low proportion. Our previous work has also shown that BTN and XO (from buttermilk) lost
their native form previously to whey proteins (Chapter 6). No decrease in the amount of the
native whey proteins or MFGM proteins was observed at pH 4.6 or during the heating at
65°C (for both pH) probably due to the poor accessibility of free thiol groups at this
temperature.
6.5.3 Confocal laser-scanning microscopy images
Even if the evolution of the protein profiles was similar (Figure 6.3), the microstructures of
non-soluble aggregates were different. As shown in Figure 6.4, non-soluble aggregates
were composed only of proteins (green) in WPI. Phospholipids (red) if present were not
integrated in the structure of the protein aggregates. Previous results showed that some
interaction protein/phospholipids or protein/MFGM proteins could be observed in heated
whey protein mixtures, but were isolated and limited. It appears that whey proteins do not
interact with MFGM proteins or phospholipids during the heating of whey at pH 4.6. At
this pH, the repulsion forces between proteins are reduced due to the absence of surface
charge. Whey proteins probably preferentially interact with each other.
109
Figure 6.4: Three-dimensional confocal laser-scanning microscope pictures of WPI heated for 25
minutes at 80°C at pH 4.6.
However, this theoretical conclusion does not explain the trend observed in the
WPI+MFGM extract mixture. As shown in Figure 6.5, MFGM proteins or phospholipids
were part of the protein aggregate structures at pH 4.6, even only after 15 minutes of
heating at 80°C. Some red dots were located on the side of aggregates (arrows I), but some
were also buried inside the structure of the protein aggregates (arrows II). Pictures were
taken under different angles in order to confirm that the red dots and green dots were
connected. Appel et al. (1982) have shown that the denaturation of butyrophiline appear at
58°C. Later, Ye et al. (2002) demonstrated that BTN and XO can form aggregates at
temperature above 60°C. Interactions between whey proteins and MFGM proteins are due
to the exposition of free thiol groups of MFGM proteins during the initial state of the whey
proteins denaturation, and involved mainly thiol/disulfide exchanges.
110
Figure 6.5: Three-dimensional confocal laser-scanning microscope pictures of the WPI+MFGM extract
heated for 15 minutes at 80°C at pH 4.6.
As shown in Figure 6.6, red dots (phospholipids) were integrated to the structure of protein
aggregates (green) at pH 4.6 in the mixture WPI+PL even during a heating at 65°C.
Phospholipids were present on the side or buried in the structure of the protein aggregates.
Our previous idea was that phospholipids could act as an aggregation nucleus during the
heat-induced denaturation of whey proteins (Chapter 6). Most of the protein aggregation
mechanisms (included -lactoglobulin) are defined as nucleation-dependent and are in
consequence initiated by the formation of an aggregation nucleus (Wang et al., 2010). In
this case, a nucleus is a small molecule often considered as the size of a dimer that is
present since the first stage of denaturation. Later in the aggregation process, non-native
monomers, denatured proteins, and/or small aggregates are incorporated to the nucleus to
form large aggregates.
111
Figure 6.6: Three-dimensional confocal laser-scanning microscope pictures of WPI+PL heated for 15
minutes at 65°C at pH 4.6
Wang et al. (2010) defined the last state of protein denaturation as the precipitation (loss of
solubility) of aggregates. As shown in Figure 6.7, this state was not reached at pH 6.8. Only
few isolated non-soluble particles were observed. As a general trend, these particles were
composed of phospholipids covered with few proteins.
112
Figure 6.7: Three-dimensional confocal laser-scanning microscope pictures of WPI heated for 15
minutes at 80°C at pH 6.8.
6.5.4 Thin layer chromatography
The evolution of the free phospholipids (not associated with the whey proteins) as a
function of the heating time is presented in Figure 6.8. Results show that at pH 6.8 the free
phospholipid profile is similar to the control at each heating time with a slight decrease in
dot sizes at 25 minutes. At pH 4.6, any free phospholipids were found at 20 and 25 minutes
in two of the three replicates (decrease in the third repetition only). A longer time than what
was expected was necessary to bind a large amount of phospholipids to whey proteins at
pH 4.6.
113
Figure 6.8: Evolution of the phospholipids profiles in WPI+PL at 0 (a), 10 (b), 15 (c), 20 (d), and 25 (e)
minutes of heating time at 80°C. Picture was converted in negative mode in order to make the spot
easier to see.
As demonstrated by the confocal images, interactions between phospholipids and whey
proteins during the early stages of the heating still occurred but involved only few
phospholipids or weak interactions (easily breakable by the TLC solvents). Unfortunately,
our TLC plates did not permit to identify the free phospholipids involved. The conclusions
that -lactoglobulin can bind long chain fatty acids in aqueous solution have been reported
since Spector et al. (1970). Cornell et al. (1989) have reported interactions between lactoglobulin and phospholipids at pH 4.4. Their results are consistent with the idea that
positively charged groups of the proteins interact with negatively charged lipids. At pH 4.6,
-lactoglobulin has a positive net charge, and phosphatidylcholine (PC) is isoelectric (other
phospholipids are ionized at pH > 4.0) (Hauser et al., 1979). As described by Cornell et al.
(1989), an electrostatic attraction between PC and -lactoglobulin could be excepted, and
few positive charges (between 5 to 7 depending of the conformation of the protein) could
welcome the negatively charge of PC. Earlier, Brown et al. (1983) have also reported
114
interactions between PC and -lactoglobulin involving both ionic and hydrophobic
interactions after a treatment with a helix-forming solvent. Ionic interactions permit to
position the lipid and protein molecules, and then hydrophobic interactions stabilize the
complex. Later, Lefèbre et al. (1999; 2000) used Fourier transform infrared spectroscopy to
better understand interactions between -lactoglobulin and phospholipids. Their results
revealed hydrophobic interactions between the proteins and SM, and electrostatic
interactions with PS. Their study also suggested that -lactoglobulin does not bind with PC.
6.6 Conclusions
The present study shows that the heat-induced denaturation of WPI proteins is modified in
presence of milk fat globule membrane extract or in presence of phospholipids. Protein
profiles as a function of heating time was similar except a delay (5 minutes) for the loss of
the native form of whey proteins and the accessibility of free thiol groups of whey proteins
was not affect by the presence of both components. Three-dimensional pictures confirmed
that only few interactions between whey proteins and MFGM proteins/phospholipids
occurred during the heating of WPI. These interactions were however frequent after the
addition of MFGM extract from buttermilk or phospholipids in the WPI. Red dots
corresponding to phospholipids were buried in the protein aggregates structure at pH 4.6
confirming the potential aggregation nucleus role of phospholipids. However, the
interactions between proteins and phospholipids seem to be weak during the early stages of
heating. A longer time (> 15 min at pH 4.6 or > 20 min at pH 6.8) was necessary to
strongly bind phospholipids to whey proteins at 80°C. TLC plates did not revealed a loss of
phospholipids at 65°C for both pH while three-dimensional images showed interactions. It
seems that phospholipids were incorporated to the structure of protein aggregates through
MFGM fragments at lower temperature (65°C), while they can directly interact with the
proteins at higher temperature (80°C).
115
6.7 Acknowledgements
This work was funded by the Le Fond Québécois de la Recherche sur la Nature et les
Technologies (FQRNT, Quebec City, Quebec, Canada) – Novalait Inc. (Sainte-Foy,
Quebec, Canada) – Ministère de l’Agriculture et de l’Alimentation du Québec (MAPAQ,
Quebec City, Quebec, Canada).
116
CHAPTER 7
EFFECT OF FREE THIOL GROUP
REACTIVITY DURING THE FORMATION OF HEATINDUCED
AGGREGATES
FROM
WHEY
AND
BUTTERMILK ON THEIR PROPERTIES
The work presented in Chapter 4 showed that: 1) the presence of buttermilk changed the
properties of the aggregates; 2) the proportion of buttermilk in the mixture controlled the
properties of the aggregates.
The work presented in this chapter aimed at understanding the impact of the free thiol
reactivity on the properties of heat-induced whey:buttermilk protein aggregates. The
preparation of the aggregates was conducted at Dairy Product Technology Center (DPTC)
of California Polytechnic State University in San Luis Obispo, but the experiments were
conducted at Laval University in Quebec City.
117
7.1 Résumé
L’objectif de cette étude était de mieux comprendre l’effet de la concentration en
groupements thiols libres lors de la formation des agrégats sur leurs propriétés. Des poudres
de babeurre issues de crème ou de crème de lactosérum ont été mises en solution dans un
concentré de protéines du lactosérum. Les mélanges ont été chauffés à 90°C à pH 4,6 en
présence ou non d’un agent bloqueur de groupements thiols: N-ethylmaleimide. Les
agrégats ont été lyophilisés. L’analyse statistique a révélé que la présence de groupements
thiols libres lors du chauffage affectait les propriétés des agrégats. Les résultats ont montré
que l’utilisation du NEM augmentait significativement le rendement d’agrégation et la
capacité de rétention d’eau des agrégats et diminuait significativement la taille moyenne
des agrégats. En solution, les agrégats exposent des groupements thiols libres même s’ils
ont été formés en présence de NEM.
118
7.2 Abstract
The objective of this study was to better understand the impact of the concentration of free
thiol groups during formation of the mixed aggregates on their properties. Spray dried
buttermilk powders from regular and whey creams (treated with CO2-SFE or not) were
hydrated in liquid whey protein concentrate and then heated up to 90°C at pH 4.6. The
dispersions were heated with or without the presence of thiol blocking N-ethylmaleimide (5
mM). The aggregated material was separated after heating by centrifugation at 15,000 g for
20 minutes and then freeze-dried. Statistical analysis of the data revealed that the presence
of NEM significantly affected the properties of the aggregates. The composition of the
mixture significantly affected the solubility of the powders only. Results showed that the
reduction of the thiol group reactivity significantly increased the aggregation yield, the
water-holding capacity of aggregates before drying, but significantly reduced the numberweighted average particle size. Aggregates exposed free thiol groups when they were in
solution even if they have been formed in the presence of NEM. Overall, results allow the
connection between our knowledge of the formation of the mixed aggregates with their
basic physical and functional properties.
7.3 Introduction
The thermal behavior of the main dairy proteins have been extensively studied since the
first report of Sawyer et al. (1963). Different mechanisms of aggregation have been
proposed for whey proteins (Gezimati et al., 1996; Havea et al., 2000; Roefs et al., 1994;
Schokker et al., 1999), whey proteins with casein micelles (Livney et al., 2004; Vasbinder
et al., 2003a), milk fat globule membrane (MFGM) proteins (Appel et al., 1982; Ye et al.,
2002), and MFGM proteins with whey proteins and caseins (Ye et al., 2004a; Ye et al.,
2004b). Heat-induced protein aggregation has been used as a basis for the development of
industrial processes to increase the retention of whey proteins in cheese matrices, or to
prepare ingredients for food formulations (Lawrence, 1993.).
119
The first approach consisted of increasing the temperature of pasteurization (above 72°C –
15s) of the cheese milk in order to promote the heat-induced interactions of whey proteins
with the casein micelles (Lawrence et al., 1993). This method has been extensively used for
the production of yogurt (85 to 95°C – several minutes), but is not optimal for the
production of cheese because it affects the enzymatic reactions and the fusion of the casein
micelles. Guyomarc’h et al. (2006) also reported excessive moisture and texture defects of
the cheeses. This method is, however, still under study, but limited due to the poor
literature regarding the properties of the whey protein/casein complexes and the absence of
industrial process for the production and isolation of the complexes on a large scale
(Donato et al., 2009).
The second approach consisted in pre-aggregating whey proteins (90 to 95°C – several
minutes) at low pH (4.2 to 4.6) or neutral pH (6.2 to 6.7) directly in liquid whey. Later, the
pre-aggregates whey proteins are incorporating in cheese milk (Lebeuf et al., 1998;
Punidadas et al., 1999). These ingredients have been widely used as fat mimetics for the
production of low fat cheese (Lawrence, 1989, 1993; Lebeuf et al., 1998; Punidadas et al.,
1999) or as texture modifiers for yogurt (Sandoval-Castilla et al., 2004; Tamine et al.,
1995). The use of these ingredients is, however, limited to 0.5 to 1.5% of the total weight
due to the high water-binding capacity of denatured proteins.
In both approaches, thiol/disulfide exchange reactions (SH/SS) are believed to direct the
formation of protein aggregates. Gilbert (1993) defined these reactions by the nucleophilic
attack of a disulfide bond (SS) of one protein by a free thiol group (SH) of another protein.
The two proteins associate through thiol-thiol oxidation and a new thiol group is released.
In heated milk, the protein aggregation is led by the exposition of free thiol groups of the
milk fat globule membrane (MFGM) proteins around 65°C (Ye et al., 2004a,b; Corredig
and Dalgleish, 1998), but mostly by the exposition of the free thiol group of β-lactoglobulin
at 78.5°C (Schokker at al., 1999). These groups will initiate interactions with whey proteins
(Schokker et al., 1999; Havea et al., 1998), caseins or casein micelles (Anema et al., 2003a;
Donato and Guyomarc’h, 2009), and MFGM proteins (Corredig and Dagleish, 1998; Ye et
al., 2004a,b). Other types of interaction such as thiol-thiol oxidation, hydrogen bonds, ionic
120
bonds, van der Waals forces, and hydrophobic interactions are less represented but seem to
play an important role for the dairy protein aggregation (Oldfield et al., 2000; Schokker et
al., 1999; Prabakaran et al., 1997).
It has been shown that it was possible to integrate buttermilk in the process of the
production of heat-induced whey protein aggregates at a low pH (Chapter 4). Our previous
results demonstrated that the mixed protein aggregates were composed of whey proteins,
caseins, MFGM proteins and phospholipids and were stabilized by both covalent and noncovalent interactions (Chapter 5 and 6). The presence of buttermilk concentrate also
changed the physical and functional properties of aggregates such as a reduction of their
water-holding capacity as a function of the level of substitution of whey concentrate by
buttermilk concentrate (Chapter 4).
In line with these observations, we hypothesized that the free thiol group reactivity of
buttermilk constituents has a direct impact on the physical and functional properties of the
mixed aggregates.
The objective of the present work was to characterize the contribution of the thiol group
reactivity during heating on some of the basic physical and functional properties of the
mixed aggregates.
7.4 Materials and methods
7.4.1 Materials
Fresh whey (unpasteurized; milk pasteurized at 74.4°C for 16.1 seconds) from TILL and
Gouda cheese production and milk cream (unpasteurized; milk pasteurized at 74.4°C for
16.1 seconds) were obtained in California Polytechnic State University’s processing plant
(San Luis Obispo, CA, USA). Whey cream (pasteurized at 82.2°C for 35 seconds; milk
pasteurized at 73.3°C for 16 seconds) from cheddar cheese production was donated by
121
Hilmar Ingredients (Hilmar, CA, USA). Elman’s reagent was from Thermo Scientific
(Rockford, IL, USA). N-ethylmaleimide and all other reagents were from Fisher Scientific
(Fair Lawn, NJ, USA).
7.4.2 Preparation of the whey and buttermilk concentrates
Fresh cheese whey was skimmed by using a pilot scale milk separator (Alfa Laval,
Uppsala, Sweden). Bacterial contamination of whey was reduced by microfiltration using a
pilot plant scale system (T-12 model, GEA-Niro Filtration, Hudson, WI, USA) through a
1.4 m membrane (Koch Membrane Systems, Wilmington, MA, USA). Microfiltered whey
was concentrated using two 10 kDa spiral polymeric ultrafiltration membranes (Koch
Membrane Systems, Wilmington, MA, USA) fitted in parallel up to a protein concentration
of 4.0 % (w/v). Whey permeate was collected and frozen until used for protein blend
formulations. The retentate was diluted sequentially in the feed tank with 3 diavolumes of
DI water. The diafiltration was stopped when a final concentration of 5.2% of protein (w/v)
was obtained. The whey protein concentrate (WPC) was then frozen to – 35°C until further
analysis. The concentration trial run was performed with whey from three different
productions.
Regular and whey cream were processed at 12°C using a continuous butter-churn for pilot
plant (Egli, Butschwil, Switzerland). Immediately after churning, regular and whey
buttermilks were filtrated through cheesecloth, and skimmed, using a pilot scale milk
separator (Alfa Laval, Uppsala, Sweden). Skim buttermilks were concentrated by UF (GEA
R-12 model, Milwaukee WI) using two 10 kDa spiral polymeric membranes fitted parallel
on the module and then the retentate was diluted sequentially in the feed tank with 3
diavolumes of DI water. The DF was conducted until a concentration of 5% of protein
(w/v) was reached. Buttermilk concentrates (BC) were spray-dried using a Niro Filterlab
Spray-drier (Hudson, WI, USA) at 35 bars, and with inlet and outlet air temperatures of
185°C and 95°C, respectively. A portion of each powder was submitted to supercritical
fluid extraction (SFE) (Thar Designs, Inc., Pittsburgh, PA, USA) to remove the non polar
lipids according to Costa et al. (2010). Circulated deionized water (+ coolant) at 3°C was
122
used for cooling different zones in the SFE apparatus. Carbon dioxide tanks were filled and
inspected by A & R Welding Supply (San Luis Obispo, CA, USA). The system conditions
were controlled manually by Windows 2000-based software. Approximately 125 g of each
sample were submitted to an extraction cycle for maximum non-polar lipid removal using
the following conditions: 7200 g of CO2 at a flow of 20 g.min-1, extraction pressure of 350
bar, and temperatures of 35°C. Powders were stored at 10°C under the following codes:
regular buttermilk concentrate (rBC), whey buttermilk concentrate (wBC), regular
buttermilk concentrate treated by SFE (rBCSFE), and whey buttermilk concentrate treated by
SFE (wBCSFE).
7.4.3 Preparation of protein aggregates powders
WPC was diluted with UF permeate to decrease the protein concentration from 5.2% to
2.6% (w/v). BC powders were then dispersed in diluted WPC to increase the total protein
concentration to 5.2% (w/v). Whey-buttermilk mixtures were stirred overnight at 4°C.
Denaturation process followed the previous proposed design (Chapter 4). Briefly, 250 mL
blends were adjusted to pH 4.6 with 1N HCL and heated from 8°C to 90°C in a
thermostatically controlled water-bath maintained at 97°C for a total time of 25 minutes.
Mixtures were constantly stirred using a caframo lab mixer during heating (speed 5). Nondiluted WPC (5.2% protein) was used as a control. The aggregated material of each mixture
was separated at the end of heating (25 minutes) by centrifugation (Beckman L7-35, Brea,
CA, USA ) at 15,000g for 20 minutes at 20°C, and then freeze-dried.
Heating experiments have also been conducted in presence of 5.0 mM of N-etylmaleimide
(NEM) in order to completely block free and exposed thiol groups during heating (Alting et
al., 2000).
Nitrogen content was determined using Kjeldahl method and converted to protein using a
conversion factor of 6.38 (AOAC, 2003). Aggregation yield was calculated before freezedrying from the protein content in the mixtures and in the supernatants after centrifugation
as follows:
123
Equation 7.1: Calculation of the aggregation yield
Aggregatio n yield (%) =
PM  PS 100
PM
where [P]M was the protein concentration in mixtures (%), and [P]S was the protein
concentration in supernatant (%).
Water-holding capacity (WHC) of aggregates was calculated before freeze-drying from the
moisture content of the centrifugation pellets, the moisture content of the powders, and the
protein content of the powders as follow:
Equation 7.2: Calculation of the water-holding capacity of the aggregates
WHC(gwater /g protein ) 
MP
PP
where [M]P was the moisture content in the centrifugation pellet (g), and [P]P was the
protein content in the centrifugation pellet (g).

7.4.4 Particle size distribution in heated liquid mixtures
The particle size distribution of mixtures (5.2% proteins (w/v)) after heating was measured
using a laser diffraction particle size analyzer in the polarization intensity differential
scattering optical mode (Beckman Coulder, Brea, CA, USA). Samples were taken after 25
minutes of heating in the thermostatically controlled water bath (set up at 97°C), shaken by
inversion for 1 minute, and diluted in the analysis chamber with one liter of DI water
(automatically filled-up by the instrument). The instrument was allowed to adjust for
electrical offsets and align of the laser prior to the measuring background. Measurements
were started after 5 minutes of agitation. Data was obtained from the average of three
measurements per sample and expressed as the number-weighted average particle size
(D[4,3]n).
124
7.4.5 Protein solubility index at pH 6.8
Protein aggregates powders were rehydrated in SMUF solution (pH = 6.8) (Jenness and
Koops, 1962) to obtain 1% (w/v) protein solutions. The pH was adjusted to 6.8 using
NaOH 1N, and solutions were stirred for an hour before to be adjusted to pH 6.8 again
using NaOH 1N or HCl 1N. Solutions were centrifuged after 2 hours of stirring (total time)
or 26 hours of stirring (total time) at ~ 3,000g for 10 min at room temperature. The protein
solubility index (PSI) was calculated from the protein concentration in solutions ([P] i) and
protein content in supernatants ([P]sup) determined by BCA microplate method as follow:
Equation 7.3: Calculation of the protein solubility index at pH = 6.8
PSI(%) 
Psup
100
Pi
7.4.6 Exposition of free thiol groups of the mixed aggregates
Accessible thiol group concentration of the soluble aggregates (from the supernatants) was
measured according to Ellman (1959). 250 µL of the solutions of soluble aggregates in
SMUF at pH 6.8 were taken and mixed with 2.5 mL of 0.1M sodium phosphate buffer
(containing 1mM EDTA; pH = 8.0), and 0.05 mL of DTNB reagent solution (4 mg in 1 mL
of sodium phosphate buffer). The tubes were shaken by inversion for 1 minute and
incubated at room temperature for 30 min. Absorbance was measured at 412 nm using a
Multiskan Spectrum (Thermo labsystems, Romeoville, IL, USA). The absorption values of
the standards (concentration of cysteine ranging from 1.5 mM to 0 mM) were plots and the
equation was extracted from the figure. The free thiol concentration of solution was
expressed as mole per gram of protein.
7.4.5 Statistical analysis
All experiments were performed in triplicate, so each value represents the mean of three
measurements. Two different and independent variables were selected and named as
follows: Composition that expressed the effect of addition of different buttermilk
125
concentrates, NEM that expressed the effect of blocking the free thiol groups during the
preparation of the powders. Data was tested according to a factorial design 5*2 except for
the nitrogen solubility index (5*2*2). In this case, a third variable named Hydration was
added in order to express the effect of a longer time of hydration of the powders (2 h vs
26h). Statistical analysis was carried out using SAS software (SAS Institute Inc., Cary, NC,
USA). Mean comparisons were performed using a Duncan post-test if appropriate. Table
7.1 summarizes significance for each treatment on each variable. Results were considered
significantly different when P < 0.05.
Table 7.1: Summary of the significance (P value) calculated by the analysis of variance of data of each
contrasts for each variables (n = 3).
Significance (P values)
Contrast
Aggregation yield* WHC* D[4,3]n* PSI
[-SH]free
Model
0.0232
0.0042 0.0011
0.0002
0.0882
Composition (A) 0.1838
0.3056 0.5053
0.0074
0.8067
NEM (B)
0.0470
0.0105 0.0075
< 0.0001 0.3528
Hydration (C)
n/a
n/a
0.0316
n/a
A*B
0.1565
0.7025 0.6818
0.0354
0.4442
A*C
n/a
n/a
n/a
0.3908
n/a
B*C
n/a
n/a
n/a
0.3388
n/a
A*B*C
n/a
n/a
n/a
0.9263
n/a
n/a
* The effect “blocs” was evaluated significant, so the software automatically re-adjusted
the model. This result means that three whey were different. The first whey was a mix from
TILL and Gouda cheeses production, while the second and third whey were from TILL
cheese production only.
7.5 Results
7.5.1 Heat-induced aggregation characterization
The presence of NEM had a significant effect (P = 0.0075) on number-weighted average
particle size distribution (D[4,3]n) of aggregates before drying (Table 7.1). The D[4,3]n was
126
1.50  0.56 µm in the absence of NEM and 0.23  0.04 µm in the presence of NEM (Figure
7.3).
Figure 7.1: Effect of the presence of NEM during formation of mixed aggregates from heating (90°C –
5min) at low pH (4.6) mixtures containing 50% of the protein from WPC and 50% from buttermilk
concentrates on the number-weighted average particle size distribution in the mixtures.
The presence of N-ethylmaleimide (NEM) had a significant effect (P = 0.0470) on the
aggregation yield (Table 7.1). The aggregation yield was 74.03  1.38% in the absence of
NEM and 78.36  1.91% in the presence of NEM (Figure 7.2).
127
Figure 7.2: Effect of the presence of NEM during formation of mixed aggregates from heating (90°C –
5min) at low pH (4.6) mixtures containing 50% of the protein from WPC and 50% from buttermilk
concentrates on the aggregation yield.
7.5.2 Properties of the mixed aggregates
The presence of NEM had a significant effect (P = 0.0105) on the water-holding capacity
(WHC) of aggregates (Table 7.1). The WHC was 2.97  0.72 gwater.gprot-1 in the absence of
NEM and 3.59  0.70 gwater.gprot-1 in the presence of NEM (Figure 7.3).
Figure 7.3: Effect of the presence of NEM during formation of mixed aggregates from heating (90°C –
5min) at low pH (4.6) mixtures containing 50% of the protein from WPC and 50% from buttermilk
concentrates on the water-holding capacity of the aggregates.
128
The interaction composition*NEM had a significant effect (P = 0.0354) on the protein
solubility index (NSI) (Table 7.1). As shown in Figure 7.4, the presence of NEM did not
affect the solubility of the aggregates powders from the heating of WPC (14.25  1.14% in
the presence of NEM vs 15.44  3.32% in the absence) and the mixtures WPC + rBC
(14.15  0.89% vs 14.97  1.49%) and WPC + wBM-SFE (11.60  2.50% vs 13.18 
1.34%). Oppositely, the presence of NEM affected the solubility of the aggregate powders
from the mixtures WPC + BM-SFE (6.52  0.46% vs 18.40  1.26%) and WPC + wBM
(6.32  0.46% vs 13.30  1.22%).
Protein Solubility Indice (%)
25
20
15
10
5
0
C
WP
rBC
w.+
_S
BC
w.+
FE
wB
w.+
C
w
w.+
E
_SF
C
B
Figure 7.4: Effect of the composition and the presence of NEM during formation of mixed aggregates
from heating (90°C – 5min) at low pH (4.6) mixtures containing 50% of the protein from WPC and
50% from buttermilk concentrates on nitrogen solubility index of the powders. Aggregates formed in
the absence of NEM are represented in black and aggregates formed in the presence of NEM are
represented in grey.
Table 7.1 shows that none of the contrasts or interactions between contrasts had a
significant effect on the accessibility of thiol groups. The concentration of thiol groups in
the soluble fraction was ranged from 1.1 ± 0.2 µmole/gprot to 1.7 ± 0.4 µmole/gprot (Figure
7.5).
129
2.5
[-SH] free (µmole.gprot-1)
2.0
1.5
1.0
0.5
0.0
WP
C
E
E
E
rBC
_SF
_SF
_S F
C
C
C
w.+
B
B
B
w
w
w.+
w.+
w.+
Figure 7.5: Effect of the composition and the presence of NEM during formation of mixed aggregates
from heating (90°C – 5min) at low pH (4.6) mixtures containing 50% of the protein from WPC and
50% from buttermilk concentrates on the accessibility of free thiol groups of the mixed aggregates.
7.6 Discussion
Statistical analysis revealed that the whey samples taken from three different productions
were significantly different. It has been reported for a long time that whey protein
concentrate (WPC) are inconsistent in their composition and functionality due to
differences in the composition of the milk, differences in the cheese-making process, and
differences in the manufacture of the WPC (Morr et al., 1993; Schmidt et al., 1984). Later,
and for the first time de la Fuente et al. (2002) investigated the structural changes in whey
proteins during the different steps of the manufacture of WPC. Their results suggested that
the differences in protein functionalities were to be attributed to the WPC production
process rather than to the cheese manufacture. Results also showed the presence of protein
aggregates likely formed during the pasteurization of milk and whey. Earlier, Schmidt et al.
(1984) suggested that factors affecting the cheese yield such as heat treatment, starter
130
culture, cooking conditions would be expected to alter the composition of the whey and
WPC. The same factors would also alter the properties of the whey proteins. Some
variations in these factors have been observed during the sampling of the whey.
N-ethylmaleimide (NEM) has been used for the understanding of the heat-induced
aggregation kinetics or mechanisms of whey proteins and especially β-lactoglobulin
(Hoffman and Van Mill, 1997; Oldfield et al., 1998, 2000; Sawyer, 1968; Xiong et al.,
1993). By its binding with the exposed free thiol groups of the protein, NEM prevents the
SH/SS exchange reactions between proteins. In consequence, aggregation occurs through
other interactions such as hydrogen bonds, ionic bonds, van der Waals forces, and
hydrophobic interactions. However, it is not evident to find clues on the properties of the
non-covalent based protein dairy aggregates because they are rarely found in normal
heating conditions (without a thiol blocking agent). Oldfield et al. (2000) reported that
“hydrophobic formed” aggregates of β-lactoglobulin were found at the beginning (under
15% of the total protein) but close to none at the end of heating at 85°C.
First, results showed that the solubility of aggregates formed in presence of NEM was
lower comparatively to aggregates formed in normal conditions except for aggregates
formed from buttermilks treated by CO2-SFE. The solubility has been measured on nonhomogenized aggregates. However, a lower solubility of the powders of aggregates formed
in the presence of NEM could be correlated to the higher level of denaturation. Results
showed that preventing the SH/SS exchange reactions significantly increased the
proportion aggregates (precipitable or sedimentable). Havea et al. (2004) observed similar
results by using DTT (dithiothreitol). Their results showed that the prevention of the SH/SS
exchange reactions offer a higher number of reactive sites and in consequence a faster
aggregation process. They explained that the lack of restriction due to movements of
disulfide bonds deeply increased the chance of proteins to associate upon collision.
Later, they found that gels formed through non-covalent interactions were strong and rigid
comparatively to gels formed in normal conditions (weak gels). Controversially, Alting et
131
al. (2000) reported that preventing the SH/SS exchange reactions significantly decreased
the hardness of cold protein gels. The weakness of gels is often attributed to the extensive
capacity of denatured proteins to hold water. Results demonstrated that aggregates were
less hydrated in normal conditions than in presence of NEM. This result could be explained
by the average particle size distribution. Results showed that the prevention of the SH/SS
exchange reactions significantly reduced the average diameter of particles. It is easy to
think that large aggregates hold less water because the hydrophilic groups of the individual
proteins become buried in the more complex structure. In normal conditions, aggregates
become larger due to addition of denatured proteins to the complex through SH/SS
exchange reactions. By preventing the formation of the SH/SS exchange reactions, the
polymerization sites are reduced so the formation of large aggregates is impossible. Results
from Alting et al. (2000) also showed that large aggregates were present after gelation only
in normal conditions.
Surprisingly, results showed that accessible thiol groups were found after hydration of the
powders of mixed aggregates. Alting et al. (2000) explained that the heat-induced
aggregation mechanism that Roefs and De Kruif (1994) proposed for the whey proteins
anticipates the presence of free thiol groups at the surface of the protein aggregates. This
result gives a strong clue for answering the question on the reactivity of protein aggregates
in a new system and the potential association with other proteins during a heat treatment. It
is, however, difficult to explain the presence of accessible thiol groups at the surface of
aggregates formed in the presence of NEM.
7.7 Conclusion
The present study showed that the reactivity of free thiol groups during the formation of
protein aggregates from whey and buttermilk has a direct impact on the properties of the
aggregates. Thiol/disulfide exchange reactions (SH/SS) are suspected to direct the
formation of the protein aggregates, but results presented showed that they also control the
size of the protein aggregates, their water-holding capacity, and their solubility. Results
132
suggest that the prevention of the SH/SS exchange reactions can be interesting for the
control of the properties of protein aggregates and offers new potential applications.
Previous results demonstrated that addition of buttermilk constituents or decreasing the pH
before heating (4.6 instead of 6.8) reduced the free thiol group reactivity. However, their
total prevention requires the use of a strong chemical that is not acceptable for food
formulation.
7.8 Acknowledgments
This work was funded by the Le Fond Québécois de la Recherche sur la Nature et les
Technologies (FQRNT, Quebec City, Quebec, Canada) – Novalait Inc. (Sainte-Foy,
Quebec, Canada) – Ministère de l’Agriculture et de l’Alimentation du Québec (MAPAQ,
Quebec City, Quebec, Canada).
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CHAPTER 8
EFFECT OF SUBSTITUTION OF SKIM
MILK POWDER BY WHEY:BUTTERMILK HEATDENATURED PROTEIN AGGREGATES IN MODEL
SET-TYPE YOGURT
The work presented in the previous chapter showed that: 1) the thiol group reactivity during
formation of the mixed aggregates has a direct impact on the properties of the aggregates;
2) thiol groups are accessible at the surface of the mixed aggregates. Results suggest that
the mixed aggregates can act as active fillers in a process involving heating due to their
accessible free thiol groups.
The work presented in this chapter aimed at understanding the behavior of whey:buttermilk
heat-denatured protein aggregates during the yogurt-making process. The effect of different
level on incorporation of aggregates on texture of yogurt was tested. Water-holding
capacity of gels, particle size distribution, liberation of thiol groups during heating, and
repartition of clusters were investigated to better understand the texture results.
Results of this part of the project will be submitted essentially in this form to the Special
Issue of Foods «Structure and Flavour of Dairy Products». Results of this paper were also
presented in poster form at 2010 STELA Colloque in Quebec City (QC, Canada) and at
2010 ADSA Join Annual Meeting in New Orleans (LA, USA).
135
8.1 Résumé
L’objectif de cette étude était d’évaluer l’impact de l’addition des agrégats formés à partir
d’un mélange protéique lactosérum (25 %) - babeurre (75 %) sur la production de yaourt
ferme. Les yaourts ont été préparés à partir de laits écrémés standardisés à 15 % (p/v) de
solides et 4,2 % (p/v) de protéines par l’ajout de différents taux de poudre de lait écrémé et
de poudre d’agrégats. L’ajout d’agrégats protéiques a affecté significativement la texture
des yaourts mais n’a pas eu d’impact sur la capacité de rétention d’eau des gels. Les images
de microscopie confocale ont montré que la présence de particules de tailles importantes
dans les laits enrichis avec les agrégats affectait directement l’homogénéité de la matrice
protéique en formation. L’analyse des groupements thiols libres des agrégats en solution
dans de l’eau suggèrent que les agrégats pourraient interagir avec les protéines du lait
écrémé lors du chauffage.
136
8.2 Abstract
The objective of this study was to assess the impact of adding heat-induced wheybuttermilk protein aggregates to acid-set type yogurt production. Whey and buttermilk
protein mixture (25:75) was adjusted to pH 4.6 and maintained at 90°C for 5 min. Set-type
yogurts were prepared from skim milks standardized to 15% (w/v) total solids and 4.2%
(w/v) proteins using different substitution levels of skim milk powder and aggregates
powders. Addition of protein aggregates significantly modified texture properties of
yogurts but did not affect water-holding capacity of gels. Confocal laser-scanning
microscope pictures showed the presence of large particles in milk fortified with protein
aggregates that directly affected homogeneity of the clusters of the protein matrix. Free
thiol groups were accessible during heating of the protein aggregates powder in a water
solution suggesting that the aggregates could interact with the milk protein during the heat
treatment of milk.
8.3 Introduction
The use of microparticulated whey proteins (MWP) as a fat replacer in yogurt systems has
been investigated. This dry dairy product was developed initially in order to provide means
of increasing the retention of whey proteins by casein during cheese curd formation
(Lawrence, 1989; Lawrence, 1993; Lelièvre, 1990). It is obtained by heating ultrafiltered
whey at 90-95°C for several minutes with the pH at acidic (4.2–4.6) or neutral (6.2–6.7)
values (Lawrence, 1993; Lebeuf et al., 1998; Punidadas et al., 1999) and spray drying. It is
generally accepted that the product does not interact with milk proteins during the cheesemaking process and that the micro-particles are only trapped within the casein network. the
cooking step. It is still unclear whether MWP acts as active or inert particles in yogurt.
Tamine et al. (1995) described the role of a fat replacer as simulating fat globules without
interacting with other milk proteins. Sandoval-Castilla et al. (2004) and Tamine et al.
(1995) concluded that MWP not only becomes part of the protein matrix, but also modifies
the microstructure of the gel The gels thus formed are lower in tension and in firmness, but
137
higher in cohesiveness (Barrantes et al., 1994; Sandoval-Castilla et al., 2004; Tamine et al.,
1995). The resulting yogurt is also more susceptible to syneresis.
The major drawback associated with MWP is the high water-holding capacity of the microparticles, which contribute directly to increasing the moisture content of the curd. Saffon et
al. (2011) proposed reducing the water-holding capacity of MWP by combining the whey
starting material with buttermilk. They showed that this significantly decreased the waterholding capacity of the resulting micro-particles, as well as modifying their properties.
Once viewed as a by-product of butter-making, buttermilk is now considered as a valuable
product because of its high content in fragments of milk fat globule membrane (MFGM), in
addition to phospholipids and whey proteins (Jiménez-Flores & Brisson, 2008; Singh,
2006). However, studies by Mistry et al. (1996), Raval et al. (1999), and Turcot et al.
(2001, 2002) indicated that the moisture content of cheese supplemented with buttermilk
remained high, due largely to phospholipids.
In the present work, less-hydrated aggregates of heat-denatured whey:buttermilk protein
were added as a substitute for skim milk powder to skim milk used for yogurt production.
The main objective of this study was to understand the behavior of these aggregates during
the yogurt-making process. The effect of different levels of skim milk powder substitution
(0 to 100%) on the textural properties of acid set-type yogurt was tested. The water-holding
capacity of the resulting gels, the particle size distribution after heating, the liberation of
thiol groups during heating, and the average size of the aggregates were investigated in
order to gain better understanding of the texture results.
8.4 Materials and methods
8.4.1 Materials
Fresh whey from mozzarella cheese production and fresh buttermilk were both obtained
from a local cheese producer (L’Ancêtre, Bécancour, QC, Canada). Powdered whey
138
permeate was obtained from Maple Leaf Foods International (Toronto, ON, Canada) and
powdered skim milk was obtained from Dairytown Products Limited (Sussex, NB,
Canada). Pasteurized skim milk was purchased at a local grocery outlet and yogurt starter
was obtained from Yogotherm (Saint-Hyacinthe, QC, Canada). Oregon Green 488 was
obtained from Life Technologies Corporation (Burlington, ON, Canada). All other reagents
were obtained from Fisher Scientific (Ottawa, ON, Canada).
8.4.2 Preparation of whey:buttermilk heat-denatured aggregates
Heat-denatured aggregates of whey:buttermilk protein were formed using our method as
described previously (Chapter 4). Briefly, cheese whey and buttermilk were skimmed using
a pilot-scale milk separator (Alfa-Laval, Uppsala, Sweden) and then concentrated
separately by ultrafiltration (UF) through a 5 kDa membrane (Romicon, Koch Membrane
Systems, Wilmington, MA, USA) to a final protein concentration of 9.5% (w/v).
Concentrates were mixed at a whey:buttermilk protein ratio of 25:75. Mixtures were
adjusted to pH 4.6 by slow addition of HCl 6N and heated from 4°C to 90°C for 16 min
(including ramp time, about 11 min at 90°C) in a stirred cooker with a steam jacket. The
heated liquid was then homogenized three times at 69 MPa using an Emulsiflex C-500
device (Avestin Canada, ON, Canada). The homogenates were freeze-dried in order to
obtain powdered whey:buttermilk aggregate (WBAP).
8.4.3 Yogurt production
Skim milk containing 8.4% (w/v) total solids and 3.6% (w/v) protein was standardized to
15% total solids and 4.2% protein using powdered skim milk and powdered permeate.
WBAP was added to replace 0, 20, 40, 60, 80 or 100% of the added powdered skim milk.
The milk thus enriched was stirred for 30 minutes at 4°C and then heated to 85°C in a
Stephan cooker with thermostatic control (Sympak France, Lognes, France), with constant
stirring for 25 minutes including ramp time. After cooling to 42°C in an ice water bath, the
milk was inoculated with a commercial yogurt starter (Streptococcus thermophilus and
Lactobacillus bulgaricus) and incubated at 42°C until a pH of 4.6 was obtained. For each
enrichment level, sixteen yogurts were analyzed after 18 hours at 4°C, eight were analyzed
139
after 7 days, and eight after 14 days. Yogurts were also prepared in falcon tubes (25 mL).
Each production was repeated three times.
8.4.4 Analytical methods
8.4.4.1
Composition analysis
Overall composition of powdered ingredients was determined using standard methods.
Total nitrogen content was determined by the Dumas combustion method (IDF, 2002)
using a LECO device (Protein Analyzer method FP-528 Leco Instruments Ltd.,
Mississauga, ON, Canada). Nitrogen values were converted into protein values using 6.38
as the nitrogen conversion factor. Fat was determined using the Mojonnier extraction
method (IDF, 2008) and lactose by phenol – sulfuric acid method. Total solids were
obtained by microwave drying (Smart System 5, CEM Corp., Matthews, NC, USA) and ash
was measured by incineration in a refractory oven at 550°C for 20 hours (AOAC, 1990).
8.4.4.2
Texture properties
The texture of the set-type yogurts was evaluated by penetration using a TA-XT2 Texture
Analyzer (Texture Technologies Corp., Scarsdale, NY, USA) connected to a computer
running the Stable Micro System (Stable Micro Systems Limited, Surrey, United kingdom),
with a cylindrical probe (diameter 12.5 mm) and 5 kg load cell. Yogurts were analyzed in
their containers (5.6 cm diameter with yogurt depth of 3.5 cm). The following parameters
were set: speed 60 mm/min, distance 20 mm, hold-up time 30 seconds, force (threshold)
0.01 Newton (N). Rupture force corresponded to the first peak, firmness to the maximum
force, and adhesiveness to the resistance to withdrawal of the penetration probe from the
sample. Relaxation was calculated from the firmness force and force after 30 seconds of
holding. Tests were carried out immediately after moving the samples from the cold room.
140
Equation 8.1: Calculation of the relaxation of acid set-type yogurt
Relaxation (%) =
Firmness (N) - Force at 30 s (N)
100
Firmness (N)
8.4.4.3
Water-holding capacity of yogurt
Yogurt prepared in Falcon tubes (25 mL) was centrifuged at 222 x g for 10 minutes at 4°C.
The clear supernatant was poured off and weighed. Water-holding capacity was calculated
from the weight of the supernatant and that of the yogurt:
Equation 8.2: Calculation of the water-holding capacity of the gel
WHC (%) = (1 -
Supernatant Weight (g)
) 100
Yogurt Wei ght (g)
8.4.4.4
Particle size distribution of milk
Skim milk was enriched with WBAP in the same proportions as for yogurt production and
stirred for 30 minutes at 4°C before analysis. Particle size distribution before and after
heating was measured using a Mastersizer 2000 laser diffraction system (Malvern
Instruments, Worcestershire, UK). Samples were shaken by inversion for 1 minute and
dispersed directly into the recirculating cell until a 12–19% obscuration value was reached.
The dispersant (deionized water) was stirred at 950 rpm and measurements were started
after 5 minutes. Data were expressed as the volume-weight average particle size, also
known as the D[4,3] value. Five measurements per sample were averaged.
8.4.4.5
Exposition of free thiol groups upon heating
Exposure of free thiol groups was measured according to Ellman (1959). A 250 L sample
of WBAP-enriched skim milk was taken every five minutes during heating and mixed with
2.5 mL of 0.1M sodium phosphate buffer (containing 1 mM EDTA; pH 8.0) and 0.1 mL of
DTNB reagent solution (4 mg in 1 mL of sodium phosphate buffer). Ammonium persulfate
(0.2000  0.0050 g) was added after 30 minutes of incubation at room temperature.
141
Absorbance was measured at 412 nm using a Multiskan Spectrum (Thermo Labsystems,
Vantaa, Finland) relative to a blank. The theoretical exposure was calculated as follows:
Equation 8.3: Calculation of the theoretical concentration of thiol group
 SH theo mole/g prot   SM -SH  PSM   WBAPSH  PWBAP 
PP
Where [-SH]theo is the concentration of exposed free thiol groups in moles per gram of
protein in the sample containing 100% WBAP substitution of the skim milk powder
supplement, SM-SH is moles of thiol group contributed by the skim milk, PSM is the protein
content in skim milk (3.6 g), WBAP-SH is moles of thiol group contributed by WBAP,
PWBAP is the protein content of powdered WBAP added (0.6 g) and PP is the protein content
of the final mixture (4.2 g).
8.4.5 Confocal laser scanning microscopy
The imaging procedure was adapted from the procedure developed by Hennessy (2011) and
was carried out using a Nikon C1 laser scanning confocal microscope 60X, water objective
(Nikon Corporation). Briefly, skim milk with 0 or 100% of the enrichment replaced with
WBAP was placed for 11 minutes in boiling water with constant stirring. After cooling to
room temperature, 10 mL aliquots were treated with 1.000  0.050 g of glucano-deltalactone and then immediately with 3 L of Oregon Green 488 (0.1 g.L-1 in
dimethylformamide) to stain proteins and with 3 L of Nile red (0.01 g.L-1 in dimethyl
sulfoxide) to stain phospholipids. A 75 L droplet of the mixture was deposited on a cover
slide placed on top of the laser, and images were captured every 15 seconds for 20 minutes
beginning at 2 min after adding glucono-delta-lactone. Files were transformed into sixframe-per-second videos using ImageJ (U.S. National Institutes of Health, Bethesda, MD,
USA; http://rsb.info.nih.gov/ij/). The videos are available at the following internet link:
https://www.dropbox.com/sh/c9gmyrv56kznu45/R1GICKO4if. Single pictures were taken
at the end of the gel formation using the following parameters: resolution = 1024/1024;
quality = 13 db; zoom = 2.31X. The dark level was set at 0 in order to limit background
142
noise and to define the clusters more clearly. Images were loaded into ImageJ, transformed
to 8-bit, and their threshold was adjusted to cover all the dark areas.
8.4.6 Statistical analysis
All experiments were performed in triplicate, each value thus representing the mean of
three measurements. All data were tested as a factorial experiment with powdered skim
milk substitution level*time. Time corresponded to storage time at 4°C for texture and
WHC analysis, and to heating time for particle size distribution and thiol group exposure in
the enriched milk. Statistical analysis was carried out using the SAS PROC GLM
procedure (SAS Institute Inc., Cary, NC, USA). Mean comparisons were performed using a
Duncan post-test. Tables 1a and 1b summarize the significance of each treatment for each
variable. Results were considered significantly different when P < 0.05.
Table 8.1: Summary of the significance of the effects of WBAP and time on the texture and waterholding capacity of yogurt gels, as calculated by analysis of variance (n = 3).
Significance (P values)
Contrast
Firmness
Rupture Force
Adhesiveness
Relaxation
WHC
Model
< 0.0001
0.0004
< 0.0001
< 0.0001
0.5114
Substitution Level (A)
< 0.0001
< 0.0001
< 0.0001
< 0.0001
0.4348
Time (B)
0.9781
0.2021
0.0604
0.7538
0.3903
A*B
0.4579
0.7150
0.2156
0.1394
0.5124
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Table 8.2: Summary of the significance of the effects of WBAP and time on the response of milk to
heating, as calculated by analysis of variance (n = 3).
Significance (P values)
Contrasts
Particle size [-SH]free
Model
0.0044
< 0.0001
Level of substitution (A) 0.0002
< 0.0001
Time (B)
0.8507
0.0174
A*B
0.6332
0.1394
8.5 Results and discussion
Heat-denatured whey protein can reinforce the yogurt gel matrix, but is also a major
contributor to increased gel water-holding capacity, firmness, and apparent/complex
viscosity (Cho et al., 1999; Sodini et al., 2004). Previous observations indicate decreased
firmness in yogurts made with MWP compared to controls (Barrantes et al., 1994;
Sandoval-Castilla et al., 2004; Tamine et al., 1995). However, Britten and Giroux (2001)
showed that the heat denaturing of the proteins can be adjusted in order to control the
texture and syneresis of yogurt
8.5.1 Whey:buttermilk protein aggregates composition and
properties
The composition of the dried dairy products is given in Table 8.3. The protein content of
WBAP was 57.3  0.1%, compared to 35.8  0.1% for powdered skim milk. Powdered
whey permeate was used to increase total solids, due to its high lactose content (85.9 
3.1%) and low protein content (2.2  0.1%). The fat content of WBAP was 12.7  0.3%.
We hypothesized that this content made little difference because of the homogenization
step (69 MPa) during the preparation of the powder. Our previous results showed that 25%
whey/75% buttermilk based on protein mass gave the least hydrated aggregates. The water144
holding capacity of WBAP aggregates was calculated at 2.04  0.18 gwater/gprotein, and the
percent of non-soluble protein at 78.5  3.4%.
Table 8.3: Composition of heat-denatured whey:buttermilk aggregates (WBAP), whey permeate (WP),
and powdered skim milk.
% Dry Matter
Protein
Lactose
Fat
Ash
WBAP
57.3  0.1
18.8  0.0
12.7  0.3
4.9  0.0
PP
2.2  0.1
85.9  3.1
0.2  0.3
6.2  0.0
SMP
35.8  0.1
56.6  3.4
1.0  0.3
8.0  0.0
The particle size distribution of whey:buttermilk aggregate during preparation (before
homogenization) varied between 0.8 m and 700 m with an average of 50.5 m (results
not shown). Lelièvre (1990) concluded that incorporation of larger heat-denatured whey
protein particles during the cheese-making process interferes with the casein network.
To facilitate particle retention and minimize product defects, aggregate particle size
distribution should range between 0.1 m and 10 m. Homogenizing at a pressure of 69
MPa prior to freeze-drying reduced WBAP particle size to an average of 2.7 m (results
not shown).
8.5.2 Particle size in skim milk
Particle size distribution, after 5 minutes at 85°C, in enriched skim milk containing various
levels of WBAP replacing added powdered skim milk, is shown in Figure 8.1. As shown in
Table 8.2, only the substitution level had a significant effect on particle size (P = 0.0002).
Average size was 22.9  19.1 m for skim milk and 159.3  36.5 m, 90.5  42.8 m,
129.6  61.6 m, 114.7  27.4 m, and 170.7  50.9 m respectively for 20%, 40%, 60%,
80%, and 100% of substitution of powdered skim milk by WBAP (results not shown).
Comparison of the means indicates that average particle size was lower in skim milk than
in skim milk + WBAP.
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The higher average size could be attributed to possible interactions between skim milk
proteins and aggregates present in WBAP. To the best of our knowledge, no conclusion has
been reached in the literature about the properties of heat-denatured milk protein
aggregates, and particularly their ability to participate in subsequent formation of heatinduced aggregates. Some researchers have concluded that added heat-denatured whey
proteins are incorporated into other protein matrices, but this idea remains a hypothesis
(Sandoval-Castilla et al., 2004; Tamine et al., 1995). The present results suggest that added
heat-denatured whey proteins do interact with milk proteins during heating. Another
plausible explanation would be that WBAP is less dispersible than skim milk protein,
especially after only 30 min of stirring.
15
Volume (%)
10
5
0
0.01
0.1
1
10
Particle Size (µm)
100
1000
Figure 8.1: Particle size distribution profile in skim milk after 5 min at 85°C, with added powdered
skim milk substituted 0% () 20% (), 40% (), 60% (), 80% () and 100% () with WBAP.
8.5.3 Exposure of thiol groups
Exposed thiol group concentration as a function of milk composition is presented in Figure
8.2. As shown in Table 8.2, both the level of substitution (P < 0.0001) and the heating time
146
(P = 0.0174) had a significant effect on the exposure of thiol groups. The concentration was
4.3  1.6 µmole/gprot in milk enriched with powdered skim milk. When heated alone in
water, WBAP had 38.3  5.3 µmole of exposed thiol groups per g of protein. The
concentration in the skim milk + WBAP mixture (17.5  2.6 µmole/gprot) was unexpectedly
higher than the calculated theoretical concentrations, 9.3  1.6 µmole/gprot. The calculated
theoretical concentrations were not significantly different from the level measured in the
control skim milk.
5
c
[-SH]free (10-5 mole/gprot)
4
3
b
2
1
a
a
a
P
A
B
W
+
sm
et
ic
al
T
he
or
Sk
im
m
ilk
+
W
B
A
P
W
B
A
P
C
on
tr
ol
0
Figure 8.2: Effect of milk composition on thiol group exposure following heating at 85°C for 25 minutes
(including ramp time). A Duncan post-test was applied to compare the means to the control.
These results suggest that re-suspended WBAP aggregates could interact with skim milk
proteins.
As reported in Chapter 5, the exposition of free thiol group increased during the first 10
minutes of heating (from 10.0  6.04 µmole/gprot to 12.1  9.97 µmole/gprot) and then stayed
constant.
147
8.5.4 Distribution of particles in enriched skim milk before
acidification
Confocal laser scanning microscope images of enriched skim milk heated to 100°C, mixed
with glucano-delta-lactone and stained are presented in Figure 8.3. These show that protein
aggregates (diameter ~ 30 m) after heating were fewer in milk enriched with powdered
skim milk than in milk enriched with WBAP, which contained larger aggregates and many
small aggregates. The amount of free phospholipids or MFGM fragments was also greater
in the WBAP-enriched milk.
Figure 8.3: Images of control (enriched with powdered skim milk) skim milk (a) and skim milk +
WBAP aggregates (0.6 g/100 mL; b) at pH 6.8, using the Nikon C1 confocal laser scanning microscope
at 60X. Proteins were dye in green and phospholipids in red.
8.5.5 Texture of yogurts
8.5.5.1
Appearance of yogurts
Lucey and Singh (1998a) defined the appearance of set-type gel as smooth with no cracks
and no surface whey Control yogurts and those enriched with WBAP both had a smooth
consistency with no whey separation. All gels were consistent and free of cracks or holes.
At all levels of substitution of powdered skim milk with WBAP, a slight sedimentation of
particles was observed. At 100% substitution, the gels were grainy. Schmidt et al. (1980)
attributed the grainy texture to the heat treatment, and particularly to treatments at 90°C for
30 minutes. Since our yogurt-making process involved heating to 85°C, these defects are
148
likely due to insufficient hydration time (30 min) for the denatured proteins in the added
WBAP.
8.5.2.2
Firmness
The effect of WBAP as a substitute for powdered skim milk on the firmness of the acid-set
type yogurt gels is presented in Figure 8.4. Table 8.1 shows that only the substitution factor
was significant (P < 0.0001). The firmness was 0.170  0.007 N for yogurts made from
milk enriched with powdered skim milk, and decreased from 0.143  0.019 N at 20%
substitution to 0.096  0.009 N at 100% substitution. Comparison of means shows that the
firmness resulting from 0% substitution was significantly higher than was obtained even in
the case of low levels of substitution.
Figure 8.4: Firmness of control set-type yogurt, and yogurts enriched with whey:buttermilk protein
aggregates. A Duncan post-test was applied to compare the means to the control.
We believe that the decrease in firmness was due mainly to the post-heating particle size.
During gelation, casein chains shrink, and the density of the matrix increases by reduction
of the pore dimension (Sodini et al., 2004). It can be hypothesized that the presence of large
aggregates prevented the contraction of the casein network, and consequently weakened the
149
gel at certain points. This idea is consistent with the trend of the particle distribution in
skim milk and enriched skim milk.
8.5.5.3
Rupture force, adhesiveness, and relaxation
The effect of WBAP as a substitute for powdered skim milk on the rupture force,
adhesiveness, and relaxation of the acid-set type gels is shown in Table 8.1.
Table 8.4: Summary of the mean rupture force, adhesiveness and relaxation of yogurt gels, as a
function of percent substitution of powdered skim milk with whey:buttermilk aggregates as enrichment
of the starting milk. A Duncan post-test was applied to compare the means to the control.
Level of Substitution
Rupture Force (N)
Adhesiveness (N/s)
Relaxation (%)
0
0.093a  0.005
-0.614a  0.083
60.2a  0.6
20
0.072b  0.010
-0.374b  0.190
56.9a,b  2.5
40
0.067b  0.005
-0.274b,c  0.071
55.7a,b  1.8
60
0.067b 0.005
-0.239b,c  0.098
52.3b,c  3.9
80
0.057c  0.011
-0,105c  0.033
49.8c,d  2.7
100
0.051c  0.009
-0.094c  0.032
47.2d  2.7
(%)
*Means within a column sharing the same letter were not significantly different (P > 0.05).
Table 8.1 again shows that only the substitution factor was significant (P < 0.0001). The
rupture force was 0.093  0.005 N for yogurts made from milk enriched with powdered
skim milk, and decreased from 0.072  0.010 at 20% substitution to 0.051  0.009 N at
100% substitution. The comparison of means showed that the yogurts made using
enrichment with WBAP were more susceptible to rupture than were those made using
enrichment with powdered skim milk only. However, no significant difference was
observed between 20%, 40% and 60% substitution. Gel relaxation was 60.2  0.6% for
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yogurts made from milk enriched with powdered skim milk, and decreased from 56.9 
2.5% at 20% substitution to 47.2  2.7 at 100% substitution. Comparison of means shows
that the relaxation resulting from 0% substitution did not differ significantly from that
obtained with 20 or 40% substitution.
Adhesiveness was -0.614  0.083 N/s for yogurts made from milk enriched with powdered
skim milk, and increased from -0.374  0.190 N.s-1 at 20% substitution to -0.094  0.032
N/s at 100% substitution with WBAP. Comparison of means shows that increasing the
level of substitution increased the tendency of the gels to adhere to the probe surface.
Similar results were obtained using fat replacers (Sandoval-Castilla et al., 2004).
8.5.6 Water-holding capacity of yogurt gels
The effect of substituting powdered skim milk with WBAP on the water-holding capacity
(WHC) of the yogurt gels is shown in Figure 8.5. Table 8.2 shows no significant
differences (P = 0.5114) between the samples. The WHC was 99.1  0.1% for yogurt made
from milk enriched with powdered skim milk, and varied from 98.6  0.1% to 98.2  0.2%
in yogurts containing WBAP.
151
Figure 8.5: Water-holding capacity of set-type yogurt, and yogurts made from milk enriched with
whey:buttermilk protein aggregates.
Gel water-holding capacity is defined as the resistance of the gel to compaction (if force >
500 g) or to whey separation (if force < 500 g). Centrifugation parameters (temperature,
time, force) differ from one study to the next. However, it is an accepted view that heating
the milk increases the water-holding capacity of the resulting set-type yogurt (Sodini et al.,
2004). Adding large amounts of denatured whey protein led to the formation of a network
composed of casein micelle chains able to immobilize large amounts of water (Dannenberg
et al., 1998). It was later confirmed that adding more whey protein before heating led to
increased immobilization of free water in the yogurt gel, due mainly to increased gel
compactness (Puvanenthiran et al., 2002). By replacing skim milk proteins with pre-formed
aggregates, the amount of denatured protein in the system was changed. This could explain
the small (not significant) decrease in the water-holding capacity of the gels. The textural
properties of the gels showed that the size of the added aggregates affects the strength and
the distribution of the protein network. Large pores allow a more open structure, facilitating
the expulsion of water.
152
8.5.7 Simulation of the gel formation
Confocal micrographic images taken when gel formation in milk was completed are
presented in Figure 8.6. The structure of the gel formed when the milk was enriched with
powdered skim milk featured small protein aggregates linked together. Heating led to the
formation of large aggregates, and fat globules were entrapped in the matrix. The images
transformed via ImageJ show that the clusters were homogenous in size, and distributed
evenly throughout the gel structure. The structure of the WBAP-enriched milk gel featured
small and large protein aggregates linked together. Fat globules were also entrapped in the
gel. The presence of yellow/orange particles suggests that the structure of these aggregates
could include both protein and fat. The size of the clusters was less homogenous than in the
gel made from milk enriched with powdered skim milk. Small clusters were present around
the large aggregates, and large clusters were present around small aggregates.
Figure 8.6: Confocal laser scanning microscope (Nikon C1) images taken of gels of control skim milk
(a) and skim milk + WBAP (0.6 g/100 mL; b) at 60X with a zoom of 2.31X; proteins were dyed green
and phospholipids are red. Images a’ and b’ were processed with ImageJ to color the cluster in red.
153
Figure 8.7 summarizes the general trends observed in videos of gel formation as a function
of pH and time in milk enriched with powdered skim milk or WBAP. As noted above, few
large particles remained in the case of powdered skim milk after the heat treatment.
However, all particles were moving for the first approximately 5 minutes (pH ranged from
5.50 to 5.29). The movement was intense, meaning that the initially observed particles
moved quickly out of the field of view and were replaced by other particles. The first gel
structure appeared about one minute after immobilization of the particles (pH ranged from
~ 5.22 to 5.16). The structure of the gel became more apparent as the pH decreased. The
matrix started to contract around pH 4.65.
The trend observed was slightly different in the WBAP-milk. As noted above, numerous
large particles and many small ones were present after heating. Unlike in the case of
powdered skim milk enrichment, these large particles were not very mobile. Small particles
were mobile but the flow was slower than in the powdered skim milk case. Particle
movement also stopped sooner (at pH ~ 5.60), and the first gel structure appeared almost
instantaneously. The matrix started to contract when the pH reached 4.91. For no obvious
reason, emission by the fluorophores (especially Oregon 488) decreased rapidly, and the
images ended up dark. However, it was still possible to see the gel using ImageJ software.
154
Figure 8.7: General trends observed in the videos of gel formation as a function of the pH and time.
155
8.7 Conclusions
The present study shows that heat-denatured whey:buttermilk protein aggregate behaves
more like a passive filler than an active filler in yogurt. However, heated skim milk proteins
may interact because of their high thiol group content. Replacing powdered skim milk with
whey:buttermilk aggregate as an enrichment for milk used for yogurt production
significantly affected certain textural properties of the gels. Fracture force, firmness, and
relaxation decreased as the level of substitution increased, while adhesive force increased.
The water-holding capacity of yogurt gels is slightly but not significantly affected. Overall,
the results show that it is possible to use heat-denatured whey:buttermilk protein aggregate
in the production of set-type yogurt, but the dispersibility of the powdered aggregate must
be strictly controlled in order to limit the presence of large particles.
8.8 Acknowledgements
The authors thank Alexandre Bastien of the department of Animal Science at Laval
University for technical support and assistance with the confocal laser scanning
microscope.
This work was funded by Le Fonds Québécois de la Recherche sur la Nature et les
Technologies (FQRNT, Quebec city, Quebec, Canada) – Novalait Inc. (Sainte-Foy,
Quebec, Canada) – Ministère de l’Agriculture et de l’Alimentation du Québec (MAPAQ,
Quebec city, Quebec, Canada).
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CHAPTER 9
GENERAL CONCLUSIONS
The goal of the work presented in this thesis was to develop a new dairy ingredient for the
utilization of buttermilk constituents. The general hypothesis of this doctoral thesis was that
a better understanding of buttermilk constituents and especially their thermal behavior
offers new possibilities for the use of buttermilk in food systems. Additionally, the
combination of whey and buttermilk can offer new ingredients with new functional
properties.
The work carried out allowed us to validate our hypotheses. The substitution of whey
proteins by buttermilk constituents during an intensive heat treatment at low pH led to the
formation of mixed aggregates including whey proteins, caseins, MFGM proteins, and
phospholipids. The physical and functional properties of aggregates can be controlled by
increasing the level of substitution of whey by buttermilk or by incorporating different
constituents from buttermilk or by promoting some interactions (covalent or non-covalent).
Finally, the mixed aggregates were integrated to the formation of the casein network during
acid set type yogurt production.
9.1 Achievements and original contributions
To our knowledge, the work presented in this thesis will be the first investigation of the
thermal behavior of constituents from buttermilk, of the formation of mixed aggregates by
combination of whey and buttermilk concentrates, and of use of mixed protein aggregates
for the production of acid set-type gels.
The first objective of this work was to develop a new approach to form protein aggregates
by combination of whey and buttermilk concentrates (Chapter 4). A process has been
developed by a combination of the widely used processes to form dairy protein aggregates
157
such as intensive heat treatment and a low pH. For the first time, buttermilk concentrate has
been used for the preparation of mixed aggregates. By studying the effect of the level of
substitution of whey concentrate by buttermilk concentrate, we developed a knowledge that
can guide the control of the physical and functional properties of the aggregates.
Essentially, we have shown that the water-holding capacity of aggregates significantly
decreased as a function of the proportion of buttermilk concentrate in the mixture.
Furthermore, rheological properties of the mixtures after heating suggested that the proteins
from whey interacted with the constituents of buttermilk. This study was critical for the
continuation of this doctoral thesis. Results of this part gave clear evidence to support our
hypothesis that a combination of whey and buttermilk offers new ingredients with new
properties and set the parameters for following works (preparation of the concentrates,
volumetric protein concentrations, level of substitution, pH, temperature, time of heating).
The second objective of this work aimed at the better understating of the effects of the
presence of buttermilk constituents on the heat-induced formation of protein aggregates
(Chapter 5). The hypothesis that constituents of buttermilk play an important role in the
formation of the mix aggregates and their properties was imposed by our previous
observations (Chapter 4). Results of this work allowed us to verify our hypothesis that
buttermilk constituents initiate the formation of aggregates and gave us new knowledge on
the thermal behavior of proteins from buttermilk. Practically, results showed that caseins,
MFGM proteins, and whey proteins from buttermilk interacted together during the
preparation of the concentrate through both covalent and non-covalent interactions.
Furthermore, confocal laser scanning microscopy images and SDS-PAGE gels showed that
whey proteins and MFGM proteins from the mixtures interacted with the pre-formed
aggregates. Results suggested that free phospholipids could be involved in the formation of
both pre-formed aggregates in buttermilk and mix aggregates. Results of this part gave
clear evidence of the heat-induced aggregation reactions that occur between whey proteins
and buttermilk constituents at a low pH.
The third objective of this work was to characterize the contribution of phospholipids from
buttermilk to the formation of the heat-induced whey-buttermilk protein aggregates
158
(Chapter 6). The hypothesis that free phospholipids are involved in the formation of protein
aggregates was imposed by our previous observations (Chapter 5). Results of this work
allowed us to verify our hypothesis that free phospholipids are involved to the aggregate
formation, but results demonstrated that a longer time than expected was necessary to bind
the phospholipids with the proteins during heating. Practically, results showed that
phospholipids were integrated to the aggregates at a low pH through the MFGM at a low
temperature (65°C), but suggested that phospholipids can directly interact with whey
proteins at a higher temperature (80°C). Results did not permit to conclude on the
interaction phospholipids – proteins at a higher pH (6.8). Results of this part gave
evidences on the role of a non-protein constituent of buttermilk on the formation of heatinduced protein aggregates at low pH.
The fourth objective of this work aimed at understating the effect of the free thiol groups
reactivity during heating on the properties of the mixed protein aggregates (Chapter 7). The
hypothesis that the properties of the aggregates can be controlled by varying the thiol group
reactivity was imposed by our previous observations (Chapter 4 to 6). Results of this work
permit to confirm our hypothesis. Practically, results demonstrated the physical and
functional properties of the mixed aggregates are controlled by the type of interactions
(SH/SS vs non-covalent) that occur during the formation of the mixed aggregates rather
than by the composition of the mixtures. Results of this part gave strong evidence on the
role of free thiol groups during formation of aggregates and their properties.
Finally, the fifth objective of this work was to evaluate the potential of the mixed
aggregates for the production of acid-set type yogurts (Chapter 8). The hypothesis that the
mixed aggregates will be integrated to the formation of a casein network after a heat
treatment was imposed by our previous observations (Chapter 7) and the literature. Results
of this work allow us to verify our hypothesis that the mix aggregates are still reactive and
can initiate the formation of a protein network. Practically, results showed that the mix
aggregates acted more like a passive than active fillers during the formation of the gels, but
some possible interactions with the proteins from the skim milk have been observed.
However, the dispersibility of the powder must be strictly improved. Results of this part
159
gave strong evidence on the potential application of the mixed aggregates for food
formulation.
Overall, according to our results the relationship between the free thiol groups reactivity in
the WPC and the properties of the heat-induced whey protein aggregates has been
summarized in Figure 9.1. First, results showed that a low portion of proteins were
denatured or aggregated during the preparation of the WPC. These pre-formed aggregates
exposed free thiol groups at their surface before the heat treatment (Chapter 5). Upon
heating, the proteins from the WPC denature and interact with the pre-formed particles to
form larger aggregates. The MFGM proteins or phospholipids if present in the WPC are
rarely involved in the formation of aggregates. At the end of heating, the protein aggregates
have an important water-holding capacity, and have some free thiol groups accessible at
their surface (Chapter 4 and 7). The prevention of the SH/SS exchange reaction led to the
formation of smaller aggregates with a higher water-holding capacity. Surprisingly,
aggregates still expose free thiol group at their surface as evidenced by the free thiol
concentration of the aggregates after dispersibility in SMUF buffer. However, non-covalent
based aggregates are rarely found in the heated WPC (Chapter 5).
160
Figure 9.1: Schematic representation of the proposed mechanism of aggregation of proteins from WPC
and the properties of the heat-induced protein aggregates.
The properties of the heat-induced aggregates formed by the mix of whey and buttermilk
concentrate (protein ratio of 50:50) are, however, probably different due to the differences
in the proportion of the major dairy proteins (caseins, whey proteins) and minor
constituents (MFGM fragments, free phospholipids). The relationship between the free
thiol reactivity in the mixture WPC + buttermilk concentrate and the properties of the heatinduced protein aggregates has been summarized in Figure 9.2.
First, addition of buttermilk constituents slightly decreased the accessibility of free thiol
groups during heating in the mixture (Chapter 5). This result is to be attributed to the
addition of the casein micelles or a more complex formation of pre-formed aggregates
during the preparation of the buttermilk concentrates. However, the composition of the preformed aggregates has no significant impact on the properties of the mixed aggregates.
These pre-formed particles exposed free thiol groups at their surface before the heat
161
treatment (Chapter 5). Upon heating, the proteins from the WPC and the buttermilk
denature and interact with the pre-formed particles (from both) to form larger aggregates.
At the end of heating, the protein aggregates have a lower water-holding capacity (WHC)
than aggregates formed in the heated WPC (Chapter 4). Moreover, the WHC of mixed
aggregates can be controlled by variation of the proportion of buttermilk constituents in the
mixtures. The aggregates are also smaller. The proportion of buttermilk constituents added
can also control the size because results from Chapter 5 showed that the size of the
aggregates is related to the number of pre-formed aggregates. The mixed aggregates also
expose free thiol groups at their surface. The prevention of the SH/SS exchange reaction
led to the formation of smaller aggregates with a higher water-holding capacity.
Surprisingly, aggregates still expose free thiol group at their surface. That kind of
aggregates is found during heating of the WPC mixed with the different buttermilks, at any
time (Chapter 5).
Figure 9.2: Schematic representation of the proposed mechanism of aggregation of constituents from
WPC + BC mixtures and the properties of the heat-induced protein aggregates.
162
9.2 Significance of the results
The work presented in this thesis is expected to have impact in various fields of dairy
science and technology. This thesis work led to several advancements in our understanding
of the thermal behavior of both whey and buttermilk constituents. For years, they have been
considered to be by-products with limited applications. Results presented in this thesis
proposed a new approach for their use and potential applications.
The thermal behavior of whey proteins has been well studied in model systems (WPI, βlactoglobulin isolate, etc). However, to our knowledge very few studies used whey directly
from cheese production. The results we have presented demonstrated that protein
aggregates are formed during the preparation of the WPC, including cheese-making
process, skimming, and filtration. The pre-formed aggregates are composed mainly from
whey proteins and involve mostly SH/SS exchanges. Upon heating, whey proteins are
denatured and associated with the pre-formed aggregates mostly through SH/SS exchanges
but also through non-covalent interactions. MFGM material is rarely involved in the
formation of the aggregates. Results also showed that aggregates formed in the WPC hold a
lot of water.
Previous results from this research group have shown that the butter-making process,
specifically the pasteurization of the cream, affects the buttermilk constituents. To our
knowledge, no study has focused at the thermal behavior of buttermilk constituents. The
results we have presented demonstrated that aggregates are formed during the preparation
of the buttermilk concentrates or powders, including pasteurization of the cream, churning,
skimming, filtration, drying, etc. The pre-formed aggregates are composed of whey
proteins, caseins, MFGM proteins and phospholipids and involve both SH/SS exchanges
and non-covalent interactions. Upon heating, the pre-formed aggregates associate with the
denatured proteins and phospholipids through both SH/SS exchanges and non-covalent
interactions. MFGM material is often involved in the formation of the aggregates. Results
have shown that the mixed aggregates hold less water comparatively to WPC aggregates.
163
It is usually admitted that dairy protein aggregates formed during heating at a low pH act
more like a passive than active filler during the production of cheese or yogurts. The results
we have presented showed that mixed aggregates are still reactive due to their high
concentration of free thiol groups. Results also identified some interactions between the
mixed aggregates and the proteins from the skim milk during a heat treatment. The
properties of the aggregates can be controlled by increasing the proportion of buttermilk
constituents in the mixture and by promoting some type of interactions. These results led to
visualize a large number of options for the use of our mix aggregates.
9.3 Question yet to be answered and perspectives
According to results presented in chapters 5 and 7, the proposed preparation of the WPC
was sufficient to standardize the composition of the concentrates (amount of protein, fat,
etc), but was insufficient to standardize the properties of the protein (size, exposure of free
thiol groups, etc.). It appears that a single change in the cheese-making process
significantly affects the properties of the subsequent.
Results presented in chapters 7 and 8 showed that dispersibilities of the powder containing
aggregates were critical and significantly affected the formation of the yogurt gels. A
homogenization step is necessary before drying in order to reduce the size of the mix
aggregates. We also suggest adding another homogenization step in the final product,
especially if the formulation involves a heating.
The results presented in this thesis demonstrate the complexity of regular and whey
buttermilk but also show promising applications for its use. This project did not focus on
incorporation of the aggregates in cheese formulation (cheddar, mozzarella, etc). This is
being performed in another research project.
164
Regarding the formation of aggregates and among the unresolved issues, the understanding
of the butter-making process and its effects on the constituents is a key issue regarding the
understanding of the properties of the buttermilk constituents and their potential uses.
Further information on the denaturation of the MFGM proteins and the release of
phospholipids from the membrane could help to understand the formation of the preformed aggregates. Further information on the denaturation of dairy protein at low pH and
the type of interactions involved is capital to understand the formation of aggregates and
their properties.
Regarding the use of aggregates and among the unresolved issues, the optimization of the
dispersibility of the aggregates powders is a key issue. Understanding the behavior of the
aggregates in a new system is necessarily to make their incorporation efficient.
Understanding the effect of addition of the mixed aggregates in a new system is important
for the control of the properties of the aggregates.
The confocal laser-scanning microscope has been a very powerful tool for this project. The
uses of this tool are, however, often inadequate and the results over discussed. There is still
a lot of work do be done for the analysis of the pictures and extraction of data.
Standardization of the techniques is a key issue regarding to the exploitation of the pictures.
With the advances in computer science and the availability of new dye and equipment for
the microscope, future work should emphasize on the use of the microscope directly
connected to the water-bath for the understanding of the heat-induced aggregation of
protein or on the formation of gels directly on top of the microscope.
Last but not least, the work presented in this thesis gave a new look regarding the use of
dairy constituents. Every year, dairy powders are produced from the surplus of milk solids
such as skim milk powders. The frame built during this project can be easily adapted to the
other milk solids products. Each major constituent of milk play a role on the formation and
on the properties of the aggregates by varying their composition and controlling their
properties. In other source of proteins (skim milk for example), the constituents may be less
165
affected by the preparation. In consequence, the thermal behavior may be slightly different,
but results of this project and the most recent literature give sufficient strong clues for the
understanding of the aggregation mechanism and the properties of the aggregates. Protein
aggregates have been mostly used as fat replacers or as water holder agents. The work
presented suggests that they can also be used for the incorporation of phospholipids in the
food matrix. The mixed aggregates also show very promising properties for the
development of products with new textures, properties, and sensory or nutritional
properties.
166
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