Development of new dairy ingredient with prebiotic and antioxidant properties through the electro

254 Pages·2017·4.79 MB·English

Development of new dairy ingredient with
prebiotic and antioxidant properties through
the electro-activation processing of sweet cheese
whey

Thèse

Ourdia Kareb

Doctorat en sciences et technologie des aliments
Philosophiae Doctor (Ph. D.)

Québec, Canada

© Ourdia Kareb, 2018
Development of new dairy ingredient with
prebiotic and antioxidant properties through
the electro-activation processing of sweet cheese
whey
Thèse

Ourdia Kareb

Sous la direction de :

Mohammed Aider, directeur de recherche
Claude P. Champagne, codirecteur de recherche
Julie Jean, codirectrice de recherche
RÉSUMÉ
Le but de ce projet était de développer la technologie d'électro-activation comme une
approche novatrice pour la valorisation intégrale des constituants du lactosérum. Ce
sous-produit est riche en composants de valeur avec des propriétés biologiques et
fonctionnelles prometteuses. La première étape de ce travail visait à déterminer les
conditions optimales pour la production du lactulose en utilisant le lactosérum comme
source de lactose. Un rendement de 35% a été obtenu avec une pureté élevée à des
températures de 0, 10 et 25°C et un court temps de réaction (≅ 40 minutes). En outre,
l'analyse protéomique a révélé l'hydrolyse des protéines de lactosérum et la formation
simultanée de produits de réaction Maillard aux propriétés antioxydantes élevées. La
deuxième étape visait à élucider les mécanismes antioxydants qui régissaient les
hydrolysats de protéines de lactosérum à réaction de Maillard (MRP-whey) et à
caractériser leurs structures. L'effet antioxydant a été attribué à de multiples propriétés
et pourrait être utilisé à la fois comme antioxydants primaires et secondaires. La
structure moléculaire du MRP-whey a été caractérisée comme étant des bases de Schiff.
De plus, des peptides bioactifs multifonctionnels, avec des propriétés biologiques ont
également été identifiés. Dans la troisième étape, l'efficacité du lactosérum électro-
activé (lactosérum-EA) possédant des propriétés prébiotique et antioxydante sur la
croissance des bactéries probiotiques testées a été démontrée. Le lactosérum-EA avait
comparativement un meilleur effet bifidogène que le lactulose. De plus, on a constaté
que le lactosérum-EA produisait un effet protecteur sur L. johnsonii La-1 durant sa
croissance en présence d'oxygène. Cet effet peut être lié, en partie, à la capacité du
lactosérum-EA à éliminer le peroxyde d'hydrogène et à prévenir son accumulation dans
le milieu de croissance. Les spectres FTIR ont montré que le lactosérum-EA empêchait
l'oxydation des lipides de la membrane cellulaire en limitant le changement dans l'ordre
structurel des acides gras. Dans l'ensemble, le lactosérum-EA comme prébiotique
possédant une capacité antioxydante a un grand potentiel d'application, non seulement
dans l'industrie laitière, mais aussi comme ingrédient fonctionnel avec diverses
bioactivités et fonctionnalités.

iii
ABSTRACT
The goal of this project was to develop the electro-activation technology as innovative
approach for an integral valorization of whey constituents. This by-product is rich in
valuable components with promising biological and functional properties. The first step
of this work aimed to determine the optimal conditions of lactulose production using
whey as a source of lactose and a yield of 35% was obtained with high purity under
temperatures of 0, 10 and 25°C and short reaction time (≅ 40 minutes). Moreover, the
proteomic analysis revealed the hydrolysis of whey proteins and simultaneous
formation of Maillard reaction products with high antioxidant properties. The second
step aimed to elucidate the antioxidant mechanisms that governed the Maillard-reacted-
whey protein hydrolysates (MRPs-whey) and to characterize their structures. The
antioxidant effect was ascribed to a multiple properties and could be used as both
primary and secondary antioxidants. The molecular structure of the MRPs-whey was
characterized to be Schiff base compounds. Additionally, multifunctional bioactive
peptides, with biological properties were also identified. In the third step, the efficacy
of electro-activated whey (EA-whey) as a combined prebiotic and antioxidant
component on the growth of tested probiotic bacteria was demonstrated. The EA-whey
was comparatively a better bifidogenic factor than lactulose. Additionally, EA-whey
was found to elicit a protective effect on L. johnsonii La-1 during its growth in the
presence of oxygen. This effect may be related, in part, to the ability of EA-whey to
scavenge hydrogen peroxide metabolites and prevent its accumulation in the growth
medium. FTIR spectra showed that EA-whey prevented the cell membrane lipids
oxidation by limiting the change in the structural order of fatty acids. Overall, EA-whey
as a prebiotic supplement possessing antioxidant capacity has great potential for
application not only in the dairy industry but also as a functional food ingredient with
potent bioactivities and functionalities.

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TABLE OF CONTENTS
RÉSUMÉ .......................................................................................................... iii
ABSTRACT ..................................................................................................... iv
TABLE OF CONTENTS .................................................................................. v
LIST OF TABLES ......................................................................................... xiii
LIST OF FIGURES ........................................................................................ xiv
LIST OF ABBREVIATIONS ...................................................................... xviii
DEDICATIONS ............................................................................................. xix
ACKNOWLEDGMENTS ............................................................................... xx
FOREWORD ................................................................................................ xxii
INTRODUCTION ............................................................................................. 1
1. CHAPTER 1: Literature review ................................................................ 4
1.1 Functional foods .................................................................................. 4
1.1.1 Probiotics ....................................................................................... 6
1.1.1.1 Probiotics concept ................................................................... 6
1.1.1.2 Commercially used probiotic foods ........................................ 6
1.1.1.3 Selection of potential probiotics ............................................. 8
1.1.1.4 Mechanisms of action of probiotics ...................................... 10
1.1.1.5 Beneficial health effects of probiotics .................................. 12
1.1.1.6 Factors affecting the survival of probiotics in dairy products ..
.............................................................................................. 14
1.1.2 Prebiotics ..................................................................................... 16
1.1.2.1 Prebiotics concept ................................................................. 16
1.1.2.2 Criteria of prebiotics ............................................................. 17
1.1.2.3 Sources, production of prebiotics ......................................... 17
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1.1.2.4 Beneficial health effects of prebiotics .................................. 20
1.1.2.5 Prebiotics as functional food ingredients .............................. 21
1.1.2.6 Synbiotic concept ................................................................. 23
1.1.2.7 Synbiotic in dairy products ................................................... 24
1.2 Whey ................................................................................................. 26
1.2.1 Whey in environmental consideration ......................................... 26
1.2.2 Whey composition ....................................................................... 27
1.2.2.1 Lactose whey ........................................................................ 28
1.2.2.2 Lactose-derived lactulose ..................................................... 28
1.2.2.2.1 General properties of lactulose ...................................... 28
1.2.2.2.2 Chemistry of lactulose synthesis ................................... 29
1.2.3 Methods of lactulose production ................................................. 31
1.2.3.1 Chemical synthesis ............................................................... 31
1.2.3.2 Enzymatic synthesis ............................................................. 31
1.2.3.3 Electro-activation synthesis .................................................. 32
1.2.4 Lactulose applications ................................................................. 33
1.2.4.1 Pharmaceutical applications ................................................. 33
1.2.4.2 Food applications .................................................................. 34
1.2.5 Whey proteins .............................................................................. 35
1.2.5.1 Nutritional value of whey proteins ....................................... 36
1.2.5.2 Whey proteins in food as a functional ingredient ................. 37
1.2.5.3 Physiological importance of whey proteins .......................... 39
1.2.5.4 Bioactive peptides generated from whey .............................. 40
1.2.5.4.1 Processing of bioactive peptides in whey ...................... 41
1.2.5.4.2 Functionalities of bioactive peptides in whey ............... 42
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1.2.5.4.2.1 Antihypertensive peptides ...................................... 43
1.2.5.4.2.2 Antioxidative peptides ............................................ 44
1.2.5.4.2.3 Antimicrobial peptides ........................................... 45
1.2.5.4.2.4 Immunomodulatory peptides .................................. 45
1.2.5.4.2.5 Other bioactive peptides ......................................... 46
1.2.5.5 Maillard reaction products from whey ................................. 47
1.2.5.5.1 Maillard reaction stages ................................................. 48
1.2.5.5.2 Maillard reaction processing ......................................... 50
1.2.5.6 Some proprieties of Maillard reaction products ................... 51
1.2.5.7 Antioxidant properties of MRPs-whey ................................. 51
1.3 Electro-activation .............................................................................. 53
1.3.1 The concept of electro-activation and devolvement .................... 53
1.3.2 Principles of electro-activated aqueous solutions ........................ 53
1.3.3 Application of electro-activation solutions .................................. 56
1.3.4 Electro-activation technology for whey valorization .................. 57
2. CHAPTER 2: Problematic, Hypothesis and Objectives ......................... 59
2.1 Problematic ........................................................................................ 59
2.2 Hypothesis ......................................................................................... 59
2.3 Main objective ................................................................................... 60
2.4 Specific objectives ............................................................................. 60
3. CHAPTER 3: Contribution to the production of lactulose-rich whey by in
situ electro-isomerization of lactose and effect on whey proteins after electro-activation
as confirmed by MALDI-TOF MS spectrometry and SDS-PAGE gel electrophoresis .
................................................................................................................. 61
3.1 RÉSUMÉ ........................................................................................... 62
3.2 ABSTRACT ...................................................................................... 63
vii
3.3 INTRODUCTION ............................................................................. 64
3.4 MATERIALS AND METHODS ...................................................... 67
3.4.1 Chemicals .................................................................................... 67
3.4.2 Electro-activation reactor and configuration ............................... 67
3.4.3 Electro-activation reaction ........................................................... 69
3.4.4 HPLC analysis of the reaction products ...................................... 69
3.4.5 Total protein content .................................................................... 70
3.4.6 Gel Electrophoresis (SDS-PAGE) ............................................... 70
3.4.7 MALDI-TOF-MS assessment ..................................................... 71
3.4.8 Determination of free amino acids by UPLC-UV florescence
detection ..................................................................................................... 71
3.4.9 Oxygene radical capacity (ORAC) of electro-activated whey .... 72
3.4.10 Statistical Analysis ...................................................................... 72
3.5 RESULTS AND DISCUSSION ....................................................... 73
3.5.1 Process of whey solution EA: Evolution of whey solution alkalinity
..................................................................................................... 73
3.5.2 Assessment of lactulose formation during whey electro-activation
..................................................................................................... 76
3.5.3 HPLC analysis of lactulose and other reaction by-products ........ 78
3.5.4 Effect of temperature ................................................................... 81
3.5.5 Effect of the electric current intensity ......................................... 83
3.5.6 Effect of the volume reaction ...................................................... 85
3.5.7 Effect of the feed whey concentration ......................................... 87
3.5.8 Proteomic analysis of the EA-whey ............................................ 89
3.5.8.1 SDS-PAGE profiles of EA-whey proteins ........................... 89
3.5.8.2 MALDI-TOF-MS analysis of whey proteins after EA ......... 91
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3.5.9 Total antioxidant capacity of EA-whey proteins ......................... 92
3.6 CONCLUSION ................................................................................. 96
4. CHAPTER 4: Impact of electro-activation on the antioxidant properties of
defatted whey .............................................................................................................. 97
4.1 RÉSUMÉ ........................................................................................... 98
4.2 ABSTRACT ...................................................................................... 99
4.3 INTRODUCTION ........................................................................... 100
4.4 MATERIALS AND METHODS .................................................... 102
4.4.1 Chemicals, reagents, membranes and electrodes ....................... 102
4.4.2 Electro-activation of whey induced Maillard reaction products 104
4.4.3 Determination of antioxidant activity ........................................ 104
4.4.3.1 Determination of reducing power of EA-whey .................. 104
4.4.3.2 Determination of 2,2-diphenyl-1-picrylhydrazyl radical-
scavenging activity of EA-whey ................................................................... 105
4.4.3.3 Determination of 2,2-diphenyl-1-picrylhydrazyl radical-
scavenging activity of EA-whey ................................................................... 105
4.4.3.4 Determination of hydroxyl radical scavenging activity of EA-
whey ............................................................................................ 106
4.4.3.5 Determination of chelating activity on Fe2+ of EA-whey ... 106
4.4.4 Measurement of browning intensity and fluorescence of electro-
activated whey ................................................................................................... 107
4.4.5 Statistical analysis ...................................................................... 107
4.5 RESULTS AND DISCUSSION ..................................................... 108
4.5.1 Oxygen radical absorbance capacity ......................................... 108
4.5.2 Reducing power ......................................................................... 110
4.5.3 Metal-chelating activity ............................................................. 112
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4.5.4 2,2-Diphenyl-1-picrylhydrazyl scavenging activity .................. 114
4.5.5 Hydroxyl radical scavenging activity ........................................ 116
4.5.6 Effect of pH on brown colour development of electro-activated
whey ................................................................................................... 119
4.6 CONCLUSION ............................................................................... 122
5. CHAPTER 5: Electro-activation of sweet defatted whey: Impact on the
induced Maillard reaction products and bioactive peptides ...................................... 123
5.1 RÉSUMÉ ......................................................................................... 124
5.2 ABSTRACT .................................................................................... 125
5.3 INTRODUCTION ........................................................................... 126
5.4 MATERIALS AND METHODS .................................................... 128
5.4.1 Chemicals and reagents ............................................................. 128
5.4.2 Maillard reaction products (MRPs) generation from EA-whey 128
5.4.3 Determination of free and sugar-bound amino acids ................. 129
5.4.4 Determination of reducing sugars in MRPs-whey .................... 130
5.4.5 Structure characterization of MRPs-whey induced by electro-
activation ................................................................................................... 130
5.4.5.1 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) ............................................................................................ 130
5.4.5.2 FT-IR measurements .......................................................... 130
5.4.5.3 LC/MS-MS analysis ........................................................... 131
5.4.5.3.1 Samples preparation .................................................... 131
5.4.5.3.2 Mass spectrometry ....................................................... 131
5.4.5.3.3 Database searching ...................................................... 132
5.4.5.3.4 Criteria for protein identification ................................. 132
5.5 Statistical analysis ........................................................................... 132
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