Boissons fermentées de type yogourt à boire enrichies

en protéines de lactosérum et en probiotiques

Mémoire

Germaine Enyoh Forkwa

Maîtrise en sciences et technologie des aliments

Maître ès Sciences (M. Sc.)

Québec, Canada

© Germaine Enyoh Forkwa, 2017

Résumé
Le but de ce projet était de développer de nouvelles boissons laitières fermentées à base de
lactosérum aux propriétés organoleptiques attrayantes. D’autres caractéristiques
recherchées pour ces boissons étaient une concentration élevée en protéines, une haute
teneur en probiotiques et sans additif (ni sucre, ni polysaccharides, ni colorants, ni arômes
artificiels). Ces boissons auraient donc un effet bénéfique potentiel sur la santé
cardiovasculaire, le poids corporel et le bien-être des consommateurs. Des boissons
contenant 10% de protéines de lactosérum ont été fabriquées par fermentation de concentré
de protéines de lactosérum (CPL) et/ou isolat de protéines de lactosérum (IPL). Dix CPL et
deux IPL ont été évalués pour leur aptitude à la fermentation lactique. Sept ferments
thermophiles et 2 souches probiotiques furent utilisés. L’impact des ferments, la stabilité
des ferments et des probiotiques, la post acidification ainsi que les propriétés
organoleptiques ont été évalués. Les conditions de fermentations ont été optimisées afin de
générer des produits ayant au moins 1 à 10 milliards de probiotiques par portion. Ce projet
a permis de développer des boissons fermentées (de type yogourt à boire) contenant 10% de
protéines de lactosérum et 2 milliards de bactéries probiotiques par mL, et ce, uniquement à
partir de dérivés de lactosérum. Par portion de 100 mL, ces boissons contiennent 200
milliards de probiotiques et seraient considérées comme les produits les plus riches en
probiotiques sur le marché québécois. Ces résultats pourraient conduire à la mise en marché
de nouveaux produits au fort potentiel de croissance qui répondent à plusieurs tendances de
marché dans le secteur des aliments santé.

iii
Abstract
The aim of this project was to develop new fermented milk beverages from whey protein
concentrates with attractive sensory properties. Other desired characteristics for these
beverages were high protein concentration, high probiotic content and no use of additives
(sugar, polysaccharides, colorants, artificial flavors). These drinks would have potential
benefits on cardiovascular health, body weight and the well-being of consumers. Beverages
containing 10% whey protein were obtained by fermenting whey protein concentrates
(WPC) and/or whey protein isolates (WPI). Ten WPCs and two WPIs were evaluated for
their ability to allow lactic fermentation by 2 types of mesophilic starter cultures. The
medium with the best fermentation kinetics and the best organoleptic properties was
selected. The impact of 7 thermophilic starter and 2 probiotic cultures on cell concentration
and stability, post acidification and organoleptic properties was studied. The fermentations
were adapted to generate products with at least 1 to 10 billion probiotics per serving. This
project allowed the development of fermented beverages (drinking yogurt type) with 10%
whey proteins and 2 billion probiotic bacteria per mL, exclusively from whey derivatives.
Each 100 mL serving contained 200 billion probiotics and would be considered as a
product with the highest probiotics counts on the Quebec market. These results could lead
to the marketing of new products with high growth potential that respond to several market
trends in the health food sector.

iv
Tables de matières
Résumé .............................................................................................................................................. iii
Abstract ............................................................................................................................................. iv
Tables de matières ............................................................................................................................. v
Liste des tableaux ............................................................................................................................. ix
Liste des figures ................................................................................................................................. x
Liste des abréviations et des sigles ................................................................................................ xii
Remerciements ................................................................................................................................ xv
Avant-propos .................................................................................................................................. xvi
Chapitre 1 : Introduction générale .................................................................................................. 1
Chapitre 2 : Revue de la littérature ................................................................................................. 3
2.1 Le lactosérum ............................................................................................................................ 3
2.2 Caractéristiques physico-chimiques des principaux constituants du lactosérum ...................... 4
2.2.1 Le lactose............................................................................................................................ 5
2.2.2 Les protéines de lactosérum (PL) ....................................................................................... 5
2.3 Propriétés des protéines de lactosérum ................................................................................... 10
2.3.1 Propriétés nutritionnelles.................................................................................................. 10
2.3.2 Propriétés santé ................................................................................................................ 11
2.3.3 Propriétés fonctionnelles .................................................................................................. 12
2.4 Ingrédients laitiers obtenus à partir du lactosérum.................................................................. 14
2.5 Ingrédients laitiers à haute teneur en protéines de lactosérum ................................................ 15
2.6 Développement de boissons à base de lactosérum .................................................................. 16
2.6.1 Boissons et jus de fruits au lactosérum non fermentées ................................................... 16
2.6.2 Boissons fermentées à base de lactosérum ....................................................................... 17
2.6.2.1 Les bactéries lactiques (BL) ...................................................................................... 18
2.6.2.2 Les probiotiques ........................................................................................................ 20
2.6.2.3 Amélioration de la croissance des probiotiques ........................................................ 22
2.6.3 Boissons au lactosérum fermentée par des bactéries lactiques ........................................ 24
Chapitre 3 : Hypothèse et objectifs ................................................................................................ 27
Chapter 4: Selection of starter cultures and whey protein sources for the production of a high
protein whey beverage .................................................................................................................... 28

v
Résumé .............................................................................................................................................. 29
Abstract ............................................................................................................................................. 30
4.1 Introduction ............................................................................................................................. 31
4.2 Materials and Methods ............................................................................................................ 33
4.2.1 Whey powders .................................................................................................................. 33
4.2.2 Rehydration of whey and derived whey powders ............................................................ 34
4.2.3 Determination of the buffering capacity (BC) ................................................................. 34
4.2.4 Lactose analysis................................................................................................................ 35
4.2.5 Bacterial strains and cultures............................................................................................ 35
4.2.6 Fermentation..................................................................................................................... 36
4.2.7 Sensory analysis of fermented solutions .......................................................................... 37
4.3 Results and Discussion ............................................................................................................ 39
4.3.1 Some chemical attributes of the different milk and whey solutions................................. 39
4.3.2 Fermentation of WPCs and WPIs .................................................................................... 41
4.3.3 Lactose supplementation .................................................................................................. 44
4.3.4 Influence of starter culture ............................................................................................... 45
4.3.5 Sensory analysis of WPIs /WPCs fermented with mesophilic culture “M1” ................... 47
4.3.6 Fermentation of YB whey concentrate ............................................................................. 48
4.3.7 Sensory analysis of WPIs / WPCs fermented with thermophilic starter cultures ............ 50
4.3.8 Commercial application ................................................................................................... 51
4.4 Conclusion............................................................................................................................... 52
4.5 Acknowledgements ................................................................................................................. 53
Chapter 5: Effect of whey protein concentrate and starter culture on the growth and stability
of probiotic bacteria in fermented high protein whey beverage ................................................. 54
Résumé .............................................................................................................................................. 55
Abstract ............................................................................................................................................. 56
5.1 Introduction ............................................................................................................................. 57
5.2. Material and Methods............................................................................................................. 59
5.2.1 Strains ............................................................................................................................... 59
5.2.2 Fermentation media .......................................................................................................... 59
5.2.3 Fermentation..................................................................................................................... 60
5.2.4 Acidification ..................................................................................................................... 60
5.2.5 Dissolved oxygen ............................................................................................................. 60

vi
 5.2.6 Microbial analysis ............................................................................................................ 60
5.2.7 Buffer capacity ................................................................................................................. 61
5.2.8 Statistical analysis ............................................................................................................ 61
5.3 Results and Discussion ............................................................................................................ 62
5.3.1 Growth during fermentation ............................................................................................. 62
5.3.2 Acidification during storage ............................................................................................. 63
5.3.3 Stability of streptococci during storage ............................................................................ 65
5.3.4 Stability of probiotic strains during storage ..................................................................... 66
5.3.5 Dissolved oxygen ............................................................................................................. 68
5.3.6 Market potential ............................................................................................................... 69
5.4 Conclusion............................................................................................................................... 71
5.5 Acknowledgments ................................................................................................................... 72
Chapter 6: Effect of growth promoting factors in a high protein probiotic whey beverage .... 73
Résumé .............................................................................................................................................. 74
Abstract ............................................................................................................................................. 75
6.1 Introduction ............................................................................................................................. 76
6. 2 Material and Methods............................................................................................................. 78
6.2.1 Starter culture ................................................................................................................... 78
6.2.2 Probiotic strains ................................................................................................................ 78
6.2.3 Whey protein concentrate and milk ingredients ............................................................... 78
6.2.4 Preparation of W and WM media..................................................................................... 79
6.2.5 Supplementation of W medium for AS screening assays ................................................ 79
6.2.6 Automated spectrophotometry assays .............................................................................. 83
6.2.7 Fermentations ................................................................................................................... 83
6.2.8 Microbial analyses............................................................................................................ 84
6.2.9 Carbohydrates and organic acid analysis ......................................................................... 84
6.2.10 Statistical analysis .......................................................................................................... 85
6. 3 Results and Discussion ........................................................................................................... 86
6.3.1 Biocompatibility assays between the starter and the probiotics ....................................... 86
6.3.2 Effect of MRS constituents on probiotic growth in fermented W medium...................... 87
6.3.3 Effect of various sources of peptones on the growth of Lactobacillus helveticus R0052 89
6.3.4 W medium fermentation by Lactobacillus helveticus R0052 .......................................... 92
6.4 Conclusion............................................................................................................................... 97

vii
 6.5 Acknowledgments ................................................................................................................... 98
Conclusion générale ........................................................................................................................ 99
Bibliographie ................................................................................................................................. 101

viii
Liste des tableaux
Tableau 2.1 Composition du lactosérum doux et du lactosérum acide………….…. 4

Table 4.1 Main composition of WPC and WPI powders ………………........……. 33

References for descriptive analysis of fermented whey protein blends in

Table 4.2 sensory analysis………...………………………….………….........…38

Composition and buffer capacities of rehydrated

Table 4.3
powders.……………………………………………………………….40

Concentrations of MRS-based ingredients added to clarified fermented

Table 6.1 “W medium” supernatant……………………………………………..81

Concentrations of protein hydrolysates added to clarified fermented W

Table 6.2 medium…………...……………………………….……………..……82

Influence of supplementation of the W medium and external pH control

(EpHC) on the growth of and acidification of Lb. helveticus R0052 and St.
Table 6.3 thermophilus …...…………………………………………………...….94

Effect of external pH control (EpHC) on levels of carbohydrates and of

Table 6.4
lactic acid during the fermentation of whey-based beverages………...96

ix
Liste des figures

Figure 2.1 Ingrédients laitiers obtenus à partir du lactosérum liquide………………..9

Valeur biologique [A] et composition en teneur en A.A essentiels [B] de

Figure 2.2
différentes sources de protéines……………………………………….....10

Figure 2.3 Les différentes étapes de gélification des protéines du lactosérum ……...13

System used to carry out fermentation and continuously monitor

Figure 4.1
pH................................................................................................................37

Figure 4.2 Fermentation profiles of whey and milk controls with mesophilic starter
“M1”……………………………………………………………….……...42

Mesophilic fermentation of whey-bases with starter culture

Figure 4.3
“M1”……………………………………………………………………....43

Supplementation of powders which could not undergo complete

Figure 4.4
acidification in lactose. Fermented with “M1”………..……………........45

Lactose supplemented protein concentrates with two types of mesophilic

Figure 4.5
cultures (“M1”compared to “M2”) ………………………………………46

Sensory analysis of fermented whey drinks with starter culture

Figure 4.6
“M1”………………………………………………………………............47

Acidification profile of YB with different commercial thermophilic starter

Figure 4.7
cultures………………………………………………………………….…49

Figure 4.8 Sensory analysis of fermented whey drinks with thermophilic starter
cultures F1 to F7……………………………………………………..……50

Viable counts of probiotic strains (Lb. helveticus R0052 and Lb. rhamnosus
Figure 5.1 R0011) after fermentation in association with a commercial yoghurt starter
culture…………………………………………………………...……..….63

x
 Effect of fermentation medium and of a probiotic culture (Lb. helveticus
R0052 and Lb. rhamnosus R0011) on viable counts of St. thermophilus
Figure 5.2
during storage at 4°C of the 10% protein fermented
drinks…………………………………………………………………...…64

Effect of medium and probiotic strains (Lb. helveticus R0052 or Lb.

rhamnosus R0011) on pH changes during 45 days of storage at 4°C of 10%
Figure 5.3
protein fermented drinks……………………………….……………….…66

Effect of fermentation medium on the viable counts of two probiotic

cultures (Lb. helveticus R0052 or Lb. rhamnosus R0011) during storage at
Figure 5.4
4°C of the 10% protein fermented drinks…………………………………67

Effect of medium and probiotic strain (Lb. helveticus R0052 or Lb.

Figure 5.5 rhamnosus R0011) on the levels of dissolved oxygen in the 10% protein
fermented drinks during storage à 4°C…...……………………………….68

Effect of a pre-fermentation by YO-Mix T11 yogurt starter and of the

addition of glucose (Glu) or MRS to W medium on the subsequent growth
Figure 6.1 of two probiotic cultures (Lb. helveticus R0052 and Lb. rhamnosus
R0011)…………………………………………………………………….86

Effect of supplementing the clarified fermented “W medium” (W) with

Figure 6.2 constituents of MRS broth……………………………..………………….88

Effect of the supplementation of the clarified pre-fermented whey-based

medium (W) with individual or combined components of the amino acid
Figure 6.3
based group of MRS broth………………………..………………….……90

Effect of the supplementation of the clarified pre-fermented whey-based

medium (W) with various protein hydrolysates on the growth of Lb.
Figure 6.4
helveticus R0052……………………………………………………..……91

Figure 6.5 Effect of the supplementation of the clarified pre-fermented whey-based

medium (W) with various dairy-based hydrolysates……..……………….92

xi
Liste des abréviations et des sigles
Chapitre 2
DBO Demande biologique en oxygène
β –lg β -lactoglobuline
α -lac α - lactalbumine
Ig Immunoglobulines
Lp Lactoperoxydase
ABS L’albumine sérique bovine
Lf Lactoferrine bovine
PL Protéines du lactosérum
IECA Inhibiteur de l'enzyme de conversion de l’angiotensine
UF Ultrafiltration
CPL Concentré protéique de lactosérum
IPL Isolat protéique de lactosérum
BL Bactéries lactiques
Lb Lactobacillus
L. Lactococcus
St. Streptococcus
EPS Exopolysaccharides
FOS Fructooligosaccahrides
CMP Caséinomacropeptides
UFC Unité formant une colonie
A.A Acides Aminés
VB Valeur Biologique
CEpH Contrôle externe de pH

Chapter 4
WPC Whey protein concentrate
WPI Whey protein isolate
BC Buffering capacity
RSM Reconstituted skim milk powder
RW Reconstituted whey powder
RWP Reconstituted whey permeate
LAB Lactic acid bacteria
SEM Standard Error of Means
WHO/FAO World Health Organisation/ Food and Agriculture organization
W media Whey protein concentrate medium

xii
WM media Whey protein concentrate and skim milk medium

Chapter 6
WPC Whey protein concentrate
WPI Whey protein isolate
W media Whey protein concentrate medium
WM media Whey protein concentrate and skim milk medium
CFU Colony Forming Unit
AS Automated spectrophotometry
EpHC External pH Control

xiii
 Je dédie ce travail
A mon mari Serges-Armand Youmbi qui sans
cesse était présent et m’encourageait tout au
long de cette expérience.

A mes parents; Raymond Taneck et Alice

Abang Forkwa de m’avoir donné
l’opportunité de connaître ce qu’est l’amour
parental, je vous serai toujours
reconnaissante.

A mes sœurs, je vous aime et vous souhaite

beaucoup de bonheurs

A tous mes ami (e) s, vous êtes précieux pour

moi

A Dieu tout puissant pour la grâce de m'avoir

accordé l'intelligence et la bonne santé sans
lesquelles je ne peux rien.

xiv
Remerciements
Je tiens à remercier de tout cœur mon directeur de recherche, le Dr Jean Christophe
Vuillemard qui m'a offert cette unique occasion d’explorer la passion et les talents cachés
que j’avais pour la recherche. Sa confiance, son calme et ses mots d’encouragements ne
seront jamais oubliés. Merci d’avoir cru en moi et de m’avoir soutenu tout au long de ces
deux années.

Je tiens également à remercier mon co-directeur, le Dr Claude Champagne de m'avoir

accueillie dans son incroyable équipe. Sa disponibilité, ses bonnes capacités d'écoute, ses
encouragements et son approche humoristique m'ont grandement aidé lors de cette
expérience. Je dirais de lui, un patron exceptionnel, car il m’a appris travailler de façon
structurée et surtout comment gérer méthodiquement les imprévus de la recherche. Merci
d’avoir fait de moi une scientifique aguerrie.

Je remercie également Marie Josée Lemay, pour sa bonne humeur, sa patience, sa

simplicité et surtout pour son aide lors de la mise au point de protocoles et la réalisation des
expériences au laboratoire. Je tiens à remercier Yves Raymond, pour sa disponibilité et son
aide technique au laboratoire et lors des analyses des résultats. Un merci à Nancy Graveline
et à mon stagiaire Philippe Bouchard qui m’a permis d’obtenir des résultats de qualité. Un
grand merci à Marc-Olivier Leroux et mes collègues à la maîtrise Andréanne, Valérie et
Noémie pour leur amour, leurs conseils et leur aide.

Je souhaiterais également remercier tous les partenaires de ce projet : le ministère de

l'Agriculture des Pêcheries et de l'Alimentation du Québec (programme InnovAction) ainsi
qu’Agriculture et Agroalimentaire Canada pour le soutien financier de ce projet.

Je tiens également à remercier le Dr Denis Roy pour avoir accepté d’évaluer ce mémoire.

xv
Avant-propos
Ce mémoire est composé de six chapitres dont trois sont rédigés sous formes d’articles
scientifiques en anglais, tous précédés d’un résumé en français.

Germaine Enyoh Forkwa, est l’auteure principale de tous les articles scientifiques qui
accompagnent ce travail. Les renseignements sur les co-auteurs (noms et adresses) sont
présentés à la page titre de chacun des chapitres rédigés sous forme de publication. Les
chapitres 4, 5 et 6 seront soumis pour publication dans des revues scientifiques.

Après une brève introduction sur le sujet, la revue de littérature présente un ensemble de
connaissances sur le lactosérum, les propriétés santé et fonctionnelles liées à ses protéines
ainsi que les produits fermentés ou non, à base de lactosérum ou enrichis en protéines de
lactosérum.

Le chapitre 4 est présenté sous forme d’un article scientifique intitulé « Selection of starter
cultures and whey protein sources for the production of a high protein whey beverage ».
L'objectif de ce chapitre était d’identifier un concentré protéique parmi douze concentrés
protéiques de lactosérum (CPL) / isolats protéiques de lactosérum (IPL) permettant une
bonne fermentation lactique ainsi que les ferments les plus performants.

Le chapitre 5, aussi rédigé sous forme d’article scientifique est intitulé: « Effect of whey
protein concentrate and starter culture on the growth and stability of probiotic bacteria in
fermented high protein whey beverage ». Ce chapitre vise le développement d’une boisson
fermentée contenant 10% de protéines et des concentrations élevées en bactéries
probiotiques.

Le sixième chapitre sous forme d’article scientifique est intitulé « Effect of growth
promoting factors in a high protein probiotics whey beverage ». Ce chapitre avait pour
objectif de déterminer l'effet de la supplémentation de la boisson laitière par des facteurs de
croissance pour les bactéries probiotiques.

xvi
Finalement une conclusion générale met en évidence la réponse à l’hypothèse de départ,
l’atteinte des objectifs, la réussite du projet et les perspectives pour la continuité du projet
sont aussi présentées.

xvii
Chapitre 1 : Introduction générale

En réponse à l’augmentation du nombre de consommateurs désirant des aliments ayant des

effets bénéfiques sur la santé, tels que l’amélioration du bien-être et la réduction du risque
de maladie, l'industrie alimentaire a développé une variété de nouveaux produits
alimentaires dits fonctionnels (Granato et al., 2010). Les aliments fonctionnels sont des
aliments ou des composants alimentaires qui sont scientifiquement reconnus comme ayant
des avantages physiologiques autres que ceux de la nutrition de base (Ozer et al., 2010). Le
marché mondial des aliments/boissons fonctionnels continue d'être un segment dynamique
et croissant de l'industrie alimentaire (Corbo et al., 2014; Ozer et al., 2010). Les laits
fermentés, en particulier les produits de type yogourt et le kéfir sont les boissons
fonctionnelles les plus populaires en Europe occidentale, en Amérique du Nord et au
Danemark (Marsh et al., 2014). La fonctionnalité d'un grand nombre de ces boissons
fermentées peut être attribuée à la présence et à l'activité de bactéries probiotiques (Verbeke
et al., 2009). Les effets bénéfiques des probiotiques sur la santé sont l'amélioration du
système immunitaire et la prévention des infections intestinales telles la diarrhée et la
gastrite tout en inhibant les pathogènes microbiens entériques et d'origine alimentaire (Cho
et al., 2015; Kasipathy, 2010; Ravinder et al., 2013). Suite au succès des produits laitiers,
de nouveaux produits contenant des probiotiques ont été mis en marché, en particulier les
boissons à base de fruits et les céréales à déjeuner (Champagne et al., 2009)

Le lactosérum, un coproduit laitier obtenu après coagulation du lait et égouttage du

coagulum est un produit riche en protéines fonctionnelles et en substances bioactives
(Pescuma et al., 2008). À cet effet, les protéines de lactosérum sont de plus en plus
reconnues et acceptées comme des ingrédients fonctionnels. L’industrie laitière utilise dans
la fabrication du yogourt et des boissons fermentées une grande variété de produits issus du
lactosérum tels que la poudre de lactosérum, les concentrés protéiques de lactosérum
(CPL), les isolats protéiques de lactosérum (IPL) et des hydrolysats protéiques de
lactosérum (HPL) (Cho et al., 2015).

Auparavant, les protéines de l’œuf étaient considérées comme les protéines ayant la plus
haute valeur biologique (VB). Toutefois, des études ont montré que les protéines de

1
lactosérum ont une VB qui dépasse d'environ 15% celle de la protéine d'œuf (Smithers,
2008). Akhavan et al. (2013) ont rapporté que la consommation de protéines de lactosérum
abaisse la glycémie. De même, Zafar et al. (2013) ont montré que l’enrichissement d’une
boisson en protéines de lactosérum permet de contrôler le poids corporel en réduisant
l’appétit, la prise alimentaire et de ce fait l’apport énergétique. Les protéines de lactosérum
sont donc une excellente source de protéines pour les personnes qui pratiquent le
culturisme, les athlètes ou les personnes présentant des problèmes de santé comme le
surpoids, etc. (Ha et al., 2003).

Les boissons lactées à base de lactosérum représentent un segment émergent de produits

laitiers non conventionnels (Almeida et al., 2009). Cependant, pour être acceptées par des
consommateurs habitués aux produits laitiers conventionnels, ces boissons santé doivent
posséder une apparence, une texture et des caractéristiques gustatives souhaitables
(Varghese et al., 2013). Les boissons à base de lactosérum liquide ou de poudre de
lactosérum réhydratée présentent parfois des saveurs désagréables (Whetstine et al., 2005).

De nombreuses formulations de boissons à base de protéines de lactosérum avec de

meilleures caractéristiques organoleptiques ont été développées par l’ajout de concentrés de
fruits (Magalhaes et al., 2011; Sabokbar et al., 2014a) ou par fermentation (Pescuma et al.,
2008). Les CPL et les IPL sont généralement utilisés dans les aliments non fermentés
(Lagrange et al., 2015). Très peu d’information n’est disponible dans la littérature au sujet
de produits fermentés à base de CPL ou d’IPL. Toutefois, Pescuma et al. (2010) ont
démontré que la fermentation d’un CPL 35% avec des souches sélectionnées de bactéries
lactiques pourrait être une nouvelle voie pour développer des boissons lactées.

L’objectif de ce travail est de développer une nouvelle boisson fermentée de type yogourt à
boire riche en protéines de lactosérum et en probiotiques.

2
Chapitre 2 : Revue de la littérature

2.1 Le lactosérum
Le lactosérum est un co-produit laitier obtenu lors d’une production fromagère. Toujours
sous forme liquide et généralement de couleur jaunâtre à verdâtre, environ 9kg de
lactosérum est généré pour 1kg de fromage pâte pressée produit. En 2015, la production
mondiale de protéines du lactosérum a été estimée à approximativement 240 millions de
tonnes pour une valeur monétaire de 5,59 milliards $. Des études estiment que ce taux
augmentera de 3,5% par an et atteindra une valeur de 8,4 milliards $ en 2020 (Global Whey
Protein Market - Growth, Trends And Forecast (2015-2020), 2016). Au Canada, environ
372,000 tonnes de fromage sont produites chaque année, y compris 200,000 tonnes au
Québec, ce qui résulte à environ 2,100,000 tonnes de lactosérum (soit environ 1,200,000
tonnes au Québec) (Quebec Dairy Council Inc; Modified Milk Ingredients Fact sheet,
2015). Selon la méthode de fabrication et du type de fromage, deux principaux types de
lactosérum peuvent être obtenus : le lactosérum doux et le lactosérum acide. Le lactosérum
doux est obtenu par coagulation enzymatique du lait ou des caséines à un pH d'environ 6,5
tandis que le lactosérum acide (pH inférieur à 5) est obtenu par la coagulation du lait par
ajout d’acides forts (phosphorique, hydrochlorique, sulfurique, etc.) ou organiques
(lactique, citrique). Les principales différences entre les deux types de lactosérum se
situent au niveau de leur teneur en minéraux, leur acidité et leur teneur en fraction protéique
(Tableau2.1) (Yadav et al., 2015). Le lactosérum doux est moins minéralisé, car lors de la
production fromagère, plus de la moitié du phosphore et des cations divalents sont retenus
dans le caillé. Au contraire, lors d’une coagulation acide, la plupart des minéraux
colloïdaux (calcium, phosphate, citrate de calcium et magnésium) se solubilisent et se
retrouvent dans le lactosérum acide (Bozanic et al., 2014; St-Gelais et al., 2010). Comparé
au lactosérum doux, le lactosérum acide a une plus faible teneur en lactose en raison du
procédé de fabrication qui implique une fermentation avant coagulation au cours de
laquelle une partie du lactose est converti en acide lactique. Cependant, le lactosérum doux
a une fraction protéique plus élevée que le lactosérum acide.

3
Tableau 2.1 : Composition du lactosérum doux et du lactosérum acide
Constituantes Lactosérum doux (g/L) Lactosérum acide (g/L)
Solides totaux 63,0-70 63,0-70,0
Lactose 46,0-52,0 44,0-46,0
Protéines 6,0-10,0 6,0-8,0
Lipides 5,0 0,4
Lactate 2,0 6,4
Cendres 5,0 8,0
Calcium 0,4-0,6 1,2-1,6
Phosphates 1,0-3,0 2,0-4,5
Chlorures 1,1 1,1
Source: (Yadav et al., 2015)
Le lactosérum contient environ 6.5% de solides totaux constitués principalement de lactose,
de protéines, de minéraux, d’acide lactique et de matière grasse (Tsakali et al., 2010). En
raison de sa haute teneur en lactose, de la présence de protéines et de matière organique, le
lactosérum a une demande biologique en oxygène très élevée (DBO > 35,000 ppm)
(Smithers, 2015) et un caractère polluant. Le lactosérum a longtemps été éliminé dans des
installations de traitement de déchets ou épandu sur les champs pour l’enrichissement des
sols (Onwulata et al., 2008). Mais en raison de sa DBO élevée, des réglementations
environnementales strictes dans de nombreuses juridictions empêchent aujourd'hui son
élimination sans un traitement préalable (Smithers, 2015). De plus, grâce au développement
et l’utilisation des nouvelles technologies de transformation et de valorisation telles que les
procédés de concentration membranaire et séchage par atomisation, le lactosérum peut être
utilisé pour l'alimentation humaine.

2.2 Caractéristiques physico-chimiques des principaux constituants du

lactosérum

Dans le cadre de cette revue de littérature, l'accent sera mis sur les principales composantes
(lactose et protéines) du lactosérum qui lui confèrent des propriétés exceptionnelles et de
multiples utilisations dans les produits alimentaires.

4
2.2.1 Le lactose
Ce disaccharide est le sucre principal du lait et par conséquent du lactosérum. Il est formé
par l’union de deux monosaccharides (le D-glucose et le D-galactose) par un lien
glycosidique C1(β)-C4 (Amiot et al., 2010). Lors d’une fermentation, le lactose est le seul
glucide fermentescible et ce, sous la dépendance des bactéries lactiques (BL). Ces BL
possèdent une enzyme (β-galactosidase), qui est capable d’hydrolyser le lactose en glucose
et galactose. Ces deux monosaccharides sont par la suite transformés en acide lactique, ce
qui entraîne une baisse du pH. En fonction des BL utilisées, d’autres produits peuvent être
aussi obtenus dont l’acide acétique, l’éthanol et le gaz carbonique (Vuillemard, 2013).
Certains de ces métabolites jouent un rôle important au niveau de la saveur et de l’arôme
typiques des produits laitiers fermentés.

Sous forme purifiée et cristallisée, le lactose est utilisé dans de nombreux produits de
confiserie, de boulangerie, dans les formulations pour enfants, boissons, etc. De même, il
peut être utilisé pour diminuer la saveur sucrée de certains aliments. En industrie
pharmaceutique, il est utilisé comme transporteur ou support pour la fabrication des
comprimés (Huffman et al., 2011).

2.2.2 Les protéines de lactosérum (PL)

Les protéines du lactosérum représentent environ 17% de l’azote protéique du lait et sont
constituées essentiellement de la β-lactoglobuline (β-lg), l’α-lactalbumine (α-lac), les
immunoglobulines (Ig), la lactoferrine bovine (Lf), l'albumine sérique bovine (ABS) et la
lactoperoxydase (LP). Un traitement thermique de ces protéines en milieu acide favorise
leur précipitation (Vuillemard, 2013). En effet, la solubilité des PL est fortement influencée
par la chaleur (dénaturation de leur structure globulaire). Les immunoglobulines sont les
plus sensibles et sont dénaturées à une température de 70°C. Toutefois, si la dénaturation
est suivie par une rupture du pont disulfure, les molécules se déplieront et deviendront
insolubles. De plus, les PL ont une valeur nutritionnelle supérieure aux caséines, car elles
contiennent en proportion relativement importante les acides aminés essentiels tels que les
acides aminés soufrés, la lysine et le tryptophane (Vuillemard, 2013). Les différents
constituants des PL sont décrits ci-dessous :

5
➢ La β -lactoglobuline: La β–lg constitue environ 50% des protéines du lactosérum
(Vuillemard, 2013). Absente dans le lait humain, la β–lg est une protéine globulaire
contenant deux ponts disulfures et une cystéine libre conférant de bonnes propriétés
fonctionnelles aux PL (Jeewanthi et al., 2015). Cette protéine a été identifiée
comme l’une des principales causes d’allergie chez les nourrissons. Cette propriété
limite l’utilisation du lait de vache dans les préparations pour nourrissons (Lucena et
al., 2006). Toutefois, des traitements industriels, tels que la stérilisation, le
chauffage ou les hautes pressions hydrostatiques améliorent sa digestibilité et une
fermentation peut potentiellement réduire son allergénicité (Pescuma et al., 2008).
La β–lg possède de bonnes propriétés gélifiantes, démontre une bonne stabilité et
solubilité sur une large gamme de pH lors de traitements à haute température. Cette
protéine a une valeur nutritionnelle élevée comme en témoigne son profil d'acides
aminés essentiels. De même, cette protéine présente une excellente aptitude au
fouettage et peut être une alternative à l’albumine d'œuf (blanc d’œuf) dans
certaines applications alimentaires (Chatterton et al., 2006). Ces propriétés de la β-
lg ont facilité son utilisation comme agent actif dans diverses boissons protéiques
enrichies, telles que les jus de fruits et boissons pour sportifs (Chatterton et al.,
2006).

➢ L’α–lactalbumine : Elle constitue environ 20% des protéines de lactosérum et a

une structure globulaire en solution aqueuse. Elle présente une forte affinité pour
des ions métalliques particulièrement le calcium. En présence de quantités
saturantes de calcium, l’α–lac est thermostable tandis qu’en absence, elle devient
très instable (Chatterton et al., 2006). L’α–lac est une protéine riche en acides
aminés (cystéine et tryptophane) précurseurs de sérotonine et du glutathion,
respectivement. L’α-lac possède des caractéristiques antimicrobiennes, anti-
cancérigènes, joue un rôle important dans l'inhibition de l'enzyme de conversion de
l’angiotensine (IECA) et dans la gestion du stress (Chatterton et al., 2006;
Madureira et al., 2007).

➢ Les immunoglobulines: Les Ig représentent environ 11% des protéines de

lactosérum et, comme leur nom l'indique, confèrent une activité immunologique au

6
 lactosérum (Vuillemard, 2013). Présentes dans le lait maternel, les Ig confèrent aux
nourrissons « une immunité maternelle passive» qui aide leur système immunitaire
(Madureira et al., 2007; Shugarman, 2016). Elles possèdent des propriétés
antimicrobiennes, antivirales et, à l’état natif, jouent un rôle important dans la
modulation du système immunitaire du nourrisson.
➢ La lactoferrine bovine: La Lf représente moins de 1% des protéines du
lactosérum. Sa bonne capacité de chélation du fer, un élément essentiel pour la
croissance des micro-organismes, lui confère des propriétés antimicrobiennes et
antivirales (Farnaud et al., 2003). Plusieurs autres études ont mis l'accent sur
l'activité biologique de la Lf et son rôle dans la lutte contre le cancer, les toxines,
son soutien au système immunitaire, la cicatrisation des plaies, et l'activité anti-
inflammatoire (Madureira et al., 2007; Shugarman, 2016).
➢ L'albumine sérique bovine: L’ABS représente environ 7% des protéines du
lactosérum et est riche en acides aminés essentiels (Vuillemard, 2013). L’ABS se lie
facilement aux acides gras libres et d’autres matières grasses et, de ce fait contribue
aux propriétés émulsifiantes des protéines de lactosérum. Elle possède aussi des
propriétés antioxydantes et joue un rôle important dans l’inhibition de la croissance
des tumeurs dans les glandes mammaires (Madureira et al., 2007).
➢ La lactoperoxydase : La Lp représente moins de 1% des protéines de
lactosérum. Elle joue un rôle important dans la prévention des infections, en
agissant comme agent antimicrobien (Yadav et al., 2015). En fait, elle n’a pas
d’activité antimicrobienne en soi, mais, en présence de cofacteurs, elle devient un
puissant catalyseur pour le système défensif. Ces cofacteurs sont, d’une part le
peroxyde d’hydrogène (H2O2) et, d’autre part, un ion, qui selon le type d’enzyme
peut être l’ion thiocyanate (SCN-), l’ion chlorure (Cl-), l’ion bromure (Br-) ou l’ion
iodure (I-) (Perraudin, 1991). Cette, réaction peut se résumer comme suit :

H2O2 + X- H2O+ OX- (où X- = SCN-, Cl-, Br-, I-)

Comme rapporté par Perraudin (1991), le lait cru à une activité bactéricide contre
Bacillus thyphosa et Bacillus rathyphosa et cette activité est liée à l’activité des
oxydases et peroxydases présentes. Par exemple, en présence de H2O2 et SCN-, la

7
 Lp produit l’anion hypothiocyanate qui est bactéricide à de très faibles
concentrations (l’ordre de quelques µmol/l).

La figure 2.1 présente quelques produits qui peuvent être obtenus à partir du lactosérum et
de ses principaux constituants, les plus utilisés étant le lactosérum en poudre, les concentrés
protéiques et le lactose.

8
 Lactosérum

Concentration Conversion
Fractionnement

Lactosérum en
poudre Protéine Lactose Minéraux
Hydrolysé Altération
chimique
Ultrafiltration Précipitation Chromatographie Cristallisation Précipitation Chromatographie
/échange d’ions /échange d’ions
Dérivés de
Lactalbumine lactose
Minéraux Lactosérum
laitiers déminéralisé

Protéines Isolat
individuelles protéique de
lactosérum

Lactosérum Protéines
Perméat Protéines de
hydrolysé hydrolysées
lactosérum

Lactose Lactosérum Adapté de : Smith (2008)

réduit en
lactose

Concentré Isolat
protéique de protéique du Figure 2.1 : Ingrédients laitiers obtenus à partir de lactosérum liquide.
lactosérum lactosérum

9
2.3 Propriétés des protéines de lactosérum
2.3.1 Propriétés nutritionnelles
De nos jours, les PL sont largement utilisées comme ingrédients alimentaires en raison de
leur propriétés nutritionnelles (Morr et al., 1993). Chaque protéine est spécifique et unique
en raison de sa séquence spécifique d’acides aminés. Les acides aminés (A.A) sont
généralement classés en deux grands groupes : les A.A non essentiels (synthétisés par le
corps) et les A.A essentiels (fournis par l’alimentation). Les protéines qui contiennent ces
A.A en proportion similaire à la quantité requise par le corps humain ont une valeur
biologique (VB) élevée. Les protéines de l’œuf ont longtemps été considérées comme la
source de protéines ayant la plus haute valeur biologique. Toutefois, des études ont montré
que les protéines de lactosérum ont une VB qui dépasse d'environ 15% celle de la protéine
d'œuf (fig. 2.2A). Comparées à d’autres types de protéines alimentaires, les protéines de
lactosérum sont riches en A.A essentiels (figure 2.2B), et en A.A à chaîne ramifiée
(leucine, isoleucine et valine) (> 20% p/p) (Smithers, 2008). Les protéines de lactosérum
sont donc une excellente source de protéines pour les sportifs, pour les personnes ayant des
problèmes de santé et pour les personnes qui pratiquent le culturisme (Ha et al., 2003).

Figure 2.2: Valeur

biologique [A] et
composition en teneur en
A.A essentiels [B] de
différentes sources de
protéines (Smithers, 2008).

10
2.3.2 Propriétés santé
Plusieurs travaux confirment les bienfaits des protéines de lactosérum sur la santé
(Smithers, 2015).

• Les protéines et les composantes bioactives du lactosérum ont la capacité de

favoriser la synthèse et la reconstruction des tissus musculaires (Paddon-Jones et
al., 2009; Reidy et al., 2013; Tipton et al., 2004). Il a été démontré que les PL
possèdent également d’autres fonctions physiologiques telles que la modulation de
la multiplication des adipocytes, l’amélioration du système immunitaire ainsi
qu’une activité anti-oxydante (Ha et al., 2003; Zemel, 2013).
• Akhavan et al. (2013) ont rapporté que la consommation de PL abaisse la glycémie
après le repas par des mécanismes dépendants et indépendants de l'insuline.
• Une autre étude réalisée sur un groupe de 158 obèses soumis 12 semaines à un
régime hypocalorique (réduction de 500 cal/jour) a montré que le groupe
expérimental qui avait ingéré un supplément de 20 g de protéines du lactosérum par
jour a montré une perte de poids (2,81 kg) supérieure au groupe placebo sans
supplément (1,62 kg) (Frestedt, 2008). De plus, les sujets ayant ingéré le
supplément protéique ont perdu moins de masse musculaire (1,07 kg) que le groupe
témoin (2,41 kg), ce qui s’est traduit par un ratio kg de gras/kg de muscle perdu de
3,75 pour le groupe supplément protéique (79% du poids perdu était du gras) contre
1,05 pour le groupe témoin. Zafar et al. (2013) ont aussi démontré que
l’enrichissement d’une boisson en protéines de lactosérum permet de contrôler le
poids corporel en réduisant l’appétit, la prise alimentaire et de ce fait l’apport
énergétique. Luhovyy et al. (2007) et Poppitt (2011) ont aussi mis en évidence
l’effet des PL sur la satiété, l’obésité et la réduction de la prise alimentaire.
• Fluegel et al. (2010) ont montré dans une étude que des sujets humains hypertendus
consommant quotidiennement pendant 6 semaines une boisson de lactosérum
contenant 28 g de protéines ont subi une diminution de pression artérielle tandis
qu’aucune variation de pression artérielle n’a été observée chez le groupe de
pression artérielle normale.

11
2.3.3 Propriétés fonctionnelles

Outre les propriétés nutritionnelles, les protéines de lactosérum possèdent des propriétés
fonctionnelles exceptionnelles, principalement en raison de leur haut degré de solubilité,
d'absorption d’eau, de gélification, émulsifiantes et moussantes (Baldasso, 2011).

a) Solubilité : La solubilité est considérée comme la propriété clé pour plusieurs

ingrédients à base de protéines du lactosérum. Elles démontrent une excellente
solubilité sur une large gamme de pH (2 à 10) (Onwulata et al., 2008). La plupart
des systèmes alimentaires ayant des valeurs de pH dans la gamme de 3 à 7, les
ingrédients à base de protéines de lactosérum sont solubles dans tous les milieux et
la plupart des aliments (sauf ceux traités thermiquement) (Huffman et al., 2011).

b) Propriétés gélifiantes : La gélification est l’aptitude des protéines du lactosérum à

former un gel cohésif stable lorsque chauffé. Soumis à une température de
chauffage appropriée, les protéines de lactosérum forment deux types de gels :
filamenteux ou agrégé. Le type de gel dépend non seulement du traitement
thermique appliqué, mais aussi du pH et de la force ionique (Bertrand, 2008). En
conditions favorisant les répulsions électrostatiques, les gels filamenteux sont
formés et ces gels présentent de bonnes capacités de rétention d’eau tandis que les
gels agrégés possèdent une capacité de rétention d’eau inférieure (Bertrand, 2008).
La gélification des protéines de lactosérum soumises à un traitement thermique a
lieu généralement en deux ou trois étapes (figure 2.3): la dénaturation de la
structure globulaire des protéines natives, un dépliement partiel, suivie par une
agrégation des protéines dénaturées sous la forme d’une matrice gélifiée qui peut
lier de grandes quantités d'eau (Huffman et al., 2011). Cependant, un équilibre entre
les liaisons attractives et répulsives est nécessaire pour obtenir le gel désiré.

12
 Tiré de Bertrand (2008)
Figure 2.3 : Les différentes étapes de gélification de protéines du lactosérum.

c) Propriétés émulsifiantes : La capacité émulsifiante peut être définie comme la

quantité d'huile pouvant être émulsionnée par une certaine quantité de protéines
avant l'inversion de phase ou l'effondrement de l'émulsion. Les protéines de
lactosérum ont à la fois des régions hydrophobes et hydrophiles qui leur confèrent
de bonnes aptitudes d’émulsification. Les protéines de lactosérum sont capables de
diminuer la tension interfaciale entre les composants hydrophiles (eau) et
hydrophobes (huile) dans les aliments.
d) Propriétés moussantes et fouettantes : Le moussage est une propriété qui peut être
désirée pour certaines applications (crème glacée) et non pour d’autres (fortification
protéique des jus de fruits). Les propriétés moussantes peuvent être définies comme
la capacité d’une protéine à former une mousse stable par incorporation d’air. Les
différentes étapes de formation de la mousse sont : la dénaturation des protéines,
l’adsorption à l’interface air-eau, le piégeage de l’air, la réparation, le contact, la
stabilisation (Huffman et al., 2011). Un traitement thermique doux favorise ou
améliore l’aptitude des protéines du lactosérum à être fouettées. La présence de
matières grasses est particulièrement néfaste pour la formation de mousses en raison
de la perturbation de la tension de surface du film (Huffman et al., 2011). Les

13
 isolats de protéines de lactosérum démontrent les meilleures propriétés moussantes
en raison de leur teneur réduite en matière grasse.
e) Viscosité et capacité de liaison de l’eau : La faible viscosité des protéines de
lactosérum favorise leur utilisation à de hautes concentrations. Cependant, soumises
à un chauffage, leur viscosité et leur capacité de liée d’eau augmentent, et leur
solubilité diminue (Huffman et al., 2011).

2.4 Ingrédients laitiers obtenus à partir du lactosérum

➢ Le lactosérum en poudre est un produit laitier obtenu par séchage du lactosérum

frais. Selon le type de lactosérum, divers types de poudre de lactosérum peuvent
être obtenus.
➢ La poudre de lactosérum doux est obtenue après écrémage et séchage du
lactosérum frais issu de la production des fromages fabriqués par une coagulation de
type présure (cheddar, mozzarella, Suisse) (Canadian Dairy Commission; Milk
Ingredients, 2011). Sur la base de la matière sèche, sa composition est la même que
celle du lactosérum liquide et son pH en solution est supérieur à 5.6. Cette poudre
de lactosérum présente une saveur douce (Huffman et al., 2011) et constitue la
principale production canadienne (Canadian Dairy Commission; Milk Ingredients,
2011).
➢ La poudre de lactosérum acide est obtenue par séchage du lactosérum frais issu de
la fabrication de fromages non affinés fabriqués principalement par coagulation
acide, tels que le cottage et la ricotta. Sa composition ressemble à celle de la poudre
de lactosérum doux à l’exception de sa teneur en lactose inférieure (Tableau 2.1) et
est une source riche en calcium laitier (Huffman et al., 2011). Son indice d’acide
plus élevé lui confère un goût plus acide (pH en solution inférieur à 5.1) (Canadian
Dairy Commission; Milk Ingredients, 2011).
➢ La poudre de lactosérum déminéralisée est un produit dont la plupart des
minéraux a été éliminée de façon sélective (Canadian Dairy Commission; Milk
Ingredients, 2011).

14
 ➢ La poudre de lactosérum délactosée est un produit dont la majorité du
lactose a été retirée par cristallisation tout en conservant les autres constituants du
lactosérum.

Les différentes poudres de lactosérum sont utilisées dans plusieurs produits alimentaires
pour leurs propriétés fonctionnelles. Dans le cas des produits dont le profil et la
concentration en minéraux sont des attributs cruciaux (aliments pour bébés), l’utilisation du
lactosérum déminéralisé et délactosé est recommandée (Canadian Dairy Commission; Milk
Ingredients, 2011).

2.5 Ingrédients laitiers à haute teneur en protéines de lactosérum

➢ Les concentrés protéiques de lactosérum (CPL) sont des ingrédients laitiers

dérivés du lactosérum obtenus après élimination d’une partie des minéraux et du
lactose par diverses techniques, telles que l’ultrafiltration et la diafiltration, suivie
d’une évaporation et d’un séchage par atomisation (Siso, 1996). Généralement, la
concentration en protéines des CPL varie entre 35 et 80%. Les CPL sont utilisés
dans plusieurs produits alimentaires pour leur haute teneur protéique et leur richesse
en acides aminés essentiels. De plus, ces produits sont utilisés comme suppléments
dans des boissons nutritionnelles, des barres nutritionnelles et des boissons fortifiées
(Onwulata et al., 2008). Comparé au lactosérum natif qui a un goût salé, les CPL
ont une saveur douce, typique du lait et dégagent des arômes sucrés, cuits et laiteux
malgré le fait que dans certains cas, l’oxydation des lipides résiduels peut donner
lieu à des odeurs désagréables (Whetstine et al., 2005). Renner et al. (1991) ont
montré que les CPL sont typiquement exempts de pathogènes, de substances
toxiques, ont des propriétés antimicrobiennes et possèdent des propriétés
recherchées en industrie alimentaire telles que leur solubilité, leur bonne capacité
émulsifiante et leur capacité de former des complexes avec d’autres ingrédients.
➢ Les isolats protéiques de lactosérum (IPL) contiennent un minimum de 90% de
protéines (Huffman et al., 2011), ce qui en fait la source de protéines la plus pure
(Hoffman et al., 2004) et par conséquent très peu de lactose et de minéraux.

15
 ➢ Les hydrolysats protéiques du lactosérum (HPL) sont des protéines hydrolysées.
Le processus d’hydrolyse est très similaire au processus de digestion et génère des
peptides facilement absorbés. Ils sont principalement utilisés dans les suppléments
de culturisme (contribue à la synthèse des protéines musculaires) (Kanda et al.,
2014) et pour l’alimentation parentérale. Toutefois, un des inconvénients des
hydrolysats réside dans leur amertume qui augmente avec le degré d’hydrolyse.

2.6 Développement de boissons à base de lactosérum

Depuis la Grèce antique, le lactosérum a été consommé pour ses effets thérapeutiques et
bénéfiques dans la prévention de maladies comme l’arthrite, l’anémie et les problèmes de
foie (Prendergast, 1985). De nos jours, le lactosérum est essentiellement utilisé comme
ingrédient dans la formulation de divers produits alimentaires. Cependant, il existe
quelques produits à base de lactosérum.

2.6.1 Boissons et jus de fruits au lactosérum non fermentées

Des études ont mis en évidence que l’ajout de concentrés/jus de fruits au lactosérum
améliore significativement les défauts de goût salé et l’arôme désagréable du lactosérum
liquide. Le lactosérum acide est recommandé, car il est compatible avec la nature et la
saveur acide des fruits (Ozer et al., 2010). Ces boissons peuvent être des substituts aux jus
de fruits santé en raison d’attributs comme bonne source de vitamines (Jelen, 2010) ou de
minéraux. Quatre types de boissons à base de lactosérum additionné de concentrés de fruits
(d’orange, de poires, des pommes et des pêches), d’acide citrique et de sucrose ont été
développées par Djurić et al. (2004). Les meilleurs résultats sur le plan de la qualité et des
propriétés organoleptiques ont été obtenus avec les concentrés de pêche, et que ceci était
dépendant de l’interaction entre la matière sèche et le taux de sucrose du milieu (Djurić et
al., 2004). Ils ont aussi constaté que les boissons à base de poires avaient une tendance à
sédimenter. Pour surmonter le problème de sédimentation, une autre étude a démontré qu’il
était possible de stabiliser un mélange de lactosérum (chauffé) et de jus de poire par ajout
de sucre et de pectine hautement méthylée (Baccouche et al., 2013). Gad et al. (2013) ont
obtenu une boisson à base de lactosérum, mangue, huile de lin et pectine montrant très peu
de sédimentation, avec d’excellentes qualités organoleptiques, une faible viscosité et une
richesse en omega-3. Cependant, d’autres types de boissons, comme dans le cas de boissons

16
obtenues avec du lactosérum et du cassis (Ribes nigrum) ont montré des problèmes de goût
et d’odeur (Jaworska et al., 2011) et que le cassis n’était pas capable de masquer les saveurs
non désirées des boissons de lactosérum. Djurić et al. (2004) rapportent que, selon la
littérature, les saveurs d'orange et d'agrumes sont des concentrés / fruits à favoriser.

2.6.2 Boissons fermentées à base de lactosérum

Le kéfir est une boisson laitière acide, légèrement carbonatée avec un faible taux d’alcool
fermenté par une microflore de bactéries lactiques, acétiques et des levures qui sont
habituellement emprisonnées dans une matrice de polysaccharides et protéines appelée
«grain de kéfir» (Londero et al., 2012). Ces microorganismes sont capables de métaboliser
le lactose, et de ce fait peuvent être utilisés pour fermenter le lactosérum (Sabokbar et al.,
2016). Cette microflore est peu sensible à l’acidité des fruits et a la capacité de fermenter
les sucres des concentrés et jus de fruits. Sabokbar et al. (2014a) ont étudié la fermentation
d’une boisson à base de lactosérum et de concentré de jus de grenade par des grains de
kéfir. L’étude a montré que le mélange de jus de grenade et de lactosérum reconstitué fut
un bon substrat de fermentation pour les grains de kéfir et de ce fait a donné lieu à la
production d’une nouvelle boisson lactée contenant des probiotiques et du jus de fruits.
Deux températures de fermentation (19 ºC et 25 ºC) avec deux concentrations (5% et 8%
p/v) d'inoculum de grains de kéfir ont été testées. La diminution du pH, la production
d'acide lactique et d'acide acétique dépendaient de la température et de la concentration des
grains de kéfir utilisés. Les boissons inoculées à 5% p/v et fermentées à 25 ºC ont été
fortement appréciées en ce qui concerne l’odeur, la consistance et l’acceptabilité globale.
Le compte microbien des BL était significativement différent pour toutes les boissons qui
étaient à l’étude avec des comptes moyens d’environ 7.5 log CFU/mL. Toutefois, dans
toutes les boissons à l’étude, aucune différence significative dans les comptes en levures
(~5.3 log CFU/mL) n’a été observée. Des résultats sensoriels similaires ont été obtenus
lorsqu'un mélange de lactosérum et de jus de pomme a été fermenté avec des grains de kéfir
(Sabokbar et al., 2015). Ces études montrent la potentialité de développer et de masquer la
saveur du lactosérum en utilisant la fermentation par les grains de kéfir. Cependant, des
chercheurs ont également exploré d'autres possibilités de fermenter seulement le lactosérum
(sans mélange avec le jus de fruit) avec des grains de kéfir.

17
Magalhaes et al. (2011) ont étudié la production d’une nouvelle boisson fermentée en
utilisant du lactosérum comme substrat principal. Comme dans les études précédentes, les
grains de kéfir étaient capables d'utiliser le lactose (standardisé à 46g/L dans toutes les
boissons) comme substrat et de ce fait produisaient des quantités similaires d'éthanol
(12g/L), d'acide lactique (6g/L) et d'acide acétique (1,5g/L) que lors de la fermentation du
lait. Les composés volatils (des alcools et des esters éthyliques) responsables des bonnes
qualités sensorielles des boissons étaient les composés les plus dominants dans les
boissons, Aucune information n'a été fournie sur les concentrations d'inoculum utilisées ni
sur les comptes microbiens obtenus après fermentation. Néanmoins, les résultats de cette
étude ont montré que la fermentation du lactosérum donnait un profil sensoriel similaire à
celui des boissons lactées fermentées. Cela indique que de nouvelles boissons aux
propriétés organoleptiques acceptables peuvent être produites par la fermentation de
lactosérum par des grains de kéfir. Il est cependant important de noter que les grains de
kéfir sont constitués de bactéries lactiques. Il est dont probable qu'une fermentation lactique
permet d’obtenir des profils sensoriels acceptables.

2.6.2.1 Les bactéries lactiques (BL)

La plupart des bactéries utilisées pour la fermentation du lait sont appelées bactéries
lactiques (BL) dû au fait qu'elles produisent principalement de l'acide lactique par le
catabolisme du lactose (Cogan et al., 1996). Sous forme de coques ou de bâtonnets, ces
bactéries Gram-positif ne produisent pas de spores, sont habituellement non mobiles et ont
des besoins de croissance importants (Jespersen, 2004). Les BL peuvent être
homofementaires ou hétérofermentaires. Les homofementaires sont des bactéries qui
produisent, après fermentation, principalement de l’acide lactique (>90%) tandis que les BL
hétérofermentaires produisent une variété de métabolites. La conversion du lactose par les
BL hétérofermentaires produit environ 50% d’acide lactique et d’autres métabolites (acide
acétique, éthanol et CO2) (Jespersen, 2004; Widyastuti et al., 2014). Les BL peuvent
améliorer la digestion du lactose (Mater et al., 2005) et ont été jugées bénéfiques pour la
santé humaine et la nutrition (Deeth et al., 1981; EFSA, 2010). Les cultures commerciales
de BL peuvent être thermophiles ou mésophiles (Lamontagne et al., 2002) ou parfois
contenir des souches des deux types.

18
 Les principales BL mésophiles sont Lactococcus lactis subsp. cremoris (L.
cremoris), Lactococcus lactis subsp. lactis (L. lactis), Lactococcus lactis subsp. lactis
biovar. diacetylactis (L. diacetylactis), Leuconostoc mesenteroides subsp. cremoris, et
Leuconostoc lactis. De plus, L. diacetylactis, Leuconostoc mesenteroides subsp. cremoris et
Leuconostoc lactis peuvent cataboliser le citrate en CO2 et en diacétyle. En fromagerie, le
CO2 est responsable de la présence d’ouvertures dans les fromages et le diacétyle est
souvent responsable du goût caractéristique du beurre et des saveurs des produits laitiers
fermentés. Ces bactéries capables de produire le diacétyle sont appelées producteurs
d’arômes, par exemple L. lactis subsp. lactis (citrate+) (Jespersen, 2004).

Ce que le secteur laitier considère comme bactérie thermophile ne correspond pas à la

nomenclature classique en microbiologie qui considère les thermophiles comme étant des
cultures pouvant se multiplier à 55°C. Cette nomenclature sied tout de même bien le secteur
pour distinguer les souches et leurs applications potentielles. Les principales BL
thermophiles sont Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus
et lactis et Lactobacillus helveticus. Lors d’une fermentation, Streptococcus thermophilus,
Lb. delbrueckii subsp. bulgaricus et certains Lb. delbrueckii subsp. lactis catabolisent le
lactose et produisent du lactate et du galactose (Jespersen, 2004). D’autre part, Lb.
helveticus utilise le galactose produit comme source de carbone et de ce fait élimine le
galactose résiduel. Certaines souches de Lb. helveticus sont très protéolytiques et peuvent
affecter le goût et la texture comme dans le cas d’un fromage (Ardö et al., 1988). Les BL
thermophiles sont capables de produire de l'acétaldéhyde qui est la saveur caractéristique
du yogourt.

En Amérique du Nord, seuls les genres Lactobacillus, Lactococcus, Leuconostoc et

Streptococcus sont communément utilisés pour la fermentation des produits laitiers. Pour
être utilisé comme culture pour la production du yogourt, le ferment doit obligatoirement
être une culture mixte constituée de St. thermophilus et Lb. delbrueckii ssp. bulgaricus
(Nauth, 2004).

19
2.6.2.2 Les probiotiques

Les BL renferment plusieurs espèces considérées comme probiotiques. Ces derniers

sont définis comme des «microorganismes vivants qui, lorsqu'ils sont administrés en
quantité suffisante, confèrent un avantage pour la santé de l'hôte» (WHO et al., 2002). Les
aliments dits «fonctionnels» sont des produits démontrant des effets santé au-delà de leur
valeur nutritionnelle de base (Ozer et al., 2010). Dans les produits laitiers fonctionnels, les
probiotiques sont les composés bioactifs qui procurent aux produits laitiers fermentés leur
fonctionnalité. Les probiotiques sont de plus en plus connus pour leurs effets positifs sur la
santé et le bien-être. Pour en citer quelques-uns, les effets bénéfiques des probiotiques
sont :

➢ amélioration de la santé du tractus intestinal (Kasipathy, 2010; McFarland, 2007) ;

➢ amélioration du système immunitaire (Ravinder et al., 2013);
➢ traitement de la colite ulcéreuse, de la maladie de Crohn et de la pouchite ;
➢ réduction de l'infection à Helicobacter pylori ;
➢ soulagement du syndrome du côlon irritable et particulièrement chez les patients
présentant des symptômes prédominants de constipation (Floch, 2014; Kasipathy,
2010; Maulik, 2011; Ravinder et al., 2013) ;
➢ traiter et/ou prévenir l’obésité et l’inflammation (Mathieu, 2015);
➢ Assimilation du lactose (Schrezenmeir et al., 2001), etc.

Les effets santé procurés par les bactéries probiotiques sont dépendants de la souche et les
effets santé documentés pour une souche ne sont pas nécessairement applicables à d'autres
(Maulik, 2011).

L’activité protéolytique de Lb. helveticus est généralement plus élevée que les autres
bactéries lactiques (Yamamoto et al., 1999), ce qui est un avantage pour la fermentation du
lait. Elle fermente le lactose et tous ses produits de dégradation (glucose, galactose) alors
que la plupart des souches de St. thermophilus n’ont pas la capacité de fermenter le
galactose (Roy et al., 1986). Au cours des 20 dernières années, l'utilisation de Lb.
helveticus est devenue plus importante en raison de sa capacité de produire des composés
bioactifs pendant la fermentation (Griffiths et al., 2013). La souche Lb helveticus R0052 a

20
le potentiel d'inhiber la croissance des pathogènes et possède des propriétés pouvant
renforcer le système immunitaire (Foster et al., 2011).

Bien que très peu d'information soit connue quant à l'utilisation de Lb. rhamnosus
en industrie alimentaire, les souches, GG et R0011 ont des propriétés probiotiques
(Champagne et al., 2008). En outre, Lb. rhamnosus R0011 est producteur
d'exopolysaccharides (Champagne et al., 2008) qui ont des effets bénéfiques sur la texture
et les rendements. Comme la souche R0052, la souche R0011 inhibe les pathogènes et
améliore le système immunitaire (Foster et al., 2011).

Les produits laitiers sont de bons vecteurs de probiotiques pour les humains (Champagne et
al., 2005). Pour obtenir des effets thérapeutiques ou des effets santé, il a été suggéré que la
portion déterminée du produit doit contenir au moins 1,0 x109 UFC de l’un ou plusieurs des
microorganismes admissibles faisant l’objet de l’allégation (Agence Canadienne
d'Inspection des Aliments: Allégations relatives aux probiotiques, 2016) et que cette
concentration soit présente à la fin du cycle de vie du produit. Dans une étude réalisée par
Shah (2000b), il a été démontré que la plupart de boissons porteuses d'allégations relatives
à la présence de probiotiques avaient en réalité des concentrations cellulaires très faibles au
moment de la vente. Pendant la fabrication et l’entreposage des produits fermentés, des
facteurs tels que l’acidité, l’oxygène dissous et le potentiel redox peuvent affecter la
croissance et la survie des probiotiques (Lankaputhra et al., 1996). Afin d’obtenir le compte
viable requis en fin de vie du produit, il faut tenir compte de la mortalité qui aura lieu lors
de l’entreposage.

Pour que les probiotiques se développent dans les produits laitiers qui contiennent
déjà des cultures lactiques, l'antibiose doit être évitée et, idéalement, une symbiose doit
s’établir entre le ferment lactique et les probiotiques. Ainsi, il faut prendre soin de choisir
des souches probiotiques compatibles avec les cultures lactiques (Champagne et al., 2005;
Tamime et al., 2007). Comme rapporté par McComas et al. (2003), les probiotiques
croissent lentement par rapport aux cultures lactiques dans un milieu laitier. Il est donc
nécessaire d’utiliser des moyens qui permettront d’augmenter et maintenir des
concentrations élevées de probiotiques.

21
2.6.2.3 Amélioration de la croissance des probiotiques

Puisque le lait ne contient que peu de peptides ou d’aminés libres, les probiotiques doivent
hydrolyser les protéines du lait. Pour des raisons technologiques, dans certains milieux
contenant des probiotiques, l’addition de cultures de yogourt peut diminuer le temps de
fermentation (Dave et al., 1998). Dans une culture de yogourt, Lb. delbrueckii ssp.
bulgaricus est la souche qui initie la protéolyse et, de ce fait, génère des peptides et acides
aminés essentiels qui sont utilisés par St. thermophilus. Cependant, lors de l’entreposage,
Lb. delbrueckii ssp. bulgaricus produit de l'acide lactique qui est responsable de la post-
acidification. Pour pallier à ce problème, la tendance actuelle est d'utiliser des cultures de
yogourt contenant des quantités négligeables de la souche Lb. delbrueckii ssp. bulgaricus.
Pour une telle culture, l'incorporation de micronutriments (peptides et acides aminés) peut
s’avérer bénéfique. Ainsi, une approche pouvant être utilisée pour promouvoir la croissance
des bactéries probiotiques dans du lait ou du lactosérum est l'utilisation de suppléments
(Gaudreau et al., 2005).

➢ Ajout de facteurs de croissances ou de suppléments

L’extrait de levure est le supplément le plus commun et le plus efficace pouvant augmenter
la croissance bactérienne (Champagne et al., 1999). Aeschlimann et al. (1990) ont étudié
l’effet de facteurs de croissance (extrait de levure, hydrolysat de caséines, CPL, liqueur de
maïs et malt) sur la croissance et la capacité productrice d’acide lactique par Lb. helveticus
dans du perméat. Ils ont obtenu les meilleurs résultats lorsque les milieux ont été
supplémentés en extrait de levure à une concentration de 10 g/L. Dans une autre étude, le
glucose, l'extrait de levure et les fractions de protéines du lait ont également amélioré la
croissance des probiotiques, suggérant que ceci pourrait être un moyen par lequel les
probiotiques pourraient être utilisés seuls dans les produits laitiers fermentés (Saxelin et al.,
1999).

Outre les peptides et acides aminés, les extraits de levure renferment plusieurs autres
ingrédients pouvant stimuler la croissance des probiotiques : nucléosides, nucléotides,
vitamines, acides gras libres. Ces ingrédients sont parfois requis dans les milieux destinés à
cultiver les lactobacilles (Elli et al., 1999; Morishita et al., 1981). Cependant, l’utilisation

22
de l’extrait de levure et d’autres substances afin de favoriser la croissance des probiotiques
présente certains inconvénients : odeurs désagréables, coût et variabilité dans la stimulation
de la croissance bactérienne. Ces inconvénients les rendent peu appropriés pour une
production industrielle dans les produits laitiers (Elli et al., 1999). Cependant, il existe
d’autres substances de grade alimentaire d’origine laitière (odeur typique des produits
laitiers) pouvant remplacer l’extrait de levure et les peptones comme facteurs de croissance.

Afin de stimuler la croissance des probiotiques, les sources d'azote provenant des protéines
du lait ou du lactosérum sont une alternative (Dave et al., 1998). Amrane et al. (1993) ont
obtenu une production d'acide lactique maximale par Lb. helveticus et suggèrent que des
hydrolysats enzymatiques de protéines de lactosérum peuvent être considérés comme
sources d'azote pour stimuler la croissance. Lucas et al. (2004), ont montré que l’ajout
d’hydrolysats au lait contenant des probiotiques pouvait avoir un effet positif ou négatif,

D’autre part, Partanen et al. (2001) ont démontré que l'acide oléique peut
efficacement remplacer le Tween 80 du milieu MRS, ce qui suggère que les acides gras
insaturés ou des huiles contenant de faibles quantités d'acides gras saturés à longue chaîne
sont aussi des bons facteurs de croissance.

Plusieurs probiotiques sont sensibles à l’oxygène (Talwalkar et al., 2004b), ce qui a

incité des chercheurs à étudier les antioxydants comme suppléments. Dave et al. (1998) ont
montré que l'ajout de cystéine, de CPL, d’hydrolysats de caséine acide ou de tryptone
améliore la viabilité des bifidobactéries. Dans le cas de la poudre de lactosérum il n’y avait
aucune amélioration de la viabilité.

➢ Fermentation en contrôle externe de pH (CEpH)

Le CEpH est une méthode communément utilisée pour la production industrielle des
ferments. Le pH est maintenu à une valeur optimale en ajoutant continuellement de
l'hydroxyde de sodium ou de l'ammoniac dans le bioréacteur (Rault et al., 2009). Le CEpH
permet une croissance prolongée des bactéries lactiques et favorise l’obtention de
populations microbiennes élevées (Champagne et al., 1995), ce qui permet d'utiliser ces
dernières à des taux d'inoculation significativement inférieurs lors d’une fermentation

23
(Savoie et al., 2007). Bien que le lait soit encore le substrat préféré pour la préparation de
ces cultures, du lactosérum commercial ou des produits à base de lait sont fréquemment
utilisés dans les fromageries (Champagne et al., 1995; Lamontagne et al., 2002).
Champagne et al. (1996) ont utilisé avec succès un milieu à base de WPC pour la
production de ferments lactiques mésophiles et thermophiles sous CEpH.

Dans la littérature, aucune étude ne rapporte la possibilité d'utiliser cette technique pour
augmenter la population de bactéries lactiques probiotiques dans des boissons fermentées.

2.6.3 Boissons au lactosérum fermentée par des bactéries lactiques

Suite aux résultats de la plupart des études qui ont mis en évidence les possibilités de
développer de nouvelles boissons par la fermentation de lactosérum à l'aide de grains de
kéfir, d'autres chercheurs ont utilisé des bactéries lactiques et probiotiques. Pescuma et al.
(2008) ont utilisé trois souches de bactéries lactiques pour développer une boisson
fermentée au lactosérum. La culture mixte à l’étude a été capable de diminuer le taux de
lactose des boissons, de dégrader la β-lactoglobuline en augmentant sa digestibilité ainsi
que la teneur en AA libres. De même, ils ont rapporté que les qualités organoleptiques de
cette boisson ont été nettement accrues. Legarová et al. (2010) ont tenté d’améliorer
l’arôme du lactosérum via une fermentation lactique (ferment de yoghurt), et par l’ajout de
25% et 50% de lait écrémé au lactosérum. La fermentation n’a pas pu améliorer de façon
significative les propriétés organoleptiques des boissons. Le facteur le plus important
influençant non seulement la qualité sensorielle globale des boissons lactées, mais aussi
l’arôme, l’aspect, la couleur, la viscosité et l’homogénéité était l’addition de lait. Bulatovic
et al. (2014b) ont utilisé du lactosérum avec 30% de lait écrémé, fermenté avec un ferment
pour yogourt et ajouté une souche probiotique. Les résultats obtenus ont montré que les
boissons présentaient des textures et des attributs sensoriels souhaitables et semblables aux
boissons laitières traditionnelles. De plus, les boissons contenaient les concentrations
cellulaires recommandées en bactéries probiotiques, possédaient des propriétés
antioxydantes et une durée de conservation d'au moins 20 jours.

24
➢ Concentrés protéiques de lactosérum (CPL et IPL) fermentés

Bien que largement utilisés comme ingrédients laitiers pour leurs caractéristiques
fonctionnelles (USDA, 2015), les CPL et IPL ont fait l’objet de peu d’études de
fermentations. Leur pouvoir tampon élevé peut favoriser la croissance des cultures lactiques
(Savoie et al., 2007). Akalin et al. (2007) ont montré que la supplémentation de laits
fermentés en CPL avait un effet positif sur la survie des probiotiques, le pouvoir tampon
des protéines sériques empêchant la post-acidification pendant l’entreposage. Pescuma et
al. (2010) ont également montré que, comparé au lactosérum, la fermentation utilisant un
CPL à 35% de protéines a été significativement améliorée. Ils ont obtenu un produit
fermenté avec une faible teneur en lactose et β-lg et une concentration élevée en acides
aminés essentiels. Après 12h de fermentation du CPL, ce dernier a été mélangé avec du jus
de pêche. Selon cette étude, le jus de pêche était ajouté pour masquer la saveur désagréable
du CPL. Par rapport au lactosérum, l'utilisation de CPL a amélioré la croissance cellulaire
(1,5 à 1,9 fois) et l'activité protéolytique des souches de lactobacilles (5 et 8 fois pour Lb.
delbrueckii ssp bulgaricus CRL 656 et Lb. acidophilus CRL 636, respectivement). La
quantité résiduelle de lactose (4 g/L) était similaire à celle des yogourts commerciaux, seuil
auquel le lactose est facilement digéré (Vesa et al., 2000). Avec le CPL, 41 -85% de la β-lg
a été hydrolysé à 37°C après 12h d’incubation (Pescuma et al., 2010) tandis qu’une
hydrolyse de 9% a été obtenue lorsque du lactosérum en poudre a été utilisé et ceci après
24h d’incubation (Pescuma et al., 2008). Les CPL semblent un substrat approprié pour la
production industrielle de boissons fermentées. Néanmoins, les analyses sensorielles de ces
boissons n’ont pas été effectuées, ce qui est une faiblesse de l’étude. De plus, seul un CPL à
35% de protéines a été étudié. Cho et al. (2015) ont produit une boisson à haute teneur en
protéines (9%) fermentée par des bactéries lactiques d'origine végétale à partir d’un CPL à
80%, de lait écrémé, de saccharose. Les attributs sensoriels de la boisson de lactosérum
fermentée étaient semblables à ceux des boissons protéiques commerciales, à l'exception de
l'acidité. Les comptes bactériens étaient élevés (109 UFC/mL) et stables.

Ces deux études ont montré la possibilité de développer des boissons à base de CPL. De
plus, les saveurs désagréables souvent attribuées au lactose et aux minéraux (en quantité
abondantes dans le lactosérum) (Morr et al., 1991; Whetstine et al., 2005) sont réduites

25
dans les concentrés protéiques de lactosérum suite au procédé de filtration sur membrane et
peuvent être masquées par les arômes produits pendant la fermentation. Cependant, il
n’existe aucun produit fermenté à base de CPL sur le marché.

L’objectif de ce projet est de développer une nouvelle boisson fermentée à base de CPL ou
d’IPL aux propriétés organoleptiques acceptables. La boisson ciblée aura un caractère
santé : riche en protéines et en probiotiques, sans sucre, ni agent épaississant, ni agent de
conservation, ni gras, ni colorant.

26
Chapitre 3 : Hypothèse et objectifs

Hypothèse

La fermentation de concentrés et d’isolats protéiques de lactosérum permet d’obtenir une

boisson fermentée riche en protéines du lactosérum et aux propriétés organoleptiques
acceptables.

Objectifs

• Sélectionner les concentrés ou isolats protéiques de lactosérum qui permettront une

vitesse d’acidification élevée;
• Sélectionner les ferments lactiques industriels qui acidifient rapidement et
conduisent à un produit fermenté de bonne saveur;
• Produire des boissons fermentées à base de CPL ou d’IPL ayant une teneur élevée
en protéines et en probiotiques.

27
Chapter 4: Selection of starter cultures and whey protein
sources for the production of a high protein whey
beverage

Germaine Enyoh Forkwa1, Claude P. Champagne1, 2*, Marie-Josée Lemay2,

Jean-Christophe Vuillemard1.

1
Institute of Nutrition and Functional Foods, Université Laval, Québec, QC, Canada
G1V 0A6
2
Saint-Hyacinthe Research and Development Centre, Agriculture and Agri-food Canada,
3600 Casavant Blvd., St. Hyacinthe, Quebec Canada J2S8E3.

* Corresponding author: claude.champagne@agr.gc.ca

28
Résumé
L'objectif de cette étude était de fabriquer des boissons à base de lactosérum par
fermentation de concentrés/isolats de protéines de lactosérum (CPL/IPL). L’objectif était de
sélectionner des cultures mésophiles (fromage frais) ou thermophiles (yogourt) aptes à
fermenter et améliorer les propriétés organoleptiques des CPL/IPL testés.

Douze CPL/IPL contenant 10% de protéines ont été fermentés. Les modèles de
fermentation mésophile et l'analyse sensorielle des boissons fermentées ont conduit à la
sélection d'un CPL. Ce CPL a ensuite été fermenté avec sept ferments commerciaux de
yogourts pour évaluer leur cinétique de fermentation ainsi que pour générer des produits
ayant de bonnes propriétés sensorielles.

Lors des fermentations mésophiles, un temps moyen de fermentation de 24 heures était

nécessaire pour que les solutions atteignent un pH d'environ 4,6-4,8. Des odeurs non
désirées comme le carton et les céréales ont été détectées dans la plupart des solutions
fermentées. La vitesse d'acidification (6h) du CPL sélectionné a été significativement
améliorée lors de la fermentation par les cultures thermophiles. Les souches de cultures
thermophiles ont influencé positivement la perception sensorielle des odeurs de type carton
et yogourt. Cette étude a permis la sélection d'une culture thermophile et d'un CPL qui
procurent de bons attributs sensoriels à une solution ayant 10% de protéines de lactosérum.
Ces résultats montrent la possibilité de développer un nouveau produit qui pourrait
répondre à plusieurs tendances du marché dans le secteur des aliments fonctionnels et de la
santé.

29
Abstract
The objective of this study was to manufacture high protein whey-based fermented
beverages by using whey protein concentrates/isolates (WPCs / WPIs) and commercial
starter cultures. The challenge was to find mesophilic (fresh cheese) or thermophilic
(yoghurt) cultures able to carry out the lactic acid fermentation and avoid sensory defects
associated with WPCs/WPIs.

Twelve WPCs/WPIs were fermented. The mesophilic fermentation patterns and sensory
analysis of fermented beverages led to the selection of one WPC. This WPC was then
fermented with seven commercial yoghurt starter cultures, for their suitability to carry out
the fermentation as well as to generate products having good sensory properties.

The rehydrated whey-based solutions differed in their lactose content and buffering
capacities. When fermented with mesophilic starter cultures, an average fermentation time
of 24 hours was needed to attain a pH of about 4.6-4.8. Off-odors like cardboard and
cereals were detected by a sensory panel in most of the fermented whey-based solutions.
The acidification rate of the selected WPC product was significantly improved when
fermented with thermophilic cultures. Moreover, the strains of thermophilic cultures
influenced positively the sensory perception of cardboard-like and yoghurt-like odors. This
process enabled the selection of a fermented WPC product (10% protein) with a
thermophilic culture that enables good sensory attributes. The results show the possibility
of developing a new product which could respond to several market trends in the functional
food and health sector.

30
4.1 Introduction
For many years, the transformation of milk into cheese has been associated with the
problem of disposal of whey, its major co-product. In 2015, Canada produced about
372,000 tons of cheese (200,000 tons from Quebec) which resulted to 2,100,000 tons of
whey (1,200,000 tons in Quebec) (Quebec Dairy Council Inc; Modified Milk Ingredients
Fact sheet, 2015). To address the significant environmental and health issues associated to
the large volumes of this by-product which has high organic content, researchers have
advanced the technological know-how of whey processing. Whey is now recognized as a
value-added ingredient because of its highly functional and nutritional properties
(Jeewanthi et al., 2015). Indeed, health benefits of whey protein are increasingly
recognized, particularly with respect to obesity (Miller et al., 2014) and cardiovascular
diseases (Pal et al., 2013). As a result, dried whey and derived dried whey products have
become very important ingredients in a wide range of food applications such as infant
formulas, health foods and drinks, gel products and frozen foods (Fuente et al., 2002).
Whey proteins have particularly been of commercial interest (Lagrange et al., 2015).

Unfortunately, dried whey-based products may bring off-flavors (Whetstine et al., 2005). It
has been suggested that these off-flavors could partially be attributed to the fact that during
production of WPC or WPI, liquid whey from different cheese are being pooled before
processing. As a result, the final pool of liquid whey will certainly have a distinct flavor
which does not characterize any specific cheese type (Park et al., 2016). Furthermore,
during cheese production, proteolytic enzymes such as chymosin may be carried over into
liquid whey. This increases levels of low molecular weight peptides and free amino acids,
which can lead to undesirable flavor formation (Holmes et al., 1977) as cited in Whetstine
et al. (2005). Also, other studies have confirmed that lipid oxidation is another source of
off-flavours in both liquid and dried whey (Park et al., 2016; Whetstine et al., 2005).
Therefore, the use of whey products in foods or beverages requires sensory studies to assess
potential off-flavors.

31
WPCs and WPIs are typically used in non-fermented foods (Lagrange et al., 2015).
However, in the search for new applications, an interesting alternative is to transform
whey-based substrates into value-added products by microbial fermentation and addition of
probiotic bacteria. The fermentation of WPCs and WPIs with commercial starter cultures or
probiotic strains could be alternatives to overcome off-flavors while aromatic compounds
released by the starters could improve the sensory qualities of the functional drinks
(Bulatovic et al., 2014b; Pescuma et al., 2015). Our hypotheses were that 1) WPCs and
WPIs have reduced levels of minerals and low molecular weight compounds which could
influence flavor than would native whey and 2) starters may contribute desirable flavors
and texture (Almeida et al., 2009; Varghese et al., 2013). The key in obtaining desirable
characteristics of health-promoting beverages depends on choosing the appropriate
commercial starter strains (Marsh et al., 2014). However, very little information was
obtained in literature regarding the fermentation of rehydrated WPCs or WPIs with cheese
or yoghurt starter cultures for the production of a functional whey drink (whey-ghurt drink).
Furthermore, there exists no scientific data on fermented, high-protein whey based dairy
product. Of particular concern are the high purity levels of WPIs, which might not contain
enough lactose or other nutrients for growth of the lactic cultures. Therefore, it is unknown
to what extent the advantage of using WPIs and WPCs for high-protein drinks to avoid
sensory problems could result in their inability in supporting a lactic fermentation.

The aim of our study was to formulate a functional fermented whey beverage with high
protein content (10%) solely from rehydrated WPIs and WPCs. The ability of 14 whey
derived powders to support the fermentation of 7 commercial lactic starter cultures was
assessed. In addition to fermentation patterns, the goal was to assess sensory properties of
the fermented drinks in order to obtain consumer acceptability.

32
4.2 Materials and Methods
4.2.1 Whey powders
Fourteen commercial whey powders were graciously provided by the following
manufacturers: Agropur (Longueuil, QC, Canada), Arla Foods Ingredients Inc. (Viby,
Denmark), Davisco (Minneapolis, MN, USA), Fonterra (NZNP, New-Zealand and
Mississauga, ON, Canada) and Hilmar Ingredients (Hilmar, CA, USA). Agropur provided:
Isochill 800, CPL 34% 25142, skim milk powder (SMP), whey powder, and permeate
powder; Arla provided: YO-8075 and YO-5088; Davisco provided: WPC 80% and WPC
HF 34%; Fonterra provided: WPC 392, WPC 480 and WPI 895 while Hilmar provided
WPI 19420, WPI 19000, WPC 8200 and WPC 8610. Amongst these powders were: whey
protein concentrates (protein content 35% - 80%) (WPC), whey protein isolates (protein
content >80%) (WPI), whey powders (< 20% protein content), whey permeate and skim
milk powder. Their composition is shown in Table 4.1 but, in order to avoid any
commercial prejudice to the suppliers who kindly provided us with these products, they
were attributed arbitrary codes which will be used throughout this work.

Table 4.1: Main composition of WPC and WPI powders

Product type Code % lactose % protein

WPC YA 1.5 77.9
WPC YB 33.3 50.8
WPI WA 0.1 90.0
WPI WB 1.0 88.0
WPC WC 5.2 76.9
WPC WD 4.5 79.1
WPC AB 6.0 75.0
WPC AC 56.7 34.9
WPC DA 7.4 76.9
WPC DB 43.7 33.8
WPC FA 4.13 74.9
WPC FB 4.63 78.0
Controls Whey 54.9 12.8
Whey permeate 88.1 2.4
SMP 50 36.2

33
4.2.2 Rehydration of whey and derived whey powders
A minimum quantity of 120 g of solution was required to ensure constant monitoring of pH
in the fermentation system used in this study. With respect to the protein content of each
powder after analysis, required quantities of powders were calculated with the aim of
obtaining solutions at 10% protein (w/w) content. However, skim milk powder (SMP),
whey powder and whey permeate were reconstituted to their typical total solid contents
(9%, 6% and 4.9% respectively). All powders were rehydrated in sterile de-ionized water
and solutions were stirred using a magnetic bar stirrer for an hour to ensure dissolution. The
pH values of all solutions was then adjusted to 6.0 using an Accumet excel XL15 pH meter,
Fisher Scientific. Subsequently, the solutions were stored for 24 hours at 4°C and then
pasteurized in a water bath at 63°C for 30 minutes. They were then cooled in an ice bath to
22°C for fermentations with the mesophilic starter (cheese) or to 40°C for fermentations by
the thermophilic starter (yoghurt).

Unless otherwise stated, the WPCs and WPIs were not supplemented. However, for some
assays, the lactose content was increased so that all solutions would have at least 1.5g/100g
lactose.

4.2.3 Determination of the buffering capacity (BC)

The BC of rehydrated solutions was assessed by determining the quantity of acid (0.1N
HCl) required to reduce the pH of the medium from pH 6.5 to pH 4.0. The titration was
carried out using a TIM865 Radiometer (Villeurbanne, France) connected to a computer for
reading and monitoring of sample pH /acid volume. Ten (10) ml of rehydrated powder
(10% protein content for WPI and WPC products) was mixed with 15 ml of de-ionized
water for sample preparation. Under constant stirring, the addition of acid to samples was
automatically stopped when a pH of 4.0 was reached. The total volume of acid added is the
amount of lactic acid required to reach the pH 4. The titration allowed an estimation of the
quantity of lactic acid that would be required to carry out the acidification by using the
following equations.

Eq 1: Mole of acid needed to drop from pH 6.5 to pH 4.0 =

[(mL of HCl titrant) X (0.1 M / 1000 mL HCl)]

34
Eq 2: Concentration of lactic acid that would be obtained (g/L) =

[(Mole of acid needed) X (90.1 g lactic acid / Mole)] / [sample volume in L]

4.2.4 Lactose analysis

Samples were prepared by adding TCA and sodium azide at a final concentration of 3.6%
and 0.015%, respectively. The blends were well mixed and incubated at 37°C overnight.
The samples were then centrifuged (Centrifuge Beckman, Rotor JLA 10.5) at 10000 g for
20 minutes at 37°C. The supernatants were incubated for 2 hours at 37°C. A second
centrifugation was carried in the same conditions. Supernatants for all samples were passed
through C18 Sep Pak column (Waters Corporation, Milford, Massachusetts, USA)
preconditioned with 5 mL of methanol and 5 mL of water and filtered on 0.45 µm Millipore
filter into HPLC vials for analysis.

Lactose, galactose, glucose and organic acids (lactic, acetic, and formic) were
analyzed by high performance liquid chromatography (HPLC) on an ICE-Ion 300
(Transgenomic, Omaha, NE, USA) separation column kept at temperature of 80°C with a
mobile phase of H2SO4 0.02 N at 0.4 mL/min and a 20 µL injection sample volume. The
HPLC system was composed of a Waters 1525 pump (Milford, MA, USA), Waters 717-
plus auto sampler Waters 2414 RI detector at 40°C and a Waters 2998 PDA detector at a
wavelength of 600 nm.

4.2.5 Bacterial strains and cultures

Lactic acid bacteria (LAB) used in this study was of two types: mesophilic cultures and
thermophilic cultures.

Two mesophilic freeze-dried starter cultures were tested; “Biena type B” (Biena, St-
Hyacinthe, QC, Canada) and “DEM3” (Centro Sperimentale del Latte, Milan, Italy).
Inocula were prepared by adding the concentration recommended by manufacturers in
media adjusted at 22°C. The two mesophilic starters contained strains of the following
species: Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus
lactis subsp. lactis biovar diacetylactis and Leuconostoc mesenteroides subsp. cremoris.

Seven commercial thermophilic starter cultures were also tested; “YO-MixTM 860”
was a frozen concentrate maintained at -80°C, “YO-Mix T11” and “YO-Mix T12” were

35
freeze dried. The YO-mix cultures were provided by DuPont Nutrition and Health
(Mississauga, Ontario, Canada). “Biena type 1” and “Biena type 2” were also freeze dried
cultures (Biena, St-Hyacinthe, QC, Canada). “Yogourmet” (Lyosan. Lachute QC, Canada)
was a commercial culture bought at the grocery store. “Rosell” was a starter culture
provided by Lallemand Health Solutions (Montréal, QC, Canada). For the freeze-dried
cultures, inocula were rehydrated in media adjusted at 40°C at concentrations that follow
manufacturers’ directives. The frozen starter was thawed by placing frozen pellets in sterile
vials at 22°C and the appropriate volume of the culture was pipetted into reconstituted
whey powder. 1ml of the latter was then used to inoculate 100ml of solutions to be
fermented in order to obtain 1 x107 CFU/ml. For YO-Mix T11, a direct inoculation at
0.01% was used.

As for the whey-based powders, in order to avoid any commercial prejudice to

companies who kindly provided us with these cultures, arbitrary codifications (F1 to F7)
were attributed to thermophilic cultures while M1 and M2 were used for mesophilic
cultures.

4.2.6 Fermentation
Fermentations were carried out in an in-house fermentation and acquisition control system
(FACS) composed of a computerized pH and temperature data acquisition system,
connected to 8 incubator jars of 250 mL which could be used simultaneously. The jars were
placed in a Forma Scientific (Ramsey, USA) programmable temperature incubator having 8
magnetic agitation units (figure 4.1). Rehydrated powders were pasteurized (63°C for 30
min), cooled to the incubation temperature (22 or 40°C) and inoculated. Mesophilic
cultures were incubated at 22°C for 24 hours while those inoculated with thermophilic
cultures were incubated at 40°C for 6 hours. In order to correctly follow the pH, the
solutions were constantly stirred at 100 RPM throughout fermentation with magnetic bars.
After fermentation, samples were cooled in an ice bath to about 4°C and then refrigerated
for viable counts or sensory analysis. The temperature and acidification data acquired by
the system were then exported to Excel spreadsheets and then analyzed using the Sigma
Plot (Systat Software, San Jose, CA, USA) program.

36
 A: Complete system with incubator B: Display of temperature and pH readings

C: electrodes in product for pH follow-up D: Screen showing pH follow-up

Figure 4.1: System used to carry out fermentation and to continuously monitor pH.

4.2.7 Sensory analysis of fermented solutions

The fermented beverages were not tasted but rather characterized by odor. A group of 6
judges met during 6 sessions to evaluate 6 different batches of fermented products. Thus,
data are the average of 6 independent trials. The judges were employees of the Food,
Research and Development Centre of St-Hyacinthe with training in describing dairy
flavors. A first sequence of analysis was carried out for samples fermented with mesophilic
starter cultures.

Judges were asked to characterize the products for 5 parameters (Table 4.2). The scale used
to qualify the intensity of flavors had 4 levels: very mild, mild, moderate and high. To
avoid judges being able to identify products on the basis of their color, they were placed in
amber bottles which were covered with perforated aluminum foil and the evaluation was
done under red light. All samples were coded with a 3-digit randomized number. The

37
samples were conditioned at 4°C and placed in styrofoam cubes for temperature
maintenance during the evaluation period.

Table 4.2: References for descriptive analysis of fermented whey protein beverages

Term Definition

Sweet yoghurt/cheese aroma Aroma associated with fermented dairy products

Cooked Aroma associated with cooked milk

Cardboard-like/ wet paper Aroma associated with wet cardboard paper

Sour, off-flavors Aroma associated with cattle or spoiled milk

Cereal-like Aroma associated with soy or other cereals

Adapted from Whetstine et al. (2005)

In combination with data from the fermentation assays (data presented in Section 4.3.2), the
first series of WPI and WPCs aroma analysis with mesophilic cultures enabled the selection
of 7 of the 12 WPC and WPI products (excluding controls). These were subsequently
characterized after fermentation with thermophilic cultures. Judges were asked to
characterise products for the detection of yoghurt-like odor, presence of cardboard-like
odor, observable smooth and milky consistency and overall acceptability. The same panel
also met 6 more times for this analysis.

38
4.3 Results and Discussion
4.3.1 Some chemical attributes of the different milk and whey solutions
No information was provided by suppliers as to whether whey powders and concentrates
were obtained from either sweet whey or acid whey or to the nature of cheese. Liquid whey
obtained by the coagulation of milk using enzymes such as chymosin is known as sweet
whey while that obtained from acidification with organic acids is known as acid whey
(Bozanic et al., 2014; Huffman et al., 2011). Chemical composition of whey greatly
depends on its method of production. The protein content of sweet and acid whey is similar
but sweet whey usually has a higher amount of free amino acids and lactose with lower
levels of phosphate and minerals (Huffman et al., 2011). Studies suggest that the amount of
free amino acids in sweet whey is about 4 times higher than that of acid whey and even 10
times higher than that of milk (Bozanic et al., 2014). However, acid whey has a higher
mineral content and acid ions, which have the capacity of changing the flavor and
properties of whey. Protein purification has even greater effect on whey composition.
Commercial WPCs and WPIs rehydrated to 10% protein content presented very different
lactose levels and buffering capacities (Table 4.3).

The buffering capacity (BC) of an aqueous system such as milk is an important physico-
chemical characteristic in lactic fermentations. The higher the buffer capacity of milk or
whey, the longer it will be to induce a unit change of pH. This affects the growth of lactic
cultures by a longer fermentation time until pH inhibition (Savoie et al., 2007). Salaün et
al. (2005) describe compositional factors such as milk proteins (caseins and whey proteins)
and presence of small constituents (inorganic phosphate, citrate, and organic acids) as the
most important parameters that determine buffer capacity of milk. In this study, there was a
statistically significant difference (P< 0.05) in the buffering capacities of skim milk powder
reconstituted (RSM) at 3.2% protein content and at 10% (Table 4.3). RSM at 10% protein
content had a higher buffer capacity (1.9% lactic acid) than its homologue at 3.2% (0.6%
lactic acid). There was also a significant difference between their lactose content. However,
there was no statistically significant difference between the buffer capacities of RW and
RWP (P>0.05). Standardised at 10% protein content, the buffer capacities obtained for

39
WPIs and WPCs were of two extremes; solutions with BC >1.4 and those with BCs <0.8.
Solutions with BC values >1.4 were characterized by a relatively high lactose content (3 -
15%). Lactose being an important nutrient for bacteria growth during fermentation, and
high BC also being favourable to growth, these data suggest that these products would be
appropriate for lactic fermentation.

Table 4.3: Composition and buffer capacities of rehydrated powders.

Product Code Composition (liquid) Buffering capacity

type % protein % lactose (equivalent
% lactic acid)
WPI AC 10 16.3a 1.48b
or DB 10 12.9c 1.36b
WPC YB 10 6.7d 1.43b
DA 10 *1.0f 0.71c
WC 10 *0.7f,g 0.77c
FA 10 *0.6g,h 0.72c
WB 10 *0.1i 0.70c
FB 10 *0.6f,g 0.70c
WD 10 *0.6f,g,h 0.69c
AB 10 *0.8f,g 0.64c
YA 10 *0.2e f 0.65cd
WA 10 *0.01i 0.65cd
Controls RSM-10% 10 13.8b 1.89a
RSM-3.2% 3.2 4.9e 0.55d
RW 0.7 4.5 0.20e
RWP 0.1 4.4 0.15e
Refer to table 4.1 for initial compositions of whey and whey products before rehydration.
*Represents solutions containing less than 1.5g/100g of lactose. RSM: Reconstituted skim milk
powder, RW: Reconstituted whey, RWP: Reconstituted whey permeate. a-i Means in a row followed
by different letters are significantly different (P< 0.05). Lactose content for RW and RWP was
provided by the manufacturer.

On the other hand, for solutions with BC < 0.8, it was noticed that these also had relatively
low lactose content. Little or no information was found in literature as to whether there is a
correlation between lactose content and buffer capacity. Presumably, the low lactose levels

40
are accompanied by low levels of other low molecular weight compounds such as amino
acids (Al-Dabbas et al., 2011; Salaün et al., 2005).

4.3.2 Fermentation of WPCs and WPIs

A concern for the development of the WPI fermented drink was insufficient lactose to
allow the fermentation to reach pH of 4.8. The first fermentation assays were therefore
carried out with mesophilic starters (M1) which have higher conversion yields of lactose
into lactic acid than do the yoghurt starters. This is because most yoghurt starters do not
convert the galactose moiety of lactose into lactic acid. The “M1”starter is considered an
aromatic starter culture (Lamontagne et al., 2002). The synthesis of aromas was desired and
fermentation at 22°C was used to promote their synthesis (Pack et al., 1968). Figure 4.2
shows the acidification profiles of “M1”starter in control solutions after 24 hours of
fermentation. There was a marked difference between the acidification profile of RSM-
3.2% and RSM-10%. During fermentation, the drop of pH was slow for RSM-10% and the
targeted final pH value of 4.8 was not attained within 24 h at 22°C. This could be attributed
to the high BC recorded with RSM-10%. In milk, the buffering actions of caseins add to
soluble citrate and phosphate (Al-Dabbas et al., 2011; Salaün et al., 2005). Reconstituted
whey powder (RW) and whey permeate had similar acidification profiles as RSM-3.2%
although it had a significantly higher pH after the 14th hour of fermentation.

41
 RSM -10%
RSM - 3.2%
7 RW
RW P

6
pH

4
0 5 10 15 20 25
Time (hours)

Figure 4.2: Fermentation profiles of whey and milk controls with mesophilic starter “M1”.
Bars represent ±SEM.

Since 12 curves could not be presented in a single figure to study the acidification patterns
for WPCs and WPIs, results were separated and presented in figures 4.3a and 4.3b.

Rehydrated powders of WA and DB did not undergo any acidification (Fig 4.3a and b,
respectively). WA contained a very low lactose content (Table 4.3) while solution DB had a
higher BC and lactose content. Thus, DB had presumably sufficient lactose to allow
complete fermentation but this did not occur. Presumably, its high BC may have resisted to
pH changes or there may be other limiting factors to growth than lactose concentration.
Although no information was provided regarding the production process of this powder by
the manufacturer, its inability to be fermented could hypothetically be attributed to its
process method. A similar slow acidification pattern in a high-lactose medium was noticed
with the solution AC. On the other hand, the poor acidification of WA could be attributed
to its little or no lactose content (Table 4.3).

42
 a
7

YA
5 YB
WA
WB
WC
WD
4
pH

b
7

AB
5 AC
DA
DB
FA
FB
4

0 5 10 15 20 25
Time (hours)
Figure 4.3: Mesophilic fermentation of whey-bases with starter culture “M1”.
Bars represent ±SEM.

43
 Some solutions underwent acidification which suddenly stopped at pH ≥ 5 after 10-
12 hours of fermentation (Fig 4.3a). A possible explanation could be that LAB was able to
convert all available lactose to lactic acid after which there was no more available substrate.
Furthermore, the inability for solutions YA and WB to attain a final pH of 4.8 could not be
attributed to BC because they had very low BCs (Table 4.3).

Solutions DA, FA, FB, WC and WD with low BCs enabled fermentation to pH 4.8
(Figure 4.3) even though they contained less than 1.0 % lactose (Table 4.3), suggesting
essential nutrients for cell growth were present. Solution YB with a high BC containing
about 6.3% of lactose was able to undergo complete acidification.

4.3.3 Lactose supplementation

YA, WA and WB solutions which contained less than 0.6 % lactose and underwent low or
no fermentation were supplemented in lactose up to 1.5 % in order to increase acidification
levels (figure 4.4). There was a marked change in the acidification profiles of YA and WB.
Lactose addition strongly improved their acidification rate. In contrast, little change was
observed in WA medium which is a WPI (Table 4.1). Its inability to undergo fermentation
could therefore be hypothetically attributed to its high protein purity, with a resulting
absence of low molecular weight growth compounds.

44
 7

6
pH

YA
5 YA lac
WA
WA lac
WB
WB lac
4
0 5 10 15 20 25
Time (hours)
Figure 4.4: Supplementation of powders which could not undergo complete acidification in
lactose. Fermented with “M1”. Bars represent ±SEM.

4.3.4 Influence of starter culture

In order to verify if the acidification profiles of lactose supplemented WPIs and WPCs were
not strain or culture-dependent, media which could not be fermented by starter “M1”, even
after lactose supplementation, were fermented with another mesophilic starter culture
“M2”. All solutions (WA, WB and YA) contained at least 1.5% lactose content, 10% whey
protein and had an initial pH of 6.5.

45
 7

6
pH

5 M1YA
M2YA
M1WA
M2WA
M1WB
M2WB

4
0 5 10 15 20 25
Time (hours)
Figure 4.5: Lactose supplemented protein concentrates with two types of mesophilic cultures
(“M1”compared to “M2”). Bars represent the ±SEM.

Figure 4.5 shows that the acidification rates were much slower with starter M2.
Nevertheless, there was very little difference in the final pH of lactose-enriched WB and
YA media fermented with “M2”, and “M1”. These data suggest that “M2” starter contained
strains which had a higher nutritional demands than the M1 culture, and confirm that the
growth of LAB in a medium is strain-dependent (Champagne et al., 2009).

Data from these assays resulted in the elimination of YA, WB, WB, AC and DB
products, either on the basis of insufficient lactose contents or low fermentation even in
presence of lactose.

46
4.3.5 Sensory analysis of WPIs /WPCs fermented with mesophilic culture “M1”
Exploratory sensory analyses were carried out to further select whey concentrates on the
basis of undesirable sensory attributes. Thus, WPIs and WPCs which underwent
acidification to pH 4.8 with the fastest mesophilic starter (M1) were further analysed.

WC
4
3
FB WD Sweet Yoghurt/cheese
2 aroma
1
cereal-like
0
FA YB
cardboard-like

AB DA

Figure 4.6: Sensory analysis of fermented whey drinks with starter culture “M1”. Attributes were
evaluated in terms of intensity: none, (0), very mild (1), mild (2), moderate (3) and high (4)

Of the 5 potential aromas (Table 4.2), three were predominantly detected by judges; sweet
yoghurt/cheese aroma; cereal-like aroma and cardboard-like (figure 4.6). The first is
associated with dairy products, but the two latter are non-dairy flavors (cardboard and
cereal) (Whetstine et al., 2005). The YB product distinguished itself from all the others by
the absence of cereal or cardboard odors, and by the highest cheese aroma (Figure 4.6).
Although many factors may be responsible for the detection of off-flavors in whey and
derived whey products, it has been suggested by many studies that two major factors are
usually involved; lipid oxidation and the Maillard reaction (Morr et al., 1991; Mortenson et
al., 2008; Whetstine et al., 2005).

Cardboard or cereal-like flavors are undesirable attributes in dairy products. It is

noteworthy that the mesophilic starter (“M1”) was not able to mask the undesirable odors,
even though it is known as an aroma-producing (diacetyl) culture. The detection of either
cardboard or cereal-like off-flavors led to the elimination of 6 out of the 7 products tested

47
and the selection of the whey protein concentrate YB. As mentioned previously, YB
enabled pasteurization (63°C – 30 min), complete acidification and was free of any visual
or textural defect.

4.3.6 Fermentation of YB whey concentrate

The YB was chosen as the best WPC blend for further studies, but the use of the mesophilic
starter resulted in 16-24 hour incubation (Figure 4a). This therefore led to attempts to
shorten the fermentation time in order to enhance the economic interest to any dairy
industry. With the objective of developing a high-protein fermented beverage, assays with
yoghurt starters are of interest for two reasons. First, the aroma profile differs from the
mesophilic cultures (Liu et al., 2004) and fermentation rates can be faster (Wouters et al.,
2002) . Yoghurt starters, on the other hand, generate galactose from lactose bioconversion
(Audet et al., 1989), which significantly reduces their lactic acid conversion yield from
lactose, as compared to mesophilic starters. It was our concern that, the limits of lactose
concentration on adequate acidification, which were noted in some products with the
mesophilic starters, would be amplified with the yogurt cultures.

When rehydrated at 10 % protein, YB contained 6.7 % lactose (Table 4.3) which

was enough for an acceptable acidification level. Since assays with mesophilic starter
cultures revealed a very slight difference in their acidification patterns, different types of
thermophilic starter cultures were also tested with YB.

48
 7 F1
F2
F3
F4
F5
F6
6 F7
pH

4
0 1 2 3 4 5 6 7
Time (hours)
Figure 4.7: Acidification profiles of YB with different commercial thermophilic starter cultures.
Bars represent ±SEM.

Figure 4.7 shows the different fermentation patterns of YB with different commercial
thermophilic starter cultures. There was no major difference in the fermentation profiles of
starters F2, F3 and F1. Starter F2 was the fastest to reach a pH of 4.8, but this was mainly
linked to a rapid initiation of acidification. Some starters (F5, F7) were slower to start, but
had the highest acidification rates between pH 6.0 and 5.0 (Figure 4.7). Although
inoculations were standardized at about 3 x 107 CFU/mL, the lag phases differed. Some
were from freshly thawed cultures while others were from freshly rehydrates starters. It is
well known that dried cultures are slower to start acidification than liquid ones (Champagne
et al., 2000). Furthermore, the detailed composition of starter cultures was not provided.
They were all composed of St. thermophilus and Lb. bulgaricus, but ratios can vary
(Caplice et al., 1999; Carminati et al., 2015; Wouters et al., 2002).

49
4.3.7 Sensory analysis of WPIs / WPCs fermented with thermophilic starter cultures
In this series of analyses, results were expressed in terms of the number of judges who
detected specific characteristics such as the presence of yoghurt-like aromas, presence of
cardboard odor, observable smooth and milky consistency and an overall acceptability.

(odor)

Figure 4.8: Sensory analysis of fermented whey drinks with thermophilic starter cultures F1 to F7.

This sensory analysis was carried out to determine the best yoghurt starter to be used in the
production of 10% protein YB whey drink without generation of off-flavors and textural
defects. There was a strong effect of starter on the sensory results. Starter F2 produced the
best yoghurt aroma and was most appreciated by judges (Figure 4.8). The high overall
acceptability could partially be linked to the lesser perception of the cardboard-like defect
(Figure 4.8). This suggests that F2 could be used to mask cardboard odor in YB medium.
The visual aspect (smoothness, milky consistency and color) of most of the products
received good remarks. However this could not be correlated with starter culture type
(Figure 4.8). The ropy nature observed in some of the samples could be a result of

50
exopolysaccharides production (Wouters et al., 2002) and this could be very interesting in
products in which thickeners are not envisaged.

As the F2 starter culture was the fastest to reach the target pH of 4.8 (Figure 4.7),
combined with the sensory results, the F2 starter was therefore considered as the best
culture for the production of a whey-based drink.

This study shows that preparing a fermented drink having high whey protein content
is possible if the WPC and the starter cultures are carefully selected.

4.3.8 Commercial application

Environmentally-friendly processes are increasingly demanded by industry and

consumers. Although the product obtained from this study is a high-protein beverage from
whey, its manufacturing process avoids the production of acid whey by-product, such as
occurred during manufacture of Greek style fermented milks. Moreover, using whey
powders has the advantage of being easy to develop for small and large manufacturing
plants. However, the challenge is to address sensory defects that are linked to the exclusive
use of whey.

51
4.4 Conclusion
Before now, no study had been carried out to describe an ecologically-friendly
process of developing a high-protein whey-based beverage solely from commercial WPCs
and WPIs powders. The results showed a significant effect of the WPCs and WPIs
chemical composition, lactose content, and buffer capacity. Furthermore, starter culture
type also affected the acidification profiles of beverages. Thermophilic cultures were found
to ferment solutions faster than mesophilic cultures and this was in agreement with
literature. In addition to WPC/WPI source, the cultures affected sensory attributes. This
could be highly dependent on each manufacturer’s production. The fermentation of WPC
with selected LAB strains could be a novel avenue for developing non-conventional high
protein dairy drinks or functional beverages. This product could also be subject to
enrichment with probiotic bacteria in order to combine the benefits of whey proteins and
beneficial bacteria. This eco-friendly, 10% whey protein drink could address several
market trends in the functional food and health sector.

52
4.5 Acknowledgements
This project was funded by the InnovAction program of the Ministère de l’Agriculture des
Pêcheries et de l’Alimentation (MAPAQ) and from Growing Forward of Agriculture and
Agri-Food Canada. Gratitude is expressed to Yves Raymond, Nancy Graveline for
technical assistance and to Mario Proulx (Aliments Ultima) for scientific and market
advice. Gratitude is expressed to the various suppliers of powders and starter cultures
(Agropur, Arla, Fonterra, Hilmar, Biena, Dupont and Lallemand Health Solutions).

53
 Chapter 5: Effect of whey protein concentrate and
starter culture on the growth and stability of probiotic
bacteria in fermented high protein whey beverage

Germaine Enyoh Forkwa1, Claude P. Champagne1,2*, Marie-Josée Lemay2,

Jean-Christophe Vuillemard1.

1
Institute of Nutrition and Functional Foods (INAF), Université Laval, Québec, QC,
Canada G1V 0A6

2
Saint-Hyacinthe Research and Development Centre, Agriculture and Agri-food Canada,
3600 Casavant Blvd., St. Hyacinthe, Quebec Canada J2S8E3.

* Corresponding author: claude.champagne@agr.gc.ca

54
Résumé
L'objectif de cette étude était de développer une boisson fermentée à base de lactosérum
contenant 10% de protéines et des concentrations élevées en bactéries probiotiques. Deux
souches de bactéries probiotiques ont été utilisées: Lactobacillus rhamnosus R0011 et
Lactobacillus helveticus R0052 en association avec un ferment pour yogourt. La
croissance, la stabilité et l'acidification des bactéries probiotiques ont été étudiées. Une
durée de fermentation moyenne de 5 h a permis d’atteindre un pH de 4,8. Lb. helveticus
R0052 s'est mieux développé que Lb. rhamnosus R0011 et les concentrations élevées de
cellules vivantes se ont maintenues pendant l’entreposage (supérieures à 6 Log CFU mL-1
pour Lb. rhamnosus R0011 et 7 Log CFU mL-1 pour Lb. helveticus R0052).

55
Abstract
The goal of this study was to develop a fermented whey-based drink containing 10 %
protein and high levels of probiotic bacteria. Two strains of probiotic bacteria were used
(Lactobacillus rhamnosus R0011 and Lactobacillus helveticus R0052) together with a
yogurt starter. The growth, stability and acidification of probiotic bacteria was examined.
The average fermentation time was 5h. Lb. helveticus R0052 grew better than Lb.
rhamnosus R0011. Probiotic cell counts in all beverages after storage were greater than 6
Log CFU mL-1 for Lb. rhamnosus R011 and 7 Log CFU mL-1 for Lb. helveticus R0052.

56
5.1 Introduction
Over the years, consumer awareness and preference for food products with health
attributes have become as important as those with nutritional claims (Verbeke et al., 2009).
As a result, the food industry has developed a variety of new functional food products,
which are increasing in demand (Granato et al., 2010). The global functional food market is
a fast growing sector of the food industry (Marsh et al., 2014) and dairy fermented based
products make up approximately 43% of this market (Ozer et al., 2010). The functionality
of many of these fermented beverages can be attributed to the presence and activity of
probiotic bacteria (Verbeke et al., 2009). In 2015, fermented milks, especially yoghurt-style
products, accounted for about 57% of the probiotic dairy products industry revenue and
contributed to over 50% of the global probiotics market size (Global Market Insights,
2016). Therefore, there is strong consumer interest in probiotic-containing dairy products.

There is no defined number of viable cells which ensures efficacy of probiotic

bacteria. However, Canada and Italy require that 1 billion living cells per portion to be able
to claim the presence of probiotics in the product (Hill et al. 2014). Yoghurts and fruit
juices meet this requirement of 1 billion/portion. However, specialty probiotic drinks may
contain 10 billion (for example DanActive™) or even 50 billion living cells per portion (for
example BioK+™). Therefore, high numbers of viable cells in a product is important from
both regulatory and marketing standpoints, and there is consumer interest for products with
10 billion cells or more per portion.

Fermented dairy products are considered excellent carriers for probiotics (Gilliland,
2002; Sodini et al., 2002). Although milk-based products have traditionally been the
carriers of probiotics, whey, a by-product of the cheese industry has received attention for
its ability to sustain the growth of probiotic cultures (Drgalic et al., 2005). Whey
ingredients, particularly proteins (Lagrange et al., 2015), purportedly have health benefits
linked to weight management (Luhovyy et al., 2007), blood pressure (Fluegel et al., 2010),
glycemia (Luhovyy et al., 2007). The addition of probiotics to a high-protein whey drink
could potentially enhance its health attributes.

57
 Fermentation with commercial starter cultures or probiotic strains designed for
yoghurt production have been proven to overcome off-flavors, increase sensory qualities or
even produce functional drinks (Bulatovic et al., 2014c; Pescuma et al., 2015). Developing
a 10 % protein fermented beverage solely from whey protein concentrates (WPC) or whey
protein isolates (WPI), in an environmentally-friendly process, poses many challenges with
respect to sensory properties and the ability of the matrix to support fermentation. In a
previous study (Chapter 4), such a product was obtained after testing 12 WPI/WPC, as well
as 7 starter cultures. However no data are available on the development of probiotic
cultures in such WPC-based products. Marsh et al. (2014) suggested that the key in
accurately reproducing desirable characteristics of health-promoting beverages depended
on choosing the appropriate starter strains and also knowing the synergistic interaction
when used in the presence of probiotics. When using native whey as a substrate, Bulatovic
et al. (2014d) reported that the addition of yeast extract and inulin improved the probiotic
growth and that enrichment increased the viable cell counts of Lactobacillus rhamnosus to
about 2.6 log units. However, in native whey matrices, literature shows that
supplementation can improve the development of probiotics, but no data concerning the
growth and viability of probiotics in a high-protein WPC-based drink is available.

The aim of this work was to study the effect of milk supplementation on probiotic
growth and stability in WPC-based beverages.

58
5.2. Material and Methods
5.2.1 Strains
A commercial yoghurt starter culture (YO-Mix T11) was graciously provided by
Danisco Inc. (DuPont Nutrition and Health, Mississauga, Ontario, Canada). In freeze-dried
form, YO-Mix T11 was stored at 4°C and used for direct inoculation (0.01%). The initial
count used as inoculum was 3 x 107 CFU mL-1.

The probiotic strains Lactobacillus helveticus R0052 and Lactobacillus rhamnosus

R0011 used in this work were provided by Lallemand Health Solutions (Montreal,
Canada). These cultures were also freeze-dried powders and stored at 4°C before use. As
practiced in industry, direct inoculation of the cultures to the fermentation medium was
carried out. Thus, 0.05g of Lb. helveticus R0052 and 0.08g of Lb. rhamnosus R0011 of
each freeze-dried probiotic culture was added to 500 mL of fermenting medium. In both
cases, the initial count used for inocula was 1 x 107 CFU mL-1.

5.2.2 Fermentation media

The 50% WPC used in this work was kindly provided by Arla Food Ingredients
(Virby, Denmark; lot F150950) and had the following composition: 50.8% proteins, 36%
lactose and 5% fat. Two types of WPC-based solutions were prepared in glass jars of 250
mL. Firstly, the WPC was rehydrated with deionized water in order to obtain a 10% protein
concentration. This will be referred to as the “W medium”. WPC was also rehydrated in a
commercial liquid skim milk (Natrel, Longueuil, QC, Canada) so that the solution
contained 6.8% proteins from WPC and 3.2% proteins from skim milk. This will be
referred to as the “WM medium”. Considering that caseins represent 80% of milk proteins,
the WM medium contained 2.5% caseins and 7.5% whey proteins. The pH was adjusted to
6.5 (Accumet excel XL 15 pH meter, Fisher scientific) using either 0.1M HCl or 4M
NaOH. After storage overnight at 4°C, solutions were pasteurized at 63°C for 30 minutes in
a water bath and cooled in an ice bath to about 43°C. Inoculation with the starter culture
and the probiotics was carried out as described previously.

59
5.2.3 Fermentation
Fermentations were carried out in an in-house fermentation and acquisition control
system (FACS) composed of a computerized pH and temperature data acquisition system,
connected to 8 electrodes which could be used simultaneously. Glass jars containing the
inoculated media were stirred at 100 RPM throughout fermentation (40°C for 6 hours) with
magnetic bars until a pH of 4.8 was reached. After fermentation, samples were rapidly
cooled in an ice bath to about 4°C. Fermented solutions were dispensed in 90 mL capped
sterile plastic containers (made from high-density polyethylene) and stored at 4°C for
further analysis. Each fermentation run was carried out in triplicate.

5.2.4 Acidification
The pH of fermented whey drinks was monitored using an Accumet excel XL 15
pH meter (Fisher scientific) after calibration with pH 4.0 and 7.0 standard buffers.

5.2.5 Dissolved oxygen

The dissolved oxygen level was measured using an oxygen conductivity salinity and
temperature device (Model 45387 FT, YSI incorporated, Ohio USA).

5.2.6 Microbial analysis

The 50 mL samples were homogenized by reciprocal shaking (about 45 times) over
10-20 seconds. A sample (1 mL) was diluted in sterile pre-heated (37°C) peptone water
diluent (1 g/L). The first dilution was submitted to a high-shear homogenization step
(OMNI THQ, 27000 RPM, 30 sec). This was carried out to break down any cell clumps
bacteria, as well as chains of cells, as recommended for the analysis of probiotic bacteria in
foods (Champagne et al., 2011). Subsequent serial dilutions were carried out in peptone
water (1 g/L) and plated onto selective media in triplicate, using the pour-plate technique.
St. thermophilus was enumerated on M17-agar media while Lactobacillus rhamnosus
R0011 and Lactobacillus helveticus R0052 strains were enumerated on MRS-agar media
added with 0.03mg/L clindamycin hydrochloride (Sigma). The Lb. delbueckii ssp
bulgaricus cell concentration was less than 1% of the total population, and did not interfere
with the L. helveticus cell counts. Petri dishes plated with St. thermophilus were incubated
in aerobic conditions at 42°C for 48 hours. Petri dishes for probiotic CFUs were incubated

60
at 45°C for 72 hours in anaerobic jars containing atmosphere controlling sachets (GasPakTM
EZ Anaerobe, Maryland, USA) and dry anaerobic indicator strips.

5.2.7 Buffer capacity

The buffering capacity (BC) of rehydrated solutions was assessed by determining
the quantity of acid (0.1N HCl) required to reduce the pH of the medium from pH 6.5 to pH
4.0. The titration was carried out using a TIM865 Radiometer (Villeurbanne, France)
connected to a computer for reading and monitoring of sample pH/acid volume. Ten (10)
ml of medium was mixed with 15 ml of de-ionized water for sample preparation. Under
constant stirring, the addition of acid to samples was automatically stopped when a pH of
4.0 was reached.

5.2.8 Statistical analysis

All experiments were repeated independently three times. Results were analysed
using SAS (SAS institute Inc., Cary, North Carolina, USA version 20.4, 2008) and Sigma
plot 13.0 to determine least significant differences (P < 0.05). An ANOVA split-plot test
was performed for means comparison.

61
5.3 Results and Discussion
The viable counts of probiotic bacteria in fermented dairy products during
fermentation and storage are strongly affected by the presence of oxygen (Talwalkar et al.,
2004b) as well as by the pH of the medium (Shori, 2015). Therefore, before presenting
CFU data, it is of interest to describe the products with respect to their acidity and dissolved
oxygen levels.

5.3.1 Growth during fermentation

The viable counts of probiotics obtained after fermentation of the whey bases are
reported in Figure 5.1. There was a statistically significant (P < 0.05) increase in cell counts
during fermentation of both Lb. rhamnosus R0011 and Lb. helveticus R0052 (Figure 5.1).
Lb. helveticus R0052 grew better than Lb. rhamnosus R0011 and, at the end of
fermentations, the observed higher cell concentrations of Lb. helveticus R0052 could
potentially be attributed to the fact that this specie has a higher proteolytic activity (Bozanic
et al., 2014). Moreover, Lb. rhamnosus R0011 poorly ferments lactose (Gaudreau et al.,
2005). The population reached in this medium by Lb. helveticus R0052 was 5 times higher
than that attained in milk or soy beverages under comparable conditions (Champagne et al.,
2009).
The caseins represented 25% of proteins in the WM medium. Since caseins have a
higher BC than whey proteins, the buffering capacity of WM was 10% higher than the W
medium. Data from the literature (Bulatovic et al., 2014b; Castro et al., 2013), report an
improvement of growth of probiotic bacteria in following the addition of milk to either
liquid or powdered whey. In this study there was no statistically significant (P > 0.05)
effect on viable probiotics counts upon addition of skim milk to WPC base (Figure 5.1).
These observations confirm the statement of Marafon et al. (2011) that growth of probiotics
is highly dependent on probiotic strain studied and its nutritional needs. Without external
pH control, an improved BC of growth media generally enables higher viable counts of
lactic cultures in the fermented broth (Champagne et al., 1995). Evidently, the 10% higher
BC of the WM did not resulted in an increase of viable counts of probiotic bacteria in the
freshly fermented media.

62
Figure 5.1 Viable counts of probiotic strains (Lb. helveticus R0052 and Lb. rhamnosus R0011)
after fermentation in association with a commercial yoghurt starter culture. Media were cooled
immediately after reaching a pH of 4.8 and microbial analyses were carried out after 24 hours of
storage at 4°C. W = whey protein concentrate medium; WM = whey protein concentrate and skim
milk medium; I = Initial viable count at inoculation; F = viable count at day 1. Error bars represent
Standard Error of the Means (SEM).

5.3.2 Acidification during storage

The viable counts of probiotic bacteria in fermented dairy products during storage
are strongly affected by the pH of the medium (Donkor et al., 2006; Shori, 2015). It was
therefore decided to standardize the pH at 4.8 for all products at the beginning of storage.
The fermentations were stopped when the designated pH been reached, which took, on the
average, 4-5 h at 40°C. The fermentations in media containing milk required slightly more
fermentation time because of the BC of caseins. Figure 5.2 shows a systematic decrease in
pH in all samples during 45 days of storage. The means of pH values varied from 4.80 to
4.47. There was no significant difference in pH (P > 0.05) values between days 1 to 3.

63
Although the BC of the media slightly differed, the amount of acid produced during the
first 3 days of storage was insufficient to significantly influence pH.

Figure 5.2: Effect of medium and probiotic strains (Lb. helveticus R0052 or Lb. rhamnosus R0011)
on pH changes during 45 days of storage at 4°C of 10% protein fermented drinks.

However, this was not the case after 10 days of storage as in a similar study carried
out by Bulatovic et al. (2014b). Indeed, on day 10, the pH of WM with strain R0052 was
significantly (P < 0.05) lower than that of strain R0011 (Figure 5.1). It is also noteworthy
that, at day 10, the pH of the WM media was lower than those of their W counterparts, but
the effect of medium disappeared after 30 and 45 days (Figure 5.2). At these latter storage
times the medium had no effect on the drop in pH. The data at days 30 and 45 are in line
with those obtained by Bulatovic et al. (2014b).

Irrespective of medium, lower pH values were recorded during storage in media

containing Lb. helveticus R0052 (Figure 5.2). The change in pH during storage of
fermented milk products is usually attributed to the production of organic acids and is strain
dependent (Widyastuti et al., 2014). As compared to strain R0011, the higher post-
acidification in media with strain R0052 could simply be linked to their higher cell
concentrations (Figures 5.1 and 5.4). Since the cell counts of St. thermophilus reached 1.6 x

64
109 CFU/mL, the proportion of probiotic bacteria in the total population was about 15% for
Lb. helveticus R0052 but only 2% for Lb. rhamnosus R0011. Thus, it could be argued that
Lb. helveticus R0052 reached a sufficiently high fraction of the total population to
significantly influence the post-acidification during storage.

5.3.3 Stability of streptococci during storage

There was no statistically significant difference (P > 0.05) in the cell counts of St.
thermophilus during storage, irrespective of the whey-based medium, time of storage or the
presence of probiotic strains (Figure 5.3). As mentioned previously, the commercial
yoghurt culture used contained more than 99% of streptococci. Thus, the lactobacilli counts
in the yoghurt starter were negligible, and only the St. thermophilus population was
ascertained in this experiment. The streptococci grew well in both media reaching CFUs
well above 1 billion per mL (Figure 5.3), which is similar to values in yoghurt (Dave and
Shah, 1997a, 1997b, 1998). There was no significant effect of the probiotic strain on St.
thermophilus counts at day 1. The literature reports instances where the growth of St.
thermophilus is affected by the presence of Lb. helveticus R0052 (Champagne et al., 2009),
but interactions between the streptococci and lactobacilli were not examined in this study.
What was noted, however, was the lack of effect of the Lb. helveticus strain on growth and
acidification of St. thermophilus. In addition, there was no effect of the presence of milk in
the medium on streptococci CFUs at day 1 (Figure 5.3).

65
Figure 5.3: Effect of fermentation medium and of a probiotic culture (Lb. helveticus R0052 and Lb.
rhamnosus R0011) on viable counts of St. thermophilus during storage at 4°C of the 10% protein
fermented drinks. W = whey protein concentrate medium; WM = whey protein concentrate and
skim milk medium. Error bars represent Standard Error of the Means (SEM).

The stability of streptococci in this study was higher than that often reported in the
literature (Oliveira et al., 2002). Since whey protein demonstrated a protective effect to St.
thermophilus in fermented milks during storage (Akalin et al., 2007), it is unknown if the
high stability noted in this study is linked to the strain or to the medium. Further studies are
needed to ascertain the effect of high whey protein content on St thermophilus viability
during storage.

5.3.4 Stability of probiotic strains during storage

With strain R0052, there was no statistically significant difference in cell counts from day 1
to 30 of storage but on the 45th day there was a very slight decrease of about 0.26 Log
CFU/mL in counts which was considered statistically as significant. However, for
probiotic R0011, the 0.37 Log CFU/mL decrease during storage was not judged to be
statistically significant. This appears illogical but is simply explained by the fact that
variability between the three independent assays with Lb. helveticus R0052 was much
lower than that obtained with Lb. rhamnosus R0011. The average viable count decrease in

66
probiotic bacteria of approximately 0.3 Log CFU/mL (50%) during the 45-day storage
period (Figure 5.4) can be considered to be low for fermented dairy products. Indeed,
although there is a wide range of responses in viability of probiotics during storage of
fermented foods (Shori, 2016) CFU drops of 1 log or more are often recorded during
storage of fermented dairy products (Odamaki et al., 2011); (Ortakci et al., 2012). This
good stability during storage could be linked to the strains (Shori, 2016), to the absence of a
negative effect of St. thermophilus (McComas et al., 2003; Ng et al., 2011), to the presence
of whey proteins (Gustaw et al., 2016) but also to the pH of the medium (Champagne et al.,
2005). A pH of 4.8 is higher than traditional yoghurt, which will typically be between pH
4.2 and 4.5. In the latter pH region, the lower the pH, the higher are viability losses
(Kailasapathy et al., 2008) .

Figure 5.4: Effect of fermentation medium on the survival of the probiotic cultures (Lb. helveticus
R0052 or Lb. rhamnosus R0011) during storage at 4°C. W = whey protein concentrate medium;
WM = whey protein concentrate and skim milk medium. Error bars represent Standard Error of the
Means (SEM).

67
 There was no effect of the growth medium on viability losses of either probiotics
during storage. This differs with data from other studies (Bulatovic et al., 2014c; Castro et
al., 2013; Legarová et al., 2010) but is in line with results of Pescuma et al. (2010). It was
also hypothesized that, the higher buffering capacity of milk would reduce the drop in pH
during storage and therefore improve bacterial stability. However, since the acidification
profiles of W and WM during storage were similar, it becomes logical that no effect of
medium on CFUs be noted as well.

5.3.5 Dissolved oxygen

During fermentation, all whey beverages were constantly agitated. This was required
to correctly ascertain the pH of the media during the fermentation, but the constant stirring
incorporated oxygen into the drinks. As a result, the fermented media contained 15%
dissolved oxygen level at the beginning of storage (Figure 5.5).

Figure 5.5: Effect of medium and probiotic strain (Lb. helveticus R0052 or Lb. rhamnosus R0011)
on the levels of dissolved during storage à 4°C.

Irrespective of medium or probiotic culture, there was no statistically significant difference

(P>0.05) in oxygen content between days 1 and 10. This tendency suddenly changed after
day 10, where there was a statistically significant (P>0.05) effect of probiotic strain. Thus,

68
at days 30 and 45, it was noted that media containing Lb. rhamnosus R0011 had a higher
content of dissolved oxygen than those with Lb. helveticus R0052. However, there was no
significant effect of growth medium on oxygen content during this period.
Literature reports that the oxygen level in fermented milk products is influenced by storage
time (Dave et al., 1998), the type of packaging (Talwalkar et al., 2004b) and the addition of
antioxidants (Dave et al., 1997a) . The data from this study are in line with the literature
whereas, in plastic containers, the oxygen content of yogurt steadily increased during
storage (Dave et al., 1997b; Miller et al., 2002; Talwalkar et al., 2004b).
The probiotic strain affected the level of oxygen increase (Figure 5.5). It is known that
oxygen consuming starter culture strains exist (Lin et al., 1999; Odamaki et al., 2011) and
that some probiotic cultures possess mechanisms to reduce oxygen toxicity (Talwalkar et
al., 2004a). To our knowledge, this study is the first to report that the species of probiotic
culture can influence the dissolved oxygen level during storage. Since CFUs of St.
thermophilus were similar in all products, even throughout storage (Figure 5.2), the
different dissolved oxygen levels would not be linked to the starter culture population.
Although the effect of probiotic bacteria on dissolved oxygen levels during storage is
not well studied, many reports exist on the effect of oxygen levels on the viability of
probiotics during storage. As a rule, the higher are oxygen levels, the higher are viability
losses in probiotics during storage (Dave et al., 1997b; Odamaki et al., 2011; Talwalkar et
al., 2004a). Thus, it would be expected that viability losses during storage would be higher
with Lb. rhamnosus than for Lb. helveticus. This was not the case, as Lb. helveticus R0052
showed good viability during storage and our data confirm the importance of the strain
regarding oxygen sensitivity (Talwalkar et al., 2003).

5.3.6 Market potential

There is no universal number of probiotics that guarantee efficacy. However, from a
regulatory standpoint in Canada and Italy (Hill et al., 2014), fermented milks containing
higher than 9 Log CFU per 100 mL portion met the requirement to allow the use of the
“probiotic” label. Furthermore, Lb. helveticus R0052 cell concentrations reached 10 Log
CFUs per 100 mL of product, which places it in a market of specialty probiotic “shots” or
drinks (Corbo et al., 2014). Combined to the 10% high protein level, these fermented whey

69
protein concentrates have potential for health attributes (Fluegel et al., 2010; Luhovyy et
al., 2007; Malkanthi et al., 2016).

70
5.4 Conclusion
This study has shown the possibility of developing fermented high probiotic whey-based
beverages from a 50% whey protein concentrate. The results showed that whey-base type
(W or WM media) did not have any effect on the growth and stability of probiotic strains
during and storage. Furthermore, it was possible to co-culture probiotic strains with
commercialized yoghurt starter culture. The high content of dissolved oxygen obtained in
beverages containing Lb. rhamnosus R011 (~55%) compared to those containing Lb.
helveticus R0052 (~28%) suggest that species of probiotic cultures can influence the
dissolved oxygen level in a high-protein dairy drink during its storage in plastic containers.
All probiotic viable cell counts exceeded 107 CFU mL-1 while counts for St. thermophilus
was constantly maintained at approximately 2.0 x 109 CFU mL-1. As these beverages
contain more than 10 billion viable cells per portion and are made up of solely whey
proteins (10%), they therefore possess a high potentiality of being termed functional drink.
They would also be conform at both regulatory and marketing standpoints.

71
5.5 Acknowledgments
This project was funded by the InnovAction program of the Ministère de l’Agriculture des
Pêcheries et de l’Alimentation (MAPAQ) and from Growing Forward of Agriculture and
Agri-Food Canada. Gratitude is expressed to Yves Raymond for technical assistance and to
Mario Proulx (Aliments Ultima), for scientific and market advice. Gratitude is expressed to
Arla and Lallemand Health Solutions, for WPC and starter cultures.

72
Chapter 6: Effect of growth promoting factors in a high
protein probiotic whey beverage

Germaine Enyoh Forkwa1, Claude P. Champagne1,2*, Marie-Josée Lemay2, Jean-

Christophe Vuillemard1

1
Institute of Nutrition and Functional Foods (INAF), Université Laval, Québec, QC,
Canada G1V 0A6

2
Saint-Hyacinthe Research and Development Centre, Agriculture and Agri-food Canada,
3600

Casavant Blvd, St. Hyacinthe, Quebec Canada J2S8E3

* Corresponding author: claude.champagne@agr.gc.ca

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Résumé
Le but de cette étude était d’obtenir une population très élevée de probiotiques dans une
boisson à haute teneur en protéines de lactosérum. L'effet bénéfique sur la croissance
cellulaire de l'addition de peptones, lipides ou minéraux a été étudié lors de fermentations
avec ou sans contrôle de pH externe. L’extrait de levure et la protéose peptone ≠ 3 ont
montré les meilleurs résultats de croissance des probiotiques. Les hydrolysats de qualité
non alimentaire ont montré un effet supérieur sur la croissance des probiotiques que leurs
homologues de grade alimentaire qui ont tout de même permis d’augmenter la
concentration cellulaire de 8 log UFC/mL à 9 log UFC/mL.

74
Abstract
The aim of this study was to increase probiotic cell concentration in a high protein whey-
based probiotic beverage by supplementation with growth factors. The growth of probiotic
strains Lb. rhamnosus R0011 and Lb. helveticus R0052 in high protein (10%) whey-based
solutions supplemented with glucose, various peptones, lipids, or minerals was monitored
using automated spectrophotometry. The effect of supplementing beverages with food
grade whey hydrolysate was further studied by fermentation under external pH control.
Yeast extract and proteose peptone ≠3 exhibited the best cell growth effect. Non-food grade
hydrolysates stimulated probiotic growth at a higher degree than their food grade
counterparts. In external pH control fermentations, the probiotic strains were combined
with a commercial yoghurt starter (YO-mix T11). External pH control did not show a
significant effect on probiotic growth. However, the growth of probiotics in high protein
whey-based beverages was enhanced from 8 Log CFU/mL to 9 Log CFU/mL by the
addition of a food grade whey hydrolysate.

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6.1 Introduction
With the increasing consumers’ awareness for food and health, the functional food and
beverage market has known significant growth over the last decades (Corbo et al., 2014;
Marsh et al., 2014; Ozen, 2012). In the U.S., there has been an increase in the market of
high protein foods and beverages as 54% of consumers try to incorporate more proteins to
their diets (Oltman et al., 2015). Being recognized as a functional ingredient (Sinha et al.,
2007), whey proteins are increasingly occupying an important part of the functional food
market. As studies have shown, whey proteins have positive effects on weight
management, satiating, obesity and other health trends (Ha et al., 2003; Oltman et al., 2015;
Zemel, 2013). Due to their functional attributes, whey proteins have become highly
desirable for protein beverage formulations (Fachin et al., 2005; Huffman et al., 2011).
Moreover, lactic fermentation has even greatly increased organoleptic properties of whey
based beverages (Bulatovic et al., 2014a; Pescuma et al., 2015; Pescuma et al., 2010).

In previous studies, we have demonstrated that probiotics added to high protein

whey beverages (10%) could grow and survive in such media (chapter 5), However, in a
study carried out by Shah (2000a), it was shown that most beverages or products containing
probiotics had relatively low counts at time of retail. At the time of consumption, products
having claims of probiotics are expected to contain a minimum amount of 106–107
CFU/mL (McComas et al., 2003; Ozer et al., 2010; Shah, 2000a) to exhibit their probiotic
effect (WHO et al., 2002). In Canada and Italy, at least 1 billion cells per portion ought to
be present in a food product which claims the presence of probiotics (Hill et al. 2014). As a
result, most probiotic-carrying foods on the market now contain at least 1 billion cells per
portion, but some products contain between 10 and 50 billion living cells per portion (e.g

DanActive™ and BioK+®). Few papers describe technological adaptations allowing to

enhance probiotic counts above 50 billion CFU per portion in fermented dairy products.

In order to ensure that these minimum amounts are present in products, monitoring
the populations of probiotic strains in food is therefore important (Colombo et al., 2014). It
has been suggested that one of the possible methods of ensuring adequate numbers of
probiotic bacteria in cultured dairy products is by adding substances that can stimulate

76
probiotic growth during manufacture or storage (McComas et al., 2003). Although in a
previous study (chapter 5), up to 108 CFU/mL probiotic cells could be obtained in a high
whey protein fermented beverage, the effect of adding dairy-based probiotic growth
stimulatory substances to such high-protein whey beverages has not yet been studied. It is
our hypothesis that the addition of selected growth factors would increase probiotic cell
counts in the fermented product above 50 billion per 100 mL portion. Since the beverage
would contain high levels of whey protein and probiotic bacteria, this new formulation
would lead to a specialty probiotic drink with health effects.

Although many dairy products have become vehicles for probiotics, little
information was found in literature on dairy supplements that would allow probiotic growth
to surpass 50 billion cells per 100 mL portion in high-protein whey-based probiotics
beverages. One of the aims of our study was to select and monitor the effect of dairy-based
ingredients on probiotic cell counts in high protein whey-based beverages.

Increasing viable counts of lactic and probiotic bacteria in fermented media can also
be achieved through technological means. The use of external pH control (EpHC) has long
been known to allow high bacterial cell counts in the production of mesophilic (Peebles et
al., 1969) or thermophilic (Champagne et al., 1996) starters, but not in the manufacturing
process itself. The second goal of this study was to examine the effect of EpHC on
probiotic cell concentration in fermented whey drinks.

Thus, the overall aim of this study is to evaluate probiotic strains, growth
supplements and EpHC in order to achieve very high probiotic cell concentrations in a
fermented high-protein whey drink.

77
6. 2 Material and Methods
6.2.1 Starter culture
A commercial yoghurt starter culture, YO-Mix T11, was graciously provided by
Danisco Inc. (DuPont Nutrition and Health, Mississauga, Ontario, Canada). Available in a
freeze-dried form, YO-Mix T11 was stored at 4°C and used for direct inoculation. In all
assays, the starter was added in order to obtain an initial inoculation level of 3x107 CFU
mL-1.

6.2.2 Probiotic strains

The probiotic strains used in this study (Lactobacillus helveticus R0052 and
Lactobacillus rhamnosus R0011) were provided by Lallemand Health solutions, Montreal,
Canada. These freeze-dried cultures were stored at 4°C before use.

For automated spectrophotometry (AS) assays, one gram of each freeze-dried

probiotic culture was first added to 9 mL of a sterile rehydration medium composed of 15
g/l of peptone, 10 g of tryptone and 5 g of yeast extract. The cell suspension was
homogenized (OMNI THQ, 27000 RPM for 30 seconds) and incubated at 37°C for 15
minutes for rehydration. Successive serial dilutions were then performed in 0.1 M
phosphate buffer (pH 6.5) in order to obtain a cell suspension of around 1x 108 CFU mL-1.
This cell suspension served to inoculate the microplates.

In fermentations assays, with or without EpHC, the lyophilized probiotic cultures

were added to 100 mL media solutions adjusted at 40°C. The amount of powder added was
designed to obtain an initial probiotic concentration of 5 x 107 CFU mL-1.

6.2.3 Whey protein concentrate and milk ingredients

The whey protein concentrate (WPC) used in this work was kindly provided by Arla
Food Ingredients (Viby, Denmark; lot F150950) and had the following composition: 50.8%
proteins, 36% lactose and 5% fat. Two types of WPC-based solutions were prepared in 100
mL glass jars. In the first, the WPC was added to deionized water in order to obtain a total
of 10% protein. This will be referred to as the “W medium”. In the second, WPC powder
was added to a commercial (Natrel, Longueuil, QC, Canada) liquid skim milk, so that the

78
solution contained 6.8% WPC proteins and 3.2% skim milk proteins; this will be referred to
as the “WM medium”. Considering that caseins represent 80% of milk proteins, the WM
medium contained 2.5% caseins and 7.5% whey proteins.

The pH of the media was adjusted to 6.5 by using either 1 M HCl or 4 M KOH and
blends were stored at 4°C overnight. The following day, solutions were pasteurized at 63°C
for 30 minutes in a water bath and cooled to about 43°C.

6.2.4 Preparation of W and WM media

The AS screening assays were carried out by using W and WM media. These assays
served two purposes: to assess biocompatibility between the starter and the probiotic
cultures, as well as to select supplements that would enhance the growth of probiotics in the
WPC-based media. In its native state, the W medium was whitish and opaque. Thus,
clarification of the media was required for AS assays.

For the biocompatibility analysis, two forms of W or WM medium were prepared:

media fermented by the yogurt starter, and unfermented media. For the “fermented
medium” treatment, the native W or WM media were inoculated with YO-mix T11 and
incubated at 40°C for approximately 6h until a pH of 4.8. The “unfermented” medium
solution was prepared by incubating non-inoculated W or WM at 4°C for 6 hours and
adjusting the pH to 4.8 by the addition of 85% DL lactic acid (Fisher, Montréal, Canada).
Both fermented and unfermented solutions at pH 4.80 were further stored overnight at 4°C.
The following day, the solutions were heated at 63°C for 30 minutes to initiate protein
precipitation. Then, samples were centrifuged (Beckman, rotor JA-18) at 30°C, 30000 x g
for 30 minutes. The pH of the resulting supernatants was adjusted to 8 with 4 M KOH, and
all samples were then incubated at 37°C for 20 minutes which allowed further precipitation,
mainly of minerals. A second centrifugation was carried out using the same centrifugation
parameters as above. The pH of supernatants was then adjusted to 6.5 with 1M HCl, and
filtered using 0.22µm Millipore units.

6.2.5 Supplementation of W medium for AS screening assays

Some probiotic cultures do not assimilate lactose rapidly (Gaudreau et al., 2005;
Hughes et al., 1995). Therefore, in the first series of AS assays, glucose was added at 5 g/L

79
in the “fermented” or “unfermented” W media described in section 6.2.4. In some
treatments, MRS was also added in these media in a ratio of 1:3.

In the second series of AS assays, the W medium contained 5 g/L glucose and
various supplements (Tables 6.1 and 6.2). Many supplements were selected on the basis of
their presence in the MRS medium formulation. MRS broth is considered to be one of the
best growth medium for probiotic bacteria (Champagne et al., 2011). Initial AS assays had
showed that growth of probiotic cultures in commercial MRS resulted in OD values above
1.4. Unfortunately, the relationship between OD and bacterial biomass was not linear at OD
values above 1.0. Thus, AS assays were conducted in media with various MRS
concentrations and it was found that an OD of approximately 0.8 was reached in MRS at
25% concentration. Thus, the MRS concentration which was further tested in clarified W
media was at an equivalent concentration of MRS-25%.

Since the MRS broth is made up of 10 major ingredients, a factorial experimental

plan based on 10 levels and their combinations was prohibitive. Thus, in order to facilitate
the identification of which ingredients could enhance growth, they were separated into
three main groups (Table 6.1). Group A contained the amino-acid based ingredients
(proteose-peptone #3, beef extract and yeast extract). Group B contained what was
considered the lipid-linked ingredients (polysorbate 80 and sodium acetate) and Group C
minerals and buffering ingredients (ammonium citrate, magnesium sulfate, and manganese
sulfate and dipotassium phosphate). Acetate can serve for the synthesis of fatty acids, and
was therefore included in the lipid-linked group (B).

80
Table 6.1: Concentrations of MRS-based ingredients added to clarified fermented “W
medium” supernatant
Group MRS Ingredients Concentration Source
(g/L)
A : Amino-acid based Proteose peptone #3 2.5 BD
ingredients Beef extract 2.5 OXOID
Yeast extract 1.25 Difco
B : Lipid-linked Polysorbate 80 0.25 Sigma
ingredients Sodium acetate 1.25 Bioshop
C : Minerals-buffers Ammonium citrate 0.5 Bioshop
linked ingredients Magnesium sulfate 0.025 Sigma
Manganese sulfate 0.0125 Bioshop
Di potassium phosphate 0.5 Bioshop
Suppliers: trademarks; BD and Difco were purchased from Beckton Dickinson and
company (Sparks, Maryland, USA), Sigma Aldrich Co., (St. Louis, Missouri, USA),
Organotechnie S.A., (La courneuve, Saint-Denis, France), Oxoid, (Basingstoke,
Hampshire, England), Bioshop (Burlington, Ontario, Canada).

In addition to the MRS-based peptones, numerous other amino-acid based ingredients were
tested at concentration varying from 1.25 to 25 g/L (Table 6.2). It is important to point out
that the assays with peptones included a series of food grade products derived from milk
proteins (Table 6.2). All these growth factors (Tables 6.1 and 6.2) were added to the W
medium supernatants. Thereafter, the pH was adjusted to 6.5 with 1N HCl or 4N KOH and
finally the solutions were filtered using a 0.22 µm Millipore units.

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Table 6.2: Concentrations of protein hydrolysates added to clarified fermented W medium

Ingredients Concentrations α-amino N level (%) Source1

(g/L)
Non Food grade protein hydrolysates
Proteose peptone #3 6.25 28
Beef extract 6.25 19
Yeast extract 6.25 60
Proteose peptone #3 + 3.12 See Table 6.1
Beef extract 3.12
Proteose peptone #3 + 3.12
yeast extract 3.12
Beef extract + 3.12
yeast extract 3.12
Proteose peptone # 3 2.5
Beef extract 2.5
Yeast extract 1.25
Casamino acids 6.25, 25 87 Difco
Trypticase peptone 6.25, 25 37 BD
Casein acid hydrolysate 6.25, 25 NS Organotechnie
Bacto tryptone 6.25, 25 40 BD
Peptone from casein (acid digest) 6.25, 25 NS Sigma
Tryptone enzymatic digest from casein 6.25, 25 NS Sigma
Lactalbumin enzymatic hydrolysate 3.1, 6.25, 12.5, 25 NS Sigma
Bacto TC lactalbumin hydrolysate 3.1, 6.25, 12.5, 25 NS Difco

Food grade dairy protein hydrolysates

Lacprodan Hydro. 365 (whey protein) 3.1, 6.25, 12.5, 25 23-29
Lacprodan DI-2021 (casein) 3.1, 6.25, 12.5, 25 8 Arla
Lacprodan DI-3071 (whey protein) 3.1, 6.25, 12.5, 25 6-10
1
Suppliers: see Table 6.1 for BD, Difco, Sigma, Organotechnie and Bioshop. Arla Foods Ingredients Group Inc., (Viby J, Denmark). α-amino N
level = amino nitrogen/total nitrogen X 100. NS: not specified by the manufacturer.

82
 To all media used for AS assays, 1 mL of a solution containing 100 g/L of sodium
ascorbate and 50 g/L cysteine (Sigma life science, St-Louis, USA) was added to 99 mL of
AS medium, prior to adjusting pH to 6.5.

Various controls were included in the experimental device. The negative control
was the unsupplemented fermented W medium supernatant (pH 6.5). Positive controls were
100% MRS medium (55 g/L) and MRS at 25% concentration (13.5 g/L). One treatment
was carried out by blending 155 µL of the negative control with 45 µL of 100% MRS. This
resulted in 25% concentration of MRS ingredients in the clarified fermented W medium.

6.2.6 Automated spectrophotometry assays

The Thermo Labsystems Bioscreen C (Helsinki, Finland) unit is essentially a

running read-through spectrophotometer with the capacity of holding 2 microplates made
up of 100 mini-wells each. To test probiotic growth, 180 μl of the various W medium
formulations was distributed, in duplicate, into the mini-wells of the microplates. Each
mini-well containing test solutions were inoculated by adding 20 μl of standardized
probiotic cell suspension (1 x 108 CFU/mL), which resulted in a 1 x 107 CFU/mL
inoculation level. Microplates were then inserted into the Bioscreen C unit which had been
previously preheated at 40°C. The incubation temperature was maintained at 40°C for 24
hours and readings were noted every 15 minutes. Before each reading, the plates were
shaken for 2 minutes, and OD values were noted at a wavelength of 600 nm. MRS broth
was used as positive control at two concentrations (25 % and 100 %). The ODmax values
were obtained by subtracting the initial OD values from the maximum values obtained
during incubation time (Max – Min).

6.2.7 Fermentations

Fermentations were carried out in an in-house fermentation and acquisition control

system (FACS) composed of a computerised pH and temperature data acquisition system.
The system had 8 jars, each having a pH electrode, which could be used simultaneously.
Glass jars containing 100 mL of inoculated media were constantly stirred at 100 RPM with
magnetic bars throughout the fermentation.

83
 In some assays, the W and WM media were supplemented with 1.25 g/10 mL of the
Lacprodan DI-3071 whey protein hydrolysate.

Fermentations were carried out at an initial inoculation count of 3 x 107 CFU/mL of

the yogurt starter as well as with 5 x 107 CFU mL-1 for Lactobacillus helveticus R0052 or
Lactobacillus rhamnosus R0011. The media were then incubated at 40°C. In a serial of
assays, once a pH of 4.8 was reached, it was maintained at that value by constant addition
of 6 N NaOH. This will be referred to as the external pH control (EpHC) treatment. The
time needed to reach this target pH varied between media and cultures, but was
approximately of 6 h. Samples (50 mL) were taken every 2 h for CFU and chemical
analyses.

6.2.8 Microbial analyses

The 50 mL samples in the 90 mL plastic cups were handshaked about 45 times over
10-20 seconds. A sample (1 mL) was diluted in sterile pre-heated (37°C) peptone water
diluent (1 g/L). The first dilution was submitted to a high-shear homogenization step using
an OMNI THQ operated at 27000 RPM for 30 seconds. This was carried out to break down
any bacterial cell clumps, as well as chains of cells, as recommended for the analysis of
probiotic bacteria in foods (Champagne et al., 2011). Subsequent serial dilutions were
carried out in peptone water (1 g/L) and plated onto selective media in duplicates, using the
pour-plate technique. Streptococcus thermophilus was enumerated on M17-agar media
while strains of R0052 and R0011 were enumerated on MRS-agar media added with
0.03mg/L clindamycin hydrochloride (Sigma). The yoghurt starter content in Lactobacillus
delbueckii ssp bulgaricus was less than 1% and their counts did not interfere with the other
cell counts. Petri dishes plated with St. thermophilus were incubated in aerobic conditions
at 42°C for 48 hours. Petri dishes for probiotic CFUs were incubated in anaerobic jars
containing atmosphere controlling sachets (GasPakTM EZ Anaerobe container system) and
dry anaerobic indicator strips at 37°C for 48 hours.

6.2.9 Carbohydrates and organic acid analysis

Samples were prepared by adding TCA and sodium azide at a final concentration of
3.6% and 0.015% respectively. The blends were well mixed and incubated at 37°C

84
overnight. The samples were then centrifuged (Centrifuge Beckman, Rotor JLA 10.5) at
10000 g for 20 minutes at 37°C. The supernatants were incubated for 2 hours at 37°C. A
second centrifugation was done as above. Supernatants for all samples were passed through
C18 Sep Pak column (Waters Corporation, Milford, Massachusetts, USA) preconditioned
with 5 mL of methanol and 5 mL of water and filtered on 0.45 µm Millipore filter into
HPLC vials for analysis.

Lactose, galactose, glucose and organic acids (lactic, acetic, and formic) were
analyzed by high performance liquid chromatography (HPLC) on an ICE-Ion 300
(Transgenomic, Omaha, NE, USA) separation column kept at a temperature of 80°C. The
mobile phase was H2SO4 0.02 N at 0.4 mL/min and the injection sample volume was 20
µL. The HPLC system was composed of a Waters 1525 pump (Milford, MA, USA), Waters
717-plus auto sampler Waters 2414 RI detector at 40°C and a Waters 2998 PDA detector at
a wavelength of 600 nm.

6.2.10 Statistical analysis

Three independent repetitions were carried out for each experimental treatment. Results
were analyzed using SAS (SAS institute Inc., Cary, North Carolina, USA version 20.4,
2008) to determine least significant differences (P < 0.05). An ANOVA split-plot test was
performed for means comparison.

85
6. 3 Results and Discussion
6.3.1 Biocompatibility assays between the starter and the probiotics
Preliminary data showed that Lb. rhamnosus R0011 did not grow well in W
medium over 6 h fermentation, while Lb. helveticus R0052 reached only 1.6 x 108 CFU/mL
(Chapter 5). These results are in line with other studies which showed that, at the opposite
of milk, whey does not contain the nutrients needed for probiotic growth (Abu-Taraboush
et al., 1998; Dave et al., 1998). As expected, there was low probiotic growth in the clarified
unsupplemented W medium (Control; Figure 6.1). This confirms the limited ability of the
W medium to support the growth of the probiotic cultures.

Figure 6.1: Effect of a prefermentation by YO-Mix T11 yogurt starter and of the addition of
glucose (Glu) or MRS to W medium on the subsequent growth of two probiotic cultures (Lb.
helveticus R0052 and Lb. rhamnosus R0011). Ctrl: control (no supplementation). The MRS
treatment consisted of a blend of the W medium and commercial MRS in a 3:1 ratio. Error bars
represent SEM.

Pre-fermentation with yoghurt starter or the addition of glucose alone did not improve
growth of the probiotics in the W medium (Figure 6.1). However, the addition of MRS

86
significantly (P < 0.05) improved growth in three of the 4 conditions (Figure 6.1). The
response to supplementation and pre-fermentation varies between probiotic cultures. The
latter has been noted frequently with lactic and probiotic bacteria (Champagne et al., 2005;
Saeed A et al., 2013). In the case of R0011 strain; the beneficial effect of MRS was
enhanced by prefermentation with the yoghurt starter. This suggests that pre-fermentation
with the starter modified the W medium by adding a growth factor for Lb. rhamnosus
R0011 which was not present in MRS. This could be an amino acid, a vitamin, a
nucleotide, formic acid or a fatty acid (Elli et al., 1999; Juillard et al., 1995; Partanen et al.,
2001; Saeed A et al., 2013; Suzuki et al., 1986). Moreover, pre-fermentation by the yogurt
starter was not detrimental to subsequent growth of the probiotics, which suggests that the
yogurt culture does not produce antagonistic compounds towards the probiotics.

An identical series of assays was carried out with clarified WM medium. The
observations were the same (data not shown). Therefore, the presence of caseins in the
medium during pre-fermentation with the starter did not substantially improve the W
medium for subsequent growth of probiotics.

6.3.2 Effect of MRS constituents on probiotic growth in fermented W medium

MRS medium is complex and expensive. Therefore, the identification of

specific constituents of MRS broth responsible for probiotic growth enhancement was
carried out. When the W medium was enriched with groups C and/or B components (Table
6.1), there was no significant improvement in the growth of either probiotic cultures
(Figure 6.2).

87
Figure 6.2: Effect of supplementing the clarified fermented “W medium” (W) with MRS
constituents (see Table 6.1). A: W + amino-based ingredients; B: W + lipid-linked ingredients; C:
W + minerals-buffers ingredients; AB: W + amino based + lipid-linked ingredients; AC: W +
amino-based + minerals-buffers ingredients; BC: W+ lipid-linked + minerals-buffers ingredients;
ABC: W + amino-based + minerals-buffers + lipid-linked ingredients; WMRS is a blend of W and
MRS medium in a 3:1 ratio. MRS 25%: positive control. All W media were supplemented by 5 g/L
of glucose. Bars represent ±SEM of 3 independent repetitions.

Group A constituents significantly enhanced the growth of both strains, particularly

strain R0052 (P<0.05) (Figure 6.2). This suggests that, group A ingredients probably
contained essential amino acids or peptides which could be easily assimilated by R0052.
Many studies have reported growth enhancement of probiotic cultures when individual
group A constituents were present in the growth medium (Champagne et al., 1999; Ibrahim
et al., 1994; Potvin et al., 1997).

When W medium was enriched with only group B or C ingredients no important

improvement of growth was noted for either strain (Figure 6.2). However, as opposed to
strain Lb. helveticus R0052, the growth of R0011 was better when W medium was enriched
with ingredients from groups A and C compared to A supplements alone. It remains to be
ascertained if this is linked to Mg or Mn minerals or to the buffer. The improvement of

88
growth of lactic cultures in dairy matrices can be enhanced by Mn or Mg addition
(Boyaval, 1989; Saeed A et al., 2013).

The positive control was MRS-25% broth. It was noteworthy that combining W
with MRS resulted in higher Lb. helveticus R0052 growth than in MRS alone (Figure 6.2).
This again points to the variable effect of nutrients on the growth of probiotic lactobacilli.

Lb. helveticus R0052 showed the highest growth in W media supplemented with
amino acid based ingredients. Thus, this strain was selected for further investigations.

6.3.3 Effect of various sources of peptones on the growth of Lactobacillus helveticus

R0052

The A group of ingredients is composed of beef extract (BE), yeast extract (YE)
and proteose peptone #3 (PP). Clarified fermented W media were supplemented with each
component as well as in combination (Figure 6.3). In order to prevent an effect of
concentration, the total quantity of ingredients added was adjusted at 6.25 g/L in all
treatments (Table 6.2).

As noted in previous assays (Figure 6.2) there was little or no growth in the
unsupplemented W medium. When the clarified fermented W medium was enriched with
the various MRS-based peptones, Lb. helveticus R0052 grew well (ODmax > 1.0) in all
media. The YE gave the highest values of the single ingredients, and pairing it with other
peptone sources did not amplify its beneficial effect. These data confirm the value of YE as
growth supplement for lactobacilli (Aeschlimann et al., 1990; Barascu, 2006; Ibrahim et
al., 1994; Potvin et al., 1997).

Protein hydrolysates distinguish themselves by the type of protein and enzyme

used and the extent of hydrolysis. To further study their bioactivity on the probiotic cell
growth, different hydrolysates were evaluated (Table 6.2). Irrespective of concentration,
there was little or no growth of Lb. helveticus R0052 in W media supplemented with a
laboratory-grade casein product, or the two ones which were highly hydrolyzed (Figure
6.4). Thus, as for lactococci (Juillard et al., 1995; St-Gelais et al., 1993), Lb. helveticus

89
R0052 growth seemed to be increased in the presence of oligopeptides rather than free
amino acids.

Figure 6.3: Effect of the supplementation of the clarified pre-fermented whey-based medium (W)
with individual or combined components of the amino acid based group of MRS broth. BE = beef
extract, YE = yeast extract, PP = proteose peptone ≠3 (See Table 6.2). Bars represent ±SEM.

Increasing “Bacto peptone” and “Trypticase peptone” concentration further

improved cell growth (Figure 6.4). But this was not the case with the “Tryptone enzymatic
digest”. These results confirm literature with respect to the source and concentration of
peptones even though, most information found with regards to the use of protein
hydrolysates as stimulatory factors for the growth of probiotic cultures were principally
carried out with milk and not on whey bases (Lucas et al., 2004; McComas et al., 2003;
Sodini et al., 2002). Moreover, in this, the evaluation of the growth of the probiotic culture
is carried out in consideration that a yoghurt starter is also present. As shown previously
(Figure 6.1), pre-fermentation with the starter improved the subsequent growth of Lb.
rhamnosus R0052. Data from this study suggest that the benefit of peptone
supplementation of a whey-based medium on a probiotic culture also occurs in association
with a yogurt starter.

90
Figure 6.4: Effect of the supplementation of the clarified pre-fermented whey-based
medium (W) with various protein hydrolysates on the growth of Lb. helveticus R0052.

As the bioactivity of peptones varies between cultures (Figure 6.2; Champagne et al.
(1999)) and is influenced by concentration, the development of a fermented beverage with a
given probiotic strain, particularly in association with a yogurt starter requires the selection
of the most appropriate peptone source and an evaluation of the concentration needed for
maximum benefit.

Since some laboratory peptones gave as high results as YE (Figure 6.3),

consideration was given to food-grade dairy-based peptones as supplements (Table 6.2).
The limited benefit of the casein-based peptone on Lb. helveticus R0052 was a concern
(Figure 6.4) Therefore, whey proteins were included in the assays (Table 6.2).

Good growth was noted with both lactalbumin-based hydrolysates and the extent of
growth was affected by concentration (Figure 6.5). At the opposite, DI-02021 did not
enhance cell growth. Compared to casein peptone (Figure 6.4) whey protein hydrolysates
seem more efficient to improve growth of Lb. helveticus R0052.

The food-grade whey protein peptone resulting in the highest cell growth was
Lacprodan DI-3071 (Figure 6.5). There was a statistically significant (p < 0.05) effect of

91
concentration, but since 25g/L DI-3071 only resulted in a 2 fold increase in OD (Figure
6.5) and would probably not be economically feasible, it was therefore decided to use a
supplementation level of 12.5 g/L for fermentation assays.

Figure 6.5: Effect of the supplementation of the clarified pre-fermented whey-based

medium (W) with various dairy-based hydrolysates.

Since the AS experiments are not carried out in the same conditions as of an actual
fermentation, the probiotic and the yogurt starter were co-cultured during fermentation in
unclarified W medium containing 10% protein concentration.

6.3.4 W medium fermentation by Lactobacillus helveticus R0052

Three whey-based beverages (W (“W medium”), WH (“W medium”+ hydrolysate)

and WMH (“WM medium” + hydrolysate)) were used to assess probiotic growth during
fermentation. The whey protein hydrolysate (H) used was LacProdan-3071 (12.5 g/L).

Without EpHC and whey peptone, Lb. helveticus R0052 reached 3.2x108 CFU/mL
in W medium at the end of the fermentation (pH 4.8) (Table 6.3). The addition of peptones

92
and milk doubled the CFUs in fermented media when the pH reached a value of 4.8.
Without EpHC, when the fermentation of media WH and WMH was extended for up to 6 h,
the pH continued to drop and CFUs increased to 2.0x109 CFU/ mL, which was more than 3
times higher than in W medium (Table 6.3). Thus, fermentation data regarding the effect of
peptones on growth of Lb. helveticus R0052 confirmed those of AS.

When EpHC was carried out to maintain the pH value at 4.8, extending the
fermentation for 6 h increased the CFUs by approximately 0.12 Log CFU/mL (39%
improvement). In the ANOVA analyses within each medium, this increase was not deemed
to be statistically significant (Table 6.3). However, examination of the data show that the
cell concentration increase in media with EpHC was systematic, and a paired t test did
show a statistically significant effect of EpHC. This was in line with the literature
(Champagne et al., 1996).

Overall data show that supplementing the W medium with the whey protein hydrolysate
was almost 7 times more efficient than EpHC in improving biomass yields under these
experimental conditions tested. Moreover, the results show the possibility to produce a
probiotic-containing beverage having 200 billion CFUs of probiotic bacteria per 100 mL of
portion.

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Table 6.3: Influence of supplementation of the W medium and external pH control (EpHC) on the growth of and acidification of Lb.
helveticus R0052 and St. thermophilus

Fermentation Analysis time

parameters
T0e1 +2h2 +4h +6h T0e +2h +4h +6h T0e +2h +4h +6h

Cell counts (Log CFU/mL)

pH
Media3 EpHCl
Lactobacillus helveticus R0052 Streptococcus thermophilus
W - 8.51 aA 8.56 aA 8.59 aA 8.60 aA 8.91aA 9.02 bA 8.93 bcA 8.90 cA 4.65 bA 4.55 cA 4.48 dA

+ 8.83 aB 8.87 aB 8.91 aB 8.75 aA 8.94 aA 8.96 aA 8.91 aA 8.94 aAB 4.79 aB 4.8 aB 4.8 aB
WH - 8.90 aB 9.12 bC 9.18 bC 9.26 bB 9.05 aB 8.96 abA 8.89 bA 8.95 bAB 4.56 bC 4.40 cC 4.30 dC
4.8
+ 8.87 aB 8.98 aC 9.29 bC 9.32 bBC 9.08 aB 8.97 abA 8.93 bA 8.93 bAB 4.81 aB 4.8 aB 4.8 aB
WMH - 8.75 aB 9.01 bC 9.18 bcC 9.28 cBC 9.00 aB 9.05 aA 9.05 aB 9.01 Ab 4.47 bD 4.27 cD 4.12 dD

+ 8.83 aB 9.06 bC 9.12 bC 9.44 cC 9.07 aB 9.06 aA 9.04 aB 9.04 aB 4.78 aB 4.8 aB 4.8aB
1
T0e =. Time at which the EpHC was initiated; this occurred when pH 4.8 had been reached, which varied between 5 and 6 h of
incubation.
2
+2h, +4h and +6h represent the incubation time (hours) after reaching T0e.
3
Media: W: Whey beverage without whey protein hydrolysate; WH: Whey beverage with whey protein hydrolysate; WMH: W+
liquid milk supplemented with whey protein hydrolysate.
4
EpHC: - : without external pH control, or + : with external pH control.
a, b,c,d
for a given row, values which are followed by the same lower-case letter are not statistically different (p > 0.05)
A, B,C,D
for a given column; values which are followed by the same capital letter are not significantly different (p>0.05).

94
It is noteworthy that supplementation of W medium with peptones and fermentations carried out
under EpHC had much lower effects on St. thermophilus than on Lb. helveticus R0052 (Table
6.3). At best, there was a 0.16 Log CFU/mL (44%) increase, and extended incubation had no
benefit in any treatment. The St. thermophilus population levels reached at pH 4.8 are in
accordance with other fermented dairy media (Tabasco et al., 2007).

Table 6.4 presents residual sugar concentrations and lactic acid production during
fermentations. In all conditions, there was residual lactose in the fermented medium. Therefore,
growth did not stop due to a lack of carbohydrates. Only small quantities of glucose were
registered, but galactose was detected at up to 11 g/L (Table 6.4). This is in accordance with the
metabolism of St. thermophilus, which uses the glucose fraction of lactose and excretes a sizeable
fraction of galactose in the medium (Hutkins et al., 1991). The lactic acid levels of the fermented
media at pH 4.8 were higher in the WH and WHM media than in the W medium. These data
suggest that the addition of peptone and milk increased the buffering capacity of the medium. In
itself, this could partially explain the beneficial effect of the supplements on the growth of the
lactobacilli. In all instances, the extended fermentations resulted in lower lactose concentrations
and higher lactic acid production. As was the case for CFUs, supplementation of the W medium
had a much greater effect on the concentrations of metabolites than did EpHC. Therefore, there
was some link between CFU increases and lactic acid production.

95
Table 6.4: Effect of external pH control (EpHC) on levels of carbohydrates and of lactic acid during the fermentation of whey-
based beverages
Fermentation
parameters Lactose (g/L) Glucose (g/L) Galactose (g/L) Lactic acid (g/L)
4 1 2
Media3 EpHC Initial T0e +4h Initial T0e +4h Initial T0e +4h Initial T0e +4h
W - 54.2aA 48.7aA 2.5aA 1.3aA 7.3aA 8.0aA 13.1aA 17.2aA
66.7
+ 52.7aAB 41.0bA 2.4aA 0.3aA 7.4aA 9.2bB 13.4aA 22.8bAB
WH - 46.1aAB 42.6aA 5.3 0.6aA 1.1aA 1.9 9.6aB 8.1bC 1.1 16.5aA 23.2bABC
66.7
+ 45.5aB 40.1aA 2.3aA 0.7aA 10.1aBC 9.7aD 16.8bA 22.3aAB
WMH - 70.4aC 63.8aB 2.9aA 0.8aA 10.5aC 11.0aE 18.4aA 24.2aBC
89.7
+ 67.1aC 54.3bC 2.8aA 0.9aA 10.8aC 11.2aE 19.2aA 29.4bC

1
T0e: time at which the EpHC, began; this occurred when the pH of the medium reached pH = 4.8, which varied between 5 and
6 h of incubation.
2
+4h represent the incubation time (hours) after reaching T0e.
3
Media: W: Whey beverage without whey protein hydrolysate; WH: Whey beverage with whey protein hydrolysate; WMH: W+
liquid milk supplemented with whey protein hydrolysate.
4
EpHC: - : without external pH control, or + : with external pH control.
a, b,c,d
for a given row, values which are followed by the same lower-case letter are not statistically different (p > 0.05)
A, B,C,D
for a given column; values which are followed by the same capital letter are not significantly different (p > 0.05).

96
6.4 Conclusion
This study reports two strategies used to obtain high viable counts of probiotic cells in a
high protein whey-based beverage. Commercial peptones (hydrolysates) added in whey-
based beverages enhanced probiotic growth from 8.2 Log CFU/mL to 9.2 Log CFU/mL.
Carrying out EpHC during the fermentation slightly improved viable counts but peptone
supplementation was much more effective. By using a whey protein hydrolysate, it was
possible to obtain a beverage entirely made of whey-based ingredients having a 10 %
protein content and 200 billion probiotic cells per 100 mL portion. This concentration is
approximately a hundred times higher than many commercial products currently on the
market.

97
6.5 Acknowledgments

This project was funded by the InnovAction program of the Ministère de l’Agriculture des
Pêcheries et de l’Alimentation (MAPAQ) and from Growing Forward of Agriculture and
Agri-Food Canada. Gratitude is expressed to Yves Raymond for technical assistance and to
Mario Proulx (Aliments Ultima) for scientific and market advice. Gratitude is expressed to
Arla and Lallemand Health Solutions, for WPC and starter cultures.

98
Conclusion générale
L’hypothèse de ce projet de recherche stipulait qu’il est possible d’obtenir une boisson
fermentée riche en protéines du lactosérum et aux propriétés organoleptiques acceptables.
Afin de vérifier cette hypothèse, différents types de concentrés de protéines ont été étudiés
afin de sélectionner les concentrés ou isolats protéiques de lactosérum qui permettent une
vitesse d’acidification élevée. Les ferments lactiques industriels qui acidifient rapidement et
conduisent à un produit fermenté de bonne saveur ont été identifiés. Enfin, différentes
approches (supplémentation et fermentation en contrôle de pH) ont été testées pour obtenir
des boissons fermentées à teneur élevée en probiotiques.

Les résultats présentés dans ce mémoire montrent qu'il est possible d'obtenir une boisson
fermentée riche en protéines de lactosérum contenant des quantités élevées en probiotiques
et ayant des propriétés organoleptiques acceptables.

Les vitesses d'acidification des concentrés protéiques utilisés dans cette étude dépendaient
fortement de leur composition chimique, de leur teneur en lactose et de leur pouvoir
tampon. En outre, le type de ferment a également influencé les profils d'acidification des
boissons. Les cultures thermophiles fermentaient les solutions plus rapidement que les
cultures mésophiles. De même que la source de CPL/IPL, les ferments affectent les
attributs sensoriels des boissons. C’est un CPL 50% a montré le meilleur potentiel pour le
développement de boissons fermentées.

Les CPL à une concentration de 10% de protéines ont été un bon milieu de fermentation et
de croissance pour les BL et probiotiques. De même, les mélanges de CPL avec du lait
écrémé étaient aussi de bons milieux de croissance des ferments. Cependant, peu importe
le type de milieu utilisé (CPL avec ou sans ajout du lait écrémé), aucune influence sur la
croissance des probiotiques n’a été observée. Indépendamment de la souche probiotique,
les comptes en cellules viables des ferments étaient supérieurs à 106 CFU mL-1. Lb.
rhamnosus R011 avait une concentration finale d’environ 2.5 x 107 CFU mL-1, Lb.
helveticus R0052 de 2.0 x 108 CFU mL-1 et 2.0 x 109 CFU mL-1 pour St. thermophilus.
Après 45 jours d’entreposage à faible pH (4.6), les concentrations de probiotiques sont
restées stables (>1 x107 CFU mL-1). Cependant, Lb. helveticus R0052 à mieux résisté que

99
Lb. rhamnosus R011. Une teneur élevée en oxygène dissous a été notée dans les boissons
contenant Lb. rhamnosus R011 comparativement à celles contenant Lb. helveticus R0052.
Ceci démontre l’effet de la souche sur la consommation d’oxygène dans le milieu.

Différents hydrolysats ont été évalués comme suppléments de croissance des probiotiques
dans les boissons fermentées et comparés à l’extrait de levure. La croissance des
probiotiques dans les milieux enrichis était fortement dépendante de la souche puisqu’il a
été observé que les concentrations cellulaires de Lb. helveticus R0052 étaient toujours plus
élevées que celles de Lb. rhamnosus R0011. Les peptones commerciales sont de bons
substituts aux extraits de levures pour favoriser la croissance des probiotiques. L’hydrolysat
de protéines de lactosérum a favorisé la croissance de Lb. helveticus R0052 de 2.0 x 108
CFU mL-1 à 2.0 x 109 CFU mL-1. La fermentation sous contrôle pH a faiblement augmenté
les concentrations cellulaires.

Ce travail a donc permis d’acquérir de nouvelles connaissances relativement au

développement de boissons à base de concentrés de lactosérum aux propriétés
organoleptiques désirables ayant une haute teneur en protéines et en bactéries probiotiques
(180 milliards de cellules probiotiques par portion de 100 ml). Par contre, une étude plus
approfondie sur la fonctionnalité et la digestibilité des peptides présents dans ces boissons
donnerait plus d'information relativement aux attributs santé. De plus, une étude de la
survie gastro-intestinale des souches probiotiques serait à réaliser afin de porter des
allégations sur cette gamme de produit. Des études sur la possibilité d'ajouter des
concentrés de jus de fruits aux boissons pourraient constituer un moyen novateur d’élargir
la diversité de cette gamme de produits.

100
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