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PIERRE-LUC CHAMPIGNY
BIOCOMPATIBILITÉ DES BACTÉRIES LACTIQUES
PROBIOTIQUES ET D’AFFINAGE AVEC DES
MYCÈTES DU CAMEMBERT ISOLÉES DE LAITS
DE TERROIR QUÉBÉCOIS
Mémoire présenté
à la Faculté des études supérieures de l’Université Laval
dans le cadre du programme de maîtrise en sciences et technologie des aliments
pour l’obtention du grade de Maître ès sciences (M. Sc.)
DEPARTEMENT DES SCIENCES DES ALIMENTS ET DE NUTRITION
FACULTÉ DES SCIENCES DE L'AGRICULTURE ET DE L'ALIMENTATION
UNIVERSITÉ LAVAL
QUÉBEC
2011
© Pierre-Luc Champigny, 2011
Résumé
L’objectif de cette étude était de vérifier la biocompatibilité entre les mycètes du fromage
Camembert et les bactéries lactiques (probiotiques ou d’affinage). La plupart des souches
fongiques utilisées ont été isolées de laits en provenance du terroir québécois et deux laits
d’origines différentes ont servi pour la fabrication de caillés modèles. La
spectrophotométrie automatisée (SA) a été employée pour présélectionner des mélanges de
souches mycéliennes et bactériennes biocompatibles. Des milieux à base de lait furent
fermentés par des mycètes et étaient ensuite inoculés avec les bactéries. La croissance
préalable des mycètes stimulait ou inhibait les bactéries, mais les effets étaient mineurs et
variaient selon les souches. Par la suite, afin de confirmer ces résultats, des caillés modèles
ont été inoculés simultanément par des combinaisons de bactéries et de mycètes. L’absence
d’inhibition des bactéries par les mycètes observée en SA a été confirmée, mais les
interactions en caillé modèle différaient de celles notées en SA en raison de l’évolution
différente du pH dans les deux séries expérimentales. Finalement, des fromages Camembert
probiotiques ont été fabriqués avec des souches du terroir et commerciales. Le Camembert
s’est révélé un aliment intéressant pour favoriser la survie des bactéries lactiques. Par
contre, aucun mélange de souches fongiques n’a été systématiquement meilleur qu’un autre
pour stimuler la viabilité des probiotiques.
ii
Abstract
This study was carried out to verify the biocompatibility between the mycetes of
Camembert cheese and lactic cultures (probiotic and ripening strains). Most of the fungi
strains used had been isolated from different milk sources over the province of Quebec
(Canada) and two different kinds of milk were used to produce cheese slurries. Automated
spectrophotometry (AS) was employed to screen some biocompatible pairings of mycete
and bacterial strains. A milk medium was fermented by yeasts and moulds and then
inoculated with bacteria. The previous growth of the mycetes was sometimes stimulatory
and sometimes inhibitory, but the effects were minor and varied as a function of the strains.
Subsequently, to confirm these AS results, cheese slurries were inoculated simultaneously
with different strains combinations. Finally, pilot scale Camembert cheese was produced to
verify its ability to support probiotic bacterial cultures viability. The absence of inhibition
of the bacteria by the mycetes in SA was confirmed, but the interactions in the cheese
slurries differed from those noted in AS because of the different pH patterns in the two
experimental series. Camembert was shown to have potential to favour the viability of
probiotic bacterial strains during ripening and storage. However, no mycete mix was
systematically better than another to stimulate this viability.
Avant-Propos
Ce mémoire de maîtrise écrit sous forme d’articles est composé de 5 chapitres dont les deux
premiers font état des connaissances, de la présentation de l'hypothèse et des objectifs à la
genèse de ces travaux. La suite de cet ouvrage est constituée de trois articles rédigés en
anglais mais tous précédés d'un résumé en français. Ces papiers seront soumis pour
publication dans des revues périodiques spécialisées en science des produits laitiers. Les
manipulations en laboratoire qui ont généré les résultats de ces publications ont été
réalisées par Pierre-Luc Champigny et deux stagiaires dont il a supervisé le travail: Mathieu
Lapointe et Mélanie Gobeil-Richard. Suite à l’obtention de ces résultats, une première
ébauche des articles a été rédigée par Pierre-Luc Champigny. De leur côté, Dr Claude
Champagne, Dr Daniel St-Gelais, Dr Ismail Fliss et Dr Steve Labrie sont les co-auteurs de
ces articles. Ils ont participé en fournissant un support scientifique lors de la planification
de l’expérimentation, de l'analyse des données et du processus de rédaction.
Le chapitre 3, «Biocompatibility between probiotic/specialty lactic cultures and mycetes
found in dairy products», porte sur une expérimentation de spectrophotométrie automatisée
réalisée afin de présélectionner des paires de mycètes et de bactéries lactiques
biocompatibles. Des caillés modèles de fromage Camembert étaient affinés pendant 12
jours par des levures et moisissures pour ensuite en extraire un lactosérum acellulaire. Par
la suite, ce lactosérum était inoculé des différentes souches bactériennes dans le but de
découvrir des interactions favorables ou défavorables entre celles-ci.
Le chapitre 4, «Biocompatibility between probiotic/specialty lactic acid bacteria and
mycetes in Camembert cheese slurry», traite encore de la biocompatibilité entre mycètes et
bactéries lactiques mais cette fois-ci dans un modèle plus près de la réalité du Camembert.
Afin de vérifier les prédictions de la méthode utilisée dans le chapitre précédent, des caillés
modèles inoculés simultanément de levures et moisissures ainsi que de bactéries lactiques
ont été affinés pendant 12 jours. La viabilité des bactéries et des mycètes a été évaluée au
iv
cours de cet affinage afin de voir si les interactions observées en spectrophotométrie
automatisée se répèteraient.
Le chapitre 5, «Viability of probiotic bacteria in Camembert cheese made with fungi strains
isolated from Quebec terroir milk», rapporte une expérimentation qui a été réalisé afin de
vérifier la capacité du fromage Camembert à optimiser la viabilité de bactéries
probiotiques. Il se distingue du chapitre 4 par la fabrication de fromages à l’échelle pilote (à
partir de 200 L de lait) au lieu de l’utilisation de caillés modèles. De plus, des suivis de
viabilité des bactéries, du pH et de la protéolyse ont été accomplis à la surface et au cœur
des fromages Camembert pendant 30 jours d’affinage et d’entreposage. Les traitements
expérimentaux étant des fromages affinés par différentes souches de levures et moisissures
du terroir, l’impact de celles-ci sur la viabilité en lien avec leur indice de protéolyse et le
pH a été étudié.
Finalement, ce mémoire se termine par une conclusion générale. Celle-ci met en évidence
l’impact possible des travaux réalisés ainsi que les résultats obtenus. Les perspectives de
recherches qu’entraînent ces résultats sont aussi exposées. Enfin, la bibliographie, indiquant
toutes les publications et les références électroniques citées au courant du texte complète
cet ouvrage.
v
Remerciements
J’aimerais par la présente remercier tout d’abord mon co-directeur, Dr Claude Champagne,
pour m’avoir accueilli à bras ouverts au sein de son équipe au CRDA (Centre de Recherche
et Développement sur les Aliments) de Saint-Hyacinthe. Sa grande disponibilité, sa
confiance en moi, son écoute et ses conseils judicieux ont beaucoup contribué à ma
formation. De plus, merci aux autres chercheurs qui étaient membres de l’équipe : mon
directeur, Dr Ismail Fliss, Dr. Daniel St-Gelais et Dr Steve Labrie. Chacun à votre façon
vous avez contribué efficacement à m’aider à réaliser ce projet autant du côté scientifique
qu’administratif. Je voulais aussi souligner l’aimable participation de Dr Jean-Christophe
Vuillemard en tant qu’évaluateur de ce mémoire.
Je remercie aussi le Fonds Québécois de Recherche sur la Nature et les Technologies
(FQRNT) pour la bourse de maîtrise qui m’a offert un bon soutien financier tout au long de
ma maîtrise.
De façon générale, mes remerciements vont également au personnel et étudiants du CRDA
pour leur bienveillance et leur gentillesse qui m’ont donné envie à chaque matin de me
lever du bon pied afin de poursuivre mes travaux de maîtrise. Plus précisément, je voulais
remercier Yves Raymond, assistant de recherche, pour ses nombreux conseils ainsi que sa
patience légendaire. De plus, merci à Nancy Guertin et Gaétan Bélanger, assistants de
recherche ainsi qu’à Mathieu Lapointe et Mélanie Gobeil-Richard, stagiaires, pour la
grande aide qu’ils m’ont fourni lors des manipulations en laboratoire et en fromagerie.
Finalement, j’aimerais remercier mes parents Marie-Claude Larrivée et Luc Champigny
pour leur support tout au long de mes études. Merci de m’avoir transmis de belles valeurs
comme la discipline, l’honnêteté et le dépassement de soi qui me permettent de prendre les
bonnes décisions autant dans ma vie personnelle que professionnelle. Mes derniers
remerciements vont bien sûr à ma muse et complice de vie Anne-Marie Desbiens. Tout au
long, de cette maîtrise tu as été auprès de moi et ton appui fut considérablement apprécié.
vi
Un petit pas pour la science
mais un grand pas pour moi!
Table des matières
Résumé ..................................................................................................................................... i
Abstract .................................................................................................................................. ii
Avant-Propos ........................................................................................................................ iii
Remerciements ........................................................................................................................ v
Table des matières ............................................................................................................... vii
Liste des tableaux ................................................................................................................... ix
Liste des figures ...................................................................................................................... x
Introduction ............................................................................................................................. 1
Chapitre 1. État des connaissances ......................................................................................... 3
1.1 Le fromage, un écosystème .......................................................................................... 3
1.2 La microbiologie de l’affinage ..................................................................................... 4
1.2.1 Les flores responsables .......................................................................................... 5
1.2.2 L’effet des conditions d’affinage ......................................................................... 10
1.2.3 Le type de lait utilisé ............................................................................................ 11
1.3 Les bactéries probiotiques .......................................................................................... 11
1.3.1 L’effet des probiotiques sur la santé .................................................................... 13
1.3.2 Les aliments probiotiques .................................................................................... 14
1.4 La biocompatibilité entre microorganismes du fromage ............................................ 17
Chapitre 2. Hypothèse et Objectifs ....................................................................................... 21
Chapitre 3 : Biocompatibilité des bactéries lactiques probiotiques et d’affinage avec des
mycètes isolées dans les produits laitiers. ............................................................................. 22
Résumé .................................................................................................................................. 23
Abstract ................................................................................................................................. 24
3.1 Introduction ................................................................................................................. 25
3.2 Materials and methods ................................................................................................ 26
3.2.1 Strains (mycetes and bacteria) and milk sources ................................................. 26
3.2.2 Inocula preparation and cultures conditions ........................................................ 28
3.2.3 Production of cheese slurries ............................................................................... 29
3.2.4 Enumeration of the mycetes ................................................................................ 30
3.2.5 CFW extraction .................................................................................................... 31
3.2.6 Automated spectrophotometry assays ................................................................. 31
3.2.6 Statistical analyses ............................................................................................... 32
3.3 Results and Discussion ............................................................................................... 33
3.3.1 Growth of mycete strains on the cheese slurries .................................................. 33
3.3.2 Growth rates of lactobacilli .................................................................................. 35
3.3.3 Biomass levels of lactobacilli and bifidobacteria ................................................ 37
3.3.4 Milk source influence on biomass levels ............................................................. 41
3.4 Conclusion .................................................................................................................. 42
Chapitre 4 : Biocompatibilité des bactéries lactiques probiotiques et d’affinage avec les
mycètes au sein de caillés modèles de fromage Camembert. ............................................... 44
Résumé .................................................................................................................................. 45
Abstract ................................................................................................................................. 46
4.1 Introduction ................................................................................................................. 47
4.2 Materials and methods ................................................................................................ 49
viii
4.2.1 Strains (mycetes and bacteria) and milk sources ................................................. 49
4.2.2 Inocula preparation and cultures conditions ........................................................ 50
4.2.3 Production of cheese slurry powder ..................................................................... 51
4.2.4 Cheese slurries assays .......................................................................................... 51
4.2.5 Enumerations of microorganisms and pH measurement ..................................... 53
4.2.6 Statistical analyses ............................................................................................... 54
4.3 Results and discussion ................................................................................................ 54
4.3.1 Probiotic culture biocompatibility and viability .................................................. 54
4.3.2 Ripening bacteria biocompatibility and viability ................................................. 57
4.3.3 Yeasts and moulds biocompatibility with bacteria .............................................. 60
4.4. Conclusion ................................................................................................................. 62
Chapitre 5 : Viabilité de bactéries probiotiques au sein de fromages Camembert fabriqués
avec des souches fongiques isolées de lait de terroir québécois. .......................................... 64
Résumé .................................................................................................................................. 65
Abstract ................................................................................................................................. 66
5.1 Introduction ................................................................................................................. 67
5.2 Materials and Methods ................................................................................................ 69
5.2.1 Strains (mycetes and bacteria) ............................................................................. 69
5.2.2 Inocula preparation and cultures conditions ........................................................ 70
5.2.3 Cheese Production Assays ................................................................................... 71
5.2.4 Enumerations of cheese microorganisms ............................................................ 72
5.2.5 Analyses ............................................................................................................... 73
5.2.6 Statistical analyses ............................................................................................... 74
5.3. Results and discussion ............................................................................................... 75
5.3.1 Yeast and mould cell counts ................................................................................ 75
5.3.2 pH and proteolysis index ..................................................................................... 76
5.3.3 Probiotic bacteria viability ................................................................................... 79
5.4 Conclusion .................................................................................................................. 81
Conclusion ............................................................................................................................ 83
Bibliographie ........................................................................................................................ 84
Liste des tableaux
Tableau 1.1. Souches de bactéries probiotiques utilisées commercialement. ...................... 12
Tableau 1.2. Tableau sommaire des allégations non spécifiques à la souche acceptées pour
les probiotiques et les espèces admissibles dans le cadre de ces allégations. ............... 13
Table 3.1. Bacterial and mycete strains used for this work .................................................. 28
Table 3.2. Growth (log CFU/g) of the mycete strains and pH of the cheese slurries after 12
days at 12˚C and 95% RH. ............................................................................................ 33
Table 3.3. Bacteria growth difference between the Milk B and the Milk A CFWs. ............ 42
Table 4.1. Bacterial and mycete strains used for this work .................................................. 49
Table 4.2. Combinations of bacteria and mycetes strains done in cheese slurries ............... 52
Table 4.3. Viability (log CFU g-1
) of Lactobacillus rhamnosus R0011 in Camembert cheese
slurries ripened up to 12 days with different yeast and mould strains. ......................... 55
Table 4.4. Variation of the homogenate pH of different Camembert cheese slurries made
with 2 different sources of milk and inoculated with Lactobacillus rhamnosus R0011
and different yeast and mould strains and ripened 12 days .......................................... 56
Table 4.5. Viability of L. casei A180 at D12 in Camembert cheese slurries ripened 12 days
with combination of different yeast and mould strains in 2 different sources of milk . 58
Table 4.6. Homogenate pH at D12 of Camembert cheese slurries inoculated with L. casei
A180 and ripened 12 days with combination of different yeast and mould strains in 2
different sources of milk ............................................................................................... 59
Table 5.1. Bacterial and mycete strains used for this work .................................................. 69
Table 5.2. Combinations of bacterial and mycete strains done in cheese ............................ 71
Table 5.3. Cell counts (log CFU g-1
) of different yeast and mould blends (Y/M) in
Camembert cheese ripened 30 days. ............................................................................. 75
Table 5.4. Viability (log CFU g-1
) of Bifidobacterium lactis BB12 and Lactobacillus
rhamnosus R0011 in Camembert cheese ripened 30 days with combination of different
yeast and mould (Y/M). ................................................................................................ 79
Liste des figures
Figure 3.1. Growth rates (μmax) of lactobacilli (A180, R0011, GG, BKA, BKB) in the Cell
Free Whey (CFW) obtained following the growth of 14 yeasts or moulds on Milk A
(Holstein) cheese slurries. ............................................................................................. 36
Figure 3.2. Growth of Lactobacillus bacterial strains (A180, R0011, GG, BKA, BKB) in
Cell Free Whey (CFW) of Milk A (Holstein). .............................................................. 38
Figure 3.3. Growth of Bifidobacteria strains (BB12 and R0175) in Cell Free Whey (CFW)
of Milk A (Holstein). .................................................................................................... 39
Figure 4.1. Effect of cheese slurry inoculation with two different bacterial strains (L.
rhamnosus R0011, and L. casei A180) on the growth of Penicillium camemberti PC
PSM2, Geotrichum candidum LMA 664 and Debaryomyces hansenii LMA 668. ...... 61
Figure 5.1. pH at the centre (1) and at the rind (2) of three Camembert cheese treatments
ripened 30 days with combination of different yeast and mould strains (A, B, C). ..... 76
Figure 5.2. Proteolysis indexes (% TCASN/TN) at the core (1) and the rind (2) of three
Camembert cheese treatments ripened 30 days with combination of different yeast and
mould strains (A, B, C). ................................................................................................ 78
1
Introduction
Actuellement, il en coûte 172 milliards de dollars par année en services de santé publique
au Canada (Conseil canadien de la santé, 2009), ce qui représente une dépense d’environ
5000 dollars par Canadien. Contrairement à la croyance populaire, ces dépenses élevées ne
seraient pas uniquement dues au vieillissement général de la population, mais
principalement à l’utilisation accrue des services (Conseil canadien de la santé, 2009).
Conséquemment, afin de réduire la fréquence d’utilisation des soins de santé, le vieux
dicton « il vaut mieux prévenir que guérir » n’a jamais été autant d’actualité. Présentement,
plusieurs recherches établissent l’importance d’effectuer divers choix alimentaires dans
l’optique de limiter les facteurs de risques entraînant certaines maladies. Ainsi, les
composés alimentaires appelés nutraceutiques, bénéfiques pour la santé au-delà de leur
aspect nutritionnel (Chen et al., 2006), suscitent un intérêt majeur. Entre autres, les acides
gras oméga 3, les polyphénols, les prébiotiques et les probiotiques en font partie.
Parallèlement à ces découvertes, le concept d’aliment fonctionnel, décrit comme un aliment
qui contient naturellement ou auquel sont ajoutés des nutraceutiques, a vu le jour.
Les bactéries probiotiques sont des microorganismes vivants qui, lorsqu’ils sont
administrés en quantités adéquates, exercent une action bénéfique sur la santé de l’hôte
(FAO/OMS, 2001). C’est pourquoi divers aliments fonctionnels existent déjà, afin de les
transporter et de maximiser leur survie. Une panoplie de conditions comme le pH, le
potentiel redox et la température d’entreposage peuvent moduler la viabilité des bactéries
au sein des aliments (Champagne et al., 2005). Assurément, le choix de l’aliment dépendra
de l’effet de ces propriétés sur le microorganisme d’intérêt. À l’heure actuelle, l’aliment
fonctionnel le plus commun pour véhiculer les probiotiques demeure le yogourt.
Cependant, cette matrice au potentiel redox positif et au pH bas n’offre pas toujours les
meilleures conditions de survie à ces bactéries avant leur ingestion (Shah, 2000).
Évidemment, d’autres aliments proposent de meilleures conditions de survie pour les
probiotiques. Certains types de fromage en font partie. Un bon exemple est le cheddar qui
2
détient un potentiel redox négatif et un pH plus près de la neutralité que celui du yogourt
(Phillips et al., 2006; Daigle et al., 1999). Également, le Camembert se révèle intéressant
pour véhiculer des probiotiques. En effet, des souches de bactéries potentiellement
probiotiques ont été isolées à partir de fromages Camembert au lait cru (Coeuret et al.,
2004). Toutefois, contrairement au cheddar, les caractéristiques qui permettent la survie des
probiotiques dans ce type de fromage sont peu connues.
Dans cette optique, l’étude de l’optimisation de la viabilité des bactéries probiotiques dans
les fromages de type Camembert doit être approfondie. Avant affinage, le Camembert ne
semble pas offrir de bonnes conditions de survie aux bactéries lactiques puisque son pH est
plus acide que ceux des fromages à pâte ferme. Par contre, lors du développement des
levures et moisissures (mycètes) qui forment la croûte fleurie, des conditions
potentiellement propices à la viabilité des probiotiques apparaîtraient. La remontée de pH
près de la neutralité provoquée par la croissance des levures et moisissures pourrait donc,
en partie, expliquer la viabilité des bactéries lactiques dans le Camembert (Spinnler et
Gripon, 2004). En plus de ce phénomène, il pourrait y avoir une véritable biocompatibilité
entre bactéries lactiques et mycètes. En fait, la nature des interactions entre ces
microorganismes semble varier d’une espèce et d’une souche à l’autre (De Freitas et al.,
2009). Également, l’activité des bactéries lactiques d’affinage pourrait aussi être favorisée
grâce à ces interactions. Un travail doit donc être fait afin de déterminer les combinaisons
idéales entre mycètes et bactéries lactiques.
Dans le cadre de travaux antérieurs, des souches de levures et moisissures ont été
identifiées et isolées à partir de laits de terroirs québécois. Ainsi, les interactions entre ces
souches, les différentes bactéries lactiques et les laits de terroir provenant de deux espèces
de vaches différentes feront l’objet de cette étude. Par conséquent, utiliser des laits et des
souches de levures et moisissures locales permettra de développer des produits avec valeur
ajoutée, issus du terroir québécois.
Chapitre 1. État des connaissances
1.1 Le fromage, un écosystème
Le fromage est souvent qualifié d’aliment « vivant », car il abrite une diversité microbienne
imposante. Tout au long de sa fabrication et de son affinage, des microorganismes se
partagent les nutriments disponibles, profitent des métabolites de certains ou meurent et
permettent la croissance d’autres. La flore microbienne du fromage varie entre autres selon
l’espèce de vaches laitières, le pâturage où celles-ci se nourrissent, les traitements
physiques que le lait subit avant de devenir fromage et le type de ferment ajouté.
Parmi tous ces facteurs, le fromager doit contrôler ceux en mesure de l’être afin d’obtenir
un produit fini de qualité constante. Ce contrôle est important, car les microorganismes en
présence, par leur métabolisme et leurs enzymes, collaborent à la succession de réactions
biochimiques qui influencent autant la texture que la flaveur du produit fini. L’aspect
microbiologique que le fromager gère le mieux est le choix du ferment. Celui-ci contient
principalement des bactéries lactiques responsables, à court terme, de l’acidification du
fromage et, à long terme, de son affinage. D’autres types de microorganismes peuvent aussi
être ajoutés au ferment. Par exemple, dans le cas des fromages à croûtes fleuries, le
fromager peut décider d’intégrer à son ferment des levures et moisissures judicieusement
choisies pour en favoriser la croissance lors de l’affinage. Enfin, l’arrivée du concept
d’aliments probiotiques incite certains fromagers à en ajouter à leurs produits.
La complexité de la maturation fromagère repose donc principalement sur sa flore
microbienne variée. Ce travail fait état des connaissances concernant la microbiologie de
l’affinage du fromage et l’ajout de probiotiques à celui-ci. Le phénomène de l’affinage du
Camembert sera d’abord éclairci et les flores microbiennes qui en sont responsables
présentées. Ensuite, les bactéries probiotiques et plus précisément le concept d’aliment
probiotique seront développés. Finalement, ce chapitre abordera les interactions entre
microorganismes et le phénomène de biocompatibilité.
4
1.2 La microbiologie de l’affinage
Un fromage en cours d’affinage est le lieu d’une pléiade de réactions biochimiques se
produisant l’une à la suite de l’autre ou simultanément. Selon le type de produit fabriqué, la
durée de la maturation peut être de quelques dizaines de jours ou de quelques années.
Comme mentionné plus haut, les flores microbiennes impliquées dans le vieillissement ne
sont pas les mêmes en fonction du produit fini désiré. Par exemple, un fabricant de cheddar
ne désire habituellement pas que des levures et moisissures œuvrent dans la maturation de
son produit tandis qu’un producteur de Camembert doit favoriser leur croissance à la
surface de ses fromages.
Lors de la fabrication et au tout début de l’affinage, la flore microbienne est d’abord
responsable de la bioconversion du citrate qui donne lieu à l’apparition de certains arômes,
en particulier l’acétaldéhyde et le diacétyle. Plus tard, lors de la maturation, les enzymes en
provenance des flores microbiennes jouent des rôles importants dans la création de flaveurs
et de textures particulières. De fait, c’est grâce à elles que des réactions comme la
protéolyse et la lipolyse modifient l’aspect sensoriel du fromage. D’une part, les enzymes
lipolytiques agissent sur les triacylglycérols du fromage en hydrolysant les liens esters des
acides gras. Ainsi, les acides gras libres contribuent à la flaveur du fromage. D’autre part,
les enzymes protéolytiques, les protéinases et les peptidases hydrolysent les liens
peptidiques des protéines ou des peptides. Ces bris de liens modifient la structure de la
matrice fromagère. De plus, la libération de certains peptides entraîne une augmentation de
l’amertume du fromage. Afin de diminuer cette amertume, des peptidases brisent les liens
entre les acides aminés de ces peptides. De la sorte, la présence d’acides aminés libres peut
entraîner des saveurs comme l’umami dans le cas de l’acide glutamique. Toutefois, c’est le
catabolisme des acides aminés plutôt que les acides aminés libres non métabolisés qui dicte
la flaveur des fromages en dégageant des composés soufrés et azotés (Peláez et Requena,
2005).
5
En résumé, la lipolyse et la protéolyse sont au premier plan lors du développement de la
texture et de la flaveur propre à un fromage affiné. L’activité de ces enzymes doit être bien
dosée et elle repose en grande partie sur les microorganismes du microbiote et les
conditions d’affinage.
1.2.1 Les flores responsables
Les microorganismes responsables de l’affinage du fromage peuvent être déjà présents dans
le ferment utilisé pour la fabrication, ajoutés spécifiquement au début de l’affinage et/ou
présents naturellement dans le lait. Dans ce dernier cas, ils font partie de la flore secondaire.
Par conséquent, l’utilisation de lait cru permet d’intégrer une plus grande variété de souches
qui, lors de l’affinage, atteignent des populations de l’ordre de 105 à 10
7 ufc g
-1 (Cogan,
2003). Pour pallier en partie la diminution de la flore secondaire causée par un traitement
thermique du lait, des bactéries constituant naturellement celle-ci peuvent être ajoutées au
ferment (Peláez et Requena, 2005). Aussi, une autre façon d’intégrer des microorganismes
est de les vaporiser à la surface des meules dans les salles de maturation. Finalement, la
flore secondaire peut provenir de l’environnement de la fromagerie. Certaines chambres
d’affinage de Camembert contiennent naturellement des souches qui dominent la flore de
surface des meules.
Les bactéries lactiques, les levures, les moisissures, les bactéries corynéformes, les
micrococacceae et les propionibactéries sont les microorganismes les plus fréquemment
retrouvés dans les microbiotes des divers fromages affinés. Ils cohabitent donc au sein de la
matrice fromagère et ils s’influencent l’un et l’autre dans leur métabolisme.
1.2.1.1 Les bactéries lactiques
Les ferments utilisés pour la fabrication de fromage comprennent principalement les genres
de bactéries lactiques Lactococcus, Leuconostocs, Streptococcus, Lactobacillus et
Enterococcus (Beresford et al., 2001).
6
Lors de la production, la croissance des bactéries du ferment, flore primaire, est
principalement responsable de l’acidification du lait en métabolisant le lactose en acide
lactique (lactate). Par la suite, lors de l’affinage, leur autolyse entraîne la libération
d’enzymes essentielles. Les souches de Lactococcus lactis ssp. cremoris sont souvent plus
autolytiques que celles de Lc. lactis ssp. lactis (Vegarud et al., 1983). C’est en partie
pourquoi Lc. lactis ssp. cremoris produit parfois plus rapidement un fromage de type
Camembert ayant un goût prononcé que Lc. lactis ssp. lactis (Cogan, 2003).
Ensuite, les bactéries lactiques de la flore secondaire participant à l’affinage du fromage
sont principalement des lactobacilles hétérofermentaires facultatives. Lactobacillus casei et
Lb. paracasei sont donc les plus présents dans le Camembert, mais le genre Pediococcus et
les lactobacilles hétérofermentaires obligatoires Lb. brevis et Lb. fermentum en font aussi
partie (Cogan, 2003). Contrairement aux bactéries de la flore primaire, les bactéries de la
flore secondaire subissent une lyse lente au sein de la matrice fromagère et le rôle de ces
bactéries dans la maturation du Camembert n’est pas totalement éclairci. Toutefois, leur
métabolisme, lorsqu’elles sont vivantes, contribue à la flaveur autrement que par leur lyse
qui libère des protéases et des lipases intracellulaires (Soda et al., 2000). Entre autres, ces
bactéries libèrent les acides aminés des peptides et les catabolisent tout en demeurant
vivantes (Peláez et Requena, 2005).
Les conditions favorisant la croissance des bactéries lactiques varient d’une espèce et d’une
souche à l’autre. Généralement, leur croissance est optimale à des pH autour de 6 et
certaines tolèrent des conditions aérobies même si la plupart préfèrent l’anaérobiose.
L’effet du sel diffère aussi d’une espèce à l’autre. La lyse de la flore primaire augmentera
avec le taux de sel aqueux tandis que les bactéries lactiques de la flore secondaire sont
assez résistantes au sel, la plupart d’entre elles se multipliant à un taux de 8% (Cogan,
2003).
7
1.2.1.2 Les levures
Les espèces de levures habituellement retrouvées dans les fromages de type Camembert
sont Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces lactis, Kluyveromyces
marxianus, Saccharomyces cerevisiae et Yarrowia lipolytica (Besançon et al., 1992;
Roosista et Fleet, 1996; Beresford et al., 2001; Boutrou et Guéguen, 2005). Geotrichum
candidum est une espèce particulière car elle est dimorphique. C'est-à-dire qu’elle adopte
des formes différentes dépendamment des souches. En effet, elle peut se répliquer en
formant un mycélium comme le fait une moisissure ou tout simplement des cellules
individuelles comme le fait traditionnellement une levure. Sur le plan écologique et
moléculaire G. candidum se comporte plutôt comme une levure (Lamontagne et al., 2002).
Les levures sont des microorganismes faisant surtout partie de la flore des fromages à
croûte fleurie (ex. Camembert ou Brie) ou lavée (ex. Oka). Reconnues pour tolérer de bas
pH, elles se développent lentement mais avec constance dans ces fromages. Selon les
souches, les levures consomment le lactose, le lactate, et/ou le galactose. Certaines espèces
comme G. candidum sont aérobies strictes; celles-ci ne peuvent donc que se développer en
surface (Boutrou et Guéguen, 2005). Les levures et les moisissures contribuent à la
remontée de pH de la matrice fromagère en métabolisant le lactate en H2O et en CO2. Leur
protéolyse participe aussi à cette remontée en libérant les groupements NH3 des acides
aminés (Beresford et Williams, 2004).
Les métabolites produits par les levures contribuant à la maturation sont l’éthanol,
l’acétaldéhyde et le CO2. De plus, certaines levures sont aptes à la protéolyse et à la
lipolyse. Toutefois, les souches de levures responsables de l’affinage doivent être bien
contrôlées. En effet, certaines souches de levures altèrent le produit en lui conférant des
saveurs fruitées ou amères parfois jugées indésirables en fonction de la concentration.
Aussi, une production trop élevée de dioxyde de carbone gazeux peut gâcher la texture d’un
Camembert. Comme les souches ne sont pas toutes tolérantes au sel, le salage du fromage
permet entre autres de les sélectionner (Beresford et Williams, 2004).
8
Enfin, l’implantation des levures à la surface d’un fromage précède souvent la venue d’un
autre type de microorganismes qui sont les moisissures. Les levures contribuent
indirectement à leur croissance en hydrolysant les protéines et la matière grasse.
1.2.1.3 Les moisissures
Semblables aux levures pour ce qui est des conditions et des substrats de croissance, les
moisissures sont cependant toutes des microorganismes aérobies obligatoires. Ainsi, elles
s’établissent surtout à la surface des fromages. Cependant, elles peuvent être présentes au
cœur d’une meule s’il y a des cavités pour l’aérer. C’est le cas de Penicillium roqueforti,
responsable des régions bleues internes du Roquefort. Penicillium camemberti est l’autre
moisissure fréquemment utilisée en fromagerie, entre autres, pour la production de
fromages à croûte fleurie de type Camembert (Aldarf et al., 2006).
Comparativement à P. roqueforti qui se développe en trois ou quatre jours, P. camemberti
se caractérise par sa faible vitesse de croissance. Son apparition à la surface d’un
Camembert autour du sixième jour d’affinage est généralement précédée par le
développement des levures (Leclercq-Perlat et al., 2004a; Cerning et al., 1987). L’activité
protéolytique et lipolytique des Penicillium influence beaucoup la maturation. Les acides
aminés issus de leur protéolyse forment ultimement des cétones, des aldéhydes, des alcools
aromatiques, des molécules soufrées de même que de l’ammoniac. Ils sont aussi
lipolytiques. Par exemple, ils métabolisent des méthylcétones à partir des gras saturés. Ces
phénomènes sont essentiels à l’apparition de composés aromatiques volatiles liés aux goûts
caractéristiques du Camembert et du fromage bleu. Par contre, ces réactions ne doivent pas
être trop poussées car elles peuvent provoquer des défauts dans certains fromages comme la
production de styrène qui, lorsque présent dans une certaine quantité, lui confère une forte
odeur de plastique (Lamontagne et al., 2002).
Les levures et les moisissures sont donc les principaux responsables de la remontée du pH
caractéristique de l’affinage d’un Camembert. La désacidification de la pâte provoquée par
9
la consommation du lactate et par la libération d’ammoniac (NH3) lors de la protéolyse
modifie les actions enzymatiques et microbiologiques au sein de celle-ci. Ce phénomène
peut être en lien avec la biocompatibilité possible des levures et moisissures avec des
bactéries acido-sensibles comme les microcoques, les corynébactéries et certaines bactéries
lactiques (Cerning et al., 1987).
1.2.1.4 Autres microorganismes
Hormis celles mentionnées plus haut, d’autres espèces de microorganismes contribuent à
l’affinage de certains fromages. Les bactéries corynéformes, les micrococacceae et les
propionibactéries en font partie.
Brevibacterium linens, est l’espèce de bactérie corynéforme la plus connue. Halotolérante
et strictement aérobe, elle ne supporte pas des conditions trop acides (Souza Motta et
Brandelli, 2008). Présente à la surface de la plupart des croûtes fleuries, elle caractérise
aussi certaines croûtes lavées comme celle du fromage Oka en lui donnant une couleur
orangée. Également, sa protéolyse contribue à des arômes soufrés (Lamontagne et al.,
2002). Lors de la maturation fromagère sa croissance suit la remontée de pH provoquée par
les levures et les moisissures. D’ailleurs, une certaine biocompatibilité existe entre ces deux
types de microorganismes (voir section 1.4).
Micrococcus et Staphylococcus sont les genres de micrococacceae les plus communs à la
surface des fromages. D’abord, Micrococcus est présent en quantité moindre que les autres
microorganismes dans les fromages à croûtes fleuries et lavées. C’est l’absence d’oxygène
et les températures d’affinage trop basses qui causent son absence ou sa présence limitée.
Toutefois, ils contribuent quand même à l’affinage (Beresford et Williams, 2004). Ensuite,
plusieurs espèces de Staphylococcus ont été isolées en quantités importantes de la flore de
surface de nombreux fromages. Heureusement, l’espèce pathogène Staphylococcus aureus
a tendance à disparaître par elle-même en cours de maturation. Anaérobes facultatifs, leur
présence principalement en surface démontre qu’ils tolèrent tout de même les conditions
10
aérobies. De plus, leur grande tolérance au sel (jusqu’à 15%) favorise aussi leur activité.
Toutefois, l’effet de la présence du genre Staphylococcus sur l’arôme est peu connu
(Beresford et Williams, 2004).
Propionibacterium est le genre de culture propionique le plus fréquemment retrouvé en
fromagerie. Au sein des fromages suisses, il métabolise le lactate pour libérer entre autres
de l’acide propionique, de l’acide acétique et du CO2 gazeux qui crée les larges ouvertures
dans la pâte (les yeux). Ces trois composés sont essentiels à la texture et au goût typique du
fromage suisse. Toutefois, les propionibactéries ne sont pas des espèces très fréquentes
dans le Camembert.
1.2.2 L’effet des conditions d’affinage
Plusieurs facteurs, intrinsèques ou environnementaux, influent sur la maturation fromagère.
L’humidité, le taux de sel en phase aqueuse, l’activité de l’eau, la flore microbienne et les
nutriments qui constituent la pâte fromagère sont les principaux facteurs intrinsèques
responsables de la sélection des microorganismes de l’affinage. D’un autre angle, la
température, l’humidité relative de l’air ambiant, les proportions des constituants de l’air et
les actions du fromager comme l’essuyage de la pâte et la vaporisation de microorganismes
sont les facteurs externes environnementaux agissants sur l’affinage du fromage.
Selon le type de fromage fabriqué, les facteurs externes en cause ne sont pas les mêmes.
Ainsi, le vieillissement du cheddar se fait dans un emballage sous vide. La température est
donc le seul facteur externe contrôlé. Elle permet de sélectionner les microorganismes selon
leur activité à une température précise. Par contre, au cours de la maturation du
Camembert, toutes ces conditions externes seront contrôlées afin d’optimiser l’affinage et
favoriser la croissance de certaines levures et moisissures en surface. Conséquemment, une
humidité relative de l’air autour de 90% et une température autour de 10°C sont
maintenues. De plus, la vaporisation de souches mycéliennes à la surface des meules peut
être réalisée. Dans le cas du Camembert, la présence d’oxygène est nécessaire au
11
développement des moisissures et favorise celui des levures. Les meules sont à l’air libre de
7 à 10 jours pour favoriser la croissance de la flore fongique. Le fromage est ensuite
emballé dans un papier perméable à l’oxygène et réfrigéré pour une vingtaine de jours (St-
Gelais et Tirard-Collet, 2002)
Conséquemment, tous les facteurs énumérés ci-dessus ont une influence sur la sélection de
la flore microbienne des fromages de type Camembert. D’ailleurs, la croissance de certains
microorganismes dépend de la présence d’autres. La section 1.2.1 sur les flores d’affinage
témoigne entre autres de plusieurs exemples de ce type de symbiose. D’ailleurs, un
microorganisme seul ne produit pas les mêmes flaveurs que lorsqu’il est combiné à d’autres
(Spinnler et Gripon, 2004).
1.2.3 Le type de lait utilisé
La physicochimie du lait utilisé pour fabriquer le fromage peut influencer l’affinage et
peut-être même les interactions entre microorganismes du fromage. Selon l’espèce de
vache, son alimentation et les saisons, la physicochimie du lait change. Les deux laits
utilisés pour les travaux proviennent de vaches Holstein et Suisse Brunes. Le lait d’Holstein
est un lait de grand mélange et le lait de Suisse Brune provient de quelques fermes d’une
région précise du Québec. La Suisse Brune est déjà reconnue pour produire un lait plus
riche en protéine et en gras que la Holstein. Ces ratios plus élevés se retrouvaient aussi dans
des fromages Cheddar et Italien fabriqués à partir de lait de Suisse Brune lorsque comparés
aux mêmes fromages de lait d’Holstein (Mistry et al., 2002, De Marchi et al., 2008). Cette
caractéristique pourrait donc influencer les interactions mycètes-bactéries lactiques.
1.3 Les bactéries probiotiques
Il y a déjà plus d’un siècle que la consommation de certaines bactéries est liée à la santé. Le
premier à avoir fait part de ce phénomène à la communauté scientifique est Metchnikoff.
12
En 1907, il établissait le lien entre la longévité des Bulgares et leur grande consommation
de produits fermentés (Richardson, 1996). Aujourd’hui, leur définition s’est raffinée.
Comme énoncé dans l’introduction, les bactéries probiotiques doivent être vivantes,
ingérées sur une base régulière et consommées en grandes quantités afin d’avoir un effet
bénéfique sur la santé (FAO/WHO, 2001).
Tableau 1.1. Souches de bactéries probiotiques utilisées commercialement.
Source : (Vasiljevic et Shah, 2008)
Une panoplie de souches de bactéries sont désormais qualifiées comme étant probiotiques
et utilisées commercialement (Tableau 1.1). Les genres Bifidobacterium et Lactobacillus
sont les plus rencontrés. De plus, au Canada, pour pouvoir faire une allégation non
spécifique à une souche sur l’emballage d’un produit, celui-ci doit contenir une des espèces
reconnues par l’Agence Canadienne d’Inspection des aliments (ACIA) comme étant
probiotiques (Tableau 1.2). Par conséquent, afin d’être qualifiée de probiotique, une souche
de bactérie doit avoir un effet démontré sur la santé et répondre à des critères précis.
13
Tableau 1.2. Tableau sommaire des allégations non spécifiques à la souche acceptées pour
les probiotiques et les espèces admissibles dans le cadre de ces allégations. Source : (ACIA,
2009)
Espèces bactériennes éligibles Allégations non spécifiques à la souche
acceptées pour les probiotiques
Bifidobacterium adolescentis
Bifidobacterium animalis subsp.
animalis
Bifidobacterium animalis subsp.
lactis -synonyme: B. lactis
Bifidobacterium bifidum
Bifidobacterium breve
Bifidobacterium longum subsp.
infantis comb. nov.
Bifidobacterium longum subsp.
longum subsp. nov.
Lactobacillus acidophilus
Lactobacillus casei
Lactobacillus fermentum
Lactobacillus gasseri
Lactobacillus johnsonii
Lactobacillus paracasei
Lactobacillus plantarum
Lactobacillus rhamnosus
Lactobacillus salivarius
Probiotique présent naturellement dans
la flore intestinale.
Fournit des microorganismes vivants
présents naturellement dans la flore
intestinale.
Probiotique contribuant à la santé de la
flore intestinale.
Fournit des microorganismes vivants
contribuant à la santé de la flore
intestinale.
1.3.1 L’effet des probiotiques sur la santé
Une fois ingérées et parvenues au milieu intestinal, les bactéries probiotiques colonisent
notamment l’iléon terminal et le côlon (Richardson, 1996). C’est rendu là qu’elles exercent
leur effet santé. Leurs principales vertus sont de diminuer l’intolérance au lactose, prévenir
14
et amoindrir les symptômes d’une diarrhée, traiter et prévenir une allergie, réduire le risque
de mutations de cellules en lien avec le cancer du côlon, inhiber des microorganismes
pathogènes intestinaux, prévenir la maladie du colon irritable et moduler le système
immunitaire (Vasiljevic et Shah, 2008). Il existe donc un lien direct entre l’administration
de certaines souches précises et leurs effets sur un problème de santé en particulier.
Toutefois, comme l’identification de ces souches au sein des aliments demeure complexe,
l’European Food Safety Authority (EFSA, 2009) a émis des réticences sur l’approbation
d’allégations santé propres à une souche particulière ou à des combinaisons de souches.
Pour corriger ce problème d’identification, cet organisme important recommande entre
autres l’instauration de divers systèmes d’identification et d’une banque de souche
internationale. Plusieurs études rapportent les bénéfices de produits laitiers avec
probiotiques, mais les résultats ne sont pas constants (Ouwehand et al., 2003). Malgré cette
variabilité, les probiotiques ont un potentiel d’effet bénéfique général sur la santé
intestinale. Ainsi, les êtres humains devraient en consommer sur une base régulière. Dans le
but de permettre cette consommation, leur incorporation aux aliments est maintenant chose
commune.
1.3.2 Les aliments probiotiques
Dans le but d’optimiser l’effet santé des probiotiques, plusieurs éléments doivent être pris
en considération lors de leur ajout aux aliments. La présence de ces bactéries dans l’aliment
ne doit pas affecter son goût et sa texture. Ensuite, l’aliment doit permettre qu’un nombre
suffisant de bactéries survive, et ce, autant lors du procédé de fabrication, de l’entreposage
du produit (date de péremption) que lors du transit gastrique.
Dans cette optique, la sélection de souches probiotiques pour fabriquer un aliment doit
respecter divers éléments. D’abord, la souche ne doit être ni pathogène ni avoir de
résistance aux antibiotiques transférable à des bactéries pathogènes. Ensuite, la souche doit
être technologiquement valable. C’est-à-dire qu’elle doit résister aux phages, capable d’être
produite en grande quantité et génétiquement stable. Elle doit aussi répondre à un critère
15
fonctionnel. Ainsi, elle doit tolérer des conditions acides comme celles de l’estomac,
résister à l’action des sels biliaires, avoir un effet documenté et valide sur la santé et doit
adhérer à la muqueuse intestinale. Finalement, les souches de bactéries sont sélectionnées
selon un critère d’effet sur la physiologie de l’être humain. Ces critères sont soit
l’immunomodulation, le métabolisme du lactose, des propriétés anticarcinogènes ou
l’inhibition de microorganismes pathogènes (Vasiljevic et Shah, 2008).
1.3.2.1 La viabilité des probiotiques dans les aliments
L’aliment doit permettre qu’un nombre suffisant de microorganismes survive jusqu’à sa
date de péremption, mais permettrait aussi, idéalement, de résister aux conditions extrêmes
qui règnent dans l’estomac (pH autour de 1.2 en absence d’aliments, sels biliaires,
enzymes). Le nombre de cellules à ingérer pour avoir un effet significatif suite au tractus
gastro-intestinal n’est pas spécifiquement connu. Selon la souche et la matrice alimentaire,
une population de 106 à 10
8 UFC par gramme de contenu intestinal semble nécessaire
(Richardson, 1996). Actuellement, la réglementation au Canada exige une quantité
minimale de 109 bactéries par portion d’aliment afin que des allégations santé non
spécifiques à une souche particulière (Tableau 1.2) soient permises sur l’emballage (Santé
Canada, 2009; ACIA, 2009).
Dans le but d’augmenter le taux survie des probiotiques dans les aliments, l’addition de
prébiotiques, l’adaptation aux différents stress et l’encapsulation font partie des méthodes à
privilégier. De plus, les différentes propriétés intrinsèques et extrinsèques à l’aliment
influencent beaucoup la survie de ceux-ci. Le pH de l’aliment, le pouvoir tampon, le
potentiel redox, la température d’entreposage, la perméabilité à l’air de l’emballage sont
quelques-unes des caractéristiques à observer lors de l’incorporation de probiotiques à un
aliment (Champagne et al., 2005). Par exemple, les probiotiques étant des bactéries
lactiques, des conditions trop acides comme un pH inférieur à 5 et un potentiel redox positif
(milieu oxygéné) ne favorisent pas leur survie à long terme dans les aliments.
16
Ce sont donc ces caractéristiques qui influencent habituellement le choix d’un aliment
comme le yogourt ou le fromage comme véhicule pour ces bactéries. D’un autre angle, des
interactions avec d’autres microorganismes pourraient aussi promouvoir leur croissance et
leur survie. Cette optique sera explorée dans la section 1.4 abordant la biocompatibilité.
1.3.2.2 Les yogourts probiotiques
Considéré comme un aliment santé, et donc en mesure d’être consommé sur une base
régulière, le yogourt est un pionnier des aliments probiotiques. Toutefois, ayant un pH aux
environs de 4 et un emballage généralement perméable à l’oxygène, il n’est pas toujours
excellent pour leur survie à long terme. Seulement les bactéries résistantes aux pH acides
peuvent survivre sur une longue période dans le yogourt. Cette tolérance à l’acide varie
selon les espèces et les souches de probiotiques utilisées (Lourens-Hattingh et Viljoen,
2001). Par exemple, une étude réalisée sur un échantillon de dix yogourts commerciaux
probiotiques a démontré que seulement la moitié d’entre eux avait encore une population
au-dessus des 106 UFC g
-1 lors de leur date d’expiration (Jayamanne et Adams, 2006). Un
choix judicieux des souches probiotiques et un taux élevé d’inoculation dès le début de la
fermentation permettrait donc d’éviter une trop grande perte de viabilité pendant
l’entreposage réfrigéré prolongé.
1.3.2.3 Les fromages probiotiques
Certains fromages sont reconnus comme étant de bons aliments supportant la viabilité des
probiotiques. Par exemple, le Cheddar (Phillips et al., 2006; Daigle et al., 1999), le fromage
de chèvre semi-ferme (Gomes and Malcata, 1998) et le Gouda (Gomes et al., 1995) en font
partie. En effet, avec un pH de 5 et plus et un potentiel redox négatif ces types de fromage
sont reconnus comme de bons milieux pour assurer la viabilité des probiotiques
(Grattepanche et al., 2008).
17
Toutefois, ce ne sont pas tous les types de fromage qui ont à première vue des propriétés
intéressantes pour véhiculer les probiotiques. Le Camembert avant affinage, étant une pâte
molle décalcifiée au pH inférieur à 5.0, en fait partie (Spinnler et Gripon, 2004). Ainsi, les
aptitudes du Camembert à promouvoir la viabilité des bactéries probiotiques ne seraient
dues ni à son pH initial, ni à son pouvoir tampon. Néanmoins, comme le pH de celui-ci
remonte vers la neutralité lors de la période d’affinage, ce serait plutôt ce stade qui
favoriserait ces bactéries bénéfiques. L’étude du microbiote d’un Camembert au lait cru a
dénoté la présence de souches potentiellement probiotiques (Coeuret, 2004). Dans ce cas,
sans en avoir ajouté dans le ferment de départ, le Camembert a permis à des souches de sa
flore naturelle ayant un potentiel probiotique de survivre. Or, les mycètes du camembert
contribuent peut-être à la viabilité de ces bactéries.
1.4 La biocompatibilité entre microorganismes du fromage
Un effet d’antagonisme, de symbiose ou de neutralité peut se manifester entre deux souches
microbiennes. D’ailleurs, les interactions entre bactéries lactiques et levures/moisissures ne
suivent pas une tendance générale. Ce sont plutôt des effets propres à certains couples de
souches qui ont été observés. Le fromage et le kéfir sont les deux principaux produits
laitiers où des souches fongiques entrent en relation avec des cultures bactériennes.
Dans le kéfir, la présence de la levure Saccharomyces delbrueckii permet à la bactérie
Lactobacillus brevis d’excréter un polysaccharide essentiel à la formation des grains de
kéfir. Ainsi, il a été démontré que la présence d’extraits de levure était nécessaire à la
production de cet exopolysaccharide (Zoukari et Anifantakis, 1988). Ceci n’est d’ailleurs
qu’un exemple parmi tant d’autres relations ou la croissance d’une souche bactérienne
dépend de celle d’une espèce de levure formant le grain de Kefir. D’une part, les levures
fournissent des facteurs de croissances essentiels pour les bactéries comme des vitamines et
des acides aminés. D’autre part, les produits métabolisés par les bactéries comme l’acide
lactique deviennent des sources d’énergie pour les levures (Farnworth et Mainville, 2003).
18
Dans le fromage, second microbiote laitier ou des souches fongiques et bactériennes se
côtoient, divers phénomènes de compatibilité ou d’incompatibilité peuvent aussi se
produire. D’abord, les mécanismes de stimulation ou d’inhibition ne sont que partiellement
compris. Dans les cas de symbiose, comme dans le cas du kéfir, les levures peuvent
synthétiser des vitamines et générer grâce à la protéolyse des acides aminés utiles aux
bactéries. Conséquemment, plusieurs souches de bactéries probiotiques incapables
d’effectuer elles-mêmes de la protéolyse en bénéficient (Champagne et al., 2005; Klaver et
al.,1993). Toutefois, cette protéolyse n’est pas toujours favorable car certains peptides émis
peuvent être antimicrobiens (Korhonen et Pihlanto, 2006). Enfin, à l’inverse, certaines
bactéries lactiques, en métabolisant le lactose, libèrent du galactose qui peut servir de
source de carbone aux levures incapables de métaboliser le lactose (Viljoen, 2001; Álvarez-
Martín et al., 2008).
Dans certains cas, les effets bénéfiques que peuvent avoir certaines espèces de levures sur
les bactéries probiotiques ou non probiotiques ont été démontrés. Un de ces exemples
d’interaction positive avec des levures implique l’espèce bactérienne Brevibacterium
linens. À la surface des fromages à croûte lavée orangée, les levures s’implantent en
premier, désacidifient la croûte et produisent des vitamines qui stimulent la croissance de
cette bactérie corynéforme (Souza Motta et Brandelli, 2008). De même, des levures
ajoutées à un yogourt incubé à 30 °C pendant plusieurs semaines ont augmenté la viabilité
des bactéries lactiques probiotiques ou non probiotiques de ce yogourt (Liu et Tsao, 2009).
Le fait que les levures aient permis la survie de bactéries dans des conditions non
réfrigérées est intéressant, car l’affinage en hâloir du Camembert se fait à des températures
variant de 12 à 15°C.
Conséquemment, les possibilités d’interactions sont nombreuses dans un fromage comme
le Camembert qui contient plusieurs souches de divers types de microorganismes. Dans
cette optique, une étude ayant tenté différentes combinaisons de souches de plusieurs
espèces présentes entre autres dans le camembert comme D. hansenii, G. candidum et K.
lactis avec L. lactis, L. paracasei et L. lactis ssp. cremoris a démontré qu’elles pouvaient se
stimuler ou s’inhiber selon les combinaisons de souches (Álvarez-Martín et al., 2008).
19
D’ailleurs, des effets contradictoires peuvent se produire au sein d’un même fromage.
Ainsi, la présence de levures stimule la croissance des espèces de lactocoques dans un
fromage de type Cantalet tandis qu’elle cause la décroissance de S. thermophilus (De
Freitas et al., 2009). Finalement, la symbiose entre deux souches peut aussi être due
indirectement à un phénomène aussi simple que la remontée de pH engendrée par le
métabolisme des levures et moisissures. Il a été observé que la flore secondaire d’affinage
d’un Camembert se développe de façon optimale à un pH plus élevé que 5.8 (Spinnler et
Gripon, 2004).
Pour ce qui est de la symbiose entre bactéries lactiques et moisissures, la littérature
scientifique ne contient pas d’exemples qui abordent la question précisément. Les
publications consultées font plutôt état d’antagonisme. Des composés antifongiques
semblables comme des peptides cycliques ou des acides organiques comme l’acide
phénylactique (Álvarez-Martín et al., 2008), métabolisés par les bactéries lactiques peuvent
inhiber la flore d’altération mycélienne (Voulgari et al., 2010). Certaines souches de
bactéries probiotiques produisent des métabolites qui inhibent la croissance des espèces
mycéliennes Aspergillus niger, Penicillium roqueforti, Fusarium spp., Candida albicans
faisant partie de flore d’altération de trempettes à base de fromage (Tharmaraj et Shah,
2009). D’un autre côté, l’acide phénylactique peut aussi être synthétisé par des souches de
G. candidum et inhiber des bactéries comme Listeria monocytogenes (Dieuxleveux et al.,
1998). Finalement, l’inhibition des bactéries peut aussi survenir lorsque des métabolites
comme les acides gras libres sont libérés par des mycètes lipolytiques. L’inhibition par les
acides gras libres dépend par contre de leur concentration et de leur structure chimique car
ils peuvent aussi stimuler la croissance des bactéries dans certains cas (Sprong et al., 2001;
Kankaanpaa et al., 2004)
Pour ces raisons, les interactions entre microorganismes d’un microbiote comme le
fromage Camembert sont intéressantes à étudier. La spectrophotométrie automatisée a déjà
été utilisée entre autre pour analyser les interactions entre différentes espèces et souches de
bactéries lactiques du cheddar (Champagne et al., 2009). Toutefois, cette technique n’a
jamais été utilisée pour confirmer la biocompatibilité entre des souches de levures,
20
moisissures et de bactéries lactiques. Cette méthode est utile, car elle permet l’essai de
plusieurs combinaisons de souches simultanément. Elle associe la densité optique d’un
milieu à la croissance du microorganisme inoculé.
21
Chapitre 2. Hypothèse et Objectifs
L’affinage d’un fromage de type Camembert génère des conditions qui permettent des
interactions positives (biocompatibilité) impliquant des levures/moisissures et des bactéries
lactiques/probiotiques et qui améliorent la viabilité ou l’activité de ces dernières.
Afin de vérifier cette hypothèse, différents objectifs devront être atteints :
- Identifier, à l’aide de la spectrophotométrie automatisée (SA), des paires de souches
de levures/moisissures et bactéries lactiques biocompatibles.
- Étudier l’effet des paramètres physicochimiques du lait de fabrication du
Camembert (grand mélange Holstein vs terroir Suisse Brune) sur les interactions
levures/moisissures et bactéries lactiques.
- Déceler des souches fongiques isolées du terroir québécois qui ont des affinités avec
les bactéries lactiques et probiotiques.
- Valider la capacité de la méthode de SA à prédire la biocompatibilité entre
levures/moisissures et bactéries lactiques à l’aide de caillés modèles.
- Étudier la viabilité de probiotiques dans des fromages.
Chapitre 3 : Biocompatibilité des bactéries lactiques
probiotiques et d’affinage avec des mycètes isolées dans
les produits laitiers.
Biocompatibility between probiotic/specialty lactic acid
bacteria and mycetes isolated from dairy products.
Pierre-Luc Champigny b, Claude P. Champagne
a, Daniel St-Gelais
a, Ismail Fliss
b, Steve
Labrie b
a Centre de recherche et développement sur les aliments, Agriculture et agroalimentaire Canada, 3600 boul.
Casavant Ouest St. Hyacinthe QC, Canada J2S 8E2
b Institut des Nutraceutiques et des Aliments Fonctionnels, Centre de recherche STELA, Université Laval,
Québec, QC, Canada G1V 0A6
23
Résumé
L’objectif de cette étude était de vérifier si la croissance de mycètes destinés à la
fabrication de fromage de type Camembert pouvait affecter celle des bactéries lactiques
(probiotiques et d’affinage) et si la provenance du lait influençait cette interaction. Des
mycètes (Geotrichum candidum, Debaryomyces hansenii, Issatchenkia orientalis et Pichia
anomala) ont été isolés de laits de terroir du Québec. Ces mycètes, ainsi que des
moisissures commerciales (Penicillium camemberti), ont été cultivés dans des caillés
modèles, et des extraits acellulaires de lactosérum (EAL) furent subséquemment préparés.
Le pH final des EAL était ajusté à 6.5. Les deux laits utilisés pour fabriquer les caillés
modèles provenaient aussi du terroir québécois. Le premier était un lait de grand mélange
de race Holstein et l’autre un lait de vache de race Suisse brune en provenance d’un terroir
spécifique. La spectrophotométrie automatisée fut utilisée afin de suivre la croissance des
cultures lactiques dans les EAL. Les plus hautes biomasses de bactéries ont été obtenues à
partir des EAL issus de G. candidum, tandis que les EAL provenant de caillés modèles de
P. camemberti donnaient les croissances bactériennes les moins élevées. La croissance des
mycètes et des bactéries était généralement meilleure dans les produits issus du lait des
vaches Suisses Brunes. Ce travail a mis en évidence que la flore de surface de fromages de
type Camembert et la source du lait de fabrication peuvent potentiellement influencer la
croissance des bactéries probiotiques et d’affinage.
24
Abstract
The purpose of this study was to determine if mycetes used in the ripening of Camembert-
type cheese can influence the quantity of lactic acid bacteria (probiotic or ripening) and if
the milk source has an effect. Mycete strains (Geotrichum candidum, Penicillium
camemberti, Debaryomyces hansenii, Issatchenkia orientalis and Pichia anomala) were
isolated from milk produced in the province of Quebec terroir. These strains and
commercial Penicillium camemberti cultures were cultivated on cheese slurries. After a
ripening of 12 days a cell free whey (CFW) was extracted from each cheese slurries. The
pH of these CFW were all adjusted to 6.5. Two different Quebec terroir milk bases were
used to produce the cheese slurries. They were from a large mixed Holstein production or
from a farm having Brown Swiss cows. Automated spectophotometry was used to follow
the growth of the different lactic cultures in the CFW. The highest bacterial biomasses
were obtained with CFW from cheese slurries containing G. candidum strains. The prior
growth of P. camemberti in the cheese slurries was either inhibitory or had no effect on
bacterial development. The milk source also influenced the growth of bacteria. It was
generally higher in CFW obtained from the Brown Swiss milk. This work showed that the
rind microbiota of Camembert-type cheese could potentially influence the growth of
probiotic and ripening bacteria.
25
3.1 Introduction
Many functional foods have been produced to deliver probiotic bacteria. Numerous
conditions such as pH, redox potential, buffering capacity and storage temperature can
affect their viability (Champagne et al., 2005). As a result, the choice of the food matrix to
be successfully enriched with probiotic bacteria depends on its chemical properties and
their effect on the added strains. Nowadays, yogurt is the most common probiotic-carrying
food matrix. However, the acid pH and the positive potential redox of this matrix do not
always offer the best conditions for the viability of probiotics (Shah, 2000).
Other foods are increasingly being tested in the hope of enabling the viability of probiotic
bacteria during storage in a better way than yogurt. Cheese is a good example. For instance,
cheddar (Phillips et al., 2006; Daigle et al., 1999), semi-hard goat cheese (Gomes and
Malcata, 1998) and Gouda (Gomes et al., 1995) have all proved to be good probiotic
carriers. Their negative redox level and higher pH than yogurt make them a logical choice
to enable the stability of the beneficial bacteria during shelf life. In this perspective,
Camembert-type cheese seems to be an interesting alternative to enable probiotic bacteria
viability. Potentially probiotic strains have been isolated from Camembert prepared with
raw milk (Coeuret et al., 2004), but the reasons why it could support probiotic cultures
were not assessed. Before ripening, Camembert does not appear to present favorable
conditions for probiotics survival because its pH is lower than most other cheeses.
However, the growth of yeasts and moulds (Y/M) at the surface of the Camembert could
enhance its capacity to support bacterial viability due to the pH raise associated with Y/M
metabolism. Furthermore, biocompatibility between lactic acid bacteria and mycetes could
occur. Such interactions seem to vary according to the strains used (De Freitas et al, 2009;
Álvarez-Martín et al., 2008). Bacterial growth stimulation may take place because Y/M
produce growth factors such as amino acids or vitamins. On the other hand, inhibition
might occur because some mycete strains are lipolytic and free fatty acid are possibly toxic
for lactic acid bacteria (Sprong et al., 2001). Moreover, some antibacterial compounds like
peptides and phenyllactic acid may appear during the ripening (Dieuxleveux et al., 1998).
26
There is, therefore, a need to assess inhibitory or synergistic effects between probiotic
bacteria and Y/M specifically used in Brie or Camembert cheese.
The aim of the present study was to examine the interactions between mycetes and lactic
acid bacteria (probiotic or ripening strains) by using an automated spectrophotometry (AS)
screening tool. Another objective of the experiment was to find new mycete strains which
could be used to manufacture typical mold-based surface-ripened terroir cheeses. For that
reason, the fungi strains used for this experiment were isolated of different milk sources
from the province of Quebec (Canada). Moreover, the bacteria/fungi interactions were
tested in two milk sources (industrial and small production) from different cow species
(Holstein and Brown Swiss).
3.2 Materials and methods
3.2.1 Strains (mycetes and bacteria) and milk sources
Bacterial and mycete strains used in this study are listed in Table 3.1. The bacterial strains
were from four commercial suppliers except for the L. rhamnosus GG that was purchased
at American Type Culture Collection (ATCC 53103) (Table 3.1). The BKA and BKB
cultures were isolated from a BioK+ commercial product (Laval, QC, Canada) which
contains a Lactobacillus acidophilus as well as a Lactobacillus casei culture. The probiotic
strains were selected because they are commercially available cultures having documented
health benefits.
The Y/M strains were isolated from seven different milk sources over the province of
Quebec (Canada). Their exact sources are confidential but in a general manner they were
obtained from different breeds of cows (Jersey, Holstein, Canadian and Brown Swiss) and
they came from the following geographical regions (Gaspésie–Îles-de-la-Madeleine,
27
Quebec city region, Montérégie) Also, two dried commercial P. camemberti preparations
were used (PC PSM2 and PC TN).
The two milk sources used to prepare the cheese slurries were from a large scale production
and from a small farm. These milks were from different breeds of cows: Holstein from the
large-scale production (multi-farm from a tanker; Milk A in Table 3.1) and Brown Swiss
milk from small producers in a particular geographical region (Milk B in Table 3.1).
Yeasts (except for G. candidum) frozen stock cultures were prepared by mixing a YM broth
solution (Becton Dickinson, Sparks, MD, USA) having 30% w/w glycerol (Sigma-Aldrich,
St-Louis, MO, USA) with a fresh liquid inoculum grown on YM broth in a 1:1 ratio. For G.
candidum and moulds, the cell suspension was prepared by recovering colonies of P.
camemberti and G. candidum from the surface of an acidified potato dextrose agar plate
(PDA; EMD Chemicals, Darmstadt, Germany) using a swab humidified in a filter-sterilized
(0.22μm Millex GP syringe filter, Millipore, Carrigtwohill, Co. Cork, Ireland) 0.05% w/v
Tween 80 (Fisher Scientific, Fairlawn, N-J, USA) solution. The cells from the swab were
resuspended in a Tween 80 solution which was then blended with the glycerol-YM broth at
the 1:1 ratio as for the other mycete cultures.
The bacterial frozen stock cultures were prepared by blending BHI broth (Becton
Dickinson) having 15% glycerol with a fresh liquid culture (pH of 4.5) in a 5:1 ratio. All
these cell suspensions were divided in aliquots of 1mL in cryovials (Nalgene, Rochester,
NY, USA) and placed in a -80˚C freezer.
28
Table 3.1. Bacterial and mycete strains used for this work
Genus Species Type Strain Source
Lactobacillus rhamnosus probiotic R0011 Institut Rosell-Lallemand,
Mtl, Canada
Lactobacillus rhamnosus probiotic GG ATCC 53103, Rockville,
MD, USA
Lactobacillus casei Ripening A180 Abiasa, St-Hyacinthe,
Canada
Bifidobacterium lactis probiotic BB12 Chr. Hansen, Barrie, On,
Canada
Bifidobacterium longum probiotic R0175 Institut Rosell-Lallemand,
Mtl, Canada
Lactobacillus ND* probiotic BKA Isolated from Bio-K+
Lactobacillus ND probiotic BKB Isolated from Bio-K+
Penicillium camemberti mould PC TN Cargill France SAS, La
Ferté sous Jouarre
Penicillium camemberti mould PC PSM2 Cargill France SAS, La
Ferté sous Jouarre
Geotrichum candidum yeast LMA 690 Milk E
Geotrichum candidum yeast LMA 317 Milk F
Geotrichum candidum yeast LMA 563 Milk A
Geotrichum candidum yeast LMA 664 Milk A
Debaryomyces hansenii yeast LMA 243 Milk A
Debaryomyces hansenii yeast LMA 395 Milk C
Debaryomyces hansenii yeast LMA 668 Milk B
Debaryomyces hansenii yeast LMA 695 Milk D
Debaryomyces hansenii yeast LMA 816 Milk E
Issatchenkia orientalis yeast LMA 696 Milk G
Issatchenkia orientalis yeast LMA 666 Milk B
Pichia anomala yeast LMA 827 Milk B *ND : Not determined
3.2.2 Inocula preparation and cultures conditions
The yeasts (except G. candidum) inocula were obtained from YM broths (Becton
Dickinson) seeded at 1% v/v with thawed stock cultures, which were incubated at 30˚C on
a shaker (250 rpm) until they reached an optical density (OD) between 0.4 and 0.8. The OD
was determined with a Beckman 7400 Spectrophotometer at 600nm (Coulter, Fullerton,
29
CA, USA). The CFU ml-1
of the liquid cultures were estimated by the OD measure after
having established an OD-CFU standard curve.
The P. camemberti and G. candidum biomass were obtained by spreading a thawed stock
culture at the surface of an acidified potato dextrose agar plate and incubating for 1 week at
room temperature (23˚C). The mould spores were collected using a sterile swab, and
suspending in a Tween 80 solution, as previously described. The concentration of the cell
suspension was determined by using an hemacytometer (Hausser Scientific, Horsham, PA,
USA).
Finally, for the automated spectrophotometry assays, bacterial inocula were prepared in
MRS broth (Becton Dickinson) supplemented with 1% v/v of a 10% w/v sodium ascorbate
(Sigma-Aldrich) and 5% w/v L-Cysteine Hydrochloride (Sigma-Aldrich) filter-sterilized
solution. This MRS medium was inoculated with 1mL of a thawed stock culture and
incubated at 37˚C until a pH of 4.5 was attained.
3.2.3 Production of cheese slurries
A cheese slurry is a model system of a cheese obtained after hydration of a lyophilized
cheese powder. To prepare the cheese slurry powder, fresh Camembert cheese was
produced with the two milk lots. Whole milk was pasteurized in batch at 65˚C for 30
minutes. The temperature of milk was then adjusted to 32˚C before inoculating at 1% w/w
with a lyophilized Flora Danica starter (Chr Hansen, Milwaukee, WI, USA). After
inoculation, maturation of milk occured for 45 minutes at 32˚C. During this time, a CaCl2
(Calsol, Danisco, Copenhagen K, Denmark) 45% w/v solution was added at 0.035% w/w.
Subsequently, the rennet (CHY-MAX extra, Chr Hansen) was added at 2.25mL/40L of
milk. 40 minutes after adding the rennet, the curd was cut into pieces of 2 cm side to release
the whey. The curd pieces were ready for molding when whey reached a pH of 6.4. Kept at
room temperature, the molds were turned over after one hour and three hours for whey
drainage. Finally, the cheese molds were placed in a chamber overnight. The curds were
30
initially at 28˚C and the chamber was programmed to gradually go down to 16˚C for the
next morning. Then, instead of carrying out the salting and ripening steps, the cheese pieces
were freeze-dried, grinded and vacuum packed to conserve them in a powder form at -40˚C.
The cheese slurries were prepared by hydrating the powder at 57% w/w solids with a
solution acidified to pH 4.8 with DL-Lactic acid (Fisher Scientific, Fair Lawn, NJ, USA)
and containing NaCl (3.5% w/w) (LaboMAT, Montreal, Qc, Canada). The paste obtained
was inoculated at 105
CFU g-1
with a fresh liquid culture of yeast or mould spores. After
inoculation, viable counts of the cell cultures were enumerated to ascertain the exact CFU
g-1
of the model cheese slurry at day 0 (D0). Non-inoculated (D0) cheese slurries were also
produced for each milk source as a control treatment (Ctrl). Two sets of slurries were made
from the two different cow milk sources (Milk A or Milk B; Table 3.1). Finally, 70g of the
slurry was placed in a 250mL glass jar, which was covered with a typical micro perforated
wrapping paper designed for Camembert. Except for the control treatment where the cell-
free whey (CFW) was extracted at D0, each cheese jar was ripened in a chamber at 12˚C
and 95% relative humidity (RH) for 12 days. Four independent fermentations were carried
out with each Y/M culture. This enabled the preparation of four separate cell-free wheys for
each mycete culture.
3.2.4 Enumeration of the mycetes
After 12 days, enumeration of the mycetes was done using a representative sample of the
slurry. The sample was diluted in a sterile 2% w/v sodium citrate (Fisher Scientific)
solution at room temperature and homogenized using a stomacher 400 unit (Seward, model
400 Circulator; Worthing, West Sussex, UK) at 260 rpm for 2 minutes. This suspension
was serially diluted in sterile 0.1% w/v peptone water (Becton Dickinson) tubes. The first
dilution of this serial was done using a bottle of 99mL peptone water 0.1% w/v (Becton
Dickinson) containing glass beads to further break curd particles as well as chains of cells.
Finally, 0.1mL of the appropriate dilutions were spread at the surface of acidified PDA
plates, which were incubated at 23˚C for 5 days.
31
3.2.5 CFW extraction
Following sampling for the enumeration, the CFW was prepared. First, the surface of the
residual cheese slurry was scraped to remove the mycete layer. Then, two parts of the slurry
were diluted with 1 part of milliQ water (Millipore) and homogenized for 1.5 minutes using
an Omni TH homogenizer (Omni TH, Omni international, Kennesaw, GA, USA ) adjusted
at 90% speed rate. Homogenate pH was then measured using a pH meter (XL15, Acumet,
Fisher Scientific) calibrated with pH 4.0 and pH 7.0 standard buffers (Fisher Scientific).
The homogenate was centrifuged using a Sorvall RC-5B (Dupont, Mississauga, Ontario,
Canada) unit at 10000g for 30 minutes. Centrifugation resulted in three phases: fat, aqueous
and solid. Only the aqueous phase was recovered. Then, the pH of aqueous phase was
adjusted to 4 with HCl (Fisher Scientific) 2N. The AS methodology requires non-turbid
media. Since precipitation of caseins occurred and, to clarify the solution, centrifugation
was repeated as above. Filtration of the supernatant was done using a Buchner with a
Whatman 42 paper filter (Whatman International, Maidstone, England). The filtrate pH was
adjusted to 6.5 using KOH (LaboMAT) 5N and clarified using a 0.45μm HVLP filter
(Millipore). Finally, the solution was sterilized by filtration at 0.22μm (Millex GP filter,
Millipore). The CFWs were kept in sterile test tubes at -40˚C until used in the AS assays.
3.2.6 Automated spectrophotometry assays
To carry out the biocompatibility screening by AS, a Bioscreen CTM
(Labsystems, Helsinki,
Finland) unit was used. The following were added in each HoneycombTM
(Labsystems)
microplate well: 180μl of a CFW, 20μl of 0.15M sterile sodium citrate buffer (Sigma-
Aldrich) and 2μl of the concentrated sodium ascorbate and L-cystein hydrochloride
solution used for the MRS media. Also, MRS broth was used as a positive control for
bacterial growth in substitution of the CFW. After preheating the system at 37˚C, the wells
were inoculated with 20μl of a bacterial culture in order to inoculate at 1 x 107 cfu mL
-1 of
medium. Each treatment was repeated in two wells. For each medium, non inoculated
blanks of each CFW and MRS were also done.
32
The Bioscreen C system was operated for 24 hour at 37˚C, taking OD readings (600 nm) of
each well every 15 minutes. Before each reading, the plate was shaken for 2 minutes with a
140 sidestep at the extra-extensive level. The BB12 and R0175 anaerobic strains did not
grow in these conditions. Therefore, with these bifidobacteria, the microplate was set in an
anaerobic environment (85%N2/10%H2/5%CO2 atmosphere) hood for 24 hours. Thence,
the growth curves of these strains were not been established. This explains why the growth
rates results (μmax) cannot be presented for these two strains. Only the difference between
the first OD and the last OD was measured to obtain the increase in OD that is related to
biomass level. Finally, each report AS data represents the average of four separate assays
on each of the two different milk sources.
3.2.6 Statistical analyses
ANOVA were carried out on SAS (SAS institute Inc., Cary, North Carolina, USA)
software with the GLM procedure and significantly differences between results were
determined using the Fisher’s least significance difference (LSD) test and Tukey test for
experiments with some missing data. Each data reported is the average of four independent
assays except for certain missing data from the first assay of the mycetes CFU g-1
after 12
days. For some analyses examining the overall effect of milk source (Milk A or Milk B) on
the growth of the various cultures, paired T tests were carried out using Instat software
(GraphPad, San Diego, CA, USA). Also, some T tests were carried out using the ttest
procedure in SAS. Each statistical test was done at a 95% confidence level.
33
3.3 Results and Discussion
3.3.1 Growth of mycete strains on the cheese slurries
All the mycete strains were grown in cheese slurries prepared from two milk sources and
viable counts as well as pH after 12 days of incubation were determined (Table 3.2).
Table 3.2. Growth (log CFU/g) of the mycete strains and pH of the cheese slurries after 12
days at 12˚C and 95% RH.*
Log CFU g-1
at D12 pH at D12
Strain Milk A Milk B Milk A Milk B
Penicillium camemberti PCTN 5.47k 5.29
k 5.73
b,c,d 5.78
b,c
Penicillium camemberti PCPSM2 6.40i,j
5.83j,k
6.11a 5.88
a,b
Geotrichum candidum LMA 690 7.84d,e,f,g,h
7.48h 5.38
d,e,f,g 5.48
c,d,e,f
Geotrichum candidum LMA 317 7.81e,f,g,h
7.53g,h
5.63b,c,d,e
5.49c,d,e,f
Geotrichum candidum LMA 563 6.64i 6.38
i,j 5.22
f,g 5.15
g
Geotrichum candidum LMA 664 7.81e,f,g,h
7.59g,h
5.63b,c,d,e
5.63b,c,d,e
Debaryomyces hansenii LMA 243 8.83a 8.68
a,b 5.20
f,g 5.26
f,g
Debaryomyces hansenii LMA 395 8.82a 8.70
a,b 5.28
f,g 5.25
f,g
Debaryomyces hansenii LMA 668 8.78a,b
8.58a,b,c
5.17f,g
5.28f,g
Debaryomyces hansenii LMA 695 8.99a 8.96
a 5.25
f,g 5.12
g
Debaryomyces hansenii LMA 816 8.75a,b
8.70a,b
5.34e,f,g
5.25f,g
Issatchenkia orientalis LMA 696 8.14b,c,d,e,f,g
7.74f,g,h
5.22f,g
5.15g
Issatchenkia orientalis LMA 666 8.36a,b,c,d,e,f
7.98c,d,e,f,g,h
5.15g 5.14
g
Pichia anomala LMA 827 8.49a,b,c,d
8.41a,b,c,d,e
5.27f,g
5.23f,g
* Values given represent the average of four independent assays. a,b,c,d,e,f,g,h
For a given variable (CFU or pH), values associated to the same letter are not significantly different
(Tukey, P>0.05).
In the ANOVA analysis, the milk source did not appear to influence the growth of any
individual Y/M strain (Table 3.2). However, closer examination of the data shows that, for
each strain, the CFU reached in Milk A was systematically higher than in Milk B (Table
3.2). A paired T test revealed that CFUs were on the average 0.25 log higher when Y/M
were grown on Milk A and that this difference was highly significant (P = 0.0004). The
fact that the statistical Tukey test is build to protect the type 1 error may be responsible of
34
causing a type II statistical error in the Table 3.2. However, this was not the case for pH.
The pH values were not different between strains in cheese slurries from different milk
sources even after a paired T test analysis. It was examined if a higher pH, which can
indicate greater consumption of lactic acid or proteolysis (Boutrou and Guéguen, 2005),
was linked to higher biomass levels. With Geotrichum candidum strains, a certain
relationship was noted (R2 = 0.60) but none was found with Debaryomyces hansenii (R
2 =
0.23).
Comparing the fungus species with respect to curd de-acidification, the cheese slurries
ripened by P. camemberti strains reached the highest pH (Table 2). Amongst the yeast, the
pH values of cheese slurries fermented by G. candidum were generally higher than those
obtained by the other yeast species (D. hansenii, I. orientalis and P. anomala). Presumably,
P. camemberti consumed lactic acid and peptides for carbon and energy source (Guéguen
and Shmidt, 1992; Aldarf et al., 2006) and carried out proteolysis. This consumption can
explain the higher pH because G. candidum strains are known to be less proteolytic than P.
camemberti (Boutrou et al., 2006) and to metabolize lactic acid only in their stationary
growth phase. In the first days of the cheese ripening, they utilize more the peptides
(Boutrou and Guéguen, 2005). In contrast, D. hansenii strains are recognized to modify the
pH in Camembert ripening mainly by lactate consumption. Since their proteolytic enzymes
are intracellular and since lysis of D. hansenii cells is not presumably significant enough in
the 12 first days of ripening, they cannot influence the cheese matrix to ultimately produce
ammonia (Roosista and Fleet, 1996; Leclercq-Perlat et al., 1999). The data on the
metabolism of I. orientalis and P. anomala in Camembert is limited, and no physiological
explanation of their smaller effects on pH is available.
The results observed for the populations of G. candidum and D. hansenii strains in cheese
slurries are in accordance with the literature on Camembert (Leclercq-Perlat et al., 1999;
Leclercq-Perlat et al., 2004a). For the P. camemberti strains, the CFU counts in this study
were 1 log CFU g-1
higher than the ones obtained by Leclercq-Perlat et al. (2004a) after 12
days of ripening, but similar after 30 days of ripening. The lower inoculation rates of the
35
cheese used in the Leclercq-Perlat et al. (2004a) study (4 x 103 g
-1 instead of 1 x 10
5 g
-1),
could potentially explain this difference.
In summary, there were differences in growth levels and pH values of the various 12 days
ripened curds as a function of the cultures used, but they were in line with the literature.
These data suggest that the cheese slurries were representative of commercial products and
they were therefore tested for potential effects on the growth of probiotic and bacterial
ripening cultures. Since differences in pH levels of the curds were noted after 12 days of
ripening of the mycetes, the CFW extracted from these curds needed to be adjusted to the
same level, i.e. pH 6.5, prior to subsequent inoculation with the lactic cultures.
3.3.2 Growth rates of lactobacilli
The μmax values (Figure 3.1) were only measured for Lactobacillus genus. Growth rates
were the same as for the control treatment (Ctrl) for most strains mixes. With the
exception of Lactobacillus rhamnosus R0011, prior growth of the fungi on the curd
resulted in lower growth rates of the lactic cultures. No pattern was detected for a
systematic beneficial or detrimental effect of a given Y/M strain on the µmax values of
probiotics. There was a noticeable strain effect with some pairings like GG and LMA 668
(positive) while BKB and PC PSM2 was negative.
36
0.0
0.1
0.2
0.3
0.4
R0011
aa,b
a,b,ca,b,c
a,b,ca,b,c a,b,ca,b,c
a,b,ca,b,c
b,c c c c b,c
0.0
0.1
0.2
0.3
0.4
A180aaaa
a aaaa
aaaaaa
0.0
0.1
0.2
0.3
0.4
GG
a a,ba,b
a,ba,b a,b,ca,b,c
a,b,ca,b,c
a,b,c a,b,ca,b,c a,b,c
b,c c
0.0
0.1
0.2
0.3
0.4
BKAa,b a,b a,b
a
a,ba,b
a,ba,b a,ba,b a,bbb b b
aa,b
c,d
0.0
0.1
0.2
0.3
0.4
BKB
Ctrl
LMA 2
43
LMA 3
95
LMA 6
68
LMA 6
95
LMA 8
16
LMA 6
96
LMA 6
66
LMA 8
27
PC T
N
PC P
SM
2
LMA 6
90
LMA 3
17
LMA 5
63
LMA 6
64
a,b a,b,ca,b,c
a,b
c,da,b
c,d
a,b
c,da,b
c,d
a,b
c,d
a,b
c,d
b,c
d,e c,d,e d,e
e
Yeast or mould strains CFW
Gro
wth
rate
(µ
max
60
0 n
m)
D. hansenii I. orientalis
& P. anomala
P.
camembertiG. candidum
0.0
0.1
0.2
0.3
0.4
R0011
aa,b
a,b,ca,b,c
a,b,ca,b,c a,b,ca,b,c
a,b,ca,b,c
b,c c c c b,c
0.0
0.1
0.2
0.3
0.4
A180aaaa
a aaaa
aaaaaa
0.0
0.1
0.2
0.3
0.4
A180aaaa
a aaaa
aaaaaa
0.0
0.1
0.2
0.3
0.4
GG
a a,ba,b
a,ba,b a,b,ca,b,c
a,b,ca,b,c
a,b,c a,b,ca,b,c a,b,c
b,c c
0.0
0.1
0.2
0.3
0.4
GG
a a,ba,b
a,ba,b a,b,ca,b,c
a,b,ca,b,c
a,b,c a,b,ca,b,c a,b,c
b,c c
0.0
0.1
0.2
0.3
0.4
BKAa,b a,b a,b
a
a,ba,b
a,ba,b a,ba,b a,bbb b b
0.0
0.1
0.2
0.3
0.4
BKAa,b a,b a,b
a
a,ba,b
a,ba,b a,ba,b a,bbb b b
aa,b
c,d
0.0
0.1
0.2
0.3
0.4
BKB
Ctrl
LMA 2
43
LMA 3
95
LMA 6
68
LMA 6
95
LMA 8
16
LMA 6
96
LMA 6
66
LMA 8
27
PC T
N
PC P
SM
2
LMA 6
90
LMA 3
17
LMA 5
63
LMA 6
64
a,b a,b,ca,b,c
a,b
c,da,b
c,d
a,b
c,da,b
c,d
a,b
c,d
a,b
c,d
b,c
d,e c,d,e d,e
e
0.0
0.1
0.2
0.3
0.4
BKB
Ctrl
LMA 2
43
LMA 3
95
LMA 6
68
LMA 6
95
LMA 8
16
LMA 6
96
LMA 6
66
LMA 8
27
PC T
N
PC P
SM
2
LMA 6
90
LMA 3
17
LMA 5
63
LMA 6
64
a,b a,b,ca,b,c
a,b
c,da,b
c,d
a,b
c,da,b
c,d
a,b
c,d
a,b
c,d
b,c
d,e c,d,e d,e
e
Yeast or mould strains CFW
Gro
wth
rate
(µ
max
60
0 n
m)
D. hansenii I. orientalis
& P. anomala
P.
camembertiG. candidum
Figure 3.1. Growth rates (μmax) of lactobacilli (A180, R0011, GG, BKA, BKB) in the Cell
Free Whey (CFW) obtained following the growth of 14 yeasts or moulds on Milk A
(Holstein) cheese slurries. “Ctrl” represents the CFW without mycete fermentation, and
constitutes the “control” treatment. a,b,c,d,e For a given histogram, the columns associated to
the same letter are not significantly different (statistical test, LSD, P>0.05). The results
represent the average of four assays. Error bars represent SEM.
37
Previous studies have shown that µmax values are not automatically linked to biomass level
(Barrette et al., 2001; Champagne et al., 2009). In this study, no interesting relationship was
observed between μmax and biomass level (Figure 3.2). R2 values of regression analyses
between µmax and ODmax data of 0.34 (GG), 0.15 (BKB), 0.01 (A180), 0.005 (BKA) and
0.00009 (R0011) were observed for the various lactobacilli strains. The pH can also be a
limiting growth factor for bacteria. This is why, after each AS run of the first assay, the
final pH of the different wells was measured (data not shown). The pH in the probiotic-
fermented CFWs from fungi strains were all between 5.2 and 6.0. This indicates that pH of
the fermented CFW was not the limiting factor in growth.
3.3.3 Biomass levels of lactobacilli and bifidobacteria
Within a certain OD range, typically 0.1 and 1.0, the increase in OD is directly proportional
to the biomass level of bacteria in CFW. In milk A, when compared to the control treatment
(Ctrl), the results showed (Figure 3.2) different relationships between the mycetes and
Lactobacillus genus bacteria.
Prior growth of the G. candidum yeasts strains enhanced the subsequent growth of the
bacterial strains R0011, GG and BKB. It had no effect on A180 and BKA strains. In
general, this yeast species was the best to stimulate bacterial growth. However, there was
only another stimulation interaction and it was between R0011 and I. orientalis LMA 666.
Prior growth of LMA 695 and the two P. camemberti cultures (PCTN and PCPSM2)
tended to inhibit the growth of A180 and R0011 cultures (Figure 3.2). For most of the other
pairings, there was no effect of yeast growth on bacterial biomass level, since their OD
levels were not significantly different from the unfermented control treatment.
38
Incr
ease
in O
D (
60
0 n
m)
a
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
R0011aa, b
a,b,ca,b,c a,b,cb,c,d
c,d c,dc,dc,d
d,ed
e,fff
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
A180
a,ba,b a,b a,b bb,cb,c b,cc
c
dd
d
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
GG
aa,b
a,b,c
a,b,ca,b
c,da,b
c,d
a,b
c,da,b
c,d
b,c
d,e
b,c
d,eb,c
d,ec,d,ed,ed,e
e
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
BKAa
a,b a,ba,b,c
a,b,ca,b
c,da,b
c,d b,c,db,c,db,c,d b,c,dc,d c,d
dd
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
Ctrl
LMA 2
43
LMA 3
95
LMA 6
68
LMA 6
95
LMA 8
16
LMA 6
96
LMA 6
66
LMA 8
27
PC T
N
PC P
SM2
LMA 6
90
LMA 3
17
LMA 5
63
LMA 6
64
BKB
a a,ba,b,c
a,b
c,db,c,db,c
d,e
b,c
d,eb,c
d,e
b,c
d,eb,c
d,e,f
c,d
e,fd,e,fd,e,f
e,f
f
Yeast or mould strains CFW
D. hansenii I. orientalis
& P. anomala
P.
camemberti
G. candidum
a
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
aa, ba,b,c
a,b,c a,b,cb,c,dc,d c,dc,d
c,d
d,ed
e,fff
aa,b
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
A180
a,ba,b a,b a,bb,c b,c
cc
dd
d
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
GG
aa,b
a,b,c
a,b,ca,b
c,da,b
c,d
a,b
c,da,b
c,d
b,c
d,e
b,c
d,eb,c
d,ec,d,ed,ed,e
e
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
BKAa
a,b a,ba,b,c
a,b,ca,b
c,da,b
c,d b,c,db,c,db,c,d b,c,dc,d c,d
dd
0,0
0,1
0,2
0,3
0,4
0.5
0.6
0.7
0.8
0.9
Ctrl
LMA 2
43
LMA 3
95
LMA 6
68
LMA 6
95
LMA 8
16
LMA 6
96
LMA 6
66
LMA 8
27
PC T
N
PC P
SM2
LMA 6
90
LMA 3
17
LMA 5
63
LMA 6
64
BKB
a a,b
b,c,db,c
d,e
b,c
d,e
b,c
d,eb,c
d,e,fd,e,fd,e,f
e,f
Yeast or mould strains CFW
D. hansenii I. orientalis
& P. anomala
P.
camemberti
G. candidum
Incr
ease
in O
D (
60
0 n
m)
a
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
R0011aa, b
a,b,ca,b,c a,b,cb,c,d
c,d c,dc,dc,d
d,ed
e,fff
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
A180
a,ba,b a,b a,b bb,cb,c b,cc
c
dd
d
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
GG
aa,b
a,b,c
a,b,ca,b
c,da,b
c,d
a,b
c,da,b
c,d
b,c
d,e
b,c
d,eb,c
d,ec,d,ed,ed,e
e
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
GG
aa,b
a,b,c
a,b,ca,b
c,da,b
c,d
a,b
c,da,b
c,d
b,c
d,e
b,c
d,eb,c
d,ec,d,ed,ed,e
e
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
BKAa
a,b a,ba,b,c
a,b,ca,b
c,da,b
c,d b,c,db,c,db,c,d b,c,dc,d c,d
dd
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
BKAa
a,b a,ba,b,c
a,b,ca,b
c,da,b
c,d b,c,db,c,db,c,d b,c,dc,d c,d
dd
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
Ctrl
LMA 2
43
LMA 3
95
LMA 6
68
LMA 6
95
LMA 8
16
LMA 6
96
LMA 6
66
LMA 8
27
PC T
N
PC P
SM2
LMA 6
90
LMA 3
17
LMA 5
63
LMA 6
64
BKB
a a,ba,b,c
a,b
c,db,c,db,c
d,e
b,c
d,eb,c
d,e
b,c
d,eb,c
d,e,f
c,d
e,fd,e,fd,e,f
e,f
f
Yeast or mould strains CFW
D. hansenii I. orientalis
& P. anomala
P.
camemberti
G. candidum
a
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
aa, ba,b,c
a,b,c a,b,cb,c,dc,d c,dc,d
c,d
d,ed
e,fff
aa,b
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
A180
a,ba,b a,b a,bb,c b,c
cc
dd
d
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
GG
aa,b
a,b,c
a,b,ca,b
c,da,b
c,d
a,b
c,da,b
c,d
b,c
d,e
b,c
d,eb,c
d,ec,d,ed,ed,e
e
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
GG
aa,b
a,b,c
a,b,ca,b
c,da,b
c,d
a,b
c,da,b
c,d
b,c
d,e
b,c
d,eb,c
d,ec,d,ed,ed,e
e
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
BKAa
a,b a,ba,b,c
a,b,ca,b
c,da,b
c,d b,c,db,c,db,c,d b,c,dc,d c,d
dd
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
BKAa
a,b a,ba,b,c
a,b,ca,b
c,da,b
c,d b,c,db,c,db,c,d b,c,dc,d c,d
dd
0,0
0,1
0,2
0,3
0,4
0.5
0.6
0.7
0.8
0.9
Ctrl
LMA 2
43
LMA 3
95
LMA 6
68
LMA 6
95
LMA 8
16
LMA 6
96
LMA 6
66
LMA 8
27
PC T
N
PC P
SM2
LMA 6
90
LMA 3
17
LMA 5
63
LMA 6
64
BKB
a a,b
b,c,db,c
d,e
b,c
d,e
b,c
d,eb,c
d,e,fd,e,fd,e,f
e,f
Yeast or mould strains CFW
D. hansenii I. orientalis
& P. anomala
P.
camemberti
G. candidum
Figure 3.2. Growth of Lactobacillus bacterial strains (A180, R0011, GG, BKA, BKB) in Cell Free Whey
(CFW) of Milk A (Holstein). “Ctrl” represents the CFW without mycete, and constitutes the “control”
treatment. a,b,c,d,e,f
For a given histogram, the columns associated to the same letter are not significantly
different (statistical test, LSD, P>0.05). The results represent the average of four assays. Error bars represent
SEM.
39
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
BB12
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
R0175
Ctrl
PC T
N
LMA 2
43
LMA 3
95
LMA 6
68
LMA 6
95
LMA 8
16
LMA 6
96
LMA 6
66
LMA 8
27
PC P
SM
2
LMA 6
90
LMA 3
17
LMA 5
63
LMA 6
64
a
a,ba,b
a,b b,c,db,c b,c,d b,c,d
b,c,d
b,c
d,e
c,d,e d,e
b,c
d,e
ee
a
bb
b
bbbbb
b
bb
c cc
Yeast or mould strains CFW
Incr
ease
in O
D (
60
0 n
m)
D. hansenii I. orientalis
& P. anomala
P.
camemberti
G. candidum
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
BB12
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
R0175
Ctrl
PC T
N
LMA 2
43
LMA 3
95
LMA 6
68
LMA 6
95
LMA 8
16
LMA 6
96
LMA 6
66
LMA 8
27
PC P
SM
2
LMA 6
90
LMA 3
17
LMA 5
63
LMA 6
64
a
a,ba,b
a,b b,c,db,c b,c,d b,c,d
b,c,d
b,c
d,e
c,d,e d,e
b,c
d,e
ee
a
bb
b
bbbbb
b
bb
c cc
Yeast or mould strains CFW
Incr
ease
in O
D (
60
0 n
m)
D. hansenii I. orientalis
& P. anomala
P.
camemberti
G. candidum
Figure 3.3. Growth of Bifidobacteria strains (BB12 and R0175) in Cell Free Whey (CFW)
of Milk A (Holstein). “Ctrl” represents the CFW without mycete, and constitutes the
“control” treatment. a,b,c,d,e For a given histogram, the columns associated to the same letter
are not significantly different (statistical test, LSD, P>0.05). The results represent the
average of four assays. Error bars represent SEM.
For the bifidobacteria, there were large differences between the two strains (Figure 3.3).
Prior culture of all the fungi strains inhibited the subsequent growth of B. longum R0175
when compared to control treatment. However, for B. lactis BB12, many of the yeast and
mould strains were stimulatory to growth (Figure 3.3). A relationship between the OD data
of the lactobacilli and the bifidobacteria could be noted. In most cases, prior growth of the
mould P. camemberti in the cheese slurry results with CFWs in which lesser growth of the
probiotic bacteria occurred.
40
To find the nature of stimulation and inhibition between Y/M and lactic acid bacteria, it
would be interesting to look at the chemical content of the CFWs. It was not investigated in
this study, but looking at data from the literature on Camembert contents (Roostita and
Fleet, 1996) and contents of some juice extracted from this type of cheese in another study
(Boutrou et al., 1999) reveal the hypothetical contain of the CFWs.
First, there is a smaller amount of fat in CFW than in cheese because, after centrifugation,
the fat supernatant is eliminated. Even if the fat is removed at this step, the lipolysis of
certain mycete strains liberates free fatty acids that are soluble in the aqueous phase at high
pH. Presumably, cheese slurries which had the highest pH values would also contain the
highest levels of hydrosoluble free fatty acids (FFA). Thus, the FFA content in the CFW
would be function of the lipolytic activity of the Y/M culture and the pH of the fermented
model cheese slurry when the first centrifugation was carried out. Generally P. camemberti
strains inhibited the most the growth of bacteria (Figures 3.2 and 3.3). Lipolysis could
explain inhibition of lactic acid bacteria by the mould but G. candidum is also recognized
as being lipolytic (Boutrou and Guéguen, 2005). Depending on the concentration of FFA,
their nature (chain length and insaturation) and the probiotic culture, FFA can either
stimulate or inhibit the growth of lactic acid bacteria (Powell and May, 1981; Partanen et
al., 2009; Sprong et al., 2001). Thus, lipolysis could be linked to either phenomena
observed in these assays.
The proteins of the cheese slurry are not completely recovered in CFW because the biggest
caseins molecules are precipitated during the neutralization step. However, smaller whey
proteins that are not precipitated under acid condition if not denatured, soluble nitrogen
(SN) and non-protein nitrogen (NPN) like peptides and amino acids resulting from
proteolysis should be in the CFWs. These two last compounds are important because they
are essential to the development of most probiotic bacteria strains (Conway et al., 2001),
and especially with those that do not show high proteolytic activities (Klaver et al., 1993).
On the other hand, some peptides resulting from the hydrolysis of milk proteins have
41
antimicrobial activities (Korhonen and Pihlanto, 2006). Therefore, proteolysis by Y/M can
potentially result in both stimulatory and inhibitory activities towards the lactic cultures.
Various other compounds could be involved. The CFW may include growth-promoting
vitamins synthesised by the rind flora (Souza Motta and Brandelli, 2008). Indeed, autolysis
of yeasts is recognized to be a good source of nutrients for lactic cultures (Smith, 1975). In
another way, inhibition could be explained by the volatile compounds only synthesized by
P. camemberti. For instance, styrene, which is recognized to give a plastic odour to cheese,
can be the responsible for inhibitory actions (Leclerq-Perlat et al., 2004b).
3.3.4 Milk source influence on biomass levels
The milk source influenced the growth of the Y/M (Table 3.2) and it was examined if the
milk source also influenced bacterial development. Milk A was from a large production of
Holstein cows and milk B came from a small production of Brown Swiss cows. The same
set of data as presented for Milk A (Figures 3.2 and 3.3) were obtained with Milk B. The
latter are not shown because the growth levels of the various lactic and probiotic cultures
generally followed the same patterns in the two milk sources. Indeed good correlations
between the two sets of ODmax data were obtained, and particularly for L. casei A180 and
B. longum R0175. The GG strain was the only one where the milk source had no significant
effect (Table 3.3).
This suggests that any inhibitory or stimulatory effects of the growth of a fungi on Milk A
on the subsequent development of probiotics generally occurs as well if the mycete is
grown on another milk source. However, there were differences between the levels of
stimulation or inhibition. In instances where a significant difference was noted in the
growth of the lactic cultures in Milk A and Milk B CFWs, biomass levels were 20 to 43%
higher when the lactic cultures were grown on Milk B. It is noteworthy that the opposite
was found with respect to the effect of milk source on Y/M growth (Table 3.2).
42
Table 3.3. Bacteria growth difference between the Milk B and the Milk A CFWs. The R2
indicates the relationship between the two milks within the various strains and “Difference
%” represent all the average difference of all the strains mixed together.*
Strain Difference % R2
BB12 21** 0.50
R0175 43** 0.85
R0011 20** 0.67
A180 31** 0.89
GG -26 0.57
General 25** 0.67 * The R
2 was calculated between the averages of four assays in all the mycetes CFW using the GLM
procedure in SAS.
** Significant difference using ttest procedure in SAS (P < 0.05)
Brown Swiss is a cow breed recognized to give richer milk than Holstein cows. Caseins
rate and fat rate are higher in milk from Brown Swiss cows. These higher rates also occur
in some Cheddar and Italian cheese made with Brown Swiss milk when compared with the
same kind of cheese made with Holstein cow milk (Mistry et al., 2002; De Marchi et al.,
2008). Proteolysis provides growth factors to bacteria, consequently a cheese with more
proteins is susceptible to provide them more amino acids. The buffering capacity of these
Brown Swiss Cheeses was also higher than the same cheeses made with Holstein milk. It
remains to be determined if to what extend these differences in fat, protein and buffering
capacity explain these results.
3.4 Conclusion
This study examined the interactions between mycetes and bacterial strains in AS. It was
found that prior growth of the yeast specie G. candidum on a milk curd simulating
Camembert cheese tended to enhance the growth of lactobacilli and bifidobacteria strains.
At the opposite, prior growth of the P. camemberti strains studied tended to inhibit the
43
bacterial strains. Finally, most of the other species tested (D. hansenii, I. orientalis and P.
anomala) did not modify the Camembert cheese slurry enough to affect the biomass level
of the bacteria.
Furthermore, an effect of the milk source on the growth of both Y/M and lactic cultures
was noted but in a different fashion. Generally, the milk B CFWs from a small production
with Brown Swiss cows conferred higher bacterial biomasses, while slurries prepared from
the Holstein large production milk source benefited most the growth of the Y/M.
Automated spectrophotometry has shown to be an effective tool to predict bacterial
biomass levels in growth media (Champagne et al., 2009b) as well as interactions between
lactic cultures in a Cheddar cheese fermentation process (Champagne et al., 2009a). It is
unknown, however, if the interactions noted in these assays will occur during Camembert
cheese production. Thus, the relationships between mycetes and bacteria found in this
research have to be tried in actual cheesemaking conditions. Indeed, the time, incubation
temperature and pH conditions used in these assays aren’t the same that would occur during
the ripening of a Camembert cheese. In the next chapter, these conditions were
experimented in cheese slurries with the probiotic strain Lactobacillus rhamnosus R0011
and the Lactobacillus casei A180 ripening strain. They were combined with a blend of
three mycete strains from different species having various effect in the AS study (P.
camemberti PCPSM2, D. hansenii LMA 668 and G. candidum LMA 664).
Acknowledgements
Yves Raymond, and Gaétan Bélanger are gratefully acknowledged for their scientific and
technical expertise. Mathieu Lapointe is also thanked for his technical assistance for the
experiment. This study was financially supported by the Fonds Québécois de Recherche sur
la Nature et les Technologies (FQRNT), NOVALAIT Inc., the Ministère de l’Agriculture
des Pêcheries et de l’Alimentation du Québec as well as Agriculture and Agri-Food
Canada. Pierre-Luc Champigny was also a recipient of an Excellence Grant from FQRNT.
44
Chapitre 4 : Biocompatibilité des bactéries lactiques
probiotiques et d’affinage avec les mycètes au sein de
caillés modèles de fromage Camembert.
Biocompatibility between Probiotic/specialty lactic acid
bacteria and mycetes in Camembert cheese slurry.
Pierre-Luc Champigny b, Claude P. Champagne
a, Daniel St-Gelais
a, Ismail Fliss
b, Steve
Labrie b
a Centre de recherche et développement sur les aliments, Agriculture et agroalimentaire Canada, 3600 boul.
Casavant Ouest St. Hyacinthe QC, Canada J2S 8E2
b Institut des Nutraceutiques et des Aliments Fonctionnels, Centre de recherche STELA, Université Laval,
Québec, QC, Canada G1V 0A6
45
Résumé
Cette étude a été réalisée dans le but de vérifier la capacité du fromage Camembert à
supporter la viabilité de cultures lactiques de spécialité (probiotique et d’affinage). Des
caillés modèles fabriqués à partir de deux sources de lait (vaches Holstein et Suisse Brunes)
ont été simultanément inoculés de souches mycéliennes et de bactéries. Les souches
fongiques inoculées étaient Penicillium camemberti PC PSM2, Geotrichum candidum
LMA 664 et Debaryomyces hansenii LMA 668. Les cultures lactiques étaient formées
d’une souches probiotique (Lactobacillus rhamnosus R0011) ou d’une souche pour
accélérer l’affinage du fromage (Lactobacillus casei A180). Les mycètes étaient inoculées
sous forme pure ou en combinaisons de deux ou trois souches. La viabilité et le pH des
différents microorganismes ont été suivis sur une période de 12 jours d’affinage à 12˚C et à
une humidité relative de 95%. Le pH de départ était de 4,8 pour tous les caillés modèles.
Pour les bactéries probiotiques, la présence de levures et moisissures peu importe l’espèce a
été positive pour leur survie. C’était aussi le cas pour la souche d’affinage mais le bénéfice
n’était pas aussi grand. De plus, le type de lait utilisé n’a eu aucun effet sur la viabilité de la
souche probiotique. Néanmoins, il a influencé celle de la souche d’affinage. De manière
générale, l’utilisation de lait de vaches Suisse Brunes a donné des comptes viables plus
élevés que l’utilisation du lait d’Holstein. Inversement, les souches bactériennes employées
n’ont pas inhibé significativement le développement de la flore de surface du fromage.
Finalement, cette recherche a démontré que le Camembert pourrait être un bon aliment pour
promouvoir la viabilité des bactéries probiotiques et que la flore fongique pouvait affecter
les comptes de ceux-ci.
46
Abstract
The purpose of this study was to ascertain the potential of Camembert-type cheese to
support the viability of specialty lactic acid bacteria (probiotic and ripening strains). Cheese
slurries made from two milk sources (Holstein or Brown Swiss cows) were simultaneously
inoculated with mycete strains and bacteria. The mycete cultures were Penicillium
camemberti PC PSM2, Geotrichum candidum LMA 664 and Debaryomyces hansenii LMA
668. The specialty lactic cultures were a probiotic strain (Lactobacillus rhamnosus R0011)
and a strain for accelerated ripening of cheese (Lactobacillus casei A180). The mycetes
were inoculated as pure cultures or in combinations of two or three strains. The viability
and pH of the different microbial strains was followed over 12 days of ripening at 12°C at
95% relative humidity. The initial pH was of 4.8 for all products. For the probiotic strain,
the presence of mycetes, whatever the specie, was positive for its development. This was
also the case for the ripening culture but the benefit of the mycetes was not as extensive.
The milk source had no effect of the viable counts of the probiotic culture, but growth of
the ripening culture was higher in the milk from Brown Swiss cows. The lactic acid
bacteria strains employed in this experiment generally did not significantly affect the
development of the rind flora strains. This study suggests that Camembert cheese can be a
good food matrix to promote probiotic viability and that mycete strain can affect the
resulting probiotic CFU levels.
47
4.1 Introduction
Formulating food with probiotic bacteria is a challenge because it is considered that
viability is an important requirement for their functionality (FAO/WHO, 2001). In foods,
factors such as pH, redox level, buffering capacity and storage temperature can influence
their viability (Champagne et al., 2005). Probiotic bacteria need to survive these
detrimental conditions all along the food processing as well as during shelf life. Hence, the
choice of the food matrix to which probiotic bacteria are added must be made taking into
account its composition.
Dairy products are often used as carriers of probiotic bacteria. Yogurt is the most popular
even though its acidity can lead to losses in viability during storage. Cheddar (Phillips et
al., 2006; Daigle et al., 1999), semi-hard goat cheese (Gomes and Malcata, 1998) and
Gouda (Gomes et al., 1995) can be good matrices for probiotics since their pH is higher
than yoghurt; this tends to minimize the losses of viability during shelf life. Bacterial
strains with some probiotic potential have been isolated from Camembert cheese made with
raw milk (Coeuret et al., 2004). However, the ability of probiotic bacteria cultures to grow
or remain viable in Camembert has not been studied.
The ripening of Camembert-type cheese involves biochemical activities from yeast and
mould (Y/M) which change the composition of the cheese, particularly at its surface. These
modifications mainly result from proteolysis, lipolysis and lactic acid assimilation.
Principally, the rind microbiota is responsible of the pH increase as well as the release of
free fatty acids and amino acids during ripening of Camembert (Spinnler and Gripon,
2004). These changes could potentially affect the probiotic cultures and, by the way,
adjunct bacterial ripening cultures. Since probiotics are sensitive to low pH and may not be
able to metabolize proteins due to a low proteolytic activity (Champagne et al., 2005;
Klaver et al., 1993), their development could be stimulated by the modifications brought by
Y/M. However, Y/M could possibly produce toxic molecules for bacteria such as free fatty
acids, antimicrobial peptides and phenylactic acid (Dieuxleveux et al., 1998)
48
Accordingly, the biocompatibility between lactic acid bacteria and mycetes in Camembert-
type cheese needs to be explored. The interactions between these microorganisms had ever
been studied in other media and it leads to a diversity of results. These interactions seem to
vary as a function of the strains used (De Freitas et al., 2009; Álvarez-Martín et al., 2008).
In some cases, probiotic and non-probiotic lactic acid bacteria were able to prevent food
spoilage from Y/M (Voulgari et al, 2010; Tharmaraj and Shah, 2009), in other cases, yeast
strains enhanced viability of probiotic bacterial strains in yoghurt (Liu and Tsao, 2009).
The aim of this study was to explore the biocompatibility of lactic acid bacteria with
mycetes species that compose the rind flora of Camembert. The Y/M strains were all
isolated from the raw milk originating from the province of Quebec (Canada) except for P.
camemberti. A screening of their biocompatibility with bacteria was previously made by
automated spectrophotometry (AS) (Chapter 3) where the development of the probiotics
was tested on a medium on which the Y/M had previously grown. In this study, the
viability of probiotic and ripening lactic acid bacteria has been tested in cheese slurry in
simultaneous growth with the Y/M cultures. Also, the effect of the milk source on the
interactions was tested as in the AS study. Two milk sources were used, the first from a
large Holstein production and the second from a small Brown Swiss cows production.
Finally, the influence of the bacterial strains used for the experiment on the Y/M biomass
was verified.
49
4.2 Materials and methods
4.2.1 Strains (mycetes and bacteria) and milk sources
Bacterial and mycete strains used in this study are listed in Table 4.1. The bacterial strains
were obtained from two commercial suppliers. The probiotic strain was selected because it
is a commercially available culture having documented health benefits. The yeast strains
were isolated from different milk sources over the Quebec province (Canada). Also, a
commercial P. camemberti strain was used; PCPSM2 from Cargill France. The two milk
sources used to prepare the cheese slurries were from a large scale production and from a
small farm. These milks were from different breeds of cows: Holstein for the large-scale
production (multi-farm from a tanker: Milk A in Table 4.1) and Brown Swiss milk from
small producers in a particular geographical region (Milk B in Table 4.1).
Table 4.1. Bacterial and mycete strains used for this work
Genus Species Type Strain Source
Lactobacillus rhamnosus probiotic R0011 Institut Rosell-Lallemand,
Mtl, Canada
Lactobacillus casei ripening A180 Abiasa, St-Hyacinthe,
Canada
Penicillium camemberti mould PC PSM2 Cargill France SAS, La
Ferté sous Jouarre
Geotrichum candidum yeast LMA 664 Milk A
Debaryomyces hansenii yeast LMA 668 Milk B
D. hansenii frozen stock culture was prepared by a YM broth (Becton Dickinson, Sparks,
MD, USA) solution having 30% w/w glycerol (Sigma-Aldrich, St-Louis, MO, USA) with a
fresh liquid inoculum grown on YM broth in a 1:1 ratio. For G. candidum and P.
camemberti the cell suspension was prepared by recovering colonies of P. camemberti and
G. candidum from the surface of an acidified potato dextrose agar plate (PDA; EMD
50
Chemicals, Darmstadt, Germany) using a swab humidified in a filter-sterilized (0.22μm
Millex GP syringe filter, Millipore, Carrigtwohill, Co. Cork, Ireland) 0.05% w/v Tween 80
(Fisher Scientific, Fairlawn, N-J, USA) solution. The cells from the swab were resuspended
in a Tween 80 solution which was then blended with the glycerol-YM broth at the 1:1 ratio
as for the other mycete cultures.
The Bacterial frozen stock cultures were prepared blending a BHI broth (Becton Dickinson)
having 15% glycerol with a fresh liquid culture (pH of 4.5) in a 5:1 ratio. All these cell
suspensions were divided in aliquot of 1mL cryovials (Nalgene, Rochester, NY, USA) and
placed in a -80˚C freezer.
4.2.2 Inocula preparation and cultures conditions
The D. hansenii inocula were obtained from YM broths (Becton Dickinson) seeded at 1%
v/v with thawed stock cultures which were incubated at 30˚C on a shaker (250 rpm) until
they reached an optical density (OD) between 0.4 and 0.8. The OD was determined with a
Beckman 7400 Spectrophotometer at 600 nm (Coulter, Fullerton, CA, USA). The CFU mL-
1 of the liquid cultures were estimated by the OD measure after having established an OD-
CFU standard curve.
The P. camemberti and G. candidum biomass were obtained by spreading a thawed stock
culture at the surface of a potato dextrose agar plate and incubating for 1 week at room
temperature (23˚C). The mould spores were collected using a sterile swab and suspending
in a Tween 80 solution as previously described. The concentration of the cell suspension
was determined by using a hemacytometer (Hausser Scientific, Horsham, PA, USA).
Finally, the bacteria inocula were prepared in MRS broth (Becton Dickinson) supplemented
with 1% v/v of a 10% w/v sodium ascorbate (Sigma-Aldrich) and 5% w/v L-Cystein
Hydrochloride (Sigma-Aldrich) filter-sterilized solution. This MRS medium was inoculated
with 1mL of a thawed stock culture and incubated at 37˚C until a pH of 4.5 was attained.
51
4.2.3 Production of cheese slurry powder
A cheese slurry is a model system of a cheese obtained after hydration of a lyophilized
cheese powder. To prepare the cheese slurry powder, fresh camembert cheese was
produced with the two milk lots. Whole milk was pasteurized in batch at 65˚C for 30
minutes. The temperature of milk was then adjusted to 32˚C before inoculating at 1% w/w
with a lyophilized Flora Danica starter (Chr Hansen, Milwaukee, WI, USA). After
inoculation, maturation of milk occured for 45 minutes at 32˚C. During this time, a CaCl2
(Calsol, Danisco, Copenhagen K, Denmark) 45% w/v solution was added at 0.035% w/w.
Subsequently, the rennet (CHY-MAX extra, Chr Hansen) was added at 2.25 mL/40L of
milk. 40 minutes after adding the rennet, the curd was cut into pieces of 2cm side to release
the whey. The curd pieces were ready for molding when whey reached a pH of 6.4. Kept at
room temperature, the molds were turned over after one hour and three hours for whey
drainage. Finally, the cheese molds were placed in a chamber overnight. The curds were
initially at 28˚C and the chamber was programmed to gradually go down to 16˚C for the
next morning. Then, instead of carrying out the salting and ripening steps, the cheeses were
freeze-dried, grinded and vacuum packed to conserve them in a powder form at -40˚C.
4.2.4 Cheese slurries assays
Cheese slurries were prepared and analyzed at different days during their ripening step (0,
3, 6 and 12) to follow the viability of lactic acid bacteria (probiotic or ripening strain) in
Camembert-type cheese. Different combinations of mycete and bacterial strains were
chosen (Table 4.2) to verify their interaction with the bacterial strains (stimulation,
inhibition or neutral).
Before hydration of cheese slurry powder, fresh cultures of each microorganisms used for
the inoculation of cheese slurries were prepared as described in 4.2.2. Then, bacterial and
D. hansenii liquid cultures were centrifuged to prevent components of the growth media to
52
influence the growth in cheese slurry. Centrifugation was carried out at 10000g for 20
minutes using a Sorvall RC-5B (Dupont, Mississauga, Ontario, Canada) centrifuge on
strains R0011 and A180. For the D. hansenii strains, centrifugation was done at 8000 g
instead of 10000 g. The pellets obtained were resuspended in a sterile 0.5% w/w NaCl
solution (LaboMAT, Montreal, Qc, Canada) to 10% of the original cell suspension
concentration.
Table 4.2. Combinations of bacteria and mycetes strains done in cheese slurries
R0011 (C*) A180 (C)
R0011/M2 A180/M2
R0011/664 A180/664
R0011/668 A180/668
R0011/664,M2 A180/664,M2
R0011/668,M2 A180/668,M2
R0011/668,664 A180/668,664
R0011/668,664,M2 A180/668,664,M2
* control treatment without Y/M
Then, the powder was hydrated at 57% w/w with physiological water acidified with DL-
Lactic acid (Fisher Scientific, Fair Lawn, NJ, USA) and salted with NaCl (3.5% w/w)
(LaboMAT). The pH was adjusted with lactic acid to pH 4.8 for each cheese slurry made
with the two different sources of milk. The slurry was inoculated at 105
CFU g-1
with the
mycete strains according to the combination used (Table 4.2). After that, the slurry was also
seeded at 108 CFU g
-1 with the appropriate bacteria. Moreover, a control treatment (C)
without rind fungus flora was done for each bacteria strains using Pimaricin (EMD
Biosciences, Darmstadt, Germany) at 40 mg/kg of cheese slurry (FAO/WHO, 2010).
Finally, 70 g of the slurry was disposed in a glass jar of 250 mL covered with micro
perforated wrapping paper for Camembert. Each sampling day was represented by a
different glass jar containing 70 g of cheese slurry. Each jar was ripened in a chamber at
53
12˚C and 95% relative humidity for 12 days. The cheese slurry jars were prepared in
aseptic conditions.
4.2.5 Enumerations of microorganisms and pH measurement
After inoculation, viable counts of the cell suspensions were carried out to ascertain the
exact CFU g-1
of cheese slurry at day 0. For the Y/M cultures, the first dilution of this serial
was done using a bottle of 99mL of peptone water 0.1% w/v (Becton Dickinson) containing
glass beads. Subsequently, this suspension was serially diluted in sterile 0.1% w/v peptone
water (Becton Dickinson) tubes and vortexed. Finally, 0.1mL of the appropriate dilution
was spread in duplicate at the surface of an acidified PDA (see 4.2.2 section). Bacterial
cultures were enumerated by homogenizing their first dilution in 0.1% (w/v) peptone water
(Becton Dickinson) during 30 seconds at 27000 rpm with Omni-Tips generator probes.
They were subsequently serially diluted and vortexed with peptone water tubes, and 1 mL
of the appropriate dilution was pour-plated in duplicate in MRS agar (Becton Dickinson).
PDA plates were incubated at 23°C for 5 days while MRS plates were incubated at 40˚C
for 48 h to prevent the growth of the Flora Danica starter. Petri plates were incubated in
aerobiosis.
For the samples taken after 3, 6 and 12 days of incubation, the CFU analyses were carried
our similarly, except for the first dilution where cheese slurry sample was diluted 9:1 in a
sterile 2% w/v sodium citrate (Fisher Scientific) solution at room temperature and
homogenized using a stomacher unit (Seward, model 400 Circulator; Worthing, West
Sussex, UK) at 260 rpm for 2 minutes
To measure the pH, two parts of the slurry were diluted with 1 part of milliQ water
(Millipore) and homogenized 1 minute using a homogenizer (Omni TH, Omni
international, Kennesaw, GA, USA). Homogenate pH was then evaluated using a pH meter
(XL15, Acumet, Fisher Scientific) adjusted with pH 4.0 and pH 7.0 standard buffers (Fisher
Scientific).
54
4.2.6 Statistical analyses
Paired T test were carried out using Instat software (Graphpad, San Diego, CA, USA).
ANOVA and T tests were carried out on SAS (SAS institute Inc., Cary, North Carolina,
USA) with the GLM and the ttest procedure. The GLM repeated procedure was also used
when the results come from repeated measures in time on the same cheese treatment batch.
Significant differences between results were determined using the Fisher’s least
significance difference (LSD) test. Each data reported is the average of 3 or more
independent assays. Each statistical test was done at a 95% confidence level.
4.3 Results and discussion
4.3.1 Probiotic culture biocompatibility and viability
The viability of the probiotic culture Lactobacillus rhamnosus R0011 was studied in
Camembert cheese slurry made from two sources of milk and ripened by different Y/M
strains combinations.
A previous study on cell-free whey extracts of the mycete-fermented slurries using
automated spectrophotometry (Chapter 3) had shown an effect of milk source on the
growth of both lactic cultures. Therefore, paired T tests were done to compare the CFU
counts between results of the two kinds of milk. However, there was no significant effect of
milk source on the growth of the probiotic strain (P = 0.16), which was not the case in
Chapter 3. The reason for the discrepancy was not determined. It must be kept in mind that,
in this study, the growth of the probiotic bacteria occurred in parallel with that of the Y/M,
while in the previous study it was sequential. Since, the pairing between CFU data of Milk
A and Milk B was also found to be effective (R = 0.7183; P = 0.02) the two series of CFUs
were combined to calculate the average values.
55
At D12, the viable counts of the probiotic culture paired with the different combinations of
Y/M were all higher than the Control (Tables 4.3). There were also some effects of Y/M
strain. The lowest CFU count was also noted with slurries obtained from the pure culture of
G. candidum. Thence, the greatest differences of viable counts for the probiotic strain were
between the control slurry (without rind flora) and the other ones ripened by the Y/M.
Lesser effects were noted between the mycete strains themselves.
Table 4.3. Viability (log CFU g-1
) of Lactobacillus rhamnosus R0011 in Camembert cheese
slurries ripened up to 12 days with different yeast and mould strains. Data are the average
of CFU in the 2 sources of milk*.
Y/M combination
Ripening time at 12°C (days)
0 3 6 12
P. camemberti PC PSM2 7.99 (a) 8.19 (a) 8.25 (a,b) 8.71 (a,b)
G. candidum LMA 664 7.99 (a) 8.16 (a) 8.23 (a,b) 8.57 (b)
D. hansenii LMA 668 7.99 (a) 8.19 (a) 8.24 (a,b) 8.74 (a,b)
664-668 7.99 (a) 8.22 (a) 8.25 (a,b) 8.74 (a)
664-PSM2 7.99 (a) 8.23 (a) 8.21 (a,b) 8.65 (a,b)
668-PSM2 8.06 (a) 8.24 (a) 8.29 (a) 8.73 (a,b)
PSM2-664-668 7.99 (a) 8.16 (a) 8.18 (a,b) 8.65 (a,b)
Control 8.01 (a) 8.11 (a) 8.05 (b) 8.24 (c) * Values given represent the average of six independent assays. a,b,c
For a given column, values associated to the same letter are not significantly different (LSD, P>0.05).
The inoculation level in the slurries was set at 108 CFU g
-1. This might appear high, but it
must be kept in mind that cheesemaking concentrates the cells, since over 80% of the
bacteria inoculated in milk are recovered in the curd and are not lost in whey (Fortin et al.,
2011). Therefore a CFU at day 0 close to 108 CFU g
-1 would result from a typical
inoculation level of 107 CFU g
-1 in milk prior to renetting.
Lactobacillus rhamnosus R0011 did not show extensive growth in the control cheese
slurry during the 12 days. These results are of interest because the scientific literature on
probiotic food suggests a recommended daily dose of probiotic bacteria around 1 billion (9
56
log CFU) (Charteris et al, 1998; Lee & Salminen, 1995). Considering 30 g as representing a
portion of cheese, the products obtained in this study would deliver 1011
CFU per portion,
which is quite high.
Table 4.4. Variation of the homogenate pH of different Camembert cheese slurries made
with 2 different sources of milk and inoculated with Lactobacillus rhamnosus R0011 and
different yeast and mould strains and ripened 12 days*.
Y/M combination
Ripening time at 12°C (days)
3 6 12
P. camemberti PC PSM2 4.86 (b) 5.05 (a) 5.70 (a)
G. candidum LMA 664 4.89 (a,b) 4.98 (a) 5.37 (b)
D. hansenii LMA 668 4.95 (a,b) 5.01 (a) 5.16 (c)
664-668 4.96 (a) 4.97 (a) 5.28 (b,c)
664-PSM2 4.95 (a,b) 5.00 (a) 5.60 (a)
668-PSM2 4.91 (a,b) 5.04 (a) 5.67 (a)
PSM2-664-668 4.90 (a,b) 5.01 (a) 5.64 (a)
Control 4.92 (a,b) 4.82 (b) 4.73 (d) * Values given represent the average of six independent assays. a,b,c,d
For a given column, values associated to the same letter are not significantly different (LSD, P>0.05).
The growth of the Y/M is accompanied by a rise in pH. This effect on pH is mostly due to
the assimilation of lactic acid in aerobic conditions, but release of ammonia following
proteolysis can also contribute. When comparing the control treatment pH to the Y/M
combinations ones (Table 4.4), a significant difference is also revealed. As noted with the
log CFU g-1
results, the more the ripening days advance, the more the control treatment pH
becomes significantly different from the other treatments. Regression analyses between pH
data at D12 (Tables 4.4) and viable counts at D12 (Tables 4.3) gave R2 values of 0.52. The
relationship between CFU and pH values at D12 was statistically significant. The higher
the pH was at D12 the higher were CFU readings of the probiotic culture. This confirms
that pH is important for probiotic cultures viability in foods (Roy, 2005; Shah, 2000), but
also shows the limit to this relationship. With an initial pH of 4.8, these slurries may be
compared with yogurt where decreases of viability following refrigerated storage were
noted by many authors (Shah et al, 1995; Micanel et al, 1997; Jayamanne and Adams,
57
2006). Although these R2
values show that pH is the main factor influencing growth of the
probiotic bacteria in the slurries, they nevertheless show that a sizeable fraction (48%) of
variations in CFU data is linked to other factors. Previous AS data show that, at equal pH
levels, the Y/M strain has a small effect on the growth of probiotic culture (Chapter 3).
Interestingly, some AS data on the effects of Y/M strain appear in disagreement with those
of this work. Indeed, prior growth of G. candidum was generally beneficial to L. rhamnosus
R0011 while that of P. camemberti was detrimental (Chapter 3). The opposite was noted in
this study (Table 4.3). This confirms that the effect of the Y/M strain on the evolution of
pH is more important than its effect on proteolysis or lipolysis of the cheese slurry.
However, the absence of strong inhibition of probiotics by Y/M observed in AS was
confirmed in cheese slurries. Also, for the probiotic culture, the incubation temperature,
incubation duration and the pH conditions in AS were not the same in this experiment,
which can potentially explain the differences between the two sets of data.
4.3.2 Ripening bacteria biocompatibility and viability
The same conditions as for the probiotic strain were tested on the L. casei A180, which is a
specialty culture for the accelerated ripening of cheese. The observations on growth (Table
4.5) were different from those of the probiotic strain. To begin, it was found that L. casei
A180 viable counts were systematically higher by 0.2 log CFU g-1
in products made with
Milk B at D12 (Table 4.5). This was in line with the data of Chapter 3. Also, in milk A, the
viable counts at day 12 were not statistically different.
58
Table 4.5. Viability of L. casei A180 at D12 in Camembert cheese slurries ripened 12 days
with combination of different yeast and mould strains in 2 different sources of milk*.
Y/M combination
Log CFU g-1
at day 12
Milk A Milk B
P. camemberti PC PSM2 9.33 (e,f) 9.57 (a,b,c)
G. candidum LMA 664 9.43 (d,e,f) 9.62 (a)
D. hansenii LMA 668 9.46 (b,c,d,e) 9.62 (a)
664-668 9.45 (c,d,e) 9.62 (a)
664-PSM2 9.36 (d,e,f) 9.57 (a,b,c)
668-PSM2 9.35 (d,e,f) 9.57 (a,b,c)
PSM2-664-668 9.31 (f) 9.58 (a,b)
Control 9.33 (e,f) 9.48 (b,d,c) * Values given represent the average of 3 independent assays for each milk source. a,b,c,d,e,f
Values associated to the same letter are not significantly different (LSD, P>0.05).
However, in milk B, the viable counts of A180 were all higher than their counterparts in
milk A. Paired T tests showed that the average difference of 0.2 log was statistically
significant (P < 0.05). The greater development of L. casei A180 in milk B products was
also noted in AS assays (Chapter 3). This could potentially be explained by higher contents
in caseins and fat in milk from Brown Swiss cows. Moreover, these rates were also higher
in some Cheddar and Italian cheese made from Brown Swiss milk when compared to the
same kind of cheese produced from Holstein milk (Mistry et al., 2002; De Marchi et al.,
2008). Proteolysis provides growth factors to lactic bacteria, so it can be hypothesized that
a cheese with more proteins is susceptible to provide them more peptides or amino acids.
The buffering capacity of the Brown Swiss cheese was also higher than the same cheese
made with Holstein milk.
59
Table 4.6. Homogenate pH at D12 of Camembert cheese slurries inoculated with L. casei
A180 and ripened 12 days with combination of different yeast and mould strains in 2
different sources of milk*.
Y/M combination
pH at day 12
Milk A Milk B
P. camemberti PC PSM2 5.59 (a) 5.42 (b,c)
G. candidum LMA 664 5.23 (d,e) 4.88 (f)
D. hansenii LMA 668 4.97 (f) 4.66 (g)
664-668 5.14 (e) 4.68 (g)
664-PSM2 5.63 (a) 5.32 (c,d)
668-PSM2 5.57 (a,b) 5.36 (c,d)
PSM2-664-668 5.60 (a) 5.34 (c,d)
Control 4.40 (h) 4.29 (h) * Values given represent the average of 3 independent assays for each milk source. a,b,c,d,e,f g,h
Values associated to the same letter are not significantly different (LSD, P>0.05).
It is noteworthy the higher results of viable counts in milk B for L. casei A180 were all
obtained at lower pH (Table 4.5 and 4.6). Therefore, in contrast with the probiotics, with L.
casei A180 the evolution of pH was not the principal factor for growth. In fact, as the
Control treatment shows there was acidification of the cheese slurry during the 12 days
ripening period. This suggests that the higher growth of L. casei A180 in cheese slurries
made from milk B could be related to acidification of the cheese slurry and explain the
lower pH values even in the presence of the Y/M. The L. casei strain used is a specialty
culture for cheese ripening. Its ability to grow in cheese having low pH in these assays
confirmed its great potential as a non-starter lactic acid bacterium (Beresford and Williams,
2004). This could explain the sharp differences in growth patterns of L. casei A180 in
cheese slurries, as compared to the probiotic L. rhamnosus R0011 culture.
60
4.3.3 Yeasts and moulds biocompatibility with bacteria
The potential effect of the bacterial cultures on the growth of the three Y/M strains used to
inoculate the cheese slurries was also investigated. In products inoculated with the pure
Y/M cultures, the log CFU g-1
values of individual Y/M strains at the last day of ripening
(day 12) were compared between the different bacterial strains and with a control treatment
without bacterial cultures. The only differences noted (figure 1) was for the G. candidum
LMA 664 strain. It grew a little better when combined with L. rhamnosus R0011 than when
combined with L. casei A180. Moreover, it had a higher biomass result in the control
treatment. Although, these differences were not very important, it cannot be related to a
severe inhibition of G. candidum by L. casei.
The biomass of G. candidum LMA 664 and D. hansenii LMA 668 strains were not affected
by the bacterial strain in presence. The log CFU g-1
between 7 and 8 for G. candidum and
around 8 for D. hansenii are often seen at day 12 in Camembert cheese (Leclercq-Perlat et
al., 1999; Leclercq-Perlat et al., 2004a). Therefore the viable counts for these two cultures
are in line with those found in the literature and observed previously (Table 3.2). For P.
camemberti, a log CFU g-1
between 6 and 6.5 is higher than in commercial cheese where
the log CFU g-1
is usually of 5 at day 12 (Leclercq-Perlat et al., 2004a). Studies which
mention that lactic acid bacteria enhance the development of mycetes are rare. Most of the
time, papers concerning fungi and lactic acid bacteria interactions report a negative impact
of the bacteria on the growth of mycetes. Some studies employed these lactic cultures to
prevent food spoilage by Y/M (Tharmaraj and Shah, 2009; Voulgari et al, 2010). Evidently,
in the case of Camembert cheese, when Y/M strains are inoculated in significant amount as
starters, probiotic and ripening bacteria do not inhibit the fungal rind flora.
Finally, with the three Y/M pure cultures, the CFU values at day 12 were 0.12 log higher in
Milk A than in Milk B, but this difference was not judged to be statistically significant (P =
0.20). In a previous study (Chapter 3), the same observation was made with respect to
slightly higher growth on Milk A but, in that instance, 9 strains had been used and the
61
difference was found to be statistically significant. Therefore data from this study are in
line with Chapter 3, and suggest that milk source has a low effect on the biomass of Y/M
reached after 12 days of ripening.
R0011 A180 Control5.5
5.7
5.9
6.1
6.3
6.5
6.7
6.9
7.1
8.1
8.2
8.3
8.4
8.5
8.6
8.7
8.8
Bacterial Strain
Lo
g C
FU
g-1
LMA 668
PC PSM2
7.2
7.3
7.4
7.5
7.6
7.7
7.8
LMA 664
a
aa
aaa
a a
b
R0011 A180 Control5.5
5.7
5.9
6.1
6.3
6.5
6.7
6.9
7.1
8.1
8.2
8.3
8.4
8.5
8.6
8.7
8.8
Bacterial Strain
Lo
g C
FU
g-1
LMA 668
PC PSM2
7.2
7.3
7.4
7.5
7.6
7.7
7.8
LMA 664
a
aa
aaa
a a
b
Figure 4.1. Effect of cheese slurry inoculation with two different bacterial strains (L.
rhamnosus R0011, and L. casei A180) on the growth of Penicillium camemberti PC PSM2,
Geotrichum candidum LMA 664 and Debaryomyces hansenii LMA 668. Values given
represent the average of six independent assays. a,b In the same histogram, values associated
to the same letter are not significantly different (LSD, P>0.05). Error bars represent SEM.
62
4.4. Conclusion
This study examined the interactions between mycete and bacterial strains in Camembert
cheese slurry. Generally, the results showed that viability of bacteria was not enhanced by a
particular yeast and mould strain alone or in a culture blend. However, for the
Lactobacillus rhamnosus probiotic strain, the presence of fungi strains, whatever the
specie, was positive for their development. On the other hand, the specialty ripening
culture, Lactobacillus casei A180, was usually not stimulated by the mycetes.
Furthermore, the effect of the milk source for cheese slurries production on the mycetes-
bacteria interactions was also ascertained. For the probiotic bacterial strain, the milk source
had no effect on its viable counts. Nevertheless, it had an influence on the ripening culture
development. The milk from a small production with Brown Swiss cows generated cheese
slurries which slightly improved viable counts of L. casei A180 when compared to the
Holstein large bulk production milk.
With the probiotic culture, growth was found to be associated with the de-acidification of
the cheese slurry. As a result, AS data on non-pH effects of the Y/M on the slurries or of
milk source did not show strong correlation with viable counts of the probiotic in the
slurries. This was not the case with the L. casei A180 ripening culture. Since the growth of
this culture was not as affected by pH as was that of the probiotic, then the beneficial effect
of milk B observed in AS studies was also noted in viable counts in the cheese slurries.
Also, inversely, the influence of the bacterial cultures on Y/M biomasses in cheese slurries
was tested. In general, the lactic acid bacteria strains employed in this experiment did not
inhibit the development of the rind flora strains.
Cheese slurries have been shown to be valuable models in predicting events under actual
cheesemaking conditions. But assays in manufacturing conditions must still be carried out
to confirm these findings. Therefore, work still has to be done in a Camembert cheese
63
process. Furthermore, the viability of the probiotic bacteria needs to be measured on a
longer period of time because Camembert-type cheese had an estimated shelf life of 2
months. In the next chapter, pilot scale Camembert cheese were produced with a blend of
two probiotic strains; L. rhamnosus R0011 and Bifidobacterium lactis BB12. This blend
was inoculated in three treatments: 1 with commercial fungi strains and 2 with different
terroir yeasts blend.
Acknowledgements
Yves Raymond, and Gaétan Bélanger are gratefully acknowledged for their scientific and
technical expertise. Mélanie Gobeil-Richard is also thanked for her technical assistance for
the experiment. This study was financially supported by the Fonds Québécois de Recherche
sur la Nature et les Technologies (FQRNT), NOVALAIT Inc., the Ministère de
l’Agriculture des Pêcheries et de l’Alimentation du Québec as well as Agriculture and
Agri-Food Canada. Pierre-Luc Champigny was also a recipient of an Excellence Grant
from FQRNT.
64
Chapitre 5 : Viabilité de bactéries probiotiques au sein de
fromages Camembert fabriqués avec des souches
fongiques isolées de lait de terroir québécois.
Viability of probiotic bacteria in Camembert cheese
made with fungi strains isolated from Quebec terroir
milk
Pierre-Luc Champigny b, Claude P. Champagne
a, Daniel St-Gelais
a, Ismail Fliss
b, Steve
Labrie b
a Centre de recherche et développement sur les aliments, Agriculture et agroalimentaire Canada, 3600 boul.
Casavant Ouest St. Hyacinthe QC, Canada J2S 8E2
b Institut des Nutraceutiques et des Aliments Fonctionnels, Centre de recherche STELA, Université Laval,
Québec, QC, Canada G1V 0A6
65
Résumé
Du fromage Camembert inoculé avec deux souches probiotiques (Lactobacillus rhamnosus
R0011 et Bifidobacterium lactis BB12) a été fabriqué à l’échelle pilote. L’effet de la
composition de la flore fongique d’affinage sur les comptes viables des probiotiques lors de
l’affinage et l’entreposage a été étudié. Trois mélanges de cultures fongiques furent
évalués. Deux mélanges étaient constitués de souches lévuriennes isolées du terroir
québécois (Canada) et le troisième était constitué d’un mélange de souches commerciales.
La viabilité des probiotiques a donc été évaluée à la surface et au cœur des meules de
fromage pour une durée de 30 jours. Le pH et la protéolyse ont aussi été mesurés à la
surface et au centre. Après 30 jours, la viabilité était élevée (entre 6 et 8 log UFC g-1
) pour
les deux souches utilisées. La population de L. rhamnosus R0011 était plus élevée en
surface qu’au cœur du fromage, et ce par plus de 1 log UFCg-1
. Les concentrations
cellulaires de B. lactis BB12 étaient similaires en surface et au centre du fromage. De plus,
les souches fongiques du terroir utilisées se sont avérées aussi efficaces que celles d’origine
commerciale pour stimuler la viabilité des probiotiques. En conclusion, cette étude tend à
confirmer que le Camembert est une matrice alimentaire facilitant la viabilité des bactéries
probiotiques.
66
Abstract
Pilot-scale Camembert cheese was manufactured and inoculated with two probiotic strains:
Lactobacillus rhamnosus R0011 and Bifidobacterium lactis BB12. The effect of the
composition of the fungi ripening flora on the probiotic cultures viability during ripening
and storage was studied. Three different blends of fungi strains were prepared. Two blends
were constituted of Québec (Canada) terroir yeasts strains and the other one was a
commercial fungi strains blend. The viability of the probiotic bacteria at the rind and at the
core of cheese pieces was followed during manufacture as well as during 30 days of
ripening and storage. pH and proteolysis were also measured at the rind and at the core.
After 30 days, the cell counts were between 6 and 8 log CFU g-1
for the two probiotic
strains used. With L. rhamnosus R0011 strain, higher viable counts were observed at the
rind than at the core. On the other hand, B. lactis BB12 cell counts were similar at the rind
and the core. Furthermore, the terroir mycete strains used were as good as the commercial
cultures to enable the viability of the probiotic cultures. Some of them showed better
development at cheese rind than commercial ones. In conclusion, this study suggests that
Camembert-type cheese is a good food matrix to support the viability of probiotic bacteria.
67
5.1 Introduction
Cheeses are increasingly considered to be probiotic carriers. Cheddar (Phillips et al., 2006;
Daigle et al., 1999), semi-hard goat cheese (Gomes and Malcata, 1998), Cottage
(Blanchette et al., 1996), Kareish (Abou-Dawood, 2002), Minas-Frescal (Fritzen-Freire et
al., 2010), Kasar (Özer et al., 2008) and Gouda (Gomes et al., 1995) have all been
considered as potential matrices for probiotic bacteria. Probiotic cultures viability is
influenced by many factors such as pH, redox level, buffering capacity and storage
temperature (Champagne et al., 2005). When formulating probiotic cheese, it is important
to follow the viability of these beneficial bacteria during manufacturing as well as during
shelf life because this criterion in combination with the amount of bacteria is important for
their functionality (FAO/WHO, 2001).
Previous studies were recently carried out by our team in which the biocompatibility
between probiotic bacteria and mycete strains and the capacity of Camembert cheese slurry
to enable the survival of probiotic bacteria were studied (Chapters 3 and 4). It was observed
that mycetes enhanced probiotic viable counts in Camembert model cheese slurry but the
use of yeast and mould (Y/M) strains alone or in blends were not the critical factor
influencing the growth of the probiotics. The de-acidification of the curd during ripening by
Y/M seemed to be responsible of enhanced viable counts in the model systems.
However, in this prior study, the cheese slurries were only ripened for 12 days and the
probiotic bacterium was inoculated after the manufacturing cheese process. Consequently,
the viability of the probiotic culture was not influenced by the production conditions. Also,
the bacteria cell count was only followed during ripening and not during the following
storage period. Third, the difference of probiotic viability between rind and core of
Camembert-type cheese was not investigated. The diversity of physicochemical factors that
take place into these two cheese sections may have a different impact on bacteria. In
surface ripened cheese, since there is always a gradient of pH, redox level and proteolysis
between the rind and the core (Spinnler and Gripon, 2004). pH and proteolysis being more
68
higher at surface of the cheese could potentially stimulate probiotic cultures cell counts at
this level. Nevertheless, the positive redox level at the rind may be detrimental for
anaerobes cultures. Finally, mycete strains isolated from raw milk originating from the
province of Quebec (Canada) were used to produce cheese. Their influence on probiotic
bacteria viability was compared with commercial strains.
The aim of this study was to confirm the ability of Camembert cheese to support the
viability of probiotic bacteria. Pilot-scale Camembert cheese was manufactured with a
blend of two probiotic bacteria strains and their viability were followed at the rind and at
the core of the cheese pieces during one month (30 days) of ripening and storage. Also, the
proteolysis and pH were measured at the rind and at the core to study their influence on the
viability.
69
5.2 Materials and Methods
5.2.1 Strains (mycetes and bacteria)
Bacterial and mycete strains used in this study are listed in Table 5.1 The yeasts LMA
strains were isolated from different confidential milk sources from geographical regions
(Gaspésie–Îles-de-la-Madeleine, Quebec city region, Montérégie) in the province of
Quebec (Canada). The other fungi strains are commercial products often used in the
industry. The probiotic strains are commercially available cultures having documented
health benefits.
Table 5.1. Bacterial and mycete strains used for this work
Genus Species Type Strain Source
Lactobacillus rhamnosus probiotic
bacteria R0011
Institut Rosell-Lallemand,
Mtl, Canada
Bifidobacterium lactis probiotic
bacteria BB12
Chr. Hansen, Barrie, On,
Canada
Penicillium camemberti Mould PC PSM2 Cargill France SAS, La
Ferté sous Jouarre
Geotrichum candidum Yeast GEO17
Danisco France, ZA de
Buxière, Dangé-Saint-
Romain
Debaryomyces hansenii Yeast LAF3 CHR Hansen France, Le
moulin d’Aulney
Geotrichum candidum Yeast LMA 563 Milk A
Debaryomyces hansenii Yeast LMA 695 Milk D
Geotrichum candidum Yeast LMA 664 Milk A
Debaryomyces hansenii Yeast LMA 668 Milk B
D. hansenii from terroir frozen stock culture was prepared by a YM broth solution (Becton
Dickinson, Sparks, MD, USA) having 30% w/w glycerol (Sigma-Aldrich, St-Louis, MO,
USA) with a fresh liquid inoculum in a 1:1 ratio. For G. candidum strains from terroir the
70
cell suspension was prepared by recovering colonies of G. candidum from the surface of an
acidified potato dextrose agar plate (PDA; EMD Chemicals, Darmstadt, Germany) using a
swab humidified in a filter-sterilized (0.22μm Millex GP syringe filter, Millipore,
Carrigtwohill, Co. Cork, Ireland) 0.05% w/v Tween 80 (Fisher Scientific, Fairlawn, N-J,
USA) solution. The cells from the swab were resuspended in a Tween 80 solution which
was then blended with the glycerol-YM broth at the 1:1 ratio as for the other mycete
cultures. All these stocks were divided in aliquot of 1mL cryovials (Nalgene, Rochester,
NY, USA) and placed in a -80˚C freezer.
5.2.2 Inocula preparation and cultures conditions
The D. hansenii terroir strains inocula were obtained from YM broths (Becton Dickinson)
seeded at 1% v/v with thawed stock culture. They were incubated at 30˚C on a shaker (250
rpm) until they reached an optical density (OD) between 0.4 and 0.8 using a Beckman 7400
Spectrophotometer at 600nm (Coulter, Fullerton, CA, USA). CFU mL-1
of the liquid
cultures was estimated by the OD measure after having established an OD-CFU standard
curve.
The G. candidum terroir strains biomass were obtained by spreading a thawed stock culture
at the surface of a potato dextrose agar plate and incubating for 1 week at room temperature
(23˚C). The mould spores were collected using a sterile swab as previously described. The
concentration of the cell suspension was determined by using an hemacytometer (Hausser
Scientific, Horsham, PA, USA).
71
5.2.3 Cheese Production Assays
Probiotic Camembert cheese was manufactured on a pilot-scale with three different
combinations of mycete strains (A, B, C) and with a blend of the two probiotic strains
(R0011 and BB12). One combination of mycetes (LAF3, GEO17, PC PSM2) was
constituted of commercial strains and the two other were from the Québec terroir (LMA)
except for the P. camemberti strain that was the same for all (PC PSM2, Cargill France)
(Table 5.2).
Table 5.2. Combinations of bacterial and mycete strains done in cheese
Cheese identification Probiotic strains Mycete strains
A R0011 and BB12 LMA 668, LMA 664 and PC PSM2
B R0011 and BB12 LMA 695, LMA 563 and PC PSM2
C R0011 and BB12 LAF3, GEO17 and PC PSM2
Whole milk (120 L) was pasteurized in batch at 65˚C for 30 minutes. After pasteurization,
milk was divided in three equal portions of 40 L to do independently the three treatments
(A, B, C). The temperature of milk was adjusted to 35˚C before inoculation at 1,5% w/w
with a lyophilized Flora Danica starter (Chr Hansen, Milwaukee, WI, USA). The milk was
also inoculated with the mycete strains and probiotic bacteria at the same time than the
starter. The two probiotic bacteria strains were inoculated using commercial lyophilized
bacteria powder to obtain a cell count in milk of 1*107
CFU mL-1
. The powder was
hydrated in pasteurized milk at 37˚C 1 hour before the inoculation in milk. The commercial
mycete cultures were also inoculated with lyophilized powder using a ratio of 2 doses/100L
of milk. This represented an inoculation level of 4 x 104 ml
-1 for P. camemberti PC PSM2,
2 x 103 ml
-1 for G. candidum GEO17 and 2 x 10
4 ml
-1 for D. hansenii LAF3. The terroir D.
hansenii liquid cultures were centrifuged to prevent the influence of culture media on the
growth in cheese. Centrifugation was carried out at 10000g for 20 minutes using a Sorvall
72
RC-5B (Dupont, Mississauga, Ontario, Canada) centrifuge. The cell pellets were
resuspended in pasteurized milk to 10% of the original cell suspension concentration. Then,
G. candidum and D. hansenii terroir strains were seeded in milk at 2*104 CFU mL
-1.
After inoculation, maturation of milk was allowed for 1 hour at 34˚C. During this time,
CaCl2 (Calsol, Danisco, Copenhagen K, Denmark) solution (45% w/v) was added at
0.035% w/w to milk. Subsequently, the rennet (CHY-MAX extra, Chr Hansen) was added
at 2.25mL/40L of milk. 27 minutes after adding the rennet, the curd was cut into pieces of 2
cm side to release the whey. The curd pieces were ready for molding when whey reached a
pH of 6.4. Kept at room temperature, the molds were turned over after 30 minutes, one
hour and three hours. Finally, the cheese molds were placed in a chamber overnight. The
curds were initially at 28˚C and the chamber was programmed to gradually go down to
16˚C for the next morning. Next morning, the pieces of cheese were salted in brine (23%
NaCl and 0.026% CaCl2) for 25 minutes before their ripening at 12˚C under 95% relative
humidity. After 10 days, the cheese pieces were wrapped in micro perforated wrapping
paper for Camembert and stored at 4˚C.
5.2.4 Enumerations of cheese microorganisms
Enumerations of probiotics bacteria were done at the surface and in the center of the
cheese. Two slices of approximately 7mm were removed from the top and bottom of the
Camembert cheese; these two mold-carrying slices will be referred to as the “rind” samples
wile the remaining central portion will be referred to as the “centre” samples. Such rind and
centre samples were taken at different moment of the production (after brining, at the end
of the 10 ripening period at 12°C and after days 16 and 30 during the storage period at
4°C). The yeasts and moulds (Y/M) were enumerated at the same moments but only in
general (the rind and center of cheese pieces was not differentiated).
For the two types of enumeration (bacteria or Y/M), a representative cheese sample was
diluted in a 9:1 ratio w/w in sterile 2% w/v sodium citrate (Fisher Scientific) solution at
73
room temperature and homogenized using a stomacher unit (Seward, model 400 Circulator;
Worthing, West Sussex, UK) at 260rpm for 2 minutes.
For the Y/M, the first dilution of this serial was done using a bottle of 99mL peptone water
0,1% w/v (Becton Dickinson) containing glass beads. Subsequently, this suspension was
serially diluted and vortexed in water tubes with the same concentration of peptone as
above. Finally, 0,1mL of the appropriate dilution was spread in duplicate at the surface of
an acidified PDA plate (see 5.2.2 section). The plates were incubated at 23˚C for 5 days.
Probiotic bacteria were enumerated by homogenizing 10mL of the stomached cheese
dilution 30 sec at 27000 rpm with Omni-Tips generator probes. They were subsequently
serially diluted and vortexed with peptone water tubes like the Y/M. 1mL of the appropriate
dilutions was pour plated in duplicate in MRS agar (Becton Dickinson). To differentiate the
two probiotic bacteria strains, antibiotics were used. Mupiricin (Sigma-Aldrich, St-Louis,
MO, USA) at 0,02g/L in MRS agar solution was used to select only BB12 strain and
Vancomycin hydrochloride (Acros Organics, NJ, USA) at 0,1g/L in MRS agar solution for
the R0011 strain. The antibiotics powders were diluted in 10ml of water and filter-sterilized
(0,22μm Millex GP syringe filter, Millipore, Carrigtwohill, Co. Cork, Ireland) in their
respective MRS agar solution. MRS plates were then incubated aerobically at 40˚C for 48
h, with the exception of the B. lactis BB12 petri plates which were incubated in an
anaerobic environment (85%N2/10%H2/5%CO2 atmosphere).
5.2.5 Analyses
The pH was measured using a pH meter (XL15, Acumet, Fisher Scientific) adjusted with
pH 4.0 and pH 7.0 standard buffers (Fisher Scientific). The rind pH of the cheese pieces
was determined by placing the electrode between two surface slices. The centre pH was
measured by placing the electrode in the middle of the remaining Camembert piece without
its surfaces.
74
The calculation of proteolysis index was done for surface and centre of cheese pieces at the
end of the ripening period at 12°C (day 10) and day 30. Primary proteolysis was assessed
by extracting and measuring the water-soluble nitrogen (WSN) of the cheese by the method
of Kuchroo and Fox (1982). The WSN phase was used to evaluate secondary proteolysis
(TCA NS) using 12% TCA (Gripon et al., 1975). The nitrogen content analysis of the TCA
NS phase and total nitrogen (TN) of the cheese were done by Kjeldhal method (AOAC,
2000). The proteolysis index is the expression of the % TCA SN/TN.
Moisture was measured by gravimetry following drying at 105˚C for 16 hours and sodium
chloride was tested using a chloride-meter analyzer Corning (Nelson-Jameson Inc.,
Marshfield, WI, USA). These two last parameters data are not published. The analyses
were done only to confirm the equivalence of each assay of the three cheese treatments.
5.2.6 Statistical analyses
ANOVA and regression test were carried out on SAS (SAS institute Inc., Cary, North
Carolina, USA) with the GLM procedure and significantly differences between results were
determined using the Fisher’s least significance difference (LSD) test and with the Duncan
test for experiments with some missing data. Each data reported is the average of three
independent assays. Each statistical test was done at a 95% confidence level.
75
5.3. Results and discussion
5.3.1 Yeast and mould cell counts
Although the specific strains were not enumerated, the total Y/M counts provide an
indication of the evolution of the mycete biomass during ripening and storage. Since
Penicillium camemberti gives much lower CFU readings than Geotrichum candidum and
Debaryomyces hansenii, the CFU readings offer a picture of the total yeast population.
Table 5.3. Cell counts (log CFU g-1
) of different yeast and mould blends (Y/M) in
Camembert cheese ripened 30 days.
Mycete strains Y/M
blend
Time (days)
D0 D10 D16 D30
LMA 668,
LMA 664 and
PC PSM2
A 5.58 a 7.49 a 7.58 a 7.65 a
LMA 695,
LMA 563 and
PC PSM2
B 5.43 b 6.76 a 6.86 b 6.76 b
LAF3, GEO17
and PC PSM2 C 5.20 c 7.23 a 7.36 a 7.16 b
Values given represent the average of 3 independent assays. a,b
For a given column, values associated to the same letter are not significantly different (LSD, P>0.05).
There were significant differences between the biomass of each Y/M blend. The blend A
had the highest CFU all along the 30 days. Its development in cheese seemed to be better
than the two other blends. Generally, the results obtained are 1 log CFU g-1
lower than
those obtained in the cheese slurries of Chapter 3 (Table 3.2). However, the inoculation
rates were approximately lower of 1 log CFU in cheesemaking.
76
5.3.2 pH and proteolysis index
The evolution of pH and proteolysis indexes were followed at different time during the
ripening and storage periods of Camembert cheese.
a a a a a a
a
a,b
b
c c c
a
bb b
a a
c c
c
a a
b
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
0 10 16 30
Days
pH
A1
B1
C1
A2
B2
C2a a a a a ac c c
a
a,b
b
bb b
a
a a
c c
c
a a
b
a a a a a a
a
a,b
b
c c c
a
bb b
a a
c c
c
a a
b
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
0 10 16 30
Days
pH
A1
B1
C1
A2
B2
C2a a a a a a
a
a,b
b
c c c
a
bb b
a a
c c
c
a a
b
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
0 10 16 30
Days
pH
A1
B1
C1
A2
B2
C2a a a a a ac c c
a
a,b
b
bb b
a
a a
c c
c
a a
b
Figure 5.1. pH at the centre (1) and at the rind (2) of three Camembert cheese treatments
ripened 30 days with combination of different yeast and mould strains (A, B, C). a,b,c
For a
given day, values associated to the same letter are not significantly different (LSD, P>0.05).
Error bars represent SEM.
From day 10 until day 30, pH at the centre of cheese was always lower than rind (Figure
5.1). At day 16, the pH at center begins to increase and it reaches around 6 at surface. The
pH values obtained in this study for the centre and the rind are similar to those of Boutrou
et al. (1999) but lower that those of Leclercq-Perlat et al. (2004a) who report a pH of 7.5 at
the rind after 10 days and a pH of 6 at the core after 30 days. In this latter study, some other
microbial strains like Brevibacterium linens and Kluyveromyces lactis were used with P.
camemberti and G. candidum. This indicates that the pH is directly influenced by the fungi
77
species and strains used which is in line with the data of cheese slurries inoculated with
pure Y/M cultures (Chapitres 3 et 4). In accordance to this, the two cheese treatments made
with terroir strains (A and B) had higher pH at day 30 (D30) than the cheese made with
commercial strains (C). The higher CFU at D30 of Y/M blend A can explain its higher pH
but the blend B with its lower CFU had a pH equal to blend A. The rind microbiota is
principally responsible of the proteolysis in Camembert cheese, which can cause the pH to
increase, particularly if there is production of ammonia (Beresford et al., 2001). When
comparing pH and the proteolysis index data from the rind and the centre, a relationship
was noted between these two parameters (R2
= 0.55). This shows that pH increase is not
only due to proteolysis. Lactate consumption by Y/M is another factor influencing pH of
Camembert cheese.
The Figure 5.2 shows the proteolysis index of the different parts (rind and centre) of the
three cheese treatments. Camembert A made with the G. candidum LMA 664 and D.
hansenii LMA 668 showed more proteolysis at D30. The three cheesemaking treatments
having all the same P. camemberti strain, the difference of proteolysis can only be
explained by the two other species in place.
At day 10 (D10), the proteolysis in the rind is recognized to be from P. camemberti and the
G. candidum species (Leclercq-Perlat et al., 1999). The latter, is known to be divided in
two categories; weak and strong proteolytic activity strains (Boutrou et al., 2006). D.
hansenii specie is not documented to be proteolytic in the first 11 days of ripening. The
only manner it can influence proteolysis after these 11 days is by autolysis (Leclercq-Perlat
et al., 1999). Subsequently, D. hansenii can contribute up to 25% of TCA SN/TN after 30
days (Leclercq-Perlat et al., 2000). The augmentation of proteolysis indexes of the three
types of cheese between D10 and 30 may therefore be partially linked with the proteolysis
contribution of the D. hansenii strains. Moreover, the higher proteolysis level of cheese A
at D30 may be explained by the presence of G. candidum LMA 664 and its higher Y/M
CFU g-1
than the G. candidum of B and C. Also, the LMA 664 strain could be in the group
of the strong proteolytic G. candidum strains. Then, the rind pH of the Camembert B that
78
was higher than Camembert C at D30 is possibly explained by lactate consumption and not
by proteolysis. Further characterization of the yeast strains are warranted in this respect.
0,00%
5,00%
10,00%
15,00%
20,00%
25,00%
30,00%
35,00%
40,00%
45,00%
10 30
Days
Pro
teo
lysis
In
dex A1
B1
C1
A2
B2
C2
a
a
a,b
b
cc
c
c cc
bb
0,00%
5,00%
10,00%
15,00%
20,00%
25,00%
30,00%
35,00%
40,00%
45,00%
10 30
Days
Pro
teo
lysis
In
dex A1
B1
C1
A2
B2
C2
a
a
a,b
b
cc
c
c cc
bb
Figure 5.2. Proteolysis indexes (% TCASN/TN) at the core (1) and the rind (2) of three
Camembert cheese treatments ripened 30 days with combination of different yeast and
mould strains (A, B, C). a,b,c
For a given day, values associated to the same letter are not
significantly different (LSD, P>0.05). Error bars represent SEM.
Finally, the Camembert cheese made with terroir strain mixes (A & B) pH and proteolysis
indexes are interesting because they are sensibly the same (B) as the commercial control
cheese (C) or higher (A). This particularity of these terroir strains may be attractive for
industrial cheese producers. Sensory analyses now need to be carried out to further asses
the commercial interest of the Y/M strains isolated.
79
5.3.3 Probiotic bacteria viability
Viability of probiotic bacteria in Camembert cheese was measured at the centre and at the
rind of the cheese pieces (Table 5.4).
Table 5.4. Viability (log CFU g-1
) of Bifidobacterium lactis BB12 and Lactobacillus
rhamnosus R0011 in Camembert cheese ripened 30 days with combination of different
yeast and mould (Y/M).
Probiotic
Sample
location
Y/M
blend
Time (days)
D0 D10 D16 D30
B. lactis
BB12
Centre
A 7.76 a 7.34 c 7.57 a,b 7.25 a,b,c
B 7.43 a,b 6.93 c 7.64 a,b 7.04 b,c,d
C 7.64 a 6.86 c 7.14 a,b,c 6.66 c,d
Rind
A 7.41 a,b 6.92 c 7.14 a,b,c 6.87 c,d
B 7.01 b 7.19 c 7.68 a,b 6.98 b,c,d
C 7.40 a,b 7.47 b,c 7.19 a,b 7.23 a,b,c
L. rhamnosus
R0011
Centre
A 7.55 a 6.86 c 6.87 a,b,c 6.39 c,d
B 7.52 a,b 6.88 c 6.13 c 6.48 c,d
C 7.63 a 7.28 c 6.57 b,c 6.16 d
Rind
A 7.64 a 8.37 a 8.14 a 7.96 a,b
B 7.68 a 8.20 a,b 8.07 a 7.97 a,b
C 7.71 a 8.40 a 8.22 a 8.13 a Values given represent the average of 3 independent assays. a,b,c,d
For a given column, values associated to the same letter are not significantly different (Duncan,
P>0.05).
Populations of each treatment were between 6 and 8 log CFU g-1
even after 30 days.
Commonly, from D16 until D30, for a particular part of the cheese (rind or core), there was
not a probiotic strain more viable than another except for the A rind part at D30 where
R0011 had a higher CFU than B. lactis BB12. The recommended daily dose is around a
billion (9 log CFU) per day for a probiotic effect (Charteris et al, 1998; Lee & Salminen,
1995). The two strains populations being combined in one piece of cheese, considering 30
g as representing a portion of cheese, the Camembert cheese produced for this study would
deliver approximately 2 X 109 CFU portion at day 30. A way to guarantee a higher portion
is to increase the inoculation rate. The L. rhamnosus R0011 and B. longum BB12 probiotic
80
cultures were each inoculated in the processing milk at log 7.0 log CFU mL-1
. Their
numbers in the cheese at D0 show an increase but this is mainly due to cell concentration in
curds during processing. Although the recovery level of the probiotic bacteria in the curd
was not established in these assays, over 80% of the bacteria inoculated in milk are
typically recovered in the curd (Fortin et al., 2011).
Concerning L. rhamnosus R0011, there were significant differences of viability between
the centre and the rind from D10 until D30. It seemed that higher pH and proteolysis
promote its viability (Figure 5.1 and 5.2). Amino acids availability as growth factors and
less acidic pH are two factors susceptible to enhance the viability of probiotic bacteria
(Champagne et al., 2005). Autolysis of yeasts like D. hansenii may also stimulate their
viability (Smith et al., 1975). On the other hand, difference of pH and proteolysis between
the rind and the core of cheese may had influenced BB12 strain viability by neutralizing the
negative effect of the oxidizing redox level at rind. Accordingly, there was no difference
between the rind and the centre for the viability of B. lactis BB12. The lower proteolysis at
centre did not influence its viability. It can be hypothesized that this species preferring an
anaerobe environment (Shimamura et al., 1992), the lower oxygen concentration at core
may compensate for the lower proteolysis index. Indeed, a study of the redox level of
Camembert cheese (Abraham et al., 2007) revealed that it was oxidizing (200 to 300mV) at
the rind from 0 to 0.4cm at day 15 and 35. Inversely, at the core, the medium was reducing
at day 15 (-300mV) and furthermore at day 35 (-350mV). The positive value at the edge of
cheese probably reflects the oxygen gradient from rind to core. The mycetes at surface,
consume oxygen to metabolize lactacte as a carbon source. Besides, during the ripening, the
lactate consumption by the rind flora induces a diffusion of this compound from the core to
the rind (Aldarf et al., 2006).
If the viability results are compared with our previous study (Chapter 4) with cheese
slurries followed on 12 days, the cell counts in cheese slurry were higher. Also, the
prediction from assays in the cheese slurries that no Y/M mix was better than another to
enhance probiotic survival was confirmed in pilot scale manufactured cheese. After 12
days, in the cheese slurries study, the log CFU g-1
were over 8,5 for L. rhamnosus R0011.
81
In this study, the cell count sample was not differentiating the core and the centre of cheese
slurries. If these results are compared to the average between core and rind of the day 10
results in real cheese (around 7,8 for R0011), the cheese slurries gives effectively higher
results. This could partially be due to the initial viable counts. The slurries were inoculated
at 0.4 log CFU g-1
higher than in cheese, and this is the approximate difference between the
viable counts in the slurries (Chapter 4) and those in the rind. These data suggest that, in
addition to the evolution of pH, the inoculation level could significantly influence the CFU
values in the cheese. More experiments in cheesemaking conditions are needed to ascertain
this hypothesis.
5.4 Conclusion
In conclusion, this study had confirmed that Camembert-type cheese could be a good food
matrix to support the viability of probiotic bacteria. After 30 days, the cell counts were at a
sufficiently high level to allow health benefits. For the L. rhamnosus R0011 strain, the rind
was shown to constitute a better environment than the core to enhance viability. On the
other side, the B. lactis BB12 strain had similar viability between the rind and the core.
Proteolysis and pH increase that took place at the rind seemed to be a factor which favoured
high L. rhamnosus R0011 viable counts.
Furthermore, the terroir mycete strains used seemed to be as effective as the commercial
strains to enhance viability of the probiotic cultures. Some of them showed better
development at cheese rind than commercial ones. It would be interesting in a further work
to characterize them for their impact on physicochemical and flavour before suggest them
to industrial cheese producers. The viability of probiotic bacteria was important after one
month but before promoting the Camembert as a good probiotic cheese, it would be
essential to study the viability of the probiotic bacteria over 2 months in Camembert to
simulate a true shelf life.
82
Acknowledgements
Yves Raymond and Gaétan Bélanger are gratefully acknowledged for their scientific and
technical expertise. Nancy Guertin and Mélanie Gobeil-Richard are also thanked for her
technical assistance. This study was financially supported by the Fonds Québécois de
Recherche sur la Nature et les Technologies (FQRNT), NOVALAIT Inc., the Ministère de
l’Agriculture des Pêcheries et de l’Alimentation du Québec as well as Agriculture and
Agri-Food Canada. Pierre-Luc Champigny was also a recipient of an Excellence Grant
from FQRNT.
83
Conclusion
Ces travaux sur la biocompatibilité des bactéries lactiques probiotiques et d’affinage avec
les mycètes de la flore du Camembert ont permis d’éclaircir certains points et d’apporter
d’autres perspectives de recherche à propos des aliments probiotiques et des fromages fins.
Le fromage Camembert s’est révélé un milieu intéressant pour y ajouter des probiotiques et
permettre leur viabilité dans le temps. L’étude en caillés modèles a révélé que les souches
de levures et moisissures utilisées ont toutes permis aux bactéries probiotiques d’augmenter
leur viabilité comparativement aux caillés modèles témoins non affinés par des mycètes. De
plus, la souche bactérienne d’affinage n’a pas semblé affectée par la présence de levures et
moisissures. Elle s’est bien développée dans le caillé qu’il soit inoculé ou non de mycètes.
Par la suite, les trois types de fromages Camembert fabriqués avec des mélanges de souches
fongiques différents ont conservé les bactéries probiotiques viables après 30 jours et en
quantité suffisante afin que l’apport quotidien d’un milliard de cellules vivantes soit
respecté avec une portion raisonnable de fromage. Toutefois, un suivi sur une plus grande
étendue de temps que seulement un mois après la fabrication permettrait de mieux évaluer
la survie des bactéries probiotiques pendant toute la durée de vie du fromage.
Finalement, les souches fongiques provenant du terroir québécois se sont comportées de
manière semblable aux souches commerciales. Par contre, avant de suggérer l’usage de ces
souches du terroir à des maîtres fromagers, il serait intéressant de caractériser autant leur
effet au niveau sensoriel que leur action sur la physicochimie du fromage. Ceci permettrait
de démontrer leur influence sur la flaveur et la texture. Enfin, cette recherche pourrait
conduire à des innovations autant du côté de la typicité du terroir québécois que des
produits à valeur ajoutée comme les aliments fonctionnels.
84
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