BIOCOMPATIBILITÉ DES BACTÉRIES LACTIQUES PROBIOTIQUES …

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


4.2.3 Production of cheese slurry powder ..................................................................... 51

4.2.4 Cheese slurries assays .......................................................................................... 51

4.2.5 Enumerations of microorganisms and pH measurement ..................................... 53


4.3 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


5.2.3 Cheese Production Assays ................................................................................... 71

5.2.4 Enumerations of cheese microorganisms ............................................................ 72

5.2.5 Analyses ............................................................................................................... 73


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 4.2. 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 5.2. 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


) 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


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


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


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


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


b,c c

0.0

0.1

0.2

0.3

0.4

BKAa,b a,b a,b

a

a,ba,b


0.0

0.1

0.2

0.3

0.4

BKAa,b a,b a,b

a

a,ba,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


Gro

wth

rate

(µ

max

60

0 n

m)


& 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



& 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


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



& 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


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


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



& 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


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


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



& 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


Incr

ease

in O

D (

60

0 n

m)


& 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


Incr

ease

in O

D (

60

0 n

m)


& 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.


a, Daniel St-Gelais

a, Ismail Fliss

b, Steve

Labrie b





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).



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


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.


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


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.


) 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


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


a, Daniel St-Gelais

a, Ismail Fliss

b, Steve

Labrie b





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.



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.


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.


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.

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B9887-50KRS41-9&_user=403646&_coverDate=08%2F31%2F2010&_rdoc=1&_fmt=high&_orig=gateway&_origin=gateway&_sort=d&_docanchor=&view=c&_acct=C000013498&_version=1&_urlVersion=0&_userid=403646&md5=6b29d9feab4140ce2f23b27b116e4c00&searchtype=a#bib25

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B9887-50KRS41-9&_user=403646&_coverDate=08%2F31%2F2010&_rdoc=1&_fmt=high&_orig=gateway&_origin=gateway&_sort=d&_docanchor=&view=c&_acct=C000013498&_version=1&_urlVersion=0&_userid=403646&md5=6b29d9feab4140ce2f23b27b116e4c00&searchtype=a#bib18

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


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).


) 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

Bibliographie

Abou-Dawood, S.A.I., 2002, Survival of nonencapsulated and encapsulated

Bifidobacterium bifidum in probiotic Kareish cheese. Egyptian Journal of Dairy

Science, 30:43-52

Abraham, S., Cachon, R., Colas, B., Feron, G., De Coninck, J., 2007, Eh and pH gradients

in Camembert cheese during ripening : Measurements using electrodes and

correlations with texture, International Dairy Journal, 17:954-960

ACIA (Agence Canadienne d’Inspection des Aliments), 2009, Allégations relatives aux

probiotiques, Chapitre 8, Section 8.7., (page web consultée en ligne le 20 avril

2011) Adresse URL :

http://www.inspection.gc.ca/francais/fssa/labeti/guide/ch8af.shtml#a8_7

Aldarf, M., Fourcade, F., Amrane, A, Prigent, Y, 2006, Substrate and metabolite diffusion

within model medium for soft cheese in relation to growth of Penicillium

camemberti, Journal of Industrial Microbiology and Biotechnology, 33: 685-692

Álvarez-Martín, P., Flórez, A. B., Hernández-Barranco, A., Baltasar, M., 2008, Interaction

between dairy yeasts and lactic acid bacteria strains during milk fermentation,

Food Control, 19: 62-70

AOAC, Dairy products. In Official methods of analysis, 17th ed., Horwitz, D. W., Latimer,

G., Eds., Arlington, VA, 2000, 2:69−82

Barrette, J., Champagne, C. P., Goulet, J., 2001, Growth-promoting properties of yeast

extracts produced at different pH values, with different autolysis promoters and

bacterial populations, Journal of chemical technology and biotechnology, 76, 203-

209

Beresford, T. P., Fitzsimons, N. A., Brennan, N. L., Cogan, T. M., 2001, Recent advances

in cheese microbiology, International Dairy Journal, 11: 259-274

Beresford, T., Williams, A., 2004, The Microbiology of Cheese Ripening – Cheese:

Chemistry, Physics and Microbiology, Third edition, Volume 1: General Aspects,

Éd. Fox P., McSweeney P., Cogan T., Guinee T. Academic press

Besançon, X., Smet, C., Chabalier, C., Rivemale, M., Reverbel, J. P., Ratomahenina, R.,

Galzy, P., 1992, Study of surface yeast flora of Roquefort cheese, International

Journal of Food Microbiology, 17: 9-18

Blanchette, L., Roy, D., Bélanger, G., Gauthier, S. F., 1996, Production of cottage cheese

using dressing fermented by bifidobacteria, Journal of Dairy Science, 79:8-15.

http://www.inspection.gc.ca/francais/fssa/labeti/guide/ch8af.shtml#a8_7

85

Boutrou, R., Gaucheron, F., Piot, M., Michel, F., Maubois, J.-L., Léonil, J., 1999, Changes

in composition of juice expressed from Camembert cheese during ripening, Lait,

79 :505-513

Boutrou, R., Guéguen, M., 2005, Interests in Geotrichum candidum for cheese technology,

International Journal of Food Microbiology, 102: 1-20

Boutrou, R., Kerriou, L., Gassi, J.-Y., 2006, Contribution of Geotrichum candidum to the

proteolysis of soft cheese, International Dairy Journal, 16:775-783

Cerning, J., Gripon, J. C., Lamberet, G. & Lenoir, J.. 1987, Les activites biochimiques des

Penicillium utilises en fromagerie, Le Lait, 67:33-39.

Champagne, C. P., Gardner, N., Roy, D., 2005, Challenges in the addition of probiotic

cultures to foods, Critical Reviews in Food Science and Nutrition, 45(1): 61-84

Champagne, C.P., Gagnon, D., St-Gelais, D., Vuillemard, J.C., 2009a, Interactions between

Lactococcus lactis and Streptococcus thermophilus strains in Cheddar cheese

processing conditions, International Dairy Journal, 19: 669-674

Champagne, C. P., Savard, T., Barrette, J., 2009b, Production of lactic acid bacteria on

spent cabbage juice, Journal of Food Agriculture & Environment, 7:82-87

Charteris, W. P., Kelly, P.M., Morelli, L., Collins, J. K., 1998, Ingredient selection criteria

for probiotic microorganisms in functional dairy foods, International Journal of

Dairy Technology 51(4):123-136

Chen, L., Remondetto, G.E., Subirade, M., 2006, Food protein-based materials as

nutraceutical delivery systems, Trends in Food Science and Technology, 17(5):

272-283

Coeuret, V., Gueguen, M., Vernoux, J.P., 2004, In vitro screening of potential probiotic

activities of selected lactobacilli isolated from unpasteurized milk products for

incorporation into soft cheese, Journal of Dairy Research, 71: 451-460

Conseil canadien de la santé, 2009, La valorisation de l’argent : Renforcer le système

canadien de soins de santé (document pdf consulté en ligne le 29 septembre 2009)

AdresseURL:http://www.healthcouncilcanada.ca/docsrpts/2009/HCC_VFMReport_

FRE _WEB.pdf

Cogan, T. M., 2003, Microbiology of cheese - Encyclopedia of Dairy Sciences, Éd.

Roginski H., Fuquay J. W., Fox P. F., Académie Press, p. 306-314

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VHY-4JFGF8T-1&_user=3914355&_coverDate=05%2F31%2F2006&_alid=750861803&_rdoc=1&_fmt=high&_orig=search&_cdi=6079&_sort=d&_docanchor=&view=c&_ct=1&_acct=C000051263&_version=1&_urlVersion=0&_userid=3914355&md5=a5522c5989c8b98084e94bd939e6c8cc

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VHY-4JFGF8T-1&_user=3914355&_coverDate=05%2F31%2F2006&_alid=750861803&_rdoc=1&_fmt=high&_orig=search&_cdi=6079&_sort=d&_docanchor=&view=c&_ct=1&_acct=C000051263&_version=1&_urlVersion=0&_userid=3914355&md5=a5522c5989c8b98084e94bd939e6c8cc

http://www.healthcouncilcanada.ca/docsrpts/2009/HCC_VFMReport_FRE%20_WEB.pdf

http://www.healthcouncilcanada.ca/docsrpts/2009/HCC_VFMReport_FRE%20_WEB.pdf

86

Conway J., Gaudreau H., Champagne C.P., 2001, The effect of the addition of proteases

and glucanases during yeast autolysis on the production and properties of yeast

extracts, Canadian Journal of Microbiology, 47:18-24

Daigle, A., Roy, D., Belanger, G., Vuillemard, J.C., 1999, Production of probiotic cheese

(Cheddar-like cheese) using enriched cream fermented by Bifidobacterium infantis,

Journal of Dairy Science, 82:1081-1091

De Freitas, I., Pinon, N., Maubois, J.-L., Lortal, S., Thierry, A., 2009, The addition of a

cocktail of yeasts species to Cantalet cheese changes bacterial survival and

enhances aroma compound formation, International Journal of Food

Microbiology, 129: 37–42

De Marchi, M., Bittante, G., Dal Zotto, R., Dalvit, C., Cassandro, M., 2008, Effect of

Holstein Friesian and Brown Swiss Breeds on Quality of Milk and Cheese, Journal

of Dairy Science, 91:1092-4102

Dieuxleveux, V. Van Der Pyl, D., Chataud, J., Guéguen, M., 1998, Purification and

characterization of Anti-Listeria compounds produced by Geotrichum candidum,

Applied and Environmental Microbiology, 64(2):p. 800-803

European Food Safety Authority (EFSA), 2009, Scientific Opinion on the substantiation of

health claims related to non characterised microorganisms pursuant to Article

13(1) of Regulation (EC) No 1924/2006 (page web consultée en ligne le 27

November 2009) Adresse URL : http://www.efsa.europa.eu/EFSA/efsa_loc ale-

1178620753812_121 1902907474.htm

Farnworth, E. R., Mainville, I., 2003, Chapter 4: Kefir: A fermented milk product –

Handbook of fermented functional foods, Ed. Farnworth E. R., CRC Press,

Washington (USA)

Food and Agriculture Organization of the United Nation/World Health Organisation

(FAO/WHO), 2001, Health and nutritional properties of probiotics in food

including powder milk with live lactic acid bacteria, a joint FAO/WHO expert

consultation, (document pdf consulté en ligne le 02/03/2011) URL:

ftp://ftp.fao.org/docrep/fao/meeting/009/y6398f.pdf

Food and Agriculture Organization of the United Nation/World Health Organisation

(FAO/WHO), 2010, Food additive details: Natamycin (Pimaricin), Codex

Alimentarius, (Page web consultée en ligne le 02/10/2011) URL:

http://www.codexalimentarius.net/gsfaonline/additives/details.html?id=208&lang=e

n

Fortin, M.-H., Champagne, C.P., St-Gelais, D., Britten, M., Fustier, P., Lacroix, M., 2011,

Effect of time of inoculation, starter addition, oxygen level and salting on the

http://www.efsa.europa.eu/EFSA/efsa_loc%20ale-1178620753812_121%201902907474.htm

http://www.efsa.europa.eu/EFSA/efsa_loc%20ale-1178620753812_121%201902907474.htm


http://www.codexalimentarius.net/gsfaonline/additives/details.html?id=208&lang=en

http://www.codexalimentarius.net/gsfaonline/additives/details.html?id=208&lang=en

87

viability of probiotic cultures during Cheddar cheese production, International

Dairy Journal, 21:75-82

Fritzen-Freire, C. B., Müller, C. M. O., Laurindo, J. B., Prudêncio, E. S., 2010, The

influence of Bifidobacterium Bb-12 and lactic acid incorporation on the properties

of Minas Frescal cheese, Journal of Food Engineering, 96:621-627

Gomes, A.M.P., Malcata, F.X., 1998, Development of probiotic cheese manufactured from

goat milk: Response surface analysis via technological manipulation, Journal of

Dairy Science, 81:1492-1507

Gomes, A.M.P., Malcata, F.X., Klaver, F.A.M., Grande, H.J., 1995, Incorporation and

survival of Bifidobacterium sp. Strain Bo and Lactobacillus acidophilus strain in Ki

in a cheese product, Netherlands Milk and Dairy Journal, 49:71-95

Grattepanche, F., Miesher-Schwenninger, S., Meile, L., Lacroix, C., 2008, Recent

developments in cheese cultures with protective and probiotics functionalities,

Dairy Science Technology, 88: 421-444

Gripon, J.C., Desmazeaud, M.J., Le Bars, D., Bergere, J., 1975, Étude du rôle des

microorganismes et des enzymes au cours de la maturation des fromages. II.

Influence de la présure commerciale, Lait, 548:502–516

Guéguen, M., Shmidt, J. L., 1992, Les Penicillium In Les Groupes Microbiens d’Intérêt

Laitier, pp. 221-257, Ed. Hermier J., Lenoir J., Weber F., Paris:CEPIL

Jayamanne, V.S., Adams, M.R., 2006, Determination of survival, identity and stress

resistance of probiotics bifidobacteria in bio-yoghurts, Letters in Applied

Microbiology, 42:189-194

Kankaanpaa, P., Yang, B., Kallio, H., Isolauri, E. & Salminen, S., 2004, Effects of

polyunsatured fatty acids in growth medium on lipid composition and on

physicochemical surface properties of lactobacilli, Applied and Environmental

Microbiology, 70:129-136.

Klaver, F. A. M., Kingma, F., Weerkamp, A. H., 1993, Growth and survival of

bifidobacteria in milk, Netherlands Milk and Dairy Journal, 47:151-164.

Korhonen, H. & Pihlanto, A., 2006, Bioactive peptides: Production and functionality,

International Dairy Journal, 16:945-960.

Kuchroo, C.N., Fox P.F., 1982, Soluble nitrogen in Cheddar cheese: Comparison of

extraction procedures, Milchwissenschaft, 37:331–335

88

Lamontagne M., Champagne C. P., Reitz-Ausseur J., Moineau S., Gardner N., Lamoureux

M., Jean J., Fliss I., 2002, Chapitre 2 : Microbiologie du lait - Science et

Technologie du lait; transformation du lait, Éd. Vignola C. L., Presses

Internationales Polytechniques, Montréal (Québec)

Leclercq-Perlat, M.-N., Oumer, A., Bergere, J.-L., Spinnler, H.E., Corrieu, G., 1999,

Growth of Debaryomyces hansenii on a bacterial surface-ripenened soft cheese,

Journal of Dairy Research, 66:271-281

Leclercq-Perlat, M.-N., Oumer, A., Bergere, J.-L., Spinnler, H.E., Corrieu, G., 2000,

Behavior of Brevibacterium linens and Debaryomyces hansenii as ripening flora in

controlled production of soft smear cheese from reconstituted milk: protein

degradation, Journal of Dairy Science, 83:1674-1683

Leclercq-Perlat, M.-N., Buono, F., Lambert, D., Latrille, E., Spinnler, H.E., Corrieu, G.,

2004a, Controlled production of Camembert-type cheeses. Part 1: Microbiological

and physiological evolutions, Journal of Dairy Research, 71:346-354

Leclercq-Perlat, M.-N., Buono, F., Lambert, D., Latrille, E., Spinnler, H.E., Corrieu, G.,

2004b, Controlled production of Camembert-type cheeses. Part II: Changes in the

concentration of the more volatile compounds, Journal of Dairy Research, 71: 346-

354

Lee Y. K., Salminen S., 1995, The coming of age of probiotics, Trends in Food Science and

Technology 6:241-245

Liu, S.-Q., Tsao, M., 2009, Enhancement of probiotic and non-probiotic lactic acid

bacteria by yeasts in fermented milk under non-refrigerated conditions,

International Journal of Food Microbiology, 135: 34-38

Lourens-Hattingh, A., Viljoen, B. C., 2001, Yogurt as probiotic carrier food, International

Dairy Journal, 11: 1-17

Micanel, N., Haynes, I.N., Playnes, M.J., 1997, Viability of probiotic cultures in

commercial Australian yogurts, Australian Journal of Dairy Technology 52:24-27

Mistry, V.V., Brouk, M.J., Kasperson, K.M., Martin, E., 2002, Cheddar cheese from milk

of Holstein and Brown Swiss cows, Milchwissenshaft, 57(1): 19-23

Organisation des Nations Unies pour l’alimentation et l’agriculture/Organisation mondiale

de la santé (FAO/OMS), 2001, Consultation mixte d’experts FAO/OMS sur

l’évaluation des propriétés sanitaires et nutritionnelles des probiotiques dans les

aliments, y compris le lait en poudre contenant des bactéries lactiques vivantes

(document .pdf consulté en ligne le 18 septembre 2009) Adresse URL :



89

Ouwehand, A. C., Salvadori, B. B., Fondén, R., Mogensen, G., Salminen, S., Sellars, R..

2003. Health effects of probiotics and culture-containing dairy products in humans,

International Dairy Federation (IDF), Brussels, 380:4-19.

Özer, B., Uzun, Y. S., Kirmaci, H. A., 2008, Effect of microencapsulation on viability of

Lactobacillus acidophilus LA-5 and Bifidobacterium bifidum BB-12 during kasar

cheese ripening, International Journal of Dairy Technology, 61:237-245

Partanen, L., Marttinen, N., Alatossawa, T., 2001, Fats and fatty acids as growth factors for

Lactobacillus delbrueckii, Systematic and Applied Microbiology, 24:500-506.

Peláez, C., Requena, T., 2005, Exploiting the potential of bacteria in the cheese ecosystem,

International Dairy Journal, 15: 831-844

Phillips, M., Kailasapathy, K., Tran, L., 2006, Viability of commercial probiotic cultures

(L. acidophilus, Bifidobacterium sp., L. casei, L. paracasei and L. rhamnosus) in

cheddar cheese, Journal of Food Microbiology, 108:276-280

Powell, H. J., May, J. T., 1981, Effect of various lipids found in human milk on the growth

of infant bifidobacteria, Journal of General and Applied Microbiology, 27:185-193.

Richardson, D., 1996, Probiotics and product innovation, Nutrition and food science, 4:

27-33

Roostita, R., Fleet, G. H., 1996, The occurrence and growth of yeasts in Camembert and

Blue-veined cheeses, International Journal of Food Microbiology, 28: 393-404

Roy, D., 2005, Technological aspects related to the use of bifidobacteria in dairy products,

Lait, 85:39-56

Santé Canada, 2009, Guide d’étiquetage et de publicité sur les aliments; Chapitre 8 :

Allégations Santé (page web consultée le 11 novembre 2009), Adresse URL:

http://www.inspection.gc.ca/francais/fssa/labeti/guide/tocf.shtml

Shah, N.P., Lankaputhra, W.E.V., Britz, M.L., Kyle, W.S.A., 1995, Survival of

Lactobacillus acidophilus and Bifidobacterium bifidum in commercial yoghurt

during refrigerated storage, International Dairy Journal 5:515-521

Shah, N.P., 2000, Probiotic Bacteria: selective enumeration and survival in dairy foods,

Journal of Dairy Science, 83: 894–907

Shimamura, S., Abe, F., Ishibashi, N., Miyakawa, H., Yaeshima, T., Araya, T., Tomita, M.,

1992, Relationship between oxygen sensitivity and oxygen Metabolism of

Bifidobacterium Species, Journal of Dairy Science, 75:3296-3306

http://www.inspection.gc.ca/francais/fssa/labeti/guide/tocf.shtml

90

Smith, J. S., Hillier, A. J. & Lees, G. J., 1975, The nature of the stimulation of the growth of

Streptococcus lactis by yeast extract, Journal of Dairy Research, 42:123-138

Soda, M. El, Madkor, S.A., Tong P. S., 2000, Adjunct cultures: Recent developments and

potential significance to the cheese industry, Journal of Dairy Science, 83: 609-619

Souza Motta, A., Brandelli, A., 2008, Properties and antimicrobial activity of the smear

surface cheese coryneform bacterium Brevibacterium linens, European Food

Research Technology, 227:1299-1306

Spinnler, H.-E., Gripon, J.-C., 2004, Surface Mould-ripened Cheeses – Cheese: Chemistry,

Physics and Microbiology, Third edition, Volume 2: Major Cheese Groups. Éd. Fox

P., McSweeney P., Cogan T., Guinee T. Academic press

Sprong, R. C., Hulstein, M. F. E., Van der Meer, R., 2001, Bactericidal activities of milk

lipid. Antimicrobial Agents and Chemotherapy, 45: 1298-1301.

St-Gelais, D., Tirard-Collet, P., 2002, Chapitre 6 : Fromage - Science et technologie du

lait; transformation du lait, Éd. Vignola C. L., Presses Internationales

Polytechniques, Montréal (Québec)

Tharmaraj, N., Shah, N. P., 2009, Antimicrobial effects of probiotic bacteria against

selected species of yeasts and moulds in cheese-based dips, International Journal of

Food Science and Technology, 44:1916-1926

Valsijevic, T., Shah, N. P., 2008, Probiotics – from Metchnikoff to bioactives, International

Dairy Journal, 18:714-728

Vegarud, G., Castberg, H. B. & Langsrud, T., 1983. Autolysis of group N streptococci.

Effects of media composition modifications and temperature, Journal of Dairy

Science, 66: 2294-2302.

Viljoen, C. B., 2001, The interaction between yeasts and bacteria in dairy environments,

International Journal of Food Microbiology, 69:37-44

Voulgari, K., Hatzikamari, M., Delepoglou, A., Georgakopoulos, P., Litopoulou-Tzanetaki,

E., Tzanetakis, N., 2010, Antifungal activity of non-starter lactic acid bacteria

isolates from dairy products, Food Control, 21:136-142

Zoukari, A., Anifantakis, E.M., 1988, Le kéfir: Caractères physico-chimiques,

microbiologiques et nutritionnels. Technologie de production. Une revue, Le Lait,

68:373-392.

BIOCOMPATIBILITÉ DES BACTÉRIES LACTIQUES PROBIOTIQUES …

Documents

Transcript of BIOCOMPATIBILITÉ DES BACTÉRIES LACTIQUES PROBIOTIQUES …