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DEVELOPPMENT ET CARACTERISATION DE
FILMS ANTIMICROBIENS POUR LA
BIOPRÉSERVATION DES PRODUITS MARINS
PRÊTS À CONSOMMER
Thèse présentée
à la Faculté des études supérieures de l'Université Laval
dans le cadre du programme de doctorat en sciences et technologie des aliments
pour l' obtention du grade de Philosophiae Doctor (Ph.D.)
2009
RÉSUMÉ
ABSTRACT
The aim of this research was to develop and characterize new antimicrobial films made
from chitosan and divergicin M35 and to evaluate their potential in biopreservation of
ready-to-eat seafood. We have shown in this work that chitosan display very interesting
antibacterial activity against Listeria monocytogenes. Three chitosans with molecular
weight (MW) of 2, 20 and 100 KDa were chosen based on high anti-listerial activity. We
have also shown that the mechanism of the antibacterial activity of chitosan may vary,
depending on its MW. The determination of minimum inhibitory concentrations of chitosan
and divergicin against L. monocytogenes has identified the additive effect of these two
antimicrobial agents against L. monocytogenes. Afterwards, films with chitosan 100 kDa
and divergicin were developed and characterized. Chitosan-divergicin films display
interesting mechanical, physical and biological properties. The incorporation of divergicin
into the chitosan films increased their antimicrobial effect to a great extent and allows a
progressive release of the active compounds from chitosan films. Our results showed that
incorporation of di vergicin did not induce significant structural change in the chitosan film
and the FTIR spectra of divergicin M35 shed from the films did not differ from those of the
original pure bacteriocin, except in peak intensity. The results obtained were validated on
cold smoked wild salmon (CSWS) during 21 days of storage at 4°C and 8°C. The
application of chitosan-divergicin film was much more effective than chitosan-divergicin
solution at inhibiting L. monocytogenes in CSWS and the film permit a complete inhibition
de L. monocytogenes during the storage. The film provided the best preservation of sample
redness, kept volatile basic nitrogen below the control value, and the firmness was
maintained substantially higher compared to the control.
IV
DÉDICACE
AVANT-PROPOS
Cette thèse comprend cInq chapitres dont trois sont présentés sous forme d' articles
scientifiques. Le chapitre l correspond à la revue de littérature permettant de faire le résumé
sur les connaissances actuelles relatives au sujet traité dans cette thèse. L ' hypothèse et les
objectifs de cette thèse sont présentés à la fin de ce chapitre.
REMERCIEMENTS
Je tiens, tout d'abord à remercier mon directeur de recherche, Dr. Ismail Fliss, ·de
m'avoir donné l'opportunité d'effectuer ma thèse dans son laboratoire. Je le remercie pour
sa grande disponibilité, son encadrement et la confiance qu'il m'a accordée tout au long de
ce travail. C ' est un grand honneur pour moi d'avoir effectué ma thèse s.ous sa direction.
Mes remerciements s'adressent aux membres du jury d'avoir accepté d'évaluer cette
thèse.
Je tiens à remercier Mr Michel Desbiens pour son implication dans ce projet ainsi
que Dr Ehab Kheader pour ses conseils scientifiques.
RÉSUMÉ .............................................................................................................................. ii
ABSTRACT ......................................................................................................................... iii
DÉDICACE ......................................................................................................................... iv
AVANT -PROPOS ....................... ~ ......................................................................................... v
REMER ClEMENTS .......................................................................................................... vi
TABLE DES MATIÈRES ................................................................................................. vii
LISTE DES T ABLEA UX ................................................................................................... xi
LIS TE DES FIGURES ...................................................................................................... xii
LISTE DES ABRÉVIATIONs .......................................................................................... xv
INTRODUCTION ................................................................................................................. 1
CHAPITRE 1......................................................................................................................... 3
REVUE DE LITTÉRA TURE .............................................................................................. 3
1. 1 LES PRODUITS MARINS PRÊTS À CONSOMMER ............................................. 3
1. 1. 1 Problématique .................................. ~ ........................................................................ 3
1. 1.2 Le saumon fumé ............................................. ~ ......................................................... 4
1. 1. 2. 1 Les caractéristiques du saumon fumé ................................................................... 4
1. 1. 2. 2 La microflore endogène du saumon fumé ............................................................ 4
1. 1. 2. 2. 1 La microflore d'altération ................................................................................. 4
1. 1.2.2.2 La microflore pathogène .................................................................................... 6
1. 1. 2. 3 Les barrières microbiologiques traditionnelles pour la conservation du saumon
fumé .................................................................................................................................... 9
1. 1.2. 3. 1 Le salage ........................................................................................................... 9
1. 1.2.3.2 Le fumage ....................................................................................................... 10
1. 1. 2. 3. 3 Les additifs chimiques ............................................................... :.................... 13
1. 1. 2. 3.4 L'emballage sous atmosphères contrôlées ..................................................... 13
1. 2 LA BIOPRÉSERVATION .......................................................................................... 14
1. 2. 1 Définition ................................................................................................................ 14
1. 2. 2 Les métabolites à activité antimicrobienne produits par les bactéries lactiques .... 15
1. 2. 2. 1 Les acides organiques ......................................................................................... 15
1.2.2.2 Le peroxyde d'hydrogène ................................................................................... 15
1.2.2.3 Le dioxyde de carbone ........................................................................................ 16
1. 2. 2.4 Le diacétyle ................................................................ ~ ....................................... 16
1. 2. 2. 5 Autres, métabolites à activité antimicrobienne .................................................. 17
1. 3 LES BACTERIOCINES ...............••................•......••.••.•••••••••••••••••••••••••••••••••••••••••••••••• 17
VIl!
Table II. 3: MIC of divergicin M35, chitosan and their combinations against divergicin-
M35-resistant L. monocytogenes strain LSD 535 ................................................ 59
Table III. 2: Water vapor permeability in g·cm- 1sec- 1pa- 1 of chitosan films at different
relative humidity ........................................................................................ 81
Table IV. 1: Residual divergicin activity (AU) in homogenate of cold-smoked wild salmon
(CSWS) and its chitosan-divergicin film covering after 14 days at 4°C followed by 7 days
at " SoC, deterinined by agar diffusion tests and the critical dilution micro-
method ................................................................................................. 104
Table IV. 2: Color parameters L, a and b for cold smoked wild salmon (CSWS) during
storage at 4°C for 14 days followe<.Ï hy gOC for 7 Jays, A: control, E: CSWS coated with
chitosan-divergicin solution (C-M35 s), F: CSWS covered with chitosan-divergicin film (C-
M35 f ) ...................................................................................................105
XlI
Figure 1. 3: Mode d'action des bactériocines de la classe lIa (Ennahar. et al. 2000) ......... 22
Figure 1. 4: Les différents modes d'incorporation des substances antimicrobiennes dans les
aliments (1) incorporation dans' l'aliment (2) immersion ou pulvérisation et (3) ·
incorporation dans un film. Les poids noirs correspondent à la substance antirnicrobienne
(d'après Coma et al. 2008) ..................................................... : ...................... 29
Figure II. 2: Agar-weIl diffusion test showing inhibition of L. m,onocytogenes LGM 6902
by different types of chitosan. (a) 2 KDa, DDA 80%, (b) 2 KDa, DDA 87.4%; Cc) 2 KDa,
DDA 94.7%; (d) 20 KDa, DDA 80%; (e) 20 KDa, DDA 87.4%; (t) 20 KDa, DDA 94.7%;
(g) 100 KDa, DDA 80%; (h) 100 KDa, DDA 87.4%; (i) 100 KDa, DDA
94.7% .................................................................................................................................... 61
Figure II. 3: Growth of L. monocytogenes LSD 532 in the presence and in the absence of
chitosan, as measured by optical density at 650 nm. 2 KDa, (B) 20 KDa, (C) 100 KDa, Ch:
chitosan .................................................................................................. 62
Figure II. 4: Scanning electron micrographs showing the action of chitosan against L.
monocytogenes LSD 532. (a) untreated ceIls; (b) treated with 2 KDa, DDA 87.4%; (c)
treated with 20 KDa, DDA 87.4% and (d) with 100 KDa, DDA 87.4%. Short arrow:
listerial ceIl; long arrows: interaction between chitosan and listerial ceIl ...................... 63
Xlll
Figure II. 5: Transmission electron micrographs showing the action of chitosan against L.
monocytogenes LSD 532. (A) untreated cells, (B) treated with 2 KDa, DDA 87.4%; (C)
treated with 20 KDa, DDA 87.4% and (D) with 100 KDa, DDA 87.4%. CW: cell wall;
arrow: pore formation ................................................................................. 64
Figure III. 1: (A) Dried chitosan film (100kDa) containing divergicin M35 (B) Agar
diffusion test showing inhibition of L. monocytogenes 532 by (a) 100 kDa chitosan film and
(b) 100 kDa chitosan film loaded with divergicin M35 ................................ ............ 82
Figure III. 2: Puncture strength of chitosan films at different relative humidity (A) chitosan
87.4%DDA and (B) chitosan 94.7% DDA: chitosan ) and chitosan + M35 (.) ......... 83
Figure III. 3: The FTIR spectra of (A) chitosan film, 87.4% DDA and (B) chitosan film,
94.7% DDA (C) chitosan film, 87.4% DDA + divergicin M35 and (D) chitosan film, 94.7%
DDA + divergicin M35 ................................... .............................................. 84
Figure 111.4: The FTIR spectra of (A) divergicin M35 (B) divergicin M35 released from
chitosan film (87.4% DDA) and (C) divergicin M35 released from the chitosan film (94.7%
DDA) ..................................................................................................... 85
Figure III. 5: Kinetics of divergicin M35 release from chitosan film into buffer: chitosan
87.4%DDA (<1 chitosan 94.7% DDA (0) and into growth medium: chitosan 87.4%DDA
(~) chitosan 94.7% DDA (0) .........................................................................86
Figure III. 6:' Effect of divergicin M35 on the evolution of L. innocua HPB 13 viable count
in TSBYE at 30°C in the presence of (A) film completely immersed in the medium and (B)
film in contact with the medium on one surface: • control ~ chitosan 94.7% DDA +
M35 ~ thitosan 94.7% DDA [3 chitosan 87.4%+ M35 2l chitosan 87.4% ........................ 87
Figure IV. 1: Agar diffusion test showing inhibition of L. monocytogenes 532 by CA)
chitosan-divergicin solution (C-M35 s) and (B) chitosan-divergicin film(C-M35 F) ....... ·... 106
Figure IV. 3: Growth of total aerobic count in cold-smoked wild salmon (CSWS) during
storage at 4°C for 14 days and at 8°C for 7 days: (fQ) un-inoculated control 1 ID) inoculated
CSWS with L. monocytogenes alone (treatment B), ( Il) inoculated CSWS coated with
chitosan-divergicin solution C-M35 s (treatment C), ( ~) covered with chitosan-divergicin
film C-M35 F (treatment D). *** The detection limit for viable cells was < 50 cfu/g.
Different letters at the same time point indicate a significant difference (P < 0.05 .......... 108
XIV
Figure IV. 4: Growth of total lactic acid bacteria count in cold-smoked wild salmon
(CSWS) during storage at 4°C for 14 days and at 8°C for 7 days: (63) un-inoculated control
( m) inoculated CSWS with L. monocytogenes alone (treatment B), (II ) inoculated CSWS
coated with chitosan-divergicin solution C-M35 s (treatment C), ( 8) covered with chitosan-
divergicin film C-M35 F (treatment D). *** The detection limit for viable cells was < 50
cfu/g. Different letters at the same time point indicate a significant difference (P <
0.05) .................................................................................................... 109
Figure IV. 5: Appearance of samples of cold-smoked wild salmon at the end of storage (A)
control, (B) CSWS coated with chitosan-divergicin solution C-M35 s, (C) CSWS covered
with chitosan-divergicin film C-M35 F (treatment F) ............................................. 110
Figure IV. 6: Evolution of firmness (A) and cohesiveness (B) of cold-smoked wild salmon
CCSWS) during storage at 4°C for 14 days followed hy goC for 7 days: oC.) control
(treatment A), (e ) CSWS coated with chitosan-divergicin solution C-M35 s (treatment E),
( • ) CSWS covered with chitosan-divergicin film C-M35 F Ctreatment F) ................... .111
xv
INTRODUCTION
La production mondiale totale des pêches et de l'aquaculture s'est chiffrée en 2004 à 104
millions de tonnes de poissons destinés à l'alimentation humaine. La contribution de
l'aquaculture aux approvisionnements mondiaux des produits marins a augmenté durant les
dernières décennies passant de 3.9% de laproduction totale en 1970, à 27.1 % en 2000 pour
atteindre 32.4% en 2004 (FAO, 2007). Ce type de production est exploité en bonne partie
par l'industrie des produits marins prêts à consommer tels que le saumon fumé dont la
consommation est en constante croissance. Cette tendance peut s'expliquer par
l'engouement des consommateurs pour le marché du produit frais. Malgré cette popularité,
les produits marins prêts à consommer constituent ~ncore un des vecteurs de, toxi-infections
alimentaires les plus redoutés. En effet, de par leur composition, les produits marins .
constituent un environnement idéal pour la survie et, la croissance de plusieurs
microorganismes indésirables incluant des pathogènes tels Listeria monocytogenes et
Clostridium botulinum type E. Ce risque de contamination est d'autant plus élevé que le
mode de préparation de ces produits ne met en jeu aucun traitement thermique en mesure
de détruire d'éventuelles bactéries pathogènes avant consommation. De plus, les tendances
actuelles du marché forcent les producteurs à réduire substantiellement le recours à des
barrières microbiologiques cl~ssiques telles le salage et les additifs chimiques en raison de
leur impact négatif sur la santé du consommateur.
À titre d'exemple, Listeria monocytogenes, une bactérie pathogène très présente dans les
produits marins frais, est responsable de 28% de cas de mortalité rapportés annuellement.
(Mead et al., 1999). La présence de L. monocytogenes dans les produits marins fumés varie
entre 0 et 75% avec une prévalence de 10% (Ben Embarek, 1994).
comme une nouvelle alternative pour pallier les inconvénients liés à l'utilisation des
barrières microbiologiques traditionnelles. La bioprésevation est une approche naturelle
basée sur l'utilisation des bactéries lactiques ou de leurs métabolites tels les acides
organiques et les bactériocines et qui a pour but de contrôler la prolifération des bactéries
pathogènes et d'altération dans les aliments (Nillson et al., 1997). Trois approches peuvent
être utilisées pour l'exploitation des bactéries lactiques comme agent de bioconservation:
l'inoculation directe des produits par la souche productrice de substance inhibitrice, l'ajout
de la substance inhibitrice comme les bactériocines sous sa forme purifiée ou semi-purifiée
ou l'incorporation des bactériocines après immobilisation dans une matrice de polymère
pour former des films antimicrobiens qui peuvent être appliqués sur la surface de l' alime!1t.
Cette dernière approche présente plusieurs avantages par rapport aux deux premières car
elle permet de diminuer les interactions de la bactériocine avec les composants de la
matrice alimentaire , de maintenir une concentration importante de la bactériocine sur la
surface, d'assurer une libération progressive de la bactériocine durant toute la période du
stockage et enfin d'avoir un effet synergique avec un polymère si ce dernier est déjà doté
d'une activité antimicrobienne tel le chitosane (Cutter et al., 2001; Coma et al., 2002;
Pranoto et al., 2005). Cependant, les connaissances actuelles en rapport avec cette approche
sont encore très fragmentaires et des données importantes sur l'incorporation de
bactériocines dans un polymère comme le chitosane de même que sur sa stabilité
physicochimique et son activité biologique sont à connaître pour espérer exploiter un jour
efficacement cette approche dans le domaine de la conservation des aliments.
À notre connaissance aucune étude n'a montré l'effet antimicrobien de films à base de
chitosane incorporant une bactériocine pour la conservation du saumon fumé à froid.
Dans le cadre de cette thèse nous nous proposons d'exploiter simultanément les propriétés
du chitosane en tant que polymère doté de propriétés antibactériennes et antifongiques
intrinsèques (Begin et Van Calsteren., 1999; Srinivasa et al., 2004) et de la divergicine
M35, nouvelle bactériocine produite par Carnobacterium divergens sous forme d'un film
d'enrobage bioactif pour la biopréservation du saumon fumé à froid.
3
CHAPITRE 1
REVUE DE LITTÉRATURE
1. 1. 1 Problématique
L'engouement des consommateurs pour des produits réfrigérés plutôt que congelés est en
hausse et imprime des tendances commerciales vers le marché du produit frais. Cette
tendance expliquerait par exemple le fait que les produits marins prêts à consommer, tels
que le saumon fumé, sont des aliments de plus en plus prisés par le consommateur. Malgré
cette popularité, ces produits constituent encore un des vecteurs de toxi-infections
alimentaires les plus redoutés. En effet, de par leur composition, les produits marins
constituent un environnement idéal pour la survie et la croissance de plusieurs
microorganismes indésirables. Ce risque de contamination est d'autant plus élevé que le
mode de préparation de ces produits ne met en jeu aucun traitement thermique en mesure
de détruire d'éventuelles bactéries pathogènes avant consommation. De plus, les tendances
actuelles du marché forcent les producteurs à réduire substantiellement le recours à des
barrières microbiologiques classiques telles le salage et les additifs chimiques en raison de
leur impact négatif sur la santé du consommateur. Au Canada, par exemple, les faibles
teneurs en sel (inférieures à 3.5%) utilisées font que plusieurs bactéries pathogènes très
redoutables sont capables de survivre dans ces produits.
4
1. 1. 2 Le saumon fumé
Le saumon fumé à froid est considéré comme l'un des produits marins prêts à consommer
les plus périssables. il est composé de: 65-78% d'eau, 8-14% de lipides, 16-22% de
protéines, 2-4.6% de sel en phase aqueuse et 0.2-1.5 mg de phénol par 100g de poisson
(Duffes, 1999). TI est aussi caractérisé par une activité de l'eau variant entre 0.983 et 0.964
et un pH de 6. Son stockage et sa distribution se font dans des emballages sous vide à des
températures de réfrigération avec une durée de vie qui varie selon les pays.
1. 1. 2. 2. 1 La microflore d'altération
Très peu de travaux portent sur la caractérisation de la microflore d'altération des produits
marins de même que sur la contribution de chaque genre bactérien à l'altération de ces
produits. Les quelques données rapportées démontrent qu'il existe une grande variation
dans la composition de la microflore d'altération des produits marins transformés et que
celle-ci est influencée par l'origine de la matière première, les différents procédés de
transformation utilisés dans les fumoirs, le type d'emballage utilisé et la température
d'entreposage (Hansen et Huss, 1998; Leroi et al., 2001).
Des études sur la microflore du saumon fumé à froid ont montré qu'après trois semaines de
stockage à 8°C sous vide, 60% de la microflore est constituée de bactéries lactiques
appartenant aux genres Carnobacterium spp. et de Lactobacillus spp., les 40 % restants
sont composés de bactéries à Gram-négatives principalement Shewanella putrefaciens,
Aeromonas spp. et Photobacterium phosphoreum (Leroi et al., 1998). Olofsson et al.,
(2007) ont fait appel à des méthodes moléculaires pour la détermination de la microflore
endogène du saumon fumé à froid. ils ont démontré que la microflore dominante du
saumon fumé juste après le conditionnement est constituée de Brochotrix thermosphacta,
5
La microflore du saumon fumé peut aussi être influencée par les conditions de stockage et
le mode d'emballage. Duffes (1999) a rapporté que cette microflore est passée de 103-104 à
107 -10 8 ufc/g après trois semaines selon les conditions de stockage. L'emballage sous vide
peut expliquer par exemple la disparition de S. putrefaciens, un microorganisme aérobie
strict, pendant l'entreposage. Ainsi durant le stockage sous vide, les concentrations
d'oxygène diminuent tandis que celles du dioxyde de carbone augmentent, ce qui permet
une sélection progressive des bactéries qui tolèrent la présence du dioxyde de carbone, ce'
dernier étant responsable de l'inhibition de plusieurs bactéries Gram-négatives et surtout les
bactéries aérobies telles que Pseudomonas et S. putrefaciens (Leroi et al., 1998). Par contre,
l'emballage sous vide a tendance à favoriser le développement d'une microflore complexe
composée principalement de bactéries lactiques mais aussi de Brochotrix thermosphacta,
d'entérobactéries, d'autres bactéries Gram-négatives et de levures (Hansen et Huss, 1998).
Contrairement aux produits carnés, le rôle des bactéries lactiques dans l'altération du
saumon fumé n'est pas très clair. Plusieurs recherches ont montré qu'il n'existe pas de
corrélation entre le compte microbien des bactéries lactiques et la durée de vie du produit
(Hansen et al., 1995; Leroi et al., 1998). En effet, les bactéries lactiques peuvent dominer
en grand nombre sans qu'il y ait altération du saumon fumé (Huss et al., 1995). Leroi et al.,
(1998) ont étudié le pouvoir d'altération de certaines bactéries sur un jus stérile du saumon
fumé et ont constaté que les bactéries S. putrefaciens, Aeromonas spp. et Brochothrix spp.
produisaient de fortes odeurs plus que celles produites par les bactéries lactiques et que P.
phosphoreum ne semble pas jouer un rôle important dans l'altération du saumon fumé. En
2001, Stohr et al., ont étudié le profil sensoriel et le potentiel d'altération de bactéries
isolées à partir de saumon fumé à froid et ils ont montré que les bactéries responsables de
l'altération du produit sont Lactobacillus sake, L. farciminis et Brochothrix thermosphacta
et que le degré d'altération par les espèces S. liquefaciens et P. phosphoreum dépend de la
souche. La microflore du saumon fumé à froid rapportée dans les études citées ci-haut
6
concerne principalement les produits européens. Aucune étude du genre n'a été réalisée au
Canada pour la caractérisation de la microflore d'altération du SFF. De plus et à notre
connaissance, les données sur le rôle des autres genres bactériens dans l'altération du SFF
sont inexistantes. La diversité de l'origine d~ la matière première, les différences au niveau
des procédés de production, des habitudes de travail et des conditions de conservation,
notamment un taux de sel très inférieur aux produits européens (1 à 2% vs 4 à 6 %),
laissent présager un écosystème différent propre aux produits canadiens. Une
caractérisation poussée de cette microflore de même que la détermination du rôle de chaque
genre bactérien identifié dans l'altération des produits fumés demeure nécessaire pour une
meilleure maîtrise de la qualité de ces produits en vue de prolonger leur vie de tablette.
1. 1. 2. 2. 2 La microflore pathogène
Le principal danger pour la santé lié à la transformation du saumon fumé est la présence de
bactéries pathogènes. En effet, le procédé de fabrication est relativement long et comporte
beaucoup de manipulations tout au long de la chaîne de production, ce qui présente un
grand risque de contamination et de développement microbien. Au Canada, les faibles taux
de sels et le fumage appliqués au saumon fumé ne permettent pas nécessairement
d'éliminer les microorganismes contaminant le produit car ils sont surtout utilisés pour des
fins organoleptiques. Le faible taux de sel ajouté et les composants résultants de l' étape du
fumage ne constituent pas un environnement fortement inhibiteur du développement
bactérien.
Les bactéries pathogènes les plus rencontrées dans le saumon fumé à froid sont Listeria
monocytogenes et Clostridium botulinum type E. Ces deux bactéries sont psychrotrophes et
capables de croître à la température de réfrigération.
Listeria monocytogenes
Listeria monocytogenes est un bacille à Gram positif, aérobie, non sporulé, hémolytique et
catalase-positif. Ce microorganisme peut tolérer jusqu'à 10% de sel et est capable de
croître dans un intervalle de température entre 1°C et 45°C, et de pH variant entre 4.5 et 9.5.
7
C'est un pathogène opportuniste se manifestant chez les vieillards, les nouveau-nés et les
sujets immunodéprimés sous la forme d'une méningo-encéphalite ou d'une septicémie.
Cette bactérie peut aussi entraîner une infection inapparente qui peut toucher des sujets de
tout âge. Cette infection entraîne des conséquences durant la grossesse et peut causer un
avortement, une mortalité à la naissance ou des maladies graves chez les nouveau-nés
(Santé Canada, 2008). Chez l'adulte, l'infection L. monocytogenes peut se manifester sous
forme de méningite, d'endocardite, de septicémie et de lésions. Aux États-Unis, ce
pathogène est responsable chaque année de 28 % des cas de mortalité dûs à l'ingestion de
produits alimentaires contaminés (Mead et al., 1999). En 2008, les autorités canadiennes
ont rapporté 57 cas de listériose au Canada dont 21 décès dus à la consommation de
produits contaminés par L. monocytogenes. Ces produits proviennent d'une usine de viande
située dans la région de Toronto et dont les produits ' ont été distribués dans plusieurs
régions du pays (Santé Canada, 2009). Ces très récents évènements survenus au Canada,
montrent que la problématique de Listeria est encore d'actualité et laissent présager un
avenir promoteur quant à l'utilisation de la biopréservation dans les produits prêts à
consommer.
L. monocytogenes peut provenir d'une contamination du poisson dans son milieu naturel
mais peut également être transmise par les manipulateurs, les surfaces de travail, l'eau, etc.
L'incidence de Listeria dépend également de la source du saumon fumé. Ainsi différentes
espèces de Listeria ont été détectées à 81 % dans les eaux froides et 30% dans les eaux
marines dont 62% étaient des L. monocytogenes (Colburn et al., 1990). La présence de
matières fécales des animaux et les effluents des eaux usées dans les habitats naturels du
saumon entraînent la contamination des écailles et de la cavité intestinale du poisson par L.
monocytogenes. Le matériel utilisé dans les unités du traitement du saumon fumé ainsi que
le personnel sont considérés comme une deuxième source de contamination du saumon
fumé par L. monocytogenes. Dans une étude réalisée par R0rvik et al., (1995), 17% des
échantillons analysés étaient contaminés par L. monocytogenes avant salage, 30% après
salage, 17% avant et 40% après filetage, 13% après l'étape de tranchage et Il % dans le
produit fini.
8
une prévalence de 10%. Cette bactérie pathogène a été détectée dans 4.31 % de produits
marins fumés au Maryland et au nord de la Californie entre 2000 et 2901 (Gombas et al.,
2003).
Au Canada, l'étude réalisée par Farber, (2000) entre 1996 et 1998 a révélé qu'environ 0,3-
0,9 % des échantillons de saumon importés des États-Unis, du Chili et de la Norvège et 13
% des échantillons de crevette et de crabe étaient contaminés par L. monocytogenes. Dillon
et al, (1994) évaluent à 27.9% l'incidence de L. monocytogenes dans le poisson fumé de
Terre-Neuve. Heinitz et Johnson (1998) rapportent la présence de L. monocytogenes dans
15.2% des échantillons de poisson fumé importé; les produits canadiens examinés étaient
contaminés dans une proportion de 14,3%. Ces chiffres indiquent que les préoccupation"s
des secteurs bio-alimentaire et de la santé au Canada envers L. monocytogenes sont bien
réelles et que des mesures plus drastiques s'imposent pour en réduire l'incidence dans les
produits marins.
1. 1. 2. 3. 1 Le salage
1. 1. 2. 3. 2 Le fumage
L'effet antimicrobien de la fumée est basé sur la diminution de l'activité de l'eau par une
déshydratation partielle ainsi que par la formation d'une croûte composée de matière grasse
et de composés de la fumée formant une barrière physique contre une post-contamination
du saumon fumé. Cependant les exigences des consommateurs pour des produits faibles en
fumée réduisent l'effet attendu de la fumée contre les bactéries pathogènes et d'altération
capables de croître dans le saumon fumé à froid.
Il
" 1
Résidus Préparation
Salage
Séchage
0
24h (10 et 18 C)}
20h (32 OC) Fumage
6h « 50 OC) 1 . . . - - -_ _ _
; Refroidissement
~. . . . _R_éS_id_U_S---'~ 1
Tranchage 1. . . . . . . ==========-'-"-____---,
., Emballage
c_o_ng_é_la_tI_.o_n_ _
L . . . - -_ _ ---'I! r-----~-------,
~ Entreposage
Expédition
Résidus Préparation
Salage
Séchage
Cuisson
I~
Refroidissement
~ Parage
'- Emballage
Congélation
I~ Entreposage
Expédition
Plusieurs chercheurs se sont penchés sur l'effet du fumage à froid sur les bactéries
pathogènes et ont rapporté que ce traitement n'est pas suffisant pour éliminer Listeria
monocytogenes (Rorvik, 2000; Leroi et al. , 2001).
Les additifs chimiques les plus utilisés dans la conservation des produits carnés et des
produits marins sont les nitrites et les sulfites. Ils permettent d ' inhiber la germination des
spores de Clostridium et de préserver la couleur des aliments. Cependant la formation de
composés cancérigènes à partir des nitrites semble inquiéter sérieusement le consommateur
sur l'utilisation de ces agents comme agents de conservation dans les aliments. Des études
se sont portées sur la substitution des additifs chimiques ou sur la diminution de leur
concentration par l'exploitation d'autres substances antimicrobiennes dans la conservation
des aliments. Ainsi Roller et al., (2002) ont montré que la combinaison du chitosane à de
faibles doses de sulfites (170ppm) a permis de réduire la croissance microbienne dans des
saucisses comparativement à l'utilisation des sulfites seuls à une concentration de 340ppm.
La législation étant devenue plus rigoureuse sur l'emploi des additifs chimiques, les
industriels sont dans l'obligation de réduire le taux d'incorporation de ces agents durant les
procédés de transformation des produits alimentaires. Notons que dans le cas des produits
marins prêts à consommer, les législations canadienne et européenne interdisent
l'utilisation des additifs chimiques.
~ L'emballage sous vide ou sous atmosphère contrôlée est le type d'emballage le plus utilisé
dans l'industrie du saumon fumé. Il permet de préserver les produits réfrigérés de la
prolifération des moisissures et de certaines bactéries aérobies strictes. Mais le risque de
croissance de L. monocytogenes et de C. botulinum type E est toujours présent.
Généralement, un emballage sous vide couplé à un stockage à la température de
réfrigération rend les produits fumés susceptibles à la germination des spores et à la
croissance de C. botulinum de type E, bactérie anaérobie stricte, ce qui présente un risque
14
de production de toxines. En Europe, l'application de taux de sel élevés dans les PMPAC
couplée à un emballage sous vide ou sous atmosphère contrôlée permet le contrôle de C.
botulinum type E. Néanmoins, au Canada, la réduction du taux de sel permet de réduire le
risque de maladies cardiovasculaires mais favorise la croissance de C. botulinum de type E.
Actuellement, l'utilisation d'emballage complètement imperméable à l'oxygène est
interdite. L'utilisation d'emballage perméable à l'oxygène permet l'inhibition de la
production de la toxine par C. botulinum de type E et permet la croissance des
microorganismes d'altération avant la production de la toxine (Dufresne et al., 2000). Dans
les travaux de cette équipe sur la toxigénèse botulique dans la truite fumée, il a été
démontré que le stockage du produit à 4°C pendant 28 jours n'a révélé aucune production
de toxine, et qu'un abus de température permettait la détection de la toxine sous emballage
sous vide ou non. Cepe"ndant l'altération du produit a été observée soit avant ou
simultanément avec la production de la toxine.
1. 2 LA BIOPRÉSERVATION
1. 2. 1 Définition
Les bactéries lactiques produisent des acides organiques tels l'acide acétique et l' acide
lactique qui possèdent une activité antimicrobienne contre plusieurs microorganismes
pathogènes et d'altération. ' L'application de sels de l'acide acétique et de l'acide lactique ou
du mélange des deux a permis de contrôler la croissance de plusieurs bactéries indésirables
dans les produits carnés (Houtsma et al., 1993; Stekelenburg et Kant-Muermans, 2001).
Strom et al., (2002) ont rapporté que pendant la fermentation du levain, certaines souches
de Lactobacillus plantarum ont une activité antifongique grâce à la production de l'acide 3-
phénylactique et de dipeptides cycliques.
1. 2. 2. 2 Le peroxyde d'hydrogène
1. 2. 2. 3 Le dioxyde de carbone
Le dioxyde de carbone est formé lors de la fermentation des sucres par les bactéries
lactiques hétérofermentaires. Ce métabolite exerce son effet antimicrobien de deux façons
différentes soit par la création d'un environnement anaérobie ou par son effet antimicrobien
direct sur les microorganismes (Lindgren et dobrogosz, 1990). Devlieghere et Debevere,
(2000) ont étudié l'effet du dioxyde de ' carbone sous atmosphère modifiée sur différents
genres de bactéries d'altération. lis ont démontré que les bactéries Gram-négatives sont
plus sensibles au dioxyde de carbone que les bactéries Gram-positives. Le dioxyde de
carbone est toxique pour un certain nombre de bactéries d'altération, cependant il peut
stimuler la croissance d'autres types de microorganismes (Holzapfel et al., 1995).
1. 2. 2. 4 Le diacétyle
Le di acétyle est produit par la dégradation de l'acide citrique par certaines Lactococcus,
Leuconostoc et Pediococcus spp. (Holzapfel et al., 1995). Le diacétyle confère à certains
produits les caractéristiques aromatiques désirables (Marshall, 1987). li possède également
une activité antimicrobienne plus importante contre les bactéries Gram-négatives, les
levures et moisissures comparativement aux bactéries Gram-positives (lay, 1982). Le
potentiel d'utilisation du diacétyle comme agent antimicrobien est limité par la
concentration du di acétyle nécessaire pour avoir un effet antimicrobien sans compromettre
la qualité organoleptique du produit fini. Ainsi, pour inhiber des bactéries Gram-négatives
il faut approximativement une concentration de di acétyle de 200 mg/kg, alors que la limite
acceptable dans les produits laitiers fermentés est de 2 à 7 mg/kg. Des concentrations
supérieures à cette limite sont souvent responsables de développement de goût indésirable
danS les produits (Earnshaw et al. 1992).
17
Certains métabolites à faibles poids moléculaires produits par les bactéries lactiques sont
dotés d'activité antimicrobienne tels la reutérine et la reutéricycline. La reutérine (3-
hydroxypropionaldéhyde), produite par Lactobacillus reuteri, est un composé dont le
pouvoir antimicrobien a été confirmé dans des matrices alimentaires (lait, fromage, produit
laitier espagnol) contre L. monocytogenes et E. coli 0157:H7 (Arques et al., 2008; EI-
Ziney et Debevere., 1998). La reutérine peut aussi être utilisée dans la bioconservation de la
viande et du poisson (Lindgren et Dobrogosz, 1990). Cependant il semble que la production
de la deutérine est restreinte à certaines souches de L. reuteri (Earnshaw, 1992).
1. 3 LES BACTÉRIOCINES
1. 3. 1 Définition et classification
La première définition générale des bactériocines a été proposée par Tagg et al., (1976). Ils
ont défini les bactériocines comme étant des substances antimicrobiennes de nature
protéique ayant un 'spectre d'inhibition restreint à des espèces étroitement apparentées à la .
souche productrice. Une définition plus récente a été proposée par Ennahar et al., (2000) et
18
décrivait les bactériocines comme étant toute molécule de nature protéique, synthétisée par
la voie ribosomique et douée d'une activité bactéricide ou bactériostatique contre certaines
. bactéries Gram-positives proches de la souche productrice sur le plan phylogénétique. Les
bactériocines sont- synthétisées au sein de la ·cellule sous la forme d'un précurseur et
subissent un processus de maturation au cours de leur exportation vers le milieu
extracellulaire (Cenatiempo et al., 1996). TI a été démontré que les bactéries productrices de
bactériocines possèdent des gènes d'immunité leur permettant de se protéger contre leurs
propres substances inhibitrices (Tagg et al., 1976; Klaenhammer, 1993; Jack et al., 1995;
Cintas et al., 2001).
En 1993, Klaenhammer a classé les bactériocines en quatre grandes classes selon leur taille,
leur structure et leur mode d'action. Cette classification a été révisée par plusieurs auteurs
(Nes et al., 1996; Eijsink et al., 2002; Drider et al., 2006) selon les propriétés génétiques et
physico-chimiques des bactériocines (Tableau 1.1).
La classe 1 : Des petits peptides dont la taille est inférieure à 4 KDa nommés lantibiotiques
et composés d'un nombre variable d'acides aminés tels que la lanthionine. Cette classe
renferme deux sous classe type A et type B. Les bactériocines de type A ont une structure
moins rigide que celle de type B avec un poids moléculaire inférieur à 4 KDa tandis que
les bactériocines de type B possèdent une structure globulaire avec un très faible poids
moléculaire 2.1 à 1.8 KDa.
La classe III : Les bactériocines de poids moléculaire supérieur à 30 KDa et instables aux
températures élevées.
Les bactériocines qui ont suscité le plus d'intérêt demeurent les bactériocines de la classe 1
dont les bactériocines modèles sont la nisine produite par Lactococcus lactis subsp. lactis
et la classe II avec la pédiocine PA-l, les carnobactériocines BMl et B2, la sakasine A et la
divercine V41 et plus récemment la divergicine M35.
Les bactériocines de la classe lIa montrent une activité anti-Listeria plus intéressante que
les bactériocines de la classe l, car elles présentent un spectre d'action plus grand (Chen et
Hoover, 2003). En plus de leurs activités anti-Listeria, les bactériocines de la classe na
présentent aussi un spectre d'action dirigé contre d'autres microorganismes pathogènes et
d'altération (Brochotrix spp., Bacillus cereus, Clostridium perfringens, Staphylococcus
aureus) (Ennahar et al., 2000; Drider et al., 2006).
o
C'l
Tableau 1. 1. Classification et caractéristiques des bactériocines des bactéries lactiques (Drider et al., 2006)
.
21
1. 3. 2 Le mode d'action
La majorité des bactériocines a pour trait commun un mode d'action agissant au niveau de
la membrane plasmique des cellules cibles (McAuliffe et al., 2001; Driessen et al., 1995).
Les bactériocines des classes 1 et II sont caractérisées par une faible taille, insuffisante pour
la perforation de la membrane plasmique par une seule molécule. li faut donc une
. association de plusieurs molécules de bactériocines pour la formation des pores dont, la
taille dépend du nombre de molécules utilisées (Cenatiempo et al., 1996).
La figure 1. 3 schématise le mode d'action des bactériocines de la classe lIa selon Ennahar
et al. (2000). La présence des segments amphiphiles contenants des hélices
transmembranaires (figure 1.3 a) permettent la formation de pores sur la membrane
cellulaire de la cellule cible. La nature cationique de la région N -terminale de ces
bactériocines favorise leur premier contact avec les phospholipides anioniques de , la
membrane cible (figure 1.3 b). Ensuite la partie hydrophobe de la région C-terminale de la
bactériocine qui intervient par une interaction hydrophobe avec la membrane et forme ainsi
les pores trans-membranaires. Cette dernière interaction a été expliquée par le fait que la
région C-terminale contient une partie hautement hydrophobe comparativement à celle de
la région N -terminale (figure 1.3 b,c). Après la formation des pores, la bactériocine induit la
mort cellulaire par déstabilisation de la membrane due au déplacement d'un flux passif
d'ions ou de petites molécules tels les acides aminés à travers la bicouche lipidique en
réduisant ainsi la force proton motrice (Montvjlle et chen, 1998; Deegan et al., 2006). Ce
mécanisme d'action peut être influencé par plusieurs facteurs, particulièrement la
composition en lipides membranaires et le pH du milieu. En 2003, Gaussier et al. ont étudié
l'effet de la Pediocine PA -1 sur des membranes lipidiques modèles et ont montré que
l'association de la bactériocine avec l'interface membranaire affecte la conformation de ce
peptide et que cette association est le résultat d'interactions électrostatiques entre le peptide
et les charges négatives de la membrane.
22
tru tu .
PrediCled main 1- 1 J 27
ic inl! ion
Figure 1. 3. Mode d'action des bactériocines de la classe lIa (Ennahar. et al. 2000)
23
Le genre Carnobacterium est le genre dominant dans la microflore du saumon fumé à froid.
C'est une bactérie Gram positif, catalase et oxydase négatives qui se présente sous forme de
bacilles minces, droits ou légèrement incurvés, en paires de deux, ou groupés ou parfois en
courte chaîne.
Plusieurs recherches ont porté sur l'étude de l'effet anti-listeria des bactéries lactiques
dans le saumon fumé à froid. Néanmoins, la caractérisation de bactériocines produites par
des espèces d'origine marine a fait l'objet de peu d'études surtout celles relatives aux
bactériocines provenant de souches de l'espèce C. divergens. Le Tableau 1.2 représente des
exemples de bactériocines, leurs souches productrices ainsi que leurs origines.
La divercine V41, produite par C. divergens V41 est la première bactériocine de C.
divergens isolée du tractus gastro-intestinal de saumon. Cette bactériocine a été séquencée,
caractérisée et étudiée pour son activité sur la flore pathogène dans le saumon (Métivier et
al., 1998; Duffes et al., 1999ab).
Parmi les souches d'origine marine qui ont montré un effet anti-listeria, on retrouve C.
piscicola. Nilsson et al., (1999) ont montré que l'effet anti-listeria de cette bactérie dans le
saumon fumé à froid n'est pas dû à la production de bactériocines mais plutôt à la
compétition de la souche productrice pour l'utilisation des nutriments essentiels. Par contre,
l'effet antibactérien de C. divergens V41 dans du saumon fumé à froid réfrigéré a été
directement associé à la production de la divercine V41, ceci dans des conditions sévères,
incluant des variations de températures et différentes concentrations en N aCI et en glucose
(Connil et al., 2002). Duffes et al., (1999b) ont observé une réduction de L. monocytogenes
de 1 log à 4°C et de 5 log à 8°C après 21 jours de stockage en présence de C. divergens
V41.
~ Tableau 1.2. : Exemple de bactériocines produites par des souches du genre Camobacterium isolées de matrices alimentaires
Les travaux récents de notre équipe (Tahiri et al., 2004) ont permis d'isoler plusieurs
souches de produits marins dont deux souches de bactéries lactiques, identifiées comme
étant C. divergens et C. piscicola, montrant une activité inhibitrice marquée contre L.
monocytogenes. Une de ces deux bactériocines produite par la souche C. divergens a fait
l'objet d'une caractérisation plus poussée à la suite de laquelle une nouvelle bactériocine, la
divergicine M35, a été identifiée. La divergicine M35 est la deuxième bactériocine
d'origine marine caractérisée après la divercine V41 produite par C. divergens V41. La
divergicine M35 a été purifiée, classée comme bactériocine de la classe lIa et séquencée en
43 acides aminés. La spectrophotométrie de masse a permis de déterminer le poids
moléculaire (4718.75 Da), la charge nette (+3) et un point isoélectrique égal à 8.6 (Tahiri et
al., 2004).
Trois approches peuvent être utilisées pour l'exploitation de l'effet antimicrobien des
bactériocines dans les aliments. La première stratégie consiste à inoculer des produits avec
la souche productrice directement. Ceci permet une production in situ continue de la
bactériocine dans la matrice alimentaire. Cette approche implique que la souche de bactérie
lactique soit capable de se reproduire dans le produit aux températures de réfrigération. Elle
peut aussi avoir certains inconvénients notamment une incompatibilité avec la flore
endogène du produit. De plus, la bactérie peut perdre sa capacité à produire la bactériocine.
Finalement, il faut s'assurer que l'utilisation de la souche bactériocinogène ne présente
aucun impact négatif sur la qualité sensorielle et organoleptique du produit fini (Holzapfel
et al. 1995, Galvez et al. 2007).
L'incorporation directe des bactériocines dans la matrice alimentaire peut être limitée par
certains facteurs tels l'interaction de la bactériocine avec certains constituants de l'aliment,
le pH et l'activité protéolytique intrinsèque du produit (Katla et al., 2001, Roller et al.,
2002, Aasen et al. 2003). Ainsi, l'application de la nisine est limitée par sa faible solubilité
à un pH autour de 6, et son inactivation dans certains types de produits (Ross et al., 2003;
Katla et al. 2001). L'ajout de la ni sine dans l'emballage du saumon fumé à froid stocké à
5°C n'a pas permis l'inactivation de L. monocytogenes (Nilsson et al 1997). Dans les
travaux antérieurs de note équipe (Tahiri et al. 2008), l'efficacité des deux premières
stratégies d'utilisation de la divergicine M35 (culture productrice, divergicine M35 pure ou'
sous forme de bio-ingrédient issu du surnageant de culture concentré du milieu à base
d'hépatopancréas de crabe) ont été comparées sur du saumon fumé à froid entreposé
pendant 21 jours à 4°C. Un effet supérieur a été obtenu contre L. monocytogenes lorsque
C. divergens M35 est ajoutée directement sur les filets de saumon comparativement à
l'ajout de la divergicine pure ou sous forme de bio-ingrédient. Cependant l'utilisation de
ces différentes stratégies n'a pas permis d'inhiber la croissance de L. monocyto genes dans
le saumon fumé 'à froid après 21 jours d'entreposage.
La diffusion des agents antimicrobiens à travers les emballages a fait l'objet de plusieurs
publications (Vojdani et Torres, 1989, 1990; Han et Floros, 1998ab). Les matériaux
d'emballage antimicrobiens doivent entrer en contact avec la surface des aliments
principalement dans le cas où les agents antimicrobiens sont non-volatiles. Ainsi, ils
28
peuvent se répandre sur la surface du produit et diffuser si nécessaire. Selon Han, (2000),
l'avantage des agents antimicrobiens volatils est qu'ils peuvent être libérés à la surface de la
matrice alimentaire sans que le polymère ne soit directement en contact avec le produit. Les
vapeurs ou les gaz antimicrobiens sont appropriés pour des applications où le contact entre
les parties exigées de l'aliment et l'emballage ne se produit pas, comme dans le cas de la
viande hachée. La figure 1.5 illustre les deux types de concepts d'emballage en présence et
en absence d'un espace libre entre le film et l'aliment (Coma, 2008).
29
Les bactériocines ont été également incorporées comme agents antimicrobiens dans les
polymères. La plupart des études rapportées dans la littérature ont fait appel à la ni sine car
c'est la seule bactériocine reconnue pour son statut GRAS et aussi pour sa disponibilité sur
le marché (Cooksey, 2000; Cha et al., 2003; Pranoto et al., 2005; Sanjurjo et al., 2006).
L'activité de la nisine dépend de la matrice utilisée comme support. · Des études visant à
développer des films antimicrobiens à base de gélatine ou de maïs ont montré que l'activité
des films contre L. monocytogenes augmentait avec la concentration de nisine et que les
films à base de maïs étaient plus efficaces contre L. monocytogenes comparativement aux
films à base de gélatine (Ku et Bin Song, 2007).
Dans ce même volet, l'équipe de Cha et al. (2002) a préparé des films à base d'alginate et
de K-carrageenane pour examiner leur effet antirnicrobien seul et en combinaison avec la
32
ni sine et autres substances microbiennes (lysozyme, extrait de raisin et EDT A). Les films
d'alginate ont montré une large activité inhibitrice comparativement aux films de K-
carrageenane avec les mêmes combinaisons et le même taux d'incorporation des agents
antimicrobiens.
Certains polymères possèdent une activité antimicrobienne intrinsèque et peuvent donc être
utilisés dans des films et des enrobages alimentaires. Les polymères cationiques tels que le
chitosane et le poly-L-lysine adhèrent facilement aux cellules microbiennes (Goldberg et
al., 1990) puisque les amines chargées positivement agissent avec les charges négatives sur
la membrane des cellules libérant ainsi les constituants intracellulaires. Les alginates de
calcium ont été aussi étudiés pour le développement de films antimicrobiens. ils ont permis
la réduction de la croissance de la flore naturelle et des coliformes sur des produits carnés
(Cuq et al., 1995). D'autre part, certains polymères nécessitent une modification afin
d'augmenter leur activité antimicrobienne. Par exemple, le potentiel antimicrobien des
films de polyamide traités par irradiation aux UV a permis d'augmenter leur activité
antimicrobienne. Ceci est probablement dû à l' augmentation de la concentration en amines
à la surface du film (Hagelstein et al., 1995). Cependant, l'utilisation de polymères
antimicrobiens dans la conservation des aliments peut être limitée par la composition de ces
derniers. En effet, certains composés comme les sels, les protéines et les acides gras
peuvent interagir avec les polymères tels le chitosane et neutraliser ainsi leur activité
biologique (Devlieghere et al. 2004b).
33
Abréviations: EVA (éthylène vinyle acétate); LLDPE (linear low density polyethylene);
LDPE (low density polyethylene); PS (polystyrène); PE (polyéthylène).
- -- ---------------------------------------~
34
1. 4. 2. 1. 1 Définition et caractéristiques
Le chitosane est un produit non toxique et présente une solubilité dans les acides
organiques à des pH<6 tels l'acide acétique, formique, lactique, succinique et malique.
Cependant, il est insoluble dans l'eau, les milieux alcalins et les solvants organiques. La
viscosité des solutions de chitosane est affectée par différents paramètres: le degré de
désacétylation, le poids moléculaire, la concentration, les liaisons ioniques, la température
et le pH (Rabea et al., 2003).
35
(a)
Chitine
Chitosane
- - - _ . _- -- '
36
'Grâce à ses propriétés filmogènes, le chitosane a suscité l'intérêt des chercheurs pour son
utilisation comme enrobage ou emballage alimentaire grâce à ses propriétés
physicochimiques et mécaniques intéressantes (Begin et Van Calsteren, 1999; Srinivasa et
al. 2004; Suyatma et al., 2004). Les films à base de chitosane sont étirables, flexibles, et ils
présentent aussi des propriétés mécaniques comparables à certains polymères
commerciaux. Le chitosane permet aussi de former des films semi-perméables, ainsi les
enrobages de chitosane ont été suggérés pour modifier l'atmosphère interne, diminuer la
respiration et retarder la maturation des fruits (Rabea et al., 2003).
Le chitosane ainsi que ses dérivés ont suscité l'intérêt de plusieurs chercheurs pour leurs
propriétés biologiques tels l'activité hypocholesterolémique (Sugano et al., 1988), l'activité
antitumorale (Tokoro et al., 1988; Jeon et Kim~ 2002) et son potentiel d'application dans les
industries agro-alimentaires comme additif stabilisateur de couleur, émulsifiant,
antioxydant et gélifiant (Agullo et al., 2003). Plus récemment, la possibilité d'utiliser le
chitosane pour ses propriétés antimicrobienne et antifongique a été démontrée (Shahidi et al.,
1999; Liu et al., 2001; Agullo et al., 2003; Liu et al., 2004; Rinaudo, 2006). Le chitosane
avec sa nature cationique permet aussi de fabriquer des films contenant des substances
antimicrobiennes chargées positivement qui peuvent migrer du film vers le produit à
conserver. Cha et al., (2003) ont montré que des films de chitosane enrichis de ni sine
(chargée positivement) présentaient une meilleure activité contre Micrococcus luteus
comparativement à des films de k-carageenan et de methylcellulose.
Micrococcus luteus 20
Pseudomonas fluorescens 500
Staphylococcus aureus 20
Xanthomonas campestris 500
Xanthomonas campes tris 500
Le mécanisme d'action antimicrobien du chitosane n'est pas encore bien connu mais
différentes hypothèses ont été proposées (Sudarshan et al., 1992; Chen et al., 1998; Rabea
et al., 2003):
1) Les charges positives du chitosane peuvent interagir avec la charge négative des
membranes de la cellule microbienne, ce qui induit la libération du matériel prot~ique et les
autres constituants intracellulaires.
En tant que polymère naturel, le chitosane peut être utilisé dans la fabrication de films
alimentaires comestibles. Agullo et al., (2003) ont étudié l'effet antimicrobien d'un film
comestible de chitosane pour la conservation de pizza prêt à consommer. Ils ont démontré
que le chitosane retardait la croissance de microorganismes (Alternaria sp, Penicillium sp
et Cladosporium sp) responsables d'altérations de ce type de produit. TI a été démontré que
le chitosane pouvait réduire le taux d'incorporation des additifs chimiques dans les produits
alimentaires. Ainsi, l'équipe de Roller et al. (2002) a montré qu'une combinaison entre le
40
1. 5 HYPOTHÈSE DE RECHERCHE
Les PMPAC sont des produits très périssables qui présentent un risque élevé de toxi-
infection. Ce risque de contamination est d'autant plus élevé que les tendances actuelles du
marché limitent substantiellement le recours à des barrières microbiologiques classiques
telles le salage et les additifs chimiques en raison de leur impact négatif sur la santé. Dans
ce contexte, la biopréservation, notamment l'utilisation de bactéries lactiques productrices
de bactériocines, est sans doute l'alternative la plus prometteuse pour assurer l'innocuité de
ce type de produits et réduire l'incidence de bactéries pathogènes redoutables telle que
Listeria monocytogenes. Cependant, les bactériocines peuvent perdre leur activité après leur
application car elles sont peu stables dans les aliments. L'incorporation des bactériocines
dans une matrice à base de polymère et son application sous forme de film sur la surface
des PMPAC permet une libération progressive de la substance antimicrobienne à travers le
film vers la surface de l'aliment et par cons~quent un meilleur contrôle de la croissance des
microorganismes visés par ce traitement. Le chitosane par sa qualité de polymère, ses
propriétés filmogènes de même que ses propriétés antibactériennes et antifongiques
intrinsèques constitue un' bon candidat pour le développement de films antimicrobiens.
Ainsi, nous avons pu émettre l'hypothèse suivante: l'exploitation simultanée des propriétés
antimicrobiennes de la divergicine M35 et des propriétés filmogènes et antibactériennes -du
chitosane permet le développement d'un film biologiquement pour le contrôle de la
microflore pathogène et d'altération du saumon fumé.
42
1. 6 OBJECTIFS DE RECHERCHE
CHAPITRE II
2 Institute of Food Science and Nutrition, Swiss Federal Institute of Technology, ETH
Zentmm, LFO FI8 CH-8092 Zurich, Switzerland
II. 1. RÉSUMÉ
II.2. ABSTRACT
The antimicrobial activities of the class IIa bacteriocin divergicin M35 and several types of
chitosan against Listeria monocytogenes were quantified by agar diffusion, critical micro-
dilution and viable count and observed by electron microscopy. Antimicrobial activity of
chitosan depended on its molecular weight (MW) and the pH. Three chitosans with MW of
2, 20 and 100 KDa and 87.4% degree of deacetylation (DDA) were chosen for further
study, based on high anti-listerial activity at pH 4.5. Electron microscopy suggested that the
mechanism of antilisterial activity also varied with the MW. Low-MW chitosan appeared to
inhibit L. monocytogenes by affecting cell permeability and growth, whereas medium and
high-MW chitosan may form a barrier on the cell surface that prevents entry of nutriments.
The minimum inhibitory concentrations (MIC) of 2, 20 and 100 KDa chitosan and
divergicin M35 against a divergicin-resistant strain of L. monocytogenes (LSD 535) were
2.5, 2.5, 0.625 and 0.25 mg/ml respectively. The combination of any of these three
chitosans and divergicinM35 appeared to have an additive effect against L. monocytogenes,
as determined by fractional inhibitory concentration index (FIC index). This study provides
useful data for the development of chitosan films incorporating divergicin M35 for
inhibiting L. monocytogenes in foods.
Key words: Chitosan; Divergicin M35; FIC index, L. monocytogenes; antibacterial effect.
46
II. 3. INTRODUCTION
Although several hypotheses have been proposed, the mechanism of the antimicrobial
activity of chitosan against bacteria is still unclear. One of the proposed mechanisms refers
to changes in cell permeability, due to interactions between the pol ycationic chitosan and
the negative charges on the cell surface (Chen et al., 1998; Shahidi et al., 1999; Rabea et
al., 2003). Unfortunately, these hypotheses' remain speculative and still need confirmation
by well-designed experiments.
treatment is usually limited or even prohibited, there is increasing necessity to apply agents
such as bacteriocins to preserve these products. Recently, we characterized a new
bacteriocin called divergicin M35, a class lIa bacteriocin produced by Carnobacterium
divergens M35 isolated from commercial frozen mussels (Tahiri et al., 2004). Divergicin
M35 has a molecular mass of 4518.75 Da, and displays inhibition activity against L.
monocytogenes known to be a serious health threat, particularly in lightly preserved
seafood (Farber and Peterkin 1991; 'R0rvik 2000; Food and Drug Administration, 2001).
The aim of the present study was therefore to 1) characterize the antimicrobial activity of
divergicin M35 and chitosans of differing MW and DDA, either alone or in combination
against L. monocytogenes and 2) elucidate the mechanism of antibacterial activity of
chitosans involved in L. monocytogenes inhibition.
Carnobacterium divergens M35 (C. divergens) , a divergicin M35 producer isolated from
commercial frozen mussels and identified by Tahiri et al. (2004), was grown aerobically at
30°C in de Man, Rogosa and Sharpe (MRS) broth (De Man et al., 1960) obtained from
Difco Laboratories (Sparks, MD, USA). L. monocytogenes strains LSD348, LSD526,
LSD532, LSD332 and LSD535, were obtained from the Laboratory Services Division,
Canadian Food Inspection Agency (Ottawa, ON, Canada). L. monocytogenes strains
LGM 16491 and LGM6902 were obtained from the Laboratorium voor Microbiologie Gent,
Culture Collection (Gent, Belgium). Listeria innocua HPB 13 CL. innocua) was obtained
from Health Protection Branch, Health and Welfare Canada (Ottawa, ON, Canada). AlI
strains were maintained as 20% glycerol stock at -80°C. AlI Listeria strains were grown
aerobically at 37°C in tryptic soy broth (Difco Laboratories) supplemented with 0.6% (w/v)
yeast extract (TSBYE).
48
II. 4. 2. Chitosan
Chitosans with different MWs (1, 2, 10, 20 and 100 KDa) and different DDA of 80,87.4 or
94.7% were obtained from ISM Biopolymer Inc. (Granby, PQ. Canada). AlI chitosans were
obtained in powder forms and were stored at refrigerated temperature during the
experiments.
The inhibitory activity of chitosan against L. monocytogenes strains (LSD 332, LSD 348,
LSD 526, LSD 532, LGM 16491, and LGM 6902) was determined for different chitosan
preparations. Chitosan was dissolved at a concentration of 1% w/v in 1% (v/v) aqueous
acetic acid and pH was adjusted to 4.5, 5.0, 5.5 or 5.9 by addition of 5N NaOH solution.
Chitosan preparations were tested using the agar-weIl diffusion method (Tagg et al. 1976).
Briefly, the solid surface of 0.75% agar-containing TSYE medium was seeded at 45°C with
1 % (v/v) overnight culture of tested strain. Seeded agar was then poured into a sterile Petri
plate and alIowed to solidify at room temperature. Wells (diameter of 7mm) were cut in the
solidified agar and filled with 80 III of chitosan solutions tested. The plates were incubated
. . . . - - - - - - - - - -- - -- - ~- - ~ ~-~ ~ ~ ---
49
aerobically at 37°C for 18 h and were visually examined for the presence of inhibition
zones around the weIl.
Chitosan solutions were added to TSBYE broth to obtain a chitosan concentration of 0.1 %
(w/v). Chitosan-containing broth was inoculated .with 1% (v/v) overnight culture of L.
monocytogenes strains (LGM 16491, LGM 6902, LSD348 or LSD532) and incubated
aerobically at 37°C for 24 h. The resulting cultures were 10-fold serially diluted in peptone
water (0.1 % w/v) and appropriate dilutions were plated onto TSBYE agar. Plates were
incubated aerobically at 37°C for 24 h and viable counts were recorded as colony forming
units (cfu) per ml of culture.
For determination the nature of effect of chitosan with different MW and DDA, the growth
of L. monocytogenes LSD532 was monitored during 30 h of incubation at 37°C. This
listeria strain was used because of its high sensitivity to divergicin M35. L. monocytogenes
(1 %) was grown in TSBYE containing 0.1 % (w/v) of tested chitosan. Samples were
withdrawn each hour during the first 10 hours and after 24 and 30 h of inoculation for the
determination of OD at 650 nm using a Thermo-max spectrophotometer (OPTI-Resources,
Quebec, PQ, Canada).
L. monocytogenes LSD532 was grown in TSBEY containing 0.1 % chitosan (2, 20 or 100
KDa and DDA 87.4%) for 18 h at 37°C. Following incubation, cells were harvested by
centrifugation, suspended in 3% (w/v) agarose and fixed for 24 h in 0.05% (w/v)
glutaraldehyde (Marivac Ltd., Halifax, NS, Canada) and 2. 5% (w/v) paraformaldehyde
solution in 0.1 M sodium cacodylate buffer at pH 7.3 (JBS-CHEM, Dorval, PQ, Canada).
Samples were washed three times (10 min each) with cacodylate buffer, post-fixed for 90
min in 1% (w/v) osmium tetroxide (Sigma-Aldrich Canada Ltd.) prepared in cacodylate
buffer, washed again three times (10 min each) with cacodylate buffer and dehydrated in a
50
graded ethanol series. For scanning electron microscopy (SEM), samples were further
steeped twice in hexamethyldisilazane (30 min each). Samples were th en dried at ambient
temperature, gilded and examined using a JEOL 6360LV scanning electron microscope
(Tokyo, J apan). For transmission electron microscopy (TEM), ethanol-dehydrated samples
were embedded in Epon (Polyscience Inc., Warrington, PA, USA) and polymerized at 60°C
for 72 h. Ultra-thin sections (60-80 nm) were prepared using an ultramicrotome (Reichert-
Jung, Vienna, Austria). Sections were collected on formvar-coated nickel grids and stained
with uranyl acetate and lead citrate. The grids were examined using a JEOL 1230 EX
transmission electron microscope (Tokyo, Japan) at 80 kV.
The MICs of chitosan and divergicin M35 against L. monocytogenes LSD535 were
determined by a Micro Test™ polystyrene micro-plate assay (96-well micro-test, Becton
Dickinson Labware, Lincoln Park, NI, USA). L. monocytogenes LSD535 was used instead
of strain 532 because it is less sensitive to divergicin M35 (Zouhir et al., 2008). This allows
a better study of the interaction (synergetic or additive effects) between chitosan and
divergicin M35. Mid-log-phase culture diluted (1/10,000) in fresh TSBYE was shown to
provide an initial- 1-5x105 CFU/well. Two-fold seriaI dilutions of chitosan or divergicin
M35 solution were made by transferring 100 III in micro-plate wells previously filled with
100 III of TSYEB. Each well then received 25 J.!l of diluted culture as described by Mota-
Meira et al. 2000. The micro-plates were incubated at 37°C for 16 h and the OD was read at
650 nm using the Thermo-max microplate reader (Molecular Devices, Opti-Resources,
Charny, PQ, Canada). Control wells (inoculated with the tested culture without added
inhibitor) and blank wells (containing un-inoculated broth with added inhibitor) were
included~ The MIC corresponds to the lowest concentration of tested inhibitor giving
complete inhibition of detectable growth (OD equal to OD of blank). The micro-dilution
assay was repeated three times and results were presented as the median of the three
repetitions.
The antimicrobial activity of chitosanldivergicin M35 combinations was measured against
L. monocytogenes .LSD 535 in 96-well polystyrene micro-plates by calculating the FIC
51
index using the checkerboard method. Briefly, each weIl contained 120 ~l of TSBYE
medium containing the antimicrobial combinations (60 ~l of chitosan and 60 ~l divergicin
M35) and 120 ~l of diluted bacterial suspension were added to each weIl.
MICchitosanlMICdivergicin M35 combinations tested were 0.0/0.0, 0.06/0.06, 0.12/0.12,
0.25/0.25, 0.5/0.5, 1.0/1.0, 1.0/0.0 and 0.0/1.0. The microplates were incubated at 37°C for
24h and the optical densities were measured at 650 nm with a Thermo-max microplate
reader.
The FIC of antimicrobial agent X was calculated as follows (Barchiesi et al. 2001):
FIC x = (MIC of agent X in combination)/(MIC of agent X alone)
The FIC index is the sum of the FIC of the agents in the combination (~ FIC = FIC A +
FIC B). The interaction is synergistic if the FIC index is< 0.5, additive if 0.5 < FIC index
::;1.0, indifferent if 1.0 < FIC index::; 4.0, and antagonistic if the FIC index> 4.0. The FIC
index determination was repeated independently three times.
II. 5. RESUL TS
Figure ILl and Table ILl show the inhibitory activity of divergicin M35 at different
stéps of purification (culture supernatant, SP Sepharose eluate and Sep-Pack C18 eluate).
Compared with a specific activityat initial step of purification, the specifie activity in SP
Sepharose eluate was increased by 4 folds and 25% of bacteriocin was eluted at this step.
The purification using column Sep-Pack C18 increased the specific activity by 5500 folds
compared with &pecific activity in supernatant culture and 96% of divergicin M35 was
eluted at this step.
52
The effect of pH on antilisterial activity of chitosan was evaluated using the agar-weIl
diffusion method. Antibacterial activity of chitosan was affected by pH with the most
significant activity being observed at lower pH values (4.5 and 5) (data not shown).
Three chitosan MW, namely 2, 20 and 100 KDa, were selected using the agar-weIl
diffusion method because of their high inhibitory activity at pH · 4.5 against L.
monocytogenes (figure 11.2). Table II.2 shows the effects of chitosan on L. monocytogenes
growth. Although the chitosans markedly inhibited the growth of most of the L.
monocytogenes strains tested, DDA seems to have a slight influence on the inhibitory effect
of low, medium or high MW chitosan based on log reduction. For example, chitosan 100
and 20 KDa with DDA vàlue of 87.4% seem to be more active than the other chitosan
preparations. Figure 11.3 shows growth curve of L. monocytogenes in the absence and in the
presence of chitosan with selected MW. A complete inhibition of L. monocytogenes was
obtained during the first 10 hours with 2 kDa chitosan, after which a substantial growth was
observed, between 10 and 24 h. Using 20 and 100 kDa chitosans, complete inhibition of L.
monocytogenes was obtained until 30 h of incubation.
Figure 11.4 shows scanning electron micrographs of L. monocytogenes strain LSD 532
incubated in the presence and in the absence of 2, 20 and 100 KDa chitosans (87.4% DDA)
at a concentration of 0.1 %. Figure 11.5 shows the same L. monocytogenes preparations
examined by transmission electron microscopy. In micrographs of L. monocytogenes
treated with 2 KDa chitosan, cells display thickened walls by interaction between chitosan
and cell wallleads to the leakage of intracellular constituents. In contrast, cells treated with
20 and 100 KDa chitosan show that chitosan form a layer on the cell surface and prevent
entry of nutrients through the cell wall.
53
II. 5. 4. MIe of chitosan and divergicin M35 and evaluation of the sensitivity of L.
monocytogenes to their combination
The MIes of divergicin M35 and chitosan 2, 20 and 100 KDa with 87.4% DDA for the
t L. monocytogenes strain LSD 535 as determined by the micro-
divergicin M35-resistanO
dilution method are shown in Table II.3. These values indicate that 100 KDa chitosan,
showing a MIe of 0.625 mg/ml, is more inhibitor than 2 and 20 KDa chitosan, with MIes
of 2.5 mg/ml, suggesting increasing antimicrobial activity with increasing MW. On the
other hand, the MIe value of divergicin M35 against L. monocytogenes LSD 535 was 0.25
mg/ml.
The results of the combinations study of chitosan 2, 20 and 100 kDa with 87.4% DDA are
given in table II.3. The combinations of chitosan and divergicin M35 appear to be additive,
regardless of chitosan MW. The inhibitory effect was obtained with concentrations of half
MIe values of divergicin M35 and chitosan.
II. 6. DISCUSSION
In this study, the method used for the purification of divergicin M35 showed that specifie
activity of divergicin M35 in the last step of purification was increased by 5500 folds
compared with specifie activity in supernatant culture and divergicin M35 was eluted after
purification with a high degree of purity (96%). This high degree of purity was essential for
the rest of our study, especially for the determination of MIe values as weIl as for the
investigation of the mechanisms involved in the inhibitory activity. In general, our results
are in good agreement with those reported by Tahiri et al. (2004).
The characterization of the antimicrobial acti vit y of chitosan has showed that this
biological activity is affected by pH. Our result revealed that activity against L.
monocytogenes was greater at lower pH values (4.5 and 5), which may be related to the
high solubility of chitosan at these pH values. No et al. (2002), evaluating the effect of
54
high-MW chitosans (470, 746 and 1,671 KDa) on L. monocytogenes as a function of pH,
concluded that growth of L. monocytogenes was suppressed by chitosan at or below pH 5.5.
Qualitative analysis of the antimicrobial activity of chitosan of different MW revealed that
the nature of this activity is MW dependent, the activities observed with 2,20 and 100 KDa
differing visibly, although all markedly inhibited the growth of various isolates of L.
monocytogens. Studying the effects of chitosan of MW varying from 1 to 10 KDa on
different Gram-negative and Gram-positive bacteria, Jeon et al. (2001) observed that MW
of 10 KDa or more was required for inhibition. No et al. (2002) investigated the
antibacterial activity of chitosan oligomers of MW ranging from 1 to 22 KDa against
various bacteria and found that 2 and 4 KDa showed relatively higher antimicrobial activity
against Gram-positive bacteria. Ueno et al. (1997) have reported that chitosan with MW
less than 2.2 KDa has a little antimicrobial activity compared with chitosan with MW of
around 5.5 KDa. Differences in effects of chitosan MW reported in the literature are
probably due to differences in types of bacteria (Gram-positive or Gram-negative) strains
tested, characteristics and concentration of the chitosan applied.
In this work, we have also evaluated the impact of DDA on the antimicrobial activity of
chitosan. Our result~ revealed that DDA does not have a major influence on the inhibitory
effect of low, medium or high MW chitosan ·against L. monocytogenes. Results also suggest
.that the antimicrobial activity of chitosan is not proportional to its DDA. This is not in
agreement with the findings of Liu et al. (2001) obtained ·with DDA ranging between 74
and 96% and which suggested that the antimicrobial activity of chitosan against E. coli
increases with increasing the DDA. However, our finding is supported by those of Park et
al. (2004), who reported that chitosan with 75% DDA showed higher antimicrobial activity
against Gram-positive and Gram-negative bacteria than chitosan with 90% and 50% DDA.
The growth of L. monocytogenes in the presence or absence of 2,20 and 100 KDa chitosan
showed that low-MW 2 KDa might have a bacteriostatic effect in contrast with medium
and higher-MW, which exhibited more bactericidal effect. This suggests that the
mechanism of antimicrobial activity of chitosan depends on its MW and that low-MW
chitosan act more like many molecules in low antimicrobial peptide family. This hypothesis
is supported by our scanning and transmission electron microscopie examination of L.
55
monocytogenes. Indeed, our study suggests that two mechanisms of action are possible,
depending -on chitosan MW. At 2 KDa, chitosan interacts via its positively charged arnino
groups with negative charges on the rnicrobial cell, increasing cell permeability by creating
pores in the cell wall and leading to cell death. In contrast, at 20 and 100 KDa, chitosan
seems to exercise its antirnicrobial action by forrning a thin layer on the cell surface, which
prevents the entry of essential nutrients and consequently causes cell death. Until recently,
the mechanism of the antirnicrobial activity of chitosan had not been clearly elucidated and
different mechanisms have been proposed in the literature (Chen et al., 1998; Shahidi et al. ,
1999; Rabea et al., 2003). However, all these mechanisms have remained theoretical and
have never been demonstrated experimentally using electron microscopy on food-borne
pathogens such as L. monocytogenes. Our study provides clear evidence of a speci fic
mechanism of the antimicrobial activity of chitosan and this mechanism varies
fundamentaIly with the MW.
FinaIly, we have evaluated possible synergistic effects between chitosan oligomers and
divergicin M35 using the FIC index. Our hypothesis is that the combination of chitosan and
divergicin M35 should inhibit L. monocytogenes in a synergistic or at least additive
manner. According to our resuits, this combination results in an additive inhibitory effect,
as indicated by a two-fold decrease in the MIC of each antimicrobial agent in combination.
This additive effect was obtained with aIl three chitosan MW tested.
56
Although we did not investigate the mechanism by which one agent can influence the
activity of the other, we speculate on the basis of the kilown mechanisms of divergicin .M35
and chitosan used in this study that using 2 KDa chitosan, the divergicin M35/chitosan
combination can act by simultaneously increasing cell permeability by creating pores in the
bacterial cell wall envelope. However, using 20 or 100 KDa chitosan, divergicin M35
forms pores and chitosan subsequently enhances this activity by forming a wall on the cell
surface, which pre vents the entry of essential nutrients.
To the best of our knowledge, this is the first study reporting the characterization of the
interaction between chitosan and a bacteriocin for inhibiting food bacteria. Also, this is the
first study using the FIC index to show an additive effect between chitosan and divergicin
M35. Results with other bacteriocins such as nisin and pediocin may corroborate our
findings and could have significant applications in the food sector.
We have shown in this work that chitosan displays very interesting antimicrobial activity
against L. monocytogenes and that this antibacterial activity depends on parameters .such as
the MW and the pH. We have also shown that the mechanism of the antibacterial activity
may vary, depending on the MW of chitosan. Finally, an additive inhibitory effect against
L. monocytogenes was observed when a combination of chitosan of any of the three MW
tested and the bacteriocin divergicin M35 was used.
Theseoriginal results may be very helpful for the development of active bio-films of
chitosan and divergicin M35 and their use for inhibiting L. monocytogenes in foods.
II. 7. ACKNOWLEDGMENTS
This research was supported by grants from the Conseil des Recherches en·Pêche et
en Agro-alimentaire du Québec (CORPAQ) and the Ministère de l'Agriculture, des
Pêcheries et de l'Alimentation du Québec (MAPAQ).
Table II. 1: Divergicin M35 activity at different stages of purification . .
eluate
C18 eluate
a Activity (AU/ml) determined by microtiter plate assay and multiplied by the volume in
millimeters.
b Specifie activity= Total activity (AU)/Total protein (mg).
58
MW (KDa) DDA(%)
20 80 TR TR TR 6.2
87.4 TR TR TR 4.56
87.4 TR TR TR TR
94.7 TR TR TR 5.18
Table II. 3: MIC of divergicin M35, chitosan and their combinations against divergicin-
M35-resistant L. monocytogenes strain LSD 535.
/ divergicin M35
/ divergicin M35
/ di vergicin M35
Figure II. 1: Agar-weB diffusion test showing inhibition of ·L. innocua HPB 13 by
divergicin M35 from C. divergens M35 culture. (A) supernatant, (B) 1.5% sodium chloride
eluate from SP-Sepharose column, (C) eluate from Sep-Pack C 18 column with 50%
acetonitrile, and (D) Sep-Pack C 18 eluate after evaporation of acetonitrile.
61
Figure II. 2: Agar-weIl diffusion test showing inhibition of L. monocytogenes LGM 6902
by different types of chitosan. (a) 2 KDa, DDA 80%, (b) 2 KDa, DDA 87.4%; (c) 2 KDa,
DDA 94.7%; (d) 20 KDa, DDA 80%; (e) 20 KDa, DDA 87.4%; (f) 20 KDa, DDA 94.7%;
(g) 100 KDa, DDA 80%; (h) 100 KDa, DDA 87.4%; (i) 100 KDa, DDA 94.7%.
62
1.20 A
1.00
0.80
0.60
0.40
0.20
0.00
1.20 B
1.00
on 0.80
--+-- Control
--0- Ch 80%
0.60 ---.- Ch 87.4%
~Ch 94.70/0 DDA
0.40
0.20
0.00
1.20 C
1.00
0.80
0.60
0.40
0.20
0.00
2 3 4 5 6 7 8 9 10 24 30
Time {hl
Figure II. 3: Growth of L. monocytogenes LSD 532 in the presence or absence of chitosan,
as measured by optical density at 650 nm.
(A) 2 KDa, (B) 20 KDa, (C) 100 KDa, Ch: chitosan
63
'Figure II. 4: Scanning electron micrographs showing the action of chitosan against
L. monocytogenes LSD 532. (a) untreated cells; (b) treated with 2 KDa, DDA 87.4%;
(c) treated with 20 KDa; DDA 87.4% and (d) with 100 KDa, DDA 87.4%.
Short arrow: listerial cell; long arrows: interaction between chitosan and listerial cell
64
CHAPITRE III
a- Dairy Research Center STELA, Pavillon Paul Comtois, Université Laval, Québec, PQ,
111.1. RÉSUMÉ
Différents types de films à base de chitosane et de divergicine M35 ont été developpés et
caractérisés sur les plans physicochimique et biologique. Pour ce faire, la divergicine M35,
une bactériocine produite par Carnobacterium divergens, a étéîncorporée dans des films de
chitosane ayant un poids moJéculaire de 2, 20 et 100 KDa et un degré de désacétylation
(DDA) de 87.4% ou de 94.7%. Seuls les films de chitosane ayant un poids moléçulaire de
100 KDa ont été retenus pour leurs propriétés filmogènes intéressantes. Nos résultats ont
montré que la couleur (~E) des films incorporant la bactériocine ainsi que leur force de
rupture et leur perméabilité à la vapeur d'eau varient en fonction du degré de
désacétylation. L'analyse par spectroscopie infrarouge à transformée de Fourier a démontré
que l'incorporation de la divergicine M35 dans les films augmente l'i1)tensité du pic de
l'amide 1 sans toutefois engendrer des changements significatifs au niveau de la structure
des films. L'analyse de la structure de la divergicine M35 libérée a montré un profil
similaire à celui de la bactériocine pure avec seulement une différence au niveau de
l'intensité des pics. L'étude de la libération de la divergicine M35 à partir des films a été
réalisée à l'aide des cellules de Franz. Les résultats obtenus ont montré une libération
graduelle de la divergicine M35 à partir des films de chitosane durant les la ou les 12
premières heures d'incubation dépendamment du degré de désacétylation et de la solution
de libération. L'étude de la libération a permis également de démontrer que l'incorporation
de la divergicine M35 dans les films de chitosane augmentait l'activité antimicrobienne de
ce dernier. Les meilleurs résultats ont été obtenus avec le chitosane avec un DDA de
94,7%.
111.2. ABSTRACT
Various formulations of chitosan film loaded with bacteriocin were developed, examined
for physicochemical and biological properties. Divergicin M35, a bacteriocin produced by
Camobacterium divergens, was incorporated into films made with 2 kDa, 20 kDa and 100
kDa chitosans deacylated either 87.4% or 94.7%. Only 100 kDa chitosan yielded films that
could be peeled and handled easily. The higher degree of deacetylation increased the total
color factor (~E) of bacteriocin-Ioaded films, their puncture strength and decreased their
water vapor permeability. Incorporation of divergicin M35 into the films increased amide 1
peak intensity but otherwise did not induce significant structural change. The FTIR spectra
of divergicin M35 shed from the films did not differ from those of the free bacteriocin,
except in peak intensity. The release kenetics of divergicin M35 from the chitosan films in
phosphate buffer and in tryptic soy broth yeast extract (TSBYE) medium was conducted
with vertical Franz cells. The results showed a graduaI release of divergicin M35 from
chitosan films between a and la or 12 hours of incubation depending on the DDA of
chitosan and the liberation solution. The two methods used to study the divergicin M35
release display different profiles on antimicrobial activity and revealed that the
incorporation of divergicin M35 into the chitosan films led to an important increase in its
antimicrobial activity. The best results were obtained with chitosan 94.7% degree of
deacetylation.
III. 3. INTRODUCTION
Ready-to-eat products such as seafood are considered high-risk for consumers because they
are capable of supporting the growth of food pathogens su ch as Listeria monocytogenes.
This rnicroorganism can affect pregnant women, fetuses, newborn infants and persons with
compromised immune systems (Rocourt et al., 2000; Katla et al., 2001). Nevertheless,
consumer demand for foods in the ready-to-eat, minimally processed and chernical-
preservative-free categories is increasing. The food industry is therefore seeking new and
efficient methods of inhibiting pathogens in these high-risk products. Among the emerging
approaches, food bio-preservation has drawn a lot of interest during the past few years and
has been proposed as a new alternative that satisfies consumers and meets industry
requirements . .
The principle of food bio-preservation is based on the use of lactic acid bacteria or their
metabolites (organic acids, diacetyl, hydrogen peroxide and bacteriocins) to control food-
borne microbial pathogens and spoilage organisms in foods. Bacteriocins produced by
lactic acid bacteria are ribosommaly synthesized peptides or proteins (Jack et al., 1995) and
inhibit microorganisms of similar phylogenetic origin. They are generaHy hypoallergenic
and degraded by enzymes in the human intestinal tract (Coma, 2008). Class lIa bacteriocins
have attracted much attention due to their potential application as natural preservatives
(Drider et al., 2006). The characteristic of class lIa bacteriocins is their anti-listerial activity
which offer an interesting preservation alternative in ready to eat products.
The efficacy of bacteriocins depends on several factors, such as how they are incorporated
into the food matrix and their interaction with the various food compounds (Benech et al.,
2002; Verluyten et al., 2004ab; Leroy and De Vuyst, 2005). Bacteriocins can be produced
in situ when the producer strain is added directly to the food matrix. This strategy has
shown a relative efficacy in different kind of foods. However, it still depends on the
capacity of the bacteriocin producing strain to proliferate and to produce the active peptide
in the food matrix (Gàlvez et al., 2007). Another strategy consists of adding the purified or
69
partially purified bacteriocin directly to the food. This method is limited by different factors
such as interaction of bacteriocins with food constituents, food pH, proteolytic activity and
other factors (Katla et al., 2001; Roller et al., 2002; Aasen et al., 2003). Another approach
is to immobilize bacteriocins in a solid support such as a pol ymer film. This new approach
offers several advantages, primarily better protection of the active peptide from inhibitors
by preventing its interaction with the food matrix, maintenance of a high concentration of
bacteriocin on the food surface due to slow and continuous release during food storage and
synergistic effects with antimicrobial polymers such as chitosan (Cutter et al., 2001; Coma
et al., 2002; Pranoto et al., 2005).
films and to study the release kinetics of divergicin M35 therefrom in different liquid
models.
III. 4. 1. Material
Chitosans of 2, 20 and 100 kDa mass and 87.4% or 94.7% deacetylation (degree of
deacetylation or DDA) were obtained from DNP Canada inc. (Granby, QC, Canada). AH
were provided in 1% (v/v) acetic acid and stored at -20°C until use.
Divergicin M35 was produced and purified using a protocol similar to that previously
described by Tahiri et al. (2004) but adapted for higher culture supernatant volume.
L. monocytogenes strain LSD532 was obtained from the Laboratory Services Division,
Canadian Food Inspection Agency (Ottawa, ON, Canada). Listeria innocua HPB13 (L.
innocua), used as indicator strain, was obtained from Health Protection Branch, Health and
Welfare Canada (Ottawa, ON, Canada). Both strains were maintained as 20% glycerol
stock at -80°C and grown aerobically at 30°C for L. innocua and 37°C for L.
monocytogenes in tryptic soy broth (Difco Laboratories, Sparks, MD) supplemented with
0.6% (w/v) yeast extract (TSBYE).
Divergicin M35 at a final concentration of 0.125 mg/ml was incorporated into the solutions
of chitosan. The antimicrobial activity of the films against L. monocytogenes 532 was
determined using . the agar diffusion method. Films were cut into 1 x 1 cm squares and
placed on the solid surface of 0.75% agar-containing medium seeded at 45°C with the
bacterium at a concentration of
5 x 106 CFU per ml. The plates were incubated aerobically at 37°C for 18 h and were
visually examined for the presence of inhibition zones around the film. The films with
acceptable physical nature and significant antimicrobial activity were used for the rest of
the study.
Film thickness was determined using a Digimatic micrometer (Mitutoyo, Japan). Five
measurements were made on each film to obtain an average, ·four around the perimeter and
one at the centre. This average was used for the water vapor permeability calculation.
Total color difference of the films was estimated by calculating the ~E value from a, band
L values measured using a Minolta Chroma Meter CR-300 using the CIE (International
Commission on lliumination) color system (CIE 1996).
Four measurements were performed on each of three samples per film and the mean value
was used for statistical analyses. The white plate was used as the color reference. ~E was
calculated by formula used by Pranoto et al. (2005):
(a = green to red, b = yellow to blue, L = whiteness) a*, b* and L* are the values of the
white plate; a, band Lare measured values of films.
72
Water vapor permeability (WVP) of the films was determined at 5°C using a modified
ASTM procedure. The film area sealed over the measurement cup was 7.92 cm2 and
moisture permeation into the cup was measured using anhydrous calcium sulfate. The cups
were placed in the chamber maintained at 75, 85 and 97% relative humidity and weighed
every 24h. WVP was then calculated when the weight of water absorbed was constant.
Puncture strength is the maximal force sustainable by the film before rupture by a piston
(3.6 mm in diameter) of standard cross-sectional area (3.14 cm2) at a constant speed of 0.5
mmJs. It was determined at 5°C and 75, 85 and 97% relative humidity using a Texture
Analyzer TA.XT2 (Stable Micro Systems, Texture Technologies Corps., Scarsdale, NY,
USA).
Infrared spectra were recorded with a Magma IR-560 Nicolet FTIR spectrometer equipped
with a mercury-cadmium-telluride detector (MCT). The sample chamber was continuously
purged with dry, carbon dioxide-free air. Spectra in the presence and absence of divergicin
73
M35 were evaluated by attenuated total reflection (ATR) using a single refraction accessory
(Harrick Scientific, USA) fitted with a Zinc selenide prism were films were pressed.
Divergicin M35 structure was evaluated before and after its release from films in buffer.
The protein solution was placed in a cell with BaF2 windows separated by 6-~m
polyethylene terephthalate spacers. Each spectrum is the result of an average of 128 scans
and apodized with a Happ-Genzel function. For the study of the amide 1 region of the
protein, subtraction of the buffer spectra contribution and Fourier self-deconvolution with a
resolution enhancement of 2 and a bandwidth of 18 cm- 1 were performed using the software
provided with the spectrophotometer (Omnic software). Water vapor subtraction was
carried out when required.
Bacteriocin release studies were conducted using vertical Franz diffusion cells with a 5.4
ml capacity receptor compartment. A circular disc of film was clamped between the
receptor and donor compartments and the disc surface area exposed to the release medium
was 0.38 cm2 . The receptor compartment was filled with aqueous buffer at pH 6 stirred .
constantly with a magnetic stirring bar and maintained at 30°C by circulating water through
the jacket of the lower compartment. Seven cells, corresponding to 2, 4, 6, 8, 10, 12 and
24h, were used. At each time interval, the entire receptor volume was withdrawn and the
divergicin M35 release into the medium was measured with L. innocua as indicator
organism using the critical dilution micro-method of Tahiri et al. (2004). Similar
experiments were done replacing the aqueous buffer by TSBYE medium previously
inoculated with L. innocua at a concentration of 105-106 CFU/ml. Viable bacterial counts
and residual bacteriocin activity were determined at 2, 4, 6, 8, 10, 12 and 24h. Cultures
were 10-fold serially diluted in peptone water (0.15%, w/v) and appropriate dilutions were
plated onto TSBYE agar. Plates were incubated aerobically at 30°C for 24 h and viable
counts were recorded as colony forming units (cfu) per ml of culture. For residual
bacteriocin activity determination, 4 ml of culture supernatant were separated by
centrifugation and heated for 10 min at 1000e prior to assay by the critical-dilution micro-
74
method. Similar assays were performed with ~hitosan films without divergicin M35 as a
control.
In a second method performed to study the release of divergicin M35 from chitosan films,
the films were immersed in TSBYE medium (5.4 ml) previously inoculated with 1% (v/v)
L. innocua at a concentration of 105-106 CFU/ml and stirred with magnetic stirring bar for
24h at 30°C. Listeria counts were determined as described previously.
Statistical analyses were performed with STATGRAPHICS plus 4.1 (Manugistics Inc.,
RockvilIe, MD, USA). Significant differences among mean values of each tested parame~er
were tested by analysis of variance. Treatment comparisons were performed using Fisher' s
least-significant differences (LSD) test with a P value of ~ 0.05 considered significant. AlI
experiments were repeated three times.
Chitosans of 100 kDa mass and 87.4% or 94.7% DDA (6.25 mg/ml) were selected because
they alIowed the formation of films with more acceptable physical properties (ease of
peeling) compared to 2 and 20 kDa. Figure III. lA shows an example of film prepared with
100 kDa chitosan.
The anti-listerial activity of 100 kDa chitosan film using agar diffusion is shown in Figure
III.IB. A clear zone of inhibition of L. monocytogenes was obtained around the film disc
indicating anti-listerial activity. Film without divergicin M35 produced a smaller inhibition _
zone. This is in agreement with results of Pranoto et al., (2005), who showed that high
molecular weight chitosan did not have any inhibitory activity against Gram (+) and Gram
(-) bacteria using agar diffusion method. When divergicin M35 was incorporated into
chitosan film, a clear inhibiton ' zone was obtained against L. monocytogenes probably due
75
ta the release of divergicin M35 and diffusion through agar medium resulting in a clear
inhibition zone around the film.
The color parameters L, a, b and the total color difference ~E of the films are shawn in
Table 111.1. Incorporation of divergicin M35 into 100 kDa, 87.4% DDA chitosan did not
affect the film transparency (L values) or ~E value significantly. However, 94.70/0 DDA
film was more colored with divergicin M35 than without. Pranoto et al., (2005) reported
that incorporating nisin into films of 900-1000 kDa and 95% DDA chitosans resulted in
increased ~E value. Our divergicin-Ioaded chitosans showed a significant increase (P <
0.05) in ~E value from 19.86 ta 24.76 for 87.4% ta 94.7% DDA, respectively. This may be
attributed ta interaction between divergicin M35 and amino groups present in higher
numbers in 94.7% DDA.
The WVP of different films is shawn in Table 111.2. In general, these values increased with
relative humidity (Kittur et al., 1998), which may be due ta the hydrophilic nature of
chitosan. The WVP also depended on the DDA, increasing at the higher value. Hwang et
al., (2003) found that the WVP of chitosan films (880 kDa) did not change over the range
of 65 ta 90% DDA. WVP values were lower when divergicin M35 was added ta chitosan at
87.4% DDA but the difference was not statistically significant. At 94.7% DDA, the
addition of divergicin M35 resulted in a significant decrease in WVP (P < 0.05), which
might be due ta greater interaction with the bacteriocin, producing a less relaxed polymer
network. Pranoto "et al., (2005) have shawn that nisin did not significantly affect WVP
when incorporated into high-molecular-weight and 95% DDA chitosan film at a
concentration of 51-153 IU/g but did sa at a concentration of 153,000 IU/g.
The puncture strength of the films is shawn in Figure 111.2. Film strength appears ta
increase with chitosan DDA, although these differences are not significant (P<0.05). The
increased strength value of films containing divergicin M35 may be attributed ta the
formation of inter-molecular interaction between chitosan NH3 + groups and functional
76
groups of divergicin M35, however the difference ofstrength value between chitosan films
containing divergicin M35 and chitosan films are not significant (P<0.05) in exception for
chitosan with 94.7% DDA at 95% RH. Pranoto et al., (2005) observed a change in
mechanical properties (tensile strength and elongation at break) after incorporation of nisin
into chitosan film with 95% DDA. Ku and Bin Song, (2007) observed that ni sin change.d
the mechanical properties of corn zein film by increasing the tensile strength.
The FfIR spectra of chitosan films with and without divergicin M35 are shown in Figure
111.3 from 1800 to 800 cm- 1• This region is characteristic for absorption bands of chitosan.
The peaks at 1030- 1155 cm-1 indicate stretching of the c-o bonds, while those at 1400 and
1550-1590 cm-1 correspond to carboxyl groups (-COO-) and ·amine groups (-NH2) (Pranoto
et al., 2005; Mathew et Abraham, 2008). The spectra indicate no major structural
difference in the chitosan filmafter incorporation of divergicin M35. However, the
divergicin M35 film showed increased peak intensity at 1636 cm- l corresponding to the
absorbance region of amide 1 due to divergicin M35. This is in agreement with the infrared
spectral results obtained by Pranoto et al., (2005) who reported that the incorporation of
nisin into chitosan·film showed an increase of pick intens'i ty at 1638 cm- l .
The FTIR. spectra of divergicin M35 between 1700 and 1600 cm- l before and after its
release from the chitosan films into buffer are shown in Figure 111.4. For pure divergicin,
the spectra are composed of four peaks at 1636, 1651, 1667 and 1683 cm-l, these bands are
usually associated to ~-sheet, helical structure and random coil, ~-turns and ~-turns
respectively (Byler and Susi., 1986; Arrondo et al., 1993; Gaussier .e t al., 2003). The
spectra of divergicin M35 after release showed the characteristic bands of pure divergicin
M35, but with a change in the intensity of the peaks after release, indicating a change of
. quantity of ~-sheet and (a-helix and random coil). The changes observed in the intensity of
the peaks of divergicin M35 after its release was probably due to conditions under which
divergicin was incorporated into chitosan films.
77
The kinetics of divergicin M35 release from the chitosan films into phosphate buffer and
TSBYE, measured as antibacterial activity, is shown in Figure III.5. In general, release was
graduaI between 0 and 10-12 hours of incubation, depending on the DDA of crutosan and
the liberation solution. The maximum release into buffer was measured after 10h of
incubation at 8.38 xl06 AU ml- 1 for both chitosanDDA levels. In the culture medium, the
activity reached a maximum at 10h for 94.7% DDA compared to 12h for 87.4% DDA,
although there was no significant difference between these activities. The final activities
measured at 24h ranged from 8.1xl03 AU ml-1 to 3.2 xl04 AU ml-1 and showed no
significant differences (P < 0.05). The changes observed in secondary structure of
divergicin M35 after release from chitosan films could be linked to the decrease of
divergicin M35 activity.
Based on these results, the divergicin M35-based film represents a high potential , for
application on food surfaces for the control of Listeria during storage. The rapid initial
release of bacteriocin from chitosan films in this study may be attributed to the high
hydrophilic nature of chitosan, which allows rapid entry of water into the film and therefore
a rapid release of the load. Chen et al., (1996) reporteâ this phenomenon for other
preservatives such as sodium benzoate and potassium sorbate.
The. effect of divergicin M35 released from chitosan films on L. innocua HPB 13 counts is
shown in Figure 111.6. The two methods used to study bacteriocin release display different
profiles. When the film without bacteriocin was immersed completely in the culture
medium (Figure 111.6A), an antimicrobial effect against L. innocua was observed during the
first four hours of incubation. No further reduction was observed after 4h, while at 24h, no
significant difference between L. innocua count in the presence or absence of chitosan film
was observed, regardless of DDA. These results confirm those reported by Tang et al.,
(2003). However, our finding brings more original information about the effect of
molecular weight and DDA on the antimicrobial activity of chitosan film. When chitosan
was used alone, our results showed that chitosan film with 87.4% DDA had higher
78
antimicrobial effect against L. innocua compared to 94.7% DDA. Park et al., (2004)
reported that 75% DDA showed higher antimicrobial activity than 90% and 50% DDA
against both Gram-positive·and Gram-negative bacteria.
When one chitosan film surface only was in contact with the TSYEB (Figure III.6B), the
application of different chitosan films resulted in reduction of L. innocua viable count
during the incubation compared with the control. This reduction was maintained during the
first 10 h of incubation with chitosan film 87.4% DDA incorporating divergicin M35 and
was significantly different after 24h with final count of 5.5x 102 cfu/ml compared to L.
innocua inoculated alone (3.43x 109 cfu/ml). When chitosan 94.7% DDA incorporating
divergicin M35 was applied, L. innocua viable counts decreased by 4.6 log cfu/ml during
the first 12h of treatment and there was no detectable L. innocua present after 24h of
incubation. On the other hand, the application of chitosan films without divergicin M35
reduce L. innocua count by 2.2 log (cfu/ml) after 8h and by 2.46 log (cfu/ml) after 6h of
incubation with the chitosan 94.7%DDA and chitosan 87.4% DDA respectively, after witch
cells continued their growth with the viable count smaller then control. Comparable results
were observed by Sanjurjo et al., (2006) for the performance of nisin incorporated into
starch and glycerol film against L. innocua, showing graduaI release of nisin during the
treatment.
79
III. 6. CONCLUSION
Chitosan films loaded with divergicin M35 display interesting mechanical, physical and
biological properties. The incorporation of divergicin M35 into the films increased their
antimicrobial effect to a great extent. The two methods used to study divergicin M35
release gave differing antimicrobial activity profiles. The divergicin-M35-loaded chitosan
films developed in this study have potential for application in food preservation. This kind
of film could be used to inhibit the growth of food pathogens su ch as L. monoytogenes in
ready-to-eat seafood, thus extending its shelf life.
III. 7. ACKNOWLEDGMENTS
We thank Stephen Davids for his critical reading of the manuscript. This research was
supported by the Conseil des Recherches en Pêches et en Agroalimentaire du Québec
(CORPAQ) and the Ministère de l'Agriculture, des Pêcheries et de l'Alimentation du
Québec (MAPAQ).
80
Table III. 2: Water vapor perme ab ility in g·cm-1sec-1pa-1 of chitosan films at different
relati ve humidity
75 85 97
DDA87.4% 1.52 ± 0.11 xl0-11etg 1.64 ± 0.20 xl0-11det 1.94 ± 0.20 xl0-11 be
87.40/0 + M35 1.36 ± 0.09 X10- 11 g 1.48 ± 0.08 xl0-1lfg 1.79 ± 0.02 Xl0-lled
DDA94.7% 2.09 ± 0.19 Xl0- llab 2.11 ± 0.05 X10- 11ab 2.22 ± 0.30 x 10-lla
94.7% +M35 1.41± 0.21 Xl0- 11g 1.43 ± 0.09 xl0-11g 1.68 ± 0.02 Xl0-11de
Figure III. 1: (A) Dried chitosan film (100kDa) ~ontaining divergicin M35.
(B) Agar diffusion test showing inhibition of L. monocytogenes 532 by (a) 100 kDa
chitosan film and (b) 100 kDa chitosan film loaded with divergicin M35.
83
50
z
:; 40
m
c
...,e 30
tn
e
.a 20
(.)
c
:::s
c.. 10
o
75 85 97
Relative humidity (%)
B
50
z
--- 40
~
m
c
~ 30
tn
.ae 20
(.)
c
~ 10
o
75 85 97
Relative humidity (%)
Figure III. 2: Puncture strength of chitosan films at different relative humidity (A) chitosan
87.4%DDA and (B) chitosan 94.7% DDA: chitosan ( ) and 'chitosan + M35 (II).
84
--.
:::J D
crS
or
()
c
ctS
..c
~
0 C
en
..c
«
\ 1 t , 1 1 t
Figure III. 3: The FTIR spectra of (A) chitosan film, 87.4% DDA anq (B) chitosan film,
94.7% DDA (C) chitosan film, 87.4% DDA + divergicin M35 and (D) chitosan film, 94.7%
DDA + divergicin M35.
85
1651
. 1636
1667 1652
1638
--:-- 1664
:::J
1683 1652
cci
'-'"
Q) 1638
U 1664
C
ctS
..0
~
0
en
..0
<{ A
c
B
Figure III. 4: The FTIR spectra of (A) divergicin M35 (B) divergicin M35 released from
chitosan film (87.4% DDA) and (C) divergicin M35 released from the chitosan film (94.7%
DDA).
86
1.00E+07
1.00E+06
-
E
1.00E+05
:5 1.00E+04
~
~
'$ 1.00E+03
~ 1.00E+02
1.00E+01
1.00E+00
2 4 6 8 10 12 24
Time (h)
Figure III. 5: Kinetics of divergicin M35 release from chitosan film into buffer: chitosan
87.4%DDA (<1 chitosan 94.7% DDA (0) and into growth medium: chitosan 87.4%DDA
(il) chitosan 94.7% DDA (0).
87
1.00E+10
A
1.00E+09
1.00E+08
1.00E+07
1.00E+06
E
Cl 1.00E+05
,.....
::::J
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
0 2 4 6 8 10 12 24
Time {hl
1.00E+10 B
1.00E+09
1.00E+08
1.00E+07
1.00E+06
E
.......
u 1.00E+05
,.....
::::J
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
0 2 4 6 8 10 12 24
Time (h)
Figure III. 6: Effect of divergicin M35 on the evolution of L. innocua HPB 13 viable count
in TSBYE at 30°C in the presence of (A) film completely immersed in the medium and (B)
film in contact with the medium on one surface: • ~ontrol ~ chitosan 94.7% DDA + M35
~ . chitosan 94.7% DDA rn chitosan 87.4%+ M35 ra chitosan 87.4%.
88
CHAPITRE IV
Running title
Anti-Listeria monocytogenes film
Institut des Nutraceutiques et des Aliments Fonctionnels, Université Laval, Sainte-Foy,
Québec, G 1V OA6
a- Dairy Research Center STELA, Pavillon Paul Comtois, Université Laval, Québec, PQ,
Canada, G lK 7P4
IV. 1. RÉSUMÉ
Des filets de saumon fumé à froid (SFF) inoculés avec Listeria monocytogenes ont été
traités avec la combinaison chitosane/divergicine M35, emballés et entreposés pendant trois
semaines à 4-8°C. Les filets ont été analysés par le suivi du compte cellulaire total, des
bactéries lactiques et de Listeria, de la couleur, de la texture et de la teneur en bases azotées
volatiles totales. La combinaison chitosane/divergicine M35 a été appliquée sur les
échantillons de SFF sous la forme d' une solution filmogène ou sous la forme d ' un film.
Nos résultats ont montré que l'application du film a réduit le nombre de cellules de L.
monocytogenes à un niveau inférieur à la limite de détection et que le compte cellulaire
total était inférieur à 104 ufc/g comparativement à 109 ufc/g pour les échantillons non
traités. Une activité anti-Listeria beaucoup moins marquée a été obtenue avec la solution
filmogène. L'activité inhibitrice du film contre L. monocytogenes a été maintenue durant
les 21 jours d'entreposage, comparativement à 14 jours pour la solution filmogène. De
plus, l'utilisation du film a permis une meilleure préservation de la couleur du SSF, a
augmenté la fermeté des filets de SFF et a maintenu le niveau des bases azotées volatiles
totales à un niveau inférieur à celui du contrôle. L'application de la solution filmogène n ' a
pas eu d'impact majeur sur les caractéristiques du SSF. Nos résultats démontrent clairement
que le film à base de chitosane et de la divergicine M35 peut être une alternative
prometteuse pour la biopréservation du poisson fumé à froid.
Mots clés: Listeria monocytogenes, divergicine M35, film de chitosane, saumon fumé à
froid.
90
IV. 2. ABSTRACT
Cold-smoked wild salmon inoculated with Listeria monocytogenes and treated with a
combination of chitosan and the bacteriocin (divergicin M35) was examined for microbial
status, color, texture, total volatile basic nitrogen and residual inhibitory activity over a
storage period of three-weeks at 4-8°C. Solution containing 100 kDa chitosan deacetylated
94.7% and divergicin M35 was spreadon the fish surface or applied as a dried film. The
film reduced L. monocytogenes to below detectable levels and kept total counts below 104
per g compared to 109 in control samples while the effectiveness of the solution was very
limited. The inhibition activity was maintained over three weeks when film was used, while
extract of samples treated with solution did not show a reduction of L. monocytogenes
count after 14 days of storage. The film provided the best preservation of sample redness,
enhanced the firmness of fillets and kept volatile basic nitrogen below the control value,
while the solution had little distinguishable impact on color, texture or volatile basic
nitrogen compared to control samples. Divergicin-Ioaded chitosan film thus may represent
an interesting alternative for the biopreservation of cold-smoked fish.
Key words: Listeria monocytogenes, divergicin M35, chitosan film, cold-smoked wild
salmon.
91
IV. 3. INTRODUCTION
Recently, bio-preservation has been proposed as a new approach to improve food quality
and safety. This new approach is based on using lactic acid bacteria and their antimicrobial
compounds including bacteriocins to control spoilage organisms and pathogens such as L.
monocytogenes in food products (Nillson et al., 1997). Among the strategies used for the
incorporation of bacteriocins into food matrices are direct inoculation of the producing
strain, adding purified or semi-purified bacteriocin and immobilizing purified bacteriocin
on solid supports such as polymer films. This latter approach offers several advantages over
the other two, primarily better protection of the active peptide from inhibitors by decreasing
its interaction with the food matrix, maintenance of a high concentration on the food
surface, slow and continuous release during food storage and synergistic effects with
antimicrobial polymers such as chitosan (Coma et al., 2001; Coma et al., 2002; Cutter et al.,
2001; Pranoto et al., 2005)
92
In a previous study, our group purified and characterized a new class lIa bacteriocin
(divergicin M35) with strong activity against L. monocytogenes, produced by a strain of
Carnobacterium divergens M35 isolated from frozen mussels (Tahiri et al., 2004). We
subsequently developed an antimicrobial film by incorporating divergicin M35 into a
chitosan matrix and studied the release of the bacteriocin into phosphate buffer and tryptic
soy broth. In the present work, we evaluate the potential of this antimicrobial film to inhibit
the growth of L. monocytogenes on cold-smoked salmon refrigerated for three weeks and
examine the impact of the film on the ph ysical and chemical properties of this seafood
product.
93
IV. 4. 1. Materials
Chitosan with a molecular weight of 100 kDa and deacetylated 94.7% was obtained from
DNP Canada Inc. (Granby, QC, Canada). The material was provided dissolved in 1% (v/v)
acetic acid and stored at -20°C until use.
Divergicin M35 was produced and purified using a protocol similar to that described
previously by Tahiri et al., (2004) but adapted for higher culture supernatant volume.
L. monocytogenes strain LSD532 was obtained from the Laboratory Services Division,
Canadian Food Inspection Agency (Ottawa, ON, Canada). Listeria innocua HPB13 (L.
innocua), used as indicator strain, was obtained from Health Protection Branch, Health and
Welfare Canada (Ottawa, ON, Canada). Both strains were maintained as 20% glycerol
stock at -80°C and grown aerobically at 30°C for L. innocua and 37°C for L.
monocytogenes in tryptic soy broth (Difco Laboratories, Sparks, MD) supplemented with
0.6% (w/v) yeast extract (TSBYE).
Cold-smoked wild Pacifie Sockeye salmon (CSWS) was provided by Fumoir Grizzly Inc.
(St-Augustin, Québec, Canada) and used immediately upon reception.
Chitosan-divergicin M35 film (C-M35 p) was prepared by pouring 145 ml of C-M35 s into
Petri dishes totaling 145 cm2 in surface area, for a distribution of 1 ml/cm2 . The liquid was
dried in a laminar flow hood for 72h at ambient temperature and the resulting film was
peeled and used' directly for each experiment. Chitosan and film without divergicin was
made for comparison. The antimicrobial activity of C-M35 s and C-M35 p against L.
monocytogenes LSD532 was confirmed using the agar diffusion method. Film was eut into
1 x 1 cm squares and placed on the solid surface of TSBYE containing 0.75% agar and
seeded at 45°C with L. monocytogenes 532 at 5 x 106 CFU per ml. For C-M35 s , 7-mm
diameter wells were eut in the solidified agar and filled with 80 ~l of tested solution. The
plates were incubated aerobically at 37°C for 18 h and examined for inhibition zones.
L. monocytogenes was grown in TSBYE at 37°C and cells were harvested by centrifugation
at 7,500 x g for 15 minat 4°C, washed twice with sterilized phosphate buffer saline (PBS ,
pH 7.2) and re-suspended in la ml of PBS. Dilutions in PBS were made to obtain a final
concentration of approximately 105 CFU/ml. Dilutions were plated on TSBYE plates and
incubated aerobically at 37°C for 24h before determining viable cell counts.
CSWS was eut into 3x3 cm squares (4 grams) and divided into six treatments as follows:
Treatment A: control samples
Treatment B: 100 ~l of L. monocytogenes suspension containing 105 cfu/ml were spread on
one side of each square for an inoculum of 2.5x 103 cfu/g of salmon and samples were dried
for la minutes in a laminar-flow biological safety cabinet.
Treatment C: 100 ~l of C-M35 s were spread on the L. monocytogenes-inoculated surface
and allowed to dry for la minutes in a laminar-flow biological safety cabinet.
2
Treatment D: samples after treatment B were covered with 9 cm of C-M35 p .
Treatment E: samples without L. monocytogenes were coated with 100 ~l of C-M35 s.
Treatment F: samples without L. monocytogenes were covered with a 9 cm2 square of C-
M35 F.
Samples of each treatment were produced in duplicate and wrapped in oxygen permeable
film obtained from Fumoir Grizzly inc. and incubated for 21 days under the conditions
suggested by AFNOR (Association Française de Normalisation NF V 45-065, 1997): 14
days at 4°C followed by seven days at 8°C. These conditions are designed to simulate
changes in temperature during storage and handling in commercial and domestic
environments.
Color, texture and total volatile basic nitrogen (TVBN) were deterrnined at 1, 7, 14 and 21
days of storage. Microbial composition, pH and antimicrobial activity were determined at
1, 3,7, 14, 21 days of storage.
Microbial analysis was determined by placing the whole 4 g sample in a sterile filtering
stomacher bag (Seward Medical, London, UK) and adding peptone water (0.1 % w/v) to
obtain a 1/10 dilution (i.e. 36 ml) . For treatment D, the C-M35 F film was removed first and
the films were processed the same way (in 36 ml of peptone water) for measurement of the
residual inhibition activity. Stomacherbag contents were homogenized for three minutes
using a Stomacher" 400 circulator (Seward, Therfford, Norfold, UK). The filtered
homogenate was 10-fold serially diluted in peptone water and dilutions were spread-plated
in duplicate on appropriate selective medium. For L. monocytogenes, CM0856 Listeria
selective medium supplemented with SR140 Listeria selective supplement (Oxoid Ltd.,
Basingstoke, Hampshire, England) was used and plates were incubated aerobically at 37°C
for 48-72h. Total lactic acid bacteria were enumerated on Nitrite Actidion Polymyxin
(NAP) agar (Davidson and Cronin, 1973). NAP medium consists of APT agar (Difco
Laboratories) supplemented with Polymyxin B (0.003 g/l) , cycloheximide (0.01 g/l) and
NaN03 (0.6 g/l), aIl obtained from Sigma-Aldrich (OakviIle, Ontario, Canada). Plates were
incubated aerobically at 25°C for 48 h. For the deterrnination of the total viable bacterial ·
counts, dilutions were spread-plated on Plate Count Agar (PCA, Difco Laboratories,
Sparks, MD) and incubated aerobically at 30°C for 48h.
96
Residual inhibitory activity was determined in both CSWS and C-M35 F homogenates
obtained from treatments C and D. Four milliliters of each homogenate were centrifuged
(7,500 x g for 15 min) at 4°C and supernatants were filtered through sterile syringe filters
with 0.45 ~m pore size (Corning, Germany). Supernatants were tested against L. innocua
HPB 13 using the qualitative agar diffusion method described above and a quantitative
critical dilution micro-method (Tahiri et al., 2004).
Physical and chemical analysis were carried for treatments A, E and F. CSWS color was
determined using a Minolta Chroma Meter CR-300 (Minolta Camera Co. Ltd., Osaka,
Japan) using the CIE color system (CIE, 1996) to de termine parameters L (lightness), a
(redness) and b (yellowness). Measurements were made after standardizing the instrument
with light source D and the measuring head was rotated 90° between duplicate
measurements at each position. Four measurements were performed on each of two
samples per treatment and the mean of the eight measurements was used for statistical
analyses.
Total volatile basic nitrogen (TVBN) production was determined by a distillation procedure
described by Pearson (1976) and the pH of the CSWS homogenate used for microbiological
analysis was measured.
Texture profile analysis was done on CSWS using a Texture Analyzer TA.XT2 (Stable
Micro Systems, Texture Technologies Corps, Scarsdale, NY, USA) with double
compression. Firmness was defined . as the peak force of the first compression and
cohesiveness was defined as the ratio of the total energy required for the second
compression to that of the first compression. The measurements were made using a 4-mm
diameter cylindrical plunger at a constant speed of 1 mm1s. Four measurements were
97
performed on each of two samples per treatment and the mean value was used for statistical
analyses.
Statistical analyses were performed with STATGRAPHICS plus 4.1 (Manugistics Inc.,
RockviIle, MD, USA). Significant differences among the treatment means of each
parameter were tested by analysis of variance. Treatment comparisons were performed
using Fi~her' s least-significant differences (LSD) test with a P value of :5 0.05 considered
significant.
IV. 5. RESULTS
the antimicrobial activity of C-M35 s and C-M35 F was confirmed using the agar diffusion
test (Figure IV.1). A clear zone of inhibition of L. monocytogenes LSD 532 was obtained
around the film and the weIl, confirming the anti-listerial activity.
Figure IV.2 represents the antimicrobial activity of C-M35 s and C-M35 F against L.
monocytogenes in CSWS stored at refrigerated temperature. On day 3, the inhibitory
solution (treatment C) resulted in a significant decrease in viable count of about 1 log
(cfu/g) of L. monocytogenes viable count as compared to no inhibitor and this difference o
remained the same throughout the storage. The C-M35 s did not show a reduction of L.
monocytogenes count after 14 days of storage. However, application of C-M35 F to CSWS
(treatment D) reduced the count to below the detection limit «50 cfu/g) from the first day
of storage.
Total aerobic counts on samples treated with C-M35 s and C-M35 F are shown in Figure
IV.3. Counts in the control (treatment A) were 3.3 log CFU/g on day 1 and finished at 9.4
98
log CFU/g after 21 days. The application of C-M35 s did not affect the total aerobic counts
on CSWS and no significant difference was observed compared to the control until the end
of storage. In contrast, the total aerobic counts in CSWS treated with C-M35 F were reduced
significantly, the difference between the C-M35 F treated CSWS and the control being 1.8
log and 5.8 log cycles by days 3 and 21 respectively.
The effect of C-M35 s and C-M35 F on totallactic acid bacteria is illustrated in Figure IV.4.
For aIl treatments except for C-M35 F, the total lactic acid bacteria counts increased
progressively during the 21 days of storage with no significant differences between
treatments, while counts were below the detection limit «50 cfu/g) throughout storage with
the C-M35 F .
Counts of L. monocytogenes, total aerobic counts and totallactic acid bacteria in C-M35 F
homogenate were zero, indicating that no viable cells remained attached to the film (data
not shown).
Residual inhibitory activity in CSWS treated with C-M35 s and C-M35 F and in C-M35 F
removed from CSWS is shown in Table IV.1. For the entire 21-day storage period, no
activity was obtained from C-M35 s-treated samples. However, residual activity was
detected in CSWS treated with C-M35 F, decreasing significantly from 4.61 x 103 AU to
5.76 X 102 AU over days 1 to 14 and ending up at a final activity of 2.3 x 103 AU on day
21. In comparison, a residual activity of 3.69 x 104AU was observed in the film removed
from CSWS after one day of storage, decreasing progressively over 14 days of storage and
then stabilizing at 4.61 x 103 AU through to day 21. CSWS treated with chitosan solution or
film without bacteriocin as controls indicated no activity, confirming that the residual
activity detected in the CSWS was due to divergicin M35 (results not shown).
99
The effect of C-M35 s and C-M35 p on the color values of un-inoculated CSWS is shawn in
Table IV.2. Differences in lightness (L) between control (treatment A) and C-M35 s-treated
CSWS (treatment E) were significant after 14 days of storage, although no significant
difference was observed after 21 days. The application of C-M35 p (treatment F) did not
produce any significant difference in the (L) value during the first week, although a
significant increase was observed thereafter. The differences in redness (a) values between
control and C-M35 s treated CSWS were not significant during the first week but later
became sa. For C-M35p-treated CSWS, redness increased significantly during the first
week compared ta the control, after which no significant difference was observed. The C-
M35 F and C-M35 s treatments respectively resulted in increased and decreased yellowness
(b) compared ta the control until 21 days of storage. Figure IV.5 shows the overall
appearance and difference in color between treatments E and F after 21 days of storage.
The difference in redness matches the measured (a) values.
Figure IV.6 shows the firmness and cohesiveness profiles of CSWS during storage at 4°C
for 14 days followed by 8°C for seven days. Firmness and cohesiveness increased in
general during the first week of storage. After seven days, the two texture profiles tended ta
decrease or remain constant and increased progressively or remained constant between days
14 and 21. Firmness increased in samples treated with C-M35 s, although no significant
difference was observed compared ta the control. For C-M35 p-treated CSWS; there was a
significant difference throughout the storage period in the firmness value compared to the
control. Cohesiveness was not significantly affected at any point by C-M35 s and for
samples treated with C-M35 F no significant difference was observed between CSWS after
14 days of storage after witch the cohesiveness value was significantly less then control.
The pH of C-M35 s-treated CSWS was constant, ranging between 5.9 and 6 during the 21
days of storage and no significant difference was observed compared ta the control (data
not shawn). The application of C-M35 p resulted in a decrease in pH ta 5.5 after one day of
storage and this value was maintained throughout the three weeks of storage. TVBN
100
production (results not shown) increased from 10.3 to 30.4 mg N/IOO g in C-M35 s-treated
samples over the 21 days ofstorage which was similar to the control (12.4 - 29.9 mg-N/I00
g). Application of C-M35 F resulted in a significantly less TVBN production, 17.5 mg-
NII00 g at the end of the 21 days of storage.
IV. 6. DISCUSSION
The combination of a natural polymer with antimicrobial activity, namely chitosan, with a
natural soluble inhibitor such as divergicin M35 is a plausible strategy for suppressing the
development of spoilage or pathogenic bacteria during the storage of ready-to-eat products
and one that may be acceptable to consumers. Our work has clearly shown that this
combination is an effective inhibitor of L. monocytogenes on cold-smoked salmon and
much more when film was applied. Application of C-M35 F showed that L. monocytogenes
counts in CSWS was lower than the detection limit «50 cfu/g) for 21 days of storage.
Since no L. monocytogenes were found in homogenate of the film, we can then conclu de
that the divergicin-Ioaded chitosan film provided complete inhibition of L. monocytogenes
in cold-smoked salmon. This inhibition was no doubt due to a continuous release of
divergicin M35, since the activity of chitosan alone was much lower. Previous studies have
shown that antimicrobial effects of active films in various food systems depend on . the
nature and concentration of the antimicrobial agent used. U sing chitosal1 as a coating on
plastic film, Ye et al. (2008b) obtained greater activity against L. monocytogenes on cold-
smoked salmon by application of chitosan-coated plastic film incorporating sodium lactate,
sodium lactate and ni sin or sodium lactate and potassium sorbate than with chitosan alone.
These investigators also observed higher activity against L. monocytogenes on ham steaks
101
The weak anti-listerial activity and absence of residual activity of the C-M35 s in CSWS
homogenate is likely due to interactions between bacteriocin and food components such as
lipids as well as partial inactivation by endogenous proteases produced in situ by other
microorganisms (Katla et al., 2001; Vaz-Velho et al., 2005). Aasen et al., (2003) showed
that more than 80% of the sakacin P added to cold-smoked salmon and chicken was
adsorbed to proteins. The antimicrobial activity of inhibitory substances may also be
affected by the presence of salt added to CSWS. Devlieghere et al., (2004b) observed that
adding NaCI to a medium diminished the antimicrobial activity of chitosan, due to
interaction between cr ions and positive charges on the chitosan and to competition of Na+
for the negative charges on the microbial cell surface and that antimicrobial activity also
depended on the isoelectric point of proteins in the food matrix and on the pH. These
phenomena probably affect bacteriocin activity, whether or not a filni is used. However,
continuous release of divergicin M35 during the storage period apparently minimized these
loss,es. Our results showed that 4.61 x 103 AU and 3.69 x 104 AU of divergicin M35
activity were found after one day of storage in homogenate of CSWS and its chitosan-
divergicin film respectively compared to an initial activity of 8 x 106 AU in the film. This
difference of divergicin M35 activity added and the activity recovered in the film and
CSWS is probably due to the phenomena previously explained.
The total aerobic and lactic acid bacteria counts as well as TVBN production suggest that
the antimicrobial sollltion was practically ineffective while the film form was quite
effective. Since initial levels of bacteria in this salmon product were low, the bacteriocin-
loaded chitosan film reduced these to below the limit of detectioll. Ye et al. (2008a) also
102
obtained a substantial reduction in total and anaerobic count using their chitosan coated
plastic film and loaded with antimicrobial agents. Our finding is also supported by Shahidi
et al. (1999); AguIlo et al. (2003) and Rinaudo (2006) who reported that chitosan was able
to inhibit the growth of a wide variety of micro-organisms. Yingyuad et al. (2006) have
reported a significant activity of chitosan against total plate count in vacuum package pork
coated with chitosan. FinaIly, Jeon et al. (2002) reported a reduction of total plate counts in
herring and Atlantic cod after application of chitosan coating prepared with glycerol as
plasticizer. The microbiological results obtained in our study demonstrate the potential of
divergicin-loaded chitosan film for extending the shelf life of CSWS.
In the present study, we were also interested in the impact of C-M35 s and C-M35 F on the
physical and chemical characteristics of CSWS. For aIl treatments, the increase in firmness
during the first week of storage is likely due to moisture loss, while changes during the
second week could be due to proteases produced by spoilage microorganisms in CSWS
(Morzel et al., 1997) or to autolytic enzymes in the fish flesh (Stoknes and Rustad, 1995).
The samples treated with C-M35 s increased in firmness, but with no significant difference
compared to the control. Yingyuad et al. (2006) reported that the application of chitosan
coating to grilled pork had no significant effect on texture compared to samples without
chitosan coating. AIso, Tahiri et al. (2008) observed that treating CSWS with divergicin
M35 had no significant effect on firmness or hardness. The significant differences observed
in CSWS covered with C-M35 F compared to control samples were probably related to the
strong affinity of chitosan for water molecules (Wu et al., 2004) through -NH2 groups. The
increase in firmness of C-M35 F-treated CSWS during the last week of storage may be due
to the onset of C-M35 F breakdown. Cohesiveness decreased slightly after treatment with C-
M35 F and significantly during the last week of storage compared to the control, which is in
relation with the changes in the muscle strength retenti on capacity after the first
compression.
Increased (L) values and decreased (a) and (b) values of C-M35 s-treated compared to
untreated CSWS are consistent with previous observations. Jo et al. (2001) also observed
similar changes in (L) and (a) values for sausage incorporating chitosan as a preservative.
103
Tahiri et al. (2008) reported a significant decrease of (b) value compared to controls after
21 days of storage of divergicin M35-treated CSWS at 4°C. The increased (a) value of C-
M35 F-treated samples is probably due to the low oxygen permeability of chitosan film (Xu
et al., 2005) and resulting decrease in oxidative breakdown of carotenoid pigments. The
loss of water from the samples may also result in increased pigment concentration
(Choubert et al., 1992).
For aIl treatments, the TVBN concentration was below the acceptable maximum proposed
by various authors (Leroi et al., 2001; Civera et al., 1995). The C-M35 F-treated samples
showed higher hygienic quality compared to the control and C-M35s-treated samples,
based on TVBN concentrations, which is considered a major quality index for cold-smoked
salmon (Brillet et al., 2005; Leroi et al., 2001). These low concentrations could be
explained by reduced counts of spoilage bacteria after C-M35 F application. J eon et al.
(2002) reported 33-50% and 26-51 % reductions of TVBN in chitosan-glycerol-coated cod
and herring respectively at the end of 12 days of storage at 4°C.
In this study, the application of C-M35 F was much more effective than C-M35 s to inhibite
L. monocytogenes in CSWS. The formulated film was shown to maintain acceptable color
and texture and extend the shelf life of refrigerated cold-smoked salmon through three
weeks of storage. Additional sensory quality studies using panels should be conducted
before commercial application of the film .
. IV. 7. ACKNOWLEDGMENTS
We thank Stephen Davids for his critical reading of the manuscript. This research was
supported by the Conseil des Recherches en Pêches et en Agroalimentaire du Québec
(CORPAQ) and the Ministère deI' Agriculture, des Pêcheries et de l'Alimentation du
Québec (MAPAQ).
~ Table IV. 1: Residual divergicin activity (AU) in homogenate of cold-smoked wild salmon (CSWS) and its chitosan-divergicin film
S covering after 14 days at 4°C followed by 7 days at 8°C, determined by agar diffusion tests and the critical dilution micro-method.
Day 1 3 7 14 21
Method a B a B a B a B a B
c Oa Oa Oa Oa
Table IV. 2: Color parameters L, a and b for cold smoked wild .salmon (CSWS) during
storage at 4°C for 14 days followed by 8°C for 7 days, A: control, E: CSWS coated with
chitosan-divergicin solution (C-M35 s), F: CSWS covered with chitosan-~ivergicin film
(C-M35 F).
Means ± sdt with different letters for the same color parameter are significantly different
(P <0.05)
106
Figure IV. 1: Agar diffusion test showing inhibition of L. monocytogenes 532 by (A)
chitosan-divergicin solution (C-M35 s) and (B) chitosan-divergicin film(C-M35 F).
107
6
a
5
b
4 a a
C)
.......
::l
. '0 3
C)
~
2
o
3 7 14 21
lime (days)
Figure IV. 3: Growth of total aerobic count in cold-smoked wild salmon (CSWS) during
storage at 4°C for 14 days and at SoC for 7 days: (~) un-inoculated control ( m )
inoculated CSWS with L. monocytogenes alone (treatment B), (II) inoculated CSWS
coated with chitosan-divergicin solution C-M35 s (treatment C), ( a) covered with
chitosan-divergicin film C-M35 F (treatment D).
*** The detection limit for viable cells was < 50 cfu/g.
Different letters at the same time point indicate a significant difference (P < 0.05).
109
a a
10 a
Cl 6
---
::J
~
U
Cl
~ 4
0
3 7 14 21
Time (days)
Figure IV. 4: Growth of totallactic acid bacteria count in cold-smoked wild salmon (CSWS)
during storage at 4°C for 14 days and at gOC ' for 7 days: (e:i) un-inoculated control ( ID )
inoculated CSWS with L. monocytogenes alone (treatment B), (II) inoculated CSWS coated
with chitosan-divergicin solution C-M35 s (treatment C), e ) covered with chitosan-divergicin
film C-M35 F (treatmènt D).
*** The detection limit for viable cells was < 50 cfu/g.
Different letters at the same time ooint indicate a significant difference (P < 0.05).
110
3.5
A
3
2.5
~
tn 2
tn
CI)
c:
E 1.5
a-
i!
0.5
0
6 11 16 21
Time (days)
0.6
U)
U)
~
Q)
.~ 0.55
U)
~
0
0.5
0.45 +-----------~----------~----------~------~--~
6 11 16 21
Time (days)
CHAPITRE V
CONCLUSION GÉNÉRALE
Au cours des dernières années, les toxi-infections d'origine alimentaire ont suscité
beaucoup de préocupations dans le secteur alimentaire ainsi que celui de la santé. Malgré
les efforts mis en place pour contrer ce fléau, le nombre de cas rapportés ne cesse
d'augmenter année après année et ce, même dans les pays développés où les conditions
d'hygiène sont souvent appliquées .avec beaucoup de rigueur. Plusieurs hypothèses ont été
avancées pour explique~ ce phénomène parmi lesquelles les changements importants au
niveau des habitudes alimentaires du consommateur notamment un engouement de plus en
plus marqué pour les produits frais tels que le saumon fumé. Malheureusement, ces
produits constituent de nos jours un des vecteurs de toxi-infections alimentaires les plus
redoutés. En effet, de par leur composition, les produits marins constituent un
environnement idéal pour la survie et la croissance de plusieurs microorganismes
indésirables incluant des pathogènes tels Listeria monocytogenes. Ce risque de
contamination est d'autant plus élevé que le mode de préparation de ces produits ne met en
jeu aucun traitement thermique en mesure de détruire d'éventuelles bactéries pathogènes
avant consommation. De plus, les tendances actuelles du marché forcent les producteurs à
réduire substantiellement le recours à des barrières microbiologiques classiques telles le
salage et les additifs chimiques en raison de leur impact négatif sur la santé du
consommateur.
L'ensemble des résultats obtenus dans la présente thèse a permis d'appuyer l'hypothèse de
recherche émise concernant l'exploitation simultanée des propriétés antimicrobiennes de la
divergicine M35 et du chitosane tout en utilisant les propriétés filmogènes de ce dernier
pour le développement d'un film biologiquement actif ayant un fort potentiel d'utilisation
113
Dans la première partie de la thèse, nous avons démontré que le chitosane et la divergicine
M35 ont une bonne activité antimicrobienne contre L. monocytogenes. Trois chitosanes
avec des poids moléculaires de 2, 20 et 100 KDa ont été choisis pour leur activité
antimicrobienne importante à pH 4.5. L'étude du mécanisme d'action du chitosane contre
L. monocytogenes a permis de mieux comprendre l'activité antimicrobienne du chitosane
sachant qu'au niveau de la littérature, le mode d'action du chitosane n'est pas clair et ceci
malgré les nombreuses hypothèses qui ont été émises. Nos résultats ont montré que le
mécanisme d'action du chitosane dépendait de son poids moléculaire. ,Ainsi le chitosane
avec un faible poids moléculaire affecte la perméabilité des cellules et par la suite, la
croissance microbienne tandis que le chitosane de poids moléculaire moyen et élevé forme
une couche sur la surface des cellules entravant le passage des nutriments vers le milieu
intracellulaire. La détermination des concentrations minimales inhibitrices des trois types
de chitosane et de la divergicine M35 contre L. monocytogenes a permis de mettre en
évidence un effet additif de ces deux composés contre L. monocytogenes. À notre
- connaissance notre étude est la première qui a pu déterminer la nature de l'effet combiné
d'une bactériocine et du chitosane pour l'inhibition de bactéries pathogènes des aliments. Il
serait intéressant d'étudier la synergie entre le chitosane et d'autres types de bactériocines
telles que la nisine et la pédiocine qui ont montré un bon potentiel quant à leur application
dans le secteur alimentaire. Un des avantages de l'utilisation de cette association de
principes actifs est la prévention du développement de variants résistants tels que déjà
décrits pour plusieurs autres substances antimicrobiennes dont les bactériocines.
Dans la deuxième partie de la thèse, nous avons utilisé les résultats obtenus dans le cadre
du premier objectif pour développer et caractériser sur les plans physico-chimique et
biologique de nouveaux films à base de chitosane et de la divergicine M35 . En général, les
résultats obtenus ont montré que l'incorporation de la divergicine M35 n'avait pas
beaucoup d'effet sur les propriétés physicochimiques des films de chitosane. Aussi l'étude
de la structure des films de chitosane en présence et en absence de la divergicine M35 a
114
Afin de valider les résultats obtenus dans le deuxième objectif, nous avons procédé à
l'application des films développés ainsi que leurs solutions filmogènes sur du saumon fumé
à froid inoculé par L.monocytogenes et ceci, dans des conditions de stockage qui simulent
la chaine du froid de ce type de produit (entreposage de 14 jours à 4°C suivi de 7 jours à
8°C). L'activité inhibitrice du film contre L. monocytogenes a été maintenue durant toute la
période du stockage, tandis qu'après 14 jours la solution filmogène n'a pas permis la
réduction du compte de L. monocytogenes. Notre étude a permis de conclure que le film à
base de chitosane et de la divergicine M35 était efficace pour inhiber la croissance de L.
monocytogenes dans le saumon fumé à froid. Ainsi des études supplémentaires sur
l'activité ·du film développé sur Clostridium botulinum type E, le deuxième pathogène
problématique des produits marins prêts à consommer, permettrait d'avoir plus
d'alternatives pour les emballages perméables à l'oxygène au Canada et de réduire le taux
élevé de sel utilisé comme agent de conservation contre Clostridium botulinum type E dans
les pays européens.
Les résultats obtenus sur l'application du film antimicrobien ont montré que cette approche
a permis une meilleure préservation de la couleur et de la qualité microbiologique du
saumon fumé à froid comparativement à la solution filmogène. Ceci permettrait d'allonger
la durée de vie de~ produits marins prêts à consommer tout en ayant un meilleur contrôle
sur le risque de présence de L. monocytogenes dans ce type de produits.
115
En considérant le nombre croissant des études portant sur le chitosane et son application
comme matrice de libération d'agents antimicrobiens, nous pouvons nous attendre à plus
d'intérêt des industriels du secteur de l' agro alimentaire en général et celui des produits
prêts à consommer en particulier pour des applications du chitosane dans la
biopréservation. Les résultats très prometteurs obtenus dans le cadre de.cette étude ouvrent
la voie pour de nouvelles études et applications visant à exploiter le potentiel des films
développés sur des produits marins autres que le saumon fumé à froid ainsi que d' autres
produits prêts à consommer qui présentent un risque de croissance de ·L. monocytogenes tels
certains produits de charcuterie.
Finalement et comme pour toute thèse de doctorat, nos travaux soulèvent un certain nombre
de questionnements et certains aspects aussi bien fondamentaux qu'appliqués qui méritent
d'être approfondis notamment:
- Mieux comprendre et caractériser le mécanisme d'action antimicrobien du chitosane.
- Élucider les mécanismes régissant l'effet additif observé entre le chitosane et la
divergicine M35.
- Évaluer l' activité antimi~robienne des films développés sur d'autres pathogènes majeurs
du saumon tel que Clostridium botulinum.
- Étudier de façon plus approfondie les effets des films antimicrobiens sur la microflore
d'altération du saumon fU!llé incluant la flore lactique endogène.
116
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