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BLAISE OUATTARA
DEVELOPPEMENT D'UN EMBALLAGE ANTIMICROBIEN POUR
LES VIANDES ET LES PRODUITS CARNÉS
These
présentée
à la Faculté des études supérieures
de l'université Laval
pour l'obtention
du grade de Philosophiae Doctor (Ph.D)
Département des Sciences des Aliments et de Nutrition
FA CUL^ DES SCIENCES DE L'AGRICULTURE ET DE
L'ALIMENTATION
UNIVERSITÉ LAVAL
Q ~ B E C
Août 1998
O BLAISE OUATTARA, 1998
National Library 1*1 of Canada Bibliothbque nationale du Canada
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AVANT-PROPOS
Rien n'ai plus agdable pour ceux qui apprennent que la disponibilitd et la sirnplicitd
de ceux qui consacrent l eu vie entière iî ai&r d'autres personnes à acqutrir de nouvelles
connaissances. I'ai eu la chance de gobter b ce fruit pendant mon sejour au Qudbec et la
saveur me restera durant toute ma carrii!rc. Je tiens iî remercier du fond du cœur tous les
professeurs du dtpartement des Sciences des Aliments et & Nutrition (ALN) de l'universitd
Laval et les chercheurs du Centre de Recherche et de Développement sur les Aliments
(CRDA)/Agriculture Canada de St-Hyacinthe qui m'ont aider B daliser ce travail.
Je remercie en particulier Dr Ronald E. Simard. L'envergure de ses connaissances
scientifiques et ses grandes qualites humaines font de lui un professeur et un chercheur a
qui on aimerait ressembler. C'est un honneur pour moi d'avoir réalisé ce travail sous sa
direction.
J'ai eu le privilège d'effectuer toute la partie expérimentale de mon doctorat dans le
laboratoire du Dr Grabiel Piette. J'ai pu alors béntficier d'un apport scientifique
considdrable de sa part. Je voudrais particuliércment le remercia pour tout le temps qu'il a
consacré pour m'accompagner dans l'apprentissage et l'arntlioration de la technique &
ddaction scientifique. A travers lui. je remercie toute l'tquipe de la section Industrie des
viandes du CRDA qui m'a apport6 chaleur et deonfort tout au long de mon stage. Merci à
France Dussault et à Yves Raymond pour leur assistance technique.
Je voudrais aussi remercier Dr André Bégin pour sa disponibilité et les conseils judicieux
qu'il m'a donnts chque fois que j'ai eu besoin de son expertise.
Les travaux antériew du Dr Rlcharù Holley ont servi de base pour le développement et
l'exécution & ce projet & recherche. J'ai pu également Mndficier à tout moment de son
expertise en microbiologie alimentaire. Je voudrais lui tkmoigner tous mes remerciements.
Je remercie Dr IsmaU Füss. pour avoir accepte malgré son programme charge & faire la
prélecture de ce travail. A travers lui. je remercie toute l'équipe de recherche qu'il dirige
avec Dr Simard pour les bons moments passe ensemble.
Je remercie aussi l'Agence Canadienne & Coopération internationale (ACDI) et le
Rograrnrne des Bourses de la Francophonie (PCBF) pour l'appui financier que j'ai reçu.
Je dédie ce travail :
À feu mon père
À ma femme Noéllie . Ce travail a CtC possible @e aux diffdrents sacrifices que tu
as consentis et au réconfort affectif que tu m'as apport6 tout au long de ce
cheminement. C'est le fruit d'un effort conjoint et je t'en remercie grandement.
À mes enfants. Nadine, Serge, Gauthier et Olivia. Vous avez kt6 peut-être sans le
savoir une ventable source d'knergie pour moi tout au long de la rbalisation de ce
travail. Je voudrais vous temoigner toute l'affection et l'amour que j'ai pour vous.
J'aimerais que ce modeste iravail vous serve plus tard d'exemple. pour faire mieux.
À mes amis, Clémence, Ali, David, Esther, Uonie, Laeticia. Merci pour tous les
bons moments passes ensemble. Je vous souhaite bon courage et bonne chance dans
la poursuite de vos progrmes respectifs.
RESm COURT
Des agents antimicrobiens ont ttC sélectionn6s sur la base de leur éfficacité h inhiber la
croissance de six souches bacttriennes d'altération des viandes et produits cambs:
BrochothrUr thennosphacta, Carnobacterium piscicola, Lactobacillus curvatus,
Lactobacillus sake, Pseudomonas fluorescens et Serratia liquefaciens. Deux acides
organiques (acides acCtique et propionique), trois huiles essentielles (de cannelle et de clou
de girofle et de romarin) et deux acides gras ik longue chaîne (acides laurique et
palmitoléique) ont et6 retenus. Ces compos6s actifs ont ét6 incorporés dans une matrice de
chitosane de rnaniére obtenir des films possCdant des propriCt6s antirnicrobiennes. Des
essais de diffusion en milieu liquide et sur des produits camés ont pemiis de montrer que la
libération des acides organiques contenu dans les films peut être réduite en abaissant la
température ou çn additionnant aux films des compos6s lipidiques tels que l'acide launque,
le cinnamaldehyde et l'eugenol. Les tests antibacttriens sur les produits camés (bologne,
jambon et pastrami) montrent que les films sont efficaces, contre la flore normale
cWnterobacteriaceae et une souche de Serratiu liquefaciens artificiellement inoculde en
surface. -
LONG
Vingt-trois composCs antimicrobiens comprenant des acides organiques. des huiies
essentielles et des acides gras ii longues chaînes ont 6th testes pour leur efficacitb
antibacterienne. Six souches bactdriennes reconnues pour leur implication dans les
prxessus de degradation des viandes et produits camés ont et6 choisies comme organismes
cibles: Brochoth ri* thennosphacta. Camobacteriwn piscicola, Laciobacillus cun>atusV
Luctobacillus sake, Pseudomonas fluorescens et Serraiia liquefaciens. D'après les résultats
obtenus, 2 acides organiques (acides acktique et propionique), 3 huiles essentielles
(cannelle, clou de girofle et romarin), et 2 acides gras il longues chaînes (acides laurique et
palrnitol6ique) ont révCle de bonnes propriétes antibactériennes.
Dans une deuxième ktape, les agents antimicrobiens les plus actifs ont dté incorporés
dans une matrice de chitosane dans le but d'obtenir des films possédant des propnétes
antibactériennes. La diffusion des acides acktique et propionique a été dtudike en milieu
liquide à différents pH (5.7, 6.4 et 7.0) et diffCrentes températures (4, 10 et 24OC). Le pH
n'avait aucun effet sur le processus de diffusion tandis que l'abaissement de la température
de 24 à 4OC provoquait une réduction des coefficients de diffusion de 2.59 à 1.19 x 10'12
m2.s-' pour l'acide acdtique et de 1.87 il 0.91 x 10'12 m2.s" pour l'acide propionique.
L'addition dacide launque ou d'huiles essentielles (cinnamald6hyde ou eughol) provoquait
dgaiement une réduction de la diffusion. Les effets maxima étaient obtenus avec l'acide
laurique et le cinnarnaldChyde respectivement pour des concentrations de 1.0% et 0.5%
(pdpd) dans la solution filmogène.
Dans une demitre Ctape, les caractéristiques de di fision des acides organiques ont CtC
detenninés sur des produits carnCs (Bologne, jambon et pastrami), suivi d'une dvaluation
d'activité antibacterienne contm la flore normale des produits c m & et de souches de
Luctobacillus s& et Serratia liquefaciens artificiellement inoculdes en surface. Quelque
soit le type de film, plus & 75% des acides acttique et propionique étaient libérés des films
dans les trois premières heures suivant leur application. Cette libération Ctait réduite quand
les films contenaient en plus de l'acide laurique ou du cinnamaldehyde. La diffusion des
acides acétique et propionique était tgalement plus faible lorsque les films etaient appliques
sur le bologne comparativement au jambon et au pastrarni. Les films ont montré de bonne
propriétés antibact6riennes particulièrement contre Serratia liquefaciens et les
En tero bacte riaceae.
vii
................................................................................................................... AVANT.PROPOS i
RÉSU& COURT ................................................................................................................. iv
..................................................................................................................... RÉsU~& LONG v
. . ................................................................................................... TABLE DES M A ~ R E S .vil
. . .................................................................................................... LISTE DES TABLEAUX xi1
.......................................................................................................... LISTE DES FIGURES xv
INTRODUCTION ............................................................................................................... 1
CHAPITRE 1 : Revue de littérature ............................. .........e..ee.....o.......m....~.~o..............o.. 5
1.1. Origines de la contamination ............................................................................................ 5
1 .1 .1 . Contamination primaire ou endogène .................................................................. 5
. . 1.1.2. Contamination seconâaire .................................................................................... 6
1.2. Types de micro-organismes ......................................................................................... 8
1.2.1 . Bactérie pathogtnes ........................................................................................... -8
1.2.2.1. Espèces impliquées ................................................................................. 8
1 .2.1. 2. Maladies occasiorin6es ........................................................................... 9
1.2.2. Bactéries d'altération ........................................................................................ 10
.......................................................................................... 1.2.2.1. Flore initiale 10
1.2.2.2. Évolution de la flore d'alttration au cours du stockage ...................... 1 1
1 .2.2.3. Phdnom2ncs d'altération microbienne ............................................... 1 2
viii
......................... 1.3. Contrôle des micro-orgamismes sur les viandes et les produits cames 15
.......................................................................................... 1.3.1. Acides organiques 1 5
..................................................................... 1.3.2. Acides gras et huiles essentielles 17
. ........................................................................................ 1.4. L'emballage antimicrobien 1 8
. . ............................................................................................. 1.4.1. MatCriaux util~sés 20
................................................... 1.4.2. Contrôle de la diffusion des antirnicmbiens 21
1 . 5 . Objectifs ............................................................................................................... 22
......... CHAPITRE 2 : Inhibitory effect of organk acids upon meat spoilage bacteria 24
2.1. Abstract ................................................................................................................................ 25
........................................................................................................................ 2.2. Introduction -26
2.3. Material and methods .......................................................................................................... 28
2.3.1. Organisms and cultures ........................................................................................... 28
2.3 .2 . Preparation of the bacterial suspensions for the growth iri hi bi tion experiments .. 29
.............................................................................. 2.3.3. Growth inhibition experiments 29
............................................................................................................... 2.3.4. pH effects 30
.......................................................................................................... 2.3.5. Data analysis 30
2.4. Results .................................................................................................................................. 31
............................................................................................................................ 2.5. Discussion 34
2.6. List of tables ......................................................................................................................... 39
2.7. List of figures ...................................................................................................................... 40
CHAPITRE 3 : Antibacterid activity ot selected fatty acids and essential oiis against
six meat s p ~ i l a p 0 ~ ~ ~ n i s ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . ~ ~ m ~ m m m m m ~ m m m m m m o o m m e m m m e m m m m e m m e m m m e m m m m m m m m a m m m e m m o ~ m m r m w e m e m m e e m m 4 8
3.1. Abstract .............................................................................................................................. 49
3.2. In~duct ion ........................................................................................................................ 50
3.3. Material and methods .............................................................................................. 51
....................................................................................... 3.3.1. ûrganisms and cultures 1
................................................................. 3.3.2. Reparation of the antibactenal media 52
............................................................................. 3.3.3. Growth inhibition experiments 53
3.3.4. Analysis of essential oils (EO) ........................ .. ............................................... 54
.................................................................................................................................. 3.4. Results 55
3.4.1. Fatty acids ............................................................................................................... 55
3.4.2. Essential oils ........................................................................................................... 55
.......................................................................................................................... 3 .5 . Discussion 57
......................................................................................................................... 3.6. List of tables 63
CHAPITRE 4 : Effect of temperature ab- on the ability of organic acids to prevent
growth of meat spoilage b ~ ~ t ~ ~ i ~ m w ~ ~ ~ ~ ~ ~ ~ m ~ ~ ~ e m m ~ ~ ~ ~ ~ ~ ~ m m m ~ ~ ~ m m a m m m e m m ~ ~ m m ~ ~ ~ ~ ~ ~ ~ e m o m m m m e ~ ~ e m m ~ ~ m e ~ ~ ~ m m m m m e m 6 7
4.1. Abstract .......................................................................................................................... 68
.................................................................................................................... 4.2. Introduction 69
.................................................................................... 4.3. Material and methods 69
4.4. Results ............................................................................................................................ 71
.................................................................................................................. 4.5. Discussion 72
............................................................................................................... 4.6. List of tables 74
5.1. Abstract .......................................................................................................................... 78
5.2. Introduction .................................................................................................................... 79
..................................................................................................... 5.3. Material and methods 81
.................................................................................................... 5.3.1. Chitosan films 81
........................................................................................ 5.3.2. Diffusion experiments 82
5.3 .3 . Fractional mass release and diffusion coefficients of acetic or propionic acid.33
...................................................................................................... 5.3.4. Data analysis 84
............................................................................................................................ 5.4. Results 85
............................................................... 5.3.1. Film preparation and film thickness 85
5.4.2. Kinetics of organic acid release from chitosan films ........................................ 85
................................................... 5.4.3. Influence of pH and temperature on diffusion 86
5.4.4. Effect of lauric acid, cinnamaldehyde, or eugenol on diffusion ........................ 87
................................................................................................................... 5.5. Discussion 88
........................................................................................ 5.6. Conclusion -91
.................................................................................................................. 5.7. List of Tables 91
5.8. List of Figures ................................................................................................................ 93
CHAPITRE 6 : Inibition of surface spoiiage bacteria on meat products by application
of antimicrobial films made with ~hitosane.e..~~~~~~~~~ee.e.aeeeeeeoeoooo~aee~eeaeoeeee~.~ l eee .IO2
........................................................................................................................... 6.1. Absrnt 103
................................................................................................................ 6.2. Introduction 104
...................................................................................................... 6.3. Material and methods 106
........................................................................................... 6.3.1. Preparation of films 106
................................................................................... 6.3.2. Organisms and cultures lû6
....................................................................................................... 6.3.3. Diffusion tests 107
............................................................................................... 6.3.4. Antirnicrobid test 109
..................................................................................... 6.3.5. Microbiological analysis 110
.......................................................................................... 6.3.6. Statistical analysis 110
............................................................................................................................... 6.4. Results 111
...................................................................................................... 6.4.1. Diffusion study 111
............................................................................................ 6.4.2. Antirnicrobial tests 112
6.5. Discussion ........................................................................................................................ 114
6.6. C O ~ C ~ U S ~ O ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ eO.ee.a.....e.a.............. 119
6.7. List of tables ................................................................................................................. 120
............................................................................................................. 6.8. List of Figures 121
CHAPITRE 7 : CONCLUSION ~ É ~ ~ ~ ~ ~ e e o e ~ e o e e . e o e e o ~ e e o e o e e o e e e e ~ e e o e e e o e e e e e o e e e e e e o e e o e e e e o e o o e 1 3 0
B I B L I O G R A P H I E e o . e e . e e e e e e e e e e o ~ e e e e e o ~ e e e e o e e e o o o e o o o e e o e e o e e o e o e o e e o e e e ~ e e e e e e e o e e e e e e e o e e o e e 1 3 3
xii
LISTE DES TABLEAUX
Tableau 1.1: Substrats et metabolites produits par les principales bactdries d'altération des viandes fraîchesaab.
Table 2.1. Relative degrees of dissociation of the various organic acids as a function of their
concentration in the growth media and of the resulting pH.
Table 2.2. Growth inhibition by organic acids of the six meat spoilage bacteria.
Table 2.3. Minimum inhibitory concentration (IWCs) of acetic, propionic, citric , and lactic
acids for growth of six meat spoilage bacteria.
Table 2.4. Differentiating the inhibitory effecis of pH and organic acids on the growth of six
meat spoilage bactena.
Tabk 3.1. Minimum inhibi tory concentration (pglml) of fatty acids against meat spoilage
bac teria.
Table 3.2. Inhibitory properties at 24 and 48 h, of diluted essential oils toward meat
spoilage bacteria.
Table 3.3. Quantitative determination of selected authentic antibacterial components in
essential oils.
Table 4.1. Total inhibition of B. themosphacta, P. jborescens, and S. liquefaciens by
organic acids at 4,8, or 20°C.
Table 4.2. Influence of temperature on the lag periods before initiation of the growth of B.
thennosphacta, P. jluorescens, and S. liquefaciens in presence of O. 1% (wlv) of
various organic acids.
Table 5.1. Summarired results of variance analysis relative to the diffusion of acetic and
propionic acids from chitosan films.
Table 5.2. Influence of temperature on diffusion of acetic and propionic acids from
chitosan films.
Table 5.3. Influence of lauric acid on the diffusion of acetic acid from chitosan films.
Table 5.4. Effects of cinnamalàehyde and eugenol on the diffusion of acetic and propionic
acids from chitosan films.
Table 6.1. Type of films and composition.
xiv
Table 6.2. Concentration of lauic acid and cinnarnaldehyde (mgkm2) in composite films
before and after 1 week application on mcat proâucts in vacuum package
conditions at 4°C.
Table 6.3. Inhibitory effects of chitosan films against L sake and S. liquefaciens inoculated
on the surface of cooked harn slices stored at 4°C.
Table 6.4. Inhibitory effect of selected chitosan films on the gmwtb of Enterobacteriaceae
present on the surfaces of bologna and pasuami.
Table 6.5. Inhibitory effect of selected chitosan films on the growth of lactic acid bactena
present on the surfaces of bologna and pastrami after 21 days storage.
LISTE DES FiGüRES
Figure 1.1. Repdsentation schdmatique & la conservation des aliments sans (A) ou avec (B)
un enrobage ou un film servant de support à l'incorporation d'agents
antirnicrobiens.
Figure 2.1. Typical growth patterns of selected meat spoilage bacteria in media containing or
not various arnounts of acetic, prupionic, lactic, and cioic acids. Values ploned
are the means of three mplicate measurements.
Fipre 2.2. Growth of BrochothBx thermospha~ta~ Pseudomonas fluoresceni, and Serratia
liquefaciens in media with or without benzoic or sorbic acid (0.15% 'wt/vol')
final concentration). Values ploned are the means of two replicate measurements.
Figure 2.3. Growth of (A) Serrafia liquefaciens and (B) Luctobacillus suke in media with or
without various amounu of organic acid or hydiochloric acid. Values plotted are
the means of three replicate measurements.
Figure 5.1. Typical curves of fractional mass release of acetic or propionic acids
incorporated in chitosan films.
Figure 5.2. Representative graphs of the influence of pH on the fractional mass dease of
acetic and propionic acids incorporated in chitosan films. Diffusion tests wen
perfomed at 10°C.
Figure 5.3. Effect of temperatun on the fractional mass release of acetic and propionic
acids incorporated in chitosan films.
Figure 5.4. Arrhenius plots and activation energies of acetic and propionic acids
incorporated in chitosan films.
Figure 6.1. Percentage of acetic or propionic acids remaining in chitosan films after
application on meat products in vacuum package conditions: influence of the
type of film.
Figure 63. Percentage of acetic acid remaining in chitosan films after application on meat
products in vacuum package conditions: influence of the type of meat products.
Figure 6.3. Percentage of propionic acid remaining in chitosan films after application on
meat products in vacuum package conditions: influence of the type of meat
produc ts.
Avec la globaiisation de l't!conornie mondiaie les viandes et les produits carnes sont
transportés sur des distances de plus en plus longues pour atteindre les marchés étrangers.
De ce fait. le problème de quaiitt dans les industries & viandes est devenu un phtnomtne
très complexe, puisque les produits doivent avoir de plus longs délais de conservation et
une protection contre la prolifbration des micro-organismes pathogénes et d'altération. Dès
lors, plusieurs travaux de recherche ont di6 entrepris afin de mieux connaître l'origine des
micro-organismes (Cmarninana et al.. 1997; Sammarco et al.. 1997), les espèces
impliqukes (Bean et al., 1997; Cabedo et al., 1997; De1aza.i et al., 1997; Karib et al., 1994)
ainsi que les mécanismes d'aldration microbiennes des viandes (Korkeala et Bjorkroth,
1997; Lambert et al.. 1991; Nychas et Tassou, 1997). On sait à travers ces études que
l'altération microbienne des viandes est un phénomène presque inévitable puisque les
espkes impliquées sont prdsentes de manière permanente dans l'environnement des
produits. De plus, les méthodes conventionnelles de conservation (réfrigkration, emballage)
ne font que remplacer des populations bactéhennes d'altération par d'autres qui s'adaptent
mieux aux nouvelles conditions écologiques.
Plusieurs travaux ont kt6 réalisés pour trouver des m6thodes additionnelles de
conservation, notamment par utilisation d'agents antirnicrobiens. Des progrès considkrables
ont 6té réalisés dans ce sens avec l'utilisation des acides organiques comme desinfectants
des carcasses (Dickson et Anderson, 1992). Ainsi, Zeitoun et Debevere (1992) et Prasai et
al. (1997) rapportent des extensions significatives de délai de conservation des viandes
fraîches de volaille et de bœuf, suite il un traitement avec de l'acide lactique ou un tampon
acide IactiquJlactate de sodium. De même, une réduction & 1.3 P 2 logio de la flore totale
de surface a d t t obtenue après une pulvdrisation des surfaces avec des solutions d'acide
acétique ou d'acide propionique @orsa et al.. 1997).
Lm acides gras à longues chaînes et les huiles e sentielles ont dgalement dté Ctuâid
pour leur propriétés antibacteriennes et antifongiques vis-A-vis de plusieurs micro-
organismes des plantes et des aliments (Kabara, 198 1; Russel, 199 1; Shelef et al., 1980).
Ces substances permettent d'inhiber les populations bactériennes de Salmonellu
typhimurium et Staphylococcus aureus (Juven et al., 1994 ; Karapinar et Aktug, 1987 ;
Paster et al., 1990), Listeriu monocytogenes (Aureli et al.. 1992 ; Wang et Johnson, 1992)
Vibrio parahaemoliticus (Kaprapinar et Aktug. 1987 ; Shelef et al., 1980) et Clostridium
botulinum (Ababouch et al., 1992).
Cependant, aussi bien pour les acides organiques que pour les acides gras et les
huiles essentielles, la plupart des études d'activitt antibactérienne ont ét6 dalisees sur des
bacteries pathogiines (Ababouch et al., 1992; Ahamad et Marth, 1989; Aureli et al., 1992;
Brocklehunt et Lund, 1990; Chung et Goepfen, 1970; Juven et al., 1994; Karapinar et
Aktug, 1987; Paster et al., 1990; Wang et Johnson, 1992), et trts peu d'informations sont
disponibles sur les bacidries responsables des phénomènes d'altération (Greer et Dilts.
1992; Ouattara et aL. 1997a; 1997b). Ceci est compktement disproportionnt5 par rapport il
l'importance grandissante des phCnom8nes d'alttration.
Compte tenu du fait que la croissance bactkrienncs sur les viandes et les produits
camés a lieu principalement en surface (Holley. 1997). les syst8mes de conservation par
utilisation des agents antirnicrobiens doivent permettre de maintenir une concentration
dlevée de ces composés il la surface des produits (Gennadios et al., 1997; Kester et
Fennema 1986; Labwa, 19%; Toms et al., 1985). Maiheuseusement, plusieurs travaux
réalises notamment sur les acides organiques rapportent que ces composts diffusent
rapidement vers l'intérieur des produits après leur application en surface (Siragusa et
Dickson, 1992).
RCcemment, le concept d'emballage antimicrobien a d t t propose comme moyen de
resoudre le probltme de diffusion d'agents antimicrobiens (Hotchkiss. 1995). Selon ce
concept, les agents actifs peuvent être incorport% dizctement dans le materiel d'emballage
partir duquel ils peuvent migrer lentement et exercer un effet de longïe durée. La
faisabilit6 de cette technologie a étd âémontde par Weng et Hotchkiss (1992, 1993) dans
des essais d'inhibition des levures et des moisissures à la surface des fromages.
Bien que les plastiques synthétiques puissent être utilisks comme matériel de base
pour l'incorporation des agents antimicrobiens, une dtude récente de Gennadios et al.
(1997) rapporte un intérêt grandissant pour les polym2res biodégradables constitues de
polysaccharides, protdines et lipides. Certains de ces polym&res comme le chitosane sont
eux-mêmes dotés d'un pouvoir antimicrobiens (Darmadji et Izumimoto, 1994; El Ghaouth
et al., 1992). L'une des ciifficultes avec l'utilisation des polysaccharides et des protdines
comme support à l'incorporation d'agents antimicrobiens est leur nature hydrophile.
Plusieurs auteurs ont ddmontrt que les films ayant un fort potentiel d'absorption d'eau
avaient de faibles propridtds de rétention des composes chimiques incorporés dans leurs
matrices (Armand et al.. 1987; Malley et ui., 1987; Shanthamurthy et Aminabhavi. 1990;
Peppas et Branon-Pepp~, 1994).
La CO-incorporation de composés lipidiques a Cté proposte comme moyen de
réduire la diffusion des agents antimicrobiens incorporés dans les films base de polymères
hydrophiles (Callagarin et al., 1997). Ainsi. Red1 et al. (1996) ont réussi iî réduire de 20 il
50% le coefficient de diffusion de l'acide sorbique incorporé dans des films de gluten & bld
en y ajoutant de la cire ou des monoglycérides acdtylds. En dCpit du fait que certains
composés possèdent des propritt6s antibactériennes (Kabara, 198 1; Ouattara et uf., 1997b),
ce paramètre n'a pas toujours t td pris en considération dans la formulation des films.
A travers les études dtjh rkalisées, il apparaît que l'emballage antimicrobien reste un
concept assez complexe. L'efficacité de ce systeme depend de plusieurs facteurs qui sont
reliés à la composition de l'aliment, aux caractéristiques physico-chimiques des matCriaux
d'emballage et à l'efficacité relative des agents antimicrobiens utilisés. Dans ce travail, nous
avons essaye d'apporter une contribution à la compréhension de ce systi?me, avec l'ultime
but d'aboutir a un film antibactdrien pour la conservation des viandes et des produits carnés.
CHAPITRE 1
REVUE DE L I ~ R A T U R E
Chez les animaux sains et bien reposés, la majoritt des tissus destin& à être
transformes en viandes et produits camés sont généralement stCriles (Dickson, 1992 ;
Nottingham, 1982 ; Rozier et al., 1985 ; Tinney et al., 1997). Cependant, au cours des
opérations d'abattage et de transformation, toutes les parties comestibles sont exposes h la
contamination par une va i t tC de microorganismes. Compte tenu de certaines
caractéristiques chimiques du muscle (Aw et pH élev&, présence de substances nutritives
facilement assimilables) (ICMSF, 1980). ces micro-organismes se multiplient rapidement et
provoquent des situations indésirables allant de la détérioration des produits il l'apparition
de maladies d'origine alimentaire chez les consommateurs humains (Korkeala et Bjorkroth,
1997 ; Roels et al., 1997).
1.1. Origines de la contamination
1.1.1. Contamination primaire ou endoghe
Ce type de contamination implique des micro-organismes initialement présents sur
l'animal vivant et qui se retrouvent ensuite sur la viande. La peau et les poils constituent les
sources les plus importantes compte tenu du contact permanent qu'ils ont avec le sol, l'eau
et la vkgdtation (Newton et al., 1978). Van Donkersgoed et al. (1997) rapportent par
exemple que la peau âes animaux de boucherie peut porter jusqutà 109 cellules
microbiennes par cm? Le tube digestif constitue Cgalemmt une source imponante de
contamination primaire, en particulier lorsqu'il subit une rupture au cours de 1'6viscdration
(Nottingham, 1982). Pour cela, il y a souvent une &mite corrélation entre le nombre
d'animaux vivants portant des Enterobucteriaceae et Salmonella spp. dans leur fkes et le
nombre de carcasses contaminées par ces micro-organismes B la fin & la chaîne d'abattage
(Berens et al., 1997 ; Canaminana et al.. 1997). De même, lors de bactéri&nie d'abattage
ou lorsque l'éviscération est faite de rnaniere tardive (plus de 30 mn aprés la saignée). les
micro-organismes peuvent traverser la paroi intestinale et se retrouver dans le muscle
(Rozier et al.. 1985). Les ganglions lymphatiques, les poumons. le foie, la vessie, l'utdrus
peuvent egalement intervenir dans la contamination primaire (Giil et Newton. 1978 ;
Dickson et Anderson, 1992 ; Easton, 1997; Simone et al., 1997 ; Moore et Madden, 1998 ).
1.1.2. Contamination secondaire
La contamination secondaire ou exogène des viandes survient au cours des diverses
manipulations qu'elles subissent au cours de leur transformation. Ce type de contamination
implique l'environnement et de nombreux vecteurs animts et inanimes (Rozier et al.,
1985 ; Samrnarco et al., 1997). D'après Jouve (1990), 80 B 90% de la microflore des
viandes qui parviennent aux consommateurs résulte des contamination survenant
l'abattoir. Le personnel humain qui travaillent dans ces industries représente la plus
importante source, en particulier lorsque les mains sont salies par des matieres souillées ou
lorsque les vêtements de travail sont mal entretenus (Jay, 1986). De plus, certaines
personnes peuvent 2m malades ou porteuses de micro-organismes pathogénes comme
Salmonella spp. et Escherichia coli 0 157 :H7 au niveau du tube digestif et de la peau, et les
transfdrer ensuite aux viandes (Buchwald et Blaser, 1984). Les matériaux inertes qui entrent
en contact avec les aliments (récipients, surfaces de travaux. couteaux. etc, ...) contribuent
également à la contamination secondaire (Sammarco et al.. 1997). Dans une 6tude réalisde
sur la prévalence de Salmonella, Listena et Yersinia dans les abattoirs, Sammarco et al.
(1997) rapportent que le plancher et les tables & travail représentent les sources de
contamination les plus importantes. De nombreuses Ctudes ont CtC faites dgaiement sur les
niveaux de contamination de l'air dans les industries de viandes (Kotula et Emswiler-rose,
1988 ; Rhalcio et Korkeala. 1997) et les rhaltats obtenus indiquent que les micro-
organismes présents dans l'air constituent une source potentielle de contamination
microbienne des viandes.
Au cours des demiéres années. des progrès considt?tables ont ktk effectués dans la
recherche de moyens de contrôle de la contamination des carcasses, notamment h travers
l'approche HACCP (Hazard Anal ysis and Critical Control Point) (Hathaway et McKenzie.
1991; Biss et Hathaway, 1995). Cette approche permet d'identifier et de comger les sources
de micro-organismes au cours des opérations d'abattage et de transformation. Cependant,
les techniques d'abattage actuellement disponibles ne peuvent pas garantir une absence
totale de micro-organisme sur les carcasses (MFSCNFPA, 1992; Dickson et Anderson,
1992; Karib et al., 1994). Par exemple, Vanderlinde et a1.(1998) rapportent un compte
bacterien moyen (flore mksophile totale) de 3.13 log CFU/cm2 sur des carcasses de bœufs
Australiens destindes il l'exportation.
1.2. Types de micro-organismes
Compte tenu de la divenit6 des sources de contamination qui interviennent au cours
' des opérations d'abattage et de transformation, plusieurs types & micro-organismes
peuvent se retrouver sur les viandes. Cependant, la majoritC des auteurs s'accordent sur le
fait que les bactdries jouent un rôle plus important comparativement au moisissures, levures
et virus. Par exemple, dans une Ctude sur les caractCristiques microbiologiques des
carcasses de bovins, Karib et al.. (1994) rapportent des charges microbiennes de 4.43 log
CFü/cm2 pour la flore bacterienne totale. contre 2.62 et 2.12 log CN/cm2, respectivement
pour les levures et les moisissures. Plus récemment, Bean et al. ( 1997) indiquent que 90%
des cas de maladies d'origine alimentaire sont dues aux bactkries, contre 6% pour les virus
et 1% pour les parasites.
1.2.1. BactCrie pathogènes
1.2.2.1. Espèces impliquées
L'incidence des bacteries pathogènes dans les abattoirs est actuellement bien
documentée. De nombreuses informations sont disponibles sur la contamination des
carcasses de bœufs, porcs. moutons et volailles par des bactéries pathogènes comme
Salmonella (Delazari et al., 1998 ; Carraminana et al., 1997; Miller et al., 1997).
Escherichia coli 0157:H7 (Cabedo et al.., 1997 ; Mermelstein, 1993), Listeria
monocytogenes (Barbosa et al., 1995 ; Dickson, 1990 ; Juven et al., 1997 ; Korsak et al.,
1998). Cumpylobacter (Cohen et al., 1984 ; Epling et al., 1993), Staphylucoccus aureus
(Mathieu et al.. 1992) et Aeromonos spp. (Cabedo et al., 1997). Les fréquences de
contamination sont variables en fonction des pays et du type de viande mais les espèces
bactdriennes le plus souvent associ&s aux maladies sont Salmonella et Escherichia coli.
Clostridium botulinum et Clostridium pewngens qui sont des bactéries pathogènes
anaérobies strictes peuvent Cgalement être présentes dans les viandes suite à une
contamination profonde (Nottingham, 1982)' ou lorsque les produits sont conditionnes sous
vide comme c'est le cas pour de nombreux produits carnes (Elkatheib, 1997 ; Rebollo et al..
1997).
1.2.1.2. Maladies occasionnées
Les bactéries pathogènes présentes sur les viandes et les produits carnCs ont et6 très
souvent associees à de nombreux cas de maladies d'origine alimentaire, gknéralement des
gastrotnt6rites. On distingue couramment deux types de maladies : i) les toxi-infections
alimentaires qui sont provoquees par la multiplication et la production de toxine à
l'intérieur de l'organisme du consommateur (toxi-infections à Clostridiwn perfrrngens,
Solmonella et E. coli entéropathoghe) ; ii) les intoxications alimentaires qui sont dues il
des toxines dCjà présentes dans l'aliment au moment ou il est consomm6 (toxine de
Cfostrîdium botulinum et de Staphylococcus aureus). Dans le deuxitme cas, la toxine peut
être active même après lg~limination de la bacterie productrice.
De nos jours, les maladies d'origine alimentaire constituent encore un problème
croissant dans tous les pays du monde (Haapapuro et al., 1997). mais en même temps de
grands progrès sont réalisés dans la recherche de moyens de contrôle, notamment ih travers
la mise au point de mdthodes rapides & détection (Czajka et Ban, 1996 ; Keith, 1997 ; Tian
et of., 1996) et l'utilisation de diverses substances antirnicrobiennes (Delami et al.. 1998 ;
Doaa et al., 1997 ; Tambl yn et Conner, 1997).
1.2.2. Bactéries d'altération
1.2.2.1. Flore initiale
Les espèces bactdriennes impliquées dans les phhomènes d'altdration des viandes
et des produits carnés ont fait l'objet de nombreuses dtudes (Holley, 1997; Korkeaia et
Bjorkroth, 1997; Renerre et Labadie, 1993). A la fin des opérations d'abattage. cette flore
est constituée en grande partie par des bacteries psychrotrophes et mésophiles compte tenu
de la prédominance de ces groupes bacteriens dans les principales sources de contamination
(Newton et al., 1978). Il apparaît egalement que les bacteries gram négatif sont plus
largement reprdsentees que les gram positif (Newton et al., 1978; Nortje et al.., 1990;
Renerre et Labadie. 1993). Parmi les espèces identifiées, on note une prédominance du
genre Pseudomonas (Greer et Dilts, 1997; Holy et Holzapfel, 1988; Newton et al., 1978;
Sundheim et al., 1998) mais aussi la prdsence des genres Acinetobacter, Ffavobacteh,
Klebsiella, Moraxella (Christopher et al., 1979; Ellerbroek, 1997).
1.2.2.2 Evolution de la flore d9alt4ration au murs du stockage
Au cours du stockage et de la distribution des viandes, il y a une variation dans la
proportion des espèces microbiennes d'alteration présentes sur les viandes en fonction des
méthodes de conservation utilisées. La rCfrigCration qui constitue actuellement le moyen le
plus répandu de conservation crée des conditions sélectives favorisant le developpement
des esp&ces bacteriemes psychrotrophes aux depends des mésophiles (ICMSF, 1980;
Ooraikul et Stiles, 1991). Cette tendance a étd aussi observée par t i e t o et al. (1991) sur des
carcasses de moutons stockées A l'air libre il des températures de réfrigération et par
Jiménez er al. (1997) sur des poitrines & poulets.
De la même maniiire, l'emballage des viandes et produits camés cr6e des
environnements gazeux qui influencent grandement le d&eloppement bactérien (Dickson
et Anderson, 1992; Labuza et al. 1992). Les films perméables il l'oxygène favorisent
nettement le dCveloppement des bactéries aérobies strictes comme Pseudomonus et
Acinefobucter (Renem et Labadie. 1993). Des espèces anaérobies facultatives
(Enrerobacferiaceae) et microaérophiles (Brochothrix thennosphacta) peuvent aussi être
présentes dans ce type d'emballage en fonction de leur proportion dans la flore initiale
(Jirnenez et al., 1997 ; Renem et Labadie, 1993).
Dans les produits emballés sous vi& et en atmosphére modifiée (MAP) ou
contrôlde (CAP), le gaz carbonique produit par le métabolisme respiratoire ou introduit
dans l'emballage inhibe la croissance de Pseudomonas et âes autres bacteries aérobies
stricts (Labuza et al., 1992 ; Renerre et Labadie, 1993 ; Jimdnez et al., 1997). Dans ces
produits, on retrouve plus fréquemment &s bacttries lactiques appartenant principalement
aux genres Lizctobacillus (L. curvatus, L plantarum, L sake) et Camobactenum (C.
divergens. C. piscicola) puisque ces bactdrie ne sont pas affectdes par le gaz carbonique
(Korkeala et Bjdrkroth, 1997; Montel et al. 1991; Ooraikul et Stiles, 1991). Plusieurs
travaux rapportent également la présence du groupe des Enterobacteriaceae (Holley, 1997 ;
McMullen et Stiles, 1993 ; Holley et McKellar, 19%).
En somme, la contamination des viandes par les bactkries d'altération apparaît
comme un phhomène inevitable, puisque Ics espèces impliquées sont pdsentes & manière
permanente dans l'environnement immédiat des produits. De plus, les méthodes
conventionnelles de conservation (réfrigdration, emballage) ne font que remplacer des
espèces bactdriennes d'alteration par d'autres espèces qui s'adaptent mieux aux nouvelles
conditions.
1.2.2.3. Phhomènes d'altération microbienne
L'altdration des viandes et produits c m & peut être définie comme un symptôme ou
un groupe de symptômes qui découlent de l'activité microbienne et qui se manifestent par
des changements de couleur, d'odeur et d'apparence. Ce phénomène a et6 ttudié aussi bien
sur la viande fraîche que sur les produits carnCs (Korkeala et Bjorkroth, 1997; Lambert et
al., 1991). Les composés chimiques de faibles poids molCculaires sont utilisds à une vitesse
variable en fonction du groupe microbien dominants et des mdtabolites indésirables sont
libérés: mucus, hydroghe sulfuré (H2S), amines volatils, esters et acktoïne (Gill et Newton,
1978; Lambert et al., 1991). Dans une ktude sur la det6noration des carcasses de poulet,
Nychas et Tassou (1997) ont dtabli une dation entre l'utilisation du glucose et du L-lactate
et la production & plusieurs protCines solubles. Le tableau 1 rtsume pour la viande fralche
les principaux substrats utilisCs par les micro-organismes d'altération ainsi que les
principaux métabolites qui sont produits.
De nos jours, les phénomhes d'alt&ation constituent une probltmatique majeure il
laquelle doivent faire face les industries de vianâe h travers le mon&. Au Canada, on
estime il plus de $200 millions. les pertes annuelles occasionnées par ce phhomène.
Tableau 1.1: Substrats et m6tabolites produits par les principales bactdnes d'altération des viandes fraîc hesaob.
Type respiratoire Substrat Métabolites
Pseudomonas Ahbic ~lucose' Mucus
Acides amines2 Sulfites
Acide lactique3 Esters
Acinetobacter
Mo raxella
Alte romonas
putrefaciens
Brochothrix
thennosphacta
Enterobacter
Lactobacillus
Anaérobie
facultatif
Anaérobie
facultatif
Anaerobie
facultatif
Anaérobie
Acides aminés' Esters
Acide lactique2 Sulfites
~ l u c o ~ e ' H2S
Acides amines2 Sulfites
~lucose ' Acide adtique
Acides amin6s2 Acé toïne
Acide butyrique
Acide
isovalérique
Acide lactique
Acides gras
volatiles
~lucose' Amines
Acides amines2 Sulfites
Acide lactique3 H2S
~lucose' Acide lactique
Acides amin6s2 Acide gras
volatils
'. Source: Lambert et al. (1991).
b. Les numérotations dans la colonne substrat indique l'ordre d'utilisation de ces substrats
par les microorganismes.
13. Contrôle des microsrgarnismes sur les viandes et tes produits camés
il existe actuellement plusieurs mCthodes acceptables qui peuvent être utilisées pour
réduire la contamination des viandes et produits c a d s par les micro-organismes et leur
prolif6ration (Dona, 1997). Ces methodes comprennent: i) le rinçage avec de l'eau froide
ou chaude (Gill et Badoni, 1997; Dorsa et al., 1998; Castillo et al., 1998); ii) la
pasteurisation la vapeur (Nutsch et al., 1997); iii) le traitement avec des solutions
antirnicrobiennes ii base de chlore (Kotula et al., 1974; Skelly et al., 1985; Marshall et al.,
1977). d'acides organiques (Cutter et al., 1997; Dorsa et d., 1998; Rasai et al., 1997) ou de
phosphate visodique (Dorsa et al., 1998). Au cours âes demiéres années, des ktudes
intensives ont 6té faites afin de d6terminer l'efficacité relative de ces diffdrentes méthodes
(Dickson et Anderson, 1992 ; Hardin et al., 1995). Parmi les antimicrobiens, les acides
organiques ont fait l'objet d'un plus grand nombre de travaux (Anderson et al., 1979 ;
Brackett et al., 1994 ; Cutter et Siragusa, 1994 ; Dickson. 1991 ; Dickson et Anderson,
1992 ; Fratarnico et al., 1996 ; Hardin et al.. 1995 ; Phebus et al., 1997). probablement h
cause de leur statut 'GRAS' (generally recognized as safe).
1J.1. Acides organiques
Plusieurs acides organiques incluant les acides acétique, ascorbique, citrique,
formique, gluconique, lactique et propionique ont t t t Cvaluds pour leurs propriétés
antimicrobiennes. La plupart de ces études indiquent que les acides acétique. lactique et
propionique sont les plus efficaces pour la décontamination des viandes (Chung et
Goepfert, 1970; Brocklehurst et Lund, 1990; Dickson et Anderson, 1992; Ouattara et al..
1997r) Des extensions significatives de délais de conservation des viandes fraîches de
volaille ou & bœuf a et6 obtenues suite à un traitement avec de l'acide lactique ou un
tampon acide 1actiqueAactate de sodium (Zeitoun et Debevere. 1992 ; Prasai et al., 1997).
De même. Reynolds et Carpenter (1974) ont pulv6risé des carcasses de porcs avec un
mélange d'acide acétique et propionique, et ont obtenu une réduction de 2 logio du nombre
total & bactéries. Dorsa et al. (1997) rapportent tgalement des rdductions de 1.3 2 logio
sur des carcasses de bccufs après pulv6risation avec des solutions d'acide adtique ou
lactique.
Des Ctudes menées sur differentes populations bactériennes cibles ont permis de
déterminer le mode d'action des acides organiques et les facteurs qui influencent leur
efficacité. Ouattara et al. (1997a) ont test6 plusieurs acides organiques contre des souches
bactériennes d'altération des viandes et rapportent une relation dtroite entre la concentration
de la forme non dissocide et l'effet inhibiteur. Sous la forme non dissociee, les acides
diffusent librement à travers la membrane cytoplasmique des bacteries. se dissocient et
provoquent une acidification du milieu interieur (Freeze et al., 1973 ; Salmond et ai., 1984 ;
Young et Foegeding, 1993). La température. le mode d'application, le temps d'exposition
et le type & tissu sont aussi des facteurs qui influencent l'efficacitd antibact6rienm des
acides organiques (Grcer et Dilts, 1992 ; Phebus et al., 1997).
1.3.2. Acides gras et les huiles essentielles
De nombreuses 6tudes ont également et6 faites sur les acides gras à longues chaînes
et les huiles essentielles extraites d'tpices. Bien que leur utilisation comme agent
antimicrobien sur les viandes et les produits cames soit peu répandue, ces composCs ont
rtvCI6 des propribtks antibactériennes et antifongiques vis-il-vis de plusieurs micro-
organismes présents dans les aliments (Kabara, 1981 ; Russel, 1991 ; Shelef et al., 1980).
Les acides gras et les huiles essentielles permettent de réduire de nanitrc significative les
populations bactériennes de Salmonella typhimurium et Stuphylococcus aureus (Juven et
al., 1994 ; Karapinar et Aktug, 1987 ; Paster et al., 1990). Listeria monocytogenes (Aureli
et al., 1992 ; Wang et Johnson, 1992) Vibrio paruhaemoliticus (Karapinar et Aktug, 1987 ;
Shelef et al., 1980) et Clostn'dium botulinum (Ababouch et al., 1992). Ouattara et al.
(1997b) rapportent dgalernent des propriétds antibactériennes contre BrochothBr
thermosphactn, Cornobacterium piscicola, Lmtobacillus curvatus, hctobacillus sake.
Pseudomonasjluorescens et Serratia liquefaciens.
On peut remarquer de la litterature que la plupart des travaux sur les substances
antimicrobiennes ont et6 rdalis4s sur les bactbries pathogènes (Aureli et al., 1992;
Brocklehurst et Lund, 1990; Chung et Goepfert. 1970; Juven et al., 1994; Paster et al.,
1990 ). Comme le mentionne Greer et Diits (1992), seulement quelques Ctudes ont étd faites
sur les bacteries d'altération. Les différentes recherches sur le contrôle des micro-
organismes des viandes et produits camés par l'utilisation d'agents antirnicrobiens ont aussi
mis en évidence certaines imperfections. Par exemple, Dickson et Anderson (1992)
rapportent que l'application directe des acides organiques il la surface des produits
s'accompagne d'une action antibactCrienne de tds cowte durée. Siragusa et Dic kson (1992)
et Toms et al. (1985) expliquent ce phtnomène par le fait que les composés actifs diffusent
rapidement vers l'intkrieur des produits, entraînant une diminution de la concentration en
surface où a lieu g6neralement la croissance microbienne.
1.4. L'emballage antimicrobien
Selon plusieurs les procedés actuels de conservation par usage de substances
antirnicrobiennes pourraient être améliorés si les composds actifs étaient maintenus à la
surface des produits pour une période suffisamment longue (Gennadios et al., 1997; Kester
et Fennema, 1986; Labuza, 1996; Torres et al., 1985). Cette idée est A la base du
développement du concept d'emballage antimicrobien qui consiste à incorporer les agents
antimicrobiens directement dans des films ou enrobages qui entrent en contact avec les
aliments (Hotchkiss, 1995). Dans ces conditions, la diffusion des composés antimicrobiens
vers l'intérieur des produits est considdrablement ralentie telle représentée schématiquement
dans la figure 1.1.
Antimicrobiens
Antimicrobiens
B
Figure 1.1: Représentation schematique de la conservation des aliments sans (A) ou avec (B) un enrobage ou un film servant de support h 1 'incorporation d 'agents antirnicrobiens.
1.4.1. Matkriaux utilisés
L'un des polymères le plus couramment utilisCs comme support à l'incorporation de
composés chimiques antimicrobiens est le polyéthylène basse densite. Weng et Hotchkiss
(1992, 1993) ont incorporé dans ce type de matériel des agents fongiques dans le but de
contrôler la croissance de levures et de moisissures h la surface des fromages. De même
Ming et al. (1997) ont fixe des bactériocine sur âes films plastiques en vue d'inhiber la
croissance de Listeria monocyzogenes sur les vianàes. D'autres types de films
antimicrobiens ont été egalement fabrique par couplage chimique de divers composks actifs
avec des films ionornCriques (Halek et Garg, 1989; Weng et al.. 1997).
Au cours des demi2res années, des efforts considérables ont dté fait dans le domaine
de la recherche pour trouver des polymères naturels biodégradable capable de jouer le
même rôle (Gennadios et al., 1997). Cette nouvelle tendance s'explique par l'apparition de
nouveaux besoins parmi lesquels il y a: i) la recherche de nouvelles méthodes de
conservation plus efficaces capables de maintenir la fraîcheurs des produits pendant des
périodes plus longues; ii) la lutte contre la pollution de l'environnement notamment par les
emballages plastiques non dégradables; iii) la recherche de marché pour des sous-produits
agro-industriels qui sont sous-utilises.
La plupart des rdsultats disponibles ont étC obtenus en utilisant des polysacchari&s
ou des protéines comme matériaux de basse. Ainsi des emballages antimicrobiens contre
Listeria monocytogenes ont et6 obtenus par fixation dt Nsine ou & pédiocine sur des films
de cellulose (Ming et uL, 1997) ou par immobilisation d'acides organiques dans un gel
d'alginate de calcium (Siragusa et Dickson, 1992).
Recemrnent. la possibi1itC d'utilisation du chitosane a 6tC aussi dtudiée. Le chitosane
est un polysaccharide amint qui possède dtjh de nombreuses applications industrielles,
notamment dans le domaine & la cosmdtologie et du traitement des eaux usCes (Demarger-
An& et Domard. 1994). Ce polymère possède aussi de bonnes propriétes d'immobilisation
(Kaya et Picard. 1996; Knorr et Teutomico. 1986) et de formation de liaison covalentes
avec des cornposks chimiques anioniques (Maxtino et al., 1996; Mi et al.. 1997; Pandya et
Knorr, 1991). Un des avantages du chitosane par rapport aux autres polymères naturels est
sans doute le fait qu'il possède lui-même des propriCt6s antibactériennes et antifongiques
@armadji et Izumimoto, 1994; El Ghaouth et al.. 1992). Toutes ces caracteristiques font du
chitosane un matériel de choix pour le ddveioppement d'un emballage antimicrobien.
1.4.2. Contrôle de la diffusion des antimicrobiens
Plusieurs auteurs ont démontré que la libération des composds chimiques incorporés
dans des films de polymére dtpend en grande partie des propriétds d'absorption d'eau des
films (Amand et al., 1987; Malley et al., 1987; Shantharnunhy et Arninabhavi. 1990;
Peppas et Branon-Peppas, 1994). Selon ces auteurs, la libération des composés incorporés
se fait simultanément avec la pénétration d'eau dans la matrice des polym&res. Compte tenu
de ce fait, les films de polysaccharides et & prottines qui sont de nature hydrophile (Kester
et Fennema, 1986; Gennadios et al., 1997), ont tendance à libérer très rapidement les
composCs incorporbs dans leur matrices.
Un des moyens utilisés pour réduire ce phtnoméne est la CO-incorporation de
composés lipidiques. Ces composés ralentissement le transport des substances
antirnicrobiennes en augmentant la tomiositd du réseau polysaccharidique ou protbique
(Callegarin et al., 1997; Redl et al., 19%) ou en réduisant la taille des pores (Papadokostaki
et al., 1997). En utilisant ce procédC, Redl et al. (19%) ont rCussi à rtduire de 20 A 50% le
coefficient de diffusion de l'acide sorbique incorporé dans des films de gluten de bld en y
ajoutant de la cire ou des monoglycCrides acCtylCs. De même, l'addition de divers acides
gras a permis de réduire la pedabilitd du potassium de sodium à travers des films de
m&hylcellulose ou d'hydroxypropyl m&hylcellulose (Vojdani et Toms, 1990) et la
permdabilité A la vapeur d'eau des films de chitosane (Wong et al.. 1992).
Ce concept d'emballage antimicrobien dans lequel des substances lipidiques sont
incorporées dans le but de réduire la diffusion des composds actifs, pourrait devenir encore
plus efficace si les substances lipidiques elles-mêmes sont dotées de propriétds
antirnicrobiennes.
1.5. Objectifs
Le but ultime de cette &ude Ctait de développer un emballage antibactdrien capable de
coctrôler la croissance microbienne à la surface des viandes et produits camés et de
prolonger la durée de conservation de ces produits. Cet objectif général a Cté realisé en trois
&tapes.
Dans un premier temps, des agents antimicrobiens comprenant des acides organiques.
huile essentielles et acides gras il longues chaînes ont et6 sélectionn6s sur la base de leur
habilite à inhiber la croissance de six bactbries dlalt&ation des viandes: Brochothrix
thenosphocta, Cantobac te rium piscicola, LactobacilIus curvatus, Lactobacillus sake,
Pseudomonas fiorescens et S e m i a liquefaciens. Les résultats de cette etape sont
pdsentts dans les chapitres 2.3 et 4.
Dans un deuxième temps, les agents antimicrobiens les plus actifs ont kt6 incorpods
dans une matrice de chitosane de maniere à obtenir des films antimicrobiens. Une étude des
caractéristiques de diffusion a étt réalisée en milieu liquide et fait l'objet du chapitre 5.
Dans la dernière étape qui fait l'objet du chapitre 6, les caractéristiques de diffusion
ont été étudiées en milieu réel sur divers produits carnés, suivi d'une évaluation de I'activitd
anti bac tdrienne des nouveaux films.
CHAPITRE 2
INHIBITORY EFFECT OF ORGAMC ACIDS UPON MEAT SPOILAGE
BACTERIA
Blaise Ouattara, Ronald E. Simard, Richard A. Holley. Gabriel LP. Piette and Andrt? Bégin
Publié dans:
Journal of Food Protection, (1997), 60: 246-253.
2,1, Abstract
The relative ability of acetic, benzoic, cibic, lactic, propionic, and sorbic acids to
inhibit the growth of six common meat spoilagc bacteria (Brochuthrir themiosphacta,
Camobactenwn piscicola, Lactobacillus curvcuu~, Lactubacilus s a k , Pseuàomonas
fluorescens, and Sem& liquefaciens) was compared under otherwise optimum conditions
(BHI or MRS broths; 20°C). Because of their low solubility in the growth media. benzoic and
sorbic acids could only be used in low concentrations (below 0.15%, w/v) and did not
efficiently inhibit bacterial growth. Al1 other acids totally inhibited growth at concentrations
ranging from 0.1% to 1% (wlv). On a weight basis, acetic acid was found to be the most
inhibitory, followed by propionic, lactic, and cihic acid, while the order of efficiency was
reveeed (citric >lactic >propionic >acetic) when the acid concentrations were expnssed on a
molar basis or when the acid effectiveness was evaiuated relative to the concentration of
undissociated molecules. Overall, the lactobacilli were the bacteria most fesistant to the action
of organic acids, followed by P. fluorescens and S. liquefaciens, while B. thennusphacta and
C. piscicola were considerably more sensitive.
Keywords
Organic acids, meat, spoilage, bacteria.
2.2. Introduction
The globalization of the world economy has resulted in meat and meat products king
s hipped over ever incrcasing distances to reach forcign markets. This has created a demand for
efficient packaging concepts and technologies which will guarantee that products remain safe
and wholesome over long periods of time. Significant propss has already been ma& in this
respect by changing the gaseous environment at the product surface, in a way that hinders the
growth of pathogenic or spoilage bacteria This is achieved, for example, in vacuum-
packaging, modified atmosphere packaging (MAP), or controlled atmosphere packaging
(CAP) (Hudson et al., 1994; Ooraikul and Stiles, 199 1 ; Renerre and Labadie, 1993; Young et
al., 1988). In al1 cases, the package has an indirect role as it merely serves to entrap the
atmosphere detnmental to bacterial growth.
According to the microbiological concept of hurdle technology (Leistner and R&l,
1976). the preservation of rneat products could M e r be improved by combining the actions
of packaging and antimicrobial treatments. Although, in theory, any antimicrobial agent could
be used in combination with packaging, organic acids have recentiy received the most
attention, ükely due to their GRAS (generally recopnized as safe) status. For exarnple. the
shelf-life of refiigerated fresh poultry un&r MAP was slightly increased after &contamination
of the meat with lactic-lactate buffer (Zeitoun and Debevere, 1992). A h , reftigerated
vacuum-packaged larnb carcasses spoiled slower when the meat had been treated with acetic
acid prior to packaging (Anderson et al., 1988). As well, treatments of prerigor cooked beef
(Abugroun et al., 1993), uncmd turkey b m t meat (Miller et al., 1993), or cold-smoked
salmon (Pelroy et al., 1994) with various organic acids improved the microbiological quality
of the packaged products during storage.
The process would be much simplified if the antimicrobial agent could be emkd&d
into the packaging film h m which it would slowly migrate to act on the product surface. The
resulting active package would thus possess antimicrobial properties of its own. From a review
of the work on the inclusion of antimicrobial agents into packaging rnatenals (Hotchkiss,
1995), it is clear that further research is needed in the area. However, the feasibility of the
technology has ken demonstrated in two studies in which the p w t h of molds on cheese was
&layed by irnizalil (Weng and Hotchkiss, 1992) or benzoic anhydride (Weng and Hotchkiss,
1993) incorporated into LDPE films.
An investigation is currently un&r way in our laboratory to develop efficient active
packages for the preservation of meat products. In order to do so, one must first know how the
regular meat flora is affected by the antibacterial agents cumntly available for food uses, in
particular the organic acids. Yet, while the effects of organic acids on meat-borne pathogens
are well documented (Ahamad and Marth, 1989; Brocklehurst and Lund, 1990; Buchanan and
Golden. 1994; Buchanan et al., 1993; Chung and Geopfert, 1970; Conner et al., 1990; Graham
and Lund, 1986; Houtsma et al., 1994; Littie et d., 1992; Ostling and Lindgnn, 1993; Young
and Foegeding, 1993), little is known about the susceptibility to acids of meat spoilage
bacteria (Gner and Dilts, 1992). The present work therefore evaluates the cfficacy of various
organic acids to control the growth of meat spoilage organisms.
23. Material and rnethods
211. Organisms and ailturm
Camobactenwn piscicola (ATCC 43224). Lactobacilfus cumutus (ATCC 25601). and
Luctobacillus sake (ATCC 15521) wen obtained from the Amencan Type Culture Collection.
Roc kville, Md, USA. Pseuubmonas fluorescens and Brochothrix thennosphoaa were isolated
from beef stored at 4°C (Farber and Idziak, 1984). Serratia liquefaciens was isolated from
vacuum packaged Bologna (Food Research and Development Centre, St-Hyacinthe, Quebec).
Lyophilized stock cultures were prepared from suspensions of bacterial cells in reconstituted
skim milk (skim milk powder in deionized water, 20% w/v final concentration) containing 5%
sucrose (w/ v). P. fluorescens, B. thennosphactu, and S. liquefaciens were grown aerobicall y in
brain heart inhision broth (BHI, Difco Laboratones, Detroit, Mi., USA). C. piscicola, L
curvatus, and L sake were grown in lactobacilli MRS broth @ifco) in an atmosphe~ enxiched
in h ydrogen and carbon dioxide (Gaspak anaerobic system; Bec ton Dickinson, Cockey sville,
Md, USA). All incubations wcrc done at 20°C without agitation. Standardized cultures were
obtained h u g h two successive 24 h growth cycles in the appropriate medium. Cells from the
standardized cultures wen subsequently inoculated in h s h medium and incubated (20°C
without agitation) for 6 h or 9 h (C. piscicola only) to obtain working cultures containing
appmximately 10' cFW.mL1.
2.3.2- Reparation of the bacterial suspeasions for the gmwth inhibition ucperiments
Anal ytical grade acetic acid glacial (99.7% wl v; Fischer Scien ti fic. Nepean, Ontario),
citric acid (monohydrate, >99% pure; Anachernia, Montreal, Quebec), DLLactic acid (88%
W/V; American Chemical, Montreal, Quebec), and propionic acid (99% wlv; Fisher Scientific)
were first added separately to sterik BHI or MRS bmth to final concentrations ranging from
O. 1 % to 1 % (w/v). Anal ytical grade benzoic (crystals. >99.5% pure; Anachernia) and sorbic
acids (>99% pue; American Chemical) were also adàed to the stenle broths but, due to their
limited solubility, only one concentration (0.15%) was usod.The growth media containing the
acids were subsequently inoculated (1: LOO) with each of the working cultures to reach final
bacterial concentrations of about 10' CN.~L- ' . Regular BHI and MRS broths (containing no
organic acid). inoculated in the same way, served as positive controls for growth.
2.3A Growth inhibition experiments
The growth inhibition experiments werc p e r f o d in % well (ü-shaped)
rnicrotitration plates (Nunc, Karnstmp, Denmark). Aiiquots of the v i o u s bacterial
suspensions in growth media with or without organic acids were fiat introduced into two to
t h m replicate wells (200 pUwell), as well as equal s i x aliquots of uninoculated media,
which serveci as negative contmls for growth. The microplates wen subsequently incubated
for 120 hours at 20°C in aerobic (P. fluorescens, B. thermosphocta and S. liquefuciens) or
anaerobic conditions (C. piscicola, L curuotus, and L suke). Water cvaporation was avoi&d
by incubating the plates in a humid atmosphere. Gmwth was evaiuatcd at regular intervals by
absorbance measwements at 540 nm in an automated plate reader (Lambda microplate reader,
Perkin Elmer, Norwalk, Ci, USA) after suspension of the sedimenteci cells on a rotary shaker
(Junior ûrbic Shaker. Iab-line Instniment, MeIrose Park, II, USA; 1500 rpm, amplitude 2
cm).
Tn order to differrntiate the inhibitory effects of organic acids h m those of pH alone,
the pH values of the p w t h media coniaining organic acids wen first measured for each
concentration used. Regular BHI and MRS meâia, containing no organic acids, were
subsequently adjusted to these pH values with HCl, the pH-adjusted media were then
inoculated with the various bacterial suspensions, and the extent of growth over time was
monitored as descnbed above. The concentrations (rnmole.~-') of undissociated acetic, lactic,
and propionic acids were calculated using the fomula: undissociated acid = total acid [l +
1 0 ' ~ ~ - The same formula was used to calculate the concentration of undissociated citric
acid, taking pKal as the relevant pKa value. This. in effect, neglects the contributions of the
second and third carboxylic groups to the equilibrium. but the resulting error is no more than
1% of the total acid concentration, in the range of pH values (4-6.5) used in the experiments.
2.35. Data Molysis
The extent of growth (AM) in the presence or absence of organic acids, afkr 24 h of
incubation and at regular time intervals thereafter, werr compared by calculating lem square
means through the GLM procedure of the SAS statistical package (SAS Institute, Cary, Nc,
USA). The Stuclent t test was used at 24, 36, 48, 72, 96, and 120 h for point-by-point
differentiation of the effects of organic acids from those of pH. Differences betwcen mean
values were considend sigrifkant whcn PQ.05.
The addition of organic acids to BHI and MRS to a final concentration of O. 1% (wiv)
caused a drop in pH ranging from 0.4 to 0.9 and from 0.6 to 0.8, respectively. &pendhg on
the acid (Table 2.1). Further dccreases in pH were obtained with inc~asing final
concentrations of the acids, to reach minimum pH values of 3.84.4 @HI) and 42-46 (MRS).
Under comparable conditions (same medium and sarne acid concentration), acetic and
propionic acids (pKa 4.8 and 4.9, respectively) were considerably less dissociated than lactic
acid (pKa 3.8). while citric acid (pKal 3.1, pKa2 4.8, pKa3 6.4) was the most dissociated of
dl.
Typical graphs illustrating the effects of acetic, citric. lactic, or propionic acid on the
growth of selected meat spoilage bactena arc shown in Figure.2.1. in generd. the presence of
organic acids in the growth media rcsultcd in growth inhibition. Inhibition took the fom of
longer lag periods, lower growth rates, andor lower bacterial numbers in stationary phase. The
extent of inhibition depended both on the bacterium and on the acid considemi. The stmgest
inhibition was observed with C. piscicola which could not grow at dl in the presence of acetic
or propionic acid (O. 1% in the p w t h medium). On the other hand, the highest concentration
of citric acid used (1%) could not prevent L curvafus or P. fluorescens h m growing for more
than 48 h and 72 h, respectively. A11 other bacterium-acid combinations yielded intermediate
âegrees of inhibition.
Large ciifferences were obxrved between the responses of the different organisms to
the presence of organic acids (Figure 2.1). In sorne cases, the extent of inhibition graduall y
increased with incmsing acid concentration in the p w t h medium (B. thermosphucta in
general; propionic acid on S. liquefaciens and on P. jluorescenr; acetic acid on L curvufus). In
other cases, the concentration of acid in the medium had to reach a cntical value before growth
inhibition staned. Beyond the critical value. inhibition generally increased in a progressive
manner (citric acid on L soke and P. /luorescens). Still in other cases, growth was unaffected
until a critical concentration of the acid was reached and inhibition was total beyond this
concentration (lactic acid on S. liquefaciens md P. fluorescens).
To faciütate the overall assessrnent of the results, the effects of organic acids on the
various bacteria were summarized in a condensed format (Table 2.2). Regardless of the
bactenum, acid concentrations of 0.5% or above always produced a significant (P4.05)
inhibition of growth over the entire time span (24-120 h) in which variance analysis was
performed. At lower acid concentrations, the various organisrns demonstrated different
degrees of sensitivity to the presence of the acids. The lactobacilli were the most resistant
organisms and they werr not affected by acid concentrations lower than 0.3%. In contrast, the
growth of C. piscicola and B. thennosphaca was always affected by the presence of acids,
even at the lowest concentration used (0.1%). Finally, P. fluorescens and S. liquefaciens
exhibited intermediate sensitivities.
The relative effectiveness of the various acids to inhibit bacterial gmwth is best
exp~ssed in tems of their minimum inhibitory concentrations. h this snidy, the minimum
inhibitory concentration of an organic acid was defined as the minimum concentration which
resulted in complete growth inhibition (no detectable increase in absorbante) during the whole
experiment (0-120 h). Accordingly, on a weight basis, acetic acid was found to be the most
inhibitory acid for al1 the bactena tested (Table 2.3), followed by propionic and lactic acids,
while citric acid was the least effective to prevent bacterial pwth . The order of effcctiveness
was reversed (citric >lactic >propionic xcetic). however, when the acid concentrations were
expressed on a molar basis or when the effectiveness was evaluated relative to the
concentration of undissociated acid molecules.
The effects of benzoic and sorbic acids on the p w t h of meat spoilage bacteria was
only tested on B. thermosphacta. P. ~uorescenî, and S. liquefaciens. Both acids partially
inhibited the growth of the three organisms at a concentration of 0.15% (Figure 2.2). Higher
concentrations could not be used due to the Limited solubility of the acids in water.
In general, growth appeared considerably mon inhibited in the presence of organic
acids than when the pH was lowered to the s m e value with hydrochloric acid : (see typical
example in Figure 2.3A: effects of acetic and hydrochloric acids on S. liquefaciens). In some
cases, however, the difference k t w c n i the effects of organic acid and of pH alone seemed less
pronounced and was only noticeable at the highest acid concentrations (for example lactic acid
on L soke; Figure 2.3B). Analysis of variance of the overall nsults confimed that, most of
the tirne, the presence of organic acid in the medium inhibited gmwth more (Pc0.05) than did
pH alonr (Table 2.4). This was always true with acid concentrations of 0.5% or higher.
2 3. Discussion
The ~su l t s of preiiminary experiments (not shown) in which the sensitivity of B.
themsphactu, P. fluorescens, and S. liquefuciens to acetic, citric, lactic, and propionic acids
w u evaluated at 4OC, 8OC, and 20°C indicated thai the extent of p w h inhibition by the acids
increased regularly with decreasing incubation temperatures. Similar results were obtained
when Listerio rnonocytogenes was grown in tryptose broth at 7OC, 13OC, and 21°C in the
presence of various concentrations of acetic, citric, and lactic afids (Ahmad and Manh,
1989). In view of these results, a 20°C incubation temperature was selected for the present
study. with the understanding that the nsults obtained would represent the sensitivity of meat
spoilage organisms to organic acids under optimal growth conditions and that a pater
inhibition might be expected to occur at the surface of meat under commercial storage
conditions, when spoilage bactena would be stressed by lower temperatures, nutrient
limitations, and cornpetition with neighbouring organisms.
The growth of B. themosphacta, P. fluorescens, and S. liquefuciens was on1 y partial1 y
inhibited when benzoic and sorbic acids were ad&d to the growth medium at a final
concentration of 0.15% (wlv). Complete inhibition would qu i r e pater concentrations of the
acids and these could not be obtained due to limited solubility. The other organic acids tested
(acetic, citric, lactic, and propionic) completely inhibited the growth of each of the six selected
meat spoilage organisms, at final concentrations ran@ng from 0.1% to 1% (w/v). This was to
be expected since the partial dissociation of the acids in solution causeâ the pH of the growth
media to drop by as much as 2.4 and 3.5 pH uni& for MRS and BHI broths, mpectively, and
since meat spoilage bactena. iike most bactena associaîed with food, are neutrophilic. As
such, they must keep an interna1 pH slightly higher than that of the p w t h medium since it is
the resulting pH gradient which causes protons to fiow through the membrane-bound
ATPases, thus producing the ce11 energy. This implies that neutrophilic bacteria must
continuously eliminate protons h m their cytoplasm in order to maintain the vital proton
gradient between the outside and the insi& of the ce11 (Booth, 1985). The process is active and
consumes energy. Increasing the acidity outside the ce11 consequently forces the bacteriurn to
spend larger arnounu of energy to elirninate the incorning protons, to the detriment of growth.
Compatison of the detrimental effects of organic acids on bacterial growth wi th that of
hydrochloric acid indicated that growth inhibition by organic acids was not entirely due to the
acidification of the gmwth media through acid dissociation. Indeed, it is well known that the
undissociated organic acid molecules, king non-ionized, diffuse through the ce11 wall and
dissociate in the cytoplasm due to its higher pH (Freeze et d, 1973; Saimond et al.. 1984;
Young and Foegeding, 1993). This generates an excess of protons in the ce11 which musi be
eliminated. Ultimately, the influx of protons surpasses the capacity of the ce11 to eliminate
them and growth stops. Active transport mechanisms also exist for the uptake of dissociated
organic acids molecules. Partially ionized citrate, in paiticular, can enter the cells of many
bac teria (e.g . Bacillus subtilis, numerous Enterobacte~aceae, Loctococci*r lactis, Leuco~~~stoc
spp.) through the action of a proton- or cation-dependent citrate pennease (David et al.. 1990,
Hugenholtz et al.. 1993; Stamnburg and Hugenholtz, 1991).
The minimum pH values at which Salmowllla<! could initiate p w t h in trytpicase soy
broth were found to k 4.05.4.40, 5.40. and 5.50 when the pH was adjusted with citric, lactic,
acetic, and propionic acids. nspectively (Chung and Geopfen, 1970). leading to the statement
that propionic acid was the most efficient to control the growth of Sulmonellue~ followed, in
that order, by acetic. lactic, and citnc acid Also, the antibacterial properties of acetic, citric,
and lactic aciL against Yersinia enterocolitica (Bmklehurst and Lund, 1990). Listeria
moriocytogenes (Ahamad and Marth, 1989; Young and Foegeding. 1993). and various
organisms endogenous to meat (Greer and Dilts, 1992; Sirami, 1987) ranked in the order
acetic >lactic >citic, based on mwic or molar total acid concentrations, with only small
differences between the efficacies of acetic and lactic acids (Ahamad and Marth, 1989;
Brocklehurst and Lund, 1990; Greer and Dilts, 1992). Similady, retic, propionic, and lactic
acids were about equally efficient in causing growth inhibition of meat spoilage organisms
(this study), when the total acid concentrations were expressed on a massic or molar b i s .
Citric acid however, was the most effective acid on a molar basis, reflecting its highcr
molecular weight.
More striking differences were observed when the respective antibacterial properties
of the acids against meat spoilage bactena were evaluated relative to the concentrations of
undissoci ated acid molecules (present study), with ci tric acid king the most effective acid,
followed by lactic acid, while propionic and acetic acids wen markedly less effective. This is
consistent with the nsults of previous studies (Buchanan and Golden, 1994; Young and
Foegeding, 1993) and likely reflects the fact that the lower capacity of citric and lactic acids to
enter bacterial cells is compensated by their greater capacity to dissociate inside the ce11 and
thus acidify the ce11 c ytoplasm (Young and Foegeding, 1993). Also, at similar concentrations.
citric and lactic acids tend to dtcrease the pH of the growth medium more than acetic and
propionic acids. In addition, citrate ions have k e n reported to chelate polyvalent cations
essential to microbiai growth (Beuchat and Golden, 1989; Russell, 1991).
As mentioned by Greer and Dilts, (1992), only sparse information exists on the effects
of organic acids on meat spoilage organisms. Several studies have âemonstrated that the
growth of spoilage bacteria on meat carcasses or cuts was slowed to various degrees after
treating the meat with organic acids, by dipping or spraying (Acuff et al., 1987; Anderson et
al., 1988; Gauthier and Jacquet, 1991; Sirami, 1987), but cornparison of the respective
susceptibility of the various spoilage bacteria to organic acids was no1 evaluated. Only in one
study were meat spoilage organisms in pure culture subrnitted to acid treatments under sirnilar
circumstances (Greer and Dilts, 1992) and the results indicated that B. thennosphacta was
more sensitive than P. fiagi to both acetic and lactic acids. The present study therefore brings
much needed information and shows large differences between the susceptibility of meat
spoilage bacteria to the antibacterial effects of organic aciL.
Of the six selected meat spoilage bactena, C. piscicola, fomerly regarded as an
atypical Luctobacillus which is often found in meat (Collins et al., 1987). was the most
affected by the presence of organic acids. This is consistent with the fact that this organism
grows best between pH 6 and pH 7 (Hu et d., 19&)), compared to pH 5.5-6.2 for L curvmus
and L soke (Kandler and Weiss, 1986). As a consequence, C. piscicola is expected to be more
sensitive than lactobacilli to extemal or intemal pH reduction. In addition, the growth of C.
piscicola in regular MRS broth, containing no organic acid, was considerably slower than the
growth of al1 the othcr organisrns, and not as profuse. Any added stress was therefon expected
to reduce growth to a large extent. Similady, the growth of B. themsphacta in regul a . BHI
broth was not as extensive and was consider5ly more affected by organic acids than the
growth of the lactobacilli, P. jZuorescenr, or S. liquefaciens. This expands on the previous
report that B. thermosph<lca was more sensitive than Pseudomonas to the action of acetic and
lactic acids (Greer and Dilts, 1992).
In the present study, the two lactobacilli were the spoilage organisms most resistant to
the action of organic acids. This is not surprising since lactobacilli, as memben of the lactic
acid bacteria group, excrete lactic acid as a result of sugar fermentation. By doing so, they
continuously acidify the surrounding medium and must therefore be equipped to survive in an
acidic environment. Inâeed, in addition to the universal cation-proton antiports for the
excretion of protons from the ceil (Booth, 1985), lactobacilli possess specific carrier-mediated
transport mechanisms for the rapid excretion of protonated lactate, as well as enzymes such as
decarboxylases and deaminases which contribute to maintain pH-homeostasis (Hutkins and
Nannen, 1993).
The present study has &ah with the susceptibility of meai spoilage bacteria to the
action of organic acids, in liquid media. In effect, the ability of the sarne acids to control
bacterial growth on meat surfaces will depend. to a great extent, on the degree of acid
dissociation. itself influenced by the buffering capacity of the smunding environment. In this
respect. the results of additional experimenu (not shown) indicated that BHI and MRS bmths
were as strongly buffered as various mat homogenates. In reality, the water film surroundhg
packaged meat producu would normally be poonr in organic matter than meat homogenates
and, therefore, less smngly buffered. This suggests that organic acids wili be less dissociated,
thus more inhibitory, at the surface of meat products than they wen in the present study.
Obviously, many other considcrations will have to be addressed in the developement of an
antimicrobial package for meats, such as the respective ease with which the different acids can
be ernbedded into a packaging film, their respective rates of diffusion from the surface of the
product to the intenor, and their effects on non-microbiological product quality ataibutes,
including the organoleptic characteristics.
2.6. List of tables
Table 2.1. Relative degrees of dissociation of the various organic acids as a function of their
concentration in the growth media and of the resulting pH
Table 2.2. Growth inhibition by organic acids of the six meat spoilage bactena
Table 2.3. Minimum inhibitory concentration (MICs) of acetic, propionic, citric . and lactic
acids for growth of six meat spoilage bacteria
Table 2.4. Differentiating the inhibitory effects of pH and organic acids on the growth of six
meat spoilage bacteria
2.7. L U of Figures
Figure 2.1. Typicd growth patterns of rlected meat spoilage bacteria in media containing or
not various amounts of acetic, propionic, lactic, and citnc acids. Values plotted are the means
of three nplicate measurements
Figure 2.2. Growth of BrochorhrLr thennosphucta, Pseudomonas fluorescens, and serratia
liquefaciem in media with or without benzoic or sorbic acid (0.15% [wt/vol] final
concentration). Values plotted are the means of two nplicate measurements.
Figure 2.3. Growth of ( A ) Serratia liquefaciem and (B) Lactobacillus sake in media with or
without various amounts of organic acid or hydrochlonc acid. Values plotted are the means of
thRe replicate measwments.
Table 2.1
Acetic acid Ropionic acid Lactic acid Citric acid (pKa=4.8) (pKa=4.9) (pKa=3.8) (pKal=3.1)
Organic Acid pH c m a pH Cd. pH Cu&. pH Cm.
Conc. (%)
BHI O 7.2 - 7.2 - 7.2 - 7.2 -
' CUHdisJ. Concentration of undissociated acid (mmole.~')
Table 2.Za
-
Organic L L C. B. P. S. acids (96) curvatw sake piscicola thermosphacta fluorescens liquefaciens
Acetic o. 1 0.2 0.3 0.5 0.75 1
Propionic O. 1 0.2 0.3 0.5 0.75 1
Lac tic O. 1 0.2 0.3 0.5 0.75 1
Citric o. 1 0.2 0.3 0.5 0.75 1
' Results indicate whether bacterial growth was significantly (@.OS) reduced (+) or not (-) in the presence of various amounts of organic acids. compved io the level observed in regular medium, containing no acid. Numbers in parentheses are ranges of t h during which growth reduction was significant When no range is specified. reduction was significant over the whole 24-120 h range.
Table 2.3"
Organic acids Lcurvar us L sake C. piscicola B. thennosphac ta P. fluorescens S. liquefaciens
Acetic (MW=60.05) Total =id (%) Total acid (mmole.~-') Undissociated acid (mmole.~-')
Propionic (MW=70.05) Total acid (%) Total acid (mmo1e.L") Undissociated acid (mmole.~'')
Lactic (MW=9û.08) Total =id (96) Toral acid (mmole.~") Undissociated acid (mmoleL1)
Citric (MW=I92.12) Total acid (%) Total acid (mmo1e.L') Undissociated acid (mmo1e.L")
M C reported (in % and mmo1e.l") are the minimum acid concentration in the growth media which completely inhibiteû bacterial growth (no deteciable absotbance) duing L e whole 120 h experiment.
b The maximum concentration of citric acid used did not completely inhibit the growth of L curvatus.
Table 2.4 '
Organic L L C. B. P. S. acids (%) curvatws sake piscicola thennosphacta fluorescens liquefaciens
Acetic o. 1 0.2 0.3 0.5 0.75 I
Ropionic o. 1 0.2 0.3 0.5 0.75 1
Lac tic o. 1 0.2 0.3 O. 5 0.75 1
Citric o. 1 0.2 0.3 0.5 O. 75 1
a ResuIts indicate whether bacterial growth was significantly (@.OS) more reduced (+) or not (-) in the presence of various amounts of organic acids than as an effect of pH alone. Numbers in parentheses are ranges of timt during which effects were significantly different. When no range is specified. effects were significantiy different over the whole 24-120 h range.
NI. NO Inhibition; Neither the presence of organic acid nor pH alone produced a signifiant growth reduction.
NA. Not Applicable; Either the presence of organic acid or pH alone caused complete growth inhibition (no detectable absorbaace) during the whole 24-120 h range.
Figure 2.1
Acetic , L. cm)att/.s
2
Propionic
P. fiorescens !
Lac t ic
P. fluorescens
Citric
2
S lique facieris I
O 30 60 90 120
Time (h)
O 30 60 90 120
Time (h)
c. piscicola
O 30 60 90 120
Tirne (h)
Figure 2.2
Time (h)
* Control (without acid) -t- Benzoic acid ++ Sorbic acid
Figure 2.3
, S iiqtiefuclem Acetic rcid
B L. sake 1 lactic acid
O 30 60 90 120 Tirne (h)
+ Control (without acid) -+- 0.3% (wtlvol) organic acid + 0.3% (wt/vd) hydrochloric acid -t 0.5% (wtfvol) organic acid - 0.5% (wtlvol) hydrochloric acid
CHAPITRE 3
ANTUBACTERIAL ACTIVITY OF SELECTED FATTY ACIDS AND ESSENTIAL
OILS AGAINST SIX MEAT SPOILAGE ORGANISMS
Blaise Ouattara, Ronald E. Simard. Richard A. Holley, Gabriel J.-P. Piette and André Begin
miblie dans :
International Journal of Food Microbiology, (1997). 37 : 155- 162.
The antibacterial activity of selected fvty acids and essential oils was examinated
against two gram-negative (Pseudomonas jluonscens and Sematia liquefaciens). and four
gram-positive (Bmhotitrir therniosphacta, Cumobacteriwn piscicda, Lactobacillus
curwatus, and Lactobacillus d e ) bacteria involved in meat spoilage. Various arnounts of eac h
preservative wen added to BHI or MRS agars, and the minimum inhibitory concentration was
determinated for each organism. Essential oils wem analysed by gas-liquid chromatography to
determine the concentration of selected components commonly found in spices. Brochothrir
~hennosphacta, Pseudomonas fluorescens and Sermtia liquefaciens were not affected by
fatty acids, and generally overcarne the inhibitory effect of essential oils after 24 hours of
exposure. Arnong the fatty acids, lauric and palmitoleic acids exhibited the greatest inhibitory
effect with minimum inhibitory concentrations of 250 to 500 pg/ml, while myristic, palmitic.
stearic and oleic acids were completely ineffective. For essential oils, clove, cinnamon,
pirnento, and rosemary were found to be the most active. The 1/100 dilution of those oils
inhibited at least five of the six tested organisms. A relationship was found between the
in hibitory effect of essentid oils and the presence of eugenol and cinnarnaldehyde.
Keywods
Fatty acids, essential oils, meat, spoilage, bactena.
3.2. Introduction
The problem of safe preservation in the meat industry has p w n to be more cornplex
as today's products require longer shelf-life and pater assurance of protection h m microbiai
spoilage. Many attempts have been made to contrd rnicrobial growth at the surface of meat
and meat products with antimicrobial chemicals. For example, significant reductions of
rnicrobial growth were obtained by dipping or spraying meat with organic a d solutions
(Abugroun et al. 1993; Anderson and Maishall, 1989). However, pmervatives could not be
stabilized at the surface of food due to evaporation, neutralization (Siragusa and Dickson,
1992), and diffusion into the matrix (Toms et al., 1985).
Fatty acids and essential oils have also been show to possess antibacterial and
antifungal activities against many plant and food rnicroorganisms (Kabara, 198 1; Shelef et al.,
1980; Russel, 1991). Gram-negative bactena were show to be generaily more resistant than
gram-positive ones to the antagonistic effects of fatty acids and essential oils because of their
ce11 wall lipopolysacharide (Kabara, 1979; Branen et al., 1980; Russel, 199 1) but this was not
always mie (Karapinar and Aknig, 1987). In addition, most shidies to date have been done
witb pathogens such as Suimonellu ryphimuriwn and Staphylococcus aureus (Karapinar and
Aktug, 1987; Paster et al., 1990; Juven et al., 1994). Listenu monoqtogenes (Aureli et al.,
1992; Wang and Johnson, 1992), Vibno parohaemoliticus (Karapinar and Aknig, 1987; Shelef
et al., 1980), and Clostridium botulinum (Ababouch et al., 1992), and little is known about the
effect of these compounds on meat spoilage bacteria such as Camobacterium piscicola,
Lactobacillus curvatw and Lactobacillus suke
An investigation is currently under way in our laboxatory to &velop active packaging
materials for the preservation of meat products. As a fiat step, it was necessary to know how
the regular meat Rora was affected by antibacterial agents currently approved for food use. in
particular fatty acids and essential oils. The purpose of the present study was therefore to
evaluate the eficacy of various fatty acids and essential oils to connol the growth of meat
spoilage organisms.
33. Materials and methods
3.3.1. Orgaisms and cultures
The following organisms were obtained from the Amencan Type Culture Collection
(Roc kvi He, MD); Camobacterium piscicola ( ATCC 43224), Lactobacillus curvufus (ATCC
2560 1), and Lncrobacillus sake (ATCC 1552 1). Pseudomonar fluorescens and Brochothrh
thennosphacta were isolated from beef stored at 4OC (Farber and Idziak, 1984). Semtia
liquefaciem was isolated from vacuum packaged bologna (Food Research and Development
Centre, St-Hyacinthe, Quebec).
P. fluorescens. B. thennosphcta, and S. lique/aciens were first inoculated and grown
aerobically on brain heart infusion agar @HI, Difco Laboratones, Detroit, Mi). C. piscicola,
L cunlatus, and L sake were sirnilarly inoculated and grown on lactobacilli MRS agar
(Difco), in an atmosphere e ~ c h e d in hydrogen and carbon dioxi& (Gaspak Anaerobic
System; Becton Dickinson, Cockeysville, MD). AU incubations were done at 20°C. Bacterial
cells were subsequently harvested and nsuspended in reconstituteâ skim milk (skim mik
powder in deionized water, 20% wlv final concentration), containing 5% sucrose (wlv), and
lyophilized to obtain stock cultures.
To prepare working cultures, stock cultures were standardized through two successive
24 h growth cycles in the appropriate broth (BHI or MRS) without agitation. Cells h m the
standardized cultures were then inoculateci in k s h medium and incubated (2m without
agitation) for 6 h (C. piscicola for 9h) to obtain working cultures containing approximately
10' CFüIrnl.
3.3.2. Preparation of the antibacterial media
Analytical grade free fatty acids [iauric (C~to), myristic (C14:0)> palmitic (f iss),
pdmiloieic (Ci6:l), stearic (Cis,o), oieic (CIS:~), linoieic (C18:2)> and iinoienic (Ci8:3)]. with a
purity 4 8 % were obtained from Sigma Chernical (St Louis, MO). The acids were first
dissolved in 95% ethanol Ababouch er al., 1992), and the sdutions were added to 250 ml
bottles of sterile BHI or MRS molten agar in concentrations ranging from 100 to 2500 pg/ml,
in increments of 50 pgml h m LOO to 500 pg/rnl, and of 100 Ciglrnl from 500 to 1 0 pg/d.
The contents of each bonle werr then dispensed into stenle peei plates and lefi to solidify.
The maximum concentration of elhanol in the agar was 2.5% (v/v). which was shown in
preliminary trials to have no inhibitory effect upon the micmrganisms used in this study.
Eight essential oils (cinnamon, ELB 40404; clove, ELB 41312; cumin, ELB 41402;
garlic. EB, 40892; omgano, ELB, 41401; black pepper, EB 33423; pimento, ELB 41441;
th yme ELB 4 1403) were provided by Food Ingredients (Mississauga, Ontario). Rosernvy oil.
8136-L was obtained h m Kalsec (Kalamazoo, MI). Oils were manually rnixed with sterile
molten BHI or MRS aga, maintained at 45OC. to dilutions of 1/10, 11100, and 111000. The
molten agars containing essential oils were poured into sterile petri plates and lefl to solidify.
3.3.3. Growth inhibition experiments
Petri plates of BHI or MRS agar containing various concentrations of fatty acids or
essential oils were inoculated with the selected organisms. The working cultures were diluted
(11100) in peptone water, and 0.1 ml of the diluted cultures was spread on the surface of the
solidified agar plates. The positive controls for p w t h consisted of BHI and MRS agar
without preservative, inoculated with the diluted working cultures. Uninoculated plates
containing either fatty acids or essential oils, served as negative controls. Test and control
plates were then incubated at 20°C under aembic conditions for B. themosphacta, P.
fluorescens, and S. Iiquefaciens, or in Hz- and Co2-eNiched amiosphere for C. piscicola, L
curvatus, and L sake.
Three petri plates were used to test the inhibitory effect for each organism and each
level of each preservative, and the expenment was performed twice. Plates were checked for
presence or absence of colonies after incubation for 24 and 48 h. The absence of colonies on
al1 the three plates of a matnient was considered as an inhibitory effect. The lowest
concentration of fatty acids or essential oils nquind to inhibit the growth of the test
microorganisms was designated as the minimum inhibitory concentration.
3.3.4. Anaiysis of essential oiis (EO)
Seventeen substances commonly found in spices were used for this expenment:
allylsulfide, carvacrol, camphor, cineole, eugenol, 4iso propylbenzaldehyde. trans-
cinnamal&hy&. myrcene, a-terpineoi, and thymol were purchased from Aldrich Chemical
(Milwaukee, m. Geraniol, linalool, and a-terpinene from Sigma Chemical (St-Louis, MO).
Carnphene, carvon, lirnonene, and y-terpinene were obtained h m Fluka Chemika-
Biochernika (Buchs, Switzerland). Camphene, linalool, a-terpinene, and y-terpinene were of
technical quali ty (90-9595 puri ty) w hile al1 the othen compounds were at least 97% pure.
A Hewlett-Packard mode1 5890 gas chromatopph equipped with a 1 -pm DB- 1 fused
siiica colurnn 30 m x 0.316 mm (J & W Scientific, Folsom, CA) was used to determine the
concentration of the 17 substances in the selected essential oils. The split injector was set at
ratio of 18: 1, and the camier gas (He) flow at 1.0 mllmin. The oven temperature was
programmed to nse 2OUmin h m 90°C to llS°C, SOUmin from 115OC to 200°C, and
nrnained isothemal at the final temperature (2ûû°C) for 4 min. Samples of EO injected in the
OC consisted of 1 pl of 250 mglrnl (roscmary), and 50 mglml (other EO) solutions in ethyl
acetate.
Al1 the fatty acids failed to inhibit B. thennosphacta. P. j?uorescens, and S.
liquefaciens at concentrations up to 2 5 0 pglml (results not shown). The inhibitory effects
against the three other bacteria (C. piscicofu. L curvatus, and L sake) are presented in Table
3.1. Al1 were unaffected by myristic. palmitic. steuic. and oleic acids ai the concentrations
tested. Lauric, palmitoleic, linoleic. and linolenic acids exhibited various inhibitory activity
with lauric and palmitoleic acids having the greatest effect. Among the organisms which were
affected by fatty acids, C. piscicola was the most susceptible.
3.4.2. Essential oils
Al1 the essential oils tested for antibacteriai activity were ineffective at the lllûûû
dilution (results no< shown). The inhibitory properties observed with the 11100 and 1/10
dilutions are shown in Table 3.2. The strongest effects were obtained with clove, cinnarnon,
pimento, and rosemas, oils, for which the 11 100 dilution inhibited at least five of the six tested
organisms. However, pimento oil was not able to maintain the inhibitory effect over 24 h. Al1
the other oils were weakly active.
Grampositive and gram-negative bacteria were generally afTected in the same manner
within 24 h of exposun, but extension of the inhibitory effects up to 48 h was less often
observed with the gram-negative bacteria (Table 3.2). For example, P. fluorescens and S.
liquefaciem, which were affected by the 11100 dilution of cinnarnon. clove, and rosemary
were no longer inhbited afier 48 h. except for S. 1ignefacien.s in the presence of clove oil. In
contrast, three of the four gram-positive organisms (C. piscicola, L curvutus, and L s&)
continued to be inhibited by the same oils at that dilution. Of the gram-positive bacteria, B.
fhennosphacta exhibited resistance similar to those of the two gram-negative bacteria tested
The contents of the essential crils in the 17 selected substances is shown in Table 3.3.
In general, the sum of selected substances which were identified and quantified constituted a
small proportion of the total mass of each essential oil, with values ranging from 0.15% for
rosemary oil to 19.94% for clove oil. In addition, t h e of the four most active essential oils
(which inhibited more than five organisms at the 111ûûû dilution) contained a significant
amount of eugenol: clove (19.81%): piment0 (9.33%); and cinnamon (5.388). Cinnarnon oil
also contained large arnounts of cinnarnaldehyde (5.37%). The least most active oil (rosemary
oil) contained camphor as its major component, but in a low amount (O. 10%).
Among the less active essential oils (those which inhibited only two or less than two
organisms at the 11 1 0 dilution), only thyme oil contained eugenol and cinarnaldehyde, but
these wen present in srna11 amounts (0.01% for each of the two components). On the other
hanci, p a t e r concentrations of other components were found in those oils: carvacrol in
oregano oil (5.19%); a-terpinene in cumin and thyme oils (1.15% and 1.64%, respectively);
and thymol in th yrne oil(2.40%).
3.5. Discussion
The two gram-negative bacteria (P. jluorescens, and S. 1~uefacien.s) were unaffected
by fatty acids at concentrations up to 2 5 0 pg/ml. This was to be expected since several other
studies (Kabara 1979; Kabara, 198 1, McKeiiar et al., 1992) reported that gram-negative
bactena were resistant to the inhibitory effects of medium and long chah fatty acids and their
derivatives. This nsistance has km attributed to the presence of ce11 wall
lipopolysacchari&s, which can scrccn out the fatty acids; the lipids are thus prevented from
accumuiating on the transporting ce11 membrane, and from entenng into the cells (Kabara,
1979; Branen et al., 1980; Russel, 1991).
B. thermosphacta, a gram-positive bacterium, dso exhibited resistance to fatty acids,
but little information is available about its sensitivity to challenge by fatty acids. Macaslue,
(1982) reported h t growth rates and numbers of B. thetmosphucta were both reduced in the
presence of 0.5 mniolll of palmitic acid. However, both the determination of palmitic acid
uptake and the determination of the inhibition of substrate uptake by palmitic acid failed to
explain the mechanism by which B. thennosphana was inhibited. Similar resistance of gram-
positive organisms was reported by Tsuchido et d. (1993) working with Bucillus subrilis.
They found mutants which were tolerant to the lytic action of sucrose esters of longchain fatty
xi&.
Arnong the saturated fatty acids under study, laurîc acid exhibited the greatest
inhibitory effect against C. piscicole, L curuutus, and L sake while d l the other saturated fatty
acids with chain length between C14 and Cii were completely ineffective. These results are!
consistent with previous reports about the antibacterial activity of saturateci fatty acids with
lauric acid king the rnost effective (Kabara., 1979. Branen et al.. 1980. Babic et al.. 1994). For
sanirated fatty acids, hydrophobic groups have k e n show to have the greatest influence on
antibacterial activity (Branen et al., l98O), but increasing hydrophobicity with longer chah
length may reduce their solubility in aqueous systems. Thus hydrophobic groups may k
pnvented from reaching sufficient concentration to interact with hydrophobic proteins or
lipids on the bacterial ceIl surface (Wang and Johnson. 1992). Lauric acid has been nported to
have the best balance between hydrophobic and hydrophilic groups (Branen et al.. 1980;
Kabara et al. 1977).
It is known that unsaturated fatty acids with chah lengths of C14 or longer are more
active against microorganisms than the comsponding satmed fatty acids (IObara, 1981).
Also, the inhibitory effects of unsaturated fatty acids are increased as the number of double
bonds in the molecule increases (Kabara, 1979). In agreement with that observation,
palrnitoleic acid was found to be more active than myristic and palmitic acids (this study), and
the antibacterial efficacies of Cla unsaturated fatty acids were in the following order: linolenic
(Ci8:3) > linoleic (Cl8:2) z oleic (Cla:i). Similar results have been reported by Wang and
Johnson. (1992) who found that linolenic acid was mon effective against Listena
monocytogenes than linoleic and oleic acids. The fact that palmitoleic acid and Cis unsaturated
fatty acids are active in spite of their long carbon chah suggests that the
hydrophobichydrophilic balance alone cannot explain the observed inhibitory effects. This
activity may be related to other factors such as a peroxidative process involving hydrogen
peroxide and bacterial iron as ~ported by Wang and Johnson (1992).
In the study on the antibacteriai activity of essential oils, no obvious diffennce in
susceptibility was found between gram-negaiive and gram-positive bacteria after 24 h of
exposw to essential 011s. Data, however, showed that the extent of the inhibitory effect up to
48 h was mostly observeci with gram-positive organisrns. This is suppoited by many oiher
repom on the pater susceptibility of gnun-positive bacteria to the inhibitory effect of
essential oils and their components (Shelef et al., 1980; Farag et al., 1989; ChaneHha et al.,
1994). As reported for fatty acids, the ce11 wall lipopolysaccharides of gram-negative bacteria
may prevent active components fmm reaching the c ytoplasmic membrane.
However. the greater resistance of gram-negative bacteria may not be an overall trend
since B. thennosphactu (gram-positive) was as resistant as S. liquefaciens (gram-negative).
Similar results have ken reported by Kim et al. (1995b) who found that L monocytogenes,
(grampositive) was more resistant to the inhibitory effects of 1 1 essential oil constituents than
the gram-negative bacteria tested under the same conditions, including Escherichia coli, E.
coli 0157:H7, Salmonella typhimurium and Vibrio vulnifcm. It seems that the variability of
the resistance of gram-positive bactena to the inhibitory effect of essential oils rnay be due to
differences between suains of the sarne bacterial species. This hypothesis was recently
confirmed by Si~opoulou et al. (1996) with two strains of Staphylococcus aureus in the
presence of carvacrol and thymol.
Zaika (1988) nviewed the litentue npotting the antimicrobid activity of many spices
and classified their activities as suong, medium, or weak. According to this ranking, several
studies (Conner, 1993; Aureli et ai., 1992; Shelef et al., 1980) showed that cinnamon, clove,
pimento, thyme, ongano? and rosemay had s tmg and consistent inhiôitory effects against
various pathogens and spoilage bacteria In agreement with this finding, four of the essential
oils testeâ in this study (cimamon, clove, pimento. and rosemary) exhibiteci a sttong inhibitory
effect toward r!ected m a t spoilage bacteria The antibacterial activities have bec9 aîtributeâ
to the prcsence of some volatile constituents in the oils. Bullemian et ui. (1977) found that
cinnarnon and clove contsined ci~amaldehyde and eugenol as major constihmts which
represented 65-75% and 9345% of the total volatile oils. respectively, and which w m
responsible for the antibacterid effect. In orcgano and thyme, the major antibacterial
consituents have k c n idcntificd as carvacrol(62-79%), and thymol (42%) respectively (Farag
et d., 1989; Sivropoulou et ul., 1996).
The means by which microorganisrns arc inhibited by essential oils seems to involvc
different modes of action. The most fiequent inhibitions involve phenoiic components of oils
which sensitize the phospholipid bilayer of the cell membrane. causing an increase of
pemeability and leaicage of vital intraccllular consituents (Kim et al., 1995b; Juven et ul.,
1994). or impairment of bacterial enzyme systerns (Wendakoon and Sakaguchi. 1995; Farag
et d. 1989). A numkr of reports indicatcâ that cssmtial oils containing cm-1, eugenol, or
thymol had the highest antibacterial perfomanccs (Kim et d., 199%; Lattaoui et Tantaoui-
Elaraki, 1994; Suresh et ul., 1992). For example, Sunsh et d. (1992) found that eugenol was
more bactericidal against E. cdi, Enteroher srrkryokii, and Klebsiella pnewnonioc than
several antibiotics including ampicillin. erythromycin, and sulphamethizole. Among non-
phenolic compounds of essential oils, cinnamaldehyck has k e n show to posscss antiiiactmal
pperties by inhibiting amino acid decarboxylase activity ( DiQy et d.. 1993; Wendakoon
and Sakaguchi, 1995). Bamowski and Nagel (1982) reponoj that aliyl hydroxycinnamates.
which are quite similar to cimamaidehyde inhbiteâ P. fluorescens by specific mode of
action related to cellular encrgy depletion.
The antibPacnd activity of eugcnol and cinnarnaldehyde was supportai by the nsults
obtained by the gas-liquid chromatographie analysis of the essential ails, although components
quantified constituted only a small proportion of the oils. Cinnamon and clove oils which wen
among the most active oils contained the largest arnounts of eugenol and cinnamaldchyde.
Also eugenol and c i~ar~idchydc w m slightly or not presmt in the oils which produced
small inhibitory efkcts (inhibition of two or less than two organism at 111000 dilution).
Therefo~. the presence of cugenol or cinnarnaldehyde was directly related ro the antibacterial
propexties of tesied essentid oils.
Our nsults. however, failed to confirm the inhibitory effect of oregano and thyme
although those oils contained high concentrations of phenolic compwnds (carvacrol and
thymol respectively). Juven et d. (1994) ceportcd that in the presence of a high oxygen
tension. thyme and oregano oils may k inactivated by oxidation of their phenolic
components. in the prcsent study, their effectiveness was not enhanced when the test was Qne
un&r anaerobic conditions (inhibition test against C. piscicola, L curuatus, and L d e ) .
Therefore, the low antibacterial activity rrportcd h m for thym and orcgano oils could not k
explained in ternis of the oxygen tension hypothesis. It is most ükely that the weak efficacy of
carvacrol and thymolantainhg oils (orcgano and thyme) found in the present study may k
due to some other factors such as insolubility in aqueous media (Juven et al., 1994), pH of the
medium (Thompson, 1990), or seasonal and intraspecific variation of essential oil composition
(McGimpsey et uf., 1994; Koklcini aml Vokou. 1989; S i ~ u l o u et al., 1996). For example.
an essential oil h m Origuwn vulgare has bcm rqorted by SiMopoulou (1996) to eliminate
S. aureus at dilutions up to 1110000, but the oil sample used contained carvacrol at a
concentration of 79.58% cornpanxi to 5.19% in the c o ~ ~ c s i a l oregano oil tested in the
present study.
In the present study, rosemary oil was as inhibitory as cinnamon and clove oils. Yet,
rosemary did not contain cinnamaldehyde nor eugenol, and al1 the other background
components mened wen present in smail amounts. Carnphor (0.10%) was the only
component which was present in rosemary oil in concentrations higher than in the other oils
under study. Therefore. the antibacterial efficacy of rosemary oil could be ai least partly
related to the presence of camphor. This is supported by the report of Lattaoui and Tantaoui-
Elaraki, (1994) who found that in some essences, minor compounds could have a huge
antibacterial impact. Also, the small arnount of the total identified components (0.15%) in
msemary oils suggests that some other components may have contributed to its high
antibacterial action.
The present study on ihe inhibitory effects of fatty aci& and essmtial oils on meat
spoilage bacteria was done under specific. controllcd conditions (BHI and MRS agan). Even
though some of these compounds showed consistent antibirtMal activities against meat
spoilage bacteria, the extrapolation of ther rcsults to mat systems must k &ne with caution.
Bacteria present on meat surfaces may attach f i d y resulting in duced expure to essential
oils or fatty acids. Roteins and lipid components of mat can also interact wiîh the active
components of antibactcrial compounds as rrponcd by Kim et d.. (1995a). A h , for
subsequent use as components of active packages. additional rxperiments must k done to
determine the case with which fatty acids and csscntial oils can be incorporated into packaging
films and their diffusion rates h m the surface of the product to the interior must be
characterized.
3.6. List of tables
Table 3.1. Minimum inhibitory concentration (Crg/mi) of fatty acids against meat spoilage
bac teria.
Table 3.2. Inhibitory properties at 24 and 48 h, of diluted essential oils toward meat
spoilage bactena.
Tabb 3.3. Quantitative detemination of selcctcd authentic amibacteriai components in
essential oils.
NOTE TO USERS
Page(s) net induded in the original manuscript and are unavailable from the author or univemity. The manuscript
was microfilrned as received.
Table 3.1
Fatty acids C. piscicola L curvatus L sake
Lauric C 12:o
Myristic C14:O
Palmitic C l6:O
Paimitoleic C 16: 1
Stcaric C 18:O
Oleic C 1 8: 1
Linoleic C 1 8: 2
Linolenic C 18: 3
NI: No inhibition at concentrations up to 2500 pg/rnl.
Table 3 3
Essential oils B. P. S. C. L L themwsphucta fluorescens liquefaciens piscicola CU watw sake
Cinnarnon ccb ++ ++ (++) (++) (*)
Cumin +c + + + + +
Black pepper + + + + ++ ++
'. Bacteria were tested ai 10' CFU/mi.
b. ++ : Inhibition by 1/1W dilution of essential oils.
'. + : Inhibition by 1/10 dilution of essential oils.
( ) inhibition extended to 48 h by 11100 dilution of essential oils.
CHAPITRE 4
EFFECT OF TEMPERATW ABUSE ON THE ABILITY OF ORGANIC ACIDS
TO PREVENT GROWTH OF MEAT SPOILAGE BACTERIA
4.1. Abstract
The effects of acetic. citric. lactic, and propionic acids on the growth of thrcc meat
spoilage bacteria. BrochothrLr thennosphacra, Pseuàomonas fluorescens, and Setrutia
liquefuciens, were detennined at 4. 8. and 20°C in Brain Heart infusion @HI) broth. The
measurements were made over a total periods of 12Oh. The organic acids produced pa t e r
inhibition of bacteriai growth and extension of lag periods at 4°C and 8OC than at 20°C.
Acetic and propionic acids were more efficient than citric and lactic acids. Among the thm
bacteria. P. jZuurescens exhibited the grcatest resistance to organic acids.
Key words
Organic acids, temperature, bacteria. rneat, spoilage
Several studies have reportai the efficacy of organic acids against many food-borne
pathogens including Escherichia coli 0 157: H7 (Conner and Kotrola, 1995), Listena
monocytugenes (Ahmad and Marth, 1989; Buchanan et al., 1993; Young and Foegeding,
1993), Salmonella (Chung and Goepfert, 1970). and Yeniniu enterocolitica (Broc klehunt
and Lund, 1990). Some investigations w m aimed at determining the efficacy of organic
acids against meat spoilage bacteria, with regard to bacteria type, acid type and
concentration. as well as temperature (Gmr and Dilts, 1992; Ouattara et al., 1997a).
Although these studies have show that many meat spoilage bacteria arc inhibited by
organic acids, it i s iilso known that antibacterial properties arc influenced by many other
factors, related to the environmental conditions, one in particular king temperature abuse
during the storage of meat and meat products (Greer and Dilts, 1992). In this context, the
pnsent study was undertaken to determine the relative efficacy of acetic, citric, lactic, and
propionic acids to inhibit three meat spoilagc bacteria (BrochuthrLr thennosphacta,
PseudomonasJluorescens, and Serratia liquefacieenr) at three different storage temperatures
(4,8, and 20°C).
4.3. Material and methods
Glacial acetic acid (99.7% wlv) and propionic acid (99% wfv) were supplied by
Fischer Scientific (Ncpean, ON). Citric acid (monohydrate,>99% pure) and DLLactic acid
(88% wlv) were obtained, respectively. from Anachemia (Montréal, QC) and Amcrican
Chernical (Montrtal, QC). Stock solutions of organic acids werc added separately to sterile
Brain Heart Infusion @HI) broth to final concentrations ranging fkom 0.1% to 0.3% (w/v).
Pseudomol~l~ fluorescens and BrocWrM thennosphacta were obtained from
Farber and Idziak (1984). These sûains were isolated h m bbef stored at 4OC. Sewutia
liquefaciens was isolated from vacuum packaged Bologna (Food Research and
Development Centre, St-Hyacinthe, QC). Lyophilized stock cultures were prepared from
suspensions of bacterial cells in skim milk powàer reconstituted in deionized water (20%
W/V) containing 5% sucrose (wlv), and grown aerobically in BHI broth (Difco Laboratones.
Detroit, MI) ai ZO'C without agitation. Standardized cultures were obtained through two
successive 24 h - growth cycles.
Growth inhibition experiments were performed in 96 well microtitration plates
(Nunc. Kamstnîp. Denmark). The growth media containing the acids were inoculated with
B. thennosphacta, P. fluorescens, or S. liquefaciens to give final bactenal concentrations of
about id CN/mL. BHI broth without any organic acid was sirnilarly inoculated to serve
as positive controls for growth at cach temperatun. Aliquots of bacterial suspensions in
growth media with or without organic acids werc introduced into three replicate wells (200
Wwell). Uninoculated medium was introduced in the same manner in microplate wells
and served as negative controls for growth. The microplates wen incubated for 120 hours
at 4OC, 8T, or 20°C under aerobic conditions in water vapor saturated air. to prcvent
evaporation. Growth was evaluated at ngular intervals by absorbance measurements at 540
nm in an automated piate reader (Lambda microplate mader. Perkin Elmer. Nonvalk. CT.
The p e n d from the inoculation of bacteria into culture media to the timc when optical
density values reached 0.02 was defined as the lag phase pend.
In media containing various concentrations of acetic, lactic, and propionic acids, the
maximum inhibition of bacterial growth was obtained at 4OC and 8°C. but when the storage
temperature was increased, the antibacterial efficacy was reduced (Table 4.1). For example,
at 4OC and 8OC al1 the bacteria wcrc completely inhibitcd with 0.2% (wlv) of acetic or
propionic acids, while incomplete inhibition of P. fluorescens or S. liquefaciens occumd at
20°C with the maximum concentration (0.3%) of the same acids. Also, lactic acid (0.3%)
inhibited P. fluorescens at 4OC but not at 8OC or 20°C. Citric acid did not produce complete
inhibition at the maximum concentration used at any temperature tested.
At the lowest concentration of organic acids used in the study (0.1%). incomplete
inhibition was obtained. The lag periods k f o ~ initiation of the growth of B.
thermosphac~a, P. fluorescens, and S. liqw$hciens is shown in Table 4.2. The influence of
temperature was function of the type of acid. With acetic and propionic acid solutions. the
reduction of incubation temperatures lcd to longer lag periods than in control solutions. For
example, at 4OC the initiation of growth for B. thennosphocta and S. liquefaciens occumd
after 60-96 h in BHI broth containing acetic or propionic Mds, compared with 24-48 h in
conwl BHI broth at the sarne temperatun. At g°C, the lag periods remained similar to
those obtained at 4OC. except for P. jluorescens. When the incubation temperatun was
20°C, lag periods werc substantially rcduced to 18 or 24 h. Contrary to acetic and propionic
acids. the inhibition patterns obtained with lactic and citric -cich at 4, 8, or 20°C werc
similar to those obtained in control BHI broth. Among the ihne bactena under study, P.
fluorescens exhibitcd the greatcst mistance. At the lowest storagc temperature (4"C), al1
the organic acids failed to give a longer lag pend for this organism than obtained in
control BHI broth.
43. Discussion
The present study complements those of previous investigations assessing the
antibacterial efficacy of organic acids against bacteria involved in meat and meat product
spoilage. Our findings with B. thennosphacta, P. jluorescens, and S. liquefaciens indicate
that organic acids produced pa t e r inhibition of bacterial growth at lower temperatures
compared with storage temperatures that reached levels of abuse. This observation can k
explained by the antibacterial hurdle theory dcscrikd by Leistner (1992). In fact, the lower
temperatures (4OC and B°C) may have enhanced the efficacy of organic acids, particularly
that of acetic and propionic aciâs.
Using similar conditions to this study, Ahamad and Martb (1989) found that an
increase in incubation temperature from f0C to 3S°C in the presence of acetic, cihic. or
lactic acids resulted in a reduction in generation time for Listeria monocytogenes. Similarly,
the resulting pH of organic acid solutions that inhibited E. coli was found to be lower when
the incubation temperature was increascd (Conner and Kotrola, 1995). indicating that
higher concentrations of organic acids iue needcd at higher temperatures to obtain a
complete inhibition of E. d i . In another sndy on p u n d k e f acidifieci with acetic, citric,
or lactic acids (Abdul-Raouf et d., 1993), the populations of E.coli were higher at 30°C
than at SOC and 2 1°C.
The weak antibactenal cffects of ci& and lactic acids have been previously
repocted (Ouanara et al., 1997a). and have partly ken explained by their low pKa values
(3.10 and 3.80, respectively). At similar pH values, citric and lactic acids are more
dissociated than acetic and propionic acids (pKa:4.80 and 4.90, respectively), and thus are
unable to diffuse through the bacterial ce11 envelope (Chemngton et al., 1991; Freeze et al.,
1973; Salmond et al., 1984, Young and Focgeding, 1993). in the present study, reducing
the incubation temperature did not enhance the antibacterial effect of citric and lactic acids.
This observation is suppoited by results from Houtsma et al. (19%). where the reduction of
incubation temperature from 37OC to 4OC was not found to have a specific effect on the
minimum concentration of sodium lactate for inhibiting various pathogens and spoilage
organisms.
The present study has dedt with the efficacy of organic acids against meat spoilage
bactena in liquid media un&r temperature abuse conditions. Taking in consideration the
results obtained hem, and those available in published liteninin (Gmr and Dilts. 1992), it
must be concludcd that application of organic acids without storage at low temperature
would lirnit the efficacy of the acid mamient end not substantiaily extend the expected
shelf-life of meat and meat products mated with organic acids. However, our results cannot
be extrapolateci to cases wherc the influence of tcmperaturc is evaiuated by applying hot
solutions of organic acids on meat surfaces. The temperatures used in these expeciments (up
to 70°C) (Anderson and Marshall. 1989. 1990; Cutter et al.. 1997) are sublcthal for many
bacteria in coneaît with the temperatures used in our experiment.
4.6. List of tables
Table 4.1. Total inhibition of B. rhemwsphacto, P. fluorescens. and S. liquefaciens by
organic acids at 4.8, or 20°C.
Table 4.2. Influence of temperatun on the lag periods (h) before initiation of the growth of
B. thennosphacta, P. fluorescens, and S. liquefaciens in presence of O. 1% (w/v) of various
organic acids.
Table 4.1
Bacteria Temp ( O C ) ûrganic acids
B. thennosphacta 4 ++ 8 ++ 20 +
P. fluorescens
S liquefaciens
'. Bacteria were tested ai the concentration of los cells/mL
2. No inhibition (O), total inhibition with the concentration of 0.2% (++), or 0.3% (+) of
organic acids.
Table 4.2
Temp.(OC) B. Thermosphacta P. fluorescens S. liquefmiem
Acetic acid 4
10
20
Citric acid 4
10
20
Lactic acid 4
10
20
Propionic acid 4
10
20
DIFFUSION OF ACETIC AND PROPIONIC ACLDS FROM CHITOSAN FILMS
iMMERSED IN WATER
5.1. Abstract
The diffusion of acetic or propionic acids from a thin (about 45 pm) chitosan film in
which they were incorporated was measurrd after immersion of the film in water, and the
effects of pH (5.7, 6.4, or 7.0) and temperature (4OC, 10°C, or 24°C) on diffusion were
investigated. The kinetics of acetic and propionic acid release deviated h m the Fickian
mode1 of diffusion. Diffusion was found to k unaffccted by pH in the range of values
tested but a âecnase in temperature from 24OC to 4OC resulted in a reduction of diffusion
coefficients from 2.59 1 ~ ' ~ m2.s" to 1.19 IO-'* m2.s" for acetic acid and from 1.87 10.12
m2.s" to 0.91 10'12 m2.s" for propionic acid The effect of temperature on diffusion was
well (+ N.9785) described by an Arrhenius-type mode1 with activation energies of 27.19
m mole" (acetic) and 24.27 mole" (propionic). Incorporation of launc acid or essential
oils (cinnarnaldehyde or eugenol) to the chitosan film at the time of preparation produced a
subsequent reduction in the diffusion of acetic or propionic acid, and maximum effects
were obtained with launc acid and cinnamaldehyde incorporated at final concentrations of
1.0% and 0.5% (wlw), respectively.
Key words
Diffusion, chitosan, acetic, propionic, films.
5.2. Introduction
Over the years, a great deal has becfi karned about microbial spoilage of meats and
its control (Greer and Dilts. 1992 ; Korkeala and Bj6rkroth. 1997; Renem and Labadie.
1993). The bacterial species responsibic for undesirable sensory changes such as soumess,
slime and gas production have been identified and found to belong to the genera
Achetobacter, BtuchuthMr, Carnobacteriwn, Enterobacter, Lactobocillus, Morarella,
Pseudomonus, and Serrutia, among others (Holley, 1997 ; Korkeala and BjUrkroth, 1997;
Renerre and Labadie, 1993). Also, antimicrobial agents such as organic acids, bacteriocins,
and spice extracts have k e n tested for their ability to contml meat spoilage (Abugroun et
al., 1993 ; Hotchkiss, 1995 ; Miller et al., 1993) and acetic acid, propionic acid, lauric acid,
clove oil, and cinnarnon oil were found to k efficient in inhibiting the growth of six
common meat spoilage bacteria in laboratory media (Ouattara et al., 1997a, 1997b).
Since microbial growth in solid and semi-solid foods such as meat and meat
products occua pnmarily at the surface, attempts have been made to delay spoilage by use
of antibacterial sprays or dips. However, direct surface application of antibacteial
substances ont0 foods was found to have limited benefits because the susbtances were
neutralized on contact or diffused rapidly into the bulk of food, away from the surface
(Siragusa and Dickson, 1992 ; Toms et al., 1985).
Cumntly, a new concept of active packaging is king developed in which
antimicrobial agents arc incorporated into packaging maienal with the ultirnate goal of
maintaining high concentrations of prcsmatives on the surfaces of foods for as long as
possible (Gennadios et d, 1997; Hotchkiss, 1995; Kcster and Fennema, 1986 ; Toms et
al., 1985). Although synthetic polymcrs can be used for this purpose. a ment rcview by
Gennadios et al. (1997) inclicated a growing interest in edible coatings due to factors such
as environmental concems, need for new storage techniques, and oppominities for creating
new markets for under-utilized agricultunl comrnodi tics with film-fomiing properties.
Edible coatings p n p d from polysacchari&s, proteins and lipids have already ken
proposed as carriers for various antimicrobial substances. For example, complete inhibition
of Listeno monocytogenes was obtained using nisin or pcdiocin fixed on a cellulose casing
(Ming et al., 1997) and organic xi& immobilizcd in a calcium alginate gel resulted in a
0.25 to 1.5 log unit reduction of L monocytogenes on lcan beef (Siragusa and
Dickson, 1992).
Chitosan, an amino polysaccharide that has found many applications in the fields of
cosmetics, wound healing, dietetics and waste water treatment (Demarger-Andre and
Domard, 1994) is another edible polymer of interest for the preparation of antirnicrobial
coatings. Due to the fb(1-4) linkages betwem nsidues, chitosan has gooà film-fomiing
properties and chitosan films an easily prcparcd by evaporation of dilute acid solution of
the polymer (Saitô et al., 1997). Chitosan has also been investigated to s m e as a matcrial
for controlled release of dxugs because of its entrapment characteristics (Kaya and Picard,
1996) and its ability to fonn covalent bonding andor cross-linking with anionic compounds
(Mi et al., 1997 ; Pandya and Knorr, 1991).
The stability of compouads incorporated in chitosan capsules or films may be.
however, compromiseci by severai factors. On one hand, chitosan films, due to their
hydrophilic nature, tend to exhibit rapid swelüng and sirnultancous release of incorporated
compounds when in contact with aqueous media or wet surfaces (Lim and Tung, 1997;
Gnanasekharan and Floros, 1997). On the other hanà, the electmstatic interactions ktween
chitosan and electrically charged molecules are much affectcd by pH (&marger-Andre and
Dornard, 1994; Pandya and Knorr, 1991). A study was thcreforc undertaken to investigate
the diffusivity of acetic and propionic acids incorporated into chitosan films in order to test
the feasibili ty of developing a chitosan-based antimicrobial film.
5.3. Material and rnethods
5.3.1. Chitosan films
Chitosan films containing organic acids were preparcd by dissolving practical grade
chitosan from crab shells (Sigma Chernical, St-Louis, MI) in aqueous solutions (1%. w/v)
of acetic or propionic acids (Fischer Scientific, Nepean, Ontario), to a final concentration of
2% (wlv). which typically nquircd ovemight stirring. Altematively, lauric acid (> 99%
pure; Sigma Chernical, St-Louis), eugenol or transcinnarnaldehydc (> 99% pure; both from
Alcirich Chernical, Milwaukee, WI) were adcied to the chitosan-acid solutions to final
concentrations (w/v) of 0.25,0.50,0.75, and 1% for lauric acid and of 0.25 and 0.50% for
cinnamaldehyde or eugenol. Al1 solutions were subsequently filtercd through a coarse glas
filter, and 100 ml of each solution was poured into a 20 cm x 20 cm x 0.5 cm Plexiglas
mould at room temperaturc (24OC î 1°C), except for solutions containing laurîc acid which
werc heatbd at 70°C kfon casting. Moulds and theû contenu wen then placed in an 80°C
oven (BT-23 Isotemp, Fischer Scientific, Nepean, Ontario) for 4-5 h to evaporate watcr,
cooled, the Med films wetc collectd, and the fihn thickncss was deterniincd with a han&
held micrometer (Model ID4 10 ME; Mitutoyo, MFG. Co, lapan).
Diffusion experiments were conducted in 500 ml glass beakers containing 200 ml of
0.2 M sodium phosphate buffer at three differcnt pH (5.7, 6.4, or 7.0) and rnaintained at
temperatures of 4*C, lO0C, or at rwm temperature (24 I 1°C). Square pieces (9 cm2) of the
films under study w m inserted and maintained between two polyethylene grids, and the
p d s were immened in the buffer which was kept agitated to obtain uniform dispersion of
acetic or propionic acid àiffusing from the chitosan film. Samples of the buffer solution
were taken periodically and the concentrations of acetic or propionic acid were determined
by high performance liquid chromatography (Waters WLC system composed of a 77 1 plus
autosarnpler, an U6K injecter, and a 600E pump; Waters Corporation, Milford, MA). Peak
separation was achieved through an Ion Guard precolumn and an Ion 3 0 polymenc
column, both from Interaction Chromatograph (San Jose, CA), using a 0.005 N sulfuric
acid solution as the mobile phase, at a flow rate of 0.5 m~.min-'. Deteetion was done at
210 nm, on a 991 Photodiode Amy W Detector (Waters Corporation).
5.3.3. Fractionai KMSS release and düMon coefficients of acetk or propionic acid
The fractional mass ~elease is the ratio M , K of the mass Mt of acid reieased in the
buffer at time t to the maximum amount of acid that can be released, i.e. the mass M, of
acid ~t~~iisxl after an infinite time penod. Lincarity of AU,& with tIn in the initial portions
of the diffusion c w e s (M,/M, d 3 ) was first verified in order to evaluate if the diffusion
of acetic or propionic acid followed the general law of diffision (Crank, 1975; Peppas,
1985). The diffusion coefficients D (m'.sa') of acetic and propionic acids wen later
calculated using the half-time mcthod equation D = 0.049h2 / t0.5 (equation 1; Lim and
Tung, 1997). where h is the film thickncss (m) and t0.3 is the time (s) at which Mt = 0.5 M,
Theoretical values of fractional mass release as a function of time t were calculated
by two methods. In the first one. the diffusion coefficients D, obtained with equation 1.
9
were substituted in equation 2. Mt / M, = I - (8 / (2n + I)' n2) expF (2n + Ir n5Dl/ h2]. n -0
given by Crank (1975). In the second one, an exponential rise to a maximum is used
(equation 3). as proposed by Lim and Tung (1997): Mt / M, = I - exp (-Kr), where t ts the
diffusion time, Mt and M, are the arnounts (mg/cm2) of organic acids released from films at
time t and at equilibnum, nspectively, and k (1s) is the rate constant. A separate relation, of
the form k = aD + b, is then established ktween al1 the conesponding calculated values of
k and D.
in order to e v d w the temperature dependance of diffusion, an Arrhenius
activation energy equation was us& D = Do exp(-Eu / RT), in which Do is the diffusion
coefficient at infinite temperature (m2.s-'), E. is the activation energy mole"), R is the
universal gas constant (8.314 mole“ "ICI), and T is the absolute temperature (OK).
5.33. Data analysb
The initial portions of the diffusion curves (h4, /h& c 2/3) w m tested for linearity
using general linear procedure (GLM) of the SAS systern (SAS Institute Inc. 1985), and a
linear gre es si on mode1 equation (Y=aX + b). The overall kinetic &ta were analysed by
using the NLIN (non-linear) procedure to &termine the rate constant of the kinetic
equation (Y= 1 - exp (-kX)). Rate constant (k) values at different temperature or
concentration of hydmphobic compounâs were tested for significant difference using the
Wald statistic (Agresti, 1996). Diffusion curves were also tested for sigrnoidal shape after a
logit-log transformation [In (Mt A&)/(l-(MtA&)) = a + b log t] using the Exel 7.0 program
from Microsoft.
A multifactor analysis of vaziance was performed to &termine the main effects of
temperature, pH and tirne, and to detennine significant interactions between factors using
GLM procedure of SAS.
5.4.1. Film pteparaüon and füm thiclmess
A precipitatc fomed when lauic acid was added to chitosan solutions prepmd in
diluted propionic acid, precluding the use of the launc-propionic acid combination for film
p~paration. Al1 other combinations of organic acids with lauic acid, cinnamaldehyde, or
eugenol lead to homogeneous chitosan solutions which yiel&d unifonn films. Films
prepared with acetic and propionic acid only were 44.4 + 3.9 pn and 44.7 t 4.7 pn thick.
respectively, while 10934% thicker films were obtained aftcr incorporation of lauric acid
(53.7 I 5.1 p), cinnamalâehyde (53.9 I 5.0 p), or eugenol (5 1.8 I 1.7 pn). Therefore,
the thickness of chitosan-acetic (or propionic) acid films was incnased in al1 experiments
involving cornparisons with films containing ad&d lauric acid, cinnarnaldehyde, or
eugenol, in order to reduce the influence of thickness on diffusion characteristics.
54.2. Kinetics of organic acid release fmm chitosan Nms
Diffusion of acetic acid from a plain chitosan film (containing no lauhc acid,
cinnamaldehyde or eugenol) immersed into pH 6.4 sodium phosphate buffer at 24°C is
represented in Figure 5.1. The diffusion rate was maximum immediately after immersion
and progressively decnased thenafter until diffusion was complete. which was achievcd in
about 200 min. Al1 other diffusion curves were similar in shapc, although diffusion rates
varied with each set of expenmental conditions (film composition. pH. and temperature). In
al1 cases, linearity of the initial portion of the curve (Mt /M* < 3 3 ) was weak (+ as low as
0.6618). In contrast, a straight line always fitted well the &ta (8 = 0.9649 t 0.0367) after a
logit-log transformation (In (Mt h&)/(l-(ii& A&)) = a + b log t ] , indicative of a sigrnoidal
shape. In addition, the kinetics of acetic or propionic acid nlease h m chitosan films was
well described (2 = 0.9184 t 0.0400) by equation 2, using D values calculated from
equation 1. A better fit (8 = 0.9760 î 0.0157) was obtained using equation 3 and rate
constants k. Calculaied rate constants were well comlatcd (? > 0.9887) with the
corresponàing diffusion coefficients D for chitosan films containing no fatty acid or
essential oil. Weaker comlations (8 S 0.8173) were obtained for films containing lauric
acid, cinnamaldehyde, or eugenol.
5 e 4 . 3 e Influence of pH and temperature on diffusion
Analysis of variance relative to the diffusion data (Table 5.1) indicated no effect (p>
0.05) of pH on diffusion of acetic and propionic acids, as illustrated in Figure 5.2.
Consequently, fractional mass release data obtained in different pH conditions were pooled
before evduating the influence of temperatun on diffusion. Increasing the temperature
from 4°C to 24T rcsulted in a faster rate of diffision for both acetic and propionic acids
(Figure 5.3). In panicular, the time necessary to release half the amount of acetic acid
initially contained in the chitosan film decnased from 77sec at 4OC to 64 sec at 10°C and
42 sec at 24OC, while the comsponding times for propionic acid were 1 I l sec (4OC), 75 sec
(lO°C), and 52 sec (24OC). Also, the difision coefficient D of acetic aciâ, calculated with
the half-time method (equation 1). and the comsponding rate constant k from equation 3
incrcastd from 1.19 10"~ to 2.59 10'12 m2.d and from 9.2 10" to 19 10" m2.s-',
rcspectively, when temperature was inmascd from 4OC to 24OC (Table 5.2). while similar
incrcases were observed with propionic acid. In addition. t~mpcrahuc dependence of the
diffusion coefficients was well &scribtd by an Arrhenius plot (Figure 5.4). with activation
energies of 27.19 mole-' and 24.27 m mole" for acetic (?= 0.9976) and propionic acid ($=
0.9785). rcspectively.
5.4.4. E f k t of lruric acid, cinnamaidehyde, or eugenol on diffbsion
Incorporating lauric acid into chitosan films at concentrations of 0.25%,0.50%, and
0.75 % (wiw) had no effect on the diffusion of acetic acid from the fiims (Table 5.3). With
the highest concentration of lauric acid (1%, wlw) a substantial reduction of D (1.84 10'12
m2.s-') and k (7.3 lu3) was observeci, cornparcd to the corresponding valws measured in
conml films, containing no lauric acid @ = 3.20 IO-'* m2.s"; k = 17.0 10-3.
The effecu of incorporating cinnarnaldehyde or eugenol into chitosan films on the
diffusion of acetic or propionic acid from the films are surnmarized in Table 5.4.
Cinnarnaldehyde (0.508, w/w) produced the maximum effect with D values of 2.02 10'"
rn2.s.' for acetic acid and 1.74 10'12 rn2.s-' for propionic acid. compared to 3.63 10"* m?d
and 2.75 1412 m2.d in control films made with acctic and propionic acid respectively.
Incorporation of eugenol (0.50%. w/w) reduccd the D value of acetic acid to 2.30 lu'*
m2.s". but no effect was observed on the ciiffision of propionic acid.
5.5. Discussion
Theontically, the releasc of acctic and propionic acids from chitosan films
immersed in water could k descriôed by the swclling-controllcd model for h g rclease
previously repocted by Mallcy et al. (1987) and Armand et al. (1987). According to this
model, water first cnters the chitosan matrix and dissolves the organic acids, thus allowing
their subsequent nlease h m the polymcr. The diffusion of acetic and propionic acids is
therefore expected to incnase with increasing pcmüation of water into the chitosan film, to
finally reach a plateau when the matrix is satufatcd with water (Amand et al. 1987). and
this was essentially confirmed by the experimental results obtained in the present study.
In reality, the situation is more complcx. Many interactions occur during diffusion
from polymen to liquids. In particular. liquid uptake gencrally causes polyrnen to swell
(Peppas and Brannon-Peppas, 1994 ; Armand et d, 1987). Also, Lim and Tung (1997)
reported a time-dependant relaxation process rcsulting from the swelling stress which
occurred during the diffusion of liquid into polymers. As a result, migration rates change
continuously and diffusion is difficult to analyse mathematically (Katan and Briston, 1974 ;
Gnanasekharan and Fioros, 1997).
In this study, the initial portions (Mt A& c 2/3) of the difhision curves wen not
found to k linear with the square mot of diffusion tirne, contrary to the predictions of the
general Fick's law of diffusion. This indicates that the rclcax of acetic and propionic acids
from chitosan films is not entinly &termincd by diffusion (Peppas, 1985). Another
evidence of the non-fickian nature of the phenomenon was proviâed by the sigrnoidal
shape of the diffusion cwes, as ahady mentioned by Lim and Tung (1997). Also. the
fractional mass release, ploued as a function of time. was better npresented by an
exponential rise to a maximum (Eq. 3) than by the classical solution (4 .2) to Fick's law,
proposed by Crank (1975). Titesc results differ h m thosc of Red et al. (19%) who
reported a typical Fickian khaviour for the diffusion of sorbic acid from wheat gluten and
lipid-based films, with cornlation coefficients over 0.99. The discrepancy is probably
related to diffennces in swelling propercies of wheat gluten (5%) and chitosan films (more
than 50% in the present study; ~ s u l t s not shown). since Piron et al. (1997) reported a
change in diffusion pattern from Fickian to non Fickian khaviour, as chitosan became
fully hydrated.
Demarger-Andre and Domard, (1994), reported that in chitosan/carboxylic acids
solutions or films. interactions wcrc purely electrostatic, without any complexation
processes. These interactions are facilitated when both chitosan and organic acids are
protonated, that is, when pH values are lower than the pK of chitosan which is 6.3 (Mi et
al., 1997), and higher than the pK of acetic and propionic acids (4.8 and 4.9. respectively).
Based on that hypothesis. the ~ l ca se of acetic and propionic acids from chitosan films
should be increased when pH incrcases from 5.7 to 7.0. This was not observed in the
present study, since no significant effoct of pH (5.7. 6.4, or 7.0) was found These rcsults
suggest that the diffusion process was not completely contmlled by the electrostatic
interactions.
The rate of diffusion of acetic and propionic acids from chitosan films incrcased
with incrcasing temperams, in the 4-24OC range ( t h stuây). Similady, increased rates of
diffusion for potassium sorbate through vanous polysaccharide films, including chitosan,
methylcellulose, and hydroxypropyl methylcellulow w m observed as temperatures were
increased from 5OC to 40°C (Vojdani and Toms, 1989; 1990). Also, in the same
temperature range (540°C), Giannakopoulos and Guilbert (1986) nported an increase in
the apparent di fision coefficients of sorbic acid incorporatcd in gel cubes from 3.57 x 10'"
to 1.50 x 10'1° m2.s-'. The dependency of diffusion on temperature is generally explained by
temperature effects on both the solubility of diffusing molecules in films and the nature of
adhesive forces at interfaces (Vojdani and Toms, 1990 ;Myint et al., 1996). The fact that
diffusion can be described by an Arrhenius equation (this study) suggests that the effect of
temperature is thermodynamic in nature, essentially controlled by the ratio of energy
provided to activation energy (Daniels and Alberty, 1972). and that no morphologicai
modification of the chitosan film is involved (Red1 et al.. 1996).
Since the release of hydrosoluble components from polymer films in which they are
incorporated is &pendent on the simultaneous entry of water, inclusion of hydrophobic
compounds into hydrophilic chitosan films was expectd to dccrease diffusion by slowing
down film hydration. Indeed, a decrcase in diffusion of acetic acîd was observed in chitosan
films containing 1.0% lauric acid or 0.5 % cinnemddchyde or eugenol, consistent with the
report of Vazquez et al. (1997) that addition of hydrophobic components into hydrophilic
polymea resulted in nduced water uptake. The influence of hydrophobic components cm
also be explained in t e n s of modifications to chitosan structure, leading to an increase in
the network tortuosity (Callegarin et d., 1997 ; Red1 et al., 1996). As a nsult, the diffusion
path is prolonged. Hydrophobie components rnay also affect other geomeüical feahires,
sucl; as porc constrictions or blind porosity. thercfon limiting molecular transport through
the network (Papadokostaki et d , 1997). This is consistent with published data on
diffusion characteristics of lipid-polysaccharide films. For example, Red et a1.(19%) found
that the addition of beeswax or acetylated monoglycende to wheat gluten films rcsultcd in a
20950% reduction in diffusion coefficients for sorbic acid. Also, the addition of various
fany acids has been found to reduce potassium sorbate pemeability of methylcellulose or
hydroxypropyl methylcellulose films (Vojdani and Toms, 1990) and water vapor
pemeability of chitosan films (Wong et al., 1992).
5.6. Conclusion
This work was concemed with the diffusion of acetic and propionic acids h m
chitosan films imrnersed in water. From the nsults obtained, two factors appear to affect
the kinetics of organic acid release: i) temperature, which affects the reaction between
molecules; low temperatures rcsulting in low diffusion coefficients. ii) presence of
hydrophobie compounds in films, which limits watcr uptake of polymers, thenby reducing
the d i h i o n of incorporated hydrosoluble molecules.
5.7. List of Tables
Table 5.1. Summarized results of variance analysis dative to the diffusion of acetic and
propionic acids from chitosan films.
Table 5.2. Influence of temperatun on diffusion of acetic and propionic acids from
chitosan films.
Table 5.3. Influence of lauric acid on the diffusion of acetic acid from chitosan films.
Table 5.4. Effects of cinnamaldehyde and eugenol on the diffusion of acetic and propionic
acids from chitosan films.
Figure 5.1. Typical c w e s of fractional mass rclease of acetic or propionic acids
incorporated in chitosan films.
Figure 5.2. Representative graphs of the influence of pH on the fractional mass release of
acetic and propionic acids incorporated in chitosan films. Diffusion tests were performed at
1O0C.
Figure 5.3. Effect of temperaturc on the fractional mass rclease of acetic and propionic
acids incorponteci in chitosan films.
Figure S A . Arrhenius plots and activation energies of acetic and propionic acids
incorporated in chitosan films.
Table 5.1
Temperature 2 0.000 1 0.000 1
PH 2 0.0930 0.0672
Time 9 0.0001 0.000 1
Table 5.2
Temperatun ha D~ kc
Acid (Ec) (1o6 m) (1012 m2/s) (10')
4 43.2 1.19 (1.16-1.22) 9.2 î O.qA
Acetic 10 44.2 1.49 (1.37- 1.68) 1 1.3 î 0.6B
24 45.8 2.59 (2.30-2.73) 19.0 t O&
4 45.3 0.91 (0.85-0.95) 6.1 î 0.3*
Propionic 10 44.2 1 .27 (1.22- 1.35) 9.3 * 0.6e
24 44.5 1.87 (1.77-1.89) 14.3 î 0.8c
' Film thickness,
Diffusion coefficient. Values in parentheses are lower and upper limits for D.
Rate factor obtained by non-linear regression. For each acid k-values with different letten
(A, 8, or C) are significantly different (p< 0.05).
Table 5 3
'The measurements were done at 24OC
Concentration of lauric acid in film-forming solutions.
Film thickness.
* Diffusion coefficient. Values in parentheses are lower and upper limits for D.
Rate factor obtained by non-liear regression. For each acid, k-values with different letters
(*, or B) are significantly different (p< 0.05).
Table 5.4
Concentration' hc Dd kC
(96 w/w) (1o6 m) (1012 m'ls) ( 10')
Acetic acid
Cinnarnaldehyde
Eugenol
Pmpionic acid
Ci nnamalde h yde
Eugenol
0 . 0 (Control) 59.0 3.63 (3.14-4.25)
0.25 59.0 2.99 (2.52-3.38)
0.50 57.0 2.02 (1.93-2.16)
0.25 51.5 2.77 (2.55-2.99)
0.50 54.0 2.30 (2.23-2.46)
0.00 (Control) 51.3 2.75 (2.5 1-2.82)
0.25 52.0 2.70 (2.46-3.05)
0.50 47.7 1.74 (1.41-2.16)
0.25 50.0 2.50 (2.45-2.57)
0.50 51.8 2.58 (1.98-3.10)
'The measurements werc done at 24°C
Concentration of cinnamaldehyde or eugenol in film fomiing solutions.
Film thickness.
Difhision coefficient. Values in parentheses are lower and upper limits for D.
Rate factor obtained by non-linear regession. Statistical analysis werc done separately for
acetic and propionic acids, and k-values with different lettca (*, B. or c) arc significantly
different (p< 0.05).
Figure 5.1.
O 100 200 300
Time (s)
Dotted and continued lines represent prediaion Born Eq. 2 and Eq. 3, respectively, using difision coefficients calculated from Eq. 1 .
l Acttic acid
Tirne (s)
O 200 400 600
Time (s)
Figure 5.3
Acetic rcid
Propionic icid
O 100 200 300 400 500 600
Tirne (s)
Figure 5.4
Acetk acid 0 Propionic acid - Linear regression plot
Activation energies of acetic acid (Ea-AA) and propionic acid (Ea-PA). Values in parentheses are the coeflficients of @on (3)
INHIBITION OF SURFACE SPûiLAGE BACTERIA ON MEAT PRODUCTS BY
APPLICATION OF ANTIMICROBIAL FILMS MADE W H CHITOSAN
dl. Abstmct
Antimicrobial films were prcparcd by incorporating acetic or propionic afids into a
chitosan matrix, with or without lauic acid or Qnnarnaldehyde. The films wen appücd on
t!uee types of meat prcxiucts (bologna, ham, or pastrami) under vacuum package conditions
and evaluated for their ability to main the antirniaobial agents. The efficacy of the films was
aiso tested against indigenous lactobact lii and Enterobactenaceae, and on k t o k i l l u s sake
or Sematia liquefucierzs artificiall y inoculatcd on the meat products surfaces. Regardless of the
types of film or meat proâuct, more than 75% of acetic or propionic acids were nleased from
the films during the fmt 3 h, while 27 to 60% of lauric acid or cinnamaldehyâe remained after
1 week. The dease of acetic or propionic acids was slower in films containing lauric acid
compared to control films and films containing cinriamalâehyde. This release was also slower
in f i h applied on bologna c o m p d to those applied on ham or pastrami. Antibacterial films
wac effective in inhibi ting bacterial grow th, paaicularl y Enterobacteriaceae and S.
liqwfaciens. Strongest antibacterial effefts were obtained with films in which cinnamaldehyde
was CO-incorporated with acetic or propionic acids.
Key words
Antibacterial films, chitosan, acetic, propionic, lauric, cinnamaldeh y&.
Microbial growth on meat products is a major cause of food spoilage. Numcrous
organoleptic changes accuring on meat and meat products have been amibutcd to the growth
of various bacterial species (Holley, 1997; Korkcala and BjUrkroth, 1997; Rencm and
Labadie, 1993). Fresh meats and meat products have often k e n trcatcd with various
antimicrobial additives by dipping, spraying, or dusting surfaces, in order to contrd rnicrobial
spoilage (Anderson et d., 1988; Abugroun et ai.., 1993). 'Ihe efflcacy of organic acids,
essential oils. and long chah fatty acids against vaious rneat spoilage organism, for potcntial
use as meat pnservatives was evaluated in controUed conditions by Ouattara et al., (1997%
1 997b).
Since bacterial growth occum mainly on meat surfaces (Holley . 1997). antimicrobial
agents incorporated into food products formulations may be diluted or inactivated by food
components, limiting their efficacy against surface contarninants (Siragusa and Dickson, 1992;
T o m et uf., 1985). The use of packaging films that contain active antimicrobial agents could
provi& more advantages by slowing the migration and maintaining high concentrations of
antimicrobials on the food surfaces. The feasibility of üiis technology has ahady been
àemonstrated with low &nsity polyethylene (LDPE) films in which iMzalil or knzoic
anhydride were incorporated (Weng and Hotschkiss, 1992; 1993). Similady, nisin and
pcdiocin were appiied to food packaging materials and significant reductions of the growth of
fistericl monocytogenes inoculatcd in meai and pwltry was found (Ming et ui., 1997).
Significant progras has also km made in this field with synthetic films. A rccent
nview by Gennadios et cil. (1997) rrpoited i n c d n g intaest in edible coatings and films.
Materials used included lipid, protein and polysaccharides. For example. cellulose-based
edible films have succcssfu11y been incorporateci with sorbates to control food surface
microbial spoilage (Vojdani and Toms, 1989; 1990, Rico Pena and Toms. 1991).
Chitosan is an arnino pol ysaccharidc that has been found to possess good film-fomiing
and antimicrobial properties (Saîto et ai., 1997; El Ghaouth et al., 1992; Damadji and
Izurnimoto, 1994). Due to its entrapment capabiüties (Knorr and Teutornimoto, 1986; Kaya
and Picard, 1990) and its ability to fom covalent bonding andor cross-linking with anionic
compounàs (Mi et al.. 1997) chitosan can also serve as a material for controlled-nlease of
dmgs.
In the present study, chitosan films were incorporatcd with acetic or propionic acids,
and applied on various meat products in vacuum package conditions. We anempted to
determine the ability of the films to main antimicmbiaî agents. Films were also tested for
antimicrobial properties against (i) indigrnous lactobacilli and Entembacteriuceae present on
meat products, and (ii) Lactobacillus su&e or Semuio liquefmiens artificially inoculateci on
meat product surfaces.
Technical grade chitosan fmm crab shells (Sigma Chemical, St-Louis, Mi) was
solubilized to a final concentration of 2% (wlv) in aqueous solutions (1 %, vlv) of acetic or
propionic acids (Fischer Scienti fic. Ne pean, Ontario). The film- fonning solutions were fil tered
through a coarse glas filter, and one hundd ml of each solution was poured on a (20 X 20
cm) plexiglass plate, and ciricd at 80°C (BT-23 [sotemp oven, Fischer Scientific, Nepean,
Ontario) for 4-5 h o w to obtain the simple films with acetic or propionic acids. Composite
films were obtained in the same manner, but lauxic acid (purity > 99%. Sigma Chemical) or
transcimarnaldehyde (Aidric h Chemical, Milwaukee, WI) wi th puri ty > 9996, were
incorporated to final concentrations of 0.5% and 1% (wlw), respectively in the simple-film
forming solutions. Three types of composite films were prepared : acetidcinnamaldehyde,
acetidlauric acid and propionic/cimama~&hy& films. Simple films neutralized according to
the procedure describeci by Vojdani and Toms (1989) serveci as controls for the determination
of the effect of chitosan alone. A complete list of the films used in this study is given in Table
6.1.
6.3.2. Organisms and culhim
kicrobocillu sake (ATCC 15521) was obtaincâ h m the Amencan Type Culture
Collection, (Rockville, MD). Serruzia IÙpefdens was isolatcd fmm vacuum packaged
bologna (Food Re-h and Developmmt Centre, St-Hyacinthe, Quebec). L sake and S.
liquefaciens were fmt grom at 20°C on &Man, Rogosa and Sharp (MRS) and brain hem
infusion @HI) agars, respectively. Both media wcn obtained from Difco laboratones (Detroit,
MI). Bacterial cells wen suspendcd in reconstituted skim milk (skim milk powder in
deionized water, 20% (wlv) final concentration) cmtaining 5% (wlv) sucrose, and lyophilized
to obtain stock culhires. Prior to the antimicrobial test, the cultures were standardized through
three successive 24 h p w t h penods in appropriate broth to obtain working cultures
containhg approximately lo9 CFü/ml.
6.33. D b i o n tests
Cooked bologna in 4 kg chubs, ham in regular mol& (5.45 kg), and cooked beef
pastrami in whole muscle pieces weighing approximately 2.5 kg were purchased locally in the
supermarket. Slices (100 mm x 15 mm, diameter x Uiickness) were cut transversally h m
meat products using a slicer (mode1 VI-34 manual slicer, Hobart Canada.. Don Mills, Ontario)
and introduced in equivalent size stcrile petn plates. Squares (9 cn?) of each tested film were
applied on the surfaces of meat slices. The slices weic placed in M e r Winpak Deli #l bags
(Winpak, Montreal, Qc) and vacuum packaged (Mode1 A-MO, Multi-vac, fepp HaggenmUller,
Wolfertschwenden, Gennany). Packages w a c stand at 4'C, and film samples were removeci
after 3,6,12,48, and 168 h storage to determine the midual concentration of the incorporatcd
compounds. Unused films were analysed to dctennine the total amount of the compounds in
the films before the diffusion test.
Al1 the compounds incorpaated in chitosan films wen quantificd using a Hewlett-
Packard mode1 5890 gas chromatograpb (J & W Scientific, Folsom, CA). Films wcre f h t
resolubilized in hydrochloric acid solution (146, wlv). For the determination of acetic and
pmpionic acids, butyric acid OS%, wlv (Sigma Chernical) was added to the nsolubiiized
solutions to serve as intemal standard, and the compounds wen extracted with ethyl acctate
(Burdick 8 Jackson. Muskegon, MI). Samples of 1 pi w e n taken from the ethyl =tate phase
and injected in a DB-FFAP column 30 m x 0.25 mm i.d (Chromatographie Specialities,
Bmckvillc, Ontario). The oven temperature was isoùicrmal at 90°C for 3.5 min and r a i d
(12*Umin) from 90°C to the final temperatun (130°C).
Cinnamaldehyde was extracted and analyzed according to the samc p r o c c d ~ ~ ~ with
camphor (Aldrich Chernical. Milwaukee, WI) as intemal standard and a 1:m DB-1 tùsed
silica column 30 m x 0.316 mm i.d (J & W Scientific, Folsom. CA). The oven temperatwe
was pmgrammed to rise ZOUmn from 90°C to 115OC, SoUmn from 115OC to 200°C, and
nmained isothennal at the final temperature (200°C) for 4 min.
Lauric acid was âettnnined using a modified saponificationtstcrification-extraction
( SE) meihod previously describeci by Dworzanski et ui. (1990). The whole ptocedurc was
carricd out in glas test tubes sealed with s m w caps. Myristic acid was addcd to the l ahc
acid film solution and served as internai standard. The mixture was thcn saponified fa 30 min
ai 100°C with methanolic sodium hydmxidc solution (3 N NaOH in 50% mthanol). To
prepare mthyl esters of the fatty acid sodium soaps, a 14% solution of boron trifluoride-
methanol (Sigma, Chernical) was added and the mixture was heated at 80 + 1°C for 10 mn.
After rapid cooiing the esters wae cxhaaed with a m i x ~ of diethyl ether and hexane (1: 1)
h m the aqueous r n e t h d c phase and adyred by gas chmatography. The analytical
conditions were similar to thosc for cirmmaldchyde except for the column tempenihuc
pmgram: starting at 15O0C for 2 min, rising 3û°C/min to 210°C and mnaining isothemial for
12 min.
63.4. Antimicrobial test
The antibacterial tests werc perfomxd on bologna and ham. Slices of 5.8 cm in
diameter and 1.5 cm in thickness w e n cut transvcrsaiîy from the mcat products as previously
âescribed. Before each use and ktwcen, the machine was washed and matcd with 80% (vlv)
ethanol. Slices were wptically introductd into equivaknt size sterile peai plates. The
antimicrobial properties of the chitosan films were first tcsted against the indigenous micro-
organisms of meat products. The upper surfaces of siices were completely coated with simple
and composite chitosan films and vacuum packaged in barria films (Skin EVA TR) using a
skin-Pack vacuum packaging machine (Moàel RM 331 M3, Triton Intact Meat slices
packed without chitosan films served as control. In a second experiment, the films wen
evaluated on artificially inoculated prducts. Stede pads were dipped in 10'~ dilutions of the
working cultures of L sake or S. IÙpef4ciens and applied on the surfeces of meat products.
After 5 min, the pads were rcmoved and mai surfaces allowed to dry un&r a biological
containment hood Package and stmge p r o c ~ s werc similar to those &scribeci pmiously
in the antimicrobial test against indigenou microorganisms. Uninoculated and inoculated
meat slices packed without chitosan fiims scrvcd as ncgative and positive controls for bacterial
(L suke and S. liquefaiem) p w t h , rcspectively. Al1 the samples wert evaiuated immediately
to determine the initial contamination and s t o d at two ciiffernt tcmpeninirics (4 and 10°C).
Other microbiological evaluations o c c d after 7.14. and 21 days storage.
At sampiing, bags were opend, and the total surfaces (26.4 cm=, 0.2-0.3 cm thick) of
the meat slices were aseptically excisai, added to 90 ml of O. 1% (wh) Bacto-Peptone (Difco)
and stomached for 120 S. S. liquefaciem and EnterobucteBaceae were enumerated on Brain
Hem Agar (BHA) and violet red bile glucose agu (VRBGA). respectively, with incubation at
35°C for 48 h. L s& was determined on MRS agar with incubation at 2S°C for 72h. Similar
conditions were used to enurnetate total Iactic afid bacteria, but thallous acetate (0.1% wfv)
was added to the original MRS medium. Dilutions w e n spread-plated on prepoured media
(0.1 ml/plate) with the exception of VRBG where the conventional oveday method was used.
The total surface of the meat samples was used to calculate the nurnber of organisms/cm2.
6.3.6. Strtirtfcal umlyrir
Experirnents were dom in duplicate and 3 samples w m analyzed at each sampling
tirne. Data were s u b j d to an analysis of main and interactions effects of time. type of meat
product, and type of film using the GLM procedure of the SAS statistical package (SAS
Institute, Cary, NC). The least significant differencc (LSD) test was used at 3, 6, 12, 48,
and 168 h for point-by-point detemination of the influence of film and meat products.
Difference between means wem considcred when pe0.05.
Regardless of the typcs of films and meat products, more than 75% of afetic or
propionic acid w m desorbted in the first 3 h foliowing the application of fiims on meat
products (Figure 6.1). After 3h, the pattcms of the dcsorption curvcs differed between acetic
and propionic acid. For acaic acid, the diffusion pmass was slowcr, and the residual amounts
found in films were higher than 10 % (bologna), and 5 46 (pastrami and h m ) over the entire
experimental pend In contrast, with pmpionic acid, the desorpion process continued and
less than 2 % of the initial concentration remaineci at the end of the experimenia period for al1
the meat products. It appears also chat films containing lauric acid retained higher percentages
of residual amounts of acetic acid compared to control films and films containing
cinnamaldehyde. In general no diffennce in the residual pemntage of organic acids was
found between films containing cinnddehydc and control films.
The influence of the type of m a t product on the diffusivity of organic acids is shown
in Figure 6.2 and 6.3. With acetic acid, midual per~entages were significantl y (@.OS) higher
when the fiims were applied on bologna c o m ~ to ham and pastrami, but no sigruficant
difference was found betweem hm and pasüami except for AAC fih (Figure 6.2). With
propionic acid, however, the type of meat product did not affkct diffûsivity (Fi grire 6.3).
For a ôetter undcrstanding of the influence of the incorporation of cinnamaldehyde and
launc acid on the diffûsivity of acetic and propionic acids. the concentration of the former two
compounds was determincd kfore and afbr 1 wcck in the same enperimntal conditions
(application of films on mcat produçts and storage in vacuum package conditions at 4OC).
With an initial concentration of 2.17 m # c d of lauric acid in fiirns, the amounts xecovd
afttr 1 wetk v a M d h m 1.29 to 1.68 rndd depending on the type of meat product as shown
in Tabk W. niose midual concentrations of lauric acid reprrsented more than 60% of the
initial concentration. With cinnamaldehyde. only 0.029 (pastrami). 0.030 (ham), and 0.053
(bologna) mg/cm2 remained in the films after 1 wetk, comparai to 0.104 mg/cm2 bcfore
application on meat products. The nsidual conceneations of lauric acid and cinnamal&hy&
nmaining wen significandy higher (p4.05) in films applied on bologna than in those applied
on ham or pastrami.
6.4.2. Antimicrobial test
The inhibitory effect of vatious chitosan films against L sake and S. liquefmiens
artificially p w n on the surfaces of meat products is presented in Table 6.3. In general, p a t e r
inhibition of bacterid growth appearcd in the presence of the films than in control samples
wherc significant p w t h was noted. At the end of the experimental pcriod, kvels of total
counts dccxeased by 0.38 to 4.13 log units &pendhg on the types of film and the bacterial
srrains.
The strctngest inhibition was observeci with films containhg cinnamaidehyde which
Rduced the total counts of S. fiquefmiem to 4.25 W c m 2 (AAC films) and 4.61 CFU/cm2
(APC films) after 21 days storage, cornparcd to the control meat slice samples which
contained 8.38 CFU/cd. Similady. 5.31 and 5.01 CFü/cm' were obtained for L sake
cornparcd to 6.10 CFUlcd in contiol samplcs. These values wem significantly ciiflemnt from
those obtained with the othcr types of fih. Aftcr 21 days AAL films provided additional
reduction of L sake (5.62 CFü/cm3 cornparcd to AA films (5.78 CNlcm'), but failed to
enhance the effectiveness of the simpk films against S. liqrref~ciens. It cm also be noted that
the neutralized fi lrns (AAN) had no antibacterial efficac y.
Among the two bacteria under study, L sokr was the most nsistant. Aftcr 14 dap. the
growth of this strain was not significantly affcctad except in the case of AP and APC films.
Furthemore. at the end of the expeiimental pniod, levels of total counts of L suke decreased
only by 0.38 to 1.09 log units. On the other han4 al1 the types of films except AAN films
inhibited the growth of S. 1iquefacien.s over al1 the cntire storage period. with reduction of
total numbers reaching a maximum of 4-13 log units.
The inhibitory effects on total Enterobucteriaceae and total lactic acid bactena in
uncontaminated meai products were iested ody with AA, AAC, and AAL films. AI1 the types
of films produced signifiant reductions of Enterobucteeriaceue present on the surfaces of
meat products with complete inhibitions occurring mainly when the films were tested on
bologna (Table 6.4). AAC fih wcrc found to k the most inhibitoiy with lowest (fl.05)
bacterial counts cornparcd to AA and AAL films, particularly in the case of pastrami. It can
also be noted that lowering of storage temperature from 10 to 4OC resulted in enhancing the
efficacy of the films.
The influence of chitosan films on the growth of total lactic acid bacteria afkr 21 days
of storage is show in Table 6.5. No complete inhibition of bacterial growth was observeù,
and no significant effect of film was obtaineâ with bologna at 4OC. In al1 the o k cases
(ôologna at 10°C; pasitram at 4 and 10°C), 1 the fih showed significant reductions in lactic
acid bacterial numkrs. However, no diffcnnce was found among the films under study.
The mechanism of the rrlease of hydrophilic compounds incorporated in a polymer
matrix can be compared to a swellingcontrolled mode1 of dnig release in which the enterance
of the diffusing liquid and the transfer of the incorporated compound out of the polymer take
place almost simultaneously (Armand et d, 1987). The observed kinetics of nlease of acetic
and propionic acids could be attributed to the hydrophiiic natue of chitosan which permits
rapid entering of water into the polyrner maaix during the earlier pend of the application of
films on meat product slices. As a consequence, the acids axe dissolved and can migrate out of
the films.
The relationship between polymer swelling and the nlease of organic acids can k
explaineci by the hypothesis of mutual npulsion of cationic p u p s as rcported by Narisawa et
al. (19%). With chitosan, mutuai rcpulsion may occur kt- the -NH3+ groups during film
formation, creating a luge number of small pons which are hydratai by the smunding
water (Okor, 1982). Also, ionic osmosis rnay play an important role because the hydration
induced by water-intake should be based on the concentration difference of ions between meat
surface and the fih. Sincc this diffe~ncc is highcr in the fkst hours following the application
of films on meat products, the diffusion praxss is more important. However, absence of
parallelism ktwem the diffusion CIVVCS of acetic and propionic acids, particularly in the latter
stage, suggests that the kinetics of rcleasc could not bc explaincd only by mutuai quision or
osmosis theories. Some &et factors rnay k involved more significantly. For exarnple,
propionic acid which has highcr mokcular weight, may affect differently the structwt of
chitosan, leading to films with diffemt flexibility than chitosanlacetic acid films. That
hypothesis is supported by the well-known effcct of cross-linking which establishes a close
relationship between film flexibility and film h ydration properties (Narisawa et al., 1996).
It is known that incorporation of lipophiüc compounâs in hydrophilic polymers rcsults
in reduction of water uptake (Vaquez et ai., 1997). The lipophilic compounds may also
modify the anangement of the structure of chitosan, leading to an increase in the network
tortwsity (Calkgarin et d., 1997; Redl et 1, 19%) or affect some geometrical feanires,
impeding transport through the network (Papadokostalâ et ai., 1997). For exarnple, Redl et al.
(1996) found that addition of beeswax or acetylated monoglyceriâe in whey gluten films
nsulted in 20-50% reduction in sorbic acid diffusivity. Similarly, the addition of various fany
acids has ben found to d u c e the pcrmcability of potassium sorbate through methylcellulose
and hyhxypropyl methylcellulose films (Vojdani and Toms, 1990) and the water
permeabiüty of chitosan films (Wong et ai., 1992). In agreement with these findings. film
containing lauiic acid were found to maintain more efficiently the incorporated organic acids,
followed by films containing ci~arnaldehyde.
During storage under vacuum, the swfaces of bologna slices kcame only slightly
wet. while h m and pastrami showed visible liquid afkr only 6h. This differcnce in water
availability on the surfaces which corne in contact with the films may have accounteà for
the higher residual concentration of acids found in films tested on bologna compared to
those tested on the othcr meat products. Bettcr performance of antirnicrobial films on the
ciria bologna surfaces is also consistent with the swelling-controlled mode1 for drug rclease
(Armand et al., 1987).
The influence of the type of meat product on the release of organic acids can be also
descrikd in tems of nsidual concentrations of lipophilic compounds recovered in the
films. Since bologna pemiits higher arnounts of lauric acid or cinnarnaldehyde to nmain in
the films. the rcsidual amounts of acetic or propionic acids were higher in films tested on
bologna cornparcd to those tested on ham and pastrami. That is also in agreement with the
fact that lipidic compounds can affect the diffusion proccss in hydrophilic polymers.
The observed kinetics of relew of acetic and propionic acids were also consistent
with the prcvious report of Demarger-Andrc and Domard (1994), that the interaction
occumng between chitosan and organic aci& is pmly electrostatic. without any
compleaation process, indicating that pH may significantly affect diffusion. Chitosan is a
glucosamine which has a pK value of 6.3 (Mi et al.. 1997). and below this value, the amino
groups of the polymer are ionized. Althougb there is somc evidence that acetic and
propionic acids arc also ionized at pK values above thcir pKa (4.76 and 4.90. respectively).
pH values found in mcat products (5.8 to 6.3) may considerably reduce the range in which
both the amino groups of chitosan and the carboxyliic p u p s of organic acids are ionized.
That is probably one of the most plausible ways to explain the important mlease of organic
acids observed in the 3 first hom following the application of film on meat products.
Furthemiore, according to the Flory's "ion-network theory" reportcd by Narisawa et al.
(19%). the pnsmce of dissociateci f o m of organic acids mates a new ionic circurnstance
nsulting in an incrcase in ionic osmosis, and hcnce hydratation is promoted by the induced
water influx. As a consequtnce, pemeability of films consiâerably incnases.
From severai previous studies, consistent inhibitory effects against many pathogens
and spoilage bacteria have been reportcd for various organic aciâs (Brocklehunt and Lund,
1990; Greer and Dilts, 1992; O u a m et d, 1997a). essential oils from spices (Aurcli et
al., 1992) and long chah fatty acids (McKellar et ul., 1992; Ouattara et al., 1997b). As a
consequence, combinations of these compounâs in a polymer matrix might provide
synergistic antibacterial effects. Positive interaction explains the strongest inhibition found
with AAC and APC films in which organic acids and cinnarnaldehyde were combined, and
complements the report of Valenta et ai. (1997) who included cinnarnaldehyâe and
lysozyme together in cnarnlgel mode1 pnparations and found an improvement of
antibacterial activity against E. coli and S. aurem. Positive interactive antibacterial effects
wcrc also obtained with AAL films. Lauric acid which is known to be inactive against gram
negative bacteria (McKellar et al., 1992 ; Ouanara et al., 1997b) could not provide AAL
films with any additional inhibitory effcct against S. liquefaciens and total
Enterobacteriaceae comparcd to AA and AP films (which contained only one organic
acid).
Our results could not establish clearly a rclationship ktwcen observed antibactcrial
properties and the ability of various films to rctain high concentrations of organic acids. A
large proportion of these compounds w m nleased imrnediately when films were placed in
contact with meat prcxiucts. Such obsmation suggests that chitosan may not possess good
carrier properties for organic acids. Weng and Hotschkiss (1993) also reported that organic
acids did not incorporate into LDPE film because of the apoiar nature of this polymer.
No antibacterial effect was obtained with AAN films, suggesting that the inhibitory
effect of chitosan alone could not be dcmonstrated. That observation was surprising since
chitosan has k e n pnviously reponed to sipificantly inhibit various meat spoilage
bacteria. The mechanisms of inhibition of chitosan molecules is mainly attributed to its
hi@ water binding capacity and its ability to inhibit various enzymes (Young et al., 1982).
Chitosan also has bio-absorption activity and can absorb nutrients normally used by
bacteria and may inhibit their growth in this rnanneflarmadji and Izumuto, 1994 ; Knom,
1991). It is possible that such mechanisms of inhibition require chitosan molecules to be
rnixed with the meat product instead of k ing applied as films on the surfaces.
L sake and total lactic acid bacteria wtrc the most rcsistant to the action of chitosan
films containing antimicrobials tested. That result is consistent with the fact that lactic acid
bacteria are generaily unaffected by organic acids (Ouattara et al., 1997a). Hutskin and
Nannen (1993) reportcd that lactobacilli possessed spccific canier-mediated transport
mechanisms which contribute to the maintenance of the intemal pH homeostasis when
bacteria are placed in acidic conditions.
6.6. Conclusion
The prcscnt study was designcd to re!srd the growth of m a t spoilagc bacteria using
various antimicrobial agents incorporattd in chitosan films. Results showed that the
antibacterial films were effective in inhibiting bacteria growth. particuiarly the growth of S.
liquefaciens and total Enterobucteriuceae. These m l t s demonstrate that incorporation of
antibacterial agents into tilm-forming solutions may be a feasible way to obtain films
capable of controlling bacterial p w t h on the surfaces of meat and meat products.
However, many other factors have to be taken into account in the development of
antibacterial packaging, such as the chernical nature of the carrier material. and how the
composition of food can affect its interactions with the antimicrobial agents. Greater
emphasis needs to k placed upon the development of inhibitor combinations which can
&lay the growth of lactic acid bacteria primarily responsable for the spoilage of vaccum
packaged meats prepared under sanitary conditions and stored pmperly under refrigeration.
6.7. List of tables
Tabk 6.1. Type of films and composition
Table 6.2. Concentration of lauric acid and cinnarnaldehyde (mglcm2) in composite films
befon and after 1 week application on rneat products in vacuum package conditions at 4°C.
Table 6.3. inhibitory effects of chitosan films against L sake and S. Iiquefaciens inoculated
on the surface of cwked ham slices stored at 4°C.
Table 6.4. Inhibitory effect of selected chitosan films on the growth of Enterobacteriaceae
present on the surfaces of bologna and pastrami.
Table 6.5. inhibitory effect of selected chitosan films on the growth of lactic acid bacteria
present on the surfaces of bologna and pastrami after 21 days storage.
6.8. List of Figures
Figure 6.1. Percentage (% of original) of acetic or propionic acids remaining in chitosan
films after application on meat products in vacuum package conditions: influence of the
type of film.
Figure 6.2. Percentage (96 of original) of acetic acid remaining in chitosan films after
application on meat products in vacuum package conditions: influence of the type of meat
products.
Fipre 6.3. Percentage (% of original) of propionic acid remaining in chitosan films after
application on meat products in vacuum package conditions: influence of the type of meat
produc t S.
Simple films Composite films
Tenn Composition Tem Composition
AA Chitosan / acetic acid AAC Chitosan 1 acetic acid 1 cinnamaldehyde
AAL Chi tosan / acctic acid 1 lauric acid
AAN' Neutralized films
AP Chitosan 1 propionic acid APC Chitosan 1 propionic acid /
cinnamaldeh y&
a' Neutraiized films : Organic acid was completely removed from these films.
Table 6.2'
Meat products Lauic acid cinnamaldehyde
After Bologna
Ham
Pastrami
'. Within each column. means folowed by different letters are significmtly different
(p4.05; LSD).
Table 6.3
conda Bacterial growth (log ~Fülcrn~)'
L. sake Control
AA
AAC
AAL
AAN
AP
APC
S. ligue faciens Control
AA
AAC
AAL
AAN
AP
APC
'. Experimental conditions. Samples noncoatcd (Control) or coated wi th acetic
acidlchitosan films (AA), acetic acidcinnamaldehydc/chitosan films (AAC), acetic
acidnauric acidlc hi tosan films (AAL), ncutralizcd acetic acidlc hi tosan films ( A M ) ,
propionic acidchitosan films (AP), propionic acidlchitosan/cinnamaldehy& films
(Am*
b. For each bacterid seain, and within each storage interval, means followed by the same
letter am not significantly differcnt ( ~ 4 . 0 5 ; LSD).
'. ND : not determined.
Table 6.4
Bacteriai p w t h (log CNlcm')'
Bologna Control 1.55 î 0.10 CF 3.20 î 0.32A 0.81 0.12* 4.5SA
AA CI 0.22 i 0.MB CIB Cb
AAC CI 0.15 î 0.07~ CIB C ~ B
AAL CI ck CIa CIB
Pastrami Controi 2.3 1 10.14 3.83 1 0.2 t A 4.95 î 0.24* 5.70 î 0.27A 6.35 * AA Cie 1.7610.51~ 0.90î0.29c 3.85î0.17c
AAC CIB 1. 50 I 0.49~ CID 3.06 O. 1 4 ~
AAL Ch 1.74 I 0.55~ 3.01 * 0.1 le 4.42 1 0.29~
'. For each storage temperature. and within rneat products. values followed by the same letter are
not significantly different ( f l .05; LSD)
b. Experimental conditions. Samples nonîoated (Control) or coated with acetic acidlchitosan films
(AA), acetic acid/cinnamaldehyde/chitosan films (AAC), acetic acidnauric acid/chitosan films
( A U *
'. CI :cornplete inhibition; no bactenal growth was observeci.
Total lactic acid bactena (log CFül~rn~)~
condb Initial 4OC 10°C
Bologna Con trol
AA
AAC
AAL
Con trol
AA
AAC
AAL
a. For each storage temperature, and within meat products, values followed by the same
letter are not significmtly different (~4 .05 ; LSD).
b. Experimental conditions. Samples non-coated (Control) or coated with acetic
ridchitosan films (AA), acetic acidlcinnamaldehydelchitosan films (AAC), acetic
acidnauric acid/chitosan films (AAL).
Figure 6.1
Bologna
1 O0 95
C1
P(
O 50 100 150 Storage time (h)
Acetic acid
O 50 100 150
Storage time (h)
Propioaic acid
-c+ Control films * Films with cinnamaldehyde Films with launc acid
Figure 6.2
- 3 6 12 48 168
Storage time (h)
O Bologna - Ham Pastrami
Acetic acidchitosan tilms (AA); Acetic acid/ciiwrmildehyde/chitosan films (AAC); Acetic aQdnauric acid/chitosan films (AAL). At =ch storage the, means with dflerent letters are significantly different (pc0.05; LSD).
Figure 6.3
Storage tirne (h)
Propionic acid/chitosan films (AP); Propionic acidlcinnamaldehyde/chitosan films. At each *orage tinte, means with diffamt laters are significantly difkrents (p~0.05; LSD).
0 Bologna B H m 30 - APC
CHAPITRE 7
CONCLUSION GÉN~RALE
Dans une première Ctape, des composés antimicmbiens ont 6tt sélectionnés sur la
base de leur efficacité contre six souches bacttriennes d'altdration des viandes et des
produits carnks : Brochoth& thennosphucta, Carnobacteriwn piscicola. kictobacillus
curvatus. Lactobacillus sake. Pseudomo~s fluorescens et Serra fia liquefaciens. Au total . 23 composés comprenant des acides organiques, des acides gras 5 longues chaines et cies
huiles essentielles ont été test&. Deux acides organiques (acides &tique et propionique).
deux acides gras (acides laurique et palrnitoldique) et trois huiles essentielles (huile de
cannelle. de clou de girofle et de romarin. Comme on pouvait s'y attenàre, les souches
bactbriennes étudides avaient des sensibilitCs variables. Compte tenu de leur pH optimal de
croissance faible, les bactdries lactiques Ctaient plus dsistantes aux acides organiques que
les autres souches. De même, les bactknes gram ndgatif Ctaient plus dsistantes aux acides
gras ii longues chaînes que les gram positif à cause de la présence d'une couche tpaisse &
lipopolysacchariâe dans leur paroi. Ceci démontre toüte l'importance du choix des
antimicrobiens.
Nous avons réussi il incorporer les agents antimicrobiens les plus actifs ou leurs
extraits dans une matrice B base de chitosanc et B obtenir &s films posstdant & bonnes
qualites mécaniques. La capacité des films h amir les antimicrobiens a kt6 ensuite Cvaluéc
en milieu liquide. D'après Ics résultats obtenus, deux facteurs influencent la cinttique de
libération des acides organiques : i) la températurc qui affecte les interactions entre les
molécules. les basses températures permettant d'obtenir les coefficients de diffusion les
plus bas ; ii) l'incorporation sirnultan& de composés lipidiques qui réduit la diffusion en
augmentant la tortuositt du &eau polysaccharidique. Aucune influence du pH n'a ttC
observée sur la performance du chitosane dans l'intervalle de pH compris entre 5.7 et 7.0.
Afin de valider les résultats sur les caractéristiques de diffusion. des tests ont ttC
réalises sur des produits camts (bologne. jambon et pastrimi) emballts sous vide durant
une période totale de 7 jours. Le contact des films avec les viandes provoque une libération
rapides des acides acetique et propionique. Plus & 75% & ces composés disparaissent
durant les 3 premières heures. Par la suite, le processus de diffusion est considérablement
ralenti ou s'arrête. Les concentrations résiduelles d'acides organique retrouvt dans les films
aprés 7 joua de conservation dependent âes facteurs suivants: i) le type d'acidc organique.
L'acide acétique est libtré moins vite que l'acide propionique; ii) la formulation âes films.
Lcs films contenant des composés lipidiques retiennent plus d'acides adtique et
propionique que les films simples; iii) le type de produits cames. Le bologne, plus sec en
surface provoque une libération plus faible des antimicrobicns que le jambon et le pastrami.
Les films ont CtC aussi test& pour leur efficacité antibactCrienne contre la flore
normale des produits camés et des souches bactCricnncs de Luctobacillus sake et Serratia
liquefuciens artificiellement inoculés en surfaces. Les résultats montrent que les films sont
efficaces. particulièrement contre Serratiu liquefuciens et les Enterobactenaceae et que les
effets antibactériens sont en relation avec l'efficacité antibactkrienne intrinsèque des
composés chimiques qu'ils contiennent. Ainsi, les taux de réduction les plus importants &
la croissance bacterienne ont et6 obtenus avec des films qui contiennent ii la fois un acide
organique (acttique ou propionique) et un extrait de Iliuile de cannelle (cinnamaldehyde),
tandis les films de chitosane neutralisés (sans agents antimicrobiens) ne possèàent aucun
effet antimicrobien.
Les résultats obtenus au corn de ce travail &montrent la faisabilité du concept
d'emballage antimicrobien pour les viandes et les produits carnds. Le syst&me reste
ndanmoins très complexe et fait intervenir plusieurs facteurs dont les effets sont encore
insufisamment optimisés. Par conséquent, avant une utilisation de ce procédé il l'échelle
industrielle des Ctudes complémentaires doivent être faites notamment pour trouver une
solution à la diffusion rapide qui a lieu après l'application des films et pour trouver des
antirnicrobiens plus efficaces contre les bactdnes lactiques d'alttration de la viande et des
produits carnes.
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