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

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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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