RECHERCHE D'ALTERNATIVES AUX NITRATES ET NITRITES DANS LES PRODUITS CARNÉS

Mémoire

Anne Patricia Kouassi

Maîtrise en génie agroalimentaire - Génie agroalimentaire

Maître ès sciences (M.Sc.)

Québec, Canada

© Anne Patricia Kouassi, 2015

iii

Résumé

L'objectif principal de cette maîtrise était de développer des formulations d'épices

capables de remplacer les nitrites et nitrates dans les produits carnés tout en gardant la

même durée de conservation que ces derniers ainsi que leurs propriétés organoleptiques,

antibactériennes et antioxydantes. Un criblage qualitatif puis quantitatif des épices a été

réalisé et trois épices ont été sélectionnées, les clous de girofle, le cumin et la cannelle.

Puis, des poudres de fruits ont été testées pour approcher la couleur rosée que donnent

les échantillons contenant les nitrites. L'analyse sensorielle effectuée par la suite a fait

ressortir le caractère sucré de la poudre de fruit et des propriétés très intéressantes de

certaines formulations par rapport à l'ajout des nitrites. Enfin, une analyse technico-

économique a été réalisée et a montré une très faible augmentation du coût de

production des produits carnés en utilisant nos formulations d'épices et de poudre de

fruit.

v

Abstract

The aim of this study was to develop formulations that can replace nitrates and nitrites

in meat-based products while keeping the same shelf life as nitrites and also, their

organoleptic, antimicrobial and antioxydant properties. A qualitative and quantitative

screening of spices have been made and three spices were selected, cloves, cumin and

cinnamon. Different fruit powders were tested to get closer to the pinkish color which

nitrites give to meats. Then, an organoleptic tests were performed on a panel of tasters

and the results have helped identify the sweetness of the variety of fruit powder and

very interesting sensory properties of several formulations in relation to the addition of

nitrites. Finally, a techno-economic analysis was performed and showed a small

increase in cost of production of meat products using spices and fruit powder, due to a

slight increase in the cost of raw materials.

vii

Table des matières

Résumé ............................................................................................................................. iii

Abstract ............................................................................................................................. v

Table des matières .......................................................................................................... vii

Liste des tableaux ............................................................................................................. xi

Liste des figures ............................................................................................................. xiii

Liste des abréviations .................................................................................................... xvii

Remerciements ............................................................................................................... xix

Avant propos .................................................................................................................. xxi

Introduction ....................................................................................................................... 1

Chapitre 1: Revue de la littérature .................................................................................... 3

1.1. Green Alternatives to Nitrates and Nitrites in Meat-Based Products – A Review ........................................................................................................................... 4

1.1.1. Résumé ........................................................................................................ 5

1.1.2. Abstract ....................................................................................................... 6

1.1.3. Introduction ................................................................................................. 7

1.1.4. Definition of nitrates and nitrites ................................................................ 9

1.1.5. Use of nitrates and nitrites as additives in food products ......................... 13

1.1.6. Physicochemical properties of Nitrites and Nitrates ................................. 14

1.1.7. Regulation of nitrite and nitrate ................................................................ 17

1.1.8. Toxico-kinetics of nitrates and nitrites ..................................................... 18

1.1.9. Effect of nitrates and nitrites ..................................................................... 20

1.1.10. Alternative of nitrites and nitrates used for preserving meat products . 24

1.1.11. Advantages in using spices as an alternative of nitrates and nitrites .... 26

1.1.12. Different types of spices ........................................................................ 28

1.1.13. Grapes ................................................................................................... 34

1.1.14. Conclusion............................................................................................. 35

1.1.15. Acknowledgements ............................................................................... 35

1.1.16. References ............................................................................................. 36

1.2. Spice use in food: Properties and benefits –A review ...................................... 42

1.2.1. Résumé ...................................................................................................... 43

viii

1.2.2. Abstract ..................................................................................................... 44

1.2.3. Introduction ............................................................................................... 45

1.2.4. General description of spices .................................................................... 46

1.2.5. Chemical properties of spices ................................................................... 49

1.2.6. Main applications of spices ....................................................................... 54

1.2.7. Advantages & Disadvantages of using spices as preservatives ................ 65

1.2.8. Conclusions and future outlook................................................................. 67

1.2.9. Acknowledgements ................................................................................... 68

1.2.10. References ............................................................................................. 69

1.3. Hypothèse et objectifs du projet ....................................................................... 77

1.3.1. Hypothèse de l'étude.................................................................................. 77

1.3.2. Objectif général ......................................................................................... 77

Chapitre 2 Use of spices as alternative of nitrites and nitrates in meat-based products . 79

2.1. Résumé ............................................................................................................. 80

2.2. Abstract ............................................................................................................. 81

2.3. Introduction ...................................................................................................... 82

2.4. Materials and methods ...................................................................................... 84

2.4.1. Preparation of meat samples ..................................................................... 84

2.4.2. Determination of thiobarbituric acid (TBA). ............................................ 85

2.4.3. Determination of p-Anisidine value .......................................................... 86

2.4.4. Microbiological analysis ........................................................................... 87

2.4.5. Statistical analysis ..................................................................................... 87

2.5. Results and discussion ...................................................................................... 87

2.5.1. Determination of p-Anisidine value .......................................................... 87

2.5.2. Determination of TBA index ..................................................................... 89

2.5.3. Microbiological analysis ........................................................................... 91

2.6. Discussion ......................................................................................................... 93

2.7. Acknowledgements .......................................................................................... 93

2.8. References ........................................................................................................ 94

Chapitre 3 Optimization of spices as alternative of nitrites and nitrates in the meat-based products ........................................................................................................................... 97

3.1. Résumé ............................................................................................................. 98

3.2. Abstract ............................................................................................................. 99

3.3. Introduction .................................................................................................... 100

ix

3.4. Materials and methods ................................................................................... 101

3.4.1. Preparation of meat extract ..................................................................... 101

3.4.2. Determination of thiobarbituric acid ....................................................... 102

3.4.3. Determination of p-anisidine value ......................................................... 103

3.4.4. Microbiological analysis ......................................................................... 103

3.4.5. Statistical analysis ................................................................................... 104

3.4.6. Experimental design and optimization ................................................... 104

3.5. Results and discussion.................................................................................... 105

3.5.1. Effect of variables on physico-chemical properties of processed meat .. 105

Bold values: Significant (p < 0.05)....................................................................... 108

3.5.2. Effects of variables on biological properties of processed meat ............ 108

3.5.3. Determination of optimal conditions and optimal responses .................. 109

3.6. Conclusions .................................................................................................... 115

3.7. Acknowledgement .......................................................................................... 115

3.8. References ...................................................................................................... 116

Chapitre 4 Color Retention in Processed Meats by Using Natural Products and Tests of Organoleptic Properties ................................................................................................ 119

4.1. Résumé ........................................................................................................... 120

4.2. Abstract .......................................................................................................... 121

4.3. Introduction .................................................................................................... 122

4.4. Materials and Methods ................................................................................... 124

4.4.1. Preparation of meat extract for color ...................................................... 124

4.4.2. Color evaluation ...................................................................................... 125

4.4.3. Measurement of different parameters ..................................................... 125

4.4.4. Organoleptic tests ................................................................................... 126

4.4.5. Statistical analysis ................................................................................... 127

4.5. Results and discussion.................................................................................... 128

4.5.1. Effect of pH on color .............................................................................. 128

4.5.2. Solution for color .................................................................................... 129

4.5.3. Organoleptic test results .......................................................................... 133

4.5.4. Discussion ............................................................................................... 153

4.5.5. Aknowledge ............................................................................................ 154

4.5.6. References ............................................................................................... 155

x

Chapitre 5 Analyse technico-économique du procédé de production des produits carnés (Utilisation des épices comme alternative aux nitrites et nitrates ................................. 157

5.1. Description des scénarios de simulation employés pour l'analyse technico-économique de la production des produits carnés ..................................................... 158

5.2. Estimation du coût des capitaux fixes ........................................................... 160

5.3. Estimation du coût d'exploitation annuel ..................................................... 165

5.4. Estimation du coût des matières premières ................................................... 166

5.5. Estimation du coût du produit ........................................................................ 169

5.6. Conclusion ...................................................................................................... 170

Conclusion générale ...................................................................................................... 171

Bibliographie ................................................................................................................. 175

xi

Liste des tableaux

Table 1.1 Summary of the properties of spices .............................................................. 33

Table 1.2 Main chemical characteristics of common spices .......................................... 49

Table 1.3 Spices Bioactivity ........................................................................................... 51

Table 1.4 List of bacterial strains inhibited by spices ..................................................... 54

Table 1.5 Use of spices as insecticides ........................................................................... 55

Table 2.1 Physicochemical properties of spices ............................................................. 83 Table 2.2 List of formulations ........................................................................................ 85

Table 3.1 Results of experimental plan by central composite design for ham and terrine ...................................................................................................................................... 102 Table 3.2 Experimental range of the three variables studied using CCD in terms of actual and coded factors ................................................................................................ 105

Table 3.3 Model coefficients estimated by central composite design and best selected prediction models .......................................................................................................... 108

Table 4.1 The 19 combinations of spices obtained by Statisticia with their pH ........... 124 Table 4.2 List of attributes for the organoleptic tests ................................................... 126

Table 4. 3 List of samples for organoleptic tests .......................................................... 127

Table 4.4 Effet of pH on colour meat ........................................................................... 128

Table 4.5 Color analysis of terrines containing different concentrations of red food chemical coloring agent ................................................................................................ 130

Table 4.6 Color analysis of terrines containing different fruits and vegetables used to improve red color .......................................................................................................... 131

Table 4.7 Analysis of variance of results of terrines' flavor ......................................... 134

Table 4.8 Analysis of variance of results of terrines' odor and texture ........................ 134

xii

Table 4.9 Results of Mean scores and Least Significant Difference for flavor of terrine ....................................................................................................................................... 135

Table 4.10 Results of Mean and Least Significant Difference for odor and texture of terrines ........................................................................................................................... 138

Table 4.11 Adjusted means of descriptors flavor, smell and texture of terrines ........... 141

Table 4.12 Analysis of variance of results of ham's flavor ........................................... 144

Table 4.13 Analysis of variance of results of ham's odor and texture .......................... 144

Table 4.14 Results of Mean and Least Significant Difference for flavor of ham ......... 145

Table 4.15 Results of Mean and Least Significant Difference for odor and texture of hams .............................................................................................................................. 148

Table 4.16 Adjusted means of descriptors flavor, smell and texture of hams .............. 151

Tableau 5.1 Capacité de production de l'entreprise des produits carnés ....................... 158 Table 5.2 Summary of cost data for food plants in 1986 (Bartholomai (1987); Clark (1997); adapted by Rouweler). ...................................................................................... 162

Table 5.3 Estimation des capitaux fixes ........................................................................ 165

Table 5.4 Coûts des matières premières utilisés dans les différents scénarios ............ 167 Table 5.5 Estimation du coût total des matières premières et du coût d'exploitation annuel ............................................................................................................................ 169

xiii

Liste des figures

Figure 1.1 Endogenous biosynthesis of nitrate ............................................................... 13 Figure 1.2 Transformation of myoglobin in meat ........................................................... 17 Figure 1.3 Toxico- kinetics of nitrates and nitrites in human body ................................ 20 Figure 1.4 Nitrate cycle in water .................................................................................... 22 Figure 1.5 Nitrogen cycle ............................................................................................... 24 Figure 1.6 World Spice production in year 2010 (FAO, 2010) ...................................... 45 Figure 1.7 Spices classification (adapted from Peter and Shylaja, 2012, Sajilata and Singhal 2012) .................................................................................................................. 48 Figure 1.8 Ranges of bacterial inhibition data for different spices (adapted from Ceylan and Fung,2004; Holley and Patel, 2005; Naidu, 2000). ................................................. 53 Figure 1.9 Main medicinal uses for several spices (modified from Peter and Shylaja, 2012) ............................................................................................................................... 56 Figure 2.1 p-anisidine values of terrines preserved using different spice formulations at 4°C for 8 weeks............................................................................................................... 88 Figure 2.2 TBA index for terrines preserved using different spice formulations at 4°C for 8 weeks ...................................................................................................................... 90 Figure 2.3 Total viable counts for terrines preserved using different spice formulations at 4°C for 8 weeks ........................................................................................................... 92 Figure 3.1 Response surface of Log10 viability in terrine obtained by varying: a) the concentration of cloves (X1) and the concentration of cumin (X2) keeping the concentration of cinnamon (X3) constant ; b) concentration of cinnamon (X3) and the concentration of cloves (X1) and keeping the concentration of cumin (X2) constant: 0.2 % w/w ........................................................................................................................... 111 Figure 3.2 Response surface of p-anisidine in terrine obtained by varying: a) the concentration of cloves (X1) and the concentration of cumin (X2) keeping the concentration of cinnamon (X3) constant ; b) concentration of cinnamon (X3) and the concentration of cumin (X2) and keeping the concentration of cloves (X1) constant: 0.2 % w/w ........................................................................................................................... 112 Figure 3.3 Response surface of TBA in terrine obtained by varying: a) the concentration of cloves (X1) and the concentration of cumin (X2) keeping the concentration of cinnamon (X3) constant ; b) concentration of cinnamon (X3) and the concentration of cloves (X1) and keeping the concentration of cumin (X2) constant: 0.2 % w/w ......... 113

xiv

Figure 4.1 Pictures of terrines uncooked and cooked at different pH ........................... 129 Figure 4.2 Pictures of coloured samples with differents percentages of fruits and vegetables ...................................................................................................................... 131 Figure 4.3 Effet of fruit powder concentration (w/w %) and associated terrines pictures: (a.) powder 1 bv (brazilian vine); (b.) powder 2 gp (grape); (c.) powder 3 st (strawberry). .................................................................................................................. 133 Figure 4.4 Sensory profile analysis of flavor sweetness, salty, bitterness, acidity and spicy of terrines ............................................................................................................. 136 Figure 4.5 Box plots of the 6 terrines following descriptors: sweet, salty, acid, bitter and spicy. A boxplot is lower than the M-1.5 (Q3-Q1) value, the first quartile (Q1), median (M) solid line, dotted average, the third quartile (Q3) and the highest value less than M +1.5 (Q3-Q1) ................................................................................................................. 137 Figure 4.6 Sensory profile analysis of odor -rancidity, aromaticity- and texture -tenderness, juiciness- of terrines ................................................................................... 139 Figure 4. 7 Box plots of odor and texture of terrines .................................................... 140 Figure 4.8 Score plot (a) and loading plot (b) of the PCA performed from the sensory analysis of flavor of terrines with nitrite (echNit), formulation 7(ech7), formulation 7+powder 3st (ech7fr), formulation 10 (ech10), formulation 12 (ech 12) and the blanc(blanc) ................................................................................................................... 142 Figure 4.9 Score plot (a) and loading plot (b) of the PCA performed from the sensory analysis of odor and texture of terrines with nitrite (echNit), formulation 7(ech7), formulation 7+powder 3st (ech7fr), formulation 10 (ech10), formulation 12 (ech 12) and the blanc(blanc) ............................................................................................................. 143 Figure 4. 10 Sensory profile analysis of flavor sweetness, salty, bitterness, acidity and spicy of tested hams ...................................................................................................... 146

Figure 4.11 The box plots of 5 tested hams following descriptors: sweet, salty, acid, bitter and spicy. ............................................................................................................. 147 Figure 4.12 Sensory profile analysis of odor -rancidity, aromaticity- and texture -tenderness, juiciness- of hams ....................................................................................... 149

Figure 4.13 The box plots of 5 tested hams with following descriptors: rancidity, aromaticity, tenderness and juiciness ............................................................................ 150 Figure 4.14 Score plot (a) and loading plot (b) of the PCA performed from the sensory analysis of flavor of hams with nitrites (hamNit), formulation 7(ham7), formulation 7+powder 3st (ham7fr), formulation 10 (ham10), formulation 12 (ham12) ................. 152 Figure 4.15 Score plot (a) and loading plot (b) of the PCA performed from the sensory analysis of odor and texture of hams with nitrites (hamNit), formulation 7(ham7),

xv

formulation 7+powder 3st (ham7fr), formulation 10 (ham10), formulation 12 (ham12) ...................................................................................................................................... 153 Figure 5.1 Scénarios 1 et 2 utilisés dans l’analyse technico-économique: Production de la terrine : pâté .............................................................................................................. 159 Figure 5.2 Scénarios 3 et 4 utilisés dans l’analyse technico-économique: Production du jambon .......................................................................................................................... 160 Figure 5.3 Estimation du coût de production des terrines de foie de porc et du jambon; Scénario1: terrines avec nitrites, Scénario2: terrines avec épices+poudre de fraise, Scénario3: jambon avec nitrites, Scénario1: jambon avec épices+poudre de fraise .... 170

xvii

Liste des abréviations

BHT-Butylated hydroxytoluene

BHA- Butylated hydroxyanisole

TBHQ- tertiary butyl hydroquinone

GRAS- Generally Recognized As Safe

w/w weight by weight

p/p poids par poids

UFC unité formant colonie

MDA malonaldehyd acid

TBA thio-barbituric acid

ppm partie par million

xix

Remerciements

Le projet d'étude arrive à son terme. A cette occasion, j'aimerais remercier et exprimer

ma gratitude à toutes les personnes qui m'ont aidées durant ces deux années d'étude.

Pour commencer, je voudrais remercier mon directeur de recherche, M. Khaled

Belkacemi, pour ses conseils, son soutien et pour m'avoir donné la chance de participer

à cette étude.

Je remercie ma co-directrice, Madame Satinder Kaur Brar, pour m'avoir accueillie dans

son équipe de recherche, pour ses conseils tant professionnels que personnels et sa

disponibilité tout au long du projet.

Ensuite, un gros merci à ma collègue de travail, mon amie, Fatma Gassara, pour sa

pédagogie, son dynamisme, ses observations, et ses conseils qui ont enrichit ces deux

années d'étude.

Je tiens à remercier également M. Mohammed Khelifi pour ses conseils et sa

convivialité pour le peu de fois où je l'ai rencontré.

J'adresse mes remerciements à toutes les personnes qui ont participé aux tests

organoleptiques, plus particulièrement à Madame Jocelyne Giasson, pour sa présence et

ses conseils.

Un merci tout particulier à Nasima Chorfa pour son aide non négligeable, à mon équipe

de recherche à l' INRS et aux stagiaires qui ont su apporter leur fraîcheur à ce projet.

Je tiens à remercier les Fonds de recherche du Québec – Nature et technologies

(FQRNT), le Ministère de l'Agriculture, des Pêcheries et de l'Alimenation (MAPAQ), le

Ministère du Développement économique, de l’Innovation et de l’Exportation

(MDEIE), Les Aliments Breton, La Maison du Gibier inc, Les Gibiers Canabec inc,

Yamaco (9138-9494 Québec inc.) et Les Spécialités Prodal (1975) ltée, pour le

financement de ce projet.

xx

Enfin, un merci et non des moindres, à ma famille, pour leur soutien indéfectible et à

mes amis, plus particulièrement Sandrine Tanoé et Ange Amon pour leur présence et

leur aide durant ce projet.

xxi

Avant propos

Ce mémoire comprend une revue de la littérature, un chapitre ``objectifs et

hypothèses`` et quatre chapitres principaux. La revue de la littérature est constituée de

deux revues qui ont été soumises, acceptées et en cours de publication. Je suis co-auteur

de la première revue et troisième auteur de la seconde revue.

La première revue : Fatma Gassara1, Anne Patricia Kouassi1 2, Satinder Kaur Brar2*,

Khaled Belkacemi1 (2013). Green Alternatives to Nitrates and Nitrites in Meat-Based

Products – A Review. (Acceptée). Elle va être publiée dans le journal Critical Reviews

in Food Science and Nutrition.

La deuxième revue : Jessica Elizabeth De La Torre Torres1, 2, Fatma Gassara1, Anne

Patricia Kouassi1, 3, Satinder Kaur Brar1*, Khaled Belkacemi3 . Properties and benefits

of spices in food. (Acceptée). Elle sera publiée également dans le journal Critical

Reviews in Food Science and Nutrition.

Les quatre chapitres principaux (2 à 5) comprennent 3 chapitres rédigés et présentés

sous forme de manuscrits dont deux ont été soumis et le troisième est en cours de

soumission. Je suis premier auteur du premier et du troisième article (chapitre 2 et 4); et

deuxième auteur du deuxième article (chapitre 3). J‘explique plus bas mes contributions

aux travaux pour lesquels je ne suis pas premier auteur.

Pour les chapitres dont je suis le premier auteur (2 et 4), j‘ai réalisé les expériences,

analysé les données et rédigé les manuscrits qui en découlent, avec l‘aide de ma

collègue associée de recherche, de mon directeur, et de ma co-directrice.

Chapitre 2: Anne Patricia Kouassi1, Fatma Gassara2, Satinder Kaur Brar2, Khaled

Belkacemi1(2014). Use of spices as alternative of nitrites and nitrates in the meat-based

products. Il a été soumis au Journal of Food Science and Technology.

Chapitre 3: Fatma Gassara, Anne Patricia Kouassi, Satinder Kaur Brar, Khaled

Belkacemi. (2014). Optimization of spices as alternatives of nitrites and nitrates in

meat-based products. J‘ai participé à la réalisation des expériences au laboratoire, j'ai

participé à la rédaction de plusieurs chapitres de l'article. Ce article est en cours de

soumission.

xxii

Le chapitre 4 prend la forme d‘un manuscrit pour une soumission prochaine : Anne

Patricia Kouassi1, Fatma Gassara2, Nasima Chorfa1, Satinder Kaur Brar2, Khaled

Belkacemi1. Color Retention in Processed Meats by Using Natural Products and Tests

of Organoleptic Properties.

1

Introduction

L'objectif principal de cette maîtrise était de développer des formulations capables de

remplacer les nitrites et nitrates dans les produits carnés tout en gardant la même durée

de conservation que ces derniers ainsi que leurs propriétés organoleptiques,

antibactériennes et antioxydantes. Un criblage qualitatif puis quantitatif de produits

naturels de faible coût comme les fruits (raisins) et les épices (la cannelle, les clous de

girofles, le cumin, le poivre noir, l’ail et le poivron rouge..) a été réalisé dans un premier

temps, afin de sélectionner les additifs assurant une meilleure activité antimicrobienne

et antioxydante dans les terrines de lapin et de porc, ainsi que dans celle du jambon. Les

résultats ont montré que les formulations 7 (Clou de girofle 0,3; Cumin 0,3; Cannelle

0,1),12 (Clou de girofle 0,2; Cumin 0,368; Cannelle 0,2) ont donné de bonnes

propriétés physico-chimiques et microbiologiques pour les terrines. De même, les

formulations 7 (Clou de girofle 0,3; Cumin 0,3; Cannelle 0,1), 10 (Clou de girofle

0,368; Cumin 0,2; Cannelle 0,2) ont été les meilleures formulations pour le jambon.

Cependant, aucune de ces formulation d'épices ne donnait de coloration rosée aux

jambons ni aux terrines, similaire à la coloration donnée par les nitrites. Ainsi, différents

agents de couleur, y compris les colorants alimentaires, les légumes (betterave) et les

fruits (fraise, raisin, melon..) ont été testés, donc ajoutés aux terrines et aux jambons

pour améliorer leur couleur. Les résultats du criblage des agents de couleur ont montré

que la poudre de fraise et la poudre de raisin sont des alternatives prometteuses aux

nitrites, et donnent une couleur rosée similaire à celle donnée par les nitrites. En outre,

l’effet des conditions avant emballage à savoir le pH, l’humidité sur les propriétés

microbiologiques, physicochimiques et la couleur des produits carnés ont été évalués.

Les résultats de ces analyses montrent que le pH alcalin donne une couleur rouge plus

prononcée pour la viande, mais une texture de la viande différente; nous avons gardé le

pH initial de la viande (qui était de 6). Par contre, la variation de l’humidité n'a pas

d'effet sur la couleur des produits carnés. Ayant obtenu des résultats satisfaisant tant

d'un point de vue physico-chimique que antimicrobien, des tests organoleptiques ont été

effectués sur un panel de dégustateur, avec les meilleures formulations d’épices

sélectionnées, étant donné que les facteurs sensoriels sont les principaux déterminants

des décisions ultérieures d'achat du consommateur. Les résultats des analyses

sensorielles ont permis de faire ressortir le caractère sucré de la poudre de fraise et les

2

propriétés très intéressantes de la formulation 7 par rapport à l'ajout des nitrites. Enfin,

une analyse technico-économique a été réalisée en vue d'évaluer l’intérêt de l'utilisation

des épices et de la poudre de fraise comme alternatives aux nitrites dans les produits

carnés, d'un point de vue industriel. Cette analyse, réalisée à partir d’une série de 4

scénarios, a montré une faible augmentation du coût de production des produits carnés

en utilisant les épices et la poudre de fraise, due à une légère augmentation du coût des

matières premières.

3

Chapitre 1: Revue de la littérature

4

1.1. Green Alternatives to Nitrates and Nitrites in Meat-Based Products – A Review

Fatma Gassara1, Anne Patricia Kouassi1 2, Satinder Kaur Brar2*, Khaled

Belkacemi1

1Département des sols et de génie agroalimentaire, Pavillon Paul-Comtois, Université

Laval, 2425, rue de l'Agriculture, Québec (Québec) G1V 0A6

2INRS-ETE, Université du Québec, 490, Rue de la Couronne, Québec, Canada G1K

9A9

5

1.1.1. Résumé

Il existe de nombreux additifs alimentaires qui, ajoutés dans les aliments, permettent

leur conservation ou encore ralentissent ou arrêtent la croissance des micro-organismes.

Les nitrites et les nitrates sont utilisés comme agents de conservation dans les viandes.

Les nitrites donnent un goût fumé à la viande, une couleur rosâtre et protégent les

consommateurs du risque de détérioration bactérienne. Leur ajout est toutefois très

limité car, à haute dose, ils peuvent être dangerux pour la santé humaine et

l'environnement. Ainsi, des alternatives aux nitrates et aux nitrites font l'objet de

nombreux travaux de recherche. En effet, les épices présentent de nombreuses

propriétés organoleptiques, anti oxydantes et anti microbiennes qui serait intéressant

d'étudier. Cette revue présente les différentes sources de nitrites et de nitrates, leur

utilisation comme additifs dans les produits alimentaires, leurs propriétés physico-

chimiques, leurs effets négatifs et l'utilisation d'alternatives dans la conservation des

produits à base de viande.

Mots-clés: nitrites, nitrates, viande, alternatives plus saines, conservation de la viande

6

1.1.2. Abstract

Several food additives are added in food for their preservation to maintain the

freshness of food (antioxidants) or to slow down or stop the growth of

microorganisms (preservative agents). Nitrites and nitrates are used as preservative

agents in meat. Nitrites give a smoked taste, a pinkish color in the meat and protect

the consumers against the risk of bacterial deterioration. Their addition is however

very limited as, in high dose, it can have risks on human health and the environment.

Thus, alternatives of nitrates and nitrites are the object of numerous research studies.

Alternatives, like spices are considered to have several organoleptic and anti-

microbial properties which would be interesting to study. This review discusses the

sources of nitrites and nitrates, their use as additives in food products, their

physicochemical properties, their negatives effects and the use of alternatives of

nitrites and nitrates in preserving meat products.

Keywords: nitrites, nitrates, meat, green alternatives, preservation

7

1.1.3. Introduction

Nitrates and nitrites are present everywhere in the environment, in water, soil, air,

plants and food. They are used as fertilizers, as explosives and also as preservative

agents in foods particularly against Clostridium botulinum. They are natural

chemical substances which are obtained from the oxidation of nitrogen by the

microorganisms. Oral reduction of nitrate is the most important source of nitrite,

accounting for approximately 70–80% of the human total nitrite exposure. About 5–

7% of all ingested nitrate is converted to nitrite at the base of the tongue, where

nitrate-reducing bacteria are present (Chan, 2011). For subjects with a high rate of

conversion, this figure may be up to 20% (Eisenbrand et al., 1980). Excessive use of

nitrates and nitrites not only presents a health hazard but may also result in nitrite

burn which is a green or white discoloration in the cured meat (Montana Meat

Processors Convention, 2001). The toxic effects of nitrates are due to its endogenous

conversion to nitrite. The Acceptable Daily Intake (ADI) for nitrate is 0-3.7 mg/kg

bw/day (expressed as nitrate ion) (Thomson, 2004). It can lead to risks to human

health and the environment. The health effect of most concern to the U.S. EPA for

children is the “blue baby syndrome” (methemoglobinemia) (Fan, Willhite & Book,

1987). The blue baby syndrome is named for the blue coloration of the skin of

babies who have high nitrate concentrations in their blood. The nitrate binds to

hemoglobin (the compound which carries oxygen in blood to tissues in the body),

and results in chemically-altered hemoglobin (methemoglobin) that impairs oxygen

delivery to tissues, resulting in the blue color of the skin (USEPA, 2007).

Nitrates and nitrites have been associated, at high level, with increased incidence of

cancer in adults, combining with secondary or tertiary amines to form N-nitroso

derivatives, and possible increased incidence of brain tumors, leukemia and

nasopharyngeal problems. The U.S. EPA concluded that there was conflicting

evidence in the literature as to whether exposure to nitrate or nitrites are associated

with cancer in adults and in children. The types of cancers studied included non-

Hodgkin’s lymphoma as well as stomach and gastric cancers in adults; and brain

tumors, leukemia, and nasopharyngeal cancers in children. Their addition is

however very limited (USEPA, 2006). Industries use nitrates and nitrites for the

8

stabilization of the red color of meats (Honikel, 2008), inhibition of the development

of toxic microorganisms, by decreasing the oxidation of lipids and to improve the

flavor (Pegg and Shahidi, 2000). Nitrates and nitrites are preferred as they are less

expensive for the properties that they offer. Meanwhile, the industries have not

found a better and economical substitute to these nitrites and nitrates. Due to the

pressure of the countries who want to establish stricter regulations, the industries are

obliged to find greener substitutes to nitrates and nitrites.

In this context, alternatives of nitrates and nitrites have been studied (Stoilova et al.,

2007; Shirin and Prakash, 2010; Stoilova et al., 2006; Grohs and Kunz, 2000). In

literature, there are chemical agents, such ascorbate and α–tocopherol, lactic-acid-

producing organisms, potassium sorbate, or treatments, such as irradiation (National

Academy of Sciences, 1982) that have been used as nitrite-nitrate substitutes. A

number of studies have been carried out to investigate the properties of aromatic

herbs, fruits, essential oils and spices and like clove, ginger, pepper or garlic

(Menon and Garg, 2001). Cinnamaldehyde, the major constituent of cinnamon

(Cinnamomum cassia) has been reported to possess antibacterial activity and

antioxidant properties (Chang et al., 2001). An essential oil is a complex mixture of

several volatile aroma compounds belonging to different classes of organic

chemistry: phenols (eg carvacrol), hydrocarbon (terpene compounds such as

limonene), alcohols (eg linalool), aldehydes (eg cinnamaldehyde), ketones (eg

menthone). Most of these compounds have antimicrobial properties, but they are

volatile compounds which contain the most important antimicrobial properties,

particularly phenols, alcohols and aldehydes: carvacrol (oregano, savory), eugenol

(sheet Ceylon cinnamon, clove), linalool (coriander), cinnamaldehyde (Chinese

cinnamon) (Oussalah, 2007). There are more than 1340 plants with defined

antimicrobial compounds, and over 30,000 components have been isolated from

phenol group-containing plant-oil compounds and used in the food industry

(Tajkarimi etal. ,2010). Researchers have found a positive linear correlation between

phenolic compounds, primarily phenolic acids and flavonoids, and the antioxidant

capacity of herbs and spices (Zheng, 2001).

The purpose of this review is to present nitrates and nitrites, their role in the

environment, later presence in natural products, their role in preservation of meat

9

and the reason why it is important to find alternatives. Some of them, such as spices

and natural products are also presented in this study, due to preservation, properties

and safety.

1.1.4. Definition of nitrates and nitrites

1.1.4.1. Nitrites and nitrates chemical’s composition Nitrates and nitrites occur naturally as compounds consisting of nitrogen and

oxygen, although in different chemical structures. The nitrogen cycle contains both

compounds. The chemical difference between nitrate and nitrite lies in one

additional oxygen atom. Nitrite is one part nitrogen and two parts oxygen. The

nitrogen cycle comprises oxidation of nitrite, NO2 into NO3, or nitrate.

1.1.4.2. Natural sources of nitrates and nitrites 1.1.4.2.1. Soil

Nitrate is a natural material in soils. Adequate supply of nitrate is necessary for good

plant growth. More than 90 percent of the nitrogen is probably absorbed by plants in the

nitrate form (Brown et al., 2007). Chemical nitrogen fertilizer is often in the

ammonium nitrogen (NH4+) form and is rapidly converted to nitrate (NO3

-) in the soil.

The amount of crop growth is essentially the same whether nitrogen fertilizer is applied

as ammonia (NH3), ammonium or nitrate (NO3-) (Brown et al., 2007). Chemical

fertilizers may be composed of ammonium nitrate, ammonium phosphates, ammonium

sulfate, various nitrate salts, urea and other organic forms of nitrogen. Soil organic

matter contains about 5 % of N. For each 1 percent of organic matter, 7-inch plow layer

of an acre (about 2,000,000 pounds of soil) contains about 1,000 pounds of N.

Microorganisms must change organic nitrogen to ammonium or nitrate before plants

can use it. Usual release of available N from soil organic matter is 1 to 4 percent

annually, depending on soil texture and weather conditions (Brown et al., 2007).

Nitrogen, present or added to the soil, is subject to several changes (transformations)

that dictate the availability of N to plants and influence the potential movement of NO3-

to water supplies (O'Leary, 1994). Animal manure is an excellent source of nitrogen

and can contribute significantly to soil improvement. Animal manure contains about 10

pounds of N per ton, poultry manure about 20 pounds; and legume residues 20 to 80

pounds. About half of this organic nitrogen may be converted to nitrate-nitrogen and

10

become available for plant use the year it is added to the soil. However, it is low in

phosphorus content. Excessive manure applications can result in toxic levels of nitrate

in forage crops the same as excessive use of chemical nitrogen fertilizer (Agbede and

Ojeniyi, 2009). Adding phosphate fertilizer to manure can reduce the nitrate content in

the crop produced. Effluent from animal waste treatment facilities may lose about 50

percent of its nitrogen to the atmosphere as it is applied to soils. However, applications

of large quantities of effluent or solid waste can add excessive amounts of nitrogen to

the soil. Applying large amounts per acre repeatedly to the same area may add more

nitrogen to the soil system than can be used. Using feed additives in livestock feeding

may contribute significant concentrations of certain elements such as copper, zinc,

arsenic or others to the solid animal waste collected in lagoons or similar facilities. Such

wastes continuously applied to soils may eventually result in soil levels toxic to plants

and possibly to animals that consume the crop.

1.1.4.2.2. The atmosphere

Nitrogen in our atmosphere is an inert molecule at ambient temperature. The nitrogen in

the air is also essential for life on Earth. It is incorporated into amino acids and proteins,

and is part of the nucleic acids, such as DNA and RNA (Encyclopedia of Earth, 2011).

However, the nitrogen atom itself is one of the chemical elements which can change its

state of oxidation widely. The outer shell of five electrons (s2p3) can take up three

additional electrons giving the nitrogen an ‘‘oxidation status’’ N3- as it exists in

ammonia (NH3) or amines or it can release five electrons forming N5+ as it exists in

nitrate NO3-. Atmospheric nitrate concentrations ranging from 0.1 to 0.4 μg/m3 have

been reported, the lowest concentrations being found in the South Pacific (Prospero and

Savoie, 1989; World Health Organization 2004). The highest levels of aerosol nitrate

measured at this northern Canadian location were about 0.40-0.55 µg/m3 between the

years 2000 and 2005 (Environment Canada, 2010). Higher concentrations ranging from

1 to 40 μg/m3 have also been reported, with annual means of 1–8 μg/m3. Mean monthly

nitrate concentrations in air in the Netherlands range from 1 to 14 μg/m3 (Janssen et al.,

1989; World Health Organization 2004). Indoor nitrate aerosol concentrations of 1.1–

5.6 μg/m3 were found to be related to outdoor concentrations (Yocom, 1982; World

Health Organization 2004).

11

1.1.4.2.3. Plant

Nitrates occur naturally in vegetables and plants. Nitrite and nitrates occur naturally in

vegetable as a consequence of the nitrogen cycle whereby nitrogen is fixed by bacteria.

Beetroot, broccoli, cabbage, celery, lettuce, radish and spinach have been reported to

contain high concentrations (greater than 1000 mg/kg) of nitrate. In contrast, nitrite

concentration in fresh vegetables is generally low (less than 1 mg/kg and not above 20

mg/kg) (Meah, 1994, Petersen and Stoltze 1999, Chung et al, 2003).

1.1.4.2.4. Water

Nitrates occur naturally in wastewater and drinking water. Nitrate is used mainly in

inorganic fertilizers. It is also used as an oxidizing agent and in the production of

explosives, and purified potassium nitrate is used for glass making. Sodium nitrite is

used as a food preservative, especially in cured meats. Nitrate is sometimes also added

to food to serve as a reservoir for nitrite. Nitrate can reach both surface water and

groundwater as a consequence of agricultural activity (including excess application of

inorganic nitrogenous fertilizers and manures), from wastewater treatment and from

oxidation of nitrogenous waste products in human and animal excreta, including septic

tanks (World Health Organization 2011). Nitrite can also be formed chemically in

distribution pipes by Nitrosomonas bacteria during stagnation of nitrate-containing and

oxygen-poor drinking-water in galvanized steel pipes or if chloramination is used to

provide a residual disinfectant and the process is not sufficiently well controlled.

1.1.4.2.5. Endogenous biosynthesis of nitrate

Nitrosating agents (NAs; NxOy form) can react under certain conditions with

nitrosatable compounds (NCs) to form N-nitrosamines and N-nitrosamides

(hereafter nitrosamines and nitrosamides), collectively called N-nitroso compounds

(NOCs): an equation showing the formation of N-Nitroso compounds (NAs + NCs

NOCs). This reaction is called N-nitrosation or simply nitrosation (Health

Canada, 2013). Human beings are exposed to various types of nitrosating agents

through diet, drinking water and tobacco smoke. These substances can also be

12

synthesised endogenously from ingested nitrate and nitrite (Bartsch et al., 1988;

Brambilla and Martelli, 2005).

Gastrointestinal infections greatly increase nitrate excretion via endogenous (non-

bacterial) nitrate synthesis, probably induced by activation of the mammalian

reticulo endothelial system (FAO/WHO, 1996; Lundberg et al., 2009). This

endogenous synthesis of nitrate, presented in figure 1.1, complicates the risk

assessment of nitrate. Increased endogenous synthesis of nitrate, as reported in

animals with induced infections and inflammatory reactions, was also observed in

humans. Infections and non-specific diarrhoea played a role in the increased

endogenous synthesis of nitrate (Gangolli et al., 1994; World Health Organization

2011). These observations are all consistent with the induction of one or more nitric

oxide synthases by inflammatory agents, analogous to the experiments described in

animals and macrophages. In humans, saliva is the major site for the formation of

nitrite. About 5% of dietary nitrate is converted to nitrite (Gangolli et al., 1994;

World Health Organization 2011). A direct correlation between gastric pH, bacterial

colonization, and gastric nitrite concentration has been observed in healthy people

with a range of pH values from 1 to 7 (Mueller et al., 1986, Viani et al., 2000;

Moigradean et al., 2008).

13

Figure 1.1 Endogenous biosynthesis of nitrate

1.1.5. Use of nitrates and nitrites as additives in food products

Nitrate and nitrite are used as food additives in processed food as preservatives and

colour fixatives in meat, poultry, fish and cheese (European Commission, 1995).

Sodium nitrite has been used extensively in curing meat and meat products, particularly

pork products, such as ham, bacon and frankfurters; certain fish and poultry products

are also cured with brines that contain sodium nitrite. The process may include dry

curing, immersion curing, or direct addition or injection of the curing ingredients.

Curing mixtures are typically composed of salt (sodium chloride), sodium or potassium

salts of nitrite and nitrate and seasonings. Sodium nitrite acts as a colour fixative and

inhibits the growth of bacteria, including Clostridium botulinum, which is the source of

botulism toxin. Nitrite is a relatively strong reducing agent that has antibacterial

properties; however, the preservation of foodstuffs can be attributed to a large degree to

the high concentration of salts (including nitrate) that are employed during the curing

endothelial cells

L- arginine +O2

NO-synthetase

NO

NO2

-

Oxidation

Urine (NO3

-;NO2-)

Oxidation

NO3-

Gastric gland

Salivary glands

NO3-

Oxidation

(Mouth) Bacterial production of NO2

-

(stomach) Bacterial production of NO2-

NO3-,NO2

-

NO3-,NO2-

Stool <0.01% (NO3

-.NO2-)

NO3-

exogenous (food)

14

process. In addition, nitrate can act as a reservoir whereby nitrite may be formed by

microbiological reduction (Pokorny et al., 2006).

1.1.6. Physicochemical properties of Nitrites and Nitrates

1.1.6.1. Role of nitrates and nitrites as antioxydant

One of the most important properties of nitrite is its ability to effectively delay the

development of oxidative rancidity. This prevention occurs even in the presence of salt,

which is a strong oxidant. Lipid oxidation is considered to be a major reason for the

deterioration of quality in meat and poultry products which often results in the

development of rancidity and subsequent warmed over flavors (Vasavada and

Cornforth, 2005). The oxidation of unsaturated fats occurs more quickly in uncured,

cooked meats than in cured meats because iron that is not bound to nitric oxide can act

as a catalyst for oxidation. The antioxidant effect of nitrite is likely due to the same

mechanisms responsible for cured color development involving reactions with heme

proteins and metal ions, chelating of free radicals by nitric oxide, and the formation of

nitriso- and nitrosyl compounds having antioxidant properties (Sebranek, 2009). The

antioxidant effect of nitrite has been well documented (Townsend and Olson, 1987;

Pearson and Gillett, 1996; Pegg and Shahidi, 2000; Honikel, 2004). Nitrite has been

shown to inhibit warmed over flavor development at relatively low levels. The addition

of nitrite to kavurma, a type of fried meat, could significantly reduce the level of

oxidation, measured by thiobarbituric acid, peroxide, and free fatty acids, as compared

to a control which did not have nitrite added (Yetim et al. (2006). Sato and Hegarty

(1971) reported significant inhibition of warmed over flavor development at a 50 ppm

nitrite level with complete inhibition at a 220 ppm level. Investigating the effect of

nitrite on lipid oxidation in various muscle systems, Morrissey and Tichivangana (1985)

reported as little as 20 ppm nitrite was sufficient to significantly (P < 0.01) inhibit

oxidation of lipid in fish, chicken, pork, and beef systems. In spite of the antioxidant

power of nitrites and nitrates, industries have to find healthier and greener alternatives

for these chemicals products

15

1.1.6.2. Antibacterial activity of nitrites and nitrates

Nitrites and nitrates play a key role in cured meat as a bacteriostatic and bacteriocidal

agent. Nitrite is strongly inhibitory to anaerobic bacteria, most importantly Clostridium

botulinum ( Sofos et al.,1979) and contributes to control of other microorganisms, such

as Listeria monocytogenes. The effect of nitrite and the likely inhibitory mechanism

differs in different bacterial species (Tompkin, 2005). The effectiveness of nitrite as an

antibotulinal agent is dependent on several environmental factors including pH, sodium

chloride concentration, reductants and iron content among others (Tompkin, 2005). The

reaction sequences involving nitric oxide are probably an important part of the

antimicrobial role of nitrite in cured meat. For example, some researchers have

suggested that nitrous acid (HNO2) and/or nitric oxide (NO) may be responsible for the

inhibitory effects of nitrite (Tompkin, 2005). As nitrite reactivity is key to microbial

inhibition (one indicator of this is the strong dependence on pH), there are questions

raised on the fact that whether ingoing or residual nitrite is most critical to antimicrobial

effects.

Tompkin (2005) concluded that residual nitrite at the time of product temperature abuse

is critical to antibotulinal effects and that depletion of residual nitrite during product

storage will reach some point at which inhibitory effects are also depleted. Other

compounds have the same antimicrobial properties as nitrites and nitrates; if the

industries use these compounds, it is also for their action on color and taste of meat.

1.1.6.3. Action of nitrites and nitrites on color and taste

The red colour of cured meat products is one of the important effects of nitrite in meat

products. The red colour develops in a number of complicated reaction steps until NO-

myglobin (Fe2+) is formed. Myoglobin exists in a muscle in three states, in which the

cofactor haem, a porphyrin ring with an iron ion in its centre binds different ligands or

in which the iron exists in the Fe2+ or Fe3+ state. In the native myoglobin, the porphyrin

moiety is supported in the ligand binding by amino acids of the protein in the

neighbourhood. In the ‘‘original’’ state, myoglobin with Fe2+ in the porphyrin cofactor

does not bind any ligand by a water molecule. In the presence of oxygen, the myoglobin

16

can bind an O2 molecule and it becomes bright red. The iron ion is in the Fe2+ state.

However, oxygen and other oxidizing agents, such asnitrite can oxidize the Fe2+ to Fe3+.

The formed metmyoglobin (MetMb) is brown. The ‘‘original’’ myoglobin (Mb),

oximyoglobin (MbO2) and the metmyoglobin occurrtogether in meat. In a muscle in a

live animal there is very little metmyoglobin which increases post-mortem with the

disappearance of oxygen except when meat is MAP-packed with high oxygen content

(Honikel, 2008).

Oxygen and NO are biatomic molecules. A similar biatomic molecule, CO also binds

very tightly to myoglobin. In some countries (e.g. USA and Norway) modified

atmospherepackaging (MAP) packaging of meat with 1–2% CO is permitted. By

reducing enzymes or chemical reactions with reducing agent, such asascorbate, Fe3+ is

reduced to Fe2+. The NO formed from N2O3 can bind to the myoglobin (Fe2+) and form

a heat stable nitrosylmyoglobin (NO-myoglobin). Oximyoglobin is not heat stable and

dissociates. The meat turns grey or brown. On heating the NO-myoglobin, the protein

moiety is denatured but the red NO-porphyrin ring system (often called nitroso-

myochromogen) still exists and is found in meat products heated to 120±1oC. This heat

stable red colour will change on bacterial spoilage and it fades in UV light. The

transformation of myoglobin have presented in details in Figure 1.2. The first one is

advantageous as the consumer recognizes spoilage in fresh meat which also changes

colour on spoilage. In most recent years, the riddle about the red colour of cured raw

hams such as Parma ham without added nitrite or nitrate has been solved. Various

authors proved that the Fe2+ in the porphyrin ring was exchanged with Zn2+ which gives

the products a pleasant red colour. Nitrite addition prevents the exchange (Adamsen,

Moller, Laursen, Olsen, & Skibsted, 2006; Moller, Adamsen, & Skibsted, 2003;

Parolari, Gabba, & Saccani, 2003; Wakamatsu, Nishimura, & Hattori, 2004;

Wakamatsu, Okui, Ikeda, Nishimura, & Hattori, 2004).

Nitrite is also responsible for the production of characteristic cured meat flavor, though

this is probably the least well understood aspect of nitrite chemistry (Pegg & Shahidi,

2000). It is easy to distinguish cooked, cured ham from fresh roast pork on the basis of

flavor but the chemical identity of distinguishing flavor components in cured meat has

eluded numerous researchers. Some of the flavor difference may be due to the

suppression of lipid oxidation by nitrite but other antioxidants do not produce cured

17

meat flavor. If nitrite does, in fact, form some volatile flavor factors, this would

represent yet another reaction product of nitrite in cured meat.

Figure 1.2 Transformation of myoglobin in meat

1.1.7. Regulation of nitrite and nitrate

Meat products manufactured in the United States are heavily regulated by the United

States Department of Agriculture (USDA) – Food Safety and Inspection Service (FSIS).

The amount of ingoing sodium or potassium nitrite in comminuted products

manufactured in the United States is 156 ppm. The amount of ingoing sodium or

potassium nitrite in comminuted products manufactured in the United States is 156 ppm

(Sebranek and Bacus, 2007). This is based on the green weight of the meat block (which

is different in other countries). According to regulations, dry cured products are

restricted to 625 ppm of ingoing sodium nitrite or potassium nitrite. Immersion cured

and massaged or pumped products are limited to 200 ppm ingoing sodium nitrite or

potassium nitrite. According to the USDA, a minimum of 120 ppm of ingoing sodium

nitrite is required for all cured “Keep Refrigerated” products. The only instance in

which the rule is not in effect is when “the establishment can demonstrate that safety is

assured by some other preservation process, such as thermal processing, pH or moisture

18

control” (Sebranek and Bacus, 2007). Neither nitrate nor nitrite is permitted in baby or

toddler food. Nevertheless, the food is safe for consumption due to the sterilization

processing to which all baby food is subjected. Health Canada has identified

maximum nitrites/nitrate levels in the Food and Drug Regulation. These levels are

maximum of 200 ppm in cured meat and meat by-products (except bacon) and maximun

of 100 ppm in bacon. These levels are well above those needed to stop the

growth Clostridum botulinum spores. A complete ban on the use of nitrates and nitrites

in foods has not been implemented because of the beneficial uses as preservatives and

particularly their prevention of Clostridium botulinum growth. There is also some

scientific evidence suggesting that low levels of nitrates and nitrites (below 200ppm)

pose no health concern (Health Canada, 2008).

Many countries have used others directives and regulations for the use of nitrites and

nitrates in meat products. The European Union (EU) have considered its regulation and

directives 2006/ 52/ DEC (Directive 2006). The use of nitrites and nitrates was limited.

In general, 150 mg nitrite/kg are allowed to be added to all meat products plus 150

mg/kg for unheated meat products. That is a maximum of 300 nitrites plus nitrates/kg. A

large number of exceptions such as dry cured bacon may have 425 mg of residual

nitrites plus nitrates / kg. The toxicity of nitrates and nitrites depend on their

concentration in meat. Due to regulations and the toxicity of nitrates and nitrites,

industries must be more stringent on their standard or find healthier alternatives for

nitrates and nitrates.

1.1.8. Toxico-kinetics of nitrates and nitrites

1.1.8.1. Absorption Nitrates and nitrites are present in the environment under ionic form, not volatile. There

are two major sources of nitrate and nitrite in the human system: endogenous l-

arginine–NO synthase pathway and the diet. The main pathway of absorption of these

substances is the ingestion. Dietary nitrate intake is considerable and nitrate is found as

a naturally occurring compound in foods such as vegetables, spinach and lettuce often

containing up to 2500 mg.kg−1 (World Health Organization 2011), fruit, cereals, fish,

milk, and walter also contain nitrate as a consequence of agricultural practices, such as

the use of nitrogen-containing fertilizers and from animal waste (Dennis and Wilson,

2003). Low levels are generally found from these sources, except in the case of some

19

vegetables. Nitrates and nitrites are also permitted as food additives in some foods,

primarily as protection against botulism.

1.1.8.2. Distribution

Concerning their distribution, nitrate is reduced to nitrite, in the oral cavity, by the

action of commensal bacteria found on the back of the tongue. The swallowed nitrite,

under the influence of the acidic conditions in the stomach, becomes protonated to

nitrous acid (NO2- + H+ HNO2). Once nitrite is formed, there are numerous

pathways in the body for its further reduction to NO, involving haemoglobin,

myoglobin, xanthine oxidoreductase ascorbate, polyphenols and protons (Lundberg et

al., 2008). After the ingestion of nitrate, either in its dietary or medicinal form, there is a

sharp rise in the salivary, plasma and urinary levels of nitrates and nitrites. In the

plasma, the levels of nitrate rise within 30 min and peak at 3 h and are sustained for up

to 24 h; In contrast, the levels of nitrites rise more gradually to a significant level by 1–

1.5 h and a plateau is reached at ∼2.5 h and remains significantly elevated for up to 6 h.

(Sami et al., 2012).

About 5% of the dietary nitrate is reduced to nitrites in the saliva and the

gastrointestinal tract. This value can reach 20% for individuals with a high rate of

conversion (Thomson et al., 2007).

Methemoglobinemia is another health hazard attributed to nitrites, a condition where

reduced iron (Fe2+) in haemoglobin is oxidized by nitrite to Fe3+, thus reducing the total

oxygen-carrying capacity of the blood (Santamaria, 2006).

Nitrites are able to be produced endogenously. Nitrites may also combine with

secondary or tertiary amines to form N-nitroso derivatives. Certain N-nitroso

compounds have been shown to produce cancers in a wide range of laboratory animals

(Codex, 1998).

1.1.8.3. Elimination

The elimination of nitrites is mainly carried out by urinary expression and maximal

urinary nitrite excretion occurred approximately 4 to 6 h after consumption of each

high-nitrate meal, with basal levels being reached by 24 h. Approximately 75% of the

total ingested nitrate was excreted via urine. The other major routes of nitrate excretion

21

The methaemoglobinaemia caused by nitrates in the drinking water was mainly

observed when the child's age was less than 3 months. Bacterial contamination of water,

gastrointestinal infections and inflammation with ensuing production of nitric oxide is

major factors that may contribute to methemoglobinemia (Fan, 2011). The blue baby

syndrome is named for the blue coloration of the skin of babies who have high nitrate

concentrations in their blood. The nitrate binds to hemoglobin (the compound which

carries oxygen in blood to tissues in the body), and results in chemically-altered

hemoglobin (methemoglobin) that impairs oxygen delivery to tissues, resulting in the

blue color of the skin. The blue coloration can be seen in the lips, nose, and ears in the

early stages of blue baby syndrome, and extend to peripheral tissues in more severe

cases (EPA, 2006).

Reduced oxygenation of the tissues can have numerous adverse implications for the

child, such as coma and death. Toxic doses of nitrites responsible for

methemoglobinemia range from 0.4 to more than 200 mg kg−1 of body weight. The

guideline value for nitrite ion in infants is 3 ppm (US EPA, 2006). Exposure to higher

levels of nitrates or nitrites has been associated with increased incidence of cancer in

adults, and possible increased incidence of brain tumors, leukemia, and nasopharyngeal

(nose and throat) tumors in children in some but not others. U.S. EPA concluded that

there was conflicting evidence in the literature as to whether exposures to nitrate or

nitrites are associated with cancer in adults and in children.

1.1.9.2. Effects on animal’s health

The effects of nitrates on animal’s health, livestock feeding contains nitrites and

nitrates. The nitrate is transformed into nitrite and in other N-nitroso compounds in the

saliva of most monogastrics or in ruminants’ rumen because of the microbial activity.

The unfavorable effects on the health of the livestock result from an acute exposure to

the nitrites due to the formation of methemoglobin in the blood. This can lead to the

cyanosis and death at very high levels.

Clinical signs of acute nitrite toxicity in a range of livestock associated with

methemoglobin are generally dose-dependent due to oxygen starvation and may include

accelerated pulse, dyspnoea, muscle tremors, weakness, vomiting, unstable gait, and

cyanosis leading to death. Symptoms of sub-chronic and chronic toxicity include

23

destruction of the ozone layer in the stratosphere. The nitrogen cycle is presented in

details in Figure 1.5.

Nitrates and nitrites are naturally occurring ions and are ubiquitous in the environment.

Nitrate contamination of surface- and groundwater is a pervasive, worldwide problem,

while nitrite is an important indicator of fecal pollution in water. According to the

National Primary Drinking Water Regulations, nitrates are highly mobile in soil and

have a higher potential to migrate to ground water due to higher solubility in water and

weak retention by soil. Nitrates and nitrites do not volatilize and therefore are likely to

remain in water until consumed by plants or other organisms. Ammonium nitrate is

taken up by bacteria, and nitrate degradation is fast under anaerobic conditions. Nitrite

is easily oxidized to nitrate, and nitrate is the more predominant compound of the two

forms detected in groundwater (EPA, 2006). Nitrates are essential for the growth of

vegetables, and are present in the composition of fertilizers natural as manure.

Fertilizers and badly used pesticides pollute subterranean waters (by infiltrating into the

ground by rainwater and watering) and surface (streaming). The excessive use of

fertilizers appreciably increases the quantity of nitrate in rivers and low depth ground

waters.

The nitrate is nevertheless a beneficial natural element integrated into the nitrogen cycle

and indispensable for the growth of vegetables. An excessive use of nitrates destabilizes

this process: the rainwater, after infiltration, entails the nitrate fertilizers, which plants

and grounds were not able to absorb, and then pollutes the fresh water.

Both nutrients, particularly in dissolved species such as nitrate, nitrite, ammonium and

phosphate, are easily assimilated by phytoplankton for growth and act as significant

factors in the regulation of primary productivity in water bodies. Nitrogen

concentrations in river and ground water increase as a response to increased agricultural

activity and point-source discharges and were one of the most implicated species in

eutrophication of water (Xinhai, 2010).

25

1.1.10.2. Vitamin

Several studies have shown that vitamins, such as -tocopherol could be an effective

alternative to nitrites and nitrates in meat products. In addition, it has been shown that

the -tocopherol can inhibit the growth of pathogens. However, vitamins are often

expensive which will make the meat process more expensive (Bhat et al., 2013).

1.1.10.3. Natural sources of nitrites and nitrates

It has been shown in the literature that there are certain products that naturally contain

nitrites such as celery, lettuce, spinach, radishes, among others. Some researchers have

used extracts of these natural products as substitutes of nitrite in meat products.

However, nitrites/nitrates, coming from natural sources or not, present a potential

harmful effect as chemical nitrites and nitrates, because even its origin is naturals, these

nitrites will be transformed to nitrosamines that have negatives effects.

1.1.10.4. Agents preventing the formation of nitrosamines

There are products that inhibit the formation of nitrosamines and nitrosamides from

nitrite and nitrate, such as ascorbate and ascorbic acid. These products can be used in

the presence of nitrites in meat products to inhibit their transformation into nitrosamines

and nitrosamides. The sequence of reactions of ascorbate preventing nitrosamine

formation has not been fully elucidated. It may be due to the reduction of residual nitrite

in meat products by ascorbate (EFSA, 2003) or the binding of NO to ascorbate and its

retarded release. In the last decades, ascorbic acid and isoascorbate (erythorbate) has

been used in cured meat batters. There is a reaction of ascorbate with oxygen forming

dehydroascorbate and thus reducing the amount of nitrite which could be oxidized to

nitrate (Bertelsen, and Quist, 1994). However, ascorbate also seems to react with nitrite

(nitrous acid or NO). Dahl, Loewe, and Bunton (1960), Fox and Ackerman (1968) and

Izumi et al. (1989) showed that ascorbate also reacts with ‘‘nitrite’’ and binding the

resulting NO. The bound NO seemed to react as NO with other meat ingredients.

Ascorbate is also added to reduce the formation of nitrosamines. The sequence of

reactions of ascorbate preventing nitrosamine formation has not been fully elucidated. It

may be due to the reduction of residual nitrite in meat products by ascorbate (EFSA,

2003) or the binding of NO to ascorbate and its retarded release together with nitrite and

26

salt. Nevertheless, it becomes clear that nitrite is a very reactive substance which

undergoes many reactions in meat products and thus its use has to be controlled. In

contrast, nitrites in meat products remain and pass through the stomach of the consumer

where they can transform under the action of acidic pH of the stomach to nitrosated

products. Similarly, nitrites themselves, without any transformation, are harmful to

consumers.

1.1.10.5. Spices and fruits

Spices are considered as good alternatives of nitrites due to their well known

antibacterial and antioxidant properties. Rosemary, mace, oregano and sage have

antioxidant properties that can delay the onset of rancidity in fats (Kim et al., 2011).

Black pepper, white pepper, garlic, mustard, nutmeg, allspice, ginger, cinnamon and red

pepper are known to stimulate the Lactobacillus bacteria producing lactic acid, which

increases the shelf life of meat products (Sallam et al. 2004). In this regard, the use of

spices and their volatile compounds as natural preservatives in food can be an

alternative to the use of chemical additives (Kim et al., 2011). The use of garlic powder

may be very important as garlic is known for its antioxidant and antimicrobial (Kim et

al., 2011). In addition, the use of garlic reduces the amount needed to ensure proper

conservation that will not alter the taste of the meat products. It has also been shown

that cloves and garlic in combination will inhibit all pathogenic bacteria

(Yadav and Singh 2004). The conjunction and the physical-chemical properties and

antimicrobial properties of garlic and cloves play an important in the use of these spices

as naturals alternatives of nitrites and nitrate in food. However, these spices if they will

be used in big quantity will damage the taste of food. Hence, the quantity of these spices

has to be minimized to maintain the organoleptic properties of food and their good

preservation in parallel.

1.1.11. Advantages in using spices as an alternative of nitrates and nitrites

Spices and herbs have been used since ancient times for different purposes. Originally,

most of them were used to preserve food. Within this group, there are spices, cinnamon,

cloves and turmeric. They possess bactericidal and fungicidal properties that can kill or

27

inhibit the growth of organisms which could spoil the food. Pepper and cinnamon are

reputed to be the best food preservatives.

Closely related to this function is the fact that the spices can disguise the bad taste or

smell of foods. This is evident especially in warm places, where the heat promotes

decomposition and is a leading cause of odors. Food processing technologies, such as

chemical preservatives cannot eliminate food pathogens, such as Listeria

monocytogenesis or delay totally the microbial spoilage (Gutierrez et al., 2009).

These constituents form the characteristic nature of the spice, and possess medicinal and

pharmacological properties with a possible impact on human health. India is known as

the “home” of spices, and is also a leading producer of major spices (Wealth of India,

2001). Among the spice-growing countries, such as India, Sri Lanka, Indonesia, and

Malaysia, these are used extensively as natural food flavorings. In addition to flavoring,

the spices help in protecting food from oxidative deterioration, thereby increasing shelf-

life, and also play a role in the body’s defenses against cardiovascular diseases, certain

cancers, and conditions, such as arthritis and asthma.

In fact, a part of the oxygen used by our body can produce “free radicals”. These

become fatal for a certain number of organic molecules as proteins, lipids and DNA of

cells. The free radicals are atoms which possess a free electron which can engender

chemical reactions found in the process of cellular oxidation. This oxidation can lead to

the development of diseases, such as cancer, cardiovascular diseases, diabetes.

Antioxidants, present in these natural plants, act to protect cells of degradation, reacting

with free radicals to make them harmless. Their properties are mainly derived from the

phenols or thiols present in these products

The first scientific studies of the preservation potential of spices, describing

antimicrobial activity of cinnamon oil against spores of anthrax bacilli, were reported in

the 1880s. Moreover, clove was used as a preservative to disguise spoilage in meat,

syrups, sauces and sweetmeats. In the 1910s, cinnamon and mustard were shown to be

effective in preserving applesauce. Since then, other spices, such as allspice, bay leaf,

caraway, coriander, cumin, oregano, rosemary, sage and thyme, have been reported to

have significant bacteriostatic properties (Burt, 2004; Ceylan and Fung, 2004; Gutierrez

et al., 2008a; Jayaprakasha et al., 2007; Tajkarimi et al., 2010).

28

These condiments can serve as disinfecting agents in the case of ground pepper and

clove, treatment of digestive disorders in the case of cinnamon, mustard, some cumin

and saffron, anti-inflammatory drug as in the case of cumin, aphrodisiac in the case of

the ginger and the hot pepper.

Natural antimicrobials have been identified in herbs and spices and several studies have

been reported on the preservative action of spices or their essential oils. Among these

natural antimicrobials are eugenol from cloves, thymol from thyme and oregano,

carvacrol from oregano, vanillin from vanilla, allicin from garlic, cinnamic aldehyde

from cinnamon, and allyl isothiocyanate from mustard (as reviewed by López-Malo et

al., 2006).

1.1.12. Different types of spices

1.1.12.1. Cinnamon (Cinnamomum zeylanicum) 1.1.12.1.1. Characteristic of Cinnamon

This spice is constituted by the internal bark of four main species of cinnamon tree

(Cinnamomum, Lauracée). C. verum allows to produce a cinnamon of very good quality

"Ceylon cinnamon". Others species lead to secondary lower-quality cinnamons called

"cassias". Cinnamomum zeylanicum (L.), commonly known as cinnamon is rich in

cinnamaldehyde as well as b-caryophyllene, linalool and other terpenes.

Cinnamaldehyde is the major constituent of cinnamon leaf oil and provides the

distinctive odor and flavor associated with cinnamon. It is used worldwide as a food

additive and flavoring agent, and the Food and Drug Administration lists it as

“Generally Recognized as Safe-GRAS. (Nikos G. Tzortzakis, 2008).

1.1.12.1.2. Role of cinnamon in preservation

This spice was found to exert antioxidant activity in the fermented meat sausage (Al-

Jalay et al., 1987), it possesses antibacterial properties against a large variety of

microorganisms. Some of them are pathogenic (Morozumi, 1978; Huhtanen 1980;

Hitokoto et al., 1980; Deans and Ritchie, 1987), inhibit growth and aflatoxin production

of molds (Bullerman et al., 1977), inhibit food spoilage by yeast (Conner and Beauchat,

29

1984) and delay acid production by the starter bacterium, Lactobacillus plantarum

(Zaika and Kissinger, 1979).

The antimicrobial and antifungal properties of cinnamon have also drawn great attention

from many researchers (Delespaul et al., 2000; Chang et al., 2001 and Kim et al., 2004).

Cinnamon possesses notable anti-allergenic, anti-inflammatory, anti-ulcerogenic, anti-

pyretic, and anaesthetic activities (Kurokawa et al., 1998; Qin et al., 2009). Some

evidence suggests that cinnamon may be effective in the supportive treatment of cancer

(Ka et al., 2003), infectious diseases, and complaints associated with modern lifestyle

due to its antioxidant (Lin et al., 2003; Okawa et al., 2001; Toda, 2003), anti-microbial

(Inouye et al., 2001; Smith-Palmer et al., 1998), and anti-inflammatory effects.

For preservation of food, cinnamon is known as an aperitif and digestion stimulant.

Cinnamaldehyde has been reported to possess antibacterial activity against a wide range

of bacteria (Chang et al., 2001), antioxidant properties (Gurdip et al., 2007).

A recent study investigated the effect of dietary supplementation with cinnamon and

garlic powder as growth promoter agents on performance, carcass traits, immune

responses, serum biochemistry, haematological parameters and thigh meat sensory

evaluation in broilers (Toghyani et al., 2010). The study concluded that cinnamon as

antimicrobial substances may inhibit intestinal pathogenic organisms and improve

digestion and absorption; particularly inclusion of 2 g/kg cinnamon proved satisfactory

results on performance indices and may have the potential to be applied as an alternative

for antibiotics growth promoter in broiler’s diet.

In another study (Tabak et al., 1999), dealing with the inhibitory effect on the growth

and urease activity of extracts from stem bark of Cinnamonum cassia on Helicobacter

pylori, it was shown that cinnamaldehyde at 200 mg/disk had the strongest inhibitory

effect (\90 mm) followed by eugenol 200 mg/disk (68 mm) and carvacrole 2000

mg/disk (66 mm). Cinnamaldehyde seems to be the main inhibitory component of

cinnamon and the utilization of cinnamon extract to inhibit both growth and urease

activity of H. pylori in vitro has proved to be more effective than the actual thyme

extract. The efficiency of cinnamon extracts in liquid medium and its resistance to lower

pH levelsmay enhance its effect in the human stomach.

30

1.1.12.2. Clove (Eugenia caryophyllus) 1.1.12.2.1. Caracteristic of cloves

The dried flower buds (Caryophylli floss) are used for medicinal and culinary purposes

and an essential oil is also distilled. The taste is hot, intense, fresh, with a peppery taste

and a touch of oriental fragrance. Not soluble in water but can be dissolved in alcohol.

Cloves control nausea and vomiting, improve digestion, protect against internal

parasites, cause uterine contractions and are strongly antiseptic. The major flavor

component is eugenol.

1.1.12.2.2. Role of cloves in preservation

The eugenol inhibits prostaglandin formation, which explains the anti-inflammatory and

analgesic effect, but the herb has further antiseptic, antispasmodic and carminative

properties. Among the many properties, it is antiplatelet, antiviral, fragrant and

flavoring properties, with a bacterial inhibition of 75-100% (Tajkarimi, 2010).

During conservation, essential oils are well known inhibitors of microorganisms

(Burt, 2004). Clove oil showed its antimicrobial activity in a study based on the

inhibitory effect of clove oil on Listeria monocytogenes in meat and cheese (Vrinda

Menon and Garg, 2001). Clove oil managed to limit the proliferation of Listeria

monocytogenes with 0.5% and 1 %, 30°C and 7°C.

Soliman & Badeaa (2002) found that 500 ppm of cinnamon oil can inhibit A. flavus, A.

parasiticus, A. ochraceus and Fusarium moniliforme on PDA. August (1978) reported

that higher concentrations of cinnamon oil and clove oil could also inhibit the asexual

spores of fungi. Mixtures of cinnamon and clove oils are therefore an interesting

alternative to use other chemical preservatives and appear well suited to use in active

packaging systems (Matan, 2006).

Many studies have shown the efficiency of clove associated with other spices, such as

cinnamon. The efficiency of cinnamon oil and clove oil, as an antibacterial agent, was

reported by Ouattara et al. (1997) and Pradsad and Seenayya (2000). The main

inhibitory components of cinnamon oil and clove oil are believed to be cinnamaldehyde

31

and eugenol, respectively (Jayatilaka et al., 1995; Porta et al., 1998), and their

effectiveness against molds and yeasts has been reported by López-Malo et al. (2002).

1.1.12.3. Cumin (Cuminum cyminum L.) 1.1.12.3.1. Caracteristic of Cumin

Sometimes spelled cumin (Cuminum cyminum L.) is a flowering annual plant of the

Umbelliferae family. This plant, which is one of the most important spices in the world,

is native to India, Iran, the Mediterranean, and Egypt. There are two varieties of cumin:

the white one (more common) and the black one. The cumin is a biennial plant, which

grows in light soils. It is sown in autumn or at the beginning of the year. The seeds

of cumin can contain 3 to 7 % of essential oil. Cumin, the aromatic seed spice, finds

wide applications in foods, beverages, and traditional medicine, and is known to possess

bioactive properties.

The cumin, pricked, spiced, flavor of hazelnut with a strong aromatic odor, has

anti-spasmodic virtues and helps to digest the heavy food. Its aroma matches with

the meat, the autumn vegetables, the salad, the cheese or the diverse pastries. It is a

carminative, an astringent, a stomachic, and is useful in dyspepsia and diarrhea.

1.1.12.3.2. Role of Cumin in preservation

Studies have illustrated its application as a preservative in foods, as well as its

antibacterial, antioxidant, hypoglycemic, hypolipidemic activities, in addition to its use

in perfumery and flavoring liquors. Cumin is traded as whole product, in the ground

form, or as an essential oil (Kanagal Sahana, 2011). Cuminaldehyde was found to be the

main component at concentrations of 53.6% for seed oil and 40.5% for herb oil (El-

Sawi et al., 2002).

Takahashi, Muraki and Yoshida (1881) reported that cumin oil contained mint sulfide as

a trace constituent. Twelve years later, (Anon, 1993) and (Shaath and Azzo, 1993)

reported that the main constituents of Egyptian cumin seed oil were cumin aldehyde, β-

pinene, γ-terpinene, ρ-mentha-1,3-dien-7-al, ρ-mentha-1,4-dien-7-al and p-cymene.

Black cumin seeds have many biological properties including anti-tumor (El-Daly,

32

1998), anti-diabetic (Al-Hader et al., 1993), diuretic (Zaoui et al., 2000) and anti-

bacterial (Kamali et al., 1998) activity.

1.1.12.4. Black pepper (P. nigrum) 1.1.12.4.1. Caracterictic of black pepper

Berry stemming from a plant native of India, called pepper plant, pepper is a part of the

family of Piperaceae and appears under the shape of clusters. Black pepper (P. nigrum)

is a perennial plant and derives its name Piper, perhaps, from the Greek name for black

pepper, Piperi (Rosengarten, 1973) and most of the European names for black pepper

were derived from the ancient Indian language, Sanskrit, such as Pippali, the name for

long pepper (P. longum). It was the great botanist Linnaeus (1753) who established the

genus Piper in his Species Plantarum.

The essential oil in the berry contributes to the aroma, while the alkaloid piperine

imparts the unique pungency. Oleoresin is extracted from the dry powdered berries by

solvents, and is the product that imparts the unique aroma, flavor and pungency in

pepper (Lewis et al, 1976) attributed blackening of pepper berries due to enzymatic

oxidation of polyphenolic substrates present in the skin of green pepper.

Black pepper is valued mostly for its spicy aroma and piquant pungent taste. Oleoresin,

produced by solvent extraction of dried powdered pepper, contains both aroma and

pungency principles (Premi, 2000). The active constituent of pepper, piperine is

sensitive to light and oxygen. Different products from black pepper available are ground

pepper, pepper oil and oleoresin (Ravindran & Johny, 2001).

Pepper is associated with a number of functional properties, such as analgesic and

antipyretic properties, antioxidant effects and antimicrobial properties (Kapoor et al.,

1993). Piperine, an active ingredient in pepper, exerts substantial analgesic and

antipyretic effects (Lee et al., 1984).

1.1.12.4.2. Role of black pepper in preservation

Cinnamon seems to have a lot of good properties for preservation in food. Nevertheless,

studies on the organoleptic properties are still required; the cinnamon having a

pronounced taste and a rather present smell. It would be necessary to measure in that

case the proportion of this spice in the formulations without changing the properties

33

looked for the preservation of meats. The organoleptic analyses are also appropriate

with cloves. Cloves, on its own, have good anti-inflammatory, analgesic and

antimicrobial properties. It can be associated with cinnamon, as mentioned above, and

maybe with other spices, due to antioxidant properties, to improve this property.

It is obvious that organoleptic analyses are important for all the formulations of spices;

in the case of black pepper, it has a spicy and strong aroma. Finally, it will be interesting

to see if the cumin powder has the same properties than cumin oil given that most of

articles deal only with cumin oil. Table 1.1 presents the summary of various properties

of spices which help to understand their eventual applications.

Table 1.1 Summary of the properties of spices

Major constituent properties References

Cinnamon

(Cinnamomum

zeylanicum)

Cinnamaldehyde

O

H

antimicrobial,

antifungal

anti-inflammatory,

anti-allergenic,

anti-ulcerogenic,

anti-pyretic,

anaesthetic

(Delespaul et al.,

2000), (Chang et

al., 2001), (Kim

et al., 2004),

(Kurokawa et al.,

1998), (Qin et al.,

2009), (Lin et al.,

2003), (Okawa et

al., 2001), (Toda,

2003), (Inouye et

al.,

2001), (Smith-

Palmer et al.,

1998)

Cloves

(Eugenia

Caryophyllus)

4-allyl-2-methoxyphenol (eugenol)

CH2

OCH3

OH

anti-inflammatory

analgesic effect,

antiplatelet,

antiviral, flavoring

properties

bacterial inhibition

antimicrobial

activity

(Tajkarimi, 2010)

(Menon and

Garg, 2001)

Cumin aldehyde anti-bacterial (Sahana, 2011)

34

1.1.13. Grapes

The grapes are good alternatives of nitrites and nitrates due to their important

antioxidant properties and presence of higher concentration of phenolic compounds

(Jayaprakasha et al., 2003), such as gallic acid, chlorogenic and caffeic acids, catechin,

etc. In addition, according to studies conducted at Oregon State University, raisins

showed conservation properties similar to those of sodium nitrite in meat products and

sausages. The combination of antioxidants, sugars and acids found in grapes proved to

be as effective as sodium nitrite in maintaining food security. The use of grapes to

replace sodium nitrite in meat has many health benefits. Firstly, while nitrite can form

carcinogenic nitrosamines, grapes do not form. Secondly, unlike sodium nitrite, the

addition of grape is not accompanied by the addition of sodium. This is important for

those who are dieting sodium hypo knowing that the addition of sodium nitrite

concentration can cause problems with hypertension. Thirdly, raisins can improve the

overall nutritional profile of meat products, because they are rich in antioxidants

(Perumall et al., 2011) and help to maintain the taste of products in addition to being

low fat. In addition, the grape is a fruit that is produced in large quantities and low cost.

Its use as an additive for preservation of meat products could be interesting especially if

Cumin

(Cuminum

cyminum L.)

CH3CH3

O

antioxidant,

hypoglycemic,

hypolipidemic

activities

anti-tumor

anti-diabetic

diuretic

anti-bacterial

(El-Daly, 1998)

(Al-Hader et al.,

1993),

(Zaoui et al.,

2000)

(Kamali et al.,

1998)

Black pepper

(Pepper

Nigrum)

Piperine

O

O

N

O

analgesic

antipyretic

antioxidant

antimicrobial

(Kapoor et al., 1993), (Lee et al., 1984)

35

it is added in the dry and ground form, which should facilitate its use and also reduce

the amount used to maintain the organoleptic properties of meat products.

1.1.14. Conclusion

Curing of meat is a process known since ancient times with the intention to prolong the

shelf-life of meat. The curing agents, nitrite and nitrate, react due to easily varying

oxidation status of nitrogen into many derivatives with meat ingredients (Honikel,

2007). Nitrites and nitrates have antioxidant, anti microbial properties, preserve the red

color of meat and are very cheap.

Nitrates have been measured in foods, and have been detected in vegetables, in

preserved meats and baby foods. Because of stricter regulations, industries have to find

a healthier alternative for these products. When ingested, they can be dangerous in high

dose and in long term for humans, animals and can also be dangerous to environment.

Among the various alternatives, the addition of spices is a very promising one; this

alternative seems healthier and could be very interesting given the numerous properties

of spices: antioxidants, antibacterial, among others. In fact, spices and herbs, which

were originally added for improving taste, can also naturally and safely improve shelf

life of food products. However, studies show that the use of essential oil is more

effective than the powder but nevertheless more expensive. Numerous spices, others

than presented here, are also being studied such as ginger, red pepper, coriander and

many others; additional analyses are necessary, such as the organoleptic, chemical and

microbiological analyses, to prove the reliability of these alternatives.

1.1.15. Acknowledgements

The authors are sincerely thankful to the Natural Sciences and Engineering Research

Council of Canada (Discovery Grant 355254), FQRNT (Programme de recherche en

partenariat visant le développement d'alternatives santé à l'ajout des nitrites et des

nitrates dans les produits carnés) for financial support. The views or opinions expressed

in this article are those of the authors.

36

1.1.16. References

Agbebe, T.M., and Ojeniyi, S.O. (2009). Tillage and poultry manure effects on soil fertility and sorghum yield in southwestern Nigeria. Soil and tillage research. 104 : 74-81.

Anon, A. (1993). Analytical methods of committee. Application of gas-liquid chromatography to analysis of essential oils Part XVI. Monography for five essential oils. Analyst. 118 : 1089–1098.

Bartsch, H., Ohshima, H., Pignatelli , B. (1988). Inhibitors of endogenous nitrosation mechanisms and implications in human cancer prevention. Mutat. Res-Fund Mol. M.. 202 : 307–324.

Bhat, R., Alias, A. K., and Paliyath, G. 2012. Progress in food preservation. Oxford, UK ; Ames, Iowa : Wiley, 2012.

Brambilla, G., and Martelli, A. (2005). Keynote comment: nitrosatable drugs, cancer, and guidelines for genotoxicity. Lancet Oncol. 6 : 538–539.

Brown, J.R., Marshall, C., and Smith, J. S. (2007). Nitrate in Soils and Plants. MU Extension G9804

Burt, S. (2004). Essential oils: their antibacterial properties and potential applications in foods—a review. Int. J. Food Microbiol. 94 : 223-253.

Ceylan, E., and Fung, D. Y. C. (2004). Antimicrobial activity of spices. J.Rapid Methods and Autom. Microbiol. 12 : 1–55.

Chan, T. Y. K. (2011). Vegetable-borne nitrate and nitrite and the risk of methaemoglobinaemia. Toxico. Lett. 200 : 107–108

Chang, S. T.; Chen, P. F., and Chang, S. C. (2001). Antibacterial activity of leaf essential oils and their constituents from Cinnamomum osmophloeum. J. Ethnopharmacol. 77 : 123–127.

Chung, S. Y., Kim, J. S., Hong, M. K., Lee, J. O., Kim, C. M., and Song, I. S. (2003). Survey of nitrate and nitrite contents of vegetables grown in Korea. Food Add. Contam. 20 : 621-628.

Cockburn, A., Brambilla, G., Fernández, M. L., Arcella, D., Bordajandi, L. R., Cottrill, B., Van Peteghem, C., and Dorne, J. L. (2010). Nitrite in feed: From Animal health to human health. Toxicol. Appl. Pharmacol.

Conner, D. E., and Beuchat, L. R. (1984). Effect of essential oils from plants on growth of spoilage yeasts. J. Food. Sci. 49 : 429-434.

Dennis, M.J., and Wilson, L.A. (2003). Nitrates and Nitrites. In : Encyclopedia of Food Sciences and Nutrition (Second Edition), pp. 4136–4141. Benjamin Caballero, York.

37

DIRECTIVE 2006/52/CE DU PARLEMENT EUROPÉEN ET DU CONSEIL du 5 juillet 2006 modifiant la directive 95/2/CE concernant les additifs alimentaires autres que les colorants et les édulcorants et la directive 94/35/CE concernant les édulcorants destinés à être employés dans les denrées alimentaires El-Sawi, S.A., and Mohamed, M.A. (2002). , Cumin herb as a new source of essential oils and its response to foliar spray with some micro-elements. Food Chem. 77 : 75–80.

Ellis, A., Li, C. G.,, and Rand, M. J. (1998). Effect of xanthine oxidase inhibition on endothelium- dependent and nitrergic relaxations. Eur. J. Pharmacol. 356 : 41-7.

Eisenbrand, G., Schmähl, D., Preussmann, R. (1980) Carcinogenicity of N-nitroso-3-hydroxypyrrolidine and dose–response study with N-nitrosopiperidine in rats. IARC. Sci. Publ. 31 : 657–666. Fan, A.M. (2011). Nitrate and Nitrite in Drinking Water: A Toxicological Review. In: Encyclopedia of Environmental Health, pp. 137–145. Jerome O. Nriagu, Oakland. Fan, A. M., Willhite, C. C., and Book, S. A. (1987). Evaluation of the nitrate drinking water standard with reference to infant methemoglobinemia and potential reproductive toxicity. Regul. Toxicol. Pharmacol. 7 : 135-48.

Food Additives (2008). Health Canada http://www.hc-sc.gc.ca/fn-an/securit/addit/index-eng.php Accessed on February 2013 Gangolli, S. D., van den Brandt, P. A., Feron, V. J., Janzowsky, C., Koeman, J. H., Speijers, G. J., Spiegelhalder, B., Walker, R., and Wisnok, J.S.(1994). Nitrate, nitrite and N-nitroso compounds. Eur. J. Pharmacol. 292 : 1-38.

Grohs, B.-M., and Kunz, B.(2000). Use of spice mixtures for the stabilisation of fresh portioned pork. Food Control . 11 : 433±436

Gutierrez, J., Barry-Ryan, C., and Bourke, P.(2009). Antimicrobial activity of plant essential oils using food model media: Efficacy, synergistic potential and interactions with food components. Food Microbiol. 26, : 142–150.

Honikel, K.-O. (2008). The use and control of nitrate and nitrite for the processing of meat products. Meat Sci. 78 : 68–76.Jayaprakasha, G. K., Selvi, T., and Sakariah, K. K. (2003). Antibacterial and antioxidant activities of grape (Vitis vinifera) seed extracts. Food Res Int. 36 : 117–122. Jensen, F.B. (2003). Nitrite disrupts multiple physiological functions in aquatic animals. Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol. 135 : 9–24. Janssen, L. H. J. M., Visser, H., and Roemer, F. G. (1989). Analysis of large scale sulphate, nitrate, chloride and ammonium concentrations in the Netherlands using an aerosol measuring network. Atmos. Environ. 23 : 2783–2796. Jayatilaka, A., Poole, S. K., Poole, C. F., and Chichila, T. M. (1995). Simultaneous micro steam distillation/solvent extraction for the isolation of semivolatile flavor

38

compounds from cinnamon and their separation by series coupled-column gas chromatography. Anal Chim Acta. 302 :147–162. Kanagal, S., Nagarajan, S., and Lingamallu, J. M. R. (2011). Cumin (Cuminum cyminum L.) Seed Volatile Oil: Chemistry and Role in Health and Disease Prevention. In: Nuts and Seeds in Health and Disease Prevention, pp. 417–427. Victor R. Preedy, V.R., Watson, R.R., and Patel, V.B., London, Tucson. Kapoor, V. K., Chawla, A. S., Manojkumar, and Pradeepkumar (1993). Search for antiinflammatory agents. India Drugs. 30 : 481–493. Kim, I.S., Yang, M-R., Lee, O.-H., and Kang, S.-N. (2011).Antioxidant activities of hot water extracts from various spices. Int J Mol Sci. 12: 4120–4131.

Kurokawa, M., A Kumeda, C., Yamamura, J.-i., Kamiyama, T., and Shiraki, K. (1998). Antipyretic activity of cinnamyl derivatives and related compounds in influenza virus-infected mice Original Research Article. Eur. J. Pharmacol. 348 : 45-51 Lee, E. B., Shin, K. H., Woo, W. S. (1984). Pharmacological Study of Piperine. Arch. Pharm. Res. 7 : 127–132

Lewis, Y. S., Krishnamurthy, N., Nambudiri, E. S., Sankarikutty, B., Shivshankar, and A. S., Mathew, A. G. (1976). The need for growing pepper cultivars to suit pepper products. Proceedings of the International Seminar on Pepper. Lundberg, J., Gladwin, M. T., Ahluwalia, A., Benjamin, N., Bryan, N. S., Butler, A., Cabrales, P., Fago, A., Feelisch, M., Ford, P. C., Freeman, B. A., Frenneaux, M., Friedman, J., Kelm, M., Kevil, C.G., Kim-Shapiro, D.B., Kozlov, A.V., Lancaster, J.R.J., Lefer, D.J., McColl, K., McCurry, K., Patel, R. P., Petersson, J., Rassaf, T., Reutov, V. P., Richter-Addo, G. B., Schechter, A., Shiva, S., Tsuchiya, K., van Faassen, E. E., Webb, A. J., Zuckerbraun, B. S., Zweier, J. L., and Weitzberg, E. (2009). Nitrate and nitrite in biology, nutrition and therapeutics. Nat. Chem. Biol. 5 : 865–869 Matan, N., Rimkeeree, H., Mawson, A. J., Chompreeda, P., Haruthaithanasan, V., and Parker, M. (2006). Antimicrobial activity of cinnamon and clove oils under modified atmosphere conditions. Int J Food Microbiol. 107 : 180–185. Meah, M. N., Harrison, N., and Davies, A. (1994). Nitrate and nitrite in foods and the diet. Food Addi. Contam. 11 : 519-532 Menon, K.V. and Garg, S.R. (2001). Inhibitory effect of clove oil on Listeria monocytogenes in meat and cheese. Food Microbiol. 18 : 647-650 Mueller, R. L., Hagel, H. J.,Wild, H.,Ruppin, H.,and Domschke, W. (1986). Nitrate and nitrite in normal gastric juice. Precursors of the endogenous N-nitroso compound synthesis. Oncology, 43 : 50–53 Moigradean, D., Lazureanu, A., Poiana, M.-A., Harmanescu, M., Gogoasa, I., and Gergen, I.. (2008). The influence of mineral fertilization about nitrogen content in soil, plant and tomato fruit. Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj Napoca Horticulture. 65: 172-177.

39

O'Leary, M., Rehm, G., and Schmitt, M. (1994). Understanding Nitrogen in Soils. WW-03770-GO Omar, A. A., Artime, E., and Webb, A. J. (2012). A comparison of organic and inorganic nitrates/nitrites. Nitric Oxide. 26 : 229–240. Ouattara, B., Simard, R. E., Holley, R. A., Piette, G. J.-P., and Bégin, A. (1997). Antibacterial activity of selected fatty acids and essential oils against six meat spoilage organisms. Int. J. Food Microbiol. 37 : 155–162. Oussalah, M., Caillet, S., Saucier, L., and Lacroix, M. (2007). Inhibitory effects of selected plant essential oils on four pathogen bacteria growth: E. coli O157:H7, Salmonella typhimurium, Staphylococcus aureus and Listeria monocytogenes. Food Control. 18 : 414-420.

Oussalah, M., Caillet, S., Saucier, L., and Lacroix, M. (2007). Inhibitory effects of selected plant essential oils on Pseudomonas putida growth, a bacterial spoilage meat. Meat Sci. 73 : 236-244. Pannala, A.S., Mani, A. R., Spencer, J. P. E., Skinner, V., Bruckdorfer, K. R.,Moore, K. P., and Rice-Evans, C. A. (2003). The effect of dietary nitrate on salivary, plasma, and urinary nitrate metabolism in humans. Free Radic. Biol. Med. 34: 576–584.

Pearson, A. M., and Gillett, T. A. (1996). Processed Meats. Chapman and Half, New York, NY

Pegg, R.B., and Shahidi, F. (2000). Nitrite Curing of Meat. The N-Nitrosamine Problem and Nitrite alternatives. Food and Nutrition Press, Inc., Trumbull, CT. Perumalla, A. V. S., and Hettiarachchy, N. S.(2011). Green tea and grape seed extracts—Potential applications in food safety and quality. Food Res. Int. 44 : 827-839. Petersen, A., and Stoltze, S. (1999). Nitrate and nitrite in vegetables on the Danish market: Content and intake. Food Addi. Contam. 16 : 291-299.

Pokorny, L., and Maturana, I., Bortle, W. H. (2006). Sodium nitrate and nitrite. In: Kirk-Othmer Encyclopedia of Chemical Technology, John Wiley and Sons, 5th Ed, New York, [online version]. Prabhakaran Nair, K.P. (2004). Advances in Agronomy, The Agronomy and Economy of Black Pepper (Piper nigrum L.) —The “King of Spices”. In : Agronomy and Economy of Black Pepper and Cardamom, pp 271–389. Prabhakaran Nair, K.P., New Delhi. Pradsad, M. M., Seenayya, G. Effect of spices on the growth of red halophilic cocci isolated from salt cured fish and solar salt. Food Res. Int. 33 : 793–798. Prospero, M., and Savoie, D. L. (1989). Nitrate in the Atmospheric Boundary Layer of the Tropical South Pacific: Implications Regarding Sources and Transport. J. Atmos. Chem. 8 : 391-415

40

Sallam, K. I., Ishioroshi, M., and Samejima, K. (2004). Antioxidant and Antimicrobial Effects of Garlic in Chicken Sausage. Lebenson Wiss Technol. 37 : 849-855.

Omar, S. A., Artime, E., and Webb, A. J. (2012). A comparison of organic and inorganic nitrates/nitrites Original Research Article. Nitric Oxide. 26 : 229-240. Rosengarten, F. Jr. (1973). The Book of Spices. Pyramid Books, New York.

Shaikh, J., Bhosale, R., and Singhal, R. (2006). Microencapsulation of black pepper oleoresin. Food Chem. 94 : 105–110.

Sebranek, J. G. (2009). Basic curing ingredients. In : Ingredients in Meat Products, pp. 1-24. Tarte, R. Eds. Springer Science+Business Media LLC, New York. Sebranek, J. G., and Bacus, J. N. (2007). Cured meat products without direct addition of nitrate or nitrite: What are the issues?. Meat Sci. 77:136–147. Shaath, N. A., and Azzo, N. R. (1993). Essential oil of Egypt. In : Food flavor ingredients and composition, pp. 591–603. Charalambous, G., Eds., Elsevier Sci. Pub, Amsterdam Shirin, A. P. R., and Prakash, J. (2010). Chemical composition and antioxidant properties of ginger root (Zingiber officinale). J. Med. Plants Res. 4 : 2674-2679

Sofos, J.N., et al. (1979). Botulism control by nitrite and sorbate in cured meats. J. Food Prot. 42:739-770.

Stoilova, I., Krastanov, A., Stoyanova, A., Denev, P., and Gargova, S. (2007). Antioxidant activity of a ginger extract (Zingiber officinale). Food Chem. 102 : 764–770

Tabak, M., Armon, R., and Neeman, I. (1999). Cinnamon extracts’ inhibitory effect on Helicobacter pylori Original Research Article. J. Ethnopharmacol. 67 : 269-277. Tajkarimi, M.M., Ibrahim, S. A., and Cliver, D. O. (2010). Antimicrobial herb and spice compounds in food. Food Control. 21 : 1199–1218.

Toghyani, M., Gheisari, A., Ghalamkari, G., and Eghbalsaied, S. (2011). Evaluation of cinnamon and garlic as antibiotic growth promoter substitutions on performance, immune responses, serum biochemical and haematological parameters in broiler chicks. Livest Sci. 138: 167-173.

Thomson, B. (2004). Nitrites and nitrates dietary exposure and risk assessment. Christchurch Science Centre Christchurch, NZ Townsend, W. E., and Olson, D. G. (1987). Cured meats and cured meat products processing. In The Science of Meat and Meat By Products, pp. 193–216, 431–456. Price, J. F., and Schweigert, B. S., Eds., Food and Nutrition Press Inc., Westport. Tu, X., Xiao, B., Xiong, J., and Chen, X.. (2010). A simple miniaturised photometrical method for rapid determination of nitrate and nitrite in freshwater. Talanta. 82 : 976–983

41

U.S. Department of Agriculture. (1995). Processing inspectors' calculation shandbook. Available at: http://www.fsis.usda.gov/OPPDE/rdad/FSISDirectives/7620-3.pdf. Accessed December 8, 2009.

U.S EPA (2006). Toxicity and Exposure Assessment for Children’s Health. http://www.epa.gov/teach/chem_summ/Nitrates_summary.pdf . Accessed on February, 2013

Vasavada, M. N., and Cornforth, D. P. (2005). Evaluation of milk mineral antioxidant activity in meat balls and nitrite-cured sausage. J. Food Sci. 70 : 250–253. Viani, F., Siegrist, H. H., Pignatelli, B., Cederberg, C., Idström, J. P., Verdu, E. F., Fried, M., Blum, A. L., and Armstrong, D. (2000). The effect of intra-gastric acidity and flora on the concentration of Nnitroso compounds in the stomach. Eur. J. Gastroenterol. Hepatol. 12 : 165-73.

World Health Organization (2004). Rolling Revision of the WHO Guidelines for Drinking-Water Quality. Nitrates and nitrites in drinking-water.

Yadav, A. S., and Singh, R. P.(2004). Natural preservatives in poultry meat. Arch. Microbiol. 181:8–1.

Yocom, J. E. (1982). Indoor/outdoor air quality relationships: a critical review. JAPCA J Air Waste Ma. 32 : 500–606. Zaika, L. L., Kissinger, J. C. (1978). Effect of major spices in Lebanon bologna on production by starter culture organisms. J. Food. Prot. 41 : 429-431.

Zheng, W., and Wang, S. Y. (2001). Antioxidant activity and phenolic compounds in selected herbs. J Agric Food Chem. 49 : 5165–70.

42

1.2. Spice use in food: Properties and benefits –A review

Jessica Elizabeth De La Torre Torres1, 2, Fatma Gassara1, Anne Patricia Kouassi1,

3, Satinder Kaur Brar1*, Khaled Belkacemi3

1 INRS-ETE, Université du Québec, 490, Rue de la Couronne, Québec, Canada G1K

9A9

2 Instituto Tecnológico y de Estudios Superiores de Monterrey (ITESM)

3 Département des sols et de génie agroalimentaire, Pavillon Paul-Comtois, Université

Laval, 2425, rue de l'Agriculture, Québec (Québec) G1V 0A6

*Corresponding Author: Tel.: (418) 654 3116 Fax : (418) 654 2600 ; E-mail

satinder.brar@ete.inrs.ca

43

1.2.1. Résumé

Les épices sont tirées des plantes qui, en raison de leurs propriétés sont utilisées comme

colorants, agents conservateurs ou médicaments. L'intérêt qu'est porté aux épices est

remarquable en raison de leur composition chimique, comprenant les

phénylpropanoïdes, les terpènes, les flavonoïdes et les anthocyanines. Les épices, telles

que le cumin (cuminaldehyde), les clous de girofle (eugénol), entre autres, sont connues

et étudiées pour leurs propriétés antimicrobiennes et antioxydantes. Ces épices sont

susceptibles d'être utilisées comme conservateurs dans de nombreux aliments à savoir

dans la viande traitée, afin de remplacer les conservateurs chimiques. Les épices ont des

effets bénéfiques, tels que lutter contre l'oxydation et sont comparables à des

antioxydants chimiques utilisés régulièrement afin d'être utilisés comme alternative

naturelle aux conservateurs de synthèse. Dans cette revue, les principales

caractéristiques des épices seront décrites ainsi que leurs différentes propriétés

chimiques, leur diverses applications et les avantages et inconvénients de leur

utilisation.

Mots-clés: Antioxidant, Antimicrobien, épices, Colorants, conservation, Propriétés.

44

1.2.2. Abstract

Spices are parts of plants that due to their properties are used as colorants, preservatives

or medicine. The interest in the potential of spices is remarkable due to their chemical

composition, as phenylpropanoids, terpenes, flavonoids and anthocyanins. Spices, such

as cumin (cuminaldehyde), clove (eugenol), among others, are known and studied for

their antimicrobial and antioxidant properties due to their main chemical compounds.

These spices have the potential to be used as preservatives in many foods namely in

processed meat to replace chemical preservatives. Spices provide beneficial effects,

such as antioxidant activity levels that are comparable to regular chemical antioxidants

used so they can be used as a natural alternative to synthetic preservatives. In this

review, the main characteristics of spices will be described as well as their chemical

properties, different applications of these spices and the advantages and disadvantages

of their use.

Key words: Antioxidant, Antimicrobial, Spices, Colorants, Preservatives, Properties.

45

1.2.3. Introduction

Spices have been used since the ancient civilization; their flavor and properties make

them important for culinary and medicinal uses (Parthasarathy et al., 2008). During

early travels to India and Africa to explore new countries, arrivals at The Spice Islands

made possible the discovery of new species into Europe and the development of trading

networks of spices. For a long time ago, several countries had fought for the control of

spice trade but the strongest nations were the ones who succeded. (World Trade

Organization, 2012). World Spice production is almost entirely achieved in India but

there are other countries that also have major spice production such as Bangladesh,

Turkey and China. Figure 1.6 presents in details the main spice producers countries

(FAO, 2010).

Figure 1.6 World Spice production in year 2010 (FAO, 2010)

Due to their important properties, spices have become essential for culinary and

medicinal proposes in several regions around the world, the trading of these spices has

been an important commercial activity since ancient times and a mean of economic

development (Tufail, 1990).

Asia is the super producer of all kinds of spices; cinnamon, pepper, nutmeg, clove and

ginger are found. There are also Latin American countries that have the leadership in

production of some trade spices, such as Brazil which stands as the major supplier of

pepper or Guatemala as the leading producer of cardamom (Parthasarathy et al., 2008).

68%

8%

7%

5%

4% 1%

1% 1% 1% 4%

World spice production

India

Bangladesh

Turkey

China

Pakistan

Nepal

Colombia

46

Due to spices properties and large applications, they have become an important

economical activity. Between the year 2000 and 2004, the value of spice imports

increased by 1.9% per year and the volume increased by 5.9%. In the year 2004, the

trade of spices was around 1.547 millions of tons with a value of US 2.97 billions. This

reflects the importance and demand of spices around the globe (International Trade

Centre, 2006).

Spices have many applications, namely as flavoring, medicinal, preservative and

coloring agent. Spices and their extracts possess preservative and natural antioxidant

properties, these extracts are popular and some of them have antibacterial, antifungal

and antiviral activities (Hernández et al., 2011). Due to the different applications

discovered in spices, research has been done over the most popular spices to determine

the chemical components that confer their properties. Main chemical compound actives

have been identified in several spices, such as cinammaldehyde in cinnamon, eugenol in

clove and cuminaldehyde in cumin which have proven to prevent food from spoilage

and inhibit the growth of pathogenic microorganisms (Carlos and Harrison, 1999).

Spice phenolic compounds are responsible for the majority of antimicrobial and

antioxidant properties, these compounds grant properties that make spices useful for

medicinal and preservative uses (Bozin et al., 2008). Nowadays, food preservation is a

main concern since most of the existing preservatives are based on synthetic chemicals.

Since spices are natural sources, the application of some as preservatives in food has

been evaluated in order to determine its efficiency and offer an opportunity to replace

synthetic preservatives such as nitrates, which have been claimed to possess negative

effect on human health (Anand and Sati, 2013). Along with this review, the main

characteristics of spices are examined as well the chemical compounds in spices, which

posses several properties that lead to wide ranging applications of spices. Finally, a brief

discussion is presented about the advantages and disadvantages of use of spices

regarding their potential.

1.2.4. General description of spices

The Geneva International Organization for Standardization defines Spices as “vegetable

products or mixtures thereof, free from extraneous matter, used for flavoring, seasoning

and imparting aroma to foods” (ISO, 1995). Spices have special properties that make

47

them useful for several proposes. Among these spices there are special characteristics

that give them distinct features, which have been given in details in Figure 1.7.

Spices incorporate leaves as mint or rosemary flowers as clove; bulbs as garlic or onion,

fruits, such as cumin or red chili, stems as cinnamon and rhizomes as ginger. Since all

spices are coming from plants they have been generally recognized as safe (GRAS).

Plants synthesize via a secondary metabolism, many compounds with complex

molecular structures. Among these metabolites are found alkaloids, flavonoids,

isoflavonoids, tannins, cumarins, glycosides, terpenes and phenolic compounds which

confer most of the properties of spices, such as flavoring, antimicrobial activity (Ceylan

and Fung, 2004), and antioxidant activity (Shobana and Akhilender, 2000; Souza et al.,

2005). Spices are well known due to their medicinal (Shan et al., 2007), preservative

and antioxidant (Burt, 2004) properties but they have been currently used for flavoring

proposes rather than for extending shelf-life of comestibles.

All spices are considered as different dried plant organs and they reside among different

taxonomical categories that correspond to several vegetal species. The wider

classification corresponds to spices that come from monocotyledoneae plants, such as

garlic, ginger, turmeric and vanilla or from dicotyledoneae plants, such as paprika,

pepper, nutmeg, cinnamon and clove (Spices Board, 2013). A more informal but

common classification of spices refers to their sensorial properties and classifies spices

within their flavor intensity or aromatic properties; for example, chili, pepper and ginger

belonging to hot spices or cinnamon clove and cumin belonging to aromatic spices

(Peter and Shylaja, 2012). Spices are defined as useful for different proposes, such as

flavoring and preserving food, these properties are due to several chemical compounds

contained in spices, namely phenylpropanoids, terpenes, flavonoids and anthocyans

(Sajilata and Singhal, 2012). All these compounds confer different properties to spices

such as antimicrobial and antioxidant activity that will be explained further in this

review.

48

Figure 1.7 Spices classification (adapted from Peter and Shylaja, 2012, Sajilata and Singhal 2012)

Spices description

Taxonomy

Angiospermae

Monocotyledoneae

Liliiflorae

Scitamineae

Orchidales

Dicotyledoneae

Archichlamydaeae

Sympetalae

Classification

Hot spices

Mild spices

Aromatic spices

Herbs

Aromatic vegetables

Main plant organs as spices

Aril

Barks

Berries

Buds

Bulbs

Pistil

Kernel

Leaf

Rhizome

Latex

Roots

Seeds

Major chemical constituents of spice essential oil

Phenylpropanoids

Terpenes

Flavonoids

Coumarins

Anthocyans

Aliphatic aldehydes

Aliphatic esters

Aliphatic ketones

Aliphatic acids

Aromatic compounds

49

1.2.5. Chemical properties of spices

There are many properties in spices that make them unique, such as their aroma but

amongst all, chemical characteristics allow spices to be used as preservatives in food.

Due to several chemical compounds, spices present antimicrobial activity and inhibit the

growth of pathogens in meat and other foods. Table 1.2 presents main chemical

characteristics that have been identified in several common spices.

Table 1.2 Main chemical characteristics of common spices

Spice Chemical profile References

Clove

Eugenia

caryophyllata

carvacrol, thymol, eugenol, cinnamaldehyde Chaieb et al., 2007

Coriander

Coriandrum sativum

linalool ,oxygenated monoterpenes ,monoterpene

hydrocarbons

Coriander seed: 60%-70% linalool 20 %

hydrocarbons

Essential oil of leaves and fruits: 2-decenoic acid

(30.8 %), E-11-tetradecenoic acid

(13.4 %), capric acid (12.7 %), undecyl alcohol

(6.4 %), tridecanoic acid (5.5 %),

undecanoic acid (7.1 %)

Coleman and Lawrence,

1992

Leung and Foster, 1996

Guenther, 1950

Bhuiyan et al., 2009

cinammon

Cinnamomum

zeylanicum

Leaves oil: eugenol (76.10 %), trans-β

caryophyllene (6.7 %),linalool (3.7 %), eugenol

acetate (2.8 %) benzyl benzoate (1.9 %).

Branches oil: linalool (10.6 %), α-pinene (9.9 %),

α-phellandrene (9.2 %)

Trajano et al., 2010

Lima et al., 2005

Indan babyleaf

Cinnamomum tejpata

Linalool (50 %) is the major compound; α-pinene,

p-cymene, β-pinene, limonene 5–10 %

Sajilata and Singhal, 2012

Nutmeg

Myristica fragrans

Nutmeg oil a-pinene, βb-pinene,

and sabinene(77.83%) in general 76.8 %

Mullavarapu and Ramesh,

1998

50

monoterpenes, 12.1 % oxygenated monoterpenes,

9.8 % phenyl propanoid ether

Gopalakrishnan, 1992

Origan

origanum vulgare

Leaf essential oil carvacrol (18.06 %)

thymol (7.36 %), g-terpinene (5.25 %), p-cymene

(5.02 %), limonene (4.68 %), caryophylene (4.12

%), cymene (3.56 %), ledene (3.41 %), linalool

(2.47 %), α-pinene (2.15 %), g-terpineol (2.10 %),

and germacrene (2.08 %).

Derwich et al., 2010

Rosemary

Rosmarinus

officinalis

a-pinene (18.25 %), followed by camphor (6.02

%), 1.8-cineole (5.25 %), camphene (5.02 %), b-

pinene (4.58 %), bornyl acetate

(4.35 %), limonene (3.56 %), borneol (3.10 %), a-

terpineol (2.89 %), and cymene

(2.02 %)

Derwich et al., 2011

The main bioactive components of all spices are mostly phenolic compounds,

flavonoids and terpenes. Table 1.3 summarizes the active compounds of the main spices

and their bioactivity. For example eugenol and cinnamaldehyde in clove are related to

their antimicrobial and antibacterial activity; however, these compounds are not

exclusive from clove, cinnamon also contains cinnamaldehyde and possesses the

antimicrobial activity but it also contains other chemical compounds, such as pinene

which confers antioxidant activity. There are a variety of phenolic compounds that hold

these properties and some of them are common among spices (Chaieb et al., 2007).

51

Table 1.3 Spices Bioactivity

Spice Active compounds Bioactivity References

Blackpepper Piperine Stimulates the digestive

enzymes of the pancreas,

enhances digestive capacity,

reduces gastrointestinal

food transit time, enhance

bioavailability of

therapeutic drugs and

phytochemicals, inhibits

hepatic and intestinal aryl

hydrocarbon hydroxylase

and glucuronyl transferase,

lower lipid peroxidation in

vivo, improves antioxidant

status, anti-mutagenic and

anti-tumor influences

Srinivasan K.,

2007

Aggarwal et al.,

2009

Han H.K., 2011

Liu Y. et al.,

2010

Cinnamon cinnamaldehyde Insulin potentiating

properties, cyclooxygenase-

2 inhibitor, reduces blood

glucose and lipids, restores

activities of plasma

enzymes, anesthetic,

antibacterial, anti-

inflammatory, anticancer,

antioxidant, antiviral,

inhibits systolic blood

pressure

Qin B. et al.,

2003

Akilen R. et al.,

2013

Aggarwal et al.,

2009

Clove Eugenol/ Isoeugenol Antioxidant, anti-

inflammatory, cytotoxic

activities, inhibition by

eugenol-related compounds

of lipopolysaccharide LPS

stimulated cyclooxygenase-

2, antimicrobial, inhibition

of platelet aggregation and

Kim S.S. et al.,

2003

Raghavendra

R.H. and Naidu

K.A., 2009

Aggarwal et al.,

2009

52

thromboxane syntheisis.

Ginger 6-gingerol Antioxidant, anti-

inflammatory, antiemetic,

antiulcer, cardiotonic,

antihypertensive,

hypoglycemic,

immunostimulant, activate

cell regulatory signals.

Ghayur M.N.

and Gilani A.H.,

2005

Nya E.J. and

Austin B., 2009

Aggarwal et al.,

2009

Rosemary Rosmarinic acid,

Carnosic acid

Antimicrobial, antioxidant,

inhibits lipoxygenase and

cyclooxygenase activity,

inhibit the Ca+2 dependent

pathways, anti-

inflammatory ,

immunomodulatory,

neuroprotective,

antiallergic, scavenge of

reactive oxygen radicals.

Kayashima T.

and Matsubara

K., 2012

Cheung S. and

Tai J., 2007

Aggarwal et al.,

2009

Different spices provide antimicrobial activity; this is due to certain chemical

compounds with the capacity to inhibit the growth of microorganisms. Figure 1.6

summarizes the range of inhibition achieved by different spices. Cinnamon clove,

rosemary and oregano have achieved levels of bacterial inhibition between 75% and

100% due to their chemical compounds, such as pinenes, eugenol and cinnamaldehyde

(Holley and Patel 2005; Naidu, 2000; Ceylan and Fung, 2004).

0

20

40

60

80

100

Perc

enta

ge

Ranges of bacterial inhibition

min

max

54

Table 1.4 List of bacterial strains inhibited by spices

Bacteria Spice with inhibitory effect Reference

Listeria monocytogenes Nisin, Origan, thymine, origan

with marjoram, thyme with sage

Clove oil, coriander, eugenol,

origan, rosemary

Burt, 2004

Du and Li, 2008

Hayouni al., 2008

Escherichia coli O157:H7 Clove,tea tree Origan, thymine,

origan with marjoram, thyme with

sage, pepper, origan with pepper

Moriera et al., 2007

Du and Li, 2008

Mosqueda-Melgar et al.,

2008

Oussalah et al., 2004

B.aereus and P. aeruginosa Origan, thymine, origan with

marjoram, thyme with sage

Du and Li, 2008

Pseudomonas ssp. Origan, pepper and origan with

pepper

Mosqueda-Melgar et al.,

2008

Oussalah et al., 2004

Aeromonas hydrophila Eugenol Burt, 2004

Salmonella typhimurium Carvacrol, citral, geraniol Burt, 2004

Photobacterium phosphoreum Oregano oil Burt, 2004

Salmonella enteritidis Mint oil Burt, 2004

Antimicrobial activity of spices depends on several factors, which include the type of

spices, composition and concentration of spices; microbial species and its occurrence

level, the substrate composition and the processing conditions and storage. Spices

stabilize foods from microbial deterioration by making the microbial growth

progressively slower and leading to complete suppression. (Souza et al., 2005).

1.2.6. Main applications of spices

Nowadays, all given properties of spices lead to numerous uses, from coloring to

flavoring spices that have been used since ancient times. The main uses of spices are

their natural colorants (Ravindran et al., 2006), flavoring, antioxidants (Shobana and

Akhilender, 2000) and antimicrobials (Ceylan and Fung, 2004). The application of

spices corresponds mainly with the food industry, but they are also used for medicine

(Shan et al., 2007), cosmetics, perfumery and nutraceuticals industry (Peter and Shylaja,

2012).

55

1.2.6.1. Insecticides Some spices have been used as insecticides as they have the potential of killing insects

in several life stages. Plants have been used as botanical insecticides as long time

traditions, for example neem is commonly applied to grain and act as a repellent and

insecticide, pyrethrum is used in flowers to control stored produced insects. These

plants, which possess insecticidal uses, contain essential oils, which have specific

chemical structures that confer this insecticidal property. Sometimes, these essential oils

are secondary metabolites that the plants produce for defense against herbivores or

disease (Suthisut et al., 2011).

The compounds that confer insecticidal properties are mostly complex mixtures of low

molecular weight, such as terpenoid compounds that give characteristic odor and flavor

to leaves, flowers, fruit, seeds bark and rhizomes (Bakkali et al., 2008). Many essential

oils of plants as spices are toxic to insects and act as fumigants, contact insecticides,

anti-feedants or repellents. It is important to mention that these essential oils are toxic to

insects but due to their low toxicity to warm blooded mammals they can be used as

sources to control produced insects (Suthisut et al., 2011). Table 1.5 summarizes the

main insecticidal uses of spices. The use of some spices as insecticides provides an

opportunity to replace the synthetic compounds from insecticides to natural alternatives,

which creates a sustainable market for this kind of products. Several chemical

insecticides in the market are claimed to have toxic compounds adverse to human

health. With the use of spices as insecticidal natural products, this problem can be

solved by the substitution of the synthetic compounds related to harmful health effects

with the main active compound of spices which are safe (Eddleston et al., 2006).

Table 1.5 Use of spices as insecticides

Spice Insects species Reference

Cardamom Tetropium castaneum,

Sitophilus zeamais,

Huang et al., 2000

Cinammon Acanthoscelides oblectus

Ceratitis capitata

Parthasarathy et al., 2008

Nutmeg/Mace Toxocara canis Nakamura et al., 1988

Curry Rhizopus stolonifer

Gloeosporium psidii

Dwivedi et al., 2002

Analgesic

coriander

peppermint

Antipyretic

dill

Anise

Anti inflammatory

Coriander

Celery

Parsley

Cumin

Ginger oil

Anticarcinogenic

Cumin

basil

57

Spices may also be used as bio enhancers; for example, piperine in black pepper has

been reported to possess bioavailability by enhancing activity with various structurally

and therapeutically diverse drugs (Singh et al., 2011). The use of spices is related to an

increased absorption of a drug in the organism due to alteration in membrane lipid

dynamics and enzymatic changes in the intestine, both of them being directly related to

several chemical structures of spices (Parthasarathy et al., 2008). Medicinal

applications of spices are important and the active compounds that provide these

properties should be further investigated to create natural based medicinal products with

a variety of uses, such as analgesic or anti-inflammatory.

1.2.6.3. Colorants

Spices are used as natural sourced colorants, bringing the advantage as against chemical

or synthetic colorants. Spices tint in different colors from yellow and orange to different

variations of red (except chlorophyll from herbs). The most common spices used for

coloring are paprika, red pepper, mustard, parsley, ginger and turmeric (Ravindran et

al., 2006).

The coloring properties of spices is due to several already mentioned chemical

compounds in spices, the principal compound responsible for the color are the

carotenoids, such as beta carotene, lutin and neoxanthin (Bartley and Scolnik, 1995).

Other compounds that provide these coloring properties to spices are flavonoids with

yellow colors, curcumin with orange and chlorophyllwith green (Ravindran et al., 2006;

Peter and Shylaja, 2012). Spices provide strong color pigments commonly between

orange, yellow and red; this can be advantageous since spices can be used as natural

colorants especially for food. Using spices as colorants in food is a natural alternative

that avoids the use of conventional synthetic colorants.

1.2.6.4. Natural flavors

Flavoring food is one of the most common uses for spices, almost each spice is related

to a specific flavor and they are basic for culinary proposes around the world.

Depending on the region, different spices are used for flavoring foods bringing a

distinguished flavor to each food style that even gives culinary identity. For example,

58

Mexico is known for its use of these flavors from cinnamon, vanilla, dried chilies and

cocoa. England uses ginger, mustard seeds, cloves, coriander and allspice. France is

known for different flavors in their foods, such as tarragon, savory marjoram, rosemary

and thyme flavor. The Arabian Peninsula is known to use a variety of spices for

flavoring proposes which include black peppercorn, caraway seed, whole cumin,

cardamom seed, fresh hot pepper garlic and coriander (Exploratorium, 2013).

Flavors given by spices are due to the certain families of chemicals, such as

phenylpropanoids, monoterpenes and other phenol compounds. Some important

chemical compounds for the flavoring potential of spices are eugenol, apiol, sufranol,

vanillin, piperine, beta caryophyllene, alfa pinene, carvacol, thymol, sabinene,

cinnamaldehyde and gingerol (Peter and Shylaja, 2012).

1.2.6.5. Natural Antioxidants

Spices are considered natural antioxidants for food. In order to preserve lipid

components from deterioration antioxidants are necessary in food. There are several

studies that consider antioxidants as defense mechanisms in the body against

cardiovascular diseases, cancer, arthritis, asthma and diabetes. Synthetic antioxidants

used nowadays in food, such as propyl gallate and hydroxyl toluene have been related to

carcinogenesis promoters so there is a strong tendency for the use of natural sources of

antioxidants (Peter and Shylaja, 2012).

The antioxidant properties of spices are due to their chemical compounds especially to

phenolic compounds, in fact there is a linear relationship between the phenolic content

and the antioxidant activity of a spice. Essential oils, oleorosin and other spice extracts

contain important antioxidant activity which can be profited by the food industry

(Wojdyło et al., 2007). Among the most important spices with antioxidant properties,

plants, such as lamiaceae, rosemary, oregano, thyme, sage, marjoram, basil, coriander

and pimento are predominant. The most common chemical compounds that provide

antioxidant properties to spices are eugenol, curcumin, gingerol, carcavol, thymol,

pimento and capsaicin (Peter and Shylaja, 2012).

59

1.2.6.6. Preservation of food

Foods most susceptible to microbial contamination are dairy products, such as

processed meat and chicken. These foods are a common vehicle for diseases and

pathogens, among them we find Escherichia coli, Salmonella, Listeria monocytogenes,

Yersinia enterocolitica, Campylobacter jejuni, Clostridium perfringen, Staphylococcus

aureus and Toxoplasma Gondi which have been isolated from meat (Reuben et al.,

2003). The meat processing industry is trying to find antimicrobial treatments to inhibit

the pathogens or decontaminate their products, these treatments can be synthetic

chemicals or antibiotics but also natural sources of antimicrobials (Hernández et al.,

2011).

Optimal microbial growth occurs at pH values between 6.5 and 7 although most

microorganisms continue to grow within the pH range of 4 and 9.5, for fresh meat, pH

varies around 5.0 and 6.5, hence microorganisms can easily grow into the meat (Tarté,

2009). Temperature is also an important factor for microorganisms and processed

meats, mesophylls replicate at temperatures between 20ºC and 40ºC, psychrotrophs

have the ability to survive and slowly replicate under refrigeration, with their optimal

growth occurring between 20ºC and 30ºC and for thermophiles, the optimal conditions

of growth are between 55-65ºC so almost at any temperature in which food can be

processed, there is a risk of contamination by one of these types of microorganisms

(Ercolini et al., 2009).

Obtaining antimicrobials from natural sources is a good alternative for preservatives in

meat products, other kinds of preservatives, such as synthetic chemicals have been

claimed to cause several adverse effects and preservatives as antibiotics produce

consequences, such as antibiotic resistance. Some natural antimicrobials studied in meat

products include bacteriocins, lactoferrin, lysozyme species, essential oils and a variety

of plant extracts. Species, such as clove cinnamon, cumin and oregano are effective

against inoculated microorganisms on meat, particularly against gram-positive and

gram-negative bacteria (Souza et al., 2006; Sema et al., 2007; Celikel and Kavas, 2008).

1.2.6.6.1. Cumin as preservative

60

Cumin (Cuminum cyminum) is a spice traditionally used as an antiseptic agent and it has

powerful antimicrobial activity in different kinds of bacteria, pathogenic and non-

pathogenic fungi for humans (Haloci et al.,2012). The cumin essential oil contains

cuminaldehyde, β-pinene, p-cymene and γ-terpinene as major chemical compounds

(Hajhashemi et al., 2004;Heinz and Varo, 1970). The main compound of the cumin’s

essential oil is cuminaldehyde which provides the antimicrobial properties. (Hernández

et al., 2011)

The alcoholic extract of cumin has been proven to present a significant inhibition of

microorganisms, such as Bacillus subtilis, Escherichia coli and Saccharomyces

cerevisiae with an outstanding antimicrobial activity for species, such as A.tumefaciens,

B. subtilis, Bacillus licheniformis, Pseudomonas oleovorans, Trichophyton rubrum, S.

cerevisiae and Saccharomyces pombe (De et al., 2003).

The antifungal properties of cumin oil have been proven in recent studies. Whole cumin

oil inhibit Aspergillus flavus and Aspergillus niger by over 90% when aldehyde fraction

of the oil containing the antimicrobial chemical compound cuminaldehyde was tested

(Balacs, 1993; Pawar and Thaker, 2006).

1.2.6.6.2. Clove as preservative

Clove (Eugenia caryophyllata) is a common spice used around the world for culinary

proposes but it also poses different properties that make cloves a potential preservative.

Clove essential oil compounds are eugenol and beta caryophyllene, both compounds

have antibacterial activity against Escherichia coli, Listeria monocytogenes, Salmonella

enterica, Campylobacter jejuni and Staphylococcus aureus (Chaieb et al., 2007).

Clove essential oil has a high concentration of eugenol of around 88.58% and it has

been proved to have diverse antimicrobial activity. Clove oil treatment in concentrations

from 1% to 2% has shown a reduction in growth rates of Listeria monocytogenes strains

(Mytle et al., 2006). Clove leaf oil has been found to inhibit Bacillus cereus with a MIC

of 39 µg/mL (Ogunwande et al., 2005).

Sensitivity of different bacterial strains to cloves essential oil have been tested and the

highest level of sensitivity was observed against five strains of Staphylococcus

epidermidis with an inhibition zone greater than 16 mm (Chaeib et al., 2007). Clove

also has fungicidal activity and their chemical compounds, such as carvacrol and

61

eugenol are known to possess fungicidal characteristics against Candida albicans and

Trichophyton mentagrophytes (Tampieri et al., 2005).

Antioxidant capacity of cloves is due to eugenol as the main chemical compound. The

main mechanisms of antioxidant activity are scavenging the radicals and chelating metal

ions and eugenol participates in photochemical reactions displaying strong antioxidant

activity (Ogata et al., 2000). Chelating potential of clove essential oil has been proven

resulting in the prevention of the hydroxyl radicals due to the eugenol in clove oil

(Jirovetz et al., 2006).

1.2.6.6.3. Cinammon as preservative

Cinnamon (Cinnamomum verum) is considered a preservative because it is an effective

antimicrobial and antibacterial which can inhibit bacterial growth, especially gram-

positive bacteria. Cinnamon oil is composed of different chemicals; amongst them the

most important are cynammyldehyde, cynammyl alcohol and eugenol (Herwita and

Idris, 2007).

Antimicrobial capacity of cinnamon has been tested against Staphylococus aureus

proving its capacity to inhibit S.aureus growth with an optimum inhibiting effort of

0.09% this result is mainly attributed to the chemical compound in cinnamon called

cynammyldehyde (Winias et al., 2011).

Cynammyldehyde inhibition to bacterial growth can be caused by inhibition of the

synthesis of cell walls, inhibition of the cell membrane function, inhibition of protein

synthesis or inhibition of the synthesis of nucleic acids (Winias et al., 2011).

Cinnamon extracts have antioxidant effects by scavenging activity against superoxide

and it has shown excellent antioxidant activities in enzymatic and non enzymatic liver

tissue oxidative systems as well as inhibition on FeCl(2)-ascorbic acid induced lipid

peroxidation of rat liver homogenate in vitro. (Aggarwal et al., 2009).

1.2.6.6.4. Black pepper as preservative

Black pepper (Piper nigrum) is a spice native from India and it’s volatile oil has been

proven to have antimicrobial activity (Dorman and Deans, 2000). The phenolic

compounds of black pepper have claimed to be responsible for the antimicrobial activity

62

by damaging the membrane of bacteria avoiding its growth (Karsha and Lakshmi,

2010).

Analysis using GC-MS has showed that black pepper essential oil contains main

chemical compounds, such as piperine, pierolein B and piperamide. This essential oil

was obtained based on acetone extraction and has proven to be effective in controlling

the mycelial growth of some fungi, such as Fusarium graminearum and Penicillum

viridcatum (Singh et al., 2004).

Most of its bioactivity is due to the main chemical compound, the alkaloid piperine.

This compound has been proven by in vitro studies to protect against oxidative damage

by inhibiting or quenching reactive oxygen species. Table 1.3 describes most of the

bioactivity of piperine in blackpepper. (Aggarwal et al., 2009)

Black pepper has been proven to have antibacterial activity with reported minimum

inhibitory concentrations of around 50-500 ppm demonstrating excellent inhibition on

the growth of gram positive bacteria, such as Staphylococcus aureus, followed by

Bacillus cereus and Streptococcus faecalis and also demonstrated inhibition against

some gram negative bacteria, such as Pseudomonas aeruginosa (Karsha and Lakshmi,

2010).

1.2.6.6.5. Rosemary as preservative

Rosemary (Rosmarinus officinalis) has been shown to possess preservative properties

for their use in foods since it’s antioxidant activity has been tested in pork products,

such as patties (Chen et al., 1999). The antioxidant properties of rosemary have been

attributed to the variety of phenolic compounds in this spice, such as carnosol, carnosic

acid, rosmarinic acid, rosmanol and tosemaridiphenol (Shahidi et al., 2003).

Carnosic acid is the main compound found in rosemary followed by other phenolic

compounds, such as carnosol. Rosemary chemical compounds are classified into three

groups, the phenolic diterpenes related to abietic acid structure, the flavonoids and the

phenolic acids (Almela et al., 2006). The main preservative properties are due to

carnosic acid in rosemary, which have a high antioxidant. The antioxidant activity of

this carnosic acid has been compared to the antioxidant activity of substances, such as

butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and tertiary butyl

63

hydroquinone (TBHQ) and the results showed that this acid has antioxidant activity

higher than BHT and BHA (Steven et al., 1996).

Carnosic acid, one of the main active compounds of rosemary, has originated from

isopentenyl diphosphate via methylerythritol phosphate and is located in chloroplasts

and intracellular membranes, such as carnosol (Almela et al., 2006). The rosemary has

been compared with other chemical preservatives and antioxidant compounds proving

efficiency that is comparable to the currently used preservatives so that the rosemary

can be used as a natural green alternative to some chemical antioxidants with

comparable results. Rosemary can be used as natural antioxidant in many foods as it

does not have a strong flavor akin to the majority of spices, namely cloves, cumin and

cinnamon among others. Hence, the use of rosemary as antioxidant will not damage the

organoleptic properties of foods.

1.2.6.6.6. Ginger as preservative

Ginger (Zingiber officinale) is a commonly used spice that contains polyphenolic

compounds, among them the 6-gingerol and its derivatives, these chemical compounds

made ginger a potent antioxidant (Stoilova et al., 2007). Fresh ginger contains moisture,

proteins, fats, fiber carbohydrates and some minerals like iron or calcium

(Govindarajan, 1982).

Ginger CO2 extracts have been proven to contain high polyphenol content and found to

have an enhanced efficiency as an antioxidant preservative at an earlier stage of fat

oxidation. The antioxidant effect of ginger is comparable to BHT, which is a chemical

antioxidant, inhibiting peroxidation in temperature range from 37°C to 80°C (Stoilova

et al., 2007).

Ginger has been shown to inhibit the multiplication of colon bacteria (Gupta and

Ravishankar, 2005) and other microorganisms, such as Escherichia coli, Proteus sp,

Staphylococci, Streptococci and Salmonella (Ernst and Pittler, 2000;White, 2007).

Ginger also has antifungal activity against some species, such as Aspergillus (Nanir and

Kadu, 1987).

The phenolic compounds in ginger are denaturing agents that avoid microbial growth by

changing the cell permeability leading to rupture of bacterial cells. Most of the phenolic

compounds are metal chelators and attach to active sites of metabolic enzymes reducing

enzyme activities and bacterial metabolism and reproduction (Ho et al., 1992).

64

Studies have showed that ginger extracts at concentrations of 0.4 mg/ml have better

antimicrobial activity than commercial antibiotics, such as Gentamicin against

Klebsiella pneumonieae, Proteus vulgaris, Streptococcus pyogenes and Staphylococcus

aureus (Ahmed et al., 2012). Ginger root extracts have been shown to be more effective

than extracts from other parts of plants, such as leaves and has been able to inhibit the

growth of Staphylococcus species with better results than common antibiotics, such

aschloramphenicol, ampicillin and tetracycline (Sebiomo et al., 2011).

1.2.6.6.7. Curry as preservative

Curry is a traditional spice used in conventional foods, the origin of curry is found in

India but nowadays, it is one of the most popular spices in the world with a

characteristic flavor and aroma (Sathaye et al., 2011). Curry has been shown to have an

important antimicrobial activity. Antimicrobial assays of coumarin extracts performed

with petroleum ether and chloroform exhibited prominent antibacterial and antifungal

activity. Chloroform extract of curry showed a good inhibitory property being effective

in species, such as Aspergillus niger and P. aeruginosa (Vats et al., 2011).

Curry contains a variety of carbazole alkaloids and coumarins that confer antimicrobial

activity. Minimum inhibitory concentrations of curry compounds have been found to be

between the range of 3.13-100µg/ml. (Rahman et al., 2005)

The antimicrobial activity of curry extracts is proportional to the concentration used and

growth inhibition has been reported against species, such as Bacillus subtilis,

Pseudomonas aeruginosa and Escherichia coli with a less minimum inhibitory

concentration (MIC) than compared to other species such as Staphylococcus aureus and

Micrococcus luteus. From these studies, E.coli has been determined as the most

resistant microorganism and higher concentrations of curry are required for its

inhibition (Vats et al., 2011).

Curry has been studied as a natural antimicrobial food preservative and also as a

detoxifying food preserving agent. Curry has been proven to be an antifungal and

antiaflatoxigenic (Murugan et al., 2013), these characteristics have set curry as an

important natural preservative with a high potential for becoming a replacement for

other types of unnatural preservatives.

65

Whole spices by themselves can be used as preservatives but their essential oils can also

be isolated and their properties determined. Essential oils from spices are homogeneous

mixtures of organic chemical compounds from the same chemical family; they are

composed of terpenoids, monoterpenes and sesquiterpenes. The antibacterial activity of

essential oils is not attributed to a specific mechanism but to several attack mechanisms

to cells with different targets (Burt, 2004). It is known that substances act on the cell’s

cytoplasmic membrane, in several cases, the presence of a hydroxyl group is related to

the deactivation of enzymes and it is probable that this group causes cell component

losses, a change in fatty acids and phospholipids and prevents energy metabolism and

genetic material synthesis (Di Pascua et al., 2005).

Antioxidant properties are important for conservation of processed meats. Nowadays,

there are several synthetic antioxidants, such as BHA, BHT and alfa tocopherol. It has

been proved that antioxidant properties from different essential oils from black pepper,

clove, geranium, Melissa, nutmeg, oregano and others show superior antioxidant

capacity to tocopherol analogue Trolox. Between all the species proven in the trial,

clove and oregano were exceptionally potent in the assay (Dorman, 2000).

In recent studies, essential oils from cumin and clove at concentrations from 500mg/L to

750 mg/L were used on meat samples at three different concentrations, 750, 1500 and

2250 microliters. The cumin essential oil produced a reduction of 3.78 log UFC/g with

the application of 750 microliters and the clove essential oil produced a reduction of

3.78 l of UFC/g with the application of 2,250 microliter, clove and cumin extracts got a

reduction of 3.6 log UFC/g demonstrating the antibacterial potential of these essential

oils (Hernández et al., 2011).

1.2.7. Advantages & Disadvantages of using spices as preservatives

Antioxidant and antimicrobial activity has been found in spices proving an important

preservative activity for food but several aspects need to be studied before assuring the

effectiveness of spices as preservatives. As it has been reviewed, spices have different

levels of aroma and flavor but most of them are characterized as being strong. If spices

are used in high quantities in order to achieve a good antioxidant or antimicrobial

activity, they can interfere with the original flavor of the food and products could be not

66

useful in the market since they would interfere with commercial desired characteristics

for several foods.

Essential oils from spice extracts are a good alternative for preservatives in meat

products but the main concern is that, when essential oils are used in meat, their

antimicrobial effect is lower because high-fat and protein levels contained in meat

protect bacteria from the essential oils' action, the essential oil is dissolved into the food

fatty phase being less available to act against the microorganisms (Rasooli, 2007).

Encapsulated rosemary essential oil has an improved antimicrobial effect than standard

rosemary essential oil against L.monocytogenes in pork liver sausage and this is

associated with the interaction of essential oils with the fatty phase of meat

(Carraminana et al., 2008). Thus, higher concentration of spices might be needed to

assure an antioxidant and antimicrobial activity but the strong flavor of spices can affect

the flavor of meat and its commercial value.

Essential oils from spices also require a process of extraction, which can make the

whole process pricier and would not result in a higher antimicrobial activity since; for

example, these essential oils can dissolve in the fatty phase of meat. Therefore, the use

of the whole spices might be a better solution for preservation in meat and other foods

since they present less complexity, less expenses and equivalent antimicrobial activity.

Another important aspect is that spice formulation effectiveness against microorganisms

differs depending on the food or media; same formulation can be effective for a specific

type of meat but not for another. A combination of clove and oregano in broth culture

showed inhibitory effect for L.monocytogenes but did not show the desired effect in

meat slurry (Lis-Balchin et al., 2003). Different spices formulations have to be tested in

vitro and in vivo in order to prove their antimicrobial effect for each type of meat.

Spices are provided from natural herbs and plants and thus do not have a synthetic

origin, essential oils of cinnamon and clove and their main active chemical compound

cinnamaldehyde and eugenol have been recognized as safe consumption products

(GRAS) by regulatory agencies of U.S. (Raybaudi et al., 2008; Turgis et al, 2009). In

contrast, nitrate and nitrite preservatives used nowadays in meat products have been

found to produce carcinogenic N-nitroso compounds, such as nitrosamines and this has

caused concerns about possible adverse health effects. (Assembly of Life Sciences U.S.,

67

1982; Anand and Sati, 2013). Therefore, spices are a safe alternative for preservatives in

food approved by regulation and with no adverse health effects reported.

1.2.8. Conclusions and future outlook

Spice uses vary from flavoring, coloring, medicinal or preservative uses and their trade

is a significant economic activity in the world. The unique properties of spices have

created a huge demand for several common spices around the world making the spices a

niche of research and economical benefits.

Several spices have been proved to have microbial growth inhibition potential to some

of the most common bacteria in food, such as L.monocytogenes, E.colli and Salmonella.

Thus, it is possible to use spices as preservatives but is necessary to prove its

antimicrobial effect on different foods, such as meat, poultry, dairy products, vegetables

and fruit to guarantee a preservative effect comparable to the conventional synthetic

preservative effect for each food prior to settle the use of spices as preservatives for

industrial or commercial proposes.

Albeit, whole spices and their essential oil have proven good antimicrobial activity, but

the use of the whole spice or essential oil is in debate due to the high purification costs

that can be involved without necessarily having an improving efficiency in the

antimicrobial or antioxidant activity. As whole spices owe this properties they can be

settled as natural preservatives and adapted to the industry for this propose.

Finally, the antimicrobial and antioxidant properties of several spices such as black

pepper, clove, nutmeg, turmeric, cumin, cinnamon among others leads to a research

field in order to use them as preservatives in food. Spices used in foods, such as meats

have a high possibility of success and potential antimicrobial activity that is comparable

with the effect of preservatives based on nitrites that are used nowadays and which

have been claimed to own negative health effects, making possible to research a way to

substitute chemical based preservatives with natural based ones for food preservation.

68

1.2.9. Acknowledgements

The authors are sincerely thankful to the Natural Sciences and Engineering Research

Council of Canada (Discovery Grant 355254), FQRNT (Programme de recherche en

partenariat visant le développement d'alternatives santé à l'ajout des nitrites et des

nitrates dans les produits carnés) for financial support. We specially thank MITACS

Globalink program for the prestigious funded internship opportunity given to Miss

Jessica Elizabeth De La Torre Torres, which made it possible for her to accomplish this

review. The views or opinions expressed in this article are those of the authors.

69

1.2.10. References

Aggarwal, B., Kunnumakkara A. (2009) Molecular Targets and Therapeutic Uses of Spices World Scientific

Ahmed, S. A., Jabbar I. I. and Abdul H. E. (2012). Study the Antibacterial Activity of Zingiber officinale roots against Some of Pathogenic Bacteria. Al- Mustansiriya J. Sci. 3(3):63-70.

Akilen R., Pimlott Z., Tsiami A. and Robinson N. (2013). Effect of short-term administration of cinnamon on blood pressure in patients with prediabetes and type 2 diabetes. Nutrition. 29(10):1192-6.

Almela, L., Sánchez-Muñoz, B., Fernández-López, J. A., Roca, M. J. and Rabe, V. (2006). Liquid chromatograpic–mass spectrometric analysis of phenolics and free radical scavenging activity of rosemary extract from different raw material. J. Chromatogr. A. 1120(1-2):221-229

Anand, S. P. and Sati, N. 2013 Artificial Preservatives and their harmful effects: Looking toward nature for safer alternatives Int. J. Pharm. Sci. Res. 4(7):2496-2501

Assembly of Life Sciences U.S. (1982). Alternatives to the current use of nitrites in food In: National Academy Press USA. Intl standard book number 0-309-03277-6 pp. 1-3. Bakkali, F., Averbeck, S., Averbeck, D. and Idaomar, M. (2008). Biological effects of essential oils a review. Food Chem. Toxicol. 46: 446- 475.

Balacs, T. (1993). Cajuput components In: Research Reports Int. J. Aromather 5(4):35

Bartley, G. E. and Scolnik, P. A. (1995). Plant carotenoids: Pigments for Photoprotection, Visual Attraction, and Human Health. Plant Cell 7(7):1027-1038

Bhuiyan, M. N. I., Begum, J. and Sultana, M. (2009). Chemical composition of leaf and seed essential oil of Coriandrum sativum L. Bangladesh J. Pharmacol. 4: 150–153. Bozin, B., Mimica-Dukic N., Samojlik I., Goran, A. and Igic, R. (2008). Phenolics as antioxidants in garlic (Allium sativum L., Alliaceae) Food Chem. 111: 925–929.

Burt, S. (2004). Essential oils: Their antibacterial properties and potential applications in foods – A review. Int. J. Food Microbiol. 94(3): 223–253. Carlos, A. M. A., and Harrison, M. A. (1999) Inhibition of selected Microorganisms in marinated chicken by pimento leaf oil and clove oleoresin J. Appl. Poult. Res. 8:100-109.

Carraminana, J. J., Rota, C., Burillo, J., and Herrera, A. (2008). Antibacterial efficiency of spanish Satureja montana essential oil against Listeria monocytogenes among natural flora in minced pork. J. Food Protect. 71(3):502–508. Celikel, N. and Kavas, G. (2008). Antimicrobial properties of some essential oils against some pathogenic microorganisms. Czech J. Food. Sci. 26:174–181.

70

Ceylan, E., and Fung, D. Y. C. (2004). Antimicrobial acitivity of spices J. Rapid Methods Autom. Microbiol. 12: 1–55.

Chaieb, K., Hajlaoui, H., Zmantar, T., Ben, A., Rouabhia, M., Mahdouani, K. and Bakhrouf, A. (2007). The chemical composition and biological activity of clove essential oil, Eugenia caryophyllata (Syzigium aromaticum L. Myrtaceae): a short review. Phytother. Res. 21: 501–506. Chattopadhyay, I., Biswas, K., Bandyopadhyay, U. and Banerjee, R. K. (2004). Turmeric and curcumin: Biological actions and medicinal applications. Current Science. 87: 1.

Chen, X. C., Jo, C., Lee, J. I. and Ahn, D. U. (1999). Lipid oxidation, volatiles and color changes of irradiated pork patties as affected by antioxidants. J. Food Sci. 64 (1): 16-19.

Cheung S. and Tai J. (2007). Anti-proliferative and antioxidant properties of rosemary Rosmarinus officinalis. Oncol Rep. 17(6):1525-31.

Coleman, W.M. and Lawrence, B.M. (1992). Comparative Automated Static and Dynamic Quantitative Headspace Analyses of Coriander Oil. J Chromatogr Sci. 30: 396–398. De, M., De, A. K., Mukhopadhyay, R., Banerjee, A. B. and Miró, H. (2003). Antimicrobial Activity of Cuminum cyminum L. Ars Pharmaceutica 44:257-269. Derwich, E., Benziane, Z. and Chabir, R. (2011). Aromatic and Medicinal Plants of Morocco: Chemical composition of essential oils of Rosmarinus officinalis and Juniperus Phoenicea Int. J. Appl. Biol. Pharm. Technol. 2(1): 145–153. Derwich, E., Benziane, Z., Manar, A., Boukir, A. and Taouil, R. (2010). Phytochemical Analysis and in vitro Antibacterial Activity of the Essential Oil of Orignum vulgare from Morocco Am.-Euras. J. Sci. Res., 5(2):120–129. Di Pascua, R., De Feo, V., Villani, F. and Mauriello, G. (2005). In vitro antimicrobial activity of essential oils from Mediterranean Apiaceae, Verbenaceae and Lamiaceae against foodborne pathogens and spoilage bacteria. Ann. Microbiol. 55:139–143. Dorman, H. J. and Deans, S. G. (2000a). Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J. Appl. Microbiol. 88(2):308-316.

Dorman, H.J.D., Surai, P. and Deans, S.G. (2000b). In vitro antioxidant activity of a number of plant essential oils and phytoconstituents. J. Essent. Oil Res. 12(2):241 248. Du, H. and Li, H. (2008). Antioxidant effect of cassia essential oil on deep-fried beef during the frying process. Meat Sci. 78:461-468. Dwivedi, B.K., Pandey, G., Pandey, R. C., Pant, H. L. and Logani, R. (2002) Evaluation of angiospermic plant extracts against Rhizopus stolonifer and Gloeosporium psidii fungi of guava. Bioved 13: 129-134.

71

Eddleston, M., Eyer, P., Worek, F., Mohamed, F., Senarathna, L., Von Meyer, L., Juszczak, E., Hittarage, A., Azhar, S., Dissanayake, W., Sheriff, M. H., Szinicz, L., Dawson, A. H. and Buckley, N. A.(2006). Differences between organophosphorus insecticides in human self-poisoning: a prospective cohort study. Lancet 367(9508): 396.

Ercolini, D., Russo, F., Nasi, A., Ferranti, P. and Villani, F. (2009). Mesophilic and Psychrotrophic Bacteria from Meat and Their Spoilage Potential In Vitro and in Beef. Appl. Environ. Microbiol. 75(7): 1990-2001.

Ernst, E. and Pittler M. H. (2000) Efficacy of ginger for nausea and vomiting. A systematic review of randomised clinical trials. Br. J. Anaesth. 84(3): 367-371.

Exploratorium (2013). Spice blends In: Spice blends of the World Science cooking California USA available online http://www.exploratorium.edu/cooking/seasoning/map/spicemap.html (assessed August 31, 2013)

FAO (2010). Spices producers. In:Food and Agriculture organization of the United States FAOSTAT

Ghayur M.N. and Gilani A.H. (2005). Ginger lowers blood pressure through blockade of voltage-dependent calcium channels. J Cardiovasc Pharmacol. 45(1):74-80.

Gopalakrishnan, M. (1992). Chemical composition of nutmeg and mace. J. Spices Aromat. Crops, 1: 49–54. Govindarajan, V. S. and Connell, D. W. (1982). Ginger: Chemistry, technology and quality evaluation (Part I). Crit. Rev. Food Sci. Nutr. 17(1): 1-96.

Guenther, E. (1950) The Essential Oils. Vol. IV. Van Nostrand, New York pp. 602-615.

Gupta, S. and Ravishankar S. (2005). A comparison of the antimicrobial activity of garlic, ginger, carrot, and turmeric pastes against Escherichia coli O157:H7 in laboratory buffer and ground beef. Foodborne Pathog. Dis. 2(4):330-40.

Hajhashemi, V., Ghannadi, A. and Jafarabadi, H. (2004) Black Cumin Seed Essential Oil, as a Potent Analgesic and Antiinflammatory Drug. Phytother. Res. 18(3):195-199.

Haloci, E., Manfredini, S., Toska,V., Vertuani, S., Ziosi, P., Topi, I. and Kolani, H. (2012). Antibacterial and Antifungal Activity Assesment of Nigella Sativa Essential Oils. World Acad. Sci. Eng. Technol. 66:1198-1200

Han H.K. (2011). The effects of black pepper on the intestinal absorption and hepatic metabolism of drugs. Expert Opin Drug Metab Toxicol. 7(6):721-9.

Hayouni, E. A., Chraief, I., Abedrabba, M., Bouix, M., Leveau, J. Y., Mohammed, H. and Hamdi, M. (2008). Tunisian Salvia officinalis L. and Schinus molle L. essential oils: Their chemical compositions and their preservative effects against Salmonella inoculated in minced beef meat. Int. J. Food Microbiol. 125(3): 242–251.

Heinz, D. E. and Varo, P. T. (1970). Volatile Components of Cumin Seed Oil J. Agric. Food Chem. 18: 234–238

72

Hernández, L., Aguirre Y.B., Nevárez G.V., Gutierrez N. and Salas E. (2011). Use of essential oils and extracts from spices in meat protection. J. Food Sci. Technol. Herwita and Idris. (2007). The impact Cinnamon Bio-Insecticide to the insect biologic aspect Epilachum varivestis, Mulsant. Jurnal akta Agrosia. 1: 99–105.

Ho, C., Lee, C. Y. and Huang M. (1992) Phenolic Compounds in Food and their effects on Health I. Am. Chem. Soc. Vol. 506.

Holley, R. A., and Patel, D. (2005). Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials. Food Microbiol. 22(4):273–292. Huang, Y., Lam, S.L., and Ho, S.H. (2000). Bioactivities of essential oil from Elletaria cardamomum (L.) Maton to Sitophilus zeamais Motschulsky and Tribolium castaneum. J. Stored Prod. Res. 36: 107-117. ISO (1995). Spices definition. In: Geneva-Based International Organization for Standarisation. ISO 676:1995. Jirovetz, L., Buchbauer, G., Stoilova, I., Stoyanova, A., Krastanov, A. and Schmidt, E. (2006). Chemical composition and antioxidant properties of clove leaf essential oil. J Agric Food Chem 54: 6303–6307.

Karsha, P. V. and Lakshmi O. (2010). Antibacterial activity of black pepper (Piper nigrum Linn.) with special reference to its mode of action on bacteria. Indian Journal of Natural Products and Resources 1(2): 213-215.

Kayashima T. and Matsubara K. (2012). Antiangiogenic effect of carnosic acid and carnosol, neuroprotective compounds in rosemary leaves. Biosci Biotechnol Biochem. 76(1):115-9.

Kim S.S., Oh O.J., Min H.Y., Park E.J., Kim Y., Park H.J., Nam Han Y. and Lee S.K. (2003). Eugenol suppresses cyclooxygenase-2 expression in lipopolysaccharide-stimulated mouse macrophage RAW264.7 cells. Life Sci. 73(3):337-48.

Leung, A. and Foster, S. (1996). Corinader. In: Encyclopedia of Common Natural Ingredients Used in Food, Drugs and Cosmetics,pp.193-195 2nd edn J. Wiley, New York. Lima, I.O., Oliveira, R.G., Lima, E., Souza, E.L., Farias, N. P., and Navarro, D.(2005). Inhibitory effect of some phytochemicals in the growth of yeasts potentially causing opportunistic infections Rev. Bras. Cienc. Farm. 41: 199–203. Lis-Balchin, M., Steyrl, H., and Krenn, E. (2003). The comparative effect of novel Pelargonium essential oils and their corresponding hydrosols as antimicrobial agents in a model food system. Phytother. Res. 17: 60–65. Liu Y., Yadev V.R., Aggarwal B.B. and Nair M.G. (2010). Inhibitory effects of black pepper (Piper nigrum) extracts and compounds on human tumor cell proliferation, cyclooxygenase enzymes, lipid peroxidation and nuclear transcription factor-kappa-B. Nat Prod Commun. 5(8):1253-7.

73

McKay, D. L. and Blumberg, J. B. (2006). A review of the bioactivity and potential health benefits of peppermint tea (Mentha piperita L.) Phytother. Res. 20(8):619-633.

Moriera, M. R., Ponce, A. G., Del Valle, C. E., and Roura, S. (2007). Effects of clove and tea tree oils on Escherichai coli O157:H7 in blanching spinach and minced cooked beef. J. Food Process. Pres. 31:379–391. Mosqueda-Melgar, J., Raybaudi-Massilia, R. M., and Martin-Belloso, O. (2008). Inactivation of Salmonella enterica Ser enteritidis in tomato juice by combining of high-intensity pulsed electric fields with natural antimicrobials. J. Food Sci. 73(2): 47–53. Mullavarapu, G. R. and Ramesh, S. (1998). Composition of essential oils of nutmeg and mace Aromat. Plant Sci. 20: 746–748. Murugan K., Anandaraj, K. and Al-Sohaibani, S. (2013). Antiaflatoxigenic food additive potential of Murraya koenigii: An in vitro and molecular interaction study. Food Res. Int. 52(1):8-16.

Mytle, N., Anderson, G. L., Doyle, M.P. and Smith, M. A. (2006). Antimicrobial activity of clove (Syzgium aromaticum) oil in inhibiting Listeria monocytogenes on chicken frankfurters. Food Control 17: 102–107.

Naidu, A. S. (2000). Overview. In: A. S. Naidu. Natural food antimicrobial systems (pp. 1–16). Boca Raton, Florida: CRC Press. Nakamura, N., Kiuchi, F., Tsuda, Y. and Kondo, K. (1988). Studies on crude drugs effective on visceral larva migrans.V. The larvicidal principle in mace Chem. Pharm. Bull 36(7):2685-2688. Nanir S. P. and Kadu B. B. (1987) Effect of medicinal plant extracts on some fungi. Acta Bot. Indica 15(2): 170-175.

Nya E.J. and Austin B.( 2009). Use of dietary ginger, Zingiber officinale Roscoe, as an immunostimulant to control Aeromonas hydrophila infections in rainbow trout, Oncorhynchus mykiss (Walbaum). J Fish Dis. 32(11):971-7.

Ogata, M., Hoshi, M., Urano, S. and Endo, T. (2000). Antioxidant activity of eugenol and related monomeric and dimeric compounds. Chem Pharm Bull 48: 1467–1469.

Ogunwande, I. A., Olawore, N.O., Ekundayo, O., Walker, T.M., Schmidt, J.M. and Setzer, W. N. (2005). Studies on the essential oils composition, antibacterial and cytotoxicity of Eugenia uniflora L. Int. J. Aromather. 15: 147–152.

Oussalah, M., Caillet, S., Salmiea, S., Saucier, L. and Lacroix, M. (2004). Antimicrobial and antioxidant effects of milk protein-based film containing essential oils for the preservation of whole beef muscle. J. Agric. Food Chem. 52(18):5598–5605.

Parthasarathy, V.A., Chempakam, B. and Zachariah, T. J. (2008). Chemistry of spices. CABI ISBN 978-1-84593-405-7 UK. Pawar, V.C. and Thaker V.S. (2006). In vitro efficacy of 75 essential oils against Aspergillus niger. Mycoses. 49(4): 316-323

74

Peter, K.V. and Shylaja, M.R. (2012). Introduction to herbs and spices: definitions, trade and applications. In: The Handbook of herbs and spices Second edition, volume 1, pp. 1-24. Woodhead Publishing. Platel, K. and Srinivasan, K. (2004). Digestive stimulant action of spices: A myth or reality?. Indian J. Med. Res. 119:167-179.

Qin B., Nagasaki M., Ren M., Bajotto G., Oshida Y. and Sato Y. (2003). Cinnamon extract (traditional herb) potentiates in vivo insulin-regulated glucose utilization via enhancing insulin signaling in rats. Diabetes Res Clin Pract. 62(3):139-48.

Raghavendra R.H. and Naidu K.A. (2009). Spice active principles as the inhibitors of human platelet aggregation and thromboxane biosynthesis. Prostaglandins Leukot Essent Fatty Acids. 81(1):73-8.

Rahman, M. M. and Gray A. I. (2005). A benzoisofuranone derivative and carbazole alkaloids from Murraya koenigii and their antimicrobial activity. Phytochem. 66(13):1601-1606.

Rasooli, I. (2007). Food preservation, a biopreservative approach. Food. Global Science Book 1:111–136. Ravindran, P.N., Nirmal Babu, K., Shiva, K. N. (2006). Genetic resources of spices and their conservation In: Ravindran, P.N., Nirmal Babu, K., Shiva, K. N. and Johny, A. K.. (eds) Advances in Spices Research. Agrobios pp. 63-91. Raybaudi, R. M. M., Rojas-Grau, M. A., Mosqueda-Melgar, J., and Martin-Belloso, O. (2008). Comparative study on essential oils incorporated into an alginate-based edible coating to assure the safety and quality of fresh-cut Fuji apples. J. Food Protect, 71:1150-1161. Reuben, A., Treminio, H., Arias, M. L., and Chaves, C. (2003) Presencia de Escherichia coli O157:H7, Listeria monocytogenes y Salmonella spp. en alimentos de origen animal en Costa Rica. Arch Latinoam Nutr 53:389–392. Richheimer, S. L., Bernart, M. W., King, G. A., Kent, M. C., Beiley, D. T. (1996). Antioxidant Activity of Lipid- Soluble Phenolic Diterpenes from Rosemary. J. Am. Oil Chem. Soc. 73(4):507-514.

Sajilata, M.G. and Singhal, R.S. (2012). Quality indices for spice essential oils Institute of Chemical Technology, India. In: the Handbook of herbs and spices Second edition, volume 1, pp.42-54. Woodhead Publishing. Sathaye, S., Bagul, Y., Gupta, S., Kaur, H. and Redkar, R. (2011). Hepatoprotective effects of aqueous leaf extract and crude isolates of Murraya koenigii against in vitro ethanol induced hepatotoxicity model. Exp. Toxicol. Pathol. 63(6): 587–591.

Sebiomo, A., Awofodu, A. D., Awosanya, A. O., Awotona F. E. and Ajayi A. J. (2011) Comparative studies of antibacterial effect of some antibiotics and ginger (Zingiber officinale) on two pathogenic bacteria. J. Microbiol. Antimicrob. 3(1):18-22.

75

Sema, A., Nursel, D. and Süleyman, A. (2007). Antimicrobial activity of some spices used in the meat industry. Bull Vet I Pulawy 51:53–57. Shahidi, F. and Marian, N. (2003) Phenolics in Food and Nutraceuticals; CRS Press LLC: Boca Raton, FL pp. 144-150.

Shan, B., Cai Y., Brooks J. D., Corke H. (2007). The in vitro antibacterial activity of dietary spice and medicinal herb extracts. Int. J. Food Microbiol. 117:112-119.

Shobana, S. and Akhilender, K. (2000). Antioxidant activity of selected Indian spices. Prostag. Leukotr. Ess. 62:107-110.

Singh, A. and Deep, A. (2011). Piperine : A Bioenhancer Review Article. Int J Pharm Res and Technol. 1(1):1-5.

Singh, G., Marimuthu, P., Catalan, C. and De Lampasona, M. P. (2004) Chemical, antioxidant and antifungal activities of volatile oil of black pepper and its acetone extract. J. Sci. Food Agric. 84(14):1878-1884.

Souza, E. L., Montenegro, Stamford, T. L. and Oliveira-Lima, E. (2006). Sensitivity of spoiling and pathogen food-related bacteria to Origanum vulgare L. (Lamiaceae) essential oil. Braz. J. Microb. 37:527–532. Souza, E. L., Montenegro, T. L., Lima, E., Trajano, V.N. and Barbosa, J. M. (2005). Antimicrobial Effectiveness of Spices: an Approach for Use in Food Conservation Systems Braz. Arch. Biol. Techn. 48(4):549-558.

Spices Board (2013). Ministry of Commerce & Industry, Goverment of India available online http://www.indianspices.com/ (assessed July 10, 2013)

Srinivasan K. (2007). Black pepper and its pungent principle-piperine: a review of diverse physiological effects. Crit Rev Food Sci Nutr. 47(8):735-48.

Stoilova, I., Krastanov, A., Stoyanova, A., Denev, P. and Gargova S.(2007) Antioxidant activity of a ginger extract (Zingiber officinale) Food Chem. 102(3):764-770.

Suthisut, D., Fields, P. G. and Chandrapatya, A. (2011). Contact Toxicity, Feeding Reduction, and Repellency of Essential Oils from Three Plants from the Ginger Family (Zingiberaceae) and their Major Components Against Sitophilus zeamais and Tribolium castaneum J. Econ. Entomol. 104(4):1445-1454.

Tajkarimi, M.M., Ibrahim, S. A., and Cliver, D. O. (2010). Antimicrobial herb and spice compounds in food. Food Control. 21 : 1199–1218. Tampieri, M. P., Galuppi, R., Macchioni, F., Carelle M. S., Falcioni L., Cioni, P.L. and Morelli, I. (2005). The inhibition of Candida albicans by selected essential oils and their major components. Mycopathologia 159: 339–345.

Tarté, R. (2009). Ingredients in Meat products: Propierties, functionality and Applications. In: Springer Science Business Media LLC page 301.

76

Trajano, V.N., Lima, E., Travassos, A. E. and De Souza, E. L. (2010). Inhibitory effect of the essential oil from Cinnamomum zeylanicum Blume leaves on some food-related bacteria. Ciênc. Tecnol. Aliment., Campinas, 30(3): 771-775. Tufail, M. (1990). Spices in Indian Economy. Academic Foundation. California University pp. 151.

Turgis, M., Han, J., Caillet, S., and Lacroix, M. (2009). Antimicrobial activity of mustard essential oil against Escherichia coli O157:H7 and Salmonella typhi. Food Control 20: 1073–1079. Vats, M., Singh, H. and Sardana S. (2011) Phytochemical screening and antimicrobial activity of roots of Murraya Koenigii (Linn) Spreng (Rutaceae). Braz. J. Microbiol. 42(4):1569-1573.

White, B. (2007). Ginger: An Overview. Am. Fam. Physician. 75(11): 1689-1691.

Winias, S., Retno, A., Magfiroh, R., Nasrulloh, M, R. and Rahayu R. (2011). Effect of cynammyldehyde from cinnamon extract as a natural preservative alternative to the growth of Staphylococcus aureus bacteria. J. Trop. Infect. Dis. 2:38-41.

Wojdyło, A., Oszmiański, J. and Czemerys R. (2007) Antioxidant activity and phenolic compounds in 32 selected herbs Food Chem. 105(3):940-949.

World Trade Organization (2012). World Trade Spices Exports by country. Retrieved by Nation Master available online http://www.nationmaster.com/graph/eco_wor_tra_exp_spi-economy-world-trade-exports-spices (assessed August 31, 2013).

77

1.3. Hypothèse et objectifs du projet

1.3.1. Hypothèse de l'étude

L'utilisation des épices peut être vue comme une alternative à l’utilisation des nitrates et

des nitrites pour la conservation des produits carnés, tout en garantissant les mêmes

propriétés anti-fongiques, antioxydantes et anti-bactériennes ainsi que la texture et les

propriétés organoleptiques allouées à ces produits chimiques de conservation, avec un

coût relativement abordable.

1.3.2. Objectif général

Plusieurs alternatives aux nitrites et nitrates ont été suggérées par des scientifiques à

savoir des produits chimiques comme le dioxyde de soufre, l’acide éthylène diamine

tétracétique, l’hydroxyanisole butylé, les esters fumarates, l’hypophosphite de sodium et

des produits naturels contenant les nitrites comme le céleri, la laitue, les épinards.

L’objectif principal de ce projet est de développer des alternatives vertes à l’utilisation

des nitrites et des nitrates comme agents de conservation dans les produits carnés

(jambon, terrines, produits saumurés, saucissons et produits fumés à chaud et à froid)

qui sont à la fois efficaces, économiques et sécuritaires pour la santé humaine étant

donné que l'on ajoute que des épices, tout en gardant la même durée de conservation et

les mêmes qualités organoleptiques, antibactériennes et antioxydantes que les nitrites.

1.3.2.1. Objectifs spécifiques

Spécifiquement, il s’agira de réaliser :

1) Un criblage qualitatif puis quantitatif des additifs alimentaires naturels de faible coût

comme les épices (la cannelle, les clous de girofles, le cumin, le poivre noir, l’ail et le

poivron rouge...) afin de sélectionner celles assurant une meilleure activité

antimicrobienne et antioxydante grâce à des analyses physico-chimiques et

microbiologiques.

78

2) Une optimisation des conditions de production et de traitement avant emballage des

produits carnés ainsi qu'une analyse sensorielle; L'optimisation se basera sur une

méthode de surfaces des réponses, un plan composite centré donnant 19 combinaisons

des trois meilleures épices criblées avec des concentrations allant de 0, 1 % à 0, 3%

(m/m).

3) Application des alternatives à l’échelle industrielle avec la réalisation d’une analyse

technico-économique qui prendra en compte l'ajout des épices dans nos viandes.

79

Chapitre 2 Use of spices as alternative of nitrites and nitrates in meat-based products

Anne Patricia Kouassi1,2, Fatma Gassara2, Satinder Kaur Brar2, Khaled

Belkacemi1

1Département des sols et de génie agroalimentaire, Pavillon Paul-Comtois, Université

Laval, 2325, rue de l'Université, Québec (Québec) G1V 0A6

2INRS-ETE, Université du Québec, 490, Rue de la Couronne, Québec, Canada G1K

9A9

(*Corresponding author, Phone: 1 418 654 3116; Fax: 1 418 654 2600; E-mail:

satinder.brar@ete.inrs.ca)

80

2.1. Résumé

L'activité antimicrobienne et antioxydante de certaines formulations d'épices utilisés

dans les produits à base de viande (les terrines) ont été étudiés. A cet effet, les valeurs

de l'indice de TBA et p-anisidine ainsi que les comptages totaux viables ont été réalisés

sur les terrines préservées avec ces formulations d'épices. Leurs activités

antimicrobiennes et anti-oxydantes sont comparées à celles des nitrites. Les résultats ont

montré que les clous de girofle, le cumin, les clous de girofle + cumin, la cannelle, les

clous de girofle + cannelle ont des valeurs de TBA et de p-anisidine inférieures à celles

des nitrites. De même, les terrines de neuf (9) formulations d'épices ont une durée de vie

égale, voire supérieure aux terrines contenant les nitrites (7 semaines). Par conséquent,

ces formulations pourraient être d'excellents agents de conservation, et ainsi empêcher

l'oxydation des lipides et prévenir la contamination microbienne des produits à base de

viande.

Mots-clés: formulations d'épices, nitrites, terrine, antimicrobien, antioxidant

81

2.2. Abstract

The antimicrobial and antioxidant activity of some spice formulations used in meat

products (terrines) were investigated. For this purpose, terrines were preserved using

these spice formulations and TBA index and p-anisidine values and total viable counts

were performed. The antimicrobial and antioxidant activities of these spices were

compared with those of nitrites. The results showed that cloves, cumin, cloves + cumin,

cinnamon, cinnamon + cloves formulations showed a TBA index and p-anisidine values

lower than those of nitrites. Similarly, eight (9) formulations of spices (cloves, cumin,

cloves + cumin, cinnamon, cloves + cumin, curry, red pepper, curry + cloves, red

pepper + cloves) gave a shelf life of terrine equal to or higher than the terrines of nitrites

(7 weeks). Hence, these formulations could be an excellent preservative that can prevent

the oxidation of lipids and prevent microbial spoilage of meat product. Spices can thus

provide an important nitrite-nitrate alternative for processed meats.

Key words: spice formulations, nitrites, terrine, antimicrobial, antioxidant

82

2.3. Introduction

Nitrates and nitrites are present everywhere in the environment. They are used as

fertilizers, explosives and preservative agents in food particularly against Clostridium

botulinum. They are natural chemical substances which are obtained from the oxidation

of nitrogen by the microorganisms in plants, soils or water. The toxic effects of nitrate

are attributed to its endogenous conversion to nitrite. At its 44th meeting (2002), JECFA

(Joint FAO/WHO Expert Committee on Food Additives) concluded that the range of

nitrate conversion is 5-7% for normal individuals and 20% for individuals with a high

rate of conversion. The acceptable daily intake (ADI)for nitrate is 0-3.7 mg/kg bw/day

(expressed as nitrate ions) (Thomson 2004). It can lead to risks to human health and the

environment. The health effect of most concern to the U.S. EPA for children is the

“blue baby syndrome” (methemoglobinemia) (Fan and others 1987). The blue baby

syndrome is named for the blue coloration of the skin of babies who have high nitrate

concentrations in their blood. The nitrate binds to hemoglobin (the compound which

carries oxygen in blood to tissues in the body), and results in chemically-altered

hemoglobin (methemoglobin) that impairs oxygen delivery to tissues, resulting in the

blue color of the skin (USEPA 2007).

At higher level, nitrates and nitrites have been associated with increased incidence of

cancer in adults, combining with secondary or tertiary amines to form N-nitroso

derivatives, and possible increased incidence of brain tumors, leukemia and

nasopharyngeal problems. Their addition in food is however very limited (USEPA,

2006). Industries use nitrates and nitrites for the stabilization of the red color of meats

(Honikel, 2008), inhibition of the development of toxic microorganisms, decreasing the

oxidation of lipids and to improve the flavor (Pegg and Shahidi, 2000). Nitrates and

nitrites are preferred as they are less expensive for the properties that they offer.

Meanwhile, the industries have not found a better and economical substitute to these

nitrites and nitrates. Due to the possibility of establishment of stricter regulations by

various countries, the industries are obliged to find greener substitutes to nitrates and

nitrites.

In this context, alternatives of nitrates and nitrites have been the subject of numerous

research studies (Stoilova et al., 2007). In literature, there are chemical agents, such

ascorbate and α–tocopherol, lactic-acid-producing organisms, potassium sorbate, or

83

treatments, such as irradiation (National Academy of Sciences, 1982) that have been

used as nitrite-nitrate substitutes. A number of studies have been carried out to

investigate the properties of aromatic herbs and spices, such as clove, ginger, pepper or

garlic (Menon and Garg, 2001). Cinnamaldehyde, the major constituent of cinnamon

(Cinnamomum cassia) has been reported to possess antibacterial activity and

antioxidant properties (Chen and Chang, 2001).

To the best of our knowledge, there is no systematic study on use of spices. With the

emergence of green alternatives and strict environmental regulations, this study is

important and aims at evaluating the best spices among 18 which can be used as

alternatives to nitrates and nitrites in terrines and ham, with chemical and

microbiological properties identical or better than nitrates and nitrites. The objective of

this study is the quantitative screening of the natural food additives, in order to choose

the best spices with good antioxidant and antimicrobial properties. Some

physicochemical properties of spices are listed in Table 2.1.

Table 2.1 Physicochemical properties of spices

Spice Physic-chemical properties References

Clove carvacrol, thymol, eugenol, cinnamaldehyde Chaieb et al., 2007

Coriander

linalool ,oxygenated mono terpenes

,monoterpene hydrocarbons

Coriander seed: 60%-70% linalool 20 %

hydrocarbons

Essential oil of leaves and fruits: 2-decenoic

acid (30.8 %), E-11-tetradecenoic acid

(13.4 %), capric acid (12.7 %), undecyl alcohol

(6.4 %), tridecanoic acid (5.5 %),

undecanoic acid (7.1 %)

Coleman and Lawrence,

1992

Leung and Foster, 1996

Guenther, 1950

Bhuiyan et al., 2009

cinammon Leaves oil: eugenol (76.10 %), trans-β

caryophyllene (6.7 %), linalool (3.7 %), eugenol

acetate (2.8 %) benzyl benzoate (1.9 %).

Branches oil: linalool (10.6 %), α-pinene (9.9

%), α-phellandrene (9.2 %)

Trajano et al., 2010

Lima et al., 2005

84

Red pepper Ascorbic acid 108.65±6.481 (mg/100 g DW)

Licopene 123.89±0.170 (mg/ Kg DW)

Carotene 2282.45±5.362 (mg/ Kg DW)

Total phenols 89.82±4.721( mg GAE g DW -1)

Ozgur et al, 2011

Garlic total phenolic compounds (6.5 mg GAE/g of

dw)

Caffeic acid ( 2.9 mg/kg of dw)

Ferulic acid (2.6 mg/kg of dw)

vanillic, p-hydroxybenzoic,

p-coumaric acids

Beato et al., 2011

Ginger

Phenols (5.69 %)

Oleoresin content (2.93 %)

Eleazu and Eleazu, 2012

Curry Antioxidant activity: Koenigine; Mukonicine;

Mahanimbinine; Murrayacinine;

Mahanimboline; Isomahanine

Antimicrobial activity : Murrayanol;

Mahanimboline; Mahanimbinine;

Murrayacinine

Ganesan et al., 2013

Cumin Total phenolic compounds (24.66 mg GAE/g)

α-pinene (0.5%), Myrcene (0.3%), limonene (0.5%), 1-8-cineole (0.2%), p-menth-3-en-7-ol (0.7%), p-mentha-1, 3-dien-7-ol (5.6%), caryophyllene (0.8%), β-bisabolene (0.9%), β-pinene (13.0%), P-cymene (8.5%), β-phellandrene (0.3%), D-terpinene (29.5%), cuminic aldehyde (32.4%), cuminyl alcohol (2.8%), β-farnesene (1.1%)

Nadeem and Riaz., 2012

2.4. Materials and methods

2.4.1. Preparation of meat samples

Samples of 200g of terrines were prepared using simple recipe of terrine of pork and

rabbit, by adding 1% w/w of various spices to be tested. The list of spices tested is

presented in details in Table 2.2. Samples were cooked in a microwave oven (Danby,

Quebec, Canada; power= 700 watts) for 30 min and then cooled in a refrigerator for 2

85

days. Each terrine was cut into several pieces using sterilized knife to avoid cross-

contamination of samples and placed under vacuum by using a vacuum food storage

system. Terrines were stored in the refrigerator during 8 weeks and samples were taken

for the analysis every week.

2.4.2. Determination of thiobarbituric acid (TBA).

The lipid oxidation was determined by 2-thiobarbituric acid according to the procedure

of Schmedes and Holmer (1989). Terrine sample (2g) was mixed with 5 ml of

trichloroacetic acid (TCA) solution (200 g/l of TCA in 135ml/l of phosphoric acid

solution) and homogenized in a blender for 30 s. After filtration with a filter paper (0.45

µm), 2 ml of the filtrate was mixed with 2ml of a solution of TBA (3g/l) in a test tube.

The tubes were incubated at room temperature in the dark for 20h. The absorbance was

measured at 532 nm using UV-Vis spectrophotometer (model UV-1200, Shimadzu,

Kyoto, Japan). TBA value was expressed as mg malonaldehyde per kg of sample and

determined by the following equation 1:

10-2 (1)

Where, A532nm is the measured absorbance, VTCA denotes the extraction solvent volume

(5ml), M is the molar mass of malonaldehyde (72g/mol) and m is the mass of the

analyzed sample (2g). TBA value was measured in triplicates and means value are

determined.

Table 2.2 List of formulations

Formulations Spices Concentration

1 Nitrite 1 % w/w

2 Ginger 1 % w/w

3 Black Pepper 1 % w/w

4 Cumin 1 % w/w

5 Coriander 1 % w/w

6 Garlic 1 % w/w

7 Cinnamon 1 % w/w

8 Curry 1 % w/w

9 Chili 1 % w/w

10 Grapes 1 % w/w

86

11 Cloves 1 % w/w

12 Cloves 1 % w/w

Ginger 1 % w/w

13 Cloves 1 % w/w

Black Pepper 1 % w/w

14 Cloves 1 % w/w

Cumin 1 % w/w

15 Cloves 1 % w/w

Coriander 1 % w/w

16 Cloves 1 % w/w

Cinnamon 1 % w/w

17 Cloves 1 % w/w

Curry 1 % w/w

18 Cloves 1 % w/w

Red Pepper 1 % w/w

2.4.3. Determination of p-Anisidine value

The hydroperoxide decomposition was determined by p-anisidine value (p-Anv)

according to IUPAC standard method (1986). Terrine sample (0.5g) was mixed with

5ml of isooctane solution. The absorbance (Ab) of this sample was measured at 350 nm

using isooctane solution as a blank reagent. Later, 1ml of p-anisidine solution (0.25

(v/v) % in glacial acetic acid) was added to the sample tube and to 5ml of isooctane test

tube. After 10 minutes, the absorbance (As) of samples was read using isooctane as

blank. The p-anisidine values were calculated using the following equation 2:

(2)

Where, As is the absorbance of the fatty solution after reaction with

p-anisidine solution, Ab is the absorbance of the fatty solution and m is the sample mass

(g).

87

2.4.4. Microbiological analysis

Total viable count (TVC) of bacteria is one of the most important indexes in evaluation

of quality and safety of meat products. The total viable count of bacteria (TVC) ufc/g on

meat product sets a limit to its shelf-life. Meat will “spoil” with TVC at 107/g because

of off-odours. Slime and discoloration appear at 108/g. The main factors determining the

time taken for the TVC to reach these levels are the initial count due to contamination

during slaughtering and processing, further contamination during storage, temperature,

pH, relative humidity and food preservative added during process.

To estimate the TVC, samples (1 g) from meat were weighed aseptically, chopped and

harvested properly and then added to 25 ml NaCl sterile solution, 0.9 % v/w (25 ml),

and homogenized for 60 s at room temperature. Decimal dilutions in NaCl sterile

solution (0.9 % v/w) were plated on plate count agar (PCA; Quelab Laboratories Inc.,

Montreal, QC, CANADA) and incubated at 30 °C for 48 h for enumerating total viable

counts (TVC). Enumeration of TVC was performed on these duplicate samples and

results are displayed as the mean of both measurements. Each sample was analyzed in

duplicate (coefficient of variation of samples from the same experiment).

2.4.5. Statistical analysis

All the experiments were assayed in replicates and an average of 3 replicates was

calculated along with the standard deviation. Data means were analyzed using

individual Student's t-tests to distinguish differences among spices formulations. The

test was performed at the level of P-value < 0.05 to determine the significance of the

difference between spices formulation for meat products preservation (SPSS 1999).

Standard errors and error bars presented in the tables and figures, respectively were

calculated using untransformed data in ANOVA. Standard error for a single proportion

was estimated as described by Gottelli and Ellison (2004).

2.5. Results and discussion

2.5.1. Determination of p-Anisidine value

88

Generally, it is possible to measure either the precursors of oxidation, such as free

radicals, hydroperoxides, or oxidation products, such as aldehydes or ketones. The p-

anisidine value essentially measures 2-alkenal. In the presence of acetic acid, p-

anisidine reacts with aldehydes producing a yellowish color and an increase in

absorbance when the aldehyde contains a double bond. The p-anisidine value is

dependent on the oxidation of fatty acids including polyunsaturated fatty acids. The p-

anisidine values measured during 8 weeks of meat product storage at 4°C are presented

in Fig. 2.1. For all the formulations tested, the value of p-anisidine increased during

storage time. This is due to the growth of lipolytic bacteria that can oxidize fatty acids

during storage. In addition, the value of p-anisidine was dependent on the spice

formulations used in terrines. From the results shown in Fig.2.1, the values of p-

anisidine in terrines preserved using cloves, cumin, cinnamon, cinnamon + cloves were

comparable and sometimes lower than the terrines conserved using nitrite (150 ppm).

This is explained by the richness of these spices with polyphenols that protect against

fatty acids oxidation contained in terrine (Kim and others 2011). The results of p-

anisidine values were consistent with the values of TBA index that showed that these

formulations had excellent antioxidant activities. Hence, cloves, cumin, cinnamon, and

the mix of these formulations could be excellent preservatives that prevent the oxidation

of lipid contained in meat products, such as terrine, bacon, salami, ham, among others.

Figure 2.1 p-anisidine values of terrines preserved using different spice formulations at

4°C for 8 weeks

0

20

40

60

80

100

120

P an

isid

ine

inde

x

5 week6 week7 week8 week

89

2.5.2. Determination of TBA index

Polyunsaturated fatty acids, such as linoleic acid are easily oxidized by the oxygen in

the air. This auto-oxidation leads to the occurrence of chain reactions with the formation

of coupled double bonds, and at a later stage also to obtain secondary products, such as

aldehydes, ketones, and alcohols. In order to assay the antioxidant effect of the spices

formulations, the experiments for inhibiting the peroxidation of the polyunsaturated

fatty acids were performed. During storage of meat products at 4°C for two months, the

lipids are oxidized and malondialdehyde (MDA), a secondary component fatty acids is

formed due to the degradation of polyunsaturated fatty acids. TBA index is able to

measure the formation of malondialdehyde. The results of the analysis of the TBA

index are presented in Fig 2.2. Black pepper, black pepper + cloves and coriander

showed a weaker effect in inhibiting lipid peroxidation. Black pepper formulation used

in this study is a commercial formulation that contains starch. Starch could be used by

microorganisms as carbon source to grow, to damage meat products by the oxidation of

lipid compounds. The presence of starch in black pepper formulation could be the cause

of weaker antioxidant activity of these formulations. Moreover, the total flavonoid

content in decreasing order was: thyme > rosemary > marjoram and oregano > basil >

cumin > clove > caraway and fennel > savory > turmeric, mace and coriander (p <

0.001) (Kim and others 2011). A significant correlation was observed between

antioxidant activity and phenol content in the plant foods studied (IJFSN 2007). Hence,

the coriander has a weaker antioxidant activity due its low content on polyphenolic

compounds. Most efficient spice formulations to inhibit lipid oxidation were cloves,

cumin, cinnamon, cumin + cloves, cinnamon + cloves. This was due to the high total

phenolic content in cloves (108.28 μg catechin equivalent (CE)/g) (Kim and others

2011), cinnamon (Muchuweti and others 2007) and cumin. Moreover, the DPPH

radical scavenging ability of these spices extracts were very important (Kim and others

2011). 2,2-diphenyl-1-picrylhydrazyl (DPPH) is widely used to test the ability of

compounds to act as free radical scavengers or hydrogen donors, and to evaluate

antioxidant activity of foods ( Frankel and Meyer 2000). In addition, a significant and

linear relationship existed between the DPPH scavenging activity and phenolic content

indicating that phenolic compounds are major contributors to antioxidant activity (Kim

and others 2011). Hence, the most efficient antioxidant activities of these spice

90

formulations were due to the high phenol content that prevented the polyunsaturated

fatty acids oxidation and the release of malonaldehyde.

Figure 2.2 TBA index for terrines preserved using different spice formulations at 4°C

for 8 weeks

0

1

2

3

4

5

6

7

TBA

Inde

x (m

g M

A/k

g)

1 week 2 week 3 week 4 week

0

2

4

6

8

10

12

TBA

Inde

x (m

g M

A/kg

)

5 week 6 week

7 week 8 week

91

2.5.3. Microbiological analysis

The enumeration of total viable counts (TVC) in terrines preserved by different

formulations of spices during 8 weeks was performed and the results of this study were

presented in Fig. 2.3. Storage time, spices formulations used for terrines preservation

and meat type had a statistically significant (P > 0.05) effect on the development of the

microbial groups determined in this study. Large microbiological changes due to

different biomass developments in the terrines resulted in higher standard deviations of

bacterial counts. TVC values will be used to determinate the shelf life of terrine. A meat

product is non-consumable if the TVC is greater than 107 UFC/g (ICMSF 1986).

Through its antimicrobial and antioxidant properties, nitrite is able to extend the shelf-

life of meat products (Jackson 2010). The results of this study have shown that the shelf

life of terrine preserved using 150 ppm of nitrates was 7 weeks. Terrines preserved by

black pepper had the shortest shelf life (5 weeks). Black pepper did not show any

antimicrobial activity. However, black pepper showed a good antimicrobial activity in

previous studies (Ghori and Ahmad 2009). The weaker antimicrobial activity of black

pepper found in our study could be due to the presence of starch in black pepper

formulations that enhanced microbial growth. Terrines preserved by, cloves (>8 weeks),

cumin (8 weeks), cloves + cumin (>8 weeks), cinnamon (7 weeks), cloves + cumin (8

weeks), curry (7 weeks), red pepper (>8 weeks), curry + cloves (8 weeks), red pepper +

cloves (>8 weeks) had a shelf life equal or higher than terrines preserved by nitrites.

These spice formulations have good antimicrobial activities compared to nitrites. The

antimicrobial potency of plants and spices is believed to be due to tannins, saponins,

phenolic compounds, essential oils and flavonoids (Aboaba and Efuwape 2001).

Moreover, naturally occurring compounds in spices, such as sulphur compounds,

terpenes and terpene derivatives, phenols, esters, aldehydes, alcohols and glycosides

have been shown to exhibit antimicrobial functions (Russel and Gold 1991; Davidson

and Baren 1993; Deis 1999). However, the inhibitory effects of spices are mostly due to

the volatile oils present in their composition (Arora-Dlijit and Kaur 1999). The result of

this study showed that cumin, cloves, cinnamon, red pepper and curry had good

antimicrobial activities. These results were also well correlated with the results of

Rahman and others (2010). Rahman and others (2010) found that cinnamon, cloves and

cumin were found to have important antimicrobial activity against Staphylococcus

aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis,

92

Micrococcus luteus, Escherichia coli and Candida albicans. In this regard, the use of

these spices and their volatile compounds as natural preservatives in food products can

offer an alternative to the use of chemical additives, such as nitrites.

Figure 2.3 Total viable counts for terrines preserved using different spice formulations

at 4°C for 8 weeks

1,E+00

1,E+01

1,E+02

1,E+03

1,E+04

1,E+05

1,E+06

1,E+07

black p

epper

red pep

per

ginger

clove

scu

min

cinnam

oncu

rry

coria

ndergarl

ic

red pep

per + c

loves

cumin +

clove

s

grapes

cinnam

on + clo

ves

coria

nder + c

loves

black

pepper

+clove

s

curry

+ clove

s

ginger+ cl

oves

Nitrite

s

Formulations

TVC

(UFC

/g)

1 week 2 week 3 week 4 week

1,0E+001,0E+011,0E+021,0E+031,0E+041,0E+051,0E+061,0E+071,0E+081,0E+091,0E+101,0E+11

black p

epper

red pep

per

ginger

clove

scu

min

cinnam

oncu

rry

coria

ndergarl

ic

red pep

per + c

loves

cumin +

clove

s

grapes

cinnam

on + clo

ves

coria

nder + c

loves

black

pepper

+clove

s

curry

+ clove

s

ginger+ cl

oves

Nitrite

s

TVC

(ufc

/g)

5 week 6 week 7 week 8 week

93

2.6. Discussion

In view of the results obtained for the antimicrobial and antioxidant activity of some

spices formulations and their use in meat products (terrines) preservation, following

conclusions can be drawn:

1) The values of p anisidine in terrines preserved using cloves, cumin, cinnamon,

cinnamon + cloves were comparable and sometimes lower than nitrite conserved

terrines (150 ppm).

2) Spices formulations that gave lowest TBA index were cloves, cumin, cinnamon,

cumin + cloves, cinnamon + cloves.

3) Terrines preserved by cloves (>8 weeks), cumin (8 weeks), cloves + cumin (>8

weeks), cinnamon (7 weeks), cloves + cumin (8 weeks), curry (7 weeks), red pepper (>8

weeks), curry + cloves (8 weeks), red pepper + cloves (>8 weeks) had a shelf life equal

or higher than terrines preserved by nitrites.

4) The use of cloves, cumin cinnamon spices and their volatile compounds as natural

preservatives in food products may be an alternative to the use of chemical additives,

such as nitrites.

2.7. Acknowledgements

The authors sincerely thank the FQRNT (programme special) in collaboration with four

adro-industries, MDEIE and MAPAQ for their financial support. We express our

gratitude to Mr. Boiteau of Aliments Breton for supplying us the raw meat to carry

out various tests. The views and the opinions expressed in this article are those of the

authors.

94

2.8. References

Aboaba O, Efuwape BM. 2001. Antibacterial Properties of Some Nigerian Species. Biol Res Commun 13:183-188. Arora-Daljit S, Kaur J. 1999. Antimicrobial activity of spices. J Antimicrob Agents 12: 257-262. Chang ST, Chen PF and Chang SC. 2001. Antibacterial activity of leaf essential oil and their constituents from Cinnamomum osmophloeum. J Ethopharmacol 77:123-127. Davidson PM, Baren AL. 1993. Antimicrobials in Foods. Marcel Dekker, New York. pp. 1-9 Deis RD. 1999. Secret world of spices. Food Product Design 5:1-7. Fan AM, Willhite CC, and Book SA. 1987. Evaluation of the nitrate drinking water standard with reference to infant methemoglobinemia and potential reproductive toxicity. Regul Toxicol Pharmacol 7(2):135–148. Frankel EN, Meyer AS. 2000. The problems of using one-dimensional methods to evaluate multifunctional food and biological antioxidants. J. Sci. Food Agric 80:1925–1941. Ghori I, Ahmad S S. 2009. Antibacterial activities of honey, sandal oil and black pepper. Pak. J. Bot 41:461-466, 2009. Gotelli N J, Ellison AM. 2004. A Primer of Ecological Statistics Sinauer Associates. Sunderland, MA, 510 pp. Honikel KO. 2008. The use and control of nitrate and nitrite for the processing of meat products. Meat Sci 78:68-76. International Commission on Microbiological Specifications for foods Sampling plans for fish and shellfish.1986. In: ICMSF (Eds.), Microorganisms in Foods. Sampling for Microbiological Analysis, Principles and Scientific Applications.Vol. 2, (2nd ed.). University of Toronto Press, Toronto, Canada. Jackson AL. 2010. Investigating the microbiological safety of uncured no nitrate or nitrite added processed meat products. Graduate Theses and Dissertations. Iowa State University. JUPAC.1986. Standard Methods for the Anal vsis of Oils, Fats and Derivatives, 7th ed. Oxford, Blackwell Scientific Publications. Kim IS, Yang MR, Lee OK, Suk-Nam Kang SN. 2011. Antioxidant Activities of Hot Water Extracts from Various Spices. Int J Mol Sci 12:4120-4131.

95

Menon KV, Garg SR. 2001. Inhibitory effect of clove oil on Listeria monocytogenes in meat and cheese. Food Microbiol 18:647-650. Muchuweti M, Kativu E, Mupure CH, Chidewe C, Ndhlala AR and Benhura MAN.2007). Phenolic composition and antioxidant properties of some spices. Am J Food Technol 2:414-420. National Academy of Sciences, Assembly of Life Sciences.1986. Alternatives to the current use of nitrite in foods. National Academy Press, Washington, D. C., p 1-3 through 1-9. Pegg RB, Shahidi F. 2000. Nitrite curing of meat. The n-nitrosamine problem and nitrite alternatives. Trumbull, CT: Food and Nutrition Press, Inc. Rahman MSA, Thangaraj S, Salique SM, Khan KF, and Natheer SE. 2010. Antimicrobial and Biochemical Analysis of Some Spices Extract against Food Spoilage Pathogens. Int J Food Nutr Saf 12:71-75. Russel RJ, Gould GW. 1991. Food Preservatives. Van Nostrand Reinhold Co., New York. Schmedes A, Holmer GA. 1989. New thiobarbituric acid (TBA) method for determination of free malonaldehyde (MDA) and hydroperoxides selectivity as a measure of lipid peroxidation. J Am Oil Chem Soc 66:813–817. Stoilova I, Krastanov A, Stoyanova A, Denev P, and Gargova S. 2007. Antioxidant activity of a ginger extract (Zingiber officinale). Food Chem:102: 764-770. SPSS Base 10.0 Application Guide SPSS, Chicago, IL (1999) Thomson B. 2004. Nitrates and nitrites dietary exposure and risk assessment. Available from: http://www.nzfsa.govt.nz/consumers/food-safety-topics/chemicals-infood/ residues-in-food/consumer-research/nitrite-nitrate-report.pdf. Accessed 2012 USEPA. 2007. National Water Quality Inventory: Report to Congress; 2002 Reporting Cycle. Document No. EPA-841-R-07-001. Washington, D.C: U.S. Environmental Protection Agency U.S. Environmental Protection Agency Ground Water and Drinking Water. 2006. Consumer Factsheet on Nitrates/Nitrites. Available from : http://www.epa.gov/safewater/dwh/c-ioc/nitrates.html. Accessed 2012

97

Chapitre 3 Optimization of spices as alternative of nitrites and nitrates in the meat-based products

Fatma Gassara1, Anne Patricia Kouassi2, Satinder Kaur Brar1*, Khaled

Belkacemi2

1INRS-ETE, Université du Québec, 490, Rue de la Couronne, Québec, Canada G1K

9A9

2Département des sols et de génie agroalimentaire, Pavillon Paul-Comtois, Université

Laval, 2325, rue de l'Université, Québec (Québec) G1V 0A6

(*Corresponding author, Phone: 1 418 654 3116; Fax: 1 418 654 2600; E-mail:

satinder.brar@ete.inrs.ca)

98

3.1. Résumé

L'utilisation des clous de girofle, du cumin et de cannelle contre l'oxydation des lipides

et la contamination microbienne a été étudiée par la méthode de réponse de surface sur

les échantillons de jambon et terrines en considérant l'effet des concentrations de ces

trois épices sur les propriétés physico-chimiques (indice de TBA et de la valeur de p-

anisidine) et les propriétés microbiologiques (TVC). Parmi elles, la concentration de

clous de girofle et de cumin ont eu un effet positif significatif sur les propriétés

microbiennes et les propriétés physico-chimiques pour les échantillons de viande (p

<0,05). Cependant, la concentration de la cannelle n'a pas eu d'effet significatif sur la

TVC, la TBA et les valeurs de p-anisidine dans les produits à base de viande (p> 0,05).

Les clous de girofle et le cumin ont joué un rôle important dans la prévention de la

croissance microbienne et l'oxydation des lipides et peuvent servir d'alternatives

intéressantes aux nitrites-nitrates dans les viandes transformées.

Mots-clés: clous de girofle, cumin, cannelle, réponse de surface, oxidation des lipides,

altération microbienne

99

3.2. Abstract

The use of three spices (cloves, cumin, and cinnamon) in the preservation of meat

products from lipid oxidation and microbial spoilage was investigated through response

surface method by using ham and terrine. The effect of concentration of cloves,

concentration of cumin and concentration of cinnamon on the physico-chemical

properties (TBA index and p-anisidine value) and microbiological properties (TVC) of

ham and terrine process were thoroughly investigated. Among them, the respective

concentration of cloves and cumin had significant positive effect on microbial (total

viable cells, TVC) and physico-chemical properties for terrine and ham (p < 0.05).

However, the concentration of cinnamon did not have a significant effect on TVC, TBA

and p-anisidine values in meat products (p>0.05). Thus, cloves and cumin played an

important role in the prevention of microbial growth and lipid oxidation and can serve

as good alternatives to nitrites-nitrates in processed meats.

Keywords: cloves, cumin, cinnamon, response surface, lipid oxidation, microbial

spoilage.

100

3.3. Introduction

Nitrites are present in environment, fertilizers, plants and soils. Nitrates and nitrites are

chemical substances also used in preservation of food. Industries use nitrates and nitrites

for the stabilization of the red color of meats (Honikel, 2008), inhibition of the

development of toxic microorganisms, by decreasing the oxidation of lipids and to

improve the flavor (Pegg & Shahidi, 2000). At higher level, nitrates and nitrites have

been associated with increased incidence of cancer in adults, combining with secondary

or tertiary amines to form N-nitroso derivatives, and possible increased incidence of

brain tumors. For all these reasons and also due to governmental restrictions, adoption

and research of a green alternative such as the addition of spice instead of nitrites, has

been carried out. It seemed important to investigate the properties of aromatic herbs and

spices, such as clove, ginger, pepper or garlic (Menon & Garg, 2001). It has been

mentioned that spices, such as cinnamon could be efficient in treatment of cancer (Ka et

al, 2003) because of its antioxidant properties (Lin et al, 2003; Okawa et al, 2001; Toda,

2003). Despite the usefulness and important physical-chemical properties of the spices,

no systematic study has been carried out so far on their use as nitrite alternatives, to the

best of our knowledge.

In this context, a qualitative analysis was carried out with terrines and ham, and three

spices were selected based on the physical-chemical and microbiological properties

imparted by them (Tajkarimi, 2010; El-Daly, 1998 ; Kamali et al., 1998; Delespaul et

al., 2000; Chang et al., 2001). In order to quantify them, the application of statistical

methodologies is helpful in defining the effects and interactions of the physiological

factors that play a role in biotechnological processes, such as in agro food production.

Response Surface Methodology (RSM) is a collection of statistical and mathematical

techniques useful for developing; improving and optimizing processes. There are

different types of RSM designs, such as 3-level factorial design, central composite

design (CCD) (Boza et. al, 2000) which was used in this study, Box-Behnken design

(Singh et. al, 1995, and D-optimal design (Sanchez-Lafuente et. al, 2002).

The aim of the present study is to evaluate the influence of the different spices which

have been chosen for the quantitative analysis using response-surface method. Response

surface methodology has been utilized to optimize the quantity of spices in terrines and

101

ham in order to find a better alternative to nitrites using the optimum quantity of

spices.To the best of our knowledge, there is no study reported on optimization, using

an experimental design, of the quantity of spices for the replacement of nitrites in meat

based product. The design used in this study is CCD which is a first-order (2N) design

augmented by additional centre and axial points to allow estimation of the tuning

parameters of a second-order model; the parameters used was p-anisidine value, TBA

index and TVC value.

3.4. Materials and methods

3.4.1. Preparation of meat extract

Samples of 200g of terrines were prepared using simple recipe of terrine of pork and

rabbit, by adding a percentage, as provided by STATISTICA 6 of STATSOFT Inc.

(Thulsa, U.S.), of various spices to be tested. Samples were cooked in a microwave for

6 min and then cooled in a refrigerator for 2 days. Each terrine was cut into several

pieces and placed under vacuum by using a vacuum food storage system.

Samples of pork were also prepared as ham with different concentrations of spices. The

respective concentrations are provided in Table 3.1.

102

Table 3.1 Results of experimental plan by central composite design for ham and terrine

3.4.2. Determination of thiobarbituric acid

The lipid oxidation was determined by 2-thiobarbituric acid according to the procedure

of Schmedes & Holmer (1989). Terrine sample (2g) was mixed with 5 ml of

trichloroacetic acid (TCA) solution (200 g / l of TCA in 135 ml / l of phosphoric acid

solution) and homogenized in a blender for 30 s. After filtration with a filter paper (0.45

µm), 2ml of the filtrate was mixed with 2ml of a solution of TBA (3g/l) in a test tube.

The tubes were incubated at room temperature in the dark for 20h. The absorbance was

measured at 532 nm using UV-Vis spectrophotometer (model UV-1200, Shimadzu,

Trial X1 X2 X3 log10 (TVC) p anisidine TBA (mg MDA/kg)

Cloves Cumin Cinnamon Terrine Ham Terrine Ham Terrine Ham

1 0.1 0.1 0.1 8.28 8.86 2.10 1.97 1.72 1.230

2 0.1 0.1 0.3 7.02 8.80 1.85 1.59 1.44 1.400

3 0.1 0.3 0.1 6.16 7.47 1.10 1.13 0.71 0.450

4 0.1 0.3 0.3 5.56 7.45 1.34 1.25 0.68 0.532

5 0.3 0.1 0.1 4.82 7.83 0.97 0.83 0.59 0.286

6 0.3 0.1 0.3 5.62 7.74 1.01 0.91 0.65 0.572

7 0.3 0.3 0.1 2.30 6.99 0.32 0.61 0.37 0.226

8 0.3 0.3 0.3 3.85 7.47 0.44 0.74 0.55 0.134

9 0.031 0.2 0.2 5.75 8.47 1.74 1.66 0.86 0.917

10 0.368 0.2 0.2 2 7.37 0.78 0.7 0.47 0.201

11 0.2 0.031 0.2 6.15 9.04 1.4 1.59 0.88 1.212

12 0.2 0.368 0.2 2 7.10 0.6 0.77 0.49 0.255

13 0.2 0.2 0.031 3.17 7.93 0.90 0.85 0.65 0.315

14 0.2 0.2 0.368 5.77 9.01 1.23 0.99 0.67 0.457

15 0.2 0.2 0.2 4.19 8.11 1.27 0.95 0.74 0.302

16 0.2 0.2 0.2 4.54 8.13 1.24 0.94 0.70 0.348

17 0.2 0.2 0.2 4.86 8.06 1.23 0.96 0.71 0.314

18 0.2 0.2 0.2 4.23 8.14 1.21 0.9 0.72 0.316

19 0.2 0.2 0.2 4.65 8.14 1.23 0.96 0.73 0.311

103

Kyoto, Japan). TBA value was expressed as mg malonaldehyde per kg of sample and

determined by the following equation 1:

10-2 (1)

Where, A532nm is the measured absorbance, VTCA denotes the extraction solvent volume

(5ml), M is the molar mass of malonaldehyde (72g/mol) and m is the mass of the

analyzed sample (2g).

3.4.3. Determination of p-anisidine value

The hydroperoxide decomposition was determined by p-anisidine value (p-Anv)

according to IUPAC standard method (1986). Terrine sample (0.5g) was mixed with

5ml of isooctane solution. The absorbance (Ab) of this sample was measured at 350 nm

using isooctane solution as a blank reagent. Later, 1ml of p-anisidine solution (0.25

(v/v) % in glacial acetic acid) was added to the sample tube and to 5ml of isooctane test

tube. After 10 minutes, the absorbance (As) of samples was read using isooctane as

blank. The p-anisidine values were calculated using the following equation 2:

(2)

Where, As is the absorbance of the fatty solution after reaction with

p-anisidine solution, Ab is the absorbance of the fatty solution and m is the sample mass

(g).

3.4.4. Microbiological analysis

Samples (1 g) from meat were weighed aseptically, added to 25 ml NaCl sterile

solution, 0.9 % v/w (25 ml), and homogenized for 60 s at room temperature. Decimal

dilutions in NaCl sterile solution (0.9 % v/w) were plated on plate count agar (PCA;

Quelab Laboratories Inc., Montreal, QC, CANADA) and incubated at 30 °C for 48 h for

enumerating total viable count (TVC). Enumeration of TVC was performed on these

duplicate samples and results are displayed as the mean of both measurements. Each

sample was analyzed in duplicate (coefficient of variation of samples from the same

experiment).

104

3.4.5. Statistical analysis

Data means were analyzed with individual Student's t-tests to distinguish differences

among spice formulations. The test was performed at P-value < 0.05 to determine the

significance of the difference between spices formulation for meat products preservation

(SPSS, 1999). Standard error for a single proportion was estimated as described by

Gottelli & Ellison (2004).

3.4.6. Experimental design and optimization

In order to identify the significant factors that affect the responses, an attempt was

made to improve the quantity of spices comparing different levels of several factors that

were found to have more influence on the antioxidant and antimicrobial properties of

spices in meat based products. The impact of three independent quantitative variables,

including cloves (X1), cumin (X2) and cinnamon (X3), was evaluated by a factorial

central composite design (CCD) to find the optimal concentrations of these three

factors. In this regard, a set of 19 experiments including, seven center points or

repetitions, six axial points (α = 1.68) and 8 points corresponding to a matrix of 23

which incorporates 8 experiments including 3 variables (+1, -1, 0), were carried out.

Each variable was studied at three different levels (−1, 0, +1) and center point (0) which

is the midpoint of each factor range. The minimum and maximum range of variables

investigated and the full experimental plan with respect to their actual and coded values

are listed in Table 3.2. A multiple regression analysis of the data was carried out by

STATISTICA 6 of STATSOFT Inc. (Thulsa, U.S.) by surface response methodology

and the second-order polynomial equation that defines predicted responses (Yi) in terms

of the independent variables (X1, X2, and X3):

Yi = b0i + b1iX1 + b2iX2 + b3iX3 + b11iX2+ b22iX2 + b33iX2 +b12iX1X2 +

b23iX2X3 + b13iX1X3 (1)

Where, Yi = predicted response, boi is intercept term, b1i, b2i, b3i linear coefficients,

b11i, b22i, b33i squared coefficients and b12i, b23i, b13i are interaction coefficients

and i refer to the response. Combination of factors (such as X1X2) represents an

interaction between the individual factors in the respective term. There are 3 different

105

responses, TBA index p-anisidine value and TVC value. These responses are a function

of the level of factors. The response surface graphs indicate the effect of variables

individually and in combination and determine their optimum levels for quantity of each

spice.

Table 3.2 Experimental range of the three variables studied using CCD in terms of

actual and coded factors Variables Symbol Coded levels

−1.682 Low (−1) Mid (0) High (1) +1. 682

Cloves (% w/w) X1 0.031 0.1 0.2 0.3 0.368

Cumin (% w/w) X2 0.031 0.1 0.2 0.3 0.368

Cinnamon(%w/w) X3 0.031 0.1 0.2 0.3 0.368

3.5. Results and discussion

3.5.1. Effect of variables on physico-chemical properties of processed meat

The central composite design was used to find the suitable values of the variables on the

physico-chemicals properties of meat products: terrine and ham. The results of CCD

experiments consisted of experimental data for studying the effects of three independent

variables; concentration of cloves, concentration of cumin and concentration of

cinnamon on the physico-chemical properties (TBA index and p-anisidine value) and

microbiological properties (TVC) of ham and terrine are presented in Table 1. The

physico-chemical properties (TBA index and p-anisidine value) and microbiological

properties (TVC) exhibited different responses in the two meat products assayed,

namely terrine and ham. The data were fitted into a second-order polynomial function

(Eq. (1)). Linear, quadratic and interaction coefficients of variables under study that

were found to be significant at p < 0.05 were retained in reduced models. Data were

best fitted by a first-order polynomial equation as it can be inferred from the good

agreement of experimental data with those predicted by the model. The quality of the

model fit was evaluated by the coefficient R2 and its statistical significance was

determined by an F-test. R2 represents the proportion of variation in the response data

106

that can be explained by the fitted model. High R2 was considered as an evidence for the

applicability of the model in the range of variables included. It should be noted that a R2

value greater than 0.75 indicates the aptness of the model. The coefficients of

determination (R2) are presented in Table 3.3. In all models, R2 is higher than 0.84 and

indicated that the model fitted well into the experimental results. The analysis of

variance (Table 3) indicated that the linear model terms of X1(the concentration of

cloves) and X2 (the concentration of cinnamon) had significant positive effect on the

physico-chemicals (TBA index and p-anisidine value) in both terrine and ham. The

values of linear coefficient related to X1 and X2 were negatives, hence when X1 and X2

increased, p anisidine, TBA values decreased. TBA index is a physico-chemical

parameter that is able to measure the formation of malondialdehyde, a secondary

product that is formed due to the degradation of polyunsaturated fatty acids.

Polyunsaturated fatty acids, such as linoleic acid are easily oxidized by the oxygen in

the air. This auto-oxidation leads to the occurrence of chain reactions with the formation

of coupled double bonds, and at a later stage also to obtaining secondary products, such

as aldehydes, ketones, and alcohols. The p-anisidine value measures also a secondary

product (adehydes) formed by secondary oxidation of polyunsaturated acid. In order to

optimise the concentrations of three spices (cloves, cumin and cinnamon), giving the

optimal antioxidant on meat product, namely terrine and ham, the experiments for

inhibiting the peroxidation of the polyunsaturated fatty acids were performed.

The concentration of cloves had a significant positive effect on physicochemical

properties of meat products. The increase of clove concentration prevents lipid

oxidation. This is due to high levels of phenolic compounds, which have antioxidant,

anti-inflammatory, and anti-clotting properties (Parle & Gurditta, 2011). The level of

phenolic compounds was a major factor in labeling cloves as the best natural

antioxidant. Clove and eugenol possess strong antioxidant activity, which is comparable

to the activities of the synthetic antioxidant, BHA (butylated hydroxyl anisole) and

Pyrogallol (Dorman et al., 2000). Clove has the highest capacity to release hydrogen

and reduce lipid peroxidation. With respect to the lipid peroxidation, the inhibitory

activity of clove oil determined using a linolenic acid emulsion system indicated a

higher antioxidant activity than the standard BHT (Butylated hydroxyl toluene). It also

showed a significant inhibitory effect against hydroxyl radicals and acts as an iron

chelator (Gulcin et al, 2004). The metal chelating activity, bleomycin dependent DNA

oxidation, diphenyl -p- picryl hydrazyl (DPPH) radical scavenging activity and the

107

ferric reducing antioxidant power (FRAP) of different spices were measured in rat

liver homogenate. Cloves showed the highest DPPH radical scavenging activity and

highest FRAP values (Yadav & Bhatnagar, 2007). The antioxidant activity of clove and

its major aroma components, eugenol and eugenol acetate were comparable to that of

the natural antioxidant α-tocopherol (Lee & Shibamoto, 2001). Moreover, cumin has a

good antioxidant potential as it contains appreciable amounts of antioxidant compounds

and its non-volatile extracts also have good inhibition properties against the free

radicals. There is also a good correlation between the total phenolic content in cumin

and its antioxidant activities and this spice can be used to produce novel natural

antioxidants as well as flavoring agents that can be used in various food products

(Annie et al., 2006). For this reason, the concentration of cumin had significant positive

effect on antioxidant activities in meat products, such as terrine and ham (p-value <

0.05).

However, the results shown in Table 3.3 indicated that the linear model terms of

X3(the concentration of cinnamon) did not have a significant effect on - p-anisidine

value in both terrine and ham and on TBA index in terrine but have significant negative

effect on TBA index in ham. These results showed that antioxidant capacities of cumin

and cloves were higher than those of cinnamon. These findings are not well correlated

with the results of Ho et al. (2008) who showed that antioxidant capacity ranked the

spices (in decreasing order): cloves > rosemary, cinnamon, turmeric > nutmeg > cumin

> paprika and cardamom.

The interactive effect of the spices on antioxidant activities has not been

described earlier in literature. The results shown in this study indicated the absence of

interactive effects of variables on p-anisidine in terrine.

108

Table 3.3 Model coefficients estimated by central composite design and best selected

prediction models

Coefficients log10 (TVC) p anisidine TBA (mg MDA/kg) Terrine Ham Terrine Ham Terrine Ham

Constant 4.44 8.13 1.23 0.94 0.72 0.31

Linear

Cloves (X1) -2.45 -0.73 -0.77 -0.65 -0.45 -0.52

Cumin (X2) -2.17 -0.95 -0.58 -0.43 -0.40 -0.55

Cinnamon (X3) 0.71 0.39 0.10

0.03

-0.003

0.10

Interactions

Cloves x Cumin -0.18 0.56 0.07 0.197 0.36 0.28 Cloves x

Cinnamon 1.054 0.27 0.05 0.117 0.14 -0.01

Cumin X Cinnamon 0.35 -0.047 0.14 0.137 0.09 -0.12

Quadratic

Cloves 0.13

-0.29

0.026

0.18 0.034

0.18

Cumin 0.27 -0.22 -0.11

0.18

0.04 0.31

Cinnamon 0.54

0.06 -0.11 -0.004 0.031153

0.060

R2 0.84 0.88 0.95 0.98 0.85 0.98 Bold values: Significant (p < 0.05).

Reduced equations for physico-chemicals (TBA index and p-anisidine value) and

microbiological properties (TVC) of ham and terrine : best selected models: Log10

TVC (terrine) = 4.44 - 2.45 X1 - 2.17 X2; Log10 TVC (ham) = 8.13 - 0.73 X1 - 0.95 X2

+ 0.39 X3 + 0.56 X1 x X2 ; p anisidine (terrine) = 1.23 - 0.77 X1 - 0.58 X2; p anisidine

(ham) = 0.94 - 0.65 X1 - 0.43 X2 + 0.18 X12 + 0.18 X2

2 + 0.197 X1 x X2 + 0.137 X2 x X3;

TBA (terrine) = 0.72 - 0.45 X1 - 0.40 X2 + 0.36 X1 x X2; TBA (ham) = 0.31 - 0.52 X1 -

0.55 X2 + 0. 10 X3 + 0.18 X12 + 0.31 X2

2 + 0.28 X1 x X2 - 0.12X2 x X3;

3.5.2. Effects of variables on biological properties of processed meat

The analysis of variance (Table 3.3) indicated that the linear model terms of

X1(concentration of cloves) and X2 (concentration of cumin) had significant positive

effect on microbial properties (TVC value) in terrine and ham. The values of linear

109

coefficient for X1 and X2 were negative, hence when X1 and X2 increased, p anisidine,

TVC values decreased. The enumeration of total viable count, TVC is an important

indicator to determinate the shelf-life of meat products. A meat product is non-

consumable, if the TVC is greater than 107 UFC/g (ICMSF, 1986).

The concentrations of cloves and cumin had a significant positive effect on microbial

properties of meat products (terrine and ham). The increase of clove concentration

prevents the microbial spoilage of meat products. This is due to high antimicrobial

activity of cloves and cumin (Parle et al., 2011). The antimicrobial potency of plants

and cumin and cloves is believed to be due to tannins, saponins, phenolic compounds,

essential oils and flavonoids (Aboaba and Efuwape, 2001). Moreover, naturally

occurring compounds in spices, such as sulphur compounds, terpenes and terpene

derivatives, phenols, esters, aldehydes, alcohols and glycosides have shown

antimicrobial functions (Russel and Gold, 1991; Davidson and Baren, 1993; Deis,

1999). However, the inhibitory effects of cumin and cloves are mostly due to the

volatile oils present in their composition (Arora-Daljit and Kaur, 1999).

The results shown in Table 3.3 indicated that the linear model terms of X3 (the

concentration of cinnamon) did not have a significant effect on TVC values in terrine,

but have significant negative effect on TVC values in ham. These results showed that

antimicrobial capacities of cumin and cloves were higher than those of cinnamon. These

findings are not well correlated with the results of Rahman et al. (2008) who showed

that cloves, cumin and cinnamon have good antimicrobial activities.

3.5.3. Determination of optimal conditions and optimal responses

By using the method of experimental factorial design and response surface analysis, the

optimal spice concentrations to obtain the best microbial and physico-chemical

properties of meat products, namely terrine and ham were determined. The validity of

the model was proved by fitting different values of the variables into the model equation

and by carrying out experiments at these values of the variables. The results of

experimental plan by central composite design for ham and terrine are presented in

details in Table 3.1. The optimal concentrations of spices giving the lowest, p-anisidine,

TBA and TVC values are determined using 3D plots presented in Figures 3.1, 3.2 and

3.3. The optimal concentrations of spices giving the lowest p-anisidine index in both

110

ham and terrine were 0.3 , 0.3 and 0.1 % w/w for cloves, cumin and cinnamon,

respectively (Table 3.2). This spice formulation rendered lowest p-anisidine values

(0.32 in terrine and 0.61 in ham) and TBA index in terrine (0.37 mg MDA/kg).

However, the optimal concentration of spices giving the lowest TBA index in ham

(0.134 mg MDA/kg) was 0.3 % w/w of cloves, cumin and cinnamon. The lowest p-

anisidine value and TBA index in terrine and ham were obtained using higher

concentration of cloves and cumin. This is due the high antioxidant potential of cumin

and cloves because it contains appreciable amounts of antioxidant compounds and

inhibition properties against the free radicals (Dorman et al., 2000; Annie et al., 2006).

However, the minimal values of TBA and p-anisidine were obtained using 0.1 % w/w

of cinnamon in terrine, but lowest TBA index in ham was obtained using 0.3 % w/w of

cinnamon. The concentration of cinnamon did not change the physicochemical

properties of meat products despite the higher anti-oxidant capacity of cinnamon

compared with cloves and cumin (Ho et al., 2008). This could be due the inefficiency of

polyphenols inside the cinnamon, that are not available in the meat products. In this

case, the use of oil or polyphenols extract could enhance the antioxydant activity of

cinnamon. The extraction of spices or the use of cinnamon oil could ameliorate the anti-

oxidant activity by the liberation of polyphenolics compounds from the spices.

111

(a)

(b)

Figure 3.1 Response surface of Log10 viability in terrine obtained by varying: a) the

concentration of cloves (X1) and the concentration of cumin (X2) keeping the

concentration of cinnamon (X3) constant ; b) concentration of cinnamon (X3) and the

concentration of cloves (X1) and keeping the concentration of cumin (X2) constant: 0.2

% w/w

112

(a)

(b)

Figure 3.2 Response surface of p-anisidine in terrine obtained by varying: a) the

concentration of cloves (X1) and the concentration of cumin (X2) keeping the

concentration of cinnamon (X3) constant ; b) concentration of cinnamon (X3) and the

concentration of cumin (X2) and keeping the concentration of cloves (X1) constant: 0.2

% w/w

113

(a)

(b)

Figure 3.3 Response surface of TBA in terrine obtained by varying: a) the

concentration of cloves (X1) and the concentration of cumin (X2) keeping the

concentration of cinnamon (X3) constant ; b) concentration of cinnamon (X3) and the

concentration of cloves (X1) and keeping the concentration of cumin (X2) constant: 0.2

% w/w

The best concentration of spices giving the lowest microbial growth in terrine

(log TVC= 2) was 0.368, 0.2 and 0.2 % w/w of cloves, cumin and cinnamon,

respectively. However, the optimal concentration of spices giving the lowest microbial

114

growth in ham (log TVC= 6.99) was 0.3, 0.3 and 0.1 % w/w of cloves, cumin and

cinnamon, respectively. The lowest TVC values have been obtained using high

concentration of cloves. Hence, cloves have the most important antimicrobial activity

when compared with cinnamon and cumin. Cloves represent one of the Mother Nature’s

antiseptic (Parle and Gurditta., 2011). Clove oil was found to be more effective than

sodium propionate (standard food preservative) against some food borne microbes.

Clove oil was found to be very effective against Staphylococcus species (Parle and

Gurditta, 2011). Clove oil showed antimicrobial activity against some human

pathogenic bacteria resistant to certain antibiotics (Lopez et al., 2005). The

antimicrobial potency of plants and spices is believed to be due to tannins, saponins,

phenolic compounds, essential oils and flavonoids (Aboaba and Efuwape, 2001).

Moreover, naturally occurring compounds in cloves, such as sulphur compounds,

terpenes and terpene derivatives, phenols, esters, aldehydes, alcohols and glycosides

have shown antimicrobial functions (Russel and Gold, 1991; Davidson and Baren,

1993; Deis, 1999). Furthermore, the inhibitory effects of spices namely cloves are

mostly due to the volatile oils present in their composition (Arora-Dlijit and Kaur,

1999). Moreover, the 3 D plot on response surface of Log10 viability presented in

Figure 1 a and Figure 3.4 a showed that when cloves and cumin concentration

increased, log 10 TVC decreased. Hence, both cumin and cloves concentration have

high antimicrobial activities. These findings are well correlated with the results of

Rahman et al (2010) who found that cloves and cumin had important antimicrobial

activity against Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas

aeruginosa, Enterococcus faecalis, Micrococcus luteus, Escherichia coli and Candida

albicans. However, the 3D plot presented in Figure 3.1a and 3.1b showed that lowest

values of TVC were obtained when the concentration of cinnamon decreased. These

results are not correlated with the results of Rahman et al (2010) who found that

cinnamon showed that cinnamon had a very good antimicrobial activity. This could be

due to the non-liberation of the components responsible for anti-microbial activity from

cinnamon. The extraction of these components or the use of cinnamon oil could increase

the antimicrobial activity of cinnamon.

115

3.6. Conclusions

Use of response surface methodology for optimization of spices concentrations giving

the best anti-oxidant and microbial activities led to following conclusions:

1) The optimal concentrations of spices giving the lowest p-anisidine index in both ham

and terrine and TBA index in ham were 0.3, 0.3 and 0.1 % w/w for cloves, cumin and

cinnamon, respectively.

2) The optimal concentration of spices giving the lowest TBA index in ham (0.37 mg

MDA/kg) was 0.3 % w/w for cloves, cumin and cinnamon.

3) The best concentration of spices giving the lowest microbial growth in terrine (log

TVC= 2) was 0.368 , 0.2 and 0.2 % w/w of cloves, cumin and cinnamon, respectively

and the optimal concentration of spices giving the lowest microbial growth in ham (log

TVC= 6.99) was 0.3, 0.3 and 0.1 % w/w of cloves, cumin and cinnamon, respectively.

4) The concentration of cloves and cumin had significant positive effect on microbial

(TVC value) and physico-chemical properties in both terrine and ham (p < 0.05).

5) The concentration of cinnamon did not have a significant effect on TVC, TBA and p-

anisidine values in meat products (p < 0.05).

3.7. Acknowledgement

The authors are sincerely thankful to the Natural Sciences and Engineering Research

Council of Canada (Discovery Grant), FQRNT (partenariat) and MAPAQ (No. 809051)

for financial support. The views or opinions expressed in this article are those of the

authors. We express our gratitude to Mr. Boiteau of Aliments Breton and La Maison du

Gibier for supplying us the raw meat to carry out various tests. The views or opinions

expressed in this article are those of the authors.

116

3.8. References

Aboaba, O., & Efuwape, B.M. (2001). Antibacterial Properties of Some Nigerian Species. Bio Research Communications, 13,183-188. Arora-Daljit, S., & Kaur, J. (1999). Antimicrobial activity of spices. International Journal of Antimicrobial Agents, 12, 257-262.

Boza, A., De la Cruz, Y., Jordan, G., Jauregui-Haza, U., Aleman, A., & Caraballo, I. (2000) Statistical optimization of a sustained-release matrix tablet of lobenzarit disodium. Drug Development and Industrial Pharmacy, 26, 1303-1307.

Chang, S. T.; Chen, P. F., and Chang, S. C. (2001). Antibacterial activity of leaf essential oils and their constituents from Cinnamomum osmophloeum. Journal of Ethnopharmacology, 77, 123–127.

Davidson, P. M., & Baren, A. L. (1993). Antimicrobials in Foods. Marcel Dekker, New York. pp. 1-9 Deis, R. D. (1999). Secret world of spices. Food Product Design, 5, 1-7. Dorman, H. J. D., Surai, D., & Deans, S. G. (2000). In vitro antioxidant activity of a number of plant essential oils and Phytoconstituents. Journal of Essential Oil Research, 12, 241–248. Gottelli, N. J., & Ellison, A. M. (2004). A Primer of Ecological Statistics Sinauer Associates. Sunderland, MA, 510 pp. Gulcin, I., Sat, I. G., Bey demir, S., Elmastas, M., & Kufrevioglu, O. I. (2004). Comparison of antioxidant activityof clove (Eugenia caryophyllata Thunb) buds and lavender (Lavandula stoechas L.). Food Chemistry, 87, 393-400. International Commission on Microbiological Specifications for foods Sampling plans for fish and shellfish (1986). In ICMSF (Eds.), Microorganisms in Foods. Sampling for Microbiological Analysis, Principles and Scientific Applications,Vol. 2, (2nd ed.). University of Toronto Press, Toronto, Canada. pp 139-147. Ka, H., Park, H-J., Jung, H-J., Choi, J-W., Cho, K-S., Ha, J., & Lee, K-T. (2003). Cinnamaldehyde induces apoptosis by ROS-mediated mitochondrial permeability transition in human promyelocytic leukemia HL-60 cells. Cancer Letters, 196, 143-152. Lee, K. G., & Shibamoto, T. (2001). Antioxidant property of aroma extract isolated from clove buds [ Syzygium aromaticum (L.) Merr. et Perry ]. Food Chemistry, 74, 443 – 448. Lopez, P., Sanchez, C., Batle, B., & Nerin, C. (2005). Solid and vapour phase antimicrobial activities of six essential oils: susceptibility of selected food borne bacterial and fungal strains. Journal of Agriculture and Food Chemistry,53, 6338 – 6346.

117

Honikel, K. O. (2008). The use and control of nitrate and nitrite for the processing of meat products. Meat Science, 78, 68-76.

Menon, K. V., & Garg, S. R. (2001). Inhibitory effect of clove oil on Listeria monocytogenes in meat and cheese. Food Microbiology, 18, 647-650.

Okawa, M., Kinjo, J., Nohara, T., & Ono, M. (2001). DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of flavonoids obtained from some medicinal plants. Biological & Pharmaceutical Bulletin, 24, 1202-1205. Parle, M., & Gurditta (2011). Basketful benefits of papaya. International Research Journal of Pharmacy, 2, 6-12.

Pegg, R. B., & Shahidi, F. (2000). Nitrite curing of meat. The n-nitrosamine problem and nitrite alternatives. Trumbull, CT: Food and Nutrition Press, Inc.

Rag havenra, H., Diwakr, B. T., Lokesh, B. R., & Naidu, K. A. (2006). Eugenol, the active principle from cloves inhibits 5 - lipoxygenase activity and leukotriene - C4 in human PMNL cells. Prostaglandins, Leukotrienes and Essential Fatty Acids, 74, 23 –27.

Rahman, M. S. A., Thangaraj, S., Salique, S. M., Khan. K. F., & Natheer. S. E. (2010). Antimicrobial and Biochemical Analysis of Some Spices Extract against Food Spoilage Pathogens. Internet Journal of Food Safety, 12, 71-75. Russel, R.J., & Gould, G. W. (1991). Food Preservatives. Van Nostrand Reinhold Co., New York.

Sanchez-Lafuente, C., Furlanetto, S., & Fernandez-Arevalo, M. (2002). Didanosine extended-release matrix tablets: optimization of formulation variables using statistical experimental design. International Journal of Pharmaceutics, 237, 107-118.

Singh, S. K., Dodge, J., Durrani, M. J., & Khan, M. A. (1995). Optimization and characterization of controlled release pellets coated with experimental latex: I. Anionic drug. International Journal of Pharmaceutics, 125, 243-255. SPSS Base 10.0 Application Guide SPSS, Chicago, IL (1999). Tajkarimi, M.M., Ibrahim, S. A., and Cliver, D. O. (2010). Antimicrobial herb and spice compounds in food. Food Control. 21 : 1199–1218 Yadav, A. S., & Bhatnagar, D. (2007). Free radical scavenging activity, metal chelation and antioxidant power of some Indian spices. Biofactors, 31, 219 -227.

119

Chapitre 4 Color Retention in Processed Meats by Using Natural Products and Tests of Organoleptic Properties

Anne Patricia Kouassi1, Fatma Gassara2, Nasima Chorfa1, Satinder Kaur Brar2*,

Khaled Belkacemi1

1Département des sols et de génie agroalimentaire, Pavillon Paul-Comtois, Université

Laval, 2325, rue de l'Université, Québec (Québec) G1V 0A6

2INRS-ETE, Université du Québec, 490, Rue de la Couronne, Québec, Canada G1K

9A9

(*Corresponding author, Phone: 1 418 654 3116; Fax: 1 418 654 2600; E-mail: satinder.brar@ete.inrs.ca)

120

4.1. Résumé

Dans la présente étude, la couleur et l'analyse sensorielle des terrines de porc et de lapin

et des échantillons de jambon ont été étudiées. Afin d'obtenir une coloration semblable

aux terrines et aux jambons industriels, deux colorants naturels nommés poudre de

raisin (2 gp) et poudre de fraise (3 st), ont donné de bons résultats proches de ceux des

nitrites pour la da (rougeur), le db (jaunissement) et le dl (luminosité). Pour voir

l'ìmpact de ces ajouts sur les propriétés organoleptiques, une analyse sensorielle a été

menée et a montré qu'il n'y avait pas de différence significative pour l'attribut épicé

entre les terrines contenant les nitrites et terrines avec la formulation 7 (0.3% clous de

girofle, 0.3% cumin, 0.1% cannelle p/p) à p <0,05, ainsi que pour les jambons. Le

tableau de moyennes ajustées et l'analyse du composant principal ont montré le

caractère sucré de la poudre de fraise.

Mot clés: Couleur, test organoleptic, épices, analyse en composantes principales,

nitrites, terrine, jambon

121

4.2. Abstract

In the present study, color and sensory analysis of pork and rabbit terrines and ham

were investigated. In order to achieve visual color appearance to the industrial product

without significantly impairing the taste of the samples, two natural coloring agents

named grape powder (2 gp) and strawberry powder (3 st) were found and gave good

results for the da (redness), db (yellowing) and dl (brightness), very closed to the

control, which is the nitrite. For sensorial analysis, nine attributes were analyzed and

according to the Tukey t-test, there was no significant difference for the spicy attribute

between the terrines containing nitrites and terrines with formulations 7 (0.3% cloves,

0.3% cumin, 0.1% cinnamon w/w) at p <0.05, neither for hams. The table of ajusted

means and analysis of Principal Component Analysis showed that the terrine with

formulation 12 has a similar profile to the one with nitrites.

Keywords: Color, organoleptic test, spices, principal component analysis , nitrites,

terrine, ham

122

4.3. Introduction

Nitrates and nitrites are used in meat-based products not only because of their

antioxidant and antimicrobial properties but also for their action on color and meat taste

(Honikel, 2008). Many alternatives, such as spices, were found to exhibit the

antioxidant and antimicrobial properties in exchange of nitrites which have toxic effects

on human health (Fan et al., 1987; USEPA, 2006). Cinnamaldehyde, for example, the

major constituent of cinnamon (Cinnamomum cassia) has been reported to possess

antibacterial activity and antioxidant properties (Chang et al., 2001); Pepper is also

associated with a number of functional properties, such as analgesic and antipyretic

properties, antioxidant effects and antimicrobial properties (Kapoor et al., 1993); clove

oil showed its antimicrobial activity in a study based on the inhibitory effect of clove oil

on Listeria monocytogenes in meat and cheese (Vrinda et al., 2001). However,

coloration of spiced meat is still a big challenge. Color has a major role in the

acceptability of the product and is related to the consumer perception of flavor,

sweetness, scents and other physical properties in relation to the quality of the product

(Roth et al., 1988; Calvo et al., 2001; MacDougall, 2002 ). Meat color is important for

industries and consumers. Thus, it necessitates a study to find a natural way to maintain

the red color of meat. In fact, studies have already been carried out to stabilize a bright

red color of beef during frozen display, on beef fed Vitamin E, beef exposed to pure

oxygen to fully bloom the meat, as well as on exposure to carbon monoxide (Huffman

et al., 1975; Liu et al., 1995; Jeong and Claus, 2011). However, none of these

approaches improved the color and shelf-life to the point of producing a commercial

viable extension (Claus and Du, 2013).

The pigment responsible for the characteristic pink color of cured meat is a ferrous

complex of myoglobin containing nitric oxide (NO), namely, nitrosylmyoglobin or NO-

Mb. The complex is formed by the reaction of myoglobin with nitrite generated NO

(Morita et al., 1998). Nitrosohemachrome is a denatured, stable form of NO-Mb in

cooked and cured meats (Martin, 2001). The chemical reactions leading to the cured

meat pigment are a complex series of processes involving microbial, enzymatic and/or

chemical catalyzed steps, which depend on pH, pigment concentration, redox potential,

curing agent distribution, temperature and relative humidity (Chasco et al. 1996). Thus,

to find a solution for the red color, fruits and vegetables were used to color the meat.

123

To measure color of different materials, various color spaces have been reported. Two

frequently used color spaces are RGB (Red, Green and Blue) and CIE Lab. RGB color

space consists of a three-dimensional rectangular coordinate system with R, G and B

axes. A color image is represented in RGB format with these three components per

pixel in the range 0–255 and their intensities are electronically combined to produce a

digital color picture (Afshari-Jouybari and Farahnaky, 2011). In this study, the CIE

L*a*b* color space was used for the measures of color of terrines and hams. It is the

most popular numerical colour space systems in food industry, which is also referred as

the CIELAB system, originally defined by the CIE in 1976 (MacCaig, 2002; CIE, 1986.

It aspires to perceptual uniformity, and its L component closely matches human

perception of lightness. CIE L*a*b* (CIELAB) is a complete color space specified by

the International Commission on Illumination (French Commission internationale de

l'éclairage, CIE). It describes all the colors visible to the human eye and was created to

serve as a device-independent model to be used as a reference.

The organoleptic tests were carried out on a trained panel in order to know if the

formulations of spices were approved and taste well. Descriptive analysis by a panel of

trained judges (also called panelists) is probably the best way to objectively assess and

compare the sensory properties of food products. The sensory quality of meat is

influenced by several factors that act before and during eating and which are often

mutually interacting (Gasperi et al., 2005). In the case of meat, the literature provides

very few useful indications about standardized references (for texture, tenderness,

juiciness and flavour) (Gorraiz et al., 2000; Byrne et al., 2001).

To the best of our knowledge, there are not many systematic studies on use extracts of

fruits and vegetables to color spicy meats. This study aims at finding the best

combination of extracts of fruits and vegetables to improve color of meat samples

containing spices. Fruits and vegetables are natural color agents, healthy products.

Moreover, the benefits of fruits have been attributed to their high phenolic compound

content, which act as antioxidants (Zuo et al., 2002). Subsequently, a trained sensory

panel was subjected to various descriptors (e.g. spicy, bitter, acid, sweet, salty, rancidity

and juiciness, among others) to judge the suitability of the finished formulations for the

industry and hence its commercialization.

124

4.4. Materials and Methods

4.4.1. Preparation of meat extract for color

In a previous study by our research group, a qualitative analysis has been made with

terrines and ham. Three spices (clove, cumin and cinnamon) were selected for their

optimal anti-microbial and anti-oxidant properties in the effective concentrations of

0.368% , 0.2% and 0.2 % w/w respectively, according to the microbial growth) (Fatma

Gassara et al., 2014). Samples of 200 g of terrines were prepared using simple recipe of

terrine of pork and rabbit, by adding a percentage, given by the statistics, of the three

spices to be tested. These concentrations are given in Table 4.1.

Table 4.1 The 19 combinations of spices obtained by Statisticia with their pH

Formulations Cloves Cumin Cinnamon pH

1 0.1 0.1 0.1 6,5

2 0.1 0.1 0.3 6,21

3 0.1 0.3 0.1 5,86

4 0.1 0.3 0.3 6,06

5 0.3 0.1 0.1 6,42

6 0.3 0.1 0.3 6,32

7 0.3 0.3 0.1 6,35

8 0.3 0.3 0.3 6,37

9 0.031 0.2 0.2 6,2

10 0.368 0.2 0.2 6,02

11 0.2 0.031 0.2 6,35

12 0.2 0.368 0.2 6,12

13 0.2 0.2 0.031 6,22

14 0.2 0.2 0.368 6,26

15 0.2 0.2 0.2 6,19

16 0.2 0.2 0.2 6,02

17 0.2 0.2 0.2 6,26

18 0.2 0.2 0.2 6,26

19 0.2 0.2 0.2 6,14

ctrl + Cloves Cumin Cinnamon 6,14

With ctrl + : terrine + nitrites

125

For color experiments, samples of 50-200 g of terrines were prepared using simple

recipe of terrine of pork and rabbit, with different percentage of red coloring agent

(Table 4.5), fruit powder, flower powders and vegetables, with nitrite as blank. The fruit

powder was mixed with 5 mL of distilled water and added to samples. Samples are

cooked in a microwave for 6 min and then cooled in a refrigerator for 2 days.

4.4.2. Color evaluation

Colorimetry is the science of measuring color. This allows separating the different color

settings: chromatic tone, chroma and lightness. Several systems for expressing color

numerically were developed by an international organization concerned with issues of

lighting and color, the International Commission on Illumination (CIE). One of the best

known of these systems is the L*a*b color space (also referred to as CIELAB) devised

in 1976 (CIE, 2007). The CIE recommends a particular illuminant/observer

combination and color spaces (CIEXYZ, CIE L*a*b) to standardize the definition of

color and provide more uniform color differences in relation to visual differences

(Korifi et al., 2013). In this system, each color can be located with its rectangular

coordinates: L* for axis of brightness, a* represent the red/green axis and b* represent

the yellow/blue axis.

4.4.3. Measurement of different parameters

4.4.3.1. Color

Colorimetric analysis on freshly cooked terrines and hams were performed using a

colorimeter , Minolta Chroma Meter Mesure CR-310 ). It is a compact tristimulus color

analyzer for measuring reflective color of surfaces. The measuring head of the chroma

meter uses wide-area illumination, a 0 degree viewing angle and a has a 50 mm-

diameter measuring area to average the reading over a wide area for measuring textured

surfaces.

4.4.3.2. Effect of color on pH

The pH value determines the quality of the meat, especially the color, tenderness,

flavor, water retention and conservation. The pH of cooked and uncooked hams was

126

determined by blending 1 g of sample with 10 ml distilled water. The pH values were

measured using an electrode attached to a digital pH meter Metler Toledo. To know the

influence of pH on terrines and hams, tests on samples without spice were made, over a

range of pH 3.5 to 11.5. The pH of all 20 terrines was taken in the first two weeks

(Table 4.1).

4.4.4. Organoleptic tests

4.4.4.1. Training

Nine panelists (6 males and 3 females) were selected from spontaneous candidature

mostly from Université Laval and INRS (Institut National de Recherche Scientifique),

for the detection, recognition and assessment of tastes, odors, flavors, and physical

characteristics, as well as for the use of classification scales. In order to train candidates

to recognize and memorize the attributes, the training was done by providing five basic

tastes, two odors and two large textures in the samples of meat. The nine attributes are

listed in Table 4.2. All of these attributes were extensively examined during group

discussions in order to reach a consensus about their meaning and to establish an

evaluation procedure and chose suitable reference standards (STONE et al. 1974). Table 4.2 List of attributes for the organoleptic tests

Attributes Adding Quantity % (w/w)

Sweety

Sugar

1

Salty Salt 0,2

Bitter Caffeine 0,05

Acid citric acid 0,04

Spicy Clove 1

Rancidity linseed oil heated

Aromaticity liquid vanilla

Tenderness Easiness with which the meat is divided into

fine particles during mastication

Juiciness Amount of wetness/juiciness released from

sample

127

4.4.4.2. Sensory evaluation

Four sessions were organized over a period of month. During each session, the panel

evaluated the 6 terrines of rabbit and pork and 5 samples of pork with our spices. All

samples were prepared with the same method to eliminate potential effects of the

preparation. Samples submitted for the tests are listed in table 4.3.

Table 4. 3 List of samples for organoleptic tests

*Powder 3st (strawberry) for coloration due to quantificationof powders (Figure 4.3)

4.4.5. Statistical analysis

The intensity of each attribute was rated in scale from 1 (not detected) to 4 (strongest).

At the end of the organoleptic test, the scores were analyzed for mean scores and

variance ratios. The least significant difference (LSD) of the Tukey test was calculated

for product comparison of each attributes. Principal Component Analysis (PCA)

highlighted similarities and differences among spices formulation for meat products

preservation. Data are presented as means±standard errors of the mean. P≤0.005 was

considered statistically significant. All calculations were performed using Sigmaplot

and SensomineR statistical package.

Nitrite

(ppm)

clove

(% w/w)

cumin

(%w/w)

cinnamon

(% w/w)

Powder 3

st (%w/w)

Sample 1 White terrine 0 0 0 0 0

Sample 2 terrine with nitrite 150 0 0 0 0

Sample 3 terrine +

formulation 7

0 0,3 0,3 0,1 0

Sample 4 terrine formulation 7 +

powder 3st*

0 0,3 0,3 0,1 0,9

Sample 5 terrine formulation 10 0 0,368 0,2 0,2 0

Sample 6 terrine formulation 12 0 0,2 0,368 0,2 0

Sample 7 ham with nitrite 150 0 0 0 0

Sample 8 ham form 7 0 0,3 0,3 0,1 0

Sample 9 Ham fomr 7 + powder 3

st*

0 0,3 0,3 0,1 0,9

Sample 10 Ham formulation 10 0 0,368 0,2 0,2 0

Sample 11 Ham formulation 12 0 0,2 0,368 0,2 0

128

In order to have a visualization of the most discriminating attributes, the box plot were

made by SigmaPlot. A comparison of several populations quickly is possible with box

plots which also allow to visually appreciate the asymmetry and the presence of atypical

individuals parameters. Each population is represented by a box whose lower bound is

the first quartile and the upper bound on the third quartile. These diagrams also allow a

glance to appreciate the essential elements of the distribution of each group and thus a

comparison.

4.5. Results and discussion

4.5.1. Effect of pH on color

Effect of pH on color was tested on ham without spice within a pH range of 3.50 to

11.5. These tests were carried out on cooked and uncooked samples as presented in

Figure 4.1 and Table 4.4. As seen in Figure 4.1, there was a change of color and texture

of meat. With the higher pH, change in meat color was more. Further away from neutral

pH to the two extremes, the texture becomes crumbly (acid pH) and elastic basic pH).

Table 4.4 Effet of pH on colour meat

Ph Sample Uncooked sample Cooked sample

de da dl db de da dl db

3,05 13,6 -5 11,2 -5,9 8,7 -3,7 4,9 -6,1

4,04 15 -2,9 14,4 -2,7 13,3 -4,9 9,7 -7,7

4,9 13 -2,3 11,8 -5 11,9 -3,6 11 -2,7

6,02 4,4 1,8 2,1 -3,4 14,4 -7,4 10,5 -6,7

7 4,7 4,1 -0,3 -2,3 15,4 -6,9 13,1 -4,2

7,97 4,4 0,5 -0,6 -4,3 9 -5,7 5,3 -3,7

9 6,6 -0,5 -2,9 -5,9 5,7 -4,7 -2,3 -2,2

10,18 3,2 2,4 1 -1,8 5 -4 -2,5 -1,6

11,5 9,7 -1,6 9,5 -0,1 4,1 -1,5 -3,7 1

Ctrl + 150

ppm

17,1 7,2 15,4 -1,2 16,6 1,8 14,3 -8,3

L* for axis of brightness, a* represent the red/green axis and b* represent the yellow/blue axis.

129

Uncooked Sample

Cooked Sample

Figure 4.1 Pictures of terrines uncooked and cooked at different pH

4.5.2. Solution for color

Out of the 19 formulations prepared, 3 formulations (formulations 7, 10 and 12

presented in Table 4.3) gave best physical and microbiological results in research of

alternatives of nitrites in meat-based products. These terrines samples were colored by

the spices. To find a solution to the coloring terrines, red coloring chemical food agent

was added at different concentrations (Table 4.5). The results showed a strong power of

red agent on terrines (without spices). It gave a "da" close to nitrites samples with one

drop per 100 g of sample. However, for greener alternatives, it was removed because it

contains chemical compounds.

130

Table 4.5 Color analysis of terrines containing different concentrations of red food

chemical coloring agent

Sample(g) Lab Color pictures

Nitrite 150 ppm dE 16,2

da 0,1

dl 13,1

db -7

4 drops (50g) dE 28,3

da 27

dl -3,4

db -7,8

1 drop (50g) dE 11,9

da 7,1

dl 3,8

db -8,7

1 drop (100g) dE 11,6

da 0,4

dl 10,5

db -4,8

Later, the coloring efficiency of red fruits powder such as strawberry, raspberry, and

vegetables such as beets, and flowers like hibiscus were tested (Table 4.6, Figure 4.2).

Beet exhibited best results in terms of meat color; however, it could not be used because

of its high nitrite content (3300 mg/kg) (IPCS).

131

Table 4.6 Color analysis of terrines containing different fruits and vegetables used to

improve red color

Sample Cooked sample

de Da dl Db

Beet 22,6 19,4 -2,5 -11,3

Hibiscus (15mL) 17,9 -3,6 15,2 -8,7

Hibiscus powder

(0.5g)

17,6 -4,4 9,7 -14

Strawberry puree 1% 21,8 -6,7 18,5 -9,5

2% raspberry

strawberry puree

(prepared in the lab)

21,4 -6,9 19,6 -5

2% raspberry

strawberry pulp

(prepared in the lab)

20,6 -6,3 19 -5,1

5% raspberry

strawberry pulp

22,9 -6,2 21,3 -5,7

10% raspberry

strawberry pulp

22,4 -5,5 20,6 -6,7

Nitrite 150ppm 16,2 0,1 13,1 -7

Figure 4.2 Pictures of coloured samples with differents percentages of fruits and

vegetables

Nitrite 150 ppm 5% raspberry strawberry pulp

10% raspberry strawberry pulp

Strawberry puree Hibiscus Hibiscus powder Beet

2% raspberry strawberry pulp

134

Table 4.7 Analysis of variance of results of terrines' flavor

Attributes

Sweetness Salty Bitterness Acidity spicy

Source of

Variation DF1 F2 P3 F P F P F P F P

Panelist 8 2.953 0.004 1.742 0.092 2.421 0.017 5.904 <0.001 5.543 <0.001

Sample 5 7.285** <0.001 3.246** 0.008 1.685 0.141 0.996 0.422 4.202** 0.001

panelist x

sample

40 0.745 0.862 0.901 0.641 0.520 0.991 0.571 0.980 0.484 0.996

Residual 162 F 5% 2.21

F 1% 3.02 Total 215

*indicates a significant difference at 5% **indicates a significant difference at 1% according to statistical tables 1Degree of freedom 2Variance ratio 3Probability

For odor and texture of terrines

The results of the ANOVA of odor and texture, presented in Table 4.8 , showed that

there was a significant difference between the samples, for the attributes aromaticity and

juiciness at 5%; and rancidity at 1%.

Table 4.8 Analysis of variance of results of terrines' odor and texture Attributes

Rancidity aromaticity tenderness juiciness

Source of

Variation DF F P F P F P F P

Panelist 8 4.129 <0.001 3.122 0.003 2.995 0.004 1.465 0.174

Sample 5 2.619* 0.026 19.02** <0.001 1.655 0.148 4.511** <0.001

panelist x

sample 40 0.681 0.923 0.848 0.724 0.240 1.000 0.461 0.997

Residual 162 F 5% 2.21

F 1% 3.02 Total 215

*indicates a significant difference at 5% **indicates a significant difference at 1%

135

4.5.3.2. Tuckey test and descriptive analysis for terrines

For flavor of terrines

The difference of sweetness, salty, acidity, bitterness and spicy in terrine containing

nitrite was not significantly different from other terrines made with different spices

according to the calculation of scores mean and LSD, presented in Table 4.9.

Table 4.9 Results of Mean scores and Least Significant Difference for flavor of terrine

Terrines Attributes1

Sweetness Salty Bitterness Acid Spicy

White (without nothing) 1.3330a 2.5830a 1.2500a 1.3610a 2.0830a

Terrine with nitrite 1.4440a 2.5830a 1.5000a 1.5830a 1.972a

Terrine with formulation 7 1.5000a 2.7780a 1.3330a 1.6390a 2.7690a

Terrine with formulation 7 +

powder 3 st

2.1670a 2.2130a 1.6110a 1.6940a 2.3330a

Terrine with formulation 10 1.6390a 2.4720a 1.3330a 1.5280a 2.4720a

Terrine with formulation 12 1.4440a 2.7410a 1.4440a 1.5000a 2.4440a

Standard error2 0.1110 0.1130 0.1020 0.1170 0.1410

LSD3 0.898 0.09027 1.120 0.818 0.943 1Mean of 9 observations 2Mean standard error of mean score based on 162 df 3Least Significant Difference

However, thanks to the descriptive analysis we can see which is the most discriminating

descriptors. The Figure 4.4 showed the descriptive analysis for flavor of our 6 terrines.

The terrine with the formulation 7 shows a barely higher salty and spicy taste than the

other terrines; the terrine with formulation 7 + powder 3 st seems to have a more

pronounced sweetness and bitterness than other terrines. There was no significant

difference of acidity on all terrines. Terrines formulation 10 and 12 showed no huge

difference in flavor compared to terrine with nitrites.

136

sweetness

salty

bitterness

acidity

spicy

0.0 0.5 1.0 1.5 2.0 2.5

blanc vs attributes terrine+nitrite vs attributes terrine 7 vs attributes terrine 7 + powder 3st vs attributes terrine 10 vs attributes terrine 12 vs attributes

Figure 4.4 Sensory profile analysis of flavor sweetness, salty, bitterness, acidity and spicy of terrines

To complete the first graph of descriptive analysis, the box plot of flavor was presented

in Figure 4.5. The 6 terrines selected were divided into 3 sub-groups (group 1: 3

<median (M) ≤ 4 in dark gray; group 2: 2 <M ≤ 3in less dark gray and hatched; Group

3: 1 <M ≤ 2 in light gray) for the descriptors tested . Indeed, the figure 4.5 shows

boxplots of the descriptor spicy is discriminant, in accordance with Figure 4.4.

137

Box plot for sweetness

score

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

ter7+powder3st

nitrite

ter 7

ter 12

blanc

ter 10

Box plot for salty

score

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

ter12

ter7+powder3st

ter7

blanc

nitrite

ter10

Box plot for bitterness

score

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

ter7+powder3st

ter12

nitrite

ter7

ter10

blanc

Box plot for acidity

score

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

nitrite

ter 7

ter7+powder3st

ter10

blanc

ter12

Box plot for spicy

score

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

ter7+powder3st

ter7

ter10

ter12

nitrite

blanc

Figure 4.5 Box plots of the 6 terrines following descriptors: sweet, salty, acid, bitter

and spicy. A boxplot is lower than the M-1.5 (Q3-Q1) value, the first quartile (Q1),

median (M) solid line, dotted average, the third quartile (Q3) and the highest value less

than M +1.5 (Q3-Q1)

138

Terrines with formulation 7, 7 + powder 3 st, 10 and 12 were in group 2 (2 <M ≤ 3)

according to figure 4.5, and were therefore spicier than terrines containing nitrites,

which is quite normal. According to the figure 4.5, among the 4 terrrines containing

spices, the formulation 7 and sample 7 + powder 3st are spiciy.

For odor and Texture

For the odor and texture, the results presented in Table 4.10 showed that there was no

difference between the terrines for the attributes rancidity, tenderness and juiciness.

However, the aromaticity of terrines with formulation 7, 7 + powder 3 st, 10 and 12 is

significantly different from the one with nitrite.

Table 4.10 Results of Mean and Least Significant Difference for odor and texture of

terrines

Terrines Attributes1

rancidity aromaticity tenderness Juiciness

Blanc 1.2220a 2.2220ab 2.9170a 2.4440a

Terrine with Nitrite 1.2220a 2.0000b 3.1110a 2.9440a

Terrine with formulation 7 1.3890a 3.3330a 3.4170a 3.3060a

Terrine with formulation 7 + powder 3st 1.6940a 2.9440ab 3.2500a 2.5090a

Terrine with formulation 10 1.2500a 2.9440ab 3.0560a 2.9170a

Terrine with formulation 12 1.3610a 2.8330ab 3.1110a 3.1110a

Standard error2 0.1050 0.1150 0.1330 0.160

LSD3 0.842 0.925 1.074 1.269 1Mean of 9 observations 2Mean standard error of mean scare on 160 ddl 3Least Significant Difference

In the descriptive analysis for odor and texture of our 6 terrines (Figure 4.7), terrine

with formulation 7 was more aromatic, slightly tender and juicy than other terrines; the

one with formulation 7 + powder 3st seems less juicy and slightly rancid compared to

that of nitrites.

139

rancidity

aromaticity

tenderness

juiciness

0.0 0.5 1.0 1.5 2.0 2.5 3.0

terrine blanc vs attributesterrine+nitrite vs attributes terrine 7 vs attributes terrine 7 + powder 3st vs attributesterrine 10 vs attributes terrine 12 vs attributes

Figure 4.6 Sensory profile analysis of odor -rancidity, aromaticity- and texture -

tenderness, juiciness- of terrines

The box plots of odor and texture (Figure 4.8) confirmed the results of Figure 4.7 and

showed that the descriptors aromaticity, juiciness and tenderness are discriminating.

Terrine with formulation 7 has a more pronounced aromaticity than other terrines,

thereafter comes formulation 10. All terrines are relatively juicy, since they are from the

group 1 and 2.

140

Box plot for rancidity

score

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

ter7+powder3st

ter7

nitrite

ter12

blanc

ter10

Box plot for aromaticity

score

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

ter7

ter10

ter7+powder3st

ter12

blanc

nitrite

Box plot for juiciness

score

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

ter7

ter10

ter12

nitrite

ter7+powder3st

blanc

Box plot for tenderness

score

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

ter12

nitrite

ter7

ter7+powder3st

ter10

blanc

Figure 4. 7 Box plots of odor and texture of terrines

141

The table 4.11 shows the adjusted means of descriptors flavor, smell and texture of

terrines, using an analysis of variance model with descriptor. This test, performed by all

products and all descriptors was introduced in the table of adjusted means via a color-

coded: blue when the coefficient is significantly larger than mean, red when it is

significantly lowerand white when it is not significantly different from zero.

Table 4.11 Adjusted means of descriptors flavor, smell and texture of terrines

Attributes

Sample Salty sweety acidity Bitter spicy Ranci

d

aromatic tender Juicy

Blanc 2.583 1.333 1.361 1.25 2.083 1.222 2.222 2.917 2.444

Nitrite 2.583 1.444 1.583 1.5 1.972 1.333 2 3.111 2.944

Sample

7 2.778 1.5 1.639 1.333 2.778 1.389 3.333 3.417 3.306

Sample

7+ 3st 2.222 2.167 1.694 1.611 2.333 1.694 2.944 3.25 2.556

Sample

10 2.583 1.639 1.528 1.333 2.472 1.25 2.944 3.056 2.917

Sample

12 2.75 1.444 1.5 1.444 2.444 1.361 2.833 3.111 3.111

4.5.3.3. Principal Component Analysis (PCA) for terrines

For flavor of terrines

The SensoMineR software made a Principal Component Analysis (PCA) with the

adjusted means by terrines and descriptors. The PCA was applied as an unsupervised

pattern recognition method to observe trends in the data and to indicate relationships

between the 6 terrines and/or between the sensory attributes. Fig. 4.8 shows the

projection of the terrines samples on the plane defined by the first and second principal

components (Fig.4.8a) and also the corresponding loading plot (Fig.4.8 b). The first and

second principal components (Dim.) describe 88.2% of the variability (60.73% Dim 1

and 27.47% Dim 2, respectively. The first dimension, highly correlated with the

sweetness of the terrines and the second dimension with the spiciness of the terrines.

142

Terrine 7+ powder 3st is is characterized by a sweeter taste and terrine with formulation

7, by a spicier taste, compared to other terrines. Terrines 10 and 12 were closer to the

terrine with nitrites in terms of flavor and were not significantly more spicy or sweet as

the nitrite one.

a)

b)

Figure 4.8 Score plot (a) and loading plot (b) of the PCA performed from the sensory

analysis of flavor of terrines with nitrite (echNit), formulation 7(ech7), formulation

7+powder 3st (ech7fr), formulation 10 (ech10), formulation 12 (ech 12) and the

blanc(blanc)

143

For odor and texture

The same analysis were made for the attributes of odor and texture (Figure 4.9). The

first and second principal components (Dim.) describe 88.55% of the variability

(59.71% Dim 1 and 28.84% Dim 2, respectively. The results showed that there was a

difference of aromaticity, especially for the terrine with formulation 7. Terrine with

formulation 12 were closer to the nitrites one in terms of smell and texture

a)

b)

Figure 4.9 Score plot (a) and loading plot (b) of the PCA performed from the sensory

analysis of odor and texture of terrines with nitrite (echNit), formulation 7(ech7),

formulation 7+powder 3st (ech7fr), formulation 10 (ech10), formulation 12 (ech 12) and

the blanc(blanc)

144

4.5.3.4. Variance analysis for hams

For flavor of hams

The same analysis were made for ham. The results of the ANOVA are presented in

Table 4.12 . Regarding the variance ratio F of samples, for a probability p <0.005, there

is no significant difference in taste between the 6 tested hams.

Table 4.12 Analysis of variance of results of ham's flavor

Attributes

sweetness salty Bitterness Acidity spicy

Source of

Variation DF F P F P F P F P F P

panelist 8 8.39 <0.001 5.701 <0.001 1.561 0.17 8.471 <0.001 9.194 <0.001

sample 4 2.648 0.051 1.054 0.395 1.626 0.19 0.471 0.757 1.500 0.226

Residual 32 F 5% 2.69

F 1% 4.02 Total 44

*indicates a significant difference at 5% **indicates a significant difference at 1%

For odor and texture of hams

Table 4.13 showed a significant difference of aromaticity and tenderness between 6

hams tested with a probability of 1% and 5% respectively.

Table 4.13 Analysis of variance of results of ham's odor and texture

Attributes rancidity aromaticity tenderness juiciness Source of

Variation DF F P F P F P F P

panelist 8 12.623 <0.001 4.894 <0.001 2.293 0.046 11.25 <0.001 sample 4 1.351 0.273 4.663** 0.004 2.862* 0.039 2.350 0.075 Residual 32 F 5% 2.69

F 1% 4.02 Total 44

*indicates a significant difference at 5% **indicates a significant difference at 1%

145

4.5.3.5. Tukey test and descriptive analysis for hams

For flavor of hams

The sweet taste of ham with formulation 7 was lower and significantly different from

other hams. There was no significant difference from the spiciness of 5 hams, although

the ham with the formulation 10 is the higher average score (1.7780). The same applies

to the bitter and acid taste, although the ham with the formulation 10 has the highest

score (1.4440) in both cases. (Table 4.14)

Table 4.14 Results of Mean and Least Significant Difference for flavor of ham

Hams Attributes1

sweetness Salty Bitterness Acid Spicy

Ham with Nitrite 1.6670a 1.5560a 1.1110a 1.3330a 1.3330a

Ham with formulation 7 1.3330b 1.7780a 1.0000a 1.2220a 1.6670a

Ham with formulation 7 + powder 3st 2.0000a 1.3330a 1.4440a 1.3330a 1.6670a

Ham with formulation 10 1.7780a 1.6670a 1.4440a 1.4440a 1.7780a

Ham with formulation 12 1.6670a 1.5560a 1.4440a 1.3330a 1.7780a

Standard error2 0.1480 0.1610 0.1700 0.1150 0.1490

LSD3 0.56 0.606 0.640 0.432 0.564 1Mean of 9 observations. 2Mean standard error of mean scare on 32 df. 3Least significant difference

The descriptive analysis presented in Fig.4.10 showed the most discriminating

descriptors of flavor for the 5 hams. There was no significant difference between the

various flavors of hams according to the Tukey test. However, the descriptive analysis

shows that the ham with formulation 7 + powder 3st appears to have a slightly

pronounced sweetness taste than other hams. Ham with formulation 12 had the same

sweetness profile as nitrites one and ham with formulation 10 and 12 seemed to be a

little bit spicier than other hams.

146

sweetness

salty

bitterness

acidity

spicy

0.0 0.5 1.0 1.5 2.0

ham 7 vs attributesham 7 + powder3st vs attributes ham Nit vs attributesham 10 vs attributes ham 12 vs attributes

Figure 4. 10 Sensory profile analysis of flavor sweetness, salty, bitterness, acidity and

spicy of tested hams

The box plot of ham's flavor , which is another representation of Tukey test, was

presented in Figure 4.11 . The 5 hams selected were divided into the same 3 sub-groups

as terrines. No descriptor is discriminative in our case. They are all from the group 3.

148

For odor and Texture

For the odor and texture, the results presented in Table 4.15 showed that hams with

formulation 10 and 7+ powder 3st are significantly different from ham with nitrite in

term of aromaticity. There is a significant difference of juiciness between the ham

formulation 7 and 10. There is no significant difference of tenderness between the 5

hams, while the ham with formulation 7 has the highest average score (2.4440). The

rancid smell of ham is not significantly different between the 5 hams, although the ham

with formulation 7 + powder 3st has the highest score (1.4440).

Table 4.15 Results of Mean and Least Significant Difference for odor and texture of

hams

Hams Attributes1

rancidity aromaticity tenderness Juiciness

Ham with Nitrite 1.3330a 1.5560b 1.7780a 1.8890ab Ham with formulation 7 1.2220a 2.1110ab 2.4440a 2.2220a Ham with formulation 7 + powder 3st 1.4440a 2.5560ac 2.1110a 1.8890ab Ham with formulation 10 1.1110a 3.0000a 2.2220a 1.5560b Ham with formulation 12 1.2220a 2.3330ab 1.6670a 1.7780ab

Standard error2 0.1090 0.2480 0.1890 0.1570

LSD3 0.413 0.936 1.353 0.595 1Mean of 9 observations. 2Mean standard error of mean scare on 32 df. 3Least significant difference

The descriptive analysis presented in Fig. 4.12 showed the most discriminating

descriptors of odor and texture for the 5 hams. According to the results, there was no

significant difference between hams containing nitrites and other hams, except for

juiciness and aromaticity descriptors. Ham formulation 7 is juicier, tenderer and more

aromatic.

150

Box plot for aromaticity of ham

score

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

ham7fr

ham10

ham12

ham 7

hamNit

Box plot for rancidity of ham

score

0.5 1.0 1.5 2.0 2.5 3.0 3.5

ham7fr

ham12

hamNit

ham 7

ham10

Box plot for juiciness of ham

score

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

ham 7

ham7fr

ham12

ham10

hamNit

Box plot for tenderness of ham

score

0.5 1.0 1.5 2.0 2.5 3.0 3.5

ham10

ham 7

ham7fr

hamNit

ham12

Figure 4.13 The box plots of 5 tested hams with following descriptors: rancidity,

aromaticity, tenderness and juiciness

The table 4.15 shows the adjusted means of descriptors flavor, smell and texture of

hams, using an analysis of variance model with descriptor thanks to the software

SensoMineR (products * descriptors). This table 16 is a summary in the scores

presented above.

151

Table 4.16 Adjusted means of descriptors flavor, smell and texture of hams

Attributes

Ham

sample

salty sweety acid bitter spicy Rancid aromatic tender juicy

Nitrite 1.556 1.667 1.333 1.111 1.333 1.333 1.556 1.778 1.889

Sample 7 1.778 1.333 1.222 1 1.667 1.222 2.111 2.444 2.222

Sample

7+3st 1.333 2 1.333 1.444 1.667 1.444 2.556 2.111 1.889

Sample 10 1.667 1.778 1.444 1.444 1.778 1.111 3 2.222 1.556

Sample 12 1.556 1.667 1.333 1.444 1.778 1.222 2.333 1.667 1.778

4.5.3.6. Principal Component Analysis (PCA)

For flavor of terrines

The first and second principal components (Dim.) describe 100% of the variability

(89.63% Dim 1 and 10.37% Dim 2, respectively). It is the reason why there are only

two attributes, sweetness and bitterness. Hams are not significantly different between

them and between the one with nitrites. However, ham 7 + powder 3st is the sweetest

because of the strawberry powder.

152

a)

b)

Figure 4.14 Score plot (a) and loading plot (b) of the PCA performed from the sensory

analysis of flavor of hams with nitrites (hamNit), formulation 7(ham7), formulation

7+powder 3st (ham7fr), formulation 10 (ham10), formulation 12 (ham12)

For odor and texture

The results of analysis of PCA for odor and texture of ham showed, in Fig.4.16 the first

and second principal components (Dim.) describe 96.34% of the variability (52.65%

Dim 1 and 43.69% Dim 2, respectively). The absence of the attribute rancidity shows

that rancidity is not significant. Ham with the formulation 12 has a profile close enough

with ham with nitrite. Ham 7, nevertheless, seems more tender and juicy and 10 more

aromatic.

153

a)

b)

Figure 4.15 Score plot (a) and loading plot (b) of the PCA performed from the sensory

analysis of odor and texture of hams with nitrites (hamNit), formulation 7(ham7),

formulation 7+powder 3st (ham7fr), formulation 10 (ham10), formulation 12 (ham12)

4.5.4. Discussion

Terrines with the best anti microbial and antioxidant results were selected for this study:

the formulation 7 (clove 0,3%; cumin 0,3%; cinnamon 0,1%), 10 (clove 0,368%; cumin

0,2%; cinnamon 0,2%) and 12 (clove 0,2%; cumin 0,368%; cinnamon 0,2%). The

results for the quantification of the addition of strawberry powder in the terrine

formulation 7 gave very satisfactory results in terms of color, as well as close to the

values of nitrite. Regarding the organoleptic tests, the Tukey test for flavor, showed that

154

there was no significant difference between the different terrines in terms of taste.

Analysis of variance and descriptive analyzes terrines showed the most discriminating

attributes which are spicy for terrine with formulation 7 and sweetness for terrine with

formulation 7 + powder 3st. The aromaticity is present in terrines containing spices,

which is normal. However, the table of ajusted means and analysis of PCA showed that

the terrine with formulation 12 has a similar profile to the one with nitrites. Moreover,

the rancid smell noticed in terrine with formulation 7 + powder 3st is probably due to

transport of samples and the composition of the strawberry powder. There has been a

breakdown in the cold chain. More cautiously, terrine with formulation 7 + strawberry

would surely have a closer nitrites profile taste. The slightly bitter taste of this terrine is

probably comes from the variety of strawberry powder incorporated into the sample.

For hams, there was also no difference in terms of taste but more in terms of texture

according to the Tukey test. Hams with spices do not seem spicier than those containing

nitrites, but more aromatic like ham containing the formulation 10. On rancidity of

hams, compared to the terrines, there was no significant difference with nitrites; this is

due to the nature of the fatty acids present in. There are more satured fatty acids in ham

than in terrine. Ham with formulation 7 is very interesting in terms of its sensory

properties compared with ham + nitrites. Terrine with formulation 7 tended to be juicier

in both terrines and hams. To quantify the addition of strawberry powder or other

natural coloring agents, it would be advisable for future studies to use the formulation

10 and 12, with average values close to the nitrites one. The strawberry powder played a

relatively important role in this organoleptic tests. It would be wise, for a future study,

to analyze the concentration of strawberry on sensory effects and thus a compromise

between color samples and sweet setting.

4.5.5. Aknowledge

The authors sincerely thank the FQRNT (programme special) in collaboration with four

agro-industries, MDEIE and MAPAQ for their financial support. We express our

gratitude to the panelists and to Mr. Boiteau of Aliments Breton for supplying us

the raw meat to carry out various tests. The views and the opinions expressed in this

article are those of the authors.

155

4.5.6. References

Afshari-Jouybari, H., Farahnaky, A. (2011). Evaluation of Photoshop software potential for food colorimetry. Journal of Food Engineering. 106 : 170–175

Byrne D.V., O’Sullivan M.G., Dijksterhuis G.B., Bredie W.L.P. and Martens M. 2001. Sensory panel consistency during development of a vocabulary for warmed-over flavour. Food Qual. Prefer. 12:171.

C. Calvo, A. Salvador, S.M. Fiszman. Eur. Food Res. Technol., 213 (2001), pp. 99–103

Chang, S. T., Chen, P. F., and Chang, S. C. (2001). Antibacterial activity of leaf essential oils and their constituents from Cinnamomum osmophloeum. J. Ethnopharmacol. 77 : 123–127

CIE, CIE 1976 L*a*b* Color Space, ISO 11664-4:2008(E)/CIE S 014-4/E (2007)

CIE (1986). Colorimetry (2nd ed.), Publication No 15.2, Wien, Austria: CIE Central Bureau Kegelgasse 27 A-1030.

Depledt, F.(2009), “Évaluation sensorielle, Manuel méthodologique”, Collection Sciences & Techniques Agroalimentaires, Éditions TEC & DOC, 3e édition

D.B. MacDougall (2002). Colour in Food: Improving Quality. Woodhead Publishing

D.L. Huffman, K.A. Davis, D.N. Marple, T.A. Mcguire. (1975). Effect of gas atmospheres on microbial growth, color and pH of beef. Journal of Food Science, 40, pp. 1229–1231

F. Gasperi*, F. Biasioli, G. Gallerani, S. Fasoli and E. Piasentier. (2005). Training of a sensory panel for quantitative descriptive analysis of lamb meat Ital. J. Food Sci. n. 3, vol. 17

Fatma Gassara1, Anne Patricia Kouassi2, Satinder Kaur Brar1, Khaled Belkacemi2. (2014). Optimisation of spices as alternative of nitrites and nitrates in the meat-based products (submitted article)

Gorraiz C., Berian M.J., Chasco J. and Iraizoz M. (2000). Descriptive Analysis of meat from young ruminants in Mediterranean systems. J. Sens. Stud. 15:137.

H.A. Roth, L. Radle, S.R. Gifford, F.M. Clydesdale. (1988). J. Food Sci., 53, pp. 1116–1162

H. Morita, R. Sakata, Y. Nagata. Nitric oxide complex of Iron (II) myoglobin converted from metmyoglobin by Staphylococcus xylosus. Journal of Food Science, 63 (1998), pp. 352–355

Honikel, K.O. (2008). The use an control of nitrate and nitrite for the processing of meat products. Meat Science, 78, 68-76.

IPCS International Programme on Chemical Safety on website: http://www.inchem.org/documents/jecfa/jecmono/v50je07.htm#1.1

156

J. R. Claus, C. Du. (2013). Nitrite-embedded packaging film effects on fresh and frozen beef color development and stability as influenced by meat age and muscle type James R. Claus, Chen Du. Meat Science. 95 : 526–535

J. Chasco, G. Lizaso, M.J. Beriain. (1996). Cured colour development during sausage processing. Meat Science, 44 (3), pp. 203–211

J. Claus, S. Mohanan, R. Russell. Biochemical and physical properties of ten different beef muscles in relation to meat color. 51st International Congress of Meat Science and Technology. Proceedings and poster, American Meat Science Association, Champaign, IL USA (2005, August 7-12) (Abstract 11611, Baltimore, MD)

J.Y. Jeong, J.R. Claus (2011). Color stability of ground beef packaged in a low carbon monoxide atmosphere or vacuum. Meat Science, 87, pp. 1–6

Q. Liu, M.C. Lanari, D.M. Schaefer (1995). A review of dietary vitamin E supplementation for improvement of beef quality. Journal of Animal Science. 73 : 3131–3140

R. Korifi, Y. Le Dréau, J-F. Antinelli, R. Valls, N. Dupuy,Talanta (2013). CIEL*a*b* color space predictive models for colorimetry devices – Analysisof perfume quality. 104 : 58–66

T.N. McCaig. (2002). Extending the use of visible/near infrared reflectance spectrophotometers to measure colour of food and agricultural products. Food Research International. 3: 731–736

U.S. Environmental Protection Agency (USEPA) (2007). Washington, D.C. "National.

Stone H., Sidel J., Oliver S., Woolsey A. and Singleton R.C. (1974). Sensory evaluation by quantitative descriptive analysis. Food Technol. 28 (11):24

X. Zhang, B. Kong, L. Y. Xiong. (2007). Production of cured meat color in nitrite-free Harbin red sausage by Lactobacillus fermentum fermentation. Meat Science. Volume 77: 593–598.

157

Chapitre 5 Analyse technico-économique du procédé de production des produits carnés (Utilisation des épices comme alternative aux nitrites et nitrates

158

5.1. Description des scénarios de simulation employés pour l'analyse technico-économique de la production des produits carnés

Dans le cadre de cette étude, l’évaluation de l’intérêt technico-économique de

l'utilisation des épices et de la poudre de fraise comme alternative aux nitrites dans les

produits carnés, été réalisée à partir d’une série de 4 scénarios. Les 4 scénarios sont

présentés en détails dans les figures 5.1 et 5.2. Les deux premiers scénarios tiennent

compte de la production de 350.000 kg/an de terrines et les deux autres scénarios

tiennent compte de la production de 1.500.000 kg/ an et de jambon. Le premier

scénario, est le scénario de référence pour la production des terrines (contenant les

nitrites) et le deuxième scénario tient compte de la production des terrines en utilisant

les épices comme agent de conservation . De même, Le troisième scénario, est le

scénario de référence pour la production des jambons (contenant les nitrites) et le

quatrième scénario tient compte de la production des jambons en utilisant les épices

comme agent de conservation. Les paramètres d’opération et les hypothèses de base

utilisés dans les 4 scénarios ont été présentés en détails dans le Tableau 5.1.

Tableau 5.1 Capacité de production de l'entreprise des produits carnés

Scénario 1 Scénario 2 Scénario 3 Scénario 4

Produits Terrines Terrines Jambon Jambon

Production 350.000 kg/an 350.000 kg/an 1.500.000 kg/ an 1.500.000 kg/ an

160

Figure 5.2 Scénarios 3 et 4 utilisés dans l’analyse technico-économique: Production du

jambon

5.2. Estimation du coût des capitaux fixes

La méthode factorielle d'estimation des capitaux fixes (Cfx) est exprimée par l'équation:

Cfx =fL *Ceq

fL : le facteur de Lang ( Sinnott, 1996) .

Ceq= coût d' achat des équipements (M $)

Le facteur de Lang dépend du type de l'industrie . Pour les entreprises alimentaires, le

facteur de Lang est de 1,8 (Maroulis & Saravacos, 2003 ).

Des divisions plus sophistiquées des capitaux fixes sont prises en compte dans la

littérature (Clark, 1997; Peters & Timmerhaus, 1991). D'après ces travaux, les capitaux

fixes sont estimés en utilisant l'équation 3.

161

Cfx = Ceq + Ccv +Cme (1)

Avec

Ccv : Coût des travaux de génie civil, y compris les aménagements, les bâtiments et les

structures.

Cme : Coût des travaux mécaniques et électriques, y compris l'installation des

équipements, la tuyauterie, l' instrumentation et le contrôle, les équipements électriques,

l'ingénierie et la supervision.

Ccv =fcv *C (2)

Cme =fme *Ceq (3)

fcv : facteur des travaux de génie civil

fme : facteur des travaux mécaniques et électriques

Cfx = (1 + fcv + fme)* Ceq (4)

Pour estimer les capitaux fixes totaux, nous étions basés sur les données de Bartholomai

(1987), qui a déterminé les capitaux fixes des différentes entreprises alimentaires y

compris l'entreprise des produis carnés. L'estimation des capitaux fixes par Bartholomai

(1987) a été présentée en détaille dans le tableau 5.3. En revanche, il est primordial de

faire la mise à jour des coûts donnés par Bartholomai (1987). Pour cette raison, nous

avons adopté l'équation 6 pour calculer les coûts des capitaux fixes d'une entreprise de

produits carnés pour l'année 2013.

Coût des capitaux fixes (2013)= Coût des capitaux fixes (1987)• (CE index 2013/CE

1987) (5)

CE index : 593,2

CE index 1987: 318,4

Coût des capitaux fixes (1987): 2000000 $: Cette valeur correspond aux coûts des

capitaux fixes d'une entreprise de produits carnés.

Les résultats de l'estimation des capitaux fixes relatifs aux différents scénarios d'analyse

ont été présentés en détail dans le Tableau 5.3.

162

Table 5.2 Summary of cost data for food plants in 1986 (Bartholomai (1987); Clark (1997); adapted by Rouweler).

Total invest-

ment cost

Raw

mate-

rials

Finis-

hed

pro-

duct

capac.

Area

(land)

Area

(land)

Equipment

cost

Wor king

hours

Investment

cost

Food production

plant

(excl. land) (excl. land)

US$ TON/h TON/h m2 ft2 US$ h/year $/ton prod/h

for details: (1986) (1986) (1986)

see Bartholomai

(1987)

[<-use for 0.6 exponent [<--Do NOT use in 0.6 exponent

calculation-->] calculation->]

1 Apple processing

(slices/sauce)

$2 923 000,00 5 4 1 860 20 000 $2006 000,00 1000 $731 000,00

2 Cannery

(community; small

scale)

$204 000,00 5 4.8 140 1 500 $123 000,00 1500 $42 500,00

3 Fruit puree

(aseptic; bag in box)

$1 100 000,00 4 3 470 5 000 $782 000,00 2000 $367 000,00

4 Multi purpose

fruit (jam; preserv)

3 2.4 95 1 000 $402 000,00

5 Orange juice

(concentration)

$2 057 000,00 20 1.6 1 860 20 000 $1116 000,00 2000 $1286 000,00

6 Baby food (glass

jars)

$200 000,00 6.6 5.5 280 3 000 $184 000,00 2000 $36 000,00

7 Tomato paste $1 837 000,00 15 2.5 $1087 000,00 2000 $735 000,00

8 Frozen vegetable $1 340 000,00 4.8 2.2 2 800 30 000 $852 000,00 2000 $609 000,00

9 Mushroom farm $116 000,00 0.04 5 600 60 000 $41 000,00 $2 900000,00

163

10 Mozzarella

cheese

$842 000,00 5.6 0.7 1 400 15 000 $350 000,00 3000 $1 203000,00

11 Blue cheese $4 093 000,00 11.5 2.1 10

500

113

000

$2908 000,00 2560 $1949 000,00

12 Dairy (fresh

milk; UHT; etc)

$13530000,00 20.8 18.1 6 510 70 000 $6400 000,00 2400 $748 000,00

13 Modular dairy $1 210 000,00 2 2 9 300 10 000 $760 000,00 250days/y $605 000,00

14 Powder milk $4 000 000,00 18 1.7 1 400 15 000 $2835 000,00 7200 $2352 000,00

15 Dried whole egg $2 287 000,00 2.5 0.25 1 120 12 000 $1357 000,00 2000 $9148 000,00

16 Yoghurt $4 286 000,00 8 8 7 900 85 000 $3 477000,00 3125 $536 000,00

17 Ice cream $2 515 000,00 2 2 1 600 17 000 $1530 000,00 2000 $1258 000,00

18 Parboiled rice $1 379 000,00 5 5 560 6 000 $1004 000,00 7200 $276 000,00

19 Corn starch $30298

000,00

8.3 5.3 2 240 24 000 $14169000,00 7920 $5717 000,00

20 Pasta $2 353 000,00 0.73 0.71 1 860 20 000 $1 760 000,00 5500 $3314 000,00

21 Precooked

lasagna

$3 261 000,00 0.7 0.67 1 070 11 500 $2 305 000,00 5700 $4867 000,00

22 Tofu 0.38 1.32 140 1 440 $350 000,00

23 Baker's yeast $26550

000,00

2.5 1.14 7 500 80 000 $11049000,00 7200 $23289000,00

24 Vinegar $750 000,00 0.033 0.31 350 3 750 $525 000,00 7200 $2 419 000,00

25 Quenelles $985 000,00 0.66 0.66 325 3 500 $600 000,00 2000 $1 492 000,00

26 Tortilla chip $1 688 000,00 0.5 0.55 900 9 500 $1 270 000,00 3500 $3 069 000,00

27 Corn snacks $310 000,00 0.18 0.27 470 5 000 $131 000,00 2000 $1 148 000,00

28 Catfish $2 400 000,00 3.2 1.8 1 120 12 000 $1 040 000,00 2000 $1 333 000,00

29 Shrimp $431 000,00 0.5 0.25 560 6 000 $204 000,00 1500 $1 724 000,00

30 Surumi $10 000

000,00

15 5.7 10

700

115

000

$6 080 000,00 3528 $1 754 000,00

31 Cattle slaughter $3 660 000,00 40 16 4 830 52 000 $930 000,00 1800 $228 000,00

32 Co-extruded

sausage

$2 000 000,00 1 1 930 10 000 $1 049 000,00 1600 $2 000 000,00

33 Protein recovery $2 671 000,00 12 6 930 10 000 $1 972 000,00 4000 $445 000,00

34 Soybean oil $24 900 00,00 42 42 2 330 25 000 $6 200 000,00 7200 $593 000,00

164

extraction

35 Vegetable oil

refinery

$2 359 000,00 2 1.8 930 10 000 $1 456 000,00 6000 $1 311 000,00

36 Pan bread $2 803 000,00 1.5 2.2 2 600 28 000 $1 795 000,00 5000 $1 274 000,00

37 Arabic bread $1 272 000,00 1.2 1.7 930 10 000 $702 000,00 6240 $748 000,00

38 Half-baked

frozen baquette

$1 953 000,00 0.48 0.54 981 10 560 $1 228 000,00 4000 $3 617 000,00

39 Seawater

desalination

$18

433000,00

3100 417 105 1 100 $9 262 000,00 7200 $44 200,00

40 Fruit juices from

concentrate

$809 000,00 2 2 675 7 240 $514 000,00 2000 $405 000,00

Soymilk 0.15 1 300 3 150 $910 000,00

Orange juice

concentrate

$2 424 000,00 20 1.5 $890 000,00 $1 616 000,00

Sausage $10

323000,00

1 1 $3 219 000,00 $10323000,00

165

Table 5.3 Estimation des capitaux fixes

Scénario 1 Scénario 2 Scénario 3 Scénario 4

Capacité (ton /

2013)

350 350 1200 1200

Capitaux fixes

(2013)

1497018 1497018 3135414 3135414

5.3. Estimation du coût d'exploitation annuel

De la même manière, le coût d'exploitation annuel peut être estimé sur la base des coûts

des matières premières et des services publics, en utilisant des facteurs appropriés. Les

coûts des matières premières et des services publics peuvent être calculés avec précision

en utilisant les bilans massiques et les bilans énergétiques. Pour le cas des entreprises

alimentaires, le coût des matériaux d'emballage est important (Clark, 1997) et il

devrait être inclus dans le coût des matières premières.

La méthode à un seul facteur pour l'estimation du coût d'exploitation annuel

est exprimée par l'équation ci-dessous:

Cop =fop *Cmu (6)

fop : le facteur de coût d'exploitation

La méthode factorielle détaillée pour l'estimation du coût d'exploitation annuel (Cop) est

résumée par les équations suivantes:

Cop = Cmu + Clabour +Cmisc (7)

Clabour =flabour *Cmu (8)

Cmisc=fmisc *Cmu (9)

Avec

Cop = Coût d'exploitation annuel

Cmu : Coût des matières premières et des services publics

166

Clabour : Coût de la main d'œuvre

Cmisc : Coût divers (entretien, les réparations, les redevances et brevets)

flabour :facteur du coût de la main d'œuvre

fmisc : facteur des coûts divers

Le modèle ci-dessus est équivalent à ce qui suit:

Cop = (1 + flabour + fmisc)* Cmu (10)

D' après les travaux de Mouralis et Mouralis (2004), dans le cas des entreprises

agroalimentaires :

Cop =1,1 *Cmu

Pour estimer le coût d'exploitation annuel, nous avons déterminé d'abord le coût des

matières premières, puis les résultats de l'estimation du coût des matières premières et

des services publics et l'estimation du coût d'exploitation annuel. Ces résultats sont

présentés dans le tableau 5.5.

5.4. Estimation du coût des matières premières

Les matières premières nécessaires pour la production des terrines et des jambons

constituent une fraction notable du coût de production. Le tableau 5.4 représente la

consommation et les coûts des matières premières utilisés dans les différents scénarios.

167

Table 5.4 Coûts des matières premières utilisés dans les différents scénarios

Scénario Matières premières quantité

utilisé (%)

Prix par tonne Références

Scénario 1 Fois de porc 25 700 Canadian $ Alibaba.com

Gras mou 40 700 Canadian $ Alibaba.com

Oeufs 5 1,92 $ / 12 gros

œufs

Agriculture

Canada, Nov 2013

Lait entier 25 958 $/ 1000 L Centre canadien

d'information

laitière, 2013

Sucre 0,4 550-700 $ Alibaba.com

Sel ordinaire 0,9 155 $ Alibaba.com

Sel nitrité 1 2000 $ Alibaba.com

Oignons 2 500 $ Agriculture canada,

2013

Ail frais 0,5 2000 $ Alibaba.com

Poivre blanc 0,15 $ 1000 $ Alibaba.com

Assaisonnement 0,05 1000- 2500 $ Alibaba.com

Scénario 2 Fois de porc 25 700 Canadian $ Alibaba.com

Gras mou 40 700 Canadian $ Alibaba.com

Œufs 5 1,92 $ / 12 gros

œufs- 2689 $

/tonne

Agriculture

Canada, Nov 2013

Lait entier 25 958 $/ 1000 L Centre canadien

d'information

laitière, 2013

168

Sucre 0,4 550-700 $ Alibaba.com

Sel ordinaire 1 $ 155 $ Alibaba.com

Oignons 2 $ 500 $ Agriculture canada,

2013

Ail frais 0,5 2000 $ Alibaba.com

Poivre blanc 0,015 1000 $ Alibaba.com

Assaisonnement 0,05 1000- 2500 $ Alibaba.com

Cumin 0,3 2000 $ Alibaba.com

Clous de girofles 0,3 8000$ Alibaba.com

Cannelle 0,1 1800$ Alibaba.com

Poudre de fraise 0,9 25000$ Alibaba.com

Scénario 3 Jambon 100 2680 $ Agriculture et

agroalimentaire

Canada, Novembre

2013

Sel nitrite 1 2000 $ Alibaba.com

Sel ordinaire 0,9

155 $ 1,395

Scénario 4 Jambon 100 2680 $ Agriculture et

agroalimentaire

Canada, Novembre

2013

Cumin 0,3 2000 $ Alibaba.com

Clous de girofles 0,3 8000$ Alibaba.com

Cannelle 0,1 1800$ Alibaba.com

Poudre de fraise 0,9 25000$ Alibaba.com

Sel ordinaire 0,9

155 $ Alibaba.com

169

Remarque: Le poids moyen de l'œuf gros est de 56 à 63 g (Fédération des

producteurs d'œufs de consommation du Québec)

Table 5.5 Estimation du coût total des matières premières et du coût d'exploitation

annuel

Scénario

Coût des matières

premières/ tonne de

produit finis ($)

Productivité

(tonne)

coût total annuel

des matières

premières ($)

coût d'exploitation

annuel ($)

1 875,22

350 306327 336959,7

2 1112,02

350 389207 428127,7

3 2701,395

1500 4052093 4457302

4 2938,195

1500 4407293 4848022

5.5. Estimation du coût du produit

L'analyse téchnico-économique réalisée dans cette étude est basée sur le model de

(Marouli et Maroulis, 2005). Le but de cette nalyse est de déterminer le coût du produit,

qui est la terrine, dans les scénarios 1 et 2, et le jambon dans les scénarios 3 et 4. Le

coût du produit est calculé en se basant sur la formule 11.

C= (e * Cfx + Cop)/F (11)

Avec

C: cout du produit ($/kg)

e : Facteur de recouvrement du capital: 5 ans

Cfx: Coût des capitaux fixes (M $ / an)

Cop: coût annuel de fonctionnement (M $ / an)

F: capacité de production annuelle (1 Gg = 106 kg)

170

Figure 5.3 Estimation du coût de production des terrines de foie de porc et du jambon;

Scénario1: terrines avec nitrites, Scénario2: terrines avec épices+poudre de fraise,

Scénario3: jambon avec nitrites, Scénario1: jambon avec épices+poudre de fraise

5.6. Conclusion

D’après les résultats trouvés dans la Figure 5.3 , l'utilisation des épices et de la poudre

de fraise comme alternatives aux nitrites à augmenté le coût de production des produits

carnés. Cette augmentation est faible dans le cas des terrines de foie de porc (8,42%) et

du jambon (7,51%). Cette faible augmentation du coût de production des produits

carnés en utilisant les épices et la poudre de fraise comme alternatives aux nitrites est

expliqué par une légère augmentation du coût des matières premières. Ceci est

principalement dû aux coûts élevés des épices, notamment les clous de girofle

(8000$/tonne) et de la poudre de fraise (25000$/tonne). Néanmoins, le coût des sels

nitrites, habituellement utilisés pour la conservation des produits carnés est relativement

faible (2000$/tonne). Par contre, cette légère augmentation est expliquée par la bonne

qualité des produits carnés contenant les épices et la poudre de fraise comme

alternatives aux épices. Ces alternatives sont sécuritaires pour la santé et leurs coûts ne

sont pas très élevés par apport à d'autres produits qui pourraient constituer un danger

pour la santé des consommateurs (cancer. etc).

0

500

1000

1500

2000

2500

3000

3500

4000

Scénario 1 Scénario 2 Scénario 3 Scénario 4

Coû

t du

prod

uit (

$/to

nne)

171

Conclusion générale

Les nitrates et les nitrites sont des substances chimiques naturelles présentes partout

dans l’environnement. Ils prolongent la durée de conservation des viandes transformées,

stabilisent la couleur des viandes rouges, ralentissent le processus d’oxydation des

lipides et inhibent le développement de microorganismes toxiques comme expliqué

dans le chapitre 1. Cependant, les nitrites, à forte dose peuvent être dangereux pour la

santé et pour l'environnement.

L'objectif général de cette étude était de trouver des alternatives vertes, sans présence de

nitrites et de nitrates au départ, dans les produits carnés, tels que la terrine faite à base

de viande de porc et de lapin et le jambon, en utilisant des épices, connues pour leurs

nombreuses propriétés antibactériennes et antioxydantes. Le premier objectif était le

criblage qualitatif puis quantitatif des additifs alimentaires naturels. Par des analyses

physico chimique et microbienne, effectuée sur une vingtaine d'épices, cinq épices ont

été sélectionnées. Les épices et combinaison d'épices ayant à la fois la meilleure activité

antimicrobienne, les meilleures propriétés anti-oxydantes et antimicrobiennes étaient : le

clou de girofle, le cumin, la cannelle, la cannelle et le clou de girofle, le cumin et le clou

de girofle. Les échantillons formulées avec ces épices avaient un durée de conservation

égale voir supérieure aux échantillons contenant les nitrites.

Dans la deuxième partie de l'étude, une optimisation quantitative a été réalisée en se

basant sur la méthode de réponse de surfaces, qui a founit un plan composite centré de

19 combinaisons de trois épices, les clous de girofle, la cannelle et le cumin, avec des

concentrations allant de 0, 1 % jusqu’au 0, 3% (m/m). Les résultats de ces

expérimentations ont montré que les activités antioxydants de la formulation 7 (clous de

girofle (0,3%) , cannelle (0,1%), cumin (0,3%)) et 15 (clous de girofle (0,2%) , cannelle

(0,2%), cumin (0,2%)) sont les plus importantes. Par contre, les formulations ayant de

très bonnes activités antimicrobiennes tant pour les terrines que le jambon, étaient la

formulation 7 (clous de girofle (0,3%) , cannelle (0,1%), cumin (0,3%)) et la

formulation 13 (clou de girofle (0,2%), cumin (0,2%), cannelle (0,031%)). De plus,

plus les concentrations de clous de girofle et de cumin étaient élevés, plus les activités

antimicrobiennes et les activités antioxydants l'étaient également. Les quantités

172

optimales des épices sélectionnées sont clous de girofle (0,3% p/p) , cannelle (0,1%

p/p), cumin (0,3% p/p) car elles assurent de bonnes qualités microbiologiques et

physico-chimiques des terrines. Ainsi, trois formulations d'épices qui donnaient les

meilleures résultats physico chimiques et microbiologiques, tant pour le jambon que

pour la terrine ,ont été sélectionnées pour la suite de l'etude: la formulation 7, la

formulation 10 (clous de girofle 0,368%, cumin 0,2% cannelle 0,2%) et la formulation

12 (clou de girofle 0,2% cumin 0,368% Cannelle 0,2%).

Voulant garder des propriétés organoleptiques semblables à celles des nitrites, la

coloration des échantillons par les épices étaient l'une des problématiques majeures de

cette étude. Pour palier à ce problème, une poudre de fraise a été ajouté à la formulation

7 . La concentration de la poudre de fraise a été quantifiée, en terme de colorimétrie par

rapport aux nitrites (0.9% p/p), et a servi pour nos tests sensoriels. Les résultats de

l'ANOVA ont montré qu'il n'y avait pas de différence significative entre les goûts des

terrines et des jambons par rapport aux nitrites. Pour obtenir des données plus

spécifiques, des tests de Tukey et une analyse en composante a fait ressortir le caractère

sucré de l'échantillon contenant la poudre de fraise. Cette analyse a révélé une légère

amertume et une certaine rancidité pour les terrined contenant cette poudre de fraise.

Ceci s'explique par un transport peu précautionneux des échantillons lors des

dégustations, une rupture de la chaîne de froid et surement à la variété de fraise utilisées

dans cette poudre. Les terrines et les jambons n'ont pas semblé très épicés dans

l'ensemble, ce qui est un résultat assez encourageant. Les échantillons contenant la

formulation 7 ont même semblés plus tendres et juteux que ceux des nitrites.

Enfin, le dernier chapitre a été consacré à l'analyse technico-économique de cette étude

avec 4 scénarios, les deux premiers concernant la terrine et les deux derniers, le jambon.

Le premier scénario, était le scénario de référence pour la production des terrines

(contenant les nitrites) et le deuxième scénario, de la production des terrines en utilisant

les épices comme agent de conservation . De même, Le troisième scénario, était le

scénario de référence pour la production des jambons (contenant les nitrites) et le

quatrième scénario, de la production des jambons en utilisant les épices comme agent de

conservation. Il en résulte une faible augmentation du coût de production des produits

carnés, 8,42% dans le cas des terrines de foie de porc et 7,51% dans le cas du jambon

étant donné le coût élevé des épices (8000$/tonne pour les clous de girofle) et de la

poudre de fraise (25000$/tonne) par rapport aux nitrites (2000$/tonne). Cette

173

augmentation serait le prix à payer pour avoir des aliments plus sains et sécuritaire pour

la santé.

D'autres études devraient être menées sur l'ajout de poudre de fruits pour la coloration

des viandes, notamment une analyse de l'impact de la concentration de fraise sur les

effets sensoriels des panélistes. Il faudrait de plus rechercher une autre variété de fraise

et faire attention lors du transport des échantillons pour obtenir des résultats plus

satisfaisants. Enfin, à des fins industrielles, une étude antibactérienne sur des bactéries

comme Listeria monocytogenes devrait être menée, étant donné le caractère assez

résistant de ces bactéries, aux traitements de nettoyage-désinfection dans les ateliers de

production de l’industrie agro-alimentaire.

175

Bibliographie

Aboaba, O. & Efuwape, B. M. (2001). Antibacterial Properties of Some Nigerian Species. Bio Research Communications, 13, 183-188. Agbebe, T.M., and Ojeniyi, S.O. (2009). Tillage and poultry manure effects on soil fertility and sorghum yield in southwestern Nigeria. Soil and tillage research. 104 : 74-81. Afshari-Jouybari, H., Farahnaky, A. (2011).Evaluation of Photoshop software potential for food colorimetry. Journal of Food Engineering. 106 : 170–175

Aggarwal, B., Kunnumakkara A. (2009) Molecular Targets and Therapeutic Uses of Spices World Scientific

Ahmed, S. A., Jabbar I. I. and Abdul H. E. (2012). Study the Antibacterial Activity of Zingiber officinale roots against Some of Pathogenic Bacteria. Al- Mustansiriya J. Sci. 3(3):63-70.

Akilen R., Pimlott Z., Tsiami A. and Robinson N. (2013). Effect of short-term administration of cinnamon on blood pressure in patients with prediabetes and type 2 diabetes. Nutrition. 29(10):1192-6.

Almela, L., Sánchez-Muñoz, B., Fernández-López, J. A., Roca, M. J. and Rabe, V. (2006). Liquid chromatograpic–mass spectrometric analysis of phenolics and free radical scavenging activity of rosemary extract from different raw material. J. Chromatogr. A. 1120 (1-2):221-229

Anand, S. P. and Sati, N. 2013 Artificial Preservatives and their harmful effects: Looking toward nature for safer alternatives Int. J. Pharm. Sci. Res. 4(7):2496-2501

Anon, A. (1993). Analytical methods of committee. Application of gas-liquid chromatography to analysis of essential oils Part XVI. Monography for five essential oils. Analyst. 118 : 1089–1098.

Assembly of Life Sciences U.S. (1982). Alternatives to the current use of nitrites in food In: National Academy Press USA. Intl standard book number 0-309-03277-6 pp. 1-3. Arora-Daljit, S. & Kaur, J. (1999). Antimicrobial activity of spices. International Journal of Antimicrobial Agents, 12, 257-262. Bakkali, F., Averbeck, S., Averbeck, D. and Idaomar, M. (2008). Biological effects of essential oils a review. Food Chem. Toxicol. 46: 446- 475.

Balacs, T. (1993). Cajuput components In: Research Reports Int. J. Aromather 5(4):35

Bartley, G. E. and Scolnik, P. A. (1995). Plant carotenoids: Pigments for Photoprotection, Visual Attraction, and Human Health. Plant Cell 7(7):1027-1038

176

Bartsch, H., Ohshima, H., Pignatelli , B. (1988). Inhibitors of endogenous nitrosation mechanisms and implications in human cancer prevention. Mutat. Res-Fund Mol. M.. 202 : 307–324.

Bhat, R., Alias, A. K., and Paliyath, G. 2012. Progress in food preservation. Oxford, UK; Ames, Iowa : Wiley, 2012.

Bhuiyan, M. N. I., Begum, J. and Sultana, M. (2009). Chemical composition of leaf and seed essential oil of Coriandrum sativum L. Bangladesh J. Pharmacol. 4: 150–153. Boza A, De la Cruz Y, Jordan G, Jauregui-Haza U, Aleman A, Caraballo I. (2000) Statistical optimization of a sustained-release matrix tablet of lobenzarit disodium. Drug Dev Ind Pharm. 26:1303-1307.

Bozin, B., Mimica-Dukic N., Samojlik I., Goran, A. and Igic, R. (2008). Phenolics as antioxidants in garlic (Allium sativum L., Alliaceae) Food Chem. 111: 925–929.

Brambilla, G., and Martelli, A. (2005). Keynote comment: nitrosatable drugs, cancer, and guidelines for genotoxicity. Lancet Oncol. 6 : 538–539.

Brown, J.R., Marshall, C., and Smith, J. S. (2007). Nitrate in Soils and Plants. MU Extension G9804.

Burt, S. (2004). Essential oils: their antibacterial properties and potential applications in foods—a review. Int. J. Food Microbiol. 94 : 223-253.

Carlos, A. M. A., and Harrison, M. A. (1999) Inhibition of selected Microorganisms in marinated chicken by pimento leaf oil and clove oleoresin J. Appl. Poult. Res. 8:100-109.

Byrne, D.V., O’Sullivan, M.G., Dijksterhuis, G.B., Bredie, W.L.P. and Martens, M. (2001). Sensory panel consistency during development of a vocabulary for warmed-over flavour. Food Qual. Prefer. 12:171.

Calvo, C., Salvador, A., Fiszman, S.M. (2001). Eur. Food Res. Technol., 213, pp. 99–103.

Carraminana, J. J., Rota, C., Burillo, J., and Herrera, A. (2008). Antibacterial efficiency of spanish Satureja montana essential oil against Listeria monocytogenes among natural flora in minced pork. J. Food Protect. 71(3):502–508.

Celikel, N. and Kavas, G. (2008). Antimicrobial properties of some essential oils against some pathogenic microorganisms. Czech J. Food. Sci. 26:174–181.

Ceylan, E., and Fung, D. Y. C. (2004). Antimicrobial activity of spices. J.Rapid Methods and Autom. Microbiol. 12 : 1–55.

Chaieb, K., Hajlaoui, H., Zmantar, T., Ben, A., Rouabhia, M., Mahdouani, K. and Bakhrouf, A. (2007). The chemical composition and biological activity of clove essential oil, Eugenia caryophyllata (Syzigium aromaticum L. Myrtaceae): a short review. Phytother. Res. 21: 501–506.

Chan, T. Y. K. (2011). Vegetable-borne nitrate and nitrite and the risk of methaemoglobinaemia. Toxico. Lett. 200 : 107–108

177

Chang, S. T., Chen, P. F., and Chang, S. C. (2001). Antibacterial activity of leaf essential oils and their constituents from Cinnamomum osmophloeum. J. Ethnopharmacol. 77 : 123–127.

Chasco, J., Lizaso, G., Beriain, M.J. (1996). Cured colour development during sausage processing. Meat Science, 44 (3), pp. 203–211

Chattopadhyay, I., Biswas, K., Bandyopadhyay, U. and Banerjee, R. K. (2004). Turmeric and curcumin: Biological actions and medicinal applications. Current Science. 87: 1.

Chen, X. C., Jo, C., Lee, J. I. and Ahn, D. U. (1999). Lipid oxidation, volatiles and color changes of irradiated pork patties as affected by antioxidants. J. Food Sci. 64 (1): 16-19.

Cheung S. and Tai J. (2007). Anti-proliferative and antioxidant properties of rosemary Rosmarinus officinalis. Oncol Rep. 17(6):1525-31.

Chung, S. Y., Kim, J. S., Hong, M. K., Lee, J. O., Kim, C. M., and Song, I. S. (2003). Survey of nitrate and nitrite contents of vegetables grown in Korea. Food Add. Contam. 20 : 621-628.

CIE (1986). Colorimetry (2nd ed.), Publication No 15.2, Wien, Austria: CIE Central Bureau Kegelgasse 27 A-1030.

Claus, J., Mohanan, S., Russell, R. Biochemical and physical properties of ten different beef muscles in relation to meat color. 51st International Congress of Meat Science and Technology. Proceedings and poster, American Meat Science Association, Champaign, IL USA (2005, August 7-12) (Abstract 11611, Baltimore, MD)

Cockburn, A., Brambilla, G., Fernández, M. L., Arcella, D., Bordajandi, L. R., Cottrill, B., Van Peteghem, C., and Dorne, J. L. (2010). Nitrite in feed: From Animal health to human health. Toxicol. Appl. Pharmacol.

Coleman, W.M. and Lawrence, B.M. (1992). Comparative Automated Static and Dynamic Quantitative Headspace Analyses of Coriander Oil. J Chromatogr Sci. 30: 396–398.

Conner, D. E., and Beuchat, L. R. (1984). Effect of essential oils from plants on growth of spoilage yeasts. J. Food. Sci. 49 : 429-434.

Davidson, P. M. & Baren, A. L. (1993). Antimicrobials in Foods. Marcel Dekker, New York.

De, M., De, A. K., Mukhopadhyay, R., Banerjee, A. B. and Miró, H. (2003). Antimicrobial Activity of Cuminum cyminum L. Ars Pharmaceutica 44:257-269.

Deis, R. D. (1999). Secret world of spices. Food Product Design, 5, 1-7.

Dennis, M.J., and Wilson, L.A. (2003). Nitrates and Nitrites. In : Encyclopedia of Food Sciences and Nutrition (Second Edition), pp. 4136–4141. Benjamin Caballero, York.

Depledt, F., “Évaluation sensorielle, Manuel méthodologique”, Collection Sciences & Techniques Agroalimentaires, Éditions TEC & DOC, 3e édition, 2009

178

Derwich, E., Benziane, Z. and Chabir, R. (2011). Aromatic and Medicinal Plants of Morocco: Chemical composition of essential oils of Rosmarinus officinalis and Juniperus Phoenicea Int. J. Appl. Biol. Pharm. Technol. 2(1): 145–153.

Derwich, E., Benziane, Z., Manar, A., Boukir, A. and Taouil, R. (2010). Phytochemical Analysis and in vitro Antibacterial Activity of the Essential Oil of Orignum vulgare from Morocco Am.-Euras. J. Sci. Res., 5(2):120–129.

Di Pascua, R., De Feo, V., Villani, F. and Mauriello, G. (2005). In vitro antimicrobial activity of essential oils from Mediterranean Apiaceae, Verbenaceae and Lamiaceae against foodborne pathogens and spoilage bacteria. Ann. Microbiol. 55:139–143.

DIRECTIVE 2006/52/CE DU PARLEMENT EUROPÉEN ET DU CONSEIL du 5 juillet 2006 modifiant la directive 95/2/CE concernant les additifs alimentaires autres que les colorants et les édulcorants et la directive 94/35/CE concernant les édulcorants destinés à être employés dans les denrées alimentaires.

Dorman, H. J. and Deans, S. G. (2000a). Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J. Appl. Microbiol. 88(2):308-316.

Dorman, H.J.D., Surai, P. and Deans, S.G. (2000b). In vitro antioxidant activity of a number of plant essential oils and phytoconstituents. J. Essent. Oil Res. 12(2):241 248.

Du, H. and Li, H. (2008). Antioxidant effect of cassia essential oil on deep-fried beef during the frying process. Meat Sci. 78:461-468.

Dwivedi, B.K., Pandey, G., Pandey, R. C., Pant, H. L. and Logani, R. (2002) Evaluation of angiospermic plant extracts against Rhizopus stolonifer and Gloeosporium psidii fungi of guava. Bioved 13: 129-134.

Eddleston, M., Eyer, P., Worek, F., Mohamed, F., Senarathna, L., Von Meyer, L., Juszczak, E., Hittarage, A., Azhar, S., Dissanayake, W., Sheriff, M. H., Szinicz, L., Dawson, A. H. and Buckley, N. A.(2006). Differences between organophosphorus insecticides in human self-poisoning: a prospective cohort study. Lancet 367 (9508): 396.

El-Sawi, S.A., and Mohamed, M.A. (2002). , Cumin herb as a new source of essential oils and its response to foliar spray with some micro-elements. Food Chem. 77 : 75–80.

Ellis, A., Li, C. G.,, and Rand, M. J. (1998). Effect of xanthine oxidase inhibition on endothelium- dependent and nitrergic relaxations. Eur. J. Pharmacol. 356 : 41-7.

Eisenbrand, G., Schmähl, D., Preussmann, R. (1980) Carcinogenicity of N-nitroso-3-hydroxypyrrolidine and dose–response study with N-nitrosopiperidine in rats. IARC. Sci. Publ. 31 : 657–666.

Ercolini, D., Russo, F., Nasi, A., Ferranti, P. and Villani, F. (2009). Mesophilic and Psychrotrophic Bacteria from Meat and Their Spoilage Potential In Vitro and in Beef. Appl. Environ. Microbiol. 75(7): 1990-2001.

Ernst, E. and Pittler M. H. (2000) Efficacy of ginger for nausea and vomiting. A systematic review of randomised clinical trials. Br. J. Anaesth. 84 (3): 367-371.

179

Exploratorium (2013). Spice blends In: Spice blends of the World Science cooking California USA available online. http://www.exploratorium.edu/cooking/seasoning/map/spicemap.html (assessed August 31, 2013)

Fan, A.M. (2011). Nitrate and Nitrite in Drinking Water: A Toxicological Review. In: Encyclopedia of Environmental Health, pp. 137–145. Jerome O. Nriagu, Oakland.

Fan, A. M., Willhite, C. C., and Book, S. A. (1987). Evaluation of the nitrate drinking water standard with reference to infant methemoglobinemia and potential reproductive toxicity. Regul. Toxicol. Pharmacol. 7 : 135-48.

FAO (2010). Spices producers. In:Food and Agriculture organization of the United States FAOSTAT.

Fatma Gassara1, Anne Patricia Kouassi2, Satinder Kaur Brar1, Khaled Belkacemi2. Optimisation of spices as alternative of nitrites and nitrates in the meat-based products (submitted article)

Frankel, E. N. & Meyer, A. S. (2000). The problems of using one-dimensional methods to evaluate multifunctional food and biological antioxidants. Journal of the Sciences of Food and Agriculture, 80, 1925–1941.

Food Additives (2008). Health Canada. Accessed on: http://www.hc-sc.gc.ca/fn-an/securit/addit/index-eng.php

Accessed on February 2013

Gangolli, S. D., van den Brandt, P. A., Feron, V. J., Janzowsky, C., Koeman, J. H., Speijers, G. J., Spiegelhalder, B., Walker, R., and Wisnok, J.S.(1994). Nitrate, nitrite and N-nitroso compounds. Eur. J. Pharmacol. 292 : 1-38.

Gasperi, F.*, Biasioli, F., Gallerani, G, Fasoli, S. and Piasentier, E. (2005). Training of a sensory panel for quantitative descriptive analysis of lamb meat Ital. J. Food Sci. n. 3, vol. 17

Ghayur M.N. and Gilani A.H. (2005). Ginger lowers blood pressure through blockade of voltage-dependent calcium channels. J Cardiovasc Pharmacol. 45(1):74-80.

Ghori, I. & Ahmad, S. S. ( 2009). Antibacterial activities of honey, sandal oil and black pepper. Pakistan Journal of Botany, 41, 461-466, 2009.

Gopalakrishnan, M. (1992). Chemical composition of nutmeg and mace. J. Spices Aromat. Crops, 1: 49–54.

Gorraiz, C., Berian, M.J., Chasco, J. and Iraizoz, M. (2000). Descriptive Analysis of meat from young ruminants in Mediterranean systems. J. Sens. Stud. 15:137.

Gottelli, N. J. & Ellison, A. M. (2004). A Primer of Ecological Statistics Sinauer Associates. Sunderland, MA.

Govindarajan, V. S. and Connell, D. W. (1982). Ginger: Chemistry, technology and quality evaluation (Part I). Crit. Rev. Food Sci. Nutr. 17(1): 1-96.

180

Grohs, B.-M., and Kunz, B.(2000). Use of spice mixtures for the stabilisation of fresh portioned pork. Food Control . 11 : 433±436

Guenther, E. (1950) The Essential Oils. Vol. IV. Van Nostrand, New York pp. 602-615.

Gulcin I, Sat IG, Bey demir S, Elmastas M, Kufrevioglu OI (2004). Comparison of antioxidant activityof clove (Eugenia caryophyllata Thunb) buds and lavender ( Lavandula stoechas L.). Food Chemistry. 87: 393- 400.

Gupta, S. and Ravishankar S. (2005). A comparison of the antimicrobial activity of garlic, ginger, carrot, and turmeric pastes against Escherichia coli O157:H7 in laboratory buffer and ground beef. Foodborne Pathog. Dis. 2(4):330-40.

Gutierrez, J., Barry-Ryan, C., and Bourke, P.(2009). Antimicrobial activity of plant essential oils using food model media: Efficacy, synergistic potential and interactions with food components. Food Microbiol. 26, : 142–150.

Hajhashemi, V., Ghannadi, A. and Jafarabadi, H. (2004) Black Cumin Seed Essential Oil, as a Potent Analgesic and Antiinflammatory Drug. Phytother. Res. 18(3):195-199.

Haloci, E., Manfredini, S., Toska,V., Vertuani, S., Ziosi, P., Topi, I. and Kolani, H. (2012). Antibacterial and Antifungal Activity Assesment of Nigella Sativa Essential Oils. World Acad. Sci. Eng. Technol. 66:1198-1200

Han H.K. (2011). The effects of black pepper on the intestinal absorption and hepatic metabolism of drugs. Expert Opin Drug Metab Toxicol. 7(6):721-9.

Hayouni, E. A., Chraief, I., Abedrabba, M., Bouix, M., Leveau, J. Y., Mohammed, H. and Hamdi, M. (2008). Tunisian Salvia officinalis L. and Schinus molle L. essential oils: Their chemical compositions and their preservative effects against Salmonella inoculated in minced beef meat. Int. J. Food Microbiol. 125(3): 242–251.

Heinz, D. E. and Varo, P. T. (1970). Volatile Components of Cumin Seed Oil J. Agric. Food Chem. 18: 234–238

Hernández, L., Aguirre Y.B., Nevárez G.V., Gutierrez N. and Salas E. (2011). Use of essential oils and extracts from spices in meat protection. J. Food Sci. Technol.

Herwita and Idris. (2007). The impact Cinnamon Bio-Insecticide to the insect biologic aspect Epilachum varivestis, Mulsant. Jurnal akta Agrosia. 1: 99–105.

Ho, C., Lee, C. Y. and Huang M. (1992) Phenolic Compounds in Food and their effects on Health I. Am. Chem. Soc. Vol. 506.

Holley, R. A., and Patel, D. (2005). Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials. Food Microbiol. 22(4):273–292.

Honikel, K.-O. (2008). The use and control of nitrate and nitrite for the processing of meat products. Meat Sci. 78 : 68–76.Jayaprakasha, G. K., Selvi, T., and Sakariah, K. K. (2003). Antibacterial and antioxidant activities of grape (Vitis vinifera) seed extracts. Food Res Int. 36 : 117–122.

181

Huang, Y., Lam, S.L., and Ho, S.H. (2000). Bioactivities of essential oil from Elletaria cardamomum (L.) Maton to Sitophilus zeamais Motschulsky and Tribolium castaneum. J. Stored Prod. Res. 36: 107-117.

Huffman, D.L., Davis, K.A., Marple, D.N., Mcguire, T.A. (1975). Effect of gas atmospheres on microbial growth, color and pH of beef. Journal of Food Science, 40, pp. 1229–1231

IPCS International Programme on Chemical Safety on website: http://www.inchem.org/documents/jecfa/jecmono/v50je07.htm#1.1

International Commission on Microbiological Specifications for foods Sampling plans for fish and shellfish (1986). In ICMSF (Eds.), Microorganisms in Foods. Sampling for Microbiological Analysis, Principles and Scientific Applications,Vol. 2, (2nd ed.). University of Toronto Press, Toronto, Canada.

ISO (1995). Spices definition. In: Geneva-Based International Organization for Standarisation. ISO 676:1995.

Jackson, A. L. (2010). Investigating the microbiological safety of uncured no nitrate or nitrite added processed meat products. Graduate Theses and Dissertations. Iowa State University.

James, R. C., Chen, D. (2013). Nitrite-embedded packaging film effects on fresh and frozen beef color development and stability as influenced by meat age and muscle type James R. Claus, Chen Du. Meat Science. 95 : 526–535

Janssen, L. H. J. M., Visser, H., and Roemer, F. G. (1989). Analysis of large scale sulphate, nitrate, chloride and ammonium concentrations in the Netherlands using an aerosol measuring network. Atmos. Environ. 23 : 2783–2796.

Jayatilaka, A., Poole, S. K., Poole, C. F., and Chichila, T. M. (1995). Simultaneous micro steam distillation/solvent extraction for the isolation of semivolatile flavor compounds from cinnamon and their separation by series coupled-column gas chromatography. Anal Chim Acta. 302 :147–162.

Jensen, F.B. (2003). Nitrite disrupts multiple physiological functions in aquatic animals. Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol. 135 : 9–24.

Jeong, J.Y., Claus, J.R. (2011). Color stability of ground beef packaged in a low carbon monoxide atmosphere or vacuum. Meat Science, 87, pp. 1–6

Jirovetz, L., Buchbauer, G., Stoilova, I., Stoyanova, A., Krastanov, A. and Schmidt, E. (2006). Chemical composition and antioxidant properties of clove leaf essential oil. J Agric Food Chem 54: 6303–6307.

JUPAC (1986). Standard Methods for the Anal vsis of Oils, Fats and Derivatives, 7th edn. Oxford: Blackwell Scientific Publications.

Ka H., Park H-J, Jung H-J, Choi J-W, Cho H-S, Ha J, Lee K-T (2003). Cinnamaldehyde induces apoptosis by ROS-mediated mitochondrial permeability transition in human promyelocytic leukemia HL-60 cells. Cancer Letters. 196 : 143–152.

182

Kanagal, S., Nagarajan, S., and Lingamallu, J. M. R. (2011). Cumin (Cuminum cyminum L.) Seed Volatile Oil: Chemistry and Role in Health and Disease Prevention. In: Nuts and Seeds in Health and Disease Prevention, pp. 417–427. Victor R. Preedy, V.R., Watson, R.R., and Patel, V.B., London, Tucson.

Karsha, P. V. and Lakshmi O. (2010). Antibacterial activity of black pepper (Piper nigrum Linn.) with special reference to its mode of action on bacteria. Indian Journal of Natural Products and Resources 1(2): 213-215.

Kapoor, V. K., Chawla, A. S., Manojkumar, and Pradeepkumar (1993). Search for antiinflammatory agents. India Drugs. 30 : 481–493.

Kayashima T. and Matsubara K. (2012). Antiangiogenic effect of carnosic acid and carnosol, neuroprotective compounds in rosemary leaves. Biosci Biotechnol Biochem. 76(1):115-9.

Kim, I.S., Yang, M-R., Lee, O.-H., and Kang, S.-N. (2011).Antioxidant activities of hot water extracts from various spices. Int J Mol Sci. 12: 4120–4131.

Kim S.S., Oh O.J., Min H.Y., Park E.J., Kim Y., Park H.J., Nam Han Y. and Lee S.K. (2003). Eugenol suppresses cyclooxygenase-2 expression in lipopolysaccharide-stimulated mouse macrophage RAW264.7 cells. Life Sci. 73(3):337-48.

Korifi, R., Le Dréau, Y., Antinelli, J.F., Valls, R., Dupuy, N. (2013). CIEL*a*b* color space predictive models for colorimetry devices – Analysisof perfume quality. Talanta. 104 : 58–66

Kurokawa, M., A Kumeda, C., Yamamura, J.-i., Kamiyama, T., and Shiraki, K. (1998). Antipyretic activity of cinnamyl derivatives and related compounds in influenza virus-infected mice Original Research Article. Eur. J. Pharmacol. 348 : 45-51 Lee, E. B., Shin, K. H., Woo, W. S. (1984). Pharmacological Study of Piperine. Arch. Pharm. Res. 7 : 127–132.

Lee, K.G, Shibamoto, T. (2001). Antioxidant property of aroma extract isolated from clove buds [ Syzygium aromaticum (L.) Merr. et Perry ]. Food Chemistry. 74(4): 443 – 448.

Leung, A. and Foster, S. (1996). Corinader. In: Encyclopedia of Common Natural Ingredients Used in Food, Drugs and Cosmetics,pp.193-195 2nd edn J. Wiley, New York.

Lewis, Y. S., Krishnamurthy, N., Nambudiri, E. S., Sankarikutty, B., Shivshankar, and A. S., Mathew, A. G. (1976). The need for growing pepper cultivars to suit pepper products. Proceedings of the International Seminar on Pepper.

Lis-Balchin, M., Steyrl, H., and Krenn, E. (2003). The comparative effect of novel Pelargonium essential oils and their corresponding hydrosols as antimicrobial agents in a model food system. Phytother. Res. 17: 60–65.

Liu, Q., Lanari, M.C, Schaefer, D.M. (1995). A review of dietary vitamin E supplementation for improvement of beef quality. Journal of Animal Science. 73 : 3131–3140

183

Liu Y., Yadev V.R., Aggarwal B.B. and Nair M.G. (2010). Inhibitory effects of black pepper (Piper nigrum) extracts and compounds on human tumor cell proliferation, cyclooxygenase enzymes, lipid peroxidation and nuclear transcription factor-kappa-B. Nat Prod Commun. 5(8):1253-7.

Lopez, P., Sanchez, C., Batle, B., Nerin, C. (2005). Solid and vapour phase antimicrobial activities of six essential oils: susceptibility of selected food borne bacterial and fungal strains. Journal of Agriculture and Food Chemistry. 53: 6338 – 6346

Lundberg, J., Gladwin, M. T., Ahluwalia, A., Benjamin, N., Bryan, N. S., Butler, A., Cabrales, P., Fago, A., Feelisch, M., Ford, P. C., Freeman, B. A., Frenneaux, M., Friedman, J., Kelm, M., Kevil, C.G., Kim-Shapiro, D.B., Kozlov, A.V., Lancaster, J.R.J., Lefer, D.J., McColl, K., McCurry, K., Patel, R. P., Petersson, J., Rassaf, T., Reutov, V. P., Richter-Addo, G. B., Schechter, A., Shiva, S., Tsuchiya, K., van Faassen, E. E., Webb, A. J., Zuckerbraun, B. S., Zweier, J. L., and Weitzberg, E. (2009). Nitrate and nitrite in biology, nutrition and therapeutics. Nat. Chem. Biol. 5 : 865–869

Matan, N., Rimkeeree, H., Mawson, A. J., Chompreeda, P., Haruthaithanasan, V., and Parker, M. (2006). Antimicrobial activity of cinnamon and clove oils under modified atmosphere conditions. Int J Food Microbiol. 107 : 180–185.

Marouli, A.Z, Maroulis, Z.B. (2005). Cost data analysis for food industry. Journal of Food Engineering. 67: 289-299

Martin, M. Meat curing technology. Y.H. Hui, Wai-Kit Nip, R.W. Rogers, O.A. Young (Eds.), Meat science and applications, Marcel Dekker, Inc., New York (2001), pp. 491–497.

MacDougall, D.B. (2002). Colour in Food: Improving Quality. Woodhead Publishing.

McCaig, T.N. (2002). Extending the use of visible/near infrared reflectance spectrophotometers to measure colour of food and agricultural products. Food Research International. 3: 731–736

McKay, D. L. and Blumberg, J. B. (2006). A review of the bioactivity and potential health benefits of peppermint tea (Mentha piperita L.) Phytother. Res. 20(8):619-633.

Meah, M. N., Harrison, N., and Davies, A. (1994). Nitrate and nitrite in foods and the diet. Food Addi. Contam. 11 : 519-532

Menon, K.V. and Garg, S.R. (2001). Inhibitory effect of clove oil on Listeria monocytogenes in meat and cheese. Food Microbiol. 18 : 647-650

Moigradean, D., Lazureanu, A., Poiana, M.-A., Harmanescu, M., Gogoasa, I., and Gergen, I.. (2008). The influence of mineral fertilization about nitrogen content in soil, plant and tomato fruit. Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj Napoca Horticulture. 65: 172-177.

Moriera, M. R., Ponce, A. G., Del Valle, C. E., and Roura, S. (2007). Effects of clove and tea tree oils on Escherichai coli O157:H7 in blanching spinach and minced cooked beef. J. Food Process. Pres. 31:379–391.

184

Morita H., Sakata, R., Nagata, Y. (1998) Nitric oxide complex of Iron (II) myoglobin converted from metmyoglobin by Staphylococcus xylosus. Journal of Food Science, 63, pp. 352–355

Mosqueda-Melgar, J., Raybaudi-Massilia, R. M., and Martin-Belloso, O. (2008). Inactivation of Salmonella enterica Ser enteritidis in tomato juice by combining of high-intensity pulsed electric fields with natural antimicrobials. J. Food Sci. 73(2): 47–53.

Mueller, R. L., Hagel, H. J.,Wild, H.,Ruppin, H.,and Domschke, W. (1986). Nitrate and nitrite in normal gastric juice. Precursors of the endogenous N-nitroso compound synthesis. Oncology, 43 : 50–53.

Muchuweti, M., Kativu, E., Mupure, C. H., Chidewe, C., Ndhlala, A. R. & Benhura, M. A. N. (2007). Phenolic composition and antioxidant properties of some spices. American Journal of Food Technology, 2, 414-420.

Mullavarapu, G. R. and Ramesh, S. (1998). Composition of essential oils of nutmeg and mace Aromat. Plant Sci. 20: 746–748.

Murugan K., Anandaraj, K. and Al-Sohaibani, S. (2013). Antiaflatoxigenic food additive potential of Murraya koenigii: An in vitro and molecular interaction study. Food Res. Int. 52(1):8-16.

Mytle, N., Anderson, G. L., Doyle, M.P. and Smith, M. A. (2006). Antimicrobial activity of clove (Syzgium aromaticum) oil in inhibiting Listeria monocytogenes on chicken frankfurters. Food Control 17: 102–107.

Naidu, A. S. (2000). Overview. In: A. S. Naidu. Natural food antimicrobial systems (pp. 1–16). Boca Raton, Florida: CRC Press.

Nakamura, N., Kiuchi, F., Tsuda, Y. and Kondo, K. (1988). Studies on crude drugs effective on visceral larva migrans.V. The larvicidal principle in mace Chem. Pharm. Bull 36(7):2685-2688.

Nanir S. P. and Kadu B. B. (1987) Effect of medicinal plant extracts on some fungi. Acta Bot. Indica 15(2): 170-175.

National Academy of Sciences (1982). Assembly of Life Sciences: Alternatives to the current use of nitrite in foods. Pp. 1-3 through 1-9. Washington, DC.: National Academy Press.

Nya E.J. and Austin B.( 2009). Use of dietary ginger, Zingiber officinale Roscoe, as an immunostimulant to control Aeromonas hydrophila infections in rainbow trout, Oncorhynchus mykiss (Walbaum). J Fish Dis. 32(11):971-7.

Ogata, M., Hoshi, M., Urano, S. and Endo, T. (2000). Antioxidant activity of eugenol and related monomeric and dimeric compounds. Chem Pharm Bull 48: 1467–1469.

Ogunwande, I. A., Olawore, N.O., Ekundayo, O., Walker, T.M., Schmidt, J.M. and Setzer, W. N. (2005). Studies on the essential oils composition, antibacterial and cytotoxicity of Eugenia uniflora L. Int. J. Aromather. 15: 147–152.

185

O'Leary, M., Rehm, G., and Schmitt, M. (1994). Understanding Nitrogen in Soils. WW-03770-GO

Omar, A. A., Artime, E., and Webb, A. J. (2012). A comparison of organic and inorganic nitrates/nitrites. Nitric Oxide. 26 : 229–240.

Omar, S. A., Artime, E., and Webb, A. J. (2012). A comparison of organic and inorganic nitrates/nitrites Original Research Article. Nitric Oxide. 26 : 229-240. Rosengarten, F. Jr. (1973). The Book of Spices. Pyramid Books, New York.

Ouattara, B., Simard, R. E., Holley, R. A., Piette, G. J.-P., and Bégin, A. (1997). Antibacterial activity of selected fatty acids and essential oils against six meat spoilage organisms. Int. J. Food Microbiol. 37 : 155–162.

Oussalah, M., Caillet, S., Saucier, L., and Lacroix, M. (2007). Inhibitory effects of selected plant essential oils on four pathogen bacteria growth: E. coli O157:H7, Salmonella typhimurium, Staphylococcus aureus and Listeria monocytogenes. Food Control. 18 : 414-420.

Oussalah, M., Caillet, S., Saucier, L., and Lacroix, M. (2007). Inhibitory effects of selected plant essential oils on Pseudomonas putida growth, a bacterial spoilage meat. Meat Sci. 73 : 236-244.

Pannala, A.S., Mani, A. R., Spencer, J. P. E., Skinner, V., Bruckdorfer, K. R.,Moore, K. P., and Rice-Evans, C. A. (2003). The effect of dietary nitrate on salivary, plasma, and urinary nitrate metabolism in humans. Free Radic. Biol. Med. 34: 576–584.

Parthasarathy, V.A., Chempakam, B. and Zachariah, T. J. (2008). Chemistry of spices. CABI ISBN 978-1-84593-405-7 UK.

Pawar, V.C. and Thaker V.S. (2006). In vitro efficacy of 75 essential oils against Aspergillus niger. Mycoses. 49(4): 316-323

Pearson, A. M., and Gillett, T. A. (1996). Processed Meats. Chapman and Half, New York, NY

Pegg, R.B., and Shahidi, F. (2000). Nitrite Curing of Meat. The N-Nitrosamine Problem and Nitrite alternatives. Food and Nutrition Press, Inc., Trumbull, CT.

Perumalla, A. V. S., and Hettiarachchy, N. S.(2011). Green tea and grape seed extracts—Potential applications in food safety and quality. Food Res. Int. 44 : 827-839.

Peter, K.V. and Shylaja, M.R. (2012). Introduction to herbs and spices: definitions, trade and applications. In: The Handbook of herbs and spices Second edition, volume 1, pp. 1-24. Woodhead Publishing.

Petersen, A., and Stoltze, S. (1999). Nitrate and nitrite in vegetables on the Danish market: Content and intake. Food Addi. Contam. 16 : 291-299.

Pleasant Hill Grain for technico-economic analysis . Accessed online: http://www.pleasanthillgrain.com/torrey_meat_grinder_m32.aspx

Platel, K. and Srinivasan, K. (2004). Digestive stimulant action of spices: A myth or reality?. Indian J. Med. Res. 119:167-179.

186

Pokorny, L., and Maturana, I., Bortle, W. H. (2006). Sodium nitrate and nitrite. In: Kirk-Othmer Encyclopedia of Chemical Technology, John Wiley and Sons, 5th Ed, New York, [online version].

Prabhakaran Nair, K.P. (2004). Advances in Agronomy, The Agronomy and Economy of Black Pepper (Piper nigrum L.) —The “King of Spices”. In : Agronomy and Economy of Black Pepper and Cardamom, pp 271–389. Prabhakaran Nair, K.P., New Delhi.

Pradsad, M. M., Seenayya, G. Effect of spices on the growth of red halophilic cocci isolated from salt cured fish and solar salt. Food Res. Int. 33 : 793–798.

Prospero, M., and Savoie, D. L. (1989). Nitrate in the Atmospheric Boundary Layer of the Tropical South Pacific: Implications Regarding Sources and Transport. J. Atmos. Chem. 8 : 391-415.

Qin B., Nagasaki M., Ren M., Bajotto G., Oshida Y. and Sato Y. (2003). Cinnamon extract (traditional herb) potentiates in vivo insulin-regulated glucose utilization via enhancing insulin signaling in rats. Diabetes Res Clin Pract. 62(3):139-48.

Raghavenra, H., Diwakr, B.T., Lokesh, B.R., Naidu, K.A. (2006). Eugenol, the active principle from cloves inhibits 5 - lipoxygenase activity and leukotriene - C4 in human PMNL cells. Prostaglandins, Leukotrienes and Essential Fatty Acids. 74: 23 –27.

Raghavendra R.H. and Naidu K.A. (2009). Spice active principles as the inhibitors of human platelet aggregation and thromboxane biosynthesis. Prostaglandins Leukot Essent Fatty Acids. 81(1):73-8.

Rahman, M. M. and Gray A. I. (2005). A benzoisofuranone derivative and carbazole alkaloids from Murraya koenigii and their antimicrobial activity. Phytochem. 66(13):1601-1606.

Rahman, M. S. A., Thangaraj, S., Salique, S. M., Khan. K. F. & Natheer. S. E. (2010). Antimicrobial and Biochemical Analysis of Some Spices Extract against Food Spoilage Pathogens. Internet Journal of Food Safety, 12, 71-75.

Rasooli, I. (2007). Food preservation, a biopreservative approach. Food. Global Science Book 1:111–136.

Ravindran, P.N., Nirmal Babu, K., Shiva, K. N. (2006). Genetic resources of spices and their conservation In: Ravindran, P.N., Nirmal Babu, K., Shiva, K. N. and Johny, A. K.. (eds) Advances in Spices Research. Agrobios pp. 63-91.

Raybaudi, R. M. M., Rojas-Grau, M. A., Mosqueda-Melgar, J., and Martin-Belloso, O. (2008). Comparative study on essential oils incorporated into an alginate-based edible coating to assure the safety and quality of fresh-cut Fuji apples. J. Food Protect, 71:1150-1161.

Reuben, A., Treminio, H., Arias, M. L., and Chaves, C. (2003) Presencia de Escherichia coli O157:H7, Listeria monocytogenes y Salmonella spp. en alimentos de origen animal en Costa Rica. Arch Latinoam Nutr 53:389–392.

187

Richheimer, S. L., Bernart, M. W., King, G. A., Kent, M. C., Beiley, D. T. (1996). Antioxidant Activity of Lipid- Soluble Phenolic Diterpenes from Rosemary. J. Am. Oil Chem. Soc. 73(4):507-514.

Roth, H.A., Radle, L., Gifford, S.R., Clydesdale, F.M. Psychophysical relationships between perceived sweetness and color in lemon- and lime-flavored drinks . J. Food Sci., 53 (1988), pp. 1116–1162.

Rouweler, J. (2013). Rapid investment Cost Estimation Methods for food production #2: Zevnik-Buchanan's method. Accessed online: http://www.academia.edu/5330084/Rapid_Investment_Cost_Estimation_Methods_for_Food_Production_Plants_2_Zevnik-Buchanans_method.xls

Russel, R. J. & Gould, G. W. (1991). Food Preservatives. Van Nostrand Reinhold Co., New York.

Sajilata, M.G. and Singhal, R.S. (2012). Quality indices for spice essential oils Institute of Chemical Technology, India. In: the Handbook of herbs and spices Second edition, volume 1, pp.42-54. Woodhead Publishing.

Sallam, K. I., Ishioroshi, M., and Samejima, K. (2004). Antioxidant and Antimicrobial Effects of Garlic in Chicken Sausage. Lebenson Wiss Technol. 37 : 849-855.

Sanchez-Lafuente, C., Furlanetto, S., Fernandez-Arevalo, M. (2002). Didanosine extended-release matrix tablets: optimization of formulation variables using statistical experimental design. Int J Pharm. 237: 107-118.

Sathaye, S., Bagul, Y., Gupta, S., Kaur, H. and Redkar, R. (2011). Hepatoprotective effects of aqueous leaf extract and crude isolates of Murraya koenigii against in vitro ethanol induced hepatotoxicity model. Exp. Toxicol. Pathol. 63(6): 587–591.

Schmedes, A., & Holmer, G. A. (1989). New thiobarbituric acid (TBA) method for determination of free malonaldehyde (MDA) and hydroperoxides selectivity as a measure of lipid peroxidation. Journal of American Oil Chemistry Society. 66, 813–817.

Sebiomo, A., Awofodu, A. D., Awosanya, A. O., Awotona F. E. and Ajayi A. J. (2011) Comparative studies of antibacterial effect of some antibiotics and ginger (Zingiber officinale) on two pathogenic bacteria. J. Microbiol. Antimicrob. 3(1):18-22.

Sebranek, J. G. (2009). Basic curing ingredients. In : Ingredients in Meat Products, pp. 1-24. Tarte, R. Eds. Springer Science+Business Media LLC, New York.

Sebranek, J. G., and Bacus, J. N. (2007). Cured meat products without direct addition of nitrate or nitrite: What are the issues?. Meat Sci. 77:136–147.

Sema, A., Nursel, D. and Süleyman, A. (2007). Antimicrobial activity of some spices used in the meat industry. Bull Vet I Pulawy 51:53–57.

Shaikh, J., Bhosale, R., and Singhal, R. (2006). Microencapsulation of black pepper oleoresin. Food Chem. 94 : 105–110.

Shahidi, F. and Marian, N. (2003) Phenolics in Food and Nutraceuticals; CRS Press LLC: Boca Raton, FL pp. 144-150.

188

Shan, B., Cai Y., Brooks J. D., Corke H. (2007). The in vitro antibacterial activity of dietary spice and medicinal herb extracts. Int. J. Food Microbiol. 117:112-119.

Shaath, N. A., and Azzo, N. R. (1993). Essential oil of Egypt. In : Food flavor ingredients and composition, pp. 591–603. Charalambous, G., Eds., Elsevier Sci. Pub, Amsterdam

Shirin, A. P. R., and Prakash, J. (2010). Chemical composition and antioxidant properties of ginger root (Zingiber officinale). J. Med. Plants Res. 4 : 2674-2679

Shobana, S. and Akhilender, K. (2000). Antioxidant activity of selected Indian spices. Prostag. Leukotr. Ess. 62:107-110.

Singh, A. and Deep, A. (2011). Piperine : A Bioenhancer Review Article. Int J Pharm Res and Technol. 1(1):1-5.

Singh, S.K., Dodge, J., Durrani, M.J., Khan, M.A. (1995). Optimization and characterization of controlled release pellets coated with experimental latex: I. Anionic drug Int J Pharm, 125: 243-255.

Singh, G., Marimuthu, P., Catalan, C. and De Lampasona, M. P. (2004) Chemical, antioxidant and antifungal activities of volatile oil of black pepper and its acetone extract. J. Sci. Food Agric. 84(14):1878-1884.

Sofos, J.N., et al. (1979). Botulism control by nitrite and sorbate in cured meats. J. Food Prot. 42:739-770.

Souza, E. L., Montenegro, Stamford, T. L. and Oliveira-Lima, E. (2006). Sensitivity of spoiling and pathogen food-related bacteria to Origanum vulgare L. (Lamiaceae) essential oil. Braz. J. Microb. 37:527–532.

Souza, E. L., Montenegro, T. L., Lima, E., Trajano, V.N. and Barbosa, J. M. (2005). Antimicrobial Effectiveness of Spices: an Approach for Use in Food Conservation Systems Braz. Arch. Biol. Techn. 48(4):549-558.

Spices Board (2013). Ministry of Commerce & Industry, Goverment of India available online http://www.indianspices.com/ (assessed July 10, 2013)

Srinivasan K. (2007). Black pepper and its pungent principle-piperine: a review of diverse physiological effects. Crit Rev Food Sci Nutr. 47(8):735-48.

Stoilova, I., Krastanov, A., Stoyanova, A., Denev, P., and Gargova, S. (2007). Antioxidant activity of a ginger extract (Zingiber officinale). Food Chem. 102 : 764–770.

Stone H., Sidel J., Oliver S., Woolsey A. and Singleton R.C. 1974. Sensory evaluation by quantitative descriptive analysis. Food Technol. 28 (11):24

Suthisut, D., Fields, P. G. and Chandrapatya, A. (2011). Contact Toxicity, Feeding Reduction, and Repellency of Essential Oils from Three Plants from the Ginger Family (Zingiberaceae) and their Major Components Against Sitophilus zeamais and Tribolium castaneum J. Econ. Entomol. 104 (4) :1445-1454.

189

Tabak, M., Armon, R., and Neeman, I. (1999). Cinnamon extracts’ inhibitory effect on Helicobacter pylori Original Research Article. J. Ethnopharmacol. 67 : 269-277. Tajkarimi, M.M., Ibrahim, S. A., and Cliver, D. O. (2010). Antimicrobial herb and spice compounds in food. Food Control. 21 : 1199–1218.

Tajkarimi, M.M., Ibrahim, S. A., and Cliver, D. O. (2010). Antimicrobial herb and spice compounds in food. Food Control. 21 : 1199–1218.

Tampieri, M. P., Galuppi, R., Macchioni, F., Carelle M. S., Falcioni L., Cioni, P.L. and Morelli, I. (2005). The inhibition of Candida albicans by selected essential oils and their major components. Mycopathologia 159: 339–345.

Tarté, R. (2009). Ingredients in Meat products: Propierties, functionality and Applications. In: Springer Science Business Media LLC page 301.

Toghyani, M., Gheisari, A., Ghalamkari, G., and Eghbalsaied, S. (2011). Evaluation of cinnamon and garlic as antibiotic growth promoter substitutions on performance, immune responses, serum biochemical and haematological parameters in broiler chicks. Livest Sci. 138: 167-173.

Thomson, B. (2004). Nitrites and nitrates dietary exposure and risk assessment. Christchurch Science Centre Christchurch, NZ

Townsend, W. E., and Olson, D. G. (1987). Cured meats and cured meat products processing. In The Science of Meat and Meat By Products, pp. 193–216, 431–456. Price, J. F., and Schweigert, B. S., Eds., Food and Nutrition Press Inc., Westport.

Trajano, V.N., Lima, E., Travassos, A. E. and De Souza, E. L. (2010). Inhibitory effect of the essential oil from Cinnamomum zeylanicum Blume leaves on some food-related bacteria. Ciênc. Tecnol. Aliment., Campinas, 30(3): 771-775.

Tu, X., Xiao, B., Xiong, J., and Chen, X.. (2010). A simple miniaturised photometrical method for rapid determination of nitrate and nitrite in freshwater. Talanta. 82 : 976–983.

Tufail, M. (1990). Spices in Indian Economy. Academic Foundation. California University pp. 151.

Turgis, M., Han, J., Caillet, S., and Lacroix, M. (2009). Antimicrobial activity of mustard essential oil against Escherichia coli O157:H7 and Salmonella typhi. Food Control 20: 1073–1079.

U.S. Department of Agriculture. (1995). Processing inspectors' calculation shandbook. Available at: http://www.fsis.usda.gov/OPPDE/rdad/FSISDirectives/7620-3.pdf. Accessed December 8, 2009.

U.S. Environmental Protection Agency (USEPA) (2007). Washington, D.C. "National

Water Quality Inventory: Report to Congress; 2002 Reporting Cycle." Document No. EPA-841-R-07-001.

190

U.S. Environmental Protection Agency Ground Water and Drinking Water. (2006). "Consumer Factsheet on: Nitrates/Nitrites." Online reference included in article [Internet document] URL

http://www.epa.gov/teach/chem_summ/Nitrates_summary.pdf.Accessed 06/18/2014.

U.S EPA (2006). Toxicity and Exposure Assessment for Children’s Health. http://www.epa.gov/teach/chem_summ/Nitrates_summary.pdf . Accessed on February, 2013

Vasavada, M. N., and Cornforth, D. P. (2005). Evaluation of milk mineral antioxidant activity in meat balls and nitrite-cured sausage. J. Food Sci. 70 : 250–253.

Vats, M., Singh, H. and Sardana S. (2011) Phytochemical screening and antimicrobial activity of roots of Murraya Koenigii (Linn) Spreng (Rutaceae). Braz. J. Microbiol. 42(4):1569-1573.

Viani, F., Siegrist, H. H., Pignatelli, B., Cederberg, C., Idström, J. P., Verdu, E. F., Fried, M., Blum, A. L., and Armstrong, D. (2000). The effect of intra-gastric acidity and flora on the concentration of Nnitroso compounds in the stomach. Eur. J. Gastroenterol. Hepatol. 12 : 165-73.

White, B. (2007). Ginger: An Overview. Am. Fam. Physician. 75(11): 1689-1691.

Winias, S., Retno, A., Magfiroh, R., Nasrulloh, M, R. and Rahayu R. (2011). Effect of cynammyldehyde from cinnamon extract as a natural preservative alternative to the growth of Staphylococcus aureus bacteria. J. Trop. Infect. Dis. 2:38-41.

Wojdyło, A., Oszmiański, J. and Czemerys R. (2007) Antioxidant activity and phenolic compounds in 32 selected herbs Food Chem. 105(3):940-949.

World Health Organization (2004). Rolling Revision of the WHO Guidelines for Drinking-Water Quality. Nitrates and nitrites in drinking-water.

World Trade Organization (2012). World Trade Spices Exports by country. Retrieved by Nation Master available online: http://www.nationmaster.com/graph/eco_wor_tra_exp_spi-economy-world-trade-exports-spices (assessed August 31, 2013).

Yadav AS, Bhatnagar D. Free radical scavenging activity, metal chelation and antioxidant power of some Indian spices. Biofactors. 2007; 31(3 -4): 219 -227.

Yadav, A. S., and Singh, R. P.(2004). Natural preservatives in poultry meat. Arch. Microbiol. 181:8–1.

Yes Group in. Accessed online: http://www.yesgroup.ca/main_site/processing.htm

Yocom, J. E. (1982). Indoor/outdoor air quality relationships: a critical review. JAPCA J Air Waste Ma. 32 : 500–606.

Zaika, L. L., Kissinger, J. C. (1978). Effect of major spices in Lebanon bologna on production by starter culture organisms. J. Food. Prot. 41 : 429-431.

191

Zhang, X., Kong, B., Xiong L. Y. (2007). Production of cured meat color in nitrite-free Harbin red sausage by Lactobacillus fermentum fermentation. Meat Science. Volume 77: 593–598.

Zheng, W., and Wang, S. Y. (2001). Antioxidant activity and phenolic compounds in selected herbs. J Agric Food Chem. 49 : 5165–70.