**10.2 Chemical composition of essential oil**

Essential oils are composed of a complex mixture. These compounds mainly belong to two families of chemicals: terpene compounds and aromatic compounds.

The terpene compounds are hydrocarbons of general formula (C5H8)n formed from isoprene units (Figure 3) and are represented in essential oils, mainly monoterpenes, sesquiterpenes and rarely few diterpenes. These compounds may be acyclic, monocyclic, bicyclic or tricyclic [Paris, 1981].

Fig. 3. Isoprene.

The aromatic compounds are derived from phenylpropane, which are characteristic of certain species, such as cinnamaldehyde in cinnamon essential oil, eugenol in the cloves, anethol and aldehyde anasique, in the essential oils of anise and fennel (Fig. 10) (Paris, 1981).

Fig. 4. Examples of aromatic compounds.

The majority of the components of EO are monoterpenes; they represent 90% of most essential oils. They are volatile usually easily driven by steam, have often pleasant odor (Lamart, 1994). By the diversity of their structure, they can be classified into several groups (Bakkali, 2008) (Table 3). Several factors may be responsible of the chemical polymorphism of essential oils. The most important are climate, soil, time harvest and method of storage and retrieval. Genetic factors (Echeverrigaray, 2001) and the growth cycle (Hance, 2003) may also influence this variability…(Bakkali, 2008). The use of essential oils is of great interest in many areas. Thus, the species most studied for their antibacterial and antifungal properties (biological activities) belong to the Lamiaceae family: Thyme, oregano, savory, lavender, mint, rosemary, sage and hyssop.

#### **10.3 Biosynthesis of essential oil**

314 Salmonella – A Dangerous Foodborne Pathogen

Fig. 2. Glandular trichomes of mint gardens observed in electron microscope scan (1000 ×)

Essential oils are composed of a complex mixture. These compounds mainly belong to two

The terpene compounds are hydrocarbons of general formula (C5H8)n formed from isoprene units (Figure 3) and are represented in essential oils, mainly monoterpenes, sesquiterpenes and rarely few diterpenes. These compounds may be acyclic, monocyclic, bicyclic or tricyclic

The aromatic compounds are derived from phenylpropane, which are characteristic of certain species, such as cinnamaldehyde in cinnamon essential oil, eugenol in the cloves, anethol and aldehyde anasique, in the essential oils of anise and fennel (Fig. 10) (Paris, 1981).

OMe

Aldehyde cinnamique Eugenol Anethol Aldehyde anisique

OMe OMe

O H

before (a) and after (b) extraction by steam distillation [Lucchesi, 2005].

families of chemicals: terpene compounds and aromatic compounds.

**10.2 Chemical composition of essential oil** 

H

Fig. 4. Examples of aromatic compounds.

OH

O

[Paris, 1981].

Fig. 3. Isoprene.

Essential oils are very complex natural mixtures that can hold about 20-60 components with very different concentrations. The main group composed by terpenoids or isoprenoids is a family of secondary metabolites widely distributed in the plant kingdom. More than 22000 compounds have been identified (Connolly 1992). Their classification is based on the number of repetition of the basic unit of isoprene: hemiterpene (C5), monoterpene (C10), sesquiterpene (C15), diterpene (C20), sesterpene (C25), triterpene (C30), tetraterpene (C40) and polyterpene. The major terpene components of essential oils are monoterpene and sesquiterpene. Isoprenoid biosynthesis of essential oils can be simplified into three phases: Isopentenyl diphosphate (IPP) Biosynthesis, condensation of IPP units and formation of prenyl diphosphates and Conversion of prenyl-diphosphates. The condensation of isopentenyl diphosphate (nucleophilic entity) to dimethylallyl diphosphate (electrophilicentity) leads to geranyl diphosphate (GPP, C10), precursor of monoterpenes. A further condensation type head-to-tail of IPP on the GPP leads to farnesyl diphosphate (FPP, C15), the precursor of sesquiterpenes. The prenyl transferases allow prenyl chain elongation by addition of one molecule of IPP.

Fig. 5. Biosynthetic precursors of the main metabolic constituents of essential oils

Use Thyme Essential Oils for the Prevention of Salmonellosis 317

composition and search for any traces of other undesirable products such as pesticides or

A pure and natural essential oil is characterized by its composition strictly 'vegetable', unlike gasoline, synthetic or "natural identical" fully reconstituted from synthetic chemicals. In aromatherapy, the use of such profiles is essential to differentiate within a species changes induced by chemical factors influencing the plant biosynthesis, such as sunshine, altitude, nature and composition of the soil. Indeed, the observation of a clump of thyme and knowledge of its origin is not sufficient to characterize its essential oil. For example, an essential oil of Thymus vulgaris can be thymol chemotype, linalool, geraniol or thuyanol.

The plant is a concentrate of active ingredients (enzymes, polysaccharides, alkaloids, terpenes, tannins, resins etc.) and often requires large quantities of plants for a few drops of essential oil (100 kilos per 200 grams of EO thyme). If scarcity is one of their attractions, it also makes the price, hence the importance of good conservation: the dark (dark bottles), air and heat. In Morocco, the development of natural resources is the integration of non-food plants in the Moroccan economy, especially their conservation and development in food, cosmetics, industrial and therapeutic. Moroccan products in the areas of essential oils and aromatic extracts are well known on the world market. For some products, Morocco has a privileged position; he is the sole or primary producer, in the case of mint, chamomile wild, thyme, Atlas cedar, etc. For other products, production Moroccan faces other competitors; this was the case for example of rosemary undergoing tough Tunisian and Spanish competition. In other cases, the position of Morocco on the world market is rather high, in

There are several techniques of extraction of essential oils (Sallé, 2004). They can be extracted simply by cold pressing, distillation, by volatile solvent or supercritical fluid such as carbon dioxide. Distillation by steam distillation of water (still) is the method most practiced in the industry. The essential oils obtained are generally low polarity products, volatile, fragrant and usually less dense than water (Valnet, 2005). They are soluble in most organic solvents and poorly soluble in water. (Sallé, 2004). Steam-hydrodistillation is the most common method of recovery of essential oils. Under the action of water vapor, gas is released by the plant tissue and effect of temperature, volatiles products are driven by the steam. The essential oils are recovered by condensing (Bruneton, 1993). Hydrodistillation (steam water 'in situ') method involves by immersing the plant material in water which is then boiled (Bruneton, 1999). The enfleurage, distillation assisted by microwaves, extraction with solvents, extraction with a supercritical fluid are among the methods used to recover

Salmonella sp. was isolated from soil argan Marrakech (Morocco). The identification was performed according to the methods described above. Inocula were prepared from liquid

Botanically, it is the same plant family Lamiaceae (or Labiatae ).

**10.5 Economic characteristics of EO** 

the case of myrtle, mint and so on. (Benjilali, 1986).

the EO of aromatic and medicinal plants (Sallé,2004, Brian, 1995).

**11. Biological activity of thymus EO** 

**11.1 Source of** *Salmonella sp*

**10.6 Methods of extraction of EO** 

chemicals added.

Conversion of prenyl- diphosphates: Under the action of terpene synthases, the acyclic isoprene precursors thus obtained may undergo transformations for the formation of other compounds by hydroxylation, oxidation, inter-conversion alcohol /aldehyde, methylation of the hydroxyl and carboxyl, acylation...

Fig. 6. Structures of monoterpenes derived from geranyl diphosphate.

#### **10.4 Quality control**

According to French and European Pharmacopoeia, the control of essential oils can be made by the miscibility of ethanol, the index of refraction, the optical rotation and gravity. The color and smell are also important parameters. The quantitative and qualitative chromatographic profile of an essential oil helps to know exactly the chemical composition and search for any traces of other undesirable products such as pesticides or chemicals added.

A pure and natural essential oil is characterized by its composition strictly 'vegetable', unlike gasoline, synthetic or "natural identical" fully reconstituted from synthetic chemicals. In aromatherapy, the use of such profiles is essential to differentiate within a species changes induced by chemical factors influencing the plant biosynthesis, such as sunshine, altitude, nature and composition of the soil. Indeed, the observation of a clump of thyme and knowledge of its origin is not sufficient to characterize its essential oil. For example, an essential oil of Thymus vulgaris can be thymol chemotype, linalool, geraniol or thuyanol. Botanically, it is the same plant family Lamiaceae (or Labiatae ).

#### **10.5 Economic characteristics of EO**

316 Salmonella – A Dangerous Foodborne Pathogen

Conversion of prenyl- diphosphates: Under the action of terpene synthases, the acyclic isoprene precursors thus obtained may undergo transformations for the formation of other compounds by hydroxylation, oxidation, inter-conversion alcohol /aldehyde, methylation

OPP

OH

Geraniol

<sup>M</sup> <sup>α</sup> -Pinene yrcene

Thymol

OH

Fig. 6. Structures of monoterpenes derived from geranyl diphosphate.

According to French and European Pharmacopoeia, the control of essential oils can be made by the miscibility of ethanol, the index of refraction, the optical rotation and gravity. The color and smell are also important parameters. The quantitative and qualitative chromatographic profile of an essential oil helps to know exactly the chemical

Citronellol Limonene

**Geranyl diphosphate** 

Borneol

**10.4 Quality control** 

OH

Linalol OH

of the hydroxyl and carboxyl, acylation...

The plant is a concentrate of active ingredients (enzymes, polysaccharides, alkaloids, terpenes, tannins, resins etc.) and often requires large quantities of plants for a few drops of essential oil (100 kilos per 200 grams of EO thyme). If scarcity is one of their attractions, it also makes the price, hence the importance of good conservation: the dark (dark bottles), air and heat. In Morocco, the development of natural resources is the integration of non-food plants in the Moroccan economy, especially their conservation and development in food, cosmetics, industrial and therapeutic. Moroccan products in the areas of essential oils and aromatic extracts are well known on the world market. For some products, Morocco has a privileged position; he is the sole or primary producer, in the case of mint, chamomile wild, thyme, Atlas cedar, etc. For other products, production Moroccan faces other competitors; this was the case for example of rosemary undergoing tough Tunisian and Spanish competition. In other cases, the position of Morocco on the world market is rather high, in the case of myrtle, mint and so on. (Benjilali, 1986).

#### **10.6 Methods of extraction of EO**

There are several techniques of extraction of essential oils (Sallé, 2004). They can be extracted simply by cold pressing, distillation, by volatile solvent or supercritical fluid such as carbon dioxide. Distillation by steam distillation of water (still) is the method most practiced in the industry. The essential oils obtained are generally low polarity products, volatile, fragrant and usually less dense than water (Valnet, 2005). They are soluble in most organic solvents and poorly soluble in water. (Sallé, 2004). Steam-hydrodistillation is the most common method of recovery of essential oils. Under the action of water vapor, gas is released by the plant tissue and effect of temperature, volatiles products are driven by the steam. The essential oils are recovered by condensing (Bruneton, 1993). Hydrodistillation (steam water 'in situ') method involves by immersing the plant material in water which is then boiled (Bruneton, 1999). The enfleurage, distillation assisted by microwaves, extraction with solvents, extraction with a supercritical fluid are among the methods used to recover the EO of aromatic and medicinal plants (Sallé,2004, Brian, 1995).

#### **11. Biological activity of thymus EO**

#### **11.1 Source of** *Salmonella sp*

Salmonella sp. was isolated from soil argan Marrakech (Morocco). The identification was performed according to the methods described above. Inocula were prepared from liquid

Use Thyme Essential Oils for the Prevention of Salmonellosis 319

Fig. 7. *T. broussonetii*

Fig. 8. *T. maroccanus*

cultures by 18 h, diluted with saline so as to contain about 108 cells / ml, at a density of between 0.08 and 0.1 at 625 nm (Careaga and al. 2003; Joffin and Leyral,2001). We evaluated the sensitivity of this strain against the essential oils of *thymus broussonetii* and *thymus maroccanus* from the region of Marrakech (Morocco). We used the Mueller Hinton medium for our study. The antimicrobial activity of essential oils, we used the method of diffusion from antibiotic susceptibility discs of pure essential oil (Jacob et al., 1979). The media poured in Petri dishes are inoculated with 1 ml of bacterial suspension of 108 cells / ml and excess inoculum was removed by aspiration (Shunying et al., 2005). The essential oil is deposited in the volume of 10 ml on Whatman sterile paper discs of 6 mm in diameter. In parallel, we use cookies to check the growth of the strain tested. The petri dishes were left 30 min at room temperature to allow complete diffusion of the product (CA-SFM, 1993). The antibacterial activity was determined in terms of diameter of inhibition zone around the discs recorded after 24 h of incubation at 37 ° C. The test is performed in three repetitions in the same experimental conditions.
