**1. Introduction**

The use of microwaves is an alternative to extract essential oils (EOs), and it can be used to assist a common method known as hydrodistillation. This method is achieved by adapting a distillation apparatus to a microwave oven, or with specialized equipment such as the NEOS System equipment (Milestone, Shelton CT, USA) [1]. The MAE is a method that uses microwave radiation as a heating source, for a mixture made with solvent and sample. This type of heating is instantaneous and occurs inside the sample, so the extraction is usually a very fast process [2].

Essential oils are substances extracted from different parts of aromatic plants (flowers, seeds, leaves, herbs, fruits, roots, and rhizomes, among others) and have antiviral, antibacterial, antifungal, and insecticidal properties [3]. EOs contain

**83**

5°C [7, 16, 17].

*Extraction, Composition, and Antibacterial Effect of Allspice (Pimenta dioica) Essential Oil…*

different components (mono- and sesquiterpenes), of which the main ones represent 85–95% of the total volume, and the others are known as minor components [4, 5]. An alternative source of EOs is the one extracted from the dried fruit of allspice. This spice has been used in meat, fish, soups, sauces, cakes, cosmetics, and drugs due to its antimicrobial and antioxidant properties [6, 7]. AEO has been used as an antimicrobial against different microbial strains, since it has been proven that the volatiles present in the EO are capable of inhibiting the growth of different bacteria and fungi [6, 8–10]. One of the limitations of the AEO is that when applied in liquid phase (directly), it generates a significant impact on the sensory attributes of food, because of its strong aroma [11]. Unlike the liquid phase, the application in vapor phase, which is achieved through the formation of atmospheres with volatilized essential oil compounds, requires lower concentrations, for its use as an antimicrobial. Therefore, the application in vapor phase could be a solution to the intense aroma characteristic of the

There are few studies on the use of allspice essential oil as an antimicrobial and its application in vapor phase. Therefore, the aim of this chapter is to extract and evaluate the composition and antibacterial effect of allspice essential oil applied in vapor phase against *Listeria monocytogenes, Salmonella* Typhimurium, and

Bacterial strains *(Salmonella enterica* serovar Typhimurium ATCC 14028, *Listeria monocytogenes* Scott A and *Pseudomonas fluorescens*) were obtained from the Food Microbiology Laboratory of Universidad de las Americas Puebla (UDLAP, Mexico, Puebla). The strains were maintained on Trypticase Soy Agar (TSA; Difco,

35°C for 24 h. Then culture inoculum cell concentration was adjusted to 107

CFU/mL for subsequent use in culture media [15, 16].

Bacteria (*L. monocytogenes*, *S.* Typhimurium, and *P. fluorescens*) were inoculated into 10 mL of Trypticase Soy Broth (TSB; Difco, BD, Sparks, MD) and incubated at

Allspice (*Pimenta dioica*) dry fruits were obtained from Condimentos Naturales

Allspice essential oil was obtained by means of MAE, using a NEOS System equipment (Milestone, Shelton CT, USA), according to the following methodology: allspice dried sample was grounded with the help of a blender (NutriBullet, Magic Bullet, USA), sieved with a mesh number 20 (850 μm), placed in a glass beaker with 2 L of distilled water, and letting the sample soak for 1.5 h. Then, the sample was introduced to the NEOS System with the following conditions: 800 W (representing 100% of the equipment power) for 30 min and at 600 W (representing 67% of the equipment's power) for another 30 min at 400 rpm. The recovered AEO was placed in hermetically sealed amber containers and stored at

*Pseudomonas fluorescens* at selected levels of pH and temperature.

*DOI: http://dx.doi.org/10.5772/intechopen.83691*

AEO [12–14].

**2. Methodology**

**2.1 Bacterial strains**

**2.2 Plant materials**

BD, Sparks, MD) slants at 5°C.

Tres Villas S.A. de C.V. Puebla.

**2.3 Extraction method**

*Extraction, Composition, and Antibacterial Effect of Allspice (Pimenta dioica) Essential Oil… DOI: http://dx.doi.org/10.5772/intechopen.83691*

different components (mono- and sesquiterpenes), of which the main ones represent 85–95% of the total volume, and the others are known as minor components [4, 5].

An alternative source of EOs is the one extracted from the dried fruit of allspice. This spice has been used in meat, fish, soups, sauces, cakes, cosmetics, and drugs due to its antimicrobial and antioxidant properties [6, 7]. AEO has been used as an antimicrobial against different microbial strains, since it has been proven that the volatiles present in the EO are capable of inhibiting the growth of different bacteria and fungi [6, 8–10]. One of the limitations of the AEO is that when applied in liquid phase (directly), it generates a significant impact on the sensory attributes of food, because of its strong aroma [11]. Unlike the liquid phase, the application in vapor phase, which is achieved through the formation of atmospheres with volatilized essential oil compounds, requires lower concentrations, for its use as an antimicrobial. Therefore, the application in vapor phase could be a solution to the intense aroma characteristic of the AEO [12–14].

There are few studies on the use of allspice essential oil as an antimicrobial and its application in vapor phase. Therefore, the aim of this chapter is to extract and evaluate the composition and antibacterial effect of allspice essential oil applied in vapor phase against *Listeria monocytogenes, Salmonella* Typhimurium, and *Pseudomonas fluorescens* at selected levels of pH and temperature.

## **2. Methodology**

#### **2.1 Bacterial strains**

Bacterial strains *(Salmonella enterica* serovar Typhimurium ATCC 14028, *Listeria monocytogenes* Scott A and *Pseudomonas fluorescens*) were obtained from the Food Microbiology Laboratory of Universidad de las Americas Puebla (UDLAP, Mexico, Puebla). The strains were maintained on Trypticase Soy Agar (TSA; Difco, BD, Sparks, MD) slants at 5°C.

Bacteria (*L. monocytogenes*, *S.* Typhimurium, and *P. fluorescens*) were inoculated into 10 mL of Trypticase Soy Broth (TSB; Difco, BD, Sparks, MD) and incubated at 35°C for 24 h. Then culture inoculum cell concentration was adjusted to 107 CFU/mL for subsequent use in culture media [15, 16].

#### **2.2 Plant materials**

Allspice (*Pimenta dioica*) dry fruits were obtained from Condimentos Naturales Tres Villas S.A. de C.V. Puebla.

#### **2.3 Extraction method**

Allspice essential oil was obtained by means of MAE, using a NEOS System equipment (Milestone, Shelton CT, USA), according to the following methodology: allspice dried sample was grounded with the help of a blender (NutriBullet, Magic Bullet, USA), sieved with a mesh number 20 (850 μm), placed in a glass beaker with 2 L of distilled water, and letting the sample soak for 1.5 h. Then, the sample was introduced to the NEOS System with the following conditions: 800 W (representing 100% of the equipment power) for 30 min and at 600 W (representing 67% of the equipment's power) for another 30 min at 400 rpm. The recovered AEO was placed in hermetically sealed amber containers and stored at 5°C [7, 16, 17].

**82**

**Chapter**

**Abstract**

this study.

vapor phase

**1. Introduction**

Extraction, Composition, and

Antibacterial Effect of Allspice

The aim of this study was to extract and evaluate the composition and antibacterial effect of allspice (*Pimenta dioica*) essential oil applied in vapor phase against *Salmonella* Typhimurium, *Listeria monocytogenes*, and *Pseudomonas fluorescens* at selected levels of pH and temperature. Microwave assisted extraction (MAE) was tested at different conditions, and it was found that the best extraction conditions were ground allspice, allspice:water relation 1:20, 90 min of soaking before extraction, 800 W for 30 min, and 600 W for 20 min. Antibacterial activity was determined through the minimal inhibitory concentration (MIC) of EO in vapor phase in culture media. Allspice essential oil (AEO) was more effective against *L. monocytogenes* despite the pH or temperature level, compared with *S.* Typhimurium and *P. fluorescens.* Allspice essential oil was able to inhibit the growth of the three bacteria tested, and it was found that both the incubation temperature and pH are the factors that could influence the inhibitory effect of the EO tested in

**Keywords:** microwave extraction, antibacterial effect, allspice essential oil,

The use of microwaves is an alternative to extract essential oils (EOs), and it can be used to assist a common method known as hydrodistillation. This method is achieved by adapting a distillation apparatus to a microwave oven, or with specialized equipment such as the NEOS System equipment (Milestone, Shelton CT, USA) [1]. The MAE is a method that uses microwave radiation as a heating source, for a mixture made with solvent and sample. This type of heating is instantaneous and occurs inside the sample, so the extraction is usually a very fast process [2].

Essential oils are substances extracted from different parts of aromatic plants (flowers, seeds, leaves, herbs, fruits, roots, and rhizomes, among others) and have antiviral, antibacterial, antifungal, and insecticidal properties [3]. EOs contain

(*Pimenta dioica*) Essential Oil

Applied in Vapor Phase

*Ana Cecilia Lorenzo-Leal, Enrique Palou* 

*and Aurelio López-Malo*

### **2.4 Essential oil yield**

The yield of AEO was calculated by means of the following equation:

$$\text{9\%}R = \frac{V}{M} \ast \mathbf{100} \tag{1}$$

where V is the final volume of essential oil, M is the initial mass used of grounded allspice dry fruit, and 100 is a mathematical factor to express it as a percentage (v/p) [18].

#### **2.5 Gas chromatography/mass spectrometry (GC/MS) analysis**

Allspice EO was analyzed by gas chromatography using a 6850 Series Network (Agilent Technologies, Santa Clara, CA), a mass selective detector (5975C VL), and with a triple-axis detector (Agilent Technologies). Component separation was accomplished by an HP-5MS (5% phenyl—95% polydimethylsiloxane) capillary column (30 m by 0.35 mm, 0.25 μm film thickness). The carrier gas used was helium with a constant flow mode of 1.5 mL/min. The temperature of the column started at 60°C for 10 min, increasing every 5 min until reaching 240°C, and maintained at 240°C for 50 min. The injector temperature was 240°C. Retention indices were calculated by a homologous series of n-alkanes C8–C18 (Sigma, St. Louis, MO). Compounds were found by comparing their retention indices from the US NIST (National Institute of Standard Technology) Library and Shimadzu retention index (RI) isothermal equation [16, 19].

$$\text{ROI} = \text{1000} \left( \frac{\log \left( t\_{\text{v}} \right) - \log \left( t\_{\text{m}} \right)}{\log \left( t\_{\text{m} + 1} \right) - \log \left( t\_{\text{m}} \right)} + n \right) \tag{2}$$

where trs is the retention time of the target component, trn is the previous alkane to the target component, trn + 1 is the alkane after the target component, and n is the number of carbons of the alkane trn + 1.

#### **2.6 Vapor phase antibacterial activity in vitro**

#### *2.6.1 Culture medium*

Trypticase soy agar was prepared adjusting its pH values (6.0 or 6.5) with hydrochloric acid (Meyer S.A. de C.V., Mexico City, Mexico), using a previously calibrated potentiometer pH 10 (Conductronic S.A. de C.V., Mexico City, Mexico). Then, the sterilized (15 min at 121°C) TSA was poured in sterile Petri dishes and allowed to solidify. Subsequently, culture media were inoculated using a spiral plater Autoplate 4000 (Spiral Biotech, Norwood, MA), applying 50 μL of inoculum of each bacteria [20].

#### **2.7 Inverted Petri dish method**

The antibacterial activity was evaluated through the minimum inhibitory concentration (MIC) that refers to the minimum concentration necessary to inhibit the visible growth of the studied strains [21], using the inverted Petri dish technique. This method consists in placing a sterile paper disc (Whatman No. 1, diameter 55 mm) impregnated with a known volume of AEO (that varied from 5 to 2000 μL) on the Petri dish lid. The culture medium was then immediately inverted on top of the lid, sealed with Parafilm®, and incubated as followed: (1) 35°C for 24 hours, (2) 25°C for 48 hours, (3) 15°C for 8 days, and (4) 10°C for 9 days [22–24]. These

**85**

*1*

**Table 1.**

*Extraction, Composition, and Antibacterial Effect of Allspice (Pimenta dioica) Essential Oil…*

incubation conditions were selected from previous experiments that corroborate that tested bacteria can grow at the studied temperatures after those incubation times. The obtained MICs were expressed as mL of EO per L of air. A Q-Count counter and software (Spiral Biotech, Norwood, MA) were used to quantify colony

To obtain the best extraction yield of the AEO, different conditions were tested: soaking times and volumes, microwave power, extraction times, and allspice dry

**Table 1** shows that whole allspice, with an allspice:water ratio of 1: 5, without soaking, at 600 W for 40 min, obtaining a yield of 0.5%. Also, it was observed that when conditions changed, yield only increased up to 0.6%. When allspice particle size decreased, yield was higher, which was also seen by Jiang et al. [7] and Chen et al. [17]. When ground sample was used, with an allspice:water relation of 1:5, without soaking in water, at 600 W for 60 min, the yield obtained was the same as the one obtained when the whole allspice was used (0.6%). However, by increasing the soaking time, the allspice:water relation and the microwave power yield increased considerably. When the extraction was made with an allspice:water relation of 1:20, a soaking time of 90 min and by using a combination of power and extraction times (800 W for 30 min and 600 W for 20 min) consecutive in the experiment, the yield increased to 2%. Therefore, the best extraction conditions determined in this study were ground allspice, allspice:water relation 1:20, 90 min

**Tested conditions Yield (%)**

Whole 1:5 No soak 600—40 0.5

Grounded (20 mesh) 1:5 No soak 300—60 0.6

1:5 90 600—40 0.6 1:10 90 600—40 0.6 1:20 90 800—30 & 600—201 0.6

1:5 90 300—60 0.8 1:5 90 600—60 0.9 1:10 90 300—60 0.8 1:10 90 600—60 0.9 1:20 90 600—60 1.1 1:20 90 800—6 & 600—541 1.3 1:20 90 800—30 & 600—201 2.0

**Allspices dry fruit size Allspice:water relation Soaking time (min) Power (W)—time (min)**

All experiments were carried out with an agitation of 400 rpm.

of soaking, and 800 W for 30 min followed by 600 W for 20 min.

*Time and power combinations were used consecutively in the experiment.*

*Conditions tested for the extraction of allspice essential oil and obtained yields.*

) when growth was observed. Tests were performed in

*DOI: http://dx.doi.org/10.5772/intechopen.83691*

**3.1 Extraction and allspice essential oil yield**

fruit size. These results are presented in **Table 1**.

forming units (CFU mL<sup>−</sup><sup>1</sup>

triplicate.

**3. Results**

*Extraction, Composition, and Antibacterial Effect of Allspice (Pimenta dioica) Essential Oil… DOI: http://dx.doi.org/10.5772/intechopen.83691*

incubation conditions were selected from previous experiments that corroborate that tested bacteria can grow at the studied temperatures after those incubation times. The obtained MICs were expressed as mL of EO per L of air. A Q-Count counter and software (Spiral Biotech, Norwood, MA) were used to quantify colony forming units (CFU mL<sup>−</sup><sup>1</sup> ) when growth was observed. Tests were performed in triplicate.
