**3. Results**

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%*R* = \_\_*<sup>V</sup>*

RI <sup>=</sup> <sup>100</sup>(

number of carbons of the alkane trn + 1.

*2.6.1 Culture medium*

**2.7 Inverted Petri dish method**

**2.6 Vapor phase antibacterial activity in vitro**

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

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

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].

*log*( *trs*)– *log*( *trn*) \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ *log*( *trn*+1)– *log*( *trn*) <sup>+</sup> *<sup>n</sup>*

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

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].

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

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

*<sup>M</sup>* <sup>∗</sup> <sup>100</sup> (1)

) (2)

**2.4 Essential oil yield**

percentage (v/p) [18].

**84**

#### **3.1 Extraction and allspice essential oil yield**

To obtain the best extraction yield of the AEO, different conditions were tested: soaking times and volumes, microwave power, extraction times, and allspice dry fruit size. These results are presented in **Table 1**.

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

**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 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.*

#### **Table 1.**

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

On the other hand, Jiang et al. [7] also used MAE to obtain EO from allspice, and the extraction time was 63 min at 1000 W, obtaining a yield of 3.25%. The yield and extraction time were greater than those obtained in this work, probably because they used higher power. Chen et al. [17] tested different conditions to extract essential oil from black pepper and observed that when the pepper was ground and soaked in water for 90 min, the amount of oil obtained was greater compared with the results obtained when it was not soaked or soaked for 30 or 60 min, which was also observed in the present work, since extraction tests made with whole raw material lower yields were obtained.

## **3.2 Chemical composition**

The main components identified by GC-MS in tested allspice EO and their calculated retention indices are reported in **Table 2**. Attokaran [25] mentions that allspice EO could contain 80–87% of eugenol, 4–8% β-caryophyllene, or 0.2–0.5% of β-phyllandrine, which coincide with the findings in the present work, especially for eugenol.

### **3.3 Antibacterial activity**

The antibacterial activity of AEO against *L. monocytogenes, S.* Typhimurium, and *P. fluorescens* was tested using the inverted Petri dish technique, and the results obtained are presented in **Figure 1**. The figure shows that the AEO had antibacterial effects (at different concentrations) against the three studied bacteria in the different conditions of pH and temperature.

Allspice EO was more effective against *L. monocytogenes*, despite the pH (6.0 or 6.5) or the evaluated temperatures (10, 15, 25 or 35° C) compared to *S.* Typhimurium and *P. fluorescens.* Du *et al. [6]* also tested the antimicrobial activity of AEO in vapor phase against *L. monocytogenes, Escherichia coli* O157:H7, and Salmonella *enterica*, being *L. monocytogenes* less resistant to the EO vapors than *E. coli* or *Salmonella* which coincide with our findings. These results could be related with the fact that Gram positive (*L. monocytogenes*) bacteria could be more susceptible to EOs than Gram negative bacteria (*S.* Typhimurium or *P. fluorescens*), as reported by various authors [5, 26, 27].

On the other hand, different pH levels did not affect much the antimicrobial effect of the AEO, but different temperature conditions did have an impact on the antimicrobial effect, especially when the temperature increases up to 25 and 35°C, as shown in **Figure 1**. It is well known that microorganisms have an optimum pH and temperature for their growth, and when these conditions move in any direction (lower or higher), microbial growth could be delayed. Furthermore, when microorganisms are exposed to different factors, such as temperature, pH or antimicrobials,


**87**

cases, the MICs were higher.

*(b) S. Typhimurium, and (c) P. fluorescens.*

**4. Conclusions**

**Figure 1.**

(*L. monocytogenes*).

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

such as EOs, an interaction between these factors could affect microbial growth. The results of these interactions could give us an idea of what microbial growth would look like in food systems; however, it is necessary to test food systems to guarantee the response of the microorganisms of interest. In this study, the pH had a lower impact, compared to the temperature, and in some cases as it increased, the MICs also did so. In summary, when the temperature and pH increased, in most

*Minimum inhibitory concentrations (MICs) of allspice essential oil against (a) L. monocytogenes,* 

Allspice essential oil was able to inhibit the growth of *L. monocytogenes*, *S.* Typhimurium, and *P. fluorescens*, 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 this study. It was also observed that lower concentrations of the AEO were

required to inhibit the growth of *L. monocytogenes*, in comparison with *S.* Typhimurium and *P. fluorescens*, which was expected, since Gram-negative bacteria (*Salmonella* or *P. fluorescens*) are more resistant to EOs than Gram positive

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

**Table 2.**

*Main components of allspice essential oil determined by gas chromatography-mass spectrometry.*

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

**Figure 1.**

*Technology, Science and Culture - A Global Vision*

rial lower yields were obtained.

**3.2 Chemical composition**

**3.3 Antibacterial activity**

ent conditions of pH and temperature.

reported by various authors [5, 26, 27].

for eugenol.

On the other hand, Jiang et al. [7] also used MAE to obtain EO from allspice, and the extraction time was 63 min at 1000 W, obtaining a yield of 3.25%. The yield and extraction time were greater than those obtained in this work, probably because they used higher power. Chen et al. [17] tested different conditions to extract essential oil from black pepper and observed that when the pepper was ground and soaked in water for 90 min, the amount of oil obtained was greater compared with the results obtained when it was not soaked or soaked for 30 or 60 min, which was also observed in the present work, since extraction tests made with whole raw mate-

The main components identified by GC-MS in tested allspice EO and their calculated retention indices are reported in **Table 2**. Attokaran [25] mentions that allspice EO could contain 80–87% of eugenol, 4–8% β-caryophyllene, or 0.2–0.5% of β-phyllandrine, which coincide with the findings in the present work, especially

The antibacterial activity of AEO against *L. monocytogenes, S.* Typhimurium, and *P. fluorescens* was tested using the inverted Petri dish technique, and the results obtained are presented in **Figure 1**. The figure shows that the AEO had antibacterial effects (at different concentrations) against the three studied bacteria in the differ-

Allspice EO was more effective against *L. monocytogenes*, despite the pH (6.0 or 6.5) or the evaluated temperatures (10, 15, 25 or 35° C) compared to *S.* Typhimurium and *P. fluorescens.* Du *et al. [6]* also tested the antimicrobial activity of AEO in vapor phase against *L. monocytogenes, Escherichia coli* O157:H7, and Salmonella *enterica*, being *L. monocytogenes* less resistant to the EO vapors than *E. coli* or *Salmonella* which coincide with our findings. These results could be related with the fact that Gram positive (*L. monocytogenes*) bacteria could be more susceptible to EOs than Gram negative bacteria (*S.* Typhimurium or *P. fluorescens*), as

On the other hand, different pH levels did not affect much the antimicrobial effect of the AEO, but different temperature conditions did have an impact on the antimicrobial effect, especially when the temperature increases up to 25 and 35°C, as shown in **Figure 1**. It is well known that microorganisms have an optimum pH and temperature for their growth, and when these conditions move in any direction (lower or higher), microbial growth could be delayed. Furthermore, when microorganisms are exposed to different factors, such as temperature, pH or antimicrobials,

**Compound Percentage in total allspice EO (%) Retention index** Eugenol 89.55 1356 α-Terpineol 2.04 1457 Caryophyllene oxide 1.48 1582 1,8-Cineole 1.06 1026 α-Cadinol 0.86 1652

*Main components of allspice essential oil determined by gas chromatography-mass spectrometry.*

**86**

**Table 2.**

*Minimum inhibitory concentrations (MICs) of allspice essential oil against (a) L. monocytogenes, (b) S. Typhimurium, and (c) P. fluorescens.*

such as EOs, an interaction between these factors could affect microbial growth. The results of these interactions could give us an idea of what microbial growth would look like in food systems; however, it is necessary to test food systems to guarantee the response of the microorganisms of interest. In this study, the pH had a lower impact, compared to the temperature, and in some cases as it increased, the MICs also did so. In summary, when the temperature and pH increased, in most cases, the MICs were higher.

### **4. Conclusions**

Allspice essential oil was able to inhibit the growth of *L. monocytogenes*, *S.* Typhimurium, and *P. fluorescens*, 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 this study. It was also observed that lower concentrations of the AEO were required to inhibit the growth of *L. monocytogenes*, in comparison with *S.* Typhimurium and *P. fluorescens*, which was expected, since Gram-negative bacteria (*Salmonella* or *P. fluorescens*) are more resistant to EOs than Gram positive (*L. monocytogenes*).

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