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Revista mexicana de ciencias pecuarias

versión On-line ISSN 2448-6698versión impresa ISSN 2007-1124

Rev. mex. de cienc. pecuarias vol.14 no.1 Mérida ene./mar. 2023  Epub 24-Mar-2023

https://doi.org/10.22319/rmcp.v14i1.5394 

Articles

Thymol and carvacrol determination in a swine feed organic matrix using Headspace SPME-GC-MS

Fernando Jonathan Lona-Ramíreza 

Nancy Lizeth Hernández-Lópeza 

Guillermo González-Alatorrea 

Teresa del Carmen Flores-Floresa 

Rosalba Patiño-Herreraa 

José Francisco Louvier-Hernándeza 

a Tecnológico Nacional de México en Celaya. Departamento de Ingeniería Química. Guanajuato, México.


Abstract

In recent years, oregano essential oil has been used as an animal food additive due to its antifungal and antibacterial properties as well as s synthetic antibiotic substitute. It is desirable to develop fast and effective thymol and carvacrol quantification method in a swine feed organic matrix. In this work, a performance comparison between the Soxhlet solvent extraction technique using petroleum ether and ethyl acetate and the head space-solid phase microextraction (HS-SPME) technique is made. A 24 design of experiments is performed for defining HS-SPME parameters: equilibrium temperature of 40 ºC, extraction temperature of 40 ºC, ionic strength of 0.57 M, and extraction time of 40 min. The HS-SPME method is more efficient for extracting thymol and carvacrol extraction from an organic matrix. Limits of detection and quantification values using Soxhlet extraction with ethyl acetate were 3.7 and 12.5 μL-1 for thymol and 1.4 and 4.7 μg L-1 for carvacrol, respectively; while LOD and LOQ for HS-SPME were 0.9 and 3.1 μg L-1 for thymol and 0.6 and 1.9 μg L-1 for carvacrol, respectively. The head space-solid phase microextraction method has the potential for quality control in the industry for active compounds present in oregano’s essential oil as an additive into an organic matrix.

Key words Essential oil; Thymol; Carvacrol; Oregano; Origanum; HS-SPME-GC-MS

Resumen:

En los últimos años, el aceite esencial de orégano se ha utilizado como aditivo en alimentos para animales debido a sus propiedades antifúngicas y antibacterianas, así como sustituto sintético de antibióticos. Es deseable desarrollar un método rápido y efectivo de cuantificación de timol y carvacrol en una matriz orgánica de alimento para cerdo. En este trabajo se realiza una comparación de rendimiento entre la técnica de extracción con disolvente Soxhlet utilizando éter de petróleo y acetato de etilo y la técnica de microextracción en fase sólida en el espacio de cabeza (HS-SPME). Se realizó un diseño de experimentos 24 para definir los parámetros de HS-SPME: temperatura de equilibrio de 40 °C, temperatura de extracción de 40 °C, fuerza iónica de 0.57 M y tiempo de extracción de 40 min. El método HS-SPME es más eficiente para la extracción de timol y carvacrol de una matriz orgánica. Los límites de los valores de detección y cuantificación utilizando la extracción Soxhlet con acetato de etilo fueron de 3.7 y 12.5 μl-1 para timol y de 1.4 y 4.7 μg l-1 para carvacrol, respectivamente; mientras que el LDD y el LDC para HS-SPME fueron de 0.9 y 3.1 μg L-1 para timol y de 0.6 y 1.9 μg L-1 para carvacrol, respectivamente. El método de microextracción en fase sólida en el espacio de cabeza tiene el potencial de control de calidad en la industria para compuestos activos presentes en el aceite esencial de orégano como aditivo en una matriz orgánica.

Palabras clave Aceite esencial; Timol; Carvacrol; Orégano; Origanum; HS-SPME-GC-MS

Introduction

People use herbal spices as food flavor enhancers and medicinal aids since antiquity1, mainly for their biological activity. Oregano is one of the most important herbs, which is the common name from a wide variety of plant genera and species worldwide, but usually referred to as Origanum in the Lamiaceae (Labiatae) family2 or Lippia graveolens in the Verbenaceae family. Oregano’s essential oil (OEO) has been used as a food additive due to its antimicrobial activity attributed to its high monoterpenes content such as thymol and carvacrol, the latter generally recognized as a safe food additive3-6. Due to the banning of antibiotics by the European Commission, OEO has received increased attention from the poultry and swine industry for improving natural defenses and strengthening animal organisms with favorable results7-10. OEO can be incorporated into the swine feed by mixing the oil and the organic matrix. However, a confident quantification method is required for quality control.

Thymol and carvacrol11 show antibacterial4,12, antioxidant13, and fungicide activity3,4 and are two of the main components of the OEO. Thus, they can serve as markers for quantification. The quality control method begins with a solvent extraction of the volatile compounds from the feed matrix; however, organic solvents are neither environmentally friendly nor acceptable for food processing. Some other extraction technologies, such as supercritical carbon dioxide extraction, require high-cost equipment and high-pressure operational conditions14,15. Thus, it is desirable to develop quick and effective thymol and carvacrol quantification method inside a swine feed organic matrix. In this paper propose the Head Space Solid Phase Micro Extraction (HS-SPME) technique along with the gas chromatography-mass spectroscopy (GC-MS) method since HS-SPME is an effective, non-expensive, and environmentally friendly technique for the detection and quantification of volatile compounds16,17.

As is known, this technique has not been used for thymol and carvacrol detection in an organic matrix added with OEO, but only to quantify these active compounds in pure oil18-20. This work, compare a solvent extraction technique using petroleum ether or ethyl acetate in a Soxhlet extractor21 with an HS-SPME technique from the swine feed flour matrix to quantify the thymol and carvacrol of the extract using a GC-MS system. It was used nitrosopiperidine (NPIP) as an internal standard for absorbing signal variations due to the extraction method and the equipment itself22 and calculate the limit of detection (LOD) and the limit of quantification (LOQ) for assessing the extraction technique’s effectiveness. Finally, it was performed a design of experiments using R23 and RStudio24 software.

Material and methods

Reagents

Thymol (100.0%), carvacrol (99.9%), nitrosopiperidine (99.9%), and sodium chloride were obtained from Sigma Aldrich (St. Louis, USA). Analytical grade (ACS) petroleum ether and ethyl acetate were obtained from Fermont (Monterrey, México). Tridistilled water from MERCK was used in HS-SPME experiments. The carrier gas used for GC-MS was ultra-high purity (grade 5.0) helium from Praxair. Polyacrylate (PA) fibers for SPME were obtained from Sigma Aldrich. A local industry provided the swine feed flour added with OEO.

Chromatographic method

An Agilent gas chromatograph model 7890A coupled with a mass spectrometer model 5975C with a positive pole ion, single quadrupole with electron impact ionization (EI) source were used for detection and identification. An HP-INNOWax capillary column (30 m, 0.25 mm ID, and 0.5 μm thickness polyethylene glycol film; Alltech) was used for compound separation. Transfer line temperature was set at 250 ºC and GC injector port at 260 °C on splitless mode. The oven temperature was initially set at 60 °C for 3 min, then raised to 250 ºC at a rate of 20 °C per minute and kept there for 3 min. MS was programmed both on scan and SIM mode, with a solvent delay time of 8 min. Scan mode was set from 20 to 300 m/z while SIM mode was set to 114 m/z (characteristic ions of nitrosopiperidine) for the time interval of 8 to 11.5 min and immediately shifted to follow the 135 and 150 m/z signals (characteristic ion of thymol and carvacrol) until the end of the analysis.

Sample preparation

A local industry provided the swine feed flour samples added with oregano’s essential oil during the manufacturing process. The samples were stored in hermetic plastic bags until used. Two different techniques for extracting thymol and carvacrol were used in this work: (i) solvent extraction using a Soxhlet distillation apparatus, and (ii) HS-SPME using a PA fiber. Three different solvents were used: ethyl acetate and petroleum ether for Soxhlet extraction and deionized water for the HS-SPME technique. 5.0 mg L-1 of NPIP was added to every sample as an internal standard.

Soxhlet solvent extraction

A sample of 10 g of swine feed was put into a Soxhlet extraction apparatus with 130 mL of solvent (either ethyl acetate or petroleum ether). The mixture was heated until five cycles were completed, then an aliquot of this extract was stored for analysis. A new fresh solvent was immediately added to perform another five cycles using the same sample, and another extract aliquot was taken. A third distillation step with more fresh solvent was made, and a third aliquot was taken. Thus, each sample (10 g of swine feed) was subjected to extraction three times using two different solvents.

Moreover, it was used two quantification methods using (a) an external standard calibration curve and (b) a standard addition method. The calibration curve for the external standard is made using known concentrations for thymol and carvacrol (2, 4, 6, 8, and 10 mg L-1). For the standard addition method, 0.5 mL of extract aliquot was mixed with 0.5 mL of solvent with different thymol and carvacrol concentrations (2, 4, 6, 8, and 10 mg L-1). In all cases, the sample amount injected into the GC was 1.0 μL.

Head space-solid phase microextraction

The HS-SPME technique involves some parameters such as equilibrium time (teq), equilibrium temperature (Teq), extraction time (text), extraction temperature, (Text), and ionic strength (I). The equilibrium time and temperature are the time and temperature at which the sample is left to reach equilibrium between the solid phase (swine feed matrix) and the vial’s headspace. The extraction time and extraction temperature correspond to the time and temperature at which the microfiber is in contact with the headspace adsorbing volatile compounds. Ionic strength is a measure of the concentration of ions in a solution and modifies the equilibrium of the system. It is necessary to determine the effect of these parameters on the signal obtained in GC-MS. To evaluate this effect, it was added thymol and carvacrol standards in water (along with 5 mg L-1 of NPIP as the internal standard) to form a 10 mg L-1 solution.

For thymol and carvacrol quantification in swine feed, a sample of 0.5 g powder swine feed was added to 15 mL glass vials with PTFE/Silicone septum with the required NaCl content and 10 mL of water with different thymol and carvacrol concentrations to perform the standard addition technique analysis. External standard calibration curves were not possible to perform in the HS-SPME due to the interactions between volatile compounds of the powder swine feed matrix in the gas phase and the fiber during the extraction. The relative area between thymol or carvacrol and the added standard (NPIP) was calculated and used as the response variable to evaluate the performance of the extraction method.

Design of experiments

A 24 factorial analysis was performed to evaluate the effect of the equilibrium temperature (40-50 ºC), extraction temperature (40-50 ºC), extraction time (20-40 min), and ionic strength (0.57-2.28 mole L-1 of NaCl). Equilibrium time is fixed at a sufficiently long time to assure equilibrium (Table 1).

Table 1 Level values of the factors for the design of the experiment 

Factor Low level -1 High level +1
Equilibrium temperature, Teq (ºC) 40 50
Extraction temperature, Text (ºC) 40 50
Extraction time, text (min) 20 40
Ionic strength, I (mole L-1) 0.57 2.28

A 24 factorial design of experiments with a single replicate consists of 16 experimental runs. The analysis of variances of the complete model (main factors and all possible interaction combinations) gives no residuals, Fo, and P-values since the degree of freedom of the error is equal to zero and there is no estimate of the internal error. So, the negligible three- and four-order interactions are used to estimate error. Moreover, after evaluating ANOVA of main effects and two-factor interactions, the significant factors are defined, and another ANOVA analysis is performed taking in account only the factors that are significant. A regression model is then evaluated, and residuals and contour plots are plotted using R-studio.

Results

Thymol and carvacrol mass spectrum identification

A sample of 0.5 g of powder swine feed was put in a vial with 10 mL of water and NPIP as the internal standard. An HS-SPME process was performed to identify the presence of thymol and carvacrol, as shown in Figure 1. Spectra from scan mode were analyzed with the NUST/EPA/NIH mass spectral library for confirmation with a 90 % concordance between the experimental and theoretical spectrum. Retention times of internal standard, thymol, and carvacrol were 10.3, 12.3, and 12.5 min, respectively. The retention time is obtained by following their respective characteristic ion: 144 for NPIP, 135 for thymol, and 150 for carvacrol. It is important to note that the HP-Innowax column was appropriate for good separation between thymol and carvacrol due to its isomeric nature.

Figure 1 Chromatograms of powder swine feed using ethyl acetate and HS-SPME 

Calibration curves

For comparison purposes, three different methodologies for thymol and carvacrol quantification were performed: (a) Soxhlet extraction using organic solvents and calibration with an external standard, (b) Soxhlet extraction using organic solvents and calibration by standard addition, and (c) HS-SPME with water as solvent and calibration using standard addition. The use of an external standard and standard addition is intended for sensibility comparison.

Figure 1 shows the comparison of SIM chromatograms using HS-SPME and Soxhlet extraction with ethyl acetate solvent. For the HS-SPME technique, the sensibility increases almost nine times when compared with the solvent extraction technique, even using less sample quantity during the micro-extraction process, which proves the effectiveness d advantage of the HS-SPME methodology.

The obtained signal [relative area = (thymol or carvacrol area) / (internal standard area)] and its relative standard deviation (RSD) for all the experiments of the factorial design is shown in Figure 2. There are seven experiments with an RSD <15 % for both analytes, but only two with an RSD <5.5 %, experiments #1 and #12. A high signal is desirable, so it was identified four experiments with a high relative area. Experiments #4, #12, and #16 show a combination of high signal (relative area) with low dispersion (RSD) values. All these experiments were performed at a salt content of 0.57 M and extraction time of 40 min, but extraction temperature and equilibrium temperature of 40 and 50 ºC. It is interesting to note that ionic strength (salt content) and extraction time are the factors in common, and they are significant factors as will be seen later.

Figure 2 Relative area and dispersions (relative standard deviation) for thymol and carvacrol for each experiment of the factorial design for improving process parameters 

In Figure 3 it can be observed the calibration curves for (a) the use of thymol and carvacrol as external standards for the Soxhlet extraction technique; (b) the use of NPIP as added standard for the Soxhlet extraction technique; and (c) the use of NPIP as added standard for HS-SPME technique. It is not possible to use external standards for the SPME technique. It is noteworthy that the signal of the relative area is in the order of tens for carvacrol either with external standard or addition standard, while the signal of relative area for thymol is in the order of units for external and addition standards. But for HS-SPME the signal of the relative area is in the order of hundreds for both thymol and carvacrol, which again confirms the increased sensibility of one order of magnitude (two orders for thymol) of this technique.

It should be noted that relative area values (y-axis) are greater for the HS-SPME technique compared to the Soxhlet extraction technique. Soxhlet extraction using ethyl acetate and HS-SPME using water as solvents.

Figure 3 Soxhlet extraction external standard calibration curves (a), Soxhlet extraction with standard addition curves (b), and HS-SPME with standard addition curves (c), for carvacrol and thymol quantification 

Discussion

Design of experiments

Table 2 shows the analysis of variance for main effects and two-factor interactions for carvacrol and thymol quantification, and it is possible to observe that only extraction time and salt content as well as the interaction between them are significant for both analytes. Table 3 shows the analysis of variance considering only ionic strength, extraction time, and ionic strength-extraction time interaction factors for carvacrol and thymol. Considering only the significant factors and interactions, the regression model for the carvacrol HS-SPME extraction is:

CarvacrolRelArea=13.9782+9.0768 I+1.3297 text-0.6242 I×text

Table 2 ANOVA of main effects and two-factor interactions for the design of the experiment 

Carvacrol Thymol
DF Sum of
squares
Mean
squared
F0 P-value DF Sum of
squares
Mean
squared
F0 P-value
Teq (ºC) 1 0.6 0.6 0.016 0.90550 1 0.2 0.2 0.011 0.92026
Text (ºC) 1 1.4 1.4 0.038 0.85218 1 0.7 0.7 0.042 0.84494
I (M) 1 1089.2 1089.2 29.208 0.00293 1 484.9 484.9 30.822 0.00261
text (min) 1 310.1 310.1 8.315 0.03444 1 109.7 109.7 6.974 0.04593
Teq x Text 1 39.0 39.0 1.046 0.35339 1 28.3 28.3 1.798 0.23760
Teq x I 1 13.7 13.7 0.368 0.57070 1 6.6 6.6 0.416 0.54715
Teq x text 1 3.6 3.6 0.096 0.76966 1 0.1 0.1 0.006 0.94100
Text x I 1 2.8 2.8 0.075 0.79541 1 1.4 1.4 0.089 0.77805
Text x text 1 65.0 65.0 1.742 0.24407 1 33.1 33.1 2.105 0.20652
text x I 1 455.8 455.8 12.222 0.01736 1 187.9 187.9 11.941 0.01813
Residuals 5 186.5 37.3     5 78.7 15.7    

DF= degrees of freedom.

Table 3 ANOVA of the significant factors for the for the design of the experiment 

Carvacrol Thymol
DF Sum of squares Mean squared F0 P-value DF Sum of squares Mean squared F0 P-value
I (M) 1 1089.2 1089.2 41.83 <0.001 1 484.9 484.9 39.065 <0.001
text (min) 1 310.1 310.1 11.91 0.00480 1 109.7 109.7 8.839 0.01163
text x I 1 455.8 455.8 17.50 0.00127 1 187.9 187.9 15.134 0.00215
Residuals 12 312.5 26.0        12 148.9 12.4       

DF= degrees of freedom.

With an R2 of 0.8558, meaning that 85.6 % of the data variability is explained by the model with a randomly distributed residuals plot (not shown). A contour plot in Figure 4, shows that when extraction time is at a high level, there is a strong negative effect of salt content, meaning that the relative area of carvacrol is higher when salt content is lower; moreover, when extraction time is at a low level, there is a still negative effect of salt content but weaker than at the high level of extraction time. Considering only the significant factors and interactions, the regression model for the thymol HS-SPME extraction is:

ThymolRelArea= 9.5790+5.5841 I+ 0.8329 text-0.4008 I×text

With an R2 of 0.8401, meaning that 84 % of the data variability is explained by the model with a randomly distributed residuals plot (not shown). A contour plot in Figure 5, shows that when extraction time is at a high level, there is a strong negative effect of salt content, meaning that the relative area of thymol is higher when salt content is lower; moreover, when extraction time is at a low level, there is a still negative effect of salt content but weaker than at the high level of extraction time. This is the same for thymol and carvacrol, the only difference is that carvacrol shows a 1.5 times higher relative area signal than thymol.

Table 3 shows the analysis of variance evaluated only with the significant factors of the DOE, i.e., ionic strength, extraction time, and ionic strength-extraction time interaction factors.

For choosing the best operational parameters, should be always select a high extraction time and low salt content. Since the other two factors are not significant, it can work at any equilibrium temperature and extraction temperature; so, was decided to work at low equilibrium and extraction temperatures for economics.

Table 4 Table of effects for the design of the experiment 

Carvacrol Thymol
Effect T-value P-value Effect T-value P-value
Ionic strength, M -16.502 -6.467 <0.001 -11.0102 -6.250 <0.001
Extraction time, min 8.804 3.451 0.00480 5.2372 2.973 0.01163
text x I -10.674 -4.183 0.00127 -6.853 -3.890 0.00215

For both analytes, an increase in salt content (or ionic strength) results in a decrement in the relative area, probably because the solution is close to saturation. Also, a high extraction time (text) benefits the relative area signal which is in good agreement with the concept that extraction increases as extraction time increments.

Table 5 shows thymol and carvacrol content in the powder swine feed when comparing the two extraction techniques and the solvents used. The total thymol and carvacrol content for each solvent is calculated by adding the measured quantity of each of the three consecutive extractions. Regarding Soxhlet solvent extraction, petroleum ether was not efficient in extracting thymol and carvacrol from the powder swine feed, as indicated by the low quantification obtained of both components, but especially for thymol. Petroleum ether extracted 52 to 55 % less thymol and 19 to 22 % less carvacrol than ethyl acetate. Interestingly, there is a selective extraction capability of both solvents for thymol over carvacrol. Again, the HS-SPME technique shows an improved extraction capacity for both thymol and carvacrol, and there are extracted with no selectivity.

Table 5 Comparison of the total thymol and carvacrol content in powder swine feed  

Technique Soxhlet extraction HS-SPME
Standard
method
Standard addition External standard Standard addition
Solvent Ethyl acetate Petroleum ether Ethyl acetate Petroleum ether water
Analyte Thymol Carvacrol Thymol Carvacrol Thymol Carvacrol Thymol Carvacrol Thymol Carvacrol
First extract, mg L-1 4.25 0.67 1.99 0.48 4.03 0.58 1.92 0.40 - -
Second extract, mg L-1 0.63 0.16 0.33 0.18 0.79 0.15 0.24 0.16 - -
Total content, mg L-1 4.88 0.82 2.32 0.67 4.82 0.73 2.16 0.56 3.25 4.17
Content in swine feed, mg kg-1 63.45 10.71 30.22 8.66 62.60 9.43 28.12 7.30 65.00 83.40

The calibration method also shows some differences. Calibration with an external standard shows a concentration value that is 9 % less on average than that using standard addition calibration; the standard addition method has an improved uncertainty, but it is an expensive method since the standard must be added to each sample.

Figure 4 Contour plot for ionic strength and extraction time for carvacrol extraction using HS-SPME 

The HS-SPME technique shows the highest concentration for thymol and carvacrol. The total thymol content agrees with the total thymol content obtained by Soxhlet extraction with ethyl acetate of about 63-65 mg/kg; however, carvacrol’s total content is very different. Carvacrol quantification by HS-SPME has a value of 83.40 mg/kg, while quantification using Soxhlet extraction with ethyl acetate is 10.71 mg/kg, which is eight times lower than the HS-SPME result. It might be related to the steric behavior of thymol and carvacrol (stereoisomers) and interactions with the fiber material (polyacrylate).

Figure 5 Contour plot for ionic strength and extraction time for thymol extraction using HS-SPME 

Method validation

Limits of detection (LOD) and quantification (LOQ) were estimated to evaluate the performance of the extraction methods and were calculated using the baseline noise and the signal, defined as three times the relation signal/noise for LOD and ten times for the LOQ (16). Figure 3 shows the calibration curves for each component (thymol and carvacrol) for the three different solvents used. LOD and LOQ values using Soxhlet extraction with ethyl acetate were 3.7 and 12.5 μL-1 for thymol and 1.4 and 4.7 μg L-1 for carvacrol, respectively. The HS-SPME technique gives better results for both substances since LOD, and LOQ values were 0.9 and 3.1 μg L-1 for thymol and 0.6 and 1.9 μg L-1 for carvacrol, respectively. The linearity of data was estimated via the linear correlation coefficient, where the lowest value found was 0.9892.

Conclusions and implications

Oregano’s essential oil is positively identified in the swine feed powder using characteristic volatile compounds thymol and carvacrol using two different extraction methods: Soxhlet and HS-SPME. Among the organic solvents for Soxhlet extraction, petroleum ether was not suitable since it only extracted about 50 and 10 % of the total thymol and carvacrol content, respectively (relative to HS-SPME quantification). Furthermore, regarding the use of ethyl acetate in Soxhlet extraction, this solvent was able to extract all the thymol, but not the carvacrol, showing some sort of selectivity. For the HS-SPME technique, a 24-factorial design of experiments was performed to evaluate process parameters and obtain the highest possible S/N ratio. The proper conditions are equilibrium temperature (Teq) of 40 ºC, extraction temperature (Text) of 40 ºC, ionic strength (I) of 0.57 M, and extraction time (text) of 40 min. HS-SPME showed a nine-times better extraction performance compared to Soxhlet extraction, even with smaller sample amounts, with a limit of detection and quantification of 0.9 and 3.1 μg L-1 for thymol, and 0.6 and 1.9 μg L-1 for carvacrol, respectively. The results show that the HS-SPME method is more efficient for thymol and carvacrol extraction from an organic matrix and has the potential for a quality-control technique in the food industry to quantify the active compounds of oregano’s essential oil when used as an additive to an organic matrix such as swine feed.

Acknowledgments

To CONACYT (The National Council of Science and Technology) for the financial support awarded to doctoral student Fernando Jonathan Lona Ramírez (grant Number: 344837) and Tecnológico Nacional de México (TecNM) for research grant number 5267.14-P to carry out this study. To Alimentos Aicansa SA for providing swine feed samples with OEO as an additive.

Literature cited

1. Sánchez-Ruíz JF, Tejeda-Rosales ME, Sánchez-Tejeda JF, Sánchez-Tejeda MG. Pharmacy, medicine and herbolary in the Florentine Codex. Rev Mex Cienc Farm 2012;43(3):55-66. [ Links ]

2. Kintzios SE. Oregano. In: Peter KV editor. Handbook of herbs and spices Vol. 2. Cambridge, UK: Woodhead; 2004:215-229. [ Links ]

3. Ahmad A, Khan A, Akhtar F, Yousuf S, Xess I, Khan LA, Manzoor N. Fungicidal activity of thymol and carvacrol by disrupting ergosterol biosynthesis and membrane integrity against Candida. Eur J Clin Microbiol Infect Dis 2011;30(1):41-50. [ Links ]

4. Liolios CC, Gortzi O, Lalas S, Tsaknis J, Chinou I. Liposomal incorporation of carvacrol and thymol isolated from the essential oil of Origanum dictamnus L. and in vitro antimicrobial activity. Food Chem 2009;112(1):77-83. [ Links ]

5. Marchese A, Orhan IE., Daglia M, Barbieri R, Di Lorenzo A, Nabavi SF, Gortzi O, Izadi M, Nabavi SM. Antibacterial and antifungal activities of thymol: A brief review of the literature. Food Chem 2016;210:402-414. [ Links ]

6. Pesavento G, Calonico C, Bilia AR, Barnabei M, Calesini F, Addona R, et al. Antibacterial activity of Oregano, Rosma-rinus and Thymus essential oils against Staphylococcus aureus and Listeria monocytogenes in beef meatballs. Food Control 2015;54:188-199. [ Links ]

7. Bozkurt M, Bintaş E, Kırkan Ş, Akşit H, Küçükyılmaz K, Erbaş G, et al. Comparative evaluation of dietary supplementation with mannan oligosaccharide and oregano essential oil in forced molted and fully fed laying hens between 82 and 106 weeks of age. Poult Sci 2016;95(11):2576-2591. [ Links ]

8. Franciosini MP, Casagrande-Proietti P, Forte C, Beghelli D, Acuti G, Zanichelli D, et al. Effects of oregano (Origanum vulgare L.) and rosemary (Rosmarinus officinalis L.) aqueous extracts on broiler performance, immune function and intestinal microbial population. J Appl Anim Res 2016;44(1):474-479. [ Links ]

9. Scocco P, Forte C, Franciosini MP, Mercati F, Casagrande-Proietti P, Dall’Aglio C, et al. Gut complex carbohydrates and intestinal microflora in broiler chickens fed with oregano (Origanum vulgare L.) aqueous extract and vitamin E. J Anim Physiol Anim Nutr (Berl) 2017;101(4):676-684. [ Links ]

10. Zeng Z, Zhang S, Wang H, Piao X. Essential oil and aromatic plants as feed additives in non-ruminant nutrition: a review. J Anim Sci Biotechnol 2015;6:7. [ Links ]

11. Russo M, Galletti GC, Bocchini P, Carnacini A. Essential oil chemical composition of wild populations of italian oregano spice (Origanum vulgare ssp. hirtum (Link) Ietswaart): A preliminary evaluation of their use in chemotaxonomy by cluster analysis. 1. Inflorescences. J Agric Food Chem 1998;46(9):3741-3746. [ Links ]

12. de Oliveira Nóbrega R, de Castro Teixeira, AP, Araújo de Oliveira W, de Oliveira Lima E, Oliveira Lima I. Investigation of the antifungal activity of carvacrol against strains of Cryptococcus neoformans. Pharm Biol 2016;54(11):2591-2596. [ Links ]

13. Safaei-Ghomi J, Ebrahimabadi AH, Djafari-Bidgoli Z, Batooli H. GC/MS analysis and in vitro antioxidant activity of essential oil and methanol extracts of Thymus caramanicus Jalas and its main constituent carvacrol. Food Chem 2009;115(4):1524-1528. [ Links ]

14. Díaz-Maroto MC, Pérez-Coello MS, Cabezudo MD. Supercritical carbon dioxide extraction of volatiles from spices: Comparison with simultaneous distillation-extraction. J Chromatogr A 2001;947(1):23-29. [ Links ]

15. Hossain MB, Barry-Ryan C, Martin-Diana AB, Brunton NP. Optimisation of accelerated solvent extraction of antioxidant compounds from rosemary (Rosmarinus officinalis L.), marjoram (Origanum majorana L.) and oregano (Origanum vulgare L.) using response surface methodology. Food Chem 2011;126(1):339-346. [ Links ]

16. Lona-Ramirez FJ, Gonzalez-Alatorre G, Rico-Ramírez V, Perez-Perez, MCI, Castrejón-González EO. Gas chromatography/mass spectrometry for the determination of nitrosamines in red wine. Food Chem 2016;196:1131-1136. [ Links ]

17. Méndez-Pérez D, González Alatorre G, Botello Álvarez E, Escamilla Silva E, Alvarado JFJ. Solid-phase microextraction of N-nitrosodimethylamine in beer. Food Chem 2008; 107(3):1348-1352. [ Links ]

18. Adams A, Kruma Z, Verhé R, De Kimpe N, Kreicbergs V. Volatile profiles of rapeseed oil flavored with basil, oregano, and thyme as a function of flavoring conditions. J Am Oil Chem Soc 2011;88(2):201-212. [ Links ]

19. Karami-Osboo R, Miri R, Asadollahi M, Jassbi AR. Comparison between head-space spme and hydrodistillation-gc-ms of the volatiles of Thymus daenensis. J Essent Oil Bear Pl 2015;18(4):925-930. [ Links ]

20. Stashenko EE, Martínez JR. Sampling volatile compounds from natural products with headspace/solid-phase micro-extraction. J Biochem Biophys Methods 2007;70(2):235-242. [ Links ]

21. Pothier J, Galand N, El Ouali M, Viel C. Comparison of planar chromatographic methods (TLC, OPLC, AMD) applied to essential oils of wild thyme and seven chemo-types of thyme. II Farmaco 2001;56(5-7):505-511. [ Links ]

22. Pawliszyn J. Solid phase microextraction: Theory and practice. New York, USA: Wiley-VCH; 1997. [ Links ]

23. R Core Team. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing, 2021. http://www.R-project.org/Links ]

24. RStudio Team. RStudio: Integrated Development Environment for R. Boston, Massachusetts, USA: RStudio. PBC; 2020. http://www.rstudio.com/ [ Links ]

Received: August 17, 2020; Accepted: September 02, 2021

*Autor de correspondencia: francisco.louvier@itcelaya.edu.mx

Conflict of interest

The authors declare no conflict of interest.

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