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Bioactivity of oregano (Origanum vulgare) essential oil against Alicyclobacillus spp.
Industrial Crops & Products 129 (2019) 345–349
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Bioactivity of oregano (Origanum vulgare) essential oil against Alicyclobacillus spp.
T
Tatiane Viana Dutraa, Juliana Cristina Castroa, Jéssica Lima Menezesa, Tatiane Rogelio Ramosa, Ivanor Nunes do Pradob, Miguel Machinski Juniorc, Jane Martha Graton Mikchad, ⁎ Benício Alves de Abreu Filhoc, a
State University of Maringa, Av. Colombo, 5790, Maringá, 87020-900, Paraná, Brazil Animal Science Department, State University of Maringa, Av. Colombo, 5790, Maringá, 87020-900, Paraná, Brazil c Department of Basic Health Sciences, State University of Maringa, Av. Colombo, 5790, Maringá, 87020-900, Paraná, Brazil d Department of Clinical Analysis and Biomedicine, State University of Maringa, Av. Colombo, 5790, Maringá, 87020-900, Paraná, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Antimicrobian activity Antioxidant activity Alicyclobacillus acidoterrestris Carvacrol acetate Essential oil Origanum vulgare
The Alicyclobacillus spp., causing deterioration in citrus beverages, has been frequently related to the use of natural antimicrobial agents in its combat; in this sense, the study sought to evaluate the activity of the essential oil of oregano (Origanum vulgare) against different isolates of this bacterium, in addition to its antioxidant activities. The minimum inhibitory concentration obtained from oregano essential oil for A. Acidiphilus and A. cycloheptanicus was 125 μg/mL; and for A. herbarius and A. acidoterrestris was 62.5 μg/mL. While the minimum bactericidal concentration obtained was 1000 μg / mL for all isolates. The combined effect of nisin and O. vulgare against A. acidoterrestris resulted in indifference. The antioxidant activity obtained was 363 μmol trolox/mg by the DPPH method and 1142 μmol trolox/mg by the ABTS method. The chemical characterization of the essential oil of oregano by GC–MS was able to identify of 93.13% of the compounds was carried out, where the major compound was carvacrol acetate represented by 59.61%. Further scanning electron microscopy was able to demonstrate damage to cells treated with the inhibitory concentrations of O. vulgare.
1. Introduction The Alicyclobacillus spp. known worldwide for causing deterioration of citrus foods such as fruit juices, teas and tomato extract; are Grampositive bacillus capable of growing at low pH and high temperatures, with a pH of 4.0–4.5 and temperature of 40 at 50 °C, thus being acidresistant and spore-forming agents (Goto, 2003). Several species of Alicyclobacillus have already been isolated in food industries, among them the most important is Alicyclobacillus acidoterrestris, due to its ability to form byproducts such as guaiacol, responsible for the astringent taste in deteriorated juices (Anjos et al., 2016). There are more than 21 species of Alicyclobacillus described, with variations of sources of isolation, morphology, pH and growth temperature; among them, A. acidiphilus presents as source acidic beverages, A. cycloheptanicus in the soil, A. herbarius in the herbal teas while A. acidoterretsris in the soil and apple juices (Sant’Ana et al., 2014). Synthetic preservatives, such as sodium and potassium benzoate,
are used to combat the Alicyclobacillus vegetative cells depending on the initial concentration (Sant’Ana et al., 2014). In search of natural alternatives against this genus of bacteria, there is the use of essential oils of plants, which may present antibacterial activity inhibiting the growth of Alicyclobacillus spp., Protecting the products of the deterioration caused by this microorganism. Calderón-Oliver et al. (2016) combined the use of the chemical preservative nisin with a natural antioxidant, avocado byproducts, in the evaluation of antimicrobial activity; resulting in synergism, allowing the reduction of costs by reducing the amount of nisin to be used. The use of natural compounds to control microorganisms is increasingly present (Castro et al., 2017). Essential oils (EOs) are liquids oily aromatic and volatile extracted from specific plant parts. Several essential oils have antibacterial, antifungal, antiviral, antioxidant and biological properties. Among these oils, the essential oil of oregano presents antioxidant and antimicrobial activities, probably due to the presence of carvacrol and thymol (Li et al., 2018; Zhou et al., 2018). In addition, recent studies report that oregano essential oil is
⁎ Corresponding author at: Department of Basic Health Science, State University of Maringá, Av. Colombo 5790, Campus Universitário, Laboratório de Análise de Água, Ambiente e Alimentos, Bloco T20, 3° andar, Sala 312, 87020-900, Maringá, Paraná, Brazil. E-mail address: baafi[email protected] (B.A.d.A. Filho).
https://doi.org/10.1016/j.indcrop.2018.12.025 Received 3 September 2018; Received in revised form 23 October 2018; Accepted 6 December 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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a standard curve with trolox solution (( ± )-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was constructed. The results were expressed as mmol trolox/mg (Rufino et al., 2007a).
antiproliferative, antiinflammatory, antidiabetic and has cancer suppressive activity (Leyva-López et al., 2017). With the growing attention given to food safety through natural alternatives, the use of plant essential oils has been highlighted because of its high potential as a preservative, as well as being generally recognized as safe (GRAS) by the WHO (World Health Organization) and still many have antimicrobial activity controlling both pathogens and spoilage food bacteria (Gutierrez et al., 2008). This study aims to characterize the essential oil of oregano, to evaluate the antioxidant activity and the antimicrobial capacity, as well as its combined use with nisin, against Alicyclobacillus spp.
2.3.2. ABTS method The antioxidant activity by the ABTS (2,2′-azino-bis-(-3-ethylbenzothiazoline-6-sulfonic acid), diammonium salt, ∼ 98%) method was performed according to Rufino et al. (2007b). In tubes protected from light, 30 μL of the diluted EO and 3 mL of the ABTS+ solution (5 ml ABTS - 7 mmol/L solution and 88 μL of potassium persulfate 140 mmol/L; reaction for 16 h in a protected environment from light) were transferred and maintained for 6 min. The read was performed in a spectrophotometer at 734 nm and a standard curve with trolox solution was constructed. The results were expressed as mmol trolox/mg.
2. Material and methods 2.1. Microbial lineage, essential oil and nisin
2.4. Antibacterial activity Strains of the species Alyciclobacillus spp. were used from the Brazilian Collection of Environmental and Industrial Microorganisms (CBMAI), located at the Center for Chemical, Biological and Agricultural Research (CPQBA/UNICAMP), with reference to this study: A. acidoterrestris 0244T; A. herbarius 0246T; A. acidiphilus 0247T; A. cycloheptanicus 0297T. The oregano essential oil used in the present study was of commercial origin (Origanum vulgare - Marca Ferquima) obtained on local commerce in the city of Maringa, Paraná. Commercial nisin (Sigma-Aldrich, St. Louis, USA) was prepared from a solution of 0.02 M hydrochloric acid (HCl); filtrate with 0.22 μm sterile membrane (Millipore, São Paulo, Brazil).
2.4.1. Minimum inhinitory concentrations (MIC) and Bactericidal concentrations (MBC) The minimum inhibitory and bactericidal concentrations was determined using the 96-well microplate microdilution technique, according to CLSI methodology (2012), with BAT broth (Bacillus Acidoterrestris) as culture medium. The microorganism was activated in BAT broth 24 h before the experiment and incubated at 45 °C. The serial dilution of oregano essential oil was performed (0.90–1000 μg/mL) and inoculating 5 μL of the standard suspension according to the McFarland scale 0.5 in each well. The EO was diluted in 1% Tween-80 and tested at concentrations of 0.90–1000 g/mL. The microplates were incubated at 45 °C for 24 h. The MIC was defined as the lowest concentration of the EO that inhibited the visual growth of the bacteria. After this period, 20 μL each well was plated in YSG agar and incubated at 45 °C for 24 h for subsequent reading, where the lowest concentration capable of inhibiting bacterial growth was considered the minimum bactericidal concentration. The experiments were performed in triplicate.
2.2. Characterization of essential oil The chemical composition of the EO was performed using gas chromatography-mass spectrometry (GC–MS) with an automatic injector (FOCUS GC – DSQ II, Thermo Electron Corp). The gas chromatograph-mass spectrometer was equipped with an Agilent DB-5 capillary column (5% phenyl/95% dimethyl siloxane stationary phase, 30 m length, 0.25 mm internal diameter, and 0.1 μm film thickness). Characterization was performed using a column temperature program that began at 60 °C, followed by a temperature increase of 3 °C/min to 230 °C. Helium was used as the carrier gas at a flow rate of 1.0 mL/min. The total analysis time was 63 min. The temperature of the injector and detector was maintained at 240 °C A 1 μL volume of the samples was injected for chromatography in split mode (1:10). EO was diluted in hexane (high-performance liquid chromatography grade; 2 μL of EO to 1000 μL of hexane) to form the stock solution (Kim et al., 2015; Castro et al., 2017, with modifications). Characterization was performed based on retention time (RT) and compared to the major compounds using Kovats retention index (KI) (Skoog et al., 2006). The compounds of the EOs were identified by analyzing the retention times of the peaks that were obtained for each EO and confirmed via a standard mixture of n-alkanes (C8–C20; SigmaAldrich). The compounds of interest were confirmed (Adams, 2007) and are presented as percentages.
2.4.2. Disc diffusion method Kirby-Bauer agar disc diffusion method was used to evaluate the inhibition growth of the bacteria. Plates with YSG were seeded with the inoculum standardized with the turbidity of the McFarland scale 0.5. These discs (5 mm diameter) were arranged around the plate with different concentrations of OE of oregano, 2000, 1000, 500, 250 μg/mL and the saline-containing control. The plate was incubated for 24 h at 45 °C and the reading was performed according to the inhibition halo formed around the disc (Prasad et al., 2001). 2.4.3. Checkerboard method Checkerboard tests were performed to evaluate the effects of drug combinations. The assay was performed in 96-well microplates to obtain the fractional inhibitory concentration (FIC) of the EO combined with nisin against A. acidoterrestris. YSG medium was used for the assay. A volume of 5 μL of the inoculum (inoculum prepared from the standard suspension according to the McFarland 0.5 scale) was added to each well, and the plates were incubated at 45 °C for 24 h. The result was analyzed by the inhibition of visible growth, the inhibitory fractional concentration index (FIC) was calculated, being FIC = FICA + FICB, where FICA = MICA combined / MICA alone, and FICB = MICB combined / MICB alone; defined as synergistic effect a result ≤ 0.5, additive 0.5 to ≤ 1, indifferent from 1 to ≤ 4 and antagonist > 4 (Schelz et al., 2006).
2.3. Antioxidant activity The extract of the essential oil was diluted in methanol (P.A., Anidrol, Brazil), 1 mg of EO in 1 mL of methanol. The extract was stored in an amber bottle to protection to the light until analysis.
2.5. Scanning electron microscopy
2.3.1. DPPH method The antioxidant activity by the DPPH (2,2-Diphenyl-1-picrylhydrazyl) method was performed according to Ma et al. (2011). In tubes protected from light, 25 μL of the diluted OE and 2 mL of the 6.25 × 10−5 mol/L DPPH solution were added and maintained for 30 min. The read was performed in a spectrophotometer at 517 nm and
The inoculum of Alicyclobacillus acidoterrestris 0244T was treated with oregano essential oil at the concentrations defined by the antimicrobial activity of the sub-mic, mic and negative control without the application of the essential oil. After incubation of samples at 45 °C for 346
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Table 1 Chemical composition of O. vulgare essential oil. Compounds
a
KI
Origanum vulgare b
β-pinene Meta-cymene α-terpinene Linalool Borneol Carvacrol Carvacrol acetate Caryophyllene < (E)- > Caryophyllene acetate Furopelargone A Total of identified compounds a b
973.53 1020.81 1053.55 1096.19 1167.66 1286.17 1296.60 1413.04 1448.79 2134.78
RT (min)
7.79 9.42 10.71 12.39 15.49 20.7 21.16 26.16 27.64 51.85 93.13%
% 1.27 5.42 4.47 1.90 1.09 2.85 69.51 4.28 1.05 1.29
KI = Kovats index. RT = Retention time (min).
Fig. 2. Antioxidant activity of O. vulgare EO for DPPH and ABTS methods. Lowercase letters in columns show significant difference (p < 0.05).
24 h, fixation with 2.5% glutaraldehyde in 0.1 M cacodylate buffer and adhesion on coverslips pretreated with poly-L-lysine (Sigma-Aldrich). Followed by dehydration with an ethanolic increasing series of 30%–100%, critical point of CO2, metallization in gold and analysis in scanning electron microscope Quanta-250 (Endo et al., 2010).
(Fig. 1). The composition of the essential oil of O. vulgare subsp glandulosum (Desf.), harvested at different times of the year in North Africa; presented major compounds as Thymol (31.80%, year 2007; 43.70%, year 2008; and 46.10%, year 2009), γ-Terpinene (24.20%, year 2007; 27.10%, year 2008; and 24.00%, year 2009) and p-Cymene (35.70%, year 2007; 12.70%, year 2008; and 11.50%, year 2009) (Mechergui et al., 2016). In O. vulgare EO purchased from Chile, the composition of the major compounds was 4-terpineol (41.17%), Thymol (21.95%), γTerpinene (5.91%) and Carvacrol (4.71%) (Brondani et al., 2018), different from the compounds found in this study. Many intrinsic and extrinsic factors such as agricultural factors, species, harvest time, plant part, season, climate, geography, extraction methods and storage care influence the composition of the EO and concentration of each compound, majority or minority (Castro et al., 2017). Cattelan et al. (2018) in its characterization of the essential oil of oregano obtained as major compound the carvacrol (65.1%), similar to the result obtained in the present work.
2.6. Statistical analysis The antioxidant data were submitted to analysis of variance by ANOVA and Tukey's test (p < 0.05) was used to compare the means by means of the statistical program SISVAR version 5.3. The figure was generated by SigmaPlot software version 11.0. 3. Results and discussion 3.1. Characterization of essential oil The characterization of O. vulgare EO by GC–MS, followed by identification and quantification is presented in Table 1. The major compound was carvacrol acetate (69.51%), of 93.13% of the total compounds identified. Meta-cymene (5.42%), α-terpinene (4.47%), Caryophyllene < (E)- > (4.28%), Carvacrol (2.85%), Linalool (1.90%), Furopelargone A (1.29%), β-pinene (1.27%), Borneol (1.09%) and Caryophyllene acetate (1.05%) were presented in smaller quantities
3.2. Antioxidant activity The antioxidant activity for O. vulgare EO (Fig. 2) showed different
Fig. 1. Chromatographic profile of essential oil of oregano (Origanum vulgare) and Fragmentation spectrum of the major compound carvacrol acetate. 347
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Fig. 3. Disk diffusion method of oregano essential oil against Alicyclobacillus acidoterrestris. Discs numbered 1 (EO concentration – 2000 μg/mL), 2 (EO concentration – 1000 μg/mL),3 (EO concentration – 500 μg/mL),4 (EO concentration – 250 μg/mL) and 5 (saline solution).
Fig. 4. Scanning Electron Microscopy. (a) Control vegetative cells of A. acidoterrestris; (b) MIC of O. vulgare EO with vegetative cells of A. acidoterrestris; (c) Sub-MIC of O. vulgare EO with vegetative cells of A. acidoterrestris.
concentrations between the tested methods, DPPH and ABTS, 363 μmol trolox/mg and 1142 μmol trolox/mg, respectively. These results suggest a good antioxidant activity by the evaluated methods. Aromatic plants such as oregano have high free radical activity (82%) (Olmedo et al., 2014). It is important to mention that the antioxidant activity of the essential oils is related to the species, time of harvest and geographical position (Mechergui et al., 2016), because the chemical composition of the oils differs due to intrinsic and extrinsic factors. The antioxidant activity found is related to compounds, such as carvacrol (Hashemi and Khaneghah, 2017), which is also related to the antibacterial activity presented in this work.
this emulsion makes the extract more stable, improving its efficiency against bacterial cells. In another application the use of essential oil was analyzed in salad dressings, where it obtained a reduction in Escherichia coli counts, demonstrating its antibacterial action (Cattelan et al., 2018). Ruiz et al. (2013), in their study, evaluated the antimicrobial actions of several fractions of Piperaceae extract against A. acidoterrestris and the results founded for MIC and MBC ranged from 15.63 to higher of 1000 μg/mL, the best result was that of the Piper aduncum fraction, with extracts of chloroform, dichloromethane fraction and the pure compound, such fractions were combined with nisin by the Checkerboard methodology, for which the result was synergism. Nisin is a bacteriocin with proven antimicrobial activity against Gram-positive bacteria, such as A. acidoterrestris; used in food industries as an antimicrobial agent approved by the Food and Drug Administration (FDA). With respect to the combination of oregano essential oil and nisin, the FICI result found for the four strains was indifferent, presenting values of 2.06; 2.12; 2.12 and 2.5 for A. acidoterrestris, A. Acidiphilus, A. cycloheptanicus and A. herbarius, respectively. It is concluded that the use of O. vulgare EO with nisin, although not synergistic, does not demonstrated the antagonism of its activities. The results of the diffusion disc method, on 5 mm diameter discs, confirmed the inhibitory activity (MIC) of O. vulgare EO against A. acidoterrestris, through formation of growth inhibition halo at different concentrations of oil used, 2000, 1000, 500 and 250 μg/mL, in addition
3.3. Antibacterial activity The MIC found for O. vulgare EO against A. Acidiphilus and A. cycloheptanicus was 125 μg/mL; and for A. herbarius and A. acidoterrestris was 62.5 μg/mL, while the MBC obtained was 1000 μg/mL, but with a reduction of 2 logs. A cellulose nanocrystals (CNCs) emulsion, prepared from microcrystalline cellulose (MCC) by hydrolysis with ammonium persulfate (APS), and oregano essential oil showed a minimum inhibitory concentration (MIC) of 12.5 μg/mL against Staphylococcus aureus, Saccharomyces cerevisiae and Escherichia coli, and the most effective antimicrobial effect was observed in relation to Bacillus subtilis with a MIC of 6.25 μg/mL (Zhou et al., 2018), this result is due to the fact that 348
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to the control in which only saline solution was plated and where there was not halo formation, as shown in Fig. 3. Bassolé and Juliani (2012), reported the production of secondary metabolites by plants, which have antimicrobial and bactericidal properties who protect them.
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3.4. Scanning electron microscopy Fig. 4 shows the scanning electron microscopy, where disruption of the cell wall and deformation of the integrity of the bacillus were observed, after with O. vulgare EO treatments, in MIC and sub-MIC concentrations, as well as the control with normal growth whole cells. Chang et al. (2017), evaluated 18 essential oils against D. Chrysanthemi ATCC 27,388 in their vapor phases, in which only 7 presented inhibition results, being the oil of oregano the one that presented greater zone of inhibition of the growth with the diameter of 44.7 ± 1.6 mm. Hernández-González et al. (2017) in their study attributed the antimicrobial capacity of oregano essential oil to the compounds thymol and carvacrol, being able to disintegrate the outer membrane of the microorganism, causing cell death. However, there are no recent studies that report the possible mechanisms of action of oregano essential oil against Alicyclobacillus spp. 4. Conclusion Carvacrol acetate was determined in the chemical characterization of the essential oil of oregano as major compound. The antimicrobial activity of EO was efficient in the control of Alicyclobacillus spp., which may be related to the good antioxidant activity of the compounds present in EO, such as carvacrol acetate. This inhibitory effect could still be observed by scanning electron microscopy through structural changes in cells. In this way, the use of O. vulgare EO becomes a viable alternative as a natural antibacterial against Alicyclobacillus spp.. However, future studies should be performed in vivo to verify such actions in food matrices. Conflict of interest The authors declare there are no conflicts of interest. Acknowledgment The authors would like to thank the Brazilian Federal Agency for the support and Evaluation of Graduate Education (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil [CAPES]). References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry. Journal of American Society for Mass Spectrometry, fourth ed., 18, 803–806. Bassolé, I.H.N., Juliani, H.R., 2012. Essential oils in combination and their antimicrobial properties. Molecules 17, 3989–4006. Brondani, L.P., Silva Neto, T.A., Freitag, R.A., Lund, R.G., 2018. Evalution of anti-enzyme properties of Origanum vulgare essential oil against oral Candida albicans. J. Mycol. Med. 28, 94–100. Calderón-Oliver, M., Escalona-Buendía, H.B., Medina-Campos, O.N., Pedraza-Chaverri, J., Pedroza-Islas, R., Ponce-Alquicira, E., 2016. Optimization of the antioxidant and
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Bioactivity of oregano (Origanum vulgare) essential oil against Alicyclobacillus spp.
T
Tatiane Viana Dutraa, Juliana Cristina Castroa, Jéssica Lima Menezesa, Tatiane Rogelio Ramosa, Ivanor Nunes do Pradob, Miguel Machinski Juniorc, Jane Martha Graton Mikchad, ⁎ Benício Alves de Abreu Filhoc, a
State University of Maringa, Av. Colombo, 5790, Maringá, 87020-900, Paraná, Brazil Animal Science Department, State University of Maringa, Av. Colombo, 5790, Maringá, 87020-900, Paraná, Brazil c Department of Basic Health Sciences, State University of Maringa, Av. Colombo, 5790, Maringá, 87020-900, Paraná, Brazil d Department of Clinical Analysis and Biomedicine, State University of Maringa, Av. Colombo, 5790, Maringá, 87020-900, Paraná, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Antimicrobian activity Antioxidant activity Alicyclobacillus acidoterrestris Carvacrol acetate Essential oil Origanum vulgare
The Alicyclobacillus spp., causing deterioration in citrus beverages, has been frequently related to the use of natural antimicrobial agents in its combat; in this sense, the study sought to evaluate the activity of the essential oil of oregano (Origanum vulgare) against different isolates of this bacterium, in addition to its antioxidant activities. The minimum inhibitory concentration obtained from oregano essential oil for A. Acidiphilus and A. cycloheptanicus was 125 μg/mL; and for A. herbarius and A. acidoterrestris was 62.5 μg/mL. While the minimum bactericidal concentration obtained was 1000 μg / mL for all isolates. The combined effect of nisin and O. vulgare against A. acidoterrestris resulted in indifference. The antioxidant activity obtained was 363 μmol trolox/mg by the DPPH method and 1142 μmol trolox/mg by the ABTS method. The chemical characterization of the essential oil of oregano by GC–MS was able to identify of 93.13% of the compounds was carried out, where the major compound was carvacrol acetate represented by 59.61%. Further scanning electron microscopy was able to demonstrate damage to cells treated with the inhibitory concentrations of O. vulgare.
1. Introduction The Alicyclobacillus spp. known worldwide for causing deterioration of citrus foods such as fruit juices, teas and tomato extract; are Grampositive bacillus capable of growing at low pH and high temperatures, with a pH of 4.0–4.5 and temperature of 40 at 50 °C, thus being acidresistant and spore-forming agents (Goto, 2003). Several species of Alicyclobacillus have already been isolated in food industries, among them the most important is Alicyclobacillus acidoterrestris, due to its ability to form byproducts such as guaiacol, responsible for the astringent taste in deteriorated juices (Anjos et al., 2016). There are more than 21 species of Alicyclobacillus described, with variations of sources of isolation, morphology, pH and growth temperature; among them, A. acidiphilus presents as source acidic beverages, A. cycloheptanicus in the soil, A. herbarius in the herbal teas while A. acidoterretsris in the soil and apple juices (Sant’Ana et al., 2014). Synthetic preservatives, such as sodium and potassium benzoate,
are used to combat the Alicyclobacillus vegetative cells depending on the initial concentration (Sant’Ana et al., 2014). In search of natural alternatives against this genus of bacteria, there is the use of essential oils of plants, which may present antibacterial activity inhibiting the growth of Alicyclobacillus spp., Protecting the products of the deterioration caused by this microorganism. Calderón-Oliver et al. (2016) combined the use of the chemical preservative nisin with a natural antioxidant, avocado byproducts, in the evaluation of antimicrobial activity; resulting in synergism, allowing the reduction of costs by reducing the amount of nisin to be used. The use of natural compounds to control microorganisms is increasingly present (Castro et al., 2017). Essential oils (EOs) are liquids oily aromatic and volatile extracted from specific plant parts. Several essential oils have antibacterial, antifungal, antiviral, antioxidant and biological properties. Among these oils, the essential oil of oregano presents antioxidant and antimicrobial activities, probably due to the presence of carvacrol and thymol (Li et al., 2018; Zhou et al., 2018). In addition, recent studies report that oregano essential oil is
⁎ Corresponding author at: Department of Basic Health Science, State University of Maringá, Av. Colombo 5790, Campus Universitário, Laboratório de Análise de Água, Ambiente e Alimentos, Bloco T20, 3° andar, Sala 312, 87020-900, Maringá, Paraná, Brazil. E-mail address: baafi[email protected] (B.A.d.A. Filho).
https://doi.org/10.1016/j.indcrop.2018.12.025 Received 3 September 2018; Received in revised form 23 October 2018; Accepted 6 December 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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a standard curve with trolox solution (( ± )-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was constructed. The results were expressed as mmol trolox/mg (Rufino et al., 2007a).
antiproliferative, antiinflammatory, antidiabetic and has cancer suppressive activity (Leyva-López et al., 2017). With the growing attention given to food safety through natural alternatives, the use of plant essential oils has been highlighted because of its high potential as a preservative, as well as being generally recognized as safe (GRAS) by the WHO (World Health Organization) and still many have antimicrobial activity controlling both pathogens and spoilage food bacteria (Gutierrez et al., 2008). This study aims to characterize the essential oil of oregano, to evaluate the antioxidant activity and the antimicrobial capacity, as well as its combined use with nisin, against Alicyclobacillus spp.
2.3.2. ABTS method The antioxidant activity by the ABTS (2,2′-azino-bis-(-3-ethylbenzothiazoline-6-sulfonic acid), diammonium salt, ∼ 98%) method was performed according to Rufino et al. (2007b). In tubes protected from light, 30 μL of the diluted EO and 3 mL of the ABTS+ solution (5 ml ABTS - 7 mmol/L solution and 88 μL of potassium persulfate 140 mmol/L; reaction for 16 h in a protected environment from light) were transferred and maintained for 6 min. The read was performed in a spectrophotometer at 734 nm and a standard curve with trolox solution was constructed. The results were expressed as mmol trolox/mg.
2. Material and methods 2.1. Microbial lineage, essential oil and nisin
2.4. Antibacterial activity Strains of the species Alyciclobacillus spp. were used from the Brazilian Collection of Environmental and Industrial Microorganisms (CBMAI), located at the Center for Chemical, Biological and Agricultural Research (CPQBA/UNICAMP), with reference to this study: A. acidoterrestris 0244T; A. herbarius 0246T; A. acidiphilus 0247T; A. cycloheptanicus 0297T. The oregano essential oil used in the present study was of commercial origin (Origanum vulgare - Marca Ferquima) obtained on local commerce in the city of Maringa, Paraná. Commercial nisin (Sigma-Aldrich, St. Louis, USA) was prepared from a solution of 0.02 M hydrochloric acid (HCl); filtrate with 0.22 μm sterile membrane (Millipore, São Paulo, Brazil).
2.4.1. Minimum inhinitory concentrations (MIC) and Bactericidal concentrations (MBC) The minimum inhibitory and bactericidal concentrations was determined using the 96-well microplate microdilution technique, according to CLSI methodology (2012), with BAT broth (Bacillus Acidoterrestris) as culture medium. The microorganism was activated in BAT broth 24 h before the experiment and incubated at 45 °C. The serial dilution of oregano essential oil was performed (0.90–1000 μg/mL) and inoculating 5 μL of the standard suspension according to the McFarland scale 0.5 in each well. The EO was diluted in 1% Tween-80 and tested at concentrations of 0.90–1000 g/mL. The microplates were incubated at 45 °C for 24 h. The MIC was defined as the lowest concentration of the EO that inhibited the visual growth of the bacteria. After this period, 20 μL each well was plated in YSG agar and incubated at 45 °C for 24 h for subsequent reading, where the lowest concentration capable of inhibiting bacterial growth was considered the minimum bactericidal concentration. The experiments were performed in triplicate.
2.2. Characterization of essential oil The chemical composition of the EO was performed using gas chromatography-mass spectrometry (GC–MS) with an automatic injector (FOCUS GC – DSQ II, Thermo Electron Corp). The gas chromatograph-mass spectrometer was equipped with an Agilent DB-5 capillary column (5% phenyl/95% dimethyl siloxane stationary phase, 30 m length, 0.25 mm internal diameter, and 0.1 μm film thickness). Characterization was performed using a column temperature program that began at 60 °C, followed by a temperature increase of 3 °C/min to 230 °C. Helium was used as the carrier gas at a flow rate of 1.0 mL/min. The total analysis time was 63 min. The temperature of the injector and detector was maintained at 240 °C A 1 μL volume of the samples was injected for chromatography in split mode (1:10). EO was diluted in hexane (high-performance liquid chromatography grade; 2 μL of EO to 1000 μL of hexane) to form the stock solution (Kim et al., 2015; Castro et al., 2017, with modifications). Characterization was performed based on retention time (RT) and compared to the major compounds using Kovats retention index (KI) (Skoog et al., 2006). The compounds of the EOs were identified by analyzing the retention times of the peaks that were obtained for each EO and confirmed via a standard mixture of n-alkanes (C8–C20; SigmaAldrich). The compounds of interest were confirmed (Adams, 2007) and are presented as percentages.
2.4.2. Disc diffusion method Kirby-Bauer agar disc diffusion method was used to evaluate the inhibition growth of the bacteria. Plates with YSG were seeded with the inoculum standardized with the turbidity of the McFarland scale 0.5. These discs (5 mm diameter) were arranged around the plate with different concentrations of OE of oregano, 2000, 1000, 500, 250 μg/mL and the saline-containing control. The plate was incubated for 24 h at 45 °C and the reading was performed according to the inhibition halo formed around the disc (Prasad et al., 2001). 2.4.3. Checkerboard method Checkerboard tests were performed to evaluate the effects of drug combinations. The assay was performed in 96-well microplates to obtain the fractional inhibitory concentration (FIC) of the EO combined with nisin against A. acidoterrestris. YSG medium was used for the assay. A volume of 5 μL of the inoculum (inoculum prepared from the standard suspension according to the McFarland 0.5 scale) was added to each well, and the plates were incubated at 45 °C for 24 h. The result was analyzed by the inhibition of visible growth, the inhibitory fractional concentration index (FIC) was calculated, being FIC = FICA + FICB, where FICA = MICA combined / MICA alone, and FICB = MICB combined / MICB alone; defined as synergistic effect a result ≤ 0.5, additive 0.5 to ≤ 1, indifferent from 1 to ≤ 4 and antagonist > 4 (Schelz et al., 2006).
2.3. Antioxidant activity The extract of the essential oil was diluted in methanol (P.A., Anidrol, Brazil), 1 mg of EO in 1 mL of methanol. The extract was stored in an amber bottle to protection to the light until analysis.
2.5. Scanning electron microscopy
2.3.1. DPPH method The antioxidant activity by the DPPH (2,2-Diphenyl-1-picrylhydrazyl) method was performed according to Ma et al. (2011). In tubes protected from light, 25 μL of the diluted OE and 2 mL of the 6.25 × 10−5 mol/L DPPH solution were added and maintained for 30 min. The read was performed in a spectrophotometer at 517 nm and
The inoculum of Alicyclobacillus acidoterrestris 0244T was treated with oregano essential oil at the concentrations defined by the antimicrobial activity of the sub-mic, mic and negative control without the application of the essential oil. After incubation of samples at 45 °C for 346
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Table 1 Chemical composition of O. vulgare essential oil. Compounds
a
KI
Origanum vulgare b
β-pinene Meta-cymene α-terpinene Linalool Borneol Carvacrol Carvacrol acetate Caryophyllene < (E)- > Caryophyllene acetate Furopelargone A Total of identified compounds a b
973.53 1020.81 1053.55 1096.19 1167.66 1286.17 1296.60 1413.04 1448.79 2134.78
RT (min)
7.79 9.42 10.71 12.39 15.49 20.7 21.16 26.16 27.64 51.85 93.13%
% 1.27 5.42 4.47 1.90 1.09 2.85 69.51 4.28 1.05 1.29
KI = Kovats index. RT = Retention time (min).
Fig. 2. Antioxidant activity of O. vulgare EO for DPPH and ABTS methods. Lowercase letters in columns show significant difference (p < 0.05).
24 h, fixation with 2.5% glutaraldehyde in 0.1 M cacodylate buffer and adhesion on coverslips pretreated with poly-L-lysine (Sigma-Aldrich). Followed by dehydration with an ethanolic increasing series of 30%–100%, critical point of CO2, metallization in gold and analysis in scanning electron microscope Quanta-250 (Endo et al., 2010).
(Fig. 1). The composition of the essential oil of O. vulgare subsp glandulosum (Desf.), harvested at different times of the year in North Africa; presented major compounds as Thymol (31.80%, year 2007; 43.70%, year 2008; and 46.10%, year 2009), γ-Terpinene (24.20%, year 2007; 27.10%, year 2008; and 24.00%, year 2009) and p-Cymene (35.70%, year 2007; 12.70%, year 2008; and 11.50%, year 2009) (Mechergui et al., 2016). In O. vulgare EO purchased from Chile, the composition of the major compounds was 4-terpineol (41.17%), Thymol (21.95%), γTerpinene (5.91%) and Carvacrol (4.71%) (Brondani et al., 2018), different from the compounds found in this study. Many intrinsic and extrinsic factors such as agricultural factors, species, harvest time, plant part, season, climate, geography, extraction methods and storage care influence the composition of the EO and concentration of each compound, majority or minority (Castro et al., 2017). Cattelan et al. (2018) in its characterization of the essential oil of oregano obtained as major compound the carvacrol (65.1%), similar to the result obtained in the present work.
2.6. Statistical analysis The antioxidant data were submitted to analysis of variance by ANOVA and Tukey's test (p < 0.05) was used to compare the means by means of the statistical program SISVAR version 5.3. The figure was generated by SigmaPlot software version 11.0. 3. Results and discussion 3.1. Characterization of essential oil The characterization of O. vulgare EO by GC–MS, followed by identification and quantification is presented in Table 1. The major compound was carvacrol acetate (69.51%), of 93.13% of the total compounds identified. Meta-cymene (5.42%), α-terpinene (4.47%), Caryophyllene < (E)- > (4.28%), Carvacrol (2.85%), Linalool (1.90%), Furopelargone A (1.29%), β-pinene (1.27%), Borneol (1.09%) and Caryophyllene acetate (1.05%) were presented in smaller quantities
3.2. Antioxidant activity The antioxidant activity for O. vulgare EO (Fig. 2) showed different
Fig. 1. Chromatographic profile of essential oil of oregano (Origanum vulgare) and Fragmentation spectrum of the major compound carvacrol acetate. 347
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Fig. 3. Disk diffusion method of oregano essential oil against Alicyclobacillus acidoterrestris. Discs numbered 1 (EO concentration – 2000 μg/mL), 2 (EO concentration – 1000 μg/mL),3 (EO concentration – 500 μg/mL),4 (EO concentration – 250 μg/mL) and 5 (saline solution).
Fig. 4. Scanning Electron Microscopy. (a) Control vegetative cells of A. acidoterrestris; (b) MIC of O. vulgare EO with vegetative cells of A. acidoterrestris; (c) Sub-MIC of O. vulgare EO with vegetative cells of A. acidoterrestris.
concentrations between the tested methods, DPPH and ABTS, 363 μmol trolox/mg and 1142 μmol trolox/mg, respectively. These results suggest a good antioxidant activity by the evaluated methods. Aromatic plants such as oregano have high free radical activity (82%) (Olmedo et al., 2014). It is important to mention that the antioxidant activity of the essential oils is related to the species, time of harvest and geographical position (Mechergui et al., 2016), because the chemical composition of the oils differs due to intrinsic and extrinsic factors. The antioxidant activity found is related to compounds, such as carvacrol (Hashemi and Khaneghah, 2017), which is also related to the antibacterial activity presented in this work.
this emulsion makes the extract more stable, improving its efficiency against bacterial cells. In another application the use of essential oil was analyzed in salad dressings, where it obtained a reduction in Escherichia coli counts, demonstrating its antibacterial action (Cattelan et al., 2018). Ruiz et al. (2013), in their study, evaluated the antimicrobial actions of several fractions of Piperaceae extract against A. acidoterrestris and the results founded for MIC and MBC ranged from 15.63 to higher of 1000 μg/mL, the best result was that of the Piper aduncum fraction, with extracts of chloroform, dichloromethane fraction and the pure compound, such fractions were combined with nisin by the Checkerboard methodology, for which the result was synergism. Nisin is a bacteriocin with proven antimicrobial activity against Gram-positive bacteria, such as A. acidoterrestris; used in food industries as an antimicrobial agent approved by the Food and Drug Administration (FDA). With respect to the combination of oregano essential oil and nisin, the FICI result found for the four strains was indifferent, presenting values of 2.06; 2.12; 2.12 and 2.5 for A. acidoterrestris, A. Acidiphilus, A. cycloheptanicus and A. herbarius, respectively. It is concluded that the use of O. vulgare EO with nisin, although not synergistic, does not demonstrated the antagonism of its activities. The results of the diffusion disc method, on 5 mm diameter discs, confirmed the inhibitory activity (MIC) of O. vulgare EO against A. acidoterrestris, through formation of growth inhibition halo at different concentrations of oil used, 2000, 1000, 500 and 250 μg/mL, in addition
3.3. Antibacterial activity The MIC found for O. vulgare EO against A. Acidiphilus and A. cycloheptanicus was 125 μg/mL; and for A. herbarius and A. acidoterrestris was 62.5 μg/mL, while the MBC obtained was 1000 μg/mL, but with a reduction of 2 logs. A cellulose nanocrystals (CNCs) emulsion, prepared from microcrystalline cellulose (MCC) by hydrolysis with ammonium persulfate (APS), and oregano essential oil showed a minimum inhibitory concentration (MIC) of 12.5 μg/mL against Staphylococcus aureus, Saccharomyces cerevisiae and Escherichia coli, and the most effective antimicrobial effect was observed in relation to Bacillus subtilis with a MIC of 6.25 μg/mL (Zhou et al., 2018), this result is due to the fact that 348
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to the control in which only saline solution was plated and where there was not halo formation, as shown in Fig. 3. Bassolé and Juliani (2012), reported the production of secondary metabolites by plants, which have antimicrobial and bactericidal properties who protect them.
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3.4. Scanning electron microscopy Fig. 4 shows the scanning electron microscopy, where disruption of the cell wall and deformation of the integrity of the bacillus were observed, after with O. vulgare EO treatments, in MIC and sub-MIC concentrations, as well as the control with normal growth whole cells. Chang et al. (2017), evaluated 18 essential oils against D. Chrysanthemi ATCC 27,388 in their vapor phases, in which only 7 presented inhibition results, being the oil of oregano the one that presented greater zone of inhibition of the growth with the diameter of 44.7 ± 1.6 mm. Hernández-González et al. (2017) in their study attributed the antimicrobial capacity of oregano essential oil to the compounds thymol and carvacrol, being able to disintegrate the outer membrane of the microorganism, causing cell death. However, there are no recent studies that report the possible mechanisms of action of oregano essential oil against Alicyclobacillus spp. 4. Conclusion Carvacrol acetate was determined in the chemical characterization of the essential oil of oregano as major compound. The antimicrobial activity of EO was efficient in the control of Alicyclobacillus spp., which may be related to the good antioxidant activity of the compounds present in EO, such as carvacrol acetate. This inhibitory effect could still be observed by scanning electron microscopy through structural changes in cells. In this way, the use of O. vulgare EO becomes a viable alternative as a natural antibacterial against Alicyclobacillus spp.. However, future studies should be performed in vivo to verify such actions in food matrices. Conflict of interest The authors declare there are no conflicts of interest. Acknowledgment The authors would like to thank the Brazilian Federal Agency for the support and Evaluation of Graduate Education (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil [CAPES]). References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry. Journal of American Society for Mass Spectrometry, fourth ed., 18, 803–806. Bassolé, I.H.N., Juliani, H.R., 2012. Essential oils in combination and their antimicrobial properties. Molecules 17, 3989–4006. Brondani, L.P., Silva Neto, T.A., Freitag, R.A., Lund, R.G., 2018. Evalution of anti-enzyme properties of Origanum vulgare essential oil against oral Candida albicans. J. Mycol. Med. 28, 94–100. Calderón-Oliver, M., Escalona-Buendía, H.B., Medina-Campos, O.N., Pedraza-Chaverri, J., Pedroza-Islas, R., Ponce-Alquicira, E., 2016. Optimization of the antioxidant and
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