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Antibacterial activity of oregano essential oil against foodborne pathogens

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Abstract

Purpose This paper aims to evaluate in vitro antibacterial activity of oregano essential oil against foodborne pathogens as a starting point for the use of spice as a natural preservative in food. Design/methodology/approach Disc and well‐diffusion assays were performed to investigate antibacterial activity of oregano essential oil against six bacteria strains: Bacillus cereus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Salmonella Typhimurium. Three concentrations of oregano essential oil were employed: 1.0 percent, 2.0 percent and 5.0 percent. Bacterial growth inhibition was determinate as the diameter of the inhibition zones. Findings Oregano essential oil showed antibacterial activity against spoilage microorganisms, at different concentrations, except for P. aeruginosa. There was a significant difference between methodologies only for the microorganism S. aureus. The results provided evidence of the existence of significant differences among the concentrations of oregano essential oil for each microorganism evaluated. Research limitations/implications Although the research for this paper involved only oregano essential oil, it provided a starting‐point for further investigations concerning spices as natural preservatives for food systems. Practical implications Disc and well‐assays were found to be simple and reproducible practical methods. Other spices, their essential oil and extracts might be researched against other micro‐organisms. Furthermore, in situ studies need to be performed to evaluate possible interactions between essential oils and compounds naturally present in food against microbial strains. Social implications The imminent adoption of measures to reduce the use of additives in foods and the reduction on using such compounds. Originality/value This study provides insights that suggest a promising exploratory development of food natural preservative against spoilage microorganisms in food systems by the use of oregano essential oil.
Nutrition & Food Science
Antibacterial activity of oregano essential oil against foodborne pathogens
Marília Gonçalves Cattelan Maurício Bonatto Machado de Castilhos Priscila Juliana Pinsetta Sales
Fernando Leite Hoffmann
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To cite this document:
Marília Gonçalves Cattelan Maurício Bonatto Machado de Castilhos Priscila Juliana Pinsetta Sales
Fernando Leite Hoffmann, (2013),"Antibacterial activity of oregano essential oil against foodborne
pathogens", Nutrition & Food Science, Vol. 43 Iss 2 pp. 169 - 174
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Antibacterial activity of oregano
essential oil against foodborne
pathogens
Marı
´lia Gonc¸alves Cattelan,
Maurı
´cio Bonatto Machado de Castilhos,
Priscila Juliana Pinsetta Sales and Fernando Leite Hoffmann
Department of Food Engineering,
Universidade Estadual Paulista Ju
´lio de Mesquita Filho, Sa
˜o Paulo, Brazil
Abstract
Purpose – This paper aims to evaluate in vitro antibacterial activity of oregano essential oil against
foodborne pathogens as a starting point for the use of spice as a natural preservative in food.
Design/methodology/approach Disc and well-diffusion assays were performed to
investigate antibacterial activity of oregano essential oil against six bacteria strains: Bacillus
cereus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and
Salmonella Typhimurium. Three concentrations of oregano essential oil were employed: 1.0 percent,
2.0 percent and 5.0 percent. Bacterial growth inhibition was determinate as the diameter of the
inhibition zones.
Findings – Oregano essential oil showed antibacterial activity against spoilage microorganisms, at
different concentrations, except for P. aeruginosa. There was a significant difference between
methodologies only for the microorganism S. aureus. The results provided evidence of the existence of
significant differences among the concentrations of oregano essential oil for each microorganism
evaluated.
Research limitations/implications Although the research for this paper involved only oregano
essential oil, it provided a starting-point for further investigations concerning spices as natural
preservatives for food systems.
Practical implications Disc and well-assays were found to be simple and reproducible practical
methods. Other spices, their essential oil and extracts might be researched against other
micro-organisms. Furthermore, in situ studies need to be performed to evaluate possible
interactions between essential oils and compounds naturally present in food against microbial strains.
Social implications – The imminent adoption of measures to reduce the use of additives in foods
and the reduction on using such compounds.
Originality/value This study provides insights that suggest a promising exploratory
development of food natural preservative against spoilage microorganisms in food systems by the
use of oregano essential oil.
Keywords In vitro, Essential oil, Oregano, Antimicrobial, Foodborne, Oils, Food preservation
Paper type Research paper
Introduction
Due the increasing public demand for safe and high-quality food, governments of
developed countries have strongly pushed into food control production
The current issue and full text archive of this journal is available at
www.emeraldinsight.com/0034-6659.htm
The authors would like to thank to Coordenac¸a
˜o de Aperfeic¸oamento Pessoal de Nı
´vel Superior,
CAPES, for financial support.
Nutrition & Food Science
Vol. 43 No. 2, 2013
pp. 169-174
qEmerald Group Publishing Limited
0034-6659
DOI 10.1108/00346651311313544
Oregano
essential oil
169
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(Ivanovic et al., 2012). The adoption of measures to reduce the use of chemical additives
speculates about the toxicity of some chemical additives in food products and
the corresponding abuse of employing such compounds worldwide (Moreira and
Ponce, 2005).
There is growing research interest in the search for alternative compounds that
could be rationally used as food preservatives. Moreover, reducing levels of salt and
sugar in food for dietary reasons also tend to increase the use of spices (Trajano et al.,
2009).
Spices are herbal products which have been safely used by people around the world
to impart desirable flavors and aromas to the local foods (Pirbalouti et al., 2010).
Antibacterial properties of spices and its essential oils against different
microorganisms have been recognized since antiquity. Natural extracts and essential
oils are used to increase shelf-life and improve the sensory characteristics of food. The
extracts and active compounds of some plants have demonstrated antibacterial
activity in laboratory studies (Burt, 2004; Ushimaru et al., 2007; Alves et al., 2008;
Yossa et al., 2010; Castilho et al., 2012).
Many antimicrobial compounds used to control foodborne pathogens target the
membrane transport activity in order to disrupt cytoplasmic homeostasis, for example,
nisin, essential oils and organic acids (McMeekin et al., 2010). Origanum oil, used as a
food-flavouring agent, possesses a broad spectrum of antimicrobial activity due, at
least in part, to its high content of phenolic derivatives, such as carvacrol and thymol
(Preuss et al., 2005). There are many reports relating chemical composition and
antimicrobial properties of the essential oils of various origanum species and their
application in various commercial preparations, as antimicrobials and antioxidants
(Castilho et al., 2012).
Although the industry does use different techniques to ensure quality and food
safety, foodborne diseases are still a serious public health problem ( Jay, 2005).
Throughout the 1990s and until today, in Europe, three major foodborne bacterial
targets have persisted, requiring high research and surveillance attention from
government agencies and food industry: Salmonella spp., E. coli and Campylobacter
spp. (Newell et al., 2010). According to Burt (2004), the most interesting area of
application for essential oils is the inhibition of growth and reduction in numbers of the
more serious foodborne pathogens such as Salmonella spp., E. coli O157:H7 and L.
monocytogenes.
In order to enhance individual antimicrobial activity against foodborne pathogens,
three concentrations (1.0, 2.0 and 5.0 percent) of oregano essential oil (OEO), has been
investigated against Bacillus cereus,Bacillus subtilis,Escherichia coli,Pseudomonas
aeruginosa,Staphylococcus aureus and Salmonella typhimurium.
Materials and methods
Microbial culture
Bacillus cereus (ATCC 11778), Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC
8739), Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 25923)
and Salmonella typhimurium (ATCC 14028) were held in Plate Count Agar (PCA)
Himedia, at 48C, reactivated overnight in PCA at 358C. Bacterial cultures were
standardized by the range of 0.5 Mc Farland resulting in a suspension containing
10
8
CFU/ml.
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Essential oil
OEO was purchased commercially from Laszlo Aromaterapia Ltd Essential oil
solutions with 1.0, 2.0 and 5.0 percent (v/v) were prepared using as solvent sterile
distilled water and 0.5 percent Tween 80.
Antimicrobial activity
The antibacterial activity of OEO was determinated against microorganisms by disc
and well-diffusion methods. The disc diffusion method of Ienette (1985) was used with
some modification to determinate the rate of inhibition growth of bacteria by essential
oil. Mu
¨eller-Hinton Agar (MHA – Himedia) was used to prepare the culture medium
and autoclaved at 1218C for 15 min. Briefly, Petri dishes (9 cm diameter) were prepared
with the same amount of MHA inoculated with 0.1 ml bacterial suspension. Sterile
paper discs (6 mm diameter) were impregnated with 40
m
l of OEO dilutions and
incubated at 358C for 24 h.
Well diffusion technique was performed according to Schillinger and Lucke (1989)
with modifications. Wells of 6 mm of diameter were cut into MHA agar plates and 40
m
l
of OEO dilution was placed into each well. The plates with the well unsealed were
incubated at 358C for 24 h. Bacterial growth inhibition was determinated as the diameter
of the inhibition zones (mm). A solution consisting of sterile distilled water and
0.5 percent Tween 80 was used as negative control. All tests were performed in triplicate.
Statistical analysis
The comparison between the screening methods used in the evaluation of antibacterial
activity was performed using the Mann-Whitney test. A significant level of 5 percent
was adopted. The Kruskal-Wallis test was used to verify the most effective
concentration of OEO to inhibit the growth of each microorganism. Tests were carried
out with Minitab Software
w
support.
Results and discussion
The essential oil studied provided an interesting inhibitory effect against B. cereus,B.
subtilis,E. coli,S. typhimurium and S. aureus.Pseudomonas aeruginosa was shown to
be resistant do OEO. Ceylan and Fung (2004) reported that bioactive components of
plant-origin antimicrobials are relatively weak against Pseudomonas spp., which
corroborates to the present results.
Data resulting from the comparison between screening techniques, Table I, suggest
a significant difference between methodologies only for the microorganism S. aureus
(p¼0.006). Even for the strain S. aureus, it was observed that there is no significant
difference among three concentrations of OEO by Kruskal-Wallis test at significant
level of 5 percent ( p¼0.058).
More precise data regarding extract antimicrobial evaluation have been obtained
with disc diffusion technique, according to Table I. Similar results are reported in
literature (Kil et al., 2009; Tajkarimi et al., 2010).
The results in Table II provide evidence of the existence of significant differences
among the concentrations of OEO for each microorganism evaluated. Moreover,
median values have shown that the concentration of 5 percent was the most effective in
the inhibition of other strain used in the study: E. coli,B. subtilis,S. typhimurium and
B. cereus.
Oregano
essential oil
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An increasing number of reports that shown from the effectiveness of oregano against
both Gram-positive and Gram-negative groups (Govakis et al., 2010; Bendahou et al.,
2008; Burt, 2004; Naidu, 2000). Carvacrol and thymol are the main phenols that
constitute about 78-85 percent of OEO. They are principally responsible for the
antimicrobial activity of the oil (Kokkini et al., 1997). Other minor constituents such as
monoterpene, hydrocarbons,
g
-terpinene and p-cymene also contribute to the
antibacterial activity of the oil (Burt, 2004).
Conclusion
Disc diffusion method was more effective in inhibiting the S. aureus strain, although
there was no significative difference among the three OEO concentrations employed.
Pseudomonas aeruginosa was resistant to all concentrations of employed essential oil.
For the inhibition of other bacteria strain (B. cereus,B. subtilis,E. coli and S.
typhimurium), concentration of 5 percent OEO was the most effective. This study
finding showed the necessity of further research in order to evaluate in situ
antibacterial properties of OEO in food systems.
Microorganism OEO concentration (%) v/v Md
a
p-value
E. coli 1 15.5 p¼0.001
2 23.0
5 30.0
B. subtilis 1 12.0 p¼0.001
2 16.5
5 28.5
S. Typhimurium 1 16.0 p¼0.005
2 17.0
5 25.0
B. cereus 1 13.0 p¼0.009
2 17.0
5 55.5
Notes:
a
Md median; p-value for the Kruskal-Wallis test
Table II.
Evaluation of OEO
concentration against
bacterial strains
Microorganism Screening method Md
a
Min. Max. p-value
E. coli Disc diffusion 25 11 32 p¼0.400
Well diffusion 18 14 35
S. aureus Disc diffusion 25 15 38 p¼0.006
Well diffusion 19 13 24
B. subtilis Disc diffusion 17 11 27 p¼0.858
Well diffusion 17 11 30
S. Typhimurium Disc diffusion 17 13 30 p¼1.000
Well diffusion 18 16 20
B. cereus Disc diffusion 22 13 90 p¼0.196
Well diffusion 19 12 21
Notes:
a
Md median; min. minimum value; max. maximum value; p-value for the Mann-
Whitney test
Table I.
Statistical results of the
screening techniques
used to assess the
antibacterial activity
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Corresponding author
Marı
´lia Gonc¸alves Cattelan can be contacted at: mariliagcattelan@gmail.com
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... Our results for the antibacterial activity of the pure OrO are in line with numerous experimental data of other authors. The effective concentrations of OrO estimated in several published studies varied between 1.25-1600 µg/mL [7,10,16,30,68,69]. Presented in percentage, the minimal inhibitory concentration (MIC) of OrO ranged depending on the strain from 0.05% to 6.23% [8,18] in broth model systems (test microorganisms S. aureus, E. faecalis, E. coli, K. pneumonia, and P. aeruginosa) and from 1% to 5% (a large set of foodborne pathogens) when the disk diffusion method was preferred as the evaluation model [10]. ...
... The effective concentrations of OrO estimated in several published studies varied between 1.25-1600 µg/mL [7,10,16,30,68,69]. Presented in percentage, the minimal inhibitory concentration (MIC) of OrO ranged depending on the strain from 0.05% to 6.23% [8,18] in broth model systems (test microorganisms S. aureus, E. faecalis, E. coli, K. pneumonia, and P. aeruginosa) and from 1% to 5% (a large set of foodborne pathogens) when the disk diffusion method was preferred as the evaluation model [10]. A recent study by Sobczyk et al. [24] reported high antibacterial activity of oregano oil and oregano leaves on chitosan-alginate-based dressings for the treatment of wounds. ...
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Oregano oil (OrO) possesses well-pronounced antimicrobial properties but its application is limited due to low water solubility and possible instability. The aim of this study was to evaluate the possibility to incorporate OrO in an aqueous dispersion of chitosan–alginate nanoparticles and how this will affect its antimicrobial activity. The encapsulation of OrO was performed by emulsification and consequent electrostatic gelation of both polysaccharides. OrO-loaded nanoparticles (OrO-NP) have small size (320 nm) and negative charge (−25 mV). The data from FTIR spectroscopy and XRD analyses reveal successful encapsulation of the oil into the nanoparticles. The results of thermogravimetry suggest improved thermal stability of the encapsulated oil. The minimal inhibitory concentrations of OrO-NP determined on a panel of Gram-positive and Gram-negative pathogens (ISO 20776-1:2006) are 4–32-fold lower than those of OrO. OrO-NP inhibit the respiratory activity of the bacteria (MTT assay) to a lower extent than OrO; however, the minimal bactericidal concentrations still remain significantly lower. OrO-NP exhibit significantly lower in vitro cytotoxicity than pure OrO on the HaCaT cell line as determined by ISO 10993-5:2009. The irritation test (ISO 10993-10) shows no signs of irritation or edema on the application site. In conclusion, the nanodelivery system of oregano oil possesses strong antimicrobial activity and is promising for development of food additives.
... Its extract was employed in traditional medicine to treat many cases such as cutaneous sores, aching muscles, asthma, cramping, diarrhea, indigestion, common colds, and boosting overall health. In addition, recent studies have suggested it as a food preservative due to its good flavor and its lethal effect against a wide range of food spoilage bacteria [13][14][15][16]. In the veterinary practice, oregano plant extracts markedly controlled the Clostridium perfringens diarrhea in boilers and enhanced intestinal mucosa healing [17]. ...
... Interestingly, the inhibition of bacterial growth in the paper disk diffusion test confirmed the oregano extract essential oils' antibacterial action besides their antioxidants and antiinflammatory effects, particularly against E. coli (the highest bacterial isolate in the present data) [13][14][15][16]. This result was supported by the observed recovery of OG lambs and their clinicopathological parameters amelioration [10][11][12]17]. ...
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... In a study examining the effect of different concentrations of oregano essential oil on biofilms of Staphylococcus aureus and Escherichia coli bacteria species, it was stated that these EOs reduce the biofilm level [55]. Likewise, the antibacterial activity of different concentrations of thyme essential oil, examined with disc and well-diffusion tests against Staphylococcus aureus and other species, was rather high [56]. Similarly, Ref. [57] reported that different O. vulgare extracts showed antimicrobial activity against many microorganism species, including S.aureus, E.faecalis bacteria species and C. albicans yeast species. ...
... In a study examining the effect of different concentrations of oregano essential oil on biofilms of Staphylococcus aureus and Escherichia coli bacteria species, it was stated that these EOs reduce the biofilm level [55]. Likewise, the antibacterial activity of different concentrations of thyme essential oil, examined with disc and well-diffusion tests against Staphylococcus aureus and other species, was rather high [56]. Similarly, Ref. [57] reported that different O. vulgare extracts showed antimicrobial activity against many microorganism species, including S.aureus, E.faecalis bacteria species and C. albicans yeast species. ...
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Overview, A.S. Naidu Lacto-Antimicrobials Lactoferrin. A.S. Naidu Lactoperoxidase, A.S. Naidu Lactoglobulins, E.F. Bostwick, J. Steijins, and S. Braun Lactolipids, M. Lampe and C. Isaacs Ovo-Antimicrobials Lysozymes, J.N. Losso, S. Nakai, and E.A. Charter Ovotransferrin, H.R. Ibrahim Ovoglobulin IgY, J.S. Sim, H.H. Sunwoo, and E.N. Lee Avidin, Y. Mine Phyto-Antimicrobials Phyto-phenols, P.M., Davidson and A.S. Naidu Saponins, W.A. Oleszek Flavonoids, A.S. Naidu, W.R. Bidlack, and A.T. Crecelius Thiosulfinates, B.B. Whitmore and A.S. Naidu Catechins, L.R. Juneja, T. Okubo, and P. Hung Glucosinolates, B.B. Whitmore and A.S. Naidu Agar, A.S. Naidu Bacto-Antimicrobials Probiotics, A.S. Naidu and R.A. Clemens Nisin, L.V. Thomas, M.R. Clarkson, and J. Delves-Broughton Pediocin, B. Ray and K. Miller Reuterin, M.G. El-Ziney, J. Debevere, and M.Jakobsen Sakacin, F. Leroy and L. De Vuyst Acid-Antimicrobials Lactic Acid, J-C. Bogeart and A.S. Naidu Sorbic Acid, J.N. Sofos Acetic Acid, D.L. Marshall, L.N. Cotton, and F.A. Bal'a Citric Acid, R.K. Sharma Milieu-Antimicrobials Sodium Chloride, R. Ravishankar and V.K. Juneja Polyphosphates, A. Prakash Chloro-cides, N. Khanna and A.S. Naidu Ozone, M. Muthukumarappin, F. Halaweish, and A.S. Naidu Appendix (Abbreviations and Symbols) Index
Chapter
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