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Antimicrobial Impacts of Essential Oils on Food Borne-Pathogens Yesim Ozogul*, Esmeray Kuley, Yilmaz Uçar and Fatih Ozogul Department of Seafood Processing Technology, Faculty of Fisheries, University of Cukurova, Adana, Turkey Received: April 16, 2015; Revised: June 12, 2015; Accepted: June 13, 2015

Abstract: The antimicrobial activity of twelve essential oil (pine oil, eucalyptus, thyme, sage tea, lavender, orange, laurel, lemon, myrtle, lemon, rosemary and juniper) was tested by a disc diffusion method against food borne pathogens (Escherichia coli, Salmonella paratyphi A, Klebsiella pneumoniae, Yersinia enterocolitica, Pseudomonas aeruginosa, Aeromonas hydrophila, Campylobacter jejuni, Enterococcus faecalis, Staphylococcus aureus). The major components in essential oils were monoterpenes hydrocarbons, -pinene, limonene; monoterpene phenol, carvacrol and oxygenated monoterpenes, camphor, 1,8-cineole, eucalyptol, linalool and linalyl acetate. Although the antimicrobial effect of essential oils varied depending on the chemical composition of the essential oils and specific microorganism tested, majority of the oils exhibited antibacterial activity against one or more strains. The essential oil with the lowest inhibition zones was juniper with the values varied from 1.5 to 6 mm. However, the components of essential oil of thyme and pine oil are highly active against food borne pathogen, generating the largest inhibition zones for both gram negative and positive bacteria (5.25-28.25 mm vs. 12.5-30 mm inhibition zones). These results indicate the possible use of the essential oils on food system as antimicrobial agents against food-borne pathogen. The article also offers some promising patents on applications of essential oils on food industry as antimicrobial agent.

Keywords: Antimicrobials, essential oils, food-borne pathogens. INTRODUCTION Plants are a source of food and medicine. Some of the plants contain bioactive constituents which take part in preventing role against the occurrence of some diseases such as cardiovascular, neurodegenerative disorders and cancer [13]. Essential oils (EOs) are considered as safe substances (ESO, GRAS-182.20) by the Food and Drug Administration [4] and some of them can be recognized as antibacterial additives [5, 6]. The primary components of essential oils are terpenes and terpenoids. They can also contain acids, alcohols, aliphatic hydrocarbons and lactones [7]. Particular interest has focused on the potential applications of plant essential oils having natural antimicrobial components with a capacity to prolong the shelf-life of food when applied alone or in conjunction with other preservation methods, such as modified atmosphere packaging (MAP), vacuum packaging (VP), heat processing or refrigeration [810]. However, these techniques do not remove hazardous pathogens such as L. monocytogenes from food products or slow down microbial spoilage. Industrially, important foodborne pathogens can survive chill temperatures, widespread in food processing environments [11]. Natural plant extracts can be used as an alternative to these preservatives. Therefore, natural antimicrobial components have been *Address correspondence to this author at the Department of Seafood Processing Technology, Faculty of Fisheries, University of Cukurova, 01330, Balcalı, Adana, Turkey; Tel: (90) 322 3386084 Ext: 2961; Fax: (90) 322 3386439; E-mail: [email protected] 1876-1429/15 $100.00+.00

investigated for food products since synthetic chemical additives have some toxic effects. Literature survey revealed that a lot of research work has been carried out on volatile oil composition [12] and antimicrobial [10, 13-16] and antifungal effects of different plants [17, 18]. Essential oils have also been used as dental and oral treatments [19], and for burns and wound healing [20, 21]. Although research on the essential oils constituents of different plant species are moderately extensive, reports on their biological activities are still limited. In this study, volatile chemical constituents of juniper, pine oil, myrtle, orange, thyme, lavender, sage tea, lemon, rosemary, French lavender, eucalyptus, and laurel have been investigated. In addition, the inhibitory effects of the essential oils were determined against food borne bacteria (E. coli, Salmonella paratyphi A, Klebsiella pneumoniae, Yersinia enterocolitica, Pseudomonas aeruginosa, Aeromonas hydrophila, Campylobacter jejuni, Enterococcus faecalis, and Staphylococcus aureus). MATERIAL AND METHODS Bacteria tested. Escherichia coli ATCC25922, Enterococus faecalis ATCC29212, Staphylococcus aureus ATCC29213, Klebsiella pneumoniae ATCC700603, Pseudomonas aeruginosa ATCC27853, Camplylobacter jejuni (ATCC 33560) and Listeria monocytogenes ATCC7677 were puchased from the American Type Culture Collection (Rockville, MD, USA). Aeromonas hydrophila NCIMB 1135, Salmonella Parathypi A NCTC13 and Yersinia enterocolitica (NCTC 11175) were obtained from © 2015 Bentham Science Publishers

54 Recent Patents on Food, Nutrition & Agriculture, 2015, Vol. 7, No. 1

Ozogul et al.

the National Collection of Type Cultures (London, UK) and the National Collections of Industrial Food and Marine Bacteria (Aberdeen, UK )

Determination of antimicrobial activity by the paper disc diffusion method. The antimicrobial activity of twelve essential oils was determined using the disc diffusion method [22] with minor modifications. Nutrient agar was employed as the standard test medium for bacteria. The agar plate was spread with the inoculum having 108 CFU/ml pathogenic bacteria. Fifty microliters of undiluted EOs were pipetted on sterile filter paper discs (diameter 6 mm), which were permitted to dry in an open sterile petri dish in a biological safety cabinet with vertical laminar flow. Paper discs were set on the inoculated agar surfaces. After incubation at 37 ± 1oC for 18-24 h for bacteria, diameters (mm) of the zones of bacterial inhibition minus the disc diameter were determined. Each test was carried out in triplicate and the results were assessed for statistical significance. Antibiotics with positive responses were utilized as the control for the plates. Tetracycline, Streptomycin and Neomycin were acted as positive controls on Gram-positive and Gram-negative bacteria.

Gas chromatography-mass spectrometry analysis (GC/MS) of essential oils. Essential oils were purchased from a national company (NURS, Adana, Turkey). These were Pinus brutia (pine oil), Eucalyptus globulus (eucalyptus), Origanum onites (oregano), Salvia officinalis (sage tea), Lavandula officinalis (lavender), Citrus sinensis (orange), Laurus nobilis (laurel), Citrus limonum (lemon), Myrtus communis (myrtle), Cuminum cyminum (lemon), Rosmarinus officinalis (rosemary), Juniperus communis (juniper). GC/MS analyses were carried out by using a Perkin Elmer Clarus 500 capillary gas chromatograph directly coupled to the mass spectrometer system (Japan). SGE, non polar fused silica capillary column (60 m x 0.25 mm, ID. BPX5 0,25um, USA) was employed under the following conditions: oven temperature program from 60°C (10 min) to 250°C at 4°C/min, and the final temperature maintained for 10 min; injector temperature 220°C; helium as carrier gas, flow rate 1.5 mL/min. The volume of injected sample was 1 μl of diluted oil in hexane; splitless injection technique; ionization energy 70eV, in the electronic ionization (EI) mode; ion source temperature 200°C; scan mass range of m/z 35-425 and interface line temperature 250°C. The composition of essential oils was determined compared to the retention time of a series of alkanes (C4- C28) as reference products and the similarity of their mass spectra with those gathered in the NIST-MS and WILEY-MS library, or reported in the literature.

Table 1.

RESULTS & DISCUSSION Composition of essential oils. The components of essential oils determined are shown in Table 1. The major component in essential oils was -pinene in juniper (90.09%), pine oil (90.18%) and myrtle oil (32.56%), 1,8-cineole in sage tea (47.51%), rosemary (52.17%), and laurel (29.60%), camphor (57.25%) in French lavender, carvacrol in oregano (71.54%) and myrtle (48.68%), linalool (43.37%) and linalyl acetate (33.60%) in lavender, limonene (71.77%) in lemon oil and orange oil (95.77%), and eucalyptol in eucalyptus oil (59.28%).

Major components (%) of essential oil identified by GC-MS.

Compounds

JUN

SAGE

ROSE

Pine oil

LAU

FLAV

THY

LAV

Lemon

MYR

Orange

EUC

-Pinene

90.09

11.97

12.0

90.18

2.67

-

-

-

1.62

32.56

-

-

1.8-Cineole

-

47.51

52.17

-

29.60

11.25

-

3.90

-

-

-

-

Camphor

-

6.37

8.53

-

-

57.25

-

4.85

-

-

-

-

Limonene

-

-

-

-

-

-

-

-

71.77

-

95.77

-

Carvacrol

-

-

-

-

-

-

71.54

-

-

-

-

-

Eucalyptol

-

-

-

-

-

-

-

-

-

48.68

-

59.28

Linalool

-

-

-

-

-

-

-

43.37

-

-

-

-

Linalyl acetate

-

-

-

-

-

-

-

33.60

-

-

-

-

Fenchone

-

-

-

-

-

25.49

-

-

-

-

-

-

Spathulenol

-

-

-

-

-

-

-

-

-

-

-

24.50

P-Cymene

-

-

-

-

-

-

11.84

-

-

-

-

-

-Pinene

1.31

3.10

1.04

-

2.09

-

-

-

6.11

-

-

-

-Myrecene

1.73

2.99

3.81

-

-

-

-

2.00

-

1.79

-

Caryophyllene

-

12.63

4.62

-

-

-

1.24

1.07

-

-

-

2.18

Camphene

-

3.01

2.92

1.56

-

1.01

-

-

-

-

-

-

Antimicrobial Effect of Essential Oils

Recent Patents on Food, Nutrition & Agriculture, 2015, Vol. 7, No. 1

55

(Table 1) contd… Compounds

JUN

SAGE

ROSE

Pine oil

LAU

FLAV

THY

LAV

Lemon

MYR

Orange

EUC

-Terpineol

-

-

2.00

1.10

11.75

-

-

-

-

5.69

-

-

-Terpinene

-

-

-

-

-

-

6.74

-

-

9.70

-

-

Borneol

-

3.65

3.55

-

-

-

-

1.61

-

-

-

-

-Humulene

-

2.45

-

-

-

-

-

-

-

-

-

-

Terpinene-4-Ol

-

-

1.07

-

-

-

-

-

-

-

-

D-Limonene

-

-

-

1.64

-

-

-

-

5.24

-

-

Bornyl Acetate

-

2.09

1.78

-

-

1.12

-

-

-

-

-

-

4-Terpineol

-

-

-

-

10.38

-

-

-

-

-

-

-

-Terpinyl acetate

-

-

-

-

18.16

-

-

-

-

-

-

-

Methyl eugenol

-

-

-

-

4.64

-

-

-

-

-

-

-

Cymol

-

-

-

-

3.81

-

-

-

-

-

-

1.02

1-P-Menthen-8-Yl Acetate

-

-

-

-

-

-

-

-

-

2.85

-

-

Eugenol

-

-

-

-

2.78

-

-

-

-

-

-

-

-Phellandrene

-

-

-

-

2.30

-

-

-

-

-

-

-

Cryptone

-

-

-

-

-

-

-

-

-

-

-

5.88

Caryophyllene oxide

-

-

-

-

-

-

1.01

-

-

-

-

2.31

Iso-Borneol

-

-

-

-

-

-

2.04

-

-

-

-

-

-Thujene

-

-

-

-

-

-

-

-

2.00

-

-

-

Geraniol

-

-

-

-

-

-

-

-

-

1.89

-

-

Lavanduyl acetate

-

-

-

-

-

-

-

1.39

-

-

-

-

P-Mentha-1.5-Dien-8-Ol

-

-

-

-

1.36

-

-

-

-

-

-

-

-3-Carene

-

-

-

-

-

-

-

1.33

-

-

-

-

Geranyl acetate

-

-

-

-

-

-

-

1.14

-

-

-

-

Sabinyl acetate

-

-

-

-

-

1.12

-

-

-

-

-

-

-4-Carene

-

-

-

-

-

-

1.11

-

-

-

-

-

Terpinyl acetate

-

-

-

-

1.10

-

-

-

-

-

-

-

Butanoic acid

-

-

-

-

-

-

-

1.08

-

-

-Bisabolene

-

-

-

-

-

-

-

-

1.03

-

-

Abbreviations: JUN: juniper, SAGE: sage tea, ROSE: rosemary, LAU: laurel, FLAV: French lavender, THY: thyme, LAV: lavender, MYR: myrtle, EUC: eucalyptus. For essential oil, compounds of < 1% were not shown.

Oregano contained 71.54% carvacrol, 11.84% p-cymene, and 6.74% -terpinene. Hussain et al. [12] reported thymol (36.5%), carvacrol (9.50%), thymyl acetate (7.30%), and bcaryophyllene (5.76%) in Tymus linearis oil, while carvacrol (44.4%), o-cymene (14.0%), -terpineol (6.47%), -pinene (6.06%) and b-caryophyllene (5.25%) were the primary compounds of Tymus serpyllum oil. Foudil-Cherif and Yassaa [23] reported great differences in chiral distribution of monoterpenes in the same plant species and between the two junipers. While needles

and berries of Juniperus communis growing in Algeria showed high amounts of -pinene, sabinene and -myrcene with 12-24%, 19-30% and 9-20%, respectively, Juniperus oxycedrus contained -pinene with 85-92% in both needles and berries. In this study, essential oil of juniper contained 90.09% -pinene. Essential oil of laurel contained 1, 8-cineole (29.60%), -terpineol (11.75%), 4-terpineol (10.38%), -terpinyl acetate (18.16%). Chalchat et al. [24] found major compounds in stems, leaves and fruits of laurel (L. nobilis) oil in the


south of Turkey as 1,8-cineole (25.72-52.79%), -terpinyl acetate (5.03-14.72%), sabinene (81.77-7.07%), -pinene (2.56-2.99%) and terpinene-4-ol (0.77-6.70%). Özcan and Chalchat [25] found 51.73-68.48% 1, 8-cineole, 4.049.87% -pinene, 2.58-3.91% -pinene, 1.33-3.24% terpinene-4-ol and 0.95-3.05% -terpineol in leaf oils of L. nobilis. The main compounds of the essential oil of sage tea (Salvia fruticosa) determined by Chalchat et al. [24] were pinene (31.05%), isoborneol (27.17%), borneol (7.64%), 1,8cineole (6.94%), camphene (6.07%), -pinene (3.88%) and bornly acetate (2.23%). Özcan et al. [26] indicated that 1, 8cineole (38.9-21.2%), camphor (18.3-24.5%), camphene (7.9-4.3%) and -pinene (4.3-3.3%) were major constituents in the oils for Salvia aucheri and Sideritis tomentosa. pinene and -pinene were also reported as main compounds in the oil of Sideritis dichotoma [27]. Bumblauskien [28] indicated that plants with same chemotypes but growing in different conditions have different composition of major components since composition of essential oil depends on geographical location and environmental factors. These sage tea species mentioned above were from the different region of Turkey. In this study, similar results were observed with the essential oil of sage tea that contained 11.97% -pinene, 47.51% 1,8-cineole, 12.63% caryophyllene and 6.37% camphor. Rosemary oil contained 1,8-cineole (52.17%), -pinene (12%), camphor (8.53%), and caryophyllene (4.62%). Rosemary oil mostly contained monoterpenes: 1, 8-cineole, limonene, and -pinene, constituting 27.6%, 13.5% and 13.0% of the essential oil, respectively [29]. Pine oil consisted of -pinene (90.18%). Zheljazkov et al. [30] showed that the major constituents of pine essential oil were -pinene and -pinene, ranging from 17% to 40% and from 21% to 29%, respectively, of the total oil. Hassiotis and Dina [31] reported that the major compounds constituting French lavender (Lavandula stoechas) contained fenchone (46.7%), camphor (9.9%), and 1,8cineole (9.0%). Danh et al. [32] reported four major compounds in lavender oil, namely linalool (43%), linalyl acetate (23%), camphor (8%) and borneol (6.6%). Similar results were found in this study. French lavender oil contained camphor (57.25%), fenchone (25.49%) and 1, 8cineole (11.25%) while lavender consisted of linalool (43.37%) and linalyl acetate (33.60%). Myrtle (Myrtus communis) oil contained -pinene (31.2%), myrtenyl acetate (19.3%), and 1, 8-cineole (16.1%) [31]. Viuda-Martos et al. [33] indicated that the principal components of myrtle leaves essential oil were 1, 8-cineole (40.37%) and -pinene (21.82%). The major components of myrtle oil (Myrtus communis) are -pinene (36.08%), 1, 8cineole (22.63%) and limonene (15.14%) [34]. However, in this work, myrtle essential oil consisted of -pinene (32.56%), eucalyptol (48.68%), -terpineol (5.69%), and dlimonene (5.24%). Franceschi et al. [35] showed that the main compounds in lemon essential oil are limonene (58.6%), -terpinene

Ozogul et al.

(14.8%), -pinene (11.2%), citral (3.3%), and -bisabolene (2.2%). Limonene (37.63-69.71%), beta-pinene (0.6331.49%), gamma-terpinene (0.04-9.96%), and p-cymene (0.23-9.84%) were reported as main compounds in lemon [36]. Lemon essential oil mainly included, limonene (71.77%) and -pinene (6.11%) in this study. Zunino et al. [37] determined that the major compound in orange oil was limonene (87.6%). Similarly, limonene (95.77%) was the major component in orange essential oil in current work. It was reported that eucalyptus oil included 98.9 % of 1, 8-cineole [37]. A report from Abravesh et al. [38] indicated that the 1,8-cineole (41.3%) was the major constituent of the oil of Eucalyptus oleosa, followed by spathulenol (11.6%) and virdiflorol (15.9%), while the main compounds of essential oil of Eucalyptus largiflorens by Rahimi-Nasrabadi et al. [39] were 1, 8-cineole (23.1%), cryptone (15.1%), 4terpineol (9.6%), and 4-allyloxyimino-2-carene (11.2%). However, eucalyptol (59.28%), spathulenol (24.50%), and cryptone (5.8%) were main compounds found in eucalyptus oil in this study. Antimicrobial effects of essential oils on gramnegative bacteria. Tables 2 and 3 show inhibition zones for essential oils against Enterobacteriaceae and other gram-negative bacteria, respectively. The antimicrobial effect of essential oils varied depending on the chemical components of the oil and specific microorganism tested. Among essential oils, oregano gave the highest antimicrobial activity against Enterobacteriaceae than that of other essentials oils. This is due to carvacrol, which is the major compound of oregano. Carvacrol was very most effective in reducing bacterial growth [40, 41]. Oregano essential oil produced inhibition zones ranging from 16 (Y. enterocolitica) to 29.75 mm (K. pneumoniae). Dobre et al. [42] reported the essential oil with the greatest spectrum of activity as following order: oregano oil > white thyme oil > clove bud oil > cinnamon oil > garlic oil > onion oil > basil oil. Celikel and Kavas [43] studied effect of the plant essential oils (thyme, myrtle, laurel, sage, and orange) against E. coli and Staph. aureus at concentrations of 5–20 μl/disc (diameter 6 mm). The results revealed the essential oil extract of thyme was more effective than that of the others, giving 23–30 mm inhibition zones. All bacteria showed low resistance to essential oil of laurel, with 12–19 mm diameter inhibition zones. In the present study, the highest inhibitory effect of laurel against gram-negative bacteria, was observed for A. hydrophila, with diameter zone of 16.25 mm. Elgayyar et al. [44] found that oil of oregano exhibited the highest antimicrobial activity against gram negative such as E. coli O157, Salmonella typhimurium, Y. enterocolitica and P. aeruginosa with inhibition zone of 46 to 87 mm compared to other essential oil (including rosemary). Hili et al. [45] indicated that P. aeruginosa proved to be the most resistant organism compared to E. coli and Staph. aureus in terms of thyme, rosemary and sage essential oil. In contrast, P. aeruginosa was very sensitive to rosemary and sage tea essential oil but it was highly resistant to thyme compared to E. coli.


Table 2.


Antimicrobial effect of essential oils on Enterobacteriaceae. Inhibition Zone Diameter (mm)

Essential Oils

x y

E. coli x

Salmonella Paratyphi A yg

1.25±0.07

f

Klebsiella Pneumoniae 5.00±0.42

Yersinia Enterocolitica

fg

5.00±0.42de

Juniper

1.50 ±0.00

Pine oil

5.25±0.35e

6.00±0.28d

20.50±0.28b

15.50±1.41a

Myrtle

3.00±0.00f

4.75±0.35de

3.25±0.21g

4.00±0.00ef

Orange

1.00±0.00g

3.00±0.00e

16.00±1.41c

9.50±0.71b

Thyme

24.00±1.41a

27.50±2.12a

29.75±2.47a

16.00±1.41a

Lavender

3.00±0.00f

3.50±0.28e

6.75±0.35f

15.00±0.99a

Sage tea

6.25±0.35e

4.5±0.42de

4.75±0.21fg

9.00±0.71bc

Lemon

5.25±0.35e

6.25±0.35d

10.75±1.06d

6.25±0.35cd

Rosemary

3.00±0.00f

5.50±0.42d

4.00±0.14g

3.15±0.21f

French lavender

9.50±0.71b

10.50±0.71c

9.50±0.71e

6.00±0.42cd

Eucalyptus

2.75±0.07f

1.00±0.00f

6.75±0.35f

7.50±0.71bc

Laurel

7.75±0.35d

5.50±0.071d

11.50±0.74d

6.50±0.14cd

Tetracycline

12.50±0.71b

9.50±0.21c

11.50±0.42d

10.00±0.71b

Streptomycin

8.50±0.57c

9.50±0.14c

15.50±0.35c

10.50±0.36b

Neomycin

5.50±0.28e

14.50±1.41b

12.50±0.78d

15.00±0.28a

Diameter of inhibition zones of essential oil including diameter of disc 6 mm. Values are given as mean ± Standard deviation (SD) of triplicate experiment.

Different lowercase letters (a–g) in a column indicate significant differences (P < 0.05) among control (antibiotics) and essential oils.

Table 3.

Antimicrobial effect of essential oils on gram-negative food-borne bacteria. Inhibition zone diameter (mm)

x

Essential Oils

Pseudomonas Aeruginosa

Aeromonas Hydrophila

Campylobacter Jejuni

Juniper

2.25±0.07ef

6.00±0.57g

3.00±0.14h

Pine oil

15.00±1.41b

24.50±2.26a

22.25±1.06b

Myrtle

3.75±0.35e

11.80±1.13d

6.00±0.00fg

Orange

2.50±0.14ef

11.00±0.85de

12.50±0.85d

Thyme

18.05±1.20a

12.50±0.57cd

30.00±2.83a

Lavender

8.00±0.71d

7.5±0.014fg

14.25±1.06cd

Sage tea

7.95±0.49d

11.00±0.71de

8.00±0.64ef

Lemon

7.50±0.57d

7.75±0.35fg

9.00±0.42e

Rosemary

11.50±1.13c

11.50±0.42d

9.00±0.00e

French lavender

12.75±0.92c

12.00±0.85d

5.50±0.57g

Eucalyptus

1.50±0.14f

9.50±0.28ef

8.75±0.49e

Laurel

9.00±0.57d

16.25±1.06b

8.00±0.71ef

Tetracycline

12.00±0.71c

14.25±0.14c

16.50±0.92c

Streptomycin

14.50±0.85b

16.50±1.13b

16.50±0.99c

Neomycin

14.50±0.57b

16.30±1.13b

15.00±0.71c

Diameter of inhibition zones of essential oil including diameter of disc 6 mm. Values are given as mean ± Standard deviation (SD) of triplicate experiment. Different lowercase letters (a–h) in a column indicate significant differences (P < 0.05) among control (antibiotics) and essential oils.

y

57


The essential oil with the lowest inhibition zones was juniper with the values which vary from 1.5 to 6 mm. K. pneumoniae was more sensitive than other Enterobacteriaceae strains in oregano, pine oil and orange essential oils. Pine oil and lavender exhibited statistically similar effect on Y. enterocolitica compared to control antibiotics. E. coli and S. paratyphi A showed a strong resistance against juniper, orange and eucalyptus essential oils, although these bacteria were more vulnerable to French lavender essential oil. Pasqua et al. [46] found that sage and lavender essential oils gave low antimicrobial activity against gram positive and negative bacteria apart from E. coli and S. Typhimurium. The result of current study indicated that inhibition zone of lavender and sage tea essential oil on gram-negative bacteria ranged from 3 to 15 mm and from 4.5 to 11 mm, respectively. Oregano essential oil produced inhibition zones ranging from 12.50 for A. hydrophila and 30 mm for C. jejuni. Pine oil also showed strong antimicrobial effect on P. aeruginosa, A. hydrophila and C. jejuni, with corresponding diameter zone of 15, 24.5 and 22.25 mm, respectively. Although pinene was the main component of juniper and pine essential oil, antimicrobial activity of juniper on these bacterial strains was weak. -pinene in essentials oils of species of different genera was reported as inhibitory effect against bacterial growth [47, 48]. Although biological properties of an essential oil are mostly attributed to its main compounds, minor components as well as possible synergistic or antagonistic effects between the substances might influence the antimiTable 4.

Ozogul et al.

crobial activity [49, 50]. This is the reason for the differences in antimicrobial activity between juniper and pine essential oil. Poor inhibition zones for eucalyptus, orange and myrtle essential oils against P. aeruginosa were observed. The antimicrobial activity of essential oils of eucalyptus (Eucalyptus oleosa) from different parts (stems, adult leaves, fruits and immature flowers) was determined at different concentrations (0.5–20 mg/mL). The results indicated that the essential oil of all the plant parts of E. oleosa had high antimicrobial activity against both gram negative and positive microorganisms [47]. Lemon, orange and rosemary oils showed strong activity against pathogen strains such as E. coli, Klebsiella pneumoniae, P. aeruginosa, whereas eucalyptus showed least inhibitory activity [51]. In the current study, weak antimicrobial activity of eucalyptus on E. coli, K. pneumoniae and P. aeruginosa were also found, even though it had medium antimicrobial activity against other gramnegative bacteria. Rosemary and French lavender exhibited statistically similar inhibition zones (11.5 mm) against P. aeruginosa and A. hydrophila. Similarly, Elgayyar et al. [44] found that oil of rosemary exhibited weak inhibition (13 mm) to P. aeruginosa. C. jejuni was more sensitive against lavender essential oils than P. aeruginosa and A. hydrophila. Antimicrobial effects of essential oils on gram-positive bacteria. Inhibition zones produced by fifteen essential oils against gram-positive bacteria are given in Table 4. The susceptibilities of gram-positive bacteria on juniper were the

Antimicrobial effect of essential oils on gram-positive food-borne bacteria. Inhibition Zone Diameter (mm)

x

Essential Oils

Enterococcus Faecalis

Staphylococcus Aureus

Juniper

5.25±0.21ı

4.25±0.35ı

Pine oil

21.25±1.06a

28.25±1.77a

Myrtle

16.50±0.71cd

7.25±0.14fg

Orange

12.00±0.99f

10.00±0.64e

Thyme

15.00±1.41de

22.25±1.20b

Lavender

17.75±1.77bc

7.25±0.07fg

Sage tea

14.50±0.71e

10.75±1.06e

Lemon

7.25±0.35h

15.00±1.41d

Rosemary

9.65±0.21g

10.00±0.99e

French lavender

19.15±0.07ab

5.50±0.14hı

Eucalyptus

12.00±0.71f

18.00±1.27c

Laurel

11.00±0.28fg

11.25±1.06e

Tetracycline

18.50±0.85b

15.00±1.27d

Streptomycin

10.00±1.06fg

6.50±0.35h

Neomycin

12.00±0.71f

9.25±0.07ef

Diameter of inhibition zones of essential oil including diameter of disc 6 mm. Values are given as mean ± S.D of triplicate experiment. Different lowercase letters (a–ı) in a column indicate significant differences (P < 0.05) among control (antibiotics) and essential oils.

y



strongest, although effectiveness of this oil to gram-negative bacteria was quite poor. Pine oil exhibited the highest antibacterial effect against Staph. aureus and Ent. faecalis, with the diameter of inhibition zones 21.25 and 28.25 mm, as compared with control antibiotics. Our findings showed that Staph. aureus was more sensitive against oregano and eucalyptus (22.25 vs.18.00) than control antibiotics. Oregano oil completely inhibited growth of S. aureus [44]. Rosemary essential oil was moderately inhibitory to Staph. aureus (inhibition zone of 10 mm). However, Kazemi et al. [52] found that the essential oil of rosemary exhibited good antibacterial activity against Staphylococcus intermedius (38 mm) and E. faecalis (30 mm).

CURRENT & FUTURE DEVELOPMENTS

French lavender also found to be more effective (with 19.15 mm inhibition diameter) than control antibiotics to inhibit Ent. faecalis growth. Lemon essential oil did not result in any remarkable inhibition zone for E. faecalis compared to antibiotics, although S. aureus growth was significantly affected from lemon essential oil in comparison to control. Sage tea essential oils also produced 14.50 (E. faecalis) and 10.75 mm (S. aureus) in diameter inhibition zones, which was higher than streptomycin and neomycin antibiotics. Similar inhibitory effects were observed in orange essential oils and neomycin but statistically significant inhibition was found compared to streptomycin. Myrtle is highly active against E. faecalis but is less active against S. aureus compared to neomycin. Inhibition zones of rosemary and streptomycin against E. faecalis are statistically non significant. Similar result was also found for neomycin and rosemary against S. aureus.

ACKNOWLEDGMENT

Earlier studies have reported that essential oils were stronger against gram-negative bacteria [46, 53] or equally strong against both gram-positive and gram-negative organisms [51]. Moreover, gram-positive bacteria were shown to be generally more susceptible than gram-negative bacteria to plant essential oils [54, 55], which supports our results showing that most of essential oils tested were more effective against gram-positive bacteria. There are also some patented researches related to essential oils for cleaning, disinfecting, sanitizing [56] and as powerful fungicides [57]. Wilson et al. [58] also patented synergistic combinations of plant essential oils and chitosan salts that control decay of fruits and vegetables and reduce contamination by foodborne human pathogens. They suggested that synergistic combinations of essential oils and chitosan salts hold promise for giving superior control of both postharvest decay organisms and foodborne human pathogens. The patent US6824795 by Khanuja et al. [59] showed a formulation contained thymol (from Trachyspermum ammi), and mint oil (from a hybrid of Mentha spicata and Mentha arvensis) which is useful in the treatment of drug-resistant bacterial infections. Patent by Ben-Yehoshua, [61] presents a microbiocidal aqueous formulation which contains a number of components : citral, 1- octanol, heptanol, nonanol, geraniol, octanal, nonanal, decanal, perillaldehyde, perillalcohol, citronellol, citronellal, carvone, carveol, linalool, vanillin, cinnamic aldehyde, cinnamic acid, eugenol, menthol, limonene, limonene hydroperoxide, carvacrol, terpineol, thymol and camphor for inhibiting microbial development. Patent by Narayanan et al. [60] includes the use of thymol and eucalyptol in addition to the mouthwash.

59

It can be also concluded that the components of essential oil of oregano and pine oil are highly active against food borne pathogen, generating the largest inhibition zones for both gram negative and positive bacteria. These results also indicate the possible use of the essential oil on food system as an effective inhibitor of food borne pathogen. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest.

The authors thank the Scientific Research Projects Unit in Cukurova University for their financial support (Research Project: SUF 2012 BAP1). REFERENCES [1] [2]

[3]

[4]

[5] [6]

[7] [8] [9]

[10]

[11] [12]

[13] [14]

Losso JN, Shahidi F, Bagchi D. Anti-Angiogenic functional and medicinal foods. 2nd ed. London: Taylor & Francis 2007. Hussain AI, Anwar F, Sherazi STH, Przybylski R. Chemical composition antioxidant and antimicrobial activities of basil (Ocimum basilicum) essential oils depends on seasonal variations. Food Chem 2008; 108: 986-95. Anwar F, Ali M, Hussain AI, Shahid M. Antioxidant and antimicrobial activities of essential oils and extracts of fennel (Foeniculum vulgare Mill.) seeds from Pakistan. Flavour Frag J 2009; 24: 170-6. FAO. Food and drug administration, code of federal regulations, Title 21 Food and drugs, Chapter I Food and Drug Administration Department of Health and Human Services, Subchapter B - Food for Human Consumption, Part 182 - Substances Generally Recognized as Safe, Sec. 182. 20 Essential oils, oleoresins (solvent free), and natural extracts (including distillates), USA 2011. Nerio LS, Olivero-Verbel J, Stashenko E. Repellent activity of essential oils. Bioresource Technol 2010; 101: 372-8. Ait-Ouazzou A, Cherrat L, Espina L, Loràan S, Rota C, Pagàn R. The antimicrobial activity of hydrophobic essential oil constituents acting alone or in combined processes of food preservation. Trends Food Sci Tech 2011; 2: 320-9. Tajkarami MM, Ibrahim SA, Cliver DO. Antimicrobial herb and spice compounds in food. Food Control 2010; 21: 1199-218. Tassou C, Koutsoumanis K, Nychas GJE. Inhibition of Salmonella enteritidis and Staphylococcus aureus in nutrient broth by mint essential oil. Food Res Int 2003; 3: 273-80. Tsigarida E, Skandamis P, Nychas GJE. Behaviour of Listeria monocytogenes and autochthonous flora on meat stored under aerobic, vacuum and modified atmosphere packaging conditions with or without the presence of oregano essential oil at 5°C. J Appl Microbiol 2000; 89: 901-9. Skandamis P, Tsigarida E, Nychas GJE. The effect of oregano essential oil on survival/death of Salmonella typhimuriumin meat stored at 5°C under aerobic, VP/MAP conditions. Food Microbiol 2002; 19: 97-10. Hansen TL, Vogel BF. Desiccation of adhering and biofilm Listeria monocytogenes on stainless steel: Survival and transfer to salmon products. Int J Food Microbiol 2011; 146: 88-93. Hossain L, Monjur-Al-Hossain ASM, Sarkar KK, Hossin A, Rahman A. Phytochemical screening and the evaluation of the antioxidant, total phenolic content and analgesic properties of the plant Pandanus foetidus (Family: Pandanaceae). Int Res J Pharm 2013; 4: 170-2. Canillac N, Mourey A. Antibacterial activity of the essential oil of Picea excelsa on Listeria, Staphylococcus aureus and coliform bacteria. Food Microbiol 2001; 18: 261-8. Gutierrez J, Barry-Ryan C, Bourke P. Antimicrobial activity of plant essential oils using food model media: Efficacy, synergistic potential and interaction with food components. Food Microbiol 2009; 26: 142-50.

60 Recent Patents on Food, Nutrition & Agriculture, 2015, Vol. 7, No. 1 [15]

[16]

[17] [18]

[19]

[20] [21]

[22] [23]

[24] [25]

[26]

[27] [28]

[29]

[30] [31]

[32]

[33]

[34]

Barker S, Altman P. A randomized, assessor blind, parallel group comparative efficacy trial of three products for the treatment of head lice in children - melaleuca oil and lavender oil, pyrethrins and piperonyl butoxide, and a “suffocation” product. BMC Dermatol 2010; 10: 6. Moore-Neibel K, Gerber C, Patel J, Friedman M, Ravishankar S. Antimicrobial activity of lemongrass oil against Salmonella enterica on organic leafy greens. J Appl Microbiol 2012; 112: 48592. Paster N, Menasherov M, Ravid U, Juven B. Antifungal activity of oregano and thyme essential oils applied as fumigants against fungi attacking store grain. J Food Protect 1995; 58: 81-5. Velluti A, Sanchis V, Ramos AJ, Turon C, Marin S. Impact of essential oils on Growth rate, zearalenone and deoxynivalenol production by Fusarium graminearium under different temperature and water activity conditions in maize grain. Food Microbiol 2004; 21: 313-8. Palombo EA. Traditional medicinal plant extracts and natural products with activity against oral bacteria: Potential application in the prevention and treatment of oral diseases. Evid Based Complement Alternat Med 2011; 680354: 1-15. Edwards-Jonesa V, Bucka R, Shawcrossa SG, Dawsona MM, Dunn K. The effect of essential oils on methicillin-resistant Staphylococcus aureus using a dressing model. Burns 2004; 30: 772-7. Thakur R, Jain N, Pathak R, Sandhu SS. Practices in wound healing studies of plants. Evid Based Complement Alternat Med 2011; 438056: 1-17. Murray PR, Baron EJ, Pfaller MA. Manual of chinical microbiology, 6th ed. Washington DC: ASM 1995. Foudil-Cherif Y, Yassaa N. Enantiomeric and non-enantiomeric monoterpenes of Juniperus communis L. and Juniperus oxycedrus needles and berries determined by HS-SPME and enantioselective GC/MS. Food Chem 2012; 135: 1796-800. Chalchat JC, Ozcan MM, Figueredo G. The composition of essential oils of different parts of laurel, mountain tea, sage and ajowan. J Food Biochem 2011; 35: 484-99. Ozcan M, Chalchat JC. Essential oil composition of Ocimum basilicum L. and Ocimum minimum L. in Turkey. Czech J Food Sci 2005; 20: 223-8. Ozcan M, Akgul A, Chalchat JC. Volatile constituents of essential oils of Salvia aucheri Benth. var. canescens Boiss. et Heldr. and S. tomentosa Mill. grown in Turkey. J Essent Oil Res 2002; 14: 339-41. Kirimer N, Baer KHC, Tumen G, Sezik E. Characterization of the essential oil of Sideritis dichotoma. J Essent Oil Res 1992; 4: 6412. Bumblauskiene L, Jakstas V, Janulis V, Mazdzieriene R, Ragazinskiene O. Preliminary analysis on essential oil composition of Perilla l. cultivated in Lithuania. Acta Pol Pharm Drug Res 2009; 66: 409-13. Nowak A, Kalemba D, Krala L, Piotrowska M, Czyzowska A. The effects of thyme (Thymus vulgaris) and rosemary (Rosmarinus officinalis) essential oils on Brochothrix thermosphacta and on the shelf life of beef packaged in high-oxygen modified atmosphere. Food Microbiol 2012; 32: 212-6. Zheljazkov VD, Astatkie T, Schlegel V. Effects of distillation time on the Pinus ponderosa essential oil yield, composition, and antioxidant activity. Horticulture Sci 2012; 47: 785-9. Hassiotis CN, Dina EI. The influence of aromatic plants on microbial biomass and respiration in a natural ecosystem. Isr J Ecol Evol 2010; 56: 181-96. Danh LT, Han LN, Triet NDA, Zhao J, Mammucari R, Foster N. Comparison of chemical composition, antioxidant and antimicrobial activity of lavender (Lavandula angustifolia L.) essential oils extracted by supercritical CO2, hexane and hydrodistillation. Food Bioprocess Tech 2012; DOI 10.1007/s11947-012-1026-z. Viuda-Martos M, Sendra E, Perez-Alvarez JA, Fernandez-Lopez J, Amensour M, Abrini J. Identification of flavonoid content and chemical composition of the essential oils of moroccan herbs: Myrtle (Myrtus communis L.), Rockrose (Cistus ladanifer L.) and Montpellier cistus (Cistus monspeliensis L.). J Essent Oil Res 2011; 23: 1-9. Kiralan M, Bayrak A, Abdulaziz OF, Ozbucak T. Essential oil composition and antiradical activity of the oil of Iraq plants. Nat Prod Res 2012; 26: 132-9.

Ozogul et al. [35]

[36] [37]

[38] [39]

[40]

[41]

[42] [43]

[44]

[45] [46]

[47]

[48]

[49] [50]

[51]

[52]

[53] [54] [55] [56] [57]

Franceschi E, Grings MB, Frizzo CD, Oliveira JV, Dariva C. Phase behavior of lemon and bergamot peel oils in supercritical CO2. Fluid Phase Equilib 2004; 226: 1-8. Bourgou S, Rahali FZ, Ourghemmi I, Tounsi MS. Changes of peel essential oil composition of four tunisian citrus during fruit maturation. Sci World J 2012; 528593: 1-10. Zunino MP, Areco VA, Zygadlo JA. Insecticidal activity of three essential oils against two new important soybean pests: Sternechus pinguis (Fabricius) and Rhyssomatus subtilis Fiedler (Coleoptera: Curculionidae). Bol Latinoam Caribe Plant Med Aroma 2012; 11: 269-77. Abravesh Z, Sefidkon F, Assareh MH. Extraction and identification of essential oil components of five Eucalyptus species in warm zones of Iran. Iran J Med Aromat Plants 2007; 23: 323-30. Rahimi-Nasrabadi, Nazarian M, Farahani S, Koohbijari GR, Ahmadi F, Batooli H. Chemical composition, antioxidant, and antibacterial activities of the essential oil and methanol extracts of Eucalyptus largiflorens F. Muell Int J Food Prop 2013; 16: 369-81. Ultee A, Kets EPW, Smid EJ. Mechanisms of action of carvacrol on the food-borne pathogen Bacillus cereus. Appl Environ Microbiol 1999; 65: 4606-10. Ouwehand AC, Tiihonen K, Kettunen H, Peuranen S, Schulze H, Rautonen N. In vitro effects of essential oils on potential pathogens and beneficial members of the normal microbiota. Vet Med 2010; 55: 71-8. Dobre AA, Gagiu V, Petru N. Antimicrobial activity of essential oils against food-borne bacteria evaluated by two preliminary methods. Rom Biotechnol Lett 2011; 16: 119-25. Celikel N, Kavas G. Antimicrobial properties of some essential oils against some pathogenic microorganisms. Czech J Food Sci 2001; 26: 174-81. Elgayyar M, Draughon FA, Golden DA, Mount JR. Antimicrobial activity of essential oils from plants against selected pathogenic and saprophytic microorganisms. J Food Protect 2001; 64: 101924. Hili P, Evans CS, Veness RG. Antimicrobial action of essential oils: The effect of dimethylsulphoxide on the activity of cinnamon oil. Lett Appl Microbiol 1997; 24: 269-75. Pasqua R, De Feo V, Villani F, Mauriello G. In vitro antimicrobial activity of essential oils from Mediterranean Apiaceae, Verbenaceae and Lamiaceae against foodborne pathogens and spoilage bacteria. Ann Microbiol 2005; 55: 139-43. Marzoug HNB, Romdhane M, Lebrihi A, Mathieu F, Couderc F, Abderraba M, et al. Eucalyptus oleosa essential oils: Chemical composition and antimicrobial and antioxidant activities of the oils from different plant parts (stems, leaves, flowers and fruits). Molecules 2011; 16: 1695-709. Buitrago D, Velasco-Díaz JT, Morales A. Chemical composition and antibacterial activity of the essential oil of Monticalia imbricatifolia Schultz (Asteraceae). Rev Latinoamer Quím 2012; 40: 1148. Cosentino S, Tuberoso CI, Pisano B, Satta M, Mascia V, Arzedi E. In-vitro antimicrobial activity and chemical composition of Sardinian thymus essential oils. Lett Appl Microbiol 1999; 29: 130-5. Vladimir-Knezevic S, Kosalec I, Babac M, Petrovic M, Ralic J, Matica B, et al. Antimicrobial activity of Thymus longicaulis C. presl essential oil against respiratory pathogens. Cent Eur J Biol 2012; 7: 1109-15. Prabuseenivasan S, Jayakumar M, Ignacimuthu S. In vitro antibacterial activity of some plant essential oils. BMC Comp Alternative Med 2006; 6: 39. Kazemi M, Rostami H, Ameri A. The study of compositions and antimicrobial properties of essential oil of Origanum vulgare and Rosmarinus officinalis on human pathogens. Curr Res Bacteriol 2012; 5: 1-12. Zaika LL. Spices and herbs: Their antibacterial activity and its determination. J Food Safety 1988; 23: 97-118. Jay JM. Modern food microbiology. 5th ed. Van Nostrand, Reinhold 1996. Reynolds JEF. Martindale the Extra Harmacopoeia. 31st ed. Royal Pharmaceutical Society of Great Britain, London 1996. Danice, D. Antimicrobial compositions containing essential oils. US2014030203 (2014). Ricardo, P.C., Eduardo, A.T., Alejandro, R.H., Rodrigo, Y.B., Sandro, B.G., Franco, D.V.L. Essential oils from Thymus vulgaris


[58]

[59]

(thyme) and Origanum vulgare (oregano) and compositions containing same. WO2014036667 (2014). Wilson, C.L., Ghaouth, A.E., Wisniewski, M.E. Synergistic combinations of natural of compounds that control decay of fruits and vegetables and reduce contamination by foodborne human pathogens. US20030113421A1 (2003). Khanuja, S.P.S., Srivastava, S., Shasney, A.K., Darokar, M., Kumar, T.R.S., Agarwal, K.K., Ahmed, A., Patra, N.K., Sinha, P.,


[60] [61]

61

Dhawan, S., Saikia, D., Kumar, S. Formulation comprising thymol useful in the treatment of drug resistant bacterial infections. US20046824795 (2004) Narayanan, K.S., Prosise, W.E., Corring, R. An alcohol-free slightly-alcoholic oral care composition and a process for preparing same. WO2012018519A1 (2012). Ben-Yehoshua, S. Microbiocidal formulation comprising essentials oils or their derivatives. US20087465469 (2008).