FOODBORNE PATHOGENS AND DISEASE Volume 12, Number 6, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/fpd.2014.1892
Effect of Essential Oils on Germination and Growth of Some Pathogenic and Spoilage Spore-Forming Bacteria Ste`ve Olugu Voundi,1,2 Maximilienne Nyegue,1,2 Iuliana Lazar,3 Dumitra Raducanu,4 Florentine Foe Ndoye,2 Marius Stamate,5 and Franc¸ois-Xavier Etoa1
Abstract
The use of essential oils as a food preservative has increased due to their capacity to inhibit vegetative growth of some bacteria. However, only limited data are available on their effect on bacterial spores. The aim of the present study was to evaluate the effect of some essential oils on the growth and germination of three Bacillus species and Geobacillus stearothermophilus. Essential oils were chemically analyzed using gas chromatography and gas chromatography coupled to mass spectrometry. The minimal inhibitory and bactericidal concentrations of vegetative growth and spore germination were assessed using the macrodilution method. Germination inhibitory effect of treated spores with essential oils was evaluated on solid medium, while kinetic growth was followed using spectrophotometry in the presence of essential oils. Essential oil from Drypetes gossweileri mainly composed of benzyl isothiocyanate (86.7%) was the most potent, with minimal inhibitory concentrations ranging from 0.0048 to 0.0097 mg/mL on vegetative cells and 0.001 to 0.002 mg/mL on spore germination. Furthermore, essential oil from D. gossweileri reduced 50% of spore germination after treatment at 1.25 mg/mL, and its combination with other oils improved both bacteriostatic and bactericidal activities with additive or synergistic effects. Concerning the other essential oils, the minimal inhibitory concentration ranged from 5 to 0.63 mg/mL on vegetative growth and from 0.75 to 0.09 mg/mL on the germination of spores. Spectrophotometric evaluation showed an inhibitory effect of essential oils on both germination and outgrowth. From these results, it is concluded that some of the essential oils tested might be a valuable tool for bacteriological control in food industries. Therefore, further research regarding their use as food preservatives should be carried out.
Introduction
G
rowth and contamination control of sporeforming bacteria remain an important objective of food industries. Indeed, bacterial spores, due to their ubiquitous character, are distributed in soil, water, and plants, so they can easily contaminate and spoil food (Hong et al., 2009; Heyndrickx, 2011). Furthermore, since spores are very resistant to physical and chemical agents, they can remain in food after sterilization (Aouadhi et al., 2013). Some spores can grow at a pH range of 3.0 to 3.5 and at low ( < 4C) or high ( £ 60C) temperatures depending on the species. Moreover, some spore-forming bacteria species such as Bacillus cereus and Geobacillus stearothermophilus are well known for their ability to cause food poisoning and spoilage, respectively (Etoa and Adegoke, 1996; Shaheen et al., 2006).
To prevent spore contamination, industries usually use high temperatures to sterilize. However, this treatment generally leads to the degradation of nutritional and organoleptic qualities of food (Islam et al., 2006). Thus, an alternative strategy is to use chemical substances with antimicrobial activities, provided that they are nontoxic. This explains all the renewed interest directed toward plant essential oils. Currently, these substances are used in aromatherapy and as additives to add flavor and preserve various foods due to their organoleptic and antibacterial properties (Ndoye, 2001; Mohammedi, 2006; Huertas et al., 2014). However, efficient use of essential oils as food preservatives requires a good knowledge about their properties (i.e., the chemical composition, the minimal inhibitory and the bactericidal concentrations [MIC and MBC], the panel of target microorganisms, and the mechanism(s) of action). To the present, many studies have been done on the chemical
1 Department of Microbiology, Laboratory of Microbiology, and 2Department of Biochemistry, Laboratory of Phytobiochemistry and Medicinal Plant Study, University of Yaounde I, Yaounde, Cameroon. 3 Departments of Food and Chemical Engineering, 4Biology, Ecology, and Environment Protection, and 5Mechanical and Environmental Engineering, Vasile Alecsandri University of Bacau, Bacau, Romania.
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composition and inhibitory effect of essential oils on the growth of a wide range of bacteria, particularly on their vegetative forms, but few have been done on their effect on bacterial spores. Nevertheless, the study of Chaibi et al. (1997) assessed the effect of essential oils extracted from Moroccan plants on spores and vegetative cells of B. cereus. The results showed an inhibitory effect on one or more stages of spore germination with sporostatic or sporocidal effect. This study, which was focused on the mechanism(s) of action of essential oils on bacterial spores, might be extended to other essential oils and more Bacillus species. The purpose of this work was to evaluate the effect of nine essential oils extracted from Cameroonian aromatic plants on growth, germination, and outgrowth of three Bacillus species and G. stearothermophilus. Materials and Methods Plant material, extraction, and chemical analyses of essential oils
For essential oil extraction, nine aromatic plants were harvested or purchased and identified at the National Herbarium of Cameroon (Table 1). Essential oil extractions were done by hydrodistillation using a Clevenger apparatus, according to the protocol described by Moni (2013). Oils were analyzed by gas chromatography–flame ionization detector and GC coupled with mass spectrometry as described earlier by Nyegue (2006). Bacterial strains, production of vegetative cells and spores
B. cereus T, B. subtilis NCTC 3610, and B. megaterium 8174 came from the collection of The Microbiology Laboratory Institute of Food Research of Reading, UK. G. stearothermophilus CNCH 5781 was obtained from the Institut Appert, Paris, France. These strains were provided in the form of spores. To obtain vegetative cells, spores were subjected to activation at 80C for 10 min. They were then grown on nutrient agar for 24 h at the optimal growth temperature of each species (35C for B. cereus, B. subtilis, and B. megaterium and 63C for G. stearothermophilus). For spore production, cells grown for 12 h on trypticase soy broth were spread on sporulation agar medium as described by Bayoı¨ et al. (2014). The plates were incubated at the aforementioned temperatures for 5 days. The spores were collected with a glass spatula, suspended in distilled water, and purified by many centrifugation cycles at 3000 · g for 15 min.
The purified spores were stored at 4C for 3 months to ensure stability of spores before use (Voundi et al., 2014). Macrodilution method
Using this method, minimal inhibitory concentration (MIC), minimal bactericidal (MBC) and minimal inhibitory concentrations for inhibition of spore germination (MICg) were assessed. The method on test tubes described by Gachkar et al. (2007) was used with the following modifications: for MIC determination, vegetative forms of Bacillus and G. stearothermophilus at 106 cells/mL were allowed to grow in 2 mL of nutrient broth (supplemented with 10 g/L of glucose, 0.2% Tween 80, and 0.05 mg/L of red phenol as growth indicator) at different concentrations of each essentials oil. The initial concentration was 20 mg/mL followed by a geometric progression of 1/2 until the 8th dilution for all the essential oils except for that of Drypetes gossweileri that went up to the 16th dilution. For MICg determination, heat-activated spores (80C, 10 min) at 106 spores/mL were inoculated in 2 mL of nutrient broth at a low concentration of essential oil to avoid its effect on the vegetative growth. The initial concentration was 1.5 mg/mL for all the essential oils except that of D. gossweileri (more active), which started at 0.004 mg/mL. A twofold dilution was made until the sixth dilution. Tubes were incubated for 24 h at the optimal growth temperature of each species, and the MICg and MIC were respectively determined as the lowest concentration where visible growth or discoloration of red phenol was not observed. MBCs were determined by inoculation of 100 lL of culture medium at greater than or equal to the MICs onto an agar plate and incubated for 24 h. The MBC was considered the lowest concentration of essential oils that allowed the survival of 0.01% of the initial inoculum (Oussou et al., 2008). Effect of combinations of essential oils on the vegetative growth
According to their effectiveness, essential oils of Ocimum gratissimum, Zingiber officinale, and Eugenia caryophyllus were each combined with that of D. gossweileri and tested again on the vegetative growth of each species. Combinations were carried out by the checkerboard assay described by Burt et al. (2005) with slight modifications as follows: using the macrodilution method, 2.5 mg/mL of each of the first three essential oils were prepared in the first line and 0.019 mg/mL
Table 1. Summary of the Conditions of Harvest and Identification of the Plants Used Name of plant Drypetes gossweileri S.Moore Ageratum conyzoı¨des L. Zingiber officinale Roscoe Eugenia caryophyllus (Spreng.) Bullock & S.G. Harrison Citrus limon (L.) Burm Citrus reticulata Blanco Ocimum gratissimum L. Cymbopogon citratus (DC.) Stapf. Thymus vulgaris L.
Part used
Origin
Voucher number
Period collected
Stem barks Entire plant Rhizomes Blossoms
Ntongo Ngoaekele-Yaounde Mfoundi market Yaounde
25749 SRF/CAM 9503 SRF/CAM 43125/HNC TSN: 506167
June 2011 June 2011 February 2012 June 2012
5817/SRF/CAM 48536/SRF/CAM 25746/SRF/CAM
July 2012 July 2012 July 2012 July 2012 October 2012
Pericap Pericap Fresh leaves Dry leaves Entire plant
ESSENTIAL OILS TO CONTROL BACTERIAL SPORES IN FOOD
of D. gossweileri were prepared in the first column. The dilution was consistent with the description of Burt et al. (2005) on microplate. Each species at 106 cells/mL was added and then incubated for 24 h at the growth temperature. The MICs were determined as the smallest concentrations of combined essential oils having inhibited visible growth. The MBCs were determined by agar subcultures as described above. The determined MICs were used to calculate the fraction inhibitory concentration (FIC) compared to individual MICs of essential oil with the formula shown below: FIC (A) ¼ MIC(A in presence of B) =MIC(A alone) ; FIC (B) ¼ MIC(B in presence of A) =MICs(B alone) : The FICs index were obtained as follows: FIC index (A/B) = FIC (A) + FIC (B). Combination of essential oils was classified as synergistic (FIC index £ 0.5), additive (0.5 £ FIC index £ 1.5), indifferent (FIC index = 2), and antagonist (FIC index > 2) (Bassole´ and Juliani, 2012). Treatment of spores with essential oil and evaluation of germination on solid medium
To perform this test, the method described earlier was used (Voundi et al., 2014). Fifty microliters of heatactivated spores (1.8 · 106 spores/mL) of each species were treated with each essential oil at the following concentrations: 20, 10, 5, 2.5, 1.25, and 0 mg/mL at 30C for 20 min. Treatments were done in 0.2% Tween 80 for good dilution of essential oils, then 100 lL of appropriate decimal dilution of treated samples were spread on Muller–Hinton agar. The plates were incubated for 24 h at the optimal growth temperature of each species. The numbers of colony-forming units (CFU) were counted and expressed in terms of percentage of germination inhibition of spores using the formula below: Percentage germination inhibition of spores (%) ¼ 100 (number of CFU in treated samples =number of CFU in control sample) · 100 Kinetic of germination and outgrowth of spores with essential oils
To assess the kinetic of germination and outgrowth of spores with essential oils, heat-activated spores (1.8 · 106 spores/mL) were transferred to nutrient broth (pH 7) on test tubes, supplemented with 0.2% Tween 80 and essential oils of D. gossweileri (0.002 mg/mL), Cymbopogon citratus, Ageratum conyzoı¨des, or Citrus limon (0.37 mg/mL). Spore germination and outgrowth kinetics were assessed by measuring the optical density using a Cary 100 UV-Vis– NIR spectrophotometer (Agilent Technologies, Les Ulis, France) at 620 nm (maximum absorption of the spores), every 10 min for 40 min at first and after every 1 h for 6 h after shaking (Maldonado et al., 2013). Control spores without essential oil were made under the same conditions. Optical densities recorded were used to draw the germination and growth kinetics representative curves of bacterial spores.
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Statistical analysis
Each experiment was conducted in triplicate. The results of percent germination and spectrophotometer measurement were expressed in terms of mean – SD. The difference between means obtained was made using GraphPad Prism version 5.03 for Windows (GraphPad Software, San Diego, CA). Results and Discussion Chemical composition of essential oils
The results of the chemical composition of the essential oils are shown in Table 2. The analysis showed a particular composition of D. gossweileri essential oil. This consists essentially of two aromatic compounds with nitrogen groups: benzyl isothiocyanate (86.7%) and benzylcyanide (12.6%). This composition is similar to that obtained previously by Eyele et al. (1997) with samples of D. gossweileri harvested in Gabon and Central African Republic, but differed from that obtained by Agnaniet et al. (2003) with benzylcyanide as the major compound, which was ascribed to a possible interconversion of both compounds during extraction of essential oil. The other essential oils generally showed common composition with two or three predominant compounds. Essential oil of O. gratissimum was mainly composed of thymol (47.1%), c-terpinene (16.6%), and p-cymene (14.1%). That of Z. officinale contains p-menth-1-en-4-ol (23.2%) and neral (15%). Germacreme (41.6%) and the trans-b-caryophyllene (24.6%) were predominantly present in the essential oil of A. conyzoı¨des. Limonene was the major compound of Citrus reticulata and C. limon essential oils, accounting for 74.8% and 86.4%, respectively. C. citratus essential oil was mainly composed of geranial (49.2%) and neral (34.4%). Eugenol (87.7%) was the major component of the essential oil of E. caryophyllus, while thymol (45%) and p-cimene (25%) were predominantly found in the essential oil of Thymus vulgaris. Effect of essential oils on vegetative growth and spore germination
This evaluation consisted of determining the inhibitory parameters (MIC, MICg, and MBC). The results summarized in Table 3 reveal a strong bacteriostatic effect of D. gossweileri essential oil, with MICs ranging from 0.0048 to 0.0097 mg/mL, and B. megaterium as the most sensitive microorganism. Essential oil of A. conyzoı¨des also exerted bacteriostatic effect with greater activity observed on B. subtilis, which exhibited MIC of 0.312 mg/mL, while MICs of 1.25 and 2.5 mg/mL were obtained from the other essential oils. The bacteriostatic effects of essential oils were due to the presence of antimicrobial compounds in major proportion in most essential oils analyzed. Indeed, thymol, caryophyllene, limonene, geranial, eugenol, and benzyl isothiocyanate are well known for their strong antibacterial effects (Hyldgaard et al., 2012). The essential oil of D. gossweileri was more active with MIC £ 0.0097 mg/mL. This MIC value was less than those previously obtained by Ngono (2008), ranging within 48.8– 390.6 lg/mL on a panel of Gram-negative bacteria. This confirms the greater susceptibility of Gram-positive bacteria to essential oils than Gram negative, as reported by many studies (Hyldgaard et al., 2012). The strong antibacterial activity of D. gosssweileri essential oil is probably related to
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Table 2. Chemical Composition of Essential Oils Determined by GC and GC-MS (%) No.
K.I.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62
928 929 936 948 977 982 985 992 994 1020 1033 1035 1039 1040 1049 1064 1071 1070 1093 1105 1101 1121 1155 1160 1165 1173 1182 1184 1186 1197 1208 1248 1252 1255 1260 1262 1264 1274 1285 1287 1293 1294 1299 1309 1310 1315 1347 1358 1389 1419 1435 1452 1468 1493 1495 1500 1504 1508 1510 1512 1517 1520
Compounds (%) a-Pinene a-Thurjene Camphene Benzaldehyde Sabinene Octene-2-ol (3E) b-Pinene a-Phellandrene Myrcene a-Terpinene P-Cimene 1,8-Cineole Limonene (Z)-b-Ocimene (E)-b-Ocimene c-Terpinene Linallool Terpinolene Pyrazine (methylthio) Phenylacetaldehyde alcohol Thujone Benzylcyanide Cryptone Camphor b-Terpineol Borneol Decanal Terpinen-4-ol a-Terpineol Citronellol Citral Ascaridol Linalol Neral Decenal (2E) Pinane-2,3-epoxy Geraniol 2-Undecanone p-Menth-1-en-4-ol Geranial Menthyl acetate Ment-1-9-ol Geranyl acetate Thymol Carvacrol Eugenol Citronellyl acetate Benzyl isothiocyanate a-Copaene b-Ylangene b-Trans caryophyllene Trans-b-farnesene b-Ionone c-Muurolene Germacreme b-Bisabolene D-Cadinene a-Zingiberene cis-c-Bisabolene Cardinene a-Sesquiphellandrene cis-Nerolidol
D.g.
O.g.
Z.o.
A.c.
C.r.
C.l.
C.c
E.c.
T.v.
— — — 0.7 — — — — — — — — — — — — — — — — — 12.6 — — — — — — — — — — — — — — — — — — — — — — — — — 86.7 — — — — — — — — — — — — — —
4.1 — 1.2 — — — 0.6 3.7 0.3 0.8 14.0 — 1.1 0.5 0.3 16.6 — — — — — — — — — 0.5 — 1.1 0.4 — — — — — — — — — — — — — — 47.1 0.6 — — — 0.4 — 0.6 — — — — — 0.9 — — 0.3 — —
2.6 0.7 9.6 — — — 0.3 1.1 0.3 0.3 — 7.0 3.7 — — — 2.2 — 0.7 — — — 0.7 — — 0.2 0.3 3.6 0.5 1.2 — — — 15.0 — 1.0 1.1 — 23.2 0.8 — — — 0.9 — — — — — — — — — 3.1 — — — 7.6 — 0.7 1.9 1.6
0.7 — 1.8 — — — — 0.7 — 0.9 1.2 — 0.5 — — — — — — — — — — — — — — — — — — — — — — — — — — — 2.1 — — — — — — — — — 24.6 0.4 4.7 — 41.6 2.9 — — 1.4 1.3 — —
0.8 — — — 1.1 — 1.1 1.1 — — — — 74.8 — — 7.3 — — — — 1.4 — — — — — — 1.8 — 2.8 — — — — — — — — — 0.4 — 0.9 — — — 1.9 1.0 — — 4.1 — — — — — — — — — — — —
0.5 — — — 1.7 — 2.7 — — — — — 86.4 — — — — 0.4 — — 0.8 — — — — — — 3.0 — 1.3 0.3 — — — — — 0.3 0.6 — — — — — — 0.8 0.2 — — — — 0.4 — — — — — — — — — — —
0.4 — — — 0.7 1.0 — — 5.9 — — — 0.6 — — — — — — — — — — 0.3 — 0.4 — 0.6 0.9 — — — 1.0 34.4 0.5 — 1.8 — — 49.2 — — 0.4 — — — — — — — — — — — 0.4 — — — — — — —
— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 0.3 — — — — — — — — — — — — 87.7 — — — — 4.3 — — — — — — — — — — —
0.8 0.5 1.0 — 5.3 — 1.1 1.0 0.9 — 25.0 — 2.6 — — — 0.3 — — 4.2 — — — — 0.5 0.7 — 2.0 — — — 1.3 — — — — — — — — — — — 45.5 0.4 3.0 — — — — 1.7 — — — — — — — — — — —
(continued)
ESSENTIAL OILS TO CONTROL BACTERIAL SPORES IN FOOD
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Table 2. (Continued) No.
K.I.
Compounds (%)
D.g.
O.g.
Z.o.
A.c.
C.r.
C.l.
C.c
E.c.
T.v.
63 64 65 66 67 68 69 70 71 72 73
1534 1536 1542 1543 1561 1572 1595 1602 1606 1609 1620
Elemol b-Thujaplicinol Sesquisabinene hydrate (trans) Dihydro eugenol acetate b-Bisabolol Geranyl isobutanoate Zingiberenol Caryophyllene oxide Eudesmol 2-Acetyl-naphtalene b-Cedene epoxide
— — — — — — — — — — —
— — — — — — — — 1.6 — —
3.2 — 0.7 — 0.5 — 0.3 — — — —
— — — — — — 3.4 0.5 — — —
— — — — — — — — — — —
— — — — — 0.5 — — — — —
— — — — — — — — — — —
— — — 6.5 — — — — — 0.2 1.3
— 0.4 — — — — — 1.3 — — —
K.I., Kovats Index; D.g., Drypetes gossweileri; O.g., Ocimum gratissimum; Z.o., Zingiber officinale; A.c., Ageratum conyzoı¨des; C.r., Citrus reticulata; C.l., Citrus limon; C.c., Cymbopogon citratus; E.c., Eugenia caryophyllus; T.v., Thymus vulgaris. GC, gas chromatography; GC-MS, gas chromatography coupled to mass spectrometry.
its isothiocyanate derivatives composition, known to have a very strong antimicrobial effect due to the R-N = C = S group in the molecule. This group has a high electrophilic central carbon, which can easily react with nucleophilic centers. Furthermore, it could cleave the disulfide bonds of proteins and could attack free amino acids by oxidative reaction (Wilson, 2011; Hyldgaard et al., 2012). Concerning the inhibitory effect of spore germination, essential oil of D. gossweileri appeared to be the most active, with inhibitory effect at 0.001 mg/mL on B. subtilis spores and 0.002 mg/mL on spores of the other Bacillus and G. stearothermophilus. For the other essential oils, the MICg ranged from 0.09 to 1.5 mg/mL, with essential oils of C. limon, C. citratus, and A. conyzoı¨des seeming to be more active, with MICg £ 0.37 on at least two of the four species
tested. The MICg of 0.37–1.5 mg/mL are close to those obtained by Chaibi et al. (1997) on spores of B. cereus T, with the essential oils of some Moroccan plants. However, the MICg shown by D. gossweileri essential oil were generally lower, further confirming its strong antimicrobial activity. On the whole, MICg were much lower than MICs (Table 2), reflecting the fact that the inhibitory action of essential oils is more effective on germination of spores compared to vegetative growth. Regarding the bactericidal effect, essential oils of C. reticulata, C. citratus, and E. caryophyllus generally showed an absolute effect. MBCs were observed at MIC concentration of 2.5 mg/mL. O. gratissimum and Z. officinale were also bactericidal but at higher concentrations, generally at 10 mg/mL.
Table 3. Minimal Inhibitory, Minimal Bactericidal (MICs and MBCs), and Minimal Concentrations of Essential Oils Inhibiting Vegetative Growth and Spores Germination Determined by Macrodilution Essential oils Drypetes gossweileri Ocimum gratissimum Zingiber officinale Ageratum conyzoı¨des Citrus reticulata Citrus limon Cymbopogon citratus Eugenia caryophyllus Thymus vulgaris
Parameter of inhibition (mg/mL)
Bacillus cereus
Bacillus megaterium
Bacillus subtilis
Geobacillus stearothermophilus
MIC/MBC MICg MIC/MBC MICg MIC/MBC MICg MIC/MBC MICg MIC/MBC MICg MIC/MBC MICg MIC/MBC MICg MIC/MBC MICg MIC/MBC MICg
0.009/– 0.002 2.5/5 1.5 0.63/2.5 0.75 1.25/– 0.37 –/– 1.5 1.25/– 0.75 5.0/5.0 0.09 2.5/2.5 – 1.25/– 0.37
0.004/– 0.002 1.25/10 0.75 1.25/10 0.75 2.5/– 0.09 2.5/2.5 0.75 2.5/– 0.37 2.5/2.5 0.37 2.5/– 0.75 1.25/– 1.5
0.009/– 0.001 2.5/10 0.75 0.63/10 – 0.312/– 0.09 2.5/– 1.5 1.25/– 0.37 2.5/– 0.37 2.5/2.5 0.75 1.25/– 0.75
0.009/– * 1.25/10 * 0.63/10 * 1.25/– * 2.5/2.5 * 2.5/– * 2.5/– * 2.5/2.5 * 1.25/– *
MIC, minimal inhibitory concentration; MBC, minimal bactericidal concentration; MICg, minimal concentration inhibiting germination of spores; (–), inactive; (*), not evaluated.
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Table 4. Effect of Combined Essential Oils on Vegetative Growth of Bacillus and Geobacillus stearothermophilus Essential oil combinations
Inhibitory parameters (mg/mL)
Bacillus cereus
Bacillus megaterium
Bacillus subtilis
G. stearothermophilus
Drypetes gossweileri/Zingiber officinale
MIC1; MIC2 MBC1; MBC2 FIC1 + FIC2 = FICindex Type of effect MIC1; MIC2 MBC1;; MBC2 FIC1 + FIC2 = FICindex Type of effect MIC1; MIC2 MBC1; MBC2 FIC1 + FIC2 = FICindex Type of effect MIC1; MIC2 MBC1; MBC2 FIC1 + FIC2 = FICindex Type of effect
0.0024; 0.31 — 0.25 + 0.5 = 0.75 Additive 0.0048; 0.62 0.0048; 0.62 0.5 + 0.5 = 1 Additive 0.0048; 0.62 0.0048; 0.62 0.5 + 0.25 = 0.75 Additive 0.0048; 0.62 0.0097; 1.25 0.5 + 0.25 = 0.75 Additive
0.0024; 0.31 — 0.5 + 0.25 = 0.75 Additive 0.0048; 0.62 — 1 + 0.25 = 1.25 Additive 0.0048; 0.62 — 1 + 0.5 = 1.5 Additive 0.0048; 0.62 — 1 + 0.25 = 1.5 Additive
0.0048; 0.62 0.0048; 0.62 0.5 + 1 = 1.5 Additive 0.0048; 0.62 — 0.5 + 1 = 1.5 Additive 0.0024; 0.31 — 0.25 + 0.12 = 0.37 Synergistic 0.0024; 0.31 — 0.25 + 0.12 = 0.37 Synergistic
0.0048; 0.62 0.0048; 0.62 0.5 + 1 = 1.5 Additive 0.0024; 0.31 0.0048; 0.62 0.25 + 0.25 = 0.5 Synergistic 0.0097; 1.25 0.0097; 1.25 1+1=2 Additive 0.0024; 0.31 0.0048; 0.62 0.5 + 0.12 = 0.37 Synergistic
Drypetes gossweileri/Ageratum conyzoı¨des D. gossweileri/Ocimum gratissimum D. gossweileri/Eugenia caryophyllus
(—), inactive; MIC1, minimal inhibitory concentration of first essential oil in combination; MIC2, minimal inhibitory concentration of second essential oil in combination; MBC1, minimal bactericidal concentration of first essential oil in combination; MBC2, minimal bactericidal concentration of second essential oil in combination; FIC1, fractions inhibitory concentration of first essential oil; FIC2, fractions inhibitory concentration of second essential oil; FICindex, fractions inhibitory concentration index.
FIG. 1. Percentage germination inhibition of spores obtained on solid medium after treatment with essential oils.
ESSENTIAL OILS TO CONTROL BACTERIAL SPORES IN FOOD
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Effect of combination of essential oils on vegetative growth
The effect of the combination of essential oils is summarized in Table 4. The results obtained show an improvement of the antibacterial effect due to the combinations of D. gossweileri and E. caryophyllus, A. conyzoı¨des, or O. gratissimum essential oils, as shown by a decrease in MICs. Furthermore, all the combinatory effects appeared to be either additive or synergistic. Combination of D. gossweileri and E. caryophyllus proved significant activity with a synergistic effect on two (B. subtilis and G. stearothermophilus) of the four species. Synergy or additive effects observed could be explained by the combined effect of benzyl isothiocyanate, major compound of D. gossweileri, and antimicrobial compounds found in high percentage in the others essential oils such as thymol, neral, trans-b-caryophyllene, and eugenol. In addition, the increased effect of the combination could be explained by the synergistic effect of benzyl isothiocyanate and the terpene hydrocarbons within the other essential oils. Nguefack et al. (2012) reported that terpene hydrocarbons have low effect, but could swell cell membranes and enable more antimicrobial compounds such as benzyl isothiocyanate to be easily transported into the cell. Treatment of spores with essential oil and evaluation of germination inhibition on solid medium
Figure 1 shows the curves representing the percentage germination inhibition of spores, at different concentrations of essential oils. The essential oil of D. gossweileri was the most effective. At 1.25 mg/mL, this oil was active on B. cereus, B. megaterium, and G. sterotherophilus spores, with germination inhibition percentages of 66.23, 48.54, and 51.66%, respectively. Furthermore, a total inhibition of germination was observed on all the spores at 20 mg/mL. The other essential oils had effect but at higher concentrations ranging from 10 to 20 mg/mL. However, Z. officinale essential oil was found to be active on spores of B. subtilis, with a total inhibition at 2.5 mg/mL. O. gratissimum was more
FIG. 2. Germination, outgrowth, and growth kinetic of spores of Bacillus cereus in the presence of essential oils. OD, optical density.
FIG. 3. Germination, outgrowth, and growth kinetic of spores of Bacillus megaterium in the presence of essential oils. OD, optical density. active on B. cereus spores where significant activity was obtained at 2.5 mg/mL and 5 mg/mL. These results might suggest a probable irreversible effect of these three essential oils as compared to others. Kinetics of spores germination and outgrowth with and without essential oils
The kinetics of the germination and outgrowth of spores of B. cereus, B. megaterium, and B. subtilis were performed in the absence (control) and the presence of essential oils using spectrophotometric measurements. Figure 2 shows the kinetics obtained with spores of B. cereus. The curves show a constancy of optical densities between 0 and 120 min for both control and tested spores. At 120 and 300 min, respectively, for control and spores in the presence of C. limon essential
FIG. 4. Germination, outgrowth, and growth kinetic of spores of Bacillus subtilis in the presence of essential oils. OD, optical density.
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oil, an increase of optical densities was recorded due to the start of vegetative growth. This observation was not revealed with spores in the presence of the other essential oils, suggesting that these essential oils probably inhibited the germination and/or outgrowth of spores. Complete inhibition of spore germination was observed in the presence of the essential oil of A. conyzoı¨des because the optical densities remained constant during the test period. Concerning the essential oils of D. gossweileri and C. citratus, slight decreases in optical densities were recorded after 100 and 180 min, respectively, characterizing spore germination. However, after 200 min, an increase in optical densities was observed, suggesting that these germinated spores might return to their initial state of dormancy. This phenomenon called microcycle of spores was already observed with B. cereus spore by Edima et al. (2010). Figures 3 and 4 show that the kinetics conducted on spores of B. megaterium and B. subtilis, respectively, revealed that from 0 to 40 min, the optical densities recorded are almost constant with control and tested spores. This period probably corresponds to the lag phase. From 60 min for the control and the spores with D. gossweileri essential oil, a decrease of optical densities was observed, probably due to the germination of spores. This germination step starts at 120 min with spores in the presence of the other essential oils. After 180 min, an exponential growth only with the control was observed, probably due to the start of vegetative growth. This observation is not shown with spores in the presence of essential oils, suggesting that the essential oil prevented the germination of spores. Conclusions
Essential oils used in this work have displayed the capacity to inhibit germination as well as vegetative growth of spores of Bacillus and G. stearothermophilus. Among the essential oils tested, D. gossweileri appears to be more active as compared to others. Furthermore, its combination with other essential oils improves the antibacterial effect. Thus, D. gossweileri essential oil could be suggested as a food preservative. Nevertheless, further studies must be conducted upon other bacterial spores. Acknowledgments
We are grateful to Professor Chantal Menut of the Institut des Biomole´cules Max Mousseron (IBMM) de Montpellier, the University of Yaounde I, and the Vasile Alecsandri University of Bacau for laboratory facilities. We thank the Romanian Government, the Agence Universitaire de la Francophonie (AUF), and the Service de la Cooperation et de l’Action Culturelle (French Embassy in Cameroon) for funding this project. We also thank the Microbiology Laboratory Institute of Food Research of Reading and the Institut Appert, Paris for providing us with bacterial spore strains. Disclosure Statement
No competing financial interests exist. References
Agnaniet H, Mounzeo H, Menut C, Bessie`re JM, Criton M. The essential oil of Rinorea subintegrifolia O.Ktze and Drypetes
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gossweileri S.Moore occurring in Gabon. Flavour Fragrance J 2003;18:207–210. Aouadhi C, Simonin H, Pre´vost H, De Lamballerie M, Maaroufi A, Mejri S. Inactivation of Bacillus sporothermodurans LTIS27 spores by high hydrostatic pressure and moderate heat studied by response surface methodology. LWT Food Sci Technol 2013;50:50–56. Bassole´ HN, Juliani HR. Essential oils in combination and their antimicrobial properties. Molecules 2012;17:3989–4006. Bayoı¨ JR, Djoulde DR, Bogne KP, Nyegue MA, Voundi OS, Bakary D, Etoa F-X. Activity of acetic acid on Bacillus stearothermophilus and Bacillus subtilis spores after sublethal pretreatments. Int J Innovative Sci Res 2014;10:570–575. Burt S, Vlielander R, Haagsman H, Veldhuizen E. Increase in activity of essential oil components carvacrol and thymol against Escherichia coli O157:H7 by addition of food stabilizers. J Food Prot 2005;68:919–926. Chaibi A, Ababouch LH, Belasri K, Boucetta S, Busta FF. Inhibition of germination and vegetative growth of Bacillus cereus T and Clostridium botulinum 62 A spores by essential oils. Food Microbiol 1997;14:161–174. Edima HC, Tadsadjieu LN, Mbofung CM, Etoa FX. Antibacterial profile of some beers and their effect on some selected pathogens. African J Biotechnol 2010;9:5938–5945. Etoa FX, Adegoke GO. Evolution of thermal resistance of spores of two Bacillus species in pepper and honey. Sci Aliments 1996;16:49–59. Eyele MC, Menut C, Bessie`re JM, Lamaty G, Nze´ EL, Denamganai J. Aromatic plants of tropical Central Africa. xxix. Benzyl isothiocyanate as major constituent of bark essential oil of Drypetes gossweileri S. Moore. J Essential Oil Res 1997;9:367–370. Gachkar L, Yadegari D, Bagher M, Taghizadeh M. Chemical and biological characteristics of Cuminum cyminum and Rosmarinus officinalis essential oils. Food Chem 2007;102: 898–904. Heyndrickx M. The importance of endospore-forming bacteria originating from soil for contamination of industrial food processing. Appl Environ Soil Sci 2011;2011:1–11. Hong A, To E, Fakhry S, Baccigalupi L, Ricca E, Cutting SM. Defining the natural habitat of Bacillus spore-formers. Res Microbiol 2009;160:375–379. Huertas JP, Esteban MD, Antolinos V, Palop A. Combined effect of natural antimicrobials and thermal treatments on Alicyclobacillus acidoterrestris spores. Food Control 2014; 35:73–78. Hyldgaard M, Mygind T, Meyer R. Essential oils in food preservation: Mode of action, synergies, and interactions with food matrix components. Frontiers Microbiol 2012;3:1–12 Islam MS, Inoue A, Igura N, Shimoda M, Hayakawa I. Inactivation of Bacillus spores by the combination of moderate heat and low hydrostatic pressure in ketchup and potage. Int J Food Microbiol 2006;107:124–130. Maldonado MC, Aban MP, Navarro AR. Chemicals and lemon essential oil effect on Alicyclobacillus acidoterrestris viability. Braz J Microbiol 2013;44:1133–1137. Mohammedi Z. Etude du pouvoir antimicrobien et antioxydant des huiles essentielles et flavonoı¨des de quelques plantes de la re´gion de Tlemcen. [Magiste`re dissertation]. University Abou Bakr Belkaı¨d of Tlemcen. 2006, (In French.) Moni NE. Etiologie bacte´rienne des gastroente´rites infantiles et activite´ antibacte´rienne de quelques huiles essentielles et leurs combinaisons. [Me´moire de Master II]. Universite´ de Yaounde I, Cameroun. 2013. (In French.)
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Ndoye´ C. Etude chimique et e´valuation des proprie´te´s anti radicalaire et antioxydante des huiles essentielles d’ espe`ces aromatiques tropicales en provenance d’Est du Cameroun. [The`se de Doctorat 3e`me Cycle]. Universite´ de Montpellier II, France, 2001. (In French.) Ngono EF. Evaluation des activites antibacterienne et antiradicalaire in vitro des huiles essentielles de Drypetes gossweileri et Pentadiplandra brazzeana. [Me´moire de Master II]. Universite´ de Yaounde I, Cameroun, 2008. (In French.) Nguefack J, Tamgue O, Dongmo JBL, Dakole CD, Leth V, Vismer HF, Amvam PH, Nkenfack AE. Synergistic action between fractions of essential oils from Cymbopogon citratus, Ocimum gratissimum and Thymus vulgaris against Penicillium expansum. Food Control 2012;23:377–383. Nyegue MA. Proprie´te´ chimiques et biologiques des huiles essentielles de quelques plantes Aromatiques et/ou me´dicinales du Cameroun: Evaluation de leurs activite´s anti radicalaires, anti-inflammatoire et antimicrobiennes. [The`se de Doctorat 3e`me Cycle]. Universite´ de Montpellier II, France. 2006. (In French.) Oussou KR, Yolou S, Boti JB, Guessennd KN, Kanko C, Ahibo C, Casanova J. E´tude chimique et activite´ antidiarrheique des huiles essentielles de deux plantes aromatiques de la pharmacope´e ivoirienne. Eur J Sci Res 2008;24:94–103.
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Address correspondence to: Ste`ve Olugu Voundi, PhD Candidate Department of Microbiology Laboratory of Microbiology University of Yaounde I P.O. Box 812 Yaounde, Cameroon E-mail: [email protected]
Effect of Essential Oils on Germination and Growth of Some Pathogenic and Spoilage Spore-Forming Bacteria Ste`ve Olugu Voundi,1,2 Maximilienne Nyegue,1,2 Iuliana Lazar,3 Dumitra Raducanu,4 Florentine Foe Ndoye,2 Marius Stamate,5 and Franc¸ois-Xavier Etoa1
Abstract
The use of essential oils as a food preservative has increased due to their capacity to inhibit vegetative growth of some bacteria. However, only limited data are available on their effect on bacterial spores. The aim of the present study was to evaluate the effect of some essential oils on the growth and germination of three Bacillus species and Geobacillus stearothermophilus. Essential oils were chemically analyzed using gas chromatography and gas chromatography coupled to mass spectrometry. The minimal inhibitory and bactericidal concentrations of vegetative growth and spore germination were assessed using the macrodilution method. Germination inhibitory effect of treated spores with essential oils was evaluated on solid medium, while kinetic growth was followed using spectrophotometry in the presence of essential oils. Essential oil from Drypetes gossweileri mainly composed of benzyl isothiocyanate (86.7%) was the most potent, with minimal inhibitory concentrations ranging from 0.0048 to 0.0097 mg/mL on vegetative cells and 0.001 to 0.002 mg/mL on spore germination. Furthermore, essential oil from D. gossweileri reduced 50% of spore germination after treatment at 1.25 mg/mL, and its combination with other oils improved both bacteriostatic and bactericidal activities with additive or synergistic effects. Concerning the other essential oils, the minimal inhibitory concentration ranged from 5 to 0.63 mg/mL on vegetative growth and from 0.75 to 0.09 mg/mL on the germination of spores. Spectrophotometric evaluation showed an inhibitory effect of essential oils on both germination and outgrowth. From these results, it is concluded that some of the essential oils tested might be a valuable tool for bacteriological control in food industries. Therefore, further research regarding their use as food preservatives should be carried out.
Introduction
G
rowth and contamination control of sporeforming bacteria remain an important objective of food industries. Indeed, bacterial spores, due to their ubiquitous character, are distributed in soil, water, and plants, so they can easily contaminate and spoil food (Hong et al., 2009; Heyndrickx, 2011). Furthermore, since spores are very resistant to physical and chemical agents, they can remain in food after sterilization (Aouadhi et al., 2013). Some spores can grow at a pH range of 3.0 to 3.5 and at low ( < 4C) or high ( £ 60C) temperatures depending on the species. Moreover, some spore-forming bacteria species such as Bacillus cereus and Geobacillus stearothermophilus are well known for their ability to cause food poisoning and spoilage, respectively (Etoa and Adegoke, 1996; Shaheen et al., 2006).
To prevent spore contamination, industries usually use high temperatures to sterilize. However, this treatment generally leads to the degradation of nutritional and organoleptic qualities of food (Islam et al., 2006). Thus, an alternative strategy is to use chemical substances with antimicrobial activities, provided that they are nontoxic. This explains all the renewed interest directed toward plant essential oils. Currently, these substances are used in aromatherapy and as additives to add flavor and preserve various foods due to their organoleptic and antibacterial properties (Ndoye, 2001; Mohammedi, 2006; Huertas et al., 2014). However, efficient use of essential oils as food preservatives requires a good knowledge about their properties (i.e., the chemical composition, the minimal inhibitory and the bactericidal concentrations [MIC and MBC], the panel of target microorganisms, and the mechanism(s) of action). To the present, many studies have been done on the chemical
1 Department of Microbiology, Laboratory of Microbiology, and 2Department of Biochemistry, Laboratory of Phytobiochemistry and Medicinal Plant Study, University of Yaounde I, Yaounde, Cameroon. 3 Departments of Food and Chemical Engineering, 4Biology, Ecology, and Environment Protection, and 5Mechanical and Environmental Engineering, Vasile Alecsandri University of Bacau, Bacau, Romania.
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composition and inhibitory effect of essential oils on the growth of a wide range of bacteria, particularly on their vegetative forms, but few have been done on their effect on bacterial spores. Nevertheless, the study of Chaibi et al. (1997) assessed the effect of essential oils extracted from Moroccan plants on spores and vegetative cells of B. cereus. The results showed an inhibitory effect on one or more stages of spore germination with sporostatic or sporocidal effect. This study, which was focused on the mechanism(s) of action of essential oils on bacterial spores, might be extended to other essential oils and more Bacillus species. The purpose of this work was to evaluate the effect of nine essential oils extracted from Cameroonian aromatic plants on growth, germination, and outgrowth of three Bacillus species and G. stearothermophilus. Materials and Methods Plant material, extraction, and chemical analyses of essential oils
For essential oil extraction, nine aromatic plants were harvested or purchased and identified at the National Herbarium of Cameroon (Table 1). Essential oil extractions were done by hydrodistillation using a Clevenger apparatus, according to the protocol described by Moni (2013). Oils were analyzed by gas chromatography–flame ionization detector and GC coupled with mass spectrometry as described earlier by Nyegue (2006). Bacterial strains, production of vegetative cells and spores
B. cereus T, B. subtilis NCTC 3610, and B. megaterium 8174 came from the collection of The Microbiology Laboratory Institute of Food Research of Reading, UK. G. stearothermophilus CNCH 5781 was obtained from the Institut Appert, Paris, France. These strains were provided in the form of spores. To obtain vegetative cells, spores were subjected to activation at 80C for 10 min. They were then grown on nutrient agar for 24 h at the optimal growth temperature of each species (35C for B. cereus, B. subtilis, and B. megaterium and 63C for G. stearothermophilus). For spore production, cells grown for 12 h on trypticase soy broth were spread on sporulation agar medium as described by Bayoı¨ et al. (2014). The plates were incubated at the aforementioned temperatures for 5 days. The spores were collected with a glass spatula, suspended in distilled water, and purified by many centrifugation cycles at 3000 · g for 15 min.
The purified spores were stored at 4C for 3 months to ensure stability of spores before use (Voundi et al., 2014). Macrodilution method
Using this method, minimal inhibitory concentration (MIC), minimal bactericidal (MBC) and minimal inhibitory concentrations for inhibition of spore germination (MICg) were assessed. The method on test tubes described by Gachkar et al. (2007) was used with the following modifications: for MIC determination, vegetative forms of Bacillus and G. stearothermophilus at 106 cells/mL were allowed to grow in 2 mL of nutrient broth (supplemented with 10 g/L of glucose, 0.2% Tween 80, and 0.05 mg/L of red phenol as growth indicator) at different concentrations of each essentials oil. The initial concentration was 20 mg/mL followed by a geometric progression of 1/2 until the 8th dilution for all the essential oils except for that of Drypetes gossweileri that went up to the 16th dilution. For MICg determination, heat-activated spores (80C, 10 min) at 106 spores/mL were inoculated in 2 mL of nutrient broth at a low concentration of essential oil to avoid its effect on the vegetative growth. The initial concentration was 1.5 mg/mL for all the essential oils except that of D. gossweileri (more active), which started at 0.004 mg/mL. A twofold dilution was made until the sixth dilution. Tubes were incubated for 24 h at the optimal growth temperature of each species, and the MICg and MIC were respectively determined as the lowest concentration where visible growth or discoloration of red phenol was not observed. MBCs were determined by inoculation of 100 lL of culture medium at greater than or equal to the MICs onto an agar plate and incubated for 24 h. The MBC was considered the lowest concentration of essential oils that allowed the survival of 0.01% of the initial inoculum (Oussou et al., 2008). Effect of combinations of essential oils on the vegetative growth
According to their effectiveness, essential oils of Ocimum gratissimum, Zingiber officinale, and Eugenia caryophyllus were each combined with that of D. gossweileri and tested again on the vegetative growth of each species. Combinations were carried out by the checkerboard assay described by Burt et al. (2005) with slight modifications as follows: using the macrodilution method, 2.5 mg/mL of each of the first three essential oils were prepared in the first line and 0.019 mg/mL
Table 1. Summary of the Conditions of Harvest and Identification of the Plants Used Name of plant Drypetes gossweileri S.Moore Ageratum conyzoı¨des L. Zingiber officinale Roscoe Eugenia caryophyllus (Spreng.) Bullock & S.G. Harrison Citrus limon (L.) Burm Citrus reticulata Blanco Ocimum gratissimum L. Cymbopogon citratus (DC.) Stapf. Thymus vulgaris L.
Part used
Origin
Voucher number
Period collected
Stem barks Entire plant Rhizomes Blossoms
Ntongo Ngoaekele-Yaounde Mfoundi market Yaounde
25749 SRF/CAM 9503 SRF/CAM 43125/HNC TSN: 506167
June 2011 June 2011 February 2012 June 2012
5817/SRF/CAM 48536/SRF/CAM 25746/SRF/CAM
July 2012 July 2012 July 2012 July 2012 October 2012
Pericap Pericap Fresh leaves Dry leaves Entire plant
ESSENTIAL OILS TO CONTROL BACTERIAL SPORES IN FOOD
of D. gossweileri were prepared in the first column. The dilution was consistent with the description of Burt et al. (2005) on microplate. Each species at 106 cells/mL was added and then incubated for 24 h at the growth temperature. The MICs were determined as the smallest concentrations of combined essential oils having inhibited visible growth. The MBCs were determined by agar subcultures as described above. The determined MICs were used to calculate the fraction inhibitory concentration (FIC) compared to individual MICs of essential oil with the formula shown below: FIC (A) ¼ MIC(A in presence of B) =MIC(A alone) ; FIC (B) ¼ MIC(B in presence of A) =MICs(B alone) : The FICs index were obtained as follows: FIC index (A/B) = FIC (A) + FIC (B). Combination of essential oils was classified as synergistic (FIC index £ 0.5), additive (0.5 £ FIC index £ 1.5), indifferent (FIC index = 2), and antagonist (FIC index > 2) (Bassole´ and Juliani, 2012). Treatment of spores with essential oil and evaluation of germination on solid medium
To perform this test, the method described earlier was used (Voundi et al., 2014). Fifty microliters of heatactivated spores (1.8 · 106 spores/mL) of each species were treated with each essential oil at the following concentrations: 20, 10, 5, 2.5, 1.25, and 0 mg/mL at 30C for 20 min. Treatments were done in 0.2% Tween 80 for good dilution of essential oils, then 100 lL of appropriate decimal dilution of treated samples were spread on Muller–Hinton agar. The plates were incubated for 24 h at the optimal growth temperature of each species. The numbers of colony-forming units (CFU) were counted and expressed in terms of percentage of germination inhibition of spores using the formula below: Percentage germination inhibition of spores (%) ¼ 100 (number of CFU in treated samples =number of CFU in control sample) · 100 Kinetic of germination and outgrowth of spores with essential oils
To assess the kinetic of germination and outgrowth of spores with essential oils, heat-activated spores (1.8 · 106 spores/mL) were transferred to nutrient broth (pH 7) on test tubes, supplemented with 0.2% Tween 80 and essential oils of D. gossweileri (0.002 mg/mL), Cymbopogon citratus, Ageratum conyzoı¨des, or Citrus limon (0.37 mg/mL). Spore germination and outgrowth kinetics were assessed by measuring the optical density using a Cary 100 UV-Vis– NIR spectrophotometer (Agilent Technologies, Les Ulis, France) at 620 nm (maximum absorption of the spores), every 10 min for 40 min at first and after every 1 h for 6 h after shaking (Maldonado et al., 2013). Control spores without essential oil were made under the same conditions. Optical densities recorded were used to draw the germination and growth kinetics representative curves of bacterial spores.
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Statistical analysis
Each experiment was conducted in triplicate. The results of percent germination and spectrophotometer measurement were expressed in terms of mean – SD. The difference between means obtained was made using GraphPad Prism version 5.03 for Windows (GraphPad Software, San Diego, CA). Results and Discussion Chemical composition of essential oils
The results of the chemical composition of the essential oils are shown in Table 2. The analysis showed a particular composition of D. gossweileri essential oil. This consists essentially of two aromatic compounds with nitrogen groups: benzyl isothiocyanate (86.7%) and benzylcyanide (12.6%). This composition is similar to that obtained previously by Eyele et al. (1997) with samples of D. gossweileri harvested in Gabon and Central African Republic, but differed from that obtained by Agnaniet et al. (2003) with benzylcyanide as the major compound, which was ascribed to a possible interconversion of both compounds during extraction of essential oil. The other essential oils generally showed common composition with two or three predominant compounds. Essential oil of O. gratissimum was mainly composed of thymol (47.1%), c-terpinene (16.6%), and p-cymene (14.1%). That of Z. officinale contains p-menth-1-en-4-ol (23.2%) and neral (15%). Germacreme (41.6%) and the trans-b-caryophyllene (24.6%) were predominantly present in the essential oil of A. conyzoı¨des. Limonene was the major compound of Citrus reticulata and C. limon essential oils, accounting for 74.8% and 86.4%, respectively. C. citratus essential oil was mainly composed of geranial (49.2%) and neral (34.4%). Eugenol (87.7%) was the major component of the essential oil of E. caryophyllus, while thymol (45%) and p-cimene (25%) were predominantly found in the essential oil of Thymus vulgaris. Effect of essential oils on vegetative growth and spore germination
This evaluation consisted of determining the inhibitory parameters (MIC, MICg, and MBC). The results summarized in Table 3 reveal a strong bacteriostatic effect of D. gossweileri essential oil, with MICs ranging from 0.0048 to 0.0097 mg/mL, and B. megaterium as the most sensitive microorganism. Essential oil of A. conyzoı¨des also exerted bacteriostatic effect with greater activity observed on B. subtilis, which exhibited MIC of 0.312 mg/mL, while MICs of 1.25 and 2.5 mg/mL were obtained from the other essential oils. The bacteriostatic effects of essential oils were due to the presence of antimicrobial compounds in major proportion in most essential oils analyzed. Indeed, thymol, caryophyllene, limonene, geranial, eugenol, and benzyl isothiocyanate are well known for their strong antibacterial effects (Hyldgaard et al., 2012). The essential oil of D. gossweileri was more active with MIC £ 0.0097 mg/mL. This MIC value was less than those previously obtained by Ngono (2008), ranging within 48.8– 390.6 lg/mL on a panel of Gram-negative bacteria. This confirms the greater susceptibility of Gram-positive bacteria to essential oils than Gram negative, as reported by many studies (Hyldgaard et al., 2012). The strong antibacterial activity of D. gosssweileri essential oil is probably related to
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Table 2. Chemical Composition of Essential Oils Determined by GC and GC-MS (%) No.
K.I.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62
928 929 936 948 977 982 985 992 994 1020 1033 1035 1039 1040 1049 1064 1071 1070 1093 1105 1101 1121 1155 1160 1165 1173 1182 1184 1186 1197 1208 1248 1252 1255 1260 1262 1264 1274 1285 1287 1293 1294 1299 1309 1310 1315 1347 1358 1389 1419 1435 1452 1468 1493 1495 1500 1504 1508 1510 1512 1517 1520
Compounds (%) a-Pinene a-Thurjene Camphene Benzaldehyde Sabinene Octene-2-ol (3E) b-Pinene a-Phellandrene Myrcene a-Terpinene P-Cimene 1,8-Cineole Limonene (Z)-b-Ocimene (E)-b-Ocimene c-Terpinene Linallool Terpinolene Pyrazine (methylthio) Phenylacetaldehyde alcohol Thujone Benzylcyanide Cryptone Camphor b-Terpineol Borneol Decanal Terpinen-4-ol a-Terpineol Citronellol Citral Ascaridol Linalol Neral Decenal (2E) Pinane-2,3-epoxy Geraniol 2-Undecanone p-Menth-1-en-4-ol Geranial Menthyl acetate Ment-1-9-ol Geranyl acetate Thymol Carvacrol Eugenol Citronellyl acetate Benzyl isothiocyanate a-Copaene b-Ylangene b-Trans caryophyllene Trans-b-farnesene b-Ionone c-Muurolene Germacreme b-Bisabolene D-Cadinene a-Zingiberene cis-c-Bisabolene Cardinene a-Sesquiphellandrene cis-Nerolidol
D.g.
O.g.
Z.o.
A.c.
C.r.
C.l.
C.c
E.c.
T.v.
— — — 0.7 — — — — — — — — — — — — — — — — — 12.6 — — — — — — — — — — — — — — — — — — — — — — — — — 86.7 — — — — — — — — — — — — — —
4.1 — 1.2 — — — 0.6 3.7 0.3 0.8 14.0 — 1.1 0.5 0.3 16.6 — — — — — — — — — 0.5 — 1.1 0.4 — — — — — — — — — — — — — — 47.1 0.6 — — — 0.4 — 0.6 — — — — — 0.9 — — 0.3 — —
2.6 0.7 9.6 — — — 0.3 1.1 0.3 0.3 — 7.0 3.7 — — — 2.2 — 0.7 — — — 0.7 — — 0.2 0.3 3.6 0.5 1.2 — — — 15.0 — 1.0 1.1 — 23.2 0.8 — — — 0.9 — — — — — — — — — 3.1 — — — 7.6 — 0.7 1.9 1.6
0.7 — 1.8 — — — — 0.7 — 0.9 1.2 — 0.5 — — — — — — — — — — — — — — — — — — — — — — — — — — — 2.1 — — — — — — — — — 24.6 0.4 4.7 — 41.6 2.9 — — 1.4 1.3 — —
0.8 — — — 1.1 — 1.1 1.1 — — — — 74.8 — — 7.3 — — — — 1.4 — — — — — — 1.8 — 2.8 — — — — — — — — — 0.4 — 0.9 — — — 1.9 1.0 — — 4.1 — — — — — — — — — — — —
0.5 — — — 1.7 — 2.7 — — — — — 86.4 — — — — 0.4 — — 0.8 — — — — — — 3.0 — 1.3 0.3 — — — — — 0.3 0.6 — — — — — — 0.8 0.2 — — — — 0.4 — — — — — — — — — — —
0.4 — — — 0.7 1.0 — — 5.9 — — — 0.6 — — — — — — — — — — 0.3 — 0.4 — 0.6 0.9 — — — 1.0 34.4 0.5 — 1.8 — — 49.2 — — 0.4 — — — — — — — — — — — 0.4 — — — — — — —
— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — 0.3 — — — — — — — — — — — — 87.7 — — — — 4.3 — — — — — — — — — — —
0.8 0.5 1.0 — 5.3 — 1.1 1.0 0.9 — 25.0 — 2.6 — — — 0.3 — — 4.2 — — — — 0.5 0.7 — 2.0 — — — 1.3 — — — — — — — — — — — 45.5 0.4 3.0 — — — — 1.7 — — — — — — — — — — —
(continued)
ESSENTIAL OILS TO CONTROL BACTERIAL SPORES IN FOOD
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Table 2. (Continued) No.
K.I.
Compounds (%)
D.g.
O.g.
Z.o.
A.c.
C.r.
C.l.
C.c
E.c.
T.v.
63 64 65 66 67 68 69 70 71 72 73
1534 1536 1542 1543 1561 1572 1595 1602 1606 1609 1620
Elemol b-Thujaplicinol Sesquisabinene hydrate (trans) Dihydro eugenol acetate b-Bisabolol Geranyl isobutanoate Zingiberenol Caryophyllene oxide Eudesmol 2-Acetyl-naphtalene b-Cedene epoxide
— — — — — — — — — — —
— — — — — — — — 1.6 — —
3.2 — 0.7 — 0.5 — 0.3 — — — —
— — — — — — 3.4 0.5 — — —
— — — — — — — — — — —
— — — — — 0.5 — — — — —
— — — — — — — — — — —
— — — 6.5 — — — — — 0.2 1.3
— 0.4 — — — — — 1.3 — — —
K.I., Kovats Index; D.g., Drypetes gossweileri; O.g., Ocimum gratissimum; Z.o., Zingiber officinale; A.c., Ageratum conyzoı¨des; C.r., Citrus reticulata; C.l., Citrus limon; C.c., Cymbopogon citratus; E.c., Eugenia caryophyllus; T.v., Thymus vulgaris. GC, gas chromatography; GC-MS, gas chromatography coupled to mass spectrometry.
its isothiocyanate derivatives composition, known to have a very strong antimicrobial effect due to the R-N = C = S group in the molecule. This group has a high electrophilic central carbon, which can easily react with nucleophilic centers. Furthermore, it could cleave the disulfide bonds of proteins and could attack free amino acids by oxidative reaction (Wilson, 2011; Hyldgaard et al., 2012). Concerning the inhibitory effect of spore germination, essential oil of D. gossweileri appeared to be the most active, with inhibitory effect at 0.001 mg/mL on B. subtilis spores and 0.002 mg/mL on spores of the other Bacillus and G. stearothermophilus. For the other essential oils, the MICg ranged from 0.09 to 1.5 mg/mL, with essential oils of C. limon, C. citratus, and A. conyzoı¨des seeming to be more active, with MICg £ 0.37 on at least two of the four species
tested. The MICg of 0.37–1.5 mg/mL are close to those obtained by Chaibi et al. (1997) on spores of B. cereus T, with the essential oils of some Moroccan plants. However, the MICg shown by D. gossweileri essential oil were generally lower, further confirming its strong antimicrobial activity. On the whole, MICg were much lower than MICs (Table 2), reflecting the fact that the inhibitory action of essential oils is more effective on germination of spores compared to vegetative growth. Regarding the bactericidal effect, essential oils of C. reticulata, C. citratus, and E. caryophyllus generally showed an absolute effect. MBCs were observed at MIC concentration of 2.5 mg/mL. O. gratissimum and Z. officinale were also bactericidal but at higher concentrations, generally at 10 mg/mL.
Table 3. Minimal Inhibitory, Minimal Bactericidal (MICs and MBCs), and Minimal Concentrations of Essential Oils Inhibiting Vegetative Growth and Spores Germination Determined by Macrodilution Essential oils Drypetes gossweileri Ocimum gratissimum Zingiber officinale Ageratum conyzoı¨des Citrus reticulata Citrus limon Cymbopogon citratus Eugenia caryophyllus Thymus vulgaris
Parameter of inhibition (mg/mL)
Bacillus cereus
Bacillus megaterium
Bacillus subtilis
Geobacillus stearothermophilus
MIC/MBC MICg MIC/MBC MICg MIC/MBC MICg MIC/MBC MICg MIC/MBC MICg MIC/MBC MICg MIC/MBC MICg MIC/MBC MICg MIC/MBC MICg
0.009/– 0.002 2.5/5 1.5 0.63/2.5 0.75 1.25/– 0.37 –/– 1.5 1.25/– 0.75 5.0/5.0 0.09 2.5/2.5 – 1.25/– 0.37
0.004/– 0.002 1.25/10 0.75 1.25/10 0.75 2.5/– 0.09 2.5/2.5 0.75 2.5/– 0.37 2.5/2.5 0.37 2.5/– 0.75 1.25/– 1.5
0.009/– 0.001 2.5/10 0.75 0.63/10 – 0.312/– 0.09 2.5/– 1.5 1.25/– 0.37 2.5/– 0.37 2.5/2.5 0.75 1.25/– 0.75
0.009/– * 1.25/10 * 0.63/10 * 1.25/– * 2.5/2.5 * 2.5/– * 2.5/– * 2.5/2.5 * 1.25/– *
MIC, minimal inhibitory concentration; MBC, minimal bactericidal concentration; MICg, minimal concentration inhibiting germination of spores; (–), inactive; (*), not evaluated.
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Table 4. Effect of Combined Essential Oils on Vegetative Growth of Bacillus and Geobacillus stearothermophilus Essential oil combinations
Inhibitory parameters (mg/mL)
Bacillus cereus
Bacillus megaterium
Bacillus subtilis
G. stearothermophilus
Drypetes gossweileri/Zingiber officinale
MIC1; MIC2 MBC1; MBC2 FIC1 + FIC2 = FICindex Type of effect MIC1; MIC2 MBC1;; MBC2 FIC1 + FIC2 = FICindex Type of effect MIC1; MIC2 MBC1; MBC2 FIC1 + FIC2 = FICindex Type of effect MIC1; MIC2 MBC1; MBC2 FIC1 + FIC2 = FICindex Type of effect
0.0024; 0.31 — 0.25 + 0.5 = 0.75 Additive 0.0048; 0.62 0.0048; 0.62 0.5 + 0.5 = 1 Additive 0.0048; 0.62 0.0048; 0.62 0.5 + 0.25 = 0.75 Additive 0.0048; 0.62 0.0097; 1.25 0.5 + 0.25 = 0.75 Additive
0.0024; 0.31 — 0.5 + 0.25 = 0.75 Additive 0.0048; 0.62 — 1 + 0.25 = 1.25 Additive 0.0048; 0.62 — 1 + 0.5 = 1.5 Additive 0.0048; 0.62 — 1 + 0.25 = 1.5 Additive
0.0048; 0.62 0.0048; 0.62 0.5 + 1 = 1.5 Additive 0.0048; 0.62 — 0.5 + 1 = 1.5 Additive 0.0024; 0.31 — 0.25 + 0.12 = 0.37 Synergistic 0.0024; 0.31 — 0.25 + 0.12 = 0.37 Synergistic
0.0048; 0.62 0.0048; 0.62 0.5 + 1 = 1.5 Additive 0.0024; 0.31 0.0048; 0.62 0.25 + 0.25 = 0.5 Synergistic 0.0097; 1.25 0.0097; 1.25 1+1=2 Additive 0.0024; 0.31 0.0048; 0.62 0.5 + 0.12 = 0.37 Synergistic
Drypetes gossweileri/Ageratum conyzoı¨des D. gossweileri/Ocimum gratissimum D. gossweileri/Eugenia caryophyllus
(—), inactive; MIC1, minimal inhibitory concentration of first essential oil in combination; MIC2, minimal inhibitory concentration of second essential oil in combination; MBC1, minimal bactericidal concentration of first essential oil in combination; MBC2, minimal bactericidal concentration of second essential oil in combination; FIC1, fractions inhibitory concentration of first essential oil; FIC2, fractions inhibitory concentration of second essential oil; FICindex, fractions inhibitory concentration index.
FIG. 1. Percentage germination inhibition of spores obtained on solid medium after treatment with essential oils.
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Effect of combination of essential oils on vegetative growth
The effect of the combination of essential oils is summarized in Table 4. The results obtained show an improvement of the antibacterial effect due to the combinations of D. gossweileri and E. caryophyllus, A. conyzoı¨des, or O. gratissimum essential oils, as shown by a decrease in MICs. Furthermore, all the combinatory effects appeared to be either additive or synergistic. Combination of D. gossweileri and E. caryophyllus proved significant activity with a synergistic effect on two (B. subtilis and G. stearothermophilus) of the four species. Synergy or additive effects observed could be explained by the combined effect of benzyl isothiocyanate, major compound of D. gossweileri, and antimicrobial compounds found in high percentage in the others essential oils such as thymol, neral, trans-b-caryophyllene, and eugenol. In addition, the increased effect of the combination could be explained by the synergistic effect of benzyl isothiocyanate and the terpene hydrocarbons within the other essential oils. Nguefack et al. (2012) reported that terpene hydrocarbons have low effect, but could swell cell membranes and enable more antimicrobial compounds such as benzyl isothiocyanate to be easily transported into the cell. Treatment of spores with essential oil and evaluation of germination inhibition on solid medium
Figure 1 shows the curves representing the percentage germination inhibition of spores, at different concentrations of essential oils. The essential oil of D. gossweileri was the most effective. At 1.25 mg/mL, this oil was active on B. cereus, B. megaterium, and G. sterotherophilus spores, with germination inhibition percentages of 66.23, 48.54, and 51.66%, respectively. Furthermore, a total inhibition of germination was observed on all the spores at 20 mg/mL. The other essential oils had effect but at higher concentrations ranging from 10 to 20 mg/mL. However, Z. officinale essential oil was found to be active on spores of B. subtilis, with a total inhibition at 2.5 mg/mL. O. gratissimum was more
FIG. 2. Germination, outgrowth, and growth kinetic of spores of Bacillus cereus in the presence of essential oils. OD, optical density.
FIG. 3. Germination, outgrowth, and growth kinetic of spores of Bacillus megaterium in the presence of essential oils. OD, optical density. active on B. cereus spores where significant activity was obtained at 2.5 mg/mL and 5 mg/mL. These results might suggest a probable irreversible effect of these three essential oils as compared to others. Kinetics of spores germination and outgrowth with and without essential oils
The kinetics of the germination and outgrowth of spores of B. cereus, B. megaterium, and B. subtilis were performed in the absence (control) and the presence of essential oils using spectrophotometric measurements. Figure 2 shows the kinetics obtained with spores of B. cereus. The curves show a constancy of optical densities between 0 and 120 min for both control and tested spores. At 120 and 300 min, respectively, for control and spores in the presence of C. limon essential
FIG. 4. Germination, outgrowth, and growth kinetic of spores of Bacillus subtilis in the presence of essential oils. OD, optical density.
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oil, an increase of optical densities was recorded due to the start of vegetative growth. This observation was not revealed with spores in the presence of the other essential oils, suggesting that these essential oils probably inhibited the germination and/or outgrowth of spores. Complete inhibition of spore germination was observed in the presence of the essential oil of A. conyzoı¨des because the optical densities remained constant during the test period. Concerning the essential oils of D. gossweileri and C. citratus, slight decreases in optical densities were recorded after 100 and 180 min, respectively, characterizing spore germination. However, after 200 min, an increase in optical densities was observed, suggesting that these germinated spores might return to their initial state of dormancy. This phenomenon called microcycle of spores was already observed with B. cereus spore by Edima et al. (2010). Figures 3 and 4 show that the kinetics conducted on spores of B. megaterium and B. subtilis, respectively, revealed that from 0 to 40 min, the optical densities recorded are almost constant with control and tested spores. This period probably corresponds to the lag phase. From 60 min for the control and the spores with D. gossweileri essential oil, a decrease of optical densities was observed, probably due to the germination of spores. This germination step starts at 120 min with spores in the presence of the other essential oils. After 180 min, an exponential growth only with the control was observed, probably due to the start of vegetative growth. This observation is not shown with spores in the presence of essential oils, suggesting that the essential oil prevented the germination of spores. Conclusions
Essential oils used in this work have displayed the capacity to inhibit germination as well as vegetative growth of spores of Bacillus and G. stearothermophilus. Among the essential oils tested, D. gossweileri appears to be more active as compared to others. Furthermore, its combination with other essential oils improves the antibacterial effect. Thus, D. gossweileri essential oil could be suggested as a food preservative. Nevertheless, further studies must be conducted upon other bacterial spores. Acknowledgments
We are grateful to Professor Chantal Menut of the Institut des Biomole´cules Max Mousseron (IBMM) de Montpellier, the University of Yaounde I, and the Vasile Alecsandri University of Bacau for laboratory facilities. We thank the Romanian Government, the Agence Universitaire de la Francophonie (AUF), and the Service de la Cooperation et de l’Action Culturelle (French Embassy in Cameroon) for funding this project. We also thank the Microbiology Laboratory Institute of Food Research of Reading and the Institut Appert, Paris for providing us with bacterial spore strains. Disclosure Statement
No competing financial interests exist. References
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Address correspondence to: Ste`ve Olugu Voundi, PhD Candidate Department of Microbiology Laboratory of Microbiology University of Yaounde I P.O. Box 812 Yaounde, Cameroon E-mail: [email protected]
