Inhibition of Bacillus subtilis and Listeria innocua by nisin in combination with some naturally occurring organic compounds

The application of combined preservative factors (hurdle technology) is very effective in controlling the growth of food spoilage and foodborne pathogenic bacteria. Antimicrobial activity of nisin alone and in combination with some natural organic compounds (carvacrol, cinnamic acid, eugenol, diacetyl, and thymol) on the growth of gram-positive bacteria Bacillus subtilis and Listeria innocua was-investigated. All the organic compounds tested exhibited antimicrobial activity against the microorganisms used; however, the MICs varied between 0.8 and 15.0 mM depending on the potency of the compound or the sensitivity of the target strain. Investigation of the interaction between the organic compounds and nisin against the test organisms revealed different patterns, varying from synergistic to antagonistic. Combinations of nisin with carvacrol, eugenol, or thymol resulted in synergistic action against both test organisms. Activity of nisin and cinnamic acid together was synergistic against L. innocua, but only additive against B. subtilis. In contrast, the combination of diacetyl and nisin resulted in an antagonistic effect against both test organisms. This study highlights the potential of the combination of these compounds with nisin to inhibit pathogen growth in food.     

Bactericidal activities of plant essential oils and some of their isolated constituents against Campylobacter jejuni,Escherichia coli,Listeria monocytogenes,and Salmonella enterica

An improved method of sample preparation was used in a microplate assay to evaluate the bactericidal activity levels of 96 essential oils and 23 oil compounds against Campylobacter jejuni, Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica obtained from food and clinical sources. Bactericidal activity (BA50) was defined as the percentage of the sample in the assay mixture that resulted in a 50% decrease in CFU relative to a buffer control. Twenty-seven oils and 12 compounds were active against all four species of bacteria. The oils that were most active against C. jejuni (with BA50 values ranging from 0.003 to 0.009) were marigold, ginger root, jasmine, patchouli, gardenia, cedarwood, carrot seed, celery seed, mugwort, spikenard, and orange bitter oils; those that were most active against E. coli (with BA50 values ranging from 0.046 to 0.14) were oregano, thyme, cinnamon, palmarosa, bay leaf, clove bud, lemon grass, and allspice oils; those that were most active against L monocytogenes (with BA50 values ranging from 0.057 to 0.092) were gardenia, cedarwood, bay leaf, clove bud, oregano, cinnamon, allspice, thyme, and patchouli oils; and those that were most active against S. enterica (with BA50 values ranging from 0.045 to 0.14) were thyme, oregano, cinnamon, clove bud, allspice, bay leaf, palmarosa, and marjoram oils. The oil compounds that were most active against C. jejuni (with BA50 values ranging from 0.003 to 0.034) were cinnamaldehyde, estragole, carvacrol, benzaldehyde, citral, thymol, eugenol, perillaldehyde, carvone R, and geranyl acetate; those that were most active against E. coli (with BA50 values ranging from 0.057 to 0.28) were carvacrol, cinnamaldehyde, thymol, eugenol, salicylaldehyde, geraniol, isoeugenol, citral, perillaldehyde, and estragole; those that were most active against L monocytogenes (with BA50 values ranging from 0.019 to 0.43) were cinnamaldehyde, eugenol, thymol, carvacrol, citral, geraniol, perillaldehyde, carvone S, estragole, and salicylaldehyde; and those that were most active against S. enterica (with BA50 values ranging from 0.034 to 0.21) were thymol, cinnamaldehyde, carvacrol, eugenol, salicylaldehyde, geraniol, isoeugenol, terpineol, perillaldehyde, and estragole. The possible significance of these results with regard to food microbiology is discussed.     

Antimicrobial Efficacy of an Array of Essential Oils Against Lactic Acid Bacteria

The essential oils of clove bud, cinnamon bark and thyme, and their individual compounds including allyl isothiocyanate (AIT), carvacrol, cinnamaldehyde, cinnamic acid, eugenol, and thymol were initially assessed for antimicrobial activity against 9 lactic acid bacteria (LAB) species. Carvacrol and thymol were the most inhibitory with MICs of 0.1% (v/v and w/v, respectively). Cinnamaldehyde, cinnamon bark oil, clove bud oil, eugenol, and thyme oil were moderately inhibitive (MICs = 0.2% v/v), while cinnamic acid required a concentration of 0.5% (w/v). AIT was not effective with MICs in excess of concentrations tested (0.75% v/v). The bactericidal capability of the oil components carvacrol, cinnamaldehyde, eugenol, and thymol were further examined against Pediococcus acidilactici, Lactobacillus buchneri, and Leuconostoc citrovorum. Thymol at 0.1% (w/v) was bactericidal against L. citrovorum (>4‐log reduction), but resulted in a 2‐log CFU/mL reduction against L. buchneri and P. acidilactici. Cinnamaldehyde at 0.2% to 0.25% (v/v) was effective against L. citrovorum, L. buchneri, and P. acidilactici, resulting in a >2‐log reduction. All 3 organisms were susceptible to 0.2% carvacrol with >3‐log reduction observed after exposure for 6 h. Eugenol was the least effective. Concentrations of 0.2% and 0.25% (v/v) were needed to achieve an initial reduction in population, >3‐log CFU/mL after 6 h exposure. However, at 0.2%, P. acidilactici and L. buchneri recovered to initial populations in 48 to 72 h. Results indicate essential oils have the capacity to inactivate LAB that are commonly associated with spoilage of shelf stable low‐acid foods.     

Antimicrobial Effects of Essential Oils,Nisin, and Irradiation Treatments against Listeria monocytogenes on Ready‐to‐Eat Carrots

The study aimed at using essential oil (EO) alone or combined EO with nisin and low dose γ‐irradiation to evaluate their antibacterial effect against Listeria monocytogenes during storage of carrots at 4 °C. Minicarrots were inoculated with L. monocytogenes at a final concentration of approximately 7 log CFU/g. Inoculated samples were coated by nisin at final concentration of 10³ International Unit (IU)/mL or individual mountain savory EO or carvacrol at final concentration of 0.35%, w/w) or nisin plus EO. The samples were then irradiated at 0, 0.5, and 1.0 kGy. The treated samples were kept at 4 °C and microbial analysis of samples were conducted at days 1, 3, 6, and 9. The results showed that coating carrots by carvacrol plus nisin or mountain savory plus nisin and then irradiating coated carrots at 1 kGy could reduce L. monocytogenes by more than 3 log at day 1 and reduced it to undetectable level from day 6. Thus, the combined treatments using nisin plus carvacrol or nisin plus mountain savory and irradiation at 1.0 kGy could be used as an effective method for controlling L. monocytogenes in minicarrots.     

Inhibitory effect of thyme and basil essential oils,carvacrol, thymol,estragol, linalool and p-cymene towards Shigella sonnei and S. flexneri

There are a few reports on the antimicrobial activity of essential oils or their major constituents towards Shigella sp. The antimicrobial effect of basil and thyme essential oil and its major constituents thymol, p-cymene, estragol, linalool, and carvacrol was determined using the agar well diffusion assay. Thyme essential oil and thymol and carvacrol showed inhibition of Shigella sp. in the agar well diffusion method. The potential of thyme essential oil, thymol and carvacrol at 0.5% and 1.0% v/v for decontamination of lettuce was evaluated. A decrease of the shigellae was observed after washing with 0.5% while at 1% Shigella numbers dropped below the detection limit. However, the antimicrobial effect on a subsequent lettuce sample in the same decontamination solution was significantly decreased. In addition, application of thyme essential oil or thymol or carvacrol for decontamination is hampered by sensoric properties of the lettuce (browning, strong odour).     

Antimicrobial activity and chemical composition of Origanum glandulosum Desf. essential oil and extract obtained by microwave extraction: Comparison with hydrodistillation

Origanum glandulosum Desf. essential oils obtained by hydrodistillation (HD), solvent-free microwave extraction (SFME) and the extract obtained by microwave-assisted extraction (MAE) were investigated by capillary gas chromatography and gas chromatography/mass spectrometry. The main components of both oils obtained by HD and SFME were thymol (41.6–81.1%) followed by γ-terpinene (27.0–3.1%), p-cymene (17.1–4.0%) and carvacrol (2.2–4.4%), respectively. In the same way, thymol (65.4%), γ-terpinene (13.1%), p-cymene (7.2%) and carvacrol (3.5%) were the main components of the extract obtained by hexane microwave extraction. The SFME method was most selective for the extraction of thymol. The examination of the antimicrobial activity of both essential oils against 10 bacteria, two yeasts and four moulds revealed that O. glandulosum oil is more antifungal than antibacterial. To our knowledge, the antifungal activity of the O. glandulosum oil obtained by HD and both antimicrobial and antifungal activities of O. glandulosum SFME oil were not yet reported. Our study suggests that O. glandulosum essential oil has the potential to be used as a food preservative and to prevent the growth of nosocomial bacteria.     

Composition and antioxidant activity of essential oils from Oregano plants grown wild in Greece

In this study, four samples of essential oils obtained from plant species with a wide distribution in Greece and economic importance for the world-wide spice market and also carvacrol and thymol were tested for their possible antioxidant activity. The four plant species wereOriganum vulgare subsp.hirtum, O. onites, Coridothymus capitatus andSatureja thymbra. The essential oils were chemically characterised by gas chromatography-ion trap detection. All the essential oils examined and also carvacrol and thymol were found to have antioxidant activity when tested on TLC plates and by measuring peroxide values of lard stored at 35° C. The results indicate that the antioxidant effect may be related to the presence of carvacrol and thymol in essential oils.     

植物精油及其活性成分的抗菌机理

芳香植物精油普遍具有良好的抗菌能力,是潜在的天然抗菌剂.本文概述山苍子、肉桂、丁香、辣木、百里香、鼠尾草、孜然、八角茴香、牛至、花椒、大蒜等24种精油的主要化学成分、抗菌作用对象和抗菌机理,并对香芹酚、肉桂醛、百里香酚、丁香酚、柠檬醛、柠檬烯等6种精油单体成分对不同微生物的抗菌作用进行分析.     

Antimicrobial Wine Formulations Active Against the Foodborne Pathogens Escherichia coli O157: H7 and Salmonella enterica

ABSTRACT:  We developed wine formulations containing plant essential oils and oil compounds effective against foodborne pathogenic bacteria Escherichia coli O157:H7 and Salmonella enterica. HPLC was used to determine maximum solubility of antimicrobials in wines as well as amounts of antimicrobials extracted by wines from commercial oregano and thyme leaves. Activity of essential oils (cinnamon, lemongrass, oregano, and thyme) and oil compounds (carvacrol, cinnamaldehyde, citral, and thymol) in wines were evaluated in terms of the percentage of the sample that resulted in a 50% decrease in the number of bacteria (BA₅₀). The ranges of activities in wines (30 min BA₅₀ values) against S. enterica/E. coli were carvacrol, 0.0059 to 0.010/0.011 to 0.021; oregano oils, 0.0079 to 0.014/0.022 to 0.031; cinnamaldehyde, 0.030 to 0.051/0.098 to 0.13; citral, 0.033 to 0.038/0.060 to 0.070; lemongrass oil, 0.053 to 0.066/0.059 to 0.071; cinnamon oil 0.038 to 0.057/0.066 to 0.098; thymol, 0.0086 to 0.010/0.016 to 0.022; and thyme oil, 0.0097 to 0.011/0.033 to 0.039. Detailed studies with carvacrol, the main component of oregano oil, showed that antibacterial activity was greater against S. enterica than against E. coli and that wine formulations exhibited high activities at low concentrations of added antimicrobials. The results suggest that wines containing essential oils/oil compounds, added or extracted from oregano or thyme leaves, could be used to reduce pathogens in food and other environments.     

Synergistic effect of nisin and garlic shoot juice against Listeria monocytogenes in milk

The aim of this research was to determine the synergistic effect of nisin and garlic shoot juice (GSJ) against Listeria monocytogenes ATCC 19118 found in whole (3.5%), low (1%) and skim (no fat content) milk. Garlic shoot juice (GSJ) at concentrations of 2.5%, 5% and 10% revealed strong and similar patterns of antilisterial effect against L. monocytogenes ATCC 19118 in all categories of milk. Nisin only at concentrations of 62.5, 125, 250 and 500 IU/ml displayed a strong antilisterial effect as compared to the control group. Also, the synergistic combinations of GSJ (2.5%, 5%) and nisin (62.5, 125, 250 and 500 IU/ml) had a remarkable antilisterial activity in all categories of whole, low and skim milk after 14 days. Results of this study indicated the synergistic effect of GSJ and nisin as a potential antilisterial agent for the food industry.     

Phytochemical composition and antimicrobial activities of the essential oils from Satureja subspicata Vis. growing in Croatia

The present study describes the phytochemical profile and antimicrobial activity of Satureja subspicata Vis. essential oils, collected in Dalmatia (Croatia). Three samples of essential oils were obtained from the aerial parts of the plant by hydrodistillation and analyzed by GC–MS. From the 24 compounds representing 97.47% of the oils, carvacrol (16.76%), α-pinene (13.58), p-cymene (10.76%), γ-terpinene (9.54%) and thymol methyl ether (8.83%) appear as the main components. The oils also contained smaller percentages of myrcene, linalool, β-caryophyllene, limonene, geranyl acetate, 1-Octen-3-ol, nerol, thymol and borneol. Furthermore, antimicrobial activity of the oil was evaluated using agar diffusion and broth microdilution methods. The antimicrobial test results showed that the oils had a great potential antimicrobial activity against all 13 bacteria and 9 fungal strains. Gram-positive bacteria are more sensitive to the investigated oil, with a range of 0.09 to 6.25 μl/ml than Gram-negative bacteria in the range which is significantly higher from 1.56 to 25.00 μl/ml. Results presented here may suggest that the essential oil of S. subspicata possesses antimicrobial properties, and is therefore a potential source of antimicrobial ingredients for the food and pharmaceutical industry.     

Antibacterial activities of essential oils and extracts of Turkish Achillea,Satureja and Thymus species against plant pathogenic bacteria

BACKGROUND: The aims of this study were to examine the chemical composition of the essential oils and hexane extracts of the aerial parts of Satureja spicigera (C. Koch) Boiss., Thymus fallax Fisch. & CA Mey, Achillea biebersteinii Afan, and Achillea millefolium L. by GC and GC–MS, and to test antibacterial efficacy of essential oils and n‐hexane, chloroform, acetone and methanol extracts as an antibacterial and seed disinfectant against 25 agricultural plant pathogens. RESULTS: Thymol, carvacrol, p‐cymene, thymol methyl ether and γ‐terpinene were the main constituents of S. spicigera and T. fallax oils and hexane extracts. The main components of the oil of Achillea millefolium were 1,8‐cineole, δ‐cadinol and caryophyllene oxide, whereas the hexane extract of this species contained mainly n‐hexacosane, n‐tricosane and n‐heneicosane. The oils and hexane extracts of S. spicigera and T. fallax exhibited potent antibacterial activity over a broad spectrum against 25 phytopathogenic bacterial strains. Carvacrol and thymol, the major constituents of S. spicigera and T. fallax oils, also showed potent antibacterial effect against the bacteria tested. The oils of Achillea species showed weak antibacterial activity. Our results also revealed that the essential oil of S. spicigera, thymol and carvacrol could be used as potential disinfection agents against seed‐borne bacteria. CONCLUSION: Our results demonstrate that S. spicigera, T. fallax oils, carvacrol and thymol could become potentials for controlling certain important agricultural plant pathogenic bacteria and seed disinfectant. Copyright © 2009 Society of Chemical Industry     

Liposomal incorporation of carvacrol and thymol isolated from the essential oil of Origanum dictamnus L. and in vitro antimicrobial activity

The chemical composition of the essential oils from Origanum dictamnus L. (wild and organic cultivated plant) was analysed by GC–MS. Carvacrol, thymol, p-cymene, and γ-terpinene were identified as major constituents and isolated from both samples. The above components were successfully encapsulated in phosphatidyl choline-based liposomes and the possible improvement of their antioxidant and antimicrobial activities was tested against selected microbia. The antimicrobial properties of the oils were tested by a diffusion technique against four Gram positive and four Gram negative bacteria and three human pathogenic fungi, as well as the food-borne pathogen, Listeria monocytogenes. The percentage of the encapsulated carvacrol, the major component of the oil, was determined by GC–FID. In order to investigate any possible synergistic or antagonistic effect between carvacrol/thymol and carvacrol/γ-terpinene, the antimicrobial activities of the mixtures, were also determined before and after encapsulation in liposomes. All tested compounds presented enhanced antimicrobial activities after the encapsulation. Finally, in all cases, their antioxidant activity using differential scanning calorimetry was studied, in order to gain knowledge about their oxidation stability.     

Short communication: Interaction of the isomers carvacrol and thymol with the antibiotics doxycycline and tilmicosin: In vitro effects against pathogenic bacteria commonly found in the respiratory tract of calves

Bovine respiratory disease is the major problem faced by cattle, specially calves, leading to reduced animal performance and increased mortality, consequently causing important economic losses. Hence, calves must be submitted to antibiotic therapy to counteract this infection usually initiated by the combination of environmental stress factors and viral infection, altering the animal's defense mechanism, and thus allowing lung colonization by the opportunistic bacteria Mannheimia haemolytica and Pasteurella multocida. Essential oils appear to be candidates to replace antibiotics or to act as antibiotic adjuvants due to their antimicrobial properties. In the present study, we aimed to evaluate the 4 essential oil components carvacrol, thymol, trans-anethole, and 1,8 cineole as antibacterial agents or as adjuvants for the antibiotics doxycycline and tilmicosin against M. haemolytica and P. multocida. Bacteria were cultured according to standard protocols, followed by the determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration. A checkerboard assay was applied to detect possible interactions between components, between antibiotics, and between components and antibiotics. Doxycycline at 0.25 and 0.125 μg/mL inhibited the growth of P. multocida and M. haemolytica, respectively, whereas tilmicosin MIC values were 1.0 and 4.0 μg/mL for P. multocida and M. haemolytica, respectively. Carvacrol MIC values were 2.5 and 1.25 mM for P. multocida and M. haemolytica, respectively, whereas thymol MIC values were 1.25 and 0.625 mM for P. multocida and M. haemolytica, respectively. Trans-anethole and 1,8 cineole did not present any antibacterial effect even at 40 mM against the investigated pathogens. All minimum bactericidal concentration values were the same as MIC, except when thymol was tested against M. haemolytica, being twice the MIC data (i.e., 1.25 mM thymol). Based on fractional inhibitory concentration checkerboard assay, no interaction was observed between doxycycline and tilmicosin. Carvacrol and thymol presented an additive effect when one of them was combined with tilmicosin. Additive effect was also observed when doxycycline was combined with thymol. Synergism was observed when carvacrol was combined with doxycycline or with thymol. Although the antibacterial effects of the tested essential oil components were observed at high concentrations for in vitro conditions, the additive and synergic effects of carvacrol and thymol with antibiotics suggest the option to apply them as antibiotic adjuvants.     

芳香精油在食品保藏中的应用性研究进展

芳香精油是重要的植物次生代谢物,药食应用历史悠久,因其多方面的生物活性,已引起人们对其在食品保藏领域替代化学防腐剂的极大兴趣。文中概述了芳香精油及其成分在采后果蔬、预切果蔬片、肉制品、乳制品和水产品等食品保鲜和贮藏领域中应用性和安全性研究的最新研究进展。许多研究表明,牛至、丁香、罗勒、胡荽、肉桂、百里香、薄荷、迷迭香、芥茉、芫荽、红根草等芳香精油以及丁子香酚、香芹酚、肉桂酸、己醛、草蒿脑、麝香草酚、香芹酮、肉桂醛、柠檬醛、香叶醇等单体成分具有优良的抗食源性腐败菌和致病菌活性。通过与食品生产前处理工艺、气调包装、配方优化等手段的结合,一些芳香精油及其成分显示出天然食品保藏剂良好的商用价值。     

The effects of essential oils carvacrol and thymol on growth of Penicillium digitatum and P. italicum involved in lemon decay

In this work the antifungal efficacy of thymol, carvacrol and the mixture of both pure essential oils has been proved against Penicillium digitatum and Penicillium italicum, by using in vitro (liquid media, PDA plates and disk growth) and in vivo (lemon) tests. Results indicated that both essential oils were effective in inhibiting fungal growth in all in vitro tests, with the highest efficacy displayed by thymol. In addition, the application of wax with thymol and carvacrol to lemons inoculated with P. digitatum demonstrated the reduction of decay (expressed as percentage of infected fruit surface) in a concentration dependent manner, as well as reduced respiration rate, ethylene production and total acidity losses. Thus, the application of these essential oils together with wax in the citrus packing lines could be considered as good alternatives to reduce the use of synthetic fungicides.     

Inhibitory effects of essential oil components on growth of food-contaminating fungi

The antifungal activity of several components of essential oils were evaluated using a paper-disk method. The substances investigated are structurally related to eugenol. Equimolar amounts were tested on more than ten fungal strains known to contaminate food. Iso-eugenol, cinnamaldehyde, carvacrol, eugenol and thymol revealed the strongest antifungal activity. The most resistant strain appeared to be Penicillium verrucosum var. cyclopium, and the most sensitive was P. viridicatum. Some of the structural effects were considered, including a free hydroxyl group in connection with an alkyl substituent which seemed to represent an especially active configuration of phenolic compounds and which rendered antimicrobial activity.     

Influence of Some Spice Essential Oils on Aspergillus Parasiticus Growth and Production of Aflatoxins in a Synthetic Medium

Gas-liquid chromatography was used to determine the essential oil compositions of thyme, cumin, clove, caraway, rosemary, and sage. The basic components of these oils were thymol, cumin aldehyde, eugenol, carvonc, borneol and thujonc, respectively. The antifungal potential of the oils against Aspergillus parasiticus were investigated. The essential oils caused complete inhibition of both mycelial growth and aflatoxin production. The effectiveness followed the sequence: thyme > cumin > clove > caraway > rosemary > sage. The major components of the essential oils produced an inhibitory effect at minimum inhibitory concentrations equal to those obtained with the oils.     

Low temperature and binding to food components inhibit the antibacterial activity of carvacrol against Listeria monocytogenes in steak tartare

Carvacrol is a major component of thyme and oregano essential oils and has potential uses as a food preservative. The effect of carvacrol on the growth of Listeria monocytogenes was investigated in vitro and in steak tartare. Carvacrol had strong antilisterial activity in growth medium (MIC = 1.6 mM), but no effect was observed when carvacrol was tested in steak tartare. There were two reasons for this reduced activity: the antilisterial activity of carvacrol was strongly reduced at lower temperatures (10 versus 30 degrees C), and the presence of food components interfered with the activity of carvacrol. Both bovine serum albumin and egg yolk inhibited carvacrol activity at > 0.2% (wt/vol) in growth medium. For the first time, carvacrol was found to bind to albumin, suggesting that the reduced antilisterial activity of carvacrol in foods such as dairy products and uncooked meats is the result of fewer free unbound carvacrol molecules available to interact with bacteria.     

Chemical composition and antioxidant and antimicrobial activity of two Satureja essential oils

Hydro-distilled volatile oils from the aerial parts of Satureja montana L., and Satureja subspicata Bartl. ex Vis., growing wild in Bosnia and Herzegovina, were analyzed by GC/MS. More than one hundred compounds were identified in both plant oils, representing 92.4–98.1% of the total oil. The major constituents of essential oils obtained from the plant material of S. montana, collected from two different localities, were thymol (31.7%), and geraniol (22.3%), respectively. The most abundant compounds in essential oils of S. subspicata, collected at two different stages of development, were thymol (28.6%), and spathulenol (37.6%), respectively. The screening of antimicrobial activity of essential oil samples was individually evaluated against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis using a paper disc diffusion method. All tested microorganisms were inhibited by essential oil samples. Antioxidant activity was tested using the DPPH radical-scavenging method. All samples showed activity comparable to thymol, which was used as a positive probe.