Enhancement of antilisterial activity of essential oil constituents by nisin and diglycerol fatty acid ester

Plant-derived essential oil components in combination with nisin and diglycerol fatty acid esters were investigated for their antibacterial activity against Listeria monocytogenes. Carvacrol and thymol were found to have the strongest antilisterial properties, followed by eugenol, cinnamaldehyde and isoeugenol. The antilisterial activity of the other essential oils (limonene, pinene, ally lisothiocyanate and linalool) was found to be low, even at the highest concentration used (0.1%). Among the diglycerol esters of fatty acids with different carbon chain lengths, diglycerol monolaurate was the most active against L. monocytogenes. A combined antilisterial effect was observed between nisin and the essential oils (carvacrol, thymol and eugenol); moreover, the addition of diglycerol monolaurate as a third preservative factor led to further combined antilisterial activities between the essential oil constituents (carvacrol, thymol and eugenol) and nisin even at lower, sub-lethal concentrations. These results indicate that nisin and diglycerol monolaurate can be used to enhance the antilisterial activity of essential oils, allowing for a reduction in the dosage used in food preservation and thereby resulting in the reduction of undesirable flavors.     

Influence of carvacrol on growth and toxin production by Bacillus cereus

The natural antimicrobial compound carvacrol was investigated for its effect on diarrheal toxin production by Bacillus cereus. Carvacrol (0-0.06 mg/ml) reduced the viable count and the maximal specific growth rate (mumax) of B. cereus in BHI broth. The total amount of protein was not affected by carvacrol. However, a sharp decrease (80%) in diarrheal toxin production was observed in the presence of 0.06 mg/ml carvacrol. Carvacrol also inhibited toxin production in soup, but approximately 50-fold higher concentrations were needed to achieve the same effect as in broth. From this study it can be concluded that carvacrol can be added to food products at doses below the MIC value, thereby reducing the risk of toxin production by B. cereus and increasing the safety of the products.     

Antibacterial Activity of Carvacrol, Citral, and Geraniol against Salmonella typhimurium in Culture Medium and on Fish Cubes

Carvacrol, citral and geraniol showed potent antibacterial activity against Salmonella typhimurium and its rifampicin-resistant (Rif^R) strain as determined in txyptic soy broth and by zone of inhibition on agar-based medium. Carvacrol had the most potent bactericidal activity, with minimum inhibitory and bactericidal concentrations (MIC and MBC) of 250 μg/mL for both tester strains. When tested at 0.5, 1.5 and 3.0% in 1% Tween 20 for bactericidal activity against Rif^R-S. typhimurium inoculated on fish cubes, carvacrol at 3.0% completely killed the inoculated bacteria, while geraniol killed most of the bacteria, and citral killed the least. Carvacrol and geranial showed potent antibacterial activity at 1.5%. Bactericidal activity became more evident as storage of fish cubes at 4°C lengthened. The comparable inhibition of these strains of Salmonella and species of Gram-negative bacteria by carvacrol and geraniol support their application as potential antibacterial agents in food systems.     

Influence of food matrix on inactivation of Bacillus cereus by combinations of nisin, pulsed electric field treatment, and carvacrol.

Carvacrol was used as a third preservative factor to enhance further the synergy between nisin and pulsed electric field (PEF) treatment against vegetative cells of Bacillus cereus. When applied simultaneously with nisin (0.04 microg/ml), carvacrol (0.5 mM) enhanced the synergy found between nisin and PEF treatment (16.7 kV/cm, 30 pulses) in potassium-N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES) buffer. The influence of food ingredients on bactericidal activity was tested using skimmed milk that was diluted to 20% with sterile demineralized water. The efficacy of PEF treatment was not affected by the presence of proteins, and results found in HEPES buffer correlated well with results in milk (20%). Nisin showed less activity against B. cereus in milk. Carvacrol was not able to enhance the synergy between nisin and PEF treatment in milk, unless used in high concentrations (1.2 mM). This concentration in itself did not influence the viable count. Carvacrol did act synergistically with PEF treatment in milk, however not in HEPES buffer. This synergy was not influenced by proteins in milk, as 5% milk still allows synergy between carvacrol and PEF treatment to the same extent as 20% milk.     

Antioxidant activity and mechanism of action of thymol and carvacrol in two lipid systems

The autoxidation of purified triacylglycerols of lard and sunflower oil (TGL and TGSO) containing 0.02, 0.05, 0.10 and 0.20% thymol and carvacrol was studied at ambient temperature. The results obtained with the inhibited lipid systems (thymol - TGL; carvacrol - TGL; thymol - TGSO and carvacrol - TGSO) showed that thymol had the highest antioxidant effectiveness and activity during TGSO oxidation. Thymol and carvacrol participated in one side reaction during inhibited TGL oxidation, and thymol took part in two side reactions during TGSO oxidation. Carvacrol molecules did not participate in side reactions during TGSO oxidation. Thymol and carvacrol contributed to chain initiation to a higher degree during TGSO oxidation than during TGL oxidation. Thymol radicals did not participate in chain propagation during TGL and TGSO oxidation. Carvacrol radicals took part in one reaction of chain propagation in both lipid systems. In general, during autoxidation of lipids at ambient temperature, thymol is a more effective and more active antioxidant than carvacrol. Both antioxidants differ in the mechanism of their inhibiting action which depends on the character of the lipid medium. Thymol is a better antioxidant in TGSO than in TGL, whereas the activity of carvacrol in the two lipid systems does not differ significantly.     

香芹酚对阴沟肠杆菌的抑菌作用及机理

香芹酚具有较好的抑菌效果,符合人们对食品防腐剂安全无毒、无副作用的要求。本研究通过最小抑菌浓度和杀菌实验分析了香芹酚对阴沟肠杆菌（Enterobacter cloacae C4）的抑菌活性,并检测其对阴沟肠杆菌细胞膜电势、胞外ATP化学发光值的影响,使用激光共聚焦显微镜观察细胞渗透性,并通过扫描电子显微镜观察香芹酚对阴沟肠杆菌细胞形态的影响,从而探究香芹酚对阴沟肠杆菌的抑菌机理。结果表明：香芹酚对阴沟肠杆菌的最小抑菌浓度为0.5 μL/mL;阴沟肠杆菌细胞膜通透性随着香芹酚含量（0.25～2 μL/mL）的增加而逐渐增加。经香芹酚处理的阴沟肠杆菌细胞膜内外电势改变,ATP大量漏出。通过扫描电子显微镜观察,发现经香芹酚处理的菌体细胞形态干瘪且明显被破坏,含量高于1 μL/mL的香芹酚几乎能够溶解菌体。推测香芹酚通过作用于细胞膜改变膜内外电势,造成细胞膜通透性改变,引起细胞ATP等内容物泄露,从而影响细胞功能,达到抑菌的作用。     

Use of carvacrol and cymene to control growth and viability of Listeria monocytogenes cells and predictions of survivors using frequency distribution functions

The antibacterial action of carvacrol and cymene on two Listeria monocytogenes strains (STCC4031 and NCTN4032) was studied. Carvacrol or cymene showed inhibitory effect on the growth of L. monocytogenes during lag and exponential growth phases and was more evident with increasing concentrations in brain heart infusion broth at 30 degrees C. Carvacrol or cymene also decreased the survival of mid-exponential-growth-phase L. monocytogenes STCC4031 cells in potassium-N-2-hydroxy-ethylpiperazine-N-ethanesulfo nic acid, at 30 degrees C. The combination of carvacrol and cymene resulted in an increased antibacterial effect on the growth and a synergistic effect on the viability of L. monocytogenes compared with the natural compounds applied separately. The analysis of survival curves by the Weibull frequency distribution function allowed an accurate prediction of the level of inactivation achieved. Interestingly, an important bactericidal effect (4.7-log reduction) of low concentrations of both antimicrobials combined (0.75 mM) was observed on L. monocytogenes in carrot juice. This study indicates the potential use of carvacrol and cymene applied simultaneously for preservation of minimally processed foods.     

Antimicrobial activity of carvacrol toward Bacillus cereus on rice

The antimicrobial activity of carvacrol, a compound present in the essential oil fraction of oreganum and thyme, toward the foodborne pathogen Bacillus cereus on rice was studied. Carvacrol showed a dose-related inhibition of growth of the pathogen. Concentrations of 0.15 mg/g and higher inhibited the growth and the extent of inhibition depended on the initial inoculum size. To decrease the input of carvacrol on the taste and flavor of the product, a combined treatment with the structure analog cymene was tested. Due to the smell and taste of carvacrol at high concentrations, carvacrol was combined with cymene, a natural antimicrobial compound with a similar structure. A synergistic effect was observed when 0.30 mg/g carvacrol was combined with 0.27 mg/g cymene. Finally it was demonstrated that a common taste enhancer like soya sauce also increased the antimicrobial action of carvacrol toward B. cereus. The antimicrobial activity of carvacrol with cymene or soya sauce was influenced by the addition of NaCl.     

Increase in activity of essential oil components carvacrol and thymol against Escherichia coli O157:H7 by addition of food stabilizers

The major components of oregano and thyme essential oils that had previously been shown to inhibit Escherichia coli O157:H7 were determined by high-performance liquid chromatography with UV detection and liquid chromatographic tandem mass spectrometry. The MICs and MBCs of carvacrol, thymol, p-cymene, and gamma-terpinene against a strain of E. coli O157: H7 phage type 34 isolated from bovine feces were determined by microdilution assay. The constituents were then tested in checkerboard assays to detect possible interactions. Carvacrol and thymol displayed bacteriostatic and bactericidal properties with MICs of 1.2 mmol/liter and were additive in combination. p-Cymene and gamma-terpinene displayed no measurable antibacterial activity up to 50 mmol/liter, and neither influenced the activity of carvacrol or thymol. Growth curves in the presence of nonlethal concentrations of carvacrol with the addition of agar (0.05%, wt/vol) or carrageenan (0.125%, wt/vol) as stabilizer were produced by optical density measurement. The stabilizers agar and carrageenan both significantly improved the effectiveness of carvacrol in broth, possibly because of a delay in the separation of the hydrophobic substrate from the aqueous phase of the medium. When carvacrol was dissolved in ethanol before addition to broth, stabilizers were not needed. Carvacrol and thymol, particularly when used in combination with a stabilizer or in an ethanol solution, may be effective in reducing the number or preventing growth of E. coli O157:H7 in liquid foods.     

Assessment of factors influencing antimicrobial activity of carvacrol and cymene against Vibrio cholerae in food

Carvacrol and cymene, phenolic compounds naturally present in the essential oil of oregano and thyme, were examined for their antimicrobial activity against Vibrio cholerae (ATCC 14033, VC1, and VC7) inoculated in carrot juice. Carvacrol exhibited a dose dependent inhibitory effect on the bacteria. Although cymene did not have antimicrobial activity against the bacteria, it enhanced the inhibitory ability of carvacrol. At 25 °C, the lowest concentrations of carvacrol and cymene required for zero detectable viable count varied depending on bacterial strains; 5 and 5 ppm, respectively, for VC7; 5 and 7.5 ppm, respectively, for VC1; and 7.5 and 7.5 ppm, respectively, for ATCC 14033. This study also examined several factors influencing the antimicrobial activity of carvacrol and cymene against V. cholerae ATCC 14033, including temperature, bacterial cell number, and food substrate. Carvacrol and cymene inhibited the bacterium in carrot juice at 25 °C more efficiently than at 15 and 4 °C. The doses of both compounds required for zero detectable viable count increased as the number of the bacterial cells in the carrot juice increased. The fat content and the complexity of foods were shown to decrease the antimicrobial activity of the compounds.     

Antifungal activities of selected aromatic plants growing wild in Greece

Essential oils of Origanum onites, Satureja thymbra, Salvia fruticosa (Greek sage), and Salvia pomifera subsp. calycina plants growing wild in Greece and their components carvacrol, camphor, and 1,8-cineole, were assayed for antifungal activity against 13 fungal species. Among the fungi tested were food poisoning, plant, animals and human pathogenic species. The oils presented various degrees of inhibition against all the fungi investigated. The highest and broadest activity was shown by the carvacrol content oils (O. onites and S. thymbra), while the oil of sage was the least effective. Carvacrol exhibited the highest and 1,8-cineole the lowest level of antifungal activity among the components tested.     

Preparation and Characterization of Carvacrol Loaded Polyhydroxybutyrate Nanoparticles by Nanoprecipitation and Dialysis Methods

In this investigation, preparation of carvacrol loaded polyhydroxybutyrate (PHB) nanoparticles was performed by nanoprecipitation and dialysis methods. PHB particles were obtained by nanoprecipitation method without and with low concentration of Tween 80 or pluronic as surfactant. Nano‐ and micro‐sized particles were formed with trimodal distribution and large aggregates. Size and distribution of nanoparticles were decreased when concentration of Tween 80 was increased to 1% (v/v) in water as polar phase. PHB nanoparticles had narrow size (157 nm) with monomodal distribution. Nanoparticles, which were prepared by dialysis method had 140 nm in diameter with monomodal distribution. Carvacrol was used as a lipophilic drug and entrapped in optimized nanoparticles formulation by nanoprecipitation and dialysis methods. Entrapment efficacy was 21% and 11%, respectively. Morphology of PHB nanoparticles was spherical. The results of kinetic release study showed that carvacrol was released for at least 3 days. Release kinetic parameters showed a simple Fickian diffusion behavior for both formulations. Carvacrol loaded PHB nanoparticles had good dispersion into the agar medium and antimicrobial activity against Escherichia coli. This study describes the 1st work on loading of carvacrol into the PHB nanoparticles by nanoprecipitation and dialysis methods.     

Antilisterial Activity of Hops Beta Acids in Broth with or Without Other Antimicrobials

ABSTRACT: Hops beta acids (HBA) are parts of hops flowers used to preserve wort and provide flavor in beer, and are reported as having antimicrobial properties. This study evaluated the antilisterial activity of HBA alone or in combination with other known antimicrobials in a culture broth medium. Listeria monocytogenes (10‐strain mixture) was inoculated (2.6 to 2.8 log CFU/mL) into tryptic soy broth supplemented with 0.6% yeast extract (TSBYE) without (control) or with HBA (0.5 to 5.0 μg/mL), potassium lactate (1.0%), sodium diacetate (0.25%), or acetic acid (0.1%), alone or in combination with HBA (0.5 to 3.0 μg/mL). Survival/growth of the pathogen during storage at 4 °C (35 d), 10 °C (20 d), or 25 °C (2 d) was periodically monitored by spiral plating onto tryptic soy agar plus 0.6% yeast extract. As expected, TSBYE without antimicrobials (control) supported rapid pathogen growth with growth rates of 0.40, 2.88, and 9.58 log CFU/mL/d at 4, 10, and 25 °C, respectively; corresponding Y_end values exceeded 9.0 log CFU/mL at 35, 20, and 2 d storage. HBA used alone (1.0 to 5.0 μg/mL) inhibited growth of L. monocytogenes at all 3 temperatures, with inhibition being more pronounced at higher concentrations and at the lower storage temperature (4 °C). The antilisterial activity of HBA (0.5 to 3.0 μg/mL) was enhanced when combined with sodium diacetate, acetic acid, or potassium lactate, achieving complete inhibition at 4 °C when 3.0 μg/mL HBA were used in combination with each of the above antimicrobials. Overall, HBA exhibited promising antilisterial activity in a broth medium and further studies are needed to investigate its potential antilisterial effects in food products.     

Inhibitory effects of Satureja hortensis L. essential oil on growth and aflatoxin production by Aspergillus parasiticus

In an effort to screen the essential oils of some Iranian medicinal plants for novel aflatoxin (AF) inhibitors, Satureja hortensis L. was found as a potent inhibitor of aflatoxins B1 (AFB1) and G1(AFG1) production by Aspergillus parasiticus NRRL 2999. Fungal growth was also inhibited in a dose-dependent manner. Separation of the plant inhibitory substance(s) was achieved using initial fractionation of its effective part (leaf essential oil; LEO) by silica gel column chromatography and further separation by reverse phase-high performance liquid chromatography (RP-HPLC). These substances were finally identified as carvacrol and thymol, based on the interpretation of 1H and 13C NMR spectra. Microbioassay (MBA) on cell culture microplates contained potato-dextrose broth (PDB) medium (4 days at 28 degrees C) and subsequent analysis of cultures with HPLC technique revealed that both carvacrol and thymol were able to effectively inhibit fungal growth, AFB1 and AFG1 production in a dose-dependent manner at all two-fold concentrations from 0.041 to 1.32 mM. The IC50 values for growth inhibition were calculated as 0.79 and 0.86 mM for carvacrol and thymol, while for AFB1 and AFG1, it was reported as 0.50 and 0.06 mM for carvacrol and 0.69 and 0.55 mM for thymol. The results obtained in this study clearly show a new biological activity for S. hortensis L. as strong inhibition of aflatoxin production by A. parasiticus. Carvacrol and thymol, the effective constituents of S. hortensis L., may be useful to control aflatoxin contamination of susceptible crops in the field.     

Synergistic effect of thymol and carvacrol combined with chelators and organic acids against Salmonella Typhimurium

To identify synergistic combinations of different food additives, the antimicrobial effects of thymol and carvacrol against Salmonella Typhimurium were assessed alone and in combination with various other preservatives including EDTA, acetic acid, lactic acid, and citric acid. Overall, growth of Salmonella Typhimurium was significantly inhibited in Mueller-Hinton broth containing thymol, carvacrol, EDTA, acetic acid, lactic acid, or citric acid at concentrations of 400 mg/liter, 400 microl/liter, 300 mg/liter, 0.2% (vol/vol), 0.2% (vol/vol), and 0.2% (wt/vol), respectively. The combination of different antimicrobials such as thymol or carvacrol with EDTA, thymol or carvacrol with acetic acid, and thymol or carvacrol with citric acid all resulted in significantly reduced populations of Salmonella Typhimurium. In samples treated with combinations, these antimicrobials had synergistic effects compared with samples treated with thymol, carvacrol, EDTA, acetic acid, or citric acid alone. However, the combined use of lactic acid with thymol or carvacrol did not produce a synergistic effect against Salmonella Typhimurium. Thus, some chelators or organic acids can be used as food preservatives in combination with thymol and carvacrol to reduce the concentrations needed to produce an adequate antimicrobial effect.     

Fine-tuning Food Safety Objectives and risk assessment

Food Safety Objectives (FSOs) have been proposed as a practical tool to translate public health targets for food safety into tolerable levels of pathogens in a food product. The FSO concept is subject to intensive debate, and has not been developed in detail. We evaluate the proposed definition of FSOs and their implementation from the perspective of Quantitative Microbiological Risk Assessment (QMRA). The relationship between QMRA and FSOs is illustrated by a model for the public health risk of Shiga-producing Escherichia coli in steak tartare. We conclude that the proposed definition of FSOs needs to be modified to properly account for variability in and uncertainty about the contamination of food with pathogenic microorganisms and emphasize that both prevalence and concentration of pathogens must be considered. For this purpose, we propose the P-D equivalence curve, a simple graphical tool to separate "tolerable" from "non-tolerable" combinations of prevalence and concentration (dose).     

Inactivation of Listeria monocytogenes on ham and bologna using pectin-based apple, carrot, and hibiscus edible films containing carvacrol and cinnamaldehyde

Edible films can be used as wrapping material on food products to reduce surface contamination. The incorporation of antimicrobials into edible films could serve as an additional barrier against pathogenic and spoilage microorganisms that contaminate food surfaces. The objective of this study was to investigate the antimicrobial effects of carvacrol and cinnamaldehyde, incorporated into apple, carrot, and hibiscus-based edible films against Listeria monocytogenes on contaminated ham and bologna. Ham or bologna samples were inoculated with L. monocytogenes and dried for 30 min, then surface wrapped with edible films containing the antimicrobials at various concentrations. The inoculated, film-wrapped samples were stored at 4 °C. Samples were taken at day 0, 3, and 7 for enumeration of surviving L. monocytogenes by plating on appropriate media. Carvacrol films showed better antimicrobial activity than cinnamaldehyde films. Compared to control films without antimicrobials, films with 3% carvacrol induced 1 to 3, 2 to 3, and 2 to 3 log CFU/g reductions on ham and bologna at day 0, 3, and 7, respectively. Corresponding reductions with 1.5% carvacrol were 0.5 to 1, 1 to 1.5, and 1 to 2 logs, respectively. At day 7, films with 3% cinnamaldehyde reduced L. monocytogenes population by 0.5 to 1.5 and 0.5 to 1.0 logs on ham and bologna, respectively. Inactivation by apple films was greater than that by carrot or hibiscus films. Apple films containing 3% carvacrol reduced L. monocytogenes population on ham by 3 logs CFU/g on day 0 which was 1 to 2 logs greater than that by carrot and hibiscus films. Films were more effective on ham than on bologna. The food industry and consumers could use these films to control surface contamination by pathogenic microorganisms. PRACTICAL APPLICATION: Antimicrobial edible, food-compatible film wraps prepared from apples, carrots, and hibiscus calyces can be used by the food industry to inactivate Listeria monocytogenes on widely consumed ready to eat meat products such as bologna and ham. This study provides a scientific basis for large-scale application of edible fruit- and vegetable-based antimicrobial films on foods to improve microbial food safety.     

Antibacterial activity against E. coli O157:H7, physical properties, and storage stability of novel carvacrol-containing edible tomato films

Edible films containing plant antimicrobials are gaining importance as potential treatment to extend product shelf life and reduce risk of pathogen growth on contaminated food surfaces. The main objective of the present study was to evaluate the antimicrobial activities, storage stabilities, and physical-chemical-mechanica1 properties of novel edible films made from tomatoes containing carvacrol, the main constituent of oregano oil. The antimicrobial activities against E. coli O157:H7 and the stability of carvacrol were evaluated during the preparation and storage of tomato-based films made by 2 different casting methods, continuous casting and batch casting. Antimicrobial assays of tomato films indicated that optimum antimicrobial effects occurred with carvacrol levels of approximately 0.75% added to tomato purees before film preparation. HPLC analysis of the films indicated that the carvacrol concentrations and bactericidal effect of the films remained unchanged over the storage period of up to 98 d at 5 and 25 degrees C. Carvacrol addition to the tomato puree used to prepare the films increased water vapor permeability of tomato films. The continuous method for casting of the films appears more suitable for large-scale use than the batch method. This 1st report on tomato-based edible antimicrobial tomato films suggests that these films have the potential to prevent adverse effects of contaminated food and promote human health associated with the consumption of tomatoes.     

Protective effect of carvacrol from <Emphasis Type="Italic">Thymus quinquecostatus</Emphasis> Celak against <Emphasis Type="Italic">tert</Emphasis>-butyl hydroperoxide-induced oxidative damage in Chang cells

Carvacrol is a monoterpenic phenol present in Thymus quinquecostatus Celak. The protective effects of carvacrol against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage were investigated in human Chang cells. Cells treated with carvacrol extracts promoted Chang cell survival and protection was associated with stabilization of the mitochondrial membrane potential (MMP), prevention of oxidative stress-triggered reactive oxygen species (ROS), and lipid peroxidation (MDA production). In addition, Annexin V/PI, observed using Hoechst staining, indicated that carvacrol inhibited t-BHP-induced cell damage and stimulated the antioxidant capability of Chang cells due to elevation of glutathione (GSH) levels, which were reduced by t-BHP treatment. Carvacrol prevented oxidative stress-induced Chang cell damage via suppression of ROS and MDA levels, and increased GSH levels.     

Effects of carvacrol, (E)-2-hexenal, and citral on the thermal death kinetics of Listeria monocytogenes

Carvacrol, (E)-2-hexenal, and citral at sublethal concentrations combined with isothermal heating between 55 and 68°C were assessed for their effects on Listeria monocytogenes 56LY. Experimental survival curves were obtained and fitted to the Weibull equation to estimate parameters describing their shape and rate. These parameters were further used to assess the impact of this combination of treatments on the cell resistance distribution during inactivation. The sublethal concentrations of the aroma compounds used (i.e., 50 mg/liter citral, 65 mg/liter (E)-2-hexenal, and 30 mg/liter carvacrol) did not prevent the growth of L. monocytogenes at 37°C but did enhance inactivation. Between 55 and 63°C, the presence of the aroma compounds reduced by about two-thirds the time needed for a 5-log reduction of the microbial counts, e.g., from 145.75 h in the control treatment (at 55°C) to 40.84 h in the presence of carvacrol (at the same temperature). The mean and variance observed in the frequency distribution of resistance were reduced as the temperature increased. The results obtained at isothermal temperatures and with single aroma components provide basic information regarding components frequently found in essential oils, which can be used in combination with less extreme thermal treatments to provide energy conservation and improve food quality.