Carvacrol Nanoemulsions Research Articles

Improving the bactericidal activity of carvacrol against Bacillus cereus by the formation of sodium casein-stabilized nanoemulsion and its application in milk preservation

Carvacrol has great antibacterial activity against Bacillus cereus, and its action mechanism was comprehensively studied in this work. It could not only control the growth of B. cereus by affecting the integrality of cellular structure and physiological metabolism, but also inhibit the swarming motility, biofilm formation, and toxins production. To increase the stability of carvacrol during the application, the sodium casein (SC)/hydroxypropyl-β-cyclodextrin (HPCD)/carvacrol nanoemulsion was fabricated. The mean particle size and polydispersity index of the nanoemulsions were 113.8 nm and 0.23, respectively, at SC and HPCD concentrations of 2% and 1%. FTIR analysis demonstrated that carvacrol was successfully encapsulated into the nanoemulsions. The MIC and MBC of nanoemulsions against B. cereus were 0.15 and 0.30 mg/mL, respectively, which were lower than those of free carvacrol (0.19 and 0.38 mg/mL). Carvacrol in nanoemulsions showed a slow and sustained release behavior. Furthermore, the nanoemulsions had good stability (particle size <200 nm) after storage at 4 °C and 25 °C for 15 days. Notably, the nanoemulsions were effective in controlling the growth of B. cereus in whole, low-fat, and skim milk and had better antimicrobial activity than free carvacrol. The results indicated that carvacrol nanoemulsions have potential in the antibacterial application of the food industry.

Succinylated starch emulsified Eugenol and Carvacrol nanoemulsions improved digestive stability, bio-accessibility and Salmonella typhimurium inhibition

The ultrasonically processed Eugenol (EU) and Carvacrol (CAR) nanoemulsions (NE) were successfully optimized via response surface methodology (RSM) to achieve broad spectrum antimicrobial efficacy. These NE were prepared using 2 % (w/w) purity gum ultra (i.e., succinylated starch), 10 % (v/v) oil phase, 80 % (800 W) sonication power, and 10 min of processing time as determined via RSM. The second order Polynomial method was suitable to RSM with a co-efficient of determination >0.90 and a narrow polydispersity index (PDI) ranging 0.12–0.19. NE had small droplet sizes (135.5–160 nm) and low volatility at high temperatures. The EU & CAR entrapment and heat stability (300 °C) confirmed by Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). Further, the volatility of EU & CAR NE was 18.18 ± 0.13 % and 12.29 ± 0.11 % respectively, being lower than that of bulk/unencapsulated EU & CAR (i.e., 23.48 ± 0.38 % and 19.11 ± 0.08 %) after 2 h at 90 °C. Interestingly, both EU & CAR NE showed sustained release behaviour till 48 h. Their digest could inhibit Salmonella typhimurium (S. typhimurium) via membrane disruption and access to cellular machinery as evident from SEM images. Furthermore, in-vivo bio-accessibility of EU & CAR in mice serum was up to 80 %. These cost-effective and short-processed EU/CAR NE have the potential as green preservatives for food industry.

Carvacrol Microemulsion vs. Nanoemulsion as Novel Pork Minced Meat Active Coatings.

Carvacrol is well documented for its antibacterial and antioxidant effects. However, its high volatility has directed researchers toward nanoencapsulation technology according to bioeconomy and sustainability trends. This study examined and compared free carvacrol (FC), carvacrol microemulsion (MC), carvacrol microemulsion busted with chitosan (MMC), and carvacrol nanoemulsions (NC) as active coatings on extending minced pork meat shelf life at 4 ± 1 °C for 9 days, focusing on microbiological, physiochemical, and sensory characteristics. The research involved pre-characterizing droplet sizes, evaluating antioxidants, and determining antibacterial efficacy. The results demonstrated that NC with a 21 nm droplet size exhibited the highest antioxidant and antibacterial activity. All coatings succeeded in extending the preservation of fresh minced pork meat in comparison to the free carvacrol sample (FC). The NC coating showed the highest extension of minced pork meat preservation and maintained meat freshness for 9 days, with a lower TBARs of 0.736 mg MDA/Kg, and effectively reduced mesophilic, lactic acid, and psychotrophic bacterial counts more significantly by 1.2, 2, and 1.3 log, respectively, as compared to FC. Sensory assessments confirmed the acceptability of NC and MCC coatings. Overall, the carvacrol-based nanoemulsion can be considered a novel antioxidant and antimicrobial active coating due to its demonstrated higher efficacy in all the examined tests performed.

CFD and experimental microfluidic study of the interaction between a natural compound nanoemulsion and E. coli bacterium considering reaction-diffusion-convection model

Essential oils have recently emerged as potent natural antibacterial agents that are effective against pathogenic bacteria. The antibacterial efficacy of a stabilized nanoemulsion of carvacrol (with a mean droplet size of 123.51 nm ± 4.2 nm) on a Gram-negative Escherichia coli (E. coli ATCC 25922) was experimentally and numerically studied utilizing a continuous-flow microfluidic system. The morphology of the destroyed bacteria was examined visually and, bacteria lysis was confirmed by measuring the release of cellular contents. After establishing the antibacterial capability kinetics of the carvacrol nanoemulsions, the acquired data from the time killing assay was used to characterize the E. coli lysis. Moreover, a computational fluid dynamics (CFD) model was implemented to evaluate the influence of altering key parameters such as the Reynolds number, flow rate ratio and inlet carvacrol concentration on the unlysed bacteria percentage at the outlet (Y1) and the mass flow rate (μg/s) of the lysed bacteria at the outlet (Y2). The maximum lysis reaction rate observed in the device was 0.96s-1 corresponding to an inlet carvacrol nanoemulsion concentration of 160μg/mL. The reaction rate (r) was established based on the carvacrol concentration (Aμg/mL) as r=-0.149A0.373. The results obtained from numerical simulations indicated that the Reynolds number causes the most changes in the average value of Y1, while carvacrol concentration impacted most on the average value of Y2 response. The findings presented in this article provide insight into the probable kinetics of carvacrol nanoemulsion antibacterial activity in the microfluidics system.

Preparation and characterization of dialdehyde starch-soybean protein isolate stabilized carvacrol emulsion composite films

In order to reduce the harm of “petroleum-based plastics” with the problem of health and environment, the emulsion composite films with carvacrol nano-emulsions as antibacterial agent and polyvinyl alcohol (PVA) as matrices were prepared via the solvent-casting method. The average particle size of nano-emulsions was 175 nm with PDI value being 0.240. The transmittance of the emulsion composite films was close to zero in the UV-B region, with obvious UV barrier performance. The DPPH free radical scavenging rate was 60.96%, and the inhibition zone of the film forming emulsions was (21.5 ± 0.87) mm for S.aureus and (26.8 ± 1.01) mm for E.coli. Compared with the pure PVA film, the tensile strength and the water contact angle of the emulsion composite films were improved, the water vapor permeability was decreased by 28.7%. X-ray Diffraction and Fourier Transform Infrared Spectroscopy results showed the successful introduction of carvacrol nano-emulsions without a changeable structure. Therefore, the emulsion composite films have shown obvious application potential as packaging materials in modern food packaging.

Antibacterial food packaging capable of sustained and unidirectional release carvacrol/thymol nanoemulsions for pork preservation

Pork is highly susceptible to microbial spoilage and oxidative deterioration. In this work, an active packaging film capable of sustained and unidirectional release natural antimicrobials was developed to prolong the shelf life of chilled pork. Thymol&carvacrol nanoemulsions (TH&CA NEs) with two active constituents were prepared by low-energy spontaneous emulsification method and showed excellent antioxidant and antibacterial abilities. A trilayer film composed of loading, adhesive and barrier layers were fabricated using polyquaternium-10 (PQ-10)&carboxymethyl chitosan (CMC) microgels (denoted as PC), mixed solution of PQ-10 and zein (Z), and zein solution as raw material of each layer by layer-by-layer casting method. TH&CA@PC-PZ-Z packaging films were obtained by adding dropwise TH&CA NEs onto PC loading layer of PC-PZ-Z film and showed good interlaminar bonding strength, suitable water retention, satisfactory biological safety, as well as great antioxidant and antibacterial activities. In addition, most of TH&CA NEs in TH&CA@PC-PZ-Z films could be released from PC loading layer in a sustainable way for at least 7 days due to the block of Z barrier layer. The preservation effects of TH&CA@PC-PZ-Z films on chilled pork were further investigated in comparison with a plastic fresh-keeping film made by low-density polyethylene (LDPE). Results showed that TH&CA@PC-PZ-Z films could effectively retard lipid and protein oxidation and inhibit microbial growth during 11 days storage, thereby prolonged the shelf-life of chilled pork. We believe that the sustainable and unidirectional drug release film can be developed as a promising packaging film in preserving fresh meat products.

Carvacrol-loaded nanoemulsions produced with a natural emulsifier for lettuce sanitization

Carvacrol is an antimicrobial agent that shows potential for eliminating microorganisms in vegetables, increasing food safety. However, intense odor and low water solubility of carvacrol are limiting factors for its application for fresh vegetables sanitization, which can be overcome by nanotechnology. Two different nanoemulsions containing carvacrol (11 mg/mL) were developed by probe sonication: carvacrol-saponin nanoemulsion (CNS) and carvacrol-polysorbate 80 nanoemulsion (CNP). Formulations presented appropriate droplet sizes (from 74.7 nm to 168.2 nm) and high carvacrol encapsulation efficiency (EE) (from 89.5 % to 91.5 %). CNS showed adequate droplet size distribution (PDI < 0.22) and high zeta potential values (around −30 mV) compared to CNP, with saponin chosen for the following experiments. Carvacrol nanoemulsions presented Bacterial Inactivation Concentration (BIC) against the Salmonella cocktail from 5.51 to 0.69 mg/mL and for the E. coli cocktail from 1.84 to 0.69 mg/mL. Among all tested nanoemulsions, CNS1 presented the lowest BIC (0.69 mg/mL) against both bacterial cocktails. Damage to bacterial cells in lettuce treated with nanoemulsion was confirmed by scanning electron microscopy. For lettuce sanitization, CNS1 showed a similar effect to unencapsulated carvacrol, with a high bacterial reduction (>3 log CFU/g) after lettuce immersion for 15 min at 2 × BIC. Using the same immersion time, the CNS1 (2 × BIC) demonstrated equal or better efficacy in reducing both tested bacterial cocktails (>3 log CFU/g) when compared to acetic acid (6.25 mg/mL), citric acid (25 mg/mL), and sodium hypochlorite solution (150 ppm). Lettuce immersed in CNS1 at both concentrations (BIC and 2 × BIC) did not change the color and texture of leaves, while the unencapsulated carvacrol at 2 × BIC darkened them and reduced their firmness. Consequently, carvacrol-saponin nanoemulsion (CNS1) proved to be a potential sanitizer for lettuce.

Suppression effect of thyme and carvacrol nano-emulsions on Aspergillus fumigatus isolated from patients in the intensive care unit of Assiut University Hospital, Egypt

Background and Aim: Aspergillus fumigatus is a zoonotic fungus that causes several diseases in humans ranging from allergic reaction to fatal disseminated invasive infection, especially in immunocompromised patients. This study aimed to investigate the incidence of invasive A. fumigatus in patients admitted to the intensive care unit (ICU) of Assiut University Hospital, highlight the factors associated with their infection, and determine the antifungal effect of thyme nano-emulsion (TNE) and carvacrol nano-emulsion (CNE) on isolated A. fumigatus strains. Materials and Methods: Mycological culture method and scanning electron microscopy (SEM) were used in the identification of A. fumigatus in 630 blood samples collected from 210 patients. TNE and CNE at five concentrations (1%, 2%, 4%, 6%, and 8%) and average sizes of 90.3 and 75.6 nm, respectively, were characterized by transmission electron microscopy. Their effect on A. fumigatus isolate growth was evaluated by the well-diffusion method and SEM, which was used for the detection of the degenerative effect of A. fumigatus ultrastructure. Results: A. fumigatus was detected in 54 of 210 (25.7%) patients in the ICU. Advanced age and chronic diseases were considered important risk factors for invasive aspergillosis, especially in patients with more than 1 clinical disease. TNE and CNE showed an inhibitory effect on A. fumigatus isolates, which significantly increased with high concentrations. The respective values for TNE at concentrations of 6% and 8% were 6±0.41 mm and 15±0.67 mm. CNE completely inhibited A. fumigatus growth at concentrations of 4%, 6%, and 8%, while mean inhibition zones of 22±0.68 mm and 30±0.32 mm appeared at concentrations of 1% and 2%. SEM demonstrated degenerative changes in A. fumigatus structure. Conclusion: TNE and CNE can be used in bioactive treatments against A. fumigatus, and additional studies are required to determine the safe and effective doses and best method for application in human and veterinary medicine.

Investigate the adverse effects of foliarly applied antimicrobial nanoemulsion (carvacrol) on spinach

Encapsulated essential oils are being developed as natural antimicrobials and insecticides for use in sustainable agriculture applications. Although these plant-based pesticides are considered to be more environmentally friendly, they can have adverse effects on agricultural crops, thereby reducing yields. Our aim was therefore to establish the potential phytotoxicity of carvacrol nanoemulsions on a commercially important crop (spinach, Spinacia oleracea). Carvacrol oil-in-water nanoemulsions were fabricated by homogenizing 10 wt% oil phase (1:1 carvacrol and medium chain triglycerides) with 90% aqueous phase (1% quillaja saponin in 5 mM sodium citrate buffer, pH 3.5). Nanoemulsions were sprayed onto the spinach leaves by foliar exposure for three days and then the health status of the plants was assessed. At relatively low carvacrol concentrations (0.005–0.5%), the plants remained healthy, but at the highest concentration employed (5%) there was a significant reduction in the biomass and chlorophyll content of the spinach, as well as an increase in electrolyte leakage and malondialdehyde formation. We demonstrated that this strong phytotoxicity was due to the carvacrol oil, rather than due to other constituents in the nanoemulsions. Our results show that there is a critical threshold concentration for use of essential oil nanoemulsions as pesticides on crop plants.

Antifungal Activity of Thyme and Carvacrol Nanoemulsions Against Aspergillus fumigatus Isolated from Different Types of Cheese

Antifungal Activity of Thyme and Carvacrol Nanoemulsions Against Aspergillus fumigatus Isolated from Different Types of Cheese

Development and characterization of corn starch/PVA active films incorporated with carvacrol nanoemulsions

An active film was prepared by corn starch (CS), polyvinyl alcohol (PVA) and carvacrol nanoemulsions (CNE). The microstructure and properties of CNE/corn starch/PVA (CNE/CSP) films were characterized and investigated. Scanning electron microscopy (SEM) revealed the uniform distribution of CNE and discontinuity of the film matrix. Fourier transform infrared (FT-IR) and rheological analysis indicated that CNE could weaken molecular interaction of the film matrix. X-ray diffraction (XRD) show that the films are amorphous and CNE has no effect on crystal structure of the films. Incorporation of CNE significantly increased the tensile strength, Young's modulus, elongation at break, barrier (water vapor and ultraviolet), antioxidant and antifungal activity. With the CNE incorporated, the optimal tensile strength, Young's modulus, elongation at break and antioxidant activity of the films can reach 12 MPa, 11 MPa, 133%, 81%, respectively. Minimum water vapor permeability was 3.1 × 10−12 gd−1m−1Pa−1. Notably, films incorporated with CNE (≥20%) had good DPPH free radical scavenging ability (>50%) when stored up to 6 days. Films with 25% CNE exhibited excellent antifungal activity against Trichoderma sp. and its inhibitory zone was 47 mm. Overall, the CSP films loaded with CNE (>15%) could be used as food packing materials with good antioxidant and antimicrobial activities.

The innovative fabrication and applications of carvacrol nanoemulsions, carboxymethyl chitosan microgels and their composite films.

Carvacrol (CA) was firstly made into physically stable nanoemulsions using an innovative spontaneous emulsification method with carvacrol itself as oil phase without introducing other carrier oils. Self-crosslinking carboxymethyl chitosan (CMC) microgels via intermolecular hydrogen bonds were then fabricated using a simple solvent exchange method with ethanol as poor solvent. The CMC microgels obtained were rapidly deposited on the surface of tin foil using an electrospray technique to produce the CMC films. The stability of the films in water was further intensified by using Ca2+ as cross-linking agent. Also, the carvacrol nanoemulsions (CA-NEs) were loaded into the CMC films by utilizing swelling property of CMC microgels in aqueous solution. The results showed that the CA-NEs@CMC composite films possess satisfactory antioxidant activity, good antibacterial activity against S. aureus and E. coli, and excellent ability to extend the shelf life of wheat bread. We believe that the present strategies to prepare CA-NEs, CMC microgels and their composite films also apply to other types of essential oils and polymers, and the CA-NEs@CMC composite film is an excellent candidate for the active packaging.

Natural antimicrobial delivery systems: Formulation, antimicrobial activity, and mechanism of action of quillaja saponin-stabilized carvacrol nanoemulsions

The objective of this research was to develop antimicrobial nanoemulsions using quillaja saponin as a natural surfactant, carvacrol as an antimicrobial agent, and medium chain triglyceride (MCT) oil as a ripening inhibitor. Oil-in-water antimicrobial nanoemulsions were fabricated by homogenizing 10 wt% oil phase (carvacrol and MCT) with 90 wt% aqueous phase (quillaja saponin in 5 mM sodium citrate buffer, pH 3.5). The physical stability and antimicrobial activity of these systems was compared to nanoemulsions formed using a synthetic non-ionic surfactant (Tween 80). Stable nanoemulsions with small mean droplet diameters (d < 150 nm) could be formed by optimizing oil phase composition and surfactant level. The antimicrobial activity of carvacrol nanoemulsions formulated with Q-Naturale® was higher than that formulated with Tween 80. Dialysis and chromatography were used to monitor the transfer of carvacrol from one hydrophobic phase to another, to mimic transport from oil droplets to bacterial cell membranes. The antimicrobial activity depended on the concentration and type of surfactant used and the ability of surfactant micelles to transfer carvacrol through the aqueous phase.

Decontamination of fresh-cut cucumber slices by a combination of a modified chitosan coating containing carvacrol nanoemulsions and pulsed light

In this study, the impact of the combination of pulsed light (PL) treatments with antimicrobial coatings, consisting of modified chitosan suspensions incorporating carvacrol nanoemulsions, was investigated on the decontamination of fresh-cut cucumber slices.The upper surface of the cucumber slices, with or without the coating deposition, was inoculated with Escherichia coli ATCC 26 to reach a final concentration of 107CFU/g of the vegetable. PL treatments were conducted at different fluence (4, 8, and 12J/cm2) on the inoculated surface of cucumber slices.Results showed that the microbial reduction was only marginally affected by the coating formulation. A slight increase was observed when the carvacrol nanoemulsions were embedded in the chitosan matrix, but microbial reduction levels remained always below 1log cycle. In contrast, the different PL treatments resulted in a statistically significant increase in inactivation with increasing the treatment fluence, reaching 2.6log cycles at the maximum fluence.Remarkably, the combination of the antimicrobial coating with the most intense PL treatments resulted in a strong synergistic effect. For example, by combining a PL treatment at 12J/cm2 with one of the antimicrobial coatings a microbial reduction >5log cycles was reached.Therefore, it can be concluded that the combination of antimicrobial coatings and PL treatment is a promising method for surface decontamination of fresh-cut vegetables, which could be exploited in view of ensuring their microbiological safety.

Inactivation of Salmonella on Sprouting Seeds Using a Spontaneous Carvacrol Nanoemulsion Acidified with Organic Acids

Over the past decade, demand has increased for natural, minimally processed produce, including sprout-based products. Sanitization with 20,000 ppm of calcium hypochlorite is currently recommended for all sprouting seeds before germination to limit sprout-related foodborne outbreaks. A potentially promising disinfectant as an alternative to calcium hypochlorite is acidified spontaneous essential oil nanoemulsions. In this study, the efficacy of an acidified carvacrol nanoemulsion was tested against mung beans and broccoli seeds artificially contaminated with a Salmonella enterica Enteritidis cocktail (ATCC BAA-709, ATCC BAA-711, and ATCC BAA-1045). Treatments were performed by soaking inoculated seeds in acidified (50 mM acetic or levulinic acid) carvacrol nanoemulsions (4,000 or 8,000 ppm) for 30 or 60 min. After treatment, the number of surviving cells was determined via plate counts and/or the most probable number (MPN) approach. Treatment for 30 min successfully reduced Salmonella Enteritidis by 4 log CFU/g on mung beans (from an initial contamination level of 4.2 to 4.6 log CFU/g) and by 2 log CFU/g on broccoli seeds (from an initial contamination level of 2.4 to 2.6 log CFU/g) to below our detection limit (≤3 MPN/g). Treated seeds were sprouted and tested for the presence of pathogens and sprout yield. The final sprout product had no detectable pathogens, and total sprout yield was not influenced by any treatment.

Understanding the effect of formulation on functionality of modified chitosan films containing carvacrol nanoemulsions

The interest towards the use of films and coatings in food preservation has been reinforced in the recent years by the development of biopolymeric matrices incorporating essential oil as antimicrobial barriers in food as an alternative to synthetic additives.This work has therefore been addressed to investigate the effect of composition parameters on antimicrobial activity and properties of films based on modified chitosan containing different types of carvacrol nanoemulsions. Specifically, response surface methodology, applied for the concentration of biopolymer and carvacrol nanoemulsion in the film forming dispersions to maximize the antimicrobial activity against two model microorganisms, Escherichia coli and Listeria innocua, as well as surface hydrophobicity, was used to determine the optimum conditions for comparison of most promising systems.Results showed that emulsion formulations had a significant effect on the intrinsic antimicrobial activity, but also that their interaction with the modified chitosan matrix affected film properties and resulting bactericidal action. The two most active emulsion formulations, based on a combination of polysorbate 20 and glycerol monooleate, and on whey protein isolates, respectively, when incorporated in modified chitosan films significantly increased the inhibition zone against E. coli and L. innocua from 7.2–7.4 mm (modified chitosan alone) to 13.4–16.1 mm, while still ensuring surface hydrophobicity of the film. In comparison to the use of pure carvacrol, the encapsulation into nanoemulsions also fostered the production of more homogeneous films with better appearance.This work hence contributes to promoting, through the advancement of the knowledge in the field, the incorporation of nanoemulsions of essential oils in edible films and coatings for integrated food preservation strategies.

Effectiveness of a spontaneous carvacrol nanoemulsion against Salmonella enterica Enteritidis and Escherichia coli O157:H7 on contaminated broccoli and radish seeds

The incidence of foodborne illness associated with the consumption of fresh produce has continued to increase over the past decade. Sprouts, such as mung bean, alfalfa, radish, and broccoli, are minimally processed and have been sources for foodborne illness. Currently, a 20,000 ppm calcium hypochlorite soak is recommended for the treatment of sprouting seeds. In this study, the efficacy of an antimicrobial carvacrol nanoemulsion was tested against Salmonella enterica subspecies enterica serovar Enteritidis (ATCC BAA-1045) or EGFP expressing Escherichia coli O157:H7 (ATCC 42895) contaminated sprouting seeds. Antimicrobial treatments were performed by soaking inoculated seeds in nanoemulsions (4000 or 8000 ppm) for 30 or 60 min. Following treatment, surviving cells were determined by performing plate counts and/or Most Probable Number (MPN) enumeration. Treated seeds were sprouted and tested for the presence of pathogens. Treatment successfully inactivated low levels (2 and 3 log CFU/g) of S. Enteritidis and E. coli on radish seeds when soaked for 60 min at concentrations ≥4000 (0.4%) ppm carvacrol. This treatment method was not affective on contaminated broccoli seeds. Total sprout yield was not influenced by any treatments. These results show that carvacrol nanoemulsions may be an alternative treatment method for contaminated radish seeds.

Effectiveness of a novel spontaneous carvacrol nanoemulsion against Salmonella enterica Enteritidis and Escherichia coli O157:H7 on contaminated mung bean and alfalfa seeds

Outbreaks of foodborne illness from consumption of sprouts have been linked to contaminated seeds prior to germination. Due to the long sprouting period at ambient temperatures and high humidity, germinating seeds contaminated with low pathogen levels (0.1logCFU/g) can result in sprouts with high numbers (≥108CFU/g) of pathogens. Currently, the recommended treatment method involves soaking seeds in 20,000ppm (2%) calcium hypochlorite prior to germination. In this study, an alternative treatment involving soaking seeds in a carvacrol nanoemulsion was tested for its efficacy against Salmonella enterica subspecies enterica serovar Enteritidis (ATCC BAA-1045) or EGFP expressing E. coli O157:H7 (ATCC 42895) contaminated mung bean and alfalfa seeds. The antimicrobial treatment was performed by soaking inoculated seed batches in the spontaneous nanoemulsion (4000 or 8000ppm) for 30 or 60min. The spontaneous nanoemulsion was formed by titrating the oil phase (carvacrol and medium chain triglycerides) and water-soluble surfactant (Tween 80®) into sodium citrate buffer. Following treatment, the numbers of surviving cells were determined by suspending the seeds in TSB and performing plate counts and/or Most Probable Number (MPN) enumeration. Treated seeds were sprouted and tested for the presence of the appropriate pathogen. This treatment successfully inactivated low levels (2 and 3logCFU/g) of S. Enteritidis and E. coli on either seed types when soaked for either 30 or 60min at nanoemulsion concentrations corresponding to 4000 (0.4%) or 8000 (0.8%) ppm carvacrol. Inoculated alfalfa seeds treated with 4000ppm nanoemulsion, required a 60min treatment time to show a similar 2–3 log reduction. Complete inactivation was confirmed by germinating treated seeds and performing microbiological testing. Total sprout yield was not compromised by any of the tested treatments. These results show that carvacrol nanoemulsions may be an alternative antimicrobial treatment method for mung bean and alfalfa seeds.

Physicochemical Properties and Antimicrobial Efficacy of Carvacrol Nanoemulsions Formed by Spontaneous Emulsification

A simple cost-effective method (spontaneous emulsification) for fabricating physically stable antimicrobial nanoemulsions from essential oils is described. These nanoemulsions (10 wt % total oil phase) were formed by titration of a mixture of essential oil (carvacrol), carrier oil (medium chain triglyceride, MCT), and nonionic surfactant (Tween) into an aqueous solution with continuous stirring. Oil phase composition (carvacrol-to-MCT mass ratio) had a major impact on initial droplet diameter, with the smallest droplets (d ≈ 55 nm) being formed at 2.5 wt % carvacrol and 7.5 wt % MCT. Surfactant type also had an appreciable impact on mean droplet diameter, with Tween 80 giving the smallest droplets (d ≈ 55 nm) from a group of food-grade nonionic surfactants (Tween 20, 40, 60, 80, and 85). The droplet size also decreased (from >5000 to <25 nm) as the total surfactant concentration was increased (from 5 to 20 wt %). The long-term stability and antimicrobial efficacy of selected nanoemulsions was examined at ambient temperature. The stability of the nanoemulsions to droplet growth during storage decreased as the carvacrol concentration in the oil phase increased. Conversely, the antimicrobial efficacy of the nanoemulsions increased as the carvacrol concentration increased. These results have important implications for the design and utilization of nanoemulsions as antimicrobial delivery systems in the food and other industries. They suggest that the carrier oil concentration must be carefully controlled to obtain good physical stability and antimicrobial efficacy.