Yuhua Chang

Influence of Surfactant Charge on Antimicrobial Efficacy of Surfactant-Stabilized Thyme Oil Nanoemulsions

Thyme oil-in-water nanoemulsions stabilized by a nonionic surfactant (Tween 80, T80) were prepared as potential antimicrobial delivery systems (pH 4). The nanoemulsions were highly unstable to droplet growth and phase separation, which was attributed to Ostwald ripening due to the relatively high water solubility of thyme oil. Ostwald ripening could be inhibited by incorporating ≥75% of corn oil (a hydrophobic material with a low water solubility) into the nanoemulsion droplets. The electrical characteristics of the droplets in the nanoemulsions were varied by incorporating ionic surfactants with different charges after homogenization: a cationic surfactant (lauric arginate, LAE) or an anionic surfactant (sodium dodecyl sulfate, SDS). The antifungal activity of nanoemulsions containing positive, negative, or neutral thymol droplets was then conducted against four strains of acid-resistant spoilage yeasts: Zygosaccharomyces bailli, Saccharomyces cerevisiae, Brettanomyces bruxellensis, and Brettanomyces naardenensis. The antifungal properties of the three surfactants (T80, LAE, SDS) were also tested in the absence of thymol droplets. Both ionic surfactants showed strong antifungal activity in the absence of thymol droplets, but no antimicrobial activity in their presence. This effect was attributed to partitioning of the antimicrobial surfactant molecules between the oil droplet and microbial surfaces, thereby reducing the effective concentration of active surfactants available to act as antimicrobials. This study shows oil droplets may decrease the efficacy of surfactant-based antimicrobials, which has important consequences for formulating effective antimicrobial agents for utilization in emulsion-based food and beverage products.

Physical properties and antimicrobial efficacy of thyme oil nanoemulsions: influence of ripening inhibitors.

Thyme oil-in-water nanoemulsions (pH 3.5) were prepared as potential antimicrobial delivery systems. The nanoemulsions were highly unstable to droplet growth and phase separation, which was attributed to Ostwald ripening due to the relatively high water solubility of thyme oil. Ostwald ripening could be inhibited by mixing thyme oil with a water-insoluble ripening inhibitor (≥60 wt % corn oil or ≥50 wt % MCT in the lipid phase) before homogenization, yielding nanoemulsions with good physical stability. Physically stable thyme oil nanoemulsions were examined for their antimicrobial activities against an acid-resistant spoilage yeast, Zygosaccharomyces bailii (ZB). Oil phase composition (ripening inhibitor type and concentration) had an appreciable influence on the antimicrobial activity of the thyme oil nanoemulsions. In general, increasing the ripening inhibitor levels in the lipid phase reduced the antimicrobial efficacy of nanoemulsions. For example, for nanoemulsions containing 60 wt % corn oil in the lipid phase, the minimum inhibitory concentration (MIC) of thyme oil to inhibit ZB growth was 375 μg/mL, while for nanoemulsions containing 90 wt % corn oil in the lipid phase, even 6000 μg/mL thyme oil could not inhibit ZB growth. This effect is also dependent on ripening inhibitor types: at the same concentration in the lipid phase, MCT decreased the antimicrobial efficacy of thyme oil more than corn oil. For instance, when the level of ripening inhibitor in the lipid phase was 70 wt %, the MICs of thyme oil for nanoemulsions containing corn oil and MCT were 750 and 3000 μg/mL, respectively. The results of this study have important implications for the design and utilization of nanoemulsions as antimicrobial delivery systems in the food and other industries.

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.

Optimization of Orange Oil Nanoemulsion Formation by Isothermal Low-Energy Methods: Influence of the Oil Phase, Surfactant, and Temperature

Nanoemulsions are particularly suitable as a platform in the development of delivery systems for lipophilic functional agents. This study shows that transparent orange oil nanoemulsions can be fabricated using an isothermal low-energy method (spontaneous emulsification), which offers the advantage of fabricating flavor oil delivery systems using rapid and simple processing operations. Orange oil nanoemulsions were formed spontaneously by titration of a mixture of orange oil, carrier oil [medium-chain triglyceride (MCT)], and non-ionic surfactant (Tween) into an aqueous solution (5 mM citrate buffer at pH 3.5) with continuous stirring. The oil/emulsion ratio content was kept constant (10 wt %), while the surfactant/emulsion ratio (SER) was varied (2.5-20 wt %). Oil-phase composition (orange oil/MCT ratio), SER, and surfactant type all had an appreciable effect on nanoemulsion formation and stability. Transparent nanoemulsions could be formed under certain conditions: 20% surfactant (Tween 40, 60, or 80) and 10% oil phase (4-6% orange oil + 6-4% MCT). Surfactant type and oil-phase composition also affected the thermal stability of the nanoemulsions. Most of the nanoemulsions broke down after thermal cycling (from 20 to 90 °C and back to 20 °C); however, one system remained transparent after thermal cycling: 20% Tween 80, 5% orange oil, and 5% MCT. The mean droplet size of these nanoemulsions increased over time, but the droplet growth rate was reduced appreciably after dilution. These results have important implications for the design and utilization of nanoemulsions as delivery systems in the food and other industries.

Fabrication, stability and efficacy of dual-component antimicrobial nanoemulsions: Essential oil (thyme oil) and cationic surfactant (lauric arginate)

The influence of a cationic surfactant (lauric arginate, LAE) on the physical properties and antimicrobial efficacy of thyme oil nanoemulsions was investigated. Nanoemulsions prepared from pure thyme oil were highly unstable due to Ostwald ripening, but they could be stabilized by adding a ripening inhibitor (corn oil) to the oil phase prior to homogenisation. The loading capacity and antimicrobial efficacy of thyme oil nanoemulsions were significantly increased by adding LAE. In the absence of LAE, at least 60 wt% corn oil had to be added to the lipid phase to inhibit Ostwald ripening; but in the presence of 0.1 wt% LAE, only 30 wt% corn oil was needed. LAE addition substantially increased the antimicrobial efficacy of the thyme oil nanoemulsions: 200 μg/ml thyme oil was needed to inhibit growth of a spoilage yeast (Zygosaccharomyces bailii) if LAE was added, whereas ⩾ 400 μg/ml was needed in the absence of LAE.

Interactions of a Cationic Antimicrobial (ε-Polylysine) with an Anionic Biopolymer (Pectin): An Isothermal Titration Calorimetry, Microelectrophoresis, and Turbidity Study

ε-Polylysine (ε-PL) is a food-grade cationic antimicrobial that is highly effective against a range of food pathogens and spoilage organisms. In compositionally complex environments, like those found in most foods and beverages, the antimicrobial activity of cationic ε-PL is likely to be impacted by its interactions with anionic components. The purpose of this study was to characterize the interactions between cationic ε-polylysine and an anionic biopolymer (high methoxyl pectin, HMP) using isothermal titration calorimetry (ITC), microelectrophoresis (ME), and turbidity measurements. ITC and ME measurements indicated that ε-PL bound to pectin, while turbidity measurements indicated that the complexes formed could be either soluble or insoluble depending on solution composition. Ionic strength and pH were also shown to affect the interactions significantly, highlighting their electrostatic origin. This study demonstrates that ε-PL can form either soluble or insoluble complexes with anionic biopolymers depending on the composition of the system. Our study provides basic knowledge that will facilitate the more rational application of ε-PL in complex food systems.

Low concentration of ethylenediaminetetraacetic acid (EDTA) affects biofilm formation of Listeria monocytogenes by inhibiting its initial adherence

The distribution and survival of the food-borne pathogen Listeria monocytogenes is associated with its biofilm formation ability, which is affected by various environmental factors. Here we present the first evidence that EDTA at low concentration levels inhibits the biofilm formation of L. monocytogenes. This effect of EDTA is not caused by a general growth inhibition, as 0.1 mM EDTA efficiently reduced the biofilm formation of L. monocytogenes without affecting the planktonic growth. Adding 0.1 mM of EDTA at the starting time of biofilm formation had the strongest biofilm inhibitory effect, while the addition of EDTA after 8 h had no biofilm inhibitory effects. EDTA was shown to inhibit cell-to-surface interactions and cell-to-cell interactions, which at least partially contributed to the repressed initial adherence. The addition of sufficient amounts of cations to saturate EDTA did not restore the biofilm formation, indicating the biofilm inhibition was not due to the chelating properties of EDTA. The study suggests that EDTA functions in the early stage of biofilm process by affecting the initial adherence of L. monocytogenes cells onto abiotic surfaces.

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 (≥10(8)CFU/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.

Cationic antimicrobial (ε-polylysine)-anionic polysaccharide (pectin) interactions: influence of polymer charge on physical stability and antimicrobial efficacy.

The cationic biopolymer ε-polylysine (ε-PL) is a potent food-grade antimicrobial that is highly effective against a range of food pathogens and spoilage organisms. In compositionally complex systems such as foods and beverages, cationic ε-PL molecules may associate with anionic substances, leading to increased turbidity, sediment formation, and reduced antimicrobial activity. This study therefore characterized the interactions between cationic ε-PL and anionic pectins with different degrees of esterification (DE) and then investigated the influence of these interactions on the antimicrobial efficacy of ε-PL. The nature of the interactions was characterized using isothermal titration calorimetry (ITC), microelectrophoresis (ME), and turbidity measurements. High (DE 61%), medium (DE 51%), and low (DE 42%) methoxyl pectins interacted with ε-PL molecules through electrostatic forces, forming either soluble or insoluble complexes with various electrical charges, depending on the relative mass ratio of pectin and ε-PL. The interaction of pectin with ε-PL increased as the negative charge density on the pectin molecules increased, that is, with decreasing DE. The antimicrobial efficacy of ε-PL against two acid-resistant spoilage yeasts (Zygosaccharomyces bailii and Saccharomyces cerevisiae) decreased progressively in the presence of increasing levels of all three pectins. Nevertheless, the low DE pectin decreased the antimicrobial efficacy of ε-PL much more dramatically, likely due to strong electrostatic binding of ε-PL onto low DE pectin molecules reducing its interaction with anionic microbe surfaces. This study provides knowledge that will facilitate the rational application of ε-PL as an antimicrobial in complex food systems.

Identification of genes involved in Listeria monocytogenes biofilm formation by mariner-based transposon mutagenesis

Listeria monocytogenes is a ubiquitous food-borne pathogen, whose distribution and survival in food-processing environments are associated with the ability to form biofilms. The process of biofilm formation is complex and its molecular mechanism is relatively poorly understood in L. monocytogenes. To better understand the genetics of this process, a mariner-based transposon mutagenesis strategy was used to identify genes involved in biofilm formation of L. monocytogenes. A library of 6,500 mutant colonies was screened for reduced biofilm formation using a microtiter plate biofilm assay. Forty biofilm-deficient mutants of L. monocytogenes were identified based on DNA sequences of the transposon-flanking regions and Southern hybridization with a transposon-based probe. The insertions harbored by these mutants led to the identification of 24 distinct loci, 18 of which, to our knowledge, have not been previously reported to function in the biofilm formation in L. monocytogenes. Genetic complementation confirmed the importance of lmo1386, a gene encoding a putative DNA translocase, for biofilm formation. Molecular analyses of mutants indicated that the majority of the 24 identified genes are related to flagella motility, gene regulation, and cell surface structures.