P. Michael Davidson

Growth Inhibition of Escherichia coli O157:H7 and Listeria monocytogenes by Carvacrol and Eugenol Encapsulated in Surfactant Micelles

Growth inhibition of four strains of Escherichia coli O157:H7 (H1730, F4546, 932, and E0019) and Listeria monocytogenes (Scott A, 101, 108, and 310) by essential oil components (carvacrol and eugenol) solubilized in nonionic surfactant micelles (Surfynol 465 and 485W) was investigated. Concentrations of encapsulated essential oil components ranged from 0.02 to 1.25% depending on compound, surfactant type, and surfactant concentration (0.5 to 5%). Eugenol encapsulated in Surfynol 485W micelles was most efficient in inhibiting growth of the pathogens; 1% Surfynol 485W and 0.15% eugenol was sufficient to inhibit growth of all strains of E. coli O157:H7 and three of four strains of L. monocytogenes (Scott A, 310, and 108). The fourth strain, L. monocytogenes 101, was inhibited by 2.5% Surfynol and 0.225% eugenol. One percent Surfynol 485W in combination with 0.025% carvacrol was effective in inhibiting three of four strains of E. coli O157:H7. Strain H1730 was the most resistant strain, requiring 0.3% carvacrol and 5% surfactant for complete inhibition. Growth inhibition of L. monocytogenes by combinations of carvacrol and Surfynol 465 ranged between 0.15 and 0.35% and 1 and 3.75%, respectively. Generally, the antimicrobial activity of Surfynol 465 in combination with eugenol was higher than that for the combination with carvacrol. The potent activity was attributed to increased solubility of essential oil components in the aqueous phase due to the presence of surfactants and improved interactions of antimicrobials with microorganisms.

Encapsulation of nisin and lysozyme in liposomes enhances efficacy against Listeria monocytogenes.

The efficacy and stability against Listeria monocytogenes of nisin and lysozyme encapsulated in phospholipid liposomes was evaluated. Antimicrobial-containing liposomes were prepared by hydrating dried lipids with buffer containing nisin, nisin plus the fluorescence probe calcein, or calcein and lysozyme. Mixtures were then centrifuged and sonicated, and encapsulated liposomes were collected using size-exclusion chromatography. Antimicrobial concentration in liposomes was determined by bicinchoninic acid assay prior to determination of antimicrobial activity against strains of L. monocytogenes. When nisin was encapsulated in liposomes, protein concentrations of 0.39, 0.27, and 0.23 mg/ml for phosphatidylcholine (PC), PC-cholesterol (7:3), and PC-phosphatidylglycerol (PG)-cholesterol (5:2:3), respectively, were obtained. Encapsulation of nisin with calcein yielded protein concentrations of 0.35, 0.39, and 0.28 mg/ml for PC, PC-cholesterol, and PC-PG-cholesterol, respectively. Encapsulation of calcein with lysozyme resulted in protein concentrations of 0.43, 0.26, and 0.19 mg/ml for PC, PC-cholesterol, and PC-PG-cholesterol, respectively. Encapsulated nisin in 100% PC and PC-cholesterol liposomes inhibited bacterial growth by >2 log CFU/ml compared with free nisin. Growth inhibition with liposomal lysozyme was strain dependent, with greater inhibition observed for strains 310 and Scott A with PC-cholesterol and PC-PG-cholesterol liposomes. Inhibition of L. monocytogenes indicated the potential of liposomes to serve as delivery vehicles for antimicrobials in foods while improving stability of antimicrobials.

Inactivation of Escherichia coli and Listeria innocua in apple and carrot juices using high pressure homogenization and nisin

High pressure homogenization has been of growing interest as a nonthermal technology for the inactivation of microorganisms in fruit and vegetable juices. Cells of Escherichia coli and Listeria innocua, used as surrogates for foodborne pathogens, were inoculated into apple or carrot juice (approximately 7 log(10) CFU/ml) containing 0 or 10 IU/ml nisin and subjected to 350 to 0 MPa high pressure homogenization. At 50 MPa homogenization pressure intervals, juice samples were collected, immediately cooled to <10 degrees C, and then serially diluted and plated on nonselective recovery media. Following incubation, survivors were enumerated. As processing pressure increased, inactivation of E. coli increased, and a >5 log reduction of cells was achieved following exposure to pressures in excess >250 MPa. In contrast, little inactivation was observed for L. innocua with pressure <250 MPa and up to 350 MPa processing pressure was required to achieve an equivalent 5 log inactivation. The addition of 10 IU nisin, together with high pressure homogenization, did not exhibit significant additional E. coli inactivation, but interactions were observed with L. innocua. Results indicate that high pressure homogenization processing is a promising technology to achieve pathogen decontamination in fruit and vegetable juices.

Thymol Nanoencapsulated by Sodium Caseinate: Physical and Antilisterial Properties

In this work, thymol was encapsulated in sodium caseinate using high shear homogenization. The transparent dispersion at neutral pH was stable for 30 days at room temperature as determined by dynamic light scattering and atomic force microscopy, which agreed with high ζ potential of nanoparticles. The slightly decreased particle dimension during storage indicates the absence of Ostwald ripening. When molecular binding was studied by fluorescence spectroscopy, thymol was observed to bind with tyrosine and possibly other amino acid residues away from tryptophan of caseins. At pH 4.6 (isoelectric point of caseins), the stabilization of thymol nanoparticles against aggregation was enabled by soluble soybean polysaccharide, resulting from the combined electrostatic and steric repulsions. The encapsulated thymol showed the significantly improved antilisterial activity in milk with different fat levels when compared to thymol crystals, resulting from the quicker mixing and increased solubility in the milk serum. The transparent thymol nanodispersions have promising applications to improve microbiological safety and quality of foods.

Naturally Occurring Antimicrobials for Minimally Processed Foods

Natural antimicrobials are gaining increased interest from researchers and food manufacturers alike seeking to discover label-friendly alternatives to the widely implemented synthetic compounds. Naturally occurring antimicrobials can be applied directly to food to protect food quality, extend food shelf life by inhibiting or inactivating spoilage microorganisms, and improve food safety by inhibiting or inactivating food-borne pathogens. There are a great number of natural antimicrobials derived from animal, plant, and microbial sources. This manuscript reviews their efficacy against spoilage and pathogenic organisms, their methods of evaluation, and their application in various foods as well as the development of novel delivery systems and incorporation with other hurdles.

Nanodispersed eugenol has improved antimicrobial activity against Escherichia coli O157:H7 and Listeria monocytogenes in bovine milk.

There has been great interest in intervention strategies based on plant essential oils to control pathogens such as Escherichia coli O157:H7 and Listeria monocytogenes (Lm). However, the poor solubility of essential oils in water makes it difficult to disperse evenly in food matrices, impacting food quality and antimicrobial efficacy. In the present study, eugenol was dispersed in nanocapsules prepared with conjugates of whey protein isolate (WPI) and maltodextrin (MD, of various chain lengths). When eugenol was encapsulated in the conjugate made with MD40 at a WPI:MD mass ratio of 1:2, the nanodispersion was transparent and was characterized for antimicrobial efficacy against E. coli O157:H7 strains ATCC 43889 and 43894, and Lm strains Scott A and 101 in tryptic soy broth (TSB) and milk with different fat levels (whole, 2% reduced fat, and skim) at 35 or 32 °C, with comparison to the same levels of free eugenol. In TSB, antimicrobial efficacy of nanodispersed eugenol against E. coli O157:H7 and Lm was not improved, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values being 0.25 g/L higher than those of free eugenol. Free eugenol performed better in TSB because there was no interfering compound and the MIC and MBC were below the solubility of eugenol. In milk, nanodispersed eugenol was consistently observed to be more effective than free eugenol, with MIC and MBC values above the solubility limit of eugenol. The nanodispersed eugenol was speculated to be evenly distributed in food matrices at concentrations above the solubility limit and supplied the antimicrobial locally when the binding caused eugenol level below the inhibition requirement. Nanodispersed eugenol thus provides a novel approach for incorporation in foods to improve antimicrobial efficacy without changing turbidity.

Spray-Dried Zein Capsules with Coencapsulated Nisin and Thymol as Antimicrobial Delivery System for Enhanced Antilisterial Properties

Food grade antimicrobial delivery systems were studied in this work to enhance the effectiveness of antimicrobials inhibiting the growth of Listeria monocytogenes during storage. Corn zein was used as a carrier biopolymer and nisin and thymol as antimicrobials. Capsules produced by spray drying demonstrated different microstructures and release characteristics of nisin at different usage levels of thymol. Better release profiles were achieved when glycerol was additionally used to prepare capsules. Capsules showing sustained release of significant amounts of both antimicrobials effectively inhibited the growth of L. monocytogenes at pH 6.0 and 30 °C in the growth medium. Capsules were also more effective than free antimicrobials in inhibiting the growth of L. monocytogenes in 2% reduced fat milk at 25 °C. Our work showed that engineered delivery systems have promise to fulfill the antimicrobial effectiveness during shelf life storage of foods to ensure microbiological safety.

Stability and Antimicrobial Efficiency of Eugenol Encapsulated in Surfactant Micelles as Affected by Temperature and pH

Growth inhibition of four strains of Escherichia coli O157:H7 (H1730, F4546, 932, and E0019) and Listeria monocytogenes (Scott A, 101, 108, and 310) by eugenol encapsulated in water soluble micellar nonionic surfactant solutions (Surfynol 485W) adjusted to pH 5, 6, and 7 and incubated at 10, 22, and 32 degrees C was determined. Concentrations of eugenol ranged from 0.2 to 0.9% at a surfactant concentration of 5%. Antimicrobial activity was assessed using a microbroth dilution assay. Eugenol encapsulated in surfactant micelles inhibited both microorganisms at pH 5, 6, and 7. At pH 5, some inhibition occurred in the absence of eugenol, i.e., by the surfactant itself (optical density at 24 h for L. monocytogenes = 0.07 and optical density at 24 h for E. coli O157:H7 = 0.09), but addition of >0.2% eugenol led to complete inhibition of both microorganisms. Inhibition of L. monocytogenes and E. coli O157:H7 decreased with increasing pH, that is, the minimum inhibitory concentration was 0.2, 0.5, and 0.5% of micellar encapsulated eugenol solutions at pH 5, 6, and 7, respectively. The encapsulated essential oil component in surfactant micelles was effective at all three temperatures tested (10, 22, and 32 degrees C), indicating that the activity of encapsulated eugenol was not affected by high or low (refrigeration) temperatures. Overall, strains of E. coli O157:H7 were more sensitive than strains of L. monocytogenes. Improved activity was attributed to increased solubility of eugenol in the aqueous phase due to the presence of surfactants and improved interactions of antimicrobials with microorganisms.

Antimicrobial properties of lauric arginate alone or in combination with essential oils in tryptic soy broth and 2% reduced fat milk

The objective of this study was to evaluate the antimicrobial activity of lauric arginate (LAE) when used alone or in combination with the essential oil (EO) from cinnamon leaf and EO components, thymol and eugenol. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) for Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella Enteritidis were determined by the microbroth dilution method in tryptic soy broth (TSB) at their optimal growth temperatures. The MIC for LAE was 11.8ppm against L. monocytogenes and E. coli O157:H7 and 23.5ppm against S. Enteritidis. Synergistic antimicrobial activity was demonstrated against L. monocytogenes with combinations of LAE and cinnamon leaf oil or eugenol, while the LAE and thymol combination showed additive antimicrobial activity. Conversely, antagonistic effects were shown for all combinations against E. coli O157:H7 and S. Enteritidis. Beef extract, at 2 or 5% w/v in TSB, showed no effects on the MIC and MBC of LAE against L. monocytogenes, while soluble starch from potato, at 2-10% w/v in TSB, increased the MIC and MBC. When tested in 2% reduced fat milk, significantly higher levels of antimicrobials were required to achieve similar inhibitions as in TSB. The growth curves of bacteria at 21°C followed similar trends as in TSB, showing synergism against the Gram-positive L. monocytogenes and antagonism against the two Gram-negative bacteria. Findings suggest that application of LAE could enhance microbial food safety, especially when used in combination with EO to inhibit the growth of Gram-positive bacteria.

Nanocapsular Dispersion of Thymol for Enhanced Dispersibility and Increased Antimicrobial Effectiveness against Escherichia coli O157:H7 and Listeria monocytogenes in Model Food Systems

ABSTRACT Essential oils are marginally soluble in water, making it challenging to evenly disperse them in foods and resulting in an increased tendency to bind with food lipids and proteins, resulting in lowered antimicrobial efficacy. In the current study, free and nano-dispersed (ND) thymol were compared in terms of their antimicrobial efficacies against Escherichia coli O157:H7 ATCC 43889 and 43894 and Listeria monocytogenes strains Scott A and 101 in apple cider and 2% reduced-fat milk. Apple cider was adjusted to pHs 5.5 and 3.5, and antimicrobial tests were performed at 0.3-, 0.5-, 0.75-, and 1.0-g/liter thymol concentrations at 35, 32, 25, and 4°C. Overall, 0.5 and 1.0 g/liter thymol in nano-dispersion and along with free thymol were inhibitory and bactericidal, respectively, against bacterial strains under all treatment conditions. At pH 5.5, 0.5 g/liter ND thymol was bacteriostatic against L. monocytogenes and E. coli for up to 48 h. At pH 3.5, L. monocytogenes controls did not survive beyond 12 h but E. coli survived and was inhibited by 0.5 g/liter ND thymol after 12 and 48 h in apple cider. E. coli strains were significantly sensitive to 4°C and pH 3.5 (P < 0.05). When bacteria were tested in 2% reduced-fat milk at 35 or 32°C, ND and free thymol demonstrated inhibition at 4.5 g/liter. Thus, the current technology seems to be promising and novel, enabling thymol-containing nano-dispersions that are not only transparent but also effective against pathogens in food applications, especially in clear beverages.