T. Matthew Taylor

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.

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.

Inactivation of Escherichia coli K-12 Exposed to Pressures in Excess of 300 MPa in a High-Pressure Homogenizer

Homogenization is used widely in the dairy industry to improve product stability and quality. High-pressure homogenization (HPH) of fluid foods up to pressures of 300 MPa has demonstrated excellent potential for microbial inactivation. Microbial inactivation can be enhanced during HPH with the inclusion of antimicrobial compounds. Escherichia coli K-12 cells, grown statically or in chemostat, were exposed to HPH processing pressures of 50 to 350 MPa in the absence or presence of the antimicrobial nisin. Valve temperature was regulated by a water bath and pressure, and temperature data were recorded continuously after process initiation. Survivors were enumerated via plating on nonselective growth media. Pressure and temperature at the valve outlet port exhibited a quadratic relationship (R(2) = 0.9617, P < 0.05). Significant HPH-induced inactivation of the gram-negative microorganism was observed in the range of 100 to 250 MPa. Above 300 MPa, heat was the main factor promoting microbial inactivation, regardless of whether cells were grown in chemostat or statically. Chemostat-grown cells were significantly (P < 0.05) more resistant to HPH processing than were statically grown cells. Data indicate potential synergistic effects of nisin and HPH on the inactivation of bacterial contaminants. This study represents the first report of inactivation of a bacterium with HPH pressures in excess of 300 MPa in the presence and absence of an antimicrobial.

Reduction of Salmonella enterica serotype Poona and background microbiota on fresh-cut cantaloupe by electron beam irradiation

The efficacy of electron beam (e-beam) irradiation processing to reduce Salmonella enterica serotype Poona on surfaces of fresh-cut cantaloupe, and the impact of e-beam irradiation processing on the numbers of indigenous microorganisms were determined. Additionally, the D10-value for S. Poona reduction on the cut cantaloupe was also determined. Fresh-cut cantaloupe pieces, inoculated with S. Poona to 7.8 log10 CFU/g, were exposed to 0.0, 0.7, or 1.5 kGy. Surviving S. Poona, lactic acid bacteria (LAB), and fungi (yeasts, molds) were periodically enumerated on appropriate media over 21 days of storage at 5 °C. Cantaloupe surface pH was measured for irradiated cantaloupe across the 21 day storage period. To determine the D10-value of S. Poona, cantaloupe discs were inoculated and exposed to increasing radiation dosages between 0 and 1.06 kGy; surviving pathogen cells were selectively enumerated. S. Poona was significantly reduced by irradiation; immediate reductions following exposure to 0.7 and 1.5 kGy were 1.1 and 3.6 log10 CFU/g, respectively. After 21 days, S. Poona numbers were between 4.0 and 5.0 log10 CFU/g less than untreated samples at zero-time. Yeasts were not reduced significantly (p ≥ 0.05) by e-beam irradiation and grew slowly but steadily during storage. Counts of LAB and molds were initially reduced with 1.5 kGy (p<0.05) but then LAB recovered grew to high numbers, whereas molds slowly declined for irradiated and control samples. Cantaloupe pH declined during storage, with the greatest decrease in untreated control cantaloupe (p<0.05). The D10-value for S. Poona was determined to be 0.211 kGy, and this difference from the reductions observed in the cut cantaloupe studies may be due to the more precise dose distribution obtained in the thin and flat cantaloupe pieces used for the D10-value experiments. The effect of e-beam irradiation at the same doses used in this study was determined in previous studies to have no negative effect in the quality of the cut cantaloupe. Therefore, incorporation of low dosage ionizing irradiation and consistent application of irradiation processing can significantly improve the microbiological safety of fresh-cut cantaloupe.

Extraction of nisin from a 2.5% commercial nisin product using methanol and ethanol solutions.

Nisin is a class Ia bacteriocin used widely in the food industry to inhibit a number of gram-positive pathogens. Although this peptide exhibits activity against many gram-positive bacteria, its effectiveness can vary significantly depending upon the food application. Encapsulation is one method that has been investigated for improving the activity of nisin. Improvement of the encapsulation efficiency of nisin requires purification of the compound, which can be accomplished utilizing organic solvents. The objective of this study was to use methanol and ethanol solutions to extract and concentrate nisin from a commercial preparation containing 2.5% nisin. Commercial nisin was extracted with different concentrations of ethanol or methanol in sterile water for up to 8 h. Approximately 75% of the nisin activity was recovered with 10 or 50% ethanol compared with less than 1% recovery with an ethanol concentration higher than 90%. Extraction with 10 or 50% methanol was approximately as effective as that with lower concentrations of ethanol. However, yields were significantly greater for extraction with methanol at concentrations greater than 90%. The solubility of the nisin likely influenced the extraction profiles for the conditions used. Purification for an 8-h extraction using 10 and 50% ethanol was 1.36 and 1.93 times, respectively. Purification was less than 0.1 at higher ethanol concentrations due to poor extraction. For methanol treatments, purification factors were all 1.09 to 5.98, and they increased as methanol concentration increased. This method for extracting and purifying nisin from dairy proteins using organic solvents may provide an alternative means for preparing and concentrating nisin for encapsulation and other applications.

Effect of chemical sanitizers on Salmonella enterica serovar Poona on the surface of cantaloupe and pathogen contamination of internal tissues as a function of cutting procedure.

Survival of Salmonella enterica subsp. enterica serovar Poona on surface and stem scar portions of inoculated cantaloupe following sanitizer application, transfer of pathogen from the rind to the flesh during cutting, and growth of Salmonella Poona on cantaloupe cubes over 15 days of refrigerated storage were investigated. Cantaloupes inoculated with a rifampin-resistant strain of Salmonella Poona (10(7) CFU/ml) for 3 min and dried for 12 h were washed with chlorine (200 mg free chlorine per liter, 3 min), lactic acid (2%, 2 min), or ozone (30 mg/liter, 5 min). Fresh-cut cantaloupe cubes were prepared by (i) cutting the cantaloupe and then removing the rind or by (ii) peeling the rind and then cutting the flesh into pieces. The numbers of Salmonella bacteria recovered were higher in the stem scar portion (6.3 ± 0.3 log CFU/cm(2)) than the surface (4.8 ± 0.2 log CFU/cm(2)). Surface treatment with tap water or chlorine did not reduce Salmonella numbers, while treatment with lactic acid or ozone reduced Salmonella by 2.5 or 2.3 log CFU/cm(2), respectively. The use of lactic acid to sanitize the cantaloupes resulted in less Salmonella transfer to flesh during cutting; Salmonella numbers decreased to below detectable levels over 9 days of refrigerated (4°C) storage. Cutting cantaloupes after peeling the rind was more effective at reducing transfer of Salmonella to the internal tissue than cutting of cantaloupes prior to rind removal. These data suggest that treatment of cantaloupe rinds with lactic acid or ozone may be effective at reducing Salmonella numbers, while lactic acid application resulted in reduction of Salmonella transfer to cantaloupe flesh.

Inhibition of Bacterial Pathogens in Medium and on Spinach Leaf Surfaces using Plant-Derived Antimicrobials Loaded in Surfactant Micelles.

UNLABELLED Encapsulation of hydrophobic plant essential oil components (EOC) into surfactant micelles can assist the decontamination of fresh produce surfaces from bacterial pathogens during postharvest washing. Loading of eugenol and carvacrol into surfactant micelles of polysorbate 20 (Tween 20), Surfynol® 485W, sodium dodecyl sulfate (SDS), and CytoGuard® LA 20 (CG20) was determined by identification of the EOC/surfactant-specific maximum additive concentration (MAC). Rheological behavior of dilute EOC-containing micelles was then tested to determine micelle tolerance to shearing. Antimicrobial efficacy of EOC micelles against Escherichia coli O157:H7 and Salmonella enterica serotype Saintpaul was first evaluated by the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Pathogen-inoculated spinach was treated with eugenol-containing micelles applied via spraying or immersion methods. SDS micelles produced the highest MACs for EOCs, while Tween 20 loaded the lowest amount of EOCs. Micelles demonstrated Newtonian behavior in response to shearing. SDS and CG20-derived micelles containing EOCs produced the lowest MICs and MBCs for pathogens. E. coli O157:H7 and S. Saintpaul were reduced on spinach surfaces by application of eugenol micelles, though no differences in numbers of surviving pathogens were observed when methods of antimicrobial micelle application (spraying, immersion) was compared (P ≥ 0.05). Data suggest eugenol in SDS and CG20 micelles may be useful for produce surface decontamination from bacterial pathogens during postharvest washing. PRACTICAL APPLICATION Antimicrobial essential oil component (EOC)-containing micelles assist the delivery of natural food antimicrobials to food surfaces, including fresh produce, for decontamination of microbial foodborne pathogens. Antimicrobial EOC-loaded micelles were able to inhibit the enteric pathogens Escherichia coli O157:H7 and Salmonella Saintpaul in liquid medium and on spinach surfaces. However, pathogen reduction generally was not impacted by the method of micelle application (spraying, immersion washing) on spinach surfaces.

Efficacy of antimicrobials for the disinfection of pathogen contaminated green bell pepper and of consumer cleaning methods for the decontamination of knives

While there is strong focus on eliminating pathogens from produce at a commercial level, consumers can employ simple methods to achieve additional pathogen reductions in the domestic kitchen. To determine the ability of antimicrobials to decontaminate peppers, samples of green bell pepper were inoculated with Salmonella enterica and Escherichia coli O157:H7 and then immersed in 3% (v/v) hydrogen peroxide (H₂O₂), 2.5% (v/v) acetic acid (AA), 70% (v/v) ethyl alcohol (EtOH), or sterile distilled water (SDW). The potential for transfer of pathogens from contaminated peppers to other non-contaminated produce items, and the effect of knife disinfection in preventing this cross contamination, were also tested. Knife disinfection procedures were evaluated by chopping inoculated peppers into 1 cm² pieces with kitchen knives. Experimental knives were then treated by either no treatment (control), wiping with a dry sterile cotton towel, rinsing under running warm water for 5 or 10s, or applying a 1% (v/v) lauryl sulfate-based detergent solution followed by rinsing with warm running water for 10s. Following disinfection treatment, knives were used to slice cucumbers. Exposure to H₂O₂ for 5 min and EtOH for 1 min resulted in reductions of 1.3±0.3 log₁₀ CFU/cm² for both pathogens. A 5 min exposure to AA resulted in a reduction of S. enterica of 1.0±0.7 log₁₀ CFU/cm² and E. coli of 0.7±0.8 log₁₀ CFU/cm². No differences (p ≥ 0.05) were found between numbers of pathogens on knives and numbers of pathogens transferred to cucumber slices, suggesting that organisms remaining on knife surfaces were transferred to cucumbers during slicing. Findings suggest that EtOH and H₂O₂ may be effective antimicrobials for in-home decontamination of peppers, and that use of detergent and warm water is effective for decontamination of implements used during meal preparation.

Synergistic inhibition of Listeria monocytogenes in vitro through the combination of octanoic acid and acidic calcium sulfate.

It has been hypothesized that inhibition of foodborne pathogens can be enhanced by using antimicrobials in combination. A broth dilution assay was devised to determine whether inhibition of Listeria monocytogenes exposed to the combination of the fatty acid octanoic acid (OCT) and the organic acid-containing antimicrobial acidic calcium sulfate (ACS) was enhanced compared with the inhibition of the pathogen exposed to either antimicrobial applied singly. MICs for OCT and ACS were 25.00 μg/g and 1.56 ml/liter, respectively, for all strains of the pathogen tested. Fractional inhibitory concentrations (FICs) from the combination exposures were calculated for use in characterizing the antimicrobial interaction as antagonistic, additive indifferent, or synergistic with respect to L. monocytogenes inhibition. Combining OCT and ACS resulted in observed synergistic inhibition of L. monocytogenes; isobolograms for all strains curved toward the origin, and FIC indices (FIC(I)s) were <1.0. Future investigations of the antimicrobial combination should focus on determining the mechanism of action of combined antimicrobials and the levels of antimicrobials required for pathogen inhibition on the surfaces of ready-to-eat meats.

Antibiotic Resistance and Growth of the Emergent Pathogen Escherichia albertii on Raw Ground Beef Stored under Refrigeration, Abuse, and Physiological Temperature

Escherichia albertii is an emerging gram-negative facultative rod that has been implicated in multiple cases of human diarrheal disease, particularly in young children. When biochemical and other typing methods have been used, this organism has often been misidentified due to similarities with other members of the family Enterobacteriaceae. Isolates have been reported to be capable of producing attachment and effacement lesions via the synthesis of intimin, cytolethal distending toxin, and a variant form of Shiga toxin. The purposes of this study were to characterize the antibiotic resistance characteristics and the growth of individual strains of E. albertii on raw ground beef at different storage temperatures. Nalidixic acid-resistant strains of E. albertii were inoculated onto raw ground beef to a target of 4.0 log CFU/g, and samples were then aerobically incubated at 5, 22, or 35°C for various time periods prior to microbiological enumeration of the pathogen on lactose-free MacConkey agar containing 50 mg of nalidixic acid per liter and 0.5% L-rhamnose. Antibiotic resistance was determined using a broth microdilution assay. E. albertii did not grow at 5°C, with populations declining slowly over 14 days of refrigerated storage. Strains of the organism grew well under abusive storage, increasing by 2.5 to 3.1 log CFU/g and 4.1 to 4.3 log CFU/g after 24 h at 22 and 35°C, respectively. All strains were resistant to tetracycline but were sensitive to tested cephalosporins and chloramphenicol. Resistance to penicillin was observed, but susceptibility to other members of the b -lactam group, including ampicillin, amoxicillin, and clavulanic acid, was recorded. E. albertii represents an emerging pathogen with a probable foodborne transmission route. Future research should focus on verifying food process measures able to inactivate the pathogen.