David J. Geveke

Selection of surrogate bacteria in place of E. coli O157:H7 and Salmonella Typhimurium for pulsed electric field treatment of orange juice.

Pulsed electric field (PEF) technology has been used for the inactivation of microorganisms and to prevent flavor loss in liquid foods and beverages in place of thermal pasteurization. When used to pasteurize orange juice, PEF may prevent loss of volatile sensory attributes. Enterohemorrhagic E. coli O157:H7 (EHEC), two strains of Salmonella Typhimurium, and twenty strains of non-pathogenic bacteria were screened for inactivation in orange juice by PEF at 22 and 20kV/cm at 45 and 55 degrees C, respectively. Higher populations of both salmonellae were inactivated (2.81 and 3.54 log CFU/ml) at 55 degrees C, in comparison with the reduction of EHEC (2.22 log). When tested under the same conditions, inactivation of EHEC was slightly greater than that of a non-pathogenic E. coli (NPEC) ATCC 35218 (2.02 log). NPEC was further tested as a surrogate for EHEC by comparing inactivation kinetics at 45, 50 and 55 degrees C at field strengths of between 7.86 and 32.55kV/cm. Statistical comparison of revealed that EHEC and NPEC inactivation curves were homogeneous at outlet temperatures of 45 and 50 degrees C; however, EHEC was slightly more sensitive to PEF than the surrogate NPEC at 55 degrees C. The higher PEF resistance of non-pathogenic E. coli 35218 at 55 degrees C may provide a desirable margin of safety when used in pilot plant challenge studies in place of E. coli O157:H7.

Inactivation of Salmonella on whole cantaloupe by application of an antimicrobial coating containing chitosan and allyl isothiocyanate.

This study investigated the antimicrobial effect of a chitosan coating+allyl isothiocyanate (AIT) and nisin against Salmonella on whole fresh cantaloupes. Cantaloupes were inoculated with a cocktail of three Salmonella strains and treated with chitosan, chitosan+AIT, chitosan+nisin, and chitosan+AIT+nisin coatings. With AIT concentrations increasing from 10 to 60 μl/ml, the antibacterial effects of coating treatments against Salmonella increased. Chitosan coatings with 60 μl/ml AIT (chitosan+60AIT) reduced more than 5 log₁₀ CFU/cm² of Salmonella. The addition of nisin to the chitosan-AIT coating synergistically increased the antibacterial effect; coatings with nisin (25 mg/ml or 25,000 IU/ml)+30 μl/ml AIT resulted in a 4.8 log₁₀ reduction of Salmonella. The chitosan+60AIT coating significantly (p<0.05) reduced populations of native bacteria on cantaloupes to ca. 2 log₁₀ CFU/cm² during the first 6 days and populations remained unchanged through day 14 at 10 °C. The same coating treatment completely inactivated mold and yeast on cantaloupe at day 1 and no regrowth occurred even up to 14 days of storage. Scanning electron microscopy revealed that cell membrane damage and leakage of intercellular components occurred as a result of the chitosan-AIT coating treatments. No visual changes in overall appearance and color of cantaloupe rind and flesh due to coating treatments were observed. These results indicate that the application of an antimicrobial coating may be an effective method for decontamination of cantaloupes.

A combined treatment of UV-light and radio frequency electric field for the inactivation of Escherichia coli K-12 in apple juice

Radio frequency electric fields (RFEF) and UV-light treatments have been reported to inactivate bacteria in liquid foods. However, information on the efficacy of bacterial inactivation by combined treatments of RFEF and UV-light technologies is limited. In this study, we investigated the relationship between cell injury and inactivation of Escherichia coli K-12 in apple juice treated with a combination of RFEF and UV-light. Apple juice purchased from a wholesale distributor was inoculated with E. coli K-12 at 7.8 log CFU/ml, processed with a laboratory scale RFEF unit at 20 kHz, 15 kV/cm for 170 micros at a flow rate of 540 ml/min followed by UV-light treatment (254 nm) for 12s at 25, 30 and 40 degrees C. Treated samples were analyzed for leakage of UV-substances as a function of membrane damage and were plated (0.1 ml) on Sorbitol MacConkey Agar (SMAC) and Trypticase Soy Agar (TSA) plates to determine the viability loss and percent injury. At 40 degrees C, UV-light treatment alone caused 5.8 log reduction of E. coli in apple juice while RFEF caused only 2.8 log reduction. A combination of the two processing treatments did not increase cell injury or leakage of intracellular bacterial UV-substances more than that from the UV-light treatment. Similarly, the viability loss determined was not significantly (P<0.05) different than UV-light treatment alone. However, the UV-substances determined in apple juice treated with RFEF was significantly (P>0.05) different than UV-light treated samples. The results of this study suggest that RFEF treatment causes more injury to the bacterial cells leading to more leakage of intracellular UV-substances than cells treated with UV-light alone. Also, the effect of the two processing treatment combination on bacterial inactivation was not additive.

UV inactivation of bacteria in apple cider.

Apple cider, inoculated with Escherichia coli and Listeria innocua, was processed using a simple UV apparatus. The apparatus consisted of a low-pressure mercury lamp surrounded by a coil of UV transparent tubing. Cider was pumped through the tubing at flow rates of 27 to 83 ml/min. The population of E. coli K-12 was reduced by 3.4 +/- 0.3 log after being exposed for 19 s at a treatment temperature of 25 degrees C. The population of L. innocua, which was more resistant to UV, was reduced by 2.5 +/- 0.1 log after being exposed for 58 s. The electrical energy for the process was 34 J/ml and is similar to that for conventional thermal processing. UV processing has the potential to improve the safety and extend the shelf life of apple cider.

Radio frequency energy effects on microorganisms in foods

Abstract Liquids containing microorganisms were exposed to radio frequency (RF) energy to study non-thermal inactivation. RF energy was applied to the liquids while heat was simultaneously removed to control temperature. Turbulent flow was maintained to minimize localized heating. An 18 MHz RF processor applied an approximately 0.5 kV/cm electric field strength to the liquids. It was capable of pasteurizing the liquids provided that cooling was minimized. There were no non-thermal effects of RF energy detected on Escherichia coli K-12, Listeria innocua , or yeast in apple cider, beer, deionized water, liquid whole egg, and tomato juice; nor were there any synergistic effects of RF energy with heat. The low temperature effects of RF energy at 18 MHz and 0.5 kV/cm were due to heat.

Shelf-life study of an orange juice-milk based beverage after PEF and thermal processing.

The effect of thermal and pulsed electric field (PEF) processing on the shelf life of an orange juice-milk beverage (OJMB) was studied. The intensities of the treatments were selected to produce similar inactivation of pectin methyl esterase (PME), an enzyme responsible for the jellification and loss of fresh juice cloudiness. Physical properties (pH, degrees Brix, and color), microbial population, PME activity, and volatile compounds of the product were analyzed during a 4-wk storage at 8 to 10 degrees C. The pH was not affected by any treatment but decreased during the storage in the untreated sample. The degrees Brix values were decreased by the 2 treatments. The thermal and PEF treatments initially inactivated PME activity by 90%. During storage, the PME activity remained constant in the 2 treated samples and decreased slightly in the untreated sample. The reductions in bacterial as well as yeast and mold counts were similar after the 2 treatments (4.5 and 4.1 log CFU/mL for thermal against 4.5 and 5 log CFU/mL for PEF). Based on the initial bacterial counts of the control, it was estimated that the shelf lives of the OJMB treated with thermal and PEF processing stored at 8 to 10 degrees C were 2 and 2.5 wk, respectively. Differences were observed in the color parameters of the OJMB between the 2 treatments in comparison with the control, with a higher difference observed in the thermally processed samples. The relative concentration of volatile compounds was higher in OJMB processed by PEF treatment than that in the thermally processed sample. During storage, the loss of volatile compounds was lower in the PEF sample while thermal and control samples had a similar rate of loss. For an OJMB, treatment with PEF achieved the same degree of microbial and enzyme inactivation as the thermal treatment, but better preserved color and volatile compounds.

Membrane damage and viability loss of Escherichia coli K-12 in apple juice treated with radio frequency electric field.

The need for a nonthermal intervention technology that can achieve microbial safety without altering nutritional quality of liquid foods led to the development of a radio frequency electric fields (RFEF) process. In order to understand the mechanism of inactivation of bacteria by RFEF, apple juice purchased from a wholesale distributor was inoculated with Escherichia coli K-12 at 7.8 log CFU/ml and then treated with RFEF. The inoculated apple juice was passed through an RFEF chamber operated at 20 kHz, 15 kV/cm for 170 micros at a flow rate of 540 ml/min. Treatment condition was periodically adjusted to achieve outlet temperatures of 40, 45, 50, 55, and 60 degrees C. Samples at each outlet temperature were plated (0.1 ml) and the number of CFU per milliliter determined on nonselective and selective agar media was used to calculate the viability loss. Bacterial inactivation and viability loss occurred at all temperatures tested with 55 degrees C treatment, leading to 4-log reductions. No significant effect was observed on bacterial population in control samples treated at 55 degrees C with a low-RFEF (0.15 kV/cm) field strength. These observations suggest that the 4-log reduction in samples treated at 15 kV/cm was entirely due to nonthermal effect. RFEF treatment resulted in membrane damage of the bacteria, leading to the efflux of intracellular ATP and UV-absorbing materials. Populations of injured bacteria recovered immediately (<30 min) from the treated apple juice averaged 0.43 log and were below detection after 1 h of RFEF treatment and determination using selective plates (tryptic soy agar containing 5% sodium chloride). The results of this study suggest that mechanism of inactivation of RFEF is by disruption of the bacterial surface structure leading to the damage and leakage of intracellular biological active compounds.

Inactivation of Saccharomyces cerevisiae with Radio Frequency Electric Fields

The application of radio frequency (RF) electric fields as a nonthermal alternative to thermal inactivation of microorganisms in liquids was investigated. A novel RF system producing frequencies in the range of 20 to 60 kHz was developed. Electric field strengths of 20 and 30 kV/cm were applied to suspensions of Saccharomyces cerevisiae in water over a temperature range of 35 to 55 degrees C. The flow rate was 1.2 liters/min. The S. cerevisiae population was reduced by 2.1 +/- 0.1 log units following exposure to a 30-kV/cm field at 40 degrees C. The results of the present study provide the first evidence that strong RF electric fields inactivate microorganisms at moderately low temperatures. Increasing the field strength, the number of treatments, and the temperature enhanced inactivation. Frequency had no effect on inactivation over the range of frequencies studied.

Effect of native microflora, waiting period, and storage temperature on Listeria monocytogenes serovars transferred from cantaloupe rind to fresh-cut pieces during preparation.

The most recent outbreak of listeriosis linked to consumption of fresh-cut cantaloupes indicates the need to investigate the behavior of Listeria monocytogenes in the presence of native microflora of cantaloupe pieces during storage. Whole cantaloupes were inoculated with L. monocytogenes (10(8)-CFU/ml suspension) for 10 min and air dried in a biosafety cabinet for 1 h and then treated (unwashed, water washed, and 2.5% hydrogen peroxide washed). Fresh-cut pieces (∼3 cm) prepared from these melons were left at 5 and 10°C for 72 h and room temperature (20°C) for 48 h. Some fresh-cut pieces were left at 20°C for 2 and 4 h and then refrigerated at 5°C. Microbial populations of fresh-cut pieces were determined by the plate count method or enrichment method immediately after preparation. Aerobic mesophilic bacteria, yeast and mold of whole melon, and inoculated populations of L. monocytogenes on cantaloupe rind surfaces averaged 6.4, 3.3, and 4.6 log CFU/cm(2), respectively. Only H(2)O(2) (2.5%) treatment reduced the aerobic mesophilic bacteria, yeast and mold, and L. monocytogenes populations to 3.8, 0.9, and 1.8 log CFU/cm(2), respectively. The populations of L. monocytogenes transferred from melon rinds to fresh-cut pieces were below detection but were present by enrichment. Increased storage temperatures enhanced the lag phases and growth of L. monocytogenes. The results of this study confirmed the need to store fresh-cut cantaloupes at 5°C immediately after preparation to enhance the microbial safety of the fruit.

Efficacy of supercritical carbon dioxide for nonthermal inactivation of Escherichia coli K12 in apple cider

This study evaluated the efficacy of a supercritical carbon dioxide (SCCO(2)) system with a gas-liquid porous metal contactor for eliminating Escherichia coli K12 in apple cider. Pasteurized, preservative-free apple cider was inoculated with E. coli K12 and processed using the SCCO(2) system at CO(2) concentrations of 0-10% (wt.%, g CO(2)/100g product), outlet temperatures of 34, 38, and 42 degrees C, a system pressure of 7.6 MPa, and a flow rate of 1L/min. Increased CO(2) concentrations and temperatures significantly (P<0.05) enhanced the bactericidal effect, resulting in a maximum reduction of 7.31 log CFU/mL at 8% CO(2) and 42 degrees C. A response surface model indicated that minimum CO(2) concentrations of 9.9% at 34 degrees C, 7.4% at 38 degrees C, and 5.4% at 42 degrees C are needed to achieve a 5-log reduction of E. coli K12 in apple cider. SEM observations showed morphological changes in the cell envelope after SCCO(2) processing. At a processing condition of 8% and 38 degrees C, the reduction of E. coli was 6.03 log and the sublethal injury of the survivors was 84%. The regrowth or survival of E. coli in SCCO(2) processed apple cider was not observed during storage for 28 days at 4, 8, and 20 degrees C. Thus this study showed the potential of SCCO(2) processing with a gas-liquid porous metal contactor for the nonthermal pasteurization of apple cider.