Dike O. Ukuku

Behavior of Listeria monocytogenes inoculated on cantaloupe surfaces and efficacy of washing treatments to reduce transfer from rind to fresh-cut pieces.

Attachment and survival of Listeria monocytogenes on external surfaces (rind) of inoculated cantaloupe, resistance of the surviving bacteria to chlorine or hydrogen peroxide treatments, transfer of the pathogen from unsanitized and sanitized rinds to fresh-cut tissues during cutting and growth, and survival of L. monocytogenes on fresh-cut pieces of cantaloupe were investigated. Surface treatment with 70% ethanol to reduce the native microflora on treated melon, followed by immersion in a four-strain cocktail of L monocytogenes (10(8) CFU/ml) for 10 min, deposited 4.2 log10 CFU/cm2 and 3.5 log10 CFU/cm2 of L monocytogenes on treated and untreated cantaloupe rinds, respectively. L. monocytogenes survived on the treated or untreated cantaloupe rinds for up to 15 days during storage at 4 and 20 degrees C, but populations declined by approximately 1 to 2 log10 CFU/cm2. Fresh-cut pieces prepared from inoculated whole cantaloupes stored at 4 degrees C for 24 h after inoculation were positive for L. monocytogenes. Washing inoculated whole cantaloupes in solutions containing 1,000 ppm of chlorine or 5% hydrogen peroxide for 2 min at 1 to 15 days of storage at 4 degrees C after inoculation resulted in a 2.0- to 3.5-log reduction in L. monocytogenes on the melon surface. Fresh-cut pieces prepared from the sanitized melons were negative for L. monocytogenes. After direct inoculation onto fresh-cut pieces, L. monocytogenes survived, but did not grow, during 15 days of storage at 4 degrees C. Growth was evident by 4 h of storage at 8 and 20 degrees C. It is concluded that sanitizing with chlorine or hydrogen peroxide has the potential to reduce or eliminate the transfer of L. monocytogenes on melon surfaces to fresh-cut pieces during cutting.

Relationship of cell surface charge and hydrophobicity to strength of attachment of bacteria to cantaloupe rind.

The cantaloupe melon has been associated with outbreaks of Salmonella infections. It is suspected that bacterial surface charge and hydrophobicity may affect bacterial attachment and complicate bacterial detachment from cantaloupe surfaces. The surface charge and hydrophobicity of strains of Salmonella, Escherichia coli (O157:H7 and non-O157:H7), and Listeria monocytogenes were determined by electrostatic and hydrophobic interaction chromatography, respectively. Initial bacterial attachment to cantaloupe surfaces and the ability of bacteria to resist removal by washing with water were compared with surface charge and hydrophobicity. Whole cantaloupes were submerged in inocula containing individual strains or in cocktails containing Salmonella, E. coli, and L. monocytogenes, either as a mixture of strains containing all three genera or as a mixture of strains belonging to a single genus, for 10 min. Inoculated cantaloupes were dried for 1 h in a biosafety cabinet and then stored for up to 7 days at 4 degrees C. Inoculated melons were washed with water, and bacteria still attached to the melon surface, as well as those in the wash water, were enumerated. Initial bacterial attachment was highest for individual strains of E. coli and lowest for L. monocytogenes, but Salmonella exhibited the strongest attachment on days 0, 3, and 7. When mixed-genus cocktails were used, the relative degrees of attachment of the three genera ware altered. The attachment of Salmonella strains was the strongest. but the attachment of E. coli was more extensive than that of L. monocytogenes on days 0, 3, and 7. There was a linear correlation between bacterial cell surface hydrophobicity (r2 = 0.767), negative charge (r2 = 0.738), and positive charge (r2 = 0.724) and the strength of bacterial attachment to cantaloupe surfaces.

Effect of Nisin in Combination with EDTA, Sodium Lactate, and Potassium Sorbate for Reducing Salmonella on Whole and Fresh-Cut Cantaloupe

Nisin (50 microg/ml), EDTA (0.02 M, disodium salt), sodium lactate (NaL, 2%), and potassium sorbate (KS, 0.02%) were tested individually and in various combinations as sanitizer treatments for reducing Salmonella on whole and fresh-cut cantaloupe. Whole cantaloupe and fresh-cut pieces were inoculated with a five-strain cocktail of Salmonella to give 4.76 +/- 0.23 log CFU/cm2 and 3.42 +/- 0.13 log CFU/g, respectively. Inoculated whole melons and fresh-cut pieces were stored at 5 degrees C for 7 days. Washing treatments were applied to inoculated whole melons at days 0, 3, and 7 of storage, and surviving bacterial populations were determined. The effect of the washing treatments on transfer of Salmonella to fresh-cut pieces prepared immediately after treatment was also determined. Directly inoculated fresh-cut pieces were treated at day 0, and surviving bacteria were enumerated at days 0, 3, and 7 of storage. The combination treatments of nisin-EDTA, nisin-NaL, nisin-KS, NaL-KS, and nisin-NaL-KS all resulted in reductions of approximately 3 log CFU/cm2 at day 0 for whole melons. When tested alone, all compounds, along with water washes, were ineffective. After 3 and 7 days of storage, the five combination washing treatments were less effective, resulting in reductions of approximately 2 log CFU/cm2. None of the combination treatments completely eliminated transfer of pathogen survivors to fresh-cut pieces. The combination treatments nisin-NaL, nisin-KS, NaL-KS, and nisin-NaL-KS, but not nisin-EDTA, gave significant (P < 0.05) reductions of Salmonella directly inoculated onto fresh-cut pieces. Washing with nisin-NaL-KS was significantly (P < 0.05) more effective than the other three combination treatments, resulting in a reduction of 1.4 CFU/g. Inhibition by the four effective treatments carried over from day 0 through day 7 of storage, with no increase in the population of Salmonella on the stored fresh-cut pieces. Sensory evaluations indicated that treatment of fresh-cut pieces with nisin-NaL and NaL-KS, but not nisin-KS or nisin-NaL-KS, were acceptable in terms of appearance, odor, and overall acceptability. After the required regulatory approval, treatment of whole cantaloupe with nisin in combination with EDTA, NaL, KS, or NaL and KS and of fresh-cut pieces with nisin-NaL or NaL-KS could help ensure the microbiological safety of fresh-cut cantaloupe.

Effect of hot water and hydrogen peroxide treatments on survival of salmonella and microbial quality of whole and fresh-cut cantaloupe.

Cantaloupe melon has been associated with outbreaks of salmonellosis. Contamination might be introduced into the flesh from the rind by cutting or by contact of cut pieces with contaminated rinds. Our objectives were to investigate the efficacy of hot water or hot 5% hydrogen peroxide treatments in reducing the population of native microflora and inoculated Salmonella on cantaloupe rind and transfer to fresh-cut tissue during cutting. Whole cantaloupes, inoculated with a cocktail of Salmonella serovars to give 4.6 log CFU/cm2 and stored at 5 or 20 degrees C for up to 5 days, were treated with hot water (70 or 97 degrees C) or 5% hydrogen peroxide (70 degrees C) for 1 min at 0, 1, 3, or 5 days postinoculation. Aerobic mesophilic bacteria and yeast and mold on treated whole melon and fresh-cut pieces were significantly (P < 0.05) reduced by all three treatments. Treatments with hot water (70 and 97 degrees C) caused a 2.0- and 3.4-log CFU/cm2 reduction of Salmonella on whole cantaloupe surfaces irrespective of days of postinoculation storage prior to treatment up to 5 days at 5 or 20 degrees C, respectively. Treatment with 5% hydrogen peroxide (70 degrees C) caused a 3.8-log CFU/cm2 reduction of Salmonella. Fresh-cut pieces prepared from untreated inoculated melons and those treated with 70 degrees C hot water were positive for Salmonella. However, fresh-cut pieces prepared from inoculated whole melon dipped in water (97 degrees C) or hydrogen peroxide (70 degrees C) for 60 s were negative for Salmonella, as determined by dilution plating onto agar medium, but were positive after enrichment at days 3 and 5 of storage at 5 degrees C. The ability to detect Salmonella in fresh-cut pieces was dependent on the initial level of inoculation. The results of this study indicate that the use of hot water (97 degrees C) or heated hydrogen peroxide to reduce the population of Salmonella on contaminated whole cantaloupes will enhance the microbial safety of the fresh-cut product.

Effects of cell surface charge and hydrophobicity on attachment of 16 Salmonella serovars to cantaloupe rind and decontamination with sanitizers.

Adherence of bacteria to cantaloupe rind is favored by surface irregularities such as roughness, crevices, and pits, thus reducing the ability of washing or sanitizer treatments to remove or inactivate attached cells. In this study, we compared the surface charge and hydrophobicity of two cantaloupe-related outbreak strains of Salmonella Poona (RM2350 and G-91-1595) to those of 14 additional Salmonella strains using electrostatic and hydrophobic interaction chromatography. The relative abilities of the 16 strains to attach to cantaloupe surfaces and resist removal by washing with water, chlorine (200 ppm), or hydrogen peroxide (2.5%) for 5 min after a storage period of up to 7 days at 5 to 20 degrees C also were determined. Whole cantaloupes were inoculated with each pathogen at 8.36 log CFU/ml, dried for 1 h inside a biosafety cabinet, stored, and then subjected to the washing treatments. Only the positive surface charge of the two cantaloupe-related strains of Salmonella Poona was significantly higher (P < 0.05) than that of the other strains. Initial bacterial attachment to cantaloupe surfaces ranged from 3.68 to 4.56 log CFU/cm2 (highest values for Salmonella Michigan, Newport, Oranienburg, and Mbandaka). The average percentage of the total bacterial population strongly attached to the cantaloupe surface for the Salmonella serovars studied ranged from 0.893 to 0.946 at 5 degrees C and from 0.987 to 0.999 at 25 degrees C. Washing inoculated melons with water did not produce a significant reduction in the concentration of the pathogens (P > 0.05). Chlorine and hydrogen peroxide treatments caused an average 3-log reduction when applied 20 to 40 min postinoculation. However, sanitizer treatments applied 60 min or more postinoculation were less effective (approximately 2.5-log reduction). No significant differences were noted in sanitizer efficacy against the individual strains (P > 0.05). The two cantaloupe-related outbreak Salmonella Poona strains did not significantly differ from the other Salmonella strains tested in negative cell surface charge or hydrophobicity, were not more effective in attaching to whole melon surfaces, and were not more resistant to the various washing treatments when present on rinds.

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.

Method of applying sanitizers and sample preparation affects recovery of native microflora and Salmonella on whole cantaloupe surfaces.

Standardized methods for applying sanitizer treatments to cantaloupes and for recovering surviving native microflora or Salmonella on inoculated cantaloupe after sanitizing are lacking. Accordingly, the objectives of this study were to compare four methods for applying sanitizers (dipping, dipping with rotation, dipping with agitation, and dipping with rubbing) using 200 ppm of chlorine or 5% H2O2, two recovery methods (homogenization of rind plugs in a stomacher or blender), and five selective recovery media for Salmonella. Whole cantaloupes were submerged in a cocktail of five strains of Salmonella (each at approximately 2 x 10(8) CFU/ml) for 10 min and allowed to dry for 1 h inside a biosafety cabinet and stored at 20 degrees C for approximately 23 h before sanitizing. The recovery of Salmonella from whole cantaloupe without sanitizing averaged 5.09 log CFU/cm2 by blending and 4.30 log CFU/cm2 by homogenization in a stomacher for the five selective agar media. Microbial populations (Salmonella or the indigenous aerobic mesophilic bacteria, gram-negative bacteria, lactic acid bacteria, Pseudomonas spp., and yeast and mold) were not significantly (P > 0.05) reduced by treating with water regardless of the treatment method used. Sanitizing with chlorine or H2O2 by dipping, with or without rotation for 2 min, also did not reduce microbial populations. However, populations of all classes of native microflora and Salmonella were significantly (P < 0.05) reduced by sanitizer treatments (2 min) applied with agitation or by rubbing. In general, sanitizer treatments applied by rubbing resulted in greater log reductions (by up to 1.7 log unit) than for treatments applied with agitation. Populations of native microflora and Salmonella recovered from cantaloupe were higher (by up to 1.8 log unit) by blending compared to homogenization in a stomacher. In most instances, selective media used did not differ significantly (P > 0.05) for recovery of Salmonella after washing treatments.

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.

ATP bioluminescence assay for estimation of microbial populations of fresh-cut melon.

Estimation of microbial numbers in foods by conventional microbiological techniques takes days, so there is a need for faster methods that can give results in minutes. Research was undertaken to investigate the use of bioluminescent ATP determination and a firefly luciferase assay to estimate the initial population of aerobic mesophilic bacteria on fresh-cut melons immediately after preparation and during storage at 5 or 15 degrees C for up to 12 days. Populations of aerobic mesophilic bacteria on fresh-cut cantaloupe prepared immediately from unsanitized whole melons averaged 3.42 log CFU/g, corresponding to an ATP value of 5.40 log fg/g. Populations for fresh-cut honeydew prepared from unsanitized whole melon averaged 1.97 log CFU/g, corresponding an ATP value of 3.94 log fg/g. Fresh-cut pieces prepared from cantaloupe or honeydew melons sanitized with either chlorine (200 ppm free chlorine) or hydrogen peroxide (2.5%) had similar ATP values: 3.1 log fg/g (corresponding to bacterial counts 1.7 log CFU/g) for cantaloupes and 2.6 log fg/g (corresponding to bacterial counts of 0.48 CFU/g) for fresh-cut honeydew. Positive linear correlations for ATP concentrations and microbial populations were found for fresh-cut cantaloupe (R2 = 0.99) and honeydew R2 = 0.95) during storage at 5 degrees C for up to 12 days. ATP values in fresh-cut melons inoculated with either aerobic mesophilic bacteria or yeast and mold were significantly higher (P < 0.05) than control values and parallel total plate counts on plate count agar. Results of this study indicate that the bioluminescent ATP assay can be used to monitor total microbial populations on fresh-cut melon after preparation and during storage for quality control purposes to establish specific sell-by or consume-by dates.

Growth Parameters of Escherichia coli O157:H7, Salmonella spp., Listeria monocytogenes, and Aerobic Mesophilic Bacteria of Apple Cider Amended with Nisin–EDTA

The effect of nisin (0 or 300 IU/mL), ethylenediamine tetraacetic acid (EDTA, 20 mM), and nisin (300 IU)-EDTA (20 mM) on growth parameters, including lag period (LP) and generation time, of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella spp. in the presence or absence of aerobic mesophilic bacteria of apple cider during storage at 5 degrees C for up to 16 days or 23 degrees C for 16 h was investigated. The growth data were analyzed and fitted to the modified Gompertz model. The LP values for aerobic mesophilic bacteria of apple cider (control) and those amended with EDTA and nisin during storage at 5 degrees C were 1.61, 1.76, and 5.45 days, respectively. In apple cider stored at 23 degrees C for 16 h, the LP values for the same bacteria and treatment were 3.24, 3.56, and 5.85 h, respectively. The LP values for E. coli O157:H7 determined in the presence of aerobic mesophilic bacteria of apple cider stored at 23 degrees C for 16 h was 1.48 h, while populations for L. monocytogenes and Salmonella in the same cider declined. In sterile apple cider left at 23 degrees C for 16 h, the LP values for E. coli O157:H7, Salmonella, and L. monocytogenes averaged 2.74, 2.37, and 3.16 h, respectively. The generation time for these pathogens were 0.402, 0.260, and 0.187 log (CFU/mL)/h, respectively. Addition of nisin and EDTA combination caused a decline in lag phase duration and the populations for all pathogens tested, suggesting possible addition of this additive to freshly prepared apple cider to enhance its microbial safety and prevent costly recalls.