INACTIVATION OF LISTERIA MONOCYTOGENES, SALMONELLA ENTERITIDIS AND ESCHERICHIA COLI O157:H7 AND SHELF LIFE EXTENSION OF FRESH-CUT PEARS USING MALIC ACID AND QUALITY STABILIZING COMPOUNDS

ABSTRACT

Inactivation of Listeria monocytogenes, Salmonella enteritidis and Escherichia coli O157:H7 and shelf life extension of fresh‐cut pears using malic acid (MA) and quality stabilizing compounds (N‐acetyl‐L‐cysteine, glutathione and calcium lactate; CGLW) were investigated. Trays of treated fresh‐cut pears were wrap sealed with a thick polypropylene film (64 µm) semipermeable to water vapor, O₂ and CO₂, and stored at 5C for 30 days. Changes in headspace gas, firmness and color of the fresh‐cut pears were also determined. Large reductions of L. monocytogenes (6.57 log₁₀ cfu/g), S. enteritidis (6.60 log₁₀ cfu/g) and E. coli O157:H7 (2.62 log₁₀ cfu/g) just after processing were achieved in those fresh‐cut pears dipped in CGLW + MA. Microbiological shelf life of pear pieces dipped in CGLW + MA was extended by more than 21 days in comparison with those cut pears immersed in water used as control sample. Lower consumption of O₂ and production of CO₂, ethylene and ethanol of fresh‐cut pears dipped in CGLW + MA were also observed. In addition, the color and firmness of pear pieces in CGLW + MA were maintained by more than 21 days in comparison with control samples. In conclusion, the combination of MA with quality stabilizing compounds can be a good alternative for assuring the safety and quality of fresh‐cut pears.

PRACTICAL APPLICATIONS

The use of natural substances generally recognized as safe (GRAS) such as malic acid and N‐acetyl‐L‐cysteine, glutathione and calcium lactate as antimicrobials and quality stabilizing compounds, respectively, can result suitable to fresh‐cut products industry, since they can assure the safety and quality of those products, while improving their sensory attributes and maintaining the fresh‐like and healthy properties of these products greatly demanded by the consumers.     

INTERACTIONS BETWEEN pH AND MALIC ACID CONCENTRATION ON THE INACTIVATION OF LISTERIA MONOCYTOGENES1

The effects and interactions of malic acid concentration and pH on the inactivation kinetics of a three strain mixture of Listeria monocytogenes was studied in brain heart infusion broth (BHI). The medium was supplemented with malic acid and monosodium malate to achieve pH levels of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 or 7.0 in conjunction with concentrations of 0.0, 0.1, 0.5, 1.0 and 2.0 M. Duplicate 20-mL portions of each pH/malate level were inoculated (ca. 10⁸ cfu/mL), stored aerobically at 28C, and assayed periodically for viable counts by plating on BHI agar. Survivor curves were generated by fitting data to a linear model that includes a lag term and used to calculate D-values and “times to 4-D inactivation”. Inactivation rates were dependent on both the pH and malic acid concentration. At the higher pH levels, malic acid appeared to provide some degree of protection compared to control cultures where the pH was adjusted with HCl. At lower pH values and at higher malic acid concentrations, a concentration-dependent anion effect was observed. The results indicate that malic acid is a relatively benign organic acid. Its antimicrobial characteristics are similar to those of citric acid and is substantially less bactericidal than lactic or acetic acids.     

DUAL-PHASIC INACTIVATION OF ESCHERICHIA COLI O157:H7 WITH PEROXYACETIC ACID, ACIDIC ELECTROLYZED WATER AND CHLORINE ON CANTALOUPES AND FRESH-CUT APPLES

The effects of peroxyacetic acid (POAA), acidic electrolyzed water (AEW) and chlorine on inactivation of Escherichia coli O157:H7 on fresh‐cut apples and cantaloupe rinds were investigated. Apple cylinders were dip‐inoculated with E. coli O157:H7 and treated with sterilized water (control), chlorine, AEW or POAA for up to 8 min. Cantaloupe cylinders were spot‐inoculated with E. coli O157:H7 to the rind and treated with sterilized water, AEW or POAA for up to 15 min. All sanitizer treatments showed a significantly (P < 0.05) higher inactivation than the control. The residual counts of E. coli O157:H7 on both fruits exhibited a dual‐phasic reduction behavior, with a fast inactivation (D values: 0.8–5.0 min) in the first minute (phase I) of treatments followed by a much slower inactivation (D values: 14.6–59.8 min) in the remaining time (phase II). The dual‐phasic inactivation seems to be related to fruit surface topography that determines the bacterial distribution.     

BEHAVIOR OF LISTERIA MONOCYTOGENES AND ESCHERICHIA COLI O157:H7 IN FRESH-SLICED CACTUS-PEAR FRUIT

Storage experiments were conducted to follow the behavior of Listeria monocytogenes and Escherichia coli O157:H7, deliberately inoculated on fresh‐cut cactus‐pear fruits before packaging under modified and control atmosphere and stored at four different temperatures (4, 8, 12 and 20C). L. monocytogenes was able to proliferate during storage at different temperature both in control and modified atmosphere. By comparing the sanitary‐risk values with those of shelf life, it is possible to conclude that the storage of cactus‐pear samples at temperatures greater than 4C, both in control and in modified atmospheres, could lead to a significant health‐time risk, and that this is strictly affected by temperature. E. coli O157:H7 was able to proliferate only in the sample stored at 4 and 8C in both package atmospheres. On the contrary, this species was completely suppressed at the higher temperatures. In our study, E. coli O157:H7 appeared to be much less suited for survival on the surface of the fruit than L. monocytogenes.     

SURVIVAL OF SALMONELLA TYPHIMURIUM, ESCHERICHIA COLI O157:H7 AND LISTERIA MONOCYTOGENES DURING STORAGE ON BEEF SANITIZED WITH ORGANIC ACIDS

Sterile beef tissue was inoculated with either Salmonella typhimurium, Escherichia coli O157:H7 or Listeria monocytogenes Scott A and washed with 23C distilled water, 1% lactic acid or 1% acetic acid. The washed tissue was subjected to simulated dry chilling or spray chilling followed by storage at 5C. The washed tissue was stored at 5C for up to 21 days at 26% relative humidity, and total bacterial populations were determined by plating on nonselective and selective agars. There was no significant difference in the surviving populations of S. typhimurium, Escherichia coli O157:H7, or L. monocytogenes after storage, irrespective of chilling method. The surviving populations of bacteria were significantly lower on acid washed adipose tissue, when compared to the comparable water washed tissue. These results indicate that although injury and recovery of pathogenic bacteria may occur as a result of organic acid carcass sanitizing treatments, there was no practical significance of this phenomenon after 3 days of storage.     

INHIBITION OF SALMONELLA SEROVARS, ESCHERICHIA COLI O157:H7 AND LISTERIA MONOCYTOGENES DURING DRY-CURING AND DRYING OF MEAT: A CASE STUDY WITH BASTURMA

Basturma, a spiced beef product intended to be cooked before consumption, was prepared in three trials by dry‐curing intact surface‐inoculated (6 log cfu per piece with Salmonella serovars, Escherichia coli O157:H7 and Listeria monocytogenes) beef eye of round subprimals for 21 days at 6.7C, and then rinsing, pressing and drying for 6–8 days at 21–27C. The finished products had percentage water‐phase salt concentrations of 8.3–18.0 and water activity of 0.84–0.95. In the finished product, no surviving Salmonella serovars and E. coli O157:H7 were detected (<1.0 log cfu) in two trials; in the third trial the reductions were 4.5 and 5.0 log cfu, respectively. L. monocytogenes populations decreased 4.0–4.9 log cfu. Basturma prepared by dry‐curing, rinsing, pressing and drying appears to present little risk of foodborne infection because this process reduces populations of Salmonella serovars, E. coli O157:H7 and L. monocytogenes. Dry‐curing and drying are potentially very important pathogen‐reduction steps.     

INHIBITION OF LISTERIA MONOCYTOGENES,SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI 0157:H7 ON BEEF MUSCLE TISSUE BY LACTIC OR ACETIC ACID CONTAINED IN CALCIUM ALGINATE GELS1

A method of applying organic acids contained in a calcium alginate gel was tested for inhibiting bacteria contaminating sterile lean beef tissue surfaces. Treated samples were incubated at 5C under controlled moisture conditions for up to 7 days and viable populations of the pathogens determined. For counts of L. monocytogenes, recovered on tryptic soy agar, alginate/lactic acid treatment reduced the log₁₀ counts 1.8 units vs 0.96 for acid treatment without alginate. With acetic acid, log₁₀ reductions were 1.51 vs 2.33 for the alginate/acetic acid vs acetic acid treatment alone. S. typhimurium was reduced 2.11 log₁₀ units vs 1.11 for alginate/lactic acid and acid treatments alone, respectively. The same trend held for E. coli 0157:H7, although the reduction was considerably less, 0.74 log₁₀ reduction vs 0.5 for alginate/lactic acid application vs acid alone. Both Gramnegative organisms were less inhibited by acetic acid treatments.     

SIMULTANEOUS DETECTION OF LISTERIA MONOCYTOGENES, STAPHYLOCOCCUS AUREUS, SALMONELLA ENTERICA AND ESCHERICHIA COLI O157:H7 IN FOOD SAMPLES USING MULTIPLEX PCR METHOD

In this study, one multiplex polymerase chain reaction (MPCR) assay was developed for simultaneous detection of four foodborne pathogens, i.e., Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica and Escherichia coli O157:H7. Five specific primer pairs were designed based on the nucleotide sequences of hemolysin gene (hly) of Listeria monocytogenes, thermostable nuclease gene (nuc) of Staphylococcus aureus, invasion gene (invA) of Salmonella enterica, shiga-like toxin gene (stx) and intimin gene (eae) of Escherichia coli O157:H7 in this assay. The specificity and sensitivity of the MPCR method were validated, and the limit of detection (LOD) of this method was about 10 copies. One cfu/mL each of these foodborne pathogens spiked in practical food samples, i.e., ground meat, beef, pork, fish, shrimp, cheese, canola leaf and cabbage, could be detected simultaneously after 24 h enrichment at a rate of 87.5%, indicating that the established MPCR detection method was effective and suitable for practical use.

PRACTICAL APPLICATIONS

This study presents a quick and effective identification method to simultaneous monitor four foodborne pathogens in food samples. The specificity and sensitivity of this method can be used to unambiguously identify these four foodborne pathogens in practical food samples based on the species-specific genes. Therefore, this detection method is applicable for surveillance measures of these four foodborne pathogens in the food production chain.     

REDUCTION OF LISTERIA MONOCYTOGENES,ESCHERICHIA COLI O157:H7 AND SALMONELLA TYPHIMURIUM DURING STORAGE ON BEEF SANITIZED WITH FUMARIC,ACETIC, AND LACTIC ACIDS1

Cylindrical samples of beef semitendinosus muscle were dipped into an inoculum of Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella typhimurium. After air drying for 10 min, samples were sanitized by dipping in fumaric acid (1.0 or 1.5%), lactic acid (1%) or acetic acid (1.0%) solutions for 15 or 30 s at 55C. The 1.5% fumaric acid was most inhibitory against L. monocytogenes, resulting in inactivation of this pathogen after 7 days of storage. Samples treated with 1.0% and 1.5% fumaric acid solutions for 30 sec gave 2.54 and 1.51 log₁₀ reductions, respectively, in S. typhimurium population compared to nontreated samples. The 1.5% fumaric acid treatments for 30 s exerted the maximum inhibition of E. coli O157:7. Acetic and lactic acid treatments were significantly less effective in reducing populations of the tested pathogens than fumaric acid treatments. As the sanitizing time in acid solutions increased from 15 to 30 s, the numbers of surviving organisms were reduced significantly.     

OCCURRENCE OF ESCHERICHIA COLIO157:H7/O157, LISTERIA MONOCYTOGENES AND SALMONELLA SPP. IN BEEF SLAUGHTERHOUSE ENVIRONMENTS, EQUIPMENT AND WORKERS

Escherichia coli O157/O157:H7, Listeria monocytogenes and Salmonella spp. are major foodborne pathogens and the emergence of these pathogens has been reported in many countries. The aim of this study was to investigate contamination of the beef slaughterhouse environment, equipment and workers with E. coli O157/O157:H7, L. monocytogenes and Salmonella spp. For this study, 500 swab samples were taken from 19 different points in five privately owned slaughterhouses, their periphery, slaughterhouse equipment and slaughterhouse employees. The presence of E. coli O157:H7/O157, L. monocytogenes and Salmonella spp. was determined with the application of the immunomagnetic separation method. Our study showed that the swabs taken from the five slaughterhouses contained E. coli O157:H7 in the environment, equipment, abattoir workers and water with a frequency 0.31, 1, 1.42 and 0%, respectively; while E. coli O157 was evident in the environment, equipment, abattoir workers and water with a ratio of 15, 10, 10 and 0%, respectively; L. monocytogenes was detected in the environment, equipment, abattoir workers and water at a ratio of 4.37, 15, 5.71 and 0%, respectively; and Salmonella spp. occurrence in the environment, equipment, abattoir workers and water at a ratio of 3.43, 16, 11.42, and 0%, respectively. Implementing hazard analysis critical control point principles in work procedures would definitely reduce the gross contamination occurring in abattoirs.

PRACTICAL APPLICATIONS

This study has revealed the effect of personnel and equipment on the contamination routes of E. coli O157:H7, Listeria monocytogenes and Salmonella spp. in meat in slaughterhouses and showed that especially L. monocytogenes and Salmonella spp. may pose a higher risk than E. coli O157:H7 in slaughterhouses.     

INACTIVATION OF ESCHERICHIA COLI O157:H7 BY THE COMBINATION OF ORGANIC ACIDS AND PULSED ELECTRIC FIELD

Inactivation of Escherichia coli O157:H7 by pulsed electric field, benzoic or sorbic acid, alone or in combination was investigated. When the cells of E. coli O157:H7 were suspended in 10% glycerol and treated with a single highvoltage (12.5 kv/cm) electric pulse at 25C, the count decreased by 1.1–1.6 log₁₀ CFU/mL. Presence of benzoic and sorbic acid (1000 ppm) in the suspending medium, at pH 3.4 without electric treatment, decreased the count by 1.9–2.5 and 0.6–1.1 log, respectively. A synergistic killing effect between the high electric pulse and organic acid was observed at pH 3.4, but not at pH 6.4. When the cell suspension of E. coli O157:H7 was treated with five electric pulses in the presence of benzoic or sorbic acid at pH 3.4, the count decreased by 5.6 and 4.2 logs, respectively. Inactivation of the pathogen by combinations of electric pulse and organic acid was enhanced by an increase in temperature, field strength, and number of electric pulses, Inactivation was greater when the cells were suspended in ionic suspending media (0.1% NaCl or 5mM phosphate buffer) than in nonionic media (10% glycerol or 1% sucrose) .     

RAPID DETECTION OF OUTBREAK ESCHERICHIA COLI O157 AND SALMONELLA ON ALFALFA SPROUTS BY IMMUNOMAGNETIC CAPTURE AND TIME‐RESOLVED FLUORESCENCE1

Commercially available alfalfa seeds were inoculated with low levels (～ 4 CFU/g) of pathogenic bacteria and sprouted at 25C. At 48 h, the spent irrigation water and sprouts were separately transferred to brain heart infusion (BHI) broth and enriched for 4 h at 37C and 160 rpm. Specific immunomagnetic beads (IMB) were then applied to capture the E. coli O157 or Salmonella in the enriched media. Separation and concentration of captured pathogens were achieved using magnetic particle concentrators (MPC). IMB captured E. coli O157:H7 and Salmonella spp. then formed sandwiched complexes with europium (Eu) labeled anti‐E. coli O157 antibodies and samarium (Sm) labeled anti‐Salmonella antibodies, respectively. After washing the complexes, the lanthanide labels were extracted out from the complexes by specific chelators to form strongly fluorescent Eu‐ and Sm‐chelates. The specific time‐resolved fluorescence (TRF) associated with Eu or Sm was measured to estimate the extent of capture of the E. coli O157 and Salmonella, respectively. The results indicated that the approach could detect E. coli O157 and many Salmonella spp. from spent irrigation water or sprouts grown from contaminated seeds. Nontargeted bacteria, e.g., native microflora present on the untreated seeds and inoculated Aeromonas and Citrobacter, exhibited no crossreactivity and counts were not significantly different from background fluorescence of the IMB alone. Since pathogen detection was achieved within 6 h, the assay could detect contamination levels as low as 4 CFU/g of seeds and it showed no cross‐reactivity with nonpathogenic microflora present on the sprouts, the developed methodology could be used as a rapid, sensitive and specific screening process for E. coli O157 and Salmonella spp. in sprouts and their irrigation water.