Patrick Marek

Detection of Penicillium expansum by polymerase chain reaction

Penicillium expansum is a major causative agent of postharvest decay in a variety of fruits, including apples, peaches, nectarines, and cherries. It causes significant economic losses to the fruit industry and is also of potential public health significance, since it produces patulin, a mycotoxin known to cause harmful effects in animals. Rapid and specific detection of P. expansum is important for ensuring microbiological quality and safety of fruits and fruit juices. The traditional methods for identification of P. expansum are time-consuming and labor-intensive. In this study, we report a polymerase chain reaction utilizing primers based on the polygalacturonase gene of P. expansum. The PCR amplified a 404-bp DNA product from all the P. expansum isolates tested, but not in other common foodborne Penicillium species and Escherichia coli. Experiments to determine the sensitivity of the PCR indicated that it can detect the DNA equivalent from as low as 25 spores of P. expansum. The PCR could potentially be used as a rapid tool for screening fruits for the presence of P. expansum.

In Vitro Inactivation of Escherichia coli O157:H7 in Bovine Rumen Fluid by Caprylic Acid

The antibacterial effect of caprylic acid (35 and 50 mM) on Escherichia coli O157:H7 and total anaerobic bacteria at 39 degrees C in rumen fluid (pH 5.6 and 6.8) from 12 beef cattle was investigated. The treatments containing caprylic acid at both pHs significantly reduced (P < 0.05) the population of E. coli O157:H7 compared with that in the control samples. At pH 5.6, both levels of caprylic acid killed E. coli O157:H7 rapidly, reducing the pathogen population to undetectable levels at 1 min of incubation (a more than 6.0-log CFU/ml reduction). In buffered rumen fluid at pH 6.8, 50 mM caprylic acid reduced the E. coli O157:H7 population to undetectable levels at 1 min of incubation, whereas 35 mM caprylic acid reduced the pathogen by approximately 3.0 and 5.0 log CFU/ml at 8 and 24 h of incubation, respectively. At both pHs, caprylic acid had a significantly lesser (P < 0.05) and minimal inhibitory effect on the population of total anaerobic bacteria in rumen compared with that on E. coli O157:H7. At 24 h of incubation, caprylic acid (35 and 50 mM) reduced the population of total anaerobic bacteria by approximately 2.0 log CFU/ml at pH 5.6, whereas at pH 6.8, caprylic acid (35 mM) did not have any significant (P > 0.05) inhibitory effect on total bacterial load. Results of this study revealed that caprylic acid was effective in inactivating E. coli O157:H7 in bovine rumen fluid, thereby justifying its potential as a preslaughter dietary supplement for reducing pathogen carriage in cattle.

Survival and growth characteristics of Escherichia coli O157:H7 in pasteurized and unpasteurized Cheddar cheese whey.

The objective of this study was to determine the survival and growth characteristics of Escherichia coli O157:H7 in whey. A five-strain mixture of E. coli O157:H7 was inoculated into 100 ml of fresh, pasteurized or unpasteurized Cheddar cheese whey (pH 5.5) at 10(5) or 10(2) CFU/ml, and stored at 4, 10 or 15 degrees C. The population of E. coli O157:H7 (on Sorbitol MacConkey agar supplemented with 0.1% 4-methylumbelliferyl-beta-D-glucuronide) and lactic acid bacteria (on All Purpose Tween agar) were determined on days 0, 1, 4, 7, 14, 21 and 28. At all storage temperatures, survival of E. coli O157:H7 was significantly higher (P<0.01) in the pasteurized whey compared to that in the unpasteurized samples. At 10 and 15 degrees C, E. coli O157:H7 in pasteurized whey significantly (P<0.05) increased during the first week of storage, followed by a decrease thereafter. However at the same temperatures, E. coli O157:H7 exhibited a steady decline in the unpasteurized samples from day 0. At 4 degrees C, E. coli O157:H7 did not grow in pasteurized and unpasteurized whey; however, the pathogen persisted longer in pasteurized samples. At all the three storage temperatures, E. coli O157:H7 survived up to day 21 in the pasteurized and unpasteurized whey. The initial load of lactic acid bacteria in the unpasteurized whey samples was approximately 7.0 log10 CFU/ml and, by day 28, greater than 3.0 log10 CFU/ml of lactic acid bacteria survived in unpasteurized whey at all temperatures, with the highest counts recovered at 4 degrees C. Results indicate the potential risk of persistence of E. coli O157:H7 in whey in the event of contamination with this pathogen.

Effect of erythorbate, storage and high-oxygen packaging on premature browning in ground beef

Premature browning (PMB) was investigated in ground beef patties with (0.04%, w/w) and without erythorbate. In Experiment 1, patties were stored at 4 °C for 48 h; at -18 °C for 21 days; or at -18 °C for 21 days, thawed at 4 °C for 24 h; and cooked. Bulk ground beef was stored at -18 °C for 24 days, thawed for 24 h at 4 °C, and patties prepared and cooked immediately. In Experiment 2, fresh patties were overwrapped with oxygen-permeable film or packaged in 80% O(2)/20% N(2) (MAP), and stored for 48 h at 4 °C, or at -18 °C for 21 days, and cooked. Total reducing activity and color (L*, a* and b* values) were measured immediately prior to cooking. Patties were cooked to internal temperatures of 60, 66, 71 and 77 °C and internal cooked color was measured. Total reducing activity was higher for the erythorbate treatment than controls for all storage conditions (P<0.05). a* Values of cooked patties were higher for erythorbate than control treatments under all storage and packaging conditions at 60 and 66 °C (P<0.05). The presence of erythorbate in ground beef patties appeared to maintain red color at cooked internal temperatures of 60 and 66 °C. Frozen bulk storage appeared to increase the susceptibility of ground beef to PMB when compared to fresh and frozen patties. Patties cooked directly from frozen state appeared less susceptible to PMB than frozen-thawed and bulk storage. Ground beef appeared predisposed to PMB when stored in high-oxygen MAP at 4 °C for 48 h.

Additive approach for inactivation of Escherichia coli O157:H7, Salmonella, and Shigella spp. on contaminated fresh fruits and vegetables using bacteriophage cocktail and produce wash.

The incidence of foodborne outbreaks involving fresh produce is of worldwide concern. Lytic bacteriophage cocktails and a levulinic acid produce wash were investigated for their effectiveness against the foodborne pathogens Escherichia coli O157:H7, Shigella spp., and Salmonella on broccoli, cantaloupe, and strawberries. Inoculated samples were treated with bacteriophage cocktails (BC) before storage at 10°C for 24 h, a levulinic acid produce wash (PW) after storage at 10°C for 24 h, or a combination of the washes (BCPW) before and after storage. All three treatments were compared against a 200-ppm free available chlorine wash. Wash solutions were prepared using potable water and water with an increased organic content of 2.5 g/liter total dissolved solids and total organic carbon. BCPW was the most effective treatment, producing the highest log reductions in the pathogens. Produce treated with BCPW in potable water with a PW exposure time of 5 min resulted in the highest reduction of each pathogen for all samples tested. The type of produce and wash solution had significant effects on the efficacy of the individual treatments. The chlorine wash in water with higher organic content was the least effective treatment tested. An additive effect of BCPW was seen in water with higher organic content, resulting in greater than 4.0-log reductions in pathogens. Our findings indicate that the combination of antimicrobial BC with a commercial produce wash is a very effective method for treating produce contaminated with E. coli O157:H7, Shigella spp., and Salmonella even in the presence of high loads of organic matter.

Inactivation of Escherichía coli O157:H7 in Cattle Drinking Water by Sodium Caprylate

Escherichia coli O157:H7 is an important foodborne pathogen. Cattle serve as one of the major reservoirs of E. coli O157:H7, excreting the pathogen in feces. Environmental persistence of E. coli O157:H7 is critical in its epidemiology on farms, and the pathogen has been isolated from cattle water troughs. Thus, there is a need for an effective method for killing E. coli O157:H7 in cattle drinking water. In this study, the efficacy of sodium caprylate for killing E. coli O157:H7 in cattle drinking water was investigated. A four-strain mixture of E. coli O157:H7 was inoculated (6.0 log CFU/ml) into 100-ml samples of well water containing 0, 75, 100, or 120 mM sodium caprylate. Water samples containing 1% (wt/vol) bovine feces or feed also were included. The samples were incubated at 21 or 8 degrees C for 21 days. Water samples were analyzed for viable E. coli O157:H7 on days 0, 1, 3, 5, and 7 and weekly thereafter. Triplicate samples of each treatment and control were included, and the study was repeated twice. The magnitude of E. coli O157:H7 inactivation in water significantly increased (P < 0.01) with increases in caprylate concentration and storage temperature. At 120 mM, sodium caprylate completely inactivated E. coli O157:H7 in all the samples after 1 to 20 days, depending on the treatments. The presence of feces or feed also had a significant effect (P < 0.01) on the antibacterial property of caprylate; the presence of feces decreased the antibacterial effect, whereas addition of feed enhanced the effect. These results indicate that sodium caprylate is effective in killing E. coli O157:H7 in cattle drinking water, but detailed cattle palatability studies of water containing caprylate are necessary.