John B. Luchansky

The effects of diacetate with nitrite, lactate, or pediocin on the viability of Listeria monocytegenes in turkey slurries

The antilisterial effects of sodium diacetate (0.1, 0.3 and 0.5%) alone or in combination with sodium nitrite (30 ppm), sodium lactate (2.5%) or pediocin (5000 arbitrary units/ml) were evaluated in slurries (25% meat in sterile deionized H2O) prepared from vacuum-packaged, ready-to-eat turkey breast meat and challenged with Listeria monocytogenes. In the absence of food additives, counts of L. monocytogenes increased from 4.5 log10 cfu/ml to ca. 8 log10 cfu/ml within 1 day at 25 degrees C and within 14 days at 4 degrees C. Similarly, the pathogen grew to ca. 8 log10 cfu/ml within 1 d at 25 degrees C and within 28 days at 4 degrees C in slurries containing nitrite or lactate. In the presence of pediocin, after an initial decrease of 0.9 log10 cfu/ml, numbers of the pathogen reached ca. 8 log10 cfu/ml within 5 days at 25 degrees C and within 28 days at 4 degrees C. However, 0.3 and 0.5% diacetate in turkey slurries were listericidal at 4 and 25 degree C, respectively. In the presence of nitrite with diacetate, there was no appreciable difference in growth of L. monocytogenes compared with diacetate alone. Antilisterial activity was potentiated in treatments containing lactate with 0.3% diacetate at 25 degrees C and lactate with 0.1% diacetate at 4 degrees C, compared to similar treatments containing diacetate or lactate alone. A listericidal effect (ca. 7 log10 cfu/ml difference compared to slurries without additives) was observed in treatments containing pediocin with 0.5% diacetate at 25 degrees C and pediocin with 0.3% diacetate at 4 degrees C. The pH of slurries containing 0.3 or 0.5% diacetate was 5.5 and 5.2, respectively, whereas nitrite (pH 6.2), lactate (pH 6.3) or pediocin (pH 6.2) in slurries had a negligible effect on pH compared to the control (pH 6.2). The increased antilisterial activity in slurries with diacetate in combination with other additives was due to synergistic effects and not just pH. Thus, sodium diacetate alone can be used to delay growth of L. monocytogenes in turkey, and an additional level of safety can be achieved using diacetate in combination with sodium lactate or pediocin.

Genomic fingerprinting of 80 strains from the WHO multicenter international typing study of Listeria monocytogenes via pulsed-field gel electrophoresis (PFGE)

An international multicenter typing study of Listeria monocytogenes was initiated by the World Health Organization (Food Safety Unit, Geneva) in order to evaluate the usefulness of various phenotypic and genotypic typing methods for L. monocytogenes, to select and standardize the most appropriate methods to define common nomenclature of varieties and to select specific reference strains. Pulsed-field gel electrophoresis was used in four laboratories for molecular characterization of a set of 80 coded strains distributed to all participating laboratories. The endonucleases ApaI and SmaI, used in all four laboratories, yielded between 21 and 28 restriction endonuclease digestion profiles (REDP). AscI was used, in addition, in laboratory A and displayed 21 REDP. The combination of ApaI, SmaI or AscI REDP established 25 to 33 genomic groups. depending on the laboratory and the number of viable strains. Agreement of typing data among the four laboratories ranged from 79 to 90%. Forty-nine (69%) of the 71 strains viable in all four laboratories were placed into exactly the same genomic groups in all four laboratories. The epidemiological relevance of the strains became known after decoding and it was shown that most of the epidemiologically related strains were correctly identified by the four groups of investigators. i.e., most related strains were placed into the same genomic groups by all four laboratories. Similar results were obtained when 11 duplicate cultures were analyzed-on average 84% of the duplicates were identified. Comparison of REDP obtained by laboratory A with REDP from previously analyzed set of 176 L. monocytogenes strains allowed the prediction of the serovar-groups of the 80 strains. These predictions of serovar-groups were later confirmed by serotyping results obtained by laboratories involved in the WHO multicenter typing study of L. monocytogenes. In general this study reconfirmed that PFGE is a very accurate and reproducible method for fine structure comparison and molecular typing of L. monocytogenes.

Comparative survival of Salmonella typhimurium DT 104, Listeria monocytogenes, and Escherichia coli O157:H7 in preservative-free apple cider and simulated gastric fluid

This study compared the survival of three-strain mixtures (ca. 10(7) CFU ml(-1) each) of Salmonella typhimurium DT104, Listeria monocytogenes, and Escherichia coli O157:H7 in pasteurized and unpasteurized preservative-free apple cider (pH 3.3-3.5) during storage at 4 and 10 degrees C for up to 21 days. S. typhimurium DT104 populations decreased by <4.5 log10 CFU ml(-1) during 14 days storage at 4 and 10 degrees C in pasteurized cider, and by > or =5.5 log10 CFU ml(-1) during 14 days in unpasteurized cider stored at these temperatures. However, after 7 days at 4 degrees C, the S. typhimurium DT104 populations had decreased by only about 2.5 log10 CFU ml(-1) in both pasteurized and unpasteurized cider. Listeria monocytogenes populations decreased below the plating detection limit (10 CFU ml(-1)) within 2 days under all conditions tested. Survival of E. coli O157:H7 was similar to that of S. typhimurium DT104 in pasteurized cider at both 4 and 10 degrees C over the 21-days storage period, but E. coli O157:H7 survived better (ca. 5.0 log10 CFU ml(-1) decrease) than S. typhimurium DT104 (> 7.0 log10 CFU ml(-1) decrease) after 14 days at 4 degrees C in unpasteurized cider. In related experiments, when incubated in simulated gastric fluid (pH 1.5) at 37 degrees C, S. typhimurium DT104 and L. monocytogenes were eliminated (5.5-6.0 log10 CFU ml(-1) decrease) within 5 and 30 min, respectively, whereas E. coli O157:H7 concentrations decreased only 1.60-2.80 log10 CFU ml(-1) within 2 h.

Viability of Escherichia coli O157:H7 in pepperoni during the manufacture of sticks and the subsequent storage of slices at 21, 4 and −20 ° C under air, vacuum and CO2

A raw, pepperoni batter (75% pork:25% beef with a fat content of about 32%) was inoculated with a pediococcal starter culture (about 10(8) cfu/g) and a five-strain cocktail of Escherichia coli O157:H7 (> or = 2 x 10(7) cfu/g), mixed with non-meat ingredients, and then hand-stuffed into 55 mm fibrous casings to form sticks. The numbers of the pathogen were determined before stuffing, after fermentation, after drying/slicing, and after periods of storage. For storage, slices were packaged under air, vacuum or CO2 and stored at -20, 4 and 21 degrees C. Sticks were fermented at 36 degrees C and 85% relative humidity (RH) to < or = pH 4.8 and then dried at 13 degrees C and 65% RH to a moisture/protein ratio (M/Pr) of < or = 1.6:1. Fermentation and drying resulted in the numbers of the pathogen decreasing by about 2 log10 units. During storage, the temperature rather than the atmosphere had the greater effect on pathogen numbers. The greatest reductions in numbers were observed during storage at 21 degrees C, when numbers decreased to about 2 and 3.8 log10 cfu/g within 14 days in product stored under air and vacuum, respectively, and a 5 log10 reduction was observed for both atmospheres within 28 days. Regardless of the storage atmosphere, numbers did not decrease below 3.6 or 3.7 log10 cfu/g after 90 days of storage at -20 or 4 degrees C, respectively. These data confirm that fermentation and drying are sufficient to eliminate only about 2 log10 cfu/g of E. coli O157:H7 from fermented sausage, and that additional strategies, such as storage for at least 2 weeks at ambient temperature in air, are required to achieve a 5 to 6 log10 reduction in the numbers of the pathogen in sliced pepperoni.

Antilisterial activity of pediocin AcH in model food systems in the presence of an emulsifier or encapsulated within liposomes

The listericidal activity of pediocin AcH was evaluated in slurries (5, 10, or 25% in dH2O) of nonfat dry milk, butterfat, beef muscle tissue, or beef tallow. Slurries were inoculated with Listeria monocytogenes (2-strain mixture; 2.5 x 10(6) cfu/ml) and then with pediocin AcH (30,000 arbitrary units (AU) per ml of slurry). Although pediocin activity was reduced in slurries, sufficient pediocin remained to decrease the listeriae population. For all slurries tested, the greatest decrease in counts of Listeria (1.2-1.8 log10 cfu decrease) and decrease in pediocin activity (12-54% recovery of original activity) occurred within 1.5 min of addition of pediocin to slurries. Thereafter, counts of Listeria did not change appreciably, but pediocin activity continued to decrease in most treatments for up to 60 min. In general, greater activity was recovered from: (i) slurries of lower (5%) compared to higher (25%) concentrations of food; and (ii) dairy- compared to meat-based slurries. Next, pediocin AcH was encapsulated within phosphatidyl-choline-based liposomes before addition to slurries (10%), or was used unencapsulated in slurries (10%) containing the emulsifier Tween 80. Greater pediocin activity (29-62% increase; average over all concentrations) was recovered from slurries containing encapsulated compared to free pediocin AcH. Likewise, greater pediocin activity was recovered from slurries containing an emulsifier (4-90% increase; average over all concentrations) compared to otherwise similar slurries without Tween 80. The additional recovery of pediocin activity afforded by liposomes or Tween 80 underscores the potential for direct application of biopreservatives to provide another hurdle for L. monocytogenes in foods.

Viability of Escherichia coli O157:H7 in ground and formed beef jerky prepared at levels of 5 and 20% fat and dried at 52, 57, 63, or 68°C in a home-style dehydrator

Beef jerky batter was prepared to fat contents of about 5 and 20% and inoculated with about 10(8) cfu g(-1) of a five-strain inoculum of Escherichia coli O157:H7. Pathogen numbers were determined in the raw batter and in the strips formed from it after drying at 52, 57, 63, and 68 degrees C for times that ranged from 2 to 20 h. For both the high and low fat products, pathogen numbers were reduced by about 5 log10 cfu g(-1) within 4 h drying at 68 degrees C and within 8 h drying at 63 degrees C. At 57 degrees C, a 5-log10-unit reduction was achieved within 10h drying for the 5% fat product and within 16 h drying for the 20% fat product. At 52 degrees C, a 5-log10-unit reduction was achieved within 10 h drying for the 5% fat product and within 20 h drying for the 20% fat product. In at least one of the three trials for all four drying temperatures tested, the pathogen was present following enrichment of the samples in synthetic media. The calculated D values decreased from 2.59, 2.48, 1.23, and 1.17 as the temperature increased from 52, 57, 63, and 68 degrees C and as the fat content decreased from 20 to 5%. However, there was no direct correlation between the moisture-to-protein ratio and either the doneness of the strips or the viability of the pathogen. These data indicate that the fat content and the time and temperature at which strips are dried directly impact the viability of E. coli O157:H7 in ground and formed beef jerky.

Characterization of bacteriocins from Enterococcus faecium with activity against Listeria monocytogenes

Laboratory cultures and environmental isolates of bacteria were screened for antagonism towards Listeria monocytogenes using an agar spot test. Seven of the 163 strains that were tested, one Streptococcus bovis, one Enterococcus casseliflavus, two E. avium and three E. faecium, consistently displayed antilisterial activity. Cell-free, pH-neutralized supernatants prepared from the three E. faecium strains (JBL1061, JBL1083 and JBL1351) exhibited strong antilisterial activity against L. monocytogenes, and were subjected to more detailed analyses. The antagonistic factors produced by these three strains were sensitive to chloroform and several proteolytic enzymes, resistant to heat (121 degrees C, 20 min), and stable over a wide pH range (3.0-10.0). Moreover, they were listericidal without causing cell lysis. These data suggest that a bacteriocin(s) is involved in the inhibition of L. monocytogenes by E. faecium JBL1061, JBL1083 and JBL1351.

Behavior of Salmonella typhimurium DT104 during the manufacture and storage of pepperoni

Pepperoni batter (ca. 70% pork:30% beef) was prepared and subsequently inoculated with a six-strain cocktail (ca. 4.4 x 10(7) per gram batter) of Salmonella typhimurium DT104. After fermentation at 36 degrees C and 92% relative humidity (RH) to < or = pH 4.8, counts of the pathogen decreased by about 1.3 log10 units. An additional 1.6 log10 unit decrease was observed following drying at 13 degrees C and 65% RH to a moisture protein ratio (M/Pr) of 1.6:1. After storage of pepperoni sticks for 56 days under vacuum at 4 or 21 degrees C, counts of the pathogen were about 4.6 and 6.6 log10 units lower, respectively, compared with starting levels in the batter. These data establish that fermentation and drying result in about a 3.0 log10 reduction in numbers of S typhimurium DT104 in pepperoni sticks and that storage of pepperoni sticks under vacuum at ambient temperature is more severe on the pathogen than refrigerated storage.

Genomic analysis using pulsed-field gel electrophoresis of Escherichia coli 0157:H7 isolated from dairy calves during the United States national dairy heifer evaluation project (1991–1992)

The genomic fingerprints of 26 Escherichia coli O157:H7 isolates from calves on 20 farms in 16 states were determined by pulsed-field gel electrophoresis (PFGE). Digestion of genomic DNAs with the restriction enzymes SfiI and XbaI yielded 14 and 18 restriction endonuclease digestion profiles (REDP), respectively. Seventeen farms (85%) had E. coli O157:H7 with a unique REDP, and when more than one calf tested positive on a farm, the isolates displayed identical REDP. Isolates from different farms within the same state displayed distinct REDP, as did most isolates from farms in different states. The exceptions were three farms in New York, Ohio, and Washington that had calves harboring E. coli O157:H7 with the same REDP. In addition to REDP, the toxin profiles of all 26 isolates were determined using oligonucleotide probes to Shiga-like toxins (SLT) I and II. Nineteen (73%) of the E. coli O157:H7 isolates harbored the genes for both SLT and II, while the remaining seven isolates (27%) had the gene for SLT II only. Also, isolates with the same REDP had the same toxin profile. The genomic relatedness among the E. coli O157:H7 isolates was also determined by principal component analysis of Dice similarity indices of REDP. Three clusters were identified, but none of these were associated with a geographic region or toxin profile.

Inhibition of microbial growth in chub-packed ground beef by refrigeration (2°C) and medium-dose (2.2 to 2.4 kGy) irradiation

Abstract Two studies were conducted to monitor microbial growth in chub-packed ground beef. In the first study, the effect of storage temperatures (2 and 7°C) on chubs was examined for up to 18 days. Storage of chubs at 2°C slowed microbial growth such that total counts exceeded 7.5 log 10 cfu/g within 10 days compared to within 4 days for chubs stored at 7°C. Microbial counts ≥7.5 log 10 cfu/g were associated with off-odors and samples were considered spoiled. The predominant microbes were homofermentative lactococci, although a gas-producing bacterium, Hafnia alvei , was prevalent in one of three trials. In the second study, medium dose (2.2 to 2.4 kGy) X-ray irradiation extended the shelf-life of chub-packed ground beef at 2°C by approximately 14 days. Microbial counts on irradiated ground beef did not exceed 7.5 log 10 cfu/g during 34 days of storage, while this level was reached in non-irradiated chubs by day 13. The predominant microbes on both non-irradiated and irradiated chubs were homofermentative lactococci; H . alvei was not isolated in the second study. These data indicated that refrigerated (2°C) storage significantly delayed microbial spoilage in chub-packed ground beef, and that spoilage was further delayed when refrigerated (2°C) ground beef chubs were exposed to medium dose irradiation.