The microbial inhibition of foodborne pathogens was determined in brain heart infusion broth with 10% (wt/vol) water-soluble extracts of green, jasmine, black, dungglre, and oolong tea against Escherichia coli O157:H7, Salmonella enterica serovar Enteritidis, Listeria monocytogenes , and Staphylococcus aureus . The mixed culture (approximately 6.0 log CFU/ml), which was composed of the four pathogens, was inoculated into brain heart infusion broth with and without tea extracts. After incubation at 35°C for 0, 1, 3, and 5 days, proper dilution of each sample was spiral plated on each selective agar. Viable cell counts were performed after incubation at 35°C for 24 to 36 h. Green, jasmine, and black tea exhibited an approximately 5.0-log suppression of S. aureus compared with the control from days 1 to 5. Green and jasmine tea also suppressed the growth of L. monocytogenes by approximately 3.0 log CFU/ml on day 5. In contrast, no tea extracts inactivated E. coli O157:H7 and Salmonella Enteritidis. Based on the result in liquid medium, green and jasmine teas of 0.1% (vol/wt) were individually evaluated for their antimicrobial activity against L. monocytogenes and S. aureus in a food model (ground beef) stored at 7°C for 0, 1, 3, 5, and 7 days. Viable cell counts of total bacteria, L. monocytogenes , and S. aureus in ground beef were not significantly different among green and jasmine tea and the control.

This study evaluated the effect of organic acids and spices, alone or combined, on Clostridium perfringens growth in cooked ground beef during alternative cooling procedures. Ground beef was inoculated with a three-strain cocktail of C. perfringens (ATCC 10388, NCTC 8238, and NCTC 8239) at 2 log spores per g and prepared following an industrial recipe (10% water, 1.5% sodium chloride, and 0.5% sodium triphosphate [wt/wt]). Treatments consisted of the base meat plus combinations of commercial solutions of sodium lactate or sodium citrate (0 or 2%, wt/wt) with chili, garlic and herbs, curry, oregano, or clove in commercial powder form (0 or 1%, wt/wt). Untreated meat was used as a control. Vacuum-packaged samples of each treatment were cooked (75°C for 20 min) and cooled from 54.4 to 7.2°C in 15, 18, or 21 h. Spore counts were estimated after inoculation, cooking, and cooling. All treatments containing sodium citrate reduced the population of C. perfringens about 0.38 to 1.14 log units during each of the three cooling procedures. No sodium citrate and spice treatment combinations showed antagonisms or synergisms. Regardless of the cooling time, the control ground beef or treatments with any of the five spices alone supported C. perfringens growth above the U.S. Department of Agriculture stabilization guidelines of 1 log unit. Except for the 21-h cooling period, addition of sodium lactate prevented C. perfringens growth over 1 log unit. Depending on the cooling time and spice, some combinations of sodium lactate and spice kept C. perfringens growth below 1 log unit.

Antimicrobial activity of water-soluble arrowroot tea extract was evaluated against Escherichia coli O157:H7, Salmonella enterica Serotype Enteritidis, Listeria monocytogenes , and Staphylococcus aureus in ground beef and mushroom soup. The concentrations of arrowroot tea used were 0, 3, and 6% (wt/wt) for ground beef and 0, 1, 5, and 10% (wt/vol) for mushroom soup. Samples without tea extract were considered controls. Each sample was stored for 0, 1, 3, 5, and 7 days at 7°C for ground beef and for 0, 1, 3, and 5 days at 35°C for mushroom soup. On each sampling time, proper dilutions were spread plated on each pathogen-specific agar. Viable cell counts of each pathogen were performed after incubation at 35°C for 24 to 48 h. For ground beef, Salmonella Enteritidis and L. monocytogenes were slightly suppressed by approximately 1.5 log, compared with the control, on day 7 at 3 and 6% arrowroot tea treatment. For mushroom soup, all test pathogens were suppressed by 6.5, 4.7, 3.4, and 4.3 log at 5% and 6.0, 4.7, 5.0, and 4.3 log at 10% against E. coli O157:H7, Salmonella Enteritidis, L. monocytogenes , and S. aureus , respectively, compared with the control on day 5. Mushroom soup with 1% arrowroot tea also showed 2.3- and 2.7-log growth suppression of Salmonella Enteritidis and S. aureus , respectively, compared with the control on day 5. This study showed that the use of arrowroot tea would effectively inhibit the microbial growth of both gram-negative and gram-positive foodborne pathogens in various foods, especially liquid foods.

Pasteurized apple juice with nisin (0, 25, 50, 100, and 200 ppm, wt/vol) and cinnamon (0 and 0.3%, wt/vol) was inoculated with Salmonella Typhimurium and Escherichia coli O157:H7 at 10 4 CFU/ml and stored at 5 and 20°C. Counts on tryptic soy agar (TSA), selective medium (xylose lysine desoxycholate agar for Salmonella Typhimurium, and MacConkey sorbitol agar for E. coli O157:H7), and thin agar layer (TAL) were determined at 1 h and 1, 3, 7, and 14 days. The TAL method (selective medium overlaid with TSA) was used for recovery of sublethally injured cells. The pathogens were gradually inactivated by the acidic pH of apple juice. Nisin and cinnamon greatly contributed to the inactivation. The killing effect was more marked at 20°C, with counts in all treated samples being undetectable by direct plating in 3 days for Salmonella Typhimurium and 7 days for E. coli O157:H7. Thus, several factors influenced the decrease in counts: low pH, addition of nisin and cinnamon, and storage temperature. The TAL method was as effective as TSA in recovering injured cells of the pathogens. The combination of nisin and cinnamon accelerates death of Salmonella Typhimurium and E. coli O157:H7 in apple juice and so enhances the safety of the product.

A 5′-nuclease (TaqMan) assay was evaluated for its capability to recover and detect stressed Yersinia enterocolitica . Sensitivity studies of a 5′-nuclease assay for detecting Y. enterocolitica O:8 in a pure culture system and spiked ground pork samples demonstrated that the assay has reliable sensitivity with a detection limit of 3 to 4 log CFU/ml or CFU/g. The PCR 5′-nuclease (TaqMan) assay was evaluated with the Thin Agar Layer Oxyrase method (TALO, overlaying 14 ml of Trypticase soy agar with a 1:30 dilution of “Oxyrase ® for Agar” onto a prepoured pathogen-specific, selective medium), and it was compared against the selective medium cefsulodin-irgasan-novobiocin (CIN) for recovering and detecting Y. enterocolitica from inoculated nonfrozen and frozen (−15°C, 2 days) ground pork samples. The TALO method showed more sensitivity (detection limit, 2 log CFU/ml), and it has greater recovery capability (0.5 to 1 log CFU/ml) than CIN ( P < 0.05). The 5′-nuclease assay provided rapid detection processing (5 versus 24 h after an 18-h enrichment). The sensitivity per PCR was calculated to as low as 0 to 1 log CFU per PCR reaction; however, in the method's current developmental stage, target pathogens should be enriched to 3 to 4 log CFU/ml or CFU/g to show consistent results. In a survey of 100 ground pork samples using TALO, CIN, and PCR methods, no Y. enterocolitica was recovered. A combined cultivation and an automated PCR TaqMan could be used as a presumptive screening test for detecting Y. enterocolitica in food samples.

This study was conducted to determine how well Clostridium perfringens spores germinate and grow in restructured roast beef treated with different commercial organic salts during an alternative chilling procedure. The meat was prepared according to an industrial recipe (10% water, 1.5% sodium chloride, and 0.5% sodium triphosphate). The base meat was treated with sodium citrate at 2 or 4.8% (wt/wt), buffered to a pH of 5.6, 5.0, or 4.4 (six treatments); a 60% (wt/wt) solution of sodium lactate at 2 or 4.8% (wt/wt); sodium acetate at 0.25% (wt/wt); or sodium diacetate at 0.25% (wt/wt). Untreated meat was used as a control. Meat samples were inoculated with a three-strain cocktail of C. perfringens spores (strains ATCC 10388, NCTC 8238, and NCTC 8239). Meat was vacuum packaged in bags and cooked in a stirred water bath to an internal temperature of 75°C for 20 min, and then the bags were cooled from 54.4 to 4.4°C within 18 h. Samples were taken after inoculation, after cooking, and after chilling. Spore and vegetative cell counts were obtained after incubation at 37°C for 8 to 10 h in Fung's Double Tubes containing tryptose sulfite agar without egg yolk enrichment. Cooking was not sufficient to eliminate C. perfringens spores. Over the 18-h cooling period, sodium citrate, sodium lactate, and sodium diacetate reduced the growth of C. perfringens to <1 log unit, a growth level that meets U.S. Department of Agriculture performance standards. The use of sodium citrate or sodium lactate at a concentration of ≥2% (wt/wt) inhibited C. perfringens growth over the 18-h cooling period.

Normal (pH 3.7) and adjusted (pH 5.0) pasteurized apple juice containing cinnamon (0, 0.1, 0.2, and 0.3%) was inoculated with Listeria monocytogenes Scott A 49594 at 10 4 CFU/ml and stored at 5 and 20°C for 7 days. Counts on tryptic soy agar (TSA), modified Oxford (MOX) medium, and thin agar layer (TAL) were determined at 1 h and 1, 3, and 7 days. The TAL method (MOX medium overlaid with TSA) was used for the recovery of injured cells. In apple juice, both at normal and adjusted pH, with any doses of cinnamon, no L. monocytogenes (a 4.6-log CFU/ml reduction) was detected after 1 h of storage at both temperatures, and no growth occurred at any points of storage. Therefore, cinnamon by itself (regardless of pH) had a pronounced killing effect. A further enrichment step with brain heart infusion agar showed that L. monocytogenes was completely inactivated in apple juice stored at 20°C, except in pH 5.0 samples with 0.1% of cinnamon. The TAL method was as effective as TSA in recovering injured cells of L. monocytogenes . Cinnamon considerably inactivates L. monocytogenes in apple juice and thus enhances the safety of this product.

The thin agar layer (TAL) method of Kang and Fung was used to enumerate acid-injured foodborne pathogens. This method involves overlaying 14 ml of nonselective medium (tryptic soy agar [TSA]) onto a prepoured and solidified pathogen-specific, selective medium in a petri dish. After surface plating, injured cells resuscitated and grew on TSA during the first few hours of incubation; then, the selective agents from the selective medium diffused to the top layer, interacted with the recovered microorganisms, and started to produce typical reactions. Foodborne pathogens were exposed to 2% acetic acid for 1, 2, or 4 min, and the recovery rate with the TAL method was compared with the rate of TSA and pathogen-specific, selective media. No significant difference occurred between TSA and TAL ( P > 0.05) for enumeration of acid-injured Escherichia coli O157:H7, Salmonella Typhimurium, Staphylococcus aureus , and Yersinia enterocolitica , and both recovered significantly higher numbers than the selective medium for each respective pathogen ( P < 0.05). For recovery of acid-injured Listeria monocytogenes , no difference ( P > 0.05) occurred among TSA, TAL, and selective media. However, fewer cells were recovered in the selective media. The TAL method is a one-step, convenient procedure for recovery of acid-injured cells.

The efficiency of Hunt broth containing Oxyrase was compared with the gas replacement method for detection of Campylobacter jejuni in inoculated ground beef and chicken skin. Five strains of C. jejuni were inoculated individually into samples and cultured with various media under conditions generated by either flushing with a mixture of gases or supplementing with Oxyrase. Oxyrase media added with 7% lysed blood, 2.5% charcoal, or 6% ground cooked meat were compared with examinations from chicken skin samples. Campylobacter counts from enrichments were performed at 6, 12, 20, and 28 h of incubation. From inoculated ground beef, counts at 20 h increased by 4 to 7 log CFU/ml depending on strains and initial concentration of inocula. The efficiencies of Hunt medium using gassing and those with Oxyrase added were similar ( P > 0.05). Broth containing 0.15 U/ml of Oxyrase without blood effectively supported the growth of all strains ( P > 0.05). From inoculated chicken skin, 20-h incubation counts increased by 3.0 to 7.5 log CFU/ml for the gassing method and by 2.7 to 7.3 log CFU/ml for supplementation with 0.6 U/ml of Oxyrase and blood. The addition of 7% lysed sheep blood provided better Campylobacter growth than supplementing with 2.5% charcoal or 6% ground cooked meat. Enrichment media incorporating with Oxyrase is a simple, convenient, and time-saving method to replace flushing with mixed gas for isolation of Campylobacter jejuni .

Culture methods for detecting virulent Yersinia enterocolitica require selective enrichment and a series of confirmatory tests that are time-consuming, costly, and laborious. The objective of this study was to evaluate a fluorogenic 5′-nuclease assay for detecting the enterotoxin yst gene of virulent Y. enterocolitica in pure cultures, inoculated ground pork samples, and naturally contaminated food samples. These results were then compared with “gold standard” methods recommended by the U.S. Food and Drug Administration in the Bacteriological Analytical Manual for detecting pathogenic Y. enterocolitica . The 5′-nuclease assay was able to identify the organism in 100% of the repetitions when 10 2 CFU/ml or more organisms were present in pure cultures and 10 3 CFU/g or more organisms were present in ground pork. Similar recovery efficiency on cefsulodin-irgasan-novobiocin (CIN) agar plates was only evident when 10 5 CFU/ml or more organisms were present in pure culture and 10 6 CFU/g or more organisms were present in inoculated ground pork. The 5′-nuclease assay indicated a contamination rate of 35.5% (94/265) in various meats and tofu, whereas the CIN plating method indicated a contamination rate of 28.3% (75/265). This resulted in 100% sensitivity and 64.5% specificity for the 5′-nuclease assay when compared with the standard culture recovery method. Only 75% (60/80) of the Yersinia spp. isolated on CIN was identified as containing a virulence plasmid by autoagglutination and crystal violet binding tests. These results indicate that the true rate of contamination of virulent Y. enterocolitica in pork and other processed meats and foods is being underestimated using current detection methods. This study demonstrates the potential of the 5′-nuclease assay for rapidly and specifically detecting virulent Y. enterocolitica in processed foods with the added advantage of being an automated detection system with high-throughput capability.

Beef strip loins were sprayed with 1.5% (vol/vol) lactic acid before and/or after 14, 28, 56, 84, and 126 days of vacuum-packaged storage at −1.1 or 2°C to assess the effects on bacteria. Five different treatment combinations including a control (no spray treatment) and one treatment with prestorage acid spray followed by poststorage water spray (A/W) were evaluated. Compared to the controls, 97% of all acid-treated loins had lower microbial counts throughout the storage period. Prestorage acid spray ( A/0 ) was more effective than poststorage acid spray (0/A)with the greatest effects ( P < 0.05) being observed on day 28, where the bacterial population was reduced by 1.9 and 1.5 log CFU/cm 2 for loins stored at −1.1 and 2°C, respectively. Microbial counts for loins with A/A(acid spray both before and after vacuum storage) or A/W treatments were lower than but not significantly different from A/0 ( P > 0.05). Poststorage washing resulted in a minimal decontaminating effect, irrespective of the washing agent used. In addition, single spray treatment immediately after fabrication seems more effective and practical than a double spray. Temperature main effects showed that colder temperature (−1.1 versus 2°C) reduced ( P < 0.03) microbial growth throughout the storage period. Salmonella contamination was not detected in any samples; however, 28% of the control and 4% of the spray-treated loins were positive for Listeria spp. Appropriate time of acid application in combination with colder storage temperature improved the microbiological quality of meat for at least four weeks.

The effects of fumaric and lactic acid on the total numbers of aerobic, psychrotrophic, and coliform bacteria on vacuum-packaged ground beef patties at 0, 1, 3, 5, 7, 10, and 14 days of storage at 4°C were studied. Fumaric acid treatments resulted in greater reductions in microbial growth than lactic acid treatments. When 5.0% fumaric acid was used, the lag phase was prolonged and microbial growth was reduced ( P < 0.05), with total aerobic, psychotrotrophic, and coliform populations reaching net maximum growths of only 1.46, 1.44, and 0.98 log units, respectively, after storage at 4°C for 10 days. Increasing the acid concentrations significantly decreased the growth of all microorganisms. The 5.0% lactic acid was the most inhibitory against coliforms, resulting in a 3.88-1og unit reduction after 10 days of storage.

Lean beef surfaces were inoculated with Escherichia coli O157:H7 and Listeria monocytogenes and then sanitized with fumaric, acetic, or lactic acid alone and in combined solutions of those acids at 55°C for 5 s. The initial inoculum level was 8.62 log CFU/cm 2 and 5.13 log CFU/cm 2 for L. monocytogenes and E. coli O157:H7, respectively. Fumaric acid at a concentration of 1% was the most effective acid in reducing the populations of L. monocytogenes by up to 1 log unit and E. coli O157:H7 by up to 1.3 log units when compared with acetic or lactic acids. The rank order of acids tested against the growth of L. monocytogenes and E. coli O157:H7 was fumaric acid followed by lactic and acetic acids. Fumaric acid at concentrations of 1.0% and 1.5% was more effective than any of the combined solutions of acids.

Beef carcass sides (n = 48) were selected randomly on three different days in a commercial processing facility and microbiologically analyzed before being moved to the cooler. Four types of samples were obtained per side from the inside round area: no trim and no wash (NTNW); trim, but no wash (TNW); trim and wash (TW), and no trim but wash (NTW). A flame-sterilized knife, forceps, and scalpel were used for each trimming treatment and sampling. Significant differences ( P < 0.05) were observed in mean aerobic plate counts (APCs) between treatments. The greatest reduction in APC (log 10 colony forming units [CFU] per cm 2 ) was observed in TNW samples followed by TW and NTW, with the corresponding mean APC reductions relative to NTNW being 3.0, 0.9, and 0.3, respectively, indicating that trimming can be an effective control point in reducing bacterial contamination in the slaughter process. Although TNW samples, had the lowest counts, samples from the same location after wash (TW) had counts 2 log cycles higher than TNW samples. These results indicate that washing spreads contamination to adjacent carcass sites. However, washing of carcasses was effective in lowering microbial populations relative to the NTNW treatment. Escherichia coli and coliform counts in all samples were low (0.03 to 0.4 log 10 CFU/cm 2 ); however, the mean E. coli or coliform count in NTNW samples was higher ( P < 0.05) than those in the rest of the treatments.

Subcutaneous and kidney-pelvic-heart fat were trimmed from one side of each beef carcass ( n = 9) immediately after cold water washing. Both sides were sampled for aerobic plate counts (APCs) before being moved to the chill room (0 h) and after 72 h of cold storage. The mean APCs (log 10 colony-forming units (CFU)/cm 2 ) of trimmed (HFT) sides at 0 or 72 h were not different ( P > 0.05) from those of the nontrimmed (NFT) sides. All sides at 72 h had reduced microbial counts compared to 0 h. By 72 h, HFT sides had numerically lower counts than NFT sides, indicating that the microbial reduction effect of the chill temperature may have been greater on fat-trimmed carcasses than on nontrimmed carcasses. Subprimals from HFT and NFT sides that were trimmed to 0.64-cm fat thickness were microbiologically analyzed before (0 days) and after (14 days) vacuum storage. APCs of all subprimals were slightly reduced after 14 d; however, no difference ( P > 0.05) occurred in treatment effect. The mean APC was higher for HFT-side subprimals than for NFT-side subprimals at both 0 and 14 days. This difference probably was due to the fat trimming required for NFT-side subprimals at day 0 as compared to minimal or no trimming of HFT-side subprimals. Those HFT subprimals which were not subsequently trimmed may have picked up additional microorganisms from contact surfaces during fabrication. Based on our trimming protocol, although HFT did not show any negative impact on the microbial quality of carcasses, the higher APC of HFT-side subprimals indicated that extensive trimming may not be effective in improving the microbial quality of meat.

We evaluated the methods of direct plate count (DPC) on Modified Oxford agar and most probable number (MPN) using Fung-Yu tubes for the detection of natural contamination by Listeria spp. by examining 100 retail samples of raw meat (beef, lamb, pork; and turkey). Listeria monocytogenes was isolated from 10 samples. Other Listeria spp. ( L. innocua , L. murrayi , and L. welshimeri ) were isolated from 27 samples. Despite the fact that very low numbers of Listeria were found on these products, the MPN method was significantly more sensitive than the DPC method. The isolation efficiencies of the MPN and DPC methods were 31 and 17%, respectively. Quantitation of indigenous Listeria showed that many meat samples contaminated with ≤ 4 colony-forming units (CFU) per g tested negative by the DPC method but positive by the MPN method.

Beef carcass sides ( n = 9 per replicate) were sprayed with water (W), 200 ppm chlorine ©, or 3% (vol/vol) lactic acid (L) immediately after rail inspection and at the end of an 8-h spray-chill cycle, resulting in a total of nine different spray combinations. All treatment combinations involving chlorine and/or lactic acid reduced carcass contamination. The reductions in mean log 10 CFU/cm 2 for carcass aerobic plate count (APC) data ranged from 0.4 to 1.8. The treatment combination using lactic acid at both spray times (L+L) resulted in the greatest reduction. Additionally, treatment combinations involving lactic acid at either time and in combination with water or chlorine tended to reduce APCs more than those treatment combinations without acid. Browning of blood splashes was observed on carcasses sprayed with lactic acid and persisted until fabrication at 72 h postmortem. A companion study was designed, in conjunction with the carcass decontamination study, to evaluate effect of carcass treatment on the microbiological quality of subprimal subdivisions derived from treated carcasses. A facet of the subprimal study evaluated chlorine spray (200 ppm) and microwave radiation as approaches to improving subprimal shelf life and safety. Cuts taken from sprayed carcasses were vacuum packaged with or without intervention treatments, stored at 1 to 2°C and evaluated for both APC and pathogen populations at specified intervals of up to 120 days. These results demonstrated that neither carcass nor intervention treatment had any significant ( P > 0.05), beneficial effect on the microbiological quality of subprimal cuts.

Manual dilutions of food samples (e.g., 1:10 dilution) for microbiological investigations are cumbersome. We tested an automatic diluter (Model GD-600 Gravimetric Diluter, Spiral System, Inc., Bethesda, MD) for accuracy and sterility for microbiological work and found that the system provided ±0.1 to +0.2 g of accuracy in delivering a commanded amount of diluent. The delivered diluent was essentially sterile, with a few samples giving 1 to 4 colonies per ml after plating. With this system, weighing and dilution are fast, easy, and accurate.

The viable cell count performance of some heat-sensitive differential agars prepared and remelted by microwave energy was evaluated for Salmonella choleraesui , Streptococcus faecalis and Escherichia coli . The conventional boiling method was used for comparison. No significant difference was found between the microwave oven processed agar and the conventional-boiling processed agar in viable cell counts of the target bacteria. Heating and reheating of violet red bile agar, bismuth sulfite agar, and KF Streptococcus agar by both methods did not change agar performance. However, remelting of desoxycholate citrate agar by both methods resulted in a substantial lowering of viable cell counts.

Stock cultures (136) and fresh isolates (163) of Enterobacteriaceae from ground beef, processed chickens, frozen pot pies and commercial poultry feeds were identified to species with the AutoMicrobic System (AMS). All stock cultures and fresh isolates were also concurrently tested with two other identification systems (Micro-ID and API), previously evaluated and proven accurate for identification of Enterobacteriaceae . The AMS correctly identified to species 135/136 (99.3%) of the stock cultures and 160/163 (98.2%) of the fresh isolates. All Salmonella cultures tested (74) were correctly identified by AMS.