Adrienne E. H. Shearer

Bacterial spore inhibition and inactivation in foods by pressure, chemical preservatives, and mild heat.

Sucrose laurates, sucrose palmitate, sucrose stearates, and monolaurin (Lauricidin) were evaluated for inhibitory effects against spores of Bacillus sp., Clostridium sporogenes PA3679, and Alicyclobacillus sp. in a model agar system. The combined treatment of sucrose laurate, high hydrostatic pressure, and mild heat was evaluated on spores of Bacillus and Alicyclobacillus in foods. The minimum inhibitory concentrations of the sucrose esters were higher than that of Lauricidin for all spores tested in the model agar system, but Lauricidin was not the most readily suspended in the test media. The sucrose laurates and sucrose palmitate were more effective and more readily suspended than the sucrose stearates. A combined treatment of sucrose laurate (<1.0%), 392 megaPascals (MPa) at 45 degrees C for 10 to 15 min provided 3- to 5.5-log10 CFU/ml reductions from initial populations of 10(6) CFU/ml for Bacillus subtilis 168 in milk, Bacillus cereus 14579 in beef, Bacillus coagulans 7050 in tomato juice (pH 4.5), Alicyclobacillus sp. N1089 in tomato juice (pH 4.5), and Alicyclobacillus sp. N1098 in apple juice. The most notable change in the appearance of the products was temporary foaming during mixing of the sucrose laurate in the foods. The effect of sucrose laurate appeared to be inhibitory rather than lethal to the spores. The inhibitory effects observed on Bacillus and Alicyclobacillus spores by the combined treatment of pressure, mild heat, and sucrose laurate appear promising for food applications where alternatives to high heat processing are desired.

Evaluation of a polymerase chain reaction-based system for detection of Salmonella enteritidis, Escherichia coli O157:H7, Listeria spp., and Listeria monocytogenes on fresh fruits and vegetables.

A polymerase chain reaction (PCR)-based detection system, BAX, was evaluated for its sensitivity in detecting Salmonella Enteritidis, Escherichia coli O157:H7, Listeria sp., and Listeria monocytogenes on fresh produce. Fifteen different types of produce (alfalfa sprouts, green peppers, parsley, white cabbage, radishes, onions, carrots, mushrooms, leaf lettuce, tomatoes, strawberries, cantaloupe, mango, apples, and oranges) were inoculated, in separate studies, with Salmonella Enteritidis, E. coli O157:H7, and L. monocytogenes down to the predicted level of 1 CFU per 25-g sample. Detection by BAX was compared to recovery of the inoculated bacteria by culture methods according to the Food and Drug Administrations (FDA) Bacteriological Analytical Manual (BAM). BAX was essentially as sensitive as the culture-based method in detecting Salmonella Enteritidis and L. monocytogenes and more sensitive than the culture-based method for the detection of E. coli O157:H7 on green pepper, carrot, radish, and sprout samples. Detection of the pathogenic bacteria in samples spiked with a predicted number of less than 10 CFU was possible for most produce samples, but both methods failed to detect L. monocytogenes on carrot samples and one of two mushroom and onion samples spiked with less than 100 CFU. Both BAX and the culture method were also unable to consistently recover low numbers of E. coli O157:H7 from alfalfa sprouts. The PCR method allowed detection of Salmonella Enteritidis, E. coli O157:H7, and L. monocytogenes at least 2 days earlier than the conventional culture methods.

Conditions for high pressure inactivation of Vibrio parahaemolyticus in oysters

The objective of this study was to identify the high pressure processing conditions (pressure level, time, and temperature) needed to achieve a 5-log reduction of Vibrio parahaemolyticus in live oysters (Crassostrea virginica). Ten strains of V. parahaemolyticus were separately tested for their resistances to high pressure. The two most pressure-resistant strains were then used as a cocktail to represent baro-tolerant environmental strains. To evaluate the effect of temperature on pressure inactivation of V. parahaemolyticus, Vibrio-free oyster meats were inoculated with the cocktail of V. parahaemolyticus and incubated at room temperature (approximately 21 degrees C) for 24 h. Oyster meats were then blended and treated at 250 MPa for 5 min, 300 MPa for 2 min, and 350 MPa for 1 min. Pressure treatments were carried out at -2, 1, 5, 10, 20, 30, 40, and 45 degrees C. Temperatures > or = 30 degrees C enhanced pressure inactivation of V. parahaemolyticus. To achieve a 5-log reduction of V. parahaemolyticus in live oysters, pressure treatment needed to be > or = 350 MPa for 2 min at temperatures between 1 and 35 degrees C and > or = 300 MPa for 2 min at 40 degrees C.

Heat resistance of juice spoilage microorganisms

The heat resistance of various yeasts (Saccharomyces cerevisiae, Rhodotorula mucilaginosa, Torulaspora delbrueckii, and Zygosaccharomyces rouxii), molds (Penicillium citrinum, Penicillium roquefortii, and Aspergillus niger), and lactic acid bacteria (Lactobacillus fermentum and Lactobacillus plantarum) obtained from spoiled acid or acidified food products was determined in 0.1 M citrate buffer at pH values of 3.0, 3.5, and 4.0. S. cerevisiae was the most heat resistant of the microorganisms in citrate buffer, and its heat resistance was further evaluated in apple, grapefruit, calcium-fortified apple, and tomato juices as well as in a juice base with high fructose corn syrup. Decimal reduction times (D-values) and changes in temperature required to change the D-value (z-values) for S. cerevisiae were higher in the juices than in citrate buffer at all pH values tested. The D57 degrees C(135 degrees F)-values varied from 9.4 min in the juice product with pH 2.8 to 32 min in a calcium-added apple juice with pH 3.9. The S. cerevisiae strain used in this study can be used in thermal-death-time experiments in acidic products to calculate process conditions and in challenge tests to validate the calculated temperatures and hold times during processing.

Survey of retail alfalfa sprouts and mushrooms for the presence of Escherichia coli O157:H7, Salmonella, and Listeria with BAX, and evaluation of this polymerase chain reaction-based system with experimentally contaminated samples

BAX, a polymerase chain reaction (PCR)-based pathogen detection system, was used to survey retail sprouts and mushrooms for contamination with Escherichia coli O157:H7, Salmonella, Listeria spp., and Listeria monocytogenes. No Salmonella or E. coli O157:H7 was detected in the 202 mushroom and 206 alfalfa sprout samples screened. L. monocytogenes was detected in one sprout sample, and seven additional sprout samples tested positive for the genus Listeria. BAX also detected Listeria species in 17 of the mushroom samples. Only 6 of 850 PCR assays (0.7%) failed to amplify control DNA, and therefore reagent failures and the inhibition of PCR by plant compounds were rare. The sensitivity of the detection system was evaluated by assaying samples inoculated with 10 CFU of each of the pathogens. One hundred seventy-two alfalfa sprout samples were inoculated with E. coli O157:H7, and two sets of 130 samples were experimentally contaminated with Salmonella Enteritidis and L. monocytogenes. The frequency of detection depended on the protocols used for inoculation and culturing. Inoculation of samples with approximately 10 CFU from frozen stocks yielded detection rates of 87.5 and 94.5% for L. monocylogenes and Salmonella Enteritidis, respectively, in mushrooms. The corresponding rates for alfalfa sprouts were 94.5 and 76.3%. The E. coli O157:H7 detection rate was 100% for mushrooms but only 48.6% for sprouts when standard BAX culture protocols were used. The substitution of an overnight incubation in modified E. coli medium for the 3-h brain heart infusion incubation increased the rate of E. coli O157:H7 detection to 75% for experimentally contaminated sprouts. The detection rate was 100% when E. coli O157:H7 cells from a fresh overnight culture were used for the inoculation. Test sensitivity is therefore influenced by the type of produce involved and is probably related to the growth of pathogens in the resuscitation and enrichment media.

High Hydrostatic Pressure and UV Light Treatment of Produce Contaminated with Eimeria acervulina as a Cyclospora cayetanensis Surrogate

The prevalence, size, genome, and life cycle of Eimeria acervulina make this organism a good surrogate for Cyclospora cayetanensis, a protozoan that causes gastroenteritis in humans, including recent outbreaks in the United States and Canada associated with contaminated raspberries and basil. Laboratory studies of C. cayetanensis are difficult because of the lack of readily available oocysts and of infection models and assays. UV radiation and high-hydrostatic-pressure processing (HPP) are both safe technologies with potential for use on fresh produce. Raspberries and basil were inoculated with sporulated E. acervulina oocysts at high (10(6) oocysts) and low (10(4) oocysts) levels, and inoculated and control produce were treated with UV (up to 261 mW/cm2) or HPP (550 MPa at 40 degrees C for 2 min). Oocysts recovered from produce were fed to 3-week-old broiler chickens, which were scored for weight gain, oocyst shedding, and lesions at 6 days postinoculation. Oocysts exhibited enhanced excystation on raspberries but not on basil. Birds fed oocysts from UV-treated raspberries had reduced infection rates, which varied with oocyst inoculum level and UV intensity. Birds fed oocysts from UV-treated raspberries (10(4) oocysts) were asymptomatic but shed oocysts, and birds fed oocysts from UV-treated basil (10(4) oocysts) were asymptomatic and did not shed oocysts. Birds fed oocysts from HPP-treated raspberries and basil were asymptomatic and did not shed oocysts. These results suggest that UV radiation and HPP may be used to reduce the risk for cyclosporiasis infection associated with produce. Both treatments yielded healthy animals; however, HPP was more effective, as indicated by results for produce with higher contamination levels.

Effect of growth and recovery temperatures on pressure resistance of Listeria monocytogenes

Experimental conditions can affect the outcome of bacterial stress-tolerance assays. Growth conditions that optimize microbial recovery should be established to help evaluate the effectiveness of treatment conditions for food safety. The objectives of this study were to determine the effects of growth and recovery temperatures on pressure resistance of early stationary-phase Listeria monocytogenes in milk. The tested conditions were the following: (1) L. monocytogenes was grown at various temperatures (10, 15, 20, 25, 30, 35, 40 and 43 degrees C), suspended in ultra-high temperature (UHT) -processed whole milk, pressure-treated at 400 MPa for 2 min at 21 degrees C and recovered on Tryptic Soy Agar supplemented with 0.6% yeast extract (TSAYE) at 35 degrees C; (2) L. monocytogenes was grown at 35 and 43 degrees C, pressure treated in milk (400 and 500 MPa, respectively, for 2 min at 21 degrees C) and recovered on TSAYE at various temperatures (4, 10, 15, 20, 25, 30, 35 and 40 degrees C); (3) L. monocytogenes originally grown at 35 degrees C, was pressure treated in milk (400 or 450 MPa for 2 min at 21 degrees C), and recovered on TSAYE at 10 degrees C for various time intervals (1, 2, 3, 6, 9 and 12 days) then at 35 degrees C for 5 days. There was no significant difference (P>0.05) in pressure-resistance of L. monocytogenes grown at 10 to 25 degrees C with approximately 6.5-log CFU/ml population reductions. At growth temperatures greater than 25 degrees C, pressure resistance increased with less than 1-log CFU/ml reduction observed for L. monocytogenes originally grown at 43 degrees C. After pressure treatment, regardless of growth temperature and pressure treatment, the greatest recovery of L. monocytogenes was within the 4 to 20 degrees C range; maximum recovery at 10 degrees C required approximately 24 days. The time for comparable post-pressure treatment recovery could be reduced by incubation at 10 degrees C for at least 2 days followed by incubation at 35 degrees C for 5 days. The findings of the present study indicate that growth and recovery temperatures affect the pressure resistance of L. monocytogenes and should, therefore, be taken into account when assessing the adequacy of inactivation treatments.

High hydrostatic pressure for development of vaccines.

Disease management in the food industry is complex and includes use of good hygienic practices, antimicrobials, and immunization. Vaccines are available against many, but not all, disease agents affecting animals reared for human food. Fewer vaccines are currently licensed and widely available for human foodborne pathogens. Increased resistance to antimicrobials provides additional impetus to develop new vaccines. In addition to the need for new vaccines, new methods of vaccine production are desired. Some current methods of vaccine production can involve use of hazardous chemicals, provide inconsistent results, or present risk to vaccine recipients with certain allergies. The efficacy of high hydrostatic pressure (HHP) for inactivation of a variety of foodborne pathogenic microorganisms has been well established, and some of these microorganisms have been demonstrated to retain immunogenic properties, suggesting HHP may have application for the development of vaccines. Studies on the effect of HHP on infectivity and immunogenicity of various viruses, a protozoan parasite, and one bacterial species are presented. Control of several of these pathogens is important for animal health and economic stability in several sectors of the food industry. The research to date on the potential for vaccine development by HHP is presented.

Influence of Growth Conditions on Pressure Resistance of Vibrio parahaemolyticus in Oysters and the Optimization of Postpressure Treatment Recovery Conditions

Vibrio parahaemolyticus ATCC 43996 was grown at 15°C for 53 h, 20°C for 24 h, 25°C for 12 h, 30°C for 9 h, 35°C for 9 h, or 40°C for 6 h to early stationary phase. Oyster meats were blended, autoclaved at 121°C for 15 min, inoculated with V. parahaemolyticus, and pressure treated at 250 MPa for 2 and 3 min and at 300 MPa for 1 and 2 min at 21°C. Overall, growth temperatures of 20 and 40°C yielded the greatest pressure resistance in V. parahaemolyticus. The effects of salt concentration and H(2)O(2)-degrading compounds on the recovery of V. parahaemolyticus also were investigated. Sterile oyster meats were inoculated with V. parahaemolyticus and treated at 250 MPa for 1, 2, or 3 min at 21°C. These meats were then blended with 0.1% peptone water supplemented with 0.5 to 1.5% NaCl and plated on tryptic soy agar (TSA) supplemented with 0 to 3.5% NaCl. For recovery of pressure-injured cells, peptone water with 1% NaCl and TSA with 0.5% NaCl were the best diluent and plating medium, respectively. Addition of sodium pyruvate (0.05 to 0.2%) or catalase (8 to 32 U/ml) did not increase the recovery of V. parahaemolyticus after pressure treatment. The effect of incubation temperature and gas atmosphere on the recovery of V. parahaemolyticus after pressure treatment also was determined. Aerobic incubation at 30°C resulted in the highest recovery of V. parahaemolyticus in sterile oyster meats. The 30°C incubation temperature was also the optimum temperature for recovery of V. parahaemolyticus in pressure-treated live oysters. The results of this study indicate that the growth conditions for V. parahaemolyticus before and after high hydrostatic pressure treatment should be taken into consideration when assessing the efficacy of pressure inactivation.

Effect of bacterial cell-free supernatants on infectivity of norovirus surrogates.

Bacterial metabolic products were evaluated for inhibitory effects on viral propagation in cell culture. Cell-free supernatants (CFS) were prepared from growth of Enterococcus faecalis ATCC 19433, Pseudomonas fluorescens ATCC 13525, Escherichia coli 08, Staphylococcus epidermidis ATCC 12228, Bacillus subtilis 168, Bacillus coagulans 185A, B. coagulans 7050, Clostridium sporogenes PA3679, and a commercial probiotic mixture of Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium bifidum, Lactobacillus salivarius, and Streptococcus thermophilus in microbiological medium or milk. The inhibitory effects of CFS on the propagation of murine norovirus 1 and Tulane virus in RAW 264.7 and LLCMK2 cells, respectively, were evaluated in the continuous presence of CFS or after exposure of host cells to CFS. Slight inhibition of viral propagation was observed for murine norovirus and Tulane virus in the continuous presence of CFS of B. subtilis 168 and E. faecalis 19433, respectively. CFS cytotoxicity was also determined by microscopic examination. Virus persisted in the CFS that demonstrated cytotoxic effects, suggesting a lack of direct effect of CFS on virions. The viral propagation indicates a general lack of competitive inhibition by bacterial extracellular products and bears significance in understanding the persistence of virus in food and human systems shared by bacteria that are recognized for their colonization and competitive capabilities.