IMPROVING THE MICROBIAL SAFETY AND QUALITY IN FRESH PRODUCE AND PROCESSED PRODUCTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0216951
• Project No.: FLA-LAL-004830
• Project Start Date: Dec 1, 2008
• Project End Date: Nov 30, 2013

Project Director
Danyluk, MI, D..

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
Citrus Research and Education Center, Lake Alfred

Non Technical Summary
The US has seen a dramatic increase in the number of foodborne illnesses attributed to contaminated produce from 0.7% of all foodborne outbreaks in the 1970?s to 6% in the 1990?s (Sivapalasingam, 2004). The top five produce items associated with 75% of produce outbreaks are lettuce and leafy greens, tomatoes, melons, green onions and leafy herbs such as basil, cilantro and parsley (Ackers et al., 2998; Campbell et al., 2001; Cummings et al., 2001; Mohle-Boetani et al., 1999). With the exception of tomatoes (CDC, 2005; Toth et al., 2002), most of these outbreaks have not originated from Florida-grown products; however, each commodity is produced commercially in Florida.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	0999	1100	30%
712	1199	1100	20%
712	1499	1100	50%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
1499 - Vegetables, general/other; 0999 - Citrus, general/other; 1199 - Deciduous and small fruits, general/other;

Field Of Science
1100 - Bacteriology;

Keywords

microbial food safety

microbial food quality

produce

processed food

Goals / Objectives
The overall goal of this project is to provide data related to the microbial safety and spoilage risks associated with fresh produce production, packing and handling practices, fresh cut, processing, retail and consumer practices. This knowledge will enable the fruit, vegetable and nut industries to provide scientific backing to risks of contamination in their production practices and facilities and documentation for methods used to reduce these risks. Additionally, results from this work will allow the the food service industry and consumers to determine appropriate responses to reduce risks from consuming these products. Specific objectives include: i. To conduct surveillance, epidemiological and transfer studies in order to determine the points and sources during production and processing of specific fruits, vegetables and nuts where foodborne pathogens may be introduced and the effect that varying production, processing, and environmental factors may have on the contamination event ii. To characterize microbial survival, growth and contamination mechanisms of foodborne pathogens on specific commodities of importance to Florida, the environment in which they are grown/processed, including microbial interactions within populations iii. To develop and test mitigation and management strategies to control foodborne pathogen on these products and there surfaces they may come into contact with during production, packing or processing iv. To evaluate the microbial causes and consequences of spoilage in processed fruit and vegetable products, and potential mitigation strategies to alleviate these causes

Project Methods
Bacterial recovery from the environment: Physical parameters at the sampling site including temperature (air and water), turbidity, pH and other appropriate will be recorded at each site. Isolates will be confirmed by growth on selective agar and by species-specific PCR. If appropriate, isolates will be further typed by conventional methods including phage typing, serotyping or sequencing. Bacterial cultures: Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes strains that have been isolated from fresh produce, production environments, juice or other outbreaks associated sources are available in the PI laboratory and will be used for all experiments. If necessary, pathogens will be requested from outside sources Pathogen recovery from fresh produce: Fresh-cut produce will be placed in a Whirl-Pak filter bag with Dey-Engley (DE) neutralizing broth and stomached at the high-speed setting. When appropriate, a rinse method will be used. In this case the produce will be placed in a stomacher bag containing DE broth and the surface of the fruit will be rubbed by hand from outside of the bag for 60 s. Dilutions will be plated on non-selective and selective agar. Plates will be incubated at the appropriate time and temperature prior to enumeration. Microscopic evaluation of dye and bacteria penetration: GFP labeled bacteria will be grown in broth medium and prepared for inoculation as described above. An aliquot of inoculum preparation used to inoculate produce that will then be left to dry as appropriate. The depth of penetration will be observed and measured using fluorescent microscopy or confocal scanning electron microscopy. Microbiological modeling and risk assessment. Growth curves data can be fit to the model of Baranyi et al (1993) using the software DMFIT to obtain growth rates and lag times. Models to predict growth rate and lag time as a function of temperature will be developing using the square root model popularized by Ratkowsky et al (1991). Models will be fit to data using Microsoft Excel. Data for Quantitative Microbial Risk Assessment (QMRA) will be collected into Excel spread sheets to perform a QMRA. It will be translated into simple probability distribution functions, and combined using reasonable assumptions in Monte Carlo simulation software (@RISK, Palisades Decision, Newfield NY, or Analytica, Lumina, Los Gatos, CA). Following completion of the model, a sensitivity analysis can be performed to identify what factors exert the greatest influence on the model. Due to the important influence these factors have on the outcomes of the model, the uncertainty in them may need to be further refined.

Progress 10/01/13 to 11/30/13

Outputs
Target Audience: Produce Researchers and Growers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Previously Reported

Publications

Progress 12/01/08 to 11/30/13

Outputs
Target Audience: Produce Growers and researchers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? All accomplishments listed in past progress reports

Publications

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Produce researchers and growers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Populations of E. coli O157:H7 and Salmonella decreased under all experimental conditions. At 2°C, E. coli O157:H7 and Salmonella populations decreases ranged from 1.2-1.6 and 1.4-1.8 log CFU/berry, respectively, over 7 days. When stored at 15.5°C, declines of E. coli O157:H7 (1.4 and 1.3 log CFU/berry) and Salmonella (1.2 and 1.1 log CFU/berry) populations were seen on light blue bruised and unblemished blueberries. Populations of E. coli O157:H7 and Salmonella decreased significantly less (P ≤ 0.05) on dark bruised blueberries (0.7 and 0.9 log CFU/berry) when compared to dark unblemished blueberries (1.6 and 1.7 log CFU/berry). Bruising did not significantly affect the fate of E. coli O157:H7 or Salmonella populations on light blueberries or on dark blueberries stored at 2°C. However; the slower population decline of E. coli O157:H7 and Salmonella on bruised dark blueberries than on unblemished berries at 15.5°C may increase risks. No significant difference existed between the individual serovar or cocktail populations at any time point. At time zero, no significant differences (P>0.05) between Salmonella populations, 7.9±0.1 log CFU/100ml, existed between water types or temperatures. Populations in DI, SS, and EPAWC were either not significantly different or significantly different at only one time point. By day three, and thereafter, in NSS populations were significantly lower than those in other waters. At six months, populations in NSS significantly decreased to 2.7±0.8, 3.8±0.7, and 0.9 log MPN/100ml, at 15°C, 21°C, and 28°C respectively. In DI, SS and EPAWC, populations at 15°C and 21°C were significantly lower than waters at 28°C. Salmonella may persist in various types of water for durations exceeding six months after initial contamination. Survival is higher in water without background populations.Populations of E. coli O157:H7 and Salmonella decreased under all experimental conditions. At 2°C, E. coli O157:H7 populations decreased by 1 and 0.7 log CFU/berry over 24 h on bruised and intact strawberries, respectively. Salmonella populations decreased by 1.3 and 1.5 log CFU/berry over 24 h on bruised and intact strawberries, respectively at 2°C. At 15.5°C, E. coli O157:H7 and Salmonella showed similar trends over 7 days, where both populations decreased by >2.3 and>2 log CFU/berry on bruised strawberries and >1.9 and >1.6 log CFU/berry on intact strawberries, respectively. Bruising did not significantly affect the fate of E. coli O157:H7 or Salmonella populations on mature strawberries. The current practice of harvesting strawberries at full ripe maturity, whether bruising occurs or not, does not impose any additional food safety risks. At 23ºC, Salmonella, E. coli O157:H7 and L. monocytogenes declined linearly at rates of -0.20, -0.33, -0.54 log CFU/g/month on peanuts and -0.16, -0.33, -1.05 log CFU/g/month on pecans, respectively. At 4°C, population of Salmonella declined on peanuts at -0.039 log CFU/g/month and remained relatively stable on pecans. E. coli O157:H7 and L. monocytogenes populations remained stable at 4ºC storage on both nut types. At -20ºC, populations of the three pathogens remained stable on both nuts with the exception of L. monocytogenes which declined at -0.06 log CFU/g/month on peanuts. Salmonella Tennessee predominated on peanuts and pecans -20, 4 and 23°C, except on peanuts stored at 23°C storage where Salmonella Enteritidis 9C was predominantly isolated. Pathogens, if present, can survive for extended periods of time at a broad range of storage temperatures on raw pecans and peanuts. Storage under refrigerated or freezing temperatures does not result in pathogen reduction.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Brar, P.K., and M.D. Danyluk. 2013. Salmonella transfer potential during hand harvesting of tomatoes under laboratory conditions. Journal of Food Protection. 76:1342-1349
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Danyluk, M.D., L.M. Friedrich, P. Sood, and E. Etxeberria. 2013. Growth or penetration of Salmonella into Citrus fruit is not facilitated by natural-light labels. Food Control. 34:398-403.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: McEgan, R., G.K. Mootian, L.D. Goodridge, D.W. Schaffner, and M.D. Danyluk. 2013. Predicting Salmonella populations from biological, chemical and physical indicators in Florida surface waters. Applied and Environmental Microbiology. 79: 4094-4105.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Vandamm, J.P., D. Li, L.J. Harris, D.W. Schaffner and M.D. Danyluk. 2013. Fate of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella on fresh-cut celery. Food Microbiology. 34:151-157.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: McEgan, R., C.A.P. Rodrigues, A. Sbodio, T.V. Suslow, L.D. Goodridge, and M.D. Danyluk. 2013. Detection of Salmonella spp. from large volumes of water by Modified Moore Swabs and Tangential Flow Filtration. Letters in Applied Microbiology. 56:88-94

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: Packing cartons may be reused during tomato harvest and packing. Presently, there is inadequate data to quantify cross-contamination risks associated with this practice. The objectives of this study were to determine Salmonella transfer coefficients (TCs) between (i) new, used, and dirty inoculated cartons and tomatoes and (ii) inoculated tomatoes and new, used, and dirty cartons, under varying inoculation conditions, contact times, and temperatures. Cartons were either touched immediately to mature green tomatoes (wet) or allowed to dry for 1 or 24 h before contact. Tomato/cartons were subjected to three different contact times (touch (0), 1, and 7 days), at 25C or 12C. The transfer direction was then reversed by inoculating the tomato and contacting the carton. Strawberries are harvested at or near full ripe maturity for superior eating quality. These fruit tend to be less firm and are more susceptible to bruising during harvest and transport, which may increase risks of foodborne pathogen proliferation. The objective of this research was to quantify the fate of Escherichia coli O157:H7 and Salmonella on bruised and intact strawberries at shipping (2C) and retail display (15.5C) temperatures. Strawberries were bruised by dropping a 32.6g steel ball into a 23 cm PVC pipe directly onto a whole strawberry. Strawberries, stored at 2C and 15.5C, were sampled at 0, 2, 5, and 24 h and days 0, 1, 3, and 7, respectively. Pathogen populations were enumerated on selective and non-selective media supplemented with rifampicin. Thermal inactivation parameters of stationary phase, non-adapted and acid adapted pathogens, primarily as cocktails of multiple strains, have been studied in various juice products. All D-values for STEC, Salmonella, and L. monocytogenes in orange juice were obtained using strain cocktails. The objective of this study was to evaluate the heat resistance of individual strains of stationary phase non-adapted and acid adapted Shiga toxin-producing Escherichia coli (STEC), Salmonella spp., and L. monocytogenes in orange juice.Three STEC, L. monocytogenes, and Salmonella strains/serotypes were evaluated. STEC and Salmonella isolates were grown in TSB, and L. monocytogenes strains grown in BHI, supplemented with 1% glucose for acid adaption, and inoculated into single-strength pasteurized orange juice without pulp. Inoculated juice was sealed into microcapillary tubes. Microtubes were immersed into water baths at 56, 58, and 60C for STEC and L. monocytogenes strains and at 55, 58, and 60C for Salmonella serotypes, removed at predetermined time intervals, and placed immediately onto ice. Thermally treated and sterilized tubes were crushed in 0.1% peptone using a sterile glass rod for microbiological analysis. Populations of STEC and Salmonella were enumerated on TSA supplemented with 0.1% sodium pyruvate; BHI agar supplemented with 0.1% sodium pyruvate was used for L. monocytogenes strains. PARTICIPANTS: Several UF and national collaborators and students were involved. TARGET AUDIENCES: Produce researchers and growers PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Salmonella populations decreased following inoculation onto new, used, and dirty cartons by 2-3 log units during 24 h drying regardless of storage temperature. In general, the highest transfer rates occurred with wet inoculum, regardless of carton type or temperature storage. The highest TCs was a wet, inoculated tomato stored 7 days at 25C, TC=14.7. Increasing contact time decreased TCs for new cartons, but increased TCs for used and dirty cartons. A greater percentage of Salmonella transferred from tomatoes to cartons than from cartons to tomatoes, regardless of carton type or storage temperature. For example, TC=0.27 from a wet, new carton, immediately touched to a tomato, ; while TC=1.38 from the tomato to the carton under the same conditions. Salmonella transfer between tomatoes and tomato cartons varies between new, used and dirty tomato cartons, indicating cross-contamination risks may increase under some conditions when cartons are dirty or reused. Populations of E. coli O157:H7 and Salmonella decreased under all experimental conditions. At 2C, E. coli O157:H7 populations decreased by 1 and 0.7 log CFU/berry over 24 h on bruised and intact strawberries, respectively. Salmonella populations decreased by 1.3 and 1.5 log CFU/berry over 24 h on bruised and intact strawberries, respectively at 2C. At 15.5C, E. coli O157:H7 and Salmonella showed similar trends over 7 days, where both populations decreased by >2.3 and>2 log CFU/berry on bruised strawberries and >1.9 and >1.6 log CFU/berry on intact strawberries, respectively. Bruising did not significantly affect the fate of E. coli O157:H7 or Salmonella populations on mature strawberries. The current practice of harvesting strawberries at full ripe maturity, whether bruising occurs or not, does not impose any additional food safety risks. Different strains in the same species respond to heat differently. Thermal tolerance was increased significantly (P < 0.05) for acid adapted STEC strains, however, acid adaptation did not improve heat resistance for Salmonlella spp., and L. monocytogenes strains at most temperatures tested. Salmonella serotypes are less heat resistant, at all temperatures tested, than L. monocytogenes and STEC. STEC, especially strain O111, are the most heat resistant at 56 and 58C; L. monocytogenes strains are the most thermal tolerance at 60C. Combining individual results of all pathogens tested, the formula of log D = 8.2 - 0.14T (C) was used to calculate a general process for orange juice at 71.1C. Using this equation, a 5-log reduction of all three pathogens in single strength orange juice requires 5.29 s at 71.1C, with a z-value of 7.1C.

Publications

• Tomas-Callejas, A., G. Lopez-Velasco, A.M. Valadez, A. Sbodio, F. Artex-Hernandez, M.D. Danyluk, and T.V. Suslow. 2012. Establishment of critical operating standards for chlorine dioxide in disinfection of dump tank and flume for fresh tomatoes. J. Food Prot. 75:304-313.
• Harris, L.J., J. Bender, E. Bihn, T. Blessington, M.D. Danyluk, P, Delaquis, L.D. Goodridge, A. Ibekwe, S. Ilic, K. Kniel, J. Lejeune, D.W. Schaffner, D. Stoeckel, and T.V. Suslow. 2012. Framework for developing research protocols: Evaluating microbial hazards and controls during production that pertain to the quality of agricultural water contacting fresh produce that may be consumed raw. Journal of Food Protection. 75:2251-2273.
• Cevallos-Cevallos, J.M., G. Gu, M.D. Danyluk, and A.H.C. van Bruggen. 2012. Adhesion and splash dispersal of Salmonella enterica Typhimurium on tomato leaflets: effect of rdar morphotype and trichome density. International Journal of Food Microbiology. 160:58-64.
• Cevallos-Cevallos, J.M., G. Gu, M.D. Danyluk, N.S. Dufault, A.H.C. van Bruggen. 2012. Salmonella can reach tomato fruits on plants exposed to aerosols formed by rain. International Journal of Food Microbiology. 158:140-146.
• Cevallos-Cevallos, J.M., E.D. Akins, L,M. Friedrich, M.D. Danyluk, and A.H. Simonne. 2012. Growth of Clostridium perfringens in refried beans. Journal of Food Protection. 75:1783-1790.
• Kimber, M., H. Kaur, L. Wang, M.D. Danyluk, and L.J. Harris. 2012. Survival of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes on inoculated almonds and pistachios stored at -19, 4, and 24C. Journal of Food Protection. 75:1394-1403.
• Cevallos-Cevallos, J.M., M.D. Danyluk, G. Gu, G.E. Vallad, A.H.C. van Bruggen. 2012. Dispersal of Salmonella by rain splash onto tomato plants. Journal of Food Protection. 75:472-479.
• Teplitski, M., J.T. Noel, A. Alagely, and M.D. Danyluk. 2012. Functional genomics studies shed light on the nutrition and gene expression of non-typhoidal Salmonella and enterovirulent E. coli in produce. Food Res Int. 45:576-586.
• Lambertini, E., M.D. Danyluk, D.W. Schaffner, C.K. Winter, and L.J. Harris. 2012. Risk of salmonellosis from consumption of almonds in the North American Market. Food Res Int. 45:1166-1174.

Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Tomatoes have associated with many multistate outbreaks of Salmonella and harvesting is suspected as a potential source of contamination. Workers prefer to wear gloves during tomato harvesting and may reuse them multiple times. Experiments were performed using mature green, round tomatoes with two types of gloves (reusable and single-use) and two hygienic conditions of reusable glove (clean and dirty). Transfer scenarios used during experiments were glove (clean and dirty) to tomato, tomato to glove (clean only) and glove (clean and dirty) to many tomatoes. Postharvest water quality management represents one of the few unit operations that approach a true critical control point for fresh tomatoes. There is a lack of performance data for sanitizers currently in use that reflect conditions of commercial systems. The objectives of this study were to develop on-site assessments of ClO2 treatment and microbiological water quality in dump tank and flume systems in FL and CA, and measure bacterial loads and pulp temperature of incoming and final washed round mature-green tomatoes within commercial tomato packing operations. Fresh-cut watermelon is considered ready to eat and does not require additional preparation by the consumer. However typical consumer handling practices may have an effect on pathogen growth and survival. The objectives of this study were to quantify the impacts of variations in storage times, temperature and types of storage containers and inoculation site on cut watermelon and on the fate of Salmonella and E. coli O157:H7, and Salmonella and L. monocytogenes, respectively. PARTICIPANTS: Several UF and national collaborators and students were involved. TARGET AUDIENCES: Produce researchers and industry members. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
No significant differences in TCs were obtained between clean reusable and single-use as well as clean and dirty reusable gloves at three drying times (0 h, 1 h and 24 h), on touching single tomato with an inoculated glove surface. Similarly, no significant differences were observed upon touching gloves with inoculated tomatoes at 0 h and 1 h drying time. Drying the inoculum on tomato surface for 24 h results in significantly more number of Salmonella positive reusable glove samples. Differences between 0 h and 1 h TCs were significant only when tomatoes were touched with inoculated gloves. When 25 tomatoes were touched with clean reusable and single-use gloves, TCs reduced significantly from third tomato and seventh tomato, respectively, while in case of dirty reusable gloves, no significant reductions were observed for all the 10 tomatoes. This study provides valuable insight into Salmonella transfer between gloves and mature, green tomatoes. Results varied among trials, due to environmental and incoming fruit conditions. Reduction in the mesophilic or coliform populations on fruit were not significant in FL; in all but one trial in CA, populations were significantly (P<0.05) higher after washing. In the dump tank, E. coli was near the limit of detection in both Florida and California and below the limit of detection in flume tank samples. ORP levels were significantly higher in flume than in dump tanks. A significant and positive correlation was established between ORP and temperature while a significant and negative correlation was established between ORP and turbidity and the population of total mesophiles and coliforms. This study provides in-plant data to develop data-based critical operating standards for ClO2 for the fresh-tomato industry. E. coli O157:H7 and Salmonella grew rapidly on all wet cut watermelon surfaces at 23C with most growth rates falling in the range of 0.20-0.30 log CFU/h regardless of packaging. Neither pathogen grew at 4C regardless of inoculation site or storage conditions. When inoculated on the rind and stored in open containers, bacterial population declined, but when stored in covered containers both pathogens grew at 0.45 and 0.29 log CFU/h for Salmonella and E. coli O157:H7 respectively and reach a population level of 106 CFU/g on the rind. Salmonella and L. monocytogenes populations decreased over 7 days at 4C, under all conditions, by ca. 1 log CFU/g and 1-2 log CFU/g, respectively. Salmonella and L .monocytogenes populations remained stable at ca. 3.0 to 3.5 log CFU/g for 7 days at 12C under all conditions. All Salmonella populations increased by ca. 1.5-2 log CFU/g over 48 h at 22C, under all conditions, with the greatest growth (2.1 log CFU/g) observed on the cut surface of samples stored in polyethylene containers. L. monocytogenes populations remained stable ca. 3 log CFU/g for 48 h at 22C, under all conditions. When held under refrigerated temperatures, Salmonella and L. monocytogenes do not grow on fresh-cut celery. Salmonella, but not L. monocytogenes, has the potential to grow on temperature-abused fresh-cut celery, especially if inoculated onto the cut surface.

Publications

• Strawn, L.K., K.R. Schneider, and M.D. Danyluk. 2011. Microbiological Safety of Tropical Fruits. Critical Reviews in Food Science and Nutrition. 51:132-145.
• Bisha,B., A. Perez-Mendez, M.D. Danyluk, L.D. Goodridge. 2011. Evaluation of modified Moore swabs and continuous flow centrifugation for concentration of Salmonella spp. and Escherichia coli O157:H7 from large volumes of water. Journal of Food Protection. 74:1934-1937.
• Perez-Cacho, P.R., M.D. Danyluk, and R. Rouseff. 2011. GC-MS quantification and sensory thresholds of guaiacol in orange juice and the relationship between guaiacol and Alicyclobacillus. Food Chemistry. 129:45-50.
• Cevallos-Cevallos, J.M., M.D. Danyluk, J. I. Reyes-De-Corcuera. 2011. GC-MS Based Metabolomics for rapid detection of Escherichia coli O157:H7 and Salmonella Typhimurium, Salmonella Muenchen and Salmonlla Hartford in beef and chicken. Journal of Food Science. 76: M238-M246.
• Danyluk, M.D., and D.W. Schaffner. 2011. Quantitative microbial assessment for E. coli O157:H7 in leafy greens from farm to consumption using published data. Journal of Food Protection. 74:700-708.
• Danyluk, M.D., L.M. Friedrich, C. Bousquet, R.M. Goodrich-Schneider, M.E. Parish, and R. Rouseff. 2011. Prevalence, Amounts, Spoilage, and Mitigation of Alicyclobacillus spp. in Tropical and Subtropical Fruit Concentrates. Food Microbiology. 28:472-477.

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: Citrus slices are commonly added to beverages in the food service industry; little is known about microbial risks associated with this practice. A study was undertaken to determine survival of Salmonella on lemon and lime stored on ice or at room temperature (RT) and transfer of Salmonella from lemon and lime slices into beverages. In natural-light labeling of fruits and vegetables the desired information is etched onto the produce surface using a low-energy carbon dioxide laser beam (10,600 nm). Etched characters are formed by surface depressions in the epidermis that seemingly facilitate entrance of decay and pathogenic organisms. We determined the fate of Salmonella on orange and tomato surfaces following the application of a natural-light label and storage at different temperatures. A number of sequences with wax application and natural light labeling were used, and influence of a soft rot spoilage organism was evaluated. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
On lemons, Salmonella inoculated onto peel and albedo decreased 2 log CFU/slice in 24 h when stored on ice. When inoculated onto lemon flesh, Salmonella decreased 1 log at time 0 h, fell below the limit of detection (1.9 log CFU/slice) from 1 to 8 h and were again detectable 24 h. When stored at RT Salmonella inoculated onto the peel and albedo decreased by 0.1 and 1.2 log CFU/slice respectively over 24 h. Salmonella on the flesh of lemons at RT decreased to below the limit of detection in 2 h, remaining undetectable through the remainder of the experiment. On limes, Salmonella inoculated onto the albedo and held at RT first decreased by 1 log CFU/slice within the first hour then grew back to 1 log CFU/slice higher than at time zero. When inoculated onto either the flesh or peel and held at RT no significant difference was observed in the counts of Salmonella over the 24 h period; counts remained at ca. 5 log CFU/slice. When stored on ice the initial Salmonella load (at time 0 h) was ca. 4.5 log CFU/slice and remained with no significant difference over the 24 h storage time. The addition of a peel inoculated lemon slice resulted in 10.2 and 8.933 MPN/ml for water and iced tea, respectively. The addition of an albedo inoculated lemon slice resulted in 0.673 and 0.246 MPN/ml of water and iced tea, respectively. The demonstrated ability of Salmonella to survive on lemon and lime slices, and transfer into beverages, suggests a potential food safety risk associated with improperly handled sliced citrus. There were no significant differences (P<0.05) between populations of Salmonella on natural light labeled or unlabeled tomatoes stored at 4, 12, or 25C over the experiment. Salmonella held at 4C decreased ca. 2 log CFU/tomato over 28 days, while those held at 12 and 25C for 28 and 14 days, respectively, remained stable. The addition of soft rot organisms at higher, lower, or equal population densities' to Salmonella did not significantly alter the behavior of Salmonella on tomatoes stored at 25C, regardless of natural light labeling. Natural light labeling tomatoes does not adversely affect the tomato surface with respect to pathogen survival, even in the presence of soft-rot pathogens and offers industry a permanent, safe alternative to traditional adhesive labeling. No set of waxing and labeling conditions promoted the growth of Salmonella on the orange surface, or allowed penetration into the orange juice. Survival of Salmonella populations on the peel surface did not differ between any of the treatment and control (unlabeled samples). In all cases, Salmonella declined between 1.5 and 3.0 log CFU/orange after 30 days, with faster declines noted at 10C. Based on the data obtained from the many combinations of treatments and under conditions extremely unfavorable and unrealistic in terms of fruit storage, natural-light labeling citrus fruit peels and subsequent waxing in any order does not allow for the growth nor influence the natural decline of Salmonella populations on citrus fruit surfaces as compared to controls.

Publications

• Danyluk. M.D., L.M. Friedrich, L.O. Interiano Villeda, K.R. Schneider, and E.J. Etxeberria. 2010. Natural-Light Labeling of Tomato does not Facilitate Growth or Penetration of Salmonella into the Fruit. Journal of Food Protection. 73:2276-2280.
• Durak, M.Z., J.J. Churey, M.D. Danyluk, R.W. Worobo. 2010. Identification and Haplotype Distribution of Alicyclobacillus spp. from Different Juices and Beverages. International Journal of Food Microbiology. 142:286-291
• Strawn, L.K. and M.D. Danyluk. 2010. Fate of Escherichia coli O157:H7 and Salmonella spp. on Fresh and Frozen Cut Pineapples. Journal of Food Protection. 73:418-424.
• Strawn, L.K. and M.D. Danyluk. 2010. Fate of Escherichia coli O157:H7 and Salmonella spp. on Fresh and Frozen Cut Mangoes and Papayas. International Journal of Food Microbiology. 138:78-84.
• Du, W.-X., M.D. Danyluk, and L.J. Harris. 2010. Efficacy of aqueous and alcohol-based quaternary ammonium sanitizers for reducing Salmonella in dusts generated in almond hulling and shelling facilities. J. Food Sci.. 75:M7-M13.

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: We investigated the fate of Salmonella and E. coli O157:H7 on fresh (23, 12 and 4C) and frozen (-20C) cut mangoes, pineapple and papaya for: 0, 1, 3, 5 and 7 days (23C); 0, 1, 3, 5, 7, 10, 14, 21 and 28 days (12 and 4C); and 0, 7, 14, 21, 28, 60, 90 and 120 days (-20C). We determined rates of Salmonella transfer from surface contaminated citrus fruit that occur as a result of hand peeling. Transfer rates were determined between the whole fruit inoculated at the stem, styler or equator and the peel, edible segments and gloved hands for three common fresh citrus varieties (Valencia oranges, Satsuma mandarins, and Marsh grapefruit). We evaluated the efficacy of aqueous and alcohol based sanitizers for reducing different Salmonella serovars in dusts generated in almond hulling and shelling facilities. PARTICIPANTS: Several UF and national collaborators and students were involved TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
We demonstrated that E. coli O157:H7 and Salmonella spp. have the potential to grow on temperature abused fresh-cut mangoes and papayas held at 23C and 12C, and survive on fresh-cut pineapples. It was also determined that E. coli O157:H7 and Salmonella spp. can survive for extended periods of time on refrigerated (4C) and frozen (-20C) cut mangoes, papayas and pineapples. Our work indicates that both fresh and frozen cut mangoes, papayas and pineapples have the potential to be vectors for E. coli O157:H7 and Salmonella spp. transmission. No significant difference was found between men and women or location of inoculum on Marsh grapefruit or Valencia oranges. The peel portion of Satsuma mandarins showed significant differences in gender and inoculation site. Percent transfer to edible segments ranged from 27.9 % - 0 % in Valencia, 5 % - 0.1% in grapefruit and 3.5% - 0% in mandarins. Transfer to gloved hands ranged from 10% - 0% in Valencia, 2.1% - 0.1% in grapefruit and 11.6% - 0% in mandarins. Pathogens transfer from the whole fruit to edible portions and hands regardless of the location of inoculation. Sterile water and aqueous based sanitizers led to a ca. 3 log CFU/ml increase in Salmonella populations after 48 hours. Alcohol based sanitizers lead to instant reduction (at 0 h) of ca. 1 log CFU/ml and a subsequent reduction to below the level of detection ( less than 1.3 log CFU/ml) after 48 hours. Alcohol based sanitizer is effective at reducing Salmonella populations, while the addition of aqueous based sanitizers and water allow for the growth of Salmonella in almond dust. The use of alcohol based sanitizer is recommended for sanitizing in Almond hulling and shelling facility.

Publications

• Friedrich, L.M., R. Goodrich-Schneider, M.E. Parish, and M.D. Danyluk. 2009. Mitigation of Alicyclobacillus spp. spores on food contact surfaces with aqueous chlorine dioxide and hypochlorite. Food Microbiol. 26:936-941. Wright, A.C., M.D. Danyluk and W.S. Otwell. 2009. Pathogens in raw foods: what the salad bar can learn from the raw bar. Curr Opin Biotech. 20:172-177.