Source: UNIV OF MINNESOTA submitted to
PRESENCE AND CONTROL OF FOOD BORNE PATHOGENS IN READY-TO-EAT FOODS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0222437
Grant No.
(N/A)
Project No.
MIN-18-069
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2010
Project End Date
Sep 30, 2014
Grant Year
(N/A)
Project Director
Diez-Gonzalez, FR.
Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
Food Science & Nutrition
Non Technical Summary
The presence of potentially pathogenic bacteria in foods is a major public health concern and is a serious threat for the delivery of safe foods by the food industry. In recent years, however, an increasing number of food-borne disease outbreaks have seriously questioned the safety of our food supply systems. Many of those outbreaks have been caused by bacteria that contaminated ready-to-eat foods. Despite the fact that the food industry and the government have implemented a series of actions to prevent contamination, new outbreaks are still frequently reported. Salmonella outbreaks have been linked to a wide variety of food products, but in recent years the occurrence of several multi-state cases related to fresh produce and dry foods have heightened the concern for transmission to these types of products and have exposed weaknesses in our food supply. Some of the implicated foods have included chocolate, dried milk, almonds, peanut butter, peanut products, toasted oats cereal and dried spices. These cases demonstrate that these pathogenic bacteria have a very unique ability to remain viable at conditions of very low moisture for long periods of time. An increased number of outbreaks caused by EHEC O157 have also been linked to fresh produce in particular lettuce and spinach. Since some of the outbreaks with fresh produce were linked to pre-washed product, it suggests that serotype O157 may have unique mechanisms of survival associated with the plant tissue. L. monocytogenes has been a concern in ready-to-eat deli meats, but recent outbreaks linked to the consumption of fresh Hispanic style cheeses have revealed the relatively high risk of transmission via these products. These emerging foodborne challenges require the re-evaluation of current practices and the development of novel strategies to have an impact on preventing more human cases. This project presents a comprehensive and broad approach to address some of these food-pathogen risks. The proposed work may lead to a better understanding of the mechanisms of pathogen survival to identify intervention methods for control. The findings generated by this project will likely contribute to the advancement of a better understanding of the survival and control of pathogenic bacteria in ready-to-eat foods. The increase in knowledge on the effectiveness of the proposed control approaches will likely contribute to finding feasible intervention methods.
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
7124010110040%
7221499110020%
7221599110020%
7223430110020%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 722 - Zoonotic Diseases and Parasites Affecting Humans;

Subject Of Investigation
1499 - Vegetables, general/other; 3430 - Cheese; 1599 - Grain crops, general/other; 4010 - Bacteria;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
The long-term goal of this project is to contribute to the understanding and control of Salmonella, Escherichia coli O157:H7 and Listeria monocytogenes in specific ready-to-eat food products. We plan to conduct a series of experiments with pathogenic strains of each of these organisms and to test their survival against different physical, chemical and biological treatments. The specific goals of this proposal are to: 1)Determine the effectiveness of bacteriophage treatment to reduce the viability of Escherichia coli O157 and Salmonella populations on the surface of lettuce, spinach and tomatoes. 2)Characterize the thermal resistance of and identify treatments that would kill. Salmonella in low water activity foods. 3)Identify combinations of antimicrobial GRAS ingredients that will inhibit and reduce the viable count of Listeria monocytogenes, E. coli O157 and Salmonella in a fresh Hispanic cheese (queso fresco). We expect that the outputs resulting from this project will include: laboratory research activities, mentoring of Food Science graduate and undergraduate students, scientific evidence that will support the idea of using bacteriophages, a time/temperature database for inactivation of Salmonella in low water activity foods, and GRAS addition combinations for treating Hispanic cheeses. This proposal will be at the core of the research program of the Principal Investigator, focused on understanding and seeking strategies to control food-borne pathogens. The PI has worked extensively in different projects related to these microorganisms, investigating pre-harvest control and prevalence of EHEC related to animal production, incidence of Salmonella and E. coli contamination in fresh vegetables, modeling the growth of L. monocytogenes in deli meats, among other projects.
Project Methods
This project will be conducted by performing a series of microbiological, biochemical and molecular biology experiments using pure cultures of Salmonella, enterohemorrhagic Escherichia coli and Listeria monocytogenes. The experimental approach is described for each of the objectives below. Objective 1: Bacteriophages specific against E. coli O157 and Salmonella have been identified and partially characterized by our research group. Phages capable of inhibiting E. coli O157 will be used to treat lettuce and spinach leaves. Phages specific for infecting Salmonella Typhimurium will be used to treat tomato plants and fruits. The impact of a variety of factors that may influence the effectiveness of phage treatments will be studied. These factors would include: stage of plant growth, bacterial concentration, bacterial pre-inoculation conditions, phage concentration, temperature, pre-inoculation treatment, lettuce variety, relative humidity, strain specificity, presence of epiphytic microorganisms, treatment of internalized bacteria, and combination of phage/chemical antimicrobial treatments. Objective 2: We have developed an inoculation system to study the viability of bacteria at water activities of toasted oats cereal and we have adapted the capillary tube technique used for thermal inactivation in liquids, for powder dry matrices. We plan to perform a series of laboratory experiments using Salmonella serovars that have been associated with outbreaks of low water activity foods. We will be using different types of low water activity model food matrices: starch, casein, commercial cereal mixtures, spices, wheat flour, corn grits and vegetable oil. These materials will be inoculated with individual Salmonella strains, ground and re-dried where necessary and subjected to thermal treatments. Surviving bacterial counts will be determined by standard microbiological techniques. The range of temperatures that we plan to investigate is from 70 to 150 degree C. The data generated will be treated as a first-degree reaction order and the kinetic parameters for death rate, D value and Z value will be calculated. Objective 3: A fresh Hispanic cheese model ("queso fresco") was developed previously to study the growth of Listeria monocytogenes and recently we have identified that combinations of nisin and caprylic acid can be very inhibitory. For this objective we propose to use a similar model to optimize the treatment to enhance its antimicrobial effects on L. monocytogenes as well as E. coli O157 and Salmonella, and to minimize its impact on sensorial characteristics. Batches of queso fresco will be inoculated with mixtures of individual bacterial species. Cheese curds will be mixed with different ingredients. Queso fresco portions will be stored at refrigeration temperatures and the count of bacterial pathogens will be monitored periodically for 3 weeks. Samples will be subjected to microbiological tests for quantification of cell count. The GRAS ingredient treatments will include nisin, caprylic acid, potassium sorbate, sodium propionate, cinnamic acid, cinnamaldehyde, carvacrol, eugenol, monolaurin, monocaprylin, and other natural extracts.

Progress 10/01/10 to 09/30/14

Outputs
Target Audience: Dairy, produce and dry food producers interested in improving food safety as well as scientist working in similar areas. These groups were reached viapeer-reviewed article publications and through presentations at scientific meetings in oral and poster formats Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? A Ph. D. student and a visiting scientist were trained in advanced genomic and transcriptomic techniques. How have the results been disseminated to communities of interest? Peer-review publications as well as presentations at scientific meetings. There publications were published in five different scientific journals (International Journal of Food Microbiology, Journal of Dairy Science, Journal of Animal Science, Journal of Food Protection and Journal of Agricultural Science). Two posters were presented at the ASM Annual Meeting, May 17-20 in Boston, MA. One oral presentation was also given at the MI ASM Branch Meeting, October 25-26 in Traverse City, MI> What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In recent years, lettuce and spinach have been linked to outbreaks of foodborne illnesses due to contamination with Escherichia coli O157:H7 (EHEC). While the molecular mechanisms allowing for the survival of EHEC in the phyllosphere of lettuce have been the focus of several recent studies, very little is known about the interaction of EHEC with spinach leaves. This study aims at identifying and analyzing EHEC genes that are involved in the interaction with spinach leaves. At 24 h, the expression of many genes (1,947) was significantly down-regulated and only 34 genes were up-regulated. Among these, ycfR, encoding for a multiple stress protein, was up-regulated 17-fold. The expression of another gene important for the interaction of EHEC to plants, ybiM, was also increased 9-fold. At 120 h, less than half of the genes that had diminished expression at 24 h were still down-regulated (927) and 21 genes were up-regulated. At 120 h ycfR expression was stimulated 6-fold, ybiM was not significantly expressed. We had previously reported that ycfR was up-regulated when EHEC interacted with lettuce leaves and roots. The role of ycfR and ybiM implicated in survival on lettuce leaves was corroborated also for spinach leaves. Salmonella has the unique ability to survive desiccation and low water activity conditions ( less than 0.6) for long periods, developing a greater tolerance to heat exposure. Divalent cations sequestering enzymes are able to affect S. enterica desiccation-triggered heat tolerance. The main objective of the present study was to investigate the impact of chelating agents on the heat resistance acquisition by S. Typhimurium. For all the treatments and temperatures tested, cells exposed to aw 0.11 had D-values at least 116[AM1]-fold longer than cells equilibrated at aw 1.0. The D-values observed for untreated cells at aw 0.11 were 125 min at 90ºC and 46 min at 95ºC. Treatment with chelating agents significantly (p < 0.05) decreased the thermal resistance of cells equilibrated at aw 0.11, but had no effect at aw 1.0. The D95 values after chelation with DIP and EDTA were 32 and 31 min, respectively. A similar trend was observed at 90ºC, when cells treated with DIP and EDTA were killed at D-values of 102 and 93 min, respectively. Our results confirmed that exposure to low aw conditions induces thermal resistance in Salmonella Typhimurium. Moreover, our results indicated that chelation affects the thermal resistance at low water activity, without effects on cells at high water activity. This finding suggests that divalent cations play a role in the cross-resistance mechanisms.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Gadotti, C., L. Nelson, and F. Diez-Gonzalez 2014. Inhibitory effect of combinations of caprylic acid and nisin on Listeria monocytogenes in queso fresco. 39: 1-6.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Akhtar, M., S. Viazis, and F. Diez-Gonzalez. 2014. Isolation, identification and characterization of lytic, wide host rang bacteriophages from waste effluents against Salmonella enterica serovars. Food Cont. 38: 67-74.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Martinez-Vaz, B., R. C. Fink, F. Diez-Gonzalez, and M. J. Sadowsky. Enteric pathogen-plant interactions: Molecular connections leading to colonization and growth and implications for food safety. Microbes and Environments. 29: 123-135.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Maserati, A., Fink, R. C., Ledgister, S. L., Stiernagle, J. L., Rappe, M. D., and F. Diez-Gonzalez 2014. Effect of chelating agents on the heat resistance development of Salmonella Typhimurium in low water activity oat cereals. American Society for Microbiology Annual Meeting, May 17-20, Boston, MA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Fink, R. C., Sadowsky, M. J., and F. Diez-Gonzalez. 2014. Analysis of the transcriptional changes induced by the association of Escherichia coli O157:H7 with spinach leaves. American Society for Microbiology Annual Meeting, May 17-20, Boston, MA.


Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Dairy and dry foods industries as well as the scientific community interested in food safety. These groups were reached via peer-reviewed article publications and through presentations at scientific meetings in oral and poster formats. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? A Research Associate continues to develop her research skills and one M. S. student was trained. How have the results been disseminated to communities of interest? Peer-review publications as well as presentations at scientific meetings. Five publications were published in five different scientific journals (International Journal of Food Microbiology, Journal of Dairy Science, Journal of Animal Science, Journal of Food Protection and Journal of Agricultural Science). Two posters were presented at the ASM Annual Meeting, May 18-21 in Denver, CO and two posters and two oral presentations were given at the IAFP Annual Meeting, July 28-31 in Charlotte, NC. One oral presentation was also given at the KY-TN ASM Branch Meeting, October 25-26 in Bowling Green, KY What do you plan to do during the next reporting period to accomplish the goals? We plan to write the manuscripts of the experimental results generated from the different projects for publication in different journals. A total of 3 publications will be written on objective 1, another 3 papers will be submitted on objective 3 and 1 publication under objective 2.

Impacts
What was accomplished under these goals? Accomplishments Impacts: this project continues to make progress to address the issue of safety of ready-to-eat foods. The three pathogen-food combinations that this project is addressing include some of the most pressing safety issues in our food supply. Fresh produce, dry foods and ready-to-eat food outbreaks account for the bulk of foodborne disease in our country. Advancement in any of those areas will certainly contribute to easing the public health burden. Change in knowledge: Objective 1: To characterize the endophytic microorganism residing in commercial lettuce leaves, two separate studies were conducted. First, a total of 30 and 25 heads of romaine and red leaf lettuce, respectively, served as the source of individual leaves which were surface sterilized, stomached, enriched in BHI broth for 24 h and plated onto BHI agar for non-selective isolation. In a separate survey, 80 heads of each the same lettuce types were similarly processed, except that GN broth and MacConkey agar (MCA) were used for isolation of Gram negative bacteria Thirty-eight out of 100 leaves were positive for internalized microorganisms, and Bacillus, Pseudomonas and Pantoea were the genera most frequently found in both types of lettuce. Members of the genus Erwinia were isolated from romaine lettuce only. In the second study, 21 and 60% of romaine and red leaf lettuce heads, respectively, had internalized bacteria capable of growing on MCA. Among the Gram negative strains, Pseudomonas and Pantoea genera were most frequently isolated. Enterobacter isolates were obtained from three red leaf samples. In summary, spore-forming bacteria and traditional epiphytic bacterial genera were frequently detected in surface-sterilized commercial lettuce leaves. Despite the common occurrence of internalized bacteria, only Enterobacter was related to E. coli O157:H7 and Salmonella. Objective 3: The purpose of this project was to assess the ability of Listeria monocytogenes to survive in low-sodium commercial sliced process cheeses. The experiments were conducted with three different brands of commercially available sliced cheeses (brands A, B, C) packaged in bulk (slice-on-slice) and as individually wrapped slices. Brands A, B and C had sodium contents 44, 34 and 29% less, respectively, than their corresponding standard formulations. The effect of salt content and temperature were measured using surface inoculation of slices with mixtures of 5 strains of L. monocytogenes and determining bacterial counts during storage. The changes in Listeria counts were correlated with those factors as well as with pH, water activity and moisture content. At 4°C, the count of L. monocytogenes remained at approximately 4 log CFU/g for the entire 60 days of storage in any of the three brands of regular and reduced salt processed cheese in both slice-on- slice or individually wrapped slices. As the storage temperature increased, the Listeria counts declined reaching undetectable levels at any condition after 25 and 15 days at 23 and 30°C, respectively. At those temperatures, the survival rate in brand C appeared to be shorter than the other two brands. The main finding of this research was that sodium reduction in processed cheese did not enhance survival nor promoted growth of L. monocytogenes at any temperature or brand tested.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Sepulveda, R., M. Shaughnessy, T. Snider, D. Boxrud, E. Cebelinski, K. Smith, J. Bender, J. Johnson, S. Holzbauer, K. Venkitanarayanan, and F. Diez-Gonzalez. 2013. Prevalence of Clostridium difficile in retail meats in minnesota and comparison of animal and human isolates. ASM Annual Meeting, May 18-21, Denver, CO
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Fink, R. C., F. H. Lee, A. Amann, C. Radtke, and F. Diez-Gonzalez. 2013.Microwave treatment of Salmonella-contaminated dry food matrices. ASM Annual Meeting, May 18-21, Denver, CO
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Akhtar, M., and F. Diez-Gonzalez. 2013. Impact of sodium reduction on survival of Listeria monocytogenes. IAFP Annual Meeting, July 28-31, Charlotte, NC.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Hystead, E., F. Diez-Gonzalez, and T. C. Schoenfuss. 2013. The effect of sodium reduction with and without potassium chloride on the survival of Listeria monocytogenes in Cheddar cheese. J. Dairy Sci. 96:6172-6185.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Lumor, S. E., B. D. Deen, I. Ronningen, K. Smith, N. R. Fredrickson, F. Diez-Gonzalez, T. P. Labuza. 2013. Assessment of the inhibition of ricin toxicity by lactose in milk, J. Food Prot. 76: 2037-39.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Compart, D. M, A. M. Carlson, G. I. Crawford, R. C. Fink, F. Diez-Gonzalez, A. Dicostanzo, and G. C. Shurson. 2013. Presence and biological activity of antibiotics used in fuel ethanol and corn co-product production. J Anim Sci. 91:2395-2404.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Pimentel-Filho, N. J., H. C. Mantovani, F. Diez-Gonzalez and M. C. Dantas Vanetti Inhibition of Listeria and Staphylococcus aureus by bovicin HC5 and nisin combination in milk J. Agric. Sci. 8:188:196.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Diez-Gonzalez, F. 2013. Microbial food safety of organic foods. KY-TN ASM Branch Meeting, October 25-26, Bowling Green, KY.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Deen, B., A. Pagel, L. He, F. Diez-Gonzalez, and T. P. Labuza. 2013. Capture and detection of Bacillus anthracis spores using aptamer based surface enhanced Raman spectroscopy. IAFP Annual Meeting, July 28-31, Charlotte, NC
  • Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Sepulveda, R. 2013. Prevalence of Clostridium difficile in retail meats from Minnesota and comparison of growth and survival of human and animal isolates. M. S. Thesis. University of Minnesota, St. Paul, MN.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Diez-Gonzalez, F. 2013. Is there scientific evidence that organic produce is as microbiologically safe as conventional produce? IAFP Annual Meeting, July 28-31, Charlotte, NC.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Hou, Z, R. C. Fink, C. Radtke, M. J. Sadowsky, and F. Diez-Gonzalez. 2013. Incidence of naturally internalized bacteria in lettuce leaves. Int. J. Food Microbiol. 162:260-265.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Hou, Z., R. C. Fink, M. Sugawara, F. Diez-Gonzalez, and M. J. Sadowsky. 2013. Transcriptional and functional responses of Escherichia coli O157:H7 growing in the lettuce rhizoplane. Food Microbiol. 35:136-142.


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: OUTPUTS: Activities: The efforts of this MAES project a have been focused to advancing objectives 2 and 3, developing alternative methods for inactivation of Salmonella in dry cereals and assessing the survival in cheese products. Experimental work was performed to study the thermal resistance and inactivation of Salmonella in dry cereals. A series of experimental trials were conducted to study the impact of temperature abuse on the germination and growth of sporeforming pathogenic bacteria in cheese sauces as well as to assess the impact of sodium reduction in Cheddar cheese. Dissemination: The findings regarding of this project were presented as posters at the 2012 American Society for Microbiology annual meeting, June 17-20 and at the 2012 American Dairy Science Association in Phoenix, AZ. Researchers attended the American Society for Microbiology annual meeting in San Francisco, CA to present the most relevant findings of. Dissemination: Researchers discussed their findings with audiences as well as distributed copies of the poster to attendees. PARTICIPANTS: PI: Francisco Diez-Gonzalez; Graduate Research Assistants: Dorey Gray, Ellen Hystead, Abena Opoku. Undergraduate Students: Alex Amman and Christie Radtke. TARGET AUDIENCES: Dairy and dry foods industries as well as the scientific community interested in food safety. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Change in knowledge: Objective 2: To determine the chemical changes caused by dehydration in dry foods, we inoculated toasted oat cereal with of Salmonella enterica ser. Typhimurium, dried overnight at 40C, and equilibrated to aw of 0.11, 0.33, 0.53, or 1.0. The cells were separated from cereal and analyzed by surface enhanced Raman scattering (SERS). Cellulose filters were inoculated with 109 CFU/mL of S. Typhimurium, dried overnight, and equilibrated to the same aw used for the inoculated cereal. Cells were harvested and total RNA was extracted for direct RNA sequencing. The analysis of the SERS spectra indicated shifts in the peaks corresponding to protein, lipids, and nucleic acids. Particularly in nucleic acids, the ratio of the peak intensities corresponding to the pyrimidines (C, T, U) compared to the purines (A, G) decreased as aw decreased in the range from 1 to 0.11. The total RNA amount decreased as aw decreased but there was no degradation. The principal component analysis showed three groupings (1) aw = 1.0, (2) aw between 0.33 and 0.53, and (3) aw = 0.11 thus indicating a difference in chemical composition of the cells. Using a simple household microwave of 1150 watts (General Electric, Inc.) generating microwaves of 2,450 MHz, we were able to treat different materials inoculated with the same Salmonella serovars. Using a toasted oat cereal inoculated with 107 CFU/g of individual strains of S. Typhimurium, Tennessee, Agona and Saintpaul. Reductions of at least 3 log CFU/g were observed in all cases after 2 minutes of exposure to microwaving. Objective 3: Stirred curd Cheddar cheese was manufactured at full, reduced, and low sodium concentrations using two separate, single-strain starter cultures. In the reduced and low sodium treatments, curds received an application of either sodium chloride only, or sodium chloride with potassium chloride (KCl) to replace the effect of sodium chloride on water activity. After manufacture, cheese was analyzed for a compositional analysis and aged. Upon completion, the cheese was separately inoculated with L. monocytogenes and stored at 4 or 12C for 27 to 63 days. The first phase of testing was run immediately after manufacturing, and simulates results of contamination during processing of cheese. During the first phase of testing, the L. monocytogenes population declined by 4 log CFU/g over 60 days of storage at 4C. The second and third phases were conducted shortly after the completion of phase one and simulate post aging contamination of the cheese. The survival of L. monocytogenes increased when the cheese was stored at 4 and 12C. In a separate project, commercial cheese sauce was inoculated with Bacillus cereus and held at temperatures of 4, 12, 23, 37, and 45C for short-term and long-term storage. B. cereus germinated well in the cheese sauce medium. At temperatures of 23C and 45C, vegetative cell populations reached 8 log CFU/g within 48 hours and at 12C reached 7 log CFU/g after 1 month. When incubated at 4C no growth was observed even after 2 months. Almost no increase in B. cereus spore count was observed at any temperature, except at 23C.

Publications

  • Hystead, E. 2012. Determining the Effect of Sodium Reduction on the Survival of Listeria monocytogenes and Bacillus anthracis in Cheddar Cheese. M. S. Thesis. University of Minnesota.
  • Gray, D. L. 2012. Survival and Germination Potential of Spore Forming Bacteria in Cheese Sauce. M. S. Thesis. University of Minnesota.
  • Opoku, A. O. 2012. Alternative Processing Methods for the Inactivation of Escherichia coli O157:H7, Salmonella and Listeria monocytogenes in Skim Milk. M. S. Thesis. University of Minnesota.
  • Ellen Hystead, F. Diez-Gonzalez, and T. C. Schoenfuss. 2012 Effect of sodium reduction on the survival of Listeria monocytogenes and Bacillus anthracis in Cheddar cheese. ADSA Annual Meeting, July 20-23, Phoenix, AZ.
  • Fink, R. C., L. He, C. L. Radtke, Z. P. Metz, A. R. Amann, T. P. Labuza, and Francisco Diez-Gonzalez. 2012. Effect of water activity on the surface enhanced Raman spectra and total RNA of Salmonella cells associated with a dry cereal. ASM Annual Meeting, June 18-20, San Francisco, CA.
  • Hou, Z. 2012. Association and Natural Internalization of Escherichia coli in the Rhizosphere of Lettuce Plants. Ph. D. Dissertation. University of Minnesota.
  • Yang, H. 2012. Evaluating the Antimicrobial Mechanism of Neutral Electrochemically Activated Water on Foodborne Pathogens and their Biofilms. Ph. D. Dissertation. University of Minnesota.
  • Hou, Z., R. C. Fink, E. P. Black, M. Sugawara, Z. Zhang, F. Diez-Gonzalez and M. J. Sadowsky. 2012. Gene expression profiling of Escherichia coli in response to interactions with the lettuce rhizosphere. J. Appl. Microbiol. 113, 1076-1086.
  • Lumor, S. E., B. D. Deen, I. Ronningen, K. Smith, N. R. Fredrickson, F. Diez-Gonzalez, T. P. Labuza. 2012. Detection and inactivation of saxitoxin in skim milk, J. Food Prot. 75: 1113-1116.
  • Lumor, S. E., N. R. Fredrickson, I. Ronningen, B. D. Deen, K. Smith, F. Diez-Gonzalez, T. P. Labuza. 2012. Comparison of the presence of Shiga toxin 1 in food matrices as determined by an enzyme-linked immunosorbent assay and a biological activity assay. J. Food Prot. 75: 1036-1042.
  • Fink, R. C., E. Black, Z. Hou, M. Sugawara, M. J. Sadowsky, and F. Diez-Gonzalez. 2012. Transcriptional responses of Escherichia coli K12 and O157:H7 in association with lettuce leaves. Appl. Environ. Microbiol. 78: 1752-1764.
  • Deen, B., T. Rodda, L. He, F. Diez-Gonzalez, and T. P. Labuza. 2012. Rapid detection and discrimination of Bacillus species using immunomagnetic separation combined with surface-enhanced Raman spectroscopy. IAFP Annual Meeting, July 22-25, Providence, RI.
  • Mahero, M., K. Hamilton, Z. Hou, M. Schermann, C. Tong, F. Diez-Gonzalez, and J. Bender. 2012. Evaluation of good agricultural practices on Minnesota vegetable growing operations. IAFP Annual Meeting, July 22-25, Providence, RI.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Activities: The efforts of this MAES project a have been focused on objectives 2 and 3, on characterizing the thermal inactivation of Salmonella in dry cereals and to assess the survival of growth in cheese products. Experimental work was performed to determine the rate of inactivation of Salmonella in dry cereals. A series of experimental trials were conducted to study the impact of temperature abuse on the germination and growth of Bacillus cereus in cheese sauces. The experimental work directed to determine the effect of antimicrobials in queso fresco to control E. coli O157:H7 and Salmonella was conducted. The findings regarding of this project were presented as posters at the 2011 American Society for Microbiology annual meeting, May 21-24, New Orleans, LA. Three M. S. student were guided and mentored and two undergraduate students were taught experimental research techniques. Events: researchers attended the American Society for Microbiology annual meeting in New Orleans, LA to present the most relevant findings of the inhibition of pathogenic bacteria in Hispanic cheeses, and dry foods. Dissemination: Researchers discussed their findings with audiences as well as distributed copies of the poster to attendees. PARTICIPANTS: PI: Francisco Diez-Gonzalez; Graduate Research Assistants: Camila Gadotti, Dorey Gray, and Matt Chick. Undergraduate Students: Phillip Kramer and Hannah Clark. TARGET AUDIENCES: Dairy and dry foods industries as well as the scientific community interested in food safety. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Change in knowledge: Objective 2: Using Salmonella serovars Typhimurium, Tennessee and Agona, at water activity (aw) of 0.53, the D-values ranged from 172 to 208 min at 65C and from 4.3 to 6.5 min at 80C among the three serovars. D-values at 80C increased 4-fold when the aw was reduced to 0.33. All serovars became more resistant to heat as the aw decreased, and at 0.11, D-values greater than 135 min were measured at 85C. The Z-values for the three serovars at 0.33 and 0.53 aw were approximately 10C, but this value increased from 20 to 50% if the serovars were tested at 0.11 aw. These findings suggest that Salmonella becomes more resistant to heat in drier foods. Objective 3: Batches of queso fresco were inoculated with mixtures of 4 log CFU/g of different Escherichia coli O157:H7 or Salmonella strains/serovars and treated with several combinations of nisin (N) from 0.4 to 0.5 g/kg, caprylic acid (CA) from 0.3 to 3.3 g/kg and trans-cinnamaldehyde (CN) from 0.3 to 1.2 g/kg. The N/CA combinations had little inhibitory effect against Salmonella and 1 log CFU/g inhibition was only seen when CA was used in the concentration of 1.6 g/kg. E. coli O157:H7 strains were somewhat more susceptible to the combination being inhibited by at least 2 log CFU/g when CA was used at 0.4 g/kg. Growth of both pathogens was inhibited by at least 1 log CFU/g when CN was used at the concentration of 0.3 g/kg and by at least 3 log CFU/g when CN was used at 1.2 g/kg. None of the treatments completely inhibited the growth of indicator lactic acid, psycrotrophic and aerobic plate count bacteria throughout storage. The N/CA/CN combination was effective to inhibit all Salmonella and E. coli O157:H7 strains tested in queso fresco at concentrations of 0.5, 0.4, and 0.3 to 0.6 g/kg of N, CA, and CN respectively. The antimicrobial combination had very little effect on growth of natural bacteria of queso fresco. The use of additional GRAS compounds such as trans-cinnamaldehyde may provide an alternative to control Salmonella and E. coli O157:H7 in queso fresco.

Publications

  • Peer-review papers: He, L., B. Deen, T. Rodda, I. Ronningen, T. Blasious, C. L. Haynes, F. Diez-Gonzalez, and T. P. Labuza. 2011. Detection of ricin in milk combined immunomagnetic separation with surface-enhanced Raman spectroscopy. J. Food Sci. 76: N49-53.
  • He, L., E. Lamont. B. Veeregoda, S. Sreevatsan, C. L. Haynes, F. Diez-Gonzalez, and T. P. Labuza. 2011. Aptamer-based surface-enhanced Raman scattering detection of ricin in liquid foods. Chem. Sci. 2: 1579-1582.
  • He, L., C. L. Haynes, F. Diez-Gonzalez, and T. P. Labuza. 2011. Rapid detection of a foreign protein in milk using IMS-SERS. J. Raman. Spec. 42: 1428-1434.
  • He, L., T. Rodda, C. Haynes, T. Deschaines, T. Strother, F. Diez-Gonzalez, and T. P. Labuza. 2011. Detection of a foreign protein in milk using surface-enhanced Raman spectroscopy coupled with antibody-modified silver dendrites. Anal. Chem. 83: 1510-1513.
  • Lumor, S. E., A. Hutt, I. Ronnigen, F. Diez-Gonzalez, and T. P. Labuza. 2011. Validation of immunodetection (ELISA) of ricin using a biological activity assay. J. Food Sci. 76: C112-C116.
  • Lumor, S. E., F. Diez-Gonzalez, and T. P. Labuza. 2011. Detection of warfare agents in liquid foods using brine shrimp lethality assay. J. Food Sci. 76: T16-T19.
  • Thesis: Chick, M. 2011. Thermal inactivation kinetics of Salmonella serovars on dry cereal. M. S. Thesis. University of Minnesota.
  • Gadotti, C. 2011. Control of pathogenic bacteria in queso fresco by using generally recognized as safe ingredients. M. S. Thesis. University of Minnesota.


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Activities: The efforts during this initial year of this MAES project a have been focused to objectives 2 and 3, on characterizing the thermal inactivation of Salmonella in dry cereals and to develop methods for control of pathogens in Hispanic cheeses. Experimental work was performed to study the parameters of bacterial killing of Salmonella dry cereals. A series of experimental trials of "queso fresco" a popular type of Hispanic cheese were conducted to identify antimicrobial ingredients that can inhibit the growth of pathogenic bacteria that included Listeria monocytogenes, Salmonella and Escherichia coli O157:H7. The findings regarding one of the objectives of this project (Develop or improve methods for control or elimination of pathogens in pre-and post harvest environments) were presented as a poster at the 2010 American Society for Microbiology annual meeting, May 23-27, San Diego, CA. One M. S. student was guided and mentored and two undergraduate students were taught experimental research techniques. Events: researchers attended the American Society for Microbiology annual meeting in San Diego, CA to present the most relevant findings of the inhibition of pathogenic bacteria in Hispanic cheeses. Dissemination: Researchers discussed their findings with audiences as well as distributed copies of the poster to attendees. PARTICIPANTS: PI: Francisco Diez-Gonzalez; Graduate Research Assistants: Camila Gadotti and Matt Chick. Undergraduate Students: Laura Nelson and Kyle Mulvey. TARGET AUDIENCES: Dairy and dry foods industries as well as the scientific community interested in food safety. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Change in knowledge: Objective 2: Using three different Salmonella serovars, Typhimurium, Tennessee and Agona, the rates of bacterial killing as D-value (time needed to reduce bacterial populations by 90%) were determined in toasted oats cereal at 0.1, 0.33 and 0.53 values of water activity. At 0.53 water activity, the D-values at 65C and 80C ranged from 153 to 207 min and from 4.3 to 6.2 min, respectively among the three serovars. D-values at 80C increased 4-fold when the water activity was reduced to 0.33. All serovars became more resistant to heat as the water activity decreased, and at 0.1, D-values greater to 135 min were measured at 85C. The Z-values for the three serovars at 0.33 and 0.53 were approximately 10C, but this value increased from 20 to 50% if the serovars were tested at 0.1 water activity. These findings suggest that Salmonella becomes more resistant to heat in drier foods. Objective 3: Batches of queso fresco were inoculated with mixtures of 4 log CFU/g of different L. monocytogenes or Salmonella strains/serovars and treated with several combinations of nisin (N) from 0.4 to 0.5 g/kg, and caprylic acid (CA) from 0.3 to 3.3 g/kg. Twenty five strains of L. monocytogenes and 6 serovars of Salmonella were tested. In addition to N and CA, several volatile compounds were also used against Salmonella. Inoculated queso fresco samples were stored at 4 or 8C for 3 weeks and the bacterial count was determined every other day by direct plating onto PALCAM or Bismuth Sulfate agar. All N/CA combinations were effective to decrease survival and growth of all L. monocytogenes and caused at least 4 log CFU/g reductions in final counts compared to controls. The levels of most strain mixtures were markedly reduced immediately after treatment and their numbers remained below 3 log CFU/g during the experiments. The combination of N/CA was only effective to reduce more than 2 logs CFU/g Salmonella if the CA concentration was greater than 2 g/kg. Treatments that included cinnamaldehyde (1 to 3 g/kg) reduced Salmonella counts almost 3 logs CFU/g. The combination of N and CA was effective to inhibit all L. monocytogenes strains in queso fresco at concentrations within regulatory levels (0.5 and 0.4 g/kg, respectively). The inhibition of Salmonella by this combination was only observed at concentrations of CA that would have a negative impact on sensory characteristics. The use of additional GRAS compounds such as cinnamaldehyde may provide an alternative to control Salmonella in queso fresco.

Publications

  • Poster: Gadotti, C., Diez-Gonzalez, F. and Nelson, L. 2010. Control of pathogenic bacteria in queso fresco by using generally recognized as safe (GRAS) ingredients. ASM annual meeting, May 23-27, San Diego, CA.