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Science Platforms > RPBIOA Completed Biotechnology Projects BT-0003-6/91 Project Leaders: M.L. Tortorello, NCFST/FDA, D. Stewart, NCFST/FDA Project Collaborators: H.J. Castillo, R&F Laboratories, L. Restaino, R&F Laboratories Project Title: Rapid Detection and Identification of Microbes in Foods Rapid screening of beef for the presence of Escherichia coli 0157:H7 was shown to be feasible using a 10 hour enrichment in modified buffered peptone water and the antibody-direct epifluorescent technique (Ab-DEFT). The Ab-DEFT involved membrane filtration, fluorescent antibody straining and epifluorescence microscopy and was accomplished in less than 1 hour. The procedure allowed detection of the pathogen artificially inoculated into beef patties at 0.1 CFU/g. The 10 hour nonselective enrichment broth supported rapid growth, which provided sufficient numbers of cells for a positive determination by the Ab-DEFT after scanning no more than 10 microscope fields using a 40X objective lens. Immunomagnetic separation using anti-E. Coli 0157 Dynabeads7 was shown to be a sensitive method that can be used to confirm presumptively positive cultures within 24 hours. The ease and rapidity of the Ab-DEFT can provide a substantial time and cost savings to the beef industry for E. coli 0157:H7 testing. BT-0006-10/94 Project Leader: P. J. Hesketh, UIC Project Collaborator: J. R. Stetter, NCFST/IIT Project Title: A Biosensor for Detecting Bacterial Toxins in Foods Food Industry needs a rapid, sensitive, selective and reliable method for the detection of toxins to ensure food safety. The objective of this research was to introduce a novel immunobiosensor technology to use in a simple hand-held type device to detect foodborne toxins. The detection of toxins by direct sensing of the specific binding reaction of the toxin (antigen) with its specific partner (antibody) is a rapid and low cost method. This is a collaborative research effort between IIT, UIC and FDA/NCFST. The NCFST project was renewed for three years to focus on applications of the sensor in food. Funding was received from the Whitaker Foundation for a period of three years. The immediate goal of the Whitaker program was to develop an understanding of the sensing mechanism of ultrathin platinum film conductimetric immunobiosensors. Stable Pt film sensors were fabricated on a Ti sublayer by sputtering onto a silica substrate. Surface cleaning and annealing of the wafers before and after deposition of the ultra thin Pt film resulted in films that had excellent adhesion for up to 24 hours in PBS solution. Initial measurements indicated the impedance responded specifically to SEB antigen in the concentration range of 0.4 to 10 ng/mL. The sensitivity of each sensor varied considerably. Monofunctional silane GOP showed a more reproducible response. Five publications resulted from this research. BT-0007-10/95 Project Leader: M.L. Tortorello, NCFST/FDA Project Title: Rapid Detection and Identification of Microbes in Food Improved methods for the microbiological analysis of foods are needed, especially quantitative methods. More efficient ways to enumerate microbial cells are essential for gathering data on incidence, exposure, and infective dose, for use in quantitative risk assessment strategies to enhance food safety. The objectives of this project included testing of molecular probes for specific enumeration of bacteria by direct epifluorescence microscopy and comparison to conventional standard methods of enumeration in food systems for sensitivity and accuracy. A fluorescent oligonucleotide probe complementary to a published 16S ribosomal RNA sequence of Escherichia coli was tested in situ in hybridizations on membrane filters. The oligonucleotide-direct epifluorescent filter technique (oligo-DEFT) was developed and involved a 2-hour hybridization with the probe after membrane filtration of water, beverages and sprouts homogenates. Enumeration was performed by semi-automated counting of cells by microscopy and image analysis. The probe showed reactivity to several genera of the Enterobacteriaceae, and was not specific for E. coli. Heat-killing of E. coli cells at 60° C did not destroy the ability of the probe to hybridize to the cells. Natural coliform populations in aquarium water were countable after a 90 minute incubation of the filter on nutrient agar (Aresuscitation@) before the hybridization step. Natural coliforms in sprouts were directly countable without the resuscitation step. Oligo-DEFT counts were correlated with standard coliform counts by membrane filtration or plating with conventional selective media. BT-0008-10/95 Project Leader: T.J. FU, NCFST/FDA Project Title: Safety Considerations for Plant Cell Cultures The objective of this research was to (1)develop expertise in plant cell culture techniques (2) to determine how stress conditions affect formation of detrimental compounds and (3) how these can be removed. Plant cell and tissue culture (PCTC) offers an alternative source for the controlled and consistent production of Anatural@ food ingredients. PCTC has been used to produce various products including flavors, colorants, essential oils, and more recently, "nutraceuticals". Many of the cell lines used to produce food ingredients come from plant families known to produce a variety of toxins or anti-nutritional agents including alkaloids, phytoestrogens, tannins, etc. Tomato and potato cell cultures were chosen as model systems for this study. Both tomato and potato are among the most consumed foods in the world and their cell cultures have been frequently used as model systems to study somaclonal variations and effects of processing conditions on secondary metabolite production. The HPLC method of Friedman et al. (1994) was adopted with modifications to determine the amount of a-tomatine in tomatoes and tomato cell cultures. The extraction and clean-up procedures were effective in removing interfering peaks. a-Tomatine was not detected in either the callus or the cell suspension cultures of Lycopersicon esculentum cv. Roma VFNT-Cherry which have been in culture for more than 10 years. Changes in lighting conditions did not activate the production of a-tomatine in these cultures. Attempts were made to initiate potato and tomato cell cultures. Potato callus culture was successfully established from explants of the tubers of Solanum tuberosum cv. Yukon Gold and grown in a modified MS medium containing 10 mg/L of NAA. BT-0010-10/96 Project Leader: T.J. Fu, NCFST/FDA Project Title: Effects of Processing Conditions on Natural Toxins Formation in Plant Cell and Tissue Culture Plant secondary metabolites are important sources of many food ingredients and disease-preventive phytochemicals. Plant cell and tissue culture offers an alternative way for controlled production of these products. The goal of this research was to determine the effect of tissue culture process on natural toxin formation and to determine whether commonly used yield improvement processes will result in higher levels of toxin. Tomato cell culture was chosen as a model system. Tomato cell culture has potential commercial applications for the production of lycopene and tomato flavor. In the mean time, tomato belongs to the family of Solanaceae known to produce toxic glycoalkaloids. The major glycoalkaloid in tomato, a-tomatine, is embryotoxic and has been shown to cause rapid death of mice. The objectives of the study were to initiate cell cultures of different tomato cultivars, to determine the level of a-tomatine in these cultures, and determine the effect of elicitation on a-tomatine production, as well as to develop analytical methods for analyzing glycoalkaloids in potato cell cultures (Solanum tuberosum cv.Yukon Gold) and determine the level of glycoalkaloids in these cultures. The results of this study indicated that tomato (or potato) cell cultures lacking morphological differentiation may not contain the natural toxin, a-tomatine (or chaconine), and elicitation may not activate the production of these glycoalkaloids in cell cultures. BT-0011-10/96 Project Leader: Steven M. Gendel Project Title: Moleclar fingerprinting Technology for Food Safety Molecular fingerprinting has emerged as an essential tool for food microbiology because it allows the identification and typing of microbial strains with a degree of precision not previously possible. This precision makes molecular fingerprinting valuable in applications such as environmental monitoring, HACCP validation, risk assessment, and epidemiological investigations. This technology can also be used to differentiate microbial strains that are highly virulent or resistant to processing. This project has made the NCFST a leader in the application of microbial fingerprinting to food safety, and will help industry and government develop a more sophisticated understanding of how microbes behave and spread in foods and food processing environments. This will translate directly into more effective and efficient strategies for microbial control, risk assessment, and food safety assurance. BT-0012-10/96 Project Leader: S.E. Keller, NCFST/FDA Project Title: Biodegradation of Fumonisin Fumonisins are mycotoxins produced primarily by Fusaria Species on corn that have been associated with human esophageal cancer where corn is a large part of the staple diet. There are no tolerance levels set for the presence of fumonisins in foods, however, the FDA may consider guidelines in the future. Little is currently known about physiological or biochemical control of fumonisin production. NCFST research has provided several insights into the control mechanisms for fumonisin production such as pH, and nitrogen levels. It has also been discovered, as a result of this research, that fumonisin can be biologically degraded. The primary objective of this research was to examine degradative pathways of fumonisin and determine the role of such pathways in the contamination or decontamination of corn and corn products. The first enzyme involved in this degradation appeared to be an esterase that hydrolyzes FB, to its polyketide backbone. Concentrated crude cell extract showed several faint bands on SDS gels. The apparent pH optimum for this enzyme is 5.0. The apparent temperature optimum for this enzyme is 50°C although stability is reduced. Higher temperatures cause a rapid loss of activity. The enzyme passes through a 100,000 mw filter but not through a 30,000 mw filter. The project was terminated with no further research planned. BT-0013-10/97 Project Leader: T.J. Fu, NCFST/FDA Project Title: Comparative Digestion and Acid Stability of Food Allergens and Non-allergenic Proteins As more foods derived from genetically modified plants are entering the marketplace, the allergenicity of the introduced proteins needs to be assessed to ensure food safety. Digestion and acid stability has been proposed as one of the factors to be considered when assessing the potential allergenicity of transgenic proteins of non-food origin. Information on the comparative digestion and acid stability of food allergens and non-allergenic proteins is crucial for such an assessment. In this work, we compared the digestion and acid stability of a number of food allergens and non-allergenic proteins. We also examined whether allergens possess higher stability than non-allergenic proteins of similar cellular functions and whether there is a correlation between the function of a protein and its stability. The stability of a group of storage proteins, plant lectins, contractile proteins, and enzymes was measured in a standard simulated gastric fluid (SGF), a standard simulated intestinal fluid (SIF) and an acidic salt solution (pH 1.2). The results showed that the stability of food allergens in SGF and SIF may be more, the same or less than that of non-allergenic proteins. There was no clear relationship between functionality of proteins and their SGF or SIF digestion stability. Therefore, it may not be possible to predict the digestion or acid stability of a certain protein based on its cellular function. This is the first study in the literature that compares the SGF, SIF and acid stability of food allergens and non-allergenic proteins in consistent and well-controlled conditions. The data collected in this study will not only help the safety assessment of transgenic proteins but will also be very useful to the food industry in general in understanding the physiochemical properties of food allergens. This one-year project resulted in three poster presentations at scientific meetings and one manuscript currently in preparation. BT-PR-0006-10/93 Project Leader: S.E. Keller, NCFST/FDA and T.M. Sullivan,NCFST/FDA Project Title: Production of Fumonisin B in Shake Flask cultures of Fusarium Proliferatum M5991 Fumonisins are mycotoxins produced primarily by Fusarium moniliforme and Fusarium proliferatum in corn. Mechanisms controlling the production of these mycotoxins in culture and field conditions have not been well established. In this study, a defined liquid medium was developed which allowed fumonisin B1 (FB1) production of up to approximately 1000 ppm by Fusarium proliferatum M5991 in shake flasks. Previously, such high concentrations could be achieved only in bioreactors. The use of this medium in shake flasks allowed rapid examination of multiple factors controlling FB1 production. Factors found to influence FB1 production include pH, glucose, and nitrogen levels. BT-0011-10/96 Project Title: Riboprint Technology for Food Safety Project Collaborators: S. Gendel, NCFST/FDA; M. Sethi, Qualicon This project used the Qualicon Riboprinter Microbial Characterization System to characterize strains of microbial pathogens found in foods and food processing environments, study the emergence and spread of new strains (particularly antibiotic resistant strains), develop procedures for sharing molecular fingerprinting data between laboratories, and develop guidelines for the use of molecular fingerprinting technology in applications such hazard identification, risk assessment, microbial challenge studies. and HACCP validation. At this time, we have analyzed over 1450 microbial strains, primarily isolates of Listeria monocytogenes, HUS-associated non-0157 E coli, Vibrio parahaemolyticus, and Clostridium botulinum. We have continued to expand and analyze our large collection of L. monocytogenes isolates. We are also screening all of these isolates for antibiotic resistance, as well as analyzing antibiotic resistant isolates obtained from other labs. One major goal of the Food Safety Initiative in the next year is to conduct a risk assessment for V. parahaemolyticus. We have begun to assemble a library of fingerprint patterns for V. parahaemolyticus. Our initial work has shown that Riboprinting can be used readily differentiate strains of this species. Although the O157:H7 serotype of E. coli is most frequently associated with HUS in the US, other serotypes have been associated with foodborne outbreaks in other countries. We have been able to show that Riboprinting can be used to subtype several of these serotypes, particularly O111 and O104. This work will be extended during the next year to more isolates, other serotypes, and to isolates of these serotypes associated with infantile diarrhea. One publication is in submission to a journal, one is in clearance, and one is in preparation. BT-0017-10/98 Project Title: Affect of Washing and/or Sanitizing Methods on the Microbial Ecology and Safety of Fresh Fruit and Vegetables Project Leader: Sue Keller, NCFST/FDA Project Collaborators: G. Fleischman, NCFST/FDA, L. Jackson, NCFST/FDA, L. Xu, Praxair, R. Merker, FDA/CFSAN, S. Chirtel, FDA/CFSAN, K. Taylor, El Dorado County Dept. of Agriculture, H. Tan, University of California, Davis The objects of this project were to determine the most effective treatment to reduces the risk of foodborne illness from fresh produce and juice, to examine the correlation between incoming microbial populations and final product quality/safety, and to examine the relationship of the natural microbial populations to general food safety and the efficacy of any sanitation methods. Determining the efficacy of a surface treatment on surface contamination required intentional inoculation of apples. Laboratory results indicated little or no effect with chlorine or ozone. Laboratory effects with surface heat treatment indicated all vegetative cells located on the surface of the fruit could be destroyed. Additional experiments were executed in a small apple cider processing facility. Results showed greater than the 1.5 log reductions obtained with natural microflora, but were not as large as laboratory experiments would have predicted. Moreover, reductions on apples reflected by reductions on pomace, were generally larger than for juice. Additional experimentation targeted sanitation procedures in the cider facility to explain the poor performance of the surface heat treatment. Results suggested that the surface treatment of apples in this type of facilities are inadequate to control pathogens. Since surface heat treatment will result in substantial visible damage to the surface of apples, it would be applicable only for fruit used for juice. Additional means of decontamination were examined. While ozone and chlorine were ineffective, 40% v/v ethanol significantly reduced surface populations of E. coli O157:H7 (3.5 to 4 log). Contact times of approximately 5 minutes were required for maximal effect. Although ethanol treatment cannot be used in a commercial setting, an ethanol rinse may have application for home use in disinfecting produce surfaces. BT-0018-10/98 Project Title: Separation/Concentration of Pathogens from Large Volumes of Fresh Produce Project Leader: T.J. Fu, NCFST/FDA Project Collaborators: K. Reinke, NCFST/IIT, T. Krupinski, NCFST/IIT, O. Vanpelt, NCFST/IIT, K. Lin, NCFST/IIT, L. Restaino, R&F Laboratories, B. Staek, IIT
BT-0018-10/99 Project Title: Process Development for Separation/Concentration of Pathogens from Large Volumes of Fresh Produce Project Leader: T.J. Fu, NCFST/FDA Several recent foodborne illness outbreaks associated with fresh produce have concerns about the microbiological safety of fresh vegetables and fruit juices. Effective monitoring of pathogens during processing is critical in ensuring the safety of these foods. Pathogen detection in foods, however, is challenging due to the low numbers of these organisms present in foods. It has been shown in the literature that tap water washing resulted in the removal of about 90 % of the microbial load from prepared salad (Adams et al., 1989). If this is also true for pathogens, the majority of the pathogens can be separated from fresh vegetables by simply rinsing the vegetables in water. A rapid sampling process capable of separating and concentrating pathogens from large volumes of fresh produce to allow the direct use of existing rapid methods for in-process monitoring of pathogens in fresh produce was developed. BT-0019-10/00 Project Title: Effects of Sub-Lethal Stress on Virulence as Related to Ribotype Lineage of Listeria monocytogenes Project Collaborators: Steve Gendel, NCFST/FDA; Peter Slade, NCFST/IIT; Paul Hall, Kraft Foods This project studied how the virulence of Listeria monocytogenes is distributed within phylogenetic divisions as determined by molecular subtyping methods, and identified the sub-lethal values of certain stress conditions, comparing the similarities and differences between strains belonging to different lineages. BT-0020-10/00 Project Title: Methods for Determining Pathogen Injury and Inactivation in Food Processing Project Project Leader: Mary Lou Tortorello, NCFST/FDA Project Collaborators: Karl Reineke, NCFST/IIT, Diane Stewart, NCFST/FDA, Larry Restaino, R & F Laboratories This project concerned the development and comparison of methods for determining sublethal injury and inactivation of E. coli O157:H7 and Listeria monocytogenes in food processing. Conventional culture, direct cellular probes, and expression of molecular constituents were compared to assess cell injury and inactivation after treatment with heat and other processing methods. Methods investigated in this project may be broadly applicable to a variety of food safety areas, e.g. rapid detection of pathogens and effects of stresses on heightenedvirulence and increased resistance to processing. |
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