Reducing Salmonella in Illinois Pigs
Our specific objectives are to estimate the importance of risk factors (hazards) for infection and, among salmonella-positive farms, define the effects of transportation on salmonella prevalence at slaughter. One-hundred-twenty farms and two slaughter plants have formally agreed to participate in the project. Farms were selected from slaughter-plant supplier lists, marketing groups, veterinary clinics and transportation companies to be broadly representative of Midwest pig production.

We have developed and pretested a 12-page survey of farm characteristics. We have refined our tissue collection and microbiologic cultural techniques to enhance sensitivity, while allowing for the testing of large numbers of pigs and farms. Samples of 1 gram of lymph node appeared to be inadequate to detect infection sensitively using our enrichment methods. Finally, we have collected samples and surveys from 40 farms and initiated retesting of farms to document repeatability of salmonella status.

Peter Bahnson
H. Fred Troutt
Veterinary Clinical Medicine
College of Veterinary Medicine
University of Illinois at Urbana-Champaign

C-FAR Fellows in Food Safety
The objective of this project is to provide support to graduate students engaged in research on food safety. Currently, stipends are provided to four students. In general, the stipends are supplemented with additional funds provided by the laboratory mentor. In addition, one student was provided funds to attend a scientific meeting. As with the stipends, matching travel funds from other sources were required.

The students receiving stipends are working on the following research projects: 1) the effect of transportation-related stress on the shedding of Salmonella typhimurium in swine, 2) the epidemiology of Toxoplasmosis gondii in swine and the effect of vaccinating cats on reduction of carrier pigs, 3) the epidemiology of salmonella on farms and risk factors associated with on farm carriage, and 4) the mechanisms promoting the long-term colonization of swine with S. typhimurium. A theme found in each research project is the interdisciplinary and collaborative nature of the projects, a requirement for the C-FAR Fellows. A graduate seminar on food safety is being developed.

Ecology of Salmonella Transmission
The purpose of this study is to identify the reservoirs of salmonella on swine farms. The initial phase of the study involves recruitment of farms with high prevalence of salmonella infection in marketed finishing pigs. The goal is to recruit 150 swine farms in Illinois, each having a minimum of 100 sows, from which 10 farms with high salmonella prevalence will be chosen for intensive on-farm epidemiologic investigation. Recruitment letters were sent to pork producers in Illinois to participate in this study.

To date, marketed finishing pigs from 50 farms have been sampled at slaughter plants. For each farm, from the cohort of marketed pigs on a given date, 30 pigs are selected at random. Lymph nodes are harvested from each pig. Laboratory analysis consists of culturing for identification of salmonella. Two grams of lymph node are sampled from each pig, and samples from six pigs are pooled for culture. Pooled samples are cultured in a tetrathionate and Rappaport-enriched broth. To date, 15 farms (30%) are positive for salmonella.

Ronald W. Weigel
Richard E. Isaacson
Veterinary Pathobiology
College of Veterinary Medicine
University of Illinois at Urbana-Champaign

Functional Foods Research and Education
C-FAR funding for the project "Stimulating Functional Foods Research and Education at the University of Illinois" has been used in FY 1996-97 to support the infrastructure of the Functional Foods for Health (FFH) program as proposed. In February 1997, the director of the FFH Program, Clare Hasler, was awarded a tenure-track position as assistant professor of nutrition in the Department of Food Science and Human Nutrition in the College of Agricultural, Consumer and Environmental Sciences. C-FAR funds are being utilized to fund that position, which is 50% outreach. During the last fiscal year, the FFH Program Industrial Affiliates Program expanded from 11 to 19 members. Due to the expanding Industrial Affiliates Program, we have been able to make $50,000 in seed grants available to FFH Program faculty. These funds will be available (following a competitive review process) on September 1. As the Affiliates Program expands, so will the FFH seed grant program. FFH faculty membership has expanded to 84 members—19 faculty having joined since July 1996. All but two of those faculty are from the Urbana-Champaign (UIUC) campus. Intense efforts were directed last year at expanding the cross-campus nature of the program at UIUC. Proposals have been reviewed by the vice-chancellors at the University of Illinois-Chicago and UIUC, who will be approaching Vice President Sylvia Manning for funding for this effort.

Clare M. Hasler
Food Science and Human Nutrition
College of Agricultural, Consumer and Environmental Sciences
University of Illinois at Urbana-Champaign

Vegetables That Prevent Cancer
We have just completed the first year of this two-year project, in which we proposed to grow 60 cruciferous vegetable cultivars and test them through chemical analysis for potential cancer chemopreventative components. A subset would be tested through bioassay for their ability to increase mouse enzyme levels for detoxification of carcinogens and other foreign chemicals in the body.

We then proposed to perform a genetic analysis of the differences between biologically effective and ineffective cultivars, with the expectation that this would lead to development of cultivars with improved potency as cancer chemopreventative agents, through genetic engineering and traditional breeding techniques.

The first year was successful, in that 72 cultivars of Brassica oleracea were grown and analyzed, and found to differ substantially in their glucosinolate and antioxidant vitamin contents. This variation is sufficiently broad to ensure that genetic manipulation of specific products is an attainable goal. In addition, we have determined glucosinolate metabolism potential (myrosinase enzyme values) in all cultivars, partially isolated one of the potent components (Crambene) that is not available commercially for bioassay, and established the mouse as a model to evaluate enhancement of the enzymes for detoxifying carcinogens. This past spring we displayed our work to the Illinois Legislature in Springfield with other C-FAR grant holders.

Making Soy Protein Tasty
Our objective is to optimize high-soy protein products for American consumers from prototypes developed for an Ohio Soybean Council project. Formulations for bread containing soy protein isolate (SPI, 15.6% of wheat flour weight), 10% vital wheat gluten, sodium stearoyl lactylate and diacetyl tartaric esters of monoglycerides (DATEM) and varying quantities of ascorbic acid were selected for testing. All additives chosen are FDA approved and used as stabilizers and/or dough conditioners in commercial breads. A descriptive sensory panel was trained. They evaluated breads to create sensory profiles of the product's characteristics and determine differences among formulations.

Volume, color and texture of the breads were measured and correlated with sensory attributes. Extruded breakfast cereals were prepared and formulations optimized to yield eight cereals containing 20% SPI. Extrusion, toasting and coating conditions were determined. Cereals with corn flour, wheat bran and SPI were prepared. Descriptive and consumer tests were completed, and selected products were optimized.

Soy yogurt optimization is continuing. Conditions for making Illinois soymilk base were standardized and effects of microorganism cultures, flavorings (sugar, vanilla, strawberry), calcium fortification and stabilizers were determined by descriptive and consumer panels. Consumers liked strawberry yogurts very much (7.8 /9.0), which is comparable to scores for dairy yogurts. Flavor volatiles produced in soy yogurts are being determined.

Use of Soy Protein to Lower Blood Cholesterol
This is a clinical intervention trial designed to determine the optimal dose of soy protein required to lower blood cholesterol in men with moderately elevated cholesterol levels (220 to 300 mg/dl). Coronary heart disease (CHD) is the leading cause of disease and death in the U.S. today. It is generally accepted that, for every 1% reduction in blood cholesterol, CHD risk is reduced by 3 to 4%. Dietary modification is one method by which individuals may favorably modify their cholesterol levels. Although the effect of soy protein on blood cholesterol has been investigated in more than 45 human clinical studies to date, no dose-response study has been conducted to determine how much soy protein one needs to eat to favorably influence cholesterol levels.

This information will be essential prior to making public health recommendations regarding the use of soy protein in CHD prevention in humans. Our overall goal is to have all of the data analyzed by early December in order to submit an abstract at that time for the annual Experimental Biology Meetings in April 1998. The results from this study will also be presented by the principal investigator at the Seventh Annual Retreat of the Functional Foods for Health Program in May 1998.

Clare M. Hasler
Ronald M. Weigel
Food Science and Human Nutrition
College of Agricultural, Consumer and Environmental Sciences
University of Illinois at Urbana-Champaign

Novel Assay for Zinc Status
A novel method to analyze nutrient status is being evaluated. Specifically, gas chromatography/mass spectrometry (GC/MS) is being used to evaluate metabolite content in urine samples, with the aim being to detect specific metabolites or a metabolite profile that can be utilized to assess zinc deficiency. During the first year, progress has consisted of utilization of the rat as a model. Diets have been prepared to be adequate or deficient in zinc. Urine has been collected and analyzed by GC/MS. At least four metabolites appear to have promise as markers for zinc deficiency. Also, we have tested the ability of zinc-deficient rats to clear a compound loaded into the diet at 1%, w/w. We have tested all 20 amino acids and thymine. We have found that some of these compounds are difficult for zinc-deficient rats to metabolize and appear in the urine in high concentrations. They may be useful as test compounds to assess zinc deficiency in humans. Further studies will extend these tests to humans.

Soy and Cardiovascular Health
Cardiovascular disease is the major killer in the United States. This project looks at the individual and combined effects of soy protein and phytoestrogens on several cardiovascular risk factors. Stable insulin-sensitive cell lines are being cultured and subjected to varying doses of genistein, diadzein or estrogen (as a control) with and without insulin. Preliminary data have been analyzed, initial animal studies have been conducted, and the data from the in vivo and in vitro studies are being studied.

Soy Protein Isolate for Patients on Peritoneal Dialysis
Sara Anderson and Trish Welchprepare a soy-protein health drink. This project was conducted to investigate the efficacy of providing isolated soy protein to individuals receiving peritoneal dialysis as a method of ameliorating protein malnutrition. In individuals (n=26) consuming isolated soy protein, indicators of protein status remained stable for one year. In addition, levels of total cholesterol and LDL-cholesterol decreased while HDL-cholesterol levels increased slightly. Only three subjects discontinued the study due to intolerance to the product. Overall satisfaction with the soy product was very good.

Alternatives to Antibiotics in Animals
The site of binding (receptor) for many bacteria to mucosal epithelium resembles the chemical structure of specific sugars. A new method for processing endometrial tissue that gives more quantitative reflection of the intrauterine environment has been developed. It is rapid and much simplified from the microscopic inspections formerly employed and shows a good deal of diagnostic promise in practical settings. Further testing and refinement of the technique is ongoing.

S. S. King
E. M. Carnevale
Animal Science, Food and Nutrition
Southern Illinois University-Carbondale

Risk for Resistant Pathogens in Cattle
A primary public concern is the frequent use of feed-grade, nontherapeutic antibiotics in livestock production. These antibiotics help prevent disease, allowing the animal to produce more quickly, efficiently and economically. Consumers, however, question the human health aspects of using antibiotics to prevent rather than treat specific disease. Low levels of antibiotics fed for a long period of time create a potential for the development of antibiotic-resistant strains of pathogenic bacteria. Because some livestock pathogens also affect humans, this causes a concern of how effectively disease can be treated in the human population.

In this study, feedlot cattle were fed either a tetracycline, bambermycin, probiotic (nonantibiotic), or control diets. Fecal samples were collected to determine resistance of gut bacteria (specifically, E. coli) to common human treatment antibiotics, for example, amikacin, ampicillin, carbenicillin, ciprofloxacin, gentamicin, and tetracycline. Fecal samples were used because fecal contamination of meat during processing is a source of bacterial contamination of food products.

Based on the results of this study, sub-therapeutic feeding of antibiotics to livestock does not increase the risk of developing antibiotic resistance in gut bacteria. Gut bacteria did increase antibiotic resistance over time, but this occurred in the nonantibiotic control and probiotic groups as well as the antibiotic bambermycin and tetracycline groups. There was, however, the greatest increase in the development of tetracycline resistance among gut bacteria collected from the tetracycline-fed cattle. This helps support the restriction of feeding sub-therapeutic levels of the same antibiotics used to treat disease.

Teresa L. North
Department of Agriculture
Kenneth C. Keudell
Department of Biology
Western Illinois University

Food Practices of Children
Over 6 million food-borne illness cases and 8,000 deaths occur each year, costing over $5 billion, according to the Centers for Disease Control. Everyone from the producer to the consumer has a role in food safety. Consumers tend to think of producers, food manufacturers and restaurants as the most likely place for food safety problems to occur and homes the least likely place. More than 80 percent of the outbreaks occur in the home and go unreported. One purpose of this research was to survey fourth- and fifth-grade children about their current food practices.

During 1997, more than 700 fourth- and fifth-grade students from west-central Illinois completed a questionnaire containing 15 statements about food practices. Fifty-three percent of the students surveyed were fourth graders. Over half of the students surveyed were males.

Results indicate that 75 percent of the children wash their hands before eating. But only 62 percent wash their hands before fixing a sandwich. Fourth graders, especially females, tend to wash their hands the most. Less healthful food practices reported by the children were eating raw cookie dough and cake batter, licking their fingers as they eat and tasting food that is being prepared. Over 70 percent of the students reported eating raw cookie dough and cake batter. Almost half of the students would eat or drink after a family member, but less than 3 percent would eat or drink after another student or eat raw or rare meat or ground beef, or raw fish. Less than 10 percent of the students ate unrefrigerated leftover sandwiches or pudding.

External Competitive Grant Program

Understanding Physical Aging of Foods
One aspect of the polymer science approach as applied to the study of food materials that needed further exploration was the problem of physical aging. In synthetic polymers, physical aging involves small-scale molecular motions below the glass transition, Tg (a temperature in which molecular mobility changes from immobile to mobile), which causes changes in the physical properties of the polymer, such as density, volume and hardness, as a function of time. Physical aging is important because changes in the physical properties of a material significantly affect its usefulness and stability.

In the original description of this work, two research strategies were proposed: 1) to probe the molecular-level behavior of carbohydrate materials (maltose, sucrose, and trehalose) as they undergo physical aging and 2) to examine the changes in the physical properties of aged carbohydrate materials. Research conducted using C-FAR funds during FY97 addressed the first of two strategies. Amy Lammert examined the molecular-level behavior of maltose glasses aged from 5 to 10,000 minutes at different temperatures below Tg (Tg - 10 degrees C and Tg - 20 degrees C) by two techniques, standard differential scanning calorimetry (SDSC) and modulated differential scanning calorimetry (MDSC). Ratchapong Wungtanagorn will examine the molecular level changes of the sucrose, trehalose and sugar blends at different temperatures below the Tg that result from physical aging.

Preventing Food Spoilage
Consumers demand minimally processed, additive-free, high-quality products with natural taste. This prompted development of non-thermal processing methods such as high-pressure processing (HPP). The goal of this research program is to evaluate the consumer safety issues associated with foods processed by HPP. The effect of HPP on the inactivation of three bacterial spores (Clostridium botulinum type E spores, Bacillus subtilis, ATCC 6633 strain and B. stearothermophilus FS1518 strain) at different temperatures and holding times was investigated.

Temperatures below 35°C for any pressurization (Maximum 828 MPa) had no effect on the reduction of viable C. botulinum spores. Log unit reduction of viable spores of Alaska and Beluga increased with increase of temperatures from 40° to 60°C and pressurization at 828 Mpa for 5 min. The reduction of the B. subtilis spores increased with an increase in the holding time for combinations of temperature (above 40°C) and pressure (275-828 MPa) treatments. Complete inactivation was obtained at 828 MPa for temperatures at and above 60°C, with holding times of 10 minutes or more. A combination of processing temperatures less than 60°C, and maximum pressurization (that is, 828 MPa) had no effect on the reduction of viable B. stearothermophilus spore samples.

The knowledge gained from this project will lead to the development of many safe, high-quality, value-added food products to meet the Illinois consumer's needs and preferences and enhance the long-term viability and competitiveness of the Illinois food industry within the global economy.

Reliable Milk Discard Times
This project is a two-year study investigating the utility of population pharmacokinetics for determination of milk discard times after antimicrobial therapy for mastitis. As stated in the original grant application, final data analysis cannot be initiated until all data are available. It was anticipated that data collection would require two years and that data analysis would require a further six months. Data collection began in January 1997. Current progress in sample and data collection indicates that these estimates are accurate.

Clostridium Perfringes in Meats
Food safety is a significant concern both in Illinois and nationwide. Clostridium perfringens is a common cause of food poisoning and ranked among the top five causes of food-borne illness in the United States. This bacterium is an inhabitant of the animal intestinal tract and can easily contaminate ground meats during processing. Traditional diagnostic methods and identification for this bacterium take several days and require various media and reagents. Currently, there is no rapid method available.

We generated monoclonal and poluclonal antibodies that can recognize a toxin produced by this bacterium once it is active to grow. We developed a direct ELISA (enzyme-linked immunosorbent assay) and a competitive ELISA, which can detect trace amounts of the bacterial toxin as an indicator for the presence of the bacterium. These assays are highly sensitive and can measure parts per million toxin, which is equivalent to less than 100 cells, in four to six hours. Such technical development provided the possibility for the future use of this system for rapid detection of the bacterium in meats and food products.

Food Safety in Small Meat-Processing Facilities
Site visits were used to assess the current operation and needs for the Hazard Analysis of Critical Control Point (HACCP) Plan, under development. Initially, a walk-through was used to assess facilities and operations and to cross-check the floor plan against blue prints. Product formulas were converted to forms required by HACCP directives. Then the products were divided into eight categories, based on similar processes used to produce them. For each product category, a separate HACCP plan was developed. The inherent hazards in the process were determined, and the level of control at that step was noted. Each processing step was then evaluated using a "decision tree" to determine whether or not it included a "critical control point." Critical control points were described in detail; this included setting critical limit, methods of assessing the degree of compliance with those critical limits, and documentation to demonstrate compliance.

The model HACCP Plan was evaluated by an USDA-FSIS representative. The revised materials were used as the basis of a multi-state HACCP workshop in which processors were trained in the methods for developing HACCP Plans for their own facilities. The HACCP Employee Training Manual should be finished by September. Employees at Thrushwood Farms Quality Meats will use the HACCP Plan, and then microbiological data will be collected over a four-month period to assess whether or not the use of the HACCP Plan reduces microbial load and presence of pathogens.

HACCP Training in Food Service
Researchers are developing Hazard Analysis of Critical Control Point training modules for food service workers; conducting training sessions; and evaluating efficacy of the modules. Some dissemination and demonstration of the modules takes place at professional meetings.

H. Ashraf
T. Welch
T. Girard
J. Corker
Animal Science, Food and Nutrition
Southern Illinois University-Carbondale

Youth Nutrition Education
The objective of this project was to evaluate the impact of an intensive nutrition, food safety, and food preparation educational intervention with youth ages 8 to 12. Evidence suggests that dietary behavior is established in childhood. Poor dietary behaviors may lead to chronic diseases or health conditions during adulthood. Therefore, an early adoption of good dietary behavior is important.

The goals of this study were to develop, implement and test the effectiveness of a youth nutrition, food preparation and food safety curriculum designed to modify and improve the dietary behavior of children. Intervention included half-day sessions held over one week during the summer. The sessions were conducted by trained workers and included hands-on activities and food preparation methods. Pre-, post- and followup tests were used to evaluate the effectiveness. Analyses of the evaluation tools were done with frequency distribution, paired t-test and McNemar's test. Classes were conducted at 15 sites throughout southern Illinois. The 227 participants ranged in age from 8 to 12 years old. The average class size was 14 (range 7 to 24). The subjects were primarily from low-income households.

Following the intervention, a higher percentage of the subjects reported to follow safe food practices, to prepare food items and to select nutritious foods according to the food guide pyramid (p<.001). The children continued to report these behaviors after three months (p<.05). The average pretest score was 4.72 (out of 9) and improved significantly on the post-test to 7.01 (p<.001, paired t-test). After three months, the average score on the follow-up test (n=132) was 7.12, significantly higher than the pretest (p<.001, paired t-test). Results illustrate the effectiveness of intervention focusing on food safety, food preparation and food selection in youth. This study supports intervention strategies for young children for the promotion of healthful food behaviors.

Value-Added Apple Products
In order to develop an apple plant with increased value, a genetically engineered apple plant is currently being developed that will produce fruit that has a medicinal value. The fruit will serve as an edible human vaccine against Respiratory Synctial Virus (RSV) disease, a serious disease of infants and the elderly that sometimes can be fatal. A series of gene constructs has been assembled in a primary plasmid pBI121; these constructs carry the F-protein coding sequence of RSV and the CaMV 35S promoter, which drives the expression of this F-protein gene into the apple plant. Apple leaves as well as tomato cotyledons (tomato is used as a model system because it produces fruit rapidly) were transformed with these gene constructs via Agrobacterium tumefacines-mediated transfer.

Following a series of gene transfer experiments, more than 50 potentially transformed organogenic calli were selected, and shoots developing from these explants were induced. Shoot regenerants are currently being analyzed to confirm the presence of the F-protein gene, using polymerase chain reaction (PCR) analysis.

In other experiments, we have been working on identifying fruit-specific promoters from the apple in order to use these promoters in our gene constructs so that the F-protein expression is targeted mainly to the fruit tissue. One such promoter has been identified and is currently being characterized. It then will be used in new gene constructs and later transferred into tomato and apple.

Breast Cancer Prevention By Soybean Isoflavones
The major objective of this project is to determine how the soybean isoflavones genistein and daidzein can protect normal human breast cancer cells against the development of cancer. Both genistein and daidzein inhibited the proliferation of normal and cancerous human breast cells. Genistein was more effective than daidzein in inhibiting cell growth and was more effective in inhibiting normal breast cell division compared to cancer cell division. In this regard, genistein inhibited cell proliferation by 50% at a concentration of 18uM in normal breast cells and at a concentration of 50uM in cancerous breast cells. Also, cultures of normal human breast cells treated with genistein exhibited less damage to their cell DNA due to cancer chemicals compared to cells not treated with genistein.

The fact that genistein can slow down cell division of normal cells is significant, because it is believed that cancer risk is higher in more rapidly dividing tissues. In addition, it appears that genistein treatment may enhance the capacity of these normal breast cells to protect against chemical carcinogens that can initiate the cancer process. Lastly, the higher sensitivity of normal human breast cells to the antiproliferative action of genistein suggests that this may be an excellent system to study how genistein can retard cell division.

Phytoestrogen and Cardiovascular Health: Friend or Foe?
Soy products contain phytoestrogens that have physiological effects on humans and animals. The goal of this project is to investigate the effects of differing doses of soy phytoestrogens on cardiovascular health and to establish the optimal level of phytoestrogens for human consumption.

William J. Banz
T. Winters
Food and Nutrition
Southern Illinois University-Carbondale