2008 Annual Report 1a.Objectives (from AD-416) Develop lactic fermentation bacteria with genetic capacity to produce natural or milk-based bioactive food ingredients to improve the health-promoting, functional and bioprotective properties of foods by utilizing microbial, molecular, gene, enzyme and proteomic technologies, and evaluate production media formulated from dairy wastes, such as whey. 1b.Approach (from AD-416) Microbial and molecular biotechnology, gene, enzyme and proteomic technologies will be used to develop selected lactic fermentation and probiotic bacteria used in dairy food production with the capacity to generate bioactive peptides. Transport systems fitted with regulatory elements will be constructed to deliver genes into food-grade bacteria (streptococci, lactococci, lactobacilli), and conditions of gene transfer and stability, nutrient requirements and other growth parameters will be optimized for gene product synthesis and secretion in selected host systems. Superior cultures will be tested for productivity in fermentation media based on whey effluents of dairy manufacturing. Cell lines and their products will be evaluated in prototype food systems. 3.Progress Report Designed improved methods to transport genes of bioactive peptides with potential for health-promoting applications in foods. The genes for antimicrobial and antihypertensive peptides were transferred to chromosomal DNA in a host bacterium where gene expression would be stabilized. Gene product synthesis was optimized in Intermediate hosts before proceeding to the food-grade yogurt fermentation bacteria as final production systems. An integration vector was developed to respond to temperature shifts to allow gene transfer to the chromosome of a yogurt culture. For several generations, the newly introduced beneficial genes were stabilized in the new host as part of the genomic DNA without negative impact on the host’s metabolic functions. Research also progressed with introducing the bioactive peptide genes into yeast cells that may be used as larger-scale production systems for peptides for the enrichment of foods. The presence of the new gene constructs in yeast was confirmed and cell lines with improved level of productivity were selected. A new approach was also developed to regulate novel gene expression in food-grade bacteria. This involved the use of special gene activating elements of the food-grade peptide nisin to turn on additional genes in the DNA transport molecule including genes for natural preservatives such as the antilisterial pediocin. Strategies were designed to transfer these structures to the more stable expression in the chromosome of yogurt bacteria. Selection of food-grade yogurt fermentation bacteria was refined to allow optimum pairing of lactose non-fermenting pediococci with various yogurt cultures to produce antilisterial peptides. The theory that simple sugars present in lactose would allow the growth and bacteriocin production by pediococci was tested under defined conditions. The amount of antilisterial pediocin produced averaged over 1.5 million units per quart of milk and resulted in a 3 log reduction of listeria count. Research on natural antimicrobial peptides of yogurt starter bacteria showed thermophilin 109 to be a two-component bacteriocin and also established in thermophilin 110 the presence of 4-5 carbohydrate groups that are required for activity. Research continued on testing nutritional supplements with impact on the production of the antipediococcus peptide bacteriocin in whey-based media. Protein and peptide degrading enzymes associated with cells of lactic fermentation bacteria were responsible for the inactivation of selected antihypertensive and antimicrobial peptides with potential as food supplements. This research demonstrated the importance of strain selection and the adjustment of conditions of fermented dairy food production to minimize the impact of microbial enzymes on health-promoting peptides. This work addressed NP306 Action Plan Component 1 (Quality, Characterization, Preservation and Enhancement) Problem Area 1d (Preservation and/or Enhancement of Quality and Marketability) and Action Plan Component 2 (New Processes, New Uses, and Value-Added Foods and Biobased Products) Problem Area 2a (New Product Technology). 4.Accomplishments 1. UPTAKE OF NEW GENES IN BACTERIAL CHROMOSOME. Keeping beneficial genes functioning in microbes used in yogurt and cheese making is limited by gene instability. Scientists designed new ways to include new genes in the chromosome of food-grade yogurt bacteria where they are stably maintained. The gene uptake can be regulated by temperature shifts, resulting in the presence of beneficial genes in dairy starter cultures that may be useful in controlling blood pressure. The new technique may be adapted to conditions of yogurt manufacture and useful in the large-scale production of these health-promoting products as food ingredients. The research addresses NP306 Action Plan Component 1 (Quality, Characterization, Preservation and Enhancement) Problem Area 1d (Preservation and/or Enhancement of Quality and Marketability) and Action Plan Component 2 (New Processes, New Uses, and Value-Added Foods and Biobased Products) Problem Area 2a (New Product Technology). 2. NOVEL ANTIMICROBIAL PEPTIDES FOR FOOD PROTECTION. Constant need exists for better antimicrobial peptides produced by yogurt starter cultures for protecting foods against pathogens. Scientists isolated two types of peptides, one of which requires carbohydrate components for antimicrobial activity. Work focused on optimizing the production of a natural inhibitor by growing food grade bacteria together with yogurt cultures. Sufficient amounts of the inhibitor were produced in milk to protect against contamination by Listeria, a human pathogen, which opens new possibilities for protecting dairy foods against bacterial contamination. The research addresses NP306 Action Plan Component 1 (Quality, Characterization, Preservation and Enhancement) Problem Area 1d (Preservation and/or Enhancement of Quality and Marketability) and Action Plan Component 2 (New Processes, New Uses, and Value-Added Foods and Biobased Products) Problem Area 2a (New Product Technology). 3. FATE OF MILK-BASED BIOACTIVE PEPTIDES IN FERMENTED DAIRY FOODS. Stability of health promoting peptides in foods is essential to have the desired impact on consumer health. Scientists developed special techniques to detect breakdown products of milk-protein based antimicrobial and blood pressure controlling peptides that are produced by enzymes of starter cultures in fermented dairy foods. Results of work help in the development of manufacturing conditions that reduce or eliminate the destruction of the beneficial peptides by bacterial enzymes. The research addresses NP306 Action Plan Component 1 (Quality, Characterization, Preservation and Enhancement) Problem Area 1d (Preservation and/or Enhancement of Quality and Marketability) and Action Plan Component 2 (New Processes, New Uses, and Value-Added Foods and Biobased Products) Problem Area 2a (New Product Technology). 4. DEVELOPMENT OF YEASTS FOR PRODUCING BENEFICIAL PEPTIDES. Yeasts have not been evaluated as producers of antimicrobial and antihypertensive peptides with potential as beneficial food supplements. Scientists developed a technique that permits the uptake of new genes by yeasts. The work lays the foundation for selecting superior yeast cultures for the large-scale production of health-promoting food grade peptides that are useful in blood pressure control and can be used as value-added ingredients in dairy foods. The research addresses NP306 Action Plan Component 1 (Quality, Characterization, Preservation and Enhancement) Problem Area 1d (Preservation and/or Enhancement of Quality and Marketability) and Action Plan Component 2 (New Processes, New Uses, and Value-Added Foods and Biobased Products) Problem Area 2a (New Product Technology). 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer Number of New Commercial Licenses Executed 3 Review Publications Somkuti, G.A., Steinberg, D.H. 2007. Molecular organization of plasmid per13 in streptococcus thermophilus st113. Biotechnology Letters. 29:1991-1999. Renye Jr, J.A., Somkuti, G.A. 2008 Cloning of milk-derived bioactive peptides in streptococcus thermophilus. Biotechnology Letters. 30:723-730.