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INVESTIGATOR: Ivy, R.

PERFORMING INSTITUTION:
CORNELL UNIVERSITY
ITHACA, NEW YORK 14853

A COMPREHENSIVE GENOMIC APPROACH TO DEVELOP A DIAGNOSTIC FOR HEAT-RESISTANT SPOREFORMERS

NON-TECHNICAL SUMMARY: Spoilage, due to the activity of microorganisms, accounts for an estimated 25% of global food loss. There is, therefore, a need for new methods to track and reduce the prevalence of spoilage organisms in food systems, including dairy foods. The presence of cold-thriving bacteria capable of forming pasteurization-resistant endospores represents a significant barrier to extending the shelf-life of fluid milk products. While the genera Bacillus and Paenibacillus are the major sporeformer presence in pasteurized fluid milk products, Paenibacillus predominates late in shelf-life of milk. Therefore, in the absence of post-pasteurization contamination, growth of Paenibacillus spp. likely represents the predominant spoilage threat to fluid milk processors. Though most Paenibacillus strains grow at refrigeration temperatures, some do not. Therefore, there is a need for new methods to specifically detect the presence of cold-thriving Paenibacillus spores in refrigerated dairy products. To this end, we propose to (i) use next generation methods to sequence the genomes of predominant Paenibacillus subtypes and identify potential gene targets (ii) screen milk isolates for these spoilage-associated genes to link them to cold spoilage ability, and (iii) use this information to develop a rapid DNA-based assay to specifically detect spores from spoilage-associated Paenibacillus strains in milk and dairy ingredients. This methodology, applied to dairy and other foods, will contribute to new understanding of the ecology of spoilage organisms and lead to new supply chain recommendations for reducing the presence of sporeformers in food systems, which will increase food availability and prolong food acceptability.

OBJECTIVES: We propose to use next generation genomic methods to design a diagnostic for psychrotolerant Paenibacillus spores in raw milk and dry ingredients. The long-term goal of this project is to develop rapid identification systems for psychrotolerant sporeformers spoilage organisms in food systems. This methodology could be widely implemented to detect spoilage organisms in raw ingredients to (i) identify of common routes of entry of spoilage organisms, (ii) optimize raw ingredient sourcing and handling, and (iii) predict the shelf-life of the product. The specific objectives of this proposed project are: OBJECTIVE 1 - SEQUENCE THE GENOMES OF PREDOMINANT PAENIBACILLUS SUBTYPES TO IDENTIFY POTENTIAL DETECTION ASSAY TARGETS: To identify genes that contribute to sporeformer growth at refrigeration temperatures and milk spoilage, we will use high throughput, deep-sequencing technologies to sequence the genomes of representatives from four prominent milk-associated Paenibacillus subtypes, including both cold-sensitive and cold-resistant Paenibacillus isolates. Comparative genomic analysis will then be used to identify candidate genes involved in cold-growth or proteolysis. OBJECTIVE 2 - SCREEN AN ISOLATE COLLECTION FOR SPOILAGE-ASSOCIATED LOCI TO ESTABLISH LINKS BETWEEN COLD SPOILAGE PHENOTYPES AND SPOILAGE-ASSOCIATED GENOTYPES: To establish links between spoilage phenotypes and spoilage associated genotypes, putative cold growth or proteolysis genes will be confirmed in representative isolates from prominent Paenibacillus ATs. Isogenenic deletion mutants will be created for putative cold growth genes. The growth of these deletion mutants at 6C in skim milk broth will be compared to the parent strain to confirm contribution of the gene to cold growth. Candidate protease genes will be sequenced in representative isolates from the prominent milk associated Paenibacillus strains. The protease activity of each genotype will be assayed to identify highly proteolytic genotypes. OBJECTIVE 3 - DEVELOP A REAL-TIME PCR-BASED ASSAY TO DETECT SPORES FROM SPOILAGE-ASSOCIATED PAENIBACILLUS SUBTYPES IN MILK AND DAIRY INGREDIENTS: A rapid DNA-based assay will be designed to detect endospores of prominent Paenibacillus cold-spoilage genotypes in dairy products. Taqman real-time PCR primers and probes will be designed to detect Paenibacillus cold spoilage and protease genes. The assay will be used to test samples of raw milk and commercially available dairy products and ingredients for the presence of psychrotolerant Paenibacillus spores.