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.
Animal Health Component
Research Effort Categories
Goals / 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.
OBJECTIVE 1: Our group has used partial rpoB gene sequencing to subtype over 700 Bacillus and over 700 Paenibacillus milk isolates. We will compare the genome of an isolate representing Paenibacillus allelic type (AT) 159, which does not exhibit cold growth, to genomes of isolates representing the other prominent ATs (i.e., AT15, AT23, and AT45), which all exhibit cold growth (CG), which will likely reveal candidate cold growth genes. Sequences will be compared to online pathway databases to identify candidate proteolysis genes. Prior to genome sequencing, phenotypes of the chosen isolates will be confirmed using previously described methods. Briefly, for CG, spores will be inoculated into ultra high temperature (UHT) milk and samples will be incubated at 6C for 28 days. Proteolytic activity will be measured by plating pure cultures on skim milk agar and quantifying clearance zones, which indicate proteolytic activity. Genomic DNA preparations will be sequenced on the Illumina/Solexa Genome analyzer. De novo assembly will be done using Velvet. Annotation each genome sequence will be obtained using the RAST server. Sequence data will be queried against the BioCyc and PMAP pathway databases to identify putative CG and proteolysis genes. OBJECTIVE 2: We anticipate creating deletion mutants in Paenibacillus AT45 for 3-4 candidate CG genes using previously described methods. CG ability of each mutant will be determined (see Obj. 1). Candidate protease genes (2 to 3 each) will be sequenced in 50-100 isolates, and a proteolysis assay (see Obj. 1) will be conducted for each of the genotypes to confirm highly proteolytic genotypes. Gene sequences and phenotypic data will be deposited into an online database, "SpoilageTracker," which will serve as a publicly available resource for the study of the ecology of spoilage organisms. OBJECTIVE 3: Taqman primers and probe will be designed to detect consensus (based on alignments of prominent Paenibacillus ATs) CG and proteolysis genes. Standard PCR will be used to test the sensitivity and inclusivity of the primers. The Taqman probe will be tested on 50-70 representatives of prominent Paenibacillus and Bacillus ATs. Parameters will be further optimized for amplification efficiency and signal strength. For application of the assay in milk, spore preparations of 2-3 representatives of Paenibacillus ATs and 2-3 representatives of Bacillus ATs will be inoculated into UHT milk at various levels. Total DNA will be isolated from 1 ml of milk using a kit. If necessary, samples will be enriched for Paenibacillus using previously described methods. At least 200 raw and 100 pasteurized milk samples and 100 ingredient samples from various dairy producers will be tested. Prior to testing, samples will be heated at 80C for 12 min to eliminate vegetative cells. After testing, milk samples will be plated on plate count agar with or without X-gal (most Paenibacillus ATs express β-galactosidase, whereas most Bacillus ATs do not). rpoB/16S rDNA sequencing will confirm positive colonies are Paenibacillus.