1 / 24

Screening and Detection of Endospore-formers in Skim Milk Powder

salena
Download Presentation

Screening and Detection of Endospore-formers in Skim Milk Powder

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

    1. Screening and Detection of Endospore-formers in Skim Milk Powder Amy J. Rife R. Jimenez-Flores Final Report ARI

    2. Outline Introduction Objectives Methodology Results Conclusions Future Research

    3. Introduction Some bacteria i.e. Bacillus sp. are able to form an endospore They are able to withstand extremes: Temperature pH Desiccation Radiation They have the ability to germinate and cause detrimental characteristics to the final product Hydrolyze lipids, proteins, starch Ferment lactose

    4. There is a need for methods of detection Current methods Standard Plate Counts Labor intensive Time consuming Poor detection limits Poor reproducibility

    5. Why milk powder? Dairy products Baked goods Confectioneries Soups and sauces Mixes Meats Animal feeds

    6. Objectives Screen Bacillus skim milk powder isolates for enzymatic activity Lipid hydrolysis Casein hydrolysis Starch hydrolysis Lactose fermentation Develop molecular methods of detection Polymerase Chain Reaction (PCR) Terminal Restriction Fragment Patterns (TRFP)

    7. Methodology Screening – DPTC skim milk powder endospore library 60 Bacillus isolates were obtained from skim milk powder from California (Barycki, 1998) Isolates identified by FAME, RAPD-PCR, and biochemical analysis (Bellenson, 1998) 15 Bacillus isolates obtained from the ATCC

    8. Methodology Screening – Disc Assay

    9. Methodology Screening – Disc Assay

    10. Methodology Screening – Disc Assay

    11. Methodology Screening - Tube assay

    12. Results Screening 75 isolate strains 48 hydrolyzed lipids 27 hydrolyzed casein 22 hydrolyzed starch 38 fermented lactose On average, each strain was positive for 1.8 of the 4 enzymes 5 strains were positive for all Model strains

    14. Methodology Detection DNA extraction methods optimized Vegetative cells Endospores PCR methods optimized for model strains Germination gene (GerC3) primers (Pitesky, 2000) Fwd: 5’-GAT GTC ATT GAT GAT-3’ Rev: 5’-CWC CWC CAY CYG GTT TYC C-3’ Temperature profiles

    15. Polymerase Chain Reaction

    16. Methodology Detection TRFP DNA extraction PCR - 16s rDNA 6-FAM® labeled primers Fwd: 5’-GTA TTA CCG CGG CTG CTG G-3’ Rev: 5’-GCY TAA CAC ATG CAA GTC GA-3’ PCR product clean-up Enzyme digestion HhaI DpnII HaeIII Ethanol precipitation 310 ABI Genetic Analyzer

    17. Terminal Restriction Fragment Patterns (TRFP)

    18. Results Detection Endospores positively identified in spiked samples during milk powder processing run

    19. Results Detection

    20. Results Detection

    21. Results Detection Model strains Identify differentiation between strains

    22. Results Detection PCR with GerC primers Identify endospore-formers during milk powder processing

    23. Conclusions TRFPs and PCR are good methods of endospore detection throughout a low heat milk powder processing run TRFPs are useful for microbial ecology studies in milk powder processing Using TRFPs in combination with a selected gene, we can differentiate between endospore species and strains

    24. Future Research Microbial ecology studies using TRFPs for low, medium and high heat milk powder processing runs. Sequencing the GerC gene for development of specific primers Using a labeled GerC primer for TRFPs

    25. Acknowledgements Agricultural Research Initiative Dairy Management Inc. California Dairy Research Foundation Friends and family

More Related