Screening and Detection of Endospore-formers in Skim Milk Powder

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Screening and Detection of Endospore-formers in
Skim Milk Powder

• Amy J. Rife
• R. Jimenez-Flores
• Final Report ARI

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Outline

• Introduction
• Objectives
• Methodology
• Results
• Conclusions
• Future Research

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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

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Introduction

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

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Why milk powder?

• Dairy products
• Baked goods
• Confectioneries
• Soups and sauces
• Mixes
• Meats
• Animal feeds

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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)

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MethodologyScreening 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

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MethodologyScreening Disc Assay
Lipid Hydrolysis Spirit Blue Agar
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MethodologyScreening Disc Assay
Casein Hydrolysis Skim Milk Agar
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MethodologyScreening Disc Assay
Starch Hydrolysis TSA 2 Starch
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MethodologyScreening - Tube assay
Lactose Fermentation
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ResultsScreening

• 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

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30 gal
Raw milk
Powdered milk
30 gal
Raw with spores
Powdered milk with spores
inoculated with approx. 107 endospores per liter
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MethodologyDetection

• 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

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Polymerase Chain Reaction
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MethodologyDetection

• 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

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Terminal Restriction Fragment Patterns (TRFP)
DNA Extraction
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ResultsDetection

• Endospores positively identified in spiked
  samples during milk powder processing run

RAW MILK S/NS ELECTOPHEROGRAM
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ResultsDetection
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ResultsDetection
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ResultsDetection

• Model strains
• Identify differentiation between strains

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ResultsDetection

• PCR with GerC primers
• Identify endospore-formers during milk powder
  processing

Positive Control
100 KB Ladder
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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

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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

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Acknowledgements

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