Evaluation of propidium monoazide real-time PCR for enumeration of probiotic lactobacilli microencapsulated in calcium alginate beads

Benef Microbes. 2015;6(4):573-81. doi: 10.3920/BM2014.0095. Epub 2015 Feb 12.

Abstract

The aim of the study was to evaluate real-time PCR coupled with propidium monoazide (PMA) treatment for enumeration of microencapsulated probiotic lactobacilli microencapsulated in calcium alginate beads. Lactobacillus gasseri K7 (CCM 7710) and Lactobacillus delbrueckii subsp. bulgaricus (CCM 7712) were analysed by plate counting and PMA real-time PCR during storage at 4 °C for 90 days. PMA was effective in preventing PCR amplification of the target sequences of DNA released from heat-compromised bacteria. The values obtained by real-time PCR of non-treated samples were in general higher than those obtained by real-time PCR of PMA-treated samples or by plate counting, indicating the presence of sub-lethally injured cells. This study shows that plate count could not be completely replaced by culture independent method PMA real-time PCR for enumeration of probiotics, but may rather complement the well-established plate counting, providing useful information about the ratio of compromised bacteria in the samples.

Keywords: Lactobacillus; calcium alginate beads; electrostatic droplet generation; propidium monoazide; viability real-time PCR.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alginates*
  • Azides / metabolism*
  • Bacterial Load / methods*
  • Cells, Immobilized
  • Drug Carriers
  • Drug Storage
  • Glucuronic Acid
  • Hexuronic Acids
  • Lactobacillus / isolation & purification*
  • Lactobacillus / metabolism
  • Probiotics*
  • Propidium / analogs & derivatives*
  • Propidium / metabolism
  • Quality Control
  • Real-Time Polymerase Chain Reaction / methods*
  • Staining and Labeling / methods*
  • Technology, Pharmaceutical / methods
  • Temperature
  • Time Factors

Substances

  • Alginates
  • Azides
  • Drug Carriers
  • Hexuronic Acids
  • propidium monoazide
  • Propidium
  • Glucuronic Acid