title: Quantification of viable bacterial starter cultures of Virgibacillus sp. and Tetragenococcus halophilus in fish sauce fermentation by real-time quantitative PCR.
abstract: Real-time quantitative polymerase chain reaction (qPCR) methods were developed for the quantification of Virgibacillus sp. SK37 and Tetragenococcus halophilus MS33, which were added as starter cultures in fish sauce fermentation. The PCR assays were coupled with propidium monoazide (PMA) treatment of samples to selectively quantify viable cells and integrated with exogenous recombinant Escherichia coli cells to control variabilities in analysis procedures. The qPCR methods showed species-specificity for both Virgibacillus halodenitrificans and T. halophilus as evaluated using 6 reference strains and 28 strains of bacteria isolated from fish sauce fermentation. The qPCR efficiencies were 101.1% for V. halodenitrificans and 90.2% for T. halophilus. The quantification limits of the assays were 10(3) CFU/mL and 10(2) CFU/mL in fish sauce samples with linear correlations over 4 Logs for V. halodenitrificans and T. halophilus, respectively. The matrix effect was not observed when evaluated using fish sauce samples fermented for 1-6 months. The developed PMA-qPCR methods were successfully applied to monitor changes of Virgibacillus sp. SK37 and T. halophilus MS33 in a mackerel fish sauce fermentation model where culture-dependent techniques failed to quantify the starter cultures. The results demonstrated the usability of the methods as practical tools for monitoring the starter cultures in fish sauce fermentation.
authors.affiliation: School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakon Ratchasima, 30000, Thailand; Microbial Cultures Research Center for Food and Bioplastics Production, Suranaree University of Technology, Thailand. Electronic address: paeng888@hotmail.com.
authors.forename: Natteewan
authors.initials: N
authors.lastname: Udomsil
authors.affiliation: Agriculture and Food Laboratory, Laboratory Services Division, University of Guelph, Ontario, Canada. Electronic address: schen@uoguelph.ca.
authors.forename: Shu
authors.initials: S
authors.lastname: Chen
authors.affiliation: School of Microbiology, Institute of Science, Suranaree University of Technology, NakonRatchasima, 30000, Thailand; Microbial Cultures Research Center for Food and Bioplastics Production, Suranaree University of Technology, Thailand. Electronic address: sureelak@sut.ac.th.
authors.forename: Sureelak
authors.initials: S
authors.lastname: Rodtong
authors.affiliation: School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakon Ratchasima, 30000, Thailand; Microbial Cultures Research Center for Food and Bioplastics Production, Suranaree University of Technology, Thailand. Electronic address: jirawat@sut.ac.th.
authors.forename: Jirawat
authors.initials: J
authors.lastname: Yongsawatdigul
mesh_terms: D000818:Animals; D056569:Enterococcaceae / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D005396:Fish Products / Q000382:microbiology*; D005399:Fishes; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D058679:Virgibacillus / Q000235:genetics / Q000378:metabolism*
keywords: Fish sauce fermentation; PMA treatment; Starter culture; T. halophilus MS33; V. halodenitrificans; Virgibacillus sp. SK37; qPCR