Genome Sequencing of Kocuria varians Strain 80, Isolated from Starter Culture for Dry Sausage.

The genome of the meat starter culture strain Kocuria varians 80 was sequenced and assembled into a single 2.82-Mb chromosome. Genome sequence comparison of K. varians strain 80 and previously sequenced strains revealed predicted proteomic differences that can impact its technological properties.

Application of EvaGreen for the assessment of Listeria monocytogenes АТСС 13932 cell viability using flow cytometry.

Determination of eukaryotic cell viability using flow cytometry is widespread and based on the use of fluorescent dyes such as SYTO, DAPI, SYBR, PI, and SYTOX. For many years, traditional microbiological methods have been used to successfully analyze prokaryotic cells, but the application of flow cytometry should be considered because it provides an opportunity for quantitative assessment. A combination of SYTO 9 or SYBR green and PI has been used successfully. DNA-binding dyes such as SYTO 9, SYBR green, and EvaGreen are used in qPCR. The aim of this study was to assess the feasibility of EvaGreen to determine the viability of Listeria monocytogenes АТСС 13932 cells using flow cytometry. RNA from Escherichia coli ATCC 25922 was isolated using the MagNA Pure LC RNA Isolation Kit-High Performance (Roche, Germany) according to the kit instructions on MagNA Pure LC® 2.0 (Roche, Switzerland). Chicken DNA was isolated using the Sorb-GMO-B kit (Syntol CJSC, Russia) according to the kit instructions. RNA from E. coli ATCC 25922, chicken DNA, a positive control, and a negative control of L. monocytogenes АТСС 13932 were stained with EvaGreen and analyzed on the Guava EasyCyte flow cytometer (Merck Millipore, Germany). Chicken DNA demonstrated both green and red fluorescence, while E. coli RNA displayed only red fluorescence. While the positive L. monocytogenes АТСС 13932 control and chicken DNA demonstrated similar fluorescence properties, the negative control showed a localization similar to that observed with E. coli RNA. Degraded ssDNA and RNA stained with EvaGreen demonstrated red fluorescence. Although EvaGreen is a class III dye, we observed fluorescence of live L. monocytogenes АТСС 13932 cells in the positive control stained with EvaGreen. The observed phenomenon was linked to the solution composition. It is necessary to repeat this analysis with various solution compositions as well as a wide range of both Gram-positive and Gram-negative bacteria to determine the effects on cell envelope permeability of EvaGreen.

Multiplex Real-Time PCR with HRM for Detection of Lactobacillus sakei and Lactobacillus curvatus in Food Samples.

Optimization of fermentation processes requires monitoring the species composition of starter cultures and their growth during fermentation. Most starter cultures contain closely related species. Nowadays, high-resolution melting (HRM) analysis is extensively used for multiplex identification of closely related species. In the present paper, we applied real-time polymerase chain reaction (PCR) with HRM analysis for the detection and differentiation of Lactobacillus sakei and L. curvatus. A primer pair was selected for the site of the rpoA gene of Lactobacillus spp. Eleven starter cultures and fifteen fermented sausages with a known bacterial composition were successfully tested using real-time PCR with HRM analysis with the developed primer pair.