Download Medicine "Lactobacterin" is a microbial mass of antagonistically

Investigation of Lactobacilli properties
E.V.Bulycheva, E.I.Korotkova, O.A.Voronova, A.A.Kustova
National Research Tomsk Polytechnic University, Russia, 634050, Lenina st. 30
E-mail: [email protected]
Key words: lactobacilli, luminescence, the luminescence spectrum, the viability of the
bacterial cell, the identification of the components.
Introduction
Lactic acid bacteria (Lactobacilli) are the microorganisms which are the main part of the normal
human intestinal micro flora. They have saccharolytic type of metabolism [1,2]. They have good
antagonistic activity against pathogenic microorganisms [3]. Lactobacilli are used not only in
medicine, as a means to improve of digestion, but also in food industry as a useful additive to
yogurts, milk, etc. Use of lactic acid bacteria as a test object for analysis of natural water toxicity is
a new approach of these microorganisms application. Elaboration of the methodology of water
toxicity determination is complicated and time consuming process, so, the first stage of this research
was investigation of the Lactobacilli culture and their metabolites by fluorescence analysis.
Obligatory condition of lactobacilli application as a test object is their viability, so, the aim of this
work was to investigate the vital activity of the Lactobacilli on the example of the medicine
"Lactobacterin". For this research was applied luminescent method that is based on the study of the
metabolism products luminescence spectra of the Lactodacilli. These metabolites can be the
markers of the bacterial viability.
Materials and methods
Reagents
Medicine "Lactobacterin" is a microbial mass of antagonistically active living lactobacilli strains
Lactobacillus plantarum 8P-A3, or L.plantarum 38 or L.fermentum 90T-C4 or L.fermentum 39,
lyophilized in the culture medium with the addition of protective sucrose or gelatin sucrose-gelatinlactose medium.
Equipment
All investigations were done on a spectrofluorometer "Fluorat-02 Panorama", produced by
"Lumex" St. - Petersburg. This device allows you to perform various investigations in a wide range
of wavelengths. As a certified analyzer spectrofluorometer "Panorama" is used for the analytical
control of the environment, health monitoring and process control.
Methods
According to instruction of the medicine "Lactobacterin" suspension was prepared by dissolving the
contents of the vial in water, 5 ml of water per 1 dose of medicine. One vial contains 5 doses. For
dissolving was taken 25 ml of water at room temperature. After dissolution of the medicine we had
homogeneous suspension wits light yellow colour.
The luminescence spectrum was obtained by synchronous scan with shift of the registration
wavelength from excitation wavelength of 20 nm.
Results and discussion
Luminescence spectrum is a graphic dependence between signal intensity of the sample and
wavelength [4]. Figure 1 shows luminescence spectrum of the medicine "Lactobacterin"
Figure 1. Luminescence spectrum of the medicine "Lactobacterin"
It is known that the peak on the wavelength 340 nm is NADH (reduced nicotinamide adenine
dinucleotide). This substance is a participant of the Lactobacilli metabolism. Its presence shows that
the bacteria is alive, so for the next study was selected wavelength 340 nm. To investigate changes
of signal intensity depending on the additives were prepared some water samples with addition of
different amounts of the Lactobacilli suspension. Figure 2 shows calibration dependence between
signal intensity and content of the bacteria in the sample.
Signal intensity, r.u
2
1,8
y = 1,6827x + 0,0423
1,6
R2 = 0,9949
1,4
1,2
1
0,8
0,6
0,4
0,2
0
0
0,2
0,4
0,6
0,8
1
1,2
Quantity of Lactobacilli, ml
Figure 2. Calibration dependence between signal intensity and content of the bacteria in the sample.
For the Lactobacilli viability investigation determination of the signals nature in the wavelength
from 380 nm to 440 nm, luminescence spectra were obtained immediately after preparation of the
suspension, and after 2 hours (Fig. 3). Luminescence spectrum is different, than previous. It is
probably due to the fact that for the analysis was taken medicine from another manufacturer, but
with the same content.
Peaks in the spectrum differ only in intensity and doesn’t differ in the position. We can use this
medicine for the next study.
Figure.3. Luminescence spectra of the medicine immediately after suspension preparation (1), and
after 2 hours (2)
It can be concluded that the signal at the wavelengths from 380 nm to 440 nm are from components
of culture medium. Luminescence spectrum of the medicine was obtained. It can be divided into
three signals areas: 280 – 300 nm - nucleic acid, 340 nm – NADH, 360 – 440 nm – components of
culture medium. Vital activity of Lactobacilli was investigated. The changing of the peak height at
340 nm, that matches NADH, with time, can be a marker of the bacterial cell metabolism and
therefore on its vital activity. In this report it is shown, that after 2 hours bacterial cells are alive,
and can be used as a test object.
This study was supported by state task “Science” № 1.1310.2014
Literature references
1.
U.O.Shulpekova, Lactic acid bacteria: their role in the regulation of gut motility, J.RJGGI. 3
(2010) 1-3.
2.
G.G.Onischenko,
V.A.Alyoshkin,
S.S.Afanasev,
V.V.Pospelova
mmunobiological
preparations and perspectives of their application in infectology, Moscow, 2002.
3.
N.A.Glushanova, Biological properties of Lactobacilli, J. Bulletin of Siberian Medicine. 4
(2003) 2-3.
4.
M.A.Konstantinova-Shlezinger, Luminescence analysis, Moscow, 1961.