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Section 3. General biology
Section 3. General biology
Tkhruni Flora, Laboratory of Microbial Preparations, Scientific and Production Center "Armbiotechnology", NAS, Yerevan, Republic of Armenia Arstamyan Lilya, Artsakh Scientific Center” SNPO, Stepanakert, Nagorno Karabakh Republic Scientific and Production Center "Armbiotechnology”, NAS, Yerevan, Republic of Armenia Balabekyan Tsovinar, Laboratory of Microbial Preparations, Scientific and Production Center "Armbiotechnology”, NAS, Yerevan, Republic of Armenia Karapetyan Kristina, Laboratory of Microbial Preparations, Scientific and Production Center "Armbiotechnology”, NAS, Yerevan, Republic of Armenia Email: floranub,@mail.ru
Antibacterial activity of single cells in the population of lactic acid bacteria
Abstract: The new endemic strains of lactic acid bacteria (LAB) were isolated from different samples of dairy products manufactured in various rural households of Armenia and Nagorno Kharabakh Republic. It was shown that population of LAB cells have variability on the expression of antimicrobial activity. Purification of cell-free cultural broth confirmed effectiveness of the used method for producing partially purified antimicrobial preparation (AMP) with high antimicrobial activity.
Keywords: antibacterial activity, random screening of single colonies of LAB
Introduction
The isolation and characterization of new strains of lactic acid bacteria from various biotopes took a great interest during last decades. Lactic acid bacteria (LAB) are widely used as starter cultures in dairy, meat, and vegetable fermentations. Lactobacillus species play a crucial role in foodstuffs because of their fermentative ability and beneficial influence on nutritional, taste, and shelf-life characteristics. One major reason for their use is the wide range of antimicrobial substances that they are able
to produce which efficiently contribute to the preservation of the fermented products. From these antimicrobial substances, bacteriocins are now being explored for their potential utility in human and animal health applications, food biopreservation and others [6, 335-351]. Production of bacteriocin by optimizing the culture conditions such as temperature, effects of pH, incubation period and substrate concentration, composition of the culture medium, and general microbial growth conditions (in vitro as well as in natural fermentations) on
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Antibacterial activity of single cells in the population of lactic acid bacteria
maximal bacteriocin production, combination of different methods for purification etc. [8, 291297]. Many protocols have been implemented, permitting separation of the fractions according to their size and/or physicochemical properties, such as salt precipitation (e. g. ammonium sulphate), acid precipitation, adsorption-desorption or by extraction with organic solvents [2, 281-305]. A rapid and two-step procedure suitable for both small- and large-scale purification of pediocin-like bacteriocins and other cationic peptides have been reported [11, 952-956].
It is known that the population of microorganisms is heterogeneous and within one population (one strain) may be contained cells with varying physiological and biochemical properties. For example, it was shown, that LAB colonies of Enterococcus faecium strain may differ from each other by the content of the plasmids. Biofilm growth causes increased average plasmid copy number as well as increased copy number heterogeneity in Enterococcus faecium cells. Single cell analysis of strains carrying two different plasmids suggested that the increase in plasmid copy number associated with biofilm growth was restricted to a subpopulation of biofilm cells [3, 612-625].
It has been suggested that the population variability on the expression of antimicrobial activity for the bacteriocin producer strain may also be heterogeneous. The aim of this work was to study the variability of the antimicrobial activity of single colonies of the endemic LAB cultures isolated from traditional lactic acid products, and increasing the yield of production of bacteriocins by using of random method for micro auto-selection of single colonies.
Materials and methods
Bacterial cultures and media. Lactobacillus rhamnosus BTK-2012, Lactobacillusplantarum BTK-66 and Enterococcus faecium BTK- 64 were isolated from different dairy products from rural households of Armenia and Streptococcus sp. K 13, Lactococcus sp. M 44 LAB strains — from Nagorno Kharabakh Republic. LAB strains were deposited with the Department of Center of Microbial Depository (CMD) under SPC “Armbiotechnology” NAS of Armenia [9, № 2924 А].
Test culture. To determine antimicrobial properties of CFC broth and AMP, conditionally pathogenic Gram-negative Salmonella typhimurium G 38 and Gram-positive Bacillus subtilis G17-89 from the depository CMD were used. Test cultures were grown on solid Nutrient agar (Himedia, India) at 37o С 16-18 h.
Random auto selection of active colonies.
Morphologically identical single colonies have been obtained from each LAB strain. Single colonies were individually inoculated in liquid MRS medium and incubated at 37oC for 48 hours. The culture broth was collected after 48 hrs and centrifuged at 6,000 g, during 20 min. To exclude inhibition effect by organic acids, the pH of obtained supernatants was adjusted to 6.0 with 4% NaOH, and their activity was tested. For further works, the active colonies with average antimicrobial activity 100 AU/ml for 48 hours in liquid MRS were selected. These colonies were then used as the parent line of pure cultures and for the inoculum preparation for the obtaining ofbacteriocins. The pure cultures ofLAB strains were maintained as frozen stocks at -20 оС in the MRS broth containing 40% glycerol. Before use, they were transferred twice into the appropriate medium and incubated during 48 hours in temperature controlled conditions at 37o С.
Inoculum preparation and obtaining of cell-free culture broth. Single colonies were grown in five ml of MRS broth (37o С, 24 h) and when were transferred into 100 ml-Erlenmeyer’s flask containing 50 ml of MRS broth and incubated overnight at 37o С for 24 hours in the thermostat. 50 ml of the obtained inoculum was transferred into 800 ml of MRS broth in 1L Erlenmeyer s flask and grown at 37o С for 48 hour in the thermostat. At the end of culture growth cell concentration achieved (7±2) х 10 8 CFU/ml (of titration) and pH reduced to 3.5-4.2. Culture broth was collected after 24 hrs and centrifuged at 6,000 g during 20 min. Antimicrobial activity of cell free culture broth (CFC), concentrated 5-fold, was determined at pH=6,0.
Detection of antimicrobial activity. The spot-on-lawn method and well diffusion (AWD) assay on the test culture plated in the solid medium was
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Section 3. General biology
applied. For spot-on-lawn method, suspension of the test culture in the Nutrient broth (containing 10 6 CFU/ml) speeded across the surface and aliquots of investigated samples of 20pl were applied above the test culture using micropipette. Plates remained at 4oC for 1-2 hours to promote diffusion of samples. Then plates were incubated in temperature-controlled conditions at 30 oC during 24-48 h. For the agar well diffusion (AWD) method pre-poured nutrient agar plates were overlain with 0.1 ml suspension of the test culture (containing 10 6 CFU/ml). Wells of 8 mm in diameter were made into the agar and 100 pi of the samples were placed into each well. Antimicrobial activity was assessed by measuring the size of the inhibition zone (diameter) of test culture growth (0, mm) after 24 h incubation in thermostat at 30o C. The antimicrobial activity is expressed in arbitrary units (AU/ml) [7, 95-108].
Purification of antibacterial compounds by ion-exchange chromatography. Culture broth was purified by combination of adsorptiondesorption [12, 3355-3359] and ion-exchange chromatography method [1, № 2925 А]. After the ion-exchange chromatography the obtained fractions were fractioned by gel filtration method, conducted on Sephadex G-25 (Superfine) equilibrated with 0.1M NaCl. Elution was carried
out with 0.1 M NaCl, or with distilled water. Antimicrobial activity was examined in each of the 2-ml fractions. Fractions having bactericidal properties were collected and vacuum evaporated at temperature 50-55o C, residual pressure
0.01 MPa. The content of DM of partially purified antimicrobial preparation (AMP) reached to approximately 30%. Antibacterial activity of AMP determined as described above.
Results
More than 200 cultures of LAB were isolated from various samples of fermented dairy products from rural households of different regions of Armenia and Nagorno Kharabakh Republic. Colonies of isolated LAB differed by color, shape and size when inoculated in agar media (MRS and/or hydrolyzed milk). The morphological study results showed that the bacteria isolated from samples were mainly represented by rod-shaped bacteria (30%) and cocci (70%). For preliminary screening ofstrains, cell free cultural broths, obtained after growth of LAB were studied for their ability to inhibit the test culture growth at pH = 6,0-6,5 to exclude impact of organic acids [5, 39-85]. The morphological and physiological features, probiotic properties, specific growth rate and antimicrobial activity of LAB were studied [4; 10, 109-115].
Figure 1. The growth inhibition of the test culture B.subtilis G 17-89 by different inoculums of L.rhamnosus BTK-2012 strain (spot-on-lawn test and AWD methods, 100 pL)
1- Inoculum from the colony with high bactericial activity (in the form of lysis);
2- Inoculum from the colony with low bactericial activity
It is known that the population ofmicroorganisms is strain may vary in their antibacterial activity. For this
heterogeneous and within one population (one purpose, we used the method of random gradual auto
strain) may contain cells with different biochemical selection ofcolonies (the standard method in genetics)
properties. A hypothesis was made that colonies ofone according to antimicrobial activity. More 100 colonies
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Antibacterial activity of single cells in the population of lactic acid bacteria
of each strain were individually inoculated in liquid MRS medium and incubated at 37°C for 48 hours. The obtained CFC broths were studied for their ability to inhibit the test culture growth at pH = 6,0-6,5. Results are shown, that among 100 colonies, supernatants of only 50-70 colonies at pH=6.0 have possessed the test culture growth inhibition property. Their activity varied from 20 to 60 AU/ml when tested on B.subtilis G17-89 test cultures. For further work, 6-10 colonies of strains with average antimicrobial activity 60 AU/ml were selected. After re-cultivation of these colonies, the colonies with antimicrobial activity more than 100 AU/ml were selected. These colonies were then used as the parent line for the inoculum in the production ofbacteriocin. It should be noted that after serial passages (>50) of these colonies in liquid MRS their antimicrobial activity did not change.
The Figure 1 shows the growth inhibition of the test culture B.subtilis G 17-89 under the influence of inoculums from initial and selected colonies at pH= 6.0.
The photo clearly demonstrates differences in antibacterial activity of selected colonies (on the example of L.rhamnosus BTK-20I2 strain) by used two methods. For the further works the inoculum 1 obtained from colony with high antimicrobial activity has been used.
Similar results were obtained by us on the Ent. faecium BTK- 64, L.plantarum BTK- 66, Streptococcus sp. K 13, Lactococcus sp. M 44 strains. This demonstrates that the LAB cell population varies in their antimicrobial activity as well (no similar data of increasing of bacteriocin yield from CFC broth are available in the literature). Results are shown in Table 1.
Table 1. - Percentage of LAB strain colonies with maximal antimicrobial activity (test culture B.subtilis G 17-89)
LAB strains Total number of colonies (N) Number of colonies with max. activity (n) Percentage, (%)
L.rhamnosus BTK-2012 100.0 80.0 80.0
Ent. faecium BTK- 64 72.0 36.0 83.3
L.plantarum BTK- 66 48.0 38.0 79.1
Streptococcus sp. K13 50.0 40.0 80.0
Lactococcus sp. M 44 50.0 35.0 70.0
According to these data, among the studied strains number of colonies with maximum antimicrobial activity are virtually the same (Xavg=80.8%).
Antimicrobial activity of CFC broth (pH 6.0), obtained by use of inoculums of selected colonies
As it seen from given results, cell-free culture broth obtained with initial strains are characterized by lower antimicrobial activity and lower protein yield as compared to these factors of L.rhamnosus BTK- 2012 grown from the inoculums obtained after colony-selection method of the studied strain.
Because LAB bacteriocins are secreted into the growth medium and many bacteriocins are not produced in high amounts by the producer strain, it is very important to concentrate the supernatant
and colonies of initial L.rhamnosus BTK- 2012 have been studied for production of bacteriocins. Antimicrobial activity was analyzed with test culture B.subtilis G 17-89 and S.typhimurium G-38 as the test culture. Results shown in Table 2.
that contains the antimicrobial substance at the very initial steps. Different methodologies have been implemented, permitting separation of the fractions according to their size and/or physicochemical properties.
For purification of the CFC broth and obtaining of AMPs both inoculums of selected colonies and colonies of initial strain were used. The results of experiments are shown on example of L.rhamnosus BTK-2012 strain and presented in Table 3.
Table 2. - Characteristics of CFC broth prepared of different inoculums of L.rhamnosus BTK-2012
CFC broth Volume, ml Activity, AU/ml Total activity, AU Protein, mg
Inoculum of initial colonies 3,000 30 0.9x10 5 1,548
Inoculum of selected colonies 3,000 50 1.5x10 5 2,200
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Section 3. General biology
Table 3. - Purification of different cell-free culture liquids of L.rhamnosus BTK-2012 (pH 6.0) by ion-exchanged chromatography
Samples Cell free cultural broth AMP Activity yield, %
Volume, ml Total activity AU Volume, ml Total activity AU
Inoculum of initial colonies 3,000 9.0x10 4 40 3.5x10 4 39.0
Inoculum of selected colonies 3,000 1.5x10 5 80 5,6 x10 4 62.0
The results of Table 3 shown, that the yield of AMP was higher when the broth was obtained after the growth of the selected colonies as compared to the broth obtained from after the growth of initial colonies (max. 62%, versus max. 39.0%).
The following purification ofAMP by gel filtration method demonstrated presence of one bacteriocin (BCN) with maximal activity for L.plantarum BTK-66 and Ent.faecium BTK- 64 strains. Two bacteriocins from Streptococcus sp. K 13 and Lactococcus sp. M 44 strains were obtained. After purification of L.rhamnosus BTK- 2012 two BCNs were obtained also which inhibited the growth of Gram-positive and Gram-negative bacteria differently [9, № 2924 А]. From antimicrobial activity of L.rhamnosus BTK- 2012, L.plantarum BTK- 66 and Ent.faecium BTK-64 strains on the Gram-negative and Gram-positive pathogenic, conditionally pathogenic bacteria, yeast belonging to different genus was shown [10, 109-115].
Discussion
Thus it was shown, that the endemic strains of LAB cell population varies in their antibacterial activity. The colonies with maximum antimicrobial activity were isolated and then used as the parent line in production of bacteriocins. Purification of cell-free cultural broth by ion-exchanging chromatography and then by gel filtration demonstrated effectiveness of the applied method for increasing of bactericidal compounds yield up to 50% (volume and antibacterial activity). Investigation of activity spectrum of obtained partially purified antimicrobial preparations proved their perspectives for use in veterinary and medicine.
Acknowledgments. This work was supported by the RA. MES State Committee of Science and Belarus National Foundation for Basic Research in the frames of the joint Armenia — Belarus joint research project 13 РБ-064.
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