OPTIMISATION OF CULTURE CONDITIONS FOR ANTIMICROBIAL COMPOUNDS PRODUCTION BY LACTOBACILLUS PLANTARUM
Vasyliuk O, Garmasheva I.
D. K. Zabolotny Institute of Microbiology and Virology of the NAS of Ukraine,
Department of physiology of industrial microorganisms
The proliferation of phytopathogenic bacteria cause plant diseases in the agriculture industry. Lactic acid bacteria produce different types of substrates with the antimicrobial properties which can be used as inhibitors of phytopathogenic bacteria. They exert a strong antagonistic activity against phytopathogenic bacteria, as a result of the production of organic acids, diacetyl, hydrogen peroxide, bacteriocins, short-chain fatty acids, inhibitory enzymes, and antimicrobial-bioactive peptides. Production of antimicrobial compounds by Lactobacillus plantarum strains may be influenced by various factors – composition and pH of medium, temperature, and time of cultivation.
The aim of this study was to investigate the effect of nutrients and cultivation conditions on the antagonistic activity of the L. plantarum against phytopathogenic bacteria.
Materials and methods. Four L. plantarum strains that have a wide spectrum of antagonistic activity against phytopathogenic bacteria were used. The influence of different carbon sources on L. plantarum antagonistic action was tested: sucrose, lactose, fructose, galactose, maltose, and arabinose. Nitrogen sources such as peptone, yeast extract, meat extract, tyrosine, and corn extract were used. Response Surface Methodology was used to optimize culture conditions such as pH, temperature, and time of cultivation. The antimicrobial activity was determined by measuring the diameter of the inhibition zone around the discs with cell-free culture supernatants (CFS).
Results. The CFS from 4 L. plantarum strains was screened for its antagonistic activity against indicator bacterial strains. The maximum inhibition zones by L. plantarum strains were observed at 3% sucrose as a carbon source. All the L. plantarum strains lost their antagonistic activity against phytopathogenic bacteria during cultivation on media with galactose and arabinose as a carbon source. Among the five nitrogen sources used, the maximum zones of growth inhibition were in the presence of 1% tyrosine in the culture medium. 15 experiments with a combination of variables were performed to examine the combined effect of three independent variables (temperature, pH, and time) on the antimicrobial activity of each L. plantarum strain against phytopathogenic bacteria. The optimized temperature, pH of the medium, and time of cultivation were 30°С, pH 7.8 and 72 hours respectively, with the maximum predicted zones of growth inhibition of L. plantarum 21с – 18.2±0.5mm, L. plantarum 1026 fc – 18.25±0.6 mm and L. plantarum 1112s – 16.94±0.5mm. It can be assumed that the increase of antagonistic activity of the strain L. plantarum 13c against phytopathogenic bacteria was highest at pH 6.8, time 48 hours and 30°С and zones of growth inhibition of the indicator strains was 22±0.5mm.
Conclusions. The highest antimicrobial action of L. plantarum strains against phytopathogenic bacteria was found at 3% sucrose and 1% tyrosine as the main source of carbon and nitrogen, respectively. For all investigated strains, the optimized temperature for the maximum antagonistic activity against phytopathogenic bacteria was 30°C. Optimum pH of medium and time of cultivation for antimicrobial production against the test strains by L. plantarum was pH 7.8 and 72 hours cultivation, respectively.