Optimization of suitable carbon source for polysaccharide production by Lactobacillus sp. UTMC ۳۹۸۳ and molecular identification of the producer strain (EPS)

BACKGROUND AND OBJECTIVESCurrently, exopolysaccharide is one of the most important and widely used biopolymers with many industrial uses in industries such as pharmaceutical, food, agriculture, etc. Lactic acid bacteria are biological factories that produce these biopolymers. With the increasing demand in the use of these biopolymers, the commercialization of the products of these bacteria and their replacement with expensive and rare products can facilitate many industrial processes and make the final product more economical. For this reason, newly isolated strains with the ability to produce more exopolysaccharide such as Limosilactobacillus using optimal culture medium have become one of the attractive fields of industrial microbiology research. The aim of this study was to propose different sources of carbon, as well as examining different concentrations of these sources, to find the optimal carbon source and the appropriate concentration for exopolysaccharide production in order to optimize the best conditions for increasing the production of lactic acid producing bacteria.MATERIALS AND METHODSIn this study, the lactic acid strain of Lactobacillus sp. UTMC ۳۹۸۳ from the Microbial Collection Center of the Division of Microbiology (University of Tehran) was used and regenerated on MRS Broth culture medium. After ensuring the purity of the colonies, bacteria were inoculated on the Erlenmeyer flask containing the MRS broth culture medium (۳۰ oC, ۱۸۰ rpm for ۲۴ hours). Then, ۱۰% of the volume of pre-cultivation flasks was replaced with other carbon sources (including lactose, whey, molasses, glucose syrup, and sucrose) in Erlenmeyer flasks containing production medium based on MRS broth and polysaccharide production, residual substrate and bacterial biomass are measured. In order to sequenceing of the strain, DNA extraction and RT-PCR technique were performed and the obtained sequences were aligned with the related sequences from NCBI and other sequences outside the genus.۲۵۵Finally, MEGA software and Maximum Parsimony criterion were used to construct a phylogeny tree and evolutionary genetics analysis.RESULTS AND DISCUSSIONBy examining the addition of ۲.۵% to ۶۰% concentrations of sucrose as a carbon source to the culture medium and examining the amount of changes in exopolysaccharide production, it was determined that the highest production rate was at a concentration of ۳۰%, ۶۷ g/liter, which was used as a control medium in the efficiency of exopolysaccharide production from different carbon sources. By replacing the concentrations of (۲۰-۲۵-۳۰-۳۵-۴۰) % whey as a carbon source, the amount of exopolysaccharide production in the highest concentration of whey (۴۰%) was obtained less than a third of the amount of exopolysaccharide production in control medium. Also, by replacing the concentrations of (۳۰-۴۰-۵۰-۶۰) % lactose as a carbon source, the amount of exopolysaccharide production at concentrations of ۳۰% and ۴۰% was reported to be very insignificant and about zero, and concentrations of ۵۰% and ۶۰% % represented lactose sediment, which caused the loss of the ability to the little exopolysaccharide production. By replacing the concentrations of (۱۰-۲۰-۳۰-۴۰)% of the glucose syrup, the average amount of exopolysaccharide production in the medium with the highest concentration (۴۰%) was detected as ۲۴.۴ g/liter, which is about half of the amount of production exopolysaccharide was optimized in control medium; However, exopolysaccharide production in the presence of beet molasses with concentrations of (۲۰-۳۰-۴۰)% in the culture medium was not detectable and the beet molasses remained unused as sediment at the end of the test process. Optimizing the production medium in terms of pH and time and conditions of bacterial growth showed that the amount of bacterial turbidity in the medium with pH=۵.۷ was higher (average: ۲.۲۱۲) compared to the medium with pH=۶.۸ on average (average: ۱.۹۹۴). The growth of bacteria was reported to increase in the first ۴۸ hours, and the shape and size of Limosilactobacillus was directly related to the amount of exopolysaccharide production.CONCLUSIONThe results of this study showed that the bacteria were unable to use lactose sugar, either in pure form or in whey, and beetroot dremelas could not grow. The results of this study showed that the bacteria could not use lactose sugar, either in pure form or in whey, and can not grow in the presence of beetroot. By examining glucose syrup as a carbon source, it was also determined that this carbon source is only suitable for the bacteria revival, although exopolysaccharide production in this medium was weak. Finally, the medium with ۳۰% sucrose (approximately ۶۷ g/L) was found to be the most suitable carbon source for the growth and production of exopolysaccharide. Our findings showed that increasing the pH of the medium as well as increasing the fermentation time did not show a positive effect on the amount of exopolysaccharide production.