Levan from Leuconostoc citreum BD1707: production optimization and changes in molecular weight distribution during cultivation

Abstract

Background Levan is a well-known homopolymer of fructose composed predominantly of β-(2, 6) fructofuranosyl linkages in the backbone with occasional β-(2, 1) linkages in the branch chains with varied applications. However, high production cost due to low yield of microbial levan has become a bottleneck for its practical applications. Furthermore, factors affecting the molecular mass of the synthesized levan by Leuconostoc spp. during prolonged cultivation is not fully elucidated. Methods The cultivation condition for Leuconostoc citreum BD1707 to synthesize levan was optimized by single-factor experiments and subsequently with response surface methodology (RSM). The average molecular weight (Mw) of levan synthesized by the strain L.citreum BD1707 under the optimized cultivation conditions was monitored by high-performance size exclusion chromatography (HPSEC). Finally, the enzyme with levan-degrading activity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results The levan yield of BD1707 reached 34.86 g/L with a corresponding productivity of 7.47 g/L/d under the optimal cultivation conditions deduced by RSM, i.e., cultivation at 26 °C and 200 rpm for 112 h in tomato juice supplemented with 172 g/L sucrose with an initial pH value of 6.12. The Mw of levan reached a peak value of 2.320 × 107 Da at 6 h of cultivation under the optimized cultivation conditions and then gradually decreased to 8.809 × 106 Da after 120 h of cultivation. Conclusion The levan yield of the strain L.citreum BD1707 could be sufficiently enhanced via cultivation condition optimization. The decrease in molecular mass of the synthesized levan was attributed predominantly to the hydrolytic activity of levansucrase secreted by L.citreum BD1707 during cultivation, with an estimated Mw of 130 KD by SDS-PAGE, while the effect of acid hydrolysis could be nearly neglected.

Levan Production by Suhomyces kilbournensis Using Sugarcane Molasses as a Carbon Source in Submerged Fermentation

“…In addition, after 9 h of cultivation, a decrease in productivity was observed at all temperatures tested. The time required for maximum EPS production (6 h) by S. kilbournensis was shorter than the time reported for other microorganisms such as Bacillus subtilis (20 h), Tanticharoenia sakaeratensis (35 h), Leuconostoc citreum BD1707 (96 h), Gluconobacter albidus (48 h), Halomonas smyrnensis (169 h), and Acetobacter xylinum NCIM2526 (122 h) [ 24 , 26 , 43 , 44 , 45 , 46 ]. On the other hand, Figure 1 shows that the highest biomass yield was present after 12 h of cultivation at 35 °C; however, the maximum production peak was not observed at any of the tested temperatures, indicating that EPSs and biomass production are not directly related.…”

“…The optimal sucrose concentration for achieving maximum synthesis efficiency varies not only among different species but also often among different strains of microorganisms [ 19 , 20 ]. In the literature, there are various reports that indicate the capacity of some microorganisms to produce levan using sucrose as the only carbon source; among these, we can highlight Bacillus subtilis [ 21 , 22 ], Lactobacillus reuteri [ 23 ], and Leuconostoc citreum [ 24 ] and Gram-negative bacteria such as Gluconobacter albidus [ 25 ], Brenneria goodwinii [ 26 ], Erwinia tasmaniensis [ 27 ], and Halomonas smyrnensis [ 28 ]. Likewise, there are reports of the overexpression of the enzymes responsible for levan production; an example is the expression of the genes of Rahnella aquatilis [ 29 ] and Leuconostoc mesenteroides in Saccharomyces cerevisiae [ 30 ] and the genes of B. subtilis expressed in Pichia pastoris [ 31 ].…”

“…In the literature, there are reports that demonstrate that agro-industrial byproducts such as beet molasses, sugar cane molasses, and syrup have been used as alternative carbon sources and have allowed for adequate microbial growth and EPS production via SmF [ 26 , 41 ]. Levan is an EPS with a high potential to be used in various industries given its physicochemical and functional characteristics [ 24 , 42 ]; however, to improve performance and quality, the design of a process allowing for large-scale, efficient, ecological, and profitable production is necessary [ 7 , 21 ]. Because of this, the objective of the present work was to evaluate the EPS production potential of the indigenous yeast strain Suhomyces kilbournensis under different process conditions.…”

Characterization of levansucrase produced by novel Bacillus siamensis and optimization of culture condition for levan biosynthesis

“…HPLC was used to determine the enzyme activity and levan production. All samples were taken at appropriate time intervals and the sugar content was determined using HPLC [ 36 , 37 ].…”

“…Levan was quantified in terms of its detected molecular weight using high-performance size exclusion chromatography (HPSEC) [ 37 ]. The size exclusion column (TSKgel Amide-80, 5 μm, 250 × 4.6 mm) was equipped with an HPLC instrument (Shimazu LC-20A series, Shimazu Corporation, Japan).…”