Structural characterization of an exopolysaccharide produced by Lactobacillus plantarum Ts

: An exopolysaccharide (EPS) was isolated from Lactobacillus plantarum Ts and purified by size exclusion chromatography train. L. plantarum showed the highest production (9.4 ± 0.8 g/l) of EPS. Furthermore, L. plantarum was cultured in medium with volume of 5 L and the EPS was extracted by ethanol precipitation. By HPLS analysis demonstrated, that the EPS has two fractions - polymer and olygomer. The molecular mass of the EPS from both fractions is respectively 8.3x104 Da for the greater pic and 5.0x102 Da. Fourier transform infrared spectroscopy analysis showed the presence of hydroxyl and carboxyl groups and glycosidic linkages. TLC analysis indicated that the EPS was a heteropolymer composed of fructose, and sucrose as monomeric constituent units. The strain L. plantarum and pathogenic E. coli 3398, St. aureus 745, B. subtilis 6633, S. typhimurium 3591, L. monocytogenes 863 and E. aerogenes 3691 were tested for their growth utilizing the EPS from L. plantarum as the sole carbon source for its possible use as a prebiotic. L. plantarum exhibited growth in the EPS supplied medium compared with sucrose as carbon source, whereas the pathogenic strains did not grow in the EPS-supplied medium. In vitro evaluations showed that, like other reported polysaccharides, this EPS displayed significant prebiotics properties.


Introduction
Biopolymers are defined as polymers that are bio-based, biodegradable, or both (Imre & Pukánszky, 2013).Biopolymers produced by live microorganisms, such as various bacteria, yeast, and mold species have gained substantial attention in recent decades due to their possible applications in industry (Moradi et al., 2021).Microbial extracellular polysaccharides are widely used in the food industry and also in the cosmetic and pharmaceutical industries (Korcz et al., 2018).Due to their applicability in the food industry, particular attention has been given to special biopolymers called exopolysaccharides (EPS) produced by lactic acid bacteria (LAB).EPS (exopolysaccharide) is a term first used by Sutherland (Arrage et al., 1995) to describe high-molecular-weight carbohydrate polymers produced by marine bacteria The physiological role of EPS depends on the ecological niches and the natural environment in which microorganisms have been isolated.Bacterial EPSs are believed to play an important role in the environment by promoting survival strategies such as bacterial attachment to surfaces and nutrient trapping, which facilitate processes of biofilm formation and development (Sauer et al., 2022).According to Moradali et al. (2019), bacterial EPSs are believed to play an important role in the environment by promoting survival strategies such as bacterial attachment to surfaces and nutrient trapping, which facilitate processes of biofilm formation and development.Most LAB, especially Fructilactobacillus, Lacticaseibacillus, Lactiplantibacillus, Lactobacillus, Lactococcus, Latilactobacillus, Lentilactobacillus, Leuconostoc, Limosilactobacillus, Pediococcus, Streptococcus, and Weissella species are capable of synthesizing a variety of EPS (Angelin & Kavitha, 2020;Zheng et al., 2020).Lactobacillus strains of food origin are regarded as safe and their EPSs can be used as prebiotics.Prebiotics are nondigestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacterial species already resident in the colon, and thus attempt to improve host health (Glenn et al., 1995).EPSs produced by the food-grade Lactic acid bacteria (LAB) with GRAS (Generally Recognized as Safe) status are an important source of natural alternatives to commercial additives of plant or animal origin.Most of those additives used are chemically modified to improve the rheological properties of the product and hence are not allowed in most European Union countries (German et al., 1999;Idrees et al., 2022).EPS produced by LAB are in a great variety, depending on the type of LAB strains, culture conditions, and medium composition (Korcz & Varga, 2021).L. plantarum is a versatile, Gram-positive, bacterium that can be found in a wide range of habitats such as dairy, meat, and many plant fermentations.The properties of EPSs depend on the composition of the monosaccharides and the connections of the monomers to build the exopolymers (Kodali et al., 2011).
For the commercial and therapeutic importance of the products, is important to provide information on the EPS isolated and purified from a Lactobacillus plantarum isolated from Bulgarian

EPS Composition Analysis
The lyophilized EPS (10 mg) was hydrolyzed with 6 M trifluoroacetic acid (TFA) for 6 h at 100°C and TFA was removed using a rotary vacuum evaporator.The hydrolyzed solution was neutralized with 15 M ammonia solution (0.32 ml) (6).The resultant hydrolysate was analyzed by thin-layer chromatography (TLC) to determine the monosaccharide composition.The monosaccharide standard was also treated with 6 M TFA and as described above, neutralized with ammonia.The EPS hydrolysates were separated using Silica Gel 60 TLC.For this we used pre-coated plates (Merck, Germany) developed with a mobile phase of ethylacetate: n-propanol: acetic acid: water (4:2:2:1 (v/v/v/v)).The separated sugars were visualized with an orcinol-sulfuric acid spray reagent.

HPLC analis
The average numerous molecular weight (Mn) and average molecular weight (Mw) of the samples were determined by molecular sieve chromatography (HPLC-SEC) at the University of Food Technology, Plovdiv.The separation was performed on an HPLC ELITE LaChrome chromatograph (VWR Hitachi, Japan) equipped with a Shodex OH PAK 806 MHQ column (8 mm x 300 mm), (Shodex Co., Tokyo, Japan) and an RI detector (VWR Hitachi, Chromaster, 5450, Japan).We used 0.1 M NaNO3 at 30 ° C as the mobile phase at a flow rate of 0.8 ml / min.The column was maintained at 30.0 ± 0,1° C, as all samples (2 mg / ml in 0.1 M NaNO 3 ) were filtered through 0.45 μm, PTFE45/ 25 mm (Isolab, Germany) before injection and 20 μl were injected at each assay.The standard curve was obtained using different standards of known molecular weight pullulins (P-5, P-10, P-20, P-50, P-100, P-200, P-400, P-800).The polydispersity index was calculated (I = Mw / Mn) and it represents a characteristic of polymers (Murdzheva et al., 2016).

Effect of EPSs on Growth of Lactic Acid Bacteria and Enteric Pathogens
The using LB medium supplemented with 1% EPS and 1% glucose (positive control) as the carbon source separately.Bacterial growth was measured by a turbidimetric method at 650 nm and calibrated against cell dry-weight using a spectrophotometer (UV/Vis Shimadzu, Japan) and for each experiment, data was analyzed using Excel statistical package.

Statistical Analyses
Experiments were repeated at least three times.The data are expressed as the mean ± standard deviation.

Screening of EPS-Producing Microbes
The presence of EPS associated with bacterial cells can be recognized by the formation of colonies in mucous solid medium (Stadler et al., 2010).Colonies showing a slimy appearance were picked and subcultured in mMRS wiht 10% glucose and incubated for 2 days at 37°C.Totally nine isolates were collected from the ganjang samples.Therefore, the presence of a translucent or creamy material involving a mucoid colony is indicative of EPS production potential.When cultivated in a media with high content of saccharides such as 10% sucrose solutions, strain L. plantarum synthesizes exopolysaccharides (Fig. 1).

Quantification of the EPS
The EPS produced by L. plntarum Ts strain was quantified by the phenol-sulfuric acid method (Dubois et al., 1956).The results of the analysis showed that starin L. plantarum produced EPS amount (9.4 ± 0.8 g/l).

FTIR Spectral Analysis of the EPS from L. plntarum
The FTIR spectrum of the EPS from L. plantarum showing the functional groups in the 4,000-400 cm -1 region is represented on Fig. 2.There is no complete match between the synthesized L. plantarum species with the two standards tested.This suggests that the synthesized FTE has a new unique structure.

Monosaccharide Composition of the EPS from L. plntarum Ts.
Hydrolysis of the EPS with 6 M TFA and subsequent analysis of the EPS hydrolysate by TLC showed the presence of mannose and glucose as principal components of the EPS from L. plntarum (Fig. 4).Galactose, 12.Lactose; 13 Maltose Hydrolysis of the EPS with 6 M TFA and subsequent analysis of the EPS hydrolysate by TLC showed the presence of fructose and glucose as principal components of the EPS from L. plantarum (Fig. 3).Comparison with selected standards shows similarity as FOS (Raftilose P95) and raffinose, FOS and GOS (Figure 4).

HPLC analysis of EPS
The average numerous molecular weight (Mn) and average molecular weight (Mw) of the purified EPS were determined by molecular sieve chromatography (HPLC-SEC) at UCT Plovdiv.The molecular weights were calculated according to the relative molecular weight of the known molecular weight Corresponding author: s.toshkova@shu.bgFull Paper DOI: 10.2478/asn-2022-0022 ©2022 Stephany Toschkova, published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License pullulans (P-5, P-10, P-20, P-50, P 100, P-200, P-400, P-800).The polydispersity index, which is a characteristic of polymers, is calculated by the formula: I = Mw / Mn (11).The results obtained are shown in Figure 5.

Figure 5. HPLC-SEC
The chromatogram of the EPS appeared in two asymetrical pics.In the sample was observed two fractionspolymer and oligomer.The molecular mass of the EPS from both fractions is respectively 8.3x10 4 Da for the greater pic 5.0x10 2 Da.

Effect of the EPS on the Growth of Lactic Acid Bacteria and Enteric Pathogens
Growth of L. plantarum strain in modified MRS broth supplemented with carbohydrates EPS utilization by LAB shown in Table 1.The growth of the pathogenic bacteria was monitored using LB medium supplemented with 1% EPS utilization by LAB and 1% sucrose (positive control) as the carbon source separately.Growth was evaluated in terms of maximum optical density at 600 nm and specific growth rate achieved during 24 h fermentation.Growth kinetics on sucrose were used as control.
L. plantarum strain fermented EPS utilization by LAB.Pathogenic strains E. coli 3398, St. aureus 745, B. subtilis 6633, S. typhimurium 3591, L. monocytogenes 863 and E. aerogenes 3691 did not show any growth in EPS-supplied medium compared with sucrose-supplied medium (Table 1), indicating that these strain could not utilize EPS as a carbon source.
The ablity of L. plantarum to sintesyze EPS is proven from longterm studies of Bulgarian authors  The technique works on the fact that bonds and groups of bonds vibrate at characteristic frequencies.A small peak around 1,530 cm -1 corresponding to an amino group was also observed in the spectrum of EPS.The obvious absorption peak 879.54 cm -1 revealed the existence of b-glycosidic bond.
The band in the range 1,030-944 cm -1 , with minimum at 998 cm -1 can be attributed to Glc.The absorption bands at 812, and 879 cm -1 indicated that the EPS contained both α and β-type glycosidic linkages in its structure.Similar results were obtained by other authors (Xu et al., 2010).The broad peak at 3331 cm -1 was an OH stretching peak, the carbonyl (C=0) stretching was at 1670 cm -1 , and the peak between 2800 and 3000 cm -1 was a C-H stretching peak.Absorption at 1110 cm -1 was typical for D-glucose in pyranose form, and that at 847 cm -1 was indicative of the presence of an R-glycosidic linkage between individual glycosyl residues in EPS.In the region (950-700 cm -1 ), polysaccharides are reported to exhibit an obvious characteristic absorption at 812 cm -1 that corresponds with the presence of mannose.Такива резултати се наблюдават и от (Kodali et al., 2011).
The carbohydrates show high absorbencies in the region 1,200-950 cm -1 , that is within the socalled fingerprint region, where the position and intensity of the bands are specific for every polysaccharide, allowing its possible identification.Polysaccharides present the highest capacity for carrying biological information since they have the greatest potential for structural variability.Presence of glucose and sucrose indicated that the EPS produced by the isolate is a heteropolysaccharide.The biological activities of glucomannans implicate the importance of this polysaccharide extracted from the flour LAB strain which showed probiotic properties.
Corresponding author: s.toshkova@shu.bgFull Paper DOI: 10.2478/asn-2022-0022 ©2022 Stephany Toschkova, published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License Mw is a major property of L. plantarum WLPL04 FTE that may affect probiotic properties.FW mw from L. plantarum WLPL04 is determined to be 6.61 × 10 4 Da, which is lower than FEV (1.1 × 10 5 Da) than L. plantarum YW11 (17) and FEV (1.15 × 10 6 Da) of L. plantarum C88 ( 24), but higher than L. plantarum BC-25 FEV (1.83 × 10 4 and 1.33 × 10 4 Da) (25).In a parallel study, the Mw of L. plantarum ZDY2013 was 5.17 The EPS from L. plantarum showed potential prebiotic property by selectively supporting the growth of LAB.The pathogenic strains were unable to grow in the medium containing EPS as a carbon source, indicating that in the presence of EPS, LAB will be selectively enriched, whereas pathogenic Enterobacteriaceae members will be suppressed.A similar result was observed in the cases of EPS from Weissella cibaria A2, W. confusa A9, L. plantarum A3, and Pediococcus pentosaceus 5S4, which selectively enhanced the growth of Bifidobacterium and Lactobacillus/ Enterococcus groups whereas Clostridia were suppressed (Hongpattarakere et al., 2012).
It has been demonstrated in this study that, in the presence of EPS strain L. plantarum is capable of fermenting it.Despite significant commercial interest in using oligosaccharides as prebiotic substrates, little is known about how these oligosaccharides are metabolized by LAB and related bacteria.LAB accumulate sugars by secondary active transport (mainly by proton motive force, PMF), the PTS, or an ATP-mediated system.However, more studies should be performed in order to elucidate the pathways of utilization of oligosaccharides in these Lactobacillus strains.

Conclusion
In conclusion, L. plantarum produced a water soluble heteropolysaccharide, which is composed of sucrose and glucose.Additionally, growth study using EPS as the sole carbon source showed that it could specifically support the growth of LAB while suppressing the growth of E. coli 3398, St. aureus 745, B. subtilis 6633, S. typhimurium 3591, L. monocytogenes 863 and E. aerogenes 3691.These results indicate that the EPS from L. plantarum has potential application as a prebiotic in the food industry.

Figure 1 .
Figure 1.EPSs (exopolysaccharides) produced by L. plantarum cultivated in a media containing 10% sucrose, which are secreted in the culture medium

Figure 2 .
Figure 2. FTIR analysis of the exopolysaccharide preparation from L. plntarum Ts

Figure 3 .
Figure 3. FTIR analysis of an EPS of L. plantarum obtained by culturing the strain on 10% sucrose compared with glucooligosaccharide-1K and raffinose-2K

Table 1 .
Oligosaccharide Utilization Optical density (OD) at 600 nm after 24 h in MRS, MRS-EPS broths.For each experiment, data were ana lyzed using the Excel statistical package.The OD readings and standard deviations were calculated from duplicate samples from three separate experiments. a