Evaluation of improved biological properties of chemically modified exopolysaccharides from Lactobacillus plantarum BR2.

This work engrosses the production and further chemical modifications of EPS produced by Lactobacillus plantarum BR2 and subsequent evaluation of their biological properties showed greater antioxidant properties for the derivatives compared to its native unmodified form. Of the three derivatives, acetylated EPS (a-EPS), carboxymethylated EPS (Cm-EPS), and sulphated EPS (s-EPS), a-EPS exhibited the highest DPPH radical scavenging and total antioxidant activity in a dose-dependent manner. At all tested concentrations, a-EPS showed higher scavenging activity, and a maximum activity of 73.81% at 2 mg/mL. Meanwhile, s-EPS showed the highest reducing power potential and hydroxyl radical scavenging activities. At 2 mg/mL concentration, the order of reducing power was observed to be s-EPS (41.39%) > a-EPS (37.43%) > Cm-EPS (24.02) > BR2 control EPS (16%) and the hydroxyl radical scavenging activity for the s-EPS was 54.43%. The highest reducing power activity exhibited by s-EPS is 2.6-fold higher and a 1.5-fold increase in the scavenging activity of native BR2 EPS after the sulphonyl group addition was observed. The increase in these activities is due to the addition of various functional groups that contributes largely to the scavenging abilities of different free radicals. The s-EPS and Cm-EPS derivatives also exhibited increased cholesterol-lowering activity of 40 and 34.5%, respectively, than the native EPS. Interestingly, there were hardly any inhibitions on cell growth and viability of normal L929 fibroblast cell lines upon treatment with these EPSes. The improved antioxidant properties resulting from chemical modification opened better avenues for EPS application in the food and pharma sectors. Thus, the potentiality of chemically modified EPS may be explored further in the development of functional foods. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03718-5.

Direct utilization and conversion of raw starch to exopolysaccharides by a newly isolated amylolytic Streptococcus sp.

A newly isolated culture is identified as Streptococcus lutetiensis with significant starch saccharifying activity. Along with considerable amylolytic property (∼ 2.71 U/mL), the culture exhibited significant production of exopolysaccharide (EPS) in starch medium. Interestingly, the glycosyl transferase activity which is essential in the biosynthesis of polysaccharide is also detected in the culture and after screening and process optimization, a maximum EPS titre of 19.92 ± 0.5 g/L was obtained from cassava starch. The crude EPS, after purification and characterization (monosaccharide analysis, FT-IR, TGA, GPC NMR, and SEM) was found to be of dextran nature with a Mw of 1275.36 kDa. Dextran type exopolysaccharide are synthesized by dextransucrase enzyme by the transfer of glucosyl residues from sucrose to dextran polymer. Interestingly, the glycosyl transferase enzyme activity which is essential in the biosynthesis of EPS is also detected in the culture. The particle size (447.8 dnm) and the zeta potential (-33.4) analysis of the purified EPS showed that the EPS produced is a stable molecule and has a random coil confirmation when exposed to alkaline condition with shear thinning property. One step conversion of sustainable low-cost starchy raw materials without adding external enzymes for hydrolysis, improved the economic viability of EPS production.

Nisin controlled homologous Over-expression of an exopolysaccharide biosynthetic glycosyltransferase gene for enhanced EPS production in Lactobacillus plantarum BR2.

Glycosyltransferases synthesize a variety of exopolysaccharides (EPS) with different properties by altering the type of glycosidic linkage, degree of branching, length, mass, and conformation of the polymers. The genome analysis of an EPS-producing, Lactobacillus plantarum BR2 (Accession No: MN176402) showed twelve glycosyltransferase genes, and the gene BR2gtf (1116 bp), annotated as an EPS biosynthetic glycosyltransferase was cloned into the pNZ8148 vector. The recombinant pNZ8148 vector along with pNZ9530, a regulatory plasmid, were electroporated to L. plantarum BR2 for the over-expression of gtf gene under a nisin-controlled expression system and the glycosyltransferase activity of the recombinant and the wild-type strains were analysed. The recombinant strain showed 54.4% increase in EPS production with the maximum EPS production of 23.2 ± 0.5 g/L in a 5 L bioreactor study after 72 h of fermentation. This study shows an effective molecular strategy possibly to be adopted in lactic acid bacteria to enhance exopolysaccharide production.

An overview of functional genomics and relevance of glycosyltransferases in exopolysaccharide production by lactic acid bacteria.

There are many reports on exopolysaccharides of lactic acid bacteria (LAB EPS) such as isolation, production and applications. The LAB EPS have been proved to exhibit significantly improved texture and rheological properties in order to prevent syneresis of fermented foods. Furthermore, they are known to have many biological properties such as mouthwatering flavors, antioxidant activity, cholesterol lowering and antimicrobial activities. Considering their GRAS status, LAB EPS need to be explored for better titre and improved biological properties, where strain improvement by genetic engineering has a major role for making tailor-made EPS. The genetic overview of the EPS production by LAB is an auxiliary area of interest as the process and the biosynthetic pathway involves numerous genes and their proteins. Among them Glycosyltransferases (gtfs) are the key enzymes involved in EPS biosynthesis. Current knowledge of gtfs of LAB and its manipulation is limited. The present review spotlights the importance of glycosyltransferases and their specific role on the biosynthesis of LAB EPS and addresses the functionality and applicability of these enzymes and their products. It enfold the available literature including some patents in recent past to underline the fact that glycosyltransferases are un-reluctantly the key proteins involved in the EPS biosynthesis.

Comparison of faecal culture and IS900 PCR assay for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faecal samples.

Comparative efficacy of faecal culture and IS900 Polymerase chain reaction (PCR) assay of faecal samples was investigated in 40 clinically suspected cases of Johne's disease in dairy cattle. The sensitivity of faecal culture and PCR assay in this study was 52.5% (21/40) and 90% (36/40) respectively. All isolates appeared only on the mycobactin J supplemented Herrold's egg yolk medium (HEYM) at 8-16 weeks post-inoculation, were acid-fast and were positive for IS900 PCR yielding a single amplicon of 217 bp. A total of 28 faecal samples out of 40 were positive by IS900 primary PCR assay for Mycobacterium avium subsp. paratuberculosis (Map) yielding an expected product of size 217 bp. Twelve faecal samples, which gave negative results in the primary PCR, were subjected to secondary PCR assay. Of the 12 samples, 8 gave positive results in the IS900 nested PCR (nPCR), which yielded a PCR product of 167 bp, proving better sensitivity of nPCR assay than single amplification PCR. PCR could detect additionally 15 samples as positive which were negative by faecal culture. The chi-square analysis showed a highly significant difference between the tests (P< 0.01). This study suggests that IS900-PCR-based detection of Map could be used as a potential diagnostic tool for rapid and effective Johne's disease surveillance.