Exopolysaccharides and uses thereof
Inventors
BIFFI, Andrea • ROSSI, Ruggero • FIORE, Walter • Guglielmetti, Simone Domenico • BALZARETTI, Silvia
Assignees
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Publication Number
US-11591416-B2
Publication Date
2023-02-28
Expiration Date
Abstract
The present invention refers to exopolysaccharide molecules, conditioned media or compositions comprising said molecules or media. Moreover, the present invention refers to use of said exopolysaccharide molecules, conditioned media or compositions as prebiotic, preferably to boost immune system.
Core Innovation
The invention relates to an exopolysaccharide (EPS) comprising repeating units of rhamnose, galactose and N-acetygalactosamine in a ratio of 4:1:1. The EPS is defined as a rhamnose/galactose/N-acetygalactosamine heteropolysaccharide with a specific repeating-unit structure corresponding to Formula I, including stereochemistry and linkage patterns and derivatized sugar forms with L-configuration for rhamnose and D-configuration for galactose and N-acetygalactosamine.
The EPS is provided as DG-EPS from Lactobacillus paracasei DG and as conditioned media and/or compositions comprising the EPS. The document reports that gene cluster regions putatively responsible for EPS biosynthesis are identified as EPS-a and EPS-b, with EPS-b including a region of approximately 13 kb and an identified unique region of approximately 7 kb. Sequence information is reported, including SEQ ID NO:1 and related sequence identities within the described gene cluster regions.
DG-EPS is characterized using NMR and GC-MS linkage/monomer analyses to support the described sugar composition and repeating-unit structure. Functional characterization is reported in THP-1 macrophage-like cells, where treatment increases expression of TNF-α, IL-6, IL-8 and CCL20 mRNA. COX-2 is reported as not significantly changed, NF-κB activation in Caco-2 is reported as having limited effects, and Caco-2 adhesion is reported as showing modest effects.
Claims Coverage
The partial claim set provided includes one independent claim. Across the dependent claims listed, at least three main inventive aspects are specified: (i) the defined repeating-unit composition ratio of rhamnose/galactose/N-acetygalactosamine; (ii) structural refinement via specified derivatized forms, stereochemical configurations, and a repeating unit defined by Formula I; and (iii) source/production restrictions tied to the Lactobacillus paracasei DG strain deposited as CNCM I-1572, including mutant and genetically engineered bacterial cells.
Rhamnose–Galactose–N-acetygalactosamine repeating-unit composition in 4:1:1 ratio
An exopolysaccharide comprising at least one repeating unit of rhamnose, galactose and N-acetygalactosamine, in a ratio of 4:1:1, respectively.
Acetylated/derivatized forms of repeating-unit sugars
The exopolysaccharide of claim 1 is defined such that rhamnose is 1,2,3,4,5-penta-O-acetyl-L-rhamnitol, galactose is 1,2,3,4,5,6-hexa-O-acetyl-D-galactitol, and N-acetygalactosamine is 2-acetamido-1,3,4,5,6-penta-O-acetyl-2-deoxy-D-galactitol.
Stereochemical configuration constraints for repeating-unit sugars
The exopolysaccharide of claim 1 is characterized by having L-configured rhamnose and/or D-configured galactose and/or D-configured N-acetygalactosamine.
Repeating unit defined by Formula I
The exopolysaccharide of claim 3 is defined such that its repeating unit conforms to Formula I.
EPS sourced from Lactobacillus paracasei DG deposited as CNCM I-1572 (or mutant)
The exopolysaccharide is obtained from the Lactobacillus paracasei DG strain deposited on May 5, 1995 at National Collection of Microorganisms Cultures of the Pasteur Institute under the code CNCM I-1572, or a mutant strain thereof.
Production using genetically engineered bacteria and/or CNCM I-1572-derived bacteria
The exopolysaccharide is produced using genetically engineered bacteria cells and/or bacteria from the Lactobacillus paracasei DG strain deposited on May 5, 1995 at National Collection of Microorganisms Cultures of the Pasteur Institute under code CNCM I-1572.
Overall, the provided claims cover an exopolysaccharide defined by a 4:1:1 repeating-unit composition of rhamnose, galactose and N-acetygalactosamine, refined by specified derivatized forms, stereochemical configurations, and a repeating unit conforming to Formula I. Coverage is further narrowed by restricting the EPS source and/or production to Lactobacillus paracasei DG deposited as CNCM I-1572, including mutant strains and genetically engineered bacteria.
Stated Advantages
Immunostimulatory activity, including increased TNF-α, IL-6, IL-8 and CCL20 mRNA in THP-1 macrophage-like cells.
Prebiotic and immune system boosting properties are described for DG-EPS.
COX-2 is reported as not significantly changed.
NF-κB activation in Caco-2 is reported as having limited effects.
Caco-2 adhesion is reported as showing modest effects.
Documented Applications
Use of DG-EPS, and conditioned media/compositions comprising DG-EPS, as prebiotic/immune-boosting agents.
Immunostimulatory activity assessment in THP-1 macrophage-like cells, including cytokine and chemokine mRNA expression (TNF-α, IL-6, IL-8, CCL20).
Assessment of inflammatory-signaling-related readouts and cellular effects in Caco-2, including NF-κB activation and Caco-2 adhesion.
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