RU2808430C2 - Prebiotic composition and its use - Google Patents

Abstract

FIELD: pharmaceuticals; medicine.

SUBSTANCE: prebiotic composition is proposed, the said composition contains an amount of sphingan in the form of an oligosaccharide with a DP from 2 to 30 effective for the manifestation of prebiotic activity. Also the following are provided: the methods of producing high/medium/low acyl sphingan as an oligosaccharide (“SOS”) (variants), a method of stimulating the growth of beneficial bacteria in the colon of a mammal, a method of reducing the level of propionate and/or increasing the level of butyrate in the colon of a mammal, a method of improving the integrity of the intestinal barrier in the mammalian colon and a method of reducing TNF-α levelsα and/or IL-8 in the mammalian colon.

EFFECT: invention provides a highly effective prebiotic composition.

16 cl, 25 dwg, 16 tbl, 4 ex

Description

Related applications

[0001] This application was filed on January 15, 2020 as an international PCT patent application and claims priority to US Provisional Patent Application No. 62/794452, filed January 18, 2019, and US Provisional Patent Application No. 62/869248 , filed July 1, 2019, the disclosures of which are incorporated herein by reference in their entirety.

Field of technology

[0002] Disclosed herein is an oral composition containing sphingan and its use as a prebiotic.

Prerequisites for creating an invention

[0003] The human gastrointestinal tract is a highly complex microbial ecosystem that has been found to be remarkably stable (Zoetendal (1998)) Many different approaches have been used to modulate intestinal flora in a manner that is beneficial to the health of the host (see for example Bielecka (2002) and Steer (2000)). These various approaches include adding live microorganisms to food (probiotics), adding dietary ingredients or dietary fiber to selectively stimulate beneficial bacteria in the host (prebiotics), and adding a combination of both probiotics and prebiotics (synbiotics).

[0004] Prebiotics are non-digestible substrates that are selectively utilized by host microorganisms to provide health benefits (Gibson (2017)). The effects of prebiotics in the gut can be assessed based on the growth of health-promoting bacteria such as lactobacilli and bifidobacteria, the reduction of intestinal pathogens, and the increase or decrease in the production of health-related bacterial metabolites. The prebiotic/bifidogenic nature of the selected prebiotics (such as inulin, fructooligosaccharides, galactooligosaccharides, lactulose and arabinooligosaccharide) has been suggested and/or confirmed in previous studies (see, for example, Guimaraes (2018), Karltohn-Senaye (2013), Patel (2013), Saavedra (2002), Tuohy (2001), Tuohy (2002), US8313789B2, US20100092440A1 and WO2004002240A2.)

[0005] In general, sphingans are polysaccharides consisting of the following substituted or unsubstituted tetrameric saccharide, usually denoted as [(→3)Glc(β1→4)GlcA(β1→4)Glc (β1→4)Rha(α1→)]n. Known sphingans include, for example, gellan (S-60), velan (S-130), ramsan (S-194) and diutan (S-657)).

[0006] Gellan (gellan gum or S-60) is produced by strains of the species Sphingomonas elodea (formerly Pseudomonas elodea ), such as strain ATCC 31461. (See, for example, Morrison (2016), Sworn (2009) and US4326053A.) Common forms of gellan gum include high acyl (also known as "natural"), unclarified (e.g., KELCOGEL ^® LT100 gellan), low acyl, unclarified (e.g., KELCOGEL ^® LT gellan), and low acyl, clarified (eg, KELCOGEL ^® and KELCOGEL ^® F gellan gums). (Sworn (2009).) A number of specialty grades are also available, such as high acyl, PHB (polyhydroxyoxybutyrate) free, clear (e.g. KELGOGEL ^® HT gellan) and low acyl, clear (double-precipitated) (e.g. GELRITE ^TM MK gellan). The natural, or high acyl, form of gellan includes two acyl substituents (acetate at the O ⁶ position and glycerate at the O ² position) in the (1→3)Glc unit, and on average there is one glycerate per tetramer, and one acetate per two tetramers . (Kuo (1986).) Low acyl gellan contains no glycerate or acetate. Gellan gums can also be produced with medium levels of glycerate and acetate. A commercial product with reduced glycerate and acetate content is KELCOGEL® DGA gellan.

[0007] Gellan gum typically acts as a gelling or suspending agent in some oral products and is present at levels that range from 0.02 to 0.5% (w/v) (See, e.g. , Fallourd (2009), Morrison (2016), Sworn (2009), US6602996B1, US6663911B2, US5342626A, US8513408B2, and US20080008814A1). Prior to its approval as a dietary supplement, studies assessed the safety of gellan gum when administered to rats and humans. (See, for example, Anderson (1988) and Edwards (1995); see also Anderson (1990).) For example, Edwards (1995) describes feeding Wistar rats for 28 days using a diet that included 50 g/kg/day gellan gum. (For reference, 50 g/kg in rats corresponds to the human equivalent amount of approximately 8 g/kg (see, for example, FDA Guidance (2005).) Interestingly, Edwards (1995) concluded that gellan gum has no sustained effect on short-chain fatty acids (SCFAs, such as acetate, propionate and butyrate) in the cecum or feces. Additionally, Anderson (1988) reports a study in which human volunteers ingested some gellan gum according to a continuous dosing regimen dose of 175 mg/kg/day for 7 days, followed by 200 mg/kg/day for an additional 16 days (For a person weighing between 60 and 75 kg, 175 mg/kg corresponds to a range of 10.5 up to 13 g, whereas 200 mg/kg corresponds to a range of 12 g to 15 g.) Based on the results presented there, Anderson (1988) concluded that gellan gum intake does not cause harmful dietary or physiological effects.In addition, Anderson (1988) concluded that gellan gum has a fecal mass enhancing effect. Consistent with the fecal bulking effect observed by Anderson (1988), a subsequent study found that gellan gum reduced diarrhea in cats. (US9028861B2.) Citing Tetsuguchi (1997), Li (2019) mentions, without explanation or evidence, that gellan as an oligosaccharide reportedly has prebiotic effects in the gut, although Tetsuguchi (1997) did not explicitly evaluate the prebiotic effects of gellan as an oligosaccharide in the intestines. To date, there are no studies that have conclusively demonstrated whether gellan gum or an oligosaccharide derived from gellan gum acts as a prebiotic.

[0008] Velan (velan gum or S-130) is produced by Sphingomonas sp. (eg ATCC 31555). (US4342866A and US5175277A.) Approximately two-thirds of the welan (1→4)Glc units are substituted at the O ³ position with an α-L-rhamnopyranosyl group (i.e., Rha(α1→)), while the remainder are (1→4)Glc The welan -units are replaced by an α-L-mannopyranosyl group (i.e. Man(α1→)). (Stankowski (1992).) In addition, the (1→3)Glc unit of welan can be replaced at the O ² position by acetyl. (Stankowski (1992).)

[0009] Ramsan (Ramsan gum or S-194) is produced by Sphingomonas sp. (eg ATCC 31961). (US4401760A.) Ramsan is replaced at the O ⁶ position of the (1→3)Glc unit by D-Glc(β1→6)-D-Glc(α1→). (Jansson (1986).) Ramsan contains one O-acetyl group per repeat unit, distributed over secondary positions. (Jansson (1986).)

[0010] Diutan (diutan gum or S-657) is produced by Sphingomonas sp. (eg ATCC 53159). (US5175278A and US20130189748A1.) The (1→4)Glc unit of diutan is replaced at the O ³ position by Rha(α1→4)-Rha(α1→), at the O ⁶ position by acetyl, and to varying degrees at the O ² and/ or O ⁶ (1→3)Glc units per acetyl. (Diltz (2001).)

Summary of the Present Invention

[0011] Disclosed herein is an oral composition containing sphingan and its use as a prebiotic.

Abbreviations

[0012] The following text includes numerous abbreviated terms. Abbreviations for selected terms disclosed herein are provided below.

[0013] A: Donor A (female, 28 years old)

[0014] Ac: acetate

[0015] B: Donor B (female, 41 years old)

[0016] b-SCFA: branched short chain fatty acids (e.g. isobutyrate, isovalerate and isocaproate)

[0017] C: Donor C (female, 34 years old)

[0018] C1: control period 1

[0019] C2: control period 2

[0020] CON(ave): average concentration for control periods 1 and 2

[0021] CD: Crohn's disease

[0022] DC: distal colon as fermenter

[0023] DP: degree of polymerization

[0024] Glc: D-glucopyranosyl

[0025] GlcA: D-glucopyranosyluronic acid

[0026] Glyc: L-glycerate

[0027] GPRs: G protein coupled receptor

[0028] HA: highly acyl

[0029] HA/LA: high acyl or low acyl

[0030] IBD: inflammatory bowel disease

[0031] IBS: irritable bowel syndrome

[0032] IFN: interferon

[0033] IL: interleukin

[0034] LA: low acyl

[0035] LCSs: long-chain sphingans

[0036] LPS: lipopolysaccharide

[0037] MAMP: microorganism associated molecular patterns

[0038] Man: L-mannopyranosyl

[0039] mM: millimolar (i.e. millimoles per liter)

[0040] MN: number average molecular weight

[0041] MW: weight average molecular weight

[0042] NaB: sodium butyrate

[0043] OTU: operational taxonomic unit

[0044] PHB: polyhydroxyoxybutyrate

[0045] PC: proximal colon

[0046] PRR: pattern recognition receptors

[0047] Rha: L-rhamnopyranosyl

[0048] ROS: reactive oxygen species

[0049] SCFA: short chain fatty acids (e.g. acetate, propionate and butyrate)

[0050] SHIME: a simulator of the microbial ecosystem in the human intestine

[0051] SOS: sphingan oligosaccharide

[0052] SPS: sphingan polysaccharide

[0053] TEER: transepithelial electrical resistance

[0054] Tetramer: [Glc(β1→4)GlcA(β1→4)Glc(β1→4)Rha], Glc, GlcA, Glc, Rha or Glc2, GlcA, Rha

[0055] Octamer: [Glc(β1→4)GlcA(β1→4)Glc(β1→4)Rha] ₂ Glc, GlcA, Glc, Rha, Glc, GlcA, Glc, Rha or Glc4, GlcA2, Rha2

[0056] SEC: gel filtration

[0057] TGF: transforming growth factor

[0058] TLR: toll-like receptor

[0059] TNF: tumor necrosis factor

[0060] TR1: processing period 1

[0061] TR2: processing period 2

[0062] TR3: processing period 3

[0063] TRT(ave): Average concentration for treatment periods 1, 2 and 3

[0064] UC: ulcerative colitis

Brief description of drawings

[0065] FIG. 1a. Gel filtration chromatogram for acid (SN9, solid line) and enzyme (SN18, dashed line) treated sphingans as poly- and oligosaccharides derived from high acyl gellan, showing elution times of pullulan molecular weight standards (namely >50 kDa (6.5 min, filled square ( )), 12 kDa (8.8 min, filled circle ( )), 5 kDa (9.3 min, filled triangle ( )), 1 kDa (10 min, empty square ( )), 342 Yes (10.65 min, empty circle ( )), and 180 Yes (11.15 min, empty triangle ( ))).

[0066] FIG. 1b. Gel filtration chromatogram for acid (SN10, solid line) and enzyme (SN17, dotted line) treated sphingans as poly- and oligosaccharides derived from low acyl gellan, showing elution times of pullulan molecular weight standards (namely, >50 kDa (6.5 min, filled square ( )), 12 kDa (8.8 min, filled circle ( )), 5 kDa (9.3 min, filled triangle ( )), 1 kDa (10 min, empty square ( )), 342 Yes (10.65 min, empty circle ( )), and 180 Yes (11.15 min, empty triangle ( ))).

[0067] FIG. 2a. Average acetate production (mM) over the control period (CON(ave.), n=6) and treatment period (TRT(ave), n=9) for the proximal colon (PC) fermenter for three different donors (A , B and C), where * indicates statistically significant differences from the previous period, while different letters indicate statistical differences between different treatments; p<0.05.

[0068] FIG. 2b. Average acetate production (mM) over the control period (CON(ave.), n=6) and treatment period (TRT(ave), n=9) for the fermenter as the distal colon (DC) for three different donors (A , B and C), where * indicates statistically significant differences from the previous period, while different letters indicate statistical differences between different treatments; p<0.05.

[0069] FIG. 3a. Average propionate production (mM) in the fermenter as the proximal colon (PC) during the control period (CON(ave), n=6) and treatment period (TRT(ave), n=9) for three different donors (A, B and C), where * indicates statistically significant differences compared with the previous period, while different letters indicate statistical differences between different treatments; p<0.05.

[0070] FIG. 3b. Average propionate production (mM) in the fermenter as distal colon (DC) during the control period (CON(ave), n=6) and treatment period (TRT(ave), n=9) for three different donors (A, B and C), where * indicates statistically significant differences compared with the previous period, while different letters indicate statistical differences between different treatments; p<0.05.

[0071] FIG. 4a. Average butyrate production (mM) in the fermenter as the proximal colon (PC) during the control period (CON(ave), n=6) and treatment period (TRT(ave), n=9) for three different donors (A, B and C), where * indicates statistically significant differences compared with the previous period, while different letters indicate statistical differences between different treatments; p<0.05.

[0072] FIG. 4b. Average butyrate production (mM) in the fermenter as distal colon (DC) during the control period (CON(ave), n=6) and treatment period (TRT(ave), n=9) for three different donors (A, B and C), where * indicates statistically significant differences compared with the previous period, while different letters indicate statistical differences between different treatments; p<0.05.

[0073] FIG. 5a. Average lactate production (mM) in the fermenter as the proximal colon (PC) during the control period (CON(ave), n=6) and treatment period (n=9) for three different donors (A, B and C), where different letters indicate statistical differences between different treatments; p<0.05.

[0074] FIG. 5b. Average lactate production (mM) in the fermenter as distal colon (DC) during the control period (CON(ave), n=6) and treatment period (n=9) for three different donors (A, B and C), where different letters indicate statistical differences between different treatments; p<0.05.

[0075] Figure 6a. Average ammonia production (mg/L) in the fermenter as the proximal colon (PC) for the control period (CON(ave), n=6) and treatment period (TRT(ave), n=9) for three different donors ( A, B and C), where different letters indicate statistical differences between different treatments; p<0.05.

[0076] FIG. 6b. Average ammonia production (mg/L) in the fermenter as distal colon (DC) for the control period (CON(ave), n=6) and treatment period (TRT(ave), n=9) for three different donors ( A, B and C), where different letters indicate statistical differences between different treatments; p<0.05.

[0077] Figure 7a . Average production of branched chain SCFAs (mM) in the fermenter as the proximal colon (DC) for the control period (CON(ave), n=6) and treatment period (TRT(ave), n=9) for three different donors (A, B, and C), where different letters indicate statistical differences between different treatments; p<0.05.

[0078] Figure 7b. Average production of branched chain SCFAs (mM) in the fermenter as distal colon (DC) during the control period (CON(ave), n=6) and treatment period (TRT(ave), n=9) for three different donors (A, B and C), where * indicates statistically significant differences from the previous period and different letters indicate statistical differences between different treatments; p<0.05.

[0079] FIG. 8. Reciprocal of Simpson's Diversity Index for Proximal Colon (PC) or Distal Colon (DC) SHIME Fermenter Lumen and Mucus at Different Time Points During Control Period (C1 and C2) and Treatment Period (TR1, TR2 and TR3) ) gellan gum for three different donors (n=1). Shading intensity indicates the overall diversity index normalized for each of the three different donors (i.e., within each row).

[0080] FIG. 9. Relative abundance (%) of dominant types in the lumen or mucus of the proximal (PC) or distal (DC) colon as SHIME fermenter at different time points during the control period (C1 and C2) and treatment period (TR1, TR2 and TR3) gellan gum for three different human donors (n=1). NB One sample was a clear outlier and was therefore removed from this control sample analysis, i.e. mucosal sample in the PC of donor A in the second control week (C2).

[0081] FIG. 10. Relative abundance (%) of different families belonging to specific phyla in the lumen of the proximal colon (PC) as SHIME fermenters at different time points during the control period (C1 and C2) and treatment period (TR1, TR2 and TR3 ) gellan gum for three different human donors (n=1). Shading intensity indicates the absolute amount normalized for each of the different families (i.e. within each row). Shading intensity indicates the absolute amount normalized for each of the different families (i.e., within each row).

[0082] FIG. 11. Relative abundance (%) of different families belonging to specific phyla in the lumen of the distal colon (DC) as SHIME fermenters at different time points during the control period (C1 and C2) and treatment period (TR1, TR2 and TR3 ) gellan gum for three different human donors (n=1). Shading intensity indicates the absolute amount normalized for each of the different families (i.e., within each row).

[0083] FIG. 12. Relative abundance (%) of different families belonging to specific phyla in proximal colon (PC) mucus as SHIME fermenters at different time points during the control period (C1 and C2) and treatment period (TR1, TR2 and TR3 ) gellan gum for three different human donors (n=1). Shading intensity indicates the absolute amount normalized for each of the different families (i.e. within each row). As a note, one sample was a clear outlier and was therefore removed from this control sample analysis, i.e. PC mucosal sample from donor A at the second control week (C2).

[0084] FIG. 13. Relative abundance (%) of different families belonging to specific phyla in distal colon (PC) mucus as SHIME fermenters at different time points during the control period (C1 and C2) and treatment period (TR1, TR2 and TR3 ) gellan gum for three different human donors (n=1). Shading intensity indicates the absolute amount normalized for each of the different families (i.e., within each row).

[0085] FIG. 14. Schematic representation of co-culture of Caco-2 and THP1 cells. Caco-2 cells are seeded onto a semi-permeable membrane, which is placed on top of wells seeded with THP1 cells. This creates an apical (AP) and basolateral (BL) compartment. The Caco-2 cell monolayer creates a barrier to macromolecules and allows passage by passive transfer of small molecules between the intercellular spaces and active transfer of micro- and macromolecules across cell membranes. Co-culture of both cell types allows for indirect interaction between the luminal contents, which are brought into contact with Caco-2 cells, and the peri-intestinal contents, which are brought into contact with immune cells (THP1). In addition, metabolites used/transformed by epithelial cells can modulate the immune cellular response, and vice versa.

[0086] FIG. 15. Signal transduction cascade activated when the intestinal epithelial barrier is damaged, resulting in a breach of the luminal contents of the intestinal cell. IFN-γ: interferon gamma; IL: interleukins; MCP-1: monocyte chemoattractant protein 1; ROS: reactive oxygen species; TGF-β: transforming growth factor beta; _TH : T helper cells; TNF-α: tumor necrosis factor alpha; T _reg : regulatory T cells.

[0087] FIG. 16. LPS and TNF-α signaling pathways leading to inflammation. AP-1: activator protein 1 (transcription factor); IL: interleukins, LPS: lipopolysaccharides; NF-κB: nuclear factor kappa B (transcription factor); TLR4: toll-like receptor 4 (LPS receptor); TNF-α: tumor necrosis factor alpha; TNFR: TNF-α receptor.

[0088] FIG. 17. Transepithelial electrical resistance (TEER) in CM and NaB control studies. TEER was measured 24 hours after treatment with Caco-2/THP1-Blue™ cocultures, and each 24 hour value was normalized to its corresponding 0 hour value and presented as a percentage of the baseline value. The dotted line represents 100% (original value). Data are plotted as mean ± standard error of the mean (SEM). (*) represents a statistically significant difference between CM and NaB. (****) = p<0.0001. CM: full Wednesday; NaB: sodium butyrate.

[0089] FIG. 18. Basolateral NF-κB activity in THP1-Blue™ cells in LPS-, LPS+, LPS+HC and LPS+NaB controls. NF-κB activity was measured after 6 hours of LPS treatment of Caco-2/THP1-Blue™ cocultures on the basolater side after pretreatment for 24 hours with NaB or complete medium on the apical side. Data are plotted as mean ± SEM. (*) represents a statistically significant difference compared to LPS+. (*) = p<0.05; (****) = p<0.0001. LPS-: cells treated with complete medium (without LPS); LPS+: cells treated with LPS; HC: hydrocortisone; NaB: sodium butyrate.

[0090] FIG. 19. Basolateral secretion of IL-6 (A) and IL-10 (B) during control studies with LPS-, LPS+, LPS+HC and LPS+NaB. Cytokines were measured after 6 hours of LPS treatment of Caco-2/THP1-Blue™ cocultures on the basolater side after pretreatment for 24 hours with NaB or complete medium on the apical side. Data are plotted as mean ± SEM. (*) represents a statistically significant difference compared to LPS+. (***) = p<0.001; (****) = p<0.0001. LPS-: cells treated with complete medium (without LPS); LPS+: cells treated with LPS; HC: hydrocortisone; NaB: sodium butyrate.

[0091] FIG. 20. Basolateral secretion of IL-1β (A), IL-8 (B), CXCL10 (C), TNF-α (D) and MCP-1 (E) during LPS-, LPS+, LPS+HC and LPS+ control studies NaB. Cytokines were measured after 6 hours of treatment with Caco-2/THP1-Blue™ cocultures on the basolater side after pretreatment for 24 hours with NaB or complete medium on the apical side. Data are plotted as mean ± SEM. (*) represents a statistically significant difference compared to LPS+. (*) = p<0.05; (**) = p<0.01; (***) = p<0.001; (****) = p<0.0001. LPS-: cells treated with complete medium (without LPS); LPS+: cells treated with LPS; HC: hydrocortisone; NaB: sodium butyrate.

[0092] FIG. 21. Effect of SHIME samples on transepithelial electrical resistance (TEER) of Caco-2/THP1-Blue™ cocultures. Results are shown for three different donors separately (A) and as an average of three donors (B). TEER was measured 24 hours after treatment of the cocultures, and each value at time point=24 hours was normalized to its corresponding value at time point=0 hours and presented as a percentage of the initial value. The gray dotted line represents 100% (original value). The dotted line corresponds to the CM experimental control (complete medium). Data are plotted as mean ± SEM. No significant differences were found between the control and the three different donor treatments. PC: proximal colon samples; DC: samples from the distal colon.

[0093] FIG. 22. Effect of SHIME samples on NF-κB activity in THP-1-Blue™ cells. Results are shown for three different donors separately (A) and as an average of three donors (B). Levels of NF-κB activity were measured 6 hours after LPS treatment on the basolateral side of Caco-2/THP-1-Blue™ cocultures after pretreatment of the apical side for 24 hours with SHIME samples. The dotted line corresponds to the LPS+ experimental control. Data are plotted as mean ± SEM. No significant differences were found between the control and the three different donor treatments. PC: proximal colon samples; DC: samples from the distal colon.

[0094] FIG. 23. Effect of SHIME samples on the secretion of IL-6 (A and B) and IL-10 (C and D). Results are shown for three different donors separately (A and C) and as an average of three donors (B and D). Cytokine levels were measured 6 hours after LPS treatment on the basolateral side of Caco-2/THP-1-Blue™ cocultures after pretreating the apical side for 24 hours with SHIME samples. The dotted line corresponds to the LPS+ experimental control. Data are plotted as mean ± SEM. (*) represents a statistically significant difference compared to control. (*) = p<0.05. PC: proximal colon samples; DC: samples from the distal colon.

[0095] FIG. 24. Effect of SHIME samples on the secretion of IL-1β (A+B) and TNF-α (C+D). Results are shown for three different donors separately (AC) and as an average of three donors (BD). Cytokine levels were measured 6 hours after LPS treatment on the basolateral side of Caco-2/THP-1-Blue™ cocultures after pretreating the apical side for 24 hours with SHIME samples. The dotted line corresponds to the LPS+ experimental control. Data are plotted as mean ± SEM. (*) represents a statistically significant difference compared to control. (****) = p<0.0001 PC: samples from the proximal colon; DC: samples from the distal colon.

[0096] FIG. 25 . Effect of SHIME samples on the secretion of IL-8 (A+B), CXCL10 (C+D) and MCP-1 (E+F). Results are shown for three different donors separately (ACE) and as an average of three donors (BDF). Cytokine levels were measured 6 hours after LPS treatment on the basolateral side of Caco-2/THP-1-Blue™ cocultures after pretreating the apical side for 24 hours with SHIME samples. The dotted line corresponds to the LPS+ experimental control. Data are plotted as mean ± SEM. No significant differences were found between control and treatment for the three different donors. PC: proximal colon samples; DC: samples from the distal colon.

Definitions

[0097] As used herein, the term "sphingan" refers to high acyl sphingan, medium acyl sphingan, low acyl sphingan, high acyl polysaccharide sphingan, medium acyl polysaccharide sphingan, low acyl polysaccharide sphingan, high acyl oligosaccharide sphingan, medium cyl sphingan in the form of an oligosaccharide, low acyl sphingan in the form of an oligosaccharide or a combination thereof.

[0098] As used herein, the term “high acyl” (or “HA”) refers to a sphingan containing an acyl group (eg, acetyl and glyceryl). High acyl sphingan includes, for example, HA gellan, HA welan, HA ramsan, HA diutan, etc.

[0099] As used herein, the term “medium acyl” (or “IA”) refers to a sphingan having less acyl group content than a high acyl sphingan but greater than the acyl group content of a low acyl sphingan. Medium acyl sphingan includes, for example, IA gellan, IA velan, IA ramsan, IA diutan, etc.

[0100] As used herein, the term “low acyl” (or “LA”) refers to sphingan in which the acyl group(s) have been substantially removed. Low acyl sphingan includes, for example, LA gellan, LA velan, LA ramsan, LA diutan, etc.

[0101] Naturally occurring sphingan may include, for example, gellan (S-60), welan (S-130), ramsan (S-194), diutan (S-657), S-88, S-198 and S- 7, consisting of a substituted or unsubstituted tetrameric saccharide ("tetramer"), usually denoted as [(→3)Glc(β1→4)GlcA(β1→4)Glc(β1→4)Rha(α1→)]n, where Glc and GlcA are D-sugars, while Rha is an L-sugar, and, where applicable, Man is an L-sugar. The chemical structures of selected sphingans are presented below by indicating abbreviated terms for individual monosaccharides (e.g. (1→3)Glc, (1→4)GlcA, (1→4)Glc and (1→4)Rha))

Sphingan R ₁ R ₂ R ₃ R ₄ R ₅ R ₆ HA gellan Ac or H Glyc M H H H LA gellan H H M H H H Diutan Ac or H Ac or H M H Rha(α1→4)-Rha(α1→) H Ramsan ^a Glc(β1→6)-Glc(α1→) H M H H H Velan H Ac M H Rha(α1→) or Man(α1→) H ^a Ramsan contains approximately one O -acetyl group per tetramer, distributed across secondary positions.

[0102] As used herein, the term "M" refers to a physiologically acceptable cation, including, for example, proton (H⁺), sodium (Na⁺), potassium (K⁺), calcium (Ca²⁺), magnesium (Mg²⁺) or combinations thereof.

[0103] The value "n" refers to an integer or fraction and refers to the number of tetrameric units that may be substituted or unsubstituted. It is understood that some naturally occurring sphingans have an n value that may correlate with the molecular weight of the naturally occurring sphingan (eg, natural gellan gum, having MW ≈ 2.5 x 10 ⁶ and MN ≈ 2.2 x 10 ⁶ ). (US6242035B1)

[0104] As used herein, the expression "degree of polymerization" or DP refers to the number of monosaccharide units in a polysaccharide or oligosaccharide chain. For example, referring to the chemical structure presented above, where n is four, DP is sixteen.

[0105] As used herein, the term "sphingan polysaccharide" (or "SPS") refers to a high/low acyl sphingan having a DP greater than 30 and less than that of natural sphingan. It is understood that SPS derived from high/medium/low acyl sphingan may include a variety of polysaccharides with different DPs.

[0106] As used herein, the term "sphingan oligosaccharide" (or "SOS") refers to a high/low acyl sphingan having a DP greater than or equal to two and less than or equal to thirty (i.e., 2≥ DP≤30) . It is understood that SOS derived from high/medium/low acyl sphingan (or HA/IA/LA sphingan) may include a variety of oligosaccharides

Detailed Description of the Present Invention

[0107] The embodiments disclosed herein generally relate to an oral composition, an oral composition and its use, methods of using an oral composition, a method for producing sphingan oligosaccharide, and sphingan oligosaccharide obtained by said method for producing sphingan in form of an oligosaccharide.

[0108] A first embodiment is directed to an oral composition containing an effective amount of sphingan to exhibit prebiotic activity.

[0109] The amount of sphingan effective to exert prebiotic activity can range from about 1 g to about 10 g, and everything in between, such as about 1.1, about 1.2, about 1.3, about 1.4 , approximately 1.5, approximately 1.6, approximately 1.7, approximately 1.8, approximately 1.9, approximately 2.0, approximately 2.1, approximately 2.2, approximately 2.3, approximately 2.4 , approximately 2.5, approximately 2.6, approximately 2.7, approximately 2.8, approximately 2.9, approximately 3.0, approximately 3.1, approximately 3.2, approximately 3.3, approximately 3.4 , approximately 3.5, approximately 3.6, approximately 3.7, approximately 3.8, approximately 3.9, approximately 4.0, approximately 4.1, approximately 4.2, approximately 4.3, approximately 4.4 , approximately 4.5, approximately 4.6, approximately 4.7, approximately 4.8, approximately 4.9, approximately 5.0, approximately 5.1, approximately 5.2, approximately 5.3, approximately 5.4 , approximately 5.5, approximately 5.6, approximately 5.7, approximately 5.8, approximately 5.9, approximately 6.0, approximately 6.1, approximately 6.2, approximately 6.3, approximately 6.4 , approximately 6.5, approximately 6.6, approximately 6.7, approximately 6.8, approximately 6.9, approximately 7.0, approximately 7.1, approximately 7.2, approximately 7.3, approximately 7.4 , approximately 7.5, approximately 7.6, approximately 7.7, approximately 7.8, approximately 7.9, approximately 8.0, approximately 8.1, approximately 8.2, approximately 8.3, approximately 8.4 , approximately 8.5, approximately 8.6, approximately 8.7, approximately 8.8, approximately 8.9, approximately 9.0, approximately 9.1, approximately 9.2, approximately 9.3, approximately 9.4 , about 9.5, about 9.6, about 9.7, about 9.8, or about 9.9.

[0110] In one aspect of the first embodiment, the amount of sphingan is selected from: about 1 g to about 10 g, about 1 g to about 9 g, about 1 g to about 8 g, about 1 g to about 7 g, from about 1 g to about 6 g, from about 1 g to about 5 g, from about 1 g to about 4 g, from about 1 g to about 3 g, or about 2 g.

[0111] The compositions of the first embodiment may contain HA/IA/LA sphingan, for example, HA gellan, IA gellan, LA gellan, HA velan, IA velan, LA velan, HA ramsan, IA ramsan, LA ramsan, HA diutan, IA diutan , LA diutan, S-88, S-198, S-7, or a combination thereof.

[0112] The compositions of the first embodiment may contain HA/IA/LA sphingan as a polysaccharide.

[0113] As explained here in more detail, HA/IA/LA sphingan as a polysaccharide can be prepared from HA/LA sphingan using, for example, a method that includes high pressure homogenization, as described, for example, in the tenth embodiment. Exemplary HA/IA/LA polysaccharide sphingans include, but are not limited to: high acyl polysaccharide gellan derived from high acyl gellan (e.g. ^KELCOGEL® LT100 and ^KELGOGEL® HT gellan), medium acyl polysaccharide gellan, derived from medium acyl gellan (e.g. KELCOGEL ^® DGA), low acyl gellan as a polysaccharide derived from low acyl gellan (e.g. KELCOGEL ^® LT gellan, KELCOGEL ^® gellan, KELCOGEL ^® F gellan and GELRITE ^TM MK gellan), high/medium -/low acyl welan as a polysaccharide derived from high/medium/low acyl welan, high/medium/low acyl diutan as a polysaccharide derived from high/medium/low acyl diutan, and high/medium/low acyl ramsan in the form of a polysaccharide obtained from high/medium/low acyl ramsan.

[0114] The compositions of the first embodiment may contain HA/IA/LA sphingan as an oligosaccharide derived from either naturally occurring HA/IA/LA sphingan or from HA/IA/LA sphingan as a polysaccharide. In one aspect, the compositions of the first embodiment may contain HA/IA/LA sphingan as an oligosaccharide derived from either naturally occurring HA/IA/LA sphingan or from HA/IA/LA sphingan as a polysaccharide having a molecular weight that , as determined by gel filtration, ranges from approximately 0.3 kDa to 12 kDa. In another aspect, the compositions of the first embodiment may contain HA/IA/LA sphingan as an oligosaccharide derived from either naturally occurring HA/IA/LA sphingan or from HA/IA/LA sphingan as a polysaccharide having a molecular weight that , as determined by gel filtration, is approximately 1 kDa.

[0115] As explained in more detail herein, HA/IA/LA sphingan oligosaccharide can be prepared from naturally occurring HA/IA/LA sphingan or HA/IA/LA sphingan polysaccharide, for example, by a process that involves hydrolyzing a glycosidic linking naturally occurring HA/IA/LA sphingan or HA/IA/LA sphingan as a polysaccharide and subjecting the hydrolyzed composition to ultrafiltration, gel filtration, precipitation, centrifugation, or a combination thereof, as described, for example, in the tenth embodiment. Exemplary HA/IA/LA sphingan oligosaccharides include, but are not limited to:

(i) a composition containing (or consisting of) Glc, GlcA, Glc, GlcA, Glyc, Glc, GlcA, Rha, Glc, GlcA, Rha, Glyc, Glc, GlcA, Rha, -H2O, Glc, Rha, Glc, Rha+28, Glc2,GlcA, Glc2,GlcA, Rha, Glc2,GlcA, Rha,+28, Glc2,GlcA, Rha,Ac, Glc2,GlcA, Rha,Glyc, Glc2,GlcA, Rha,Glyc,+28, Glc2,GlcA, Rha,Glyc.-H2O, Glc2,GlcA, Rha,-H2O, Glc2,GlcA, Rha2,Glyc, Glc2,GlcA2,Rha, Glc2,GlcA2,Rha2,Ac2,Glyc2,-H2O, Glc2,Rha , Glc3,GlcA, Rha, Glc3,GlcA, Rha2, Glc3,GlcA, Rha2, Glc3,GlcA, Rha2, Glc3,GlcA, Rha2,Glyc, Glc3,GlcA2,Rha, Glc3,GlcA2,Rha, Glyc, Glc3,GlcA2 ,Rha2,Glyc, Glc3,GlcA3,Rha2, Glc3,GlcA3,Rha2, Glc4,GlcA, Rha2,+43, Glc4,GlcA, Rha2,Ac, Glyc, Glc4,GlcA2,Rha, Glc4,GlcA2,Rha, Ac, Glyc,-H2O, Glc4,GlcA2,Rha, Ac,Glyc2, Glc4,GlcA2,Rha2,Ac, Glyc, Glc4,GlcA2,Rha2,Glyc, Glc4,GlcA3,Rha2, Glc4,GlcA2,Rha3,Ac, Glc4,GlcA3 ,Rha2/Glc4,GlcA2,Rha2,Glyc2, Glc5,GlcA2,Rha2, Glc5,GlcA2,Rha2, Glc5,GlcA2,Rha2,Ac, Glc5,GlcA4,Rha2, Glc6,GlcA3,Rha3, Glc(Ac/Glyc)x , GlcAx, Glcx, Rhax (where x is from 4 to about 25), Glcx, GlcAx, Glcx, Rhax (where x is from 4 to about 25), or a combination thereof;

(ii) a composition containing (or consisting of) a tetramer (Glc, GlcA, Glc, Rha), a tetramer (Glc, GlcA, Glc, Rha) with acetate and/or glycerate, an octamer (Glc, GlcA, Glc, Rha, Glc , GlcA, Glc, Rha), octamer (Glc, GlcA, Glc, Rha, Glc, GlcA, Glc, Rha) with acetate and/or glycerate, Glc, GlcA, Glc, Rha, Glc, GlcA, Glc, Rha, or their combination;

(iii) a composition containing (or consisting of) a tetramer (Glc, GlcA, Glc, Rha), an octamer (Glc, GlcA, Glc, Rha, Glc, GlcA, Glc, Rha), a pentamer (Glc, GlcA, Glc, Rha ,Glc), GlcA, Glc,Rha, Glc, GlcA,Glc, Glc, GlcA, or a combination thereof;

(iv) a composition containing (or consisting of) Glc(Glc-Glc), GlcA, Glc(Glc-Glc), GlcA, Glc, Glc, Glc, or a combination thereof;

(v) a composition containing (or consisting of) a tetramer (Glc, GlcA, Glc, Rha), GlcA, Glc, (Rha-Rha), Glc, (Rha-Rha), Rha, GlcA, Glc, Rha, Glc, GlcA, Glc, Rha, Glc, GlcA, Glc;

(vi) a composition containing (or consisting of) Glc, GlcA, Glc, GlcA, Glyc, Glc, GlcA, Rha, Glc, GlcA, Rha, Glyc, Glc, Rha, Glc, Rha+28, Glc2, GlcA, Glc2 ,GlcA, Rha, Glc2,GlcA, Rha,+28, Glc2,GlcA, Rha,Ac, Glc2,GlcA, Rha,Glyc, Glc2,GlcA, Rha,Glyc,+28, Glc3,GlcA, Rha, Glc3,GlcA , Rha2, Glc3, GlcA, Rha2, Glc3, GlcA, Rha2, Glc3, GlcA, Rha2, Glyc, Glc3, GlcA2, Rha, Glyc, Glc3, GlcA2, Rha2, Glyc, Glc3, GlcA3, Rha2, Glc4, GlcA, Rha2 ,Ac, Glyc, Glc4,GlcA2,Rha2,Ac, Glyc, Glc4,GlcA2,Rha2,Glyc, Glc4,GlcA2,Rha3,Ac, Glc4,GlcA3,Rha2/Glc4,GlcA2,Rha2,Glyc2, Glc5,GlcA2,Rha2 , Glc5, GlcA2, Rha2, Ac, Glc(Ac/Glyc)x, GlcAx, Glcx, Rhax (where x is from 4 to about 25), or a combination thereof;

(vii) a composition containing (or consisting of) Glc, GlcA, Glc, GlcA, Rha, Glc, Rha, Glc, Rha+28, Glc2,GlcA, Rha, Glc2,GlcA, Rha,+28, Glc2,GlcA2, Rha, Glc3,GlcA, Rha, Glc3,GlcA, Rha2, Glc3,GlcA2,Rha, Glc3,GlcA3,Rha2, Glc3,GlcA3,Rha2, Glc4,GlcA, Rha2,+43, Glc4,GlcA2,Rha, Glc4,GlcA3 ,Rha2, Glc5,GlcA2,Rha2, Glc5,GlcA2,Rha2, Glc5,GlcA4,Rha2, Glc6,GlcA3,Rha3, Glcx, GlcAx,Glcx, Rhax (where x is from 4 to about 25), or a combination thereof;

(viii) a composition containing (or consisting of) Glc, GlcA,Rha,-H2O, Glc, Rha, Glc2,GlcA, Rha,-H2O, Glc2,Rha, or a combination thereof;

(ix) a composition containing (or consisting of) Glc, GlcA, Glc, GlcA, Glyc, Glc, GlcA, Rhaa, Glc, GlcA, Rha, Glyc, Glc, Rha, Glc, Rha+28, Glc2, GlcA, Glc2 ,GlcA, Rha, Glc2,GlcA, Rha,+28, Glc2,GlcA, Rha,Ac, Glc2,GlcA, Rha,Glyc, Glc2,GlcA, Rha,Glyc,+28, Glc2,GlcA, Rha,Glyc.- H2O, Glc2,GlcA, Rha2,Glyc, Glc2,GlcA2,Rha2,Ac2,Glyc2,-H2O, Glc3,GlcA, Rha, Glc3,GlcA, Rha2, Glc3,GlcA, Rha2,Glyc, Glc3,GlcA2,Rha, Glyc , Glc3,GlcA2,Rha2,Glyc, Glc3,GlcA3,Rha2, Glc4,GlcA, Rha2,+43, Glc4,GlcA, Rha2,Ac, Glyc, Glc4,GlcA2,Rha, Ac,Glyc,-H2O, Glc4,GlcA2 ,Rha, Ac,Glyc2, Glc4,GlcA2,Rha2,Ac, Glyc, Glc4,GlcA2,Rha2,Glyc, Glc4,GlcA3,Rha2, Glc4,GlcA2,Rha3,Ac, Glc4,GlcA3,Rha2/Glc4,GlcA2,Rha2 ,Glyc2, Glc5,GlcA2,Rha2, Glc5,GlcA2,Rha2,Ac, Glc(Ac/Glyc)x, GlcAx,Glcx, Rhax (where x is from 4 to about 25), or a combination thereof;

(x) a composition containing (or consisting of) any of samples Nos. 1-18; or

(xi) a composition containing (or consisting of) any of samples Nos. 9, 10, 17 and 18.

[0116] As noted above, some sphingans may be substituted with an acyl substituent, a monosaccharide, or a disaccharide side chain (e.g., the (1→4)Glc of diutan is substituted at positionO ³ side chain Rha(α1→4)-Rha(α1→)). A substituted oligosaccharide having a saccharide side chain is designated using parentheses, for example, GlcA, Glc,(Rha-Rha) and Glc,(Rha-Rha),Rha.

[0117] And reference to HA/IA/LA sphingan oligosaccharide is intended to mean any of the exemplary HA/IA/LA sphingan oligosaccharides or a combination thereof.

[0118] The compositions may be in the form of liquids, semi-solids or solids. The compositions may be in the form of cereals, snacks, or other oral forms. The compositions may be fruit based, such as juice or smoothie, or dairy based, such as milk, ice cream or yoghurt. The compositions may suitably be in the form of drinks. The term "beverage" includes the ready-to-drink liquid form, as well as the concentrate and powder form for dissolution. The ready-to-drink drink can be still or carbonated.

[0119] The compositions may be unsweetened or sweetened with sugar or potent sweeteners such as sucralose, ammonium glycyrrhizinate, acesulfame-K, aspartame, saccharin, salt (e.g., sodium, potassium, calcium, etc.) of saccharin, sodium cyclamate, stevia , other non-sugar sweeteners and mixtures thereof. The compositions may also contain other conventional additives such as flavoring agents, coloring agents, stabilizers, etc.

[0120] The compositions may be stored as a powder in a sealed container or package, which may include instructions for use.

[0121] Alternatively, the compositions may be formulated as a tablet or capsule product, which may contain, in addition to sphingan, other suitable excipients such as a binder, filler, lubricant, disintegrating agent, glidant, flow additive, agent anti-caking agent, sorbent, preservative, wetting agent, sweetener, flavoring agent, glazing agent, etc. The tablets may be coated according to methods well known in the art. Examples of fillers include, but are not limited to, alkaline earth metal carbonate (eg, magnesium carbonate, calcium carbonate, etc.); cross-linked polymer (eg cross-linked polyvinylpyrrolidone (crospovidone) and cross-linked sodium carboxymethylcellulose (croscarmellose sodium)); fatty acid; fumed silicon dioxide; lubricant (eg, stearic acid, stearin, magnesium stearate); a pH adjusting agent (eg, an acid (eg, hydrochloric acid) and a base (eg, sodium hydroxide)); plant fiber (such as corn protein zein); polysaccharide and derivatives thereof (eg starch, cellulose or modified cellulose such as microcrystalline cellulose and cellulose ethers such as hydroxypropylcellulose and hydroxypropylmethylcellulose); protein (eg gelatin); saccharide and its derivatives (eg disaccharide, eg sucrose, lactose, etc.); shellac; silica; sodium starch glycolate; sugar alcohol (eg isomaltitol, xylitol, sorbitol and maltitol); synthetic polymer (eg polyvinylpyrrolidone and polyethylene glycol), talc and wax.

[0122] The composition may also contain a probiotic and an additional prebiotic.

[0123] Examples of probiotics include, but are not limited to, Lactobacillus rhamnosus GG, Bifidobacterium infantis , Lactobacillus acidophilus , Bifidobacterium lactis HN019, Bifidobacterium longum (including strain 35624), Lactobacillus salivarius , Bifodobacterium bifidu m, Lactobacillus plantarum , Lactob acillus paracasei , Bifidobacterium breve , Lactobacillus gasseri KS-13, Bacillus coagulans (GBI-30, 6086), Bacillus subtilis DE111, each of which can be used alone or in combination.

[0124] Examples of additional prebiotics include, but are not limited to, inulin, fructooligosaccharide, galactooligosaccharide, guar gum, tara gum, xanthan gum, xanthan polysaccharide, xanthan oligosaccharide, konjac gum, karaya gum, arabinogalactan, lactulose, psyllium, pectin, pectin polysaccharide , pectin oligosaccharide, tragacanth, gum arabic, carrageenan, etc.

[0125] The results presented here indicate that sphingan (A) stimulates the growth of beneficial bacteria in the human colon; (B) reduces propionate levels and/or increases butyrate levels in the human colon; (C) improves the integrity of the intestinal barrier in the human colon; and/or (D) reduces the levels of TNF-α and/or IL-8 in the human colon. Accordingly, the embodiments disclosed herein relate to an oral composition for:

(A) stimulating the growth of beneficial bacteria in the colon of a mammal, wherein the composition contains an amount of sphingan effective to stimulate the growth of beneficial bacteria and an oral medium (second embodiment);

(B) reducing the level of propionate and/or increasing the level of butyrate in the colon of a mammal, wherein the composition contains an effective amount of sphingan and an oral medium (third embodiment);

(C) improving the integrity of the intestinal barrier in the colon of a mammal, wherein the composition contains an effective amount of sphingan to improve the integrity of the intestinal barrier and an oral medium (fourth embodiment); or

(D) reducing the levels of TNF-α and/or IL-8 in the colon of a mammal, wherein the composition contains an amount of sphingan effective to reduce the levels of TNF-α and/or IL-8 and an oral medium (fifth embodiment).

[0126] With respect to any of the second, third, fourth and fifth embodiments, the calculated amount of sphingan (i.e., (i) an effective amount of sphingan to promote the growth of beneficial bacteria (second embodiment), (ii) an effective amount of sphingan ( third embodiment), (iii) an amount of sphingan effective to improve intestinal barrier integrity (fourth embodiment), and (iv) an amount of sphingan effective to reduce TNF-α and/or IL-8 levels (fifth embodiment)) may be between about 1 g to about 10 g sphingan, and all values in between, such as about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, approximately 1.8, approximately 1.9, approximately 2.0, approximately 2.1, approximately 2.2, approximately 2.3, approximately 2.4, approximately 2.5, approximately 2.6, approximately 2.7, approximately 2.8, approximately 2.9, approximately 3.0, approximately 3.1, approximately 3.2, approximately 3.3, approximately 3.4, approximately 3.5, approximately 3.6, approximately 3.7, approximately 3.8, approximately 3.9, approximately 4.0, approximately 4.1, approximately 4.2, approximately 4.3, approximately 4.4, approximately 4.5, approximately 4.6, approximately 4.7, approximately 4.8, approximately 4.9, approximately 5.0, approximately 5.1, approximately 5.2, approximately 5.3, approximately 5.4, approximately 5.5, approximately 5.6, approximately 5.7, approximately 5.8, approximately 5.9, approximately 6.0, approximately 6.1, approximately 6.2, approximately 6.3, approximately 6.4, approximately 6.5, approximately 6.6, approximately 6.7, approximately 6.8, approximately 6.9, approximately 7.0, approximately 7.1, approximately 7.2, approximately 7.3, approximately 7.4, approximately 7.5, approximately 7.6, approximately 7.7, approximately 7.8, approximately 7.9, approximately 8.0, approximately 8.1, approximately 8.2, approximately 8.3, approximately 8.4, approximately 8.5, approximately 8.6, approximately 8.7, approximately 8.8, approximately 8.9, approximately 9.0, approximately 9.1, approximately 9.2, approximately 9.3, approximately 9.4, approximately 9.5, approximately 9.6, approximately 9.7, approximately 9.8 and approximately 9.9 g.

[0127] In one aspect of any of the second, third, fourth and fifth embodiments, the mammal is, for example, a human, a dog, a cat, a rat, a mouse, a hamster, a guinea pig, a cow, a bison, a pig, a sheep, a horse, a goat, deer, llama, alpaca, etc.

[0128] In one aspect of any of the second, third, fourth and fifth embodiments, the amount of sphingan is selected from: about 1 g to about 10 g, from about 1 g to about 9 g, from about 1 g to about 8 g, from about 1 g to about 7 g, from about 1 g to about 6 g, from about 1 g to about 5 g, from about 1 g to about 4 g, from about 1 g to about 3 g, or about 2 g.

[0129] And, in one aspect of any of the second, third, fourth and fifth embodiments, the amount of sphingan is sufficient to achieve an effective concentration of sphingan in the colon, wherein said concentration of sphingan in the colon ranges from about 1 mg/ml to approximately 10 mg/ml, and all values in between, for example, approximately 1.5 mg/ml, approximately 2 mg/ml, approximately 2.5 mg/ml, approximately 3 mg/ml, approximately 3.5 mg/ml , approximately 4 mg/ml, approximately 4.5 mg/ml, approximately 5 mg/ml, approximately 5.5 mg/ml, approximately 6 mg/ml, approximately 6.5 mg/ml, approximately 7 mg/ml, approximately 7.5 mg/ml, approximately 8 mg/ml, approximately 8.5 mg/ml, approximately 9 mg/ml or 9.5 mg/ml.

[0130] The compositions of any of the second, third, fourth and fifth embodiments may contain any of naturally occurring HA/IA/LA sphingan, HA/IA/LA sphingan polysaccharide, HA/IA/LA sphingan oligosaccharide, or a combination thereof, and optionally further contain a probiotic or additional prebiotic as described in the first embodiment.

[0131] In addition, embodiments disclosed herein relate to either a method for or use for the manufacture of a drug or dietary supplement for:

(A) stimulating the growth of beneficial bacteria in the colon of a mammal, the method comprising ingesting, in an effective regimen, an amount of sphingan and an oral medium effective in stimulating the growth of beneficial bacteria (sixth embodiment);

(B) reducing the level of propionate and/or increasing the level of butyrate in the colon of a mammal, the method comprising administering an effective regimen of a composition containing an effective amount of sphingan and an oral medium (seventh embodiment);

(C) improving the integrity of the intestinal barrier in the colon of a mammal, the method comprising ingesting an effective regimen of a composition containing an effective amount of sphingan to improve the integrity of the intestinal barrier and an oral medium (eighth embodiment);

(D) reducing the levels of TNF-α and/or IL-8 in the colon of a mammal, the method comprising ingesting in an effective regimen a composition containing an amount of sphingan effective to reduce the levels of TNF-α and/or IL-8 and a medium for oral administration (ninth embodiment);

(E) using the composition of any of the first, second, third, fourth and fifth embodiments alone or in combination with a probiotic or additional prebiotic described herein to produce a composition for (i) stimulating the growth of beneficial bacteria in the colon of a mammal (tenth embodiment embodiment), (ii) reducing the level of propionate and/or increasing the level of butyrate in the colon of the mammal (eleventh embodiment), (iii) improving the integrity of the intestinal barrier in the colon of the mammal (twelfth embodiment), or (iv) reducing the levels of TNF-- α and/or IL-8 in the colon of a mammal (thirteenth embodiment); or

(F) In one aspect of any of the sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth embodiments, the mammal is, for example, a human, a dog, a cat, a rat, a mouse, a hamster, a guinea pig, a cow, a bison, pig, sheep, horse, goat, deer, llama, alpaca, etc.

[0132] For these and other embodiments described and claimed herein, an effective oral regimen may include, for example, (i) daily dosing, eg, once, twice, three times a day, etc.; (ii) weekly dosing, for example every day for seven days, every other day for seven days, etc.; (iii) monthly dosing, such as daily dosing for a desired period of time followed by a period of rest, with continued dosing daily for the desired period of time.

[0133] With respect to any of the sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth embodiments, the calculated amount of sphingan (i.e., (i) the amount of sphingan effective for promoting the growth of beneficial bacteria (sixth embodiment) , (ii) an effective amount of sphingan (seventh embodiment), (iii) an effective amount of sphingan to improve intestinal barrier integrity (eighth embodiment), and (iv) an amount of sphingan effective to reduce TNF-α and/or IL-8 levels ( ninth embodiment)) may be from about 1 g to about 10 g of sphingan, and all values in between, such as about 1.1, about 1.2, about 1.3, about 1.4, about 1, 5, approximately 1.6, approximately 1.7, approximately 1.8, approximately 1.9, approximately 2.0, approximately 2.1, approximately 2.2, approximately 2.3, approximately 2.4, approximately 2, 5, approximately 2.6, approximately 2.7, approximately 2.8, approximately 2.9, approximately 3.0, approximately 3.1, approximately 3.2, approximately 3.3, approximately 3.4, approximately 3, 5, approximately 3.6, approximately 3.7, approximately 3.8, approximately 3.9, approximately 4.0, approximately 4.1, approximately 4.2, approximately 4.3, approximately 4.4, approximately 4, 5, approximately 4.6, approximately 4.7, approximately 4.8, approximately 4.9, approximately 5.0, approximately 5.1, approximately 5.2, approximately 5.3, approximately 5.4, approximately 5, 5, approximately 5.6, approximately 5.7, approximately 5.8, approximately 5.9, approximately 6.0, approximately 6.1, approximately 6.2, approximately 6.3, approximately 6.4, approximately 6, 5, approximately 6.6, approximately 6.7, approximately 6.8, approximately 6.9, approximately 7.0, approximately 7.1, approximately 7.2, approximately 7.3, approximately 7.4, approximately 7, 5, approximately 7.6, approximately 7.7, approximately 7.8, approximately 7.9, approximately 8.0, approximately 8.1, approximately 8.2, approximately 8.3, approximately 8.4, approximately 8, 5, approximately 8.6, approximately 8.7, approximately 8.8, approximately 8.9, approximately 9.0, approximately 9.1, approximately 9.2, approximately 9.3, approximately 9.4, approximately 9, 5, approximately 9.6, approximately 9.7, approximately 9.8 and approximately 9.9 g.

[0134] In one aspect of any of the sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, and thirteenth embodiments, the amount of sphingan is selected from: from about 1 g to about 10 g, from about 1 g to about 9 g, from about 1 g to about 8 g, about 1 g to about 7 g, about 1 g to about 6 g, about 1 g to about 5 g, about 1 g to about 4 g, about 1 g to about 3 g, or approximately 2 g.

[0135] And, in one aspect of any of the sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth embodiments, the amount of sphingan is sufficient to achieve an effective concentration of sphingan in the colon as described herein.

[0136] Alternatively, and with respect to any of the sixth, seventh, eighth and ninth embodiments, the mammal is a human and the calculated amount of sphingan (i.e., (i) an amount of sphingan effective to promote the growth of beneficial bacteria (sixth embodiment implementation), (ii) an effective amount of sphingan (seventh embodiment), (iii) an amount of sphingan effective to improve the integrity of the intestinal barrier (eighth embodiment), and (iv) an amount effective to reduce the levels of TNF-α and/or IL-8 sphingan (ninth embodiment)) may be from about 10 mg/kg to about 150 mg/kg body weight of the person ingesting the composition. In addition, the amount of sphingan is intended to include all values between the stated limits, such as, for example, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, approximately 45 mg/kg, approximately 50 mg/kg, approximately 55 mg/kg, approximately 60 mg/kg, approximately 65 mg/kg, approximately 70 mg/kg, approximately 75 mg/kg, approximately 80 mg /kg, approximately 85 mg/kg, approximately 90 mg/kg, approximately 95 mg/kg, approximately 100 mg/kg, approximately 105 mg/kg, approximately 110 mg/kg, approximately 115 mg/kg, approximately 120 mg/kg , approximately 125 mg/kg, approximately 130 mg/kg, approximately 135 mg/kg, approximately 140 mg/kg, or approximately 145 mg/kg.

[0137] In one aspect of any of the sixth, seventh, eighth and ninth embodiments, the mammal is a human and the amount of sphingan is selected from: about 10 mg/kg to about 150 mg/kg, about 10 mg/kg to about 140 mg/kg, from about 10 mg/kg to about 130 mg/kg, from about 10 mg/kg to about 120 mg/kg, from about 10 mg/kg to about 110 mg/kg, from about 10 mg/kg to approximately 100 mg/kg, approximately 10 mg/kg to approximately 90 mg/kg, approximately 10 mg/kg to approximately 80 mg/kg, approximately 10 mg/kg to approximately 70 mg/kg, approximately 10 mg/kg kg to about 60 mg/kg, 10 mg/kg to about 50 mg/kg, about 10 mg/kg to about 40 mg/kg, or about 20 mg/kg to about 30 mg/kg body weight of the person taking inside the composition.

[0138] The compositions of any of the sixth, seventh, eighth and ninth embodiments may contain any of naturally occurring HA/LA sphingan, HA/LA sphingan polysaccharide, HA/LA sphingan oligosaccharide, or a combination thereof, and optionally additional contains a probiotic or additional prebiotic as described in the first embodiment.

[0139] The results disclosed herein indicate that sphingan (eg, gellan gum) increased levels of Bifidobacteriaceae in a human proximal and distal colon model. At the operational taxonomic unit (“OTU”) level, the major changes are found to relate to increased levels of Bifidobacteriaceae OTU 2 (related to Bifidobacterium adolescentis) . Therefore, in one aspect of the second, sixth or tenth embodiments, the bacteria are Bifidobacteriaceae. Additionally, in another aspect of the second, sixth, or tenth embodiments, the bacteria are Bifidobacteriaceae OTU 2. The increase in levels of Bifidobacteriaceae in the lumen of the proximal colon ranges from about 20% to about 180% during treatment compared to the untreated control, whereas the increase in levels of Bifidobacteriaceae in the lumen of the distal colon ranged from approximately 330% to approximately 590% during treatment compared to untreated controls. In yet another aspect of the second, sixth, or tenth embodiments, levels of Bifidobacteriaceae are increased in the lumen of the proximal colon by about 20% to about 180% during treatment compared to an untreated control. And, in a further aspect of the second, sixth or tenth embodiments, levels of Bifidobacteriaceae are increased in the lumen of the distal colon by approximately 330% to approximately 590% during treatment compared to untreated controls.

[0140] In addition, the results disclosed herein indicate that sphingan oligosaccharide at a concentration of approximately 4 mg/ml increased levels of bacteria (eg, Blautia , Parabacteroides , Faecalibacterium , Clostridium XVIII ) in vitro based on stool samples from healthy adults. Blautia levels in vitro were increased at least approximately 5-fold compared to untreated controls. Parabacteroides levels in vitro increased approximately 2-fold to approximately 40-fold compared to untreated controls. Faecalibacterium levels in vitro increased approximately 10-fold to approximately 190-fold compared to untreated controls. Clostridium XVIII levels in vitro increased approximately 12-fold to approximately 60-fold compared to untreated controls.

[0141] In addition, the results disclosed herein indicate that sphingan oligosaccharide at a concentration of approximately 4 mg/ml increased levels of bacteria (eg, Parabacteroides , Faecalibacterium , Clostridium XVIII ) in vitro based on stool samples from patients with inflammatory bowel disease . Blautia levels in vitro were increased by at least approximately 8-fold compared to untreated controls. Faecalibacterium levels in vitro were increased by at least approximately 8-fold compared to untreated controls. Clostridium XVIII levels in vitro increased approximately 20-fold to approximately 100-fold compared to untreated controls.

[0142] The results disclosed herein indicate that oral sphingan (eg, gellan gum) increased propionate levels in both a proximal and distal human colon model, and that oral gellan gum increased butyrate levels in both a proximal and distal human colon model. distal parts of the colon. Therefore, in one aspect of the third, seventh, or eleventh embodiments, wherein the mammal is a human, the reduction in proximal colon propionate levels ranges from about 8% to about 21% during treatment compared to control. In one aspect of the third, seventh, or eleventh embodiments, wherein the mammal is a human, the reduction in propionate levels in the distal colon ranges from about 8% to about 11% during treatment compared to control. In one aspect of the third, seventh, or eleventh embodiments, wherein the mammal is a human, the increase in proximal colon butyrate levels ranges from about 15% to about 24%. In one aspect of the third, seventh, or eleventh embodiments, in which the mammal is a human, the increase in butyrate levels in the distal colon ranges from about 4% to about 13%.

[0143] A fourteenth embodiment is directed to a method for producing sphingan polysaccharide (“SPS”) and/or sphingan oligosaccharide (“SOS”).

[0144] The method for producing SPS includes: hydrating the naturally occurring HA/IA/LA sphingan in water and reducing the molecular weight of the naturally occurring HA/IA/LA sphingan by homogenization, sonication, irradiation, oxidation and/or hydrolysis.

[0145] Reducing the molecular weight (i.e., reducing the chain length) of naturally occurring HA/IA/LA sphingan can be achieved using high pressure homogenization by a method that includes: (i) hydrating the HA/LA product powder sphingan in deionized water to obtain a solution of hydrated HA/IA/LA sphingan (approximately 1% (w/v)); (ii) passing the hydrated HA/IA/LA sphingan solution through a homogenizer 1 to 10 times at an operating pressure of about 8,500 psig to about 12,000 psig (and everything in between) to produce a homogenized HA/IA solution /LA SPS; (iii) adding a sufficient amount of a suitable organic solvent to the homogenized solution to obtain an HA/IA/LA SPS precipitate; (iv) collecting the HA/IA/LA SPS precipitate by centrifugation; and (v) drying and grinding the collected HA/IA/LA SPS powder.

[0146] In one aspect of the method for producing HA/IA/LA SPS, HA/IA/LA sphingan may be, for example, high acyl gellan, medium acyl gellan, low acyl gellan, high acyl diutan, medium acyl diutan, low acyl diutan, high acyl ramsan, medium acyl ramsan and low acyl ramsan. In another aspect of the method for producing HA/IA/LA SPS, said passing is performed 1-10 times (eg, 1, 2, 3, 4, etc.) at a pressure of approximately 8500 pounds per square inch. In yet another aspect of the method for producing SPS, said transmission is performed 1-10 times (eg, 1, 2, 3, 4, etc.) at a pressure of approximately 12,000 pounds per square inch. In a further aspect of the SPS production process, said passing is performed 10 times at a pressure of approximately 12,000 pounds per square inch. And, in yet another aspect of the method for preparing SPS, a suitable organic solvent is one that promotes precipitation of the thus obtained HA/IA/LA sphingan as a polysaccharide, including, for example, isopropanol.

[0147] A method for producing HA/IA/LA SOS includes: preparing a first composition containing naturally occurring HA/IA/LA sphingan or HA/IA/LA SPS and a liquid medium; hydrolyzing the glycosidic bond of HA/IA/LA sphingan or HA/IA/LA SPS to obtain a second composition; subjecting the second composition to ultrafiltration, gel filtration, precipitation, centrifugation, or a combination thereof to obtain a third composition containing HA/IA/LA SOS; and optionally isolating or recovering the third composition using a suitable method, such as, for example, lyophilization.

[0148] In one aspect of the method for producing HA/IA/LA SOS, said hydrolysis can be achieved by acid, enzyme, sonication, high pressure homogenization, irradiation, or a combination thereof.

[0149] In one aspect of the process for producing HA/IA/LA SOS, said hydrolysis can be achieved using an aqueous medium having a pH of about 1 to about 3. In another aspect, said hydrolysis can be achieved using an aqueous medium having a pH of about 1 to about 3 3 (or a pH of approximately 2), which aqueous medium may contain a suitable inorganic or organic acid. Examples of suitable acids include, but are not limited to, sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, citric acid, oxalic acid, formic acid, acetic acid, trifluoroacetic acid, or a combination thereof.

[0150] In one aspect of the process for producing HA/IA/LA SOS, said hydrolysis can be achieved by hydrolysis with formic acid at a pH of about 2 and a temperature of about 95 ^ο C for a period of time sufficient to hydrolyze the glycosidic bond in HA /IA/LA sphingan or HA/IA/LA SPS.

[0151] In one aspect of the method for producing HA/IA/LA SOS, said hydrolysis can be achieved using an enzyme, wherein the enzyme is capable of cleaving one or more glycosidic bonds in a sphingan, including, but not limited to, gellanase, rhamnogalacturonan endolyase (EC 4.2.2.23 ), rhamnogalacturonan exolyase (EC 4.2.2.24), gellan lyase (EC 4.2.2.25) described by Hashimoto, gellan lyase described by Kennedy (1994), or a combination thereof. It is understood that the expression "gellanase" refers to an enzyme that is capable of cleaving one or more glycosidic bonds in sphingan.

[0152] In one aspect of the method for producing HA/IA/LA SOS, said treatment comprises filtering a second composition through a membrane having a molecular weight cutoff of either about 5 kDa or about 10 kDa to produce a filtrate comprising the third composition.

[0153] The fifteenth embodiment is directed to a composition containing sphingan as an oligosaccharide prepared according to the fourteenth embodiment.

[0154] Except where otherwise noted, all technical and scientific terms used herein have the same meaning as they would normally be understood by one of ordinary skill in the art. The following examples are intended only to further illustrate the embodiments claimed and disclosed herein and are not intended to limit the scope of the claimed subject matter.

Examples

I. Example 1. Receiving HA/LA SPS and SOS

Preparation of sphingan in the form of a polysaccharide

[0155] The chain length of naturally occurring sphingan can be reduced using high pressure homogenization by a method that includes: (i) hydrating a powdered sphingan product (e.g., gellan, diutan, and ramsan) in 1 L of deionized water at a concentration of 1% (weight to volume), to obtain a solution of hydrated sphingan; (ii) mechanically treating the hydrated sphingan solution in an APV Model 1000 homogenizer at approximately 12,000 psi (x 10) to produce homogenized solutions; (iii) adding a sufficient amount of isopropyl alcohol to the homogenized solution to obtain a precipitate of sphingan as a polysaccharide; (iv) collecting the sphingan precipitate as a polysaccharide by centrifugation; and (v) drying and grinding the collected sphingan powder into a polysaccharide. Using this method with selected sphingans (e.g., high acyl gellan, low acyl gellan, high acyl diutan, and high acyl ramsan), the following samples of sphingans were obtained as polysaccharides, presented in Table 1, which were hydrated in water at a concentration of 0. 8% (weight to volume), for subsequent studies.

Table 1 . Summary of sphingans in the form of polysaccharides (Samples from Nos. 1-7)

Sample with no. Comments 1 Gellan as a polysaccharide derived from KELCOGEL ^® LT100 gellan (high acyl gellan, unbleached). 2 Gellan (high acyl) as a polysaccharide derived from KELCOGEL ^® HT gellan (high acyl, enzyme treated, PHB free). 3 Gellan in the form of a polysaccharide obtained from gellan GELRITE ^TM MK (low acyl, clarified, doubly precipitated). 4 Diutan is a polysaccharide derived from PHB-free diutan. 5 Ramsan is a polysaccharide derived from naturally occurring ramsan. 6 Gellan (from strain 438) as a polysaccharide. ^a 7 Gellan (from strain 438) as a polysaccharide. ^b Notes :
^a Sample #6 is gellan as a polysaccharide derived from gellan, which was obtained from strain 438, a strain derived from wild-type Sphingomonas elodea . Gellan from strain 438 was isolated by treating the fermentation broth with protease, EDTA, SDS, lysozyme and glucoamylase; followed by recovery of gellan gum by isopropanol-induced precipitation of the treated and heated broth.
^b Sample #7 is gellan as a polysaccharide derived from gellan, which was obtained from strain 438. Gellan from strain 438 was isolated by centrifuging the fermentation broth to obtain pelleted cells and supernatant, treating the collected supernatant with glucoamylase and protease, and extracting gellan from heated broth using isopropanol precipitation.

[0156] The polysaccharide gellan produced herein differs from commercially available gellan products in that the chain length is reduced by high pressure homogenization. Indeed, previous studies have shown that high-pressure homogenization reduces the chain length (and thus molecular weight) of naturally occurring gellan. (See US6242035B1, which is incorporated by reference in its entirety, wherein high pressure homogenization of natural gellan gum (MW ≈ 2.5×10 ⁶ ; MN ≈ 2.2×10 ⁶ ) produces gellan gum with an MW less than or equal to approximately 1.7 x 10 ⁶ as determined by gel filtration/multiple angle laser scattering.)

Preparation of sphingans in the form of oligosaccharides (SOS)

[0157] Preparation of SOS typically involves: (i) preparing a 2% (w/v) solution of naturally occurring HA/IA/LA sphingan (or HA/IA/LA SPS); (ii) hydrolysis with formic acid (pH 2) at 95 ^o C, overnight to obtain a hydrolyzate; (iii) filtering the hydrolysis using an ultrafiltration membrane with a molecular weight cutoff of either 5 kDa or 10 kDa to obtain a filtrate; (iv) lyophilizing the filtrate to obtain a lyophilisate; (v) washing the lyophysilate with anhydrous ethanol (x 3) to obtain a washed powder; and (vi) drying the washed powder to obtain SOS. (Alternatively, hydrolysis may occur by using: (i) a suitable enzyme such as gellanase; (ii) sonication; (iii) high pressure homogenization; (iv) irradiation; or (v) other known methods). Using an acid hydrolysis procedure (e.g. using formic acid) or enzymatic (e.g. using Japan gellanase (EC 4.2.2.25) or gellanase from strain 438), the following samples of HA/LA sphingans were obtained as oligosaccharides, presented in Table 2a , in which the percentage monosaccharide content refers to the monosaccharide content (glucose and rhamnose) divided by sample concentration, monosaccharide composition, oligosaccharide content and molecular weight are those described below.

Table 2a . Summary of sphingans in the form of oligosaccharides (samples from Nos. 8-18)

Sample with no. Comments 8 SOS derived from gellan KELCOGEL ^® LT100; acid hydrolysis; molecular weight cut-off - 5 kDa (monosaccharide content - 1.7%). 9 SOS derived from KELCOGEL ^® HT gellan; acid hydrolysis; molecular weight cut-off - 5 kDa (monosaccharide content - 1.5%).
Monosaccharide composition: Rha, Glc, GlcA, Glyc in an approximate ratio of 3:5:2:2 with unknown uronic acid present.
Oligosaccharide content: Gellan-like oligosaccharides (both acetylated and glycerinated) having a DP of about 2 to about 9.
Molecular Weight: Sample molecular weight ranges from approximately 0.5 kDa to approximately 4 kDa (a double peak observed corresponding to sizes smaller and larger than approximately 1.2 kDa). 10 SOS derived from gellan GELRITE™ MK; acid hydrolysis; molecular weight cut-off - 5 kDa (monosaccharide content - 1.8%).
Composition of monosaccharides: Rha, Glc, GlcA in an approximate ratio of 3:5:2. Only trace amounts of glycerate.
Oligosaccharide content: Gellan-like oligosaccharides (mostly non-esterified) having a DP of about 2 to about 12.
Molecular Weight: Sample molecular weight ranges from approximately 0.5 kDa to approximately 4 kDa (a double peak is observed corresponding to sizes smaller and larger than 1.2 kDa). eleven SOS derived from natural, PHB-free diutan; acid hydrolysis; molecular weight cut-off - 5 kDa (monosaccharide content - 0.7%). 12 SOS derived from naturally occurring ramsan; acid hydrolysis; molecular weight cut-off - 5 kDa (monosaccharide content - 2.2%). 13 SOS derived from KELGOGEL ^® HT gellan; acid hydrolysis; molecular weight cut-off is 10 kDa. 14 SOS obtained from gellan GELRITE ^TM MK; acid hydrolysis; molecular weight cut-off is 10 kDa. 15 SOS derived from natural, PHB-free diutan; acid hydrolysis; molecular weight cut-off is 10 kDa. 16 SOS derived from naturally occurring ramsan; acid hydrolysis; molecular weight cut-off is 10 kDa. 17 SOS derived from gellan GELRITE™ MK; enzymatic (using gellanase Japan EC 4.2.2.25) hydrolysis; molecular weight cut-off is 5 kDa.
Monosaccharide composition: Rha to Glc ratio approximately 1 to approximately 2 (negligible amounts of glucuronic acid and large amounts of unknown compound 1 and 2).
Oligosaccharide content: approximately 50% unsaturated GlcA, Glc,Rha, Glc and approximately 10% unsaturated GlcA, Glc,Rha.
Molecular weight: narrow size distribution - approximately 1 kDa 18 SOS derived from KELCOGEL ^® HT gellan; enzymatic (from strain 438) hydrolysis, molecular weight cutoff - 5 kDa.
Monosaccharide composition: Rha, Glc, GlcA, Glyc in an approximate ratio of 3:5:2:2 with unknown uronic acid present.
Oligosaccharide Content: Gellan-like oligosaccharides (both acetylated and glycerinated) having a DP of about 2 to about 9, with minor amounts of unsaturated compounds.
Molecular Weight: The sample has an observed molecular weight of approximately 0.5 kDa to approximately 4 kDa (a double peak observed corresponding to a size smaller and larger than approximately 1.2 kDa, similar to SN9).

[0158] Monosaccharide content was determined for selected SOS samples by dissolving the SOS sample in deionized water and analyzing the glucose and rhamnose contents using a Thermo Fisher ion chromatography system. Total monosaccharide content is calculated as the total concentration of glucose and rhamnose divided by the concentration of the sample.

[0159] The composition of monosaccharides (for samples No. 9-10 and 17-18) was determined as follows. SOS samples were dissolved in 4% sulfuric acid to a concentration of 3.5 g/L and autoclaved at 121 ^ο C for one hour. Monosaccharides were quantified using a Dionex ICS-5000 system according to Zeuner (2016). Glycerate was quantified using external standards. Unknown compound 1 (“UNK1”), unknown compound 2 (“UNK2”), and unknown uronic acid 1 (“UNK URON1”) were quantified as glucuronic acid units. Table 2b summarizes the monosaccharide composition for each of samples No. 9-10 and 17-18

Table 2b . Summary of sphingans in the form of oligosaccharides (composition of monosaccharides, samples No. 9-10 and 17-18

Sample with no. UNK1 UNK2 Rha Glc Glyc GlcA UNK URON1 mol% (standard deviation) 9 0.44 (0.07) 1.58 (0.47) 29.22 (1.61) 50.22 (1.05) 21.56 (1.32) 20.57 (0.33) 8.02 (0.44) 10 0.42 (0.09) 1.60 (0.51) 27.45 (1.60) 53.16 (0.43) 0.00 (0.00) 19.40 (0.16) 7.87 (0.16) 17 21.26 (0.90) 70.71 (2.15) 34.09 (0.52) 65.91 (1.83) 0.00 (0.00) 0.00 (0.00 ) 0.00 (0.00) 18 0.48 (0.04) 1.18 (0.13) 29.14 (1.36) 50.86 (4.44) 23.17 (1.33) 20.00 (2.04) 7.85 (0.45)

[0160] The content of oligosaccharides (for samples No. 9-10 and 17-19) was determined as follows. Identification and determination of relative abundance of oligosaccharides was performed using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) on an Amazon SL ion trap (Bruker Daltonics, Bremen, Germany) coupled with a UHPLC UltiMate 3000 (Dionex, Sunnyvale, CA , USA). 5 μl of sample in 50% ACN (final concentration=5 g/l) was injected onto a TSKgel Amide 80 HILIC column (150 mm x 2 mm; 2 μm, TOSOH, Greisheim, Germany). Chromatography was carried out at a rate of 0.2 ml/min at 45 ^ο C in a system with three eluents consisting of eluent A (water), eluent B (acetonitrile) and C (100 mM ammonium formate, pH 5). Eluent C was maintained at 5% at all times. The elution profile was as follows (times in minutes): 0-5, isocratic 75% B; 5-25, linear gradient up to 25% B; 25-30, isocratic 5% B; 30-40, isocratic 75% B. Electrospray was operated in negative mode with UltraScan mode and scanning range from 100 to 2000 m/z (mass/charge), intelligent parameter setting - 1000 m/z. Automated tandem mass spectrometry (MS ² ) events were performed on the two most abundant precursor ions. Capillary voltage 4.5 kV, end plate displacement 0.5 kV, nebulizer pressure 3.0 bar, dry gas flow 12.0 L/min, and dry gas temperature 280 ^ο C. Compounds were identified using MS (mass spectrometry) and MS ⁿ and quantified by relative intensity in data analysis 4.2 SR2

Table 2c . Summary of sphingans in the form of oligosaccharides (oligosaccharide content, samples from Nos. 9-10 and 17-18

SOS Sample No./Content SOS, % 9 10 17 18 Glc, GlcA 3.24 2.37 - 2.71 Glc, GlcA, Glyc 3.28 - - 3.06 Glc, GlcA, Rha ^a 0.80+14.25 0.79+16.85 - 1.22+12.79 Glc, GlcA, Rha, Glyc 1.02 - - 0.65 Glc, GlcA,Rha,-H2O - - 11.92 - Glc, Rha 4.33 1.74 1.30 3.39 Glc, Rha+28 0.89 0.39 - 0.96 Glc2,GlcA 0.88 - - 0.84 Glc2, GlcA, Rha 11.95 15.84 - 10.07 Glc2,GlcA,Rha,+28 7.52 8.83 - 8.11 Glc2, GlcA, Rha, Ac 1.36 - - 1.04 Glc2, GlcA, Rha, Glyc 10.86 - - 10.20 Glc2,GlcA, Rha,Glyc,+28 4.22 - - 5.25 Glc2, GlcA, Rha, Glyc.-H2O - - - 0.50 Glc2, GlcA, Rha, -H2O - - 54.88 - Glc2, GlcA, Rha2, Glyc - - - 1.47 Glc2,GlcA2,Rha - 0.33 - - Glc2,GlcA2,Rha2,Ac2,Glyc2,-H2O - - - 3.29 Glc2,Rha - - 23.00 - Glc3, GlcA, Rha 0.97 2.10 - 0.78 Glc3, GlcA, Rha2 0.56 - - - Glc3, GlcA, Rha2 1.53 2.79 - 0.56 Glc3, GlcA, Rha2 2.79 - - - Glc3, GlcA, Rha2, Glyc 2.79 - - 2.42 Glc3,GlcA2,Rha - 1.81 - - Glc3, GlcA2, Rha, Glyc 1.81 - - 1.81 Glc3,GlcA2,Rha2,Glyc 5.47 - - 4.82 Glc3,GlcA3,Rha2 - 2.90 - - Glc3,GlcA3,Rha2 3.36 11.59 - 3.19 Glc4,GlcA, Rha2,+43 - 5.40 - 1.57 Glc4, GlcA, Rha2, Ac, Glyc 2.70 - - 2.72 Glc4,GlcA2,Rha - 2.59 - - Glc4,GlcA2,Rha,Ac,Glyc,-H2O - - - 0.87 Glc4, GlcA2, Rha, Ac, Glyc2 - - - 0.89 Glc4, GlcA2, Rha2, Ac, Glyc 2.04 - - 1.68 Glc4,GlcA2,Rha2,Glyc 5.49 - - 3.97 Glc4,GlcA3,Rha2 - 13.12 - 2.58 Glc4,GlcA2,Rha3,Ac 0.95 - - 0.88 Glc4,GlcA3,Rha2/Glc4,GlcA2,Rha2,Glyc2 2.79 - - 2.20 Glc5,GlcA2,Rha2 0.69 0.69 - 0.44 Glc5, GlcA2, Rha2 - 0.69 - - Glc5,GlcA2,Rha2,Ac 1.57 - - 1.57 Glc5,GlcA4,Rha2 - 0.24 - - Glc6, GlcA3, Rha3 - 0.07 - - derivative 1513 with unknown m/z ratio 3.24 - - - unknown 379z1 - - 2.86 - unknown 597z2 - 1.24 - - unknown 668z2 - 7.40 - - unknown 719z1+Glyc - - - 0.63 unknown 719z1 - - - 0.89 derivative of Glc2,GlcA, Rha,-H2O with unknown m/z ratio - - 6.04 - derivative of Glc3,GlcA2,Rha2 with unknown m/z ratio - 0.93 - - ^a Glc, GlcA,Rha could be Rha-Glc-GlcA or GlcA-Glc-Rha.

[0161] SOS, identified as Glc2,GlcA, Rha,Glyc, is a tetrameric gellan unit with one glycerate, while SOS, identified as Glc2,GlcA, Rha,Ac, is a tetrameric gellan unit with one acetyl. Some SOS include multiple sugar components (namely Glc5, GlcA2, Rha2, Ac) - the oligosaccharide can be set based on a certain number of saccharides. For example, Glc5,GlcA2,Rha2,Ac includes two tetrameric units (namely, Glc-GlcA-Glc-Rha) with an additional glucopyranosyl (Glc) and acetyl (Ac). In addition, Glc6,GlcA3,Rha3 is an oligosaccharide that includes three tetrameric units (namely, 3X Glc-GlcA-Glc-Rha). SOS, identified by loss of water (“-H2O”, see e.g. Glc2,GlcA, Rha,Glyc.-H2O), are an unsaturated lyase/β-radiation product. In some cases, for longer gellan-like oligomers (e.g., Glc4,GlcA3,Rha2/Glc4,GlcA2,Rha2,Glyc2), two structures are proposed because mass spectral fragmentation is insufficient to distinguish the presence of one glucuronic acid from two glycerate substituents. Not all observed SOS could be structurally identified. Based on fragmentation, some components can be partially identified due to similarity in fragmentation patterns, so they are designated as “derivative of unknown m/z ratio” after the most similar identified compound. Other compounds could not be identified due to poor fragmentation or because they were compounds of a different type than the expected gellan-derived SOS. These unknown SOS are labeled "unknown (observed m/z ratio) z1 or z2" depending on whether one or two charges were observed. As evidenced by the gel filtration data below, analyzed samples may include sphingans as polysaccharides (DP >30, but less than naturally occurring sphingans and sphingans as oligosaccharides (2≥DP≤30).

[0162] The present molecular weight of the SOS samples was determined as follows. High efficiency gel filtration was performed using an Ultimate iso-3100 SD pump with a WPS-3000 sampler (Dionex) connected to an RI-101 refractive index detector (Shodex). 100 μL of sample was loaded onto a TSKgel G3000PW (300 × 7.5 mm) column equipped with a TSKgel PWH (7.5 × 7.5 mm) guard column (Tosoh Bioscience). Elution was performed using 100 mM sodium nitrate at a flow rate of 1.0 ml/min at 40 ^ο C. Pullulan standards were used as standards.

[0163] In FIG. 1a shows the gel filtration chromatogram (“SEC”) for acid (SN9, solid line) and enzyme (SN18, dashed line) treated sphingans as poly- and oligosaccharides derived from high acyl gellan, while FIG. Figure 1b shows the SEC for acid-treated (SN10, solid line) and enzyme-treated (SN17, dashed line) sphingans as poly- and oligosaccharides derived from low acyl gellan. As in fig. 1a and in Fig. Figure 1b shows the elution time of pullulan molecular weight standards (namely, >50 kDa (6.5 min, filled square ( )), 12 kDa (8.8 min, filled circle ( )), 5 kDa (9.3 min, filled triangle ( )), 1 kDa (10 min, empty square ( )), 342 Yes (10.65 min, empty circle ( )), and 180 Yes (11.15 min, empty triangle ( ))). SEC data for Fig. 1a show a comparable distribution of sphingans as polysaccharides (SPS) and sphingans as oligosaccharides (SOS) derived from high acyl sphingan. These should be compared with the SEC data for FIG. 1b, where the SPS and SOS distributions for the acid-treated sample (SN10) are different from the SPS and SOS distributions for the enzyme-treated sample (SN17). The SEC data also shows a molecular weight range from approximately 0.5 kDa to approximately 4 kDa (and possibly up to approximately 12 kDa) for samples Nos. 9, 10, and 18. Interestingly, sample (SN17) derived from the low acyl sphingan by enzyme treatment, demonstrates initial elution of SOS having a molecular weight ranging from approximately 0.5 kDa to approximately 1 kDa (with a narrow peak size distribution of approximately 1 kDa).

[0164] The oligomer content of SOS samples was determined using mass spectral analysis. Typically, an SOS sample was prepared by dissolving SOS at a concentration of 0.4% using a water/acetonitrile (1:1) mixture containing 1 mM NaCl. Samples were filtered through a 0.22 micron filter before being introduced into the MSQ plus Single Quad Mass Spec from Thermo Fisher. The mass spectrometer was operated in negative electrospray ionization mode, scanning from 150 to 1000 m/z. Based on the intact mass of oligomers, various oligosaccharides were detected in the SOS samples. Table 3 summarizes the oligomers observed for selected SOS samples

Table 3. Identified oligomers in SOS samples

Sample with no. Identified oligomers 9 Tetramer (663), tetramer with glycerate (751), octamer (654, two charges, Glc, GlcA, Glc (517), Rha, Glc, GlcA (501), Glc, Rha (361, chloride addition product) 10 Tetramer (663), octamer (654, two charges), pentamer (Glc, GlcA, Glc, Rha, Glc, 825), GlcA, Glc, Rha (501), Glc, GlcA, Glc (517), Glc, GlcA ( 355) eleven Glc(Glc-Glc), GlcA (679), Glc(Glc-Glc) (539, chloride addition product), GlcA, Glc (391, chloride addition product), Glc, Glc (377, chloride addition product) 12 Tetramer (663), GlcA, Glc,(Rha-Rha) (683, chloride addition product), Glc-(Rha-Rha),Rha (654, chloride addition product), GlcA, Glc,Rha (501), Glc, GlcA,Glc (517), Rha, Glc (361, chloride addition product), GlcA, Glc (355) () denotes a side chain.

II. Example II. Effect of sphingans (e.g., naturally occurring sphingans, SPS and SOS) on the activity of selected gut microbiota samples

[0165] Samples containing 8 mg/mL (0.8% (w/v) SPS or SOS were diluted twofold to provide samples containing 4 mg/mL (0.4% (w/v) )) SPS or SOS The effect of samples #1-12 at 4 mg/mL on a panel of over twenty-five gut microbiota samples was assessed after 24 hours fermentation using an in vitro fermentation screening platform (i-screen ", as described by Fehlbaum (2018). Specifically, a standard pool of fecal microbiota samples obtained from 5-6 healthy adults (health status based on exclusion criteria) was used, which was pre-cultured overnight from frozen material. Dilution was then performed in microtiter plates into which samples were added and subsequently incubated under anaerobic conditions for 24 hours at 37 ^o C. After incubation, culture samples were collected and processed for further analysis.In a 96-well plate, some wells were used for technical controls, controls without microbiota (n=3) and negative control with microbiota only (n=3), leaving 80 wells available for experiments. A number of sphingan samples and comparison samples of plant extracts (e.g. pectins, pectin oligosaccharides and carrageenans) and vermicomposts (e.g. xanthan and xanthan oligosaccharides)) were analyzed at a concentration of approximately 4 mg/ml, corresponding to a dose of approximately 4 g/day. (Van den Abbeele (2011).)

[0166] Changes in microbiota composition were determined using next-generation sequencing, which recognizes bacteria at the genus level and in many cases (but not all) at the species level. To ensure uniform distribution of samples in the sequencing pool, total bacterial load was determined by quantitative polymerase chain reaction (“PCR”) using a universal primer-probe set. Amplicons of the V4 region of 16S rDNA were obtained by PCR, thereby standardizing the level of template DNA and using unique barcoded error-correcting primers and avoiding over-amplification. The amplicons were then gel purified, quantified, and pooled. Sequence analysis was then performed on the llumina MiSeq® device by paired-end sequencing (2x250 bp). 3′ sequence analysis was performed using a standardized sequencing pipeline developed by the Netherlands Organization for Applied Scientific Research. The pipeline includes paired-end read assembly, high-quality filtering, chimera removal, and taxonomic classification + clustering of processed reads.

[0167] Standard controls were performed in triplicate. Specifically, the panel of microorganisms included Bacteroides , Coprococcus , Lachnospiraceae unclassified, Megasphaera , Escherichia / Shigella , Clostridium XlVa, Allisonella , Bifidobacterium , Dorea , Collinsella , Mogibacterium , Sutterella , Bilophila , Blautia , Clostridium sensu stricto , Phascolarctobacterium , Faec alibacterium , Clostridium XlVb, Clostridium XI, Acidaminococcus , Gemmiger , Lachnospira , Parabacteroides , Paraprevotella and Butyricicoccus . The effect was determined relative to an untreated control. For the first i-screen analysis of samples Nos. 1-12, Table 4a summarizes the observed effect on the growth of Bifidobacterium and Faecalibacterium , where the results presented are for the untreated control (growth indicated as 1.0).

Table 4a. Data on the activity of selected samples of intestinal microbiota observed for samples No. 1-12

Sample with no. Bacterial growth Bifidobacterium Blautia Faecalibacterium Parabacteroides 1 1.42 1.09 0.90 1.80 2 1.11 0.87 0.71 1.63 3 1.23 0.85 0.83 4.03 4 1.16 0.84 0.51 0.93 5 1.32 1.60 0.43 2.51 6 0.85 0.81 1.58 2.37 7 0.88 0.92 0.78 4.62 8 0.96 3.23 112.77 9.47 9 1.00 2.42 58.31 5.37 10 0.78 4.78 188.91 21.11 eleven 0.91 2.58 41.57 32.57 12 0.96 3.49 70.44 38.69

[0168] Values in bold represent significant changes in bacterial growth compared to the untreated control. According to the results presented in Example III (below), sphingans in the form of polysaccharides (namely, samples No. 1-3) stimulated the growth of Bifidobacterium . Surprisingly, sphingans in the form of oligosaccharides (namely, samples nos. 8-12) stimulated the growth of each of Faecalibacterium , Blautia , and Parabacterioides to a significant extent compared to the untreated control. It is well known that Bifidobacterium and Faecalibacterium (e.g. Faecalibacterium prausnitzii ) are butyrate-producing bacteria. Accordingly, the results obtained using i-screen showing that sphingans in the form of oligosaccharides stimulate the growth of Faecalibacterium indicate that these compositions exhibit prebiotic activity. And, since Faecalibacterium prausnitzii is known to be associated with anti-inflammatory effects, the results obtained using i-screen suggest that SOS act as anti-inflammatory agents by stimulating the growth of Faecalibacterium prausnitzii.

[0169] Additional i-screen analyzes were performed on samples Nos. 9, 10, 17 and 18 using three different fecal pools, namely two pools obtained from healthy adults (H1 and H2) and one pool obtained from patients with irritable bowel syndrome ("IBD"), for three or four bacteria (viz. Blautia , Parabacteroides , Faecalibacterium , Clostridium XVIII ). Specifically, the fecal pools used included: (i) the H1 pool was obtained from six healthy volunteers (Caucasian, 25-60 years old, European lifestyle and diet, self-reported health status, no antibiotic use in the past 3 months), (ii ) the H2 pool was obtained from 5 healthy adult volunteers (20–65 years, no antibiotic use in the past 3 months, self-reported health status) and (iii) the IBD pool was obtained from four patients with IBD, namely ulcerative colitis. For additional analyzes using i-screen (namely, first i-screen (No. 1-2), second i-screen (No. 3-4) and third i-screen (No. 5-16)) samples with No. 9 -10 and 17-18, Table 4b summarizes the observed effect on the growth of three or four bacteria (namely, Blautia (" Blaut ."), Parabacteroides (" Para ."), Faecalibacterium (" Faecal ."), Clostridium XVIII ( "ClXVIII" )), where the results presented are for the untreated control (growth indicated as 1.0).

Table 4b. Data on the activity of selected samples of intestinal microbiota observed for samples Nos. 9-10 and 17-18.

No. Sample with no. Pool Blaut. Para. Faecal. ClXVIII 1 9 ^a H1 2.42 5.37 58.31 - 2 10 ^a H1 4.78 21.11 188.91 - 3 9 ^b H1 2.21 6.79 30.71 - 4 10 ^b H1 3.20 15.99 51.54 - 5 9 H1 1.91 3.56 3.75 - 6 10 H1 2.67 8.37 23.19 - 7 17 H1 2.54 4.34 7.86 - 8 18 H1 1.90 3.82 5.16 - 9 9 H2 1.10 3.64 6.76 16.63 10 10 H2 1.79 7.25 21.93 31.13 eleven 17 H2 2.18 6.84 1.39 55.91 12 18 H2 1.27 3.38 7.47 12.66 13 9 IBD - 1.74 5.11 31.39 14 10 IBD - 6.92 6.06 30.84 15 17 IBD - 6.79 2.45 98.28 16 18 IBD - 1.63 5.26 32.58 ^a ResponsesFaecal. are those presented in Table 4a.
^b ResponsesFaecal. Are those presented in Table 5.

[0170] Regarding elements 1-4 in Table 4b, it can be seen that SOS created favorable conditions for increasing the fold change of Blautia , Parabacteroides and Faecalibacterium in the fecal pools of healthy adults compared to untreated controls. SOS obtained from low acyl gellan oligomers showed the highest fold change values for Faecalibacterium (188.91), as well as Parabacteroides (21.11) and Blautia (4.78)

[0171] With respect to elements 5-8 in Table 4b, the following observations were made. SOS (acid and enzyme treated) stimulated the growth of Blautia , Parabacteroides and Faecalibacterium in the fecal pools of healthy adults compared to untreated controls. These results confirm the results of the first and second i-screen for samples of acid-treated high- and low-acyl gellans in the form of oligomers. Large fold change values in the three genera were obtained with acid-treated low acyl gellans in the form of oligomers. In addition, high- and low-acyl gellans as oligomers produced by enzymatic treatment were also effective in increasing the growth of three genera in the same fecal pool.

[0172] Using the fecal H2 pool (elements 9-12 of Table 4b), it can be seen that SOS (acid and enzyme treated) increased the fold changeBlautia,Parabacteroides,Faecalibacterium and bacteria from the cluster XVIII Clostridium. SOS (acid treated) created favorable conditions for the highest fold change valuesFaecalibacterium (21.93) andParabacteroides (7.25). Cluster fold change values XVIII Clostridium ranged from 12.66 to 55.91. In comparison, the majority of Clostridium clusters XVIII and XIVa found in the gut produce acetate (several strains in Clostridium cluster XIVa also produce butyrate along with acetate), and based on genomic (metabolic network) analysis, both clusters do not produce toxins. Narushima (2014).

[0173] Using the IBD fecal pool (elements 13-16 of Table 4b), the results indicate that SOS (from low acyl gellan) provided favorable conditions for the largest fold change values of Parabacteroides . Acid-treated low acyl gellan oligomers showed the highest fold change value in Faecalibacterium . The fold change of Clostridium cluster XVIII also increased from 30.84 to 98.28. It is interesting to note the significant increase in Parabacteroides in the case of SOS. Parabacteroides break down healthy foods high in fiber and protect against inflammation. These bacteria are absent in patients suffering from inflammatory bowel disease. Martínez (2010), Noor (2010), Segata (2012), and Zitomersky (2013).

[0174] Table 5 summarizes the observed effect for the second analysis using i-screen samples Nos. 9-16 (and comparison samples 1-16, plus Livaux™ supplement, inulin and amoxicillin) on a panel of eight bacteria ( Lachnospiraceae unclassified (“ Lachn.U.” ), Clostridium XlVa (“ ClX1Va” ), Bifidobacterium (“ Bifid.” ), Coprococcus (“ Copro.” ) Blautia (“ Blaut. ”), Phascolarctobacterium , (“ Phasc.” ), Faecalibacterium ("Faecal." ), Butyricicoccus ("Butyr." ), and Parabacteroides (" Para .")), where the results presented are for the untreated control (growth indicated as 1.0).

Table 5 . Effect of selected SOS (SN9-SN16) and comparison samples (CS) relative to a panel of eight bacteria.

Sample Lachn.U. ClX1Va Bifid. Copro. Blaut. Phasc. Faecal. Butyr. Para. SN9 2.42 1.00 0.65 1.02 2.21 1.08 30.71 0.95 6.79 SN10 1.19 0.93 0.73 0.83 3.20 1.37 51.54 0.93 15.99 SN11 1.20 1.07 0.38 1.22 2.56 1.97 30.24 1.12 26.30 SN12 0.53 1.17 1.34 0.31 2.28 2.90 48.73 0.73 37.79 SN13 2.06 0.96 0.80 0.88 2.12 1.04 17.49 0.91 9.36 SN14 1.16 0.83 0.86 0.78 3.00 1.29 43.63 0.99 17.67 SN15 1.14 0.88 0.67 1.12 4.96 1.24 29.05 0.84 6.59 SN16 1.08 0.80 1.32 0.82 2.30 2.11 11.24 0.92 31.01 CS1 3.90 0.62 1.01 0.52 1.86 1.21 3.74 0.55 0.49 CS2 3.54 0.52 0.62 0.39 1.26 1.31 3.09 0.67 0.53 CS3 3.08 0.54 0.83 0.56 1.91 1.58 3.63 0.77 0.52 CS4 2.82 0.54 0.88 0.51 1.68 1.64 3.20 0.71 0.55 CS5 1.67 0.55 0.43 0.71 1.31 1.94 3.49 0.91 0.71 CS6 2.45 0.54 0.89 0.50 1.74 1.67 2.26 0.84 0.47 CS7 1.52 0.71 0.53 0.93 1.15 1.09 1.35 1.49 0.75 CS8 1.13 0.51 0.65 0.58 0.55 1.27 1.13 1.19 0.46 CS9 1.22 1.02 0.74 1.27 0.92 0.98 1.46 1.25 0.98 CS10 2.03 0.66 0.84 0.87 1.49 1.53 3.64 0.71 0.80 CS11 1.20 1.07 0.60 0.84 1.13 1.86 1.77 0.72 1.38 CS12 2.35 0.54 2.13 0.57 2.72 1.79 2.18 0.67 0.58 CS13 0.72 0.59 1.95 0.56 5.15 2.17 1.81 0.68 0.52 CS14 1.17 1.05 0.56 1.07 1.31 1.24 1.04 0.68 2.80 CS15 1.23 0.81 1.05 0.83 1.79 1.21 1.15 1.33 7.16 CS16 1.38 0.73 1.52 0.82 2.52 1.50 1.25 1.00 8.26 CS17 1.19 0.82 1.11 1.03 1.79 1.28 0.94 1.07 7.01 CS18 0.97 0.72 1.68 1.15 1.53 1.07 1.14 0.74 0.79 CS19 0.73 0.81 3.35 0.82 3.64 0.78 0.93 0.89 0.62 CS20 0.04 0.03 0.24 0.04 0.23 0.08 0.37 0.00 12.10

[0175] Table 6 summarizes the compositional composition of comparison samples 1-16 used in the second screen.

Table 6 . Comparison Sample Summary (“CS”) 1-16

C.S. Comments CS1 Semi-finished lemon pectin (67.3% DE; IV 5.3 dl/g). ^a CS2 Lime Pectin (55.5% DE; IV 5.0 dL/g; random esterification (“EP”)). ^a CS3 Semi-finished orange pectin (55.7% DE; IV 3.1 dl/g). ^a CS4 Orange pectin (28.3% DE; IV 3.0 dl/g), random EP. ^a CS5 Semi-finished sugar beet pectin (53.0% DE, IV 2.4 dl/g, 18.0% DAc). ^a CS6 Orange pectin (55.1% DE, IV 1.7 dl/g). ^a CS7 Sugar beet oligosaccharide (“POS”) pectin obtained by treating sugar beet pectin with pectin lyase and polygalacturonase; passing through a 0.2 micron filter and subjecting the permeate to passing through a 3 kDa molecular weight cutoff filter. CS8 Lemon POS (methylated), obtained by treating lemon pectin with pectin lyase; filtration through a filter with a molecular weight cutoff of 70 kDa; and then subjecting the permeate to passing through a filter with a molecular weight cutoff of 3 kDa. CS9 Lemon POS (methylated), obtained by treating lemon pectin with pectin methylesterase and pectin lyase; filtration through a filter with a molecular weight cutoff of 70 kDa; and then subjecting the permeate to passing through a filter with a molecular weight cutoff of 3 kDa. CS10 Insoluble citrus fiber. CS11 κ-Carrageenan (partially modified: typically 17-18% nu). CS12 Pectin extracted from waste in the form of peels. CS13 HR (“hairy” part, or RG1, rhamnogalacturonan 1) of sugar beet, obtained by treating HR of sugar beet with pectin lyase and polygalacturonase; passing through a 0.2 micron filter and subjecting the retentate to passing through a 10 kDa molecular weight cutoff filter. CS14 Xanthan is an oligosaccharide derived from non-pyrulylated xanthan (“NPX”). CS15 An NPX oligosaccharide prepared from CS14 by treatment with xanthase followed by passage through a filter with a molecular weight cutoff of 5 kDa. CS16 Xanthan is an oligosaccharide derived from clarified xanthan gum powder (KELTROL ^® T xanthan). CS17 Xanthan oligosaccharide, obtained from xanthan polysaccharide (cf. CS16) by further digestion with xanthase followed by passage through a filter with a molecular weight cutoff of 5 kDa. CS18 Livaux™ Kiwi Powder (a commercial product that claims to stimulate F. prausnitzii ). CS19 Inulin (commercially available from Sigma). CS20 Amoxicillin (commercially available from Sigma). ^a For processed pectins or pectin oligosaccharide (POS), pectin samples were processed using either pectin methylesterase for lower degrees of esterification (DE) or polygalacturonase and pectin lyase for pectins with lower MW/IV (molecular weight/internal viscosity).

[0176] Based on the results of Table 5, it can be seen that all sphingan oligosaccharides showed the greatest growth of Faecalibacterium compared to all comparison samples. In particular, the greatest growth of Faecalibacterium was demonstrated by gellan oligosaccharides (approximately 52 times) derived from gellan GELRITETM MK (molecular weight cut-off - 5 kDa (SN10)), ramsan oligosaccharides (approximately 49 times) derived from naturally occurring ramsan (SN12), and gellan oligosaccharides (43-fold) obtained from GELRITE™ MK (molecular weight cut-off - 10 kDa (SN14)). Interestingly, the product Livaux™, which is advertised as having Faecalibacterium growth-promoting activity (see, for example, livaux.com/livaux-gi-problem/), showed only a 1.14-fold increase in Faecalibacterium growth activity compared to the untreated control. The relatively low Faecalibacterium growth activity for Livaux™ is consistent with published data. (US20170326190A1).

[0177] Comparison of the results from Table 4 and Table 5 shows that in some cases, variability for the selected samples (compare SN10 (188.31 vs. 51.54) and SN12 (70.44 vs. 48.73)). Additional analysis of the selected data shows that the coefficient of variation (namely, the ratio of the standard deviation to the mean) for the selected sphingans can vary from approximately 7% to approximately 32%, and in some cases up to approximately 80%.

[0178] Based on the results of Table 5, it can be seen that all sphingans in the form of oligosaccharides showed an increase in Blautia growth activity (namely, a 2-5-fold increase compared to the untreated control).

[0179] Data not shown indicate that all sphingans in the form of oligosaccharides showed a decrease in growth activityEscherichia/Shigella (approximately 9-36% reduction compared to untreated control). These will differ from Livaux™, which has demonstrated increased growth activityEscherichia/Shigella (increase by approximately 45% compared to untreated control).

III. Example III. Effect of gellan gum on the activity and composition of the gut microbiome in the lumen and mucosa of the human gastrointestinal tract .

A. Materials and Methods, SHIME Experimental Design, and Typical SHIME Fermenter Setup

[0180] Aspects of a human intestinal microbial ecosystem mimic (or SHIME) are known (see, for example, Molly (1993), Possemiers (2004), Possemiers (2017), Van de Wiele (2013), Van den Abbeele (2012), and Van den Abbeele (2013).)

[0181] A typical SHIME fermenter device representing the adult human gastrointestinal tract was described by Molly (1993). It consists of a sequence of five fermenters that mimic different parts of the human gastrointestinal tract (e.g. stomach (V1), small intestine (V2), ascending colon (V3), transverse colon (V4) and descending colon (V5)) . The first two fermenters operate on a fill-and-pull principle to simulate the various stages of food absorption and digestion, using peristaltic pumps to add a specified amount of SHIME feed material (140 ml 3 times daily), as well as pancreatic fluid and bile (60 ml 3 times daily). day), respectively, into the stomach (V1) and small intestine (V2) compartments and emptying the corresponding fermenters at regular intervals. The last three compartments mimic the large intestine. These fermenters involve constant stirring; they have constant volume and pH control. The retention times and pH of the various vessels are chosen to resemble in vivo conditions in various parts of the colon. Once inoculated with fecal microbiota, these fermenters mimic the ascending (V3), transverse (V4) and descending (V5) colon. Inoculum preparation, retention time settings, pH, temperature, and fermenter feed composition were previously described by Possemiers (2004). Once the microbial community has stabilized in different parts of the colon, a representative microbial community is established in the three compartments of the colon, which differs in both composition and functionality in different parts of the colon.

[0182] The human intestinal tract is home to a large and complex community of microorganisms that participate in maintaining human health by preventing colonization by pathogens and producing nutrients. Microorganisms are not randomly distributed throughout the intestine, and those that adhere to the intestinal wall play an important role as a “barrier” against pathogens, controlling mucosal immune responses and occupying a niche at the expense of potentially harmful invaders. However, current in vitro strategies do not allow the cultivation of some of the microorganisms that adhere to the intestinal mucosa and are limited to modeling the luminal microbial community. This means that an important part of the gut ecosystem is not taken into account and potentially important information is lost.

[0183] To solve this problem, the SHIME system was modified to take into account the colonization of the mucous layer. (See, for example, Van den Abbeele (2012) and Van den Abbeele (2013).) A modified SHIME system is known as M-SHIME, which allows the cultivation of both the luminal and mucus-associated microbial communities over several weeks.

[0184] The inclusion of a mucosal compartment increases the value and capabilities of SHIME modeling and allows assessment of whether a particular treatment is also capable of modulating the microbial community associated with the mucosa.

I. Adapted SHIME device for research

[0185] The SHIME device of the TWINSHIME configuration was adapted to the TripleSHIME configuration, which included a vessel (or fermenter) for the stomach, small intestine, proximal colon, and distal colon for each of the donors. The TripleSHIME configuration allowed for side-by-side comparisons of three different conditions. The potential fermentation of gellan gum by the microbiota of three different human donors (Donor A: female, 28 years old; Donor B: female, 41 years old; Donor C: female, 34 years old) was assessed. Colon parts were limited to two parts compared to three parts in TWINSHIME. Retention times and pH ranges were optimized to obtain results that are representative of the entire gastrointestinal tract. In practice, in the TripleSHIME experiments, instead of working with 2 blocks, each consisting of an AC-TC-DC configuration (ascending, transverse, and descending colon), 3 PC-DC blocks were used. After inoculation with adult fecal microbiota, these fermenters mimic the proximal colon (PC; pH 5.6-5.9; retention time = 20 hours; volume 500 ml) and distal colon (DC; pH 6.6-6. 9; retention time=32 hours; volume 800 ml).

[0186] The SHIME experiment for this study consisted of three phases (stabilization, control, and treatment) that lasted seven weeks.

[0187] Stabilization Period : After inoculating the colonic fermenters with a suitable stool sample, a two-week stabilization period allowed the microbial community to differentiate in different fermenters depending on local environmental conditions. During this period, SHIME was fed a minimal nutrient matrix to maintain maximum diversity of gut microbiota initially present in the fecal inoculum.

[0188] Control Period : During this two-week control period, standard SHIME nutrient matrix was further dosed into the model over a period of 14 days. Analysis of samples during this period allows us to determine the composition and activity of the microbial community at baseline levels in the various fermenters that were used as controls to evaluate treatment effects.

[0189] Treatment Period : During this three-week period, the SHIME fermenter was operated at nominal conditions, but using a diet supplemented with the test product. Samples collected from colonic fermenters during this period allow investigation of the specific effect on the composition and activity of the resident microbial community.

B. Analysis of the composition and activity of the microbial community

[0190] A special feature of SHIME is the ability to work with a stable community - the microbiota and regularly collect samples from different parts of the intestine for further analysis. The large volumes of the colon parts allow sufficient volumes of fluid to be collected each day without disturbing the microbial community or compromising the rest of the experiment.

[0191] A number of microbial parameters are monitored throughout the experiment as part of a standard SHIME experiment. These measurements are necessary to evaluate the performance of the model and allow monitoring of major changes in microbial community composition and activity caused by prebiotic treatment.

1. Analysis of the composition and activity of the microbial community

[0192] Acid/base consumption : Microbial production of metabolites in colonic fermenters changes the pH. Without constant pH control (by adding acid or base), the pH will exceed specified ranges. Acid/base consumption is constantly monitored during the SHIME experiment.

[0193] Total gas production : The assessment of total gas production is an important aspect associated with potential end-use tolerance issues. However, online measurements of gas production are difficult in continuous intestinal models due to the continuous influx and outflow of masses. Therefore, overall gassing analysis is typically assessed in batch units.

2. Microbial community activity (3 times a week)

[0194] Short chain fatty acids (SCFA) : Concentrations of acetic acid, propionic acid and butyric acid were analyzed.

[0195] Lactate : SCFA precursor and potential antimicrobial agent.

[0196] Ammonia and branched chain SCFAs (isobutyric acid, isovaleric acid and isocaproic acid) are markers of proteolytic fermentation, with rather adverse effects on host health.

[0197] Microbial community composition (1 time per week); Samples were selected for 16S rRNA-targeted Illumina sequencing.

C. Gellan gum used for research

[0198] The product tested included a food grade gellan gum, KELCOGEL® LT100-P Gellan Gum (“Gellam Gum”). KELCOGEL® LT100-P Gellan Gum is a natural (high acyl) gellan gum. The product was studied at a dose of 1 g/day in vitro, which corresponds to a dose of 2 g/day in vivo.

D. SHIME Device Stability

[0199] During the control period, SCFA levels were very stable across the three SHIME blocks (average levels were 94.4% similar between consecutive time points in the control period), clearly indicating stability of the microbial community in terms of activity and composition. Stable fermenter conditions increase confidence that any effect observed during processing is truly the result of the test product introduced.

E. Total enzymatic activity

1. Acid/base consumption

[0200] Acid and base consumption reflects the overall microbial activity throughout the SHIME experiment. To ensure that optimal environmental conditions are maintained, pH in the SHIME system is controlled by pH regulators ranging from 5.6 to 5.9 in the proximal colon and 6.6 to 6.9 in the distal colon. Once the microbial community has stabilized in the various fermenters (starting 2 weeks after inoculation), alkali and acid consumption is generally low. However, during processing, the bacteria may produce increased amounts of SCFA. As a consequence, the environment in the fermenters will become acidified, requiring the introduction of a base into the respective fermenters to maintain them within preset pH ranges. As a result, acid/base consumption will increase. By measuring the acid/base consumption throughout the experiment, the potential effect of the test product on the activity of the microbial community can be assessed. However, it should be noted that acid/base consumption is only a rough indicator of fermentation by microorganisms, since not all acids produced through fermentation cause a similar decrease in pH (acids with a lower pKa, such as acetate, effectively reduce pH), while the conversion acids into each other can also affect pH (for example, the conversion of acetate/lactate to propionate/butyrate increases pH). Actual measurement of microbial metabolites (such as SCFA and lactate) provides more accurate readings.

[0201] The data in Table 7 shows that the overall fermentation of the test product showed similar trends across the three donors tested in both the proximal and distal colon.

Table 7 . Average weekly base and acid consumption (ml/day) during two control periods (C1 and C2) and three treatment weeks (TR1-TR3) for gellan gum treatment for three different donors (A, B and C) in fermenters as proximal colon (PC) and average acid/base consumption for the entire control period (n=6) and treatment period (n=9).

Periods PC DC A B C A B C C1 1.1 2.2 0.9 15.9 13.8 18.5 C2 0.8 0.5 1.1 17.3 15.4 16.1 TR1 -0.4 -0.7 0.0 21.6 18.2 19.6 TR2 1.6 1.4 0.5 15.0 15.1 17.5 TR3 4.3 3.1 2.9 19.3 17.2 21.1 CON(ave) 1.0 1.3 1.0 16.6 14.6 17.3 TRT(ave) 1.8 1.3 1.1 18.6 16.8 19.4

[0202] In the proximal colon, acidification was very limited, but in the last week of treatment, there was a trend toward increased base consumption in all dons studied. In the distal colon, acidification was more pronounced during the control period compared to the proximal colon. This is explained by the fact that the physical transfer of a more acidic suspension from the proximal colon (pH=5.6-5.9) to the distal colon automatically causes more base consumption in that distal colon to maintain the pH in the correct range ( pH=6.6-6.9). The addition of the test product resulted in a slight increase in base consumption immediately after the start of treatment for all donors tested.

Gas formation

[0203] Since gases are the primary endpoint of fermentation activity by intestinal microorganisms, changes in gas production serve as an indicator of the overall fermentation profile. Since gas production is not monitored in the continuous SHIME model, given the regular flushing of the headspace with nitrogen gas (to ensure anaerobiosis), gas production is assessed during individual short-term batch incubations. During these incubations, the same dose of test product is delivered to the proximal colon-derived SHIME microbiota during the control period, thereby mimicking the processes occurring at the beginning of treatment in the continuous SHIME model.

[0204] Donor-dependent effects were observed in relation to gas formation (data not shown). While donor B experienced a slight increase in gas production after treatment with the test product, treatment resulted in a slight decrease in gas production in other donors. In general, gas production was most intense in the time interval from 6 to 24 hours for all conditions. Only in the time interval from 4 to 6 hours was there a consistent (but moderate) increase in gas production in all donors after treatment with the study product, while other time intervals were characterized by donor-specific differences.

[0205] Overall, gellan gum treatment had little effect on gas production by the intestinal microbiota in the three donors studied.

F. Analysis of microbial community activity

Short Chain Acid (SCFA) Products

[0206] The following information describes the effect of the test product on SCFA production in the Triple-SHIME experiment. SCFA production results from carbohydrate metabolism in the colon and is associated with various health effects. The most common SCFAs are acetate, propionate and butyrate. It is well known that SCFAs play a critical role in gut health. Acetate can be used as an energy source for the host and as a potential substrate for lipid synthesis in the body. In addition, it is an important byproduct of butyrate synthesis and may have antimicrobial effects against pathogens. However, the health-promoting effects are mainly attributed to propionate and butyrate, which act as major energy sources for the intestinal epithelium and have demonstrated protective effects against inflammation and colon cancer. Cummings (1987). Propionate is known to be transported to the liver, where it has plasma cholesterol-lowering effects and a positive effect on glycemic control. (See Wright (1990), Demigne (1995) and Wong (2006)).

[0207] In summary, the beneficial effects of the tested substrates on SCFA production therefore include increased production of acetate, propionate and/or butyrate. The information below takes into account a direct comparison of results for three donors.

[0208] For optimal comparison of different donors, the average SCFA levels for all three of them are presented for each of the different SCFAs (per week and per period).

2. Acetate production

[0209] Acetate can be produced by a wide range of intestinal microorganisms, including, among many others, Bacteroides spp. (phylum Bacteroidetes) and Bifidobacteria. It followed that although gellan gum significantly increased acetate levels in the proximal colon of donors A and C, there was no effect on acetate levels in donor B (Fig. 2a, Table 8). The largest average increase was observed in donor A (ie, an increase of 1.7 mM or +21%). In contrast, in the distal colon, increased acetate levels after gellan gum treatment were observed only in donor B (Fig. 2b, Table 8), with an average increase of 2.1 mM (+6%) compared to the control period.

Table 8. Effect of gellan gum treatment on acetate production (in mM) in proximal (PC) and distal (DC) colon fermenters for three different donors (A, B and C) and on average weekly acetate production during controls periods (C1 and C2) and treatment weeks (TR1-TR3) (see also Fig. 1a-1b).

Periods PC DC A B C A B C C1 8.5 11.7 7.4 38.9 36.0 37.7 C2 8.0 7.4 7.4 35.9 34.8 36.5 TR1 8.5 9.6 8.3 36.4 36.7 36.8 TR2 9.5 8.0 7.5 35.2 37.2 35.5 TR3 11.6 10.1 10.8 39.3 38.5 38.5 CON(ave) 8.2 9.6 7.4 37.4 35.4 37.1 TRT(ave) 9.9 9.2 8.9 37.0 37.5 36.9

3. Propionate production

[0210] Propionate can be produced by a wide range of intestinal microorganisms, the most common propionate producers being Bacteroides spp. (phylum Bacteroidetes), Veillonella (phylum Firmicutes) and Akkermansia muciniphila (phylum Verrucomicrobia). For all three donors studied, administration of gellan gum resulted in a significant reduction in propionate levels in response to treatment in both colons (Figures 3a-3b, Table 9). In the proximal colon, a strong immediate decrease was observed for donors A and B, whereas the effect was less pronounced for donor C (i.e., a decrease of 1.7 mM (-8%) for donor C compared to -4 .3 mM (-18%) and -4.8 mM (-20%) for donors A and B, respectively). On the other hand, a more gradual decrease in propionate levels was observed in the distal colon of all donors. These results are surprising given the studies of Edwards (1995) and Anderson (1988). For example, Edwards (1995) stated that in Wistar rats gellan gum had no consistent effect on SCFA levels, while Anderson (1988) reported that ingestion of large amounts of gellan gum resulted in a decrease in fecal propionate levels in female volunteers by 23% and an increase in the content of propionate in feces in male volunteers by 33%.

Table 9. Effect of gellan gum treatment on propionate production (in mM) in proximal (PC) and distal (DC) colon fermenters for three different donors (A, B and C) and on average weekly propionate production during controls periods (C1 and C2) and treatment weeks (TR1-TR3) (see also Fig. 2a-2b).

Periods PC DC A B C A B C C1 24.2 23.5 21.3 34.9 34.5 34.4 C2 23.1 23.2 22.6 33.2 36.0 35.0 TR1 19.3 19.1 21.5 32.7 33.9 33.9 TR2 19.1 16.9 18.7 28.4 29.9 30.9 TR3 19.5 19.6 20.4 29.8 30.3 30.7 CON(ave) 23.6 23.4 21.9 34.1 35.2 34.7 TRT(ave) 19.3 18.6 20.2 30.3 31.4 31.8

4. Butyrate products

[0211] Butyrate is produced by members of clusters IV and XIVaClostridium(phylum Firmicutes). In a process called cross-feeding, these microorganisms convert acetate and/or lactate (along with other substrates) into the health-related butyrate. Butyrate levels gradually increased after the addition of gellan gum in the proximal colon and to a lesser extent in the distal colon for all donors studied. (Fig. 4a-4b, table 10). The effect was most pronounced in the proximal region with significant increases of 2.3 mM (+24%), 1.9 mM (+21%), and 1.4 mM (+15%) for Donor A, Donor B, and Donor C , respectively. In the distal colon, butyrate levels in the donor alone were significantly increased following the addition of gellan gum (i.e., increased by 1.4 mM (+13%)).

Table 10. Effect of gellan gum treatment on butyrate production (in mM) in proximal (PC) and distal (DC) colon fermenters for three different donors (A, B and C) and on average weekly butyrate production during controls periods (C1 and C2) and treatment weeks (TR1-TR3) (see also Fig. 4a-4b).

Periods PC DC A B C A B C C1 9.4 9.2 9.4 10.6 12.2 11.3 C2 9.4 9.2 8.9 10.9 11.3 10.7 TR1 10.8 9.1 9.0 11.4 11.5 10.1 TR2 12.2 11.5 10.4 12.1 13.5 12.1 TR3 12.0 12.8 12.2 13.0 13.2 12.2 CON(ave) 9.4 9.2 9.1 10.8 11.8 11.0 TRT(ave) 11.7 11.1 10.5 12.2 12.7 11.4

5. Lactate production

[0212] The human intestine contains both lactate-producing and lactate-utilizing bacteria. Lactate is produced by lactic acid bacteria and lowers the pH of the environment. Lactate can have potent antimicrobial effects against pathogens, especially at low pH values. Another beneficial effect of lactate is due to its conversion to butyrate and/or propionate. Thus, since different types of microorganisms produce and convert lactate, an increase in lactate concentration may result from either an increase in production or a decrease in conversion. Therefore, caution must be exercised in interpreting data regarding lactate results.

[0213] In the proximal colon, lactate concentration increased in the last week of treatment for all donors studied, reaching significance only for donor A (Table 11). However, for other donors, high standard deviations could be observed in the last week of treatment as lactate concentrations gradually increased in this week, i.e. from 0.19 mM at the beginning of the last week of treatment to 0.73 mM at the end of the week for donor B and from 0.13 mM to 0.46 mM for donor C. In the distal colon, significantly increased lactate concentrations were observed in the last week of treatment for Donor C. For Donor A, there was a trend toward higher lactate concentrations after the addition of gellan gum, whereas the treatment had no effect on lactate concentrations for Donor B.

Table 11. Effect of gellan gum treatment on lactate production (in mM) in proximal (PC) and distal (DC) colon fermenters for three different donors (A, B and C) and on average weekly lactate production during controls periods (C1 and C2) and treatment weeks (TR1-TR3) (see also Fig. 5a-5b).

Periods PC DC A B C A B C C1 0.03 0.10 0.06 0.61 0.84 0.62 C2 0.02 0.01 0.01 0.65 0.65 0.45 TR1 0.03 0.05 0.05 0.57 0.84 0.60 TR2 0.06 0.15 0.11 0.64 0.74 0.66 TR3 0.32 0.45 0.30 0.79 0.67 0.96 CON(ave) 0.02 0.06 0.04 0.63 0.75 0.54 TRT(ave) 0.14 0.22 0.15 0.67 0.75 0.74

6. Ammonia and BCFA products

[0214] The production of both ammonia (NH ₄ ⁺ ) and branched chain SCFA (b-SCFA=sum of isobutyrate, isovalerate and isocaproate) results from protein degradation and reflects the proteolytic activity of the gut microbiota. Because the latter has been associated with direct and indirect detrimental health effects (eg, colon carcinogenesis), reducing ammonia/b-SCFA production is considered beneficial. In fig. 6a-6b (Table 12) presents the average ammonia production (in mg/ml) associated with different treatments in two parts of the colon, while FIG. 7a-7b (Table 13) presents the average branched chain SCFA production (in mM) associated with different treatments in two parts of the colon.

[0215] Ammonia levels were not affected by gellan gum treatment in either the proximal or distal colon for all donors studied, except for a slight increase in the proximal colon in the last week of treatment for donor C. These results were supported by SCFA levels with branched chain, where by the end of treatment there was only a slight increase in both the proximal and distal colon for all donors studied.

Table 12. Effect of gellan gum treatment on ammonia production (mg/L) in fermenters as proximal (PC) and distal (DC) colons for three different donors (A, B and C), and on average weekly ammonia production (mg/L) during control periods (C1 and C2) and treatment weeks (TR1-TR3) (see also Fig. 6a-6b).

Periods PC DC A B C A B C C1 102 110 82 271 280 281 C2 92 85 90 265 285 245 TR1 72 54 89 240 225 281 TR2 120 95 92 268 273 291 TR3 110 114 129 251 299 250 CON(ave) 97 98 86 268 283 263 TRT(ave) 101 88 103 253 266 274

Table 13. Effect of gellan gum treatment on branched chain SCFA production (mM) in proximal (PC) and distal (DC) colon fermenters for three different donors (A, B and C) and on average weekly SCFA production with branched chain (mM) during control periods (C1 and C2) and treatment weeks (TR1-TR3) (see also Fig. 7a-7b)).

Periods PC DC A B C A B C C1 1.9 1.7 1.7 2.3 2.3 2.3 C2 1.8 1.8 1.7 2.3 2.3 2.3 TR1 1.7 1.6 1.7 2.2 2.4 2.4 TR2 2.1 1.8 1.7 2.4 2.4 2.5 TR3 2.0 2.1 2.1 2.5 2.7 2.7 CON(ave) 1.9 1.8 1.7 2.3 2.3 2.3 TRT(ave) 1.9 1.8 1.8 2.4 2.5 2.5

G. Analysis of microbial community composition

[0216] Illumina 16S rRNA-targeted sequencing is a molecular method based on amplification of the 16S rRNA gene. Because Illumina's sequencing method is PCR-based, microbial sequences are amplified until saturation levels are reached. Therefore, when obtaining information on a wide range of (not pre-specified) OTUs (> 100 different from the most dominant OTUs), the results are presented as proportional values relative to the total number of sequences in each sample, allowing semi-quantitative results to be obtained. The methodology used here involves primers that span the two hypervariable regions (V3-V4) of the 16S rDNA. Using a paired-end sequencing approach, 2x250 bp sequencing. produces 424 bp amplicons. Such fragments are taxonomically more useful than smaller fragments that are taxonomically less informative. In addition to processing data at the phylum and family level, specific altered OTUs can be identified, and the Simpson diversity index can be calculated as a measure of both diversity and evenness of distribution. The smallest possible index value is 1, which corresponds to a community consisting of only one OTU. The maximum possible value is equal to the total number of OTUs. The index will move closer to its maximum value when the distribution of OTUs is more even, whereas a community dominated by a small number of OTUs will produce values closer to 1. The higher the index, the greater the diversity and the greater the evenness of the distribution.

1. Diversity Index

[0217] The reciprocal of the Simpson Diversity Index was calculated as a measure of diversity, in terms of both the abundance and evenness of species distribution. Based on the diversity indices, during the control period, each of the three SHIME blocks was colonized by reproducible luminal and mucosal microbial communities in both PC and DC. Diversity was higher in DC, although it was also significantly higher for luminal microbiota compared to mucosal microbiota in both PC and DC (Table 14).

Table 14. Mean reciprocal of Simpson's diversity index in lumen (L) and mucus (M) of proximal (PC) and distal (DC) colons as three SHIME blocks during the control period (n=6). In addition, there were also significant differences (p<0.05) for the reciprocal of Simpson's diversity index between L and M or between PC and DC, as evidenced by their p-values calculated using Student's t test.

L M PC vs C L versus M PC DC PC DC L M PC DC Diversity Index 4.8 11.3 3.5 7.7 0.000 0.002 0.012 0.037

[0218] Additionally, regarding treatment effects, gellan gum increased gut microbiota diversity compared to control for all three donors tested (Figure 8). Only the diversity of luminal microbiota in PC decreased slightly after treatment with gellan gum.

2. Type level

[0219] In addition, phylum-level microbiota composition indicated that three different SHIME blocks were colonized by reproducible luminal and mucosal microbial communities in both the proximal and distal colon. As a result, means were calculated for each of the four environmental conditions, while statistical tests were performed to understand the preference for a particular type for any of the four environmental conditions (Table 15).

Table 15. Mean relative abundance (%) by type level of microorganisms in the lumen (L) and mucus (M) of the proximal colon (PC) and distal colon (DC) as three SHIME blocks during the control period (n=6). In addition, also significant differences (p<0.05) for a particular type between L and M or between PC and DC are highlighted in bold and underlined using their p-values calculated using Student's t test.

Type Relative content (%) p-value L M PC vs DC L versus M PC DC PC DC L M PC DC Actinobacteria 34% 2% 44% 5% 0.000 0.000 0.124 0.096 Bacteroidetes 17% 47% 12% 17% 0.001 0.234 0.508 0.000 Firmicutes 44% 38% 43% 40% 0.107 0.579 0.913 0.586 Lentisphaerae 0% 0% 0% 0% 0.060 0.080 >0.05 0.063 Proteobacteria 6% 3% 1% 5% 0.123 0.034 0.044 0.108 Synergistetes 0% 10% 0% 32% 0.000 0.000 0.537 0.000 Verrucomicrobia 0% 0% 0% 0% 0.091 0.287 0.866 0.929

[0220] This revealed type-specific colonization of the lumen compared to the mucus layer with: (i) higher levels of Bacteroidetes in the lumen (significant only in DC); (ii) higher levels of Proteobacteria in the lumen (significant only in PC); and (iii) higher levels of Synergistetes in mucus (present only in DC). In addition, the following longitudinal differences were observed along the colon: (i) increased levels of Actinobacteria in PC; (ii) increased levels of Bacteroidetes in DCs (significant only in the lumen); (iii) presence of Synergistetes in DC; and (iv) lower levels of Proteobacteria in PC in the mucus layer, whereas the opposite trend was observed in the lumen.

[0221] Regarding processing, it followed that at the main site of fermentation, i.e. in the proximal colon lumen (Figure 9), gellan gum strongly increased Actinobacteria levels at the expense of Bacteroidetes and Firmicutes for all three donors tested. Similar observations were made for luminal samples from the distal colon (Fig. 9). Additionally, in the distal colon, luminal levels of Synergistetes and Lentisphaerae were increased following gellan gum treatment. In the mucosal compartment (Fig. 9), sample variability tended to be greater over time. This could be explained by the more heterogeneous composition of the biofilm that forms on top of the mucus layer compared to the homogeneous luminal suspension. Actinobacteria in the mucosa as well as in the lumen were enriched in both the proximal and distal colon following gellan gum treatment (with the exception of donor C in PC, which showed very strong stimulation of Synergistetes), however this was not accompanied by a decrease in Bacteroidetes and Firmicutes, as in lumen. In fact, in the mucosal compartment, differences in Firmicutes levels were observed between individuals following gellan gum treatment, i.e. donor A showed a decrease in Firmicutes levels, whereas donors B and C showed an increase. Finally, gellan gum treatment generally increased the relative abundance of Proteobacteria in proximal colon mucosal samples.

3. Family and OTU level

[0222] At the family level, the effects of gellan gum processing will be primarily discussed for the primary site of fermentation, i.e. lumen of the proximal colon (Fig. 10). For other conditions in the colon (distal colon lumen (Figure 11), proximal colon mucosa (Figure 12), and distal colon mucosa (Figure 13)) many similar observations have been made and will therefore be discussed. only specific and distinct changes from the main site of fermentation.

[0223] Gellan gum strongly increased Bifidobacteriaceae levels for all three donors studied. Shown in FIG. 10-11 information shows that the levels of Bifidobacteriaceae in the lumen of the proximal colon as fermenters for the two control periods averaged 24.7 ± 5.5%, while the levels of Bifidobacteriaceae in the lumen of the proximal colon as fermenters for three treatment periods averaged 39.0±8.8%. In addition, shown in FIG. 10-11 information shows that the levels of Bifidobacteriaceae in the lumen of the distal colon as fermenters for the two control periods averaged 1.85 ± 1.0%, while the levels of Bifidobacteriaceae in the lumen of the distal colon as fermenters for three treatment periods averaged 8.3±2.3%. At the OTU level, the main changes were found to relate to increased levels of Bifidobacteriaceae OTU 2 (related to Bifidobacterium adolescentis ). This strong bifidogenic effect fits well with the significant increase in acetate levels observed for all three donors following gellan gum treatment.

[0224] Gellan gum treatment strongly reduced Bacteroidaceae levels for all three donors studied. The family Bacteroidaceae contains many known propionate producers, which explains the strong reduction in propionate levels that was observed after the addition of gellan gum. In addition, a decrease in the relative abundance of Veillonellaceae was observed after gellan gum treatment, which was mainly due to a decrease in the abundance of Veillonellaceae OTU 1 (related to Megamonas sp.). Because this OTU is a potent propionate producer (upon lactate consumption), its reduction likely contributed to the decrease in propionate concentrations observed during the treatment period.

[0225] Gellan gum also slightly increased Lachnospiraceae levels over the three-week treatment period for the three donors studied, which may be related to the increased butyrate concentrations observed during the same period. In contrast, in the lumen of the distal colon, levels of Lachnospiraceae were reduced, although levels of other butyrate-producing families were increased after gellan gum treatment, e.g. Acidaminococcaceae, Eubacteriaceae and Ruminococcaceae. However, in the last week of treatment, Ruminococcaceae levels again decreased in the distal colon, while Veillonellaceae were stimulated in the same week. The latter explains the increased propionate production observed in the distal colon in the last week of treatment and is mainly attributed to stimulation of Veillonellaceae OTU 1 (related to Megamonas sp.).

[0226] Another butyrate-producing family that was exceptionally enriched in mucosal conditions following gellan gum treatment was the family Clostridiaceae, with a clear increase in Clostridiaceae OTU 23 (related to Clostridium butyricum ) in the proximal colon compared to Clostridiaceae OTU 17 ( related to Clostridium tertium ) in the distal colon.

[0227] Another finding consistent with previous findings following gellan gum treatment was an increase in several families within the phylum Proteobacteria, such as an increase in Enterobacteriaceae and Xanthomonadaceae. These families are mainly known because they contain several opportunistic species, however these families also contain many commensals that are known to ferment proteins in various parts of the colon, but mainly in the distal colon. Indeed, similar observations were made in the distal colon, where several families of the phylum Proteobacteria were slightly increased after gellan gum treatment. These data may correlate with the slight increase in BCFA levels that was observed towards the end of the treatment period.

[0228] Finally, several donor-specific changes were noted following gellan gum treatment in the proximal colon lumen: (i) increased levels of Microbacteriaceae for donors B and C; (ii) increased levels of Micrococcaceae for donors A and C; (iii) increased levels of Enterococcaceae, especially observed for donor C (similar observations were made in the distal colon, which could explain the increased lactate concentration in the last week of treatment that was noted for this donor); and (iv) increased levels of Synergistaceae for donor C. Because Synergistaceae primarily colonize the distal colon, stronger effects were observed in samples from the lumen of the distal colon, where a strong enrichment of Synergistaceae was observed for all three donors studied.

H. Summary of the Results of Example III

[0229] Acid/base consumption, gas production, SCFA, lactate and ammonia production were very stable in three different SHIME units during the control period. This indicated that the SHIME model operated under the most optimal conditions leading to a stable microbiota in the colon. This stability is a precondition that any effect observed during treatment is truly the result of the test product administered at a concentration corresponding to the 2 g/day dose in vivo.

[0230] After initiation of gellan gum treatment, base consumption increased in the proximal colon (indicating microbial fermentation via SCFA/lactate production) in the last week of treatment for all donors studied. In addition, a modest immediate increase in base consumption was observed in the distal colon. In terms of gas production, donor dependent effects were observed, with a slight increase in gas production for Donor B, while gas production for the other donors decreased after product addition.

[0231] While base consumption and gas production are only a rough indication of microbial fermentation, SCFA measurements provide more detailed insight into saccharolytic fermentation processes. They showed that gellan gum was primarily fermented in the proximal colon, where it immediately decreased propionate levels while acetate and butyrate levels gradually increased. The donor A microbiota produced the most pronounced increases in both acetate and butyrate levels following gellan gum treatment. Also in the distal colon, acetate and butyrate levels gradually increased over the course of treatment, whereas propionate levels gradually decreased, followed by an increase in the final week of treatment. The greatest increase in acetate production was observed for donor B, whereas donor A produced the greatest increase in butyrate levels. In addition, lactate concentrations generally remained very stable. In the proximal colon, lactate levels increased significantly only in the last week of treatment for donor A. In the distal colon, significantly increased lactate concentrations were noted in the last week of treatment for donor C.

[0232] With regard to markers of proteolytic fermentation, it follows that ammonia levels were not affected for all donors studied, in both the proximal and distal colon, with the exception of a slight increase in the proximal colon in the last week of treatment for donor C. These results were supported by BCFA levels, with only small increases observed towards the end of treatment in both the proximal and distal colon for all donors studied.

[0233] 16S rRNA-targeted sequencing analysis showed that the SHIME model supported a diverse luminal and mucosal microbiota in both the proximal and distal colon for the three donors studied. Interestingly, the mucosal microbiota was, consistent with data from adult humans, highly enriched in families containing well-known butyrate-producing species. In addition to this species-specific mucosal colonization, longitudinal differences in microbial colonization (proximal versus distal colon) were also identified.

[0234] In terms of treatment effects on microbial community composition, gellan gum was found to increase the diversity of the gut microbiota of the three study donors compared to the control period. In addition, it followed that at the main site of fermentation (the lumen of the proximal colon), gellan gum strongly increased the levels of Actinobacteria at the expense of Bacteroidetes and Firmicutes. The increase in Actinobacteria levels was mainly associated with blooms caused by Bifidobacteriaceae, which closely corresponded to the increase in acetate levels for all three donors studied. Interestingly, the bifidogenic effect following gellan gum supplementation was associated only with increases in levels of OTU related to Bifidobacterium adolescentis . Reductions in Bacteroidetes and Firmicutes levels were primarily attributed to decreases in Bacteroidaceae and Veillonellaceae levels for all three donors studied. Both families contain several strong propionate producers, which correlates with the decreases in propionate concentrations observed during the treatment period. Finally, the increase in butyrate production over the 3-week gellan gum treatment period was potentially associated with an increase in butyrate-producing species belonging to several Firmicutes families, such as Lachnospiraceae in the proximal colon lumen, Acidaminococcaceae, Eubacteriaceae and Ruminococcaceae in the distal colon lumen. intestines, and Clostridiaceae in mucosal conditions.

IV. Example IV. The effect of gellan gum on the functions of the intestinal wall

A. Introduction

[0235] Microorganisms in the intestine are a biologically active community that lies at the interface between the host and its nutrient medium. As a consequence, they strongly influence several aspects of host physiology and metabolism. A wide range of structural components and metabolites of microorganisms directly interact with host intestinal cells, affecting nutrient absorption and epithelial health. Both microorganism-associated molecular patterns (MAMPs) and bacterial-derived metabolites (e.g., short-chain fatty acids (SCFAs)) activate various signaling pathways such as lymphocyte maturation, epithelial health, and pattern recognition receptor-mediated neuroendocrine signaling ( PRR) and G protein-coupled receptor (GPR)-mediated signaling. In turn, these signaling pathways will determine inflammatory tone, energy balance, intestinal contractility, and appetite regulation (reviewed in Ha (2014)). Dysregulation of microbiome-host interactions is now believed to contribute to a variety of diseases (Groschwitz (2009)), including metabolic syndrome and obesity, inflammatory bowel diseases (IBD) such as Crohn's disease (CD) and ulcerative colitis (UC) , irritable bowel syndrome (IBS), celiac disease, diabetes, allergies, asthma and autoimmune diseases. Common to these disorders is dysregulation of the intestinal epithelial barrier (more permeable), initiating pathology (Fasano (2011)). When intestinal barrier function is compromised, the movement of molecules is no longer under control, so luminal contents can leak into the lamina propria and activate the immune system, thereby leading to uncontrolled immune responses (a process known as “leaky gut”). The intestinal epithelial barrier is formed by intercellular tight junctions, a complex protein-protein network that mechanically links adjacent cells and closes the intercellular space. Therefore, the intestinal epithelial barrier controls the balance between immunological tolerance and immune system activation, and therefore plays an important role in the pathogenesis of leaky gut. Improper functioning or regulation of these tight junctions appears to be responsible for the large intercellular spaces allowing luminal elements to pass through the barrier, with subsequent local and systemic inflammation.

B. In vitro Caco-2/THP1 co-culture model

[0236] To mimic the interface between the gut microbiome and the host, several in vitro models have been developed in recent years, which include the use of intestinal epithelial cell-like cells and human-derived immune cells. The model used here is a co-culture model of intestinal epithelial cell-like cells (Caco-2 cells) and human monocytes/macrophages (THP1 cells). (See FIG. 14; see also Possemiers (2013) Satsu (2006).) When plated on suitable supports, Caco-2 spontaneously differentiates into mature enterocyte-like cells characterized by polarization, villi, coning, tight junctions, and infection, and expression of brush border enzymes on the apical surface (reviewed by Sambuy (2005)). THP1 monocytes isolated from a human patient with acute leukemia differentiate into macrophage-like cells after treatment with phorbol 12-myristate 13-acetate (PMA). PMA-activated THP1 cells acquire morphological features characteristic of macrophages, are able to adhere to a substrate, develop lamellipodia necessary for migration and phagocytosis, and become primed for toll-like receptor (TLR) responses. (Dumrese (2009).) Tight junction proteins hold adjacent epithelial cells together, thereby forming a virtually impenetrable barrier to macromolecules. The "tightness" of these junctions can be measured as transepithelial electrical resistance (TEER), with a high TEER corresponding to a tighter barrier. When barrier function is lost, transcellular fluid transfer (between cells) increases, which can be defined as a decrease in TEER. When Caco-2 cells are placed on top of PMA-activated THP1 cells, which secrete cytokines into the supernatant, their monolayer is disrupted. This is possibly due to cytokine-mediated disruption of tight junctions and can be defined as a decrease in TEER.

[0237] Within the intestine, chemical, mechanical, or pathogen-triggered disruption of the barrier can result in an influx of bacteria from the lumen into the lamina propria (FIG. 15). This will activate the immune system, which will switch from physiological “tolerogenic” inflammation to harmful pathological inflammation. The inflammatory signaling cascade will be initiated by the production of signaling molecules such as proinflammatory cytokines (eg, tumor necrosis factor (TNF)-α and interleukin (IL)-1β). TNF-α, together with interferon (IFN)-γ, is produced by leukocytes and CD4 ⁺ T _H helper cells type 1, important cellular defenders against invading microorganisms. These proinflammatory cytokines will induce the production of chemokines (eg, IL-8 and chemokine (CXC motif) ligand (CXCL)-10) and adhesion molecules) necessary for neutrophil recruitment and reactive oxygen species (ROS) generation. ROS production is necessary to kill invading bacteria and close gaps in the epithelial wall. However, they can also cause tissue destruction and inflammation, leading to the need to eliminate inflammation through the production of anti-inflammatory cytokines such as IL-6 and IL-10.

[0238] IL-6 has both pro-inflammatory and anti-inflammatory properties. Scheller (2011). IL-6 leads to the recruitment of monocytes/macrophages through the activation of monocyte chemoattractant protein (MCP)-1, which promotes the removal of neutrophils. IL-6 is also capable of inhibiting the production of proinflammatory cytokines such as IL-1. In addition, IL-6 has a positive effect on intestinal epithelial regeneration and wound healing. Dann (2008). On the other hand, IL-6, together with transforming growth factor (TGF)-β), induces the differentiation of an important subpopulation of CD4 ⁺ T cells, T _H 17 cells, which play a key role in host defense against extracellular microorganisms in mucosal tissue.

[0239] IL-10 is an anti-inflammatory cytokine that can suppress certain cell types of the innate and adaptive immune response. In addition, IL-10 induces the activation of pro-inflammatory molecules and enhances the function of regulatory T cells (Treg), which will restore immune homeostasis. Lyer (2012). When these shutdown mechanisms are disrupted and immune homeostasis cannot be restored, gut pathology can occur, which can lead to chronic inflammation (as seen for example in IBD, which is characterized by excessive activation of T _H1 -mediated responses, namely overproduction of TNF-α ).

[0240] With regard to inflammation, TNF-α is one of the most potent and dangerous cytokines produced by the immune system, as it has pleiotropic effects and is capable of enhancing inflammatory signaling (Figure 16). Unchecked, TNF-α can lead to chronic inflammation and even death in cases of acute inflammation. For this reason, anti-TNF-α therapy is widely used in chronic inflammatory conditions, including IBD and rheumatoid arthritis.

[0241] The Caco-2/THP-1 coculture model exhibits some features also observed in patients with IBD and is therefore proposed as an IBD-like model that can be used to study the effect of substances that can both protect the integrity of the intestinal epithelial barrier. and reduce inflammation. Satsu (2006). As discussed, in this model, protection of intestinal barrier function is defined as an increase in TEER, while anti-inflammatory potential is determined through analysis of the cytokine profile (increase in anti-inflammatory cytokines and decrease in pro-inflammatory cytokines).

[0242] Colonic suspensions collected from SHIME are brought into contact with the apical side of the cocultures (Caco-2 cells). The effects observed in the basolateral chamber (where THP1 cells reside) are then mediated indirectly by signals generated by Caco-2 cells and/or by the transport of micro- and macromolecules. A unique aspect of this approach is that it allows the assessment of the effect induced by the product and fermentation-derived metabolites produced by the gut microbiota during the stages of digestion (i.e. not just the pure product). Daguet (2016).

C. Purpose of the study

[0243] The purpose of this part of the study was to examine the potential beneficial effects of a gellan gum product and its metabolites on intestinal wall function in three different donors. Bacteria interact closely with the intestinal wall, so modulation of microbial activity is likely to affect intestinal wall function. This will be assessed by assessing intestinal epithelial permeability and specific immune markers in vitro.

D. Materials and Methods

[0244] Samples collected from the SHIME experiment described above were used to evaluate the in vitro effect of fermented foods on intestinal epithelial barrier function and immune markers. They include samples from fermenters as proximal and distal colons from three different donors, collected at the end of the control and treatment periods.

E. Caco-2 cells

[0245] The coculture experiment was performed as previously described. Daguet (2016). Briefly, Caco-2 cells (HTB-37; American Type Culture Collection) were seeded in 24-well semipermeable inserts. Caco-2 monolayers were cultured for 14–21 days with three media changes per week to obtain a functional transepithelial electrical resistance (TEER) cell monolayer. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing glucose and glutamine and supplemented with HEPES and 20% (v/v) inactivated (HI) fetal bovine serum (FBS).

F. THP1-Blue™ Cells

[0246] THP1-Blue™ cells (InvivoGen) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium containing glucose and glutamine supplemented with HEPES, sodium pyruvate, and 10% (v/v) HI-FBS. THP1-Blue™ are human THP1 monocytes stably transfected with a reporter construct expressing the secreted alkaline phosphatase (SEAP) gene under the control of the transcription factor nuclear factor kappa B (NF-κB)-inducible promoter. Upon activation by TLR (eg, lipopolysaccharide (LPS); isolated from Gram-negative bacteria), NF-κB is activated and induces SEAP expression and secretion. SEAP activity can then be measured in the supernatants using QUANTI-Blue reagent (InvivoGen). THP1-Blue™ cells were seeded in 24-well plates and treated with PMA, which induces the cells to differentiate into macrophage-like cells that are able to adhere and are primed for TLR signaling.

G. Co-cultivation of Caco-2/THPl-blue™

[0247] Before setting up coculture, the TEER of Caco-2 monolayers was measured (time point=0 hour). The TEER of the empty liner was subtracted from all readings to account for the residual electrical resistance of the liner. Inserts containing Caco-2 were then placed on top of PMA-differentiated THP1-Blue™ cells for further experiments as previously described. Possemiers (2013) and Lyer (2012).

[0248] Briefly, the apical compartment (containing Caco-2 cells) was filled with filter-sterilized (0.22 μm) colonic suspensions from SHIME or various concentrations of live bacteria. Cells were also treated apically with sodium butyrate (NaB) (Sigma-Aldrich) as a positive control. The basolateral compartment (containing THP1-Blue™ cells) was filled with complete Caco-2 medium. Cells were also exposed to complete Caco-2 medium in both chambers as a control. Cells were treated for 24 hours, after which TEER was measured (time point=24 hours). After subtracting the empty liner's TEER, all values at 24 hours were normalized to its own value at 0 hours (to account for differences in the initial TEER of different liners) and presented as a percentage of the baseline value. The basolateral supernatant was then removed and cells were stimulated basolaterally with complete Caco-2 medium containing ultrapure LPS ( Escherichia coli K12, InvivoGen). Cells were also stimulated basolaterally with LPS in combination with hydrocortisone (HC) (Sigma-Aldrich) and medium without LPS (LPS-) as controls. After LPS stimulation, basolateral supernatants were collected for cytokine measurements (IL-Iβ, IL-6, IL-8, IL-10, TNF-α, CXCL10 and human MCP-1 using Luminex® multiplex (Affymetrix-eBioscience)) and NF-κB activity, according to the manufacturers' instructions. Cells were incubated at 37 ^ο C in a humidified air/CO ₂ atmosphere (95:5, v/v).

H. Statistics

[0249] Experimental controls were presented first in separate graphs; these refer to cells treated with complete medium as control (CM or LPS−), cells treated with lipopolysaccharide (LPS+) and sodium butyrate (NaB), and hydrocortisone (HC) as controls. For TEER, CM and NaB conditions were compared, and statistical significance was calculated using an unpaired two-tailed Student's t test. For immune markers (cytokines/chemokines and NF-κB activity), all conditions (LPS−, LPS+HC, and LPS+NaB) were compared with LPS+. Statistical significance was calculated using one-way analysis of variance using Dunnett's multiple comparison test with LPS+. (*), (**), (***) and (****) represent p < 0.05, p < 0.01, p < 0.001 and p < 0.0001, respectively.

[0250] Results regarding the SHIME samples are presented separately. Control (C) and post-treatment (T) samples submitted for both fermenters as colon (proximal (PC) and distal (DC) colon) were collected as three biological replicates in the SHIME experiment. Results for three different donors separately are shown, as well as the average for the three donors. To evaluate differences in TEER, NF-κB activation, and cytokine production between each treated sample and the control, ordinary one-way analysis of variance (ANOVA) was performed using Tuckey's multiple comparison test (significance indicated by an asterisk (*)). (*), (**), (***) and (****) represent p<0.05, p<0.01, p<0.001 and p<0.0001, respectively. All statistics were performed using GraphPad Prism™ software version 7.02 for Windows (GraphPad Software, San Diego, CA, USA).

I. Results for controls

1. Transepithelial electrical resistance (TEER)

[0251] After co-culture for 24 hours, complete medium (CM) as a control showed a reduction in TEER of almost 40% due to damage caused by PMA-activated THP1 cells on Caco-2 cells (Figure 17). As expected, sodium butyrate (NaB; positive control) was able to protect Caco-2 cells from this damage and maintain monolayer TEER. Peng (2007). Note that LPS is added only after TEER measurement at time = 24 hours. However, preliminary experiments showed that the dose of LPS used did not significantly affect the integrity of the Caco-2 cell barrier.

2. Immune markers

[0252] The results obtained for various immune markers can be seen in FIG. 18, fig. 19 and fig. 20. As expected, LPS was able to increase NF-κB activation (Figure 18), as well as the secretion of all cytokines tested (IL-6 and IL-10 (Figure 19) and IL-Iβ, IL-8, CXCL10, TNF-α and MCP-1 (Fig. 20)). In addition, hydrocortisone (HC), being a corticosteroid, acts as a broad-spectrum immunosuppressant by suppressing LPS-induced cytokines and chemokines (Figs. 19 and 20) and inhibiting LPS-induced NF-κB transcriptional activity (Fig. 18). In contrast, sodium butyrate (NaB) showed a marker-dependent effect. NaB increased the transcriptional activity of NF-κB (Fig. 18), an effect that is possibly mediated by attenuating the inhibitory activities of histone deacetylases (HDACs) on non-histone proteins such as NF-κB. Glozak (2005) and Vinolo (2011). In addition, NaB demonstrated clear selective post-transcriptional inhibitory activity against several immune mediators. More specifically, NaB selectively increased LPS-induced secretion of IL-6 and IL-10 (involved in immune homeostasis) (Fig. 19), although it selectively inhibited LPS-induced production of TNF-α (pro-inflammatory cytokines) and IL-8, CXCL10 and MCP-1 (chemokines involved in the recruitment of immune cells) (Fig. 20).

[0253] In conclusion, all controls in this experiment behaved as expected, and the results obtained for the SHIME samples are presented below. Note that in this experiment, HC and NaB unexpectedly did not reduce LPS-induced IL-1β expression.

J. Results for SHIME samples

1. Transepithelial electrical resistance (TEER)

[0254] SHIME samples collected during the final weeks of control and treatment from all fermenters as colons were diluted (1:5, v/v) in complete Caco-2 media after filtration (0.22 µm) and applied apically into cocultures for 24 hours.

[0255] Compared to complete medium (CM) as a control, where TEER was reduced by approximately 40% (FIG. 17), all control and treated samples selected from SHIME were able to maintain TEER at near baseline levels (FIG. 21). Although not significant, a small increase in TEER was observed following gellan gum treatment in distal colon samples from all three donors compared to controls. Given the fact that this increase was consistently observed for all three donors, it can be concluded that gellan gum fermentation has the potential to improve intestinal epithelial barrier function in the in vitro model used.

2. Immune markers

[0256] After apically pretreating Caco-2/THP-1-Blue™ cocultures with SHIME samples for 24 hours, the basolateral supernatant was removed and cells were stimulated with LPS. After 6 h of stimulation, the basolateral supernatant was collected to measure cytokines and chemokines secreted into the medium and to determine NF-κB activity.

[0257] Compared to the LPS+ control (red dotted line), all SHIME samples increased LPS-induced NF-κB transcriptional activity (Figure 22). However, there was no statistically significant difference between control and treated samples. Therefore, the increase in NF-κB activity most likely reflects the effect on cells of the SHIME suspension rather than the test compound.

[0258] Similar to the results obtained for NF-κB activity, all SHIME samples increased the levels of LPS-induced IL-6 and IL-10 compared to the LPS+ control (Figure 23). Although not significant, small increases in IL-6 and IL-10 levels compared with controls were consistently observed for all donors in distal colon samples. It was only statistically significant for IL-6 levels in donor A. This increase in IL-6 and IL-10 levels was also observed when analyzing the average of the three donors. Interestingly, when paired Student's t test was applied to all proximal and distal colon samples, there was a significant increase in IL-10 levels (p<0.05) for the three donors studied.

[0259] The results obtained for IL-1β and TNF-α are presented in FIG. 24. All SHIME samples clearly increased IL-1β secretion compared to the LPS+ control (red dotted line), however, no differences in IL-1β levels were found between control and treatment, with the exception of Donor B, where a significant increase in IL-1β levels was observed after processing for the fermenter as the proximal colon. When analyzing the mean of the three donors, no significant differences in IL-1β secretion were observed between control and treatment.

[0260] Compared to their controls, LPS-induced TNF-α levels were reduced in samples from the proximal and distal colon of Donor A and Donor B, but not Donor C. When the average of the three donors was observed, a slight decrease in secretion was observed TNF-α in proximal colon samples compared with controls, but no statistically significant differences were observed.

[0261] As seen in FIG. 25, IL-8 secretion was generally reduced after the addition of gellan gum compared with control for samples from the distal colon of two of three donors. However, this difference was not significant.

[0262] Levels of LPS-induced CXCL10 were generally slightly increased in distal colon samples from all donors following gellan gum treatment. For proximal colon samples, only one donor showed a slight decrease in CXCL10 expression after treatment. MCP-1 levels tended to decrease slightly following treatment for the proximal colon. In contrast, a clear increase was observed for fermenters as the distal colon for all donors. However, significance was not achieved.

[0263] In conclusion, although gellan gum had little effect on intestinal epithelial barrier function, it tended to increase the expression of the anti-inflammatory cytokines IL-6 and IL-10. In some conditions, the production of proinflammatory cytokines and chemokines tended to be reduced. However, only some statistically significant differences were observed between treatment and control.

[0264] To have an overview of the changes caused by the treated samples compared to the controls, the mean of the treated SHIME samples from the three donors was normalized for the fermenters as proximal and distal colon to the mean of the SHIME control samples. and presented in the form of table 16.

Table 16. Results of cell experiments based on the mean of treated SHIME samples from three donors, normalized to the mean of SHIME control samples.

Colon TEER NF-κB IL-6 IL-10 IL-1β TNF-α IL-8 CXCL10 MCP-1 Proximal section 1.00 1.01 0.90 1.06 1.28 0.88 1.06 0.97 0.85 Distal section 1.04 0.99 1.16 1.09 0.93 0.98 0.93 1.12 1.31

[0265] In general, it is reasonable to state that the changes in immune markers in the treated samples compared to the controls are quite small. As shown in Table 16, gellan gum appears to enhance IL-10 secretion and reduce TNF-α secretion in both colonic fermenters. Secretion of IL-8 was slightly decreased, whereas IL-6 was increased only for samples from the distal colon. IL-Iβ secretion appears to be increased for proximal colon samples, but these results are influenced by a significant increase in IL-1β in only one donor. MCP-1 secretion is reduced by gellan gum treatment for proximal colon samples but increased for distal colon samples.

K. Summary of the Results of Example IV

[0266] The purpose of this part of the study was to examine the potential beneficial effects of gellan gum and its metabolites on intestinal wall function in terms of modulating leaky gut conditions. This was done by assessing intestinal epithelial permeability and specific immune markers in vitro.

[0267] Following colonic fermentation, gellan gum generally increased intestinal barrier integrity as measured by TEER. Although the increases were modest, consistent increases were noted for all three donors when distal colon specimens were positioned in the in vitro model. In addition, the product generally had immunosuppressive properties, resulting in a trend toward decreased levels of several immune mediators, including the proinflammatory cytokine TNF-α and the chemoattractant protein IL-8, which are known to play a role in neutrophil recruitment. MCP-1, which promotes neutrophil clearance, was generally increased in distal colon fermenters following gellan gum treatment. On the other hand, IL-10, a true anti-inflammatory cytokine, tended to increase, as did IL-6, a cytokine involved in wound healing. All of these reported changes were mainly observed in distal colon fermenters, suggesting a more pronounced effect of gellan gum fermentation products on host immune cells in the distal colon.

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[0324] Alternative embodiments, examples and modifications that would still be covered by the present invention may be conceived by those skilled in the art, particularly in light of the foregoing teachings. Moreover, it should be understood that the terminology used to describe the present invention is intended in accordance with the spirit of the words to be descriptive and not limiting.

[0325] The subject matter of US provisional patent applications Nos. 62/794452 and 62/869248 is hereby incorporated by reference in its entirety. In addition, reference information provided herein is incorporated by reference in its entirety where appropriate. In the event of a difference in meaning between included terms and terms disclosed herein, the meaning of the terms disclosed herein will control.

[0326] Those skilled in the art will also appreciate that various adaptations and modifications of the preferred and alternative embodiments described above may be formed without departing from the scope and spirit of the present invention. It should therefore be understood that, within the scope of the appended claims, the present invention may be practiced differently than specifically described herein.

Claims (53)

1. A prebiotic composition containing an amount of sphingan in the form of an oligosaccharide with a DP of 2 to 30 that is effective for the manifestation of prebiotic activity. 2. Prebiotic composition according to claim 1, in which the amount of sphingan is selected from 1 g to 10 g, from 1 g to 9 g, from 1 g to 8 g, from 1 g to 7 g, from 1 g to 6 g, from 1 g to 5 g, from 1 g to 4 g, from 1 g to 3 g or 2 g. 3. The prebiotic composition according to claim 1 or 2, in which the sphingan includes a high-acyl sphingan in the form of an oligosaccharide, a medium-acyl sphingan in the form of an oligosaccharide, a low-acyl sphingan in the form of an oligosaccharide, or a combination thereof. 4. The prebiotic composition according to claim 1 or 2, wherein the sphingan oligosaccharide comprises a high-, medium- or low-acyl sphingan oligosaccharide (SOS) obtained by a method that includes obtaining a first composition containing high/medium/low acyl sphingan or high/medium/low acyl sphingan in the form of a polysaccharide and a liquid medium; hydrolyzing the glycosidic bond of the high/medium/low acyl sphingan or the high/medium/low acyl sphingan as a polysaccharide to produce a second composition; performing ultrafiltration, gel filtration, precipitation, centrifugation of the second composition or a combination thereof to obtain a third composition containing high/medium/low acyl SOS; And, optionally, isolating or retrieving a third composition. 5. Prebiotic composition according to any one of paragraphs. 1, 2 and 3, 4, in which the sphingan oligosaccharide comprises a high/medium/high acyl sphingan oligosaccharide selected from: (i) Glc,GlcA, Glc,GlcA,Glyc, Glc,GlcA,Rha, Glc,GlcA,Rha,Glyc, Glc,GlcA,Rha,-H2O, Glc,Rha, Glc,Rha+28, Glc2,GlcA, Glc2,GlcA,Rha, Glc2,GlcA,Rha,+28, Glc2,GlcA,Rha,Ac, Glc2,GlcA,Rha,Glyc, Glc2,GlcA,Rha,Glyc,+28, Glc2,GlcA,Rha,Glyc. -H2O, Glc2,GlcA,Rha,-H2O, Glc2,GlcA,Rha2,Glyc, Glc2,GlcA2,Rha, Glc2,GlcA2,Rha2,Ac2,Glyc2,-H2O, Glc2,Rha, Glc3,GlcA,Rha, Glc3 ,GlcA,Rha2, Glc3,GlcA,Rha2, Glc3,GlcA,Rha2, Glc3,GlcA,Rha2,Glyc, Glc3,GlcA2,Rha, Glc3,GlcA2,Rha,Glyc, Glc3,GlcA2,Rha2,Glyc, Glc3,GlcA3 ,Rha2, Glc3,GlcA3,Rha2, Glc4,GlcA,Rha2,+43, Glc4,GlcA,Rha2,Ac, Glyc, Glc4,GlcA2,Rha, Glc4,GlcA2,Rha,Ac,Glyc,-H2O, Glc4,GlcA2 ,Rha,Ac,Glyc2, Glc4,GlcA2,Rha2,Ac,Glyc, Glc4,GlcA2,Rha2,Glyc, Glc4,GlcA3,Rha2, Glc4,GlcA2,Rha3,Ac, Glc4,GlcA3,Rha2/Glc4,GlcA2,Rha2 ,Glyc2, Glc5,GlcA2,Rha2, Glc5,GlcA2,Rha2, Glc5,GlcA2,Rha2,Ac, Glc5,GlcA4,Rha2, Glc6,GlcA3,Rha3, Glc(Ac/Glyc)x,GlcAx,Glcx,Rhax (where x is from 4 to about 25), or a combination thereof; (ii) tetramer (Glc, GlcA, Glc, Rha), tetramer (Glc, GlcA, Glc, Rha) with acetate and/or glycerate, octamer (Glc, GlcA, Glc, Rha, Glc, GlcA, Glc, Rha), octamer (Glc, GlcA, Glc, Rha, Glc, GlcA, Glc, Rha) with acetate and/or glycerate, Glc, GlcA, Glc, Rha, Glc, GlcA, Glc, Rha; (iii) tetramer (Glc,GlcA,Glc,Rha), octamer (Glc,GlcA,Glc,Rha,Glc,GlcA,Glc,Rha), pentamer (Glc,GlcA,Glc,Rha,Glc), GlcA,Glc, Rha, Glc, GlcA, Glc, Glc, GlcA; (iv) Glc(Glc-Glc),GlcA, Glc(Glc-Glc), GlcA,Glc, Glc,Glc; (v) tetramer (Glc,GlcA,Glc,Rha), GlcA,Glc,(Rha-Rha), Glc,(Rha-Rha),Rha, GlcA,Glc,Rha, Glc,GlcA,Glc, Rha,Glc, GlcA,Glc; (vi) Glc,GlcA, Glc,GlcA,Glyc, Glc,GlcA,Rha, Glc,GlcA,Rha,Glyc, Glc,Rha, Glc,Rha+28, Glc2,GlcA, Glc2,GlcA,Rha, Glc2,GlcA ,Rha,+28, Glc2,GlcA,Rha,Ac, Glc2,GlcA,Rha,Glyc, Glc2,GlcA,Rha,Glyc,+28, Glc3,GlcA,Rha, Glc3,GlcA,Rha2, Glc3,GlcA,Rha2 , Glc3,GlcA,Rha2, Glc3,GlcA,Rha2,Glyc, Glc3,GlcA2,Rha,Glyc, Glc3,GlcA2,Rha2,Glyc, Glc3,GlcA3,Rha2, Glc4,GlcA,Rha2,Ac, Glyc, Glc4,GlcA2 ,Rha2,Ac,Glyc, Glc4,GlcA2,Rha2,Glyc, Glc4,GlcA2,Rha3,Ac, Glc4,GlcA3,Rha2/Glc4,GlcA2,Rha2,Glyc2, Glc5,GlcA2,Rha2, Glc5,GlcA2,Rha2,Ac , Glc(Ac/Glyc)x, GlcAx, Glcx, Rhax (where x is from 4 to about 25), or combinations thereof; (vii) Glc,GlcA, Glc,GlcA,Rha, Glc,Rha, Glc,Rha+28, Glc2,GlcA,Rha, Glc2,GlcA,Rha,+28, Glc2,GlcA2,Rha, Glc3,GlcA,Rha, Glc3,GlcA,Rha2, Glc3,GlcA2,Rha, Glc3,GlcA3,Rha2, Glc3,GlcA3,Rha2, Glc4,GlcA,Rha2,+43, Glc4,GlcA2,Rha, Glc4,GlcA3,Rha2, Glc5,GlcA2,Rha2 , Glc5,GlcA2,Rha2, Glc5,GlcA4,Rha2, Glc6,GlcA3,Rha3, Glc(Ac/Glyc)x,GlcAx,Glcx,Rhax (where x is from 4 to 25), or combinations thereof; (viii) Glc,GlcA,Rha,-H2O, Glc,Rha, Glc2,GlcA,Rha,-H2O, Glc2,Rha; (ix) Glc,GlcA, Glc,GlcA,Glyc, Glc,GlcA,Rha, Glc,GlcA,Rha,Glyc, Glc,Rha, Glc,Rha+28, Glc2,GlcA, Glc2,GlcA,Rha, Glc2,GlcA ,Rha,+28, Glc2,GlcA,Rha,Ac, Glc2,GlcA,Rha,Glyc, Glc2,GlcA,Rha,Glyc,+28, Glc2,GlcA,Rha,Glyc.-H2O, Glc2,GlcA,Rha2, Glyc, Glc2, GlcA2, Rha2, Ac2, Glyc2,-H2O, Glc3, GlcA, Rha, Glc3, GlcA, Rha2, Glc3, GlcA, Rha2, Glyc, Glc3, GlcA2, Rha, Glyc, Glc3, GlcA2, Rha2, Glyc , Glc3,GlcA3,Rha2, Glc4,GlcA,Rha2,+43, Glc4,GlcA,Rha2,Ac, Glyc, Glc4,GlcA2,Rha,Ac,Glyc,-H2O, Glc4,GlcA2,Rha,Ac,Glyc2, Glc4 ,GlcA2,Rha2,Ac,Glyc, Glc4,GlcA2,Rha2,Glyc, Glc4,GlcA3,Rha2, Glc4,GlcA2,Rha3,Ac, Glc4,GlcA3,Rha2/Glc4,GlcA2,Rha2,Glyc2, Glc5,GlcA2,Rha2 , Glc5, GlcA2, Rha2, Ac, Glc(Ac/Glyc)x, GlcAx, Glcx, Rhax (where x is from 4 to 25), or combinations thereof; or combinations thereof. 6. A method of stimulating the growth of beneficial bacteria in the colon of a mammal, the method comprising oral administration in an effective regimen, which may include (i) daily oral administration, such as once, twice, three times a day, etc.; or (ii) weekly oral administration, eg every day for seven days, every other day for seven days, etc.; or (iii) monthly dosing, such as daily dosing for a desired period of time followed by a rest period followed by daily dosing for a desired period of time; an amount of a prebiotic composition effective for stimulating the growth of beneficial bacteria, containing a prebiotically effective amount of sphingan in the form of an oligosaccharide according to any one of claims. 1-5 and medium for oral administration. 7. A method of reducing the level of propionate and/or increasing the level of butyrate in the colon of a mammal, the method comprising: oral administration in an effective regimen, which may include (i) daily administration, for example one, two, three times a day, etc. .; or (ii) weekly oral administration, eg every day for seven days, every other day for seven days, etc.; or (iii) monthly dosing, such as daily dosing for a desired period of time followed by a rest period followed by daily dosing for a desired period of time; a composition containing an effective amount of a prebiotic composition containing a prebiotically effective amount of sphingan in the form of an oligosaccharide according to claims. 1-5, and medium for oral administration. 8. A method of improving the integrity of the intestinal barrier in the colon of a mammal, the method comprising: oral administration in an effective regimen, which may include (i) daily administration, for example one, two, three times a day, etc.; or (ii) weekly oral administration, eg every day for seven days, every other day for seven days, etc.; or (iii) monthly dosing, such as daily dosing for a desired period of time followed by a rest period followed by daily dosing for a desired period of time; a composition containing an amount of a prebiotic composition effective for the integrity of the intestinal barrier, containing a prebiotically effective amount of sphingan in the form of an oligosaccharide according to claims. 1-5, and medium for oral administration. 9. A method of reducing the levels of TNF-α and/or IL-8 in the colon of a mammal, the method comprising: oral administration in an effective regimen, which may include (i) daily oral administration, for example one, two, three times a day and etc.; or (ii) weekly oral administration, eg every day for seven days, every other day for seven days, etc.; or (iii) monthly dosing, such as daily dosing for a desired period of time followed by a rest period followed by daily dosing for a desired period of time; a composition containing an amount of a prebiotic composition effective for reducing TNF-α and/or IL-8, containing a prebiotically effective amount of sphingan in the form of an oligosaccharide according to claims. 1-5, and medium for oral administration. 10. Method according to paragraphs. 6-9, wherein the mammal is a human and the amount of sphingan is selected from 10 mg/kg to 150 mg/kg, 10 mg/kg to 140 mg/kg, 10 mg/kg to 130 mg/kg, 10 mg/kg to 120 mg/kg, from 10 mg/kg to 110 mg/kg, from 10 mg/kg to 100 mg/kg, from 10 mg/kg to 90 mg/kg, from 10 mg/kg to 80 mg /kg, from 10 mg/kg to 70 mg/kg, from 10 mg/kg to 60 mg/kg, from 10 mg/kg to 50 mg/kg, from 10 mg/kg to 40 mg/kg or from 20 mg /kg to 30 mg/kg body weight of the person taking the composition orally. 11. Method according to paragraphs. 6-10, in which the sphingan includes high-, medium- or low-acyl sphingan in the form of an oligosaccharide (SOS), obtained by a method that includes: obtaining a first composition containing high/medium/low acyl sphingan or high/medium/low acyl sphingan in the form of a polysaccharide and a liquid medium; hydrolyzing the glycosidic bond of the high/medium/low acyl sphingan or the high/medium/low acyl sphingan as a polysaccharide to produce a second composition; performing ultrafiltration, gel filtration, precipitation, centrifugation of the second composition or a combination thereof to obtain a third composition containing high/medium/low acyl SOS; And, optionally, isolating or retrieving a third composition. 12. Method according to any one of paragraphs. 6-11, in which the sphingan includes a high/medium/high acyl sphingan in the form of an oligosaccharide selected from: (i) Glc,GlcA, Glc,GlcA,Glyc, Glc,GlcA,Rha, Glc,GlcA,Rha,Glyc, Glc,GlcA,Rha,-H2O, Glc,Rha, Glc,Rha+28, Glc2,GlcA, Glc2,GlcA,Rha, Glc2,GlcA,Rha,+28, Glc2,GlcA,Rha,Ac, Glc2,GlcA,Rha,Glyc, Glc2,GlcA,Rha,Glyc,+28, Glc2,GlcA,Rha,Glyc. -H2O, Glc2,GlcA,Rha,-H2O, Glc2,GlcA,Rha2,Glyc, Glc2,GlcA2,Rha, Glc2,GlcA2,Rha2,Ac2,Glyc2,-H2O, Glc2,Rha, Glc3,GlcA,Rha, Glc3 ,GlcA,Rha2, Glc3,GlcA,Rha2, Glc3,GlcA,Rha2, Glc3,GlcA,Rha2,Glyc, Glc3,GlcA2,Rha, Glc3,GlcA2,Rha,Glyc, Glc3,GlcA2,Rha2,Glyc, Glc3,GlcA3 ,Rha2, Glc3,GlcA3,Rha2, Glc4,GlcA,Rha2,+43, Glc4,GlcA,Rha2,Ac, Glyc, Glc4,GlcA2,Rha, Glc4,GlcA2,Rha,Ac,Glyc,-H2O, Glc4,GlcA2 ,Rha,Ac,Glyc2, Glc4,GlcA2,Rha2,Ac,Glyc, Glc4,GlcA2,Rha2,Glyc, Glc4,GlcA3,Rha2, Glc4,GlcA2,Rha3,Ac, Glc4,GlcA3,Rha2/Glc4,GlcA2,Rha2 ,Glyc2, Glc5,GlcA2,Rha2, Glc5,GlcA2,Rha2, Glc5,GlcA2,Rha2,Ac, Glc5,GlcA4,Rha2, Glc6,GlcA3,Rha3, Glc(Ac/Glyc)x,GlcAx,Glcx,Rhax (where x is from 4 to 25), Glcx, GlcAx, Glcx, Rhax (where x is from 4 to 25), or a combination thereof; (ii) tetramer (Glc,GlcA,Glc,Rha), tetramer (Glc,GlcA,Glc,Rha) with acetate and/or glycerate, octamer (Glc,GlcA,Glc,Rha,Glc,GlcA,Glc,Rha), octamer (Glc, GlcA, Glc, Rha, Glc, GlcA, Glc, Rha) with acetate and/or glycerate, Glc, GlcA, Glc, Rha, Glc, GlcA, Glc, Rha; (iii) tetramer (Glc,GlcA,Glc,Rha), octamer (Glc,GlcA,Glc,Rha,Glc,GlcA,Glc,Rha), pentamer (Glc,GlcA,Glc,Rha,Glc), GlcA,Glc, Rha, Glc, GlcA, Glc, Glc, GlcA; (iv) Glc(Glc-Glc),GlcA, Glc(Glc-Glc), GlcA,Glc, Glc,Glc; (v) tetramer (Glc,GlcA,Glc,Rha), GlcA,Glc,(Rha-Rha), Glc,(Rha-Rha),Rha, GlcA,Glc,Rha, Glc,GlcA,Glc, Rha,Glc, GlcA,Glc; (vi) Glc,GlcA, Glc,GlcA,Glyc, Glc,GlcA,Rha, Glc,GlcA,Rha,Glyc, Glc,Rha, Glc,Rha+28, Glc2,GlcA, Glc2,GlcA,Rha, Glc2,GlcA ,Rha,+28, Glc2,GlcA,Rha,Ac, Glc2,GlcA,Rha,Glyc, Glc2,GlcA,Rha,Glyc,+28, Glc3,GlcA,Rha, Glc3,GlcA,Rha2, Glc3,GlcA,Rha2 , Glc3,GlcA,Rha2, Glc3,GlcA,Rha2,Glyc, Glc3,GlcA2,Rha,Glyc, Glc3,GlcA2,Rha2,Glyc, Glc3,GlcA3,Rha2, Glc4,GlcA,Rha2,Ac, Glyc, Glc4,GlcA2 ,Rha2,Ac,Glyc, Glc4,GlcA2,Rha2,Glyc, Glc4,GlcA2,Rha3,Ac, Glc4,GlcA3,Rha2/Glc4,GlcA2,Rha2,Glyc2, Glc5,GlcA2,Rha2, Glc5,GlcA2,Rha2,Ac , Glc(Ac/Glyc)x,GlcAx,Glcx,Rhax (where x is from 4 to 25), or combinations thereof; (vii) Glc,GlcA, Glc,GlcA,Rha, Glc,Rha, Glc,Rha+28, Glc2,GlcA,Rha, Glc2,GlcA,Rha,+28, Glc2,GlcA2,Rha, Glc3,GlcA,Rha, Glc3,GlcA,Rha2, Glc3,GlcA2,Rha, Glc3,GlcA3,Rha2, Glc3,GlcA3,Rha2, Glc4,GlcA,Rha2,+43, Glc4,GlcA2,Rha, Glc4,GlcA3,Rha2, Glc5,GlcA2,Rha2 , Glc5,GlcA2,Rha2, Glc5,GlcA4,Rha2, Glc6,GlcA3,Rha3, Glc(Ac/Glyc)x,GlcAx,Glcx,Rhax (where x is from 4 to 25), or combinations thereof; (viii) Glc,GlcA,Rha,-H2O, Glc,Rha, Glc2,GlcA,Rha,-H2O, Glc2,Rha; (ix) Glc,GlcA, Glc,GlcA,Glyc, Glc,GlcA,Rha, Glc,GlcA,Rha,Glyc, Glc,Rha, Glc,Rha+28, Glc2,GlcA, Glc2,GlcA,Rha, Glc2,GlcA ,Rha,+28, Glc2,GlcA,Rha,Ac, Glc2,GlcA,Rha,Glyc, Glc2,GlcA,Rha,Glyc,+28, Glc2,GlcA,Rha,Glyc.-H2O, Glc2,GlcA,Rha2, Glyc, Glc2, GlcA2, Rha2, Ac2, Glyc2,-H2O, Glc3, GlcA, Rha, Glc3, GlcA, Rha2, Glc3, GlcA, Rha2, Glyc, Glc3, GlcA2, Rha, Glyc, Glc3, GlcA2, Rha2, Glyc , Glc3,GlcA3,Rha2, Glc4,GlcA,Rha2,+43, Glc4,GlcA,Rha2,Ac, Glyc, Glc4,GlcA2,Rha,Ac,Glyc,-H2O, Glc4,GlcA2,Rha,Ac,Glyc2, Glc4 ,GlcA2,Rha2,Ac,Glyc, Glc4,GlcA2,Rha2,Glyc, Glc4,GlcA3,Rha2, Glc4,GlcA2,Rha3,Ac, Glc4,GlcA3,Rha2/Glc4,GlcA2,Rha2,Glyc2, Glc5,GlcA2,Rha2 , Glc5, GlcA2, Rha2, Ac, Glc(Ac/Glyc)x, GlcAx, Glcx, Rhax (where x is from 4 to 25), or combinations thereof; or combinations thereof. 13. The method of claim 6, wherein the sphingan comprises naturally occurring sphingan and the bacteria are Bifidobacteriaceae. 14. The method according to claim 6, in which the mammal is a human, and the sphingan includes a high/low acyl sphingan in the form of an oligosaccharide, and the bacteria are Blautia, Parabacteroides, Faecalibacterium, Clostridium XVIII or a combination of them. 15. A method for producing high/medium/low acyl sphingan in the form of an oligosaccharide (“SOS”) according to any one of claims. 1-5, which includes: obtaining a first composition containing high/medium/low acyl sphingan or high/medium/low acyl sphingan in the form of a polysaccharide and a liquid medium; hydrolyzing the glycosidic bond of the high/medium/low acyl sphingan or the high/medium/low acyl sphingan as a polysaccharide to produce a second composition; performing ultrafiltration, gel filtration, precipitation, centrifugation of the second composition or a combination thereof to obtain a third composition containing high/medium/low acyl SOS; And optionally isolating or extracting a third composition; wherein said hydrolysis is mediated by an enzyme which is gellanase, rhamnogalacturonan endolyase (EC 4.2.2.23), rhamnogalacturonan exolyase (EC 4.2.2.24), gellanlyase (EC 4.2.2.25) or a combination thereof. 16. A method for producing high/medium/low acyl sphingan in the form of an oligosaccharide (“SOS”) according to any one of paragraphs. 1-5, which includes: obtaining a first composition containing high/medium/low acyl sphingan or high/medium/low acyl sphingan in the form of a polysaccharide and a liquid medium; hydrolyzing the glycosidic bond of the high/medium/low acyl sphingan or the high/medium/low acyl sphingan as a polysaccharide to produce a second composition; performing ultrafiltration, gel filtration, precipitation, centrifugation of the second composition or a combination thereof to obtain a third composition containing high/medium/low acyl SOS; And optionally isolating or recovering the third composition, said embodiment comprising filtering the second composition through a membrane having a molecular weight cutoff of either about 5 kDa or about 10 kDa to obtain a filtrate comprising the third composition.

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