Prebiotic composition comprising pea fibre and uses thereof

Abstract

The present invention relates to a prebiotic composition for promoting the growth or increasing the relative abundance of health-beneficial bacteria in the gastro-intestinal tract of an individual, the composition comprising a dietary pea fibre. The prebiotic composition also increases the production of short chain fatty acids in the intestinal tract of an individual. The present invention can be used as an additive to improve the physical properties of a food stuff in addition to modulating the microbiome.

Description

[0001] PREBIOTIC COMPOSITION AND USES THEREOF

[0002] Field

[0003] The present invention relates to the provision of a prebiotic composition that can promote the growth of health-beneficial bacteria and the production of short chain fatty acids in the intestinal tract of an individual. In particular, it relates to a prebiotic composition that can be used as an additive to improve the physical properties of a food stuff in addition to modulating the microbiome.

[0004] Background

[0005] Probiotics are microorganisms that, when consumed, act to supplement or modulate the microbiota composition in the gut of an individual so as to confer health benefits. These health benefits may include, among others, the reduction of the incidence of cancer, diarrhoea, diabetes, and irritable bowel syndrome of an individual.

[0006] Prebiotics are non-digestible fibres that can be included into the diet of an individual to serve as a substrate for colonic microorganisms in the gut of an individual. Prebiotics are selectively metabolisable by a subset of microbiota populations and, therefore, act to alter the relative abundance of microbiota in the gut. Health benefits can be conferred by consuming a prebiotic that is selectively degraded by probiotic microorganisms. The degradation product of prebiotics may also confer health benefits.

[0007] There exists a need for ‘clean label’ non allergenic food additives that can promote the growth of health-beneficial bacteria in an individual and improve the physical properties of a food stuff by, for example, acting as a preservative or consistency adjuster.It is an object of the present invention to provide a composition comprising a water-soluble polysaccharide for use in promoting the growth or increasing the relative abundance of health-beneficial bacterial in the intestinal tract of an individual. It would be advantageous if the composition could be easily incorporated into a food stuff (for example, as a foam stabiliser, anti-coagulant, preservative, consistency adjuster, gluten dispersibility improver or bulking agent). It would further be beneficial if the composition could be useful in the treatment or prevention of obesity or in the control of weight in an individual.

[0008] Summary of the Invention

[0009] We provide a prebiotic composition comprising a soluble pea fibre that can be used to promote the growth or increase the relative abundance of health-beneficial bacteria in the gastrointestinal tract of an individual.

[0010] According to a first aspect of the present invention, there is provided a composition for use as a prebiotic in promoting the growth or increasing the relative abundance of health-beneficial bacteria in the gastro-intestinal tract of an individual, the composition comprising a dietary pea fibre.

[0011] In one embodiment the dietary pea fibre is extracted from pea seed.

[0012] In one embodiment the dietary pea fibre is extracted from the yellow pea seed.

[0013] In one embodiment the dietary pea fibre is extracted from P. sativum.

[0014] In one embodiment the dietary pea fibre is a homogalacturonan.In one embodiment the dietary pea fibre comprises a negatively charged backbone region and at least one neutral polysaccharide branched regions.

[0015] In one embodiment the dietary pea fibre comprises a negatively charged backbone comprising galacturonic acid.

[0016] In one embodiment the dietary pea fibre comprises a negatively charged backbone further comprising a repeating unit of 1,2-a-L-Rhamnose and 1,4-a-D-Galacturonic acid; Xylogalacturonan, homogalacturonan with a-D-Xylose branched at 0-2 of galacturonic acid.

[0017] In one embodiment a single molecule of the pea fibre has a molecular diameter of more than 100 nm and equal to or less than 200 nm.

[0018] In one embodiment a single molecule of pea fibre has a molecular diameter of more than 125 nm and equal to or less than 200 nm.

[0019] In one embodiment a single molecule of pea fibre has a molecular diameter of more than 150 nm and equal to or less than 200 nm.

[0020] In one embodiment a single molecule of pea fibre has a molecular diameter of more than 175 nm and equal to or less than 200 nm.

[0021] In one embodiment a single molecule of pea fibre has a molecular diameter of more than 125 nm and equal to or less than 175 nm.

[0022] In one embodiment the degree of methyl esterification of constituent galacturonic acid is 45% or lessIn one embodiment the degree of methyl esterification of constituent galacturonic acid is 30% or less.

[0023] In one embodiment the degree of methyl esterification of constituent galacturonic acid is 20% or less.

[0024] In one embodiment the degree of methyl esterification of constituent galacturonic acid is 10% or less.

[0025] In one embodiment the degree of methyl esterification of constituent galacturonic acid is between 0% and 10%.

[0026] In one embodiment a single molecule of pea fibre has a star shaped structure.

[0027] In one embodiment a single molecule of pea fibre has a structure in which about 3 to 20 linear side chains of similar length are branched from the main chain.

[0028] In one embodiment the composition comprises polymeric components with a molecular rotation radius of 25 nm to 40 nm.

[0029] In one embodiment the composition comprises polymeric components with a molecular rotation radius of 30 nm to 40 nm.

[0030] In one embodiment the composition includes polymeric components with a molecular weight greater than or equal to 10,000.In one embodiment the composition includes polymeric components with an absolute molecular weight of 500,000 to 1 ,000,000.

[0031] In one embodiment the composition includes polymeric components with an absolute molecular weight of 800,000 to 900,000.

[0032] In one embodiment all components of the composition have a molecular weight below 5,000,000.

[0033] In one embodiment, the composition does not comprise pea starch and / or pea protein.

[0034] In one embodiment, the pH of the composition is about pH 3 to about pH 12.

[0035] In one embodiment, the pH of the composition is about pH 4 to about pH 10.

[0036] In one embodiment, the composition comprises at least 50% dietary pea fibre.

[0037] In one embodiment, the composition comprises at least one of galacturonic acid, arabinose, glucose, rhamnose, galactose and / or xylose

[0038] In one embodiment, the composition comprises about 35-50 (w / w %) Arabinose, about 20-35 (w / w %) Glucose, about 1-10 (w / w%) Galactose, 2-3 (w / w %) Xylose, 3-5 (w / w %) Rhamnose, and / or about 17-20 (w / w%) Galacturonic acid.

[0039] In one embodiment, the dietary pea fibre is water soluble.

[0040] In one embodiment, the dietary pea fibre is incorporated into a food stuff or food additive.In one embodiment, the composition is incorporated into a snack bar.

[0041] In one embodiment, the composition is incorporated into flour and / or rice.

[0042] In one embodiment, the total weight percentage of dietary pea fibre in flour and / or the total weight percentage of dietary pea fibre in cooked rice is at least 0.4%.

[0043] In one embodiment, the composition is incorporated into a beverage.

[0044] In one embodiment, the beverage is a meal replacement drink or an acidified milk beverage.

[0045] In one embodiment, the composition is incorporated into pet food.

[0046] In one embodiment, the pet food is in the form of dry food or paste.

[0047] In one embodiment, the pea fibre is incorporated into a food or beverage as one or more of the following: an acidified food stabilizer, a foam reducer and / or stabiliser, an anti-coagulant, a preservative consistency adjuster, a gluten dispersibility improver, a bulking agent, a sedimentation inhibitor, a syneresis reducer, and / or a consistency regulator.

[0048] In one embodiment, the composition is incorporated into a dietary supplement. The dietary supplement may be for human or animal consumption.

[0049] In one embodiment, the dietary supplement is in the form of a powder, a gel, gummy, a liquid or a paste.In another embodiment, the dietary supplement further comprises a probiotic.

[0050] In one embodiment, the dietary supplement is incorporated into a form of a dissolvable dry drinks powder.

[0051] In one embodiment, the dietary supplement is combined with a gelling agent.

[0052] In one embodiment, the gelling agent is selected from gelatine, pectin, agar or carrageenan.

[0053] In one embodiment, the composition comprising a dietary supplement combined with a gelling agent is in the form of a gummy.

[0054] In one embodiment, the composition according to any previous embodiment is combined with additional probiotics, fibres or other active microbiome modulating ingredients.

[0055] In one embodiment, the dietary pea fibre is present in the amount of 1-3 grams per portion

[0056] In one embodiment, the dietary pea fibre is present in the amount of 2 grams per portion

[0057] In one embodiment, the health-beneficial bacteria are selected from the Lachnospiraceae family and / or one or more of any one of the following genera of bacteria: Anaerostipes, Blautia, Monoglobus, Roseburia, Bifidobacterium, Bacteroides and / or Lactobacillus.

[0058] In one embodiment, the beneficial bacteria are from one or more of any one of the following species of bacteria: Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, Blautia faecis, Roseburia faecis, and / or Monoglobus pectinilyticus Eubacterium hallii,Eubacterium eligens, Lachnospiraceae NC2004, Lachnospiraceae NK4A136, Lachnospiraceae ND3007, Lachnospiraceae UCG-001 and / or Lachnospiraceae UCG-004.

[0059] In one embodiment, the composition increases small chain fatty acid (SOFA) production in the proximal and distal colon, preferably the SOFA are at least one of butyric acid, propionate, and / or acetate.

[0060] In one embodiment, the composition prevents or decreases the relative abundance of healthdetrimental bacteria in the gastro-intestinal tract of an individual.

[0061] In one embodiment, the health-detrimental bacteria is from the genera Clostridiaceae and Clostridium.

[0062] In one embodiment, the use of the dietary pea fibre is a non-therapeutic use.

[0063] In one embodiment, the composition is for use in the treatment, management, or prevention of disease in a subject.

[0064] According to a second aspect of the present invention, there is provided a method of treating, managing, or preventing a disease in a subject comprising administering the composition of any previous embodiment.

[0065] In one embodiment, the disease according to any previous embodiment is a dysbiosis of the gastrointestinal tract in an individual.

[0066] In one embodiment, the dysbiosis is associated with metabolic syndrome, obesity, or in the weight management of an individual.In one embodiment, the dysbiosis is associated with Crohn’s disease, Ulcerative Colitis (UC), Irritable Bowel Syndrome (IBS) or constipation.

[0067] In one embodiment, the dysbiosis is associated with Behcet syndrome, systemic sclerosis, or rheumatic arthritis.

[0068] In one embodiment, the dysbiosis is associated with an immune mediated disease.

[0069] In one embodiment, the immune mediated disease is an allergy or asthma.

[0070] In one embodiment, the dysbiosis is associated with Parkinson’s disease.

[0071] In one embodiment, the dysbiosis is associated with coronary artery disease, atherosclerosis or hypertension, the composition comprising pea fibre.

[0072] In a third aspect of the present invention, there is provided a method of weight management in an individual, the method comprising administering the composition of any previous embodiment up to 4 times a day.

[0073] In a fourth aspect of the present invention, there is provided a symbiotic composition comprising a prebiotic comprising dietary pea fibre; and a probiotic comprising an isolated bacterial strain from the Lachnospiraceae family and / or one or more of any one of the following genera of bacteria: Anaerostipes, Blautia, Monoglobus, Roseburia, Bifidobacterium, Bacteroides and / or Lactobacillus.In one embodiment, the probiotic isolated bacteria are selected from a list consisting of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, Blautia faecis, Roseburia faecis, and / or Monoglobus pectinilyticus Eubacterium hallii, Eubacterium eligens, Lachnospiraceae NC2004, Lachnospiraceae NK4A136, Lachnospiraceae ND3007, Lachnospiraceae UCG-001 and / or Lachnospiraceae UCG-004.

[0074] In one embodiment, the prebiotic and probiotic bacterial strain are provided together in a single formulation.

[0075] In one embodiment, the prebiotic and probiotic bacterial strain are provided separately for administration to an individual sequentially.

[0076] In a fifth aspect of the present invention there is provided a method of enhancing the in vitro growth of one or more bacterial genera of interest, the method comprising adding an amount of the composition according to the composition of any previous embodiment to a growth medium and growing the bacterial genera of interest in the supplemented growth medium.

[0077] In a sixth aspect of the present invention, there is provided a method of increasing the relative abundance of health-beneficial bacteria in an individual, the method comprising administering an effective amount of the composition of any previous embodiment.

[0078] In one embodiment of the present aspect, the health-beneficial bacteria are selected from the family of Lachnospiraceae and / or one or more of any one of the following genera of bacteria: Anaerostipes, Blautia, Monoglobus, Roseburia, Bifidobacterium, Bacteroides and / or Lactobacillus.In another embodiment of the present aspect, the health-beneficial bacteria are selected from the Lachnospiraceae family and / or one or more of any one of the following genera of bacteria: Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, Blautia faecis, Roseburia faecis, and / or Monoglobus pectinilyticus Eubacterium hallii, Eubacterium eligens, Lachnospiraceae NC2004, Lachnospiraceae NK4A136, Lachnospiraceae ND3007, Lachnospiraceae UCG-001 and / or Lachnospiraceae UCG-004.

[0079] Description of the Figures

[0080] Embodiments of the invention are described below, by way of example only, with reference to the accompanying figures in which:

[0081] Figure 1 is a schematic drawing showing the proposed molecular structure of pea fibre (Nakamura et al. 2022). Homogalacturonan, the main chain being composed of 1,4-a-D-Galacturonic acid; Rhamnogalacturonan, the main chain being composed of the repeating unit of 1,2-a-L-Rhamnose and 1 ,4-a-D-Galacturonic acid; Xylogalacturonan, homogalacturonan with a-D-Xylose branched at 0-2 of galacturonic acid.

[0082] Figure 2 is a line graph showing the differences in viscosity (mPa x S) between pea fibre (FIPEA-D®) and a selection of known food stabilising dietary fibres (SOYAFIBE-S CA100, Guar Gum, Gum Arabic and HM- Pectin).

[0083] Figure 3 comprises two line graphs that show the rate of precipitation (Figure 3A) and viscosity (Figure 3B) of high milk solids non-fat (MSNF) acidified milk beverages prepared with Pectin, SOYA-S CA100, or pea fibre (FIPEA-D®) fibres at different pH levels. Each beverage comprised 3% w / v MNSF and 0.2% w / v fibre and were stored at 4°C for seven days beforeanalysis. Precipitation was measured by calculating the percentage of sedimentation produced after samples were centrifuged at 3,000 rpm for 20 minutes.

[0084] Figure 4 is a schematic flow chart taken from (Food Fund, 2014, 5, 1113) showing an overview of the simulated in vitro digestion method (INFOGEST protocol) used throughout the examples of the present application. The numbers in circles indicate points during the in vitro digestion protocol in which samples of each reaction were taken.

[0085] Figures 5A, 5B and 50 comprise three separate High Performance Size Exclusion Chromatography (HPSEC) spectra showing the molecular weight distribution of different fibre type samples taken before, during and after in vitro digestion reactions using the INFOGEST protocol. Sample numbers before the decimal point represent the stage of the digestion protocol that the sample was taken. Sample numbers after the decimal point number represent the replicate number. Samples were mixed in 1:1 (wt / wt) ratios with simulated salivary fluid (SSF) (Samples 1.1 and 1.2) before they were mixed with salivary amylase to a final concentration of 75 U / rnL. Samples were then incubated for two minutes, mixed with simulated gastric fluid (SGF) in a 1:1 (wt / wt) ratio and adjusted to pH 3 (Samples 3.1 and 3.2). Once adjusted to pH 3, samples were mixed with pepsin in a 1:1 (1:1) ratio and incubated for 2 hours. Once incubated, samples were mixed in a 1:1 (wt / wt) ratio with simulated intestinal fluid and adjusted to pH 7 (Samples 5.1 and 5.2). Samples were then mixed with 100 U / rnL pancreatin and 10 mM Bile and incubated for 2 hours (Samples 7.1 and 7.2). Figure 5A is a HPSEC chromatography spectrum of samples taken in an empty digest control in vitro digestion reaction as described above. Figures 5B and 5C are HPSEC chromatography spectra of samples taken from an in vitro digestion reaction of pea fibre. Figure 5B shows samples taken directly from the in vitro digestion reactions. Figure 5C shows samples taken from the in vitro digestion reaction and dialysed to remove lower molecular weight moieties such as salts and digestion enzymes. Pea 2 is a spectrum from freshly dissolved pea fibre samples, used forcomparison. Peaks visible in figures 5B and 5C at a retention time of ~ 8-10 minutes are a result of intact fibre.

[0086] Figure 6 is a schematic diagram showing the overview of in vitro anaerobic fermentation reactions to determine the effects of pea fibre, soy fibre (SOYAFIBE-S CA100) and pectin on the composition of colonic bacteria. A sample of microbiota was isolated from the faecal material of a single donor (Donor 1) and used to inoculate a SHIME® system. The inoculated SHI ME® system was provided with adult feed with starch (prodigest) three times a day for four days before microbiota derived from either the proximal (PC) or distal (DC) compartments were separately used to inoculate fermentation vessels containing either basal medium (2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3 and 1 g mucin / liter) (control), or basal medium further comprising preparations of in vitro digested pea Fibre (Pea), Soy Fibre (SOYAFIBE-S CA100) (Soy) or Pectin (Pectin).

[0087] Figures 7A, 7B, 7C and 7D are a series of line graphs showing the head space pressure and pH changes of sealed vessels used during separate fermentation reactions of pea fibre (circle), soy fibre (SOYAFIBE-S CA100) (square), pectin fibre (triangle), FUJI CD (control digest, inverted triangle) and buffer (control, diamond). Measurements were taken 6 hours, 24 hours, and 48 hours after the beginning of the fermentation reactions. Statistically significant differences compared to control digests are indicated in the boxes to the left of each fibre name in the legend (*t). Figures 7A and 7B show headspace pressure measurements taken from fermentation reactions inoculated with proximal colon microbiota and distal colon microbiota, respectively. Figures 7C and 7D are line graphs showing the pH measurements offermentation reactions with proximal colon microbiota and distal colon microbiota respectively.

[0088] Figures 8A and 8B are line graphs that show changes in alpha diversity at the start, and during fermentation reactions inoculated with proximal colonic microbiota (Figure 8A) or distal colonicbacteria (Figure 8B). Fermentation reactions were performed in the presence of pea fibre (circle), soy fibre (SOYAFIBE-S CA100) (square), pectin fibre (triangle), Fuji CD (control digest, inverted triangle) or a medium-only control (diamond). Alpha diversity was measured and plotted using the Shannon Index, which is a measure of both the number of species and the inequality between species abundance. Values range from 1 to the number of species in the sample, wherein a value of 1 would indicate that a single species is dominant in a sample and a high number would indicate that all those species have an equal abundance.

[0089] Figure 9 shows a Bray Curtis diversity analysis of identified microbiota species present before, during and after fermentation of SHIME® system-derived proximal colon microbiota fermentation reactions in the presence of pea fibre, soy fibre (SOYAFIBE-S CA100), pectin fibre, FUJI CD (control digest) and a medium only control. Axes represent the two principal coordinates (PCo; showing the percentage of variance indicated in parentheses on the X and Y axes labels) of samples taken at the start and after 6 hours, 24 hours, 48 hours of fermentation (indicated to the right of each data point). Circled data points indicate areas individual points relating to one type of sample are clustered. The lowest circled data points represent medium and FUJI digest controls. The second set of circled data points from the bottom of the graph represent soy fibre (SOYAFIBE-S CA100). The third set of circled data points from the top of the graph represent pea fibre. The top set of circled data points represent pectin fibre.

[0090] Figure 10 is a Bray Curtis beta diversity analysis of identified microbiota species present before, during and after fermentation reactions of SHIME® system-derived distal colon microbiota with culturing media alone, or further containing pea fibre, soy fibre (SOYAFIBE-S CA100), pectin fibre, or FUJI CD (control digest). Axes represent the two principal coordinates (PCo; showing the percentage of variance indicated in parentheses on the X and Y axes labels)of samples taken at the start (t=0) and after 6 hours (t=6), 24 hours (t=24), 48 hours (t=48) of fermentation (indicated to the right of each data point).

[0091] Figure 11 is a schematic diagram showing the overview of in vitro fermentation reactions performed using undigested pea fibre, soy fibre (SOYAFIBE-S CA100), pectin and inulin alongside a control. A sample of microbiota was isolated from the faecal material of a single donor (Donor 1) and used to inoculate a SHIME® system. The inoculated SHIME® system was provided with adult feed with starch (prodigest) three times a day for four days before microbiota samples taken from the distal colon compartment and used to inoculate separate vessels containing basal medium alone (2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3 and 1 g mucin / liter) (CNTRL) or basal medium further supplemented with pea fibre (Pea), soy fibre (SOYAFIBE-S CA100) (Soy), Pectin (Pectin), or Inulin (Inulin). This experiment was repeated twice more using the faecal material from a second (Donor 2) and third donor (Donor 3).

[0092] Figures 12A, 12B, 12C and 12D comprise a heatmap diagram (Figure 12A) and isolated sections of the same (Figure 12B, 12C, and 12D), showing the hierarchical clustering (Euclidean complete linkage) of the different phylogenetic compositions identified in microbiota samples taken from fermentation experiments. Fermentation experiments were set up by inoculating vessels of media using microbiota samples isolated from the distal colon compartment of three separate SHIME® systems. Each SHIME® system was itself inoculated with a microbiota sample isolated from faecal material of a different donor. Vessels either contained basal medium (2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3 and 1 g mucin / liter) with dialysis buffer (CNTRL) or basal medium further supplemented with pea fibre, soy fibre (SOYAFIBE-S CA100), Pectin, or Inulin. Once set up, fermentation reactions were incubated anaerobically at 37°C with continuous shaking at 120 rpm. Samples were taken at 0 (t=0), 6 (t=6) and 24 (t=24) hourtimepoints. The microbiota composition of each sample was determined through deep sequencing of the 16S rRNA gene. Sequencing data was quality controlled and aligned to the 16S reference database, Silva. The genus of each sequenced microbe was identified, and their relative abundance was calculated. Each column of the heat map represents a different sample type, and each row represents a different microbial genus which is represented by number as follows: Anaerovoracaceae (1), Christensenellaceae (2), Veillonellaceae (3), Lachnospiraceae (4), Sutterellaceae (5), {unknown order} Bacilli (6), Bacillaceae (7), Family XI (8), Uncultured (9), Planococcaceae (10), {Unknown Family} Oscillospirales (11), Erysipelotrichaceae (12), Lactobacillaceae (13), {Unknown Order} Clostridia (14), {Clostridium} methylpentosum group (15), {Eubacterium} coprostanoligenes group (16), Oscillospiraceae (17), Ruminococcaceae (18), Paenibacillaceae (19), Marinifilaceae (20), Clostridiaceae (21), {Unknown Family} Lactobacillales (22), Enterococcaceae (23), {Unknown Family} Enterobacterales (24), Enterobacteriaceae (25), Bacteroidaceae (26), Erwiniaceae (27), Morganellaceae (28), Desulfovibrionaceae (29), Butyricicoccaceae (30), Peptostreptococcaceae (31), Coriobacteriaceae (32), Rikenellaceae (33), Eggerthellaceae (34), Puniceicoccaceae (35), Ethanoligenenaceae (36), Acidaminococcaceae (37), Selenomonadaceae (38), Fusobacteriaceae (39), Akkermansiaceae (40), Atopobiaceae (41), Victicallaceae (42), Corynebacteriaceae (43), Pseudomonadaceae (44), Bifidobacteriaceae (45), Tannerellaceae (46). Darker cells within the heat map represent a microbial genus that is more relatively abundant in each sample. Relative frequencies can be calculated by comparing the shade of a given cell with the scale bar to the right-hand side of the heatmap. The three rows of coded blocks at the top of the heat map represent time (hours, top), Donor (middle, 1,2 or 3). Fibre sample types are provided on the third row, wherein A = soy fibre (SOYAFIBE-S CA100), B = Inulin, C= pea fibre, D= pectin and E = the dialysis buffer control. Each column represents a single sequenced sample. Samples are clustered based on their Euclidean complete linkage, and conclusions can be drawn on the impact of time, donor type and sample type by how closely samples of the same type are clustered together.Figure 13 is a box and whisker plot showing the alpha diversity of species identified in microbiota samples taken from fermentation experiments containing either basal culturing medium alone (2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3 and 1 g mucin / liter) (CNTRL), or basal cultural medium further supplemented with pea fibre (Pea), soy fibre (SOYAFIBE-S CA100) (Soy), Pectin (Pectin), or Inulin (Inulin). Fermentation experiments were set up by inoculating vessels of media with microbiota isolated from the distal colon compartment of three separate SHI ME® systems. Each SHIME® system was itself inoculated with isolated microbiota from faecal material of a different donor. Once set up, fermentation reactions were incubated anaerobically for 4 days at 37°C with continuous shaking at 120 rpm. Samples were taken at 0, 6 and 24 hour timepoints. The microbiota composition of each sample was determined through the deep sequencing of the 16S rRNA gene and alignment to the Silva 16S reference database. Each plot represents the data gathered from fermentation experiments of the different donors (D1, D2, and D3 respectively). Each dot represents an individual sample. The shading of each dot represents the fibre type used in the sample corresponding to the key in the bottom right corner of the graph. The size of the dot represents the different time points, wherein the smallest dots represent samples taken after 0 hours of fermentation, medium dots represent samples taken 6 hours after fermentation, and the largest dots represent samples taken after 24 hours of fermentation.

[0093] Figure 14 is a Bray Curtis beta diversity analysis of identified microbiota species present before, during and after fermentation reactions of SHIME® system-derived distal colon microbiota with basal culturing media alone (2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3 and 1 g mucin / liter), or basal culturing medium containing one of pea fibre, soy fibre (SOYAFIBE-S CA100), pectin fibre, inulin or FUJI CD (control digest). Each SHIME® system was itself inoculated with a microbiotasample isolated from faecal material of a different donor. Once established, fermentation reactions were incubated anaerobically for 4 days at 37°C with continuous shaking at 120 rpm. Samples were taken at 0, 6 and 24 hour timepoints and the microbiota composition of each sample was determined through deep sequencing of the 16S rRNA gene. The principal Coordinate (PCo) analysis plots for all donors, timepoints and fibre samples are plotted and axes represent the two principal coordinate values (PCo; showing the percentage of variance indicated in parentheses on the X and Y axes labels). Data point symbols represent different timepoints, as indicated in the key located at the bottom left of the graph. The shade of each plotted sample represents donor group from which each sample is derived. The lightest sample shading represents non-fermented samples. The second lightest sample shading represents samples of Donor 1, the darkest sample shading represents samples of Donor 2 and medium shading represents samples from Donor 3. The type of fibre used in each sample is written next to each plotted point.

[0094] Figure 15 is a Venn diagram showing a comparison of the changes in microbiota composition of separate SHIMEO-derived distal colon fermentation reactions incubated for 24 hours in basal culturing media (2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3 and 1 g mucin / liter) further supplemented with pea fibre, soy fibre (SOYAFIBE-S CA100), or inulin. Changes in microbiota composition were calculated relative to a no fibre control fermentation. The differential analysis was performed for all donors, and differences were determined using a False Discovery Rate (FDR) with a p-value of <0.05. Fold changes in taxa abundance equal or greater than 1.5 are indicated in the diagram with an upward arrow or a downward arrow, representing higher or lower abundances relative to the control fermentation reactions respectively.

[0095] Figure 16 is a Venn diagram showing the change in microbiota composition of SHIME(B>-derived distal colon samples before, during and after fermentation reactions with basal medium(2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3 and 1 g mucin / liter) further supplemented with pea fibre. Results are an analysis of the average of three different fermentation reactions using SHIMEO-derived colon bacteria in which each fermentation was inoculated with microbiota from faecal material of a different donor. Each differential bacterial genus for t=6h vs t = Oh, overlap and t = 24h vs t=Oh is shown in the table as a / og2fold change, where t = time of fermentation (hours, h). The False Discovery Rate (FDR) p-value was < 0.05 and the minimum presented fold change was >1.5. Arrows up and down show upregulated and downregulated compared to a control fermentation reaction performed in basal culturing media (2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3 and 1 g mucin / liter) alone at t = 24 h.

[0096] Figure 17 is a series of bar graphs showing the concentration of short chain fatty acids detected in fermentation experiments. Fermentation experiments were set up using fresh faecal samples from three different donors (Donor 1 , Donor 2 and Donor 3) to separately inoculate flasks of basal medium (2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3 and 1 g mucin / liter) supplemented with 0.5 mg / mL bile (control, CNTL) and further containing 10 mg / mL pea fibre, pectin or fructooligosaccharide (FOS). Two further fermentation experiments were set up using fresh faecal samples from donor 1 to separately inoculate basal medium supplemented with 5 mg / mL or 3.6 mg / mL pea fibre. Samples were collected and centrifuged before the supernatant from each sample was diluted 1:1 with 16.6 mM H2SO4 and filtered with a 0.2 pm filter. The short chain fatty acid samples were measured on an HPLC, AMINEX HPX-87H column (Biorad) (eluent buffer 8, 3 mM H2SO4 (444 pL / litre milli-Q), flowrate 0.5 mL / min, run time: 42 minutes, HPLC Water 2414 with Rl detection, Sample chamber 15°C, Detector Rl at 35°C). An scFA standard was run after every 20 samples and was used for calculations of the unknown scFA in the samples by using the peak area. Each bar graph represents a different timepoint:scFA_tOh = samples taken at the start at fermentations, scFA_t3h represents samples taken after 3 hours of fermentation, scFA_6h represents samples taken after 6 hours of fermentation, sc_24 hours represents samples taken after fermentation and scFA_t48h represents samples taken after 48 hours of fermentation.

[0097] Figure 18 is a series of bar graphs showing the concentration of Butyrate detected in fermentation experiments. Fermentation experiments were set up using fresh faecal samples from three different donors (Donor 1, Donor 2 and Donor 3) to separately inoculate flasks of basal medium (2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3 and 1 g mucin / liter) supplemented with 0.5 mg / mL bile (control, CNTL) and further containing 10 mg / mL pea fibre, pectin or fructo-oligosaccharide (FOS). Two further fermentation experiments were set up using fresh faecal samples from donor 1 to separately inoculate basal medium supplemented with 5 mg / mL and 3.6 mg / mL pea fibre. To determine the concentration of each scFA, the supernatant from each sample was diluted 1:1 with 16.6 mM H2SO4 and filtered with a 0.2 pm filter. The short chain fatty acid samples were measured on an HPLC, AMINEX HPX-87H column (Biorad) (eluent buffer 8, 3 mM H2SO4 (444 pL / litre milli-Q), flowrate 0.5 mL / min, run time: 42 minutes, HPLC Water 2414 with Rl detection, Sample chamber 15°C, Detector Rl at 35°C). An scFA standard was run after every 20 samples and was used for calculations of the unknown scFA in the samples by using the peak area. Each bar graph represents a different timepoint, as indicated at the top of each graph.

[0098] Figure 19 is a heat map diagram showing the hierarchical clustering (Euclidean complete linkage) of the different phylogenetic compositions identified in microbiota samples taken from fermentation experiments. Fermentation experiments were set up using fresh faecal samples from three different donors to separately inoculate flasks of basal medium (2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3and 1 g mucin / liter) supplemented with 0.5 mg / mL bile (control, CNTL) and further containing 10 mg / mL pea fibre, pectin or fructo-oligosaccharide (FOS). Samples were taken at 0 hour and 48 hour time points. The microbiota composition of each sample was determined through deep sequencing of the 16S rRNA gene. Sequencing data was quality controlled and aligned to the 16S reference database Silva. The genus of each sequenced microbe was identified, and their relative abundance was calculated. Each column of the heat map represents a different sample type, and each row represents one of 132 different microbial genera which is represented by number as follows: Campylobacter (1), Lactobacillus (2), Enterorhabdus (3), Bifidobacterium (4), Subdoligranulum (5), Phascolarctobacterium (6), Desulfovibrio (7), Sutterella (8), Uncultured-2 (9), {Unknown Genus} VadinBE97 (10), {Unknown Genus} UCG-010 (11), {Unknown Family} Gastranaerophilales (12), Holdemanella (13), Paraprevotella (14), UCG-004 (15), {Unknown Genus} Erysipelatoclostridiaceae (16), Oxalobacter (17), GCA-900066575 (18), {Unknown Family} Clostridia vadinBB60 group (19), {Unknown Family} RF39 (20), Peptococcus (21), {Unknown Class} Firmicutes (22), Neisseria (23), Phocea (24), Acidaminococcus (25), {Unknown Class} Proteobacteria (26), Family XIII UCG-001 (27), [Eubacterium] ventriosum group (28), Colidextribacter (29), Akkermansia (30), Uncultured-5 (31), Bilophila (32), Bacteroides (33), Tyzzerella (34), Alistipes (35), UCG-003 (36), Christensenellaceae R-7 group (37), Odoribacter (38), Barnesiella (39), {Unknown genus}, Uncultured (40), Butyricimonas (41), Candidatus Soleaferrea (42), Butyricicoccus (43), Escherichia-Shigella (44), UGC-002 (45), Uncultured-6 (46), NK4A214 group (47), Family XIII AD3011 group (48), Asteroleplasma (49), {Unknown Genus} [Clostridium] methylpentosum group (50), Uncultured-7 (51), Victivallis (52), UCG-005 (53), Coprococcus (54), Lachnospiraceae FCS020 group (55), Marvinbryantia (56), [Ruminococcus] gauvreauii group (57), CAG-56 (58), Haemophilus (59), Uncultured-8 (60), Megamonas (61), Romboutsia (62), [Eubacterium] ruminatium group (63), Fusicatenibacter (64), Faecalibacterium (65), {Unknown Genus} Ruminococcaceae (66), Eisenbergiella (67), {Unknown Genus} UCG-011 (68), Intestimonas (69), Oscillibacter (70), Flavonifractor (71), Slackia (72), {Unknown Genus}Muribaculaceae (73), [Ruminococcus] gnavus group (74), Uncultured-1 (75), Uncultured-3 (76), Asaccharobacter (77), Agathobacter (78), Ruminococcus (79), [Eubacterium] siraeum group (80), Parasutterella (81), Dialister (82), Streptococcus (83), Lachnospira (84), Incertae Sedis (85), Anaerofilum (86), Uncultured-4 (87), Lachnoclostridium (88), Lachnospiraceae UCG-004 (89), Blautia (90), Lachnospiraceae UCG-008 (91), {Unknown Genus} Lachnospiraceae (92), {Unknown Order} Clostridia (93), Monoglobus (94), [Eubacterium] oxidoreducens group (95), Coprobacter (96), Lactococcus (97), Adlercreutzia (98), Collinsella (99), Lachnospiraceae NC2004 group (100), [Ruminococcus] torques group (101), Veillonella (102), Lactonifactor (103), {Unknown Family} Oscillospirales (104), [Eubacterium] nodatum group (105), Pseudoflavonifractor (106), [Anaerorhabdus] furcosa group (107), Lachnospiraceae UCG-010 (108), DTU089 (109), Defluviitaleaceae UCG-011 (110), Merdibacter (111), Negativibacillus (112), Oscillospira (113), Erysipelotrichaceae UCG-003 (114), Rosaburia (115), Lachnospiraceae ND3007 group (116), Dorea (117), Parabacteroides (118), [Eubacterium] eligens group (119), UCG-008 (120), Intestinibacter (121), {Unknown Genus} Oscillospiraceae (122), UBA1819 (123), Anaerostipes (124), [Eubacterium] hallii group (125), Lachnospiraceae NK4A136 group (126), {Unknown Family} Clostridia UCG-014 (127), [Eubacterium] xylanophilum group (128), {Unknown Genus} [Eubacterium] coprostanoligenes group (129), Lachnospiraceae UCG-001 (130), Turicibacter (131), and Clostridium sensu stricto 1 (132). Darker cells within the heat map represent a microbial genus that is more relatively abundant in each sample. Relative frequencies can be calculated by comparing the shade of a given cell with the scale bar to the right-hand side of the heatmap. The three rows of coded blocks at the top of the heat map represent time (h, top), Donor (middle), and fibre sample type (bottom). Each column represents a single sequenced sample. Samples are clustered based on their Euclidean complete linkage, and conclusions can be drawn on the impact of time, donor type and sample type by how closely samples of the same type are clustered together.Figure 20 is a box and whisker plot showing the alpha diversity of species identified in microbiota samples taken from fermentation experiments. Fermentation experiments were set up using fresh faecal samples from three different donors to separately inoculate flasks of basal culturing medium (2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3 and 1 g mucin / liter) supplemented with 0.5 mg / mL bile (control, CNTL) alone, or further containing 10 mg / mL pea fibre, pectin or fructooligosaccharide (FOS). Each plot represents the data gathered from fermentation experiments of the different donors (D1, D2, and D3). Each dot represents an individual sample. The shading of each dot represents the fibre type used in the sample according to the key in the bottom right corner of the graph. The size of the dot represents the different time points, wherein the smallest dots represent samples taken after 0 hours of fermentation and the largest dots represent samples taken after 24 hours of fermentation.

[0099] Figure 21 is a Bray Curtis beta diversity analysis of identified microbiota species present before, during and after fermentation reactions. Fermentation experiments were set up using fresh faecal samples from three different donors to separately inoculate flasks of basal medium (2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3 and 1 g mucin / liter) supplemented with 0.5 mg / mL bile (control, CNTL) and further containing 10 mg / mL pea fibre, 5 mg / mL pea fibre (Donor 1 only), 3.6 mg / mL pea fibre (Donor 1 only), 10 mg / mL pectin or 10mg / mL fructo-oligosaccharide (FOS). Samples were taken at 0 and 24 hour timepoints and microbiota compositions of each sample was determined through deep sequencing of the 16S rRNA gene and mapping to the Silva 16S reference database. The principal Coordinate (PCo) analysis plots for all donors, timepoints and fibre samples are plotted, and axes represent the two principal coordinate values (PCo; showing the percentage of variance indicated in parentheses on the X and Y axes labels, PCo1 =43% and PCo2 = 28%). Dot types represent different timepoints, as indicated in the key located at the bottom left of the graph. Samples from each different donor, Donor 1, Donor 2, and Donor3 have been circled and labelled as D1 , D2 and D3 respectively. The type of fibre used in each sample is written next to each plotted point. Bray Curtis beta diversity compares abundance or read count data between samples. If samples have a similar abundance for the same species they will cluster together and the more dissimilar the samples are, the further apart they will be in the graph. Primary grouping occurs based on donor differences between samples, rather than in relation to timepoint or sample type.

[0100] Figure 22 is a Bray Curtis beta diversity analysis of identified microbiota species present before, during and after fermentation reactions with varying concentrations of pea fibre. Fermentation experiments were set up using fresh faecal samples from a single donor (Donor 1) to separately inoculate flasks of basal culturing medium (2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3 and 1 g mucin / liter) supplemented with 0.5 mg / mL bile and 10mg / mL, 5 mg / mL, or 3.6 mg / mL pea fibre. The lightest sample shading represents samples of fermentation reactions in the presence of 10 mg / mL (10) pea fibre, the medium shading represents samples of fermentation reactions in the presence of 3.6 mg / mL (3) pea fibre and the darkest shading represents samples of fermentation reactions in the presence of 5 mg / mL (5) pea fibre. Samples were taken immediately before fermentation (t=0) and after 24 hours of fermentation at 37°C (t =24).

[0101] Figure 23 is a Venn diagram showing changes in microbiota composition found in separate fermentation reactions inoculated with fresh faecal material and incubated for 24 hours in basal culturing media (2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3 and 1 g mucin / liter) further supplemented with 10 mg / mL FOS, pectin, or pea fibre. Changes in microbiota composition were calculated relative to a no fibre control fermentation. The differential analysis was performed for all donors, and differences were determined using a False Discovery Rate (FDR) with a p-value of <0.05. Fold changes in taxa abundance equal or greater than 1.5 are indicated in the diagram with anupward arrow or a downward arrow, representing higher or lower abundances relative to the control fermentation reactions respectively.

[0102] Figures 24A, 24B and 24C are three bar graphs that show changes in the relative abundance of Blautia during fresh faecal fermentation reactions performed in basal culturing media (2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3 and 1 g mucin / liter) (control, CNTL), or basal culturing media further supplemented with pea fibre, FOS, or pectin. Separate fermentation reactions were inoculated with fresh faecal material derived from one of three donors (labelled as D1, D2 or D3) and samples were taken at the start of fermentation (t=0), and after 24 hours of fermentation (t=24). Figure 24A shows the changes in the relative abundance across all fibres and the control over time and across all three donors. Figure 24B shows the changes in the relative abundance of Blautia for fermentation reaction containing 10 mg / mL pea fibre over time across all three donors. Figure 24C shows the changes in relative abundance of Blautia over time in donor one when fermented with one of three different concentrations of pea fibre (3.6 mg / mL, 5 mg / mL, or 10 mg / mL).

[0103] Figures 25A and 25B are two bar graphs showing the changes in the relative abundance of Blautia during SHI ME®- derived microbiota fermentation reactions performed in basal culturing media (2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3 and 1 g mucin / liter) further supplemented with 3.6 mg / mL pea fibre or 3.6 mg / mL pectin. Samples were taken at the start (t=0), after 6 hours (t=6), 24 hours (t=24) and after 48 hours (t=48) of fermentation. Pea fibre and pectin sample data points taken from the same conditions are coupled together, with pectin samples on the left-hand side (light shaded bars), and pea fibre samples on the right-hand side (dark shaded bars). Figure 25A shows the changes in relative abundance in Blautia observed during fermentation reactions inoculated with microbiota from the distal compartment a single SHIME® system. Figure 25Bshows the changes in relative abundance in Blautia observed during fermentation reactions separately inoculated with microbiota from the distal compartment of three different SHIME® systems. Each SHIME® system was itself inoculated with isolated microbiota of a different donor (D1, D2, or D3).

[0104] Figure 26 is a bar graph that shows changes in the relative abundance of bacteria of the Monoglobus genus during fresh faecal fermentation reactions performed in basal culturing media (2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3 and 1 g mucin / liter) (control, CNTL), or basal culturing media further supplemented with pea fibre (Pea), FOS, or pectin. Separate fermentation reactions were inoculated with fresh faecal material derived from one of three donors (labelled as D1, D2 or D3) and samples were taken at the start of fermentation (t=0), and after 24 hours of fermentation (t=24).

[0105] Figure 27 is a bar graph that shows changes in the relative abundance of bacteria of the Roseburia genus during fresh faecal fermentation reactions performed in basal culturing media (2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3 and 1 g mucin / liter) (control, CNTL), or basal culturing media further supplemented with pea fibre (Pea), FOS, or pectin. Separate fermentation reactions were inoculated with fresh faecal material derived from one of three donors (labelled as D1, D2 or D3) and samples were taken at the start of fermentation (t=0), and after 24 hours of fermentation (t=24).

[0106] Figure 28A-H are bar graphs showing changes in the growth of different isolated bacterial strains cultured in basal media (2g peptone, 2g yeast extract, 0.5 g L-cysteine, 5.22 g dipotassium phosphate, 16.32 g potassium phosphate, 2 mL TWEEN 80, 2 g sodium bicarbonate and 1 g mucin / Litre) (Control, CNTL), or basal media further supplemented witheither 3.6g / L or 10 g / L of pea fibre, dialysed pea fibre, or FOS. Bacterial growth was measured by analysing the optical density (O.D.) of each culture in a Tecan Spectrophotometer at the start of fermentation, and after 96 hours of fermentation. O.D measurements taken at the start offermentation were subtracted from the O.D measured after 96 hours offermentation in order to correct for variance in starting concentrations of bacteria. Each experiment was repeated three times and the average O.D was plotted for each strain. The separately tested bacterial strains were Akkermansia muciniphila (Figure 28A), Bifidobacterium adolescentis (Figure 28B), Bifidobacterium bifidum (Figure 28C), Bifidobacterium longum (Figure 28D), Blautia faecis (Figure 28E), Roseburia faecis (Figure 28F), Lactobacillus plantarum (Figure 28G), Monoglobus pectinilyticus (Figure 28H).

[0107] Figure 29 shows a principal component analysis (PCA) of normalized microbiota composition data obtained from all samples collected in the 10 mg / mL fibre experiments described in Example 7. The PCA reflects the combined influence of donor identity, treatment, and timepoint on overall microbial community composition. Visual inspection indicates that donor-specific effects account for the largest proportion of variation, supporting the use of subsequent partial redundancy analyses (pRDA) to evaluate treatment- and time-related effects after conditioning for inter-donor variability.

[0108] Figure 30 shows a partial redundancy analysis (pRDA) of normalized microbiota composition data obtained from the 10 mg / mL fibre experiments described in Example 7, with donor identity included as a covariate. After removal of inter-donor variability, the time x treatment interaction explained 53% of the observed variation and was statistically significant (P = 0.002), indicating a treatment-associated modulation of microbiota composition attributable to fibre addition.

[0109] Figure 31 shows a Partial redundancy analysis (pRDA) performed on log2fold change (log2FC) microbiota composition data, calculated from paired samples collected at t = 0 and t = 24 h,obtained in the 10 mg / mL fibre experiments described in Example 7, with donor identity included as a covariate. Normalized short-chain fatty acid (SCFA) data were included as supplementary variables, with pectin-derived data excluded. After conditioning for inter-donor variability, the time x treatment interaction explained 47% of the observed variation and was statistically significant (P = 0.002). The ordination indicates treatment-associated differences in microbiota configuration, with propionate and butyrate showing the strongest correlation with pea fibre addition, consistent with fibre-specific modulation of microbial metabolic output.

[0110] Figure 32 shows a heatmap depicting log2fold changes of microbial taxa identified as the most important contributors to treatment-related separation in the redundancy analysis (RDA). Taxa were selected based on the ten highest absolute CFIT2 values driving separation of pea fiber-treated samples relative to control medium (CNTL) and fructo-oligosaccharide (FOS) conditions. Representative taxa were further constrained to exhibit negative Respl and Resp2 scores, corresponding to their positioning on the first and second ordination axes and aligning with the location of the pea fiber group (negative x- and y-axis) in Figure 32a. Overall, pea fiber treatment induced higher log2fold changes in the selected taxa compared with both CNTL and FOS treatments.

[0111] Figures 33 shows a partial redundancy analysis (pRDA) of of log2fold change (log2FC) microbiota composition data, calculated from paired samples collected at t = 0 and t = 24 h for all treatments in the 10 mg / mL fiber experiments described in Example 7, with donor identity included as a covariate. Normalized short-chain fatty acid (SCFA) concentrations were included as supplementary variables and did not contribute to the ordination constraints. After conditioning for inter-donor variability, the time x treatment interaction explained 44% of the observed variation (P = 0.02). Separation along the first ordination axis was primarily driven by opposing microbiota responses to pea fiber versus FOS treatment, which positioned on opposite sides of the axis. Notably, the health-associated SCFAs propionate and butyratealigned in the same direction as the pea fiber treatment, consistent with a microbiota configuration associated with enhanced production of beneficial fermentation metabolites.

[0112] Figure 34A shows Partial redundancy analysis (pRDA) performed on log2fold change (log2FC) microbiota data, calculated from paired samples collected at t = 0 and t = 24 h for all treatments in the 10 mg / mL fibre experiments described in Example 7. The analysis was restricted to genera belonging to the Lachnospiraceae family, with donor identity included as a covariate. Normalized short-chain fatty acid (SCFA) concentrations were included as supplementary variables. Following removal of inter-donor variability, treatment-related separation was observed, with pea fiber-treated samples positioned in the upper left quadrant of the ordination space (negative Respl, positive Resp2; FIG34B). Notably, butyrate aligned in the same direction as pea fibre, indicating an association between the pea fibre-induced microbiota configuration and increased production of this health-associated SCFA.

[0113] Figure 34B shows a heatmap depicting log2fold changes of the most influential Lachnospiraceae genera contributing to the treatment-related clustering observed in panel A. Taxa were selected based on their contribution to the ordination (CFIT2 values). Consistent with the ordination results, pea fibre samples clustered distinctly and showed increased log2fold changes of several Lachnospiraceae genera, including Eubacterium hallii, a known butyrate-producing taxon, relative to control and FOS treatments. show a partial RDA analysis of normalised short chain fatty acid data collected from the 10 mg / mL fibre experiments of example 7, excluding pectin data , controlling for donor effect and reduced to Lachnospiracaea genera.

[0114] Figure 35A shows a Partial redundancy analysis (pRDA) of log2fold change (log2FC) microbiota composition data, calculated from paired samples collected at t = 0 and t = 24 h, obtained from the 10 mg / mL fibre experiments described in Example 7. The analysis wasrestricted to genera belonging to the Lachnospiraceae family, control medium samples were excluded, and donor identity was included as a covariate. After conditioning for inter-donor variability, separation was observed primarily along the first ordination axis, with pea fiber-treated samples clustering in the lower-right quadrant of the ordination space. The separation was mainly driven by opposing microbiota responses to pea fibre versus FOS treatment, which positioned on opposite sides of the axis.

[0115] Figure 35B shows a heatmap depicting log2fold changes of the most influential Lachnospiraceae genera contributing to the treatment-related clustering observed in panel A. Taxa were selected based on their contribution to the ordination (CFIT2 values). Consistent with the pRDA results, the heatmap highlights distinct abundance patterns differentiating pea fiber and FOS treatments, underlying the observed separation in ordination space.

[0116] Figure 36A shows a Redundancy analysis (RDA) of normalized short-chain fatty acid (SCFA) concentration data obtained from the 10 mg / mL fibre experiments described in Example 7, assessing the combined time x treatment effect across all sampling timepoints. The constrained model explained 98% of the observed variation and was statistically significant (P = 0.002), with a clear separation of t = 24 h samples along the primary ordination axis. At 24 h, FOS-treated samples were characterized by a strong association with lactate accumulation, whereas pectin and pea fiber treatments aligned with increased levels of acetate, propionate, and butyrate, indicating treatment-specific fermentation profiles.

[0117] Figure 36B shows an RDA of normalized SCFA data restricted to t = 24 h samples only, highlighting treatment-dependent differences in fermentation end products at the endpoint of incubation. The constrained model explained 97% of the observed variation and was statistically significant (P = 0.002), with control medium samples associated with branched-chain fatty acids, indicative of protein-derived fermentation, and FOS treatment againcharacterized by lactate enrichment. In contrast, pectin- and pea fiber-treated samples aligned with propionate, acetate, butyrate, and formate, reflecting distinct carbohydrate-driven metabolic outputs.

[0118] Figures 37A and 37B display line charts showing the change in propionate and butyrate concentration, respectively over time, for each fiber treatment in the 10 mg / mL fiber experiments described in Example 7. Data points represent group mean concentrations, with error bars indicating standard deviation (SD). Lines connect mean values at successive sampling timepoints to illustrate temporal trends in SCFA production for each treatment.

[0119] Figures 38A to 38F display line charts showing the change in lactate, formate, acetate, isovalerate, iso-butyrate, valerate concentration, respectively, over time, for each fiber treatment in the 10 mg / mL fiber experiments described in Example 7. Data points represent group mean concentrations, with error bars indicating standard deviation (SD). Lines connect mean values at successive sampling timepoints to illustrate temporal trends in SCFA production for each treatment.

[0120] Figures 39A and 39B show two images taken with an atomic force microscope of molecular structures of pectic polysaccharides obtained from pea seeds (39A) and of pectin molecules derived from citrus fruit peel (39B).

[0121] Detailed description

[0122] The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, anyfeature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0123] Generally, nomenclatures used in connection with, and techniques of microbiology, cell and tissue culture, pathology, molecular biology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012).

[0124] The nomenclatures used in connection with, and the laboratory procedures and techniques of analytical chemistry, microbiology, bioinformatics and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0125] The invention relates to a composition for use as a prebiotic in promoting the growth or increasing the relative abundance of health-beneficial bacteria in the gastro-intestinal tract of an individual, the composition comprising a dietary pea fibre. The prebiotic composition of the present invention which promoting the growth or increasing the relative abundance of health-beneficial bacteria in the gastro-intestinal tract of an individual also increases the production of short chain fatty acids in the intestinal tract of an individual. The present invention therefore can be used as an additive to improve the physical properties of a food stuff in addition to modulating the microbiome.

[0126] The prebiotic composition comprising a dietary pea fibre of the present invention is advantageous because it increases the relative abundance of health-beneficial bacteria fromthe Lachnospiraceae family and the genera of Anaerostipes, Blautia, Monoglobus, Roseburia, Bifidobacterium, Bacteriodes and or Lactobacillus in the gastro-intestinal tract of an individual. The prebiotic composition comprising a dietary pea fibre of the present invention is further advantageous because it increases the relative abundance of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, Blautia faecis, Roseburia faecis, Monoglobus pectinilyticus, Eubacterium hallii, Eubacterium eligens, Lachnospiraceae NC2004, Lachnospiraceae NK4A136, Lachnospiraceae ND3007, Lachnospiraceae UCG-001 and / or Lachnospiraceae UCG-004.

[0127] In one embodiment, the dietary pea fibre is extracted from pea seed. In a preferred embodiment, the dietary pea fibre is extracted from the yellow pea seed. In a further preferred embodiment, the pea seed is derived from P. sativum. The skilled person would be aware of other types of peas which would be equally suitable for use in the preparation of dietary pea fibre.

[0128] In one embodiment, the dietary pea fibre is a homogalacturonan. The homogalacturonan dietary pea fibre may comprises a negatively charged backbone region and at least one neutral polysaccharide branched regions. The negatively charged backbone region may comprises galacturonic acid. In one preferred embodiment, the dietary pea fibre comprises a repeating unit of 1,2-a-L-Rhamnose and 1,4-a-D-Galacturonic acid; Xylogalacturonan, homogalacturonan with a-D-Xylose branched at 0-2 of galacturonic acid. In a particularly preferred embodiment, the structure of the pea fibre is according to the structure shown in Figure 1. Figure 1 is a schematic drawing showing the proposed molecular structure of pea fibre (Nakamura et al. 2022). In another particularly preferred embodiment, the dietary pea fibre is FIPEA-D® (FUJI OIL HOLDINGS INC.).In one embodiment a single molecule of the pea fibre has a molecular diameter of more than 100 nm and equal or less than 200 nm, or is between 125 nm and 200 nm, 150 nm and 200 nm, 175 nm and 200 nm, or 125 nm and 175 nm.

[0129] In one embodiment the degree of methyl esterification of constituent galacturonic acid is 45% or less, 30% or less, 20% or less, 10% or less, or is between 0% and 10%.

[0130] In one embodiment a single molecule of pea fibre has a star shaped structure.

[0131] In one embodiment a single molecule of pea fibre has a structure in which about 3 to 20 linear side chains of similar length are branched from the main chain.

[0132] In one embodiment the composition comprises polymeric components with a molecular rotation radius of 25 nm to 40 nm or 30 nm to 40 nm.

[0133] In one embodiment the composition includes polymeric components with a molecular weight greater than or equal to 10,000. In one embodiment the composition includes polymeric components with an absolute molecular weight of 500,000 to 1 ,000,000 or 800,000 to 900,000.

[0134] In one embodiment all components of the composition have a molecular weight below 5,000,000.

[0135] The dietary pea fibre may not comprise pea starch and / or pea protein. The starch and protein can be isolated from the dietary pea fibre during its extraction. Preferred processes for separating the dietary pea fibre from starch and protein are exemplified below but the skilled person would be aware of other methodologies for providing a dietary pea fibre isolated frompea starch and pea protein. This is advantageous because certain individuals can be allergic to pea protein. Thus, the composition is non-allogenic.

[0136] The pH of the composition may be about pH 3 to about pH 12. In further embodiments, the pH of the composition may be any value above about pH 3 to any value below about pH 12. In yet further embodiments, the pH of the composition may be about pH 3 to about pH 11, about pH 4 to about pH 11, about pH 4 to about pH 10, about pH 5 to about pH 9, about pH 6 to about pH 7. In one preferred embodiment, the pH of the composition is about pH 4 to pH 10.

[0137] In some embodiment, the composition comprises at least 30%, 35%, 40%, 45%, 50% 55%, 60%, 65%, 70%, or 75% dietary pea fibre. Preferably, the composition comprises at least 50% dietary pea fibre. In one embodiment, the composition comprises 50% or more dietary pea fibre.

[0138] In one embodiment, the composition according to anyone of the preceding claims, wherein the composition comprises at least one of galacturonic acid, arabinose, glucose, rhamnose, galactose and / or xylose. In a further embodiment, the composition comprises about 35-50 (w / w %) Arabinose, about 20-35 (w / w %) Glucose, about 1-10 (w / w%) Galactose, 2-3 (w / w %) Xylose, 3-5 (w / w %) Rhamnose, and / or about 17-20 (w / w%) Galacturonic acid.

[0139] In some embodiments, the composition may comprise no other ingredients other than the dietary pea fibre. In other embodiments, the composition may comprise other ingredients. For example, in some embodiments, the composition may comprise other types of prebiotics. Nonlimiting examples of other prebiotics include fructooligosaccharides such as oligofructose, inulin, and mannan oligosaccharides and galactooligosaccharides, such as lacto-N-tetraose, lacto- N-neotetraose, lacto- N-fucopentaose, and oligofructose-enriched inulin.In some embodiment, the composition of the present invention is for a non-therapeutic use. Therefore, the composition comprising dietary pea fibre of the invention may be used as a non-therapeutic prebiotic.

[0140] Use of the dietary pea fibre in food and beverages form part of the invention. In one embodiment, the dietary pea fibre may be incorporated into a food stuff or food additive. For example, in one embodiment, the composition may be incorporated into a snack bar.

[0141] In one embodiment, the dietary pea fibre is water soluble. This is advantageous as it makes the dietary pea fibre easy to form part of food and beverages.

[0142] The composition comprising the dietary pea fibre may therefore be incorporated into a food stuff as one or more of the following: an acidified food stabilizer, a foam reducer and / or stabiliser, an anti-coagulant, a preservative consistency adjuster, a gluten dispersibility improver, a bulking agent, a sedimentation inhibitor, a syneresis reducer, and / or a consistency regulator. The pea fibre of the present invention may therefore be used as a functional ingredient whilst conferring a desirable prebiotic attribute. For example, in one embodiment, the composition may be incorporated into flour and / or rice. The total weight percentage of dietary pea fibre in flour and / or the total weight percentage of dietary pea fibre in cooked rice is at least 0.4%.

[0143] The pea fibre may therefore be used in a wide variety of foodstuffs, such as acidified foodstuffs (such as acidified milk beverages containing live lactic acid bacteria, acidified plant-based protein drinks), frozen foodstuffs (such as frozen foam-containing frozen beverages, frozen desserts (including frozen cheesecakes and gluten-free cheesecakes and other frozen desert foodstuffs)) and effervescent and / or carbonated liquids (such as soft drinks, beer and beer likebeverages). It will be apparent to the skilled addressee that the functional use in liquids could be to reduce and / or stabilize foaming.

[0144] When used in beverage (such as tea), the pea fibre may have the additional functional benefit of being a sedimentation inhibitor. The pea fibre may also be used to reduces syneresis in viscous liquids containing water (such as jams and preserves). The pea fibre may be used in foodstuffs where the main ingredient is wheat and can be combined with wheat flour. The pea fibre may be used as a dispersion stabilizer for fine particulate matter, a gluten dispersibility modifier, a loosening improver (for noodles / pasta) and a consistency regulator for starch glue. The pea fibre may also be used as a humectant in a range of pastry and dumpling style products (such as shumai, dumplings, wontons, and xiaolong bao). In one embodiment, the composition may be incorporated into a meal replacement drink or an acidified milk beverage.

[0145] The dietary pea fibre may be formulated into a dietary supplement. Treatments or specific processes can be applied to improve such a dietary supplement. The composition may be in the form of a capsule, tablet, gel or liquid. The capsule or tablet may be enteric-coated, pH dependant, slow-release, timed release, and / or gastro-resistant.

[0146] In one embodiment, the dietary pea fibre may be incorporated into pet food. Advantageously, many of the health benefits of the pea fibre composition presented herein may also be conferred to animals when similarly administered with the dietary pea fibre. In a preferred embodiment, the pet food is in the form of dry food or a paste.

[0147] The composition may include an acceptable carrier or vehicle so that the compositions are, for example, in powder form. The term "carrier" refers to a diluent, adjuvant or excipient, with which the composition is administered. The composition, particularly when formulated into a dietary supplement, can be formulated into a powder, granule, tablet, pill, capsule, wafer or the like.The preparation of suitable powder, granule, tablet, pill, capsule, wafer or the like is well-known in the art.

[0148] The composition may be provided in the form of a dissolvable dry drinks powder or combined with a gelling agent. Preferably, the gelling agent is selected from gelatine, pectin, agar or carrageenan. Preferably, the composition, the gelling agent, or the combination of both, may be in the form of a gummy.

[0149] The compositions can take the form of one or more dosage units. Where the composition comprises a protein according to an aspect of the present invention, the dose unit comprises at least 1-3 grams per portion. Preferably, the dietary pea fibre is present in the amount of 2 grams per portion. When administered in a liquid form, the dose range may be about 3 mg / mL to about 10 mg / mL, 4 mg / mL to about 9 mg / mL, 5 mg / mL to about 8 mg / mL, 6 mg / mL to about 7 mg / mL. Preferably, in some preferred embodiments, the dosage is 3.6 mg / mL, 5 mg / mL, or 10 mg / mL.

[0150] In one embodiment, the invention relates to a composition for use as a prebiotic for promoting the growth or increasing the relative abundance of health-beneficial bacteria in the gastrointestinal tract of an individual. The health-beneficial bacteria may be selected from one or more of any one of the following genera of bacteria: Anaerostipes, Blautia, Monoglobus, Roseburia, Bifidobacterium, Bacteroides and / or Lactobacillus. In particular, the composition may highly promote the growth of beneficial bacteria from the genera of Blautia and / or Bifidobacterium. Further, the beneficial bacteria may be from one or more of any one of the following species of bacteria: Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, Blautia faecis, Roseburia faecis, and / or Monoglobus pectinilyticus Eubacterium hallii, Eubacterium eligens, Lachnospiraceae NC2004, LachnospiraceaeNK4A136, Lachnospiraceae ND3007, Lachnospiraceae UCG-001 and / or Lachnospiraceae UCG-004.

[0151] As used herein, the term “species” refers to a taxonomic entity as conventionally defined by genomic sequence and phenotypic characteristics. A “strain” is a particular instance of a species that has been isolated and purified according to conventional microbiological techniques. It will be understood that the terms bacteria and bacterial isolates as used herein refer to a plurality of bacteria, that is a bacterial population.

[0152] The composition may increase small chain fatty acid (SOFA) production in the proximal and distal colon, preferably the SOFA are at least one of butyric acid, propionate, and / or acetate. Increased amounts of these SOFA, particularly butyric acid, is advantageous because they have been shown to have health benefits, such as improved barrier function and antiinflammatory effects.

[0153] As disclosed herein, the pea fibre composition of the present invention (FIPEA-D ®) comprises a highly unique structure and composition when compared to other fibres, including other pea fibres. Without wishing to be bound by theory, it is thought that the specific structure and composition of FIPEA-D®, leads to the unique accessibility (or 3D-access) for bacterial derived enzymes, or CAZymes to prey on specific carbohydrate moieties. These carbohydrate moieties may otherwise be inaccessible to these enzymes, or accessible to a lesser extent.

[0154] Thus, when administrated to an individual, FIPEA-D ® provides a unique profile of upregulated microbiota. Similarly, FIPEA-D ® produces a unique profile of changes to short chain fatty acid production. The unique profile of upregulated microbiota and short chain fatty acids provided by FIPEA-D ® provide numerous health benefits to the individual.In addition, administration of FIPEA-D ® to an individual has been shown herein to reduce the breakdown of proteins / peptides into branched-chain-fatty-acids. This reduction in breakdown products of proteins / peptides in an individual is known in the art to confer health benefits to an individual.

[0155] Accordingly, in one embodiment there is provided a pea fibre composition for use in the increased production of any one or more of acetate, butyrate, valerate or propionate in the gastrointestinal tract of an individual.

[0156] In another embodiment there is provided a pea fibre composition for use in the increased production of butyrate in the gastrointestinal tract of an individual.

[0157] In another embodiment there is provided a pea fibre composition for use in the increased production of propionate in the gastrointestinal tract of an individual.

[0158] In another embodiment there is provided a pea fibre composition for use in the increased production of acetate in the gastrointestinal tract of an individual.

[0159] In another embodiment there is provided a pea fibre composition for use in the increased production of valerate in the gastrointestinal tract of an individual.

[0160] In another embodiment there is provided a pea fibre composition for use in the increased production of acetate and butyrate in the gastrointestinal tract of an individual.

[0161] In another embodiment there is provided a pea fibre composition for use in the increased production of acetate and propionate in the gastrointestinal tract of an individual.In another embodiment there is provided a pea fibre composition for use in the increased production of acetate and valerate in the gastrointestinal tract of an individual.

[0162] In another embodiment there is provided a pea fibre composition for use in the increased production of butyrate and propionate in the gastrointestinal tract of an individual.

[0163] In another embodiment there is provided a pea fibre composition for use in the increased production of butyrate and valerate in the gastrointestinal tract of an individual.

[0164] In another embodiment there is provided a pea fibre composition for use in the increased production of propionate and valerate in the gastrointestinal tract of an individual.

[0165] In another embodiment there is provided a pea fibre composition for use in the increased production of acetate, butyrate and propionate in the gastrointestinal tract of an individual.

[0166] In another embodiment there is provided a pea fibre composition for use in the increased production of acetate, butyrate and valerate in the gastrointestinal tract of an individual.

[0167] In another embodiment there is provided a pea fibre composition for use in the increased production of acetate, propionate and valerate in the gastrointestinal tract of an individual.

[0168] In another embodiment there is provided a pea fibre composition for use in the increased production of butyrate, propionate and valerate in the gastrointestinal tract of an individual.

[0169] In another embodiment, there is provided a pea fibre composition for use according to any previous embodiment, wherein the pea fibre further reduces the breakdown of proteins / peptides into branched-chain-fatty-acids in the gastrointestinal tract of an individual.The composition also prevents or decreases the relative abundance of health-detrimental bacteria in the gastro-intestinal tract of an individual. For example, decreasing the relative abundance of health-detrimental bacteria is from the genera Clostridiaceae and / or Clostridium.

[0170] In some embodiments, the composition may be used as a pharmaceutical composition. In further embodiments the composition may be used as a therapeutic composition. The composition may further comprise a pharmaceutically acceptable excipient. Excipients in the composition, dosage forms of the composition and administration routes would be known to the skilled person in the art. The composition may be administered in a therapeutically effective amount.

[0171] The term “therapeutically effective amount” is defined herein as an amount of the compound being administered which will relieve to some extent one or more of the symptoms of the disorder being treated, or result in inhibition of the progress or at least partial reversal of the condition.

[0172] In one aspect of the invention, there is provided a composition according to an aspect of the invention that may be for use in the treatment, management, or prevention of disease in a subject.

[0173] In another aspect of the invention, there is provided a method of treating, managing, or preventing a disease in a subject comprising administering the composition according to an aspect of the invention.

[0174] As used herein, "treat", "treating" or "treatment" means inhibiting or relieving a disease or disorder. For example, treatment can include a postponement of development of the symptomsassociated with a disease or disorder, and / or a reduction in the severity of such symptoms that will, or are expected, to develop with said disease. The terms include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result is being conferred on at least some of the mammals, e.g., human patients, being treated. Many medical treatments are effective for some, but not all, patients that undergo the treatment.

[0175] The term "subject" or "patient" refers to an animal which is the object of treatment, observation, or diagnosis. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline. In one embodiment, the subject is a patient disease associated with intestinal barrier permeability.

[0176] In one embodiment, the disease is a dysbiosis of the gastrointestinal tract in an individual. Dysbiosis of the gastrointestinal tract are associated with many diseases.

[0177] Dysbiosis is defined herein as an imbalance in bacterial composition which results in changes in bacterial microbiome composition, bacterial metabolic activities, and / or changes in bacterial distribution within the gut.

[0178] The dysbiosis may be associated with many different diseases. In an embodiment, the dysbiosis is associated with metabolic syndrome, obesity, or in the weight management of an individual. In one embodiment, the dysbiosis is associated with Crohn’s disease, Ulcerative Colitis (UC), Irritable Bowel Syndrome (IBS), or constipation. In one embodiment, the dysbiosis is associated with Behget syndrome, systemic sclerosis, or rheumatic arthritis, the composition comprising pea fibre. In one embodiment, the dysbiosis is associated with Parkinson’s disease, the composition comprising pea fibre. In one embodiment, the dysbiosis is associated withcoronary artery disease, atherosclerosis or hypertension, the composition comprising pea fibre.

[0179] The invention may also relate to a method of weight management in an individual, the method comprising administering the composition of any one of claims 1-9 up to 4 times a day.

[0180] The invention may also relate to a synbiotic composition comprising (a) a prebiotic comprising dietary pea fibre; and (b) a probiotic comprising an isolated bacterial strain from the Lachnospiraceae family and / or one or more of any one of the following genera of bacteria: Anaerostipes, Blautia, Monoglobus, Roseburia, Bifidobacterium, Bacteroides and / or Lactobacillus. The probiotic may specifically comprise any one of the following species: Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, Blautia faecis, Roseburia faecis, Monoglobus pectinilyticus, Eubacterium hallii, Eubacterium eligens, Lachnospiraceae NC2004, Lachnospiraceae NK4A136, Lachnospiraceae ND3007, Lachnospiraceae UCG-001 and / or Lachnospiraceae UCG-004.

[0181] It will be apparent to the skilled addressee that the composition may comprise a bacterial species in an isolated form. The term “isolated” encompasses bacteria that have been separated from other species or strains that were present in samples taken from a natural environment. Isolation may be achieved by means of streaking for single colonies, antibiotic or auxotrophic selection, or any other method / combination of methods that are routine to a person skilled in the art. In some embodiments, isolation may be performed to the extent that the presence of other bacterial species cannot be detected through the use of sensitive molecular biology techniques, such as polymerase chain reaction (PGR) or real time-polymerase chain reaction (RT-PCR).In one embodiment, the bacteria may be senescent or otherwise inactivated prior to long term storage or administration. Senescence or inactivation may be induced through lyophilisation, spray drying or any other method routine to a person skilled in the art. It will be apparent to a person skilled in the art that inactivated bacteria may, advantageously, survive for long periods in a lyophilised or otherwise dried state. It will be further apparent that these inactivated or senescent bacteria may return to a metabolically active state upon their introduction to a suitable environment, such as the gastrointestinal gut of an individual.

[0182] The composition may comprise a live bacterial product. The composition may preferably comprise a live bacterial product wherein a substantial proportion of the individual bacteria are viable. “Viable” in this context refers to bacteria capable of colonising the gastrointestinal gut of a subject administered with the composition.

[0183] In some embodiments, the live bacterial product may be further classed as a live biotherapeutic product, i.e. live bacteria that are capable of alleviating a disease, or symptoms thereof. The therapeutic effect of the bacteria may be direct or indirect. For example, the administration of the live bacteria may directly induce changes in symptoms. Alternatively, the composition may act to increase the therapeutic effect of a separately administered therapeutic composition.

[0184] As described herein, the presence or proportion of each bacterial species or strain in a composition may be selected according to the needs of the subject being so administered. For example, an overweight subject may be administered a composition with a higher proportion of isolated bacteria that have been shown to alleviate the comorbidities associated with being overweight.

[0185] It will be apparent to the skilled addressee that there are multiple methods for determining the sequence identity of bacterial species or strains present in both mixed and isolatedpopulations. Sequencing methods include, but are not limited to sequencing by synthesis methods, such as Sanger sequencing, Illumina® sequencing, or PacBio® sequencing. Sequencing may also be carried out using other methods which include, but are not limited to, Nanopore® sequencing.

[0186] Sequencing methods may be used to determine the entire genome sequence of each strain. Alternatively, sequencing methods may be used to target a single gene or other small proportion of the genome. Bacterial species and strains may be identified through the sequencing of their 16S ribosomal DNA. The 16S ribosomal DNA (rDNA) is approximately 1500bp in length and encodes the 30S ribosomal subunit. 16S ribosomal DNA is widely conserved among even distantly related bacteria and may be present in multiple copies in some bacterial cells. Advantageously, 16S rDNA comprises hypervariable regions (V1-V9 regions) that are sufficiently diverse so as to enable the differentiation of genera and species for most bacteria.

[0187] By way of example, the use of standard bacteriology and molecular biology techniques may be used to isolate the bacterial genomic DNA of a given sample. The purified bacterial genomic DNA may be subject to polymerase chain reaction (PCR) using primers designed to specifically amplify the 16S rDNA region. The resultant PCR product may be washed or otherwise purified of residual free dNTP’s, primers and any other buffer components that may otherwise interfere with downstream sequencing protocols. Sequencing may occur across the entire 16S rDNA region, or across any one of the 9 hypervariable regions (V1-V9). Once retrieved, these sequences may be delineated and used to determine the genetic composition of the 16S rDNA gene or subdomain thereof. Each bacterial species may finally be classified and compared to reference sequences to elucidate their identities.Methods of classifying bacterial species, and determining their relatedness according to percentage identity are known in the art. As used herein, the terms “homology” or “identity” typically refer to the percentage similarity in nucleic acid sequence between a reference sequence and a given sequence of interest, wherein the percentage is determined by calculating the number of identical nucleic acids in identical positions. It may be necessary to account for gaps or breaks in sequences that would otherwise better align. It will be apparent to a person skilled in the art that there exists multiple methods and computer programs for such calculations.

[0188] In one embodiment, it is possible to align a sequence of interest with a reference sequence using a commercially available alignment tool, according to their default scoring matrix and gap penalties. One such commercially available programme is the NCBI’s BLAST tool (blast.ncbi.nlm.nih.gov).

[0189] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of a Blautia bacterial strain.

[0190] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Monoglobus bacterial strain.

[0191] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of a Roseburia bacterial strain.According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of a Bifidobacterium bacterial strain.

[0192] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Blautia and Monoglobus bacterial strains.

[0193] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Blautia and Roseburia bacterial strains.

[0194] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Blautia and Bifidobacterium bacterial strains.

[0195] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Monoglobus and Roseburia bacterial strains.

[0196] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Monoglobus and Bifidobacterium bacterial strains.According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Roseburia and Bifidobacterium bacterial strains.

[0197] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Blautia, Roseburia and Monoglobus bacterial strains.

[0198] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Blautia, Roseburia and Bifidobacterium bacterial strains.

[0199] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Monoglobus, Roseburia and Bifidobacterium bacterial strains.

[0200] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of a bacterium from the Lachnospiraceae and / or any one of the following genera of bacteria: Anaerostipes, Blautia, Monoglobus, Roseburia, Bifidobacterium, Bacteroides and / or Lactobacillus, bacterial strains.According to specific embodiment of the present invention, there is provided a composition comprising a mixture of a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises one or more bacterial strains selected from one or more of the following Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, Blautia faecis, Roseburia faecis or Monoglobus pectinilyticus Eubacterium hallii, Eubacterium eligens, Lachnospiraceae NC2004, Lachnospiraceae NK4A136, Lachnospiraceae ND3007, Lachnospiraceae UCG-001 and / or Lachnospiraceae UCG-004. According to another specific embodiment of the present invention, there is a composition comprising a prebiotic and a probiotic comprising a combination of any number of any one or more of the bacterial species of any previous embodiment.

[0201] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of a Blautia faecis bacterial strain.

[0202] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of a Monoglobus pectinilyticus bacterial strain.

[0203] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of a Roseburia faecis bacterial strain.

[0204] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of a Bifidobacterium adolescentis, Bifidobacterium bifidum, or Bifidobacterium longum bacterial strain.According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Blautia faecis and Monoglobus pectinilyticus bacterial strains.

[0205] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Blautia faecis and Roseburia faecis bacterial strains.

[0206] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Blautia faecis and Bifidobacterium adolescentis, Bifidobacterium bifidum or Bifidobacterium longum bacterial strains.

[0207] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Monoglobus pectinilyticus and Roseburia faecis bacterial strains.

[0208] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Monoglobus pectinilyticus and Bifidobacterium adolescentis, Bifidobacterium bifidum or Bifidobacterium longum bacterial strains.According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Roseburia faecis and Bifidobacterium adolescentis, Bifidobacterium bifidum or Bifidobacterium longum bacterial strains.

[0209] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Blautia faecis, Roseburia faecis and Monoglobus pectinilyticus bacterial strains.

[0210] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Blautia faecis, Roseburia faecis and Bifidobacterium adolescentis, Bifidobacterium bifidum or Bifidobacterium longum bacterial strains.

[0211] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Monoglobus pectinilyticus, Roseburia faecis, and Bifidobacterium adolescentis, Bifidobacterium bifidum or Bifidobacterium longum strains.

[0212] According to another specific embodiment of the present invention, there is provided a composition comprising a prebiotic and a probiotic, wherein the prebiotic comprises pea fibre and wherein the probiotic comprises or consists of Blautia faecis, Monoglobus pectinilyticus, and Bifidobacterium adolescentis, Bifidobacterium bifidum or Bifidobacterium longum bacterial strains.In one embodiment, the prebiotic and probiotic bacterial strain are provided together in a single formulation. In another embodiment, the prebiotic and probiotic bacterial strain are provided separately for administration to an individual sequentially. In one embodiment, the bacteria can be viable bacteria that are capable of colonising and / or engraftment of the gastrointestinal gut of a subject.

[0213] The bacterial strains used in the live bacterial products provided herein generally are isolated from the microbiome of healthy individuals. In some embodiments, the live bacterial products include strains originating from a single individual. In some embodiments, the live bacterial products include strains originating from multiple individuals. In some embodiments, the bacterial strains are obtained from multiple individuals, isolated and grown up individually. The bacterial compositions that are grown up individually may subsequently be combined to provide the compositions of the disclosure. It should be appreciated that the origin of the bacterial strains of the live bacterial products provided herein is not limited to the human microbiome from a healthy individual.

[0214] In some embodiments in which the composition of the invention comprises more than one bacterial strain, species or genera, the individual bacterial strains, species or genera may be for separate, simultaneous or sequential administration. In some embodiments, the more than one bacterial strain, species or genera are stored separately but are mixed together prior to use.

[0215] The invention may relate to a composition according to previous embodiments that may be for use in the production of isolated bacteria, for example probiotic strains for commercial products.The demand for probiotics is steadily increasing, making the improved growth of probiotic strains highly commercially relevant. The inventors have shown that the pea fibre of the present invention is, advantageously, capable of selectively enhancing the growth of certain beneficial bacterial species during fermentation. The pea fibre of the present invention can therefore be useful in the production of products containing probiotic compositions.

[0216] The pea fibre of the present invention may, for example, be included into dry media ingredients prior to preparation for fermentation. Alternatively, the pea fibre may be included during the fermentation process.

[0217] The probiotic of interest may be an isolated strain, or a stock of multiple different bacteria strains from, for example, a crude sample. The in vitro reaction may comprise a fermentation reaction, or any other method of growing bacteria that will be apparent to a person skilled in the art. The grown probiotic may then be purified and dried using methods known to a person skilled in the art.

[0218] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.All documents mentioned in this specification are incorporated herein by reference in their entirety, including any references to gene accession numbers and references to patent publications.

[0219] "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0220] The invention is further described in the non-limiting examples.

[0221] Examples

[0222] Previous experiments had determined the physical properties of pea fibre and established methods of its isolation. Experiments were then conducted to determine whether pea fibre could be classed as a dietary fibre. According to the EU definition a dietary fibre is a:

[0223] ‘carbohydrate polymers with three or more monomeric units, which are neither digested nor absorbed in the human small intestine and belong to the following categories:

[0224] -edible carbohydrate polymers naturally occurring in the food as consumed,

[0225] -edible carbohydrate polymers which have been obtained from food raw material by physical, enzymatic or chemical means and which have a beneficial physiological effect demonstrated by generally accepted scientific evidence,-edible synthetic carbohydrate polymers which have a beneficial physiological effect demonstrated by generally accepted scientific evidence’.

[0226] First, pea fibre was investigated using in vitro digestion methods to determine its ability to survive digestion and reach the colon intact. Further experiments were then conducted to determine whether pea fibre had any prebiotic properties. Prebiotic activity was assessed by analysing changes in microbiota composition as a result of fermentation reactions in the presence of pea fibre.

[0227] Example 1: Preparation and assessment of physical properties of pea fibre compared to other commonly used food stabilisers

[0228] of pea fibre

[0229] The pea fibre used in the experiments is a water-soluble polysaccharide, sold under the brand name FIPEA-D® (FUJI OIL HOLDINGS INC.), extracted from pea seed and preferably extracted from the yellow pea seed derived from P. sativum.

[0230] The production method of pea fibre is described in the method below, or in methods outlined in, for example, WO2012176852A1, EP2939548A1, EP2997831A1, EP3135124B1, EP3329786A1 or EP2725038B1.

[0231] The production of pea fibre can be achieved using pea seed as a raw material. Preferably the pea seed is produced by yellow pea P. sativum. It is preferable that the fibre fraction is isolated from protein and starch fractions present in the raw material. Extraction of the fibre fraction is achieved through the addition of water at a ratio of 5- to 20-parts water to 1-part raw material. The pH of the resultant mixture is adjusted to a pH above 3 and below pH 12 through the addition of an acid or an alkali. This pH range avoids the hydrolysis or decomposition the fibre. Preferably, the pH of the fibre fraction is maintained at a pH of between 4 and 10.The resultant mixture is heated for 2 to 3 hours at a temperature between 60°C and 150°C, preferably not lower than 80°C and not higher than 130°C. In one embodiment it is preferably not lower than 60°C and not higher than 130°C. At a temperature less than 60°C, the extraction efficiency of pectic polysaccharides is poor, and less practical. At a temperature more than 150°C, there may be a case where pectic polysaccharides are hydrolysed during the extraction process and it is impossible to maintain a star structure. It is possible to optionally adjust the period depending on the condition of the raw material and the temperature or the like. There is no particular limitation on acids and alkalis to be used. It is possible to use acids such as hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, and formic acid, and alkalis such as sodium hydroxide, calcium hydroxide, sodium hydrogen carbonate, sodium carbonate, and ammonia. Additionally, extraction may be performed by using highly-pure cellulases, hemicellulases, or pectinases with which the target pectin having a star structure is not hydrolyzed, alone or in combination.

[0232] After heating, the soluble polysaccharide can be isolated through centrifugation methods. For example, isolation of the pea fibre can occur through centrifugation at 1 ,000 rpm for 5 minutes is sufficient to remove protein and starch dispersed in the water mixture.

[0233] Purification

[0234] Although it is possible to dry the pectic polysaccharides as they are, it is desirable to perform purification such as removal of protein, desalination, removal of pigment components in order to allow functions to be more developed.

[0235] In some embodiments, the isolated fibre fraction can be further purified using additional centrifugation steps. This can be achieved through the addition of 5-parts water to 1-partisolated fibre fraction followed by stirring and then centrifugation. Stirring can be achieved through the use of a homogeniser at 3,000 rpm for 30 minutes and centrifugation through a centrifugal filter at 1 ,000 rpm for 5 minutes. It will be apparent to the skilled addressee that this additional step may be carried out multiple times so as to further isolate the fibre fraction from the protein and starch fractions.

[0236] As for protein removal methods, it is possible to aggregate protein by pH adjustment, and then, to remove the protein by physical separation means such as pressure filtration separation, centrifugation, and membrane separation. Alternatively, it is possible to decompose protein using optional proteolytic enzymes, and then, to adsorb and remove the decomposed protein using dialysis membranes, activated carbon, ion exchange, or hydrophobic resins. As for desalination methods, any methods such as electrodialysis, ion exchange resins, or UF membrane separation can be used, if the methods are those for removing these. As for methods for removing pigment components, in addition to methods for decomposing pigment components, such as ozone treatment or UV irradiation, any methods such as partition with hydrophilic polar solvents such as ethanol or isopropanol can be used. It is preferred that one or more of these methods is used in combination. Pectic polysaccharides subjected to the purification treatment are subjected to optional sterilization treatment to thereby provide dried polysaccharides by means of methods such as freeze drying, spray drying, and hot air drying of ethanol precipitates.

[0237] It is preferred to remove starch in the stage of the raw materials, in the stage of fibre separated from the raw materials, in the stage of extraction of pectic polysaccharides, or in the stage after extraction. It is possible to remove starch by combining one or more of methods for amylase decomposition, cooling precipitation, and aggregate precipitation with an emulsifier. Although dry fractionation is feasible if in the stage of raw materials, wet fractionation is suitable, wherein it is possible that the crushed raw material is added with water and heated to a temperature atwhich starch is not gelatinized to thereby allow the starch to be separated as starch particles by the centrifugation. Alternatively, it is also possible to decompose and remove starch by heating the raw material added with water to a temperature higher than the temperature at which the starch is gelatinized and then treating the raw material with amylase. If in the stage of fibre separated from beans raw materials, it is possible to decompose and remove starch by dispersing the fibre in water, heating the fibre to a temperature at which the starch is gelatinized, and then treating the fibre with amylase. Examples of methods for removing starch in the processes of extracting pectic polysaccharides or after extraction include adding amylase to a raw material added with water before extraction, adding amylase to extracted slurry before solid-liquid separation, or adding amylase to filtrate after solid-liquid separation.

[0238] Amylase is a generic name of enzymes that hydrolyse starch, and examples of amylase include p-amylase, a-amylase, glucoamylase, and pullulanase. Although these highly purified amylases may be used for this purpose, commercially available amylase formulations in which one or more of these are mixed may be used. In this context, it is possible to decompose and remove starch by chemical approaches such as acid hydrolysis in the stage raw material, fibre, or before or after extraction of pectic polysaccharides, but starch removal treatment with enzyme treatment is preferred because pectic polysaccharides of the present invention are concurrently decomposed.

[0239] Preferably, the amylase will be present in an amount of 0.1 % by mass of solid treated material. Optionally, the amylase may be inactivated by heating in water for 15 minutes. Preferably, the amylase is an a-amylase (4-a-d-glucan glucanohydrolase; EC 3.2.1.1) from the Bacillus amyloliquefaciens strain NZYM-WR.The isolated and / or purified pea fibre mixture may be dried to produce a dry fibre. Drying may be achieved using one or more common industrial methods, such as freeze-drying, spray drying or hot air drying of an ethanol precipitate.

[0240] The resultant dried pea fibre may be sterilised by undergoing any standard sterilisation treatments. Sterilisation treatment may include sterilisation through heating with saturated or super-heated steam.

[0241] Esterolysis

[0242] In some embodiments, the pectic polysaccharides of the present invention may undergo a process to reduce the degree of methyl esterification to 45% or less, preferably 30% or less.

[0243] Any method may be used to remove methyl esters provided that the methods are those with which decomposition of sugar chains of the pectic polysaccharides is suppressed and the ester can be decomposed. When the treatment is performed on pectic polysaccharides after extraction, optional alkalis are added to a 1% to 5% by weight pectic polysaccharides aqueous solution to adjust the pH preferably to 8 or higher, more preferably to 12 or higher such that esterolysis is feasible. The heating temperature is preferably 20°C or more, more preferably 40°C or more, and the heating period is preferably 10 minutes or more, more preferably 30 minutes or more, and also preferably 4 hours or less. By setting the pH at extraction to the alkaline side, it is possible to perform extraction and esterolysis concurrently. Alternatively, it is also possible to perform esterolysis using commercially available pectin methyl esterases or commercially available enzyme formulations containing the enzyme. In this context, esterolysis can be performed in any stages for preparing pectic polysaccharides. Examples of the stages for ammonia treatment include the raw material before extraction, the slurry after extraction, the pectic polysaccharides solution subjected to solid-liquid separation, purified treatmentliquids, and the powder after drying. In this context, as alkalis, any alkalis such as sodium hydroxide, calcium hydroxide, sodium hydrogen carbonate, sodium carbonate, and ammonia can be used. Additionally, the degree of methyl esterification is calculated, after the amounts of galacturonic acid and methyl-esterified galacturonic acid are quantified by Doesburg titration method, with the following formula:

[0244] methyl-esterified galacturonic acid / the total galacturonic acid x 100 (%)

[0245] Physical properties of pea fibre

[0246] The final composition of the FIPEA-D ® pea fibre is high in fibre (more than 50 %w / v), and possesses a branched chain, spherical structure composed of a negatively charged backbone comprising galacturonic acid and branched chains that further comprise neutral polysaccharides.

[0247] Characteristics of the pectic polysaccharides are that their molecular shape is different from that of pectin. A single molecule observed with an atomic force microscope has a star structure with a molecular diameter of more than 100 nm and equal to or less than 200 nm, and additionally that the degree of methyl esterification of constituent galacturonic acid is 45% or less, preferably 30% or less. The star structure is a counterpart of a linear chain structure or a spherical structure and has a structure in which about 3 to 20 linear side chains having a comparable length are branched from the main chain, for example. The pectic polysaccharides of FIPEA-D® are between 100 nm and 200 nm and the degree of methyl esterification is 45% or less, preferably 30% or less. It is not possible to achieve dispersion stabilising effects of FIPEA-D® using pectic polysaccharides less than 100 nm or more than 200 nm, or by spherical or linear pectic polysaccharides, or the like, which do not have a star structure, as well as by polysaccharides whose degrees of methyl esterification are more than 45%.FIPEA-D® includes polymeric components whose molecular weights are 10,000 or more. The polymeric components are defined as fractions of those whose molecular weights are recognised to be 10,000 or more as analysed by gel filtration. The molecular rotation radius of these polymeric fractions is preferably 25 nm to 40 nm, more preferably 30 nm to 40 nm, and their average absolute molecular weight is preferably 500,000 to 1,000,000, more preferably 800,000 to 900,000. Additionally, it is preferred that the molecular weight does not exceed 5,000,000. The above molecular weights are calculated as follows: PLC (TSK-gel G-5000PWXL: TOSOH CORPORATION cp 7.8 mm x 300 mm) is used, and the molecular weight is determined by calculation from a standard pullulan P-82 (Showa Denko K. K.). Additionally, the molecular rotation radius is determined by means of static light scattering (HPLC- MALLS). The analysis is performed under the following conditions; an eluant: a 50 mM sodium acetate aqueous solution (pH 5.0) and a flow rate: 1.0 ml / min on a Rl detector and an MALLS detector.

[0248] Although a carboxyl group in the 6-position in galacturonic acid has been methyl- esterified, it is important that the degree of methyl esterification that indicates methyl-esterified galacturonic acid included in all the galacturonic acid molecules is 45% or less, preferably 30% or less.

[0249] The total sugar content of the pea fibre was determined using the phenol-sulfuric acid method and the galacturonic acid content was determined using the Blumenkantz method. The composition of pea fibre was analysed in samples of pea fibre wherein polysaccharides had been hydrolysed into monosaccharides with sulfuric acid and neutralising barium carbonate. The abundance of each monosaccharide was determined through ion exchange chromatography with a pulsed ampomeric detector using the Dionex ISC-3000 ion chromatography system equipped with a Dionex Carbopack PA-1 column. The monosaccharide composition is presented in Table 1. Analysis showed that the composition ofpea fibre comprised, in descending order of abundance, galacturonicacid, arabinose, glucose, rhamnose, galactose and xylose.

[0250]

[0251] Table 1: The sugar composition (w / w%) of pea fibre

[0252] The molecular structure of the pea fibre was determined through scanning probe microscopy according to the method of Ikeda et al. (Ikeda, Gohtani, Nishirinari and Zhong, 2013). The beam shaped Si Cantilever (spring constant 20N / m) was excited to a frequency close to a resonant frequency of 0.5-1.0 Hz. The structure shows a straight chain structure with small branches. The proposed molecular structure of the pea fibre is Homogalacturonan, the main chain being composed of 1,4-a-D-Galacturonic acid; Rhamnogalacturonan, the main chain being composed of the repeating unit of 1,2-a-L-Rhamnose and 1,4-a-D-Galacturonic acid; Xylogalacturonan, homogalacturonan with a-D-Xylose branched at 0-2 of galacturonic acid (as shown in Figure 1).

[0253] The viscosity and precipitation rates of pea fibre, SOYAFIBE-S CA100, Guar Gum and HM-Pectin were also assessed. Pea fibre exhibited a similar viscosity profile to SOYAFIBE-S CA100, whereas Guar Gum and HM-Pectin exhibited higher levels of viscosity at low concentrations. Gum Arabic was the least viscous at all concentrations (as shown in Figure 2). SOYAFIBE-S CA100 (square) and pea fibre (diamond) demonstrated very similar rates of precipitation of about 1% at pH levels between 3.6 to 4.4. which increased to about 5% precipitation at pH 4.6. Pectin (triangle) exhibited much higher rates of precipitation of around 12-13% at pH levels of 3.6 to 3.8, decreasing to 1% precipitation between pH levels 4 and 4.4and increasing to 5% precipitation at pH 4.6. The viscosity of milk beverage samples prepared with soy fibre (SOYAFIBE-S CA100), and pea fibre were both consistently between 4 and 6 mPas between pH levels 3.6 and 4.6, whereas beverage samples prepared with Pectin exhibited high viscosity level of 14 mPa at pH 3.8 and 6-8 mPa between pH levels of 4 and 4.6 (as shown in Figures 3A and 3B).

[0254] Example 2: HPSEC analysis shows that soluble pea fibre is not digested during in vitro digestion reactions.

[0255] The digestion characteristics of pea fibre, as well as soy fibre (SOYAFIBE-S CA100), pectin and milli-Q-water controls, were determined through in vitro digestion reactions according to the digestion protocol developed by the COST INFOGEST network. Duplicate samples were taken from each reaction at 7 predefined timepoints (as illustrated in Figure 4). In brief, samples were mixed in a 1:1 (wt / wt) ratio with simulated salivary fluid (SSF) (Samples 1.1 and 1.2), before they were mixed with salivary amylase to a final concentration of 75 U / rnL. Samples were then incubated for two minutes, mixed with simulated gastric fluid (SGF) in a 1:1 (wt / wt) ratio and adjusted to pH 3 (Samples 3.1 and 3.2). Samples were mixed with pepsin in a 1:1 (1:1) ratio and incubated for 2 hours. Once incubated, samples were mixed in a 1:1 (wt / wt) ratio with simulated intestinal fluid and adjusted to pH 7 (Samples 5.1 and 5.2). Samples were then mixed with 100 U / rnL pancreatin and 10 mM Bile and incubated for 2 hours (Samples 7.1 and 7.2).

[0256] Results were analysed using High Performance Size Exclusion Chromatography (HPSEC), performed on an UltiMate 3000 system (Dionex, Sunnyvale, CA, USA) equipped with TSK-Gel super AW guard column (6 mm ID x 40 mm) (Tosoh Bioscience, Tokyo, Japan) and three TSK-Gel super AW columns 4000, 3000, 2000 (6 mm x 150 mm), used in series. Measurements were conducted at 55°C and samples (2 mg / mLor4 mg / mLfor digests, 10 pl) were eluted with0.2M NaNOsat a flow rate of 0.6 mL / min. The elution was monitored with a Shodex® Rl - 101 detector (Showa Denko K.K., Tokyo, Japan) and UV detector set at 280 nm wavelength (UltiMate™ 3000 VWD Variable wavelength Detectors, Thermo Fisher Scientific Waltham, USA). The molecular weight estimation was based on pullalan standards ranging from 0.3 -708 kDa, which yielded the following equation y= -1.2058x + 23.275.

[0257] The first in vitro digestion reaction performed was a control reaction containing milli-Q water (as shown in Figure 5A). This experiment was conducted prior to any HPSEC runs of pea fibre samples so that the spectra of the various reagents and enzymes used in the COST INFOGEST protocol could be established. HPSEC analysis of the digested milli-Q water showed that this control reaction contained no identifiable polymers, as determined by a lack of any peaks at a retention time of 8-10 minutes, but contained small molecules such as salts and digestive enzymes (retention time (RT) -13.75-14.75 minutes).

[0258] Pea fibre was subsequently digested using the COST INFOGEST digestion protocol as described above. HPSEC analysis of the pea fibre samples taken at different stages of the digestion process demonstrated that digestion did not result in a detectable loss of intact fibre (RT of -8-10 minutes) (as shown in Figure 5B). HPSEC analyses of both pectin and soy fibres (SOYAFIBE-S CA100) similarly showed no detectable loss of intact fibre.

[0259] Samples 1.1, 1.2, 7.1 and 7.2 were then extensively dialysed against distilled water in dialysis membranes with a molecular weight cut off at 12-14 kDa (Medicell Membranes Ltd., London, UK). HPSEC analysis of these dialysed samples was performed alongside a control of freshly diluted pea fibre (pea 2) (as shown in Figure 5C). The HPSEC spectra of the dialysed pea fibre samples were very similar to that of the dissolved pea fibre control, but the pea fibre control had significant peaks at an RT of -13-15 minutes. These peaks correspond to small molecules present in the fibre prior to digestion.Example 3: Free glucose detection and HPAEC monosaccharide composition analysis further shows that pea fibre is not digested during in vitro digestion reactions

[0260] The production of free glucose is an indicator that successful digestion has occurred. Therefore, the abundance of free glucose was determined in in vitro digested samples of pea fibre alongside similarly treated samples of pectin and soy fibre (SOYAFIBE-S CA100). Crude samples of each fibre were mixed in a 1 : 1 (wt / wt) ratio with simulated salivary fluid (SSF) (Step 1), before they were mixed with salivary amylase to a final concentration of 75 U / rnL (Step 2). Samples were then incubated for two minutes, mixed with simulated gastric fluid (SGF) in a 1:1 (wt / wt) ratio and adjusted to pH 3 (Step 3). Samples were mixed with pepsin in a 1:1 (1:1) ratio (Step 4) and incubated for 2 hours. Once incubated, samples were mixed in a 1:1 (wt / wt) ratio with simulated intestinal fluid and adjusted to pH 7 (Step 5). Samples were then mixed with 100 U / rnL pancreatin and 10 mM Bile (Step 6) and incubated for 2 hours (Step 7).

[0261] Free glucose content was determined using a glucose oxidase / peroxidase (GOPOD) colorimetric assay (Megazyme, Wicklow, Ireland) according to the manufacturer’s instruction. Free glucose was measured using absorbance at 510 nm each independent sample was measured in triplicate against the reagent blank. The amount of free glucose detected in each step of the pea fibre, soy fibre (SOYAFIBE-S CA100) and pectin in vitro digestion reactions are presented in Table 2.

[0262]

[0263]

[0264] Table 2: GOPOD analyses of digested samples to quantify free glucose in digested samples at different stages of in vitro digestion (average and standard deviation (STD) of duplicate analyses).

[0265] As shown in Table 2, the release of free glucose was not detected for pectin or soy fibre (SOYAFIBE-S CA100), and there was only a limited release of glucose release during the digestion of pea fibre, which occurred after the addition of pancreatic enzymes. This observed release of glucose is likely to be a result of residual starch within the pea fibre. These results are consistent with HPSEC analyses of Example 2, which showed no evidence of a loss or shifting of high molecular weight populations.

[0266] The sugar moiety composition of each crude undialysed fibre was subsequently determined through High Performance Anion Exchange Chromatography (HPAEC) monosaccharide composition analysis. HPAEC was also performed on fibres taken during in vitro digestion steps 1 (after dialysis, but before the addition of amylase) and step 7 (after full digestion anddialysis) in order to determine changes in the sugar moiety composition that take place during digestion. A reduction of sugar moieties in samples taken at step 7 when compared those taken at step 1 would be a clear indicator of successful digestion because sugar moieties released as a result of step 7 would be below the molecular weight cut off of the dialysis membrane.

[0267] To perform HPAEC analysis, dried samples of each fibre were subjected to methanolysis by the addition of 2M HCI in anhydrous methanol followed by incubation for 16 hours at 80°C. Released methyl glycosides were then hydrolysed by the addition of 2M TFA for 1 hour at 121 °C. The resultant monosaccharides were quantified on an ISC 3000 system (Dionex, Sunnyvale, CA, USA) coupled to the ICS5000 ED pulsed ampomeric detector (Dionex) equipped in Dionex CarboPac PA-1 guard column (50 x 2 mm) connected to Dionex CarboPac PA-1 (2x250 mm) column. Three mobile phases A) 0.1 M NaOH B) 1 M sodium acetate in 0.1 M sodium hydroxide C) Milli-Q water and post column addition of E) 0.5 M NaOH were combined into the following elution profile: 0.4 mL / min 0-32 min isocratic C + 0.1 mL / min post column addition E, 0.4 mL / min: 32.1-45 min 50.1-58 min, isocratic 100% A, 58.1-73 min, isocratic 100% C + post column addition E. The injection volume of all samples was 10 pL.

[0268] The sugar moieties detected in step 7 differ between fibre types. Pectin contains a majority of uronic acid moieties and a lower abundance of galactose, arabinose and other moieties. Pea and soy fibres have lower uronic acid and higher arabinose content than pectin, which may be related to hemicellulose content. Soy has the highest galactose content of all three materials in the undigestible fraction.

[0269] As shown in Table 3, monosaccharide glucose content is detected in the undialysed crude samples of all fibres, but not in steps 1 or 7 for soy and pectin fibres. This is likely to be related to low molecular weight molecules containing glucose monomer units that have been dialysed out.HPAEC analysis of the pea fibre monosaccharide composition revealed the presence of glucose in the crude materials and after Step 1, but not after Step 7. This observation could indicate that pea fibre is being digested, but it may also indicate the presence of digestible starch in the sample. To investigate the source of loss of glucose moieties, glucose was excluded from the mol / mol% calculation in the additional analysis shown below in Table 4. As can be seen in this table, the abundance of other sugar moieties in pea fibre remained constant between Steps 1 and 7, suggesting that the pea fibre remains intact, and that starch was present in the sample. It was concluded that 7% mol / mol of starch was degraded by digestive enzymes during the in vitro digestion reaction.

[0270]

[0271] Table 3: Monosaccharide composition of crude samples and dialysed samples of pea, pectin and soy fibres before and after digestion. Compositions were determined using HPAEC analysis after samples were dissolved and acid hydrolysed.

[0272]

[0273] Table 4: Monosaccharide composition of crude samples and dialysed samples of pea pectin and soy fibres as shown in Table 3, with glucose excluded from the %mol / mol calculationIn summary, the GOPOD and HPAEC analyses further confirm that pea fibre is resistant to digestion, has a high probability of reaching the colon intact and, therefore, fulfils this characteristic of a dietary fibre.

[0274] Example 4: Pea fibre shown to act as a substrate for colonic bacteria during fermentation

[0275] Once it was established that pea fibre could survive to reach the colon undigested (as described in Example 2), experiments were designed to determine if pea fibre could act as a substrate for proximal and distal colonic bacteria.

[0276] Samples of pea fibre, soy fibre (SOYAFIBE-S CA100) and pectin were used for fermentation reactions after they were first digested according to the COST INFOGEST digestion method (as described in Examples 2 and 3). Samples were then centrifuged, and the supernatant was filtered using crossflow filtration with a 5 kD filter. The crossflow dialysate buffer was the same buffer as the composition of final INFOGEST digestion buffer. After filtration, the retentate was preserved and used to resuspend the centrifuged pellet. 20 mL of this reconstituted retentate was used in fermentation reactions.

[0277] The fermentation reactions were set up as follows: 43 mL of basal medium (2g peptone, 2g yeast extract, 0.5 g L-cysteine, 5.22 g dipotassium phosphate, 16.32 g potassium phosphate, 2 mL TWEEN 80, 2 g sodium bicarbonate and 1 g mucin / Liter), 20 mL retentate and 7 mL microbiota. The microbiota inoculum was isolated from the proximal and distal colon compartments of a simulator of Human Intestinal Microbial Ecosystem (SHIME ®), which was derived from a single donor and provided with adult feed with starch (prodigest) three times a day for four days. The pH of the basal medium for the proximal colon and distal colon samples were 5.8 and 6.8 respectively. Fermentation vessels were made anaerobic by flushing with100% N2 in 5 cycles of 2 minutes 500 mbar over pressure followed by 800 mbar under pressure and ending with 500 mbar over pressure.

[0278] Duplicate fermentation reactions were separately performed with distal and proximal microbiota for all fibre samples, a FUJI CD control sample (control digest performed according to the COST INFOGEST digestion method (Minkus et al, 2014 and Brodkorp et al, 2019), and a media only control containing 20 mL of dialysis buffer in place of a retentate (as shown in Figure 6). Vessels were incubated at 37°C with continuous shaking at 120 rpm. Samples were taken at the start of fermentation and 6 hours, 24 hours, and 48 hours after fermentation. The fermentation vessels were maintained in anaerobic conditions during sample collection. Collected samples were centrifuged at 10,000 rpm for 3 minutes to obtain a supernatant for pH measurement, and both the supernatant and cell pellet were stored at -80°C for further analysis.

[0279] Increase in fermentation vessel headspace pressure as an indicator of successful fermentation

[0280] The fermentation of substrates such as fibres, carbohydrates and or medium components, generate metabolic by-products. These by-products include volatiles such as methane, sulphur dioxide and carbon dioxide, ammonia and hydrogen. The production of these volatiles can be tracked by measuring the headspace air pressure of the sealed fermentation vessels. If volatiles are being produced, pressure will increase in the fermentation vessel.

[0281] Headspace air pressure for each sample was measured after 6 hours, 24 hours and 48 hours of fermentation (as shown in Figures 7A and 7B). The highest production of volatiles was induced by pectin for both proximal and distal colon microbiota fermentations. The patterns observed for soy (SOYAFIBE-S CA100) and pea fibres in both proximal and distal microbiota fermentations were comparable. A significant difference in the production of volatiles betweenpea fibre and the control was observed after 24 hours in proximal colon fermentations, and after 6 hours in distal colon fermentations.

[0282] pH decrease as a further indicator of successful fermentation

[0283] Soluble fibres are typically degraded by the obligate anaerobes inhabiting the colon which results in the production of short chain fatty acids (SCFAs). As SCFAs are weak acids, the pH of fermentation reactions drop as a consequence of their production. pH may, however, increase or stabilise during fermentation once the fibres are exhausted and the bacteria switch to proteins as a source of energy and / or the secondary metabolism of lactate and acetate.

[0284] The pH of each fermentation reaction after 6 hours, 24 hours and 48 hours was measured and plotted (as shown in Figure 7C and 7D). Statistically significant differences in pH were observed after 6 hours of fermentation for soy (SOYAFI BE-S CA100) and pectin and at 24 and 48 hours of fermentation for all fibres when compared to the control digests, consistent with fermentation. All fibres exhibited decreases in pH during fermentation from 0 to 24 hours, at which point the pH levels appeared to plateau. The most rapid decline was exhibited in soy fibre, followed by pea fibre. Soy fibre and pea fibres showed a similar pH profile during fermentation reaction with distal colon microbiota.

[0285] Example 5: Microbiota analysis of fermentation reactions with pea fibre

[0286] The collected cell pellet samples from the fermentation reactions of Example 4 were used to study the different effects that each fibre had on microbiota composition. Factors such as utilisation of the fibres as substrate, acidification of the medium through production of SCFA and other metabolites can influence growth and survival of bacterial species present in theSHIME(B^derived starting cultures. The composition and diversity of the microbiota were analysed at different fermentation timepoints.

[0287] Cell pellets were processed and sent for deep sequencing of the 16s rRNA. Raw sequence data was obtained from Baseclear in Fastq files. The Fastq file, containing sequence reads and their quality scores, was uploaded in CLC genomic workbench to be quality control checked and aligned to the 16S reference database, Silva. All samples passed the quality checks. All fermentations were performed in duplicate with the exception of the medium control which was run once. Three samples were not sequenced due to technical difficulties, these were: proximal colon medium control 0 hours, one replicate of proximal colon Fuji CD control after 24 hours and one replicate of the distal colon pea fibre fermentation taken after 24 hours.

[0288] Obtained sequence data was binned into Operational taxonomic units (OTU). A representative sequence was then taken for taxonomic classification resulting in a table with the identified taxa and their relative abundance per sample. Separate OTU relative abundance tables were created and analysed for distal and proximal colon samples, as shown in Table 5 (Proximal Colon), and Table 6 (Distal Colon).

[0289]

[0290] Table 5: The phylogenetic composition of the proximal colon samples at the bacterial class level. The description of each sample code is provided in Table 7.

[0291]

[0292] Table 6: The phylogenetic composition of the distal colon sample at the bacterial class level. The description of each sample code is as provided in Table 7.

[0293]

[0294] Table 7: Sample codes used for individual fermentation experiments presented in tables 5 and 6. Sample codes are provided in the left-hand column (ID), the other columns, from left to right, indicate type of fibre used (Sample), choice of distal or proximal SHIME® compartment (DC and PC respectively) (Colon) and fermentation time in hours (Time, h).

[0295] Alpha-diversity metrics provide information about the total number of species or species richness detected within one sample (total OTU count) and how diverse and evenly distributed they are, as determined using the Shannon Index (Jost 2007).The fermentation reactions, described in Example 4, should provide a closed ecosystem in which the total number of species cannot be altered. However, deep sequencing provides a detection limit in which certain bacteria may only be abundant enough for detection during fermentation after having a competitive advantage specific to the fibre provided. Alpha diversity will, therefore, decrease through the domination of a bacterial species capable of hydrolysing a specific fibre.

[0296] A consequence of the background ‘flora’ of plant material is that a decrease is observed in microbiota diversity at the start of fermentation (t=0). As a result, the analysis of alpha diversity has been focussed on changes over time between fibres.

[0297] The alpha diversity analyses of distal and proximal samples were analysed separately and plotted as Shannon Index plots (as shown in Figure 8A and 8B). Values of the Shannon Index can range from 1 to the total number of species, where 1 means that there is a single dominant species, and a high number means that all of those species have equal abundances. Overall, alpha diversity analyses of the proximal or distal samples did not show clear differences between fibre and control samples. Of the fibres, the results for pea and soy fibres (SOYAFIBE-S CA100) are more similar to each other than pectin results.

[0298] Bray Curtis beta diversity was also assessed and plotted in order to determine abundances of different microbiota detected between different samples. Samples with a similar abundance for the same species will cluster together, and the more dissimilar the samples are, the further apart they will be on the graph. Proximal and distal colon samples were analysed separately, as they were when assessing alpha diversity. For the proximal colon (as shown in Figure 9), a clustering per type of sample was observed, with the control samples residing at the bottom of the graph, indicating stable microbiota during fermentation. The addition of the fibres caused the samples to separate from each other. This separation is driven by the background floracausing the t=0 samples to move right (soy fibre, SOYAFIBE-S CA100) and upwards (pea fibre and pectin) in the PCo plot. During fermentation, the soy (SOYAFIBE-S CA100) and pea fibre samples start clustering together in the middle of the plot, suggesting that they have a similar effect on microbiota composition. Pectin dives the composition in a similar direction (leftwards), but the separate clustering suggests a differential shift in microbiota composition.

[0299] The Bray Curtis analysis of the distal colon samples (as shown in Figure 10) shows a similar pattern to that seen in the proximal colon. The added background ‘flora’ introduced by the fibres drive the t=0 samples to the bottom of the plot and, in the pea fibre and soy fibre (SOYAFIBE-S CA100) samples, this effects wanes as the samples return to the starting composition of the negative controls. Pectin drives the microbiota composition into a different state.

[0300] In summary, the addition of the fibres resulted in a change in phylogenetic composition compared to the controls from the start of fermentation. This is likely due to the background bacterial DNA originating from the three fibre materials tested. Taking this into account, alpha diversity analyses did not show clear differences between fibre and control samples, and the results for pea and soy fibre (SOYAFIBE-S CA100) results were more similar to each other than to pectin. Beta-diversity analysis showed clear time-dependent effects for all fibre samples, showing interaction between fermentation processes and microbiota composition as expected, as well as clustering driven by the background ‘flora’. Fermentation results of the soy (SOYAFIBE-S CA100) and pea fibre samples in the proximal and distal colon suggest similar shifts in microbiota composition, whereas pectin appears to drive the microbiota composition differently.Example 6: Further analysis of the effects of pea fibre on distal colon microbiota compositions

[0301] Further in vitro batch fermentation reactions were performed with a specific focus on changes in microbiota composition as a result of pea fibre (as illustrated in Figure 11). Similar to the experiments laid out in Example 2, pectin and soluble soy fibre (SOYAFIBE-S CA100) were included for comparison with pea fibre. Inulin was also included as a further example of a well-known dietary fibre. Both the pea fibre and the reference fibres were introduced into the fermentation reactions without pre-digestion because earlier experiments had established that none of the tested fibres were digested during in vitro digestion reactions. Each fermentation reaction was performed separately with a microbiota sample from one of three separate SHI ME® systems. Each system was inoculated with faecal material from a different doner. Microbiota samples were exclusively isolated from the distal compartment of each SHIME® system because previous experiments had demonstrated that this is where most fermentation was observed. The final composition of each of the composition is outlined in Table 8.

[0302]

[0303] Table 8: Final composition of fermentation reactions used in Example 6.Fermentation vessels were incubated at 37°C with continuous shaking at 120 rpm. Samples were taken at the start, after 6 hours and after 24 hours of fermentation while maintaining anaerobic conditions. Collected samples were centrifuged at 10,000 rpm and both the supernatant and cell pellet were separately stored at -80°C for further analysis.

[0304] Collected cell pellet samples from the fermentation reactions were used to study the different effects that each fibre had on microbiota composition. Factors such as utilisation of the fibres as a substrate, acidification of the medium through production of SCFA and other metabolites can differentially influence growth and survival of bacterial species present in the SHIME®-derived starting cultures. The composition and diversity of the microbiota were analysed at different fermentation timepoints.

[0305] As in Example 5, cell pellets were processed and sent for deep sequencing of the 16s rRNA. Raw sequence data was obtained from Baseclear in Fastq files. The Fastq file, which contains reads and their quality scores, was uploaded in CLC genomic workbench to be quality control checked and aligned to the 16S reference database, Silva. All samples passed the quality checks. Fermentations were performed in duplicate, resulting in a total of 94 samples. Obtained sequence data was binned into Operational taxonomic units (OUT) before representative sequences were taken for taxonomic classification. The relative abundance of bacteria present in all samples at the start of fermentation is presented at the phylum level in Table 9. Overall, the phylogenetic composition between technical duplicates is highly similar and addition of the fibres resulted in a change in phylogenetic composition compared to the controls.

[0306]

[0307]

[0308] Table 9: A summary of the relative bacterial abundance detected in samples of each donor al the start of fermentation on the phylum level.

[0309] A hierarchical clustering (Euclidean complete linkage) was plotted as a heat map to show the results on a genus level for all donors, time and fibre samples (as illustrated in Figures 12A-D). The hierarchical clustering shows a clear difference between the donors as indicated with the different coded blocks around the highest level of clustering (Figure 12A: left-hand side: donor 3, middle: donor 2, right-hand side: donor 1). Figures 12B, C and D are magnified sections of the heat map of Figure 12A showing individual clustering for Donor 1, Donor 2, and Donor 3 respectively. A minor clustering in relation to time can be observed, but no clear clustering per type of fibre is apparent.

[0310] Alpha diversity analysis was then performed and plotted as three individual box and whisker plots representing samples of each donor (as shown in Figure 13). No clear pattern is observed across sample type or time point, other than showing more variability at later time points. In the starting materials (dissolved fibre samples prior to fermentation, Table 10 below) a background of 16S rRNA is present that is higher in pea and soy fibres (SOYAFIBE-S CA100) compared to inulin or pectin. This background is hypothesised to relate to DNA from a diverse set of dead bacteria or bacterial fragments in the sample materials which may be present in low absolute amounts. The background alpha diversity is lower than the alpha diversity of the microbiota from the three donors and the addition of the fibre samples to the donor samples for the fermentations does not obviously affect the alpha diversity in the various samples from the three different donors. This suggests that the 16S rRNA background in the starting materials did not affect the microbiota diversity analysis after the fermentations.

[0311]

[0312] Table 10: Summary of the alpha diversity detected in starting fibres prior to fermentation.

[0313] Beta diversity analysis was also assessed in order to analyse inter sample differences in microbiota diversity. A Bray Curtis beta diversity was plotted onto a principle coordinate analysis plot (as shown in figure 14), which compares abundances / read count data between samples. If samples have a similar abundance for the same species then they will cluster together. Conversely, if samples are dissimilar in their composition, they will be further apart in the graph. The Bray Curtis shows that grouping occurs based on donor and not on samples or time. At the start of fermentation (t=0), the samples form clusters dependent on which donor the samples are derived from. This indicates that the background diversity observed in the fibre samples do not have a major impact on the overall microbiota diversity. After 6 hours (t=6) and 24 hours (t=24), clustering based on the donor is not clear, indicating that time and / or fermentation effects affect the microbiota composition. No clustering was observed based on the sample type (fibre type or control) at any of the time points, and no ‘consistent direction of travel’ was observed over time.

[0314] In summary, fermentation of the fibres using SHIMEO-derived distal colon microbiota from three donors resulted in clear differences in microbiota composition between analysed samples, but diversity analysis and clustering patterns of the samples showed that these relate more clearly to the different donors rather than the fibre type or the timepoint of analysis. No clear differences between the fibres at the different timepoints were observed, although pea and soy fibre (SOYAFIBE-S CA100) samples were particularly proximal to each other.The pea fibre fermentation effects on the microbiota composition from the above study was further analysed, and the abundance of each bacterial class is presented in Table 11 below. The abundance data demonstrated an increase in Firmicutes overtime, and a decrease in the class of Actinobacteria, which were mostly Bifidobacteria.

[0315]

[0316] Table 11 : Phylogenetic composition of the pea fibre samples at the bacterial class level. The description of each sample code is provided in Table 12.

[0317]

[0318] Table 12: Sample codes used for the individual fermentation experiments presented in table 11. Sample codes are provided in the left-hand column (ID), the other columns, from left to right, indicate type of fibre used (Sample), choice of distal or proximal SHIME® compartment (DC and PC respectively) (Colon) and fermentation time in hours (Time, h).Changes in microbiota composition were also observed across the different timepoints for the control samples so that differential analysis between fibre and control samples could be performed. This allowed an analysis of the effect of fibre fermentation relative to the control samples after 24 hours (as shown in Figure 15).

[0319] Increases in Bifidobacterium compared to the no fibre control was observed for both pea and soy fibre (SOYAFIBE-S CA100) after 24 hours of fermentation. An increase in Lactobacillus but not Bifidobacterium compared to the control was observed after fermentation with inulin. Notably, the Blautia genus of bacteria was upregulated for pea and inulin. The Blautia genus is well known for having probiotic characteristics.

[0320] Pea fibre fermentation experiments were further assessed to compare bacterial genus abundance between the different time points (as shown in Figure 16). Pea fibre induces an increase in fibre fermenting bacteria such as Phascolarctobacterium at 24 hours, but most differences were observed after 6 hours of fermentation. Most strictly anaerobic bacteria were downregulated at this timepoint, but Clostridium sensu stricto 1 and 13 showed a significant increase after 6 hours. After 24 hours of fermentation, some anaerobic bacteria genera show a moderate increase in abundance. Notably, there was an increase in an unknown family of Lactobacillales after 6 hours and after 24 hours when compared to the start of fermentation.

[0321] In summary, distal colonic microbiota fermented with each of the test fibres exhibited a clear change in microbiota composition, but no clear differences between the fibres at the different timepoints were observed. Similar to previous examples, fermentation with pea and soy fibre (SOYAFIBE-S CA100) samples drove very similar changes in microbiota composition. Furthermore, fermentation of distal colonic bacteria with pea fibre led to a significant increase in beneficial colonic bacteria, including those of the Blautia genus, Phascolarctobacterium, Clostridium sensu stricto 1 and 13, and an unknown family of Lactobacillales.Example 7: Analysis of the effects of pea fibre on microbiota composition and short chain fatty acid production in fermentation reactions inoculated with fresh faecal samples

[0322] Pea fibre effects on microbiota composition were further assessed using fermentation reactions inoculated with fresh faecal samples. A summary of the fermentation reaction components is provided in Tables 13 and 14 below. Fermentation reactions were also performed using Pectin fibre, fructo-oligosaccharides (FOS) with a low degree of polymerisation, alongside a buffer only control. FOS was included in this round of experiments as a rapidly fermentable positive control. FOS is known to increase the abundance of Bifidobacteria (Arboleya etal. 2013) under anaerobic conditions.

[0323]

[0324] Table 13: Final concentrations of medium components used for fermentation reactions performed in Example 7.

[0325] Fermentation reactions were performed in triplicate and each replicate was inoculated with faecal material derived from a different donor (Donor 1 , Donor 2, or Donor 3, details of each Donor are presented in Table 15 below). Two additional fermentation reactions inoculated withdonor 1 faecal material were performed using different concentrations of pea fibre (as outlined in Table 11 below), in order to determine if there was a dose response in changes to microbiota compositions.

[0326]

[0327] Table 14: List of samples identifying each fibre used and their concentrations.

[0328]

[0329] composition in faecal samples.Fibres were dissolved in a buffer solution used in the INFOGEST protocol which is composed of all buffers used in the mouth, stomach and intestine steps, with the addition of bile and in the absence of digestive enzymes.

[0330] Fresh faecal samples were collected in a bucket with a closing lid with an additional anaerobic bag (Anaerogen 2.5 L, VWR) to absorb all of the oxygen. The faecal samples were transferred into an aerobic chamber comprising 2% Fhand 98% N2. Faecal samples were then diluted 1:5 with dilution buffer (8.8g / L K2HPO4, 6.8g / L KH2PO4, 1 g / L sodium thioglycolate). Glass beads were added to the diluted faecal samples prior to agitation into a homogenous suspension.

[0331] Fermentation vessels were incubated at 37°C with continuous shaking at 120 rpm. Samples were taken at the start, after 6 hours, after 24 hours, and after 48 hours of fermentation while maintaining anaerobic conditions. Collected samples were centrifuged at 10,000 rpm and both the supernatant and cell pellet were separately stored at -80°C for further analysis.

[0332] Short chain fatty acid analysis

[0333] Short chain fatty acids (scFA) are produced by the gut microbiota and can be used as a substrate by the microbiota for secondary metabolism. They can also be absorbed in the colon by the epithelium. scFAs, such as butyrate, can be used by epithelial cells as an energy source and have a multitude of effects on intestinal and immune function. The major scFAs produced during fermentation are acetic acid, propionic acid and butyric acid.

[0334] To determine the concentration of each scFA, the supernatant from each sample was first diluted 1:1 with 16.6 mM H2SO4 and filtered with a 0.2 pm filter. The short chain fatty acid samples were measured on an HPLC, AMINEX HPX-87H column (Biorad) (eluent buffer 8, 3 mM H2SO4 (444 pL / litre milli-Q), flowrate 0.5 mL / min, run time: 42 minutes, HPLC Water 2414with Rl detection, Sample chamber 15°C, Detector Rl at 35°C). An scFA standard was run after every 20 samples and was used for calculations of the unknown scFA in the samples by using the peak area. scFA standards include: acetic acid, propanoic acid, butyric acid, lactic acid, valeric acid, iso-butyric acid, iso-valeric acid, formic acid in mM, as shown in Table 16. Standards were freshly made.

[0335]

[0336] Table 16: Standard mM scFA used during HPLC runs for the determination of scFA

[0337] An overview of total scFA concentrations across all fibres and timepoints for the three donors is provided in Figure 17. Total scFA levels increase until they reach a plateau at 24 hours. Anincrease in the production of scFA levels is observed in donor 1 results when pea fibre concentrations are increased for 3.6 to 5 mg / mL of pea fibre. A further increase was observed across all donors provided with 10 mg / mL pea fibre.

[0338] Butyric acid is the scFA for which most beneficial effects have been described in the literature. The butyric acid results are shown separately in Figure 18. Consistently, butyric acid levels are higher for the 10 mg / mL doses than the lower 3.6 and 5 mg / mL pea fibre doses in donor 1.

[0339] At 24-hour and 48-hour time points, all fibres consistently show higher levels of butyric acid than the control medium, but only pea fibre and pectin show significant differences at individual time points.

[0340] Determination of changes in Microbiotia Composition

[0341] Samples taken at the start of fermentation, and after 24 hours of fermentation were sequenced as follows: First, DNA was isolated from the microbiota pellets of each sample and the 16S rRNA gene (V3-V4 region) was amplified. These PCR fragments were then sequenced using an Illumina MySeq sequencer, resulting in 300 bp paired reads with a minimum of 10,000 MiSeq reads per sample.

[0342] Raw sequence data was obtained from Baseclear in Fastq files. Quality filtering was performed using PhiX and sequences were trimmed of the Illumina adapter sequences. These trimmed Fastq files were uploaded to CLC genomic workbench (V21.03) with the microbial genomics module (V21.0), where the paired reads were merged, and the sequence reads were mapped to the 16S region from the Silva reference database (Silva 16S and 18S v13299%). Microbiota derived from the fresh faecal samples organised into an Operational taxonomic Units (OUT)abundance table, in which abundance levels per taxon is shown for each sample including the most precise taxonomic classification was created and analysed separately for each donor.

[0343] Statistical analysis was performed with GraphPad Prism (v10.1.0), using 2-way ANOVA (when dataset contains no missing values) or mixed effects analysis (when dataset contains missing values) with post-hoc Turkey’s test to correct for multiple comparisons. Relative abundance of the taxa identified in the treatments were compared to the control at the same timepoint. Within treatments, paired statistical testing was performed with ANOVA and 0-hour timepoints as the comparator value to all subsequent timepoints. Across all analyses, p-values, p-values<0.05 are considered statistically significant. P-values were rounded to a single digit and used indicators of significance levels are *p<0.05; ** p<0.01; *** p<0.001; ****p<0.0001.

[0344] The relative abundance of different microbiota present at the start of fermentation for all samples was calculated at the phylum level for each donor (as shown in Table 17). As can be seen in Table 17 below, the most prominent phylum are Firmicutes and Bacteroides, which are both common for faecal microbiota.

[0345]

[0346]

[0347] Table 17: Abundance data at the Phylum level at the start of fermentation for each donor (t=0 hours).

[0348] An overview of the microbiota composition for the different, most dominant taxa detected over different donors and timepoints is presented in Table 18 below. For the control medium (CNTL) relatively minor changes are observed at the start of fermentation and after 24 hours of fermentation, and these were largely related to the phylum Proteobacteria and class Gammaproteobacteria, which is attributed to composition changes due to low substrate availability. More consistent changes were observed for FOS, pea and pectin fibres, where Actinobacteria abundance increased from the start of fermentation to after 24 hours of fermentation.

[0349]

[0350] Table 18: Phylogenetic composition of the fermentation samples of Example 7 for each donor at the bacterial class level.

[0351]

[0352] Table 18 (Continued): Phylogenetic composition of the fermentation samples of Example 7 for each donor at the bacterial class level. The description of each sample code is presented in Table 19

[0353]

[0354] Table 19: Sample codes used for the individual fermentation experiments presented in table 11 and outlined in Example 7. Sample codes are provided in the left-hand column (ID), the other columns, from left to right, indicate type of fibre used (Sample), concentration of each dietary fibre (Concentration) (mg / mL), Donor number (Donor) and fermentation time in hours (Time, h).

[0355] A hierarchical clustering (Euclidean complete linkage) was performed and plotted onto a heat map to show the abundance of each different microbial genus for all donors, time points and fibres sampled (as illustrated in Figure 19). The hierarchical clustering shows a clear differencebetween the donors, as indicated with the different blocks around the highest level of clustering. Fermentation time can be seen to affect clustering, as most 0 hour and 24 hour timepoints cluster together within each donor. Fibre samples do not cluster together within each donor.

[0356] Minor differences between the start of fermentation and after 24 hours of fermentation were observed in the control, and these differences mostly related to the Proteobacteria phylum and the Gammaproteobacteria, which is attributed to compositional changes due to low substrate availability. For the FOS, pea and pectin fibres, more consistent changes are observed, including an increase in Actinobacteria phylum between the start of fermentation and after 24 hours of fermentation.

[0357] Alpha diversity analysis was performed and plotted as three individual box and whisker plots representing samples of each donor (as shown in Figure 20). Overall, alpha diversity differs between donors, with the lowest alpha diversity in Donor 3. No clear pattern was observed across sample type or timepoint, except for a lower alpha diversity for FOS after 24 hours of fermentation compared to the start of fermentation for Donors 1 and 2.

[0358] Beta diversity analysis was also assessed in order to analyse inter-sample differences in microbiota diversity. A Bray Curtis beta diversity was plotted onto a principal coordinate analysis plot (as shown in Figure 21), which compares abundances / read count data between samples. If samples have a similar abundance for the same species, then they will cluster together. Conversely, if samples are dissimilar in their composition, they will be further apart in the graph. Grouping occurs based on donor and not on samples or timepoint, which means that the main diversity between the samples is related to donor differences. After 24 hours of fermentation, a change in microbiota diversity can be seen by a slight sub-clustering for fibre-fermented samples. This is clearer when magnifying Donor 1 results for the three doses of peafibre (as shown in Figure 22), where clustering occurs with samples taken at the start of fermentation and a shift is seen after 24 hours of fermentation. Also seen was a dose-response effect in the vertical direction related to PCo2 (explaining 22% of variation), with 3.6 mg / mL pea fibre being the lowest, 5 mg / mL in the middle, and 10 mg / mL pea fibre being the highest.

[0359] In summary, fermentation of the fibres using fresh faecal sample-derived microbiota from three donors resulted in a difference in microbiota composition between analysed samples. Similar to the results of previous examples, diversity analysis and clustering patterns of the samples showed that these relate more clearly to the different donors rather than the fibre type or the timepoint of analysis. No clear differences between the fibre types were observed after 24 hours of fermentations in the beta diversity analysis.

[0360] A closer inspection of the pea fibre fermentation effects on the microbiota composition showed that Firmicutes decreased over time and that the phylum of Actinobacteria, particularly Bifidobacteria increased after fermentation for 24 hours.

[0361] Changes in microbiota composition were also observed over 24 hours in the control sample (not shown) and differential analysis between the pea and fibre control samples were performed to determine the effects of fibre fermentation relative to the media only control (as shown in Figure 23). Analysis showed that 12 genera are up or down regulated by all three fibres, out of a total of 115 genera that are detectable after 24 hours of fermentation. This equates to 10.4% of genera being up or down regulated by all the fibres. Bifidobacterium is increased by all fibres after 24 hours of fermentation relative to the control, as are Phascolarctobacterium and Colli nsella.

[0362] The relative abundances of genera at the start of fermentation and after 24 hours of fermentation were further assessed (as shown in Figure 23). The Lachnospiracaeae ND3007group is upregulated by pea fibre as well, but with lower abundance. The Lachnospria genus has been associated with healthy dietary patterns and with pectin degradation (Ericson et al., 2020).

[0363] Pea fibre was the only fibre to consistently increase the relative abundance of Blautia and Roseburia, and both pea fibre and pectin fibre increased the abundance of Monoglobus. The abundance of Blautia is increased by fermentation with pea fibre in all three donors (Figures 24A, 24B 24C, 25A and 25B). When analysing the individual donors, pea fibre is the only fibre in which the relative abundance of Blautia species increased in all three donors between the start of fermentation and after 24 hours of fermentation, while only one donor showed an increase with FOS, and two donors showed an increase with pectin fibre. No increase in Blautia was observed in the control group.

[0364] No clear dose-response effect is observed in pea fibre fermentation reactions Donor 1, but an increase in Blautia species is observed at all doses, including the lowest one of 3.6 mg / mL. Earlier fermentation reactions with pea fibre also showed that 3.6 mg / mL of pea fibre led to increases of Blautia species after 24 hours of fermentation in one experiment (as shown in Figure 24C), and in one of three donors in another experiment (Blautia are strict anaerobes, so low exposure to oxygen in this experiment is hypothesised to have had an effect on two other fermentation reactions derived from other donors).

[0365] Figure 25A shows that pea fibre induced an increase in the relative abundance bacteria of the Blautia genus after 24 hours but not after 48 hours. Figure 25B shows that pea fibre induced an increase in the relative abundance after 24 hours of fermentation with donor 1 microbiota, but not donor 2 or donor 3 microbiota.Similar to the other fibres, FOS and Pectin, pea fibre increased Bifidobacterium species compared to the control after 24 hours of fermentation. While this was not observed in previous examples with the lower test doses of 3.6 mg / mL for all fibres). Similar to Blautia, Bifidobacterium is a strictly anaerobic genus, which may explain differences between this result and those in previous examples. Furthermore, previous experiments were using SHIME® derived microbiota, whereas the experiments of the present example were using fresh faecal material to inoculate fermentation reactions. Bifidobacterium is highly abundant in SHIME®-derived microbiota and is considered an artefact of the system. It may, therefore, be that it was difficult to detect changes in Bifidobacterium abundance when it was already very high.

[0366] Roseburia, was increased by fermentation with pea fibre in one donor (as shown in Figure 27), but not increased by any other fibre.

[0367] Pea fibre uniquely upregulated the relative abundance of Monoglobus across all three donors after 24 hours of fermentation (as shown in figure 26). The relative abundance of Roseburia was also uniquely increased by fermentation with pea fibre in one donor (as shown in Figure 27).

[0368] Example 8: Growth Rate determination of isolated bacterial strains cultured in the presence of Pea fibre and FOS.

[0369] The experiments presented in the above examples show that the presence of pea fibre can increase the relative abundance of Bifidobacteium, Blautia, Roseburia and Monoglobus bacteria in fermentation reactions that were inoculated with mixed populations of microbiota. Described below are the results of further fermentation reactions conducted using single strains of bacteria from each of the above genera as well as commonly used probiotic bacterial strains. These experiments were performed in order to confirm the findings of the aboveexamples and to determine whether the identified growth of these bacteria was actually reliant on the pea fibre rather a than cross-feeding or interaction with other bacteria present in the mixed microbiota populations.

[0370] Preparation of isolated Bacterial Strains for single strain fermentation experiments

[0371] Isolated Bacterial strains of Akkermansia muciniphila (DSM 22959), Bifidobacterium adolescentis (DSM 20083), Bifidobacterium bifidum (DSM 20082), Bifidobacterium longum (DSM 20219), Blautia faecis (DSM 27629), Roseburia faecis (DSM 16840), Lactobacillus plantarum (WCFS1), and Monoglobus pectinilyticus (DSM 104742) were obtained from the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Leibniz Institute). Once obtained, each strain was grown in appropriate culture medium and stored as a 15% v / v glycerol stock. A sample of each glycerol stock was diluted in appropriate media, and the resulting culture was incubated anaerobically for 24 hours at 37°C. Appropriate culture medium and dilution factors for each strain are presented in table 20.

[0372]

[0373]

[0374] Table 20: List of strains used for single strain fermentation reactions with their appropriate medium and inoculation dilutions.

[0375] Incubated cultures were used to inoculate 96 well plate fermentation reactions containing basal medium (2 g peptone, 2 g yeast extract, 0,5 g L-Cysteine, 5,22 g K2HPO4, 16,32g KH2PO4, 2 ml Tween 80, 2 g NaHCO3 and 1 g mucin / liter), or basal medium further supplemented with either 10 or 3.6 g / L of dialysed pea fibre, pea fibre or fructo-oligosaccharide (FOS). -Bacterial growth was measured by analysing the optical density (O.D.) of each culture in a Tecan Spectrophotometer at the start of fermentation, and after 96 hours of fermentation. O.D measurements taken at the start of fermentation were subtracted from the O.D measured after 96 hours of fermentation in order to correct for variance in starting concentrations of bacteria. Each experiment was repeated three times and the average O.D was plotted for each strain. The separately tested bacterial strains were Akkermansia muciniphila (Figure 28A), Bifidobacterium adolescentis (Figure 28B), Bifidobacterium bifidum (Figure 28C), Bifidobacterium longum (Figure 28D), Blautia faecis (Figure 28E), Roseburia faecis (Figure 28F), Lactobacillus plantarum (Figure 28G), Monoglobus pectinilyticus (Figure 28H).

[0376] The results of these isolated strain fermentations containing pea fibre clearly demonstrated that pea fibre is capable of stimulating the growth of Bifidobacterium adolescentis (DSM20083), Bidobacterium longum (DSM 20219), Roseburia faecis (DSM 27629), Blautia faecis (DSM 27629) and Monoglobus pectinyticus (DSM 104782). Similar fermentation reactions containing FOS demonstrated that FOS supports the growth of Bifidobacteriumadolescentis (DSM20083), Bidobacterium longum (DSM 20219), Roseburia faecis (DSM 27629), Blautia faecis (DSM 27629) and Akekermansia muciniphilia (DSM 22959). Notably, pea fibre was the only fibre to support the growth of Monoglobus pectinilyticus, whereas FOS was the only fibre that could support the growth of Akkermansia municiphilia.

[0377] This data supports the findings outlined in the previous examples, and clearly demonstrates that the identified increase in relative abundance during mixed population microbiota experiments was not due to cross feeding or other interactions with other bacteria.

[0378] Example 9: Analysis of Microbiota upregulated by Pea Fibre

[0379] The above examples have outlined experiments that show that several genera of bacteria were upregulated in fermentation reactions comprising pea fibre. A summary of the probiotic effects of each identified genera is as follows:

[0380] Bifidobacterium

[0381] Bifidobacteria are known to express a “general” set of enzymes (CAZymes) that can break down different types of carbohydrates (Bedu-Ferrari et al. 2022). Bacteria of the Bifidobacterium genus are well known in the field to have potential health benefits. It is, therefore, widely considered as a genus with probiotic properties (Slavin, 2013).

[0382] Blautia

[0383] The probiotic properties of species of the Blautia genus have recently been reported (Benitez-Paez., et al 2020). In one study, fewer Blautia species were discovered in the gut microbiota of obese children than in children of a healthy weight, and there was a further decrease in Blautia observed in obese children exhibiting insulin resistance. Also observed was a parallel increase in the proinflammatory cytokines and chemokines: gamma interferon [INF-y], tumournecrosis factor alpha [TNF-a] and monocyte chemoattractant 1 [MCP-1]. In vitro studies showed that two species of Blautia, B. luti and B. wexlerae exert an anti-inflammatory effect on peripheral blood mononuclear cell cultures, leading to the conclusion that the depletion of B. luti and B. wexlerae species in the gut microbiome of obese individuals can contribute to inflammation leading to insulin resistance.

[0384] Another study investigated the association between visceral fat accumulation (VFA) and the intestinal microflora composition in Japanese men and women who were 20 — 76 years of age. In this study, Blautia was identified as the only gut microbe that was significantly and inversely associated with VFA regardless of the sex of the individual (Ozato., et al 2022). Further investigation determined that B. producta and B. hansenii species were significantly and negatively associated with VFA.

[0385] Roseburia

[0386] The Roseburia intestinalis species has been identified as the most abundant butyrate-producing bacterium in human faeces, which contributes to an average of 2.3% of bacteria faecal microbiota populations (Duncan, et al. 2002, Hold, et al 2003). Changes in the relative abundance of Roseburia intestinalis within the intestinal microbiota have been implicated in a number of different human diseases (Nie et al., 2021):

[0387] Digestive Diseases

[0388] A study of the microbiome in 668 patients with Crohn’s Disease and healthy subjects revealed a lower abundance of Roseburia intestinalis in patients with Crohn’s Disease (Gevers et al., 2014). Roseburia was similarly reduced in paediatric patients with ileal Crohn’s disease (Morgan et al., 2012), and patients with Ulcerative Colitis (UC) and Irritable Bowel Syndrome (IBS) exhibited similar decreases in Roseburia intestinalis (Kumari et al., 2013, Rajilic-Stojanovic et al., 2013, Chassard et al., 2012).Autoimmune Disorders

[0389] Roseburia intestinalis was found to be in lower abundance in adults with Behcet syndrome and systemic sclerosis, particularly in those patients with systemic sclerosis and gastrointestinal involvement (Consolandi etal., 2015). Sequencing results of the faecal samples identified that rheumatic arthritis patients had a lower abundance of Roseburia than the healthy control group, leading to the suggestion that Roseburia could be used as a health marker (Forbes et al. 2018).

[0390] Metabolic Diseases

[0391] Butyrate levels have been strongly linked with glucose homeostasis, insulin resistance and appetite, implicating a role for Roseburia in diabetes (Canfora et al., 2015). Furthermore, a large-scale population study in China discovered that there was a decreased abundance in those with T2DM, and a set of 26 studies assessing bacterial alterations in T1DM in 2,600 and 189 adults discovered that the most severe alterations were in the abundance of Roseburia.

[0392] Additionally, those with a higher BMI carried a higher abundance of Roseburia, and children and adolescents with primary hyperlipidaemia had a lower abundance of Roseburia. These findings suggest a correlation between the abundance of Roseburia and changes in blood lipid concentrations (Gargari et al., 2018).

[0393] Nervous System

[0394] The abundance of Roseburia intestinalis was shown to be less abundant in patients with a major depressive disorder (MDD), and faecal microbiota transplantation from MDD patients to germ-free recipient mice could induce depression-like behaviour (Zheng et al., 2016). It was further shown that Roseburia can influence the 5-HT level and GFAP expression in colonic tissue, leading to the alleviation of depression-like behaviour in mice (Xu etal., 2021).Faecal and mucosal 16S rRNA sequencing revealed that Roseburia was lower in abundance in patients with Parkinson’s disease than in control groups, and that Roseburia associated gut dysbiosis promoted the aggregation of a-synuclein (Keshavarzian et al., 2015).

[0395] Circulation and Haematology

[0396] Metabolites derived from Roseburia intestinalis can cross the gut barrier and circulate in the bloodstream. A study revealed that patients with atherosclerosis had a lower abundance of Roseburia than in a healthy subject (Karlsson et al., 2012). A negative correlation between Roseburia and the development of atherosclerotic plaques was determined in mouse models fed with a high concentration of plant polysaccharides (Kasahara et al., 2018). Similarly, two independent studies determined that patients with coronary artery disease (CAD) had significantly depleted levels of Roseburia (Lui etal., 2019; Zhu, Q. et al., 2018).

[0397] Infection and Cancer

[0398] An investigation determined that the abundance of R. intestinalis was inversely correlated with immune activation and vascular inflammation (Dillon et al., 2017). Patients with tuberculosis (TB) had an enriched abundance of Roseburia which promoted TB pathophysiology by enhancing the anti-inflammatory environment of the host (Maji et al., 2019).

[0399] Monoglobus

[0400] Monoglobus is a pectin degrading bacterium in the human colon. Pectin is not digestible by the small intestine be ordinarily digested in the colon, but it can be digested by colonic bacteria in the large intestine to provide beneficial metabolites such as short chain fatty acids (X. Tang and P. de Vos 2023). The breakdown of Pectin can also lower the pH in the large intestine, which inhibits the adhesion and growth of pathogenic bacteria.This genus may also mediate a link between high fibre intake and a lower risk of hypertension (Li eta!., 2023).

[0401] Example 10: Further Analysis of the Unique Microbiota Modulating Properties of FIPEA-D®

[0402] This example presents a state-of-the-art re-analysis of an in vitro fermentation experiment evaluating the microbiota-modulatory effects of a proprietary soluble pea fibre (FIPEA-D®), benchmarked against fructo-oligosaccharides (FOS), pectin, and a no-fibre control. The use of an updated QIIME-based bioinformatics pipeline and multivariate statistical analyses has provided refined insights into the compositional and functional impacts of pea fibre on gut microbial communities.

[0403] Pea fibre induced the strongest and most distinct microbiota shifts among all tested substrates, exceeding those observed for both pectin and FOS. While FOS elicited the expected bifidogenic and lactate-driven response, pea fibre promoted a broad and complex restructuring of the microbial ecosystem, characterised by enrichment of multiple genera within the Lachnospiraceae family. This family comprises metabolically versatile plant polysaccharide specialists equipped with extensive carbohydrate-active enzyme repertoires, enabling degradation of structurally complex plant fibres.

[0404] FIPEA-D® stimulated Lachnospiraceae taxa which are functionally diverse, implying an interactive microbial network of primary degraders, secondary fermenters, and cross-feeding taxa, resulting in balanced production of key health-relevant short-chain fatty acids, including acetate, propionate, and notably butyrate. The observed butyrate production appears to arise from co-ordinated community-level metabolism rather than reliance on a single dominantproducer, suggesting a robust, resilient microbiota response even in diverse, host-specific microbiota constellations as tested here.

[0405] Lachnospiraceae have emerged as a major focus of contemporary microbiome research, as multiple population-based and disease-association studies have consistently reported their reduced abundance across a broad spectrum of metabolic, immunological, and gastrointestinal disorders. This growing body of evidence has positioned specific Lachnospiraceae members as key indicators of gut ecosystem health and resilience. In parallel, several taxa within this family are actively being developed as next-generation probiotics, also referred to as live biotherapeutic products, for a wide range of clinical indications, underscoring their translational relevance.

[0406] Collectively, these findings position FIPEA-D® as a promising candidate for next-generation, “precision prebiotic” strategies, by selectively stimulating a wide range of functionally different Lachnospiracaea taxa linked to proprionate and butyrate production. Based upon recent microbiome based insights with regard to Lachnospiraceae members, FIPEA-D® is positioned as a promising dietary ingredient for targeting these taxa linked in literature to metabolic health, immune regulation, gut function, and long-term resilience against lifestyle-related diseases.

[0407] Summary of Analysis Methods

[0408] Raw Sequencing data was collected from the in vitro microbiota experiments obtained from the presented in example 7 and used to perform the following steps:Organisation of the Data

[0409] Metadata reformatted: experimental details, donors, treatment - integrated with measured data (pH, SCFAs, gas etc.)

[0410] Re-analyses 16S sequencing data (QIIME pipeline)

[0411] o Hampered sequence quality (end of reads; typical for MiSeq platform) -> fixed (DADA2)

[0412] o Qiime ASV calling -> genus and species classification (poor)

[0413] Multivariate analyses of the microbiota data

[0414] Focus on controlling for donor effects (3)

[0415] Elucidating of unique features of microbiota shifts induced by Pea Fiber

[0416] Short-chain fatty acids plotted as supplementary variables

[0417] Two types of analyses performed:

[0418] o Relative abundance data at baseline (tO) versus / and end of fermentation (t24) o Log2 - fold change data (TO -> T24) focused on unique features treatments (All, Fibers_only, unique Pea features)

[0419] In -depth characterization microbiota effects of Pea Fibre (FIPEA-D®)

[0420] Focus on the short-chain fatty acids stimulated superiorly by Pea Fiber: Propionate and Butyrate

[0421] In-depth characterization stimulation of Lachnospiraceae genera by Pea Fiber and their relation with human health

[0422] Multivariate Statistics: Short description of the multivariate models used (PCA and RDA)Principal Component Analysis (PC A)

[0423] PCA is an exploratory method that summarises the largest overall variation in the data. It does not use information on treatment, donor, or time. Patterns are descriptive and useful for data exploration but treatment effects may be obscured.

[0424] (partial) Redundancy Analysis (RDA)

[0425] Hypothesis-driven method explaining variation attributable to known variables (e.g. treatment)

[0426] Partial RDA removes variation linked to ‘masking’ factors; e.g. donor-specific variation is typically very large and thereby obscuring treatment-specific effects

[0427] Displays only the variation explained by the variables of interest; The first constrained axis (RDA1, x-axis) captures the main treatment-associated gradient

[0428] Monte Carlo permutation testing assesses whether the variation explained along the first constrained axis is greater than expected by chance (p-value)

[0429] How to read the RDA plots

[0430] Samples (points)

[0431] Each point represents one sample based on microbiota composition

[0432] Samples closer together have more similar microbiota profiles

[0433] Separation along the x-axis (RDA1) reflects the main treatment-associated effect (p- value)

[0434] Separation along the y-axis (RDA2) reflects additional, secondary variation not fully explained by the primary treatmentEllipses

[0435] Samples in the same Ellipse share similar microbial signatures

[0436] Opposite quadrants (horizontal) indicate contrasting community compositions related to treatments

[0437] Interpretation and statistics are primarily based on horizontal (RDA1) separation

[0438] Group centroids

[0439] Centroids indicate the average position of each treatment group

[0440] Distance between centroids reflects the magnitude of treatment-associated differences Overlap suggests limited or variable treatment effects

[0441] Taxa arrows

[0442] - Typically, the 15 most contributing taxa driving the treatment separation are presented as Arrows

[0443] - Arrow direction indicates association with samples and treatment groups in that direction

[0444] - Arrow length reflects the strength / importance of that taxon in explaining the observed variation.

[0445] Supplementary SCFA arrows

[0446] SCFAs are included as supplementary variables and do not define the ordination Their arrows show how SCFA levels co-vary with microbiota patterns

[0447] Alignment with treatment centroids or taxa arrows suggests functional associationSCFAs pointing in the same direction as specific taxa indicate linked microbial activity Important: interpretation is associative, not causal.

[0448] Key takeaway

[0449] The RDA plot shows how treatment-associated microbiota shifts (x-axis) relate to specific taxa and functional readouts (SCFAs), with secondary variation captured on the y-axis.

[0450] How to read Heatmaps

[0451] Show the most important taxa contributing to the Clustering. CFit2 represents importance (i.e. the length of the arrow) of the taxon Respl is the position on the first axis (X-axis). Respl is the vertical position on the second axis (Y-axis). The heatmap show the log2FC change after 24 hours of fermentation (blue is a decrease, red is increase).

[0452] QC: Microbiota Classification

[0453] The average microbiota composition of all samples resemble, on average, an adult microbiome. There is no apparent shift into an artificial gut microbiota system. Few sequences (ASVs) are classified (reliably) down to the species level. Core expected microbial species (commensals) are detected as previously reported.

[0454] The Donor Effect is Dominant

[0455] As best shown in Figure 29, 68% of the microbiota variation in the experiment is explained by the donor, highlighting the individuality of the microbiome. This individuality is maintained throughout the experiment. This presents a need to control for donor-specific effects byintroducing covariate (donor) analyses to enable a zoomed in review of ‘homogenous’ fibrespecific microbiota shifts across donors.

[0456] Dietary Fiber addition modulates microbiota composition

[0457] As best shown in Figure 30, after removing variation attributable to donor differences, 24 hours of fermentation modulates the microbiota composition significantly. Fiber addition is the primary driver of the significance observed in this RDA analyses and characterised by an increase in Bifidobacterium spp. (bottom right quadrant of Figure 30). A Focus on the difference obtained at 24 hours only by calculating Log2 Fold-change (0->24 hours). Log2-transformed Data is then analyzed by RDA to identify unique microbiota signatures related to the different fiber treatments.

[0458] Microbiota shifts after 24 hours fermentation are fiber-specific

[0459] Short-chain fatty acids measured are included as supplementary variables to aid biological interpretation and to discover taxa and treatment associations. As best shown in Figure 31 , fibers are significantly altering microbiota shifts after 24h of fermentation; largest effects are observed by Pea Fiber addition. All fibers increased levels of Bifidobacterium and short-chain fatty acids and decreased in branched-chain fatty acids (marker of proteolytic fermentation). FOS addition is characterized by Adlercreutzia specifically and lactate accumulation. Pectin is characterized by stimulation of the Lachnospiraceae ND3007 cluster and Acetate accumulation.

[0460] Pea fibre supplementation was most strongly associated with an increased relative abundance of Monoglobus, alongside consistent stimulation of Lachnospira and Blautia. Concomitantly, Pea Fiber significantly enhanced the production of the beneficial short-chain fatty acidsPropionate and Butyrate. Importantly, Pea Fiber supplementation counteracted microbiota shifts observed under control conditions, which are likely driven by the protein-rich basal fermentation medium. This is supported by the enrichment of potentially proteolytic taxa and the accumulation of branched-chain fatty acids (BCFA; iso-forms of the short-chain fatty acids) - indicative of increased proteolytic fermentation in the control treatment.

[0461] Pea fibre is superior in shifting Microbiota composition compared to FOS

[0462] As shown by Figure 32, when removing pectin from the analyses, the microbiota modulatory capacity of Pea Fiber addition is becoming increasingly apparent. The major signal (and corresponding) p-value is driven by Pea Fibre addition to the control medium.

[0463] The shared signal between FOS and Pea Fibre is the stimulation of Bifidobacterium and the accompanied acetate accumulation.

[0464] A distinctive feature of the Pea fibre-induced microbiota shifts is the stimulation of Monoglobus, a well-established pectin-degrading specialist, together with numerous taxa belonging to the Lachnospiraceae family that have not yet been cultured (highlighted in green across the different group definitions), as well as well-characterized cultured genera such as Lachnospira and the Eubacterium eligens group. Consistent with these compositional changes, fibre supplementation promoted increased propionate and butyrate production, while simultaneously inhibiting branched-chain fatty acid (BCFA) formation, an effect that was not observed upon FOS supplementation. Also, several Lachnospiraceae taxa were also associated with the control medium, suggesting a metabolic capacity to utilize mucin (major carbohydrate source), or minor carbohydrate components of the yeast extract (P-glucans or manno-oligosaccharides), which together constitute the only available carbohydrate substrates in the control condition. Alternatively, these taxa may adopt a predominantlyproteolytic lifestyle, deriving energy from amino acids supplied by the peptones and yeast extract. Support for this latter metabolic strategy is provided by the pronounced accumulation of BCFAs and valerate in the control medium, indicating dominant amino acid fermentation (typically by specialized anaerobic bacteria, e.g. Dorea).

[0465] Is the non-pectin part or structural properties of FIPEA-D® feeding the propionate and butyrate producing members of the microbiota?

[0466] When comparing Fiber addition only the microbiota induced shifts are still significantly different, mainly driven by the difference between FOS and Pea Fiber. Interestingly, Bacteroides now emerges as a strong shared signal between both Pectin and Pea, likely linked to the broad saccharolytic capacity, as evidenced by the rich CAZYME profile of several Bacteroides species.

[0467] Lachnospiracea taxa only: identify unique taxa stimulated by Pea Fiber

[0468] When reducing the information in the RDA model (Lachnospiracaea genera), the differences observed by Fiber addition is still statistically significant, mainly driven by the difference between FOS and Pea Fiber.

[0469] Interestingly, Eubacterium halli, a species well known for its butyrate producing capacity, now emerges as a taxon linked to Pea Fiber.

[0470] Pea Fibre is the most effective stimulator of proprionate and butyrate production

[0471] As best shown in Figures 35A, 35B, 36A and 36B, RDA of SOFA composition reveals distinct fermentation profiles across treatments. Butyrate and propionate production are more stronglyassociated with pectin and pea fibre supplementation. Pea fibre induced the most rapid and efficient increase in propionate and butyrate over time, with levels approximately two-fold higher (48h) than those observed with FOS. Pea fibre appears to support both primary fermentation and direct stimulation of butyrogenic taxa, thereby accelerating propionate and butyrate production

[0472] Pea Fiber is the most effective stimulator of propionate and butyrate production

[0473] As shown in Figure 38A, lactate accumulation was observed exclusively following FOS supplementation; in all other conditions, lactate was either not produced (control) or rapidly consumed (>6 h) by secondary fermenters

[0474] As shown in Figure 38B, formate concentrations increased across all fiber-supplemented conditions; however, formate remained stable under FOS treatment and was subsequently consumed in the pea fiber- and pectin-supplemented conditions, consistent with uptake by acetogenic populations (potentially explaining the further increase in acetate from 24h to 48h).

[0475] As best shown in Figures 38D to 38F, branched-chain fatty acids and valerate reached their highest levels under the experimental control conditions and were most strongly reduced following FOS supplementation, likely as a consequence of pH reduction driven by excessive lactate accumulation.

[0476] Interpretation of Lachnospiraceae taxa associated with dietary treatments

[0477] Lachnospiraceae constitute a highly diverse family displaying context-dependent functional profiles. Taxa associated with the control treatment are linked in the literature to proteolytic metabolism and the production of potentially pro-inflammatory metabolites and have beenassociated with inflammatory and metabolic disease states Pea fibre supplementation stimulated a broad and largely non-overlapping set of Lachnospiraceae genera across donors, indicating a robust response despite pronounced inter-individual microbiome variability. This donor-specific yet consistent responsiveness suggests strain-level functional redundancy, supporting good translational potential and compliance in heterogeneous real-world populations. The stimulated Lachnospiraceae taxa encompass a wide range of metabolic roles, including primary polysaccharide degradation, primary fermentation, secondary fermentation (lactate and acetate conversion), acetogenesis, and most important (direct) butyrate production. This functional diversity supports the establishment of a resilient ecosystem that promotes both direct and indirect butyrate production, contributing to metabolic stability and host health. Consistent with this interpretation, reduced abundance or absence of several of these taxa has been associated with obesity, type 2 diabetes, inflammatory bowel disease, irritable bowel syndrome, cardiovascular disease and COVID-19 severity. Recent reviews position Lachnospiraceae as emerging next-generation probiotics (live biotherapeutics), with reported disease indications ranging from colonization resistance against enteric pathogens (VRE, C. difficile) to inflammatory, allergic, metabolic diseases and preclinical work showing beneficial roles in cancer treatment

[0478] Summary of further analysis

[0479] The experiments of the previous examples compared soluble pea fibre (FIPEA-D ®) with established reference substrates, including fructo-oligosaccharides (FOS), pectin, and a basal-medium control, focusing on microbial composition and short-chain fatty acid (SCFA) production. In this example, the raw 16S rRNA gene sequencing data from the previous examples were re-analysed using a state-of-the-art bioinformatics workflow based on QIIME, combined with updated taxonomic annotation and multivariate statistical analyses. This reanalysis aimed to extract deeper insights into community-level shifts and to identify uniquemicrobiota signatures associated with pea fiber, particularly in comparison with the well-characterized fermentable fibers such as FOS and pectin.

[0480] Overall Impact of Fiber Addition on Microbiota Composition

[0481] Addition of fermentable fibres induced pronounced changes in microbiota composition compared with the control condition, confirming the strong selective pressure exerted by dietary carbohydrates on gut microbial ecosystems. Among the tested substrates, pea fibre induced the most substantial compositional shifts, exceeding those observed for pectin and markedly surpassing the effects of FOS. Multivariate analyses demonstrated clear separation of the pea fiber condition from all other treatments, indicating that its microbiota-modulatory effects are not merely quantitative (i.e. stronger fermentation), but also qualitative in nature, affecting a broader and distinct range of microbial taxa.

[0482] Reference Responses: FOS and Pectin

[0483] As expected, FOS supplementation resulted in a classical bifidogenic response, characterized by increased abundance of Bifidobacterium spp. and enhanced lactate production. This response is well documented in the literature and reflects the narrow substrate specificity of FOS, which is primarily utilized by a limited group of fast-growing saccharolytic bacteria. Pectin induced a broader microbial response than FOS, consistent with its more complex polysaccharide structure. However, the magnitude and diversity of the compositional changes remained lower than those observed for pea fiber, suggesting that FIPEA-D ® offers a wider spectrum of fermentable carbohydrates for ‘plant specialists’ in the human microbiome.Pea Fibre Induces a Complex Lachnospiraceae-Dominated Shift

[0484] The most distinctive feature of the FIPEA-D® addition was a pronounced and complex shift within the family Lachnospiraceae. Multiple genera belonging to this family were selectively enriched, highlighting a coordinated response of a functionally diverse group of plant polysaccharide specialists. Lachnospiraceae represent a phylogenetically and functionally heterogeneous family that plays a central role in the degradation of complex dietary carbohydrates in the human gut. Members of this family are known to encode a broad array of carbohydrate-active enzymes (CAZymes), including glycoside hydrolase families such as GH13 and GH31 (starch and oligosaccharide degradation), GH43 (arabinoxylan and hemicellulose breakdown), and GH28 and polysaccharide lyases (pectin degradation). This enzymatic versatility enables Lachnospiraceae to access complex plant-derived substrates that are inaccessible to more specialized fermenters.

[0485] Functional Diversity and SCFA Production Capacity

[0486] The enrichment of Lachnospiraceae under pea fibre supplementation reflects stimulation of a highly interactive microbial network comprising primary degraders, secondary fermenters, and cross-feeding taxa. Primary degraders initiate the breakdown of complex polysaccharides, releasing intermediate metabolites such as oligosaccharides, acetate, and lactate.

[0487] These intermediates are subsequently utilized by secondary fermenters and acetogens, which convert acetate / lactate and hydrogen + carbon dioxide, respectively, into butyrate. This functional characterisation of the FIPEA-D® increase Lachnospiraceae genera is consistent with the observed SCFA profile, where all major health-relevant SCFAs acetate, propionate, and notably butyrate are increased. Among these, butyrate is of particular interest due to its central role in colonic epithelial energy metabolism, gut barrier integrity and systemic healthbenefits (metabolic). The data suggest that the complex Lachnospiraceae community stimulated by FIPEA represents an efficient butyrate-producing ecosystem rather than reliance on a single dominant butyrate producer.

[0488] Health Relevance of Lachnospiraceae enrichment

[0489] Although Lachnospiraceae remain a relatively under-characterized family at the species and strain level, accumulating evidence links their abundance and activity to beneficial health outcomes. Increased representation of specific Lachnospiraceae taxa has been associated with improved metabolic health, enhanced satiety signalling via SCFA-mediated gut-brain pathways, and improved insulin sensitivity.

[0490] In addition, butyrate produced by Lachnospiraceae plays a key role in immunomodulation, including promotion of regulatory T cell (Treg) differentiation and maintenance of immune tolerance. These effects contribute to reduced low-grade inflammation and improved resilience of the gut ecosystem against pathogenic colonization (colonization resistance). Such mechanisms are increasingly relevant for conditions such as irritable bowel syndrome (IBS), susceptibility to infections (VRE, Cdif), and chronic non-communicable diseases often associated with Western-style diets.

[0491] Beyond the production of SCFAs, Lachnospiraceae are increasingly recognized as a source of a broad and still incompletely characterized repertoire of microbial metabolites with potential health relevance. These include organic acids, alcohols, phenolic and indole-derived compounds, and small bioactive molecules arising from complex cross-feeding networks.Summary and Implications

[0492] In summary, re-analysis of the in vitro fermentation data demonstrates that soluble pea fibre exerts a uniquely strong and complex modulatory effect on gut microbiota composition compared with FOS and pectin. This effect is characterized by broad stimulation of functionally diverse Lachnospiraceae taxa, supporting efficient and balanced production of health-relevant SCFAs, particularly butyrate. Literature also reports on certain Lachnospiraceae taxa being stimulated by Pea Fiber consumption / addition, but not in a broader family context as reported here. Stimulation of a wide range, of functionally different Lachnospiracaea taxa seems to be a unique feature of FIPEA-D ® compared to the prior art, and it might also explain the butyrate stimulating capacity, which has not frequently been reported previously for pea fibre. These findings position FIPEA-D ® as a promising dietary ingredient for targeting microbial ecosystems linked to metabolic health, immune regulation, gut function, and long-term resilience against lifestyle-related diseases.

[0493] Conclusions

[0494] The present inventors have successfully demonstrated the prebiotic potential of pea fibre. Firstly, they have shown that pea fibre can be considered a dietary fibre. Additionally, it has been shown that pea fibre can increase the relative abundance of several beneficial bacteria species. In particular, fermentation with pea fibre has been shown to increase the concentration of specific (unknown) Lachnospiracaea genera, Monoglobus, Roseburia, Blautia and Bifidobacterium, and these bacterium have a wide range of documented positive effects on health and well-being in an individual, such as obesity or in the control of weight management.The optional features set out herein may be used either individually or in combination with each other where appropriate and particularly in the combinations as set out in the accompanying claims. The optional features for each aspect or exemplary embodiment of the invention as set out herein are also to be read as applicable to any other aspect or exemplary embodiments of the invention, where appropriate. In other words, the skilled person reading this specification should consider the optional features for each exemplary embodiment of the invention as interchangeable and combinable between different exemplary embodiments.

[0495] Attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0496] All of the features disclosed in this specification (including any accompanying claims, and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0497] Each feature disclosed in this specification (including any accompanying claims, and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0498] The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.References

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Claims

Claims1. A composition for use as a prebiotic in promoting the growth or increasing the relative abundance of health-beneficial bacteria in the gastro-intestinal tract of an individual, the composition comprising a dietary pea fibre.

2. The composition for use according to claim 1 , wherein the dietary pea fibre is extracted from pea seed.

3. The composition for use according to claim 2, wherein the dietary pea fibre is extracted from the yellow pea seed.

4. The composition for use according to any one of claims 1 to 3, wherein the dietary pea fibre is extracted from P. sativum.

5. The composition for use according to any one of the preceding claims, wherein the dietary pea fibre is a homogalacturonan.

6. The composition for use according to any one of the preceding claims, wherein the dietary pea fibre comprises a negatively charged backbone region and at least one neutral polysaccharide branched regions.

7. The composition for use according to claim 5, wherein the negatively charged backbone region comprises galacturonic acid.

8. The composition for use according to claim 6, wherein the dietary pea fibre comprises a repeating unit of 1,2-a-L-Rhamnose and 1,4-a-D-Galacturonic acid; Xylogalacturonan, homogalacturonan with a-D-Xylose branched at 0-2 of galacturonic acid.

9. The composition according to any preceding claim, wherein a single molecule of the pea fibre has a molecular diameter of more than 100 nm and equal to or less than 200 nm.

10. The composition according to claim 9, wherein a single molecule of pea fibre has a molecular diameter of more than 125 nm and equal to or less than 200 nm.

11. The composition according to either one of claims 9 or 10, wherein a single molecule of pea fibre has a molecular diameter of more than 150 nm and equal to or less than 200 nm.

12. The composition according to any one of claims 9 to 11, wherein a single molecule of pea fibre has a molecular diameter of more than 175 nm and equal to or less than 200 nm.

13. The composition according to any one of claims 9 to 11, wherein a single molecule of pea fibre has a molecular diameter of more than 125 nm and equal to or less than 175 nm.

14. The composition according to any preceding claim, wherein the degree of methyl esterification of constituent galacturonic acid is 45% or less15. The composition according to claim 14, wherein the degree of methyl esterification of constituent galacturonic acid is 30% or less.

16. The composition according to either of claims 14 or 15, wherein the degree of methyl esterification of constituent galacturonic acid is 20% or less.

17. The composition according to any one of claims 14 to 16, wherein the degree of methyl esterification of constituent galacturonic acid is 10% or less.

18. The composition according to any one of claims 14 to 17, wherein the degree of methyl esterification of constituent galacturonic acid is between 0% and 10%.

19. The composition according to any previous claim, wherein a single molecule of pea fibre has a star shaped structure.

20. The composition according to any previous claim, wherein a single molecule of pea fibre has a structure in which about 3 to 20 linear side chains of similar length are branched from the main chain.

21. The composition according to any previous claim, wherein the composition comprises polymeric components with a molecular rotation radius of 25 nm to 40 nm.

22. The composition according to any previous claim, wherein the composition comprises polymeric components with a molecular rotation radius of 30 nm to 40 nm.

23. The composition according to any previous claim, wherein the composition includes polymeric components with a molecular weight greater than or equal to 10,000.

24. The composition according to any previous claim, wherein the composition includes polymeric components with an absolute molecular weight of 500,000 to 1,000,000.

25. The composition according to claim 24, wherein the composition includes polymeric components with an absolute molecular weight of 800,000 to 900,000.

26. The composition according to any previous claim, wherein all components of the composition have a molecular weight below 5,000,000.

27. The composition for use according to any one of the preceding claims, wherein the composition does not comprise pea starch and / or pea protein.

28. The composition for use according to any one of the preceding claims, wherein the pH of the composition is about pH 3 to about pH 12.29 The composition for use according to claim 27, wherein the pH of the composition is about pH 4 to pH 10.

30. The composition for use according to any one of the preceding claims, wherein the composition comprises at least 50% dietary pea fibre.

31. The composition for use according to anyone of the preceding claims, wherein the composition comprises at least one of galacturonic acid, arabinose, glucose, rhamnose, galactose and / or xylose32. The composition for use according to claim 31 , wherein the composition comprises about 35-50 (w / w %) Arabinose, about 20-35 (w / w %) Glucose, about 1-10 (w / w%) Galactose, 2-3 (w / w %) Xylose, 3-5 (w / w %) Rhamnose, and / or about 17-20 (w / w%) Galacturonic acid.

33. The composition for use according to any one of the preceding claims, wherein the dietary pea fibre is water soluble.

34. The composition for use according to any one of the preceding claims, wherein the dietary pea fibre is incorporated into a food stuff or food additive.

35. The composition for use according to claim 34, wherein the composition is incorporated into a snack bar.

36. The composition for use according to any one of the preceding claims, wherein the composition is incorporated into flour and / or rice.

37. The composition for use according to claim 36, wherein the total weight percentage of dietary pea fibre in flour and / or the total weight percentage of dietary pea fibre in cooked rice is at least 0.4%.

38. The composition for use according to any one of claims 1 to 33, wherein the composition is incorporated into a beverage.

39. The composition for use according to claim 38, wherein the beverage is a meal replacement drink or an acidified milk beverage.

40. The composition for use according to any one of claims 1 to 33, wherein the composition is incorporated into pet food.

41. The composition for use according to claim 40, wherein the pet food is in the form of dry food or paste.13342. The composition for use according to any one of claims 34 to 39, wherein the pea fibre is incorporated into a food or beverage as one or more of the following: an acidified food stabilizer, a foam reducer and / or stabiliser, an anti-coagulant, a preservative consistency adjuster, a gluten dispersibility improver, a bulking agent, a sedimentation inhibitor, a syneresis reducer, and / or a consistency regulator.

43. The composition for use according to any one of claims 1 to 33, wherein the composition is incorporated into a dietary supplement.

44. The composition for use according to claim 43, wherein the dietary supplement is in the form of a powder, a gel, a gummy, a liquid, or a paste.

45. The composition for use according to claim 43 or 44, wherein the dietary supplement further comprises a probiotic.

46. The composition for use according to claim 44, wherein the dietary supplement is incorporated into a form of a dissolvable dry drinks powder.

47. The composition for use according to claim 44, wherein the dietary supplement is combined with a gelling agent.

48. The composition for use according to claim 47, wherein the gelling agent is selected from gelatine, pectin, agar or carrageenan.

49. The composition for use according to claim 44 or 47 to 48, wherein the gelling agent is in the form of a gummy.13450. The composition for use according to any one of the preceding claims, wherein the composition is combined with additional probiotics, fibres or other active microbiome modulating ingredients.

51. The composition for use according to any one of the preceding claims, wherein the dietary pea fibre is present in the amount of 1-3 grams per portion.

52. The composition for use according to claim 50, wherein the dietary pea fibre is present in the amount of 2 grams per portion53. The composition for use according to claims 51 or 52, wherein the health-beneficial bacteria are selected from the Lachnospiraceae family and / or one or more of any one of the following genera of bacteria: Anaerostipes, Blautia, Monoglobus, Roseburia, Bifidobacterium, Bacteroides and / or Lactobacillus.

54. The composition for use according to any one of claims 51 to 53, wherein the beneficial bacteria are from one or more of any one of the following species of bacteria: Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, Blautia faecis, Roseburia faecis, Monoglobus pectinilyticus, Eubacterium hallii, Eubacterium eligens, Lachnospiraceae NC2004, Lachnospiraceae NK4A136, Lachnospiraceae ND3007, Lachnospiraceae UCG-001 and / or Lachnospiraceae UCG-004.13555. The composition for use according to any preceding claims, wherein the composition increases small chain fatty acid (SCFA) production in the proximal and distal colon, preferably the SCFA are at least one of butyric acid, propionate, valeric acid and / or acetate.

56. The composition for use according to any of the preceding claims, wherein the composition prevents or decreases the relative abundance of health-detrimental bacteria in the gastrointestinal tract of an individual.

57. The composition for use according to claim 56, wherein the health-detrimental bacteria is from the genera Clostridiaceae and Clostridium.

58. The composition for use according to any of the preceding claims, wherein the use of the dietary pea fibre is a non-therapeutic use.

59. A composition according to any one of claims 1 to 33 for use in the treatment, management, or prevention of disease in a subject.

60. A method of treating, managing, or preventing a disease in a subject comprising administering the composition of any one of claims 1 to 33.

61. The composition for use according to claim 59 or method according to claim 60, wherein the disease is a dysbiosis of the gastrointestinal tract in an individual.

62. The composition for use, or method, according to claim 61, wherein the dysbiosis is associated with metabolic syndrome, obesity, or in the weight management of an individual.13663. The composition for use, or method, according to claim 61, wherein the dysbiosis is associated with Crohn’s disease, Ulcerative Colitis (UC), Irritable Bowel Syndrome (IBS) or constipation.

64. The composition for use, or method, according to claim 61, wherein the dysbiosis is associated with Behget syndrome, systemic sclerosis, or rheumatic arthritis, the composition comprising pea fibre.

65. The composition for use, or method, according to claim 61, wherein the dysbiosis is associated with an immune mediated disease.

66. The composition for use, or method, according to claim 65, wherein the immune mediated disease is an allergy or asthma.

67. The composition for use, or method, according to claim 61, wherein the dysbiosis is associated with Parkinson’s disease, the composition comprising pea fibre.

68. The composition for use, or method, according to claim 61, wherein the dysbiosis is associated with coronary artery disease, atherosclerosis or hypertension, the composition comprising pea fibre.

69. A method of weight management in an individual, the method comprising administering the composition of any one of claims 1-33 up to 4 times a day.

70. A synbiotic composition comprising:(a) a prebiotic comprising dietary pea fibre; and137(b) a probiotic comprising an isolated bacterial strain from the Lachnospiraceae family and / or one or more of any one of the following genera of bacteria: Anaerostipes, Blautia, Monoglobus, Roseburia, Bifidobacterium, Bacteroides and / or Lactobacillus.

71. The synbiotic composition according to claim 70, wherein the probiotic isolated bacteria are selected from a list consisting of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, Blautia faecis, Roseburia faecis, and / or Monoglobus pectinilyticus Eubacterium hallii, Eubacterium eligens, Lachnospiraceae NC2004, Lachnospiraceae NK4A136, Lachnospiraceae ND3007, Lachnospiraceae UCG-001 and / or Lachnospiraceae UCG-004..

72. The synbiotic composition according to claim 71, wherein the prebiotic and probiotic bacterial strain are provided together in a single formulation.

73. The synbiotic composition according to claim 71, wherein the prebiotic and probiotic bacterial strain are provided separately for administration to an individual sequentially.

74. A method of enhancing the in vitro growth of one or more bacterial genera of interest, the method comprising adding an amount of the composition according to any one of claims 1 to 33 to a growth medium and growing the bacterial genera of interest in the supplemented growth medium.

75. A method of increasing the relative abundance of health-beneficial bacteria in an individual, the method comprising administering an effective amount of the composition of any one of claims 1-33.13876. The method according to claim 75, wherein the health-beneficial bacteria are selected from the family of Lachnospiraceae and / or one or more of any one of the following genera of bacteria: Blautia, Monoglobus, Roseburia, Bifidobacterium, Bacteroides and / or Lactobacillus.

77. The method according to claims 74 or 75, wherein the health-beneficial bacteria are selected from the Lachnospiraceae family and / or one or more of any one of the following genera of bacteria: Anaerostipes, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, Blautia faecis, Roseburia faecis, and / or Monoglobus pectinilyticus Eubacterium hallii, Eubacterium eligens, Lachnospiraceae NC2004, Lachnospiraceae NK4A136, Lachnospiraceae ND3007, Lachnospiraceae UCG-001 and / or Lachnospiraceae UCG-004.139

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