Compositions and uses

Abstract

A method for improving gut microbiota population in a subject by administering a composition comprising allulose, erythritol or a combination thereof. A method for increasing one or more colonic bacterial populations from the families Lachnospiraceae or Eubacteriaceae by administering a composition comprising allulose, erythritol or a combination thereof. A method for preventing, treating or ameliorating a disease in a subject by administering a composition comprising allulose, erythritol or a combination thereof. The disease may be one or more of metabolic syndrome, obesity, type 2 diabetes, insulin resistance, hyperlipoproteinemia, hyperuricemia, hepatic steatosis, hypercholesterolemia, hypertriglyceridemia, irritable bowel syndrome (IBS), colon cancer, allergy, non-alcoholic fatty liver disease, inflammatory bowel disease, cardiovascular disease, or inflammation. The bacterial population may be the species Anaerostipes hadrus, Blautia obeum, Anaerobutyricum hallii, Eubacterium rectale (Agathobacter rectalis), Eubacterium callanderi, Eubacterium limosum and Eubacterium maltosivorans. A composition comprising allulose, erythritol or a combination thereof and one or more colonic bacterial populations from the families Lachnospiraceae or Eubacteriaceae is also provided. The composition may in the form of a food composition.

Description

Field of the Invention [1] The present invention relates to methods for improving gut microbiota population, increasing one or more colonic bacterial populations from the families Lachnospiraceae or Eubacteriaceae in a subject, and treating, preventing or ameliorating a disease, the methods comprising administering to the subject compositions comprising allulose, erythritol or a combination thereof; compositions comprising allulose, erythritol or a combination thereof for use in such methods; and compositions comprising allulose, erythritol or a combination thereof and one or more colonic bacterial populations from the families Lachnospiraceae or Eubacteriaceae. Background of the Invention [2] The gut microflora form a complex ecosystem that interacts with host cells and nutrients. An adult human body is estimated to contain a living bacterial biomass of greater than 1014 and more than 400 different species, which represents the largest, densest, and most diverse microbial community in the human body. The presence of the gut bacteria is a part of normal human physiology and is important for the development of gut functions, harvesting energy from dietary carbohydrates, harvesting essential vitamins and metabolising environmental chemicals in the gut. Recent studies further suggested that gut bacteria may be involved in fat storage and affect weight gain and loss. Gut bacteria are also involved in maturation of the immune system, is in constant communication with the immune system, and protection against pathogens. Given the importance of gut bacteria in health and wellness, a strong interest in functional food ingredients to enhance the populations of beneficial gut bacteria has emerged. [3] High sugar intake has been linked to an increased risk of developing various non-communicable diseases, including insulin resistance, type 2 diabetes (T2D), and obesity (Gillespie et al., 2023, Nutrients 15 (4): 889; Kokubo et al., 2019, Environmental Health and Preventive Medicine 24 (1): 13). In line with the observation that the human gut microbiota plays a critical role in health and disease (Gomaa, 2020, Antonie Van Leeuwenhoek 113 (12): 2019-40), the gut microbiota of type-2-diabetic (T2D) subjects has a particular composition, characterised by lower levels of bacteria capable of producing butyrate (Arora and Tremaroli 2021). Butyrate is the preferred energy source of epithelial cells and plays a protective role against colon cancer and colitis (Riviere et al., 2016, Frontiers in Microbiology 7:979). It is one of the three most abundant short chain fatty acids (SOFA) (along with acetate and propionate) and is typically produced by the gut microbiota upon colonic fermentation of indigestible substrates. Strategies aiming to improve the health status of T2D subjects could seek to address the gut microbiota and boosting SCFA alongside traditional dietary strategies including lowering sugar intake. When designing such strategies, it is important to consider the interplay between gut microbiota, enterotypes and geographical-related differences (such as environmental factors and / or diet) (Costea et al., 2018, Nature Microbiology 3 (1): 8-16; Yatsunenko et al., 2012, Nature 486 (7402): 222-27). [4] Low-no-calorie sweeteners (LNCS) provide sweetness and have been increasingly used as sugar substitutes. Many LNCS exist, each having specific sweetness and pharmacokinetics. Based on their structure and / or sweetness, LNCS might include rare sugars, polyols, and high-intensity sweeteners. Depending on their application, LNCS can further be divided in bulking agents and high-potency sweeteners. While bulking agents are often less sweet than sucrose and provide bulk and texture to foods, high-potency sweeteners are used in small quantities as they impart intense sweetness. [5] Certain LNCS are known to have prebiotic effects. The potential prebiotic activity of allulose (also known as D-allulose or D-psicose) has been discussed (Ozgur et al., Turkiye Klinikleri J Health Sci 2022, 7(2), 573). Some studies have shown a prebiotic effect of allulose with other food ingredients, such as p-glucans (Rugji et al., Food Sci Techno / , 2022, 42, e07022) and probiotics (Choi et al., Nutrients, 2018, 10, 1797). [6] Several studies have investigated the effect of LNCS on the human gut microbiota. As reviewed recently (Hughes et al., 2021, Nutrition Today 56 (3): 105-13), there is currently no evidence that LNCS adversely impact the gut microbiota when consumed at approved levels. Moreover, it is of importance to generate sweetener-specific evidence. While fermentation by gut microbes has been demonstrated for rare sugars (Roy et al., 2018, Journal of Food Science 83 (11): 2699-2709) and specific high-intensity sweeteners (Samuel et al., 2018, The Journal of Nutrition 148 (7): 1186S-1205S), there is no such evidence for polyols, which partially reach the colon where they could also be fermented by gut microbes (Ruiz-Ojeda et al., 2019, Advances in Nutrition 10 (suppl_1): S31-48). Further, despite that health benefits have been described for LNCS, some negative (Suez et al., 2014, Cell 185 (18): 3307-3328.e19; 2022, Nature 514 (7521): 181-86) or contradictory results have been reported as well. A first problem is the wide variation of test doses, which are sometimes irrelevantly high. Secondly, many of the studies reporting conflicting data are performed with animal models and doses (at or below the Acceptable Daily Intake (ADI)) in such models may not be relevant to humans due to the fundamental differences in gastrointestinal physiology. [7] There is increasing demand for functional food ingredients, both for human and animal consumption, that deliver health benefits in addition to nutrition. One class of such health-related food ingredients are known as “prebiotic” compounds, namely substrates that are selectively utilised by host microorganisms conferring a health benefit (Gibson, G., Hutkins, R., Sanders, M. etal. Expert consensus document: The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics. Nat Rev Gastroenterol Hepatol 14, 491-502 (2017)). Representative examples of prebiotic compounds that are currently used in food products include a wide variety of carbohydrates, such as dietary fibers. In addition to “prebiotic” ingredients, there is a growing market for foodstuffs and animal feeds that are formulated with “probiotic” bacteria. For example, there is increasing interest in the use of probiotic organisms as feed additives for animals, primarily as possible replacements for antibiotics. Many bacteria that are considered to have beneficial probiotic properties are normal commensal bacteria present in the healthy human or animal gut microflora. The most frequently used human probiotics include the Lactobacilli and Bifidobacteria. However, use of either of these bacterial groups as probiotic food or feed additives is fraught with difficulties. In order to be of benefit, the bacteria must survive the manufacturing process as viable cells, and be formulated as stable products (i.e., no significant loss of viability on storage for long periods and sometimes under adverse conditions). In addition, the bacteria must survive passage through the extreme acidity of the stomach and exposure to bile salts in the upper small intestine. It is hypothesised that the beneficial effects of probiotics are due to brief colonisation of the small intestine and / or the colon which entails successfully competing with the existing microbiota that number some 107 to 1011 living bacteria per gram of luminal contents. [8] There remains a need for greater understanding about how gut microbiota is affected by dietary factors and for products to improve gut microbiota. Summary of the Invention [9] The following summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.

[10] In a first aspect, the present invention provides a method for improving gut microbiota population in a subject, the method comprising administering to the subject a composition comprising allulose, erythritol or a combination thereof.

[11] Ina second aspect, the present invention provides a method for increasing one or more colonic bacterial populations from the families Lachnospiraceae or Eubacteriaceae in a subject, the method comprising administering to the subject a composition comprising allulose, erythritol or a combination thereof.

[12] In a third aspect, the present invention provides a method for preventing, treating or ameliorating a disease in a subject, the method comprising administering to the subject a composition comprising allulose, erythritol or a combination thereof.

[13] In a fourth aspect, the present invention provides a method for increasing colonic short chain fatty acid levels in a subject, the method comprising administering to the subject a composition comprising allulose, erythritol or a combination thereof.

[14] In a fifth aspect, the present invention provides a composition comprising allulose, erythritol or a combination thereof and one or more colonic bacterial populations from the families Lachnospiraceae or Eubacteriaceae.

[15] In a sixth aspect, the present invention provides a composition comprising allulose, erythritol or a combination thereof for use in a method according to any of the first to sixth aspects.

[16] In a seventh aspect, the present invention provides a use of the composition or composition for use according to any of the fifth or sixth aspects, for the manufacture of a medicament for improving gut microbiota population.

[17] In an eighth aspect, the present invention provides a use of the composition or composition for use according to any of the fifth or sixth aspects, for the manufacture of a medicament for increasing one or more colonic bacterial populations from the families Lachnospiraceae or Eubacteriaceae.

[18] In a ninth aspect, the present invention provides a use of the composition or composition for use according to any of the fifth or sixth aspects, for the manufacture of a medicament for preventing, treating or ameliorating a disease.

[19] In a tenth aspect, the present invention provides a use of the composition or composition for use according to any of the fifth or sixth aspects, for improving gut microbiota population.

[20] In an eleventh aspect, the present invention provides a use of the composition or composition for use according to any of the fifth or sixth aspects, for increasing one or more colonic bacterial populations from the families Lachnospiraceae or Eubacteriaceae.

[21] Ina twelfth aspect, the present invention provides a use of the composition or composition for use according to any of the fifth or sixth aspects, for preventing, treating or ameliorating a disease.

[22] In a thirteenth aspect, the present invention provides a composition comprising allulose, erythritol or a combination thereof to increase colonic bacterial populations. Brief Description of the Drawings

[23] The invention set forth herein can be advantageously understood with regard to the drawings.

[24] Figs. 1A and 1B show the concentrations (peak area) of D-allulose and erythritol respectively at 6, 24 and 48 hrs for the whole sample.

[25] Figs. 2A-2I show the effects of erythritol and D-allulose on fermentation parameters for the whole sample.

[26] Figs. 3A-3C show the cell count for Actinobacteria (cells / mL), Bacteroidetes (cells / mL) and Firmicutes (cells / mL), respectively, after 6, 24, and 48 hrs of incubation with erythritol or D-allulose for the whole sample.

[27] Fig. 4 shows the significant treatment effects on microbial families and species in the wholesample analysis at 48 hrs.

[28] Fig. 5 shows correlations between fermentation parameters (gas production, SCFA, and BCFA) and microbial composition at 48 hrs.

[29] Fig. 6 shows the experimental design set up of Example 1.

[30] Figs. 7A-7I show the effects of erythritol and D-allulose on fermentation parameters by donor group.

[31] Figs. 8A-8C show the cell count for Actinobacteria (cells / mL), Bacteroidetes (cells / mL) and Firmicutes (cells / mL), respectively, after 6, 24, and 48 hrs of incubation with erythritol or D-allulose by donor group.

[32] Fig. 9 shows the significant treatment effects on microbial families and species by donor group analysis at 48 hrs. Detailed Description of the Invention

[33] Before the disclosed methods and materials are described, it is to be understood that the aspects described herein are not limited to specific embodiments, methods, or compositions, and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.

[34] Throughout this specification, unless the context requires otherwise, the word “comprise” and “include” and variations (e.g., “comprises,” “comprising,” “includes,” “including”) will be understood to imply the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other integer or step or group of integers or steps.

[35] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[36] Ranges can be expressed herein as from “about” one particular value and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[37] The application provides novel compositions and methods for improving gut microbiota population. The application identifies bacteria populations that are closely related to butyrate production and compositions and methods for increasing those colonic bacterial populations.

[38] In view of the present disclosure, the methods and compositions described herein can be configured by the person of ordinary skill in the art to meet the desired need. In general, the disclosed methods and compositions provide improvements in gut microbiota population.

[39] For example, in certain aspects, the methods of the disclosure increase one or more colonic bacteria populations that are capable of fermentation and short chain fatty acid production.

[40] In one aspect, the application provides methods for improving gut microbiota population. In one embodiment, the method may include selectively enriching a first gut microbiota population. The first gut microbiota population may include, for example, a short-chain fatty acid (SCFA)-producing bacterium.

[41] In one embodiment, the method may include the step of administering to a subject a composition. The composition may be an oral or parenteral formulation. The composition may selectively increase one or more populations of gut microbiota.

[42] For example, in certain embodiments of the methods and compositions described herein, administering a composition comprising allulose, erythritol or a combination thereof, improves the gut microbiota population in a subject. In particular, the methods and compositions described herein increase the population of one or more colonic bacteria populations, each selected from the families Lachnospiraceae or Eubacteriaceae, and any combination thereof. For example, in one embodiment of the methods and compositions described herein, administering allulose, erythritol or a combination thereof increases the population of Lachnospiraceae. In another embodiment of the methods and compositions described herein, administering allulose, erythritol or a combination thereof increases the population of Eubacteriaceae. In another embodiment of the methods and compositions described herein, administering allulose, erythritol or a combination thereof increases the population of one or more colonic bacteria populations, each selected from the genera Anaerostipes, Blautia, Anaerobutyricum, and Eubacterium, and any combination thereof. In another embodiment of the methods and compositions described herein, administering allulose, erythritol or a combination thereof increases the population of one or more colonic bacteria populations, each selected from the species Anaerostipes hadrus, Blautia obeum, Anaerobutyricum hallii, Eubacterium hallii, Eubacterium rectale, Eubacterium callanderi, Eubacterium limosum and Eubacterium maltosivorans, and any combination thereof.

[43] The one or more colonic bacteria may be selected from any selected from the families Bifidobacteriaceae, Coriobacteraceae, Eggerthellaceae, Bacteroidaceae, Barnesiellaceae, Prevotellaceae, Rikenellaceae, Tannerellaceae, Acidaminococcaceae, Clostridia_u_f, Clostridiaceae, Clostridiales_u_f, Erysopelotrichaceae, Eubacteriaceae, Firmicutes_u_f, Lachnospiraceae, Oscillospiraceae, Ruminococcaceae, Veillonellaceae, Burkholderialies_u_f, Desulfovibrionaceae, and Akkermannsiaceae. The one or more colonic bacteria populations may preferably be any selected from the families Lachnospiraceae or Eubacteriaceae. For instance, the one or more colonic bacteria may be any selected from the genera Abyssivirga, Acetatifactor, Acetitomaculum, Aequitasia, Agathobacter, Alitiscatomonas, Anaerobium, Anaerobutyricum, Anaerocolumna, Anaeromicropila, Anaeropeptidovorans, Anaerosacchariphilus, Anaerosporobacter, Anaerostipes, Anaerotaenia, Anaerotignum, Anthropogastromicrobium, Bariatricus, Blautia, Bilifractor, Brotolimicola, Brotonthovivens, Butyribacter, Butyrivibrio, Caecibacterium, Catenibacillus, Catonella, Cellulosilyticum, Chordicocus, Coprococcus, Cuneatibacter, Diplocoster, Dorea, Eisenbergiella, Enterocloster, Eubacterium, Extibacter, Faecalicatena, Faecalimonas, Falcatimonas, Frisingicoccus, Fusicatenibacter, Fusimonas, Gallinestinimicrobium, Gluceribacter, Herbinix, Hespellia, Hominisplanchenecus, Hoministercoradaptatus, Howardella, Jingyaoa, Johnsonella, Jutongia, Kineothrix, Konateibacter, Lachnoanaerobaculum, Lachnobacterium, Lachnoclostridium, Lachnospira, Lachnotalea, Lacrimispora, Laedolimicola, Lientehia, Luxibacter, Marvinbryantia, Mediterraneibacter, Merdimonas, Mobilisporobacter, Mobilitalea, Moryella, Muricomes, Muricoprocola, Murimonas, Niameybacter, Novisyntrophococcus, Natranaerovirga, Ohessyouella, Oliverpabstia, Ori bacterium, Otoolea, Parablautia, Parasporobacterium, Petralouisia, Porcincola, Pseudobutyrivibrio, Qiania, Robinsoniella, Roseburia, Schaedlerella, Sellimonas, Shuttleworthia, Simiaoa, Sporobacterium, Sporofaciens, Stomatobaculum, Suilimivivens, Suipraeoptans, Suonthocola, Syntrophococcus, Tyzzerella, Variimorphobacter, Velocimicrobium, Waltera, Wansuia, Wujia, Zhenhengia, Acetobacterium, Alkalibacter, Alkalibaculum, Aminicella, Anaerofustis, Eubacterium, Gallibacter, Garciella, Intestinibacillus, Irregularibacter, Mogibacterium, Pseudorami bacter, Rhabdanaerobium and combinations thereof.

[44] In certain embodiments, the one or more colonic bacteria populations includes one selected from the genus Anaerostipes, for example A. amylophilus, A. butyraticus, A. caccae, A. faecalis, A. faecis, A. hadrus, A. hominis, A. rhamnosivorans, and combinations thereof.

[45] In certain embodiments, the one or more colonic bacteria populations includes one selected from the genus Blautia, for example B. acetigignens, B. ammoniilytica, B. argi, B. brookingsii, B. caecimuris, B. celeris, B. coccoides, B. difficilis, B. faecicola, B. faecis, B. glucerasea, B. hansenii, B. hominis, B. hydrogenotrophica, B. intestinalis, B. lenta, B. liquoris, B. luti, B. marasmi, B. massiliensis, B. obeum, B. phocaeensis, B. producta, B. provencensis, B. pseudococcoides, B. schinkii, B. segnis, B. stercoris, B. tarda, B. wexlerae, and combinations thereof.

[46] In certain embodiments, the one or more colonic bacteria populations includes one selected from the genus Anaerobutyricum, for example A. butyricum, A. hallii, A soehngenii, A. faecale, A stercoripullorum, A. stercosis, and combinations thereof.

[47] In certain embodiments, the one or more colonic bacteria populations includes one selected from the genus Eubacterium, for example Eubacterium acidaminophilum, Eubacterium aggregans, Eubacterium albensis, Eubacterium angustum, Eubacterium barkeri, Eubacterium brachy, Eubacterium budayi, Eubacterium callanderi, Eubacterium cellulosolvens, Eubacterium combesii, Eubacterium coprostanoligenes, Eubacterium difficile, Eubacterium dolichum, Eubacterium eligens, Eubacterium hallii (now renamed A. hallii), Eubacterium homisis, Eubacterium infirmum, Eubacterium limosum, Eubacterium maltosivorans, Eubacterium minutum, Eubacterium multiforme, Eubacterium nitritogenes. Eubacterium nodatum, Eubacterium oxidoreducens, Eubacterium plexicaudatum, Eubacterium pyruvativorans, Eubacterium ramulus, Eubacterium rangiferina, Eubacterium rectale, Eubacterium ruminantium, Eubacterium saphenum, Eubacterium segne, Eubacterium siraeum, Eubacterium sulci, Eubacterium tarantellae, Eubacterium tenue, Eubacterium thermomarinus, Eubacterium tortuosum, Eubacterium uniforme, Eubacterium ventriosum, Eubacterium xylanophilum, Eubacterium yurii, and combinations thereof.

[48] In certain embodiments, the one or more colonic bacteria populations includes one selected from the phyla Actinobacteria, Bacteroidetes, and Firmicutes.

[49] In certain embodiments, the one or more colonic bacteria populations includes one or more selected from the species Dysmosmobacter weibionis, Oscillospiraceae_u_s, Oscillibacter u_s, Clostridiaceae u_s, Lachnospiraceae_u_s, Anaesrostipes hadrus, Collinsella_u_s, Alistipes onderdonkii, Blautia wexlerae, Bacteroides xylanisolvens, Collinsella aerofaciens. Alistipes finegoldii, Enterocloster aldensis, Bacteroides_u_s, Enterocloser clorstridioformis, Enterocloster bolteae, Dorea longicatena, Blautia massiliensis, Eubacterium_u_s, Blautia obeum, Blautia_u_s, Faecalibacterium prasunitzii, Subdoligranulum_u_s, and Ruminicoccus_u_s. In certain such embodiments, the method or composition comprises allulose.

[50] In certain embodiments, the one or more colonic bacteria populations includes one or more selected from the species Blautia massiliensis, Alistipes finegoldii, Lawsonibacter asaccharolyticus, Dorea longicatena Faecalibacterium_u_s, Subdoligranulum_u_s, Anaerostipes hadrus, Alistipes onderdonkii, Collinsella_u_s, Blautia wexlerae, Lachnospiraceae_u_s, Bifidobacterium longum, Enterocloster bolteae, Enterocloster clostridioformis, Bacteroides xylanisolvens, Bacteroides_u_s, Enterocloster aldensis, Eubacterium_u_s, Clostribium_u_s, and Alistipes putredinis. In certain such embodiments, the method or composition comprises erythritol.

[51] In certain embodiments of the methods and compositions described herein, administering a composition comprising allulose improves the gut microbiota population in a subject. In particular, the methods and compositions described herein increase the population of one or more colonic bacteria populations, each selected from the families Lachnospiraceae, and any combination thereof. For example, in one embodiment of the methods and compositions described herein, administering allulose, erythritol or a combination thereof increases the population of Lachnospiraceae. In another embodiment of the methods and compositions described herein, administering allulose, erythritol or a combination thereof increases the population of one or more colonic bacteria populations, each selected from the genera Anaerostipes, Blautia, and Anaerobutyhcum, and any combination thereof. In another embodiment of the methods and compositions described herein, administering allulose, erythritol or a combination thereof increases the population of one or more colonic bacteria populations, each selected from the species Anaerostipes hadrus, Blautia obeum, and Anaerobutyhcum hallii, and any combination thereof.

[52] In certain embodiments of the methods and compositions described herein, administering a composition comprising erythritol improves the gut microbiota population in a subject. In particular, the methods and compositions described herein increase the population of one or more colonic bacteria populations, each selected from the families Eubacteriaceae, and any combination thereof. In another embodiment of the methods and compositions described herein, administering allulose, erythritol or a combination thereof increases the population of Eubacteriaceae. In another embodiment of the methods and compositions described herein, administering allulose, erythritol or a combination thereof increases the population of one or more colonic bacteria populations, each selected from the genus Eubacterium. In another embodiment of the methods and compositions described herein, administering allulose, erythritol or a combination thereof increases the population of one or more colonic bacteria populations, each selected from the species Eubactehum hallii, Eubacterium rectale, Eubacterium callanderi, Eubacterium limosum and Eubacterium maltosivorans.

[53] In certain embodiments of the methods and compositions described herein, administering a composition comprising combination of allulose and erythritol improves the gut microbiota population in a subject. In particular, the methods and compositions described herein increase the population of one or more colonic bacteria populations, each selected from the families Lachnospiraceae or Eubacteriaceae, and any combination thereof. For example, in one embodiment of the methods and compositions described herein, administering allulose, erythritol or a combination thereof increases the population of Lachnospiraceae. In another embodiment of the methods and compositions described herein, administering allulose, erythritol or a combination thereof increases the population of Eubacteriaceae. In another embodiment of the methods and compositions described herein, administering allulose, erythritol or a combination thereof increases the population of one or more colonic bacteria populations, each selected from the genera Anaerostipes, Blautia, Anaerobutyricum, and Eubacterium, and any combination thereof. In another embodiment of the methods and compositions described herein, administering allulose, erythritol or a combination thereof increases the population of one or more colonic bacteria populations, each selected from the species Anaerostipes hadrus, Blautia obeum, Anaerobutyricum hallii, Eubacterium hallii, Eubacterium rectale, Eubacterium callanderi, Eubacterium limosum and Eubacterium maltosivorans, and any combination thereof. In preferred embodiments, of the methods and compositions described herein, more than one (i.e. two or more, three or more, etc.) colonic bacteria populations, each selected from the families Lachnospiraceae or Eubacteriaceae, the genera Anaerostipes, Blautia, Anaerobutyricum, and Eubacterium, and the species Anaerostipes hadrus, Blautia obeum, Anaerobutyricum hallii (formerly Eubacterium hallii), Eubacterium rectale, Eubacterium callanderi, Eubacterium limosum and Eubacterium maltosivorans are increased.

[54] The improving of the gut microbiota and / or the increasing one or more bacterial populations selected from the families Lachnospiraceae or Eubacteriaceae may result in the prevention, treatment or amelioration of a disease including, without limitation, metabolic syndrome, obesity, type 2 diabetes (T2D), insulin resistance, hyperlipoproteinemia, hyperuricemia, hepatic steatosis, hypercholesterolemia, hypertriglyceridemia, irritable bowel syndrome (IBS), colon cancer, allergy, non-alcoholic fatty liver diseases (NAFLD), inflammatory bowel disease (IBD), cardiovascular disease (CVD), inflammation, and other disorders.

[55] In certain embodiments of the methods and compositions described herein, one or more of the colonic bacteria populations (e.g., as described above) are increased by at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 80%, or even at least about 100% as compared to a non-treated subject. In certain such embodiments, the colonic bacteria population is increased by no more than about 500%. In other such embodiments, the colonic bacteria population is increased by no more than about 400%. In other such embodiments, the colonic bacteria population is increased by no more than about 300%. In other such embodiments, the colonic bacteria population is increased by no more than about 200%. In other such embodiments, the colonic bacteria population is increased by no more than about 100%. In certain embodiments of the methods and compositions described herein, each of the one or more of the colonic bacteria populations (e.g., as described above) are increased by at least about 5%, at least about 10%, at least about 20%, at least about 50%, or even at least about 100% as compared to a non-treated subject. This means that there are instances where each of these bacteria may be affected independently of each other at different rates (e.g., one bacteria may increase by 50% in population, whereas another bacteria may only increase 25%). In certain such embodiments, each colonic bacteria population is increased by no more than about 500%. In other such embodiments, each colonic bacteria population is increased by no more than about 400%. In other such embodiments, each colonic bacteria population is increased by no more than about 300%. In other such embodiments, each colonic bacteria population is increased by no more than about 200%. In other such embodiments, each colonic bacteria population is increased by no more than about 100%.

[56] In certain embodiments of the methods and compositions described herein, the proportion of one or more of the colonic bacteria populations (e.g., as described above) as a percentage of total colonic bacteria is increased by at least about 20%, at least about 25%, at least about 50%, at least about 100%, at least about 200% or even at least about 300% as compared to a non-treated subject. In certain such embodiments, the proportion of one or more of the colonic bacteria populations as a percentage of total colonic bacteria is increased by no more than about 700%. In other such embodiments, the proportion of one or more of the colonic bacteria populations as a percentage of total colonic bacteria is increased by no more than about 600%. In other such embodiments, the proportion of one or more of the colonic bacteria populations as a percentage of total colonic bacteria is increased by no more than about 500%. In other such embodiments, the proportion of one or more of the colonic bacteria populations as a percentage of total colonic bacteria is increased by no more than about 400%. In certain embodiments of the methods and compositions described herein, the proportion (i.e., as a percentage of total colonic bacteria) of each of the one or more of the colonic bacteria populations (e.g., as described above) is increased by at least about 20%, at least about 25%, at least about 50%, at least about 100%, at least about 200% or even at least about 300% as compared to a non-treated subject. This means that there are instances where each of these bacteria may be affected independently of each other at different rates (e.g., one bacteria population may increase by 50% in proportion, whereas another bacteria population may only increase 25%). In certain such embodiments, each proportion is increased by no more than about 500%. In other such embodiments, each proportion is increased by no more than about 400%. In other such embodiments, each proportion is increased by no more than about 300%. In other such embodiments, each proportion is increased by no more than about 200%. In other such embodiments, each proportion is increased by no more than about 100%.

[57] In certain embodiments of the methods and compositions described herein, the allulose, erythritol or the combination thereof is administered at a rate of at least about 0.1 g / day. For example, in certain embodiments the allulose, erythritol or the combination thereof is administered at a rate of at least about 0.3 g / day, at least about 0.5 g / day, at least about 0.7 g / day, at least about 1.0 g / day, at least about 1.2 g / day, at least about 1.3 g / day, at least about 1.5 g / day, at least about 2.0 g / day, at least 3.0 g / day, at least 5.0 g / day, at least 7 g / day, at least 10 g / day, at least 12 g / day, at least 13 g / day, at least 15 g / day, or even at least 20 g / day and no more than about 100 g / day, or no more than 75 g / day, perhaps no more than 25 g / day, 10 g / day or even 5.0 g / day .

[58] For example, in certain embodiments of the methods and compositions described herein, the allulose, erythritol or the combination thereof is administered at a rate in the range of about 3 g / day to about 100 g / day. In other embodiments of the methods and compositions described herein, the allulose, erythritol or the combination thereof is administered at a rate in the range of about 0.1 g / day to about 35 g / day, or about 0.12 g / day to about 25 g / day. In other embodiments of the methods and compositions described herein, the allulose, erythritol or the combination thereof is administered at a rate in the range of about 0.5 to about 6.5 g / day, about 0.5 to about 4.0 g / day, about 0.5 to about 3.0 g / day, about 0.5 to about 2.0 g / day, about 1.0 to about 6.5 g / day, about 1.0 to about 4.0 g / day, about 1.0 to about 3.0 g / day, about 1.5 to about 6.5 g / day, about 1.5 to about 4.0 g / day, about 1.5 to about 3.0 g / day, about 0.5 to about 1.5 g / day, about 0.7 to about 1.5 g / day, about 0.9 to about 1.5 g / day, or about 1.0 to about 1.5 g / day, about 1.2 to about 2.0 g / day, about 1.3 to about 2.0 g / day, about 1.4 to about 2.0 g / day, about 1.5 to about 2.0 g / day, about 1.6 to about 2.0 g / day, about 1.7 to about 2.0 g / day, about 1.8 to about 2.0 g / day, or about 1.9 to about 2.0 g / day. In certain embodiments of the methods and compositions described herein, the allulose, erythritol or the combination thereof is administered at a rate of about 0.1 g / day, 0.3 g / day, 0.5 g / day, 0.7 g / day, 1.0 g / day, 2.0 g / day, 3.0 g / day, 4.0 g / day, 5 g / day, about 6 g / day, about 7 g / day, about 8 g / day, about 9 g / day, or about 10 g / day. In other embodiments of the methods and compositions described herein, the allulose, erythritol or the combination thereof is administered at a rate in the range of about 11 to about 20 g / day. In other embodiments of the methods and compositions described herein, the allulose, erythritol or the combination thereof is administered at a rate of about 11 g / day, or about 12 g / day, or about 13 g / day, or about 14 g / day, or about 15 g / day, or about 16 g / day, or about 17 g / day, or about 18 g / day, or about 19 g / day, or about 20 g / day.

[59] In a given day, the administration can be broken up into any number of dosages. For example, in one embodiment of the methods and compositions described herein, the allulose, erythritol or the combination thereof is administered once per day (e.g., in a single serving). In other embodiments of the methods and compositions described herein, the allulose, erythritol or the combination thereof is administered a plurality of times a day, for example, twice per day or three times per day (e.g., in a plurality of servings, for example, in two servings or in three servings per day). When a plurality of administrations or servings is to be used, the amounts per day described above can be divided among the number of administrations or servings to provide acceptable amounts per serving that are well tolerated (i.e., does not cause severe bloating, flatulence, stomach noises, abdominal cramps, diarrhoea, nausea, and / or vomiting).

[60] In another embodiment, the disclosure provides a method of increasing one or more colonic bacteria populations and increasing SCFA (e.g. butyrate) production in a subject, where the method includes orally administering allulose, erythritol or the combination thereof. The administration can, in certain embodiments, be as otherwise described herein.

[61] To improve the gut microbiota population, the compositions may be administered orally or parentally. In one embodiment, the composition may be a food, a drink, a supplement, or a pharmaceutical formulation. In one embodiment, the composition may be in the form of suppository, tablet, pill, granule, powder, film, microcapsule, aerosol, spirit, tincture, tonic, liquid suspension, or syrup. The composition may be administered at a dosage of from about 5.0 mg / kg body weight to about 40.0 mg / body weight.

[62] In certain embodiments of the methods and compositions described herein, the subject is a mammal. In one embodiment of the methods and compositions described herein, the subject is a human, for example, a non-adult human (e.g., in the range of about 2 years old to about 20 years old, or about 13 years old to about 19 years old), or an older human (e.g., at least about 45 years old, at least about 50 years old, at least about 60 years old, at least about 70 years old, at least about 80 years old or even at least about 90 years old, especially an older female human). Accordingly, in certain embodiments the methods and compositions described herein can be used with subjects who are especially likely to benefit from increased SCFA (e.g. butyrate) production (e.g., type-2 diabetic individuals).

[63] In certain embodiments of the methods and compositions described herein, butyrate production is increased by at least about 0.5% as compared to a non-treated subject. In certain embodiments of the methods and compositions described herein, butyrate production is increased by at least about 1%, at least about 2%, at least about 3%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, or at least about 14%, or at least about 15%, as compared to the non-treated subject. In other embodiments of the methods and compositions described herein, butyrate production is increased by at least about 20%, or at least about 25% as compared to a non-treated subject. In certain embodiments of the methods and compositions described herein, butyrate production is increased by at least about 20%, at least about 25%, at least about 30%, or at least about 35% as compared to a non-treated subject. In certain such embodiments, butyrate production is increased by no more than about 200% as compared to a non-treated subject. In other such embodiments, butyrate production is increased by no more than about 100% as compared to a non-treated subject. In other such embodiments, butyrate production is increased by no more than about 50% as compared to a non-treated subject. The timing for butyrate production can be, for example, in the range of 0-24 h, 24-48 h, 48-72 h. In preferred embodiments, the increase in butyrate production is in the range of 0-48 hours after administration.

[64] It is envisioned that the effects of improving gut microbiota population, increasing one or more colonic bacteria populations and / or increasing butyrate production relate to both humans and animals, and thus can be applied to foodstuffs and animal feed. Representative non-human animals include, livestock, such as horse, chicken, turkey, cattle, cow, swine, sheep, goat, llama and bison, cats and dog, rodent, rabbit, hamster and bird.

[65] The administration can be performed over an extended time period, for example, over the course of at least about a week, over the course of at least about two weeks, of at least about three weeks over the course of at least about four weeks, of at least about seven weeks or even over the course of at least about 52 weeks. The person of ordinary skill in the art that in such long-term administrations, days of administration may be "missed"; desirably the number of days missed is less than about 10% of the total number of days over which the administration is performed.

[66] Another embodiment of the invention is a composition that includes at least about 0.1 g of allulose, erythritol or a combination thereof per serving. For example, certain embodiments of compositions as described herein include at least about 0.25 g, at least about 0.5 g, at least about 1 g, at least about 2 g, at least about 3 g, at least about 4 g, at least about 5 g, at least about 6 g, at least about 8 g, at least about 10 g, or even at least about 20 g of allulose, erythritol or a combination thereof per serving. In certain such embodiments, the composition includes no more than 100 g, no more than about 50 g, or even no more than about 40 g of allulose, erythritol or a combination thereof per serving. The compositions can, for example, be provided as food compositions as described below. In other embodiments, a composition is provided as a nutritional supplement. Such compositions can be useful in performing the methods described herein.

[67] In certain such embodiments of the compositions as described herein, the size of the serving can be, for example, at least about 75 g, at least about 150 g, or even at least about 200 g. In certain embodiments, the size of the serving is no more than about 1000 g, or even no more than about 500 g. For example, in one embodiment, the serving size in the range of about 75 mL to about 1000 mL. In certain embodiments, each serving is separately packaged. In other embodiments, multiple servings are packaged together, and provided with instructions relating a serving size and / or an amount of allulose, erythritol or a combination thereof per serving as described herein.

[68] Another embodiment of the invention is a composition that includes allulose, erythritol or a combination thereof in an amount of at least about 0.1%, at least about 0.25%, at least about 0.5%, at least about 1%, at least about 2.5%, at least about 3%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, or even at least about 40% by weight. However, in certain such embodiments, the composition has a maximum amount of allulose, erythritol or a combination thereof that is no greater than about 75%, or even no greater than about 50% by weight. The compositions can, for example, be provided as food compositions as described below. The compositions can, for example, be provided with the serving sizes and / or the amounts of allulose, erythritol or combination thereof per serving as described herein.

[69] Another embodiment of the invention is a composition that includes one or more (e.g., two or more, or three or more) bacterial populations, each from a family selected from the group consisting of Lachnospiraceae and Eubacteriaceae, from a genus selected from the group consisting of Anaerostipes, Blautia, and Anaerobutyricum, or from a species selected from the group consisting of species Anaerostipes hadrus, Blautia obeum, Anaerobutyricum hallii (formerly Eubacterium hallii), Eubacterium rectale, Eubacterium callanderi, Eubacterium limosum and Eubacterium maltosivorans. One or more of the bacterial populations, can, for example, act as probiotics. The compositions described herein can, in certain embodiments, include allulose, erythritol or a combination (for example, in an amount as described above). But in other embodiments, the composition does not include allulose, erythritol or a combination thereof. Such embodiments can be useful, for example, for addition to or coadministration with compositions including allulose, erythritol or a combination, such that the bacterial populations of the composition are present in the colon at the same time as the allulose, erythritol or the combination. Accordingly, the subject can in certain embodiments enjoy the benefits of the combination of allulose, erythritol or the combination with the bacterial populations identified herein without being administered a single composition that includes both the allulose, erythritol or the combination therefore and the bacterial populations. Similarly, products suitable for the enjoyment the benefits of the combination of the allulose, erythritol or the combination thereof with the bacterial populations identified herein can be formulated that do not include both the allulose, erythritol or the combination thereof and the bacterial populations.

[70] For example, in certain embodiments of the compositions described herein, the composition includes one or more (e.g., two or more, or three or more) bacteria populations, each from a species (e.g., each from a different genus) selected from the families Lachnospiraceae and Eubacteriaceae, the genera Anaerostipes, Blautia, and Anaerobutyricum, or from a species Anaerostipes hadrus, Blautia obeum, Anaerobutyricum hallii (formerly Eubacterium hallii), Eubacterium rectale, Eubacterium callanderi, Eubacterium limosum and Eubacterium maltosivorans, and any combination thereof.

[71] In embodiments of the invention, administering to the subject a composition comprising allulose increases one or both colonic bacterial populations selected from the species A. hadrus and B. obeum.

[72] In embodiments of the invention, administering to the subject a composition comprising erythritol increases one or more colonic bacterial populations selected from the species A. hallii, E. callendari, E. limosum, .and E maltosivorans.

[73] In embodiments of the invention, administering to the subject a composition comprising allulose and erythritol increases one or both colonic bacterial populations selected from the species A hadrus and B. obeum and one or more colonic bacterial populations selected from the species A. hallii, E. rectale, E. callendari, E. limosum, .and E maltosivorans.

[74] Compositions including both the prebiotic (allulose, erythritol or combination thereof) and the probiotic (one or more (e.g., two or more, or three or more) bacteria populations) are known as synbiotic compositions. The synbiotic products of the present invention are novel combinations of probiotic live bacteria and prebiotic compounds that are believed to achieve a synergistic effect. In certain embodiments of the present invention, a symbiotic product is a combination of at least one bacteria selected from the families Lachnospiraceae and Eubacteriaceae and allulose, erythritol or a combination thereof. Such synbiotic products can be used as ingredients in foodstuffs and animal feed.

[75] In certain embodiments, the synbiotic product has less than about 10% moisture content. In certain embodiments, the synbiotic product has less than about 9% moisture content. In certain embodiments, the synbiotic product has less than about 8% moisture content. In certain embodiments, the synbiotic product has less than about 7% moisture content. In certain embodiments, the synbiotic product has less than about 6% moisture content. In certain embodiments, the synbiotic product has less than about 5% moisture content. In certain embodiments, the synbiotic product has from about 1% to about 10% moisture content. In certain embodiments, the synbiotic product has from about 5% to about 10% moisture content. As used herein, moisture content is measured by ASTM standard D6869 American Society for testing and materials: "Karl Fisher" titration.

[76] The synbiotic product composition can be prepared by blending an amount of a dry spore component with an amount of a dry prebiotic component. Alternatively, liquid forms of prebiotic components can be mixed with liquid forms of probiotic spore components and the mixture can bedded, such as by spray drying. One of skill in the art will recognise other alternative methods common to commercial manufacture are practical and useful.

[77] Of course, as the person of ordinary skill in the art will appreciate, the compositions including specific combinations of bacteria populations as described herein can further include other bacteria populations, either as elsewhere described herein or otherwise. For example, the compositions can further include one or more bacteria populations selected from the genera Bacteroides, Butyricicoccus, Oscillibacter, Dialister, Parabacteroides, Alistipes, Anaerococcus, Catenibacterium, Clostridiales, Lactobacillus, Bifidobacterium, Ruminococcaceae, and Akkermansia.

[78] The compositions can, for example, be provided as food compositions as described below. In other embodiments, a composition is provided as a nutritional supplement. In still other embodiments, a composition is provided as an ingredient to be mixed with a food composition, for example, during processing or cooking, or at the time of serving or eating. The compositions can, for example, be provided with the allulose, erythritol or combination thereof, concentrations, the serving sizes and / or the amounts of allulose, erythritol or combination thereof per serving as described herein. The amount of the bacterial populations added to the composition may be adjusted by the person of skill in the art to meet the desired need. In general, each of the bacterial populations may be in the amount of about 1 xio3 to about 1xio10 CFU (colony-forming unit). In certain embodiments, the bacterial population is in the amount of about 1xio5 to about 1xio10 CFU, or about 1xio6 to about 1xio10 CFU, or about 1x107 to about 1x1010CFU, or about 1x108 to about 1x101°CFU, or about 1xio3 to about 1xio8 CFU, or about 1x104 to about 1xio8 CFU, or about 1xio5 to about 1x-|08 CFU, or about 1xio6 to about 1x108 CFU, or about 1x105 to about 1x107 CFU, or about 1x104 CFU, or about 1xio5 CFU, or about 1x106 CFU, or about 1x107 CFU or about 1x108CFU, or about 1x109 CFU, or about 1x101°CFU.

[79] The synbiotic products of the invention may be used as an ingredient or component of food products such as human foodstuffs or animal feed. Such food products contain an amount of the synbiotic product such that the food products can be consumed in amount that provides an effective amount of the synbiotic product. Incorporation of ingredients into food products is known in the art and will vary according to the type of food product prepared. Although there are certain food products that have been traditionally marketed as containing prebiotics or probiotics, the types of food products contemplated herein are not limited to a particular type of food product. Representative, non-limiting examples of certain embodiments of food products containing the synbiotic product include supplements, nutritional bars, eternal clinical nutrition preparations, baby food, and coatings for dry foodstuffs, such as coating bakery goods or chewing gum.

[80] In embodiments of the present invention, the compositions are preferably edible compositions.

[81] Another embodiment of the invention is a composition as described above that further includes one or more mineral species. Each mineral species can, for example, be a divalent mineral species, or a species selected from a calcium species, a magnesium species, a copper species, a potassium species, a zinc species and an iron species. For example, in one embodiment, the composition includes calcium. In another embodiment, the composition includes calcium and / or magnesium. In another embodiment, the composition includes calcium, magnesium, and / or iron. The mineral species can be provided, for example, as a salt, such as a carbonate salt, a halide salt, or a bicarbonate salt. Calcium, for example, can be provided as, e.g., calcium carbonate or calcium gluconate. The mineral (e.g., the calcium) can be provided, for example, at an amount of at least about 50 mg per dose or serving, at least about 100 mg per dose or serving, at least about 250 mg per dose or serving, at least about 500 mg per dose or serving, or even at least about 1000 mg per dose or serving. In certain such embodiments, the calcium is included at less than about 2000 mg per dose or serving or even less than about 1000 mg per dose or serving. The compositions can, for example, be provided as food compositions as described below. In other embodiments, a composition is provided as a nutritional supplement. The compositions can, for example, be provided with the allulose, erythritol or combination thereof concentrations, the serving sizes and / or the amounts of allulose, erythritol or combination thereof per serving as described herein.

[82] In other embodiments, the composition of the disclosure does not include a mineral species as described above.

[83] Another embodiment of the invention is a composition as described above that further includes one or more additional prebiotics. Examples of prebiotics include, but are not limited to, inulin, lactulose, fructooligosaccharide, galactooligosaccharide, mannooligosaccharide, larch arabinogalactan, xylooligosaccharide, polydextrose, resistant maltodextrin, and tagatose. In certain embodiments, the disclosure provides compositions as described above, wherein the prebiotic is in the range of 0.025 g to 30 g. In certain embodiments, the prebiotic is in the amount of about 0.1 to about 20 g, or about 1 to about 10 g, or about 0.1 to about 5 g, or about 1 to about 5 g, or about 5 to about 10 g, or about 5 to about 8 g, or about 2 to about 8 g, or about 2 to about 5 g, or about 2 to about 8 g, or about 0.05 g, or about 0.1 g, or about 1 g, or about 2 g or about5 g, or about 8 g, or about 10 g.

[84] In one embodiment, the composition of the disclosure does not include one or more additional prebiotics as described above. For example, in one embodiment, the compositions of the disclosure do not include one or more of the prebiotics selected from the group consisting of inulin, lactulose, fructooligosaccharide, galactooligosaccharide, mannooligosaccharide, larch arabinogalactan, xylooligosaccharide, polydextrose, resistant maltodextrin, and tagatose. In another embodiment, the compositions of the disclosure does not include inulin. In yet another embodiment, the compositions of the disclosure does not include pullulan.

[85] As the person of skill in the art will appreciate, the compositions described herein can be used in practicing the methods described elsewhere herein.

[86] The terms "edible" and "edible composition" are used in a broad sense herein to include a variety of substances that can be ingested by humans, such as food, beverages and medicinal and nutritional supplement dosage forms such as syrups, powders, capsules or tablets. The terms “food” and “food composition” are used more narrowly to mean foods and beverages and ingredients therefor. Suitable food compositions can be in a variety of forms including, but are not limited to baked foods, breakfast cereal, dairy products, soy products, confections, jams and jellies, beverages (powdered and / or liquid), shakes, fillings, yogurts (dairy and nondairy yogurts), kefirs, extruded and sheeted snacks, gelatin desserts, snack bars, meal replacement and energy bars, cheese and cheese sauces (dairy and non-dairy cheeses), edible and water-soluble films, soups, syrups, table top sweeteners, nutritional supplements, sauces, dressings, creamers, icings, ice cream, frostings, glazes, pet food, tortillas, meat and fish, dried fruit, infant and toddler food, and batters and breadings.

[87] In order to make the food product suitable for use as a flavor enhancer in food compositions, in many cases it will be desirable for it also to include a natural and artificial flavors. Suitable examples of such flavors include apple, citrus, grape, orange, cherry, lemon, lime, vanilla, peach, peanut butter, pineapple, pomegranate, blueberry, raspberry, blackberry, jasmine, lavender, mint, strawberry, banana, mango, passion fruit, dragon fruit, kiwi, chocolate, maple, rum, butter, and combinations thereof.

[88] In certain embodiments, the composition is in the form of an agglomerated powder, for example, like those used in making powdered drinks and nutritional supplements.

[89] In order to make the food product suitable for use as a sweetener composition in food, in many cases it will be desirable for it also to include a non-nutritive high-intensity sweetener. Suitable examples of such non-nutritive high-intensity sweeteners include, but are not limited to sucralose, acesulfame potassium, aspartame, monkfruit, Stevia, and combinations thereof.

[90] The person of ordinary skill in the art will appreciate that the allulose, erythritol or combination thereof can be provided in any of several different physical forms, such as powder, granules, agglomerated powder, syrup or concentrated syrup solids. In one embodiment, allulose, erythritol or combination thereof is in particulate form. The particulates can be held together by a binder, such as a binder composition that comprises a major amount of maltodextrin.

[91] Examples Example 1 - Investigation of the interplay of non-nutritive sweeteners with gut microbiota

[92] Allulose (DOLCIA PRIMA® DS NG Crystalline) and erythritol (ERYTESSETM 20-60M Erythritol) were subjected to simulations of human oral, gastric and small intestinal digestion procedures. To test these products at doses that are as representative as possible for the in vivo situation, several criteria were considered including the maximum use levels, estimated daily intake, acceptable daily intakes (ADIs) and gastrointestinal tolerance. This allowed selection of representative in vivo oral test doses of 5g / d. To further enhance the biorelevance of the test doses, the in vivo oral dose was then corrected for the fraction of each test product that is absorbed along the upper gastrointestinal tract in vivo, thus allowing calculation of the fraction of the oral dose that reaches the colon (= ex vivo colonic test doses). Such absorption levels were 80% for allulose and 90% for erythritol.

[93] Fig. 6 summarises the experimental design setup • (A) Both test products were subjected to 48h colonic incubations and compared to untreated test arms (NSC). • (B) Sampling and analysis performed to assess the impact of the test products on metabolite production and microbial composition of T2D subjects and co-living healthy human adults (n = 6 per group) (from Van den Abbeele et al. 2023). 1.1 Oral, gastric and intestinal simulation

[94] Digestion in the upper GIT and colonic fermentation of D-allulose and erythritol were examined using the SIFR® model. D-allulose and erythritol were exposed to oral, gastric, and small intestinal digestion. The oral, gastric and small intestinal digestion procedures were simulated according to the latest standardised INFOGEST 2.0 method (Brodkorb etal. 2019) with modifications to ensure that this method was compatible with the subsequent colonic fermentation.

[95] The INFOGEST 2.0 method is a static digestion method that uses constant ratios of meal to digestive fluids and a constant pH for each step of digestion. Samples are subjected to sequential oral, gastric and intestinal digestion while parameters such as electrolytes, enzymes, bile, dilution, pH and time of digestion are based on available physiological data. Small modifications were implemented to this protocol, i.e., an implementation of six enzyme assays (amylase, pepsin activity, lipase, trypsin, and chymotrypsin) together with an assay to quantify bile acids, and the removal of oxygen along the small intestinal incubation, to tailor the method to the further colonic fermentation experiments. 1.2 Dose selection

[96] Dose selection was based on a literature review performed prior to the study to identify in vivo and ex vivo test doses that are representative of real-world usage and intake, and were physiologically relevant. To identify the in vivo oral doses, the following criteria were considered: maximum use levels set by ESFA and FDA, adult estimated daily intake, acceptable daily intake (Joint FAO / WHO Expert Committee on Food Additives), and gastrointestinal tolerance (Table 1). Therefore representative in vivo oral test doses were selected (g / day; Table 2). The in vivo oral dose was adjusted for the portion of D-allulose and erythritol absorbed in the upper GIT to calculate the proportion of the oral dose that reaches the colon, which determined the ex vivo colonic test dose (Table 2). For D-allulose, previous studies indicate urinary excretion rates ranging from 66-86% after ingestion of doses between 5-20g / day (Iida et al. 2010; Williamson et al. 2014). Accordingly, this study assumed that 80% of D-allulose is absorbed in the upper GIT, resulting in 20% reaching the colon. For erythritol, previous studies indicate urinary excretion rates ranging from 78%-90% after ingestion of doses between 18g-60g / day (Noda et al. 1994; Bornetetal. 1996). Toxicokinetic studies included in EFSA’s scientific opinion of the safety of erythritol indicate that 80-90% of an oral dose is eliminated in the urine within 24 hrs (EFSA 2015). Thus, the present study assumed that 90% of erythritol is absorbed in the upper GIT, resulting in 10% reaching the colon.

[97] Table 1. Summary of the dose selection criteria to identify in vivo test doses of D- allulose and erythritol Dose selection criteria D-allulose Erythritol Maximum use levels in selected categories (specified by EFSA and / or FDA) GRAS levels: chewing gum 50% (500g / kg); beverages 3.5% (35g / kg) No added sugar chewing gum: quantum satis; flavoured drinks: 16,000 mg / L as flavour enhancer only Sweetness intensity vs. sucrose 0.7 0.6-0.8 Adult population intake Estimated intakes are below 500mg / d but when used as a sugar replacement, intakes may reach 10-30g / d depending on the food consumed (Daniel et al. 2022) Average daily total polyols intakes were 3.5g (0.09g kg-1 BW) and increased to 10.4g (0.27g kg-1 BW) at the 95th percentile. Average total polyols intakes per meal were 1.9g (0.05g kg-1 BW) and increased to 5.6g (0.15g kg-1 BW) at the 95th percentile (Tennant 2014) ADI (JECFA) Not specified Not specified Gl Tolerance Up to 30g / d (FDA, GRAS notice 400) Up to 35g / d (Storey et al. 2007) Selected in vivo oral dose 5g 5g

[98] Abbreviations: EFSA: European Food Safety Authority; FDA: Food and Drug Administration; ADI: acceptable daily intake; JECFA; Gl: Gastrointestinal; GRAS: Generally Recognized as Safe; BW: body weight.

[99] Table 2. In vivo oral dose (g / day), in vivo small intestinal absorption (%), and ex vivo colonic test doses (g / day) for D-allulose and erythritol. Study condition In vivo oral dose (g / day) Small intestinal absorption in vivo Ex vivo colonic test dose (g / day) D-allulose 5 80% 1 Erythritol 5 90% 0.5

[100] 1.3 Colonic incubations

[101] A kinetic, ex vivo study was implemented, simulating the colonic fermentation of test products by the gut microbiota derived from type 2 diabetes patients (T2D) and co-living healthy human adults (H) (n = 6 per donor group). Colonic incubations of D-allulose and erythritol were conducted for 12 participants and samples were taken at 6 hrs, 24 hrs and 48 hrs.

[102] Systemic Intestinal Fermentation Research (SIFR®) technology was used. SIFR® is a validated, miniaturised, bioreactor-based, high-throughput, ex vivo gut microbiome platform that accurately predicts in vivo results (Van den Abbeele et al. 2023).

[103] Individual bioreactors were processed in parallel (managed simultaneously) in a bioreactor management device (Cryptobiotix, Ghent, Belgium). Each bioreactor contained 5mL of nutritional medium-fecal inoculum blend supplemented with digested test products (D-allulose or erythritol) derived from the small intestinal digestion. Each bioreactor was sealed individually, before being rendered anaerobic. Blend M0017 was used for preparation of the nutritional medium (Cryptobiotix, Ghent, Belgium). Each bioreactor contained 5 mL of nutritional medium-fecal inoculum blend supplemented with digested test products derived from the simulated small intestinal digestion protocol, then sealed individually, before being rendered anaerobic. Blend M0017 was used for the preparation of the nutritional medium (Cryptobiotix, Ghent, Belgium). After preparation, bioreactors were incubated under continuous agitation (140 rpm) at 37°C for 48h (MaxQ 6000, Thermo Scientific, Thermo Fisher Scientific, Merelbeke, Belgium). Upon gas pressure measurement in the headspace, liquid samples were collected for subsequent analysis. Fresh fecal samples were collected according to a procedure approved by Ethics Committee of the University Hospital Ghent (reference number BC-09977). 1.4 Donor sourcing

[104] The selection criteria for all donors were: age 25-65, no antibiotic use in the past 6 months, no gastro-intestinal disorders (cancer, ulcers, IBD), no use of probiotics, nonsmoking, alcohol consumption <3 units / d and BMI <30. Six pairs of co-living human adults consuming a similar diet were sourced. One donor of each pair was previously diagnosed with T2D. The co-living donors consumed a similar diet to lower the potential variation introduced by differences in long-term diet (a major driver of microbiota composition) on baseline microbiota composition, thus allowing a more optimal study of the impact of differences in gut microbiota composition and metabolite production between T2D subjects and healthy adults.

[105] The 6 healthy subjects consisted of 4 male and 2 female subjects. The T2D subjects consisted of 2 male and 4 female subjects.

[106] For each test subject, a no substrate control (NSC) was initiated simultaneously to the test product incubations. This NSC consisted of a background medium and microbiota without test product so that any of the changes observed between the NSC and test product incubations could be attributed to the presence of the test products. 1.5 Quality Control

[107] For each donor, a NSC incubation was conducted concurrently to the D-allulose and erythritol incubations. The NSC ensured that differences observed between the NSC and D-allulose and erythritol incubations could be attributed to the presence of D-allulose and erythritol. For quality control (QC) purpose, NSC incubations were run in technical triplicate. The coefficients of variation for fundamental fermentation parameters [pH, gas production, and the three main SCFAs (acetate, propionate and butyrate)] were on average 1.75%, thus confirming the high technical reproducibility of the SIFR® technology as previously established (Van den Abbeele et al. 2023). 1.6 Fundamental Fermentation Parameters

[108] Upon gas pressure measurement in the closed reactors, liquid samples were collected for subsequent analysis. Following this, liquid samples were obtained and analysed. One hundred and twenty samples were analysed (3 study conditions, 12 donors, 3 time points) in addition to baseline samples (0 hrs) of the NSC for 12 donors as the levels of both lactate and SCFA were the same at 0 hrs in the study conditions. Twenty-four samples were also analysed for QC (2 additional NSC replicates at 48 hrs to assess technical reproducibility; n = 3). SCFAs (acetate, propionate, butyrate, and valerate) and branched chain fatty acids (bCFAs) (total of isobutyrate, isovalerate and isocaproate) were quantified by gas chromatography (GC) coupled to flame ionisation detection (Trace 1300, Thermo Fisher Scientific, Merelbeke, Belgium), upon diethyl ether extraction as described previously (De Weirdt et al. 2010), while lactate was measured with an enzymatic method according to manufacturer's instructions (EnzytecTM, R-Biopharm, Darmstadt, Germany). pH was measured using an electrode (Hannah Instruments Edge HI2002, Temse, Belgium). Extraction was performed from the samples with diethyl ether, after addition of 2-methyl hexanoic acid as an internal standard. Briefly, samples of 0.5 mL were diluted (1:3) in distilled water then acidified with 0.5 mL of 48% sulphuric acid. Subsequently, an excess of sodium chloride was incorporated and 0.2 mL of 2-methylhexanoic acid (internal standard) and 2 mL of diethyl ether. Following homogenisation and separation of the diethyl ether and water layer, extracts of diethyl ether were obtained and analysed by a Trace 1300 chromatograph (Thermo Fisher Scientific, Merelbeke Belgium) with a Stabilwax-DA capillary GC column, a flame ionisation detector and a split injector and the makeup and carrier gas used was nitrogen gas. The injection volume was 1 pL and the temperature was 110-240 °C. The temperature of the injector was 240°C and the temperature of the detector was 250 °C. The sample pH was measured using an electrode (Hannah Instruments Edge HI2002, Temse, Belgium). Lactate was measured by an enzymatic method as stated by the manufacturer’s directions (EnzytecTM, R-Biopharm, Darmstadt, Germany). 1.7 Metaqenomic Analysis

[109] For microbial composition analysis, quantitative data was obtained by correcting abundances (%; shallow shotgun sequencing (3M reads)) with total cell counts for each sample (cells / mL; flow cytometry), resulting in estimated cell counts / mL of different taxonomic groups, overall allowing to obtain more representative insights in the impact of interventions on the gut microbiota (Vandeputte et al. 2017). One hundred and twenty samples were analysed (3 study conditions, 12 donors, 3 time points) in addition to baseline samples (0 hrs) of the NSC for 12 donors as the microbiota composition at 0 hrs were the same in the study conditions. Abundances (%; shallow shotgun sequencing; 5.54 M reads per sample [average]) were corrected with total cell counts per sample (cells / mL; flow cytometry) to generate estimated cell counts / mL.

[110] First, a bacterial cell pellet was obtained by centrifugation of 1mL sample during 5 min at 9000 g. DNA was extracted via the SPINeasy DNA Kit for Soil (MP Biomedicals, Eschwege, Germany), according to manufacturer’s instructions.

[111] Subsequently, DNA libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, United States) and IDT Unique Dual Indexes with total DNA input of 1ng. Genomic DNA was fragmented using a proportional amount of Illumina Nextera XT fragmentation enzyme. Unique dual indexes were added to each sample followed by 12 cycles of PCR to construct libraries. DNA libraries were purified using AMpure magnetic Beads (Beckman Coulter, Brea, CA, United States), eluted in QIAGEN EB buffer, quantified using a Qubit 4 fluorometer and a Qubit dsDNA HS Assay Kit, and sequenced on an Illumina Nextseq 2000 platform 2x150bp. Unassembled sequencing reads were converted to relative abundances (%) using the CosmosID-HUB Microbiome Platform (CosmosID Inc., Germantown, MD, United States) (Agarwal et al. 2022; Hasan et al. 2014). Reads were retained if a Phred score was higher than 17 for a minimum of 80% of the read lengths (Brumfield et al. 2021). Removal of host DNA sequences was carried outby mapping reads to a human DNA sequences database which was custom-made in the CosmosID-HUB (Kalan et al. 2019). The CosmosID in-house K-mer-based algorithm and database was used for taxonomic identification as reported by Hourigan et al. (2018) and Leonard et al. (2021). 1.8 Cell count

[112] For total cell count analysis, liquid samples were diluted in anaerobic phosphate-buffered saline (PBS), after which cells were stained with SYTO 16 at a final concentration of 1pM and counted via a BD FACS Verse flow cytometer (BD, Erembodegem, Belgium). Data was analysed using FlowJo, version 10.8.1. Due to the differences in cell numbers between samples, proportional data (%) were corrected to absolute levels (cells / mL). Data processing at phylum, species, and family level was performed on the absolute level (cells / mL) results. 1.9 Metabolomics

[113] Liquid chromatography-mass spectrometry (LC-MS) allowed relative quantification of each test product while providing insights in a broad range of microbial metabolites. Liquid chromatography-mass spectrometry (LC-MS) allowed quantification of D-allulose and erythritol in the study arms where they were dosed to understand the time intervals during which they were fermented. There was background detection for D-allulose, such that there was an interference with other hexoses that are part of the background medium (likely fructose). Nevertheless, dosing with D-allulose increased the detection of the metabolite identified as D-allulose / hexose, well above levels of the background medium. Moreover, background hexoses were rapidly fermented within 6h of incubation unlike D-allulose. As the only difference between NSC and D-allulose is the presence of D-allulose, this suggests the specific detection of D-allulose from 6h onwards. The analysis was carried out using a Vanquish UHPLC (Thermo Scientific, Germering, Germany) coupled to a Orbitrap Exploris 240 MS (Thermo Scientific, Bremen, Germany), using an electrospray ionisation source, applied both in negative and positive ionisation mode. The UPLC was performed using a slightly modified version of the protocol described by Doneanu et al. (2011). Peak area extraction and pre-processing were performed using Compound Discoverer 3.3 (Thermo Scientific), along with a manual extraction based on an in-house library using Skyline 21.1 (MacCoss Lab Software) (Adams et al. 2020). Annotation of compounds was communicated at different confidence levels (CL), i.e., level 1 (retention times (compared against in-house authentic standards), accurate mass (with an accepted deviation of 3ppm), and MS / MS spectra), level 2a (retention times and accurate mass), level 2b (accurate mass and MS / MS spectra) and level 3 (accurate mass alone). Technical variability was confirmed by running a QC sample (pooled aliquots of all samples) every mixed injection to verify technical variability. 1.10 Data analysis

[114] All univariate and multivariate analyses were performed using R (version 4.2.2; www.r-project.org). For the PCA analysis, the FactoMineR package was used (Husson et al. 2014). Linear mixed models were fit using the Ime4 package (Bates et al. 2014), using the following syntax: lmer(Short chain fatty acid ~ Treatment * Donor group + (1 | Donor), while the effects were visualised using the ggeffects package (Llidecke 2018). Regularised Canonical Correlation Analysis (rCCA) was executed using the mixOmics package with the shrinkage method for estimation of penalisation parameters (version 6.20.3) (Rohart et al. 2017). Statistical significance of supplementation effects between the test products (D-allulose and erythritol) compared with the NSC at each time point (6, 24, 48 hrs) on metabolites or specific microbial taxa were assessed via repeated measures ANOVA analyses (based on paired testing among the 12 human adults), with p-value-correction according to Benjamini-Hochberg (Benjamini and Hochberg 1995). For analysis of microbial composition, three measures were taken. First, statistical analysis was performed on the log 10-transformed values. Second, a value of a given taxonomic group below the limit of detection (LOD) was considered equal to the overall LOD as described recently (Van den Abbeele et al. 2023). Finally, a threshold was set to retain the 100 most abundant species in the analysis, to avoid excessive p-value corrections. Treatment effects on microbial families and species at 48 hrs are reported. Correlations between metabolites and microbial taxa at 48 hrs were identified using Regularised Canonical Correlation Analysis (rCCA). rCCA was performed using the mixOmics R package and the estimation of penalisation parameters was done using the shrinkage method following a central log-ratio transformation of the microbiota matrix. To generate the correlation matrix, the first three components of the rCCA were used (Rohart et al. 2017; version 6.20.3). 1.11 Results 1.11.1 Fermentation Kinetics of Erythritol and D-allulose

[115] LC-MS analysis allowed for the detection of D-allulose and erythritol in the study arms where they were dosed to elucidate the fermentation kinetics. Fig. 1A and B show the concentration (peak area) of D-allulose and erythritol respectively at 6, 24 and 48 hrs for the whole sample. D-allulose was rapidly fermented between 6-24 hrs. Erythritol was slowly fermented with a high concentration still evident at 24 hrs. Erythritol was mostly fermented between 24-48 hrs.

[116] Fig. 1A shows D-allulose and Fig. 1B shows erythritol at 6, 24, and 48 hrs of incubation for the whole sample (n=12) following treatment with erythritol or D-allulose. There was background detection for D-allulose, such that there was an interference with other hexoses that are part of the background medium. 1.11.2 Effects of Erythritol and D-allulose on Fermentation Parameters

[117] The effects of erythritol and D-allulose on fermentation parameters are shown in Figs. 2A-I for the whole sample and by donor group in Figs. 7A-I. There was a significant increase in total SCFAs after D-allulose treatment, which was largely driven by a significant increase in butyrate and acetate production at both 24 and 48 hrs compared to the NSC in the analysis of the whole sample (Figs. 2D and 1F). These effects of D-allulose on butyrate and acetate were consistent across healthy participants and participants with T2DM in the donor-specific analysis (Figs. 7D and 7F). Erythritol had no effect on total SCFA concentration at any time point across all analyses (Fig. 2C and Fig. 7C). However, the analysis of individual SCFAs demonstrated significant effects of erythritol treatment. Similar to the effects of D-allulose treatment, erythritol significantly increased butyrate at 24 and 48 hrs after treatment compared with the NSC in the whole-sample analysis (Fig. 2F). In the donor-specific analysis, the significant increase in butyrate production from erythritol treatment vs. the NSC was consistent across healthy participants and participants with T2DM at 24 hrs and 48 hrs (Fig. 7F). Other changes in the production of SCFAs following D-allulose and erythritol treatment were noted, but they were not consistent across different time points and / or healthy and T2DM individuals (Figs. 2D-E and Figs. 7D-E).

[118] Figs. 2A-I show (A) pH, (B) gas (mbar), (C) total SCFAs (mM), (D) acetate (mM), (E) propionate (mM), (F) butyrate (mM), (G) BCFA (mM), (H) lactate (Mm), and (I) valerate (mM) levels after 6, 24, and 48 hrs of incubation with erythritol or D-allulose for the whole sample (n=12). Statistical differences between test products vs. the NSC are indicated by * (0.01 <Padjusted<0.05), ** (0.001 <p <0.01) or*** (p <0.001).

[119] Figs. 7A-I show (A) pH, (B) gas (mbar), (C) total SCFA (mM), (D) acetate (mM), (E) propionate (mM), (F) butyrate (mM), (G) BCFA (mM), (H) lactate (Mm), and (I) valerate (mM) levels after 6, 24, and 48 hrs of incubation with erythritol or D-allulose by donor group (n=6 healthy participants; n=Q participants with T2DM). Statistical differences between test products vs. the NSC are indicated by * (0.01 <Padjusted <0.05), ** (0.001 <padlusM<0.01) or *** (Padjusted <0.001). Abbreviations: H: Healthy participants: T2D: Type 2 Diabetic participants; NSC: no-substrate control. 1.11.3 Effects of Erythritol and D-allulose on Microbial Composition: Phylum-, Family- and Species-level

[120] There were three main phyla detected across the adult donors: Actinobacteria, Bacteroidetes, and Firmicutes. As a result, a targeted analysis was performed for each of these three phyla. At the phylum level, both D-allulose and erythritol significantly increased Firmicutes compared with the NSC. D-allulose significantly increased Firmicutes at 24 and 48 hrs and erythritol significantly increased Firmicutes at 48 hrs compared to the NSC in the whole-sample analysis (Fig. 3C). In the analysis by donor group, D-allulose significantly increased Firmicutes in healthy participants at 24 hrs and in participants with T2DM at 48 hrs compared to the NSC (Fig. 8C). In the analysis by donor group, there were no significant effects of erythritol on Firmicutes. There were no effects of D-allulose and erythritol on Actinobacteria and Bacteroidetes across all analyses, excluding a significant increase in Actinobacteria following erythritol treatment compared to the NSC at 48 hrs in healthy participants in the donor-specific analysis (Fig. 3A-B and Fig. 8A-B).

[121] Figs. 3A-C show (A) Actinobacteria (cells / mL), (B) Bacteroidetes (cells / mL) and (C) Firmicutes (cells / mL) after 6, 24, and 48 hrs of incubation with erythritol or D-allulose for the whole sample (n=12). Statistical differences between test products vs. the NSC are indicated by * (0.01 <padjUSted <0.05), ** (0.001 <p <0.01) or *** (p <0.001).

[122] Figs. 8A-C show (A) Actinobacteria (cells / mL) (B) Bacteroidetes (cells / mL) and (C) Firmicutes (cells / mL) after 6, 24, and 48 hrs of incubation with erythritol or D-allulose by donor group (n=Q healthy participants; n=Q participants with T2DM). Statistical differences between test products vs. the NSC are indicated by * (0.01 <Padjusted <0.05), ** (0.001 <p^O.OI) or *** (p.»steJ <0.001). Abbreviations: H: Healthy participants: T2D: Type 2 Diabetic participants; NSC: no-substrate control.

[123] Fig. 4 shows the significant treatment effects on microbial families and species in the whole-sample analysis at 48 hrs. D-allulose significantly increased the Lachnospiraceae family at 48 hrs in the whole-sample analysis (Fig. 4) and in healthy participants in the donorspecific analysis (Fig. 9). The increase in Lachnospiraceae family at 48 hrs was due to the specific and significant increase of two species: Anaerostipes hadrus and Lachnospiraceae_ unclassified_species (u_s) along with a marked but non-significant increase in Blautia obeum demonstrated in the analysis of the whole sample at 48 hrs (Fig. 4). The same pattern of effects for these specific species were demonstrated following D-allulose treatment in healthy participants and participants with T2DM in the donor-specific analysis, but the differences were not significant (Fig. 9).

[124] Fig. 4 shows microbial families and species expressed as Iog2 (treatment / NSC) that were significantly affected after 48 hrs of colonic incubations with erythritol or D-allulose compared to the NSC (FDR= 0.10) for the whole sample (n=12). Significant differences between test products vs. the NSC are indicated in bold. Abbreviations: AL: D-allulose; ER: Erythritol; u_f: unclassified family; u_s: unclassified species.

[125] Fig. 9 shows microbial families and species expressed as Iog2 (treatment / NSC) that were significantly affected after 24 and 48 hrs of colonic incubations with erythritol or D-allulose compared to the NSC (FDR= 0.10) by donor group (n=6 healthy participants; n=Q participants with T2DM). Significant differences between test products vs. the NSC are indicated in bold. Abbreviations: AL: D-allulose; ER: Erythritol; H: Healthy participants; T2D: Type 2 Diabetic participants; u_f: unclassified family; u_s: unclassified species. 1.11.4 Effects of Erythritol and D-allulose on Microbial Species and Related SCFA Production

[126] Correlations between fermentation parameters (gas production, SCFA, and BCFA) and microbial composition at 48 hrs are shown in Fig. 5. There was a strong correlation between both Anaerostipes hadrus and Lachnospiraceae_u_s and butyrate production following D-allulose treatment. There was also a marked correlation between Blautia obeum and acetate production following D-allulose treatment. Following erythritol treatment, there was a correlation between Eubacterium_u_s and butyrate production.

[127] Fig. 5 is a Regularised Canonical Correlation Analysis (rCCA) between fermentation parameters (gas production, SCFA, and BCFA) and microbial composition at 48 hrs after treatment with D-allulose or erythritol for the whole sample (n=12). Threshold >0.4. 1.12 Discussion

[128] The impact of representative and physiologically relevant doses of D-allulose and erythritol on the gut microbiota and metabolite production in adults with T2DM and co-living healthy adults was investigated using a validated ex vivo methodology. The results of this study provide important insights into the compound-specific effects of D-allulose and erythritol on the gut microbiota, two LNCS that are unexplored in the current literature. The inclusion of adults with T2DM and co-living healthy adults provides important insights on the impact of diabetes-induced alterations in gut microbiota composition on the fermentation of D-allulose and erythritol.

[129] This study revealed that both D-allulose and erythritol selectively increase the abundance of specific gut microbes in both healthy individuals and those with T2DM (Anaerostipes hadrus, Anaerobutyricum hallii, Blautia obeum, Eubacterium_u_s, and Lachnospiraceae_u_s). The D-allulose induced-increase in Anaerostipes hadrus and Lachnospiraceae_u_s was correlated with butyrate production. Anaerostipes hadrus are known butyrate-producing microbiota (Allen-Vercoe et al. 2012). There was also a correlation between Blautia obeum and acetate production following D-allulose treatment, in line with its metabolic capability to produce acetate (Oliphant and Allen-Vercoe 2019). Correspondingly, D-allulose increased butyrate and acetate production between 24-48 hrs in both healthy individuals and those with T2DM. Similarly, erythritol significantly increased butyrate 24-48 hrs after treatment in both healthy individuals and co-living individuals with T2DM. In line with this finding, erythritol induced a highly specific increase of Eubacterium_u_s and Anaerobutyricum hallii. While Anaerobutyricum hallii is a potent butyrate producer (Shetty et al. 2018), the current study demonstrated that Eubacterium_u_s was correlated with butyrate production. Previous studies on the effect of erythritol on human gut microbiota using in vitro models have demonstrated both increased production of SCFAs (Mahalak et al. 2020) and null effects (Hiele et al. 1993; Arrigoni et al. 2005). However, in the studies demonstrating null findings, the erythritol dose was either very low (e.g., 50mg) or not reported. Studies in animal models (mice and rats) have demonstrated dose-dependent increases in fecal SCFAs with D-allulose consumption (Matsuo et al. 2003), but no effects on fecal SCFAs (Han et al. 2020b). SCFAs are rapidly absorbed in the large intestine and therefore fecal SCFA concentrations are a less accurate reflection of SCFA production and may account for the null findings observed by Han et al. (2020b). Whilst further studies are needed to confirm the findings, the results of the current study indicate that D-allulose and erythritol increase butyrate and acetate production 24-48 hrs after treatment due to the selective modulation of the microbial composition.

[130] The butyrate-production kinetics were different for D-allulose and erythritol, such that D-allulose treatment significantly increased butyrate within 24 hrs whereas erythritol treatment significantly increased butyrate to a greater degree between 24-48 hrs. This corresponds with the observed fermentation kinetics of D-allulose and erythritol. D-allulose was rapidly fermented between 6-24 hrs whilst erythritol was slowly fermented between 24-48 hrs. This suggests complementary effects of D-allulose and erythritol on butyrate production kinetics, via the modulation of different gut microbiota species. These findings highlight the potential for a synergistic combination of D-allulose and erythritol, leading to sustained butyrate production over a 48 hr period. Utilising a blend of D-allulose and erythritol could be an effective approach of increasing butyrate production, particularly in individuals with T2DM exhibiting reduced abundance of butyrate-producing microbiota. A blend of erythritol and D-allulose may also increase the likelihood of sweetness synergism and positive sensory properties combined with weak bitterness (Jang et al. 2021). Moreover, in people with T2DM, an acute dose of D-allulose has been shown to reduce postprandial glucose response following an oral glucose tolerance test (OGTT) (Noronha et al. 2018) and chronic consumption of erythritol for four weeks improved small vessel endothelial function and aortic stiffness (Flint et al. 2014). Due to the very low energy density of D-allulose and erythritol, they can be incorporated into the diet at doses that would confer benefits to butyrate production without adding substantial calories.

[131] Restoration of butyrate levels in the large intestine through the intake of D-allulose and erythritol might be a novel approach for the treatment of T2DM, alongside lifestyle modification and glucose lowering drugs (Arora and Tremaroli 2021). Butyrate is the primary energy source for colonocytes (Koh et al. 2016). SCFAs also signal through free fatty acid receptors 2 and 3 (FFAR2 / 3; type of G-coupled protein receptors), which are widely expressed in diverse cell types in human, and therefore are key signalling molecules between the gut microbiota and the host (Koh et al. 2016; Mishra et al. 2020). Butyrate plays a role in glucose homeostasis and the pathophysiology of diabetes (Puddu et al. 2014; Arora and Tremaroli 2021; Mayorga-Ramos et al. 2022). A host-genetic-driven increase in gut production of butyrate was associated with improved insulin response after an OGTT in normoglycemic individuals (Sanna etal. 2019). Anaerostipes hadrus, a species selectively increased by D-allulose treatment in the current study, was associated with an increase in serum butyrate and increased insulin sensitivity following an OGTT in healthy individuals (Cui et al. 2022). In type-2 diabetic rats, treatment with butyrate significantly reduced plasma glucose, HbA1c, insulin-resistance, and gluconeogenesis (Khan and Jena 2016). In enteroendocrine cells, butyrate activates the FFAR2 and 3, stimulating the release of peptide YY and glucagon like peptide-1 thereby affecting glucose regulation and satiety (N0hr et al. 2013; Christiansen et al. 2018; Larraufie et al. 2018). Following absorption and utilisation by colonocytes, any surplus butyrate is transported into the portal circulation and subsequently into the systemic circulation (Koh et al. 2016). Within the systemic circulation, butyrate can modulate [3-cell function in the pancreas via FFAR2 / 3, monocarboxylate transporters, and inhibition of histone deacetylases (Mayorga-Ramos et al. 2022).

[132] The International Scientific Association for Probiotics and Prebiotics defines a prebiotic as a substrate that is selectively utilised by host microorganisms conferring a health benefit (Gibson et al. 2017). The selective utilisation of D-allulose and erythritol by specific gut microbiota observed in the current study is in line with this definition, particularly for individuals with T2DM. However, human clinical studies are required to demonstrate an associated health benefit to confirm prebiotic effects consistent with the ISAPP definition. Previous studies in mice have demonstrated prebiotic effects of D-allulose. In particular, two previous studies have demonstrated that D-allulose modulates specific microbiota species and these changes were correlated with improvements in high fat diet-induced obesity and fasting blood glucose (Han et al. 2020a, 2020b). Moreover, synbiotic mixtures combining two probiotic species with D-allulose as a prebiotic suppressed diet-induced obesity in mice via the regulation of lipid metabolism (Choi et al. 2018). In the current study, D-allulose and erythritol increased butyrate production due to the selective increase in the abundance of specific gut microbiota, highlighting their prebiotic potential as substrates selectively utilised by host microorganisms. The potential associated health benefits may include butyrate-mediated effects on glycemic control and insulin sensitivity (Arora and Tremaroli 2021; Mayorga-Ramos et al. 2022).

[133] Representative and physiologically relevant doses of D-allulose and erythritol were demonstrated to increase SCFAs (mostly butyrate and acetate), due to the selective modulation of the microbial composition. These findings suggest a potential prebiotic effect of D-allulose and erythritol as substrates that are selectively utilised by host microorganisms. While only the aspect of selective utilisation was demonstrated in this study, the health benefits could follow from the production of SCFAs, and particularly from the production of butyrate in those with T2DM. Combining (blending) D-allulose and erythritol could be a promising strategy to increase butyrate production in individuals with T2DM, resulting in benefits related to glycemic control.

[134] At 48h, both allulose and erythritol positioned distinctly different from the NSC, which correlated with higher acetate / propionate / butyrate levels, enhanced gas production and lower proteolytic activity (bCFA levels), suggesting that both ingredients enhanced microbial activity compared to the NSC.

[135] Allulose was strongly fermented between 6-24h. At 48h, it significantly increased acetate, propionate, butyrate (and thus also total SCFA) levels, decreased pH and significantly increased gas production. Allulose consistently increased butyrate levels, both for T2D subjects and healthy donors.

[136] Erythritol was slowly fermented between 24-48h. While it did not significantly affect total SCFA levels, it boosted butyrate levels to levels that were remarkably high.

[137] Microbial compositional analyses further showed that: • Allulose strongly and highly specifically increased the bacterial family Lachnospiraceae from 24h onwards, which followed a remarkably specific increase of two species: Anaerostipes hadrus, and Blautia obeum. A marked correlation between Anaerostipes hadrus / Lachnospiraceae and butyrate production was found-Anaerostipes hadrus is indeed a dominant lactate consuming, butyrate-producing species (Allen-Vercoe et al. 2012); • Erythritol highly specifically boosted butyrate production. In line with such specific effect, a highly specific increase of the following species was observed, i.e., Eubacterium hallii, Eubacterium rectale, Eubacterium callanderi, Eubacterium limosum and Eubacterium maltosivorans along with Anaerobutyricum hallii. Both Anaerobutyricum hallii and Eubacterium are indeed butyrate producers (Shetty et al. 2018; Ryu et al. 2022), and it was particularly Eubacterium that related with butyrate levels; • There is complementarity between allulose and erythritol in terms of kinetics, as allulose increased butyrate between 6-24h, while erythritol increased it between 24-48h, acting via different bacterial species.

[138] The foregoing description of embodiments of the present invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. As the person of skill in the art will recognise, many modifications and variations are possible in light of the above teaching. It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the claims and their equivalents. The invention will now be described with reference to the following clauses. 1. A method for improving gut microbiota population in a subject, the method comprising administering to the subject a composition comprising allulose, erythritol or a combination thereof. 2. A method for increasing one or more colonic bacterial populations from the families Lachnospiraceae or Eubacteriaceae in a subject, the method comprising administering to the subject a composition comprising allulose, erythritol or a combination thereof. 3. A method for preventing, treating or ameliorating a disease in a subject, the method comprising administering to the subject a composition comprising allulose, erythritol or a combination thereof. 4. The method according to clause 3, wherein the disease is one or more of metabolic syndrome, obesity, type 2 diabetes (T2D), insulin resistance, hyperlipoproteinemia, hyperuricemia, hepatic steatosis, hypercholesterolemia, hypertriglyceridemia, irritable bowel syndrome (IBS), colon cancer, allergy, non-alcoholic fatty liver diseases (NAFLD), inflammatory bowel disease (IBD), cardiovascular disease (CVD) or inflammation. 5. The method according to any of clauses 1-4, wherein at least one of the one or more bacterial populations is from the family Lachnospiraceae. 6. The method according to any of clauses 1-5, wherein at least one of the one or more colonic bacterial populations is from the genus Anaerostipes. 7. The method according to any of clauses 1-6, wherein at least one of the one or more bacterial populations is of the species Anaerostipes hadrus. 8. The method according to any of clauses 1-7, wherein at least one of the one or more colonic bacterial populations is from the genus Blautia. 9. The method according to any of clauses 1-8, wherein at least one of the one or more bacterial populations is of the species Blautia obeum. 10. The method according to any of clauses 1-9, wherein at least one of the one or more colonic bacterial populations is from the genus Anaerobutyricum. 11. The method according to any of clauses 1-10, wherein at least one of the one or more bacterial populations is from of the species Anaerobutyricum hallii. 12. The method according to any of clauses 1-11, wherein at least one of the one or more bacterial populations is from the family Eubacteriaceae. 13. The method according to any of clauses 1-12, wherein at least one of the one or more colonic bacterial populations is from the genus Eubacterium. 14. The method according to any of clauses 1-13, wherein at least one of the one or more colonic bacterial populations is from the species Eubacterium rectale, Eubacterium callanderi, Eubacterium limosum and Eubacterium maltosivorans. 15. The method according to any of clauses 1-14, wherein one or more of the colonic bacterial populations is increased by at least about 0.5% as compared to a non-treated subject. 16. The method according to any of clauses 1-15, wherein one or more of the colonic bacterial populations is increased by at least about 10% as compared to a non-treated subject. 17. The method according to any of clauses 1-16, wherein one or more of the colonic bacterial populations is increased by at least about 15% as compared to a non-treated subject. 18. The method according to any of clauses 1-17, wherein one or more of the colonic bacteria populations is increased by at least about 50% as compared to a non-treated subject. 19. The method according to any of clauses 1-18, wherein each of the colonic bacteria populations is increased by at least about 0.5% as compared to a non-treated subject. 20. The method according to any of clauses 1-19, wherein each of the colonic bacteria populations is increased by at least about 10% as compared to a non-treated subject. 21. The method according to any of clauses 1-20, wherein colonic butyrate levels are increased as compared to a non-treated subject. 22. The method according to any of clauses 1-21, wherein colonic butyrate levels are increased by 5% as compared to a non-treated subject. 23. A method for increasing colonic short chain fatty acid levels in a subject, the method comprising administering to the subject a composition comprising allulose, erythritol or a combination thereof. 24. The method according to any of clauses 1-23, wherein allulose is administered at a rate of at least about 0.1 g / day. 25. The method according to any of clauses 1-24, wherein allulose is administered at a rate of at least about 0.25 g / day. 26. The method according to any of clauses 1-25, wherein allulose is administered at a rate of at least about 0.5 g / day. 27. The method according to any of clauses 1-26, wherein allulose is administered at a rate of at least about 1.0 g / day. 28. The method according to any of clauses 1-27, wherein allulose is administered at a rate of at least about 2.0 g / day. 29. The method according to any of clauses 1-28, wherein erythritol is administered at a rate of at least about 0.1 g / day. 30. The method according to any of clauses 1-29, wherein erythritol is administered at a rate of at least about 0.25 g / day. 31. The method according to any of clauses 1-30, wherein erythritol is administered at a rate of at least about 0.5 g / day. 32. The method according to any of clauses 1-31, wherein erythritol is administered at a rate of at least about 1.0 g / day. 33. The method according to any of clauses 1-32, wherein erythritol is administered at a rate of at least about 2.0 g / day. 34. The method according to any of clauses 1-33, wherein the subject is a human. 35. The method according to any of clauses 1-34, wherein the subject is a human suffering from diabetes. 36. The method according to any of clauses 1-35, wherein the subject is a human suffering from Type II diabetes. 37. The method according to any of clauses 1-36, wherein administration is performed over the course of at least two weeks. 38. The method according to any of clauses 1-37, wherein the allulose, erythritol or the combination thereof is administered orally. 39. The method according to any of clauses 1-38, wherein the allulose, erythritol or the combination thereof is administered in the form of an edible composition. 40. The method according to any of clauses 1-39, wherein the allulose, erythritol or the combination thereof is administered in the form of a synbiotic composition. 41. The method according to clause 40, wherein the synbiotic composition comprises the one or more bacterial populations from the families Lachnospiraceae or Eubacteriaceae. 42. A composition comprising allulose, erythritol or a combination thereof and one or more colonic bacterial populations from the families Lachnospiraceae or Eubacteriaceae. 43. A composition comprising allulose, erythritol or a combination thereof for use in a method according to any of clauses 1 to 41. 44. The composition according to clause 42 or the composition for use according to clause 43, wherein at least one of the one or more bacterial populations is from the family Lachnospiraceae. 45. The composition or composition for use according to any of clauses 42-44, wherein at least one of the one or more colonic bacterial populations is from the genus Anaerostipes. 46. The composition or composition for use according to any of clauses 42-45, wherein at least one of the one or more bacterial populations is of the species Anaerostipes hadrus. 47. The composition or composition for use according to any of clauses 42-46, wherein at least one of the one or more colonic bacterial populations is from the genus Blautia. 48. The composition or composition for use according to any of clauses 42-47, wherein at least one of the one or more bacterial populations is of the species Blautia obeum. 49. The composition or composition for use according to any of clauses 42-48, wherein at least one of the one or more colonic bacterial populations is from the genus Anaerobutyricum. 50. The composition or composition for use according to any of clauses 42-49, wherein at least one of the one or more bacterial populations is from of the species Anaerobutyricum hallii. 51. The composition or composition for use according to any of clauses 42-50, wherein at least one of the one or more bacterial populations is from the family Eubacteriaceae. 52. The composition or composition for use according to any of clauses 42-51, wherein at least one of the one or more colonic bacterial populations is from the genus Eubacterium. 53. The composition or composition for use according to any of clauses 42-52, wherein at least one of the one or more colonic bacterial populations is from the species Eubacterium rectale, Eubacterium callanderi, Eubacterium limosum and Eubacterium maltosivorans. 54. The composition or composition for use according to any of clauses 42-53, wherein the composition or composition for use comprises at least 0.1 g of allulose per serving. 55. The composition or composition for use according to any of clauses 42-54, wherein the composition or composition for use comprises at least 0.25 g of allulose per serving. 56 The composition or composition for use according to any of clauses 42-55, wherein the composition or composition for use comprises at least 0.5 g of allulose per serving. 57. The composition or composition for use according to any of clauses 42-56, wherein the composition or composition for use comprises at least 0.1 g of erythritol per serving. 58. The composition or composition for use according to any of clauses 42-57, wherein the composition or composition for use comprises at least 0.25 g of erythritol per serving. 59. The composition or composition for use according to any of clauses 42-58, wherein the composition or composition for use comprises at least 0.5 g of erythritol per serving. 60. The composition or composition for use according to any of clauses 42-59, wherein the composition or composition for use is in the form of a food composition. 61. The composition or composition for use according to any of clauses 42-60, wherein the food composition is in a form selected from baked foods, breakfast cereal, dairy products, soy products, confections, jams and jellies, beverages (powdered and / or liquid), shakes, fillings, yogurts (dairy and non-dairy yogurts), kefirs, extruded and sheeted snacks, gelatin desserts, snack bars, meal replacement and energy bars, cheese and cheese sauces (dairy and non-dairy cheeses), edible and water-soluble films, soups, syrups, table top sweeteners, nutritional supplements, sauces, dressings, creamers, icings, ice cream, frostings, glazes, pet food, tortillas, meat and fish, dried fruit, infant and toddler food, and batters and breadings. 62. The composition or composition for use according to any of clauses 42-61, wherein the food composition is in the form of an agglomerated powder, a nutritional supplement or a medicinal dosage form. 63. The method according to any of clauses 1-41, wherein the allulose, erythritol or the combination thereof is provided as a composition or composition for use according to any of clauses 42 to 62. 64. The method according to any of clauses 1-41 or 63, wherein elevated short chain fatty acid (e.g. butyrate) production is observed in the period of 1-24 hours after administering the composition. 65. The method according to any of clauses 1-41, 63 or 64, wherein elevated short chain fatty acid (e.g. butyrate) production is observed in the period of 24-48 hours after administering the composition. 66. The method according to any of according to any of clauses 1-41 or 63-65, wherein method is therapeutic. 67. The method according to any of according to any of clauses 1-41 or 63-66, wherein method is non-therapeutic. 68. Use of the composition or composition for use according to any of clauses 41 to 62, for the manufacture of a medicament for improving gut microbiota population. 69. Use of the composition or composition for use according to any of clauses 41 to 62, for the manufacture of a medicament for increasing one or more colonic bacterial populations from the families Lachnospiraceae or Eubacteriaceae. 70. Use of the composition or composition for use according to any of clauses 41 to 62, for the manufacture of a medicament for preventing, treating or ameliorating a disease. 71. Use of the composition or composition for use according to any of clauses 41 to 62 for improving gut microbiota population. 72. Use of the composition or composition for use according to any of clauses 41 to 62 for increasing one or more colonic bacterial populations from the families Lachnospiraceae or Eubacteriaceae. 73. Use of the composition or composition for use according to any of clauses 41 to 62 for preventing, treating or ameliorating a disease.

Claims

1. A method for improving gut microbiota population in a subject, the method comprising administering to the subject a composition comprising allulose, erythritol or a combination thereof.

2. A method for increasing one or more colonic bacterial populations from the families Lachnospiraceae or Eubacteriaceae in a subject, the method comprising administering to the subject a composition comprising allulose, erythritol or a combination thereof.

3. A method for preventing, treating or ameliorating a disease in a subject, the method comprising administering to the subject a composition comprising allulose, erythritol or a combination thereof.

4. The method according to claim 3, wherein the disease is one or more of metabolic syndrome, obesity, type 2 diabetes (T2D), insulin resistance, hyperlipoproteinemia, hyperuricemia, hepatic steatosis, hypercholesterolemia, hypertriglyceridemia, irritable bowel syndrome (IBS), colon cancer, allergy, non-alcoholic fatty liver diseases (NAFLD), inflammatory bowel disease (IBD), cardiovascular disease (CVD) or inflammation.

5. The method according to any of claims 1-4, wherein at least one of the one or more bacterial populations is of the species Anaerostipes hadrus, Blautia obeum, Anaerobutyricum hallii, Eubacterium rectale, Eubacterium callanderi, Eubacterium limosum and Eubacterium maltosivorans.

6. The method according to any of claims 1-5, wherein one or more of the colonic bacterial populations is increased by at least about 0.5% as compared to a non-treated subject.

7. The method according to any of claims 1-6, wherein colonic butyrate levels are increased as compared to a non-treated subject.

8. A method for increasing colonic short chain fatty acid levels in a subject, the method comprising administering to the subject a composition comprising allulose, erythritol or a combination thereof.

9. The method according to any of claims 1-8, wherein allulose, erythritol or the combination thereof is administered at a rate of at least about 0.1 g / day.

10. The method according to any of claims 1-9, wherein the subject is a human.

11. The method according to any of claims 1-10, wherein the subject is a human suffering from Type II diabetes.

12. The method according to any of claims 1-11, wherein administration is performed over the course of at least two weeks.

13. The method according to any of claims 1-12, wherein the allulose, erythritol or the combination thereof is administered orally.

14. The method according to any of claims 1-13, wherein the allulose, erythritol or the combination thereof is administered in the form of an edible composition.

15. The method according to any of claims 1-14, wherein the allulose, erythritol or the combination thereof is administered in the form of a synbiotic composition.

16. A composition comprising allulose, erythritol or a combination thereof and one or more colonic bacterial populations from the families Lachnospiraceae or Eubacteriaceae.

17. A composition comprising allulose, erythritol or a combination thereof for use in a method according to any of claims 1 to 15.

18. The composition or composition for use according to claim 16 or 17, wherein at least one of the one or more bacterial populations is of the species Anaerostipes hadrus, Blautia obeum, Anaerobutyricum hallii, Eubacterium rectale, Eubacterium callanderi, Eubacterium limosum and Eubacterium maltosivorans.

19. The composition or composition for use according to any of claims 16-18, wherein the composition or composition for use comprises at least 0.1 g of allulose, erythritol, or the combination thereof per serving.

20. The composition or composition for use according to any of claims 16-19, wherein the composition or composition for use is in the form of a food composition.

21. The composition or composition for use according to any of claims 16-20, wherein the food composition is in a form selected from baked foods, breakfast cereal, dairy products, soy products,confections, jams and jellies, beverages (powdered and / or liquid), shakes, fillings, yogurts (dairy and non-dairy yogurts), kefirs, extruded and sheeted snacks, gelatin desserts, snack bars, meal replacement and energy bars, cheese and cheese sauces (dairy and non-dairy cheeses), edible and water-soluble films, soups, syrups, table top sweeteners, nutritional supplements, sauces, dressings, creamers, icings, ice cream, frostings, glazes, pet food, tortillas, meat and fish, dried fruit, infant and toddler food, and batters and breadings.

22. The composition or composition for use according to any of claims 16-21, wherein the food composition is in the form of an agglomerated powder, a nutritional supplement or a medicinal dosage form.

23. The method according to any of claims 1-15, wherein the allulose, erythritol or the combination thereof is provided as a composition or composition for use according to any of claims 16-22.

24. The method according to any of claims 1-15 or 23, wherein elevated short chain fatty acid (e.g. butyrate) production is observed in the period of 1-24 hours after administering the composition.

25. The method according to any of claims 1-15,23 or 24, wherein elevated short chain fatty acid (e.g. butyrate) production is observed in the period of 24-48 hours after administering the composition.

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