Use of allulose or erythritol in improving gut microbiota and medicine

Abstract

Methods for improving the gut microbiota of a subject, increasing one or more bacterial communities belonging to the Helicobacterium or Euglobulaceae families in their colonic flora, for the prevention, treatment, or relief of disease, and for increasing the level of short-chain fatty acids in the colon, comprising administering to the subject a composition containing allulose, erythritol, or a combination thereof. The composition used in the above methods comprises allulose, erythritol, or a combination thereof. The composition comprises allulose, erythritol, or a combination thereof, and one or more colonic bacterial communities from the Helicobacterium or Euglobulaceae families.

Description

Technical Field

[0001] This invention relates to improving the gut microbiota and increasing the number of microbiota from the Trichophytonceae family in subjects. Lachnospiraceae ) or Eubacteriaceae ( Eubacteriaceae The invention relates to one or more colonic bacterial populations, and methods for treating, preventing or improving diseases, the methods comprising administering to a subject a composition comprising allulose, erythritol or a combination thereof; a composition comprising allulose, erythritol or a combination thereof for use in the above methods; and a composition comprising allulose, erythritol or a combination thereof, and one or more colonic bacterial populations from the Trichophyceae or Euglenomycetes families. Technical Background The gut microbiota constitutes a complex ecosystem that interacts with host cells and nutrients. It is estimated that an adult human contains more than 10¹¹ 4 CFU (Cauliflower Fuel Cells) contains bacterial biomass encompassing over 400 different species, forming the largest, densest, and most diverse microbial community in the human body. The presence of gut bacteria is an integral part of normal human physiological functions, playing a crucial role in intestinal development, energy acquisition from dietary carbohydrates, absorption of essential vitamins, and metabolism of environmental chemicals within the gut. Recent studies further suggest that gut bacteria may be involved in fat storage and influence weight gain and loss. Gut bacteria also participate in the maturation of the immune system, maintaining a continuous connection with it and defending against pathogens. Given the importance of gut bacteria to health and wellness, there is significant interest in functional food ingredients that can increase the number of beneficial gut bacteria.

[0003] High sugar intake has been proven to be associated with an increased risk of several 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). Given the crucial role of the human gut microbiota in health and disease (Gomaa, 2020, Antonie Van Leeuwenhoek (113(12): 2019-40) The gut microbiota of patients with type 2 diabetes (T2D) has a unique composition characterized by low levels of butyrate-producing bacteria (Arora and Tremaroli 2021). Butyrate is the preferred energy source for epithelial cells and plays a protective role in preventing colon cancer and colitis (Rivière et al., 2016, 2019-2019).Frontiers in Microbiology 7:979). It is one of the three most abundant short-chain fatty acids (SCFAs) (the other two being acetic acid and propionic acid), typically produced by the gut microbiota through colonic fermentation of indigestible substrates. Strategies aimed at improving the health of patients with type 2 diabetes, in addition to traditional dietary strategies such as reducing sugar intake, can also focus on modulating the gut microbiota and promoting the production of SCFAs. When designing such strategies, the interactions between the gut microbiota, enterotype, and geographically relevant differences (such as environmental factors and / or diet) must be considered (Costea et al., 2018, ). Nature Microbiology 3 (1): 8-16; Yatsunenko et al., 2012, Nature 486 (7402): 222-27).

[0004] Low-calorie or zero-calorie sweeteners (LNCS) provide sweetness and are increasingly used as sugar substitutes. Currently, various types of LNCS exist, each with specific sweetness and pharmacokinetic properties. Based on their structure and / or sweetness, LNCS can be classified into rare sugars, polyols, and high-intensity sweeteners. Depending on their specific application, LNCS can be further classified into building agents and high-performance sweeteners. Building agents are typically less sweet than sucrose and are mainly used to provide volume and texture in food; while high-intensity sweeteners, because they impart a strong sweetness, are usually only needed in small amounts.

[0005] Some LNCs are known to have prebiotic effects. The potential prebiotic activity of allulose (also known as D-allulose or D-psicose) has been discussed (Özgür et al., Turkiye Klinikleri J Health Sci 2022, 7(2), 573). Some studies have shown that allulose, along with other food components such as β-glucan (Rugji et al., Food Sci Technol , 2022, 42, e07022) and probiotics (Choi et al., Nutrients (2018, 10, 1797) When combined, it has a prebiotic effect.

[0006] Several studies have explored the impact of LNCS on the human gut microbiota. As a recent review (Hughes et al., 2021, ...) has shown... Nutrition TodayAs noted in 56 (3): 105-13), there is currently no evidence that LNCS have an adverse effect on the gut microbiota within the approved intake range. Furthermore, establishing evidence for specific sweeteners is crucial. Although studies have confirmed the effects of rare sugars (Roy et al., 2018, Journal of Food Science 83(11): 2699-2709) and certain high-intensity sweeteners (Samuel et al., 2018, The Journal of Nutrition Evidence exists that polyols (148(7): 1186S-1205S) are fermented by gut microbes, but there is no such evidence for polyols, which can reach the colon and may also be fermented by gut microbes there (Ruiz-Ojeda et al., 2019). Advances in Nutrition 10 (suppl_1): S31-48). Furthermore, while the health benefits of LNCS have been described, some negative effects have also been reported (Suez et al., 2014, Cell 185 (18): 3307-3328.e19; 2022, Nature (514 (7521): 181–86) or contradictory results. The primary problem is the huge, sometimes even meaningless, differences in experimental doses. Secondly, many studies reporting contradictory data use animal models and doses (equal to or below the acceptable daily intake (ADI)), which may not be comparable to humans due to fundamental differences in gastrointestinal physiology.

[0007] The market demand for functional food ingredients (both for human and animal consumption) that offer both nutritional and health benefits is growing. Among these health-related food ingredients is a class of compounds called "prebiotics," which are substrates that can be selectively utilized by the host microbiome to provide health benefits (Gibson, G., Hutkins, R., Sanders, M., et al., Expert Consensus Paper: Consensus Statement of the International Scientific Association for Probiotics and Prebiotics (ISAPP) on the Definition and Scope of Prebiotics. Nat Rev Gastroenterol Hepatol 14, 491–502 (2017)). Typical prebiotic compounds currently used in food include various carbohydrates, such as dietary fiber. In addition to prebiotic ingredients, the market for foods and animal feeds containing prebiotic bacteria is also expanding. For example, there is growing interest in using probiotics as animal feed additives, primarily as a potential alternative to antibiotics. Many bacteria considered to possess beneficial probiotic properties are actually normal commensal bacteria present in the gut microbiota of healthy humans or animals. The most commonly used probiotics for humans include lactic acid bacteria (Lactobacillus). Lactobacilli) and Bifidobacteria ( Bifidobacteria However, there are many difficulties in using these two types of bacteria as probiotic foods or feed additives. For them to be effective, these bacteria must survive as living cells during the production process and be produced as stable products (i.e., their activity does not significantly decrease during long-term storage, even under harsh conditions). Furthermore, these bacteria must be able to tolerate the highly acidic environment of the stomach and withstand the erosion of bile salts in the upper small intestine. It is hypothesized that the benefits of probiotics stem from their transient colonization in the small intestine and / or colon, which requires successful competition with the existing microbiota. This process implies that probiotics must successfully compete with the existing microbiota, which can number as high as 10 per gram of intestinal contents. 7 -10 11 Live bacteria.

[0008] Further understanding of how dietary factors affect the gut microbiota is still needed, and products that can improve the gut microbiota should be developed. Summary of the Invention

[0009] The following overview is for illustrative purposes only and is not intended to limit the invention in any way. Other aspects, embodiments, and features of the invention will become apparent from the accompanying drawings and the detailed description below, in addition to the exemplary aspects, implementations, and features described above.

[0010] In a first aspect, the present invention provides a method for improving the gut microbiota of a subject, the method comprising administering to the subject a composition comprising allulose, erythritol, or a combination thereof.

[0011] In a second aspect, the present invention provides a method for increasing the colonic bacterial community of one or more species from the Trichophyceae or Eurybacterialaceae families in a subject, the method comprising administering to the subject a composition comprising allulose, erythritol, or a combination thereof.

[0012] In a third aspect, the present invention provides a method for preventing, treating or improving a disease in a subject, the method comprising administering to the subject a composition comprising allulose, erythritol or a combination thereof.

[0013] In a fourth aspect, the present invention provides a method for increasing the level of short-chain fatty acids in the colon of a subject, the method comprising administering to the subject a composition comprising allulose, erythritol, or a combination thereof.

[0014] In a fifth aspect, the present invention provides a composition comprising allulose, erythritol or a combination thereof, and one or more colonic bacterial communities from the Trichophyceae or Eurybacterialaceae families.

[0015] In a sixth aspect, the present invention provides a composition for use in the methods of any of the first to sixth aspects, comprising allulose, erythritol, or a combination thereof.

[0016] In a seventh aspect, the present invention provides the use of a composition or use of any of the compositions in the fifth or sixth aspects in the preparation of a medicament for improving the gut microbiota.

[0017] In an eighth aspect, the present invention provides a composition or use of any of the fifth or sixth aspects in the preparation of a medicament for improving one or more colonic bacterial communities derived from the Trichophyceae or Euglenomycetes families.

[0018] In a ninth aspect, the present invention provides a composition or use of any of the fifth or sixth aspects in the preparation of a medicament for the prevention, treatment or improvement of a disease.

[0019] In a tenth aspect, the present invention provides a composition or use of any of the fifth or sixth aspects in improving the gut microbiota.

[0020] In an eleventh aspect, the present invention provides a composition or use of any of the fifth or sixth aspects of the composition in improving one or more colonic bacterial communities derived from the Trichophyceae or Euglenomycetes families.

[0021] In a twelfth aspect, the present invention provides a composition or use of any of the fifth or sixth aspects in the prevention, treatment or improvement of a disease.

[0022] In a thirteenth aspect, the present invention provides a composition comprising allulose, erythritol, or a combination thereof, for improving the colonic bacterial community. Attached Figure Description

[0023] The invention can be more clearly understood in conjunction with the accompanying drawings.

[0024] Figure 1A and 1B The concentrations (peak areas) of D-allulose and erythritol in the whole sample at 6, 24, and 48 hours are shown.

[0025] Figures 2A-2I show the effects of erythritol and D-allulose on the fermentation parameters of the whole sample.

[0026] Figures 3A - 3C The figures show the number of actinomycetes ( ) in whole samples after co-culturing with erythritol or D-allulose for 6, 24, and 48 hours, respectively. Actinobacteria (cells / mL), Bacteroides ( Bacteroidetes (cells / mL) and Firmicutes ( Firmicutes Cell count (cells / mL).

[0027] Figure 4 The study demonstrated the significant effects of treatment on microbial families and species in a 48-hour full-sample analysis.

[0028] Figure 5 The correlation between fermentation parameters (gas production, short-chain fatty acids (SCFA), and branched-chain fatty acids (BCFA)) and microbial composition over 48 hours was shown.

[0029] Figure 6 The experimental design scheme of Example 1 is shown.

[0030] Figures 7A - 7I The effects of erythritol and D-allulose on fermentation parameters of each donor group were shown.

[0031] Figures 8A - 8C The cell counts of Actinomycetes (cells / mL), Bacteroides (cells / mL), and Firmicutes (cells / mL) in different donor groups after 6, 24, and 48 hours of culture with erythritol or D-aloxose are shown.

[0032] Figure 9 The study showed the significant effects of treatments on microbial families and species obtained from donor group analysis at 48 hours. Detailed Implementation

[0033] Before describing the disclosed methods and materials, it should be noted that the various aspects described herein are not limited to specific implementations, methods, or combinations, and therefore variations are naturally possible. Furthermore, it should be noted that the terminology used herein is for describing specific aspects only and should not be considered restrictive unless expressly defined herein.

[0034] In this specification, unless the context otherwise requires, the terms “comprise” and “include” and their variations (e.g., “comprises”, “comprising”, “includes”, “including”) should be understood to mean including the said components, features, elements or steps, or combinations of components, features, elements or steps, but do not exclude any other unmentioned integers, steps or combinations of steps.

[0035] In this specification and the appended claims, unless the context clearly requires it, the singular forms “a”, “an” and “the” all have the meaning of the plural.

[0036] In this invention, a range may be expressed as “about” a specific value and / or “about” another specific value. When expressing such a range, the other side includes from that specific value and / or to another specific value. Similarly, when a numerical value is expressed as an approximation using the preposition “about”, it should be understood that the specific value constitutes the other side. Furthermore, it should be understood that the endpoints of each range are significant both relative to and independent of the other endpoint.

[0037] This invention provides novel compositions and methods for improving the gut microbiota. The invention identifies bacterial communities closely associated with butyrate production, and compositions and methods for increasing these colonic bacterial communities.

[0038] In view of the content of this invention, those skilled in the art can configure the methods and compositions described herein to meet desired needs. Overall, the disclosed methods and compositions can improve the gut microbiota.

[0039] For example, in some aspects, the method of the present invention can increase one or more colonic bacterial communities with fermentation capabilities and short-chain fatty acid production capabilities.

[0040] In one aspect, the present invention provides a method for improving the gut microbiota. In one embodiment, the method may include selectively enriching a first gut microbiota. For example, the first gut microbiota may include short-chain fatty acid (SCFA) producing bacteria.

[0041] In one embodiment, the method may include the step of administering a composition to a subject. The composition may be an oral or passivated formulation. The composition may selectively enhance one or more gut microbiota.

[0042] For example, in some embodiments of the methods and compositions described herein, administration of a composition comprising allulose, erythritol, or a combination thereof to a subject improves the subject's gut microbiota. In particular, the methods and compositions of the present invention can increase the number of one or more colonic bacterial communities, each selected from the families Trichophyceae or Eubacteriaceae, or any combination thereof. For example, in one embodiment of the methods and compositions of the present invention, administration of allulose, erythritol, or a combination thereof can increase the number of Trichophyceae bacteria. In another embodiment of the methods and compositions of the present invention, administration of allulose, erythritol, or a combination thereof can increase the number of Eubacteriaceae bacteria. In yet another embodiment of the methods and compositions of the present invention, administration of allulose, erythritol, or a combination thereof can increase the number of one or more colonic bacterial communities, each selected from the genus *Corynebacterium* (anaerobic bacteria). Anaerostipes ), Broutella spp. Blautia ), Anaerobic butyric acid bacteria ( Anaerobutyricum) and Eubacterium spp. ( Eubacterium ), and any combination thereof. In another embodiment of the method and composition of the present invention, application of allulose, erythritol, or a combination thereof may increase the number of one or more colonic bacterial communities, each selected from the following species: Hadrus anaerobic cyclophosphamide ( Anaerostipes hadrus ), Broutbacterium ovale ( Blautia obeum ), Halibutyric acid bacteria ( Anaerobutyricum hallii ), Halibut ( Eubacterium hallii ), Proctobacterium rectum ( Eubacterium rectale ), Kalandella ( Eubacterium callanderi ), Mycobacterium mucosae ( Eubacterium limosum ) and maltobacterium ( Eubacterium maltosivorans ), and any combination thereof.

[0043] One or more colonic bacteria may be selected from the following families: Bifidobacteriaceae ( Bifidobacteriaceae ), Rhodotorulaceae ( Coriobacteraceae ), Egertaceae ( Eggerthellaceae Bacteroidetes ( Bacteroidaceae Barnesidae ( Barnesiellaceae ), Prevotellaceae ( Prevotellaceae ), Riken Mycaceae ( Rikenellaceae Tanneraceae ( Tannerellaceae ), Amino acid cocci ( Acidaminococcaceae Clostridium class unclassified families ( Clostridia_u_f Clostridium ( Clostridiaceae Clostridium orders unclassified families ( Clostridiales_u_f ), Erysipelothrix family ( Erysopelotrichaceae ), Eubacteriaceae ( Eubacteriaceae Unclassified families within the phylum Firmicutes ( Firmicutes_u_f ), family Trichophyceae ( Lachnospiraceae ), family Oscillatoria ( Oscillospiraceae ), Rumenaceae ( Ruminococcaceae Veillonaceae ( Veillonellaceae ), Unclassified families in the order Burgholdales ( Burkholderialies_u_f ), Desulfuric Vibrio ( Desulfovibrionaceae ) and Argmanaceae ( Akkermannsiaceae One or more colonic bacterial communities are preferably selected from any bacteria of the Laminariaceae or Euglenomycetes families. For example, one or more colonic bacteria may be selected from the following genera: *Aquaporinus* (…). Abyssivirga Acetic acid factor bacteria ( ) Acetatifactor ), Polyacetic acid bacteria ( Acetitomaculum ), genus Equisetum ( Aequitasia ), genus of bacteria ( Agathobacter ), genus *Femmomonas* Alitiscatomonas ), Anaerobic bacteria ( Anaerobium ), Anaerobic butyric acid bacteria ( Anaerobutyricum ), Anaerobic column bacteria ( Anaerocolumna ), Anaerobic microtrichum ( Anaeromicropila ), Anaerobic Peptidates ( ) Anaeropeptidovorans ), Anaerobic saccharophiles ( Anaerosacchariphilus ), Anaerobic Bacillus spp. Anaerosporobacter ), Anaerobic Corynebacterium ( Anaerostipes ), Anaerobic zoster bacteria ( Anaerotaenia ), Anaerobic marker bacteria ( Anaerotignum Human gastric microorganisms ( ) Anthropogastromicrobium ), weight loss bacteria ( Bariatricus ), Broutella spp. Blautia ), Bile-decomposing bacteria ( Bilifractor ), genus *Plasma* ( Brotolimicola ), genus *Hydrocetes* ( Brotonthovivens ), Butyric acid bacteria ( Butyribacter ), Vibrio butyricum ( Butyrivibrio ), spp. of cecum ( Caecibacterium ), Streptomyces ( Catenibacillus ), Catanella spp. Catonella ), Cellulose-degrading bacteria ( Cellulosilyticum ), genus *Streptococcus* Chordicocus ), genus *Plasmodium* Coprococcus ), Cuneiformis ( Cuneatibacter ), genus *Dystrophus* Diplocoster ), Dorebrospinal fungi ( Dorea ), Eisenbergella spp. Eisenbergiella ), Clostridium ( Enterocloster ), Eubacterium spp. ( Eubacterium ), Exobacteria ( Extibacter ), fecal streptococci ( Faecalicatena ), genus *Plasmodium* Faecalimonas ), Fusarium spp. Falcatimonas ), Frisiencoccus ( Frisingicoccus ), Clostridium spp. Fusicatenibacter ), Clostridium ( Fusimonas ), Chicken intestinal microorganisms ( Gallinestinimicrobium ), Gumobacterium ( Gluceribacter Herbaceous fungi ( Herbinix ), Hesperia ( Hespellia ), human visceral bacteria ( Hominisplanchenecus ), Human fecal adaptable bacteria ( Hominisplanchenecus ), Howard spp. Howardella ), Genus *Jingyao* ( Jingyaoa ), genus Johnson ( Johnsonella ), Polytonymycium ( Jutongia ), genus *Ketromynium* Kineothrix ), genus Conate ( Konateibacter ), genus *Hymenobacter* ( Lachnoanaerobaculum ), genus Trichophyton ( Lachnobacterium ), Clostridium ( Lachnoclostridium ), genus *Trichophyton* Lachnospira ), genus Trichophyton ( Lachnotalea ), Lacrimal spp. Lacrimispora ), genus *Sphagnum* ( Laedolimicola ), genus *Lendrobium* Lientehia ), genus *Photobacterium* Luxibacter ), Malvin Bryantella ( Marvinbryantia ), Mediterranean bacteria ( Mediterraneibacter ), genus *Plasmodium* Merdimonas ), Animalia spp. Mobilisporobacter ), Genus *Aeromonas* ( Mobilitalea ), Morilla spp. Moryella ), Mullicomes genus ( Muricomes ), Muricola genus ( Muricoprocola ), Mullie ( Murimonas ), Niameyella ( Niameybacter ), Neosynostococcus ( Novisyntrophococcus ), spp. of halophilic anaerobic bacteria ( Natranaerovirga ), genus *Euphorbia* Ohessyouella Oliver Pabstella ( ) Oliverpabstia ), Oral bacteria ( Oribacterium ), Ottura ( Otoolea Parabroutella ( Parablautia ), Parasporobacterium spp. Parasporobacterium Petra Luteinella spp. Petralouisia ), Pigsty Fungi ( Piglet ), Vibrio pseudobutyricum ( Pseudobutyrivibrio ), Qiania, Robinsonia Robinsonella ), genus Rocheria ( Rosemary ), genus *Schedlerus* Schaedlerella ), spp. of Celimonales Sellimonas ), Shatterworthia spp. Shuttleworthia ), genus *Sima* ( Monkey ), Bacillus spp. Sporobacterium ), spore-forming bacteria ( Sporoforming ), stomatitis ( Stomatobacillus ), genus *Stachys* ( Self-sufficient ), Pig-preferred bacteria ( Self-willed ), spp. of porcine coli Swanthocolla ), symbiotic cocci ( Syntrophococcus ), genus *Tizella* ( Tyzzerella ), Polyformobacteria ( Variimorphobacter ), genus *Vescirrhosa* Velocimicrobium ), Walterbacterium ( Walter ), Vansuga ( Wansuia ), genus *Wu* ( Wujia ), Genus *Zhenheng* ( Zhenhengia Acetobacter spp. Acetobacterium ), Alkalibacterium spp. Alkalibacter ), basophilic bacilli ( Alkali stick ), Aminomonas spp. Little girl ), Anaerobic Species ( Anaerobic ), Eubacterium spp. ( Eubacterium ), Bacillus spp. Gallibacter ), Garcia genus ( Garciella ), Enterobacteriaceae ( Intestinal bacillus ), Irregular Bacillus spp. Irregularibacter ), genus *Morgiella* ( Mogibacterium ), Pseudomycium spp. Mogibacterium ), rod-shaped anaerobic bacteria ( Rhabdanaerobium ) and its combinations.

[0044] In some embodiments, one or more colonic bacterial groups include a species selected from the genus *Corynebacterium*, such as *Amylophilus*, *Butyrate-producing*, etc. A. butyricus ), fecal anaerobic bacteria ( A. poop ), fecal enteroanaerobic corynebacteria ( A. faecalis ), fecal anaerobic corynebacteria ( A. faeces ), strong anaerobic corynebacteria ( A. hadrus ), human anaerobic corynebacteria ( A. human ), Rhamnopyrenoidospora ( A. rhamnose-eating ), and their combinations.

[0045] In some embodiments, one or more colonic bacterial groups include a species selected from the genus *Brutella*, such as *Brutella acetogenifer*. B. acetigignens ), ammoniabroutella ( B. ammoniolytica Arginine Brontë ( B. argi ), Brookings Broutbacterium ( B. brookingsii ), Brautella cecum ( B. blind people ), Rapid Broutella ( B. celeris ), Brutus cocci ( B. coccoides ), Broutbacillus diversicolor ( B. difficult ), Brautella fecalis ( B. faecalis ), fecal braurates ( B. faeces ), glucosbroutella ( B. gluceracea ), Hansenbroutella B. hansenii ), human Brontë (B. of man ), Blotchella hygrophila ( B. hydrogenotrophica ), Enterobacter baumannii ( B. intestinalis ), Broutella dysenteriae ( B. slow ) 、 liquid Brontë bacteria ( B. liquor ), Mubrautella ( B. clay ), Broutbacterium burmannii ( B. marasmi ), Masseybroutella ( B. massiliensis ), Brutus obese B. I will die. ), Forsabroutella B. phocaeensis ), product Broutella ( B. products ), Provence Broutella ( B. provencensis ), Pseudococcus Broutella ( B. pseudococcoides ), Schenckbroutella ( B. skink ), Lazybroutella ( B. lazy ), fecal braurates ( B. dung ), Broutella dysenteriae ( B. tarda ), Wexlerbroutella B. wexlerae ), and their combinations.

[0046] In some embodiments, one or more colonic bacterial communities include a species selected from the genus *Anaerobes*, such as butyrate-producing anaerobic butyrate bacteria (A. butyrate-producing anaerobic butyrate bacteria). A. butyricum ), Harvey anaerobic butyric acid bacteria ( A. hallii ), Pinellia anaerobic butyric acid bacteria ( A. soehngenii ), fecal anaerobic butyric acid bacteria ( A. faecalis ), chicken manure anaerobic butyric acid bacteria ( A. stercoripullorum ), fecal anaerobic butyric acid bacteria ( A. dung beetle ) and their combinations.

[0047] In some embodiments, the one or more colonic bacterial communities include a species selected from the genus Eubryl, such as Eubryl acidophilus (… E. acidaminophilum Aggregates ( ) E. aggregans ), Leukobacterium ( E. from Albania ), narrow-banded bacillus ( E. angustum Pasteurella multocida ( ) E. barkeri ), Proteus mirabilis ( E. brachy Budae bacteria ( E. budayi ), Kalandella ( E. callanderi ), fibrinolytic bacteria ( E. cellulolytic ), Combibloc ( E. combesii ), coprosterol-producing bacteria ( E. coprostanologens ), Escherichia coli ( E. difficile ), Longerobacterium ( E. dolichum ), and preferred Eubryl bacillus ( E. choosing ), Halibut ( E. hallii It has now been renamed A. hallii ), Human Eugenics ( E. of man ), Weak Eubacterium ( E. weak ), Limiting bacteria ( E. limosum ), Maltodactylophilic bacteria ( E. maltose-eating ), Microbes ( E. minute ), Polymorphonuclear Bacillus ( E. multiforme ), Nitrite-producing Eubryl bacillus ( E. nitritogenes ), Nodularia esculenta ( E. knotted ), Oxidoreduction Escherichia coli ( E. oxidation-reduction ), Eucommia ulmoides ( E. plexicaudatum ), E. pyruvate ( E. pyruvate-eating ), Microbacterium tumefaciens ( E. ramulus ), Reindeer Bacterium ( E. rangiferina ), Proctobacterium rectum ( E. rectal ), Rumeneobacterium ( E. ruminantium Cryptococcus ( ) E. saphenous ), Delayed Escherichia coli ( E. slowly ), Cystobacter sylvatinib ( E. siraeum ), Gyreobacterium glomerulosa ( E. sulci ), Tarantella eugenol ( E. tarantellas ), Bacillus subtilis ( E. thin ), Thermopylae Herba ( E. thermomarine ), Eutrophus truncatum ( E. tortuous ), uniform Eubacterium ( E. uniform ), Abdominal Bacterium ( E. ventriosum ), xylanophilic bacteria ( E. xylanophilum ), Euribum E. yurii ), and their combinations.

[0048] In some embodiments, the one or more colonic bacterial communities comprise a subset of Actinobacteria (…). Actinobacteria Bacteroidetes ( Bacteroidetes ) and Firmicutes ( Firmicutes One of them.

[0049] In some embodiments, the one or more colonic bacterial communities include one or more species selected from the following: *Vibrio wiltii* (… Dysmosmobacter weibionisUnclassified oscillating fungi ( Oscillospiraceae_u_s Unclassified Oscillatoria species ( Oscillibacter_u_s ), unclassified Clostridium species ( Clostridiaceae_u_s Unclassified species of the Trichophyceae family ( Lachnospiraceae_u_s ), Hachodon anaerobic corynebacterium ( Anaerostipes hadrus Unclassified Collins species ( Collinsella_u_s ), Alisteria wengdedon g ( Alistipes onderdonkii ), Broutbacterium velutipes ( Blautia wexlerae ), xylobiobacterium lysozyme ( Bacteroides xylanisolvens ), Collins gas-producing bacteria ( Collinsella aerofaciens ), Allisteria farungii ( Alistipes finegoldii ), Clostridium adrenaeus ( Enteroclostrum from Alden Unclassified Bacteroides ( Bacteroides_u_s ), Clostridium enterobacteriaceae ( Enteroclostrum clostridial ), Clostridium botulinum ( Enteroclostridium boltea ), long chain Dorsetella ( Golden long-chain ), Masseybroutella ( Blautia massiliensis Unclassified Eubacterium species ( Eubacterium_u_s ), Brutus obese I am dying. Unclassified Broutella species ( Blautia_u_s ), Prevotella ( Faecalibacterium prausnitzii Unclassified Bacteroides ( Subdoligranulum_u_s ), and unclassified rumenococcal species ( Ruminococcus_u_s In some such embodiments, the method or composition comprises allulose.

[0050] In some embodiments, one or more colonic bacterial groups include one or more species selected from the following: *Brutella marsii* (… Blautia massiliensis ), Allisteria farungii ( Common grebe finegolds Sugar-free Lawsonia ( Lawsonibacter asaccharolyticus ), long chain Dorsetella ( Golden long-chain Unclassified fecal bacteria ( Faecalibacterium_u_s Unclassified Bacteroides ( Subdoligranulum_u_s ), Hachodon anaerobic corynebacterium ( Anaerostipes hadrus ), Alisteria wenddonkie ( Alistipes onderdonkii Unclassified Collins spp. Collinsella_u_s ), Broutbacterium velutipes ( Blautia wexlerae Unclassified Trichophyceae ( Lachnospiraceae_u_s Bifidobacterium longum ( Bifidobacterium longum), Clostridium botulinum ( Enterocloster bolteae ), Clostridium enterobacteriaceae ( Enterocloster clostridioformis ), xylobiobacterium lysozyme ( Bacteroides xylanisolvens Unclassified Bacteroides ( Bacteroides_u_s ), Clostridium adrenaeus ( Enterocloster aldensis Unclassified Eubacterium genus ( Eubacterium_u_s ), unclassified Clostridium species ( Clostridium_u_s ), and Alisteria putrefaction ( Alistipes putredinis In some such embodiments, the method or composition comprises erythritol.

[0051] In certain embodiments of the methods and compositions of this invention, administration of a composition containing allulose to a subject can improve their gut microbiota. Specifically, the methods and compositions of this invention can increase the number of one or more colonic bacterial communities selected from the Trichophyceae family, or any combination thereof. For example, in one embodiment of the methods and compositions of this invention, administration of allulose, erythritol, or a combination thereof can increase the number of Trichophyceae bacteria. In another embodiment of the methods and compositions of this invention, administration of allulose, erythritol, or a combination thereof can increase the number of one or more colonic bacterial communities, each selected from the genera *Corynebacterium*, *Brouteria*, and *Anaerobacter*, and any combination thereof. In another embodiment of the methods and compositions described herein, administration of allulose, erythritol, or a combination thereof can increase the number of one or more colonic bacterial communities, each selected from the genera *Hardrus*, *Brouteria*, and *Haloxybutyricum*, and any combination thereof.

[0052] In some embodiments of the methods and compositions of the present invention, administration of a composition comprising erythritol can improve the gut microbiota of a subject. Specifically, the methods and compositions of the present invention can increase the number of one or more colonic bacterial communities selected from the Eubacteriaceae family, or any combination thereof. In another embodiment of the methods and compositions of the present invention, administration of allulose, erythritol, or a combination thereof can increase the number of Eubacteriaceae. In another embodiment of the methods and compositions of the present invention, administration of allulose, erythritol, or a combination thereof can increase the number of one or more colonic bacterial communities, each selected from the Eubacteria genus. In another embodiment of the methods and compositions of the present invention, administration of allulose, erythritol, or a combination thereof can increase the number of one or more colonic bacterial communities, each selected from the following species: *Eubacterium halibutyricum*, *Eubacterium rectum*, *Eubacterium karlandii*, *Eubacterium mucinum*, and *Eubacterium maltose*.

[0053] In certain embodiments of the methods and compositions of the present invention, administration of a composition comprising allulose and erythritol can improve the gut microbiota of a subject. Specifically, the methods and compositions of the present invention can increase the number of one or more colonic bacterial communities, each selected from the families Trichophyceae or Eubacteriales, or any combination thereof. For example, in one embodiment of the methods and compositions of the present invention, administration of allulose, erythritol, or a combination thereof can increase the number of Trichophyceceae. In another embodiment of the methods and compositions of the present invention, administration of allulose, erythritol, or a combination thereof can increase the number of Eubacteriales. In yet another embodiment of the methods and compositions of the present invention, administration of allulose, erythritol, or a combination thereof can increase the number of one or more colonic bacterial communities, each community being selected from the genera *Corynebacterium*, *Brutella*, *Butyricum*, and *Eubacterium*, and any combination thereof. In another embodiment of the method and composition of the present invention, application of allulose, erythritol, or a combination thereof may increase the number of one or more colonic bacterial communities, each selected from the following species: *Hadrossella anaerobicis*, *Brutella ovalis*, *Helella anaerobicbutyricum*, *Helella harlequinae*, *Helella rectovalis*, *Helella karlandella*, *Helella myxobacterium*, and *Helella maltose*, and any combination thereof. In a preferred embodiment of the method and composition of the present invention, the colonic bacterial community comprises one or more (i.e., two or more, three or more, etc.), each selected from the family Helicobacteriaceae or Helicobacteraceae, and the genera *Hadrossella*, *Brutella*, *Helella anaerobicbutyricum*, and *Helella harlequinae*, and the following species: *Hadrossella anaerobicis*, *Brutella ovalis*, *Helella anaerobicbutyricum* (formerly known as *Helella harlequinae*). Eubacterium hallii The number of *E. rectum*, *E. karland*, *E. mucosa*, and *E. maltose* increased.

[0054] An improved gut microbiota and / or an increased population of one or more bacteria selected from the families Trichophyceae or Eurybacteriaceae may help prevent, treat, or improve diseases, including but not limited to metabolic syndrome, obesity, type 2 diabetes (T2D), insulin resistance, hyperlipoproteinemia, hyperuricemia, hepatic steatosis, hypercholesterolemia, hypertriglyceridemia, irritable bowel syndrome (IBS), colon cancer, allergies, non-alcoholic fatty liver disease (NAFLD), inflammatory bowel disease (IBD), cardiovascular disease (CVD), inflammation, and other diseases.

[0055] In certain embodiments of the methods and compositions of the present invention, compared with untreated subjects, one or more colonic bacterial communities (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%. In some such embodiments, the increase in colonic bacterial communities does not exceed about 500%. In other such embodiments, the increase in colonic bacterial communities does not exceed about 400%. In other such embodiments, the increase in colonic bacterial communities does not exceed about 300%. In other such embodiments, the increase in colonic bacterial communities does not exceed about 200%. In other such embodiments, the increase in colonic bacterial communities does not exceed about 100%. In certain embodiments of the methods and compositions of the present invention, compared with untreated subjects, one or more colonic bacterial communities (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%, respectively. This means that in some cases, these bacteria may be affected independently of each other at different rates (e.g., the community of one bacterium may increase by 50%, while another bacterium may only increase by 25%). In some such embodiments, the increase in the number of each colonic bacterium population does not exceed about 500%. In other such embodiments, the increase in the number of each colonic bacterium population does not exceed about 400%. In other such embodiments, the increase in the number of each colonic bacterium population does not exceed about 300%. In other such embodiments, the increase in the number of each colonic bacterium population does not exceed about 200%. In other such embodiments, the increase in the number of each colonic bacterium population does not exceed about 100%.

[0056] In certain embodiments of the methods and compositions of the present invention, the proportion of one or more colonic bacterial communities (e.g., as described above) to 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%. In some such embodiments, the increase in the proportion of one or more colonic bacterial communities as a percentage of total colonic bacteria does not exceed about 700%. In other such embodiments, the increase in the proportion of one or more colonic bacterial communities (as a percentage of the total colonic bacteria) does not exceed about 600%. In other such embodiments, the increase in the proportion of one or more colonic bacterial communities (as a percentage of the total colonic bacteria) does not exceed about 500%. In other such embodiments, the increase in the proportion of one or more colonic bacterial communities (as a percentage of the total colonic bacteria) does not exceed about 400%. In certain embodiments of the methods and compositions of the present invention, compared with an untreated subject, the proportion of each of one or more colonic bacterial communities (e.g., as described above) (i.e., as a percentage of the 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%. This means that in some cases, these bacteria may be affected independently of each other at different rates (e.g., the proportion of one bacterial community may increase by 50%, while another bacterial community may increase by only 25%). In some such embodiments, the increase in each proportion does not exceed about 500%. In other such embodiments, the increase in each proportion does not exceed about 400%. In other such embodiments, the increase in each proportion does not exceed about 300%. In other such embodiments, the increase in each proportion does not exceed about 200%. In other such embodiments, the increase in each proportion does not exceed about 100%.

[0057] In certain embodiments of the methods and compositions of the present invention, the dosage of allulose, erythritol, or combinations thereof is at least about 0.1 g / day. For example, in some embodiments, the dosage of allulose, erythritol, or combinations thereof is 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 not exceeding about 100 g / day, or not exceeding 75 g / day, perhaps not exceeding 25 g / day, 10 g / day, or even 5.0 g / day.

[0058] For example, in some embodiments of the methods and compositions of the present invention, the dosage range of allulose, erythritol, or combinations thereof is from about 3 g / day to about 100 g / day. In other embodiments of the methods and compositions of the present invention, the dosage range of allulose, erythritol, or combinations thereof is from about 0.1 g / day to about 35 g / day, or from about 0.12 g / day to about 25 g / day. In other embodiments of the methods and compositions of the present invention, the dosage range of allulose, erythritol, or combinations thereof is from about 0.5 to about 6.5 g / day, from about 0.5 to about 4.0 g / day, from about 0.5 to about 3.0 g / day, from about 0.5 to about 2.0 g / day, from about 1.0 to about 6.5 g / day, from about 1.0 to about 4.0 g / day, from about 1.0 to about 3.0 g / day, from about 1.5 to about 6.5 g / day, from about 1.5 to about 4.0 g / day, from about 1.5 ... 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 of the present invention, allulose, erythritol, or combinations thereof are administered at doses 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 of the present invention, the dosage range of allulose, erythritol, or combinations thereof is about 11 to 20 g / day. In other embodiments of the methods and compositions of the present invention, the dosage of allulose, erythritol or combinations thereof is 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.

[0059] Within a specific day, the dosage can be divided into any number of doses. For example, in one embodiment of the method and composition of the invention, allulose, erythritol, or a combination thereof are administered once daily (e.g., in a single dose). In other embodiments of the method and composition of the invention, allulose, erythritol, or a combination thereof are administered multiple times daily, such as twice or three times daily (e.g., divided into multiple doses, such as in two or three doses daily). When multiple administrations or divided doses are required, the above-mentioned daily dosage can be allocated according to the number of administrations or doses to provide a moderate and well-tolerated dose per dose (i.e., without causing severe bloating, flatulence, borborygmus, abdominal cramps, diarrhea, nausea, and / or vomiting).

[0060] In another embodiment, the present invention provides a method for increasing one or more colonic microbiota and enhancing SCFA (e.g., butyrate) production in a subject, the method comprising oral administration of allulose, erythritol, or a combination thereof. In some embodiments, the administration may be as described herein.

[0061] To improve the gut microbiota, the composition can be administered orally or non-orally. In one embodiment, the composition can be a food, beverage, dietary supplement, or pharmaceutical preparation. In one embodiment, the composition can be in the form of suppositories, tablets, pills, granules, powders, films, microcapsules, aerosols, liniments, tinctures, tonics, liquid suspensions, or syrups. The dosage of the composition can be from about 5.0 mg / kg body weight to about 40.0 mg / kg body weight.

[0062] In some embodiments of the methods and compositions of the present invention, the subjects are mammals. In one embodiment of the methods and compositions of the present invention, the subjects are humans, such as non-adults (e.g., aged about 2 to 20 years, or about 13 to 19 years), or older humans (e.g., at least about 45 years, at least about 50 years, at least about 60 years, at least about 70 years, at least about 80 years, or even at least about 90 years, especially older women). Therefore, in some embodiments, the methods and compositions of the present invention can be used in subjects (e.g., patients with type 2 diabetes) who are particularly likely to benefit from increased SCFA (e.g., butyrate) production.

[0063] In some embodiments of the methods and compositions of the present invention, butyric acid production is increased by at least about 0.5% compared to an untreated subject. In some embodiments of the methods and compositions of the present invention, butyric acid 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%. In other embodiments of the methods and compositions of the present invention, butyric acid production is increased by at least about 20%, or at least about 25%, compared to an untreated subject. In some embodiments of the methods and compositions of the present invention, butyric acid production is increased by at least about 20%, at least about 25%, at least about 30%, or at least about 35%, compared to an untreated subject. In some such embodiments, butyric acid production is increased by no more than about 200% compared to an untreated subject. In other such embodiments, butyric acid production is increased by no more than about 100% compared to an untreated subject. In other such embodiments, butyric acid production is increased by no more than about 50% compared to an untreated subject. The time for butyric acid production can be, for example, within the range of 0-24 hours, 24-48 hours, or 48-72 hours. In a preferred embodiment, the increase in butyric acid production occurs within the range of 0-48 hours after application.

[0064] The effects of improving the gut microbiota, increasing one or more colonic bacterial communities, and / or increasing butyrate production are expected to apply to both humans and animals, thus enabling applications in food and animal feed. Representative non-human animals include livestock such as horses, chickens, turkeys, cattle, cows, pigs, sheep, goats, camels, bison, cats, dogs, rodents, rabbits, hamsters, and birds.

[0065] Application can be carried out for a long period of time, such as at least about one week, at least about two weeks, at least about three weeks, at least about four weeks, at least about seven weeks, or even at least about 52 weeks. Those skilled in the art will understand that during such a long period of application, some application days may be “missed”; ideally, the number of missed days should be less than about 10% of the total number of application days.

[0066] Another embodiment of the invention is a composition comprising at least about 0.1 g of allulose, erythritol, or a combination thereof per serving. For example, certain embodiments of the compositions of the invention comprise 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 some such embodiments, the composition comprises 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. For example, the composition may be provided as a food composition as described below. In other embodiments, the composition is provided as a nutritional supplement. Such compositions can be used to carry out the methods of the invention.

[0067] In some embodiments of such compositions of the present invention, the amount of each serving may be, for example, at least about 75 g, at least about 150 g, or even at least about 200 g. In some embodiments, the amount of each serving does not exceed about 1000 g, or even not more than about 500 g. For example, in one embodiment, the amount of each serving is in the range of about 75 mL to about 1000 mL. In some embodiments, each serving is individually packaged. In other embodiments, multiple servings are packaged together and accompanied by a description of the edible serving size of each serving and / or the amount of allulose, erythritol, or a combination thereof contained in each serving as described in the present invention.

[0068] Another embodiment of the invention is a composition comprising allulose, erythritol, or combinations thereof in amounts 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%, and even at least about 40% (by weight). However, in some such embodiments, the maximum content of allulose, erythritol, or combinations thereof in the composition does not exceed about 75%, and even does not exceed about 50% (by weight). For example, the composition may be provided as a food composition as described below. For example, the composition may be provided per serving and / or per serving containing allulose, erythritol, or combinations thereof as described in the invention.

[0069] Another embodiment of the invention is a composition comprising one or more (e.g., two or more, or three or more) bacterial communities, each selected from the group consisting of *Trichophyton* and *Eucrobacter* families, the group consisting of the genus *Anaerobacter*, *Brutella*, and *Anaerobacter*, or selected from the following species: *Hardrus*, *Brutella ovalis*, *Haloxybutyricum* (formerly *Eucrobacter hali*), *Eucrobacter rectum*, *Eucrobacter karlandii*, *Eucrobacter mucosae*, and *Eucrobacter maltose*. One or more bacterial communities may function as probiotics, for example. In some embodiments, the composition of the invention may contain allulose, erythritol, or a combination thereof (e.g., in the amounts described above). However, in other embodiments, the composition does not contain allulose, erythritol, or a combination thereof. Such embodiments can be used, for example, added to or co-administered with compositions containing allulose, erythritol, or a combination thereof, such that the bacterial community in the composition is present in the colon simultaneously with allulose, erythritol, or a combination thereof. Therefore, in some embodiments, subjects can enjoy the benefits of allulose, erythritol, or a combination thereof with the bacterial community described herein, without taking a single formulation containing allulose, erythritol, or a combination thereof, and the bacterial community. Similarly, products suitable for enjoying the benefits of allulose, erythritol, or a combination thereof, with the bacterial community described herein, can be formulated without containing allulose, erythritol, or a combination thereof, and the bacterial community.

[0070] For example, in some embodiments of the compositions of the present invention, the compositions comprise one or more (e.g., two or more, or three or more) bacterial communities, each community being selected from the families Trichophyceae and Eubacteriaceae, the genera *Anaerobic Entomopathogenicus*, *Broutella*, and *Anaerobic Butyric Acid Bacteria*, or selected from the following species or any combination thereof: *Anaerobic Entomopathogenicus Hadrus*, *Broutella ovalis*, *Anaerobic Butyric Acid Bacteria* (formerly known as *E. halibutyricum*), *E. rectum*, *E. karland*, *E. mucin*, and *E. maltose*.

[0071] In embodiments of the present invention, administration of a composition containing allulose to a subject can enhance one or both colonic bacterial communities selected from Hadrus anaerobic cyclophosphamide and Broutella ovalis.

[0072] In embodiments of the present invention, administration of a composition containing erythritol to a subject can enhance the colonic bacterial community selected from one or more strains of Hale anaerobic butyric acid bacteria, E. karlandella, E. myxobacterium, and E. maltose.

[0073] In embodiments of the present invention, administration of a composition comprising allulose and erythritol to a subject may increase one or two colonic bacterial communities selected from *Hadross anaerobic Entomopathogenicus* and *Brutella ovalis*, as well as one or more colonic bacterial communities selected from *Haylorhizium anisopliae*, *Eucalyptus karlandii*, *Eucalyptus myxobacterium*, and *Eucalyptus maltose*.

[0074] A composition comprising both a prebiotic (alokulose, erythritol, or a combination thereof) and a probiotic (one or more (e.g., two or more, or three or more) bacterial communities) is called a synbiotic composition. The synbiotic products of this invention are innovative combinations of probiotic bacteria and prebiotic compounds, believed to produce a synergistic effect. In some embodiments of this invention, the synbiotic product is a composition comprising at least one bacterium selected from the families Trichophyceae and Euglenomycetes, and allulose, erythritol, or a combination thereof. Such synbiotic products can be used as ingredients in food and animal feed.

[0075] In some embodiments, the moisture content of the Biostime product is less than about 10%. In some embodiments, the moisture content of the Biostime product is less than about 9%. In some embodiments, the moisture content of the Biostime product is less than about 8%. In some embodiments, the moisture content of the Biostime product is less than about 7%. In some embodiments, the moisture content of the Biostime product is less than about 6%. In some embodiments, the moisture content of the Biostime product is less than about 5%. In some embodiments, the moisture content of the Biostime product is from about 1% to about 10%. In some embodiments, the moisture content of the Biostime product is from about 5% to about 10%. As used herein, moisture content is determined according to the Karl Fischer titration method of ASTM D6869.

[0076] Synbiotic product compositions can be prepared by mixing a certain amount of dried spore component with a certain amount of dried prebiotic component. Alternatively, liquid prebiotic component can be mixed with liquid probiotic spore component, and the mixture can be dried, for example by spray drying. Those skilled in the art will recognize that other alternative methods commonly used in commercial manufacturing are also practical and effective.

[0077] Of course, those skilled in the art will understand that compositions comprising specific bacterial community combinations of the present invention may further comprise other bacterial communities, whether described in other parts of the invention or in other forms. For example, the composition may also comprise one or more bacterial communities selected from the genera Bacteroides (Bacteroides). Bacteroides ), Butyric acid bacteria ( Butyricicoccus ), Oscillatoria ( Oscillibacter ), Diplococcus ( Dialister ), Parabacterium genus ( Parabacteroides ), Aristippus ( Alistipes ), Anaerobic cocci ( Anaerococcus ), Streptomyces ( Catenibacterium ), Clostridiales ( Clostridiales Lactobacillus ( ) Lactobacillus Bifidobacterium spp. Bifidobacterium Rumenococci ( Ruminococcaceae ) and Omani genus ( Akkermansia ).

[0078] For example, the composition may be provided as a food composition as described below. In other embodiments, the composition is provided as a nutritional supplement. In still other embodiments, the composition is provided as an ingredient for mixing with a food composition during processing or cooking, or when consumed. For example, the composition may contain allulose, erythritol, or a combination thereof at the concentrations described in this invention, and in portions and / or in portions of allulose, erythritol, or the combination thereof. Those skilled in the art can adjust the amount of bacterial communities added to the composition to meet desired requirements. Generally, the content of each bacterial community may be from about 1 × 10³ to about 1 × 10¹. 0 CFU (colony-forming units). In some embodiments, the bacterial community content is approximately 1 × 10⁻⁶. 5 To approximately 1×10¹ 0 CFU, or approximately 1×10 6 To approximately 1×10 10 CFU, or approximately 1×10 7 To approximately 1×10 10 CFU, or approximately 1×10 8 To approximately 1×10 10 CFU, or approximately 1×10 3 To approximately 1×10 8 CFU, or approximately 1×10 4 To approximately 1×10 8 CFU, or approximately 1×10 5 To approximately 1×10 8 CFU, or approximately 1×10 6 To approximately 1×10 8 CFU, or approximately 1×10 5 To approximately 1×10 7 CFU, or approximately 1×10 4 CFU, or approximately 1×10 5 CFU, or approximately 1×10 6 CFU, or approximately 1×10 7 CFU, or approximately 1×10 8 CFU, or approximately 1×10 9 CFU, or approximately 1×10 10CFU.

[0079] The synbiotic products of this invention can be used as ingredients or components in food products (such as human food or animal feed). Such foods contain a certain amount of the synbiotic product, allowing for the intake of an effective dose of the synbiotic product when consumed. Methods for adding ingredients to food are known in the art and vary depending on the type of food being prepared. Although some foods are traditionally advertised as containing prebiotics or probiotics, the types of food involved in this invention are not limited to a specific type. Representative, non-limiting examples of certain embodiments of foods containing synbiotic products include dietary supplements, nutrition bars, external clinical nutrition preparations, infant foods, and coatings for dried foods, such as baked goods or chewing gum.

[0080] In embodiments of the present invention, the composition is preferably an edible composition.

[0081] Another embodiment of the invention further comprises a composition as described above containing one or more mineral species. For example, each mineral may be a divalent mineral species, or a mineral selected from calcium, magnesium, copper, potassium, zinc, and iron species. For example, in one embodiment, the composition comprises calcium. In another embodiment, the composition comprises calcium and / or magnesium. In yet another embodiment, the composition comprises calcium, magnesium, and / or iron. The mineral species may be provided, for example, in the form of salts, such as carbonates, halides, or bicarbonates. For example, calcium may be provided in the form of calcium carbonate or calcium gluconate. The content of the mineral (e.g., calcium) may be: at least about 50 mg, at least about 100 mg, at least about 250 mg, at least about 500 mg, or even at least about 1000 mg per dose or serving. In some such embodiments, the calcium content per dose or serving is less than about 2000 mg, or even less than about 1000 mg. For example, the composition may be provided as a food composition as described below. In other embodiments, a composition as a nutritional supplement is provided. For example, the composition may contain the concentration, amount per part, and / or content of allulose, erythritol, or a combination thereof as described in this invention.

[0082] In other embodiments, the compositions of the present invention do not contain the mineral species described above.

[0083] Another embodiment of the invention further comprises the composition described above containing 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 some embodiments, the invention provides the composition described above, wherein the prebiotic content is in the range of 0.025 g to 30 g. In some embodiments, the prebiotic content is about 0.1g to about 20g, or about 1g to about 10g, or about 0.1g to about 5g, or about 1g to about 5g, or about 5g to about 10g, or about 5g to about 8g, or about 2g to about 8g, or about 2g to about 5g, or about 2g to about 8g, or about 0.05g, or about 0.1g, or about 1g, or about 2g, or about 5g, or about 8g, or about 10g.

[0084] In one embodiment, the composition of the present invention does not contain one or more of the aforementioned additional prebiotics. For example, in one embodiment, the composition of the present invention does not contain one or more prebiotics selected from the group consisting of inulin, lactulose, fructooligosaccharides, galactooligosaccharides, mannose, larch arabinogalactan, xylooligosaccharides, polydextrose, resistant maltodextrin, and tagatose. In another embodiment, the composition of the present invention does not contain inulin. In yet another embodiment, the composition of the present invention does not contain pullulan.

[0085] Those skilled in the art will understand that the compositions of the present invention can be used to implement the methods described in other parts of the present invention.

[0086] The terms "edible" and "edible composition" are used broadly in this invention to encompass a wide range of substances that can be ingested by humans, such as foods, beverages, and pharmaceutical and nutritional supplement dosage forms such as syrups, powders, capsules, or tablets. The terms "food" and "food composition" are used more narrowly to specifically refer to foods, beverages, and their ingredients. Applicable food compositions may take many forms, including but not limited to baked goods, breakfast cereals, dairy products, soy products, confections, jams and jellies, beverages (powdered and / or liquid), smoothies, fillings, yogurt (milk-containing and non-milk-containing yogurt), kefir, extruded and compressed snacks, gelatin desserts, snack bars, meal replacements and energy bars, cheese and cheese sauces (milk-containing and non-milk-containing cheeses), edible and water-soluble films, soups, syrups, table sweeteners, nutritional supplements, sauces, dressings, creamers, icing, ice cream, frosting, glazes, pet food, tortillas, meat and fish, dried fruit, infant food, and batter and breading coatings.

[0087] To make this food product suitable for use as a flavor enhancer in food formulations, it is often best to also include natural and artificial flavorings. 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 syrup, rum, butter, and combinations thereof.

[0088] In some embodiments, the composition is in the form of an aggregated powder, such as a powder similar to that used to make powdered beverages and nutritional supplements.

[0089] In order to make the food product suitable for use as a sweetener composition in food, in many cases it is preferable to also contain 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.

[0090] Those skilled in the art will understand that allulose, erythritol, or combinations thereof can be provided in a variety of different physical forms, such as powders, granules, agglomerated powders, syrups, or concentrated syrup solids. In one embodiment, allulose, erythritol, or combinations thereof are in granular form. These granules may be held together by an adhesive, such as an adhesive composition containing a large amount of maltodextrin.

[0091] Example Example 1 - Study on the interaction between non-nutritive sweeteners and gut microbiota Allulose (DOLCIA PRIMA® DS NG crystalline form) and erythritol (ERYTESSETM 20-60M erythritol) underwent experiments simulating the human oral, gastric, and small intestinal digestive processes. To test these products at doses that best represent in vivo conditions, several criteria were considered, including maximum dosage, estimated daily intake, acceptable daily intake (ADI), and gastrointestinal tolerability. Based on these criteria, 5 g / day was selected as a representative in vivo oral test dose. To further improve the biorelevance of the test dose, the oral dose was subsequently corrected for the proportion of each test product absorbed in the upper gastrointestinal tract in vivo, thereby calculating the proportion of oral dose reaching the colon (i.e., the in vitro colonic test dose). The absorption rate of allulose was 80%, and that of erythritol was 90%.

[0092] Figure 6 The experimental design scheme was summarized.

[0093] (A) Both test products were incubated in the colon for 48 hours and compared with the untreated control group (NSC).

[0094] (B) The effects of the test product on metabolite production and microbial composition in subjects with type 2 diabetes and healthy adults living with them (n=6 per group) were assessed by sampling and analysis (cited in Van den Abbeele et al., 2023).

[0095] 1.1 Simulation of the oral cavity, stomach, and intestines The digestion of D-allulose and erythritol in the upper gastrointestinal tract (GIT) and their fermentation in the colon were investigated using the SIFR® model. D-allulose and erythritol underwent oral, gastric, and small intestinal digestion processes. The oral, gastric, and small intestinal digestion procedures were simulated according to the latest standardized INFOGEST 2.0 method (Brodkorb et al., 2019), modified to ensure compatibility with subsequent colonic fermentation.

[0096] The INFOGEST 2.0 method is a static digestion method that uses a constant diet-to-digestive fluid ratio and a constant pH value in each digestion step. Samples undergo sequential digestion in the mouth, stomach, and intestines, with parameters such as electrolytes, enzymes, bile, dilution, pH value, and digestion time all set based on existing physiological data. This protocol has been fine-tuned by adding six enzyme activity assays (amylase, pepsin activity, lipase, trypsin, and chymotrypsin) and bile acid quantification, and by removing oxygen during small intestinal incubation to adapt the method for subsequent colonic fermentation experiments.

[0097] 1.2 Dosage Selection Dosage selection was based on a pre-study literature review aimed at identifying physiologically relevant in vivo and in vitro test doses that were representative of actual use and intake. To determine the in vivo oral dose, the following criteria were considered: the upper limits of use set by the European Scientific Committee on Food Additives (ESFA) and the US Food and Drug Administration (FDA), the estimated daily intake for adults, the acceptable daily intake (ADI) (FAO / WHO Joint FAO / WHO Expert Committee on Food Additives), and gastrointestinal tolerability (Table 1). Therefore, representative in vivo oral test doses (g / day; Table 2) were selected. For the in vivo oral dose, adjustments were made based on the absorption ratios of D-allulose and erythrose in the upper gastrointestinal tract (GIT) to calculate the proportion of oral dose reaching the colon, thus determining the in vitro colonic test dose (Table 2). Regarding D-allulose, previous studies have shown that urinary excretion rates after ingestion of doses of 5–20 g / day range from 66% to 86% (Iida et al., 2010; Williamson et al., 2014). Therefore, this study hypothesizes that 80% of D-allulose is absorbed in the upper gastrointestinal tract, resulting in 20% reaching the colon. For erythritol, previous studies have shown that after ingestion of doses of 18-60 g / day, urinary excretion rates are between 78% and 90% (Noda et al., 1994; Bornet et al., 1996). Toxicokinetic studies included in the European Food Safety Authority's (EFSA) scientific opinion on the safety of erythritol indicate that 80%-90% of oral doses are excreted in the urine within 24 hours (EFSA 2015). Therefore, this study hypothesizes that 90% of erythritol is absorbed in the upper gastrointestinal tract, resulting in 10% reaching the colon.

[0098] Table 1. Summary of Dosage Selection Criteria for Determining In Vivo Test Doses of D-Allulose and Erythritol

[0099] Abbreviations: EFSA: European Food Safety Authority; FDA: U.S. Food and Drug Administration; ADI: Acceptable Daily Intake; JECFA; GI: Gastrointestinal; GRAS: Generally Recognized As Safe; BW: Body Weight.

[0100] Table 2. In vivo oral dose (g / day), in vivo small intestinal absorption rate (%) and in vitro colon test dose (g / day) of D-allulose and erythritol.

[0101]

[0102] 1.3 Colon incubation An in vitro kinetic study was conducted to simulate the colonic fermentation of the test product by the gut microbiota of patients with type 2 diabetes (T2D) and cohabiting healthy adults (H) (n = 6 per donor group). Colonic incubation with D-allulose and erythritol was performed on 12 subjects, and samples were collected at 6, 24, and 48 hours.

[0103] The experiment employed the whole-body gut fermentation study (SIFR®) technique. SIFR® is a validated, miniaturized, bioreactor-based, high-throughput in vitro gut microbiome platform that accurately predicts in vivo outcomes (Van den Abbeele et al., 2023).

[0104] Each bioreactor was processed in parallel (simultaneously managed) within a bioreactor management system (Cryptobiotix, Ghent, Belgium). Each bioreactor contained 5 mL of nutrient medium-fecal inoculum mixture supplemented with pre-digestion test products derived from the small intestinal digestion process (D-allulose or erythritol). Each bioreactor was individually sealed and then placed in an anaerobic environment. The nutrient medium was prepared using Blend M0017 (Cryptobiotix, Ghent, Belgium). Each bioreactor contained 5 mL of nutrient medium-fecal inoculum mixture supplemented with digestion test products from a simulated small intestinal digestion protocol, and then each bioreactor was individually sealed and placed in an anaerobic environment. The nutrient medium was prepared using Blend M0017 (Cryptobiotix, Ghent, Belgium). After preparation, the bioreactors were incubated at 37°C with continuous stirring at 140 rpm for 48 hours (MaxQ 6000, Thermo Scientific, ThermoFisher Scientific, Merelbeck, Belgium). After measuring the gas pressure in the headspace, liquid samples were collected for subsequent analysis. Fresh fecal samples were collected following procedures approved by the Ghent University Hospital Ethics Committee (reference number BC-09977).

[0105] 1.4 Donor sourcing The selection criteria for all donors were: age 25-65 years, no antibiotic use in the past 6 months, no gastrointestinal diseases (cancer, ulcers, IBD), no probiotic use, non-smoking, alcohol consumption <3 units / day, and BMI <30. Six pairs of cohabiting adults with similar dietary habits were recruited. One participant in each pair was previously diagnosed with type 2 diabetes (T2D). Cohabiting participants adopted similar diets to reduce the potential impact of long-term dietary differences (an important factor influencing gut microbiota composition) on baseline gut microbiota composition, thus allowing for a more accurate study of the effects of differences in gut microbiota composition and metabolite production between T2D participants and healthy adults.

[0106] The six healthy participants included four men and two women. The participants with type 2 diabetes included two men and four women.

[0107] For each subject, a substrate-free control (NSC) was initiated simultaneously with the start of the test product culture. This NSC consisted of a background culture medium and microbiome without the test product, so that any changes observed between the substrate-free control and the test product culture could be attributed to the presence of the test product.

[0108] 1.5 Quality Control For each donor, NSC culture was performed concurrently with D-allulose and erythrose cultures. NSC ensured that the differences observed between NSC, D-allulose, and erythrose cultures were attributable to the presence of D-allulose and erythrose. For quality control (QC) purposes, NSC cultures were technically triple replicated. The average coefficient of variation for the basic fermentation parameters (pH, gas production, and the three major short-chain fatty acids (acetic acid, propionic acid, and butyric acid)) was 1.75%, confirming the previously established high technical reproducibility of the SIFR® technology (Van den Abbeele et al., 2023).

[0109] 1.6 Basic Fermentation Parameters After measuring the gas pressure in a closed reactor, liquid samples were collected for subsequent analysis. The liquid samples were then acquired and analyzed. A total of 120 samples were analyzed (3 study conditions, 12 donors, 3 time points). Additionally, baseline NSC samples (0 hours) from 12 donors were analyzed, as lactate and SCFA levels were identical at 0 hours under the study conditions. Furthermore, quality control analyses were performed on 24 samples (two additional NSC replicates were added at 48 hours to assess technical reproducibility). n= 3). SCFAs (acetic acid, propionic acid, butyric acid, and valeric acid) and branched-chain fatty acids (bCFAs) (a collective term for isobutyric acid, isovaleric acid, and isohexanoic acid) were detected by gas chromatography-flame ionization (Trace 1300, Thermo Fisher Scientific, Merelbeck, Belgium), with extraction methods as previously reported (De Weirdt et al., 2010); lactic acid was determined enzymatically according to the manufacturer's instructions (Enzytec™, R-Biopharm, Darmstadt, Germany). pH was measured using an electrode (Hannah Instruments Edge HI2002, Termesser, Belgium). After adding 2-methylhexanoic acid as an internal standard, the sample was extracted with diethyl ether. Briefly, 0.5 mL of the sample was diluted 1:3 with distilled water and then acidified with 0.5 mL of 48% sulfuric acid. Subsequently, excess sodium chloride, 0.2 mL of 2-methylhexanoic acid (internal standard), and 2 mL of diethyl ether were added. After homogenization and separation of the diethyl ether and aqueous layers, the diethyl ether extract was obtained and analyzed using a Trace 1300 chromatograph (Thermo Fisher Scientific, Merelbeck, Belgium). This instrument is equipped with a Stabilwax-DA capillary gas chromatograph column, flame ionization detector, and split injector. Nitrogen was used for both the makeup gas and the carrier gas. The injection volume was 1 μL, and the column temperature range was 110–240°C. The injection port temperature was 240°C, and the detector temperature was 250°C. The pH of the sample was determined using an electrode (Hannah Instruments Edge HI2002, Termesser, Belgium). Lactic acid content was determined enzymatically, following the manufacturer's instructions (Enzytec™, R-Biopharm, Darmstadt, Germany).

[0110] 1.7 Metagenomic Analysis In microbial composition analysis, quantitative data were obtained by correcting abundance data (%%); shallow random sequencing (3M reads per sample) for the total cell count (cells / mL; flow cytometry) of each sample, thus estimating the cell count / mL for different taxa. This method generally reveals the impact of interventions on the gut microbiota more accurately (Vandeputte et al., 2017). A total of 120 samples were analyzed (3 study conditions, 12 subjects, 3 time points). In addition, baseline NSC samples (0 hours) from 12 subjects were analyzed because the microbial community composition was identical at 0 hours under the study conditions. Abundance (%%); shallow random sequencing; average 5.54M reads per sample) was corrected for the total cell count (cells / mL; flow cytometry) of each sample to obtain the estimated cell count / mL.

[0111] First, centrifuge 1 mL of the sample at 9000 g for 5 minutes to obtain a bacterial cell pellet. Then, extract DNA using the SPINeasy Soil DNA Extraction Kit (MP Biomedicals, Eschwerg, Germany) according to the manufacturer's instructions.

[0112] Subsequently, DNA libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) and the IDT Unique dual index, with an input of 1 ng of total DNA. Genomic DNA was fragmented using Illumina Nextera XT fragmentase in a proportional manner. Unique dual index was added to each sample, followed by 12 PCR cycles to construct the library. The DNA library was purified using AMPure magnetic beads (Beckman Coulter, Brea, CA), eluted with QIAGEN EB buffer, quantified using a Qubit 4 fluorometer and a Qubit dsDNA HS assay kit, and sequenced at 2 × 150 bp on the Illumina NextSeq 2000 platform. Unassembled sequencing reads were converted to relative abundance (%) using the CosmosID-HUB microbiome platform (CosmosID Inc., Germantown, MD, USA) (Agarwal et al., 2022; Hasan et al., 2014). If at least 80% of the length of the read sequence has a Phred score higher than 17, the read sequence is retained (Brumfield et al., 2021). Host DNA sequence removal was accomplished by mapping the read sequence to a customized human DNA sequence database within CosmosID-HUB (Kalan et al., 2019). Classification and identification employed a K-mer-based algorithm and database developed internally by CosmosID; specific methods are described in the reports of Hourigan et al. (2018) and Leonard et al. (2021).

[0113] 1.8 Cell Count For total cell count analysis, liquid samples were diluted with anaerobic phosphate-buffered saline (PBS), followed by staining with SYTO 16 to a final concentration of 1 µM, and cell counting was performed using a BD FACS Verse flow cytometer (BD, Ehrenbergheim, Belgium). Data were analyzed using FlowJo software (version 10.8.1). Due to differences in cell counts between samples, percentage data (%) were corrected and converted to absolute values ​​(cells / mL). Data processing at the phylum, genus, and family levels was based on absolute value (cells / mL) results.

[0114] 1.9 Metabolomics Liquid chromatography-mass spectrometry (LC-MS) not only enabled relative quantification of each test product but also revealed extensive information on microbial metabolites. In the application group, LC-MS was used to quantify D-allulose and erythrose to determine their fermentation time intervals. A background signal of D-allulose was detected, causing interference with other hexoses (likely fructose) in the background medium. Nevertheless, the detection levels of metabolites identified as D-allulose / hexoses significantly increased after the addition of D-allulose, far exceeding the levels in the background medium. Furthermore, unlike D-allulose, the background hexose was rapidly fermented within 6 hours of incubation. Given that the only difference between NSC and D-allulose is the presence of D-allulose, this indicates that D-allulose can be specifically detected from 6 hours onwards. Analysis was performed using a Vanquish UPLC (Thermo Scientific, Gömmerling, Germany) coupled with an Orbitrap Exploris 240 mass spectrometer (Thermo Scientific, Bremen, Germany) with an electrospray ionization source and both negative and positive ion modes. UPLC was performed using a slightly modified version of the protocol described by Doneanu et al. (2011). Peak area extraction and pretreatment were performed using CompoundDiscoverer 3.3 (Thermo Scientific) software, combined with manual extraction using Skyline 21.1 (MacCoss Lab Software) software based on an internal library (Adams et al., 2020). Compound identification results were reported according to different confidence levels (CL): Level 1 (retention time (compared to internal standards), accurate mass (tolerance of 3 ppm), and MS / MS spectrum), Level 2a (retention time and accurate mass), Level 2b (accurate mass and MS / MS spectrum), and Level 3 (accurate mass only). Technical variability was verified by running a quality control sample (a mixture of all samples) for each pooled injection.

[0115] 1.10 Data Analysis All univariate and multivariate analyses were performed using R software (version 4.2.2; www.r-project.org). Principal component analysis (PCA) was performed using the FactoMineR package (Husson et al., 2014). Linear mixed models were fitted using the lme4 package (Bates et al., 2014) with the following syntax: lmer(short-chain fatty acid ~ treatment * donor group + (1| donor); while effect visualization was performed using the ggeffects package (Lüdecke, 2018). Regularized canonical correlation analysis (rCCA) was performed using the mixOmics package (version 6.20.3) combined with the shrinkage method to estimate the regularization parameter (Rohart et al., 2017). The statistical significance of the supplemental effects of the tested products (D-allulose and erythritol) on metabolites or specific microbial groups at each time point (6, 24, 48 hours) compared to non-carbohydrate (NSC) was assessed by repeated measures ANOVA (based on paired tests in 12 adult subjects), and according to the Benjamin and Hochberg method. p-value correction was performed using Hochberg (1995). Three measures were taken for the microbial composition analysis. First, statistical analysis was performed on the log10 transformed values. Second, following recent research (Van den Abbeele et al., 2023), if a taxa's value was below the limit of detection (LOD), it was considered equal to the overall LOD. Finally, a threshold was set to retain the 100 most abundant species in the analysis, thus avoiding excessive p-value correction. The effects of treatment on 48-hour microbial families and species were reported. Regularized canonical correlation analysis (rCCA) was used to identify correlations between 48-hour metabolites and microbial taxa. rCCA was performed using the mixOmicsR package, and the penalty parameter was estimated using a shrinkage method after central log-ratio transformation of the microbial community matrix. The first three components of rCCA (Rohart et al., 2017; version 6.20.3) were used to generate the correlation matrix.

[0116] 1.11 Results 1.11.1 Fermentation kinetics of erythritol and D-allulose LC-MS analysis revealed the presence of both D-allulose and erythritol in the study group where they were added, thus elucidating the fermentation kinetics. Figure 1A and 1B The concentrations (peak areas) of D-allulose and erythrose in the whole sample at 6, 24, and 48 hours are shown. D-allulose fermented rapidly within 6–24 hours. Erythrose fermented slowly, with high concentrations still observed after 24 hours. Erythrose was mainly fermented within 24–48 hours.

[0117] Figure 1A The D-allulose levels of the entire sample (n=12) after treatment with erythritol or D-allulose were shown at 6 hours, 24 hours and 48 hours of incubation. Figure 1B This shows the situation with erythritol. D-allulose has a background detection signal, which causes it to interfere with other hexoses in the background culture medium.

[0118] 1.11.2 Effects of erythritol and D-allulose on fermentation parameters The effects of erythritol and D-allulose on fermentation parameters are shown in Figures 2A-I for all samples, and are also presented separately for each donor group. Figure 7A In the I-I group, treatment with D-allulose significantly increased the total SCFA content, primarily due to a significant increase in butyrate and acetate production compared to NSC at 24 and 48 hours (see Figures 2D and 1F). In donor-specific analyses, the effects of D-allulose on butyrate and acetate were consistent in both healthy subjects and subjects with type 2 diabetes mellitus (T2DM). Figure 7D and 7F In all analyses, erythritol did not affect the total SCFA concentration at any time point (Figure 2C and 2D). Figure 7C However, analysis of individual SCFAs showed that erythritol treatment had a significant effect. Similar to the effect of D-allulose treatment, in the whole-sample analysis, erythritol significantly increased butyrate levels at 24 and 48 hours post-treatment compared to NSC (Figure 2F). In donor-specific analyses, erythritol treatment led to a significant increase in butyrate production compared to NSC, a phenomenon consistent in both healthy and type 2 diabetes subjects at 24 and 48 hours (Figure 7F). Although other changes in SCFA production were also observed after D-allulose and erythritol treatment, these changes were not consistent across different time points and / or between healthy subjects and patients with type 2 diabetes (T2DM) (Figures 2D-E and 2E). Figure 7D -E).

[0119] Figures 2A-I show the levels of (A) pH, (B) gas (mbar), (C) total SCFA (mM), (D) acetic acid (mM), (E) propionic acid (mM), (F) butyric acid (mM), (G) BCFA (mM), (H) lactic acid (mM), and (I) valerate (mM), from whole samples (n=12) after co-culturing with erythritol or D-allulose for 6, 24, and 48 hours. Statistical differences between the test product and NSC are indicated by * (0.01). <p 校正值 <0.05), **(0.001) <p 校正值 <0.01) or *** (p 校正值<0.001) indicates.

[0120] Figure 7A -I shows the levels of (A) pH, (B) gas (mbar), (C) total SCFA (mM), (D) acetic acid (mM), (E) propionic acid (mM), (F) butyric acid (mM), (G) BCFA (mM), (H) lactic acid (mM), and (I) valerate (mM), respectively. Data were obtained from donor groups (n=6 healthy subjects; n=6 type 2 diabetic subjects) after 6, 24, and 48 hours of culture in erythritol or D-allulose. Statistical differences between the tested products and NSCs are expressed as * (0.01). <p 校正值 <0.05), **(0.001) <p 校正值 <0.01) or *** (p 校正值 <0.001) indicates a healthy subject; T2D indicates a subject with type 2 diabetes; NSC indicates a substrate-free control group.

[0121] 1.11.3 Effects of erythritol and D-allulose on microbial composition (phylum, family, and species levels) Three major phyla were detected in all adult subjects: Actinobacteria, Bacteroidetes, and Firmicutes. Therefore, targeted analyses were performed for each of these phyla. At the phylum level, both D-allulose and erythritol significantly increased Firmicutes compared to NSC. In the whole-sample analysis, D-allulose significantly increased Firmicutes at both 24 and 48 hours compared to NSC, while erythritol significantly increased Firmicutes at 48 hours (Figure 3C). In the analysis by subject group, D-allulose significantly increased Firmicutes at 24 hours in healthy subjects compared to NSC, and significantly increased Firmicutes at 48 hours in subjects with type 2 diabetes mellitus (T2DM). Figure 8C In analyses conducted by subject group, erythritol had no significant effect on Firmicutes. In all analyses, neither D-allulose nor erythritol had any effect on Actinomycetes or Bacteroidetes; however, in participant-specific analyses, at 48 hours, the erythritol-treated group in healthy subjects showed a significant increase in Actinomycete counts compared to the NSC group (Figures 3A-B and 3B). Figure 8A -B).

[0122] Figures 3A-C show the condition of (A) Actinobacteria (cells / mL), (B) Bacteroidetes (cells / mL), and (C) Firmicutes (cells / mL) of the whole sample after co-culturing with erythritol or D-allulose for 6 hours, 24 hours, and 48 hours. n =12). Statistical differences between the tested product and the NSC are represented by *(0.01). <p 校正值 <0.05), **(0.001) <p 校正值<0.01) or *** (p 校正值 <0.001) indicates.

[0123] Figure 8A -C shows the abundance of (A) Actinobacteria (cells / mL), (B) Bacteroidetes (cells / mL), and (C) Firmicutes (cells / mL) after 6, 24, and 48 hours of erythritol or D-allulose culture, categorized by donor group (healthy subjects n=6; type 2 diabetic subjects n=6). Statistical differences between the test product and NSC are indicated by * (0.01). <p 校正值 <0.05), **(0.001) <p 校正值 <0.01) or *** (p 校正值 <0.001) indicates a healthy subject; T2D indicates a subject with type 2 diabetes; NSC indicates a substrate-free control group.

[0124] Figure 4 The study demonstrated the significant effects of treatment on microbial families and species in the 48-hour whole-sample analysis. In the 48-hour whole-sample analysis ( Figure 4 ) and donor specificity analysis of healthy subjects ( Figure 9 D-allulose significantly increased the abundance of Trichophyceae. The increase in Trichophyceae at 48 hours was attributed to a significant increase in the specificity of two species: *Hadrossella anaerobicis* and unclassified Trichophyceae species (…). Lachnospiraceae_u_s Meanwhile, *Brutella ovalis* also showed a significant but not statistically significant increase, which was confirmed in the 48-hour whole sample analysis. Figure 4 In donor-specific analyses, both healthy subjects and those with type 2 diabetes mellitus (T2DM) showed similar patterns of influence after D-allulose treatment, but the differences were not statistically significant. Figure 9 ).

[0125] Figure 4 The table shows the families and species of microorganisms significantly affected by erythritol or D-allulose culture in the colon for 48 hours in the whole sample (n=12) compared to NSC (expressed as log2(treatment / NSC)) (FDR=0.10). Significant differences between the test product and NSC are indicated in bold. Abbreviations: AL: D-allulose; ER: erythritol; u_f: unclassified family; u_s: unclassified species.

[0126] Figure 9The table shows the families and genera of microorganisms significantly affected after incubation in the colon with erythritol or D-allulose for 24 and 48 hours compared to NSC (FDR=0.10), (expressed as log2(treatment group / NSC)). Results are categorized by donor group (healthy subjects n=6; type 2 diabetic subjects n=6). Significant differences between the test product and NSC are indicated in bold. Abbreviations: AL: D-allulose; ER: erythritol; H: healthy subjects; T2D: type 2 diabetic subjects; u_f: unclassified family; u_s: unclassified species.

[0127] 1.11.4 Effects of erythritol and D-allulose on microbial species and the yield of related SCFAs Figure 5 The correlation between 48-hour fermentation parameters (gas production, SCFA, and BCFA) and microbial composition was demonstrated. Following D-allulose treatment, *Hadrossella anaerobicis* and unclassified *Trichophyton* species (…) were observed. Lachnospiraceae_u_ s A significant correlation was found between butyric acid production and erythritol treatment. Furthermore, a significant correlation was also found between *Brutella ovalis* and acetic acid production after D-allulose treatment. Unclassified *Eucrobacter* species ( Eubacterium_u_s There is a correlation between ) and butyric acid production.

[0128] Figure 5 The regularized canonical correlation (rCCA) analysis is presented for the fermentation parameters (gas yield, SCFA, and BCFA) and microbial composition after 48 hours of treatment with D-allulose or erythrose for all samples (n=12). Threshold > 0.4.

[0129] 1.12 Discussion This study employed a validated in vitro method to investigate the effects of representative and physiologically consistent levels of D-allulose and erythritol on the gut microbiota and metabolite production in adults with type 2 diabetes mellitus (T2DM) and their cohabiting healthy adults. The results provide important insights into the compound-specific effects of D-allulose and erythritol, two LNCs that have not been adequately studied in the existing literature, on the gut microbiota. Including adults with T2DM and their cohabiting healthy adults contributes to a deeper understanding of the impact of diabetes-induced changes in gut microbiota composition on D-allulose and erythritol fermentation.

[0130] This study found that both D-allulose and erythritol selectively increased the abundance of specific gut microbiota (including *Clostridium harlequinae*, *Clostridium butyricum*, *Brutella ovalis*, and unclassified *Eubacterium* species) in healthy subjects and patients with type 2 diabetes. Eubacterium_u_s ) and unclassified species of the Trichophyceae family ( Lachnospiraceae_u_sD-allulose-induced increases in abundance of *Hadrossella anaerobicis* and unclassified *Trichophyton* species were positively correlated with butyrate production. *Hadrossella anaerobicis* is known to be a butyrate-producing group (Allen-Vercoe et al., 2012). D-allulose treatment of *Brutella ovalis* was also correlated with increased acetate production, consistent with its acetate-producing metabolic capacity (Oliphant and Allen-Vercoe, 2019). Accordingly, D-allulose increased butyrate and acetate production within 24–48 hours in both healthy subjects and patients with type 2 diabetes mellitus (T2DM). Similarly, erythritol significantly increased butyrate production within 24–48 hours after administration in both healthy subjects and T2DM patients. Consistent with this finding, erythritol induced increased abundance of unclassified *Eubacterium* species (…). Eubacterium_u_s ) and Harry's Anaerobic Butyric Acid Bacteria ( Anaerobutyricum hallii The high specificity of D-allulose was enhanced. Although *Haloxylon ammodendron* is a potent butyrate-producing bacterium (Shetty et al., 2018), this study shows that unclassified *Eucrobacter* species are associated with butyrate production. Previous studies using in vitro models to investigate the effects of erythritol on the human gut microbiota have shown both increased SCFA production (Mahalak et al., 2020) and no effect (Hiele et al., 1993; Arrigoni et al., 2005). However, in those studies where no significant effect was observed, the dose of erythritol was either very low (e.g., 50 mg) or not reported. Studies in animal models (mice and rats) have shown that D-allulose intake leads to a dose-dependent increase in fecal SCFA (Matsuo et al., 2003), but other studies have shown no effect on fecal SCFA (Han et al., 2020b). SCFAs are rapidly absorbed in the large intestine, therefore the concentration of SCFAs in feces cannot accurately reflect SCFA production, which may explain the lack of significant results observed by Han et al. (2020b). Although further research is needed to confirm these findings, the results of this study indicate that D-allulose and erythritol can increase the production of butyric acid and acetic acid within 24–48 hours after administration by selectively modulating microbial composition.

[0131] The butyrate production kinetics of D-allulose and erythritol differ: D-allulose treatment significantly increased butyrate levels within 24 hours, while erythritol treatment significantly increased butyrate levels within 24–48 hours. This is consistent with observations of the fermentation kinetics of D-allulose and erythritol. D-allulose ferments rapidly within 6–24 hours, while erythritol ferments slowly within 24–48 hours. This suggests that D-allulose and erythritol have complementary effects on butyrate production kinetics by modulating different gut microbiota. These findings highlight the potential of a synergistic combination of D-allulose and erythritol to achieve sustained butyrate production for up to 48 hours. Utilizing a mixture of D-allulose and erythritol may be an effective method to increase butyrate yield, particularly suitable for patients with type 2 diabetes mellitus (T2DM) who have reduced butyrate-producing bacterial abundance. A mixture of erythritol and D-allulose may also enhance the synergistic effect of sweetness and provide positive sensory properties while imparting a slight bitterness (Jang et al., 2021). Furthermore, in patients with type 2 diabetes mellitus (T2DM), studies have shown that a single dose of D-allulose can reduce postprandial glycemic response after an oral glucose tolerance test (OGTT) (Noronha et al., 2018), while four consecutive weeks of erythritol intake can improve small vessel endothelial function and reduce aortic stiffness (Flint et al., 2014). Because D-allulose and erythritol have extremely low energy densities, they can be incorporated into the diet at doses that promote butyrate production without significantly increasing caloric intake.

[0132] Restoring butyrate levels in the large intestine through the intake of D-allulose and erythritol may be a novel approach to treating type 2 diabetes mellitus (T2DM), which can be used in combination with lifestyle modifications and hypoglycemic agents (Arora and Tremaroli, 2021). Butyrate is a major energy source for colonic cells (Koh et al., 2016). SCFAs also exert signaling effects through free fatty acid receptors 2 and 3 (FFAR2 / 3; a type of G protein-coupled receptor), which are widely expressed in various cell types in the human body and are therefore key signaling molecules between the gut microbiota and the host (Koh et al., 2016; Mishra et al., 2020). Butyrate plays an important role in glucose homeostasis and the pathophysiology of diabetes (Puddu et al., 2014; Arora and Tremaroli, 2021; Mayorga-Ramos et al., 2022). In normoglycemic individuals, increased intestinal butyrate production, driven by host genetic factors, is associated with improved insulin response after an oral glucose tolerance test (OGTT) (Sanna et al., 2019). In this study, selectively proliferated *Hadrossella harzianum* after D-allulose treatment was associated with elevated serum butyrate levels and enhanced insulin sensitivity in healthy subjects after an OGTT (Cui et al., 2022). In type 2 diabetic rats, butyrate treatment significantly reduced plasma glucose, glycated hemoglobin, insulin resistance, and gluconeogenesis (Khan and Jena, 2016). In enteroendocrine cells, butyrate can activate FFAR2 and FFAR3, thereby stimulating the release of peptide YY and glucagon-like peptide-1, which in turn affects glycemic regulation and satiety (Nøhr et al., 2013; Christiansen et al., 2018; Larraufie et al., 2018). After being absorbed and utilized by colonic cells, excess butyrate is transported to the portal venous circulation and then into the systemic circulation (Koh et al., 2016). In the systemic circulation, butyrate can regulate the function of pancreatic β cells through FFAR2 / 3, monocarboxylic acid transporters, and inhibition of histone deacetylases (Mayorga-Ramos et al., 2022).

[0133] The International Society for the Study of Probiotics and Prebiotics (ISAPP) defines prebiotics as substrates that can be selectively utilized by host microorganisms to provide health benefits (Gibson et al., 2017). The selective utilization of D-allulose and erythritol by specific gut microbiota observed in this study conforms to this definition, particularly in patients with type 2 diabetes mellitus (T2DM). However, further human clinical studies are needed to demonstrate their associated health benefits and confirm their prebiotic effect as defined by ISAPP. Previous studies in mice have confirmed the prebiotic effect of D-allulose. In particular, two previous studies have shown that D-allulose can modulate specific microbial community species, and these changes are positively correlated with high-fat diet-induced obesity and improved fasting blood glucose levels (Han et al., 2020a, 2020b). Furthermore, a synbiotic mixture combining two probiotics with D-allulose as a prebiotic inhibited diet-induced obesity in mice by regulating lipid metabolism (Choi et al., 2018). In this study, D-allulose and erythritol increased butyrate production by selectively increasing the abundance of specific gut microbiota, highlighting their prebiotic potential as substrates selectively utilized by host microbes. Related potential health benefits may include butyrate's mediating effects on glycemic control and insulin sensitivity (Arora and Tremaroli, 2021; Mayorga-Ramos et al., 2022).

[0134] Studies have confirmed that representative and physiologically appropriate doses of D-allulose and erythritol can increase the production of short-chain fatty acids (primarily butyrate and acetic acid) by selectively modulating microbial composition. These results suggest that D-allulose and erythritol, as substrates selectively utilized by host microorganisms, may have a prebiotic effect. Although this study only confirmed the selective utilization aspect, their health benefits may stem from the production of SCFAs, particularly butyrate production in patients with type 2 diabetes mellitus (T2DM). Mixing D-allulose with erythritol (in a specific ratio) may be an effective strategy to increase butyrate production in T2DM patients, thereby leading to glycemic control-related benefits.

[0135] At 48 hours, allulose and erythritol showed distinctly different performance from NSC, which was associated with higher levels of acetate / propionate / butyrate, enhanced gas production, and lower levels of proteolytic activity (bCFA), indicating that both components enhanced microbial activity compared to NSC.

[0136] Allulose was vigorously fermented within 6–24 hours. At 48 hours, it significantly increased levels of acetic acid, propionic acid, butyric acid (and total SCFA), decreased pH, and significantly increased gas production. Allulose consistently increased butyric acid levels in both T2DM subjects and healthy subjects.

[0137] Erythritol ferments slowly over 24–48 hours. While it did not significantly affect the total SCFA content, it did raise butyrate levels to a significantly higher level.

[0138] Microbial composition analysis further indicates that: From 24 hours onwards, allulose significantly and highly specifically increased the levels of Trichophyton (family Trichophyton). Lachnospiraceae The number of bacteria was initially measured, followed by highly specific increases in two species (Hadross anaerobic Confederates and Broutella ovalis). Studies have found a significant correlation between Hadross anaerobic Confederates / Chaetocerocephala and butyrate production—Hadross anaerobic Confederates is indeed a major lactic acid-consuming, butyrate-producing bacterium (Allen-Vercoe et al., 2012). Erythritol exhibits a highly specific effect on butyrate production. Consistent with this specificity, highly specific increases were observed in the following bacterial species: *Haloxylon ammodendron*, *Haloxylon rectum*, *Haloxylon karlandii*, *Haloxylon myxobacterium*, and *Haloxylon maltose*, as well as *Haloxylon ammodendron* anaerobic butyrate bacteria. *Haloxylon ammodendron* and *Haloxylon* are indeed butyrate-producing bacteria (Shetty et al., 2018; Ryu et al., 2022), with a particularly significant association between butyrate levels and *Haloxylon* spp. In terms of kinetics, allulose and erythritol are complementary: allulose increases butyrate content within 6-24 hours, while erythritol increases butyrate content within 24-48 hours. They exert their effects through different bacterial species.

[0139] The foregoing description of embodiments of the present invention is intended for illustration and explanation, and is not intended to be exhaustive or to limit the invention to the specific forms disclosed. Those skilled in the art will recognize that many modifications and variations can be made based on the foregoing teachings. It will be readily apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope. Therefore, the present invention is intended to cover these modifications and variations, provided they fall within the scope of the claims and their equivalents.

Claims

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

2. A method to improve the levels of bacteria from the Trichophytonceae family in subjects ( Lachnospiraceae ) or Eubacteriaceae ( Eubacteriaceae A method for producing one or more colonic bacterial communities, the method comprising administering to a subject a composition comprising allulose, erythritol, or a combination thereof.

3. A method for preventing, treating, or improving 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 the following: 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 disease (NAFLD), inflammatory bowel disease (IBD), cardiovascular disease (CVD), or inflammation.

5. The method according to any one of claims 1-4, wherein, At least one of the bacteria in one or more bacterial communities comes from the family Trichophyceae ( Lachnospiraceae ).

6. The method according to any one of claims 1-5, wherein, At least one of the one or more colonic bacterial communities is derived from the genus *Corynebacterium* (anaerobic bacteria). Anaerostipes ).

7. The method according to any one of claims 1-6, wherein, At least one of the bacteria in one or more bacterial communities is *Hadrossella anaerobicis* ( Anaerostipes hadrus )kind.

8. The method according to any one of claims 1-7, wherein, At least one of one or more colonic bacterial communities is derived from the genus *Brutella*. Blautia ).

9. The method according to any one of claims 1-8, wherein, At least one of the bacteria in one or more bacterial communities is *Brutella ovalis* ( Blautia obeum )kind.

10. The method according to any one of claims 1-9, wherein, At least one of the one or more colonic bacterial communities is derived from the genus *Butyric acid bacteria*. Anaerobutyricum ).

11. The method according to any one of claims 1-10, wherein, At least one of one or more bacterial populations comes from *Harley anaerobic butyric acid bacteria* (Haloxylon ammonium). Anaerobutyricum hallii )kind.

12. The method according to any one of claims 1-11, wherein, At least one of the bacteria in one or more bacterial communities comes from the family Eubacteriaceae ( Eubacteriaceae ).

13. The method according to any one of claims 1-12, wherein, At least one of the one or more colonic bacterial communities is derived from the genus Eubryl ( Eubacterium ).

14. The method according to any one of claims 1-13, wherein, One or more colonic bacterial communities may contain at least one of the following species: Escherichia coli (E. coli) Eubacterium rectale ), Kalandella ( Eubacterium callanderi ), Mycobacterium mucosae ( Eubacterium limosum ) and maltobacterium ( Eubacterium maltosivorans ).

15. The method according to any one of claims 1-14, wherein, Compared with untreated subjects, one or more colonic bacterial communities were increased by at least about 0.5%.

16. The method according to any one of claims 1-15, wherein, Compared with untreated subjects, one or more colonic bacterial communities were increased by at least about 10%.

17. The method according to any one of claims 1-16, wherein, Compared with untreated subjects, one or more colonic bacterial communities were increased by at least about 15%.

18. The method according to any one of claims 1-17, wherein, Compared with untreated subjects, one or more colonic bacterial communities were each increased by at least approximately 50%.

19. The method according to any one of claims 1-18, wherein, Compared with untreated subjects, each colonic bacterial community increased by at least approximately 0.5%.

20. The method according to any one of claims 1-19, wherein, Compared with untreated subjects, each colonic bacterial community increased by at least approximately 10%.

21. The method according to any one of claims 1-20, wherein, Compared with untreated subjects, colonic butyrate levels were increased.

22. The method according to any one of claims 1-21, wherein, Compared with untreated subjects, colonic butyrate levels increased by 5%.

23. A method for increasing the level of short-chain fatty acids in the colon of a subject, the method comprising administering to the subject a composition comprising allulose, erythritol, or a combination thereof.

24. The method according to any one of claims 1-23, wherein, The dosage of allulose is at least about 0.1 g / day.

25. The method according to any one of claims 1-24, wherein, The dosage of allulose is at least about 0.25 g / day.

26. The method according to any one of claims 1-25, wherein, The dosage of allulose is at least about 0.5 g / day.

27. The method according to any one of claims 1-26, wherein, The dosage of allulose is at least about 1.0 g / day.

28. The method according to any one of claims 1-27, wherein, The dosage of allulose is at least about 2.0 g / day.

29. The method according to any one of claims 1-28, wherein, The dosage of erythritol is at least about 0.1 g / day.

30. The method according to any one of claims 1-29, wherein, The dosage of erythritol is at least about 0.25 g / day.

31. The method according to any one of claims 1-30, wherein, The dosage of erythritol is at least about 0.5 g / day.

32. The method according to any one of claims 1-31, wherein, The dosage of erythritol is at least about 1.0 g / day.

33. The method according to any one of claims 1-32, wherein, The dosage of erythritol is at least about 2.0 g / day.

34. The method according to any one of claims 1-33, wherein, The subjects were humans.

35. The method according to any one of claims 1-34, wherein, The subjects were humans with diabetes.

36. The method according to any one of claims 1-35, wherein, The subjects were humans with type 2 diabetes.

37. The method according to any one of claims 1-36, wherein, Administer a course of treatment lasting at least two weeks.

38. The method according to any one of claims 1-37, wherein, Allulose, erythritol, or combinations thereof are administered orally.

39. The method according to any one of claims 1-38, wherein, Allulose, erythritol, or combinations thereof are administered in the form of an edible composition.

40. The method according to any one of claims 1-39, wherein, Allolose, erythritol, or combinations thereof are administered in the form of a synbiotic composition.

41. The method according to claim 40, wherein, The synbiotic composition comprises one or more bacterial communities from the Trichophyceae or Euglenomycetes families.

42. A composition comprising allulose, erythritol or a combination thereof, and one or more colonic bacterial communities from the family Trichophyceae or Eubacteriales.

43. A composition for use in the method of any one of claims 1-41, comprising allulose, erythritol, or a combination thereof.

44. The composition according to claim 42 or the composition for use according to claim 43, wherein, At least one of the bacteria in one or more bacterial communities comes from the family Trichophyceae.

45. The composition or use composition according to any one of claims 42-44, wherein, At least one of the one or more colonic bacterial communities is derived from the genus *Corynebacterium*, an anaerobic bacterium.

46. ​​The composition or use composition according to any one of claims 42-45, wherein, At least one of the bacteria in one or more bacterial communities is a Hadrus anaerobic clump-shaped bacterium.

47. The composition or use composition according to any one of claims 42-46, wherein, At least one of the one or more colonic bacterial communities is derived from the genus *Brutella*.

48. The composition or use composition according to any one of claims 42-47, wherein, At least one species of bacteria in one or more bacterial communities is an Ovobroutella species.

49. The composition or use composition according to any one of claims 42-48, wherein, At least one of one or more colonic bacterial communities originates from the genus *Butyric acid bacteria*.

50. The composition or use composition according to any one of claims 42-49, wherein, At least one of one or more bacterial communities originates from the anaerobic butyric acid bacterium species *Harleyella*.

51. The composition or use composition according to any one of claims 42-50, wherein, At least one of the bacteria in one or more bacterial communities comes from the Eubacteriaceae family.

52. The composition or use composition according to any one of claims 42-51, wherein, At least one of the one or more colonic bacterial communities is derived from the genus Eubryl.

53. The composition or use composition according to any one of claims 42-52, wherein, At least one of the colonic bacterial communities is derived from the bacterial species Eubrilia rectum, Eubrilia karlandii, Eubrilia mucina, and Eubrilia maltose.

54. The composition or use composition according to any one of claims 42-53, wherein, Each composition or use composition contains at least 0.1g of allulose.

55. The composition or use composition according to any one of claims 42-54, wherein, Each composition or use composition contains at least 0.25g of allulose.

56. The composition or use composition according to any one of claims 42-55, wherein, Each composition or use composition contains at least 0.5g of allulose.

57. The composition or use composition according to any one of claims 42-56, wherein, Each composition or use composition contains at least 0.1 g of erythritol.

58. The composition or use composition according to any one of claims 42-57, wherein, Each composition or use composition contains at least 0.25 g of erythritol.

59. The composition or use composition according to any one of claims 42-58, wherein, Each composition or intended use composition contains at least 0.5 g of erythritol.

60. The composition or use composition according to any one of claims 42-59, wherein, The composition or its use is in the form of a food composition.

61. The composition or use composition according to any one of claims 42-60, wherein, The food composition is selected from baked goods, breakfast cereals, dairy products, soy products, confectionery, jams and jellies, beverages (powdered and / or liquid), milkshakes, fillings, yogurt (milk-containing and non-milk-containing yogurt), kefir, extruded and compressed snacks, gelatin desserts, snack bars, meal replacements and energy bars, cheese and cheese sauces (milk-containing and non-milk-containing cheeses), edible and water-soluble films, soups, syrups, table sweeteners, nutritional supplements, sauces, seasonings, creamer, coatings, ice cream, frosting, syrups, pet food, tortillas, meat and fish, dried fruit, infant food, and batter and breadcrumb coatings.

62. The composition or use composition according to any one of claims 42-61, wherein, The food composition is in the form of aggregated powder, nutritional supplement, or pharmaceutical preparation.

63. The method according to any one of claims 1-41, wherein, Allulose, erythritol, or combinations thereof are provided in the form of the composition or use composition as described in any one of claims 42-62.

64. The method according to any one of claims 1-41 or 63, wherein, Increased production of short-chain fatty acids (e.g., butyric acid) was observed within 1–24 hours after application of the composition.

65. The method according to any one of claims 1-41, 63 or 64, wherein, Increased production of short-chain fatty acids (e.g., butyric acid) was observed within 24–48 hours after application of the composition.

66. The method according to any one of claims 1-41 or 63-65, wherein, This method is therapeutic.

67. The method according to any one of claims 1-41 or 63-66, wherein, This method is non-therapeutic.

68. The use of the composition or use of any one of claims 41-62 in the preparation of a medicament for improving the gut microbiota.

69. The composition or use of any one of claims 41-62 in the preparation of a medicament for improving one or more colonic bacterial communities derived from the Trichophyceae or Eurybacterialaceae families.

70. The use of the composition or use of any one of claims 41-62 in the preparation of a medicament for the prevention, treatment or improvement of a disease.

71. Use of the composition or use of any one of claims 41-62 in improving the gut microbiota.

72. The composition or use of any one of claims 41-62 in improving one or more colonic bacterial communities derived from the family Helicobacterceae or Eubacteriaceae.

73. The use of the composition or use of any one of claims 41-62 in the prevention, treatment or improvement of a disease.

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