Synbiotic Compositions for Metabolic Management, Particularly for Glucose Metabolism Management and for Modulation of Satiety Hormones Levels - Patent application

Abstract

The present invention relates to a synbiotic formulation comprising at least one probiotic strain and at least one amino acid or derivative, which has an effect in controlling glucose metabolism, e.g. preventing diabetes or treating / restoring pre-diabetes to health.

Description

[Technical field]

[0001] The present invention relates to a synbiotic formulation for use in modulating satiety hormone levels in a subject, comprising at least one probiotic Bacillus subtilis strain and at least one dipeptide containing a glutamine or glutamic acid unit, the satiety hormone being selected from CCK, GLP-1 and PYY.Furthermore, the formulation is effective in glucose metabolism management, e.g., diabetes prevention or pre-diabetes treatment / restoration to health.

[0002] The gut microbiota is a key regulator of health effects induced by dietary components. The microbial metabolite butyrate appears as an important and targetable mediator of such effects, while a relative deficiency of butyrate is associated with several intestinal and metabolic diseases. Available prebiotic strategies to stimulate butyrate production in the human gut, such as the application of FODMAPs, can cause undesirable side effects, such as diarrhea, abdominal pain and flatulence, especially in individuals with food intolerance or irritable bowel syndrome, resulting in limited availability. Therefore, we aimed to develop a FODMAP-free synbiotic composition as a novel means to shift both the composition and activity of the biota towards butyrate production.

[0003] The gastrointestinal microbiota forms an interesting interrelated relationship with orally ingested substances (which may be food or pharmaceutical ingredients) and human physiology. In that sense, the composition and activity of the microbiota are influenced by the diet, while food molecules are converted into partly well-absorbed metabolites through numerous (microbe-specific) metabolic pathways. Examples of metabolites derived from the gut microbiota with known effects on the host include phenolic acids, indole derivatives, and the short-chain fatty acids (SCFAs) acetate, propionate, and butyrate. Butyrate is an important energy source and differentiation factor for colonic epithelial cells. It also assists in the formation of mucins as well as tight junction component proteins, thereby contributing to the integrity of the intestinal barrier [1]. Moreover, butyrate can induce anti-inflammatory signaling through binding to aryl hydrocarbon, GPR41, GPR109, and PPARγ receptors, and as a result, it has been implicated in the pathogenesis of inflammatory bowel disease (IBD), which shows reduced levels of butyrate and butyrate-producing bacteria [1].

[0004] A proportion of about 2% of intraluminal butyrate enters the circulation via the portal vein [2], thereby affecting tissues other than the digestive tract, such as the liver, adipose tissue, and pancreas. The systemic actions of a sufficient butyrate supply can be summarized as a (beneficial) regulation of cardiometabolic health (increased insulin sensitivity, reduced plasma glucose and cholesterol levels, as well as glucose and lipid metabolism leading to increased satiety and reduced blood pressure [3]). Although the available studies do not establish a causal role for butyrate or other SCFAs in the pathophysiology of metabolic disorders, they probably contribute at least in part to the health benefits of, for example, diets rich in dietary fiber [4,5]. In conclusion, increased colonic butyrate levels are an attractive target for developing microbiota-targeted intervention strategies to achieve targeted health outcomes, especially for the prevention and treatment of IBD and metabolic disorders, e.g., type 2 diabetes, characteristics of the metabolic syndrome, and cardiovascular disease. Of particular interest would be the management of borderline diabetes (prediabetic pathology) to prevent the development of full-blown diabetes. This is because such a condition is kind of reversible. Once full-blown diabetes (definition) develops, recovery becomes much more difficult.

[0005] Prediabetes is when blood sugar levels are higher than they should be, but not high enough to be diagnosed as diabetes. It is sometimes called impaired fasting glucose or impaired glucose tolerance. Most people with type 2 diabetes first have prediabetes, which usually has no symptoms. A significant proportion of people, especially in Western countries, have prediabetes, but 90% do not know they have it. Treating prediabetes can prevent more serious health problems, such as type 2 diabetes and heart, blood vessel, eye and kidney problems.

[0006] Colonic butyrate levels can be targeted by prebiotics, probiotics, their combinations, and also by direct ingestion of butyrate in the form of salts or butyrate precursors, such as tributyrin. Sodium butyrate has been clinically evaluated as a concomitant treatment in IBD patients, supporting, for example, the efficacy of topical application of 5-ASA in refractory distal ulcerative colitis [6]. However, oral interventions are preferred, since a convenient and patient-friendly form of administration is an important compliance factor, even more so in preventive approaches. In the case of butyrate, this is achieved by a sustained-release coated tablet formulation that targets butyrate to the distal small intestine / colon [7]. The kinetic profile shows a sudden intraluminal release of butyrate from the carrier, which is probably less favorable than a more sustained uptake of butyrate formed continuously in the digestive tract. Moreover, the strong odor of butyrate-containing supplements limits their applicability.

[0007] Perhaps the most well-established means of inducing this formation is the ingestion of prebiotic fibers, either through a normal diet or through fortified foods or dietary supplements. The most studied and applied of prebiotics are fructooligosaccharides (FOS), galactooligosaccharides (GOS), arabinoxylan oligosaccharides (AXOS), xylooligosaccharides (XOS), and β-glucans. These carbohydrates form part of the larger group of FODMAPs (fermentable oligosaccharides, disaccharides, monosaccharides, and polyols). Because FODMAPs often cause undesirable side effects, such as diarrhea, constipation, and flatulence, diets low in FODMAP content are becoming increasingly popular [8]. In patients with irritable bowel syndrome, a low FODMAP diet has effectively alleviated these symptoms [9]. Thus, the use of fiber as a source of intestinal SCFAs is of limited applicability, at least for certain populations. An alternative approach is the direct application of butyrate-producing bacteria as adjunctive or therapeutic probiotics. The human gut microbiota is known to use four catabolic pathways that result in butyrate

[10] , three of which process proteins / amino acids as free forms, and the pyruvate / acetyl-CoA pathway (Ac pathway), which processes carbohydrates as well as proteins, the latter being the most abundant in a multi-ethnic metagenomic analysis

[11] . Faecalibacterium prausnitzii, Oscillibacter, and Clostridium XIVa constitute the major core community of all bacteria associated with the Ac pathway.Although species from these taxa do not belong to the commonly used probiotic bacteria of the genera Lactobacillus, Bifidobacterium, and Bacillus, they have recently been investigated and applied as so-called next-generation probiotics, e.g., Akkermansia muciniphila, Faecalibacterium prausnitzii, Clostridium butyricum, Clostridium beijerinckii, and Eubacterium hallii. Although these developments are clearly interesting, a recent pilot study evaluating a cocktail containing the four above-mentioned next-generation probiotic strains failed to achieve a significant increase in fecal butyrate levels

[12] . Although different Clostridium butyricum strains have been characterized as butyrate producers in vitro and in rodents, their ability to produce butyrate in the human intestine has not yet been established

[13] . 8 A 4-week intervention with a Butyricicoccus pullicaecorum strain at 1 CFU / day did not affect fecal butyrate.

[14] Overall, the application of next-generation / butyrate-producing probiotics in humans is interesting, but they also face technical challenges, such as being strictly anaerobic, making them difficult to produce, and making it difficult to ensure the long-term stability of the formulated final product.

[0008] A nutritional product containing Bacillus subtilis DSM 32315 and alanyl-glutamine in a colon-targeted capsule formulation that results in the production of SCFAs is disclosed in EP 3784807 A1.

[0009] In the present invention, a clinical pilot study was conducted on 18 healthy volunteers, showing that the formulation can safely improve butyrate levels in humans. Four weeks of supplementation was well tolerated and significantly increased fecal butyrate levels accompanied by changes in the microbiota. In addition, circulating lipid parameters were significantly improved, GLP-1 and PYY were significantly reduced, and effects on fasting blood glucose were detected, with subjects with prediabetes fasting blood glucose levels reduced to zero after four weeks of supplementation compared to three prediabetes subjects before starting.

[0010] Moreover, surprisingly, the improvement in glycemic metabolism, indicated by the reduction in the number of patients with prediabetes after the intervention, was accompanied by a significant reduction in GLP-1 and PYY levels. Normally, a (postprandial) increase in satiety hormones would be expected

[15] . This could be an indicator of improved metabolic health comparable to weight loss, since it is one of the very rare conditions where a reduction in the total amount of circulating satiety hormones has been reported in association with a positive effect [16,17]. The significant reduction with supplementation could be evaluated as an indicator for improvement. One hypothesis for the observed reduction in fasting GLP-1 could have been that GLP-1 secretion could be inhibited by circulating nonesterified fatty acids, as reported by Ranganath et al.

[18] . In this case, Bacillus subtilis, together with the substrate dipeptide (alanyl-glutamine), could have increased the production of SCFAs, especially in the upper gastrointestinal tract, supporting this hypothesis. Few studies have focused on fasting plasma concentrations of PYY and GLP-1. One of them is the study by Luis et al.

[16] . In an obese study population, a significant decrease in basal GLP-1 levels was observed in subjects who lost weight after a hypocaloric diet. This was accompanied by a significant improvement in anthropometric parameters and cardiovascular risk factors, such as LDL-cholesterol, triglycerides, insulin and a decrease in the HOMA index. Furthermore, a significant correlation was found in the study by Luis et al. between basal insulin and GLP-1 levels. Although fasting insulin was not measured in the present study, a slight decrease in blood glucose levels was observed even in the normoglycemic group. A decrease in GLP-1 concentrations after weight loss has also been reported by other researchers

[17] , although some publications show an increase in GLP-1 concentrations after weight loss

[19] . Thus, various mechanisms are thought to be involved, which may lead to differences during weight loss and even during weight maintenance. Nevertheless, we disclose here for the first time a clear decrease in GLP-1 and PYY in a normoweight study population in response to a synbiotic dietary supplement.

[0011] Lipid status, especially the blood biomarkers total cholesterol and LDL cholesterol, showed significant reductions throughout the study. This is in line with previous in vivo studies that showed that administration of, for example, Lactobacillus probiotics was effective in improving lipid profiles, including lowering total and LDL cholesterol

[20] . These cholesterol-lowering effects may be partially attributable to bile salt hydrolase (BSH) activity. Deconjugated bile salts are reabsorbed less efficiently than conjugated bile salts, resulting in higher amounts of free bile acids excreted in the stool. Free bile salts are also less efficient at solubilizing and absorbing lipids in the intestine. Thus, deconjugation of bile salts may lead to a reduction in serum cholesterol by increasing the demand for cholesterol for de novo synthesis of bile acids to compensate for bile acids lost in the stool, or by reducing cholesterol solubility and thereby decreasing cholesterol absorption from the intestinal lumen. Additional modes of action of probiotics have also been reported.

[0012] Collinsella, especially Collinsella aerofaciens, may be of further interest in health applications in the future. Recent studies have identified butyrate-producing species of the genus Collinsella

[21] . In addition, Collinsella has been described as beneficial in several further publications, for example, WO 2010125421, which describes the use of Collinsella aerofaciens to reduce bloating, as well as WO 2016038198, which discloses the use of Collinsella for the treatment of inflammatory bowel disease.

[0013] In conclusion, this intervention shows that this synbiotic composition is an effective and safe tool to stimulate intestinal butyrate production with subsequent positive effects on the regulation of satiety hormone concentrations.

[0014] Accordingly, the present invention provides a formulation for use in regulating satiety hormone levels in a subject, comprising: - at least one probiotic strain of Bacillus subtilis, and - at least one dipeptide containing a glutamine or glutamic acid unit Including, The present invention relates to a formulation wherein the satiety hormone is selected from CCK, GLP-1 and PYY.

[0015] In a preferred configuration, the formulation further comprises one or more plant extracts selected from curcuma extract and green tea extract.

[0016] In a preferred configuration, the formulation should be administered to a subject at least one oral dose per day, preferably at least two oral doses per day, of at least 1 billion CFU of the probiotic Bacillus subtilis strain and at least 250 mg of the dipeptide.

[0017] In specific configurations, the modulation of satiety hormones is a decrease in satiety hormone concentrations of at least 5%, or at least 10%, or at least 20%, or at least 30% after 4 weeks of administration of the formulation.

[0018] In a specific configuration, the dosage is at least 1 billion CFU of the probiotic Bacillus subtilis strain and at least 250 mg of the dipeptide per day.

[0019] However, in the fasting state, plasma GLP-1 concentrations were significantly lower in individuals with normal glucose tolerance than in those with type 2 diabetes.[24,25] In case-control studies, GLP-1 levels were positively correlated with characteristics of the metabolic syndrome.

[25] However, fasting GLP-1 was reduced in obese subjects after a weight-reduction diet.[26,27]

[0020] Such a reduction in (potentially elevated) satiety hormone concentrations is beneficial because it indicates a rebalancing of the metabolic state seen with weight loss diets, a potential sign of the onset of metabolic syndrome [26,27].

[0021] When the formulation is administered in capsule or tablet form, it is further preferred that it includes an enteric coating.

[0022] According to the invention it is preferred if the probiotic strain is selected from Bacillus subtilis DSM 32315, Bacillus subtilis DSM 32540, Bacillus subtilis DSM 32592, preferably Bacillus subtilis DSM 32315.

[0023] The dipeptide is preferably selected from glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid, and their acetylated forms.

[0024] In a specific configuration, the dipeptide is L-alanyl-L-glutamine.

[0025] In a preferred embodiment, the total amount of probiotic strains and amino acids or oligopeptides is at least 40% by weight, preferably at least 50% by weight, more preferably at least 60% by weight, most preferably at least 70% by weight of the total weight of the formulation.

[0026] It is further preferred if the total amount of plant extracts is at least 10% by weight, preferably at least 20% by weight, more preferably between 20 and 40% by weight of the total weight of the formulation.

[0027] In another preferred embodiment, the formulation comprises an enteric coating, wherein the enteric coating comprises one or more of the following: methyl acrylate-methacrylic acid copolymer, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymer, shellac, cellulose acetate trimellitate, sodium alginate, zein, preferably methyl acrylate-methacrylic acid copolymer.

[0028] In another preferred embodiment, the formulation comprises an enteric coating comprising a polymer composition, wherein the polymer is polymerized from 20-30% by weight of methyl methacrylate, 60-70% by weight of methyl acrylate, and 8-12% by weight of methacrylic acid. It is even more preferred if the polymer is polymerized from 25% by weight of methyl methacrylate, 65% by weight of methyl acrylate, and 10% by weight of methacrylic acid.

[0029] In another aspect of the invention, the formulation according to the invention is for use in the prevention and treatment of obesity, adiposity, type 2 diabetes, metabolic syndrome.

[0030] Another aspect of the invention is a formulation for use in reducing fasting blood glucose in a subject, preferably at a fasting blood glucose level of less than 100 mg / dl, comprising: - at least one probiotic strain of Bacillus subtilis, and - at least one dipeptide containing a glutamine or glutamic acid unit The present invention relates to a formulation comprising:

[0031] In a preferred configuration, the probiotic Bacillus subtilis strain is Bacillus subtilis DSM 32315 and the dipeptide is alanine-glutamine, preferably L-alanyl-L-glutamine.

[0032] In a specific configuration, fasting blood glucose is reduced after 4 weeks of administration of at least 1 oral dose per day, preferably at least 2 oral doses per day, of at least 1 billion CFU of the probiotic Bacillus subtilis strain and at least 250 mg of the dipeptide.

[0033] In another specific configuration, the glycemic response in the subject is preferably decreased in response to the glucose test meal.

[0034] Glycemic responses can be measured two hours after a standardized glucose test meal.

[0035] In a particular configuration, the subject is a human with a fasting blood glucose level above 100 mg / dl and is in a pre-diabetic state.

[0036] A fasting blood glucose level of 99 mg / dL or less is considered normal, 100-125 mg / dL indicates that the subject has prediabetes, and 126 mg / dL or greater indicates that the subject has diabetes.

[0037] Fasting blood glucose levels may be determined as baseline blood glucose levels predicted from an individual's daily 24-hour glucose profile and medical history data.

[0038] Another possibility for determining whether a subject is in a pre-diabetic state is to determine the % glycated hemoglobin (HbA1c) level, in which case a subject is considered to be pre-diabetic with an HbA1c level above 5.7%.

[0039] In certain configurations, glycemic response is reduced after 4 weeks of administration of at least 1 oral dose per day, preferably at least 2 oral doses per day, of at least 1 billion CFU of the probiotic Bacillus subtilis strain and at least 250 mg of the dipeptide.

[0040] In a particular configuration of the present invention, the subject's body weight is reduced by at least 1 kg 4 weeks after administration of the formulation, said reduction in body weight being independent of the subject's caloric intake and physical activity.

[0041] Thus, the formulation is administered to the subject at least one oral dose per day, preferably at least two oral doses per day, of at least 1 billion CFU of the probiotic Bacillus subtilis strain and at least 250 mg of the dipeptide. [Brief description of the drawings]

[0042] [Figure 1] A scatter plot of the distribution of fasting blood glucose levels [mg / dl] with mean ± 95% CI. [Diagram 2] FIG. 1 shows a scatter plot of the distribution of total GLP-1 levels [pmol / L] with mean ± 95% CI. [Diagram 3] Scatter plot of distribution of PYY levels [pg / mL] with mean ± 95% CI. [Figure 4] FIG. 1 shows a scatter plot of the distribution of total cholesterol levels [mg / dL] with mean ± 95% CI. [Diagram 5] FIG. 1 is a scatter plot of the distribution of HDL cholesterol levels [mg / dL] with mean ± 95% CI. [Figure 6] FIG. 1 shows a scatter plot of the distribution of triglyceride levels [mg / dL] with mean ± 95% CI. [Figure 7] FIG. 1 is a scatter plot of the distribution of LDL / HDL cholesterol ratios with mean±95% CI. [Figure 8] Scatter plot of Collinsella aerofaciens [relative abundance] with mean ± 95% CI. [Figure 9] FIG. 1 illustrates fasting blood glucose before and after supplementation with SAMANA® FORCE. [Figure 10] FIG. 1 shows the correlation between fasting glucose at baseline and change in fasting glucose (delta %). [Figure 11] FIG. 1 shows PPGR (median and IQR) of glucose test meal pre- and post-intervention in non-prediabetic participants. [Figure 12] FIG. 1 shows PPGR (median and IQR) of glucose test meal pre- and post-intervention in pre-diabetic participants. [Figure 13] FIG. 1 shows butyrate production in an in vitro gut model by synbiotic combinations containing different combinations of Bacillus subtilis strains and dipeptides. [Figure 14] FIG. 1 shows butyrate production in an in vitro intestinal model by combination of Bacillus subtilis DSM 32315 with various dipeptides. [Figure 15] FIG. 1 shows butyrate production in an in vitro intestinal model with 100% or 50% dosing with various coatings.

[0043] Working Example Human Studies I The study was conducted in an open-label format with a 2-week run-in period (basic characterization of subjects regarding occurrence of gastrointestinal symptoms and bowel function) followed by a 4-week intervention period. After informed consent, healthy subjects were screened for eligibility to participate in the study. Stool and blood samples were collected for biomarker measurements and microbiome analysis at the start, after 14 days and at the end of the intervention (28 days). To control for confounding factors, diet was recorded using a food frequency protocol 3 days before each visit. Blood routine parameters were measured at each visit after an overnight fast of at least 10 hours.

[0044] The composition of the synbiotic product in colonic coated HPMC capsules (daily dose = 2 capsules) was as follows: Each capsule contains Bacillus subtilis DSM 32315 spore powder (containing approximately 2 billion CFU), L-alanyl-L-glutamine (290 mg), 90 mg curcuma extract, 90 mg green tea extract, D- and B-vitamins and minerals.

[0045] The capsule includes a colonic coating with EUDRAGUARD® biotic that may begin to disintegrate at the pH conditions of the lower small intestine and colon (pH>7.0). The coating includes a polymer composition, where the polymer is polymerized from 20-30% by weight methyl methacrylate, 60-70% by weight methyl acrylate, and 8-12% by weight methacrylic acid. It is more preferred if the polymer is polymerized from 25% by weight methyl methacrylate, 65% by weight methyl acrylate, and 10% by weight methacrylic acid.

[0046] Ingestion of study products The above product composition was taken without chewing with water as one capsule in the morning (variable depending on whether breakfast was eaten or not) and one capsule in the evening (variable depending on whether dinner was eaten or not). On the study day prior to the study visit, the intake of the evening capsule was standardized to be taken 12±2 hours before the scheduled visit at the study site.

[0047] The following parameters were measured: Fasting blood glucose was measured (as well as differential hemograms, liver enzymes (GPT, GOT, γ-GT, AP), creatinine, and uric acid).

[0048] Routine parameters were determined in routine laboratories. Analysis of blood routine parameters / differentiated hemograms was performed within 24 hours after blood collection.

[0049] Blood samples for routine parameters and hemograms and lipid profiles were transported to the hematology routine laboratory on the same day.

[0050] Blood routine parameters were checked at screening, visits 1, 2 and 3. For the study, blood was drawn after an overnight fast of at least 10 hours. Preparation: clotting for 30 minutes at room temperature, centrifugation for 10 minutes at 3000xg and 4°C. Blood samples for routine parameters and hemograms as well as lipid profiles were transported to the blood routine laboratory on the same day.

[0051] Serum lipid profile (triglycerides, total cholesterol, HDL- and LDL-cholesterol) was measured in a routine laboratory on the same day after each visit. Total cholesterol, HDL-cholesterol and triglycerides were measured photometrically. LDL-cholesterol was calculated according to the Friedewald calculation.

[0052] To assess total GLP-1 and PYY, DPP-IV and AEBSF inhibitors were added to EDTA plasma tubes prior to blood collection. The prepared tubes were stored frozen until blood collection.

[0053] Total GLP-1 and PYY were analyzed in plasma at BioTeSys GmbH using ELISA kits (Merck Millipore EZGLP1T-36K for total GLP-1 and EZHPYYT66K for PYY).

[0054] For GLP-1 and PYY analysis, samples were aliquoted and stored below −70°C until transport or analysis.

[0055] Example 1: Effect of 4 weeks of supplementation with a synbiotic product on the prediabetic subject population [Table 1]

[0056] Three subjects had elevated / prediabetic fasting glucose levels at baseline. At the end of the intervention after 4 weeks, all subjects' fasting glucose levels were within the (healthy) reference range (see Table 1).

[0057] During the course of the study for glucose, a decline was observed over the study period (see Table 2). [Table 2]

[0058] Example 2: Effects of 4 weeks of supplementation on GLP-1 and PYY levels accompanied by metabolic improvements (cholesterol and glucose) but not directly correlated with significant changes in butyrate levels In addition, the gut hormones total GLP-1 and PYY were measured in fasting blood samples at each visit. Both hormones showed a significant decrease from baseline to the end of the intervention. Gut hormones play an important role in communication between the gut and the brain, mediating hunger and satiety signals. GLP-1 and PYY are anorectic hormones secreted from the gut into the circulation in response to a meal, decreasing appetite and food intake. PYY and GLP-1 play important roles in regulating food intake and insulin secretion and have attracted translational interest in the field of obesity and diabetes. PYY production is highest in enteroendocrine cells located distally in the intestine, reflecting the site where high concentrations of short-chain fatty acids (SCFAs) are produced by the gut microbiota (Larraufie et al. 2018). During the study, the mean level of total GLP-1 significantly decreased from 23.11 pmol / L to 14.89 pmol / L. In addition, mean PYY levels decreased from 96.44 pg / mL to 57.52 pg / mL from baseline to the end of the 4-week intervention.

[0059] One hypothesis for the observed decrease in fasting GLP-1 could be that circulating non-esterified fatty acids could inhibit GLP-1 secretion, as reported by

[18] . In this case, Bacillus subtilis, together with the substrate dipeptide (alanyl-glutamine), could increase the production of SCFAs, especially in the upper gastrointestinal tract, supporting this hypothesis. Few studies have focused on fasting plasma concentrations of PYY and GLP-1. One of them is the study by Luis et al.

[16] . In an obese study population, a significant decrease in basal GLP-1 levels was observed in subjects who lost weight after a hypocaloric diet. This was accompanied by a significant improvement in anthropometric parameters and cardiovascular risk factors, such as a decrease in LDL-cholesterol, triglycerides, insulin and HOMA index. Furthermore, a significant correlation was found in the study by Luis et al. between basal insulin levels and GLP-1 levels. Although fasting insulin was not measured in the present study, a slight decrease in blood glucose levels was observed even in the normoglycemic group. A decrease in GLP-1 concentrations after weight loss has also been reported by other researchers

[17] , while some publications have shown an increase in GLP-1 concentrations after weight loss

[19] . Thus, various mechanisms are thought to be involved, and there may be differences during weight loss and weight maintenance. In any case, the interesting finding of a clear decrease in GLP-1 and PYY in a normal-weight study population in response to SAMANA® FORCE should be further investigated. As a next step, we propose to also evaluate the effect on postprandial secretion levels of GLP-1 and PYY after a meal.

[0060] GLP-1 GLP-1 is an intestinal hormone released from endocrine cells in the intestine to control appetite. Fasting blood levels were measured at baseline (V1), 2 weeks after intervention (V2), and at the end of the intervention 4 weeks later (V3). Total GLP-1 levels were significantly decreased from baseline to the end of the intervention 4 weeks later (p<0.001). Already 2 weeks after intervention, total GLP-1 levels were significantly decreased (p<0.001) (see Table 3). [Table 3]

[0061] PYY PYY is an anorectic gut hormone for controlling appetite. Consistent with total GLP-1, PYY levels were significantly decreased from baseline to the end of the intervention after 4 weeks (p<0.001). Already after 2 weeks of intervention, PYY levels were significantly decreased (p<0.0139) (see Table 4). [Table 4]

[0062] Example 3: Effect of 4 weeks of supplementation on lipid parameters Lipid status, particularly the blood biomarkers total cholesterol and LDL cholesterol, showed significant reductions throughout the study.

[0063] All blood samples were collected under fasting conditions. Total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides were measured. The majority of subjects showed levels within the reference range (see dotted lines). For total cholesterol and triglycerides, fasting blood reference values ​​are less than 190 mg / dL and less than 150 mg / dL, respectively. For HDL cholesterol, a healthy blood level is indicated by >40 mg / dL.

[0064] Total and LDL cholesterol levels were significantly reduced from baseline to the end of the 4-week intervention (see Figures 4 and 5 and Tables 5 and 6). On average, HDL cholesterol levels increased slightly, but not significantly, from 46.06 mg / dL to 47.33 mg / dL throughout the study (see Figure 5 and Table 6).

[0065] Furthermore, triglyceride levels did not change significantly over the study period (see FIG. 6 and Table 7). [Table 5] [Table 6] [Table 7] [Table 8]

[0066] In the literature, the LDL / HDL cholesterol ratio is often calculated to estimate the risk of atherosclerosis and coronary heart disease. The ideal ratio is indicated by less than 3.0, with higher values ​​indicating a higher risk of heart disease

[22] . In the present study, the LDL / HDL cholesterol ratio was significantly reduced from baseline to the end of the intervention after 4 weeks (see Figure 7 and Table 8).

[0067] Example 4: Increased abundance of Collinsella aerofaciens in fecal samples An increase in the phylum Actinobacteria was confirmed at the class level. This increase can be attributed to both the orders Coriobacteriales and Bifidobacteriales as well as the families Coriobacteriaceae and Bifidobacteriaceae. Confirming the results at the level of Coriobacteriales and at the level of Coriobacteriaceae, a significant increase was also observed in the genus Collinsella. The significant change in the genus Collinsella can be attributed to the species Collinsella aerofaciens (see Figure 8). Surprisingly, this increase in Collinsella by the combination of B. subtilis and alanyl-glutamine was detected both in in vitro gut microbiota and intestinal models and in this human study (see FIG. 8).

[0068] Human Studies II Research Process At the beginning of the study, participants applied the MillionFriends program (www.millionfriends.de) provided by Perfood GmbH. Continuous tissue glucose monitoring (Abbott Freestyle Libre system) was therefore performed for 14 days, including one day of sensor calibration. Baseline values ​​of the parameters of interest were measured within these 14 days. This included the participants' glycemic response to a prescribed and standardized test meal, as well as other parameters to describe glycemia. Participants also answered questions about their digestive and general health. In addition, participants collected stool samples and sent them for analysis.

[0069] Following the first sensor-assisted trial phase, participants began taking SAMANA® FORCE. They were advised to take two capsules daily without chewing. They were free to decide when to take the capsules and whether to take them with or without food. Otherwise, participants continued to eat as usual.

[0070] After two weeks of regular intake of SAMANA® FORCE and completion of questionnaires, patients entered the second sensor-assisted study phase. Continuous tissue glucose monitoring (Abbott Freestyle Libre system) was again performed for 14 days, including one day of sensor calibration. Intake of SAMANA® FORCE continued during the second sensor phase. Glycemic response to the test meal and parameters representative of blood glucose were again measured. In addition, a second stool sample was collected and digestive and health questionnaires were again completed.

[0071] Only after completing the second sensor phase did participants receive a nutrition report with personalized dietary recommendations based on their glycemic response during the first sensor phase.

[0072] The composition of the synbiotic product in colonic coated HPMC capsules (daily dose = 2 capsules) was as follows: Each capsule contains Bacillus subtilis DSM 32315 spore powder (containing approximately 2 billion CFU), L-alanyl-L-glutamine (290 mg), 90 mg curcuma extract, 90 mg green tea extract, D- and B-vitamins and minerals.

[0073] The capsule includes a colonic coating with EUDRAGUARD® biotic that may begin to disintegrate at the pH conditions of the lower small intestine and colon (pH>7.0). The coating includes a polymer composition, where the polymer is polymerized from 20-30% by weight methyl methacrylate, 60-70% by weight methyl acrylate, and 8-12% by weight methacrylic acid. It is more preferred if the polymer is polymerized from 25% by weight methyl methacrylate, 65% by weight methyl acrylate, and 10% by weight methacrylic acid.

[0074] Standardized glucose meal testing Participants were asked to drink a standardized glucose test meal during both sensor-assisted test phases. For this, they received a weighed packet of glucose (60 g) along with the test kit. Participants should dissolve the packet in 200–300 ml of water and drink this on an empty stomach at the start of each sensor phase in the morning.

[0075] Measurement of fasting glucose, HbA1c and glycemic response Fasting glucose was determined as the baseline blood glucose level using a proprietary algorithm developed in-house. Briefly, the baseline was predicted from daily individual 24-h glucose profiles as well as medical history data. HbA1c (%) was calculated by multiplying the mean blood glucose value of the entire study phase by 0.03 and adding 2.6. Of note, the HbA1c calculated in this study is not of the same quality as standardized laboratory-measured HbA1c.

[0076] To analyze the glycemic response to the test meal, AUCi (area under the glucose curve) was measured over a 120-minute period after recording the meal intake. The trapezoidal rule was applied for the calculation. Participants were instructed to maintain a minimum of 2 hours between two meals and / or between a meal and physical activity.

[0077] During the first two weeks, subjects had their blood glucose levels tracked by non-invasive continuous blood glucose measurements (read via a mobile application) to detect the glycemic response to daily diet and nutrition, which was recorded in a diary. In addition, they performed three challenges with a standard diet (glucose (adaptive glucose tolerance test), white bread with protein and fat spread, and whole wheat bread). This phase was followed by a 4-week intervention phase, during which they consumed the symbiotic products (daily) for 14 days without blood glucose measurements, followed by 14 days of similar non-invasive continuous blood glucose measurements (read via a mobile application) to detect the glycemic response to daily diet and nutrition in a diary. They performed the three challenges again with a standard diet (glucose (adaptive glucose tolerance test), white bread with protein and fat spread, and whole wheat bread). Stool samples for microbiota analysis were taken before and after the study.

[0078] Example 5: Weight Loss Data Results showed that significant weight loss, independent of nutritional adaptation (as dietary patterns remained the same as indicated by unchanged macronutrient supply and distribution), improved the glycemic response in area under the curve in an adapted glucose tolerance test.

[0079] One hundred and ninety-two study participants were recruited through social media. The study cohort was 65.1% female (n=125) and 34.9% male (n=67). The mean age was 42.86 years (±12.48). The mean weight was 80.28 kg (±17.88), and the mean BMI was 26.88 kg / m 2 (±6.17).

[0080] Neither daily energy nor mean amounts of protein and fat were significantly different between both sensor phases. Regarding physical activity, participants were less active during the second sensor phase (p<0.001).

[0081] Prediabetes Subgroups A closer look at the glycemic parameters measured in the first sensor phase revealed that several participants had higher fasting glucose and HbA1c values. In 99 participants, calculated HbA1c was above 5.7%, and 62 participants had fasting glucose above 100 mg / dl. Fasting glucose above 100 mg / dl and HbA1c values ​​above 5.7% indicate the presence of prediabetes, so participants could be divided into prediabetic (n=62) and non-prediabetic (n=118) subgroups based on these two criteria.

[0082] These two groups were analyzed separately, therefore participants' eating behaviors and levels of physical activity should also be analyzed separately to identify these factors as possible confounding factors within these groups.

[0083] Sixty-two study participants were considered to have prediabetes. These participants were 53.23% (n = 33) female and 46.03% (n = 29) male. The mean age was 42.52 years (± 12.18). The mean weight was 84.18 kg (± 16.20), and the mean BMI was 27.60 kg / m 2 (±6.45).

[0084] Neither daily energy nor the mean amounts of carbohydrate, protein, fat and fiber were significantly different between both sensor phases. Regarding physical activity, prediabetic participants were less active during the second sensor phase (p=0.016).

[0085] Non-prediabetic subgroup The 118 study participants had no signs of prediabetes. These participants were 69.49% (n = 82) female and 30.51% (n = 36) male. The mean age was 41.16 years (± 12.10). The mean weight was 78.13 kg (± 18.35), and the mean BMI was 26.45 kg / m 2 (±5.98).

[0086] Regarding macronutrient intake, no significant differences were found in mean daily fat and protein intake and kilocalories per day intake. However, carbohydrate (p=0.001) and fiber (p<0.001) intakes decreased during the second sensor phase. Regarding physical activity, participants without prediabetes were less active during the second sensor phase (p<0.001).

[0087] Comparison of weight and composition In addition to possible changes in glycemic response, changes in body weight were also of interest. Participants were asked to report their current weight and waist-to-hip ratio (whr) at the beginning and end of the study. For this purpose, participants were provided with a tape measure included in the study kit. Only participants with known fasting blood glucose and who reported weight or WHR at both time points were included in the analysis.

[0088] Comparing weight at the start and end of the study showed that participants lost significant amounts of weight (p<0.001). Significant weight loss was also seen in pre-diabetic (p<0.001) and non-pre-diabetic (p<0.001) participants.

[0089] In addition to weight, we also looked at whether there was a change in waist-to-hip ratio. In contrast to weight, no significant changes were observed in WHR. This was applied to both the prediabetic and non-prediabetic study populations and subgroup analyses.

[0090] The following table summarizes the body weight and composition analyses (see Table 9). [Table 9]

[0091] The absolute weight loss over the course of the study is summarized in Table 10. [Table 10]

[0092] Fasting blood glucose analysis Fasting blood glucose in the first and second sensor phases was compared to answer the question of whether SAMANA® FORCE intake affects fasting blood glucose (n=180). Fasting blood glucose was determined as the baseline blood glucose value using a proprietary algorithm developed in-house. Briefly, the baseline is predicted from daily individual 24-hour glucose profiles as well as medical history data.

[0093] The results showed a significant reduction in fasting blood glucose (p<0.001) with supplementation with SAMANA® FORCE and are summarized in Table 11. [Table 11]

[0094] Subanalysis of participants with prediabetes The previous results raised the question of whether fasting glucose was reduced in all participants or whether this benefit was seen in specific groups of participants. Therefore, we investigated whether any of the participants had fasting glucose levels indicating the presence of prediabetes (>100 mg / dl) at the start of the intervention.

[0095] First, data from participants with prediabetes (n=62) and those without prediabetes (n=118) were analyzed separately. Looking more closely at participants with prediabetes, we found that fasting blood glucose was significantly reduced after regular intake of SAMANA® FORCE (p<0.001), which is shown in Figure 9.

[0096] Thirty-three people had fasting plasma glucose levels below 100 mg / dl and were therefore no longer in the prediabetic category at the end of the study.

[0097] In contrast, no significant change in fasting glucose was observed in participants without prediabetes (p=0.582).

[0098] Additionally, the percentage change in fasting glucose (delta%) over the course of the study was compared between prediabetic and non-prediabetic participants. Consistent with previous results, there was a significantly greater decline in fasting glucose in the prediabetic group (p<0.001). The results are summarized in Table 12. [Table 12]

[0099] These results suggest that the potential effect of SAMANA® FORCE on fasting glucose may depend on baseline fasting glucose levels. To get to the bottom of this assumption, a correlation analysis was performed. The analysis showed a significant correlation between baseline fasting glucose levels and the percentage change in fasting glucose (R=-0.38, p<0.001). The higher the initial value, the greater the reduction in fasting glucose. This analysis is shown in Figure 10.

[0100] Glucose Testing: Analysis of Postprandial Glycemic Responses To answer the question of whether daily ingestion of SAMANA® FORCE makes a difference in postprandial glycemic response, responses 2 hours after a standardized glucose test meal were compared.

[0101] Glucose response was described by AUCi. AUCi (area under the glucose rise curve) was measured for 120 minutes after recording meal intake. The trapezoidal rule was applied for the calculation. Comparison of glycemic response was limited to participants who completed the glucose test meal in both sensor phases (n=168).

[0102] Results showed that glycemic response was reduced following supplementation with SAMANA® FORCE (p=0.012).

[0103] Subanalysis of participants with prediabetes The previous results raised the question of whether the glycemic response to the glucose test meal after ingestion of SAMANA® FORCE was reduced in all participants or whether the effect was only seen in those with prediabetes. Therefore, the glycemic response to the glucose test meal was analyzed separately in those with prediabetes (n=57) and those without prediabetes (n=110). Fasting blood glucose at the start of the intervention indicated the presence (>=100 mg / dl) or absence (<100 mg / dl) of prediabetes. The responses were then compared between both groups. Only data from participants who completed the glucose test meal in both sensor phases were used in the analysis. Data from one prediabetic participant were removed because the glycemic response indicated that the glucose test meal was eaten in error (pre=13.71 mg / dl, post=125.88 mg / dl).

[0104] Analysis of prediabetic participants showed a significant reduction in glycemic response to a glucose test meal following supplementation with SAMANA® FORCE (p=0.012). Results are summarized in Table 13. [Table 13]

[0105] In contrast, as shown in Table 14, no significant changes were observed in the glycemic response to the standardized glucose test meal in non-prediabetic participants (p=0.345). [Table 14]

[0106] Additionally, the change in glycemic response (delta%) over the course of the study was compared between prediabetic and non-prediabetic participants. The percent change was not significantly different between both groups (p=0.072).

[0107] Furthermore, the time course of postprandial blood glucose levels (PPGL) during an oral glucose tolerance test (OGTT) was compared from -20 min before glucose ingestion to 140 min after the meal. A significant reduction in postprandial blood glucose levels could be detected after the supplementation period in all participants, as shown in Table 15. However, the effect was even stronger in prediabetic participants, as shown in Figures 11 and 12. [Table 15]

[0108] Analysis of HbA1c and average blood glucose levels HbA1c was calculated by multiplying the mean blood glucose value over the entire study phase by 0.03 and adding 2.6. The results are summarized in Table 16. Mean blood glucose values ​​were measured by continuous glucose monitoring and are summarized in Table 17. [Table 16] [Table 17]

[0109] Analysis of butyrate production in an in vitro intestinal model Example 6: Butyrate production by various combinations of bacterial strains and dipeptides Intestinal Screening Model To measure the effect of the probiotic strain Bacillus subtilis DSM 32315 on the adult colonic microbiota, an intestinal screening model was used (i-screening, TNO, The Netherlands). Thus, the i-screening model was inoculated with standard human adult fecal microbiota material, consisting of feces collected from six healthy adult volunteers (Caucasian, European lifestyle and nutrition). This fecal material was mixed and grown in a fed-batch fermenter for 40 h to generate a standardized microbiota as previously described

[23] . These standard adult gut microbiota sets were stored at -80 °C in 12% glycerol.

[0110] The intestinal microbiota was cultured in vitro in modified standard ileal emptying medium (SIEM) with the following modified composition: pectin 0.047 g / l, xylan 0.047 g / l, arabinogalactan 0.047 g / l, amylopectin 0.047 g / l, starch 0.392 g / l, casein 24.0 g / l, bactopeptone 24.0 g / l, bovine bile 0.4 g / l and cysteine ​​0.2 g / l.

[0111] All components were supplied by Trititium Microbiology (Veldhoven, The Netherlands). The pH of the medium was adjusted to 5.8.

[0112] For the i-screening fermentation, the pre-cultured standardized fecal inoculum was diluted 50-fold in 1350 μl of modified SIEM. All experiments were performed in triplicate. Bacillus subtilis (DSM 32315) and other strains were pre-cultured separately in 50 ml of LB Kelly medium for approximately 16 h. Incubation was performed in shake flasks at 37°C under aerobic conditions. After incubation, the bacterial density was determined by optical density measurement at 600 nm. 1 × 10 10 A final stock solution of cells / ml was prepared in 1 ml of buffer (0.1 mM MES pH 6). The suspension of each strain was diluted with approximately 10 9 Cells / ml were introduced into the i-screen to a final level of 100 μg / ml.

[0113] The i-screening incubation was carried out under the following gas conditions: 0.2% O2, 0.2% CO2, 10% H2, 89.6% N2.

[0114] Although the probiotic strain Bacillus subtilis DSM 32315 and further control strains (Bacillus strain B and Bacillus strain C) do not produce detectable levels of n-butyrate after 24 hours of exposure in the SIEM, they have a significant positive effect on the production levels of n-butyrate by the combination of the human microbiota with the dipeptide Ala-Gln (p-value < 0.05). The results are summarized in Figure 13.

[0115] FIG. 13 shows the n-butyrate concentration (mM) measured in the presence of colonic microbiota containing Bacillus subtilis DSM 32315 or the control strains Bacillus strain B or Bacillus strain C in combination with the dipeptide Ala-Gln after 24 hours of incubation in SIEM.

[0116] Comparison of the best B. subtilis strain DSM 32315 in combination with different dipeptides showed that the combination with the dipeptide Ala-Gln had the best effect on butyrate production, the results of which are summarized in Figure 14.

[0117] FIG. 14 shows the n-butyrate concentration (mM) measured in the presence of colonic microbiota containing Bacillus subtilis DSM 32315 in combination with the various dipeptides Ala-Gln, Ac-Gly-Glu, Gly-Gln and Gly-Tyr after 24 h of incubation in SIEM.

[0118] Example 7: Butyrate Production for Synbiotic Products with Various Coating Compositions Additionally, the effect of different coating compositions on butyrate production was analyzed: the colonic coating contained EUDRAGUARD® biotic, whereas the enteric coating contained EUDRAGUARD® natural.

[0119] EUDRAGUARD® biotic may begin to disintegrate at the pH conditions of the lower small intestine and colon (pH>7.0). The coating comprises a polymer composition, where the polymer is polymerized from 20-30% by weight methyl methacrylate, 60-70% by weight methyl acrylate, and 8-12% by weight methacrylic acid. It is more preferred if the polymer is polymerized from 25% by weight methyl methacrylate, 65% by weight methyl acrylate, and 10% by weight methacrylic acid.

[0120] EUDRAGUARD® natural is a corn starch-based coating that contains modified starch.

[0121] The results for butyrate production are summarized in Table 18 and FIG. [Table 18]

[0122] FIG. 15 shows butyrate production in an in vitro intestinal model with various coating compositions using two capsules per day (100% dose) or one capsule per day (50% dose). * = significantly different from control.

[0123] A 100% daily dose (=2 capsules) contains: Bacillus subtilis DSM 32315 spore powder (containing approximately 2 billion CFU), L-alanyl-L-glutamine (290 mg), 90 mg curcuma extract, 90 mg green tea extract, D- and B-vitamins and minerals.

[0124] References 1. Gasaly N, Hermoso MA, Gotteland M: Butyrate and the Fine-Tuning of Colonic Homeostasis: Implication for Inflammatory Bowel Diseases. Int J Mol Sci 2021, 22(6). 2. Boets E, Gomand SV, Deroover L, Preston T, Vermeulen K, De Preter V, Hamer HM, Van den Mooter G, De Vuyst L, Courtin CM et al: Systemic availability and metabolism of colonic-derived short-chain fatty acids in healthy subjects: a stable isotope study. J Physiol 2017, 595(2):541-555. 3. Nogal A, Valdes AM, Menni C: The role of short-chain fatty acids in the interplay between gut microbiota and diet in cardio-metabolic health. Gut Microbes 2021, 13(1):1-24. 4. Salamone D, Rivellese AA, Vetrani C: The relationship between gut microbiota, short-chain fatty acids and type 2 diabetes mellitus: the possible role of dietary fibre. Acta Diabetol 2021. 5. Hou JK, Abraham B, El-Serag H: Dietary intake and risk of developing inflammatory bowel disease: a systematic review of the literature. Am J Gastroenterol 2011, 106(4):563-573. 6. Vernia P, Annese V, Bresci G, d’Albasio G, D’Inca R, Giaccari S, Ingrosso M, Mansi C, Riegler G, Valpiani D et al: Topical butyrate improves efficacy of 5-ASA in refractory distal ulcerative colitis: results of a multicentre trial. Eur J Clin Invest 2003, 33(3):244-248. 7. Roda A, Simoni P, Magliulo M, Nanni P, Baraldini M, Roda G, Roda E: A new oral formulation for the release of sodium butyrate in the ileo-cecal region and colon. World J Gastroenterol 2007, 13(7):1079-1084. 8. Kaminski M, Skonieczna-Zydecka K, Nowak JK, Stachowska E: Global and local diet popularity rankings, their secular trends, and seasonal variation in Google Trends data. Nutrition 2020, 79-80:110759. 9. Halmos T, Suba I: [Physiological patterns of intestinal microbiota. The role of dysbacteriosis in obesity, insulin resistance, diabetes and metabolic syndrome]. Orv Hetil 2016, 157(1):13-22. 10. Boucher J, Kleinridders A, Kahn CR: Insulin receptor signaling in normal and insulin-resistant states. Cold Spring Harb Perspect Biol 2014, 6(1). 11. Vital M, Karch A, Pieper DH: Colonic Butyrate-Producing Communities in Humans: an Overview Using Omics Data. mSystems 2017, 2(6). 12. Perraudeau F, McMurdie P, Bullard J, Cheng A, Cutcliffe C, Deo A, Eid J, Gines J, Iyer M, Justice N et al: Improvements to postprandial glucose control in subjects with type 2 diabetes: a multicenter, double blind, randomized placebo-controlled trial of a novel probiotic formulation. BMJ Open Diabetes Res Care 2020, 8(1). 13. Stoeva MK, Garcia-So J, Justice N, Myers J, Tyagi S, Nemchek M, McMurdie PJ, Kolterman O, Eid J: Butyrate-producing human gut symbiont, Clostridium butyricum, and its role in health and disease. Gut Microbes 2021, 13(1):1-28. 14. Boesmans L, Valles-Colomer M, Wang J, Eeckhaut V, Falony G, Ducatelle R, Van Immerseel F, Raes J, Verbeke K: Butyrate Producers as Potential Next-Generation Probiotics: Safety Assessment of the Administration of Butyricicoccus pullicaecorum to Healthy Volunteers. mSystems 2018, 3(6). 15. Reidelberger R, Haver A, Anders K, Apenteng B, Lanio C: Effects of solid-phase extraction of plasma in measuring gut metabolic hormones in fasted and fed blood of lean and diet-induced obese rats. Physiol Rep 2016, 4(10). 16. de Luis DA, Gonzalez Sagrado M, Conde R, Aller R, Izaola O: Decreased basal levels of glucagon-like peptide-1 after weight loss in obese subjects. Ann Nutr Metab 2007, 51(2):134-138. 17. Adam TC, Jocken J, Westerterp-Plantenga MS: Decreased glucagon-like peptide 1 release after weight loss in overweight / obese subjects. Obes Res 2005, 13(4):710-716. 18. Ranganath L, Norris F, Morgan L, Wright J, Marks V: Inhibition of carbohydrate-mediated glucagon-like peptide-1 (7-36)amide secretion by circulating non-esterified fatty acids. Clin Sci (Lond) 1999, 96(4):335-342. 19. Verdich C, Toubro S, Buemann B, Lysgard Madsen J, Juul Holst J, Astrup A: The role of postprandial releases of insulin and incretin hormones in meal-induced satiety--effect of obesity and weight reduction. Int J Obes Relat Metab Disord 2001, 25(8):1206-1214. 20. Wu Y, Zhang Q, Ren Y, Ruan Z: Effect of probiotic Lactobacillus on lipid profile: A systematic review and meta-analysis of randomized, controlled trials. PLoS One 2017, 12(6):e0178868. 21. Qin P, Zou Y, Dai Y, Luo G, Zhang X, Xiao L: Characterization a Novel Butyric Acid-Producing Bacterium Collinsella aerofaciens Subsp. Shenzhenensis Subsp. Nov. Microorganisms 2019, 7(3). 22. Millan J, Pinto X, Munoz A, Zuniga M, Rubies-Prat J, Pallardo LF, Masana L, Mangas A, Hernandez-Mijares A, Gonzalez-Santos P et al: Lipoprotein ratios: Physiological significance and clinical usefulness in cardiovascular prevention. Vasc Health Risk Manag 2009, 5:757-765. 23. Ladirat SE, Schols HA, Nauta A, Schoterman MH, Keijser BJ, Montijn RC, Gruppen H, Schuren FH: High-throughput analysis of the impact of antibiotics on the human intestinal microbiota composition. J Microbiol Methods 2013, 92(3):387-397. 24. Faerch, K.; Torekov, S.S.; Vistisen, D.; Johansen, N.B.; Witte, D.R.; Jonsson, A.; Pedersen, O.; Hansen, T.; Lauritzen, T.; Sandbaek, A.; et al. GLP-1 Response to Oral Glucose Is Reduced in Prediabetes, Screen-Detected Type 2 Diabetes, and Obesity and Influenced by Sex: The ADDITION-PRO Study. Diabetes 2015, 64, 2513-2525. [CrossRef] 25. Seon, M.J.; Hwang, S.Y.; Son, Y.; Song, J.; Kim, O.Y. Circulating GLP-1 Levels as a Potential Indicator of Metabolic Syndrome Risk in Adult Women. Nutrients 2021, 13, 865. [CrossRef] 26. Adam, T.C.; Jocken, J.; Westerterp-Plantenga, M.S. Decreased glucagon-like peptide 1 release after weight loss in overweight / obese subjects. Obes. Res. 2005, 13, 710-716. [CrossRef] 27. De Luis, D.A.; Gonzalez Sagrado, M.; Conde, R.; Aller, R.; Izaola, O. Decreased basal levels of glucagon-like peptide-1 after weight loss in obese subjects. Ann. Nutr. Metab. 2007, 51, 134-138. [CrossRef]

[0125] Figure 1: Distribution of fasting plasma glucose levels [mg / dl]; scatter plot with mean ± 95% CI Figure 2: Distribution of total GLP-1 levels [pmol / L]; Scatter plot with mean ± 95% CI; Baseline vs. 2 weeks p<0.001 (paired t-test); Baseline vs. 4 weeks p<0.001 (paired t-test) Figure 3: Distribution of PYY levels [pg / mL]; Scatter plot with mean ± 95% CI; Baseline vs. 2 weeks p=0.0139 (paired t-test); Baseline vs. 4 weeks p<0.001 (paired t-test) Figure 4: Distribution of total cholesterol levels [mg / dL]; Scatter plot with mean ± 95% CI; Baseline vs. 4 weeks p=0.0037 (paired t-test) Figure 5: Distribution of HDL cholesterol levels [mg / dL]; scatter plot with mean ± 95% CI Figure 6: Distribution of triglyceride levels [mg / dL]; scatter plot with mean ± 95% CI Figure 7: Distribution of LDL / HDL cholesterol ratios; Scatter plot with mean ± 95% CI; Baseline vs. 4 weeks p=0.0022 (paired t-test) Figure 8: Collinsella aerofaciens [relative abundance] in stool samples by time point; scatter plot with mean ± 95% CI; baseline vs. 4 weeks p=0.0179 (Wilcoxon signed rank test) Figure 9: Fasting blood glucose before and after supplementation with SAMANA® FORCE. Paired t-test was used for analysis. Statistics shown. Figure 10: Correlation between fasting glucose at baseline and change in fasting glucose (delta %). Spearman correlation was used for analysis. Statistics shown. Figure 11: PPGR (median and IQR) of glucose test meal pre- and post-intervention in non-prediabetic participants Figure 12: PPGR (median and IQR) of glucose test meal before and after intervention in participants with prediabetes FIG. 13: Butyrate production in an in vitro gut model by synbiotic combinations containing different combinations of B. subtilis strains and dipeptides. Figure 14: Butyrate production in an in vitro intestinal model by combination of B. subtilis DSM 32315 with various dipeptides FIG. 15: Butyrate production in an in vitro intestinal model with different coating compositions using two capsules per day (100% dose) or one capsule per day (50% dose), *= significantly different from control.

Claims

1. A formulation for use in regulating satiety hormone concentration in a subject, comprising: - at least one probiotic Bacillus subtilis strain, and - at least one dipeptide containing glutamine or glutamate units , wherein the satiety hormone is selected from CCK, GLP-1 and PYY, the formulation.

2. The formulation according to claim 1, wherein the formulation should be administered to the subject at an oral dosage of at least once a day of at least 1 billion CFU of the probiotic Bacillus subtilis strain and at least 250 mg of the dipeptide.

3. The formulation according to claim 2, wherein the regulation of the satiety hormone is a decrease in satiety hormone concentration of at least 5%, or at least 10%, or at least 20%, or at least 30% four weeks after the administration of the formulation.

4. The formulation according to claim 1 or 2, further comprising one or more plant extracts selected from curcuma extract and green tea extract.

5. The formulation according to claim 1 or 2, wherein the probiotic Bacillus subtilis strain is selected from Bacillus subtilis DSM 32315, Bacillus subtilis DSM 32540, Bacillus subtilis DSM 32592.

6. The formulation according to claim 1 or 2, wherein the dipeptide is selected from glycine-glutamine, glycine-glutamate, alanine-glutamine, alanine-glutamate and their acetylated forms.

7. The formulation according to claim 1 or 2, wherein the total amount of the probiotic Bacillus subtilis strain and amino acids or oligopeptides is at least 40% by weight of the total weight of the formulation.

8. The formulation according to claim 4, wherein the total amount of the plant extract is at least 10% by weight of the total weight of the formulation.

9. The formulation according to claim 1 or 2, wherein the formulation comprises an enteric coating, where the enteric coating comprises one or more of the following: methyl acrylate-methacrylic acid copolymer, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymer, shellac, cellulose acetate trimellitate, sodium alginate, zein.

10. The formulation according to claim 1 or 2, wherein the formulation comprises an enteric coating comprising a polymer composition, wherein the polymer composition is polymerized from 20 to 30% by weight of methyl methacrylate, 60 to 70% by weight of methyl acrylate, and 8 to 12% by weight of methacrylic acid.

11. The formulation according to claim 1 or 2, for use in the prevention and treatment of obesity, adiposis, type 2 diabetes, and metabolic syndrome.

12. A formulation for use in reducing fasting blood glucose in a subject, - at least one probiotic Bacillus subtilis strain, and - at least one dipeptide containing a glutamine or glutamate unit comprising the formulation.

13. The formulation according to claim 12, wherein the probiotic Bacillus subtilis strain is Bacillus subtilis DSM 32315 and the dipeptide is alanyl-glutamine.

14. The formulation according to claim 12 or 13, wherein the blood glucose response in the subject is reduced.

15. The formulation according to claim 12 or 13, wherein the subject is a human and is in a prediabetic state and has a fasting blood glucose level above 100 mg / dl.

16. The formulation according to claim 15, wherein the weight of the subject is reduced by at least 1 kg four weeks after administration of the formulation.

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