HK1218839B - Treatment of obesity, the metabolic syndrome, type 2 diabetes, cardiovascular diseases, dementia, alzheimer's disease and inflammatory bowel disease by using at least one bacterial strain from prevotella - Google Patents

Description

Treatment of obesity, metabolic syndrome, type 2 diabetes, cardiovascular disease, dementia, Alzheimer's disease, and inflammatory bowel disease by using at least one bacterial strain from the genus Prevotella

Field of the Invention

The present invention relates to a product for treating obesity, metabolic syndrome, type 2 diabetes, cardiovascular disease, dementia, Alzheimer's disease and inflammatory bowel disease, comprising at least one isolated bacterial strain from the species Prevotellaceae, wherein the strain is selected from the group consisting of Prevotella copri, Prevotellastercorea, Prevotella histicola, Prevotella ruminicola, Prevotella Bryantii 25A and Prevotella distasonis. The product may be a food product.

Background of the Invention

Many food and ingredient companies in this area have increased their activities of identifying ingredients and food products, and described ingredients and food products, when in use, except providing basic nutrients, will also provide the health effects favorable to the consumer, and these ingredients and food products are so-called functional foods.Example is live microorganisms, i.e. so-called probiotics, which have shown the special health benefits to intestinal health and are included in food products, such as different dairy products, for example fermented milk, yogurt, and different beverages.Other examples of food products with health benefits are foods comprising dietary fiber (DF), prebiotic carbohydrates, stannol, ω-3 fatty acids, vitamins, polyphenols, hypoglycemic effect foods etc.

From the perspective of public health, there is a particular need to develop new food products that can be used to improve glucose metabolism and reduce obesity and related conditions. The impairment of glucose metabolism is also relevant to impaired cognitive function. The prevalence of lifestyle-related conditions (such as obesity and type 2 diabetes (T2D)) is growing globally, and it has been proposed that by 2030, the number of people suffering from T2D in the whole world will increase to 552 million by the current 366 million. Therefore, there is an urgent need for preventive strategies. Dietary-based prevention is considered to be the most effective strategy in the struggle with lifestyle-related diseases, and epidemiological studies show that, for example, a large amount of intake of whole grain foods and legumes is useful in preventing and managing diabetes and weight control.

Obesity is a major factor contributing to cardiometabolic disorders, but its mechanisms are not fully understood. However, the key features appear to be "metabolic inflammation" and activation of innate systems. Dietary patterns (e.g., high GI foods and energy-dense foods) are increasingly considered to predict future risk of cardiovascular disease. Currently, a growing body of knowledge supports the importance of healthy intestinal microbiota in the fight against cardiometabolic disorders, and the intestinal ecosystem is recognized as a regulator of host metabolism, appetite, and weight regulation. It has been shown that the metabolic "crosstalk" between the intestinal microbiota and peripheral tissues is regulated by intestinal fermentation of indigestible dietary components (e.g., indigestible carbohydrates).

A large body of evidence for the effects of fermentable gut substrates on host metabolism comes from studies using inulin, which has shown benefits on glucose metabolism and body weight regulation in animal experimental models.

Recently, the importance of the gut microbiota has been documented in studies based on fecal transplants from lean human donors to subjects with metabolic syndrome (MetS); MetS represents a group of risk factors that identify subjects at high risk for developing T2D and cardiovascular disease. Fecal transplants resulted in increased insulin sensitivity in T2D subjects 6 weeks after the fecal infusion. These new findings are exciting and increase knowledge about the importance of a healthy microbiota. However, fecal transplants can be complex and risky.

Therefore, strategies to increase the number of beneficial bacteria and to promote a healthy balance of the intestinal microbial composition by other means are needed. To date, most studies demonstrating an increase in beneficial bacteria following dietary manipulation have been cross-sectional and therefore cannot prove causality. This can be achieved by providing live bacteria (including so-called probiotics) via enemas or via food products, or by dietary supply of adequate colonic substrates (e.g., specific prebiotic substrates in the usual diet), or by combining probiotics with prebiotics.

Despite attempts in the food industry to develop new food products or functional food-type products with improved health properties, there are still huge problems with obesity and the increasing number of subjects suffering from MetS or T2D. These conditions are usually treated with medication. However, if healthy subjects or people at risk of the disease are provided with food products specifically designed to resist the early stages of the disease process, this will prevent the development of obesity and MetS (including impairment of cognitive function). In addition, such food products can also promote disease management in patients with overt diseases associated with MetS.

SUMMARY OF THE INVENTION

The present invention relates to the unique discovery that species belonging to the family Prevotellaceae have beneficial effects on host health. Species belonging to the family Prevotellaceae can be used in any kind of food product, or as an ingredient in a food product, as a probiotic (with or without added indigestible carbohydrates) enclosed in a capsule for administration, as an enema or suppository, thereby improving the health of the consumer.

In a first aspect, the present invention relates to a product for treating obesity, metabolic syndrome, type 2 diabetes, cardiovascular disease, dementia, Alzheimer's disease, and inflammatory bowel disease, comprising at least one isolated bacterial strain from the species Prevotellaceae, wherein the strain is selected from Prevotella enterica, Prevotella stercorea, Prevotellahisticola, Prevotella rumenicola, Prevotella briseae 25A, and Prevotella distichiae. Such a product makes it possible for the first time to provide new health food ingredients and food products, capsules, enemas, or suppositories that can be used to treat the various diseases mentioned above, thereby improving glucose metabolism and reducing risk factors in metabolic syndrome. The product can be provided together with one or more dietary fibers and/or resistant starch.

Further benefits and objects of the present invention will be described in more detail with particular reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Blood glucose and serum insulin responses (incremental change (Δ)) to a standardized breakfast after 3 days of consumption of barley-based bread (BB) and white wheat bread (WB). Compared with the reference WB, consumption of BB resulted in lower postprandial peak glucose and insulin concentrations (P < 0.01 and P < 0.001, respectively), as well as lower glucose and insulin areas under the curve (0-150 min: P < 0.01 and P < 0.001, respectively) (n = 39).

Figure 2. Blood glucose and serum insulin responses (incremental change (Δ)) to a standardized breakfast after 3 days of consumption of barley-based bread (BB) and white wheat bread (WB). Compared with the reference WB, consumption of BB resulted in lower postprandial peak glucose and insulin concentrations (both P < 0.05), as well as lower areas under the glucose and insulin curves (0-150 min: P < 0.05 and P < 0.01, respectively) (n = 20).

Figure 3. Exhaled _H2 excretion with a standardized breakfast after 3 days of consumption of barley-based bread (BB) and white wheat bread (WB). Compared with the reference meal WB, the consumption of BB resulted in a significant increase in exhaled _H2 (mean concentrations during the experimental days, P<0.01) (n=20).

Figure 4. Plasma PYY concentrations after 3 days of consumption of barley-based bread (BB) and white wheat bread (WB) at a standardized breakfast. Compared with 3 days of WB intake, BB consumption resulted in significantly higher plasma PYY concentrations (0-120 min) (main effect of experimental day, P < 0.05) (n = 20).

Figure 5. Glycemic response in responders (n=10) and non-responders (n=10) after 3 days of consumption of barley-based bread (BB) or white wheat bread (WB) after a standardized breakfast. In the responder group, 3 days of BB consumption resulted in a significantly lower glucose response compared to 3 days of WB intake (area under the curve 0-150 min, P<0.0001). In the non-responder group, no improvement in glucose tolerance (area under the curve) was seen with BB.

Figure 6. Serum insulin response in responders (n=10) and non-responders (n=10) after 3 days of consumption of barley-based bread (BB) and white wheat bread (WB) after a standardized breakfast. In the responder group, 3 days of BB consumption resulted in a significantly lower insulin response compared to 3 days of WB intake (area under the curve 0-120 min, P<0.01). In the non-responder group, no improvement was seen with BB.

Figure 7. Glycemic response in responders (n=7) and non-responders (n=7) after 3 days of consumption of barley-based bread (BB) and white wheat bread (WB), respectively, after a standardized breakfast. In the responder group, 3 days of BB consumption resulted in a significantly lower glucose response compared to 3 days of WB intake (area under the curve 0-150 min, P<0.0001). In the non-responder group, no improvement in glucose tolerance (area under the curve) was seen with BB.

Figure 8 shows the taxonomic variation of the main gut microbial species between the different groups involved in this study. Data were generated after 454 pyrosequencing analysis. The six bars represent the groups involved in this study: R-control (responder control), R-WB (responder white wheat bread), and R-BB (responder barley-based bread). NR = non-responder. Control refers to samples collected without consuming the test bread (BB or WB).

Figure 9 shows that mice colonized with intestinal Prevotella for 2 weeks (n = 6) exhibited improved oral glucose tolerance and lower serum insulin levels compared to mice colonized with Bacteroides thetaiotaomicron alone (n = 6) (P = 0.0076). Mice colonized with intestinal Prevotella and Bacteroides thetaiotaomicron dual colonization (n = 7) also showed better oral glucose tolerance compared to mice colonized with Bacteroides thetaiotaomicron alone (P = 0.0426).

Detailed Description of the Invention

definition

In the context of this application and invention, the following definitions apply:

The term "metabolic syndrome" or MetS refers to a group of risk factors, such as obesity, hyperlipidemia, hypertension, and glucose intolerance, that identify subjects at high risk for developing type 2 diabetes (T2D) and cardiovascular disease.

The term "probiotic" means live microorganisms, such as bacteria, that confer a health benefit on the host after colonizing the intestinal tract.

The term "dietary fiber" or "DF" means a carbohydrate comprising three or more monomers that resists digestion and absorption in the small intestine and is fully or partially fermented by intestinal bacteria in the colon. In the present application, the term DF refers to indigestible polysaccharides of non-starch origin, such as beta-glucans, arabinoxylans, cellulose, oligosaccharides, fructans, pectins, guar gums. Indigestible substrates closely related to indigestible polysaccharides in plants (e.g., indigestible protein fractions, phenolic compounds, lignin, waxes, phytates/inositol hexaphosphates, phytosterols) are also included in the definition of DF.

The term "resistant starch" (RS) refers to starch products and starch degradation products that escape digestion in the small intestine of healthy individuals, i.e., starch-derived DF. RS can achieve some of the benefits of insoluble DF and some of the benefits of soluble DF. RS can be derived from plant-encapsulated starch, non-gelatinized starch, or retrograded starch.

The term "retrograded starch" means that starch molecules have recrystallized after cooking and cooling, resulting in a structure that resists digestion and absorption in the small intestine.

The term "phytically encapsulated starch" means starch that is physically trapped within a food matrix or plant cells, thereby making it inaccessible to digestive enzymes in the small intestine.

The term "non-gelatinized starch" means unprocessed starch granules in their native form, such as starch in uncooked potatoes or uncooked grains, or starch granules that resist gelatinization after processing, resulting in an intact crystalline structure that remains indigestible.

The term "prebiotic" means an indigestible food ingredient, preferably an indigestible carbohydrate such as DF and RS, that stimulates bacterial growth and/or activity in the digestive system in a manner associated with health benefits.

The term "synbiotic" means a combination of probiotics and prebiotics.

The term "SCFA" means short-chain fatty acids produced from microbial intestinal fermentation of indigestible carbohydrates.

The term "encapsulated" means that the Prevotella strain can be encapsulated to protect it from the environment (e.g. oxygen and acidity) and will therefore remain intact and maintain quality and activity in the food product and during passage through the intestine. The technology used for encapsulation can be based on, for example, alginate/salt-, guar gum-, xanthan gum-, locust bean gum-, carrageenan- or other dietary fibers, casein-, prebiotic- or starch-based edible films, Pickering emulsions, etc. Different encapsulation technologies can be used, some of which are described by R Vidhyalakshmi et al., 2009[ 1 ].

The term "responder" means a human subject who experiences improved glucose regulation after consuming a prebiotic barley grain product.

The term "non-responder" means a human subject who shows no signs of improved glucose regulation after consuming a prebiotic barley grain product.

The term "enema" is used to describe the procedure by which a solution containing Prevotella species is introduced into the intestine via the rectum with or without a prebiotic substrate in order to favorably modulate the intestinal microbial composition.

The term "metabolic inflammation" is used to describe low-grade chronic inflammation orchestrated by metabolic cells in response to excess nutrients and energy associated with insulin resistance and metabolic dysfunction.

The present invention

The DF and RS inherent in barley grain products have been found to have beneficial effects on cardiometabolic risk markers and appetite regulation, which may also be true for other fiber groups. Thus, after the ingestion of barley grain products, glucose tolerance and effective insulin utilization were improved over a time perspective of 11-14 hours, which is an overnight span from a barley grain dinner to a subsequent standardized breakfast in healthy subjects.

Another important finding was the increase in plasma concentrations of GLP-1 and other appetite-regulating hormones and the reduction in spontaneous energy intake, while at the same time reducing perceived hunger. Furthermore, barley indigestible carbohydrates reduced markers of metabolic inflammation. An enhanced metabolic inflammatory state is a well-established cardiometabolic risk factor, making dietary approaches that reduce metabolic inflammation promising in the prevention of MetS and T2D. Increased breath hydrogen (a marker of intestinal fermentation) and SCFA production were observed, indicating increased intestinal fermentation activity following the ingestion of barley indigestible carbohydrates. Significant associations were found between markers of intestinal fermentation and improved glucose regulation, demonstrating the prebiotic-mediated effects of barley kernel-based products on metabolic risk markers, i.e., the observed beneficial effects are related to a mechanism of intestinal bacterial fermentation of indigestible carbohydrates. SCFAs produced during fermentation by the intestinal microbiota provide energy to the enterocytes of the colon and, in addition, act as signaling molecules.

Thus, it has been demonstrated that SCFAs produced by bacterial fermentation can trigger a signal transduction cascade by acting on SCFA receptors on L cells, leading to an increased release of intestinal peptides such as GLP-1 and PYY (in vitro model). The results obtained clearly demonstrate the prebiotic potential of DF and RS in cereal products such as barley grain products.

Surprisingly, it has been found that in healthy subjects, the benefits of cardiometabolic risk markers and appetite regulation observed after consuming barley grain products (bread) may be related to specific changes in the intestinal microbiome, and we have identified an increase in Prevotella species. In addition, transferring a specific Prevotella strain (intestinal Prevotella) to germ-free mice can improve glucose tolerance in animals. The results propose the probiotic effect of the identified bacterial strains that have benefits for glucose metabolism. One of the criteria for selecting Prevotella strains is the presence of the glycoside hydrolase required for degrading complex sugars. All selected strains have glycoside hydrolases and are capable of degrading complex sugars of huge scope. Another criterion is that the selected strains produce succinate/salt as the main product by degrading complex sugars. The examples of strains are intestinal Prevotella, Prevotella stercorea, Prevotella histicola, rumen-dwelling Prevotella, Prevotella brisebonae 25A and Prevotella distichi. Also possible are mixtures of one or more different strains, for example 2, 3, 4, 5 or 6 different strains. The strains may be present in amounts of 10 ⁷ or more, for example 10 ⁷ , 10 ⁸ , 10 ⁹ or even higher.

Furthermore, the results show for the first time that the studied prebiotic substrates (i.e., DF and RS present in barley grain products) combined with strains of the genus Prevotella can be particularly valuable in a synbiotic approach, as these intestinal substrates favor the expansion of Prevotella. The results were obtained in healthy middle-aged/elderly subjects in the fasting state and after a standardized breakfast after 3 days of ingestion of a barley grain-based product (based on 100 g of available carbohydrates/day). Using a crossover design, the barley grain product was compared with a white wheat bread reference product. By influencing the composition of the gut microbiota and increasing the proportion of Prevotella species, barley kernel-based products resulted in increased intestinal fermentation activity (breath hydrogen ( _H2 ) as a test marker) (P<0.001), increased SCFA concentrations (P<0.05), improved glucose regulation (reduced glucose and insulin concentrations) (P<0.05), increased PYY (a satiety hormone released in the gut) concentration (P<0.05), increased fasting GLP-1 (an anti-obesity and anti-diabetic gut hormone) concentration, increased perceived fasting satiety, and decreased fasting hunger. By altering the composition of the gut microbiota and increasing the proportion of Prevotella species, the consumption of barley kernel-based foods enriched with barley DF and RS increased the intestinal fermentation metabolite succinate in humans and mice.

The results further showed that among the test subjects, some individuals (approximately 15%) did not achieve improved glucose regulation after consuming the test product based on barley grains (measured by glucose- and/or insulin response to a standardized meal after a 3-day intervention with barley). Interestingly, these "non-responders" also did not achieve increased intestinal Prevotella concentrations after consuming the barley grain product. This emphasizes the causal relationship between improved blood glucose regulation and increased abundance of Prevotella species, which is completely new knowledge in the public domain. In addition, these results show for the first time that some individuals do not easily change their intestinal microbiota composition and can particularly benefit from the oral intake of the proposed barley prebiotic (DF+RS) in combination with Prevotella species; or the intake of Prevotella species alone.

Benefits on glycemic control, metabolic inflammatory markers and appetite regulation have been identified using, for example, barley grain food products enriched with DF and RS or DF or RS. Metabolic benefits were associated with markers of intestinal fermentation (i.e., breath hydrogen), plasma SCFAs (which indicate a prebiotic mechanism).

Gut microbial profiling of fecal samples from healthy humans who consumed the aforementioned barley grain product demonstrated that benefits on metabolic risk markers and appetite regulation were associated with specific changes in the composition of microorganisms present in the gut. Compared to subjects who did not consume the barley grain food product, subjects who consumed the aforementioned barley grain food product showed an increase in Prevotella species in their gut microbiota and improved cardiometabolic risk markers (see Examples below).

Furthermore, it has been shown that colonizing germ-free mice with a single strain of Prevotella species improves the animals' glucose metabolism. Therefore, the above results suggest that species related to Prevotella are suitable for use as probiotics with, for example, antidiabetic properties and weight-regulating potential. The close relationship between glucose metabolism impairment and impaired cognitive function also makes Prevotella species relevant for preventing cognitive impairment associated with MetS. Other diseases or conditions are described above.

Based on the above experiments and conclusions, it was determined that the present invention relates to a product, such as a food product or food ingredient, or a formulation to be used as an enema, comprising at least one bacterial strain isolated from humans from the family Prevotellaceae. The bacteria may be genetically modified, for example, to produce succinates. Examples include Prevotella enterica, Prevotella stercorea, Prevotella histicola, Prevotella rumen-dwelling, Prevotella briseae 25A, and Prevotella distichiae.

Probiotic food products can be used alone or in combination with other ingredients, such as at least one type of dietary fiber (e.g., DF and RS or DF or RS), and in some instances, such as succinic acid to regulate glucose metabolism and reduce risk factors for metabolic syndrome in mammals (e.g., humans). Examples of diseases or conditions treated include treatment of obesity, metabolic syndrome, type 2 diabetes, cardiovascular disease, dementia, Alzheimer's disease, and inflammatory bowel disease, such as obesity, metabolic syndrome, and type 2 diabetes.

The probiotic food product can also include at least one DF and/or one RS, its natural or synthetic, purified, mixture or variant. Examples of DF and RS are DF and RS from cereals or legumes, such as viscous and non-viscous DF from barley, rye, wheat, oats, legume seeds, beta-glucans, guar gum, lignin, lignans and oligosaccharides (such as galacto-oligosaccharides and fructo-oligosaccharides), plant-encapsulated RS, retrograded RS, chemically modified starch or non-gelatinized RS. The product can include at least one type of resistant starch (RS), its natural, synthetic, purified, mixture or variant. Examples of RS are plant starch, such as retrograded starch, plant-encapsulated starch, non-gelatinized starch and cyclodextrin, or chemically modified starch. Fiber can be, for example, from barley grains. The ratio of RS/DF can be, for example, 10.8 (RS)/13.04 (DF). DF can be dietary fiber from barley, wheat, rye, oats or extracted β-glucans, guar gum, lignin, lignans and oligosaccharides (e.g., galacto- and fructo-oligosaccharides), plant-encapsulated RS, retrograded RS, chemically modified starches or non-gelatinized RS. The product can contain at least one type of resistant starch (RS), natural, synthetic, purified, mixtures or variants thereof. Examples of RS are plant starches, such as retrograded starch, plant-encapsulated starch, non-gelatinized starch and cyclodextrins, or chemically modified starches. The fiber can, for example, be derived from barley grains.

The product may, for example, comprise one or more bacterial strains and RS and/or DS to achieve the desired result.

In another embodiment, the product of the present invention comprises other bacterial strains, wherein the bacterial strain can produce succinate/salt.The example of such bacterial strain includes any beneficial bacterial strain, and includes for example Lactobacillus (Lactobacillus), such as Lactobacillus reuteri (Lactobacillus reuteri), Lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus plantarum (Lactobacillus plantarum); Roseburia (Roseburia) and Bifidobacteria (Bifidobacteria), such as Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium lactis (Bifidobacterium lactis), Bifidobacterium animalis (Bifidobacterium animalis) and Bifidobacterium bifidum (Bifidobacterium bifidum).Other bacterial strains can also be genetically modified, for example to produce succinate/salt.

The product may also contain succinate in free form or with the addition of a bacterial strain that produces succinate. The bacterial strain may be naturally producing succinate or may be genetically modified to produce succinate.

Thus, the product may be encapsulated or lyophilized, as is well known to those skilled in the art.

In another embodiment, the bacterial strain from the family Prevotellaceae is encapsulated in any form to protect the strain from oxygen. Examples of encapsulation techniques include stabilized Pickering emulsions using starch granules as a barrier material, edible films, encapsulation based on polysaccharides such as β-glucan, guar gum, xanthan gum, locust bean gum, carrageenan, prebiotics, alginates/salts, milk proteins, etc. Other techniques include the formation of interpolymer complexes in supercritical carbon dioxide. Various encapsulation techniques can be used, some of which are described by R Vidhyalakshmi et al., 2009.

In another embodiment, one or more bacterial strains from the family Prevotellaceae may be provided by enema alone or in combination with a prebiotic substrate (eg, DF and/or RS).

The product can be, for example, any suitable food product and includes any food product that is not heat treated or has been mildly heat treated, or into which Prevotella can be introduced after heat treatment, such as beverages, shots (e.g., fruit or milk based), smoothies, drinks, juices, drinking water, cold soups/soup mixes, oil products, spreads, condiments, cold sauces/sauce mixes, salsa, dairy products, ice cream, and cereal-based beverages containing or supplemented with Prevotella or a combination of Prevotella and prebiotics. Prevotella can also be encapsulated and included in the foods described above, thereby increasing resistance to environmental and processing conditions.

The food product can also be the food of heat treatment, for example soft bread, crisp bread, flat bread, corn tortilla, porridge, breakfast cereals, cereal bar or other snacks, potato flour or other fast food products, instant meals, they comprise for example prebiotics from barley, to supplement or not supplement when Prevotella is edible.The capsule that comprises Prevotella bacterial strain is taken in together with the product listed above, or capsule is edible for providing Prevotella.Capsule can be prepared by prebiotic carbohydrate (for example by barley) additionally.Prevotella can also for example be encapsulated and be included in food by stable emulsion technology.

Prevotella species can also be provided in dry powder form and packaged in disposable containers to be added to meals (e.g., breakfast cereals). The food product can also be, for example, a drink, soup, yogurt, cold cereal pudding, or pellets provided in a two-chamber package, with the Prevotella species, with or without prebiotics, in one chamber and the food product in the other, which are mixed when consumed. The chamber with the Prevotella species can also be composed of a straw, which is included in the package or dispensed separately. The Prevotella species is then consumed by drinking through the straw.

The products can be stable emulsions in which Prevotella with or without prebiotics are encapsulated in microdroplets of the emulsion. These products can be administered, for example, in the form of pellets.

In case Prevotella is contained in a capsule, the capsule should preferably be in the form of an enteric-coated capsule.Prevotella may also be contained in an enteric-coated tablet, a depot tablet, a depot (extended release) capsule or an extended release granulate.

The product may also be a food ingredient to be added to a food product.

The present invention also defines products that can be used to treat many diseases or conditions, including those mentioned below. The products can be used to treat obesity and related conditions (e.g., type 2 diabetes) related to impaired blood sugar regulation, as well as provide protection for subclinical inflammation and related diseases (e.g., cardiovascular disease). In addition, the protected regimen resists the characteristics of the pathogenesis of dementia. Furthermore, the imbalance of blood sugar is associated with inflammation and endothelial damage, thereby also providing a connection between the protected regimen and the prevention of cardiovascular disease.

Thus, even mild impairment of glucose regulation within the normal range is associated with significantly lower performance on cognitive tests, and the regimens described herein improve glucose regulation, suggesting an additive benefit in dementia. Furthermore, the protected regimens described herein stimulate incretin hormones, such as GLP-1. GLP-1 exerts neuroprotective and anti-apoptotic effects, reducing beta-amyloid (Aβ) plaque accumulation, regulating long-term potentiation and synaptic plasticity, and promoting the differentiation of neuronal progenitor cells, thereby preventing dementia and Alzheimer's disease. Neuroprotective effects of GLP-1: Cognitive deficits can be treated in individuals with mood disorders. Furthermore, stimulation of GLP-2 observed using the protected regimens protects against inflammatory bowel disease, and exogenous GLP-2 is known to protect the mucosa from chemotherapy-induced mucositis in rats.

The present invention also relates to the identified products (e.g., food products or food ingredients), and the use of the products for improving glucose metabolism, reducing metabolic inflammation, promoting appetite regulation, and reducing risk factors in metabolic syndrome. This can be used to prevent and regulate conditions associated with MetS, such as obesity, glucose intolerance, diabetes, or cardiovascular disease, as well as conditions in subjects with cognitive impairment associated with MetS. The probiotics of the present invention can also be used in enema formulations or suppositories (with or without a prebiotic substrate).

The invention also relates to the use of a product as defined above for reducing risk factors in metabolic syndrome, improving glucose metabolism, promoting weight regulation and reducing the risk of cognitive impairment associated with MetS.

Finally, the present invention relates to the use of succinates or succinate-producing bacterial strains for improving glucose metabolism, promoting body weight regulation and reducing risk factors in metabolic syndrome.

The product can be used on humans, horses, as well as dogs and cats.

The following examples are intended to illustrate but not to limit the invention in any way, shape or form, either explicitly or implicitly.

Example

Example 1

Prebiotic carbohydrates in barley grain-based products beneficially affect metabolic risk markers, appetite-regulating hormones, and perceived satiety in healthy subjects

Overview of research content and research design

The aim of this study was to evaluate the effect of inherently indigestible carbohydrates (DF and RS present in barley kernel-based products) on risk markers associated with MetS. In a crossover design, 39 healthy middle-aged subjects (age 64.5 ± 5.6 years) with a normal body mass index (mean ± SD = 23.6 ± 2.3 kg/m ² ) were provided with either barley kernel-based bread or white wheat bread (WB, reference product) for 3 consecutive days. On the following day (day 4), a standardized breakfast was provided, and physiological test markers were repeatedly measured in the fasting state and in the postprandial time (0-180 min). The study was divided into 3 sub-studies (Studies AC). Physiological test markers in Study A included measurements of blood glucose, serum insulin, breath hydrogen (H ₂ ) excretion (a marker of colonic fermentation), and recording of the subjects' appetite. Venous blood was repeatedly collected in a subgroup of the cohort for further evaluation (see below). In addition, fecal samples were collected before the start of the study and after each intervention period for characterization of the gut microbiota (Study A).

Based on the above cohort, additional physiological test markers were determined in 20 randomly selected subjects (64.1±5.9 years, 23.5±2.2 kg/m ² ) (Study B). In addition to blood glucose, serum insulin and breath H ₂ , appetite-regulating hormones (PYY, GLP-1) and plasma short-chain fatty acids (SCFA) were measured.

In Study C, 20 subjects from the Study A cohort were selected based on the extent to which glucose regulation improved with a standardized breakfast over the overnight span after consuming a barley grain-based product (BB) the previous night. The 10 subjects in whom BB had the most pronounced effect on glucose regulation and the 10 subjects in whom BB had the least pronounced effect on glucose regulation were included. Study C is reported separately below.

Materials and methods

Test subjects

Inclusion criteria were age 50-70 years, normal to slightly overweight (BMI 18-28 kg/ ^m² ), fasting blood glucose ≤ 6.1 mmol/L, nonsmokers, generally healthy, and without known metabolic disorders or food allergies. Antihypertensive agents without any anti-inflammatory properties and over-the-counter analgesics were acceptable. The study was approved by the Regional Ethical Review Board in Lund, Sweden (reference 2010/457).

Study A: Six male and 33 female healthy volunteers, aged 64.5±5.6 years and with normal body mass index (mean±SD=23.6±2.3 kg/m ² ), participated in this study.

Study B: Twenty healthy volunteers, 3 males and 17 females, aged 64.1 ± 5.9 years and with normal body mass index (mean ± SD 23.5 ± 2.2 kg/m ² ), were randomly selected from the cohort in Study A.

Test meals

The test diets were barley-based bread (BB) and white wheat bread (WB; reference diet). Each test product was consumed on three consecutive days at intervals of two weeks. The amounts of the test and reference products were calculated to provide 100 g of potentially available starch per day, as analyzed by Holm et al. [2] (Table 1). On the first two days, the daily intake was divided into three equal portions to be consumed at approximately 0800, 1400, and 2100. On the third day, half of the daily intake (50 g of available starch) was divided equally between the 0800 and 1400 diets, and the other half was consumed at 2100.

Table 1. Composition of barley grain bread (BB) and white wheat bread reference (WB) in studies A-C.

¹ Calculated by difference; total starch minus RS (3-5).

² The DF mentioned in this application does not contain physically unavailable or indigestible starch, ie RS.

Standardized breakfast

After 3 days of intervention with BB or WB, respectively, a standardized breakfast was consumed and consisted of 122.9 g WB (corresponding to 50 g available carbohydrates) and 2.5 dl of drinking water.

Recipes and preparation of test products

White wheat bread (WB, reference meal and standardized breakfast) : WB was baked in a home baker according to a standardized program (Tefal home bread model nr. 573102; menu selection, program 2 [white bread, 1000 g, fast (time 2:32)]). Bread was made from 540 g white wheat flour (Ab, Sweden), 360 g water, 4.8 g dry yeast, 4.8 g NaCl (iodine-free). After cooling, the bread was sliced and divided into portions, wrapped in aluminum foil, placed in plastic bags and stored in the refrigerator (-20°C). One day before consumption, the testers were instructed to thaw the bread at ambient temperature, still wrapped in aluminum foil and plastic bags.

Bread (BB) based on barley grains : 595g barley grains were boiled in 520g water for 12min, then cooled at ambient temperature for 30min. All water was absorbed into the barley grains during cooking. 105g wheat flour, 6g dry yeast, 5g salt and 300g water were added to the barley grains. The dough was kneaded for 4min (Electrolux AKM 3000, N23N25) and proofed for 30min in a bowl, then proofed again in a baking tray (35min). The baking tray was covered with aluminum foil and baked at 225°C in a household oven, wherein a tray of water was retained to maximize the steam present, until the internal temperature of the bread reached 96°C. After baking, the bread was cooled at ambient temperature in a damp towel without a baking tray. After cooling, the bread was placed in a plastic bag and placed at room temperature at night. One day after the bread was sliced and divided into portions and wrapped in aluminum foil, it was placed in a plastic bag and stored in a refrigerator (-20°C).

Analysis of total starch, RS and DF in test products

The test products were analyzed for total starch [3], RS [4], and DF [5]. The bread was air-dried and ground before analysis for total starch and DF. RS in the test products was analyzed as consumed. Available starch was calculated by subtracting RS from total starch (Table 1).

Experimental procedures

This study is a randomized (order of test products) crossover study, which means that each subject participates in 2 3-day interventions, consuming BB or WB respectively, with an interval of about 2 weeks. In those days of consuming test or reference products, encourage the subject to standardize their usual meals and meal patterns, and avoid alcohol, excessive physical exercise or foods rich in DF. In addition, in the previous 2 weeks and throughout the research process, they should not consume antibiotics or probiotics. After the last test dinner of consuming barley grain bread or WB respectively, the subject is fasted, and standardized breakfast is provided until the next morning. The subject arrives at the experimental department at 0730, and an intravenous cannula (BD Venflon, Becton Dickinson) is inserted into the antecubital vein for blood sampling. Collect fasting blood test, and record satiety and exhaled H ₂ before consuming breakfast. At～0800 and in 13min, consume breakfast. In addition, the measurement of appetite before and after breakfast is obtained using 100mm visual analogue scale (VAS). During the 2.5 h period of repeated blood sampling, subjects were instructed to maintain a constant and low level of physical activity.

Sampling and analysis of physiological variables and breath hydrogen in exhaled breath

Finger prick capillary blood samples were obtained for the determination of blood glucose (B-glucose, HemoCue AB, Sweden). Venous blood samples were collected for the determination of physiological test markers in serum (s) (s-insulin) and plasma (p) (p-SCFA, p-GLP-1 and p-PYY). Serum and plasma were separated by centrifugation and immediately stored in a refrigerator (-40°C) until analysis. Prior to blood sampling, blood collection tubes intended for the analysis of plasma GLP-1 and PYY were prepared using an inhibition mixture consisting of DPPIV (10 μl/ml blood) (Millipore, StCharles, USA) and 10000 KIE/ml aprotinin (50 μl/ml blood) (Bayer HealthCare AG, Leverkusen, Germany). The tubes containing the inhibition mixture were kept on ice until use, but for a maximum of 6 days. Commercial enzyme-linked immunosorbent assay (ELISA)-based kits were used to determine s-insulin (Mercodia, Uppsala, Sweden), PYY (3-36 and 1-36), and GLP-1 (active 7-36) (Alpco Diagnostics, Salem USA). SCFA (acetate, propionate, butyrate) were determined using GC [6]. Breath hydrogen (H ₂ ) was measured in exhaled breath as an indicator of colonic fermentation activity using a Gastro+ (Bedfont EC60 Gastrolyzer, Rochester, England). Fecal samples were collected before and on day 4 of the dietary intervention (i.e., after 3 days of consumption of the test and reference products, respectively). Subjects were instructed to collect feces from the first bowel movement occurring on day 4 and to freeze the samples immediately and submit them to the laboratory within 24 h for continuous storage at -80°C until analysis. The times used to determine the physiological parameters are shown in Table 2.

Table 2. Schedule for determination of test markers.

Computational and statistical methods

GraphPad Prism (5th edition, GraphPad Software, San Diego, CA, USA) is used for graphing. Trapezoidal model is used to calculate the blood glucose and serum insulin area under the curve (iAUC, concentration as a function of time) for each subject and test meal. The increasing peak (iPeak) concentration of glucose and insulin is measured as the individual maximum postprandial increment from baseline. ANOVA (general linear model) is used to assess the significant differences of the test variables after different test meals in MINITAB statistical software (the 16th edition of release; Minitab, Minitab Inc, State College, PA). In the case of unevenly distributed residuals (using Anderson-Darling test, and when P < 0.05, it is considered to be unevenly distributed), Box Cox conversion is performed on the data before ANOVA. Pearson correlation is used to perform correlation analysis between parameters in MINITAB statistical software (the 14th edition of release; Minitab, Minitab Inc, State College, PA). The value of P < 0.05 is considered to be significant. Data are expressed as mean ± SEM, and values of P < 0.05 were considered significant.Example 1: Study A: n = 39, Study B: n = 20, Study C: n = 20.

result

Study A (n=39)

Blood glucose and serum insulin

Consuming BB for 3 days significantly improved the glycemic response to a standardized breakfast compared to the reference WB regarding postprandial peak concentrations (iPeak, P < 0.01) and iAUC 0-150 min (P < 0.01) (Table 3). Furthermore, BB resulted in significantly lower insulin iAUC (0-150 min, P < 0.001) and insulin iPeak (P < 0.001, Figure 1, Table 3).

Table 3. Blood glucose and serum insulin responses to a standardized breakfast after 3 days of consumption of barley kernel-based bread (BB) or white wheat bread (WB).

*; P < 0.05, **; P < 0.01, ***; P < 0.001 (difference between WB and BB).

Exhaled _H2

Compared with the reference WB, consumption of BB significantly increased exhaled H ₂ , indicating increased intestinal fermentation activity after BB (P<0.001, Table 4).

Table 4. Exhaled _H2 output and appetite perception after fasting and after a standardized breakfast, after 3 days of consumption of barley kernel-based bread (BB) or white wheat bread (WB), respectively.

*; P < 0.05, ***; P < 0.001 (difference between WB and BB).

Appetite sensation

Compared with consuming WB for 3 days, consuming BB for 3 days resulted in increased satiety and decreased hunger on an empty stomach the next day (P<0.05, Table 4).

Study A: Overview and Conclusions: Consumption of a barley kernel-based product resulted in improved postprandial glucose regulation 11-14 hours after the last serving. Furthermore, the barley kernel-based product increased satiety and reduced hunger over the same timeframe. Concurrently, breath hydrogen excretion increased, indicating increased colonic fermentation activity. The results suggest that the beneficial effects achieved after consumption of the barley kernel-based product are mediated by increased intestinal fermentation of the intrinsic DF and RS present in the barley kernel-based product and activation of specific microbiota.

Study B (additional markers tested, n=20, randomly selected from the cohort in Study A)

Blood glucose and serum insulin response

Consuming BB for 3 days beneficially affected the glycemic response at the subsequent standardized breakfast compared to consuming WB for 3 days, with lower iPeak (P < 0.05) and iAUC 0-150min (P < 0.05). In addition, s-insulin iAUC (0-150min) and insulin iPeak were significantly reduced (P < 0.01 and P < 0.05, respectively) (Figure 2).

Markers of intestinal fermentation: exhaled _H2 and SCFA

Compared with the reference diet, WB, consuming BB for 3 days resulted in a significant increase in exhaled _H₂ (P < 0.01, Figure 3, Table 5). Furthermore, BB ingestion significantly increased the concentrations of s-acetate and total SCFA (P < 0.05, Table 5). No significant increase in circulating s-butyrate was observed after BB compared with WB (BB 16.1 ± 0.7; WB 14.2 ± 0.9; P = 0.11). These results suggest increased intestinal fermentation activity after BB.

Table 5. SCFA concentrations in fasting plasma after 3 days of consumption of barley kernel-based bread (BB) or white wheat bread (WB), respectively.

*; P<0.05, ¹ P=0.11.

Gut-related hormones

GLP-1

GLP-1 is a hormone produced by L cells in the intestine that increases insulin secretion from the pancreas and increases insulin sensitivity of both alpha and beta cells in a glucose-dependent manner. GLP-1 also increases beta cell mass, inhibits acid secretion and gastric emptying in the stomach, and reduces food intake by enhancing satiety signals in the brain.

Consumption of the BB test diet resulted in significantly increased p-GLP-1 concentrations under fasting conditions on day 4 after BB (1.6 ± 0.5 pmol/L) compared to after WB (1.0 ± 0.4 pmol/L) (P < 0.01).

GLP-2

The concentration of p-GLP-2 during the experimental days increased significantly after 3 days of BB consumption (average 0-150 min: 3.5 ± 0.5 ng/ml) compared with after WB consumption (average 0-150 min: 3.1 ± 0.4 ng/ml, P < 0.05).

PYY

PYY is also produced by L cells and inhibits gastric motility and has been shown to reduce appetite.

A significant main effect of the test diet was observed after 3 days of BB consumption compared to 3 days of WB intake, showing an increase in the plasma concentration of PYY (0-120 min) (P<0.05, FIG4 ).

The relationship between gut-related hormones and SCFA

After 3 days of WB or BB consumption, the plasma concentration of PYY correlated with the plasma concentration of total SCFA (r = 0.51, P < 0.05 and r = 0.57, P = 0.01), respectively.

Study B: Overview and Conclusions: This extended study showed that, in addition to the results presented in Study A, the barley grain-based product increased plasma concentrations of gut hormones important for appetite and glucose regulation over a time span of 11-14 hours after consumption. Increased intestinal fermentation activity was measured as increased breath hydrogen excretion and increased plasma concentrations of SCFAs. Furthermore, the barley grain product increased plasma concentrations of GLP-2, a gut hormone important for intestinal barrier function by reducing the permeability of the intestinal wall to, for example, endotoxins.

The present study suggests that intestinal fermentation of inherently indigestible carbohydrates present in barley kernel-based products may constitute the mechanism of a promising approach aimed at preventing and/or treating obesity and related metabolic disorders.

Study C (n=20, selected from the Study A cohort based on the degree of improvement in glucose regulation with a standardized breakfast)

Based on the cohort of Study A (n=39, Example 1), 20 subjects (18 women and 2 men, age (mean±SD) 64.9±5.1 years, body mass index (mean±SD) 23.2±2.4 kg/m ² ) were included in a further study. The subjects were selected based on the efficacy of the barley grain product in improving individual glucose regulation compared to WB. The 10 subjects (8 women and 2 men, 64.0±4.6 years, BMI 23.9±2.7 kg/m ² ) with the most pronounced effect of BB on glucose regulation in Study A were included and designated "responders", and the 10 subjects (10 women, 65.7±5.3 years, BMI 22.5±1.7 kg/m ² ) with the lowest improvement in glucose regulation by BB were included and designated "non-responders". The criteria used to define responders and non-responders are described below. In the responder group, 3-day consumption of BB resulted in significantly improved glucose tolerance after a standardized breakfast (iAUC 0-150 min after BB and WB: 156 ± 20 and 251 ± 26 mmol*min/L, respectively, P < 0.0001, Figure 5) and reduced insulin response (iAUC 0-120 min after BB and WB: 10.3 ± 1.5 and 15.3 ± 2.1 nmol*min/L, respectively, P < 0.01, Figure 6). In contrast, no significant improvement was observed in the non-responder group in either glucose tolerance (iAUC 0-150 min after BB and WB: 173 ± 23 and 191 ± 24 mmol*min/L, respectively, P = 0.11) or insulin response (iAUC 0-120 min after BB and WB: 15.8 ± 1.8 and 16.3 ± 1.5 nmol*min/L, respectively, P = 0.51).

Fecal samples collected before the study and after consumption of BB and WB, respectively, were characterized with respect to the gut microbiota (see Example 2).

Definition of “Responders” and “Non-Responders”

Responders were defined as the 10 subjects in the cohort of Study A (n=39) who achieved the most significant beneficial effects of the barley grain product on glucose regulation. Among responders, the barley grain product reduced the area of incremental glucose after a standardized breakfast (iAUC, 0-90 min) by at least 25% (ranging from -25 to -67%, with a mean of -39%), reduced the total AUC over the same period, and reduced the insulin iAUC after a standardized breakfast by at least 15% (ranging from -15 to -69%, with a mean of -33%). Many of the remaining 29 subjects had a reduced glucose response or a reduced insulin response. However, the 10 subjects with the lowest improvement in glucose and/or insulin response were designated "non-responders." In the non-responder group (n=10), 4 subjects achieved unreduced glucose and insulin responses after barley, 3 subjects had a slightly reduced glucose response but no improvement in insulin response, and 3 subjects achieved a slightly reduced insulin response after breakfast but no improvement in glucose response.

Study C: Overview and Conclusions: Results showed individual differences in the beneficial efficiency of barley grains on glucose regulation over a span of 11-14 hours. The reason for the individual discrepancies was suggested to be related to differences in the composition of the gut microbiota.

A follow-up to Study C was performed. The aim was to confirm the results previously obtained on glucose regulation and gut microbiota after consuming BB versus WB. The study design was similar to the one described previously. All subjects in Study C (10 responders and 10 non-responders) were asked if they would like to repeat the study. Seven responders (6 females and 1 male, 65.6 ± 4.3 years old, BMI 23.5 ± 3.2 kg/m ² ) and seven non-responders (7 females, 64.7 ± 6.1 years old, BMI 21.7 ± 1.2 kg/m ² ) received results.

The results of the follow-up study were consistent with those obtained previously. Thus, in the responder group (n = 7), BB significantly improved glucose tolerance to a standardized breakfast (iAUC 0-150 min after BB and WB: 239 ± 32 and 295 ± 28 mmol*min/L, respectively, P < 0.0001, Figure 7), while no improvement in glucose tolerance was observed in the non-responder group (iAUC 0-150 min after BB and WB: 277 ± 20 and 287 ± 36 mmol*min/L, respectively, P = 0.81).

Summary and conclusions of the follow-up study: The results obtained in the follow-up study thus confirm the results obtained in the previous above-mentioned studies (Studies A-C) regarding the beneficial effects of DF and RS in BB on glucose regulation and indicate the individual components of the metabolic response.

Example 2

Characterization of the human gut microbiota after ingestion of WB or BB products; and inoculation experiments in mice to confirm causality

Fecal DNA extraction, amplification, pyrosequencing, and data analysis

Genomic DNA was isolated from 100–150 mg of fecal material per individual using the repeated bead beating (RBB) method previously described by Salonen et al. [7]. Fecal DNA was quantified using a NanoDrop ND-1000 spectrophotometer (Nano-Drop Technologies), and genomic DNA quality was assessed by gel electrophoresis using 1% agarose-GelRed gels. Aliquots of genomic DNA were diluted to 10 ng/1 μl and then used for PCR.

Amplification of the V1-V2 variable regions of the 16 rRNA gene was performed using 27F and 338R primers fused to 454 titanium sequencing adapters to assess fecal microbiome diversity. The 338R primer contains a unique error-correcting 12-base barcode that allows analysis of multiple samples in a single sequencing run. Each sample was amplified in triplicate in a 25 μL reaction volume containing 1.5 U FastStart Taq DNA polymerase (Roche), 0.2 μM of each primer, and 1 ul (~10 ng) of extracted genomic DNA. PCR was performed under the following conditions: initial denaturation at 95°C for 3 min; followed by denaturation at 95°C for 20 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 60 seconds for 25 cycles; and a final extension step at 72°C for 10 min. The PCR products were analyzed by PCR agarose gel electrophoresis (ELISA) and agarose gel electrophoresis (ELISA) kit (Invitrogen, Carlsbad, CA). The PCR products were diluted to a concentration of 20 ng/1 μl and then amplified using Ampure magnetic purification beads (Agencourt, Danvers, MA). The resulting products were analyzed by 454GS FLX titanium chemistry sequencing at National Genomics Infrastructure (Stockholm).

The raw data were quality filtered to remove sequences shorter than 200 nucleotides, longer than 1000 nucleotides, containing primer mismatches, ambiguous bases, uncorrectable barcodes, or homopolymer runs of more than 6 bases. The read lengths of the quality filters were trimmed by their 454 adapters and barcode sequences and analyzed using the software package QuantitativeInsightsIntoMicrobialEcology (QIIME) (version 1.5.0). The number of read lengths assessed by the quality filter totaled 755,963 (an average of 12,599 sequences/sample). Denoising of the sequencing data was performed using denoise_wrapper.py (a wrapper algorithm available in QIIME).

Sequences were assigned to operational taxonomic units (OTUs) using UCLUST with a pairwise identity threshold of 97%. The most abundant sequence was selected as the representative of each OTU and taxonomically assigned using the Ribosomal Database Project (RDP) classifier. Representative OTUs were aligned using Pynast and used to construct a phylogenetic tree using FastTree, which estimated the β-diversity of the sample (weighted UniFrac).

Extraction and quantification of short-chain fatty acids

120-190 mg of frozen feces were transferred to glass tubes (16 x 125 mm) fitted with screw caps, and 100 μl of stock solutions of internal standards (1 M concentration of [1- ¹³ C] acetate and [ ² H ₆ ] propionate, 0.5 M concentration of [ ¹³ C ₄ ] butyrate, 0.1 M concentration of [1- ¹³ C ₁ ] isobutyrate and [1- ¹³ C] isovalerate, 40 mM concentration of [1,2- ¹³ C ₂ ] hexanoate, [ ¹³ C] lactate, and [ ¹³ C ₄ ] succinate) were added. Prior to extraction, the samples were lyophilized at -50°C for 3 h (yield 28-78 mg/dry weight). After acidification with 50 μl 37% HCl, the organic acid was extracted (2 ml diethyl ether/extraction; 2 rounds). A 500 μl aliquot of the extracted sample was mixed with 50 μl N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA; Sigma) at room temperature. An aliquot (1 μl) of the resulting derivatized material was injected into a gas chromatograph (Agilent Technologies 7890A) coupled to a mass spectrometer detector (Agilent Technologies 5975C). A linear temperature gradient was used. The initial temperature of 65°C was maintained for 6 min, increased to 260°C (15°C/min), and further increased to 280°C for 5 min. The syringe and transfer line temperature was 250°C. Quantitation was accomplished by comparison with labeled internal standards in selected ion monitoring acquisition mode (valerate versus [1- ¹³ C]isovalerate, heptanoate and octanoate versus [1,2- ¹³ C ₂ ]hexanoate, fumarate versus [ ¹³ C ₄ ]succinate). The m/z ratios of the monitored ions are as follows: 117 (acetic acid), 131 (propionic acid), 145 (butyric acid), 146 (isovaleric acid), 159 (isovaleric and valeric acid), 173 (hexanoic acid), 187 (heptanoic acid), 201 (octanoic acid), 261 (lactic acid), 287 (fumaric acid), 289 (succinic acid), 121 ([ ² H ₂ ]- and [1- ¹³ C]acetates), 136 ([ ² H ₅ ]propionate), 146 ([1- ¹³ C ₁ ]isobutyrate), 149 ([ ¹³ C ₄ ]butyrate), 160 ([1- ¹³ C]isovaleric acid), 175 ([1,2- ¹³ C ₂ ]hexanoate), 264 ([ ¹³ C] lactate) and 293 ([ ¹³ C ₄ ]succinate).

Results on human fecal microbial patterns

Pyrosequencing of 16S rRNA gene barcoded amplicons resulted in 755,963 high-quality sequences, with an average of 12,599 sequences per sample (range 7,018-21,116), of which the amount of sequences generated in responders (R) and non-responders (NR) after each treatment was similar. Firmicutes (~70%) was the most abundant phylum in each study group, followed by Bacteroidetes (~20%). However, no significant differences in the relative abundance of Firmicutes and Bacteroidetes were observed between responders or non-responders or by any treatment. However, in the responder group, a non-significant increase in the abundance of Bacteroidetes was recorded after supplementation with the barley grain bread diet (Figure 8A).

The Bacteroidetes phylum includes the genera Prevotella and Bacteroides, and interestingly, Prevotella was found to have a significantly higher relative abundance in the responder group (Figure 8B, two-way ANOVA, P < 0.01). In addition, the level of Prevotella increased in the responder group after consumption of barley grain bread, which was not observed in the non-responders.

Furthermore, in the responder group, significantly elevated levels of fecal succinate were measured after the diet supplemented with barley grain bread (Table 6). Succinate is known to be the major metabolite from the fermentative activity of Prevotella species.

Table 6. Concentration of organic acids in feces

Inoculation experiments in mice

10 to 12 week old sterile Swiss Webster male mice were inoculated with a single oral gavage of 10 ⁸ CFU of Bacteroides thetaiotaomicron strain VPI-5482 (overnight culture in YCFA medium) or Prevotella intestinalis strain DSM18205 (overnight culture in PYG medium) alone or together. Both strains were isolated from human excreta. Both strains were cultured and transferred to a mouse device in a 15ml Hungate tube. Single- and dual-colonized mice were raised in an isolation cage system for 14 days. Before killing the mice, an oral glucose tolerance test (OGTT) was performed. Before the OGTT, all mice were fasted for 4h. Colonization density was verified using qPCR assay (which uses species-specific primers). Age-matched sterile male Swiss Webster control group mice were also fed the same autoclaved feed diet.

Mice were fasted for 4 hours and then orally gavaged with 60% D-glucose (3 g/kg body weight). Blood was drawn from the tail vein at 0, 30, 60, 90, and 120 minutes, and blood glucose levels were measured using a glucometer. Additional blood was collected from the tail vein at 0, 15, and 30 minutes for analysis of serum insulin levels using an insulin ELISA assay (Crystal Chem, Inc.).

result

In order to study whether Prevotella affects the improvement of glucose tolerance, we used human excreta-derived Prevotella strains (intestinal Prevotella) to single-plant colonized mice, and compared the glucose tolerance of these mice with the glucose tolerance of mice that were single-planted with Bacteroides thetaiotaomicron and dual-planted with two strains. Compared with mice that were colonized with Bacteroides thetaiotaomicron, colonization with Bacteroides thetaiotaomicron caused glucose tolerance to be impaired (Fig. 9A-B). In addition, compared with mice that were single-planted with Bacteroides thetaiotaomicron, after oral gavage with glucose, at 15 and 30 min, the serum insulin levels in mice that were single-planted with Bacteroides thetaiotaomicron were lower (Fig. 9C). Importantly, when mice colonized with Bacteroides thetaiotaomicron were colonized with intestinal Prevotella, they showed improved glucose tolerance. These data show that although Bacteroides thetaiotaomicron impairs glucose tolerance, intestinal Prevotella prevents this damage.

Example 3

Product Examples

A) A beaker containing a portion of yogurt with barley DF and/or RS and Prevotella spp. (10 ⁷ CFU or more) sealed in a lid and separated by a membrane.

B) Bottle containing a serving of drinking yogurt with barley DF, RS and Prevotella spp. (10 ⁹ CFU) sealed in an accompanying drinking straw.

C) A portion of barley DF, RS, and Prevotella hermetically sealed in a single-dose package.

D) Bottle containing a serving of fruit drink with barley DF and/or RS and Prevotella spp. ( ¹⁰⁹ CFU) sealed in an accompanying drinking straw.

E) Bottle containing a serving of fruit drink with barley DF and RS and Prevotella spp. ( ¹⁰⁹ CFU) sealed in the cap and separated by a membrane.

F) Emulsified food product comprising Prevotella encapsulated in a matrix with or without a prebiotic carbohydrate component.

AF) One portion of barley DF and RS and Prevotella (7 g insoluble barley DF, 3.3 g soluble barley DF, 8.5 g RS and 10 ⁹ CFU Prevotella).

References

1.Vidhyalakshmi, R., R.Bhakyaraj, and R.S.Subhasree, Encapsulation "The Future of Probiotics"-A Review.Advances in Biological Research 2009.3(3-4):p.96-103.

2.Holm, J., et al., A rapid method for the analysis of starch.1986.38:p.224-226.

3.I.M.E.and M.A.In-vivo and in-vitrodigestability of starch in autoclaved pea and potato products. Journal of theScience of Food and Agriculture, 1992.58:p.541-553.

4.A.K., et al., An in vitro method, based on chewing, topredict resistant starch content in foods allows parallel determination ofpotentially available starch and dietary fiber. The Journal of Nutrition, 1998.128(3):p.651-60.

5.Asp, N.-G., et al., Rapid enzymatic assay of insoluble and solubledietary fiber. Journal of Agricultural and Food Chemistry, 1983.31:p.476-482.

6.Brighenti, F., Summary of the conclusion of the working group onProfibre interlaboratory study on determination of short chain fatty acids inblood, in Functional properties of non-digestible carbohydrates, F.Gullion, etal., Editors.1998, European Comission, DG XII, Science, Research and Development: Brussels, Belgium.p. 150-153.

7. Salonen, A., et al., Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis. J Microbiol Methods, 2010.81(2):p.127-34.

Claims (14)

1. Use of at least one isolated bacterial strain from the species Prevotellaceae in the preparation of a product for the treatment of obesity, metabolic syndrome, type 2 diabetes and obesity-related cardiovascular disease, wherein said strain is Prevotella copri. 2. The use according to claim 1, wherein the product comprises at least one type of dietary fiber. 3. The use according to claim 2, wherein the product comprises at least one resistant starch. 4. The use according to claim 1, wherein the bacterial strain is genetically modified. 5. The use according to claim 3, wherein the dietary fiber and resistant starch are related to any form of cereal selected from: barley fiber, wheat fiber, rye fiber, oat fiber and β-glucan. 6. The use according to claim 5, wherein the dietary fiber is derived from barley. 7. The use according to claim 3, wherein the resistant starch is selected from any of the following forms: starch inherent in barley grains, retrograde starch, plant-encapsulated starch, natural non-gelatinized starch, cyclodextrin, and chemically modified starch. 8. The use according to claim 1, wherein the product comprises other bacterial strains, wherein the other bacterial strains are strains that produce succinates/salts. 9. The use according to claim 1, wherein the product further comprises a succinate/salt. 10. The use according to any one of claims 1-9, wherein the bacterial strain from the Prevotaceae family is encapsulated or freeze-dried. 11. A product comprising at least one isolated bacterial strain from the Prevotaceae family, wherein the strain is *Prevotella enterica*, and wherein the product further comprises at least one type of dietary fiber. 12. The product of claim 11, wherein the product comprises an isolated bacterial strain from the Prevotaceae family as the sole bacterial strain, wherein the strain is *Prevotella enterica*. 13. The product of claim 11, wherein the product further comprises a bacterial strain that produces succinate/salt. 14. The product according to any one of claims 11-13, wherein the product further comprises a succinate/salt.