Compositions and methods using a combination of at least one type of fiber and at least one type of probiotic to regulate the relationship between intestinal bacteria and cholesterol.

Abstract

A composition containing a combination of at least one type of fiber and at least one type of probiotic can be orally administered to a subject in an amount effective in modulating the relationship between the target intestinal bacteria and blood cholesterol levels. This method can achieve at least one outcome, which is improvement of blood cholesterol levels, and prevention and / or treatment of cardiovascular, circulatory, endocrine, and / or nervous system conditions and / or diseases.

Description

Technical Field

[0001]

[0001] This disclosure generally relates to compositions and methods using at least one fiber and at least one probiotic to modulate the relationship between the gut bacteria of a subject and blood cholesterol levels. This disclosure further relates to methods of using and manufacturing such compositions.

Background Art

[0002]

[0002] The human gut microbiome is an ecosystem of trillions of bacteria that contributes to overall metabolism and health, as well as to specific metabolic processes.

[0003]

[0003] The correlation between gut bacteria and blood cholesterol levels has been previously elucidated. In particular, a study on adult Dutch individuals identified 34 bacteria in the gut microbiome that are associated with changes in cholesterol levels (Norton A. "Gut Bugs May Affect Body Fat, "Good" Cholesterol", published online in Circulation on September 10, 2015, issued on September 10). Although the nature of the relationship between cholesterol changes and gut bacteria is unknown, gut bacteria may not only directly affect cholesterol, but cholesterol may also affect the microbiome composition. Some bacterial species have previously been associated with the metabolism of bile acids, and this metabolism can also affect cholesterol levels.

[0004]

[0004] Therefore, the burden that can be caused by one or more of an unhealthy diet, antibiotics, other drugs, infections, intense exercise, or alcohol on gut microbiome taxa related to cholesterol may lead to an increase in blood cholesterol levels that can further cause metabolic disorders, particularly in the cardiovascular and circulatory systems, endocrine system, and nervous system conditions and / or diseases.

[0005]

[0005] Given the unexplained mechanisms that regulate these specific bacteria in the gut microbiome, it is necessary to identify the nutrients required to regulate the relationship between the above gut bacteria and cholesterol in order to further improve blood cholesterol levels and further prevent and / or treat cholesterol-related conditions.

[0006]

[0006] Furthermore, there is a need to provide nutritional compositions and methods for regulating the relationship between target intestinal bacteria and blood cholesterol levels. [Overview of the project]

[0007]

[0007] Clinical studies disclosed herein demonstrate that the combined intake of fiber blends and probiotic mixtures modulates the relationship between the target gut bacteria and blood cholesterol levels.

[0008]

[0008] In particular, the relationship between at least one type of intestinal bacteria selected from the group consisting of Clostridium species, Coprococcus catus, Ruminococcus torques, and Slackia isoflavoniconvertens and LDL (low-density lipoprotein) cholesterol is eliminated, and Bacteroides coprocola, Bacteroides faecis, Bacteroides massiliensis, Bacteroides vulgatus, Clostridium species, Firmicutes bacteria, Parabacteroides distasonis It promotes a positive correlation between at least one type of intestinal bacterium selected from the group consisting of *Distasonis*, *Parabacteroides goldsteinii*, *Ruminococcus bicirculans*, *Ruminococcus lactaris*, and *Slachia isoflavoniccomvertens* and HDL (high-density lipoprotein) cholesterol.

[0009]

[0009] Accordingly, the present disclosure relates, in general terms, to novel compositions and methods for regulating the relationship between intestinal bacteria and target blood cholesterol levels by using a combination of fiber blends and probiotic mixtures.

[0010]

[0010] Benefits from this improvement include, for example, an improvement in the subject's blood cholesterol levels, and as a result, the prevention and / or treatment of cardiovascular and circulatory, endocrine and nervous system conditions and / or diseases in subjects whose gut microbiome is under strain.

[0011]

[0011] Further features and advantages are described herein and will become apparent from the following drawings and embodiments for carrying out the invention. [Brief explanation of the drawing]

[0012] [Figure 1] This graph shows the results of experimental examples disclosed herein, demonstrating that the relationship between gut bacteria and LDL cholesterol levels is eliminated when nutritional intervention is performed. [Figure 2] This graph shows the results of experimental examples disclosed herein, demonstrating that a positive correlation between gut bacteria and HDL cholesterol levels is promoted when nutritional interventions are performed. [Modes for carrying out the invention]

[0013]

[0014] definition

[0015] The following are some definitions. However, definitions may also be found in the "Embodiments" section below, and the above heading "Definitions" does not mean that such disclosures in the "Embodiments" section are not definitions.

[0014]

[0016] All percentages stated herein are by total weight of the composition unless otherwise specified. As used herein, “about,” “approximately,” and “substantially” are understood to refer to a numerical range, for example, a range of -10% to +10% of the reference digit, preferably -5% to +5%, more preferably -1% to +1%, and most preferably -0.1% to +0.1%. All numerical ranges herein should be understood to include all integers or fractions within that range. Furthermore, these numerical ranges should be interpreted as supporting claims that cover any number or subset of a number within that range. For example, a disclosure of 1 to 10 should be interpreted as corresponding to ranges such as 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0015]

[0017] When used in this disclosure and the appended claims, the singular forms "a," "an," and "the" also refer to multiple subjects unless otherwise indicated by the context. Thus, for example, a reference to "a vitamin (a vitamin or the vitamin)" includes both embodiments having a single vitamin and embodiments having two or more vitamins.

[0016]

[0018] The terms “comprise,” “comprises,” and “comprising” should be interpreted as not being exclusive but potentially encompassing others. Similarly, the terms “include,” “including,” and “or” should all be interpreted as potentially encompassing others unless such interpretation is clearly prevented by the context. However, compositions disclosed herein may not include elements not specifically disclosed herein. Therefore, disclosures of embodiments using the term “comprising” include disclosures of embodiments that “essentially include / consistently consist of” the specified components, and disclosures of embodiments that “consist of” the specified components.

[0017]

[0019] The terms "at least one of X or Y" and "and / or Y" as used in their respective contexts should be interpreted as "X" or "Y" or "X and Y". For example, "at least one of resistance or recovery" and "resistance and / or recovery" should be interpreted as "resistance" or "recovery" or "both resistance and recovery".

[0018]

[0020] As used herein, the terms “example” and “such as” are merely illustrative and descriptive, and should not be considered exclusive or comprehensive, especially when followed by a list of terms. As used herein, “associated with” or “linked with” another state means that these states occur simultaneously, preferably that they are caused by the same underlying condition, and most preferably that one of the specified states is caused by the other specified state.

[0019]

[0021] "Prevention" includes reducing the risk, incidence, and / or severity of a condition or disorder. The terms "treatment" and "to treat" include both preventive or deterrent treatments (treatments that prevent and / or delay the onset of a diseased condition or disorder) and curative, therapeutic, or disease-modifying treatments, such as therapeutic measures for the cure, delay, reduction of symptoms, and / or cessation of progression of a diagnosed diseased condition or disorder, as well as treatment for patients at risk of developing a disease, suspected of having a disease, and patients who are unwell or diagnosed with a disease or medical condition. The terms "treatment" and "to treat" do not necessarily mean treating until the subject is fully recovered. The terms "treatment" and "to treat" also refer to maintaining and / or promoting the health of individuals who are not diseased but are susceptible to unhealthy conditions. The terms “treatment / therapy” and “to treat / therapy” are also intended to include the synergistic effect, or otherwise enhancement, of one or more primary preventive or therapeutic measures. In non-exclusive examples, treatment / therapy may be performed by a patient, caregiver, physician, nurse, or other healthcare professional.

[0020]

[0022] As used herein, a prophylactic or therapeutic "effective amount" is an amount that, in an individual, prevents a deficiency, treats a disease or medical condition, or more generally, reduces symptoms, manages disease progression, or provides a nutritional, physiological, or medical benefit to the individual.

[0021]

[0023] As used herein, the terms "food", "food product", and "food composition" mean a product or composition intended for oral ingestion by a human or other mammal and containing at least one nutrient for a human or other mammal.

[0022]

[0024] As used herein, "nutritional composition" and "nutritional product" include any number of food raw materials and optionally additional raw materials, based on the functional requirements in the product and in full compliance with all applicable regulations. Optional raw materials can include, but are not limited to, conventional food additives such as one or more acidulants, additional thickeners, pH-adjusting buffers or pH-adjusting agents, chelating agents, colorants, emulsifiers, excipients, flavorings, minerals, osmotic agents, pharmaceutically acceptable carriers, preservatives, stabilizers, sugars, sweeteners, conditioning agents and / or vitamins. Optional raw materials can be added in any suitable amount.

[0023]

[0025] "Probiotics" means a preparation of microbial cells or components of microbial cells that beneficially affect the health or well-being of a host. (Salminen S, Ouwehand A. Benno Y. et al., "Probiotics: how should they be defined", Trends Food Sci. Technol., 1999:10 107-10).

[0024]

[0026] As used herein, the term "unit dosage form" refers to physically discrete units suitable as unit dosages for human and animal subjects, each unit containing a predetermined quantity of a composition disclosed herein in an amount sufficient to produce the desired effect, together with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage form are determined by the particular compound employed, the effect to be achieved, and the pharmacodynamics associated with each compound in the host body.

[0025]

[0027] A "subject" or "individual" is a mammal, preferably a human.

[0026]

[0028] Embodiments

[0029] The inventors investigated how the constituent bacteria of the microbiota are related to the amount of blood cholesterol and whether the constituent bacteria are also related to the level of resilience.

[0027]

[0030] On the one hand, the inventors found that the species CAG138 of the genus Clostridium, Coprococcus catus, Lachnococcus torques, Slackia isoflavoniconvertens are positively related to the level of LDL cholesterol. On the other hand, Bacteroides coprocola, Bacteroides faecis, Bacteroides massiliensis, Bacteroides vulgatus, the species CAG43 of the genus Clostridium, Firmicutes bacterium AM55-24TS, Firmicutes bacterium CAG145, Parabacteroides distasonis, Parabacteroides goldsteinii, Lachnococcus biscirculans, Lachnococcus lactaris, Slackia isoflavoniconvertens were negatively correlated with the level of HDL cholesterol.

[0028]

[0031] The inventors investigated whether short-term nutritional load affects the relationship between the above intestinal bacteria and cholesterol.

[0029]

[0032] The inventors have found that nutritional intervention eliminates the relationship between LDL cholesterol and at least one intestinal bacterium selected from the group consisting of Clostridium species (e.g., Clostridium species CAG138), Coprococcus catus, Ruminococcus turchis, and Slacchia isoflavonicombotens, as well as Bacteroides coprocola, Bacteroides phaesis, Bacteroides masiliensis, Bacteroides vulgatus, and Clostridium species (e.g., We observed that at least one type of intestinal bacterium selected from the group consisting of Clostridium species (CAG43), Firmicutes (e.g., Firmicutes AM55-24TS, Firmicutes CAG145), Parabacteroides distasonis, Parabacteroides goldsteenii, Ruminococcus biscirculans, Ruminococcus lactalis, and Slachia isoflavoniccombertens promotes a positive correlation with HDL cholesterol.

[0030]

[0033] Accordingly, one aspect of the present disclosure is a method for regulating the relationship between a target intestinal bacterium and blood cholesterol levels, wherein a composition comprising an effective amount of a combination of at least one type of fiber and at least one type of probiotic is administered to the target.

[0031]

[0034] In one embodiment, the intestinal bacteria are selected from the group consisting of Clostridium species CAG138, Coprococcus catus, Ruminococcus turchis, Slacchia isoflavonicombotens, Bacteroides coprocola, Bacteroides phaesis, Bacteroides masiliensis, Bacteroides bulgatus, Clostridium species CAG43, Firmicutes AM5524 TS, Firmicutes CAG145, Parabacteroides distasonis, Parabacteroides goldsteenii, Ruminococcus biscirculans, Ruminococcus lactalis, and Slacchia isoflavonicombotens.

[0032]

[0035] In one embodiment, regulating the relationship between the gut and cholesterol involves eliminating the relationship between LDL cholesterol and at least one type of intestinal bacteria selected from the group consisting of Clostridium species (e.g., CAG138), Coprocococcus catus, Ruminococcus turchis, and Slachia isoflavonicombotens.

[0033]

[0036] In one embodiment, regulating the gut relationship involves a step of promoting a positive correlation between HDL cholesterol and at least one intestinal bacterium selected from the group consisting of Bacteroides coprocola, Bacteroides phaesis, Bacteroides maciliensis, Bacteroides vulgatus, Clostridium species (e.g., CAG43), Firmicutes bacteria (e.g., AM55-24TS, CAG145), Parabacteroides distasonis, Parabacteroides goldsteenii, Ruminococcus biscirculans, Ruminococcus lactalis, and Slachia isoflavoniccomvertens.

[0034]

[0037] In one embodiment, the method is for achieving at least one outcome selected from improvement of blood cholesterol levels, and prevention and / or treatment of cardiovascular, circulatory, endocrine, and / or nervous system conditions and / or diseases.

[0035]

[0038] In some embodiments, the subject ingests one of the compositions disclosed herein daily, for example, for at least one week or even more than one month prior to the stressor.

[0036]

[0039] Another embodiment is a unit dosage form of a composition comprising a combination of at least one type of fiber and at least one type of probiotic, wherein the combination (of at least one type of fiber and at least one type of probiotic) is effective in modulating the relationship between the intestinal bacteria and blood cholesterol levels of a subject to whom the unit dosage form is administered.

[0037]

[0040] In the embodiments disclosed herein, each of at least one type of fiber is edible, i.e., all components of the fiber are safe and suitable for human and / or animal consumption. At least one type of fiber comprises insoluble fiber and / or soluble fiber, preferably a blend of insoluble and soluble fiber. In some embodiments, at least one type of fiber can be selected from the group consisting of xylooligosaccharides, flaxseed, partially hydrolyzed guar gum (PHGG), glucomannan, cellulose, prune powder, pectin, e.g., apple peel pectin, and mixtures thereof. In some embodiments, at least one type of fiber is at least two types of fiber, e.g., two, three, four, five, six, or seven types of fiber, and optionally more types of fiber. Optionally, one or more of Luo Han Guo fruit powder, xylitol, or magnesium (e.g., magnesium citrate) may be included with at least one type of fiber.

[0038]

[0041] In some embodiments, at least one probiotic may be selected from the group consisting of Lactobacillus acidophilus, Bifidobacterium lactis, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus plantarum, Bifidobacterium bifidum, Lactobacillus gasseri, and mixtures thereof. In some specific embodiments, at least one probiotic may be a strain selected from the group consisting of Lactobacillus acidophilus La-14, Bifidobacterium lactis Bl-04, Lactobacillus rhamnosus GG, Bifidobacterium longum BL-05, Lactobacillus plantarum Lp-115, Bifidobacterium bifidum Bb-06, Lactobacillus gasseri Lg-36, and mixtures thereof. In some embodiments, at least one probiotic may be at least two probiotic strains, for example, two, three, four, five, six, or seven probiotic strains, and optionally more probiotic strains.

[0039]

[0042] At least one type of fiber may be administered to an individual in a total daily dose of about 5g to 40g, preferably about 15g to 25g. At least one type of fiber may be administered in a composition containing about 300mg to 1000mg of total fiber per gram of dry composition.

[0040]

[0043] At least one type of probiotic, 1 x 10 3 cfu~1×10 12 cfu (cfu = colony-forming unit), preferably 1 × 10 7 cfu~1×10 11It can be administered to an individual as a daily dose of cfu. At least one type of probiotic may be 1 × 10⁶ 3 cfu / g ~ 1 × 10 12 It may be administered in a composition containing a dry composition of cfu / g. At least one probiotic may be in the form of a viable, fragmented, or fermentation product (e.g., supernatant) or metabolite, or a mixture of any or all of these states.

[0041]

[0044] The combination of at least one type of fiber and at least one type of probiotic is preferably administered orally in the form of a food composition.

[0042]

[0045] The subjects to be administered a combination of at least one type of fiber and at least one type of probiotic can be selected from groups consisting of human infants, human children, human adolescents, human adults, and human elderly.

[0043]

[0046] A combination of at least one type of fiber and at least one type of probiotic may be administered to an individual by at least one route selected from the group consisting of oral, topical, enteral, and parenteral administration. For example, a combination of at least one type of fiber and at least one type of probiotic may be administered in the form of a composition selected from the group consisting of complete nutritional products, beverages, dietary supplements, meal replacements, food additives, supplements for food products, dissolvable powders, enteral nutrition products, infant formulas, capsules, and combinations thereof.

[0044]

[0047] Optionally, a combination of at least one fiber and at least one probiotic is administered in a composition further comprising at least one component selected from the group consisting of amino acids, proteins, nucleotides, fish oil, non-marine omega-3 fatty acid sources, phytonutrients, antioxidants, and mixtures thereof.

[0045]

[0048] Another embodiment is a method for producing a composition for regulating the relationship between a target intestinal bacterium and blood cholesterol levels, comprising the step of adding at least one type of fiber to at least one type of probiotic and optionally at least one additional component.

[0046]

[0049] The composition may be a food product, an animal food product, or a pharmaceutical composition. For example, the product may be a nutritional composition, a nutraceutical, a beverage, a food additive, or a pharmaceutical. Food additives or pharmaceuticals may be in the form of, for example, tablets, capsules, lozenges, liquids, or powders in sachets.

[0047]

[0050] In some embodiments, at least one probiotic is administered simultaneously with at least one fiber in a separate composition, for example, in separate compositions administered to the same individual within 1 hour, preferably within 30 minutes, more preferably within 10 minutes, and most preferably within 1 minute.

[0048]

[0051] A composition comprising a combination of at least one type of fiber and at least one type of probiotic is preferably selected from the group consisting of milk powder-based products; instant beverages; ready-to-drink formulations; nutritional powders; nutritional liquids; dairy products, especially yogurt or ice cream; cereal products; beverages; water; coffee; cappuccino; malt beverages; chocolate-flavored beverages; cooked products; soups; tablets; and / or syrups.

[0049]

[0052] The composition may optionally contain one or more milks obtained from animal or plant sources, such as cow's milk, human milk, sheep's milk, goat's milk, horse's milk, camel's milk, rice milk, or soy milk. Additionally or alternatively, milk protein fractions or colostrum may be used.

[0050]

[0053] A composition comprising a combination of at least one fiber and at least one probiotic may further contain protective hydrophilic colloids (e.g., gum, protein, modified starch), binders, film-forming agents, encapsulants / encapsulating materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizers (oils, fats, waxes, lecithin, etc.), adsorbents, carriers, fillers, co-compounds, dispersants, wetting agents, processing aids (solvents), fluidizers, flavoring agents, bulking agents, gelling agents, gel-forming agents, antioxidants, and antimicrobial agents.

[0051]

[0054] A composition comprising a combination of at least one fiber and at least one probiotic may also contain conventional pharmaceutical additives and adjuvants, excipients and diluents, examples of which include, but are not limited to, water, gelatin derived from any raw material, plant gum, lignin sulfonate, talc, sugar, starch, gum arabic, vegetable oil, polyalkylene glycol, flavorings, preservatives, stabilizers, emulsifiers, buffers, lubricants, colorants, wetting agents, and fillers. Furthermore, the composition may also contain organic or inorganic carrier materials suitable for oral or enteral administration, as well as vitamins, minerals, trace elements and other micronutrients, in accordance with the recommendations of government agencies such as the USRDA.

[0052]

[0055] A composition comprising a combination of at least one type of fiber and at least one type of probiotic may optionally contain one or more amino acids, protein sources, carbohydrate sources, and / or lipid sources, particularly in embodiments of the composition that are food products.

[0053]

[0056] Any suitable food-derived protein, such as animal protein (e.g., milk protein, meat protein, and egg protein); plant protein (e.g., soy protein, wheat protein, rice protein, and pea protein); a mixture of free amino acids; or a combination thereof may be used. Milk protein, such as casein and whey, and soy protein are particularly preferred.

[0054]

[0057] A composition comprising a combination of at least one type of fiber and at least one type of probiotic may be administered to humans or animals, particularly companion animals, pets, or livestock. This composition has beneficial effects on any age group. Preferably, the composition is formulated for administration to infants, young people, adults, or the elderly. In some embodiments, the composition may be administered to pregnant and lactating mothers to treat infants.

[0055]

[0058] A composition comprising a combination of at least one type of fiber and at least one type of probiotic can be administered at least one day per week, preferably at least two days per week, more preferably at least three or four days per week (e.g., every other day), most preferably at least five days per week, six days per week, or seven days per week. The duration of administration may be at least one week, preferably at least one month, more preferably at least two months, most preferably at least three months, for example, at least four months. In one embodiment, administration is at least daily, for example, the subject may receive administration once or more times a day. In some embodiments, administration is continued for the remainder of the individual's life. In other embodiments, administration is continued until there are no detectable symptoms of the medical condition. In specific embodiments, administration is continued until there is a detectable improvement in at least one symptom, and in further cases, it is continued to maintain remission. [Examples]

[0056]

[0059] Examples

[0060] The following non-limiting embodiments generally illustrate the concepts underlying the embodiments disclosed herein.

[0057]

[0061] Materials and methods

[0062] This trial was randomized, controlled, open-label, and based on a 2x2 crossover design. Twenty eligible male and female participants aged 18–45 years were randomly assigned to one of two intervention sequences: 1) control (no resilience enhancement) followed by resilience enhancement; or 2) resilience enhancement followed by control.

[0058]

[0063] Each participant, regardless of cohort, followed the following study design: Participants were followed for 21 days. Their diet was monitored but not controlled during the first 10 days. After 10 days (starting from day 0), all participants also received a 5-day dietary load consisting of 60% fat, 15% carbohydrates, 25% protein, and 10g fiber. Following the above dietary disturbance period, participants returned to their normal diets, which were monitored daily. Participants continuing the intervention period received a combination of fiber and probiotics for 22 days. Fecal and plasma samples were collected at various schedules throughout the study period. Additionally, questionnaires regarding food and comfort were collected throughout the study period. The trial was registered on clinicaltrials.gov under the number NCT04424329.

[0059]

[0064] The loading diet was designed to provide 60% fat, 25% protein, 15% carbohydrates, and 10g of fiber per 2000 calories of total daily calorie intake. Three different meal plans were created, and two of them were repeated during a 5-day loading period. The same meal plan was served in the same order during both study periods.

[0060]

[0065] I designed the diet load using the nutrition software "diet builder" (http: / / dnh-portal.dor.ch.nestle.com / ). Diet builder allows you to select one or more food composition databases (FCDBs), choose foods from them, create meals for each eating occasion (e.g., breakfast, lunch), and determine the total nutritional composition for the day (e.g., kcal, fat weight (g), protein weight (g)).

[0061]

[0066] The estimated energy requirement (EER) for each study participant was calculated using the Institute of Medical Science (IOM) formula. The nutrition software "diet builder" estimates the EER using the IOM formula when the participant's demographic characteristics, including self-reported physical activity levels, are entered into the tool. Based on the estimated EER, participants were grouped into four different energy requirement clusters (2000 kcal / day, 2500 kcal / day, 3000 kcal / day, and 3750 kcal / day) according to their individual energy needs.

[0062]

[0067] Food was prepared and given to the subjects to take home. The subjects were instructed to consume all of the food if possible and not to consume any additional items.

[0063]

[0068] Before and after the food load, participants were asked to record their food intake daily using a proprietary application developed by the company (Nestle Research app ref). During the food load, participants were asked to record the amount of load food consumed and any additional items they consumed. The entered data was converted into nutritional information by the application.

[0064]

[0069] The inventors used a combination of probiotics and fiber as an intervention product. Regarding probiotics, participants consumed 5 × 10⁶ units of fiber. 9Participants ingested one capsule containing a combination of CFUs (Bifidobacterium longum, Bifidobacterium bifidum, Lactobacillus gasseri), Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus, and Bifidobacterium lactis. For fiber, participants consumed 14 g of powder containing xylooligosaccharides, apple pectin, partially hydrolyzed guar gum, glucomannan, flaxseed powder, cellulose, and prune powder (see Supplementary Table 01 for the percentages of each probiotic and fiber). All combinations are currently marketed by Pure Encapsulations (USA).

[0065]

[0070] DNA was extracted using 200ug of fecal matter. 100ng of DNA was sequenced using an Illumina HiSeq 3000 to obtain 10Gb of data. The raw data was then processed using FastQC for quality assessment and annotated using the Atlas pipeline (Kieser, BMC Bioinformatics, 2020). Taxonomic annotation was used with alpha, beta, and gamma diversity indices to characterize intra- and inter-participant differences. Community structure was constructed using taxonomic covariance. Functional variability was extracted from gene annotation using RefSeq, KEGG, and eggNOG.

[0066]

[0071] The tests were conducted by Metabolon (Morrisville, North Carolina, USA) using their in-house HD4 platform. Briefly, the samples were extracted with methanol to precipitate proteins and dissociate small molecules bound to the proteins. The resulting extracts were divided into five fractions: two samples for analysis by two separate reversed-phase (RP) / UPLC-MS / MS methods using cationic electrospray ionization (ESI), one sample for analysis by RP / UPLC-MS / MS using anionic ESI, one sample for analysis by HILIC / UPLC-MS / MS using anionic ESI, and one was kept as a backup. Metabolites were identified using proprietary software to match ions with Metabolon's standard library. All methods underwent different quality controls, including technical replicates, negative controls (water and solvent), and known controls (cocktails of known metabolites; a list of QC standards is shown in Supplementary Table 02). Metabolites were quantified by integrating peak areas. Metabolites were mapped against a proprietary reference library to characterize identified peaks. Data were analyzed using two-way repeated measures ANOVA and principal component analysis (PCA). For all analyses, missing values ​​were substituted with the observed minimum value for the specific compound. Statistical analysis was performed on naturally logarithmic transformed data. Data curation was performed as needed to remove system artifacts, misassignments, redundancy, and background noise.

[0067]

[0072] Fasting EDTA plasma samples for metabolomics analysis were collected from 20 subjects in both study groups at three time points (day 0, day 4, and day 11) across two periods corresponding to the start, end, and post-loading events.

[0068]

[0073] 55 μL of plasma sample was centrifuged (1.5 min, 21130 rcf, 4°C) and divided into two 20 μL aliquots. The remainder was used as the pooled sample. For quality control, 5 μL, 10 μL, 15 μL, 20 μL, and 25 μL aliquots were obtained from the pool. All aliquots were maintained at -80°C until extraction. Before extraction, the sample was thawed on ice and 500 μL of extraction solution (40:40:20 ACN:MeOH:H2O) containing an isotope-labeled internal standard was added. After mixing by vortexing, the extract was centrifuged at 21130 rcf, 4°C for 10 minutes. The supernatant was dried overnight in a vacuum centrifuge at 4°C and 5 mbar. The dried sample was resuspended in 50 μL of 70% (v / v) acetonitrile aqueous solution and centrifuged at 21130 rcf, room temperature for 1 minute. The obtained supernatant was transferred to a glass vial for liquid chromatography-coupled mass spectrometry (LC-MS).

[0069]

[0074] A 3 μL sample was injected into a Vanquish UHPLC (Thermo Scientific) equipped with a 100 × 2.1 mm, 5 μm hydrophilic liquid chromatography (HILIC) ZIC-pHILIC column (Merck Sequant) and a ZIC-pHilic guard column (20 × 2.1 mm, 5 μm, Merck Sequant), and the samples were separated. Separation was achieved by applying a linear solvent gradient in normal phase mode at a flow rate of 0.2 mL / min and a temperature of 35°C. Solvent A as the mobile phase was H2O (pH approximately 9.3) containing 10 mM ammonium acetate (NH4Ac) and 0.04% (v / v) ammonium hydroxide (NH4OH), and solvent B was acetonitrile (can). The gradient, started with 90% solvent B, was increased to the initial conditions within 0.1 minutes, and the column was equilibrated for 8.9 minutes. The total analysis time was 30 minutes.

[0070]

[0075] The eluted metabolites were analyzed using an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) with a heated electrospray ionization (H-ESI) source, at spray voltages of 3500V and 3000V in positive and negative modes, respectively. The sheath gas was set to 20 AU, and the auxiliary gas was maintained at 15 AU. The vaporizer temperature was set to 280°C, and the ion transport tube temperature to 310°C. Full scans were measured by scanning four mass windows, switching between positive and negative modes alternately on the fly. The windows covered the m / z ranges of 73–300 and 195–1000, respectively. The resolution of the low-mass window was set to 50,000, and the resolution of the high-mass window was set to 60,000. Instrument control was performed using Xcalibur software (Thermo Scientific).

[0071]

[0076] An automated R pipeline (AlpsLCMS-version 0.0.0.9009)1, which combines multiple R packages, was applied for preprocessing, peak detection, feature reduction, and inferential annotation of metabolomics data. For data normalization and semi-quantification, an approach based on the linearity of signals from pooled QC samples was applied. Specifically, metabolic features from five different plasma volumes (5 μL, 10 μL, 15 μL, 20 μL, and 25 μL, n=4) were normalized against each internal standard, and correlation analysis was performed. The internal standard with the highest correlation between the normalized feature and plasma volume was selected for subsequent normalization. Features that did not produce a linear response after normalization following correlation analysis (cutoff r² > 0.8) were excluded from further data analysis. After filtering out CVs of less than 30% in 20 μL QC, isotopic data were manually removed based on feature mass differences and retention times, and redundant annotations (i.e., when the compound signaled in both modes) were removed from both the positive and negative modes.

[0072]

[0077] Univariate and multivariate statistical analyses were performed using the statistical software R (version 3.6.2). Principal component analysis (PCA) and heatmaps were performed on the data for research purposes. To investigate differences between visits, baseline correction was performed for all subjects by dividing the value of each metabolite by the corresponding metabolite value at day 0 (pre-load). After discarding the assumption of normality, univariate statistical significance was assessed using the Kruskal-Wallis test for multiple independent samples between groups (control and nutritional intervention) at each visit. The Benjamini-Hochberg III method was used to control for the false detection rate (FDR) in all comparisons. A FDR-corrected p-value < 0.05 was considered statistically significant.

[0073]

[0078] Fecal samples were homogenized with an orthophosphate solution containing S-labeled internal standards (acetic acid-D3, propionic acid-D5, isobutyric acid-D7, butyric acid-D5, isovaleric acid-D9, and valeric acid-D9), and then centrifuged at 2000 g to obtain fecal water. The fecal water samples were deproteinized with 5-sulfosalic acid, and short-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs) were extracted with chloroform and subsequently derivatized with tert-butyl-dimethylsilyl-imidazole (TBDMSIM). (REF) Analysis of SCFAs and BCFAs was performed by gas chromatography coupled to a mass spectrometer (GC-MS, Agilent Technologies, 6890 series XL MSD 5975 C). Chromatographic separation was performed using DB-5MS (J&W Scientific, Folsom, CA) for a run time of 15 minutes. The mass spectrometer was operated in selected ion monitoring (SIM) mode. Quantitative values ​​were calculated by comparing the peak area ratio (unlabeled / labeled) with the corresponding calibration curve. The following SCFAs were analyzed: acetic acid (C2:0), propionic acid (C3:0), butyric acid (C4:0), and valeric acid (C5:0). For BCFAs, isobutyric acid (iC4:0), isovaleric acid (iC5:0), and 2-methylbutyric acid (2-methylC4:0) were measured (quantified using isovaleric acid-D9 as an internal standard). SCFAs and BCFAs were expressed as the number of μmoles per gram of wet feces. For values ​​below the limit of quantification (LLOQ), half of the LLOQ value was substituted.

[0074]

[0079] Fecal consistency was assessed by participants using the Bristol Stool Scale (ref), which ranks all excreted feces from 1 (hard lumps) to 7 (watery stool). The number of bowel movements was also recorded by participants. Both were recorded daily throughout the study period.

[0075]

[0080] Blood samples were collected on days 0, 4, and 11. Serum samples obtained by blood coagulation and spinning at 1500g, 10 minutes, and 4°C were rapidly frozen at -80°C and sent to MLM Medical Labs GmbH (Monchengladbach, Germany) in dry ice for evaluation of permeability markers. Enteric fatty acid-binding proteins and IgM-type anti-endotoxin core antibodies were analyzed by ELISA using human iFABP (KBH1541) and human EndoCAb® IgM (HK504-IGM) ELISA kits manufactured by HycultBiotech (Beutelsbach, Germany) according to the manufacturer's instructions.

[0076]

[0081] Fresh blood samples were collected in BD Vacutainer plasma tubes (VWR BDAM368495) either before breakfast (fasting state) or 5 hours after a high-fat meal (postprandial state) on days 0, 4, and 11. Plasma was isolated by centrifugation at 2000g at 4°C for 10 minutes and used for subsequent measurements of hsCRP, IL-1β, IL-6, IL-8, and TNFα. Plasma hsCRP levels were measured using a commercially available kit (Abbott Laboratories) and analyzer (Architect C8000; Abbott Laboratories). For cytokine analysis, plasma samples were diluted 1:2 and analyzed for IL-1β, IL-6, IL-8, and TNFα using the Mesoscale Custom V-PLEX human cytokine assay (Meso Scale Discovery (MSD), Inc., K151AOH-2) according to the manufacturer's instructions. Briefly, samples were incubated on MSD plates at room temperature for 2 hours with shaking. The plates were washed and incubated with the detection antibody for a further 2 hours. After washing, the samples were analyzed in a double-row using MSD Workbench software to display the difference between the treatment, intervention, and fasting periods (logarithmic scale).

[0077]

[0082] Bowel comfort was rated on a visual analog scale from 0 to 10, with 0 representing no symptoms and 10 representing the worst possible symptoms. Participants recorded levels of nausea, vomiting, audible abdominal sounds, abdominal cramps, and flatulence daily throughout the study period.

[0078]

[0083] All analyses were performed in R. Unless otherwise specified, two-tailed tests were used for all statistical tests. Microbiome data were analyzed and defined at the species level using genome-based mapping aggregates. Unless otherwise noted, microbiome data were evaluated using non-parametric tests (Wilcoxon rank test and Mann-Whitney test). Multiple testing was performed using the Benjamini-Hochberg method (FDR) whenever applicable (q-value). Statistical analysis of fecal metabolites was performed using naturally log-transformed data.

[0079]

[0084] result

[0085] Figure 1 shows that the relationship between the target gut bacteria and blood LDL cholesterol levels is eliminated when nutritional intervention is performed.

[0080]

[0086] Figure 2 shows that nutritional interventions enhance the correlation between the target gut bacteria and blood HDL cholesterol levels.

[0081]

[0087] This rigorous randomized controlled clinical trial demonstrates that the combined intake of a fiber blend and a probiotic mixture modulates the relationship between target gut bacteria and blood cholesterol levels.

[0082]

[0088] It should be understood that various changes and modifications to the preferred embodiments described herein will be obvious to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of this subject matter and without impairing the intended advantages. Accordingly, such changes and modifications are intended to be covered by the appended claims.

Claims

1. A method for regulating the relationship between target intestinal bacteria and blood cholesterol levels, wherein a composition containing an effective amount of a combination of at least one type of fiber and at least one type of probiotic is administered to the target.

2. Intestinal bacteria include species of the genus Clostridium, Coprococcus catus, Ruminococcus torques, Slackia isoflavoniconvertens, Bacteroides coprocola, Bacteroides faecis, Bacteroides massiliensis, and Bacteroides vulgatus. The method according to claim 1, selected from the group consisting of *Clostridium vulgatus*, species of the genus *Clostridium*, *Firmicutes*, *Parabacteroides distasonis*, *Parabacteroides goldsteinii*, *Ruminococcus bicirculans*, *Ruminococcus lactaris*, and *Slachia isoflavoniccombertens*.

3. The method according to claim 1 or 2, wherein the at least one type of fiber is selected from the group consisting of insoluble fibers, soluble fibers, and mixtures thereof, and preferably is a mixture thereof.

4. The method according to any one of claims 1 to 3, wherein the at least one type of fiber is selected from the group consisting of xylooligosaccharides, flaxseed, partially hydrolyzed guar gum (PHGG), glucomannan, cellulose, prune powder, pectin, for example apple peel pectin, and mixtures thereof.

5. The method according to any one of claims 1 to 4, wherein the at least one type of fiber comprises at least two types of fibers, for example, two, three, four, five, six, or seven types of fibers, and optionally more types of fibers.

6. The above at least one type of probiotic is Lactobacillus acidophilus, Bifidobacterium lactis, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus plantarum, Bifidobacterium bifidum, Lactobacillus gasseri The method according to any one of claims 1 to 5, selected from the group consisting of gasseri, and mixtures thereof.

7. The method according to any one of claims 1 to 6, wherein the at least one probiotic comprises a strain selected from the group consisting of Lactobacillus acidophilus La-14, Bifidobacterium lactis Bl-04, Lactobacillus rhamnosus GG, Bifidobacterium longum BL-05, Lactobacillus plantarum Lp-115, Bifidobacterium bifidum Bb-06, Lactobacillus gasseri Lg-36, and mixtures thereof.

8. The method according to any one of claims 1 to 7, wherein the at least one probiotic comprises at least two probiotic strains, for example, two, three, four, five, six, or seven probiotic strains, and optionally more probiotic strains.

9. The method according to any one of claims 1 to 8, wherein the combination of at least one type of fiber and at least one type of probiotic is administered orally to the subject.

10. The method according to any one of claims 1 to 9, wherein the subject is selected from the group consisting of human infants, human children, human adolescents, human adults, human elderly, and animals, such as companion animals.

11. The method according to any one of claims 1 to 10 for achieving at least one outcome selected from improving blood cholesterol levels, and preventing and / or treating conditions and / or diseases of the cardiovascular, circulatory, endocrine, and / or nervous systems.

12. A method for producing a composition for regulating the relationship between intestinal bacteria and blood cholesterol levels in a subject to which the composition is administered, the method comprising the step of adding at least one type of fiber to at least one type of probiotic and optionally at least one additional component.

13. To regulate the relationship between the aforementioned intestinal bacteria and blood cholesterol levels, i. A step of eliminating the relationship between LDL cholesterol and at least one type of intestinal bacteria selected from the group consisting of Clostridium species, Coprococcus catus, Ruminococcus turchis, and Slacchia isoflavonicombotens. and / or ii. A step to promote a positive correlation between HDL cholesterol and at least one intestinal bacterium selected from the group consisting of Bacteroides coprocola, Bacteroides phaesis, Bacteroides masiliensis, Bacteroides vulgatus, species of Clostridium, Firmicutes, Parabacteroides distasonis, Parabacteroides goldstainii, Ruminococcus biscirculans, Ruminococcus lactalis, and Slachia isoflavonicovertens. A method according to any one of claims 1 to 12, including the method described in any one of claims 1 to 12.

14. A unit dosage form of a composition comprising a combination of at least one type of fiber and at least one type of probiotic, wherein the combination of at least one type of fiber and at least one type of probiotic is contained in an amount effective for regulating the relationship between intestinal bacteria and blood cholesterol levels in a subject to whom the unit dosage form is administered.

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