Compositions and methods using a combination of at least one fiber and at least one probiotic to improve and / or maintain optimal cholesterol levels.

Abstract

The composition contains a combination of at least one type of fiber and at least one type of probiotic, and the combination of at least one type of fiber and at least one type of probiotic is formulated for administration to a subject with a stressed gut microbiome to improve and / or maintain optimal cholesterol levels in the subject with a stressed gut microbiome. Preferably, the subject is experiencing one or more microbiome stressors, e.g., dietary stressors, antibiotics, other drugs, infections, strenuous exercise, stress, alcohol, travel, and combinations thereof; more preferably, at least dietary stressors; most preferably, at least a high-fat diet, e.g., a Western high-fat diet or a ketogenic diet. The method can achieve at least one outcome, which is improving and / or maintaining optimal cholesterol levels in a subject with a stressed gut microbiome.

Description

[Technical Field]

[0001]

[0001] The disclosure generally relates to compositions for use in improving and / or maintaining optimal cholesterol levels in subjects with a stressed gut microbiome. The disclosure further relates to methods of using and manufacturing such compositions. [Background technology]

[0002]

[0002] The human gut microbiome is an ecosystem of trillions of bacteria that contribute to overall metabolism and health. Throughout life, the gut microbiome is subjected to one or more of the following loads: unhealthy diet, antibiotics, other medications, infections, strenuous exercise, or alcohol.

[0003]

[0003] Such a burden on the gut microbiome can lead to metabolic disorders and negatively impact overall health.

[0004]

[0004] At the same time, various factors such as lack of exercise and physical activity, stress and hormones, obesity or being overweight, smoking, alcohol, drugs, medical conditions, and unhealthy diets, such as high-fat diets, can lead to increased blood cholesterol levels, which can further cause cardiovascular and circulatory, endocrine and nervous system conditions and / or diseases.

[0005]

[0005] As the microbiome becomes more stressed, cholesterol levels may increase more rapidly. Methods and compositions that have been sufficiently reported may not be effective in improving cholesterol to optimal levels, or may be ineffective, and this has not been addressed to date.

[0006]

[0006] Therefore, it is necessary to identify the nutrients required to improve and / or maintain optimal cholesterol levels when the gut microbiome is under stress.

[0007]

[0007] Furthermore, there is a need to provide nutritional compositions and methods for improving and / or maintaining optimal cholesterol levels when the intestinal microbiome is under stress. [Overview of the project]

[0008]

[0008] Clinical studies disclosed herein demonstrate that in subjects subjected to a high-fat, low-fiber diet, resulting in a stressed gut microbiome, the intake of a combination of fiber blends and probiotic mixtures improves and / or maintains optimal cholesterol levels. Accordingly, this disclosure relates, in general, to novel compositions and methods for improving and / or maintaining optimal cholesterol levels in subjects with a stressed gut microbiome by using a combination of fiber blends and probiotic mixtures.

[0009]

[0009] Benefits of this improvement include, for example, the prevention and / or treatment of cardiovascular and circulatory, endocrine and nervous system conditions and / or diseases in subjects whose gut microbiome is under stress.

[0010]

[0010] 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]

[0011] [Figure 1] This graph shows the results of experimental examples disclosed herein, demonstrating that cholesterol levels decrease when nutritional interventions are performed. [Figure 2] This graph shows the results of experimental examples disclosed herein, demonstrating that the frequency of subjects with near-optimal cholesterol levels decreases when nutritional intervention is performed. [Modes for carrying out the invention]

[0012]

[0013] definition

[0014] 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.

[0013]

[0015] 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.

[0014]

[0016] 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.

[0015]

[0017] 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.

[0016]

[0018] 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".

[0017]

[0019] 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.

[0018]

[0020] "Prevention" includes reducing the risk, incidence rate, and / or severity of a condition or disorder. The terms "treatment" and "treating" include both prophylactic treatment or inhibitory treatment (treatment that prevents and / or delays the onset of a targeted pathological condition or disorder) and curative treatment, therapeutic treatment, or disease-modifying treatment, for example, therapeutic means for curing, delaying, reducing symptoms, and / or halting the progression of a diagnosed pathological condition or disorder, as well as treatment of patients at risk of developing a disease, or suspected of having a disease, and patients with poor health, or patients diagnosed with a disease or medical condition. The terms "treatment" and "treating" do not necessarily mean treating until the subject is completely cured. The terms "treatment" and "treating" also refer to maintaining and / or enhancing the health of an individual who is not suffering from a disease but is potentially prone to an unhealthy condition. The terms "treatment" and "treating" are also intended to include synergistic effects of one or more primary preventive or therapeutic means, or otherwise enhancement. As a non-limiting example, treatment can be performed by a patient, caregiver, physician, nurse, or another medical professional.

[0019]

[0021] As used herein, a "therapeutically effective amount" for prevention or treatment is an amount that, in an individual, prevents a deficiency, treats a disease or medical condition, or, more generally, reduces symptoms, manages the progression of a disease, or provides a nutritional, physiological, or medical benefit to the individual.

[0020]

[0022] As used herein, the terms "food", "food product", and "food composition" mean a product or composition intended for oral ingestion by humans or other mammals and containing at least one nutrient for humans or other mammals.

[0021]

[0023] As used herein, "nutritional composition" and "nutritional product" include any number of food raw materials and optionally additional raw materials, based on the functional needs in the product and in full compliance with all applicable regulations. Optional raw materials may include, but are not limited to, conventional food additives such as one or more acidulants, additional thickeners, pH-adjusting buffers or pH adjusters, chelating agents, colorants, emulsifiers, excipients, flavorings, minerals, permeating agents, pharmaceutically acceptable carriers, preservatives, stabilizers, sugars, sweeteners, texturizers and / or vitamins, etc. Optional raw materials can be added in any suitable amount.

[0022]

[0024] "Probiotics" means a preparation of microbial cells or components of microbial cells that beneficially affect the health or well-being of the host. (Salminen S, Ouwehand A. Benno Y. et al., "Probiotics: how should they be defined", Trends Food Sci. Technol., 1999:10 107-10).

[0023]

[0025] As used herein, the term "unit dosage form" refers to a physically discrete unit suitable as a dosage unit for human and animal subjects, each unit containing a predetermined amount of the composition disclosed herein in an amount sufficient to produce the desired effect, together with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications of the unit dosage form are determined by the specific compound used, the effect to be achieved, and the pharmacodynamics associated with each compound in the host body.

[0024]

[0026] "Subject" or "individual" is a mammal, preferably a human.

[0025]

[0027] Embodiments

[0028] High-fat diets have been shown to disrupt blood lipid levels and alter lipoprotein metabolism (dyslipidemia). The inventors investigated whether short-term nutritional loads induce dyslipidemia in participants and whether microbiota resilience is related to plasma lipids. Plasma cholesterol and triglyceride levels were measured at baseline, after nutritional load (day 4), and after the recovery period (day 11). The inventors observed a significant increase in total cholesterol and LDL cholesterol levels after nutritional load (Wilcoxon rank test P<0.01, Figure 1), which returned to baseline levels after the recovery period (P<0.001). HDL cholesterol levels did not change significantly with the load but decreased after the recovery period (P=0.038). However, triglyceride levels showed the opposite trend, significantly decreasing after nutritional load (P=0.027) and then recovering after the recovery period (P=7.2×10⁻⁶). -4 Interestingly, the inventors observed that changes in LDL and total cholesterol were negatively correlated with microbiome resistance (rho=-0.63, P=0.0035), but not negatively correlated with recovery (rho=0.02, P=0.87). This suggests that the lower the microbiome's resistance to nutrient load, the higher the cholesterol levels in the blood.

[0026]

[0029] A key observation was that when participants were supplemented with a combination of probiotics and fiber, their total cholesterol and LDL cholesterol levels significantly decreased (Wilcoxon test P<0.01). To further understand the promising effects of supplementation, the inventors classified participants based on whether their individual blood lipid levels were considered "optimal" or "near-optimal," and then evaluated the development of the proportion of participants in each category before and after the high-fat diet and after the recovery period. During the control phase, 50% of participants temporarily shifted their total cholesterol levels from the optimal category to the near-optimal category after the high-fat load (Figure 2). The reduction in the effect of the high-fat diet on cholesterol levels was negatively correlated with microbiome resilience (Spearman correlation rho=-0.53, P=0.0036). Similarly, the percentage of participants who moved from the optimal to near-optimal LDL cholesterol category was 10% during the control period compared to 0% during the intervention (Wilcoxon rank test P=0.073). In summary, this suggests that increased microbiome resilience associated with probiotic and fiber supplementation may help reduce acute dyslipidemia induced by high-fat loading. In investigating possible mechanisms of action, the inventors hypothesized that enhanced resilience might affect microbial cholesterol metabolism, and consequently, cholesterol transport from the gut to the bloodstream. The inventors measured the difference in ApoB48 levels, a marker of lipid absorption, before and after high-fat loading. The inventors found this difference to be significantly smaller during the intervention (Wilcoxon rank test P=0.038). This suggests that supplementation and associated microbial changes may affect cholesterol transport from the gut to the bloodstream, thereby maintaining cholesterol levels within the optimal range. Finally, in the control group, the increased ketone body levels after the high-fat diet did not change during the intervention (Wilcoxon test Pfdr > 0.05) and were significantly lower than the levels during the control period (Pfdr < 0.05, same test).

[0027]

[0030] Accordingly, one aspect of the present disclosure is a composition for use in improving and / or maintaining optimal cholesterol levels in subjects with a stressed gut microbiome, comprising an effective amount of a combination of at least one type of fiber and at least one type of probiotic.

[0028]

[0031] In one embodiment, the composition is intended for use in the prevention and / or treatment of cardiovascular, circulatory, endocrine, and / or nervous system conditions and / or diseases.

[0029]

[0032] In one embodiment, the stressed gut microbiome is brought about by microbiome stressors experienced by the subject.

[0030]

[0033] In one embodiment, the microbiome stressor is selected from the group consisting of dietary stressors; antibiotics; other drugs; infections; strenuous exercise; stress; alcohol; travel; parenteral nutrition; enteral nutrition; short bowel syndrome; enteritis; chemotherapy; colon cancer; diarrhea; proton pump inhibitors; gluten-free diets; diets free of fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAPs); and combinations thereof; preferably, at least a dietary stressor; more preferably, at least a high-fat diet; most preferably, a Western-style high-fat, low-fiber diet or a ketogenic diet.

[0031]

[0034] In preferred embodiments, the stressor is a dietary stressor, such as a high-fat diet, for example, a Western diet or a ketogenic diet, or a low-carbohydrate diet. As used herein, a high-fat diet is a daily calorie intake in which more than 35% of the daily calorie intake comes from dietary fat, for example, at least about 40% of the daily calorie intake comes from dietary fat, at least about 45% of the daily calorie intake comes from dietary fat, at least about 50% of the daily calorie intake comes from dietary fat, at least about 55% of the daily calorie intake comes from dietary fat, or at least about 60% of the daily calorie intake comes from dietary fat.

[0032]

[0035] Western diets are characterized by highly processed and refined foods; high levels of sugar, salt, and fat; protein derived from red meat; and low levels of fiber. A "low-fiber" diet is considered to have less than 15 grams of fiber per 2000 calories per day.

[0033]

[0036] As used herein, a low-carbohydrate diet means that approximately 15% or less of the daily calorie intake comes from carbohydrates, for example, approximately 10% or less of the daily calorie intake comes from carbohydrates, or approximately 5% or less of the daily calorie intake comes from carbohydrates.

[0034]

[0037] In some embodiments, subjects have consumed a high-fat diet (e.g., a Western diet or a ketogenic diet) or a low-carbohydrate diet for at least one day (e.g., at least one week or at least one month) prior to the first administration of the combination of at least one fiber and at least one probiotic, and optionally thereafter administer the combination of at least one fiber and at least one probiotic (e.g., daily administration over a period of at least one week or at least one month).

[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 contained in an amount effective to improve and / or maintain an optimal cholesterol level in a subject to whom the unit dosage form is administered, in a state in which the intestinal microbiome is under load.

[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] Another embodiment is a method for improving and / or maintaining optimal cholesterol levels in a subject with a stressed gut microbiome, the method comprising the step of administering the composition to the subject.

[0040]

[0043] In one embodiment, the present disclosure is a method for achieving at least one outcome selected from the prevention and / or treatment of cardiovascular, circulatory, endocrine and / or nervous system conditions and / or diseases, comprising administering a composition described herein to a subject in a state of a strained gut microbiome, thereby improving and / or maintaining optimal cholesterol levels in the subject.

[0041]

[0044] 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.

[0042]

[0045] At least one type of probiotic is 1 x 10 3 cfu~1×10 12 cfu (cfu = colony-forming unit), preferably 1 × 10 7 cfu~1×10 11 It can be administered to an individual as a daily dose of cfu. At least one type of probiotic is 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.

[0043]

[0046] 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 composition such as a food composition.

[0044]

[0047] 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.

[0045]

[0048] 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.

[0046]

[0049] 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.

[0047]

[0050] Another embodiment is a method for producing a composition for improving and / or maintaining an optimal cholesterol level in a subject to which the composition is administered, in a state in which the intestinal microbiome is under stress, 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.

[0048]

[0051] 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.

[0049]

[0052] 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.

[0050]

[0053] 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.

[0051]

[0054] The composition may optionally include 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.

[0052]

[0055] A composition comprising a combination of at least one fiber and at least one probiotic may further contain protective hydrophilic colloids (such as gum, protein, or modified starch), binders, film-forming agents, encapsulants / encapsulating materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizers (such as oils, fats, waxes, or lecithin), 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.

[0053]

[0056] 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.

[0054]

[0057] 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.

[0055]

[0058] Any suitable food-derived protein, such as animal protein (milk protein, meat protein, and egg protein, etc.); plant protein (soy protein, wheat protein, rice protein, and pea protein, etc.); 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.

[0056]

[0059] 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.

[0057]

[0060] 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]

[0058]

[0061] Examples

[0062] The following non-limiting examples generally illustrate the concepts underlying the embodiments disclosed herein.

[0059]

[0063] Materials and methods

[0064] 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.

[0060]

[0065] 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 number NCT04424329.

[0061]

[0066] 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 the 5-day loading period. The same meal plan was served in the same order during each test period.

[0062]

[0067] 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)).

[0063]

[0068] 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.

[0064]

[0069] 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.

[0065]

[0070] 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.

[0066]

[0071] 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).

[0067]

[0072] 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.

[0068]

[0073] 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.

[0069]

[0074] 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.

[0070]

[0075] 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).

[0071]

[0076] 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.

[0072]

[0077] 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).

[0073]

[0078] 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 of 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.

[0074]

[0079] 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.

[0075]

[0080] 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.

[0076]

[0081] 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.

[0077]

[0082] 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.

[0078]

[0083] 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 in treatment, intervention, and fasting days (logarithmic scale).

[0079]

[0084] Intestinal comfort was evaluated on a visual analog scale of 0 - 10, where 0 was asymptomatic and 10 was the worst possible symptom. Participants recorded the levels of nausea, vomiting, audible abdominal sounds, abdominal cramps, and flatulence daily throughout the study period.

[0080]

[0085] All analyses were performed in R. Unless otherwise specified, two-sided tests were used for all statistical tests. Microbiome data were analyzed using genome-based mapping aggregates and defined at the species level. Unless otherwise stated, microbiome data were evaluated using non-parametric tests (Wilcoxon rank sum test and Mann-Whitney). Whenever applicable, multiple testing was performed by the Benjamini-Hochberg method (FDR) (q-value). For fecal metabolites, statistical analysis was performed using natural logarithm-transformed data.

[0081]

[0086] Results

[0087] Figure 1 shows the change in plasma cholesterol levels over time. The x-axis represents time (days). The y-axis represents the level of the biomarker. The color represents the intervention status of the population as described in the legend below. Significance was defined by a paired two-sided Wilcoxon rank sum test ( * =Pfdr < 0.05; ** =Pfdr < 0.01; *** =Pfdr < 0.005). Panel A) shows the level of total cholesterol in plasma; Panel B) shows the level of LDL-cholesterol in plasma; Panel C) shows the level of HDL cholesterol in plasma.

[0082]

[0088] As shown in Figure 1, the levels of total cholesterol and LDL cholesterol were significantly lower when the claimed composition was supplemented to the participants.

[0083]

[0089] Figure 2 shows that the frequency of subjects with near-optimal cholesterol levels was lower when the claimed composition was supplemented to the participants.

[0084]

[0090] This rigorous randomized controlled clinical trial demonstrates that combined intake of a fiber blend and a probiotic mixture improves and / or maintains optimal cholesterol levels in subjects with a stressed gut microbiome.

[0085]

[0091] 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 composition comprising an effective amount of a combination of at least one type of fiber and at least one type of probiotic, for use in improving and / or maintaining optimal cholesterol levels in subjects with a stressed gut microbiome.

2. The composition according to claim 1, wherein the intestinal microbiome under the aforementioned load is brought about by the microbiome stressors experienced by the subject.

3. The composition according to claim 2, wherein the microbiome stressor is selected from the group consisting of dietary stressors, antibiotics, other drugs, infections, strenuous exercise, stress, alcohol, travel, parenteral nutrition, enteral nutrition, short bowel syndrome, enteritis, chemotherapy, colon cancer, diarrhea, proton pump inhibitors, gluten-free diets, diets that do not contain fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAPs), and combinations thereof, and is preferably at least a dietary stressor, more preferably at least a high-fat diet, most preferably a Western-style high-fat low-fiber diet or a ketogenic diet.

4. The composition according to any one of claims 1 to 3, 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.

5. The composition according to any one of claims 1 to 4, 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.

6. The composition according to any one of claims 1 to 5, 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.

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

8. The composition according to any one of claims 1 to 7, 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.

9. The composition according to any one of claims 1 to 8, 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.

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

11. The composition according to any one of claims 1 to 10, 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.

12. A composition according to any one of claims 1 to 11, for use in the prevention and / or treatment of conditions and / or diseases of the cardiovascular, circulatory, endocrine, and / or nervous systems.

13. A method for improving and / or maintaining an optimal cholesterol level in a subject whose intestinal microbiome is under stress, comprising the step of administering to the subject a composition according to any one of claims 1 to 11.

14. A method for achieving at least one outcome selected from the prevention and / or treatment of cardiovascular, circulatory, endocrine and / or nervous system conditions and / or diseases, comprising the step of improving and / or maintaining optimal cholesterol levels in a subject whose intestinal microbiome is under strain by administering a composition according to any one of claims 1 to 11.

15. A method for producing a composition for improving and / or maintaining optimal cholesterol levels in a subject to which the composition is administered, in a state of a stressed intestinal microbiome, 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.

16. 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 to improve and / or maintain the optimal cholesterol level in a subject to whom the unit dosage form is administered, in a state in which the intestinal microbiome is under load.

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