METHOD FOR MEASURING AND IMPROVING GUT HEALTH
20210239696 · 2021-08-05
Inventors
Cpc classification
G01N2469/10
PHYSICS
Y02A90/10
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61P1/14
HUMAN NECESSITIES
A61K31/702
HUMAN NECESSITIES
G16H20/10
PHYSICS
G01N2800/52
PHYSICS
International classification
G16H20/10
PHYSICS
Abstract
The present invention relates to a method for measuring gut health comprising the steps of: a) collecting or receiving at least one, preferably two faecal samples, preferably three or more faecal samples from a human or animal, and the sample comprises markers; b) using the sample of step a), to generate output databased on a composition and/or function and/or metabolic activity of gut microbiota; c) measuring output data in relation to level and/or stability of one or more marker sand/or relation between different markers to generate a result on gut health.
Claims
1. Method for measuring gut health comprising the steps of: a) collecting or receiving at least one, preferably two faecal samples, preferably three or more faecal samples from a human or animal, and the sample comprises markers; b) using the sample of step a), to generate output data based on a composition and/or function and/or metabolic activity of gut microbiota; c) measuring output data in relation to level and/or stability of one or more markers and/or relation between different markers to generate a result on gut health.
2. Method according to claim 1, further comprising the step: d) using the result on gut health to diagnose, prevent or treat disease, improve the quality of life, recommend a diet, lifestyle change, supplement or medication.
3. Method according to claim 2, wherein the output data comprises quantities of at least one bacteria, gene and/or metabolite, preferably two, preferably three or more.
4. Method according to claim 3, wherein the at least one bacteria are a member of the microbiota, preferably member of the fiber degrading or metabolizing bacteria, preferably belonging to Bacteroidetes, Firmicutes, Actinobacteria or Verrucomicrobia.
5. Method according to claim 3, wherein the marker is gene activity or metabolic activity displayed by any of the microbiota members, preferably gene or metabolic activity related to fiber metabolism, preferably gene or metabolic activity related to short chain fatty acid biosynthesis, preferably gene or metabolic activity related to propionic and/or butyric acid production.
6. Method according to claim 1, wherein one marker is used in conjunction with at least one other marker, preferably to monitor interactions between different bacteria, preferably to monitor cross-feeding interactions, preferably cross-feeding of organic acids such as short chain fatty acids and lactic acid between different bacteria, preferably cross-feeding interaction between acetate and lactic acid producing bacteria, and butyric producing bacteria, preferably cross-feeding interaction between Bifidobacterium or Lactobacilli and Faecalibacterium prausnitzii.
7. Method according to claim 1, wherein the result is grouped into different categories based on results from an intervention with a fiber and/or probiotic and/or dietary change and/or lifestyle change to determine an individual's response and optimal gut health.
8. Method according to claim 1, wherein the result is used to determine a baseline and/or an acute inflammation.
9. Method according to claim 8, wherein a change in the level and/or stability and/or relation between different markers is indicative of inflammation in the host.
10. Method according to claim 4, wherein the level of Faecalibacterium prausnitzii is used to indicate an optimal gut health in the intervals 50-0.1%, preferably 35-0.1%, preferably 20-0.5%.
11. Method according to claim 4, wherein the level of Bifidobacterium is used to indicate an optimal gut health in the intervals 0.1-50%, preferably 1-50%, preferably 2-50%, preferably 5-50%, preferably 10-50%.
12. Method according to claim 6, wherein the relation between Faecalibacterium prausnitzii and Bifidobacterium is used to indicate an optimal gut health and the ratio between Faecalibacterium prausnitzii and Bifidobacterium is between 100-0.01 preferably 50-0.02, preferably 25-0.04, preferably 10-0.1, preferably 5-0.2.
13. Method according to claim 8, wherein the inflammation is indicative of development or onset of metabolic or chronic disease.
14. Method according to claims 1 and 13, wherein the results on gut health are used for diagnosis and/or prevention, and/or treatment of a disease, wherein the disease is selected from the group consisting of metabolic disease, gastrointestinal health, Crohn's disease, Ulcerative colitis, Multiple sclerosis, IBS, IBD, ADHD, Alzheimer's disease, muscle disease, non-alcoholic fatty liver disease, cardiovascular disease, allergy, asthma, diabetes, eczema and skin diseases, obesity, cancer, neurological health issues, endocrine system conditions, clostridium difficile associated conditions, locomotor system conditions, cutaneous condition, autoimmune system conditions, mental health associated conditions, skin related conditions, infectious disease and other health conditions associated with antibiotic usage, thyroid health issues, cerebro-craniofacial health, arthritis, dementia, and kidney disease.
15. Method according to claim 1, wherein the result is used to base a recommendation, where in the recommendation is a healthier diet, preferably a more fiber-rich diet or a fiber supplement or a probiotic supplement or a medication.
16. Method according to claim 1, wherein the recommended fiber supplement comprises non- or partially digestible polysaccharides and/or oligosaccharides and/or disaccharides consisting of modified or unmodified starch and partial hydrolysates thereof, inulin or partially hydrolyzed inulin, natural oligofructoses, fructo-oligosaccharides (FOS), lactulose, lactosucrose, soybean-oligosaccharides (SOS), galactomannan and suitable partial hydrolysates thereof, manno-oligosaccharides (MOS), indigestible polydextrose, acemannan, various gums and pectin and partial hydrolysates thereof, indigestible dextrin and partial hydrolysates thereof, trans-galacto-oligosaccharides (GOS), xylo-oligosaccharides (XOS), xylan, arabinoxylan, arabinogalactan, arabino-xylooligosaccharides (AXOS), beta-glucan and partial hydrolysates thereof, chito-oligosaccharides (COS), glucomano-oligosaccharides (GMOS), arabinooligosaccharides (AOS), pectin-oligosaccharides (POS), laminar-oligosaccharides, human milk oligosaccharides (HMO), bovine milk oligosaccharides (BOS), cellulose derived oligosaccharides.
17. Method according to claim 10, wherein the level of Faecalibacterium prausnitzii is used to diagnose diabetes, where Faecalibacterium prausnitzii is less than 10%, more specifically less than 7%.
18. Method according to claim 10, wherein the level of Faecalibacterium prausnitzii is used in the treatment of diabetes, where Faecalibacterium prausnitzii level is increased to more than is more than 7%, specifically more than 10%, specifically more than 14%.
19. Method according to claim 10, wherein the level of Faecalibacterium prausnitzii is used to diagnose arthritis, where Faecalibacterium prausnitzii is less than 10%.
20. Method according to claim 10, wherein the level of Faecalibacterium prausnitzii used in the treatment of arthritis, where Faecalibacterium prausnitzii level is increased to more than is more than 10%, specifically more than 14%.
21. Method according to claim 11, wherein the level of Bifidobacterium is used to diagnose IBD, where the level of Bifidobacterium is unstable and is reduced below 4%, more specifically 3%, more specifically below 2%, more specifically in 3 month's time.
22. Method according to claim 11, wherein the level of Bifidobacterium is used in the treatment of IBD, where the level of Bifidobacterium is stabilized above 2%, more specifically above 3%, more specifically above 4%.
23. Method according to claim 10, wherein the level of Faecalibacterium prausnitzii is used to diagnose cutaneous condition, more specifically acne, where the level of Faecalibacterium prausnitzii is unstable and is reduced below 14%, more specifically in 3 month's time.
24. Method according to claim 10, wherein the level of Faecalibacterium prausnitzii is used in the treatment of cutaneous condition, more specifically acne, where the level of Faecalibacterium prausnitzii is stabilized above 14%, more specifically in 3 month's time.
25. Method according to any claim 17-24 where soluble fiber, more specifically soluble fiber mentioned in claim 16 is used to treat the aforementioned conditions in claims 17-24.
Description
SHORT DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0048] Soluble fibers reach the colon where they are converted to short chain fatty acids to support the growth of a diverse flora of good bacteria, reducing the gut permeability and reducing inflammation. Therefore, an optimal gut health can be considered as a microbiota that has access to plenty of fiber through the diet. However, people are consuming too little fiber especially soluble fiber meaning there is a fiber deficiency and most people have not reach a state of optimal or even good gut health. There has been no definition of a normal, good or healthy microbiota due to the fact that most people have not optimized their microbiota with fiber through their diet.
[0049] In order to address the problem of measuring and improving gut health for an individual, a measurement of different bacteria present in faecal samples was performed. A test group of 7 people took a soluble fiber supplement consisting of 2-5 g arabinogalactan every day for a total period up to 4 months. The test subjects sent in a fecal sample before starting the daily consumption of arabinogalactan. Bacterial DNA was extracted and QPCR was used to measure the level of total bacteria, F. prausnitzii, Bifidobacteria and Prevotella present in each sample. All results were stored together with each user's profile in a database. Based on the Ct values the relative percentage of each bacteria or bacteria group was determined (
[0050] The pattern of butyric acid producing bacteria was only possible to discover due to the intervention with a soluble fiber known to increase bacterial diversity in the microbiota and following the microbiota development during several months. It took between 2 and 4 months to reach a stable microbiota depending on the individuals gut microbiome at the beginning of the study demonstrating that repeated measurements over a long period of time is important for measuring and improving gut health (
[0051] Further, it was only possible to see change in the marker after increasing the fiber dose for some individuals showing the difficulties in discovering these patterns of gut health. Stabilizing the marker at an optimal level for gut health is therefore very individual based and should distinguish this invention from previous work in the area of microbiome diagnosis and drug development. The fact that it is possible with the current invention to measure an individual's response to an intervention or medication and to optimize their gut health makes it possible to classify an individual's microbiome as poor, sub optimized or optimized from a gut health perspective not possible before.
[0052] Furthermore, this invention demonstrates that by analysing the dynamics to discover patterns of bacteria in the microbiota after introducing a soluble fiber it was possible to draw conclusion about the impact on gut health of that particular individual. Moreover, the methodology can be used to monitor gut health once an individual has reach their optimal level of gut health due to the sensitivity of the microbiota to change in inflammation (
[0053] It was surprising to discover that a single member of the core microbiome F. prausnitzii together with Bifidobacteria could be used as markers for both gut health, gut dysbiosis and a suboptimal gut flora. There is a clear correlation between the amount of fiber and the diversity of the microbiome and therefore the markers interplay discovered in this invention can be used as an indicator of bacterial diversity and gut health.
[0054] Surprisingly, even in otherwise healthy individuals there was an initial relative decrease of F. prausnitzii and/or Bifidobacteria by introducing fiber which shows a clear improvement in bacterial diversity making this method ideal for recommending diets, supplements, probiotics or medications for optimizing each individuals gut health. This is somewhat counterintuitively since a decrease F. prausnitzii and/or Bifidobacteria would usually be interpreted as something undesirable but with this current invention clearly demonstrates that a more normalized level of F. prausnitzii and/or Bifidobacteria to other gut bacteria can be more beneficial to gut health than simply a very high relative amount of one beneficial bacteria.
[0055] Reducing the amount of F. prausnitzii seems counterintuitive since it is regarded as a peace keeping bacteria or a new type of probiotic. It only makes sense in the light of keeping the absolute numbers constant while increasing other bacteria able to produce e.g. acetate to improve butyric acid production, diversity and bacteria load. Further, tracking a member of the core microbiome assuming it is rather constant such as F. prausnitzii, and the fluctuations is in the other microbes, makes it possible to correct or compensate for increasing total bacteria for e.g. measuring good probiotic bacteria impact in the total microbiome.
[0056] The results from the test group can be interpreted as three phases. The first phase with a relative decrease of the bacterial markers is indicative of a mobilization phase where the gut microbiota community of bacteria is adapting to a new environment of more available carbon sources in the form of fiber. This phase is then followed by a growth phase of good bacteria e.g. Bifidobacteria. Finally, there is a stabilization of the gut microbiota community at this new level of improved gut health called the stabilization phase.
[0057] How quickly an individual move between these three phases depends largely on the individual's unique microbiota as well as how much fiber is consumed through the diet. However, it is evident from the results that most people have the capacity to restore their microbiota and gut health through increasing their fiber intake.
[0058] Another surprising discovery was that by analysing multiple samples during a longer time frame it was possible to discover bacteria to bacteria interactions. For example, the cross-feeding with e.g. Bifidobacteria and butyrate producing bacteria belonging to clostridial clusters IV or XIVa. This cross-feeding produce butyric acid through breakdown of fibers to acetate by e.g. Bifidobacteria. Butyrate is well recognized as an important anti-inflammatory substance which helps to maintain a healthy gut barrier function. Measuring the cross-feeding and therefore the potential for short chain fatty acid production is another use of this method to monitor gut health together with monitoring core gut microbes.
[0059] For people, skilled in the art it should be obvious that other bacteria present in the microbiota could be used in a similar fashion as F. prausnitzii as a marker of gut health, flora diversity and fiber intake. However, F. prausnitzii belonging to the core microbiome (present in most individuals) and fairly stable during the life of a healthy individual would serve as one of the best markers of gut health. Further, F. prausnitzii is a single species making it ideal for detection with a fast and accurate detection methods such as QPCR. In people with disease it was possible to detect levels of bacteria with or without a fiber intervention that restored the gut microbiota composition to a healthy state. By measuring the levels of F. prausnitzii and Bifidobacteria , it was possible both to diagnose and treat conditions of IBD, diabetes, arthritis and cutaneous condition (Acne) (Table 1 A-D). It was surprising to find out that different levels of each biomarker was linked to a specific conditions and in the case of IBD and acne that the biomarker had to be tracked up to 3 months in order to see a pattern of unstability in these conditions, while in the case of diabetes and arthritis it was possible to with one sample see reduced levels of F. prausnitzii to diagnose and treated these two conditions. In the case of IBD and acne a stable level of Bifidobacteria and F. prausnitzii respectively was the indicator of a good treatment for each condition. Hence multiple samples are needed to diagnose and treat IBD and Acne since it is the stability of Bifidobacterium in the case of IBD and F. prausnitzii in the case of Acne which is not evident from only one sample. However, in the case if diabetes and arthritis a single sample seems to be enough to diagnose these conditions although multiple samples are needed to properly track and treat these conditions. The methods for measuring gut health herein described can be used for treatment of the subjects suffering from the diseases or syndromes herein described.
[0060] As mentioned above, the level of Faecalibacterium prausnitzii is used to indicate an optimal gut health in the intervals 50-0.1%. In one embodiment the level of Faecalibacterium prausnitzii is used to diagnose diabetes, where Faecalibacterium prausnitzii is less than 10%, more specifically less than 7%; or the level of Faecalibacterium prausnitzii is used in the treatment of diabetes, where Faecalibacterium prausnitzii level is increased to more than is more than 7%, specifically more than 10%, specifically more than 14%; or the level of Faecalibacterium prausnitzii is used to diagnose arthritis, where Faecalibacterium prausnitzii is less than 10%; or the level of Faecalibacterium prausnitzii used in the treatment of arthritis, where Faecalibacterium prausnitzii level is increased to more than is more than 10%, specifically more than 14%. Further, the level of Faecalibacterium prausnitzii is used to diagnose cutaneous condition, more specifically acne, where the level of Faecalibacterium prausnitzii is unstable and is reduced below 14%, more specifically in 3 month's time. The level of Faecalibacterium prausnitzii may also be used in the treatment of cutaneous condition, more specifically acne, where the level of Faecalibacterium prausnitzii is stabilized above 14%, more specifically in 3 month's time.
[0061] Also as mentioned above, the level of Bifidobacterium is used to indicate an optimal gut health in the intervals 0.1-50%. In one embodiment the level of Bifidobacterium is used to diagnose IBD, where the level of Bifidobacterium is unstable and is reduced below 4%, more specifically 3%, more specifically below 2%, more specifically in 3 month's time; or level of Bifidobacterium is used in the treatment of IBD, where the level of Bifidobacterium is stabilized above 2%, more specifically above 3%, more specifically above 4%;
The methods defined above includes that soluble fiber, more specifically soluble fiber mentioned as defined as the recommended fiber supplement is used to treat the aforementioned conditions.
EXAMPLES
Example 1: Measuring and Improving Gut Health in a Test Group After Intervention with a Soluble Fiber
[0062] Human fecal samples were collected from a test group of adult men and women every month up to 4 months including a base line sample taken before starting with the supplement. The fecal samples were bead beaten in a lysis buffer for 20 minutes. Bacterial DNA was isolated with magnetic beads and eluted in RNase free water. Total DNA was quantified using 260 nm using a nano-drop spectrophotometer. Quantitative PCR amplification and detection were carried out using primers for F. prausnitzii 5′-3′ (GGAGGAAGAAGGTCTTCGG & AATTCCGCCTACCTCTGCACT), Bifidobacteria 5′-3′ (CTCCTGGAAACGGGTGGT & GCTGCCTCCCGTAGGAGT), Prevotella 5′-3′ (CAGCAGCCGCGGTAATA & GGCATCCATCGTTTACCGT) and total bacteria 5′-3′ (ACTCCTACGGGAGGCAGCAGT & ATTACCGCGGCTGCTGGC),PCR amplification and detection was performed using an Quantstudio 3 (Applied Biosystems, Darmstadt, Germany) in optical-grade 96-well plates sealed with optical sealing tape. Each reaction mixture (22.5 μl) was composed of 10 μl of SYBR Green PCR Master Mix (Applied Biosystems, Darmstadt, Germany), 2 μl primer mix (10 pmol/μl each), 9 μl sterile distilled H2O, and 1.5 μl stool DNA (10 ng/μl). For the negative control, 2 μl of sterile distilled H2O was added to the reaction solution instead of the template DNA solution. A melting curve analysis was carried out following amplification to distinguish the targeted PCR product from the nontargeted PCR product. Each real-time PCRs were performed in triplicate, and average values were used for calculations. PCR conditions consisted of one cycle of 50° C. for 2 min, 95° C. for 2 min and then 40 cycles of 95° C. for 30s, 60° C. for 30s, and 72° C. for 60s. The fraction of each bacteria was calculated as 1/(2{circumflex over ( )}(delta Ct)), where delta Ct is the difference between the cycles for total bacteria and the target bacteria. The fraction of each bacteria was plotted in a graph to determine the pattern for each test subject. Example 2: F. prausnitzii and Bifidobacterium levels in people with IBD, diabetes, arthritis and cutaneous condition (acne) with or without an intervention with fiber. In order to find out what levels of F. prausnitzii and Bifidobacterium can be used to diagnose people with a disease, participants with IBD, diabetes, arthritis and cutaneous condition (acne) were tested for their levels of F. prausnitzii and Bifidobacterium with or without intervention with a soluble fiber (arabinogalactan). Fecal samples were collected, processed and analyzed as mentioned in example 1 up to 2 months with participants consuming non (control group) or up to 5 g per day of arabinogalactan (study group). The results average values were calculated for each bacterial marker for each group (with n participants) (Table 1A-D). It was possible both to diagnose and treat conditions by evaluating the level of each bacterial marker. For IBD Bifidobacterium was the best marker, however multiple tests were needed to detect the unstability in the level of Bifidobacterium in IBD cases (Table 1A). For diabetes F. prausnitzii was the best marker (Table 1B) where a low level of F. prausnitzii was evident in the control group vs. the fiber group. For Arthritis F. prausnitzii was the best marker (Table 1C) where a reduced level of F. prausnitzii was evident in the control group vs. the fiber group. For Acne F. prausnitzii was the best marker (Table 1D) where a reduced stability in the level of F. prausnitzii was evident from multiple samples in the control group vs. the fiber group. Table 1 A Relative percentages of F. prausnitzii (FP) and Bifidobacterium (BB) in a study group with people with IBD. Cont. is a control group with participants not consuming any fiber, while Fiber are participants consuming up to 5 g per day of soluble arabinogalactan fiber.
TABLE-US-00001 IBD 0 IBD 1 IBD 2 IBD month month months values Cont. FP % 17.7 12.8 15.0 15.2 (n = 22) BB % 5.1 3.7 1.8 3.5 Fiber FP % 14.2 11.1 12.4 12.6 (n = 57) BB % 5.8 4.2 4.4 4.8
Table 1 B Relative percentages of F. prausnitzii (FP) and Bifidobacterium (BB) in a study group with people with diabetes. Cont. is a control group with participants not consuming any fiber, while Fiber are participants consuming up to 5 g per day of soluble arabinogalactan fiber.
TABLE-US-00002 Diabetes 0 Diabetes 1 Diabetes 2 Average month month months values Cont. FP % 5.1 7.3 8.1 6.8 (n = 10) BB % 4.4 3.1 4.3 3.9 Fiber FP % 10.4 16.1 16.4 14.3 (n = 43) BB % 3.1 5.8 2.8 3.9
Table 1 C Relative percentages of F. prausnitzii (FP) and Bifidobacterium (BB) in a study group with people with arthritis. Cont. is a control group with participants not consuming any fiber, while Fiber are participants consuming up to 5 g per day of soluble arabinogalactan fiber.
TABLE-US-00003 Arthritis 0 Arthritis 1 Arthritis 2 Average month month months values Cont. FP % 7.2 9.9 11.2 9.4 (n = 16) BB % 5.9 4.6 4.7 5.1 Fiber FP % 11.9 13.1 18.8 14.6 (n = 56) BB % 4.9 2.6 2.7 3.4
Table 1 D Relative percentages of F. prausnitzii (FP) and Bifidobacterium (BB) in a study group with people with Acne. Cont. is a control group with participants not consuming any fiber, while Fiber are participants consuming up to 5 g per day of soluble arabinogalactan fiber.
TABLE-US-00004 Acne 0 Acne 1 Acne 2 Average month month months values Cont. FP % 22.5 6.4 8.7 12.5 (n = 20) BB % 7.5 2.9 5.1 5.2 Fiber FP % 14.9 16.5 14.6 15.3 (n = 50) BB % 7.9 4.0 4.0 5.3