Bifidobacteria for use in affecting low-grade inflammation

Abstract

The present invention relates generally to the human therapeutic effects of Bifidobacterium adolescentis, and in particular to the alleviation of low-grade inflammation in the population of elderly people.

Description

Technical Field

[0001] The present invention relates generally to the therapeutic effects of Bifidobacterium adolescentis and in particular to the relief of low-grade inflammation in the elderly. Technical Background

[0002] Bifidobacteria are natural inhabitants of the gastrointestinal tract and possess genetic adaptations that enable them to colonize this harsh and complex habitat. Bifidobacteria interact with key elements of intestinal function and contribute to the maintenance of homeostasis. Recent scientific advances have shown that bifidobacteria can reduce mucosal antigen load, improve the intestinal barrier, and induce modulation of local and systemic immune responses through strain-dependent interactions with the host.

[0003] Bifidobacteria are used as probiotics due to their recognized benefits to human health. Probiotics are "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host" (FAO / WHO, 2001). About 12 strains of Bifidobacteria are commercially available with clinically proven effects.

[0004] Low-grade inflammation is associated with an increased risk of cardiovascular disease (CVD) (Biasucci, 2004; Kaptoge et al., 2010), type 2 diabetes (Barzilay et al., 2001; Dehghan et al., 2007), rheumatoid arthritis (Nielen et al., 2004), and cancer (Taniguchi and Karin, 2018). Low-grade inflammation is defined as a slightly elevated C-reactive protein (CRP) level above 3 mg / L and below 10 mg / L (Imhof et al., 2003; Pearson et al., 2003; Rifai and Ridker, 2003). As an acute phase protein, CRP is a marker for a variety of pathological processes such as infection, cancer, tissue damage, and chronic inflammatory diseases (Dinh et al., 2019). Studies have reported an association between aging and increased C-reactive protein and proinflammatory cytokines (Ferrucci and Fabbri, 2018; Franceschi et al., 2017; Li et al., 2011).

[0005] In healthy adults, Bifidobacterium adolescentis is one of the two dominant bifidobacterium species in the intestine (Matsuki et al., 2004). However, the abundance of Bifidobacterium decreases with age (Arboleya et al., 2016). Specifically, the abundance of Bifidobacterium adolescentis significantly decreased with age compared with young, middle-aged, and elderly groups aged ≥60 years (Chen et al., 2021).

[0006] Data from animal and human studies suggest that high levels of Bifidobacterium adolescentis in the microbiome are associated with a lower risk of acquiring age-related diseases (Haro et al., 2016; Hevia et al., 2016; Kato et al., 2017; Lim and Kim, 2017; Nobili et al., 2018; Ouwehand et al., 2008). Therefore, it has been suggested that a decrease in Bifidobacterium adolescentis may have a negative impact on inflammation and health.

[0007] In healthy adults, Bifidobacterium adolescentis one of the two dominant bifidobacterium species in the intestine (Matsuki et al., 2004). However, the abundance of Bifidobacterium adolescentis decreases with age (Arboleya et al., 2016). Specifically, when comparing young, middle-aged, and elderly groups aged ≥60 years, the abundance of Bifidobacterium adolescentis significantly decreased with age (Chen et al., 2021).

[0008] SUMMARY OF THE DISCLOSURE

[0009] The present disclosure relates to dietary manipulation of the intestinal microbiota, preferably in the elderly, such as supplementation with probiotics, and how this represents a method for protecting healthy gastrointestinal microflora and influencing inflammatory responses in the elderly population. To test this, the inventors designed a protocol to study the effect of supplementation with two doses of Bifidobacterium adolescentis (DSM 29103) for 12 weeks compared to placebo in a 3-group parallel design, placebo-controlled and randomized study in an elderly population aged ≥65 years.

[0010] According to a first aspect, the present disclosure provides a method of ameliorating a condition characterized by elevated levels of hs-CRP or TNF-α in a subject by administering to the subject a therapeutically effective amount of Bifidobacterium adolescentis.

[0011] In a preferred embodiment, the Bifidobacterium adolescentis is Bifidobacterium adolescentis DSM 29103.

[0012] Conditions characterized by elevated levels of hs-CRP or TNF-α may be low-grade inflammation.

[0013] Subjects may be selected based on one or more of the following criteria:

[0014] a) Aged between 65 and 85;

[0015] b) plasma hs-CRP level is 3.0 mg / L-10.0 mg / L; and

[0016] c) BMI is 18.5 kg / m 2 -32 kg / m 2 .

[0017] The therapeutically effective amount may be 1 billion CFU or more per day, 10 billion CFU or more per day.

[0018] For example, a therapeutically effective amount may be administered as 2 capsules per day.

[0019] The present disclosure provides Bifidobacterium adolescentis for use in alleviating low-grade inflammation.

[0020] For example, the Bifidobacterium adolescentis may be Bifidobacterium adolescentis DSM 29103.

[0021] Low-grade inflammation is characterized by plasma hs-CRP levels of 3.0 mg / L-10.0 mg / L.

[0022] For example, low-grade inflammation occurs in people aged 65 to 85 years with a BMI of 18.5 kg / m 2 -32 kg / m 2 among the subjects.

[0023] The present disclosure also provides a composition comprising Bifidobacterium adolescentis and a pharmaceutically acceptable excipient. In one embodiment, the amount of live Bifidobacterium adolescentis is 1 billion CFU or 10 billion CFU.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a graphical timeline summarizing the research protocol.

[0026] Figure 2 is a table showing an overview of biological samples, specimens, and measurement schedules.

[0027] Figure 3 is a table showing the ingredients of the test product.

[0028] Detailed description

[0029] As used herein, a bacterial "strain" refers to a bacterium that remains genetically unchanged when grown or propagated. This includes multiple identical bacteria.

[0030] "Wild-type strain" refers to the non-mutated form of the bacterium found in nature.

[0031] In this context, the term "derivative strain" is understood to be a strain derived from a parent strain by means such as genetic engineering, radiation and / or chemical treatment and / or selection, adaptation, screening, etc. In a specific embodiment, the derivative strain is a functionally equivalent mutant, for example, a mutant having substantially the same or improved properties as the parent strain (e.g., with respect to probiotic properties). Such derivative strains are part of the present invention. The term "derivative strain" includes strains obtained by subjecting the strain of the present invention to any conventionally used mutagenic treatment (including chemical mutagens such as ethyl methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV light treatment), or spontaneously generated mutants.

[0032] "Mutant bacteria" or "mutant strains" refer to natural (spontaneous, naturally occurring) mutant bacteria or induced mutant bacteria, containing one or more mutations in their genome (DNA) that are not present in wild-type DNA. "Induced mutants" are bacteria in which mutations are induced by human treatment, such as treatment with any conventionally used mutagenic treatment, including treatment with chemical mutagens, such as those selected from the group consisting of: (i) mutagens that bind to or incorporate into DNA, such as base analogs, for example 2-aminopurine or chelators such as ICR-191, (ii) mutagens that react with DNA, including alkylating agents, such as nitrosoguanidine or hydroxylamine, or ethyl methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV radiation or gamma radiation, etc. In contrast, "spontaneous mutants" or "naturally occurring mutants" have not been induced by humans.

[0033] Derivative strains such as mutants may have been subjected to several mutagenesis treatments (a single treatment is understood as a mutagenesis step followed by a screening / selection step), but typically no more than 20, no more than 10, or no more than 5 treatments are performed. In specific embodiments of derivative strains (such as mutants), less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, or even less than 0.0001% of the nucleotides in the bacterial genome have been altered (e.g., by substitution, insertion, deletion, or a combination thereof) compared to the parent strain.

[0034] As described above the mutant bacteria are non-GMO, i.e. have not been modified by recombinant DNA technology. As an alternative to the preferred method of providing mutants by random mutagenesis described above, such mutants may also be provided by site-directed mutagenesis, for example by using appropriately designed PCR techniques or by using transposable elements that can be integrated into bacterial replicons.

[0035] When mutants were provided as spontaneously arising mutants, the above-mentioned wild-type strains were subjected to the selection step without any previous mutagenesis treatment.

[0036] Any mutant strain of the Bifidobacterium adolescentis strains having accession numbers DSM 29103, DSM 29104, DSM 29106, DSM 29107, DSM 29111, DSM 29102 and DSM 29105 can be obtained by subjecting the strain to the mutagenesis treatment described above to harvest mutant strains and selecting mutant strains having desired properties. Alternatively, spontaneously generated mutants can be selected.

[0037] One embodiment of the present disclosure relates to an isolated strain selected from the group consisting of DSM 29103, DSM 29104, DSM 29106, DSM 29107, DSM 29111, DSM 29102 and DSM 29105, and a mutant of any of said deposited strains capable of modulating elevated levels of hs-CRP or TNF-α in a subject. A particularly preferred embodiment is Bifidobacterium adolescentis with accession number DSM 29103, or a mutant thereof, which is capable of modulating elevated levels of hs-CRP or TNF-α in a subject.

[0038] The term "probiotic product" refers to any product comprising probiotics. Probiotic products comprising the strains according to the invention can be administered in the form of food or dietary supplements. For example, Bifidobacterium adolescentis can be incorporated into dairy products, such as milk, and in particular fermented dairy products, optionally in combination with other lactic acid bacteria, for example with yogurt starters, or into other foods such as snack bars, or beverages such as juices.

[0039] Probiotic products comprising Bifidobacterium adolescentis may also be provided as dietary supplements in the form of powders, tablets such as lozenges or effervescent tablets, lozenges, capsules, chewing gum, in individual sachets, or as a component of more conventional compositions such as oil drops, emulsions or pastes, or in any other suitable carrier determined by a person skilled in the art to be an effective carrier of live microorganisms.

[0040] Probiotics are live microorganisms, and this can be a challenge during the formulation and storage of probiotic products. Probiotics are particularly sensitive to temperature, moisture content, oxygen, and other ingredients in the formulation matrix. Preferably, the bacteria of the present invention remain viable after long-term storage so that when the probiotic product of the present invention is administered to an individual in need thereof, the bacteria are able to impart its beneficial effects.

[0041] The term "viable" means that the cells are alive and are able to form colonies in the culture dish during pour plate or spread plate. The number of viable probiotics is determined as the number of colony forming units (CFU) by incubation under conditions suitable for the growth of the probiotic strain by pour plate or spread plate culture method. By this method, cells that are able to grow and form colonies will be counted. When numerical values ​​are given in this specification and claims, they should be understood as CFU / g unless the context indicates otherwise. In some embodiments, the probiotic product of the present invention comprises at least 10 at the end of shelf life (EOS). 9 CFU / unit. The end of shelf life may be at least 3 months, such as at least 6 months, at least 9 months, at least 12 months, at least 18 months or at least 24 months.

[0042] Using low water activity ensures better survival of the probiotics during product storage.

[0043] Water activity (aW) is defined as the partial pressure of water vapor in a composition at a specific temperature divided by the partial pressure of water vapor in a standard state at the same temperature. Therefore, water activity is a measure of the amount of free (i.e., unbound) water in a composition. It can be calculated as: aW = p / p0, where p is the partial pressure of water vapor in the composition, and p0 is the vapor pressure of pure water at the same temperature. In probiotic products, it is generally preferred that the water activity (aw) is in the range of 0.1-0.2.

[0044] The probiotics used in the probiotic product of the present invention are usually frozen or freeze-dried. In order to obtain a higher survival rate, the bacteria are mixed with a cryoprotectant before freezing or freeze-drying.

[0045] The term "cryoprotectant" refers to an excipient that is capable of increasing the survival rate and improving the storage stability of bacteria during freezing and / or drying. Cryoprotectants as used herein typically include sugars.

[0046] The sugar may be a monosaccharide, a disaccharide, an oligosaccharide or a polysaccharide, or a mixture of at least two sugars. The composition may even comprise three, four or more sugars.

[0047] In some embodiments, the composition comprises a mixture of at least one monosaccharide or disaccharide and at least one oligosaccharide. In other embodiments, the composition comprises a mixture of at least one monosaccharide or disaccharide and at least one polysaccharide.

[0048] Monosaccharides that can be used in the probiotic products of the present disclosure include glucose (also known as dextrose), fructose, ribose and galactose. Disaccharides useful in the probiotic products of the present disclosure include sucrose, trehalose, maltose and lactose. The composition may contain one or more monosaccharides or disaccharides, such as one, two or three or even more different sugars.

[0049] In some embodiments, the probiotic product of the present disclosure comprises at least one oligosaccharide. Oligosaccharides are carbohydrate polymers containing three to nine monosaccharides. Fructooligosaccharides (FOS) are present in many vegetables and consist of short-chain fructose molecules. Galactooligosaccharides (GOS) are also naturally occurring and consist of short-chain galactose molecules. The human body can only partially digest these compounds. The composition can comprise one, two or even more different oligosaccharides.

[0050] In some embodiments, the probiotic product of the present disclosure comprises at least one polysaccharide. Polysaccharides are polymeric carbohydrate molecules composed of more than 10 monosaccharide units bound together by glycosidic bonds, and produce monosaccharides or oligosaccharides after hydrolysis. Their structures range from linear to highly branched. Examples of polysaccharides used in the probiotic product of the present disclosure are maltodextrin, cyclodextrin, alginate, pectin, chitosan, starch and inulin. The composition can comprise one, two, three or even more different polysaccharides.

[0051] For example, the cryoprotectant may comprise a disaccharide such as sucrose or a mixture of glucose and a polysaccharide such as maltodextrin.

[0052] The addition of oligo- or polysaccharides such as FOS, GOS, inulin and other polysaccharides may help to reduce the water activity and have the further advantage that oligo- and polysaccharides are not as sweet as mono- and disaccharides, and they also add fibre to the composition.

[0053] Polyols (sugar alcohols) have the general formula HOCH2(CHOH)nCH2OH. They are often added to foods because they are lower in calories and less sweet than sugar. In addition, they are not broken down or metabolized into acid by bacteria in the mouth and therefore do not cause tooth decay.

[0054] Said composition can also comprise at least one polyol, such as erythritol, inositol, isomalt, mannitol, maltitol, sorbitol or xylitol, or its mixture.Preferred polyol is xylitol, sorbitol and mannitol.Said composition can comprise one, two, three or even more different polyols.

[0055] The cryoprotectant may further comprise peptides, proteins, protein hydrolysates or mixtures thereof. Examples of peptides and proteins used herein are casein, pea, whey, albumin, soy protein, glutamic acid or gelatin, and any isolate or hydrolysate thereof. Other additives may also be present, for example antioxidants such as ascorbate, sodium citrate, propyl gallate.

[0056] The present disclosure also relates to a probiotic comprising an isolated strain according to the present disclosure and a cryoprotectant such as a saccharide.

[0057] Combinations of multiple probiotic species or strains can be used, i.e., 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or even more of the species and strains listed herein. In a currently preferred embodiment, only one, two, three, four or five different strains are present in a probiotic product according to the present disclosure.

[0058] In addition to probiotics, one or more other active ingredients, for example one, two, three, four or more active ingredients selected from the group consisting of vitamins (such as vitamin A, D, E, K2, C, B2, B6, B12, biotin, niacin, folic acid); minerals (such as zinc, selenium, chromium, copper, calcium, chloride) and vegetable extracts, such as cranberry extract / juice, royal jelly can be included in the probiotic product.

[0059] It is contemplated that in order to obtain a therapeutic effect, the probiotic product should be administered once daily for at least one week, and advantageously for longer periods of time, such as at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 9 weeks, preferably at least 12 weeks, in an amount equivalent to at least 10 6 CFU, for example at least 10 7 CFU, preferably at least 10 8 CFU, usually 10 9 CFU and 10 12 CFU of Bifidobacterium adolescentis.

[0060] In this study, the probiotic product contained Bifidobacterium adolescentis as an active ingredient. Bifidobacterium adolescentis can be used as the only active ingredient. Alternatively, the probiotic product as described herein may also contain compounds of interest, such as other bacterial strains, vitamins, prebiotics, fibers, or other compounds that may have a beneficial health effect.

[0061] The other bacterium may be selected from Bifidobacterium lactis, Lactobacillus rhamnosus, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Leuconostoc lactis, Leuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactobacillus casei subsp. casei, Streptococcus thermophilus, Bifidobacterium longum, Lactobacillus lactis, Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus lactis ... fermentum, Lactobacillus salivarius, Lactobacillus delbrueckiisubsp.bulgaricus and Lactobacillus acidophilus.

[0062] In the context of describing the present invention (especially in the context of the following claims), the use of the terms "a, an" and "said / the" and similar references should be interpreted as covering the singular and plural, unless otherwise indicated herein or clearly contradicted by the context. Unless otherwise indicated, the terms "comprising", "having", "including" and "containing" should be interpreted as open terms (i.e., meaning "including, but not limited to"). Unless otherwise indicated herein, the description of the numerical range herein is intended only as a shorthand method for referring to each individual numerical value within the range individually, and each individual value is incorporated into the specification as if it were described separately herein. Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention, rather than to limit the scope of the present invention, unless otherwise required. Any language in the specification should not be interpreted as indicating that any unrequired element is essential to the practice of the present invention.

[0063] Preservation and Expert Solutions

[0064] The applicant requests that, in accordance with existing regulations of industrial property offices of contracting states of the Budapest Treaty, samples of the deposited microorganisms described below may only be provided to experts before the date of grant of the patent.

[0065] Table 1. Deposits made with a depository that has been granted the status of an international depository institution under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz-Institut DSMZ - German Collection of Microorganisms, Inhofenstrasse 7B, 38124 Braunschweig, Germany.

[0066] strain Accession Number Date of deposit Bifidobacterium adolescentis DSM 29103 2014.07.16 Bifidobacterium adolescentis DSM 29104 Bifidobacterium adolescentis DSM 29106 Bifidobacterium adolescentis DSM 29107 Bifidobacterium adolescentis DSM 29111 Bifidobacterium adolescentis DSM 29102 Bifidobacterium adolescentis DSM 29105 Example

[0067] Example 1. Effect of Bifidobacterium adolescentis (DSM 29103) on the levels of inflammatory markers in the elderly population - a A randomized, double-blind, placebo-controlled, parallel-group pilot study with a 12-week intervention

[0068] Purpose

[0069] The primary objective of the study was to evaluate the effectiveness of two doses of Bifidobacterium adolescentis in altering hs-CRP, a marker of low-grade inflammation, in an elderly population.

[0070] The secondary objectives of the study were to evaluate the effectiveness of two doses of Bifidobacterium adolescentis:

[0071] -Changes in blood-borne biomarkers of systemic inflammation, intestinal barrier function, and immunity;

[0072] - Changes in biomarkers of intestinal inflammation;

[0073] - Changes in the microbiome;

[0074] - Changes in health-related quality of life, mental health, and physical well-being were assessed using the Short Form 36 (SF-36), Depression and Anxiety Scale-21 (DASS-21), and the World Health Organization (5) Well-Being Index, respectively.

[0075] Test products

[0076] Bifidobacterium is a genus of anaerobic, Gram-positive, non-spore-forming, non-motile bacteria that produce lactic and acetic acid. It is a common component of the human intestinal microbiota. The experimental product was a vegetable capsule containing high or low doses of Bifidobacterium adolescentis DSM29103. In this article, the name of the experimental product is shortened to Bif-038.

[0077] The reference product (placebo) is the same capsule but without Bif-038. All products will be manufactured at Chr. Hansen Laboratories in Horsholm, Denmark, which is certified for food production.

[0078] The production batch of Bifidobacterium adolescentis DSM 29103 used in this study had a minimum of 0.5*10 9 CFU / capsule or 5*10 9 CFU / capsule. The daily dose is 2 capsules in the three groups of the trial. In parallel with this trial, the CFU stability of the trial product batches will be analyzed and these data will be available and included in the final clinical trial report.

[0079] In Europe, EFSA has granted QPS status to strains of Bifidobacterium belonging to the species adolescentis (EFSA, 2007). This means that the strains used in this trial are safe for use in food and as food supplements.

[0080] The specific ingredients of the test product are shown in Figure 3 .

[0081] The test capsules and placebo capsules were similar in smell, taste, and appearance. All study products were packaged in identical vials with identical labels except for the randomization number.

[0082] Study subjects, investigators, study staff, statisticians and sponsor will be blinded throughout the study and until database lock. Only the study supply coordinator designated by Chr. Hansen A / S will be unblinded and have access to the randomization list when necessary for packaging, labeling and resupply of study product.

[0083] Methodology

[0084] The study was a randomized, double-blind, 3-group, placebo-controlled pilot study conducted in an elderly population with mild inflammation (hs-CRP 3mg / L-10mg / L) comparing two daily doses of 1 billion CFU and 10 billion CFU of Bifidobacterium adolescentis to placebo for 12 weeks.

[0085] Subjects received one of the two test products or a placebo product daily for 12 weeks.

[0086] Five visits are planned throughout the study period. Subjects will attend two screening visits, a baseline visit, a 6-week post-intervention visit, and an end-of-study visit after the 12-week intervention.

[0087] During screening, the subjects are assigned a screening number starting from 1001 according to the order in which they enter the trial. For example, the screening number of the first subject is 1001.

[0088] If subjects are eligible for participation in the trial, they receive their randomization number at Visit 2. The randomization number includes the stratification number (female = 5; male = 9) and will be assigned sequentially in the order in which subjects complete Visit 2.

[0089] For example, the first woman included would receive random number: 5001, and the fifth woman included would receive random number: 9005.

[0090] The SAS proc plan program will be used to develop a randomization list for N = 54 subjects per stratum.

[0091] Visit 1 and Visit 2 are screening and recruitment visits at least two weeks apart. At the screening visit, eligibility will be checked after the participant has given their written informed consent.

[0092] Vaccination status will be obtained as part of the medical history.

[0093] Repeated CRP measurements are necessary to differentiate between short-term and more persistent CRP elevations.

[0094] Only participants with hs-CRP between 3.0 mg / L and 10.0 mg / L were invited to the second screening visit.

[0095] Participants with repeat hs-CRP levels in the range of 3.0 mg / L-10.0 mg / L will be invited to baseline visit 3, included in the study and randomized.

[0096] Subjects will be asked to complete a diet diary prior to Visit 3, Visit 4, and Visit 5. Subjects will be instructed on how to use the diary. Stool sample kits will be distributed and subjects will be instructed on how to collect samples prior to the baseline visit.

[0097] Visit 3 is the baseline visit. Stool samples and blood samples will be collected. Questionnaires will be provided for completion on site. Prior to this visit, subjects have already collected stool samples and completed a diet diary.

[0098] Visit 4, after 6 weeks of intervention. Blood samples will be collected. Compliance will be assessed by counting returned unused capsules, and capsules for the remaining 6 weeks of the study will be provided. Questionnaires will be provided for on-site completion. Stool sample kits will be distributed and subjects will be instructed on how to collect samples before the next visit.

[0099] Visit 5 is the end-of-study visit. Stool samples and blood samples will be collected. Questionnaires will be provided for on-site completion. Compliance will be assessed by counting the number of unused capsules returned. Prior to this visit, subjects have already collected stool samples and completed a diet diary.

[0100] Participants who report being ill before the visit will have their new visit rescheduled at least 5 days after their last symptom, as CRP levels typically remain elevated for several days after an acute inflammatory state. In addition, participants should avoid any type of vaccination for at least ±3 days before / after the visit.

[0101] At each visit, participants assessed cold or flu-like symptoms with the Jackson Questionnaire and had their temperature taken.

[0102] Visits 1 and 2, Screening Visits:

[0103] Blood sample

[0104] Questionnaire

[0105] ·temperature

[0106] Visit 3, Baseline Visit; Day 0:

[0107] Blood sample

[0108] Fecal sample

[0109] Questionnaire

[0110] 3-day food diary

[0111] ·Anthropometric measurements

[0112] ·temperature

[0113] Visit 4, 6 weeks after intervention; Week 6:

[0114] Blood sample

[0115] Adherence

[0116] Questionnaire

[0117] 3-day food diary

[0118] ·Anthropometric measurements

[0119] ·temperature

[0120] Visit 5, End of Study Visit; Week 12:

[0121] Blood sample

[0122] Fecal sample

[0123] Adherence

[0124] Questionnaire

[0125] 3-day food diary

[0126] ·Anthropometric measurements

[0127] ·temperature

[0128] A graphical representation of the study protocol is provided in Figure 1 .

[0129] Planned number of subjects

[0130] Due to the exploratory nature of the pilot study design, no sample size calculation was performed. Each study group is planned to include 20 subjects, so a total of 60 subjects. During randomization, subjects will be stratified by sex to ensure equal gender distribution between study groups. At least 1 / 3 of the members of each group will be male and female. Randomized: n = 60. Evaluable: n = 51 (15% dropout rate; dropouts will not be replaced).

[0131] Inclusion Criteria

[0132] Be willing to participate in the study and comply with its procedures, and be able to give written informed consent.

[0133] The researchers determined that healthy adults:

[0134] Age: ≥65 years and ≤85 years

[0135] hs-CRP: 3.0mg / L-10.0mg / L

[0136] BMI: 18.5 kg / m 2 -32 kg / m 2

[0137] Controlled, stable hypertension as determined by the investigator

[0138] Temperature between ≥36.5℃ and ≤37.3℃

[0139] ·Stable body weight (≤5% change) within 3 months before screening.

[0140] Test products

[0141] Active product group 1: Bifidobacterium adolescentis, DSM 29103, 1 billion CFU, 2 capsules 0.5B orally per day.

[0142] Active product group 2: Bifidobacterium adolescentis, DSM 29103, 10 billion CFU, 2 capsules of 5B orally per day.

[0143] Placebo group: 2 placebo capsules were taken orally daily.

[0144] The placebo capsules and the low-dose capsules (0.5B) will be enriched with sodium ascorbate according to the level in the high-dose capsules (5B).

[0145] Choice of dose and dosing regimen

[0146] In the low-dose intervention group and the high-dose intervention group, the daily active test product contained a minimum of 1*10 9 CFU and 10*10 9 At these two doses, we aimed to evaluate whether the potential effects of Bif-038 were dose-dependent.

[0147] Each subject was given the study dose

[0148] The subjects were provided with the study product at Visit 3 (4 bottles) and Visit 4 (3 bottles).

[0149] Subjects returned the empty bottles and any unused product at Visit 4 and Visit 5 (end-of-study visit), and compliance was assessed by counting the returned capsules.

[0150] Subjects received 168 capsules to be taken over 12 weeks, with an additional 24 days of extra product provided in case of product loss or visit delays.

[0151] Subjects were instructed to take two capsules daily with breakfast, preferably before breakfast (on an empty stomach), for 12 weeks.

[0152] The study product should be swallowed with liquids, but hot or acidic liquids should be avoided when swallowing the capsules.

[0153] If a subject forgets to take a dose of the investigational product, it may be taken later that day, with or without a meal, but an additional dose should not be taken the next day to make up for the missed dose from the previous day.

[0154] Subjects should take the first dose on Visit 3 and the last dose on the day before Visit 5.

[0155] Subjects should take the capsules after their study visit on the day of Visit 4. They should not take the capsules on an empty stomach.

[0156] If new GI symptoms or other AEs that are significantly worse, severe, or intolerable occur after taking / using the product or overdose, the dosing can be interrupted after consulting the medical monitoring representative and PI / assistant investigator of the sponsor. Without increasing the risk to the subjects, the dosing can be temporarily interrupted for the shortest possible time and then continue to participate in the trial safely. No more than 3 consecutive days. If the patient subject needs to interrupt the medication for more than 3 days and the GI symptoms and / or other AEs do not improve, the sponsor representative and PI / assistant investigator must be consulted. The patient participant can withdraw from the interrupted IP and can choose to continue the trial without IP or continue the early withdrawal of the participant.

[0157] Duration of intervention

[0158] Duration of test product / placebo intervention: 12 weeks

[0159] Evaluation Criteria

[0160] The primary endpoint is the difference in the change in hs-CRP from Visit 3 (baseline) to Visit 5 (end of study) with daily intake of 1 billion or 10 billion CFU of Bifidobacterium adolescentis compared with placebo. Levels of s-CRP, a marker of low-grade inflammation, will be measured at Visits 1 to 5. Subjects must have an hs-CRP between 3.0 mg / L and 10.0 mg / L at Visits 1 and 2 to be considered eligible for randomization.

[0161] The secondary endpoint was the effect of daily intake of 1 billion or 10 billion CFU of Bifidobacterium adolescentis on the difference in TNF-α from Visit 3 (baseline) to Visit 5 (end of study) compared with placebo.

[0162] Figure 2 An overview of the biological samples, specimens, measurement schedule, and content of measurements is provided.

[0163] References

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Claims

1. A method of ameliorating a condition characterized by elevated levels of hs-CRP or TNF-α in a subject by administering to the subject a therapeutically effective amount of Bifidobacterium adolescentis. 2 . The method according to claim 1 , wherein the Bifidobacterium adolescentis (B. adolescentis) is Bifidobacterium adolescentis DSM29103.

3. The method according to any one of the preceding claims, wherein the condition characterized by elevated levels of hs-CRP or TNF-α is low-grade inflammation.

4. The method according to any one of the preceding claims, wherein the subject is selected by at least one of the following criteria: a) Aged between 65 and 85; b) plasma hs-CRP level is 3.0 mg / L-10.0 mg / L; and c) BMI is 18.5 kg / m 2 -32 kg / m 2 .

5. The method according to any one of the preceding claims, wherein the therapeutically effective amount is 1 billion CFU or 10 billion CFU per day.

6. The method of claim 5, wherein the therapeutically effective amount is administered as 2 capsules per day.

7. Bifidobacterium adolescentis for use in reducing low-grade inflammation.

8. The Bifidobacterium adolescentis for use according to claim 7, wherein the Bifidobacterium adolescentis is Bifidobacterium adolescentis DSM29103.

9. Bifidobacterium adolescentis for use according to claim 7 or 8, wherein the low-grade inflammation is characterized by hs-CRP levels in plasma of 3.0 mg / L-10.0 mg / L.

10. Bifidobacterium adolescentis for use according to claims 7 to 9, wherein the low-grade inflammation occurs in individuals aged 65 to 85 years and optionally with a BMI of 18.5 kg / m 2 -32 kg / m 2 of the subjects.

11. A composition comprising Bifidobacterium adolescentis and a pharmaceutically acceptable excipient.

12. The composition according to claim 11, wherein the amount of live Bifidobacterium adolescentis is 1 billion CFU or 10 billion CFU.

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