Live saccharomyces cerevisiae strains for the treatment and / or prevention of bone-loss disorders

Abstract

The present invention pertains to the use of a live Saccharomyces cerevisiae strain for the treatment and / or prevention of bone-loss disorders. The present invention further pertains to a combination of a live Saccharomyces cerevisiae strain with vitamin K2 for the treatment and / or prevention of bone- loss disorders such as osteoporosis. The present inventors have shown that such a combination allows significantly improving bone health markers while reducing negative markers associated with osteoclastogenic activity.

Description

DescriptionTitle: LIVE SACCHAROMYCES CERE VISIAE STRAINS FOR THE TREATMENT AND / OR PREVENTION OF BONE-LOSS DISORDERSTechnical Field

[0001] The present invention pertains to the medical field, in particular to the treatment of bone disorders. The present invention particularly relates to the use of live Saccharomyces cerevisiae strains for the treatment of bone-loss disorders such as osteoporosis.Backg ound Art

[0002] Bones provide a rigid framework and are responsible for the mechanical support of the body. They are dynamic tissues that are tightly regulated and under constant remodeling. Bone remodeling is the result of a strict balance between bone resorption (mediated by osteoclasts) and bone formation (mediated by osteoblasts) to maintain an appropriate bone density and mechanical strength. This balance relies on multiple mediators and a large number of coordinated signaling mechanisms which are essential for maintaining a proper bone function.

[0003] Unfortunately, under certain pathological conditions, this balance can be deregulated and lead to abnormal bone remodeling and the development of bone-loss disorders.

[0004] Osteoporosis is one of the most common bone-loss disorders. It occurs when bone mineral density and bone mass decrease. It is defined as low bone mineral density caused by altered bone microstructure ultimately predisposing patients to low-impact, fragility fractures and ultimately loss of mobility and poor quality of life. According to the International Osteoporosis Foundation, over 32 million individuals, including 25.5 million women and 6.5 million men, aged 50 and above suffered from osteoporosis in Europe in 2019. This represents approximately 5.6% of the total European population aged 50 and above. The cost of osteoporosis in the EU in 2019 was estimated at €56.9 billion. Worldwide, more than 200 million people have osteoporosis causing more than 8.9 million fractures annually. Over 70% of those over age 80 are affected.

[0005] The two main mechanisms by which osteoporosis develops are excessive bone resorption by osteoclast cells and / or insufficient formation of new bone tissue by osteocytes / osteoblast cells. Calcium and vitamin D depletion worsen the disease. Hormones play an active role in bone remodeling, and hormone level variations can therefore directly have an impact on bone formation. For example the lack of estrogen resulting from menopause increases bone resorption. In the same vein, the thyroid hormone calcitonin increases bone deposition, and the parathyroid hormone (PTH), secreted by the parathyroid glands in response to low-calcium levels induces higher osteoclastic activity to induce calcium release in blood.

[0006] Nutrition and dietetic measures, including food supplementation with Vitamin D and calcium have been proposed in prevention of osteoporosis. Vitamin D helps calcium fixation and phosphorus. Some studies show that a combination of Vitamin D with calcium allows limiting osteoporotic fracturesin patients (see e.g. Feng et al. Orthop. Surg. 2021 ; 13:1262-1268). However, such supplementation strategies remain ineffective in patients who are not depleted in calcium and / or vitamin D.

[0007] Probiotics comprising bacteria such as Lactobacillus strains have been identified as potentially useful in the prevention of osteoporosis. Recently, a combination of vitamin D and Lactobacillus reuteri probiotic showed interesting results in terms of bone health. However, further research needs to be carried out to evaluate whether it has any effect on fracture risk (Rizzoli and Biver. Current osteoporosis reports 18 (2020): 273-284).

[0008] Alternative therapeutic strategies involve bisphosphonates administration to reduce osteoclast activity or selective estrogen receptor modulators (SERMs) to increase bone formation. Unfortunately, these treatments may be associated with various side-effects and none of them are fully satisfying (Migliaccio, Silvia, Marina Brama, and Giovanni Spera. Clinical Interventions in Aging 2.1 (2007): 55-64).

[0009] Accordingly, there is still a need for the provision of novel therapies that would allow preventing and reducing bone loss during osteoporosis. Such therapies could be useful more generally in the treatment of various bone disorders associated with bone loss.Summary of the invention

[0010] Using a specific model of the human intestinal microbial ecosystem, the present inventors have shown that it is possible to stimulate various bone-formation markers upon administration of a composition comprising a live Saccharomyces cerevisiae strain. The inventors have particularly shown that such a composition allows inducing a significant increase in propionate and butyrate levels. Propionate and butyrate are two short-chain fatty acids that are known to be correlated with bone formation. The results provided herein therefore show that it is possible to improve bone formation upon administration of a composition comprising a live Saccharomyces cerevisiae strain, and, accordingly, that such a composition can be useful for the treatment of disorders associated with bone loss.

[0011] Accordingly, a first aspect of the present invention pertains to a live Saccharomyces cerevisiae strain for use in the treatment and / or prevention of a bone-loss disorder.

[0012] The live Saccharomyces cerevisiae strain can preferably be obtained from the strain deposited on October 17, 2007, at the Collection Nationale de Cultures de Micro-organismes (CNCM) pursuant to the treaty of Budapest under No. CNCM I-3856.

[0013] More surprisingly, the present inventors have shown that the live Saccharomyces cerevisiae strain can synergistically interact with vitamin K, in particular vitamin K2, and strongly promotes the production of positive bone markers, including propionate, butyrate, indole-3-ethanol and kynurenic acid, while at the same time reducing negative markers associated with osteoclastogenic activity (such as indole-carboxaldehyde). The present invention therefore also relates to a combination therapy comprising a live Saccharomyces cere visiae strain and vitamin K2 for the treatment of boneloss disorders.

[0014] In this context, the present invention pertains to a kit of parts comprising a live Saccharomyces cerevisiae strain and vitamin K2. The invention also relates to the use of said kit of parts in therapy, particularly in the treatment and / or treatment of bone-loss disorders, including osteoporosis.Brief Description of Drawings

[0015] Figure 1 : relative short-chain fatty acids (SCFA) concentration after 48 hours of in vitro human fermentation, normalized for the blank. N=2 donors.

[0016] Figure 2: Production of indole-3-ethanol after 48h of in vitro fermentation with Vitamin K2, CNCM I-3856 and the combo. N=2 donors.

[0017] Figure 3: Production of kynurenic acid after 48h of in vitro fermentation with Vitamin K2, CNCM I-3856 and the combo. N=2 donors.

[0018] Figure 4: Production of indole-carboxaldehyde after 48h of in vitro fermentation with Vitamin K2, CNCM I-3856 and the combo. N=2 donors.

[0019] Figure 5: Schematic mode of action of the combo Vitamin K2 and CNCM I-3856. Green arrow in bone formation indicated positive effect with production of kynurenic acid (KA), Short-Chain fatty acids (SCFAs) and Amino butyric acid (ABA), while blocking the negative effects in bone resorption by Indole-Acetaldehyde (ICA) and adrenocorticotropin hormone (ACTH). The combination also inhibits bone resorption by induction of production of SCFAs and indole-3-ethanol (I3E).Detailed description of the invention

[0020] According to a first embodiment, the present invention pertains to a live Saccharomyces cerevisiae strain for use in the treatment and / or prevention of a bone-loss disorder in a patient in need thereof.

[0021] Saccharomyces cerevisiae, also referred to as “brewer's yeast” or “baker's yeast” is a well-known species of yeast which is commonly used in winemaking, brewing and baking processes (see for review Parapouli et al. AIMS microbiology 6.1 (2020): 1). Saccharomyces cerevisiae strains all possess a nuclear genomic DNA of 12068 kilobases (kb) organized in 16 chromosomes (Goffeau A et al. Science. 1996;274:563-547). In addition to this nuclear genomic DNA, Saccharomyces cerevisiae strains also possess extra chromosomal elements which vary between the various strains including mitochondrial DNA (mtDNA) molecules, double-stranded DNA elements and single- and double-stranded RNA molecules and retroviruses.

[0022] In the context of the present invention, the Saccharomyces cerevisiae strain used can be isolated / obtained from any known Saccharomyces cerevisiae. According to a preferred embodiment, the Saccharomyces cerevisiae strain can be obtained from the strain deposited on October 17, 2007, at the Collection Nationale de Cultures de Micro-organismes (CNCM, 25 / 28 rue du docteur ROUX, F-75724 Paris Cedex 15) pursuant to the treaty of Budapest under No. CNCM I-3856. The strain can e.g. be obtained by isolating a yeast from strain CNCM I-3856 and culturing it. Indeed, as shown in the experimental section below, this specific strain is particularly efficient for inducing a strong increase in the synthesis of positive bone markers such as the short-chain fatty acids propionate and butyrate, as well as indole-3-ethanol, which is correlated with bone formation, and kynurenic acid, which is a negative marker of bone resorption. This strain has been described e.g. in patent applications published under references W02009 / 103884 (for the prevention / treatment of gastrointestinal disorders), W02014 / 009656 (for preventing / treating vaginal mycoses),WO2020 / 127136 (for the treatment / prevention of oropharyngeal candidiasis) and W02020 / 245057 (for the treatment / prevention of simple and / or recurring cystitis). The Saccharomyces cerevisiae strain according to the present invention may be cultured by means of any suitable method known by the skilled person. Methods for culture of yeasts are known in the prior art, and a person skilled in the art knows how to optimize the culture conditions for each strain as a function of its nature. A yeast is obtained by multiplication of a yeast strain in a culture medium, for example as described in the reference book "Yeast Technology", 2nd edition, 1991 , G. Reed and T. W. Nagodawithana, published by Van Nostrand Reinhold, ISBN 0-442-31892-8. Thus, for example, on an industrial scale, yeast cells usable in the context of the present invention may be obtained by a method comprising the following steps:- culturing a yeast strain in a culture medium in several stages, firstly in semi-anaerobiosis, then in aerobiosis (oxygen-rich medium / atmosphere) to obtain multiplication of the starting yeast;- separating, by centrifugation, the yeast cells obtained; and- prepare the yeasts in an appropriate form such as a liquid yeast cream containing between 12% and 25% of yeast dry matter.

[0023] In the context of the present invention, the strain used is alive, i.e. a live Saccharomyces cerevisiae strain. By a “live Saccharomyces cerevisiae strain” is meant that the metabolism of the Saccharomyces cerevisiae strain is active or reactivatable or capable of multiplying.

[0024] The live Saccharomyces cere visiae strain can be in any form suitable for administration in a patient. The strain is advantageously formulated as a probiotic, i.e. formulated for oral administration. The skilled person knows how to formulate live Saccharomyces cerevisiae strains for oral administration. The strain can e.g. be in a fresh form such as a cream yeast or compressed yeast, or in a dry form such as a dry yeast or frozen yeast.

[0025] Fresh yeasts are characterized by a high water-content compared with dry yeasts. Fresh yeasts encompass cream yeasts and compressed or crumbled yeasts.

[0026] Cream yeasts, also known as "liquid yeasts", are aqueous suspensions of yeast cells having a cream-type viscosity. These aqueous suspensions of living yeast cells generally have a solid content of at least 12% by weight, particularly from 12 to 40 % by weight. A cream yeast may have, for example, a solid content of between 12 and 25% by weight, preferably between 14 and 22% by weight. In such a case, the yeast is preferably encapsulated. The encapsulation methods and the different types of capsules are well known to one skilled in the art.

[0027] Compressed yeasts comprise yeasts compressed into a compact block and crumbled compressed yeasts. Compressed yeasts in a compact block, also known as "yeast bars", are characterized by a solids content of between 26% and 35%. Crumbled compressed yeasts have a water content of between 21% and 35%.

[0028] In the context of the present invention, the live yeast is preferably in the form of a dry yeast. When present in a dry form, the yeast can be in the form of an instantaneous dry or active dry form. Dry forms generally comprise a dry material level above 90%, preferably ranging from about 92%-96%.

[0029] Frozen yeasts are characterized by a solid content of between 74% and 80%.

[0030] Typically, the daily dosage of live Saccharomyces cerevisiae yeast administered to the patient in the context of the present invention is comprised between 107and 6x1010CFU (Colony Forming Unit), preferably between 108and 2x1010CFU.

[0031] The present inventors have shown that the use of a live Saccharomyces cerevisiae strain alone, without requiring the presence of any other microorganism such as bacteria (including Lactobacillus stains), allows improving bone health markers. Therefore, in the context of the present invention, the live Saccharomyces cerevisiae strain does not need to be administered in combination with another microorganism. Accordingly, according to a further embodiment, the live Saccharomyces cerevisiae is not administered in combination with a microorganism that does not belong to the Saccharomyces cerevisiae species. This means that the treatment / prevention of the bone-loss disorder according to the present invention does not comprise the administration of another microorganism than the live Saccharomyces cere visiae strain.

[0032] In the context of the present invention, the “bone-loss” disorder refers to a skeletal disorder comprising a decrease in bone density, strength and / or mass. Bone-loss disorders are typically correlated with an imbalance in bone remodeling, in particular with an increased osteoclastogenic activity and a reduced osteoblastogenic activity, thereby leading to bone resorption and / or increased fragility. Bone-loss disorders include osteoporosis, osteopenia, osteoarthritis, Paget’s disease, osteomyelitis, osteomalacia, degenerative joint disease, musculoskeletal pathologies, osteophytes, rickets and delayed or non-union fractures.

[0033] According to a specific embodiment, the bone-loss disorder treated in the context of the present invention is a low bone-density disorder such as osteoporosis and osteopenia. Low bonedensity disorders are well characterized. Bone density is generally measured with dual-energy X-ray absorptiometry (DEXA). It allows providing a score referred to as a T-score. A T-score comprised between +1 and -1 indicates normal bone density, between -1 to -2.5 indicates osteopenia and of -2.5 and below indicates osteoporosis.

[0034] In the context of the present invention, the “patient” or “subject” refers to any human or animal suffering from a bone-loss disorder. Typically, the patient is a mammal. The patient can e.g. be a human, a feline such as a cat, a canine such as a dog or an equid such as a horse. Preferably, the patient is human.

[0035] According to the present invention the term “preventing” a disease or disorder means avoiding its onset, particularly in individuals at risk of developing it, or delaying its symptoms or repercussions. In this context, it is therefore referred to the preventive or prophylactic treatment of this disorder. In particular, “preventing” consists in administering the strain before the disorder appears.

[0036] The term “treating” a disease or disorder means treating said disease / disorder in order to cure it, alleviate its symptoms or repercussions, or at least stabilize it. In this context, it is therefore referred to the curative treatment of this disorder.

[0037] In particular, “treating” consists in administering the strain when the disease or disorder has set in.

[0038] The terms “reduce the severity of symptoms” of a disease / disorder and “reduce the symptoms” of a disease / disorder are used interchangeably and refer to reducing the abnormal manifestation / perceptible clinical sign(s) caused by said disease / disorder.

[0039] As explained above, the present inventors have shown that it is possible to significantly potentiate the beneficial effects of the live Saccharomyces cerevisiae strain on bone health by combining it with vitamin K2. Indeed, the experimental data provided below shows that the live Saccharomyces cerevisiae strain and vitamin K2 act synergistically for stimulating the expression of positive bone formation markers while at the same time reducing the expression of negative markers associated with bone resorption.

[0040] Those skilled in the art will understand that vitamin K and derivatives thereof refer to one or more compounds of Formula 1 and their pharmaceutically or nutritionally acceptable salts:

[0041] O Formula 1

[0042] wherein R may be any covalently linked organic group including polyisoprenoid residues, esters, ethers, and thiol adducts.

[0043] According to some aspects, the vitamin K may be a vitamin K2, i.e., a menaquinone, selected from the group consisting of short-chain menaquinones (i.e., MK-1 , MK-2, MK-3, and MK- 4), long-chain menaquinones (i.e., MK-5, MK-6, MK-7, MK-8, and MK-9), and combinations thereof. Those skilled in the art will understand that menaquinones are abbreviated MK-n, wherein M represents menaquinone, K represents vitamin K, and n represents the number of isoprenoid side chain residues. According to some aspects, the vitamin K2 may comprise or consist of MK-7 or MK-4.

[0044] Sources of vitamin K2 which may be useful according to aspects of the present disclosure include, but are not limited to different forms of vitamin K2 including synthetic MK-4, MK-5, MK-6, MK-7, MK-8, MK-9, MK-10, MK-11 , MK12, and MK-13, natto (i.e., food prepared from fermented soybean), fermented foods, dairy products, meat, fish and combinations thereof.

[0045] Vitamin K2 promotes bone health and acts in concert with vitamin D to support bone physiology and reduces some side effects of high vitamin D intake like soft tissue (e.g. blood vessel) calcification. It’s necessary for blood clotting and promotes the accumulation of calcium in bones and teeth. Vitamin K2 activates osteocalcin, a protein that promotes specific bone and teeth calciumaccumulation. There are reports showing poor vitamin K2 status in frail individuals and association with bone fragility. Therefore, a higher intake of vitamin K2 is also recommended for bone health.

[0046] Therefore, according to a further embodiment, the present invention pertains to a combination of a live Saccharomyces cerevisiae strain with vitamin K2, particularly for the treatment and / or prevention of bone-loss disorders.

[0047] In the context of the present invention the live Saccharomyces cerevisiae strain and vitamin K2 can be formulated as a 2-in-1 product, comprising both said strain and vitamin K2. Alternatively, the live Saccharomyces cerevisiae strain and vitamin K2 can also be administered separately, as two distinct products.

[0048] Accordingly, the present invention also pertains to a kit of parts comprising a live Saccharomyces cerevisiae strain and vitamin K2.

[0049] The term “kit of parts” herein refers to a combined preparation wherein the active ingredients are physically separated for use in a combined therapy by simultaneous administration or sequential administration to the patient.

[0050] The present invention particularly pertains to a kit of parts comprising a live Saccharomyces cere visiae strain and vitamin K2, for use in therapy, such as in the treatment and / or prevention of a bone-loss disorder in a patient in need thereof. According to the present invention, the live Saccharomyces cerevisiae strain and vitamin K2 are administered to the patient in a separate form, either simultaneously, separately or sequentially in any order to the patient for treating said boneloss disorder.

[0051] According to a specific embodiment, the form of the vitamin K2 used in the context of the present invention is MK-7 or MK-4. Vitamin K2 is formulated to be administered at a daily dosage comprised between 5 to 100 pg per day, preferably 10 to 50 pg per day, more preferably around 20 pg per day (i.e. between 18 and 22 pg per day). This amount may be administered in a single dose or in more than one dose which may be taken at different times throughout the day.

[0052] The skilled person knows how to formulate vitamin K2 for administration in a patient in the context of the present invention. Vitamin K2 may be administered orally and be formulated in a solid form such as a tablet, capsule or chewable tablet or may be in a liquid form such as a solution, suspension, dispersion or syrup. Soft-gel capsules, in which the active ingredients are dissolved or dispersed in a liquid non-aqueous solution, are particularly suitable for administering vitamin K in the context of the present invention. Soft gel capsules may be prepared by dissolving or suspending vitamin K2 and any excipients or other desirable formulation aids in an oily medium which is then encapsulated in the soft gel capsule. Alternatively, vitamin K2 may also be formulated for transdermal administration, e.g. in the form of a cream or gel, or of a patch which may be adhesively attached to the skin and contains a reservoir of the vitamin K2 optionally in combination with a penetration enhancer or other suitable excipients. Vitamin K2 can also be administered intravenously, and formulated as a solution suitable for i.v. administration. Finally, it is also possible to deliver or administer vitamin K2 in nutritional products such as a fortified food or beverage product. Preferrednutritional product formats include: juice drinks, dairy drinks, powdered drinks, sports drinks, mineral water, soy beverages, hot chocolate, malt drinks, biscuits, bread, crackers, confectioneries, chocolate, chewing-gum, margarines, spreads, yogurts, breakfast cereals, snack bars, meal replacements, protein powders, desserts, and medical nutrition tube feeds and nutritional supplements.

[0053] These dosage forms may be prepared by methods which are well-known to those skilled in the art.

[0054] Conventional additives, but are not limited, may be included in the kit of the invention, including any of those selected from preservatives, chelating agents, effervescing agents, natural or artificial sweeteners, flavoring agents, coloring agents, taste masking agents, acidulants, emulsifiers, thickening agents, suspending agents, dispersing or wetting agents, antioxidants, and the like.

[0055] The present invention will now be illustrated by means of the following examples.EXAMPLESExample 1 : Effects of a combination of a live Saccharomyces cerevisiae strain and vitamin K2 on bone formation markers

[0056] Introduction

[0057] Osteoporosis is a worldwide problem affecting more than 200 million people. In this invention, a combination of yeast and vitamin K2 was found to result in the production of beneficial bone health markers (i.e. metabolites measured during an in vitro human fermentation study). The yeast probiotics corresponding to the S. Cerevisiae strain deposited on October 17, 2007, at the Collection Nationale de Cultures de Micro-organismes (CNCM) pursuant to the treaty of Budapest under No. CNCM I-3856 in combination with Vitamin K2 menaquinone-7 (VitaMK-7® - Gnosis by Lesaffre, FRANCE) showed beneficial combined effects in an in vitro human batch fermentation model of 48 hours, in terms of metabolite profile. Specific markers known to promote bone health were significantly increased with the combo during colonic fermentation as compared to CNCM I-3856 or Vitamin K2 alone. To our surprise the combo maintains or improves the positive bone markers associated to probiotic intake while it reduces or erase the negative markers observed with CNCM I-3856 alone. Altogether the results provided herein show a strong impact of CNCM I-3856 on positive bone markers, and a biological synergy of the combo to promote bone health.

[0058] Materials and methods

[0059] The SHIME (Simulator of the Human Intestinal Microbioal Ecosystem, ProDigest) model was used in a batch fermentation set-up, where 200mL of sugar-depleted growth medium (1.2 g / LArabinogalactan, 2 g / L Pectin, 0.5 g / L xylan, 0.4 g / L glucose, 3 g / L yeast extract, 1 g / L special pepton, 3 g / L mucin and 0.5 g / L L-cystein-HCI) was supplemented with the combination of CNCM I-3856 (the strain strain deposited on October 17, 2007, at the Collection Nationale de Cultures de Micro-organismes (CNCM) pursuant to the treaty of Budapest under No. CNCM I-3856) (1g / intake)and Vitamin K2 MK7 Menaquinone (10mg / intake) and inoculated with a 10% fecal slurry of 2 individual donors (experiment repeated twice).

[0060] The reactors were individually and daily flushed with nitrogen to maintain anaerobic conditions. Samples were collected at TO, T24h and T48h and the short-chain fatty acid (SCFA), ammonium, lactate and branched-chain fatty acids (BCFA) were measured, as well as the microbial composition (16S metabarcoding) after 48h of fermentation. On a selection of samples, bone health biomarkers related to tryptophan metabolism were quantified using a targeted metabolomics approach.

[0061] Results

[0062] Results show an increase in the SCFAs, especially butyrate and propionate, when CNCM I-3856 was combined with Vitamin K2, compared to both products similarly dosed as single supplements (Figure 1). Propionate is known to trigger bone formation through osteocalcin gene expression modulation.

[0063] The metabolomics analysis conducted in those biological samples after human fermentation under additional Vitamin K2 and CNCM I-3856 showed that anti-inflammatory marker indole-3-ethanol was increased when the combo was supplemented compared to the Vitamin K2 and CNCM I-3856 alone (Figure 2). Besides, kynurenic acid, a biomarker known for bone formation and remodeling was also increased when comparing the combo with Vitamin K2 alone (Figure 3). On the other hand, the pro-osteoclastogenic biomarker indole-carboxaldehyde was decreased when the combo was supplemented compared to the supplementation of CNCM I-3856 alone, showing a corrective beneficial effect unexpected from the combo (Figure 4).

[0064] Conclusion

[0065] In conclusion this invention has shown that the use of CNCM I-3856 alone allows strongly promoting several markers involved in bone formation. The results further shown that supplementation of the combo Vitamin K2 and CNCM I-3856 synergistically interact so as to positively promote several markers involved in bone formation while mitigating expression of several markers of bone resorption (Figure 5). The supplementation of CNCM I-3856 alone, and more importantly of the combination CNCM I-3856 and Vitamin K2, can therefore result in a net positive role in bone health and prevention of osteoporosis.Example 2: Impact a live Saccharomyces cerevisiae strain on gut microbial activity and community composition in healthy and osteoporosis patients.

[0066] The aim of this project was to assess the impact of a live Saccharomyces cerevisiae strain on gut microbial activity and community composition in healthy individuals, and in individuals having been diagnosed with osteoporosis. The Colon-on-a-plate® technology was used to simulate colonic fermentation, and at the end of the simulation an assessment was made of treatment impact on production of saccharolytic (SCFA and lactate) and proteolytic markers (branched CFA and ammonium). In addition, LA-REIMS metabolic fingerprinting was performed because of the significantly higher resolution this technique offers as compared to mentioned metabolic markers alone (SCFA, BCFA, ammonium, and lactate). Changes in community composition were assessedwith shallow shotgun sequencing. Twelve donors were included per donor group, to account for interindividual differences.

[0067] Materials and Methods

[0068] Materials and Methods used are generally disclosed in Caron, Juliette, et al. "In vitro human gastrointestinal digestibility and colonic fermentation of edible yeast-based protein: A comparative study with whey and casein." Food Research International 208 (2025): 116098.

[0069] Preservation of fecal inocula

[0070] Stool samples of twelve healthy adult donors were collected and stored in an ultra-freezer (-80°C). Prior to cryopreservation, faecal suspensions were prepared under anaerobic conditions and mixed with an optimized cryoprotectant (ProDigest), i.e. a modified version of the cryoprotectant developed by Hoefman et al., 2013. Before addition, the cryoprotectant was sparged with nitrogen gas until anaerobiosis. The obtained suspensions were flash frozen and then preserved at 80 °C (cryostock) under anaerobic atmosphere for long-term storage. Just before the experiment, an aliquot was defrosted and immediately added to the colonic fermentation wells.

[0071] Short-term colonic simulation

[0072] A short-term screening assay typically consists of the incubation of a single dose of a test compound under conditions representative for the large intestine, using fecal inocula of selected donors as microbial sources.

[0073] At the start of the experiment, wells were filled with a background nutritional medium representative for the colon environment (ProDigest’s nutritional medium PD01 (fiber-depleted)). Prior to addition, the nutritional media were made anaerobic by boiling, to drive out oxygen. Then, test products were added to respective reactors to reach final concentrations of 5 mg / mL CNCM I-3856. Finally, 10% (v / v) of a fecal inoculum containing 7.5% (w / v) cryopreserved fecal material of beforementioned donors was added to respective reactors and served as microbial source. The total volume in each well was 7 mL. Incubation temperature was 37°C, under continuous mild shaking (90 rpm) and anaerobic atmosphere.

[0074] Endpoints of the study

[0075] An assessment was made of pH, and of SCFA-, lactate-, and ammonium-concentrations 48h after start of incubation. In addition, metabolic fingerprinting was performed (LA-REIMS), and samples were subjected to shallow shotgun sequencing to obtain detailed insights into microbial community composition and changes induced by treatment.

[0076] Overall fermentative activity

[0077] pH: the pH in the incubations is an indirect result of bacterial metabolism and can be used to predict the effect a treatment will have on intestinal pH. Indeed, pH is amongst others determined by the production of SCFA / BCFA / lactate / NH4+and can therefore quickly provide insight in whether treatment effects in terms of these endpoints are expected. Each measurement was done in single replicate.

[0078] Changes in microbial metabolite production

[0079] Short chain fatty acid analysis: The pattern of SCFA production is an assessment of the microbial carbohydrate metabolism (acetate, propionate, and butyrate) or protein metabolism (branched CFA) and can be compared to typical fermentation patterns for normal Gl microbiota. The method is based on a liquid-liquid extraction sample preparation; analysis is with Gas Chromatography (GC), and detection with a Flame Ionization Detector (FID). Each measurement was done in single replicate.

[0080] Lactate analysis: the human intestine harbors both lactate-producing and lactate-consuming bacteria. Lactate is produced by lactic acid bacteria and decreases the pH of the environment, thereby also acting as an antimicrobial agent. It can also be rapidly converted into propionate and butyrate by other microorganisms. Determination of lactate concentrations was performed using the Enzytec™ kit (R-Biopharm). Each measurement was done in single replicate.[00811 Ammonium analysis: Ammonium is a product of proteolytic degradation and is typically produced by urease-producing bacteria. Urease converts urea into ammonium / ammonia. Ammonia can be absorbed through the gut wall to be detoxified in liver and kidneys. Especially in persons suffering impaired ammonium detoxifying capabilities (for instance liver cirrhosis patients), ammonium production can be toxic. Determination of ammonium concentrations in the samples was done by colorimetric analysis, using the indophenol blue spectrophotometric (IPB) method. Each measurement was done in single replicate.

[0082] Changes in metabolic fingerprints (LA-REIMS)

[0083] Sample preparation

[0084] Samples were thawed at 4 °C and shortly vortexed (1 min, 400 rpm, 20 °C). Per sample, 100 pL was transferred into a well of a 96-well plate and subjected to LA-REIMS analysis.

[0085] LA-REIMS analysis

[0086] The LA-REIMS platform used a MID infrared laser system (Opolette™ HE2940, OPOTEK, LLC, USA) that consisted of a Q-switched Nd:YAG laser pumping Optical Parametric Oscillator (OPO). Transmission of the laser energy into the sample was achieved through free space optics, including a series of metallic-coated mirrors (OptoSigma Global Top, France) and a Plano-convex lens (Thorlabs, GmbH, Germany). Hereby, the process of laser ablation is being initiated, based on the laser-emitted infrared wavelength regime that excites the most intense vibrational band (oxygenhydrogen stretching mode) of the water molecules that are contained by the sample under investigation. This induces so-called matrix-assisted desorption and ionization of intact biomolecules. Subsequently, the aerosol that is produced, is transferred to the REIMS platform.

[0087] Mass analysis was carried out on a Xevo G2-XS Quadrupole Time-of-Flight (QToF) mass spectrometer (Waters Corporation, UK), being operated in negative ionization mode and applying an m / z scan range from 50 to 1200 Da. For sampling ( / '.e., performing the process of laser ablation foreach individual sample), an automated versatile 96-multi-well plate-based platform was incorporated in the standard LA-REIMS workflow (Plekhova, V. et al. (2021). Nature Protocols 16:4327-4354).

[0088] When performing LA-REIMS analysis, the process of laser ablation takes place for 3 s during which metabolites are released from the sample and measured by REIMS. This process of laser ablation is associated with a burn signal, with underlying this burn a rich mass spectrum that includes m / z features from the sample as well as general background noise. This noise can also be observed during the preceding and subsequent cool down periods (total of 20.6 s).

[0089] Quality assurance

[0090] The REIMS instrument was calibrated prior to analysis, in sensitivity mode, according to the manufacturer’s standard instructions (Waters Corporation, UK).

[0091] LA-REIMS data processing

[0092] Data were analysed using MassLynx® Progenesis® Bridge V4.1 and Progenesis® QI V2.3 (Waters Corporation, UK). ProGenesis Bridge and Progenesis QI (Water Corporation). Detailed information on the LA- REIMS platform was previously described by Plekhova et al., 2021 .

[0093] Changes in microbial community composition

[0094] Shallow shotgun sequencing

[0095] DNA libraries were prepared using the xGen DNA Library Prep Kit (IDT) and xGen Normalase UDI Primers with total DNA input of 1.5 ng. Genomic DNA was fragmented using a proportional amount of IDT xGEN fragmentation enzyme. Unique dual indexes were added to each sample followed by 10 cycles of PCR to construct libraries. DNA libraries were purified using AMpure magnetic Beads (Beckman Coulter) and eluted in QIAGEN EB buffer. DNA libraries were quantified using Qubit fluorometer and Qubit™ dsDNA HS Assay Kit. Libraries were then sequenced on an Illumina NovaSeq X Plus platform at 2x150 bp.

[0096] Unassembled sequencing reads were directly analyzed as described elsewhere (Ottensen et al., 2016, Ponnusamy et al., 2016, Hasan et al., 2014, Lax et al., 2014) for multi-kingdom microbiome analysis and quantification of organisms' relative abundances. Briefly, the system utilizes curated genome databases and a high-performance data-mining algorithm that rapidly disambiguates hundreds of millions of metagenomic sequence reads into the discrete microorganisms engendering the particular sequences.

[0097] Quantification of total bacterial cells by flow cytometry

[0098] Samples that were analyzed with shallow shotgun sequencing to map shifts in community composition were also analyzed with flow cytometry (FC), to determine the number of total bacterial cells per sample, thus allowing to assess treatment impact on bacterial biomass.

[0099] Samples were analyzed on a BD Accuri C6 Plus Flow Cytometer. The samples were run using the high flow rate. Bacterial cells were separated from medium debris and signal noise byapplying a threshold level of 700 on the SYTO channel. Proper parent and daughter gates were set to determine all populations.

[0100] Statistics

[0101] Fermentative activity and metabolite production

[0102] Paired two-sided T-tests were performed to evaluate whether treatment effects in terms of the investigated metabolites were statistically significant across the various donors of a given donor group (n=12), using per-donor measurements as replicate values (resulting in twelve replicate measurements, i.e. one per donor). By applying this approach, an effect is considered significant only if it is observed across multiple donors of the given population, thus accounting for interindividual differences. The test product was compared with the negative control. In addition, treatment impact in the healthy group was compared with treatment impact in the osteoporosis group, to assess whether the response to treatment (in terms of metabolite production) was different in either group. To do so, treatment / control ratios (fold changes) were calculated for each treatment, for each donor. Then unpaired two-sided T-tests were applied on these fold changes (osteoporosis group was compared with healthy group for a given treatment), to determine whether the treatment effect was different in the osteoporosis group from the healthy group. The p-value was determined using the abovementioned statistical tests. The cut-off for statistical significance was set at p < 0.05.

[0103] Then, redundancy analysis (RDA) was performed to assess how much of the variation in one set of variables (metabolites) is explained by the variation in another set of variables (treatment).

[0104] Metabolic fingerprints

[0105] Normalized LA-REIMS data were subjected to multivariate statistical analysis using SIMCA 17 (Sartorius, Germany). Here, data were pre-processed, thereby performing log-transformation to induce normal distributions and unit variance scaling (1 / SD, with SD being the standard deviation) to standardize the range of signal intensities. Unsupervised Principal Component Analysis (PCA-X) was executed to assess the natural patterning of samples and reveal potential outliers (based on the Hoteling’s T2criterion). Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) was used to differentiate samples according to experimental conditions in a supervised fashion. Validity of the OPLS-DA models was verified by permutation testing (n = 100), cross-validated analysis of variance (p-value < 0.05), and the quality parameter Q2(Y) (> 0.5). In general, the model performance is described by R2(X) (the predictive and orthogonal variation in X-values, i.e. m / z features), R2(Y) (the ability to predict the Y-data for the specifically used dataset, i.e. predicting the sample classification), and Q2(Y) (the ability to correctly predict the Y-data when an external dataset would be considered) (MKS Umetrics (2012). “User Guide to SIMCA”).

[0106] Microbial community composition

[0107] Biomass

[0108] The approach was identical to the approach followed to evaluate changes in metabolite production. In short, paired two-sided T-tests were performed for comparisons within the same donorgroup (healthy group and osteoporosis group), considering each donor a replicate measurement, thus resulting in 12 replicate measurements for each condition (12 donors). Unpaired two-sided T-tests were performed for the comparisons between donor groups. An effect was considered significant if the p-value was below 0.05.

[0109] Alpha diversity

[0110] Alpha diversity is used to express bacterial diversity in a sample in terms of species richness and / or evenness. Four alpha-diversity measures were calculated: (1) ‘Observed taxa’ (measure for species richness), (2) ‘Chaol ’ (measure for species richness), (3) ‘Shannon’, (measure for species richness and evenness), and (4) ‘Simpson’ (measure for species richness and evenness, giving more weight to common or dominant species (rare species with only a few representatives will not impact diversity)). Paired two-sided T-tests were performed to examine whether, in a given donor group, treatment effects in terms of species richness and evenness were statistically significant across the various donors (n=12), using per-donor measurements as replicate values (resulting in 12 replicate measurements, i.e., one per donor). The cut-off for statistical significance was set at p < 0.05.

[0111] Beta diversity

[0112] Whether or not treatment affects overall community composition was assessed using Discriminant Analysis of Principal Components (DAPC) and Hierarchical clustering. DAPC joins two analysis methods to assess effects on population structure. In this approach, sequence data are transformed using principal component analysis (PCA), and subsequently clusters are identified with discriminant analysis (DA - Miller, J.M., Cullingham, C.l. & Peery, R.M. Heredity 125, 269-280(2020)). https: / / doi.org / 10.1038 / s41437-020-0348-2. The DA aims to maximize among-group variation and minimize within-group variation. In this approach, the groups (treatments) used in the DA were a priori defined. Hierarchical clustering expresses dissimilarities in community composition between the various conditions in the dendrogram, where the sum of the horizontal lines separating two conditions is a measure for dissimilarity in terms of community composition between respective conditions.

[0113] Differential abundance analysis

[0114] As for metabolites, conditions were compared by using biological replicates as input values (resulting in 12 replicate measurements per condition, i.e., one per donor), thus enabling to assess whether an effect is consistent across the donors. Differential abundance analysis was performed using statistical methods LEfSe and treeclimbR.

[0115] LEfSe analysis (Segata N, Izard J, Waldron L, et al. Metagenomic biomarker discovery and explanation. Genome Biol. 2011 ;12(6):R60. Published 2011 Jun 24. doi:10.1186 / gb-2011 -12-6-r60) was performed on relative abundance data (obtained by total sum scaling), to identify the bacterial taxa with significantly different abundances between conditions. By measuring the extent and statistical significance of differences in bacterial abundances between two conditions, LEfSe enables to identify treatment-induced community shifts. In order to do so, the algorithm couples statistical significance with biological consistency and effect size estimation, and thus provides in-depth insightin the biological relevance and magnitude of bacterial enrichments. All features shown in the LEfSe plots meet p <0.05 for Kruskal-Wallis and Wilcoxon tests. No restrictions were put forward with respect to minimal LDA scores, but in general, LDA scores > 2.0 are considered biologically relevant. LDA scores express the extent of differences in taxon abundances between conditions. The higher the LDA score, the higher the difference in abundance between the two biological conditions. The 20 features with highest LDA scores, meeting the threshold for statistical significance, were plotted in the LEfSe plots.

[0116] treeclimbR analysis (Huang, R., Soneson, C., Germain, PL. et al. Genome Biol 22, 157(2021) doi :10.1186 / s13059-021 -02368-1) was performed on relative abundance data (obtained by total sum scaling) to identify the differentially abundant taxa between two conditions. Bacterial enrichments exceeding a fold change of 4 (Iog24= 2 in chart) as compared to a reference condition are considered biologically relevant by consensus; the cut-off for statistical significance is set at a p-value of 0.05.

[0117] Redundancy analysis (RDA)

[0118] Redundancy analyses (RDA) was performed to complement differential abundance analysis. RDA correlates metagenomic shifts to treatments; it is a multivariate statistical tool which explores the relationship between microbial community composition and experimental variables (treatments), without providing info on statistical significance. Prior to analysis, data were transformed to enable paired analysis. This was done by calculating differences in relative abundance between treatments and blanks for each taxon and donor. These values were used as input values for the statistical test.

[0119] Results

[0120] pH

[0121] Monitoring the pH during a colonic simulation provides a good indication of the production of SCFA, lactate and ammonium (NH4+). In general, a pH drop is observed initially due to the production of SCFA / lactate. This pH drop is often followed by a pH increase due to proteolytic fermentation, which results in the production of amongst others NH4+, and due to conversion of strong acids into weaker acids through cross-feeding (for instance acetate / lactate-to-propionate / butyrate conversion).

[0122] In both donor populations, CNCM I-3856 treatment was characterized by a significantly lower pH as compared to the untreated control 48h after start of incubation, indicating that production of acid metabolites SCFA and / or lactate was stimulated by treatment, thereby confirming the results shown in Example 1 .

[0123] Short-chain fatty acids (SCFA)

[0124] SFCA production results from carbohydrate metabolism in the colon and is related with various health effects. The dominant SCFAs are acetate, propionate, and butyrate. Acetate can be used as an energy source for the host and as a potential substrate for lipid synthesis in the body. Propionate reduces cholesterol and fatty acid synthesis in the liver (beneficial effect on metabolic homeostasis), and is a dietary factor that counters obesity by inducing a feeling of satiety. Butyrate is a major energy source for colonocytes and induces differentiation in these cells (related to cancerprevention), and it plays a key role in immune regulation. Positive effects of a product on SCFA production therefore include increases of acetate, propionate and / or butyrate.

[0125] Acetate can be produced by many different gut microbes (including amongst others Bifidobacterium spp. , Bacteroides spp. and Lactobacillus spp.) and is a primary metabolite generated from substrate fermentation. Acetate production was strongly stimulated by CNCM I-3856 treatment in the healthy (+40%) and osteoporosis (+34%) groups. In both donor groups, significance was reached across donors (p<0.001 ), indicating that the effect was highly consistent across donors. The impact on acetate production was comparable in the healthy population and the osteoporosis population.

[0126] Propionate can be produced by different gut microbes, with the most abundant propionate producers being Bacteroides spp., Akkermansia muciniphila and Veillonellaceae. Propionate production was doubled by CNCM I-3856 treatment in the healthy group (+100%) and in the osteoporosis group (+96%). In both donor groups, significance was reached across donors (p<0.001 ), indicating that the effect was highly consistent across donors. The impact on propionate production was comparable in the healthy population and the osteoporosis population.

[0127] Butyrate is mostly produced by members of the Lachnospiraceae and Ruminococcaceae families. In a process called cross-feeding, these microbes convert acetate and / or lactate (along with other substrates) to the health-related butyrate. Butyrate production significantly increased by CNCM I-3856 treatment in the healthy group (+89%) and in the osteoporosis group (+64%). In both donor groups, significance was reached across donors (p<0.001), indicating that the effect was highly consistent across donors. The impact on butyrate production was comparable in the healthy population and the osteoporosis population.

[0128] Lactate production

[0129] Lactate is produced by lactic acid bacteria and decreases the pH of the environment. In doing so, it can inhibit pathogenic growth, as pathogens typically favour neutral pH milieus. Another beneficial effect of lactate results from its conversion into butyrate and / or propionate through crossfeeding by specialized micro-organisms. These cross-feeding interactions imply that lactate is not only produced, but also consumed by the intestinal microbiota.

[0130] Residual lactate was detected. The outcome was comparable to the SCFA, in the sense that residual lactate levels were significantly higher than control in CNCM I-3856 treatment, both in the healthy and osteoporosis groups. In each donor group, significance was reached across donors (p<0.001), indicating that the effect was highly consistent across donors. The impact on lactate production was comparable in the healthy population and the osteoporosis population.

[0131] Marker for protein metabolism: branched CFA and ammonium

[0132] Less abundant fatty acids include branched CFA (isobutyrate, isovalerate and isocaproate). Production of BCFA and ammonium results from proteolytic microbial activity, which is associated with production of toxic by-products such as p-cresol. Therefore, high BCFA and ammoniumproduction in the colon has been associated with detrimental health effects. As a result, products that reduce BCFA and ammonium production are considered beneficial for health.

[0133] CNCM I-3856 treatment tended to moderately increase the production of BCFA (+11% in healthy group and +26% in osteoporosis group). In the osteoporosis group, CNCM I-3856 treatment stimulated the production of BCFA (p<0.001). The impact of CNCM I-3856 treatment on BCFA production was different in the healthy group from the osteoporosis group, with the stimulatory effect on BCFA production being more pronounced in the osteoporosis population.

[0134] Ammonium production was stimulated by CNCM I-3856 treatment in the healthy (+26%) and osteoporosis (+25%) groups. In both donor groups, significance was reached across donors (p<0.001), indicating that the effect was highly consistent across donors. The impact on ammonium production was comparable in the healthy population and the osteoporosis population.

[0135] Redundancy analysis

[0136] Redundancy analysis (RDA) showed highly similar fermentation profiles in the osteoporosis and healthy groups, suggesting that production of investigated metabolites (SCFA, BCFA, ammonium and lactate) was similar in both target groups, regardless of treatment. Interindividual variation was somewhat more pronounced in the osteoporosis group.

[0137] Metabolic shifts at 48h were mostly defined by acetate, propionate, butyrate, lactate, and ammonium. CNCM I-3856 treatment was strongly correlated with production of acetate, propionate, butyrate, lactate, and ammonium in both donor groups.

[0138] LA-REIMS metabolic fingerprinting

[0139] Upon analysis of the biological samples and associated iQC samples, metabolic fingerprints were composed by using Progenesis® QI software. More specifically, this software lists the m / z-values of all signals ( / '.e., metabolic features) that are being detected upon LA-REIMS analysis, together with the signal intensities. It should be noted that data acquisition was performed in negative ionization mode only, typically associated with lower levels of analytical noise compared to the positive ionization mode (Cameron, S.J.S. (2019). Analytical Chemistry 91 : 13448-13457). In total, 1999 unique m / z-features were defined as constituents of the metabolic fingerprints. This number is in line what is typically observed for this type of matrix ( / '.e., 1500 to 2000 metabolic features).

[0140] Multivariate statistics

[0141] General data exploration

[0142] In first instance, PCA-X modelling was performed to assess the natural patterning of samples and define the analytical performance of the LA-REIMS analysis. Data were scaled (unit variance; ^SD, with SD being the standard deviation) to standardize the range of signal intensities, and log-transformed to induce normal data distributions. A PCA-X model was constructed based on the biological samples, as well as the iQC samples and was composed out of 18 principal components, whereby the first two principal components explained 32.60% of the present X-variance ( / '.e., across the m / z-features). Based on the clustering of the iQC-samples, acceptable instrumental stability wasconcluded. This was also confirmed by calculating the percentage of features {i.e., 76.5%) that had a coefficient of variance below 30% across the entire set of iQC-samples. This percentage is in line with other studies.

[0143] Assessment of CNCM I-3856 effects compared to the blank

[0144] Next, an PCA-X score plot was constructed based on the biological samples only, thereby excluding the iQC-samples. The results suggested that CNCM I-3856 was able to induce a metabolic shift compared to the blank.

[0145] Additional PCA-X plots were constructed to evaluate the treatment effect in relation to the health state of the donors {i.e., healthy or diagnosed with osteoporosis). It was observed that for both populations, a metabolic shift occurred upon the CNCM i-3856 treatment, confirming the previous findings.

[0146] Based on these observations, several statistical comparisons were performed for in-depth assessment and significance determination of the treatment effect. This approach indicated that the effects for the CNCM l-3856-based treatment towards the blank were significant, and present in both the healthy and osteoporosis population.

[0147] Impact of health status on the treatment effects

[0148] Having obtained insights about the impact of the assessed treatment in the various donor populations, the following section aimed at assessing differences in these impacts between the two populations. In first instance, based on the PCA-X plot and corresponding OPLS-DA model, it was defined that under conditions of no treatment, the metabolic fingerprints were not significantly different between the healthy and osteoporosis-diagnosed donor group. Indeed, no (valid) OPLS-DA model could be constructed. Based on the PCA-X plots, no distinct segregation was observed for the healthy versus osteoporosis-diagnosed population under treatment. This was confirmed by the OPLS-DA modelling, where no (valid) OPLS-DA models could be generated.

[0149] Donor effect

[0150] An extra PCA-X plot was generated to define any metabolic differences in the treatment effect according to the donor, across both donor groups. Overall, four clusters were observed when considering all samples of the healthy and osteoporosis group, indicating different metabolic fingerprints across the donors. Even with a relatively high level of variability within the donor population, the treatment effect as seen for the CNCM i-3856 treatment were manifested in a similar way across all these donors, indicating robustness for this treatment.

[0151] Microbial community composition

[0152] Microbial community composition was assessed using shallow shotgun sequencing. With this technique, 3M reads are generated from the entire microbiome in a sample, providing resolution at the species-to-strain level. It allows to evaluate how a specific treatment modifies microbial community composition, and as such to explain changes in metabolite production.

[0153] Within this study, two techniques were combined according to Vandeputte et al. (2017) to map the community shifts induced by the different treatments in large detail:

[0154] Shallow shotgun sequencing, providing relative abundances of different taxa at different phylogenetic levels (microbial phylum, family, genus, and species level).

[0155] Accurate quantification of bacterial cells in the samples through flow cytometry.

[0156] Community composition 48h post-treatment

[0157] An assessment was made of treatment impact on luminal microbial community composition, by analysing samples collected 48h after start of incubation. To do so, effects on biomass and on alpha- and beta-diversity were examined, and differential abundance (LEfSe and treeclimbR) and redundancy analyses were performed to identify treatment-induced bacterial shifts.

[0158] Alpha-diversity was calculated to examine the CNCM I-3856 treatment on bacterial diversity within a sample (species richness and evenness), using the various indices indicated in Statistics. Beta-diversity, or differences in community composition between conditions, was assessed and presented in a hierarchical clustering dendrogram and using DAPC. LEfSe and treeclimbR analysis were used to identify which bacterial groups were significantly altered by the treatment across the donors (per donor group). Both analysis tools are used in parallel, as they make use of different statistical methods, and as such complement each other. By looking at consistencies across donors (in this analysis, each donor is considered a ‘replicate’ measurement), the effect becomes independent of interindividual differences.

[0159] Effects on bacterial biomass

[0160] CNCM I-3856 treatment resulted in significantly higher biomass levels compared to the untreated control, both in the healthy (+81% or +3.17E+09 cells / mL) and osteoporosis (+94% or +3.73E+09 cells / mL) groups. In both donor groups, significance was reached across donors (p<0.001), indicating that the effect was highly consistent across donors. The impact on biomass production was comparable in the healthy population and the osteoporosis population.

[0161] Effects in alpha-diversity

[0162] Species richness, the measure for the number of different bacterial taxa in a sample, was significantly reduced by CNCM I-3856 treatment in the healthy population. In the osteoporosis population, CNCM I-3856 treatment lowered species richness. The reduction of species richness associated with CNCM I-3856 treatment could be because the yeast outcompeted specific community members in competition for nutrients.

[0163] Species evenness, the measure for the distribution of bacterial groups, was overall unaffected by treatment in both donor populations. This implies that CNCM I-3856 treatment did not disrupt the microbial balance that was established in these bacterial communities.

[0164] Finally, no significant differences in terms of alpha-diversity were found between the healthy and osteoporosis populations, demonstrating that the treatment’s impact on alpha-diversity was comparable in both populations.

[0165] Effects on beta-diversity

[0166] Assessments of beta-diversity offer a comprehensive perspective on the influence of treatments on microbial community composition and the interrelations between conditions. Such evaluations yield insights into the impact of treatments and the variations in their effects across populations. The results show a distinct separation of conditions representing the healthy population and conditions representing the osteoporosis population, implying a distinct microbial community composition for both donor groups. However, both populations responded in a similar fashion to the treatment. Indeed, a comparable clustering pattern of treatment was observed for both populations: the separation of CNCM I-3856 treatment from the untreated control indicates a measurable impact of this treatment on the gut microbial community of each donor group.

[0167] Differential abundance analysis

[0168] LEfSe and treeclimbR are unpaired statistical analysis tools, used to detect which gut bacteria are differentially abundant in treated conditions as compared to a reference. Both analysis tools are used in parallel, as they make use of different statistical methods, and as such complement each other. By looking at consistencies across donors (in this analysis, each donor is considered a replicate measurement), the effect becomes independent of interindividual variation. In this analysis, two questions were answered:

[0169] How does the healthy population differ from the osteoporosis population in terms of community composition, 48h after experimental start?

[0170] How do both communities respond to treatment?

[0171] Difference in community composition between the healthy and osteoporosis population

[0172] Community composition in the untreated controls (blank) of the healthy and osteoporosis groups were compared to identify population-specific differences. LEfSe and treeclimbR identified 20 taxa that were differentially abundant in both populations, with 8 taxa being more abundant in the healthy population and 12 taxa being more abundant in the osteoporosis population.

[0173] Treatment-induced bacterial enrichments in the healthy and osteoporosis populations

[0174] Using the untreated control as a reference, differential abundance analysis detected a total of 11 statistically enhanced taxa in the CNCM I-3856 treated condition in the healthy and osteoporosis populations combined. Interestingly, the identity of the enriched taxa was strongly dependent on the donor group, resulting in 4 healthy-specific enrichments and 6 osteoporosisspecific enrichments.

[0175] In the healthy population, bacterial enrichments involved Dialister pneumosintes, Eubacterium ventriosum, Peptostreptococcus anaerobius, an unclassified Anaeromassilibacillus, and Roseburia hominis.

[0176] In the osteoporosis population, CNCM I-3856 treatment significantly stimulated seven bacterial taxa, among which four Bacteroides species. CNCM l-3856-enriched bacterial taxa involvedBacteroides ovatus, Bacteroides rodentium, Bacteroides thetaiotaomicron, an unclassified Bacteroides, an unclassified Ruminococcus, and members of the Streptococcus genus.

[0177] Redundancy analysis

[0178] Community-based redundancy analysis (RDA) gives insight into associations between treatment and microbial community shifts, in addition to the differential abundance analysis. By default, treeclimbR and LEfSe use unpaired statistical tests, which, considering the paired nature of this experimental setup, implies a loss of resolution. For redundancy analysis, data were transformed to enable paired analysis. Metagenomic RDA analysis was performed using the twelve donors per donor group as biological replicates.

[0179] RDA found that CNCM I-3856 treatment was positively correlated with Bacteroides thetaiotaomicron in both the healthy and osteoporosis populations.

[0180] Conclusion

[0181] CNCM I-3856 had strong stimulatory effects on production of acetate (+40% in healthy and +34% in osteoporosis group), propionate (+100% in healthy and +96% in osteoporosis group), butyrate (+89% in healthy and +64% in osteoporosis group), and lactate. The effect was highly consistent across donors in both donor groups, and therefore these results are expected to be representative for a broad population. Considering that propionate and butyrate are superior in terms of impact on gut health, it can be concluded that CNCM I-3856 is an effective probiotic to promote gut-health in healthy persons and persons diagnosed with osteoporosis. The response to treatment was similar for both donor groups, except for BCFA, for which the stimulatory effect was more pronounced in the osteoporosis group. Ammonium (+26% in healthy and +25% in osteoporosis group) and BCFA (+11% in healthy and +26% in osteoporosis group) were increased by CNCM I-3856 treatment, indicative of elevated proteolytic fermentation. This can be due to a protein fraction in the product (eventually derived from the yeast), or could be inherent to the selected simulation model. CNCM I-3856 may have boosted saccharolytic fermentation, which led to faster depletion of carbohydrates in the nutritional medium as compared to the untreated control (the nutritional medium is not refreshed during the incubation), which finally resulted in an earlier onset of proteolytic fermentation.

[0182] CNCM I-3856 treatment reduced species richness, enhanced bacterial biomass production (+81% in healthy and +94% in osteoporosis group), and resulted in the enrichment of important SCFA-producing species such as Roseburia hominis and Bacteroides thetaiotaomicron in both donor groups. Several bacterial enrichments were specific for the healthy group, including Anaeromassibacillus_u_s, Dialister pneumosintes, Eubacterium ventriosum, and Peptostreptococcus anaerobium. Other enrichments were specific for the osteoporosis group, including Bacteroides ovatus, Bacteroides rodentium, Bacteroides_u_s, Ruminococcus_u_s and members of the Streptococcus genus. The reduction in species richness may have been due to nutrient competition between CNCM I-3856 yeast and the gut microbiota. Importantly, species evenness - indicative of the balance of the community - was unaffected by CNCM I-3856 treatment,indicating that CNCM 1-3856 did not negatively impact the established gut balance in the healthy and osteoporosis populations.

[0183] These findings indicate that CNCM I-3856 treatment demonstrates a promising health-beneficial impact on the gut microbiota of healthy individuals, and individuals suffering from osteoporosis, through the enrichment of health-promoting SCFA, as induced by the enrichment of several health-promoting microbes.

[0184] Based on the generated LA-REIMS metabolic fingerprints, major impact was associated with the CNCM I-3856 treatment, and this in both the healthy and osteoporosis population.PCT(Original in Electronic Form)(This sheet is not part of and does not count as a sheet of the international application) 0-1 Form PCT / RO / 134Indications Relating to DepositedMicroorganism(s) or Other BiologicalMaterial (PCT Rule 13bis)0-1-1 Prepared Using ePCT-Filing-EmbeddedVersion 4.15.022 MT / FOP 20251014 / 2.80-2 International Application No.0-3 Applicant's or agent's file reference B2400225EPWO1 The indications made below relate tothe deposited microorganism(s) orother biological material referred to inthe description on:1-1 Paragraph number 121-3 Identification of deposit1-3-1 Name of depositary institution CNCM Collection nationale de cultures de micro- organismes1-3-2 Address of depositary institution Collection nationale de cultures de micro- organismes (CNCM)Institut Pasteur, 25-28rue du Docteur Roux ,75724 Paris Cedex 15France1-3-3 Date of deposit 17 October 2007 (17.10.2007)1-3-4 Accession Number CNCM 1-38561-4 Additional Indications1-5 Designated States for Which All designationsIndications are Made1-6 Separate Furnishing of IndicationsThese indications will be submitted to theInternational Bureau laterFOR RECEIVING OFFICE USE ONLY0-4 This form was received with theinternational application: yes(yes or no)0-4-1 Authorized officer Benzler, AnnemarieFOR INTERNATIONAL BUREAU USE ONLY0-5 This form was received by theinternational Bureau on:0-5-1 Authorized officer

Claims

Claims

1. A live Saccharomyces cerevisiae strain for use in the treatment and / or prevention of a bone-loss disorder in a patient in need thereof.

2. The live Saccharomyces cerevisiae strain for use according to claim 1, wherein said Saccharomyces cere visiae strain is not administered in combination with a microorganism that does not belong to the Saccharomyces cerevisiae species.

3. A kit of parts comprising:- a live Saccharomyces cerevisiae strain; and- vitamin K2.

4. The kit of parts according to claim 3, for use in therapy.

5. The kit of parts according to claim 3, for use in the treatment and / or prevention of a boneloss disorder in a patient in need thereof.

6. The live Saccharomyces cerevisiae strain for use according to claim 1 or 2, or the kit of parts according to any one of claims 3 to 5, wherein said live Saccharomyces cerevisiae strain comprises the strain deposited on October 17, 2007, at the Collection Nationale de Cultures de Micro-organismes (CNCM) pursuant to the treaty of Budapest under No. CNCM I-3856.

7. The live Saccharomyces cerevisiae strain for use according to claim 1 or 2, or the kit of parts for use according to any one of claims 4 to 6, wherein said strain is administered to the patient at a daily dosage of between 107and 6x1010CFU (Colony Forming Unit) of Saccharomyces cerevisiae, preferably between 108and 2x1010CFU.

8. The live Saccharomyces cerevisiae strain for use according to claim 1 or 2, or the kit of parts for use according to any one of claims 4 to 7, wherein said bone-loss disorder is selected from the group consisting of osteoporosis, osteopenia, osteoarthritis, Paget’s disease, osteomyelitis, osteomalacia, degenerative joint disease, musculoskeletal pathologies, osteophytes, rickets and delayed or non-union fractures.

9. The live Saccharomyces cerevisiae strain for use according to claim 1 or 2, or the kit of parts for use according to any one of claims 4 to 8, wherein said bone-loss disorder is selected from the group consisting of osteoporosis and osteopenia.

10. The live Saccharomyces cerevisiae strain for use according to claim 1 or 2, or the kit of parts for use according to any one of claims 4 to 9, wherein said bone disorder is osteoporosis.

11. The kit of parts for use according to any one of claims 4 to 10, wherein vitamin K2 is administered at a daily dosage comprised between 5 to 100pg per day, preferably 10 to 50 pg per day.

12. The kit of parts for use according to any one of claims 4 to 11 , wherein said vitamin K2 is MK-7 and / or MK-4.

13. The kit of parts for use according to any one of claims 4 to 12, wherein the live Saccharomyces cerevisiae strain and vitamin K2 are administered simultaneously.

14. The kit of parts for use according to any one of claims 4 to 12, wherein the live Saccharomyces cerevisiae strain and vitamin K2 are administered subsequentially.

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