Beneficial effect of a probiotic bacterium Bacillus subtilis in the fight against infections

Abstract

The present invention relates to a strain of Bacillus subtilis, in particular the strain of Bacillus subtilis CU1 (CNCM I-2745), or cells obtained by culturing said strain, or a composition comprising said strain or said cells, said composition advantageously being a functional food or food supplement, for its use in preventing and / or treating an infection via modulation of peripheral phagocytic activity in a subject, in particular in a young subject.

Description

Title of the invention: Beneficial effect of a probiotic bacterium Bacillus subtilis in the fight against infections FIELD OF THE INVENTION

[0001] The present invention relates to the field of immunity, in particular the prevention of infections and / or the improvement of the response to infections, in particular via modulation of phagocytic activity, including in young people, by a specific probiotic strain of Bacillus subtilis, administered in the form of a functional food or food supplement. STATE OF THE PRIOR ART

[0002] An infection is an invasion of the body or a site of the body by a pathogenic agent which can be a bacterium, a virus, a fungus or a parasite. These microorganisms can cause disease by disrupting normal body functions.

[0003] The immune response provides defense against infections and differs depending on the pathogen involved.

[0004] The innate immune response is rapid and non-specific. It is activated as soon as the body detects a foreign presence, but it is not adapted to a particular pathogen. Innate immune cells such as macrophages, natural killer (NK) cells and neutrophils are responsible for this immune reaction. In contrast, the acquired immune response, which takes longer, is more specific and adapted to the particular pathogen encountered by the body. It is mainly orchestrated by adaptive immune cells, including T and B lymphocytes.

[0005] Intrinsic factors such as aging, fatigue, chronic illness or treatment of certain diseases, and / or extrinsic factors such as temperature or humidity level, can weaken the immune system, thus increasing the risk of infection.

[0006] It is recommended, particularly for the elderly or for subjects undergoing post-antibiotic treatment, to take food supplements, particularly based on probiotics, to reduce the risk of contracting common infectious diseases (CIDs) caused in particular by respiratory or gastrointestinal viruses.

[0007] Probiotics are defined as "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host." Lactobacillus and Bifidobacterium are the most common bacterial probiotics. more commonly used. Fermented milk or dairy products containing Lactobacillus have been shown to affect the duration or frequency of respiratory and gastrointestinal infections and reduce the risk of colds in healthy older adults. Some trials have shown that Lactobacillus and Bifidobacterium probiotics may increase immune responses to influenza vaccination in older adults.

[0008] Spore-forming bacteria such as Bacillus species are of interest because their spores (or endospores) are resistant to the stomach acid barrier and are stable for long periods in commercial food products. Bacteria of the genus Bacillus, considered commensal microorganisms of the intestine, have been used as probiotics for the prophylaxis of human gastrointestinal disorders, to prevent recurrent respiratory infections or as an adjuvant to the use of antibiotics.

[0009] Probiotics have been suggested to protect against infectious diseases by several strain-dependent mechanisms, including secretion of anti-pathogenic substances, competitive exclusion of pathogens, maintenance of mucosal integrity, and stimulation of systemic or mucosal immune responses. Bacz7 / u5 species have been shown to produce antimicrobial substances, improve epithelial intestinal barrier functions, and stimulate the production of cytokines and secretory immunoglobulin A (or SIgA) in humans.

[0010] Thus, different strains of Bacillus, notably Bacillus subtilis (B. subtilis), are currently marketed for use as probiotics in humans and animals to improve their health.

[0011] In particular, the strain of B. subtilisCUl (or BSCU1), deposited at the CNCM (National Collection of Cultures of Microorganisms, Pasteur Institute, 28 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-2745 dated October 25, 2001, is known for its use as a beneficial probiotic.

[0012] Document WO2014 / 091160 describes the use of this strain for the prevention and / or treatment of diarrhea via its ability to act on water absorption in the colon and to limit the anchoring sites of pathogenic bacterial elements on the intestinal barrier.

[0013] Lefevre et al. (Immunity & Ageing, 2015, 12:24) showed that consumption of this strain (BSCU1), in a cohort of elderly people, significantly reduced the average number of days with symptoms of infection during the consumption period, and that participants had increased levels of SIgA in their feces and saliva after taking the probiotic, demonstrating a local action on the mucous membranes.

[0014] It should be noted, however, that it is apparent from the state of the art that certain oral compositions based on probiotics do not have a systemic effect given the intestinal mucosa and its barrier function which ensures confinement of these compositions in the intestinal lumen / an absence of absorption and translocation of the luminal contents towards the circulatory system. This absence of systemic effect of compositions of the state of the art is probably due to the choice of strains of bacteria and / or concentrations administered.

[0015] Furthermore, studies have shown differences in the effectiveness of these probiotics depending on the age of the subject, i.e. depending on their physiological / cellular maturity. In other words, certain strains of probiotic bacteria show a differential effect depending on the age of the subject.

[0016] In any event, there is a clear need to find solutions to prevent and / or combat infections.

[0017] DESCRIPTION OF THE INVENTION

[0018] Definitions

[0019] The definitions below correspond to the meaning generally used in the context of the invention and are to be taken into account, unless another definition is explicitly indicated.

[0020] For the purposes of the invention, the articles "a" and "an" are used to refer to one or more (e.g., at least one) units of the grammatical object of the article. For example, "an element" designates at least one element, i.e., one or more elements.

[0021] The terms "about" or "approximately", used in reference to a measurable value such as a quantity, a time, and the like, should be understood to encompass measurement uncertainties of ± 20% or ± 10%, preferably ± 5%, even more preferably ± 1%, and particularly preferably ± 0.1% of the specified value.

[0022] Ranges: Throughout this description, the various features of the invention may be presented as a range of values. It should be understood that the description of values as a range is for the sole purpose of making reading easier and should not be interpreted as a rigid limitation of the scope of the invention. Accordingly, the description of a range of values should be considered as specifically disclosing all possible intermediate ranges as well as each of the values within that range. For example, the description of a range from 1 to 6 should be considered as specifically describing each of the ranges it comprises, such as the ranges from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as each of the values in this interval, for example, 1; 2; 2.7; 3; 4; 5; 5.3 and 6. This definition holds regardless of the range of the interval.

[0023] The term "isolated" should be understood in the context of the invention as synonymous with "removed" or "extracted from its natural environment or state." For example, an isolated strain of bacteria or peptide is a strain of bacteria or peptide extracted from the natural environment in which it is usually found, whether a plant or a living animal, for example. Thus, a strain of bacteria or peptide naturally present in a living animal is not an isolated strain of bacteria or peptide for the purposes of the invention, while the same strain of bacteria or peptide, partially or completely separated from other elements present in its natural context, is "isolated" for the purposes of the invention. An isolated strain of bacteria or peptide may exist in a substantially purified form, or may exist in a non-native environment such as, for example, a host cell.

[0024] Surprisingly, the Applicant has identified a specific probiotic bacterial strain whose use makes it possible to modulate (immunomodulation), or even stimulate innate immunity, in particular via modulation, or even stimulation or increase, of phagocytic activity, and thus to prevent and / or treat an infection and / or reduce the symptoms of an infection, in a subject including a young subject.

[0025] As already stated, the term "probiotic" is used to define live microorganisms which, when ingested in sufficient quantity, exert a positive effect on health, comfort and well-being, beyond traditional nutritional effects.

[0026] For the purposes of the invention, the terms "modulation of phagocytic activity" and "modulating phagocytic activity" designate a modification of phagocytic activity, i.e. an increase or a decrease in this activity. In the context of the invention, this advantageously involves an increase or a stimulation of phagocytic activity, i.e. the observation of increased phagocytic activity via an increase in the number and / or activity of the cells involved in phagocytosis, as demonstrated in the examples.

[0027] Thus and according to a first aspect, the invention relates to the strain of Bacillus subtilis, deposited with the CNCM (National Collection of Cultures of Microorganisms, Pasteur Institute, 28 rue du Docteur Roux, 75724 Paris Cedex 15) on October 25, 2001 under number 1-2745, for use in preventing and / or treating an infection via modulation of peripheral phagocytic activity in a subject.

[0028] According to a particular embodiment, the modulation of peripheral phagocytic activity in a subject is an increase or stimulation of this activity.

[0029] In the remainder of the description, the expressions “Bacillus subtilis strain deposited with the CNCM on October 25, 2001 under number 1-2745”, “CNCM 1-2745 strain”, “Bacillus subtilis CU1 strain”, “(probiotic) CU1 strain” and “(probiotic) BSCU1 strain” are used interchangeably.

[0030] In other words, the present invention relates to: - the use of the B. subtilis strain, deposited with the CNCM on October 25, 2001 under number 1-2745, to prevent and / or treat an infection via modulation of peripheral phagocytic activity in a subject; - the use of the B. subtilis strain, deposited with the CNCM on October 25, 2001 under number 1-2745, to prepare a medicament, advantageously a functional food or a food supplement, to prevent and / or treat an infection via modulation of peripheral phagocytic activity in a subject; - a method for preventing and / or treating an infection, via modulation of peripheral phagocytic activity in a subject comprising the administration, advantageously orally, of the strain of B. subtilis, deposited with the CNCM on October 25, 2001 under number 1-2745.

[0031] In other words, the specific strain in question is intended to prevent or treat an infection and / or reduce the symptoms of an infection. In the context of the application, the terms "reduce", "lessen" or "mitigate" are used interchangeably.

[0032] In the context of the present application and in a conventional manner, the term "infection" means an invasion of the organism or a site of the organism by an infectious agent. This infectious agent, also called a pathogenic agent, is advantageously an agent exogenous to the organism, present in the environment, or even at the level of the skin or mucous membranes of the subject, including a pathobiont agent. These are advantageously microorganisms which can be bacteria, viruses, fungi or parasites. It should be noted that certain pathogenic agents are not organisms (prions), while others are not microscopic (parasitic worms).

[0033] The pathogenic power of an infectious agent corresponds to its capacity to cause a disease in a host organism. The virulence of an infectious agent measures its capacity to develop in an organism (invasive power) and to secrete toxins there (toxic power).

[0034] For illustration purposes only, an infection may be caused by the following bacteria:

[0035] Certain strains of Escherichia coli are sources of diarrhea, urinary tract infections, nosocomial infections, septicemia, and newborn meningitis.

[0036] Staphylococcus aureus and Pseudomonas aeruginosa are sources of eye infections, wounds and acute gastroenteritis.

[0037] Salmonella Yersinia enterocolitica causes gastroenteritis.

[0038] Campylobacter jejuni has become the leading source of enteric diseases bacterial in industrialized countries.

[0039] Shigella dysenteriae causes dysentery.

[0040] Infections can also be viral in origin, which can result in headaches, fever, body aches, fatigue, etc.

[0041] Thus, gastroenteritis or intestinal flu can be caused by different viruses such as norovirus, adenovirus, rotavirus, astrovirus, cytomegalovirus, enterovirus and can result in nausea, diarrhea, vomiting, abdominal cramps and possibly fever, congestion of the respiratory tract, etc.

[0042] Viral infections also commonly affect the upper and / or lower respiratory tract. Upper respiratory tract infections, including the common cold and influenza, primarily affect the nose and throat. Lower respiratory tract infections, including croup, bronchiolitis, and pneumonia, affect the trachea, airways, and lungs. Although these respiratory infections can be classified according to the causative virus (e.g., influenza for influenza; rhinovirus and coronavirus for the common cold; respiratory syncytial virus (RSV) for bronchiolitis), they are generally classified according to clinical syndromes.

[0043] A parasite is an organism that lives on or in another organism (the host) and benefits, for example by obtaining nutrients, from the host at its expense. Thus, infections can also be of parasitic origin, which can result in abdominal pain, diarrhea, appetite disorders, nausea, etc. Examples of parasites include pinworm, giardiasis, tapeworm (or "tapeworm") or roundworm.

[0044] It should be noted that acute infections and chronic infections can be distinguished according to their duration, typically less than and more than 2 weeks, respectively. In the context of the invention, acute infections are advantageously targeted.

[0045] According to one embodiment, the infection does not cause diarrhea, in particular diarrhea of bacterial origin.

[0046] According to another embodiment, the infection is not a respiratory tract infection.

[0047] According to another embodiment, the infection is not of viral origin.

[0048] “Preventing a disease or condition”, here an infection, means avoiding its occurrence, particularly in subjects at risk of developing it, or delaying the symptoms or repercussions. In this context, we speak of preventive or prophylactic treatment of this infection.

[0049] In particular, “preventing” means administering such a strain upstream of the infection.

[0050] By "treating a disease or condition", here an infection, is meant the act of treating it in order to cure it, to relieve its symptoms or repercussions, or to at least stabilize it. In this context, we speak of curative treatment of this infection.

[0051] In particular, "treating" or "fighting" means administering such a strain when the infection has set in.

[0052] The expressions "reduce the severity of symptoms of an infection" and "reduce the symptoms of an infection" are used interchangeably and refer to reducing the abnormal manifestation / perceptible clinical sign(s) caused by an infection (e.g., discomfort, pain, burning, feeling of suffocation, etc.).

[0053] It should be noted that the detection of an infection can be based on the analysis of the symptoms observed or on the search for and / or identification of the microorganism(s) involved by any technique known to those skilled in the art (PCR, ELISA test, etc.).

[0054] According to one embodiment, the infections targeted within the scope of the invention are viral or bacterial or parasitic infections.

[0055] According to another embodiment, the infection targeted within the framework of the invention is a viral or bacterial or parasitic infection, advantageously a gastrointestinal, respiratory, urinary, dental, dermal, cardiac, cerebral, renal, blood or lymphatic infection; advantageously gastrointestinal or respiratory.

[0056] As demonstrated in the present application, the prevention and / or treatment of an infection and / or the reduction of the symptoms of an infection results from the administration of the specific probiotic bacterial strain targeted, which has the capacity to modulate phagocytic activity, advantageously to stimulate or even increase the already activated phagocytic function. By definition, phagocytosis is a cellular process allowing the ingesting and elimination of particles with a diameter greater than 0.5 μm, in particular microorganisms. Cells specialized in phagocytosis include macrophages, neutrophils, monocytes, dendritic cells and osteoclasts. Thus, the administration of this strain modulates, advantageously stimulates, the peripheral innate immune system.

[0057] According to a preferred embodiment, the strain used within the framework of the invention is intended for human and / or veterinary use, advantageously for human use.

[0058] According to an advantageous embodiment, the strain used in the context of the invention is intended for a young subject or an elderly subject. In the context of the invention, - the term "young subject" means a man or a woman, under 60 years of age, or even under 55 years of age, or even 50 years of age or younger. This may be children as well as adults; - “elderly subject” means a man or woman over 60 years old, or even over 65 years old.

[0059] According to a particular embodiment, the subject receiving the administration of the strain used in the context of the invention has at least one or even all of the following characteristics: - age less than 60 years or more than 60 years; - body mass index (BMI) between 17 and 40 kg / m2; - good general condition; - non-smoker.

[0060] According to another particular embodiment, the subject receiving the administration of the strain used in the context of the invention has at least one or even all of the following characteristics: - age between 18 and 79 years; - body mass index (BMI) between 18.5 and 40 kg / m2, advantageously between 18.5 and 25 kg / m2 or between 22 and 28 kg / m2; - good general condition; - non-smoker.

[0061] According to one embodiment, the strain used within the framework of the invention is intended for a so-called healthy subject.

[0062] In the context of the invention, the term "healthy subject" means a healthy man or woman, in whom (z) no pathology or disease other than the possible infection has been diagnosed and / or declared (i.e., BSCU1 for use in treating an infection or reducing the symptoms of an infection); or (zz) no pathology or disease has been diagnosed and / or declared (i.e., BSCU1 for use in preventing an infection) in particular chosen from the following group: cardiovascular diseases, metabolic diseases in particular type II diabetes and obesity, cancers, autoimmune diseases, depression, and neurological or neuropsychiatric diseases such as Alzheimer's, Parkinson's and bipolar disorders.

[0063] Alternatively, the strain used in the context of the invention may be intended for a subject suffering from a disease or condition chosen from the following group: cardiovascular diseases, metabolic diseases, in particular type II diabetes and obesity, cancers, depression, and neurological or neuropsychiatric diseases such as Alzheimer's, Parkinson's and bipolar disorders.

[0064] According to another aspect, the invention relates to cells obtained by culturing the strain of Bacillus subtilis, deposited with the CNCM (National Collection of Microorganism Cultures, Pasteur Institute, 28 rue du Docteur Roux, 75724 Paris Cedex 15) on October 25, 2001 under number 1-2745, for use in preventing and / or treating an infection via modulation of peripheral phagocytic activity in a subject.

[0065] According to a particular embodiment, the modulation of peripheral phagocytic activity in a subject is an increase or stimulation of this activity.

[0066] In other words, the present invention relates to: - the use of cells obtained by culturing the B. subtilis strain, deposited with the CNCM on October 25, 2001 under number 1-2745, to prevent and / or treat an infection via modulation of peripheral phagocytic activity in a subject; - the use of cells obtained by culturing the B. subtilis strain, deposited with the CNCM on October 25, 2001 under number 1-2745, to prepare a medicament, advantageously a functional food or a food supplement, to prevent and / or treat an infection via modulation of peripheral phagocytic activity; - a method for preventing and / or treating an infection via modulation of peripheral phagocytic activity in a subject comprising the administration, advantageously orally, of cells obtained by culturing the strain of B. subtilis, filed with the CNCM on October 25, 2001 under number 1-2745.

[0067] In practice, the cells are obtained by culturing the bacterial strain in a culture medium, according to methods well known to those skilled in the art, for example described in the book “Biotechnology, 5th edition, R. Scriban, Edition Tec. & Doc., 1999, ISBN: 2-7430-0309-X”.

[0068] Typically, a method for producing cells by culturing the strain implemented within the framework of the invention comprises the following steps: - inoculation of a culture medium with an inoculum of the strain; - culture in aerobic conditions, to obtain cell multiplication; - separation of the biomass from its culture medium, to obtain cells.

[0069] The cells thus obtained are predominantly in vegetative form. The “vegetative form” of a bacterium designates the form of a bacterium placed in favorable conditions. The expression “predominantly in vegetative form” means that at at least 70% of the cells are in vegetative form, preferably at least 80%, and more preferably at least 90%. An example of favorable conditions is a non-limiting culture medium at a temperature and pH favorable to bacterial multiplication. A non-limiting culture medium contains all the nutrients necessary for cell multiplication.

[0070] The cell production method may further comprise an intermediate step, between the aerobic culture step and the step of separating the biomass from its culture medium, of placing the cells in unfavorable conditions. The cells thus obtained are then mainly in sporulated form.

[0071] The "sporulated form" of a bacterium refers to the form of a bacterium placed in unfavorable conditions. The expression "predominantly in sporulated form" means that at least 70% of the cells are in sporulated form, preferably at least 80%, and more preferably at least 90%. The sporulated form is a form of resistance which allows the cells to resist a hostile environment such as a lack of nutrients, i.e. a limiting nutrient environment, water stress, a significant variation in pH or temperature, or even crossing the digestive tract.

[0072] The placing of the cells in unfavorable conditions is for example obtained by: non-renewal of the bacteria culture medium, stopping the supply of culture medium, use of a limiting culture medium, change of temperature and / or pH, control of aeration and / or agitation so as to maintain an O2 pressure greater than 20% in the culture medium and a CO2 pressure less than 1.5% in the outgoing gases or their combination. It is also possible to obtain cells predominantly in sporulated form by adding glutamic acid to the culture medium in an amount of the order of 0.75g / L.

[0073] In a preferred embodiment, the cells are predominantly in sporulated form, i.e. approximately 90%, or even 95% or even 100% of the cells are in sporulated form.

[0074] In a preferred embodiment, the cells are in sporulated and / or vegetative form, advantageously sporulated, preferably sporulated and freeze-dried.

[0075] The cell production method may also comprise a subsequent step of drying the cells to obtain cells in dry form. The drying is for example freeze-drying, fluidized bed drying or spray drying.

[0076] In a particular embodiment, the cells are in dry form. “Cells in dry form” advantageously means that the biomass obtained at the end of the cell production process advantageously comprises more than 90% dry matter, preferably more than 95% dry matter.

[0077] According to another aspect, the invention relates to a composition comprising the strain of Bacillus subtilis, deposited with the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15) on October 25, 2001 under number 1-2745, or cells obtained by culturing said strain, for use in preventing and / or treating an infection via modulation of peripheral phagocytic activity.

[0078] According to a particular embodiment, the modulation of peripheral phagocytic activity in a subject is an increase or stimulation of this activity.

[0079] In other words, the present invention relates to: - the use of a composition comprising the strain of B. subtilis, deposited with the CNCM on October 25, 2001 under number 1-2745, or cells obtained by culturing said strain, to prevent and / or treat an infection via modulation of peripheral phagocytic activity in a subject; - the use of a composition comprising the strain of B. subtilis, deposited with the CNCM on October 25, 2001 under number 1-2745, or cells obtained by culturing said strain, to prepare a medicament, advantageously a functional food or a food supplement, to prevent and / or treat an infection via modulation of peripheral phagocytic activity; - a method for preventing and / or treating an infection via modulation of peripheral phagocytic activity in a subject comprising the administration, advantageously orally, of a composition comprising the strain of B. subtilis, deposited with the CNCM on October 25, 2001 under number 1-2745, or cells obtained by culture of said strain

[0080] A composition implemented within the framework of the invention may be a pharmaceutical composition, which may contain pharmaceutically acceptable excipients, or a nutraceutical composition, which may contain nutraceutically acceptable excipients.

[0081] According to a preferred embodiment, the composition used in the context of the invention is a nutraceutical composition, including a product comprising a food additive (functional food), or a food supplement. Nutraceutically acceptable excipients are, for example, maltodextrin and magnesium stearate.

[0082] A food product can be any type of food, beverage or confectionery. For example, it can be a drink, a cereal bar, chewing gum, of a possibly fermented dairy product, in which the strain used in the context of the present invention is added.

[0083] By definition, a food supplement is defined as a foodstuff whose purpose is to supplement the normal diet and which constitutes a concentrated source of nutrients or other substances having a nutritional or physiological effect, alone or in combination.

[0084] In practice, a food supplement is marketed in dose form, for example with presentation forms such as capsule, lozenge, tablet, pill and other similar forms, sachet of powder, ampoule of liquid, bottle equipped with a dropper and other similar forms of liquid or powder preparations intended to be taken in measured units of small quantity.

[0085] In a known manner, the composition used in the context of the invention may comprise the usual adjuvants or excipients used in the field in question, such as hydrophilic or lipophilic thickeners or gelling agents, appetizers, hydrophilic or lipophilic additives, preservatives, antioxidants, diluents, vitamins, minerals, suspending agents, cellulose derivatives, absorbents, cryoprotective agents or even colorants.

[0086] Of course, the person skilled in the art will take care to choose this or these possible adjuvants or excipients, and to adjust their quantity, in such a way that the advantageous properties of the composition according to the invention are not, or not substantially, altered by the envisaged addition.

[0087] According to one embodiment, the composition according to the invention comprises nutritive substances usable as a support and / or prebiotic substances advantageously chosen from fructo-oligosaccharides, inulins, isomalto-oligosaccharides, lactitol, lactosaccharose, lactulose, pyrodextrins, soy oligosaccharides, transgalacto-oligosaccharides, xylo-oligosaccharides, yeast walls, vitamins, in particular vitamin E.

[0088] According to another embodiment, the composition according to the invention comprises at least one compound chosen from the following group: zeolites, calcium carbonate, calcium sulfate, magnesium carbonate, talc, trehalose, chitosan, shellac, albumin, starch, skimmed milk powder, whey, whey powder, maltodextrins, lactose, inulin, dextroses, celluloses, clays including sepiolite, yeast and cereal derivatives, vegetable oils or a solvent chosen from water or a physiological solution.

[0089] According to a particular embodiment, the composition according to the invention comprises a coating material advantageously chosen from maltodextrins, guar seed flour, gum arabic, alginates, modified starch and starch derivatives, dextrins, cellulose derivatives such as cellulose ester and ether cellulose, proteins such as gelatin, albumin, casein, gluten, gum arabic, gum tragacanth, lipids such as waxes, paraffin, stearic acid, mono- and diglycerides.

[0090] The composition according to the invention may be in the form of a powder, a capsule, a spray, a solution, an emulsion, a suspension or a dispersion.

[0091] Advantageously, the composition according to the invention is in dry or liquid form, in particular in lyophilized, dried, pressed, liquid or frozen form, advantageously in lyophilized form.

[0092] According to another preferred embodiment, the composition used in the context of the invention is in a form suitable for oral administration, whether solid or liquid. Thus, such a composition may be in the form of a capsule, pill, lozenge, tablet, capsule, powder, suspension, solution, syrup or granule.

[0093] According to another embodiment, the composition used in the context of the invention is devoid of any other active ingredient, advantageously of any other microorganism, in particular of any other probiotic, in particular of bacterial origin. In other words, the composition may contain only, as living microorganism, the strain targeted in the present invention.

[0094] Alternatively, the strain targeted by the invention can be combined with other microorganisms, advantageously probiotics, in particular of bacterial origin such as the genera Bacillus, Bifidobacterium, Lactobacillus, Lactiplantibacillus, Ligilactobacillus, Lactococcus and Bifidobacterium, or yeasts such as Saccharomyces, Kluyveromyces, Pichia and Torulaspora.

[0095] Suitably, a composition according to the invention contains a quantity of cells corresponding to the daily dose recommended for the intended application.

[0096] Note that the daily dose may depend on the type of subject, its weight, the method of administration and the type of curative or preventive treatment. Thus, such a daily quantity may range from 105 to 1011 CFU (for “Colony Forming Unit”), for example 105, 106, 107, 108, 109, 1010, 1011 CFU per day.

[0097] Suitably for human use, a composition used in the context of the invention comprises from 1.108 to 1.10" CFU, advantageously from 1.109 CFU to 1.1010 CFU, for example 2.109, 3.109, 4.109, 5.109, 6.109, 7.109, 8.109 or 9.109 CFU, even more advantageously 2.109 CFU.

[0098] Alternatively, in a manner suitable for human use, a composition implemented within the framework of the invention comprises the strain or cells in an amount greater than or equal to 1.109 CFU, advantageously between 1.109 and 1.1010 CFU, for example 2.109 CFU.

[0099] According to one embodiment, the strain, cells or composition used in the context of the present invention are administered daily, advantageously once or twice a day, in particular before taking a meal, for example breakfast.

[0100] According to another embodiment, the strain, cells or composition used in the context of the present invention are administered continuously and regularly for several weeks, namely at least one week, or even 2, 3 or 4 weeks, or even at least 1 month or more (several months to several years).

[0101] Thus and according to a particular embodiment, the daily intake of the preventive or curative treatment is done without interruption and / or in a non-sequential manner (for example successive cycles of 10 days of intake, followed by 18 days of break).

[0102] The manner in which the invention can be implemented and the advantages which result therefrom will become more apparent from the following examples of implementation, given for informational and non-limiting purposes, with the support of the appended figures. FIGURES

[0103] [Fig.l]: Determination of the frequency of certain innate immune cells by flow cytometry before administration of the probiotic composition of the invention (“Reference”) and then 4 weeks after its daily administration (“Week 4”) in PBMCs of adult (30 < age < 49 years) and elderly (65 < age < 79 years) subjects. The data indicate the mean values + / - standard deviation. Mo: monocytes; DC: dendritic cells; pDC: plasmacytoid dendritic cells. p-values: *: [0.01; 0.05[; **: [0.001; 0.01[; ***: <0.001.

[0104] [Fig.2]: Determination of the expression of the activation marker CD69 on the surface of monocytes (“CD69+ on CD 14”) by flow cytometry before administration of the probiotic composition of the invention (“Reference”) then 4 weeks after its daily administration (“Week 4”) in PBMCs of adult (30 < age < 49 years) and elderly (65 < age < 79 years) subjects. The data indicate the mean values + / - standard deviation. p values: *: [0.01; 0.05[; **: [0.001; 0.01[; ***: <0.001.

[0105] [Fig.3A]: Determination of the differential expression of genes in PBMCs of adult (30 < age < 49 years) and elderly (65 < age < 79 years) subjects before administration of the probiotic composition of the invention (“Reference”) compared to 4 weeks after its daily administration (“Week 4 4”). Volcano-plot representation.

[0106] [Fig.3B]: Determination of differential gene expression in PBMCs of adult (30 < age < 49 years) and elderly (65 < age < 79 years) subjects before administration of the probiotic composition of the invention (“Reference”) compared to 4 weeks after its daily administration (“Week 4 4”). Representation in Gene Ontology Terms

[0107] [Fig.4]: Determination of the phagocytosis capacity of granulocytes and monocytes from whole blood collected before administration of the probiotic composition of the invention (“Reference”) then 4 weeks after its daily administration (“Week 4”).

[0108] (AC) mean values + / - standard deviation of the frequency of monocytes having phagocytosed bacteria (A), and mean fluorescence intensity indicating the amount of bacteria phagocytosed by monocytes (B) and granulocytes (C). p-values: *: [0.01; 0.05[; **: [0.001; 0.01[; ***: <0.001. EXAMPLES OF ACHIEVEMENT

[0109] All experiments were carried out using the B. subtilis CU1 strain deposited on October 25, 2001 at the CNCM (National Collection of Microorganism Cultures, Pasteur Institute, 28 rue du Docteur Roux, 75724 Paris Cedex 15) under number 1-2745. 1. Materials and methods

[0110] 1.1. Populations

[0111] Subjects were recruited from the healthy general population.

[0112] Subjects were recruited from the healthy general population.

[0113] The study was a single-center trial.

[0114] To participate in this study, subjects had to meet all of the following criteria: - adult subjects (29 people): 30 < age < 49 years and 18.5 < BMI < 25.0 kg / m2; - elderly subjects (30 people): 65 < age < 79 years and 22.0 < BMI < 28.0 kg / m2; and - Non-smokers

[0115] 1.2. Administration

[0116] The probiotic composition according to the invention comprises BSCU1 cells mixed with excipients (maltodextrin and magnesium stearate; see Table 1). The cells come from the product LifeinU® BSCU1 marketed by GNOSIS by LESAFFRE (Marcq en Baroeul, France).

[0117] Each probiotic capsule contains 2.109 CFU of B. subtilis, the majority of which is in sporulated form (i.e., spores of B. subtilis CU1).

[0118] The capsules were taken orally, as a dietary supplement.

[0119] The subjects were instructed to consume one capsule of the study product daily for 4 weeks, in the morning (before breakfast), i.e. a daily dose of 2.109 CFU of B. subtilisCUl (CNCM 1-2745).

[0120] [Tables 1] Ingredients Quantity (mg) Quantity (%) LifeinU™ BSCU1 (BSCU1 CNCM 1-2745) 20 6.67 Maltodextrin glucidex 19 (Roquette) 270 90 Magnesium stearate 10 3.33 Total 300 100

[0121] 1. 3. Analysis of the phenotype of immune cells

[0122] Blood samples were collected into BD Vacutainer CPT tubes and then stored at room temperature. The tubes were centrifuged for 25 minutes at 1800 xg and room temperature in a shaker rotor (with interruptions). The cell fraction was poured into 50 ml tubes. The cells were washed twice with PBS. The washed PBMCs were diluted in 1 ml of PBS for cell counting. For cell phenotyping, PBMCs were stained with fluorochrome-conjugated antibodies against extracellular markers. The isolated PBMCs (1.106 per well) were placed in a 96-well NUNC plate (Thermo Fisher Scientific, US), and the cells were washed twice with 100 μl of PBS, followed by centrifugation of the plate for 4 minutes at 400 xg and RT.

[0123] For live / dead cell staining, Zombie AquaTM Viability Solution (Biolegend, US) was diluted 500 times in PBS, 100 µl was added per well and incubated for 30 minutes at room temperature. The cells were washed with FACS buffer (200 µl) containing PBS (1X) with BSA (0.5% v / v), ethylenediaminetetraacetic acid (EDTA, 2.5 mM) and sodium azide (NaN3, 10% v / v). The plate was then centrifuged at 400 x g for 4 minutes (RT) and the liquid was discarded. The antibody mixture (see Table 2 below) was diluted in FACS buffer and added to the cells. The plate was incubated for 30 minutes at 4°C in the dark. Cells were washed three times by adding cold FACS buffer (200 μL) and centrifuged at 400 × g (4°C) for 4 minutes. The supernatant was discarded and the cell pellet was resuspended in FACS buffer (300 μL) and measured on the CytoFLEX LX (Beckman Coulter, US).The generated flow cytometry data were analyzed using Flowjo vlO (FlowJo LLC, US).

[0124] [Tables2] Immunity Fluorochrome Marker Supplier Reference # Adaptive CD3 AF700 Biolegend 344822 Innate CD3 FITC Biolegend 344804 Adaptive CD4 BUV395 BD 563550 Adaptive CD8 FITC Biolegend 344704 Native CD 11c PE Biolegend 337206 Adaptive CD 14 PerCPCy5.5 Biolegend 325622 Native CD 14 APC-Fire750 Biolegend 301854 Native CD16 BV785 Biolegend 302046 Adaptive CD 19 BV785 Biolegend 302240 Native CD 19 FITC Biolegend 302206 Adaptive CD25 BV421 Biolegend 302630 Adaptive CD45 APC-Fire750 Biolegend 304062 Adaptive CD56 BV605 Biolegend 318334 Native CD56 FITC Biolegend 318304 Native CD68 PE-Cy7 Biolegend 333816 Native CD86 APC Biolegend 305412 Native CD 123 PE-Dazzle594 Biolegend 306034 Adaptative CD 127 PE / Dazzle594 Biolegend 351336 Innée CD206 BV421 Biolegend 321126 Adaptative / 1 year HLA-DR PerCPCy5.5 Biolegend 307630 Adaptative / 1 year Human TruStain Fc X Biolegend 422302 Adaptative / 1 year True-Stain Monocyte Blocker Biolegend 426101 Adaptive NKG2D PE Biolegend 320806

[0125] 1.4. Analysis of gene expression in PBMC

[0126] Blood was collected into BD Vacutainer 10 ml PET tubes containing heparin. PBMCs were isolated using Leucosep® tubes (Greiner bio-one, Austria) filled with separation medium according to the manufacturer's instructions. Heparinized blood (10 ml) was diluted 1:2 with Dulbecco's phosphate-buffered saline (DPBS) and poured into the Leucosept® tube. The sample was centrifuged at 1000 x g for 10 minutes (brakes off). The enriched cell fraction was poured out of the tube and washed twice with DPBS for 10 minutes at 250 x g. Cells were counted using a TC20 automatic cell counter. After the last wash, the cells were suspended in RLT buffer (Qiagen RNease Kit, Qiagen, Netherlands) containing 1% [3-mercaptoethanol] according to the manufacturer's instructions. Samples were stored at -80°C until RNA isolation.

[0127] RNA isolation was performed using the RNeasy Mini Kit (Qiagen, USA) according to the manufacturer's instructions. After lysis and precipitation, samples were loaded onto the RNeasy column and subjected to DNAse treatment to remove DNA. RNA was eluted in RNase-free water and stored at -80°C in several aliquots for quality control and library preparation. RNA concentration was measured using the NanoDrop ND -1000 (Thermo Scientific, Waltham, USA). In addition, 10% of the samples were analyzed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany).

[0128] RNA samples were shipped on dry ice to Novogene UK. Directional mRNA libraries were prepared by Novogene (poly A enrichment) and sequenced on an Illumina NovaSeq 6000 PE150 platform to obtain at least 6 GB of PE150 raw data per sample. Quality-controlled FASTQ format data files were transferred to NIZO for bioinformatics analysis.

[0129] For the analysis of sequencing results, a total of 118 RNA samples were collected from two different populations, namely adults and elderly people. These samples were analyzed using the nf-core / maseq pipeline version 3.12.0 (https: / / nf-co.re / rnaseq / 3.12.0), which includes a series of steps. First, a preprocessing step was performed using tools such as FastQC, TrimGalore, BBsplit, and SortMeRNA to ensure quality control. Then, the reads were aligned to the reference genome, and gene expression values were quantified using the STAR tool. Post-processing steps included generating counts using featureCounts and Kalisto, followed by further processing and final quality control using RSeqQC, DESEq2, and MultiQC. The resulting data were then subjected to differential analysis using DESEq2 version 1.38.3, where genes with a logarithmic change greater than 1.5 or less than -1.5 and a p-value less than 0.05 were considered differentially expressed. Gene set enrichment analysis (GSEA) was also performed using the fgsea package in R version 4.1.0.

[0130] 1.5. Ex vivo evaluation of phagocytosis

[0131] Ex vivo phagocytosis in whole blood was assessed using pHrodo™ Green E. coli BioParticles™ (ThermoFischer Scientific, US) according to the manufacturer's instructions. Fluorescence was measured using FACSCanto II (BD, US). Samples were analyzed using FlowJoTM version 10. 2. Results

[0132] 2.1. Evaluation of the effect of BSCU1 consumption on the distribution of innate immune cells and the expression of activation markers

[0133] The effect of 4 weeks of daily BSCU1 supplementation on the representativeness of innate immune cells in PBMCs from adult and elderly subjects was analyzed by flow cytometry. The results are presented in [Fig.l].

[0134] The results show, in the adult and elderly groups, respectively, an increase of 6.6 and 8.2 percentage points in the relative abundance of monocytes (Mo), accompanied by a decrease of 4 and 5.2 percentage points in the relative abundance of conventional dendritic cells (DC) and a decrease of 1 percentage point in the relative abundance of plasmacytoid dendritic cells (pDC) after 4 weeks of daily supplementation with BSCUL

[0135] The results presented in [Fig.2] show, in the adult and elderly groups, respectively, an increase of 41.9% and 52.4% in the expression of the activation marker CD69 on the surface of monocytes after 4 weeks of daily supplementation with BSCU1.

[0136] These results clearly show immunomodulation by BSCU1, mainly affecting myeloid cells.

[0137] 2.2. Evaluation of the effect of BSCU1 consumption on the program immune cell gene expression

[0138] To explore how BSCU1 treatment affects the gene expression program of immune cells, RNA sequencing was performed on PBMCs (peripheral blood mononuclear cells) obtained at baseline and after 4 weeks of BSCU1 treatment from the adult and elderly groups.

[0139] Analysis of genes differentially expressed in PBMCs from adults and represented by the volcano plot ([Fig.3A]) shows an enrichment (variation > 2) of the expression of transcripts coding for the cytokine IL-8, the transcription factor EGR1 and the protein G0S2 involved in the inflammatory response of mononuclear cells.

[0140] Analysis of Gene Ontology (GO) terms in adults showed enrichment in functional pathways such as type I IFN response, complement activation, oxidative phosphorylation, and phagocytosis engulfment process ([Fig.3B]).

[0141] Thus, treatment with BSCU1 resulted in changes in the gene expression program associated with immune functions in the test subjects.

[0142] 2.3. Evaluation of the effect of BSCU1 consumption on phagocytic function

[0143] The phagocytic capacity of granulocytes and monocytes obtained from whole blood was evaluated before administration of the probiotic composition of the invention and then 4 weeks after this daily administration in adults and the elderly.

[0144] The results are illustrated in [Fig.4].

[0145] The results show, in the groups of adults and elderly people who consumed BSCU1, an increase of 6 and 8.2 percentage points in the total frequency of monocytes that phagocytosed bacteria, respectively ([Fig.4] (A)). In addition, the average fluorescence intensity, which measures the amount of phagocytosed bacteria, increased by 77.2% and 91.2% in monocytes and by 13.1% and 7.9% in granulocytes, in adult and elderly subjects respectively ([Fig.4] (B) and (O).

[0146] These results clearly show that BSCU1 consumption promotes the phagocytic capacity of myeloid cells, particularly monocytes, in adults and the elderly. 3. Conclusion

[0147] Supplementation with BSCU1 according to the invention influences the representativeness of immune cells, including an increase in the frequency of monocytes and a decrease in the frequency of dendritic cells and plasmacytoid dendritic cells in adult and elderly groups. Phagocytosis is an important function of the innate immune system that aims to fight and eliminate microbes and an increase in this phagocytic activity of monocytes and granulocytes was observed in subjects who consumed the probiotic BSCU1 according to the invention.

[0148] In conclusion, it emerges from these data that the ingestion of BSCU1 according to the invention induces a better reactivity to infections by an infectious agent linked to an increased phagocytic activity. The combination of these effects is at the origin of a lower incidence of infections in subjects supplemented with BSCU1.

Claims

Claims

1. Strain of Bacillus subtilis, deposited with the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15) on October 25, 2001 under number 1-2745, for use in preventing and / or treating an infection via an increase in peripheral phagocytic activity in a subject.

2. Cells obtained by culturing the B. subtilis strain, filed with the CNCM on October 25, 2001 under number 1-2745, for use in preventing and / or treating an infection via an increase in peripheral phagocytic activity in a subject.

3. Cells for their use according to claim 2, characterized in that they are in sporulated and / or vegetative form, advantageously sporulated, preferably sporulated and freeze-dried.

4. Composition comprising the strain of Bacillus subtilis, deposited with the CNCM on October 25, 2001 under number 1-2745, or cells obtained by culturing said strain for use in preventing and / or treating an infection via an increase in peripheral phagocytic activity in a subject.

5. Composition for its use according to claim 4, characterized in that it comprises the strain or cells in an amount greater than or equal to 1.109 CFU, advantageously between 1.109 and 1.1010 CFU, for example 2.109 CFU.

6. Composition for its use according to one of claims 4 to 5, characterized in that it is in a form suitable for oral administration, advantageously in the form of a capsule, pill, lozenge, tablet, capsule, powder, suspension, solution or granule.

7. Composition for its use according to one of claims 4 to 6, characterized in that said composition is a functional food or a food supplement.

8. Strain for its use according to claim 1, cells for their use according to one of claims 2 to 3, or composition for its use according to one of claims 4 to 7 characterized in that the subject is a young subject, advantageously aged less than 60 years, preferably a child or an adult under 50 years.

9. Strain for its use according to claim 1, cells for their use according to one of claims 2 to 3, or composition for its use according to one of claims 4 to 7 characterized in that the subject is an elderly subject, advantageously over 60 years old, preferably over 65 years old.

10. Strain for its use according to claim 1, 8 or 9, cells for their use according to one of claims 2 to 3, 8 or 9, or composition for its use according to one of claims 4 to 9 characterized in that the infection is a viral or bacterial or parasitic infection, advantageously a gastrointestinal, respiratory, urinary, dental, dermal, cardiac, cerebral, renal, blood or lymphatic infection.

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