ES2908480A1 - Aerobic microorganism of the genus bifidobacterium spp (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Aerobic microorganism of the genus Bifidobacterium spp. The present invention is related to a microorganism of the genus Bifidobacterium spp. which includes a promoter that comprises the Gaaagga sequence (SEQ ID no: 1) at a distance between 12 and 31 nucleotides of the start -up codon responsible for the transcription of a gene, where said microorganism is aerotolerant and belongs to species B. bifidum. The present invention is also related to the composition that includes this microorganism and the uses of the same in health due to its beneficial health properties and in the manufacture of fermented dairy products. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

Aerobic microorganism of the genus Bifidobacterium spp

The present invention relates to an aerobic microorganism of the genus Bifidobacterium spp., compositions comprising it, and the use thereof in the production of fermented dairy products such as cheese and yogurt. Therefore, the present invention falls within the field of biotechnology, preferably in the field of the food industry.

STATE OF THE ART

The Bifidobacterium genus includes at least 48 different species, of which 40 can be isolated from the gastrointestinal tract of mammals, birds, and insects, and the rest come mainly from sewage. They all have a fermentative metabolism characterized by a specific glycolytic pathway called "ruta bifida" (Sánchez et al., 2004). The strains of bifidobacteria of intestinal origin have been the subject of numerous studies that link their presence, or their metabolites, with benefits for human health. Specific strains of bifidobacteria that exert beneficial effects on the host are considered probiotics and are included as health-promoting agents in a wide variety of dietary supplements and so-called functional foods. As an example, in the dairy industry, probiotic strains belonging to the Bifidobacterium genus are used as an adjunct component to the starter (or starter) culture intended for the production of fermented beverages, cheeses, ice creams, and frozen desserts; on some occasions bifidobacteria may even be responsible for the fermentation process. However, its inclusion in functional foods constitutes a challenge for the industry from a technological point of view. On the one hand, companies that produce and market starter and adjunct cultures have to work with "robust" strains that achieve minimum production yields (obtaining biomass at an affordable cost), that are capable of withstanding freeze-drying or drying processes at high temperature (among others) to obtain a stable and viable concentrate, both during these processes and during the subsequent storage of the concentrated crops until the supply to the demanding companies. On the other hand, companies in the food sector or the pharmacy-parapharmacy sector that use these cultures in their processes have to control viability losses during manufacturing and processing. storage of the food or food supplement, respectively, containing probiotic strains. This entire process limits the application of most bifidobacteria species. In this way, Bifidobacterium animalis subsp. lactis is almost the predominant species in the vast majority of fermented products and is present in many preparations in supplement form, and is supplied by a few companies specializing in the production of starter or adjunct cultures.

Among the most important limiting factors for the production, use and commercialization of bifidobacteria of intestinal origin is their low tolerance to oxygen. The absence of molecular detoxification mechanisms prevents these bacteria, which are strict anaerobes, from eliminating reactive oxygen species (ROS) such as hydrogen peroxide (H ² O ² ) or superoxide anion ( Oí). In fact, normal concentrations of oxygen in the air (21% v/v) are toxic to the most abundant microorganisms in the human intestinal environment because their high reactivity induces damage to biological molecules, especially DNA and phospholipids. However, other aerobic, microaerophilic, and aerotolerant microorganisms have developed protective responses that allow tolerance to ambient oxygen concentration. In the presence of oxygen, ROS species accumulate both in anaerobic microorganisms and in by-products of aerobic metabolism. In general, anaerobic bacteria do not harbor (or harbor but do not express) genes encoding the reducing enzyme complex of these ROS species in their genomes (mainly catalase, peroxidase, or superoxide dismutase), or other related activities, such as NAD(P )H peroxidases or alkylhydroperoxide (Ahp) reductase systems that detoxify H ² O ² to H ² O using NAD(P)H.

In recent years, several research groups have studied the response of different species of Bifidobacterium to oxygen. In general, it has been found that members of this genus are sensitive to oxygen and cannot form colonies on agar plates in a normal atmosphere because, as a general rule, growth is arrested in the presence of oxygen by cytoplasmic accumulation of ^H2 . OR ² .

Therefore, there is a need in the state of the art to provide microorganisms of the genus Bifidobacterium spp. aerotolerant to oxygen capable of being used in industrial processes in the food sector, or other similar ones, where the presence of oxygen is unavoidable.

DESCRIPTION OF THE INVENTION

The inventors of the present invention have isolated a bacterium of the genus Bifidobacterium spp., in particular of the species Bifidobacterium bifidum, aerotolerant, that is, it is capable of growing in the presence of oxygen, after being subjected to freezing and/or lyophilization processes, when most of the strains belonging to this genus are sensitive to oxygen, it is toxic to them.

The inventors isolated the aerotolerant B. bifidum strain CECT30106 from the wild or parental strain of B. bifidum Wt (CECT30105) isolated from a biopsy of the ileum of a healthy individual, which, after being subjected to a mutagenesis process by light ultraviolet was able not only to grow on the surface of an agar plate of a defined medium, that is, in a specific solid medium under both aerobic and anaerobic conditions, but in fermented products it was able to do so for longer than other strains, surviving longer. beyond 15 days, specifically, up to 28 days (something never before described in the state of the art), after being subjected to a freezing process at -80oC or a freezing process at -80oC followed by lyophilization by emptiness.

The genomic study of this strain revealed that it showed differentially over-expressed genes related to oxygen resistance and redox reactions, and that the promoters that regulate the expression of these genes showed a common motif located between 12 and 31 nucleotides from the start codon. of the transcript. Based on this discovery, the inventors have developed a series of inventive aspects that will be explained in detail below.

Microorganism of the invention and composition comprising it

In one aspect, the present invention relates to a microorganism of the genus Bifidobacterium spp. comprising a promoter comprising the sequence GAAAGGA (SEQ ID NO: 1) at a distance of between 12 and 31 nucleotides from the start codon responsible for the transcription of a gene. Hereinafter, this microorganism will be called "microorganism of the invention".

Taxonomically, the microorganism of the invention is a microorganism of the genus Bifidobacterium spp., which is a genus of Gram-positive, anaerobic, non-motile bacteria, with varied morphology and frequently branching. However, in the present invention, the bacteria of the genus Bifidobacterium spp. it is aerobic, that is, it can grow and multiply in the presence of oxygen. Therefore, in a particular embodiment, the microorganism of the invention has the ability to grow under aerobic conditions.

In the present invention, "aerobic conditions" are understood as those conditions in which there is the presence of oxygen in the medium surrounding the microorganism.

The microorganism of the invention is characterized by containing a promoter comprising the sequence GAAAGGA (SEQ ID NO: 1) at a distance of between 12 and 31 nucleotides from the start codon responsible for the transcription of a gene.

In the present invention, "promoter" is understood as the DNA region that controls the initiation of transcription of a certain portion of DNA to RNA. A promoter therefore promotes the transcription of a gene. In the present invention, the promoter comprises the sequence GAAAGGA (SEQ ID NO: 1) at a distance of between 12 and 31 nucleotides from the start codon responsible for the transcription of a gene. In a particular embodiment, said gene that is regulated by said promoter is the gene that encodes the protein alkyl hydroperoxide reductase C and/or the gene that encodes the protein alkyl hydroperoxide reductase F.

Protein alkyl hydroperoxide reductase C or AhpC (in English alkyl hydroperoxide reductase C or peroxiredoxirí) is a bacterial enzyme responsible for the reduction of organic hyperoxides directly to their reduced dithiol form. In a particular embodiment, the AhpC protein is the AhpC protein from B. bifidum. In another particular embodiment, the AhpC protein comprises an amino acid sequence that has a sequence identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% with the sequence SEQ ID NO : 2. In another particular embodiment, the AhpC protein comprises, or consists of, an amino acid sequence that has 100% sequence identity with the sequence SEQ ID NO: 2 (GeneBankAccess number SFC21796.1).

SEQ ID NO: 2

001 MTLLQHELTD FTVQAFQNNE FHEVTKADVL GHWSVFFFYP ADFTFVCPTE LEDLAAKYED 061 FKKIGCEIYS VSCDTHFVHKAWHDANEKIAKIQYPMLADP TALLAKDLDT YNEADGVAER 121 GDFIVNPEGKVVAYEVISSN VGRNADELLR RVQASQFVYE HGDQVCPAKW TPGEETIEPS 181 LDLVGLL

Protein alkyl hydroperoxide reductase F or AhpF is a homodimeric flavoenzyme that catalyzes the NADH-dependent reduction of protein AhpC, which in turn catalyzes the reduction of hydrogen peroxide and organic hydroperoxides.

In a particular embodiment, the AhpF protein is the AhpF protein from B. bifidum. In another particular embodiment, the AhpF protein comprises an amino acid sequence that has a sequence identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% with the sequence SEQ ID NO : 3. In another particular embodiment, the AhpF protein comprises, or consists of, an amino acid sequence that has 100% sequence identity with the sequence SEQ ID NO: 3 (GeneBank Access number WP_047290408).

SEQ ID NO: 3

1 MTQINRSDLY DVVVIGGGPA GLTAGLYLARARYRVLILEK DDFGGQITIT DEVVNYPGVG

61 HTSGRALTQT MRNQAKDFGA EFLSAEATGL DVNGDIKTVH TSRGDLKTFG ILIATGASPR 121 KLGFAGEAEYAGRGVAYCAT CDGEFFTGRE VLVVGGGFAAAEESVFLTKYASKVTVLVRE 181 PDFTCDAAVS AEAKNNPKID VRYNVELKGVTAGKGGLREA TILDRGTGET ESWKPSDDGT 241 FGVFVFAGYV PATELVRGVV ELDDHGYVVT RGYLETSVPG VYAAGDLRVK NLRQVVTATA 301 DGAIAAVELE RYAKQMSEKT GLVPPRPTAS AYEQAEAQTAAAVNSAAAAG TTPAPAPVKR 361 SADTAAATAAAKKPGELFSAAIKQQLGVVF GRMTRPVTLV LELDDTPLST ELQGFIGEMV 421 ALSNGKLNSVAVDAAGVITA EDGSSAPTSL TVGEPLAVTL PDGAELPVYG SLDDSGRAQF 481 DVAGVLPSAR PVVLMCVPAE NSEAGTLLFT GLAFHGVPSG HEFNSFVLGL YNAAGPGQPL 541 DDDLKARAEA IDTPIDVMIL VSLTCTMCPE TVLAAQRIAS LNPNVRAEAY DVAHFPELKD 601 QYGAMSVPCI VINQPGGEQK VEFGKKSVPQ MLTLLGA

Another characteristic of the microorganism of the invention is that it has overexpressed genes related to resistance to oxygen and redox reactions, with respect to a microorganism of the genus Bifidobacterium spp. not aerotolerant. In the present invention, "non-aerotolerant" is understood as that bacterium of the genus Bifidobacterium spp. It cannot grow in the presence of oxygen. In a particular embodiment, the over-expressed genes in the aerotolerant strain of Bifidobacterium spp.

they are the gene encoding AphC and the gene encoding AphF. These proteins have been described in previous paragraphs of this description.

The microorganism of the invention can belong to any microorganism of the genus Bifidobacterium spp. Thus, examples of microorganisms of the genus Bifidobacterium include, but are not limited to, B. adolescentis, B. angulatum, B. animalis, B. asteroides, B. bifidum, B. boum, B. breve, B. catenulatum, B. choerinum, B. coryneforme, B. cuniculi, B. denticolens, B. dentium, B. gallicum, B. gallinarum, B. indicum, B. infantis, B. inopinatum, B. lactis, B. longum, B. magnum, B. merycicum, B. minimum, B. pseudocatenulatum, B. pseudolongum, B. pullorum, B. ruminantium, B. saeculare, B. subtile, B. suis, B. thermacidophilum, and B. thermophilum.

In a particular embodiment, the microorganism of the invention is B. bifidum which, in another even more particular embodiment, is the strain B. bifidum IPLA60003. The B. bifidum IPLA60003 strain was deposited under the Budapest Treaty on May 26, 2020 in the Spanish Type Culture Collection as International Depository Authority, located at Building 3 CUE, Pare Científic Universitat de Valencia, Catedrático Agustín Escardino, 9, 46980 Paterna, Valencia, Spain. The deposit number assigned was CECT30106. The depositor of the strain was the Higher Council for Scientific Research.

Any strain derived from the B. bifidum IPLA60003 strain is also contemplated in the present invention, where said strain maintains or improves the ability to grow and develop in the presence of oxygen, that is, it is aerotolerant. The derived microorganism can be produced naturally or intentionally, by mutagenesis methods known in the state of the art, such as, but not limited to, the growth of the original microorganism in the presence of mutagenic or stress-causing agents, or by genetic engineering directed at the modification of specific genes. According to a preferred embodiment, the strain derived from the B. bifidum strain IPLA60003 is a genetically modified mutant. The terms mutant strain or derivative strain may be used interchangeably.

The present invention also relates to the cellular components, metabolites, secreted molecules or any combination thereof, obtained from the B. bifidum IPLA60003 strain, or from a combination of microorganisms comprising at least the B. bifidum IPLA60003 strain.

Cellular components of bacteria could include cell wall components (such as, but not limited to, peptidoglycan), nucleic acids, membrane components, or others such as proteins, lipids, and carbohydrates and their combinations, such as lipoproteins, glycolipids or glycoproteins, or polysaccharides. Metabolites include any molecule produced or modified by the bacterium as a consequence of its metabolic activity during its growth, its use in technological processes (for example, but not limited to, food or drug manufacturing processes), during product storage or during gastrointestinal transit. Examples of these metabolites are, but are not limited to, organic and inorganic acids, proteins, peptides, amino acids, enzymes, lipids, carbohydrates, lipoproteins, glycolipids, glycoproteins, vitamins, salts, metals, or nucleic acids. Secreted molecules include any molecule exported or released abroad by the bacterium during its growth, its use in technological processes (for example, food or drug manufacturing), product storage, or gastrointestinal transit. Examples of these molecules are, but are not limited to, organic and inorganic acids, proteins, peptides, amino acids, enzymes, lipids, carbohydrates, lipoproteins, glycolipids, glycoproteins, polysaccharides, vitamins, salts, metals, or nucleic acids.

As the person skilled in the art understands, the microorganism of the invention can be contained within a composition. Thus, in another aspect, the present invention refers to a composition comprising the microorganism of the invention, hereinafter "composition of the invention", including all the particular embodiments defined in the preceding paragraphs.

As is known, the Bifidobacterium genus is one of the genera of autochthonous commensal or mutualistic bacteria present in the intestinal flora or microbiota, that is, the microorganisms that reside in the colon. They aid in digestion, and are associated with a lower epidemiological incidence of allergies and also prevent some forms of tumor growth. Bifidobacteria can be used as probiotics, playing an important role in the beneficial effect of certain foods or drugs.

Thus, in a particular embodiment, the composition of the invention is a functional nutritional composition, or a nutraceutical.

In the event that the composition of the invention is formulated as a functional nutritional composition, said nutritional composition can be a food or be incorporated into a food or food product intended for both human and animal feed. Thus, in a particular embodiment, the functional nutritional composition is selected from a food (which can be a food for specific nutritional purposes or a food supplement or a functional food) and a nutritional supplement.

The term "functional nutritional composition" or "functional nutritional composition" of the present invention refers to that composition that comprises an ingredient or compound that may be present in a food or in a food supplement, and that beneficially affects one or more functions of the body, so that it provides a better state of health and well-being. Examples of foods that may comprise the microorganism of the invention, or the composition containing the microorganism of the invention, include, but are not limited to, animal feed, dairy products, vegetable products, meat products, snacks, chocolates, beverages, baby foods, cereals , fried, industrial pastries, cookies, etc. Examples of dairy products include, but are not limited to, products derived from fermented (for example, but not limited to, yogurt or cheese) or non-fermented (for example, but not limited to, ice cream, butter, margarine, or whey) milk. . The plant product is, for example, but not limited to, a cereal in any presentation form, fermented or unfermented, or a snack. The beverage may be, but is not limited to, milk or a non-fermented plant-based beverage. However, in a particular embodiment, the food product is selected from the group consisting of a dairy product, a meat product, a vegetable product, a fodder and a beverage.

In another more particular embodiment, the food is a dairy product, even more particularly, a fermented dairy product. In another particular embodiment, the dairy product is selected from the list consisting of milk, cheese, yogurt and kefir.

The term "supplement", synonymous with any of the terms "dietary supplement", "nutritional supplement", "dietary supplement" or "dietary supplement", is a component or components intended to supplement food. Some examples of dietary supplements include, but are not limited to, vitamins, minerals, botanicals, amino acids, and components of food, such as enzymes and glandular extracts. They are not presented as substitutes for a conventional food or as the only components of a meal or a nutritional diet, but rather as a complement to the diet.

The term "nutraceutical" as used in the present invention refers to substances isolated from a food and used in a dosage form that have a beneficial effect on health. Said nutraceutical may be a supplement.

In another particular embodiment, the composition of the invention is administered to a subject through the diet.

As understood by those skilled in the art, the microorganism of the invention must be present in the composition of the invention in a therapeutically effective amount so that they can exert their beneficial effect when administered to a subject. In the present invention, "therapeutically effective amount" is understood as that amount of the component of the pharmaceutical composition that, when administered to a subject, is sufficient to produce the effect. Said component of the pharmaceutical composition refers to the microorganism of the invention. The therapeutically effective amount will vary depending on, for example, the subject's age, body weight, general health, gender and diet, as well as the mode and timing of administration, the rate of excretion or the combination of drugs among others. Thus, in a particular embodiment, the total concentration of microorganisms of the microorganism of the invention in the composition is between 106 and 1012 CFU/mL or grams (colony-forming units = bacteria), preferably 109 CFU/mL or grams. In another particular embodiment, the administration dose of the microorganism of the invention in the composition is between 106 and 1012 CFU/day, preferably 109 CFU/day, and in another even more particular embodiment, the administration regimen is, at least once a day, in particular twice a day, and more particularly three times a day, once with each meal (breakfast, lunch and dinner).

The composition of the invention, in addition to the microorganism of the invention, may comprise other microorganisms or bioactive components.

Other microorganisms that may accompany the microorganism of the invention in the composition include, without limitation, microorganisms of phylogenetic groups, genera or species of prokaryotes of intestinal, food or environmental origin, such as example but not limited to Archaea, Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Verrucomicrobia, Fusobacteria, Metanobacteria, Spirochaetes, Fibrobacteres, Deferribacteres, Deinococcus, Thermus, Cyanobacteria, Methanobrevibacterium, Peptostreptococcus, Ruminococcus, Coprococcus, Subdolingranulum, Dorea, Bulleidia, Anaerofustis, Gemella , Roseburia, Catenibacterium, Dialister, Anaerotruncus, Staphylococcus, Micrococcus, Propionibacterium, Enterobacteriaceae, Faecalibacterium, Bacteroides, Parabacteroides, Prevotella, Eubacterium, Akkermansia, Bacillus, Butyrivibrio or Clostridium.

In a particular embodiment, the composition of the invention additionally comprises a microorganism selected from the group consisting of a yeast, a mold, and a lactic acid bacterium other than Bifidobacterium spp.

Examples of molds that can be combined with the microorganism of the invention include, without being limited to, those belonging to the genus Penicillium (for example, the species P. roqueforti, P. candidum). Examples of yeasts that can be combined with the microorganism of the invention include, but are not limited to, such as Saccharomyces cerevisiae, Geotrichum candidum, Debaromyces hansenii, or the like.

Examples of lactic acid bacteria and other Gram-positive bacteria that can be combined with the microorganism of the invention include, without limitation, bacteria of the genus Lactobacillus, Lactococcus, Leuconostoc, Weissella, Pediococcus, Streptococcus, Propionibacterium, Bacillus.

Examples of bacteria of the above genera include, without limitation, bacteria belonging to the genus Lactobacillus such as Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus gallinarum, Lactobacillus amylovorus, Lactobacillus brevis, Lactobacillus rhamnosus, Lactobacillus kefir, Lactobacillus paracasei , Lactobacillus crispatus, Lactobacillus zeae, Lactobacillus helveticus, Lactobacillus salivalius, Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus xeuteri, Lactobacillus crispatus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus johnsonii, Lactobacillus pentosus, Lactobacillus mali and the like; bacteria of the genus Streptococcus such as Streptococcus thermophilus ; bacteria of the genus Lactococcus such as Lactococcus lactis subsp. lactis, Lactococcus lactis subsp.

cremoris and the like; Enterococcus genus bacteria such as Enterococcus faecalis, Enterococcus faecium and the like; Bacillus genus bacteria such as Bacillus subtilis; bacteria of the genus Propionibacterium, such as Propionibacterium freudenreichii, Propionibacterium jensenii, Propionibacterium acidipropionic, and the like.

The composition according to any of those defined above, may also comprise at least one bioactive component (active substance, active ingredient or therapeutic agent), such as other food components, plant and/or animal products, minerals, vitamins, or the like. .

The term "bioactive component" refers to a compound with biological activity within the scope of the patent application that can improve or complement the activity of the microorganism of the invention, including ingredients or components of foods (for example and without limiting : polyunsaturated fatty acids, conjugated linoleic acid, prebiotics, fiber, Guar gum, glucomannan, chitosan, copper picolinate, calcium, etc.), plants, extracts or components of plants (for example and without limitation polyphenols, ephedrine or extracts of Ephedra spp., green tea [Camellia sinensis], or bitter orange [Citrus aurantium]).

Due to the beneficial effects that microorganisms of the genus Bifidobacterium have on the health and well-being of a subject, the present invention also contemplates that the microorganism of the invention can be used in a pharmaceutical composition. Thus, in another particular embodiment, the composition of the invention is a pharmaceutical composition.

The "pharmaceutical composition" is a set of components that is formed by at least the microorganism of the invention in any concentration, which has at least one application in the improvement of the physical or physiological or psychological well-being of a subject, which implies an improvement of the state overall health or reduced risk of disease. Said pharmaceutical composition may be a medicament.

The medicament to which the present invention refers can be for human or veterinary use. "Medicine for human use" is any substance or combination of substances that is presented as having properties for the treatment or prevention of diseases in humans or that can be used in humans or administered to humans in order to restore, correct o modify physiological functions by exerting a pharmacological, immunological or metabolic action, or to establish a medical diagnosis. The "veterinary medicine" is any substance or combination of substances that is presented as having curative or preventive properties with respect to animal diseases or that can be administered to the animal in order to restore, correct or modify its physiological functions exercising a pharmacological, immunological or metabolic action, or to establish a veterinary diagnosis. "Veterinary medicines" will also be considered "premixes for medicated feed" prepared to be incorporated into a feed.

In addition to the requirement of therapeutic efficacy where said pharmaceutical composition may necessitate the use of other therapeutic agents, there may be additional rationales that strongly mandate or recommend the use of a combination of the microorganism of the invention and a bioactive component as described above. previously defined.

In a particular embodiment, the pharmaceutical composition also comprises at least one pharmaceutically acceptable carrier and/or excipient.

The term "excipient" refers to a substance that helps the absorption of any of the components of the composition of the present invention, stabilizes said components or helps to prepare the pharmaceutical composition in the sense of giving it consistency or providing flavors that make it more enjoyable. Thus, the excipients could have the function of keeping the components together, such as starches, sugars or cellulose, sweetening function, colorant function, drug protection function such as to isolate it from air and/or humidity, function filling of a pill, capsule or any other form of presentation such as dibasic calcium phosphate, disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph. Therefore, the term "excipient" is defined as that material which, included in galenic forms, is added to the active ingredients or their associations to enable their preparation and stability, modify their organoleptic properties or determine the physical-chemical properties of the substance. Pharmaceutical composition and its bioavailability. The "pharmaceutically acceptable" excipient must allow the activity of the compounds of the pharmaceutical composition, that is, it must be compatible with said components.

The "galenic form or pharmaceutical form" is the disposition to which the active principles and excipients are adapted to constitute a medicine. It is defined by the combination of the form in which the pharmaceutical composition is presented by the manufacturer and the form in which it is administered.

The "vehicle" or carrier is preferably an inert substance. The function of the vehicle is to facilitate the incorporation of other compounds, allow a better dosage and administration or give consistency and shape to the pharmaceutical composition. Therefore, the vehicle is a substance that is used in the medication to dilute any of the components of the pharmaceutical composition of the present invention to a certain volume or weight; or that even without diluting said components it is capable of allowing a better dosage and administration or giving consistency and shape to the medication. When the presentation form is liquid, the pharmaceutically acceptable carrier is the diluent.

In addition, the excipient and the vehicle must be pharmacologically acceptable, that is, the excipient and the vehicle is allowed and evaluated so that it does not cause harm to the organisms to which it is administered.

In each case, the presentation form of the pharmaceutical composition will be adapted to the type of administration used, therefore, the composition of the present invention can be presented in the form of solutions or any other clinically permitted form of administration and in a therapeutically effective amount. . The pharmaceutical composition of the invention can be formulated in solid, semi-solid, liquid or gaseous forms, such as tablet, capsule, powder, granule, ointment, solution, suppository, injection, inhalant, gel, microsphere or aerosol. According to an even more preferred embodiment of the present invention, the pharmaceutical composition is presented in a form adapted for oral administration.

The form adapted to oral administration refers to a physical state that can allow oral administration. Said form adapted to oral administration is selected from the list that includes, but is not limited to, drops, syrup, tisane, elixir, suspension, extemporaneous suspension, drinkable vial, tablet, capsule, granules, seal, pill, tablet, troche or lyophilized.

Another possibility is that the pharmaceutical composition is presented in a form adapted to sublingual, nasal, intracatecal, bronchial, lymphatic, rectal, transdermal administration, inhaled or parenteral. The strain of the invention; the cellular components, metabolites, secreted molecules or any of their combinations, obtained from the strain of the invention, or the composition of the invention; they can be associated, for example, but not limited to, with liposomes or micelles.

In the sense used in this description, the expression "therapeutically effective amount" refers to that amount of the component of the pharmaceutical composition that when administered to a mammal, preferably a human, is sufficient to produce the prevention and/or treatment , as defined below, of a disease or pathological condition of interest in the mammal, preferably a human. Said component of the pharmaceutical composition refers to the strain of the invention; or to cellular components, metabolites, secreted molecules; or a combination thereof, and which may optionally be included in said composition in combination with an additional bioactive component, and which contribute to the therapeutic effect of the pharmaceutical composition. The therapeutically effective amount will vary, for example, according to the activity of the microorganism of the invention; of the additional microorganism or additional microorganisms that comprise the composition of the invention; excretion rate, drug combination; the severity of the particular disorder or pathological condition; and the subject undergoing therapy, but may be determined by one skilled in the art on the basis of his or her own knowledge and this disclosure.

Uses of the microorganism and of the composition of the invention

As previously explained, the microorganism of the invention, because it belongs to the genus Bifidobacterium spp., has beneficial effects on the health and well-being of a subject to whom the microorganism of the invention is administered.

Thus, in another aspect, the present invention relates to the microorganism of the invention, or the composition of the invention, for its use as a medicine.

In another aspect, the present invention relates to the microorganism of the invention, or the composition of the invention, for its use in the treatment and/or prevention of constipation, Helicobacter pylori infection, irritable bowel syndrome (IBS ), inflammatory bowel disease (such as ulcerative colitis, pouchitis after surgery for ulcerative colitis, or Crohn's disease), autoimmune diseases, inflammatory conditions, and diarrhea.

The term "treatment", as understood in the present invention, refers to combating the effects caused by a disease or pathological condition of interest in a subject (preferably a mammal, and more preferably a human) including:

(i) inhibit the disease or pathological condition, that is, stop its development; (ii) alleviate the disease or pathological condition, that is, cause regression of the disease or pathological condition or its symptomatology;

(iii) stabilize the disease or pathological condition.

The term "prevention" as understood in the present invention consists of avoiding the appearance of the disease, that is, preventing the disease or pathological condition from occurring in a subject (preferably a mammal, and more preferably a human), in particular, when said subject has a predisposition for the pathological condition.

In the present invention, the term "constipation" refers to that process suffered by a subject who has three or fewer stools in a week. The stools may be hard and dry.

In the present invention, " H. pylori infection" is understood as a bacterial infection that causes inflammation of the stomach (gastritis), gastroduodenal ulcer and certain types of stomach cancer.

In the present invention, "inflammatory bowel disease" (ulcerative colitis and Crohn's disease) is understood to mean an inflammatory disease of the colon (the large intestine) and/or the rectum. It is characterized by inflammation and ulceration of the inner wall of the colon. Typical symptoms include diarrhea (sometimes bloody) and often abdominal pain.

In the present invention, "irritable bowel syndrome" is understood as that syndrome that affects the large intestine and that can cause abdominal cramps, bloating and changes in bowel habits, giving rise to both constipation and diarrhea depending on the affected subject.

In the present invention, "pouchitis" or "pouchitis" is understood as the disease of unknown cause that consists of a non-specific inflammation of the ileoanal reservoir.

The ileoanal reservoir is performed in those subjects who have required a panproctocolectomy, regardless of the underlying pathology.

In addition to the characteristics of the Bifidobacterium spp. genus, the microorganism of the invention has the additional capacity to tolerate oxygen and to remain viable under aerobic conditions for more than 15 days, even reaching 28 days, in a fermented milk product. . This makes its use in the food industry in the production of fermented dairy foods, or functional foods, where most of the processes are carried out under aerobic conditions, of special interest.

Therefore, in another aspect, the present invention relates to the use of the microorganism of the invention, or of the composition of the invention, in the production of fermented milk derivatives.

In the present invention, "fermented dairy derivative" is understood as dairy products from lactic cultures due to the action of lactic acid bacteria (Lactobacillales) such as Lactobacillus, Lactococcus, and Leuconostoc.

There are many types of cultured or fermented dairy products and their variants can be found throughout the world. Examples of fermented milk products include, but are not limited to, cheeses, yogurt, cottage cheese, buttermilk (United States, Canada), acidophilus milk (United States, Canada), kiselo mlyako (Bulgaria), sauermilch or dickmilch (Germany), zure melk (Netherlands), lapte bátut (Romania), filmjólk or fil (Sweden), surmelk or kulturmelk (Norway), piimá and viili (Finland), amasi (South Africa), calpis (Japan), buttermilk (Venezuela) and pilfrut (Bolivia).

The production of the vast majority of fermented dairy products is based on the use of industrially produced starter cultures, forced in part by the use of pasteurized milk in which the original milk microbiota has been partially destroyed. The use of this type of culture, together with the use of more hygienic processing methods and controlled ripening conditions, has had an enormously positive effect on quality, providing microbiologically safer products with more reproducible organoleptic and theological properties.

Methods of the invention

In another aspect, the present invention relates to a method for producing a fermented milk product that comprises incubating the microorganism of the invention, or the composition according to the invention, with a milk product under the appropriate conditions for carrying out the fermentation. industrial fermentation process. By way of example, but not limited to, in a typical fermentation process pasteurized milk would be used, which would be inoculated with a starter culture and/or with the microorganism of the invention, incubated at 37°C until reaching a pH less than or equal to 4.5 and cooled to 4°C for subsequent refrigerated storage during its useful life, which could extend beyond 28 days.

The microorganism of the invention may have previously undergone a lyophilization and/or freezing process prior to incubation with the dairy product, without this altering its ability to carry out the fermentation of the dairy product in the presence of oxygen.

Examples of dairy products that can be fermented with the microorganism or composition of the invention include, without limitation, milk.

All the particular embodiments previously described for the microorganism of the invention and the composition of the invention throughout the description, both individually and in combination, are applicable to the present inventive aspect.

In another aspect, the present invention relates to a method for conferring air tolerance to a microorganism of the genus Bifidobacterium spp. comprising transforming a microorganism of the genus Biofidobacterium spp. oxygen intolerant with a promoter comprising the sequence GAAAGGA (SEQ ID NO: 1) at a distance of between 12 and 31 nucleotides from the start codon responsible for the transcription of a gene.

In the present invention, "bacterial transformation" is understood as a process of genetic material transfer through which certain bacteria, for example, those of the genus Bifidobacterium spp, can collect genetic material from other organisms. Bacterial transformation methods are known in the state of the art and are routine practice for those skilled in the art.

All the terms used in the present inventive aspect, as well as those of its particular embodiments, have been explained for previous inventive aspects, and are applicable to the present inventive aspect.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Aerotolerant phenotype of B. bifidum CECT30106. CECT30106 colonies growing on the surface of agar plates in aerobiosis and anaerobiosis.

Figure 2. Metabolic pathways affected by aeration. A) Different metabolic pathways (FDR <0.05; with two exceptions marked with an asterisk) belonging to the central metabolism that are overexpressed during the growth of strain CECT30106 under aeration compared to anaerobic conditions. B) In addition, genes encoding activities involved in drug metabolism are also over-expressed

Figure 3. Common motif shared by the promoters of the overexpressed genes in strain CECT30106. The relative expression levels of these genes under aerated and anaerobic conditions, obtained from the RNAseq data, are presented on the right. The first ATG is marked in bold. This motif is proposed as the target of three regulatory proteins that present changes in their amino acid sequence in the CECT30106 strain with respect to the wild-type Wt strain (CECT30105).

Figure 4. Changes induced in strain CECT30106 in the presence of oxygen. (A) ATP content normalized by protein concentration. (B) Plot of redox ratios calculated as the ratio of FAD to the sum of FAD plus NADH fluorescent signals after growth of strain CECT30106 under anaerobiosis, aerobiosis, and aeration. (C) Total and specific FiFo-ATPase activities (type F) in membranes of strain CECT30106 obtained from cells grown aerobically or anaerobically.

Figure 5. Growth (Log CFU/ml) of the three strains of B. bifidum in different experimental conditions: fresh standardized cultures, cultures of strains subjected to freezing (direct seeding after the process, or cultivated for 24 hours in the presence of O ² or in anaerobiosis), and cultures of strains subjected to lyophilization (idem).

Figure 6. Evolution of pH and counts (Log CFU/ml) of milk fermented with three strains of B. bifidum. For pH and counts, only the mean values of three biological replicates are shown; coefficients of variation for these data (% mean/standard deviation) ranged from 0.01-6.49% for pH and 1.53-45.0% for counts. The mean and standard deviation of the final survival (%CFU 27 days/CFU final fermentation) of the strains after refrigeration for 27 days are shown.

EXAMPLES

Next, the invention will be illustrated by tests carried out by the inventors, which show the effectiveness of the product of the invention.

Example 1. Characterization of aerotolerant B. bifidum CECT30106

1. MATERIAL AND METHODS

1.1 Bacterial strains and growth conditions.

Bifidobacterium bifidum LMG11041 [Type strain (ATCC29521T) purchased from Belgian collection], CECT30105 and CECT30106 were routinely cultured in MRS (Biokar Diagnostics, Beauvais, France) supplemented with 0.05% (w/v) L-cysteine (Sigma Chemical Co. ., St. Louis, MO, USA) (MRSC) or in BHI (Oxoid, Ltd., Baingstoke, Hampshire, UK) supplemented with 0.05% (w/v) L-cysteine HCl monohydrate (BHIC , Sigma, St. Louis, MO) and 2% (w/v) glucose (Sigma, St. Louis, MO). Depending on the aerotolerance phenotype, these strains were incubated for 24 hours at 370C in i) an MG500 anaerobic chamber (Don Whitley Scientific, West Yorkshire, UK) under 10% (v/v) H ² , 10% CO ² and 80% N ² (referred to as anaerobic conditions), ii) a conventional incubator (referred to as aerobic or aerobic conditions), or iii) an Excella E24 rotary shaker-incubator (New Brunswick Scientific, Edison, NJ) (referred to as aeration conditions) . In the latter case, 200 mL liquid cultures were grown in 500 mL flasks under agitation (at 250 rpm). Growth in liquid cultures was monitored by following OD600nm at different incubation times.

1.2. Generation of an aerotolerant strain of B. bifidum.

To generate mutants, strain CECT30105, isolated from the mucosa of the ileum of a healthy individual, was propagated in MRSC. Cultures were incubated at 37°C under anaerobic conditions and an overnight culture was used to inoculate 50 mL of fresh MRSC (1% v/v). Exponential phase cultures (OD600nm = 0.4) were washed twice with PBS and resuspended in 5 mL of PBS that were placed in a sterile Petri dish; finally, they were exposed to UV light (ultraviolet radiation sterilization table, JP Selecta, Barcelona, Spain) for 3 minutes. Subsequently, they were seeded on the surface of MRSC and after incubation in aerobiosis a single colony was observed that was able to form colonies in the Petri dish I. This strain was named CECT30106.

1.3. Genome sequencing.

Total DNA from CECT30105 and CECT30106 was extracted using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany), with a modified protocol for Gram-positive bacteria. Genome sequencing was performed at GenProbio SRL, (Italy) using the Illumina NextSeq paired-end method, according to standard Illumina protocols, using the Illumina HiSeq2000 sequencer. Both genomes were uploaded and assembled into PATRIC using the SPADES built-in assembler (https://www.patricbrc.org). The genome sequence of CECT30105 consisted of 1,850,464 reads of 250 bp of paired ends according to the Illumina HiSeq2000 sequencer. The result of the final assembly was a set of 32 unoriented contigs, with a total length of 2,168,250 bp and a GC content of 62.6%. Open Reading Frames (ORF) and tRNA were calculated generating a total number of 1808 and 55, respectively. In the case of strain CECT30106, a total number of 1,572,920 paired-ended 250-bp reads were assembled in 54 unoriented contigs, for a total length of 2,185,465 bp and a GC content of 62.5%. , obtaining 1817 ORF and 56 tRNA-encoding genes.

1.4. Comparison of the genomes of the and analysis of single nucleotide polymorphism.

Genome comparison between strains CECT30105 and CECT30106 was performed using the Mauve v2.4.0 tool (Darling, et al. 2004, Genome Research, 14(7):1394-403). Genome contigs from both strains were sequenced and aligned against the reference genome of Bifidobacterium bifidum strain PRL2010 (NCBI RefSeq sequence: NC_014638.1). The Muscle 3.6 aligner was selected (Edgar, 2004 Nucleic Acids Res. 32(5):1792-1797; Edgar, 2004 BMC Bioinformatics, (5) 113) with default parameters.

In addition, single nucleotide polymorphism (SNP) analysis was carried out to discover gene mutations in strain CECT30106. The alignment of the assembly files of both strains was performed using the LAST web service (Kielbasa, S. M., et al. 2011 Genome Res. 21,487-493). The highest scoring alignment type was selected to align the assembly files of both strains. The alignment of the first and last 300 base pairs of each with you has not been taken into account.

1.5. Cell lysis and RNA extraction

All the material used in this stage was previously autoclaved twice for 15 minutes at 1210C. Cell cultures of strain CECT30106 grown under anaerobic or aerobic conditions in the exponential growth phase (OD600nm « 0.6) were collected, centrifuged (10,000 x g, 5 minutes, room temperature) and treated with RNA-Protect following the instructions. from the manufacturer (Qiagen). Bacterial pellets were subsequently subjected to acidic phenol extraction, resuspending cells in 400 pL TE, 250 pL acidic phenol (Calbiochem), 150 pL chloroform (Merck), 50 pL 10% (w/v) SDS ) and 500 pL of zirconia/silica beads (0.5 - 0.1 mm; BioSpec). The mixture was kept on ice and treated in a FastPrep-24 (MP) device with 4 pulses of 30 seconds at maximum speed, with interspersed 1-minute incubations on ice. The lysed cells were centrifuged (10,000 x g, 10 minutes, 40C) and the upper phase was transferred to cold tubes containing 500 pL of chloroform. The tubes were centrifuged again (10,000 x g, 5 minutes, 40C) and the resulting upper phase transferred to a clean tube. These resulting supernatants were used to obtain at least 20 pg of total RNA using the RNeasy kit following the manufacturer's instructions (Qiagen) and including column digestion with the RNAse-free DNAse kit (Qiagen). Total RNA concentration was estimated using a NanoDrop device.

1.6. RNA sequencing and RNASeq analysis - Aliquots of 20 pg of total RNA were sent for sequencing to the GenProbio SRL facility, where the rRNA was depleted using the riboZero kit (lllumina). Library preparation, sequence execution, and raw data were obtained on an Illumina Myseq machine following the manufacturer's instructions.

Finally, 4 million reads of 75 nt were obtained for each sample. For RNASeq analysis, the genomic sequence of strain B. bifidum CECT30106 and its format annotation were previously obtained from the PATRIC server. Raw RNASeq data was first quality checked with FASTQC and filtered if necessary using the FASTX toolkit. A genomic index of strain CECT30106 was made with Bowtie2, and RNASeq reads were aligned. Mapped reads were converted to *.bam format and counted using the Counts tool. The output of this software was used as input for DESeq2 from library R, which was used to calculate differential gene expression between aerobic and anaerobic conditions. All plots were obtained in the R environment using the following libraries: RcolorBrewer, gplots, genefilter, and calibrate.

1.7. Differential analysis of gene expression

Differential analysis of expression between aerobic and anaerobic growth conditions of strain CECT30106 was performed with the Hisat2+HTseq+DESeq2 procedure (Wen, G. A Simple Process of RNA-Sequence Analyzes by Hisat2, Htseq and DESeq2. in Proceedings of the 2017 International Conference on Biomedical Engineering and Bioinformatics - ICBEB 2017 11-15 (ACM Press, 2017). do¡:10.1145/3143344.3143354). This protocol uses HISAT2 (Kim, D., et al. 2015. Nat. Methods 12, 357-360) to align sample reads, HTSeq (Anders, S., et al.

2015. Bioinformatics 31, 166-169) to normalize raw counts and DEseq (Love, MI, et al. 2014. Genome Biol. 15, 550) to perform differential expression analysis. The genome of CECT30106 was selected to align and annotate twelve samples from each condition. Genes with q value <0.05 and |log2(fc)|>1 were defined as significantly differentially expressed.

1.8. Enrichment of differentially expressed enzyme pathways.

The EC2KEGG v1 tool (Porollo, A. 2014. Source Code Biol. Med.

9, 19) to identify enriched pathways between the two growth conditions. For said enrichment, significantly differentially expressed enzymes, ie genes with Enzyme Commission (EC) number, were selected. Bifidobacterium bifidum BGN4 (bbf; in KEGG database: Kyoto Encyclopedia of Genes and Genomes) was selected as the reference genome.

1.9. Discovery of motifs in gene promoters.

The identification of common motifs shared by the promoters of the differentially expressed (overexpressed) genes of strain CECT30106 was performed using the Multiple Em for Motif Elicitation (MEME) Suite 5.0.3 tool (Bailey, T. L. et al.

2009. Nucleic Acids Res. 37, W202-W208). Classic discovery mode was selected to find motifs between 6 and 50 amino acids in length.

1.10. Estimation of the redox equilibrium by fluorescence spectroscopy.

The fluorescence properties of buffered cell suspensions (OD600nm 0.6) in 50 mM Tris-HCl buffer pH 7.0 were monitored in an Eclipse fluorescence spectrophotometer (Varian, Inc., Palo Alto, CA). To estimate the redox balance of cells, the inventors used the method described by Ammor et al. (Ammor, M. S et al. 2007. J. Microbiol. Methods 69, 100-6), in which the intensity values of fluorescence corresponding to NADH were calculated from the emission at 413 nm after applying an excitation wavelength (Aex) of 316 nm, while for FAD+ the intensity values were calculated from the emission at 436 nm at an Aex 380nm. The redox ratio was deduced from the NADH and FAD+ data obtained, using the following equation: redox ratio = FAD+ Intensity/(FAD+ Intensity NADH Intensity).

1.11. ATPase activity.

ATPase activity present in flipped membranes was calculated from the release of inorganic phosphate, measured colorimetrically after its reaction with sodium molybdate and malachite green, as previously described (Sánchez, B., et al. 2006 Environ. Microbiol. 8, 1825-1833). The membranes of the CECT30106 strain were obtained from independent cultures (50 mL) under the conditions listed above (aeration, aerobiosis and anaerobiosis). Bacterial pellets were resuspended in 4 mL of 100 mM potassium phosphate buffer, pH 7.0, supplemented with 10 mM MgSÜ ⁴ and treated with 10 mg/mL lysozyme and 50 units/mL mutanolysin. The suspensions were incubated at 300C for 4 hours with constant agitation. Cells were disrupted by passing them once through a cell disruptor (Constant Systems Ltd., Daventry, UK) at 2.06 X 108 Pa. Cells without break down and cellular debris. The protein concentration of the membrane extract was measured using a BCA protein assay kit (Pierce, Rockford, II) following the manufacturer's instructions. The specific activity of FiFo-ATPase was calculated by preincubating the membranes for 10 minutes at 37C and subsequently for 60 minutes. minutes on ice with dicyclohexylcarbodiimide (DCCD) (Sigma; final concentration 0.2 mM), which is capable of inhibiting all ATPases with the exception of the F ¹ F ⁰ class (Sánchez, B., et al. 2006. Environ. Microbiol. 8, 1825-1833). Membrane samples without inhibitor were measured for activity and used as a control for total ATPase activity.

1.12. Measurement of intracellular ATP content

Intracellular ATP content of anaerobically or aerobically grown strain CECT30106 was determined by fluorimetric assay on cell lysates using the ATP assay kit (Abcam Cambridge, MA, USA) following the manufacturer's instructions. Briefly, the amount of ATP present in cell lysates obtained by sonication was measured using serial dilutions of an ATP standard that was correlated with arbitrary bioluminescence units to obtain a calibration line for calculating ATP molarity. The bioluminescence of ATP standards and cell lysates was measured on a Cary Eclipse fluorescence spectrophotometer (Varian, Palo Alto, CA) with an integration time of 1 s. ATP bioluminescence intensity values were calculated from the emission wavelength of 535 nm after applying an excitation wavelength of 380 nm. ATP levels were corrected for protein concentration, which was estimated with a BCA protein assay kit (Pierce, Rockford, II). The results were expressed as nmoles of ATP per mg of total protein.

2. RESULTS

2.1 Generation of the aerotolerant strain of B. bifidum.

The CECT30105 strain, which is called non-mutant or wild type (wt), was isolated from a biopsy of the ileum of a healthy individual. This strain, which also showed a slight aerotolerance phenotype unprecedented in the literature, was subjected to random mutagenesis by UV irradiation. Mutants capable of forming colonies on the surface of agar plates were selected by incubating the plates under a normal atmosphere (aerobiosis or aerobic) at 37 C. After this procedure, the inventors were able to isolate a single colony that was named CECT30106 , which was properly identified as B. bifidum after amplification and sequencing of the 16S ribosomal DNA gene. Interestingly, both MRSC and BHI supported growth of strain CECT30106 in liquid, but the presence of visible colonies on agar plates was only observed on BHI-agar. supplemented with 1.8% (w/v) glucose and 0.25% (w/v) cysteine (Figure 1). The strain was able to grow up to an OD600nm of 2.36 ± 0.17 in static liquid cultures (aerobiosis) and up to OD600nm values of 1.34 ± 0.28 when 200 mL cultures were subjected to constant agitation in 500 mL flasks (200 rpm; aeration).

The genome of strain CECT30106 shows the typical genetic traits of a bifidobacterium, such as the presence of the typical glucose fermentation pathway called “bifido pathway”, a complete F ¹ F ⁰ -ATP synthase cluster, or a salt hydrolase gallstones, as well as 141 genes involved in carbohydrate metabolism. However, 57% of the genes were not assigned to a specific functional category corresponding to 528 hypothetical protein-coding genes. Within the genome, genes encoding homologues of oxygen detoxification enzymes such as BatA, BatB, alkylhydroxyperoxide reductase protein C and F, and protoporphyrinogen oxidase proteins were detected.

2.2. Comparison of genomes and identification of single nucleotide polymorphisms.

Single nucleotide polymorphism (SNP) analysis was performed to detect UV-induced mutations in the CECT30106A strain with respect to the non-mutant (wt or CECT30105), which could be responsible for its aerotolerance phenotype. Twenty-six SNPs were identified in the CECT30106 strain with respect to the non-mutant CECT30105 strain (Table 1), three of them being located in intergenic regions and the rest in open reading frames, which corresponded in most cases to hypothetical proteins. . The remaining genes affected by UV radiation encoded 5,10-methylenetetrahydrofolate reductase, a TPR repeat-containing protein, a multi-antimicrobial extrusion protein, ADP-glucose transglucosylase, isoleucyl-tRNA synthetase, an LSU ribosomal protein and a regulatory protein with BlastP similarity to regulators of arsenic sensing.

Table 1. SNPs in the CECT30106 genome with respect to the CECT30105 strain.

2.3 Changes in gene expression profiles.

Differential gene expression between aerobic and anaerobic growth conditions of strain CECT30106 was analyzed by RNAseq. This analysis showed that under aerated conditions, hundreds of genes were overexpressed (553, FDR < 0.05), in particular several involved in oxygen resistance and redox reactions (e.g., oxygen-insensitive NADPH nitroreductase, thioredoxin, alkylhydroxyperoxide reductase proteins C and F), most of the “bifido pathway” genes, and the FiF0-ATPase operon. In general, the different gene expression profiles of strain CECT30106 grown in aeration with respect to anaerobiosis allowed pooling of RNASeq experiments by condition using an unsupervised PCA.

2.4. Enrichment of differentially expressed enzyme pathways.

The clustering of differentially expressed genes between the two growth conditions (aeration versus anaerobiosis) in strain CECT30106 revealed changes mainly related to the central metabolism of the bacterium. they only had take into account pathways with FDR values equal to or less than 0.05 (Figure 2A). The high concentration of oxygen in the growth medium increased the expression of genes that belong to pathways related to glycolysis/gluconeogenesis, utilization of carbohydrates, amino- and nucleotide sugars, nicotinate and nicotinamide, energy, metabolism of xenobiotics (drugs, Figure 2B) and microbial metabolism, along with others related to the biosynthesis of secondary metabolites. In terms of pathways with decreased expression, the number was markedly lower and mainly affected metabolic and biosynthetic pathways of secondary metabolites. Notably, most of the "bifido pathway" genes were overexpressed, including NAD-dependent glyceraldehyde-3-phosphate dehydrogenase and acetate kinase, suggesting that growth on aeration forces the bacteria to synthesize more ATP and binding agents. redox to fuel the cellular machinery responsible for overcoming oxidative stress. This observation was reinforced by the fact that the gene encoding L-lactate dehydrogenase was reduced in expression by 2.4-fold in aeration compared to anaerobiosis. Many other genes encoding enzymes whose activities feed into the central energy harvesting pathway were overexpressed, although genes encoding many glycolytic enzymes and sugar transporters were repressed by oxygen. Finally, genes encoding enzymes involved in the synthesis of surface-associated and structural components, such as peptidoglycan, teichoic acids, and lipids, were affected by the presence of oxygen during CECT30106 growth.

2.5. Over-expressed gene promoters that share a genetic motif.

An analysis was carried out to identify motifs shared by the promoters of the most significantly over-expressed genes in strain CECT30106 grown aerobically. Sixteen gene promoters related to the aerotolerance phenotype of this strain were selected. Eleven of these 16 promoters shared a common motif (GAAAGGA (SEQ ID NO: 1); e value = 0.0031) at a distance between 12 and 31 nucleotides from the initiation codon. This fact was associated with the presence of a ribosomal protein and a genetic regulator in which the SNPs induced by UV radiation determined an amino acid change in the coding sequence of the final protein (Figure 3), and this motif is proposed as a target of the two mutated genetic regulators. In particular, genes encoding alkylhydroperoxide reductase protein C and F, known to function in endogenous hydrogen peroxide detoxification, were among the genes most upregulated in their expression (histogram in the figure). A simple sequence analysis showed that these proteins they are not present in all Bifidobacterium species, and their upregulation of their expression seems crucial for aerotolerance.

2.6 Physiological and biochemical changes in response to oxygen.

To corroborate some of the results obtained after the global analysis of gene expression by RNASeq, different biochemical and enzymatic tests were performed on strain CECT30106 grown under aerated, aerobic or anaerobic conditions. First, the intracellular ATP concentration in CECT30106, measured as nmoles of ATP normalized by mg of total cytoplasmic protein, was significantly higher in aeration than in aerobiosis or anaerobiosis (Figure 4A). Second, the redox ratio calculated from the fluorescence values of the cofactors FAD+ and NADH did not show perceptible changes between the three conditions, although the individual signals of FAD+ and NADH decreased in the aerobic and aerated conditions (Figure 4B). . The present inventors finally measured the F-type ATPase activity present in B. bifidum membranes. For this, the inventors included the F ¹ F ⁰ type inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) in the experiments (Solioz, 1984. Trends Biochem. Sci. 9, 309-312). No differences were found in the total activity or in the activity of type F ATPase between the aerobic and anaerobic conditions (Figure 4C), suggesting that although all the genes included in the FiF0-ATPase cluster were found to be over-expressed by the technique of RNASeq, this did not translate into higher activity, at least under our experimental conditions.

Example 2. Technological properties of Bifidobacterium bifidum strains.

1. MATERIALS AND METHODS

1.1. Bacteria and culture conditions

In this study, 3 strains of Bifidobacterium bifidum have been used, whose codes and origin are shown in Table 2.

C. IPLA C. CECT Observations

LMG11041 - Type strain (= ATCC29521T) acquired from Belgian collection

IPLA60008 CECT30105 Parental strain, new isolate from human biopsy

IPLA60003 CECT30106 Derived strain, adapted in the laboratory to

or 2

Table 2. Codes of the B. bifidum strains used in this study.

The B. bifidum IPLA60008 strain was deposited under the Budapest Treaty on May 26, 2020 in the Spanish Type Culture Collection as International Depository Authority, located at Building 3 CUE, Pare Científic Universitat de Valencia, Catedrático Agustín Escardino, 9, 46980 Paterna, Valencia, Spain. The deposit number assigned was CECT30105. The depositor of the strain was the Higher Council for Scientific Research.

Routinely, unless otherwise specified, strains LMG11041 and IPLA60008 were grown in MRSc (Biokar's MRS 0.25% L-cysteine-HCI monohydrate) from stocks stored at -80oC which were seeded on surface of MRSc-agar and incubated at 37°C under anaerobic conditions in the Mac500 cabinet (Down Whitley Scientific, West Yorkshire, UK) with an atmosphere of 10% H2, 10% C02 and 80% N2, for at least 3 days. Subsequently, colonies of each strain were picked to inoculate 10 ml of MRSc broth that was incubated overnight under the same conditions. However, the CECT30106 strain was thawed directly in the BHIGc culture medium (Oxoid BHI, 2% glucose 0.25% L-cysteine-HCI monohydrate), before proceeding to inoculate 10 mL of fresh medium corresponding to each strain to generate the pre-culture.

The liquid pre-cultures were used to inoculate different volumes of different media as indicated in the following sections, with the aim of standardizing the processes. In general, the counts for strains LMG11041 and CECT30105 were made on MRSc-agar and for strain CECT30106 on BHIGc-agar.

1.2. Survival to freezing and lyophilization

With this experimental design we intend to verify the behavior of the three strains under study against technological processes used to obtain biomass from bacterial cultures that, later, can be used in companies in the food, pharmaceutical or biotechnology sector. The resistance, and subsequent ability to grow in the presence of 02, of the three strains against freezing (at -80oC) and against lyophilization (which also includes a prior freezing step at -80oC) have been studied. To compare the results, standardized fresh (active) cultures have been used. As starting material, 50 ml of the medium corresponding to each strain were used, which were inoculated (2%) with the pre-cultures described in the previous section, which were incubated under standard anaerobic conditions for 19±1 hours. The 50 mL grown cultures were the inoculum used for the following procedures, which were performed in triplicate (3 biological replicates).

To carry out the "fresh cultures" (F), 2 tubes (1%) were inoculated with 10 mL of fresh medium: the FA tube was incubated under anaerobic conditions and the FO tube was incubated at 37°C in a standard oven (therefore in the presence of air and oxygen). After 24 hours of incubation, the viable cells were counted, using the corresponding media, incubating the plates under anaerobic conditions for at least 3 days.

To carry out the "cultures after freezing" (C), 24 mL of the starting culture were used, washed with PBS and resuspended in 2.4 mL (concentration factor x10) of the corresponding medium with 30% glycerol as agent. cryoprotectant, freezing it at -80oC (New Brunswick U500 freezer) for 24 hours. After this period, the tubes were thawed at room temperature; On the one hand, direct seeding was carried out after freezing and, on the other hand, 2 tubes were inoculated (1%) with 10 mL of fresh medium: the CA tube was incubated under anaerobic conditions and the CO tube was incubated at 37°C in a standard oven. After 24 hours of incubation, the viable cells were counted, using the corresponding media, incubating the plates under anaerobic conditions for at least 3 days.

To carry out the "cultures after lyophilization" (L), the remaining 24 mL of the starting culture were used, washed with PBS and resuspended in 2.4 mL (concentration factor x10) of milk (BD skim-milk, Difeo, reconstituted at 11% and tindalized) as a cryoprotective agent, freezing it at -80oC for 24 hours. After this period, the tubes were lyophilized in a lyophilizer (Freezemobile 12EL, Virtis) for an additional 24 hours. Subsequently, the lyophiles were resuspended in the same starting volume (2.4 mL) of PBS; on the one hand, direct seeding was carried out after lyophilization and, on the other hand, 2 tubes were inoculated (1%) with 10 mL of fresh medium: the LA tube was incubated under anaerobic conditions and the LO tube was incubated at 37°C in a standard oven. After 24 hours of incubation, the viable cells were counted, using the corresponding media, incubating the plates under anaerobic conditions for at least 3 days.

In all cases, the pre-cultures (= “initial” sample) were counted, as well as the direct cultures after stress conditions (= sample “after x treatment”) or after being incubated for 24 hours. in the presence of oxygen (=sample “culture-02”) or in the absence of oxygen (=sample “culture-ANA), as described above.

1.3. dairy fermentation

Sterilized semi-skimmed commercial cow's milk (Alimerka S.A., Lugo de Llanera, Asturias) was used to evaluate the fermentation capacity and survival in the fermented product of the 3 strains under study. For this, the pre-cultures of the bacteria grown overnight were used to inoculate (2%) 100 mL of fresh MRSc that was incubated for 19±1 hours. The cultures were washed with 50 mL of sterile PBS and resuspended in 10 mL of milk (initial culture concentration factor x10) with which 100 mL of commercial milk was inoculated. After correct homogenization by shaking, they were divided into 10x10 10 mL Falcon tubes, 9 of which were incubated in a bath at 37°C for 22 ± 2 hours (tube number 10 was used as a sample fermentation start time = IF) . After this time, after checking if a coagulum had formed, the fermentation was stopped, allowing it to cool at room temperature for 2 hours before placing 8 tubes in a refrigerated chamber at 80C (tube number 9 was used as a sample, final fermentation time = FF ). To monitor the shelf life of the fermented milks, one tube per week was used for approximately 1 month (samples d4, d8, d11, d15, d18, d21, d24 and d27).

For the analysis of the samples collected at the 10 sampling points indicated above, 1 mL was separated in a sterile eppendorf tube to proceed with the counts and the rest to measure the pH using a pH-meter (Crison basic 20). For the counts, serial dilutions were made in % Ringer (Merck) which were seeded on the surface of MRSc-agar (strains LMG11041 and CECT30105) or on BHIc-agar (strain CECT30106) and incubated for at least 3 days under the conditions of anaerobiosis described above. Finally, after counting the plates from each sampling point, colonies were randomly picked from some of the plates and from the sampling points to confirm the identity of the strains. For this, DNA was extracted directly from the colonies, following standardized methods in the group using the commercial GenElute Bacterial Genomic DNA kit (Sigma, St. Louis, MO), and the 16S ribosomal RNA gene was amplified. using the universal primers 27F (5-AGAGTTTGATCCTGGCTCAG-3' [SEQ ID NO: 4]) and 1492R (5'-CTACGGCTACCTTGTTACGA-3' [SEQ ID NO: 5]). The PCR was performed under the following conditions: initial step at 95°C for 5 minutes; followed by 30 cycles of 94°C for 30 s, 55°C for 45 s, and 72°C for 1 minute; and a final extension at 72°C for 10 minutes and 4°C for maintenance. Finally, the amplification products were sequenced in Macrogen and the sequences obtained were compared with those deposited in the Genbank database using the Blastn tool (NCBI, National Center of Biotechnology Information).

2. RESULTS

2.1. Survival to freezing and lyophilization

Figure 5 shows the number of viable cells (Log CFU/ml) recovered in each of the experimental conditions described in section 1.2 above.

The "fresh cultures", that is, made from an active standardized pre-culture, show that only the strain CECT30106 adapted in the laboratory to O ² is capable of growing under aerobic conditions, since no viable strains have been detected for the other strains. two strains (detection level <100 CFU/mL).

The viability study of the three strains after freezing shows similar results with respect to the initial reference culture, since no variations were detected in the count of viable cells obtained from the strains (concentrated stock x10) before freeze-drying (8.67±0 .32, 7.86±0.82 and 7.92±0.80 Log CFU/mL, for CECT30106, CECT30105 and LMG11041, respectively) compared to those obtained after direct seeding of the stocks after thawing (8.57 ±0.37, 7.95±0.74 and 8.09±0.86 Log CFU/mL, respectively). When these frozen stocks were used to inoculate new fresh medium, it was observed that under anaerobic conditions they reached similar counts (7.65±0.80, 7.14±0.38 and 6.86±0.60 Log CFU/mL , respectively) to those of the fresh reference culture (8.19±0.31, 7.10±0.02 and 7.51±0.14 Log CFU/mL, respectively). Again, when the cultures generated from the frozen inoculum were incubated in the presence of oxygen, the only strain capable of growing and achieving a similar number of viable cells (7.48±0.99 Log CFU/mL) to that incubated in anaerobiosis was the CECT30106.

Finally, the study of survival to lyophilization and subsequent growth capacity in the presence and absence of oxygen showed similar results to those of freezing for strains CECT30106 and CECT30105 (viable concentrated stock x10 before lyophilization 8.46±0.18 and 7.67±0.82, viable after direct seeding of lyophiles 8.22±0.41 and 7.54±0.82, viable after culture after lyophilization and anaerobic incubation 7.46±0.19 and 7 0.05±0.61) being the one adapted to O ² the only one able to grow in its presence (7.20±0.27). In the case of the LMG11041 strain, no reproducible results have been achieved with respect to the viable ones after lyophilization (in two biological replicates the counts were below the detection limit (2 Log CFU/mL) and in another 8.27 Log CFU were obtained). /mL). However, the 3 lyophiles of this strain were able to grow after being inoculated in fresh medium and incubated in anaerobiosis while, in agreement with the results obtained in the other experimental procedures, it was not able to grow in the presence of oxygen.

In summary, the strains CECT30106 and CECT30105 are capable of surviving the freezing and lyophilization processes used in this study, and only the strain that has been adapted in the laboratory to oxygen, CECT30106, is capable of growing in the presence of oxygen after having subjected to freezing and lyophilization processes. Collection strain LMG11041 resists freezing well, but no conclusive data was obtained on lyophilization, and in no case is it able to grow in the presence of oxygen.

2.2. dairy fermentations

Figure 6 shows the mean values (of triplicates) of pH, counts and survival after the end of the shelf life study period (27 days) under refrigeration of the fermented milks with the three strains of B. bifidum under study.

Regarding the acidification capacity of the strains in milk, it should be noted that the strains CECT30106 (adapted to 02) and CECT30105 (parental wt) reached lower pH values at the end of fermentation than the collection strain LMG11041 (4.45 ±0.01, 4.56±0.17 and 5.04±0.20, respectively), which maintained the highest pH values throughout the refrigerated storage period (Figure 6). The 10 tubes with the control sterilized milk (not inoculated) maintained their stable pH values (6.62±0.01) throughout the study period and no counts of microorganisms were detected in MRSc (detection limit 10 CFU/mL ) at any of the sampling points.

Regarding the counts obtained during fermentation (Figure 6), the O ² -adapted strain (CECT30106) was able to increase by about one logarithmic unit (FF - IF : 0.832 Log CFU/mL) the counts after its inoculum in milk, while that its parent strain (CECT30105, which is capable of growing microaerophilically) did so slightly (0.232 Log CFU/mL) and the collection strain (strictly anaerobic) was not able to increase the number of viable cells compared to those inoculated (-0.031 Log CFU/mL), despite starting with slightly higher counts in freshly inoculated milk (IF: 7.71±0.35 Log CFU/mL) compared to the strains obtained in the IPLA (IF: 7.26±0 0.24 and 7.10±0.18 Log CFU/mL for CECT30106 and CECT30105, respectively).

The evolution of the fermented milks stored in refrigeration for 27 days shows, as expected, a continuous decrease in the viable count in the fermented products. However, despite the higher acidity of milk fermented with the CECT30106 and CECT30105 strains, which represents an additional stress condition for them, the decrease in the number of viable cells follows the same trend as that of the LMG11041 collection strain. The final count (Log CFU/mL) of the cells that survived after 27 days of cold storage were: 3.87±0.19, 2.16±0.45 and 3.05±0.01 for CECT30106, CECT30105 and LMG11041, respectively. Taking into account the initial number of cells starting after fermentation, these data show that the survival percentage of the CECT30106 strain was higher than that of the other two strains under study (Figure 6).

In general, the data obtained in this study show that the strains of B. bifidum obtained at the IPLA are capable of growing and fermenting milk, achieving its coagulation by generating a pH value equal to or lower than the isoelectric point of the milk. casein. Therefore, the milks fermented with these two strains presented a semi-solid consistency, while those inoculated with the collection strain were liquid. This strain (LMG11041) was not able to grow in milk, although the drop in pH indicates that it shows some metabolic activity to maintain its viability under these conditions. Finally, the three strains were able to maintain the number of viable cells above the minimum recommended dose (7 Log CFU/mL og) by some authors for probiotic strains for at least 4 days in refrigeration after fermentation: 7.93± 0.62, 7.61±1.22 and 7.43±1.06 for CECT30106, CECT30105 and LMG11041, respectively.

Finally, to verify that the viable microorganisms that we recovered from the fermented milk collected at the different sampling points corresponded to the species B. bifidum, 17 colonies were randomly collected (Table 3) from which DNA was isolated to carry out 16S ribosomal RNA gene sequencing.

Table 3. Colonies isolated from the count plates of the fermented milks at different sampling points (end of fermentation = FF and day 27), which are shown in parentheses.

After sequencing of the PCR products, it was confirmed that all the strains belonged to the species B. bifidum , showing 100% coverage and 100% identity with the sequences of the strains deposited in the Genbank database (NCIB). and with that of the original strains.

Claims (15)

1. A microorganism of the genus Bifidobacterium spp. comprising a promoter comprising the sequence GAAAGGA (SEQ ID NO: 1) at a distance of between 12 and 31 nucleotides from the start codon responsible for the transcription of a gene. 2. Microorganism according to claim 1, wherein the microorganism of the genus Bifidobacterium spp. It is Bifidobacterium bifidum. 3. Microorganism according to claim 2, wherein the B. bifidum microorganism is the B. bifidum strain with deposit number CECT30106. 4. Microorganism according to any one of claims 1 to 3, wherein the microorganism comprises over-expressed genes encoding the protein alkyl hydroperoxide reductase C and/or alkyl hydroperoxide reductase F with respect to a bacterium of the genus Bifidobacterium spp. not aerotolerant. 5. Composition comprising a microorganism according to any one of claims 1 to 4. 6. Composition according to claim 5, wherein the composition further comprises, a microorganism selected from the group consisting of a yeast and a lactic acid bacterium other than Bifidobacterium spp. 7. Composition according to claim 6, wherein the composition is a functional nutritional composition, or a nutraceutical. 8. Composition according to claim 7, wherein the functional nutritional composition is a food or a nutritional supplement. 9. Composition according to any one of claims 8, wherein the food is a dairy product. 10. Composition according to claim 9, wherein the dairy product is selected from the list consisting of milk, cheese, yogurt and kefir. 11. Use of a microorganism according to any one of claims 1 to 4, or a composition according to claim 5 or 6, in the production of fermented milk derivatives. 12. A microorganism according to any one of claims 1 to 4, or a composition according to claim 5 or 6, for use as a medicament. 13. A microorganism according to any one of claims 1 to 4, or a composition according to claim 5 or 6, for use in the treatment and/or prevention of constipation, Helicobacter pylori infection, irritable bowel syndrome ( SCI), ulcerative colitis, pouchitis after surgery for ulcerative colitis, a respiratory tract infection, and diarrhea. 14. Method for producing a fermented dairy product comprising incubating a microorganism according to any one of claims 1 to 4, or a composition according to claim 5 or 6, with a dairy product under suitable conditions for the process to be carried out fermentation industry. 15. Method for conferring aerotolerance to a microorganism of the genus Bifidobacterium spp. comprising transforming a microorganism of the genus Biofidobacterium spp. oxygen intolerant with a promoter comprising the sequence GAAAGGA (SEQ ID NO: 1) at a distance of between 12 and 31 nucleotides from the start codon responsible for the transcription of a gene.