MX2011002080A - Immunomodulatory extracts from lactobacillus bacteria and methods of manufacturing and use thereof. - Google Patents

Abstract

The present invention includes extracts from Lactobacillus bacteria, which may produce immunomodulatory effects in subjects. Embodiments of the invention may be used, for example, as nutraceuticals or pharmaceuticals for treatment of diseases or as adjuvants in medical treatment, such as those related to an imbalance of the production of anti-inflammatory or proinflammatory cytokines. Conditions for which extracts of the invention may be useful include infections, allergies, autoimmunity disorders, and inflammation, or as adjuvants providing healthful benefits in subjects. The invention also includes, inter alia, methods of making and using such extracts. The invention also relates to particular strains of Lactobacillus bacteria.

Description

IMMUNOMODULATING EXTRACTS OF LACTOBACILLUS BACTERIA AND METHODS OF MANUFACTURING AND USE OF THE SAME FIELD OF THE INVENTION The embodiments of the present invention include extracts of Lactobacillus bacteria, which can produce immunomodulatory effects in subjects. The embodiments of the invention can be used, for example, as nutraceuticals or pharmaceuticals for the treatment of diseases, such as those related to an imbalance of the production of anti-inflammatory or proinflammatory cytokines, such as infections, allergies, alterations of autoimmunity and inflammation, or as adjuvants providing healthy benefits in subjects. The invention also includes, inter alia, methods of making and using such extracts. The invention is also concerned with particular strains of Lactobacillus bacteria.

BACKGROUND AND BRIEF DESCRIPTION OF THE INVENTION Immunomodulation is a global term that refers to a broad range of immune intervention that alters normal or abnormal immune responses. Microbes produce and secrete a wide range of molecules that can modulate eukaryotic immune responses (Lavelle et al., Curr Top Med Chem. 2004, 4 (5), 499-508). These include factors that subvert protective mechanisms in order to facilitate the colonization of pathogens and persistence. Viral, bacterial and parasite-derived molecules that can inhibit inflammatory responses have been identified. In addition to microbial factors that can suppress immune responses, potent immune activators may also be of microbial origin by themselves. These include bacterial enterotoxins, excretory-secretory products derived from parasites and viral nucleic acids.

A family of at least 11 receptors, called toll-like receptors (TLR) and expressed by the host organism, is considered to play a key role in immunological detection and innate sensitivity to microbes. Figure 1 provides a list of TLR ligands (Gay and Gangloff, Ann. Rev. Biochem., 2007, 76: 141-65). TLRs recognize a wide range of molecules also known as pathogen-associated molecular patterns (PAMP) produced by viruses, bacteria and fungi (Tse and Homer, Ann Rheum Dis. 2007 Nov; 66 Suppl 3: iii77-80). TLR-linked immunomodulation has been applied in the development of new therapies for a wide spectrum of pathologies, including infectious, malignant, autoimmune and allergic diseases.

TLR agonists and antagonists have been studied as potential therapeutics for the prevention and treatment of diseases. In present relatively small chemistries, TLR agonists have been used as adjuvants for vaccines against the objective of preventing infections, extinguish allergic hypersensitivities and clear malignant cells. TLR agonists have also been investigated as monotherapies and adjunctive therapies for the treatment of patients with infectious, allergic and malignant diseases. The use of TLR antagonists has also been studied in preclinical studies and clinical trials as potential therapeutics for autoimmune diseases and sepsis.

Probiotics are live microorganisms that can provide healthy benefits to a subject when administered in appropriate amounts (Mottet et al., Digestive and Liver Disease, 2005, 37: 3-6, Ezendam et al., Nutr Rev, Jan. 2006, 64 (1): 1-14; Gilí and Prasad, Adv Exp Med Biol, 2008, 606: 423-54). The biological mechanisms involved in the stimulation of immune response by probiotic microorganisms and certain cellular components of those microorganisms have been the subject of study. For example, gram-positive bacteria have a characteristic cell wall comprising macromolecules such as lipoteichoic acid (LTA). LTA can be associated with immunostimulatory activity (eg, Bhakdi et al., Infect. Inrniun., 1991, 59: 4614-4620; Setoyama et al., J Gen Microbiol, 1985, 131 (9): 2501-2503; Cleveland et al., Infect Immun, 1996, 64 (6): 1906-1912). See also (Deininger et al., Clin Vaccine Immunol, 2007, 14 (2): 1629-1633). In addition, bacteria Probiotics may contain a variety of TLR ligands with immunomodulatory characteristics. It was found that cell wall fragments of several bifidobacterial strains stimulate the production of interferon-gamma (IFN-γ) in vitro in mouse splenocytes (T. Ambrouche, "Cont ibution a I'ethe du pouvoir immunomodulatory des bifidobacteries: Analyze in vitro et etude ex vivo des mecanismes moleculaires impliques ", Ph.D. Thesis, Universite Laval, Quebec, 2005). Capsules made from particle wall fragments of particular lactic acid bacteria (Del-Immune V®, Pure Research Products, LLC, Colorado) are also designed to stimulate the immune system.

The ingestion of probiotic bacteria in living or dead form or ingestion of cell wall fragments into particles of such bacteria, however, may not be the most effective way to provide an immunomodulatory effect in a subject, however. For example, extracts of living cells may comprise large proteins and lipopeptides whose size prevents efficient absorption by the subject, thus limiting the local concentration of auxiliary molecules of probiotic bacteria in the body. Conditions inside the body can also destroy active bacterial components or otherwise modify the chemical structures of those components, rendering them inactive. The risks associated with the oral administration of microorganisms Live probiotics (Lactobacillus) include bacteremia and sepsis (Lactobacillus Sepsis Associated With Probiotic Therapy, Pediatrics, Jan. 2005, 115 (1): 178-181). Hence, there is a need for other means to administer the beneficial effects of probiotic bacteria to subjects in need thereof.

The present invention is concerned with extracts of Lactobacillus, some modalities of which may show strong immunomodulatory activities. For example, embodiments of the present invention are concerned with extracts of bacterial strains which may be useful as nutraceuticals or as pharmaceuticals, in some cases, to treat infectious diseases, allergy, respiratory disorders and inflammatory pathologies, or to act as an adjunct in connection with a treatment protocol. The present invention is also concerned with compositions comprising the extracts and manufacturing processes of the extracts, for example using means that do not pose a risk of prion diseases. Processes according to the invention include, for example, lysis of cells under alkaline conditions, or under alkaline conditions followed by acidic conditions. In some embodiments, the extracts of the invention are soluble extracts, which means qpae does not contain significant amounts of solid or particulate matter. In some embodiments, the extracts contain chemically modified TLR ligands. In some For example, alkaline treatment can cause chemical modification of cellular materials including TLR ligands, cell wall components, proteins, lipoteichoic acids, lipopeptides and phospholipids.

Some embodiments of the invention may comprise extracts obtained from one or more of the following species: Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus casei defensis, Lactobacillus casei ssp. casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus lactis, and Lactobacillus delbrueckii.

In some embodiments, the extracts comprise at least one strain of each of the above species of bacteria, while in other embodiments, one or more specific strains of the above list may be removed or substituted with one or more different strains. Some embodiments of the present invention comprise an extract obtained from one or more of the following bacterial strains: Lactobacillus fermentum 1-3929, Lactobacillus rhamnosus 71.38, Lactobacillus plantarum 71.39, Lactobacillus johnsonii 103782, and Lactobacillus helveticus 103146. The above strains are deposited according to with the Budapest Treaty. Lactobacillus fermentum 1-3929, Lactobacillus rhamnosus 71.38, Lactobacillus plantarum 71.39, Lactobacillus johnsonii 103782, and Lactobacillus helveticus 103146 are each deposited in the National Collection of Cultivation of Microorganisms at the Pasteur Institute, 25 Doctor Roux Street, 75724 Paris, France. Lactobacillus fermentum 1-3929 was deposited on February 27, 2008. The other strains are among the deposit collections and can be obtained by contacting the depositor.

The invention is also concerned, inter alia, with the strain Lactobacillus fermentum 1-3929, extracts obtained from that strain, methods of making such extracts, and uses thereof. That strain was obtained by allowing strains of Lactobacillus plantarum and Lactobacillus fermentum to undergo chromosomal exchange, thus producing a new strain of Lactobacillus. It was found that the extracts obtained from Lactobacillus fermentum 1-3929 were active in several different in vivo and in vi tro models that correlate with infection and immunological alterations.

In some embodiments, an extract is obtained from only a specific bacterial strain. Alternatively, more than one strain can be used. In other embodiments, one or more extracts of a different type of microorganisms, such as from a bacterial species without Lactobacillus can be added.

Extracts can be obtained by lysis Bacterial cells under specific conditions after the cells are cultured at an appropriate concentration in a culture medium. In some embodiments, the bacteria are grown in a medium that does not pose a risk of prion-related diseases or a risk of other diseases that can be transmitted by ingesting products obtained from animal-based media. For example, in some embodiments a plant-based medium is used to grow the cells, such as a soy-based medium.

The Used ones (that is, the products of cell lysis) can also be filtered to remove the larger nucleic acids and cell fragments, such as insoluble matter or particulate matter. In some embodiments, the amount of nucleic acids present in the extracts is less than 100 g / ml. Hence, in some embodiments, the resulting extract comprises soluble molecular components and does not contain significant amounts of insoluble material or particulate material.

The membrane and cell wall molecules can be dissolved or suspended in the extracts, including lipoproteins, lipopeptides, peptidoglycans, lipooligo-saccharides, lipoteichoic acids, and theitoic acids. During the lysis process, the molecules in the cells, such as in membranes and cell walls, can be chemically modified, for example, cleaved into more complex structures. small, by alkaline treatment. Despite such chemical modifications, the embodiments of the invention may retain their biological activity compared to whole cells, or such embodiments may still demonstrate improved biological activities compared to whole cells.

For example, the alkaline treatment can be used to lyse cells or it can be applied to cells that have previously been lysed by another method. During the alkaline treatment process according to some embodiments of the invention, the L-amino acids found in natural proteins and lipopeptides are at least partially racemized to D-amino acids. The D-amino acids can be beneficial to increase the time of effectiveness of the extracts, since they are not efficiently digested in the intestine of the mammal. D-amino acids can also protect the smaller peptides and proteins from degradation during digestion. Examples of D-amino acids include protein-linked D-amino acids, and to a lesser extent lysinoalanin (from Vrese et al., J Nutrition, 2000, 2026-2031). Thus, the antigenic molecules in the chemically modified extracts during lysis to contain D-amino acids may remain in the patient's body for a longer time, potentially allowing a stronger immunostimulatory effect in some embodiments.

In some modalities, a filtration process It can also influence the properties of the. Resulting extracts, such as the pore size of the filter, and in some cases, the chemical properties of the filter surface (that is, its polarity), can alter the type of materials that are removed and retained. By. example, some embodiments of the present invention use a filtration process designed to retain molecules of interest but remove other molecular components such as nucleic acids or insoluble or particulate matter.

The filtered extracts can also be further purified by organic extraction, organo-aqueous extraction, chromatography, ultracentrifugation, ultrafiltration or a combination thereof.

BRIEF DESCRIPTION OF THE FIGURES Figure 1: Ligands for the family of 11 toll-like receptors (TLR), expressed by the host organism.

Figure 2: A diagram of a tangential flow filtration system (TFF) for the preparation of bacterial extracts following the lysis of bacteria. The diagram shows two different configurations for filters: a parallel mode where all the filters work simultaneously and a coil mode where the filters are configured in a serial mode.

Figure 3: Generalized correlation enters the operating and flow parameters, which indicates the areas of pressure control and mass transfer control for the tangential flow filtration process (TFF).

Figure 4a-4b: Stimulation of spleen cells cultured for 48 hours in the presence of different dilutions of the lysates AFer300, CFer300, and DFer300, and ARahr300, CRahr300, and DRahr300. After the addition of 30 μl / cavity of Alamar blue® solution diluted 1: 1 in cell culture medium, the cells were further incubated (a) 8.5 hours (first experiment); (b) 24 hours (second experiment). The average emission value is shown at 590 nm ± standard deviation of crops in duplicate.

Figure 5a-5b: Induction of nitric oxide (NO) production in mice treated with extracts of Lactobacillus fermentum 1-3929 and Lactobacillus rhamnosus 71.38 in (a) the first analysis, and (b) the second analysis. The results are expressed in nitric oxide μ? (NO) as average value ± standard deviation.

Figure 6: Effect of extracts of the invention on airway hypersensitivity (AHR) by whole body plethysmography (Emka) with increased methacholine concentrations inhaled one day after the last antigen attack. The results are shown (in improved pause value by means of +/- standard error of the mean) for the negative control animals treated with pH saline Regulated phosphate (PBS) (n = 4), animals attacked with untreated LACK (as the positive control group, n = 8), mice attacked with LACK treated with OM-1009a (n = 8), and mice attacked with LACK treated with OM-1009B (n = 7).

DETAILED DESCRIPTION OF THE INVENTION Definitions Extract: An extract, as defined herein, means a material obtained following the lysis of: one or more bacterial strains. In some cases, the extract is obtained from only one strain, while in others the extract is obtained from a mixture of several different strains.

In some cases, the extract is a soluble extract, which means that it does not contain significant amounts of particulate materials and insoluble materials, such as particles or solid cell wall fragments. Instead of this, the components of cell walls, organelles and cell membranes may be included in the extracts to the extent that they are dissolved or suspended. For example, the extract can be treated to remove particulate materials and insoluble materials, such as via filtration, centrifugation or other separation technique.

Chemical lysis: This is a method of lysing bacterial cells under basic, acid and / or osmotic conditions.

Lysate: As used herein, this term means an extract of bacteria obtained from a cell lysis procedure.

Filtration: A filtration process, as described herein, means the passage of an extract or a mixture of extracts, through one or more filters such as microfilters (eg, microfiltration) and / or ultrafilters (ie, ultrafiltration). Such filtration may not necessarily remove 100% of the components that it is designed to remove, but may in some embodiments return the extracts substantially free of those components. In some cases, the filtration is repeated in several steps or cycles.

Initial pH: That term means the pH measured at the start of a procedure, such as bacterial lysis or filtration.

Saccharides: A saccharide, as defined herein, includes monosaccharides, disaccharides, as well as larger saccharides such as linear and branched polysaccharides. The saccharides also include substituted or chemically modified saccharides, such as lipopolysaccharides (LPS) and their chemically modified variants.

Lipoproteins: The term refers to macromolecules that comprise both protein and peptide chains and lipids, for example, a protein or peptide covalently bound to a lipid. Lipoprotein, as used herein, also includes lipopeptides.

Peptidoglycans: This term refers to polymers that include sugars and amino acids.

Lipoteichoic acid (LTA): This term refers to an amphiphilic surface-associated adhesion molecule present in gram-positive bacterial strains.

Teicoic acid: This term refers to polymers of glycerol phosphate or ribitol phosphate linked together via phosphodiester bonds.

D-amino acids: This term refers to amino acids that exist in dextra-rotating isomeric forms, as opposed to biosynthetically produced L-amino acids that exist in levo-rotating isomeric forms.

Racemization: This term indicates at least partial chemical modification of L-amino acids to D-amino acids.

The means that avoids the risk of prion-based diseases means a culture medium used at any stage of. the preparation of the extracts that does not comprise materials such as serum or meat extracts taken from animals such as cows or sheep, or from any other animal that can transmit prion-based diseases. Examples of such media include plant-based media or synthetic media and also media using horse serum or media that they comprise materials taken from animal species that do not transmit prion diseases. Examples of prion-based diseases include, for example, mad cow disease, lumbar pruritus and Creutzfeld-Jaco disease.

A medium without animal is a medium that does not include components derived from animals. Examples include a vegetable-based (ie, vegetable) medium, such as a soy medium and a synthetic medium.

Nutraceutical, as used herein, means any composition that may have a healthy effect on a subject after administration wherein the composition is for example, available to the subject without the prescription of the physician.

Treatment, as used in a therapeutic context in the present, means both treatment of current diseases or alterations as well as the prevention of or protection from the development of new diseases or alterations, for example.

Adjuvant, as used herein to refer to embodiments of the invention, refers to a method of the invention provided to a subject in conjunction with a medical treatment plan.

Immuno-modulation, immuno-modulatory and like terms, as used herein, refer to the ability to modify immune responses in a subject of a way that may have health benefits, such as to produce an anti-inflammatory or immunostimulatory effect.

Anti-inflammatory and similar terms, as used herein, refer to immunomodulatory effects that serve to reduce inflammation.

Immuno-stimulator and like terms, as used herein, refer to the stimulation of the immune system.

Protective immunity, as used herein, means that a modality is provided to a subject to provide protection from subsequent attack with an infectious agent or allergen. As a consequence, during the attack, the level of the infectious agent or allergen in the subject is sufficiently low in concentration so as not to significantly compromise the health of the subject. The duration of time in which such attack protection is effective may be limited, such as for a period of hours, days or weeks.

Subject, as used herein, refers to any animal subject, including mammalian subjects, such as humans and domestic animals. Domestic animals for example, may include mammals such as dogs, cats, horses, pigs, cows, sheep, goats and other winged and may also include non-mammals such as birds, eg chickens, ducks, geese, turkeys and other livestock birds .

It will be understood that the specific bacterial strains • identified herein and used in the invention may include the strain obtained from the original deposit referred to herein or a genetic clone thereof, including a strain that has been re-deposited at a later time with a deposit code name different, but that is genetically considered the same strain as the originally deposited version.

All numbers used here are approximate, taking into account errors inherent in their measurement, rounding and significant figures.

Preparation of extracts The present invention includes an extract of one or more bacterial Lactobacillus strains, wherein the extract is a soluble extract and wherein the extract comprises chemically modified bacterial molecules.

The extracts of the present invention can be prepared, for example by cell culture followed by harvesting the resulting biomass, lysis and purification. For each strain, to obtain a sufficient amount of material, fermentation cultures can be initiated from a batch of working seeds followed by inoculation to larger fermentation containers.

The media used can be the same for each species. In some modalities, a means that avoids the risk of prion-based diseases can be used for culture of all the strains to be used.

After fermentation, the biomass resulting from a strain or from a set of strains can be deactivated by heat treatment, concentrated and frozen. From here, the match material used to form the extracts, in some embodiments, may consist of whole cells without lysis.

In other embodiments, the starting material used to prepare the extracts may be biomass obtained from cells already subjected to lysis at least partially mechanically, enzymatically or chemically. In still other embodiments, the starting material may be a fraction of such cells previously lysed, such as a fraction containing cell wall.

In some embodiments, the starting material is treated with an alkaline medium, such as a strong base, such as hydroxide or other strong mineral or organic bases. In this lysis or basic treatment step, the cells without lysis in the starting material are subjected to lysis whereas in some embodiments, the cellular components can be chemically modified. Hence, in some embodiments, the chemically modified bacterial molecules are obtained by basic treatment, such as treatment with strong base of one or more of the bacterial strains of lactobacilli from which the extract is obtained (ie, basic treatment of the cells or components or fractions without subjecting them to the lysis of bacterial cells, as explained).

In some embodiments, a dry weight concentration of biomass of 2 to 90 g / L may be subjected to a basic treatment, such as from about 2 to about 80 g / L or about 3 to about 40 g / L, such as 3. , 5, 10, 15, 20, 25, 30, 35 or 40 g / L or even approximately 5 to 50 g / L or other intervals limited by the concentrations listed above. In some embodiments, about 40 to about 80 g / L are subjected to basic treatment, such as 40, 50, 60, 70 or 80 g / L or ranges limited by the concentrations listed above.

The biomass dry weight is defined herein by the dry weight of the material in grams per liter of sample. It can be measured by drying the sample on a small porcelain plate at a temperature of approximately 1052C until it reaches a constant mass.

The temperature can be from 30 to 60 ° C, such as from 30 to 552, 30 to 502C, 30 to 45aC, 30 to 40SC or 30 to 352C. In some embodiments, the basic treatment temperature may be from 35 to 60aC, such as 35 to 55aC, 35 to 50aC, 35 to 45aC or 35 to 40SC, for example. In some embodiments, the basic treatment temperature may be 31SC, 322C, 332C, 34eC, 35SC, 36eC, 37aC, 382C, 39aC or even 40aC or intervals limited by the temperatures listed above.

The basic treatment time can vary from 2 hours to several days, such as 1, 2, 3, 4, 5 even 10 days or from 3 to 120 hours or from 3 to 48 hours, such as 3, 5, 8, 15 , 14, 16, 18, 20, 22, 24, 26, 28, 30, 36, 40, 44 or 48 hours or 15 to 120 hours, such as 60 to 120 hours, such as 60, 72, 84, 96, 108 or 120 hours or intervals limited by the times listed above. It will be understood that these time intervals include any fractional number of days, hours or minutes in them.

In some embodiments, a strong base concentration of 0.001 N to 0.1 N is used, such as from 0.001 N to 0.6 N or from 0.10 N to 0.8 N, or from 0.6 N to 1.0 N or an interval starting or ending 0.001 , 0.002, 0.003 or 0.1 N or from 0.1 N to 0.6 N or an interval starting from the end of 0.6, 0.7, 0.8, 0.9, 1.0 or 1.0 N or other intervals limited by the concentrations listed above. In some embodiments, a base concentration is used to obtain an initial pH greater than 9.0 or a third greater than 9.5, a pH greater than 10.0 and less than 13.5, such as greater than 11.5, greater than 12.0, greater than 12.5, higher of 13.0 or of pH 9.0 to pH 13.5. In still other embodiments, a base concentration can be used to obtain an initial p'H greater than 10.0 and less than 13.0 or from pH 9.0 to pH 13.0, for example.

In some modalities, the pH during the treatment Basic can be decreased after extraction of soluble components. For example, the initial pH may be a basic pH, such as pH 9.0 at pH 13.0 or pH 9.5 at pH 12.5. The basic treatment may be allowed to proceed for a certain period of time, such as 3 to 120 hours, such as 3 to 48 hours or for a period of time as listed above, at a temperature as listed above. Then, in some embodiments, the pH may optionally be rendered acidic by the addition of for example hydrochloric acid to obtain a pH of between 2.0 and 4.5 or a pH of between 2.5 and 4.5 or a pH of between 2.5 and 4.0, such as 2.5 , 3.0, 3.5, 4.0 or a limited range by any of the pHs listed above. The second low pH treatment can be carried out at a temperature of 30 to 60aC, between 5 to 55 or 35 to 452C, such as 352C, 36aC, 372C 38SC 392C 402C, 412C, 422C, 432C, 442C or even 452C. The acid treatment time can vary from one hour to several hours to 72 hours, for example between 1 and 24 hours or between 1 hour and 6 hours or between 3 hours and 48 hours or between 3 hours and 24 hours or between 4 and 72 hours hours or even between 24 hours and 72 hours or any time interval limited by the times listed above.

In some embodiments of the invention, an alkaline treatment is carried out on a bacterial biomass comprising, for example, Lactobacillus fermentum material, having a biomass dry weight of between 10 g / L to 40 g / L. In other embodiments, the alkaline treatment is carried out on a bacterial biomass comprising a mixture of Lactobacillus strain and having a biomass dry weight between 10 g / L and 40 g / L. In such embodiments, the alkaline treatment can be carried out at a hydroxide ion concentration of between 0.025 N and 0.25 N or at a pH of between 9.5 and 12..5 at a temperature of 35 to 45 ° C for a time of between 3 hours and 48 hours. hours. In some embodiments, alkaline treatment is carried out on bacterial biomass comprising material from one or more strains of Lactobacillus at a hydroxide ion concentration of between 0.025 N and 0.20 N between 0.025 and 0.15 N, between 0.025 and 0.10 N, between 0.05 N and 0.25 N, between 0.05 N and 0.20 N, between 0.05 and 0.15 N, between 0.05 N and 0.10 N, between 0.10 N and 0.25 N, between 0.10 N and 0.20 N, between 0.10 N and 0.15 N, between 0.15 N and 0.25 N, between 0.15 N and 0.20 N, or even between 0.20 N and 0.25 N. Such modalities can have for example a pH between 9.5 and 12.0, between 9.5 and 11.5, 9.5 and 11.0, between 9.5 and 10.5, between 9.5 and 10.0 , between 10.0 and 12.5, between 10.0 and 12.0, between 10.0 and 11.5, between 10.0 and 11.0, between 10.0 and 10.5, between 10.5 and 12.5, between 10.5 and 12.0, between 10.5 and 11.5, between 10.5 and 11.0, between 11.0 and 12.5, between 11.0 and 12.0, between 11.0 and 11.5, between 11.5 and 12.5, between 11.5 and 12.0, or even a pH between 12.0 and 12.5. The alkaline treatment time for such modalities can be between 3 hours and 36 hours, between 3 hours and 24 hours, between 3 hours and 18 hours, between 3 hours and 12 hours, between 3 hours and 6 hours, between 6 hours and 48 hours, between 6 hours and 36 hours, between 6 hours and 24 hours, between 6 hours and 18 hours, between 6 hours and 12 hours, between 6 hours and 8 hours, between 8 hours and 48 hours, between 8 hours and 36 hours, between 8 hours and 24 hours, between 8 hours and 18 hours, between 8 hours and 12 hours, between 12 hours and 48 hours hours, between 12 hours and 36 hours, between 12 hours and 18 hours, between 18 hours and 48 hours, between 18 hours and 36 hours, between 18 hours and 24 hours, between 24 hours and 48 hours, between 24 hours and 36 hours, or between 36 hours and 48 hours. The alkaline treatment can be carried out for any of the time periods limited to the previous intervals, for example 3, 6, 8, 12, 18, 24, 36 or even 48 hours. Such conditions can provide a moderate alkaline treatment.

In other embodiments, 10 g / L and 40 g / L of biomass dry weight of one or more strains of Lactobacillus can be subjected to a hydroxide ion concentration of between 0.15 N and 0.50 N or a pH of between 11.5 and 13.5, at a temperature of 35 to 45SC for a time of between 15 hours and 120 hours. For example, in some embodiments, the hydroxide concentration can be between 0.15 N and 0.45 N, between 0.15 N and 0.40 N, between 0.15 N and 0.35 N, between 0.15 N and 0.30 N, between 0.15 N and 0.25 N, between 0.15 N and 0.20 N, between 0.20 N and 0.50 N, between 0.20 N and 0.40 N, between 0.20 N and 0.30 N, between 0. 25 N and 0.50 N, between 0.30 N and 0.50, between 0.30 N and 0.40 N, or between 0.40 N and 0.50. Such modality can have a pH between 11.5 and 13.0, between 11.5 and 12.5, between 11.5 and 12.0, between 12.0 and 13.5, between 12.0 and 13.0, between 12.0 and 12.5, between 12.5 and 13.5, between 12.5 and 13.0, between 13.0 and 13.5, for example. The period of time for the alkaline treatment can be between 15 hours and 100 hours, between 15 hours and 90 hours, between 15 hours and 75 hours, between 15 hours and 60 hours, between 15 hours and 48 hours, between 15 hours and 36 hours, between 24 hours and 120 hours, between 24 hours and 100 hours, between 24 hours and 90 hours, between 24 hours and 75 hours, between 24 hours and 60 hours, between 24 hours and 48 hours, from 36 hours to 120 hours, from 36 hours to 100 hours, from 36 hours to 90 hours, from 36 hours to 75 hours, from 36 hours to 60 hours, from 36 hours to 48 hours, from between 48 hours and 120 hours, between 48 hours and 100 hours, between 48 hours and 90 hours, between 48 hours and 75 hours, between 48 hours and 60 hours, between 60 hours and 120 hours, of between 60 hours and 100 hours, between 60 hours and 90 hours, between 60 hours and 75 hours, between 75 hours and 120 hours, between 75 hours and 100 hours, between 75 hours and 90 hours, between 90 hours and 120 hours, between 100 hours and 120 hours, for example. Periods of time are also contemplated for alkaline treatment in such modalities include 15, 24, 48, 60, 75, 90, 100 and 120 hours.

Such conditions can provide a strong alkaline treatment.

In other embodiments, between 10 g / L and 40 g / L dry weight of starting biomass can be treated with a hydroxide concentration between 0.025 N and 0.25 N or a pH of between 9.5 and 12.5, at a temperature of 35 to 45SC for a period of time between 3 hours and 48 hours. The pH can then be adjusted to between 2.5 and 4.0 by the addition of acid, such as hydrochloric acid (HCl) comprising an acid treatment. The acid treatment can be carried out at a temperature of between 35eC and 452C for a time between one hour and 24 hours. For example, in such embodiments, the alkaline treatment of bacterial biomass comprising one or more Lactobacillus strains can be effected with a hydroxide concentration of between 0.025 N and 0.20 N, between 0.025 and 0.15 N, between 0.025 and 0.10 N , between 0.05 N and 0.25 N, between 0.05 N and 0.20 N, between 0.05 and 0.15 N, between 0.05 N and 0.10 N, between 0.10 N and 0.25 N, between 0.10 N and 0.20 N, between 0.10 N and 0.15 N, between 0.15 N and 0.25 N, between 0.15 N and 0.20 N, or even between 0.20 N and 0.25 N. During alkaline processing, such modalities can have for example a pH between 9.5 and 12.0 , between 9.5 and 11.5, between 9.5 and 11.0, between 9.5 and 10.5, between 9.5 and 10.0, between 10.0 and 12.5, between 10.0 and 12.0, between 10.0 and 11.5, between 10.0 11.0, between 10.0 and 10.5, between 10.5 and 12.5, between 10.5 and 12. 0., between 10.5 and 11.5, between 10.5 and 11.0, between 11.0 and 12.5, between 11.0 and 12.0, between 11.0 and 11.5, between 11.5 and 12.5, between 11.5 and 12.0, or even a pH of between 12.0 and 12.5. The alkaline treatment time for such modalities can be between 3 hours and 36 hours, between 3 hours and 24 hours, between 3 hours and 18 hours, between 3 hours and 12 hours, between 3 hours and 6 hours, between 6 hours and 48 hours, between 6 hours and 36 hours, between 6 hours and 24 hours, between 6 hours and 18 hours, between 6 hours and 12 hours, between 6 hours and 8 hours, between 8 hours and 48 hours, between 8 hours and 36 hours, between 8 hours and 24 hours, between 8 hours and 18 hours, between 8 hours and 12 hours, between 12 hours and 48 hours, between 12 hours and 36 hours, between 12 hours and 18 hours, between 18 hours and 48 hours, between 18 hours and 36 hours, between 18 hours and 24 hours, between 24 hours and 48 hours, between 24 hours and 36 hours hours, or between 36 hours and 48 hours. The alkaline treatment can be carried out for any of the time periods limited by the above ranges, for example, 3, 6, 8, 12, 18, 24, 36 or even 48 hours. The pH can then be adjusted to between 2.5 and 3.5, between 2.5 and 3.0, between 3.0 and 4.0, between 3.0 and 3.5 or between 3.5 and 4.0 by the addition of acid, for acid treatment following lysis alkaline The acid treatment can be carried out for a time between 1 and 18 hours, between 1 hour and 12 hours, between 1 hour and 6 hours, from 1 hour and 3 hours, from 3 hours and 24 hours, from 3 hours and 18 hours, from 3 hours and 12 hours, from 3 hours and 6 hours, from 6 hours and 24 hours , between 6 hours and 18 hours, between 6 hours and 12 hours, between 12 hours and 24 hours, between 12 hours and 18 hours, between 18 hours and 24 hours. Times also contemplated for acid treatment include 1, 3, 6, 12, 18 and 24 hours.

The lysates obtained immediately after the basic treatment described above can then be purified by centrifugation and / or filtration, for example to remove particles and insoluble components. For example, lysates can be centrifuged at nine hundred times the gravity, followed by one or more rounds of filtration on a 0.2 micron filter. In some cases, successive rounds of filtration over larger pore filters followed by filtration over a 0.2 micron filter can be used. Ultra-filtration methods can also be employed in order to help extract soluble materials from the extract, for example, re-circulating the ultra-filtration permeate for additional micro-filtration. In some embodiments, a tangential flow filtration (TFF) method can be used to filter the lysates and extract soluble molecules from larger cell fragments (Figure 2). See, for example, Separations Technology, Pharmaceutical and Biotechnology Applications, Wayne P. Olson, Editor. Interpharm Press, Inc., Buffalo Grove, IL, U.S.A., P. 126 to 135 - ISBN: 0-935184-72-4. At the beginning of such a process, a diluted bacterial lysate can be stored in a first tank. In TFF, for example, the extract can be exposed to both a micro-filter and an ultra-filter. For example, a circuit or micro-filtration loop (MF) is started and the product is pumped. The resulting MF retentate is recycled, while the MF permeate is transferred to a second tank.

After reaching an appropriate degree of concentration, a loop or ultrafiltration circuit (UF) is initiated. The ultrafiltration permeate can be re-circulated back to the first tank for continuous extraction of solubilized extracts from the lysate while the ultrafiltration retentate is stored in the second tank. During continuous extraction, the volumes in tanks 1 and 2 can be adjusted by regulating the flow rates of the permeate micro filtration and ultrafiltration.

Several such extraction cycles can be performed, either with TFF (tangential flow filtration) or other filtration method. In modalities that use TFF, at the end of the last cycle, the ultra-filtration circuit can be closed and the micro-filtration circuit can be put into operation alone and the micro-filtration permeate transferred to tank 2., The cross-flow and transmembrane pressure conditions are defined by the independent pressure controlled regions and controlled regions of mass transfer in theory of films described for example by M. Cheryan (Ultrafiltration and Microfiltration Handbook, 2nd Ed., Ch. 4, 1998). The permeate flow and extraction performance are affected by the filtration conditions (trans-membrane pressure (TMP), cross flow, temperature, etc.). The type of filter can also affect the filtration performance, as well as the type of plate system (cassette filter). Different configurations can be used, in which parallel mode and coil mode are included (see Figure 2). Specific conditions are developed for optimized performances for each combination of type of mode and type of filter used.

The micro-filtration circuit can be equipped with filters from 1.2 microns to 0.1 microns, such as filters from 0.65 to 0.2 microns or 0.45 microns. The cross flow can be between 100 and 3000 liters / hours m2 (LHM), such as between 300 and 2500 LHM or 2000 LHM with a TMP of 0.3 to 2 bar. The ultrafiltration circuit can be equipped with filters from 10 KDa to 1000 KDa, such as from 10 KDa to 100 KDa or from 10 KDa to 30 KDa or from 30 KDa to 100 KDa. The cross flow may be between 30 and 1000 LHM, such as between 20 and 500 LHM with a TMP of 0.2 to 1.5 bar.

Between 5 and 20 volumes of diafiltration can be used to extract soluble components from bacterial cell walls. In some modalities, between 8 and 15 volumes are used. From here, for example, in some modalities, between 5 and 15 filtration cycles can be used and in some cases between 8 and 15 cycles.

Following filtration, the extract can be further concentrated or centrifuged, if desired. For example, an additional micro filtration using a smaller pore filter can be effected, such as a 0.2 micron filter. Following filtration, the extract can be lyophilized before being formulated for use.

In some embodiments, following the filtration, the extract can be purified in order to separate, eliminate and increase the concentration of one or more modified components in the extract. For example, a strong ion chromatography step can be used in order to remove charged components. Other purification processes can be used, such as gel filtration, chromatography, ultra-centrifugation, extraction and precipitation.

Chemical Properties of Bacterial Extracts The basic treatment can result in a variety of chemical modifications to cellular components. For example, in proteins: (1) the bonds of peptide may undergo partial cleavage generating smaller polypeptides; (2) natural L-amino acids can be at least partially racemized to D-amino acids; and (3) asparagine and glutamine residues can be deaminated, leading to changes in the isoelectric point of the protein. Molecules such as lipbteic acids, lipopeptides and phospholipids may undergo base-catalyzed hydrolysis of the ester bonds and / or amide bonds leading to modified amphiphilic structures which may have new physicochemical and immunological properties. Examples of other possible chemical modifications include partial solubilization of cell wall polysaccharides and complete hydrolysis of ribonucleic acid (AR) to individual ribonucleotides, including rearrangement of phosphate groups.

Hence, some or all of those chemical modifications may occur during the basic treatment of Lactobacillus cells as described herein. Such molecular modifications can affect the biological activities of the extracts.

For example, the basic treatment of bacteria according to the present invention may result in the partial hydrolysis of proteins, also as deamination, deamidation and / or partial racemization of amino acids from L to D. In analytical study of an extract in accordance with the invention, peaks representing D-acid were observed aspartic acid, D-glutamic acid, D-serine, D-methionine, D-histidine, D-alanine, D-arginine, D-phenylalanine, D-tyrosine, D-leucine and D-lysine. The percentage of D-amino acids of those species in that study fluctuated from 3% to 40%. Hence, some of the embodiments of the invention allow the racemization of one or more of serine, threonine, histidine, alanine, arginine, tyrosine, phenylalanine, leucine and lysine, such as all of the above amino acids or any selection of more than one but less than all of the above amino acids, such as, for example, alanine, phenylalanine and lysine. In some embodiments, at least 10% of one or more of the above amino acids can be racemized to D to L. In other embodiments, at least 40% of one or more of the above amino acids can be racemized.

Thus, the extracts of the present invention can comprise between 1 and 90% of D-amino acids, such as between 1 and 80% or between 1 and 60%. In some embodiments, the extract comprises between 10 and 45% of D-amino acids, such as between 25 and 35% of D-amino acids. The extracts of the invention may comprise at least one D-amino acid selected from the group consisting of D-aspartic acid, D-asparagine, D-glutamic acid, D-glutamine, D-serine, D-methionine, D-histidine, D-alanine, D-arginine, D-phenylalanine, D-tyrosine, D-leucine, D-lysine, D-valine and D-threonine. In some embodiments, the concentration of any D-amino acid comprises between 1 and 50%, such as between 10 and 40%, or even between 15 and 35%.

Some extracts of the present invention comprise cell wall components and bacterial membrane of Lactobacillus, such as lipoteichoic acids, teichoic acid, peptidoglycan or a combination thereof. In some modalities, those components are chemically modified. Some extracts also comprise cell wall and / or cell membrane components such as lipoproteins, which can also be chemically modified. In some embodiments, the cell wall components or cell membrane components, for example lipoproteins, are discrete or suspended in the extracts and thus are not present in the form of particles or insoluble form.

In addition, an extract according to the present invention may comprise; for example, from 10 to 100 mr / ml of soluble dry weight (SDW) of material, 1 to 30 mg / ml of protein (Prot.), 0.5 to 4.0 mg / ml of sugar and less than 100 ig / ml of DNA. For example, some embodiments contain approximately 15 to 35 mg / ml of soluble dry weight, 3 to 7 mg / ml of protein, 1.0 to 3.0 mg / ml of sugar and 10 to 40 g / ml of DNA. An extract according to the present invention may contain, for example, 30 mg / ml of soluble dry weight, 9.6 mg / ml of protein, 2.4 mg / ml of sugar and 33 g / ml of DNA or another example containing 32.4 mg / ml of soluble dry weight, 5.8 mg / ml of protein, 2.3 mg / ml of sugar and less than 100 pg / ml of DNA. The Soluble Dry Weight (SDW) in g / L or mg / ml is determined by obtaining 5 ml of the soluble fraction resulting from the lysis or basic treatment and drying it at a constant mass on a porcelain disk at 105 ° C.

In some embodiments, the extracts comprise at least 0.3 mg / ml of saccharides, such as between 0.3 and 4.5 mg / ml of saccharides. In some embodiments, at least one saccharide is chosen from monosaccharides, disaccharides and polysaccharides. Some extracts of the invention comprise at least one branched polysaccharide. In some embodiments, at least one saccharide is chemically modified.

The lysis or basic treatment of the bacteria according to the present invention can result in a decrease in the average molecular weight of the component macromolecules at a range of, for example, 1 kDa to between 300 kDa and 100 kDa, or at a range from 1 kDa to between 60 kDa and 10 kDa. In some embodiments, the extract comprises at least one protein with a molecular weight of less than 50 kDa or less than 30 kDa, such as less than 10 kDa.

Biological Activities of Bacterial Extracts The extracts according to the invention can have immunomodulatory activities. For example, some extracts can stimulate the immune system. Some extracts may have anti-inflammatory activities. The specific effects of an extract may depend on the conditions of manufacture and the species or strain of Lactobacillus or mixture of species or strains, of which the extract is obtained. Thus, some extracts according to the invention may show potent immunostimulatory activity, and may thus be useful in the treatment of infections or as adjuvants to such treatment, while other modalities may show weaker immunostimulatory activity but show anti-inflammatory activity, being thus useful in the treatment of inflammatory disorders such as allergies, asthma, autoimmune diseases, colitis and inflammatory bowel diseases or as adjuvants to such treatment.

Thus, some extracts according to the invention can be effective to treat patients suffering from alterations in which they include, but are not limited to, microbial infections, allergic diseases and alterations of the digestive system. Some extracts according to the invention may also be provided to a patient as nutraceuticals, for example, as adjuvants in the treatment of a variety of conditions including, but not limited to, microbial infections, allergic diseases and system disorders. digestive.

The range of the biological activities of the extracts can be determined by several in vitro and in vivo analyzes. For example, the AlamarBlueTM analysis incorporates a fluorometric / colorimetric growth indicator based on the detection of metabolic activity by an oxidation-reduction indicator (REDOX) in response to chemical reduction resulting from cell growth (Example 4).

Cellular analyzes in vitro test the production of nitric oxide (NO) from primary murine macrophages and can select the ability of an extract to stimulate the immune system in order to kill invading bacteria (Figure 5). In some embodiments, the extracts can stimulate NO production in murine macrophages, leading to measured NO concentrations ranging from 3 μ? at 60 μ? , such as 5 μ? at 40 μ? . In some modalities, the concentration of NO can be greater than 30 μ? . The type of bacterial species can also affect these results. For example, extracts of Lactobacillus fermentum can induce a production of nitric oxide larger than Lactobacillus rahmnosus (see, for example Example 5 below). In order to select embodiments of the invention in terms of immunostimulatory or anti-inflammatory potential in vivo, tests of the bacterial extracts of the invention can be performed on human peripheral blood mononuclear cells (PBMC). See Foligne et al. . { World J Gastroenterol, 2007, 13 (2): 236-243). The release of both ILl2p70 (an inflammatory cytokine) and IL10 (an anti-inflammatory cytokine) can be measured, and the ratio of IL-10 / IL-12 can be calculated (Example 6). Some embodiments of the invention produce a IL10 / IL12 ratio higher than a live Lactobacterium fermentum control, thus suggesting that some extracts of the invention may exhibit equivalent anti-inflammatory properties, or even stronger than the live parent microorganism when administered in vivo. Hence, the invention includes extracts capable of obtaining a proportion of IL10 / IL12 that can be calculated in mononuclear cells of human peripheral blood, where the proportion is greater than or equal to the proportion of IL10 / IL12 obtained by a strain of live Lactobacillus of which The extract is obtained.

The immune responses of the extracts of the invention can also be tested by examining their effects on Toll-like receptors (TLR), for example, extracts. can be tested in HEK293 cells in the presence or absence of the TLR2 agonist Pam3Cys, or in the presence or absence of the TLR4 agonist LPS (Example 7). The HEK293 cell line allows efficient monitoring of TLR activity using ELISA analysis such as IL-8 titration or reporter-based systems that monitor NF- activation. induced by TLR. Some embodiments of the invention can act as TLR 2/6 antagonists in HEK TLR 2/6 cells. Thus, some modalities may be useful for combating infections in a subject, while other modalities may be developed for use against inflammation and / or alterations of autoimmunity.

The extracts currently disclosed can also be selected for TLR and N0D2 receptor activity (Example 8). Some embodiments of the invention activate the TLR and / or N0D2 receptors in vitro, indicating that they may be able to activate the immune system, via TLR and / or N0D2.

The technique of plaque-forming cells (PFC) can be used to evaluate a non-specific determination of B-lymphocytes (Example 9). Certain lymphoid cells release hemolytic antibodies that diffuse and cause the lysis of neighboring red blood cells to form a lysis plate in the presence of complement. Some embodiments of the invention can increase the secretion of immunoglobulins by B cells, and thus can potentially be used prophylactically for priming the immune system in subjects suffering from recurrent infections.

The anti-infective efficacy of the extracts of the present invention can be tested, for example after infection of Salmonella in subjects, such. as infection of mice (Example 10). Some embodiments of the invention can provide protective immunity against infections, such as bacterial infections, that is, Salmonella infections. For example, some modalities can decrease the mortality induced in mice by injection of Salmonella thyphimurium.

The combination of the in vitro activity of NO with the Determination of an in vivo activity measured, for example, in a murine model of allergen-induced asthma (Example 11) can provide a more complete view of the potential clinical activity of the extracts disclosed herein. In the LACK model (major Leishmania parasite protein), the number of eosinophils found in broncho-alveolar lavage fluids when compared to asthmatic control animals (untreated) can be decreased by a factor of between 1 and 10, such like 1.5 times to 5 times decreased. Thus, in some embodiments, the extract decreases the number of eosinophil cells, number of neutrophil cells, number of lymphocyte cells, or any combination thereof, in an asthmatic murine subject by a factor of at least 1.5 with respect to to an untreated asthmatic witness. Some embodiments of the invention may decrease eosinophilia in asthmatic subjects, and concomitantly decrease the level of Th2 cytokines (such as IL4, IL5, IL13), which are asthma markers. Thus, such modalities, for example, may have anti-inflammatory activities in subjects suffering from immunological alterations, such as allergic disorders, including asthma.

Compositions that comprise Bacterial Extracts The extracts according to the invention can be formulated in a variety of different ways to eventual administration. For example, oral tablets, capsules, pills, as well as liquid formulations or aerosols can be prepared. Formulations can also be prepared for infusion or injection.

Modes of this invention can be formulated, for example, as solid dosage forms or liquid dosage forms. Exemplary solid dosage forms may include, for example, a tablet, for example a coated tablet, chewable tablet, effervescent tablet, sublingual tablet, granules, powder or a capsule) containing the extract.

The solid dosage forms may also contain diluents, fillers and / or other excipients. Other excipient components may be added such as preservatives, colorants, flavors and sweeteners.

As an alternative to capsules and tablets, it is possible to develop powder or granule formulations. Liquid dosage forms such as solution or syrup, suspension and drops can also be developed for the oral route.

The extracts of the present invention can be included in one or more nutraceutical compositions, such as nutritional and / or dietary supplements and food additives or one or more pharmaceutical compositions.

Administration of Bacterial Extracts to a Subject A dose comprising at least one extract of the present invention can be administered to a subject suffering from or at risk of developing at least one alteration chosen from alteration of the digestive system, alteration of the respiratory system, alteration of the urinary system and allergic conditions. For example, in some embodiments, the extracts may be administered to subjects suffering from, or at risk of developing, upper and lower pulmonary infections, obstructive pulmonary disease with acute lower respiratory infection, obstructive pulmonary disease with acute exacerbation, nasopharyngitis, sinusitis , pharyngitis, tonsillitis, laryngitis, tracheitis, laringof ringitis, influenza, pneumonia, bronchopneumonia, bronchitis, rhinitis, nasopharyngitis, pharyngitis, sinusitis, tonsillitis, laryngitis, laryngotracheitis, bronchitis, allergic rhinitis, allergic asthma, atopic dermatitis, urinary tract infections due to obstructive and reflux uropathies, urethritis, tubulo-interstitial nephritis, obstructive pyelonephritis, cystitis including chronic cystitis, male pelvic pain syndrome including prostatitis and chronic prostatitis, prostatocystitis, female pelvic inflammatory diseases, Crohn's disease and / or intimal syndrome irritable estino.

In some modalities, the extracts are administered to a subject in the form of a nutraceutical composition such as a nutritional supplement and / or food additive. In other embodiments, the extracts are administered to a subject in the form of a pharmaceutical composition. The administration may comprise a single dose or administration of multiple doses.

In some embodiments, an extract may be provided in a therapeutically effective dose to treat a subject suffering from one or more of the above conditions. In some modalities, an extract may be provided as an adjunct to other medical treatment.

WORK EXAMPLES Example 1; Bacterial crops Example 1.1: Culture of Lactobacillus fermentum 1-3929 Initial conditions of Cultivation The culture medium was prepared by dissolving the following components in purified water: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone: 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L '; pyruvate: 0.1 g / L; glutamate: 0.2 g / L; solution of; metal (copper sulfate: 3 mg / L; iron chloride: 830 mg / L; zinc sulfate: 860 mg / L; sulfuric acid: 1.1 mg / L): 0.5 mL / L. Then polypropylene glycol (0.02 ml / L, density 1005 g / ml) was added.

After dissolution, the pH was not adjusted. After sterilizing the medium, small Erlenmeyer flasks were inoculated individually with the contents of the frozen flasks (containing 1.5 ml of frozen bacteria) and incubated at 37 ° C for 8 hours. Then, aliquots of 20 ml of. This culture was transferred to larger Erlenmeyer flasks containing 1000 ml of culture medium, and incubated again under the same conditions. After 16 hours of culture, the contents of a 1 liter Erlenmeyer flask was transferred to the pre-fermentor.

Growing conditions in pre-fermenters 20 liters of culture medium were prepared by dissolving in purified water the following components: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone: 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: 0.1 g / L; glutamate: 0.2 g / L; metal solution (copper sulfate: 3 mg / L, iron chloride: 830 mg / L, zinc sulfate: 860 mg / L, sulfuric acid: 1.1 mg / L): 0.5 mL / L. Then polypropylene glycol (0.02 ml / L) was added. After dissolution, the pH was not adjusted. The incubation temperature was regulated at 37 ° C, with agitation of 100 rpm and without aeration. The pH was not regulated during cultivation. After 24 hours, 7 liters of the pre-fermenters were transferred to a thermenator (optical density (OD) at 700 nm of pre-fermentor culture after 24 hours = 1.24). The cultures of the pre-fermenters were transferred under sterile conditions to the fermenters.

Growing Conditions in Fermenters 70 liters of culture medium were prepared by dissolving in purified water the following components: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone: 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: 0.1 g / L; glutamate: 0.2 g / L; metal solution (copper sulfate: 3 mg / L, iron chloride: 830 mg / L, zinc sulfate: 860 mg / L, sulfuric acid: 1.1 mg / L): 0.5 mL / L. Then polypropylene glycol (0.02 ml / L) was added. After dissolution, the pH was not adjusted.

After sterilization, 8 g / L of glucose was added to the culture. The incubation temperature was regulated at 37 ° C, with agitation at 100 rpm and without aeration. The pH was regulated at 5.7 during the culture. After 16 hours, the cultures (OD at 700 nm of culture 2.30) were deactivated by heat treatment at 65 ° C for 35 minutes and transferred to a harvest tank. Once deactivated, the Cultures were transferred to an ultrafiltration spar with the purpose of separating the biomass from the culture medium, concentrates and washed with NaCl (9 g / L) in purified water. The harvested biomass was taken in aliquots (mass of the concentrated bacterial suspension 2000 g to 31.8 mg of biomass of dry weight per gram of concentrated bacterial suspension) and then frozen at -15 ° C.

Example 1.2: Cultivation of Lactobacillus helvetícus 103146 Initial Conditions of Cultivation The culture medium was prepared by dissolving the following components in purified water: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone: 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: 0.1 g / L; glutamate: 0.2 g / L; metal solution (copper sulfate: 3 mg / L, iron chloride: 830 mg / L, zinc sulfate: 860 mg / L, sulfuric acid: 1.1 mg / L): 0.5 mL / L. Then polypropylene glycol (0.02 ml / L) was added. After dissolution, the pH was not adjusted. After sterilization of the medium, the small Erlenmeyer flasks were individually inoculated with the contents of the frozen flasks (containing 1.5 ml of frozen bacteria) and incubated at 37 ° C for 9 hours. Then, aliquots of 20 ml of this culture were transferred to large Erlenmeyer flasks containing 1000 My culture medium and incubated again under the same conditions. After 15 hours of culture, the contents of the 1 liter Erlenmeyer flask was transferred to the pre-fermentor.

Growing conditions in pre-fermenters 20 liters of culture medium were prepared by dissolving in purified water the following components: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone: 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: 0.1 g / L; glutamate: 0.2 g / L; metal solution (copper sulfate: 3 mg / L, iron chloride: 830 mg / L, zinc sulfate: 860 mg / L, sulfuric acid: 1.1 mg / L): 0.5 mL / L. Then polypropylene glycol (0.02 ml / L) was added. After dissolution, the pH was not adjusted. The incubation temperature was regulated at 37 ° C, with agitation at 100 rpm and without aeration. The pH was not regulated during cultivation. After 9 hours, 7 liters of the pre-fermenters were transferred to a thermenator. (OD at 700 nm of pre-fermenter culture after 9 hours: 0.14). The cultures of the pre-fermenters were transferred under sterile conditions to the fermenters.

Growing Conditions in Fermenters 70 liters of culture medium were prepared by dissolving in purified water the following components: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone: 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: 0.1 g / L; glutamate: 0.2 g / L; metal solution (copper sulfate: 3 mg / L, iron chloride: 830 mg / L, zinc sulfate: 860 mg / L, sulfuric acid: 1.1 mg / L): 0.5 mL / L. Then polypropylene glycol (0.02 ml / L) was added. After dissolution, the pH was not adjusted.

After sterilization, 8 g / L of glucose was added to the culture. The incubation temperature was regulated at 33 ° C, with agitation at 100 rpm and without aeration. The pH was adjusted to 6.7 at the beginning of the culture with CH3COOH. After 24 hours, the cultures (OD 700 nm culture 4.17) were deactivated by heat treatment at 65 ° C for 35 minutes and transferred to a harvest tank. Once deactivated, the cultures were transferred to an ultrafiltration beam in order to separate the biomass from the culture medium, concentrates and washed with NaCl (9 g / L) in purified water (9 g / L). The biomass harvested was taken in aliquots (mass of concentrated bacterial suspension 440 g to 19.1 mg of dry weight biomass per gram of suspension bacterial concentrate) and then frozen at -15 ° C.

Example 1.3; Cultivation of Lactobacillus plantarum 71.39 Initial Conditions of Cultivation The culture medium was prepared by dissolving the following components in purified water: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone: 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: 0.1 g / L; glutamate: 0.2 g / L; metal solution (copper sulfate: 3 mg / L, iron chloride: 830 mg / L, zinc sulfate: 860 mg / L, sulfuric acid: 1.1 mg / L): 0.5 mL / L. Then polypropylene glycol (0.02 ml / L) was added. After dissolution, the pH was not adjusted. After sterilization of the medium, the small Erlenmeyer flasks were individually inoculated with the contents of the frozen flasks (containing 1.5 ml of frozen bacteria) and incubated at 35 ° C for 9 hours. Then, 20 ml aliquots of this culture were transferred to large Erlenmeyer flasks containing 1000 ml of culture medium, and incubated again under the same conditions. After 15 hours of culture, the contents of the 1 liter Erlenmeyer flask was transferred to the pre-fermentor.

Growing conditions in pre-fermenters 20 liters of culture medium were prepared by dissolving in purified water the following components: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone: 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: 0.1 g / L; glutamate: 0.2 g / L; metal solution (copper sulfate: 3 mg / L, iron chloride: 830 mg / L, zinc sulfate: 860 mg / L, sulfuric acid: 1.1 mg / L): 0.5 mL / L. Then polypropylene glycol (0.02 ml / L) was added. After dissolution, the pH was not adjusted. The incubation temperature was regulated at 37 ° C, with agitation at 100 rpm and without aeration. The pH was not regulated during cultivation. After 9 hours, 7 liters of the pre-fermenters were transferred to a thermenator. (OD at .700 nm of pre-fermenter culture after 9 hours = 1.62). The cultures of the pre-thermenters were transferred under sterile conditions to the termendores.

Growing Conditions in Fermenters 70 liters of culture medium were prepared by dissolving in purified water the following components: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone: 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: 0.1 g / L; glutamate: 0.2 g / L; metal solution (copper sulfate: 3 mg / L, iron chloride: 830 mg / L, zinc sulfate: 860 mg / L, sulfuric acid: 1.1 mg / L): 0.5 mL / L. Then polypropylene glycol (0.02 ml / L) was added. After dissolution, the pH was not adjusted.

After sterilization, 8 g / L glucose is added to the culture. The incubation temperature was regulated at 35 ° C, with agitation at 100 rpm and without aeration. After 24 hours, the cultures (OD at 700 nm of culture = 6.36) were deactivated by heat treatment at 65 ° C for 35 minutes and transferred to a harvest tank. Once deactivated, the cultures were transferred to an ultrafiltration beam in order to separate the biomass from the culture medium, concentrates and washed with NaCl (9 g / L) in purified water. Aliquots of the harvested biomass (mass of concentrated bacterial suspension 600 g to 60.7 mg of biomass of dry weight per gram of concentrated bacterial suspension) were taken and then frozen at -15 ° C.

Example 1.4; Culture of Lactobacillus rhamnosus 71.38 Initial Conditions of Cultivation The culture medium was prepared by dissolving the following components in purified water: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone: 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: 0.1 g / L; glutamate: 0.2 g / L; metal solution (copper sulfate: 3 mg / L, iron chloride: 830 mg / L, zinc sulfate: 860 mg / L, sulfuric acid: 1.1 mg / L): 0.5 mL / L. After dissolution, the pH was not adjusted. After sterilization of the medium, the small Erlenmeyer flasks were individually inoculated with the contents of the frozen flasks (containing 1.5 ml of frozen bacteria) and incubated at 35 ° C for 9 hours. Then, 20 ml aliquots of this culture were transferred to larger Erlenmeyer flasks containing 1000 ml of culture medium, and incubated again under the same conditions. After 15 hours culture, the contents of the 1 liter Erlenmeyer flask was transferred to the pre-fermentor.

Growing conditions in pre-fermenters 20 liters of culture medium were prepared by dissolving in purified water the following components: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone: 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: 0.1 g / L; glutamate: 0.2 g / L; metal solution (copper sulfate: 3 mg / L; iron chloride: 830 mg / L; zinc sulfate: 860 mg / L; sulfuric: 1.1 mg / L): 0.5 ml / L. Then polypropylene glycol (0.02 ml / L) was added. After dissolution, the pH was not adjusted. The incubation temperature was regulated at 35 ° C, with agitation at 100 rpm and without aeration. The pH was not regulated during cultivation. After 9 hours, 7 liters of the pre-fermenters were transferred to a fermenter. (OD at 700 nm of pre-fermentor culture after 9 hours: 3.75). The cultures of the pre-fermenters were transferred under sterile conditions to the fermenters.

Growing Conditions in Fermenters 70 liters of culture medium were prepared by dissolving in purified water the following components: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone: 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: 0.1 g / L; glutamate: 0.2 g / L; metal solution (copper sulfate: 3 mg / L, iron chloride: 830 mg / L, zinc sulfate: 860 mg / L, sulfuric acid: 1.1 mg / L): 0.5 mL / L. Then polypropylene glycol (0.02 ml / L) was added. After dissolution, the pH was not adjusted.

After sterilization, 8 g / L of glucose was added to the culture. The incubation temperature was regulated at 35 ° C, with agitation at 100 rpm and without aeration. After 14 hours, cultures (OD 700 nm culture 5.43) were deactivated by heat treatment at 65 ° C for 35 minutes and transferred to a harvest tank. Once deactivated, the cultures were transferred to an ultrafiltration spar with the purpose of separating the biomass from the culture medium, concentrates and washed with NaCl (9 g / L) in purified water. Aliquots were taken from the harvested biomass (mass of concentrated bacterial suspension 2798 g to 51.7 mg dry weight of biomass per gram of concentrated bacterial suspension) and then frozen at -15 ° C.

Example 1.5; Culture of Lactobacillus johnsonii 103782 Initial Conditions of Cultivation The culture medium was prepared by dissolving the following components in purified water: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone: 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: 0.1 g / L; glutamate: 0.2 g / L; metal solution (copper sulfate: 3 mg / L, iron chloride: 830 mg / L, zinc sulfate: 860 mg / L, sulfuric acid: 1.1 mg / L): 0.5 mL / L. Then polypropylene glycol (0.02 ml / L) was added. After dissolution, the pH was not adjusted. After sterilization of the medium, small Erlenmeyer flasks were inoculated individually with the contents of the frozen flasks (which contain 1.5 ml of frozen bacteria) and incubated at 33 ° C for 10 hours. Then, aliquots of 20 ml of this culture were transferred to larger Erlenmeyer flasks containing 1000 ml of culture medium, and incubated again under the same conditions. After 14 hours of culture, the contents of the 1 liter Erlenmeyer flask were transferred to the pre-fermentor.

Growing Conditions in the Pre-fermentors 20 liters of culture medium were prepared by dissolving in purified water the following components: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone: 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: 0.1 g / L; glutamate: 0.2 g / L; metal solution (copper sulfate: 3 mg / L, iron chloride: 830 mg / L, zinc sulfate: 860 mg / L, sulfuric acid: 1.1 mg / L): 0.5 mL / L. Then polypropylene glycol (0.02 ml / L) was added. After dissolution, the pH was not adjusted. The incubation temperature was regulated at 35 ° C, with agitation at 100 rpm and without aeration. The pH of the culture was adjusted to 5.6 with acetic acid. After 24 hours, 7 liters of the pre-fermenters were transferred to a thermenator. (OD at 700 nm of pre-fermenter culture after 24 hours: 0.47). The crops of the Fermenters were transferred under sterile conditions to the fermenters.

Growing Conditions in Fermenters 70 liters of culture medium were prepared by dissolving in purified water the following components: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone: 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: 0.1 g / L; glutamate: 0.2 g / L; metal solution (copper sulfate: 3 mg / L, iron chloride: 830 mg / L, zinc sulfate: 860 mg / L, sulfuric acid: 1.1 mg / L): 0.5 mL / L. Then polypropylene glycol (0.02 ml / L) was added. After dissolution, the pH was not adjusted.

After sterilization, 8 g / L of glucose was added to the culture. The incubation temperature was regulated at 35 ° C, with agitation at 100 rpm and without aeration. After 24 hours, the cultures (OD at 700 nm, 0.174) were deactivated by heat treatment at 70 ° C for 30 minutes and transferred to a harvest tank. Once deactivated, the cultures were transferred to an ultrafiltration spar with the purpose of separating the biomass from the culture medium, concentrates and washed with NaCl (9 g / L) in purified water. Aliquots of the harvested biomass were taken (mass of suspension Bacterial concentrate 61.5 g to 20.2 mg. of biomass dry weight per gram of concentrated bacterial suspension) and then frozen at -15 ° C.

Example 2: Bacterial Lysates Example 2.1 An aliquot of Lactobacillus fermentum I-3929 biomass of Example 1.1 containing 6 g of dry bacterial weight was thawed at room temperature, then diluted with purified water to reach 12 g / L dry weight of bacterial biomass. The alkalinization was carried out with 0.03 M sodium hydroxide. The pH measured at the beginning of the lysis was 10.3. Then, the lysis was incubated for 6 hours at 40 ° C under continuous agitation. After incubation, the pH was 9.9.

Example 2.2 An aliquot of the biomass of Lactobacillus rhamnosus 71.38 of Example 1.4 containing 20 g of bacterial dry weight was thawed at room temperature, then diluted with purified water to reach 40 g / L dry weight of bacterial biomass. The alkalinization was carried out with 0.03 M sodium hydroxide. The pH measured at the beginning of the lysis was 10.3. Then, the lysis was incubated for 6 hours at 40 ° C under continuous agitation. After incubation, the pH was 9.7.

Example 2.3 An aliquot of Lactobacillus fermentum I-3929 biomass of Example 1.1 containing 6 g of bacterial dry weight was thawed at room temperature, then diluted with purified water to reach 12 g / L dry weight of bacterial biomass. The alkalinization was carried out with 0.06 M sodium hydroxide. The pH measured at the beginning of the lysis was 12.3. Then the lysis was incubated for 6 hours at 40 ° C under continuous agitation. After incubation, the pH was 11.8.

Example 2.4 An aliquot of Lactobacillus fermentum I-3929 biomass of Example 1.1 containing 6 g of bacterial dry weight was thawed at room temperature, then diluted with 0.2 N NaCl solution to reach 12 g / L dry weight of bacterial biomass. The final concentration of the NaCl solution was 0.15 N. The pH measured at the beginning of the lysis was 6.4. Then the lysis was incubated for 6 hours at 40 ° C under continuous agitation. After incubation, the pH was 6.3.

Example 2.5 An aliquot of the lysate of Example 2.1 was taken to continue the lysis process. The volume was adjusted to pH 3.6 with 25% HCl and then incubated in a water bath at 40 ° C for 1 hour under static conditions.

Example 2.6 An aliquot of the lysate of Example 2.4 was taken to continue the lysis process. The volume was adjusted to pH 12.4 with 10 N NaOH and then incubated in a water bath at 40 ° C for 1 hour under static conditions.

Example 2.7 An aliquot of Lactobacillus biomass would plant 71.39 of Example 1.3 containing 0.4 g of bacterial dry weight was thawed at room temperature and then diluted with purified water to reach 7 g / L of dry weight of bacterial biomass. The alkalinization was carried out with 0.06 M sodium hydroxide. The pH measured at the beginning of the lysis was 12.6. Then the lysis was incubated for 2 hours at 40 ° C under continuous agitation. After incubation, the pH was 12.4.

Example 2.8 An aliquot of Lactobacillus johnsonii 103782 biomass of Example 1.5 containing 0.3 g of bacterial dry weight was thawed at room temperature and then diluted with purified water to reach 6 g / L dry weight of bacterial biomass. The alkalinization was carried out with 0.06 M sodium hydroxide. The pH measured at the beginning of the lysis was 12.5. Then the lysis was incubated for 2 hours at 40 ° C under continuous agitation. After incubation, the pH was 12.3.

Example 2.9 An aliquot of the biomass of Lactobacillus helveticus 103146 of Example 1.2 containing 0.4 g of bacterial dry weight was thawed at room temperature, then diluted with purified water to arrive at 8 g / L dry weight of bacterial biomass. The alkalinization was carried out with 0.06 M sodium hydroxide. The pH measured at the beginning of the lysis was 12.8. Then the lysis was incubated for 2 hours at 40 ° C under continuous agitation. After incubation, the pH was 12.2.

Example 2.10 An aliquot of the biomass of Lactobacillus fermentum 1-3929 of Example 1.1 containing 6.8 g of bacterial dry weight was thawed at room temperature, then diluted with purified water to arrive at 7 g / L dry weight of bacterial biomass. The alkalinization was carried out with 0.08 M sodium hydroxide. The pH measured at the beginning of the lysis was 12.2. Then the lysis was incubated for 7 hours at 40 ° C under continuous agitation. After incubation, the pH was 12.0. The pH of the lysis was adjusted to 9.8 with HCl.

The lysis comprised proteins in solubilized dry weight (SDW): 20.5 mg / g of proteins (Prot): 4.9 mg / g.

Example 2.11 An aliquot of the biomass of Lactobacillus fermentum 1-3929 of Example 1.1 containing 6.8 g of bacterial dry weight was thawed at room temperature, then diluted with purified water to arrive at 7 g / L dry weight of bacterial biomass. The alkalinization was carried out with 0.15 M sodium hydroxide. The pH measured at the beginning of the lysis was 13.0. Then the lysis was incubated for 63 hours at 40 ° C under continuous agitation. After incubation, the pH was 12.6. The lysis pH was adjusted to 9.9 with HC1.

The lysis comprised SDW: 23.7 mg / g, Prot: 5.0 mg / g.

Example 2.12 An aliquot of Lactobacillus fermentum I-3929 biomass of Example 1.1 containing 98 g of bacterial dry weight was thawed at room temperature, then diluted with purified water to reach 10 g / L dry weight of bacterial biomass. The alkalinization was carried out with 0.08 M sodium hydroxide. The pH measured at the beginning of the lysis was 12.0. Then the lysis was incubated for 24 hours at 40 ° C under continuous agitation. After incubation, the pH was 11.1. The lysis pH was adjusted to 11.5 with NaOH.

The lysis comprised SDW: 23.7 mg / g.

Example 2.13 An aliquot of Lactobacillus fermentum I-3929 biomass from Example 1.1 containing 39 g of bacterial dry weight was thawed at room temperature, then diluted with a 0.2 N NaCl solution to reach 7.6 g / L dry weight of bacterial biomass with In order to produce an osmotic lysate. The pH measured at the beginning of the lysis was 4.4. Then, the bacterial osmotic lysate was incubated for 24 hours at 40 ° C under continuous agitation. After incubation, the pH was 4.2. Then the bacterial lysate was made alkaline with the addition of NaOH to reach 0.06 N. The pH measured at the start of the alkaline lysis was 10.6. The lysis was re-incubated for 2.25 hours at 40 ° C under continuous agitation. After incubation, 1 the pH was 10.2.

Example 3; Lysate Purification Example 3.1 The lysate of Example 2.5 was centrifuged for 20 minutes at 3000 times the force of gravity. Then, the supernatant was adjusted to pH 6.8 with 10 N NaOH and filtered through successive filters with porosities of 0.45 μ ??? and 0.2 μp? and finally with a sterile filter of 0.22 μp ?. The concentrate comprised Prot: 2.8 mg / ml; DNA: 17.4 g / ml. Percentage of D-amino acid: 14.9% D-Ala, 14.4% D-Pro, 14.2% D-Asp.

Production of NO in mg dry weight / ml: 0.003 mg / ml (Cl) - 0.03 mg / ml (C2) - 0.3 mg / ml (C3) active dry weight / ml: Cl: 3.3 μ ?, C2: 33.2 μ, C3: 52.5 μ ?. Active dry weight in g / L or mg / ml is the dry weight soluble in g / L or mg / ml minus the chloride content in g / L or mg / ml.

Example 3.2 The lysate of Example 2.6 was centrifuged for 20 minutes at 3000 times the force of gravity. Then, the supernatant was adjusted to pH 7 with HC1 and filtered through successive filters with porosities of 0.45 μp? and 0.2 μp ?, and finally with a 0.22 μp sterile filter ?. The concentrate comprised Prot: 12.3 mg / ml; DNA: 27.7 μg / ml. Percentage of D-amino acid: 15.9% D-Ala, 24.4% D-Pro, 17.4% D-Asp, 45.1% D-Ser, 10.1% D-Met.

Production of NO in active dry weight mg / ml :: 0.003 mg / ml (Cl) - 0.03 mg / ml (C2) - 0.3 mg / ml (C3) active dry weight / ml: Cl: 8.0 μ ?, C2 : 56.0 μ ?, C3: 94.3 μ ?.

Example 3.3 300 ml of the lysate of example 2.1 were centrifuged first at 3000 times the force of gravity for 20 minutes. The supernatant was adjusted to pH 7.1 with HC1. The extract was concentrated through a 0.45 micron membrane in poly ether sulfone (PES) (Sartorius Stedim Biotech GmbH) with a constant flow of 330-350 mL / min in a cross reference flow Sartoflow® Slice 200 Benchtop. The volume of performance was 75%. The concentrate was finally sterile filtered through a memorandum of PES of 0.2 micras (Nalgene).

The concentrate comprised SDW: 30.0 mg / g; Prot: 9.6 mg / mL; AD: 32.8 g / ml. Percentage of D-amino acid: 14.3% D-Ala, 13.9% D-Pro, 14.1% D-Asp, 36.9% D-Ser. Production of NO in mg active dry weight / mL: 0.003 mg / mL (Cl) - 0.03 mg / mL (C2) -0.3 mg / mL (C3) active dry weight / mL: Cl: 5.1 μ ?, C2: 30.0 μ ?, C3: 64.0 μ ?.

Example 3.4 300 ml of the lysate from example 2.2 were adjusted to pH 7.0 with HC1. The extract was filtered as described in Example 3.3 with a constant flow at 350 mL / min. The volume yield was more than 75%. The concentrate was finally sterile filtered through a 0.2 micron PES membrane (Nalgene).

The concentrate comprised SDW: 49.2 mg / g; Prot: 14.2 mg / mL; DNA: 34.1 μg / ml. Percentage of D-amino acid: 16.0% D-Ala, 13.3% D-Pro, 14.2% D-Asp.

Production of NO in mg of active dry weight / mL: 0.003 mg / mL (Cl) - 0.03 mg / mL (C2) - 0.3 mg / mL (C3) active dry weight / mL: Cl: 0 μ ?, C2: 4.1 μ ?, C3: 26.3 μ ?.

Example 3.5 300 ml of the lysate from example 2.3 were centrifuged first at 3000 times the force of gravity for 20 minutes. The supernatant was adjusted to pH 7.1 with HC1. The extract was filtered as described in Example 3.3 with a constant flow at 330-350 mL / min. The volume yield was more than 75%. The concentrate was finally filtered sterile through a PES membrane of 0.2 micras (Nalgene).

The concentrate was composed of SDW: 34.5 mg / g; Prot: 8.9 mg / mL; DNA: 16.8 μg / mL. Percentage of D-amino acid: 16.4% D-Ala, 24.3% D-Pro, 17.3% D-Asp.

Production of NO in active dry weight mg / mL: 0.003 mg / mL (Cl) - 0.03 mg / mL (C2) - 0.3 mg / mL (C3) active dry weight / mL: Cl: 3.0 μ ?, C2: 36.4 μ ?, C3: 69.5 μ ?.

Example 3.6 1000 mL of the lysate of Example 2.13 were centrifuged for 20 minutes at 9384 times the force of gravity. The supernatant was filtered directly through a sterile 0.22 micron filter.

The concentrate comprised SDW: 32.7 mg / g; Prot: 6.5 mg / g; Sugar: 2.4 mg / g. The concentration of sugar was analyzed according to the procedure of Herber et al. (Meth Microbiol, 1971, 5B: 266 et seq.).

The selection of biological activity was carried out in human peripheral blood mono-nuclear cells (PBMC) at different concentrations as described in example 6. The results obtained at 0.5 mg of active dry weight / mL were TNF-a: 83 pg / mL; IL-6; 898 pg / mL; IL-12: 140 pg / mL (in the presence of 10 ng / mL) of IF-?) And IL-10: 221 pg / mL (in the presence of lFN-?).

Example 3.7 The bacterial lysate mixture from Example 2.10 was transferred to a micro-filtration tank (MF) after being adjusted to pH 10.2. The micro-filtration unit (MF) used a 0.45 micron (Sartocon Slice Sartorius) tangential flow filtration (TFF) filter. The cross flow was adjusted to 290 L / h m2 (LHM) and the trans-membrane pressure (TMP) to 0.4-0.5 bar. The permeate was transferred to an ultra-filtration (UF) tank.

Once the volume of the lysate in the micro-filtration tank had reached half the initial volume, the UF unit was started. The permeate of the ultrafiltration filters was used as a washing buffer in the micro-filtration tank. The volumes of both the micro-filtration and ultra-filtration tanks were maintained at the same level. After 10 volumes of diafiltration, the ultrafiltration was stopped and the bacterial lysate was concentrated in the micro-filtration tanks.

The recovered volumetric efficiency was 83%. The extract recovered in the ultrafiltration tank was adjusted to pH 7.3 with HCl and then filtered through a sterile 0.2 micron filter.

The concentrate comprised SDW: 17.5 mg / g; Prot: 4.3 mg / g.

Example 3.8 The lysate of Example 2.7 was centrifuged for 15 minutes at 9384 times the force of gravity. Then, the supernatant was adjusted to pH 7.3 with HCl and filtered through successive filters with porosities of 0.45 microns and 0.2 microns and finally with a sterile filter of 0.22 microns.

Peripheral blood mono-nuclear cells (PBMC) with a 10-fold diluted product: 777 pg / mL IL-10, 26 pg./mL IL-12, 13183 pg / mL IL-6.

Example 3.9 The lysate from example 2.8 was centrifuged for 15 minutes at 9384 times the force of gravity. Then the supernatant was adjusted to 7.4 with HCl and filtered through successive filters with porosities of 0.45 microns and 0.2 microns and finally with a sterile filter of 0.22 microns.

PBMC with 10-fold diluted product: 904 pg / mL IL-10, 0 pg / mL IL-12, 4995 pg / mL IL-6.

Example 3.10 The lysate from Example 2.9 was centrifuged for 15 minutes at 9384 times the force of gravity. Then, the supernatant was adjusted to pH 7.6 with HCl and filtered through successive filters with porosities of 0.45 microns and 0.2 microns and finally with a sterile filter of 0.22 microns.

The PBMC with product diluted 10 times: 476 pg / mL IL-10, 0 pg / mL IL-12, 4924 pg / mL IL-6.

Example 3.11 The lysate from Example 2.11 was transferred to a micro-filtration tank after being adjusted to pH 10.4. The installation of TFF was similar to example 3.7. The cross flow was adjusted to 2909 L / h m2 and the TMP to 0.4-0.5 bar. The ultrafiltration was stopped after 10 volumes of diafiltration. The yield in recovered volume was 86%. The final product was adjusted to pH 7.3 with HCl.

The concentrate obtained comprised SDW: 24.2 mg / g; Prot: 4.8 mg / g.

Example 3.12 The lysate from example 2.12 was transferred to a micro-filtration tank after being adjusted to pH 11.5. The installation of TFF was similar to example 3.7. The cross flow was adjusted to 290 L / h m2 and the TMP to 0.4 bar. The ultra- filtration was stopped after 10 volumes of diafiltration. The yield in recovered volume was 74%. The final product was adjusted to pH 7.2 with HCl.

The concentrate obtained comprised SDW: 32.4 mg / g; Prot: 5.8 mg / g; Sugar: 2.3 mg / g.

The selection of the biological activity was carried out on human mono-nuclear peripheral blood cells (PBMC) at different concentrations as described in example 6. The results obtained at 0.5 mg active dry weight / mL were TNF-OI: 37 pg / mL; IL-6: 1092 pg / mL; IL-12: 81 pg / mL (in the presence of 10 ng / ml IFN-?) And IL-10: 160 pg / mL (in the presence of 10 ng / ml IFN-?). Protein concentration was measured by DC protein analysis (BioRad, DC protein analysis, kit No. 500-0116). The micro-plate analysis protocol was carried out. The samples to be analyzed (0.1 mL) were diluted with 1.9 mL of pH-regulating solution of 25 mM phosphate pH 11. A standard curve of protein was prepared each time the analysis was carried out with a solution of bovine serum albumin at 2 mg. of BSA / ml (BSA, Pierce ref 23210). The BSA solution was diluted with a buffer solution of 25 mM phosphate pH 1.1 to obtain the following concentrations: 0.18, 0.24, 0.3, 0.36 and 0.42 mg BSA / mi. Samples (20 μ?) And BSA standards (20 μ?) Were d in quadruplicate to a dry clean micro-titer. Reagent A (25 μ?) Of the analysis kit of DC protein was d to each cavity. After 10 minutes, 200 of reagent B were d to each well. The micro-title plate was mixed on a rotating plate stirrer for 5 seconds. After 20 minutes at room temperature the absorbance at 750 nm was recorded. The protein concentration of each mixture was calculated using the slope of the linear regression on a standard protein curve: OD at 750 nm = a * (protein concentration) + b mg protein in sample = [20 x (OD a- 750 nm - b)] / a The soluble dry weight (SDW) was determined by obtaining 5 mL of the soluble fraction resulting from the lysis and drying it at a constant mass on a porcelain plate at 105 eC.

Example 4; Activation of murine spleen cells in vitro The immuno-stimulating effect of the embodiments of the invention was analyzed in vitro by measuring the activation of murine spleen cells (Alamar Blue analysis) Materials and methods Stimulation of spleen cells The Alamar Blue ™ analysis is designed to quantitatively measure the growth of human or animal cells. This analysis incorporates a fluorometric / colorimetric growth indicator based on activity detection metabolic by means of an oxidation-reduction indicator (REDOX) in response to chemical reduction resulting from cell growth. Related to growth, the REDOX indicator causes changes from the oxidized form (without fluorescence, blue) to the reduced form (fluorescent, red).

Balb / c mice (female, 6-8 weeks old) were received from Charles River Laboratories, Sulzfeld, Germany and were sacrificed by cervical dislocation. The vessels were homogenized using a potter device; cell suspensions were washed by centrifugation (280 x g, 4eC, 10 min) and re-suspended in 5 ml of RPMI 1640 containing 5% FCS, 100 U / ml penicillin and 100 g / ml streptomycin. 100 μ? of spleen cells (2 x 106 / ml) were incubated with 50 μ? of dilutions of bacterial extract in plates of 96 cavities (Falcon 3072) for 48 hours at 372C and 5% of C02 in the cell culture medium.

After the addition of 30 μ? of AlamarBlue ™ solution diluted 1: 1 with the culture medium, the chemical reduction of AlamarBlue ™ was measured with a Fluoroskan Ascent reader (ThermoLabsystems, Frankfurt, Germany) at excitation wavelengths of 544 nm and 590 nm emission.

Test articles The following extracts were tested: Afer300: extract of Lactobacillus fermentum 1-3929 (12 g / L) obtained as described in example 3.5 (31.6 mg active dry weight / g); Cfer300: extract of Lactobacillus fermentum 1-3929 (12 g / L) obtained as described in example 3.3 (28.4 mg active dry weight / g); Dfer30: extract of Lactobacillus fermentum 1-3929 (12 g / L) obtained as described in example 2.4 and purified using the same conditions as example 3.3 (29.5 mg active dry weight / g). This example was used as a control subjected to non-alkaline lysis.

ARahr300: Lactobacillus rhamnosus extract 71.38 (40 g / L) obtained by alkaline lysis with 0.03 M NaOH at 402C for 6 hours (49.3 mg active dry weight / g); CRahr300: Lactobacillus rhamnosus extract 71.38 (40 g / L) obtained as described in Example 3.4 (46.4 mg active dry weight / g); DRahr300: extract of Lactobacillus rhamnosus 71.38 (40 g / L) subjected to lysis by osmotic tension in 0.15 N NaCl solution at 40aC for 6 hours (46.2 mg active dry weight / g). This example was used as a control subjected to non-alkaline lysis.

Negative witnesses Dest. Water (distilled water) or phosphate buffered saline (PBS).

Results In vitro studies to determine splenocyte activation The increase in metabolic activity of mouse spleen cells after treatment with the extract was determined in 2 independent experiments by the analysis of Alamar Blue (see Figures 4a and 4b).

Spleen cells were cultured for 48 hours in the presence of the extract. After the addition of Alamar blue® solution, the emission values were measured. As shown in Figures 4a-b, the bacterial extracts were effective at dilutions about 1: 300. All the groups were statistically compared with the control groups using the student's T test.

Table 1: p value for bacterial extracts in two independent experiments by Alamar Blue analysis (see Figures 4a and 4b).

Lot Dilution Value p test 1 Value p test 2; Afer300 (dilution 1: 300) 0.000016 (Fig. 4a) 0.00016 (Fig. 4b) Cfer300 (dilution 1: 300.}. 0.00021 (Fig. 4a) 0.048 (Fig. 4b) Dfer300 (dilution 1: 300) 0.00033 (Fig. 4a) 0.00016 (Fig. 4b) ARahr300 (dilution 1: 300) 0.0029 (Fig. 4a) 0.0012 (Fig. 4b) CRahr300 (dilution 1: 300) 0.0031 (Fig. 4a) 0.12 (Fig. 4b), 0. 0025 (Fig. 4a) 0.08 (Fig. 4b).

DRahr300 (dilution 1: 300) conclusion The immuno-stimulating effect of bacterial extracts was analyzed in vitro by measuring the activation of spleen cells moved by the Alamar Blue analysis. Here, in two independent experiments, an extract obtained by osmotic lysis (Dfer300) was compared, which includes cell wall components in intact particles with two extracts according to this invention (AFer300 and CFer300). Those three extracts were obtained from Lactobacillus fermentum 1-3929. The extracts of AFer300, CFer300 and DFer300 were each effective in stimulating the metabolism of the cells at a dilution of 1: 300. No difference was observed between the osmotic lysis control DFer300 and the AFer300 and CFer3Q0.

Three extracts obtained from a second strain, Lactobacillus rhamnosus 71.39 (ARahr300, CRahr300, DRahr300), were also compared. As in the previous assembly, ARahr300 and CRahr300 are within the scope of the invention, while DRahr is an osmotic lysis control. Here, the extract of ARahr300 was effective in two independent analyzes at a dilution of 1: 300, while the extracts of CRahr300 and DRahr300 showed a weak but significant stimulation in one of the analyzes at the same dilution. When comparing the results of all the six extracts, it is appreciated that the extracts of Lactobacillus fermentum 1-3929 were more effective in stimulating the growth of spleen cells in comparison with the extracts of Lactobacillus rhamnosus 71:38.

Example 5: Production of nitric oxide by macrophages derived from bone marrow The immunostimulating potential of a series of embodiments according to the invention was tested by measuring the production of nitric oxide (NO) by murine bone marrow-derived macrophages.

Materials and methods Male C57 / BL6 mice of 6 weeks of age (6 weeks male, quality 'SPF, Charles Rivier, FR) were killed by inhalation of C02. The hip, femur and tibia of the posterior flap were removed. The bone marrow was removed from the lumen by injecting Dulbecco's modified Eagle (DH) medium through the bone after cutting both ends. After washing, the stem cells were re-suspended (40,000 cells / ml) in DH medium supplemented with 20% horse serum and 30% L929 cell supernatant. The cell suspension was incubated for 8 days in an incubator at 372C under 8% C02 and saturated humidity atmosphere. The macrophages were then detached with ice-cold PBS, washed and re-suspended in DH medium supplemented with 5% fetal calf serum (FCS), amino acids and antibiotics (DHE medium). The cell density was adjusted to 700000 cells / mL. The Aqueous solutions of the extracts were serially diluted in the DHE medium directly into micro-titer plates. The extracts of the invention were tested in triplicate and each micro-titer plate comprised the medium as a negative control. The final volume in each cavity was 100 uL. 100 μ ?? of the cell suspension were added to the diluted extracts and the cells were incubated for 22 hours in an incubator at 372C, under 8% C02 and saturated humidity atmosphere. At the end of the incubation period, 100 μ ??? of the supernatant were transferred to another micro-titer plate and the concentration of nitrite produced in each supernatant was determined by carrying out a Griess reaction. 100 μ ?. of Griess reagent (5 mg / mL of sulfanilamide + 0.5 mcr / mL of N- (l-naphthyl) ethylene-diamine hydrochloride) in 2.5% aqueous phosphoric acid was added to each well. The micro-titer plates were read with a spectrophotometer (SpectraMax Plus, Molecular Devices) at 562 nm against a reference at 690 nm. The nitrite concentration was proportional to the nitric oxide content that is formed. The nitrite content was determined based on a standard curve of sodium nitrite (NaN02 1 to 70 uM). The results were given in nitric oxide uM (NO) as mean value ± standard deviation and plotted as a dose response curve.

Test articles The following extracts were tested: First analysis: OP070lB4_Cfer300: extract of Lactobacillus fermentum 1-3929 (12 g / L) obtained as described in example 3.3; OP0701B4_Dfer300: extract of Lactobacillus fermentum 1-3929 (12 g / L) obtained as described in example 2.4 and purified using the same conditions as in example 3.3. This example was used as a control subjected to non-alkaline lysis.

OP070lB4_CRahr300: extract of Lactobacillus rhamnosus 71.38- (40 g / L) obtained as described in example 3.4.

OP070lB4_DRahr300: Lactobacillus rhamnosus extract 71.38 (40 g / L) obtained by osmotic tension in 0.15 N NaCl solution at 40 aC for 6 hours. This example was used as a control subjected to non-alkaline lysis.

Second analysis: OP0701C_10G0.5P4H: extract of Lactobacillus fermentum 1-3929 at 10 g / 1 dry weight of biomass with 0.037 M NaOH included at 402C for 4 hours.

OP0701C_10G1P4H: extract of Lactobacillus fermentum 1-3929 at 10 g / 1 dry weight of biomass with 0.075 M NaOH incubated at 40aC for 4 hours.

OP0701C_10G2P4H: extract of Lactobacillus fermentum 1-3929 at 10 g / 1 dry weight of biomass with 0.150 M NaOH incubated at 402C for 4 hours.

OP0701C_10G1P21H: extract of Lactobacillus fermentum 1-3929 at 10 g / 1 dry weight of biomass with 0.075 M NaOH incubated at 40eC for 21 hours.

OP070lC_10G0.5P21H: extract of Lactobacillus fermentum 1-3929 at 10 g / 1 dry weight of biomass with 0.037 M NaOH incubated at 40eC for 21 hours.

OP0701C_10G2P21H: extract of Lactobacillus fermentum 1-3929 at 10 g / 1 dry weight of biomass with 0.150 M NaOH incubated at 402C for 21 hours.

OP0701C_10G2P45H: extract of Lactobacillus fermentum 1-3929 at 10 g / 1 dry weight of biomass with 0.1.50 M NaOH incubated at 40aC for 45 hours.

Results of the first analysis In the first analysis (Figure 5a), the extracts obtained from the alkaline lysis according to the present invention were observed to have the same activity as the extracts obtained from an osmotic lysis.

It was also observed that the immuno-stimulation activity was related to the selected strain. The extracts obtained from Lactobacillus fermentum 1-3929 induced higher production of O2 and N03 than the extracts obtained from the strain Lactobacillus rhamnosus 78.31.

Results of the second analysis In the second analysis (Figure 5b) it was observed that the in vitro activity of the extract was correlated with the initial conditions of the lysis. The activities of the following bacterial extracts are shown for the same strain Lactobacillus fermentum 1-3929 and for the same amount of 10 g of biomass dry weight / liter of lysis: OP0701C_10G0.5P4H, OP0701C_10G1P4H, OP0701C_10G2P4H, OP0701C_10G1P21H, OP0701C_10G0.5P21H, OP0701C_10G2P21H and OP0701C_10G2P45H. In vitro activity depended on the initial concentration of NaOH and the duration of lysis. conclusion The results of these experiments showed that in spite of the chemical modifications generated by the alkaline process, the activity of the modalities is not diminished when compared to a control extract obtained by osmotic lysis or in comparison with the respective live bacteria (see example 6). below for this aspect).

Example 6: In vitro selection for pro- and anti-inflammatory activity In order to select embodiments of the invention in terms of its immuno-stimulant or anti-inflammatory potential in vitro, tests were carried out on a series of extracts Bacterial in human PBMC. The release of both ILl2p70 (an inflammatory cytokine) and IL-10 (an anti-inflammatory cytokine) was measured and the proportion of IL-10 / IL-12 was reported, as described by Foligne et al. (World J Gastroenterol 2007 January 14; 13 (2): 236-243).

The objective was to compare the production of IL-12p70 and IL-10 in a series of 6 extracts obtained via different extraction / purification methods. The proportion of IL-10 / IL-12p70 obtained for each extract can be correlated with the anti-inflammatory potential of the extracts. Live bacteria were used as control against which to compare the activities of the extracts (2xl07 cfu / ml of Lactobacillus fermentum 1-3929).

Materials and methods Graded quantities of the 6 bacterial extracts were diluted in cell culture medium in the presence of 10 ng / ml of IFN- ?, a cytokine that is known to improve the production of IL-12p70, starting with an initial dose of 1 mg / ml (highest final dose tested). PBMC isolated from the blood of healthy donors were tested as serial dilutions of active dry weight extracts of 100 ng / ml. at 1 mg / ml.

IFN-? at least 3 hours before the lysates with the bacterial strain alive in the medium. They get reported data from two independent experiments.

Preparation of human PBMC Human PBMC were isolated from peripheral blood. Briefly, after a Ficoll gradient centrifugation (Pharmacia, Uppsala, Sweden), mononuclear cells were collected, washed in RPMI 1640 medium (Live technologies, Paisley, Scotland) and adjusted to 2 x 106 cells / ml in RPMI 1640 supplemented with gentamicin (150 pg / mL), L-amine (2 mmol / L) and 10% fetal calf serum (FCS) (Gibco-BRL).

Cytokine induction PBMC (2 x 106 cells / mL) were seeded in culture plates of. tissue of 24 cavities (Corning, NY). The cells were stimulated as described above. After 24 hours of stimulation at 37 aC in an atmosphere of air with 5% CO2, the culture supernatants were collected, clarified by centrifugation and stored at -20 ° C until the cytokine analysis. Cytokines were measured by ELISA using pairs of anti-body pharmingen (BD Biosciences, San Jose, Ca, United States of America) in terms of IL-10 and IL-12p70, according to the manufacturer's recommendations.

Test articles OP0701C_10G2P45H (A): Lysing extract of Lactobacillus fermentum 1-3929 at 10 g of biomass dry weight / liter lysis, 0.15 M NaOH at 402 C for 45 hours.

OP0701C_10G1P4H (B): Lysis extract from Lactobacillus fermentum 1-3929 at 10 g of biomass dry weight / liter of lysis, 0.075 M NaOH at 402 C for 4 hours.

OP0701C_5G4P21H (C): Lysing extract of Lactobacillus fermentum 1-3929 at 5 g dry weight biomass / liter lysis, 0.300 M NaOH at 40 ° C for 21 hours.

OP0701C_40G0.5P4H (D): Lysing extract of Lactobacillus fermentum 1-3929 at 40 g of biomass dry weight / liter of lysis, 0.037 M NaOH at 40 aC for 6 hours.

OP070lB4_CFerl50 (E): Lysing extract of Lactobacillus fermentum 1-3929 (12 g / L) obtained as described in example 3.1.

OP070lB4_BFer300 (F): Lysing extract of Lactobacillus fermentum 1-3929 at 6 g dry weight of biomass / liter of lysis, 0.075 M NaOH at 402C for 6 hours.

Lactobacillus fermentum 1-3929, live bacteria frozen at -809C at 2 x 107 cfu / ml in 20% glycerol.

Results The aim of this study was to compare the immuno-stimulation or anti-inflammatory potential in vitro of embodiments of the invention with a live Lactobacillus strain.

In order to analyze the different extracts and live bacteria, the amount of each cytokine IL-10 and IL-12 released from the "PBMCs was compared, also as the ratio of IL10 / IL12.

If either of the IL-10 or IL-12 concentrations or both values were close to unstimulated levels (ie, less than 10 pg / ml), the proportion was not considered as calculable and marked as NC (value not calculated).

The results obtained from the PBMC expressed in pg / ml are shown in Tables 2 and 3.

In the presence of IFNy, the effect of 6 bacterial extracts of the invention (extract A to extract F) were compared with a form of Lactobacillus fermentum (2 x 107 bacteria / ml, tested twice as live sample 1 and live sample 2) in Human PBMC (IL-10 and IL-12p70 responses in pg / ml). The IL10 / IL12 ratios are reported only for the doses of 100 ug / ml and 1 mg / ml extracts.

Table 2: Pro-inflammatory potential (immuno-stimulant) or anti-inflammatory (immuno-modulator) in vitro of bacterial extracts in human PBMC.

N. C: Value not calculable In the presence of IFN-α, the effect of 6 extracts of the invention (extract A to extract F) were compared with a live form of Lactobacillus fermentum (2 x 107 bacteria / ml, tested twice as live sample 1 and live sample 2 ) in human PBMC (response of IL10 and IL1 2 p70 in pg / ml). The IL10 / IL12 ratios are reported only for the dose of 100 μg / ml and 1 mg / ml extracts.

Table 3: In vitro pro-inflammatory (immuno-stimulant) anti-inflammatory (immuno-modulator) potential of the strain Lactobacillus fermentum viva 1-3929 in human PBMC.

Based on the results presented in Table 2 and Table 3, the production of IL-10 was descending in the following order: Live Lactobactus > A > C Based on the results presented in Table 2 and Table 3, the production of IL-12 was decreasing in the following order: Live Lactobacillus > E > B > D > F > A > C When the proportion is considered, the general pattern was observed: B = D > E = F > LactobaciBus live The results of the proportions of IL10 / IL12 for extracts A and C are not shown since it was not found that these extracts were effective inducers of cytokines.

At lower concentrations of 100 ug / ml, it was observed that the cytokine concentrations were too low to draw any conclusion conclusion The proportions obtained suggest that, under the experimental conditions used in example 6, some bacterial extracts of the invention have greater immuno-modulatory effects than those of live Lactobacillus fermentum. For example, extracts B, D, E and F showed higher IL10 / IL12p70 ratios than the live bacterium control. Thus, such extracts could be more active than live pro-biotic bacteria against conditions described herein, such as inflammatory conditions.

Example 7; Action on receptors similar to Toll Modes of the invention are obtained from gram-positive bacteria and consequently, they are expected to act via TLR2 receptors. TLR receptors are expressed principally but not exclusively by immune cells such as monocytes, macrophages, dendritic cells, T cells, etc. and they are key detectors of microbial products, which can be recognized as signal hazards by the host. Although they trigger a non-specific innate immunity first, the Activation of TLR initiates a full immunological cascade that results, in the presence of antigens, in the development of acquired immunity.

Cells that express a given functional TLR gene are valuable tools for many applications, such as the study of the mechanisms involved in the recognition or signaling of TLR and the development of new potential therapeutic drugs. The experiments described below tested the activity of three bacterial extracts in these key immune response adapters.

Materials and methods The responses of the extracts of the invention were tested (either per se to verify their agonist effect or in the presence of the TLR2 agonist Pam3Cys or in the presence of the TLR4 agonist LPS to verify the antagonistic activities) in the following two cellular systems: a) HEK-TLR2 / 6 (IL-8 ELISA after 24 hours) b) HEK-MD2-TLR4-CDl4 (IL-8 ELISA after 24 hours) a) HEK-TLR2 / 6 The HEK293 cell line was chosen for its basal or low basal expression of the TLR genes. These cells allow efficient monitoring of TLR activity using ELISA analysis such as IL-8 titration or reporter-based systems that monitor the activation of TLR-induced NF-kB.

HEK-TLR2 / 6 cells (invivogen, Toulouse, France) are designed. HEK293 cells stably transfected with multiple genes from the TLR2 / 6 pathway that include TLR2, TLR6 and genes involved in recognition or involved in the signaling cascade. These cells secrete IL-8 after stimulation of TLR2 / 6. The experiments were carried out according to the manufacturer's instructions.

Briefly, 2 x 10 4 cells / well (200 μ? Of RPMI) were incubated at 37 aC for 3 days (5% C02). The medium was removed and 90 μ? of RPMI + FCS at 5% to the cavities. Then, the agonists and controls were added (10 μ? / Cavity). The cells were returned to the incubator for 24 hours. The supernatants were collected and the IL-8 ELISA was carried out according to the manufacturer's instructions.

Test articles OP0700lB4_Afer50: Lysing extract of Lactobacillus fermentum 1-3929 at 12 g dry weight biomass / liter lysis, 0.075 M NaOH at 40 SC for 4 hours, purified as described in Example 3.6.

OP0701C-BtlLAC: Lysing extract of Lactobacillus fermentum 1-3929 obtained as described in example 3.7.

OP0701C-Bt2LAC: Lysing extract of Lactobacillus fermentum 1-3929 obtained as described in example 3.11.

Results Secretion of IL-8 The results for the controls (negative = ultra-pure K12 LPS, TLR4 agonist and PAM3CSK4 = positive, TLR2 agonist) are provided in Tables 4-6. The results (expressed as pg / ml of IL-8) show the average values of IL-8 secretion determined by ELISA 24 hours after stimulation with the controls.

The HEK TLR2 / 6 cell line responded to the TLR2 agonist Pam3Cys. In contrast, the TLR 4 agonist of E coli (LPS K12) was inactive, even at the high dose of 0.01 ug / ml.

Experiments with the 3 bacterial extracts alone The 3 bacterial extracts tested in the present (OP0701B4 Afer50, OP070lC-BtlLAC, and OP0701C-Bt2LAC) exhibited higher immunostimulatory properties than the TLR2 agonist Pam3Cys.

Experiments with 3 bacterial extracts + Pam3Cys As mentioned above, the three bacterial extracts were also tested for their antagonistic or additive properties against Pam3Cys added just after of the extracts.

Bacterial extract OP070lB4_AFer50 Table 4: Secretion of IL-8 (pg / ml) of supernatants obtained from HEK TLR2 / 6 cells stimulated at the concentrations indicated with the controls (medium, LPS or Pam3Cys) with the bacterial extract OP070lB4_AFer50 or with the extract and Pam3Cys.

The results in Table 4 indicate that the extract of OP070lB4_AFer50 induced the production of high levels of IL-8 (TLR 2/6 agonist).

Bacterial extract OP0701C-BtlLAC Table 5: Secretion of IL-8 (pg / ml) of supernatants obtained in HEK TLR2 / 6 cells stimulated at the concentrations indicated with the controls (medium, LPS or Pam3Cys), with the bacterial extract OP0701C-BtlLAC or with the extract and Pam3Cys.

The results in Table 5 indicate that the extract of OP0701C-BtlLAC induced the production of high levels of IL-8 (TLR 2/6 agonist).

Bacterial extract OP0701C-Bt2LAC Table 6: Secretion of IL-8 (pg / ml) of supernatants obtained in HEK TLR2 / 6 cells stimulated at the concentrations indicated with the controls (medium, LPS or Pam3Cys) with the bacterial extract OP0701C-Bt2LAC or with the extract and Pam3Cys .

The results in Table 6 indicate that the OP0701C-Bt2LAC extract did not induce IL-8 production (TLR 2/6 agonist) and in the presence of Pam3Cys had an antagonistic effect on TLR2 / 6. conclusion Depending on the conditions of the alkaline lysis (initial concentration of biomass dry weight, initial base concentration and duration of the basic treatment), the bacterial extracts could have different modes of action.

OP070lB4_Afer50 and OP070lC-BtlLAC were agonists to TLR2 / 6 but stronger alkaline lysis performed on the same bacterial strain resulted in an antagonist TLR2 / 6 activity (OP0701C-Bt2LAC). b) HEK-TLR4-MD2-CD14 TLR4 was studied extensively since it is the main receptor involved in the recognition of lipo-polysaccharide (LPS) responsible for septic shock.

HEK-TLR4-MD2-CD14 cells are highly sensitive to LPS. They were obtained by stable transfection of HEK293 cells with TLR4, MD2 and CD14 genes and an NF-kB-inducible reporter system. These cells secrete IL-8.

The same experimental procedure was used as for the other HEK TLR2 / 6 cell line described above. The results for the controls and the 3 bacterial extracts of the invention are shown in Tables 7-9.

Results; Secretion of IL-8 The results for the witnesses (positive = LPS K12C ultra-pure, TLR4 agonist and PAM3CSK4 = negative, TLR2 agonist) are provided in Tables 7-9. The results (expressed as pg / ml of IL-8) show the average values of IL-8 secretion 24 hours after stimulation with the controls.

The cell line clearly responds only to the TLR4 agonist LPS K12. In contrast, as expected, the TLR2 agonist of Pam3Cys was inactive even at the high dose of 0.01 iig / ml.

Experiments with the 3 bacterial extracts alone As expected from the gram-positive bacterial agonists, the 3 bacterial extracts tested in the present (OP0701B4 Afer50: Table 7, OP070lC-BtlLAC: Table 8 and OP0701C-Bt2LAC: Table 9) did not exhibit any clear immuno-stimulatory property via the TLR4 route.

Experiments with the 3 bacterial extracts + LPS K12 As mentioned above, the three extracts of the invention were also tested for their supposed antagonistic or additive properties against LPS added just after the extracts. Again, no effect on the level of the TLR4 receptor was observed (Tables 7-9).

Bacterial extract OP0701B4 AFer50 Table 7: Secretion of IL-8 (pg / ml) of supernatants obtained in HEK TLR4 cells stimulated at the concentrations indicated with the controls (medium, LPS or Pam3Cys) with the bacterial extracts OP0701C-AFer50 or with the extracts and LPS K12.

The results indicate that the extract of OP070lB4_AFer50 did not activate the TLR4 receptor (Pam3Cys: 138 pg / mL).

Bacterial extract of OP0701CBtl-LAC Table 8: Secretion of IL-8 (pg / ml) of supernatants obtained on HEK TLR4 cells stimulated at the concentrations indicated with the controls (medium, LPS or Pam3Cys) with the bacterial extracts OP070lC-BtlLAC or with the LPS K12 extracts.

LPS 12 0.0003 UQftlll 0.001 tra / ml 0.003 uo / ml 0.01 m 251 477 802 1333 Bacterial extract of OP0701CBtl2-LAC Table 9: Secretion of IL-8 (pg / ml) of supernatants obtained in HEK-TLR4 cells stimulated at the concentrations indicated with the controls (medium, LPS or Pam3Cys) with the bacterial extracts OP0701C-Btl2LAC or with the extracts and LPS K12 . conclusion Taken together, the results presented herein with respect to HEK cells, and those obtained in human PBMC cells, suggest that Afer50 and BTlLAC are suitable for stimulate the immune system via the TLR2 pathway. In addition, BT2LAC can behave as a TLR2 / 6 antagonist. From here, the extracts according to the invention can stimulate the immune system via the TLR2 pathway or act as an antagonist of TLR2 / 6, thus correlating the anti-infection and anti-inflammatory activities in vivo.

Example 8: Cells that express a given functional TLR gene are valuable tools for many applications, such as the study of the mechanisms involved in the recognition or signaling of TLR, and the development of new potential therapeutic drugs. Therefore, it was the objective of the experiments described below to test the activity of 4 bacterial extracts in these key adapters of the immune response.

In this test, 8 TLR and NOD2 receptors were selected by 4 bacterial extracts, including extracts according to the present invention.

Method Bacterial extracts according to the invention were tested in 96-well microplates. The extracts were diluted in DMEM culture medium and 20 μ? of each dilution was tested in duplicate. A volume of 180 μ? from suspensions of HEK293 cells containing 25,000 or 50000 cells in DMEM culture medium + 10% FCS (a cell line HEK293 specified for each TLR with reporter gene secreting alkaline phosphatase under the control of NF- ??) were added to each cavity in duplicate. After 16 hours of incubation with each cell line, 20 to 50 μ? of each supernatant were transferred to 96-well microplates and completed with 200 μ? of Quantiblue (InVivoGen not REP-QBl). The enzymatic reaction with secreted alkaline phosphatase was carried out for 30 to 60 minutes for the different series of cell lines expressing TLR. The reading was made using a microplate reader at 630 nm. The results are expressed in OD at 630 nm. materials List of positive control agonists (and their respective concentrations) used in this selection analysis TLR2 PAM2 100 ng / ml; TLR3 Poly (I: C) 100 ng / ml; TLR4 E. coli K12 LPS 1 μg / ml; TLR5 S. typhimurium flagellin 1 g / ml; TLR7 R848 10 g / ml; TLR8 R848 10 pg / ml; TLR9 CpG ODN 2006 10 μg / ml; NOD2 Muramildipeptide 1 g / ml.

Negative Witnesses A recombinant HEK-293 cell line for the reporter gene only (NFkB) was used as a negative control for TLR cell lines. The negative control value for each clone was the background signal of these non-induced clones. TNF-alpha was used as a positive control for this cell line that does not express TLR.

Test Items The following bacterial extracts were tested: OP070lB4_CFer300: extract of Lactobacillus fermentum 1-3929 (12 g / L) obtained as described in Example 3.3 (F); OP070lB4_CRahr300: extract of Lactobacillus rhamnosus 71.38 (40 g / L) obtained as described in Example 3.4 (G); OP070lD_10Ll PswitchA: extract of Lactobacillus fermentum 1-3929 (10 g / L) obtained as described in Example 3.12 (I); Y OP070lD_5LOSMOConc: osmotic lysis extract of Lactobacillus fermentum 1-3929 (7.6 g / L) obtained by osmotic tension in 0.16 N NaCl solution at 40 ° C for 24 hours (H) as a control.

Preparation of bacterial extracts 20 μ? of each sample to be tested were used to stimulate all cell lines in a 200 μL reaction volume.

The selection was made at a single concentration, commonly at 0.5 mg dry weight / ml. The tests were carried out in duplicate.

Results The bacterial and control extracts were tested in duplicate in the recombinant HEK-293 cell line that functionally expresses a TLR or NOD2 protein also given as a reporter gene driven by the NFkB promoter. The activation results of TLR and NOD2 are given as optical density (OD) values. The results are shown in Tables 10-14, and summarized in Table 15.

Bacterial Extract F (OP0701B4Cfer300) Table 10: Activation of TLR and NOD2 receptors by extracts of OP070lB4Cfer300.

Bacterial extract F specifically activated hTLR2, hTLR4 and Nod2, and to a lesser extent, hTLR5.

Bacterial Extract G (OP070lB4CRahr300) Table 11: activation of TLR and N0D2 receptors by extracts of OP070lB4CRahr300.

Bacterial extract G activated hTLR2, h OD2, and to a much smaller extent, hTLR4.

Bacterial Extract H (OP0701D_5LOSMOConc) Table 12: activation of TLR and NOD2 receptors by extracts of OP070lD_5LOSMOConc.

The Bacterial H extract specifically activated hTLR2, hTLR4 and Nod2.

Bacterial Extract I (OP0701D_10L1 PswitchA) Table 13: Activation of TLR and NOD2 receptors through extracts of OP070lD_10LPswitchA.

Bacterial Extract I strongly activates hTLR2, hTLR5, h 0D2, and to a lesser extent, hTLR4 and hTLR9.

Table 15: Activation of TLR and N0D2 receptors by different bacterial extracts obtained from different process conditions according to the present invention.

Thus, bacterial extracts according to the present invention can help to activate the immune system via the various TLRs and also Nod2. Accordingly, the extracts of the invention can be good activators of the immune response.

Comparing the extract H with the extract G and extract F, it becomes evident that the extracts according to the invention have the same TLR and Nod route as the extracts obtained from osmotic lysis. Comparing extract H and extract I, it can be seen that the TLR5 pathway was activated, and the TLR9 pathway to a lesser extent, by the addition of an acid lysis followed by alkaline. Even extract F, resulting from a weak alkaline process, showed activation of the TLR5 pathway as opposed to extract H.

Extract I acted on TLR5, which indicates a potential application in radiotherapy. Of course, radiation therapy is a well-established and highly effective treatment for certain types of cancer. Its side effects can be devastating, however, as it can destroy healthy cells in the body, especially bone marrow cells and cells in the gastrointestinal system. Burdelya et al. recently reported that a peptide CBLB502 binds to TLR5 and activates the signaling pathway of the factor ?? nuclear, a pathway that cancer cells often activate to prevent cell death (Burdelya et al., "An agonist of toll-like receptor 5 has radioprotective activity in mouse and primate models", Science, 2008, 320: 226-30 ). Mice and rhesus monkeys treated with CBLB502 briefly before exposure to fatal doses of total body irradiation exhibited less damage to bone marrow cells and healthy gastrointestinal cells and survived significantly longer than controls. Importantly, in tumor-bearing mice, CBLB502 does not compromised the antitumor efficacy of radiation therapy.

Example 9; Ability of the extract to produce plaque-forming cells (against red blood cells of sheep) in mice The plate-forming cell (PFC) technique allows the evaluation of a non-specific stimulation of B-lymphocytes. This technique was first described by Cunningham and Seenberg (Immunology, 1968, 14, 599). The presence of hemolytic antibodies around antibody-forming cells was shown as described below. Murine lymphoid cells, also as a dense population of foreign erythrocytes (sheep), were simultaneously introduced onto a microscope slide. Certain lymphoid cells release hemolytic antibodies that diffuse and cause lysis of neighboring red blood cells by forming a lysis plate in the presence of complement. At the end of the experiment, the number of reported PFCs was counted at 106 cells or at the spleen.

Materials and methods Test articles Bacterial extract 1 obtained as described in Example 3.5, and bacterial extract 2 obtained as described in Example 3.4.

Animals 40 male Balb / C mice / experiment (IFFA-CREDO, St. Germain sur I'Arbresle Cedex, France), from 5 to 6 weeks of age and with an average weight of 20 +/- 2 g were distributed in 5 groups of 8 animals each.

Experimental design The animals were divided into 5 groups as follows: Group (a) 8 mice received 1.2 mg / mouse extract 1 obtained as described in Example 3.5; volume = 0.2 mi Group (b) 8 mice received 0.6 mg / reiton extract 1 obtained as described in Example 3.5.

Group (c) 8 mice received 1.2 mg / mouse extract 2 obtained as described in Example 3.4; volume = 0.2 mi Group (d) 8 mice received 0.6 mg / mouse extract 2 obtained as described in Example 3.4; volume = 0.2 mi Group (e) 8 mice received 0.2 ml of isotonic saline.

The mice received the extracts described above, at the indicated doses, daily per os (via the oral route) for 5 consecutive days (day 1 to day 5). Two more intubations occurred on days 19 and 20. On day 29, the antigen (106 red blood cells of sheep) in 0.2 ml of isotonic saline solution (0.9% NaCl, Alsever's solution) was injected intravenously through the caudal vein of the animal; 4 days later (day 33) suspensions of spleen cells were prepared.

Reagents and equipment Solution of Alsever, free of endotoxin, (Sigma, 38299 St Quentin Fallavier, Cedex France, ref A 3351) Basal Eagle Medium (EME, lOx concentrate) in bicarbonate-free Earle solution (Bio-Merieux, 69280 Marcy l'Etoile, France ref: 8 210 2) Freeze-dried guinea pig serum (Bio-Merieux, Marcy l'Etoile, France ref: 7 212 2) Red blood cells of sheep (Bio-Merieux, Marcy l'Etoile, France ref: 7214 1) Trypan blue, sterile distilled water, 0.9% NaCl, tissue homogenizer, centrifuge, Neubauer chamber for cell numbering, glass tubes.

Treatment with test article or witness The control animals received sheep red blood cells (SRBC) only. Animals from other groups received the bacterial extracts per os as indicated previously. For this purpose, each extract was dissolved in water Suspension of Spleen Cells 4 days after the last antigen injection (SRBC), a suspension of spleen cells was prepared for each mouse according to the following procedure: The animal was anesthetized with ether and then sacrificed by cervical dislocation. The spleen was removed and crushed in a glass tissue homogenizer with 2 ml of BME at pH 6.75-6.80. Then, 3 ml of sterile distilled water was added, and the mixture was stirred for 20 seconds. The suspension obtained was further diluted with 10 ml of BME and centrifuged at 1200 rpm for 10 minutes at 4 ° C. The supernatant was discarded and the precipitate was suspended in -2 ml of BME. The cells are allowed to recover 5-10 minutes on ice. A viability count of the mononuclear cells was carried out in the following manner: A 1/20 dilution of an aliquot of the cell suspension was prepared with saline solution containing 0.1% trypan blue solution. The cells without color, this is the living cells, were counted in a Neubauer chamber.

The cell suspension was kept on melted ice during counting and then immediately used for the lysis plates. This technique prevents a loss in feasibility that can happen if the wait is too long.

Preparation of slides On an ordinary microscope slide, carefully dedusted and with any traces of grease removed, 3 parallel strips (0.1 mm thick) of double-sided tape were glued at intervals of approximately 1.5 cm. The bands were then covered with pre-cleaned coverslips (22 mm x 22 mm) to form two "chambers" between the slide and coverslips.

A fraction of the spleen cell suspension was adjusted to 25,000 mononuclear cells per mm 3. In a small glass tube, a mixture of the following elements (aggregates in the order described) was prepared by means of an automatic pipette: 1) 7.5 mi from BME 2) 0.5 ml of the SRBC suspension washed and centrifuged twice with saline, at 50% residue 3) 0.4 ml of normal guinea pig serum used as a source of complement.

Finally, at 50 μ? of the spleen cell suspension at 25,000 cells / mm3 were added to 200 μ? of the preceding mixture. After a gentle homogenization, the suspension was introduced into "Cunningham chambers" by capillary action before sealing of the free slides with paraffin. Then the slides were placed in a humid chamber in an oven at 37 ° C.

Reading After one hour incubation in the oven, the slides were examined under the 100-fold magnified microscope (10 X ocular, 10 X objective).

The lysis plates were easy to recognize. 5 vertical optical bands were examined on each slide and the number of lysis plates counted. The number of cells (N) that form direct lysis plates per 106 cells was calculated by extrapolation. If x is the number of plates observed and X the number of cells examined, the number of direct lysis plates per 106 cells is equal to: N = (X / X) * 106 where X = C * V = C * (n * e * 1 * L) C = final concentration of spleen cells V = observed volume n = number of optical bands e = thickness of the adhesive tape 1 = optical field width L = length of an optical band.

The number of direct lysis plates per 106 cells was obtained from the following equation: PFC (by 106 cells) = (x * 106) / (C * n * e * l * L) By knowing the number of cells collected per spleen, it was possible to deduce the number of cells that form lysis plaques per spleen.

In the "absolute reference" mice, no spontaneous lysis plaque was observed.

Statistical Analysis and Results The results were considered significant when p < 0.05 (Student's t test). The results are shown in Table 16.

Table 16: Ability of bacterial extracts 1 and 2 to produce plaque-forming cells (against red blood cells of sheep) in mice GRM PFC / 106 PFC / proportion Cells Extract 1; see Example 3.5. 100% 124% * 1 19% * 1. 2 mg / mouse / administration) 100% 123% * 143% * 100% 1 14% * 113% Extract 1; see Example 3.5 100% 117% * 103% (0.6 mg / mouse / administration) 100% 118% * 139% * 100% 1 12% * 128% * Extract 2; see Example 3.4 100% 119% * 109% (1.2 mg / mouse administration) 100% 124% * 131% * 100% 1 17% * 120% * Extract 2; see Example 3.4 100% 1 17% * 112% * (0.6 mg / mouse / administration) 100% 114% * 119% * 100% 113% * 111% * The extracts of the invention appeared to be B cell activators when tested at concentrations of 1.2 mg / mouse / d at or 0.6 mg / mouse / day. A slight dose-dependent effect was observed. Hence, some extracts of the invention could potentially be used to prime the immune system in patients suffering from recurrent infections.

Example 10; Effect of CFer300 on intraperitoneal Salmonella typhi uri m infection in balb / c mice The protection of mice infected with Salmonella typhimurium was tested by bacterial lysates of Lactobacillus origin after oral administration.

Materials and methods Animals and Domestic Animal Breeding Balb / c mice were housed in the facilities of the Institute for Immunology, Moscow. For the in vivo protection experiment, laboratory grade white mice without cross were purchased from Stolbovaya, the Russian State Scientific Center for Biomedical Technologies (Russian Academy of Medical Sciences). Upon arrival, the mice had a body weight of 12-14 g. In all experiments, mice were kept under pathogen-free conditions in standard rodent diet and water.

Study Groups (main experiment) Two groups of 22 mice per group were used to test the anti-infective efficacy of bacterial extracts prepared using an experimental model of Salmonella typhimurium infection in mice.

One group was treated orally with a solution of CFer300, and the second group received a treatment of sham (water) as a negative control. 0. 5 ml of the solution were administered to each mouse orally once a day for 10 consecutive days before all the mice were attacked with Salmonella typhimurium: Group 1: Mice treated with CFer300 given per os a single dose 2 mg (0.5 ml).

Group 2: The mice received a sham treatment using oral administration of 0.5 ml of water daily for 10 days.

Test Article The bacterial extract tested was OP0701B4CFer300 ("CFer300"); the extract of Lactobacillus fermentu 1-3929 (12 g / L) obtained as described in Example 3.3 (28.4 mg active dry weight / g).

Preliminary experiment The purpose of the preliminary experiment was to determine the dose of the infectious agent that would induce mortality close to 50% three weeks after the attack. As an attack, a suspension of enteric Salmonella, serovar typhimurium strain 415 (I. Mecnnikov, Institute for Vaccines and Serum, Russian Academy of Medical Sciences) was injected intraperitoneally to each mouse. The attack dose ranged from 103 to 105 CFU of Salmonellae per mouse.

Observations and death record After the attack, the mice were kept under standard conditions for laboratory animals. Daily observations and death records were recorded during a period of 21 days post-infection. The anti-infective efficacy of the preparations was estimated according to the proportion of post-infection survival (SR), the average post-infection duration of life (ADL), the defense factor (DF), and the efficiency index of preparation ( El), calculated for each experimental group. The SR was taken as percent of live animals in the experimental groups on day 21 post-infection. The ADL, DF and El were calculated as follows: ADL = (XI + X2 + ... + Xn) / N where: ADL is the average life span, XI to Xn are the post-infection life durations for experimental mice 1 to n, and N is the total number of animals in the experimental group.

DF = CD / ED where: DF is the defense factor, CD is the percent of death in the control group, and ED is the percent of death in the experimental group.

El = [(DF - I) / DF] x 100% where: The is the preparation efficiency index, and DF is the defense factor.

Results; drug tolerance The preparation was administered orally once a day for 10 days and was well tolerated. No evidence of toxicity or side effects was observed during the 10-day pretreatment period.

Titration of Salmonellae A preliminary experiment was carried out in order to determine the attack dose of Salmonella typhimurium which results in approximately 50% death rate.

The results are shown in Table 17.

Table 17: Preliminary experiment to determine the attack dose of Salmonella typhimurium corresponding approximately 50% death rate.

The underlined numbers represent the number of animals found dead on the indicated day (postinfection). conclusion Based on the data obtained, the dose of 105 CFU of Salmonella typhimurium strain 415 (67% of dead animals, in bold) was chosen for the subsequent study.

Main experiment; Attack of mice treated with CFer300 Mice pretreated for 10 days with the preparation of CFer300 (n = 22) or in the control group with water (n = 22) were attacked the day after the end of the pretreatment.

A suspension of Salmonella typhimurium was administered intraperitoneally as an attack dose of 105 CFU per mouse. A follow-up observation was carried out 21 days post-infection. The proportion of death of the groups is shown in Table 18.

Table 18: Follow-up observation 21 days post-infection The numbers in italics represent the number of animals found dead on the indicated day (postinfection). These data were used to calculate SR, ADL, DF and El (Table 19).

Table 19: proportion of survival during the observation period (21 days) control group pre-treated with water, The survival rate during the observation period (21 days) was 55%, and the ADL was 14.7 days. At a dose of 2 mg / day, CFer300 exhibited a protective effect, resulting in SR = 73% and ADL = 17.1 days, that is, DF = 1.67 and El = 40%.

• In summary, a 10-day course of daily oral treatment with CFer300 given in individual doses of 2 mg offered partial protection in mice infected with Salmonella typhimurium. As shown in this example, and as previously shown in vi tro and ex-vivo, the extracts currently revealed can be used in the development of additional therapeutic drugs.

Example 11: Effect of two extracts of Lactobacillus in an asthma model induced by IACK Two embodiments of the invention were tested in a murine model of allergen-induced asthma after oral administration (Julia et al., Immunity, 2002, 16: 271-283).

Materials and methods Animals and domestic animals BALB / c ByJ female 6-week-old mice were purchased from Janvier, France. They were maintained and fed under standard conditions.

Study groups A total of 27 mice were divided into 4 groups as follows: Group A: untreated, LACK-sensitized and mice attacked with saline; LACK is a protein of the parasite Leishmania major (4 mice).

Group B: LACK-sensitized mice untreated and attacked (8 mice) Group C: mice treated with OM-1009A (8 mg of dry weight residue per administration), LACK-sensitized and attacked (8 mice) Group D: mice treated with OM-1009B (8 mg of dry weight residue per administration), LACK-sensitized and attacked (7 mice) Treatment and schedule with article of test and article witness Mice were treated from day -3 to day 22 .. On day 0 and day 7, mice were sensitized intraperitoneally (ip) with 10 μg of LACK in the presence of 2 mg of alum 3. From day 17-21, mice were exposed to an aerosol attack of 20 minutes a day of a LACK solution (0.15%) (Groups B, C, and D), or a saline solution (Group A) as a control. On day 22, the mice were analyzed for their ability to develop AHR after inhalation of methacholine. On day 23, the mice were sacrificed and the inflammation of the lung was determined.

Methodology Mice were treated therapeutically three times as described above. Washes were performed in individual mice bled with a cannula inserted into their trachea. The lungs were washed 3 times with 1 ml of warmed PBS. Cells were washed with PBS, resuspended in 300 μ ?, and counted using a Burker-Türk camera. For differential BAL cell counts, cytospin preparations were prepared and stained with Wright / Giemsa stain. The groups tested were wild type mice (wt) LACK-sensitized and PBS-attacked (control), mice wt LACK-sensitized and attacked (asthmatic animals), mice wt OM-1009A treated, and OM-1009B-treated. The total cell number in BAL was determined by microscope examination of cytospin preparations stained with Wright / Giemsa stain.

Since a decrease in airway inflammation was observed in the treated mice (see results below), lung extracts were prepared, and IL-4, IL-5, and IL-13 were quantified by multiplex analysis (arrangement of CBA).

Test Items The two bacterial lysates tested were: OM-1009A extract of Lactobacillus fermentum 1-3929 obtained as described in Example 3.7 and OM-1009B extract of Lactobacillus fermentum 1-3929 obtained as described in Example 3.11.

Results a) Airway hypersensitivity On day 22, the mice were analyzed for their ability to develop AHR after inhalation of methacholine at the indicated doses. The results are reported in Figure 5. The results show that OM-1009-B restored the basal Penh values similar to the non-asthmatic group without treating with PBS. OM-1009-A was more efficient, since the Penh values obtained were even lower than those observed in the non-asthmatic control group. b) Total and differential number of cells in bronchioalveolar lavage On day 23, mice were sacrificed and lung inflammation was determined. The total number of cells in BAL reported in Table 20 (right column), and also the numbers of differential cells: number of Eosinophils (Eo), number of Neutrophils (Neutral), number of Lymphocytes (Lympho), and number of other cells (Other).

Table 20: Total and differential cell number in BAL after treatment with two extracts of the invention (Groups C and D). The values of the mean and standard error of the mean are provided.

Both extracts significantly decreased the total number of cells in BAL (p <0.01 for OM-1009A, and p <0.03 for OM-1009B when compared to group B). The numbers of eosinophils were decreased 3- and 1.7 times respectively. The numbers of neutrophils were decreased 1.8- and 3.6 times respectively. The reduction in the numbers of eosinophils and neutrophils indicates a lower concentration of inflammatory cells in BAL of animals pretreated with the extracts c) Th2 cytokines detected in lung extracts and quantified by multiplex analysis (CBA array) Table 21 Individual lung IL4 levels in mice.

Table 22: individual lung IL5 levels in mice. pg / mg mouse 1 mouse 2 mouse 3 mouse 4 mouse 5 mouse 6 mouse 7 mouse 8 Media (A) PBS 0 0.0004 0.0043 0.0012 (B) LACK (mice 0.1 141 0.1002 0.1532 0.1 1 15 0.3098 0.5173 0.5385 0.4547 0.2874 asthmatics) (C) PO OM 1009A 0.1817 0.0445 0.0572 0.0364 0.1643 0.0488 0.1917 0.1057 0.1038 (D) PO OM 1009B 0.1518 0.2798 0.1797 0.5439 0.3685 0.1320 0.1705 0.2609 Table 23: Individual lung IL13 levels in mice.

The levels of the Th2 cytokines (IL4, IL5 and IL13), when compared to the control group, asthmatic (B), were decreased by the extract of OM-1009A, but not the extract of OM-1009B. Hence, bacterial extracts obtained by a strong base treatment (OM-1009-B) may be less active than those obtained under a moderate basic treatment (OM-1009-A). Although both extracts were active in the analysis of the Penh factor (Figure 6), OM-1009A may be a better candidate than OM-1009B for the treatment or prevention of conditions related to Th2-related diseases, such as allergic diseases and atopy or symptoms of them.

Claims (28)

1. An extract of one or more bacterial strains of Lactobacillus, characterized in that the extract is a soluble extract and wherein the extract comprises chemically modified bacterial molecules.

2. The extract according to claim 1, characterized in that the chemically modified bacterial molecules result from the exposure of the one or more bacterial Lactobacillus strains to an alkaline medium.

3. The extract according to claim 1 or 2, characterized in that the extract has immunomodulatory activity in a subject.

4. The extract according to claim 3, characterized in that the extract has immunostimulatory activity in a subject.

5. The extract according to claim 3, characterized in that the extract has anti-inflammatory activity in a subject.

6. The extract according to any of claims 1-5, characterized in that the one or more strains of Lactobacillus comprise one or more of Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus casei defensis, Lactobacillus casei ssp. casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus lactis, and Lactobacillus delbrueckii.

7. The extract according to any of claims 1-6, characterized in that the one or more bacterial strains of Lactobacillus comprise one or more of Lactobacillus fermentum 1-3929, Lactobacillus rha nosus 71.38, Lactobacillus plantarum 71.39, Lactobacillus johnsonii 103782, and Lactobacillus helveticus 103146.

8. The extract according to any of claims 1-7, characterized in that one or more of aspartic acid, glutamic acid, serine, histidine, alanine, arginine, tyrosine, methionine, phenylalanine and lysine in said extract are racemized by at least 10% .

9. The extract according to any of claims 1-8, characterized in that the extract is capable of obtaining a proportion of IL10 / IL12 that can be calculated in human peripheral blood mononuclear cells, the proportion is greater than or equal to the proportion of IL10 / IL12 obtained by a strain of live Lactobacillus from which the extract is obtained.

10. The extract according to claim 1, characterized in that the extract decreases the number of eosinophil cells, number of neutrophil cells, number of lymphocyte cells or any combination thereof, in an asthmatic mu'rino subject by a factor of at least 1.5 with respect to an untreated asthmatic witness.

11. A process for the preparation of the extract according to claim 1, characterized in that it comprises: (a) culture one or more bacterial strains of Lactobacillus in a culture medium; (b) exposing each bacterial strain of Lactobacillus to an alkaline medium; Y (c) treating the product of step (b) to remove insoluble material and particulate matter.

12. The process according to claim 11, characterized in that step (c) is carried out by tangential flow filtration.

13. The process according to claim 11 or 12, characterized in that the exposure of each bacterial strain of Lactobacillus to a pH greater than 9.0 is sufficient for the chemical modification of bacterial molecules.

14. The process according to claim 11, 12 or 13, characterized in that it also comprises treating each strain at a pH of less than 4.5 next to part (b) and before part (c).

15. The process according to any of claims 11-14, characterized in that the chemical modification comprises racemization of one or more of aspartic acid, glutamic acid, serine, histidine, alanine, arginine, tyrosine, methionine, phenylalanine and lysine in the extract by at least 10%.

16. A nutraceutical composition characterized in that it comprises the extract according to any of claims 1-10 or the extract prepared according to any of claims 11-15.

17. A pharmaceutical composition characterized by comprising the extract according to any of claims 1-10 or the extract prepared according to any of claims 11-15.

18. A method for reducing at least one symptom associated with at least one selected condition of a respiratory disturbance, an allergic condition, an alteration of the urinary system, and a digestive disturbance, comprising the administration of a therapeutically effective amount of the compliance extract with any of claims 1-15 or the composition according to claim 16 or 17 to a subject.

19. The method according to claim 18, characterized in that the respiratory alteration is chosen from upper and lower pulmonary infections, nasopharyngitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, laryngopharyngitis, influenza, pneumonia, bronchopneumonia and obstructive pulmonary disease with acute exacerbation.

20. The method in accordance with the claim 18, characterized in that the allergic condition is chosen from allergic rhinitis, allergic asthma and atopic dermatitis.

21. The method according to claim 18, characterized in that the alteration of the urinary system is chosen from urethritis, tubulo-interstitial nephritis, obstructive pyelonephritis, cystitis including chronic cystitis, male pelvic pain syndrome including prostatitis and chronic prostatitis, prostatocystitis and pelvic inflammatory diseases of woman.

22. The method in accordance with the claim 18, characterized in that the digestive alteration is chosen from Crohn's disease and irritable bowel syndrome.

23. The method according to claim 18, characterized in that the subject is a human or a domestic animal.

24. The use of the extract according to any of claims 1-15 or the composition according to claim 16 or claim 17, characterized in that it is used as a medicament for reducing at least one symptom associated with at least one selected condition of a respiratory alteration, an allergic condition, an alteration of the urinary system and a digestive alteration.

25. The use of the extract according to any of claims 1-15 or the composition according to claim 16 or claim 17, characterized because it is used as an immunomodulatory agent.

26. A strain of isolated microorganism, characterized in that it is Lactobacillus fermentum 1-3929.

27. An extract characterized in that it is obtained from the strain according to claim 25.

28. The extract according to claim 26, characterized in that the extract is a soluble extract.