RU2500412C2 - IMMUNOMODULATORY EXTRACTS OF BACTERIA Lactobacillus AND METHODS FOR PREPARING AND USING THEM - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely to an extract of one or more bacterial strains Lactobacillus. The extract of one or more bacterial strains Lactobacillus representing a soluble extract, wherein the extract contains chemically modified bacterial molecules prepared by the action of an alkaline medium on one or more bacterial strains Lactobacillus; the extract is effective in treating diseases associated with the anti-inflammatory cytokine production imbalance. A method for preparing the extract of one or more bacterial strains Lactobacillus. A pharmaceutical composition effective for reducing at least one symptom associated with at least one condition specified in a respiratory disorder, an allergic condition, an urinary disorder and a gastric disorder, containing the extract. A nutritional composition. A pharmacological composition effective in treating the diseases associated with the anti-inflammatory cytokine production imbalance, containing the extract. A method of relieving the above symptoms. The extract prepared by the above method.

EFFECT: extract is effective in treating the diseases associated with the anti-inflammatory cytokine production imbalance.

21 cl, 7 dwg, 23 tbl, 5 ex

Description

FIELD OF THE INVENTION

Embodiments of the present invention include extracts from Lactobacillus bacteria, which may have immunomodulatory effects in individuals. Embodiments of the present invention can be used, for example, as nutraceuticals or pharmaceuticals for the treatment of diseases, such as diseases associated with an imbalance in the production of anti-inflammatory or pro-inflammatory cytokines, such as infections, allergic conditions, autoimmune disorders and inflammation, or as adjuvants providing Wellness benefits for individuals. Along with other aspects, the invention also includes methods for the preparation and use of such extracts. The invention also relates to specific strains of Lactobacillus bacteria.

BACKGROUND AND SUMMARY OF THE INVENTION

Immunomodulation is a global term that refers to a wide range of immune interventions that alters normal or pathological 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 disrupt defense mechanisms to promote colonization and persistence of the pathogen. Viruses, bacteria, and parasite-derived molecules that can inhibit inflammatory reactions have been identified. In addition to microbial factors that can suppress immune responses, potent immune activators themselves can also be of microbial origin. These include bacterial enterotoxins, excretory-secretory products of parasitic origin, and viral nucleic acids.

It is believed that a family of at least 11 receptors called toll-like receptors (TLRs) and expressed by the host organism plays a key role in immunological detection and innate microbial reactivity. In FIG. 1 shows 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-related molecular types (PAMPs) produced by viruses, bacteria and fungi (Tse and Horner, Ann Rheum Dis. 2007 Nov; 66 Suppl 3: iii77-80). TLR-related immunomodulation has been used to develop new methods of treating a wide range of pathological conditions, including infectious, malignant, autoimmune and allergic diseases.

TLR agonists and antagonists have been investigated as potential therapeutic agents for the prevention and treatment of diseases. In relatively small clinical trials, TLR agonists have been used as adjuvants for vaccines designed to prevent infections, eliminate allergic hypersensitivity, and remove malignant cells. TLR agonists have also been investigated as monotherapy and adjuvant therapies for treating patients with infectious, allergic and malignant diseases. The use of TLR antagonists has also been investigated in preclinical studies and clinical trials as potential therapeutic agents for autoimmune diseases and sepsis.

Probiotics are living microorganisms that can have healing effects on an individual when administered in adequate amounts (Mottet et al., Digestive and Liver Disease, 2005, 37: 3-6; Ezendam et al., Nutr Rev, Jan. 2006, 64 (1): 1-14; Gill and Prasad, Adv Exp Med Biol, 2008, 606: 423-54). The biological mechanisms involved in stimulating the immune response by probiotic microorganisms and certain cellular components of these microorganisms have been the subject of research. For example, gram-positive bacteria have characteristic macromolecules that make up the cell wall, such as lipoteichoic acid (LTA). LTA may be associated with immunostimulatory activity (e.g., Bhakdi et al., Infect. Immun., 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, probiotic bacteria may contain a variety of TLR ligands with immunomodulatory characteristics. It was found that cell wall fragments from various bifidobacterial strains stimulate the production of interferon-gamma (IFN-γ) in vitro in murine splenocytes (T. Ambrouche, "Contribution a l'etude du pouvoir immunomodulateur des bifidobacteries: Analyze in vitro et etude ex vivo des mecanismes moleculaires impliques, "Ph.D. Thesis, Universite Laval, Quebec, 2005). Capsules made from fragments of the cell wall in the form of particles of certain lactic acid bacteria (Del-Immune V®, Pure Research Products, LLC, Colorado) are also intended to stimulate the immune system.

However, ingestion of probiotic bacteria in live or killed form or ingestion of fragments of the cell wall in the form of particles of such bacteria may not be the most effective way of providing an immunomodulating effect in individuals. For example, living cell extracts may contain large proteins and lipopeptides, the size of which prevents the individual from efficiently absorbing them, thereby limiting the local concentration of beneficial molecules from probiotic bacteria in the body. Conditions within the body can also destroy active bacterial components or otherwise modify the chemical structures of these components, making them inactive. Risks associated with the oral administration of live probiotic microorganisms (Lactobacillus) include bacteremia and sepsis (Lactobacillus Sepsis Associated With Probiotic Therapy, Pediatrics, Jan. 2005, 115 (1): 178-181). Therefore, there is a need for other agents to provide the beneficial effects of probiotic bacteria in individuals in need thereof.

The present invention relates to extracts of Lactobacillus, some embodiments of which may exhibit high immunomodulatory activity. For example, embodiments of the present invention relate to extracts from bacterial strains that may be useful as nutraceuticals or as pharmaceuticals, in some cases for the treatment of infectious diseases, allergies, respiratory disorders and inflammatory pathological conditions, or act as an adjunct in connection with the treatment protocol. The present invention also relates to compositions containing extracts and methods for producing extracts, for example, using media that do not pose a risk of prion diseases. Methods in accordance with 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, meaning that they do not contain significant amounts of solid or particulate matter. In some embodiments, implementation, the extracts contain chemically modified TLR ligands. In some embodiments, 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 include extracts obtained from one or more of the following types:

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 contain at least one strain from each of the above types of bacteria, while in other embodiments, one or more specific strains from the above list can be removed or replaced by one or more other strains. Some embodiments of the present invention include an extract obtained from one or more of the following bacterial strains: Lactobacillus fermentum I-3929, Lactobacillus rhamnosus 71.38, Lactobacillus plantarum 71.39, Lactobacillus johnsonii 103782, and Lactobacillus helveticus 103146. The above strains were deposited. Each of Lactobacillus fermentum I-3929, Lactobacillus rhamnosus 71.38, Lactobacillus plantarum 71.39, Lactobacillus johnsonii 103782, and Lactobacillus helveticus 103146 was deposited in the Pasteur Institute's Collection Microorganism Institute at the Institute of Organisation at the Institute of Organisation at Culture Roux, 75724 Paris, France. Lactobacillus fermentum I-3929 was deposited on February 27, 2008. Other strains are among the collections of the depository and can be obtained by contact with the depository.

The present invention also, along with other aspects, relates to a strain of Lactobacillus fermentum I-3929, extracts obtained from this strain, methods for producing such extracts and their uses. This strain was obtained by allowing these strains from Lactobacillus plantarum and Lactobacillus fermentum to undergo chromosome exchange, thereby obtaining a new strain of Lactobacillus. It was found that extracts obtained from Lactobacillus fermentum I-3929 are active in several in vivo and in vitro models that correlate with infection and immunological disorders.

In some embodiments, the extract is obtained from only one specific bacterial strain. Alternatively, more than one strain may be used. In other embodiments, one or more extracts from another type of microorganism, for example, a bacterial species other than Lactobacillus, may be added.

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

Lysates (i.e., cell lysis products) can also be filtered to remove nucleic acids and larger cell debris, such as insoluble or particulate material. In some embodiments, the amount of nucleic acids present in the extracts is less than 100 μg / ml. Therefore, in some embodiments, the obtained extract contains soluble molecular components and does not contain significant amounts of insoluble or particulate material.

Membrane and cell wall molecules, including lipoproteins, lipopeptides, peptidoglycans, lipopoligosaccharides, lipoteichoic acids and teichoic acids, can be dissolved or suspended in extracts. During the lysis process, molecules in cells, such as membranes and cell walls, can become chemically modified, for example, cleaved into smaller structures by treatment with alkali. Despite such chemical modifications, embodiments of the invention may retain their biological activity compared to whole cells, or such embodiments may even exhibit increased biological activity compared to whole cells.

For example, alkaline treatment can be used to lyse cells or can be applied to cells that were previously lysed in a different way. During the alkaline treatment process in accordance with some embodiments of the invention, the L-amino acids found in natural proteins and lipopeptides are at least partially racemicized into D-amino acids. D-amino acids can have a beneficial effect on increasing the duration of the effectiveness of the extracts, since they do not undergo efficient digestion in the gastrointestinal tract of mammals. D-amino acids can also protect smaller peptides and proteins from degradation during digestion. Examples of D-amino acids include protein-bound D-amino acids and, to a lesser extent, lysinalanine (de Vrese et al., J Nutrition, 2000, 2026-2031). Thus, antigenic molecules in extracts chemically modified during lysis to contain D-amino acids can remain in the human body for a longer time, potentially providing the possibility of a stronger immunostimulating effect in some embodiments.

In some embodiments, the filtration process may also affect the properties of the extracts obtained, since the pore size of the filter, and in some cases the chemical properties of the filter surface (i.e., its polarity), can change the type of materials that are removed and retained. For example, in some embodiments, a filtering process is used to hold molecules of interest, but to remove other molecules, such as nucleic acids or insoluble or particulate materials.

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

Brief Description of the Drawings

Figure 1: Ligands for a family of 11 toll-like receptors (TLRs) expressed by the host.

Figure 2: Drawing of a tangential flow filtering device (TFF) for producing bacterial extracts after bacterial lysis. The drawing shows two different configurations for filters: a parallel type, where all filters work simultaneously, and a coil type, where the filters are configured according to the serial type.

Figure 3: Generalized correlation between functional parameters and flow, indicating areas of pressure regulation and mass transfer adjustment for a tangential flow filtration method (TFF).

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

Figure 5: Induction of nitric oxide (NO) production in mice treated with extracts of Lactobacillus fermentum I-3929 and Lactobacillus rhamnosus 71.38, in (a) the first analysis, and (b) the second analysis. Results are expressed in μM nitric oxide (NO) as mean ± standard deviation.

6: Effect of extracts of the invention on airway hyperreactivity (AHR) as determined by whole body plethysmography (Emka) at increasing concentrations of respirable metacholine one day after the last antigenic stimulation. Results (mean increased pause ± standard error of the mean) are shown for animals from the negative control group treated with saline with phosphate buffer (PBS) (n = 4), untreated animals with antigenic stimulation of LACK (as a positive control group, n = 8), mice treated with OM-1009A treated with LACK antigenic stimulation (n = 8), and mice treated with OM-1009B treated with LACK antigenic stimulation (n = 7).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Extract: Extract, as defined herein, means material obtained after 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, meaning that it does not contain significant amounts presented in the form of particles and insoluble materials, such as fragments in the form of particles or solid cell walls. Instead, components from cell walls, organelles, and cell membranes can be contained in the extracts to the extent that they are dissolved or suspended. For example, the extract may be processed to remove particulate and insoluble materials, for example, by filtration, centrifugation, or other separation technique.

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

Lysate: Used in the present description, this term refers to an extract of bacteria obtained from the cell lysis procedure.

Filtration: The filtration process, as described herein, means passing an extract or mixture of extracts through one or more filters, such as microfilters (i.e., microfiltration) and / or ultrafilters (i.e., ultrafiltration). Such filtration may optionally remove 100% of the components for which it is intended to be removed, but may, in some embodiments, render extracts substantially free of these components. In some cases, filtering is repeated in several passes or cycles.

Starting pH: This term means the pH measured at the beginning of the procedure, such as lysis or filtration.

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

Lipoproteins: This term refers to macromolecules that include both protein or peptide chains and lipids, for example, a protein or peptide covalently linked to a lipid. As used herein, the term lipoprotein also includes lipopeptides.

Peptidoglycans: This term refers to polymers. Containing sugar and amino acids.

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

Teichoic acid: This term refers to polymers of glycerol phosphate or ribitol phosphate joined together through phosphodiester bonds.

D-amino acids: This term refers to amino acids that exist in dextrorotatory isomeric forms, as opposed to biosynthetically derived L-amino acids that exist in levorotatory isomeric forms.

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

An environment that avoids the risk of prion-based diseases means a culture medium used at any stage of extracts that do not contain materials, such as whey or meat extracts taken from animals such as cows or sheep, or any other animal, which can transmit prion-based diseases. Examples of such media include plant-based synthetic media, as well as media using horse serum or media containing materials from animal species that do not transmit prion diseases. Examples of prion-based diseases include, for example, bovine spongiform encephalopathy, pruritus pruritus, and Creutzfeldt-Jakob disease.

A non-animal medium is a plant-based (i.e., plant) medium, such as soybean medium and synthetic medium.

Used in the present description, the term nutraceutical means any composition that can have healing effects in the individual after administration, where the composition, for example, is available to the individual without a doctor’s prescription.

The term “treatment” as used in the therapeutic context in the present description also means the treatment of current diseases and disorders, as well as, for example, prevention or protection against the development of new diseases or disorders.

As used herein, the term adjuvant to describe embodiments of the invention refers to embodiments of the invention carried out in an individual in combination with a medical treatment plan.

As used herein, the terms immunomodulation, immunomodulatory, and the like, refer to the ability to modify the immune responses of an individual in a manner that can have healing effects, for example, providing an anti-inflammatory or immunostimulating effect.

Used in the present description, the terms anti-inflammatory and the like refer to immunomodulatory effects that serve to reduce inflammation.

As used herein, immunostimulatory and the like terms refer to stimulation of the immune system.

As used herein, the term “protective immunity” means that an embodiment of the invention is used in an individual in order to provide protection against subsequent antigenic stimulation by an infectious agent or allergen. As a result, during antigenic stimulation, the level of the infectious agent or allergen in the individual has a sufficiently low concentration so as not to cause significant damage to the health of the individual. The length of time over which such protection against antigenic stimulation is effective may be limited, for example, to a period of several hours, days or weeks.

As used herein, the term “individual” means any animal individual, including mammalian individuals, such as humans and domestic animals. Pets may, for example, include mammals such as dogs, cats, horses, pigs, cows, sheep, goats or other livestock, and may also include non-mammals such as birds, such as chickens, ducks, geese, turkeys and others species of farm birds.

It is understood that certain bacterial strains identified in the present description and used in the invention may include a strain obtained from the initial deposit specified in the present description, or its genetic clone, including a strain that was re-deposited at a later time under a different deposit code name, but which is considered genetically the same strain as the originally deposited variant.

All numbers used in the present description are approximate, taking into account errors inherent in their measurement, rounding and figures of statistical significance.

Getting extracts

The present invention includes an extract of one or more bacterial strains of Lactobacillus, where the extract is a soluble extract, and where the extract contains chemically modified bacterial molecules.

The extracts of the present invention can be obtained, for example, by culturing cells, followed by collection of the resulting biomass, lysis and purification. For each strain to obtain a sufficient amount of material, fermentation cultures can begin with a lot of working sowing, followed by the introduction of seed in larger fermentation containers.

The media used may be the same for each species. In some embodiments, a medium that avoids the risk of prion-based diseases can be used to grow all strains to be used.

After fermentation, the resulting biomass from a single strain or from a set of strains can be inactivated by heat treatment, concentrated and frozen. Therefore, in some embodiments, the starting material used to form the extracts may be non-lysed whole cells.

In other embodiments, the starting material used to produce the extracts may be biomass derived from cells already at least partially lysed mechanically, enzymatically, or chemically. In still other embodiments, the starting material may be a fraction of such pre-lysed cells, such as a fraction containing cell walls.

In some embodiments, the starting material is treated with an alkaline medium, such as consisting of a strong base, such as hydroxide or other strong mineral or organic bases. At this stage of lysis or base treatment, non-lysed cells in the starting material are lysed, while in some embodiments, the cellular components can be chemically modified. Therefore, in some embodiments, chemically modified bacterial molecules are obtained by treatment with a base, such as strong base treatment of one or more bacterial strains of Lactobacillus, from which the extract is obtained (i.e., treatment with a base of non-lysed cells or components or fractions from bacterial cells, such as just been explained).

In some embodiments, a dry weight concentration of biomass of from 2 to 90 g / l, for example, from about 2 to about 80 g / l, or from about 3 to about 40 g / l, for example, 3, 5, can be treated with a base. 10, 15, 20, 25, 30, 35, or 40 g / l, or even from 5 to 50 g / l, or other ranges limited to the above concentrations. In some embodiments, a base is treated with from about 40 to about 80 g / l, for example, 40, 50, 60, 70, or 80 g / l or other ranges limited to the concentrations listed above.

The dry mass of biomass is defined herein as the dry mass of material in g per liter of sample. It can be measured by drying the sample in a small porcelain dish at about 105 ° C until it reaches a constant weight.

The temperature can be from 30 to 60 ° C, for example, from 30 to 55 ° C, from 30 to 50 ° C, from 30 to 45 ° C, from 30 to 40 ° C, or from 30 to 35 ° C. In some embodiments, the base treatment temperature may be from 35 to 60 ° C, for example, from 35 to 55 ° C, from 35 to 50 ° C, from 35 to 45 ° C, or, for example, from 35 to 40 ° C. In some embodiments, the base treatment temperature may be 31 ° C, 32 ° C, 33 ° C, 34 ° C, 35 ° C, 36 ° C, 37 ° C, 38 ° C, 39 ° C, or even 40 ° C , or ranges limited to the temperatures listed above.

The base treatment time can vary from 2 hours to several days, for example, 1, 2, 3, 4, 5 or even 10 days, or from 3 to 120 hours, or from 3 to 48 hours, for example, 3, 5, 8, 15, 14, 16, 18, 20, 22, 24, 26, 28, 30, 36, 40, 44 or 48 hours, or from 15 to 120 hours, for example, from 60 to 120 hours, for example, 60, 72, 84, 96, 108 or 120 hours, or ranges limited to the above time intervals. It is understood that these time ranges include any fractional number of days, hours, or minutes within them.

In some embodiments, a strong base concentration of from 0.001 N to 1.0 N is used, for example, 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 a range starting or ending from 0.001, 0.002, 0.003 or 0.1 N, or from 0.1 N to 0.6 N, or a range starting or ending from 0.6, 0.7, 0, 8, 0.9, 1.0, or 1.0 N, or other ranges limited to the concentrations listed above. In some embodiments, such a base concentration is used. In order to achieve an initial pH of greater than 9.0, or a pH of greater than 9.5, a pH of greater than 10.0 and less than 13.5, for example, greater than 11.5, greater than 12.0, greater than 12.5 more than 13.0 or from pH 9.0 to pH 13.5. In still other embodiments, a base concentration such that, for example, an initial pH of greater than 10.0 and less than 13.0, or from pH 9.0 to pH 13.0, can be used.

In some embodiments, the pH during base treatment may be lowered after extraction of the soluble components. For example, the starting pH may be a basic pH, such as from pH 9.0 to pH 13.0, or from pH 9.5 to pH 12.5. The base treatment may be allowed to continue for a certain period of time, for example, from 3 to 120 hours, for example, from 3 to 48 hours, or for the time periods listed above at the above temperatures. Then, in some embodiments, it is possible to acidify the pH by adding, for example, hydrochloric acid so as to obtain a pH from 2.0 to 4.5, or a pH from 2.5 to 4.5, or a pH from 2.5 to 4 , 0, for example, 2.5, 3.0, 3.5, 4.0, or in the range limited to any of the pH values listed above. The second treatment at low pH can be carried out at a temperature of from 30 to 60 ° C, from 35 to 55, or from 35 to 45 ° C, for example, 35 ° C, 36 ° C, 37 ° C, 38 ° C, 39 ° C , 40 ° C, 41 ° C, 42 ° C, 43 ° C, 44 ° C or even 45 ° C. The acid treatment time may vary from 1 hour to several hours or up to 72 hours, for example, from 1 hour to 24 hours or from 1 hour to 6 hours, or from 3 hours to 48 hours, or from 3 hours to 24 hours or from 4 to 72 hours, or even from 24 hours to 72 hours, or in any time range limited to the time periods listed above.

In some embodiments, the alkaline treatment is performed on a bacterial biomass containing, for example, material from Lactobacillus fermentum having a dry biomass of 10 g / L to 40 g / L. In other embodiments, alkaline treatment is performed on a bacterial biomass containing a mixture of Lactobacillus strains and having a dry biomass of 10 g / l to 40 g / l. In such embodiments, alkaline treatment may be performed at a concentration of a hydrochloride ion of from 0.025 N to 0.25 N or at a pH of from 9.5 to 12.5 at a temperature of from 35 to 45 ° C for a period of time from 3 hours to 48 hours In some embodiments, the alkaline treatment is performed on a bacterial biomass containing material from one or more strains of Lactobacillus at a concentration of a hydrochloride ion from 0.025 N to 0.20 N, from 0.025 to 0.15 N, from 0.025 to 0.10 N, from 0.05 N to 0.25 N, from 0.05 N to 0.20 N, from 0.05 to 0.15 N, from 0.05 N to 0.10 N, from 0.10 N to 0 , 25 N, from 0.10 N to 0.20 N, from 0.10 N to 0.15 N, from 0.15 N to 0.25 N, from 0.15 N to 0.20 N, or even from 0.20 N to 0.25 N. In such embodiments, the pH may, for example, be from 9.5 to 12.0, from 9.5 to 11.5 , from 9.5 to 11.0, from 9.5 to 10.5, from 9.5 to 10.0, from 10.0 to 12.5, from 10.0 to 12.0, from 10.0 to 11.5, from 10.0 to 11.0, from 10.0 to 10.5, from 10.5 to 12.5, from 10.5 to 12.0, from 10.5 to 11.5, from 10.5 to 11.0, from 11.0 to 12.5, from 11.0 to 12.0, from 11.0 to 11.5, from 11.5 to 12.5, from 11.5 to 12.0 or even a pH of 12.0 to 12.5. The alkaline treatment time for such embodiments may be from 3 hours to 36 hours, from 3 hours to 24 hours, from 3 hours to 18 hours, from 3 hours to 12 hours, from 3 hours to 6 hours, from 6 hours to 48 hours , from 6 hours to 36 hours, from 6 hours to 24 hours, from 6 hours to 18 hours, from 6 hours to 12 hours, from 6 hours to 8 hours, from 8 hours to 48 hours, from 8 hours to 36 hours, from 8 hours to 24 hours, from 8 hours to 18 hours, from 8 hours to 12 hours, from 12 hours to 48 hours, from 12 hours to 36 hours, from 12 hours to 18 hours, from 18 hours to 48 hours, from 18 hours to 36 hours, from 18 hours to 24 hours, from 24 hours to 48 hours, from 24 hours to 36 hours or from 36 hours to 48 hours. Alkaline treatment can be performed for any time periods bordering the above ranges, for example, 3, 6, 8, 12, 18, 24, 36, or even 48 hours. Such conditions They can provide moderate alkaline treatment.

In other embodiments, 10 g / L and 40 g / L dry mass of biomass from one or more strains of Lactobacillus can be exposed to a hydroxide ion concentration of 0.15 N to 0.50 N or a pH of 11.5 to 13.5, at a temperature of from 35 to 45 ° C for a period of time from 15 hours to 120 hours. For example, in some embodiments, the concentration of hydroxide may be from 0.15 N to 0.45 N, from 0.15 N to 0.40 N , from 0.15 N to 0.35 N, from 0.15 N to 0.30 N, from 0.15 N to 0.25 N, from 0.15 N to 0.20 N, from 0.20 N up to 0.50 N, from 0.20 N to 0.40 N, from 0.20 N to 0.30 N, from 0.25 N to 0.50 N, from 0.30 N to 0.50 N, from 0.30 N to 0.40 N or from 0.40 N to 0.50. In such an embodiment, the pH may be, for example, from 11.5 to 13.0, from 11.5 to 12.5, from 11.5 to 12.0, from 12.0 to 13.5, from 12.0 to 13.0, from 12.0 to 12.5, from 12.5 to 13.5, from 12.5 to 13.0, from 13.0 to 13.5. The time period for alkaline treatment can be from 15 hours to 100 hours, from 15 hours to 90 hours, from 15 hours to 75 hours, from 15 hours to 60 hours, from 15 hours to 48 hours, from 15 hours to 36 hours, from 24 hours to 120 hours, 24 hours to 100 hours, 24 hours to 90 hours, 24 hours to 75 hours, 24 hours to 60 hours, 24 hours to 48 hours, 36 hours to 120 hours, 36 h to 100 h, from 36 h to 90 h, from 36 h to 75 h, from 36 h to 60 h, from 36 h to 48 h, from 48 h to 120 h, from 48 h to 100 h, from 48 h up to 90 hours, from 48 hours to 75 hours, from 48 hours to 60 hours, from 60 hours to 120 hours, from 60 hours to 100 hours, from 60 hours to 90 hours, from 60 hours to 75 hours, from 75 hours to 120 hours, from 75 hours to 100 hours, from 75 hours to 90 hours, from 90 hours to 120 hours, or, for example, from 100 to 120 hours. Time periods also provided for alkaline treatment in such embodiments thereof include 15, 24, 48, 60, 75, 90, 100, and 120 hours. Such conditions may provide strong alkali treatment.

In other embodiments, 10 g / L to 40 g / L of the original dry biomass can be treated with a hydroxide concentration of 0.025 N to 0.25 N or a pH of 9.5 to 12.5, at a temperature of 35 to 45 ° C. over a period of time from 3 hours to 48 hours. Then, the pH is adjusted to a level of 2.5 to 4.0 by the addition of an acid such as hydrochloric acid (HCl), which constitutes an acid treatment. Acid treatment can be performed at a temperature of from 35 to 45 ° C. for a period of time from 1 hour to 24 hours. For example, in such embodiments, alkaline treatment of a bacterial biomass containing one or more strains of Lactobacillus can be performed at a hydroxide concentration of from 0.025 N to 0.20 N, from 0.025 to 0.15 N, from 0.025 to 0.10 N, from 0.05 N to 0.25 N, from 0.05 N to 0.20 N, from 0.05 to 0, 15 N, from 0.05 N to 0.10 N, from 0.10 N to 0.25 N, from 0.10 N to 0.20 N, from 0.10 N to 0.15 N, from 0, 15 N to 0.25 N, from 0.15 N to 0.20 N, or even from 0.20 N to 0.25 N. During alkaline treatment in such embodiments, the pH may, for example, be set from 9.5 to 12.0, from 9.5 to 11.5, from 9.5 to 11.0, from 9.5 to 10.5, from 9.5 to 10.0, from 10.0 to 12.5, from 10.0 to 12.0, from 10.0 to 11.5, from 10.0 to 11.0, from 10.0 to 10.5, from 10.5 to 12.5, from 10.5 to 12.0, from 10.5 to 11.5, from 10.5 to 11.0, from 11.0 to 12.5, from 11.0 to 12.0, from 11.0 to 11.5, 11.5 to 12.5, 11.5 to 12.0, or even a pH of 12.0 to 12.5. The alkaline treatment time for such embodiments may be from 3 hours to 36 hours, from 3 hours to 24 hours, from 3 hours to 18 hours, from 3 hours to 12 hours, from 3 hours to 6 hours, from 6 hours to 48 hours , from 6 hours to 36 hours, from 6 hours to 24 hours, from 6 hours to 18 hours, from 6 hours to 12 hours, from 6 hours to 8 hours, from 8 hours to 48 hours, from 8 hours to 36 hours, from 8 hours to 24 hours, from 8 hours to 18 hours, from 8 hours to 12 hours, from 12 hours to 48 hours, from 12 hours to 36 hours, from 12 hours to 18 hours, from 18 hours to 48 hours, from 18 h to 36 h, 18 h to 24 h, 24 h to 48 h, 24 h to 36 h, or 36 h to 48 h. Alkaline treatment can be performed for any period of time bordering the above ranges for example, 3, 6, 8, 12, 18, 24, 36 or even 48 hours. Then the pH m can be brought to the level from 2.5 to 3.5, from 2.5 to 3.0, from 3.0 to 4.0, from 3.0 to 3.5, or from 3.5 to 4.0 by the addition of acid for acid treatment after alkaline lysis. Acid treatment can be performed over a period of time from 1 hour to 18 hours, from 1 hour to 12 hours, from 1 hour to 6 hours, from 1 hour to 3 hours, from 3 hours to 24 hours, from 3 hours to 18 hours, from 3 hours to 12 hours, from 3 hours to 6 hours, from 6 hours to 24 hours, from 6 hours to 18 hours, from 6 hours to 12 hours, from 12 hours to 24 hours, from 12 hours to 18 hours, from 18 hours to 24 hours. The time periods also provided for acid treatment include 1, 3, 6, 12, 18 and 24 hours.

The lysates obtained after the main treatment described above can then be purified by centrifugation and / or filtration, for example, to remove particulate and insoluble components. For example, lysates can be centrifuged at 9,000 x gravity followed by one or more filtration cycles through a 0.2 μm filter. In some cases, sequential filtration cycles through filters with a large pore size can be used, followed by filtration through a filter with a pore size of 0.2 μm. Ultrafiltration methods can also be used to facilitate the extraction of soluble materials from the extract, for example, by recirculating ultrafiltration permeate for further microfiltration.

In some embodiments, a filtering method can be used to filter lysates and to extract soluble molecules from larger cell fragments (FIG. 2). See, for example, Separations Technology, Pharmaceutical and Biotechnology Applications, Wayne P. Olson, Editor, lnterpharm Press, Inc., Buffalo Grove, IL, U.S.A., p.126-135 - ISBN: 0-935184-72-4. At the beginning of this process, the diluted bacterial lysate can be stored in the first tank. For example, in TFF, the extract can be filtered both through a microfilter and through an ultrafilter. For example, a microfiltration loop (MF) is turned on and the product is pumped. The resulting MF retentate is recycled, while the MF permeate is transferred to a second tank.

After reaching the appropriate degree of concentration, the ultrafiltration loop (UF) is turned on. UF permeate can be recycled back to the first tank to continuously extract solubilized extracts from the lysate, while UF retentate is stored in the second tank. During continuous extraction, volumes in reservoirs 1 and 2 can be selected by adjusting the flow rates of microfiltration and ultrafiltration permeates.

Several such extraction cycles, or TFF, or another filtering method may be performed. In embodiments using TFF, at the end of the last cycle, the microfiltration loop can be turned off, and the microfiltration loop can be activated separately, and the MF permeate can be transferred to tank 2.

Transverse flow and transmembrane pressure conditions are determined by pressure-independent and mass-controlled regions in the film theory described, for example, by M. Cheryan (Ultrafiltration and Microfiltration Handbook, 2 ^nd Ed., Ch. 4, 1998). The permeate flow and extraction yield are affected by filtration conditions (transmembrane pressure (TMP), transverse flow, temperature, etc.). The type of filter can also affect the filtration efficiency, as can the type of plate system (cassette filter). Various configurations may be used, including parallel type and coil type (see FIG. 2). Certain conditions are designed for optimized operation of each combination of mode type and filter type used.

The microfiltration loop can be equipped with filters with a pore size of from 1.2 μm to 0.1 μm, such as filters with a pore size of from 0.65 to 0.2 μm or 0.45 μm. The cross-flow can range from 100 to 3000 l / h m ² (LHM), for example, from 300 to 2500 LHM or 2000 LHM at a TMP of 0.3 to 2 bar (31.5 to 210 N / m ² ). The ultrafiltration loop can be equipped with filters from 10 kDa to 1000 kDa, for example, from 10 kDa to 100 kDa, or from 10 kDa to 30 kDa, or from 30 kDa to 100 kDa. The cross-flow can range from 30 to 1000 LHM, for example, from 20 to 500 LHM at a TMP of 0.2 to 1.5 bar (157.5 N / m ² ).

From 5 to 20 volumes of diafiltration can be used to extract soluble components from the walls of bacterial cells. In some embodiments, 8 to 15 volumes are used. Therefore, for example, in some embodiments, from 5 to 15 cycles of filtration can be used, in some cases from 8 to 15 cycles.

If desired, after filtration, the extract can be further concentrated or centrifuged. For example, further microfiltration can be performed using a filter with a smaller pore size, such as a filter with a pore size of 0.2 μm. After filtration, the extract can be lyophilized before being included in the composition for use.

In some embodiments, after filtration, the extract may be purified to separate, eliminate, or increase the concentration of one or more modified components in the extract. For example, a strong ion chromatography step may be used to remove charged components. Other purification methods may be used, such as gel filtration, chromatography, ultracentrifugation, extraction, and precipitation.

Chemical properties of bacterial extracts

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

Therefore, some or all of these chemical modifications can occur during base treatment with Lactobacillus cells, as described in this application. Such molecular modifications may affect the biological activities of the extracts.

For example, treatment with a base of bacteria in accordance with the present invention can lead to partial hydrolysis of proteins, as well as deamination, deamidation and / or partial racemization of amino acids from L to D. In one analytical study of the extract in accordance with the invention, each of the peaks representing D aspartic acid, D-glutamic acid, D-serine, D-methionine, D-histide, D-alanine, D-arginine, D-phenylalanine, D-tyrosine, D-leucine and D-lysine. The percentage of D-amino acids of these species in this study ranged from 3% to 40%. Therefore, some embodiments of the invention allow racemization of one or more of serine, threonine, histidine, alanine, arginine, tyrosine, phenylalanine, leucine and lysine, for example, all of the above amino acids, or any choice of more than one, but less than all of the above higher amino acids, such as, for example, alanine, phenylalanine and lysine. In some embodiments, at least one or more of the above amino acids may become racemicized from D to L. In other embodiments, at least 40% of one or more of the above amino acids may become racemicized.

Thus, the extracts of the present invention may contain from 1 to 90% D-amino acids, for example, from 1 to 80% or from 1 to 60%. In some embodiments, implementation, the extract contains from 10 to 45% D-amino acids, for example, from 25 to 35% D-amino acids. The extracts of the invention may contain 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 implementation, the concentration of any of the D-amino acids is from 1 to 50%, for example, from 10 to 40% or even from 15 to 35%.

Some extracts of the present invention contain a bacterial cell wall and membrane components of Lactobacillus, such as lipoteic acid, teichoic acid, peptidoglycan, or a combination thereof. In some embodiments, implementation, these components are chemically modified. Some extracts also contain cell wall and / or cell membrane components, such as lipoproteins, which can also be chemically modified. In some embodiments, implementation, cell wall components or cell membrane components, for example, lipoproteins, are dissolved or suspended in extracts and thus are not present in particulate or insoluble form.

In addition, the extract in accordance with the present invention may contain, for example, from 10 to 100 mg / ml soluble dry mass (SDW) of the material, from 1 to 30 mg / ml protein (Prot.), From 0.5 to 4.0 mg / ml sugar and less than 100 mg / ml DNA. For example, some embodiments contain from about 15 to 35 mg / ml soluble dry weight, from 3 to 7 mg / ml protein, from 1.0 to 3.0 mg / ml sugar, and from 10 to 40 μg / ml DNA. The extract in accordance with the present invention may contain, for example, 30 mg / ml soluble dry weight, 9.6 mg / ml protein, 2.4 mg / ml sugar and 33 μg / ml DNA, or in another example, contains 32.4 mg / ml soluble dry weight, 5.8 mg / ml protein, 2.3 mg / ml sugar and less than 100 μg / ml DNA. Soluble Dry Mass (SDW) in g / l or mg / ml is determined by obtaining 5 ml of the soluble fraction by lysis or treatment with a base and drying to constant weight in a porcelain cup at 105 ° C.

In some embodiments, the extracts contain at least 0.3 mg / ml saccharides, for example, 0.3 to 4.5 mg / ml saccharides. In some embodiments, at least one saccharide is selected from monosaccharides, disaccharides, and polysaccharides. Some extracts of the invention contain at least one branched polysaccharide. In some embodiments, the implementation of the at least one saccharide is chemically modified.

Lysis or treatment with a base of bacteria in accordance with the present invention can lead to a decrease in the average molecular weight of the component macromolecules to a range of, for example, from 1 kDa to 300 kDa and 100 kDa, or to a range from 1 kDa to 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, for example, less than 10 kDa.

Types of biological activity of bacterial extracts

Extracts in accordance with the invention may have immunomodulatory activity. For example, some extracts may stimulate the immune system. Some extracts may have anti-inflammatory activity. Certain effects of the extract may depend on the conditions of manufacture and the type or strain of Lactobacillus or a mixture of species and strains from which the extract is derived. Accordingly, some extracts in accordance with the invention can exhibit potent immunostimulating activity and, thus, can be used in the treatment of infections or as additions to such treatment, while other embodiments may exhibit weaker immunostimulatory activity, but exhibit anti-inflammatory activity, thus being useful in the treatment of inflammatory disorders such as allergies, asthma, autoimmune diseases, colitis and inflammatory bowel disease I, or in addition to such treatment.

Thus, some extracts in accordance with the invention may be effective in treating patients suffering from disorders including, but not limited to, microbial infections, allergic diseases, and digestive tract disorders. Some extracts in accordance with the invention can be provided to the patient in the form of nutraceuticals, for example, as supplements in the treatment of a variety of conditions, including, without limitation, microbial infections, allergic diseases and digestive tract disorders.

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

In vitro cell assays test the production of nitric oxide (NO) from murine macrophages and are used in screening to determine the ability of the extract to stimulate the immune system to kill an invasive bacterium (Figure 5). In some embodiments, the extracts can stimulate NO production in murine macrophages, resulting in measured NO concentrations of 3 μM to 60 μM, for example, 5 μM to 40 μM. In some embodiments, the implementation, the concentration of NO may be higher than 30 μm. The type of bacterial species may also affect these results. For example, extracts from Lactobacillus fermentum can cause greater nitric oxide production than Lactobacillus rahmnosus (see, for example, Example 5 below). To screen for embodiments of the invention to detect their immunostimulating or anti-inflammatory potential in vitro, tests of the bacterial extracts of the invention can be performed on human peripheral blood mononuclear cells (PBMCs). See publication Foligne et al. (World J Gastroenterol, 2007, 13 (2): 236-243). The release of both IL12p70 (inflammatory cytokine) and IL10 (anti-inflammatory cytokine) can be measured and the ratio of IL-10 / IL-12 calculated (Example 6). Some embodiments of the invention give a higher IL10 / IL12 ratio than the control of live Lactobacteria fermentum, thus indicating that some extracts of the invention may exhibit equivalent or even stronger anti-inflammatory properties than the live parent microorganism when administered in vivo. Accordingly, the invention includes extracts capable of achieving a calculated IL10 / IL12 ratio in human peripheral blood mononuclear cells, where the ratio is equal to or greater than the IL10 / IL12 ratio achieved by the live Lactobacillus strain strain from which the extract is derived.

The immune responses of the extracts of the invention can also be tested by examining their effects on Toll-like receptors (TLRs). For example, extracts can be tested on HEK293 cells in the presence or absence of a TLR2 Pam3Cys agonist, or in the presence or absence of a TLR4 LPS agonist (Example 7) . The HEK293 cell line provides the ability to effectively monitor TLR activity using ELISA assays, such as IL-8 titration or reporter-based systems that monitor TLR-induced NF-κB activation. Some embodiments of the invention may act as TLR 2/6 antagonists in HEK TLR 2/6 cells. Thus, some embodiments may be used to fight infections in an individual, while other embodiments may be used against inflammation and / or autoimmune disorders.

The screening of the extracts described herein may also be conducted for TLR and NOD2 receptor activity (Example 8). Some embodiments of the invention activate m / OR or NOD2 TLR receptors in vitro, indicating that they may be able to activate the immune system of TLRs and / or NOD2.

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

The anti-infective efficacy of the extracts of the present invention can be tested, for example, after Salmonella infection in individuals, such as a mouse infection (Example 10). Some embodiments of the invention can provide protective immunity against infections, such as bacterial infections, i.e., Salmonella infections. For example, some embodiments may reduce mortality in mice caused by injection of Salmonella thyphimurium.

A combination of in vitro NO activity with in vivo determination of activity, as measured, for example, in a mouse model of allergy-induced asthma (Example 11), can provide a more complete view of the potential clinical activity of the extracts described herein. In the LACK model (a protein from the parasite Leishmania major), the number of eosinophils found in broncho-alveolar lavage fluids can be reduced by a factor of 1 to 10 compared to control animals with asthma (not treated), for example, reduced from 1, 5 times to 5 times. Accordingly, in some embodiments, the extract reduces the number of eosinophilic cells, the number of neutrophilic cells, the number of lymphocyte cells or any combination thereof in an asthmatic mouse individual by a factor of at least 1.5 relative to the untreated asthma control. Some embodiments may reduce eosinophilia in asthmatic individuals and at the same time lower the level of Th2 cytokines (such as IL4, IL5, IL13), which are markers of asthma. Thus, such embodiments, for example, may have anti-inflammatory activities in individuals suffering from immunological disorders, such as allergic disorders, including asthma.

Compositions containing bacterial extracts

The extracts in accordance with the invention can be compiled in a number of different ways for the final introduction. For example, oral tablets, capsules, pills, as well as liquid formulations or aerosols can be prepared. Infusion or injection formulations may also be prepared.

Embodiments of the present invention may be formulated, for example, in the form of solid dosage forms or liquid dosage forms. Illustrative solid dosage forms may include, for example, a tablet (eg, a coated tablet, a chewable tablet, a tablet that emits gas upon dissolution, a sublingual tablet, granular material, powder or capsule) containing the extract.

Solid dosage forms may also contain diluents, excipients and / or other excipients. Other excipients may be added, such as preservatives, coloring agents, flavors and sweeteners.

As an alternative to capsules and tablets, they can also be developed for the oral route of administration.

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 in one or more pharmaceutical compositions.

The introduction of bacterial extracts to the individual

A dose containing at least one extract of the present invention may be administered to an individual suffering from at least one disorder selected from a digestive tract disorder, respiratory tract disorder, urinary tract disorder and allergic conditions, or at risk of their development. For example, in some embodiments, the extracts may be administered to individuals suffering from infections of the upper and lower lungs, obstructive pulmonary disease with acute lower respiratory tract infection, obstructive pulmonary disease in the acute stage, nasopharyngitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, laryngari , flu, pneumonia, bronchopneumonia, bronchitis, rhinitis, nasopharyngitis, pharyngitis, sinusitis, tonsillitis, laryngitis, laryngotracheitis, bronchitis, alle asthma, atopic dermatitis, urinary tract infections due to obstructive and reflux uropathy, urethritis, tubulo-interstitial nephritis, obstructive pyelonephritis, cystitis, including chronic cystitis, pelvic pain syndrome in men, including prostatitis and chronic prostatitis, prostatitis cystitis, organs in women or at risk of their development.

In some embodiments, the extracts are administered to the individual in the form of a nutraceutical composition, such as a nutritional supplement and / or food supplement. In other embodiments, the extracts are administered to an individual in the form of a pharmaceutical composition. Administration may include administration of a single dose or multiple doses.

In some embodiments, the extract may be provided in a therapeutically effective dose for treating an individual suffering from one or more of the above conditions. In some embodiments, an extract may be provided as a complement to another medical treatment.

WORKING EXAMPLES

EXAMPLE 1: Bacterial cultures

EXAMPLE 1.1: Culture of Lactobacillus fermentum I-3929

Initial cultivation conditions

The culture medium was obtained 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, density 1.005 g / ml) was added. After dissolution, the pH was not adjusted. After sterilizing the medium, small Erlenmeyer flasks were separately inoculated with the contents of frozen vials (containing 1.5 ml of frozen bacteria) and incubated at 37 ° C for 8 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 16 hours of growth, the contents of the 1 L Erlenmeyer flask were transferred to the exciter.

The cultivation conditions in the exciters

20 liters of culture medium were obtained 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. The incubation temperature was regulated at 37 ° C, with stirring at a speed of 100 rpm. During cultivation, the pH was not adjusted. After 24 hours, 7 liters of the exciters were transferred to the fermenter (optical density (OD) at 700 nm of the exciter culture after 24 hours = 1.24). The cultures of the pathogens under sterile conditions were transferred to fermenters.

The cultivation conditions in the fermenters

70 liters of culture medium were obtained in purified water of 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 was added to the culture. The incubation temperature was regulated at 37 ° C with stirring at a speed of 100 rpm and without aeration. During cultivation, the pH was adjusted to 5.7. After 16 h, cultures (OD cultures at 700 nm 2.30) were inactivated by heat treatment at 65 ° C for 35 minutes and transferred to a cell culture collection tank. After inactivation, the cultures were transferred to an ultrafiltration rack to separate biomass from the culture medium, concentrated and washed with NaCl (9 g / l) in purified water. The collected biomass was divided into aliquots (mass of concentrated bacterial suspension 2000 g at 31.8 mg dry weight of biomass per 1 gram of concentrated bacterial suspension) and then frozen at -15 ° C.

EXAMPLE 1.2: Culture of Lactobacillus helveticus 103146

Initial cultivation conditions

The culture medium was obtained 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 sterilizing the medium, small Erlenmeyer flasks were separately inoculated with the contents of frozen vials (containing 1.5 ml of frozen bacteria) and incubated at 37 ° 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 of growth, the contents of a 1 L Erlenmeyer flask were transferred to the exciter.

The cultivation conditions in the exciters

20 liters of culture medium were obtained 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. The incubation temperature was regulated at 37 ° C, with stirring at a speed of 100 rpm without aeration. During cultivation, the pH was not adjusted. After 9 hours, 7 liters of the exciters were transferred to the fermenter (OD at 700 nm culture of the exciter after 9 hours: 0.14). The cultures of the pathogens under sterile conditions were transferred to fermenters.

The cultivation conditions in the fermenters

70 liters of culture medium were obtained in purified water of 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 was added to the culture. The incubation temperature was regulated at 33 ° C, with stirring at a speed of 100 rpm and without aeration. At the start of cultivation, the pH was adjusted to 6.7 CH ₃ COOH. After 24 h, cultures (OD cultures at 700 nm 4.17) were inactivated by heat treatment at 65 ° C for 35 min and transferred to a cell culture collection tank. After inactivation, the cultures were transferred to an ultrafiltration rack to separate biomass from the culture medium, concentrated and washed with NaCl (9 g / l) in purified water (9 g / l). The collected biomass was divided into aliquots (mass of concentrated bacterial suspension 440 g at 19.1 mg dry weight of biomass per 1 gram of concentrated bacterial suspension) and then frozen at -15 ° C.

EXAMPLE 1.3: Culture of Lactobacillus plantarum 71.39

Initial cultivation conditions

The culture medium was obtained 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 sterilizing the medium, small Erlenmeyer flasks were separately inoculated with the contents of frozen vials (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 of growth, the contents of a 1 L Erlenmeyer flask were transferred to the exciter.

The cultivation conditions in the exciters

20 liters of culture medium were obtained 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. The incubation temperature was regulated at 37 ° C, with stirring at a speed of 100 rpm without aeration. During cultivation, the pH was not adjusted. After 9 hours, 7 liters of the exciters were transferred to the fermenter (OD at 700 nm of the culture of the exciter after 9 hours = 1.62). The cultures of the pathogens under sterile conditions were transferred to fermenters.

The cultivation conditions in the fermenters

70 liters of culture medium were obtained in purified water of 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 was added to the culture. The incubation temperature was regulated at 35 ° C, with stirring at a speed of 100 rpm and without aeration. After 24 hours, cultures (OD cultures at 700 nm = 6.36) were inactivated by heat treatment at 65 ° C for 35 minutes and transferred to a cell culture collection tank. After inactivation, the cultures were transferred to an ultrafiltration rack to separate biomass from the culture medium, concentrated and washed with NaCl (9 g / l) in purified water. The collected biomass was divided into aliquots (mass of concentrated bacterial suspension 600 g at 60.7 mg dry weight of biomass per 1 gram of concentrated bacterial suspension) and then frozen at -15 ° C.

EXAMPLE 1.4: Culture of Lactobacillus rhamnosus 71.38

Initial cultivation conditions

The culture medium was obtained 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 sterilizing the medium, small Erlenmeyer flasks were separately inoculated with the contents of frozen vials (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 of growth, the contents of a 1 L Erlenmeyer flask were transferred to the exciter.

The cultivation conditions in the exciters

20 liters of culture medium were obtained 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. The incubation temperature was regulated at 35 ° C, with stirring at a speed of 100 rpm without aeration. During cultivation, the pH was not adjusted. After 9 hours, 7 liters of the exciters were transferred to the fermenter (OD at 700 nm culture of the exciter after 9 hours: 3.75). The cultures of the pathogens under sterile conditions were transferred to fermenters.

The cultivation conditions in the fermenters

70 liters of culture medium were obtained in purified water of 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 was added to the culture. The incubation temperature was regulated at 35 ° C, with stirring at a speed of 100 rpm and without aeration. After 14 hours, cultures (OD cultures at 700 nm 5.43) were inactivated by heat treatment at 65 ° C for 35 minutes and transferred to a cell culture collection tank. After inactivation, the cultures were transferred to an ultrafiltration rack to separate biomass from the culture medium, concentrated and washed with NaCl (9 g / l) in purified water. The collected biomass was divided into aliquots (mass of concentrated bacterial suspension 2798 g at 51.7 mg dry weight of biomass per 1 gram of concentrated bacterial suspension) and then frozen at -15 ° C.

EXAMPLE 1.5: Culture of Lactobacillus iohnsonii 103782

Initial cultivation conditions

The culture medium was obtained 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 sterilizing the medium, small Erlenmeyer flasks were separately inoculated with the contents of frozen vials (containing 1.5 ml of frozen bacteria) and incubated at 33 ° C for 10 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 14 hours of growth, the contents of a 1 L Erlenmeyer flask were transferred to the exciter.

The cultivation conditions in the exciters

20 liters of culture medium were obtained 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. The incubation temperature was regulated at 35 ° C, with stirring at a speed of 100 rpm without aeration. The pH of the culture was adjusted to 5.6 with acetic acid. After 24 hours, 7 liters of the pathogens were transferred to the fermenter (OD at 700 nm of the pathogen culture after 24 hours: 0.47). The cultures of the pathogens under sterile conditions were transferred to fermenters.

The cultivation conditions in the fermenters

70 liters of culture medium were obtained in purified water of 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 was added to the culture. The incubation temperature was regulated at 35 ° C, with stirring at a speed of 100 rpm and without aeration. After 24 h, cultures (OD cultures at 700 nm 0.174) were inactivated by heat treatment at 70 ° C for 39 min and transferred to a cell culture collection tank. After inactivation, the cultures were transferred to an ultrafiltration rack to separate biomass from the culture medium, concentrated and washed with NaCl (9 g / l) in purified water. The collected biomass was divided into aliquots (weight of concentrated bacterial suspension 61.5 g at 20.2 mg dry weight of biomass per 1 gram of concentrated bacterial suspension) and then frozen at -15 ° C.

EXAMPLE 2: Bacterial Lysates

EXAMPLE 2.1

One aliquot of the Lactobacillus fermentum I-3929 biomass from Example 1.1, containing 6 g of bacterial dry matter, was thawed at room temperature, then diluted with purified water to achieve 12 g / L dry weight of the bacterial biomass. Alkalization was performed with 0.03 M sodium hydroxide. The pH measured at the start of the lysis was 10.3. The lysate was then incubated for 6 hours at 40 ° C. with continuous stirring. After incubation, the pH was 9.9.

EXAMPLE 2.2

One aliquot of the Lactobacillus rhamnosus 71.38 biomass from Example 1.4, containing 20 g of bacterial dry matter, was thawed at room temperature, then diluted with purified water to achieve 40 g / L dry weight of the bacterial biomass. Alkalization was performed with 0.03 M sodium hydroxide. The pH measured at the start of the lysis was 10.3. The lysate was then incubated for 6 hours at 40 ° C. with continuous stirring. After incubation, the pH was 9.7.

EXAMPLE 2.3

One aliquot of the Lactobacillus fermentum I-3929 biomass from Example 1.1, containing 6 g of bacterial dry matter, was thawed at room temperature, then diluted with purified water to achieve 12 g / L dry weight of the bacterial biomass. Alkalization was performed with 0.06 M sodium hydroxide. The pH measured at the start of the lysis was 12.3. The lysate was then incubated for 6 hours at 40 ° C. with continuous stirring. After incubation, the pH was 11.8.

EXAMPLE 2.4

One aliquot of the Lactobacillus fermentum I-3929 biomass from Example 1.1, containing 6 g of bacterial dry matter, was thawed at room temperature, then diluted with 0.2 N NaCl solution to achieve 12 g / L of dry bacterial biomass. The final concentration of the NaCl solution was 0.15N. Alkalization was performed with 0.06 M sodium hydroxide. The pH measured at the start of the lysis was 6.4. The lysate was then incubated for 6 hours at 40 ° C. with continuous stirring. 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 pH of the volume was adjusted to 3.6 with 25% HCl and then incubated in a water bath at 40 ° C for 1 h under static conditions.

EXAMPLE 2.6

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

EXAMPLE 2.7

One aliquot of the Lactobacillus plantarum 71.39 biomass from Example 1.3, containing 0.4 g of the bacterial dry mass, was thawed at room temperature, then diluted with purified water to achieve 7 g / L dry mass of the bacterial biomass. Alkalization was performed with 0.06 M sodium hydroxide. The pH measured at the start of the lysis was 12.6. The lysate was then incubated for 2 hours at 40 ° C. with continuous stirring. After incubation, the pH was 12.4.

EXAMPLE 2.8

One aliquot of the Lactobacillus johnsonii 103782 biomass of Example 1.5 containing 0.3 g of bacterial dry matter was thawed at room temperature, then diluted with purified water to achieve 6 g / L dry weight of the bacterial biomass. Alkalization was performed with 0.06 M sodium hydroxide. The pH measured at the start of the lysis was 12.5. The lysate was then incubated for 2 hours at 40 ° C. with continuous stirring. After incubation, the pH was 12.3.

EXAMPLE 2.9

One aliquot of the Lactobacillus helveticus 103146 biomass of Example 1.2, containing 0.4 g of bacterial dry matter, was thawed at room temperature, then diluted with purified water to achieve 8 g / L of dry bacterial biomass. Alkalization was performed with 0.06 M sodium hydroxide. The pH measured at the start of the lysis was 12.8. The lysate was then incubated for 2 hours at 40 ° C. with continuous stirring. After incubation, the pH was 12.2.

EXAMPLE 2.10

One aliquot of the Lactobacillus fermentum I-3929 biomass from Example 1.1, containing 6.8 g of bacterial dry matter, was thawed at room temperature, then diluted with purified water to achieve 7 g / L dry weight of the bacterial biomass. Alkalization was performed with 0.08 M sodium hydroxide. The pH measured at the start of the lysis was 12.2. The lysate was then incubated for 7 hours at 40 ° C. with continuous stirring. After incubation, the pH was 12.0. The pH of the lysate was adjusted to 9.8 HCl.

The lysate contained solubilized dry weight (SDW): 20.5 mg / g, protein (Prot): 4.9 mg / g.

EXAMPLE 2.11

One aliquot of the Lactobacillus fermentum I-3929 biomass from Example 1.1, containing 6.8 g of bacterial dry matter, was thawed at room temperature, then diluted with purified water to achieve 7 g / L dry weight of the bacterial biomass. Alkalization was performed with 0.15 M sodium hydroxide. The pH measured at the start of the lysis was 13.0. The lysate was then incubated for 63 hours at 40 ° C. with continuous stirring. After incubation, the pH was 12.6. The pH of the lysate was adjusted to 9.9 HCl.

The lysate contained SDW: 23.7 mg / g, Prot: 5.0 mg / g.

EXAMPLE 2.12

One aliquot of the Lactobacillus fermentum I-3929 biomass from Example 1.1, containing 98 g of bacterial dry matter, was thawed at room temperature, then diluted with purified water to achieve 10 g / L of dry bacterial biomass. Alkalization was performed with 0.08 M sodium hydroxide. The pH measured at the start of the lysis was 12.0. The lysate was then incubated for 24 hours at 40 ° C. with continuous stirring. After incubation, the pH was 11.1. The pH of the lysate was adjusted to 11.5 NaOH.

The lysate contained SDW: 23.7 mg / g.

EXAMPLE 2.13

One aliquot of the Lactobacillus fermentum I-3929 biomass of Example 1.1, containing 39 g of bacterial dry matter, was thawed at room temperature, then diluted with 0.2 N NaCl solution to achieve 7.6 g / L of dry bacterial biomass to obtain an osmotic lysate. The pH measured at the start of the lysis was 4.4. Then, the bacterial osmotic lysate was incubated for 24 hours at 40 ° C. with continuous stirring. After incubation, the pH was 4.2. Then, the bacterial lysate was made alkaline by adding NaOH to achieve 0.06 N. The pH measured at the beginning of alkaline lysis was 10.6. The lysate was re-incubated for 2.25 hours at 40 ° C. with continuous stirring. After incubation, the pH was 10.2.

EXAMPLE 3: Cleaning lysates

EXAMPLE 3.1

The lysate of example 2.5 was centrifuged for 20 minutes at 3000 × g. Then the pH of the supernatant was adjusted to 6.8 10 N NaOH and filtered through successive filters with pore sizes of 0.45 μm and 0.2 μm and, finally, a sterilization filter with pore size of 0.22 μm. The concentrate contained Prot: 2.8 mg / ml; DNA: 17.4 μg / ml. Percentage of D-Amino Acids: 14.9% D-AIa, 14.4% D-Pro, 14.2% D-Asp.

NO production in mg dry weight / ml: 0.003 mg / ml (C1) - 0.03 mg / ml (C2) - 0.3 mg / ml (C3) active dry weight / ml: C1: 3.3 μM, C2 : 33.2 μM; C3: 52.5 μM. Active Dry Mass in g / l or mg / ml is the soluble dry mass 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 × g. Then the pH of the supernatant was adjusted to 7 HCl and filtered through successive filters with pore sizes of 0.45 μm and 0.2 μm and, finally, a sterilization filter with pore size of 0.22 μm. The concentrate contained Prot: 12.3 mg / ml; DNA: 27.7 μg / ml. Percentage of D-Amino Acids: 15.9% D-Ala, 24.4% D-Pro, 17.4% D-Asp, 45.1% D-Ser, 10.1% D-Met.

NO production in mg dry weight / ml: 0.003 mg / ml (C1) - 0.03 mg / ml (C2) - 0.3 mg / ml (C3) active dry weight / ml: C1: 8.0 μM, C2 : 56.0 μM; C3: 94.3 μM.

EXAMPLE 3.3

300 ml of the lysate of example 2.1 was first centrifuged at 3000 × g for 20 minutes. The pH of the supernatant was adjusted to 7.1 with HCl. The extract was concentrated through a Polyester Sulfone (PES) membrane (Sartorius Stedim Biotech GmbH) with a pore size of 0.45 μm with a constant flow of 330-350 ml / min on a cross flow system on a Sartoflow® Slice 200 Benchtop Crossflow System. The output was 75%. Finally, the concentrate was subjected to sterilization filtration through a 0.2 μm PES membrane (Nalgene).

The concentrate contained SDW: 30.0 mg / g; Prot: 9.6 mg / ml; DNA: 32.8 μg / ml. Percentage of D-Amino Acids: 14.3% D-Ala, 13.9% D-Pro, 14.1% D-Asp, 36.9% D-Ser. NO production in mg of active dry weight / ml: 0.003 mg / ml (C1) - 0.03 mg / ml (C2) - 0.3 mg / ml (C3) active dry weight / ml: C1: 5.1 μM, C2: 30.0 μM; C3: 64.0 μM.

EXAMPLE 3.4

The pH of 300 ml of the lysate of Example 2.2 was adjusted to 7.0 with HCl. The extract was filtered as described in example 3.3 at a constant flow rate of 350 ml / mn. The output amounted to more than 75%. Finally, the concentrate was subjected to sterilization filtration through a 0.2 μm PES membrane (Nalgene).

The concentrate contained SDW: 49.2 mg / g; Prot: 14.2 mg / ml; DNA: 34.1 μg / ml. Percentage of D-Amino Acids: 16.0% D-Ala, 13.3% D-Pro, 14.2% D-Asp.

NO production in mg of active dry weight / ml: 0.003 mg / ml (C1) - 0.03 mg / ml (C2) - 0.3 mg / ml (C3) active dry weight / ml: C1: 0 μM, C2: 4.1 μM, C3: 26.3 μM.

EXAMPLE 3.5

300 ml of the lysate of example 2.3 was first centrifuged at 3000 × g for 20 minutes. The pH of the supernatant was adjusted to 7.1 with HCl. The extract was filtered as described in example 3.3 at a constant flow rate of 330-350 ml / mn. The output amounted to more than 75%. Finally, the concentrate was subjected to sterilization filtration through a 0.2 μm PES membrane (Nalgene).

The concentrate contained SDW: 34.5 mg / g; Prot: 8.9 mg / ml; DNA: 16.8 μg / ml. Percentage of D-Amino Acids: 16.4% D-Ala, 24.3% D-Pro, 17.3% D-Asp.

NO production in mg active dry weight / ml: 0.003 mg / ml (C1) - 0.03 mg / ml (C2) - 0.3 mg / ml (C3) active dry weight / ml: C1: 3.0 μM, C2: 36.4 μM; C3: 69.5 μM.

EXAMPLE 3.6

1000 ml of the lysate of example 2.13 was centrifuged for 20 minutes at 9384 × g. The supernatant was filtered directly through a sterilization filter with a pore size of 0.22 μm.

The concentrate contained SDW: 32.7 mg / g; Prot: 6.5 mg / g; Sugar: 2.4 mg / g. Sugar concentration was analyzed in accordance with the procedure described by Herbert et al. (Meth. Microbiol, 1971, 5B: 266 et seq.).

Screening for biological activity was performed on Human Peripheral Blood Mononuclear Cells (PBMCs) at various concentrations as described in Example 6. The results obtained with 0.5 mg dry weight / ml were as follows: TNF-α: 83 pg / ml ; IL-6: 898 pcg / ml; IL-12: 140 pg / ml (in the presence of 10 ng / ml IFN-γ); and IL-10: 221 pg / ml (in the presence of IFN-γ).

EXAMPLE 3.7

The mixture of bacterial lysates of Example 2.10 was transferred to a microfiltration (MF) tank after adjusting the pH to 10.2. A microfiltration unit (MF) used a tangential flow (TF) filter with a pore size of 0.45 μm (Sartocon Slice Sartorius). The cross-flow was adjusted to 290 l / h • mc ² (LHM) and the Transmembrane Pressure (TMP) to 0.4-0.5 bar (42-52.5 N / m ² ). Permeate was transferred to an ultrafiltration tank (UF).

After the lysate volume in the microfiltration tank reached half the original volume, the UF block was started. Permeate UF filters were used as wash buffer in the MF tank. Tank volumes of both MF and UF were maintained at the same level. After passing 10 volumes of diafiltration, UF was stopped and the bacterial lysate was concentrated in MF tanks. The recovered volumetric yield was 83%. The pH of the extracted extract in the UF tank was adjusted to 7.3 with HCl and then filtered through a sterilization filter with a pore size of 0.2 μm.

The concentrate contained 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 × g. Then the pH of the supernatant was adjusted to 7.3 HCl and filtered through successive filters with pore sizes of 0.45 μm and 0.2 μm, and finally a sterilization filter with a pore size of 0.22 μm.

Peripheral Blood Mononuclear Cells (PBMC) with 10-fold diluted product: 777 pg / ml IL-10, 26 pg / ml IL-12, 13183 pg / ml IL-6.

EXAMPLE 3.9

The lysate of example 2.8 was centrifuged for 15 minutes at 9384 × g. Then the pH of the supernatant was adjusted to 7.4 HCl and filtered through successive filters with pore sizes of 0.45 μm and 0.2 μm, and finally a sterilization filter with a pore size of 0.22 μm.

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 of example 2.9 was centrifuged for 15 minutes at 9384 × g. Then the pH of the supernatant was adjusted to 7.6 HCl and filtered through successive filters with pore sizes of 0.45 μm and 0.2 μm, and finally a sterilization filter with a pore size of 0.22 μm.

PBMC with 10-fold diluted product: 476 pcg / ml IL-10, 0 pcg / ml IL-12, 4924 pcg / ml IL-6.

EXAMPLE 3.11

The lysate of example 2.11 was transferred to the MF tank after adjusting the pH to 10.4. The TFF setting was similar to Example 3.7. The transverse flow was adjusted to 290 l / h m ² and the TMP to 0.4-0.5 bar (42-52.5 N / m ² ). UF was stopped after 10 volumes of diafiltration. The output of the extracted volume was 86%. The pH of the final product was adjusted to 7.3 with HCl.

The resulting concentrate contained SDW: 24.2 mg / g; Prot: 4.8 mg / g.

EXAMPLE 3.12

The lysate of example 2.12 was transferred to the MF tank after adjusting the pH to 11.5. The TFF setting was similar to Example 3.7. The cross-flow was adjusted to 290 l / h m ² and the TMP to 0.4 bar (42 N / m ² ). UF was stopped after 10 volumes of diafiltration. The output of the extracted volume was 74%. The pH of the final product was adjusted to 7.2 with HCl.

The resulting concentrate contained SDW: 32.4 mg / g; Prot: 5.8 mg / g; Sugar: 2.3 mg / g.

Screening for biological activity was performed on human Peripheral Blood Mononuclear Cells (PBMCs) at various concentrations as described in Example 6. The results obtained with 0.5 mg dry weight / ml were as follows: TNF-α: 37 pg / ml ; IL-6: 1092 pg / ml; IL-12: 81 pg / ml (in the presence of 10 ng / ml IFN-γ); and IL-10: 160 pg / ml (in the presence of 10 ng / ml IFN-γ). Protein concentration was measured using the DC Protein assay protein assay kit (BioRad, DC Protein assay, kit no. 500-0116). A microplate assay protocol was performed. Samples for analysis (0.1 ml) were diluted with 1.9 ml of 25 mM phosphate buffer at pH 11. A protein standard curve was constructed each time an analysis was performed with bovine serum albumin solution at 2 mg BSA / ml (BSA, Pierce ref 23210). The BSA solution was diluted with 25 mM pH 11 phosphate buffer to obtain the following concentrations: 0.18, 0.24, 0.3, 0.36 and 0.42 mg BSA / ml. Samples (20 μl) and BSA standards (20 μl) were added in four repetitions to a clean, dry microtiter plate. Reagent A (25 μl) from the DC Protein assay kit protein assay kit was added to each well. After 10 minutes, reagent B was added to each well. The contents of the lunar microtiter plate were mixed on a rotary plate shaker for 5 seconds. After 20 minutes at room temperature, the absorbance at 750 nm was recorded. The protein concentration in each sample was calculated using the slope of linear regression on the protein standard curve:

OD at 750 nm = a * (protein concentration) + b mg of protein in the sample = [20 × (OD at 750 nm - b)] / a

Soluble dry mass (SDW) was determined by obtaining 5 ml of the soluble fraction by lysis and drying to a constant mass in a porcelain cup at 105 ° C.

EXAMPLE 4: Activation of mouse splenic cells In Vitro

The immunostimulating effect of the embodiments of the invention was analyzed in vitro by measuring activation of Mouse Spleen Cells (analysis using the dye Alamar blue).

Material and methods

Splenic cell stimulation

The AlamarBlueTM-Assay assay is designed to quantify the growth of human or animal cells. The analysis includes a fluorometric / colorimetric growth indicator based on the detection of metabolic activity by the oxidation-reduction indicator (REDOX) in response to chemical recovery from cell growth. Due to growth, the REDOX indicator causes changes from the oxidized (non-fluorescent, blue) form to the restored (fluorescent, red) form.

Balb / c mice (females, 6-8 weeks old) were obtained from Charles River Laboratories, Sulzfeld, Germany, and were killed by cervical vertebrae displacement. The spleens were homogenized using a ceramic homogenizer; cell suspensions were washed by centrifugation (280 × g, 4 ° C, 10 min) and resuspended in 5 ml of RPMI 1640 medium containing 5% FCS, 100 IU / ml penicillin and 100 μg / ml streptomycin. 100 μl of spleen cells (2 × 10 ⁶ / ml) were incubated with 50 μl of dilutions of the bacterial extract in 96-well plates (Falcon 3072) for 48 h at 37 ° C and 5% CO ₂ in cell culture medium.

After adding 30 μl of an AlamarBlueTM solution diluted 1: 1 with culture medium, the chemical reduction of AlamarBlueTM was measured with a Fluoroskan Ascent Reader (ThermoLabsystems, Frankfurt, Germany) at an excitation wavelength of 544 nm and an emission wavelength of 590 nm.

Test products

The following extracts were tested:

Afer300: extract from Lactobacillus fermentum I-3929 (12 g / l), obtained as described in example 3.5 (31.6 mg active dry weight / g);

Cfer300: extract from Lactobacillus fermentum I-3929 (12 g / l), obtained as described in example 3.3 (28.4 mg active dry weight / g);

Dfer300: extract from Lactobacillus fermentum I-3929 (12 g / l), obtained as described in example 2.4, and purified using the same conditions as in example 3.3 (29.5 mg active dry weight / g). This example was used as a non-alkaline lysed control.

ARahr300: extract from Lactobacillus rhamnosus 71.38 (40 g / l), obtained by alkaline lysis of NaOH 0.03 M at 40 ° C for 6 hours (49.3 mg active dry weight / g);

CRahr300: extract from Lactobacillus rhamnosus 71.38 (40 g / l) as described in Example 3.4 (46.4 mg active dry weight / g);

DRahr300: extract from Lactobacillus rhamnosus 71.38 (40 g / l) lysed by osmotic stress in a 0.15 N NaCl solution at 40 ° C for 6 hours (46.2 mg active dry weight / g). This example was used as a non-alkaline lysed control.

Negative controls

Aqua dest. (distilled water) or phosphate buffered saline (PBS).

results

In vitro studies to determine splenocyte activation

The increase in the metabolic activity of murine splenic cells after treatment with the extract was determined in 2 independent experiments by analysis using Alamar Blue (see figa and 4b and table 1).

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

Table 1 The p value for bacterial extracts in 2 independent experiments by analysis using Alamar Blue dye (see figa and 4b) The consignment Breeding P value test 1 P value test 2 Affer300 (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) DRahr300 (dilution 1: 300) 0.0025 (FIG. 4a) 0.08 (FIG.4b)

Conclusion

The immunostimulating effect of bacterial extracts was analyzed in vitro by measuring activation of murine splenic cells by Alamar Blue analysis. Moreover, in two independent experiments, the applicants compared the extract obtained by osmotic lysis (Dfer300), which includes intact, particle-shaped components of the cell walls. With two extracts in accordance with the present invention (AFer300 and CFer300). These three extracts were obtained from Lactobacillus fermentum I-3929. Each of the extracts AFer300, CFer300 and DFer300 were effective in stimulating cell metabolism at a dilution of 1: 300. Applicants did not observe a difference between the osmotic lysis control of DFer300 and Afer300 and CFer300.

Applicants also compared three extracts obtained from a second strain, Lactobacillus rhamnosus 71.38 (ARahr300, CRahr300, DRahr300). As in the previous kit, ARahr300 and CRahr300 are within the scope of the invention, while DRahr is the control of osmotic lysis. In this case, the ARahr300 extract was effective in two independent assays at a dilution of 1: 300, while the CRahr300 and DRahr300 extracts showed weaker but more significant stimulation in one of the two assays with the same dilution. When comparing the results for the six extracts, it also appears that Lactobacillus fermentum I-3929 extracts were more effective in stimulating splenic cell growth compared to Lactobacillus rhamnosus 71.38 extracts.

EXAMPLE 5: Production of nitric oxide by macrophages obtained from bone marrow

The immunostimulating potential of a series of embodiments in accordance with the invention was tested by measuring the production of nitric oxide (NO) by mouse macrophages obtained from bone marrow.

Materials and methods

Six week old male C57 / BL6 mice (six week old males, quality SPF, Charles Rivier, FR) were killed by inhalation of CO ₂ . The hip joint was removed, the femur and the tibia were removed from the back of the body. The bone marrow was removed from the lumen by injection of Dulbecco's Modified Eagle Medium (DH) through the bone after cutting both ends. After washing, the stem cells were resuspended (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 37 ° C in an atmosphere of 8% CO ₂ and saturated with moisture. Then the macrophages were separated by ice-cold PBS, washed and resuspended in DH medium supplemented with 5% fetal calf serum (FCS), amino acids and antibiotics (DHE medium). The cell density was adjusted to 700,000 cells / ml. Aqueous solutions of the extracts were serially diluted in DHE medium directly in microtiter plates. The extracts of the invention were tested in triplicate, and each microtiter plate contained medium as a negative control. The final volume in each well was 100 μl. 100 μl of the cell suspension was added to the diluted extracts, and the cells were incubated for 22 hours in an incubator at 37 ° C, in a moisture-saturated atmosphere of 8% CO ₂ . At the end of the incubation period, 100 μl of the supernatant was transferred to another microtiter plate, and the concentration of nitrite produced in each supernatant was determined by the Griss reaction. 100 μl of Griss reagent (5 mg / ml sulfanilamide + 0.5 mg / ml N- (1-naphthyl) ethylenediamine hydrochloride) in 2.5% aqueous phosphoric acid was added to each well. The microtiter plates were read with a spectrophotometer (SpectraMax Plus, Molecular Devices) at 562 nm compared to a reference at 690 nm. The nitrite concentration was proportional to the content of the nitrite oxide formed. The nitrite content was determined on the basis of a standard curve of sodium nitrite (from 1 to 70 μm NaNO ₂ ). The results were presented in μM nitric oxide (NO) as mean ± standard deviation and plotted as a dose-response curve.

Test products

The following were tested:

First analysis:

OP0701B4_CFer300: extract from Lactobacillus fermentum I-3929 (12 g / l), obtained as described in example 3.3.

OP0701B4_Dfer300: extract from Lactobacillus fermentum I-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 non-alkaline lysed control.

OP0701B4_CRahr300: extract from Lactobacillus rhamnosus 71.38 (40 g / l), obtained as described in example 3.4.

OP0701B4_ DRahr300: extract from Lactobacillus rhamnosus 71.38 (40 g / L) obtained by osmotic stress in 0.15 N NaCl at 40 ° C for 6 hours. This example was used as a non-alkaline lysed control.

Second analysis:

OP0701C_10G0.5P4H: extract from Lactobacillus fermentum I-3929 at a concentration of 10 g / L dry weight of 0.037M NaOH biomass, incubated at 40 ° C for 4 hours.

OP0701C_10G1 P4H: extract from Lactobacillus fermentum I-3929 at a concentration of 10 g / L dry weight of 0.075M NaOH biomass, incubated at 40 ° C for 4 hours.

OP0701C_10G2P4H: extract from Lactobacillus fermentum I-3929 at a concentration of 10 g / L dry weight of 0.150M NaOH biomass, incubated at 40 ° C for 4 hours.

OP0701C_10G1P21H: extract from Lactobacillus fermentum I-3929 at a concentration of 10 g / L dry weight of 0.075M NaOH biomass, incubated at 40 ° C for 21 hours.

OP0701C_10G0.5P21H: extract from Lactobacillus fermentum I-3929 at a concentration of 10 g / L dry weight of 0.037M NaOH biomass, incubated at 40 ° C for 21 hours.

OP0701C_10G2P21 H: extract from Lactobacillus fermentum I-3929 at a concentration of 10 g / l dry weight of 0.150 M NaOH biomass, incubated at 40 ° C for 21 hours.

OP0701C_10G2P45H: extract from Lactobacillus fermentum I-3929 at a concentration of 10 g / l dry weight of 0.150 M NaOH biomass, incubated at 40 ° C for 45 hours.

First analysis results

In the first analysis (Fig. 5a), it was observed that extracts obtained by alkaline lysis in accordance with the present invention have the same activity as extracts obtained by osmotic lysis.

Applicants also observed that immune stimulation activity was associated with the selected strain. Extracts obtained from Lactobacillus fermentum I-3929 caused the production of more NO ₂ and NO ₃ than extracts obtained from Lactobacillus rhamnosus 78.31 strain.

Results of the second analysis

In a second analysis (Fig. 5b), applicants observed that the activity of the in vitro extract was correlated with the initial conditions of lysis. The activity of these bacterial strains is shown for the same strain of Lactobacillus fermentum I-3929, and for the same amount of 10 g dry biomass weight per liter of lysate: 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 the experiments presented showed that despite the chemical modifications generated by the alkaline treatment process, the activity of the embodiments did not decrease compared to the control extract obtained by osmotic lysis or compared to the corresponding live bacteria (for this aspect, see Example 6 below )

EXAMPLE 6: In Vitro Screening for Pro- and Anti-Inflammatory Activity

To screen for embodiments of the invention to detect their immunostimulating or anti-inflammatory potential in vitro, tests were performed on a series of bacterial extracts on human PBMCs. The release of IL12p70 (inflammatory cytokine) and IL10 (anti-inflammatory cytokine) was measured, and the ratio of IL-10 / IL-12 was presented as described in Foligne et al. (World J Gastroenterol 2007 January 14; 13 (2): 236-243).

The aim was to compare the production of IL-12p70 and IL-10 on a series of 6 extracts obtained by extraction / purification methods. The ratio of IL-10 / IL-12p70 obtained for each extract may correlate with the anti-inflammatory potential of the extracts. Live bacteria were used as a control with which the activity of extracts was compared (2x10 ⁷ cfu / ml (colony forming units / ml) Lactobacillus fermentum I-3929)).

Materials and methods

Stepwise amounts of 6 bacterial extracts were diluted in cell culture medium in the presence of 10 ng / ml IFN-γ, a cytokine that is known to enhance IL-12p70 production, starting with an initial dose of 1 mg / ml (highest final tested dose). PBMCs isolated from the blood of healthy donors were tested as serial dilutions of the active dry weight of the extracts from 100 ng / ml to 1 mg / ml.

IFN-γ was added to the medium at least 3 hours before the lysates or live bacterial strain. The data of two independent experiments are presented.

Obtaining human PBMC

PBMCs were isolated from peripheral blood. Briefly, after centrifugation in a ficoll gradient (Pharmacia, Uppsala, Sweden), mononuclear cells were harvested, washed in RPMI 1640 medium (Live technologies, Paisley, Scotland) and their contents were adjusted to 2 × 10 ⁶ cells / ml in RPMI 1640 supplemented with gentamicin (150 μg / ml), L-glutamine (2 mmol / L) and 10% fetal calf serum (FCS) (Gibco-BRL).

Cytokine Induction

PBMC (2 × 10 ⁶ cells / ml) were plated in 24-well tissue culture plates (Corning, NY). The cells stimulated. As described above. After 24 h of stimulation at 37 ° C in an atmosphere of air with 5% CO ₂ , the culture supernatants were collected, clarified by centrifugation and stored at -20 ° C until analysis of cytokines. Cytokines were measured by ELISA using pairs of antibodies from Pharmingen (BD Biosciences, San Jose, Ca, USA) to IL-10 and IL-12p70, in accordance with the manufacturer's recommendations.

Test products

OP0701C_10G2P45H (A): Lactobacillus fermentum I-3929 lysate extract at 10 g dry weight of biomass / liter of lysate, 0.15 M NaOH at 40 ° C for 45 hours

OP0701C_1OG1P4H (B): Lactobacillus fermentum I-3929 lysate extract at 10 g dry weight of biomass / liter of lysate, 0.075 M NaOH at 40 ° C for 4 hours

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

OP0701C_40G0.5P4H (D): Lactobacillus fermentum I-3929 lysate extract at 40 g biomass dry weight / liter lysate, 0.037 M NaOH at 40 ° C for 6 hours

OP0701B4_CFer150 (E): Lactobacillus fermentum I-3929 lysate extract (12 g / L), prepared as described in Example 3.1.

OP0701B4_BFer300 (F): Lactobacillus fermentum I-3929 lysate extract at 6 g biomass dry weight / liter lysate, 0.075 M NaOH at 40 ° C for 6 hours

Lactobacillus fermentum I-3929, live bacteria frozen at -80 ° C at 2 × 10 ⁷ cfu / ml in 20% glycerol.

results

The purpose of this test was to compare the immunostimulating and anti-inflammatory potential in vitro of embodiments of the invention with a live strain of Lactobacillus. To analyze various extracts and live bacteria, the amount of each of the cytokines IL-10 and IL-12 released from PBMC, as well as the ratio of IL10 / IL12, were compared.

If the concentrations of either IL-10, or IL-12, or both values were close to the levels without stimulation (i.e., below 10 pg / l), then the ratio was not considered calculated and was marked with the letters N.C. (not calculated value).

The results obtained by PBMC, expressed in PCG / ml, are shown in tables 2 and 3.

In the presence of IFNγ, the effect of 6 bacterial extracts according to the invention (from extract A to extract F) was compared with the live form of Lactobacillus fermentum (2 × 10 ⁷ bacteria / ml, tested twice as Live sample 1 and Live sample 2) on human PBMC (reactions IL10 and IL12p70 in PCG / ml). The ratios of IL10 / IL12 are presented only for extract doses of 100 μg / ml and 1 mg / ml.

table 2 Pro-inflammatory (immunostimulating) or anti-inflammatory (immunomodulating) potential of in vitro bacterial extracts on human PBMC A B C D E F IL10 (+ IFNγ) 1 mg / ml 146 1089 9 953 961 549 100 mcg / ml 3 232 one 121 106 6 IL12 (+ IFNγ) 1 mg / ml 8 78 one 94 77 48 100 mcg / ml one 110 one 59 162 eleven IL10 / IL12 (+ IFNγ) 1 mg / ml N.C. 14.03 N.C. 10.17 12.49 11.40 100 mcg / ml N.C. 2.11 N.C. 2.05 0.65 0.52 N.C. Not calculated value

In the presence of IFNγ, the effect of 6 bacterial extracts according to the invention (from extract A to extract F) was compared with the live form of Lactobacillus fermentum (2 × 10 ⁷ bacteria / ml, tested twice as Live sample 1 and Live sample 2) on human PBMC (reactions IL10 and IL12p70 in PCG / ml). The ratios of IL10 / IL12 are presented only for extract doses of 100 μg / ml and 1 mg / ml.

Table 3 Pro-inflammatory (immunostimulating) or anti-inflammatory (immunomodulating) potential in vitro of a live strain I-3929 Lactobacillus fermentum on human PBMC Lactobacillus fermentum I-3929
Live sample 1 Lactobacillus fermentum I-3929
Live sample 2 IL10 (+ IFNγ) 2 × 10 ⁷ CFU / ml 386 355 IL12 (+ IFNγ) 2 × 10 ⁷ CFU / ml 187 191 IL10 / IL12 (+ IFNγ) 2 × 10 ⁷ CFU / ml 2.06 1.86

Based on the results presented in table 2 and table 3, the production of IL10 decreased in the following order:

B≅E≅D> F> live Lactobacillus> A> C

Based on the results presented in table 2 and table 3, the production of IL12 decreased in the following order:

live Lactobacillus> E> B> D> F> A> C

When considering the ratio, the following general type was observed:

B≅D> E≅F> live Lactobacillus

The results of the ratios of IL10 / IL12 for extracts A and C are not shown, since it was found that these extracts are effective inducers of cytokines.

At concentrations lower than 100 μg / ml, the observed cytokine concentrations were too low to draw any conclusions.

Conclusion

The obtained ratios may indicate that under the experimental conditions used in example 6, some bacterial extracts according to the invention have more pronounced immunomodulating effects than the effects of live Lactobacillus fermentum. For example, extracts B, D, E, and F showed higher IL10 / IL12p70 ratios than the control in the form of live bacteria. Thus, such extracts may be more active. Than live probiotic bacteria, against the conditions described in this application, such as inflammatory conditions.

EXAMPLE 7: Effect on Toll-like receptors

Embodiments of the invention are derived from gram-positive bacteria, and therefore, it is expected that they act through TLR2 receptors. TLR receptors are expressed mainly, but not exclusively, by immune cells such as monocytes, macrophages, dendritic cells, T cells, etc., and they are key sensors of microbial products that can be recognized by the host as hazard signals. Even though TLR receptors first trigger nonspecific innate immunity, their activation initiates a complete immunological cascade, which in the presence of antigens leads to the development of acquired immunity.

Cells that express this functional TL gene are valuable tools for many uses, such as researching the mechanisms involved in recognizing or transmitting TLR signals, and developing new potential therapeutic drugs. The Experiments described below tested the activity of three bacterial extracts on these key immune response adapters.

Materials and methods

The reactions of the extracts according to the invention were tested (or as such to check their agonistic effect, or in the presence of a TLR2 Pam3Cys agonist, or in the presence of a TLR4 LPS agonist to check antagonistic activity) in the following two cell systems:

a) HEK-TLR2 / 6 (ELISA IL-8 after 24 hours)

b) HEK-MD2-TLR4-CD14 (ELISA IL-8 after 24 hours)

a) HEK-TLR2 / 6

The HEK293 cell line was chosen for their zero or low basal expression of TLR genes. These cells provide effective monitoring of TLR activity using ELISA assays, such as IL-8 titration or receptor-based systems that monitor TLR-induced NF-κB activation.

HEK-TLR2 / 6 cells (Invivogen, Toulouse, France) are engineered HEK293 cells. Stably transfected with multiple genes from the TLR2 / 6 pathway, which include TLR2, TLR6, and genes involved in recognition or involved in the signaling cascade. These cells secrete IL-8 after stimulation with TLR2 / 6. The experiments were carried out in accordance with the manufacturer's instructions.

Briefly, 2 × 10 ⁴ cells / well (200 μl of RPMI medium) were incubated at 37 ° C for 3 days (5% CO ₂ ). The medium was removed and 90 μl RPMi + 5% FCS was added to the wells. Then agonists and controls (10 μl / onion) were added. Cells were returned to the incubator for 24 hours. Supernatants were harvested and IL-8 ELISA was performed according to the manufacturer's instructions.

Test products

OP0701B4_Afer50: Lactobacillus fermentum I-3929 lysate extract at a concentration of 12 g of dry biomass weight / liter of lysate, 0.075 M NaOH at 40 ° C for 4 hours, purified as described in Example 3.6.

OP0701C-Bt1LAC: extract from Lactobacillus fermentum I-3929, obtained as described in Example 3.7.

OP0701C-Bt2LAC: extract from Lactobacillus fermentum I-3929, obtained as described in example 3.11.

Results: secretion of IL-8

The results for the controls (negative = LPS K12 ultrapure, TLR4 agonist; and PAM3CSK4 = positive, TLR2 agonist) are presented in Tables 4-6. Results (expressed in PCG / ml IL-8) show the average IL-8 secretion values determined by ELISA 24 hours after stimulation with controls.

The HEK TLR2 / 6 cell line responded to the TLR2 Pam3Cys agonist. In contrast, an E coli TLR4 agonist (LPS K12) was inactive even at a high dose of 0.01 μg / ml.

Experiments with the separate use of 3 extracts

The 3 bacterial extracts tested in this case (OP0701B4Afer50, OP0701C-Bt1LAC and OP0701C-Bt2LAC) showed higher immunostimulating properties than the TLR2 Pam3Cys agonist.

Experiments with three bacterial extracts + Pam3Cvs

As indicated above, three bacterial extracts were also tested to determine their antagonistic or additive properties compared to Pam3Cys added immediately after the extracts.

Bacterial extract OP0701 B4 AFer50

The results presented in table 4 indicate that the extract OP0701B4_AFer50 caused the production of high levels of IL-8 (agonist TLR 2/6).

Bacterial extract OP0701C-BtILAC

The results presented in table 5 indicate that the extract OP0701C-Bt1LAC caused the production of high levels of IL-8 (agonist TLR 2/6).

Bacterial Extract OP0701C-Bt2LAC

The results presented in table 6 indicate that the extract OP0701C-Bt2LAC did not cause the production of IL-8 (agonist TLR 2/6), and in the presence of Pam3Cys had an antagonistic effect on TLR2 / 6.

Conclusion

Depending on the conditions of alkaline lysis (initial concentration of dry weight of biomass, initial concentration of base and duration of treatment with base), bacterial extracts could have different types of action.

OP0701B4_Afer50 and OP0701C-Bt1LAC were TLR2 / 6 agonists, but stronger alkaline lysis performed on the same bacterial strain led to antagonistic activity against TLR2 / 6 (OP0701C-Bt2LAC).

b) HEK-TLR4-MD2-CD14

Extensive studies have been performed on TLR4 because it is the primary receptor involved in the recognition of lipopolysaccharide (LPS), which is 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 a NF-κB-induced 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 3 tested bacterial extracts according to the invention are shown in tables 7-9.

Results: Secretion of IL-8

The results for the controls (positive = LPS K12C ultrapure, TLR4 agonist; and PAM3CSK4 = negative, TLR2 agonist) are presented in Tables 7-9. Results (expressed in PCG / ml IL-8) show the average IL-8 secretion values determined by ELISA 24 hours after stimulation with controls.

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

Experiments with the separate use of 3 extracts

As expected from agonists of gram-positive bacteria, these 3 tested bacterial extracts (OP0701B4 Afer50: Table 7, OP0701C-Bt1LAC: Table 8 and OP0701C-Bt2LAC: Table 9) did not show distinct immunostimulatory properties through the TLR4 pathway.

Experiments with 3 bacterial extracts + LPS K12

As indicated above, the three extracts according to the invention were tested only to identify their alleged antagonistic or additive properties in comparison with the LPS added immediately after the extracts. Again, no effect was observed at the TLR4 receptor level (Tables 7-9).

Bacterial extract OP0701B4 AFer50

The results indicate that the extract OP0701B4_AFer50 did not activate the TLR4 receptor (Pam3Cys: 138 pg / ml).

Bacterial Extract OP0701CBtI-LAC

Bacterial Extract OP0701CBt2LAC

Conclusion

Taken together, the results presented here on HEK cells and those obtained on human PBMC cells indicate that Afer50 and BT1LAC are able to stimulate the immune system via the TLR2 pathway. In addition, BT2LAC may act as a TLR2 / 6 antagonist. Therefore, the extracts in accordance with the invention can stimulate the immune system via the TLR2 pathway or act as antagonists of TLR2 / 6, thus correlating with anti-infective and anti-inflammatory activity in vivo.

EXAMPLE 8

Cells that express this functional TLR gene are valuable tools for many uses, such as investigating the mechanisms involved in recognizing or transmitting TLR signals, and developing new potential therapeutic drugs. Therefore, the purpose of the experiments described below is to test the activity of 4 bacterial extracts against these key adapters of the immune response.

In this test, 8 TLR and NOD2 receptors were screened for 4 bacterial extracts, including extracts in accordance with the present invention.

Method

Bacterial extracts in accordance with the invention were tested in 96-well microplates. The extracts were diluted in DMEM culture medium, and 20 μl of each dilution was tested in duplicate. A volume of 180 μl of HEK293 cell suspensions containing 25,000 or 50,000 cells in DMEM + 10% FCS culture medium (one HEK293 cell line specific for each TLR with a reporter gene secreted alkaline phosphatase under the control of NF-kB) was added to each well in duplicate. After 16 hours of incubation with each cell line, 20 to 50 μl of each supernatant was transferred to 96-well microplates and 200 μl of Quantiblue was completed (InVivoGen no REP-QB1). An enzymatic reaction with secreted alkaline phosphatase was performed for 30-60 minutes for various series of cell lines expressing TLR. Reading was performed using a microplate reader at 630 nm.

Material

List of positive control agonists (and their respective concentrations) used in this screening assay

TLR2 PAM2 100 ng / ml; TLR3 PoIy (I: C) 100 ng / ml; E. coli K12 TLR4 LPS1 μg / ml; flagellin TLR5 S. typhimurium flagellin 1 μg / ml; TLR7 R848 10 μg / ml; TLR8 R848 10 μg / ml; TLR9 CpG ODN 2006 10 μg / ml; NOD2 Muramyldipeptide 1 μg / ml.

Negative controls

The reporter gene only (NFkB) HEK-293 recombinant cell line 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 not expressing TLR.

Tested Products

The following bacterial extracts were tested:

OP0701B4_CFer300: extract from Lactobacillus fermentum I-3929 (12 g / l), obtained as described in example 3.3 (F);

OP0701B4_CRahr300: extract from Lactobacillus rhamnosus 71.38 (40 g / l), obtained as described in example 3.4 (G);

OP0701D_10L1 PswitchA: extract from Lactobacillus fermentum I-3929 (10 g / l), obtained as described in example 3.12 (I); and

OP0701D_5LOSMOConc: osmotic extract of Lactobacillus fermentum I-3929 (7.6 g / L) obtained by osmotic stress in 0.16 N NaCl at 40 ° C for 24 h (H) as a control.

Obtaining bacterial extracts

20 μl of the sample to be tested was used to stimulate all cell lines in 200 μl of reaction volume.

Screening was performed in a single concentration, typically 0.5 mg dry weight / ml. Tests were performed in duplicate.

results

Bacterial extracts and controls were tested in duplicate on the HEK-293 recombinant cell line that functionally express the TLR or NOD2 protein, as well as the reporter gene, driven by the NFkB promoter. TLR and NOD2 activation results are presented as absorbance values (OD). The results are shown in tables 10-13 and summarized in table 15.

Bacterial Extract F (OP0701B4Cfer300)

Table 10
Activation of TLR and NOD2 receptors with OP0701B4Cfer300 extracts hTLR2 hTLR3 hTLR4 hTLR5 hTLR7 hTLR8 hTLR9 hNOD-2 Sample Comparison 3,493 2,052 1,341 3,657 1,480 2,786 1,955 0.576 Extract F 3,963 0.051 0.735 0,304 0,070 0,080 0,026 0.503

Bacterial extract F specifically activated hTLR2, hTLR4 and hNod2, and to a lesser extent, hTLR5.

Bacterial Extract G (OP0701B4CRahr300)

Table 11
TLR and NOD2 receptor activation with OP0701 B4CRahr300 extracts hTLR2 hTLR3 hTLR4 hTLR5 hTLR7 hTLR8 hTLR9 hNOD-2 Sample Comparison 3,493 2,052 1,341 3,657 1,480 2,786 1,955 0.576 G extract 0.808 0.003 0.177 0.017 0,028 0,044 -0.032 0.369

Bacterial extract G activated hTLR2, hNOD2, and to a much lesser extent hTLR4.

Bacterial Extract H (OP0701D 5LOSMOConc)

Table 12
Activation of TLR and NOD2 receptors with extracts OP0701D_5LOSMOConc hTLR2 hTLR3 hTLR4 hTLR5 hTLR7 hTLR8 hTLR9 hNOD-2 Sample Comparison 3,493 2,052 1,341 3,657 1,480 2,786 1,955 0.576 H extract 4,537 0,057 0.638 0,050 -0.072 0,054 0.011 0.892

Bacterial extract H specifically activated hTLR2, hTLR4 and Nod2.

Bacterial Extract I (OP0701D_10L1PswitchA)

Table 13
Activation of TLR and NOD2 receptors with extracts OP0701D_10LPswitchA hTLR2 hTLR3 hTLR4 hTLR5 hTLR7 hTLR8 hTLR9 hNOD-2 Sample Comparison 3,493 2,052 1,341 3,657 1,480 2,786 1,955 0.576 Extract I 4,168 0,096 0.342 3,490 -0.024 0.132 0.410 0.790

Bacterial extract I strongly activates hTLR2, hTLR5, hNOD2, and to a lesser extent hTLR4 and hTLR9.

Table 15
Activation of TLR and NOD2 receptors by various extracts resulting from other conditions of the method in accordance with the present invention hTLR2 hTLR3 hTLR4 hTLR5 hTLR7 hTLR8 hTLR9 hNOD-2 Extract F + - + ± - - - + G extract + - ± - - - - + H extract + - + - - - - + Extract I + - ± + - - ± +

Thus, the bacterial extracts of the present invention can help activate the immune system through various TLRs as well as Nod2. Therefore, the extracts of the invention can be good activators of the immune response.

When comparing extract H with extract G and extract F, it becomes apparent that the extracts have the same TLR and Nod paths as those obtained by osmotic lysis. When comparing extract H and extract I, it was possible to observe the activation of the TLR5 pathway and, to a lesser extent, the TLR9 pathway with the addition of an acid lysate after an alkaline lysate. Even the F extract obtained as a result of the weak alkali method showed activation of the TLR5 pathway, in contrast to the H extract.

Extract I acted on TLR5, which indicates the potential for use in radiation therapy. Indeed, radiation therapy is a well-established and highly effective treatment for certain types of cancer. However, its side effects can be very serious, as it can destroy healthy cells in the body, in particular bone marrow cells and cells in the gastrointestinal tract. Recently, Burdelya et al. reported that the CBLB502 peptide binds to TLR5 and activates nuclear factor-κB signaling, 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). Rhesus mice and macaques treated with CBLB502 shortly before exposure to lethal doses of the whole body showed less damage to healthy bone marrow and gastrointestinal cells, and they survived significantly longer than controls. Importantly, in tumor-affected mice, CBLB502 did not impair the antitumor efficacy of radiation therapy.

EXAMPLE 9: The ability of the extract to produce plaque-forming cells (against sheep red blood cells) in mice

The plaque-forming cell technique (PFC) provides an opportunity to evaluate 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 has been demonstrated, as described below. Murine lymphoid cells, as well as a dense population of foreign (mutton) red blood cells, were simultaneously placed on a microscope slide. Certain lymphoid cells release hemolytic antibodies that diffuse and cause lysis of neighboring red blood cells to form a lysis plaque in the presence of complement. At the end of the experiment, the number of PFCs per 10 ⁶ cells or per spleen was counted.

Materials and methods

Tested Products

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) aged 5 to 6 weeks and with an average body weight of 20 ± 2 g were divided into 5 groups of 8 animals each .

Experiment structure

The animals were divided into 5 groups as follows:

Group (a) 8 mice receiving 1.2 mg / mouse of extract 1, as described in example 3.5; volume = 0.2 ml.

Group (b) of 8 mice treated with 0.6 mg / mouse of extract 1, as described in example 3.5.

Group (c) of 8 mice treated with 1.2 mg / mouse of extract 2, as described in example 3.4; volume = 0.2 ml.

Group (d) of 8 mice receiving 0.6 mg / mouse of extract 2 as described in Example 3.4; volume = 0.2 ml.

Group (e) 8 mice receiving 0.2 ml of isotonic saline.

Mice received the extracts described above at the indicated doses daily per os (by oral route) for 5 consecutive days (from day 1 to day 5). Two more intubations occurred on days 19 and 20. On the 29th day, the antigen (10 ⁶ sheep red blood cells) in 0.2 ml of isotonic saline (0.9% NaCl, Olsver's solution) was injected intravenously through the tail vein of the animal; after 4 days (33rd day), splenic cell suspensions were obtained.

Reagents and equipment

Endotoxin Free Olsver Solution (Sigma, 38299 St Quentin Fallavier, Cedex France, ref. A 3351)

Basic Medium Needle (BME concentrated 10 times) in a bicarbonate-free Needle solution (Bio-Merieux, 69280 Marcy I'Etoile, France ref: 8 210 2)

Lyophilized Guinea Pig Serum (Bio-Merieux, Marcy I'Etoile, France ref: 7 212 2)

Lamb erythrocytes (Bio-Merieux, Marcy I'Etoile, France ref: 7214 1)

Trypan blue, sterile distilled water, NaCl 0.9%, tissue homogenizer, centrifuge, Neubauer chamber for cell counting, glass tubes.

Test or Control Product Treatment

Control animals received only lamb erythrocytes (SRBC). Animals of other groups received oral bacterial extracts as previously indicated. For this, each extract was dissolved in water.

Splenic cell suspension

4 days after the last injection of antigen (SRBC), a suspension of splenic cells was obtained for each mouse in accordance with the following procedure:

The animal was anesthetized with ether and then killed by displacement of the cervical spine. The spleen was removed and crushed in a glass tissue homogenizer with 2 ml of BME at a pH of 6.75-6.80. Then 3 ml of sterile distilled water was added, and the mixture was stirred for 20 seconds. The resulting suspension was then diluted with 10 BME and centrifuged at 1200 rpm for 10 min at 4 ° C. The supernatant was removed and the precipitate was suspended in 2 ml of BME. Cells were allowed to recover for 5-10 minutes on ice. Illumination of viable mononuclear cells was performed as follows:

A dilution of a 1/20 aliquot of the cell suspension was obtained with saline containing 0.1% trypan blue solution. Unstained cells, i.e., living cells, were counted in a Neubauer chamber.

The cell suspension was kept in melting ice during counting and then immediately used for lysis of plaques. This technique prevents the loss of viability that can occur when waiting too long.

Obtaining slides

On an ordinary microscope slide, carefully sprinkled with powder and with any trace of fat removed, 3 parallel strips (0.1 mm thick) of double-sided tape were glued at intervals of about 1.5 cm. Then the strips were covered with pre-cleaned coverslips (22 mm × 22 mm) to form two “chambers” between the slide and the coverslip.

The splenic cell suspension fraction was adjusted to 25,000 mononuclear cells per mm ³ . In a small glass tube, a mixture of the following elements (added in the indicated order) was obtained using an automatic pipette:

1) 7.5 ml BME

2) 0.5 ml of a suspension of SRBC, washed and centrifuged twice with saline, at 50% residue

3) 0.4 ml of normal guinea pig serum used as a complement source

Finally, 50 μl of a suspension of splenic cells at a density of 25,000 cells / mm ^{3 was} added to 200 μl of the previous mixture. After gentle homogenization, the suspension was introduced into the Cunningham Chambers by capillary action before sealing the free sides with paraffin. Then the slides were placed in a humid chamber in a thermostat at 37 ° C.

annotation

After incubation for 1 hour in a thermostat, the slides were examined under a microscope with a magnification of 100 times (10X eyepiece, 10X objective).

Lytic plaques were easily recognized. Five vertical optical strips were examined on each slide and the number of lytic plaques was counted. The number of cells (N) forming direct lytic plaques per 10 ⁶ cells was calculated by extrapolation. If x represents the number of plaques observed, and X represents the number of cells examined, then the number of direct lytic plaques per 10 ⁶ cells is:

N = (x / X) * 10 ⁶

where X = C * V = C * (n * e * l * L)

C = final concentration of splenic cells

V = observed volume

n = number of optical strips

e = adhesive tape thickness

I = optical field width

L = optical strip length

The number of direct lytic plaques per 10 ⁶ cells was obtained from the following equation:

PFC (per 10 ⁶ cells) = (x * 10 ⁶ ) / (C * n * e * I * L)

Knowing the number of collected cells per spleen, it was possible to derive the number of cells per spleen forming lytic plaques.

In mice, which were the "absolute standard", spontaneous lytic plaques were not observed.

Statistical Analysis and Results

The results were considered significant when p <0.05 (t student test). The results are shown in table 16.

Table 16
The ability of bacterial extracts 1 and 2 to produce plaque-forming cells (against mutton erythrocytes) in mice GRM PFC / 10 ⁶ cells PFC Frequency Extract 1; see Example 3.5
(1.2 mg / mouse / administration) one hundred%
one hundred%
one hundred% 124%
123%
114% 119%
143%
113% Extract 1; see Example 3.5
(0.6 mg / mouse / administration) one hundred%
one hundred%
one hundred% 117%
118%
112% 103%
139%
128% Extract 2; see Example 3.4
(1.2 mg / mouse / administration) one hundred%
one hundred%
one hundred% 119%
124%
117% 109%
131%
120% Extract 2; see Example 3.4
(0.6 mg / mouse / administration) one hundred%
one hundred%
one hundred% 117%
114%
113% 112%
119%
111%

It turned out that the extracts according to the invention are activators of B-cells when tested at concentrations of 1.2 mg / mouse / per day or 0.6 mg / mouse / per day. An insignificant dose-dependent effect was observed. Therefore, some extracts of the invention could potentially be used to antigenically stimulate the immune system in patients suffering from recurrent infections.

EXAMPLE 10: Effect of CFer300 with intraperitoneal infection of Salmonella typhimurium in balb / c mice

Protection of mice infected with Salmonella typhimurium was tested with bacterial lysates obtained from Lactobacillus after oral administration.

Materials and methods

Animals and their contents

Balb / c mice were kept in the laboratories of the Institute of Immunology in Moscow. For an in vivo protection experiment, non-inbred white mice of laboratory breed were purchased at Stolbovaya at the Russian State Scientific Center for Biomedical Technologies (Russian Academy of Medical Sciences). On delivery, the body weight of the mice was 12-14 g. During all experiments, the mice were kept in the absence of pathogens in a standard rodent ration and water supply.

Study groups (main experiment)

Two groups of 22 mice were used to test the anti-infective efficiency of bacterial extracts obtained using an experimental model of Salmonella typhimurium infection in mice.

One group was orally treated with CFer300 solution, and the second group received a false treatment (water) as a negative control.

0.5 ml of the solution was administered to each mouse orally once a day for 10 consecutive days before all mice were subjected to antigenic stimulation of Salmonella typhimurium:

Group 1: Mice treated with CFer300 administered orally in a single dose of 2 mg (0.5 ml).

Group 2: False treatment mice using oral administration of 0.5 ml of water daily for 10 days.

Product under test

The tested bacterial extract was OP0701B4CFer300 ("CFer300"); extract from Lactobacillus fermentum I-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 an infectious agent that would cause mortality close to 50%. 3 weeks after antigenic stimulation. As antigenic stimulation, a suspension of Salmonella enterica, serovar typhimurium strain 415 (I. Mechnikov Institute of Vaccines and Serums, Russian Academy of Medical Sciences) was injected intraperitoneally with each mouse. The dose of antigenic stimulation ranged from 10 ³ to 10 ⁵ CFU Salmonellae per mouse.

Observations and death registration

After antigenic stimulation, the mice were kept under standard conditions for laboratory animals. Daily observation and registration of death was carried out for a period of 21 days after infection. The anti-infective efficacy of the drugs was evaluated according to survival after infection (SR), life expectancy after infection (ADL), protection factor (DF) and drug efficacy index (El) calculated for each experimental group. SR was taken as the percentage of animals living in the experimental groups on the 21st day after infection. ADL, DF and EI were calculated as follows:

ADL = (X1 + X2 + ... + Xn) / N

Where:

ADL represents life expectancy,

X1 to Xn represent the post-infection period for experimental mice 1 through n, and

N represents the total number of animals in the experimental group.

DF = CD / ED

Where:

DF represents a protection factor,

CD represents the percentage of dead animals in the control group, and

ED represents the percentage of dead animals in the experimental group.

EI = [(DF-1) / DF] × 100%

Where:

EI represents the index of the effectiveness of the drug, and

DF represents a protection factor.

Results: drug tolerance

The drug was administered orally once a day for 10 days. No evidence of toxicity or side effects was observed during the 10-day pretreatment period. And he was well tolerated.

Titration Salmonellae

A preliminary experiment was performed to determine the dose of antigenic stimulation of Salmonella typhimurium in order to cause a mortality of approximately 50%. The results are shown in table 17.

Table 17
Preliminary experiment to determine the dose of antigenic stimulation of Salmonella typhimurium, corresponding mortality of approximately 50% Dose of S.typhi Mouse / group Days after infection Dead mouse / total Dead (%) one 2 3 four 5 6 7 8 9 10 eleven 12 13 10 ⁵ 6 - - - - one 2 - - - - one - - 4/6 67 10 ⁴ 6 - - - - - - - one - - - - - 1/6 17 10 ³ 6 - - - - - - - - - - - - - 0/6 0

The underlined numbers represent the number of animals found dead on the indicated day (after infection).

Conclusion

Based on the data obtained, a dose of 10 ⁵ CFU of strain 415 Salmonella typhimurium strain (67% of dead animals, in bold) was selected for further study.

Main experiment: Antigenic stimulation of mice treated with CFer300

Mice receiving pretreatment with CFer300 (n = 22) for 10 days or in the control group with water (n = 22) were subjected to antigenic stimulation a day after the end of pretreatment.

A suspension of Salmonella typhimurium was administered intraperitoneally as a dose of 10 ⁵ CFU antigenic stimulation per mouse. Control observation was performed within 21 days after infection. Mortality in groups of animals is shown in table 18.

The numbers in italics represent the number of animals found dead on the indicated day (after infection). These data were used to calculate SR, ADL, DF, and EI (Table 19).

Table 19
Survival over the observation period (21 days) Preliminary treatment with substances Mortality (%) Survival rate (%) ADL (days) Df EI (%) CFer 300 27 73 17.1 1,67 40% H ₂ O 45 55 14.7 one 0

In the control group that received pretreatment with water, survival during the observation period (21 days) was 55%, and ADL was 14.7 days. At a dose of 2 mg / day, CFer300 showed a protective effect, which led to SR = 73% and ADL = 17.1 days, i.e., DF = 1.67, and EI = 40%.

In summary, a 10-day course of daily oral administration of CFer300, administered in a single dose of 2 mg, provided partial protection in mice infected with Salmonella typhimurium. As shown in this experiment, and previously shown in vitro and ex-vivo, the extracts disclosed herein can be used in the further development of therapeutic drugs.

EXAMPLE 11: Effect of two Lactobacillus extracts on a LACK-induced asthma model

Two embodiments of the invention were tested in a mouse model of allergen-induced asthma after oral administration (Julia et al., Immunity, 2002, 16: 271-283).

Material and methods

Animals and content

6-week-old female BALB / c ByJ mice were purchased from Janvier, France. They were kept and fed under standard conditions.

Study groups

A total of 27 mice were divided into 4 groups as follows:

Group A: untreated, sensitized LACK, and stimulated with saline mouse; LACK is a protein from the parasite Leishmania major (4 mice).

Group B: untreated, sensitized LACK and antigen-stimulated mice (8 mice).

Group C: Treated with OM-1009A (8 mg dry weight of the remainder per administration), LACK sensitized and antigenically stimulated mice (8 mice).

Group D: Treated with OM-1009B (8 mg dry weight of the remainder per administration), sensitized LACK and antigen-stimulated mice (7 mice).

Treatment and regimen with test or control product

Mice were treated from -3 to the 22nd day. On day 0 and on day 7, mice were sensitized intraperitoneally (ip) with 10 μg LACK in the presence of 2 mg of alum 3. From day 17 to day 21, the mice were exposed to a 20-minute exposure to antigenic stimulation of aerosol LACK solution (0.15%) ( Groups B, C and D), or saline (Group A) as a control. On day 22, an analysis was made of the ability of mice to respond to methacholine inhalation by the development of AHR. On day 23, mice were sacrificed and pneumonia was assessed.

Methodology

Mice were treated with medication three times as described above. Lavage by cannula inserted into the trachea was performed in individual mice after bloodletting. The lungs were washed 3 times with 1 ml of warmed PBS. Cells were washed with PBS, resuspended in 300 μl and counted using a Barker-Türk chamber. For differential counting of BAL cells (broncho-alveolar lavage), cell preparations after centrifugation were prepared and stained according to Wright / Giemsa. The tested groups were: wild-type (wt) mice, LACK sensitized and PBS-stimulated (Control), LACK sensitized and antigen-stimulated wt mice (animals with asthma), wt mice treated with OM-1009A, and wt mice treated OM-1009B. The total number of cells in the BAL was determined by microscopic examination of cell preparations after centrifugation and Wright / Giemsa staining.

Since the treated mice (see results below) showed a decrease in airway inflammation, pulmonary extracts were obtained, and IL-4, IL-5, and IL-13 were quantified by multiplex analysis (using CBA software).

Tested Products

The following two bacterial lysates were tested: OM-1009A extract from Lactobacillus fermentum I-3929, obtained as described in Example 3.7, and OM-1009B extract from Lactobacillus fermentum I-3929, obtained as described in Example 3.11.

results

a) Airway hyperreactivity

On the 22nd day, an analysis was performed to identify the ability of mice to develop AHR after inhalation of methacholine in the indicated doses. The results are presented in figure 5. The results show that OM-1009-B restored Penh basal values similar to the untreated, asthmatic group treated with PBS. OM-1009-A was more effective because the Penh values obtained were even lower than those observed in the non-asthma control group.

b) Total and differential cell numbers in broncho-alveolar lavage fluids

On day 23, mice were euthanized and pneumonia evaluated. The total number of cells in the BAL is presented in Table 20 (right column), as well as the differential cell numbers: Number of eosinophils (Eo), number of neutrophils (Neutro), number of lymphocytes (Lympho) and number of other cells (Other).

Table 20
The total and differential number of cells in the BAL after treatment with two extracts of the invention (Groups C and D). The mean values and standard error of the mean are presented. Average Eo Neutro Lympho Other Total number of cells (A) Ctrl PBS 901 3178 11796 49125 65,000 (B) Ctrl LACK = mice with asthma 347691 51317 85609 301097 785714 (C) OM-1009A + LACK 115227 27879 54725 158419 356250 (D) OM-1009B + LACK 203834 14210 34523 204575 457143

Sem Eo Neutro Lympho Other Total number of cells (A) Ctrl PBS 166 906 6255 5568 2887 (B) Ctrl LACK 78464 14392 17736 78575 135275 (C) OM-1009A + LACK 36741 10444 27024 34296 94905 (D) OM-1009B + LACK 57955 3220 6731 25311 74992

Both extracts significantly reduced the total number of cells in BAL (p <0.01 for OM-1009A; and p <0.03 for OM-1009B. Compared to group B). The number of eosinophils decreased by 3 and 1.7 times, respectively. The number of neutrophils decreased by 1.8 and 3.6 times, respectively. A decrease in the number of eosinophils and neutrophils indicates a lower concentration of inflammatory cells in the BAL of animals that received preliminary treatment with extracts.

c) Th2 cytokines detected in pulmonary extracts and quantified by multiplex analysis (CBA software)

Table 21
Individual lung IL4 levels in mice pkg / mg mouse 1 mouse 2 mouse 3 mouse 4 mouse 5 mouse 6 mouse 7 mouse 8 Wednesday (A) PBS 0 0.0053 0.0071 0.0157 0.0070 (B) LACK
(mice with asthma) 0,0395 0.0141 0,0279 0,0203 0,0353 0,0574 0.0949 0,0806 0.0462 (C) PO OM 1009A 0,0379 0.0120 0.0066 0.0056 0.0181 0.0146 0,0396 0.0463 0,0226 (D) PO OM 1009B 0,0409 0,0515 0.0414 0,0511 0,0484 0,0300 0.0270 0.0415

Table 22
Individual lung IL5 levels in mice pkg / mg mouse 1 mouse 2 mouse 3 mouse 4 mouse 5 mouse 6 mouse 7 mouse 8 Wednesday (A) PBS 0 0,0004 0.0043 0 0.0012 (B) LACK
(mice with asthma) 0.1141 0,1002 0.1532 0.1115 0.3098 0.5173 0.5385 0.4547 0.2874 (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 IL13 levels in mice lungs pkg / mg mouse 1 mouse 2 mouse 3 mouse 4 mouse 5 mouse 6 mouse 7 mouse 8 Wednesday (A) PBS 0 0 0 0,0208 0.0052 (B) LACK
(mice with asthma) 0.2727 0,0896 0.3542 0.2303 0.6178 1,1306 1.1768 1,0244 0.6120 (C) PO OM 1009A 0.3598 0,1231 0,0888 0,0995 0.2975 0.1909 0.5213 0.5463 0.2784 (D) PO OM 1009B 0.3628 0.8919 0.4291 0.7699 0.6441 0.4981 0.4972 0.5847

Th2 levels of cytokines (IL4, IL5 and IL13) (see Tables 21, 22, 23), when compared with the asthmatic control group (B), were reduced by OM-1009A extract, but not OM-1009B extract. Therefore, bacterial extracts obtained by treatment with a strong base (OM-1009-B) may be less active than extracts obtained under conditions of treatment with a moderate base (OM-1009-A). Even though both extracts were active in Penh factor analysis (FIG. 6), OM-1009A may be a better candidate than OM-1009B for treating or preventing conditions related to Th2-related diseases such as allergic diseases and atopy, or their symptoms.

Claims (21)

1. An extract of one or more bacterial strains of Lactobacillus, where the extract is a soluble extract, and where the extract contains chemically modified bacterial molecules, the extract obtained by exposure to an alkaline medium on one or more bacterial strains of Lactobacillus, and where the extract is useful in the treatment diseases associated with an imbalance in the production of anti-inflammatory cytokines. 2. The extract according to claim 1, where the extract has immunomodulatory activity in an individual. 3. The extract according to claim 2, where the extract has immunostimulating activity in an individual. 4. The extract according to claim 2, where the extract has anti-inflammatory activity in an individual. 5. The extract according to claim 1, where one or more bacterial strains of Lactobacillus contains one or more of Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus casei defensis, Lactobacillus casei defensis, Lactobacillus casei. casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus lactis and Lactobacillus delbrueckii. 6. The extract according to claim 1, where one or more bacterial strains of Lactobacillus contains one or more of Lactobacillus fermentum 1-3929, Lactobacillus rhamnosus 71.38, Lactobacillus plantarum 71.39, Lactobacillus johnsonii 103782 and Lactobacillus helveticus 103146. 7. The extract according to claim 1, where the specified chemical modification leads to the fact that one or more of aspartic acid, glutamic acid, serine, histidine, alanine, arginine, tyrosine, methionine, phenylalanine and lysine in the extract are racemicized, at least , on 10%. 8. The extract according to claim 1, where the extract is able to achieve the calculated ratio of IL10 / IL12 in mononuclear cells of human peripheral blood, and the ratio is equal to or greater than the ratio of IL10 / IL12 achieved by the live strain of Lactobacillus from which the extract is obtained. 9. The extract according to claim 1, where the extract reduces the number of eosinophilic cells, the number of neutrophilic cells, the number of lymphocyte cells or any combination thereof in asthmatic mice by a factor of at least 1.5 relative to an untreated asthma control. 10. The method of obtaining the extract according to claim 1, including:
(a) culturing one or more bacterial strains of Lactobacillus in a culture medium;
(b) the effect of an alkaline environment on each bacterial strain of Lactobacillus; and
(c) treating the product of step (b) to remove insoluble and particulate material. 11. The method according to claim 10, where stage (c) is performed by filtering in a tangential flow. 12. The method of claim 10, wherein step (b) comprises exposing each bacterial strain of Lactobacillus to a pH of greater than 9.0, sufficient for chemical modification of the bacterial molecules. 13. The method of claim 10, further comprising treating each strain at a pH of less than 4.5 after step (b) and before step (c). 14. The method of claim 10, where the chemical modification includes 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%. 15. A pharmaceutical composition useful for reducing at least one symptom associated with at least one condition selected from a respiratory disorder, an allergic condition, an urinary tract disorder, and a digestive disorder, comprising the extract of claim 1. 16. A nutraceutical composition useful in the treatment of diseases associated with an imbalance in the production of anti-inflammatory cytokines, containing the extract according to claim 1. 17. A pharmaceutical composition useful in the treatment of diseases associated with an imbalance in the production of anti-inflammatory cytokines, containing the extract according to claim 1. 18. A method of reducing at least one symptom associated with at least one condition selected from a respiratory disorder, an allergic condition, an urinary tract disorder, and a digestive disorder, comprising administering to a subject a therapeutically effective amount of an extract according to claim 1. 19. The method of claim 18, wherein the individual is a human or a pet. 20. The extract obtained using the method according to any one of claims 10-14. 21. The extract according to claim 20, where the extract is a soluble extract.

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