JP2012502026A - Immunoregulatory extract of Lactobacillus bacteria and its production and use - Google Patents

Abstract

  The present invention includes an extract of Lactobacillus bacteria that can produce an immunomodulatory effect in a subject patient. Embodiments of the invention may be used, for example, as a dietary supplement or pharmaceutical to treat a disease, or as an adjuvant in medical treatment such as a disease caused by an imbalance in anti-inflammatory or inflammatory cytokine production. Conditions in which the extract of the present invention may be useful include infections, allergies, autoimmune diseases, and inflammation, but may also be useful as adjuvants that provide health benefits to the subject patient. The present invention also includes a method for producing and using such an extract, and particularly relates to a specific strain of the genus Lactobacillus.

Description

  Embodiments of the invention include extracts of Lactobacillus bacteria that can produce an immunomodulatory effect in a subject patient. For example, embodiments of the present invention include nutritional supplements and pharmaceuticals for treating diseases such as infections, allergies, autoimmune diseases, and diseases caused by an imbalance in anti-inflammatory or inflammatory cytokine production such as inflammation. It can also be used as an adjuvant that provides health benefits to the subject patient. The present invention also includes a method for producing and using such an extract, and relates to a specific strain of the genus Lactobacillus.

  Immunomodulation is a generic term that refers to a wide range of immune interventional treatments that alter normal or abnormal immune responses. Microorganisms produce and secrete a wide range of molecules that can modulate the eukaryotic immune response (Lavelle et al., Curr Top Med Chem. 2004, 4 (5), 499-508). And include factors that reverse protection mechanisms to promote persistence. Viral molecules, bacterial molecules and parasite-derived molecules that can inhibit the inflammatory response have been identified. In addition to microbial factors that can suppress the immune response, powerful immune activators can themselves be of microbial origin. These include bacterial enterotoxins, parasite-derived excretion / secretions, and viral nucleic acids.

  Called Toll-like receptors (TLRs), a family of at least 11 receptors expressed by its host organism is believed to play an important role in immunological detection and innate responses to microorganisms . FIG. 1 provides a list of TLR ligands (Gay and Gangloff, Ann. Rev. Biochem., 2007, 76: 141-65). TLR recognizes a wide range of molecules, also known as pathogen-related molecular patterns (PAMPs) produced by viruses, bacteria and fungi (Tse and Horner, Ann Rheum Dis. 2007 Nov; 66 Suppl 3: iii77-80) . TLR-mediated immunomodulation has been applied to the development of new therapies in a wide range of pathologies including infectious diseases, malignant diseases, autoimmune diseases and allergic diseases.

TLR agonists and antagonists are being investigated as potential treatments for the prevention and treatment of disease. In relatively small clinical trials, TLR agonists are used as vaccine adjuvants for the purpose of preventing infections, eliminating allergic hypersensitivity and eliminating malignant cells. TLR agonists are also being investigated as monotherapy and adjuvant therapy for the treatment of patients with infectious diseases, allergic diseases and malignant diseases. The use of TLR antagonists has also been studied in preclinical and clinical trials as a potential treatment for autoimmune diseases and sepsis.
Probiotics are live microorganisms that can provide health benefits to target patients when administered in appropriate doses (Mottet et al. Digestive and Liver Disease, 2005, 37: 3-6; Ezendam et al. Nutr (2006 (Revised January), 64 (1): 1-14; Gill and Prasad, Adv Exp Med Biol, 2008, 606: 423-54). Biological mechanisms involved in stimulating immune responses by probiotic microorganisms and specific cellular components of these microorganisms are the subject of research. For example, Gram positive bacteria have a characteristic cell wall that contains macromolecules such as lipoteichoic acid (LTA). LTA can be associated with immunostimulation (eg, 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 can include various TLR ligands with immunomodulatory properties. Cell wall fragments obtained from various Bifidobacterial strains were found to stimulate interferon gamma (IFN-γ) production in vitro in mouse 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 of particulate cell wall fragments of certain lactic acid bacteria (Del-Immune VR, Pure Research Products, Colorado, USA) are also intended to stimulate the immune system.

Lavelle et al. Curr Top Med Chem. 2004, 4 (5), 499-508 Gay and Gangloff, Ann. Rev. Biochem., 2007, 76: 141-65 Tse and Horner, Ann Rheum Dis. 2007 Nov; 66 Suppl 3: iii77-80 Mottet et al. Digestive and Liver Disease, 2005, 37: 3-6 Gill and Prasad, Adv Exp Med Biol, 2008, 606: 423-54 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 Deininger et al., Clin Vaccine Immunol, 2007, 14 (2): 1629-1633 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

  Ingestion of probiotic bacteria in active or inactive forms, or uptake of particulate cell wall fragments of such bacteria, may not be the most effective way to provide an immunomodulatory effect to a subject patient. In fact, live cell extracts can contain so much protein and lipopeptides that they prevent efficient absorption by the subject patient, thus limiting the local concentration of useful molecules from probiotic bacteria in the subject. The condition in the subject can also destroy the active bacterial components, or conversely, modify the chemical structure of those components to inactivate the active bacterial components. Risks associated with oral administration of raw probiotic microorganisms (Lactobacillus) include bacteremia and sepsis (Lactobacillus Sepsis Associated With Probiotic Therapy, Pediatrics (January 2005) 115 (1): 178-181 ). Therefore, there is a need for other means of providing the beneficial effects of probiotic bacteria to target patients in need thereof.

  The present invention relates to Lactobacillus extracts, and some embodiments of the present invention may exhibit potent immunomodulatory activity. For example, embodiments of the invention relate to extracts from bacterial strains that may be useful as dietary supplements or pharmaceuticals, in some cases treating infectious diseases, allergies, respiratory diseases, and inflammatory pathologies. Or an extract that may serve as an adjunct in connection with a treatment protocol. The present invention also relates to a composition comprising the extract and a process for producing the extract, for example, a process using a medium that does not pose a risk of prion disease. The process according to the present invention includes, for example, cell lysis under alkaline conditions or alkaline conditions following acidic conditions. In some embodiments, the extract of the present invention is a soluble extract, ie, an extract that does not contain a significant amount of solids or particulate matter. In some embodiments, the extract contains a chemically modified TLR ligand. In some embodiments, alkaline treatment may cause chemical modification of cellular material including TLR ligands, cell wall components, proteins, lipoteichoic acid, lipopeptides and phospholipids.

Some embodiments of the invention may include extracts obtained from one or more of the following species:
Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus casei (defensis), Lactobacillus casei subspecies casei, Lactobacillus paracasei, Lactobacillus Bulgaricus, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus salivaius, Lactobacillus lactis, and Lactobacillus delbrecchi.
In some embodiments, the extract comprises at least one strain of the bacterial species, while in other embodiments one or more specific strains of the list are removed or one or more Of different strains. Some embodiments of the present invention include extracts 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 strain is stored according to the Budapest Treaty. Lactobacillus fermentum I-3929, Lactobacillus rhamnosus 71.38, Lactobacillus plantarum 71.39, Lactobacillus johnsonii 103782, and Lactobacillus helveticus 103146, Collection Nationale de Culture des Microorganismes at the Institut Pasteur ( 25 rue du Dr. Roux, 75724 Paris, France). Lactobacillus fermentum I-3929 was deposited on February 27, 2008. Other strains are included in the collection of the repository and can be obtained by contacting the repository.

  In addition, the present invention particularly relates to the strain Lactobacillus fermentum I-3929, an extract obtained from the strain, a method for producing the extract, and a method for using the extract. The strain was obtained by exchanging chromosomes of strains from Lactobacillus plantarum and Lactobacillus fermentum to produce a novel Lactobacillus genus strain. The extract obtained from Lactobacillus fermentum I-3929 was found to be active in several different in-vivo and in-vitro models that correlate with infectious and immunodeficiency diseases.

  In some embodiments, the extract is obtained from only one specific bacterial strain. Alternatively, one or more strains may be used. In other embodiments, one or more extracts from different types of microorganisms, such as non-lactic bacteria species may be added.

  The extract can be obtained by lysing bacterial cells under specific conditions after growing them to an appropriate concentration in a medium. In some embodiments, the bacteria are grown in media that does not pose a risk of prion-related disease or other diseases that can be transmitted by ingesting products from animal-derived media. For example, in some embodiments, the cells are grown using a vegetable-derived medium, such as a soybean-derived medium.

  In addition, the solubilized solution (lysate) (that is, the product of cell lysis) can be filtered to remove large cell debris such as nucleic acids, insoluble substances and particulate substances. In some embodiments, the amount of nucleic acid present in the extract is less than 100 · g / mL. Thus, in some embodiments, the resulting extract contains soluble molecular components and does not contain significant amounts of insoluble or particulate matter.

  Membrane molecules and cell wall molecules may be dissolved or suspended in extracts containing lipoproteins, lipopeptides, peptidoglycans, lipooligosaccharides, lipoteichoic acid, and teichoic acid. During the lysis treatment, intracellular molecules such as membrane molecules and cell wall molecules can be chemically modified by alkali treatment (eg, cleaved into small structures). Despite such chemical modifications, embodiments of the invention may retain biological activity comparable to whole cells or exert enhanced biological activity over whole cells.

  For example, alkaline treatment can be used to lyse cells, or alkaline treatment can be applied to cells previously lysed by another method. During alkaline treatment according to some embodiments of the invention, L-amino acids found in natural proteins and lipopeptides are at least partially racemized to D-amino acids. D-amino acids are beneficial in increasing the shelf life of the extract because they are not effectively digested in the mammalian intestine. D-amino acids can also protect small peptides and proteins from degradation during digestion. Examples of D-amino acids include D-amino acids bound to proteins, to a lesser extent lysinoalanine (de Vrese et al. J Nutrition, 2000, 2026-2031). Thus, in some embodiments, antigen molecules in extracts that are chemically modified upon dissolution to contain D-amino acids remain longer in the patient's body for a more potent immunostimulatory effect. Can demonstrate.

  In some embodiments, the filtering may result in extraction of the filter because the pore size of the filter, and in some cases, the chemical properties of the filter surface (ie, its polarity) may change the type of material that is removed and retained. It can also affect the properties of things. For example, some embodiments of the invention use a filtering process that is designed to retain certain molecules, but to remove other molecular components such as nucleic acids and insoluble or particulate matter.

  The filtered extract can be further purified by organic extraction, organic aqueous extraction, chromatography, ultracentrifugation, ultrafiltration, or a combination thereof.

11 shows 11 Toll-like receptor (TLR) family ligands expressed by the host organism. Continuation of Figure 1a Figure 2 shows a diagram of a tangential flow filtration (TFF) system for the preparation of bacterial extracts after lysis of bacteria. This diagram shows two different filter configurations: a parallel mode in which all filters operate simultaneously, and a serpentine mode in which the filters are configured in series mode. General correlation between flow parameters and operating parameters indicating areas of pressure control and mass transfer control for tangential flow filtration (TFF). Figure 5 shows stimulation of splenocytes cultured for 48 hours in the presence of different dilutions of lysate (lysate) AFer300, CFer300, and DFer300, and ARahr300, CRahr300, and DRahr300. After addition of 30 μl / well Alamar blue® solution diluted 1: 1 in cell culture medium, the cells were further incubated (a) 8.5 hours (first experiment); (b) 24 hours (second experiment). . The figure shows the average luminescence value at 590 nm ± overlap culture standard deviation. Continuation of Figure 4a (A) Induction of nitric oxide (NO) production in mice treated with extracts of Lactobacillus fermentum I-3929 and Lactobacillus rhamnosus 71.38 in first assay and (b) second assay . The results were expressed as nitric oxide (NO) amount in μM as an average value ± standard deviation. The result which measured the effect with respect to airway hypersensitivity (AHR) of the extract of this invention by whole body plethysmography (Emka) in the state which increased the inhaled methacholine concentration on the 1st after the last antigen challenge. Results (mean Penh +/− standard error of mean) are negative control animals (n = 4) treated with phosphate buffered saline (PBS), untreated LACK challenge animals (positive control group) , N = 8), OM-1009A-treated LACK challenged mice (n = 8), and OM-1009B-treated LACK challenged mice (n = 7).

Definition

  Extract: An extract refers to a substance obtained after lysis of one or more bacterial strains, as defined herein. In some cases, the extract is obtained from only one strain, while in other cases, the extract is obtained from a mixture of some different strains.

  In some cases, the extract is a soluble extract, meaning that it does not contain significant amounts of particulate matter or insoluble matter such as particulate fragments or solid cell wall fragments. Instead, components from cell walls, organelles, and cell membranes can be included in the extract as long as they are lysed or suspended. For example, the extract may be processed to remove particulate matter or insoluble matter, such as by filtration, centrifugation, or another separation technique.

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

  Lysate: As used herein, the term refers to an extract obtained from a bacterial cell lysis procedure.

  Filtration: Filtering, as described herein, is an extract or extraction with one or more filters, such as a microfiltration membrane (ie, microfiltration) or an ultrafiltration membrane (ie, ultrafiltration). It means the passage of a mixture of products. Such filtration does not necessarily remove 100% of the components as designed, but in some embodiments may provide an extract that is substantially free of those components. In some cases, the filtration is repeated in multiple passes or cycles.

  Initial pH: This term refers to the pH value measured at the beginning of a procedure such as lysis or filtration of bacteria.

  Saccharides: Saccharides include not only monosaccharides and disaccharides, but also relatively large saccharides such as linear polysaccharides and branched polysaccharides, as defined herein. Sugars also include substituted or chemically modified sugars such as lipopolysaccharide (LPS) and chemically modified variants thereof.

  Lipoprotein: This term refers to a macromolecule comprising both a protein chain or peptide chain and a lipid, eg, a protein or peptide covalently linked to a lipid. Lipoproteins also include lipopeptides as used herein.

  Peptidoglycan: This term refers to a polymer containing sugars and amino acids.

  Lipotycoic acid (LTA): This term refers to a surface-associated adherent amphiphilic molecule present in Gram-positive bacterial strains.

  Teicoic acid: This term refers to a polymer of glycerol phosphate or ribitol phosphate linked via a phosphodiester bond.

  D-amino acid: This term refers to an amino acid that is present in the dextrorotatory isomer as opposed to a biosynthetically produced L-amino acid that is present in the levorotatory isomer.

  Racemization: This term refers to at least partial chemical modification of an L-amino acid to a D-amino acid.

  A medium that avoids the risk of prion disease is any stage of preparation of extracts that do not contain substances such as serum or meat extract taken from animals such as cattle or sheep, or any other animal that can transmit prion disease Means the medium used in Examples of such a medium include a medium derived from vegetables and a synthetic medium, and further include a medium using horse serum or a medium made of a substance collected from an animal species that does not transmit prion disease. Examples of prion diseases include, for example, mad cow disease, scrapie, and Creutzfeldt-Jakob disease.

  A non-animal-derived medium is a medium that does not contain components derived from animals. Examples include vegetable-derived (ie, vegetable) media such as soy media, and synthetic media.

  As used herein, a dietary supplement is any composition that can provide a health benefit to a subject patient immediately after administration to the subject patient, such as without a doctor's prescription. It means any composition characterized by being available to the subject patient.

  Treatment, as used herein in connection with treatment, means not only the treatment of an existing disease or disorder, but also the prevention or protection of the occurrence of a new disease or disorder.

  An adjuvant, as used herein with reference to embodiments of the present invention, refers to embodiments of the present invention that are provided to a subject patient in conjunction with a medical plan.

  “Immunomodulation”, “immunomodulatory” and like terms, as used herein, refer to subjects in a manner that provides health benefits to produce anti-inflammatory and immunostimulatory effects. By the ability to modify the patient's immune response.

  Anti-inflammatory and like terms, as used herein, refers to an immunomodulatory effect that functions to reduce inflammation.

  Immunostimulatory and like terms mean the stimulatory action of the immune system, as used herein.

  Protective immunity, as used herein, means that an embodiment is provided to a subject patient to protect the subject patient against subsequent infectious agent and allergen exposure. As a result, during exposure, the concentration of infectious agents and allergens in the subject patient is sufficiently low so that the health of the subject patient is not significantly impaired. The effective time of protection from such exposure may be limited to hours, days, weeks, or the like.

  A subject patient, as used herein, refers to any animal subject, including mammalian subjects such as humans and livestock. Livestock can also include mammals such as dogs, cats, horses, pigs, cows, sheep, goats, or other livestock, and also non-mammals such as birds such as chickens, ducks, geese, turkeys and others Can also include other livestock birds.

  The specific bacterial strain identified herein and used in the present invention is a strain obtained from the original repository described herein or its gene clone (different storage for later use) Naturally, it is common knowledge that it may include a strain that has been re-stored under the code name but is found to be genetically the same strain as the originally stored version.

  All numbers used herein are approximations taking into account the measured values, rounding off, and errors inherent in significant figures.

Extract preparation The present invention comprises an extract of one or more Lactobacillus strains, characterized in that the extract is a soluble extract, and the extract is chemically modified It contains bacterial molecules.

  The extract of the present invention can be prepared, for example, by culturing cells and then collecting, lysing and purifying the resulting biomass. To obtain a sufficient amount of material for each strain, the fermentation culture can be started from an effective seed lot and subsequently seeded in a large fermentation vessel.

  The medium used may be the same for each bacterial species. In some embodiments, media that avoids the risk of prion disease may be used for the growth of all strains used.

  After fermentation, the resulting biomass from a strain or series of strains can also be inactivated by heat treatment, concentration, and freezing. Thus, the starting material used to form the extract may be insoluble whole cells in some embodiments.

  In other embodiments, the starting material used to prepare the extract can be biomass obtained from cells that have been at least partially mechanically, enzymatically or chemically lysed. In yet other embodiments, the starting material can be a fraction of previously lysed cells, such as a cell wall containing the fraction.

  In some embodiments, the starting material is treated with an alkaline medium such as a strong base, a hydroxide, or other strong inorganic or organic base. In this lysis or base treatment step, undissolved cells in the starting material are lysed in some embodiments while cellular components can be chemically modified. Thus, in some embodiments, chemically modified bacterial molecules are obtained by base treatment, such as strong base treatment of one or more Lactobacillus strains, from which said extract is Obtained (ie, base treatment of components and fractions of undissolved or bacterial cells as described above).

  In some embodiments, a biomass dry weight concentration of 2-90 g / L (eg, 3, 5, 10, 15, 20, 25, 30 such as about 2 to about 80 g / L, or about 3 to about 40 g / L). , 35, or 40 g / L, or about 5-50 g / L, or other concentration ranges limited by the above concentrations) may be subject to base treatment. In some embodiments, about 40 to about 80 g / L (e.g., 40, 50, 60, 70, or 80 g / L, or a concentration range limited by the above concentrations) is subject to base treatment.

  The biomass dry weight is here defined by the dry weight of the substance (grams per liter of sample). Such dry weight can be measured by drying the sample in a small earthenware dish at about 105 ° C. until a constant mass is reached.

  30-60 degreeC (For example, 30-55 degreeC, 30-50 degreeC, 30-45 degreeC, 30-40 degreeC etc., or 30-35 degreeC) may be sufficient as the said temperature. In some embodiments, the base treatment temperature may be 35-60 ° C. (eg, 35-55 ° C., 35-50 ° C., 35-45 ° C., or 35-40 ° 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. Moreover, it may be a temperature range defined by the above temperature.

  Base treatment time ranges from 2 hours to several days (eg, 1, 2, 3, 4, 5 days or 10 days, etc., or 3 to 120 hours, or 3 to 48 hours, 3, 5, 8, 15, 14, 16 , 18, 20, 22, 24, 26, 28, 30, 36, 40, 44, or 48 hours, or 15-120 hours, 60-120 hours, 60, 72, 84, 96, 108 hours, or 120 hours Or a time defined in the above range). It will be appreciated that these time ranges include fractions of days, hours, or minutes.

  In some embodiments, a strong base concentration of 0.001N to 1.0N (eg, 0.001N to 0.6N, or 0.10N to 0.8N, or 0.6N to 1.0N, or the start of a range Value or end value is in the range of 0.001, 0.002, 0.003, or 0.1N, or 0.1N to 0.6N, or the start or end value of the range is 0.6, 0.7, 0.8, 0.9, 1.0, or 1.0 N range, or other ranges limited by the above concentrations, etc.) are used. In some embodiments, an initial pH greater than 9.0, or an initial pH greater than 9.5, an initial pH greater than 10.0 but less than 13.5 (eg, an initial pH greater than 11.5, 12. A base concentration is used that achieves an initial pH greater than 0, an initial pH greater than 12.5, an initial pH greater than 13.0, or an initial pH between pH 9.0 and pH 13.5. In still other embodiments, base concentrations can be used, for example, to achieve an initial pH of greater than 10.0 but less than 13.0, or an initial pH of pH 9.0 to pH 13.0.

  In some embodiments, the pH during base treatment can be reduced at the time of extraction of the soluble component. For example, the initial pH may be a base pH (eg, pH 9.0 to pH 13.0, or pH 9.5 to pH 12.5). The base treatment is allowed to be performed only for a certain period (for example, 3 to 120 hours, 3 to 48 hours, or the above period at the above temperature). Then, in some embodiments, the pH can also be acidified if desired. For example, 2.0 to 4.5 pH, or 2.5 to 4.5 pH, or 2.5 to 4.0 pH (2.5, 3.0, 3.5, 4.0 pH, etc.), or the above pH Hydrochloric acid may be added so as to achieve the range defined by any of the above. The second treatment is a low pH, temperature range 30-60 ° C, 35-55 ° C, or 35-45 ° C (eg, 35 ° C, 36 ° C, 37 ° C, 38 ° C, 39 ° C, 40 ° C, 41 ° C, 42 ° C, 43 ° C, 44 ° C) or 45 ° C. The acid treatment time may range from 1 hour to several hours (up to 72 hours), for example, 1 hour to 24 hours, or 1 hour to 6 hours, or 3 hours to 48 hours, or 3 hours to It may be 24 hours, or 4 to 72 hours, or 24 hours to 72 hours, or any time range limited by the above times.

  In some embodiments of the invention, alkaline treatment is performed on bacterial biomass (eg, consisting of material from Lactobacillus fermentum and having a biomass dry weight of 10 g / L to 40 g / L). In another embodiment, the alkali treatment is performed on bacterial biomass comprising a mixture of Lactobacillus strains and having a biomass dry weight of 10 g / L to 40 g / L. In such an embodiment, the alkali treatment may be performed at a hydroxide ion concentration of 0.025N to 0.25N or at a temperature of 35 to 45 ° C. and a pH of 9.5 to 12.5 for 3 hours to 48 hours. . In some embodiments, the alkali treatment comprises one or more Lactobacillus strains (hydroxide ion concentrations of 0.025N to 0.20N, 0.025 to 0.15N, 0.025 to 0.10N). 0.05N to 0.25N, 0.05N to 0.20N, 0.05 to 0.15N, 0.05N to 0.10N, 0.10N to 0.25N, 0.10N to 0.20N, 0 .10N to 0.15N, 0.15N to 0.25N, 0.15N to 0.20N, or 0.20N to 0.25N). The pH of such embodiments is, for example, 9.5 to 12.0, 9.5 to 11.5, 9.5 to 11.0, 9.5 to 10.5, 9.5 to 10.0, 10 1.0 to 12.5, 10.0 to 12.0, 10.0 to 11.5, 10.0 to 11.0, 10.0 to 10.5, 10.5 to 12.5, 10.5 -12.0, 11.5 to 11.5, 10.5 to 11.0, 11.0 to 12.5, 11.0 to 12.0, 11.0 to 11.5, 11.5 to 12 1.5, 11.5 to 12.0, or pH 12.0 to 12.5. The alkali treatment time of this embodiment is 3 hours to 36 hours, 3 hours to 24 hours, 3 hours to 18 hours, 3 hours to 12 hours, 3 hours to 6 hours, 6 hours to 48 hours, 6 hours to 36 hours. 6 hours to 24 hours, 6 hours to 18 hours, 6 hours to 12 hours, 6 hours to 8 hours, 8 hours to 48 hours, 8 hours to 36 hours, 8 hours to 24 hours, 8 hours to 18 hours, 8 Time ~ 12 hours, 12 hours ~ 48 hours, 12 hours ~ 36 hours, 12 hours ~ 18 hours, 18 hours ~ 48 hours, 18 hours ~ 36 hours, 18 hours ~ 24 hours, 24 hours ~ 48 hours, 24 hours ~ It may be 36 hours, or 36 to 48 hours. The alkali treatment may be performed for any period (eg, 3, 6, 8, 12, 18, 24, 36, or 48 hours) limited to the above time range. Under these conditions, a moderate alkali treatment is obtained.

  In another embodiment, biomass dry weights of 10 g / L and 40 g / L from one or more Lactobacillus strains have a hydroxide ion concentration of 0. May be exposed to 15N to 0.50N or pH 11.5 to 13.5. For example, in some embodiments, the hydroxide concentration is 0.15N to 0.45N, 0.15N to 0.40N, 0.15N to 0.35N, 0.15N to 0.30N, 0.35N. 15N to 0.25N, 0.15N to 0.20N, 0.20N to 0.50N, 0.20N to 0.40N, 0.20N to 0.30N, 0.25N to 0.50N, 0.30N It may be 0.50N, 0.30N to 0.40N, or 0.40N to 0.50. Such embodiments include, for example, pH 11.5 to 13.0, 11.5 to 12.5, 11.5 to 12.0, 12.0 to 13.5, 12.0 to 13.0, 12.0. ˜12.5, 12.5 to 13.5, 12.5 to 13.0, 13.0 to 13.5. The alkali treatment period is, for example, 15 hours to 100 hours, 15 hours to 90 hours, 15 hours to 75 hours, 15 hours to 60 hours, 15 hours to 48 hours, 15 hours to 36 hours, 24 hours to 120 hours, 24 hours. Time-100 hours, 24 hours-90 hours, 24 hours-75 hours, 24 hours-60 hours, 24 hours-48 hours, 36 hours-120 hours, 36 hours-100 hours, 36 hours-90 hours, 36 hours- 75 hours, 36 hours to 60 hours, 36 hours to 48 hours, 48 hours to 120 hours, 48 hours to 100 hours, 48 hours to 90 hours, 48 hours to 75 hours, 48 hours to 60 hours, 60 hours to 120 hours 60 hours to 100 hours, 60 hours to 90 hours, 60 hours to 75 hours, 75 hours to 120 hours, 75 hours to 100 hours, 75 hours to 90 hours, 90 hours to 120 hours During or a 100 to 120 hours. In addition, the alkali treatment period in such an embodiment includes 15, 24, 48, 60, 75, 90, 100, and 120 hours. Under such conditions, strong alkali treatment can be achieved.

  In other embodiments, the starting biomass dry weight of 10 g / L to 40 g / L is a hydroxide concentration of 0.025 N to 0.25 N or a pH of 9.5 to 12 at a temperature of 35 to 45 ° C. for 3 hours to 48 hours. 5 can also be processed. The pH may then be adjusted to 2.5-4.0 (including acid treatment) by adding an acid such as hydrochloric acid (HCl). The acid treatment may be performed at a temperature of 35 to 45 ° C. for 1 to 24 hours. For example, in such embodiments, alkaline treatment of bacterial biomass comprising one or more Lactobacillus strains may be performed at hydroxide concentrations of 0.025N to 0.20N, 0.025 to 0.15N, or 0.025 to 0. .10N, 0.05N to 0.25N, 0.05N to 0.20N, 0.05 to 0.15N, 0.05N to 0.10N, 0.10N to 0.25N, 0.10N to 0.20N , 0.10N to 0.15N, 0.15N to 0.25N, 0.15N to 0.20N, or 0.20N to 0.25N. During the alkali treatment, such embodiments have pH 9.5 to 12.0, 9.5 to 11.5, 9.5 to 11.0, 9.5 to 10.5, 9.5 to 10.0, 10.0 to 12.5, 10.0 to 12.0, 10.0 to 11.5, 10.0 to 11.0, 10.0 to 10.5, 10.5 to 12.5, 10. 5-12.0, 10.5-11.5, 10.5-11.0, 11.0-12.5, 11.0-12.0, 11.0-11.5, 11.5 It may have 12.5, 11.5 to 12.0, or pH 12.0 to 12.5. The alkali treatment time of this embodiment is 3 hours to 36 hours, 3 hours to 24 hours, 3 hours to 18 hours, 3 hours to 12 hours, 3 hours to 6 hours, 6 hours to 48 hours, 6 hours to 36 hours. 6 hours to 24 hours, 6 hours to 18 hours, 6 hours to 12 hours, 6 hours to 8 hours, 8 hours to 48 hours, 8 hours to 36 hours, 8 hours to 24 hours, 8 hours to 18 hours, 8 Time ~ 12 hours, 12 hours ~ 48 hours, 12 hours ~ 36 hours, 12 hours ~ 18 hours, 18 hours ~ 48 hours, 18 hours ~ 36 hours, 18 hours ~ 24 hours, 24 hours ~ 48 hours, 24 hours ~ It may be 36 hours, or 36 to 48 hours. The alkali treatment may be performed for any period (eg, 3, 6, 8, 12, 18, 24, 36, or 48 hours) limited to the above time range. The pH is then adjusted to 2.5-3.5, 2.5-3.0, 3.0-4.0, 3.0-3.0 by adding an acid for acid treatment after alkali dissolution. You may adjust to 3.5 or 3.5 to 4.0. The acid treatment is performed for 1 hour to 18 hours, 1 hour to 12 hours, 1 hour to 6 hours, 1 hour to 3 hours, 3 hours to 24 hours, 3 hours to 18 hours, 3 hours to 12 hours, 3 hours to 3 hours to It can be performed over 6 hours, 6 hours to 24 hours, 6 hours to 18 hours, 6 hours to 12 hours, 12 hours to 24 hours, 12 hours to 18 hours, 18 hours to 24 hours. In addition, 1, 3, 6, 12, 18, and 24 hours are also included in the consideration of the acid treatment time.

  The solubilized solution (lysate) obtained as a result of the base treatment can then be purified by, for example, centrifugation or filtration in order to remove particulate components and insoluble components. For example, the lysate (lysate) can be centrifuged at 9000 × gravity and then filtered one or more times with a 0.2 micron filter. In some cases, a method of filtering with a large pore filter several times continuously and then filtering with a 0.2 micron filter may be used. In order to support the extraction of soluble substances from the extract, an ultrafiltration method, for example, a method of recirculating the ultrafiltration permeate for further microfiltration can be employed.

  In some embodiments, tangential flow filtration (TFF) methods can be used to filter lysate (lysate) and extract soluble molecules from relatively large cell debris (FIG. 2). See, for example, Separations Technology, Pharmaceutical and Biotechnology Applications, Wayne P. Olson (Editor) Interpharm Press, Inc., (Buffalo Grove, Ill., USA), pages 126-135-ISBN: 0-935184-72-4 . At the beginning of such treatment, the diluted bacterial lysate may be stored in the first tank. In TFF, for example, the extract may be exposed to both a microfiltration membrane and an ultrafiltration membrane. For example, a microfiltration (MF) loop is started and the product is pumped out. The resulting MF residue is recycled while the MF permeate is moved to the second tank.

  After reaching a moderate concentration, an ultrafiltration (UF) loop is started. Since the UF residue is stored in the second tank and at the same time, the extract solubilized from the solubilized liquid is continuously extracted, the UF permeate can be recycled and returned to the first tank. During the continuous extraction, the amount of liquid in the tank 1 and the tank 2 can be adjusted by controlling the flow rates of the microfiltration permeate and the ultrafiltration permeate.

  Such multiple extraction cycles can be performed using either TFF or another filtration method. In an embodiment using the TFF method, at the end of the final cycle, the ultrafiltration loop can be interrupted, the microfiltration loop can be run alone, and the MF permeate can be moved to vessel 2.

  The conditions for crossflow and transmembrane pressure are determined according to the membrane theory explained above, for example, by M.Cheryan (Ultrafiltration and Microfiltration Handbook, 2nd edition, Chapter 4, 1998). It is prescribed. The permeation flow rate and extraction yield are affected by filtration conditions (transmembrane pressure (TMP), crossflow, temperature, etc.). The filter type can affect not only the filtration performance but also the type of plate system (cassette filter). Various configurations can be used including parallel mode and serpentine mode (see FIG. 2). Specific conditions have been developed to achieve optimized performance for each combination of mode type and filter type used.

  The microfiltration loop can also be attached to a 1.2 micron to 0.1 micron filter (eg, a 0.65 to 0.2 micron or 0.45 micron filter). The cross-flow may be 100-3000 liters / hour m 2 (LHM) (eg, 300-2500 LHM, or 2000 LHM with a TMP of 0.3-2 bar). The ultrafiltration loop may be attached to a 10 KDa to 1000 KDa filter (for example, 10 KDa to 100 KDa, or 10 KDa to 30 KDa, or 30 KDa to 100 KDa). The cross-flow may be 30-1000 LHM (eg, 20-500 LHM at a TMP of 0.2-1.5 bar).

  Soluble components can also be extracted from the bacterial cell wall using a diafiltration volume of 5-20. In some embodiments, a liquid volume of 8-15 is used. Thus, for example, in some embodiments, 5-15 cycles of filtration can be used, and in some cases, 8-15 cycles of filtration can be used.

  After filtration, the extract can be further concentrated or centrifuged if desired. For example, further microfiltration may be performed using a small pore filter, such as a 0.2 micron filter. After filtration, the extract may be lyophilized prior to formulation for use.

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

Chemical properties of bacterial extracts Various chemical modifications can also be made to cellular components by base treatment. For example, in proteins: (1) peptide bonds can undergo partial cleavage to produce smaller polypeptides; (2) natural L-amino acids can be at least partially racemized to D-amino acids; and (3 ) Asparagine and glutamine residues can be deaminated to cause a change in protein isoelectric point. Molecules such as lipoteichoic acid, lipopeptides, and phospholipids undergo hydrolysis catalyzed by ester- or amide-bonded bases to obtain new physicochemical and immunological properties Can cause a modified amphiphilic structure. Other examples of possible chemical modifications include partial solubilization of cell wall polysaccharides, complete hydrolysis of ribonucleic acid (RNA) to individual ribonucleotides, including rearrangement of phosphate groups.

  Therefore, some or all of these chemical modifications can occur during base treatment of Lactobacillus cells, as described herein. Such molecular modifications can affect the biological activity of the extract.

  For example, the bacterial base treatment according to the present invention results in not only partial hydrolysis of proteins but also deamination of amino acids, deamination, or partial racemization from L-amino acids to D-amino acids. Can bring as. In the analytical study of the extract according to the present invention, D-aspartic acid, D-glutamic acid, D-serine, D-methionine, D-histidine, D-alanine, D-arginine, D-phenylalanine, D-tyrosine, D Respective peaks of -leucine and D-lysine were observed. The range of the proportion of D-amino acids in the bacterial species in such studies was 3% to 40%. Thus, some embodiments of the invention provide one or more selected from serine, threonine, histidine, alanine, arginine, tyrosine, phenylalanine, leucine, and ricin (eg, all of the above amino acids, or the above amino acids). Amino acids arbitrarily selected from one or more and less than all, such as alanine, phenylalanine and lysine). In some embodiments, at least 10% of one or more of the amino acids can be racemized from D-amino acids to L-amino acids. In other embodiments, at least 40% of one or more of the amino acids can be racemized.

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

  Some extracts of the present invention include Lactobacillus bacterial cell wall components and membrane components such as lipoteichoic acid, teichoic acid, peptidoglycan, or combinations thereof. In some embodiments, the components are chemically modified. Some extracts contain cell wall components or cell membrane components such as lipoproteins, and these components can be chemically modified. In some embodiments, the cell wall component or cell membrane component, eg, lipoprotein, is dissolved or suspended in the extract and is therefore not present in particulate or insoluble form.

  In addition, the extract according to the present invention comprises, for example, 10 to 100 mg / mL soluble dry weight (SDW) substance, 1 to 30 mg / mL protein (Prot.), 0.5 to 4.0 mg / mL sugar. And less than 100 μg / mL of DNA. For example, some embodiments contain about 15-35 mg / mL soluble dry weight, 3-7 mg / mL protein, 1.0-3.0 mg / mL sugar and 10-40 μg / mL DNA. . The extract according to the invention can also 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 32.4 mg / mL There are also other examples containing mL soluble dry weight, 5.8 mg / mL protein, 2.3 mg / mL sugar and less than 100 μg / mL DNA. Soluble dry weight (SDW) (g / L or mg / mL unit) is obtained by performing dissolution treatment or base treatment and obtaining 5 mL of soluble fraction obtained by drying to a constant mass in a ceramic dish at 105 ° C. Quantified.

  In some embodiments, the extract comprises at least 0.3 mg / mL saccharide (eg, 0.3-4.5 mg / mL saccharide). In some embodiments, the at least one saccharide is selected from monosaccharides, disaccharides, and polysaccharides. Some extracts of the present invention contain at least one branched polysaccharide. In some embodiments, at least one saccharide is chemically modified.

  The lysis treatment or base treatment of the bacterium according to the present invention can result in a decrease in the average molecular weight of the polymer component, for example, from 1 KDa to 300 KDa to 100 KDa, or from 1 KDa to 60 KDa to 10 KDa. In some embodiments, the extract comprises at least one protein having a molecular weight of less than 50 KDa or less than 30 KDa (eg, less than 10 KDa).

Biological activity of bacterial extracts The extract according to the invention may have immunomodulatory activity. For example, some extracts can stimulate the immune system. Some extracts may have anti-inflammatory activity. The specific effect of the extract may depend on its production conditions and the strain or strain of Lactobacillus, or the mixture of strains or strains from which the extract is obtained. Thus, some extracts according to the present invention may exhibit strong immunostimulatory properties, and thus may be useful in treating infections or assisting in such treatments, while other embodiments are weakly immunostimulatory. However, since it exhibits anti-inflammatory activity, it is useful for the treatment of inflammatory disorders such as allergies, asthma, autoimmune diseases, colitis, and inflammatory bowel diseases, or for assisting such treatment.

  Thus, some extracts according to the present invention may be effective in treating patients suffering from disorders including, but not limited to, microbial infections, allergic diseases, and gastrointestinal disorders. Also, some extracts according to the present invention may be administered to patients as a dietary supplement (eg, treatment of various conditions including, but not limited to, microbial infections, allergic diseases, and gastrointestinal disorders) As an adjuvant).

  The range of biological activity of the extract can also be quantified by several in-vitro and in-vivo test methods. For example, the Alamar blue ™ assay incorporates a fluorometric / colorimetric growth index based on detection of metabolic activity by a redox (REOX) index in response to chemical reduction resulting from cell proliferation. (Example 4).

  The in-vitro cell assay tests nitric oxide (NO) production from primary mouse macrophages and screens the extract's ability to kill invading bacteria to stimulate the immune system (FIG. 5). In some embodiments, the extract may stimulate NO production in mouse macrophages, causing a measured NO concentration range of 3 μM to 60 μM (eg, 5 μM to 40 μM). In some embodiments, the nitric oxide concentration can be 30 μM or higher. Also, the type of bacteria can affect these results. For example, an extract from Lactobacillus fermentum can induce higher nitric oxide production than Lactobacillus rhamnosus (see, eg, under Example 5). In order to screen in-vitro for possible immunostimulation or anti-inflammatory of embodiments of the present invention, the bacterial extract of the present invention can also be tested on human peripheral blood mononuclear cells (PBMC). See Foligne et al. (World J Gastroenterol, 2007, 13 (2): 236-243). While the luminescence of both IL12p70 (inflammatory cytokine) and IL10 (anti-inflammatory cytokine) can be measured, the IL10 / IL12 ratio can also be calculated (Example 6). Some embodiments of the present invention produce higher IL10 / IL12 ratios than raw Lactobacillus fermentum control, so when some extracts of the present invention are administered in-vivo, Suggests that it may be equivalent to or more potent anti-inflammatory properties of the parental microorganism. Thus, the present invention includes an extract that can achieve a calculated IL10 / IL12 ratio in human peripheral blood mononuclear cells, wherein the ratio is achieved by a live Lactobacillus strain from which the extract can be obtained. It is characterized by being equal to or exceeding the IL10 / IL12 ratio.

  The immune response of the extract of the present invention can also be tested by examining its effect on Toll-like receptors (TLR). For example, extracts can also be tested in HEK293 cells in the presence or absence of the TLR2 agonist Pam3Cys, or in the presence or absence of the TLR4 agonist LPS (Example 7). The HEK293 cell line allows for efficient monitoring of TLR activity using ELISA assays such as IL8 titration and reporter-based systems that monitor TLR-induced NF-B activation. Some embodiments of the invention may serve as a TLR2 / 6 antagonist in HEKTLR2 / 6 cells. Thus, while some embodiments may be useful in combating infection in a subject patient, other embodiments may be developed for use in the treatment of inflammation or autoimmune diseases.

  The extracts disclosed in the present invention can also be screened for TLR and NOD2 receptor activity (Example 8). Some embodiments of the present invention activate TLR and / or NOD2 receptors in-vitro, but this points out that they can also activate the immune system via TLR or NOD2. is there.

  Non-specific stimulation of B lymphocytes can also be assessed using the plaque forming cell (PFC) technique (Example 9). Certain lymphoid cells diffuse and form lytic plaques in the presence of complement, causing the hemolytic antibody responsible for adjacent erythrocyte lysis to emit light. Some embodiments of the invention may be able to be used prophylactically to prime the immune system of a subject patient suffering from recurrent infections, because B cells may increase immunoglobulin secretion. .

  The anti-infective effect of the extract of the present invention can be tested, for example, immediately after a Salmonella food poisoning in a subject patient or immediately after such an infection in a mouse (Example 10). Some embodiments of the present invention may provide protective immunity against bacterial infections, ie infections such as Salmonella food poisoning. For example, some embodiments may reduce mortality in mice induced by infusion of Salmonella typhimurium.

  A combination of quantification of measured in-vivo activity without in-vitro activity, such as in a mouse model of allergen-induced asthma (Example 11), is the potential clinical activity of the extract disclosed in the present invention. Can provide a more complete view of In the LACK (protein from large parasite Leishmania) model, the number of eosinophils found in bronchoalveolar lavage fluid is 1-10 times (eg, 1) compared to asthmatic control (untreated) animals. (5 times to 5 times). Thus, in some embodiments, the extract provides the number of eosinophil cells, neutrophil cells, lymphocyte cells, or any combination thereof in an asthmatic mouse subject as untreated asthma. Reduce by at least 1.5 times relative to the control group. Some embodiments of the present invention may lower eosinophils in the subject asthma patient and concomitantly reduce the concentration of Th2 cytokines (IL4, IL5, IL13, etc.) that are asthma markers. Thus, such embodiments may have anti-inflammatory activity in a subject patient suffering from an immunodeficiency such as, for example, an allergic disorder including asthma.

Compositions Containing Bacterial Extracts Extracts according to the present invention can be prepared in a number of final modes of administration. For example, not only oral tablets, capsules and pills but also liquids or aerosols can be prepared. Infusions and injectable preparations can also be prepared.

  Embodiments of the invention can be prepared, for example, as a solid dosage form or a liquid dosage form. Typical solid dosage forms can include, for example, tablets (eg, coated tablets, chewable tablets, effervescent tablets, sublingual tablets, granules, powders, or capsules) containing the extract.

  The solid dosage form can also contain diluents, fillers, or other excipients. Other excipient components such as preservatives, colorants, seasonings, and sweeteners can also be added.

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

  The extract of the present invention may also include one or more dietary supplement compositions, such as nutritional supplements or dietary supplements and food additives, or one or more pharmaceutical compositions.

Administration of a bacterial extract to a subject patient A dose comprising at least one extract of the present invention suffers from or develops at least one disorder selected from gastrointestinal disorders, airway disorders, urinary tract diseases, and allergic conditions. May be administered to a subject patient at risk of For example, in some embodiments, the extract comprises upper and lower respiratory tract infection, acute obstructive pulmonary disease with lower respiratory tract infection, obstructive pulmonary disease with acute exacerbation, nasopharyngitis, sinusitis, pharyngitis , Tonsillitis, laryngitis, tracheitis, pharyngopharyngitis, influenza, pneumonia, bronchial pneumonia, bronchitis, rhinitis, nasopharyngitis, pharyngitis, sinusitis, tonsillitis, laryngitis, pharyngeal tracheitis, bronchi Bladder including inflammation, allergic rhinitis, allergic asthma, atopic dermatitis, obstructive and reflux urinary tract disease due to urinary tract infection, urethritis, tubulointerstitial nephritis, obstructive pyelonephritis, chronic cystitis Target patients suffering from or at risk of developing male pelvic pain syndrome, including prostatitis and chronic prostatitis, prostate cystitis, female pelvic inflammatory disease, Crohn's disease, or irritable bowel syndrome Can be administered.

  In some embodiments, the extract is administered to a subject patient in the form of a dietary supplement composition, such as a nutritional supplement or food additive. In another embodiment, the extract is administered to a subject patient in the form of a pharmaceutical composition. Said administration may comprise a single dose or multiple doses.

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

Example 1: Bacterial culture
Example 1.1: Culture of Lactobacillus fermentum I-3929
Initial culture conditions A culture medium was prepared by dissolving the following ingredients in purified water: sodium chloride: 3 g / L; sodium monohydrogen phosphate: 2 g / L; sodium acetate: 1 g / L; soy peptone 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; polypropylene glycol ( 0.02 mL / L, density 1.005 g / mL) was then added. The pH was not adjusted after dissolution. After sterilization of the medium, small Erlenmeyer flasks were individually inoculated with the contents of frozen vials (containing 1.5 mL of frozen bacteria) and incubated at 37 ° C. for 8 hours. A 20 mL aliquot of this culture was then transferred to a large Erlenmeyer flask containing 1000 mL of culture medium and incubated again under the same conditions. After 16 hours of growth, the contents of the 1 liter Erlenmeyer flask were transferred to the pre-fermentor.

Culture conditions in the pre-fermentor A 20 liter culture medium was prepared by dissolving the following ingredients in purified water: sodium chloride: 3 g / L; sodium monohydrogen phosphate: 2 g / L; sodium acetate: 1 g / L; soybean 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): then 0.5 mL / L of polypropylene glycol (0.02 mL / L) was added. The pH was not adjusted after dissolution. The incubation temperature was adjusted to 37 ° C. with 100 rpm stirring and no aeration. The pH was not adjusted during the culture. After 24 hours, 7 liters were transferred from the pre-fermentor to the fermentor (24 hours later, pre-fermentor culture, optical density (OD) = 1.24 at 700 nm). The pre-fermentor culture was transferred to the fermentor under sterile conditions.

Culture conditions in the fermentor 70 liters of culture medium was prepared by dissolving the following components in purified water: sodium chloride: 3 g / L; sodium monohydrogen phosphate: 2 g / L; sodium acetate: 1 g / L Soy peptone, 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: 0.1 g / L 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): then 0.5 mL / L Of polypropylene glycol (0.02 mL / L) was added. The pH was not adjusted after dissolution.

  After sterilization, 8 g / L glucose was added to the culture. The incubation temperature was adjusted to 37 ° C. with 100 rpm stirring and no aeration. The pH was adjusted to 5.7 during the culture. After 16 hours, the culture (culture, OD = 2.30 at 700 nm) was inactivated by heat treatment at 65 ° C. for 35 minutes and transferred to a collection tank. Immediately after inactivation, the culture was transferred to an ultrafiltration skid to concentrate the biomass from the medium, concentrated, and washed with sodium chloride (9 g / L) in purified water. The recovered biomass was aliquoted (mass of concentrated bacterial suspension 2000 g, 31.8 mg of biomass dry weight per gram of concentrated bacterial suspension) and then frozen at −15 ° C.

Example 1.2: Culture of Lactobacillus helveticus 103146
Initial culture conditions A culture medium was prepared by dissolving the following ingredients 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): then 0.5 mL / L polypropylene glycol (0.02 mL) / L) was added. The pH was not adjusted after dissolution. After sterilization of the medium, small Erlenmeyer flasks were individually inoculated with the contents of frozen vials (containing 1.5 mL of frozen bacteria) and incubated at 37 ° C. for 9 hours. A 20 ml aliquot of this culture was then transferred to a large Erlenmeyer flask containing 1000 mL of culture medium and incubated again under the same conditions. After 15 hours of growth, the contents of the 1 liter Erlenmeyer flask were transferred to the pre-fermentor.

Culture conditions in the pre-fermentor A 20 liter culture medium was prepared by dissolving the following ingredients in purified water: sodium chloride: 3 g / L; sodium monohydrogen phosphate: 2 g / L; sodium acetate: 1 g / L; soybean 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): then 0.5 mL / L of polypropylene glycol (0.02 mL / L) was added. The pH was not adjusted after dissolution. The incubation temperature was adjusted to 37 ° C. with 100 rpm stirring and no aeration. The pH was not adjusted during the culture. After 9 hours, 7 liters were transferred from the pre-fermentor to the fermentor. (9 hours later, prefermentor culture, OD at 700 nm: 0.14). The prefermentor culture was transferred to the fermentor under sterile conditions.

Culture conditions in the fermentor 70 liters of culture medium was prepared by dissolving the following components in purified water: sodium chloride: 3 g / L; sodium monohydrogen phosphate: 2 g / L; sodium acetate: 1 g / L Soy peptone, 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: 0.1 g / L 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): then 0.5 mL / L Of polypropylene glycol (0.02 mL / L) was added. The pH was not adjusted after dissolution.

  After sterilization, 8 g / L glucose was added to the culture. The incubation temperature was adjusted to 33 ° C. with 100 rpm stirring and no aeration. The pH was adjusted to 6.7 with CH3COOH at the beginning of the culture. After 24 hours, the culture (culture, OD = 700 at 700 nm) was inactivated by heat treatment at 65 ° C. for 35 minutes and transferred to a collection tank. Immediately after inactivation, the culture was transferred to an ultrafiltration skid to concentrate the biomass from the medium, concentrated, and washed with sodium chloride (9 g / L) in purified water. The recovered biomass was aliquoted (mass of concentrated bacterial suspension 440 g, 19.1 mg dry weight of biomass per gram of concentrated bacterial suspension) and then frozen at −15 ° C.

Example 1.3: Cultivation of Lactobacillus plantarum 71.39
Initial culture conditions A culture medium was prepared by dissolving the following ingredients 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): then 0.5 mL / L polypropylene glycol (0.02 mL) / L) was added. The pH was not adjusted after dissolution. After sterilization of the medium, small Erlenmeyer flasks were individually inoculated with the contents of frozen vials (containing 1.5 mL of frozen bacteria) and incubated at 35 ° C. for 9 hours. A 20 ml aliquot of this culture was then transferred to a large Erlenmeyer flask containing 1000 mL of culture medium and incubated again under the same conditions. After 15 hours of growth, the contents of the 1 liter Erlenmeyer flask were transferred to the pre-fermentor.

Culture conditions in the pre-fermentor A 20 liter culture medium was prepared by dissolving the following ingredients in purified water: sodium chloride: 3 g / L; sodium monohydrogen phosphate: 2 g / L; sodium acetate: 1 g / L; soybean 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): then 0.5 mL / L of polypropylene glycol (0.02 mL / L) was added. The pH was not adjusted after dissolution. The incubation temperature was adjusted to 37 ° C. with 100 rpm stirring and no aeration. The pH was not adjusted during the culture. After 9 hours, 7 liters were transferred from the pre-fermentor to the fermentor. (9 hours later, prefermentor culture, 700 nm OD = 1.62) The prefermentor culture was transferred to the fermentor under sterile conditions.

Culture conditions in the fermentor 70 liters of culture medium was prepared by dissolving the following components in purified water: sodium chloride: 3 g / L; sodium monohydrogen phosphate: 2 g / L; sodium acetate: 1 g / L Soy peptone, 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: 0.1 g / L 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): then 0.5 mL / L Of polypropylene glycol (0.02 mL / L) was added. The pH was not adjusted after dissolution.

  After sterilization, 8 g / L glucose was added to the culture. The incubation temperature was adjusted to 35 ° C. with 100 rpm stirring and no aeration. After 24 hours, the culture (culture, OD = 6.36 at 700 nm) was inactivated by heat treatment at 65 ° C. for 35 minutes and transferred to a collection tank. Immediately after inactivation, the culture was transferred to an ultrafiltration skid to concentrate the biomass from the medium, concentrated, and washed with sodium chloride (9 g / L) in purified water. The recovered biomass was aliquoted (mass of concentrated bacterial suspension 600 g, 60.7 mg biomass dry weight per gram of concentrated bacterial suspension) and then frozen at −15 ° C.

Example 1.4: Cultivation of Lactobacillus rhamnosus 71.38
Initial culture conditions A culture medium was prepared by dissolving the following ingredients 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): pH is not adjusted after dissolution of 0.5 mL / L It was. After sterilization of the medium, small Erlenmeyer flasks were individually inoculated with the contents of frozen vials (containing 1.5 mL of frozen bacteria) and incubated at 35 ° C. for 9 hours. A 20 ml aliquot of this culture was then transferred to a large Erlenmeyer flask containing 1000 mL of culture medium and incubated again under the same conditions. After 15 hours of growth, the contents of the 1 liter Erlenmeyer flask were transferred to the pre-fermentor.

Culture conditions in the pre-fermentor A 20 liter culture medium was prepared by dissolving the following ingredients in purified water: sodium chloride: 3 g / L; sodium monohydrogen phosphate: 2 g / L; sodium acetate: 1 g / L; soybean 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): then 0.5 mL / L of polypropylene glycol (0.02 mL / L) was added. The pH was not adjusted after dissolution. The incubation temperature was adjusted to 35 ° C. with 100 rpm stirring and no aeration. The pH was not adjusted during the culture. After 9 hours, 7 liters were transferred from the pre-fermentor to the fermentor. (9 hours later, pre-fermentor culture, 700 nm OD: 3.75). The pre-fermentor culture was transferred to the fermentor under sterile conditions.

Culture conditions in the fermentor 70 liters of culture medium was prepared by dissolving the following components in purified water: sodium chloride: 3 g / L; sodium monohydrogen phosphate: 2 g / L; sodium acetate: 1 g / L Soy peptone, 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: 0.1 g / L 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): then 0.5 mL / L Of polypropylene glycol (0.02 mL / L) was added. The pH was not adjusted after dissolution.

  After sterilization, 8 g / L glucose was added to the culture. The incubation temperature was adjusted to 35 ° C. with 100 rpm stirring and no aeration. After 14 hours, the culture (culture, OD = 5.43 at 700 nm) was inactivated by heat treatment at 65 ° C. for 35 minutes and transferred to a collection tank. Immediately after inactivation, the culture was transferred to an ultrafiltration skid to concentrate the biomass from the medium, concentrated, and washed with sodium chloride (9 g / L) in purified water. The recovered biomass was aliquoted (mass of concentrated bacterial suspension 2798 g, 51.7 mg biomass dry weight per gram of concentrated bacterial suspension) and then frozen at −15 ° C.

Example 1.5: Cultivation of Lactobacillus johnsonii 103782
Initial culture conditions A culture medium was prepared by dissolving the following ingredients 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): then 0.5 mL / L polypropylene glycol (0.02 mL) / L) was added. The pH was not adjusted after dissolution. After sterilization of the medium, small Erlenmeyer flasks were individually inoculated with the contents of frozen vials (containing 1.5 mL of frozen bacteria) and incubated at 33 ° C. for 10 hours. A 20 mL aliquot of this culture was then transferred to a large Erlenmeyer flask containing 1000 mL of culture medium and incubated again under the same conditions. After 14 hours of growth, the contents of the 1 liter Erlenmeyer flask were transferred to the pre-fermentor.

Culture conditions in the pre-fermentor A 20 liter culture medium was prepared by dissolving the following ingredients in purified water: sodium chloride: 3 g / L; sodium monohydrogen phosphate: 2 g / L; sodium acetate: 1 g / L; soybean 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): then 0.5 mL / L of polypropylene glycol (0.02 mL / L) was added. The pH was not adjusted after dissolution. The incubation temperature was adjusted to 35 ° C. with 100 rpm stirring and no aeration. The pH of the culture was adjusted to 5.6 using acetic acid. After 24 hours, 7 liters were transferred from the pre-fermentor to the fermentor. (24 hours later, pre-fermentor culture, 700 nm OD: 0.47). The pre-fermentor culture was transferred to the fermentor under sterile conditions.

Culture conditions in the fermentor 70 liters of culture medium was prepared by dissolving the following components in purified water: sodium chloride: 3 g / L; sodium monohydrogen phosphate: 2 g / L; sodium acetate: 1 g / L Soy peptone, 50 g / L; glucose: 12 g / L; calcium chloride: 0.1 g / L; potassium chloride: 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: 0.1 g / L 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): then 0.5 mL / L Of polypropylene glycol (0.02 mL / L) was added. The pH was not adjusted after dissolution.

  After sterilization, 8 g / L glucose was added to the culture. The incubation temperature was adjusted to 35 ° C. with 100 rpm stirring and no aeration. After 24 hours, the culture (OD = 0.174 at 700 nm) was inactivated by heat treatment at 70 ° C. for 30 minutes and transferred to a collection tank. Immediately after inactivation, the culture was transferred to an ultrafiltration skid to concentrate the biomass from the medium, concentrated, and washed with sodium chloride (9 g / L) in purified water. The recovered biomass was aliquoted (mass of concentrated bacterial suspension 61.5 g, 20.2 mg dry weight of biomass per gram of concentrated bacterial suspension) and then frozen at −15 ° C.

Example 2: Bacterial lysate
Example 2.1
The Lactobacillus fermentum I-3929 biomass from Example 1.1 containing 6 g bacterial dry weight was thawed to room temperature and then with purified water to reach 12 g / L bacterial biomass dry weight. Diluted. Alkalization was performed with 0.03 M sodium hydroxide. The pH measured at the start of dissolution was 10.3. The lysis was then incubated at 40 ° C. for 6 hours under continuous stirring. The pH after incubation was 9.9.

Example 2.2
Lactobacillus rhamnosus 71.38 biomass from Example 1.4 containing 20 g bacterial dry weight was thawed to room temperature and diluted with purified water to reach 40 g / L bacterial biomass dry weight did. Alkalization was performed with 0.03 M sodium hydroxide. The pH measured at the start of dissolution was 10.3. The lysis was then incubated at 40 ° C. for 6 hours under continuous stirring. The pH after incubation was 9.7.

Example 2.3
The Lactobacillus fermentum I-3929 biomass from Example 1.1 containing 6 g bacterial dry weight was thawed to room temperature and then with purified water to reach 12 g / L bacterial biomass dry weight. Diluted. Alkalization was performed with 0.06M sodium hydroxide. The pH measured at the start of dissolution was 12.3. The lysis was then incubated at 40 ° C. for 6 hours under continuous stirring. The pH after incubation was 11.8.

Example 2.4
The Lactobacillus fermentum I-3929 biomass from Example 1.1 containing 6 g bacterial dry weight was thawed to room temperature and then 0.2 N to reach a bacterial biomass dry weight of 12 g / L. Diluted with sodium chloride solution. The final concentration of the sodium chloride solution was 0.15N. The pH measured at the start of dissolution was 6.4. The lysis was then incubated at 40 ° C. for 6 hours under continuous stirring. The pH after incubation was 6.3.

Example 2.5
An aliquot of the solubilized solution of Example 2.1 was collected and the dissolution process continued. The liquid volume was adjusted to pH 3.6 using 25% hydrochloric acid and then incubated under static conditions at 40 ° C. for 1 hour in a water bath.

Example 2.6
An aliquot of the solubilized solution of Example 2.4 was collected and the dissolution process continued. The solution was adjusted to pH 12.4 with 10N sodium hydroxide and then incubated under static conditions at 40 ° C. for 1 hour in a water bath.

Example 2.7
The Lactobacillus plantarum 71.39 biomass from Example 1.3 containing 0.4 g bacterial dry weight was thawed to room temperature and then purified water to reach 7 g / L bacterial biomass dry weight. Diluted with Alkalization was performed with 0.06M sodium hydroxide. The pH measured at the start of dissolution was 12.6. The lysis was then incubated at 40 ° C. for 2 hours under continuous stirring. The pH after incubation was 12.4.

Example 2.8
Lactobacillus johnsonii 103782 biomass from Example 1.5 containing 0.3 g bacterial dry weight was thawed to room temperature and diluted with purified water to reach 6 g / L bacterial biomass dry weight did. Alkalization was performed with 0.06M sodium hydroxide. The pH measured at the start of dissolution was 12.5. The lysis was then incubated at 40 ° C. for 2 hours under continuous stirring. The pH after incubation was 12.3.

Example 2.9
Lactobacillus helveticus 103146 biomass from Example 1.2 containing 0.4 g bacterial dry weight was thawed to room temperature and diluted with purified water to reach 8 g / L bacterial biomass dry weight did. Alkalization was performed with 0.06M sodium hydroxide. The pH measured at the start of dissolution was 12.8. The lysis was then incubated at 40 ° C. for 2 hours under continuous stirring. The pH after incubation was 12.2.

Example 2.10
The Lactobacillus fermentum I-3929 biomass from Example 1.1 containing 6.8 g bacterial dry weight was thawed to room temperature and then purified to reach 7 g / L bacterial biomass dry weight Dilute with water. Alkalization was performed with 0.08M sodium hydroxide. The pH measured at the start of dissolution was 12.2. The lysis was then incubated at 40 ° C. for 7 hours under continuous stirring. The pH after incubation was 12.0. The pH of the dissolution was adjusted to 9.8 using hydrochloric acid.

  Soluble dry weight (SDW) contained in the lysate: 20.5 mg / g, protein (Prot): 4.9 mg / g.

Example 2.11.
The Lactobacillus fermentum I-3929 biomass from Example 1.1 containing 6.8 g bacterial dry weight was thawed to room temperature and then purified to reach 7 g / L bacterial biomass dry weight Dilute with water. Alkalization was performed with 0.15 M sodium hydroxide. The pH measured at the start of dissolution was 13.0. The lysis was then incubated at 40 ° C. for 63 hours under continuous stirring. The pH after incubation was 12.6. The pH of the dissolution was adjusted to 9.9 using hydrochloric acid.

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

Example 2.12.
The Lactobacillus fermentum I-3929 biomass from Example 1.1 containing 98 g bacterial dry weight was thawed to room temperature and then with purified water to reach 10 g / L bacterial biomass dry weight. Diluted. Alkalization was performed with 0.08M sodium hydroxide. The pH measured at the start of dissolution was 12.0. The lysate was then incubated at 40 ° C. for 24 hours under continuous stirring. The pH after incubation was 11.1. The dissolution pH was adjusted to 11.5 using sodium hydroxide.

  SDW contained in the lysate: 23.7 mg / g.

Example 2.13
The Lactobacillus fermentum I-3929 biomass from Example 1.1 containing 39 g bacterial dry weight was thawed to room temperature and then 0 to reach a bacterial biomass dry weight of 7.6 g / L. Dilute with 2N sodium chloride solution. The pH measured at the start of dissolution was 4.4. The bacterial osmotic lysate was then incubated at 40 ° C. for 24 hours under continuous stirring. The pH after incubation was 4.2. The bacterial lysate was then made alkaline by adding sodium hydroxide to reach 0.06N. The pH measured at the start of alkali dissolution was 10.6. The lysis was reincubated at 40 ° C. for 2.25 hours under continuous stirring. The pH after incubation was 10.2.

Example 3: Purification of lysate (lysate)
Example 3.1
The solubilized solution of Example 2.5 was centrifuged at 3000 × gravity for 20 minutes. The supernatant was then adjusted to pH 6.81 with sodium hydroxide, filtered through a continuous filter having a porosity of 0.45 μM to 0.2 μM, and finally filtered through a 0.22 μM sterile filter. Protein contained in the concentrate: 2.8 mg / mL; DNA: 17.4 μg / mL. D-amino acid ratio: 14.9% D-Ala, 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 : 3.3 μM, C2: 52.5 μM. Active dry weight (g / L or mg / mL unit) = soluble dry weight (g / L or mg / mL unit) -chloride content (g / L or mg / mL unit).

Example 3.2
The solubilized solution of Example 2.6 was centrifuged at 3000 × gravity for 20 minutes. The supernatant was then adjusted to pH 7 with 10N sodium hydroxide, filtered through a continuous filter having a porosity of 0.45 μM to 0.2 μM, and finally filtered through a 0.22 μm sterile filter. Protein contained in the concentrate: 12.3 mg / mL; DNA: 27.7 μg / mL. D-amino acid ratio: 15.9% D-Ala, 24.4% D-Pro, 17.4% D-Asp, 45.1% D-Ser, 10.1% D-Met .

  No production at 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: 8.0 μM, C2: 56.0 μM, C3: 94.3 μM.

Example 3.3
The 300 mL lysate of Example 2.1 was first centrifuged at 3000 × gravity for 20 minutes. The supernatant was adjusted to pH 7.1 using hydrochloric acid. The extract was concentrated on a Sartoflow RSlice 200 Benchtop Crossflow System with a 0.45 μM membrane of polyethersulfone (PES) (Sartorius Stedim Biotech GmbH) at a constant flow rate of 330-350 mL / min. The yield was 75%. The concentrate was finally sterile filtered through a PES membrane 0.2 μM (Nalgene).

  SDW contained in the concentrate: 30.0 mg / g; Prot: 9.6 mg / mL; DNA: 32.8 μg / mL. D-amino acid ratio: 14.3% D-Ala, 13.9% D-Pro, 14.1% D-Asp, 36.9% D-Ser. No production at 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: 5.1 μM, C2: 30.0 μM, C3: 64.0 μM.

Example 3.4
300 mL of the solubilized solution of Example 2.2 was adjusted to pH 7.0 using hydrochloric acid. The extract was filtered at a constant flow rate at 350 mL / min as described in Example 3.3. The yield was over 75%. The concentrate was finally sterile filtered through a PES membrane 0.2 μM (Nalgene).

  SDW contained in the concentrate: 49.2 mg / g; Prot: 14.2 mg / mL; DNA: 34.1 · g / mL. D-amino acid ratio: 16.0% D-Ala, 13.3% D-Pro, 14.2% D-Asp.

  No production at 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: 0 μM, C2: 4.1 μM, C3: 26.3 μM.

Example 3.5
The 300 mL lysate of Example 2.3 was first centrifuged at 3000 × gravity for 20 minutes. The supernatant was adjusted to pH 7.1 using hydrochloric acid. As described in Example 3.3, the extract was filtered at a constant flow rate from 330 to 350 mL / min. The yield was over 75%. The concentrate was finally sterile filtered through a PES membrane 0.2 μM (Nalgene).

  SDW contained in the concentrate: 34.5 mg / g; Prot: 8.9 mg / mL; DNA: 16.8 μg / mL. D-amino acid ratio: 16.4% D-Ala, 24.3% D-Pro, 17.3% D-Asp.

  No production at 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 from Example 2.13 was centrifuged at 9384 × gravity for 20 minutes. The supernatant was filtered by passing directly through a 0.22 μM sterile filter.

  SDW contained in the concentrate: 32.7 mg / g; Prot: 6.5 mg / g; Sugar: 2.4 mg / g. The sugar concentration was measured according to the procedure by Herbert et al. (See Meth. Microbiol, 1971, 5B: 266, below).

  As described in Example 6, screening of biological activity at various concentrations was performed on human peripheral blood mononuclear cells (PBMC). The results obtained at 0.5 mg active dry weight / mL were: TNF-α: 83 pg / mL; IL6: 898 pg / mL; IL12: 140 pg / mL (in the presence of 10 Ng / mL IFN-γ); and IL10 : 221 pg / mL (in the presence of IFN-γ).

Example 3.7
The bacterial lysate mixture of Example 2.10 was transferred to a microfiltration (MF) bath after adjusting the pH to 10.2. In the microfiltration (MF) unit, a 0.45 · M tangential flow filtration (TFF) filter (manufactured by Sartocon Slice Sartorius) was used. The cross flow was adjusted to 290 L / hm2 (LHM), and the transmembrane pressure difference (TMP) was adjusted to 0.4 to 0.5 bar. The permeate was transferred to an ultrafiltration (UF) bath.

  When the amount of the solubilized liquid in the microfiltration tank reaches half of the initial liquid volume, the UF unit is started. The permeate of the UF filter is used as a wash buffer in the MF tank. The liquid volume of both the MF tank and the UF tank was kept the same. After 10 diafiltration volumes, the UF was stopped and the bacterial lysate was concentrated in the MF tank. The recovery volume ratio was 83%. The extract recovered in the UF tank was adjusted to pH 7.3 using hydrochloric acid, and then filtered through a 0.2 · M sterilizing filter.

  SDW contained in the concentrate: 17.5 mg / g; Prot: 4.3 mg / g.

Example 3.8
The lysate of Example 2.7 was centrifuged at 9384 × gravity for 15 minutes. Then, the supernatant was adjusted to pH 7.3 with hydrochloric acid, filtered through a continuous filter having a porosity of 0.45 μM to 0.2 · M, and finally filtered through a 0.22 μM sterilizing filter.

  Peripheral blood mononuclear cells (PBMC) with 10-fold diluted product: 777 pg / mL IL10, 26 pg / mL IL12, 13183 pg / mL IL6.

Example 3.9
The lysate of Example 2.8 was centrifuged at 9384 × gravity for 15 minutes. The supernatant was then adjusted to pH 7.4 with hydrochloric acid, filtered through a continuous filter having a porosity of 0.45 μM to 0.2 μM, and finally filtered through a 0.22 μM sterile filter.

  PBMC with 10-fold diluted product: 904 pg / mL IL10, 0 pg / mL IL12, 4995 pg / mL IL6.

Example 3.10.
The lysate of Example 2.9 was centrifuged at 9384 × gravity for 15 minutes. The supernatant was then adjusted to pH 7.6 with hydrochloric acid, filtered through a continuous filter having a porosity of 0.45 μM to 0.2 μM, and finally filtered through a 0.22 μM sterile filter.

  PBMC with 10-fold diluted product: 476 pg / mL IL10, 0 pg / mL IL12, 4924 pg / mL IL6.

Example 3.11.
The solubilized solution of Example 2.11 was transferred to the MF tank after adjusting the pH to 10.4. The TFF installation was similar to Example 3.7. The cross flow was adjusted to 290 L / hm2 and the TMP was adjusted to 0.4-0.5 bar. The UF was stopped after 10 diafiltration volumes. The recovery volume ratio was 86%. The final product was adjusted to pH 7.3 using hydrochloric acid.

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

Example 3.12.
The solubilized solution of Example 2.12 was transferred to the MF tank after adjusting the pH to 11.5. The TFF installation was similar to Example 3.7. The cross flow was adjusted to 290 L / hm2 and the TMP was adjusted to 0.4 bar. The UF was stopped after 10 diafiltration volumes. The recovery volume ratio was 74%. The final product was adjusted to pH 7.2 using hydrochloric acid.

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

As described in Example 6, screening of biological activity at various concentrations was performed on human peripheral blood mononuclear cells (PBMC). The results obtained at 0.5 mg active dry weight / mL are: TNF-α: 37 pg / mL; IL6: 1092 pg / mL; IL12: 81 pg / mL (in the presence of 10 Ng / mL IFN-γ); and IL10 : 160 pg / mL (in the presence of 10 Ng / mL IFN-γ). Protein concentration was measured by DC protein assay (BioRad, DC protein assay, kit number 500-0116). A microplate assay protocol was performed. The analysis sample (0.1 mL) was diluted with 1.9 mL of 25 mM phosphate buffer pH 11. A protein standard curve was prepared each time the assay was performed with bovine serum albumin solution at 2 mg BSA / mL (BSA, Pierce reference number 23210). The BSA solution was diluted with 25 mM phosphate buffer pH 11 to obtain the following concentrations: 0.18, 0.24, 0.3, 0.36-0.42 mg BSA / mL. Sample (20 μl) and BSA standard solution (20 μl) were added in quadruplicate to a clean and dry microtiter plate. Reagent A (25 μl) from the DC protein assay kit was added to each well. After 10 minutes, 200 μl of Reagent B was added to each well. The microtiter plate was mixed for 5 seconds on a rotary shaker. After standing at room temperature for 20 minutes, the absorbance was recorded at 750 nm. The protein concentration of each sample was calculated using the linear regression slope on the protein standard curve:
OD at 750 nm = a * (protein concentration) + b
Protein in sample (mg) = [20 × (OD-b at 750 nm)] / a

  Soluble dry weight (SDW) was quantified by obtaining 5 mL of soluble fraction resulting from dissolution and drying to constant mass in a ceramic dish at 105 ° C.

Example 4: Activation of mouse splenocytes in-vitro The immunostimulatory effect in the embodiment of the present invention was measured in-vitro by measuring the activation of mouse splenocytes (Alamar blue assay).

Materials and methods
Spleen cell stimulation
The Alamar blue ™ assay is designed to quantitatively measure the proliferation of human or animal cells. This assay incorporates a fluorometric / colorimetric growth index based on the detection of metabolic activity by a redox (REOX) index in response to chemical reduction as a result of cell proliferation. The REOX index associated with growth causes a change from the oxidized (non-fluorescent, blue) form to the reduced (fluorescent, red) form.

Mouse Balb / c mice (female, 6-8 weeks old) were obtained from Charles River Laboratories (Suerfeld, Germany) and sacrificed by cervical dislocation. The spleen was homogenized using a Potter Elbegem homogenizer; the cell suspension was washed by centrifugation (280 × gravity, 10 minutes at 4 ° C.), 5% FCS, 100 U / mL penicillin and 100 μg / mL. Resuspended in 5 mL RPMI 1640 containing streptomycin. 100 μl of splenocytes (2 × 10 ⁶ / mL) were incubated for 48 hours at 37 ° C. in 96-well plates (Falcon 3072) with 50 μl of bacterial extract dilution and 5% carbon dioxide in cell medium.

  After addition of 30 μl Alamar blue ™ solution diluted 1: 1 with medium, Alamar blue ™ using Fluoroskan Ascent Reader (ThermoLabsystems, Frankfurt, Germany) with excitation at 544 nm and emission wavelength of 590 nm The chemical reduction of was measured.

Test substances 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 (29.5 mg of Lactobacillus fermentum I-3929 (12 g / L) obtained as described in Example 2.4 and purified under the same conditions as Example 3.3 Active dry weight / g). This example was used as a non-alkali dissolution control.

  ARahr300: Extract from Lactobacillus rhamnosus 71.38 (40 g / L) obtained by alkaline dissolution method with 40 M for 6 hours using 0.03 M sodium hydroxide (49.3 mg active dry weight / g);

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

  DRahr 300: Extract from Lactobacillus rhamnosus 71.38 (40 g / L) (46.2 mg active dry weight / L) dissolved in 0.15 N sodium chloride solution by osmotic stress for 6 hours at 40 ° C. g). This example was used as a non-alkali dissolution control.

Negative control distilled water or phosphate buffered saline (PBS).
result
In-vitro study to quantify splenocyte activation Increased metabolic activity of mouse splenocytes after treatment with the extract was quantified in two independent experiments with the Alamar blue assay (see FIGS. 4a and 4b).

    Spleen cells were cultured for 48 hours in the presence of the extract. Luminescence values were measured after addition of Alamar blueR solution. As shown in FIGS. 4a-4b, the bacterial extract was effective at a dilution of approximately 1: 300. All groups were statistically compared to the control group using Student's t test.

Conclusion
The immunostimulatory effect of the bacterial extract was measured in vitro by measuring the activation of mouse splenocytes using the Alamar blue assay. Here, in two independent experiments, the inventors obtained an extract containing intact particulate cell wall components (DFer300) obtained by osmotic lysis and two extracts (AFer300 and CFer300) according to the present invention. ). These three extracts were obtained from Lactobacillus fermentum I-3929. The AFer300 extract, CFer300 extract, and DFer300 extract were each effective in stimulating the metabolism of the cells at a dilution of 1: 300. The inventors have observed that there is no difference between the DFer300 osmotic dissolution control and the AFer300 and CFer300.

  The inventors also compared three extracts obtained from the second strain, Lactobacillus rhamnosus 71.38 (ARahr300, CRahr300, DRahr300). As in the previous set, ARahr300 and CRahr300 are within the scope of the present invention, while DRahr is an osmotic lysis control. Here, the ARahr300 extract was effective in two independent assays at a dilution of 1: 300, while the CRahr300 and DRahr300 extracts showed a lower effect, but at the same dilution, the 2 One of the two assays showed significant irritation. A comparison of the results of all six extracts also showed that the Lactobacillus fermentum I-3929 extract compared to the Lactobacillus rhamnosus 71.38 extract in stimulating splenocyte proliferation. , Showed more effective.

Example 5 Production of Nitric Oxide by Bone Marrow-Derived Macrophages Regarding the possibility of immunostimulation of a series of embodiments according to the present invention, measuring the production amount of nitric oxide (NO) using mouse bone marrow-derived macrophages The test was carried out.

Materials and Methods Six-week-old male C57 / BL6 mice (6-week-old male, SPF quality, manufactured by Charles Rivier, France) were killed by carbon dioxide inhalation. The tibia from its buttocks, thighs and hind limbs was removed. After cutting both ends, bone marrow was extracted from the lumen by injecting Dulbecco's modified Eagle medium (DH) through the bone. After washing, the stem cells were resuspended (40000 cells / mL) in DH medium supplemented with 20% horse serum and 30% L 929 cell supernatant. The cell suspension was incubated for 8 days at 37 ° C. in an incubator under saturated atmosphere with 8% carbon dioxide. Macrophages were then separated using 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. An aqueous solution of the extract was serially diluted in DHE medium directly in a microtiter plate. The extract of the present invention was tested in triplicate, and each microtiter plate containing the medium was used as a negative control. The final volume in each well was 100 μl. 100 μl of the cell suspension was added to the diluted extract and the cells were incubated for 22 hours at 37 ° C. in an incubator under an atmosphere saturated with 8% carbon dioxide. At the end of the incubation period, 100 μl of the supernatant was transferred to another microtiter plate and the nitrite concentration produced in each supernatant was quantified by performing a grease reaction. 100 μl of grease reagent (5 mg / mL sulfanilamide + 0.5 mg / mL N- (1-naphthyl) ethylene-diamine hydrochloride) in 2.5% aqueous phosphoric acid was added to each well. The microtiter plate was read using a spectrophotometer (SpectraMax Plus, Molecular Devices) at 562 nm against a 690 nm reference. The nitrite concentration was proportional to the nitric oxide content formed. The nitrite content was quantified based on a sodium nitrite standard curve (1-70 μM NaNO 2). Results were expressed as nitric oxide (NO) in μM as mean ± standard deviation and plotted as a dose response curve.

Test substances The following extracts were tested:

First assay :
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 under the same conditions as Example 3.3. This example was used as a non-alkali dissolution 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 sodium chloride solution at 40 ° C. for 6 hours. This example was used as a non-alkali dissolution control.

Second assay :
OP0701C — 10 g 0.5P4H: Extract from Lactobacillus fermentum I-3929 at 10 g / L biomass dry weight with 0.037 M sodium hydroxide incubated at 40 ° C. for 4 hours.

  OP0701C — 10 g 1P4H: Extract from Lactobacillus fermentum I-3929 at 10 g / L biomass dry weight with 0.075 M sodium hydroxide incubated at 40 ° C. for 4 hours.

  OP0701C — 10 g 2P4H: Extract from Lactobacillus fermentum I-3929 at 10 g / L biomass dry weight with 0.150 M sodium hydroxide incubated at 40 ° C. for 4 hours.

  OP0701C — 10 g 1P21H: Extract from Lactobacillus fermentum I-3929 at 10 g / L biomass dry weight with 0.075 M sodium hydroxide incubated at 40 ° C. for 21 hours.

  OP0701C—10 g 0.5P21H: Extract from Lactobacillus fermentum I-3929 at 10 g / L biomass dry weight with 0.037 M sodium hydroxide incubated at 40 ° C. for 21 hours.

  OP0701C — 10 g 2P21H: Extract from Lactobacillus fermentum I-3929 at 10 g / L biomass dry weight with 0.150 M sodium hydroxide incubated at 40 ° C. for 21 hours.

  OP0701C — 10 g 2P45H: Extract from Lactobacillus fermentum I-3929 at 10 g / L biomass dry weight with 0.150 M sodium hydroxide incubated at 40 ° C. for 45 hours.

Results of the first assay In the first assay (Figure 5a), it was observed that the extract obtained from the alkaline lysis method according to the present invention had the same activity as the extract obtained from osmotic lysis.

  The inventors also observed that the immunostimulatory activity was related to the selected strain. The extract obtained from Lactobacillus fermentum I-3929 induced more NO2 and NO3 production than the extract obtained from Lactobacillus rhamnosus 78.31.

Results of the second assay In the second assay (Figure 5b), we observed that the in-vitro activity of the extract correlated with its initial conditions of lysis. The activity of the following bacterial extracts is shown against the same strain Lactobacillus fermentum I-3929 and also against the same amount of 10 g biomass dry weight / liter of lysis: OP0701C_10g 0.5P4H, OP0701C_10g1P4H, OP0701C_10g2P4H, OP0701C_10g1P21H, OP0701C_10g0.5P21H, OP0701C_10g2P21H, and OP0701C_10g2P45H. The in-vitro activity varies depending on the initial concentration and dissolution period of sodium hydroxide.

Conclusion The results of these experiments show that, regardless of the chemical modification produced by the alkaline treatment, the activity of the embodiment is compared to the aa control extract obtained by osmotic lysis, or compared to the respective live bacteria. (See Example 6 below for this embodiment).

Example 6: In-Vitro Screening for Inflammatory and Anti-inflammatory Activity In order to screen in-vitro for immunostimulation or anti-inflammatory potential of embodiments of the present invention, the test was performed with human PBMC on a series of bacterial extracts. went. As described by Foligne et al., The luminescence of both IL12p70 (inflammatory cytokine) and IL10 (anti-inflammatory cytokine) was measured and the IL10 / IL12 ratio was reported. (World J Gastroenterol (January 14, 2007); 13 (2): 236-243).

  The aim was to compare IL12p70 and IL10 production in six extracts obtained through various extraction / purification methods. The IL10 / IL12p70 ratio obtained for each extract can be correlated with the anti-inflammatory potential of the extract. Live bacteria were used as a control against which the activity of the extract (2 × 10 7 cfu / ml Lactobacillus fermentum I-3929) was compared.

Materials and Methods Step-wise amounts of the six bacterial extracts are diluted in cell culture medium in the presence of 10 Ng / ml IFN-μ (a cytokine known to enhance IL12p70 production) to give an initial dose of 1 mg Administration was started at / ml (highest final dose tested). PBMCs isolated from healthy donor blood were tested as serial dilutions of active dry weight extract from 100 Ng / ml to 1 mg / ml.

  IFN-μ was added at least 3 hours before the lysate and the live bacterial strain were placed in the medium. Data from two independent experiments are reported.

Preparation of human PBMC PBMC were separated from the peripheral fluid. After a short period of time, Ficoll gradient centrifugation (Pharmacia, Uppsala, Sweden), mononuclear cells were collected, washed with RPMI 1640 medium (Live technologies, Paisley, Scotland), gentamicin (150 g / mL), L -Adjusted to 2 · 10 ⁶ cells / mL in RPMI 1640 supplemented with glutamine (2 mmol / L) and 10% fetal calf serum (FCS) (Gibco-BRL).

Cytokine induction PBMC (2 × 10 ⁶ cells / mL) were seeded in 24-well tissue culture plates (Corning, New York, USA). Cells were stimulated as described above. After stimulation for 24 hours at 37 ° C. in an air atmosphere containing 5% carbon dioxide, the culture supernatant was collected, purified by centrifugation, and stored at −20 ° C. until cytokine analysis. In accordance with manufacturer's recommendations, cytokines were measured against IL10 and IL12p70 by ELISA using a Pharmingen antibody pair (BD Biosciences, San Jose, Calif.).

Test substance OP0701C — 10 g 2P45H (A): Extract from Lactobacillus fermentum I-3929 lysis. 10 g biomass dry weight / liter (dissolved), 0.15 M sodium hydroxide at 40 ° C. for 45 hours.

  OP0701C — 10 g 1P4H (B): Extract from Lactobacillus fermentum I-3929 lysis. 10 g biomass dry weight / liter (dissolved), 0.075 M sodium hydroxide at 40 ° C. for 4 hours.

  OP0701C — 5G4P21H (C): Extract from Lactobacillus fermentum I-3929 lysis. 5 g biomass dry weight / liter (dissolved), 0.300 M sodium hydroxide at 40 ° C. for 21 hours.

  OP0701C — 40 g 0.5P4H (D): Extract from Lactobacillus fermentum I-3929 lysis. 40 g biomass dry weight / liter (dissolved), 0.037 M sodium hydroxide at 40 ° C. for 6 hours.

  OP0701B4_CFer150 (E): Extract from Lactobacillus fermentum I-3929 (12 g / L) obtained as described in Example 3.1.

  OP0701B4_BFer300 (F): Extract from Lactobacillus fermentum I-3929 lysis. 6 g biomass dry weight / liter (dissolved), 0.075 M sodium hydroxide 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 aim of this study was to compare the potential for immunostimulation or anti-inflammation of embodiments of the present invention in vitro with live Lactobacillus strains. In order to analyze the various extracts and the live bacteria, not only the amount of each cytokine IL10 and IL12 released from PBMC was compared, but also the ratio of IL10 / IL12.

  If either IL10 or IL12 concentration, or both concentration values are close to the non-stimulatory concentration (ie, less than 10 pg / ml), the ratio is not considered calculable and C. Labeled (non-calculated).

The results obtained from PBMC expressed in pg / ml are shown in Tables 2 and 3.
In the presence of IFNμ, the action of the six bacterial extracts of the present invention (Extract A to Extract F) was compared with the live form of Lactobacillus fermentum (in IL10 and IL12p70 response, pg / ml unit) in human PBMC (IL10 and IL12p70 reaction). 2 × 10 ⁷ bacteria / ml, tested twice as raw sample 1 and raw sample 2). The IL10 / IL12 ratio is reported only for extract doses of 100 μg / ml and 1 mg / ml.

ＩＦＮμの存在下で、本発明の６つの細菌抽出物（抽出物Ａ〜抽出物Ｆ）の作用を、ヒトＰＢＭＣ（ＩＬ１０及びＩＬ１２ｐ７０反応、ｐｇ/ｍｌ単位）でラクトバチルス・ファーメンタムの生形態（２ｘ１０⁷細菌/ｍｌ、生サンプル１及び生サンプル２として２回試験済み）と比較した。ＩＬ１０/ＩＬ１２の比率は、１００μｇ/ｍｌ及び１ｍｇ/ｍｌの抽出物用量に対してのみ報告される。

In the presence of IFNμ, the action of the six bacterial extracts of the present invention (Extract A to Extract F) was compared with the live form of Lactobacillus fermentum (in IL10 and IL12p70 response, pg / ml unit) in human PBMC (IL10 and IL12p70 reaction). 2 × 10 ⁷ bacteria / ml, tested twice as raw sample 1 and raw sample 2). The IL10 / IL12 ratio is reported only for extract doses of 100 μg / ml and 1 mg / ml.

Based on the results presented in Tables 2 and 3, IL10 production decreased in the following order:
B≈E≈D>F> Raw Lactobacillus>A> C

Based on the results presented in Tables 2 and 3, IL12 production decreased in the following order:
Raw Lactobacillus>E>B>D>F>A> C

When examining the ratio, the following general pattern was observed:
B ≒ D> E ≒ F> Raw Lactobacillus

  The IL10 / IL12 ratio results for Extract A and Extract C are not shown because these extracts were found not to be effective inducers of cytokines.

  At concentrations below 100 μg / ml, it was observed that the cytokine concentration was too low to conclude.

Conclusion The obtained ratio suggests that under the experimental conditions used in Example 6, the immunomodulatory effect of some bacterial extracts of the present invention is greater than that of raw Lactobacillus fermentum Can do. For example, extracts B, D, E, and F showed a higher IL10 / IL12p70 ratio than the live bacterial control. Thus, such extracts are more active against the conditions described herein, such as inflammatory conditions, than live probiotic bacteria.

Example 7: Action on Toll-like Receptors Embodiments of the present invention are obtained from Gram positive bacteria and are therefore thought to act through the TLR2 receptor. TLR receptors are expressed primarily by immune cells such as, but not limited to, monocytes, macrophages, dendritic cells, T cells, are important sensors of microbial products, and are recognized as dangerous signals by the host. Although it can trigger non-specific immunity, TLR activation initiates a complete immunological cascade, thus fostering acquired immunity in the presence of antigen.

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

Materials and Methods The response of the extract of the present invention was tested in the following two cellular systems (confirmed agonistic action or antagonistic activity in the presence of the TLR2 agonist Pam3Cys or in the presence of the TLR4 agonist LPS) One of the checks):
a) HEK-TLR2 / 6 (IL8 ELISA after 24 hours)
b) HEK-MD2-TLR4-CD14 (IL8 ELISA after 24 hours)

a) HEK-TLR2 / 6
The HEK293 cell line was selected based on no or low basal expression level of the TLR gene. These cells allow for efficient monitoring of TLR activity using ELISA analysis such as IL8 titration and reporter-based systems that monitor TLR-induced NF-μB activity.

  HEK-TLR2 / 6 cells (Invivogen, Toulouse, France) are stably transfected with multiple genes from the TLR2 / 6 pathway including TLR2, TLR6, and genes that recognize or participate in the signaling cascade. HEK293 cells using advanced technology. These cells secrete IL8 after TLR2 / 6 stimulation. The experiment was performed according to the manufacturer's instructions.

Briefly, 2 × 10 ⁴ cells / well (200 ul RPMI) were incubated at 37 ° C. for 3 days (under 5% carbon dioxide). The medium was removed and 90 μL RPMI + 5% FCS was added to the wells. The agonist and control were then added (10 μl / well). The cells were returned to the incubator and incubated for 24 hours. The supernatant was collected and an IL8 ELISA was performed according to the manufacturer's instructions.

Test substance OP0701B4_AFer50: Extract from Lactobacillus fermentum I-3929 lysis. 12 g biomass dry weight / liter (dissolved), purified with 0.075 M sodium hydroxide at 40 ° C. for 4 hours 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: Results of IL8 secretion control (negative = LPSK12 ultrapure, TLR4 agonist; and PAM3CSK4 = positive, TLR2 agonist) are shown in Tables 4-6. This result (expressed in IL8 in pg / ml) shows the mean secreted IL8 quantified by ELISA 24 hours after stimulation with the control.

  The cell line HEKTLR2 / 6 responded to the TLR2 agonist Pam3Cys. In contrast, the TLR4 agonist from E. coli (LPSK12) was inactive even at a high dose of 0.01 μg / ml.

Three bacterial extracts (OP0701B4Afer50, OP0701C-Bt1LAC, and OP0701C-Bt2LAC) that were experimentally tested with only three bacterial extracts showed higher immunostimulatory properties than the TLR2 agonist Pam3Cys.

Three bacterial extracts + Pam3Cys experiments As described above, the three bacterial extracts were also tested for putative antagonistic or additive properties compared to Pam3Cys added immediately after extraction.
Bacterial extract OP0701B4_AFer50

表４の結果は、前記ＯＰ０７０１Ｂ４_ＡＦｅｒ５０抽出物が高濃度のＩＬ８（アゴニスト（ＴＬＲ２/６））産生を誘導したことを指摘する。
細菌抽出物ＯＰ０７０１Ｃ-Ｂｔ１ＬＡＣ

The results in Table 4 indicate that the OP0701B4_AFer50 extract induced high concentrations of IL8 (agonist (TLR2 / 6)) production.
Bacterial extract OP0701C-Bt1LAC

表５の結果は、前記ＯＰ０７０１Ｃ-Ｂｔ１ＬＡＣ抽出物が高濃度のＩＬ８（アゴニスト（ＴＬＲ２/６））の産生を誘導したことを指摘する。
細菌抽出物ＯＰ０７０１Ｃ-Ｂｔ２ＬＡＣ

The results in Table 5 indicate that the OP0701C-Bt1LAC extract induced the production of high concentrations of IL8 (agonist (TLR2 / 6)).
Bacterial extract OP0701C-Bt2LAC

Conclusion Depending on the conditions of the alkaline lysis method (initial concentration of biomass dry weight, initial base concentration and base treatment period), the bacterial extract can have various modes of action.

  Although OP0701B4_AFer50 and OP0701C-Bt1LAC were agonists (TLR2 / 6), antagonistic TLR2 / 6 activity was obtained (OP0701C-Bt2LAC) when a strong alkaline lysis method was used for the same bacterial strain.

b) HEK-TLR4-MD2-CD14
TLR4 has been extensively studied because it is a major receptor involved in the recognition of lipopolysaccharide (LPS) that is responsible for septic shock.

  HEK-TLR4-MD2-CD14 cells were very sensitive to LPS and were obtained by stable transfection of HEK293 cells with TLR4, MD2 and CD14 genes and an NF-μB-inducible reporter system. These cells secrete IL8.

  The same experimental procedure was adopted for TLR2 / 6 of the other HEK cell lines described above. The results for the tested controls and the three bacterial extracts of the present invention are shown in Tables 7-9.

Results: Results of IL8 secretion control (positive = LPSK12C ultrapure, TLR4 agonist; and PAM3CSK4 = negative, TLR2 agonist) are shown in Tables 7-9. This result (expressed in IL8 in pg / ml) represents the mean secretory level of IL8 24 hours after stimulation with the control.

  The cell line clearly responded only to the TLR4 agonist LPSK12. In contrast, as expected, the TLR2 agonist from Pam3Cys was inactive even at a high dose of 0.01 μg / ml.

As expected for experiments with only three bacterial extracts , Gram-positive agonists, the three bacterial extracts tested (OP0701B4Afer50: Table 7, OP0701C-Bt1LAC: Table 8, and OP0701C-Bt2LAC: Table 9) are the TLR4 pathway. Showed no apparent immunostimulatory properties.

Three bacterial extracts + LPSK12 experiments As previously described, the three extracts of the present invention were also tested for putative antagonistic or additive properties compared to LPS added immediately after extraction. Again, no effect was observed at the TLR4 receptor concentration (Tables 7-9).
Bacterial extract OP0701B4_AFer50

この結果は、前記ＯＰ０７０１Ｂ４_ＡＦｅｒ５０抽出物がＴＬＲ４受容体（Ｐａｍ３Ｃｙｓ：１３８ｐｇ/ｍＬ）を活性化しなかったことを示す。
細菌抽出物ＯＰ０７０１ＣＢｔ１-ＬＡＣ

This result indicates that the OP0701B4_AFer50 extract did not activate the TLR4 receptor (Pam3Cys: 138 pg / mL).
Bacterial extract OP0701CBt1-LAC

Bacterial extract OP0701CBt2-LAC

結論
総合すれば、ＨＥＫ細胞、及びヒトＰＢＭＣ細胞で得られた分泌物に関するこの結果は、ＡＦｅｒ５０及びＢｔ１ＬＡＣが、ＴＬＲ２経路を介して免疫系を刺激できることを示唆する。また、Ｂｔ２ＬＡＣは、ＴＬＲ２/６アンタゴニストとして機能し得る。それゆえ、本発明に係る抽出物は、ＴＬＲ２経路を介して免疫系を刺激することや、ＴＬＲ２/６アンタゴニストとしての機能を果たすこともできるため、ｉｎ-ｖｉｖｏで抗感染活性や抗炎症活性に相関する。

Conclusions Taken together, this result on secretions obtained in HEK cells and human PBMC cells suggests that AFer50 and Bt1LAC can stimulate the immune system via the TLR2 pathway. Bt2LAC can also function as a TLR2 / 6 antagonist. Therefore, the extract according to the present invention can stimulate the immune system via the TLR2 pathway and can also function as a TLR2 / 6 antagonist, so that it has anti-infection activity and anti-inflammatory activity in-vivo. Correlate.

Example 8 :
Cells that express a given functional TLR gene are valuable tools for many applications such as the study of mechanisms involved in TLR recognition or signaling and the development of new potential therapeutics. Therefore, the aim of the following experiment was to test the activity of four bacterial extracts with these important adapters of the immune response.
In this test, 8 bacterial TLR and NOD2 receptors were screened against 4 bacterial extracts containing the extract according to the present invention.

Methods Bacterial extracts according to the present invention were tested in 96 well microplates. The extract was diluted with DMEM medium and 20 μl of each dilution was tested in duplicate. 180 μl volume containing 25000 or 50000 cells in DMEM medium + 10% FCS (one HEK293 cell line with reporter gene secreting alkaline phosphatase under the control of NF-kB specific for each TLR) HEK293 cell suspension was added in duplicate to each well. After 16 hours incubation of each cell line, 20-50 μl of each supernatant was transferred to a 96 well microplate and completed with 200 μl Quantiblue (Invivogen number REP-QB1). Various series of TLRs expressing cell lines were subjected to enzymatic reaction with secreted alkaline phosphatase for 30-60 minutes. Reading was performed using a microplate reader at 630 nm. Results were expressed as OD at 630 nm.

material
List of positive control agonists (and their respective concentrations) used in this screening assay TLR PAM2 100 Ng / ml; TLR3 poly (I: C) 100 Ng / ml; TLR4 E. coli K12LPS 1 μg / ml; TLR5 Salmonella typhimurium flagellin 1 μg / ml TLR7 R848 10 μg / ml; TLR8 R848 10 μg / ml; TLR9CpG ODN 2006 10 μg / ml; NOD2 muramyl dipeptide 1 μg / ml.

A negative control reporter gene only (NFkB) recombinant HEK-293 cell line was used as a negative control for the TLR cell line. The negative control value for each clone was the background signal of these non-induced clones. TNF- 痾 was used as a positive control for this non-TLR expressing cell line.

The following bacterial extracts were tested:

  OP0701B4_CFer300: Extract (F) from Lactobacillus fermentum I-3929 (12 g / L) obtained as described in Example 3.3;

  OP0701B4_CRahr300: Extract (G) from Lactobacillus rhamnosus 71.38 (40 g / L) obtained as described in Example 3.4;

  OP0701D — 10L1PswitchA: Extract (I) from Lactobacillus fermentum I-3929 (10 g / L) obtained as described in Example 3.12.

  OP0701D_5LOSMOConc: Extract from osmotic dissolved Lactobacillus fermentum I-3929 (7.6 g / L) obtained by osmotic stress for 24 hours at 40 ° C. in 0.16 N sodium chloride solution as control (H ).

Preparation of the bacterial extract In a reaction volume of 200 μl, all cell lines were stimulated with 20 μl of each sample to be tested.

  Screening was performed at a single concentration, typically 0.5 mg dry weight / mL. The test was done in duplicate.

Results The bacterial extracts and controls were tested in duplicate on a recombinant HEK-293 cell line that functionally expresses not only a given TLR or NOD2 protein but also a reporter gene driven by the NFkB promoter. TLR and NOD2 activation results are expressed as optical density (OD) values. The results are shown in Table 10-14 and summarized in Table 15.
Bacterial extract F (OP0701B4CFer300)

細菌抽出物Ｆは、ｈＴＬＲ２、ｈＴＬＲ４及びｈＮＯＤ２を特異的に活性化し、より少ない程度でｈＴＬＲ５も活性化した。
細菌抽出物Ｇ（ＯＰ０７０１Ｂ４ＣＲａｈｒ３００）

Bacterial extract F specifically activated hTLR2, hTLR4 and hNOD2, and to a lesser extent hTLR5.
Bacterial extract G (OP0701B4CRahr300)

細菌抽出物Ｇは、ｈＴＬＲ２、ｈＮＯＤ２を活性化し、かなり少ない程度ではｈＴＬＲ４も活性化した。
細菌抽出物Ｈ（ＯＰ０７０１Ｄ_５ＬＯＳＭＯＣｏｎｃ）

Bacterial extract G activated hTLR2, hNOD2, and to a lesser extent hTLR4.
Bacterial extract H (OP0701D_5LOSMOConc)

細菌抽出物Ｈは、ｈＴＬＲ２、ｈＴＬＲ４及びＮＯＤ２を特異的に活性化した。
細菌抽出物Ｉ（ＯＰ０７０１Ｄ_１０Ｌ１ＰｓｗｉｔｃｈＡ）

Bacterial extract H specifically activated hTLR2, hTLR4 and NOD2.
Bacterial extract I (OP0701D_10L1PswitchA)

細菌抽出物Ｉは、ｈＴＬＲ２、ｈＴＬＲ５、ｈＮＯＤ２を強力に活性化し、より少ない程度では、ｈＴＬＲ４とｈＴＬＲ９を活性化する。

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

かくして、本発明に係る細菌抽出物は、様々なＴＬＲ、さらにはＮＯＤ２を介して前記免疫系の活性化に役立ち得る。従って、本発明の抽出物は、免疫応答の優れた活性化因子であり得る。

Thus, the bacterial extract according to the present invention may help to activate the immune system through various TLRs and even NOD2. Therefore, the extract of the present invention can be an activator with excellent immune response.

  Comparing extract H with extract G and extract F reveals that the extract according to the invention has the same TLR and NOD pathways as the extract obtained from osmotic lysis. Comparing extract H and extract I, the inventors activated the TLR5 pathway by adding acid lysis followed by alkali lysis, and activated the TLR9 pathway to a lesser extent. Can be observed. Even extract F resulting from the weak alkaline treatment showed activation of the TLR5 pathway, in contrast to extract H.

  Extract I acted on TLR5 and showed potential application in radiotherapy. In fact, radiation therapy is a well-established therapy that is extremely effective in treating certain types of cancer, and its side effects are destructive because it can destroy healthy somatic cells, especially bone marrow and gastrointestinal cells is there. Have reported that the CBLB502 peptide binds to TLR5 and activates the nuclear-B signaling pathway that cancer cells frequently activate to avoid cell death (Burdelya et al., “An agonist of toll-like receptor 5 has radioprotective activity in mouse and primate models, "Science, 2008, 320: 226-30). Mice and rhesus monkeys treated with CBLB502 just prior to exposure to a lethal dose of whole body irradiation showed less damage to healthy bone marrow and digestive cells and survived significantly longer than controls. Importantly, in tumor-bearing mice, CBLB502 did not impair the antitumor effect of radiation therapy.

Example 9: The ability of an extract to produce plaque forming cells (relative to sheep erythrocytes) in mice. The plaque forming cell ( PFC) technology allows the evaluation of non-specific stimuli of B lymphocytes. This technique was first described by Cunningham and Seenberg (Immunology, 1968, 14, 599). The presence of hemolytic antibodies around antibody-forming cells was demonstrated as follows. Not only mouse lymphoid cells, but also a dense population of foreign (sheep) erythrocytes was co-introduced onto the microscope slide. Certain lymphoid cells diffuse and form lytic plaques in the presence of complement, causing the hemolytic antibody responsible for adjacent erythrocyte lysis to emit light. At the end of the experiment, the number of PFCs reported per to 10 ⁶ cells or splenocytes was measured.

Materials and methods
Test substance 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 l'Arbresle, France), 5-6 weeks old, average body weight 20 +/- 2g divided into 5 groups, 8 per group It was.

The experimental animals were divided into five groups as follows:
Group (a) 8 mice, feeding amount 1.2 mg / mouse, extract 1 obtained as described in Example 3.5; liquid volume = 0.2 ml
Group (b) Eight mice, feeding amount 0.6 mg / mouse, extract 1 obtained as described in Example 3.5.
Group (c) 8 mice, feeding amount 1.2 mg / mouse, extract 2 obtained as described in Example 3.4; liquid volume = 0.2 ml
Group (d) Eight mice, feeding amount 0.6 mg / mouse, extract 2 obtained as described in Example 3.4; liquid volume = 0.2 ml
Group (e) 8 mice, 0.2 ml isotonic saline

Mice are orally administered daily with the above extract at the above dose (via oral route) for 5 consecutive days (Day 1 to Day 5). Two intubations were added on days 19 and 20. On day 29, the antigen (10 ⁶ sheep erythrocytes) in 0.2 ml of isotonic saline (0.9% sodium chloride, Alsever solution) was injected intravenously (via the tail vein of the animals); 4 days later ( On day 33), a spleen cell suspension was prepared.
Reagents and equipment <br/> Alsever fluid, endotoxin-free (Sigma, 38299 St Quentin Fallavier, Cedex France, ref. A 3351)
Eagle basal medium (BME, 10 times concentration), lead carbonate-free Earl solution (Bio-Merieux, 69280 Marcy l'Etoile, France ref: 8 210 2)
Lyophilized guinea pig serum (Bio-Merieux, Marcy l'Etoile, France ref: 7 212 2)
Sheep red blood cells (Bio-Merieux, Marcy l'Etoile, France ref: 7214 1)
Trypan blue, sterile distilled water, 0.9% sodium chloride, tissue homogenizer, centrifuge, Neubauer chamber (for cells and glass tubes)

Control animals treated with test or control substances received only sheep erythrocytes (SRBC). Other animal groups were orally administered the bacterial extract described above. For this purpose, each extract was dissolved in water.

Spleen cell suspension Four days after the last antigen (SRBC) injection, a spleen cell suspension was prepared for each mouse by the following procedure:

  The animals were anesthetized with ether and then sacrificed by cervical dislocation. The spleen was removed and ground with a glass tissue homogenizer (pH 6.75-6.80 in 2 ml BME). Then 3 ml of sterile distilled water was added and the mixture was stirred for 20 seconds. The resulting suspension was further diluted with 10 BME and centrifuged at 4 ° C. and 1200 rpm for 10 minutes. The supernatant was discarded and the precipitate was suspended in 2 ml BME. The cells were allowed to recover on ice for 5-10 minutes. The number of viable mononuclear cells was measured as follows:

  A 1/20 dilution of an aliquot of the cell suspension was prepared in saline containing a 0.1% solution of trypan blue. Colorless cells, ie the living cells, were counted in a Neubauer chamber.

  The cell suspension was kept on thawed ice during counting and used for lysis spots immediately after counting. This technique prevents a decrease in survival rate when the counting time is too long.

Slide adjustment Using a normal microscope slide, carefully remove dust, remove any traces of fat, and apply three pieces of double-sided tape (thickness 0.1 mm) in parallel at intervals of about 1.5 cm. The three pieces of double-sided tape are covered with a pre-cleaned cover glass (22 mm × 22 mm) to form two “chambers” between the slide and the cover glass.

The spleen cell suspension fraction was adjusted to 25,000 mononuclear cells per mm 3. In small glass tubes, a mixture consisting of the following elements (added in the order given) was prepared by automatic pipette:
1) 7.5 ml of BME
2) 0.5 ml of SRBC suspension (washed with physiological saline and centrifuged twice. Residue 50%)
3) 0.4 ml normal guinea pig serum (used as complement source)

  Finally, 50 μl of the splenocyte suspension (25,000 cells / mm 3) was added to 200 μl of the mixture. After homogenization, the suspension was introduced into “Cunningham Chambers” by capillary action before sealing the open side with paraffin. The slide was then placed in a humid chamber in a dryer at 37 ° C.

After incubation for 1 hour in the lecture dryer, the slides were examined with a microscope (magnification 100 ×: eyepiece 10 ×, objective 10 ×).

The lysis spots were easily recognized. Five pieces of vertical light strip were examined on each slide and the number of lysed spots was counted. The number of cells (N) forming direct lysis spots per 10 ⁶ cells was calculated by extrapolation. If x is the number of spots observed and X is the number of cells examined, the number of directly dissolved spots is determined by the following formula:
n = (x / X) * 10 ⁶
Where X = C * V = C * (n * e * l * L)
C = final concentration of splenocytes V = observed liquid volume n = number of light strips e = adhesive tape thickness I = light field width L = light strip length

The number of direct lysis spots per 10 ⁶ cells was obtained from the following equation:
PFC (per 10 ⁶ cells) = (x * 10 ⁶ ) / (C * n * e * I * L)

  By knowing the number of cells recovered per spleen, it is possible to deduce the number of cells that form lytic spots per spleen.

  Spontaneous lytic plaques were not observed in “absolute reference” mice.

If statistical analysis and result p <0.05, the result is accepted as significant (Student t test). The results are shown in Table 16.

本発明の抽出物は、１.２ｍｇ/マウス/日または０.６ｍｇ/マウス/日の濃度で試験される場合、Ｂ細胞活性化因子であるよう思われる。わずかな用量依存性効果が観察された。それゆえ、本発明の一部の抽出物を用いて、反復性感染に苦しむ患者においてその免疫系を刺激する可能性もあり得る。

The extract of the present invention appears to be a B cell activator when tested at a concentration of 1.2 mg / mouse / day or 0.6 mg / mouse / day. A slight dose-dependent effect was observed. Therefore, some extracts of the present invention may be used to stimulate the immune system in patients suffering from recurrent infections.

Example 10: Effect of CFer300 on intraperitoneal S. typhimurium infection in Balb / c mice The protection of mice infected with S. typhimurium was tested with a bacterial lysate from the genus Lactobacillus after oral administration.

Materials and methods
Animal and livestock Balb / c mice were housed in the Institute of Immunology in Moscow. For in-vivo protection experiments, outbred laboratory-grade white mice were purchased from Stolbovaya, Russian State Scientific Center for Biomedical Technologies (Russian Academy of Medical Sciences). The weight of the mouse immediately after arrival was 12-14 g. Throughout all experiments, mice were maintained sterile with a standard rodent diet and water.

Research group (main experiments)
Two groups (22 mice per group) were used to test the anti-infective effect of bacterial extracts prepared using an experimental model of Salmonella typhimurium infected mice.

  Group 1 was treated by oral administration of CFer300 solution, and Group 2 received sham treatment (water) as a negative control.

0.5 ml of the solution was orally administered to each mouse once a day for 10 consecutive days before all mice were exposed to Salmonella typhimurium:
Group 1: Mice treated orally with a single dose of 2 mg (0.5 ml) of CFer300.
Group 2: Mice treated sham by oral administration of 0.5 ml water daily for 10 days.

Test substance The bacterial extract tested was OP0701B4CFer300 (“CFer300”); an extract (28.4 mg of Lactobacillus fermentum I-3929 (12 g / L) obtained as described in Example 3.3. Active dry weight / g).

Preliminary Experiment The purpose of the preliminary experiment was to quantitatively determine the dose of infectious agent that induces approximately 50% mortality in 3 weeks after bacterial exposure (challenge). As a challenge, a suspension of Salmonella, serotype Salmonella strain 415 (I. Mechnikov, Institute for Vaccines and Sera, Russian Academy of Medical Sciences) was injected intraperitoneally into each mouse. The challenge dose was 103-105 CFU Salmonella per mouse.

Observations and death records After the challenge, mice were maintained under standard conditions of experimental animals. Daily observations and death records were recorded for 21 days after infection. The anti-infective effect of the formulation was calculated as the survival rate after infection (SR), average life span after infection (ADL), protection rate (DF), and formulation efficacy index (EI) calculated for each experimental group. Estimated based on. The SR was the proportion of animals surviving in the experimental group on day 21 after infection. The ADL, DF, and EI were calculated as follows:
ADL = (X1 + X2 + ... + Xn) / N
here:
ADL is the average life,
X1-Xn is the life span after infection of experimental mice 1-n,
N is the total number of animals in the experimental group.
DF = CD / ED
here:
DF is the defense rate,
CD is the mortality rate in the control group, and ED is the mortality rate in the experimental group.
EI = [(DF-1) / DF] x100%
here:
EI is the drug product efficacy index, and DF is the protection rate.

Results: Drug resistance The preparation was orally administered once a day for 10 days, and the drug resistance was good. No signs of toxicity or side effects were observed during the 10 day pretreatment.

Preliminary titration of Salmonella was performed to determine the challenge dose of Salmonella typhimurium that resulted in approximately 50% mortality. The results are shown in Table 17.

下線を引いた数字は、各日数（感染後）ごとに死亡が発見された動物数を表す。

Underlined numbers represent the number of animals found dead on each day (after infection).

Conclusion Based on the data obtained, 10 ⁵ CFU of Salmonella typhimurium 415 (67% dead animals, bold) was selected as the dose to be used in subsequent studies.

Main experiment: Challenge of mice treated with CFer300 (exposure to bacteria)
For 10 days, mice pre-treated with CFer300 formulation (n = 22) and mice in the control group given water (n = 22) were challenged (bacterial exposure) one day after the end of pre-treatment.

A suspension of Salmonella typhimurium was administered intraperitoneally at a challenge dose of 10 ⁵ CFU per mouse. Follow-up was performed for 21 days after infection. Table 18 shows the mortality rate for each group.

イタリック体の数字は、各日数（感染後）ごとに死亡が発見された動物数を表す。これらのデータを用いてＳＲ、ＡＤＬ、ＤＦ及びＥＩを算出した（表１９）。

Numbers in italics represent the number of animals found dead on each day (after infection). SR, ADL, DF and EI were calculated using these data (Table 19).

水で前治療を施した対照群では、観察期間中（２１日間）の生存率は５５%、及びＡＤＬは１４.７日間であった。２ｍｇ/日の用量でＣＦｅｒ３００は保護作用を示し、ＳＲ=７３%及びＡＤＬ=１７.１日間、ＤＦ=１.６７及びＥＩ=４０%であった。

In the control group pretreated with water, the survival rate during the observation period (21 days) was 55%, and the ADL was 14.7 days. CFer300 showed a protective effect at a dose of 2 mg / day, SR = 73% and ADL = 17.1 days, DF = 1.67 and EI = 40%.

  In summary, a single oral dose of 2 mg per day over 10 days of CFer300 provided partial protection to mice infected with Salmonella typhimurium. As shown in this example, or as previously shown in-vitro and ex-vivo, the extracts disclosed in the present invention may be useful for future therapeutic drug development.

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

Materials and methods
Animals and livestock 6-week-old female Balb / cByJ mice were purchased from Jumbar, France, maintained under standard conditions and fed.

Study groups A total of 27 mice were divided into 4 groups as follows:
Group A: untreated, LACK sensitized and saline challenged mice; LACK is a protein from the parasite Leishmania (4 mice).
Group B: Untreated LACK sensitized and challenged mice (8 mice)
Group C: OM-1009A-treatment (8 mg dry weight residue per dose), LACK sensitized and challenged mice (8 mice)
Group D: OM-1009B-treatment (8 mg dry weight residue per dose), LACK sensitized and challenged mice (7 mice)

Treatment with test substance or control substance and schedule mice were treated from day 3 to day 22. On days 0 and 7, mice were sensitized by intraperitoneal injection (ip) with 10 · g LACK in the presence of 2 mg alum3. On days 17-21, mice are aerosol challenged with LACK solution (0.15%) per day for 20 minutes (Group B, Group C, and Group D), or saline (Group A) as a control. Gave. On day 22, the ability of mice to develop AHR immediately after inhalation of methacholine was analyzed. On day 23, mice were sacrificed and lung inflammation was assessed.

Methodology Mice were treated therapeutically three times as described above. Cannulas were inserted into the trachea and bled, and individual mice were washed (perfused). The lungs were washed 3 times with 1 ml of warm PBS. Cells were washed with PBS, resuspended in 300 μl, and counted using a Burker-Turk chamber. To obtain the percentage of cells in BAL, a cytospin preparation was made and stained with Wright Giemsa stain. The test groups are as follows: LACK sensitized and PBS challenged wild type (wt) mice (control), LACK sensitized and challenge wtus (asthmatic animals), OM-1009A-treated wtus, and OM-1009B- Treatment wt us. The total number of cells in BAL was quantified by microscopic examination of cytospin preparations stained with Wright-Giemsa stain.

  Since reduction of airway inflammation was observed in treated mice (see results), lung extracts were prepared and IL4, IL5, and IL13 were quantified by multiple analysis (CBA array).

The two bacterial lysates tested were as follows: OM-1009A extract from Lactobacillus fermentum I-3929 obtained as described in Example 3.7, and Example 3 OM-1009B extract from Lactobacillus fermentum I-3929 obtained as described in .11.

result
a) On day 22 of airway hyperresponsiveness, the ability of mice to develop AHR immediately after inhalation of methacholine was analyzed at each dose. This result is reported in FIG. 5 and shows that OM-1009-B, similar to the PBS-untreated non-asthma group, restored basal Penh values. OM-1009-A was more efficient because the obtained Penh value was even lower than that observed in the non-asthma control group.

b) On the 23rd day of total cell number and cell percentage in bronchoalveolar mice, mice were sacrificed and lung inflammation was evaluated. The total number of cells in the BAL is reported in Table 20 (right column) and the cell percentages are as follows: eosinophil count (Eo), neutrophil count (Netro), lymphocyte count (Lympho) ), And other cell numbers (Other).

Both extracts significantly reduced the total number of cells in the BAL (p <0.01 (OM-1009A); and p <0.03 (OM-1009B) when compared to group B). The number of eosinophils decreased by 3 to 1.7 times. The number of neutrophils decreased by 1.8 times to 3.6 times, respectively. A decrease in the number of eosinophils and neutrophils indicates a decrease in the concentration of inflammatory cells in the BAL of animals pretreated with the extract.
c) Th2 cytokines were quantified by detecting multiple extracts in the lung and multiplex analysis (CBA array)

  Th2 cytokine (IL4, IL5, and IL13) concentrations were decreased by the OM-1009A extract but not by the OM-1009B extract as compared to the asthma control group (B). Therefore, the bacterial extract obtained by the strong base treatment (OM-1009-B) may be less active than the bacterial extract obtained under moderate base treatment (OM-1009-a). Even though both extracts are active in the Penh factor assay (FIG. 6), OM-1009A is an OM− for the treatment or prevention of conditions related to Th2-related diseases such as allergic diseases, atopy, or signs thereof. Can be a better candidate than 1009B.

Claims (28)

An extract according to one or more Lactobacillus strains, characterized in that it is a soluble extract and further comprising a chemically modified bacterial molecule. 2. The extract of claim 1, wherein the chemically modified bacterial molecule results from exposing the one or more Lactobacillus strains to an alkaline medium. The extract according to claim 1 or 2, which has immunomodulating activity in a subject patient. 4. The extract according to claim 3, wherein the extract has immunostimulatory activity in the subject patient. 4. The extract according to claim 3, which has anti-inflammatory activity in the subject patient. The extract according to any one of claims 1 to 5, wherein the one or more Lactobacillus strains are one or more Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus casei defensis, Lactobacillus casei subspecies casei, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus reuteri, An extract comprising Lactobacillus salivaius, Lactobacillus lactis, and Lactobacillus delbrecchi. The extract according to any one of claims 1 to 6, wherein the one or more Lactobacillus strains are one or more Lactobacillus fermentum I-3929, Lactobacillus rhamnosus 71. 38, an extract comprising Lactobacillus plantarum 71.39, Lactobacillus johnsonii 103782, and Lactobacillus helveticus 103146. The extract according to any one of claims 1 to 7, wherein one or more aspartic acid, glutamic acid, serine, histidine, alanine, arginine, tyrosine, methionine, phenylalanine, and lysine contained in the extract. Extract characterized in that it is at least 10% racemized. 9. The extract according to any one of claims 1 to 8, wherein an IL10 / IL12 ratio that can be calculated in human peripheral blood mononuclear cells can be achieved, said ratio being the raw Lactobacillus from which said extract is obtained An extract characterized by being equal to or exceeding the IL10 / IL12 ratio realized by the strain. The extract according to claim 1, wherein the number of eosinophils, neutrophils, lymphocytes, or any combination thereof in an asthmatic mouse subject is at least compared to an untreated asthma control group. An extract characterized in that it can be reduced by a factor of 1.5. The extract preparation step according to claim 1, wherein the step comprises:
(A) culturing one or more Lactobacillus strains in a medium;
(B) exposing each Lactobacillus strain to an alkaline medium; and (c) treating the product of step (b) to remove insoluble and particulate matter. . 12. The process according to claim 11, wherein step (c) is performed by tangential flow filtration. The process according to claim 11 or 12, wherein the chemical modification of the bacterial molecule can be sufficiently performed only by exposing each Lactobacillus genus bacterial strain to a pH exceeding 9.0. 14. The process according to claim 11, 12, or 13, further comprising the step of treating each strain at a pH of less than 4.5 between process step (b) and step (C). Process. 15. The process according to any one of claims 11 to 14, wherein the chemical modification comprises one or more aspartic acid, glutamic acid, serine, histidine, alanine, arginine, tyrosine, methionine, phenylalanine in the extract, and Comprising at least 10% racemization of lysine. A dietary supplement composition comprising the extract according to any one of claims 1 to 10, or the extract prepared according to the method according to any one of claims 11 to 15. A pharmaceutical composition comprising the extract according to any one of claims 1 to 10, or the extract prepared according to the method according to any one of claims 11 to 15. 16. Extract according to any one of claims 1 to 15, in a method for alleviating at least one symptom associated with at least one condition selected from respiratory diseases, allergic conditions, urinary tract diseases and digestive disorders. Or a therapeutically effective amount of the composition of claim 16 or claim 17 to a subject patient. The method according to claim 18, wherein the respiratory disease is an upper or lower respiratory tract infection, nasopharyngitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, pharyngopharyngitis, influenza, pneumonia, bronchopneumonia , And is selected from obstructive pulmonary diseases with acute exacerbations. 19. The method of claim 18, wherein the allergic condition is selected from allergic rhinitis, allergic asthma, and atopic dermatitis. 19. The method of claim 18, wherein the urinary tract disease is urethritis, tubulointerstitial nephritis, obstructive pyelonephritis, chronic cystitis including cystitis, prostatitis including male pelvic pain syndrome, and chronic prostatitis , Selected from the group consisting of prostatic cystitis and female pelvic inflammatory disease. The method of claim 18, wherein the digestive disorder is selected from Crohn's disease and irritable bowel syndrome. The method according to claim 18, wherein the target patient is a human or a domestic animal. Use of the extract according to any one of claims 1 to 15 or the composition according to claim 16 or claim 17 from respiratory diseases, allergic conditions, urinary tract diseases and digestive disorders. A use characterized in that it is used as a drug for alleviating at least one symptom associated with at least one selected condition. Use of the extract according to any one of claims 1 to 15, or the composition according to claim 16 or claim 17 as an immunomodulatory agent. An isolated microbial strain, Lactobacillus fermentum I-3929. An extract obtained from the strain of claim 25. The extract according to claim 26, wherein the extract is a soluble extract.

Images