KR20110051268A - Immunomodulatory extracts from lactobacillus bacteria and methods of manufacturing and use thereof - Google Patents

Abstract

The present invention includes an extract derived from Lactobacillus bacteria, which may exhibit an immunomodulatory effect in a subject. Embodiments of the present invention may be used as an adjuvant in medical treatment, such as, for example, as a nutraceutical or drug for the treatment of a disease or as an adjuvant associated with an imbalance in the production of anti-inflammatory or anti-inflammatory cytokines. have. Conditions in which the extract of the present invention may be used include infections, allergies, autoimmune diseases, and inflammation, and the extract of the present invention may be used as an adjuvant to provide health benefits in a subject. In addition, the present invention includes a method for producing such an extract and a method of using the same. The invention also relates to certain strains of Lactobacillus bacteria.

Description

ImmunoOMULULATORY EXTRACTS FROM LACTOBACILLUS BACTERIA AND METHODS OF MANUFACTURING AND USE THEREOF}

Field of invention

Embodiments of the present invention include extracts from Lactobacillus bacteria that can produce an immunomodulatory effect in a subject. Embodiments of the invention provide, for example, for the treatment of diseases such as diseases associated with an imbalance in the production of anti-inflammatory or proinflammatory cytokines, such as infections, allergies, autoimmune diseases, and inflammation. It can be used as nutraceuticals or pharmaceuticals or as an adjuvant to provide health benefits to a subject. In addition, the present invention is inter alia ), a method of making the extract and a method of using the extract. The invention also relates to certain strains of Lactobacillus bacteria.

Background and Summary of the Invention

Immunomodulation is an international term used to mean a wide range of immune interventions that modify normal or abnormal immune responses. Microorganisms produce and secrete a wide range of molecules that can modulate eukaryotic immune responses (Lavelle et al., Curr Top Med Chem. 2004, 4 (5), 499-508). These include factors that overturn protective mechanisms to promote pathogen colonization and persistence. Viruses, bacteria and parasite-derived molecules have been identified that can inhibit the inflammatory response. In addition to microbial factors capable of inhibiting the immune response, potent immune activators may also be of microbial origin. These include bacterial enterotoxins, parasite-derived excretion-secreting products, and viral nucleic acids.

It is believed that a family of at least eleven receptors, called toll-like receptors (TLRs) and secreted by the host individual, play a key role in immunological sensing and innate responsiveness to microorganisms. Figure 1 provides a list of TLR ligands (Gay and Gangloff, Ann Rev Biochem , 2007, 76:... 141-65). TLRs recognize a wide range of molecules, also known as pathogen-associated molecular patterns (PAMPs) produced by viruses, bacteria and fungi (Tse and Homer, Ann Rheum Dis . 2007 Nov; 66 Suppl 3: iii77-80). TLR-associated immunomodulation has been applied to the development of novel therapeutics for a wide range of pathologies, including infectious, malignant, autoimmune and allergic diseases.

TLR agonists and antagonists have been studied as potential therapies for the prevention and treatment of diseases. In relatively small clinical trials, TLR agonists have been used as adjuvants for vaccines aimed at preventing infections, eliminating allergic hypersensitivity, and removing malignant cells. TLR agonists were also investigated as monoapies and adjunct therapies for the treatment of patients with infectious, allergic and malignant diseases. The use of TLR antagonists has also been studied in preclinical and clinical studies as potential therapies for autoimmune diseases and sepsis.

Probiotics are living microorganisms that can provide health benefits to a subject when administered in an appropriate amount (Mottet et al., Digestive and Liver Disease , 2005, 37: 3-6; Ezendam et al., Nutr Rev , Jan. 2006, 64 (1): 1-14; Gill and Prasad, Adv Exp Med Biol , 2008, 606: 423-54). Probiotic microorganisms and biological mechanisms involved in stimulating immune responses by certain cellular components of such microorganisms have been the subject of research. For example, Gram-positive bacteria have characteristic cell walls that include macromolecules such as lipoteichoic acid (Lipoteikosan, LTA). LTA may be associated with immunostimulatory activity (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 may contain various TLR ligands with immunomodulatory characteristics. Cell wall fragments from various Bifidobacteria strains have been found to stimulate in vitro interferon-gamma (IFN-γ) production in mouse splenocytes (T. Ambrouche, "Contribution a I'etude du pouvoir immunomodulateur des bifidobacteries: Analyse in in vitro et etude ex vivo des mecanismes moleculaires impliques, "Ph.D. Thesis, Universite Laval, Quebec, 2005. Also capsules made of particulate cell wall fragments of certain lactic acid bacteria (Del-Immune V®, Pure Research Products, LLC , Colorado) is intended to stimulate the immune system.

However, the injestion of probiotic bacteria in live or killed form, or the injection of particulate cell wall fragments of such bacteria may not be the most effective way to provide an immunomodulatory effect in a subject. For example, live cell extracts may include giant proteins and lipopeptides, the size of which interferes with efficient uptake by the subject and thus, localization of useful molecules from probiotic bacteria in the body. Limit the concentration. The state of the body can also destroy the active bacterial components or otherwise modify the chemical structure of those components, thereby inactivating them. Risks associated with oral administration of live probiotic microorganisms (Lactobacillus) are bacteremia and sepsis (Lactobacillus Sepsis Associated With Probiotic Therapy, Pediatrics , Jan. 2005, 115 (1): 178-181). Thus, there is a need for other means of delivering the beneficial effects of probiotic bacteria to a subject in need thereof.

The present invention relates to a Lactobacillus extract, some embodiments may exhibit strong immunomodulatory activity. For example, embodiments of the invention may, in some cases, be useful as nutraceuticals or drugs that treat infectious diseases, allergies, respiratory disorders, and inflammatory pathologies, or act as ancillary products in conjunction with treatment protocols. Related to extracts from bacterial strains. The invention also relates to a composition comprising an extract, and to a method for preparing the extract, for example, a method for preparing the extract using a medium that does not pose a risk of prions disease. . The method according to the invention comprises lysis of cells, for example, under alkaline conditions or under acidic conditions after alkaline conditions. In some embodiments, the extracts of the present invention are soluble extracts, meaning that such extracts do not contain significant amounts of solid or particulate matter. In some embodiments, the extract contains a chemically modified TLR ligand. In some embodiments, alkaline treatment can result in 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 SSP . Casei ( Lactobacillus) casei ssp. casei ), Lactobacillus paracasei ), Lactobacillus bulgaricus), Lactobacillus para Kasei (Lactobacillus paracasei), Lactobacillus even know Phil Ruth (Lactobacillus acidophilus ), Lactobacillus reuteri), Lactobacillus salivarius (Lactobacillus salivarius), Lactobacillus lactis (Lactobacillus lactis ), and Lactobacillus delbrueckii ).

In some embodiments, the extract comprises one or more strains from each of the bacterial species, while in other embodiments, one or more specific strains from the list may be removed or replaced with one or more other 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 strains have been deposited according to the Budapest Treaty. Lactobacillus fermentum ) I-3929, Lactobacillus rhamnosus ) 71.38, Lactobacillus plantarum) 71.39, Lactobacillus zone Sony (Lactobacillus johnsonii) 103782, and Lactobacillus helveticus (Lactobacillus helveticus ) 103146 has been deposited in the Collection Nationale de Culture des Microorganismes in Paris, France, 752 \ 724, Rousseau Dr. Roux 25 and Institute Pasteur, respectively. Lactobacillus fermentum I-3929 was deposited on February 27, 2008. Other strains are on the depository of the depository and can be obtained by contacting the depositary.

The invention also relates, among other things, to strain Lactobacillus fermentum I-3929, extracts obtained from this strain, methods of making such extracts, and uses thereof. Such strains include Lactobacillus plantarum and Lactobacillus The strain from fermentum ) was obtained by undergoing chromosomal exchange and thus creating a new Lactobacillus strain. Extracts obtained from Lactobacillus fermentum I-3929 were found to be significantly differently active in vivo and in vitro models with respect to infections and immunological disorders.

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

Extracts can be obtained by growing the cells to a suitable concentration in the culture medium and then lysing the bacterial cells under certain conditions. In some embodiments, the bacteria are grown in a medium that does not pose a risk of prion-related diseases or other diseases that can be delivered through ingesting products obtained from animal-based media. For example, in some embodiments, the cells are cultured using a vegetable-based medium, such as a soya-based medium.

Lysates (ie, products of cell lysis) can also be filtered to remove nucleic acids and larger cell debris, such as insoluble or particulate matter. In some embodiments, the amount of nucleic acid present in the extract is less than 100 μg / mL. Thus, in some embodiments, the resulting extract contains soluble molecular components and does not contain significant amounts of insoluble or particulate matter.

Membrane and cell wall molecules, including lipoproteins, lipopeptides, peptidoglycans, lipopolysaccharides, lipoteichoic acid, and teicosane, may be dissolved or suspended in the extract. During the lysis process, molecules within cells, such as membranes and cell walls, can be chemically modified, for example by alkaline treatment, to be cut into smaller structures. Despite such chemical modifications, embodiments of the present invention may maintain their biological activity compared to whole cells, or such embodiments may even exhibit improved biological activity compared to whole cells.

For example, alkaline treatment may be used to lyse the cells or may be applied to previously lysed cells by another method. During the alkali treatment process according to some embodiments of the present invention, L-amino acids found in natural proteins and lipopeptides are at least partially racemized to D-amino acids. D-amino acids can be beneficial for increasing the shelf life of extracts because they are not digested efficiently in the intestines of mammals. D-amino acids can also protect smaller peptides and proteins from degradation during digestion. Examples of D-amino acids include protein-linked D-amino acids and less to lysinoalanine (de Vrese et al., J Nutrition, 2000, 2026-2031). Thus, antigenic molecules in extracts chemically modified during the dissolution process to contain D-amino acids can remain in the patient's body for longer periods of time, potentially in some embodiments exerting a stronger immune stimulating effect. .

In some embodiments, the filtration process may also affect the properties of the resulting extract, because the pore size of the filter and, in some cases, the chemical properties of the filter surface (ie its polarity) are removed and maintained. This is because the shape of the material can be modified. For example, some embodiments of the present invention utilize a filtration process designed to retain the molecule of interest but remove other molecular components such as nucleic acids or insoluble or particulate matter.

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

Brief description of the drawings

1: Ligands for a family of eleven toll-like receptors (TLRs) expressed by a host individual.

2: Diagram of a tangential flow filtration (TFF) system for the preparation of bacterial extracts after dissolution of bacteria. This diagram shows two different arrangements for the filters: in parallel mode all the filters operate at the same time, in serpentine mode the filters are arranged in serial mode.

3: Generalized correlation between operating parameters and flux, representing regions of pressure regulation and mass transfer regulation for tangential flow filtration processes (TFF).

Figure 4: Stimulation of splenocytes incubated for 48 hours in the presence of different dilutions of AFer300, CFer300, and DFer300, and ARahr300, CRahr300, and DRahr300 lysates. After addition of a 30 μl / well Alamar Blue® solution diluted 1: 1 in the cell culture medium, cells were further (a) 8.5 h (first experiment); (b) Incubate for 24 h (second experiment). Mean release values of duplicate cultures are shown at 590 nm ± standard deviation.

Figure 5: Nitric oxide (NO) in mice treated with extracts of Lactobacillus fermentum I-3929 and Lactobacillus rhamnosus 71.38 in (a) the first assay, and (b) the second chewing gum. A) induction of production. Results are expressed as mean ± standard deviation.

Figure 6: Effect of the extract of the present invention on airway hyperresponsiveness (AHR) by whole-body plethysmography (Emka) with increasing concentrations of inhaled methacholine one day after the last antigen challenge . Negative control animals treated with phosphate buffered saline (PBS) (n = 4), untreated LACK-attack animals (positive control group, n = 8), OM-1009A-treated LACK-attack mice (n = 8), and results for OM-1009B-treated LACK-attack mice (n = 7) (mean augmentation stop value +/− standard error of mean).

Detailed description of the invention

Justice

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

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

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

Lysate: As used herein, the term refers to an extract of bacteria obtained from the cell lysate section.

Filtration: As described herein, the filtration process refers to the passage of an extract or mixture of extracts through one or more filters, such as microfilters (ie, microfiltration) and / or ultrafilters (ie, ultrafiltration). Such filtration may not necessarily remove 100% of the components designed to be removed, but in one embodiment, allows the extract to be substantially free of such components. Filtration is repeated several times or several times.

 Initial pH: The term refers to the pH measured at the start of a procedure, such as bacterial dissolution or filtration.

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

Lipoproteins: The term refers to macromolecules comprising both protein or peptide chains, eg, proteins or peptides covalently bound to lipids. As used herein, lipoproteins include lipopeptides.

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

Lipoteichoic acid (LTA): This term refers to surface-associated attached amphiphilic molecules present in Gram-positive bacterial strains.

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

D-amino acids: The term refers to amino acids that exist in the preferential isomeric form, as opposed to biosynthetically produced L-amino acids, which exist in the form of the enantiomers.

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

Any stage of preparation of an extract that does not include a substance such as serum or meat extract obtained from an animal, such as a cow or sheep, or any other animal capable of carrying the prion-based disease, may be a medium that avoids the risk of prion-based disease. Means the culture medium used in the. Examples of such media include media using vegetable-based or synthetic media and horse serum or media comprising materials obtained from animal species that are not right for prion diseases. Examples of prion-based diseases include, for example, mad cow disease, scrapie, and Creutzfeld-Jacob disease.

Non-animal media are media that do not include components derived from animals. Examples include vegetable-based (ie, vegetable) media such as soybean media, and synthetic media.

As used herein, nutraceutical refers to any composition that may have a beneficial effect on health in a subject upon administration, wherein the composition is usable, for example, in a subject without a doctor's prescription.

As used herein in the therapeutic context, treatment refers to the treatment of an existing disease or disorder, as well as the prevention or protection against, for example, the development of a new disease or disorder.

As used herein, an adjuvant refers to an embodiment of the invention that refers to an embodiment of the invention and is provided to a subject in conjunction with a medical treatment plan.

As used herein, immunomodulation, immunomodulation, and other similar terms refer to an activity that modulates an immune response in a subject, such as an anti-inflammatory or immunostimulatory effect, in a manner that may indicate a beneficial health benefit. Means.

As used herein, anti-inflammatory and similar terms mean an immunomodulatory effect that serves to reduce inflammation.

As used herein, the terms of immunostimulatory and similar terms mean stimulation of the immune system.

As used herein, protective immunity means that an embodiment is provided to a subject to provide protection from subsequent attacks with an infectious agent or allergen. As a result, during the attack, the level of the infectious agent or allergen in the subject is sufficiently low to a concentration that does not significantly degrade the subject's health. The duration for which such protection from an attack will be effective may be limited, for example, to a period of hours, days, or weeks.

As used herein, subject refers to any animal subject, including mammalian subjects, such as humans and livestock. For example, livestock may include mammals such as dogs, cats, horses, pigs, cattle, sheep, goats, or other domestic animals, and may also include non-mammals such as birds, such as chickens, ducks, Geese, turkeys and other livestock birds.

Certain bacterial strains presented herein and used in the present invention are derived from the original deposits cited herein, including strains that were later re-deposited under different deposit code names but are considered to be genetically identical to the original deposited strains. It should be understood that the strains obtained or clones thereof may be included.

All numbers used herein are estimates that take into account the errors, rounding, and significant figures inherent in their measurements.

Preparation of Extract

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

Extracts of the invention can be prepared, for example, by culturing cells and subsequently harvesting, lysing and purifying the resulting biomass. For each strain, fermentation cultures can be inoculated into larger pre-fermentation baths, starting with a working seed lot, to obtain a sufficient amount of material.

The medium used may be the same for each species. In some embodiments, a medium free of risk of prion-based disease can be used to culture all strains to be used.

After fermentation, the resulting biomass from one strain or a set of strains can be inactivated, concentrated and frozen by heat treatment. As such, in some embodiments, the starting material used to make the extract may be whole cells that have been injured.

In other embodiments, the starting material used to prepare the extract may be at least in part a biomass obtained from a mechanically, enzymatically, or chemically lysed cell. In yet other embodiments, the starting material may be any fraction of such prelyzed cells, such as cell wall-containing fractions.

In some embodiments, the starting material is treated with an alkaline medium, for example from strong bases such as hydroxides, or other strong mineral or organic bases. In this lysis or base treatment step, undissolved cells in the starting material are lysed, whereas in some embodiments, cellular components can be chemically modified. Thus, in some embodiments, chemically modified bacterial molecules are subjected to base treatment, such as strong base treatment (ie, from uninjured cells or bacterial cells, as described above) for one or more Lactobacillus bacterial strains from which the extract is obtained. Base treatment on the components or fractions of

In some embodiments, 2 to 90 g / L, such as about 2 to about 80 g / L, or about 3 to about 40 g / L, such as 3, 5, 10, 15, 20, 25, 30, 35 Alternatively, 40 g / L, or even about 5-50 g / L or other range of biomass dry weight concentrations bounded by the concentrations listed above may be applied to the base treatment.

Biomass dry weight is defined herein as the dry weight of a material in grams per liter of sample. This can be measured by drying the sample in a small porcelain fold at about 105 ° C. until a constant weight is reached.

The temperature may be 30 to 60 ° C, for example 30 to 55 ° C, 30 to 50 ° C, 30 to 45 ° C, 30 to 40 ° C, or 30 to 35 ° C. In some embodiments, the base treatment temperature may be, for example, 35 to 60 ° C, such as 35 to 55 ° C, 35 to 50 ° C, 35 to 45 ° C, or 35 to 40 ° C. In some embodiments, the base treatment temperature is bounded by 31 ° C., 32 ° C., 33 ° C., 34 ° C., 35 ° C., 360 C, 37 ° C., 38 ° C., 39 ° C., or even 40 ° C., or the temperatures listed above. It may be a temperature in the range.

The time of base treatment can be from 2 hours to several days, such as 1, 2, 3, 4, 5 or even 10 days, or 3 to 120 hours or 3 to 48 hours, such as 3, 5, 8, 15, 14, 16 , 18, 20, 22, 24, 26, 28, 30, 36, 40, 44, or 48 hours, or 15 to 120 hours, such as 60 to 120 hours, such as 60, 72, 84, 96, 108, Or 120 hours, or a time in a range that is demarcated by the time listed above. It is to be understood that this range of times includes any partial value of days, hours, or minutes herein.

In some embodiments, a range starting or ending from 0.001 N to 1.0 N, such as 0.001 N to 0.6 N, or 0.10 N to 0.8 N, or 0.6 N to 1.0 N, or 0.001, 0.002, 0.003, or 0.1 N, Or a strong base concentration in the range starting or ending from 0.1 N to 0.6 N, or 0.6, 0.7, 0.8, 0.9, 1.0, or 1.0 N, or other range bounded by the concentrations listed above. In some embodiments, the base concentration is at least 9.0 initial pH, or at least 9.5, at least 10.0 and at most 13.5, such as at least 11.5, at least 12.0, at least 12.5, at least 13.0, or at pH 9.0 to pH. It is used to ensure that 13.0 is achieved. In still other embodiments, the base concentration may be used, for example, to achieve an initial pH of at least 10.0 and a pH of less than 13.0, or pH 9.0 to pH 13.0.

In some embodiments, the pH during base treatment may be reduced upon extraction of soluble components. For example, the initial pH can be a basic pH such as pH 9.0 to pH 13.0, or pH 9.5 to pH 12.5. Base treatment can proceed at a temperature listed above for a specific time period, such as 3 to 120 hours, such as 3 to 48 hours, or a time period as listed above. Then, in some embodiments, the pH is bounded by a pH of 2.0 to 4.5, or a pH of 2.5 to 4.5, or a pH of 2.5 to 4.0, such as 2.5, 3.0, 3.5, 4.0, or any of the pH listed above. May be made acidic, for example by addition of hydrochloric acid, in order for the range to be obtained. The second treatment at low pH is 30 to 60 ° C, 35 to 55, or 35 to 45 ° C, such as 35 ° C, 36 ° C, 37 ° C, 38 ° C, 39 ° C, 40 ° C, 41 ° C, 42 ° C, 43 ° C, 44 It may be carried out at a temperature of < RTI ID = 0.0 > The time of acid treatment can 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 24 hours, or 4 hours to 72 hours, or even 24 hours to 72 hours, or any time range bounded by the times listed above.

In some embodiments of the invention, the alkali treatment is applied to bacterial biomass comprising a material from, for example, Lactobacillus fermentum with a biomass dry weight of 10 g / L to 40 g / L. Is executed for. In another embodiment, the alkaline 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 embodiments, the alkali treatment may proceed for 3 to 48 hours, at a hydroxide ion concentration of 0.025 N to 0.25 N, or at a pH of 9.5 to 12.5, at a temperature of 35 to 45 ° C. In some embodiments, the alkaline treatment is 0.025 N to 0.20 N, 0.025 to 0.15 N, 0.025 to 0.10 N, 0.05 N to 0.25 N, 0.05 N to 0.20 N, 0.05 to 0.15 N, 0.05 N to 0.10 N, 0.10 N to Substances from one or more Lactobacillus strains at hydroxide ion concentrations of 0.25 N, 0.10 N to 0.20 N, 0.10 N to 0.15 N, 0.15 N to 0.25 N, 0.15 N to 0.20 N, or even 0.20 N to 0.25 N It can be performed on a bacterial biomass comprising a. Such embodiments include, for example, 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 to 12.0, 10.5 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 12.5, 11.5 to 12.0, or even pH 12.0 to 12.5. The time of alkali treatment for such embodiments 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 to 24 hours, 6 to 18 hours, 6 to 12 hours, 6 to 8 hours, 8 to 48 hours, 8 to 36 hours, 8 to 24 hours, 8 to 18 hours, 8 hours to 12 hours, 12 hours to 48 hours, 12 hours to 36 hours, 12 hours to 18 hours, 18 hours to 48 hours, 18 hours to 36 hours, 18 hours to 24 hours, 24 hours to 48 hours, 24 hours To 36 hours, or 36 to 48 hours. Alkali treatment may be carried out for any time period demarcating the above range, eg, for 3, 6, 8, 12, 18, 24, 36, or even 48 hours. Such conditions can provide adequate alkali treatment.

In another embodiment, a biomass dry weight of 10 g / L to 40 g / L from at least one Lactobacillus strain is 0.15 N to 0.50 N hydroxide at a temperature of 35 to 45 ° C. for 15 to 120 hours. May be subjected to ion concentration or pH 11.5-13.5. For example, in some embodiments, the hydroxide concentration is 0.15 N to 0.45 N, 0.15 N to 0.40 N, 0.15 N to 0.35 N, 0.15 N to 0.30 N, 0.15 N to 0.25 N, 0.15 N to 0.20 N, 0.20 N to 0.50 N, 0.20 N to 0.40 N, 0.20 N to 0.30 N, 0.25 N to 0.50 N, 0.30 N to 0.50 N, 0.30 N to 0.40 N, or 0.40 N to 0.50. Such embodiments may have, 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 to 12.5, 12.5 to 13.5, 12.5 to 13.0, 13.0 to 13.5. The time period for alkali treatment 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 to 100 hours, 24 to 90 hours, 24 to 75 hours, 24 to 60 hours, 24 to 48 hours, 36 to 120 hours, 36 to 100 hours, 36 to 90 hours Hour, 36 hours to 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, or 100 To 120 hours. Also in such embodiments the time periods contemplated for alkali treatment include 15, 24, 48, 60, 75 90, 100, and 120 hours. Such conditions can provide a strong alkali treatment.

In another embodiment, a dry biomass dry weight of from 10 g / L to 40 g / L has a hydroxide concentration of 0.025 N to 0.25 N or a pH of 9.5 to 12.5 at a temperature of 35 to 45 ° C. for a time of 48 to 48 hours. Can be treated as The pH can then be adjusted to 2.5 to 4.0 by addition of acid, such as hydrochloric acid (HCl), including acidic treatment. This acidic treatment can be carried out at a temperature of 35 to 45 ° C. for 1 to 24 hours. For example, in such embodiments, the alkaline treatment of bacterial biomass comprising one or more Lactobacillus strains may comprise 0.025 N to 0.20 N, 0.025 to 0.15 N, 0.025 to 0.10 N, 0.05 N to 0.25 N, 0.05 N to 0.20 N, 0.05 to 0.15 N, 0.05 N to 0.10 N, 0.10 N to 0.25 N, 0.10 N to 0.20 N, 0.10 N to 0.15 N, 0.15 N to 0.25 N, 0.15 N to 0.20 N, or even 0.20 N to 0.25 N It can be run at a hydroxide concentration of. In the course of alkali treatment, such embodiments are, for example, pH 9.5-12.0, 9.5-11.5, 9.5-11.0, 9.5-10.5, 9.5-10.0, 10.0-12.5, 10.0-12.0, 10.0-11.5, 10.0-11.0, 10.0 to 10.5, 10.5 to 12.5, 10.5 to 12.0, 10.5 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 12.5, 11.5 to 12.0, or even pH 12.0 to 12.5. . Alkali treatment times of such embodiments are 3 to 36 hours, 3 to 24 hours, 3 to 18 hours, 3 to 12 hours, 3 to 6 hours, 6 to 48 hours, 6 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 hours to 12 hours, 12 hours to 48 hours, 12 hours to 36 hours, 12 hours to 18 hours, 18 hours to 48 hours, 18 hours to 36 hours, 18 hours to 24 hours, 24 hours to 48 hours, 24 hours to 36 hours Or 36 hours to 48 hours. Alkali treatment may be performed for any time period bounded by the above ranges, for example 3, 6, 8, 12, 18, 24, 36, or even 48 hours. The pH can then be adjusted to 2.5 to 3.5, 2.5 to 3.0, 3.0 to 4.0, 3.0 to 3.5, or 3.5 to 4.0 by acid treatment, in the case of acid treatment after alkali dissolution. Acid treatment is 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 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. Also considered times for acid treatment include 1, 3, 6, 12, 18, and 24 hours.

The lysate obtained after the base treatment can then be purified, for example, by centrifugation and / or filtration to remove particulate and insoluble components. For example, the lysate can be centrifuged at 9000 x g (gravity) and subsequently subjected to one or more rounds of filtration in a 0.2 micron filter. In some cases, filtration on a 0.2 micron filter may be used after a continuous round of filtration on a larger porous filter. Ultrafiltration methods can also be used to aid in the extraction of soluble substances from the extract, for example, this ultrafiltration recycles the permeate (permeate) for further microfiltration.

In some embodiments, tangential flow filtration (TFF) methods can be used to filter lysate and extract soluble molecules from larger cell debris (FIG. 2). See, for example, Separations Technology, Pharmaceutical and Biotechnology Applications, Wayne P. Olson, Editor, lnterpharm Press, Inc., Buffalo Grove, IL, USA, p.126 to 135-ISBN: 0-935184- 72-4. At the start of such a method, the diluted bacterial lysate may be stored in a first tank. In TFF, for example, the extract can be exposed to both microfilters and ultrafilters. For example, a microfiltration (MF) loop is initiated and the product is pumped. The resulting MF retentate is recycled while the MF permeate is transferred to a second tank.

After reaching the appropriate concentration, the ultrafiltration (UF) loop is started. The UF permeate can be returned to the first tank and recycled for continuous extraction of the dissolved extract from the lysate, while the UF permeate is stored in a second tank. During continuous extraction, the volumes of tanks 1 and 2 can be adjusted by controlling the flow rates of microfiltration and ultrafiltration permeate.

Several such extraction cycles can be performed with TFF or another filtration method. In embodiments using TFF, at the end of the last cycle, the ultrafiltration loop can be stopped, the microfiltration loop can run alone and the MF permeate can be transferred to tank 2.

The conditions of crossflow and transmembrane pressures are determined by pressure dependent mass transfer in the film theory described by M. Cheryan in M. Cheryan (Ultrafiltration and Microfiltration Handbook, 2nd Ed., Ch. 4, 1998). Defined by the control region. Permeate intake and extraction yield are affected by filtration conditions (membrane pressure (TMP), crossflow, temperature, etc.). The type of filter may also affect the type of plate system (cassette filter), including filtration performance. Different arrangements may be used, including parallel mode and serpentine mode (see FIG. 2). Specific conditions are developed for optimized performance for each combination of type of mode and filter type used.

The microfiltration loop is adapted to filters from 1.2 microns to 0.1 microns, such as 0.65 to 0.2 microns, or 0.45 microns. The crossflow can be 100 to 3000 liters per hour m 2 (LHM), such as 300 to 2500 LHM, or 2000 LHM with 0.3 to 2 bar TMP. The ultrafiltration loop is adapted to a filter of 10 KDa to 1000 KDa, such as 10 KDa to 100 KDa, or 10 KDa to 30 KDa, or 30 KDa to 100 KDa. The crossflow can be 30-1000 LHM, such as 20-500 LHM, with a TMP of 0.2-1.5 bar.

5 to 20 dialysis volumes can be used to extract soluble components from bacterial cell walls. In some embodiments, 8 to 15 volumes are used. Thus, for example, in some embodiments, 5 to 15 cycles of filtration can be used, and in some cases 8 to 15 cycles can be used.

After filtration, the extract can be further concentrated or centrifuged if necessary. For example, additional microfiltration using a smaller porous filter, such as a 0.2 micron filter, can be performed. After filtration, the extract can be lyophilized prior to formulating it for use.

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

Chemical Properties of Bacteria Extracts

Base treatment can result in various chemical modifications to cellular components. For example, in proteins: (1) peptide bonds may undergo partial cleavage to produce smaller polypeptides; (2) the natural L-amino acid can be at least partially racemized with D-amino acid; (3) Asparagine and glutamine residues are deaminoated, resulting in changes in protein isoelectric point. Molecules such as lipoteichoic acid, lipopeptides, and phospholipids are modified amphiphilic structures that can undergo base-catalytic hydrolysis of ester bonds and / or amide bonds and have new physicochemical and immunological properties. May be caused. Examples of other possible chemical modifications include partial hydrolysis of cell wall polysaccharides and complete hydrolysis of ribonucleic acid (RNA) into individual ribonucleotides, including rearrangement of phosphate groups.

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

For example, base treatment of bacteria according to the present invention can result in partial hydrolysis of proteins as well as deaminoation, deamides, and / or partial racemization of amino acids from L to D. In one analytical study for extracts according to the invention, D-aspartic acid, D-glutamic acid, D-serine, D-methionine, D-histidine, D-alanine, D-arginine, D-phenylalanine, D-tyrosine, D Peaks representing -leucine and D-lysine were observed respectively. The proportion of D-amino acids of such species in the study ranged from 3% to 40%. Accordingly, some embodiments of the present invention provide for the racemization of one or more serine, threonine, histidine, alanine, arginine, tyrosine, phenylalanine, leucine, and lysine, such as racemization of all of the above amino acids, or for example alanine, phenylalanine and Like lysine, it allows racemization of any selected amino acid of one or more but less than all of said amino acids. In some embodiments, at least 10% of one or more of these amino acids may be racemized from D to L. In other embodiments, 40% of one or more of the amino acids can be racemized.

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

Some extracts of the present invention include Lactobacillus bacterial cell walls and membrane components such as lipoteichoic acid, teichoic acid, peptidoglycan, or combinations thereof. In some embodiments, such components are chemically modified. Some extracts also include cell wall and / or cell membrane components, such as lipoproteins, which may also be chemically modified. In some embodiments, cell wall components or cell membrane components, such as lipoprotein, are dissolved or suspended in an extract and are therefore not in particulate or insoluble form.

In addition, extracts according to the invention can be prepared by, for example, 10 to 100 mg / mL soluble dry weight (SDW) of substance, 1 to 30 mg / mL of protein (Prot.), 0.5 to 4.0 mg / mL of sugar and It may contain up to 100 μg / mL of DNA. For example, some embodiments may contain about 15 to 35 mg / mL soluble dry weight, 3 to 7 mg / mL protein, 1.0 to 3.0 mg / mL sugar and 10 to 40 μg / mL DNA. have. Extracts according to the invention may contain, for example, 30 mg / mL of soluble dry weight, 9.6 mg / mL protein, 2.4 mg / mL of sugar and 33 μg / mL of DNA, another example being 32.4 It may contain soluble dry weight of mg / mL, 5.8 mg / mL protein, 2.3 mg / mL sugar and up to 100 μg / mL DNA. Soluble dry weight (SDW) of g / L or mg / mL is measured by obtaining 5 mL of the resulting soluble fraction resulting from dissolution or base treatment and drying it to constant mass in a ceramic dish at 105 ° C.

In some embodiments, the extract comprises 0.3 mg / mL or more saccharides, such as 0.3 to 4.5 mg / mL saccharide. In some embodiments, at least one saccharide is selected from monosaccharides, disaccharides and polysaccharides. Some extracts of the present invention comprise at least one branched polysaccharide. In some embodiments, at least one saccharide is chemically modified.

Dissolution or base treatment of bacteria according to the present invention results in decreasing the average molecular weight of the component macromolecules, for example, between 1 kDa and 300 kDa and 100 kDa, or between 1 kDa and 60 kDa and 10 kDa. Can cause. In some embodiments, the extract comprises one or more proteins having a molecular weight of 50 kDa or less or 30 kDa or less, such as 10 kDa or less.

Biological Activity of Bacterial Extracts

Extracts 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 particular effect of the extract may depend on the conditions of preparation and the species or strain of Lactobacillus, or a mixture of species or strains, from which the extract is obtained. Thus, some extracts according to the present invention may exhibit potent immunostimulatory activity, and thus may be useful in treating infections or as an ancillary for such treatment, while other embodiments exhibit weaker immunostimulatory activity. It may exhibit anti-inflammatory activity and thus be useful for treating or as an accessory for treating inflammatory disorders such as allergies, asthma, autoimmune diseases, colitis, and inflammatory bowel disease.

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, digestive tract disorders. In addition, some extracts according to the present invention are functional foods, such as, but not limited to, microbial infections, allergic diseases, and digestive tract disorders, in the treatment of various conditions, provided as an adjuvant to a patient. Can be.

The range of biological activity of the extract can be determined by several in vitro and in vivo assays. For example, AlamarBlue ™ -Assay includes fluorescence / chromic growth indicators based on the detection of metabolic activity by redox indicators in response to chemical decreases excited by cell growth (Example 4).

In vitro cell assays can assess the production of nitric oxide (NO) from primary murine macrophages and screen the activity of extracts that stimulate the immune system to kill invading bacteria (FIG. 5). In some embodiments, the extract can stimulate NO production in murine macrophages such that measured NO concentrations range from 3 μM to 60 μM, such as 5 μM to 40 μM. In some embodiments, the NO concentration may be greater than 30 μM. The type of bacterial species may also influence these results. For example, Lactobacillus The extract from fermentum is Lactobacillus rahmnosus ) can lead to more production of nitric oxide (see, eg, Example 5 below). In order to screen embodiments of the invention with respect to their immunostimulatory or anti-inflammatory potential in vitro, evaluation of the bacterial extracts of the invention can be performed on human peripheral blood mononuclear cells (PBMCs). See, eg, Foligne et al., World J Gastroenterol , 2007, 13 (2): 236-243). Release of both IL12p70 (inflammatory cytokine) and IL10 (anti-inflammatory cytokine) can be measured and the IL-10 / IL-12 ratio calculated (Example 6). Some embodiments of the present invention exhibit a higher IL10 / IL12 ratio than the live lactobacterial fermentum control, so that when some extracts of the present invention are administered in vivo, they are equivalent to or even stronger than the living parental microorganisms. -Suggests inflammatory properties. Accordingly, the present invention encompasses extracts capable of achieving a calculable IL10 / IL12 ratio in human peripheral blood mononuclear cells, wherein the ratio is in combination with the IL10 / IL12 ratio achieved by the live Lactobacillus strain from which the extract is obtained. Same or exceed the above ratio.

The immune response of the extracts of the invention can also be tested by examining their effects on toll-like receptors (TLRs). For example, extracts can be tested in HEK293 cells in the presence or absence of TLR2 agonist Pam3Cys, or in the presence or absence of TLR4 agonist LPS (Example 7). HEK293 cell lines allow efficient monitoring of TLR activity using ELISA, such as receptor-based systems that monitor IL-8 titration or TLR-induced NF-κB activation. Some embodiments of the invention may serve as TLR 2/6 antagonists in HEK TLR 2/6 cells. Thus, some embodiments may be useful for combating infection in a subject, while other embodiments may be developed for use against inflammation and / or autoimmune diseases.

The extracts disclosed herein can be screened for TLR and NOD2 receptor activity (Example 8). Some embodiments of the present invention activate TLRs and / or NOD2 receptors in vitro, suggesting that they can activate the immune system via TLRs and / or NOD2.

Plaque Forming Cells (PFC) technique can be used to assess nonspecific stimulation of B-lymphocytes (Example 9). Certain lymphoid cells form lytic plaques in the presence of complement to disperse neighboring erythrocytes and release hemolytic antibodies that cause lysis of these erythrocytes. Some embodiments of the present invention can increase the secretion of immunoglobulins by B cells and thus can be used to preventively prime the immune system in subjects with potentially recurrent infections.

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

For example, in allergen-induced asthma (Example 11), the combination of in vitro NO activity with measurement of measured in vivo activity may provide a more complete review of the potential clinical activity of the extracts disclosed herein. Can be. In the LACK (protein from parasitic Leishmania major) model, the levels of eosinophils found in bronchial-alveolar lavage fluids, as compared to asthmatic control (untreated) animals, ranged from 1 to 10 factors, such as 1.5. It can be reduced from 5 to 5 times. Thus, in some embodiments, the extract may reduce eosinophil cell count, neutrophil cell count, lymphocyte cell count, or any combination thereof in asthmatic murine subjects by an index of at least 1.5, compared to an asthma untreated control. Some embodiments of the present invention may reduce eosinophilia in asthmatic subjects and may concomitantly reduce the level of Th2 cytokines (eg, IL4, IL5, IL13), markers of asthma. Thus, such embodiments may exhibit anti-inflammatory activity in a subject suffering from an immunological disorder, such as, for example, an allergic disorder, including asthma.

Compositions Containing Bacteria Extracts

Extracts according to the invention can be formulated for final administration in a number of different ways. For example, oral tablets, capsules, pills, as well as liquid formulations or aerosols can be prepared. Injectable or injectable formulations may also be prepared.

Embodiments of the invention may be formulated, for example, in solid dosage form or liquid dosage form. Representative solid dosage forms include, for example, tablets, such as coated tablets, chewable tablets, effervescent tablets, sublingual tablets, granulates, powders, or capsules containing extracts. Can be.

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

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

Extracts of the invention may be included in one or more nutraceutical compositions, such as nutritional and / or dietary supplements and food additives, or one or more pharmaceutical compositions.

On subject  Dosing of bacterial extracts

Dosages comprising one or more extracts of the invention may be administered to a subject suffering from or at risk of developing one or more disorders selected from digestive tract disorders, respiratory disorders, urinary tract disorders, and allergic diseases. For example, in some embodiments, the extract may be used for treating upper and lower lung infections, obstructive pulmonary disease with acute lower respiratory infection, obstructive pulmonary disease with acute exacerbation, nasopharyngitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, Laryngary-pharyngitis, influenza, pneumonia, bronchial pneumonia, bronchitis, rhinitis, nasopharyngitis, pharyngitis, sinusitis, tonsillitis, laryngitis, laryngeal-bronchiolitis, bronchitis, allergic rhinitis, allergic asthma, atopic dermatitis, obstructive and reflux uropathy Urinary tract infections, urethritis, tubular interstitial nephritis, obstructive pyelonephritis, cystitis including chronic cystitis, male pelvic pain syndrome, including prostatitis and chronic prostatitis, prostate cystitis, female pelvic inflammatory disease, Crohn's disease, and / or hypersensitivity It may be administered to a subject suffering from colorectal syndrome or at risk of developing such a disease.

In some embodiments, the extract is administered to the subject in the form of a nutraceutical composition, such as a nutritional supplement and / or a food additive. In another embodiment, the extract is administered to the subject in the form of a pharmaceutical composition. Administration can include single dose or multiple dose administrations.

In some embodiments, the extract may be provided in a therapeutically effective dosage for treating a subject suffering from one or more of the conditions described above. In some embodiments, the extract may be provided as an adjunct for other medical treatments.

Example

Example  One; Bacterial culture

Example 1.1 Lactobacillus fermentum ) I-3929 culture

Initial culture condition

Culture medium was prepared by dissolving the following components in purified water: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone 50 g / L; Glucose: 12 g / L; Calcium chloride: 0.1 g / L; Potassium chloride: 0.1 g / L; Sodium bicarbonate: 0.5 g / L; Pyruvate: 0.1 g / L; Glutamate: 0.2 g / L; Metal solution (copper sulfate: 3 mg / l; iron chloride: 830 mg / l; zinc sulfate: 860 mg / l; sulfuric acid: 1.1 mg / L): 0.5 mL / L. Polypropylene glycol (0.02 mL / L, density 1.005 g / mL) was then added. After dissolution, no pH was adjusted. After sterilizing the medium, the small Elenmeyer flasks were individually inoculated with the contents of the frozen vial (containing 1.5 mL of frozen bacteria) and incubated at 37 ° C. for 8 hours. An aliquot of this culture was then transferred to a larger Elenmeyer 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 Ellenmeyer flask were transferred to a prefermenter.

In pre-fermentation tank  Culture condition

20 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; 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) was then added. After dissolution, no pH was adjusted. The incubation temperature was adjusted to 37 ° C. while stirring at 100 rpm without venting. pH was not adjusted during incubation. After 24 hours, 7 liters of the prefermentation tank was transferred to the fermenter (optical density (OD) = 1.24 at 700 nm after 24 hour incubation). The culture of the prefermentation tank was transferred to the fermenter under sterile conditions.

Culture conditions in fermenter

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; 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) was then added. After dissolution, no pH was adjusted.

After sterilization, 8 g / L glucose was added to the culture medium. The incubation temperature was adjusted to 37 ° C. while stirring at 100 rpm without venting. The pH was adjusted to 5.7 during incubation. After 16 hours, the culture (OD of culture at 700 nm = 2.30) was inactivated by heat treatment at 65 ° C. for 35 minutes and transferred to a harvest tank. Once inactivated, the cultures were transferred to ultrafiltration skids to separate the biomass from the culture medium, concentrated and washed with NaCl (9 g / L) in purified water. The collected biomass was aliquoted (weight of bacterial suspension concentrated to 2000 g was 31.8 mg dry weight biomass per gram of concentrated bacterial suspension) and then frozen at -15 ° C.

Example  1.2: Lactobacillus Helvetica ( Lactobacillus helveticus Culture of 103146

Initial culture condition

Culture medium was prepared by dissolving the following components in purified water: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone 50 g / L; Glucose: 12 g / L; Calcium chloride: 0.1 g / L; Potassium chloride: 0.1 g / L; Sodium bicarbonate: 0.5 g / L; Pyruvate: 0.1 g / L; Glutamate: 0.2 g / L; Metal solution (copper sulfate: 3 mg / l; iron chloride: 830 mg / l; zinc sulfate: 860 mg / l; sulfuric acid: 1.1 mg / L): 0.5 mL / L. Polypropylene glycol (0.02 mL / L) was then added. After dissolution, no pH was adjusted. After sterilizing the medium, the small Elenmeyer flasks were individually inoculated with the contents of the frozen vial (containing 1.5 mL of frozen bacteria) and incubated at 37 ° C. for 9 hours. An aliquot of this culture was then transferred to a larger Elenmeyer 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 Ellenmeyer flask were transferred to a prefermentation bath.

In pre-fermentation tank  Culture condition

20 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; 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 A. Polypropylene glycol (0.02 mL / L) was then added. After dissolution, no pH was adjusted. The incubation temperature was adjusted to 37 ° C. while stirring at 100 rpm without venting. pH was not adjusted during incubation. After 9 hours, 7 liters were transferred from the pre-fermentation tank to the fermentation tank. (OD: 0.14 at 700 nm after incubation for 9 hours). The culture of the prefermentation tank was transferred to the fermenter under sterile conditions.

Culture conditions in fermenter

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; 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) was then added. After dissolution, no pH was adjusted.

After sterilization, 8 g / L glucose was added to the culture medium. The incubation temperature was adjusted to 37 ° C. while stirring at 100 rpm without venting. The pH was adjusted to 6.7 at the start of the culture with CH ₃ COOH. After 124 hours, the culture (OD of culture at 700 nm = 4.17) was inactivated by heat treatment at 65 ° C. for 35 minutes and transferred to a collection tank. Once inactivated, the cultures were transferred to ultrafiltration skids to separate the biomass from the culture medium, concentrated and washed with NaCl (9 g / L) in purified water (9 g / L). The collected biomass was aliquoted (weight of bacterial suspension concentrated to 440 g was 19.1 mg dry weight biomass per gram of concentrated bacterial suspension) and then frozen at -15 ° C.

Example  1.3: Lactobacillus Planta Room ( Lactobacillus plantarum 71.39 incubation

Initial culture condition

Culture medium was prepared by dissolving the following components in purified water: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone 50 g / L; Glucose: 12 g / L; Calcium chloride: 0.1 g / L; Potassium chloride: 0.1 g / L; Sodium bicarbonate: 0.5 g / L; Pyruvate: 0.1 g / L; Glutamate: 0.2 g / L; Metal solution (copper sulfate: 3 mg / l; iron chloride: 830 mg / l; zinc sulfate: 860 mg / l; sulfuric acid: 1.1 mg / L): 0.5 mL / L. Polypropylene glycol (0.02 mL / L) was then added. After dissolution, no pH was adjusted. After sterilizing the medium, the small Elenmeyer flasks were individually inoculated with the contents of the frozen vial (containing 1.5 mL of frozen bacteria) and incubated at 37 ° C. for 9 hours. An aliquot of this culture was then transferred to a larger Elenmeyer 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 Ellenmeyer flask were transferred to a prefermentation bath.

In pre-fermentation tank  Culture condition

20 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; 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) was then added. After dissolution, no pH was adjusted. The incubation temperature was adjusted to 37 ° C. while stirring at 100 rpm without venting. pH was not adjusted during incubation. After 9 hours, 7 liters were transferred from the pre-fermentation tank to the fermentation tank. (OD = 1.62 at 700 nm after incubation for 9 hours). The culture of the prefermentation tank was transferred to the fermenter under sterile conditions.

Culture conditions in fermenter

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; 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) was then added. After dissolution, no pH was adjusted.

After sterilization, 8 g / L glucose was added to the culture medium. The incubation temperature was adjusted to 37 ° C. while stirring at 100 rpm without venting. After 24 hours, the cultures (OD of 6.36 cultures at 700 nm) were inactivated by heat treatment at 65 ° C. for 35 minutes and transferred to a collection tank. Once inactivated, the cultures were transferred to ultrafiltration skids to separate the biomass from the culture medium, concentrated and washed with NaCl (9 g / L) in purified water. The collected biomass was aliquoted (weight of bacterial suspension concentrated to 600 g was 60.7 mg dry weight biomass per gram of concentrated bacterial suspension) and then frozen at -15 ° C.

Example  1.4: Lactobacillus Ramnosus ( Lactobacillus rhamnosus 71.38 incubation

Initial culture condition

Culture medium was prepared by dissolving the following components in purified water: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone 50 g / L; Glucose: 12 g / L; Calcium chloride: 0.1 g / L; Potassium chloride: 0.1 g / L; Sodium bicarbonate: 0.5 g / L; Pyruvate: 0.1 g / L; Glutamate: 0.2 g / L; Metal solution (copper sulfate: 3 mg / l; iron chloride: 830 mg / l; zinc sulfate: 860 mg / l; sulfuric acid: 1.1 mg / L): 0.5 mL / L. Polypropylene glycol (0.02 mL / L) was then added. After dissolution, no pH was adjusted. After sterilizing the medium, the small Elenmeyer flasks were individually inoculated with the contents of the frozen vial (containing 1.5 mL of frozen bacteria) and incubated at 37 ° C. for 9 hours. An aliquot of this culture was then transferred to a larger Elenmeyer 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 Ellenmeyer flask were transferred to a prefermentation bath.

In pre-fermentation tank  Culture condition

20 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; 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) was then added. After dissolution, no pH was adjusted. The incubation temperature was adjusted to 37 ° C. while stirring at 100 rpm without venting. pH was not adjusted during incubation. After 9 hours, 7 liters were transferred from the pre-fermentation tank to the fermentation tank. (OD: 3.75 at 700 nm after incubation for 9 hours). The culture of the prefermentation tank was transferred to the fermenter under sterile conditions.

Culture conditions in fermenter

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; 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) was then added. After dissolution, no pH was adjusted.

After sterilization, 8 g / L glucose was added to the culture medium. The incubation temperature was adjusted to 37 ° C. while stirring at 100 rpm without venting. After 14 hours, the culture (OD of culture at 700 nm = 5.43) was inactivated by heat treatment at 65 ° C. for 35 minutes and transferred to a collection tank. Once inactivated, the cultures were transferred to ultrafiltration skids to separate the biomass from the culture medium, concentrated and washed with NaCl (9 g / L) in purified water. The collected biomass was aliquoted (weight of bacterial suspension concentrated to 2798 g was 51.7 mg dry weight biomass per gram of concentrated bacterial suspension) and then frozen at -15 ° C.

Example  1.5: Lactobacillus John sony ( Lactobacillus johnsonii Culture of 103782

Initial culture condition

Culture medium was prepared by dissolving the following components in purified water: sodium chloride: 3 g / L; Sodium monohydrogen phosphate: 2 g / L; Sodium acetate: 1 g / L; Soy peptone 50 g / L; Glucose: 12 g / L; Calcium chloride: 0.1 g / L; Potassium chloride: 0.1 g / L; Sodium bicarbonate: 0.5 g / L; Pyruvate: 0.1 g / L; Glutamate: 0.2 g / L; Metal solution (copper sulfate: 3 mg / l; iron chloride: 830 mg / l; zinc sulfate: 860 mg / l; sulfuric acid: 1.1 mg / L): 0.5 mL / L. Polypropylene glycol (0.02 mL / L) was then added. After dissolution, no pH was adjusted. After sterilizing the medium, the small Elenmeyer flasks were individually inoculated with the contents of the frozen vial (containing 1.5 mL of frozen bacteria) and incubated at 33 ° C. for 10 hours. An aliquot of this culture was then transferred to a larger Elenmeyer 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 Ellenmeyer flask were transferred to a prefermentation bath.

In pre-fermentation tank  Culture condition

20 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; 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) was then added. After dissolution, no pH was adjusted. The incubation temperature was adjusted to 35 ° C. while stirring at 100 rpm without venting. The pH of the culture was adjusted to 5.6 with acetic acid. After 24 hours, 7 liters from the prefermentation tank were transferred to the fermentor. (OD: 0.47 at 700 nm after incubation for 24 hours). The culture of the prefermentation tank was transferred to the fermenter under sterile conditions.

Culture conditions in fermenter

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; 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) was then added. After dissolution, no pH was adjusted.

After sterilization, 8 g / L glucose was added to the culture medium. The incubation temperature was adjusted to 35 ° C. while stirring at 100 rpm without venting. After 24 hours, the culture (OD = 0.174 of the culture at 700 nm) was inactivated by heat treatment at 70 ° C. for 30 minutes and transferred to a collection tank. Once inactivated, the cultures were transferred to ultrafiltration skids to separate the biomass from the culture medium, concentrated and washed with NaCl (9 g / L) in purified water. The collected biomass was aliquoted (61.5 g of bacterial suspension concentrated in 20.2 mg of dry weight biomass per gram of concentrated bacterial suspension) and then frozen at -15 ° C.

Example  2: bacteria Melt

Example  2.1

6 g Lactobacillus buffer lactofermentum from Example 1.1 containing the bacterial dry weight (of Lactobacillus fermentum ) An aliquot of I-3929 biomass was thawed at room temperature and diluted with purified water to a dry weight of 12 g / L bacterial biomass. Alkalinization was performed with 0.03 M sodium hydroxide. The pH measured at the start of dissolution was 10.3. The lysates were then incubated at 40 ° C. for 6 hours under continuous stirring. After incubation, the pH was 9.9.

Example  2.2

20 g Lactobacillus ramno Versus (Lactobacillus from Example 1.4 containing the bacterial dry weight of rhamnosus ) An aliquot of 71.38 biomass was thawed at room temperature and diluted with purified water to a dry weight of 40 g / L bacterial biomass. Alkalization was performed with 0.03 M sodium hydroxide. The pH measured at the start of dissolution was 10.3. The lysates were then incubated at 40 ° C. for 6 hours under continuous stirring. After incubation, the pH was 9.7.

Example  2.3

6 g Lactobacillus buffer lactofermentum from Example 1.1 containing the bacterial dry weight (of Lactobacillus fermentum ) An aliquot of I-3929 biomass was thawed at room temperature and diluted with purified water to a dry weight of 12 g / L bacterial biomass. Alkalization was carried out with 0.06 M sodium hydroxide. The pH measured at the start of dissolution was 12.3. The lysates were then incubated at 40 ° C. for 6 hours under continuous stirring. After incubation, the pH was 11.8.

Example  2.4

6 g Lactobacillus buffer lactofermentum from Example 1.1 containing the bacterial dry weight (of Lactobacillus fermentum ) An aliquot of I-3929 biomass was thawed at room temperature and diluted with 0.2 N NaCl to dry weight 12 g / L of bacterial biomass. The final concentration of NaCl solution was 0.15N. The pH measured at the start of dissolution was 6.4. The lysates were then incubated at 40 ° C. for 6 hours under continuous stirring. After incubation, the pH was 6.3.

Example  2.5

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

Example  2.6

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

Example  2.7

0.4 g Lactobacillus Planta room (Lactobacillus from Example 1.3 containing the bacterial dry weight of plantarum ) An aliquot of 71.39 biomass was thawed at room temperature and then diluted with purified water to a dry weight of 7 g / L bacterial biomass. Alkalization was carried out with 0.06 M sodium hydroxide. The pH measured at the start of dissolution was 12.6. The lysates were then incubated at 40 ° C. for 2 hours under continuous stirring. After incubation, the pH was 12.4.

Example  2.8

0.3 g Lactobacillus zone Sony (Lactobacillus from Example 1.5 containing the bacterial dry weight of johnsonii ) An aliquot of 103782 biomass was thawed at room temperature and then diluted with purified water to a dry weight of 6 g / L bacterial biomass. Alkalization was carried out with 0.06 M sodium hydroxide. The pH measured at the start of dissolution was 12.5. The lysates were then incubated at 40 ° C. for 2 hours under continuous stirring. After incubation, the pH was 12.3.

Example  2.9

0.4 g Lactobacillus helveticus (Lactobacillus from Example 1.2 containing the bacterial dry weight of helveticus) diluted 103 146 Bio Math was thawed at room temperature and then the aliquot of the purified water is made to be 8 g / L dry weight of bacteria bio Math. Alkalization was carried out with 0.06 M sodium hydroxide. The pH measured at the start of dissolution was 12.8. The lysates were then incubated at 40 ° C. for 2 hours under continuous stirring. After incubation, the pH was 12.2.

Example  2.10

Lactobacillus fermentum from Example 1.1 containing 6.8 g of bacterial dry weight (Lactobacillus fermentumAn aliquot of I-3929 biomass was thawed at room temperature and diluted with purified water to a dry weight of 7 g / L bacterial biomass. Alkalization was carried out with 0.08 M sodium hydroxide. The pH measured at the start of dissolution was 12.2. The lysates were then incubated at 40 ° C. for 7 hours under continuous stirring. After incubation, the pH was 12.0. Lysate pH was adjusted to 9.8 with HCl.

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

Example  2.11

6.8 g Lactobacillus buffer lactofermentum from Example 1.1 containing the bacterial dry weight (of Lactobacillus fermentum ) An aliquot of I-3929 biomass was thawed at room temperature and diluted with purified water to a dry weight of 7 g / L bacterial biomass. Alkalization was performed with 0.15 M sodium hydroxide. The pH measured at the start of dissolution was 13.0. The lysates were then incubated at 40 ° C. for 63 hours under continuous stirring. After incubation, the pH was 12.6. Lysate pH was adjusted to 9.9 with HCl.

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

Example  2.12

98 g Lactobacillus buffer lactofermentum from Example 1.1 containing the bacterial dry weight (of Lactobacillus fermentum ) An aliquot of I-3929 biomass was thawed at room temperature and diluted with purified water to a dry weight of 10 g / L bacterial biomass. Alkalization was carried out with 0.08 M sodium hydroxide. The pH measured at the start of dissolution was 12.0. The lysates were then incubated at 40 ° C. for 24 hours under continuous stirring. After incubation, the pH was 11.1. Lysate pH was adjusted to 11.5 with NaOH.

The lysate consisted of SDW: 23.7 mg / g.

Example  2.13

39 g Lactobacillus buffer lactofermentum from Example 1.1 containing the bacterial dry weight (of Lactobacillus fermentum ) An aliquot of I-3929 biomass was thawed at room temperature and then diluted with 0.2 N NaCl solution to produce a dry weight of bacterial biomass of 7.6 g / L to produce purified osmotic lysate. 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. After incubation, the pH was 4.2. The bacterial lysates were then alkaline by adding NaOH to 0.06 N. The pH measured at the start of alkaline dissolution was 10.6. This lysate was reincubated at 40 ° C. for 2.25 h under continuous stirring. After incubation, the pH was 10.2.

Example  3: Lysate  refine

Example  3.1

The lysate of Example 2.5 was centrifuged at 3000 x g for 20 minutes. The supernatant was then adjusted to pH 6.8 with NaOH 10N and filtered through a continuous filter with 0.45 μm and 0.2 μm porosity and finally filtered with a sterile filter 0.22 μm. Concentrate was Prot: 2.8 mg / mL; DNA: 17.4 μg / mL. D-amino acid ratio: D-Ala 14.9%, D-Pro 14.4%, D-Asp 14.2%.

NO production in dry weight mg / mL: active dry weight / mL: 0.003 mg / mL (C1)-0.03 mg / mL (C2)-0.3 mg / mL (C3): C1: 3.3 μM, C2: 33.2 μM, C3: 52.5 μM. The active dry weight in g / L or mg / mL is the soluble dry weight in g / L or mg / mL minus the chloride content in g / L or mg / mL.

Example  3.2

The lysate of Example 2.6 was centrifuged at 3000 x g for 20 minutes. The supernatant was then adjusted to pH 7 with HCl, filtered through a continuous filter with 0.45 μm and 0.2 μm porosity and finally filtered with a sterile filter 0.22 μm. Concentrate was Prot: 12.3 mg / mL; DNA: 27.7 μg / mL. D-amino acid ratio: D-Ala 15.9%, D-Pro 24.4%, D-Asp 17.4%, D-Ser 45.1%, D-Met 10.1%.

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

Example  3.3

300 ml of the lysate of Example 2.1 were first centrifuged at 3000 x g for 20 minutes. The pH of the supernatant was adjusted to 7.1 with HCl. The extract was concentrated through a 0.45 μm membrane in polytersulfone (PES) (Sartorius Stedim Biotech GmbH) at a constant flow rate of 330-350 mL / min in the Sartoflow® Slice 200 Benchtop Crossflow System. The volume obtained was 75%. The concentrate was finally sterile filtered by passing through a 0.2 μm PES membrane (Nalgene).

Concentrate was SDW: 30.0 mg / g; Prot: 9.6 mg / mL; DNA: 32.8 μg / mL. D-amino acid ratio: D-Ala 14.3%, D-Pro 13.9%, D-Asp 14.1%, D-Ser 36.9%. NO production in mg active dry weight per mL: active dry weight / mL: 0.003 mg / mL (C1)-0.03 mg / mL (C2)-0.3 mg / mL (C3): C1: 5.1 μM, C2: 30.0 μΜ, C3: 64.0 μΜ.

Example  3.4

The pH of the lysate of 300 ml of Example 2.2 was adjusted to pH 7.0 with HCl. The extract was filtered at a constant flow rate of 350 mL / min as described in Example 3.3. The volume obtained was greater than 75%. The concentrate was finally sterile filtered through a 0.2 μm PES membrane (Nalgene).

Concentrate was SDW: 49.2 mg / g; Prot: 14.2 mg / mL; DNA: 34.1 μg / mL. D-amino acid ratio: 16.0% D-Ala, 13.3% D-Pro, 14.2% D-Asp.

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

Example  3.5

300 ml of the lysate of Example 2.3 were first centrifuged at 3000xg for 20 minutes. The pH of the supernatant was adjusted to pH 7.1 with HCl. The extract was filtered at a constant flow rate of 330-350 mL / min as described in Example 3.3. The volume obtained was greater than 75%. The concentrate was finally sterile filtered through a 0.2 μm PES membrane (Nalgene).

Concentrate was SDW: 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 in mg of active dry weight per mL: active dry weight / mL: 0.003 mg / mL (C1)-0.03 mg / mL (C2)-0.3 mg / mL (C3): C1: 3.0 μM, C2: 36.4 μΜ, C3: 69.5 μΜ.

Example  3.6

100 ml of the lysate of Example 2.13 was first centrifuged at 9384 x g for 20 minutes. The supernatant was filtered by direct passage through a 0.22 μm sterile filter.

Concentrate was SDW: 32.7 mg / g; Prot: 6.5 mg / g; Sugar: consisted of 2.4 mg / g. Sugar concentrations are described by Herbert et al., Meth. Microbiol, 1971, 5B: 266 et seq.].

Screening for biological activity was performed on human peripheral blood mononuclear cells (PBMC) at different concentrations as described in Example 6. Results obtained with 0.5 mg of active dry weight / mL yielded TNF-α: 83 pg / mL; IL-6: 898 pg / mL; IL-12: 140 pg / mL (with 10 ng / ml IFN-γ); And IL-10: 221 pg / mL in the presence of IFN-γ.

Example  3.7

The bacterial lysate mixture of Example 2.10 was adjusted to pH 10.2 and then transferred to a microfiltration (MF) tank. The microfiltration (MF) unit used was a 0.45 μm tangential flow filtration (TFF) filter (Sartocon Slice Sartorius). The cross flow was adjusted to 290 L / h m 2 (LHM) and the Trans Membrane Pressure (TMP) to 0.4-0.5 bar. Permeate was transferred to an ultrafiltration (UF) tank.

When the lysate in the microfiltration tank reached half the initial volume, the UF unit was run. The permeate of the UF filter was used as wash buffer in the MF tank. The volumes of both the MF and UF tanks were kept at the same level. After 10 dialysis volumes, UF was stopped and bacterial lysates were concentrated in the MF tank. The recovered volume yield was 83%. The pH of the extract recovered in the UF tank was adjusted to HCl and then the extract was filtered through a 0.2 μm sterile filter.

Concentrate was SDW: 17.5 mg / g; Prot: 4.3 mg / g.

Example  3.8

The lysate of Example 2.7 was centrifuged at 9384 x g for 15 minutes. The pH of the supernatant was then adjusted to pH 7.3 with HCl, filtered through a continuous filter with porosity of 0.45 μm and 0.2 μm, and finally with a 0.22 μm sterile filter.

10-fold diluted product and peripheral blood mononuclear cells (PBMC): 777 pg / mL IL-10, 26 pg / mL IL-12, 13183 pg / mL IL-6.

Example  3.9

The lysate of Example 2.8 was centrifuged at 9384 x g for 15 minutes. The pH of the supernatant was then adjusted to pH 7.4 with HCl, filtered through a continuous filter with porosities of 0.45 μm and 0.2 μm, and finally with a 0.22 μm sterile filter.

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

Example  3.10

The lysate of Example 2.8 was centrifuged at 9384 x g for 15 minutes. The pH of the supernatant was then adjusted to pH 7.6 with HCl, filtered through a continuous filter with porosities of 0.45 μm and 0.2 μm, and finally with a 0.22 μm sterile filter.

10-fold diluted product and PBMC: 476 pg / mL IL-10, 0 pg / mL IL-12, 4924 pg / mL IL-6.

Example  3.11

The bacterial lysate mixture of Example 2.11 was adjusted to pH 10.4 and then transferred to the MF tank. TFF installation was similar to Example 3.7. The cross flow was adjusted to 290 L / h m 2 (LHM) and the TMP to 0.4-0.5 bar. UF was stopped after 10 diafiltration volumes. The recovered volume yield was 86%. The pH of the final product was adjusted to pH 7.3 with HCl.

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

Example  3.12

The pH of the lysate of Example 2.12 was adjusted to pH 11.5 and then transferred to the MF tank. TFF installation was similar to Example 3.7. The cross flow was adjusted to 290 L / h m 2 and the TMP to 0.4 bar. UF was stopped after 10 diafiltration volumes. The volume yield recovered was 74%. The pH of the final product was adjusted to pH 7.2 with HCl.

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

Screening for biological activity was performed on human peripheral blood mononuclear cells (PBMC) at different concentrations as described in Example 6. Results obtained with an active dry weight of 0.5 mg per mL yielded TNF-α: 37 pg / mL; IL-6: 1092 pg / mL; IL-12: 81 pg / mL (in the presence of 10 ng / ml IFN- [gamma]); and IL-10: 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 no. 500-0116). The microplate assay protocol was run. Samples to be analyzed (0.1 mL) were diluted with 1.9 mL of 25 mM phosphate buffer at pH 11. Protein standard curves were plotted for each assay time performed with bovine serum albumin solution at 2 mg BSA / ml (BSA, Pierce ref 23210). The BSA solution was diluted with 25 mM phosphate buffer at pH 11 to give the following concentrations: 0.18, 0.24, 0.3, 0.36 and 0.42 mg BSA / ml. Samples (20 μl) and BSA standards (20 μl) were added four times to a clean, dried 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. Microtiter plates were mixed on a rotary plate for 5 seconds. After 20 minutes at room temperature, the absorbance was recorded at 750 nm. The protein concentration of each sample was calculated using the linear regression slope for the protein standard curve:

OD = a * (protein concentration) + b at 750 nm

Mg of protein in the sample = [20 x (OD-b at 750 nm)] / a

Soluble dry weight (SDW) was determined by obtaining 5 mL of soluble fraction obtained from the dissolution and drying it to constant weight in a porcelain dish at 105 ° C.

Example  4: In vitro Murine  Activation of Spleen Cells

The immunostimulatory effects of embodiments of the invention were assayed in vitro by measuring the activation of murine splenic cells (Alamar Blue Assay).

Materials and methods

Stimulation of Spleen Cells

The AlamarBlue ™ assay was designed to quantitatively measure the growth of human or animal cells. The assay includes fluorescence / chromic growth indicators based on the detection of metabolism activity by redox indicators in response to compound reduction resulting from cell growth. Associated with growth, the REDOX indicator causes a change from the oxidized (non-fluorescent, blue) form to the reduced (fluorescent, red) form.

Mouse Balb / c mice (females, 6-8 weeks old) were obtained from Charles River Laboratories, Sulzfeld, Germany, and were rescued by cervical spine separation. Homogenizing the spleen using a potter; The cell suspension was washed by centrifugation (280 × g, 4 ° C., 10 min) and resuspended in 5 ml RPMI 1640 containing 5% FCS, 100 U / ml penicillin and 100 μg / ml streptomycin. 100 μl splenocytes (2 × 10 ⁶ / ml) were incubated for 48 hours at 37 ° C. with 50 μl of bacterial extract dilution in 96-well plates (Falcon 3072) and under 5% CO ₂ in cell culture medium.

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

Test article

The following extracts were tested:

Afer300: carried by Lactobacillus spread obtained as described in Example 3.5 lactofermentum (Latobcillus fermentum ) extract from I-3929 (12 g / L) (31.6 mg active dry weight / g);

Cfer300: carried by Lactobacillus spread obtained as described in Example 3.3 lactofermentum (Latobcillus fermentum ) extract from I-3929 (12 g / L) (28.4 mg active dry weight / g);

Dfer300: Example 2.4 as obtained in Example 3.3 and a Lactobacillus buffer lactofermentum (Latobcillus purified using the same conditions described in fermentum ) extract from I-3929 (12 g / L) (29.5 mg active dry weight / g). This example was used as an alkali free lysis control.

ARahr300: alkali dissolution obtained at 40 ℃ with NaOH for 6 hours, the Lactobacillus ramno Versus (Lactobacillus rhamnosus ) extract from 71.38 (40 g / L) (49.3 mg active dry weight / g);

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

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

Negative control group

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

result

Spleen cells  To measure activation In vitro  Research

The increase in metabolic activity of mouse spleen cells after treatment with the extract was measured in two independent experiments by the Alamar Blue assay (see FIGS. 4A and 4B).

Splenocytes were incubated for 48 hours in the presence of extract. After addition of the Alamar Blue® solution, the release value was measured. As shown in Figures 4A-B, the bacterial extracts were effective at a dilution ratio of about 1: 300. All groups were statistically compared to controls using Student's T-assessment.

Table 1: p-values for bacterial extracts in two independent experiments by Alamar Blue assay (see FIGS. 4A and 4B).

conclusion

The immunostimulatory effect of the bacterial extracts was assayed in vitro by measuring the activation of murine spleen cells by the Alamar Blue assay. Here, in two independent experiments, we compared two extracts (AFer300 and CFer300) according to the present invention with an extract obtained by osmotic lysis (Dfer300), comprising intact, particulate cell wall components. These three extracts are Lactobacillus ( Lactobacillus) fermentum ) I-3929. AFer300, CFer300, and DFer300 extracts were each effective at stimulating cell metabolism at a dilution of 1: 300. We did not observe a difference between the DFer300 osmotic lysis control and Afer300 and CFer300.

We 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 ratio of 1: 300, while the CRahr300 and DRahr300 extracts showed weaker but significant irritation in one of the two assays at the same dilution ratio. When comparing the results of all six extracts, Lactobacillus fermentum ) I-3929 extract is Lactobacillus rhamnosus ) is thought to be more effective at stimulating splenic cell growth compared to 71.38 extract.

Example  5: Production of Nitric Oxide by Bone Marrow-derived Macrophages

The immunostimulatory potential of a series of embodiments according to the invention was tested by measuring the production of nitric oxide (NO) by murine bone marrow derived macrophages.

Materials and methods

Six week old male C57 / BL6 mice (6 week old male, SPF quality, Charles Rivier, FR) were digested via CO2 inhalation. Tibia from the hip, femur, and posterior appendages were extracted. After cutting both ends, Dulbecco's Modified Eagle Medium (DH) was injected through the bone to extract bone marrow from the lumen. After washing, stem cells were resuspended in DH medium supplemented with 20% horse serum and 30% L929 cell supernatant. Cell suspensions were incubated for 8 days in an incubator under 37 ° C., 8% CO ₂ and a moisture-saturated atmosphere. Macrophages were then removed with ice-cold PBS, washed and resuspended in DH medium supplemented with 5% fetal bovine serum (FCS), amino acids and antibiotics (DHE medium). Cell density was adjusted to 700000 cells / mL. An aqueous solution of the extract was serially diluted in DHE medium directly on a microtiter plate. The extract of the present invention was tested in triplicates and each microtiter plate contained the medium as a negative control. The final volume of each well was 100 μL. 100 μL of the cell suspension was added to the diluted extracts and the cells were incubated for 22 hours in an incubator at 37 ° C. under 8% CO 2, moisture-saturated atmosphere. 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 measured by running a Greries reaction. 100 μL of a grease reagent (5 mg / mL sulfanilamide + 0.5 mg / mL N- (1-naphthyl) ethylene-diamine hydrochloride) in 2.5% aqueous solution of phosphoric acid was added to each well. Microtiter plates were read with a spectrophotometer (SpectraMax Plus, Molecular Devices) at 562 nm for a reference at 690 nm. Nitrite concentration was proportional to the nitric oxide content formed. Nitrite content was determined based on a standard curve of sodium nitrite (1-70 μΜ NaNO ₂ ). Results were presented in μM nitric oxide (NO) as mean ± standard deviation and plotted as dose response curves.

Test item

The following extracts were tested:

First test :

OP0701B4_CFer300: Example 3.3 a Lactobacillus buffer lactofermentum (Lactobacillus obtained as described in fermentum ) extract from I-3929 (12 g / L);

OP0701B4_Dfer300: Example 2.4 to give as described in Example 3.3 and with a Lactobacillus bacteria and purified using the same buffer conditions lactofermentum (Lactobacillus fermentum ) extract from I-3929 (12 g / L). This example was used as a non-alkali lysis control.

OP0701B4_CRahr300: a Lactobacillus ramno obtained as described in Example 3.4 Versus (Lactobacillus rhamnosus ) extract from 71.38 (40 g / L).

OP0701B4_DRahr300: obtained by osmotic stress in 0.15 N NaCl solution for from 40 ℃ 6 sigan Lactobacillus ramno Versus (Lactobacillus rhamnosus ) extract from 71.38 (40 g / L). This example was used as a non-alkali lysis control.

Second test :

OP0701C_10G0.5P4H: 10 g / l with a bio Math dry weight at 40 ℃ with 0.037M NaOH incubated for 4 hours Lactobacillus buffer lactofermentum (Lactobacillus of fermentum ) extract from I-3929.

OP0701C_1OG1P4H: 10 g / l with a bio Math dry weight at 40 ℃ with 0.075M NaOH incubated for 4 hours Lactobacillus buffer lactofermentum (Lactobacillus of fermentum ) extract from I-3929.

OP0701C_10G2P4H: 10 g / l dry weight with 0.150 M Bio Math a Lactobacillus buffer lactofermentum (Lactobacillus incubated at 40 ℃ with NaOH for 4 hours in fermentum ) extract from I-3929.

OP0701 C_1OG1P21H: Extract from Lactobacillus fermentum I-3929 incubated for 21 hours at 40 ° C. with 0.075 M NaOH at a dry weight of biomass of 10 g / l.

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

OP0701C_10G2P21H: Extract from Lactobacillus fermentum I-3929 incubated for 21 hours at 40 ° C. with 0.150 M NaOH at a biomass dry weight of 10 g / l.

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

First test result

In the first assay (FIG. 5A), the extract obtained by alkali dissolution according to the present invention was observed to have the same activity as the extract obtained by osmotic dissolution.

We also observed that immunostimulatory activity is associated with the selected strain. Lactobacillus fermentum ) I-3929 The extract obtained from Lactobacillus rhamnosus ) resulted in more NO ₂ and NO ₃ production than the extract obtained from 78.31 strain.

Second test result

In the second assay (FIG. 5B), we found that the in vitro activity of the extract correlated with the initial conditions and state of dissolution. Identical Lactobacillus fermentum ) I-3929 strain and the same amount of biomass dry weight per liter of lysate showed the activity of the following bacterial extracts: OP0701C_10G0.5P4H, OP0701C_10G1P4H, OP0701C_10G2P4H, OP0701C_10G1P21H, OP0701 C_10G0.5P21H And OP0701C_10G2P45H. In vitro activity depends on the initial concentration of NaOH and the duration of dissolution.

conclusion

The results of these experiments revealed that, despite the chemical modifications produced by the alkaline process, the activity of the embodiments was not reduced compared to the control extracts obtained by osmotic dissolution or compared to each living bacterium (in this aspect See Example 6 below).

Example  6: relating to fragrance- and anti-inflammatory activity In vitro  Screening

In order to screen the immunostimulatory or anti-inflammatory potential of embodiments of the invention in vitro, tests were conducted on a series of bacterial extracts against human PBMCs. The release of both IL12p70 (inflammatory cytokine) and IL10 (anti-inflammatory cytokine) was measured, and the IL-10 / IL-12 ratio was determined by Foligne et. al ., World J Gastroenterol 2007 January 14; 13 (2): 236-243.

The goal was to compare the production of IL-12p70 and IL-10 for a series of six extracts obtained through different extraction / purification methods. The IL-10 / IL-12p70 ratio obtained for each extract may be related to the anti-inflammatory potential of the extract. Live bacteria were used as a control for comparing the activity of extracts ( Lactobacillus at 2 × 10 ⁷ cfu / ml fermentum ) I-3929).

Materials and methods

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

IFN- [gamma] was added at least 3 hours before adding lysate or live bacterial strains to the medium. Data from two independent tests was recorded.

human PBMCs Manufacture

PBMCs were isolated from peripheral blood. In summary, after Ficoll gradient centrifugation (Pharmacia, Uppsala, Sweden), mononuclear cells are collected, washed with RPMI 1640 medium (Live technologies, Paisley, Scotland), gentamicin (150 μg / mL), L- Adjusted to 2 × 10 ⁶ cells / mL in RPMI 1640 supplemented with glutamine (2 mmol / L), and 10% fetal bovine serum (FCS) (Gibco-BRL).

Induction of Cytokines

PBMCs (2 × 10 ⁶ cells / mL) were seeded in 24-well tissue culture plates (Corning, NY). Cells were stimulated as described above. After stimulation at 37 ° C. for 24 hours in air containing 5% CO ₂ , the culture supernatants were collected, cleared by centrifugation and stored at −20 ° C. until cytokine analysis. Cytokines were measured by ELISA using pharmingen antibody pairs (BD Biosciences, San Jose, Ca, USA) for IL-10 and IL-12p70, according to manufacturer's recommendations.

Test item

OP0701C_10G2P45H (A): Extract from Lactobacillus fermentum I-3929 dissolution at 0.037 M NaOH for 45 hours at 40 ° C., at a dry weight of 10 g biomass per liter of lysate.

OP0701C_1OG1P4H (B): Extract from Lactobacillus fermentum I-3929 dissolution at 0.075 M NaOH for 4 h at 40 ° C., at a biomass dry weight of 10 g per liter of lysate.

OP0701C_5G4P21H (C): Extract from Lactobacillus fermentum I-3929 dissolution at 0.300 M NaOH for 21 h at 40 ° C., at a dry weight of 5 g biomass per liter of lysate.

OP0701C_40G0.5P4H (D): Extract from Lactobacillus fermentum I-3929 dissolution at 0.037 M NaOH for 6 h at 40 ° C., at a dry weight of 40 g biomass per liter of lysate.

OP0701B4_CFer150 (E): it carried by a Lactobacillus buffer lactofermentum (Lactobacillus obtained as described in Example 3.1 fermentum ) extract from I-3929 (12 g / L).

OP0701B4_BFer300 (F): Extract from Lactobacillus fermentum I-3929 dissolution at 0.075 M NaOH for 6 hours at 40 ° C., at a dry weight of 6 g biomass per liter of lysate.

20% glycerol from 2x10 ⁷ cfu / ml to, was frozen at -80 ℃, live bacterium, Lactobacillus buffer lactofermentum (Lactobacillus in fermentum ) I-3929.

result

The purpose of this test was to compare the in vitro immunostimulatory or anti-inflammatory potential of embodiments of the invention with live Lactobacillus strains. To analyze different extracts and living bacteria, the IL10 / IL12 ratios were compared, as well as the amount of each cytokine IL-10 and IL-12 released from PBMCs.

If either or both of the IL-10 or IL-12 concentrations are close to unstimulated levels (i.e. less than 10 pg / ml), this ratio is considered uncalculated and results in a non-calculated value (NC). Is indicated.

Results from PBMCs expressed in pg / ml are shown in Tables 2 and 3.

In the presence of IFNγ, the effects of six bacterial extracts (Extracts A to F) of the present invention were tested twice as Lactobacillus fermentum ( probiotic sample 1 and probiotic sample 2) in viable form on human PBMCs, 2 × 10 ⁷ bacteria / ml) (IL10 and IL12p70 reactions in pg / ml). IL10 / IL12 ratios were recorded only for doses of 100 μg / ml and 1 mg / ml extract.

Table 2: In vitro perfume-inflammatory (immunostimulatory) or anti-inflammatory (immunoregulated) potential of bacterial extracts in human PBMCs.

In the presence of IFNγ, the effects of six bacterial extracts (Extracts A to F) of the present invention were tested twice as Lactobacillus fermentum ( probiotic sample 1 and probiotic sample 2) in viable form on human PBMCs, 2 × 10 ⁷ bacteria / ml) (IL10 and IL12p70 reactions in pg / ml). IL10 / IL12 ratios were recorded only for doses of 100 μg / ml and 1 mg / ml extract.

Table 3: In Vitro Anti-inflammatory (Immunostimulatory) or Anti-inflammatory (Immunomodulatory) Potential of Lactobacillus fermentum Strain I-3929 Living in Human PBMCs.

Based on the results presented in Tables 2 and 3, the production of IL10 was reduced in the following order:

Based on the results presented in Tables 2 and 3, the production of IL12 was reduced in the following order:

Considering the ratio, the following general pattern was observed:

The results of the IL10 / IL12 ratios for extracts A and C were not presented because these extracts were not found to be effective inducers of cytokines.

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

conclusion

The ratio obtained was determined under the experimental conditions used in Example 6 that some of the bacterial extracts of the present invention were lactobacillus fermentum ( Lactobacillus). fermentum ) may have a much better immunomodulatory effect than live bacteria. For example, extracts B, D, E, and F showed higher IL10 / IL12p70 ratios than live bacterial controls. Thus, such extracts may be more active than probiotic live bacteria against the conditions described herein, such as inflammatory conditions.

Example  7: Action on toll-like receptors

Embodiments of the invention are obtained in Gram positive bacteria and are expected to act via the TLR2 receptor accordingly. TLR receptors are mainly expressed by, but not limited to, immune cells such as monocytes, macrophages, dendritic cells, T-cells, and the like, of microbial products that can be recognized as signal dangers by the host. Is the main sensor. They first trigger nonspecific innate immunity, but TLR activation initiates a complete immune cascade, resulting in the development of acquired immunity in the presence of an antigen.

Cells expressing the corresponding functional TLR genes are valuable tools for various applications, such as the study of mechanisms involved in TLR recognition or signaling, and the development of novel potent therapeutic drugs. The following experiments test the activity of three bacterial extracts against this major adapter of immune response.

Materials and methods

The response of the extracts of the invention was tested in the following two cell lines (to confirm their agonist effect or to confirm antagonistic activity in the presence of TLR2 agonist Pam3Cys or in the presence of TLR4 agonist LPS): a) HEK- TLR2 / 6 (IL-8 ELISA after 24 hours) b) HEK-MD2-TLR4-CD14 (IL-8 ELISA after 24 hours)

a) HEK - TLR2 / 6

HEK293 cell line was selected for null or low basal expression of the TLR gene. These cells allow for the efficient monitoring of TLR activity using an ELISA assay, such as a reporter-based system that monitors IL-8 titration or TLR-induced NF-κB activation.

HEK-TLR2 / 6 cells (Invivogen, Toulouse, France) were stably transfected with a number of genes from the TLR2 / 6 pathway, including TLR2, TLR6 and genes involved in cognition or involved in the signaling cascade Genetically engineered HEK293 cells. These cells secrete IL-8 after TLR2 / 6 stimulation. The experiment was conducted according to the manufacturer's instructions.

In summary, 2 × 10 ⁴ cells / well (20 ul RPMI) were incubated at 37 ° C. for 3 days (5% CO ₂ ). The medium was transferred and 90 μl RPMi + 5% FCS was added to the wells. Then, agonist and control were added (10 μl / well). Cells were returned back to the incubator for 24 hours. Supernatants were pooled and IL-8 ELISA was performed according to the manufacturer's instructions.

Test item

OP0701B4_Afer50: Example 3.6 for a, for 4 hours at 40 ℃ purified as described in, in 0.075 M NaOH, 12 g per liter of lysate with Math bio dry weight Lactobacillus buffer lactofermentum (Lactobacillus for fermentum ) I-3929 extract from lysis.

OP0701C-Bt1LAC: embodiment the Lactobacillus buffer lactofermentum (Lactobacillus obtained as described in Example 3.7 fermentum ) extract from I-3929.

OP0701C-Bt2LAC: Example 3.11 the Lactobacillus buffer lactofermentum (Lactobacillus obtained as described in fermentum ) extract from I-3929.

result: IL -8 secretion

Results for the control (negative = LPS K12 ultrapure, TLR4 agonist; and PAM3CSK4 = positive, TLR2 agonist) are shown in Tables 4-6. The results (il-8 expressed in pg / ml) show the mean value of IL-8 secretion measured by ELISA after 24 hours of stimulation with the control.

Cell line HEK TLR2 / 6 responded to the TLR2 agonist Pam3Cys. In contrast, E coli (LPS K12) derived TLR 4 agonists showed no activity, even at high doses of 0.01 μg / ml.

Experiment using three bacterial extracts alone

The three bacterial extracts tested here (OP0701B4Afer5O, OP0701C-Bt1LAC, and OP0701C-Bt2LAC) showed higher immunostimulatory properties than the TLR2 agonist Pam3Cys.

3 bacterial extracts + Pam3Cvs Experiment

As mentioned above, the three bacterial extracts were tested for their presumed antagonistic or additional properties compared to Pam3Cy added just after the extract addition.

Bacterial extract OP0701B4 AFer5O

Table 4: Secretion of IL-8 (pg / ml) from supernatants obtained from control (medium, LPS, or Pam3Cys), bacterial extract OP0701B4_AFer50 or HEK TLR2 / 6 cells stimulated with said bacterial extract and Pam3Cys

The results in Table 4 indicate that OP0701B4_AFer50 extract induced high levels of IL-8 production (agonist TLR 2/6).

Bacterial extract OP0701C - Bt1LAC

Table 5: Secretion of IL-8 (pg / ml) from supernatants obtained from control (medium, LPS, or Pam3Cys), bacterial extract OP0701C-Bt1LAC or HEK TLR2 / 6 cells stimulated with these bacterial extracts and Pam3Cys .

The results in Table 5 show that OP0701C-Bt1LAC extract induced high levels of IL-8 production (agonist TLR 2/6).

Bacterial extract OP0701C - Bt2LAC

Table 6: Secretion of IL-8 (pg / ml) from supernatants obtained from control (medium, LPS, or Pam3Cys), bacterial extract OP0701C-Bt2LAC or HEK TLR2 / 6 cells stimulated with these bacterial extracts and Pam3Cys .

The results in Table 6 show that OP0701C-Bt2LAC extract did not induce IL-8 production (agonist TLR 2/6) and showed antagonistic effects on TLR2 / 6 in the presence of Pam3Cys.

conclusion

Depending on the conditions of alkali dissolution (initial concentration of biomass dry weight, initial base concentration and base treatment duration), bacterial extracts may exhibit different modes of action.

OP0701B4_Afer50 and OP0701C-Bt1LAC were agonist TLR2 / 6, but stronger alkali lysis performed against the same bacterial strain eventually showed antagonistic TLR2 / 6 activity (OP0701C-Bt2LAC).

b) HEK - TLR4 - MD2 - CD14

TLR4 has been studied extensively because it is a major receptor involved in the recognition of lipopolysaccharides (LPS) involved in sceptic shock.

HEK-TLR4-MD2-CD14 cells are very sensitive to LPS. They were obtained by stable transfection of HEK293 cells with the TLR4, MD2 and CD14 genes and the NF-κB-induced reporter system. These cells secrete IL-8.

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

Result: IL-8 secretion

The results for the control (negative = LPS K12 ultrapure, TLR4 agonist; and PAM3CSK4 = positive, TLR2 agonist) are shown in Tables 7-9. The results (il-8 expressed in pg / ml) show the mean value of IL-8 secretion measured by ELISA after 24 hours of stimulation with the control.

The cell line reliably responds only to the TLR4 agonist LPS K12. In contrast, TLR2 agonists from Pam3Cys did not show activity even at high doses of 0.01 μg / ml.

Experiment using three bacterial extracts alone

As expected from the Gram positive bacterial agonists, the three bacterial extracts tested here (OP0701B4Afer5O: Table 7, OP0701C-Bt1LAC: Table 8, and OP0701C-Bt2LAC: Table 9) did not show definite immunostimulatory properties via the TLR4 pathway. .

3 bacterial extracts + LPS K12 Experiment

As mentioned above, the three bacterial extracts of the present invention were tested for their presumed antagonistic or additional properties compared to LPS added immediately after the addition of the extract. Likewise, there was no effect observed at the TLR4 receptor level (Table 7-9).

Bacterial extract OP0701B4 AFer50

Table 7: Secretion of IL-8 (pg / ml) from supernatants obtained from control (medium, LPS, or Pam3Cys), bacterial extract OP0701C-AFer50 or HEK TLR4 cells stimulated with these bacterial extracts and LPS K12.

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

Bacterial extract OP0701CBt1 - LAC

Table 8: Secretion of IL-8 (pg / ml) from supernatants obtained from control (medium, LPS, or Pam3Cys), bacterial extract OP0701C-Bt1LAC or HEK TLR4 cells stimulated with these bacterial extracts and LPS K12.

Bacterial extract OP0701CBt2 - LAC

TABLE 9 Secretion of IL-8 (pg / ml) from supernatants obtained from control (medium, LPS, or Pam3Cys), bacterial extract OP0701C-Bt2LAC or HEK TLR4 cells stimulated with these bacterial extracts and LPS K12.

conclusion

Taken together, the results presented here for HEK cells, and the results obtained in human PBMC cells, suggest that Afer50 and BT1LAC can stimulate the immune system via the TLR2 pathway. Moreover, BT2LAC can act as a TLR2 / 6 antagonist. Therefore, extracts according to the invention can stimulate the immune system via the TLR2 pathway or act as a TLR2 / 6 antagonist and thus be associated with anti-infective and anti-inflammatory activity in vivo.

Example  8:

Cells expressing the corresponding functional TLR genes are valuable tools for various applications, such as the study of mechanisms involved in TLR recognition or signaling, and the development of novel potent therapeutic drugs. Thus, the experiments below aimed to test the activity of four bacterial extracts against this major adapter of immune response.

In this test, eight TLRs and NOD2 receptors were largely screened for four bacterial extracts, including the extracts according to the present invention.

Way

The bacterial extracts according to the invention were tested in 96 well microplates. Extracts were diluted in DMEM culture medium and 20 μl of each dilution was tested in duplicate. A 180 μl volume of HEK293 cell suspension containing 25000 or 50000 cells (HEK293 cells specific for each TLR with a reporter gene secreting alkaline phosphatase under the control of NF-kB) in DMEM culture medium + 10% FCS To each well. After 16 hours of incubation of each cell line, 20-50 μl of each supernatant was transferred to 96-well microplates and made complete with 200 μl of Quantiblue (InVivoGen no REP-QB1). Enzymatic reaction with secreted alkaline phosphatase was performed for 30-60 minutes against different series of TLRs expressing cell lines. Reading was performed using a microplate reader at 630 nm. The results are shown as OD at 630 nm.

material

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

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

Negative control group

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

Test item

The following bacterial extracts were tested:

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

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

OP0701D_10L1PswitchA: Example 3.12 a a Lactobacillus buffer lactofermentum (Lactobacillus obtained as described in (I) fermentum ) extract from I-3929 (10 g / L); And

OP0701D_5LOSMOConc: obtained by osmotic stress in 0.16 N NaCl solution for 24 hours at 40 ℃ as a control group (H) osmotic dissolved Lactobacillus buffer lactofermentum (Lactobacillus fermentum ) extract from I-3929 (7.6 g / L).

Preparation of Bacteria Extract

All cell lines were stimulated with 200 μL reaction volume using 20 μl of each sample to be tested.

Screening was carried out at a single concentration, typically at 0.5 mg dry weight / mL. The test was conducted in duplicate.

result

Bacterial extracts and controls were tested in duplicate against recombinant HEK-293 cell lines functionally expressing reporter genes induced by the NFkB promoter, including the given TLR or NOD2 protein. TLR and NOD2 activation results are expressed as optical density (OD) values. The results are shown in Tables 10-14 and summarized in Table 15.

Bacteria Extract F ( OP0701B4Cfer300 )

Table 10: Activation of TLR and NOD2 receptors by OP0701 B4Cfer300 extract.

Bacterial extract F specifically activated hTLR2, hTLR4 and hNod2 and activated hTLR5 on a smaller scale.

Bacteria Extract G ( OP0701B4CRahr300 )

Table 11: Activation of TLR and NOD2 Receptors by OP0701B4CRahr300 Extract.

The bacterial extract G specifically activated hTLR2, hTLR4 and hNod2 and activated hTLR5 on a much smaller scale.

Bacteria Extract H ( OP0701D5LOSMOConc )

TABLE 12 Activation of TLR and NOD2 Receptors by OP0701D_5LOSMOConc Extract.

Bacterial extract H specifically activated hTLR2, hTLR4 and hNod2.

Bacterial Extract I ( OP0701  D 10 L1 PswitchA )

Table 13: Activation of TLR and NOD2 Receptors by OP0701D_10LPswitchA Extract.

Bacterial extract I strongly activated hTLR2, hTLR5, hNOD2 and activated hTLR4 and hTLR9 on a smaller scale.

Table 15: Activation of TLR and NOD2 receptors by different bacterial extracts obtained with different treatment conditions according to the invention.

Thus, bacterial extracts according to the present invention may be helpful for activating the immune system through various TLRs, and also through Nod2. Therefore, the extracts of the present invention are good activators of the immune response.

When comparing extracts H to G and extract F, it is clear that the extracts according to the invention follow the same TLR and Nod pathways as the extracts obtained by osmotic dissolution. When comparing Extract H and Extract I, we could observe that by addition of acid lysates followed by alkaline lysates, the TLR5 pathway is activated and the TLR9 pathway is activated on a smaller scale. Even extract F obtained from the weak alkaline process showed activation of the TLR5 pathway in contrast to extract H.

Extract I acts on TLR5, suggesting potent utility in radiotherapy. Indeed, radiation therapy is a well-established and very effective treatment for certain types of cancer. However, its side effects can be devastating because they can destroy healthy cells in the body, especially bone marrow cells and cells in the gastrointestinal tract. Burdelya et al. Recently reported that CBLB502 peptides bind to TLR5 and activate the nuclear factor-κB signaling pathway (the pathway that cancer cells commonly 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 lethal dose of systemic showed less damage to healthy bone marrow and gastrointestinal tract cells and survived significantly longer than controls. Importantly, in tumor-bearing mice, CBLB502 did not reduce the antitumor efficacy of radiotherapy.

Example  9: Activity of Extracts Producing Plaque-forming Cells (on Sheep Red Blood Cells) in Mice

Plaque forming cell (PFC) technology can be evaluated for nonspecific stimulation of B-lymphocytes. This technique was first introduced in Cunningham and Seenberg, Immunology, 1968, 14, 599. The presence of hemolytic antibodies around antibody-forming cells was demonstrated as follows. Murine lymphocytes, as well as a dense foreign (sheep) erythrocyte population, were simultaneously applied on microscope slides. Certain lymphoid cells release lysing antibodies that, in the presence of complement, form lytic plaques which expand and result in lysis of neighboring red blood cells. At the end of the experiment, the number of PFCs recorded for 10 ⁶ cells or spleen was counted.

Materials and methods

Test item

Bacterial extract 1 obtained as described in Example 3.5 and bacterial extract 2 obtained as described in Example 3.4.

animal

40 male Balb / C mice / experiment (IFFA-CREDO, St. Germain sur I'Arbresle Cedex, France), 5-6 weeks old, with an average body weight of 20 +/− 2 g, were divided into five groups of eight each.

Experimental Design

The animals were divided into five groups as follows:

Group (a): 8 mice receiving 1.2 mg / mouse of Extract 1 obtained as described in Example 3.5; Volume = 0.2 ml

Group (b): 8 mice receiving 0.6 mg per mouse of the extract 1 obtained as described in Example 3.5.

Group (c): 8 mice receiving 1.2 mg / mouse of Extract 2 obtained as described in Example 3.4; Volume = 0.2 ml

Group (d): 8 mice receiving 0.6 mg per mouse of Extract 2 obtained as described in Example 3.4; Volume = 0.2 ml

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

Mice received the above extracts at the indicated doses, daily per os (via the oral route) for 5 consecutive days (1-5 days). Two or more intubations were performed on days 19 and 20. On day 29, antigen (10 ⁶ sheep red blood cells) in 0.2 ml of isotonic saline (0.9% NaCl, Alsever solution) was injected intravenously through the tail vein of the animal; After 4 days (33 days), splenic cell suspensions were prepared.

Reagents and Equipment

Alsever solution without endotoxin (Sigma, 38299 St Quentin Fallavier, Cedex France, ref. A 3351)

Eagle base medium (Basal Medium Eagle (BME), 10 × concentrated) in bicarbonate-free eagle solution (Bio-Merieux, 69280 Marcy I'Etoile, France ref: 8 210 2)

Lyophilized guinea pig serum (Bio-Merieux, Marcy I'Etoile, France ref: 7 212 2)

Positive red blood cells (Bio-Merieux, Marcy I'Etoile, France ref: 7214 1)

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

Treatment of Test or Control Items

Control animals received only red blood cells (SRBC). Different groups of animals were injected with bacterial extracts per os as previously described. To this end, each extract was dissolved in water.

Spleen Cell Suspension

Four days after the last antigen (SRBC) injection, splenic cell suspensions for each mouse were prepared according to the following procedure:

Animals were anesthetized with ether and then sliced by cervical spine separation. The spleens were extracted and ground in a glass tissue homogenizer with 2 ml BME at pH 6.75-6.80. Then 3 ml of sterile distilled water were added and the mixture was stirred for 20 seconds. The resulting suspension was further diluted with 10 BME and centrifuged at 1200 rpm for 10 minutes at 4 ° C. The supernatant was discarded and the precipitate suspended in 2 ml BME. Cells were recovered by placing on ice for 5-10 minutes. The viability count of monocytes was performed in the following manner:

An aliquot of the cell suspension diluted to 1/20 was prepared with saline containing 0.1% trypan blue solution. Uncolored cells, ie live cells, were counted in a Neubauer chamber.

The cell suspension was kept in melting ice during counting and immediately used for plaque lysis. This technique prevents the loss of survival that can occur with longer wait times.

Preparation of the slide

In a conventional microscope slide, the dust was carefully shaken off, removing any grease marks, and three parallel strips (0.1 mm thick) of double sided tape were applied at about 1.5 cm intervals. The strip was then covered with pre-cleaned coverslips (22 mm x 22 mm) to form two "chapters" between the slides and coverslips.

The fraction of splenic cell suspensions was adjusted to 25,000 mononuclear cells per cell. In a small glass tube, a mixture of the following ingredients (added in the order described) was prepared using an automatic pipette:

1) 7.5 ml BME

2) 0.5 ml SRBC suspension washed with 50% residue and centrifuged twice

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

Finally, 200 μl of the mixture was added to 50 μl splenic cell suspension at 25,000 cells / dl. After mild homogenization, the suspension was charged to "Cunningham Chambers" by capillary action and then the slides were sealed with paraffin. The slides were then placed in wet chapters in an oven at 37 ° C.

Explanation( Lecture )

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

Dissolve plaques were easily recognized. Five vertical optical strips were examined on each slide and the number of lysis plaques was counted. The number of cells (N) which formed direct lysis plaques per 10 ⁶ cells was calculated by extrapolation. If x is the number of plaques observed and X is the number of cells examined, the number of direct soluble plaques per 10 ⁶ cells is according to the following equation:

here,

C = final concentration of splenic cells

V = observed volume

n = number of optical strips

e = thickness of the adhesive tape

I = width of the optical field

L = length of the optical strip

The number of direct lysing plaques per 10 ⁶ cells was calculated according to the following equation:

By subtracting the number of cells collected per spleen, it is possible to infer the number of cells that form spleen lysis plaques.

In "absolute reference" mice, no spontaneous lysis plaques were observed.

Statistical Analysis and Results

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

Table 16: Activity of bacterial extracts 1 and 2 to produce plaque forming cells (for positive red blood cells) in mice.

The extract of the present invention was found to be B-cell activator when tested at a concentration of 1.2 mg / mouse / day or 0.6 mg / mouse / day. Some dose-dependent effect was observed. Thus, some extracts of the present invention can potentially be used to prime the immune system in patients with recurrent infections.

Example  10: balb / c on mouse Intraperitoneal  Salmonella typhimurium ( Salmonella  typhimurium) for infection CFer300 Effect

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

Materials and methods

Animals and Breeding

Balb / c mice were raised in a facility at the Institute for Immunology, Moscow. For in vivo protection experiments, non-inbreeding experimental grade white mice were purchased from the Russian State Scientific Center for Biomedical Technologies (Russian Academy of Medical Sciences). Upon arrival, the mice weighed 12-14 g. Throughout the experiment, mice were maintained under pathogen-free conditions on standard rodent diet and drinking water.

Research group (main experiment)

Salmonella typhimurium in mice using two groups of 22 mice per group herd Stadium (Salmonella typhimurium ) was used to test the anti-infective efficacy of the bacterial extracts prepared.

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

After injecting 0.5 ml of solution into each mouse once a day for 10 consecutive days, all mice were attacked with Salmonella typhimurium :

Group 1: Mice treated with CFer300 injected os in a single 2 mg (0.5 ml) dose.

Group 2: mice receiving sham treatment with oral administration of 0.5 ml of water daily for 10 days.

Test item

The bacterial extracts tested were OP0701B4CFer300 ("CFer300"); Example 3.3 a Lactobacillus buffer lactofermentum (Lactobacillus obtained as described in fermentum ) extract from I-3929 (12 g / L) (28.4 mg active dry weight / g).

Preliminary experiment

The purpose of the preliminary experiments was to determine the dose of infectious material that could cause mortality close to 50% three weeks after challenge. Upon challenge, suspensions of Salmonella enterica, serovar typhimurium strain 415 (I. Mechnikov, Institute for Vaccines and Sera, Russian Academy of Medical Sciences) were injected intraperitoneally in each mouse. Attack dose ranges from Salmonellae 10 ³ to 10 ⁵ CFU per mouse.

Observation and death record

After challenge, mice were maintained under standard conditions for experimental animals. Records of daily observations and deaths were recorded for a period of 21 days after infection. Post-infection survival (SR), average duration of life (ADL), defense factor (DF), and product efficacy index, calculated for the anti-infective efficacy of the product for each test group (efficacy index, EI) was evaluated. SR is expressed as the percentage of animals surviving in the experimental group 21 days after infection. ADL, DF, and EI were calculated as follows:

here,

ADL is the average life duration,

X1 to Xn is the life time post infection following experimental mice 1 to n,

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

here,

DF is the defense index,

CD is the rate of death in the control group,

ED is the death rate in the experimental group.

here:

EI is the product efficacy index,

DF is the defense index.

Result: drug resistance

The preparation was injected orally once daily for 10 days and was well tolerated. No evidence of toxicity or side effects was observed during the 10 days pretreatment period.

Salmonella titration ( titration )

Preliminary experiments were conducted to determine the attack capacity of Salmonella typhimurium, which caused approximately 50% mortality. The results are shown in Table 17.

Table 17: Preliminary experiments to determine the attack capacity of Salmonella typhimurium corresponding to approximately 50% mortality.

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

conclusion

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

Main experiment: CFer300 Attack on Killed Mouse

Mice pretreated with CFer300 preparation for 10 days (n = 22) or mice in control group pretreated with water (n = 22) were attacked the day after pretreatment.

Salmonella typhimurium suspensions were administered intraperitoneally at a 10 ⁵ CFU challenge dose per mouse. Follow-up was performed for 21 days after infection. The mortality rates for the groups are shown in Table 18.

Table 18: Follow-up 21 days after infection

Numbers italicized indicate numbers of animals found dead on the date indicated. This data was used to calculate SR, ADL, DF and EI (Table 19).

Table 19: Survival rate for observation period (21 days)

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

In summary, daily daily oral treatment with CFer300 given at a single dose of 2 mg provided partial protection in mice infected with Salmonella typhimurium . As shown in this example, previously in vitro and ex vivo, the extracts disclosed herein may be useful for further therapeutic drug development.

Example  11: LACK Two induction-induced asthma models Lactobacillus  Effect of Extract

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

Materials and methods

Animals and Breeding

Six-week old female BALB / c ByJ mice were purchased from Janvier, France. These mice were maintained and fed under standard conditions.

Research group

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

Group A: untreated, LACK-sensitized and saline-attack mice; LACK is a protein derived from the parasite Leishmani a major (4 mice).

Group B: untreated, LACK-sensitized and challenge mice (8 mice)

Group C: OM-1009A-treated (8 mg dry weight residue per dose), LACK-sensitized and challenge mice (8 mice)

Group D: OM-1009B-treated (8 mg dry weight residue per dose), LACK-sensitized and challenge mice (7 mice)

Treatment and Schedule with Test or Control Items

Mice were treated from -3 to 22 days. On days 0 and 7 mice were sensitized intraperitoneally (i.p.) with 10 μg of LACK in the presence of 2 mg of alum 3. From days 17-21, mice were exposed to aerosol challenge (groups B, C, and D) for 20 minutes daily with LACK solution (0.15%), or saline solution (group A) as a control. On day 22, mice were analyzed for their ability to develop AHR upon inhaling methacholine. On day 23, mice were excised and lung inflammation evaluated.

Methodology

Mice were treated three times therapeuticly as described above. Washing was performed on individual mice bleeding with their cannula inserted into their airways. The lungs were washed three times with 1 ml of warm PBS. Cells were washed with PBS, resuspended in 300 μl and counted using a Bucker-Turk chamber. For differentiated BAL cell counts, cytospin preparations were made and stained with Wright / Giemsa staining. The following groups were tested: LACK-sensitized and PBS- attacked wild-type (wt) mice (control), LACK-sensitized and attacked mice (asthma), OM-1009A-treated wt mice, and OM-1009B. Treated wt mice. Total cell number in BAL was determined by microscopic examination of cytospin preparations stained with light / zemsa staining.

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

Test item

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

result

a) airway hypersensitivity

On day 22, mice were analyzed for their ability to develop AHR upon inhalation of methacholine at the doses described. The results are recorded in FIG. The results suggest that OM-1009-B restored baseline Penh values similar to PBA untreated asthma group. OM-1009-A was more efficient because the Penh values obtained were much lower than those observed in the non-asthmatic control group.

b) total in bronchial-alveolar lavage Cell number  And discrimination Cell number

On day 23, mice were excised and lung inflammation evaluated. The total cell number of BAL is reported in Table 20 (right column), and also differentiated cell numbers: eosinophil count (Eo), neutrophil count (Neutro), lymphocyte count (Lympho), and other cell counts (Others). have.

Table 20: Total cell numbers and differential cell numbers in BAL when treated with two extracts of the invention (groups C and D). Mean and standard error values are shown.

Both extracts significantly reduced total cell numbers in BAL (p <0.01 for OM-1009A; and p <0.03 for OM-1009B, compared to group B). Eosinophil counts were reduced 3- and 1.7-fold, respectively. Neutrophil counts decreased 1.8- and 3.6-fold, respectively. A decrease in the number of eosinophils and neutrophils means a lower concentration of inflammatory cells in BAL of animals pretreated with the extract.

c) detected in lung extracts and multiplexed To analysis (CBA array)  Quantified Th2  Cytokine

Table 21: Individual IL4 lung levels in mice.

Table 22: Individual IL5 Lung Levels in Mice.

Table 23: Individual IL13 lung levels in mice.

Th2 cytokine (IL4, IL5, and IL13) levels were reduced 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 strong base treatment (OM-1009-B) may have less activity than the bacterial extract obtained by medium treatment of base strength (OM-1009-A). Although both extracts showed activity in the Penh factor assay (FIG. 6), OM-1009A was used in the treatment or prophylaxis of Th2-related diseases such as allergic diseases and atopies, or conditions associated with their symptoms. It may be a better candidate.

Institute Pasteur CNCMI03929 20080227

Claims (28)

An extract of at least one Lactobacillus bacterial strain, wherein said extract is a soluble extract and comprises a chemically modified bacterial molecule. The extract of claim 1, wherein the chemically modified bacterial molecule is produced by exposing the one or more Lactobacillus bacterial strains to alkaline medium. The extract according to claim 1 or 2, wherein the extract exhibits immunomodulatory activity in the subject. The extract of claim 3, wherein the extract exhibits immunostimulatory activity in the subject. The extract of claim 3, wherein the extract exhibits anti-inflammatory activity in the subject. The method of claim 1, wherein the one or more Lactobacillus strains are Lactobacillus fermentum ), Lactobacillus rhamnosus ), Lactobacillus plantarum ), Lactobacillus johnsonii ), Lactobacillus helveticus ), Lactobacillus casei defensis , Lactobacillus casei SSP . Casei ( Lactobacillus) casei ssp. casei ), Lactobacillus paracasei ), Lactobacillus bulgaricus), Lactobacillus para Kasei (Lactobacillus paracasei), Lactobacillus even know Phil Ruth (Lactobacillus acidophilus ), Lactobacillus reuteri), Lactobacillus salivarius (Lactobacillus salivarius), Lactobacillus lactis (Lactobacillus lactis ), and Lactobacillus delbrueckii ). The method according to any one of claims 1 to 6, wherein the one or more Lactobacillus bacterial strains are Lactobacillus fermentum ) I-3929, Lactobacillus rhamnosus ) 71.38, Lactobacillus plantarum) 71.39, Lactobacillus zone Sony (Lactobacillus johnsonii) 103782, and Lactobacillus helveticus (Lactobacillus helveticus ) extract comprising at least one of 103146. 8. The extract of claim 1, wherein at least 10% of at least one of aspartic acid, glutamic acid, serine, histidine, alanine, arginine, tyrosine, methionine, phenylalanine, and lysine in the extract is racemized. . The method according to any one of claims 1 to 8, wherein the extract can achieve a calculable IL10 / IL12 ratio in human peripheral blood mononuclear cells, the ratio being due to the live Lactobacillus strain from which the extract was obtained. An extract equal to or greater than the IL10 / IL12 ratio achieved. The extract of claim 1, wherein the extract reduces eosinophil count, neutrophil count, lymphocyte count, or any combination thereof in the asthma murine subject as compared to the asthma untreated control. (a) culturing at least one Lactobacillus bacterial strain in culture medium;
(b) exposing each Lactobacillus bacterial strain to alkaline medium; And
(c) treating the product of step (b) to remove insoluble and particulate material. The method of claim 11, wherein step (c) is performed by tangential flow filtration. The method of claim 11 or 12, wherein exposing each Lactobacillus bacterial strain to a pH above 9.0 will chemically sufficiently modify the bacterial molecule. The method of claim 11, 12, or 13, further comprising treating each strain at a pH of less than 4.5 after step (b) and before step (c). The method according to any one of claims 11 to 14, wherein said chemical modification comprises at least 10% of at least one of aspartic acid, glutamic acid, serine, histidine, alanine, arginine, tyrosine, methionine, phenylalanine, and lysine in said extract. Comprising semi-finishing. A nutraceutical composition comprising an extract of any one of claims 1 to 10 or an extract prepared according to any one of claims 11 to 15. A pharmaceutical composition comprising an extract of any one of claims 1 to 10 or an extract prepared according to any one of claims 11 to 15. A respiratory disorder, allergic disease, urinary system disorders, comprising administering to a subject a therapeutically effective amount of an extract according to any one of claims 1 to 15 or a composition according to claim 16 or 17, And reducing one or more symptoms associated with one or more diseases selected from digestive disorders. 19. The method of claim 18, wherein the respiratory disorders include upper and lower infectious diseases of the lungs, nasopharyngitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, laryngitis, influenza, pneumonia, bronchial pneumonia, and acute exacerbation. The method is selected from the accompanying obstructive pulmonary disease. The method of claim 18, wherein the allergic disease is selected from allergic rhinitis, allergic asthma, and atopic dermatitis. 19. The male pelvic pain of claim 18, wherein the urinary tract disorder comprises urethritis, tubulo-interstitial nephritis, obstructive pyelonephritis, cystitis including chronic cystitis, prostatitis and chronic prostatitis The method is selected from male pelvic pain syndrome, prostate cystitis, and female pelvic inflammatory diseases. The method of claim 18, wherein the digestive disorder is selected from Crohn's disease and irritable bowel syndrome. The method of claim 18, wherein the subject is a human or domestic animal. An extract according to any one of claims 1 to 15 or 16 or as a medicament for reducing one or more symptoms associated with one or more diseases selected from respiratory disorders, allergic diseases, urinary system disorders, and digestive disorders. Use of the composition according to claim 17. Use of an extract according to any one of claims 1 to 15 or a composition according to claim 16 or 17 as an immunomodulator. Isolated microbial strain, Lactobacillus fermentum ) I-3929. An extract obtained from the strain of claim 25. The extract of claim 26, wherein the extract is a soluble extract.

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