TW201010711A - Immunomodulatory extracts from lactobacillus bacteria and methods of manufacturing and use thereof - Google Patents

Abstract

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

Description

201010711 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION A specific example of the present invention includes an extract from a milled U«(10)(10)7/like bacteria which can produce an immunomodulatory effect in an individual. Specific examples of the present invention can be used, for example, as health foods or medicines for treating diseases such as those associated with anti-inflammatory or pro-inflammatory mediator production imbalances, such as infection, allergy, autoimmune A condition and inflammation, or an adjuvant that provides a health benefit in an individual. The invention also includes (i.e., S) the manufacture and use of such extracts. The invention is also directed to a particular strain of bacteria. [Prior Art] Immunomodulation refers to the overall term for a wide range of immune interventions that alter normal or abnormal immune responses. Microorganisms produce and secrete a wide range of molecules that regulate eukaryotic immune responses (Lavelle et al, Cw " r〇/? 2〇〇4, 4(5), 499-508). These include disrupting protective mechanisms to promote the colonization and persistence of pathogens+. Molecules that inhibit inflammatory reactions, scorpion venom, bacteria and parasites have been identified. In addition to the microbial factors that inhibit the immune response, the effective immune activating factor itself may be of microbial origin. These include bacterial enterotoxin, excretion-secretion products produced by parasites, and viral nucleic acids. It is believed that at least 11 families of receptors called tol-like receptors (TLRs) and represented by host organisms play a key role in immunological testing and initiate responses to microorganisms. Figure 丨 provides a list of TLR ligands (Gay and Gangloff, known, 2〇〇7, 76:141 65). 201010711 The TLR identifies a wide range of molecules, also known as pathogen-associated molecular patterns (PAMP), which are produced by viruses, bacteria and genus (Tse and Horner, d«« and November 2007; 66 Supplement 3: iii77-80). Immunoregulation of TLR linkages has been used in the development of novel therapies for broad-spectrum pathology including infection, malignancy, autoimmune and allergic diseases. TLR agonists and antagonists have been investigated as potential therapeutic agents for the prevention and treatment of diseases. In relatively small clinical trials, TLR agonists have been used as adjuvants for beta vaccines designed to prevent infection, eliminate allergies, and eliminate malignant cells. TLR agonists have also been studied as monotherapy and adjuvant therapies for the treatment of patients with infections, allergies and malignant diseases. TLR antagonists have also been investigated as potential therapeutic agents for autoimmune diseases and sepsis in preclinical and clinical trials. Probiotics are living microorganisms that provide health benefits to individuals when administered in appropriate amounts (Mottet et al., hver Dz. jeaie, 2005, 37:3-6; Ezendam et al., iVwir and ev, January 64, 2006 ( 1).1-14; GlU and Prasad, Ji/v Med 2008, 606: 423-54). The biological mechanisms involved in the immune response stimulation of probiotic microorganisms and certain cellular components of their microorganisms have been the subject of research. For example, gram-positive bacteria have characteristic cell walls that contain macromolecules such as lipoteichoic acid 'LTA' [i can be associated with immunostimulatory activity (e.g., Bhakdi et al, ^(4)., 1991,59:4614-4620; Setoyama et al., Gew Microho/, 1985, 131(9).2501-2503 'Cleveland et al., /«/eei 1996, 201010711 64(6):1906- 1912). See also (Deininger et al, C7z « Immunol, 2007, 14(2): 1629-1633). In addition, probiotics may contain a variety of TLR ligands with immunomodulatory characteristics. It was found that cell wall fragments from a variety of bifidobacterial strains stimulated the production of ex vivo interferon-7* (IFN-γ) in mouse spleen cells (T. Ambrouche, r Contribution a l'etude du pouvoir immunomodulateur des bi fidobacteries : Analyse in vitro et etude ex vivo des mecanismes moleculaires impliqu0s," Ph.D. Thesis, # Universite Laval, Quebec, 2005 ). A capsule made of a cell wall fragment of a specific lactic acid bacterium (Del-Immune V9, Pure Research Products, LLC, Colorado) is also intended to stimulate the immune system. However, injecting live or killed forms of probiotics or microparticle cell wall fragments injected with the bacteria may not be the most effective means of providing immunomodulatory effects in an individual. For example, living cell extracts can contain large proteins and lipopeptides that are sized to impede effective absorption by the individual, thus limiting the local concentration of beneficial molecules from the probiotics in the body. Conditions within the body can also destroy 活性 active bacterial components or otherwise alter the chemical structure of their components to inactivate them. Risks associated with oral administration of probiotic microorganisms (lactobacilli) include pain and sepsis (Lactobacillus Sepsis Associated With

Probiotic Therapy, Shan Ruguang/I, January 2005, 115 (1): 178_181). Therefore, there is a need for other ways of delivering the beneficial effects of probiotics to individuals in need. SUMMARY OF THE INVENTION The present invention relates to a sputum extract, some of which may exhibit strong immunomodulatory activity of 201010711. By way of example, specific examples of the invention relate to pharmaceuticals useful as therapeutic foods or in some cases for the treatment of infectious diseases, allergies, respiratory disorders, and inflammatory pathologies or as adjuvants associated with treatment regimens. Extract from bacterial strains. The invention is also directed to compositions comprising such extracts, and methods of making such extracts, for example, using a medium that does not cause the risk of pathogenic protein particles (pri〇n). The methods of the invention include, for example, lysing cells under alkaline conditions, or subsequently lysing cells under acidic conditions under acidic conditions. In some specific ® examples, the extract of the present invention is a soluble extract, meaning that it does not contain large amounts of solid or particulate matter. In some embodiments, the extract contains a chemically modified TLR ligand. In some embodiments, the qualitative treatment can result in chemical modification of cellular material including TLR ligands, cell wall components, proteins, lipoteichoic acid, lipopeptides, and phospholipids. Some specific examples of the invention may comprise extracts obtained from one or more of the following species: Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, yo. Lactobacillus johnsonii, Lactobacillus helveticus, Lactobacillus casei defensis, Lactobacillus casei ssp. casei, Lactobacillus paracasei, Lactobacillus Bulgaricus), Lactobacillus acidophilus, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus salivarius 201010711 (Lactobacillus salivarius), Lactobacillus lactis, anti-Bai's milk Bacterium (Lact〇bacillus delbrueckii) 一些 In some embodiments, the extract comprises at least one strain from each of the above bacterial species, and in other specific examples, one or more of the particular strains from the above list may be In addition to or substituted one or more different strains. Some specific examples of the present invention include extracts obtained from one or more of the following bacterial strains: 肄舁 肄舁 shore mill /-3929, A. faecalis, Lactobacillus plantarum η, 39, Lactobacillus johnsonensis ι〇3782 , anti-Swiss ❹ and shore 6 . The above strains are deposited under the Budapest Treaty (Budapest 酦. Lactobacillus brevis 1-3929, Lactobacillus rhamnosus n 38, Lactobacillus plantarum 71, Lactobacillus johnsonii 103782, Lactobacillus reuteri 7 each deposited in France, 75724 Paris, 25 rue du Dr. R〇ux, Collection Nationale de Culture des Microorganismes, Institut Pasteur. The invention is also (especially) about strains of sputum and axe produced by The extract obtained by the strain, the method for producing the extract, and the use thereof. The halogen strain is obtained by subjecting the strains of Lactobacillus plantarum and Lactobacillus utilis to undergo chromosome exchange, thereby producing a novel Lactobacillus strain. #磨1-3929 The extract obtained is active in several different in vivo and in vitro models associated with infectious and immunological conditions. In some embodiments, the extract is obtained from only one particular bacterial strain. More than one strain is used. In other specific examples, microorganisms from different types can be added (such as from non-cluster species) - or a variety of extracts. 11 201010711 Extracts can be obtained by lysing bacterial cells under specific conditions after the cells are grown in the medium to a suitable concentration. In some embodiments, the bacteria are not causing pathogenic protein particle-related diseases. The risk may be increased in a medium that is at risk of other diseases transmitted by the product obtained from the animal-based medium. For example, 'in some embodiments, the plant-based medium (such as a soybean-based medium) is used to grow the cells. The cytoplasm (ie, the product of cell lysis) can also be filtered to remove nucleic acids and larger cell debris, such as insoluble or particulate matter. In some instances, the amount of nucleic acid present in the extract is less than 100 pg. /mL·. Thus 'in some specific examples' the extract contains soluble molecular components and does not contain large amounts of insoluble or particulate material. Membrane and cell wall molecules can be dissolved or suspended in extracts, including lipoproteins, peptides, peptides Glycan, lipooligosaccharide, lipoteichoic acid, and teich〇ic acid. During the dissolution process In the meantime, molecules in cells (such as membranes and cell walls) can be chemically modified by alkaline treatment, such as cleavage into smaller structures. Despite the presence of such chemical modifications, the specific examples of the present invention are relatively fine. • The cells may retain their biological activity, or such specific examples may even exhibit enhanced biological activity compared to whole cells. For example, alkaline treatment may be used to lyse cells, or may be applied to another method previously dissolved. During the process of the assay according to some embodiments of the present invention, the amino acid found in the native protein and lipid is at least partially racemic to D. amino acid. D• Amino acid may be beneficial for increasing The usefulness of the extract is because it does not digest effectively in the gut of the mammal. D. Amino acid can also protect the smaller form and the egg does not fall during digestion. 12 201010711 Solution. Examples of D-amino acids include protein-bound d-amino acids, and a smaller range includes apo-alanine (de Vrese et al., J. J., 2, 2026-2031). Thus, antigenic molecules that are chemically modified during solubilization in extracts containing D-amino acids can remain in the patient for long periods of time, potentially allowing for stronger immunostimulatory effects in some specific examples. In some embodiments The filtration method may also affect the characteristics of the resulting extract due to the pore size of the filter, and in some cases, the chemical characteristics of the surface of the filter (i.e., its polarity) may change the type of material removed and retained. In some aspects, some embodiments of the invention employ filtration methods designed to retain molecules of interest but to remove other molecular components, such as nucleic acids or insoluble or particulate matter. The filtered extract can also be organically extracted, organically _Aqueous extraction, chromatography, ultracentrifugation, ultrafiltration, or a combination thereof for further purification. [Embodiment] Definition "ExtraCt": An extract as defined herein means dissolved sputum - or a plurality of fine g The material obtained after the plant. In some cases, the extract is obtained from only the sorghum strain, while in other cases, the extract is obtained from a mixture of several different strains. Under these conditions, the extract is a soluble extract, It means that it does not contain a large amount of microparticles and non-cold materials, such as microparticles or solid cell wall fragments. The extract may contain components from the cell wall, organelles and cell membranes dissolved or suspended. For example, extraction The material may be treated (such as by enthalpy; deviate ~ or another separation technique) to remove particulates and insoluble materials 13 201010711 m. Chemical lysis: This is in alkaline, acidic and / or infiltration conditions A method of dissolving bacterial cells. Lysate: As used herein, this term means a bacterial extract obtained from a cell lysis procedure. Filtration: A filtration method as described herein means an extract or extraction. The mixture passes through one or more filters, such as a micro-periator (ie, 'microfiltration') and/or an ultra-filter (ie, 'ultrafiltration). The filtration can be removed without removing _% The components to be removed are counted, but in some embodiments, the extract may be substantially free of its components. In some cases, the filtration is repeated in several passes or cycles. Initial pH The term "the term" means the pH measured at the beginning of a procedure such as bacterial dissolution or over/under. Saccharide: A sugar as defined herein includes monosaccharides, disaccharides, and larger sugars, such as linear and Branched polysaccharides. Sugars also include substituted or chemically modified sugars, such as lipopolysaccharide (LPS) and its chemically modified variants. Lipoprotein: This term refers to a protein or peptide bond and a New Moon. A macromolecule, such as a protein or peptide covalently bound to a lipid. Lipoproteins as used herein also include lipopeptides. Peptidoglycan: This term refers to a sugar and amino acid <polymer. Lipoteichoic acid (LTA): This term refers to a surface-associated adherent amphiphilic molecule present in a Gram-positive strain. Teichoic acid: This term refers to a polymer that is linked to glycerol phosphate or phosphoribosyl phosphate via bismuth diacetate 201010711. D-amino acid (D_aminGaeid): This term refers to the fact that the amino acid present in the dextrorotatory form is the opposite of the L-amino acid which is produced by the biosynthesis in the left-handed isomeric form. Racemization: This term indicates that at least a portion of the L-amino acid is chemically modified to a D-amino acid. • A medium that avoids the risk of disease based on pathogenic protein particles means preparing serum or meat extracts that do not contain any other animal, such as from animals such as cattle or sheep, or diseases that can transmit disease-based protein particles. The medium used in any stage of the extract of the material. Examples of the medium include a plant-based medium or a synthetic medium, and a medium using horse serum or a medium containing materials derived from animal species which do not transmit pathogenic protein particle diseases. Examples of diseases based on pathogenic protein particles include, for example, mad cow disease, scrapie (like) and subacute spongiform encephalopathy (Creutzfeld_Jac〇bdisease). The non-animal medium is a medium that does not include components produced by the animal. Examples include plant-based (i.e., botanical) media (such as soy media) and synthetic media. β. Health foods as used herein means any composition that has a beneficial health effect in an individual when administered, wherein The composition, for example, can be used by an individual without a doctor's prescription. Treatment as used herein in the therapeutic context means, for example, /D treating a current disease or condition and preventing the development or protection of a new disease or condition from the development of a new disease or condition. 15 201010711

Ad Adjuvant as used herein in connection with a specific embodiment of the invention refers to a specific example of the invention provided to an individual in connection with a medical treatment plan. Immunomodulation, immunomodulatory, and the like, as used herein, refers to the ability to alter an immune response in an individual, such as to produce an anti-inflammatory or immunomodulatory effect, in a manner that has a health benefit. As used herein, anti-inflammatory and the like are used to reduce the immunomodulatory effects of inflammation. Immunostimulatory as used herein and its like terms refer to stimulation of the immune system. Protective immunity, as used herein, means providing a specific instance to an individual to provide protection against subsequent infection or allergen challenge. Therefore, during the attack, the concentration of the infectious agent or allergen in the individual is sufficiently low' to not significantly impair the health of the individual. The length of time for effective protection against an attack can be limited to, for example, hours, days, or weeks. A subject, as used herein, means any animal individual, including mammalian individuals, such as humans and domestic animals. Livestock, for example, may include mammals such as dogs, cats, horses, pigs, cow 'sheep, goats or other animals' and may also include non-mammals, such as blacks, such as hard, duck, goose 'turkey and others Livestock and poultry. It is to be understood that the particular bacterial strains identified herein and used in the present invention may include strains obtained from the initial registrations listed herein or genetically pure lines including 16 201010711, which are subsequently re-registered under different accession number names, but are considered genetically The same strain as the originally deposited version. All numbers used herein are approximations, taking into account the inherent errors, the Preparation of extracts The present invention comprises an extract of - or a plurality of strains of Lactobacillus bacteria, wherein the extract is a soluble extract, and wherein the extract contains bacterial molecules of the chemical substance. The extract of the present invention can be prepared, for example, by culturing cells, followed by collecting the resulting biomass' to dissolve and purify. For each strain, t, in order to obtain a sufficient amount of material, the fermentation culture can be started from batch working seeds and subsequently inoculated into larger fermentation vessels. The medium used can be the same for each species. In some specific examples, a medium that avoids the risk of disease based on pathogenic protein particles can be used to grow all of the strains to be used. After the fermentation, the resulting biomass from the strain or group of strains can be inactivated by heat treatment to concentrate and freeze. Thus, in some embodiments, the starting material used to form the extract may not dissolve whole cells. In other embodiments, the starting material used to prepare the extract may be a biomass obtained from cells that are at least partially mechanically, enzymatically or chemically dissolved. In other specific realities, the starting material may be part of such previously dissolved cells, such as those containing cell walls. In some embodiments, the starting materials are treated with an inert medium such as a strong test (such as hydroxide) or other strong inorganic or organic test. 17 201010711 In this dissolution or alkali treatment step, undissolved cells in the starting material are dissolved, and in some embodiments, the 1J Ύ cell component can be chemically modified. Therefore, in some specific examples, the chemically modified bacterial molecules are obtained by alkali treatment, such as by using one or more kinds of extracts obtained by invigorating the virgins of the horses, and extracting the bacterial strains (that is, 'as before Zhang Wei 1 Illustrated 'alkali treatment of undissolved cells or components or parts from bacterial cells) was obtained. In some specific examples,

经受The raw materials with a dry weight concentration of 2 g/L to 90 g/L are subjected to alkali treatment, and the acne is acne

Let $/morning be such as from about 2 g/L to about 80 g/L or from about 3 g/L to about 40 g/L, such as 1 /τ & such as 3 g/L, 5 g/L, 10 g/L, I5 g/L, 2〇g/L, 25 g/L, 3〇g/L, 35 g/L or 40 g/L, or even about 5 g/L to 50 g/L or Other ranges of concentration constraints listed above. In some embodiments, from about 4 〇g/L to about 8 〇g/L is subjected to an alkali treatment, such as 4 〇 / / 50 g / L 60 g / L, 70 g / L or 80 g / L or by The range of concentration constraints listed above is subjected to alkali treatment. The dry weight of the biomass is defined herein by the number of grams of dry weight of the material per liter of sample. It can be taken at about 105. (: The sample is dried in a small porcelain plate until it reaches a constant weight. The temperature can be 30 ° C to 60 ° C, such as 30. (: to 55 ° C, 30 ° C to 50 ° C, 30 C to 45. (:, 30 ° C to 4 ° t:, or 30 ° C to 35 ° C. In some embodiments, the alkali treatment temperature may be, for example, 35 ° C to 60 ° C, such as 35 ° C to 55 ° C '35 ° C to 5 (TC, 35 ° C to 45 t:, or 35 t to 4 (TC. In some specific examples, the alkali treatment temperature can be 3 It:, 32 ° C, 33 ° C , 34 ° C, 35 ° C, 36 ° C, 37 ° C, 38 ° C, 39 ° C or even 40 ° C, or the range of temperature constraints listed above. 18 201010711 alkali treatment time can be from 2 hours Up to several days, such as i, 2, 3 4, 5 or even 10 days, or 3 to 120 hours, or 3 to 48 hours, 3, such as 3, 5, 8, 15, 14, 16, 18, 20, 22, 24, 26, 28, %, cut, 44 or 48 hours, or 15 to 12 hours, such as 6 to 30% hours, such as 60, 72, 84, 96, 1 to 8 or 12 hours, Or the range of time constraints listed. It should be understood that these time ranges include any scores of days, hours, or minutes. In the specific examples, a strong alkalinity of 0·001 N to i 〇N, such as 0.001 N to 0.6 N, or 〇, 10 N to 〇8 N, or 〇6 N to 1· 〇Ν, or from 0.001 N, 〇·〇〇2Ν, 〇〇〇3>^ or 〇"^open=beam range' or 0.1 Ν to 0.6 Ν, or 〇6 Ν, 〇7Ν, 〇8 ν , Ό Ν·9Ν, ^ or ^ range of beginning or ending, "other ranges constrained by the concentrations listed above. In some embodiments, the alkali concentration is used such that an initial pH greater than 9.0 is reached, or a large M952 pH is greater than And a pH value of less than 13.5, such as a pH greater than u.5, greater than 12 〇, greater than Μ, greater than 13.0, or pH 9.0 to pH 13 5. In other embodiments, the alkali concentration may be used such that greater than 10.0 is achieved. And an initial pH of less than 13 Torr, or for example, pH 9 〇 to pH 13.0. In some embodiments, the value of 11 during the alkali treatment may be reduced when extracting the soluble component. For example, the initial pH may be Alkaline {) 11 values, such as 1^9.〇 to 1) 1113.0, or 1) 119.5 to 1^125. Alkali treatment at temperatures listed above may be allowed for a specific time. For example, 3 to 12 hours, such as 3 to 48 hours, or a period as listed above. Next, in some embodiments, the pH may be made acidic by adding, for example, hydrochloric acid, as appropriate, in order to obtain a pH between 2.0 and 4.5, or a pH between 2.5 and 4.5, or a pH between 2 and 5 and 4., such as a pH of 2.5, 3.0, 3.5, 4.0 , or a range bound by any of the pH values listed above. The second treatment at low pH may range from (10) to 6 (rc, 35. to 55C, or 35C to 45°C, such as 35°C, 36°C, 37°C, 38°C, 39°). C, 4 (TC, 4TC, 42 ° C, 43 ° C, 44. (:, or even 45t temperature. The acid treatment time can be from 丨 hours to several hours, up to 72 hours change, for example between i Between hours and 24 hours, or between i hours and 碜6 hours, or between 3 hours and 48 hours, or between 3 hours and 24 hours, or between 4 hours and 72 hours Between, or even between 24 hours and 72 hours, or a time frame bound by the times listed above.

. 'only·,... The inclusion of Qiaogongyue between 10 g /L and wide g/L (for example) from Lactobacillus fermentum = quality for qualitative treatment. In other specific examples, an experimental treatment is carried out on a bacterial biomass comprising (for example, a mixture of bacteria: a mixture of bacteria and having a weight between ...). In the case of (4), the treatment may be between 0.025. N with 0.25 N under the test 'or between ..5. The value of H is between 3 hours and 48 hours ^ in the C body example, between 〇·〇25 ^^ Between 〇2〇—, OWN, between . and .2q 于. Then 〇.. between ,··05Ν and ..10N: ': between one and N弋, between 〇_1〇 N between 20 and 201010711 0.25 Ν, between 0.10 Ν and 0.20 ,, 4 # between 0.10 Ν盥 0.15 Ν, between 0.15 Ν and 0.25 ,, ” between 0.15 ν and 0.20 Ν Or, even between 〇20 Ν and 0.25 Ν 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性 碱性Handle 1, and twin ^) have a range of 12.0 between 9 s, between 9.5 and 11.5, and 9.5 and 9.5 are between 9.5 and 10.5, between 9.5 and 10.5. Ι0 Λ , · Between '. υ, between 12.5, between 10.0 and 12.0, confession; 10·0 and 1 1 〇.0 Ja. 1 j ^ between 10.0 and 11.0, between 10.0 and Between j 0,

• Between 5, between *\ 1 Λ C and 12.5, between 10.5 and 12.0, 乂^ 10·5 between 10.5 and ns, between 10.5 and 11.0, between 1ΐ Λ κ Between U.5 and 121 and 12·5 between 11.0 and 12_0, between 11.0 and 11.5, between, between 11 and 5 fish? The pjj between 1 1.5 and 12.0 is enclosed with a value of 12.5, or even between pH and I2.5. These examples are based on 12.0 alkaline treatment of 拄 between 3 hours and 36 hours 'between 3 hours and between 8 hours and 8 hours Between Between 3 hours and 18 hours, between 3 + machi < between ' ^ 12 ], η main between 3 hours and ό hours, between ό small &, 、, between 巧 and 48 hours „ . between 6 hours and 36 hours , between 6 + < between 'media - and 24 hours „ between 6 hours and 18 hours, between 6 hours 'media 1 2 small _ > pq a between 6 hours and 8 hours, Between 8 hours and 3 times x 48 /]> gi ^ Ββ between 8 hours and hours, between 8 hours and Β 1 ; between hours and 18 hours, between 8 hours and ΐ 2 / hour Between 8 hours and 48 hours, between 12 hours and 】, between 12 hours and 18 hours, between 18 hours and 4 J, between 'between 12 hours, between 18 21 201010711 Between hour and 36 hours, between the hour and the hour of the hour... between 24 hours and between 24 hours, between 2 hours and % hours, or between, ^, m sex Material riding calendar (four) bundle above range Any period '3,6,8,12 e.g., |' or eye Co. francium ~ * 18,24,36 or even 48 hours. These conditions provide a mildly acceptable treatment. Between 0.35 N and 舆0.25 N and 0 _ 5 0 N between 〇·30 N and 0.5 0 Ν

:: In his specific example, the dry weight of IOg/L and 4〇g/L from different strains of Lactobacillus can be subjected to ❿, 〇 5 N and 0.50 N at a temperature of hunger to listen. The hydroxide ion concentration or the pH between "$ and .5 lasts between 15 hours and 12 hours. For example, in some specific real money, the hydroxide soil can be between 015 N and (4) N, between 〇·15Ν and 〇 between 〇”5N Between 0.15 Ν and 0.20 Ν Between 0.20 Ν between 0.30 Ν between 0.15 N and 〇, between 3〇N between 0.15 Ν and 0.20 介于 between 0.20 Ν and 0.40 介于 between 0.25 0.5 and 0.50 ---- --~-Between 〇_30 Ν and 〇.4〇Ν, or between 〇4〇Ν and 0.50. This particular example can have, for example, u. Between ΐ·5 and 12.5 is between Between U.5 and 12 ,, between 12.0 and 13.5, between 12.0 and 13 '' between 12 〇 and 12.5 Between 12.5 and 13.5, between 12.5 and 13 、, between 13.0 and 13.5. The period of alkaline treatment can be, for example, between 15 hours and 100 hours. , between 15 hours and 9 hours, between 15 hours and 75 hours, between 15 hours and 6 hours, between 15 hours and 48 hours, between 15 hours and 36 hours , 22 201010711 " between 24 hours and 120 hours, between 24 hours and 1 hour, between 24 hours and 90 hours, between 24 hours and 75 hours, between 24 Between hours and 60 hours, between 24 hours and Ming hours, between 36 hours and 12 hours, between % hours and ι hours, between 36 hours and 90 hours, Between 36 hours and 75 hours, between 36 hours and 60 hours, between % hours and 48 hours, between 48 hours and 120 hours, between 48 hours and 1 hour Between hours and hours, between 48 hours and 9 hours, between 48 hours and 75 hours, between 48 hours and 6 hours between between hours and 120 hours, between 6 Between hours and hours, between hours and 90 hours, between 6 hours and 75 hours between 75 hours and 120 hours, between 75 hours and 1 hour Between 75 hours and 90 hours, between 9 hours and 12 hours or between 100 hours and 120 hours. The alkaline treatment periods also contemplated in these specific examples include 15, 24, 48, 6 〇, 75, 9 〇, 1 〇〇, and 12 〇 hours. These conditions provide a strong alkaline treatment. _ In other specific examples, the initial raw shell dry weight between 1〇g/L and 4〇g/L can be between G()25 N and 〇25 N at a temperature of 35C to 45°C. The hydroxide concentration between the two or the pH between 9 and 15 is treated between 3 hours and 48 hours. The ruthenium 1511 can then be adjusted to between 2.5 and 4.0 by the addition of an acid (such as hydrochloric acid '110:1)' comprising an acid treatment. Acid treatment can be carried out at temperatures between hunger and price; between hours and 24 hours. For example, in such specific examples, the assay for bacterial biomass comprising - or a plurality of Lactobacillus strains may be between 23 201010711 m 0.025 N and 0.20 N, between 0.025 N and 0.15 N, Between 0_025 N and 0.10 N, between 0.05 N and 0.25 N, between 0.05 N and 0.20 N, between 0.05 N and 0.15 N, between 0.05 N and 0_10 N, Between 0.10 N and 0.25 N, between 0_10 N and 0.20 N, between 0.10 N and 0.15 N, between 0.15 N and 0.25 N, between 0.15 N and 0.20 N, Or even at a hydroxide concentration between 0.20 N and 0.25 N. During alkaline treatment, such specific examples may, for example, have between 9.5 and 12.0, φ between 9.5 and 11.5, between 9.5 and 11.0, between 9.5 and 10.5, Between 9_5 and 10·0, between 10.0 and 12.5, between 10.0 and 12.0, between 10.0 and 11.5, between 10.0 and 11.0, between 10.0 and 10.5, Between 10.5 and 12.5, between 10.5 and 12.0, between 10.5 and 11.5, between 10.5 and 11.0, between 11 _0 and 12.5, between 11.0 and 12.0, Between 11.0 and 11.5, between 11.5 and 12.5, between 11.5 and 12.0, or even between 12.0 and 12.5. The alkaline treatment time of these specific examples may be between 3 hours and 36 hours, between 3 hours and 24 hours, between 3 hours and 18 hours, between 3 hours and 12 hours. Between 3 hours and 6 hours, between 6 hours and 48 hours, between 6 hours and 36 hours, between 6 hours and 24 hours, between 6 hours and 18 hours, Between 6 hours and 12 hours, between 6 hours and 8 hours, between 8 hours and 48 hours, between 8 hours and 36 hours, between 8 hours and 24 hours, Between 8 hours and 18 hours 24 201010711, between 8 hours and 12 hours, between 12 hours and 48 hours, between 12 hours and 36 hours, between 12 hours and 8 hours Between 18 hours and 48 hours, between 18 hours and % hours, between 8 hours and 24 hours, between 24 hours of gold hour, between 24 hours and 36 hours Between hours, or between % hours and 48 hours. The alkaline treatment can be carried out for any period of time, such as 3, 6, 8, 12, 18, 24, 36 or even 48 hours. The pH can then be adjusted to between 2.5 and 3.5, with an acid (for acidic treatment after alkaline dissolution), between 2.5 and 3.0, between 3 and 4.0. Between 3, 〇 and 3 5, or between 3 5 and 4 。. Acid treatment can be carried out between i hours and 18 hours, between 〖hours and Μ hours, between! Between hours and 6 hours, between hrs and 3 hours, between 3 hours and 24 hours, between 3 hours and hours, between 3 hours and 12 hours, between 3 Between 6 hours and 24 hours between 6 hours and 6 hours, between 6 hours and 8 hours, between 6 hours and 12 hours, between 12 hours and hours "at 12 hours" The time between 8 hours and 24 hours between 8 hours. The expected time for acid treatment includes i, 3, 6, 1, 18 and 24 hours. The lysis obtained after the treatment described above The material can then be purified by centrifugation and/or; for example, to remove particulates and insoluble components. For example, the lysate can be centrifuged at 9000 χ gravity and subsequently called at 〇2; On the device, the "multiple rounds of the data. In some cases, you can use the larger holes & the continuous number of transitions on the filter, and then on the 0,2 μιη filter over 25 201010711 filter. Can also use super Filtration to aid in the extraction of soluble materials from the extract, for example, recycling the ultrafiltration permeate for further microfabrication. In some embodiments, the tangential flow filtration (TFF) method can be used to filter lysates and extract soluble molecules from larger cell debris (Figure 2). See, for example, Separations Technology, Pharmaceutical and

Biotechnology Applications, edited by Wayne P_ Olson, Interpharm Press, Inc., Buffalo Grove, IL, U.S.A., pp. 126-135 -ISBNW-9351 84-72-4. At the beginning of the process, the diluted bacterial lysate can be stored in the first storage tank. For example, in TFF, the extract can be exposed to microfilters and ultrafilters. For example, a microfiltration (MF) cycle is initiated and the product is pumped out. The resulting MF retentate is recycled and the MF permeate is transferred to a second storage tank. After reaching the appropriate concentration, an ultrafiltration (UF) cycle is initiated. The UF permeate can be recycled back to the first storage tank for continuous extraction of the dissolved extract from the lysate while the UF retentate is stored in the second storage tank. During continuous extraction, the volume in tanks 1 and 2 can be adjusted by adjusting the flow rate of the microfiltration and ultrafiltration permeate. This extraction cycle can be carried out several times using TFF or another filtration method. In the specific example of using TFF, at the end of the last cycle, the ultrafiltration cycle can be shut down and the microfiltration cycle can be operated separately and the MF permeate can be transferred to storage tank 2. The conditions of cross-flow and membrane pressure are defined by the pressure-independent and mass transfer control zone in thin film theory, as described, for example, by M. Cheryan {Ultrafiltration and Microfiltration Handbook, 2nd edition, special 26 201010711 4, ed.). Permeate flux and extraction yield are affected by filtration conditions (transmembrane pressure (Τ Μ P ), cross flow, temperature, etc.). Filter types and board system types (cassette fUter) can also affect performance. Different configurations including parallel mode and 蜿蜒 mode can be used (see Figure 2). The study uses specific conditions for the best performance of each combination of mode type and filter type. The microfiltration cycle can be equipped with a 1.2 μm to 〇”μηι filter, such as a 〇 65 μηι to 〇·2 μιη filter or a 〇 45 μηι filter. The cross flow may be between ❹ L/h.m^ 3000 L/h.m2 (LHM), such as between 3 〇〇 LHM and 2500 LHM, or 2000 LHM, where TMp is 〇 3 to 2 bar. The ultrafiltration cycle can be equipped with 10 KDa to 1 KDa, such as 1 KDa to 100 Da or 1 KDa to 30 KDa, or 30 KDa to 1 KDa. The cross flow may be between 30 LHM and 1 〇〇〇 LHM, such as between 2 〇 LHM and 500 LHM where τ Μ ρ is 0.2 bar to 1.5 bar. A diafiltration volume between 5 and 20 can be used to extract from the bacterial cell wall. In some embodiments, a volume between 8 and 15 is used. Thus, for example, in some embodiments, a filtration cycle between 5 and 15 may be used, and in some cases between 8 and 15 cycles. The extract may be further concentrated or centrifuged if needed after filtration. For example, 迨e m can be used. Further microfiltration using a smaller pore size filter (such as a 0. 2 μιη filter) is used. After filtration, the extract can be lyophilized' subsequently formulated for use. In some embodiments, after the mashing, the extract can be purified to separate, increase or increase the concentration of one or more of the modified components in the extract. By way of example, a strong ionic chromatography step can be used to remove the exchanged components. Other purification methods can be used, such as condensate: a, a k sin gel filtration, chromatography, ultracentrifugation, extraction and precipitation. The servant of bacterial extracts, which can cause a variety of chemical modifications of the cell components. For example, in a protein: (1) a peptide bond can undergo partial cleavage, resulting in a smaller polymorphism; (2) a natural, L-amino acid can be at least partially racemic to a D-amino acid; and (3) 〇天$proline and (iv) Aminic acid residue* deamination, resulting in changes in the isoelectric point of the protein. For example, molecules such as phospholamic acid, lipopeptide and phospholipid can undergo base-catalyzed hydrolysis of ester bonds and/or guanamine bonds. A modified two-parental structure that has new physicochemical properties and immunological properties. Examples of other possible chemical modifications include partial solubilization of cell wall polysaccharides and complete hydrolysis of ribonucleic acid (RNA) into individual ribonucleotides, including phosphate rearrangements. Thus, some or all of these chemical modifications can occur during base treatment of the Lactobacillus cells as described herein. These molecular modifications can affect the biological activity of the extract. For example, the alkaline treatment of the bacteria of the present invention can result in partial hydrolysis of the protein as well as partial exotherlation of the deamination, deamidamine and/or amino acid from L to D. In an analytical study of the extract of the present invention, D-aspartate, D-glutamic acid, D-serine, D-guanidine thiol, D-histamine, D-propylamine were respectively observed. Acid, D-arginine, D-phenylalanine, D-tyramine, D-leucine and D-lysine. The percentage of D-amino acids in their species in the study ranged from 3% to 40%. Thus, in some embodiments of the invention 28 201010711 allows for the opening of one of the amines, ursolic acid, histidine, alanine, phenylalanine, leucine and lysine, face "^ + or more 'For example, all of the above 2-based acids, or more than all of the above-mentioned amino acids, such as alanine, phenylalanine, and lysine, are racemized. In this case, At least (10) of - or more of the amino acids may be racemized. In other embodiments, at least 40% of one of the above amino acids may be racemized. Thus, the extract of the present invention The material may comprise a D-amino acid such as between 1% and 80% or between 1% and 6% between (4) and (4).

In some embodiments, the extract comprises a D-amino acid between (10) and Na, such as between 25% and 35%, and the extract of the present invention may comprise at least one selected from the group consisting of · Aspartic acid, d• Asparagine, D-glutamic acid, D_glutamic acid, D' lysine, 1 methionine, D_histidine, D-alanine, D _ arginine, amphetamine, D tyrosine, D-leucine, D_ lysine, D_proline acid u threonate group of d_ amino acids. In some specific examples The concentration of any of the D-amino acids is comprised between 1% and 50%, such as between 1% and 4%, or even between 15% and 35%. Some extracts comprise Lactobacillus bacterial cell walls and membrane components, such as lipoteichoic acid, teichoic acid, peptidoglycan, or combinations thereof. In some embodiments, the components are chemically modified. Including 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 (eg, lipoproteins) are dissolved or suspended in the extract, and thus It does not Microparticles or insoluble 29 201010711 % form is present. Further, the extract of the present invention may comprise, for example, 1 〇 mg/mL to 1 〇〇 mg/mL soluble dry weight (SDW) material, 丨 mg/mL to 3 〇 mg/ mL protein (Prot.), 〇·5 mg/mL to 4.0 mg/mL sugar and less than 1 μμ§/πιΕ DNA. For example, some specific examples contain about 15 mg/mL to 35 mg/mL solubility. Dry weight, 3 mg/mL to 7 mg/mL protein, 1 〇 mg/mL to 3.0 mg/mL sugar, and 1 〇pg/mL to 40 pg/mL DNA. The extract of the present invention may contain, for example, 30 mg /mL soluble dry weight, 96 mg/mL ginseng white matter, 2.4 11^/11^ sugar and 33 called /11^0>^, or another example containing 324 mg/mL soluble dry weight, 5.8 mg/mL protein, 2 3 mg/mL sugar and less than 100 gg/mL DNA. Soluble dry weight (SDW) in g/L or mg/mL by obtaining 5 mL of soluble fraction produced by dissolution or alkali treatment at 1 05° It is determined by drying it to a constant weight in a porcelain dish under C. In some embodiments, the extract comprises at least mg3 mg/mL., such as between 〇.3 1^/1^ and 4,51^/„ ^Between the sugar. In some embodiments, the at least one sugar is selected from the group consisting of a monosaccharide, a disaccharide, and a polysaccharide. Some of the extracts of the present invention comprise at least one branched polysaccharide. In some embodiments, at least one of the sugars is chemically modified. Dissolution or alkali treatment of the bacteria of the present invention may result in a decrease in the average molecular weight of the component macromolecules to, for example, lkDa to a range between 3〇〇_ and 1〇〇_ or 1 kDa to between 60 kDa and 1 Between the heart and the heart. In some embodiments, the extract comprises at least one protein having a molecular weight of less than 50 kDa or less than 30 kDa' such as less than 1 〇 kDa. Bacterial extract organisms n 30 201010711 钃 The extract of the present invention may have immunomodulatory activity. For example, some extracts can stimulate the immune system. Some extracts may have anti-inflammatory activity. The specific action of the extract may depend on the conditions of manufacture and the species or strain of the lactobacillus from which the extract is obtained, or the mixture of species or strains. Thus, some of the extracts of the present invention may exhibit potent immunostimulatory activity, and thus may be suitable for treating infections or as adjuvants for such treatments, while other specific examples may exhibit weaker immunostimulatory activity but exhibit anti-inflammatory activity, thus being suitable For the treatment of inflammatory conditions such as allergies, asthma, autoimmune diseases, colitis and ® inflammatory bowel disease or as an adjuvant for this treatment. Thus, some of the extracts of the present invention are effective for treating patients suffering from conditions including, but not limited to, microbial infections, allergic diseases, and digestive tract disorders. Some of the extracts of the present invention may also be provided to a patient as a health food', for example, as an adjuvant for treating various conditions including, but not limited to, microbial infections, allergic diseases, and digestive disorders. The biological range of the extract can be determined by several in vitro and in vivo assays. For example, the AlamarBlueTM-assay is based on the incorporation of a fluorescent/colorimetric growth indicator by an oxidative-reduction (rED〇x) indicator that reacts to chemical reduction produced by cell growth (Example 4) . In vitro cell assays test for nitric oxide (NO) production in major murine macrophages and screen the extract to stimulate the immune system to kill invading bacteria (Figure 5). In some embodiments, the extract stimulates NO production in murine animal macrophages, resulting in a concentration of NO ranging from 3 μΜ to 60 μΜ, such as from 5 μΜ to 40 μΜ. In some embodiments, 31 201010711 NO concentration may be higher than 30 μΜ. The type of bacterial species can also affect these choices. For example, extracts from Lactobacillus fermentum can induce greater nitrogen oxide production than extracts of Lactobacillus rhamnosus (see, e.g., Example 5, below). Specific Examples of the Invention for Screening for In Vitro Immunostimulatory or Anti-Inflammatory Potential The test of the bacterial extract of the present invention can be carried out on human peripheral blood mononuclear cells (PBMC). See Foligne et al.' (fTor/i/ J Gasiroewiero/, 2007 13(2): 236-243). The release of iL12P70 (inflammatory interleukin) and IL1〇© (anti-inflammatory mediator) was measured and the IL-10/IL-12 ratio was calculated (Example 6). Specific examples of the invention produce a higher ratio of IL 1 /IL12 than the live rubbing shore mill control, thus indicating that some of the extracts of the invention may exhibit equivalent or even equivalent to living parent microorganisms when administered in vivo. Strong anti-inflammatory properties. Accordingly, the present invention includes an extract capable of achieving a calculable IL 10/IL12 ratio in human peripheral blood mononuclear cells, wherein the ratio is equal to or greater than the IL10/IL12 ratio achieved by the Lactobacillus livea strain from which the extract is obtained. The immune response of a substance can also be tested by examining its effect on a Toll-like receptor (TLR), for example, in the presence or absence of the TLR2 agonist Pam3Cys, or in the presence or absence of a TLR4 agonist. The extract was tested in HEK293 cells in the case of LPS (Example 7). The HEK293 cell line enables efficient monitoring of TLR activity using ELISA assays such as il-8 titration or monitoring of TLR-induced NF-κΒ activation based reporter-based systems. Some specific examples of the invention can serve as TLR 2/6 antagonists in HEK TLR 2/6 cells. Thus, some specific examples can be used to combat infection in an individual, while other specific examples can be developed for combating inflammation and/or autoimmune disorders. The extracts disclosed in the present invention can also be screened for TLR and NOD2 receptor activity (Example 8). Some specific examples of the invention activate the trl and/or NOD2 receptor in vitro, indicating that it activates the immune system via TLR and/or NOD2.

Non-specific stimulation of sputum lymphocytes can be assessed using plaque forming ceU (PFc) techniques (Example 9). Certain lymphocytes release hemolytic antibodies that are diffuse and cause adjacent red globules to dissolve by forming plaques in the presence of complementary sequences. Some embodiments of the invention may increase the secretion of immunoglobulins by B cells, and thus may potentially preventive use to elicit the immune system of an individual suffering from a recurrent infection. The anti-infective efficacy of the extract of the present invention can be tested, for example, when an individual is infected with 汐/7 (heart/wowe/M), and the mouse is infected with the infection (Example 1). Some embodiments of the invention may provide protective immunity against infections such as bacterial infections (i.e., stenosis). For example, some specific examples can be reduced by injecting the 7 殇 7 7 7 7 7 ( 10 10 10 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、. The combination of NO in vitro activity with the assay of in vivo activity as measured in a murine allergen-induced asthma model (Example n) provides a more comprehensive view of the potential clinical activity of the extracts disclosed herein. . The number of eosinophils found in the bronchoalveolar lavage fluid in the LACK (protein from the parasite Leishmania () adult) model was reduced by t and ι compared to asthma control (untreated) animals. The factor between ' is reduced by 1-5 times to 5 times. Therefore, in some specific examples, 201010711, extracts reduced the number of eosinophils, neutrophilic white blood cells, lymphocyte counts, or any combination thereof in asthmatic murine individuals relative to asthma untreated controls. A factor of at least 1.5. Some specific examples of the present invention can reduce eosinophils' in the body of asthmatic individuals and are accompanied by a decrease in the content of asthma marker Th2 interleukins (such as IL4, IL5, IL13). Thus, such specific examples, for example, may have anti-inflammatory activity in an individual suffering from an immunological disorder, such as an allergic condition, including asthma. The content of the bacterial extract is fine. The extract of the present invention can be formulated in a variety of different ways for final administration. For example, oral rotatory agents, capsules, pills, and liquid sputum formulations or aerosols can be prepared. Formulations for infusion or injection can also be prepared. Specific examples of the invention may be formulated, for example, as a solid dosage form or a liquid dosage form. Exemplary solid dosage forms can include, for example, a binder containing an extract (e.g., a sputum lotion, a sirloin tablet, an effervescent tablet, a sublingual lozenge), granules, powders, or capsules. The solid dosage form can also contain diluents, fillers, and/or other excipients. Other sizing agent components such as preservatives, colorants, flavoring agents, and sweeteners may be added. As an alternative to capsules and lozenges, powder or granule formulations can be developed which can also be developed as a liquid dosage form for solutions or syrups, suspensions and drops for oral administration. The extract of the present invention can be contained in one or more health food compositions such as nutraceutical and/or dietary supplements and food additives, or in a pharmaceutical composition. 34 201010711 Bacterial extracts to individuals

An agent comprising at least one extract of the invention may be administered to an individual suffering from or at risk of developing at least one condition selected from the group consisting of: a digestive tract disorder, a respiratory tract disorder, a urinary tract disorder ) and allergic conditions (allergic condition). For example, in some embodiments, the extract may be administered to a sputum that is at risk of developing or at risk of developing a pulmonary upper and lower respiratory infection, having an acute lower respiratory tract infection. Obstructive pulmonary disease with acute lower respiratory infection, obstructive pulmonary disease with acute exacerbation, nasopharyngitis, sinusitis, pharyngitis, almond Adenitis (tonsillitis), laryngitis (raryngitis), tracheitis (tracheitis), laryngopharyngitis, influenza, pneumonia, bronchopneumonia, bronchitis, rhinitis (rhinitis), nasopharyngitis, pharyngitis, sinusitis, masculinitis, laryngitis, laryngotracheitis, bronchitis, allergic rhinitis, allergic asthma (allergi c asthma ), atopic dermatitis, urinary tract infections due to obstructive and reflux uropathy, urethritis, tubulo-interstitial nephritis 35 201010711 ( Tubulo-interstitial nephritis), obstructive pyelonephritis, cystitis including chronic cystitis, male pelvic pain syndrome (including prostatitis and chronic prostate) Chronic prostatitis, prostatocystitis, female pelvic inflammatory disease, Crohn's disease, and/or irritable bowel syndrome. Φ In some embodiments, the extract is administered to the individual in the form of a health food composition, such as a health food supplement and/or a food additive. In other embodiments, the extract is administered to the individual in the form of a pharmaceutical composition. The administration can include single or multiple dose administration. In some embodiments, the extract can be provided in a therapeutically effective dose to treat an individual suffering from one or more of the above conditions. In some embodiments, the extract can be provided as an adjuvant to other medical treatments. Working examples Refer to the examples? :釭a换屏龅例1.1: Mushroom gallium 啬 I-392Q^^^ Initial culture conditions Prepare the medium by dissolving the following components in pure water: sodium chloride: 3 g/L; Hydrogen diamine: 2g / L; sodium acetate: lg / L; soy protein gland: 5 〇 g / L, glucose. 12 g / L; gasification mother: 〇" g / L; gasification unloading:; sodium bicarbonate : 0.5 g / L; propyl salt: 〇" g / L; glutamate: 〇 2 g / L; metal solution (sulfate steel: 3 mg / 1; gasification iron: 83 〇 mg / 1; sulfuric acid Zinc: 36 201010711 860 mg/1; sulfuric acid: η mg/L): 〇5 mL/L. Then, polypropylene glycol (0.02 mL/L, density 1.005 g/mL) was added. After dissolution, the pH was not adjusted. After sterilizing the medium, the Erlenmeyer flask was individually incubated with cold bundle vial contents (containing 1 > 5 mL of frozen bacteria) and incubated for 8 hours at m. A 20 mi aliquot of this culture was then transferred to a larger Erlenmeyer flask containing 1 mL of medium and incubated again under the same conditions. After 16 hours of growth, the contents of the 1 L Erlenmeyer flask were transferred to the pre-fermenter. • Pelletizer in heavy A fermenter # Prepare 20 L medium by dissolving the following components in pure water: sodium vaporification: 3 g/L; disodium hydrogen phosphate: 2 g/L; sodium acetate: 1 g/L; soy protein gland 5 〇g/L 'glucose. 12 g / L, gasification about: 〇. 1 g / L; gasification unloading: 0 · 1 g / L ·; sodium bicarbonate: 〇 · 5 g/L; pyruvate: 〇1 g/L; glutamate: 〇·2 g/L; metal solution (copper sulfate: 3 mg/1; gasified iron: 83 〇mg/1; zinc creatinate) · 860 mg/1; sulfuric acid: 1.1 mg/L): 〇_5 mL/L. Then add para-propylene glycol (〇_〇2 mL/L). After dissolution, the pH was not adjusted. The incubation temperature was adjusted to 37 ° C, stirred at 100 rpm and not aerated. The pH was not adjusted during the cultivation. After 24 hours, 7L from the former decimator was transferred to the masher (the optical density (〇D) ~ ^24 at 700 nm before the masher culture was obtained after 24 hours). The culture of the pre-fermenter is transferred to the fermenter under aseptic conditions. In the hall of the instrument, the cultivar is made of 7 〇L medium by dissolving the following components in pure water: sodium chloride: 3 g/L; disodium hydrogen phosphate: 2 g/L; sodium acetate: 丨g /L; soy protein gland: 50 g / L; glucose: 12 g / L; gasification calcium: (M g /) L; gasification unloading: 37 201010711 0.1 g / L; sodium bicarbonate: 0.5 g / L; Pyruvate: 〇1 g/L; glutamate. 0.2 g/L; metal solution (copper sulfate: 3 mg/1; gasified iron: 83 ......; sulphuric acid: 860 mg/l; sulfuric acid:丨"mg/L): 〇 5 mL / L. Then, polypropylene glycol (0.02 mL / L) was added. After dissolution, the pH was not adjusted.

After sterilization, 8 g/L of glucose was added to the culture. The incubation temperature was adjusted to 37 ° C, stirred at 1 rpm and not aerated. The pH was adjusted to 5.7 during the culture. After 16 hours, the culture (the culture had an OD of 2.30 at 7 〇〇 nm) was at 65. The underarm heat treatment was inactivated for 35 minutes and transferred to a collection tank. After inactivation, the culture was transferred to an ultrafiltration chute to separate the biomass from the medium, concentrated and washed with NaCl (9 g/L) in pure water. The collected biomass was aliquoted (the mass of the concentrated bacterial suspension was 2,000 mg per gram of concentrated bacterial suspension under 318 mg dry weight) and then at -15. Frozen under the armpits. f Example L2: Beef broth from Switzerland 取3 146 _ initial culture conditions Prepare the medium by dissolving the following components in pure water: sodium chloride: 3 g/L, disodium hydrogen phosphate: 2 g /L; sodium acetate: 1 g / L; soy protein gland: 50 g / L, glucose: 12 g / L; gasification calcium: 〇" g / L; gasification potassium: 〇" g / L; sodium bicarbonate .0·5 g/L; pyruvate: 〇1 g/L; glutamate: 〇2 g/L; metal solution (copper sulfate: 3 mgM; gasified iron: 83 〇mg/1; zinc sulfate : 860 mg/1, sulfuric acid: hl mg/L) : 〇.5 mL/L. Then, polypropylene glycol (0.02 mL/L) was added. After dissolution, the pH was not adjusted. After sterilizing the medium, the small conical flask was frozen in vial contents (containing 1.5 mL of frozen bacteria) and at 37. (: incubation for 9 hours. Then 20 ml aliquots of this culture were transferred to larger conical flasks containing 1 〇〇〇 medium, and 38 201010711 was incubated again under the same conditions. After 15 hours of growth, The contents of the 1 L Erlenmeyer flask were transferred to the pre-fermenter. The replacement parts in the JL Fermenter were prepared by dissolving the following components in pure water to prepare 20 L medium: sodium chloride. 3 g/L Disodium hydrogen sulphate: 2 g / L; sodium acetate: 1 g / L; soy peptone: 50 g / L; glucose: 12 g / L; calcium carbonate: ο.! g / L; : 0-1 g/L; sodium cesium carbonate: 0·5 g/L; pyruvate: 〇·ι g/L, glutamine: 〇.2 g/L; metal solution (copper sulfate: 3 mgM; ferric chloride: 830 mg/!; zinc sulfate: 860 mg]; sulfuric acid: 1.1 mg/L): 0.5 mL/L, followed by addition of polypropylene glycol (〇.〇2 mL/L). pH value. The incubation temperature was 37 ° C, stirred at 1 rpm and not ventilated. The pH was not adjusted during the culture. After 9 hours, the 7 L from the former decimator was transferred to the decimator. The OD of the former fermenter culture at 7 〇〇 nm after 9 hours: 0.14). The culture was transferred to a fermenter under aseptic conditions. The culture medium in the fermenter was prepared by dissolving the following components in pure water to prepare 70 L medium··gasification nano: 3 g/L; hydrogen phosphate Disodium: 2 g/L; sodium acetate: 1 g/L; soy protein gland: 50 g/L; glucose: 12 g/L; vaporized calcium: 0.1 g/L; potassium carbonate: 0·1 g/ L; sodium bicarbonate: 〇.5 g / L; pyruvate: 0.1 g / L; glutamate. 0.2 g / L; metal solution (copper sulfate: 3 mgH; ferric chloride: 830 mg / l; Zinc sulfate: 860 mg/1; sulfuric acid: η mg/L): 0.5 mL/L. Then add polypropylene glycol (〇_〇2 mL/L). After dissolution, the pH value is not adjusted. 8 g/L glucose was added. The incubation temperature was adjusted to 33 ° C, stirred at 100 rpm and not aerated. The pH was adjusted to 6.7 at the beginning of the culture with 39 201010711 CH3COOH. After 24 hours, the culture was cultured at 700 The enthalpy D at nm is 4.17) is inactivated by heat treatment at 6 VC for 35 minutes and transferred to a collection tank. After inactivation, the culture is transferred to an ultrafiltration slide to separate the biomass from the medium, concentrated and purified. (9 g/L) in NaCl (9 g/L) The collected biomass was aliquoted (the mass of the concentrated bacterial suspension was 14. g per gram of concentrated bacterial suspension under 19.1 mg dry weight) and then frozen at -15 °C. Compound_Example 1 _3: Lactobacillus plantarum 71.39. The initial cultured bee was prepared by dissolving the following components in pure water: sodium vaporification: 3 g/L 'disodium hydrogenate: 2 g/L; sodium acetate: 1 g/L; soybean protein gland: π g/L; glucose: 12 g/L; gasification five: 〇1 g/L; gasification _: 〇i; sodium bicarbonate: 0.5 g/L; pyruvate: 〇.! g/L; glutamate: 〇·2 g/L; metal solution (copper sulfate: 3 mg/1; gasified iron: 83 〇 mg/1; ·· 860 mg/i; sulfuric acid ·· K1 mg/L) : 〇5 mL/L. Then, polypropylene glycol (〇.〇2 mL/L) was added. After dissolution, the pH was not adjusted. After the medium was sterilized, the small conical flask was incubated with the contents of the cold cuvette (containing 15 cold-cold bacteria) and cultured at 35T:. An n aliquot of this culture was then transferred to a larger Erlenmeyer flask containing mL of medium and incubated again under the same conditions. Growing. After an hour, transfer the contents of the [conical flask to the former decanter. Former 酦毪 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Lg/L; soy protein 201010711 脒: 50 g/L; glucose: 12 g/L; calcium carbonate: 0.1 g/L; potassium hydride: 〇_ 1 g/L; sodium bicarbonate: 〇_5 g/ L; pyruvate: 0.1 g/L; proline: 0.2 g/L; metal solution (copper sulfate: 3 mg/1; iron oxide: 830 mg/Ι; zinc sulfate: 860 mg/1; sulfuric acid :li mg/L) : 0.5 mL/L. Then, polypropylene glycol (〇.〇2 mL/L) was added. After dissolution, the pH was not adjusted. The incubation temperature was adjusted to 37 ° C, stirred at 100 rpm and not aerated. The pH was not adjusted during the cultivation. After 9 hours, 7 L from the former denitrator was transferred to the fermenter. (OD = 1.62 at 7 〇〇 nm for the previous masher culture after 9 hours). © The culture of the pre-fermenter is transferred to the fermenter under aseptic conditions. The waxed bee pieces in the fermenter were prepared by dissolving the following components in pure water to prepare 70 L medium: sodium vaporification: 3 g/L; disodium hydrogen phosphate: 2 g/L; sodium acetate: 1 g/ L; soy protein gland: 50 g / L; glucose: 12 g / L; gasification calcium: 〇. 1 g / L; gasification potassium: 〇. 1 g / L; sodium bicarbonate: 〇 · 5 g / L ; pyruvate: 〇.1 g / L; valine: 0.2 g / L; metal solution (copper sulfate: 3 mg / Ι; ferric chloride: 830 mg / Ι; sulfuric acid: 860 mg / 1; Sulfuric acid: li mg/L) : 〇 5 mL/L. Then, polypropylene glycol (〇·〇 2 mL/L) was added. After dissolution, the pH was not adjusted. After sterilization, 8 g/L glucose was added to the culture. Adjust the incubation temperature to 35. (: 'Stirring at 100 rpm without aeration. After 24 hours, the culture (culture OD = 6.3 6 at 700 nm) was inactivated by heat treatment at 65 °C for 35 minutes and transferred to a collection tank. After inactivation, the culture was transferred to an ultrafiltration chute to separate the biomass from the culture medium, concentrated and washed with NaCl (9 g/L) in pure water. The collected biomass was aliquoted (in per gram concentrated bacterial suspension) The mass of the concentrated bacterial suspension under 60.7 mg dry weight was 6〇0 201010711 g) and then frozen at -15 ° C. Example 1.4: Initial culture conditions for culture of R. rhamnoides 71.38

The medium was prepared by dissolving the following components in pure water: sodium gasification: 3 g/L; disodium hydrogen phosphate: 2 g/L; sodium acetate: 1 g/L; soy peptone: 50 g/L; Glucose: 12 g / L; Calcified calcium: 〇.1 g / L; Potassium chloride: 0.1 g / L; Sodium bicarbonate · · 0.5 g / L; Pyruvate: oi g / L; guanamine : 0.2 g/L; metal solution (copper sulfate: 3 mg/1; gasified iron: 830 mg/1; zinc sulfate: 860 mg/Ι; sulfuric acid: 1.1 mg/L): 〇_5 mL/L. After dissolution, the pH was not adjusted. After the medium was sterilized, the small conical flask was individually cultured in a frozen vial contents (containing 1.5 mL of cold bed bacteria) and incubated at 35 ° C for 9 hours. A 20 ml aliquot of this culture was then transferred to a larger Erlenmeyer flask containing 1 mL of medium and incubated again under the same conditions. After 5 hours of growth, the contents of the 1 L Erlenmeyer flask were transferred to the pro-acid feeder. The crucible in the front crucible prepares 2〇L medium by dissolving the following components in pure water: sodium vaporification: 3 g/L; disodium hydrogen phosphate: 2 g/L; sodium acetate·· g/L; soy protein 脒.50 g / L; glucose: 12 g / L; gasification calcium: 01 g / L; gasification potassium · 〇. 1 g / L; sodium bicarbonate: 0 · 5 g / L ; pyruvate: 〇jg/L; glutamine. 0.2 g / L; metal solution (copper sulfate: 3 mg / 1; gasification iron: 83 〇 eight; zinc sulfate: 860 mg > a; sulfuric acid: 1.1 Mg/L) : 〇5 mL/L. Then, polypropylene glycol (0.02 mL/L) was added. After dissolution, not adjusted? 11 values. Adjust the temperature of the nursery and the farm to 35. (:, stir with 1〇〇rPm and not ventilate. In the culture ^ 2: adjust the pH value..., after the transfer from the former acidifier 7 [transfer to acid: 42 201010711. (9 hours before the acidifier The culture was centrifuged at 700 nm D. 3.75. The culture of the pre-fermenter was transferred to the fermenter under aseptic conditions. The Petri kit in the fermenter was dissolved in pure water by the following components. Preparation of 70 L medium: gasification nano: 3 g / L; disodium hydrogen phosphate: 2 g / L; sodium acetate: 1 g / L; soy peptone: 50 g / L; glucose: 12 g / L; Calcium: 0.1 g / L; potassium carbonate: 0_ 1 g / L; sodium bicarbonate: 〇. 5 g / L; pyruvate: 0.1 g / L; glutamic acid • Compliance: 0.2 g / L; Solution (copper sulfate: 3 mg/1; ferric chloride: 830 mg/1; zinc bracate: 860 mg/1; sulfuric acid: ll mg/L): 0.5 mL/L. Then add polypropylene glycol (〇_〇2) mL/L). After dissolution, the pH was not adjusted. After sterilization, 8 g/L glucose was added to the culture. The incubation temperature was adjusted to 35 ° C, stirred at 100 rpm and not ventilated. After 14 hours, Culture (culture with an OD of 5.43 at 700 nm) by at 65. (: heat treatment 35 Inactivated in minutes and transferred to the collection tank. After inactivation, the culture was transferred to an ultrafiltration chute to separate the biomass from the culture medium, concentrated and washed with pure NaCU (9 g/L) in pure water. Qualitative aliquot (mass of 2798 g of concentrated bacterial suspension per 5 g of concentrated bacterial suspension 51.7 mg dry weight) and then frozen at -15 ° C. t-mask 1.5: culture of Lactobacillus johnsonii n7S2 Initial training course

: Gasification sodium: 3 nano·· 2 g/L ·, sodium acetate: ig/L; soy protein: 5〇12g/L; gasification: 〇”g/L; gasification: 〇^ g/ L; g / L; pyruvate: 〇. lg / L; valine: 〇 2 § several; 43 201010711 metal solution (copper sulfate: 3 mg / 1; ferric chloride: 83 〇 mg / 1; sulfuric acid Zinc: Vice; Sulfuric acid: i.丄mg/L) : 〇5 mL/L. Then, polypropylene glycol (0.02 mL/L) was added. After dissolution, the pH was not adjusted. After the medium was sterilized, the small conical flask was incubated for 1 hour at 33 C with the contents of the cold conical vial (containing i 5 red cold bacteria). Then, 2 aliquots of this culture were transferred to a larger conical flask containing 1000 ng medium and incubated again under the same conditions. After 14 hours of growth, the contents of the i L conical flask were Transfer to the former decanter. ❹ The handle of the ILM fermenter is prepared by dissolving the following components in pure water to prepare 2〇L medium: sodium carbonate dg/L; disodium hydrogen phosphate: 2g/L; Sodium acetate: 1 g/L; soy protein 脒.50 g/L; glucose: 12 g/L; calcium carbonate: 〇1 g/L; potassium hydride: 0.1 g/L; sodium bicarbonate: 〇5 g /L; pyruvate: 〇1 g /; L; glutamine melon. 0 · 2 g / L, metal solution (copper sulfate: 3 mg / 1; gasification iron: 83 〇 mg / 1; Word: 860 mg / 1 ; sulfuric acid: u mg / L) : 〇 · 5 mL / L. Then add polypropylene glycol (0. 02 mL / L). After dissolution, the PU value is not adjusted. The incubation temperature is adjusted to 35 ° C, stirred at 1 rpm and not ventilated. After adjusting the pH of the culture to 5.6 « 24 hours with acetic acid, transfer 7 l from the former decimator to the decimator. (After 24 hours, the former decimator Culture at 700 nm OD · 〇 · 47). Pre-fermenter culture in sterile conditions Transfer to the fermenter. The broth in the salt shaker is prepared by dissolving the following components in pure water to prepare 7〇L medium: sodium chloride. 3 g/L; disodium hydrogen phosphate: 2 g/ L; sodium acetate: 1 g / L; soy protein 44 201010711 脒: 50 g / L; glucose: 12 g / L; calcium chloride: oj g / L; gasification unloading · 0.1 g / L; sodium bicarbonate: 0.5 g / L; pyruvate: oi g / L; valine: 0 · 2 g / L; metal solution (copper sulfate: 3 mg / l; gasification iron: 830 rng / j; sulfuric acid: 860 Mg/1; sulfuric acid ·· 1 ·丨mg/L) : 〇5 mL/L. Then add polypropylene glycol (〇.〇2 mL/L). After dissolution, the pH value is not adjusted. After sterilization, in the culture 8 g/L glucose was added. The incubation temperature was adjusted to 35 ° C, stirred at 1 rpm and not ventilated. After 24 hours, the culture (〇 D at run D was 〇 174) at 7 〇 β (: heat treatment for 2 minutes inactivation and transfer to the collection tank. After inactivation, the culture is transferred to the ultrafiltration chute to separate the biomass from the medium, concentrated and washed with Nac丨 (9 g/L) in pure water. Divide the raw material collected by the household (in each gram of concentrated bacterial suspension 2〇.2 The mass of the concentrated bacterial suspension under mg dry regenerating is 61.5 g) and then cooled at -15 °C; East. Suitable for example 2丄|mineral grinding preparation example 2.1 • I 392^ S has 6 § bacteria dry An aliquot of the heavy granules from Example 1, 1 was thawed to room temperature, and then diluted with pure water as far as 12 g/L of bacterial 妒壬 玍 玍 dry weight. Alkalization with 〇.〇3 Μ sodium hydroxide. The measured value at the beginning of dissolution was up to a P value of 10.3. The lysate was then incubated at 40 ° C under continuous mixing for 6 hours. After incubation, the pH was 9.9. Example 2.2_ Two Si V V dry weights from the Μ 荐 荐 到 到 到 到 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 Basification with 〇.03 M sodium hydroxide. At 45 201010711 When the dissolution starts, the amount of thirst is continued.

The pH is 1〇.3. The lysate was then incubated at 4 ° C for 6 hours and the pH was 9.7. There are 6 g of dry weight of the bacteria from Example 1 · 1 of the prawn #磨 1-3929 皙 $ , . I etc. The sample is thawed to room temperature' and then diluted with pure water

The test was carried out by measuring the dry weight of m.G6M sodium hydroxide at 12 g/L. The pH measured at the beginning of the dissolution was 123. The lysate was then incubated for 6 hours under constant agitation. After incubation, the pH was 1 h8. f Example 2 4 An aliquot of the granules from Example 1.1 with a dry weight of g. 1 3929 raw f was unrolled to room temperature' followed by dilution with G.2 N NaCl solution to 12 g/ L bacteria dry weight. Final concentration of NaC1 solution: 〇·15Ν. The positive value measured at the beginning of dissolution was 6.4. The lysis was then incubated at 4 ° C for 6 hours under continuous (four). Positive value after incubation Example 2.5 An aliquot of the lysate of Example 2.i was continued to dissolve. The volume was adjusted to pH 36 with HC1 25% and then incubated for 1 hour under static conditions in a water bath. Example 2.6 The lysate of Example 2.4 was incubated for 10 hours under static conditions by adjusting the volume to the enthalpy. The aliquots are followed by a dissolution process. At 12.4 and then in a water bath at 4 Torr. Example 2.7 An i aliquot of the corpus sylvestris 46 201010711 7 1 ·39 from Example 13 containing 0.4 g of dry weight of bacteria was decanted to room temperature' and then diluted with pure water to reach the biomass. Dry weight. The test was carried out with QG6% sodium hydroxide. The value of ρ measured at the beginning of dissolution was 12 6 . The lysate is then placed at 4 Torr. The armpits were incubated for 2 hours under continuous disturbance. After incubation, the pH was 12.4.

Re-enactment? R I3 aliquots of the #78 Griffin 13782 from Example 15 were thawed to room temperature, followed by dilution with pure water to § L, T. The weight was measured by G.G6 Μ sodium hydroxide. The enthalpy measured at the beginning of the dissolution was Ι2·5β and the lysate was incubated for 2 hours under continuous scrambling. Cultivate 彳4, ρΗ^ 12 3.

Don't you Q

Winter a had an average of 0.4 g of dry weight of the gentleman's dry mash from the i 2 sample; the east to room temperature' was then diluted with pure water to the dry weight of the 生g L bacterial biomass. Basification was carried out with 0.06 Μ sodium hydroxide. The value of ρ measured at the beginning of dissolution was 128. The lysate was then incubated under machine for 2 hours with constant agitation. After incubation, the ΡΗ value was 12.2. Example 2.1 η 3 has a dry weight of 6.8 g of bacteria. An aliquot of the pruning material from Example 1.1 was dissolved at room temperature, followed by dilution with pure water to 7 to obtain bacterial biomass. Dry weight. Inspected with sodium hydroxide. The value of η Η μ & , measured at the beginning of dissolution was 12.2. The lysate was then incubated at 4 〇t with 7 ^, 眭 _ _ y. J under continuous stirring. After the month of culture, the pH was 12.0. The pH of the lysate was adjusted to 9.8 with HC1. The lysate contained $ i / / dry weight (s〇lub Uised dry weight, 47 201010711 SDW) · 2〇, 5 mg/g, protein (Prot): 4.9 mg/g. Appropriate Example 2 1 Ί 1 aliquot of the 3.9 g of the dry weight of the bacteria from Example hl was thawed to room temperature, followed by dilution with pure water g/L Bacterial biomass is dry weight. The alkalization was carried out with 〇15 M sodium hydroxide. The pH measured at the beginning of each solution was 13 〇. The lysate was then incubated for 63 hours at 4 Torr with continuous stirring. After incubation, the pH was 12 6 . The pH of the lysate was adjusted to 9.9 with HC1. The β lysate contained SDW: 23 7 mg/g, pr〇t: 5 〇mg/g. _Exception 2 · 1 9 One of the aliquots of the 1-3929 biomass from Example i丨 was thawed to room temperature, followed by dilution with pure water. g/L bacterial dry weight. Basification with 〇 8 M sodium hydroxide. The pH measured at the beginning of the solution was 12 〇. The lysate is then placed. The underarms were incubated for 24 hours under continuous mixing. After incubation, the pH is 1]. The pH of the lysate was adjusted to 11.5 with Ν & ® Lysate contains SDW: 23.7 mg/g. f Example 2.n will contain 39 g of bacterial dry weight from the example of the 骅摩舁 shore mill 1-3929 biomass! Aliquots were thawed to room temperature and then diluted to 7.6 g/L bacterial dry weight in 〇2 n Naci solution to produce osmotic lysates. The pH measured at the beginning of the dissolution was 4.4. The bacterial permeable lysate is then at 40. . Incubate for 24 hours with continuous stirring. After incubation, the pH was 4·2. The bacterial lysate is then made alkaline by adding Na0H to reach 〇6N. . The pH measured at the beginning of alkaline dissolution was 1〇6. The lysate was incubated for a second 2 hours at 4 °C under continuous mixing. After cultivating, 10.2. Example of Lysate u The lysate of Example 2.5 was centrifuged at 30 Torr for 2 minutes. The supernatant was then adjusted to pH 6.8 with NaOH 1 〇 N and filtered through a continuous filter with a porosity of 0.45 μηη and 〇·2 μιη, and finally with a sterile filter 参.22 μιη. The concentrate contained Prot: 2.8 mg/mL; DNA: 17.4 gg/mL. Percentage of D-amino acid: 14 9% D Ala, 14 4% D_pr〇, 14 2% D-Asp 〇 in mg dry weight/mL no Yield: 〇.〇〇3 mg/mL (C1) - 〇 〇3 mg/mL (C2) 〇·3 mg/mL (C3) active dry weight/mL: C1: 3.3 μΜ, C2. 33.2 μΜ, C3: 52.5 μΜ. 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.实施 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 HC1 and filtered through a continuous filter having a porosity of 0.45 μm and 0.2 μηη, and finally filtered through a sterile filter of 0.22 μηη. The concentrate contained Prot: 12.3 mg/mL; DNA: 27.7 pg/mL. Percentage of D-amino acid·· 15.9% D-Ala, 24.4% D-Pro, 17.4% D-Asp, 45·10/〇D-Ser, 1〇·”/〇D-Met. NO production in /mL: 0_003 mg/mL (Cl)- 49 201010711 0.03 mg/mL (C2) - 0.3 mg/mL (C3) active dry weight / mL : Cl : 8.0 μΜ, C2 : 56·0 μΜ C3: 94.3 μΜ. Example 3.3 First, 300 ml of the lysate of Example 2.1 was centrifuged at 3000 xg for 20 minutes. The supernatant was adjusted to pH 7.1 with HC1. The extract was in Sartoflow® Slice 200 Benchtop Crossflow System It was concentrated at a constant pressure of 330-350 mL/min through a 0.45 μm polythersulfone (PES) membrane (Sartorius Stedim Biotech® GmbH). The volumetric yield was 75%. The concentrate was finally passed through a PES membrane of 0.2 μm ( Nalgene ) Sterile filtration. Concentrate containing SDW ··30.0 mg/g; Prot: 9·6 mg/mL; DNA: 32.8 pg/mL. D-amino acid percentage: 14.3% D-Ala, 13.9% D-Pro , 1 4.1 % D-Asp, 3 6.9% D-Ser. NO production in mg active dry weight/mL: 0.003 mg/mL (Cl) - 0·03 mg/mL (C2) - 0.3 mg/mL ( C3) Active dry weight / mL : Cl : 5 · 1 μΜ, C2 : 30_0 μΜ, C3 : 64.0 μΜ. Example 3.4 300 300 ml of the lysate of Example 2.2 was adjusted to pH 7.0 with HC1. The extract was passed at 350 mL/min as described in Example 3.3. 75%. The concentrate was finally sterile filtered through a PES membrane 0.2 μm (Nalgene). The concentrate contained SDW: 49.2 mg/g; Prot: 14.2 mg/mL; DNA: 34.1 pg/mL. D-amino acid percentage: 16.0 % D-Ala, 13.3% D-Pro, 14.2% D-Asp. NO production in mg active dry weight/mL: 0.003 mg/mL (Cl) - 50 201010711 0.03 mg/mL (C2) - 0.3 mg/ Active dry weight of mL (C3) / mL : Cl : 0 μΜ, C2 : 4.1 μΜ, C3 : 26.3 μΜ. Example 3.5 300 ml of the lysate of Example 2.3 was first centrifuged at 3000 x g for 20 minutes. The supernatant was adjusted to pH 7.1 with HC1. The extract was filtered at a constant flow of 330-350 mL/min as described in Example 3.3. The volumetric yield is over 75%. The concentrate was finally aseptically passed through a PES membrane at 0.2 μm (Nalgene). ❹ Concentrate containing SDW: 34.5 mg/g; Prot: 8.9 mg/mL; DNA: 16.8 pg/mL. Percentage of D-amino acid: 16.4% D-Ala, 24.3% D-Pro, 17.3% D-Asp. NO production in mg active dry weight/mL: 0_003 11^/1111^((:1)-0.03 mg/mL (C2) - 0.3 mg/mL (C3) active dry weight/mL : C 1 : 3.0 μΜ, C2: 36.4 μΜ, C3: 69.5 μΜ Example 3.6 1000 mL of the lysate of Example 2.13 was firstly centrifuged at 9384 X g for 20 minutes. The supernatant was directly filtered through a sterile filter 0.22 μηη. The material contained SDW: 32.7 mg/g; Prot: 6.5 mg/g; sugar: 2.4 mg/g. The sugar concentration was determined according to the procedure of Herbert et al. (MeM. MicroZu'o/, 1971, 5B: 266 and below). Different concentrations of human peripheral blood mononuclear cells (PBMC) were screened for biological activity as described in Example 6. The result obtained at 0.5 mg active dry weight/mL was TNF-α: 83 pg/mL; IL- 6 : 898 pg/mL ; IL -12 :

140 pg/mL (in the presence of 10 ng/ml IFN-γ); and IL-10: 221 pg/mL 51 201010711 (in the presence of IFN-γ). Example 3.7 After the bacterial lysate mixture of Example 2.10 was adjusted to a pH of 10.2, it was transferred to a microfiltration (MF) storage tank. The microfiltration (MF) unit used a 〇.45 μηι tangential flow filtration (TFF) filter (Sartocon Slice Sartorius). Adjust the cross flow to 290 L/h.m2 (LHM) and adjust the transmembrane pressure (ΤΜΡ) to 0.4-0_5 bar. The permeate is transferred to an ultrafiltration (UF) storage tank. After the volume of the lysate in the microfiltration tank reaches half of the initial volume, the UF unit is started. The permeate of the UF filter was used as a wash buffer in the MF sump. The volume of the MF storage tank and the UF storage tank are kept at the same level. 1 After 〇 diafiltration volume, 'stop UF' and concentrate the bacterial lysate in the MF sump. The volumetric yield recovered was 83%. The extract recovered in the UF storage tank was adjusted to a ΡΗ value of 7.3 with HC1 and then filtered through a 〇·2 μηι sterile filter. The concentrate contained SDW: 17.5 mg/g; Prot: 4.3 mg/g. Example 3.8 Lu The lysate of Example 2.7 was centrifuged at 9384 X g for 15 minutes. The supernatant was then adjusted to pH 73 with HC1 and filtered through a continuous filter having a porosity of 〇 45 and 〇·2 μηι, and finally passed through a sterile filter 〇 22 μηη. Peripheral blood mononuclear cells (pBMC) with 10-fold dilutions: 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 supernatant was then adjusted to pH 7.4 with HC1 and filtered through a continuous filter with porosity 〇 45 52 201010711 and 0.2 μηη, and finally with a sterile filter 〇 22 μιη transition. PBMC with 10-fold dilution product: 904 pg/mL IL-i〇, 〇pg/mL IL-12, 4995 pg/mL IL-6. Example 3.10 The lysate of Example 2.9 was centrifuged at 9384 x g for 15 minutes. The supernatant was then adjusted to pH 7_6 with HC1 and filtered through a continuous filter having a porosity of 〇 45 μηη and 0.2 μηη, and finally passed through a sterile filter 〇.22 〇. PBMC with 10-fold dilution product: 476 pg/mL IL-10, 0 pg/mL IL-12, 4924 pg/mL IL-6. Example 3.11 After the lysate of Example 2·11 was adjusted to a pH of 10.4, it was transferred to an MF storage tank. The TFF device is similar to embodiment 3.7. Adjust the cross flow to 290 L/h.m2 and adjust TMP to 〇_4-〇·5 bar. The UF was stopped after 10 times the diafiltration volume. The volumetric yield recovered was 86%. The final product was adjusted to a pH of 7.3 with JJC1. The concentrate obtained contained SDW: 24.2 mg/g; Prot: 4.8 mg/g. Example 3.1 2 After adjusting the lysate of Example 2.12 to pH η.5, transfer it to the MF sump. The TFF device is similar to Example 3 7 ^ The cross flow is adjusted to 290 L/h, m2 and will be ΤΜΡ Adjust to 〇.4 bar. Stop UF after 1〇 diafiltration volume. The volumetric yield recovered was 74%. The final product was adjusted to a value of 7.2 with HC1. 53 201010711 The concentrate obtained contains SDW: 32.4 mg/g; Prot: 5.8 mg/g; sugar: 2.3 mg/g. For different concentrations of human peripheral blood mononuclear cells (PBMC) as described in Example 6 Screening for activity. The results obtained at 0.5 mg dry weight/mL were TNF-α: 37 pg/mL; IL-6: 1092 pg/mL; IL-12: 81 pg/mL (in the presence of 10 ng/ml IFN-γ) And iL-i〇: 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 sample to be analyzed (0.1 mL) was diluted in 1.9 mL of 25 mM phosphate buffer (pH 11). Each time a protein standard curve was prepared with 2 mg BSA / ml of bovine serum albumin solution (BSA, Pierce ref 23210). The BSA solution was diluted with 25 mM phosphate buffer (卩11 value 11) to obtain the following indifference: 181.181118 3|§8/1111, 0.24 111§ BSA/ml, 0.3 mg BSA/ Ml, 0.36 mg BSA/ml and 0.42 mg BSA/nU. Samples (20 μΐ) and BSA standards (20 μΐ) were added in quadruplicate to a clean dry microtiter plate. Reagent A (25 M1) was added to each well. After 1 minute, 200 μM reagent Β was added to each well. The microtiter plate was mixed on a rotary disk shaker for 5 seconds. After 20 minutes of warming, the absorbance at 75 〇 nm was recorded. The protein concentration of each sample was calculated using the linear regression slope on the protein standard curve: 75 nm 下 D = a * (protein concentration) + b in the sample The number of mg of protein = [20 X (〇 D - b at 750 nm)] I a 54 201010711 Soluble dry weight (SDW) by obtaining 5 mL of soluble fraction from dissolution and at 10 ° C in porcelain In the dish, it was measured by dryness to constant weight. Example 4: SL accretion of sputum cells The activation of spleen cells of murine animals was used to measure the immunostimulatory effect of the specific examples of the present invention in vitro. (Alamar Blue assay) ° Materials and Methods Stimulation of Spleen Cells O AlamarBlueTM-tests are designed to quantify the growth of human or animal cells. This assay is based on chemical reduction from cell growth. The reactive redox (REDOX) indicator detects metabolic activity incorporated into the glory/colorimetric growth indicator. Associated with growth, the REDOX indicator causes oxidation (non-fluorescent, blue) form reduction (fluorescence, red) Form Variable since Charles River Laboratories, Sulzfeld, Germany mice received Balb / c mice (female, 6-8 weeks old), and to sacrifice by cervical dislocation. The spleen cells were homogenized using a clay pot; the cell suspension was washed by separation from the heart (280 X g, 4 ° C, 10 min) and resuspended in 5 ml of 5% FCS, 100 U/ml penicillin (penicillin) And 100 pg/ml streptomycin in RPMI 1640. 100 μΐ of spleen cells (2×10 6 /ml) were incubated in a 96-well culture dish (Falcon 3072) in a cell culture medium at 37 ° C and 5% CO 2 for 48 hours. After adding 30 μΐ of AlamarBlueTM solution diluted 1:1 in medium, Fluoroskan Ascent reader (ThermoLabsystems, 55 201010711)

Frankfurt, Germany ) measured the chemical reduction of AlamarBlueTM at 544 nm excitation wavelength and 590 nm emission wavelength. Test items Test the following extracts:

Afer300: extract from pramiprin/-3929 (12 g/L) obtained as described in Example 3.5 (31.6 mg active dry weight/g);

Cfer300: Extract obtained from the prawn / 3929 (12 g / L) obtained as described in Example 3.3 (28.4 mg active dry weight / g);

Dfer300: Extract from pramipexole/exo 29 (I2 g/L) obtained as described in Example 2.4 and purified using the same conditions as in Example 3.3 (29.5 mg active dry weight/g) This example was used as a non-alkaline dissolution control. ARahr3 00: An extract from 虞李溏涊#磨71.38 (40 g/L) was obtained by alkaline dissolution at NaOH 0.03 for 6 hours at 40 ° C (49 3 mg active dry weight / g); CRahr300: as implemented The extract obtained from Example 3·4 from 虞李潜和#磨 71.3 8 (40 g/L) (46·4 mg active dry weight/g); DRahr300: by 在 at 4 °C • An extract (46.2 mg active dry weight/g) from 虞 褚 71 71 71.38 (4 〇g/L) dissolved in osmotic pressure for 6 hours in a 15 N NaCl solution. This example was used as a non-alkaline dissolution control. Negative control Pure water (Aqua dest) (distilled water) or phosphate buffered saline (PBS). 56 201010711 Results In vitro study to determine spleen cell activation The increase in metabolic activity of mouse spleen cells after extract treatment was determined by two independent experiments by Alamar Blue assay (see, Figures 4a and 4b). Spleen cells were cultured for 48 hours in the presence of extract. After adding the Alamar blue® solution, measure the emission value. As shown in Figures 4a-b, the bacterial extract is effective at about 1 - 300 dilutions. All groups were statistically compared to the control group using the student's test (student's Τ-test). 〇 Table 1: P values of bacterial extracts in 2 independent experiments of Alma Blue assay (see Figure 4a and Figure 4b) Batch dilution P value test 1 p value test 2 Afer300 (dilution 1:300) 0.000016 (Figure 4a) 0.00016 (Fig. 4b) Cfer300 (diluted 1:300) 0.00021 (Fig. 4a) 0.048 (Fig. 4b) Dfer300 (diluted 1:300) 0.00033 (Fig. 4a) 0.00016 (Fig. 4b) ARahr300 (diluted 1:300) 0.0029 ( Figure 4a) 0.0012 (Fig. 4b) CRahr300 (diluted 1:300) 0.0031 (Fig. 4a) 0.12 (Fig. 4b) DRahr300 (dilution 1:300) 0.0025 (Fig. 4a) 0.08 (Fig. 4b) 〇Conclusions by Alma Blue The activation of murine spleen cells was measured to in vitro assay the immunostimulatory effects of bacterial extracts. Herein, we compared the extract obtained by osmotic dissolution (Dfer300) (which includes the complete particulate cell wall component) with the two extracts of the present invention (AFer3 00 and CFer3 00 ) in two independent experiments. These three extracts were obtained from the prawn 1-3929. The AFer300, CFer300 and DFer300 extracts each effectively stimulate the metabolism of the cells at 1:300 dilution. No differences were observed between the DFer300 57 201010711 osmotic dissolution control and Afer300 and CFer300. We also compared the three extracts (ARahr300, CRahr300, DRahr300) obtained from the second strain, recommended by ####71.38. As previously set, 'ARahr 300 and CRahr 300 are within the scope of the invention, and DRahr is a osmotic dissolution control. Herein, the ARahr300 extract was effective in two independent assays at 1 j〇〇 dilution, while the CRahr300 and DRahr300 extracts at the same dilution exhibited weak but significant stimulation in one of the two assays. In the comparison of the results of all six extracts, it was also shown that the 肄舁 1-3 莽 1-3 1-3929 extract was more effective in stimulating spleen cell growth than the 虞 舁 舁 71 71 71 71.38 extract. Example 5: Gasification of Macrophage Cell Produced by Osteoblasts The immunostimulatory potential of a series of specific examples of the present invention was tested by measuring the nitric oxide (NO) production of macrophages produced by murine bone marrow. Materials and Methods Six-week-old male C57/BL6 mice (6-week-old male 'SPF quality, Charles Rivier, FR) were killed by C〇2 inhalation. Remove the buttocks, leg joints and cheekbones from the hind limbs. Bone marrow was extracted from the lumen by bone injection of Dulbecco's Modified Eagle Medium (DH) after excision of both ends. After washing, the stem cells were resuspended (4 cells/ml) in DH medium supplemented with 20% horse serum and 30% L929 cell supernatant. The cell suspension was incubated in an incubator at 37 ° C under 8% CO 2 under a humidified atmosphere for 8 days. The macrophages were then separated by ice-cold pBS separation and resuspended in DH medium (DHE medium) supplemented with 5% fetal bovine serum (FCS), amino acids and antibiotics. Adjust the cell density 58 201010711 to 70000 cells/ml. The aqueous extract was serially diluted in DHE medium placed directly in a microtiter plate. The extract of the present invention was tested in triplicate and each microtiter plate contained the medium as a negative control. The final volume of each well is 1 〇〇. The iOO 41^ cell suspension was added to the diluted extract and the cells were incubated in an incubator at 3 rc under s% eh and under a humidified atmosphere for 22 hours. At the end of the incubation period, 1 〇〇 pL of the supernatant was transferred to another microtiter plate and the concentration of nitrite produced in each supernatant was determined by running the Griess reaction. ® Add Griess reagent in a 2.5% aqueous solution of disc acid to each well (5 mg/mL sulfonamide + 0.5 mg/mL N-(l-naphthyl)) Amino hydrochloride) 〇 Relative to the reference at 69 〇 nm The microtiter plate was read at 562 nm with a knife luminosity § ten (. SpectraMax Plus, Molecular Devices). The nitrite concentration is proportional to the amount of nitrogen oxide formed. The nitrite content was determined based on a standard curve of arsenic acid (1 μΜ to 70 μΜ NaN02 ). The results are given as μΜ nitric oxide (NO) as the mean standard deviation and plotted as a dose response curve. The following extracts were tested: First assay: OP0701B4 - CFer300: Extract from Lactobacillus reuteri 1-3929 glL obtained as described in Example 3.3., OP0701B4_Dfer3 00: obtained from the prawn as described in Example 2.4 The extract of -3929 (12 g/L) was purified using the same conditions as in Example 313. This example was used as a non-alkaline dissolution control. 59 201010711 OP0701B4_CRahr3 00: Extract obtained from the 虞 莩 舁 磨 #磨 71.38 (40 g/L) as described in Example 3.4 OP0701B4_DRahr3 00: at 40. . The extract from S. chinensis #71.38 (40 g/L) was obtained by osmotic pressure for 6 hours in a 〇15 N NaCl solution. This example was used as a non-alkaline dissolution control. The second test: OP0701C_10G0.5P4H: 10 g / Ι dry weight of 10 g / Ι dry weight of dried 苈 苈 / -3929 was incubated with 0.037 M NaOH for 4 hours to obtain the extract. OP0701C_10G1P4H: At 40. . 10 g / Ι 质 干 下 # # # # # # # 392 392 392 392 392 392 392 392 392 392 392 392 392 392 392 392 392 392 392 392 392 392 392 OP0701C—10G2P4H: 10 g/Ι dry weight of 蓊 涊 彦 #-3/3P29 was incubated at 0.150 M NaOH for 4 hours at 40 °C to obtain the extract. OP0701C_10G1P21H: The extract obtained by incubating 10 g/Ι dry weight of Tsai ® Moji #磨/-3P2P at 0.075 M NaOH for 21 hours at 40 °C. OP0701C_10G0.5P21H: The extract obtained by incubating 10 g of dry weight of 10 gA and 40 苈/-3P2P at 0·037 M NaOH for 21 hours at 40 °C. OP0701C_10G2P21H: The extract was obtained by incubating l〇g/l of dry weight at 40 ° C. ##乙# grinding with 0.150 M NaOH for 21 hours. 60 201010711 Revision OP0701C_10G2P45H: 10 g/1 dry weight of 鏺 摩 Momo #卢/-3P2P was incubated at 0.150 M NaOH for 45 hours at 40 °C to obtain the extract. Results: First assay In the first assay (Fig. 5a), it was observed that the extract obtained from the alkaline dissolution of the present invention has the same activity as the extract obtained by the osmotic dissolution. We have also observed that the immunostimulatory activity is related to the selected strain. The extracts obtained from φ 舁 舁#磨磨1-3929 are more than the extracts obtained from the strains #潜为#彦 78.3 1 The extracts obtained are N02 and N〇3. Results: Second assay In the second assay (Fig. 5b), we observed that the in vitro activity of the extract was related to the initial conditions of dissolution. For the same strain, 磨 磨 929929 and the activity of the following bacterial extracts for the same amount of 10 g of dry weight/liter of lysate: OP0701C_10G0.5P4H, OP0701C_10G1P4H, ® OP0701C_10G2P4H, OP0701 C_10G1P21Η, OP0701C_10G0.5P21H , OP0701 C_10G2P2 1H and OP0701C_10G2P45H. In vitro activity depends on the initial concentration of NaOH and the duration of dissolution. Conclusions The results of these experiments show that although the alkaline treatment produces a chemical modification, the activity of the specific examples does not decrease when compared to the control extract obtained by osmotic dissolution or when compared to the individual living bacteria. 61 201010711 ψ f Example 6: E. Inflammation and Anti-Inflammatory Activity Method for Exogenous Screening for Immunological Stimulation or Anti-Inflammatory Potential of Specific Examples of the Invention 'Testing a Series of Bacterial Extracts on Human PBMC . The release of IL12p70 (inflammatory interleukin) and IL10 (anti-inflammatory mediator) was measured as described by Folignie et al. (J January 14, 2007. 13(2): 23 6-243), and IL1 was reported. 〇 / IL12 ratio. The goal was to compare the production of IL-12p70 and IL-10 from a series of six extracts obtained by different extraction/purification methods. The ® IL_ 1 〇/IL-12p70 ratio obtained for each extract is related to the anti-inflammatory potential of the extract. The viable bacteria were used as a control' to compare the activity of the extract (2X 107 cfu/ml Life 4 Yananyan 1-3929). Materials and Methods Six fractions of bacterial extracts were diluted in cell culture medium at a starting dose of 1 mg/ml (the highest final dose tested) in the presence of 1 ng/mi ΙΡΝγ, IFN-γ is known. Increase the IL-I2p70 production of interleukins. Blood isolated PBMCs from healthy donors were tested in serial dilutions of active dry weight extracts from 100 ng/ml to 1 mg/ml. IFN-γ was added at least 3 hours before the lysate or live bacterial strain in the medium. Report data from two independent experiments. Human PBMC _ $ PBMC isolated from peripheral blood. Briefly, after Ficoll gradient centrifugation (Pharmacia, Uppsala, Sweden), monocytes were collected, washed in RPMI 1640 medium (Live technologies, Paisley, Scotland) and supplemented with gentamicin (150,000). Pg/mL), L-62 201010711 glutamine amine (2 mmol/L) and 10% fetal bovine serum (FCS) (Gibco-BRL) in RPMI 1640 adjusted to 2x1 06 cells/ml. Induction of interleukins PBMCs (2 x 106 cells/ml) were seeded in 24-well tissue culture dishes (Corning, NY). Stimulate the cells as described above. After 24 hours of stimulation at 37 ° C in a 5% CO 2 atmosphere, the culture supernatant was collected, clarified by centrifugation and stored at -20 ° C until interleukin analysis. Interleukins IL_10 and IL-12p70 were measured by ELISA using a pharmingen antibody pair (BD Biosciences, 〇 San Jose, Ca, USA) according to the manufacturer's recommendation. Test article OP0701C_10G2P45H (A): 10 g of dry weight of raw biomass / liter of lysate splash #其#磨/-392P, 0.15 M NaOH at 40 ° C for 45 hours to obtain the extract. OP0701C_10G1P4H(B): 10g of dry weight/liter of lysate. The extract obtained by NaOH/-3929, 0_075 M NaOH at 40 ° C for 4 hours. ® 〇P〇701C_5G4P21H (C): 5 g of dry weight/liter of lysate. Waste 舁# grinding/-3P2P, 0.300 M NaOH The extract was obtained at 40 ° C for 21 hours. OP0701C_40G0.5P4H (D): 40 g of dry weight/liter of lysate. #磨/-392P, 0.03 7 M NaOH at 40 ° C for 6 hours to obtain the extract. OP0701B4_CFerl50 (E): Extract obtained from 罄肄舁# mill (12 g/L) as obtained in Example 3.1. 63 201010711 OP0701B4 - BFer300 (F): 6 g dry weight of raw biomass / liter of lysate 镕 旄 磨 # 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -3929, live bacteria chilled at 2xl 〇7 cfu/ml in 20% glycerol at -80 ° C. Results The objective of this test was to compare the in vitro immune stimulation of the specific examples of the invention with the live strain of Agrobacterium Or anti-inflammatory potential. To analyze different extracts and β live bacteria, compare the amount of interleukin il-10 and iL_12 released from PBMC, and IL10/IL12 ratio. If IL-10 or IL-12 concentration or two values The ratio is not close to the unstimulated level (ie, less than 10 pg/ml) and the ratio is considered uncalculated and labeled as NC (uncalculable value). The results obtained from pBMc in pg/ml are shown in Table 2 and Table 3 compares the effects of the six bacterial extracts of the present invention (extraction A to extract F) and the live form of human PBMC in the presence of IFNY (2xl〇7 bacteria/ The effect of milliliters (tested twice as live sample i and live sample 2) (IL1〇 and IL12p7〇 in Pg/ml). Reported only 100 pg/ The ratio of ml and 1 mg/ml extract dose iLi〇/ili2. Table 2. In vitro pre-inflammatory (immunostimulation) or anti-inflammatory (immunomodulatory) potential of bacterial extracts in human pbMC 64 201010711

11,1» (+ LFNy) lmg/ml 100pg/ml IU2(+IFiN'y) ~ lmg/ml_ 100pg/ml lmg/ml 100pg/ml NC 14.03 NC 10.17 12.49 11.40 NC 2.11 NC 2.05 0.65 0.52 NC: Not Calculatable value

φ In the presence of IFNy, compare the effects of the six extracts of the present invention (Extract A to Extract F) with the live form of human PBMC (2χ1 07 bacteria/ml as a live sample) 1 and live sample 2 tested twice) (IL10 and IL12p70 reactions in pg/ml). Only the ratio IL1 0/IL12 at the dose of 100 μg/m\A 1 mg/ml extract was reported. Table 3: Live sputum 1-3929 in human PBMC in vitro anterior inflammation (immunostimulation) or anti-inflammatory (immunomodulatory) potential Lactobacillus brevis 1-3929 Lactobacillus brevis 1-3929 live sample 1_ live sample 2 ______

Based on the results presented in Tables 2 and 3, IL 10 production is reduced in the following order:

BsEsD>F> Live milk sample 谪>A>C 65 201010711 Low based on the results presented in Table 2 and Table 3, lLu yields subcutaneously lowering the milk sample 镝>E>B>D>F>A> When considering the ratio, the following general pattern was observed: 82〇>£兰罗> The live milk sample did not show the ratio of extract A and extract C to iu〇/ili2 because the extract was not found to be an interleukin Effective inducer. At concentrations below 100 pg/ml, it is low and no conclusion can be drawn. in conclusion

The ratio of the observed interleukin concentrations too much indicates that under the experimental conditions used in Example 6, the bacterial extracts #immunomodulatory & For example, extracts B, D, and M F exhibited higher IL10/IL12P70 t匕 than live bacterial control. Thus, for the conditions described herein (such as inflammatory conditions), the extracts may be more active than live probiotic bacteria. Example 7: 赍Τη丨丨枰.

Specific examples of the present invention are obtained from Gram-positive bacteria, and thus it is expected to function via the TLR2 receptor. The TLR receptor is in principle (but not exclusively) expressed by immune cells such as monocytes, macrophages, dendritic cells, tau cells, etc. and is a key sensor of microbial products that can be recognized by the host as a signal hazard. Although it first elicits non-specific innate immunity, TLR activation initiates a complete immunological cascade, which leads to the development of acquired immunity in the presence of antigen.

Cells that express a given functional TLR gene are valuable tools for many applications, such as the study of mechanisms involved in identification or signal transduction in tlR 66 201010711 and the development of new potential therapeutics. The experiments described below tested the activity of the three bacterial extracts as key adapters for these immune responses. Materials and Methods The reaction of the extract of the present invention was tested in two cell systems (the effect of its agonist was tested by itself, or the antagonist activity was tested in the presence of the TLR2 agonist Pam3Cys or in the presence of the TLR4 agonist LPS) : a) HEK-TLR2/6 (IL-8 ELISA after 24 hours) ❿ b) HEK-MD2-TLR4-CD14 (IL-8 ELISA after 24 hours) a) HEK-TLR2/6 against the TLR gene The HEK293 cell line was selected for low base performance. Such cells enable efficient monitoring of TLR activity using ELISA assays such as IL-8 titration or monitoring of TLR-induced NF-κΒ activation based reporter-based systems. HEK-TLR2/6 cells (Invivogen, Toulouse, France) are engineered for stable transfection of genes from the TLR2/6 pathway (including TLR2, TLR6) and participating in the identification or involved in the signal transduction cascade. HEK293 cells. These cells secrete IL8 after TLR2/6 stimulation. Experiments were performed according to the manufacturer's instructions. Briefly, 2χ104 cells/well (200 μΐ RPMI) were incubated at 37 °C over 3 days (5% CO 2 ). The medium was removed and 90 μΐ RPMi + 5 % FCS was added to each well. An agonist and control (10 [mu]l/well) were then added. Return the cells to the incubator for 24 hours. The supernatant was collected and subjected to IL-8 ELISA according to the manufacturer's instructions. 67 201010711 Test article OP0701B4_Afer50 : 12 g of dried stem/liter of lysate Lactobacillus 1-3929, 0.075 M NaOH was dissolved at 40 ° C for 4 hours, and the obtained extract was purified as described in Example 3.6. OP0701C-BULAC: Extract from Lactobacillus fermentum 1-3929 obtained as described in Example 3.7. OP0701C-Bt2LAC: Extract from Lactobacillus fermentum 1-3929 obtained as described in Example 3.11. ❹ Result: IL-8 secretion The results of the control (negative = LPS K12 ultrapure, TLR4 agonist; and PAM3CSK4 = positive, TLR2 agonist) are provided in Tables 4-6. The results (expressed as pg/ml of IL-8) show the average value of IL-8 secretion by ELISA after 24 hours of stimulation with the control. The cell line HEK TLR2/6 reacted with the TLR2 agonist Pam3Cys. In contrast, the TLR 4 agonist from E. coli (LPS K12) was inert even at high doses of 0.01 pg/ml. ® Experiment using only 3 bacterial extracts The three bacterial extracts tested in this paper (OP0701B4 Afer50, OP0701C-BtlLAC and OP0701C-Bt2LAC) exhibited higher immunostimulatory properties than the TLR2 agonist Pam3Cys. Experiment with 3 bacterial extracts + Pam3Cys As mentioned above, the assumed antagonistic or additive properties of the three bacterial extracts compared to Pam3Cys added just after the extract were also tested. Bacterial extract OP0701B4 AFer50 68 201010711 Table 4: IL obtained from control (medium, LPS or Pam3Cys), bacterial extract OP0701 B4_AFer50 or supernatant extracted from Pam3Cys-stimulated HEK TLR2/6 cells at the indicated concentrations -8 (pg/ml) secretion

Pam3Cys 0.0003 μβ/ml 0.001 μ^ιηΐ 0.003 μβ/ml 0.01 us/ml 18 88 269 696 Medium na 2 pg/ml IL~8 0.01 LPSEcoli K12 pg/ml 2 pg/ml IL-8 OP0701B4_Afer50 pg/ml Bacterial extract + Bacterial extract + Bacterial extract + Bacterial extract IL-8 Pam3Cys Pam3Cys 0.001 Pam3Cys 0.003 +Pam3Cys 0.01 0.0003 μ^ιηΐ με/ml pg/ml pg/ml pg/ml IL-8 pg/ml IL-8 pg/ mlIL-8 pp/ml IL-8 0.2 mg/ml 132 180 314 566 1095 0.5 mg/ml 462 546 713 900 1742 1 mg/ml 1218 1243 1307 1980 2297 2 mg/ml 1830 2314 2595 2639 2683 Results in Table 4 It was shown that OP0701B4_AFer50 extract induced high level of IL-8 (agonist TLR 2/6) production.

Bacterial extract OP0701C-BtlLAC Table 5: at the indicated concentrations with control (medium, LPS or Pam3Cys), bacterial extract OP0701C-BtlLAC or the extract with

Secretion of IL-8 (pg/ml) from the supernatant obtained on Pam3Cys-stimulated HEK TLR2/6 cells

Pam3Cys 0.0003 pg/ml 0.001 "g/ml 0.003 "g/ml 0.01 pg/ml 18 88 269 696 medium na 2 pg/ml IL-8 LPS E coli K12 0.01 Jig/ml 2 pg/ml IL-8 69 201010711 OP0701C -BtlLAC pg/ml IL-8 bacterial extract + Pam3Cys 0.0003 μς/ηαΐ pg/ml IL-8 bacterial extract + Pam3Cys 0.001 pg/ml pg/ml IL-8 bacterial extract + Pam3Cys 0.003 μς/ιηΐ pg/ml IL-8 bacterial extract + Pam3Cys 0.01 μ^ιηΐ pg/ml IL-8 0.2 mg/ml 347 499 488 824 1311 0.5 mg/ml 1504 1447 1577 1889 2213 1 mg/ml 2740 2966 2295 3023 2649 2 mg/ml 4306 4318 3744 3540 3454 The results in Table 5 indicate that OP0701C-BULAC extract induces high levels of IL-8 (agonist TLR 2/6) production.

Bacterial extract OP0701C-Bt2LAC Table 6: Supernatant obtained on control (medium, LPS or Pam3Cys), bacterial extract OP0701C-Bt2LAC or HEK TLR2/6 cells stimulated with Pam3Cys at the indicated concentrations IL-8 (pg/ml) secretion

Pam3Cys 0.0003 pg/ml 0.001 pg/ml 0.003 Mg/ml 0.01 pg/ml 18 88 269 696 Medium na 2 pg/ml IL-8 LPS E coli K12 0.01 Ag/ml 2 pg/ml IL-8 OP0701C-Bt2LAC pg/ Ml IL-8 Bacterial extract + Pam3Cys 0.0003 pg/ml pg/ml IL-8 Bacterial extract + Pam3Cys 0.001 pg/ml pg/ml Π^-8 Bacterial extract + Pam3Cys 0.003 μ^/πιΐ pg/ml IL- 8 Bacterial extract + Pam3Cys 0.01 μς/ml pg/ml IL-8 0.2 mg/ml 9 17 60 209 571 0.5 mg/ml 8 12 41 175 448 1 mg/ml 11 18 36 120 361 2 mg/ml 13 15 24 59 206

The results in Table 6 indicate that OP0701C-Bt2LAC extract does not induce IL-8 (agonist TLR 2/6) production and has an antagonistic effect on TLR2/6 70 201010711 in the presence of Pam3Cys. Conclusion Depending on the alkaline dissolution conditions (initial concentration of dry weight of the biomass, initial alkali concentration and duration of alkali treatment), bacterial extracts may have different modes of action. OP0701B4_Afer50 and OP0701 C-BtlLAC are agonists TLR2/0, but the strong positive solubility on the same bacterial strain leads to antagonism of TLR2/6 activity (OP0701C-Bt2LAC). ❿ b) HEK-TLR4-MD2-CD14 TLR4 has been extensively studied for its primary receptor involved in the identification of lipopolysaccharide (LPS) for septic shock. HEK-TLR4-MD2-CD14 cells are highly sensitive to LPS. It was obtained by stably transfecting HEK293 cells with the TLR4, MD2 and CD14 genes and the NF-κΒ-induced reporter system. These cells secrete IL-8. The same experimental procedure as the one used for the other HEK cell line TLR2/6 described above was used. The results of the controls tested and the three ® bacterial extracts of the invention are shown in Tables 7-9. Results: IL_8 secretion The results of the control (positive = LPS K12C ultrapure, TLR4 agonist; and PAM3CSK4 = negative, TLR2 agonist) are provided in Tables 7-9. The results (expressed as pg/ml of IL-8) show the mean of IL-8 secretion after 24 hours of stimulation with the control. The cell line only responded significantly to the TLR4 agonist LPS K12. In contrast, as expected, the TLR2 agonist from Pam3Cys was inert even at a high dose of 0.01 pg/ml 71 201010711. Experiments using only three bacterial extracts, such as Gram-positive bacteriostats, are expected to be tested in the three bacterial extracts (OP0701B4 Afer50: Table 7; OP0701C-BULAC: Table 8 and OP0701C-Bt2LAC: Table 9) No clear immunostimulatory properties were demonstrated via the TLR4 pathway. Experiment using 3 bacterial extracts + LPS K12 As mentioned above, the assumed antagonistic or additive properties of the three extracts of the present invention compared to the LPS added just after the extract were also tested. Similarly, no effect was observed at the TLR4 receptor level (Tables 7-9). Bacterial extract OP0 701B4 AFer5 0 Table 7: Supernatant obtained on control (medium, LPS or Pam3Cys), bacterial extract OP0701C-AFer50 or HEK TLR4 cells stimulated with LPS K12 at the indicated concentrations IL-8 (pg/ml) secretion

LPS K12 0.0003 pg/ml 0.001 pg/ml 0.003 pg/ml 0.01 pg/ml 251 477 802 1383 Medium na 0 pg/ml IL-8 Pam3Cys 0.01 pg/ml 138 pg/ml IL-8 OP0701B4_Afer50 pg/ml IL-8 Bacterial extract + LPS K12 0.0003 μ^ηιΐ pg/ml IL-8 Bacterial extract + LPS K12 0.001 μς/ιηΐ pg/ml IL-8 Bacterial extract + LPS K12 0.003 pg/ml pg/ml EL-8 Bacterial extraction +LPS K12 0.01 μ^ηιΐ pg/ml IL-8 0.2 mg/ml 152 1 502 856 1446 0.5 mg/ml 176 335 458 736 1511 1 mg/ml 192 391 529 956 1580 2 mg/ml 198 422 736 935 1553 72 201010711 The results indicate that the OP0701B4_AFer50 extract does not activate the TLR4 receptor (Pam3Cys · 13 8 pg/mL ) °

Bacterial extract OP0701CBtl-LAC Table 8: IL obtained from the control (medium, LPS or Pam3Cys), bacterial extract OP0701C-BULAC or HEK TLR4 cells stimulated with LPS K12 at the indicated concentrations -8 (pg/ml) Secreted LPS K12 0.0003 pg/ml 0.001 pg/ml 0.003 pg/ml 0.01 pg/ml 251 477 802 1383 Medium na 0 pg/ml IL-8 0.01 Pam3Cys pg/ml 138 pg/ml IL- 8 pg/ml bacterial extract + bacterial extract + bacterial extract + bacterial extract IL-8 LPS K12 LPS K12 LPS K12 +LPS K12 0.0003 pg/ml 0.001 μ^ιηΐ 0.003 pg/ml 0.01 μς/ηιΐ OP0701C-BtlLAC Pg/ml IL-8 pg/ml IL-8 pg/ml IL-8 pg/ml IL-8 0.2 mg/ml 140 282 471 795 1345 0.5 mg/ml 152 292 502 778 1537 1 mg/ml 152 318 616 952 1576 2 mg/ml 162 322 632 1144 1716

Bacterial extract OP0701CBt2-LAC Table 9: Controls (medium, LPS or at indicated concentrations)

Pam3Cys), bacterial extract OP0701C-Bt2LAC or IL-8 (pg/ml) of the supernatant obtained from this extract and LPS K12-stimulated HEK TLR4 cells. LPS K12 0.0003 pg/ml 0.001 pg/ml 0.003 pg/ Ml 0.01 pg/ml 251 477 802 1383 | Medium | na |〇pg/ml IL-8 | 73 201010711 0.01 Pam3Cys pg/ml 138 pg/ml IL-8 OP0701C-Bt2LAC pg/ml IL-8 Bacterial Extract + LPS K12 0.0003 pg/ml pg/ml IL-8 bacterial extract + LPS K12 0.001 μ^ιηΐ pg/ml IL-8 bacterial extract + LPS K12 0.003 pg/ml pg/ml IL-8 bacterial extract + LPS K12 0.01 ^^ηιΐ pg/ml IL-8 0.2 mg/ml 148 213 449 713 1111 0.5 mg/ml 154 225 333 622 1228 1 mg/ml 125 219 375 782 1306 2 mg/ml 105 259 391 799 1164 Conclusion In summary, this article presents The results of HEK cells and their results on human PBMC cells indicate that Afer50 and BT1LAC can stimulate the immune system via the TLR2 pathway. In addition, BT2LAC can be expressed as a TLR2/6 antagonist. Therefore, the extract of the present invention can stimulate the immune system or act as a TLR2/6 antagonist via the TLR2 pathway, and thus is associated with anti-infective and anti-inflammatory activities in vivo. Example 8: Cells that represent a given functional TLR gene are valuable tools for many applications, such as studies of mechanisms involved in TLR identification or signal transduction, and the development of new potential therapeutics. Therefore, the objective of the experiments described below was to test the activity of the key adapters of the four bacterial extracts for these immune responses. In this test, eight TLR and NOD2 receptors were screened against four bacterial extracts, including the extract of the present invention. method

The bacterial extract of the present invention was tested in a 96-well microplate. The extract was diluted in DMEM medium and 20 μM of each dilution was tested in duplicate. Adding 180 μΐ volume to each well in duplicate containing 25000 or 50000 cells in DMEM medium + 10% FCS (a HEK293 cell line specific for each TLR 74 201010711, in which the conductor gene is controlled in NF-kB A HEK293 cell suspension that secretes alkaline phosphatase). After incubation for 16 hours in each cell strain, each supernatant of 20 μM to 50 μM was transferred to a 96-well microplate and refined with 200 μΐ Quantiblue (InVivoGen no REP-QB1). For different series of TLR-expressing cell lines, the enzymatic reaction of the secreted alkaline phosphatase is carried out for 30 to 60 minutes. Reading was performed at 630 nm using a microwell plate reader. The results are expressed in OD at 630 nm. ❹ Materials 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 ; TLR4 乂广禅崴 K12 LPS 1 Pg/ml; TLR5 S. typhimurium flagellin 1 pg/ml; TLR7 R848 10 pg/ml; TLR8 R848 10 pg/ml; TLR9 CpG ODN 2006 10 pg/ml; NOD2 cell wall 醯 dipeptide 1 pg/ml. Negative control A recombinant HEK-293 cell line reporting only the conductor gene (NFkB) was used as a negative control for the TLR cell line. The negative control values for each pure line are background signals for these non-inducible lines. TNF-α was used as a positive control for this non-TLR-expressing cell line. Test article The following bacterial extracts were tested: OP0701B4_CFer3 00: Extract obtained from Splash 75 201010711 ▲ 鏔荔#崴/-3P2P (12 g/L) as described in Example 3.3; OP0701B4_CRahr3 00: as in the example Extracted from 虞李潜#乙#磨71.38 (40 g/L) as described in 3.4; OP0701D_10LlPswitchA: obtained as described in Example 3.12 from ###/-3929 (10 g/ Extract of L) (I); and OP0701D_5LOSMOConc: obtained from osmotic dissolution and rubbing #磨/-JPM (7.6 g/L) obtained by osmotic pressure in a 0.16 N NaCl solution at 40 ° C for 24 hours. The extract (H) was used as a control. % Preparation of bacterial extracts Each 20 μM sample to be tested was used to stimulate all cell lines in a 200 pL reaction volume. Screening is performed at a single concentration, typically 0.5 mg dry weight/mL. Test in duplicate. Results: Bacterial extracts and controls were tested in duplicate on recombinant HEK-293 cell lines functionally displaying a given TLR or NOD2 protein 10 and a reporter gene driven by the NFkB promoter. The results of TLR and NOD2 activation are given in terms of optical density (OD) values. The results are shown in Tables 10-14 and are summarized in Table 15. Bacterial extract F (QP0701B4Cfer3 00 ) Table 10: Activation of TLR and NOD2 receptors by OP0701B4Cfer300 extract 76 201010711 hTLR2 hTLR3 hTLR4 hTLR5 hTLR7 hTLR8 hTLR9 hNOD-2 Reference 3.493 2.052 1.341 3.657 1.480 2.786 1.955 0.576 Extract F 3.963 0.051 0.735 0.304 0.070 0.080 0.026 0.503 Bacterial extract F specifically activates hTLR2, hTLR4 and hNod2, and activates hTLR5 to a lesser extent. Bacterial extract G (OP0701B4CRahr300) Table 11: TLR and NOD2 receptors were extracted by OP0701B4CRahr300. Activation of hTLR2 hTLR3 hTLR4 hTLR5 hTLR7 hTLR8 hTLR9 hNOD-2 Reference 3.493 2.052 1.341 3.657 1.480 2.786 1.955 0.576 Extract G 0.808 0.003 0.177 0.017 0.028 0.044 -0.032 0.369 Bacterial extract G activates hTLR2, hNOD2 and activates hTLR4 to a lesser extent.细菌 Bacterial extract H (OP0701D 5LOSMOConc) Table 12: Activation of TLR and NOD2 receptors by OP0701D_5LOSMOConc extract hTLR2 hTLR3 hTLR4 hTLR5 hTLR7 hTLR8 hTLR9 hNOD-2 Reference 3.493 2.052 1.341 3.657 1.480 2.786 1.955 0.576 Extract H 4.537 0.057 0.638 0.050 -0.072 0.054 0.011 0.892 The bacterial extract specifically activated hTLR2, hTLR4 and Nod2. 77 201010711 Bacterial extract I ( OP0701D 1〇LlPswitchA) Table 13: TLR and NOD2 receptor activation by 〇p〇701D_10LPswitchA extract hTLR2 hTLR3 hTLR4 hTLR5 hTLR7 hTLR8 hTLR9 hNOD-2 Reference 3.493 2.052 1.341 3.657 1.480 2.786 1.955 0.576 Extract I 4.168 0.096 0.342 3.490 -0.024 0.132 0.410 0.790 Bacterial extract I strongly activated hTLR2, hTLR5, hNOD2, and activated hTLR4 and hTLR9 to a lesser extent. Table 1 5: TLR and NOD2 receptors Activation of different bacterial extracts obtained from different treatment conditions of the invention hTLR2 hTLR3 hTLR4 hTLR5 hTLR7 hTLR8 hTLR9 hNOD-2 extract F + - + +/- - + extract G + - +/- - - _ + extract H + - + - - - - + extract I + - +/- + + / _ + Therefore, the bacterial extract of the present invention can help activate immunity via various TLRs and Nod2 system. Therefore, the extract of the present invention can be a good activator for the immune response.

Comparing the extract mash with the extract G and the extract F, it is apparent that the extract of the present invention has the same TLR 78 201010711 it path and Nod path as the extract obtained by the osmotic dissolution. Comparing extract H with extract, we can observe that TLR5 pathway is activated by increasing acidic solubilization after assay dissolution, and to a lesser extent TLR9 pathway is activated. Even the extract F produced by the weak alkaline treatment exhibited activation of the TLR5 pathway opposite to the extract. Extract I acts on TLR5, indicating potential applications in radiation therapy. In fact, radiation therapy is a well established and highly effective treatment for certain types of cancer. However, its side effects can be devastating because it can destroy healthy cells in the body, especially bone marrow cells and cells in the gastrointestinal tract. ® Burdelya et al. recently reported that the CBLB502 peptide binds to TLR5 and activates the nuclear factor-kB k transduction pathway, a pathway in which cancer cells are normally activated to avoid cell death (Burdelya et al., "An agonist 〇ft〇li_iike receptor 5 Has radioprotective activity in mouse and primate models,"We" 2008, 320:226-30). Mice and geese monkeys treated with CBLB5〇2 before the total body irradiation to be exposed to a lethal dose show healthy bone cells And less damage to the gastrointestinal cells and a significant longer survival than the control. Importantly, in mice bearing tumors, CBLB502 does not impair the anti-tumor efficacy of radiation therapy. Implementation ϋ ^ ^ in the mouse | | The ability to produce plaque- or electro-optic plaque-forming cells (PFC) technology enables the assessment of non-specific stimuli in sputum-lymphocytes. This technique is best practiced by Cunningham and Seenberg (/wwwwo/o) , 1968, 14, 599) First described. Proof of the presence of hemolytic antibodies around antibody-forming cells as follows. Will be murine lymphocytes as well as foreign (sheep) red blood cells The dense population was simultaneously introduced into the microscope glass 79 201010711. Some lymphocytes released hemolytic antibodies, which were diffuse and caused the dissolution of adjacent red blood cells by forming lysis plaques in the presence of complement. At the end of the experiment, the number of PFCs was reported to be 1〇6. Cells or counted spleen cells. Materials and Methods Test article Bacterial extract 1 was obtained as described in Example 3.5, and Bacterial Extract 2 was obtained as described in Example 3.4. Animal β will be 5 to 6 weeks old and 40 males with an average body weight of 20 +/- 2 g

Balb/C mice/experiments (IFFA-CREDO, St. Germain sur l'Arbresle

Cedex, France) is distributed in 5 groups of 8 animals each. Experimental Design Animals were divided into 5 groups as follows: Group (a) 8 mice, each receiving 1.2 mg of extract 1 obtained as described in Example 3.5; volume = 〇·2 ml group (b) 8 Mice, each mouse received 0.6 mg of extract 1 obtained as described in Example 3.5®. Group (c) 8 mice, each receiving 1 · 2 mg of extract 2 obtained as described in Example 3.4; volume = 0.2 ml (d) 8 mice 'received per mouse 6.6 mg of extract 2 obtained as described in Example 3.4; volume = 0.2 ml group (e) 8 mice 'received 〇. 2 ml isotonic saline mice at the indicated doses daily ( The extract as described above was received via the oral route for 5 consecutive days (Day 1 to Day 5). 201010711 • Two intubations occurred on the 19th and 20th days. On the 29th day, the antigen in the 0.2 ml isotonic saline (0.9% NaCl, Alsever solution) (106 sheep red blood cells) was intravenously injected through the hip vein of the animal; the spleen cells were prepared 4 days later (day 33). suspension. Reagents and equipment Endotoxin-free Alsever's solution (Alsever's solution,

Sigma, 38299 St Quentin Fallavier, Cedex France, ref. A 3351 ) ® basal medium in Bacteria-free EarleS solution (BME, 1 〇x concentration) (Bio-M6rieux, 69280 Marcy l'Etoile, France ref : 8 210 2 ) Cambodian vein rat jk clear (Bio-M0rieux, Marcy l'Etoile, France ref: 7 212 2 ) Weft red jk ball (Bio-M6rieux, Marcy l'Etoile, France ref: 7214 1 ) Cone Insect blue, sterile distilled water, NaCl 0.9%, tissue homogenizer, centrifuge 'Neubauer chamber for counting cells, glass tube. The plexus Ji test hangs the item processing. The control animal only receives the sheep red blood cell (SRBC). Animals of other groups received bacterial extracts orally as indicated by the sputum. For this purpose, each extract was dissolved in water. Cell suspension 4 days after the last antigen (SRBC) injection, a suspension of spleen cells of each mouse was prepared according to the following procedure: 201010711 A suspension of spleen cells of each mouse was prepared: The animals were anesthetized with ether and then sacrificed by cervical dislocation. Spleen cells were removed and comminuted in a glass tissue homogenizer at 2 6 BME at pH 6 75 6 8 . Then 3 ml of sterile distilled water was added and the mixture was stirred for 20 seconds. The resulting suspension was further diluted with 1 〇 BME and centrifuged at 1200 rpm for 10 minutes at 4 times. The supernatant is discarded and the pellet is suspended in 2, such as BME. The cells were allowed to recover on ice for 51 minutes. The viability count of monocytes was counted as follows: β A 1/20 dilution of an aliquot of the cell suspension was prepared in physiological saline containing a G.1% trypan blue solution. Uncolored cells (i.e., living cells) were counted in a Neubauer chamber. The cell suspension was stored on the molten ice during the counting and then immediately used to dissolve the plaques. This technique prevents loss of survivability, which can occur when the waiting time is too long. On the ordinary microscope slides, carefully remove the dust and remove any traces of fat, and stick the three parallel strips (〇1 and 2 thick) at intervals of about 1.5 cm. The strip is then covered with a pre-cleaned coverslip (22 mm x 22 melon) to form two "chambers" between the slide and the coverslip. A portion of the spleen cell suspension was adjusted to 25,0 monocytes per square millimeter. In a small glass tube, a mixture of the following elements (added in the stated order) was prepared by means of an automatic pipette:

1) 7.5 ml BME 2) 0.5 ml SRBC suspension is washed with physiological saline 82 201010711 and centrifuged twice under 5〇% residue. 3) 0.4 ml of normal guinea pig serum is used as the source of complement ^most, to 5〇 2 GG μΐ of the aforementioned mixture was added to a s1 25,000 cells/mm 3 spleen cell suspension. After mild homogenization, the suspension was introduced into the "Cunningham Chamber" by capillary action and then the free side was sealed with money. The slides were then placed in a humid chamber in the oven below s m:. Lecture

The slides were incubated for 1 hour in an oven, such as from J, after a microscope at a magnification of 100 times (eyepiece 10 x, objective lens 10 X). Dissolved plaques are easy to identify. In 夂 and 4. Five vertical optical strips were examined on each slide and the number of dissolved plaques was counted. By counting, the number of cells that directly dissolve plaques per 106 cells is calculated by inference (Ν,. The right X is the number of observed plaques and is also the number of cells tested. The number of cells directly in the cymbal, Λ 6 & J 6 & 10 cells directly dissolved in the plaque is equal to N = (x / X) * 1 〇 6 Where X = C*V = C*(n*e C = final concentration of spleen cells V = volume observed n = number of optical strips e = tape thickness I = width of the optical field L = length of the optical strip 1 The number of direct lysis plaques of one cell is obtained by the following equation: PFC (per 1 〇 6 cells) = (x * 1 〇 6) / ne ^ ; 83 201010711 By knowing the number of cells collected by each spleen, The number of cells that form plaques in each spleen can be inferred. In the "absolute reference" mice, spontaneous dissolution plaques were not observed. Statistical analysis and results When p < 0.05, the results were considered significant (student t test). The results are shown in Table 16. Table 16: Ability of bacterial extracts 1 and 2 to produce plaque forming cells (for sheep red blood cells) in mice ❹ GRM PFC/106 cells PFC / ratio extract 1 ; See Example 3.5 100% 124%* 119%* (1.2 mg per mouse per dose) 100% 100% 123%* 114%* 143%* 113% Extract 1; see Example 3_5 100% 117%* 103% (0.6 mg per mouse per dose) 100% 100% 118%* 112%* 139%* 128%* Extract 2; see example 3.4 100 % 119%* 109% (1.2 mg per mouse per dose) 100% 100% 124%* 117%* 131%* 120%* Extract 2; see example 3.4 100% 117%* 112%* (0.6 mg per mouse per dose) 100% 100% 114%* 113%* 119%* 111% Like when tested at 1.2 mg per mouse per day or 0.6 mg per mouse per day, The extract of the present invention is presented as a B cell activator. A slight dose-dependent effect is observed.Thus, some of the extracts of the present invention can potentially be used to elicit the immune system of a patient suffering from a recurrent infection. Example 10: CFer300 The effect of intraperitoneal Salmonella typhimurium in ba丨b/c small SLII was orally administered and tested by infected lysate from its source of grinding. #磨的鼠的保护. 84 201010711 Materials and Fangmu Animals and ^^ alb'c mice were housed in the Institute for Immunology, -SC〇W. For in vivo protection experiments, t, non-innate experiments to grade mice are from Stolb〇vaya, Russian state Scientific Centre for Biomedical Technologies (Russian Academy of Medical Sciences). Upon arrival, the mice weighed l2_i4 y throughout the experiment, the mice were maintained under pathogen free conditions on a standard rodent diet and water. ❹ To experiment) Two groups of 22 mice per group were used to test the anti-infective efficacy of bacterial extracts prepared using an experimental model of mouse sputum sputum axe infection. One group was treated with σ with a CFer3G solution and the second group received a pseudo treatment (water) as a negative control. Each mouse was orally administered with a solution of 5 ml of sputum once a day for 1-5 days, and then all mice were challenged with a sputum: #1咏, a single 2 mg (〇.5 ml) was orally administered. Give CFer3〇〇® processing. Group 2: The mice received a pseudo-treatment with daily oral administration of 〇·5, such as water, for 10 days. The bacterial extract tested on the test article was 〇P0701B4CFer3(10) ("CFer300"); the extract from 罄摩莫淖彦 (12 g/L) obtained as described in Example 3.6 (28.4 mg active dry weight/g) Preliminary Experiment 85 201010711 The purpose of the preliminary experiment was to determine the dose of the infectious agent that would induce nearly 5% mortality after 3 weeks of challenge. Each mouse was intraperitoneally injected with Salmonella enterica, Enterobacter serovar typhimurium ^ 415 ( I. Mechnikov, Institute for Vaccines and Sera

Suspension of Russian Academy of Medical Sciences as an attack. The attack dose ranged from 1 〇 3 to 1 〇 5 CFU per mouse. Observations and death records After the challenge, the mice were kept under standard conditions in laboratory animals. Sense ® records daily observations and death records during 2 days. The anti-infective efficacy of the formulation was estimated based on the post-infection survival rate (SR), the mean life duration after infection (ADL), the defense factor (DF), and the Formulation Efficacy Index (EI) calculated for each experimental group. SR is the percentage of surviving animals in the experimental group on day 21 post infection. ADL, DF and EI are calculated as follows: ADL = (X1 + χ2 + ... + χη) /N where: ADL is the average life duration, ❹ X1 to Xn is the life duration of infection of experimental mice 1 to η, and Ν The total number of animals in the experimental group. DF = CD / ED where: DF is the defense factor, CD is the percentage of death in the control group and ED is the percentage of death in the experimental group. 86 201010711 EI = [(DF - 1) / DF] x 100% where:

El is the formulation efficacy index and DF is the defense factor. The sex-accepting preparation was orally administered once a day for 1 day and was well tolerated. No evidence of toxicity or side effects was observed during the 10 day pretreatment. After the preliminary experiment to measure the attack dose of approximately 〇% death rate. The results are shown in Table 17. Table 17: Preliminary experimental death mice/total death (%) for the challenge dose of (4) feedstock corresponding to approximately 50% mortality 12 13 - _ 4/6 67 — L__ - 1/6 17 I_ 0/6 0 I typhoid Sha's #彦 dose

The underlined numbers indicate the number of animals found to have died on the indicated day (post-infection). Conclusions _ Based on the data obtained, the selection of 1〇5 CFu dose of 虞 裳 # 择 株 415 (67% dead animals, _) was used for subsequent studies. Test: Treatment with CFer300 The mice pretreated with CFeBOO preparation for 10 days (n=22), or the water treated mice (n=22) of the group 87 201010711 were challenged at the end of pretreatment. The 4 #幕夕戌# grinding suspension was intraperitoneally administered with 1 〇 5 CFU of challenge per mouse. Follow-up observations were made 21 days after infection. The mortality rates of each group are shown in Table 18. Table 1 8: Follow-up observation of drug injection 21 days after infection - i number of deaths (%) survival 1 2 4 1 b Ί 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 \/yiJ \/0) CFer 300 1 1 1 1 - 27 73 H2O 1 2 2 2 _ — - 1 Peak - 1 1 - - - - - 45 ~~55~~

The number in italics indicates the number of animals found to have died on the first indicated day (post-infection). These data were used to calculate Sr, ADL, DF, and 耵 (Table 19). Table 19. Survival rate during the observation period (21 days)

In the control group pretreated with water, the survival rate during the observation period (21 days) was 55/. 'And the ADL is 14.7 days. At a dose of 2 mg per day, CFer3〇〇 exhibited a protective scent, resulting in SR = 73% and ADL = 171 days, ie DF = 1.67 and EI = 4〇〇/0. In summary, the 1 G-day course of oral administration of CFer3 in a single dose of 2 mg provided partial protection in infected mice. As shown in this example, and previously shown in vitro and ex vivo, 88 201010711 The extracts disclosed herein are applicable to further therapeutic drug development. Example 11: Two 瘌.霣as篕 "τ from * - — Chat —^

Role m. After oral administration, two specific examples of the invention were tested in a murine model of allergen-induced asthma (Julia et al., 2, 2, 16:271-283). Materials and Methods Animals and Feeds ® 6-week-old female BALB/c ByJ mice were purchased from janvier, France. Keep it under standard conditions and keep it in silver. The study group divided a total of 27 mice into 4 groups as follows: Group A: untreated LACK-sensitized mice challenged with physiological saline; LACK is a protein-only mouse from the parasite Leishmania adults ). Group B: untreated LACK-sensitized and challenged mice (8 small® rats) group (::〇)\/[-1009 human treatment (each dose of 8 1^ dry weight residue), LACK-sensitized and challenged mice (8 mice) Group D: 〇M-1009B treatment (8 mg dry weight residue per administration), LACK-sensitized and challenged mice (7 small Rats were treated with test or control articles and mice were treated from day 3 to day 22. On days 0 and 7, mice were intraperitoneally (iP) at 10 pg LACK in the presence of 2 mg 矾3. ·) Min 89 201010711. On days 17-21, the mice were exposed to LACK solution (0.15%) for 20 minutes per day, aerosol challenge (groups B, C and D), or physiological saline (group a) as a control. On day 22, the mice were analyzed for their ability to develop AHR upon inhalation of methacholine. On day 23, the mice were sacrificed and the lungs were inflamed. Methods The mice were treated therapeutically as described above. Three times. Irrigation was performed in individual mice that were bled by a cannula inserted into the trachea. The lungs were washed 3 times with 1 ml of warm PBS. The cells were washed with PBS, resuspended at 300 μΐ and used Burker-T. Tirk chamber count. For differentiated BAL cell counts, Cytospin preparations were prepared and stained with Wright/Giemsa staining. The tested groups were: wild type (wt) mice (control) sensitized by LACK and challenged with PBS, Lock-sensitized and challenged wt mice (asthmatic animals), 0M- 1 009A-treated wt mice, and 0M-1009B-treated wt mice. The cytospin preparation stained with Wright/Giemsa by microscopic examination The total number of cells in the BAL was determined. ® Since the tracheal inflammation was observed to decrease in the treated mice (see results below), lung extracts were prepared and quantified by multivariate analysis (CBA array) of IL-4, IL- 5 and IL-13. The two bacterial lysates tested for the test article were: OM-1009A extract from prawn # 1-3929 obtained as described in Example 3.7 and obtained as described in Example 3.11. The OM-1009B extract from Momoqi 3-5929 was extracted from 201010711. Results: a) On the 22nd day of gas camp high reactivity, the ability of mice to develop AHR at the indicated dose of B-methyl inhalation was analyzed. . The results are reported in Figure 5. The results show that the basic Penh value of OM-1009-B recovery is similar to the non-puncture group without pbs treatment. OM-1009-A is more effective because the penJl values obtained are even lower than those observed in the non-asthmatic control group. b) Total number of cells in bronchoalveolar lavage fluid ^ & Ο On day 23, the mice were sacrificed and the lungs were inflamed. The total number of cells in bAL and the number of differentiated cells: eosinophil count (Eo), neutrophil white number (Neutro), lymphocyte count (Lympho), and other cell numbers (〇ther) are reported in Table 20. (right bar). Table 20. Total cell number and number of differentiated cells in B AL when treated with the two extracts of the invention (groups c and d). Provide mean and standard error mean mean Eo Neutro Lympho 蟪 雎 (A) CtrlPBS 901 3178 11796 49125 65000 (B) CtrlLACK> asthma mice 347691 51317 85609 301097 785714 (C) OM-1009A+LACK 115227 27879 54725 158419 356250 (D) OM-1009B+LACK 203834 14210 34523 204575 457143 SEM Eo Neutro Lympho Other Int cell (A) Ctrl PBS 166 906 6255 5568 2887 (B) Ctrl LACK 78464 14392 17736 78575 135275 (C) OM- 1009A+ LACK 36741 10444 27024 34296 94905 (D) OM-1009B+ LACK 57955 3220 6731 25311 74992 Both extracts significantly reduce the total number of cells in the BAL (when compared to Group B compared to 91 201010711 j, for OM-1009A p <0.01; and p < 0.03) for OM-1009B. The number of eosinophils was reduced by a factor of 3 and 1.7, respectively. The number of neutrophils was reduced by 1.8 times and 3.6 times, respectively. A decrease in the number of eosinophils and the number of neutrophils indicates a lower concentration of inflammatory cells in the BAL of the animal pretreated with the extract. c) Th2 interleukins detected in lung extracts and quantified by multivariate analysis (CBA display) Table 21: Individual IL4 lung content in mice Spring pg/mg Mice 1 Mice 2 Mice 3 Mice 4 mouse 5 mouse 6 mouse 7 mouse 8 mean (A) PBS 0 0.0053 0.0071 0.0157 0.0070 (B) LACK (asthma mice) 0.0395 0.0141 0.0279 0.0203 0.0353 0.0574 0.0949 0.0806 0.0462 (C) POOM1009A 0.0379 0.0120 0.0066 0.0056 0.0181 0.0146 0.0396 0.0463 0.0226 (D) POOM 1009B 0.0409 0.0515 0.0414 0.0511 0.0484 0.0300 0.0270 0.0415 Table 22: Individual IL5 lung content in mice pg/mg mouse 1 mouse 2 mouse 3 mouse 4 mouse 5 mice 6 mouse 7 mice 8 mean (A) PBS 0 0.0004 0.0043 0 0.0012 (B) LACK (asthma mice) 0.1141 0.1002 0.1532 0.1115 0.3098 0.5173 0.5385 0.4547 0.2874 (C) POOM1009A 0.1817 0.0445 0.0572 0.0364 0.1643 0.0488 0.1917 0.1057 0.1038 ( D) POOM1009B 0.1518 0.2798 0.1797 0.5439 0.3685 0.1320 0.1705 0.2609 Table 23: Individual IL13 lung content in mice pg/mg mouse 1 mouse 2 mouse 3 mouse 4 mouse 5 mouse 6 mouse 7 small Rat 8 mean (A) PBS 0 0 0 0.0208 0.0052 (B) LACK (asthma mice) 0.2727 0.0896 0.3542 0.2303 0.6178 1.1306 1.1768 1.0244 0.6120 (C) POOM1009A 0.3598 0.1231 0.0S88 0.0995 0.2975 0.1909 0.5213 0.5463 0.2784 (D) POOM1009B 0.3628 0.8919 0.4291 0.7699 0.6441 0.4981 0.4972 0.5847 92 201010711 When compared with the asthma control group (B), the content of Th2 interleukin (iL4, IL5 and IL13) was decreased by OM-1009A extract, but not by 〇m_1〇〇9b extract. reduce. Therefore, the bacterial extract obtained by the strong alkali treatment (OM-1009-B) can be less active than the one obtained by the mild alkali treatment (OM-1009-A). Although both extracts are active under the Penh factor assay (Figure 6)', OM-1 is used to treat or prevent conditions associated with Th2-related diseases such as allergic diseases and atopic dermatitis or its symptoms. 009A can be a better candidate than OM-1009B. Φ [Simplified illustration] Figure 1. Eleven ligands of the family of toll-like receptors (TLRs) expressed by host organisms. Figure 2: A graph of a tangential flow filtration (tangential fl〇w fiitratio I1, TFF) system for preparing bacterial extracts after bacterial dissolution. The chart shows two different filter configurations: Parallel mode, where all filters operate simultaneously, and 蜿蜒 mode, where the filters are configured in continuous mode. Figure 3: General relationship between operating parameters and flux, indicating the area of pressure control and mass transfer control for tangential flow filtration (TFF). Figure 4: Stimulation of spleen cells cultured for 48 hours in the presence of lysates AFer300, CFer300 and DFer300 and different dilutions of ARahdOO, CRahr3〇〇 and DRahr3〇〇. After adding 30 μM of Alamar blue8 solution diluted 1:1 in the cell culture medium per well, the cells were further cultured (a) for 8.5 hours (first experiment); (b) 24 hours (second experiment). The average emission value of the double replicate culture at 59 〇 nm is shown as the standard deviation of the soil. Figure 5: In the (a) first test and (b) the second test, induced nitrogen oxides in mice treated with 鏺肄旄93 201010711 #彦1-3929 and 4#潜舁#磨71.38 extracts ( NO) is produced. The average soil standard deviation is expressed as μΜnitrogen oxide (NO). Figure 6. Airway hyperresponsiveness (ahr) of the extract of the invention by inhaled acetonitrile methylcholine by sputum concentration by whole body plethysmography (Emka) 1 day after the last antigen challenge. effect. Display with phosphate buffered saline (PBS)

Animals (η=4), untreated LACK-attacked animals (as positive control, n=8), OM-1009A·treated LACK tappings >, & , Jiudan mice (n= =8) and OM-1009B-treated LACK-challenged mice (n=7) J, and results (average enhanced pause value +/- standard error of mean). [Main component symbol description] None _ 94

Claims (1)

201010711 X. Patent Application Range: 1. One or more itching mills ((10)) extracts of bacterial strains wherein the extract is a soluble extract' and wherein the extract contains chemically modified bacterial molecules. 2. The extract of claim 1, wherein the chemically modified bacterial molecules are produced by exposing the one or more strains of the sputum bacteria to an assay medium. 3. The extract of claim 1 or 2, wherein the extract has immunomodulatory activity in the individual. 4. The extract of claim 3, wherein the extract has immunostimulatory activity in the body. 5. The extract of claim 3, wherein the extract has anti-inflammatory activity in the individual body. 6. The extract of claim 1, wherein the one or more 舁 囷 囷 & & & & La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La La ❹ { Lactobacillus plantarum), Lactobacillus johnsonii), Lactobacillus helveticus, Lactobacillus casei defensis, Lactobacillus casei ssp. casei, deputy dry milk Bacillus subtilis Q Lactobacillus par acasei, Lactobacillus bvgaricus'), Lactobacillus paracasei, Lactobacillus acidophilus 'Lactobacillus reuteri, Lactobacillus salivarius, milk Si One or more of Lactobacillus lactis 95 201010711 i Lactobacillus lactis, Lactobacillus delbrueckii. 7. The extract of claim 1 of the patent scope, wherein the one or more of the bacillus sinensis is found in Lactobacillus brevis, Lactobacillus rhamnosus Ί%, Lactobacillus plantarum, Lactobacillus helveticus One or more. 8. The extract of the scope of the patent application, wherein the extract is in the day; the amino acid, amylin, serine, histidine, alanine, arginine, guanamine, guanamine At least 10°/ of one or more of acid, phenylalanine, and lysine. Racemized. 9. The extract of claim 1 of the patent, which enables the extract to achieve a calculable IL1〇/ILl2 ratio in human peripheral blood mononuclear cells, the ratio being equal to or greater than the activity of obtaining the extract. The strain reached the ili〇/ili2 ratio. An extract of the first aspect of the patent, wherein the extract comprises the number of eosinophils, the number of neutrophils, the number of lymphocytes, or the number of lymphocytes in the individual of the genus Any combination reduces the factor by at least 1.5 relative to the untreated control. U.-species preparation as claimed in the scope of patents! The extract of the item comprises: 乇 (a) cultured in a medium - or a plurality of milled g strains; (b) exposing each of the selected strains of the bacterial strain to an alkaline medium; and treating the product of the "U" by shifting In addition to the insoluble and particulate I2. The method of claim (1), wherein the step (7) is carried out by tangential flow filtration by means of 96 201010711 j-. 13. The method of claim U or i2 , wherein each of the exposed bacteria strains at a pH greater than 9,0 is sufficient to chemically modify the bacterial molecules. 14. The method of claim 5, further comprising after part (b) and in part (c) Before the treatment of each strain at a pH of less than 4.5%, the method of claim 5, wherein the chemical modification package & contains the aspartic acid, bran in the extract At least 丨〇0/〇 消 in one or more of aminic acid, serine, histidine, alanine, arginine, tyrosine, methionine, phenylalanine, and lysine旋化. 1 6. A health food composition, which includes, for example, a patent application The extract of any one of items 1 to 0, or the extract prepared according to any one of claims 1 to 5 of the patent application. 17. A pharmaceutical composition comprising the scope of the patent application The extract of any one of the above items, or the extract prepared according to any one of claims 1 to 15 of the patent application. 1 8. The extract of any one of claims 1 to 15. Or a composition according to claim 6 or claim 7, which is suitable for use in alleviating at least one symptom associated with at least one condition selected from the group consisting of a respiratory condition, an allergic condition, a urinary tract condition, and a digestive condition. Pharmaceuticals. 1 9. The extract of claim 18, wherein the respiratory condition is selected from the group consisting of upper respiratory tract and lower respiratory tract infection, nasopharyngitis, sinusitis, pharyngitis, tonsillitis, laryngitis, bronchitis, Pharyngitis, influenza, pneumonia, 97 201010711 Radionome pneumonia & an obstructive pulmonary disease with acute exacerbations. The extract of claim 18, wherein the allergic condition is selected from allergic rhinitis, allergies Asthma, and ectopic 1. The extract of claim 18, wherein the urinary tract disorder is selected from the group consisting of urethritis, renal tubule_interstitial nephritis, obstructive pyelonephritis, cystitis including slow I. cystitis, including prostatitis And male pelvic pain syndrome of chronic prostatitis, prostatic cystitis, and female pelvic inflammatory disease. 22. The extract of claim 18, wherein the digestive disorder is selected from Crohn's disease (Crohn.s disease) And large intestine irritability. 23. The extract of claim 18, wherein the individual is a human or a domestic animal. An use of an extract according to any one of the items of the present invention, or a composition according to claim 16 or 17 for use in mitigating, and at least one selected from the group consisting of respiratory diseases, A pharmaceutical product of at least one symptom associated with a condition of an allergic condition, a urinary tract condition, and a digestive condition. 25. The use of an extract according to any one of claims -15, or a composition of claim 16 or claim 7, which is used as an immunomodulator. 26. An isolated microbial strain which is sputum mill 1 3929. 27. An extract which is obtained from a strain of claim 25 of the patent application. 28. The extract of claim 26, wherein the extract is a soluble extract. 98