JP2020015757A - Pharmaceutical composition and food composition for inducing satiation and extending satiety in object in need thereof - Google Patents

Abstract

To provide a composition for inducing satiation and extending satiety in an object in need thereof.SOLUTION: The present invention provides a composition used for extending satiety or decreasing food intake in an object in need thereof, containing an effective amount of ClpB protein containing a specific amino acid sequence.SELECTED DRAWING: None

Description

  The present invention relates to pharmaceutical and food compositions that induce satiety and prolong satiety in a subject in need thereof.

  The composition of the gut flora is related to the metabolic phenotype of the host (Ley et al., 2006), and translocation of the "obese" flora leads to steatosis (Turnbaugh et al., 2006) and overeating (Vijay-Kumar et al., 2010), which suggests that intestinal flora may affect host feeding events. Although the mechanisms underlying the effects of enterobacteria on host appetite are unknown, it is possible that host molecular pathways that control food intake could be used.

  Current food intake control models suggest that gut-derived fasting and satiety hormones signal some of the brain networks that regulate the homeostatic and pleasurable aspects of eating (Berthoud, 2011; Inui, 1999; Murphy and Bloom, 2006). Of these, in particular, the hypothalamic arcuate nucleus (ARC), which contains pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) / agouti-related protein (AgRP) neurons, each relaying the paraventricular nucleus (PVN). Anorexiagenic and anorectic pathways of origin are important (Atasoy et al., 2012; Cowley et al., 1999; Garfield et al., 2015; Shi et al., 2013). The ARC and PVN pathways focus on the outer parabrachial nucleus, which sends anorexia-inducing projections to the central amygdala (CeA), which express calcitonin gene-related peptide (CGRP) (Carter et al., 2013; Garfield). et al., 2015).

  A hypothetical mechanism of the effect of the gut microbiota on the control of host appetite is energy harvesting activity (Turnbaugh et al., 2006), and the production of transmitters and metabolites of neuroactive activity ((Dinan et al., 2006). 2015; Forsythe and Kunze, 2013; Sharon et al., 2014) The present inventors have determined that bacterial proteins that act directly in pathways that control appetite locally or systemically in the intestine are involved. Indeed, some bacterial proteins have been shown to exhibit sequence homology to peptide hormones that regulate appetite (Fetissov et al., 2008), and in recent years, the intestinal symbiotic E. coli ( Produced by Escherichia coli (E. coli) The identified ClpB protein was identified as an antigen mimic of α-melanocyte stimulating hormone (α-MSH) (Tennoune et al., 2014) α-MSH is activated by melanocortin receptor 4 (MC4R) A POMC-derived neuropeptide that plays a key role in signaling satiety (Cone, 2005) The anorexic-inducing effects of MC4R-mediated α-MSH are primarily a central site of action of α-MSH (Mul et al., 2013), and recent studies have shown that activation of MC4R in intestinal endocrine cells of the intestine indicates that the satiety hormones glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). Stimulated release (Panaro et al., 2014). alpha-MSH-like molecules from can act directly on the intestinal endocrine cells synthesizing satiety hormone.

  The present invention relates to a food composition and a pharmaceutical composition that induce satiation in a subject in need thereof and prolong satiety. In particular, the invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION We have found that ClpB protein or a bacterium that expresses ClpB protein induces satiety, prolongs satiety, reduces food intake, and controls weight gain in subjects in need thereof. And / or demonstrated that weight loss can be stimulated.

  Accordingly, one aspect of the present invention pertains to a method of inducing satiety in a subject in need thereof, comprising administering to the subject an effective amount of a ClpB protein or a bacterium that expresses a ClpB protein. .

  A further aspect of the invention pertains to a method of extending satiety in a subject in need thereof, comprising administering to the subject an effective amount of a ClpB protein or a bacterium that expresses a ClpB protein.

  Thus, one aspect of the present invention is a method of reducing the amount of meal in a subject in need thereof, comprising administering to the subject an effective amount of ClpB protein or an effective amount of bacteria that express ClpB protein. About.

  A further aspect of the present invention is a method of reducing food intake in a subject in need thereof, comprising administering to the subject an effective amount of a ClpB protein or a bacterium that expresses a ClpB protein. About.

  Yet another aspect of the invention is a method of controlling weight gain in a subject in need thereof, comprising administering to the subject an effective amount of a ClpB protein or an effective amount of a bacterium that expresses a ClpB protein. About.

  Yet another aspect of the invention is a method of stimulating weight loss in a subject in need thereof, comprising administering to the subject an effective amount of a ClpB protein or a bacterium that expresses a ClpB protein. About.

  The method of the invention is intended for humans, pets, or livestock, where the pets or livestock consist of dogs, cats, guinea pigs, rabbits, pigs, cows, sheep, goats, horses, and / or poultry. It can be selected from a group. In some embodiments, the subject is a male or female subject.

  In some embodiments, the subject is not obese. In some embodiments, the subject is obese. "Obesity" as used herein refers to a medical condition, preferably wherein the subject has a BMI greater than 30. “BMI” or “Body Mass Index” is defined as the subject's weight divided by the subject's height squared. Equations commonly used in medicine yield units of measurement of kg / m2. Generally, non-obese subjects have normal weight. "Normal" and "simple overweight" herein refer to the weight that results in a BMI of 18.5-30.

  As used herein, the term "satiety" is meant to refer to an essentially constant state in which an individual feels that his or her needs are being met or minimized. Many physiological factors are thought to contribute to an individual's satiety. For example, taste or taste, smell or smell, and stomach satiety may all contribute to whether an individual feels "satisfied." More specifically, "satiety" is a condition in which further eating is inhibited and determines the interval between meals and the amount of food consumed in the next meal. "Enhanced satiety" and the like mean more apparent and / or longer satiety as compared to a control situation. As used herein, the term "saturation" refers to a condition that terminates eating during a meal, and generally occurs after a period of time (eg, within 20-30 minutes) after consumption of the meal has commenced. / Indicates the observed state. Therefore, in this specification, the phrase "induces satiation" or the like means that the subject tends to stop consuming food during a meal. The effect on satiation can be determined by scoring the end point of the meal. The effect of satiation is when the amount of calories consumed at the end of a meal is, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 20% or more, significantly less than the control Is recognized. Over an extended period of time (1, 2, 3, 4, 5 weeks or more), weight loss or weight change compared to the control diet can also be scored. The subject's body weight to which a normal amount of the test composition is administered (eg, once a day, twice a day, or more) is preferably significantly controlled (decreased or increased) compared to the control body weight. Is reduced). As used herein, "control subject" refers to a subject who has not received a probiotic bacterial strain of the invention.

As used herein, the term "ClpB" has its general meaning in the art and refers to the heat shock protein F84.1, which is a member of the Hsp100 / ClpB family of hexameric AAA + -ATPases. Is also known. ClpB can be found in Staphylococcus aureus, Francisella tularensis, Listeria monocytogenes, Yersinia enterolyticum and some Yersinia enterolyticum, and other Yersinia enterolyticum bacteria. It has been described as an essential factor in the acquisition of thermotolerance of Gram-negative and Gram-positive pathogenic bacteria, as well as virulence and infectivity. In E. coli K12, the chaperone protein ClpB, also known as heat shock protein F84.1 or htpM, is a 857 amino acid protein. In general, the chaperone protein ClpB has SEQ ID NO: 1 (NCBI reference number: NP — 417083.1, available as of November 6, 2013, and / or UniProtKB / Swiss-plot number: P63284, November 6, 2013). (As long as available) comprising or consisting of the amino acid sequence of the chaperone protein ClpB from E. coli K12 having (where available). Generally, the amino acid sequence of the chaperone protein ClpB comprises or consists of an amino acid sequence that is 96-100% identical to the amino acid sequence of SEQ ID NO: 1. Preferably, the amino acid sequence of ClpB is 96, 97, 98, 99, or 100% identical to amino acid positions 540-550 of SEQ ID NO: 1 (ARWTGIPVSR). In the context of the present application, the percentage of identity is calculated using a global alignment (ie comparing two sequences over their entire length). Methods for comparing the identity of two or more sequences are well known in the art. For example, when considering the total length of two sequences, a Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48: 443-453) may be used. The needle program is, for example, ebi. ac. It is available on the uk world wide web site. The percentage of identity according to the present invention is preferably obtained by using a needle (global) program including a "Gap Open" parameter corresponding to EMBOSS: 10.0, a "Gap Extend" parameter corresponding to 0.5, and a Blosum62 matrix. Use to calculate. In the present invention, the ClpB protein mimics the α-MSH protein in inducing satiation. Thus, in some embodiments, a ClpB protein of the invention is recognized by an anti-α-MSH antibody. Generally, this antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody, eg, a polyclonal rabbit anti-α-MSH IgG (1: 1000, Peninsula Laboratories, San Carlos, Calif., USA). The amino acid sequence of α-MSH preferably comprises or consists of the amino acid sequence SYSMEHFRWGKPV (SEQ ID NO: 2) (Gen Pept Sequence ID, PRF: 223274, as available on December 2, 2013).
SEQ ID NO: 1:

  In some embodiments, a ClpB protein is administered to a subject in the form of a pharmaceutical composition. In some embodiments, a ClpB protein is combined with a pharmaceutically acceptable excipient and, optionally, a sustained release matrix, such as a biodegradable polymer, to form a pharmaceutical composition. The terms "pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce a deleterious, allergic, or other untoward reaction when administered to a mammal, particularly a human, as appropriate. Represent. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid. In the pharmaceutical compositions of the present invention, the active ingredients can be administered to animals and humans alone or in combination with other active ingredients in unit dosage form as a mixture with conventional pharmaceutical auxiliaries. Suitable unit dosage forms include oral dosage forms such as tablets, gel capsules, powders, granules, and oral suspensions or solutions, sublingual and buccal dosage forms, aerosols, implants, subcutaneous, transdermal, topical, Includes intraperitoneal, intramuscular, intravenous, subcutaneous, transdermal, intrathecal, and intranasal dosage forms, as well as rectal dosage forms. Preferably, the pharmaceutical composition contains a vehicle that is pharmaceutically acceptable for an injectable formulation. These may be in particular isotonic and sterile physiological saline solutions (such as mono- or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixtures of such salts), or Alternatively, it may be a dry, especially lyophilized composition, which can be made up by the addition of sterile water or saline to make up an injectable solution. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil or aqueous propylene glycol; and sterile solutions for extemporaneous preparation of sterile injectable solutions or dispersion. Sexual powders. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. Pharmaceutical compositions must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Solutions containing a compound of the present invention as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and can be prepared in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The active ingredient can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed by the free amino groups of proteins), formed by inorganic acids such as, for example, hydrochloric acid or sulfuric acid, or by organic acids, such as acetic acid, oxalic acid, tartaric acid, and the like. It is formed. Salts formed with free carboxyl groups can be used, for example, with inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine. It can also be derived. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersants, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars and sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptide in the required amount in the appropriate solvent with various other ingredients enumerated above and, if necessary, sterile filtering. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying which results in a powder of the active ingredient being added to the active ingredient powder in addition to the powder of the active ingredient, which is pre-filtered and sterilized. Technology and freeze-drying technology. After formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are readily administered in a variety of dosage forms, such as the types of injectable solutions described above, but drug release capsules and the like can also be used. For parenteral administration in an aqueous solution, for example, the solution should be buffered as appropriate and the liquid diluent should be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 100 ml of subcutaneous infusion fluid or injected at the proposed infusion site. Any variation in dosage will occur as necessary, depending on the condition of the subject being treated. The person responsible for administration will, in each case, determine the appropriate dose for the individual subject.

  As used herein, the expression "bacteria expressing ClpB" refers to the chaperone protein ClpB as defined above, or 96-100% identical to the amino acid sequence of SEQ ID NO: 1, more preferably SEQ ID NO: A bacterium that expresses or overexpresses a polypeptide comprising or consisting of an amino acid sequence that is 96, 97, 98, 99, or 100% identical to one amino acid sequence.

  In some embodiments, the bacterium expressing the ClpB protein is a probiotic bacterial strain.

  As used herein, the term "probiotic" is incorporated in sufficient quantities to exert a positive effect on health, comfort, and wellness beyond traditional nutritional effects. , Meaning live microorganisms. Probiotic microorganisms have been defined as "living microorganisms that, when administered in appropriate amounts, provide a host with a health benefit" (FAO / WHO 2001). As used herein, the expression "probiotic bacterial strain" refers to a bacterial strain that has a beneficial effect on the health and well-being of the host.

  In some embodiments, a probiotic bacterial strain of the invention is a live probiotic bacterial strain. The expression "living probiotic bacterial strain" refers to a microorganism that is metabolically active and can colonize the digestive tract of a subject.

  In some embodiments, a probiotic bacterial strain of the invention is a non-viable probiotic bacterial strain consisting of a mixture of bacterial fragments. In some embodiments, the mixture of bacterial fragments of the invention consists of proteins from a bacterial strain.

  In some embodiments, the probiotic bacterial strain of the invention is selected from food-grade bacteria. "Food-grade bacteria" means bacteria that are used in food products and are generally recognized as safe for use in food products.

  The bacterial strain may be a naturally occurring bacterial strain or a genetically modified bacterial strain.

  In some embodiments, a probiotic bacterial strain of the invention is a bacterium that constitutively expresses a ClpB protein.

  In some embodiments, the ClpB protein is overexpressed in bacteria. Generally, protein expression is "up-regulated" or when the protein is expressed or produced in greater amounts or yields than a given baseline yield that naturally occurs under standard conditions Are "overexpressed". Protein overexpression is used, for example, to control (a) the growth or survival conditions of the host cell, (b) the polynucleotide encoding the protein, (c) the expression of the polynucleotide in the cell and its copy number. This can be achieved by altering any one or more of the promoter, and (d) the host cell itself.

  In some embodiments, the bacterium was subjected to stress so that expression of the ClpB protein is up-regulated in the bacterium. The stress may be selected from the group consisting of exposure to heat, temperature changes, mechanical stress, or long term storage, storage at low humidity, and / or lyophilization or spray drying.

  In some embodiments, the bacteria have been fed at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times. . Generally, this nutrient is provided by a culture medium convenient for bacterial growth, such as the Mueller Hinton described in the Examples. In some embodiments, the bacteria are isolated at a stationary stage following the repetitive nutrient supply described above. This is because the concentration of ClpB protein is at a maximum during this stationary phase.

  In some embodiments, the bacterium comprises at least one point mutation such that expression of the ClpB protein is up-regulated. As used herein, the term "point mutation" refers to a substitution and / or deletion of a nucleic acid. Alternatively or simultaneously, at least one mutation is located in a regulatory DNA sequence of the ClpB gene, for example, in a transcriptional and translational control sequence, and preferably the mutation regulates protein expression. Mutations in the regulatory DNA sequences can act to up-regulate protein expression.

  In some embodiments, a probiotic bacterial strain of the invention is a bacterium that has been genetically modified to express a ClpB protein. Generally, bacterial strains have been transformed with nucleic acids encoding ClpB proteins. The term `` transformation '' refers to a host cell that expresses an introduced gene or sequence to produce a desired substance, generally a protein or enzyme encoded by the introduced gene or sequence. Refers to the introduction of a "foreign" (ie, exogenous or extracellular) gene, DNA or RNA. A host cell that receives and expresses the introduced DNA or RNA has been “transformed”. The nucleic acid may be retained extrachromosomally during transformation of the parent microorganism, or may be modified to integrate into the genome of the microorganism. Thus, nucleic acids may be integrated (eg, regions that allow for homologous recombination and targeted integration into the host genome) of extracellular chromosomal constructs (eg, origins of replication, promoters, and other regulatory sequences), or stable expression and It may include additional nucleotide sequences that have been modified to aid replication. In some embodiments, the nucleic acid is a nucleic acid construct or vector. In some embodiments, the nucleic acid construct or vector is an expression construct or vector, but other constructs and vectors, such as those used for cloning, are included in the invention. In some embodiments, the expression construct or vector is a plasmid. Generally, the expression construct / vector further comprises a promoter, as previously described herein. In some embodiments, the promoter allows for constitutive expression of the gene under its control. However, an inducible promoter may be used. The expression constructs / vectors of the present invention may include any number of regulatory elements in addition to a promoter and additional genes suitable for expression of the ClpB protein, if desired. Methods for transforming bacterial cells with extracellular nucleic acids are well known in the art.

  In some embodiments, the probiotic cell line is a Gram-negative strain.

  In some embodiments, the probiotic bacterial strain is a member of the Enterobacteriaceae family.

  In some embodiments, the probiotic bacterial strain is an E. coli strain. In some embodiments, the probiotic E. coli strain used according to the teachings of the present invention comprises a non-pathogenic E. coli strain that exerts probiotic activity. An example of a probiotic E. coli strain is Escherichia coli strain BU-230-98, ATCC Deposit No. 202226 (DSM 12799), which is a known and commercially available probiotic E. coli strain (Escherichia). coli) strain M-17. An example of a non-pathogenic bacterium is Escherichia coli Nissle 1917. An example of an E. coli strain that was not known as a probiotic is the laboratory E. coli strain K12.

  Generally, the probiotic bacterial strains of the present invention are produced using any suitable culture medium well known in the art. For example (but not limited to) various fermentation media, such as industrial media, in which the strain is first grown and used as such or after concentration (eg, drying), or after addition of another food base or food. Are suitable for the present invention. Alternatively, bacterial cells, bacterial cells containing a medium (eg, fermentation broth), or a fraction of the cells containing the medium (ie, a medium containing the bacterial strain) may be used. Cells or cells containing media include live or viable bacterial cells and / or dead or non-viable bacterial cells of the strain. Thus, this medium may be treated by, but not limited to, heating or sonication. Also, lyophilized or frozen bacteria and / or cell-free media (which may be concentrated) are included in the method of preparing a probiotic bacterial strain of the invention.

  Generally, the probiotic strains of the present invention are administered to a subject by ingestion (ie, oral route).

In some embodiments, a probiotic bacterial strain of the invention is encapsulated to protect it from the stomach. Thus, in some embodiments, the probiotic bacterial strains of the invention are formulated into a composition in an encapsulated form to significantly improve the survival time of the bacterial strain. In such cases, the presence of the capsule may delay or prevent the breakdown of microorganisms, especially in the digestive tract. It is understood that the composition of this embodiment is encapsulated in an enteric coated, sustained release capsule or tablet. The enteric coating can leave the capsule / tablet intact (ie, undissolved) as it passes through the digestive tract until the time the capsule / tablet reaches the small intestine. Methods for encapsulating live bacterial cells are well known in the art (see, eg, US Patents to General Mills Inc, such as US Patent No. 6,723,358). For example, microencapsulation with alginate and Hi-Maize ™ starch followed by lyophilization has been successful in extending the shelf life of bacterial cells in dairy products (eg, Kailasapathy et al. Curr. Issues Intest Microbiol. 2002 September; 3 (2): 39-48). Alternatively, encapsulation can be performed using glucomannan fibers, such as fibers extracted from konjac (Amorphophallas konjac). Alternatively, encapsulation of live probiotics in a sesame oil emulsion may be used (see, for example, Hou et al. J. Dairy Sci. 86: 424-428). In some embodiments, the agent for enteric coating is preferably a copolymer of methacrylic acid-alkyl acrylate, such as a Eudragit® polymer. Poly (meth) acrylates have proven to be particularly suitable as coating materials. EUDRAGIT® is the trade name for copolymers derived from esters of acrylic and methacrylic acids, the properties of which are determined by functional groups. The individual EUDRAGIT® grades differ in the properties of the neutral, alkaline or acidic groups and thus in the physicochemical properties. The successful use and combination of different EUDRAGIT® polymers provides an ideal solution for the controlled release of drugs in various pharmaceutical and technical applications. EUDRAGIT® can provide a functional film for the coating of sustained release tablets and pellets. This polymer is available from Ph. Eur. , USP / NF, DMF, and JPE. EUDRAGIT® polymers offer the following possibilities for controlled release of drugs: targeting of the gastrointestinal tract (tolerance in the stomach, release in the colon), protective coatings (masking of taste and smell, protection from moisture), And delayed release of the drug (sustained release formulation). EUDRAGIT® polymers are available in a wide range of different concentrations and physical forms, including aqueous solutions, aqueous dispersants, organic solutions, and solid materials. The pharmaceutical properties of a EUDRAGIT® polymer are determined by the chemical properties of its functional groups. Classified as follows:
Poly (meth) acrylates soluble in digestive juices (due to salt formation): EUDRAGIT® L (methacrylic acid copolymer), S (methacrylic acid copolymer), FS and E (basic) with acidic or alkaline groups The butylated methacrylic acid copolymer) polymer allows for a pH dependent release of the active ingredient. Active Ingredient Application: Controlled release of drug in all parts of the gut from simple taste masking by mere resistance to gastric juice.
Poly (meth) acrylates insoluble in digestive juices: EUDRAGIT® RL and RS (ammonio methacrylate copolymer) polymers with alkaline groups and EUDRAGIT® NE polymers with neutral groups are pH-dependent The expansion of the active ingredient allows for a controlled release of the active ingredient.

  The EUDRAGIT® enteric coating provides protection against drug release in the stomach and allows for controlled release in the intestine. The criterion governing release is the pH-dependent dissolution of the coating that occurs in certain parts of the intestine (above pH 5-7) rather than in the stomach (pH 1-5). For these applications, grades of anionic EUDRAGIT® containing carboxyl groups can be mixed with one another. This allows fine adjustment of the dissolution pH and thus the definition of the site of drug release in the intestine. EUDRAGIT® L and S grades are suitable for enteric coatings. EUDRAGIT® FS 30D (aqueous dispersant of methyl acrylate, methyl methacrylate, and methacrylic acid-based anionic copolymer) is used for controlled release, especially in the colon.

  Generally, the probiotic bacterial strains of the invention are administered to a subject in the form of a food composition. Thus, a further aspect of the invention relates to a food composition comprising an amount of a probiotic bacterial strain of the invention.

  In some embodiments, a food composition comprising a probiotic bacterial strain of the invention is selected from a complete food composition, a food supplement, a nutritional composition, and the like. The compositions of the present invention may be used as food ingredients and / or serving ingredients.

  The components of the food may be in the form of a solution or as a solid, depending on the use and / or the mode of application and / or the mode of administration.

  The probiotic bacterial strains of the invention may generally be added at any time during the production process of the composition, for example, may be added to the food substrate at the beginning of the production process, or may be added to the final food product. May be added.

  "Food" refers to a liquid (i.e., beverage), solid, or semi-solid edible composition, particularly an entire food composition (a substitute food), and does not require additional nutritional or food supplement compositions. Food supplement compositions do not completely replace nutritional intake with other means. Food compositions and food supplement compositions are, for example, fermented dairy products or dairy-based products, and are preferably administered or ingested once or more times a day. Fermented milk products can be made directly using the bacteria according to the invention in the production process, for example by adding them to a food substrate, using methods known per se. In such methods, the bacterial strains of the present invention may be used in addition to commonly used microorganisms and / or may be replaced with one or more or a portion of commonly used microorganisms. For example, in the preparation of fermented dairy products such as yogurt or yogurt-based beverages, the bacteria of the present invention may be added to or used as part of the starting culture, Alternatively, it may be appropriately added during fermentation or the like. Optionally, the bacteria may be inactivated or killed later in the production process. Fermented milk products include, but are not limited to, milk-based products such as, but not limited to, desserts, yogurt, yogurt beverages, quarks, kefir, fermented milk-based beverages, buttermilk, cheese, dressings, low fat spreads , Fresh cheese, soymilk-based beverages, ice cream and the like. Alternatively, the food composition and / or food supplement composition may be a non-dairy or non-fermented dairy product (eg, a bacterial strain or cell-free medium in non-fermented milk or another food medium). In some embodiments, a probiotic bacterial strain of the invention is encapsulated and dispersed in a food (eg, in milk) or a non-food medium. Non-fermented dairy products may include ice cream, nutrition bars and dressings and the like. Non-dairy products may include powdered beverages and nutrition bars and the like. This product uses a known method such as adding an effective amount of a bacterial strain and / or a cell-free culture medium to a food substrate, such as a skim milk or milk-based composition, and known fermentation. Then, it may be manufactured. Other food bases to which (including compositions) bacterial cells and / or cell-free culture media can be added may be meat, meat substitutes, or plant bases.

  Compositions comprising a probiotic bacterial strain of the invention may be solid, semi-solid, or liquid. The composition may be in the form of a food or food supplement, such as tablets, gels, powders, capsules, beverages, bars and the like. For example, the composition may be in the form of a powder enclosed in a sachet dissolvable in water, fruit juice, milk, or another beverage.

  As used herein, the term “food ingredient” or “supply ingredient” includes formulations that are or can be added to foods as functional foods or nutritional supplements.

  “Nutritive food” or “dietary supplement” or “functional” food means a food that contains ingredients that have a beneficial effect on health or can improve physiology.

  "Food supplement" means a food product that has the purpose of completing a normal diet. Food supplements are a concentrated source of nutrients or other substances that have nutritional or physiological effects when consumed in small quantities, alone or in combination.

  In the present invention, "functional foods" refer to recently developed foods and corresponding products, whose importance is attributed not only to their increased value with respect to nutrition and taste, but also to the substances of the particular ingredients. Represents According to the invention, the maintenance and promotion of medium or long term health is important. In this context, non-therapeutic use is preferred. The terms "nutritical" or "food shootative", and "designer food", which also describe embodiments of the present invention, are used interchangeably, but in part, as synonyms. Also used in the method. However, the preventive aspects and promotion of health, as well as the food characteristics of the product, are most clearly defined by the term functional food. In many cases, they relate to products which have been increasingly accumulated by classification and selection (which is also an example of the invention), purification, concentration and by addition. It does not include isolated active substances, especially substances in the form of tablets or pills. Although there is no legal definition of a functional food, most organizations interested in this area agree that they are foods that are sold as foods with certain health benefits beyond basic nutritional benefits. ing. Thus, functional foods are typically those that incorporate components or ingredients (such as those described herein) that provide the food with a particular function, such as medical or physiological benefits other than pure nutritional effects. Food.

  In some embodiments, the beverage is a functional or therapeutic beverage, a third-quencher, or a regular beverage. By way of example, the compositions of the present invention may be used as soft drinks, fruit juices, or beverages containing whey protein, health tea, cocoa beverages, dairy beverages, and lactic acid bacteria beverages, yogurt and drinking yoghurt, cheese, ice cream, ice cream and Desserts, confectionery, biscuit cakes and cake mixes, snack foods, nutritionally balanced foods and beverages, fruit fillings, care glazes, chocolate fillings for baking, cheesecake fillings, fruity fillings, cakes And donut icing, instant baking filling cream, filling for cookies, ready-to-use baking filling, low calorie filling, adult nutritional beverage, acidified soy / juice beverage, sterile Retort chocolate drink, bar mixes, beverage powders, calcium fortified soy / plain and chocolate milk drinks, can be used as a component for calcium-enriched coffee drinks.

  The composition further comprises an American cheese sauce, a coagulant for grated and finely ground cheese, cheese dip, cream cheese, non-fat sour cream for dry-mix whip topping, frozen / thawed whipped cream, frozen / thawed stable whipped cream, low fat And light natural cheddar cheese, low-fat Swiss yogurt, inflated frozen desserts, hard-pack ice cream, hard-pack ice cream with label-friendly and inexpensive taste foods, low-fat ice Creams: soft serve, barbecue sauce, cheese dip sauce, cottage cheese dressing, dry mixed alfredo sauce, mixed cheese sauce, dry mixed tomato sauce, and more It can be used as a food ingredient.

  In some embodiments, compositions comprising the probiotic bacterial strains of the present invention are used with yogurt products such as fermented yogurt beverages, yogurt, drinking yogurt, cheese, fermented cream, milk-based desserts, and others. Optionally, the composition can be further used as a component in one or more of a cheese application, a meat application, or an application involving a protective culture.

  In some embodiments, a food composition comprising a probiotic bacterial strain of the invention is suitable for preparing a food replacement product. As used herein, the term "food replacement product" includes, unless otherwise specified, any nutritional product, including proteins, carbohydrates, lipids, vitamins, and minerals, and combinations thereof. Suitable as the sole or main nutritional source for meals. Generally, food replacement products include at least one carbohydrate source, at least one lipid source, and / or at least one protein source. Sources of proteins include, for example, animal proteins (such as milk, meat, and egg proteins); vegetable proteins (such as soy, wheat, rice, and pea proteins); mixtures of free amino acids; Any suitable dietary protein may be used, such as a combination of Milk and soy proteins such as casein and whey are particularly preferred. The protein may be intact or hydrolyzed, or may be a mixture of intact and hydrolyzed protein. For example, for animals believed to be at risk for developing milk allergy, it would be desirable to provide partially hydrolyzed proteins (2-20% degree of hydrolysis). If a hydrolyzed protein is required, the hydrolysis step may be performed as desired and as known in the art. For example, a whey protein hydrolyzate can be prepared by enzymatically hydrolyzing the whey fraction in one or more steps. If the whey fraction used as the starting material is substantially free of lactose, the protein is found to have less lysine loss during the hydrolysis step. This can reduce the degree of lysine loss from about 15% by weight of total lysine to less than about 10% by weight of lysine, for example, about 7% by weight loss of lysine reduces the nutritional quality of the protein source. Greatly improve. If the composition comprises a fat source, the fat source preferably provides 5% to 40% of the energy of the composition, for example 20% to 30% of the energy. A suitable fat profile can be obtained using a mixture of canola oil, corn oil, high oleic sunflower oil. The carbohydrate source preferably provides between 40% and 80% of the energy of the composition. Any suitable carbohydrate can be used, such as, for example, sucrose, lactose, glucose, fructose, corn syrup solids, maltodextrin, and mixtures thereof. In general, replacing daily diets with energy-restricted diets during meal replacement contributes to maintaining weight after weight loss.

  Food compositions comprising the probiotic bacterial strains of the invention generally include a carrier or vehicle. "Carrier" or "vehicle" means a substance suitable for administration and is non-toxic and harmful to any component of the composition, e.g., any liquid, gel, solvent, liquid diluent, solubilizer, and the like. And any substance known in the art that does not interact with. Examples of nutritionally acceptable carriers include, but are not limited to, water, saline, alcohol, silicone, wax, petrolatum, vegetable oil, polyethylene glycol, propylene glycol, liposomes, sugar, gelatin, lactose, amylose, Examples include magnesium stearate, talc, surfactant, silicic acid, viscous paraffin, balm, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxylmethylcellulose, polyvinylpyrrolidone, and the like.

  In some embodiments, a food composition comprising a probiotic bacterial strain of the invention comprises an amount of dietary fiber. Dietary fiber passes through the small intestine, is not digested by enzymes, and functions as a natural bulking and laxative. Dietary fiber can be insoluble or soluble, so a mixture of the two is generally preferred. Suitable sources of dietary fiber include soybeans, peas, oats, pectins, guar gum, gum arabic, fructooligosaccharides, galactooligosaccharides, sialyl-lactose, and oligosaccharides from animal milk. In some embodiments, the dietary fiber is selected from mannans. Mannans such as guar gum, locust bean gum, konjac, and xanthan gum (such as glucomannan and galactomannan) are present in some plant cell walls. Glucomannan is generally composed of (1-4) -β linked glucose and mannose units, and galactomannan is generally replaced by (1-6) -α-galactose 1 unit. (1-4) -β-mannan skeleton. The endosperm of many legumes, such as guar and locust beans, contains galactomannan in the endosperm during seed development. Glucomannan has also been found as a minor component of grain.

  In some embodiments, food compositions comprising the probiotic bacterial strains of the present invention comprise trace elements and minerals and trace elements such as vitamins, according to recommendations of government agencies such as the USRDA. For example, the composition may contain, in a given range, one or more of the following micronutrients per daily dose: 300-500 mg calcium, 50-100 mg magnesium, 150-250 mg phosphorus, 5-20 mg iron, 1-7 mg zinc, 0.1-0.3 mg copper, 50-200 μg iodine, 5-15 μg selenium, 1000-3000 μg β-carotene, 10-80 mg vitamin C, 1-80 mg 2 mg vitamin B1, 0.5-1.5 mg vitamin B6, 0.5-2 mg vitamin B2, 5-18 mg niacin, 0.5-2.0 μg vitamin B12, 100-800 μg folic acid, 30- 70 μg biotin, 1-5 μg vitamin D, 3-10 μg vitamin E.

  In some embodiments, a composition comprising a probiotic bacterial strain of the invention comprises an emulsifier. Examples of food-grade emulsifiers generally include diacetyltartaric acid esters of mono- and diglycerides, lecithin, and mono- and diglycerides. Similarly, suitable salts and stabilizers may be included.

  In some embodiments, a food composition comprising a probiotic bacterial strain of the invention comprises at least one prebiotic. “Prebiotic” is a food substance intended to promote the growth of a probiotic bacterial strain of the invention in the intestine. The prebiotic may be selected from the group consisting of oligosaccharides and optionally comprises fructose, galactose, mannose, soybean, and / or inulin; and / or dietary fiber.

  In some embodiments, a composition comprising a probiotic bacterial strain of the invention comprises a protective hydrocolloid (gum, protein, modified starch, etc.), a binder, a film former, an encapsulant / material, a wall. / Shell material, matrix compound, coating agent, emulsifier, surfactant, solubilizer (oil, fat, wax, lecithin, etc.), adsorbent, carrier, filler, co-compound, dispersant, wetting agent, processing aid Agents (solvents), glidants, taste masking agents, bulking agents, jelling agents, gel formers, antioxidants, and antimicrobial agents. The composition also includes, but is not limited to, water, gelatin of any origin, vegetable gum, lignin sulfonate, talc, sugar, starch, gum arabic, vegetable oil, polyalkylene glycol, flavoring agents, preservatives Pharmaceutical additives and adjuvants, including stabilizers, emulsifiers, buffers, lubricants, colorants, wetting agents, excipients, and the like, may also be included. In all cases, such additional ingredients are selected for their suitability for the intended recipient.

  In some embodiments, administration of the ClpB protein (or a probiotic bacterial strain that expresses the protein) is interrupted for one or more periods as appropriate, eg, for a period of one day or more, Is repeated 2-3 times a day for an extended period of at least 4 days, even for 4-15 weeks. In some embodiments, the ClpB protein is administered concomitantly or sequentially with the subject's diet. In some embodiments, the ClpB protein is administered prior to a meal in the subject.

As used herein, the term “effective amount” refers to a beneficial effect (eg, stimulating satiety, prolonging satiety, reducing food intake, controlling weight gain, and / or stimulating weight loss). Represents a sufficient amount of ClpB protein to achieve. In the context of the present invention, the amount of ClpB protein administered to a subject will depend on individual characteristics, such as general health, age, sex, weight, and the like. One skilled in the art can appropriately determine the dose depending on these and other factors. For example, when the ClpB protein is administered to a subject in a probiotic form, the strains of the present invention are capable of producing enough colonies to produce a beneficial effect in the subject. When the probiotic bacterial strain is administered in the form of a food product, it may generally contain from 10 ³ to 10 ¹² cfu of the probiotic bacterial strain of the invention per gram dry weight of the food composition.

  The present invention is further described by the following figures and examples. However, these examples and figures should not be construed as limiting the scope of the invention in any way.

Body weight analyzed at the start and at the end of 3 weeks of intragastric delivery of E. coli K12 wild-type (WT) or E. coli K12 lacking the ClpB gene (ΔClpB) by gavage in normal adult C57B16 mice, Effects on 24-hour food intake and dietary patterns. Control mice (Ctr) received no oral gavage. The meal pattern was measured as the amount of meal corresponding to the average amount of food consumed during one meal and the frequency of meals corresponding to the number of meals per 24 hours separated by at least 5 minutes. A decrease in the amount of meal reflects faster satiation, and a decrease in the frequency of meal reflects a longer satiety period. Two-way repeated measures ANOVA, Bonferroni post-hoc test, * p <0.05. B, D, G, H, ANOVA, Tukey's post-test, * p <0.05, ** p <0.01, *** p <0.001. E. FIG. Student's t-test, * p <0.05.

Example 1
Materials and Methods Growth of E. coli in vitro after routine feeding E. coli K12 bacteria were placed in 50 ml Falcon vials, 30% broth, 1.75% casein hydrolyzate, and 0.15% The cells were cultured at 37 ° C in 40 ml of MH medium containing starch at 25 ° C and pH 7.3 (Becton, Dickinson, MD). Bacteria were given fresh MH medium every 12 hours for 5 consecutive days to model the planned twice daily diet in humans. Bacterial growth was measured as OD at λ = 600 nm by a spectrophotometer every 2 hours after the first supply of MH medium, every hour after the third supply, and every 10 minutes after the fifth supply. At the end of each 12 hour cycle, the bacteria were centrifuged at 6,000 rpm for 5 minutes at room temperature (RT). The supernatant was discarded and replaced with an equivalent (〜40 ml) of fresh MH medium. After the last supplementation of MH medium, bacteria were sampled for protein extraction in the log phase and then in the stationary phase.

Protein extraction E. coli K12 bacteria were centrifuged at 4,000 g for 30 minutes at 4 ° C. The bacterial residue was dissolved in 2 ml of Trishydroxymethylaminomethane (TRIS) buffer (pH 7.4) and homogenized by sonication at room temperature for 3 minutes. To separate proteins from unlysed cell fragments, bacterial homogenates were centrifuged at 10,000 g for 30 minutes at 4 ° C. The supernatant was collected and then ultracentrifuged at 60,000 g for 45 minutes at 4 ° C. to further separate the protein into cytoplasmic (supernatant) and membrane (residue) fractions. The membrane protein was dissolved in TRIS buffer (pH 7.4). Protein concentration was measured using a 2-D Quant Kit (GE Healthcare, Piscataway, NJ).

Two-dimensional polyacrylamide gel electrophoresis For 2D-PAGE, rehydrate a fixed pH gradient (IPG) strip (pH 4-7; 18 cm; BIO-RAD, Hercules, CA) using 300 μg of E. coli protein extract. did. The proteins were then separated in the first dimension by isoelectric focusing at a total of 85,000 V-h by using the IPGphor isoelectric focusing system (GE Healthcare). After this, the IPG strip was washed with an equilibration buffer (urea 6 mol / L, glycerol 30% (volume: volume), 2% (weight: volume) sodium dodecyl sulfate (SDS), Tris-HCl 50 mmol / L (pH 8.8), Incubate in 0.25% (weight: volume) bromophenol blue containing 2% (weight: volume) dithiothreitol for 15 minutes, then equilibrate with 4% (weight: volume) iodoacetamide Alkylation in buffer for 15 minutes. The IPG strips were then immobilized on a 10% polyacrylamide gradient gel (20 cm 18 cm 1 mm) for SDS-PAGE. The second dimension was performed overnight at 25 ° C., 12 mA / gel on an Etan Daltsix vertical electrophoresis system (GE Healthcare). After SDS-PAGE, the 2D gel was fixed in 2% (vol: vol) orthophosphoric acid and 50% (vol: vol) methanol at room temperature for 2 hours. The gel is then rinsed with water and the protein spots stained with CBB G-250 (BIO-RAD) [34% (vol: vol) methanol, 17% (weight: vol) ammonium sulfate, 2% (vol: vol) And 0.66 g of CBB G-250 / L].

Analysis of Different Protein Expression Images of stained 2D gels were scanned with ImageScanner II (GE Healthcare) calibrated with gray scale markers (Kodak, Rochester, NY) and digitized with Labscan 6.0 software (GE Healthcare). . Analysis of different protein expression, including spot detection, quantification, matching, and comparative analysis was performed using ImageMaster 2D Platinum 5.0 software (GE Healthcare). Samples of each protein were subjected to at least 3 (membrane protein) and 4 (cytoplasmic protein), 2D-PAGE and 3 (or 4) gels to minimize inter-experiment variability. The sets were compared using ImageMaster to ensure that no statistically different spots appeared in the set of gels. The most representative gel of each set (gel migration, spot definition, and spot number) was used to compare log phase and stationary phase E. coli proteins. The expression level was determined by the relative amount of each spot in the gel, expressed as% volume, and calculated as the spot volume / sum of all spots separated in the gel. This normalized spot volume accounts for variations due to protein loading and staining by considering the total volume across all spots present on the gel. The variation in volume was calculated as the ratio of the mean% volume for the group of spots between the two periods. Only spots with a volume variation of more than 1.5 were considered valid. The absence of spots in the gel indicated that no detectable expression could be recorded for the protein under the chosen experimental conditions. The corresponding p-value was determined by the Student's t-test (significance level p <0.05) after log transformation of the spot volume.

Identification of Proteins by Liquid Chromatography-Electrospray Ionization MS / MS Protein spots of interest were excised from 2D gels stained with CBB G-250 using Ettan Spot Picker (GE Healthcare) and automated. Digestion of the proteins in the gel was performed with the Ettan Digester (GE Healthcare) described above (Goichon et al., 2011). The protein extract is then resuspended in 10 μl of 5% (vol: vol) acetonitrile / 0.1% (vol: vol) formic acid, 6340 Ion Trap mass with nanospray source and HPLC chip cube interface Analysis was performed using a nano-LC1200 system connected to an analyzer (Agilent Technologies, Courtaboeuf, France). Briefly, peptides were concentrated, desalted on a 40 nL RP-C18 trap column, and Zorbax (30-nm pore size, 5-μm particle size) C18 column (43 mm length × 75 μm inner diameter, Agilent Technologies) ). The eluent was analyzed on an ion trap mass spectrometer using a 9 minute linear gradient (0.1% formic acid with 3% to 80% acetonitrile) at a flow rate of 400 nl / min.

  For protein identification, a list of MS / MS peaks was extracted and compared to the protein database by using the MASCOT Daemon version 2.2.2 (Matrix Science) search engine. The search was performed using the following specific parameters: enzyme specificity, trypsin: percentage of remaining peptide 1 allowed; no fixed modification; variable modification, oxidation of methionine, carbamide methylation of cysteine, serine, tyrosine, and Phosphorylation of threonine; monoisotopic; charge of peptide, 2+ and 3+; mass tolerance for precursor ion, 1.5 Da; mass tolerance for fragment ion, 0.6 Da; ESI-TRAP as instrument; Using the database of the National Center for Bioinformatics (NCBI) [NCBInr 201220531 (18280215 sequences, 6265275233 residues)] (Bethesda, MD). The following criteria: at least two top ranked peptides (bold and red) each containing a MASCOT score greater than 54 (p <0.01), or at least two top each containing a MASCOT score greater than 47 A protein hit was automatically verified if it met one of the peptides (bold and red) ranked (p <0.05). To assess the false positive rate, all initial database searches were performed using the MASCOT "decoy" option. If the false positive rate did not exceed 1%, the results were considered appropriate.

ATP Assay ATP production in vitro was measured using an ATP colorimetric / fluorometric assay kit (BioVision, CA) according to the manufacturer's instructions. Briefly, log phase or stationary phase bacterial proteins were added in duplicate wells for each concentration (1, 10, and 25 μg / ml in ATP assay buffer) to 50 μl / well in ATP assay buffer. Adjusted. Next, 10 μl of different nutrient solutions, 15% sucrose or MH medium were added to the corresponding wells and adjusted to 50 μl / well with ATP assay buffer; only 50 μl / well ATP buffer was added to control wells. The plate was incubated at 37 ° C. for 2 hours. After incubation, 50 μl of ATP reaction mixture (containing a mixture of ATP assay buffer, ATP probe, ATP converter and developing agent) was added to each well. After a 30 minute incubation at room temperature protected from light, the OD was measured at 570 nm and protected from sunlight.

Development and Validation of ClpB Immunoassay The design of the ClpB detection assay was based on several criteria, such as detection of specific and sensitivity in the linear concentration range. A particular condition was the detection of ClpB without α-MSH cross-reactivity, which can occur due to the presence of an α-MSH-like epitope on the ClpB molecule. To simplify this technique and signal detection, we used the option of reading the OD with a standard 96-well ELISA plate and spectrophotometer. Detailed protocols for ClpB ELISA and Western Blot (WB) are presented in a separate paragraph.

  To prevent binding of the exposed antibody (Ab) to the captured Ab, we created a ClpB capture antibody (Capture Ab) and a ClpB detection antibody (Detection Ab) in different species, rabbits and mice, respectively. To most efficiently capture ClpB protein from complex biological samples, we coated ELISA plates with rabbit polyclonal antibodies having multiple anti-ClpB epitopes. To avoid cross-reactivity between ClpB and α-MSH, we selected a high sensitivity that recognizes ClpB but not α-MSH, pre-selected by ELISA screening of some Ab clones. A mouse monoclonal anti-ClpBAb characterized by and specificity was used as the detection Ab. Alkaline phosphatase conjugated anti-mouse exposed antibody was used as a general ELISA tool to obtain a chromogenic enzyme reaction readable as OD proportional to analyte concentration. Linear changes in OD resulting from the detection of seven serial dilutions of the recombinant E. coli ClpB protein ranging from 2 pM to 150 were obtained without reaching a plateau and without saturation of the OD signal.

  To verify the specificity of the developed ClpB assay, we measured the concentration of ClpB in protein samples extracted from 10 different cultures of E. coli K12 WT and a ΔClpB mutant E. coli culture. The ΔClpB mutant and the corresponding wild-type (WT) strain are described in Dr. It was kindly provided by Axel Mogk (ZMBH, Heidelberg University, Germany). In addition, we analyzed the presence of ClpB in these bacterial protein samples by WB using an anti-ClpB polyclonal rabbit antibody and compared the signal intensity values between the WB band and the concentration in the ELISA. ClpB was detected in all wild-type E. coli cultures, where seven cultures had concentrations of ClpB greater than 1000 pM, but ClpB could not be detected in protein samples extracted from ΔClpB E. coli. Was. WB revealed a major band of the expected 96 kDa size in the WT, but not in ΔClpB E. coli. The OD intensity of these bands varied between individual samples and showed a positive correlation with the concentration of ClpB measured by ELISA in samples of the same E. coli culture. Thus, the absence of ClpB in the protein regulation of ΔClpB E. coli confirms the specificity of the assay, and a good match between ELISA and WB indicates a cross-validation of both ClpB immunodetection techniques. Offered.

  To verify that the ClpB plasma assay detects ClpB from intestinal bacteria, we fed WT or ΔClpB E. coli by gavage for 3 weeks using a ClpB ELISA. ClpB in the plasma of the mouse was measured. Plasma samples were obtained from a test previously published by the inventors (Tennoune et al., 2014). We have found that ClpB is normally present in the plasma of mice, including both controls and mice that have been gavaged with culture broth without bacteria. Importantly, plasma levels of ClpB were increased in mice given WT E. coli, but remained unchanged in mice given ClpB-deficient E. coli, with ClpB in plasma originating from intestinal bacteria. It was confirmed.

ClpB ELISA
Rabbit polyclonal anti-E. Coli ClpB antibody (custom generated by Delphi Genetics, Gosselies, Belgium) was used at 4 ° C. on a 96-well Maxisorp plate using a 100 μl concentration of 2 μg / ml in 100 mM NaHCO 3 buffer (pH 9.6). Coated for 12 hours. The plate was washed (5 min × 3 times) with phosphate buffered saline (PBS) (pH 7.4) containing 0.05% Tween 20. Serially dilute recombinant E. coli ClpB protein (custom generated by Delphi Genetics) to 5, 10, 25, 50, 70, 100, and 150 pM in sample buffer (PBS, 0.02% sodium azide, pH 7.4). , Two samples were added to each well to prepare a standard substance. Sample samples included colonic mucosa and plasma samples from mice and rats, or proteins extracted from E. coli K12 cultures. Sample samples were added to the remaining wells, two per sample, and incubated at room temperature for 2 hours. The plate was washed (5 min × 3 times) with phosphate buffered saline (PBS) (pH 7.4) containing 0.05% Tween 20. Mouse monoclonal anti-E. Coli ClpB antibody (1: 500 in sample buffer, custom generated by Delphi Genetics) was added to the wells and incubated for 90 minutes at room temperature. The plate was washed with PBS containing 0.05% Tween 20 (pH 7.4) (5 minutes × 3 times). Jackson ImmunoResearch Laboratories, Inc. Alkaline phosphatase-conjugated goat anti-mouse IgG (1: 2000 in sample buffer) from (West Grove, PA) was added to the wells and incubated at room temperature for 90 minutes. Plates are washed (5 min × 3 times) with PBS (pH 7.4) containing 0.05% Tween 20 and then 100 μl of p-nitrophenyl phosphate solution (Sigma, St. Louis, Mo.) Was added as a substrate for alkaline phosphatase. After incubation for 40 minutes at room temperature, the reaction was stopped by adding 50 μl of 3N NaOH. The OD was determined at 405 nm using a microplate reader Metertech 960 (Metertech Inc., Taipei, Taiwan). Blank OD values resulting from plate readings without the addition of plasma samples or standard dilutions of ClpB protein were subtracted from the sample OD values.

C. Western blot of ClpB. Western blots were performed using proteins extracted from E. coli K12. Protein samples (10 μg) were separated on a 20% acrylamide SDS gel in Tris-glycine buffer, transferred to a nitrocellulose membrane (GE Healthcare, Orsay, France) and TBS (10 mmol / L Tris, pH 8; 150 mmol) / L NaCl) + 5% (w / v) skim milk in 0.05% (w / v) Tween 20 for at least 1 hour at room temperature. The membrane was then incubated with rabbit polyclonal anti-E. Coli ClpB antibody (1: 2000, Delphi Genetics) at 4 ° C. overnight. After washing three times in a blocking solution of 5% (w / v) skim milk powder in TBS / 0.05% Tween 20, the membrane was washed with a peroxidase-conjugated anti-rabbit IgG (1: 3000, SantaCruz Biotechnology) for 1 hour. Incubated. After washing three times, the peroxidase reaction was developed using an ECL detection kit (GE Healthcare). Protein bands are compared to molecular weight standards (Precision Plus, BioRad), films are scanned using ImageScanner III (GE Healthcare), and bands are scanned using ImageQuant TL software 7.0 (GE Healthcare). The pixel density was analyzed.

Intestinal administration of E. coli proteins in rats Animals Animal husbandry and experiments were in accordance with guidelines established by the National Institutes of Health in the United States and were in compliance with both French and European regulations (Official Journal of the European Community). L 358, 18/12/1986). Female Sprague-Dawley rats (Janvier, Genest-Saint-Isle, France) weighing 200-250 g under controlled environmental conditions (22 ± 1 ° C., 12 hour light cycle with 7:30 am lighting). In a fully equipped animal facility, the animals were housed in breeding cages (3 rats per cage) for one week to acclimate the rats to the housing conditions. Standard pelleted rodent chow (RM1 diet, SDS, UK) and drinking water were provided ad libitum.

Experiment # 1
This experiment was designed to evaluate the relevance of our in vitro E. coli growth model to the in vivo context of bacterial growth in the gut. Rats were anesthetized with ketamine (75 mg / kg, Virbac, Carros, France) / xylazine (5 mg / kg, Bayer, Leverkusen, France) solution (3: 1 vol., 0.1 mL / 100 g body weight IP). After laparotomy, the colon was prepared by placing two ligatures: one: the caecocolonic junction and the second: 4 cm below. Injection into the colon and sampling of the contents of the lumen was performed using a polypropylene catheter inserted into the ascending colon and fixed with a first ligation. 2 ml of MH medium or water was slowly injected into the colon and immediately thereafter collected for OD measurement. After the OD measurement, all of the colon content samples were returned to the colon. Such sampling of colon contents without fresh MH medium or water was repeated every 5 minutes for the first 20 minutes, then at 30 and 60 minutes. Bacterial density was measured by spectrophotometer as OD at λ = 600 nm. Blood samples were taken from the portal vein before the first injection, 30 minutes and 60 minutes after the injection. Fecal samples were taken from the colon at the end of the experiment for ClpB DNA extraction and PCR.

Real-Time Quantitative Polymerase Chain Reaction Quantitative PCR (qPCR) was performed using a CFX 96 q-PCR instrument (BioRad, CA) to analyze the density of bacteria expressing ClpB DNA. Total DNA was extracted from rat feces using the QAMP DNA tool mini kit (QIAGEN Venlo, Netherlands). The qPCR mixture contained 5 μl of SYBR Green Master (QIAgen, West Sussex, UK), 0.5 μM each of forward and reverse primers, DNA from the sample, and water to bring the total volume to 10 μl. Primers were purchased from Invitrogen (Cergy-Pontoise, France). The three-step PCR was performed for 40 cycles. The sample was denatured at 95 ° C for 10 minutes, annealed at 60 ° C for 2 minutes, and extended at 95 ° C for 15 seconds.

Experiment # 2
This experiment aimed to evaluate the effect of E. coli proteins on the release of intestinal peptides (GLP-1 and PYY) into the systemic circulation. Rats are anesthetized and colons are prepared as described above and colonic injections of E. coli proteins (0.1 μg / kg protein in 2 ml PBS) extracted during log (n = 6) or stationary (n = 6) phases are made. , Once every 20 minutes. Blood samples were taken from the portal vein before and after a 20-minute colonic infusion for GLP-1, PYY and ClpB assays. A sample of colonic mucosa was taken at the end of the ClpB assay experiment. GLP-1 and PYY assays were performed using a fluorescent enzyme immunoassay kit (Phoenix Pharmaceutical Inc., CA) according to the manufacturer's instructions. Fluorescence was measured at 325 nm for excitation and 420 nm for emission using a microplate reader, Chameleon (HIDEX Inc., Turku, Finland).

Administration of Escherichia coli protein, food intake, and brain c-fos test in rats Animals Male Wistar rats weighing 200-250 g (Janvier, Genest-Saint-Isle, France) were acclimated to the accommodation conditions described above. Feeded like. Three days prior to the experiment, rats were transferred to individual metabolic cages (Tecniplast, Lyon France), where they were given the same RM1 diet, but freely in powdered form (SDS). Drinking water was always provided ad libitum. The rats were gently handled for a few minutes each day during the acclimatization period in order to familiarize them with the treatment. At the end of acclimation, rats were divided into three groups with similar average body weight and used for experiments 1-3. Two experiments involving food restriction were performed on the same rats, separated by four days. A third experiment in freely-fed rats involved a new species.

Experiment 1
The first experiment aimed to compare the effects of E. coli membrane proteins extracted in log phase and stationary phase. Rats were fasted overnight (18.00-10.00 hours) with free access to water. After fasting, E. coli proteins were injected at 10.00 hours with I.D. Immediately after the P injection, the rats were returned to metabolic cages with pre-weighed amounts of food. Food intake was measured at 1 hour, 2 hours, and 4 hours. The first group of rats (n = 6) received 0.1 mg / kg of membrane protein extracted from log phase E. coli in 300 μl of PBS and the second group of rats (n = 6) received stationary phase Was administered with 0.1 mg / kg of membrane protein extracted from E. coli, and the control group (n = 6) was administered with 300 μl of PBS.

Experiment # 2
The second experiment aimed to compare the effects of E. coli cytoplasmic proteins extracted in log phase and stationary phase. An experimental protocol similar to Experiment # 1 was used.

Experiment # 3
This experiment was designed to evaluate the effect of total E. coli protein on food intake in freely-fed rats. E. coli proteins (0.1 mg / kg protein in 300 μl of PBS, IP) extracted in log phase (n = 6) or stationary phase (n = 6) or PBS alone as control (n = 6) Was injected at 19.30 hours and the animals were returned to metabolic cages with pre-weighed amounts of food. Cumulative food intake was measured after 2 hours. Immediately thereafter, rats were anesthetized with sodium pentobarbital (0.2 mg / kg, IP) and perfused for immunohistochemical examination of c-fos expression in the brain.

Tissue preparation and immunohistochemistry. Brains were fixed by perfusion / immersion with 4% paraformaldehyde, frozen and cut (14 μm) on a cryostat (Leica Microsystems, Nanterre, France) and then tyramide signal Processed for immunohistochemistry using amplification (TSA) + fluorescein kit (NEN, Boston, MA). For single staining, a rabbit polyclonal antiserum to cfos (1: 4,000, Ab-5, Calbiochem, Merck KGaA, Darmstadt, Germany) was used. In the case of double staining, after TSA, a rabbit monoclonal antibody against β-endorphin (β-end) developed by anti-rabbit cyanine-3 antibody 1: 200 (Jackson ImmunoResearch, West Grove, PA) (Life Technologies, Frederick, MD) or a mouse monoclonal IgG (Santa Cruz Biotechnology, inc., TX) against calcitonin gene-related peptide (CGRP) developed by anti-mouse rhodamine red conjugate antibody 1: 200 (Jackson ImmunoResearch). The direct immunofluorescence technique was applied using any of the 1,000-fold dilutions. In the hypothalamic arcuate and hypothalamic ventromedial nuclei and the central amygdala, positive cells were counted at 20 × magnification from six consecutive sections. The average number of positive cells per rat was used to calculate the group mean. Digital images were optimized for brightness and contrast in Adobe Photoshop 6.0 software (Adobe Systems, San Jose, CA).

Long-Term Administration of E. coli Protein in Mice Two-month-old male C57B16 mice (n = 32) were purchased from Janvier Labs and acclimated to the animal facility for one week with a 12-hour lighting cycle at 8:00. The mice were then individually housed in BioDAQ mouse cages (Research Diets, Inc., New Brunswick, NJ) each equipped with an automatic feeding monitor. After acclimation to BioDAQ cages for 3 days, mice were divided into three groups (n = 8), each containing (i) PBS, (ii) bacterial proteins extracted at log phase (0.1 mg / kg body weight), (Iii) Bacterial protein (0.1 mg / kg body weight) extracted in the stationary phase was administered twice daily at 9:00 and 18:30. P. Different treatments consisted of injections. Food (SERLAB, Montaire, France) and drinking water were provided ad libitum and body weights were measured daily. Feeding data was monitored continuously for one week and analyzed using BioDAQ data viewer 2.3.07 (Research Diets). The meal interval was set to 300 seconds for the analysis of the meal pattern. Satiety ratio was calculated as the time (s) of the postprandial interval divided by the amount of food consumed in the previous meal (g). After the experiment, the mice were sacrificed by decapitation, the brain was excised, and the hypothalamus was probed for a microarray of neuropeptide mRNA.

Microarray of Hypothalamic Neuropeptide mRNA Total RNA was analyzed using the NucleoSpin® RNA II kit (Macherey-Nagel, Duren, Germany) according to the manufacturer's protocol, for thalamic administration of long-term administration of E. coli proteins. Extracted from the bottom. The digested RNA was reverse transcribed using an ImProm-II ™ Reverse Transcription System kit (Promega, Madison, Wis.) At 42 ° C. for 60 minutes. The obtained cDNA was used for a real-time PCR reaction. A panel of nine primer pairs was designed using Primer express software (Life Technologies, Saint-Aubin, France) and verified for efficacy and specificity. A PCR reaction consisting of 6 μl of cDNA and 6 μl of Fast SYBR Green Master Mix (Life Technologies) containing the specific reverse and forward primers at a concentration of 100 nM, was applied to a Bravo liquid handling system (Agilent 96 well) using a Bravo liquid handling system. Partitioned and amplified with QuantStudio 12K Flex (Life Technologies). The cDNA production signal for the target gene was internally corrected by a reference gene signal for variations in the amount of input mRNA. The gene expression levels were then compared to a group of control samples and modulation was determined using the 2- ^ΔΔCq method.

Electrophysiological recordings. Brain sections (250 μm) were prepared as previously described (Fioramonti et al., 2007) in adult POMC-eGFP mice (5-7 weeks old, Ref: C57BL / 6J-Tg (Pomc). -EGFP) 1 Low / J, prepared from The Jackson Laboratory. _{Sections, 118mM NaCl, 3mM KCl, 1mM} MgCl 2, 25mM NaHCO 3, 1.2mM NaH 2 PO 4, 1.5mM CaCl 2, 5mM Hepes, molar was adjusted to 310 mOsM by 2.5 mM D-glucose (sucrose penetration Incubation was carried out in oxygenated extracellular medium containing a pressure concentration of pH 7.4) at room temperature for a recovery period of at least 60 minutes. After being placed in the recording chamber, the sections were refluxed at 2-3 ml / min in the same extracellular medium. Sections were viewed using a Nikon microscope EF600 with fluorescence (fluorescein filter) (Nikon France, Champigny sur Marne) and an IR-DIC video microscope. Living ARC POMC neurons were visualized using a fluorescent video camera (Nikon) using a 40 × water immersion objective (Nikon). A borosilicate pipette (4-6 MΩ; 1.5 mm OD, Sutter Instruments, Novato, CA) was filled with the filtered extracellular medium. Cell attach recordings were performed using a Multiclamp 700B amplifier, digitized using a Digidata 1440A interface, and obtained at 3 kHz using pClamp 10.3 software (Axon Instruments, Molecular Devices, Sunnyvale, CA). Pipette and cell volumes were completely offset. After establishing a stable baseline, 1 nM ClpB (Delphi Genetics) was perfused for 5-10 minutes. The firing rate of POMC neurons was measured over the last 3 minutes of perfusion of ClpB, 7-10 minutes after perfusion, and compared to the firing rate measured 3 minutes before perfusion.

Statistical Analysis Data was analyzed using GraphPad Prism 5.02 (GraphPad Software Inc., San Diego, CA) and plotted graphs. Normality was assessed by the Kolmogorov-Smirnov test. Group differences were analyzed according to normality results by analysis of variance (ANOVA) or non-parametric Kruskal-Wallis (K-W) test with Tukey or Dan post-hoc test. Where appropriate, individual groups were compared using the Student's t-test and Pearson correlation or Mann-Whitney (MW) test according to the normality results. The effect of successive experiments was analyzed using repeated measures (RM) ANOVA and Bonferroni's post test. Data are presented as mean ± standard error of the mean (sem), and for all tests, p <0.05 was considered statistically significant.

Results Growth of Escherichia coli after regular feedings After the first supply of Mueller-Hinton (MH) nutrient medium to E. coli cultures, three E. coli growth phases: 1) 2 hour induction phase; 2) 4 hours A logarithmic growth phase was observed, and 3) a stationary phase that maintained an optical density (OD) of 0.35 stably for 6 hours. After the third and fifth supply of MH medium, only two growth phases were found: log phase and stationary phase, the first growth phase started immediately after feeding. Each new feeding cycle was characterized by a shorter duration logarithmic growth phase, 2 hours after the third feeding and 20 minutes after the fifth feeding. After the fifth feeding, bacterial proteins were extracted, separated into membrane and cytoplasmic fractions, and used for proteomic analysis and in vivo experiments in fasted rats. In a new experiment, Escherichia coli K12 was fed 9 times in order to verify whether continuous regular feeding could further enhance the kinetics of bacterial growth. We found that after the 7th and 9th feeding, the logarithmic growth phase did not change anymore and lasted 20 minutes with the same relative increase in OD (Δ0.3). This reflected that bacterial growth after each nutrient supply was identical. With the McFarland turbidity standard, an increase in OD of 0.3 corresponds to an increment of 10 ⁸ -10 ⁹ bacteria. After the ninth nutrient feed, bacterial proteins were extracted in the logarithmic and stationary phases and showed total protein concentrations of 0.088 mg / ml and 0.15 mg / ml, respectively. The extracted proteins were tested for ClpB levels and tested in ATP production assays and in vivo experiments involving intracolonic injection and systemic injection in freely-fed mice and rats, followed by detection of c-fos in the brain. used.

Proteomic analysis To analyze whether the expression profile of proteins fluctuates with the growth phase of bacteria induced by nutrients, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was performed corresponding to the log and stationary phases. The membrane and cytoplasmic fractions of Escherichia coli K12 extracted 10 minutes and 2.3 hours after the fifth addition of the MH medium were individually performed. The total number of protein spots detected was 2895 (1367 in the membrane and 1528 in the cytoplasm).

  Comparison of 2D-PAGE of membrane proteins during log and stationary phases revealed 20 proteins that were differentially (at least 1.5-fold) expressed. Of these, 17 proteins showed increased expression during the log phase, and 15 were identified by mass spectrometry. Comparison of 2D-PAGE of cytoplasmic proteins indicated 20 proteins that were differentially (at least 1.5-fold) expressed. In contrast to the membrane proteins, the majority (19) of the cytoplasmic proteins showed increased expression in the stationary phase. Only one protein spot corresponding to flagellin had high expression in log phase. Most of the proteins identified were involved in either the assimilation or catabolism process, and exhibited an overall mixed metabolic profile during both growth phases.

Production of ATP by E. coli Proteins In Vitro To test whether changes in the bacterial proteome during the growth phase could affect its energy extraction capacity, we tested the ability of logarithmic and stationary phase E. coli K12 proteins to ATP production from nutrients was tested in vitro. We have found that proteins from both growth phases can increase ATP production from different energy sources. The concentration of ATP was higher when using a protein-containing mixed energy source, such as MH medium, as compared to the sucrose solution. ATP production increased in a dose-dependent manner with bacterial protein concentration, but no significant difference was seen between the ATP-producing effects of the protein from the log or stationary phase.

Production of ClpB by E. coli in vitro The present inventors have developed and validated an enzyme-linked immunosorbent assay (ELISA) for the detection of E. coli ClpB used in this test. Whether the production of ClpB protein was different during the growth phase of the bacteria was tested in four separate E. coli K12 cultures. Western blot detected a 96 kDa band corresponding to ClpB in all protein preparations and levels increased during the stationary phase. These changes were further confirmed using ClpB ELSA in preparations of the same bacterial protein, indicating that the concentration of ClpB almost doubled in the stationary phase.

Injection of nutrients and E. coli proteins into the intestinal tract To test whether our in vitro model of nutrient-induced E. coli growth is related to the kinetics of intestinal bacterial growth in vivo, MH medium Alternatively, water was injected into the colon of anesthetized rats. We have found that instillation of MH medium, rather than water, induces bacterial growth in the intestine, and a logarithmic growth phase lasts for 20 minutes, which is consistent with in vitro data. ClpB levels in plasma measured at the portal vein were not significantly different 30 or 60 minutes after MH infusion. However, the concentration of plasma ClpB showed a positive correlation with the stool ClpB DNA content.

  Next, to determine whether the growth-dependent changes in the E. coli proteome could affect host mechanisms of appetite control locally in the intestine, anesthetized rats were tested in separate experiments. A 20 minute colonic infusion of 0.1 mg / kg log phase or stationary phase E. coli protein was performed. The concentration of ClpB in the colonic mucosa, measured 20 minutes after injection, was higher in rats receiving stationary phase protein, but the plasma level of ClpB was higher for bacterial proteins from either logarithmic or stationary phase. Was not affected either. Consistent with our hypothesis that the effect of E. coli proteins on host appetite signaling may depend on the growth phase of the bacteria, we found that colonization of E. coli proteins from log phase rather than stationary phase. It was found that instillation of GLP-1 stimulated plasma levels of GLP-1, whereas in contrast, increased plasma levels of PYY were detected following injection of proteins from the stationary phase but not the log phase.

Food intake and brain c-fos after acute administration of E. coli protein in rats
In all the rats and mice tested, ClpB was present in plasma, so E. coli proteins in plasma could affect appetite through systemic effects, and this effect was due to the bacterial growth phase. Could be different for proteins related to By testing this possibility in rats fasted overnight, we found that a single intraperitoneal (IP) administration of a membrane fraction of E. coli protein extracted in the stationary phase (0.1 mg / kg body weight) decreased food intake for 1 hour and 2 hours before re-feeding compared to the control group. In contrast, administration of the cytoplasmic fraction of E. coli protein (0.1 mg / kg body weight, IP) extracted during the log phase increased food intake for 4 hours before feeding again.

  Eat free to further test whether E. coli total protein can affect spontaneous food intake in a growth phase-dependent manner and to activate central sites such as ARC Rats were injected with a bacterial protein (0.1 mg / kg body weight, IP) before the onset phase began. Food intake was measured for 2 hours after injection, and rats were then sacrificed for analysis of c-fos expression in the brain. We found that rats injected with bacterial proteins from stationary phase consumed less than controls but food intake was not significantly affected by injection of bacterial proteins from log phase. I found that.

  E. coli protein on freely-fed rats. P. Two hours after injection, c-fos expression was analyzed by immunohistochemistry in the hypothalamus ARC and ventromedial nucleus (VMN), and in CeA. An increase in the number of c-fos positive cells was found in ARC and VMN of mice that received stationary phase derived bacterial proteins. The majority of cells expressing c-fos in the ARC were β-endorphin (control, 71.31 ± 12.81%, E. coli log phase, 73.56 ± 10.45%, E. coli stationary phase, 80.50). ± 9.68%, ANOVA p = 0.36), ie identified as anorexia-inducing POMC neurons. Thus, the percentage of c-fos neurons remaining in the ARC was not significantly different between the groups (control, 28.69 ± 12.81%, E. coli log phase, 26.44 ± 10.45%, E. coli Stationary phase, 19.5 ± 9.68%, ANOVA p = 0.36). The total number of β-endorphin positive cells was not significantly different between groups (control, 54.82 ± 10.67 cells, log phase of E. coli, 66.03 ± 11.43 cells, steady state of E. coli). Phase, 66.03 ± 5.06 cells, ANOVA p = 0.09), the relative number of activated β-endorphin neurons indicates the protein in stationary phase compared to control and rats receiving log phase protein. Increased in rats treated with. Furthermore, the number of activated β-endorphin neurons was inversely proportional to food intake (Pearson r = −0.57, p = 0.018).

  For CeA, the number of c-fos positive cells was increased in rats injected with protein from stationary phase compared to the other two groups. The phenotype of almost all c-fos positive cells in CeA was determined to be CGRP-expressing neurons (control, 100 ± 0.01%, log phase of E. coli, 100 ± 0.01%, stationary phase of E. coli). , 100 ± 0.01%, ANOVA p = 0.92). The total number of CGRP-positive neurons in CeA was similar between groups (control, 123.8 ± 13.15 cells, E. coli log phase, 118.3 ± 25.59 cells, E. coli stationary phase). , 126.1 ± 6.64 cells, ANOVA p = 0.85), the percentage of c-fos activated CGRP neurons increased only in rats receiving stationary phase protein. Activation of CGRP neurons was inversely proportional to food intake (Pearson r = -0.89, p = 0.001).

Eating pattern and hypothalamic neuropeptides after prolonged injection of E. coli protein in mice To determine if bacterial proteins can influence the feeding pattern, twice daily of total E. coli protein Injections (0.1 mg / kg body weight, IP) were given to mice on free access for one week. One day post-injection was characterized by significantly lower body weight and food intake in mice that received bacterial proteins from stationary but not log phase compared to controls. Although the daily diet was not significantly different between the groups, this decrease compared to the day before the injection was observed after one week in mice receiving the stationary phase protein. An increasing trend was observed in mice receiving stationary phase-derived bacterial proteins, but the frequency of diet was not significantly different between the groups. The total food intake among the three groups was not significantly different during the 6-day injection, but when analyzed separately for light and dark, mice injected with the log phase protein showed that food intake during the light phase It increased and decreased during the light-out period. In contrast, mice receiving stationary phase protein had no effect during the light phase, and had less food intake during the light off phase than the control. During the first day post-injection, the satiety ratio increased in mice receiving the stationary phase derived protein, and the same group showed a decreasing trend after one week.

  To gain insight into the molecular changes that underlie the changes in feeding patterns observed after six days of bacterial protein injection, the inventors hypothesized that some neuropeptides involved in the control of appetite, the thalamus The underlying mRNA expression levels were analyzed. We show that mice receiving stationary phase protein showed elevated brain-derived neurotrophic factor (BDNF) and orexin precursor mRNA levels as compared to controls; and It was found that compared to mice injected with a log phase protein that showed increased BDNF levels but reduced QRFP levels, they showed elevated adrenocorticotropic hormone releasing hormone (CRH) precursor mRNA levels.

Electrophysiological activation of hypothalamic POMC neurons by ClpB Determines whether ClpB, a marker for E. coli proteins up-regulated in stationary phase and a mimic of α-MSH, activates ARC POMC neurons To this end, the effect of ClpB was tested on brain sections from POMC-eGFP mice using cell-attached patch clamp electrophysiology. When ClpB (1 μM) was applied to the bath, the frequency of action potential of 〜50% of ARC POMC neurons (n = 7/13) was 229 ± 109% (basal value: 2.02 ± 0.78 Hz vs. ClpB: 3. 82 ± 1.36 Hz). In general, POMC neurons did not completely return to basal firing frequency at least 10 minutes after application of ClpB.

DISCUSSION From our studies, bacterial proteins have shown that intestinal bacterial growth is important in controlling host appetite, including both short- and long-term mechanisms associated with nutrient-induced bacterial growth in the gut and their systemic effects, respectively. Reveal that can be physiologically related. The following key results support this conclusion: 1) Regular nutrient feeding promotes and stabilizes the logarithmic growth of E. coli lasting 20 minutes, consistent with in vivo data. 2) Stationary phase E. coli was characterized by different proteome profiles, including increased total bacterial protein content and ClpB; 3) E. coli proteins from both growth phases were responsible for ATP production in vitro. Stimulated in a dose-dependent manner; 4) Plasma levels of ClpB did not change after nutrient-induced bacterial growth in the intestine, but correlated with ClpB DNA in gut flora; 5) Log phase and stationary phase Intestinal infusion of E. coli protein from the subject stimulated GLP-1 and PYY in plasma, respectively. 6) Systemic injection of E. coli protein significantly reduced food intake only by proteins from stationary phase, with activation of c-fos in anorexia-induced ARC and CeA neurons. 7) ClpB stimulated firing frequency of ARC POMC neurons.

Regular nutrition and bacterial growth Among the wide variety of bacteria that colonize the human digestive tract, Escherichia coli is the most common facultative anaerobic bacterium and should be used as a model organism for commensal enterobacteria. Is legal (Foucault et al., 2009). Here, we see that E. coli altering the kinetics of growth during periodic nutrient feeding is the fifth in a cycle in which an immediate logarithmic growth phase lasting 20 minutes followed by a stationary phase. After the supply cycle. Next, the growth cycle was replicated identically after the next nutrient supply, suggesting that it could play a role of a pacemaker that is set endogenously to bacterial mechanisms. A similar kinetics of bacterial growth in response to nutrient infusion has been observed in the colon of rats, indicating that our in vitro data indicate that in vivo situations, such as human normal dietary intake. Support what can be relevant. The increase in 10 ⁸ -10 ⁹ bacterial counts remains stable after each new supply, indicating that the stable production of the corresponding bacterial biomass, including an increase in protein content in the intestine, is not May play a role as a diet-inducible regulator for humans. If the duration of the average human diet is similar to the duration of logarithmic growth of E. coli, which is regularly fed, the host's satiety will reach a stationary phase 20 minutes after contact with the ingested nutrients. It is speculated that this can be caused by intestinal bacteria reaching. However, the bacterial content in the gastrointestinal tract is 10 ^{3 in} the stomach and 10 ^{12 in} the colon. In addition, it takes about 2 hours for nutrients ingested through the stomach and small intestine to pass, and about 10 hours to pass through the large intestine. Thus, due to the delay in nutrient delivery to most intestinal bacteria, bacterial growth during meals, due to the Pavlovian head-phase reflex to ingestion, compared to direct contact with large amounts of nutrients, May be initiated by nutrients released into the environment.

Detection of bacterial protein expression and intestinal tract during the growth phase Since the growth kinetics of E. coli, which is regularly fed, can be related to the host meal and the post-meal period, we have identified bacterial protein expression. Was tested if its expression could potentially be associated with regulation of gut bacteria and host appetite. First. We compared the proteome of E. coli in log phase versus stationary phase. For this analysis, E. coli proteins were extracted during the logarithmic phase, i.e., 10 minutes after nutrient supply, and at a stationary phase, 2 hours after, which is a time usually characterized by satiety. The finding of at least 40 differentially expressed proteins between the two growth phases indicates that they differ not only quantitatively by the nearly doubled protein content after bacterial growth, but also qualitatively. It was confirmed that they were different from each other. Second, the potential relevance of the modified protein expression profile to the control of host appetite is to: i) compare the ability of bacterial proteins to make energy substrates; Has been tested by determining the potential direct effects of The latter potential is supported by the recent proteomic identification of Escherichia coli ClpB as a mimic of a protein on the conformation of α-MSH (Tennoune et al., 2014). This data supports our earlier hypothesis that enteric bacterial proteins presenting epitopes homologous to anorexiagenic or anorectic peptides may be responsible for the production of cross-reactive autoantibodies. Verified (Fetissov et al., 2008). Thus, it can be taken into account that such a combination of bacterial mimetic proteins can directly affect appetite depending on the expression profile of the protein associated with the bacterial growth phase. By analyzing E. coli cultures and intestinal mucosa, we found an increase in ClpB levels associated with the stationary phase. Thus, increased ClpB may contribute to activation of the anorexia-inducible pathway and increased bacterial protein production following nutrient-induced growth of E. coli. An important issue is that bacterial proteins such as ClpB can act on the appetite regulation pathway.

  Although bacterial proteins are present in the intestinal mucosa (Haangel et al., 2012), their passage through the intestinal barrier has not been well studied (Lim and Rowley, 1985). Theoretically, after spontaneous and induced bacteria lyse in the intestine (Rice and Bayles, 2008), bacterial components can be absorbed by intestinal cells regulated by the enteric nervous system and spread by paracellular cells. It can cross the epithelial barrier of the mucosa (Neunlist et al., 2012). For example, lipopolysaccharide (LPS) released upon lysis of Gram-negative bacteria is normally present in the plasma of healthy humans and mice and has high basal levels after consuming a high fat diet (Cani et al., 2007). Plasma levels of LPS also increase after a meal (Hart et al., 2012), but there is no such data for bacterial proteins. Here we show that plasma ClpB levels remain stable in rats after bacterial growth in the intestine or after intestinal infusion of bacterial proteins. This is because ClpB in plasma, and most likely other bacterial proteins present in plasma, are not rapidly affected by nutrient-induced bacterial growth and therefore have a short period of satiety to the brain. Indicates that it cannot participate in signaling.

  However, the concentration of ClpB in plasma correlates with ClpB DNA in the gut flora, indicating that ClpB should be relatively independent of its variability associated with nutrient bacterial growth over time. It is suggested that the number of producing bacteria may be a major factor responsible for the long-term maintenance of plasma levels of ClpB. This conclusion is further supported by our data obtained in a validation of the ClpB ELISA, where mice treated with E. coli by long-term oral gavage show increased plasma ClpB levels, whereas ClpB No increase is observed in mice to which mutant E. coli was administered. Thus, enterobacteria present in plasma containing ClpB may be able to systematically act on the composition of enterobacteria in connection with long-term control of energy metabolism.

Effect of E. coli proteins on ATP production in vitro Changes in energy through the food chain represent a universal link between all organisms (Yun et al., 2006). ATP from nutrient catabolism in both bacteria and animals serves as the main energy substrate in the assimilation process. Animals can detect changes in ATP through the activity of adenosine-5'-monophosphate-activated protein kinase (AMPK) resulting in increased food intake when ATP levels are low, and vice versa. (Dzamko and Steinberg, 2009). Thus, in this study, we compared the ability of log phase and stationary phase E. coli proteins to produce ATP in vitro. In fact, many of the identified proteins exhibited anabolic or catabolic properties. The present inventors have found that E. coli proteins can stimulate ATP production in vitro, suggesting that they can continue to catalyze ATP production following bacterial lysis in the gut. No difference in the ability to produce ATP was found between the log phase and stationary phase derived proteins, but ATP production dependent on bacterial protein concentration was dependent on bacterial protein content after nutrient-induced bacterial growth. Indicate that more ATP synthesis should occur with an increase in. The relevance of the efficiency of gut flora to harvest energy for host metabolism has been previously established by comparing obese humans and rats to lean humans and rats (Turnbaugh et al., 2006). Our data further substantiate these results by showing that E. coli proteins can produce ATP. Diet-induced bacterial growth also results in an increase in intestinal ATP, suggesting that it should contribute to energy utilization and luminal detection of intestinal relaxation (Glasgow et al.). , 1998).

Intestinal Effects of E. coli Proteins on Satiety Hormone Next, we tested whether intestinal E. coli proteins could stimulate systemic release of intestinal satiety hormones such as GLP-1 and PYY ( Adrian et al., 1985; Batterham et al., 2002; Beglinger and Degen, 2006; Flint et al., 1998). In fact, both hormones are produced by the same or separate intestinal endocrine L cells, which are abundant in the entire gut and in the colon (Eissele et al., 1992), so that the L cells are directly exposed to bacterial proteins. ing. The present inventors have found that proteins from the log phase have different effects on E. coli cells on the release of GLP-1 and PYY, indicating stimulation of GLP-1 and PYY from the stationary phase. These results point to some similarities between nutrient-induced bacterial growth and the known kinetics of diet-induced release of GLP-1 and PYY. Indeed, as shown in humans, a sharp peak of GLP-1 in plasma occurs 15 minutes after intragastric infusion of a liquid meal, and a sustained rise in plasma PYY over a long period of time 15 to 30 minutes after (Edwards et al., 1999; Gerspach et al., 2011). Longer release of GLP-1 was associated with fat intake (van der Klaauw et al., 2013). Thus, the growth kinetics of regularly fed enteric bacteria is transiently compatible with the kinetics of GLP-1 and PYY release, which is a meal-induced signaling of satiety in the intestinal tract. Suggests an inducible role for intestinal bacteria in E. coli, especially for E. coli proteins. The different effects of log phase-derived E. coli proteins that stimulate GLP-1 may likely reflect the role of GLP-1 incretin in glycemic control (Edwards et al., 1999; Steinert et al., 2014). . The recent demonstration of a functional MC4R expressed by L cells (Panaro et al., 2014) provides a background for potential activation by α-MSH mimetic bacterial proteins. Increased ClpB production during the stationary phase of E. coli and increased levels of ClpB in the intestinal mucosa are associated with increased levels of PYY in plasma, thereby directing ClpB in activating PYY producing L cells in the colon. Role is suggested. On the other hand, previously unidentified E. coli proteins potentially up-regulated during the logarithmic growth phase can preferentially stimulate secretion of GLP-1.

Systemic Effects of E. coli Proteins on Brain Pathways that Regulate Food Intake and Appetite Herein, we presently demonstrate the peripheral effects of hungry or freely-fed rats and freely-fed mice. Injection of E. coli protein into E. coli showed that their food intake changed depending on the growth phase of E. coli. Given that plasma ClpB was not affected by intestinal infusion of nutrients, but was stable for a short period of time, the systemic effects of bacterial proteins should be interpreted as being related to long-term regulatory effects on appetite. In addition, due to the short duration of the logarithmic phase, bacterial proteins up-regulated during the long-lasting stationary phase are dominant in plasma, and their systemic administration improves physiological conditions better Can be expressed as The 0.1 mg / kg concentration of E. coli protein used in these experiments was similar to an effective satietygenic dose of a peptide hormone such as leptin or PYY after peripheral administration in humans or rodents. (Batterham et al., 2002; Halaas et al., 1995; Heymsfield et al., 1999).

  Increased food intake in fasted rats during re-feeding during the lighting phase was observed after administration of cytoplasmic proteins from the log phase and a decrease in (after administration of) membrane proteins from the stationary phase. This experiment confirmed that different protein mixtures from the same bacteria could increase or decrease food intake by their systemic effects. However, in a more physiological situation, by examining the effects of total E. coli proteins in rats fed freely during the light-out phase, only a reduction in food intake induced by proteins from the stationary phase was observed.

  The results of repeated injections in freely-fed mice further support the role of E. coli proteins in promoting a negative energy balance. In fact, the first day of bacterial protein injection was accompanied by a decrease in food intake and body weight, which was significant in mice receiving stationary phase protein, characterized by an increased percentage of satiety. Food intake in these mice then normalized, but a progressive decrease in food intake was accompanied by an increase in the number of meals, most likely as a complementary mechanism for maintaining food intake. High (Meguid et al., 1998). Furthermore, it was observed that the action of Escherichia coli protein was different between the lighting period and the lighting period. Thus, the expression pattern of the neuropeptide mRNA that controls appetite in the hypothalamus showed a mixed response with activation of both anorexiagenic and stimulatory pathways. Of note, both groups of mice that received E. coli protein showed a similar increase in BDNF mRNA, a downstream anorexia-inducing pathway to the MC4R in VMN (Xu et al., 2003). This pathway may be the basis for the observed decrease in food intake during the light-out period in both groups, with a logarithm indicating low levels of the anorectic QRFP (Chartrel et al., 2003) and NPY (Herzog, 2003). This was even more pronounced in the mice injected with the nascent protein. In contrast, mice receiving stationary phase-derived bacterial proteins show an enhanced anorexic profile with elevated levels of CRH mRNA and are most likely to involve MC4R expressing PVN neurons (Lu et al., 2003). These changes (Baird et al., 2009) combined with increased expression of orexin A pre-mRNA, which stimulates feeding frequency, indicate the percentage of food and satiety, respectively, in these mice after 6 days of injection. Can be reduced.

  Consistent with our hypothesis that bacterial proteins produced during the stationary phase could activate key key anorexic pathways, we found that rats fed freely Found an increase in c-fos expression in anorexiagenic ARC POMC neurons and VMN, long known as satiety center and interconnected with ARC POMC neurons (Sternson et al., 2005). The resulting c-fos pattern was induced during food intake (Johnstone et al., 2006) or by satiety hormones such as PYY or pancreatic polypeptides (Batterham et al., 2002; Challis et al. Lin et al., 2009) Similar to a satietygenic response. Relatively small amounts (％ 10%) of c-fos activated POMC neurons may have circulating E. coli proteins having a regulatory effect on appetite and body weight acting via this hypothalamic pathway Suggests. Determining the activation of c-fos by NPY / AgRP neurons was not feasible, but their contribution to signaling by bacterial proteins could not be ruled out and these neurons also expressed MC3R and MC4R (Mounien et al., 2005). Moreover, c-fos activation in CeA CGRP neurons was stronger than ARC POMC activation (-40%) from ARC POMC and NPY / AgRP neurons, and possibly other unanalyzed sources such as the solitary nucleus. May exhibit downstream effects that converge from regions of the brain that control appetite.

  Finally, to determine whether the activation of appetite-controlling brain sites such as ARC POMC neurons by bacterial proteins could be triggered by these local effects, we examined these neurons. It was tested whether the application of ClpB to the cells could activate the electrical activity of neurons. Our results showed that about half of the tested neurons increased their action potential frequency and remained activated for at least 10 minutes. The sustained action of ClpB is consistent with the action of α-MSH on POMC neurons expressing functional MC3R and MC4R (Smith et al., 2007), which indicates that ClpB is anorexic. It has been suggested that it may be a physiologically active agent of hypothalamic POMC neurons, somewhat similar to PYY and leptin (Batterham et al., 2002; Cowley et al., 2001). However, we did not know if ClpB could activate POMC neurons directly or via a local network.

  Taken together, these data support a role for systemic E. coli proteins, such as ClpB, whose expression increases in the stationary phase in promoting negative energy balance through activation of the anorectic pathway in the brain. Becomes It also suggests that alterations in the composition of the microflora that result in low or high amounts of E. coli, and possibly other bacteria from Enterobacteriaceae, can positively or negatively affect the energy balance of the host, respectively.

Example 2
One-month old male C57B16 mice (Janvier Laboratories) were acclimated to the animal facility for one week and maintained as described above. Mice are grouped into the following three groups: (i) a group that administers 10 ⁸ E. coli K12 bacteria by gavage; (ii) a group that administers 10 ⁸ gavages of ClpB-deficient Escherichia coli K12 bacteria; Were also divided into a control group that was not treated. The ClpB mutant strain was produced in Bernd Bukau's Laboratory (ZMBH, Heidelberg University, Heidelberg, Germany) and was provided by Dr Axel Mogk along with the corresponding wild-type (WT) E. coli 5. Mice were individually housed in BioDAQ cages (Research Diets), and 0.5 ml of LB medium containing bacteria was administered by oral gavage in the stomach daily for 21 days before the light-out period. The first day of gavage was accompanied by a decrease in body weight and food intake in mice given WT E. coli, in contrast to bacteria that do not express ClpB protein (FIG. 1).

REFERENCES Throughout this application, various references describe the state of the art to which the invention pertains. The disclosures of these references are incorporated herein by reference in the present disclosure.

Claims (6)

  A composition for use in extending the satiety of a subject in need thereof, comprising a ClpB protein comprising an amino acid sequence of SEQ ID NO: 2.   A composition for use in reducing food intake in a subject in need thereof, comprising a ClpB protein comprising the amino acid sequence of SEQ ID NO: 2.   3. The composition of claim 1 or 2, wherein the subject is obese.   The composition according to any one of claims 1 to 3, which is a pharmaceutical composition.   The composition according to any one of claims 1 to 3, which is a food composition.   The composition of claim 5, wherein the food composition further comprises at least one prebiotic.

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