WO2011071370A1 - Bactéries lactiques probiotiques - Google Patents

Bactéries lactiques probiotiques Download PDF

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Publication number
WO2011071370A1
WO2011071370A1 PCT/NL2010/050647 NL2010050647W WO2011071370A1 WO 2011071370 A1 WO2011071370 A1 WO 2011071370A1 NL 2010050647 W NL2010050647 W NL 2010050647W WO 2011071370 A1 WO2011071370 A1 WO 2011071370A1
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Prior art keywords
lactic acid
cells
subject
acid bacterium
composition
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PCT/NL2010/050647
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English (en)
Inventor
Johannes Snel
Michiel Kleerebezem
Renate Maria Louise Zwijsen
Bart Smit
Wouter Herman Noordman
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Campina Nederland Holding B.V.
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Publication of WO2011071370A1 publication Critical patent/WO2011071370A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the present invention relates to immunomodulating probiotic lactic acid bacteria, to methods wherein the bacteria are used to reduce allergy, and to food products wherein the bacteria may be incorporated to reduce allergy upon consumption of the product. Background of the invention
  • IgE-mediated allergy also referred to as type I hypersensitivity
  • type I hypersensitivity is the most important hypersensitivity reaction in the body. It is induced by a strong reaction against certain types of environmental compounds (food molecules, pollen, house dust mite, bee venom, etc) referred to as allergens.
  • Typical type I hypersensitivities associated with IgE antibodies include hay fever, asthma, urticaria, anaphylaxic shock and the like.
  • T-lymphocytes play an important role in directing the immune response. Different subsets of T-lymphocytes can be found in the human body. Allergy is the result of a T-helper type 2-(Th2)-mediated immune response and is characterized by the production of interleukins IL-4, IL-5 and IL-13.
  • IL-4 is the cytokine responsible for the formation of IgE-producing cells.
  • IL-5 stimulates B cell growth and increases immunoglobulin secretion. B cells synthesize IgE under certain conditions.
  • IL-13 contributes to isotype switching in B cells to promote IgE production.
  • Th2 responses are physiologically normal responses that can exist in the absence of allergy, as well as in the presence of strong Thl responses. Therefore, a role for T regulatory cells (Treg or Th3) and induction of tolerance has been postulated to control the development of allergy.
  • Allergens do not directly target T-lymphocytes, but are phagocytized by antigen presenting cells such as dendritic cells that can be found throughout the intestinal tract, the respiratory tract and underneath the skin. Antigen presenting cells process the allergens and express parts of it at their cell surface. These are recognized by T lymphocytes in association with other cell surface markers such as MHC-II and B7-2 molecules.
  • Non-activated dendritic cells are recognized by a low expression of the surface markers MHC-II and B7-2. These cells produce IL-10, and stimulate regulatory T cells (also called T- suppressor cells or T-helper 3 cells; Th3 cells) leading to tolerance induction. Activated dendritic cells have increased expression of MHC-II and B7-2. These cells may produce high levels of IL-12 and IFN- ⁇ that stimulate formation of T-helper 1 cells (Thl cells), or produce IL-6 and IL-10 to stimulate T-helper 2 cells (Th2 cells). The latter cell type may lead to allergy induction.
  • the gastro-intestinal tract contains the largest outer surface area of the human body, and has a dense concentration of non-self molecules, such as food molecules and commensal bacteria, that is in close contact with the locally present mucosal immune system. It is thus not surprising that the intestinal microbiota can play a role in the development of allergy. Indeed different colonization patterns have been found in allergic children compared to normal healthy children. Lactobacilli are an important component of the commensal microflora of humans and are frequently used as probiotics. Therefore Lactobacilli have been investigated for their ability to play a role in the prevention of allergy.
  • EP1364586 discloses lactic acid bacterial strains belonging to the Lactobacillus genus, and in particular Lactobacillus paracasei strains that are able to promote the induction of oral tolerance in mice to the cow's milk protein beta-lactoglobulin.
  • US2005214270 discloses anti-allergic agents comprising as an active ingredient one or more lactic acid bacterial strains of the species Lactobacillus acidophilus or Lactobacillus fermentum that suppress the IgE level in an ovalbumin hypersensitized mouse model.
  • EP 1634600 discloses anti-allergic compositions comprising as active ingredient the Lactobacillus paracasei KW 3110 strain that induces IL 12 production and reduces IL 4 production in vitro in mouse spleen lymphocytes sensitized with ovalbumin.
  • PBMCs Peripheral Blood Mononuclear Cells
  • Nonaka et al. describe antiallergic effects of orally administered Lactobacillus pentosus strain S-PT84 in an OVA-induced mouse allergy model (Int. Arch. Allergy Immunol, 2008. 145:249-257). There is however still a need for further probiotic lactic acid bacterial strains with demonstrated immunomodulating capabilities on humans that may be used in methods for reducing allergy, and that may e.g. be in incorporated into food products to reduce allergy.
  • the present invention relates to a lactic acid bacterium that upon consumption by a subject: a) reduces IgE levels in a blood sample of said subject; and b) stimulates T-helper 3 cells (Th3 cells) and/or a Th3 response in ex vivo stimulated PBMCs of said subject; after daily consumption of said lactic acid bacterium, compared to a reference strain L. plantarum WCFS 1 consumed in the same form and level by a subject.
  • the invention is concerned with a lactic acid bacterium that upon consumption by a subject: a) reduces IgE levels in a blood sample of said subject; and b) stimulates T-helper 3 cells (Th3 cells) and/or a Th3 response in ex vivo stimulated PBMCs of said subject; after daily consumption of said lactic acid bacterium.
  • a lactic acid bacterium that upon consumption by a subject: a) reduces IgE levels in a blood sample of said subject; and b) stimulates T-helper 3 cells (Th3 cells) and/or a Th3 response in ex vivo stimulated PBMCs of said subject; after daily consumption of said lactic acid bacterium.
  • said lactic acid bacterium further c) represses T-helper 2 cells (Th2 cells) and/or a Th2 response in ex vivo stimulated PBMCs of said subject.
  • stimulation of Th3 cells and/or a Th3 response induces IL-10 production.
  • repression of Th2 cells and/or a Th2 response repressed induction of IL-5 and/or IL-13.
  • the bacterium is consumed by the subject in an amount of 10 8 - 10 12 colony- forming units per day.
  • the bacterium is consumed in the form of a fermented food product, in particular a fermented dairy product.
  • the bacterium belong to the genus Lactobacillus, preferably the species Lactobacillus plantarum. In a particularly advantageous embodiment, the bacterium is Lactobacillus plantarum strain CBS 125632. In a second aspect the present invention is concerned with a lactic acid bacterium as defined hereinabove for use as a medicament, in particular for use in the treatment of allergy, preferably an IgE -mediated allergy.
  • the invention also pertains to a composition
  • a composition comprising a lactic acid bacterium as defined above, wherein the composition is at least one of a food composition (i.e. food for all human lifestages), a food supplement, a nutraceutical composition, a pharmaceutical composition and animal feed.
  • the composition is a dairy product, preferably a dairy product selected from fermented dairy products, yoghurt, milk, milk-based drinks and powders, buttermilk, yoghurt-based drinks, cheese, butter, ice-cream, a milk-derived dessert, soft curd cheese and fresh cheese.
  • a dairy product selected from fermented dairy products, yoghurt, milk, milk-based drinks and powders, buttermilk, yoghurt-based drinks, cheese, butter, ice-cream, a milk-derived dessert, soft curd cheese and fresh cheese.
  • the present invention relates to the use of a lactic acid bacterium as defined above for the manufacture of a composition for the treatment or prevention of allergy, preferably an IgE-mediated allergy.
  • the composition may be administered in an effective amount, the effective amount comprising between about lx 10 6 and about lx 10 12 colony forming units per day.
  • the composition may be a nutraceutical composition, a pharmaceutical composition, animal feed or a dairy product, preferably a dairy product selected from fermented dairy products, yoghurt, milk, milk-based drinks and powders, buttermilk, yoghurt-based drinks, cheese, butter, ice-cream, a milk-derived dessert, soft curd cheese and fresh cheese.
  • the present invention provides for a method for identifying a lactic acid bacterium that upon consumption by a subject: a) reduces IgE levels in a blood sample of said subject; b) stimulates T-helper 3 cells (Th3 cells) and/or a Th3 response in ex vivo stimulated PBMCs of said subject; and c) optionally, represses T- helper 2 cells (Th2 cells) and/or a Th2 response in ex vivo stimulated PBMCs of said subject; after daily consumption of said lactic acid bacterium, compared to a reference strain L.
  • plantarum WCFS 1 consumed in the same form and level by a subject said method comprising the steps of: allowing a subject to daily consume a lactic acid bacterium; determining the IgE levels in a blood sample of said subject, IL-10 levels, and, optionally, IL-5 levels and/or IL-13 levels, in ex vivo stimulated PBMCs prior to and after daily consumption of said lactic acid bacterium; and selecting a lactic acid bacterium effecting reduced IgE levels, increased IL-10 levels, and, optionally, reduced IL-5 and/or IL-13 levels after consumption.
  • Figure 1 provides an overview of the experimental design and tested parameters.
  • Figure 2 shows changes in immunoglobulins during the intervention period. Mean values ⁇ standard error are presented. * Value before intervention different from value after intervention (P ⁇ 0.05).
  • Figure 3 demonstrates cytokines (a. IL-5; b. IL-10; c. IL-12; d. IL-13; e. IL-17; f. IFN- ⁇ ) in culture medium of PBMC stimulated with aCD3/aCD28 antibodies after 4 days of growth. Mean values ⁇ standard error before intervention (dashed bars), and after intervention (black bars) are represented. * Values before and after intervention are different (P ⁇ 0.05). Description of the invention
  • the present invention relates to lactic acid bacterial strains with immunomodulating capabilities.
  • the lactic acid bacterium has the following immunomodulating capabilities upon consumption by a subject: a) reduces IgE levels in a blood sample of said subject; b) stimulates T-helper 3 cells (Th3 cells) and/or a Th3 response in ex vivo stimulated PBMCs of said subject; after daily consumption of said lactic acid bacterium, compared to a reference strain L. plantarum WCFS1 consumed in the same form and level by a subject.
  • the present invention relates to a lactic acid bacterium that upon consumption by a subject: a) reduces IgE levels in a blood sample of said subject; and b) stimulates T-helper 3 cells (Th3 cells) and/or a Th3 response in ex vivo stimulated PBMCs of said subject; after daily consumption of said lactic acid bacterium.
  • a lactic acid bacterium that upon consumption by a subject: a) reduces IgE levels in a blood sample of said subject; and b) stimulates T-helper 3 cells (Th3 cells) and/or a Th3 response in ex vivo stimulated PBMCs of said subject; after daily consumption of said lactic acid bacterium.
  • the lactic acid bacterial strains may be used as a medicament, preferably they may be used in the treatment of allergy, or prevention of allergy.
  • lactic acid bacteria is used herein to refer to bacteria, which produce lactic acid as a product of fermentation, including e.g. bacteria of the genus Lactobacillus, Streptococcus, Lactococcus, Oenococcus, Leuconostoc, Pediococcus, Carnobacterium, Propionibacterium, Enterococcus and Bifidobacterium.
  • the lactic acid bacterial strains of the invention are "probiotics” or “probiotic strains”, which term herein refers to strains administered in live, sublethal, or dead form, and includes crude extracts of the lactic acid bacteria, killed lactic acid bacterial cells, permeabilized lactic acid bacterial cells, and cell fractions of such lactic acid bacteria, which have a beneficial effect on the host when ingested (e.g., enterally or by inhalation) by a subject.
  • probiotics or “probiotic strains”
  • probiotic strains which term herein refers to strains administered in live, sublethal, or dead form, and includes crude extracts of the lactic acid bacteria, killed lactic acid bacterial cells, permeabilized lactic acid bacterial cells, and cell fractions of such lactic acid bacteria, which have a beneficial effect on the host when ingested (e.g., enterally or by inhalation) by a subject.
  • the effect of the strains presented herein to stimulate the immune system are achieved mainly by the
  • the bacterial strains of the present invention are live strains.
  • a "subject" refers herein to a human or non- human animal, in particular a vertebrate.
  • the human may be a baby, an infant, a child, teenager, an adolescent, or an adult.
  • the subject is a human adult.
  • the allergy that may be treated by the lactic acid bacterial strains of the invention preferably is an IgE-mediated allergy, also referred to as type I hypersensitivity.
  • the treatment may comprise the prevention and/or reduction of environmental allergies such as hay fever, topic dermatitis, bronchial asthma, chronic allergic rhinitis, urticaria, and anaphylactic shock.
  • the allergy to be treated may be caused by a large variety of allergens including: airborne particles (hay fever): (pollen from) grass, weeds, timothy grass, birch trees and mould spores; drugs: penicillin, sulfonamides, salicylates (also found naturally in numerous fruits) and local anaesthetics; foods (food allergy): nuts, peanuts, sesame, seafood, eggs (typically albumen,), peas, beans, soybeans and other legumes, celery and celeriac, soy, milk, wheat (gluten) and corn or maize; insect stings: bee sting venom and wasp sting venom; and animal products (animal allergy): animal hair and dander, cockroach calyx, and dust mite excretion.
  • airborne particles hay fever
  • drugs penicillin, sulfonamides, salicylates (also found naturally in numerous fruits) and local anaesthetics
  • foods food allergy
  • the lactic acid bacterial strains that are used as active ingredient in the treatment of allergy have immunomodulating capabilities.
  • the lactic acid bacterial strains have one or more of the following immunomodulating capabilities that are advantageous in the treatment of allergy:
  • Th3 cells T-helper 3 cells
  • Th3 cells are also referred to as regulatory T cells or T-suppressor cells and favour induction of tolerance;
  • Th2 cells T-helper 2 cells
  • Th2 response which may lead to induction of allergy
  • Thl cells No or moderate stimulation of T-helper 1 cells (Thl cells) and/or a Thl response.
  • a lactic acid bacterium having the capability to reduce induction of an inflammatory response in a subject reduces IgE levels in a blood sample of said subject.
  • IgE levels may be measured by methods which are known to the skilled person. For example, IgE levels may be determined using standard ELISAs, solid-phase displacement radioimmunoassays, double-antibody radioimmunoassays, solid-phase sandwich radioimmunoassays, and nephelometry.
  • a lactic acid bacterium with the capability to stimulate T-helper 3 cells (Th3 cells) and/or a Th3 response preferably is a lactic acid bacterium that upon consumption by a subject induces IL-10 production.
  • the lactic acid bacterium induces significantly more IL-10 than a reference strain does.
  • the lactic acid bacterium induces at least 110%, 115%, 120%, 125%, 130%, 135%, 140%, 150%, or more, of the amount of IL-10 that is induced under the same conditions by a reference strain L. plantarum WCFSl or in the absence of any reference strain..
  • the induction is preferably measured in ex vivo stimulated PBMCs of said subject.
  • the IL-10 levels may be measured by any means known in the art, for example, a commercially available ELISA kit for determination of IL-10 levels, such as from R&D Systems, Minneapolis, M.N., or other manufacturers.
  • a further preferred lactic acid bacterium with the capability to stimulate T-helper 3 cells (Th3 cells) and/or a Th3 response is a lactic acid bacterium that upon consumption daily by a subject, preferably an allergic subject, more preferably from a subject with an IgE-mediated allergy, most preferably a subject with a pollen allergy (of which birch pollen are most preferred), significantly induces IL-10 production compared to a reference strain, or compared to the absence of a reference strain.
  • said lactic acid bacterium induces at least 10, 20, 30, 40, 50, 60, 70, 80 or 90%, or more, more IL-10 after daily consumption of said lactic acid bacterium than a reference strain L.
  • the induction is preferably measured in ex vivo stimulated PBMCs of said subject.
  • the subject may be an allergic subject, preferably a subject with an IgE- mediated allergy, more preferably a subject with a pollen allergy (of which birch pollen are most preferred).
  • said lactic acid bacterium further c) represses T-helper 2 cells (Th2 cells) and/or a Th2 response in ex vivo stimulated PBMCs of said subject.
  • a lactic acid bacterium with the capability to repress and/or at least lacking the capability to stimulate T-helper 2 cells (Th2 cells) and/or a Th2 response (leading to induction of allergy) preferably is a lactic acid bacterium that upon consumption daily by a subject represses or at least does not stimulate the induction of IL-5 significantly.
  • the lactic acid bacterium represses IL-5 to such extent upon ex vivo stimulation of PBMCs of a subject about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% or less of the amount of IL-5 is formed as is formed upon ex vivo stimulation of PBMCs under the same conditions of a subject having consumed a reference strain L. plantarum WCFS1, or in the absence of any reference strain.
  • the subject is a subject with an IgE- mediated allergy, most preferably a subject with a pollen allergy (of which birch pollen are most preferred).
  • a lactic acid bacterium with the capability to repress and/or at least lacking the capability to stimulate T-helper 2 cells (Th2 cells) and/or a Th2 response (leading to induction of allergy) preferably is a lactic acid bacterium that upon consumption daily by a subject represses or at least does not stimulate the induction of IL-13 significantly.
  • the lactic acid bacterium represses IL-13 to such extent upon ex vivo stimulation of PBMCs of a subject about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% or less of the amount of IL-13 is formed as is formed upon ex vivo stimulation of PBMCs under the same conditions of a subject having consumed a reference strain L. plantarum WCFS1, or in the absence of any reference strain.
  • the subject is a subject with an IgE- mediated allergy, most preferably a subject with a pollen allergy (of which birch pollen are most preferred).
  • the lactic acid bacterium of the present invention may further have the capability to stimulate T-helper 1 cells (Thl cells) and/or a Thl response, or increase the Thl/Th2 ratio.
  • the comparison with the reference strain is not absolutely required; it may suffice to measure the levels referred to above prior to and after ingestion of the lactic acid bacterium of the present invention. Similar repressions and stimulations may thus be achieved, albeit in the same subject prior to and after ingestion of the lactic acid bacterium of the present invention. In an embodiment, the levels are measured after at least 4 weeks ingestion of the lactic acid bacterium of the present invention.
  • the term “consumption” equals the term “ingestion”, and refers to oral intake. Consumption may take place during any length of time, and may take place during 2, 3, 4, 5 , 6, or more weeks to yield the immunomodulatory effect. In an embodiment, consumption takes place during at least 4 weeks to yield the immunomodulatory effect, and such effect can be seen within those 4 weeks.
  • the term "significantly” refers to statistically significant. Significance levels show how likely a result is due to chance. As used herein, the level used to mean something is good enough to be believed, is 0.90. This means that the finding has a 90% chance of being true.
  • a reference strain refers to the Lactobacillus plantarum WCFS1 strain. However, the results may also be compared to results obtained in the absence of any (reference) strain, as Lactobacillus plantarum WCFS1 was found not to show any significant effect (see group 3).
  • the levels of cytokines as induced after consumption of the above described lactic acid bacteria of the invention were measured after ex vivo stimulation of PBMCs by routine methods known in the art, exemplified in the Examples section. Using such ex vivo stimulation method, the capacity of immune cells to respond to antigens can be measured.
  • the response of immune cells to anti-CD3 antibodies and anti-CD28 antibodies and the response of immune cells to Bet v 1 was measured.
  • Anti-CD3 antibodies and anti-CD28 antibodies provides an indication of the general allergy response, whereas Bet v 1 is a well accepted inducer of birch pollen allergy.
  • the PBMCs of the subjects have been subjected to certain potentially immunomodulating bacteria. Ex vivo, in blood drawn from the subjects, it can be measured to what extent the contact with the potentially immunomodulating bacteria has altered its response to allergens.
  • PBMCs may be isolated from blood samples prior to consumption of the lactic acid bacteria of the invention and after daily consumption, for example, during at least 4 weeks, of the lactic acid bacteria of the invention.
  • the PBMCs may be stimulated by addition of anti-CD3 and anti-CD28 antibodies, or Bet v 1.
  • Anti-CD3 and anti-CD28 antibodies are indicative of general allergy responses, whereas Bet v 1 is indicative of birch pollen responses.
  • Cytokine levels in samples before and after consumption of the lactic acid bacterium of the invention may be compared for changes in cytokine levels.
  • the lactic acid bacterial strains of the invention preferably are of the genus Lactobacillus.
  • the bacteria should be food-grade, i.e. they should be considered as not harmful, when ingested by a human or animal subject. It is understood that non-food grade bacteria, for example pathogenic bacteria, which have been modified so that they are no longer harmful when ingested by a subject, are included within the scope of the invention.
  • the Lactobacillus strains may be of the following species: L. rhamnosus, L. casei, L. paracasei, L. helveticus, L. delbrueckii, L. reuteri, L. brevis, L. crispatus, L. sakei, L. jensenii, L.
  • sanfransiscensis L. fructivorans, L. kefiri, L. curvatus, L. paraplantarum, L. kefirgranum, L. parakefir, L. fermentum, L. plantarum, L. acidophilus, L. johnsonii, L. gasseri, L. xylosus, L. salivarius etc.
  • Preferred species of lactic acid bacterial strains are of a species selected from L. acidophilus, L. plantarum, L. fermentum, L. rhamnosus, L. paracasei, L. acetotolerans, L. reuteri, L. casei, and L. paracasei subsp., more preferred are L. plantarum and L.
  • a lactic acid bacterial strain of the present invention is robust, meaning that its viability upon storage in the final product is higher than 50% after at least 7 days.
  • replicates and/or derivatives of the deposited strain or any other strain according to the invention are encompassed by the invention.
  • the term “replicate” refers to the biological material that represents a substantially unmodified copy of the material, such as material produced by growth of micro-organisms, e.g. growth of bacteria in culture media.
  • the term “derivative” refers to material created from the biological material and which is substantially modified to have new properties, for example caused by heritable changes in the genetic material. These changes can either occur spontaneously or be the result of applied chemical and/or physical agents (e.g. mutagenesis agents) and/or by recombinant DNA techniques as known in the art.
  • the invention relates to the use of a lactic acid bacterial strain as described above for the preparation (or manufacture) of a composition for the treatment allergy as defined herein above.
  • the composition that is manufactured using one or more strain(s) according to the invention may be any type of composition, which is suitable for consumption by, or administration to a subject, preferably a human subject suffering from an allergy as defined above.
  • the composition may be a food (including formulae), a food supplement composition, a nutraceutical, a pharmaceutical composition, or a feed composition.
  • the components and texture of the composition may vary.
  • a food or food/nutritive composition comprises besides the bacterial strain(s) of the invention also a suitable food base.
  • a food or food composition is herein understood to include solids (for example powders, such as baby formula powders), semi-solids and/or liquids (e.g. a drink or beverage) for human or animal consumption.
  • a food or food/nutritive composition may be a dairy product, such as fermented dairy products, yoghurt, milk or milk-based drinks or powders, buttermilk, yoghurt-based drinks, cheese and butter, ice-cream, desserts (e.g. milk-derived desserts like custards), soft curd cheese, fresh cheese (e.g. cottage cheese), etc.
  • Such foods or food compositions may be prepared in a manner known per se, e.g.
  • the strain(s) of the invention are used in or for the preparation of a food or food/nutrient composition, e.g. by fermentation.
  • examples of such strains include probiotic lactic acid producing bacteria of the invention.
  • the strain(s) of the invention may be used in a manner known per se for the preparation of such fermented foods or food/nutrition compositions, e.g. in a manner known per se for the preparation of fermented dairy products using lactic acid producing bacteria.
  • the strain(s) of the invention may be used in addition to the microorganism usually used, and/or may replace one or more or part of the micro-organism usually used.
  • a live food grade lactic acid producing bacterium of the invention may be added to or used as part of a starter culture or may be suitably added during or after such a fermentation.
  • flavourings, anti-oxidants, vitamins, minerals, colouring agents, etc may be present.
  • living cells are used, dead or non-viable cells may also be used in some compositions.
  • a preferred lactic acid bacterial strain of the invention is a strain that is stable in a dairy product, particularly in a fermented dairy product. Stability of the strain is herein understood as survival of viable bacteria after prolonged storage in a (fermented) dairy product.
  • a lactic acid bacterial strain of the invention is stable in a (fermented) dairy product under refrigeration conditions ( ⁇ 7°C) for at least 1, 2, 3, or 4 weeks whereby it is understood that the survival log of the bacterial strain (i.e. cfu after storage / cfu at start) is at least 0.5.
  • a food supplement may comprise one or more carriers, stabilizers, prebiotics and the like.
  • the composition is in powder form, for enteral (preferably oral) administration, although nasal administration or inhalation may also be suitable.
  • enteral preferably oral
  • the cells may be present in an encapsulated form in order to be protected against the stomach juices which means that in this case the cells do not need to be resistant to digestive juice.
  • the composition may e.g. be in the form of a powder packed in a sachet which can be dissolved or dispersed in water, fruit juice, milk or another beverage.
  • the dose of cells is preferably at least at least lxlO 6 cfu, preferably between about lxlO 6 - lxlO 12 cfu (colony forming units) per day, more preferably between about lxlO 7 - lxlO 12 cfu/day, more preferably about
  • the effective dose may be provided as a single dosage or may be subdivided into several smaller dosages and administered for example in two, three or more portions per day.
  • a nutritional composition preferably comprises carbohydrates and/or proteins and/or lipids suitable for human and/or animal consumption.
  • the compositions may or may not contain other bioactive ingredients, such as other (probiotic) strains, and prebiotics, which support the probiotic strains.
  • the cells When using living cells of the strain(s), the cells may be present in an encapsulated form in order to be protected against the stomach juice.
  • the dose of living cells per strain is preferably at least lxlO 6 cfu, preferably between about lxlO 6 - lxlO 12 cfu (colony forming units) per day, more preferably
  • the nutritional composition may replace the normal food/drink intake of a subject, or may be consumed in addition thereto.
  • dosages equivalent to the above cfu's are used. These equivalent dosages may be e.g. be determined using optical density (e.g. OD 6 oo) or by protein, lipid or DNA quantification.
  • nutraceutical/pharmaceutical compositions will usually be used for enteral, for example oral application.
  • Nutraceutical/pharmaceutical compositions will usually comprise a pharmaceutical carrier in addition to the strain(s) of the invention. The preferred form depends on the intended mode of administration and (therapeutic) application.
  • the pharmaceutical carrier can be any compatible, non-toxic substance suitable to deliver the strains(s) to the desired body cavity, e.g. the intestine of a subject. E.g.
  • nutraceutical/pharmaceutical compositions may further comprise additional biologically or pharmaceutically active ingredients.
  • the invention pertains to a method for preparing a composition for the treatment of allergy, comprising the steps of: a) growing at least one lactic acid bacterial strain as defined herein in a suitable liquid or solid medium; b) optionally isolating the strain from the medium, for example by centrifugation and/or filtration and performing down stream processing as known in the art, for example lyophilisation, spray drying and/or freezing; and, c) formulating the strain into a form suitable for administration to a subject.
  • the strains of the invention may be grown on artificial media or on natural media, such as (low fat) milk, yoghurt, and the like. It may then be used directly to make a composition according the invention, or the bacteria may be concentrated or isolated by centrifugation and/or filtration from the medium and then formulated into suitable compositions.
  • the present invention provides for a method for identifying a lactic acid bacterium that upon consumption by a subject: a) reduces IgE levels in a blood sample of said subject; b) stimulates T-helper 3 cells (Th3 cells) and/or a Th3 response in ex vivo stimulated PBMCs of said subject; and c) optionally, represses T-helper 2 cells (Th2 cells) and/or a Th2 response in ex vivo stimulated PBMCs of said subject; after daily consumption of said lactic acid bacterium, for example, during at least 4 weeks, compared to a reference strain L.
  • plantarum WCFS 1 consumed in the same form and level by a subject said method comprising the steps of: allowing a subject to daily consume a lactic acid bacterium, for example, during at least 4 weeks; determining the IgE levels in a blood sample of said subject, IL-10 levels, and, optionally, IL-5 levels and/or IL-13 levels, in ex vivo stimulated PBMCs prior to and after daily consumption, for example, during at least 4 weeks of said lactic acid bacterium; and selecting a lactic acid bacterium effecting reduced IgE levels, increased IL-10 levels, and, optionally, reduced IL-5 and/or IL-13 levels after consumption, for example during 4 or more weeks.
  • indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements.
  • the indefinite article “a” or “an” thus usually means “at least one”.
  • Yoghurt drinks were prepared by inoculating pasteurized skimmed milk with a commercial yogurt starter (placebo) and in addition one of the following strains:
  • Lactobacillus casei Shirota L. plantarum WCFS1, L. plantarum NIZO3400, L.
  • PBMC Human peripheral blood mononuclear cells
  • GlutaMAX GlutaMAX (IMDM) supplemented with 1% Penicillin-Streptomycin and 2% human AB serum (all from Gibco-BRL, Paisley, Scotland) with additions according to previously described procedures [28].
  • Cells were cultured in 48-well plates at 1 x 10 6 cells/well and incubated at 37 °C in a humidified atmosphere with 5% C0 2 . The supernatants were stored at -20 °C and overnight transferred to -80 °C before analysis Direct blood and serum analysis
  • the erythrocyte sedimentation analysis was carried out using the StaRRsed Compact Blood Sedimentation Instrument (Sysmex) according to routine procedures. C-reactive protein, bilirubin, creatinine, ALT, AST and GGT were analyzed using automated clinical chemistry analyzers (Roche).
  • Peripheral blood was centrifuged at 800x g for 10 min at room temperature, and the serum was removed for storage at -80 °C until analysis.
  • Birch pollen-specific IgE, IgG and IgG4 antibodies were determined using ImmunoCAP (Phadia AB) according to the instructions of the manufacturer.
  • Immunological phenotyping was performed on the cells after thawing. Surface antigens were identified by the following monoclonal antibodies: anti-human CD3, CD4, CD8, CD14, CD19, CD25, CD16, and anti-human CD56 (BD Pharmingen, San Diego, USA). Appropriate isotype-matched controls were included.
  • Two monoclonal antibody mixtures were used: 1) a-hCD3 (PE-Cy7), a-hCD4 (PE), a- hCD8 (APC), a-hCD25 (APC-Cy7); and 2) a-hCD14 (APC), a-hCD16 (PE), a-hCD19 (APC-Cy7) and a-hCD56 (PE).
  • the procedure was performed according to the instructions of the manufacturer.
  • Four-color flowcytometric acquisition was performed on a FACSCanto II (BD Biosciences), using the BD FACSDiva software.
  • a gate was set to exclude debris, and the percentages of cells expressing CD3, CD4, CD8, CD25, CD16/CD56, CD14, and CD19 were then calculated. The percentage of positive cells was corrected for values of the isotype control.
  • PBMC stimulation and cytokine analysis After overnight adaptation to the culture conditions, the cells were stimulated either polyclonally using aCD3/aCD28 antibodies (BD Biosciences; 150 ng/ml and 100 ng/ml, respectively) or antigen-specifically with rBet v 1.0101 (Bet v 1; Biomay, 10 ⁇ g/ml).
  • Culture supernatants were harvested after 4 days of culture for aCD3/aCD28 stimulated cells and after 1 and 7 days for Bet v 1 stimulated cells.
  • PMA Sigma, 2 ⁇ g/ml
  • Ca-I Sigma, 1 ⁇ g/ml
  • IL-1B The production of the innate and adaptive cytokines IL-1B, IL-4, IL-5, IL-10, IL- 12p70, IL-13, IL-17A, IFN- ⁇ and TNF-a was detected using Cytometric Bead Array (CBA, BD Biosciences, San Diego, CA). All buffers used in this protocol were obtained from the BD CBA Soluble Protein Master Buffer Kit (BD Pharmingen) and the procedure was performed according to the manufacturer's protocol.
  • CBA Cytometric Bead Array
  • the detection limits according to the manufacturer were as follows: 1.1 pg/ml IL- ⁇ , 1.1 pg/ml IL-5, 2.3 pg/ml IL-10, 2.2 pg/ml IL-12, 0.6 pg/ml IL-13, 0.3 pg/ml IL-17, 0.3 pg/ml IFN- ⁇ and 0.7 pg/ml TNF-a.
  • the samples were measured on the FACSCanto II, using FCAP software (BD Biosciences).
  • probiotics The safety of the probiotics was confirmed by clinical testing of serum parameters and by the absence of reported adverse effects during the last study visit. As anticipated, probiotic treatment did not result in increases of erythrocyte sedimentation, leukocyte counts and serum C-reactive protein concentration. The safety markers serum creatinine, bilirubin, ALT, AST and GGT were not affected in any of the treatment groups. Taken together none of these parameters pointed to any adverse effect, which was in agreement with the absence of adverse effects of the probiotic products indicated by the subjects.
  • Probiotics reduce birch pollen-specific IgE, but not IgG and IgG4
  • L. casei Shirota affects PBMC cell composition by lowering CD16+/CD56+ cells
  • Human PBMC were characterized for surface markers, to evaluate whether the tested probiotics had a direct effect on the composition of blood cell populations. Large individual differences in PBMC cell composition were observed at the start of the study but all values were within the normal ranges as reported by others [29].
  • differences between probiotic and placebo group were observed for percentage of CD3+ cells (higher for the group receiving NIZ02877), percentage of CD16+/CD56+ cells (lower for NIZ02877), CD8+ fraction of the CD3+ cells (lower for WCFS 1 and NIZO3400), and CD4+ fraction of the CD3+ cells (higher for WCFS1).
  • L. casei Shirota induced a decrease in CD16/CD56-positive cells. No effect of probiotic intervention was seen in any of the other groups (Table 1).
  • L. plantarum CBS125632 affects cytokine production of aCD3/aCD28 stimulated PBMC
  • PBMC Human PBMC were subjected to polyclonal or antigen-specific stimulation in order to induce cytokine production, and in this way evaluate the responsiveness of the immune system.
  • cytokine production varied between individuals, stimulation with aCD3/aCD28 antibodies for 4 days resulted in a clear cytokine production in all PBMC preparations, and detectable levels of IL-5, IL-10, IL-13, IL-17 and IFN- ⁇ .
  • the cytokine IL-12 was not detectable in 5 samples before intervention and in 3 samples after intervention.
  • Probiotic treatment with L. plantarum CBS 125632 led to a significant change in IL-5, IL-13, and IL-10 induction (Figure 3).
  • IL-5 was reduced by 29% ⁇ 11% and IL-13 by 21% ⁇ 1 1%.
  • IL-10 was increased by 24% ⁇ 13%. Such an effect was not seen with cells from the placebo group, or any of the other probiotic strains.
  • hPBMC were antigen-specifically stimulated with Bet v 1. These cells produced low but detectable levels of IL- ⁇ , TNF-a and IL-10 within 24 h. Cytokines IL-12, IL-17 and IFN- ⁇ were only detectable in 16%>, 8% and 33% of the samples respectively. Due to relatively high variation between samples and subjects, no effect of probiotic intervention was seen in any of the groups (data not shown).
  • cytokine concentrations were in general 2-fold to 10-fold lower than from
  • CD3+ pre 51.9 ⁇ 2.0 57.5 ⁇ 2.2 53.3 ⁇ 3.0 53.7 ⁇ 4.1 59.0 ⁇ 2.4 56.8 ⁇ 3.0
  • CD4-/CD8+ 36.7 ⁇ 2.6 34.0 ⁇ 3.2 29.7 ⁇ 2.1 29.0 ⁇ 2.3 32.5 ⁇ 1.8 31.0 ⁇ 1.4
  • CD4-/CD8+ 37.7 ⁇ 2.7 33.1 ⁇ 3.2 28.9 ⁇ 1.4 29.8 ⁇ 2.5 32.5 ⁇ 1.8 31.3 ⁇ 1.7
  • probiotic strains that were selected on basis of in vitro data. We observed an effect on plasma IgE levels after consumption of four candidate probiotic strains that was not
  • liver function parameters ALT, AST, GGT and bilirubin
  • IgE is an antibody isotype with a relatively short half life of only a few days.
  • IgE bound to mast cells is substantially longer than that of free serum IgE, the observed changes in serum do not necessarily predict changes in allergy symptoms within the time span of the study.
  • Th2 responses are physiologically normal responses that can exist in the absence of allergy, and that IFN- ⁇ secreted by Thl cells can contribute to allergy by increasing inflammatory reactions, and promoting survival and functionality of eosinophils and mast cells. Consumption of L. casei Shirota led to a reduction of CD16+/CD56+ cells. This population of NK cells is known for its ability to produce IFN- ⁇ , which contributes to an increased Thl profile.
  • probiotic bacteria alter the microbiota composition since bacterial cell numbers and diversity of the microbiota were significantly different between allergic subjects and healthy controls, even outside the pollen season.

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Abstract

La présente invention concerne des bactéries lactiques probiotiques immunomodulatrices, des méthodes dans lesquelles les bactéries sont utilisées pour réduire l'allergie et des produits alimentaires dans lesquels les bactéries peuvent être incorporées pour réduire l'allergie après consommation du produit. Les bactéries lactiques probiotiques préférées stimulent les réponses Th1 et/ou Th3 et/ou répriment les réponses Th2 in vivo, comme il peut être déterminé par les profils des cytokines qui sont induites chez les êtres humains après la consommation des bactéries lactiques.
PCT/NL2010/050647 2009-12-08 2010-10-05 Bactéries lactiques probiotiques WO2011071370A1 (fr)

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EP1364586A1 (fr) 2002-05-24 2003-11-26 Nestec S.A. Produits probiotiques et tolérance orale
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EP1634600A1 (fr) 2003-03-13 2006-03-15 Kirin Beer Kabushiki Kaisha Composition antiallergique
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