• 专利附录
  • 专利说明
  • 权利要求
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    • 专利摘要:
    BIFIDOBACTERIUM LONGUM AND FUNCTIONAL GI DISORDERS. The present invention relates, in general, to the field of probiotic bacteria. In particular, it refers to Bifidobacterium longum, such as Bifidobacterium longum ATCC BAA-999 and its use in ingestible compositions. The composition described in the present invention can be used to treat or prevent functional GI disorders.
    公开号:BR112012004046B1
    申请号:R112012004046-4
    申请日:2010-08-24
    公开日:2021-02-17
    发明作者:Peter McLean；Stephen Michael Collins；Premysl Bercik；Elena Verdu de Bercik；Gabriela Bergonzelli de Gonda
    申请人:Société des Produits Nestlé S.A.；
    IPC主号:
    专利说明:

    [0001] [001] The present invention refers, in general, to the field of probiotic bacteria. In particular, it refers to Bifidobacterium longum NCC3001 (ATCC BAA-999), subsequently referred to as Bifidobacterium longum ATCC BAA-999 and its use in ingestible compositions. The composition described in the present invention can be used to treat or prevent functional gastrointestinal (GI) disorders.
    [0002] [002] Functional GI disorders (FGIDs) is a group of disorders that includes irritable bowel syndrome (IBS) and functional dyspepsia, which are chronic conditions associated with high morbidity, characterized by abdominal discomfort or pain, abdominal distention and, in the IBS, altered bowel habits (diarrhea and / or constipation).
    [0003] [003] The prevalence of FGIDs in the general population is relatively high, ranging from 15% to 30%, presenting a substantial economic burden. It has been suggested that direct annual costs with IBS alone are now around $ 41 billion USD in the 8 most industrialized countries, with considerable additional indirect costs (eg absence from work, etc.). Current treatments for FGIDs demonstrate at most marginal effectiveness, which has historically been linked to a poor understanding of the pathogenesis of the disease.
    [0004] [004] The pathophysiology needs of IBS remains to be elucidated.
    [0005] [005] Recent studies have described inflammation of the mucosa and changes in intestinal microflora in patients with IBS and a correlation of the disease with intestinal infections.
    [0006] [006] The fact that some probiotics exhibit antibacterial, antiviral and anti-inflammatory properties and that they can restore the balance of the intestinal microbiota, suggested that they may become suitable therapeutic agents for IBS.
    [0007] [007] So far, several studies on the effect of different probiotics on individuals with IBS have been published. These studies suggest that the use of probiotics may be associated with improved symptoms of IBS, but also that not all probiotics are equally effective in IBS.
    [0008] [008] Several meta-analyzes carried out recently concluded that there is inadequate data to comment on the efficacy of other probiotics (Lynne V McFarland, et al., World J Gastroenterol 2008 May 7; 14 (17): 2650-2661; Nourieh Hoveyda, et al. , BMC Gastroenterology 2009, 9:15).
    [0009] [009] Consequently, it was an objective of the present invention to improve the state of the art and, in particular, to provide the technique with a composition comprising an alternative bacterial strain that is effective, easily available, low cost and safe for administration without effects unwanted side effects that can be used to treat or prevent functional GI disorders.
    [0010] [0010] The present inventors have addressed this need. They were surprised to see that they could achieve this goal for the sake of the independent claims. The dependent claims further develop the present idea of the invention.
    [0011] [0011] It has been found that the effectiveness in the treatment and / or prevention of functional GI disorders depends on the bacterial genus, the species and the strain used.
    [0012] Accordingly, one embodiment of the present invention is a composition comprising Bifidobacterium longum ATCC BAA-999 and / or its fermented growth medium for use in the treatment and / or prevention of functional GI disorders.
    [0013] [0013] The present invention also relates to the use of Bifidobacterium longum ATCC BAA-999 for the preparation of a composition to treat and / or prevent functional GI disorders.
    [0014] [0014] Bifidobacterium longum ATCC BAA-999 is a strain of the species Bifidobacterium longum which has been found to be, in particular, effective in obtaining the present invention.
    [0015] [0015] Advantageously, Bifidobacterium longum ATCC BAA-999 is commercially available and has already been tested and found to be acceptable for addition to food products, for example.
    [0016] [0016] Bifidobacterium longum ATCC BAA-999 (BL999) can be obtained commercially from specialized suppliers, for example, from Morinaga Milk Industry Co. Ltd. in Japan under the trademark BB536.
    [0017] [0017] Bifidobacterium longum ATCC BAA-999 (BL999) can be grown according to any suitable method. It can be added to products in a lyophilized or spray-dried form, for example.
    [0018] [0018] The term "Bifidobacterium longum ATCC BAA-999 (BL999)" is intended to include the bacterium, parts of the bacterium and / or a growth medium fermented by the bacterium.
    [0019] The composition can be any composition, but is preferably a composition to be administered orally, enterally or rectally.
    [0020] [0020] For example, the composition may be an edible composition.
    [0021] [0021] "Edible" means a material that is approved for human or animal consumption.
    [0022] [0022] Typically, the composition can be selected from the group consisting of a food composition, a pet food composition, a dietary supplement, a drink and / or a medical composition.
    [0023] [0023] If the composition of the present invention is a food composition, it has the advantage that such a composition can be distributed in pharmacies, drugstores, but also in normal supermarkets, where the compositions are easily available to everyone.
    [0024] [0024] The generally pleasant taste of food compositions will additionally contribute to the acceptance of the product. In particular, young children or pets are much more likely to readily consume compositions with a flavor that is generally accepted.
    [0025] [0025] Examples of food products that are applicable to the present invention are yogurts, milk, flavored milk, ice cream, ready-to-eat desserts, powders for reconstitution, for example, with milk or water, chocolate milk drinks, malted drinks, dishes ready for consumption, instant meals or drinks for humans or food compositions that represent a complete or partial diet intended for pets or farm animals.
    [0026] [0026] Consequently, in one embodiment, the composition according to the present invention is a food product intended for humans, pets or livestock.
    [0027] [0027] The composition can be intended for animals selected from the group consisting of dogs, cats, pigs, cattle, horses, goats, sheep or poultry.
    [0028] [0028] In a preferred embodiment, the composition is a food product intended for adult species, in particular, human adults.
    [0029] [0029] The composition of the present invention may additionally contain protective hydrocolloids (such as gums, proteins, modified starches), binders, film forming agents, encapsulating agents / materials, barrier / capsule materials, matrix compounds, coatings , emulsifiers, surfactants, solubilizing agents (oils, fats, waxes, lecithins, etc.), adsorbents, vehicles, fillers, co-compounds, dispersing agents, wetting agents, processing aids (solvents), fluent agents, agents that mask the flavor, agents for assigning weight, gelling agents, gel-forming agents, antioxidants and antimicrobials. The composition may also contain conventional pharmaceutical additives and adjuvants, excipients and diluents including, but not limited to water, gelatin of any origin, vegetable gums, lingninosulfonate, talc, sugars, starch, arabic gum, vegetable oils, polyalkylene glycols, flavoring agents, preservatives, stabilizers, emulsifying agents, buffers, lubricants, dyes, wetting agents, similar charges. In all cases, such additional components will be selected taking into account their suitability for the intended recipient.
    [0030] [0030] The composition can be a nutritionally complete formula.
    [0031] [0031] The composition according to the invention can comprise a protein source.
    [0032] [0032] Any suitable dietary protein can be used, for example, animal proteins (such as milk proteins, meat proteins and egg proteins); vegetable proteins (such as soy protein, wheat protein, rice protein and pea protein); mixtures of free amino acids; or their combinations. Milk proteins such as casein and whey and soy proteins are particularly preferred.
    [0033] [0033] Proteins can be intact or hydrolyzed or a mixture of intact and hydrolyzed proteins. It may be desirable to provide partially hydrolyzed proteins (degree of hydrolysis between 2 to 20%), for example, to human and / or animal individuals at risk of developing allergy to cow's milk.
    [0034] [0034] Additionally, sources of pre-hydrolyzed protein are generally more easily digested and absorbed by an impaired gastrointestinal tract.
    [0035] [0035] If hydrolyzed proteins are necessary, the hydrolysis process can be carried out as desired and as is known in the art. It may be desirable to provide partially hydrolyzed proteins (degree of hydrolysis between 2 and 20%).
    [0036] [0036] For example, a whey protein hydrolyzate can be prepared by enzymatic hydrolysis of the whey fraction in one or more steps. If the whey protein used as the starting material is substantially lactose-free, it has been found that the protein undergoes much less blockage of lysine during the hydrolysis process. This allows the extent of the lysine blockade to be performed to be reduced from about 15% by weight of the total lysine to about 10% by weight of the lysine; for example, about 7% by weight of lysine, which greatly improves the nutritional quality of the protein source.
    [0037] [0037] The composition can also contain a source of carbohydrates and a source of fat.
    [0038] [0038] If the composition includes a fat source, the fat source provides, preferably, 5% to 40% of the energy of the composition; for example, 20% to 30% of energy. A suitable fat profile can be obtained using a mixture of canola oil, corn oil and sunflower oil rich in oleic acid.
    [0039] [0039] A source of carbohydrates can be added to the composition.
    [0040] [0040] The carbohydrate source preferably provides 40% to 80% of the composition's energy. Any suitable carbohydrate can be used, for example, sucrose, lactose, glucose, fructose, corn syrup solids, maltodextrins and mixtures thereof. Dietary fiber can also be added. Dietary fiber passes through the small intestine undigested by enzymes and functions as a natural bulking and laxative agent. Dietary fiber can be soluble or insoluble and in general a mixture of the two types is preferred. Suitable sources of dietary fiber include soy, peas, oats, pectin, guar gum, partially hydrolyzed guar gum, gum arabic, fructooligosaccharides, acid oligosaccharides, galactooligosaccharides, sialylactose and oligosaccharides derived from animal milks. A preferred fiber mixture is a mixture of inulin with short chain fructooligosaccharides. Preferably, if the fiber is present, the fiber content is between 2 and 40 g / l of the composition as consumed, more preferably between 4 to 10 g / l.
    [0041] [0041] The composition may also contain minerals and micronutrients, such as residual elements and vitamins according to the recommendations of government agencies such as USRDA. For example, the composition may contain one or more of the following micronutrients in the given ranges per daily dose: 300 to 500 mg of calcium, 50 to 100 mg of magnesium, 150 to 250 mg of phosphorus, 5 to 20 mg of iron, 1 to 7 mg of zinc, 0.1 to 0.3 mg of copper, 50 to 200 µg of iodine, 5 to 15 µg of selenium, 1000 to 3000 µg of beta carotene, 10 to 80 mg of Vitamin C, 1 to 2 mg Vitamin B1, 0.5 to 1.5 mg of Vitamin B6, 0.5 to 2 mg of Vitamin B2, 5 to 18 mg of niacin, 0.5 to 2.0 µg of Vitamin B12, 100 to 800 µg of folic acid, 30 to 70 µg of biotin, 1 to 5 µg of Vitamin D and 3 to 10 µg of Vitamin E.
    [0042] [0042] One or more food-grade emulsifiers can be incorporated into the composition, if desired; for example, diacetyl esters of tartaric acid of mono and diglycerides, lecithin and mono and diglycerides. Similarly, suitable salts and stabilizers can be included.
    [0043] [0043] The composition can be administered orally and / or enterally; for example, in the form of a powder for reconstitution with milk or water.
    [0044] [0044] According to a preferred embodiment of the present invention, the composition comprises at least one other type of other food-grade microorganism.
    [0045] [0045] "Food grade" microorganisms are microorganisms that are safe for use in food.
    [0046] [0046] Food grade microorganisms are preferably food grade bacteria or food grade yeast. Food-grade bacteria can be selected from the group consisting of lactic acid bacteria, bifidobacteria, propionibacteria or their mixtures. As food-grade yeasts, for example, Saccharomyces cerevisiae and / or Saccharomyces boulardii can be used.
    [0047] [0047] Food-grade bacteria can be probiotic bacteria.
    [0048] [0048] "Probiotic" means cell preparations or components of microbial cells with a beneficial effect on the health or well-being of the host (Salminen S., Ouwehand A., Benno Y. and others "Probiotics: how should they be defined "Trends Food Sci. Technol. 1999: 10 107-10).
    [0049] [0049] Probiotic bacteria are preferably selected from the group consisting of lactic acid bacteria, bifidobacteria, propionibacteria or mixtures thereof. Probiotic bacteria can be any lactic acid bacteria or bifidobacteria with established probiotic characteristics. For example, they may be able to promote the development of an intestinal bifidogenic microbiota.
    [0050] [0050] Suitable probiotics can be selected from the group consisting of Bifidobacterium longum, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillusacculususaculususaculusaculusaculusaculusaculusaculusaculusaculusaculusacrusaculus selected from the group consisting of Lactobacillus johnsonii (NCC533; CNCM I-1225), Bifidobacterium longum (NCC490; CNCM I-2170), Bifidobacterium longum (NCC2705; CNCM I2618), Bifidobacterium lactis (2818; CNCM I-3446us), Lactobacill (NCC2461; CNCM I-2116), Lactobacillus rhamnosus GG (ATCC53103), Lactobacillus rhamnosus (NCC4007; CGMCC 1.3724), Enterococcus faecium SF 68 (NCIMB10415) and mixtures thereof.
    [0051] [0051] In a preferred embodiment of the present invention, the composition additionally contains at least one prebiotic. "Prebiotic" means food substances designed to promote the growth of probiotic bacteria in the intestines.
    [0052] [0052] Prebiotics can promote the growth of certain food-grade bacteria, in particular probiotic bacteria, in the intestines and, therefore, can intensify the effect of Bifidobacterium longum ATCC BAA-999. In addition, several prebiotics have a positive influence, for example, on digestion.
    [0053] [0053] Preferably, the prebiotic can be selected from the group consisting of oligosaccharides and, optionally, contain fructose, galactose, mannose, soy and / or inulin; dietary fibers; or their mixtures.
    [0054] [0054] Bifidobacterium longum ATCC BAA-999 can be used as a live bacterium as well as an inactivated non-replicating bacterial species.
    [0055] [0055] "Non-replicating" means that no viable cells and / or units that form colonies can be detected by classical plating methods. Such classic plating methods are summarized in the microbiology book: James Monroe Jay, Martin J. Loessner, David A. Golden. 2005. Modern food microbiology. 7th edition, Springer Science, New York, N.Y. 790 p. Typically, the absence of viable cells can be shown as follows: no visible colony on the agar plates or turbidity in the liquid growth medium after inoculation with different concentrations of bacterial preparations ("non-replicating" samples) and incubation under conditions appropriate (aerobic and / or anaerobic atmosphere for at least 24 h).
    [0056] [0056] It is preferable that at least part of the Bifidobacterium longum ATCC BAA-999 are alive in the composition and, preferably, arrive alive in the intestine. In this way they can persist in the intestine and can increase their effectiveness by multiplying. They can also be effective by interacting with commensal bacteria and / or the host.
    [0057] [0057] For food products or special sterile medicines, for example, it may be preferable that Bifidobacterium longum ATCC BAA-999 is present in a non-replicating form in the composition. Therefore, in one embodiment of the present invention at least a part of Bifidobacterium longum ATCC BAA-999 is not replicating in the composition.
    [0058] [0058] In therapeutic applications, the compositions are administered in an amount sufficient to at least partially cure or stop the symptoms of the disease and its complications. An adequate amount to achieve this is defined as "a therapeutically effective dose". Amounts effective for this purpose will depend on several factors known to those skilled in the art, such as the severity of the disease and the weight and general condition of the patient.
    [0059] [0059] In prophylactic applications, the compositions according to the invention are administered to a patient susceptible or at risk of another way of developing a particular disease, in an amount sufficient to at least partially reduce the risk of developing the disease. Such an amount is defined as "an effective prophylactic dose". Again, the precise amounts depend on several patient-specific factors such as the patient's health and weight.
    [0060] [0060] Generally, Bifidobacterium longum ATCC BAA-999 will be administered in a therapeutically effective dose and / or an effective prophylactic dose.
    [0061] [0061] If Bifidobacterium longum ATCC BAA-999 is present in a viable form, it is theoretically effective in any concentration, considering the fact that these bacteria can colonize the intestine and multiply.
    [0062] [0062] For the composition of the present invention, it is generally preferred that the daily dose of the composition comprises between 104 and 1012 cfu (colony forming units) of Bifidobacterium longum ATCC BAA-999. A particular suitable daily dose of Bifidobacterium longum ATCC BAA-999 is between 104 to 1011 cfu, more preferably between 104 to 1010 cfu.
    [0063] The composition of the present invention may also comprise between 102 and 1010 cfu, preferably 102 to 109 colony forming units, more preferably 102 to 108 cfu of Bifidobacterium longum ATCC BAA-999 per gram of dry weight of the composition.
    [0064] [0064] In the case of inactivated and / or non-replicating Bifidobacterium longum ATCC BAA-999, it is generally preferred that the composition of the present invention comprises between 102 and 1010 non-replicating cells of Bifidobacterium longum ATCC BAA-999 per gram of dry weight of the composition . A particular suitable daily dose of Bifidobacterium longum ATCC BAA-999 is between 103 to 108 non-replicating cells, more preferably between 105 to 108 non-replicating cells per gram of dry weight of the composition.
    [0065] [0065] Obviously, microorganisms that do not replicate do not form colonies, therefore, the expression cells should be understood as the amount of non-replicating microorganisms that is obtained from the specified amount of bacterial cells that replicate. This includes microorganisms that are inactivated, not viable or dead or present as fragments such as DNA or cell wall materials.
    [0066] [0066] The composition of the present invention can be provided in powder form which has a water activity of less than 0.2, for example, in the range of 0.19 to 0.05, preferably less than 0.15.
    [0067] [0067] The composition can be a powder that does not need refrigeration. The low water activity provides this stability without refrigeration and ensures that the probiotic microorganism, for example, Bifidobacterium longum ATCC BAA-999, will remain viable even after long periods of storage. Water activity or aw is a measure of the state of water energy in the system. It is defined as the water vapor pressure divided by that of pure water at the same temperature; therefore, pure distilled water has a water activity of exactly one.
    [0068] [0068] Additionally or alternatively, the probiotic microorganism Bifidobacterium longum ATCC BAA-999 can be supplied in encapsulated form.
    [0069] [0069] It has been found that the encapsulation of bacteria has therapeutic and technical advantages. Encapsulation increases the survival of bacteria and, therefore, the number of live bacteria that reach the intestine. In addition, the bacteria are gradually released, allowing a prolonged action of the bacteria on the health of the individual. The bacteria can be micro-encapsulated, for example, as described by FR2443247 (Société DES Produits Nestlé), incorporated by reference. Briefly, the bacteria can be lyophilized or spray dried and incorporated into a gel.
    [0070] [0070] The present inventors were surprised, in particular, to find that the composition of the present invention can be used successfully to significantly increase the expression of BDNF by the hippocampus.
    [0071] [0071] BDNF (brain-derived neurotrophic factor) is a growth factor in a unique family of polypeptide growth factors. BDNF and other neurotrophic factors, for example, NGF (nerve growth factor), NT-3 (neurotrophin-3) and NT-4 (neurotrophin-4) are essential for the health and well-being of the nervous system.
    [0072] [0072] This effect could possibly explain the effect observed against functional disorders of the intestine.
    [0073] [0073] The term "functional GI disorder" refers to a group of intestinal disorders that are characterized by abdominal complaints without a structural or biochemical cause that can explain the symptoms.
    [0074] [0074] Functional GI disorders are well known to those skilled in the art and are described and defined, for example, as functional gastrointestinal disorders with symptoms attributable to the middle or lower gastrointestinal tract by Longstrecht and others, in GASTROENTEROLOGY 2006; 130: 1480-1491, for example.
    [0075] [0075] Functional GI disorder can be selected from the group consisting of irritable bowel syndrome; functional dyspepsia; functional constipation; functional diarrhea; functional abdominal pain; functional distension, epigastric pain syndrome, postprandial discomfort syndrome or their combinations.
    [0076] [0076] Functional GI disorders that can be treated or prevented by the subject matter of the present invention comprise anxiety linked to functional GI disorders, for example.
    [0077] [0077] Anxiety is a psychological or physiological state that results in an unpleasant sensation that is typically associated with discomfort, fear or distress. Anxiety is, for example, a normal response to stress. It can help a person deal with a difficult situation at work or school, but - when excessive - it results in anxiety disorders, a group of which is anxiety linked to functional GI disorders.
    [0078] [0078] The inventors - without wishing to be linked to theory - currently believe that the underlying mechanism by which the compositions of the present invention are effective, is related to the modulation of the microbial-intestinal-brain bi-directional axis, possibly significantly associated with psychological factors .
    [0079] [0079] It is clear to those skilled in the art that any features described in that application can be combined freely without departing from the scope of the present invention as described. In particular, all the features described for the composition of the present invention are applicable to the use of the present invention and vice versa.
    [0080] [0080] Additional features and advantages of the present invention result from the following Examples and Figures.
    [0081] [0081] Figure 1 shows the result of a test with a dark box / light box: total time spent in the light box, latency to re-enter the light box and latency for descent in mice infected with Trichuris muris (Tm). Tm-means and Tm-B. longum are mice infected with Tm treated with fresh medium (negative control) and Bifidobacterium longum ATCC BAA-999, respectively; a Tm group treated with the L. rhamnosus strain (L.rh) is shown for comparison.
    [0082] [0082] Figure 2 shows the results of in situ hybridization in the cerebral hippocampal region of mice infected with Trichuris muris (Tm). Tm-B. longum are mice infected with Tm treated with Bifidobacterium longum ATCC BAA-999; a Tm group treated with the L. rhamnosus strain is shown for comparison. Quantification of 35S signals was performed by autoradiography and image analysis (upper right panel).
    [0083] [0083] Figure 3 shows the results on the colon inflammation measured by the assay of the myeloperoxidase activity (left panel) and mononuclear cell infiltration (right panel) in mice infected with Trichuris muris (Tm). Tm-means and Tm-B. longum are mice infected with Tm treated with fresh medium (negative control) and Bifidobacterium longum ATCC BAA-999, respectively; a Tm group treated with the L. rhamnosus strain is shown for comparison. EXAMPLES: Materials and methods Bacterial culture conditions:
    [0084] [0084] Probiotics (Bifidobacterium longum ATCC BAA-999 and L. rhamnosus NCC4007 for comparison) were grown under anaerobic conditions in Man-Rogosa-Sharpe broth (MRS, BioMerieux) (0.5% cysteine bifidobacterium). After 24 h at 37ºC, the number of bacteria was evaluated by measuring the optical density at 600 nm (1 OD600 = 108 bacteria / mL). The bacterial cells were pelleted and subsequently resuspended at a concentration of 1010 / mL in their used culture medium. Aliquots of 1 mL were kept frozen until use. Animals:
    [0085] [0085] Male BALB / c or AKR mice (Harlan, Canada) were acquired at the age of 6 to 8 weeks and housed in a specific pathogen-free conventional unit at McMaster University Central Animal Facility. All experiments were carried out with the approval of the McMaster University Animal Care Committee. Experiment: Chronic infection with T. muris:
    [0086] [0086] Male AKR mice were fed by gavage with T. muris (300 eggs / mouse) (n = 26) or with placebo (n = 9). Infected mice were fed by gavage daily with L. rhamnosus, B. longum or fresh MRS from day 30 for 10 days. Uninfected mice were fed by gavage with fresh MRS on a daily basis from day 30 to day 40. At the end of the probiotic or placebo administration, the mice were subjected to the dark box / clear box and reduction tests. The mice were then sacrificed and tissue samples were obtained. Colon samples were fixed in formalin for histological analysis or were quickly frozen for MPO determination. The brains were instantly frozen in liquid nitrogen and stored for in situ hybridization. Behavior test: Dark box / light box test:
    [0087] [0087] Anxious behavior was assessed individually in mice using the dark box / light box as described in the literature. Briefly, each mouse was placed in the center of a box with bright lighting (30x30 cm) connected by an opening (10x3 cm) to a smaller dark box (30x15 cm). The locomotor behavior of each mouse in the clear box was recorded for 10 min by a digital video camera and stored on a computer for offline analysis. Various parameters were assessed by a blind observer including the total time spent in the light box, the latency to re-enter the light box (time spent in the dark box after the first entry) and the number of passes (number of passes from the dark box to the clear box). Descent test:
    [0088] [0088] Anxious behavior was assessed using the descent test as described in the literature. Briefly, each mouse was placed in the center of an elevated platform (7.5 cm in diameter, 3 cm in height) positioned in the center of a black floor. The latency to descend from the pedestal was measured by a stopwatch; the maximum test duration was 5 min. Histology:
    [0089] [0089] Colon samples were fixed in 10% formalin and then stained with hematoxylin-eosin. The slides were examined under a microscope to assess the acute and chronic inflammatory infiltrate. Myeloperoxidase activity assay:
    [0090] [0090] In order to assess acute intestinal inflammation, the myeloperoxidase (MPO) activity assay was performed on frozen tissues as previously described. MPO activity is expressed in units per mg of tissue, where a unit of MPO is defined as the amount of enzyme capable of converting 1 µmol of hydrogen peroxide into water in 1 minute at room temperature. In situ hybridization in the CNS:
    [0091] [0091] BDNF levels in the hippocampus and CRH in the hypothalamus (paraventricular nucleus) were assessed by in situ hybridizations using 35S-labeled RNA probes in frozen brain sections as previously described (Whitfield et al. 1990; Foster et al., 2002 ). Briefly, the brains were removed and frozen quickly by immersion in 2-methylbutane at -60ºC and stored at -70ºC. The cryostatic section of coronal sections 12 µm thick were mounted thawed on slides covered with gelatin, dried and stored at -35ºC. The tissue sections were fixed with 4% formaldehyde, acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine-HCl, pH 8.0, dehydrated and degreased with chloroform. A BNDF antisense ribonucleotide probe (gift from Dr. James Lauterborn and Dr. C. Gall, University of California Irvine) and a CRH antisense ribonucleotide probe (gift from Dr. James Hermam, University of Cincinnati) were transcribed from the plasmid linearized using the Riboprobe system (Promega Biotech, Burlington, ON) with α-35S-UTP (specific activity ˃ 1000 Ci / mmol; Perkin-Elmer, Boston, MA) and the polymerases T3 and T7, respectively. The radiolabeled probes were diluted in a hybridization buffer (0.6 M NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0, 10% dextran sulfate, 0.01% salmon sheared sperm DNA, total RNA yeast 0.05%, type XI, yeast tRNA 0.01%, 1X Denhardt's solution) and brain sections (approximately 500,000 COM / section) applied. The slides were incubated overnight at 55 ° C in a humidified chamber. To reduce the non-specific binding of the probe, the slides were washed in a RNase solution of 20 µg / mL for 30 min at room temperature, followed by 1 h each in 2x SSC at 50ºC, 0.2X SSC at 55ºC and 60ºC. The slides were dehydrated and air dried for autoradiography. The slides and 14C plastic models were placed on X-ray cassettes, placed in front of a film (BioMax MR; Eastman Kodak, Rochester, NY) for 5 days and developed (Kodak Medical X-Ray Processor). Auto-radiographic images of the brain section film and the models were digitized with a compact camera with 60 mm Nikon lenses using the QCapture program (Qicam; Quorum Technologies Inc., Guelph, ON) and an image-based analysis system Macintosh computer with the Image program (http: //rsb.info.nih.gov.nihimage). The light transmittance through the film was measured outlining the structure on the monitor. For the BDNF mRNA, the transmittance was converted to levels of radioactivity using the Rodbard curve applied to the models. For CRH mRNA, the level slicing feature was used to measure light transmittance and mRNA signal area. The calculated DPM was then multiplied by the area to produce an integrated density measure. The illustrations were made directly from the captured images, Statistical analysis:
    [0092] [0092] Data are presented as mean ± standard deviation or medians with interquartile ranges as appropriate. Data were analyzed using two-way ANOVA, t-test or unpaired t-test as appropriate. A p-value of ˂ 0.05 was considered to be statistically significant. Results:
    [0093] [0093] Mice chronically infected with the parasite Trichuris muris showed an increase in anxiety-like behavior in two behavior tests: 1) In the dark box / light box test, infected animals showed a decrease in time spent in the light box and one increased latency to re-enter the clear box; 2) In the descent test, the infection increased the latency to descend from the pedestal (Figure 1). Treatment with Bifidobacterium longum ATCC BAA-999, but not with L. rhamnosus NCC4007, induced a reduction in anxiety-like behavior towards normality. The effect on behavior was correlated with a normalization of the decrease mediated by Trichuris muris in BDNF levels in the hippocampus by B. longum only (Figure 2). In contrast, treatment with B. longum as well as L. rhamnosus resulted in a reduction in myeloperoxidase activity and mononuclear infiltration previously induced by infection with Trichuris muris (Figure 3), indicating that normalization of behavior was independent of the anti-inflammatory effect. -inflammatory bacteria.
    权利要求:
    Claims (14)
    [0001]
    Composition for use in the treatment and / or prevention of a functional gastrointestinal (GI) disorder linked to anxiety, selected from the group consisting of irritable bowel syndrome, functional dyspepsia, functional constipation, functional diarrhea, functional abdominal pain, functional swelling, epigastric pain, postprandial distress syndrome and their combinations, characterized by the fact that it comprises Bifidobacterium longum ATCC BAA-999 and an acceptable vehicle; at least 5% of Bifidobacterium longum ATCC BAA-999 are viable in said composition; and said composition comprising between 104 and 1010 cells of Bifidobacterium longum ATCC BAA-999 per daily dose.
    [0002]
    Composition according to claim 1, characterized by the fact that it is selected from the group consisting of a food composition, a food composition for pets, a dietary supplement, a drink and / or a medical composition.
    [0003]
    Composition according to claim 1 or 2, characterized by the fact that it still contains at least one prebiotic.
    [0004]
    Composition according to claim 3, characterized by the fact that the prebiotic is selected from the group consisting of oligosaccharides, and, optionally, contains fructose, galactose, mannose, soy and / or inulin; dietary fibers; or mixtures thereof.
    [0005]
    Composition according to any one of claims 1 to 4, characterized by the fact that at least 10% of Bifidobacterium longum ATCC BAA-999 are viable in the composition.
    [0006]
    Composition according to any one of claims 1 to 4, characterized by the fact that at least 15% of Bifidobacterium longum ATCC BAA-999 are viable in the composition.
    [0007]
    Composition according to any one of claims 1 to 6, characterized by the fact that at least 80% of the Bifidobacterium longum ATCC BAA-999 are not replicating in the composition.
    [0008]
    Composition according to any one of claims 1 to 6, characterized by the fact that at least 90% of Bifidobacterium longum ATCC BAA-999 are not replicating in the composition.
    [0009]
    Composition according to any one of claims 1 to 6, characterized by the fact that at least 95% of Bifidobacterium longum ATCC BAA-999 are not replicating in the composition.
    [0010]
    Composition according to any one of claims 1 to 9, characterized in that it comprises between 102 and 108 cells of Bifidobacterium longum ATCC BAA-999 per g dry weight of the composition.
    [0011]
    Composition according to any one of claims 1 to 10, characterized in that it also contains a source of protein, which comprises hydrolyzed whey protein.
    [0012]
    Composition according to any one of claims 1 to 11, characterized by the fact that it is in the form of powder, which has a water activity of less than 0.2.
    [0013]
    Composition according to any one of claims 1 to 11, characterized by the fact that it is in the form of a powder, which has a water activity of 0.19 to 0.05.
    [0014]
    Composition according to any one of claims 1 to 11, characterized by the fact that it is in the form of powder, which has a water activity of less than 0.15.
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    法律状态:
    2017-12-12| B07D| Technical examination (opinion) related to article 229 of industrial property law|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |
    2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: A61K 35/74 (2015.01), A23K 10/18 (2016.01), A23L 3 |
    2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|
    2018-05-02| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa)|
    2018-10-09| B06T| Formal requirements before examination|
    2019-10-15| B25A| Requested transfer of rights approved|Owner name: SOCIETE DES PRODUITS NESTLE S.A. (CH) |
    2020-03-31| B07A| Technical examination (opinion): publication of technical examination (opinion)|
    2020-12-22| B09A| Decision: intention to grant|
    2021-02-17| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 17/02/2021, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    EP09168590.9|2009-08-25|
    EP09168590.9A|EP2289527B1|2009-08-25|2009-08-25|Bifidobacterium longum and functional GI disorders|
    PCT/EP2010/062320|WO2011023689A1|2009-08-25|2010-08-24|Bifidobacterium longum and functional gi disorders|
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