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    • 专利摘要:
    PROBIOTIC COMPOSITION FOR USE IN TREATING INTESTINAL INFLAMMATION. The invention relates to a composition comprising an effective amount of Lactobacillus glantarum CECT 7484, Lactobacillus giantarum CECT 7485, and Pediococcus acidilactici CECT 7483. This composition is useful in the treatment of gastrointestinal diseases or conditions such as inflammatory bowel disease, irritable bowel or abdominal distension and bloating.
    公开号:BR112012018813B1
    申请号:R112012018813-5
    申请日:2011-01-27
    公开日:2020-12-08
    发明作者:Jordi Espadaler Mazo；Jordi Cuñé Castellana
    申请人:Ab-Biotics S.A.；
    IPC主号:
    专利说明:

    [0001] This application claims the benefit of US provisional application No. 61 / 299,116, and European patent application EP10151998, both filed on January 28, 2010, and which are hereby incorporated by reference.
    [0002] The present invention relates to the fields of medicine, microbiology and nutrition and, in particular, to a new probiotic composition. In particular, new strains of Lactobacillus plantarum and Pediococcus acidilactici have been isolated and combined in a formulation useful in the treatment of gastrointestinal diseases, such as intestinal inflammation (eg inflammatory bowel disease) and irritable bowel syndrome. Background of the Invention
    [0003] Ulcerative colitis (UC), bolsite and Crohn's disease are examples of inflammatory bowel diseases (IBD) characterized by chronic inflammation in the intestine. Clinical symptoms are diarrhea, abdominal pain, occasional anal bleeding, weight loss, tiredness and sometimes fever. Although it occurs at any age, IBD is more common in adolescents and young adults, who as a consequence may show delayed development and stunted growth. The frequency of the disease is similar to that of type 1 diabetes in Europe and the United States. The clinical evolution of IBD varies considerably. Patients with mild to moderate symptoms can be treated without hospitalization. However, 10-15% of patients have a severe course of the disease, which in many cases is accompanied by surgery.
    [0004] IBD is treated clinically by reducing inflammation and thereby controlling gastrointestinal symptoms. However, there is still no medical cure for IBD. Colectomy can eliminate UC but impairs quality of life and increases the risk of complications. Available medical treatments include the use of 5-aminosalicylic acid (5-ASA), corticosteroids and immunomodulatory drugs. Prolonged treatment of mild to moderate symptoms of IBD is usually done with the use of 5-ASA while corticosteroids and immunomodulatory drugs are used to treat severe symptoms. Diarrhea and abdominal pain appear as side effects of 5-ASA whereas long-term use of corticosteroids often shows serious side effects that include reduced bone mass, infection, diabetes, muscle wasting and psychiatric disorders. Immunomodulatory drugs suppress the immune system, which controls the symptoms of IBD. However, the immunocompromised state leaves the patient susceptible to many diseases.
    [0005] Irritable bowel syndrome (IBS) is a condition characterized by abdominal pain and / or discomfort that is associated with bowel movement or altered bowel habit, where symptoms are not explained by structural or biochemical abnormalities. Urgency, distension or sensation of incomplete bowel movements are also common in IBS. Therefore, it is classified among functional gastrointestinal disorders that include diseases such as functional distension, non-cardiac chest pain, non-ulcer dyspepsia, and chronic constipation or diarrhea (Longstreth G. H. et al., 2006). It is worth noting that IBS has a substantial impact on morbidity and quality of life that goes beyond abdominal pain and discomfort, as the associated symptoms affect the patient's sense of well-being and their ability to act normally (Dean BB et al ., 2005).
    [0006] There is a huge movement in the field of drug development for the treatment of IBS. In this context, several antidepressants have become popular although their effectiveness in clinical trials has been modest and their clinical usefulness is limited by unpleasant side effects. Serotonergic agents have demonstrated effectiveness in the overall symptoms of IBS. However, recent safety concerns have significantly restricted its use. It is therefore of great interest to develop new therapies for IBS.
    [0007] Probiotics are defined as "living microorganisms" that, when ingested in certain amounts, have beneficial health effects that go beyond the inherent basic nutrition "(Araya M. et al., 2002; Guarner F. et al ., 1998). Several lactic acid bacteria and species of the genus Bifidobacterium are probiotics, which suggests that they have been shown to promote a specific health effect. Probiotic bacteria must satisfy several requirements related to lack of toxicity, viability, adhesion and beneficial effects. These probiotic aspects depend on the strain, even among bacteria of the same species, so it is important to find the strains that perform best in all probiotic requirements. Clinical trials in humans using probiotics alone or in combination with antibiotics have been carried out in order to to identify strains and / or formulations for the treatment of patients with IBD or IBS, or to keep already treated IBD patients in remission.
    [0008] WO 96/29083 and EP 554418 disclose two strains of Lactobacillus that colonize the intestine including Lactobacillus plantarum 299v (DSM 6595) and Lactobacillus casei ssp. rhamnosus 271 (DSM 6594). EP 415941 discloses methods for preparing a nutrient composition comprising treating porridge with enzymes before mixing with lactobacilli. US Patent 7195906 discloses a Bifidobacterium strain isolated from the dried and washed human gastrointestinal tract for the treatment of inflammatory diseases, especially inflammatory gastrointestinal activity, such as IBD and IBS.
    [0009] Despite promising potential, a considerable improvement in the effect of probiotics for use in the treatment of inflammatory bowel diseases (such as IBD) as well as other gastrointestinal diseases (such as IBS) is still necessary. Summary of the Invention
    [00010] The inventors of the present invention have found that a composition comprising strains of Lactobacillus and Pediococcus is effective in the treatment of intestinal inflammation. In particular, three new probiotic strains belonging to the genus Lactobacillus plantarum and to the genus Pediococcus acidilactici were isolated, the said strains enabling the effective treatment of intestinal inflammation when combined in the form of a single formulation.
    [00011] Therefore, in a first aspect the present invention offers a composition comprising an effective amount of Lactobacillus plantarum CECT 7484, Lactobacillus plantarum CECT 7485, and Pediococcus acidilactici CECT 7483, or mutants or variants thereof.
    [00012] The strains of CECT 7485 and CECT 7484 Lactobacillus plantarum, and the strain of CECT 7483 Pediococcus acidilactici were deposited in the Type Culture Collection of Spain (Valencia, Spain) on 02.04.2009. All three strains deposited are viable and retain all aspects related to the deposit.
    [00013] The term "effective amount", as used in this report, means an amount of an active agent high enough to provide the desired benefit, but low enough to avoid serious side effects according to medical criteria.
    [00014] It is clear that when using the strains deposited as starting material, versed in the technique, as usual, by conventional mutagenesis or re-isolation techniques, obtain new mutants or derivatives thereof that preserve or improve the relevant aspects and advantages described in this report of the strains that form the composition of the invention. The person skilled in the art is the one who will decide which method is appropriate to be used to determine the anti-inflammatory, immunomodulatory or anti-IBS activity or abdominal anti-distension of the strains. Examples of possible methods for measuring this activity are shown in the examples below.
    [00015] In one embodiment, the mutant is a genetically modified mutant.
    [00016] In another embodiment of the first aspect of the invention, the variant is a naturally occurring variant.
    [00017] The strains that form part of the composition of the first aspect of the invention may be in the form of viable cells. Alternatively, the strain may be in the form of non-viable cells.
    [00018] Generally, the use of the CECT 7483 strain of P. acidilactici, as well as the CECT 7484 and CECT 7485 strains of L. plantarum is done as viable cells. However, it can extend to non-viable cells such as dead cultures or compositions containing beneficial factors produced by P. acidilactici CECT 7483, as well as by L. plantarum CECT 7484 and CECT 7485. This includes thermally destroyed or microorganisms organisms destroyed by exposure to altered pH, sonication, radiation or subjected to pressure. With non-viable cells the preparation of the product is simpler, since the cells can be easily incorporated into drugs and the storage requirements are much less restricted than those of viable cells.
    [00019] When used in the form of the composition of the invention, the strains are preferably in a 1: 1: 1 concentration ratio.
    [00020] CECT 7483, CECT 7484, and CECT 7485 strains have significant inhibitory activity against several pathogenic and potentially pathogenic bacterial strains, while simultaneously showing minimal antagonism against common communal strains of the human gastrointestinal flora. In addition, these three strains show a lack of significant inhibitory activity between them, thus allowing their combined use in a single formula. This is quite relevant because it means that the composition, defined in the first aspect of the invention, has a beneficial effect on the intestine due to the "intact" effect of each of the three strains. The combination of these strains in a single formula (i.e., the composition of the invention) shows the ability to improve clinical symptoms (such as weight loss and diarrhea) in different animal models of intestinal inflammation. According to these results, the composition of the invention has the unique ability to significantly reduce acute (IL-6) and chronic (IFNy) cytokines.
    [00021] A wide variety of lactic acid bacterial species has a long history of evident safe use. The European Food Safety Authority has developed a system that grants "Qualified Safety Assumption" (QPS) status to taxonomic units with a long and proven track record of evident safe use. The strains CECT 7483, CECT 7484 and CECT 7485 belong to bacterial species that have QPS status (Andreoletti O. et al., 2008).
    [00022] The strains of the present invention have the advantage that they are particularly useful as probiotics. As mentioned above, probiotic bacteria must satisfy several requirements related to the absence of toxicity, viability, adhesion and beneficial effects. These probiotic aspects depend on the strain, even among bacteria of the same species. Therefore, it is important to find the strains that perform best in all probiotic requirements. The examples below offer (by way of example) protocols for determining each of the probiotic aspects and it has also been shown that the said strains have excellent probiotic aspects.
    [00023] The emergence and spread of resistance to antimicrobials in bacteria represents a threat to the health of man and animals and has a great financial and social cost. When resistance to an antimicrobial is inherent in a bacterial species, it is often called 'intrinsic resistance' (sometimes called 'natural resistance'). It is assumed that intrinsic resistance has minimal potential to spread horizontally, whereas acquired resistance mediated by added genes is considered to have high potential for lateral propagation. The inventors of the present invention have discovered that the strains that form the composition of the invention do not have any significant resistance to antibiotics of human and / or veterinary importance (ampicillin, gentamicin, streptomycin, erythromycin, tetracycline, clindamycin, and chloramphenicol) according to the guidelines in the Authority European Food Security Agency (Anadón A. et al., 2005; Bories G. et al., 2008), thus excluding the risk of a potential transfer of antibiotic resistance to pathogenic species.
    [00024] Additionally, the inventors of the present invention have found that the strains CECT 7483, CECT 7484, and CECT 7485 can be co-administered with other drugs used for the treatment of IBD (such as mesalazine). As shown below, the growth of said strains is not completely inhibited even when saturated concentrations of mesalazine are used. In other words, even when high concentrations of mesalazine are used, the effectiveness of the composition of the invention comprising the probiotic strains is not compromised and, therefore, it can have both probiotic and anti-inflammatory functions.
    [00025] The strains of the invention have been shown to be highly resistant to the conditions of the mammalian gastrointestinal environment (acidic environment, bile salts, and high concentrations of lysozyme and oxygen peroxide), thus being able to survive passage through the gastrointestinal tract (hereinafter also called "GIT"). The strains also have good adhesion to the intestinal epithelium, which allows them to remain in the intestinal tract and exert their probiotic effects.
    [00026] In addition, the present strains have several beneficial effects on the host. In addition to the anti-inflammatory activity in the intestine, they benefit the intestinal microbiota balance due to their antagonistic activity. The term "antagonistic activity" refers to the inhibition of the growth of non-beneficial gastrointestinal bacteria by the activity of probiotic bacteria. The condition of having an inadequate gastrointestinal microbial balance is known as dysbiosis and has multiple negative consequences for human well-being. It will be shown below that strains have a great ability to inhibit the growth of pathogenic strains when compared to other commercial strains. In addition, as mentioned below, the inventors have found that the new strains of the invention do not exhibit significant inhibitory capacity between them.
    [00027] Additionally, it was discovered that the strains that form the composition of the first aspect of the invention produce large amounts of short-chain fatty acids (SCFA). The production of SCFA from non-digestible fibers is an interesting probiotic trait. This is a desirable trait in a probiotic because the SCFA produced shows several beneficial properties in the host. Among its various properties, SCFAs, especially butyric acid, are easily absorbed by the intestinal mucosa, stimulate the absorption of sodium and water in the colon, and are trophic to the intestinal mucosa (D'Argenio G. et al., 1999; Tedelind S . et al., 2007). In addition, butyric acid is used as a fuel by colonocytes. Each strain in the formula is a strong producer of a different SCFA, be it acetic, propionic or butyric, which are the three main SCFAs found in the intestine. A better understanding of how short-chain fatty acids act in the inflammatory process can help increase the effectiveness of current treatments for intestinal inflammation. In this context, the relationship between SCFAs and the regulation of inflammatory conditions through receptors coupled to protein G has already been reported (Maslowski K. M. et al., 2009).
    [00028] In addition, the CECT 7483, CECT 7484 and CECT 7485 strains promote immunomodulatory effects on the host, as they induce an improved cytokine pattern of the intestinal mucosa. These immunomodulatory effects are beneficial to the host because they help to achieve greater resistance to the disease and a reduced risk of allergies. Gram-negative bacteria in the GIT are known to exhibit the lipopolysaccharide (LPS) molecule on their surface, which induces the production of pro-inflammatory signals by intestinal mucosa cells. Probiotic supplementation can change this situation favoring a greater presence of Gram-positive bacteria in the GIT (grouped in the group of lactic acid bacteria), with better ecological suitability or with antagonistic properties against some Gram-negative microorganisms. However, some probiotic microorganisms show the ability to modulate the production of cytokines per se, which are messenger molecules that regulate the inflammatory and immune responses in the body. In particular, some probiotic bacteria induce a more balanced pattern between pro / anti-inflammatory signaling in the intestinal mucosa (regardless of the effect on Gram-negative bacteria). As will be illustrated later, strains of the composition of the invention have been found to promote a reduction in the levels of inflammatory cytokines (IFN-y and IL-6), thereby inducing an improved pattern of cytokines from the intestinal mucosa. This immunomodulatory effect is complemented by the antagonistic properties of strains in reducing the presence of pathogenic Gram-negative bacteria in the GIT.
    [00029] It is known that non-viable bacteria as well as bacterial components can have immunomodulatory effects per se. For example, it has been reported that cellular components of the Lactobacillus species induce anti-inflammatory cytokines (Pathmakanthan S. et al., 2004) or reduce anti-inflammatory cytokines (Zhang L. et al., 2005). With the isolation of these components, it is possible to anticipate pharmaceutical grade manipulation.
    [00030] Considering the results shown below, it is clear that the strains CECT 7483, CECT 7484 and CECT 7485, which form the composition of the first aspect of the invention, are characterized by specific traits such as: survival to gastrointestinal passage, adherence to intestinal mucosa, resistance to oxidative stress, production of metabolites with anti-inflammatory activity (be it short-chain fatty acids or other products with said activity) and absence of antagonism between them. The composition and isolated strains of the present invention are obviously not derived from the prior art because they are the result of complex research and the results that have been obtained in terms of activity in intestinal inflammation are surprising. Protocols for determining each of these features are included below. According to the content of the present application, the person skilled in the art can find other strains belonging to the genera Lactobacillus and Pediococcus, and more particularly to the species Lactobacillus plantarum and Pediococcus acidilactici which, when administered alone or combined in a single composition, present the same probiotic and therapeutic effects than those described in this application.
    [00031] In a second aspect, the invention offers a composition comprising an effective amount of the strains of the invention, or mutant strains thereof, for use as a medicament.
    [00032] In particular, it has been found that the composition comprising the strains CECT 7483, CECT 7484 and CECT 7485 has an anti-inflammatory activity in the intestine in IBD models. As explained above, intestinal inflammation is one of the main characteristics of IBD. Therefore, the composition of the first aspect of the invention is useful in preventing or treating said disease.
    [00033] Therefore, in a third aspect, the invention offers the composition defined in the first aspect of the invention for use in preventing or treating intestinal inflammation in an animal, including man. This aspect can alternatively be formulated as the use of a composition defined in the first aspect of the invention for the production of a medicament for the prevention and / or treatment of intestinal inflammation. This can alternatively be formulated as a method for the prevention and / or treatment of intestinal inflammation in an animal, including man, comprising administering to said animal in need thereof an effective amount of the composition defined in the first aspect of the invention.
    [00034] In an embodiment of the third aspect of the invention, the composition is used for the treatment or prevention of inflamed bowel disease.
    [00035] From the data obtained using the IBD models reported in the examples (see below), it appears that the administration of the strains of the invention, which are effective in the treatment of conditions characterized by intestinal inflammation and diarrhea, can also be useful to treat other conditions characterized by inflammation of the mucosa or submucosa of the intestine and where diarrhea predominates, such as enteritis caused by radiotherapy or chemotherapy. Enteritis is a common side effect of abdominal and pelvic radiotherapy, affecting 60-70% of patients. Enteritis can force changes in the radiotherapy program agenda to reduce side effects, potentially leading to suboptimal anti-tumor efficacy of treatment. There are currently no preventive strategies for radiotherapy-induced enteritis. However, some probiotics have shown promise in randomized controlled trials (RCTs). A probiotic composition, such as that of the present invention, combining the health-promoting effects of SCFA production, the ability to resist the reactive oxygen and nitrogen species found in the inflamed mucosa, and the antimicrobial activity against opportunistic pathogens, may be useful for treat radiotherapy and chemotherapy-induced enteritis.
    [00036] On the other hand, the present inventors have found that the strains of the present invention are efficient in the treatment of IBS. As will be shown later, the composition of the first aspect of the invention is useful in the treatment of IBS, as assessed by a randomized, double-blind, placebo-controlled trial.
    [00037] Therefore, in a fourth aspect the present invention offers the composition of the first aspect of the invention for use in preventing and / or treating IBS. This aspect can alternatively be formulated as the use of a composition defined in the first aspect of the invention for the production of a medicament for the prevention and / or treatment of IBS. This can alternatively be formulated as a method for the prevention and / or treatment of IBS in an animal, including man, comprising administering to said animal in need thereof an effective amount of the composition defined in the first aspect of the invention.
    [00038] Furthermore, the present inventors have found that due to the characteristics of the strains, the composition of the first aspect of the invention is useful in the treatment of bloating and bloating. As will be shown later, when the composition of the invention is administered to persons suffering from bloating and bloating, a surprising improvement is observed.
    [00039] Therefore, in a fifth aspect the present invention offers the composition of the first aspect of the invention for use in the treatment of bloating and bloating. This aspect can alternatively be formulated as the use of a composition defined in the first aspect of the invention for the production of a medicament for the treatment of bloating and bloating. This can alternatively be formulated as a method for the treatment of bloating and bloating in an animal, including man, comprising administering to said animal in need thereof an effective amount of the composition defined in the first aspect of the invention.
    [00040] The surprising beneficial effects observed in people suffering from IBS, and / or bloating and bloating may occur due to the fact that the strains of the invention CECT 7483, CECT 7484 and CECT 7485 are capable of producing the SCFAs listed in the Table 6 and the antagonistic activity shown in Table 3.
    [00041] It is already known in the prior art that SCFAs modulate intestinal activity. In particular, SCFAs are known to stimulate the release of serotonin (5-HT) in the colon of rats (Fukumoto S. et al., 2003; Tazoe H. et al., 2008) which plays a key role in regulating motility and bowel sensitivity. Similarly, butyric acid has been reported to decrease intestinal visceral sensitivity in human volunteers (Vanhoutvin S. A. et al., 2009). From there, it can be concluded that the strains that form the composition of the invention can be useful to treat not only IBS or abdominal pain, but also other conditions related to gastrointestinal motility and / or gastrointestinal pain, such as functional constipation or functional diarrhea .
    [00042] The composition and isolated strains of the present invention are obviously not derived from the prior art because they are the result of complex research and the results that have been obtained in terms of efficiency in treating IBS and bloating and bloating are surprising.
    [00043] Surprisingly, the present inventors have discovered, for the first time, a strain of Pediococcus acidilactici capable of treating IBD and IBS. This capacity, without wishing to stick to the theory, is due, as is believed, to the specific properties, pointed out throughout this report, of the isolated strain of Pediococcus. In view of the teachings and protocols provided in this report, the person skilled in the art is able to find other strains of P. acidilactici with the same probiotic and therapeutic aspects as the subject of this application.
    [00044] The composition according to the invention comprising an effective amount of the strains of the invention, or mutants thereof, can be formulated as edible pharmaceutical or veterinary products, in which said strains are the only active agents or are mixed with a or more other active agents and / or are mixed with acceptable pharmaceutical or veterinary excipients (in the case of a pharmaceutical or veterinary product) or with suitable additives (in the case of an edible product). In a particular embodiment of the invention, the products additionally contain one or more other active agents. Preferably, the additional active agent or agents are other probiotic bacteria that are not antagonistic to the strains that form the composition of the invention. Depending on the formulation, strains can be added as purified bacteria, as a bacterial culture, as part of a bacterial culture, as a bacterial culture that has been post-treated, and alone or together with suitable carriers or ingredients. Prebiotics can also be added.
    [00045] In other respects the invention offers pharmaceutical and veterinary products that contain an effective amount of the composition of the invention together with suitable amounts of acceptable pharmaceutical or veterinary excipients. In this context, the pharmaceutical product can be prepared in any suitable form that does not negatively affect the bioavailability of the strains that form the composition of the invention. Accordingly, the composition of the invention can be formulated to be administered orally in the form, for example, of a freeze-dried powder, capsules, liquid preparations etc. The choice of excipients and the most appropriate methods for formulation in view of the particular purpose of the composition is known to those skilled in the field of pharmaceutical technology. Although oral administration is preferred, other forms are possible, such as injectable, rectal or topical.
    [00046] The term "pharmaceutically acceptable" as used in this report refers to compounds, materials, compositions, and / or dosage forms that, according to sensible medical criteria, are suitable for use in contact with an individual's tissues (eg human being) without excessive toxicity, irritation, allergic response, or other problem or complication, consistent with a reasonable risk / benefit ratio. Every carrier, excipient, etc. it has to be "acceptable" in the sense of being compatible with the other ingredients in the formulation. Such carriers, excipients, etc. can be found in standard pharmaceutical texts. Also, the term "acceptable veterinarian" means suitable for use in contact with the tissues of a non-human animal.
    [00047] Strains of the invention can also be included in a variety of edible products, such as dairy products, yogurt, curd, cheese (e.g., quark, creamy, processed, soft and hard), fermented milk, powdered milk, fermented product based on milk, ice cream, fermented product based on cereal, powder based on milk, soft drink, sauce and animal feed. The term "edible product" is used in this report in its broadest sense, including any type of product, in any form of presentation, that can be ingested by an animal, but excluding pharmaceutical and veterinary products. Examples of other edible products are meat products (eg liver paste, frankfurter and salami sausages or meat pastes), chocolate pastes, fillings (eg truffles, creams) and toppings, chocolate, confectionery products (eg caramels, fondants or toffee), baked goods (cakes, sweets), sauces and soups, fruit juices and coffee bleaches. Particularly interesting edible products are dietary supplements and baby formulas. In the context of the present invention, dietary supplements also include nutraceuticals, which are known as food extracts that have some medicinal effect on man's health. Animal fodder is also included in the scope of the invention. The compositions of the invention could also be used as an ingredient in other food products.
    [00048] Therefore, in another aspect of the invention, an edible product is offered which contains the composition of the invention together with appropriate amounts of edible ingredients. Preferably, the composition of the invention is a dietary supplement.
    [00049] The effective amount of colony forming units (cfu) for each strain in the composition will be determined by those skilled in the art and will depend on the final formulation. For example, in edible products, the strain or strains are present in an amount of about 105 cfu / g to about 1012 cfu / g, preferably in an amount of about 107 cfu / g to about 1012 cfu / g, of current legislation. The term "colony-forming unit" ("cfu") is defined as the number of bacterial cells revealed by microbiological counts on agar plates.
    [00050] Dietary supplements usually contain probiotic strains in an amount ranging from 107 to 1012 cfu / g. In a particular embodiment, the composition of the invention is a dietary supplement comprising between 109 and 1011 cfu / g.
    [00051] The strains of the invention are produced by cultivating the bacteria in a suitable medium and under suitable conditions. Strains can be grown individually to form a pure culture, either as a mixed culture together with other microorganisms, or by growing bacteria of different types separately and then combining them in the desired proportions. After cultivation, the cell suspension is recovered and used as such or treated in the desired manner, for example, by concentration or freeze drying, to be further employed in the preparation of pharmaceutical or edible products. Sometimes the probiotic preparation is subjected to an immobilization or encapsulation process in order to improve the shelf life. Several techniques of immobilization or encapsulation of bacteria are known in the literature.
    [00052] If the composition according to the invention is used as a dietary supplement, it can be administered as such, it can be mixed with a suitable drinkable liquid, such as water, yogurt, milk or fruit juice, or it can be mixed with a solid or liquid food. In this context the dietary supplement can be in the form of tablets, pills, capsules, granules, powders, suspensions, sachets, lozenges, sweets, bars, syrups and corresponding forms of administration, usually in the form of a unit dose. Preferably, the composition of the invention is administered in the form of tablets, capsules or powders, produced by conventional pharmaceutical preparation processes.
    [00053] Throughout this report and in the claims the word "comprises" and its variations are not intended to exclude other technical aspects, additives, components or stages. Objectives, advantages and additional aspects of the invention will become apparent to those skilled in the art upon reading the report or can be inferred from the practice of the invention. In addition, the present invention covers all possible combinations of the particular and preferred embodiments described in this report. Brief Description of Drawings
    [00054] Figure 1 represents the pulsed field gel electrophoresis patterns for the genomic DNA restricted Not-I or Sfi-I (left) and Sma-I (right) of: 1, Pediococcus acidilactici CECT 7483; 2, Lactobacillus plantarum CECT 7484; 3, Lactobacillus plantarum CECT 7485. As controls: 4, commercial strain P. acidilactici Rossell1001 (Institut Rossell, Canada); 5, L. plantarum 299v (Probi AB, Sweden); and 6, L plantarum strain isolated from the commercial product VSL # 3 (VSL Pharmaceuticals, USA). This figure refers to the section "genotyping strains".
    [00055] Figure 2 represents the disease activity index (Y axis) in a group of mice suffering from intestinal inflammation induced by DSS. The X axis represents the administration of: a, the probiotic formulation of the invention to a group of mice suffering from intestinal inflammation induced by DSS; b, a commercial probiotic formulation (VSL # 3) for a group of mice suffering from DSS-induced intestinal inflammation; c, vehicle to a group of mice suffering from intestinal inflammation induced by DSS; and d, vehicle to a healthy control group. This figure refers to the section "Effect In Vivo on Chemically Induced Intestinal Inflammation".
    [00056] Figure 3 represents the levels of IL-6 (Y axis) in a group of mice suffering from intestinal inflammation induced by DSS. The X axis represents: a, the probiotic formulation of the invention administered to a group of mice suffering from DSS-induced intestinal inflammation; b, a commercial probiotic formulation (VSL # 3) administered to a group of mice suffering from DSS-induced intestinal inflammation; c, vehicle administered to a group of mice suffering from intestinal inflammation induced by DSS; and d, vehicle to a healthy control group. This figure refers to the section "Effect In Vivo on Chemically Induced Intestinal Inflammation".
    [00057] Figure 4 represents the number of weeks without symptoms (ie, the number of weeks before the onset of the first symptom, the disease activity index being therefore equal to zero) (Y axis) in a doped mouse model with IL-10. The X-axis represents the administration of: a, the probiotic composition of the invention to a group of IL-10 doped mice; b, the commercial probiotic formulation VSL # 3 to a group of IL-10 doped mice; c, PBS to a group of IL-10 doped mice; and d, vehicle to a healthy control group. This figure refers to the section "Effect In Vivo on Spontaneous Intestinal Inflammation".
    [00058] Figure 5 represents the levels of IFN- (Y axis) in a mouse model stunned with IL-10. The X-axis represents: a, probiotic formulation of the invention to a group of IL-10 doped mice; b, a commercial probiotic formulation (VSL # 3) to a group of IL-10 doped mice; c, vehicle to a group of mice doped with IL-10; and d, vehicle to a healthy control group. This figure refers to the section "Effect In Vivo on Chemically Induced Intestinal Inflammation".
    [00059] Figure 6 represents the levels of IL-6 (Y axis) in a mouse model stunned with IL-10. The X-axis represents: a, probiotic formulation of the invention to a group of IL-10 doped mice; b, a commercial probiotic formulation (VSL # 3) to a group of IL-10 doped mice; c, vehicle administered to a group of IL-10 doped mice; and d, vehicle to a healthy control group. This figure refers to the section "Effect In Vivo on Chemically Induced Intestinal Inflammation".
    [00060] Figure 7 represents the variation of the IBSQoL score compared to the baseline (Y axis) of volunteers treated with capsules including the composition (black bars) or placebo (white bars). The X axis represents the variation in the score 21 days and after 42 days of treatment. This figure refers to "Improving the Quality of Life Associated with Health" in the "In Vivo Effectiveness in Individuals with IBS" section.
    [00061] Figure 8 represents the variation of the VSI score compared to the baseline (Y axis) of capsule-treated volunteers including composition (black bars) or placebo (white bars). The X axis represents the variation in the score after three weeks and after six weeks of treatment. This figure refers to the "Improved Visceral Sensitivity" in the "In Vivo Efficacy in Individuals with IBS" section. EXAMPLES
    [00062] The following examples describe the characterization of the strains of the invention, their specific probiotic aspects and their physiological effects on the gastrointestinal and immune systems. As used in this report, strain F1033 corresponds to Pediococcus acidilactici CETC 7483, strain F2064 to Lactobacillus plantarum CECT 7484, and strain F2076 to Lactobacillus plantarum CECT 7485. 1. Isolation of microorganisms A) Methods
    [00063] For the isolation of microorganisms, fresh feces and saliva (Daniel C. et al., 2006) were collected from children 0-5 years old and dissolved in PBS buffer (pH 7.4), divided into aliquots and plated on MRS supplemented with various combinations of antibiotics. The strains were grown under microaerophilic conditions (5% CO2) at 37 ° C. The incubation time depended on the growth rate, but was usually between 24 hours and 3 days. Gram stain was performed to obtain a first identification. Once developed, the isolated strains were stored by lyophilization in PBS 0.1X with 15% skimmed-milk powder. B) Results
    [00064] The new strains F2064, F2076 and F1033 were developed on MRS agar supplemented with 10 μg / ml of vancomycin. Microscopic examination revealed that strains F2064 and F2076 are Gram-positive bacilli, whereas strain F1033 is a Gram-positive with coccid morphology. 2. Identification A) Methods
    [00065] Genomic DNA was extracted using the Wizard genomic DNA purification kit (Promega). For each isolated strain, the 16S gene was amplified by PCR, using the universal primers 27f, 357f, 907r and 1492r (Weisburg WG et al., 1991), which generated a nearly full length (1465 bp) 16S rRNA fragment. The DNA was washed using the Quiaquick kit (Quiagene, GmbH, Hilden, Germany) and four sequencing reactions were performed per sample, using the BigDye v.3.1 kit, on a 3130 genetic analyzer (Applied Biosystems). The software "Selected sequencing primers DNA Sequence Analysis v.5.2" (Applied Biosystems) was used for data collection and for the construction of chromatograms, which were analyzed using the software Chromas (Technelysium Pty Ltd.) and BioEdit (Ibis Biosciencies ). The identification of the genus and species was made by comparing the sequence obtained with 16S sequences of known organisms contained in both the RefSeq database (http://www.ncbi.nlm.nih.gov/RefSeq/) using a search BLASTN (Altschul SF et al., 1990), as well as the Ribosomal Database Project (Wang Q. et al., 2007). TABLE 1: Primers used to amplify and sequence the 16S gene.
    B) Results
    [00066] Strains F2064 and F2076 have been identified as members of the Lactobacillus plantarum group. The F1033 strain was identified as Pediococcus acidilactici. 3. Survival of the gastrointestinal tract A) Methods
    [00067] To assess tolerance to acidic environments, 20 μl aliquots of each bacterial strain culture were placed in 96-well plates, along with 200μl aliquots of MRS medium with the pH adjusted to 2 and 3 with HCl (Panreac) . The plates were kept at 37 ° C for 1 hour and the optical density at 620 nm was measured. Finally, viable cells were determined by counting the plaque and compared with the number of viable cells in the inoculum.
    [00068] To assess tolerance to bile salts, 20 μl aliquots of each bacterial strain culture were placed on a 96-well plate along with 200μl of MRS medium supplemented with 0.5% Oxgall (Sigma). The plates were incubated at 37 ° C and 5% CO2 for 3 hours, and then the optical density was measured. Finally, viable cells were determined by counting the plaque and compared with the number of viable cells in the inoculum. B) Results
    [00069] All three strains showed good ability to survive acidic environments, with less than a log reduction in the number of viable cells after 1 hour of incubation in MRS at pH = 2 or pH = 3. The strains also have a marked resistance to bile salts, with a reduction of less than 50% in the number of viable cells after 3 hours of incubation in MRS supplemented with 0.5% bile salts. 4. Adherence A) Methods
    [00070] Pork intestine washed with PBS pH 7.4, containing 0.01% gelatin and a cocktail of protease inhibitors (Complete®, Sigma). The mucosa was scraped and dissolved in HEPES-Hank buffer (10 mM HEPES, pH 7.4) (Collado M. et al., 2007) containing the inhibitors mentioned above. Then, the mucus was centrifuged at 13000 rpm for 10 minutes using the same buffer. The supernatants were recovered and the protein content was determined by the Bradford protocol. 24 hours before the assay, 1 ml of a 0.5 mg / ml mucus solution was incubated in the wells of a 24-well ELISA plate.
    [00071] Each strain to be tested was grown overnight in MRS medium supplemented with tritium-labeled thymidine (5 μl in 3 ml of MRS). The cultures were centrifuged and adjusted to 108 cfu / ml in PBS by counting in a Neubauer chamber and samples from each culture were collected to determine the amount of tritium-labeled thymidine incorporated using a scintillation reader. Then, 0.5 ml was added to the wells containing mucus from the 24-well plate and incubated at 37 ° C for 60 minutes. The supernatant from each well was removed, and the wells were washed twice with MEM Alpha (Gibco) medium to remove bacteria that did not adhere well. Finally, the wells were scraped to recover the mucus along with the adherent bacteria, and radioactivity was measured. The specific activity (cpm / CFU) of each culture was calculated from the total radioactivity incorporated in the PBS suspension adjusted to 108 cfu / ml. Lactobacillus rhamnosus GG (Valio Ltd, Finland) was used as a positive control, because of its remarkably high adherence to the intestinal epithelium (Jacobsen C. N. et al., 1999).
    [00072] Caco-2 cells were purchased from the ATCC (ECACC No.: 86010202). The cells were seeded in 24-well plates and allowed to grow in DMEM until reaching confluence (37 ° C, 5% CO2). The experimental procedure for obtaining the number of bacteria that adhere per unit area of shard-2 cells is essentially the same as that explained above for mucus adhesion. B) Results
    [00073] The adhesion capacity of strains F1033, F2064 and F2076 was measured by scintillation of thymidine marked with tritium and compared with those of the commercial strain L. rhamnosus GG. Adhesion to epithelial cells using the Caco-2 model is a common assay for probiotic strains. Compared to L. rhamnosus GG, strains F2064 and F2076 show an affinity for epithelial cells 60% lower. However, considering the high affinity of L. rhamnosus GG for epithelial cells, these values are comparable to other well-known probiotics such as L. plantarum 299v, and superior to many other probiotic strains (Jacobsen CN et al., 1999). On the other hand, the adhesion of the F1033 strain to epithelial cells is 2.5 times more than that of L. rhamnosus GG. In addition, the F2064 and F2076 strains showed a much higher affinity for intestinal mucus than for epithelial cells, whereas the F1033 strain showed an opposite behavior. The results are shown in the table below. TABLE 2: Adhesion of probiotic bacterial strains to mucus. [* Of a total bacterial concentration of 108 cfu].
    5. Capacity for Antagonism A) Methods
    [00074] The following indicator strains were used: P. mirabilis CECT 4557, K. oxytoca CIP 103434, C. perfringens ATCC 13124, C. ramosum ATCC 25582, E. faecalis CETC 795, Y. pseudotuberculosis ATCC29833, B. vulgatus ATCC 8482 and B. thetaiotaomicron ATCC2079 were collection strains. C. albicans, S. enterica thyphimurium, S. enterica cholerasuis, C. jejuni, E. coli and P. aeruginosa were isolated from the laboratory. The indicator strains were spread evenly on plates containing the appropriate medium (oxoid) and allowed to develop until confluence at the appropriate temperatures under microaerophilic conditions (5% CO2). Then, 6 mm (diameter) cylindrical sections of confluent agar plates with F1033, F2064 or F2076 were placed upside down on the plate with the indicator strain and incubated overnight at 37 ° C. The following day, the zones of inhibition were measured by placing the agar plate on a flat ruler. Growth inhibitory activity (GI) was calculated as follows:
    where IZD is the diameter of the inhibition zone and CD is the diameter of the cylinder, measured in millimeters. B) Results TABLE 3: Growth inhibitory activity (GI) of probiotic strains against 12 pathogenic or potentially pathogenic strains, and against 2 common communal strains of the gastrointestinal flora.

    [00075] Strains F2064, F2076 and F1033 showed significant inhibitory activity against Candida albicans and several potentially pathogenic bacteria. On the other hand, the strains showed minimal activity against commensal strains commonly found in the gastrointestinal flora native to the genus Bacteroides. Also, strains F2064, F2076 and F1033 did not show significant inhibitory activity between them. It is worth noting that the F1033 strain is the only strain that has high inhibitory activity against Campylobacter jejuni, while the F2076 strain excels in inhibiting Escherichia coli and the F2064 strain in inhibiting Candida albicans and Proteus mirabilis. 6. Antioxidant Capacity A) Methods
    [00076] 20 μl aliquots of overnight cultures of each strain (109 cfu / ml approximately) were placed in a 96-well plate. 200 μl of MRS supplemented with 10 mM paraquat (C12H14Cl2N2, a superoxide anion donor) or 10 mM sodium nitroprusside (Na2 [Fe (CN) 5NO], a nitric oxide donor) were added to the wells and the plates were incubated at 37 ° C and 5% CO2. Optical densities at 620 nm were read 6 hours later. The results are expressed as a percentage of growth in relation to growth in the traditional MRS medium. The same protocol was followed with the L. rhamnosus GG strain and with the L. plantarum strain isolated from the commercial formulation VSL # 3 (isolation was performed using traditional procedures). B) Results
    [00077] Oxidative stress is defined as an imbalance between the generation of reactive oxygen species (ROS) and reduced antioxidant defense systems. Oxidative stress develops particularly in inflammatory reactions because inflammatory cells, neutrophils and macrophages produce large amounts of ROS (Rezaie A. et al., 2007; Roessner A. et al., 2008). Strains F1033, F2064 and F2076 showed survivability in strong oxidizing conditions comparable to that of the known probiotic strain L. rhamnosus GG, as well as that of the strain L. plantarum isolated from the formula VSL # 3. It is worth noting that the F2076 strain showed the highest resistance to both paraquat (superoxide anion donor) and sodium nitroprusside (nitric oxide donor). Resistance to oxidative stress is a desirable trait for probiotic strains that are expected to survive in the environment of an inflamed mucosa. TABLE 4: Percentage growth in medium containing 10 mM of paraquat or sodium nitroprusside, in relation to the traditional MRS medium.
    7. Strain Genotyping A) Methods
    [00078] Strains F1033, F2064 and F2076 were submitted to a previously described protocol (Rodas A. M. et al., 2005) with minimal modifications. The strains were grown on MRS agar plates and incubated at 37 ° C and 5% CO2 for 18 hours. The cells were harvested and washed 3 times in 8 ml of PET (10 mM Tris pH 7.6, 1 M NaCl) and then centrifuged at 6000 rpm for 10 minutes. The pellets were resuspended in 700 ml of lysis buffer (6 mM Tris, 1 M NaCl, 0.1 M EDTA, 0.5% SLS, 0.2% deoxycholic acid; 1 mg / ml lysozyme ; 40 U / ml mutanolysin; 20 mg / ml RNase). An equal volume of 1.6% low melting point agarose (FMC BioProducts, Rockland, ME, USA) was added to the resuspended cells and solidification was allowed to occur at 4 ° C for 1 hour. The inserts were transferred to 2 ml of lysis buffer II (0.5 M EDTA pH 9.2, 1% N-lauryl sarcosine and 1 mg / ml pronase) and incubated at 50 ° C for 48 hours. Then the inserts were washed at room temperature with TE buffer (10 mM Tris, 1 mM EDTA pH 8.0). The digestion of total DNA was carried out separately by the restriction enzymes Sfi-I and Sma-I (Roche Diagnostics).
    [00079] Pulsed field electrophoresis was performed using the CHEF DRIII device (BioRad Laboratories). The inserts were loaded onto a 1% agarose gel (SeaKem ME agarose, FMC BioProducts, ME, USA). The DNA MW markers were the Lambda ladder marker PFG Marker and the Low Range marker PFG Marker (New England Biolabs). After electrophoresis, the gels were colored with ethidium bromide and UV using the GelDoc system (BioRad). B) Results
    [00080] Figure 1 shows the pulsed field electrophoresis profiles obtained. The F1033 strain shows a genomic restriction profile similar to P. acidilactici R1001 after digestion with Sma-I. However, the genomic profile obtained after digestion with the Not-I enzyme is markedly different. On the other hand, the genomic restriction profiles of strains F2064 and F2076 are clearly different from each other and also in relation to that of the strain L. plantarum 299v and the strain L. plantarum contained in the formula VSL # 3. 8. Production of Short-Chain Fatty Acids A) Methods
    [00081] The strains were incubated for one night in a basal medium (see TABLE 5) supplemented with different fibers, each (inulin, pectin and FOS) in a specific amount, under microaerophilic conditions (5% CO2) at 37 ° Ç. Then, the cells were removed by centrifugation at 12,000 rpm for 10 minutes and the supernatants were filtered and frozen in liquid nitrogen and kept at -80 ° C until analyzed by gas chromatography, focusing on the amount of acetic acid, propionic acid and butyric acid. TABLE 5
    B) Results
    [00082] Short-chain fatty acids (SCFAs) are the end products of the breakdown of carbohydrates by anaerobic bacteria in the large intestine. SCFAs, mainly acetate, propionate and butyrate are responsible for approximately 80% of the concentration of anions in the colon and are produced in a practically constant molar ratio of 62:22:15. Among its various properties, SCFAs, especially butyric acid, but also acetic acid and propionic acid, are easily absorbed by the intestinal mucosa, are relatively high in caloric content, are metabolized by colonocytes and hepatocytes, stimulate the absorption of sodium and water in the colon and are trophic to the intestinal mucosa (D'Argenio G. et al., 1999). On the other hand, high amounts of acetic acid have long been known to be irritating to the intestinal mucosa (Yamada Y. et al., 1992). The strains F1033, F2064 and F2076 are strong producers of acetic acid, propionic acid or butyric acid. TABLE 6: Production of acetic acid, propionic acid and butyric acid by strains developed in basal medium enriched with inulin, pectin and FOS.
    9. Compatibility with treatments for IBD A) Method
    [00083] Supplemented broth was prepared by dissolving 5-aminosalicylic acid (Pentasa®, Ferring Pharmaceuticals) at the maximum soluble concentration (0.84 gr / L) and half of this concentration (0.42 gr / L) in liquid MRS broth . The strains of the invention were grown in standard MRS broth or in broth supplemented with 5-aminosalicylic acid for 4 hours at 37 ° C in microaerophilic conditions (5% CO2), and growth was evaluated by measuring the optical density at 620 nm . The results are expressed as a percentage of growth in traditional MRS medium. B) Results
    [00084] Prolonged treatment of mild to moderate symptoms of IBD is usually accomplished by the use of oral aminosalicylates (derived from 5-ASA) (Katz JA, 2007). It is therefore interesting to assess whether the probiotic strains of the invention can be co-administered with 5-ASA derivatives. Considering that the growth of none of these strains is completely inhibited by the high stringency of the conditions, it can be concluded that the co-administration of mesalazine possibly will not compromise the efficacy of the probiotic, even using saturated concentrations of mesalazine (0.84 g / L) as shown in TABLE 7: TABLE 7
    10. In Vivo Effect on Chemically Induced Intestine Inflammation A) Methods
    [00085] The therapeutic effect of the composition of the invention on mild intestinal inflammation was investigated with a repetitive oral administration for 5 days of sodium dextran sulfate (DSS) to the mouse (Okayasu I. et al., 1990). When used in a low dose (2.5-3%) for a short period of time (5 days), DSS produces mild colitis, with intestinal inflammation at the histological level but without significant macroscopic changes (for example, shortening of the colon, adhesions mesenteric).
    [00086] External symptoms include weight loss and diarrhea, with rare occurrence of blood in the stool. Therefore, this model is representative of low-grade ulcerative colitis.
    [00087] Strains F1033, F2064 and F2076 were lyophilized in sterile water with 15% skimmed milk and 4% sucrose as cryoprotectants and mixed in equal amounts (ratio in concentration 1: 1: 1).
    [00088] Eight-week-old Balb / c mice (Charles River, Barcelona, Spain), weighing 20-25 g, were kept under specific pathogen-free (SPF) conditions in an isolator (Harlan Iberica, Barcelona, Spain) at constant temperature (22 ° C) in a 12 hour light / dark cycle. Two mice acted like brothers in the same litter. The mice had free access to sterile diets (standard laboratory diet; Harlan Iberica, Barcelona, Spain) and drinking water. The mice were kept in the unit for 7 days before the beginning of the experiments (quarantine). The mice were divided into one of four groups: a) probiotic composition of the invention + DSS (n = 8); b) VSL # 3 (VSL Pharmaceuticals, USA) + DSS (n = 8); c) vehicle + DSS (n = 8); and d) vehicle + healthy controls (n = 6).
    [00089] Probiotics (or vehicle) were administered by oral gavage for ten days (day 10) before DSS administration was started (day 0). Each mouse received 2.5 x 108 cfus of probiotic daily in 0.1 mL of sterile water (vehicle) per gavage. Mice not treated with probiotic received the same volume of vehicle (distilled water with 15% skimmed milk and 4% sucrose).
    [00090] The mice were fed 3% (w / v) DSS (40kD molecular weight, Applichem Lifescience, VWR, Barcelona) in their water bowl for 5 days (days 0 to 4, followed by three days without DSS) according to a method previously described with minor modifications (Okayasu I, et al. Gastroenterol 1990). Healthy controls never received DSS.
    [00091] Clinical signs were monitored daily. The disease activity index was calculated according to the following formula and the interpretation table: DAI = ScoreWeight loss + Blood score in stool + Consistency score
    [00092] The results are shown in TABLE 8: TABLE 8 of feces

    [00093] The disease activity index score used in this invention was first described by Cooper et al. and combines several clinical symptoms into a normalized score (Cooper H. S. et al., 1993). The maximum score is 12 points. This score has been widely used to evaluate the effectiveness of experimental treatments - probiotics among them - in animal models of IBD (Fitzpatrick LR et al., 2007; Grabig A. et al., 2006; Sasaki M. et al., 2005) .
    [00094] After being sacrificed with an overdose of the inhaled halothane anesthetic (Fluotane®, Zeneca Ltd, UK), colon samples from the animals were collected and washed in cold PBS. The colon weight / length ratio was noted. The samples for cytokine measurement were frozen in liquid nitrogen and homogenized in 1 mL of cold PBS with a cocktail of inhibitory proteins (Sigma-Aldrich Chem., Spain) and centrifuged (15000 x g, 10 min). The concentrations of IL-6, IL-10, IL23p19, IFN-y and TNF-α were measured in colonic supernatants using the "Cytokine 6-Plex Assay" assay (ProcartaTM Cytokine Profiling Kit, PANOMICS, Spain) for the Luminex® platform (Luminex® Co, Austin, USA). Globules of fluorescent microparticles, previously labeled with specific antibodies to cytokines, were incubated with 50 μL of supernatant diluted 1: 5. Specific biotinylated secondary antibodies and streptavidin-phycoerritrin (S-PE) were added in sequence. Data were expressed as pg of cytokine per mg of protein (Quick Start Bradford Protein Assay, BIO-RAD, CA, USA). All measurements were made in duplicate. B) Disease Activity Index Results
    [00095] As shown in figure 2, the group that received the probiotic formula of the invention showed a significant improvement in clinical symptoms when compared to controls treated with DSS, as assessed by the disease activity index (p <0.05, ANOVA bilateral with Tukey-Kramer post-hoc test). Healthy controls also had a lower disease activity index (p <0.05). Cytokine levels
    [00096] Analysis of various cytokines in the intestinal mucosa revealed that the probiotic formula of the invention significantly decreased levels of IL-6 when compared to controls treated with DSS (p <0.01, bilateral ANOVA with Tukey-Kramer post-hoc test ), while the effect of the commercial probiotic formula VSL # 3 failed to reach significance (p> 0.05). IL-6 is a marker of acute inflammation (figure 3). As expected, IL-6 levels in healthy controls were also significantly lower than in controls treated with DSS (p <0.05). A statistically significant correlation was found between clinical symptoms (AID scores) and IL-6 levels in the intestinal mucosa (p <0.05, Spearman's correlation test) (data not shown). On the other hand, the correlation between clinical symptoms and levels of IL-10, IL-23, TNFa or IFNy was not statistically significant, and the probiotic formula of the invention did not significantly affect the levels of these cytokines. 11. In Vivo Effect on Spontaneous Intestinal Inflammation A) Methods
    [00097] The therapeutic effect of the formula of the invention was also investigated in the model of IL-10 doped mice. This model spontaneously develops intestinal inflammation at 8 to 12 weeks of age, with 80-90% penetrance (Scheinin T. et al., 2003). Interleukin 10 (IL-10) is an important regulatory cytokine that suppresses macrophage / manocyte effector functions, helper T cells (Th1), and natural killer cells. In addition, IL-10 increases B cell proliferation and differentiation. Murine models without the IL-10 gene spontaneously develop inflamed bowel disease and gastrointestinal tumors. The gastrointestinal flora was involved in the pathogenesis of these diseases since animals without germs do not develop the disease. IL-10 doped mice are widely used to evaluate new therapeutic options for IBD.
    [00098] IL-10-deficient or wild-type C57B6J mice at six weeks of age (Charles River, Barcelona, Spain) were kept under specific pathogen-free (SPF) conditions in an isolator (Harlan Ibérica, Barcelona, Spain) at constant temperature (22 ° C) in a 12 hour light / dark cycle. The mice had free access to sterile diets (the diet based on the AIN-93 standard for maintaining mice was composed of 12% water, 14.5% protein, 4% fat, 4.5% fiber and 4, 7% ash; Harlan Interfauna Ibérica SA, Barcelona, Spain) and drinking water.
    [00099] The mice were divided into one of three groups: a) probiotic formula I.3.1 (n = 12 IL-10 - / -; n = 5 wild type); b) VSL # 3 (n = 12 IL-10 - / -; n = 5 wild type); and c) vehicle (n = 12 IL-10 - / -; n = 5 wild type). Each mouse in groups "a" and "b" received 109 CFU of probiotic in sterile drinking water (vehicle) daily. Mice not treated with probiotic (placebo group) received only vehicle. Probiotics (or vehicle) were administered for ten weeks. Clinical signs were monitored daily. The disease activity index (Cooper H. S. et al., 1993) was calculated in the same way as in the DSS-induced intestinal inflammation model (see above).
    [000100] Sixteen-week-old mice were sacrificed with an overdose of the inhaled halothane anesthetic (Fluotane®, Zeneca Ltd, UK). Colon samples from the animals were collected and washed in cold PBS. Blood samples were also collected by cardiac puncture to analyze the concentration of hematocrit and hemoglobin (Analyzer with Coulter MaxM automatic loader, Izasa, Spain). The colon weight / length ratio was noted. Then the colons were frozen in liquid nitrogen and the cytokines IL-6, and IFNy were measured using the same protocol as that used in the DSS-induced intestinal inflammation model (see above). B) Disease Activity Index Results
    [000101] As shown in figure 4, a significant delay in the appearance of clinical symptoms was observed both in the group treated with the composition of the invention and in the group treated with the commercial formula VSL # 3 when compared to vehicle-treated controls (p <0 , 01, bilateral ANOVA with Tukey-Kramer post-hoc test). In addition, the treated groups tended to have lower scores on the disease activity index, although the difference did not reach significance (data not shown). Cytokine Levels
    [000102] Analysis of various cytokines revealed that the probiotic composition of the invention significantly reduced levels of IFNy in doped mice when compared to doped mice treated with vehicle (p <0.01, bilateral non-parametric ANOVA with Dunn's post-hoc test ) and the commercial formula VLS # 3 (p <0.05). In fact, as shown in figure 5, IFNy levels reached the same levels as those of healthy wild-type controls. Additionally, as inferred from figure 6, there was also a clear tendency for probiotic formulas to reduce IL-6 levels, although the results have not reached significance due to the large standard deviation among vehicle-doped mice.
    [000103] A significant correlation was observed between the severity of clinical symptoms (disease activity index) and IFNy levels at the end of the study in the colon mucosa measured after sacrifice (p <0.05, Spearman's correlation test ) (data not shown). Probiotic Formula Safety
    [000104] Clinical signs (weight loss, altered behavior, appearance of hair, diarrhea and blood in the stool) were monitored daily in wild-type mice receiving daily doses of the invention's probiotic formula, VSL # 3 formula or vehicle for 10 weeks. No signs of morbidity were detected during the study. After sacrificed, the animals were submitted to gross necropsy. Analysis of all important cavities and organs did not reveal any pathological changes (data not shown). 12. In Vivo Efficacy in Individuals with IBS A) Study Design Methods
    [000105] A multicenter, randomized, double-blind, placebo-controlled clinical trial to study the effect of the composition on patients with IBS was conducted.
    [000106] Hydroxymethyl propyl cellulose capsules were filled with: (1) 150 mg of maltodextrin, (2) 5 mg of magnesium stearate, (3) 5 mg of silicon dioxide and (4) 200 mg of a mixture 1: 1: 1 of the three strains of the invention (at a concentration of 5 ^ 101 ° cfus / capsule). In addition, a placebo was made with the same list of excipients and amounts but without including the composition of the invention. The contents of the capsules during the study ranged from 5 ^ 101 ° to M ° 1 ° cfus.
    [000107] 33 eligible adult patients of both sexes meeting the Rome III criteria for irritable bowel syndrome (Longstreth GF et al., 2 °° 6) were admitted and randomly assigned to one of the following treatments for 6 weeks: a) the capsule including the composition of the invention once daily (n = 18); and b) the placebo capsule once a day (n = 15). The study was conducted in accordance with the Helsinki Declaration for Clinical Trials and approved by the appropriate Ethics Committee. Evaluation of Effectiveness
    [000108] The main objective of this study was the global effect on health-related quality of life (hereinafter also called "HRQOL"), assessed using a specific questionnaire for IBS: the validated Spanish version of the IBSQOL questionnaire (Badia X. et al., 2000). Obeying the guidelines of the Spanish Association of Gastroenterology, the scores were standardized on a scale of 0-100. The secondary objective was to assess anxiety associated with gastrointestinal sensations and symptoms using a questionnaire validated for the visceral sensitivity index (hereinafter also called "VSI") (Labus J. S. et al., 2004). Volunteers were instructed to complete these questionnaires at baseline (day 1), on day 21 and day 42. Data were assessed by analyzing intention to treat. The results are shown in figures 7 and 8. B) Characteristic Results at Baseline
    [000109] No significant difference was evident between groups in terms of characteristics at baseline, as can be seen in Table 9, indicating that individuals in both groups were comparable in terms of the variables assessed. The groups were also comparable in terms of baseline blood biochemical parameters, anthropometric parameters, age and sex. TABLE 9: Baseline scores for both treatment groups

    [000110] The composition of the invention significantly improved the health-related quality of life over placebo when assessed after 21 days and after 42 days of treatment (p <0.05, T test). Therefore, it has been shown that the composition of the present invention significantly reduces morbidity and improves the quality of life of individuals with IBS far more than the placebo effect. The positive effects of the composition include mastery of discomfort associated with food, anxiety, interference with daily activities and sleep disturbances in the HRQOL questionnaire. The improvements in these items suggest a reduction in abdominal pain, discomfort and altered bowel habits. As far as we know, this is the first time that a probiotic composition has been shown showing a significant effect on the general quality of life associated with the health of patients with IBS. Improvement in the Visceral Sensitivity Index (figure 8)
    [000111] The composition of the present invention significantly reduced the specific visceral sensitivity of gastrointestinal symptoms of individuals with IBS compared to placebo. The effect was almost significant after 21 days of treatment, and clearly significant after 42 days of treatment (p <0.01, T test), further confirming the usefulness of the composition of the present invention in the treatment of IBS. The most marked improvement was seen in abdominal discomfort and in items associated with swelling in the questionnaire. In particular, TABLE 10 shows the number of individuals who reported significant improvement in relation to swelling and distention (defined by an increase of at least two points compared to baseline on the 6-point scale of the VSI questionnaire that measures anxiety associated with swelling and distention) at the end of treatment. The difference between the two groups is statistically significant (p <0.05, Fisher's exact test). TABLE 10. Effect on anxiety associated with bloating and bloating, according to the VSI questionnaire, after 42 days of treatment

    [000112] From the results obtained, it is therefore concluded that the composition of the invention is effective in the treatment of abdominal distension and bloating. 13. Effect on Abdominal Bloating and Reduced Intestinal Function
    [000113] A 25-year-old woman suffered from chronic abdominal swelling and altered intestinal motility, sometimes reporting only one bowel movement per week. The diagnosis revealed a hypotonic and hypokinetic stomach, with no evidence of other structural changes in the gastrointestinal tract.
    [000114] The patient underwent a treatment of one capsule a day (like those described in example 12). After a week of treatment, the patient reported a significant reduction in abdominal swelling and distension and a normalization of bowel habits. The symptoms reappeared after the treatment was stopped for a few days. After restarting the treatment in the form of a capsule every two days, the patient again reported a noticeable and lasting positive effect on both bloating and bowel habits.
    [000115] This example further confirms the use of the composition of the invention to treat abdominal bloating and altered intestinal motility in individuals who are not classified as having irritable bowel syndrome. BIBLIOGRAPHIC REFERENCES
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    权利要求:
    Claims (7)
    [0001]
    1. Composition, characterized by the fact that it comprises: (a) from 105 cfu to 1012 cfu of the isolated strain of Lactobacillus plantarum deposited in the Spanish Type Culture Collection - Colección Espanola de Cultivos Tipo (CECT) under access number CECT 7484, by gram weight of the composition; (b) from 105 cfu to 1012 cfu of the isolated strain of Lactobacillus plantarum deposited in the Spanish Type Culture Collection (CECT) under accession number CECT 7485, per gram of weight of the composition; and 105 cfu to 1012 cfu of the isolated strain of Pediococcus acidilactici deposited in the Spanish Type Culture Collection (CECT) under accession number CECT 7483, per gram of weight of the composition, and (c) at least one pharmaceutically acceptable excipient, at least a pharmaceutically acceptable excipient for veterinary use and / or at least one edible ingredient.
    [0002]
    2. Composition according to claim 1, characterized by the fact that the isolated strain was grown in a suitable culture medium and post-treated after cultivation to obtain a biologically pure culture, and in which the resulting biologically pure culture is in the form of a liquid or solid.
    [0003]
    3. Composition, according to claim 2, characterized by the fact that the post-treatment is lyophilization.
    [0004]
    4. Use of the composition as defined in claim 1, characterized by the fact that it is in the manufacture of a medicine for the prevention and / or treatment of inflammation of the intestine, in which the inflammation of the intestine is Inflammatory Bowel Disease or Irritable Bowel Syndrome .
    [0005]
    5. Pharmaceutical product, characterized in that it comprises the composition as defined in claim 1, together with pharmaceutically acceptable excipients.
    [0006]
    6. Veterinary product, characterized by the fact that it comprises the composition as defined in claim 1, together with acceptable veterinary excipients.
    [0007]
    7. Edible product, characterized by the fact that it comprises the composition as defined in claim 1, together with other edible ingredients.
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    同族专利:
    公开号 | 公开日
    CN102905712A|2013-01-30|
    AU2011209407A1|2012-09-20|
    ZA201205683B|2013-08-28|
    UA109123C2|2015-07-27|
    EP2528610A1|2012-12-05|
    CL2012002062A1|2013-10-04|
    MX2012008817A|2012-11-23|
    BR112012018813A2|2017-06-20|
    KR20130002372A|2013-01-08|
    KR101840239B1|2018-03-20|
    CO6511276A2|2012-08-31|
    US20160220620A1|2016-08-04|
    CA2787544C|2019-07-09|
    EP2528610B1|2013-11-13|
    JP2013517784A|2013-05-20|
    CN102905712B|2015-12-16|
    RU2563525C2|2015-09-20|
    US10155015B2|2018-12-18|
    AU2011209407B2|2014-02-27|
    PL2528610T3|2014-02-28|
    RU2012136615A|2014-03-10|
    US20130101566A1|2013-04-25|
    JP5777640B2|2015-09-09|
    ES2437940T3|2014-01-15|
    MY162344A|2017-06-15|
    CA2787544A1|2011-08-04|
    WO2011092261A1|2011-08-04|
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    法律状态:
    2017-10-24| B07D| Technical examination (opinion) related to article 229 of industrial property law|
    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)|
    2019-05-14| B06T| Formal requirements before examination|
    2019-09-17| B07A| Technical examination (opinion): publication of technical examination (opinion)|
    2020-03-10| B07A| Technical examination (opinion): publication of technical examination (opinion)|
    2020-08-18| B09A| Decision: intention to grant|
    2020-12-08| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 27/01/2011, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US29911610P| true| 2010-01-28|2010-01-28|
    EP10151998.1|2010-01-28|
    EP10151998|2010-01-28|
    US61/299,116|2010-01-28|
    PCT/EP2011/051170|WO2011092261A1|2010-01-28|2011-01-27|Probiotic composition for use in the treatment of bowel inflammation|
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