USE OF A PROBIOTIC FORMULATION COMPRISING A BIFIDOBACTERIUM BIFIDUM CEPA

专利摘要:
use of a probiotic formulation of a bifidobacterium bifidum strain for the treatment of gastrointestinal diseases. the present invention relates to a strain of bifidobacterium bifidum which shows at least an adhesion of about 10 bacterial cells per mm² of epithelial cell monolayer or having at least an adhesion index of 1.5 and a strain of bifidobacterium bifidum which is bifidobacterium bifidum mimbb75 deposited under deposit no. dsm 24514 for use as a probiotic in food and/or as a medicine. A probiotic formulation is provided which comprises any of the aforementioned strains, mutants or variants, uses of said probiotic formulation, strains, mutants and variants thereof, and a method of producing said probiotic formulation.
公开号:BR112013019258B1
申请号:R112013019258-5
申请日:2012-01-27
公开日:2021-09-08
发明作者:Simone Guglielmetti；Diego Mora
申请人:Synformulas Gmbh；
IPC主号:

专利说明:

[0001] To date, diseases which are correlated with a functional disorder of the gastrointestinal tract and/or which are correlated with undesirable gastrointestinal inflammatory activity are common diseases with widespread prevalence. Diseases of both groups are characterized by abdominal pain, discomfort, distension, flatulence, diarrhea, constipation, digestive disorder, urgency and/or reduction and/or increase in the number of bowel movements, feeling of incomplete bowel movement and/or combinations of these, leading to significantly reduced overall well-being of affected people. In particular, in relation to Irritable Bowel Syndrome (IBS) an effective treatment is still lacking (Brenner and Chey, 2009). Several drugs for IBS have been withdrawn or restricted due to adverse health effects. In addition, current therapies are primarily aimed at individual symptoms and do not alleviate IBS and improve quality of life (QoL) in general.
[0002] Diseases that are correlated with a functional disorder of the gastrointestinal tract, such as IBS or inflammatory bowel disease, are believed to be associated (i) with an imbalance of the gut microbiota (Kassinen et al., 2007) and/ or (ii) a dysfunctional intestinal barrier in the gastrointestinal tract (Marshall et al., 2004). This is believed to result in the reduction of probiotic bacteria, such as Bifidobacteria, in the gastrointestinal tract. Furthermore, it is believed that a dysfunctional intestinal barrier causes the undesirable entry of facultative pathogenic bacteria into the mucosa, which can lead to gastrointestinal inflammatory activity subsequently. Recent studies propose that this inflammation causes symptoms typical of IBS, such as pain, motility and bowel dysfunction.
[0003] An imbalance of the intestinal microbiota is mainly characterized by a reduction in the number of non-pathogenic microorganisms of human origin, such as Bifidobacteria, while the number of pathogenic microorganisms, such as facultative Enterobacteria, is increased (Jian-Min et al. , 2004). This imbalance is believed to lead to increased production of gaseous compounds and short-chain fatty acids, gastroparesis, flatulence and pain in affected people. Thus, by bringing the intestinal microbiota back into balance, the probiotic bacteria came into focus. They likely have beneficial effects on immune function through competitive inhibition and may beneficially affect the host, improving the intestinal microbial balance. "Probiotics" comprising probiotic bacteria of non-pathogenic human origin were defined as live microbial foods and drugs which, when ingested in a certain number, exert effects on health, in addition to inherent basic nutrition. Mixtures of various microorganisms, particularly Lactobacillus and Streptococcus species, have traditionally been used in fermented dairy products or pharmaceuticals to promote health.
[0004] Many studies have been conducted on this issue, however, since an imbalance of the gut microbiota is only one side of the coin, it is not surprising that most therapies which focus simply on delivering probiotics to the gastrointestinal tract have not been successful in alleviating the symptoms of patients suffering from a functional disorder of the gastrointestinal tract. For example, IBS is diagnosed by the Rome III criteria, which are basically: abdominal pain or discomfort on at least 3 days a month for the past three months with symptoms which started at least 6 months ago combined with at least two criteria of ( a) ease through defecation, (b) onset associated with a change in stool frequency or (c) in stool consistency and is often accompanied by swelling or distension and difficulty in evacuating or constipation and/or diarrhea. Along with these symptoms, patients may suffer from compromised social and personal function and decreased quality of life. However, even if symptoms such as pain/discomfort, distension/swelling, urgency are largely removed, an improvement in the quality of life (QoL) related to the patient's overall health is very desirable. To date, none of the available treatments, including probiotics and others, have been able to improve symptoms of pain/discomfort, distension/swelling, and digestive upset simultaneously with QoL.
[0005] In this regard, the second important aspect in the development of a functional disorder of the gastrointestinal tract has to be considered, which refers to a dysfunctional intestinal barrier of the gastrointestinal tract characterized by an increase in its permeability. A very popular theory in this regard is that changes in the gut and systemic immune system allow pathogenic microorganisms - which could dominate in the event of an imbalance in the gut microbiota - to cross the barrier and penetrate the mucosa, causing inflammatory reactions. Based on this hypothesis, several studies have been conducted that focus on the treatment and relief of the inflammatory reaction and its effects on the gastrointestinal tract. EP 1141235, for example, describes a UCC35624 strain of Bifidobacterium longum infantis (B. infantis) for application in nutritional supplements and drugs that have demonstrated a strong ability to stimulate an anti-inflammatory response in the host by decreasing the levels of IL-8 (Whorwell et al., 2006) and normalization of the IL-10/IL-12 ratio (Mahony et al., 2005). It is said that this ability may be the main reason for the effect of the UCC35624 strain of Bifidobacterium longum infantil on IBS and symptoms of inflammatory bowel disease. However, different sources question whether the effect of B. infantis can be attributed to direct anti-inflammatory properties. Furthermore, although Lactobacillus salivarius has also shown strong anti-inflammatory effects in the in vitro and mouse model - similar to B. infantis - it does not show significant effects in relieving IBS and its symptoms.
[0006] More importantly, the effectiveness of probiotics is strongly strain-dependent and only certain strains may be able to improve IBS and its symptoms. To date, several studies have examined the effects of probiotics on symptoms of diseases which are correlated with a functional disorder of the gastrointestinal tract, such as IBS (Mahony et al., 2005; Kajander et al., 2005; Williams et al., 2008; Guyonnet et al., 2007). However, only a few could show significant benefit. Furthermore, no probiotic strain has shown a significant effect in alleviating the symptoms of irritable bowel syndrome and simultaneously improving quality of life. These results could be attributed to the fact that the effectiveness of probiotics is strongly strain-dependent. It is widely accepted that the properties of one probiotic strain cannot be transferred to another (Brenner and Chey, 2009). Therefore, it is important to recognize that the effectiveness and effects of a single probiotic strain cannot be predicted, which highlights the importance of testing and proving the effectiveness of a single strain. For this purpose, methods are described in the experimental section.
[0007] Never before has a probiotic study shown a reduction in the symptoms mentioned above (pain / distension / bloating / urgency / digestive disturbance) and a simultaneous improvement in quality of life in a patient with a disease such as IBS. Thus, there is a significant need for a therapy that improves the symptoms of pain/distension/bloating, urgency and digestive upset and the overall quality of life of a patient who is suffering from a functional disorder of the gastrointestinal tract, where the intestinal microbiota is brought back to balance and also the functionality of the intestinal wall is restored.
[0008] Therefore, the present invention provides a strain of Bifidobacterium bifidum (B. bifidum) or a mutant or variant thereof for use as a probiotic in food and/or as a medicine that shows at least an adhesion of about 10 bacterial cells per mm2 of epithelial cell monolayer or having at least an adhesion index of 1.5 and a strain of Bifidobacterium bifidum or mutant or variant thereof for use as a probiotic in food and/or as a medicine which is Bifidobacterium bifidum MTMBb75 deposited under DSM Deposit No. 24514 or a mutant or variant thereof that shows not only a significant improvement in symptoms of abdominal pain/discomfort, distension/swelling, urgency and/or digestive disorder of a gastrointestinal disease, in particular of IBS but, on the other hand, it simultaneously leads to a significant increase in the patient's quality of life. A probiotic formulation and uses of the strain and probiotic formulation are further provided. The relief of IBS in general, together with an improvement in symptoms of pain/discomfort, distension/swelling, urgency and/or digestive disturbance with a simultaneous improvement in QoL with the strains of the invention, such as a strain of Bifidobacterium bifidum or mutant or variant thereof showing at least an adhesion of about 10 bacterial cells per mm2 of epithelial cell monolayer or having at least an adhesion index of 1.5 or a strain of Bifidobacterium bifidum or mutant or variant which is deposited Bifidobacterium bifidum MIMBb75 under DSM Deposit No. 24514 or a mutant or variant thereof was an unexpected result, as it is known and accepted in the prior art that the efficacy of a probiotic strain is highly specific and even closely related members of a probiotic strain. Bifidobacterium strains typically do not have comparable properties or are even inactive in gastrointestinal diseases (Brenner and Chey, 2009). Without intending to be bound by theory, the inventors believe that the superior and unexpected efficacy of the Bifidobacterium bifidum strain of the invention, and in particular of Bifidobacterium bifidum MIMBb75, is due to its ability to prevent the passage of pathogenic bacteria through the intestinal barrier of the gastrointestinal tract and its penetration into the mucosa. Admitting that the dysfunction of the intestinal barrier is due to "holes" or weakened parts in the intestinal wall through which pathogenic microorganisms can enter the mucosa, the inventors believe that the adhesion of non-pathogenic cells to the intestinal wall reduces the permeability of the barrier, thus, improving the intestinal wall in a "hole-closure" mode of action. In this way, the probiotic strain and formulation of the invention have a double positive effect on the gastrointestinal flora and health not only because mucosal inflammatory reactions are prevented, but also the intestinal microbiota is brought back into balance. Furthermore, it is very important that a reduction in symptoms or the disease itself is also maintained during the discontinuation phase, that is, after the final phase of therapy where, for example, no more strains, mutants or variants of the present invention or a probiotic formulation comprising the strain, mutant or variant of the invention is administered to the individual or patient. In the case of the strain, mutant or variant of the invention, the positive effect of the strain, mutant or variant of the invention is maintained in the case of global symptoms of IBS, abdominal pain/discomfort, distension/swelling and digestive disturbance during the interruption phase, as will be explained in detail below and also presented in the Examples and Figures section.
[0009] Thus, the present invention provides a strain of Bifidobacterium bifidum or a mutant or variant thereof for use as a probiotic in food and/or as a medicine, the strain, mutant or variant thereof showing at least an adhesion of about 10 bacterial cells per mm2 of epithelial cell monolayer or having at least an adhesion index of 1.5.
Preferably, said strain is Bifidobacterium bifidum MIMBb75 deposited under Deposit No. DSM 24514 or a mutant or variant thereof. Also, a strain of Bifidobacterium bifidum or mutant or variant thereof for use as a probiotic in food and/or as a medicine is provided, the strain being Bifidobacterium bifidum MIMBb75 deposited under Deposit No. DSM 24514 or a mutant or variant thereof . Said strain, mutant or variant is especially for use as a probiotic in food and/or as a medicine for the prophylaxis and/or treatment of a disease related to a functional disorder of the gastrointestinal tract, undesirable gastrointestinal inflammatory activity, an imbalance of intestinal microbiota, a reduction of Bifidobacteria in the gastrointestinal tract and/or a dysfunctional intestinal barrier in the gastrointestinal tract and/or for use in the relief, prevention and/or treatment of an individual who is suffering from abdominal pain, discomfort, distension, swelling, urgency, digestive disturbance and/or reduced and/or increased number of bowel movements, feeling of incomplete bowel movement, global symptoms of IBS and/or combinations thereof, or improvement in a patient's quality of life. The usefulness of the strain, mutant or variant as a probiotic in food and/or as a medicine in the relief, prevention and/or treatment of an individual who is suffering from abdominal pain, discomfort, distension, digestive disorders, bloating, urgency and/or number of reduced and/or increased bowel movements, feeling of incomplete bowel movement, global symptoms of IBS and/or combinations thereof can be assessed using a 7-point Likert scale, which is commonly used in clinical studies and will be further described below. The patient's quality of life (QoL) can be assessed using the standard SF-12 questionnaire, which is also common for those skilled in the art in clinical trials and an accepted standard for measuring QoL. Furthermore, in case of global symptoms of IBS, abdominal pain/discomfort, distension/swelling, the positive effect of the strain, mutant or variant of the present invention is maintained during the discontinuation phase, as will be explained in detail below and is also presented in the Examples and Figures section.
[00011] In one embodiment, the disease is a member selected from the group consisting of irritable bowel movement, inflammatory bowel disease, eg, ulcerative colitis or Crohn's disease, irritable bowel syndrome, pouchitis or post-infection colitis, gastrointestinal cancer, a systemic disease such as rheumatoid arthritis, an autoimmune disorder due to undesirable inflammatory activity, diarrheal diseases due to undesirable inflammatory activity such as diarrhea associated with Clostridium difficile, diarrhea associated with antibiotics, infectious diarrhea associated with Rotavirus or post-infection diarrhea and combinations thereof; preferably the disease is irritable bowel movement.
[00012] In one embodiment, the strain, mutant or variant of the invention is a genetically modified mutant or the strain, mutant or variant is a naturally occurring variant. In alternative embodiments, cells of the strain, mutant or variant are viable or not viable. With non-viable cells, product preparation is simpler, cells can be easily incorporated into drugs, and storage requirements are much less limited than in the case of viable cells. Cells can be thermally inactivated or through exposure to an altered pH or subjection to pressure. However, viable cells are capable of inhabiting the gastrointestinal system of an individual who is suffering from a disease or symptom as described herein and therefore have greater effectiveness in alleviating, preventing and/or treating an individual who is suffering from it. disease or symptom.
[00013] The invention further provides a probiotic formulation comprising the strain of Bifidobacterium bifidum or mutants or variants thereof for use as a probiotic in food and/or as a medicine, the strain or mutant or variant showing at least an adhesion of about 10 bacterial cells per mm2 of epithelial cell monolayer or having at least an adhesion index of 1.5, such as Bifidobacterium bifidum MIMBb75, deposited under Deposit No. DSM 24514 or a mutant or variant thereof. Preferably, the probiotic formulation is for use as a probiotic in food and/or as a medicine.
[00014] In a preferred embodiment, the probiotic formulation further comprises at least one prebiotic. Preferably, the prebiotic is inulin or a fructooligosaccharide.
[00015] In any of the modalities mentioned above, the probiotic formulation further comprises at least one pharmaceutically acceptable compound, an ingestible carrier, an adjuvant, a bacterial component, a drug entity, a biological compound and/or a protein and/or peptide , in particular a protein and/or peptide which is rich in glutamine/glutamate, a lipid, a carbohydrate, a vitamin, minerals and/or trace elements; optionally, said at least one pharmaceutically acceptable compound is a member selected from the group consisting of one or more vitamins, such as group B vitamins, one or more minerals, such as calcium or magnesium, one or more carbohydrates, such as lactose , maltodextrin, inulin, dextrose, mannitol, maltose, dextrin, sorbitol, fructose and a mixture thereof. Preferably, the ingestible carrier is a capsule, tablet, powder or food product; optionally, the food product is a dairy product, sour milk, yogurt, frozen yogurt, yogurt production, such as fermented yogurt drink, drinking yogurt, cheese, fermented cream, desserts based on fermented milk or humanized milk, milk in powder, milk concentrate, curd, sauce, beverages and others.
[00016] In one embodiment, the probiotic formulation further comprises a protein and/or peptide, in particular a protein and/or peptide that is rich in glutamine/glutamate, a lipid, a carbohydrate, a vitamin, minerals and/or trace elements.
[00017] In any of the above embodiments, the strain, mutant or variant may be present in the probiotic formulation in more than 101 cfu per capsule or tablet or per unit of food product or powder, optionally at least 102, 103, 104 105 or 106 Colony Forming Units (cfu) per capsule or tablet or per unit of food product or powder, preferably the strain, mutant or variant thereof is present at more than 107 cfu per capsule or tablet or per unit food product or powder, more preferably the strain, mutant or variant is present at more than 108 cfu per capsule or tablet or per unit food product or powder, even more preferably the strain, mutant or variant is present in more than 109 cfu or more than 1010 cfu per capsule or tablet or per unit of food product or powder. A "powder unit" indicates an amount of powder comprising more than 101 cfu, optionally at least 102, 103, 104, 105 or 106 Colony Forming Units (cfu), preferably more than 107 cfu, more preferably more than 108 CFU, even more preferably more than 109 cfu or more than 1010 cfu. Thus, a unit of powder represents the amount which comprises a patient's normal daily dose; however, the daily dose can be increased or decreased for various reasons and in various ways which are described below. A typical amount by weight of a unit of powder should be easily digestible and ingestible, ranging from mg to several grams, eg 5 g, 10 g, 15 g or more. A powder unit can also be mixed with any food product mentioned herein and also with another food product which is considered suitable by those skilled in the art. Thus, the amount of cfu per weight amount of powder can be much greater than the standard daily dose in an amount of powder which is considered to be easily ingestible and digestible. Here, the powder has to be diluted with other desired components in the probiotic formulation as described above. A "food product unit" or "food product" generally indicates a typical quantity, by weight or volume, which is considered to be the normal or typical quantity of the particular food product used as an ingestible vehicle, a typical proposed quantity. as a consumption portion to be food or drink, a packaging unit or comparable. An "edible vehicle unit" therefore denotes a tablet, capsule, suppository or the like or a powder unit or food product unit, which denotes a typical quantity, by weight or volume, which is considered to be the normal quantity. or typical of the particular food product used as an ingestible vehicle, the typical amount proposed as the consumable portion to be food or drink, a packaging unit, or the like.
[00018] In any of the above embodiments, the daily dose of the strain, mutant or variant administered to a patient in the probiotic formulation is not less than 101 cfu per capsule or tablet or per unit of food product or powder, optionally at least 102 , 103, 104, 105 or 106 cfu, preferably not less than 107 cfu, more preferably not less than 108 cfu, even more preferably not less than 109 cfu or not less than 1010 cfu.
[00019] The probiotic formulation according to any of the above modalities is useful and provided for use as a probiotic in food and/or as a medicine for the prophylaxis and/or treatment of a disease related to a functional disorder of the gastrointestinal tract, to inflammatory activity undesirable gastrointestinal, to a reduction of Bifidobacteria in the gastrointestinal tract and/or a dysfunctional intestinal barrier in the gastrointestinal tract and/or for the relief, prevention and/or treatment of an individual who is suffering from abdominal pain, discomfort, distension, bloating, digestive disorder , urgency and/or reduced and/or increased number of bowel movements, feeling of incomplete bowel movement, global symptoms of IBS and/or combinations thereof. Optionally, the disease is a selected member of the group consisting of irritable bowel movement, inflammatory bowel disease, eg ulcerative colitis or Crohn's disease, irritable bowel syndrome, pouchitis or post-infection colitis, gastrointestinal cancer, a disease systemic, such as rheumatoid arthritis, an autoimmune disorder due to undesirable inflammatory activity, diarrheal disease due to undesirable inflammatory activity, such as Clostridium difficile-associated diarrhea, antibiotic-associated diarrhea, rotavirus-associated diarrhea or post-infection diarrhea, and combinations of same. Preferably, the disease is irritable bowel movement.
[00020] The invention also provides uses of the strain, mutant or variant of the invention and the probiotic formulation of the invention as a probiotic in food and/or as a medicine for the prophylaxis and/or treatment of a disease associated with a functional disorder of the gastrointestinal tract , unwanted gastrointestinal inflammatory activity, a reduction of Bifidobacteria in the gastrointestinal tract and/or a dysfunctional intestinal barrier in the gastrointestinal tract and/or to relieve, prevent and/or treat an individual who is suffering from abdominal pain, discomfort, distension, bloating , digestive disturbance, urgency and/or reduced and/or increased number of bowel movements, feeling of incomplete bowel movement and/or combinations thereof. Optionally, the disease is a selected member of the group consisting of irritable bowel movement, inflammatory bowel disease, eg ulcerative colitis or Crohn's disease, irritable bowel syndrome, pouchitis or post-infection colitis, gastrointestinal cancer, a disease systemic, such as rheumatoid arthritis, an autoimmune disorder due to undesirable inflammatory activity, diarrheal disease due to undesirable inflammatory activity, such as Clostridium difficile-associated diarrhea, antibiotic-associated diarrhea, rotavirus-associated diarrhea or post-infection diarrhea, and combinations of same.
[00021] Also provided is a method for producing the probiotic formulation of the invention comprising at least the following steps: a) fermentation and/or growth of the strains or mutants or variants of the invention, preferably a strain or mutant or variant showing at least an adhesion of about 10 bacterial cells per mm2 of epithelial cell monolayer or having at least an adhesion index of 1.5 or the MIMBb75 strain of Bifidobacterium bifidum, deposited under Deposit No. DSM 24514 or a mutant or a variant of the same, in a protein-rich liquid growth medium, b) collection of the cells by means of centrifugation, stabilization, lyophilization, grinding and sieving of the cells, c) optionally mixing/combining the cells with one or more members of the group consisting of in a prebiotic, a pharmaceutically acceptable compound, an adjuvant, a bacterial component, a drug entity, a biological compound, a protein and/or peptide or the like, and d)int production of the cells from step b) or the mixture from step c) in an ingestible vehicle.
[00022] Bifidobacterium bifidum strain or mutant or variant thereof for use as a probiotic in food and/or as a drug showing at least an adhesion of about 10 bacterial cells per mm2 of epithelial cell monolayer or having at least one index of adhesion of 1.5, such as Bifidobacterium bifidum MTMBb75 or a mutant or variant thereof, deposited under Deposit No. DSM 24514 and Bifidobacterium bifidum strain or mutant or a variant thereof for use as a probiotic in food and/ or as a drug which is Bifidobacterium bifidum MIMBb75 deposited under Deposit No. DSM 24514 or a mutant or variant thereof (both strains are referred to as the "strains" or "cells" of the invention) belong to the genus of bifidobacteria, as which are gram-positive, non-motile, often branched anaerobic bacteria usually present in the gastrointestinal tract and vagina. They are generally isolated from the feces of healthy newborns and adults. When referring to strains, cells from the strain are also considered. Bifidobacteria have been described to aid digestion, are associated with a lower incidence of allergies, and may also play a role in preventing some forms of tumor growth. Bifidobacteria belong to the Bifidobacteriaceae family of Actinobacteria. Bifidobacterium bifidum MIMBb75 is obtained from the Industrial Microbiology Culture Collection, DiSTAM, University of Milan, Milan, Italy and a deposit of Bifidobacterium bifidum MIMBb75 was made at the DSMZ (Deutsche Sammlung von und Mikroorganismen Zellkulturen GmbH, Braunschweig, Germany) on 26 January of 2011 and deposited under Deposit No. DSM 24514. The strains or mutants or variants of Bifidobacterium bifidum of the invention have inhibitory activity against a wide range of Gram positive and Gram negative bacteria. The strains, mutants or variants of Bifidobacterium bifidum herein invention exhibit a broad spectrum of activity against bacteria, including Staphylococcus, Pseudomonas, coliforms and Bacillus species. A "mutant" of the strain of the present invention denotes a strain of Bifidobacterium bifidum which has been modified, i.e. mutated, by conventional molecular biology methods which are known to those skilled in the art. A "variant" of the strain of the invention is a naturally occurring variant, i.e. a closely related strain of Bifidobacterium bifidum that occurs in the gastrointestinal tract and vagina of healthy infants and adults. The mutant and variant strain of the invention have the same beneficial properties as the Bifidobacterium bifidum strain of the invention, and in particular the MIMBb75 strain of Bifidobacterium bifidum, for patients suffering from any disease or symptom described herein. The variant and the mutant are also considered to be the strain's DNA homologues. Within the scope of the present invention, the term "homologous" is used in reference to strains that share a certain degree of "homology", i.e. "identity" or "similarity" at the level of chromosomal DNA. There are several algorithms to determine this degree of homology or similarity. Preferably, homology can be determined by means of Lasergene software from DNA star Inc., Madison, Wisconsin (USA) using the CLUSTAL method (Higgins et al., 1989, Comput. Appl. Biosci., 5(2)) , 151). An organism that is "homologous" (= essentially similar) at the chromosomal DNA level has at least 55% or 60%, preferably at least 65% or 70%, more preferably at least 75% or 80%, even more preferably at at least 85% or 90% and more preferably at least 92%, 94%, 95%, 96%, 97%, 98% or 99% homology at the chromosomal DNA level.
[00023] A "disease" in the sense of the invention is any condition of an individual, also referred to as a patient or human being, which is altered compared to the condition of an individual who is considered healthy. In general, a disease of the invention is correlated with a functional disorder of the gastrointestinal tract, undesirable gastrointestinal inflammatory activity, an imbalance of the intestinal microbiota, reduction of bifidobacteria in the gastrointestinal tract, a dysfunctional intestinal barrier of the gastrointestinal tract and/or combinations thereof . Typical diseases which are targeted by the strains, mutants, variants, probiotic formulation and the uses thereof are all diseases of the gastrointestinal tract or digestive system, such as irritable bowel movement, inflammatory bowel disease, eg ulcerative colitis or Crohn's disease, irritable bowel syndrome, pouchitis or post-infection colitis, gastrointestinal cancer, a systemic disease such as rheumatoid arthritis, an autoimmune disorder due to undesirable inflammatory activity, diarrheal disease due to undesirable inflammatory activity such as diarrhea associated with Clostridium difficile, antibiotic associated diarrhea, rotavirus associated diarrhea or post-infection diarrhea or similar and combinations thereof. A "symptom" or "individual symptom" in the sense of the invention is the way in which a disease manifests itself in an individual, such as abdominal pain, discomfort, distension, bloating, digestive disturbances, urgency and/or reduced number of bowel movements and/ or increased, feeling of incomplete evacuation, global symptoms of IBS and/or combinations thereof, and a reduction in overall quality of life. "Global symptoms of IBS" comprises a global assessment of all individual symptoms mentioned above in relation to IBS. A "patient" (= person or human or animal) is a healthy or unhealthy individual who ingests the probiotic formulation of the invention or the strain or mutant or variant of Bifidobacterium bifidum that shows at least an adhesion of about 10 per cell bacterial epithelial cell monolayer mm2 or having at least an adhesion index of 1.5 or a therapy with the strain or mutant or a variant of Bifidobacterium bifidum which is Bifidobacterium bifidum MIMBb75 deposited under Deposit No. DSM 24514 or a mutant or variant thereof for preventive reasons, which is not suffering from any disease or any symptom mentioned herein or which ingests the probiotic formulation of the invention or the strain of Bifidobacterium bifidum or mutant or variant which shows at least an adhesion of about 10 cells bacteria per mm2 of epithelial cell monolayer or having at least an adhesion index of 1.5 or a therapy with the Bifidobacterium bifidum strain or mutant or a var as Bifidobacterium bifidum MIMBb75 deposited under Deposit No. DSM 24514 or a mutant or variant thereof, for reasons of treatment of the patient, person, human or animal who is suffering from a disease or any symptom mentioned herein, such as a disease of the gastrointestinal tract or correlated with a functional disorder of the gastrointestinal tract, undesirable gastrointestinal inflammatory activity, an imbalance of the intestinal microbiota, reduction of Bifidobacteria in the gastrointestinal tract and/or a dysfunctional intestinal barrier of the gastrointestinal tract or a symptom, such as abdominal pain, discomfort, distension, flatulence, digestive disturbances, urgency and/or reduced and/or increased number of bowel movements, feeling of incomplete bowel movement and/or combinations thereof, or a reduced quality of life. Thus, a patient (= person) who needs a new therapy, such as a therapy with the strain of Bifidobacterium bifidum or mutant or variant thereof that shows at least an adhesion of about 10 bacterial cells per mm2 of epithelial cell monolayer or having at least an adhesion index of 1.5 or a therapy with the Bifidobacterium bifidum strain or variant thereof or mutant which is Bifidobacterium bifidum MTMBb75 deposited under Deposit No. DSM 24514 or a mutant or variant thereof. A patient, person or individual can also be a person without any of the symptoms or illnesses mentioned above.
[00024] An "adhesive strain", in the sense of the invention, is a strain of Bifidobacteria bifidum or a mutant or variant thereof that has adhesive properties towards human colon cells and is therefore capable of binding to cells of the colon. human or animal intestinal wall, thereby reducing the permeability of the gastrointestinal barrier and improving the intestinal wall in a "hole-closure" mode of action. A simple method for testing a strain of Bifidobacterium bifidum or mutant or variant thereof for this property is provided in the Examples section. In this context, a strain of Bifidobacterium bifidum or mutant or variant thereof which shows at least an adhesion of about 10, preferably of about 20, more preferably of about 30, even more preferably of about 40, most preferably of about 50 or 55 bacterial cells per mm2 of epithelial cell monolayer or having at least an adhesion index of 1.5, preferably of at least 2, more preferably of at least 2.5, even more preferably of at least 3 , is considered to be an adhesive strain or mutant or variant thereof.
[00025] The probiotic formulation of the present invention is for use as a probiotic in food and/or as a medicine and comprises a strain or an adhesive strain of Bifidobacterium bifidum or a mutant or variant thereof that shows at least an adhesion of about 10 , preferably from about 20, more preferably from about 30, even more preferably from about 40, more preferably from about 50 or 55 bacterial cells per mm2 of epithelial cell monolayer or has at least an adhesion index of 1 .5, preferably at least 2, more preferably at least 2.5, even more preferably at least 3 or the probiotic formulation of the present invention is for use as a probiotic in food and/or as a medicine and comprises a strain of Bifidobacterium bifidum or mutant or variant which is Bifidobacterium bifidum MIMBb75 deposited under Deposit No. DSM 24514 or a mutant or variant thereof. The probiotic formulation of the present invention can also be considered as a pharmaceutical formulation. "Food" denotes, in principle, any form of substance or material that is ingestible and digestible by humans and, preferably, also by animals. In general, this is composed of substances or materials that can be eaten or drunk to provide nutritional support for the body and/or leisure. In the case of humans, these substances can be of plant, microbial or animal origin. In the case of the present invention, food products are in particular intended to comprise microorganisms, in particular at least the strains of the present invention, as well as other substances which are mentioned herein as being optionally compounded in the probiotic formulation, such as at least a pharmaceutically acceptable compound, an ingestible carrier, an adjuvant, a bacterial component, a drug entity, a biological compound and/or protein and/or peptide, i.e. a protein and/or peptide that is rich in glutamine/glutamate, a lipid, a carbohydrate, a vitamin, minerals and/or trace element; optionally, said at least one pharmaceutically acceptable compound is a member selected from the group consisting of one or more vitamins, such as group B vitamins, one or more minerals, such as calcium or magnesium, one or more carbohydrates, such as lactose , maltodextrin, inulin, dextrose, mannitol, maltose, dextrin, sorbitol, fructose and mixtures thereof. A "drug" in general is not understood as a food product, as a drug is usually only administered to a human or patient in a certain condition which is not considered a healthy condition, but a disease. However, a medication can also provide nutritional support for the body and/or leisure, as in the case of a person who is suffering from depression. Medicines and therapeutic products in general are subject to more stringent regulations and approval processes. These approval processes are not necessary in the case of a foodstuff, which is understood as the main form of administration and main application of the strains or mutants or variants of the invention. As explained above, a "probiotic" in the sense of the present invention comprises probiotic bacteria of non-pathogenic human origin and has been defined as microbial live food and medical supplements which, upon ingestion in a certain number, have health effects in addition to of inherent basic nutrition. Mixtures of various microorganisms, particularly Lactobacillus and Streptococcus species, have traditionally been used in fermented dairy products to promote health. The probiotic of the present invention comprises at least the adhesive Bifidobacterium bifidum strains of the invention or mutants or variants thereof or the MIMBb75 strain of Bifidobacterium bifidum of the invention or mutants or variants thereof. However, also other strains, cells, mutants or variants of other species which have been found to be useful probiotics may be present in the probiotic formulation of the present invention as a bacterial component. These are, for example, probiotic strains of Lactobacillus, Bifidobacterium, Saccharomyces, Streptococcus species or mixtures thereof. Thus, a "bacterial component" indicates a viable or non-viable strain or cells of a probiotic strain as mentioned above and can further be used for compounds such as biological molecules, polysaccharides, lipids or even of bacterial origin or be produced by means of fermentation or expression of bacteria. Even dead material from any species of bacteria can be understood as a bacterial compound.
[00026] The probiotic formulation of the invention may further comprise at least one prebiotic or a combination of two, three, four or even more different prebiotics. A "prebiotic" in the sense of the invention is intended to serve as nutrients for the strains of the invention and keep them in a viable and healthy state after freeze-drying thawing. Per gram of microbial cells, i.e. per gram of strain, in general 2-5 g preferably ~2.5 g, more preferably at least 2.5 g, even more preferably at least 3 g, most preferably at least 4 g of the prebiotic is needed. In general, for humans, non-digestible food ingredients, such as certain carbohydrates, are used as a prebiotic. Examples of a prebiotic comprise carbohydrates, preferably oligosaccharides, inulin, fructo-oligosaccharides, galacto-oligosaccharides, psyllium, oligofructose, isomalto-oligosaccharides xylo-oligosaccharides, sojo-oligosaccharides, maltodextrin, gluco-oligosaccharides, mannanoligosaccharides, mannanoligosaccharides, mannanoligosaccharides lactulose, polydextrose, oligodextran, gentioligosaccharide, pectic oligosaccharides, xanthan gum, arabic gum, hemicellulose, resistant starch and its derivatives and mixtures and/or combinations thereof.
[00027] The probiotic formulation of the invention may further comprise at least one pharmaceutically acceptable compound, at least one ingestible vehicle, at least one adjuvant, at least one bacterial component, at least one drug entity, at least one biological compound and/or at least one protein and/or peptide, in particular a protein and/or peptide which is rich in glutamine/glutamate, a lipid, a carbohydrate, a vitamin, minerals and/or trace elements.
[00028] A "pharmaceutically acceptable compound" means a liquid, solid or gaseous chemical or a biological compound that is acceptable in a pharmaceutical composition or formulation characterized by good tolerability by human and animal body and physiology and being pharmacologically inactive or not having any detrimental effect on the physiology of the recipient. At least one, two, three, four, five or even more different pharmaceutically acceptable compounds may be present in the probiotic formulation of the invention, in different amounts. The amount can be adjusted by the manufacturer according to the specific needs of the strain of Bifidobacterium bifidum that is used and according to a particular application, mode of administration or dosage regimen. Examples of pharmaceutically acceptable compounds include prebiotics, carbohydrates, lipids, vitamins, minerals, trace elements, amino acids, nucleic acids, maltodextrin, inulin, lactose, glucose, sucrose, maltose, dextrin, dextrose, fructose, sorbitol, fructooligosaccharide, mannitol, corn starch, crystalline cellulose, gum arabic, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose, cellulose, polyvinylpyrrolidone, gum tragacanth, gelatin, preferably bovine gelatin, syrup, aminosalicylate, sulfasalazine, 5-aminosalicylic acid, acid 4 -aminosalicylic, benzalazine, dihydrochloride salt, olsalazine, balsalazide, bismuth subsalicylate, methyl cellulose, carboxy methyl cellulose, methyl hydroxy benzoic acid esters, propyl hydroxy benzoic acid esters, talc, magnesium stearate, inert polymers, water and mineral oils, iodine, magnesium, magnesium aspartate-ascorbate complex, magnesium amino acid chelate, zinc, steel chelate zinc minoacid, selenium, selenium amino acid complex, copper, copper amino acid chelate, manganese, manganese amino acid chelate, chromium, chromium polynicotinate, molybdenum, molybdenum amino acid chelate, potassium, aspartate-ascorbate complex potassium, choline, choline bitartrate, inositol, vanadium, vanadium sulfate, boron, boron citrate-aspartate, citrus bioflavinoids, modified cellulose gum, silica, vegetarian stearin, titanium dioxide, magnesium stearate. Preferably, said at least one, two, three, four, five or even more pharmaceutically acceptable compounds are a member selected from the group consisting of one or more vitamins such as B vitamins, one or more minerals such as calcium, magnesium and one or more carbohydrates, gelatin, preferably bovine gelatin, mannitol, dextrin, fructose, sorbitol, a prebiotic, maltose, maltodextrin, inulin, dextrose, iron, lactose and a mixture thereof. In addition, the probiotic formulation may comprise pharmaceutically acceptable fillers, binders, lubricants, wetting agents, disintegrants, emulsifying agents, suspending agents, preservatives, sweetening agents and flavoring agents that are known to those skilled in the art.
[00029] The probiotic formulation and the strains or mutant or variant thereof of the present invention can be formulated in such a way that, after administration to a patient, the strain or cells are released rapidly, slowly or continuously. Examples of vitamins that may be comprised in the probiotic formulation of the invention are water-soluble and water-insoluble vitamins such as vitamin A (for example, retinol, retinal and carotenoids, including beta-carotene), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin, eg niacinamide, nicotinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, eg pyridoxamine, pyridoxal), vitamin B7 (biotin), vitamin B9 (eg acid folic, folinic acid), vitamin B12 (eg cyanocobalamin, hydroxycobalamin, methylcobalamin), vitamin C (ascorbic acid), vitamin D (eg ergocalciferol, cholecalciferol), vitamin E (eg tocotrienols, tocopherols), vitamin K (eg phylloquinone, menaquinones). Preferably, the group B vitamins are composed in the probiotic formulation of the invention, such as vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin, for example, niacinamide, nicotinamide), vitamin B5 (pantothenic acid), vitamin B6 (eg pyridoxine, pyridoxamine, pyridoxal), vitamin B7 (biotin), vitamin B9 (eg folic acid, folinic acid), vitamin B12 (cyanocobalamin,eg hydroxycobalamin, methylcobalamin) and mixtures thereof. Examples of mineral salts comprised in the probiotic formulation of the invention are magnesium, calcium, zinc, selenium, iron, copper, manganese, chromium, molybdenum, potassium, vanadium, boron, titanium, preferably magnesium and/or calcium are present. A "trace element" is a chemical element that is only needed in small amounts for the growth, development and/or physiology of the organism, preferably a human organism. "Carbohydrates" are organic compounds consisting only of carbon, hydrogen and oxygen and having the empirical formula Cm(H20)n, where the ratio of hydrogen atoms to oxygen is 2: 1. "Lipids" are biological compounds by less partially insoluble in water due to a long hydrophobic carbohydrate moiety. Lipids are a very important part of cell membranes in biological systems.
[00030] An "edible vehicle", in the sense of the present invention, is a vehicle which is used for administering the probiotic formulation or the same strain or mutant or variant of the invention to an individual or patient to ingest by aiding the probiotic formulation or the strain or mutant or variant thereof of the invention. The term "dairy product", as used herein, is intended to include a medium comprising milk of animal and/or vegetable origin. Animal milk includes cow, sheep, goat and buffalo milk. As milk of vegetable origin, any fermentable substances of vegetable origin can be mentioned which can be used according to the invention, in particular from soy, rice or cereals. A possible ingestible carrier is a member selected from the group comprising a capsule, tablet, powder, granules, troches, capsules, wafer, elixir, emulsion, solution, syrup, suspension, soft and hard gelatin capsule, suppositories, aseptic packaged powders or a food product; optionally, the food is a dairy product, sour milk, yogurt, frozen yogurt, yogurt production, such as fermented yogurt drink, drinking yogurt, cheese, fermented cream, milk-based desserts, fermented milk or humanized milk, milk powder, milk concentrate, curd, sauces, beverages and others. An "adjuvant", in the sense of the invention, is a pharmacological or immunological agent that modifies the effect of other agents, such as drugs or vaccines or of the probiotic formulation, strain or mutant or variant of the present invention, while having few - if any - direct effects when administered alone.
[00031] "Drug entity" refers to a chemical or biological compound that is pharmaceutically active while being pharmaceutically acceptable to the patient, such as bisacodyl, loperamide, aminosalicylate, sulfasalazine, 5-amino acid -salicylic, 4-aminosalicylic acid, benzalazine, dihydrochloride salt, olsalazine, balsalazide, bismuth subsalicylate or mixtures thereof. Such a compound may be any compound that promotes beneficial effects of the probiotic formulation or strain and mutants or variants thereof of the invention, either directly or indirectly. Directly means that the Bifidobacterium bifidum strain or mutant or variant thereof is itself protected, its growth or adhesion to the intestinal barrier is supported. Indirectly means that additional substances or compounds, i.e. drug entities, which are known to be useful in the prophylaxis and/or treatment of a disease related to a functional disorder of the gastrointestinal tract, to undesirable gastrointestinal inflammatory activity, to a intestinal microbiota imbalance, a reduction of Bifidobacteria in the gastrointestinal tract and/or a dysfunctional intestinal barrier in the gastrointestinal tract or for use in the relief, prevention and/or treatment of an individual who is suffering from abdominal pain, discomfort, distension, swelling, digestive disorders, urgency reduced and/or increased number of bowel movements, feeling of incomplete bowel movement and/or combinations thereof or to improve a patient's quality of life, may be comprised alone or in combination with other drug entities in the probiotic formulation. In particular, a drug entity may be comprised in the probiotic formulation, which is a medicine known for the treatment of diseases of the gastrointestinal tract, such as irritable bowel movement, inflammatory bowel disease, for example ulcerative colitis or Crohn's disease. , irritable bowel syndrome, post-infection pouchitis or colitis, gastrointestinal cancer, a systemic disease such as rheumatoid arthritis, an autoimmune disorder due to undesirable inflammatory activity, diarrheal disease due to undesirable inflammatory activity such as Clostridium-associated diarrhea difficile, antibiotic associated diarrhea, rotavirus associated diarrhea or post-infection diarrhea and combinations thereof. Examples of such a drug entity known to those skilled in the art can be found in Rote Liste, 2010, Germany or any other pharmaceutical register. A "biological compound" can be any biological compound, such as a carbohydrate, amino acid, lipid, nucleic acid, protein, peptide, cell compartment, phospholipid, polyether, plant, animal or microbial compound. "Protein", "peptide" or "polypeptide" shall be understood, according to their general meaning, as being a linear composition of proteinogenic or non-proteinogenic amino acids, where the proteins may also comprise one or more catalytically relevant subunits and substances, such as vitamins or mineral substances.
[00032] "Colony Forming Unit" (cfu) is a measure of viable bacterial cells of the strain of Bifidobacterium bifidum or a mutant or variant thereof of the invention. The probiotic formulation of the invention comprises the strain, mutant or variant thereof in more than 101 cfu, optionally at least 102, 103, 104, 105 or 106 colony forming units (cfu) per capsule or tablet or per unit of food product or powder; preferably the strain, mutant or variant is present at greater than 107 cfu per capsule or tablet or per unit of food product or powder, more preferably the strain, mutant or variant is present at greater than 108 cfu per capsule or tablet or per unit food product or powder, even more preferably the strain, mutant or variant is present at more than 109 cfu per capsule or tablet per unit food product or powder. The daily dose of the strain, mutant or variant administered to a patient in the probiotic formulation is not less than 101 cfu, optionally at least 102, 103, 104, 105 or 106 cfu, preferably not less than 107 cfu, more preferably not less of 108 cfu, even more preferably not less than 109 cfu or less than 1010 cfu. The daily dose can be increased depending on the patient's physiological condition, that is, in case of severe symptoms. Here, the amount can be increased by the fact that the daily dose is doubled, tripled or multiplied. On the contrary, the daily dose can be halved or divided into two, three or four, for example in the case of intolerance or additional medication or the daily amounts as mentioned above may not be administered daily, but every two or three days or weekly. The strain, mutant or variant thereof or the probiotic formulation can be taken at any time which is deemed convenient by the person or patient, such as in the morning, at noon, in the afternoon or at night; preferably, the time is in the morning, for example after waking up, or at night, for example, before going to sleep. If the strain, mutant or variant thereof or the probiotic formulation comprises an ingestible vehicle which is a food product, administration is carried out in accordance with the nature of the ingestible vehicle, i.e. the food product. If the strain, mutant or variant thereof or the probiotic formulation comprises an ingestible vehicle, such as a tablet or capsule or comparable ingestible vehicle, administration is carried out orally, accompanied by a drink or water, preferably water.
[00033] In general, the probiotic formulation and the strains of the invention are administered according to the specific form of the probiotic, foodstuff or drug. Thus, normally, the probiotic formulation and strains of the invention are administered orally, preferably parenterally, or via the rectum.
[00034] The method for producing the probiotic formulation of the invention comprises at least the following steps: a) fermentation and/or culture of a strain of Bifidobacterium bifidum or mutant or variant that shows at least an adhesion of about 10 bacterial cells per mm2 of epithelial cell monolayer or having at least an adhesion index of 1.5 or a strain of Bifidobacterium bifidum or mutant or variant which is Bifidobacterium bifidum MIMBb75 deposited under Deposit No. DSM 24514 or a mutant or variant thereof in a protein-rich liquid growth medium, b) collection of cells from the aforementioned strains by means of centrifugation, stabilization, lyophilization, grinding and sieving of the cells, c) optionally, mixing/combining the cells with one or more members of the group consisting of in a prebiotic, a compound, an adjuvant, a bacterial component, a drug entity, a biological compound, a pharmaceutically acceptable protein and/or peptide, and d) introducing the cells from step b) or the mixture from step c) into an ingestible vehicle.
[00035] First, it should be noted that the fermentation and growth of Bifidobacterium bifidum strains, mutants or variants of the invention can be carried out by standard means and methods which are known to those skilled in the art, as described in "Probiotics and Health Claims", Wolfgang Kneifel, Seppo Salminen, John Wiley & Sons, 1st Edition (January 7, 2011).
[00036] In general, a medium for the culture and/or fermentation of Bifidobacterium bifidum strains, mutants or variants of the invention comprises at least water, dextrose, yeast extract and mineral salts. The standard medium used for the culture and/or fermentation of the strains or variants or mutants of the invention consists of MRS broth (Disco, Detroit, MI, USA) supplemented with 0.05% L-cysteine hydrochloride (cMRs). Those skilled in the art are aware of the fact that also other media can be used for culturing, fermentation and pre-culture of bacterial organisms, such as strains of Bifidobacterium bifidum. Optionally, prior to step a), the strains or variants or mutants thereof are cultivated in a shake flask preculture. For this purpose a volume of at least 200 ml, preferably at least 300 ml, more preferably at least 400 ml, even more preferably at least 500 ml and most preferably at least 600 ml of standard medium as per mentioned above are inoculated with at least a single cell of the strain or variant or mutant. This preculture is generally grown overnight under anaerobic conditions at 37°C at 220 rpm.
[00037] The pre-culture can then be totally or partially used for inoculating a larger fermentation in a pre-fermenter in a volume of at least 500 L, preferably of at least 600 L, more preferably of at least 700 L, even more preferably at least 800 L, more preferably at least 1000 L. After fermentation of the strains, variants and mutants of the invention in a prefermenter, the cell culture containing said strains, variants and mutants is at least partially transferred for a main fermenter having at least a volume of at least 5,000 L, preferably at least 7,500 L, more preferably at least 10,000 L, even more preferably at least 15,000 L, and even more preferably at least 20,000 L. Also in the pre-fermentor and main fermenter, the standard medium mentioned above is used for fermentation and growth of the strains, mutants and variants of Bifidobacterium bifidum of the invention. Fermentation and growth are stopped and cells are harvested after depletion of the carbohydrates comprised in the medium which serve as nutrients for the strains, mutants and variants of the invention. In general, cell density after depletion is at least 101 cfu, optionally at least 102, 103, 104, 105 or 106 cells/ml, preferably at least 107 cells/ml, more preferably at least 108 cells/ml, still more preferably at least 109 cells/ml or at least 1010 cells/ml. Those skilled in the art will have knowledge of methods for determining cell density or biomass, such as determining biomass and dry weight using standard drying procedures and gravimetric determination of pre-weighed glass tubes or measuring optical density using a spectrophotometer (Hitachi as U1100, Japan) at an absorption of 578 nm.
[00038] The pre-fermentor and main fermenter can be derived from a standard bioreactor suitable for culture, fermentation, growth and/or transformation of a strain, mutants and/or variants of a strain of Bifidobacterium bifidum of the invention. Standard bioreactor suppliers are, for example, Applikon Biotechnology BV (Schiedam, Netherlands), Infors (Bottmingen, Switzerland), Bioengineering (Wald, Switzerland) and Sartorius Stedim Biotech GmbH (Gottingen, Germany). Pre-fermentation and main fermentation are carried out in batch mode, which generally means that no medium or other components are replenished during fermentation.
[00039] After main fermentation, cells are collected by means of centrifugation, for example, using a disc centrifuge or separator having an operating capacity of around 2000-15,000 L/h, as provided by GEA Westfalia Separator Group GmbH (Oelde , Germany) or similar. The cells are stabilized, lyophilized, ground and sieved using conventional applications and means, which are well known to those of skill in the art thereafter. Freeze drying provides a concentrate having a volume of 500 L to 1000 L, preferably 600 L to 800 L and having a weight of approximately 100 kg and 200 kg, preferably ~150 kg.
[00040] Thereafter, the cells are optionally mixed with a prebiotic and/or at least one or more members of the group consisting of a compound, an ingestible vehicle, an adjuvant, a bacterial component, a drug entity, a biological compound and /or a pharmaceutically acceptable protein and/or peptide and/or mixtures thereof, in particular a protein and/or peptide which is rich in glutamine/glutamate, a lipid, a carbohydrate, a vitamin, minerals and/or trace elements; optionally, said at least one pharmaceutically acceptable compound is a member selected from the group consisting of one or more vitamins, such as group B vitamins, one or more minerals, such as calcium or magnesium, one or more carbohydrates, such as lactose , maltodextrin, inulin, dextrose, mannitol, maltose, dextrin, sorbitol, fructose and a mixture thereof, in different amounts.
[00041] To obtain the probiotic formulations of the invention, in general, 15 - 40 g, preferably 18 - 35 g, more preferably 20 - 30 g, even more preferably 22 - 28 g, more preferably 25 g of cells of Bifidobacterium bifidum, strain, mutants or variant thereof of the invention are mixed with 50 - 100 g, preferably with 60 - 90 g, more preferably with 65-85 g, even more preferably with 70 - 80 g, most preferably with 75 g of one or more members of the group consisting of a prebiotic, carbohydrate, a pharmaceutically acceptable compound or mixtures thereof as a nutrient for the cell, the strain, mutant or variant thereof and are optionally mixed with 30 - 70 g, preferably with 35 - 65 g, more preferably with 40 - 60 g, even more preferably with 45 - 55 g, more preferably with 50 g of one or more members of the group consisting of a compound, an ingestible carrier, an adjuvant, a bacterial component , a drug entity , a biological compound and/or a pharmaceutically acceptable protein and/or peptide, in particular a protein and/or peptide which is rich in glutamine/glutamate, a lipid, a carbohydrate, a vitamin, minerals, trace elements and/or mixtures of the to improve the flowability of the probiotic formulation. Preferably 15 - 40 g, preferably 18 - 35 g, more preferably 20 - 30 g, even more preferably 22 - 28 g, more preferably 25 g of Bifidobacterium bifidum cells, strain, mutant or variant of the present invention are mixed with 50 - 100 g, preferably with 60 - 90 g, more preferably with 65-85 g, even more preferably with 70 - 80 g, more preferably with 75 g of a carbohydrate, a mixture of carbohydrates or mixture of these prebiotics and optionally with 30 - 70 g, preferably with 35-65 g, more preferably with 40 - 60 g, even more preferably with 45 - 55 g, more preferably with 50 g of one or more members of the group consisting of a compound, a carbohydrate, a vitamin, minerals, pharmaceutically acceptable trace elements and/or mixtures thereof. More preferably 15 - 40 g, preferably 18 - 35 g, more preferably 20 - 30 g, even more preferably 22 - 28 g, more preferably 25 g of Bifidobacterium bifidum cells, strain, mutants or variants thereof of the invention are mixed with 50-100 g, preferably with 60 - 90 g, more preferably with 65-85 g, even more preferably with 70 - 80 g, more preferably with 75 g of a carbohydrate, inulin, a fructooligosaccharide or a mixing thereof and optionally with 30 - 70 g, preferably with 35-65 g, more preferably with 40-60 g, even more preferably with 45-55 g, most preferably with 50 g of one or more group members which consists of a vitamin, such as a group B vitamin, one or more minerals, such as calcium or magnesium, one or more carbohydrates, such as lactose, maltodextrin, inulin, dextrose, mannitol, maltose, fructooligosaccharide, maltose, dextrin, sorbitol, fructose and a mixture thereof. Even more preferably, 25 g of Bifidobacterium bifidum cells, strain, mutant or variant thereof of the present invention are mixed with 75 g of a member selected from the group consisting of a carbohydrate, prebiotic, maltodextrin, inulin, dextrose, mannitol, maltose , dextrin, sorbitol, fructose and a mixture thereof are mixed with 50 g of a member selected from the group consisting of cellulose, compound, an ingestible carrier, an adjuvant, a bacterial component, a drug entity and a biological compound/protein or a pharmaceutically acceptable peptide, in particular a protein and/or peptide which is rich in glutamine/glutamate, a lipid, a carbohydrate, a vitamin, minerals, trace elements and a mixture thereof. More preferably, 25 g of Bifidobacterium bifidum cells, strain, mutant or variant of the present invention are mixed with 75 g of maltodextrin and are mixed with 50 g of cellulose. Mixing/combining the components with the cells, strain, mutants or variants thereof of the invention after fermentation and/or growth of the strain, collection, stabilization, lyophilization, grinding and sieving can be carried out by means of conventional techniques, applications and means that are known to those skilled in the art, preferably a column mixer is used. Naturally, also larger or smaller amounts can be used in a mix/blending, depending on the volume of the device used for mixing/blending, while the proportion of the components corresponds to the proportions mentioned above. A typical proportion, for example, is 25% strain, 75% prebiotic or carbohydrate or maltodextrin and 50% pharmaceutically acceptable compound such as cellulose.
[00042] For example, from 50mg - 300mg, preferably 75mg - 250mg, more preferably 100mg-200mg, even more preferably 120mg -175mg, most preferably 150mg of Bifidobacterium bifidum cells, strain, mutants or variants of the same invention or of the above-mentioned mixtures or probiotic formulations of the invention are introduced into an ingestible carrier unit, for example, in a capsule, a tablet, troches, a capsule, wafer, elixir, emulsion, solution, syrup, suspension, hard and soft gelatin capsule, suppository, aseptic packaged powder or a food product such as a dairy product, sour milk, yogurt, frozen yogurt, yogurt production such as fermented yogurt drink, drinking yogurt, cheese, fermented cream, desserts based on fermented milk or humanized milk, powdered milk, concentrated milk, cottage cheese, sauce and/or drinks, preferably 50 mg, more preferably 75 mg, more preferably 100 mg, even more is preferably 120 mg and more preferably 150 mg of Bifidobacterium bifidum cells, strain, mutants or variant of the invention or of the mixtures mentioned above are introduced in a capsule, troche, tablet, wafer, soft and hard gelatin capsule, suppository, more preferably in a capsule, a hard and soft gelatin capsule. Thus, after introduction into the ingestible vehicle unit, i.e., the capsule itself, tablet, powder, granules, troches, wafer, elixir, emulsion, solution, syrup, suspension, soft and hard gelatin capsule, suppositories, aseptic packaged powders or food products such as a dairy product, sour milk, yogurt, frozen yogurt, yogurt production such as fermented yogurt drink, drinking yogurt, cheese, fermented creams, desserts based on fermented milk or humanized milk, milk powder , concentrated milk, cottage cheese, sauce and/or beverage comprises more than 101 cfu, optionally at least 102, 103, 104, 105 or 106 cfu, preferably more than 107 cfu, more preferably more than 108 cfu, even more preferably more than 109 cfu or more than 1010 cfu. In any case, the amount by weight can be adjusted according to the concentration of cfu, so that the amount of Bifidobacterium bifidum cells, strain, mutant or variant of the invention in the final product, such as capsule, tablet, powder, granule , troche, wafer, elixir, emulsion, solution, syrup, suspension, soft and hard gelatin capsule, suppositories, aseptic packaged powders or food product, corresponds to more than 101 cfu, optionally at least 102, 103, 104 105 or 106 cfu , preferably more than 107 cfu, more preferably more than 108 cfu, even more preferably more than 109 cfu or more than 1010 cfu. In the case where the amount of cfu is less than 101 cfu, optionally not less than 102, 103, 104 105 or 106 cfu, 107 cfu, 108 cfu, even 109 cfu or less than 1010 cfu, the patient can adapt to the amounts mentioned above of cfu upon administration of more than one ingestible vehicle unit.
[00043] In an exemplary embodiment, 25 mg of Bifidobacterium bifidum MIMBb75 cells deposited under Deposit No. DSM 24514 i.e. at least 109 cfu of Bifidobacterium bifidum MEVIBb75 deposited under Deposit No. DSM 24514, 75 mg of maltodextrin and 50 mg of cellulose are introduced into a gelatin capsule, preferably in a bovine gelatin capsule. Preferably, the above-mentioned capsule with 25 mg of Bifidobacterium bifidum MEVIBb75 cells deposited under Deposit No. DSM 24514 comprising at least 109 cfu of Bifidobacterium bifidum MEVIBb75 deposited under Deposit No. DSM 24514, 75 mg of maltodextrin and 50 mg of cellulose is administered once a day. Ingestible vehicles in general must be of standard to high quality, must meet the hygiene requirements for food supplements, and may be obtained from a variety of manufacturers. Capsules, for example bovine gelatine capsules, can for example be obtained from Capsugel (Bornem, Belgium).
[00044] In another preferred embodiment, 25 mg of Bifidobacterium bifidum MEVIBb75 cells deposited under Deposit No. DSM 24514, preferably comprising at least 109 cfu of Bifidobacterium bifidum MEVIBb75 deposited under Deposit No. DSM 24514 and 2 g of maltodextrin are mixed together and are considered as a unit of powder. Preferably, the above-mentioned powder unit with 25 mg of Bifidobacterium bifidum MIMBb75 cells deposited under Deposit No. DSM 24514, i.e. at least 109 cfu of Bifidobacterium bifidum MEVIBb75 deposited under Deposit No. DSM 24514, is administered once a day.
[00045] Regardless of the particular ingestible vehicle chosen by the person or patient, the daily dose of Bifidobacterium bifidum cells, strain, mutant or variant administered to a patient in the probiotic formulation is not less than 101 cfu, optionally at least 102, 103, 104, 105 or 106 cfu, preferably not less than 107 cfu, more preferably not less than 108 cfu, even more preferably not less than 109 cfu or not less than 1010 cfu. Alternative doses are discussed above.
[00046] The invention is not limited to the embodiments described above, which can be varied in detail. EXAMPLES 1. Bacterial Culture Conditions
[00047] In general, Bifidobacterium species, such as Bifidobacterium bifidum MIMBb75, were routinely grown overnight under anaerobic conditions at 37°C in MRS broth (Disco, Detroit, MI, USA) supplemented with L-cysteine hydrochloride (cMRS) at 0.05%. 2. Test for Adhesive Properties of Bifidobacterium bifidum MLMBb75 to Caco-2 Human Intestinal Epithelial Cell Lines 2.1 Bacterial Adhesion to Caco-2 Cells
[00048] This test can be performed to test whether a particular strain of Bifidobacteria has adhesive properties to human colon cells and therefore should be understood as an adhesive strain in the sense of the present invention. Caco-2 human colon adenocarcinoma cells (ATCC HTB-37) were routinely cultured in 3 cm Petri dishes on glass microscope slides in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (vol/ vol) thermally inactivated (30 min at 56 °C), 100 U/ml-1 penicillin, 100 mg/ml-1 streptomycin, 0.1 mM non-essential amino acids and 2 mM L-glutamine and incubated at 37 °C in a water jacketed incubator in an atmosphere of 95% air and 5% carbon dioxide. For this assay, epithelial cells from any in vivo source including, but not limited to, epithelial cells derived from the human gastrointestinal tract, are suitable; however, Caco-2 cells or derivatives thereof are more preferred and are commercially available from culture collection banks such as ATCC and ECACC. Culture medium was changed twice a week. For adhesion assays, cells were used 15 days after confluence (fully differentiated cells). Cell monolayers were carefully washed twice with phosphate buffered saline (Phosphate Buffered Saline - PBS) (pH 7.3) before bacterial cells were added. The concentration of an overnight bacterial cell culture was determined microscopically after staining with DAPI (4',6'-diamidino-2-phenylindole). Approximately 2 x 108 cells from each strain resuspended in PBS (pH 7.3) were incubated with a fully differentiated Caco-2 cell monolayer. After 1 h at 37 °C under anaerobic conditions, all monolayers were washed three times with PBS to release unbound bacteria. Cells were then fixed with 3 ml of methanol and incubated for 8 minutes at room temperature. After methanol was removed, cells were stained with 3 ml of Giemsa staining solution (1:20) (Carlo Erba, Milan, Italy) and allowed to rest for 30 minutes at room temperature. The wells were then washed until no more color was seen in the wash solution and dried in an incubator for 1 h. Glass microscope slides were then removed from the Petri dish and examined microscopically (magnification, x100) immersed in oil.
[00049] Adherent bacteria in 20 randomly selected microscopic fields were counted and the mean calculated. An unpaired Student t test was performed for statistically significant differences.
[00050] Bifidobacterium bifidum MIMBb75 was found to be well adherent to Caco-2 human colon cells, as a significant proportion of cells from this bacterial strain remained attached to the Caco-2 cell monolayer, providing evidence that adhesion did not it was just nonspecific to the physical envelope. In particular, about 55 bacterial cells adhered to a 1 mm2 monolayer of epithelial cells, resulting in an adhesion index (bacterial cells/100 cells Caco-2) of 3.874 (P < 0.026). In this regard, cells from a strain of Bifidobacterium showing at least an adhesion of about 10 bacterial cells per mm2 of epithelial cell monolayer or having at least an adhesion index of 1.5 are considered to be an adhesive strain. 2.2 Preparation of Bacterial Cell Wall Extract
[00051] Bacterial cells of 0.2 L of liquid culture were collected by means of centrifugation and processed according to methods previously described by Mattarelli et al. (1993) with a modified use of the French press (12,000 lbs/inch2, ~ 2,142kg/cm2) to break the cells. 3. Double-Blind, Randomized, Placebo-Controlled Study to Assess the Efficiency of Bifidobacterium Bifidum MIMBb75 in Patients With Irritable Bowel Syndrome 3.1 Overview
[00052]122 patients were randomized successfully to receive placebo (N = 62) or Bifidobacterium bifidum MIMBb75 (N=60) (also considered as "Bifdobacteria group"). Subjects consumed 1 x 109 cfu/capsule or placebo once daily for four weeks. IBS severity and individual IBS symptoms were recorded daily on a 7-point Likert scale. 3.2 Study Population
[00053] Patients were recruited by principal investigators and by advertising. The nutritional study protocol was presented to the Ethics Committee of the Bavarian Chamber of Physicians. For inclusion, clinically discrete individuals aged between 18 and 68 years, with mild to moderate IBS (Rome III criteria) were considered. Individuals with inflammatory organic gastrointestinal disease, systemic diseases, cancer, autoimmune diseases, diabetes, known lactose intolerance or immunodeficiency, in addition to other abdominal surgery other than appendectomy, without sigmoidoscopy or colonoscopy disease in the last five years when over 50 years of age age, diagnosed with hyperthyroidism, using antipsychotics or systemic corticosteroids for at least 3 months before the start of the study, major psychiatric disorder, celiac disease or pregnancy were excluded. 3.3 Study Design
[00054] This study was performed as a prospective, multicenter, randomized, double-blind, placebo-controlled, two-group nutritional study. Throughout the study, patients recorded their overall IBS symptoms as well as individual IBS symptoms using a patient diary. In addition, patients were questioned at the health post about global and individual symptoms of IBS (visits 2-4) and quality of life (visits 3 and 4).
[00055] Medical consultations took place at screening, after two weeks of (implementation phase), after 6 weeks (end of treatment) and after 8 weeks (end of interruption phase) (Figure 1).
[00056] After patients had provided their written consent, they were eligible for the screening examination on day 1 (visit 1), which included a complete medical history and physical examination. central laboratory, including a pregnancy test. At the screening visit, patients were instructed to maintain their eating habits and lifestyle throughout the study. A patient diary was delivered.
[00057] At the second visit (day 15), the diaries were reviewed. Patients who had at least 2 days of mild to moderate pain during the second week of running and met all inclusion criteria and did not violate any of the exclusion criteria were randomly assigned 1:1 to receive B. bifidum MIMBb75 or placebo. Treatment was allocated according to a computer-generated blocked randomization list with a block size of 4. The block size was not disclosed to investigators. During the intervention period, patients received one capsule comprising the probiotic formulation daily for 4 weeks or an identical looking placebo. The allocation was made blindly to patients and health post staff.
[00058] At the end of the treatment phase (visit 3, day 43), investigators collected unused study product and empty bags in order to confirm compliance. Diaries were collected and analyzed.
[00059] After the nutritional supplement withdrawal phase (visit 4, day 57), a complete physical examination was performed and a blood sample was collected. Bisacodyl and loperamide were authorized as recovery medications. Other probiotics and medications that might influence the efficacy of the study product were not authorized. 3.4 Preparation of Probiotic
[00060] Nutritional supplement was prepared under conditions of good manufacturing process (Good Manufacturing Process - GMP).B. bifidum MIMBb75 was grown in protein-rich liquid growth medium, collected by centrifugation, stabilized, lyophilized, ground and sieved. Dry powder bacteria were mixed with a pharmaceutically acceptable compound and introduced into capsules at 1 x 109 cfu.Capsules. placebos were identical in appearance and contained maltodextrin. 3.5 Endpoint Definitions
[00061] The prospectively defined primary efficacy variable was the assessment of global symptoms of IBS using a 7-point Likert scale. Patients were asked to answer the daily question "Considering your IBS symptoms (eg, abdominal pain/discomfort, distension/bloating, digestive disturbance, urgency, bowel habit) in general, how have you been affected by these symptoms over the past few years 24 hours " Possible responses ranged from 0 (not all), 1 (very mild), 2 (mild), 3 (moderate), 4 (strong), 5 (very strong) to 6 (intolerable).
[00062] Secondary efficacy variables included individual symptoms of IBS alone, such as "abdominal pain/discomfort", "bloating/bloating", "digestive disturbance" and "urgency", recorded on the same 7-point Likert scale. Individual symptom scores were also combined into a composite symptom score as the arithmetic mean of the three individual symptom scores. In addition, reduced and/or increased number of bowel movements, feeling of incomplete bowel evacuation, and intake of other medication were reported daily in the diary.
[00063] At the end of treatment and again at the end of the study, clinicians asked patients for overall assessment of efficacy and tolerability. The effectiveness was assessed by the following question: "Please, considering how you felt during the four week treatment, your well-being and symptoms of abdominal discomfort/pain and bowel habits changed compared to how you normally felt before taking study medication and how would you rate your symptom relief during the last 4 weeks " Possible answers are: "completely relieved (1), considerably relieved (2), somewhat relieved (3), unchanged (4) or worst (5)". "Completely relieved" and "considerably relieved" were defined as "adequate relief". Health-related quality of life was assessed using the SF-12 questionnaire before treatment and at the end of treatment.
[00064] Adverse events were recorded throughout the study and global assessment of tolerance was asked by the physician at visits 3 and 4. Laboratory values and vital signs were examined at the screening visit and at the end of the study. 3.6 Statistical Methods 3.6.1 Sample Size Estimation
[00065] A reduction in the Subject's Global Assessment (SGA) of at least 20% on the 7-point Likert scale was considered a relevant treatment effect. Based on published data (Whorwell, 2006), a 0.6-point difference in the SGA of IBS symptoms between B. bifidum MIMBb75 and placebo on the 7-point Likert scale was predicted (eg, 3.0 in the group with placebo and 2.4 in the bifidobacteria group). The standard deviation was estimated at 1.0 using the same data. With these assumptions, a Wilcoxon-Mann-Whitney test with a two-sided significance level of a = 0.05 and a power of l-β = 0.8, a sample size of 47 patients per group was required. With an estimated dropout rate of 15-20% after randomization, 110 randomized patients were planned and 132 patients were recruited to account for possible withdrawals before the start of the study. 3.6.2 Statistical Analysis
[00066] The primary objective of this study was to demonstrate a significant SGA reduction of overall IBS symptoms at the end of treatment in the placebo versus Bifidobacteria group. The SGA was calculated for each subject as the arithmetic mean at baseline, during the treatment period, and during the discontinuation phase. To explain possible differences in baseline values, change from baseline, calculated as the mean score over 4 weeks of treatment minus the mean score during the run phase (1-2 weeks), was defined as the primary target criterion. The non-parametric Van Elteren test stratified by study centers was used to compare treatment groups. P < 0.05 was considered statistically significant.
[00067] The primary analysis was based on the intent-to-treat population, where all patients successfully randomized were included. Missing post-baseline values were imputed by the baseline value for the primary target criterion and these patients were evaluated as non-responders. An additional per-protocol analysis was performed for support purposes.
[00068] Descriptive analyzes of secondary target criteria were based on available data.
[00069] Treatment differences were tested using Wilcoxon's nonparametric test for continuous variables or by means of Fisher's exact test for binary variables. All p-values are two-sided.
[00070] Secondary efficacy variables included response based on a rule of 50% symptom relief during treatment (at least improvement in two out of four weeks during the treatment period and improvement defined as at least one point reduction in relative to baseline). All statistical analyzes were performed using SAS version 9.1.3 for Windows, SAS Institute Inc., Cary, NC, USA. 3.7 Results 3.7.1 Individuals
[00071] A total of 132 patients were enrolled in the study and 122 patients were successfully randomized to receive either placebo (N = 62) or B. bifidum MIMBb75 (N = 60). All randomized patients were analyzed for intention to treat (Intent To Treat - ITT) (N = 122). One patient with no post-randomization visit was excluded from the adverse event analysis. A total of 103 patients (49 placebo, 54 verum) were analyzed according to the protocol (Figure 2). 3.7.2 Baseline Characteristics
[00072] In terms of baseline characteristics, there were no significant differences between groups. 21.5% were classified as diarrhea-predominant IBS, 19.8% as constipation-predominant IBS, and 58.7% as alternating IBS, with no significant difference between the Bifidobacteria group and the placebo group.
[00073] Demographics were well balanced between treatment groups, with approximately 67% female patients and mean weight of 71 kg, corresponding to a BMI of 24. Patients were, on average, 41 years old. in the placebo group and 37 years of age in the Bifidobacteria group (Table 1).Table 1: Demographic characteristics of the intention-to-treat population (Intent To Treat - ITT). SD - standard deviation; BMI - body mass index.
3. 7.3 Subject's Global Assessment (SGA) of IBS Symptoms
[00074] The primary endpoint was SGA reduction of IBS symptoms in the subject's global assessment diary. B. bifidum MIMBb75 significantly improved overall IBS symptoms by -0.88 points [95% Confidence Interval: -1.07, -0.69] (from 2.95 in the run phase to 2.07 in the treatment phase) compared with only -0.16 points [95% CI: -0.32; 0.00] (from 2.79 in the execution phase to 2.63 in the treatment phase) in the placebo group (p<0.0001) using the 7-point Likert scale. Assessment of SGA on a weekly basis showed a significant benefit for patients in the Bifidobacteria group for each week from the second week of treatment to the end of the study (Figure 3). 3.7.4 Secondary Objectives
[00075] Secondary endpoints included changes in individual IBS symptoms such as "pain/discomfort", "bloating/bloating", "digestive disorder", "urgency", "reduced and/or increased number of bowel movements" and "feeling of incomplete evacuation" on a 7-point Likert scale. B. bifidum MIMBb75 showed a significant reduction in pain/discomfort by -0.82 points [95% CI: -1.01; -0.63] vs -0.18 [95% CI: -0.35; -0.01] in the placebo group (p < 0.0001) and distension/swelling of -0.92 points [95% CI: -1.15; -0.69] vs -0.21 [95% CI: -0.37; -0.05] in the placebo group (p < 0.0001) during treatment. The reduction persisted during the interruption phase. Urgency was significantly reduced by -0.67 points [95% CI: -0.86; -0.48] vs -0.21 [95% CI: -0.35; -0.07] in the placebo group (p = 0.0001) during treatment but not during the discontinuation phase. No effect could be detected for bowel movement frequency and feeling of incomplete bowel evacuation (Figure 4).
[00076] Assessment of individual IBS symptoms of pain/discomfort and distension/swelling on a weekly basis showed a significant benefit for patients in the Bifidobacteria group compared to placebo for every week from the second week of treatment to the end of study. A significant difference in urgency between the bifidobacteria and placebo group was shown between four and six weeks (Figures 5 and 6). Digestive disturbance was measured by the item "number of bowel movements" in the questionnaire at the health post. The number of bowel movements decreased from 3.89 to 2.44 in the Bifidobacteria group vs 3.69 to 6.47 in the placebo group (p = 0.0002) after treatment. The reduction persisted during the discontinuation phase (2.33 in the Bifidobacteria group vs 3.47 in the placebo group, p<0.0001). 3. 7.5 Composite Score
[00077] A composite score was calculated for the individual symptoms of IBS (pain/discomfort, distension/swelling, urgency). During the execution phase, the score was comparable in the two groups. Patients in the bifidobacteria group benefited significantly from consumption of B. bifidum MIMBb75 vs placebo (0.80 in the Bifidobacteria group; -0.20 in the placebo group; p<0.0001). This improvement was also preserved during the discontinuation phase (-0.85 in the Bifidobacteria group; -0.31 in the placebo group; p<0.0001). 3.7.6 Treatment Responders
[00078] Global responders were defined as patients who experience an improvement from the mean weekly score of at least one point on the Likert scale for the primary endpoint (IBS symptom SGA) in at least two of the 4-week treatment period (rule 50%). Responders with abdominal pain were defined using the same 50% rule for at least an average improvement of one point for the "pain/discomfort" assessment.
[00079] The overall responder rates were 56.7% in the bifidobacteria group and only 21.0% in the placebo group (p = 0.0001). The difference between treatment groups was only slightly less pronounced when considering only the symptom of "pain/discomfort", where responder rates were calculated to be 48.3% in the bifidobacteria group and only 24.2% in the placebo group (p = 0.008) (Figure 7). 3.7. 7 Global Effectiveness at the Health Post
[00080] The overall assessment of efficacy was significantly better in the bifidobacteria group compared to placebo. At the end of treatment, 43.3% of patients in the Bifidobacteria group achieved adequate relief compared to only 8.1% in the placebo group (p<0.0001). At the end of the study, adequate relief was reported for 46.7% in the Bifidobacteria group and 11.3% of patients in the placebo group (p<0.0001; Figure 8). 3.7.8 Health-Related Quality of Life
[00081] Assessment of the sum of the SF-12 scores showed a significant gain in quality of life within the bifidobacteria group. The sum of physical health improved by 3.99 in the bifidobacteria group and only 1.08 in the placebo group compared to the baseline value (P = 0.0185). The mental health sum improved by 5.78 in the bifidobacteria group and only 1.58 in the placebo group compared to the baseline value (p = 0.0083). 3.7.9 Adverse Events
[00082] Only 36 adverse events were reported with suspected relationship to the study product, 13 in the placebo group and 23 in the treatment group, but no significant difference could be detected in the side effect profile of B. bifidum MIMBb75 versus placebo. 3.7.10 Summary
[00083] B. bifidum MIMBb75 significantly reduced the subject's global assessment (SGA) of IBS symptoms by -0.88 points [95% CI: -1.07, -0.69] compared to only -0.16 points [95% CI: -0.32; 0.00] in the placebo group (p < 0.0001). B. bifidum MIMBb75 also significantly improved individual IBS symptoms of pain/discomfort, distension/swelling, digestive upset, and urgency. Assessment of the sum of the SF-12 scores showed a significant gain in quality of life within the bifidobacteria group. Furthermore, adequate relief was reported by 46.7% of patients in the Bifidobacteria group and only 11.3% of patients in the placebo group (p<0.0001).
[00084] The overall responder rates were 56.7% in the bifidobacteria group and only 21.0% in the placebo group (p = 0.0001). B. bifidum MIMb75 was well tolerated and adverse events were no different from placebo. 3.8 Conclusion
[00085] B. bifidum MIMBb75 effectively relieves global IBS as well as improves individual IBS symptoms. Considering the high efficacy of B. bifidum MIMBb75 in IBS, together with the good side effect profile, B. bifidum MIMBb75 is a promising candidate for IBS therapy.
[00086] This randomized, double-blind, placebo-controlled study indicates that B. bifidum MIMBb75 has beneficial effects in the treatment of IBSI. In this study, B. bifidum MIMBb75 significantly improved overall IBS and its related symptoms such as pain/discomfort and swelling compared to placebo. In addition, B. bifidum MIMBb75 also significantly improved quality of life. These benefits persisted in the non-consuming interruption phase. Overall response rates were predominantly high, with 56.7% in the Bifidobacteria group compared to only 21.0% in the placebo group (p = 0.0001). At the end of the study, adequate relief was reported for 46.7% in the Bifidobacteria group and only 11.3% of patients in the placebo group (p<0.0001).
[00087] To date, several studies have examined the effects of bifidobacteria on IBS and its symptoms. However, only a few could show significant benefit. Furthermore, as far as we know, no probiotic strain has been able to show significant relief from irritable bowel syndrome and simultaneously improve quality of life. Although some studies may not have shown efficacy due to sample size and small randomization errors, several different probiotic strains have repeatedly shown no significant improvement in IBS. Recently, Brenner et al. (2009) published a systematic review of randomized controlled trials (Randomised Controlled Trials - RCTs) that aim to assess the efficacy, safety and tolerability of probiotics in the treatment of IBS. A total of 16 RCTs were included in the analysis. Of these, only one strain of Bifidobacteria showed efficacy in improving IBS symptoms in two properly designed studies. These results can be attributed to the fact that the effectiveness of probiotics is strongly strain-dependent and only a few strains would be able to demonstrate efficacy in IBS.
[00088] In conclusion, B. bifidum MIMBb75 improves IBS as well as its overall symptoms, along with a good side effect profile. BRIEF DESCRIPTION OF THE DRAWINGS
[00089] Figure 1: Study Scheme.
[00090] Figure 2: Study flowchart.
[00091] Figure 3: Comparison of the effects of placebo and B. bifidum MIMBb75 on overall IBS symptoms (by SGA, re-registered on a scale of 0-6) on a weekly basis. Significant improvement in overall IBS symptoms in bifidobacteria vs placebo group.
[00092] Figure 4: Comparison of IBS symptom reduction (B. bifidum MIMBb75 vs placebo) over mean score changes from baseline to treatment phase.
[00093] Figure 5: Comparison of the effects of placebo and B. bifidum MIMBb75 on pain/discomfort (recorded on a Likert scale of 0-6) on a weekly basis. Significant improvement in bifidobacteria group vs placebo group.
[00094] Figure 6: Comparison of the effects of placebo and B. bifidum MIMBb75 on distension/swelling (recorded on a scale of 06) on a weekly basis. Significant improvement in bifidobacteria group vs placebo group.
[00095] Figure 7: Global responders during treatment (ITT).
[00096] Figure 8: Adequate relief after treatment (ITT).

权利要求:
Claims (9)
[0001]
1. Use of a probiotic formulation comprising a strain of Bifidobacterium bifidum MIMBb75, deposited under Deposit No. DSM 24514, characterized in that it is for the preparation of a pharmaceutical composition for the treatment of irritable bowel syndrome.
[0002]
2. Use of a probiotic formulation according to claim 1, characterized in that said treatment comprises alleviating, preventing and/or treating abdominal pain, bloating, constipation, reduced number of bowel movements, increased number of bowel movements, feeling of incomplete evacuation, or combinations thereof.
[0003]
3.Use of the probiotic formulation according to claim 1 or 2, characterized in that it further comprises at least inulin and/or a fructooligosaccharide.
[0004]
4.Use of the probiotic formulation, according to any one of claims 1 to 3, characterized in that it further comprises at least one pharmaceutically acceptable compound; an ingestible vehicle; an adjuvant; a bacterial component; a drug entity; a biological compound and/or a protein; and/or a peptide; a lipid; a carbohydrate; a vitamin and/or mineral.
[0005]
5. Use of the probiotic formulation according to claim 4, characterized in that said at least one pharmaceutically acceptable compound is a member selected from the group consisting of B complex vitamins, minerals selected from calcium minerals; and magnesium minerals, carbohydrates selected from lactose, maltodextrin, inulin, dextrose, mannitol, maltose, dextrin, sorbitol, fructose, and a mixture thereof; and/or glutamine/glutamate rich protein, and a glutamine/glutamate rich peptide.
[0006]
6.Use of the probiotic formulation, according to claim 4 or 5, characterized by the fact that the ingestible vehicle is a capsule, tablet, powder or a food product.
[0007]
7. Use of the probiotic formulation, according to claim 6, characterized in that the food product is a dairy product, sour milk, yogurt, frozen yogurt, powdered milk, concentrated milk, curd, sauce or drinks.
[0008]
8.Use of the probiotic formulation according to claim 6 or 7, characterized in that the strain is present in more than 101 cfu per capsule or tablet or per unit of food product or powder.
[0009]
9. Use of the probiotic formulation according to claim 6 or 7, characterized in that the strain is present in more than 102, 103, 104 105 or 106 cfu per capsule or tablet or per unit of food product or powder .

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法律状态:
2017-09-05| B25A| Requested transfer of rights approved|Owner name: DR. FISCHER GESUNDHEITSPRODUKTE GMBH (DE) |

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2017-09-12| B25B| Requested transfer of rights rejected|Owner name: SYNFORMULAS GMBH (DE) |

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2017-09-19| B25A| Requested transfer of rights approved|Owner name: SYNFORMULAS GMBH (DE) |

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2017-10-24| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: A61K 35/745 (2015.01), A23L 33/135 (2016.01) |

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-05-02| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette]|

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2019-09-03| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-03-16| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-06-08| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-07-13| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-09-08| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 27/01/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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EP11000744.0|2011-01-31|

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EP11000744.0A|EP2481299B1|2011-01-31|2011-01-31|Bifidobacterium bifidum strains for application in gastrointestinal diseases|

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PCT/EP2012/051369|WO2012104226A1|2011-01-31|2012-01-27|Bifidobacterium bifidum strains for application in gastrointestinal diseases|

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