Abstract:
The present invention provides a method and composition for ameliorating or reducing the symptoms and signs and for the treatment of obesity, diabetes, and related conditions in a mammal in need thereof, said method comprising administering effective amounts of a pharmaceutically acceptable composition comprising a mixture of probiotic microorganisms with distinct but complementary pathways of carbohydrate metabolism, for a time sufficient to ameliorate, reduce or treat at least one sign or symptom of obesity, diabetes or cardiovascular disease. Compositions having the desired properties, and methods for their use in pharmaceutical and nutritional formulations, are provided.

Description:
[0001]    This application claims the priority benefit under 35 U.S.C. section 119 of U.S. Provisional Patent Application No. 61/703,257 entitled “Probiotic Compositions And Methods For The Treatment Of Obesity And Obesity-Related Conditions” filed on Sep. 20, 2012; which is in its entirety herein incorporated by reference. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates, in general, to combinations of probiotic organisms for the treatment of obesity, diabetes and obesity-related conditions. Also, this invention relates to methods for combining and using probiotic organisms to prevent and treat obesity, diabetes and cardiovascular disease. 
       BACKGROUND OF THE INVENTION 
       [0003]    Obesity, once an uncommon condition, is now pandemic. The World Health Organization currently estimates 1.4 billion adults worldwide are overweight. Of these, an alarming 200 million men and 300 million women are obese. Obesity is associated with a constellation of physiological disorders such as insulin resistance, type 2 diabetes mellitus, hypertension, dyslipidemia, cardiovascular disease, and metabolic syndrome. The medical costs associated with obesity in the U.S. have been estimated at $147 billion per year. Safe and effective interventions are urgently needed to combat the medical problems and costs associated with obesity. 
         [0004]    Once simplistically considered a disorder caused by an imbalance of energy intake (caloric consumption) versus energy expenditure (physical activity/exercise), obesity is now viewed as a complex, multifactorial disorder. Among other factors, the increased use of high-fructose corn syrup in the United States mirrors the rapid increase in obesity (Bray et al., 2004). In fact, studies in rats have shown that type II diabetes and hypertension can be induced by feeding a high-fructose diet (Hwang et al., 1987). The digestion, absorption and metabolism of fructose differ from those of glucose. Hepatic metabolism of fructose favors de novo lipogenesis. In addition, unlike glucose, fructose does not stimulate insulin secretion or enhance leptin production. Because insulin and leptin act as key signals in the regulation of food intake and body weight, this suggests that dietary fructose may contribute to increased energy intake and weight gain. 
         [0005]    Many previous studies have shown that probiotic bacteria support the growth of beneficial gut bacteria colonies but it also seems that certain beneficial probiotic strains can also alter host metabolism pathways for the better. Microbial organisms produce bioactive substances that influence carbohydrate and lipid metabolism, and modulate both intestinal and systemic inflammatory processes. Thus, there has been increasing interest in identifying nutritional supplements and probiotic foods that are effective for the control of obesity and diabetes (for a review, see Mallappa et al., 2012). In particular, methods are needed to identify probiotic organisms that be combined to produce effective treatments for these serious conditions. 
         [0006]    It has long been known that the gut microbiota extracts energy from dietary substances indigestible by the host. Dietary components that escape digestion by endogenous enzymes in the upper gastrointestinal tract become available as substrates in the large intestine. These non-digestible dietary carbohydrates include resistant starch, plant cell wall material, and oligosaccharides. Also, several studies indicate that fructose is not completely absorbed in the small intestine; undigested fructose is transported into the large intestine, where it is fermented by the colonic flora. In addition, several heterofermentative bacteria are capable of converting fructose to mannitol (Wisselink et al., 2002). 
         [0007]    Yadav et al. (2007) studied the progression of type II diabetes in rats fed high-fructose diets; they observed that a diet supplemented with  Lactobacillus acidophilus  and  Lactobacillus casei  delayed the onset of glucose intolerance, hyperglycemia, and hyperinsulinemia. Andreasen et al. (2010) reported that a strain of  Lactobacillus acidophilus  preserved insulin sensitivity among volunteers with type II diabetes, whereas insulin sensitivity decreased in the placebo group. Kadooka et al. (2010) observed a slight but statistically significant effect of a strain of  Lactobacillus gasseri  on abdominal adiposity, body weight and other body measures in adults with obese tendencies. However, Arora et al. (2012) found no effect of a single probiotic agent,  Lactobacillus acidophilus  NCDC 13, on weight loss in obese subjects. Also, Murphy et al. (2012) observed no improvement in metabolic profiles in obese mice fed  Lactobacillus salivarius  strain UCC118. 
         [0008]    Studies performed to date have focused primarily on single probiotic species. To date, no group has described a systematic method for combining probiotic microorganisms to improve the efficacy of probiotic compositions. However, we have observed that certain mixtures of two or more probiotic microorganisms, if given together, are more effective than individual species. In particular, we have found that a probiotic microorganism that metabolizes carbohydrates via the Embden-Myerhof pathway (EMP) or a phosphoketolase pathway (PKP) can be combined with a probiotic microorganism that metabolizes carbohydrates via a fructose-6-phosphate pathway (F6PPK) to produce synergistic effects. These metabolic pathways are known to those skilled in the art, but the use of complementary metabolic pathways to design effective treatments for obesity has not been described in the prior art. 
         [0009]    In sum, there has gone unmet a need for improved methods, compositions, etc. that can prevent weight gain and ameliorate one or more symptoms and signs associated with obesity. Effective dietary and/or pharmaceutical interventions for these conditions could have a major public health impact. The present systems and methods, etc., provide these and/or other advantages. 
       OBJECTS OF THE INVENTION 
       [0010]    It is an object of the present invention to provide mixtures of probiotic microorganisms that are safe and effective for—the prevention of weight gain and the treatment of obesity, diabetes, hypertension and cardiovascular disease. 
         [0011]    Another object of the invention is to teach methods for combining probiotic microorganisms to create synergistic compositions. 
         [0012]    These and other objects of the present invention will become more readily apparent from the description and examples which follow. 
       SUMMARY OF THE INVENTION 
       [0013]    In one aspect, the compositions, methods, systems, etc., herein are directed to providing probiotic compositions that are capable of maintaining or reducing body weight or body mass index (BMI), preventing or treating obesity and/or obesity-related conditions. The compositions can also be used to prevent excessive weight gain during pregnancy. 
         [0014]    The compositions, formulations, methods, etc., provided herein can be used as dietary supplements or as food additives or as pharmaceutical agents or otherwise as desired to achieve these aims. The methods, etc., herein include methods, kits, labels, systems, etc., directed to labeling, marketing and otherwise providing the compositions to health care professionals and/or to consumers for use in this application. 
         [0015]    The compositions may be used as dietary supplements, food and beverage additives, and as pharmaceutical agents for reducing the symptoms of obesity, diabetes and/or obesity-related conditions in a human in need thereof. 
         [0016]    The inclusion of a first probiotic microorganism that metabolizes carbohydrates via a homofermentative or heterofermentative pathway (EMP or PKP) and a second probiotic microorganism that metabolizes carbohydrates via a fructose-6-phosphate pathway (F6PPK) is essential for this invention. The first microorganism may be a homofermentative or heterofermentative lactic acid bacterium, preferably a species of  Lactobacillus.  In a further embodiment, the compositions, etc., are provided in capsules or other suitable administration formats, and a single capsule provides a full serving or dose. Generally speaking, a serving is an individual, full quantity of food or drink. Nutritional supplements and the like are typically considered foods, and thus herein the term “serving” is the term used for a full portion of supplement, which can be, for example, 1 capsule, ¼ teaspoon, or 6 tablets. Dose is a full quantity of medication to be taken at one time. As used herein, both indicate a full portion to be taken by or administered to a recipient at a single time. 
         [0017]    In general, probiotic yields are 100-450 billion Colony Forming Units (CFU) per gram. In one example, each serving or dose comprises at least about 1 billion and up to 50 billion Colony Forming Units (CFU) of active microorganisms per 1 capsule serving. For higher serving doses, powders can be used. For example, Ther-Biotic Complete Powder (ProThera, Inc.) has 400 billion CFU per teaspoon. 
         [0018]    In a further embodiment, the first microorganism is one or more of  Lactobacillus acidophilus  ( L. acidophilus ),  L. brevis, L. bulgaricus, L. casei, L. crispatus, L. curvatus, L. fermentum, L. gasseri, L. helveticus, L. johnsonii, L. paracasei, L. pentosus, L. plantarum, L. reuteri, L. rhamnosus, L. salivarius, L. sakei;  and the second microorganism is one or more of  B. bifidum, B. breve, B. lactis, B. longum,  or  B. infantis.  Alternatively, the second microorganism may be  Leuconostoc mesenteroides  (and subspecies thereof, such as  Leuconostoc pseudomesenteroides  and  Leuconostoc mesenteroides  ssp.  cremoris ). 
         [0019]    In one embodiment, the selected species of  Lactobacillus  and  Bifidobacterium  or  Leuconostoc  is combined with one or more further probiotics. The additional probiotic may be any microorganism that has a beneficial effect on obesity and/or obesity-related conditions. Typically, the additional probiotic is one or more of:  Lactobacillus acidophilus, L. brevis, L. bulgaricus, L. casei, L. crispatus, L. curvatus, L. fermentum, L. gasseri, L. helveticus, L. johnsonii, L. paracasei, L. humans paraplantarum, L. pentosus, L. plantarum, L. reuteri, L. rhamnosus, L. salivarius, L. sakei, B. animalis, B. bifidum, B. breve, B. lactis, B. longum, B. infantis, Streptococcus thermophilus, Saccharomyces boulardii,  and  Saccharomyces cereviseae.    
         [0020]    In a further embodiment, the composition can be a dietary supplement which is administered as a dried powder, a tablet, a hydroxypropyl methylcellulose capsule, or a gelatin capsule. Exemplary methods for encapsulation of probiotics can be found, e.g., in US Patent Appl. 2007/0122397. 
         [0021]    In a further embodiment, the composition can be provided within a food or beverage suitable for human consumption. For the purpose of this invention, exemplary food and beverage products include a cereal based product, rice cake, soy cake, food bar product, cold formed food bar product, custard, pudding, gelatin, rice milk, soy milk, almond milk, yogurt, kefir, juice, mashed fruit product, candy, candy bar, and applesauce. 
         [0022]    In one embodiment, none of the probiotic organisms in the composition have been or are propagated or grown in media containing casein or gluten. 
         [0023]    In an additional embodiment, the composition can be a pharmaceutical composition, subject to FDA approval. The pharmaceutical compositions, capsules, etc., herein are contained in a pharmaceutically acceptable container. As a pharmaceutical composition, the product can be marketed and dispensed together with the written description, brochure, information sheet, catalog, or label explaining the product can reduce one or more symptoms of obesity and/or the product is free of casein and gluten. In an additional embodiment the product is marketed together with a written description, brochure, information sheet, catalog, or label explaining that the product is hypoallergenic. The label can be an FDA approved label. 
         [0024]    The nutritional and/or pharmaceutical composition that is the subject of the present invention further can comprise at least one prebiotic agent that promotes the growth of probiotic microorganisms in the gastrointestinal tract. The prebiotic agent can comprise at least one of a fructooligosaccharide, galactooligosaccharide, lactulose, beta-glucan, inulin, pectin and resistant starch. 
         [0025]    The nutritional and/or pharmaceutical composition further can comprise conjugated linoleic acid (CLA) isomers, containing conjugated double bonds. Conjugated linoleic acid (CLA) is a group of polyunsaturated fatty acids found in beef, lamb, and dairy products that exist as positional and stereoisomers of octadecadienoate (18:2) (Caescu et al., 2004). Various health benefits have been attributed to CLA in experimental animal models including actions to reduce carcinogenesis, atherosclerosis, onset of diabetes, and body fat mass. The most bioactive CLA isomers are cis-9, trans-11, trans-10 and cis-12. 
         [0026]    The nutritional and/or pharmaceutical composition further can contain chromium. Chromium, as a nutritional supplement, is used to improve blood sugar control in people with prediabetes, type 1 and type 2 diabetes, and high blood sugar due to taking steroids. Two easily absorbed forms of chromium are chromium polynicotinate or chromium picolinate or chromium histidinate. As an example, the compositions contemplated herein can contain chromium polynicotinate at a dose of approximately 500-1000 μg per capsule or dose. 
         [0027]    The obesity-related condition that is the subject of the invention can be one or more of hyperglycemia, insulin resistance, diabetes, hypertriglyceridemia, hypercholesterolemia, atherosclerosis, angina pectoris, arterial occlusion, myocardial infarction and/or stroke. 
         [0028]    In addition, the compositions and methods, etc. of the invention can be used to ameliorate or prevent excessive weight gain during pregnancy. The Institute of Medicine recommends a weight gain of 25-35 pounds for women of normal weight, 28-40 pounds for those considered underweight, 15-25 pounds for overweight women and no more than 15 pounds for obese women. A woman with a normal BMI (Body Mass Index) of 20-25 should attempt to follow this weight gain schedule: 
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 WEEK TOTAL 
                 CUMULATIVE GAIN 
                 EXAMPLE (140 pound woman) 
               
               
                   
               
             
             
               
                 15 weeks 
                  2-5 pounds 
                 Total Weight: 142-145 pounds 
               
               
                 20 weeks 
                  6-11 pounds 
                 Total Weight: 146-151 pounds 
               
               
                 25 weeks 
                 11-17 pounds 
                 Total Weight: 151-157 pounds 
               
               
                 30 weeks 
                 16-23 pounds 
                 Total Weight: 156-163 pounds 
               
               
                 35 weeks 
                 20-28 pounds 
                 Total Weight: 160-168 pounds 
               
               
                 40 weeks 
                 25-35 pounds 
                 Total Weight: 165-175 pounds 
               
               
                   
               
             
          
         
       
     
         [0029]    Accordingly, “excessive weight gain” can be defined as weight gain that exceeds the guidelines shown above. 
         [0030]    The compositions and methods, etc. can be used to ameliorate or prevent gestational diabetes. Gestational diabetes is defined as “any degree of glucose intolerance with onset or first recognition during pregnancy” (Metzger et al., 1998). 
         [0031]    In still other aspects, this application is directed to isolated and purified compositions as described herein for use in the manufacture of a medicament for reducing or maintaining body mass index (BMI) or for inhibiting, preventing, or treating obesity or an obesity-related condition, as well as methods of manufacturing such medicaments, which can comprise combining a pharmaceutically effective amount of the composition and a pharmaceutically acceptable capsule, tablet, powder or liquid. 
         [0032]    These and other aspects, features and embodiments are set forth within this application, including the following Detailed Description. Unless expressly stated otherwise, all embodiments, aspects, features, etc., can be mixed and matched, combined and permuted in any desired manner. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0033]    The body mass index (BMI) (calculated as weight in kilograms divided by the square of height in meters) is the most commonly accepted measurement for overweight and/or obesity. In adults, a BMI exceeding 25 is considered overweight, while obesity is defined as a BMI of 30 or more, with a BMI of 35 or more considered as serious co-morbidity and a BMI of 40 or more considered morbid obesity. For the purposes of this invention, “obesity” shall mean a BMI of 30 or more. 
         [0034]    One out of every five overweight people is affected by the “metabolic syndrome”. Metabolic syndrome is one of the fastest growing obesity-related health concerns in the United States and is characterized by a cluster of health problems including obesity, hypertension, abnormal lipid levels, and high blood sugar. According to the Centers for Disease Control and Prevention (CDC), the metabolic syndrome affects almost one quarter (22 percent) of the American population—an estimated 47 million people. The assemblage of problems characterized as comprising the metabolic syndrome can increase a patient&#39;s risk for developing more serious health problems, such as diabetes, heart disease, and stroke. 
         [0035]    Overweight and obese people have an increased incidence of heart disease, and thus fall victim to heart attack, congestive heart failure, sudden cardiac death, angina, and abnormal heart rhythm more often than those that maintain a healthy body mass index. Obesity often increases the risk of heart disease because of its negative effect on blood lipid levels, which increase in obese patients and then, in turn, increase triglyceride levels and decrease high-density lipoprotein—which is also known as HDL. People with an excessive amount of body fat have higher levels of triglycerides and low-density lipoprotein—which is also known as LDL or “bad cholesterol”—as well as lower levels of HDL cholesterol in the blood. This combination creates optimal conditions for developing atherosclerotic heart disease. 
         [0036]    Being overweight or obese increases the risk of developing high blood pressure. Hypertension, or high blood pressure, greatly raises the risk of heart attack, stroke, and kidney failure. In fact, blood pressure rises as body weight increases. Losing even 10 pounds can lower blood pressure—and losing weight has the biggest effect on those who are overweight and already have hypertension. 
         [0037]    Obesity is associated with the development of diabetes. More than 80 percent of people with type 2 diabetes, the most common form of the disease, are obese or overweight. Type 2 diabetes develops when either there is impaired insulin production by the pancreas in the setting of insulin resistance in the tissues and organs in the body. As obesity diminishes insulin&#39;s ability to control blood sugar (glucose), there is an increased risk of developing diabetes because the body begins overproducing insulin to regulate blood sugar levels. Over time, the body is no longer able to keep blood sugar levels in the normal range. Eventually the inability to achieve healthy blood sugar balance results in the development of type 2 diabetes. Furthermore, obesity complicates the management and treatment of type 2 diabetes by increasing insulin resistance and glucose intolerance, which makes drug treatment for the disease less effective. In many cases, a reduction of body weight to a normal range normalizes blood glucose and restores insulin sensitivity. 
         [0038]    Childhood obesity is also a major public health problem, particularly in Western countries. Children 2-18 years of age are considered obese if the BMI is greater than the 95th percentile. Despite policies targeted at reducing its prevalence, childhood obesity has more than doubled in children and tripled in adolescents in the past 30 years. As with adults, obesity in childhood causes hypertension, dyslipidaemia, chronic inflammation, increased blood clotting tendency, endothelial dysfunction, and hyperinsulinemia. This clustering of cardiovascular disease risk factors has been identified in children as young as 5 years of age. Thus there is an urgent need for safe effective interventions, including nutritional interventions, to combat the epidemic of obesity in children as well as in adults. 
         [0039]    The present compositions, medicaments, therapeutics, systems, methods, etc., are directed to the prevention, inhibition and treatment of obesity and obesity-related conditions. Said obesity-related conditions are selected from the group consisting of insulin resistance, hyperglycemia, diabetes, hypertriglyceridemia, atherosclerosis, angina pectoris, myocardial infarction and/or stroke. 
       Probiotic Compositions 
       [0040]    “Probiotics” within the context of the present invention is used in accord with its usual meaning, for example as selected, viable microbial dietary supplements that, when introduced in sufficient quantities, beneficially affect the human organism via their effects in the gastrointestinal tract (Holzapfel et al., 2001; Holzapfel &amp; Schillinger, 2002). The FAO/WHO has adopted the definition of probiotics as “Live microorganisms which when administered in adequate amounts confer a health benefit on the host” (FAO/WHO guidelines, 2002). These beneficial bacteria may be found for example in milk or in milk processing factories, living or decaying plants, and also in the intestines of man and animals. 
         [0041]    Currently, the best-studied probiotics are the lactic acid bacteria, particularly  Lactobacillus  spp. and  Bifidobacterium  spp.  Lactobacillus  is a genus of Gram-positive facultative anaerobic bacteria. The genus  Lactobacillus  currently comprises over 100 species and encompasses a wide variety of organisms. They are common and usually benign. In humans they are present in the vagina and the gastrointestinal tract, where they are symbiotic and make up a small portion of the gut flora (Tannock, 1999). Lactobacilli that have been used in humans include  L. acidophilus, L. salivarius, L. johnsonii, L. casei, L. lactis, L. reuteri, L. plantarum, L. rhamnosus, L. brevis, L. gasseri,  and other species and subspecies. The use of  Lactobacillus  species in humans has been extensively reviewed in the scientific literature, including the references provided herein. These ingredients are readily available from commercial suppliers, including Danisco-Dupont (US); Chr. Hansen (Denmark); Institut Rosell Lallemand (Montreal, Canada); and others. Exemplary species and strains of  Lactobacillus  for the present invention include the following well-known strains:  L. acidophilus  NCFM,  L. acidophilus  La-14,  L. bulgaricus  Lb-64,  L. brevis  Lbr-35,  L. casei  Lc-11,  L. lactis  L1-23,  L. plantarum  Lp-115,  L. paracasei  Lpc-37,  L. rhamnosus  Lr-32 and  L. salivarius  Ls-33, which are well known to those skilled in the art. 
         [0042]      Bifidobacterium  is a genus of Gram-positive anaerobic bacteria, currently comprised of 31 characterized species, 11 of which have been detected in human feces (Tannock, 1999). Bifidobacteria are Gram-positive, irregular or branched rod-shaped bacteria that are commonly found in the intestines of humans and most animals and insects. Probiotic  Bifidobacterium  strains that are useful for the present invention include but are not limited to the following strains which are well known to those skilled in the art:  B. breve  Bb-03,  B. lactis  Bi-07 and Bi-04,  B. longum  Bi-05. 
         [0043]      Leuconostoc  is a genus of Gram-positive bacteria, placed within the family of Leuconostocaceae. All species within this genus are heterofermentative.  Leuconostoc,  along with other lactic acid bacteria such as  Pediococcus  and  Lactobacillus,  is responsible for the fermentation of cabbage making sauerkraut. For the purposes of the present invention, one exemplary strain of  Leuconostoc  is  L. mesenteroides  ATCC 13146. 
       Carbohydrate Metabolism in Lactic Acid Bacteria 
       [0044]    Lactic acid bacteria (LAB) are capable of generating energy by homo- or heterofermentative metabolism of sugars. During anaerobic growth of obligately homofermentative LAB in the presence of excess substrate, energy sources like glucose are converted into pyruvate via the Embden-Meyerhoff-Parnas pathway, and the pyruvate is further metabolized to lactate (see FIG. 1). Homofermentative LAB include most species of  enterococci, lactococci, pediococci, streptococci, tetragenococci,  and  vagococci.    
         [0045]    Early work demonstrated that fructose 1,6 bisphosphate aldolase (EC 4.1.2.13) and isomerase enzymes were absent in heterofermentative organisms, suggesting that the pathway does not follow the usual Embden-Meyerhof pattern of glycolysis (DeMoss et al., 1951). As more research was conducted, it was realized that these organisms utilize a different pathway, named the phosphoketolase pathway (PKP; EC 4.1.2.9), which produces equimolar amounts of CO 2 , lactate, and acetate-ethanol (FIG. 2). 
         [0046]    Heterofermentative LAB can be divided into obligately heterofermentative species, in which both hexoses and pentoses are fermented via the PKP, and facultatively heterofermentative organisms, which degrade hexoses via the Embden-Meyerhoff-Parnas pathway and pentoses via the PKP. Many of the enzymes used in the latter pathway are shared with the pentose phosphate pathway. 
         [0047]    Xylulose 5-phosphate phosphoketolase (XPK; EC 4.1.2.9) is the central enzyme of the PKP of heterofermentative and facultative homofermentative lactic acid bacteria. XPK prefers xylulose 5-phosphate to fructose 6-phosphate. In the presence of inorganic phospate this enzyme converts xylulose 5-phosphate (X5P) into glyceraldehyde 3-phosphate and acetylphosphate. Some taxa known to possess the PKP pathway include  Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus reuteri, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc mesenteroides  ssp.  cremoris,  and some species of  Weissella.    
         [0048]    Lactobacilli can be grouped into one of these categories:
   1) Obligately homofermentative (Group I) including:  L. acidophilus, L. delbrueckii, L. helveticus, L. salivarius      2) Facultatively heterofermentative (Group II) including:  L. casei, L. curvatus, L. plantarum, L. sakei      3) Obligately heterofermentative (Group III) including:  L. brevis, L. buchneri, L. fermentum, L. reuteri      
 
         [0052]    Bifidobacteria are considered key commensals in human-microbe interactions and they contribute to the degradation of undigested polysaccharides in the human colon (Suzuki et al., 2010). Bifidobacteria utilize a unique pathway of hexose catabolism which produces primarily acetate and lactate (de Vries and Stouthamer, 1967). This fermentation pathway, which is known as the “Bifid shunt” or the “fructose-6-phosphate pathway” yields 3 mols of acetate and 2 mols of lactate for 2 mols of glucose, with production of 5 mols of ATP. The key enzyme in the pathway is xylulose-5-phosphate phosphoketolase/fructose-6-phosphate phosphoketolase (Xfp; EC 4.1.2.22), which catalyzes two important steps: splitting D-fructose 6-phosphate into D-erythrose 4-phosphate and acetylphosphate, and splitting D-xylulose 5-phosphate into D-glyceraldehyde 3-phosphate and acetylphosphate. This enzyme has often been used as a tool in the identification of bifidobacteria. More recently, such dual substrate-specificity enzymes have been found in other organisms including  Leuconostoc mesenteroides  and  Lactobacillus paraplantarum  (Lee et al. 2005; Jeong et al., 2007). 
         [0053]    In addition,  Bifidobacterium longum,  which metabolizes intracellular fructose via the fructose-6-P phosphoketolase pathway, contains a fructokinase (Frk; EC 2.7.1.4) (Caescu et al. 2004). Fructokinases have also been found in  Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Lactobacillus plantarum,  and  Lactococcus lactis.  The presence of fructokinase enables these organisms to grow using fructose as a unique carbon source. Furthermore, a number of heterofermentative lactic acid bacteria (LAB), yeasts, and filamentous fungi also are known to convert fructose into mannitol in significant quantities, including  Leuconostoc mesenteroides.  The reduction of fructose to mannitol in heterofermentative lactic acid bacteria is catalyzed by an NADH-linked mannitol dehydrogenase (EC 1.1.1.67) (Wisselink et al., 2002; Saha &amp; Racine, 2011). 
         [0054]    In clinical practice we have discovered that certain combinations of lactic acid bacteria, if given together, are far more effective than if either species is given alone. The basis of the present invention is the observation that compositions comprising two or more probiotic organisms, with distinct pathways of carbohydrate metabolism, produce synergistic results with respect to weight loss and diabetes. Specifically, we have discovered that combination products containing at least one species of  Lactobacillus  and at least one species of  Bifidobacterium  or  Leuconostoc  are effective in the treatment of obesity, diabetes, and obesity-related conditions. 
         [0055]    No one has previously recognized that distinct carbohydrate metabolic pathways can be used to design synergistic compositions for the treatment of obesity. Without being bound by theory, we propose that the beneficial effects of our compositions may result from the efficient metabolism of carbohydrates by the combined action of these particular species; from the combination of short-chain fatty acids or other metabolites produced by the complementary species; or by another mechanism. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Compositions for the treatment of obesity and diabetes 
               
             
          
           
               
                   
                   Lactobacillus  sp. (per capsule 
                   Bifidobacterium  sp. (per 
                 Other sp. (per 
               
               
                 Composition 
                 or serving) 
                 capsule or serving) 
                 capsule or serving) 
               
               
                   
               
               
                 Composition 1 
                   L. acidophilus ,  L. rhamnosus , 
                   B. bifidum ,  B. longum ,  B. lactis , 
                 
                   Streptococcus 
                 
               
               
                   
                   L. casei ,  L. plantarum ,  
                   B. breve  (1.0+ billion CFU of 
                   thermophilus  (1.0+ 
               
               
                   
                   L.   salivarius  (2.5+ billion CFU of  
                 each) 
                 billion CFU) 
               
               
                   
                 each) 
                   
                   
               
               
                 Composition 2 
                   L. rhamnosus ,  L. casei ,  
                   B. bifidum ,  B. longum ,  B. breve , 
                   
               
               
                   
                   L. salivarius ,  L. paracasei  (2.0+  
                   B. infantis  (2.0+ billion CFU of 
                   
               
               
                   
                 billion CFU of each) 
                 each) 
                   
               
               
                 Composition 3 
                   L. acidophilus ,  L. rhamnosus , 
                   B. bifidum ,  B. lactis ,  B. infantis   
                   
               
               
                   
                   L. brevis  (1.0+ billion CFU of  
                 (1.0+ billion CFU of each) 
                   
               
               
                   
                 each) 
                   
                   
               
               
                 Composition 4 
                   L. acidophilus ,  L. rhamnosus   
                   B. bifidum ,  B. lactis  (1.5+  
                 
                   Leuconostoc 
                 
               
               
                   
                 (1.5+ billion CFU of each) 
                 billion CFU of each) 
                   mesenteroides  (1.0+ 
               
               
                   
                   
                   
                 billion CFU) 
               
               
                 Composition 5 
                   L. acidophilus , 2.5+ billion  
                   B. bifidum , 2.5+ billion CFU 
                 — 
               
               
                   
                 CFU 
                   
                   
               
               
                 Composition 6 
                   L. acidophilus , L. rhamnosus , 
                   B. bifidum ,  B. lactis , 1.25+ 
                 — 
               
               
                   
                 1.25+ billion CFU of each 
                 billion CFU of each 
                   
               
               
                 Composition 7 
                   L. acidophilus  (6.3+ billion  
                   B. bifidum , 6.3+ billion CFU 
                 
                   Streptococcus 
                 
               
               
                   
                 CFU),  L. rhamnosus  (9.4+  
                   
                   thermophilus  1.5+ 
               
               
                   
                 billion CFU),  L. bulgaricus   
                   
                 billion CFU 
               
               
                   
                 (1.5+ billion CFU) 
                   
                   
               
               
                 Composition 8 
                   L. rhamnosus ,  L. casei ,  
                   B. bifidum ,  B. longum ,  B. breve , 
                 — 
               
               
                   
                   L. salivarius ,  L. paracasei  (17+ 
                   B. infantis  (8+ billion CFU of 
                   
               
               
                   
                 billion CFU of combined 
                 combined  Bifidobacterium   
                   
               
               
                   
                   Lactobacillus  species) 
                 species) 
                   
               
               
                 Composition 9 
                   L. acidophilus ,  L. rhamnosus , 
                   B. bifidum ,  B. longum ,  B. lactis   
                 — 
               
               
                   
                   L. paracasei  (100+ billion CFU 
                   
                   
               
               
                   
                 of combined  Lactobacillus  and 
                   
                   
               
               
                   
                   Bifidobacterium  species) 
                   
                   
               
               
                 Composition 10 
                   L. acidophilus  (1.85+ billion 
                   B. bifidum  (0.70+ billion CFU), 
                 
                   Streptococcus 
                 
               
               
                   
                 CFU),  L. rhamnosus ,  
                 
                   B. lactis 
                 
                 
                   thermophilus 
                 
               
               
                   
                   L. bulgaricus ,  L. brevis ,  L. casei , 
                   
                   
               
               
                   
                   L. salivarius ,  L. plantarum   
                   
                   
               
               
                   
                 (4.9+ billion CFU combined 
                   
                   
               
               
                   
                 with  S. thermophilus ) 
                   
                   
               
               
                 Composition 11 
                   L. rhamnosus  (2.5+ billion 
                   B. bifidum ,  B. breve  (1.25+ 
                 
                   Saccharomyces 
                 
               
               
                   
                 CFU) 
                 billion CFU) 
                   boulardii  (5.0+ 
               
               
                   
                   
                   
                 billion CFU) 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 1  
       [0056]    A 47 year-old man presents for evaluation of obesity. He has been gaining weight since his early 40s. He weighs 280 pounds and is 5 foot 11 inches tall with a body mass index (BMI) of 39. His blood pressure is 140/90. Laboratory testing is remarkable for a fasting blood glucose of 136 mg/dL and triglycerides of 220 mg/dL. A diet is recommended consisting of high protein, reduced refined carbohydrates, and 2200 calories per day. A 4-day-per-week minimum program of aerobic exercise is prescribed. The patient is given a multispecies  Bifidobacterium/Lactobacillus  probiotic formula, (Composition 1; see Table 1) in the amount of two capsules per day to be taken with meals. When the patient is seen in follow up after 3 months, his weight is 232 with a BMI of 32.4. His blood pressure is now 130/84 and his glucose and triglycerides are normal. He is advised to continue his diet, exercise and probiotics. When he is seen again in 6 months, he weighs 189 pounds with a BMI of 26.8. 
       EXAMPLE 2  
       [0057]    A 62-year old woman presents for evaluation of obesity. She weighs 191 pounds and is 5 foot 6 inches tall with a body mass index (BMI) of 31. She reports consuming a diet consisting of approximately 2200 calories per day and walking for 30 minutes three to four days per week. For the past three months she has been consuming 3 billion CFU/day of a commercial  Lactobacillus acidophilus  supplement; however, she has been unable to lose weight. The patient is given a multispecies probiotic formula containing  Bifidobacterium  and  Leuconostoc  in addition to  Lactobacillus  (Composition 4; see Table 1) in the amount of one capsule per day to be taken with a meal. When the patient is seen at followup in 3 months her weight is 175 pounds with a BMI of 28. 
       EXAMPLE 3  
       [0058]    A 30-year-old pregnant woman presents for evaluation of excessive weight gain and gestational diabetes in her 28 th  week of pregnancy. She is 5′4″ tall and weighs 163 pounds, having gained 40 pounds during her pregnancy. An oral glucose tolerance test reveals a plasma glucose level of 12 mmol/L when measured 2 hours after the challenge, suggesting overt diabetes. The patient is advised to avoid high-sugar foods, like sweets and desserts; to increase her daily intake of dietary fiber and protein; and to incorporate at least 40 minutes per day of gentle exercise in her routine. In addition, the patient is given a  Lactobacillus/Bifidobacterium  probiotic formula, consisting of  L. acidophilus, L. rhamnosus, B. bifidum, and B. lactis  (Composition 3; see table 1) and advised to take one capsule per day with a meal. When the patient is seen at followup in 2 weeks her weight has stabilized at 163 pounds and her plasma glucose level has decreased to 10.5 mmol/L when measured 2 hours after a glucose challenge. 
       EXAMPLE 4  
       [0059]    A 10-year-old female child is seen at a pediatric clinic for evaluation of obesity. At birth, she weighed 9 pounds and was 20 inches in length. Even in infancy the child had risk factors for obesity as a result of a family history of the disease. Also, the patient&#39;s mother had gestational diabetes, which can predispose a child to overweight/obesity. Children 2-18 years of age are considered obese if the BMI is greater than the 95th percentile. During early childhood, the patient&#39;s weight was maintained in the 90 th  to 95 th  percentile. However, she continued to grow, and by her 10 th  birthday she was considered overweight with a height of 50 inches, weight of 85 pounds, and BMI of 24 which is in the 96 th  percentile according to CDC guidelines (Centers for Disease Control). A dietitian advises the mother to modify the child&#39;s diet by limiting snack foods and providing fresh fruit for dessert instead of cookies. The dietitian also advises a probiotic supplement, (Composition 2; see Table 1), which contains a mixture of  Lactobacillus  and  Bifidobacterium,  in the amount of one capsule per day to be consumed with a meal. When seen at follow-up 6 months later the patient&#39;s height is 52 inches and her weight is 74 pounds, which represents a weight loss of 11 pounds. The patient&#39;s BMI is now 19.2, placing the BMI-for-age at the 76th percentile. The patient&#39;s mother is advised to continue the diet and probiotic regimen and to encourage the child to participate in a sports, dance or an exercise program. 
         [0060]    The entire contents including the references cited therein and the following patents, published applications including all their foreign equivalents and journal publications are incorporated by reference in their entirety for all purposes to the same extent as if each individual patent, patent application or publication were so individually denoted. 
       REFERENCES 
     US Patent Documents 
       [0061]      
         [0000]    
       
         
               
               
               
               
             
           
               
                   
               
               
                   
                 US Patent 
                 Date 
                 Inventor 
               
               
                   
               
             
             
               
                   
                 U.S. Pat. No. 6,641,808  
                 November 2003 
                 Bojrab 
               
               
                   
                 U.S. Pat. No. 6,942,857  
                 September 2005 
                 Song, et al. 
               
               
                   
               
             
          
         
       
     
       US Patent Applications 
       [0062]      
         [0000]    
       
         
               
               
               
               
             
           
               
                   
               
               
                   
                 Applications 
                 Publication Date 
                 Inventor 
               
               
                   
               
             
             
               
                   
                 2011/0123501 
                 May 26, 2011 
                 Chou, et al. 
               
               
                   
                 2010/0111915 
                 May 6, 2010 
                 Isolauri, et al. 
               
               
                   
                 2005/0112112 
                 May 26, 2005 
                 Park, et al. 
               
               
                   
                 2012/0121753 
                 May 17, 2012 
                 Kim, et al. 
               
               
                   
                 2010/0061967 
                 Mar. 11, 2010 
                 Rautonen 
               
               
                   
                 2012/0058094 
                 Mar. 8, 2012 
                 Blaser, et al. 
               
               
                   
               
             
          
         
       
     
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