Compositions and methods for promoting nitric oxide production through an oral delivery system

Abstract

Probiotic compositions, particularly oral compositions, comprising one or more bacteria that are capable of producing nitrite and/or nitric oxide in a subject are provided. Some compositions further comprise nitrate/nitrate, such as a botanical source of nitrate. Some compositions further comprise botanical sources of nitrate reductase. Also, provided are methods of improving the oral and/or vascular health of a subject by orally administering a composition comprising one or more bacteria capable of producing nitric oxide. Methods of repopulating the nitric oxide producing microflora and bacterial environment in the oral cavity of a subject are also provided.

1 . A method of establishing or enhancing a bacteria population in the oral cavity comprising administering a probiotic composition, where the probiotic composition comprises bacteria selected from a group consisting essentially of  Neisseria, Veillonella, Haemophilus, Actinomyces, Granulicatella, Prevotella, Leptotrichia, Brevibacillus, Porphyromonas, Fusobacterium , and clade Gemellaceae, where the composition is configured to release at least a portion of the bacteria in the oral cavity. 
     
     
         2 . The method of  claim 1 , wherein the bacteria are selected from a group consisting essentially of  Granulicatella, Neisseria, Prevotella, Haemophilus, Fusobacterium, Brevibacillus , and clade Gemellaceae. 
     
     
         3 . The method of  claim 1 , wherein the bacteria comprise one or more of the following:  Granulicatella adiacens, Haemophilus parainfluenzae, Actinomyces odontolyticus, Actinomyces viscosus, Actinomyces oris, Neisseria flavescens, Neisseria mucosa, Neisseria sicca, Neisseria subflava, Prevotella melaninogenica, Prevotella salivae, Veillonella dispar, Veillonella parvula, Veillonella atypical, Fusobacterium nucleatum , and  Brevibacillus brevis.    
     
     
         4 . The method of  claim 1 , wherein the bacteria consists essentially of nitrite-accumulating bacteria, where the nitrite-accumulating bacteria are bacteria that allow nitrite levels in a bacteria culture to increase and nitrate levels to decrease when the bacteria culture comprises bacteria isolate or mixture consisting essentially of the nitrite-accumulating bacteria. 
     
     
         5 . The method of  claim 1 , wherein the bacteria consists essentially of strains that contain nitrate-reductase encoding genes and do not contain nitrite-reductase encoding genes. 
     
     
         6 . The method of  claim 1 , wherein the bacteria consists essentially of strains that express one or more of the following nitrate reductase genes: narG, narL, narJ, narQ, narI, napC, napB, napH, napD, napA, napG, and napF. 
     
     
         7 . The method of  claim 1 , wherein the isolated bacteria consists essentially of strains that do not express the following nitrite reductase genes: nirK, nirB, nirD, nrfF, nrfA, and nrfH. 
     
     
         8 . The method of  claim 1 , wherein the bacteria consists essentially of strains that do not express nitric oxide reductase. 
     
     
         9 . The method of  claim 1 , wherein the bacteria consists essentially of weak or non-acid producing bacterial strains. 
     
     
         10 . The method of  claim 1 , wherein the composition comprises 1 mg to 100 g of the bacteria. 
     
     
         11 . The method of  claim 1 , where the composition comprise an activity of 5 billion to 20 billion colony forming units. 
     
     
         12 . The method of  claim 1 , wherein the composition is in the form of a tablet, a granule, powder, a gum, a biofilm, an oral liquid preparation, a food product, or a lozenge. 
     
     
         13 . The method of  claim 1 , wherein the probiotic composition is stored at or below 45° C. 
     
     
         14 . The method of  claim 1 , wherein the bacteria are freeze-dried. 
     
     
         15 . The method of  claim 1 , wherein the bacteria are genetically-modified or recombinant bacteria, where a gene encoding nitric oxide reductase is suppressed. 
     
     
         16 . The method of  claim 1 , wherein the probiotic composition further comprises nitrate. 
     
     
         17 . The method of  claim 1 , wherein the probiotic composition further comprises a botanical source of nitrate. 
     
     
         18 . The method of  claim 17 , wherein the botanical source of nitrate comprises one or more of beet root, kale, artichoke, holy basil,  gymnema sylvestre , ashwagandha root,  salvia , St. John wort, broccoli,  stevia , spinach, gingko, kelp,  tribulus , eleuthero,  epimedium, eucommia , hawthorn berry,  rhodiola , green tea, codonopsys,  panax ginseng, astragalus , pine bark, dodder seed,  Schisandra, cordyceps , and mixtures thereof. 
     
     
         19 . The method of  claim 1 , the probiotic composition further comprises the one or more excipients, wherein the one or more excipients comprises a substance having a pH buffering capacity. 
     
     
         20 . The method of  claim 19 , wherein the pH buffering substance is selected form the group consisting of bicarbonates, carbamides, phosphates, proteins, salts, and combinations of two or more thereof. 
     
     
         21 . A probiotic composition comprising:
 an effective amount of nitrate and   a bacteria isolate or mixture that comprises one or more of  Neisseria, Veillonella, Haemophilus, Actinomyces, Granulicatella, Prevotella, Leptotrichia, Brevibacillus, Porphyromonas, Fusobacterium , and oral bacteria of the Gemellaceae family, wherein the composition does not contain a contaminating amount of  Lactobacillus.      
     
     
         22 . The probiotic composition of  claim 21 , wherein the bacteria are selected from a group consisting essentially of  Granulicatella, Neisseria, Prevotella, Haemophilus, Fusobacterium, Brevibacillus  and clade Gemellaceae. 
     
     
         23 . The probiotic composition of  claim 21 , wherein the bacteria comprise one or more of the following:  Granulicatella adiacens, Haemophilus parainfluenzae, Actinomyces odontolyticus, Actinomyces viscosus, Actinomyces oris, Neisseria flavescens, Neisseria mucosa, Neisseria sicca, Neisseria subflava, Prevotella melaninogenica, Prevotella salivae, Veillonella dispar, Veillonella parvula, Veillonella atypical, Fusobacterium nucleatum , and  Brevibacillus brevis.    
     
     
         24 . The probiotic composition of  claim 21 , wherein the bacteria consists essentially of nitrite-accumulating bacteria, where the nitrite-accumulating bacteria are bacteria that allow nitrite levels in a bacteria culture to increase and nitrate levels to decrease when the bacteria culture comprises bacteria isolate or mixture consisting essentially of the nitrite-accumulating bacteria. 
     
     
         25 . The probiotic composition of  claim 21 , wherein the bacteria consists essentially of bacteria that comprise nitrate-reductase encoding genes and do not comprise nitrite-reductase encoding genes. 
     
     
         26 . The probiotic composition of  claim 21 , wherein the isolated bacteria consists essentially of strains that express one or more of the following nitrate reductase genes: narG, narL, narJ, narQ, narI, napC, napB, napH, napD, napA, napG, and napF. 
     
     
         27 . The probiotic composition of  claim 21 , wherein the isolated bacteria consists essentially of strains that do not express the following nitrite reductase genes: nirK, nirB, nirD, nrfF, nrfA, and nrfH. 
     
     
         28 . The probiotic composition of  claim 21 , wherein the isolated bacteria consists essentially of strains that do not express nitric oxide reductase. 
     
     
         29 . The probiotic composition of  claim 21 , wherein the isolated bacteria consists essentially of weak or non-acid producing bacterial strains. 
     
     
         30 . The probiotic composition of  claim 21 , wherein the isolated bacteria are naturally present in oral microflora of a mammal. 
     
     
         31 . The probiotic composition of  claim 21 , wherein the composition is in the form of a tablet, a capsule, a granule, powder, a gum, a biofilm, an oral liquid preparation, a food product, or a lozenge. 
     
     
         32 . The probiotic composition of  claim 21 , wherein the probiotic composition is stored at or below 45° C. 
     
     
         33 . The probiotic composition of  claim 21 , wherein the bacteria are freeze-dried. 
     
     
         34 . The probiotic composition of  claim 21 , wherein the bacteria are genetically-modified or recombinant bacteria, where a gene encoding nitric oxide reductase is suppressed. 
     
     
         35 . The probiotic composition of  claim 21 , where the effective amount of nitrate is from a botanical source. 
     
     
         36 . The probiotic composition of  claim 21 , wherein the botanical source comprises one or more of beet root, kale, artichoke, holy basil,  gymnema sylvestre , ashwagandha root,  salvia , St. John wort, broccoli,  stevia , spinach, gingko, kelp,  tribulus , eleuthero,  epimedium, eucommia , hawthorn berry,  rhodiola , green tea, codonopsys,  panax ginseng, astragalus , pine bark, dodder seed,  Schisandra, Cordyceps , and Mixtures thereof. 
     
     
         37 . The probiotic composition of  claim 21 , wherein the probiotic composition is an oral composition. 
     
     
         38 . The probiotic composition of  claim 21 , further comprising one or more excipients, wherein the one or more excipients comprises a substance having a pH buffering capacity. 
     
     
         39 . The probiotic composition of  claim 38 , wherein the pH buffering substance is selected form the group consisting of bicarbonates, carbamides, phosphates, proteins, salts, and combinations of two or more thereof. 
     
     
         40 . Use of a probiotic composition according to any one of  claims 21  to  39  in oral form for the enhancement of nitric oxide (NO) production in subjects suffering from one or more of cardiovascular disease, atherosclerosis, stroke, ischemic injury, peripheral artery disease, congestive heart failure, hypertension, pulmonary arterial hypertension, hypertension associated with urea cycle disorders and pre-eclampsia, vascular dementia, Alzheimer's Disease, metabolic syndrome, and type 2 diabetes. 
     
     
         41 . A method of enhancing nitrate reduction in the oral cavity comprising
 administering a probiotic composition,   where the probiotic composition comprises a bacteria isolate or mixture consisting essentially of nitrite-accumulating bacteria, where the nitrite-accumulating bacteria are bacteria that allow nitrite levels in a bacteria culture to increase and nitrate levels to decrease when the bacteria culture comprises nitrate and a bacteria isolate or mixture consisting essentially of the nitrite-accumulating bacteria and   where the composition is configured to release at least a portion of the bacteria in the oral cavity.   
     
     
         42 . The method of  claim 41 , where the nitrite-accumulating bacteria are bacteria that allow nitrite levels in a bacteria culture to increase by at least two-fold when the bacteria culture comprises nitrate and a bacteria isolate or mixture consisting essentially of the nitrite-accumulating bacteria. 
     
     
         43 . The method of  claim 41 , where the nitrite accumulating bacteria are naturally occurring in the oral cavity of a human. 
     
     
         44 . The method of  claim 41 , where the nitrite accumulating bacteria are bacteria that reduce nitrate at a higher rate than reducing nitrite. 
     
     
         45 . The method of  claim 41 , where the probiotic composition is a probiotic composition according to any one of  claims 21  to  39 . 
     
     
         46 . A method of enhancing nitrate reduction in the oral cavity comprising
 administering a probiotic composition,   where the probiotic composition comprises a bacteria isolate or mixture that comprises one or more of  Neisseria, Veillonella, Haemophilus, Actinomyces, Granulicatella, Prevotella, Leptotrichia, Brevibacillus, Porphyromonas, Fusobacterium , and clade Gemellaceae,   wherein the composition does not contain a contaminating amount of  Lactobacillus.      
     
     
         47 . The method of  claim 46 , wherein the bacteria are selected from a group consisting essentially of  Granulicatella, Neisseria, Prevotella, Haemophilus, Fusobacterium, Brevibacillus  and clade Gemellaceae. 
     
     
         48 . The method of  claim 46 , where the probiotic composition is a probiotic composition according to any one of  claims 21  to  39 . 
     
     
         49 . A method of making a probiotic composition comprising
 combining a bacteria isolate or mixture consisting essentially of nitrite-accumulating bacteria and a carrier to form a probiotic composition,   where the nitrite-accumulating bacteria are bacteria that allow nitrite levels in a bacteria culture to increase and nitrate levels to decrease when the bacteria culture comprises nitrate and a bacteria isolate or mixture consisting essentially of the nitrite-accumulating bacteria.   
     
     
         50 . The method of making the probiotic composition of  claim 49 , further comprising
 making a dosage form of the probiotic composition, where the dosage form is a tablet, a capsule, a granule, powder, a gum, a biofilm, an oral liquid preparation, a food-product serving, or a lozenge.   
     
     
         51 . The method of making the probiotic composition of  claim 49 , further comprising
 combining nitrate, the bacteria isolate or mixture consisting essentially of nitrite-accumulating bacteria, and a carrier to form a probiotic composition.   
     
     
         52 . The method of  claim 49 , where the probiotic composition is a probiotic composition according to any one of  claims 21  to  39 . 
     
     
         53 . A method of making a probiotic composition comprising
 combining a bacteria isolate or mixture and a carrier to form a probiotic composition,   where bacteria isolate or mixture comprises one or more of  Neisseria, Veillonella, Haemophilus, Actinomyces, Granulicatella, Prevotella, Leptotrichia, Brevibacillus, Porphyromonas, Fusobacterium , and clade Gemellaceae and does not contain a contaminating amount of  Lactobacillus.      
     
     
         54 . The method of making the probiotic composition of  claim 49 , further comprising
 making a dosage form of the probiotic composition, where the dosage form is a tablet, a capsule, a granule, powder, a gum, a biofilm, an oral liquid preparation, a food-product serving, or a lozenge.   
     
     
         55 . The method of making the probiotic composition of  claim 49 , further comprising
 combining nitrate, the bacteria isolate or mixture consisting essentially of nitrite-accumulating bacteria, and a carrier to form a probiotic composition.   
     
     
         56 . The method of  claim 55 , where the probiotic composition is a probiotic composition according to any one of  claims 21  to  39 . 
     
     
         57 . A method of screening a bacteria isolate or mixture for nitrite-accumulating capacity comprising
 anaerobically culturing a bacteria sample in a medium comprising a predetermined concentration of nitrate;   detecting the levels of nitrate and the levels of nitrite in the medium after at least 10 hour of culturing; and   selecting the bacteria sample from a medium where the nitrate levels are decreased and the nitrite levels are increased to make a probiotic composition.   
     
     
         58 . The method of  claim 57 , further comprising selecting the bacteria sample from a medium where the nitrate levels are decreased and the nitrite levels are increased two-fold after 48 hours of culturing. 
     
     
         59 . The method of  claim 57 , further comprising selecting the bacteria sample from a medium where the nitrate levels are decreased by at least 50% and the nitrite levels are increased after 48 hours of culturing. 
     
     
         60 . The method of  claim 57 , where the bacteria sample consists essentially of bacteria that are naturally occurring in the oral cavity of a human. 
     
     
         61 . The method of  claim 57 , where the probiotic composition is a probiotic composition according to any one of  claims 21  to  39 . 
     
     
         62 . A method of measuring levels of nitric oxide comprising
 administering a probiotic composition to an oral cavity of a subject;   measuring the levels of nitrite in the saliva or breath of the subject after 10 minutes to 3 hours of the administration;   where the probiotic composition comprises a bacteria isolate or mixture comprising one or more of  Neisseria, Veillonella, Haemophilus, Actinomyces, Granulicatella, Prevotella, Leptotrichia, Brevibacillus, Porphyromonas, Fusobacterium , and clade Gemellaceae and not containing a contaminating amount of  Lactobacillus.      
     
     
         63 . The method of  claim 62  further comprising measuring the levels of nitrite in the saliva or breath of the subject before the administration. 
     
     
         64 . The method of  claim 62 , where the probiotic composition further comprises an effective amount of nitrate. 
     
     
         65 . The method of  claim 63 , whereby an increase in nitrite levels is observed. 
     
     
         66 . The method of  claim 63 , where the probiotic composition is a probiotic composition according to any one of  claims 21  to  39 .