JP2014001245A - Block of pathogen by bacillus coagulans - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a highly effective, non-antibiotic base treatment curing method functioning in the conditions of an acute treatment, and also prophylactically functioning in such a manner that an antibiotic resistant pathogen is moved in both of the human being and animals.SOLUTION: This invention provides a composition, a treatment system and a using method utilizing the discovery that, in the relaxation and obstruction of pathogenic bacterium propagation and/or colony formation velocity by new lactic acid production bacterial strain (such as Bacillus coagulans indicated in this specification) or its extracellular product, and in the relaxation of the toxic physiological effect of infection by these pathogens, they have the ability to offer block activity. This invention provides a composition including lactic acid production bacterial strain (such as Bacillus coagulans) for blocking pathogenic bacterial infection.

Description

(Field of Invention)
The present invention relates to treatments and compositions using the extracellular products thereof in new strains and / or therapeutic compositions of probiotic organisms.

  More particularly, the present invention relates to the use of one or more species or strains of probiotic cells and / or their extracellular products for the control of gastrointestinal pathogens, including antibiotic resistant species.

(Background of the Invention)
The gastrointestinal plexus has been shown to play a number of important roles in maintaining gastrointestinal liver function and general physiological health. For example, the growth and metabolism of many individual bacterial species that inhabit the gastrointestinal tract depends primarily on available substrates for such bacterial species, most of which are derived from diet. For example, see:
Gibson et al. , 1995. Gusteroenterology I06: 975-982;
Christ, et al. 1992. Gut 33: 1234-1238;
Gorbach, 1990. Ann. Med. 22: 37-41;
Reid et al, 1990. Clin. Microhiol. Rev. 3: 335-344. These findings have attempted to alter the structure and metabolic activity of the fungal assembly through a diet with live microbial food supplements, primarily with probiotics. The best known probiotics are lactic acid producing bacteria (ie Lactobacilli and Bifiaohacteria). They are widely used in yogurt and other dairy products. These probiotic organisms are non-pathogenic and non-toxin producing, remain viable during storage, and survive the route through the stomach and small intestine. Since probiotics do not constitutively colonize the host, they need to be taken on a regular basis for any health-promoting properties to continue. Commercial probiotic preparations generally include a mixture of Lactobacilli and Bifidobacterium, but yeast species (eg, Saccharontices) have also been utilized.

  A highly effective, non-antibiotic-based that functions in acute treatment situations and functions prophylactically to displace antibiotic-resistant pathogens (eg, antibiotic-resistant enterococci) in both humans and animals There remains a need for the development of treatment regimens.

Gibson et al. , 1995. Gusteroenterology I06: 975-982 Christ, et al. 1992. Gut 33: 1234-1238 Gorbach, 1990. Ann. Med. 22: 37-41 Reid et al, 1990. Clin. Microhiol. Rev. 3: 335-344

(Summary of the Invention)
The present invention provides that this novel lactic acid-producing bacterial strain (eg, Bacillus coagulans disclosed herein), or its extracellular product, is a pathogenic bacterium (especially an antibiotic-resistant pathogenic bacterial species (Enterococcus, Clostridium, Escherichia, (Klebsiella, Cumpvlobacter, Peptococcus, Heliobacter, Hemophyllus, Staphylococcus, Yersinia.Vibrio, Shigella, Salmonella, Streptococcus, Proteus, Proteus, Proteus, Proteus Also Compositions, therapeutic systems and methods of use that take advantage of the discovery that has the ability to exhibit inhibitory activity in mitigating and preventing the rate of colonization and in mitigating the toxic physiological effects of infection by these pathogens I will provide a. As currently defined, a probiotic microorganism is a microorganism that confers a benefit when grown in a particular microenvironment, for example, direct growth of other organisms within the same microenvironment. By inhibiting or interfering. Probiotic organisms include, but are not limited to, bacteria that have the ability to grow in the gastrointestinal tract, at least temporarily move or destroy pathogen organisms, and provide other benefits to the host. For example, see: Salminen et al. 1996. Antonio Van Leeuwenhoek 70: 347-358; Elmer et al, 1996. JAMA
275: 870-876; Rafter, 1995. Scand. J. et al. Gastroenterol. 30: 497-502; Perdigon et al, 1995. J. et al. Dairv Sci. 78: 1597-1606; Gandi, Toxvnsenal Lett. Doctors & Patents, pp. 108-110, Jan. 1994; Lidbeck et al, 1992. Eur. J. et al. Cancer Prev. 1: 341-353.

  Further, the Bacillus coagulans disclosed herein have the following biochemical and physiological properties, but are not limited to: (i) Production of (L) + optical isomers of lactic acid (propionic acid); (Ii) having an optimal growth temperature between 20-44 ° C; (iii) up to about 90 ° C, which can be propagated in the human or animal body without specific induction (eg heat shock or other environmental factors) (Iv) production of one or more extracellular products exhibiting probiotic activity that inhibits the growth of bacteria, yeast, fungi, viruses or any combination thereof; And / or (v) the ability to utilize a broad spectrum of substrates for growth. Preferably, the purified population of Bacillus coagulans has an optimal growth temperature of less than 45 ° C. For example, a purified population of Bacillus coagulans has an optimal growth temperature of 20 ° C, more preferably 30 ° C, more preferably 35 ° C, more preferably 36 ° C and most preferably 37 ° C. In contrast, the previously identified population of Bacillus coagulans has an optimal growth temperature above 37 ° C (eg, an optimal growth temperature of 45 ° C). This strain grows at low pH, such as the pH found in the mammalian gastrointestinal tract (eg, pH 2-5).

  A purified or isolated preparation of a cell line means that the preparation is in an amount sufficient to prevent replication of the preparation at a particular temperature and does not contain another bacterial species or strain. Preparations of purified or isolated cell lines are made by standard methods (eg limited dilution and temperature selection).

  In one embodiment of the present invention, a therapeutic composition is disclosed comprising Bacillus coagulans in a pharmaceutically acceptable carrier suitable for oral administration to the human or animal gastrointestinal tract. In another embodiment, the Bacillus coagulans strain is included in the therapeutic composition in the form of spores. In another embodiment, the Bacillus coagulans strain is included in the composition in the form of a dried or lyophilized cell mass.

Certain embodiments of the present invention have about 1 × 10 ³ to 1 × 10 ¹⁴ CFU of live Bacillus coagulans or spores, preferably about 1 × 10 ⁵ to 1 × 10 ¹¹ CFU, preferably about 1 × 10 ³ to 1 × 10 ¹⁴ CFU per day. About 5 × 10 ⁸ to 10 ¹⁰ CFU, Bacillus
Includes administration of live vegetative bacteria or spores of coagulans. If the condition to be treated is an antibiotic resistant digestive pathogen and the patient is an adult, a typical dosage is per day. About 1 × 10 ² to 1 × 10 ¹⁴ CFU of viable vegetative bacteria or spores, preferably about 1 × 10 ⁸ to 1 × 10 ¹⁰ CFU, and more preferably about 2.5 × 10 ⁸ to 1 × 10 ¹⁰ CFU of surviving vegetative bacteria or spores.

  In another aspect of the invention, a composition comprising an extracellular product of Bacillus coagulans in a pharmaceutically acceptable carrier suitable for oral administration to a human or animal is disclosed. In one embodiment, the extracellular product is a purified supernatant or filtrate of a culture of a purified Bacillus coagulans strain. In another embodiment, the extracellular product is a semi-purified or purified lyophilized supernatant or filtrate of a culture of an isolated Bacillus coagulans strain. In a preferred embodiment, the extracellular product is an active agent having antimicrobial activity. This is separated or purified from the supernatant or filtrate of the isolated Bacillus coagulans strain culture.

  The extracellular product comprises a total concentration ratio of Bacillus coagulans extracellular product in the range of about 1% to 90%, and the remainder is administered to the subject in a composition comprising a carrier or delivery component. The subject is preferably a mammal (eg, a human). The bacteria and / or products derived from the bacteria are also suitable for veterinary use (eg to treat animals such as dogs and cats). Preferred embodiments include a composition comprising an extracellular product with a total concentration of extracellular product of Bacillus coagulans ranging from about 10% to 75%, with the remainder comprising a carrier or delivery component.

  The present invention is not limited to oral administration of the therapeutic ingredients disclosed herein. The skin and / or mucous membrane is treated with a composition comprising Bacillus coagulans vegetative cells or an extracellular composition produced by vegetative cells. For example, administration of Bacillus coagulans strains and / or their extracellular products can help reduce vaginal pathogens and reduce the incidence of recurrence through vaginal repopulation with these probiotic lactic acid producing bacteria. Useful. This composition is used to treat conditions characterized by a reduction or absence of lactic acid producing bacteria in the vagina. This condition is a common etiology of both vaginal yeast infections and bacterial vaginosis. Furthermore, the use of such probiotic cell lines is effective in reducing or preventing pathogens that are resistant to one or more antibiotics. Skin creams, lotions, gels, etc., including Bacillus coagulans disclosed herein and / or their extracellular products, are effective in reducing or preventing pathogenic organisms in skin, mucous membranes and keratinous tissue. Yes, and further reduce the occurrence of antibiotic resistant pathogens. In addition to topical and oral administration, the composition is administered intravaginally, intraocularly, intranasally, otically or buccally (orally).

  Further embodiments of the invention include the use of probiotic organisms in the production of livestock where antibiotics such as vancomycin and gentamicin are commonly used to promote health and weight gain. Most if not all probiotic organisms are sensitive to these two antibiotics, and this fact has limited the possible use of such microorganisms in the livestock industry. Furthermore, there are many environmental problems associated with the use of antibiotics in livestock production. For example, the excrement of an animal loaded with antibiotics degrades very slowly, and the antibiotic residue can remain, further delaying biodegradation. As bacterial species resistant to vancomycin, gentamicin and other antibiotics increases, biodegradation is enhanced.

The present invention describes compositions, treatment regimens and uses for inhibiting pathogens and / or parasites in the gastrointestinal tract and feces of animals. In accordance with the present invention there is provided a composition comprising Bacillus coagulans vegetative cells or spores in a pharmaceutically or nutritionally acceptable carrier suitable for oral administration to the digestive tract of an animal. In another embodiment, the extracellular product from the Bacillus coagulans culture is Bacillus
utilized with or without coagulans vegetative cells or spores.

In one embodiment, the bacteria are about 1 × 10 ³ −1 × 10 ¹⁴ colony forming units (
CFU) / g, preferably in the composition at a concentration of CFU / g of about 1 × 10 ⁵ −1 × 10 ¹² . On the other hand, in another preferred embodiment, the concentration is about 1 × 10 ⁹ −1 ×.
10 ¹³ CFU / g, about 1 × 10 ⁵ −1 × 10 ⁷ CFU / g, or about 1 × 10 ⁸ −1
× 10 ⁹ CFU / g.

  In one embodiment, the bacteria are present in a pharmaceutically acceptable carrier suitable for oral administration to animals, preferably present as a powdered edible supplement, various pelleted formulations or liquid formulations.

  The present invention also describes a therapeutic system for inhibiting the growth of pathogens and / or parasites in the gastrointestinal tract and / or feces of animals. This includes a container comprising a label and the composition described herein. Here, the label comprises instructions for the use of the composition to inhibit the growth of pathogens and / or parasites.

  Advantages of such non-antibiotic probiotic bacteria-based therapeutic regimens include, but are not limited to: (i) administration of the composition of Enterococcus in the gastrointestinal tract Results in a decrease in the rate of colonization; (ii) does not contribute to the development of antibiotic resistance; (iii) the composition can be used in the hospital to prevent enterococci pools prophylactically, Reduce the chance of high-risk patients from acquired VRE; (iv) the dosage of the composition may vary depending on the age of the patient, etc .; and (v) the composition may contain additional antibiotics It can be utilized in food animals to reduce the development of resistance.

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. Unless otherwise stated, the techniques used or contemplated herein are standard methodologies well known to those skilled in the art. It should be understood that the foregoing general description and the following detailed description are only exemplary and illustrative of the claimed invention and are not intended to limit it.
The present invention provides the following.
(Item 1) A composition comprising an isolated Bacillus strain, the strain comprising:
(A) producing lactic acid;
(B) has an optimal growth temperature in the range of 20-44 ° C, and (c) grows in the pH range of 2-5.
Composition.
2. The composition of claim 1, wherein the strain produces L (+) dextrorotatory lactic acid and produces spores that are resistant to temperatures up to about 90 ° C.
(Item 3) The above strains are Bacillus coagulans, Bacillus
selected from the group consisting of stereothermophilus, Bacillus thermoacidurans, Lactobacillus spogenes, Bacillus smithii, Bacillus dextrolacticus, Lactobacillus cereal, and cecillus
(Item 4) The composition according to item 1, wherein the strain is Bacillus coagulans.
(Item 5) The composition according to item 1, wherein the strain is selected from the group consisting of Bacillus coagulans GBI-1, Bacillus coagulans GBI-20, Bacillus coagulans GBI-30, and Bacillus coagulans GBI-40.
(Item 6) An extracellular product derived from the composition according to Item 1.
7. A composition comprising an isolated Bacillus strain, wherein the strain produces lactic acid and has an optimal growth temperature in the range of 20-25 ° C.
8. The composition of claim 7, wherein the strain produces L (+) dextrorotatory lactic acid and produces spores that are resistant to temperatures up to about 90 ° C.
(Item 9) The above strains are Bacillus coagulans, Bacillus
from the group consisting of stereothermophilus, Bacillus thermoacidurans, Lactobacillus spogenes, Bacillus smithii, Bacillus dextrolyticus, Lactobacillus cereal and Bacillus rececil.
(Item 10) The composition according to item 7, wherein the strain is Bacillus coagulans.
(Item 11) The composition according to item 7, wherein the strain is Bacillus coagulans GBI-20.
(Item 12) An extracellular product derived from the composition according to item 7.
(Item 13) A composition comprising an isolated Bacillus strain, wherein the strain produces lactic acid and has an optimal growth temperature in the range of 25-35 ° C.
14. The composition of claim 13, wherein the strain produces L (+) dextrorotatory lactic acid and produces spores that are resistant to temperatures up to about 90 ° C.
(Item 15) The above strain is selected from Bacillus coagulans, Bacillus stereothermophilus, Bacillus thermoacidurans, Lactobacillus sporegenes, Bacillus smithii, Bacillus smithii, Bacillus smithii, Bacillus smithi, and Bacillus succinicus
(Item 16) The composition according to item 13, wherein the strain is Bacillus coagulans.
(Item 17) The composition according to item 13, wherein the strain is Bacillus coagulans GBI-30.
(Item 18) An extracellular product derived from the composition according to item 13.
19. A composition comprising an isolated Bacillus strain, wherein the strain produces lactic acid and has an optimal growth temperature in the range of 35-40 ° C.
20. The composition of claim 19, wherein the strain produces L (+) dextrorotatory lactic acid and produces spores that are resistant to temperatures up to about 90 ° C.
(Item 21) The above strains are selected from Bacillus coagulans, Bacillus stereothermophilus, Bacillus thermoacidurans, Lactobacillus sporegenes, Bacillus smithii, Bacillus smithii, Bacillus smithii, Bacillus smithi, and Bacillus stalks.
(Item 22) The composition according to item 19, wherein the strain is Bacillus coagulans.
(Item 23) The composition according to item 19, wherein the strain is Bacillus coagulans GBI-40.
(Item 24) An extracellular product derived from the composition according to item 19.
(Item 25) A method for inhibiting pathogenic bacterial infection, comprising the step of contacting an infection site with the composition according to item 1.
26. A method of inhibiting a pathogenic bacterial infection comprising contacting an infected site with a Bacillus coagulans composition.
(Item 27) The method according to item 26, wherein the infection site is a gastrointestinal tract.
(Item 28) The method according to item 26, wherein the infected site is skin or mucous membrane.
29. The method of claim 26, wherein the composition comprises viable vegetative bacterial cells.
30. The method of claim 26, wherein the composition comprises bacterial spores.
31. The method of claim 26, wherein the composition comprises an extracellular product of Bacillus coagulans.
32. The method of claim 26, wherein the composition is administered at a dose of 10 mg-10 g / day.
33. The method of claim 29, wherein the composition is administered at a dose of 1 × 10 ² −1 × 10 ¹⁴ viable vegetative bacterial cells / day.
34. The method of claim 30, wherein the composition is administered at a dose of 1 × 10 ² −1 × 10 ¹⁴ spores / day.
35. The method of claim 26, wherein the composition is administered orally, buccally, topically, vaginally, nasally, ocularly or otically.

Bacillus coagulans 1% isolate (GBI-1); ATCC-99% isolate (ATCC 31284) in either medium of trypticase soy broth (TSA) or glucose yeast extract (GYE); 5937- FIG. 5 is a bar graph showing the minimum and optimum culture temperatures for 20 ° C. isolates (GBI-20); and 5937-30 ° C. (GBI-30). FIG. 2 is a bar graph showing the reaction kinetics of the end points of 1% Bacillus coagulans strain (GBI-1). FIG. 3 is a bar graph showing the end point reaction kinetics of the ATCC-99% Bacillus coagulans strain (ATCC No. 31284). FIG. 4 is a bar graph showing the kinetics of end points of 5937-20 ° C. Bacillus coagulans strain (GBI-20) and 5937-30 ° C. Bacillus coagulans strain (GBI-30) with TSB and GYE medium. FIG. 5 is a bar graph showing the reaction kinetics of end points of 5937-20 ° C. Bacillus coagulans strain (GBI-20) and 5937-30 ° C. Bacillus coagulans strain (GBI-30) with NB and BUGMB media. FIG. 6 shows the results of alignment with other Bacillus species, proximity binding phylogenetic tree and simple alignment analysis for Bacillus coagulans ATCC-99% isolate (ATCC No. 31284). FIG. 7 shows Bacillus coagulans 20 ° C. isolate (GBI-20). FIG. 5 shows the results of alignment with other Bacillus species, proximity-coupled phylogenetic tree and simple alignment analysis. FIG. 8 shows Bacillus coagulans 30 ° C. isolate (GBI-30). FIG. 5 shows the results of alignment with other Bacillus species, proximity-coupled phylogenetic tree and simple alignment analysis. FIG. 9 is a bar graph showing the results of aminopeptidase profiles for Bacillus coagulans ATCC-99% isolate (ATCC No. 31284). FIG. 10 is a bar graph showing aminopeptidase profile results for Bacillus coagulans ATCC-1% isolate (GBI-1). FIG. 11 is a bar graph showing the results of aminopeptidase profiles for Bacillus coagulans ATCC-30 ° C. isolate (GBI-30). FIG. 12 is a bar graph showing the results of aminopeptidase profiles for Bacillus coagulans ATCC-20 ° C. isolate (GBI-20).

(Detailed description of the invention)
Lactic acid producing bacterial species (eg, Lactobacillus, Bifidiobacterium and most of Bacillus) are generally due to their instability in the harsh (ie acidic) pH environment of bile (especially human bile), It has been considered inappropriate for intestinal colonization. However, Bacillus coagulans (including the new strains disclosed herein) have been found to survive and colonize in the bile-like gastrointestinal tract and grow in this low pH range. In particular, this human bile environment is different from the animal model bile environment, and thus there has never been an accurate description of Bacillus coagulans in the human gastrointestinal tract model.

  With the current dramatic increase in the number of bacterial strains resistant to one or more antibiotics, the development of non-antibiotic-based therapeutic regimens has become very important. Prior to the disclosure of the present invention, antibiotic-resistant pathogens (eg, antibiotic-resistant enterococci) in acute therapeutic situations and in highly effective biorational therapies that function therapeutically and in both humans and animals There remains a need for development in prophylactic and vector control applications to alleviate. This is due to colonization (or recolonization) of the gastrointestinal tract with probiotic microorganisms. This colony formation serves to reduce or prevent both the rate of colonization and possible physiological adverse effects due to the colonization of antibiotic resistant gastrointestinal pathogens.

  Lactic acid producing bacteria are Gram positive and their morphology varies from long slender cocoons to short spheres, which often form “chains”. Its metabolism is fermentable and some species are oxygen-tolerant (ie, oxygen can be utilized through the enzyme flavoprotein oxidase), while other species are very anaerobic. Spore-forming lactic acid producing bacteria are conditionally anaerobic bacteria, while the rest are anaerobic. Growth of these bacteria is optimal at pH 5-5.5, and this organism has complex auxotrophy for amino acids, peptides, nucleotide bases, vitamins, minerals, fatty acids and carbohydrates. This lactic acid bacterium has the property of producing lactic acid from fermentable sugars. Lactobacillus. Leuconostoc. The genera of Pediococcus and Streptococcus are important members of this group. The taxonomy of lactic acid producing bacteria is based on the gram reaction and the production of lactic acid from various fermentable carbohydrates. These groups include:

  Homofermentative: produces over 85% lactic acid from glucose.

  Heterofermentative: produces only 50% lactic acid and produces significant amounts of ethanol, acetic acid and carbon dioxide. Heterofermenting species that produce DL-lactic acid, acetic acid and carbon dioxide are well known. These species include the following: Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus brevis, Lactobacillus sslbrückii, and Lactobacillus slbrucekii, and Lactobacillus sulbracili.

  Other probiotic preparations were initially systematically evaluated for their effects on health and longevity in the early 1900's (see, eg, Mechinikoff, Prolongation of Life, William Heinermann, London 1910). Their use has been significantly limited since the advent of antibiotics to treat pathogenic microorganisms in the 1950s. For example, see: Winberg, et al, 1993. Pediatr. Nephrol. 7: 509-514; Malin et al, Aiii. Nutr. Metab. 40: 137-145; and US Pat. No. 5,176,911. Unfortunately, most of these early studies on probiosis are observable rather than mechanistic in nature, and thus the processes responsible for many probiotic phenomena have not been shown quantitatively .

  There is an increasing interest in the link between the gut microbiota and its effect on the health of the human host. The human gastrointestinal tract ecosystem is colonized by over 500 species of bacteria and represents a very complex microenvironment. The composition of the gut microbiota is constantly changing and is affected by factors such as diet, stress, age and treatment with antibiotics and other drugs.

  Many manufacturers have marketed various probiotic preparations to provide the beneficial effects of lactic acid producing bacteria. The reported health benefits of these preparations include efficacy in the treatment of various disorders. These include, but are not limited to: enteritis, constipation, diarrhea, (abdominal) bloating, hyperacidity, gastroenteritis, gingivitis, anticholesterolemia, liver brain damage, and tumor formation, and antibiotics Intestinal recolonization at favorable plexus after treatment with. However, these reports are very problematic due to factors such as differences in the viability of the plexus transplanted into the gastrointestinal tract. Successful use depends on the following factors: (i) high counts of living organisms during manufacture and subsequent storage in dosage forms; (ii) acidic gastric secretions once ingested And the viability of these lactic acid producing bacteria in their intestinal pathways; and (iii) producing sufficient quantities of antagonistic metabolites against pathogens such as L (+) dextrorotatory lactic acid and bacteriocin.

So far, many species of Lactobacillus have been tested, including but not limited to: Lactobacillus bulgaricus, Lactobacillus bifidus, Lactobacillus aclophilus, Lactobacillus casei, and Lactobacillus il.
brevis. Interestingly, however, Lactobacillus acidophilus, which has long been regarded as the best candidate for therapeutic use, has since been found to be totally ineffective as a probiotic organism for recolonization of the gastrointestinal tract and in alleviating gastrointestinal disorders. Indicated. In addition, this bacterial species produces D (-) (left-handed) lactic acid. This lactic acid is not an effective antagonist agent and may induce metabolic disorders. In view of this fact, the World Health Organization (WHO) has recommended limiting intake of D (-) lactic acid for adults and complete avoidance in infant nutrition.

  It has now been found that probiotic bacteria reduce or prevent the growth of spoilage or pathogenic microorganisms through a process of competitive inhibition. This may be via a non-physiologically induced acidic environment (ie through the production of lactic acid or other biological acids) and / or antibiotic-like substances responsible for bacterial antimicrobial effects (ie bacteriophages). Shin) production. For example, see: Klaenhammer, 1993. FEMS Microbiol. Rev. 12: 39-85; Barefoot et al. 1993. J. et al. Diarv Sci. 76: 2366-2379. For example, selected Lactobacillus strains that produce antibiotics have proven effective for the treatment of infection, sinusitis, hemorrhoids, dental inflammation and various other inflammatory conditions. For example, see: US Pat. No. 5,439,995. Similarly, Lactobacillus reuteri has been shown to produce antibiotics with antimicrobial activity against Gram negative and Gram positive bacteria, yeast and various protozoa. For example, U.S. Pat. Nos. 5,413,960 and 5,439,678. In addition, probiotic bacterial proteolytic, lipolytic and galactosidase activities have also been shown to improve the digestibility and assimilation of digested nutrients, which allows these bacteria to be in convalescent / senile In nutrition and as an adjunct to antibiotic treatment.

  Probiotics have also been shown to have antimutagenic properties. For example, gram positive and gram negative bacteria have been demonstrated to bind to mutagenic pyrolysis products that are produced during cooking at high temperatures. Studies done with lactic acid producing bacteria indicate that these bacteria are alive or dead due to the fact that the process results from absorption of mutagenic pyrolysis products to carbohydrate polymers present in the bacterial cell wall It can be either. For example, see: Zang, et al. 1990. J. et al. Dairy Sci. 73: 2702-2710. Lactobacillus has also been shown to degrade carcinogens (eg, N-nitrosamines) that can play an important role if the process is found to occur subsequently at the mucosal surface level. For example, see: Rowland and Grasso, 1986. Appl. Microbiol. 29: 7-12. Furthermore, co-administration of lactulose and Bifidobacterium longum to rats injected with the carcinogen azooxymethane was demonstrated to reduce abnormal intestinal crypt lesions. This lesion is generally considered to be a preneoplastic marker. For example, see: Challa, et al. 1997. Cuccinogenesis 18: 5175-21. The purified cell wall of Bifidobacteria can also have anti-tumorigenic activity in that the cell wall of Bifidobacteria infantis induces phagocytic activation that destroys growing tumor cells. For example, see: Sekin, et al. 1994. Bifidobacterium and Microflora 13: 65-77. Bifidobacteria probiotics also have 1,2-dimethylhydrazine in mice when co-administered with fructo-oligosaccharides (FOS; see, eg, Koo and Rao, 1991. Nltrit. Rev. 51: 137-146). Has been shown to reduce colon carcinogenicity induced by and liver and mammary tumors in rats (see, eg, Reddy and Ravenson, 1993. Cascer Res. 53: 3914-3918). . Interestingly, populations at high risk for colon cancer have been shown to have intestinal plexus. The intestinal plexus efficiently metabolizes steroids and hydrolyzes glucuronides, while at the same time producing carcinogens (eg nitrosamines). A diet containing a high concentration of live lactic acid producing bacteria has been found to significantly reduce these harmful bacterial-mediated activities in such individuals.

  It has also been demonstrated that the gastrointestinal microbiota affects mucosal and systemic immunity in the host. See, for example, Famularo, et al. , Stimulation of Immunity by Probiotics. In: Probiotics: Therapeutic and Other Beneficial Effects. pg. 133-161. (Fuller, R., ed. Chapman and Hall, 1997). Intestinal epithelial cells, blood leukocytes, B lymphocytes, T lymphocytes, and accessory cells of the immune system all showed a correlation in the immunity. See, for example: Schiffrin, et al. 1997. Anl. J. et al. Clin. Nutr. 66 (suppl): 5-20S. Other bacterial metabolites that have immunomodulatory properties include endotoxin lipid polysaccharides, peptidoglycans and lipoteichoic acid. For example, see: Standardford, 1994. Infect. Linmun. 62: 119-125. Thus, probiotic organisms are believed to interact with the immune system at many levels including but not limited to: cytokine production, mononuclear cell proliferation, macrophage phagocytosis and killing, regulation of autoimmunity, Such as immunity against bacterial and protozoan pathogens. For example, see: Matsumura, et al. 1992. Animal Sci. Technol. (Jpn) 63: 1157-1159; Solis-Pereyra and Lemmonia, 1993. Nutr. Res. 13: 1127-1140. Lactobacillus strains have also been shown to produce significant changes in inflammatory and immunological responses. These include, but are not limited to, decreased colonic inflammatory infiltration without causing a similar decrease in the number of B and T lymphocytes. For example, see: De Simone, et al. 1992. Immunopharmacol. Immunotoxicol. 14: 331-340.

  While the attachment of probiotics to the gastrointestinal epithelium is an important determinant of their ability to modify host immunoreactivity, on the other hand, this is not a general property of Lactobacillus or Bifidobacterium, but also to a successful probiosis It's not essential. For example, see Fuller, 1989. J. et al. Appl. Bacteriol. 66: 365-378. For example, adhesion of Lactobacillus acidophilus and Bifidobacterium to human intestinal cell-like CACO-2 cells has been demonstrated to interfere with enterotoxigenic and enteropathogenic Escherichia coli and Salmonella typhimurium and Yersinia pseudotuberculosis. See, for example: Bernet, et al. 1994. Gut 35: 483-489; Bernet, et al. 1993. Appl. Environ. Microbiol. 59: 4121-4128.

  The gastrointestinal microflora presents a microbial-based barrier to invading organisms, but pathogens are damaged by the integrity of their microbiota by stress, disease, antibiotic treatment, dietary changes or physiological changes in the gastrointestinal tract. Is often established when. For example, Bifidobacterium is known to be involved in opposing pathogen colonization in the large intestine. For example, see: Yamazaki, et al. 1982. Bifidobacterium and Microflora 1: 55-60. Similarly, Bifidobacterium breve is administered to children with gastroenteritis to eliminate causative pathogenic bacteria (ie, Campylobacter jejuni) from its feces (see, for example, Tojo, 1987. Acta Pediatr. Jpn. 29: 160-167) and supplementing infant formula milk with Bifidobacterium bifidini and Streptococcus thermophilus has been found to reduce rotavirus and diarrhea symptoms in hospitalized children (see, eg, below) (Saavedra, 1994. The Laizcette 344 1046-109).

  Furthermore, lactic acid producing bacteria can also colonize the skin and mucous membranes and can be used either prophylactically or therapeutically to control bacterial infections. For example, lactic acid producing bacteria can utilize glycogen in vaginal epithelial cells to produce lactic acid, which maintains this environment at a pH in the range of 4.0-4.5. This production environment is not feasible for the growth of pathogens such as Candida albicans, Gardnerella vaginalis, and various non-specific bacteria responsible for vaginal infection. There is a great deal of qualitative evidence that demonstrates the depletion of these lactic acid producing bacteria is a causal and effective relationship in fungal and bacterial gynecological diseases.

(Antibiotic administration and multidrug antibiotic resistant pathogen bacterial strain)
Antibiotics are used to control pathogenic microorganisms in both humans and animals. Unfortunately, extensive use of antimicrobial agents (especially broad spectrum antibiotics) has produced a number of serious clinical consequences. For example, antibiotics often kill beneficial and non-pathogenic microorganisms (ie flora) that contribute to digestive function and health in the gastrointestinal tract. Thus, recurrence (infection and return of symptoms associated with it) and secondary opportunistic infections often result from depletion of the lactic acid production and other beneficial flora in the gastrointestinal tract.

  Unfortunately, most if not all lactic acid production or probiotic bacteria are very sensitive to common antibiotic compounds. Thus, during the course of normal antibiotic levels, many individuals produce a number of harmful physiological side effects. These include the following: diarrhea, intestinal cramps, and sometimes diarrhea. These side effects are mainly due to the non-selective action of antibiotics. This is because antibiotics do not have the ability to distinguish between beneficial and non-pathogenic bacteria and pathogenic bacteria, as both bacterial types are killed by these factors. Thus, individuals who have taken antibiotics often suffer from gastrointestinal problems as a result of killing or severely weakening the beneficial microorganisms that normally colonize the gastrointestinal tract (ie, the gut). Changes that occur in the composition of the intestinal plexus are more severe than diarrhea and dehydration when the vitamin-producing enterobacteria are killed, the vitamin deficiency is replaced, the pathogenic products are overgrown, and the remaining beneficial gastrointestinal bacteria are replaced Can cause disease.

  Another deleterious consequence of indiscriminate use of antimicrobial agents is the occurrence of multidrug antibiotic resistant pathogenic bacterial strains. For example, see: Mitchell, 1998. The Lancet 352: 462-463. For example, the methicillin-resistant Staphylococcus aureus (MRSA) strain was responsible for over 50 deaths in a single Australian hospital. See: Shannon, 1998. Lancet 352: 490-491. However, early reports of these MRSA infections have been diminished by the more recent spread of enterococcal multidrug resistant (MDR) strains, including vancomycin-resistant enterococci (VRE). Vancomycin is generally regarded as a “last resort” antibiotic. The development of such resistance has led to many reports of systemic infections that remain untreatable with conventional antibiotic therapy.

(Multi-drug resistant enterococci)
Vancomycin-resistant enterococci (VRE) have emerged as an important nosocomial pathogen in the past decade. First reported in the United States in 1989, these organisms quickly spread throughout the United States. In particular, the Enterococcus jaecium strain VRE is often resistant to all antibiotics effective in the treatment of receptive enterococci. This situation has led clinicians treating VRE infections to lack suboptimal bactericides (eg, chloramphenicol) or therapeutic options. Efforts to control the spread of VRE through reduced infection control measures and vancomycin use have been less effective.

  The intestine colonized by VRE is the most important source for the spread of these organisms. Most patients with VRE have asymptomatic intestinal colonization. This can last for months. These patients are at risk of developing a VRE infection and are a possible source of spread to health care workers, the environment and other patients. Infection control measures that implement minimizing the spread of VRE are expensive and inconvenient for patients, family members, and healthcare providers.

  Recent studies have shown that lactic acid producing Bacillus coagulans species, particularly the new strains of Bacillus coagulans disclosed herein, for use in biorational therapy for the prevention or treatment of antibiotic-resistant digestive pathogens. Profound potential has been demonstrated. In view of the current situation of rapidly emerging diseases and antibiotic resistance, new therapies and new thinking about controlling pathogens are required. In some applications, antibiotics are useful in considering large reservoirs of new and antibiotic-resistant strains resulting from misuse of antibiotics in the context of medical institutions and as "growth factors" in livestock production. Became a thing of the past.

  Enterococcus, the major cause of nosocomial bacteremia, surgical wound infections and urinary tract infections, is becoming resistant to many and sometimes all standard treatments. New rapid assessment methods are drawing attention to the importance of testing enterococcal isolates at the species level. Most enterococcal infections are caused by Enterococcus faecalis. This is likely to develop characteristics related to overt toxicity, but at least for now it also seems to retain sensitivity to at least one effective antibiotic. The rest of the infection is mostly caused by Eterococcus faecium. This is a species with virtually no known overt virulence characteristics, but even last resort antibiotics appear to be resistant. Effective control of multi-drug resistant enterococci requires: (i) a better understanding of the interaction between enterococci, environment and human; (ii) use of much more sensible antibiotics; (iii) Better contact isolation in hospitals and other patient care environments; (iv) improved research; and, most importantly, (v) the development of new treatment paradigms (eg, non-antibiotic-based), The cycle between drug introduction and drug resistance is not very fragile.

  Two types of enterococci cause infection: (i) derived from the patient's native flora, do not appear to have resistance beyond that inherent in the genus, and do not appear to be infected Type. And (ii) isolates having multidrug resistant antibiotic resistance properties and capable of nosocomial infection. The therapeutic challenge of multi-drug resistant (MDR) enterococci (ie, strains with significant resistance to two or more antibiotics, often including but not limited to vancomycin) is an important hospital-acquired infection for a sharper perspective Brought their role as pathogens.

  During the last decade, enterococci have been recognized as a leading cause of nosocomial bacteremia, surgical wound infections, and urinary tract infections. Two types of enterococci are generally found to be associated with causative infections: (i) derived from the patient's native flora and not appear to have resistance beyond that inherent in the genus And a type that does not appear to spread between beds, and (ii) a isolate with multidrug antibiotic resistance properties and capable of nosocomial infection. The therapeutic challenge of multidrug resistant (MDR) enterococci (ie, those strains that have significant resistance to two or more antibiotics, often including but not limited to vancomycin) Brought a sharper perspective.

Enterococci usually inhabit the bowel and can be found in the intestines of almost all animals, from cockroaches to humans. In humans, typical concentrations of enterococci in feces are up to 10 ⁸ CFU / g. For example, see: Rice, ei al. , 1995. Occurrence of high-level amino acid resistance in environmental isolates of enterococci. Appl. Envinon. Microbiol. 61: 374-376. The main species that inhabit the gut vary. In Europe, the United States and the Far East, Enterococcus faecalis is dominant in some cases, and in other cases Enterococcus faecium is dominant. In addition, among four or more known enterococcal species (see, for example, Devriese, et al., 1993. Phenotypic identifying of the genocentococcus and differential of splicens.
groups. J. et al. Appl. Bacteriol. 75: 399-408), only Enterococcus faecalis and Enterococcus faecium colonize and infect humans in detectable numbers. Here, Enterococcus faecalis is isolated from approximately 80% of human infections, and Enterococcus faecium is isolated from the rest of the individuals.

  Enterococci are very robust and tolerant to a wide variety of growth conditions, including temperatures from 10 ° C. to 45 ° C. and hypotonic, hypertonic, acidic or alkaline environments. Sodium azide and concentrated bile salts interfere with or kill most microorganisms, whereas enterococci are tolerant to this, and in fact are used as selection agents in viewpoint-based media. As a conditional organism, enterococci grow under reducing or oxidizing conditions, but enterococci are usually considered strict fermenters. Because they lack the Krebs cycle and respiratory chain. However, Enterococcus faecalis is an exception. This is because exogenous hemin can be used to produce d, b and o type cytochromes. Enterococcus jaecalis cytochrome is expressed under anaerobic conditions only in the presence of exogenous hemin and can therefore promote colonization of inappropriate sites.

  Enterococci are also inherently resistant to many antibiotics. Unlike the acquired resistance and virulence properties normally encoded by transposons or plasmids, intrinsic resistance is based on chromosomal genes, which are typically non-transferable. Penicillin, ampicillin, piperacin, imipenem, and vancomycin are among the few antibiotics that are consistently inhibitory, but do not exhibit bacterial killing activity against Enterococcus faecalis. Enterococcus faecium is not as sensitive to β-lactam antibiotics as Enterococcus faecalis. This is because the former penicillin binding protein has a significantly lower affinity for antibiotics. The first reports of strains highly resistant to penicillin first appeared in the 1980s. For example, see: Bush, et al. 1989. High-level penicillin resistance ammonitions of enterococci: implications for treatment of enterococcal effects. Ann. Intern. Med. 110: 515-520; Sapico, et al. 1989. Enterococci high resistant to penicillin and ampicillin an emerging clinical probe. J. et al. Clin. Microbiol. 27: 2091-2095.

  As discussed in more detail below, enterococci often acquire antibiotic resistance through exchange of resistance-encoding genes performed in mating transposons, pheromone-responsive plasmids, and other broad host range plasmids. In the past 20 years, we have seen a rapid appearance of MDR enterococci. High levels of gentamicin resistance were first reported in 1979 (see, for example, Horodicianu, et al., 1979. High-level, plasmid-born resistance to gentamicin in Streptococcus faecalis. Agents Chemother. 16: 686-689), and numerous reports of nosocomial infections were immediately followed in the 1980's (see, eg, Zervos, et al., 1987. : An epidemi logic study.Ann.Intern.Med.106: 687-691). At the same time, the sporadic spread of nosocomial Enterococcus faecalis and Euterococcus facilun infections appears to be accompanied by penicillin resistance, which is due to β-lactamase production. However, such separators remain relatively rare. Finally, MDR enterococci that have lost sensitivity to vancomycin have been reported in Europe and the United States. For example, see: Sahm, et cil. 1989. In-vitro sustainability studies of vancomycin-resistant Enterococcus faecalis. Antimicrob. Agents Chemother. 33: 1588-1591.

  Of several phenotypes for vancomycin-resistant enterococci, VanA (resistant to vancomycin and teicoplanin) and VanB (resistant to vancomycin alone) are the most common. In the United States, VanA and VanB wet about 60% and 40% of vancomycin-resistant enterococci (VRE) isolates, respectively. See, for example, Clark, et al. 1993. Charactorization of glycopeptide-resistant Enterococci from U. S. hospitals. Aitinimicrob. Agents Chemother. 37: 2311-2317. Inducible genes encoding these phenotypes alter cell wall synthesis and make the strain resistant to glycopeptides. In the laboratory, it has been demonstrated that these genes can be transferred from enterococci to other bacteria. For example, see: Arthur, et al. 1993. Genetics and mechanisms for glycopeptide-resistance in Enterococci. Antimicrob. Agents Chenother. 37: 1563-1571. For example, Staphylococcus aureus became vancomycin resistant through transfer of resistance from Enterococcus faecalis.

  As previously discussed, most enterococci occur naturally or are inherently resistant to a variety of drugs, including cephalosporin and rice-resistant penicillinase-resistant penicillin (e.g., Oxacillin) and clinically achievable concentrations of clindamycin and aminoglycoside systems. Compared to Streptococcus, most enterococci are relatively resistant to penicillin, ampicillin and pseudopenicillin. Many enterococci are also tolerant to the killing effects of cell wall activators (eg, ampicillin, vancomycin, etc.); recent data may not be unique and acquired after exposure to antibiotics Suggest that it may have been. For example, the inherent in vivo resistance of Enterococcus faecalis to trimethoprim-sulfamethoxazole may explain the lack of efficacy in animal models. Furthermore, the recent killing activity against Enterococcus faecalis appears unreliable and just depends on the method. In animal models, this combination does not show good activity and is generally not generally accepted as an effective anti-enterococcal therapy, especially for systemic infections.

  In addition to natural resistance to many drugs, enterococci are also plasmid- and transposon-mediated tetracyclines (eg, minocycline and doxycycline); erythromycin (eg, azithromycin and clarithromycin); clamphenicol; Developed resistance to trimethoprim; and high levels of clindamycin. The tendency for Enterococcus faecalis to acquire multidrug antibiotic resistance properties can arise from a variety of significantly different mechanisms for conjugation.

  The best characterized system of conjugation involves oligopeptides that are pheromones and pheromone-responsive plasmids. For example, see: Clewell and Keith, 1989. Sex pheromones and plasmid transferrin Enterococcus faecalis. Plasmid 21: 175-184. In this mating system, Enterococcus faecalis strains typically secrete a number of different low oligopeptide pheromones into the culture medium. This pheromone is specific for different types of plasmids. When a cell containing a pheromone-responsive plasmid (ie a possible donor cell) comes into contact with its corresponding pheromone, transcription of the gene on the plasmid is switched on, resulting in the synthesis of aggregates on the surface of the cell membrane. Then, when the donor comes into contact with another Enterococcus faecalis bacterium, the aggregate (which contains two Arg-Gly-Asp motifs) adheres to the binding agent on the surface of most Enterococcus faecalis cells, Aggregate the cells. The pheromone-responsive plasmid can then be transferred from the donor bacterium to another (recipient) bacterium by a process that has not yet been well characterized. Once the recipient cell has acquired this particular plasmid, the synthesis of its corresponding sex pheromone is shut off, preventing self-aggregation. This mating system occurs primarily in Tenenococcus faecalis, but is a highly efficient means of plasmid transfer.

  Another system of conjugation that is also not well characterized includes a broad host range plasmid that can transfer between species of enterococci and other gram positive organisms (eg, streptococci and staphylococci). . For example, see: Clewell, 1981. Plasmids, drug resistance, and gene transfer in the genus Streptococcus. Microbiol. Rev. 45: 409-436. This frequency is generally much lower than the pheromone system. Because staphylococci, streptococci and enterococci share a number of resistance genes, these broad host range plasmids may be a mechanism in which some of these resistance genes are spread among different genera.

The third type of conjugation involves mating transposons, which may also explain the spread of resistance genes to many different species. For example, see: Clewell, 1986. Conjugative transposons and the discussion of antibiotic resistance in streptococci. Atiniii. Rev. Microbial. 40: 635-659. Contrary to normal transposons, which can jump intracellularly from one DNA position to another, conjugating transposons also encode the ability to produce conjugation between different bacterial cells. Plasmids typically acquire a rather complex mechanism for replication (often depending on successful interactions with host proteins) and are faced with additional problems of surface exclusion and improper synthesis. Transposons (which do not replicate but instead insert into the chromosome or into a new host plasmid) appear to be an even more efficient and distant way of delivering resistance genes interspersed. This may explain why the tetM gene of the conjugated transposon Tn916 spreads across gram-positive species into gram-negative organisms (including Neisseria gonorrhoeae and meningococci), as well as mycoplasma and ureaplasma. For example, see: Roberts, 1990. Characterisation of the TetM determineds
in urogenital and respiratory bacteria. Antimicrob. Agetits Clinioother. 34: 476-478. Other resistance genes (including those encoding resistance to erythromycin and kanamycin) have also been found in conjugated transposons; these frequently contain or are associated with Tn916. Such transposons may have evolved from the ancestors of Tn916. Their appearance suggests the possibility of further spread of resistance among Gram positive organisms. Particularly ominous is the report of a vanB gene cluster in a large zygotic chromosomal element that appears to be at least functionally similar to a conjugated transposon.

(Epidemiology of multi-drug resistant enterococci)
Colonization and infection of MDR enterococci occurs worldwide. According to the report of the secretary, the proportion of nosocomial infections caused by VRE increased more than 20 times between 1989 and 1993 (ie, 0.3% to 7.9%) in the United States. This indicates rapid diffusion. New database technologies (e.g., The Survey Network (TSN) Database USA) now allow assessment of resistance profiles by species. TSN Database collects and compiles daily data from over 100 clinical laboratories in the United States, identifies possible laboratory test errors, and resistance profiles and mechanisms that threaten public health (eg, vancomycin resistant yellow The appearance of staphylococci) is detected. Analyzing data collected by TSN Database between 1995 and September 1, 1997, whether the initial increase in vancomycin resistance was specific to vancomycin and whether it represented a continuous trend And whether speciation is qualitatively important in analyzing this trend. There was little change in resistance spread from 1995 to 1997. In contrast, Enterococcus faecium's resistance to vancomycin and ampicillin was increased to an alarming level. For example, in 1997, 771 (52%) of 1482 Enterococcus faecium isolates were vancomycin resistant and 1220 (83%) of 1474 isolates were ampicillin resistant. Despite Enterococci faecum resistance, Enterococci faecalis remained the most commonly encountered by two Enterococcus species in TSN Database. The total isolate of Enterococcus faecalis vs. Enterococcus faecium was approximately 4: 1, the blood separator was 3: 1 and the urine isolate was 5: 1. This observation underestimates important differences in survival strategies and the probability of therapeutic success, and important factors are usually ambiguous by bringing these organisms together with Enterococcus species or enterococci.

  The widespread emergence and spread of ampicillin and vancomycin resistance in Enterococcus faecalis is quite disruptive to the current therapeutic dilemma. There is little reason that vancomycin and ampicillin resistance is expected to provide only a selective advantage for the species faecium but not for the species faecalis. The relatively lack of these resistances in Enterococcus faecalis may simply reflect the current lack of permeability and the balance of its properties. Because of these important differences between the two species, a meaningful assessment of Enterococcus resistance must include species identification.

  Enterococci are responsible for about 110,000 urinary tract infections annually in the United States, responsible for 25,000 bacteremias, responsible for 40,000 wound infections, and 1,100 cases. It has been demonstrated to be the cause of endocarditis. Most cholera infections occur in the hospital. Enterococcal infection-related deaths are difficult to establish due to the fact that serious concurrent illnesses are common. However, enterococcal sepsis has been correlated to up to 50% of fatal cases. However, several recent case control and historical cohort studies have shown that the risk of death associated with antibiotic-resistant enterococci is significantly higher than susceptible enterococci bacteremia. This trend is expected to increase as the MDR separator becomes more diffuse.

  Although the precise aspects of nosocomial infections for MDR enterococci are difficult to determine, molecular microbiological and epidemiological evidence can be disseminated between patients (probably in the hands of healthcare providers or medical devices). Strongly suggests spread between hospitals by patients with long-term intestinal colonization. Many outbreaks of MDR enterococci have been reported. And all but 2 were attributed to Enterococctls faecium. This disparity, particularly in view of the larger number of clinical Enterococcus faecalis isolates, may reflect reporting bias due to the novel combination of resistances that occur in Enterococcus faecium. When isolates from the outbreak of MDR enterococci were analyzed by DNA sequencing, it was demonstrated that over half included clonally related isolates.

  Previous treatment with antibiotics is common in almost all patients colonized or infected with MDR enterococci. For example, see: Montecalvo, et al. 1994. Outbreak of vancomcinin-, ampicillin-, and aminoglycosides-resistant Enterococcus faecium bacteria in an adult oncology unit. Antimicrob. Agents Chemother. 38: 1363-1367. Other risk factors include long-term hospitalization; high severity of disease score; intra-abdominal surgery; renal failure; intestinal feeding; Exposure to the surface.

Antibiotics can promote colonization and infection with MDR enterococci by at least two mechanisms. First, many broad-spectrum antibiotics have little or no anti-enterococcal activity, and administration generally results in the growth of susceptible (or resistant) enterococci at risk sites for infection . Second, most antibiotics substantially reduce the normal resistance of the intestine to colonization by exogenous organisms. Colony resistance arises primarily from the “limited action” of the normal anaerobic flora and to a lesser extent intact mucosa, gastric acid secretions, intestinal uncopper and intestinal associated immunity. Antibiotic-induced changes in the gut protection flora act as a catalyst for colonization of exogenous pathogens such as MDR enterococci. Antibiotic restriction programs are more effective when they include a wise prescription of all antibiotics, not just one drug (eg, vancomycin). For example, the use of this approach substantially reduced VRE intestinal colonization in certain hospital pharmacies that restricted the use of vancomycin, cefotaxime and clindamycin. For example, see: Quale, et al. , 1996. Manipulation of a
hospital antimicrobiological formal control an outbreak of vancomcinin-resistant enterococci. Clin. Infect. Dis. 23: 1020-1025.

(Vancomycin resistant genetic element)
In recent years there has been a warning outbreak in acquired vancomycin-resistant enterococci. Vancomycin has been used clinically since 1950, but it was not as prominently used until the late 1970s and especially until the 1980s. Since multiple bacterial genes are involved in the development of vancomycin resistance, the development of such resistance has been neither simple nor recent.

Vancomycin resistance in enterococci is heterogeneous at many levels. Three phenotypes of vancomycin resistance (VanA, VanB and VanC), each associated with a different ligase, are now well described. A fourth VanD type was recently reported. For example, see: Noble, et al. 1992. Co-transfer
of vancomycin and other resistance genes from Enterococcus faecalis NCTC 12201 to Staphlococcus aureus. FEMS Microbiol. Lett. 93: 195-198. VanA and VanB resistances are acquired (ie not part of the normal genome of enterococci) and are often encoded by transposable gene clusters. VanA type strains are typically highly resistant to vancomycin and moderate to highly resistant to tycoplanin. This phenotype is often mediated by plasmids or transposons and is inducible (ie, recent exposure to vancomycin results in the induction of the synthesis of several proteins that confer resistance together). For example, see: Hiramatsu, et al. 1997. Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J. et al. Antimicrob. Chemother. 40: 135-146. The VanA gene cluster is found in Tn1546, a small Tn3-like transposon, and in elements that appear to be closely related (eg, Tn5488 with an insertion sequence (IS1251) within Tn1546). For example, see: Eliopoulos, et
al. 1994. In vitro activities of two glycycyllines against gram-positive bacteria. Antimicrob. Agents Chemother. 38: 534-541. These elements were then found in both transferable and non-transferable plasmids, as well as in the host strain chromosome.

  VanB-type resistance was not initially found to be metastasizable, but in at least some cases, the VanB gene cluster is a transposable element located on a large (ie, 90 kb to 250 kb) chromosome. Found, these secret baths contain a 64 kb complex transposon (ie, Tn1547) therein. The VanB-containing 64 kb transposon is part of a 250 kb migratory element that was shown to have migrated from one enterococcal chromosome and inserted into another. Although not demonstrated, circularization of vanB containing large mobile elements can be excised from one strain's chromosome, can be circularized, can be transferred from one enterococci to another enterococci, and recipients Similar to the mechanism described for mating transposons that can be reinserted into the chromosome of. The 64 kb of this transposon can also jump to another plasmid within the host enterococci, and the plasmid can then be transferred to another recently by conjugation and can incorporate the VanB resistance gene into it.

  In contrast, VanC1 and VanC2 are normally present genes, respectively. gaiiitalttm and F.M. A gene that is characteristic of the endogenous species of Casseriflavus and is not transposable.

(Therapeutic approach)
Antibiotics suitable for treating MDR enterococcal infections (eg, endocarditis or bacteremia) are often not available in the presence of neutropenia. Combinations of penicillin and vancomycin, ciprofloxacin and ampicillin, or novobiocin and doxycycline have been used inter alia but may be unpredictable and remain clinically unproven. A substantial drawback of the broad spectrum approach is that more organisms are infected (ie, both protective probiotic organisms and pathogens), and more resistance evolves. Broad spectrum antibiotics allow empirical treatment in the absence of specific diagnoses and generate a more substantial return on short-term investment. However, broad spectrum antibiotics affect not only disease-causing organisms, but also probiotic organisms that are present in large enough numbers to develop resistance due to rare mutations or gene exchange events.
As long as the medical and pharmaceutical academia continues to rely on the use and development of broad-spectrum therapy as the primary therapeutic modality, there are other therapeutic aspects (e.g. targeted therapies) in development, but drug introduction The subsequent cycle of resistance emerges without doubt.

  With the current dramatic increase in the number of bacterial strains that are resistant to one or more antibiotics, the development of non-antibiotic-based therapeutic regimens is particularly important. Prior to the disclosure of the present invention, colonization (or recolonization) of the gastrointestinal tract using probiotic microorganisms (which is due to colonization of antibiotic-resistant digestive pathogens and the possible physiological It functions therapeutically in acute treatment scenarios by functioning to reduce or prevent both adverse effects) and in both humans and animals in prophylactic and vector controlled applications, for example antibiotic resistance pathogens (e.g. antibiotics) There remains a need for the development of highly effective biorational therapies that function to reduce or delay the development of substance-resistant enterococci).

  In addition to enterococci, the probiotic compositions of the invention are effective against other common or antibiotic resistant strains of pathogens including, but not limited to: Candicla, Clostridim, Escherichia. Klebsiella, Cantpylobactej-, Peptococcus, Heliobacter, Hemophyllus, Staphylococcus, Yersinia, Vibrio, Shigella, Salmonella, Streptococcus, Protecoscus Advantages of such non-antibiotic probiotic bacteria-based therapeutic regimens include, but are not limited to: (i) administration of this composition is the rate of colonization of enterococci in the gastrointestinal tract (Ii) does not contribute to the development of antibiotic resistance; (iii) the composition can be used to preventively reduce nosocomial enterococcal reservoirs, which are high-risk Simultaneously reduces the chance that the patient will acquire a VRE; (iv) the dosage of the composition may vary depending on the patient, condition, etc .; and (v) the composition is utilized in food animals , Can reduce the occurrence of further antibiotic resistance.

  In further embodiments, skin creams, lotions, gels, including the novel strains of Bacillus coagulans disclosed herein and / or their extracellular products, are pathogenic organisms in skin, mucosa and keratinous tissue. Are useful in reducing or preventing, and further reduce the appearance of antibiotic-resistant pathogens. By way of example and not limitation, cells, spores and / or extracellular products from these new Bacillus coagulans strains can be incorporated into skin products for this express purpose. For example, Pseudomorzas, Staphylococcus and / or Enterococcus pathogenic antibiotic resistant strains are often associated with severe burn infection. Thus, saliva, lotion, gel, etc. are effective in reducing or preventing these pathogenic organisms in combination with Bacillus coagulans strains and / or their extracellular products as disclosed in the present invention. Furthermore, the administration of these probiotic bacteria helps to achieve an appropriate biodiversity state of the skin in burn cases. Because, in general, such biodiversity is not associated with pathogenic overgrowth.

  Probiotic Lactic Acid Producing Bacterial Strain As used herein, “probiotic” refers to forming at least part of a transient or endogenous flora and thereby to a host organism A microorganism that exhibits an advantageous prophylactic and / or therapeutic effect. Probiotics are generally known to be clinically safe (ie non-pathogenic) by those skilled in the art. By way of example and not limitation of any particular mechanism, the effect of prevention and / or treatment of acid-producing bacteria of the present invention results in part from competitive inhibition of pathogen growth due in part to: (i) Its excellent colony-forming ability; (ii) unwanted microbial parasitism; (iii) production of acids (eg lactic acid compounds, acetic acid compounds and other acid compounds) and / or other extracellular products with antimicrobial activity; And (iv) various combinations thereof. It should be noted that the above products and activities of the acid producing bacteria of the present invention act synergistically to produce the advantageous probiotic effects disclosed herein.

  Probiotic bacteria suitable for use in the methods and compositions of the invention are: (i) have the ability to produce and secrete acid compounds (eg, lactic acid, acetic acid, etc.); (ii) gastrointestinal Demonstrates advantageous function in the tube; and (iii) is non-pathogenic. By way of illustration and not limitation, a number of suitable bacteria have been identified and disclosed herein, but the invention is not limited to the bacterial species currently classified as long as it is disclosed and intended. Should be noted. The physiochemical results from in vivo production of lactic acid are important for the effectiveness of the probiotic lactic acid producing bacteria of the present invention. Lactic acid production significantly reduces pH (ie increases acidity) within the local microbiota environment and does not contribute to the growth of many undesirable physiologically harmful bacteria and fungi. Thus, due to the mechanism of lactic acid production, probiotics inhibit the growth of competing pathogenic sanctions.

  Representative lactic acid producing bacteria useful as probiotics of the present invention are effective lactic acid producers, including non-pathogenic members of the genus Bacillus, which are bacteriocins that inhibit the growth of pathogenic organisms. Or produce other compounds.

  Bacillus species, particularly those species that have the ability to form spores (eg, Bacillus coagulans) are preferred embodiments of the present invention. The sporulation capacity makes these bacterial species relatively resistant to heat and other conditions, provides long life in product formulations, and tissue survival and colonies under conditions such as pH, salt, etc. in the gastrointestinal tract It is a treatment for formation. Furthermore, additional useful properties of many Bacillus species include non-pathogenic, anaerobic, conditional and heterotrophic, thus making these bacterial species safe And enables easy colonization in the gastrointestinal tract.

  Preferred methods and compositions disclosed herein utilize new strains of Bacillus coagulans and / or their extracellular products as probiotics. Prior to the present invention, various "classical" Lactobacillus and / or Bifidobacterium species generally were found to be Inadequate for colony formation. The purified Bacillus coagulans strain of the present invention can survive in the gastrointestinal tract and can colonize. This is because the optimum temperature for growth is lower than the standard known strain of Bacillus coagulans. Further, probiotic Bacillus coagulans are non-pathogenic and are generally considered safe (ie, GRAS classification) by the US Federal Drug Administration (FDA) and the US Department of Agriculture (USDA) and by those skilled in the art.

  Since Bacillus coagulans has the ability to purify heat-resistant spores, it is particularly useful for producing pharmaceutical compositions that require heat and pressure in their production. Accordingly, the use of viable Bacillus coagulans spores in a pharmaceutically acceptable carrier is particularly preferred for making and using the compositions disclosed in the present invention.

  Growth to form cell cultures, cell pastes and spore preparations of these various Bacillus species is generally well known in the art. In addition, the present invention discloses a method for the separation and partial purification of extracellular products produced by Bacillus coagulans cultures.

  (Commercial source of traditional strains of Bacillus coagulans) Bacillus coagulans Gram-positive bacilli have cells with a diameter greater than 1.0 μm, with variable swelling of the spore sac and no paraspore crystal formation. . Bacillus coagulans is a non-pathogenic Gram-positive spore-forming bacterium that produces L (+) lactic acid (dextrorotatory) under homofermentation conditions. This was isolated from a natural source (eg, a heat treated soil sample inoculated in a nutrient medium) (see, eg, Bergey's Manual of Systemic Bacteriology, Vol. 2, Sneath, P HA et al., Eds., Williams & Wilkins, Baltimore, MD, 1986). Purified Bacillus coagulans strains include endonucleases (eg, US Pat. No. 5,200,336); amylases (US Pat. No. 4,980,180); lactases (US Pat. No. 4,323,651) and It has served as a source of enzymes including cyclomaltodextrin glucanotransferase (US Pat. No. 5,102,800). Bacillus coagulans has also been utilized to produce lactic acid (US Pat. No. 5,079,164). A strain of Bacillus coagulans (which was also referred to as Lactobacillus spogenes; Sakaguchi & Nakayama, ATCC No. 31284) was combined with Bacillus natto to produce fermented food from other lactic acid producing bacteria and steamed soybeans. (US Pat. No. 4,110,477). The Bacillus coagulans strain has also been used as an animal feed additive for poultry and livestock to reduce disease and improve food utilization and thus enhance animal growth rates (International PCT patent application no. WO9314187 and WO9411492). In particular, Bacillus coagulans strains have been used as a general nutritional supplement and drug to control constipation and diarrhea in humans and animals.

Bacillus coagulans cultures have been deposited with the following major international culture collection agencies: Agricultural Research Service.
Culture Collection; Russian Collection of Microorganisms; Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures, VKM DSMZ); American Type Culture Collection (ATCC); Finnish Microorganism Collection (University of Goteborg, Sweden); Japan RIKEN microorganisms Strain Conservation Facility (JCM); and Japan Federation for Culture Collage tion).

  There were a total of eight lactic acid producing bacterial species from the deposits, which were either: (i) previously classified and deposited as Bacillus coagulans, but another related Bacillus species Or (ii) has been deposited as another closely related species but has recently been reclassified to Bacillus coagulans. These related species include, but are not limited to: Bacillus coagulans, Bacillus stereothermophilus. , Bacillus thermoacidurans, Lactobacillus spogenes, Bacillus smithii, Bacillus dextrolyticus, Lactobacillus cereale, and Bacillus recelyticus. However, there is currently some confusion regarding the classification of these related bacterial strains. This is because there are no defined rules even among similar strains, even for optimal or appropriate growth parameters. For example, Bacillus stereothermophilus is known to have an optimal growth of about 55 ° C.

  A variety of Bacillus coagulans bacterial strains are currently commercially available from the American Type Culture Collection (ATCC, Rockville, MD), which includes the following accession numbers: Bacillus coagulans Hammer NRS 727 (ATCC 11014; ATCC 11014) coagulans Hammer strain C (ATCC No. 11369); Bacillus coagulans Hammer (ATCC No. 31284); and Bacillus coagulans Hammer NCA 4259 (ATCC No. 15949). Purified Bacillus coagulans bacterium is also available from Deutsche Sammlung von Mikroorganismen und Zellkuturen GmbH (Birschweig, Germany) using the following accession number: (Corresponding to DSM 2383, ATCC 11014); Bacillus coagulans Hammer (corresponding to DSM 2384, ATCC 11369); and Bacillus coagulans Hammer (corresponding to DSM 2385, ATCC 15949). Bacillus coagulans bacteria are also found in Sabinsa Corporation (Piscataway, NJ) or K. et al. K. It can be obtained from commercial sources such as Fermentation (Kyoto, Japan).

  These above-mentioned Bacillus coagulans strains and their growth requirements have been described previously (see, eg, Baker. D. et al, 1960. Cai. J. Alibiobiol. 6: 557-563; Nakamura H. et al, 1988. Iit. J Sist. Bacteriol 38: 63-73). In addition, various strains of Bacillus coagulans can also be obtained from natural sources (eg, heat-treated soil samples) from well-known means (see, eg, Bergey's Manual of Systemic Bacteriology, Vol. 2, 1117, Sneath, PHA et al., eds., Williams & Wilkins, Baltimore, MD, 1986).

  Bacillus coagulans was originally mischaracterized as Lactobacillus, as originally described, as evidenced by the fact that this bacterium is labeled Lactobacillus spogenes (see below: Nakamura et al. 1988). Vint. J. Last. Bacteriol. 38: 63-73). However, the initial classification was inaccurate due to the fact that Bacillus coagulans form spores and secrete L (+) lactic acid through metabolism. Both of these aspects provide important features for their usefulness. Instead, these developmental and metabolic aspects required that the bacteria be classified as lactobacillus and therefore re-named.

(Biochemical characterization of Bacillus coagulans)
Bacillus coagulans is a member of the genus Bacillus and is sporulated, which is activated in the acidic environment of the stomach and can germinate and grow in the intestine, and is the preferred L (+) optical isomer of lactic acid And can effectively interfere with the growth of many bacterial and fungal pathogens. Table 1 below is a comparison chart showing the biochemical attributes of lactic acid producing bacteria and the like.

公知のＬａｃｔｏｂａｃｉｌｌｕｓ種は、一般的に消化管の過酷な（すなわち酸性）ｐＨ環境（例えば、胆汁、特にヒト胆汁の存在下）における不安定性のため腸の集落形成に不適切と考えられている。この不安定性は、プロバイオティック物として乳酸産生細菌株の使用がより活発に探求されない主な理由の１つである。

Known Lactobacillus species are generally considered unsuitable for colonization due to instability in the harsh (ie, acidic) pH environment of the digestive tract (eg, in the presence of bile, particularly human bile). This instability is one of the main reasons why the use of lactic acid producing bacterial strains as probiotics is not explored more actively.

In contrast to these bacterial species, Bacillus coagulans can survive, colonize, and grow in the gastrointestinal tract. In particular, the human bile environment is different from the animal model bile environment, and the growth of Bacillus coagulans in the human gastrointestinal tract model has not been described. The following growth characteristics indicate that the strength of Bacillus coagulans over other species of lactic acid producing bacteria includes, but is not limited to:
Facultative aerobic organisms: Bacillus coagulans have the ability to grow well in either free oxygen containing environments or obligate anaerobic environments. This is important due to the fact that Lactobacilli and Bifidobacterium are not aerobic. Thus, in essence, these above bacterial species are obligately anaerobic and do not grow well in environments containing free oxygen. Bacillus coagulans can be used in surface active formulations (eg, skin powders, creams, ointments, etc.) and can act prophylactically against pathogen overgrowth because they can survive in a free oxygen environment.

  Thermotolerance: Bacillus coagulans resting cells have the ability to grow at temperatures as high as 65 ° C, while endospores can withstand temperatures in excess of 100 ° C. Indeed, Bacillus coagulans together with Bacillus stereothermophilus is used for quality control purposes in autoclaves. This fact is significant due to the shortcomings of all Lactobacillus and Bifidobacterium. For bacteria with commercial viability, it must be stable and viable at the time of packaging. This viability must be maintained in order to deliver an effective product to the consumer.

  Salt tolerance: Bacillus coagulans have the ability to grow in highly alkaline environments containing 7% NaCl or 10% caustic soda.

  Characteristics of Bacillus coagulans cited in Bergey's Manual (7th edition) include the following: Gram-positive sporulation rods having a size of about 0.9 μm × 3.0 to 5.0 μm, aerobic to slightly preferred Production of L (+) (dextrorotatory) lactic acid in a temper, homofermentation mode. Due to the fact that Bacillus coagulans has the typical characteristics of both Lactobacillus and Bacillus, its taxonomic position between the Lactobacillusae family and the Bacillaceae family is often discussed.

  It is often very difficult to classify between two species of bacteria. The two species are morphologically similar and have similar physiological and biological properties. DNA homology analysis is a useful technique to solve this difficulty. The base composition (ie% GC content) and the specific nucleotide sequence of bacterial DNA generally differ between bacterial species and subspecies. In addition, closely related bacterial DNA hybridizes more effectively to each other. In the present invention, these previously described methodologies are effectively used, distinguished and understand the essential similarities between Bacillus coagulans and members of the genus Lactobacillus, and their classification under the genus Bacillus. Check academic position.

  Table 2 below discusses the colony morphology of Bacillus coagulans.

下の表３は、Ｂａｃｉｌｌｕｓ ｃｏａｇｕｌａｎｓを利用する炭水化物発酵のメカニズムを考察する。

Table 3 below discusses the mechanism of carbohydrate fermentation utilizing Bacillus coagulans.

（４．Ｂａｃｉｌｌｕｓ ｃｏａｇｕｌａｎｓの生物学的「安全性」）
Ｂａｃｉｌｌｕｓ ｃｏａｇｕｌａｎｓは、ほとんどの一般的なＬａｃｔｏｂａｃｉｌｌｕｓおよびＢｉｆｉｄｏｂａｃｔｅｒｉｕｍ種の使用より長い安全の歴史を享受する。ＬａｃｔｏｂａｃｉｌｌｕｓおよびＢｉｆｉｄｏｂａｃｔｅｒｉｕｍ種は、健康食品店で一般に「栄養性補助」として販売されているか、または培養された乳製品の生成において使用される。

(4. Biological “safety” of Bacillus coagulans)
Bacillus coagulans enjoy a longer safety history than the use of most common Lactobacillus and Bifidobacterium species. Lactobacillus and Bifidobacterium species are commonly sold in health food stores as “nutritional supplements” or are used in the production of cultured dairy products.

The general perception of biological safety can be based solely on the perspective of skilled personnel qualified through scientific training and experience in assessing the safety of substances added directly or indirectly to food. The basis for such a perspective can be obtained through:
(1) Scientific procedure.

  (2) In the case of substances used in food prior to January 1, 1985 through experience based on general use in food. The general perception of safety requires general knowledge about substances in the scientific community that are informed about the safety of substances that are added directly or indirectly to food.

  (3) The general perception of safety based on scientific procedures would require scientific evidence of the same quantity and quality needed to obtain food additive regulations for ingredients. ing. The general perception of safety through scientific procedures is usually based on published studies that can be confirmed by unpublished studies and other data and information.

  (4) The general awareness of safety through experience based on general use in food prior to January 1, 1958 is without the quantity or quality of scientific procedures that require the approval of food additive regulations. Can be determined. The general perception of safety through experience based on general use in food prior to January 1, 1958 is based solely on food use of substances prior to January 1, 1958. , And usually based on generally available data and information.

  Lactic acid producing bacteria are necessary ingredients in fermented dairy products. Due to the fact that Bacillus coagulans was first isolated in 1932, used in the production of food products prior to January 1, 1958, and has not been associated with any pathogen or opportunistic disease since its isolation, GRAS (general Is approved under nine sections and subsections of the US Federal Register. The GRAS inventory simply states that food additives are not considered to be any fraudulent toxicogenic or pathogenic response and are considered safe by those skilled in food science, biochemistry, and microbiology. Show.

  Bacillus coagulans, subspecies Hammer (ATCC-31284) was first isolated by Nakayama as a soil separation in 1933 Yamanashi University. Bacillus coagulans species are usually soil separation. With the exception of Bacillus cereus and Bacillus anthraces, Bacillus species are known to be environmentally friendly. To date, there has been no reference of any species of Bacillus coagulans involved in pathogenicity or opportunistic diseases. Similarly, in the analysis of published data, there were also no clinical trials at risk due to the pathogenesis by lactic acid producing bacteria. In view of these facts not discussed in the relevant scientific field, Bacillus coagulans are safe as therapeutic compositions.

(Sensitivity of Bacillus coagulans to antibodies)
Organisms listed in the GRAS are safe for use in “normal” immune responsive individuals, while sensitive individuals (eg, immunosuppressed, immunocompromised, organ transplants, etc.) are biologically safe. It can be at risk to become bacteremia or septic through the ingestion of bacterial products that are believed to be. Although there is an evaluation paper by a companion who is associated with severe systemic infection (ie, opportunistic pathogenesis) of Lactobacilli, there is no report showing a Bacillus coagulans-mediated pathogenesis. Despite the foregoing, research is currently underway in immunocompromised mice / rats to determine whether these new strains of Bacillus coagulans are arbitrarily potent against such opportunistic pathogenesis. is there.

Analysis of antibiotic susceptibility of Bacillus coagulans subspecies Hammer (ATCC 31284) was performed using the Kirby-Bauer (colony counting on plate) and Vitek (absorbance of culture) high sensitivity test methodologies and, if necessary, rational A specific antibiotic compound that was effective in inducing the formation of Bacillus coagulans colonies regardless of gender was ascertained. Using the Kirby-Bauer test, Bacillus
coagulans was found to be sensitive to ampicillin; ciprofloxacin; trimethoprim-sulfamethoxazole; rifampin; erythromycin; vancomycin; gentamicin; oxacillin and had intermediate sensitivity to tetracycline . Using the Vitek test, Bacillus coagulans was found to be sensitive to penicillin; vancomycin; gentamicin (500 μg / ml); streptomycin (2,000 μg / ml); nitrofurantoin; norfloxacin; chloramphenicol And was resistant to tetracycline. In addition, a Nitrocefin test was performed and showed that Bacillus coagulans was positive for low-level β-lactamase production.

(Production of antimicrobial substances by Bacillus coagulans)
Bacteriocins are proteins or protein particulate complexes that have bactericidal activity against species closely related to producer bacteria. The inhibitory activity of lactic acid-producing bacteria (eg, Bacillus coagulans) against spoilage organisms can be attributed in particular to the production of bacteriocin.

  Table 4 below lists some of the various bacteriocins. This bacteriocin was isolated and characterized from a lactic acid producing bacterial species.

さらに、乳酸産生細菌はまた、他の代謝産物（例えば、過酸化水素、二酸化炭素、およびジアセチル）を介して病原性／腐敗性微生物の増殖を阻害する。

In addition, lactic acid producing bacteria also inhibit the growth of pathogenic / septic microorganisms through other metabolites such as hydrogen peroxide, carbon dioxide, and diacetyl.

  Metabolites of lactic acid producing bacteria that exert antagonism against pathogenic bacteria are summarized in Table 5 below.

生成された乳酸の光学異性体形態のレベルは、細菌の特定の種に依存する。これらの異性体の構造的立体配置は、以下のとおりである：

The level of optical isomer form of lactic acid produced depends on the specific species of bacteria. The structural configuration of these isomers is as follows:

ヒトにおいて、両方の異性体は、腸管から吸収される。Ｌ（＋）乳酸は、グリコーゲン合成において完全かつ迅速に代謝されるが、それに対してＤ（−）乳酸はより低速で代謝され、そして代謝されない酸は、尿中に排出される。代謝されない乳酸の存在は、胎児において代謝性アシドーシスを生じる。Ｌａｃｔｏｂａｃｉｌｌｕｓ ａｃｉｄｏｐｈｉｌｕｓは、Ｄ（−）形態を生成し、従って、臨床的利益は疑わしい。対照的に、Ｂａｃｉｌｌｕｓ ｃｏａｇｕｌａｎｓは、Ｌ（＋）乳酸のみを生成し、従って、Ｄ（−）形態を生成する乳酸産生細菌の他の種より好ましい。

In humans, both isomers are absorbed from the intestinal tract. L (+) lactic acid is metabolized completely and rapidly in glycogen synthesis, whereas D (-) lactic acid is metabolized more slowly and unmetabolized acid is excreted in the urine. The presence of unmetabolized lactic acid results in metabolic acidosis in the fetus. Lactobacillus acidophilus produces the D (−) form, and thus clinical benefit is questionable. In contrast, Bacillus coagulans produces L (+) lactic acid only and is therefore preferred over other species of lactic acid producing bacteria that produce the D (-) form.

(A new strain of purified Bacillus coagulans)
Previously available strains of lactic acid producing bacteria (including Bacillus coagulans ATCC type strain # 31284) were not effective as probiotics due to various factors. These factors include, but are not limited to: their high optimal growth temperature (ie> 40 ° C.) requirements and 80 ° C. “spore shock” requirements for their spore germination. These requirements are incompatible with the use of these previously available Bacillus coagulans strains as probiotics, such as in therapeutic compositions (eg, in the treatment of antibiotic resistant gastrointestinal pathogens).

  The Bacillus coagulans described herein have biochemical and physiological characteristics. These features include, but are not limited to: (i) production of the (L) + optical isomer of lactic acid (propionic acid); (ii) having an optimal growth temperature of less than 45 ° C .; iii) generation of spores resistant to temperatures up to about 90 ° C. that can germinate in the human or animal body without specific induction (eg spore shock or other environmental factors); (iv) bacteria, yeast, fungi, Production of one or more extracellular products exhibiting probiotic activity that inhibits the growth of the virus, or any combination thereof; and / or (v) the ability to utilize a wide range of substrates for growth. These new strains are discussed more fully below.

  Three previously uncharacterized strains of Bacillus coagulans that have the ability to produce lactic acid and other extracellular products under laboratory fermentation conditions while at the same time having significantly lower growth temperature optimums Disclosed in the specification. These new strains share some characteristics with the ATCC type strain (ATCC-31284), but they differ in their increased effectiveness for use as probiotics (eg, lower growth temperatures). Optimal condition).

These new strains were originally discovered in a mixed microbiota of Bacillus coagulans colonies. In this colony, these strains differ from the Bacillus coagulans ATCC type strain (hereinafter “ATCC-31284” or “ATCC-99%”) in both colony morphology and optimal growth temperature. It was. These new strains are characterized as follows:
Bacillus coagulans 1% isolate-GBI-1 Bacillus coagulans 20 ° C isolate-GBI-20 Bacillus coagulans 30 ° C isolate-GBI-30 Bacillus coagulans 40 ° C-I

  Bacillus coagulans 1% isolate strain (hereinafter referred to as “GBI-1”); Bacillus coagulans strain with optimal growth temperature of 20 ° C. (hereinafter referred to as “GBI-20”); 30 ° C. Bacillus coagulans strain (hereinafter referred to as “GBI-30”) having an optimal growth temperature of 40 μC; and Bacillus coagulans strain (hereinafter referred to as “GBI-40”) having an optimal growth temperature of 40 ° C. Of these new strains. The biochemical, physiological, and morphological characteristics of these novel strains of Bacillus coagulans are fully discussed in the specific examples section below.

(Treatment of gastrointestinal infection of antibiotic-resistant bacteria)
The present invention contemplates methods of treating, reducing or controlling gastrointestinal infections of antibiotic resistant bacteria using the therapeutic compositions or therapeutic systems disclosed herein. The disclosed methods of treatment function to inhibit the growth of pathogenic bacteria associated with gastrointestinal infections and concomitantly mitigate the deleterious physiological effects / symptoms of these pathogenic bacterial infections.

  The new strains of Bacillus coagulans disclosed herein are generally considered safe for those skilled in the art (ie, GRAS approved by the FDA) and are therefore for direct consumption in food or for nutrition Suitable as a food supplement. The methods of the invention include administering a therapeutic composition comprising one or more Bacillus coagulans strains and / or extracellular products thereof to the gastrointestinal tract of a human or animal to treat or prevent a bacterial infection. . Administration is preferably with a formulation compatible with oral administration such as liquids, powders, solid foods, etc. (formulated to contain the therapeutic composition of the present invention by using methods well known in the art). Done.

  The method of the invention comprises administering to a human or animal a composition comprising one or more of the following to treat or prevent colonization of antibiotic resistant pathogens to the gastrointestinal tract: Bacillus coagulans bacterial cells (Ie, vegetative bacterial cells); spores; and / or isolated Bacillus coagulans extracellular products (including metabolites with antibiotic-like properties). In particular, with respect to VRE, VISA, PRP, and other pathogens, the method includes administering Bacillus coagulans to a patient, for example, in a food or as a dietary supplement. Oral administration is in aqueous suspensions, emulsions, powders or solids, either already formulated in the food or as a composition added to the food prior to consumption by the user. Administration to the gastrointestinal tract can also be in the form of a rectal suppository (eg, in a gel or semi-solid formulation). All such formulations are performed using standard methodologies.

  Administration of the therapeutic composition is preferably a gel, suspension, aerosol spray, capsule, tablet, comprising the therapeutic composition of the present invention (all formulated using methods well known in the art) It is performed on the gastrointestinal tract using powders, semi-solid formulations (eg suppositories).

  Administration of compositions comprising active probiotic lactic acid producing bacteria that are effective in preventing or treating pathogenic bacterial infections generally ranges from about 10 mg per dosage over a period ranging from 1 day to 1 month. Consists of 1 to 10 doses of 10 g therapeutic composition. Administration is (generally) once every 12 hours and once every 4 hours. A preferred embodiment is the administration of the therapeutic composition from about 0.1 to 5 g per dose for 2 to 4 times per day for 1 to 7 days. This preferred dose is sufficient to prevent or treat infection with pathogenic bacteria. It will be appreciated that the particular route, dosage and timing of administration will depend, in part, on the particular pathogen and / or condition being treated and the extent of this condition.

Embodiments of the present invention provide about 1 × 10 ³ to 1 × 10 ¹⁰ CFU viable vegetative bacteria or spores per day, more preferably about 1 × 10 ⁵ to 1 × 10 ¹⁰ CFU per day. Administration of viable vegetative bacteria or spores, and most preferably about 5 × 10 ⁸ to 1 × 10 ⁹ CFU of viable vegetative bacteria or spores per day. If the condition being treated includes antibiotic-resistant gastrointestinal pathogens and the patient is an adult, a typical dosage is about 1 × 10 ² to 1 × 10 ¹⁴ CFU of viability per day. Vegetative bacteria or spores, preferably about 1 × 10 ⁸ to 1 × 10 ¹⁰ CFU of viable vegetative bacteria or spores per day, and more preferably about 2.5 × 10 ⁸ to 1 per day × 10 ¹⁰ CFU viable vegetative bacteria or spores.

  Another embodiment of the present invention discloses the administration of a composition comprising Bacillus coagulans extracellular product and the remainder comprising a carrier or delivery component in a total concentration ratio range of about 1% to 90%. Preferred embodiments include compositions comprising Bacillus coagulans extracellular product in the total concentration ratio range of about 10% to 75% and the balance comprising a carrier or delivery component.

  The present invention further contemplates a therapeutic system for treating, reducing and / or controlling pathogenic bacterial infections. Typically, this system is in the form of a package containing the therapeutic composition of the present invention or in combination with a packaging material. The packaging material includes indicia or instructions for use of the packaging components. This instruction indicates the intended use of the packaged ingredients as described herein for the method or composition of the invention.

  By way of illustration and not limitation, the system may include one or more unit dosages of a therapeutic composition according to the present invention. Alternatively, the system can instead include a bulk amount of the therapeutic composition. This indication includes instructions to use the therapeutic composition in either unit dose or bulk form, as appropriate, and includes information regarding storage of the composition, indications, dosages, routes, and modes of administration, etc. Information can be included.

  In addition, depending on the particular intended use, the system may optionally include one or more of the following ingredients, either in a combined package or in separate packages: bifidogenic) components such as oligosaccharides, flavoring agents and carriers. One particularly preferred embodiment includes unit dose packaging of Bacillus coagulans spores for use in combination with conventional liquid products, along with instructions for combining formulations and probiotics for use in therapeutic methods.

(Inhibition of pathogens and parasites in animals)
The present invention also discloses compositions and methods of use for inhibiting the growth of parasites and / or antibiotic resistant pathogenic organisms in the gastrointestinal tract of animals. As used herein, the terms “pathogen” and “parasite” are used interchangeably in the context of harmful organisms that grow in the gastrointestinal tract and / or feces of an animal, although these terms are It is understood that it has a different meaning.

  The present invention describes compositions and methods of use for inhibiting or preventing the growth of pathogens in the gastrointestinal tract of an animal, the method comprising in the gastrointestinal tract of the animal one or more of the following inventions: Administering or treating a colony of antibiotic resistant pathogens in the gastrointestinal tract, comprising: Bacillus coagulans bacterial cells (ie, proliferating bacterial cells); spores; and / or isolated Bacillus coagulans extracellular product (including metabolizing antibiotic-like properties). Specifically, for VRE, VISA, PRP, and other pathogens, the method includes administering to an animal, for example, as a Bacillus coagulans or food supplement in food. Oral administration is preferably in an aqueous suspension, emulsion, powder or solid, already formulated in food, or as a composition (added to food by the user prior to consumption) is there. Administration to the gastrointestinal tract may also be in the form of a suppository (eg, a gel or semi-solid formulation). All such formulations are made using standard methodologies.

  The methods of the present invention include the administration of a composition of the present invention comprising an active ingredient to an animal in various dosage regimes as described herein to obtain nutritional results. Administration of a composition comprising an active ingredient effective to inhibit parasite growth in the intestine and feces is from 10 mg to 10 g per dose of the composition from 1 day to 1 month for animals weighing about 100 kg. Generally composed of 1 to 10 dosage units. The dosage unit is generally administered once every 12 hours to once every 4 hours. Preferably from 2 to 4 doses of this composition per day from 1 to 7 days (each containing about 0.1 to 50 g per dose) are sufficient to achieve the desired result.

Preferred methods are 1 × 10 ² to 1 × 10 ¹⁰ viable bacteria or spores per day, in some embodiments 1 × 10 ³ to 1 × 10 ⁶ per day, and in other embodiments 1 × 10 ^6. Includes administration of ˜1 × 10 ⁹ , and more preferably approximately 5 × 10 ⁸ to 1 × 10 ⁹ viable bacteria or spores into the gastrointestinal tract. Typical dosage ranges range from approximately 1 × 10 ³ to 1 × 10 ⁶ viable bacteria per day, or approximately 1 × 10 ⁶ to 1 × 10 ⁹ viable bacteria per day.

  Another embodiment of the present invention discloses administration of a composition comprising a total concentration ratio of Bacillus coagulans extracellular product ranging from about 1% to 90% extracellular product relative to a residue comprising a carrier or delivery component. . A preferred embodiment comprises a composition with a total concentration ratio of Bacillus coagulans extracellular product ranging from about 10% to 75% extracellular product relative to the residue comprising the carrier or delivery component.

  The method is typically practiced on any animal where inhibition of pathogens or parasites is desired. The animal can be any domestic or zoological species for which such inhibition of parasites / pathogens provides economic and health benefits. Any animal may be advantageous by the claimed method. The animals include birds, reptiles, mammals (eg, domestic animals such as horses, cows, sheep, goats, pigs) or any of a variety of animals of zoological interest. Other objectives will be readily apparent to those skilled in the art of nutrient absorption, food utilization, and bioavailability.

  The invention further encompasses therapeutic systems that treat, attenuate, and / or control pathogenic bacterial infections. Typically, this system is in the form of a package containing the therapeutic composition of the present invention, or in combination with a packaging material. The packaging material includes a label or instructions for use of the package contents. This instruction (instruction) indicates the intended use of the packaging composition described herein for the method or composition of the invention.

  For example, without limitation, the system may include more than a unit dose of the therapeutic composition according to the present invention. Alternatively, the system can instead include a bulk amount of the therapeutic composition. This label includes instructions, where appropriate, for using the therapeutic composition in either unit dose or bulk form, and also information regarding storage of the composition, disease efficacy, dosage, route And information such as mode of administration.

  Further, depending on the particular intended application, the system may optionally include one or more of the following components, either in combination or in separate packages: bifidogenic Compositions such as oligosaccharides, fragrances and carriers. One particular preferred embodiment is a unit dose package of Bacillus coagulans spores for use in combination with conventional liquid products, along with instructions in combination with probiotic bacteria using a formulation for use in therapeutic methods. including.

  Controlling and / or inhibiting the growth of parasites and pathogenic organisms in stool is produced, accumulated and / or stored as long as stool provides growth and breeding soil to organisms where it is not desired Inhibits the growth and propagation of these unwanted parasites in the region. For example, parasites / pathogens stimulate and propagate to other hosts in barns or livestock sheds, in animal cages, in feed lots, in animal display enclosures, and in similar areas where animals are raised and feces accumulate There is an opportunity to propagate, propagate and / or infect. Various undesirable problems caused by these environments are solved by the present invention. For example, it is not desirable for parasites or pathogens to be transmitted to the host and further infected. And if multiple animals are held together in a cage, some means of controlling the transmission of the infection is very advantageous. In addition, in many situations where the host animal is bitten by a parasite or flying insect, the animal is stimulated and / or irritated, causing behavioral problems including excessive kicking, biting, and related behavior. This is dangerous for nearby animals and those who handle animals.

  In another embodiment, the present invention contemplates a method for reducing and / or controlling the population of insects flying in the cage / ori / enclosure of the animal in which the animal is housed. The method includes administering the composition of the present invention to the gastrointestinal tract of an animal in a cage.

  The present invention is useful for controlling many types of parasites and pathogenic organisms, and therefore the present invention need not be limited to inhibiting any particular genus or species of an organism. For example, based on the mechanisms described herein for the effectiveness of this composition, it is understood that all insect species that can act as animal parasites can be targeted by the methods of the invention. Parasites can infect any of a variety of animals (including mammals, reptiles, birds, etc.), and therefore the present invention is considered not limited to any particular animal. Examples of well-known or important parasites are described herein for purposes of illustration of the invention, but are not to be construed as limiting the invention. Representative parasites and animal and / or human hosts are described in great detail in various veterinary literature, such as “Merck's Veterinary Manual” and “Cecils' Human Diseases”. Horse parasites include Horse bots, lip bots or throat Hotts (e.g., G. intestinalis, G. haemorrhiodalis, and G. nasalis), throat Hotts, For example, a gastric parasite, Parascaris species (e.g., caused by H. muscae or H. microsoma mulus), or by a Crascia species (e.g., C. mepastomoma), or caused by a Trichostronglus species (e.g., T. axei) Roundworms (white parasites) produced by P. eciourum), Stroncvlus species (eg S blood parasites (palisada worms, red parasites, or genus Sclerostoma), cecum and colon caused by Tridontophorus species (eg, T. tenucollis) caused by Vulcriris, S. epeunus or S. edentus Small roundworms, helminths caused by Oxvulis species (eg, O. euui), intestinal nematode infections caused by Stroncivloides westeri, Anonocephala species (eg, A. macma and A. perfoliata), and caused by Paranonlocephala malam The resulting tapeworm is mentioned.

  Various other parasites, including wire worms caused by Haemonchus species (ie, barber's pole worms or large stomach worms), are ruminants, typically Cause disease in cattle. Parasites that occur in ruminants, typically pigs, include gastric parasites caused by Hvostromumulus species.

  Additional parasites are known to infect various animal hosts and are therefore targets for treatment by the methods of the present invention. For example, gastrointestinal parasites can include Spirocerca species (eg, S. lupi) that infect various animals and produce esophageal parasites in dogs, and Physolooptera species that produce gastroparasites in dogs and cats.

  If the animal is fed a spheronized or granulated food, the composition can be included in the spheronized or granulated food, or the spheronized composition of the invention and It may comprise a mixture of foods that are spheronized in combination. The mixing of the spheronized formulation of the composition of the present invention with the spheronized food is a conventional diet that uses a commercial diet and adjusts the amount of the composition of the present invention to be administered simultaneously. As long as a system is provided, it is a particularly preferred method for practicing the present invention.

  Administration of the therapeutic composition may be a gel, suspension, aerosol spray, capsule, tablet, granule, pellet, wafer, powder or semi-solid formulation (eg, suppository) (all suppositories) comprising the nutritional composition of the invention. Preferred for the intestine using a method well-known in the art.

  The present invention further contemplates a system that inhibits the growth of parasites and / or pathogens in the gastrointestinal tract of an animal or in the faeces of an animal. This comprises a container containing a label and a composition according to the invention, wherein the label comprises instructions (instructions) for the use of the composition to inhibit the growth of pathogens / parasites.

  Typically, this system exists in the form of a package containing the composition of the present invention or in combination with a packaging material. The packaging material includes a label or instructions for use of the composition of the package. This instruction indicates the intended use of the packaging composition described herein for the method or composition of the invention.

  For example, the system may include one or more unit doses of the therapeutic composition according to the present invention. Alternatively, the system can include a bulk amount of the composition. The label includes instructions for using the composition in either unit dose or bulk form, where appropriate, and information regarding storage of the composition, feeding instructions, health and diet instructions, dosage, Information such as route of administration, method of mixing the composition with a preselected food may be included.

(A. Culture of Bacillus coagulans)
Bacillus coagulans are aerobic and facultative and are typically grown in nutrient broth containing up to 2% by weight NaCl at pH 5.7-6.8, but both NaCl and KCl grow. Is not necessary. A pH of about 4.0-7.5 is optimal for initiation of sporulation (ie, sporulation). The new strain of Bacillus coagulans disclosed herein grows optimally between 20 ° C. and 40 ° C., and the spores can withstand pasteurization. In addition, the bacteria exhibit aeration and organic nutrient growth by utilizing a nitric acid source or a sulfuric acid source.

Bacillus coagulans can be cultured in a variety of media,
Certain growth conditions have been demonstrated to be more efficient where they produce cultures that produce high levels of sporulation. For example, sporulation has been demonstrated to be enhanced when the culture medium contains 10 mg / l MgSO ₄ sulfate. Here, a ratio of spores to proliferating cells is produced at about 80:20. Furthermore, certain culture conditions produce bacterial spores whose metabolic enzyme spectrum is particularly suitable for the present invention (ie, production of lactic acid and enzymes for enhanced probiotic activity and biodegradability). Although spores produced by these aforementioned culture conditions are preferred, a variety of other suitable conditions that produce viable Bacillus coagulans spores can be utilized in the practice of the invention.

  Suitable media for cultivation of Bacillus coagulans include: PDB (potato dextrose broth); TSB (trypsin soy broth); and NB (nutritive broth) (all of which are well known in the art and are Available from suppliers). In one embodiment of the invention, a medium supplement comprising enzyme digests of poultry and / or fish tissue and comprising food yeast is particularly preferred. A preferred medium supplement yields a medium containing at least 60% protein, about 20% complex carbohydrates, and about 6% lipid. Media can be obtained from various commercial suppliers, notably DIFCO (Newark, NJ); BBL (Cockeysville, MD); Advanced Microbiological Systems (Shakopee, MN); and Troy Biologicals (Troy, MD). An effective growth medium for Bacillus coagulans is Glucose Ysast extract (GYE) medium. The GYE recipe is shown in Table 6 below.

次いで、この培地のｐＨを約６．３に調節し、次に１２０℃で１５分間、１．２ｋｇ／ｃｍ²で蒸気滅菌した。

The pH of the medium was then adjusted to about 6.3 and then steam sterilized at 1.2 kg / cm ² at 120 ° C. for 15 minutes.

  The formulation of the trace mineral solution used for the analysis of the Bacillus coagulans bacterial strain of the present invention is shown in Table 7 below.

（ｉ）スモールスケールの培養
Ｂａｃｉｌｌｕｓ ｃｏａｇｕｌａｎｓのスモールスケールの培養は、上述のグルコース酵母エキス（ＧＹＥ）培地を用いることにより達成し得る。この培地に播種し、そして細胞密度が約１×１０⁸〜１×１０⁹細胞／ｍｌになるまで増殖させた。この細菌を標準的なエアリフト発酵槽を利用して３０℃で培養した。胞子形成のために受容可能なＭｎＳＯ₄の範囲を、１．０ｍｇ／ｌ〜１．０ｇ／ｌに見出した。この栄養細菌細胞は、６５℃ま
で活性を有して繁殖し得、そして胞子は９０℃まで安定である。

(I) Small scale culture Small scale culture of Bacillus coagulans can be achieved by using the above-described glucose yeast extract (GYE) medium. This medium was seeded and grown to a cell density of about 1 × 10 ⁸ to 1 × 10 ⁹ cells / ml. The bacteria were cultured at 30 ° C. using a standard airlift fermenter. The range of acceptable MnSO ₄ for sporulation was found to be 1.0 mg / l to 1.0 g / l. This vegetative bacterial cell can propagate with activity up to 65 ° C and the spores are stable up to 90 ° C.

  After incubation, the Bacillus coagulans bacterial cells or spores are recovered using standard methods (eg, filtration, centrifugation), and the recovered cells and spores are then lyophilized, spray dried, air dried or frozen. Can do. As described herein, the supernatant from the cell culture can be collected and used as an extracellular material with useful antibacterial activity secreted by Bacillus coagulans in the formulations of the invention.

Typical yields obtained from the culture methods described above are in the range of about 1 × 10 ⁹ to 1 × 10 ¹³ viable spores, and more typically about 10 to 15 × 10 6 per gram before drying. ¹⁰ cells / spore. It is also noted that Bacillus coagulans spores maintain at least 90% viability for up to 7 years when stored at room temperature after the drying step. Therefore, the effective shelf life at room temperature of a composition comprising Bacillus coagulans Hammer spores is about 10 years.

(Ii) Large scale culture Large scale batch fermentation of Bacillus coagulans can be achieved using the glucose yeast extract (GYE) medium described above. The fermentor may comprise: a 500 liter 314 series stainless steel airlift fermentor with a 60 psi pressure grade; a Hanna duel set pH value control system with in-process electrodes; 0 for sterile air supply High pressure turbine blower with 2 μm in-line filter; 10 kw process temperature controller; and appropriate high burst pressure stainless steel sanitary hoses and fittings.

  Batch fermentation includes the following procedures. A single colony of Bacillus coagulans was selected from the petri dish colonies using a sterile curved tube. This single colony was then used to inoculate a 2 liter Erlenmeyer flask containing GYE medium, dextrose, and minerals. The culture was incubated on an orbital shaker (with 2 "orbital) for about 18 hours at 35 ° C. Using this 2 liter culture, a 500 liter sterilized batch containing GYE medium, dextrose, and minerals. The batch fermenter was sown for about 30 hours at 35 ° C. under high aeration (36-38 LPM) After this incubation, the batch fermenter was turned off and for 4 hours. The temperature was lowered to 20 ° C. to facilitate sedimentation of the contained bacterial cells.The fermentation broth was harvested using an Alpha-Laval Sharples continuous feed centrifuge at 12,000 rpm at 10 ° C. and the bacterial sediment. Was recovered for subsequent lyophilization.

(Preparation of Bacillus coagulans spores)
A dry culture of Bacillus coagulans spores can be prepared, for example, as follows. About 1 × 10 ⁷ spores were seeded in 1 liter of culture medium containing 30 g (wt / vol) GYE medium, dextrose, and minerals. This culture was maintained at 37 ° C. for 72 hours in a high oxygen environment to produce a culture having about 15 × 10 ¹⁰ cells / g (medium). The culture was then filtered to remove the liquid culture medium and the resulting bacterial pellet was resuspended in water and lyophilized. The lyophilized bacteria were made into a fine “powder” using standard and good manufacturing experiment (cGMP) methods.

(Preparation of extracellular product of Bacillus coagulans)
Bacillus coagulans cultures were prepared as described in specific Examples A (i)-(ii). Cultures were maintained for 5 days as described. The culture was first autoclaved at 250 ° F. for 30 minutes and then 4000 r. p. m. For 15 minutes. The resulting supernatant was collected and subjected to submicron filtration through a Buchner funnel equipped with an unused 0.8 μm filter. The filtrate was then collected and further filtered through a 0.2 μm Nalge vacuum filter. The resulting filtrate was then collected (approximately 900 ml capacity per liter of culture medium). This consisted of a liquid containing extracellular products, was quantitatively analyzed and used in subsequent inhibition experiments.

  The following method was used to characterize this supernatant.

(Liquid chromatography of proteins)
20 ml of the culture supernatant was loaded onto an analytical Mono9 chromatography column (Pharmacia) equilibrated with buffer A (0.25 M Tris HCl; pH 8.0) and BioCAD Sprint chromatography system (Perseptive).
Biosystems, Inc. ) At 2 ml / mm. The column was washed with 15 ml of buffer A and eluted with a linear gradient ranging from 0% B (ie, buffer B is a 3M aqueous NaCl solution) to 40% B over a 12 minute time frame. . The column was then washed with 100% B for 5 minutes. The column was then re-equilibrated with buffer A. Absorbance was monitored at 280 nm to detect elution of aromatic amino acids (ie, tyrosine) found in bacterial proteins.

  This result demonstrates that the majority of the protein mixture eluted at 0.1 M to 0.8 M NaCl and a small fraction of mineral eluted at a NaCl concentration of 3.0 M. Fractions were collected and stored and dialyzed against water with a Spectrapor dialysis membrane ("truncated" at about 1,000 Daltons MW) to facilitate subsequent analysis.

(Ultraviolet spectroscopy and visible spectroscopy)
Using a Uvikon 930 scanning spectrophotometer (Kontron Instruments), placed in a 1 cm quartz cuvette, the absorption spectra differing between wavelengths of 200 nm and 600 nm were determined. Baseline was determined using water or culture medium.

  The water (blank) results show an absorption peak for Bacillus coagulans from 290 nm to 305 nm and a significant amount of additional absorbing material found between 210 nm and 400 nm. It has also been shown that there is significant absorption mainly due to the presence of proteins at UV wavelengths.

(SDS polyacrylamide gel electrophoresis)
Electrophoresis was performed by the method of Laemmli (see Laemmli, 1970, Nature 227: 680-685). The acrylamide gel was run into a 1 mm cassette (Novex) and run according to the recommendations of the commercial supplier (ie 120 volts, 90 minutes [12% gel] and 2 hours [16%]). The gel was then silver stained by the method of Blum et al. (Blum et al., 1987. Electrophoresis 8: 93-99). 16% acrylamide was found to isolate the most Bacillus coagulans protein. All samples were dialyzed against water prior to preparation for electrophoresis to improve salt-bound electrophoresis artifacts. A wide range of protein markers (BioRad) were used for protein molecular weight determination.

  The electrophoresis results demonstrated a significant number of protein bands for Bacillus coagulans ranging from less than 4,000 to 30,000 daltons.

(High pressure liquid chromatography)
5 ml of culture supernatant was extracted with 2 ml of acetonitrile, benzene or 24: 1 (v / v) chloroform: isoamyl alcohol for about 2 hours. This phase became separable in 4 hours and was further centrifuged at 5,000 × g for 10 minutes. The organic phase was then filtered through a 0.2 μm PVDF filter (Gehnan Acrodisc LC-13) and loaded onto an Econosil C-18 10U HPLC column (Altech) in a mobile phase of 20 mM Tris-HCl, pH 7.5. Elution started after a total of 5 minutes (15 minute linear gradient to 60% acetonitrile (ACN) in water). Elution was continued with 60% ACN for 5 minutes, then the column was washed with 20 mM Tris-HCl (pH 7.5) and equilibrated again.

  Reversed phase HPLC results on the ACN-extracted Bacillus coagulans supernatant showed that as the organic properties of the solvent increased, the “organic nature” of the HPLC (ie, the increase in material eluting at a higher percentage of ACN) ) And pigmented molecules (ie, molecules that absorb visible light) have been shown to lead to an increase in capture. These molecules described above were isolated and further characterized.

  The results of these analytical methods described above demonstrated that the culture supernatant from Bacillus coagulans is very heterogeneous in nature and contains multiple protein and organic molecules. However, it is proteins that predominate in this molecule, and there are a total of 20 different types in each sample. These protein species can be further subdivided by the use of ion exchange chromatography, thus allowing further characterization. In addition, many dyes that are both highly complex (based on absorption at high wavelengths) and hydrophobic (based on their preference for retention on nonpolar solvents and C-18 HPLC columns) There are also sex molecules (ie, molecules that absorb visible light).

  In embodiments of the present invention, the liquid containing the extracellular product can be formulated into a liquid ointment composition for use for direct application in transdermal, keratinous, or mucosal tissue.

(Isolation and characterization of a new Bacillus coagulans strain)
(Isolation and characterization of viable bacterial colonies)
(Dilution and heat treatment)
Approximately 1 g of lyophilized Bacillus coagulans sample was placed in a homogenizing container with a sterilized surface. Approximately 200 ml of sterile saline diluted solution (containing 8.5 mg sodium chloride and 25 mg sodium lauryl sulfate per liter) was added and the mixture was homogenized at 12,000-15,000 rpm for 5 minutes.

1 ml of the homogenized suspension was then transferred to 9.0 ml saline in a screw capped tube (25 mm x 150 mm size) and mixed thoroughly. This serial dilution was repeated to a final 2 × 10 ⁻⁸ fold dilution to obtain what was termed “dilution factor”. The final dilution tube was then heat treated in a 70 ° C. water bath for 30 minutes and then immediately cooled to 45 ° C.

(Plating)
Glucose yeast extract (GYE) agar was dissolved and then cooled to 45 ° C. in a water bath. A total of 5 Petri dishes were used per sample. 1 ml of the heat-treated final dilution tube was added to each petri dish, then 5 ml of the above-dissolved GYE agar medium was added to the petri dish and mixed thoroughly. Once set, the plates were incubated in an inverted position at 40 ° C. for a total of 48 hours.

(Count of bacterial surviving colonies)
Plates showing 30-300 colonies were selected for counting. Plates with little change in total number of colonies were counted and then the average number per plate was calculated. Multiply the average number of colonies counted per plate by the reciprocal of the dilution factor to obtain the number of viable cells per gram of sample (eg, the average number of colonies per plate is 90 and the final dilution factor is If it was 2 × 10 ⁻⁶ , then the number of surviving spores was 90 × (2 × 10 ⁶ ) or 1.8 × 10 ¹⁰ viable spores per gram)
.

As discussed later, subsequent gas chromatographic fatty acid methyl ester (GC-Fame) and Biolog ^™ analysis indicated that these bacteria were Bacillus coagulans strains that had not been characterized previously. Table 8 below shows the new Bacillus coagulans strain described herein (ie, 20 ° C.
Bacillus coagulans isolate (5937-20 ° C.); 30 ° C. Bacillus coagulans isolate (5937-30 ° C.); ATTC 99% Bacillus coagulans isolate (ATTC 99%); and ATTC 1% Bacillus coagulans isolate (here) (−) Indicates no growth; (+) indicates little or minimal growth; and (++) indicates significant or optimal growth). Glucose yeast extract (GYE) medium and trypticase soy broth (TSB) culture medium were used.

図１は、棒グラフの形式で、トリプチカーゼダイズブロス（ＴＳＡ）培地かまたはグルコース酵母エキス（ＧＹＥ）培地のいずれかでの、Ｂａｃｉｌｌｕｓ ｃｏａｇｕｌａｎｓ １％分離体（ＧＢＩ−１）；ＡＴＣＣ−９９％分離体；５９３７−２０℃分離体（ＧＢＩ−２０）；および５９３７−３０℃分離体（ＧＢＩ−３０）の最小培養温度および最適培養温度を説明する。

FIG. 1 shows, in bar graph format, Bacillus coagulans 1% isolate (GBI-1) in either trypticase soy broth (TSA) medium or glucose yeast extract (GYE) medium; ATCC-99% separation. The minimum and optimum culture temperatures of the body; 5937-20 ° C. isolate (GBI-20); and 5937-30 ° C. isolate (GBI-30) are described.

(Research on pH fluctuation)
(Materials and methods)
A total of four cultures of Bacillus coagulans strains were tested for pH permeation, organic nutrient plate counts, and optical transmission (%) of culture growth at 28 hour intervals in tryptic soy broth (TSB) medium. ) And the optical density (OD). These strains include: 20 ° C. Bacillus coagulans isolate (GBI-20 ° C.); 30 ° C. Bacillus coagulans isolate (GBI-30 ° C.); ATTC 99% Bacillus coagulans isolate (ATCC 99%); and 1 % Bacillus coagulans isolate (GBI-1).

  Each of the above bacterial strains was added to a 50 ml Erlenmeyer flask containing 20 ml TSB medium. Seven flasks were prepared for each of the four separators (separators for the study at 28 hours each 4 hour interval). The initial seeding culture is a broth culture in a test tube, which has a permeability (%) of 10%. 1.0 ml of this culture was then added to each of a total of 28 flasks (meaning 7 flasks for each strain). The seeded flask was incubated at 45 ° C. for 28 hours on a rotary invitromental shaker. The shaker was stopped every 4 hours and new cultures were collected for evaluation. OD readings for% optical transmission, pH, and total organic nutrient plate counts by 3M Petri film spread plate method were performed to monitor changes in bacterial cell density and pH at different time intervals. Table 9, Table 10, and Table 11 show the pH evaluation, OD for transmittance (%), and total organic nutrient plate count, respectively.

（実験結果）
表９〜１１から確認され得るように、異なる間隔でのＬ（＋）乳酸産生に関して、すべての分離体の間に顕著な変化がある。細胞密度に対応するこの間隔を、Ｖｉｔｅｋ機を５４０ｎｍまたは６８０ｎｍのいずれかで稼動させて、光透過率を用いて、およびＴＳＢでの標準のプレートカウントを用いて決定した。２０℃ Ｂａｃｉｌｌｕｓ ｃｏａｇｕｌａｎｓ分離体（ＧＢＩ−２０℃）および３０℃ Ｂａｃｉｌｌｕｓ ｃｏａｇｕｌａｎｓ分離体（ＧＢＩ−３０℃）は、１％ Ｂａｃｉｌｌｕｓ ｃｏａｇｕｌａｎｓ分離体（ＧＢＩ−１）およびＡＴＴＣ ９９％ Ｂａｃｉｌｌｕｓ ｃｏａｇｕｌａｎｓ分離体よりも、高増殖速度を提供し、それは８時間の間隔およびその後の発酵ブロスの低下したｐＨにおいてより顕著に有効であった（ＴＳＢを発酵基質として用いた）。これらの結果は、上述のこれらの株が、１％ Ｂａｃｉｌｌｕｓ ｃｏａｇｕｌａｎｓ分離体（ＧＢＩ−１）およびＡＴＴＣ ９９％ Ｂａｃｉｌｌｕｓ ｃｏａｇｕｌａｎｓ分離体のいずれかよりも、Ｅｓｃｈｅｒｉｃｈｉａ、Ｃａｍｐｙｌｏｂａｃｔｅｒ、Ｃａｎｄｉｄａ、ＣｌｏｓｔｒｉｄｉｕｍおよびＳｔａｐｈｙｌｏｃｏｃｃｕｓのようなｐＨ特異的な疾患を和らげることについて有効であることを示すようだ。

(Experimental result)
As can be seen from Tables 9-11, there is a significant change between all isolates with respect to L (+) lactic acid production at different intervals. This interval corresponding to cell density was determined using the Vitek machine at either 540 nm or 680 nm, using light transmission, and using standard plate counts with TSB. 20 ° C. Bacillus coagulans isolate (GBI-20 ° C.) and 30 ° C. Bacillus coagulans isolate (GBI-30 ° C.) are also 1% Bacillus coagulans isolate (GBI-1) and ATTC 99% Bacillus coagulans isolate. It provided a growth rate, which was more markedly effective at the 8 hour interval and subsequent reduced pH of the fermentation broth (TSB was used as the fermentation substrate). These results indicate that these strains described above are more likely to be found in Escherichia, Campylobacter, Candida, Clostridium and staphlocos, than either 1% Bacillus coagulans isolate (GBI-1) or ATTC 99% Bacillus coagulans isolate. It seems to show effectiveness in relieving specific diseases.

(Proliferation / endpoint kinetic study)
(GBI-1 and ATCC-99% Bacillus coagulans isolates)
Two cultures of Bacillus coagulans species were analyzed using a growth / endpoint kinetic test. These species include: 1% Bacillus coagulans isolate (GBI-1) and ATCC-99% Bacillus coagulans isolate.

  In kinetic assay number 1, a 1% Bacillus coagulans species (GBI-1) was tested. In kinetic assay number 2, the ATCC-99% Bacillus coagulans species was tested. Specific species of Bacillus coagulans to be tested were grown at 45 ° C. for a total of 48 hours in trypticase soy broth (TSB) medium. Following incubation, the culture was suspended in sterile saline until turbidity of approximately 40-50% T (turbidity). The diluted culture was placed in a 96-well microtiter plate well containing a specific growth medium containing one of the following: TSB, glucose yeast extract (GYE) medium, or further oxygenated or not oxygenated either. To oxygenate the growth medium, 100 ml of each medium was placed in an oxygen concentrator with a flow rate of 4 l / min for a total of 15 minutes. In addition, each microplate well also contains a tetrazolium dye / redox indicator system. Bacterial growth (ie, metabolic respiration or carbon source oxidation) was monitored by tetrazolium reduction measured at 590 nm in a spectrophotometric microplate reader.

  Bacterial growth was measured every 20 minutes during a total incubation of 22 hours at 32 ° C. Kinetic data was processed and background blank values were subtracted.

  Following completion of the kinetic proliferation assay referenced above, the reduction of tetrazolium measured in a microplate at 590 nm is read as an endpoint kinetic assay. FIGS. 2 and 3 show the endpoint kinetics for both 1% Bacillus coagulans species (GBI-1) and ATCC-99% Bacillus coagulans species, respectively.

(5937-20 ° C and 5937-30 ° C Bacillus coagulans isolates)
Two cultures of Bacillus coagulans species were analyzed using a growth / endpoint kinetic test. These species include: 20 ° C. Bacillus coagulans isolate (GBI-20) and 30 ° C. Bacillus coagulans isolate (GBI-30).

    In kinetic assay number 3, a 20 ° C. Bacillus coagulans isolate (GBI-20) was tested. In kinetic assay number 4, a 30 ° C. Bacillus coagulans isolate (GBI-30) was tested. The particular species of Bacillus coagulans to be tested was grown at 35 ° C. for a total of 48 hours in glucose yeast extract (GYE) medium. Following incubation, the culture was suspended in sterile saline until turbidity of approximately 40-50% T (turbidity). The diluted culture was placed in the wells of a 96-well microtiter plate containing a specific growth medium containing one of the following: GYE or trypticase soy broth, nutrient broth (NB) or biolog universal growth medium (Biolog Universal Growth Medium (BUGMB)) or either further oxygenation or no oxygenation. To oxygenate the growth medium, 100 ml of each medium was placed in an oxygen concentrator with a flow rate of 4 l / min for a total of 15 minutes. In addition, each microplate well also contains a tetrazolium dye / redox indicator system. Bacterial growth (ie, metabolic respiration or carbon source oxidation) was monitored by tetrazolium reduction measured at 590 nm in a spectrophotometric microplate reader.

  Bacterial growth was measured every 20 minutes during a total incubation of 18 hours at 32 ° C. Kinetic data was processed and background blank values were subtracted.

  Following completion of the kinetic proliferation assay referenced above, the reduction of tetrazolium measured in a microplate at 590 nm is read as an endpoint kinetic assay. 4 and 5 represent the endpoint kinetic histograms of the 5937-20 ° C. Bacillus coagulans isolate (GBI-20) and 5937-30 ° C. Bacillus coagulans isolate (GBI-30), respectively.

(Biolog ^™ analysis of Bacillus coagulans isolate)
To distinguish between the ATCC-type species Bacillus coagulans Hammer (ATCC No. 31284) and the new species of Bacillus coagulans disclosed herein, the biolog micros were used for the identification and characterization of microorganisms by carbon source recognition patterns. A Plate System ^™ (Biolog Microplate System ^™ ) was used. An inoculum of ATCC type seed (ATCC No. 31284) was placed in a flask containing three trypticase soy broths. These flasks were then incubated at different temperatures to compensate for any bacterial selection due to temperature. After 30 hours of incubation, aliquots from each broth flask were aseptically transferred in a laminar flow biological cabinet and plated onto TSA medium in a pre-prepared and dried petri dish. After incubation for 24 and 48 hours at 30 ° C., 35 ° C. and 40 ° C., a survey on colony forming units (CFU) is performed.

The Biolog Microplate System ^™ was used for the identification and characterization of microorganisms by the carbon source recognition pattern of the Bacillus coagulans species disclosed in the present invention. The microplate technology described above allows characterization of microorganisms through the use of 95 different analytical methods. Thus, a total of 4 × 10 ²⁸ possible patterns produced from one microplate are obtained. Discernable pattern obtained for the species of the microorganism, and different species of bacteria gives "family" of different patterns, this pattern is recognized by the bio-log micro log ^TM (Biolog Microlog ^TM) software and distinguish Can be done. Microplate analysis for the Biolog Microlog ^™ system includes Gram negative bacteria, Gram positive bacteria, yeast, lactic acid producing bacteria, and E. coli. It can be used for the analysis of E. coli / Salmonella. In addition, further analysis can also be performed by the use of additional selective media.

  Briefly, a given microbial isolate is characterized by streaking the organism on a nutrient medium (eg, GYE or TSA) that supports the growth and growth of a large number of microorganisms. However, more unfavorable organisms may require chocolate or BIER agar for growth, while many “environmental” have been found to grow better with minimal media. Culture plates were incubated at 28-35 ° C for 4-18 hours.

  Following incubation, bacterial colonies were removed from the culture plates by use of a cotton swab moistened with saline. A uniform turbidity suspension was then prepared in 0.85% saline by comparison with the known standard turbidity. The bacterial suspension was inoculated into microplate wells (150 μl / well) and the plates were covered with corresponding microplate lids. The covered plates were then incubated at 28 ° C. to 35 ° C. for 4 hours or overnight (16-24 hours).

  The microplate was then read using a microplate reader at 590 nm. The absorbance and transmittance (ie color) in each well is referenced to a negative control well (A-1) and any purple recorded above this control level is positive for the use of a particular carbon source. I was able to read it. Data was reported as percent color change when compared to well A-1 using the following formula:

一般的に、パーセント色変化が４０と等しいかもしくはそれより大きい値であると見出された場合、所定のウエル内の反応を「陽性」と考えた。しかし、各基質が異なり得、そして４０より小さい値の陽性試験が可能であり得るパラメーターとしてこの値を経験的に決定しなければならない。使用されたコンピュータアルゴリズムは、再現性およびオペレーターによる偏りの回避を確実にする標準化設定を提供する。使用されたすべての炭素源の名前は、応答とは関係なしに結果として提供される。

In general, a reaction in a given well was considered “positive” if the percent color change was found to be equal to or greater than 40. However, this value must be determined empirically as a parameter where each substrate can be different and a positive test with a value of less than 40 can be possible. The computer algorithm used provides a standardized setting that ensures reproducibility and avoidance of operator bias. The names of all carbon sources used are provided as a result regardless of the response.

  Table 12 below shows total heterotrophic plate counts using trypticase soy agar (TSA) for the novel Bacillus coagulans species disclosed herein.

以下の表１３は、本明細書中で開示された新規なＢａｃｉｌｌｕｓ ｃｏａｇｕｌａｎｓ種と比較する各サンプルにおける好気性型種のおおよそのパーセンテージを示す。

Table 13 below shows the approximate percentage of aerobic type species in each sample compared to the novel Bacillus coagulans species disclosed herein.

（ＧＣ−ＦＡＭＥプロセシング）
細菌種をトリプチカーゼダイズ寒天（ＴＳＡ）プレートに画線接種した。次に２４時間のインキュベーションに続いて、ＴＳＡプレートを刊行された標準的なＧＣ−ＦＡＭＥ方法論によりガスクロマトグラフィー脂肪酸メチルエステル（ＧＣ−ＦＡＭＥ）分析のために調製した。続いて、細菌種を好気性菌（ＴＳＢＡ）および臨床的好気性菌（ＣＬＩＮ）コンピュータデータベースの両方に対して調べた。ＧＣ−ＦＡＭＥ分析の結果を以下の表１４に示す。

(GC-FAME processing)
Bacterial species were streaked on trypticase soy agar (TSA) plates. Following the 24 hour incubation, TSA plates were then prepared for gas chromatography fatty acid methyl ester (GC-FAME) analysis by published standard GC-FAME methodology. Subsequently, the bacterial species were examined against both aerobic bacteria (TSBA) and clinical aerobic bacteria (CLIN) computer databases. The results of GC-FAME analysis are shown in Table 14 below.

（１６ＳリボソームＲＮＡ（ｒＲＮＡ）配列分析）
（材料および方法）
１６ＳリボソームＲＮＡ（ｒＲＮＡ）配列分析を、Ｂａｃｉｌｌｕｓ ｃｏａｇｕｌａｎｓ菌株：ＧＢＩ−１；ＡＴＣＣ−９９％；ＧＢＩ−４０；ＧＢＩ−３０；およびＧＢＩ−２０に関して実施した。

(16S ribosomal RNA (rRNA) sequence analysis)
(Materials and methods)
16S ribosomal RNA (rRNA) sequence analysis was performed on Bacillus coagulans strains: GBI-1; ATCC-99%; GBI-40; GBI-30; and GBI-20.

The protocol used to generate 16S rRNA gene sequence data is shown below. The 16S rRNA gene was PCR amplified from genomic DNA isolated from bacterial colonies. Primers used for amplification are E. coli for the 500 bp package. corresponded to E. coli positions 005 and 531. Excess primer and dNTPs were subsequently removed from the amplification product by use of a Microcon 100 ^™ (Amicon) molecular weight cut-off membrane. The PCR amplification products were then subjected to agarose gel electrophoresis analysis to confirm both the quality and quantity of these products.

Cycle sequencing of 16S rRNA amplification products was performed using AmpliTaq FS ^™ DNA polymerase and drhodamine dye terminator. Excess dye-labeled terminator was removed from the sequencing reaction using a Sephadex G-50 spin column. The amplification product was then recovered by centrifugation, dried under vacuum, and stored at −20 ° C. until use. These products were resuspended in a solution of formamide / blue dextrin / ETDA and heat denatured before electrophoresis. Samples were electrophoresed on an ABI Prism 377 DNA Sequencer for about 6 hours using pre-poured 5% Long Ranger ^™ (RMC) polyacrylamide / urea gel. The resulting sequence data was analyzed using PE / Applied Biosystems DNA editing and assembly software.

The identification of the designated bacteria was based on 16S rRNA gene sequence homology. The sample sequence was identified by comparison against the PE Applied Biosystem's MicroSeq ^™ database using MicroSeq ^™ sequence analysis software. Sequence alignments that provide the highest degree of sequence homology are indicated as a percentage of the genetic distance format (ie, the percent difference between the two aligned sequences). It should be noted that in this format, a low percentage indicates a high degree of sequence homology. FIGS. 6-8 provide results obtained by alignment of the novel Bacillus coagulans strain disclosed in the present invention with various other Bacillus species, and by Neighbor Joining Tree and Consistion Alignment analysis. The results for the ATCC-99% isolate are shown in FIG. 6; the results for GBI-20 are shown in FIG. 7; and the results for GBI-30 are shown in FIG.

  Closest neighbors (see Saitou and Nei, 1987. Mol. Biol. Evol. 4: 406-425) and / or UPGMA (Waterman, 1995, Introduction to Computational Biology 360-365 (Chapman and Hall Publishing) Reference is also provided herein. Similarly, a “genealog” was generated using aligned sequence matches that provided the highest degree of sequence homology.

(Experimental result)
Note that all experimental results are presented in a genetic distance format, which is essentially the exact opposite of the percent homology.

Species level: This indicates species level agreement. 16S rRNA sequence homology greater than 99% is an indicator of species-level concordance (Staekebrandt and Goebel, 1994. Taxonomic Note: A Place for DNA-DNA
Association and 16S rRNA Sequence Analysis in the Present Specialties Definition in Bacteriology. Int. J. et al. Syst. Bacteriol. 44: 846-849).

  Genus level: This indicates that the sample appears to be grouped within a particular genus, but the alignment did not produce a species level match. A genus level match indicates that the sample species is not included in the MicroSeq database.

  No match: This indicates that the sample did not group well within any particular genus found in the MicroSeq database. In such cases, a search of both the GenBank and ribosome database project (RDP) databases with the sample sequences was subsequently performed to obtain closer matches. If the sample sequence does not match well in any of these databases, this may indicate a new species whose 16S rRNA gene sequence is not present in any of the databases.

  Table 15 below provides the results of a percent genetic difference study in a tabular format.

  (Table 15)

１６Ｓ ｒＲＮＡ配列相同性は、９９％よりも大きく、そして種レベルの一致の指標であることを見出された。

16S rRNA sequence homology was found to be greater than 99% and an indicator of species level concordance.

(Aminopeptidase profiling)
Aminopeptidase profiling or activity is used to distinguish bacteria and fungi into species and subspecies (see, for example, Hughes et al., 1988, LacZY gene modified peptideid activity in Pseudomonas aureofaciens. 502: Hytopathophys. Immobilized bacteria by aminopeptidase profiling. See Anal. Chem. 61: 1656-1660), as well as defining the ecological status of parasites and developing media for preference organisms. Recent developments in time-resolved 96-well plate fluorometers provide a fast and sensitive method for obtaining peptidase profiles for bacterial identification. Mossman et al., 1997. Aminopeptidase profiling using a time-resolved, 96-well plate filter fluorometer. Appl. See Spectroscopy 51: 1443-1446.

Aminopeptidase profiling is disclosed in Bacillus disclosed herein.
It has been shown that this is an effective procedure for distinguishing new strains of coagulans from previously known and characterized strains (eg, ATCC type strains).

(Materials and methods)
Profiling analysis of the disclosed aminopeptidases is described by Mossman et al., 1997, Appl. Follow the methodology as shown by Spectroscopy 51: 1443-1446. Each Bacillus coagulans isolate was first incubated on a Tryptic Soy Broth (TSB) agar plate for 24 hours before the plate was washed from 10 mM pH 7 phosphate buffer. Table 16 below shows the culture conditions of the various Bacillus coagulans strains used in the present invention.

  (Table 16)

培養後、９６ウェルの平底で黒色のポリスチレンプレート（ＦｌｕｏｒｏＮｕｎｃ；Ｎａｌｇｅ−Ｎｕｎｃ，Ｎａｐｅｒｖｉｌｌｅ，ＩＬ）の各セルに０．５ｍｌを置く前に、細胞密度を、５４０ｎｍ（８５％ 透過率）での分光測光法によって２．５×１０⁶細胞
／ｍｌに調整した。各ウェルは、２０個の非蛍光Ｌ−アミノ酸−β−ナフチルアミド基質（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ ＣＯ．，Ｓｔ．Ｌｏｕｉｓ，ＭＩ）のうちの１つを、１×１０^-4Ｍの終濃度で含んだ。３００μｌのマイクロプレートウェル容積の平衡は、２５０μｌの１０ｍＭ リン酸緩衝液からなった。

After incubation, the cell density was measured spectrophotometrically at 540 nm (85% transmittance) before placing 0.5 ml in each cell of a 96 well flat bottom black polystyrene plate (FluoroNunc; Nalge-Nunc, Naperville, IL). It was adjusted to 2.5 × 10 ⁶ cells / ml by the method. Each well contained one of 20 non-fluorescent L-amino acid-β-naphthylamide substrates (Sigma Chemical CO., St. Louis, MI) at a final concentration of 1 × 10 ⁻⁴ M. The 300 μl microplate well volume equilibration consisted of 250 μl of 10 mM phosphate buffer.

  The 20 different peptidase substrates used to generate this profile include the following amino acids β-naphthylamide: L-alanine (ALA), L-arginine (ARG), L-asparagine (ASN), L -Aspartic acid (ASP), L-cysteine (CYS), glycine (GLY), L-glutamic acid (GLU), L-histidine (HIS), L-isoleucine (ILE), L-leucine (LEU), L-lysine (LYS), DL-methionine (MET), L-phenylalanine (PHE), L-proline (PRO), L-serine (SER), transhydroxy-L-proline (HPR), L-tryptophan (TRP), L -Tyrosine (TYR), and L-valine (VAL). β-naphthylamine alone was also used as a positive control. Bacterial blanks, substrate blanks, and buffer blanks were also included in the assay procedure as negative controls. Four replicates of each bacterial isolate were run after a 4 hour incubation period.

Aminopeptidase profiles were constructed using data obtained from time-determined laser fluorometer assays of enzymatically hydrolyzed fluorescent β-naphthylamide products from non-fluorescent β-naphthylamide substrates. The time determination 96 well plate fluorometer consists of a sealed tube, a nitrogen laser guided in a blank, a flat bottom FluoroNunc 96 well plate through an excitation portion of a bifurcated optical fiber. Fluorescence was collected at an angle of ⁰ to the excitation light using the light emitting portion of the bifurcated optical fiber. A desired emission wavelength was selected prior to detection using a 931A photomultiplier tube using a 389 nm cut-on filter. A total of 25 fluorescence decays were averaged by a Tektronix DSA 602 digital oscilloscope and transferred to a PC computer via an IEEE-488 interface card to obtain a relative fluorescence reading after blank subtraction.

(Experimental result)
Significant differences were detected in the enzyme profiles of these Bacillus coagulans strains, which are identical in other methods for 16S rRNA sequencing, GC-FAME, and Biolog identification. This data is shown for each of the four Bacillus coagulans strains in a histogram format plotting the fluorescence intensity for each aminopeptidase enzyme activity listed below. FIG. 9 shows a histogram plot of fluorescence intensity for each of the aminopeptidase enzyme activities for the Bacillus coagulans 99% ATCC isolate; FIG. 10 shows a histogram of fluorescence intensity for each of the aminopeptidase enzyme activities for the Bacillus coagulans GBI-1 isolate. FIG. 11 shows a histogram plot of fluorescence intensity for each of the aminopeptidase enzyme activities for the Bacillus coagulans GBI-30 isolate; and FIG. 12 shows the Bacillus coagulans.
Shown is a histogram plot of fluorescence intensity for each of the aminopeptidase enzyme activities for the GBI-20 isolate. As shown in FIGS. 9-12, each specific aminopeptidase and control is identified using numbers 1-24. These numbers are:

番号１２（リジンアミノペプチダーゼ）および番号２２（β−ナフチルアミン、１００％コントロール）に対する活性は、「オフスケール（ｏｆｆ−ｓｃａｌｅ）」であることが見出された場合、プロットしない。全ての細胞密度を、８５％Ｔで標準化した。

Activity against number 12 (lysine aminopeptidase) and number 22 (β-naphthylamine, 100% control) is not plotted if found to be “off-scale”. All cell densities were normalized at 85% T.

  The results shown in FIGS. 9-12 show the differences present in the aminopeptidase profiles of these Bacillus coagulans isolates, regardless of the general similarity within this profile. For example, the 20 ° C. isolate GBI-20 (see FIG. 12) is most similar to the 99% isolate (see FIG. 9) with a dramatic deviation in the relative amount of proline aminopeptidase. On the other hand, the 30 ° C. isolate GBI-30 (see FIG. 11) is very similar to the pattern of 1% isolate GBI-1 (see FIG. 10), but phenylalanine The relative amount of aminopeptidase deviates. This methodology can therefore be used to identify these Bacillus coagulans strains both rapidly and effectively.

(Use of Bacillus coagulans in the inhibition of gastrointestinal VRE)
The ability of Bacillus coagulans vegetative bacteria and spores to inhibit vancomycin-resistant Enterococci (VRE) colony formation was tested. Prior to the present disclosure, there was no effective treatment available to reduce either the amount or duration of intestinal colonization by VRE. For example, many antibiotics have been shown to have only a very transient effect on VRE colony formation. Therefore, the development of safe and effective therapeutic agents for improving VRE colony formation reduces the potentially fatal consequences of VRE infections, VRE infections between patients, hospital costs, and inconvenience for patients and health care workers. Plays a significant mitigating role.

(Materials and methods)
A mouse model (originally developed to study the effect of various antibiotics on the persistence of VRE intestinal colony formation) was used for these experiments. Two sets of experiments were performed using a total of 33 mice. High levels of VRE colony formation were established in all 33 mice by administering approximately 5 × 10 ⁸ VRE by oral gavage while clindamycin was administered subcutaneously daily for 5 days simultaneously. This method consistently results in the occurrence of high levels of lethal colonization of VRE in mice (mean = 9 log ₁₀ CFU / 1 g feces).

These mice were then divided into three experimental groups and administered the following drugs: Group 1 = saline, oral gavage for 4 days (total 11 control mice); Group 2 = Bacillus Coagulans Overnight culture, approximately 1 × 10 ⁷ vegetative bacterial organisms, oral gavage for 4 days (total of 17 control mice); and group 3 = Bacillus Coagulans spores, approximately 1 × 10 ⁷ organisms, oral gavage 4 days (total 5 control mice). Fecal samples were collected at 3-5 day intervals during the experiment to determine the levels of VRE and Bacillus coagulans. Fecal samples are homogenized, serially diluted in saline, and plated on BHI agar containing 6 μg / ml aztreonam and 6 μg / ml nystatin for enterococcosel selection agar for Bacillus coagulans quantification for VRE quantification did. If VRE was not detected in the sample, a lower limit of detection was assigned.

(Preliminary microbiology results)
Kirby-Bauer antimicrobial susceptibility test:
Sensitivity to:
Intermediate susceptibility to ampicillin, ciprofloxacin, trimethoprim-sulfamethoxazole, rifampin, erythromycin, vancomycin, gentamicin, and oxacillin below: Sensitivity test based on Tetracycline Vitek instrument (Machine):
Sensitivity to:
Resistance to penicillin, vancomycin, gentamicin (500 μg / ml), streptomycin (2,000 μg / ml), nitrofurantoin, norfloxacin, and chloramphenicol:
Tetracycline nitrocefin test:
Positive low level β-lactamase production (mouse colonization)
Bacillus coagulans were given to 8 mice to determine the dose to use in subsequent formal experiments. All mice were colonized with high levels of VRE (> 9 log ₁₀ CFU / g of feces) prior to administration of Bacillus coagulans. Control mice received no treatment. Bacillus coagulans were administered daily by gastric gavage at three different doses: 1.5 × 10 ⁶ CFU / kg = normal human dose, 2.5 × 10 ⁸ CFU / kg, and 3.5 × 10 ⁹ C
FU / kg. The level of VRE in the feces was determined after 5 days. The results of these preliminary studies are shown in Table 17 below.

^*ＶＲＥのレベルを５×１０⁸ＣＦＵ Ｂａｃｉｌｌｕｓ ｃｏａｇｕｌａｎｓ／ｋｇ／日を用いて処置した２／３マウスについて、検出のレベルより下（＜１．７ｌｏｇ₁₀ＣＦＵ／ｇ）だった。

^* VRE levels were below the level of detection (<1.7 log ₁₀ CFU / g) for 2/3 mice treated with 5 × 10 ⁸ CFU Bacillus coagulans / kg / day.

By using the colony formation methodology described above, high levels of VRE colony formation were established in all of the mice (ie 7.1 to 10.2 log ₁₀ VRE / feces 1 g). The initial levels of VRE present in saline control mice and Bacillus coagulans mice were not significantly different. VRE levels gradually decreased in all saline control mice after withdrawal of clindamycin (in agreement with previous experiments).

Compared to the saline control, the level of VRE declined more rapidly in mice that received Bacillus coagulans. 5 days after discontinued clindamycin (Bacillus coagulans treatment of 4 days), the average level of VRE, compared with feces 6.7log ₁₀ VRE / 1g in saline control, 5.3log ₁₀ VRE / 1g I found that it was feces. This indicated a 25-fold decrease in VRE levels (p <0.05). After 8 days was discontinued clindamycin (Bacillus coagulans 4 days after completed treatment), the average level of VRE, compared with feces 4.3log ₁₀ VRE / 1g in saline control, 2.9Log ₁₀ VRE / G of feces was found. This indicated a 28-fold decrease in VRE levels (p <0.05). While 35% (6/17 animals) of Bacillus coagulans-treated mice failed to detect VRE levels 8 days after discontinuing clindamycin, the saline control could detect no VRE levels at this time. None (p <0.05). The mean level of VRE present in the feces of 5 mice fed Bacillus coagulans spores was also significantly lower (p <0.05) than that in saline control mice, but these 5 mice VRE level could not be detected.

All of the mice given Bacillus coagulans were unable to detect the level of Bacillus coagulans in these feces one day after 4 days of treatment (range 3.1-6.4 log ₁₀ CFU / 1 g feces) and these All of the mice still had low levels of Bacillus coagulans detectable in their feces 4 days after completion of treatment.

  These studies demonstrate that oral administration of Bacillus coagulans (both vegetative and spore forms) resulted in a significant reduction in the level of VRE in the feces of colonized mice compared to saline controls. did. This result (which was obtained through the use of this mouse model) correlates well with findings in various studies that tested VRE colonized human patients. Thus, this established mouse model provides a means to study the effects of drugs designed to eliminate VRE colony formation. 35% of mice given Bacillus coagulans failed to detect VRE levels 4 days after completion of treatment. As a comparison, all mice that received saline did not contain VRE. On average, a 25- to 28-fold reduction in the level of VRE was observed in Bacillus coagulans treated mice compared to saline treated mice. Furthermore, 65% of the mice fed Bacillus coagulans had a reduction in VRE equal to about 50 times the original inoculation. Thus, all of the test mice had a significant reduction in VRE load (60% of mice showing 2-logVRE reduction, as well as complete eradication of Enterococci with a statistically 0% recovery of VRE in the mouse feces 40 %). These results suggest that Bacillus coagulans treatment is an effective means to improve both the level and persistence of VRE colonization in human patients.

Inhibition of VRE by Bacillus coagulans does not appear to involve any of the traditional mechanisms of inhibition despite the use of probiotic bacteria such as Bacillus coagulans. As previously discussed, there are two main mechanisms using acid producing Bacillus for the elimination of microorganisms. These mechanisms are the following:
Competitive inhibition or elimination: This is the ability of most Bacillus to compete with other organisms for substrates and trace minerals.
Micro-Environment Modification: This changes the physical or biochemical properties or activities of bacterial cell membranes by the production of acids (eg lactic acid, acetic acid, etc.) or other agents with antibacterial properties Usually works to let you.

  There was a dramatic decrease in VRE levels (ie 2-log in 60% of the effective group and 40% of the total eradication group), but the result was no increase corresponding to the Bacillus coagulans concentration in the treatment group. (Shown as CFU per gram of mouse feces). It is believed that one experimental group showed substantially better results than another successful experimental group (but without a corresponding Bacillus count to justify it). Thus, these results suggest that competitive inhibition by Bacillus coagulans is not a mechanism that results in a reduction in VRE levels in this study.

Furthermore, it is also known that Enterococci is not inhibited by changes in the pH of this micro-environment. For example, Enterococcus faecium (which is an Enterococcus species responsible for most (if not all) VRE transport and infection) is used as a probiotic in the animal production industry. This organism, itself, produces the D-enantiomer of lactic acid and is commonly co-administered with Lactobacillus and Bifidobacterium, which yields the L-enantiomer of lactic acid. Therefore, Enterococcus faecium is not affected by lactic acid producing organisms (produced regardless of optical isomers of lactic acid). Thus, the second method used by probiotic bacteria (which alters the microenvironment) to inhibit bacterial colonization is Bacillus.
It is not thought to play a role in the inhibition of VRE by coagulans. Due to the above experimental results, it is believed that the improvement in VRE by Bacillus coagulans is due to the production of one or more antibacterial reagents by Bacillus. The antibacterial reagent may be an organic molecule (s) and / or a heat resistant protein (s).

A composition for inhibiting the growth of VRE is a Bacillus coagulans vegetative bacterium and / or spore in combination with a culture medium (supernatant) in either a crude or semi-purified form. Of a wide concentration (ie, 1 × 10 ⁹ to 1 × 10 ¹¹ CFU). This is Bacillus coa
When present with galans vegetative cells and / or spores, this culture medium was also named GRAS classification by the FDA. To reduce the total volume, the medium can be partially or fully lyophilized. Thus, co-administration of both this vegetative bacterium / spore and some types of supernatant components would result in all possible probiotic inhibition mechanisms (ie, antibiotic action, parasitism, competitive inhibition and microenvironment / It serves to ensure that the (pH change) is encompassed by the administration of the therapeutic composition described above.

As previously discussed above, the Bacillus coagulans culture medium has been shown to contain extracellular product (s). This product is produced and secreted by bacteria. It has significant antibacterial properties against bacteria, fungi, yeast and viruses. A methodology for the purification of one or more reagents responsible for these antimicrobial properties is also currently under development. Accordingly, preferred embodiments of the present invention provide a wide concentration of Bacillus coagulans vegetative bacteria and / or spores (ie, in combination with either unpurified or semi-purified forms of these extracellular product (s) (ie, 1 x 10 ⁹ to 1 x
10 ¹¹ CFU).

  Bacillus coagulans treatment is also useful for inhibiting other strains of VRE. Similarly, Bacillus coagulans can be used to colonize pathogenic organisms (eg, Candida species, Salmonella, clotting enzyme negative Staphylococci, and multiresistant Gram negative rods (eg, Klebsiella species and Escherichia coli.)). Prevent or improve the level of

(Equivalent)
From the foregoing detailed description of particular embodiments of the present invention, a unique methodology for the use of lactic acid producing bacteria (preferably Bacillus coagulans) for the prevention and treatment of gastrointestinal etiology and diseases associated therewith. However, it should be readily apparent that it is disclosed. Although specific embodiments have been disclosed in detail herein, this is done by way of example only for purposes of illustration and is not intended as a limitation on the appended claims. In particular, it is contemplated by the inventors that various substitutions, changes and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims. . For example, the selection of a particular antibiotic to be utilized in the therapeutic composition of the present invention is considered routine for those skilled in the art in view of the knowledge of the embodiments described herein.

  Other embodiments are within the scope of the appended claims.

Claims (1)

Invention described in this specification.

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