Probiotic composition based on the enterococcus strain and used as a treatment means and method for the production thereof



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[0032] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and

materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.


[0034] It must be noted that as used herein and in the appended claims, the singular forms"a", "and", and"the"include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to"a probiotic bacterium"includes a plurality of such bacteria and reference to"the dosage unit"includes reference to one or more dosage units and equivalents thereof known to those skilled in the art, and so forth.
[0035] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
DETAILED DESCRIPTION OF THE INVENTION [0036] The present invention provides formulations comprising inactivated probiotic bacteria, and various therapeutic methods using the formulations. Subject formulations are useful for treating any disorder that is amenable to treatment with viable probiotic bacteria. Thus, the present invention provides methods of treating gastrointestinal inflammation; microbial infections; diarrheal diseases; allergic disorders; non-alcoholic liver disease; and the like.
[0037] The invention is based on the observation that irradiated probiotic bacteria, but not probiotic bacteria inactivated by extreme heat, are efficacious in treating colitis in an animal model of colitis. Thus, contrary to what has been reported in the art, probiotic bacteria need not be alive to exert a beneficial effect. Without wishing to be bound by theory, the reason that irradiate probiotic bacteria are effective, while bacteria killed by treatment at 100 C for 30 minutes are not, may relate to maintenance of structural integrity (e. g. , of the cell wall and/or cytosolic components) in the former, but not in the latter, bactericidal method.
100331 Currently available probiotic formulations are typically stored at a temperature of no higher than 45 C, and usually are either lyophilized, or are stored in an aqueous or other non- frozen medium at refrigeration temperatures (e. g. , at about 4 C), because at higher temperatures, viability of probiotic bacteria is reduced. The formulations of the present invention are advantageous over currently available probiotic formulations in that they need not be maintained within a particular temperature range, as required of live probiotic cultures.
The subject probiotic formulations are storage stable over a wide temperature range.

[0039] A further advantage of the formulations of the instant invention lies in the fact that, because the inactivated probiotic bacteria formulations of the invention are non-viable or have reduced viability, they can be administered safely to immunocompromised individuals, and to infants.


PROBIOTIC FORMULATIONS [0040] The present invention provides formulations comprising inactivated probiotic bacteria, and methods of making the formulations. The formulations are suitable for human consumption, and may include one or more pharmaceutical excipients, including food-grade excipients. As such, the subject invention further provides food products that include inactivated probiotic bacteria.
[0041] Probiotic bacteria included in the formulations of the present invention are non- pathogenic and non-toxigenic when viable. Such bacteria may be found as part of the bacterial flora of the normal, healthy human intestine. In many embodiments, inactivated probiotic bacteria included in the formulations of the present invention are or have been isolated from their natural environment, e. g. , or are variants of bacteria isolated from their natural environment. A number of probiotic bacteria are commercially available. Variants include bacteria with naturally-occurring mutations; bacteria that have been manipulated in the laboratory to differ genetically from a naturally occurring bacteria (e. g. , by the introduction of one or more mutations, by the introduction of exogenous polynucleotides (e. g.,"recombinant" or genetically modified bacteria, etc. ). Typically, subject bacteria are grown in in vitro culture before being inactivated.
[0042] Suitable bacteria for inclusion in the instant formulations include, but are not limited to, bacteria of various species, including lactobacillus species, e. g., Lactobacillus acidophilus, L. plantarum, L. casei, L. rhamnosus, L. delbrueckii (including subspecies bulgaricus), L. reuteri, L. fermentum, L. brevis, L. Iactis, L. cellobiosus, L. GG, L. gasseri, L. johnsonii, and L. plantarum ; bifidobacterium species, e. g., Eifdobacteriuyn bifidum, B. infaiitis, R longum, R enMopMM, B. adolescentis, B. breve, B. auimalis ; streptococcus species, e. g., Streptococcus lactis, S. cremoris9 S. salivarius (including subspecies thezrmophilus), and S'. intermedius ;

Leuconostoc species; Pediococcus species; Propionibacterium species; Bacillus species; non- enteropathogenic Escherichia species, e. g. , non-enteropathogenic Escherichia coli, e. g., E. coli

Nissle, and the like ; and Enterococcus species such as Enterococcus faecalis, and E. faecium.
Other suitable probiotic bacteria are known in the art, and have been described. See, e. g. , U. S.
Patent No. 5,922, 375. The person skilled in the art would understand and recognize those microorganisms which may be included in the compositions of the invention.

[0043] Bacteria other than the bacteria that are commonly considered as probiotic bacteria can also be used in a subject formulation. For example, bacteria that are pathogenic when viable can also be used, since the bacteria are inactivated before use. Essentially any bacteria that is capable, when inactivated, of alleviating the symptoms of a disorder amenable to treatment with viable probiotic bacteria, e. g. , a gastrointestinal inflammatory disorder, a microbial infection, an allergic disorder, and the like, can be included in a subject formulation.


[0044] In some embodiments, a subject formulation includes two or more different inactivated probiotic bacteria, e. g. , the bacteria may differ in strain, species, or genus. The bacteria may differ in, e. g. , strain, species, or genus. As one non-limiting example, a formulation comprises S. salivarius subsp. the mophilus, B. breve, B. infantis, B. lofigum, L. acidophilus, L. casei, and

L. delbrueckii subsp. bulgaricus. For example, the combinations of bacteria found in a commercially available product such as Kyo-Dophilus capsules (Wakunaga Probiotics), Kyo-

Dophilus tablets (Wakunaga Probiotics), Acidophilasg (Wakunaga Probiotics), Probiata (l tablets, Flora Grow (Arise & Shine), Bifa 15 (Eden Foods), TH1 Probiotics (Jarrow Formulasa), Replenish (Innercleanse 2000), Flora Bac (PDI Labs), Subalin, Colinfant,

Mutaflor, and the like.


[0045] As another non-limiting example, a subject probiotic formulation comprises two different Lactobacillus strains, e. g. , different isolates of the same species that are genetically diverse. As another non-limiting example, a subject probiotic formulation comprises from one to four Lactobacillus strains and from one to four Bifidobacterium strains. As another non- limiting example, a subject probiotic formulation comprises from one to four lactobacillus strains, from one to four bifidobacterium strains, and a non-enteropathogenic E. coli strain. As another non-limiting example, a subject probiotic formulation comprises from one to four lactobacillus strains and a non-enteropathogenic E. coli strain. As another non-limiting example, a subject probiotic formulation comprises from one to four bifidobacterium strains, and a non-enteropathogenic E. coli strain.
[0046] The probiotic bacteria in the subject formulations are inactivated. As used herein, the term"inactivated9'refers to non-viable bacteria or bacteria with reduced viability. The probiotic bacteria of the subject formulations are inactivated such that bacterial growth in vitro is inhibited. In many embodiments, inactivated bacteria are unable to grow in in vitro culture, e. g., growth in in vitro culture is undetectable. Whether bacterial growth is inhibited in vitro can be determined using well-known methods, e. g. , plating the bacteria on agar supplemented with suitable growth medium (e. g. , Luria-Bertani broth, DeMan, Rogosa, Sharpe (MRS) broth, and the like); and counting the number of colonies formed after overnight (e. g. , 12-16 hours)

culture at 37 C. The number of colony-forming units (cfu) is a measure of the viability of the culture. Bacterial growth in vitro can also be determined by culturing the bacteria in liquid medium containing appropriate nutritional supplements, and, after a period of about 12-16 hours at 37 C, the turbidity of the culture medium is measured, e. g. , absorbance at, e. g. , 570-

600 nm.
[0047] The probiotic bacteria of the subject formulations are inactivated by a process other than extreme heat inactivation, e. g. , the inactivated probiotic bacteria are not inactivated by heating to 100 C for 30 minutes. Subject inactivated bacteria are inactivated in such a manner such that they cannot replicate, and in such a manner that allows for the release of DNA from the bacteria following introduction into the gastrointestinal tract of an individual. The probiotic bacteria of the subject formulations are not inactivated by infection with bacteriophage. Inactivation can be achieved by various processes other than heat inactivation, including, but not limited to, irradiation; treatment with microwaves (e. g. , treatment with 915

MHz or 2450 MHz); treatment with radio frequencies; treatment with antibiotics; pasteurization; and treatment with chemical agents that reduce viability. In many embodiments, the inactivated bacteria remain intact, e. g. , the cell wall of the bacteria remains relatively intact following the inactivation procedure.


[0048] In other embodiments, the cell wall does not remain intact following the inactivation procedure. In these embodiments, the inactivation process may result in holes in the cell wall, or may result in partial or complete breakdown of the cell wall. Disruption of the integrity of the cell wall may occur following certain inactivation procedures, such as freeze-thaw inactivation; and the like.
[0049] Chemical agents include, but are not limited to, aldehydes, e. g., fonnaldehyde, glutaraldehyde, and the like; food preservative agents such as SO2, sorbic acid, benzoic acid, nitrate, and nitrite salts; gases such as ethylene oxide; halogens, such as iodine, chlorine, and the like; peroxygens, such as ozone, peroxide, peracetic acid; bisphenols; phenols ; phenolics; biguanides, e. g., chlorhexidine; and the like.
[0050] Antibiotics include, but are not limited to, Gentamicin ; Vancomycin ; Oxacillin ;

Tetracyclines; Nitroflurantoin; Chloramphenicol; Clindamycin; Trimethoprim- sulfamethoxasole ; a member of the Cephlosporin antibiotic family (e. g. , Cefaclor, Cefadroxil,

Cefixime, Cefprozil, Ceftriaxone, Cefuroxime, Cephalexin, Loracarbef, and the like); a member of the Penicillin family of antibiotics (e. g. , Ampicillin, Amoxicillin/Clavulanate,

Bacampicillin, Cloxicillin, Penicillin VK, and the like); with a member of the Fluoroquinolone family of antibiotics (e. g. , Ciprofloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin,


Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, and the like); or a member of the

Macrolide antibiotic family (e. g. , Azithromycin, Erythromycin, and the like); and neomycin.


[0051] In some embodiments, the probiotic bacteria are irradiated. The probiotic bacteria are irradiated at an energy and for a period of time sufficient to inhibit bacterial growth in vitro and/or to render the probiotic bacteria non-viable, e. g. , such that growth in in vitro culture is undetectable using standard methods. In some embodiments, the irradiation is ionizing radiation. Gamma radiation is an example of ionizing radiation. For example, the bacteria are irradiated using gamma irradiation in an amount of from about 5 kiloGray (kGy) to about 50 kGy, from about 10 kGy to about 20 kGy, from about 20 kGy to about 40 kGy, or from about

25 kGy to about 35 kGy. Bacteria are irradiated for a period of time of from about 15 seconds to about 48 hours, e. g. , from about 15 seconds to about 1 minute, from about 1 minute to about

15 minutes, from about 15 minutes to about 30 minutes, from about 60 minutes to about 90 minutes, from about 90 minutes to about 2 hours, from about 2 hours to about 4 hours, from about 4 hours to about 8 hours, from about 8 hours to about 12 hours, from about 12 hours to about 16 hours, from about 16 hours to about 24 hours, from about 24 hours to about 36 hours, or from about 36 hours to about 48 hours. The total amount of irradiation and the duration of irradiation can be adjusted, depending on various factors, e. g. , the number of bacteria being irradiated. The total amount of irradiation and the duration of irradiation that results in bacteria that have reduced viability or are non-viable (e. g. , are unable to grow in in vitro culture) are readily determined by those of ordinary skill in the art.
[0052] In other embodiments, the radiation is ultraviolet (UV) radiation. For example, the probiotic bacteria are exposed to UV radiation of from about 2000 wu sec/cm2 to about 1,000 , uW sec/cm2.
[0053] In some embodiments, the probiotic bacteria are inactivated by pasteurization. The process of pasteurization is well known in the art of food sciences. Any method for pasteurization can be used for the current invention. Pasteurization generally involves heating the material to be pasteurized at one of the following temperatures, for the following time period: at about 60 C for at least about 30 minutes ; at 72 C for at least about 15 seconds; at

88 C for at least about 1 second; at 90 C for at least about 0.5 second; at 94 C for about 0.1 second; at 96 C for about 0.05 second; or 100 C for about 0.01 second. Standard pasteurization conditions are found in the literature, e. g. , in U. S. Patent Nos. 6,475, 545,

4,438, 147, and 6,528, 085. For example, in the present invention, pasteurization of liquids or solids comprising a suitable probiotic bacterium is carried out by heating the liquid or solid under conventional pasteurization conditions such as, for example, but not limited to, about

72 C to about 85 C for from about 15 seconds to about 10 minutes, e. g. , about 72 C to about 85 C for from about 15 seconds to about 30 seconds, from about 20 seconds to about 40 seconds, from about 30 seconds to about 60 seconds, from about 1 minute to about 2 minutes, from about 2 minutes to about 5 minutes, or from about 5 minutes to about 10 minutes.


Generally, temperatures above 90 C are not used to inactivate bacteria in the present invention.
[0054] Viability is reduced by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%, or more, such that fewer than about 50%, fewer than about 40%, fewer than about 30%, fewer than about

20%, fewer than about 10%, fewer than about 5%, or fewer than about 1%, or fewer, of the bacteria in the formulation are viable. In some embodiments, 100% of the bacteria are non- viable, e. g. , are unable to grow in in vitro culture.


[0055] Viability of bacteria is determined using any known method. For example, bacteria are contacted with a membrane-permeant fluorescent dye (e. g., SYTO 9, SYTOX, and the like) that labels live bacteria with green fluorescence; and membrane-impermeant propidium iodide that labels membrane-compromised bacteria with red fluorescence. Roth et al. (1997) Appl.
Environ. Microbiol. 63: 2421-2431 ; Lebaron et al. (1998) Appl. Environ. Microbiol. 64: 2697-

2700; and Braga et al. (2003) Antimicrob. Agents Chemother. 47: 408-412. Bacterial viability is also determined by plating the bacteria on an agar plate containing requisite nutritional supplements, and counting the number of colonies formed (colony forming units, cfu).


[0056] Inactivated probiotic bacteria of the invention are stable at temperatures from about 10 C to about 80 C, from about 15 C to about 75 C, from about 20 C to about 70 C, from about 25 C to about 65 C, from about 30 C to about 60 C, or from about 35 C to about 55 C.
For example, inactivated probiotic bacteria of the invention are stable at temperatures from about 10 C to about 60 C, from about 20 C to about 60 C, or from about 30 C to about 60 C.
In many embodiments, the inactivated probiotic bacteria are storage stable for a period of weeks, months, or years at the indicated temperature ranges.
[0057l A subject formulation comprises from about 5% to about 90%, from about 10% to about 85%, from about 15% to about 80%, from about 20% to about 75%, from about 25% to about 70%, from about 30% to about 65%, or from about 35% to about 60%, by weight or by volume, inactivated probiotic bacteria.
[0058] Formulations according to the present invention are prepared so that a liquid unit form contains from about 1 x 105 to about 1 x 1014, from about 5 x 105 to about 5 x 1013, from about

1 x 106 to about 1 x 1012, from about 5 x 106 to about 5 x 1011, or from about 1 x 107 to about 1 x 101 bacteria per unit dosage form, e. g. , per ml, per gram, per tablet, per capsule, per packet,


per serving size, etc. Formulations according to the present invention are prepared so that a solid, semi-solid, or gel unit form contains from about 1 x 105 to about 1 x 1014, from about 5 x

105 to about 5 x 1013, from about 1 x 106 to about 1 x 1012, from about 5 x 106 to about 5 x 1011, or from about 1 x 107 to about 1 x 101 bacteria per unit dosage form, e. g. , per gram, per tablet, per packet, per capsule, per serving size, etc.
[0059] The following are non-limiting examples of unit dosage forms of a subject formulation:

1-5 x 101 inactivated bacteria per packet, tablet, or capsule; 1-5 x 1011 inactivated bacteria per packet, tablet, or capsule; 1-5 x 1012 inactivated bacteria per packet, tablet, or capsule; 1-5 x 1013 inactivated bacteria per packet, tablet, or capsule; 1-5 x 1014 inactivated bacteria per packet, tablet, or capsule; 1-5 x 101 inactivated bacteria per ml liquid formulation; 1-5 x 1011 inactivated bacteria per ml liquid formulation; 1-5 x 1012 inactivated bacteria per ml liquid formulation; 1-5 x 10l3 inactivated bacteria per ml liquid formulation; 1-5 x 1014 inactivated bacteria per ml liquid formulation. The unit dosage forms can be packaged in multiples, e. g., the formulation is provided in a package of 4, 8, 12,16, or 20 unit dosage forms.


[0060] The term"unit dosage form, "as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of inactivated probiotic bacteria of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular bacteria or combination of bacteria employed and the effect to be achieved. The instant formulations are typically provided in unit dosage forms. In such form, the formulation is subdivided into unit doses containing appropriate quantities of the inactivated probiotic bacteria. The unit dosage form can be a packaged preparation, the package containing discrete quantities of formulation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, suppository, or lozenge itself, or it can be the appropriate number of any of these in packaged form. A"unit dosage form"may be in the form of a table or capsule; a unit amount of a liquid or gel formulation; or, where the formulation is in the form of a food product or a nutraceutical, a serving size.
Formulations [0061] In general, inactivated probiotic bacteria are formulated in a pharmaceutically acceptable composition for delivery to a host. In some embodiments, inactivated probiotic bacteria are formulated with a pharmaceutically acceptable carrier suitable for a solid or semi-

solid formulation. In some embodiments, inactivated probiotic bacteria are formulated with a pharmaceutically acceptable carrier suitable for a liquid or gel formulation.


[0062] Inactivated probiotic bacteria formulations of the invention are typically formulated for enteral delivery, e. g. , oral delivery, or delivery as a suppository, but can also be formulated for parenteral delivery, e. g. , vaginal delivery, inhalational delivery (including oral delivery, nasal delivery, and intrapulmonary delivery), and the like.
[0063] The inactivated probiotic bacteria of the present invention may be formulated in a wide variety of oral administration dosage forms, with one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. In powders, the carrier is a finely divided solid which is a mixture with the inactivated probiotic bacteria. In tablets, the inactivated bacteria are mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term"preparation"is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the inactivated probiotic bacteria, with or without carriers, is surrounded by a carrier, which is in association with it.
Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be as solid forms suitable for oral administration.
[0064] Other forms suitable for oral administration include liquid form preparations such as emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, or solid form preparations which are intended to be converted shortly before use to liquid form preparations. Emulsions may be prepared in solutions in aqueous propylene glycol solutions or may contain emulsifying agents such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by mixing the inactivated probiotic bacteria with water and adding suitable colorants, flavors, stabilizing and thickening agents. Aqueous suspensions can be prepared by dispersing the inactivated probiotic bacteria in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well known suspending agents. Solid form preparations include solutions, suspensions, and emulsions, and
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