Acetobacter and gluconacetobacter strains and their metabolites for use in inhibiting xanthine oxidase

Abstract

A method for inhibiting xanthine oxidase and for reducing uric acid levels using a pharmaceutical composition or a food product obtained by culturing Gluconacetobacter hansenii or Acetobacter pasteurianus in a medium. Also disclosed is a pharmaceutical composition and a food product that each include a metabolite of Gluconacetobacter hansenii or Acetobacter pasteurianus for reducing uric acid levels in a subject and methods for producing the pharmaceutical composition and the food product.

Claims

1. A pharmaceutical composition for reducing uric acid levels in a subject, the composition comprising dextrin and a culture of an acetic acid bacteria in a medium that contains glucose, soy peptone, and yeast extract, wherein the acetic acid bacteria is Acetobacter pasteurianus strain AHU01, deposited under Accession No. DSM 28893, and the pharmaceutical composition is in lyophilized form.

2. The composition of claim 1, further comprising at least one microorganism selected from the group consisting of Lactobacillus spp., Bifidobacterium spp., and Saccharomyces spp.

3. The composition of claim 2, wherein the at least one microorganism is Lactobacillus fermentum, Lactobacillus pentosus, Lactobacillus gasseri, Lactobacillus oris, Bifidobacterium longum, or Saccharomyces cerevisiae.

4. A food product for reducing uric acid levels in a subject, the food product comprising in lyophilized form an acetic acid bacteria and dextrin, wherein the acetic acid bacteria is Acetobacter pasteurianus strain AHU01, deposited under Accession No. DSM 28893, and the food product is a vinegar, a health drink, a yogurt, a beverage, an ice cream, sour milk, or a cheese.

5. The food product of claim 4, further comprising a food ingredient.

6. The food product of claim 5, wherein the food ingredient is one or more of a colorant, an acidity regulator, an anticaking agent, an antioxidant, a bulking agent, a carrier, an emulsifier, a flavor enhancer, a glazing agent, a preservative, a stabilizer, a sweetener, a thickener, a nutrient additive, and a flavoring agent.

7. The food product of claim 6, further comprising at least one microorganism selected from the group consisting of Lactobacillus spp., Bifidobacterium spp., and Saccharomyces spp.

8. The food product of claim 7, wherein the at least one microorganism is Lactobacillus fermentum, Lactobacillus pentosus, Lactobacillus gasseri, Lactobacillus oris, Bifidobacterium longum, and Saccharomyces cerevisiae.

9. The food product of claim 4, further comprising at least one microorganism selected from the group consisting of Lactobacillus spp., Bifidobacterium spp., and Saccharomyces spp.

10. The food product of claim 9, wherein the at least one microorganism is Lactobacillus fermentum, Lactobacillus pentosus, Lactobacillus gasseri, Lactobacillus oris, Bifidobacterium longum, or Saccharomyces cerevisiae.

11. A method for reducing uric acid levels in a subject, the method comprising identifying a subject in need of reduced uric acid levels and administering the pharmaceutical composition of claim 1 to a subject in need thereof in an amount effective for reducing uric acid levels.

12. The method of claim 11, wherein the subject suffers from gout or hyperuricemia.

13. The method of claim 12, wherein the pharmaceutical composition further comprises at least one microorganism selected from the group consisting of Lactobacillus fermentum, Lactobacillus pentosus, Lactobacillus gasseri, Lactobacillus oris, Bifidobacterium longum, and Saccharomyces cerevisiae.

14. A method for reducing uric acid levels in a subject, the method comprising identifying a subject in need of reduced uric acid levels and administering the food product of claim 4 to a subject in need thereof in an amount effective for reducing uric acid levels.

15. The method of claim 14, wherein the food product further comprises at least one microorganism selected from the group consisting of Lactobacillus fermentum, Lactobacillus pentosus, Lactobacillus gasseri, Lactobacillus oris, Bifidobacterium longum, and Saccharomyces cerevisiae.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention description below refers to the accompanying drawings, of which:

(2) FIG. 1 is a bar graph showing xanthine oxidase inhibitory activity of acetic acid bacteria strains;

(3) FIG. 2 is a bar graph showing xanthine oxidase inhibitory activity of Acetobacter pasteurianus strain AHU02 grown in different media; and

(4) FIG. 3 is a bar graph showing xanthine oxidase inhibitory activity of Acetobacter pasteurianus strain AHU02 grown in different volumes of media for specific periods of time.

DETAILED DESCRIPTION

(5) As set forth above, a method for reducing uric acid levels in a subject is disclosed that includes a step of culturing the acetic acid bacteria Gluconacetobacter hansenii or Acetobacter pasteurianus in a medium to form a composition. The acetic acid bacteria can be selected from Acetobacter pasteurianus strains AHU01 and AHU02, deposited under Accession Nos. DSM 28893 and DSM 28894, respectively. Alternatively, the Acetobacter pasteurianus strains can be strains AHU03 and AHU04. In a particular embodiment, the acetic acid bacteria is Gluconacetobacter hansenii strain AHU06, deposited under Accession No. DSM 28902.

(6) The culturing step is carried out in a medium. The medium can be, but is not limited to, M1A broth, a rice extract, a sorghum extract, grape juice, and plum juice. The medium is free of apple juice. In a particular embodiment, the method includes a step of removing the acetic acid bacteria from the medium after culturing and prior to administering the composition.

(7) The composition can be a vinegar or a health drink. In a specific embodiment, the method includes a step of lyophilizing the composition to form a powder.

(8) In an embodiment, the composition is administered orally to the subject. In a specific embodiment, the subject suffers from gout or hyperuricemia.

(9) The amount of the composition administered is effective for reducing uric acid levels in the subject. A skilled artisan can easily determine the effective amount by, e.g., measuring changes in the concentration of uric acid in the blood of the subject.

(10) A method for inhibiting xanthine oxidase is also provided. The method, as mentioned above, requires culturing an acetic acid bacteria in a medium to form a composition. The acetic acid bacteria can be Gluconacetobacter hansenii or Acetobacter pasteurianus. In an embodiment, the acetic acid bacteria is selected from Acetobacter pasteurianus strains AHU01, AHU02, AHU03, and AHU04. In another embodiment, the acetic acid bacteria is Gluconacetobacter hansenii strain AHU06.

(11) As set forth above, the culturing step is carried out in a medium. The medium can be, but is not limited to, M1A broth, a rice extract, a sorghum extract, grape juice, and plum juice. The medium is free of apple juice. In a particular embodiment, the method includes a step of removing the acetic acid bacteria from the medium after culturing and prior to contacting the composition with the xanthine oxidase.

(12) In one embodiment, the contacting step can be performed in vitro. For example, a preparation of xanthine oxidase can be placed in a vessel together with the composition. In another embodiment, the contacting step is accomplished by administering the composition orally to a subject having xanthine oxidase.

(13) The method set forth above for producing a composition for reducing uric acid levels in a subject includes, among others, a step of inoculating a medium with an acetic acid bacteria. The acetic acid bacteria is Gluconacetobacter hansenii or Acetobacter pasteurianus. In one embodiment, the acetic acid bacteria is selected from Acetobacter pasteurianus strains AHU01, AHU02, AHU03, and AHU04. In a specific embodiment, the acetic acid bacteria is Gluconacetobacter hansenii strain AHU06.

(14) The method also includes a step of culturing the acetic acid bacteria in the medium to form the composition. The medium can be, but is not limited to, M1A broth, a rice extract, a sorghum extract, grape juice, and plum juice. The medium is free of apple juice.

(15) In a particular embodiment, the method includes a step of removing the acetic acid bacteria from the medium after culturing and prior to administering the composition. In a preferred embodiment, the culture density of the acetic acid bacteria prior to the removing step is 110.sup.7 to 110.sup.8 cells/ml.

(16) The composition obtained by culturing the acetic acid bacteria in a medium can be sterilized by methods including but not limited to pasteurization, irradiation, autoclave, and filtration. For example, the composition can be sterilized by filtration through a 0.2 m filter. In a particularly preferred embodiment, the sterilized liquid broth is first filtered or centrifuged to remove the bacteria and then concentrated.

(17) The composition thus formed can be a food product such as a vinegar or a health drink. In further embodiments, the composition can be a yogurt, a beverage, an ice cream, sour milk, a biozyme (an enzyme mixture extracted from fermented fruits or vegetables), or a cheese.

(18) In a specific embodiment, the method includes a step of lyophilizing the composition to form a powder.

(19) A composition for reducing uric acid levels in a subject is disclosed which contains a metabolite of Gluconacetobacter hansenii or Acetobacter pasteurianus. As mentioned above the acetic acid bacteria can be selected from Acetobacter pasteurianus strains AHU01, AHU02, AHU03, and AHU04. In one embodiment, the acetic acid bacteria is Gluconacetobacter hansenii strain AHU06. The composition can be in powder form.

(20) The compositions described above can also contain one or more food ingredients, e.g., a colorant, an acidity regulator, an anticaking agent, an antioxidant, a bulking agent, a carrier, an emulsifier, a flavor enhancer, a glazing agent, a preservative, a stabilizer, a sweetener, a thickener, a nutrient additive, and a flavoring agent.

(21) In a particular embodiment, the composition includes a pharmaceutically acceptable carrier or excipient.

(22) The term carrier or excipient as used herein refers to any substance, not itself a therapeutic agent, used as a carrier, diluent, adjuvant, or vehicle (i) for delivery of a therapeutic agent to a subject, (ii) for adding to a formulation to improve its handling or storage properties, and/or (iii) to facilitate formation of a dosage unit of the composition into a discrete article such as a capsule or tablet suitable for oral administration.

(23) Suitable carriers or excipients are well known in the art of manufacturing pharmaceutical formulations or food products. Carriers or excipients can include, by way of illustration and not limitation, buffers, diluents, disintegrants, binding agents, adhesives, wetting agents, polymers, lubricants, glidants, substances added to mask or counteract a disagreeable taste or odor, flavors, dyes, fragrances, and substances added to improve appearance of the composition.

(24) Acceptable carriers or excipients include citrate buffer, phosphate buffer, acetate buffer, bicarbonate buffer, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, magnesium carbonate, talc, gelatin, acacia gum, sodium alginate, pectin, dextrin, mannitol, sorbitol, lactose, sucrose, starches, cellulosic materials (e.g., cellulose esters of alkanoic acids and cellulose alkyl esters), low melting wax cocoa butter, amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), ethylenediamine tetraacetic acid (EDTA), dimethyl sulfoxide (DMSO), sodium chloride or other salts, liposomes, mannitol, sorbitol, glycerol or powder, polymers (e.g., polyvinyl-pyrrolidone, polyvinyl alcohol, and polyethylene glycols), and other pharmaceutically acceptable materials. The carrier does not destroy the pharmacological activity of the therapeutic agent and is non-toxic when administered in doses sufficient to deliver a therapeutic amount of the agent.

(25) In another embodiment, the composition can include, in addition to the metabolite of the acetic acid bacteria, probiotic microorganisms including but not limited to Lactobacillus spp., Bifidobacterium spp., and Saccharomyces spp. For example, one or more of Lactobacillus fermentum, Lactobacillus pentosus, Lactobacillus gasseri, Lactobacillus oris, Bifidobacterium longum, and Saccharomyces cerevisiae can be included in the composition. In a particular aspect, the composition contains one or more of the above-mentioned probiotic microorganisms and a metabolite of an acetic acid bacteria selected from Acetobacter pasteurianus strains AHU01, AHU02, AHU03, AHU04, and Gluconacetobacter hansenii strain AHU06.

(26) Without further elaboration, it is believed that one skilled in the art can, based on the disclosure herein, utilize the present invention to its fullest extent.

(27) The following specific examples are, therefore, to be construed as merely descriptive, and not limitative of the remainder of the disclosure in any way whatsoever.

EXAMPLES

Example 1: Acetic Acid Bacteria Produce a Xanthine Oxidase Inhibitory Activity

(28) Fifty-one acetic acid bacteria strains were separately inoculated onto M1A plates (2.5% mannitol, 0.5% yeast extract, 0.3% peptone, and 2% agar) and the plates incubated for 2 days at 30 C. to form colonies.

(29) Xanthine oxidase inhibitory activity was measured as follows. First, 10 l of each strain was scraped from the M1A plate and added to a well in a 96 well plate. Then 150 l of 50 mM phosphate-buffered saline (PBS) and 80 l of 150 M xanthine was added to each well. An initial absorbance value at 290 nm (OD.sub.before) was determined before adding 10 l of xanthine oxidase (0.1 U) into each well. After incubating the plate at 25 C. for 30 min., the absorbance value was measured again at 290 nm (OD.sub.after). The xanthine oxidase inhibitory activity of each sample was calculated according to the following formula:

(30) $\mathrm{XOI}\left(\%\right)=\frac{100\left[1-\left({\mathrm{OD}}_{\mathrm{after}}-{\mathrm{OD}}_{\mathrm{before}}\right)\right]}{\left(\mathrm{Blank}{\mathrm{OD}}_{\mathrm{after}}-\mathrm{Blank}{\mathrm{OD}}_{\mathrm{before}}\right)}$

(31) The results are shown in FIG. 1. Among the 51 distinct acetic acid bacterial strain examined, only seven strains inhibited xanthine oxidase by more than 30%. In particular, Acetobacter pasteurianus strain AHU01 inhibited xanthine oxidase activity by 73.6%.

(32) Applicants deposited Acetobacter pasteurianus strains AHU01 and AHU02 on Jun. 5, 2014 under the terms of the Budapest Treaty with the International Strain Depositary Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture, Inhoffenstr. 7 B, D-38124 Braunschweig GERMANY. Acetobacter pasteurianus strains AHU01 and AHU02 were assigned Accession Nos. DSM 28893 and DSM 28894, respectively. Applicants also deposited on Jun. 5, 2014 Gluconacetobacter hansenii strain AHU06 in the above repository under Accession No. DSM 28902.

Example 2: Effect of Media on Acetic Acid Bacteria Xanthine Oxidase Inhibition

(33) Acetobacter pasteurianus strain AHU02 was inoculated onto M1A plates and cultured at 30 C. for 4 days. Each plate was washed with 7 ml of sterile M1A seed broth. The seed broth containing cells (1 ml) was inoculated into 50 ml of various media in a 250 ml triangular flask. The inoculated media were incubated at 30 C. with shaking at 125 rpm for 7 days. Samples of each media was assayed for xanthine oxidase inhibition as described above. The results are shown in FIG. 2.

(34) Acetobacter pasteurianus strain AHU02 produced the highest level of xanthine oxidase inhibitory activity, reaching 60% inhibition. By contrast, no inhibition of xanthine oxidase activity was detected after growing Acetobacter pasteurianus strain AHU02 in apple juice. Culturing Acetobacter pasteurianus strain AHU02 in sorghum, grape juice, rice extract and plum juice resulted in intermediate levels of inhibitory activity ranging from 15% to 50%.

Example 3: Effect of Culturing Time and Volume on Acetic Acid Bacteria Xanthine Oxidase Inhibition

(35) A seed broth containing Acetobacter pasteurianus strain AHU02 was prepared as described in Example 2 above. Seed broth was added at 2% v/v to 200, 300, and 400 ml of SPS medium (1% sucrose, 1% peptone, 1% soy peptone, and 0.2% sodium nitrate) in a 1 L triangular shaker flask and incubated with shaking at 125 rpm for 3-10 days at 30 C. Xanthine oxidase inhibition was measured as set forth in Example 1 supra. The results are shown in FIG. 3.

(36) Acetobacter pasteurianus strain AHU02 grown in a culture volume of 200 ml produced the highest level of xanthine oxidase inhibitory activity at each time point as compared to this strain grown in 300 ml or 400 ml of media. It is known that the smaller culture volume results in more efficient oxygenation of the media during culture. Without being bound by theory, it is likely that efficient production of xantine oxidase inhibitory activity by Acetobacter pasteurianus requires a high level of oxygen.

(37) The highest level of xanthine oxidase inhibitory activity was obtained after 3 days of culturing Acetobacter pasteurianus strain AHU02 in a 200 ml volume. This level decreased upon prolonged culturing, falling off by nearly 65% after 10 days of culture. A similar reduction in xanthine oxidase inhibitory activity over time was observed in the 300 ml and 400 ml cultures.

Example 4: Effect of Glucose Concentration on Production of Xanthine Oxidase Inhibitory Activity by Acetic Acid Bacteria

(38) A seed broth containing Acetobacter pasteurianus strain AHU01 was prepared as described in Example 2 above. In a 250 ml triangular flask, 0.5 ml of the seed broth was inoculated into 50 ml of media each containing a different concentration of glucose ranging from 8% to 16% (w/v). In addition to glucose, the media contained 1.5% soy peptone and 3% yeast extract. The cultures were incubated at 30 C. with shaking at 150 rpm for 7 days.

(39) Xanthine oxidase inhibitory activity was measured by HPLC by the following procedure. In a reaction tube, 880 l of xanthine (50 g/ml in 100 mM PBS) and 40 l of 50 mM PBS or 40 l of the culture supernatants were premixed, and 80 l of xanthine oxidase (0.1 U) was added to initiate the reaction. The reaction was incubated at 30 C. for 30 min., after which an equal volume of absolute ethanol was added to terminate the reaction. The terminated reaction was filtered through a 0.22 m membrane filter and the content of xanthine in the reactions was analyzed by HPLC. Xanthine oxidase inhibitory activity of the samples was calculated as follows:

(40) $\mathrm{XOI}\left(\%\right)=\frac{100{\left[\mathrm{xanthine}\right]}_{\mathrm{initial}}-{\left[\mathrm{xanthine}\right]}_{\mathrm{after}\mathrm{sample}}}{{\left[\mathrm{xanthine}\right]}_{\mathrm{initial}}-{\left[\mathrm{xanthine}\right]}_{\mathrm{after}\mathrm{control}}}$

(41) The results are shown in Table 1 as follows:

(42) TABLE-US-00001 TABLE 1 Inhibition of xanthine oxidase activity glucose concentration xanthine oxidase inhibition .sup.8.sup.a .sup.32.74.sup.b 10 41.97 12 55.84 16 68.12 .sup.avalues expressed as w/v % of glucose in the media. .sup.bvalues expressed as percentage inhibition of xanthine oxidase activity.

(43) A clear correlation exists between the glucose content of the growth media and the level of xanthine oxidase activity produced by Acetobacter pasteurianus grown in the media.

Example 5: Treatment of Experimental Uricemia

(44) Acetobacter pasteurianus strain AHU01 was inoculated onto an M1A plate and cultured for 2 days at 30 C. The plate was washed with 7 ml of sterile water as seed broth. 0.5 ml of the seed broth was inoculated into 50 ml of a custom media (1% soy peptone, 0.2% yeast extract, 3% glucose, 0.2% malt extract, and 3% fructose) in a 250 ml triangular flask and incubated with shaking at 150 rpm for 7 days at 30 C. The medium was then collected and centrifuged at 3000 rpm for 15 minutes. Following centrifugation, the supernatant was collected, lyophilized, and freeze-dried to form a solid fermentation product for use in animal experiments.

(45) ICR mice were used as experimental animals. Potassium oxonate, a uricase inhibitor, was used to induce a high level of uric acid in the serum of the mice. Mice were fasted for one hour and then fed saline or potassium oxonate (400 mg/kg) via a feeding tube. After one hour, potassium oxonate-treated mice were fed saline, allopurinol (10 mg/kg), or the Acetobacter pasteurianus strain AHU01 fermentation product (150 mg or 200 mg resuspended in saline per mouse) prepared as described above. Ten animals were used for each experimental group and for the control group. The animals were sacrificed after one hour and the level of uric acid in their serum was analyzed. The results are shown in Table 2 below.

(46) TABLE-US-00002 TABLE 2 A fermentation product of Acetobacter pasteurianus strain AHU01 can reduce serum uric acid levels in experimental animals. Experimental group.sup.a serum uric acid concentration saline control 3.51 0.02 mg/dL potassium oxonate (400 mg/kg) 4.91 0.08 mg/dL potassium oxonate + allopurinol 2.82 0.28 mg/dL (10 mg/kg) potassium oxonate + 150 mg fermentation 2.98 0.13 mg/dL product potassium oxonate + 200 mg fermentation 2.94 0.12 mg/dL product .sup.amice (N = 10 per condition) fed saline or the compounds indicated in a total volume of 200 l

Other Embodiments

(47) All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

(48) From the above description, a person skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the present invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.