U.S. patent application number 15/483575 was filed with the patent office on 2017-08-03 for novel strain of lactobacillus rhamnosus and its metabolites for use in inhibiting xanthine oxidase and treating gout.
The applicant listed for this patent is Food Industry Research and Development Institute. Invention is credited to Mei-Huei Chen, Siao-Jhen Chen, Yen-Lin Chen, Hsun-Yin Hsu, Shy-Yunn Wann, Li-Wen Yu.
Application Number | 20170216376 15/483575 |
Document ID | / |
Family ID | 55347337 |
Filed Date | 2017-08-03 |
United States Patent
Application |
20170216376 |
Kind Code |
A1 |
Chen; Siao-Jhen ; et
al. |
August 3, 2017 |
NOVEL STRAIN OF LACTOBACILLUS RHAMNOSUS AND ITS METABOLITES FOR USE
IN INHIBITING XANTHINE OXIDASE AND TREATING GOUT
Abstract
A method for inhibiting xanthine oxidase and for reducing uric
acid levels using a composition obtained by culturing Lactobacillus
rhamnosus in a medium. Also disclosed is a composition including a
metabolite of Lactobacillus rhamnosus for reducing uric acid levels
in a subject and a method for producing the composition.
Inventors: |
Chen; Siao-Jhen; (Tainan
City, TW) ; Chen; Yen-Lin; (Hsinchu City, TW)
; Hsu; Hsun-Yin; (Hsinchu City, TW) ; Wann;
Shy-Yunn; (Hsinchu City, TW) ; Chen; Mei-Huei;
(Hsinchu City, TW) ; Yu; Li-Wen; (Hsinchu City,
TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Food Industry Research and Development Institute |
Hsinchu City |
|
TW |
|
|
Family ID: |
55347337 |
Appl. No.: |
15/483575 |
Filed: |
April 10, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14823585 |
Aug 11, 2015 |
9636368 |
|
|
15483575 |
|
|
|
|
62040616 |
Aug 22, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23Y 2220/73 20130101;
C12Y 117/03002 20130101; C12N 1/02 20130101; C12P 1/04 20130101;
A61K 35/747 20130101; A61K 9/0053 20130101; A61K 9/0095 20130101;
C12N 1/20 20130101; A23L 33/135 20160801; A61K 35/745 20130101;
A23L 2/382 20130101; A61K 9/19 20130101; A23L 2/52 20130101; A61K
36/064 20130101; A23V 2002/00 20130101; C12N 2500/72 20130101; C12N
9/0093 20130101; A61P 19/06 20180101; C12R 1/225 20130101 |
International
Class: |
A61K 35/747 20060101
A61K035/747; A23L 33/135 20060101 A23L033/135; A61K 35/745 20060101
A61K035/745; A61K 36/064 20060101 A61K036/064; A61K 9/19 20060101
A61K009/19; A61K 9/00 20060101 A61K009/00 |
Claims
1. A composition for reducing uric acid levels in a subject, the
composition comprising a metabolite of Lactobacillus rhamnosus
mixed with a pharmaceutically acceptable carrier selected from the
group consisting of ethylenediamine tetraacetic acid, dimethyl
sulfoxide, polyvinyl-pyrrolidone, polyvinyl alcohol, polyethylene
glycol, and a combination thereof, wherein the metabolite is an
inhibitor of xanthine oxidase activity and the Lactobacillus
rhamnosus is Lactobacillus rhamnosus strain I21 deposited under
Accession No. DSM 28876.
2. The composition of claim 1, wherein the composition is free of
Lactobacillus rhamnosus.
3. The composition of claim 2, wherein the composition is
sterile.
4. The composition of claim 1, wherein the composition is
sterile.
5. The composition of claim 1, further comprising a probiotic
microorganism selected from the group consisting of Lactobacillus
spp., Bifidobacterium spp., Saccharomyces spp, and a combination
thereof.
6. The composition of claim 5, wherein the probiotic microorganism
is one or more of Lactobacillus fermentum, Lactobacillus pentosus,
Lactobacillus gasseri, Lactobacillus oris, Bifidobacterium longum,
and Saccharomyces cerevisiae.
7. The composition of claim 2, further comprising a probiotic
microorganism selected from the group consisting of Lactobacillus
spp., Bifidobacterium spp., Saccharomyces spp, and a combination
thereof.
8. The composition of claim 7, wherein the probiotic microorganism
is one or more of Lactobacillus fermentum, Lactobacillus pentosus,
Lactobacillus gasseri, Lactobacillus oris, Bifidobacterium longum,
and Saccharomyces cerevisiae.
9. The composition of claim 1, wherein the composition is a liquid
solution, a suspension, an emulsion, a syrup, a tablet, a pill, a
capsule, a sustained release formulation, a powder, a granule, an
ointment, a lotion, a liniment, or a cream.
10. The composition of claim 2, wherein the composition is a liquid
solution, a suspension, an emulsion, a syrup, a tablet, a pill, a
capsule, a sustained release formulation, a powder, a granule, an
ointment, a lotion, a liniment, or a cream.
11. A composition for reducing uric acid levels in a subject, the
composition comprising a culture of Lactobacillus rhamnosus mixed
with a pharmaceutically is acceptable carrier selected from the
group consisting of ethylenediamine tetraacetic acid, dimethyl
sulfoxide, polyvinyl-pyrrolidone, polyvinyl alcohol, polyethylene
glycol, and a combination thereof, wherein the composition is an
inhibitor of xanthine oxidase activity and the Lactobacillus
rhamnosus is Lactobacillus rhamnosus strain I21 deposited under
Accession No. DSM 28876.
12. The composition of claim 11, wherein the composition is a
liquid solution, a suspension, an emulsion, a syrup, a tablet, a
pill, a capsule, a sustained release formulation, a powder, a
granule, an ointment, a lotion, a liniment, or a cream.
13. The composition of claim 12, wherein the composition is
sterile.
14. The composition of claim 11, wherein the composition is
sterile.
15. The composition of claim 11, further comprising a probiotic
microorganism selected from the group consisting of Lactobacillus
spp., Bifidobacterium spp., Saccharomyces spp, and a combination
thereof.
16. The composition of claim 15, wherein the probiotic
microorganism is one or more of Lactobacillus fermentum,
Lactobacillus pentosus, Lactobacillus gasseri, Lactobacillus oris,
Bifidobacterium longum, and Saccharomyces cerevisiae.
17. The composition of claim 12, further comprising a probiotic io
microorganism selected from the group consisting of Lactobacillus
spp., Bifidobacterium spp., Saccharomyces spp, and a combination
thereof.
18. The composition of claim 17, wherein the probiotic
microorganism is one or more of Lactobacillus fermentum,
Lactobacillus pentosus, Lactobacillus gasseri, Lactobacillus oris,
Bifidobacterium longum, and Saccharomyces cerevisiae.
19. The composition of claim 11, wherein the culture of
Lactobacillus rhamnosus includes a culture medium selected from de
Man-Rogosa Sharpe broth, milk, and juice.
20. The composition of claim 11, further comprising one or more
food ingredient selected from the group consisting 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, a flavoring agent, and a
combination thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/823,585 filed on Aug. 11, 2015, which
claims the benefit of U.S. Provisional Application Ser. No.
62/040,616, which was filed on Aug. 22, 2014. The content of both
prior applications is hereby incorporated by reference in its
entirety.
BACKGROUND
[0002] Field of the Invention
[0003] The invention relates to inhibition of xanthine oxidase
activity by lactic acid bacteria and their fermentation
metabolites.
[0004] Background Information
[0005] Uric acid is the end product of purine metabolism in the
body. A high level of uric acid in the blood leads to the formation
and deposition of uric acid crystals in the joints, kidneys, and
other organs. A blood uric acid concentration higher than 7 mg/dL
is considered to be hyperuricemia.
[0006] Hyperuricemia is a common metabolic disorder that is
associated with gout, hypertension, cardiovascular disease,
diabetes, and kidney disease. An epidemiological survey performed
in Taiwan from 1993 to 2008 indicated that the percentage of male
and female patients demonstrating hyperuricemia was 21.6% and
9.57%, respectively.
[0007] Xanthine oxidase is a key enzyme in the synthesis of uric
acid. As a result, inhibition of xanthine oxidase activity can
reduce the production of uric acid. Indeed, the xanthine oxidase
inhibitor, uricase, is effective for lowering the concentration of
uric acid in the blood. Uricase is an enzyme not found in humans.
It is typically isolated as a recombinant mammalian protein and
administered by IV infusion. As such, it can be expensive to
produce and difficult to administer.
[0008] Allopurinol is also a xanthine oxidase inhibitor. This
compound is administered clinically to lower serum uric acid
levels. However, allopurinol has side effects, such as allergic
reactions, gastrointestinal discomfort, leukopenia and
thrombocytopenia, hepatitis, nephropathy, and 6-mercaptopurine
toxicity, which in certain cases can lead to death.
[0009] In view of the drawbacks of existing therapies for
hyperuricemia, many biopharmaceutical companies focused on the
development of new uric acid-lowering agents. For example, Izumida
et al., J. Antibiotics 50:916-918, isolated a compound that can
lower uric acid levels, namely, hydroxyakalone, from the marine
bacterium Agrobacterium aurantiacum.
[0010] Other microbial species have also been shown to possess
uric-acid lowering capability, including strains of Lactobacillus
fermentum, Lactobacillus pentosus, Lactobacillus gasseri,
Lactobacillus oris, Bifidobacterium longum, and Saccharomyces
cerevisiae. See, e.g., US Patent Application Publications
2010/0316618, 2011/0014168, is and 2013/0330299; and European
Patent Application Publications 2457576 and 1649863.
[0011] The need still exists to develop new xanthine oxidase
inhibitors from natural sources which can be easily produced and
safely administered.
SUMMARY
[0012] To meet this need, inhibitors of xanthine oxidase produced
by lactic acid bacteria are provided.
[0013] A method for inhibiting xanthine oxidase is also provided.
The method includes the steps of culturing Lactobacillus rhamnosus
in a medium to form a composition and contacting the xanthine
oxidase with the composition.
[0014] Also disclosed is a method for reducing uric acid levels in
a subject. The method includes culturing Lactobacillus rhamnosus in
a medium to form a composition and administering the composition to
a subject having elevated uric acid levels. The amount administered
is effective for reducing uric acid levels.
[0015] Within the scope of the invention is a method for producing
a composition for reducing uric acid levels in a subject. The
method is carried out by inoculating a medium with Lactobacillus
rhamnosus and culturing the Lactobacillus rhamnosus in the medium
to form a composition.
[0016] Further disclosed is a composition for reducing uric acid
levels in a subject. The composition includes a metabolite of
Lactobacillus rhamnosus.
[0017] The details of one or more embodiments of the invention are
set forth in the description and the examples below. Other
features, objects, and advantages of the invention will be apparent
from the detailed description of several embodiments and also from
the claims. All publications and patent documents cited herein are
incorporated by reference in their entirety.
DETAILED DESCRIPTION
[0018] As mentioned above, a method is disclosed for reducing uric
acid levels in a subject by administering a composition containing
Lactobacillus rhamnosus. In a particular embodiment, the
Lactobacillus rhamnosus is Lactobacillus rhamnosus I21 deposited
under Accession No. DSM 28876.
[0019] In one embodiment, the subject is hyperuricemic. In another
embodiment, the subject suffers from gout.
[0020] The method, as set out supra, includes a step of forming a
composition by culturing Lactobacillus rhamnosus in a medium. The
medium can be, but is not limited to, de Man-Rogosa-Sharpe (MRS)
broth, milk, and juice. In specific embodiments, the medium is
grape juice, mango juice, or orange juice. In a particular
embodiment, the method includes a step of removing the
Lactobacillus rhamnosus from the medium after culturing and prior
to administering the composition.
[0021] The composition described above can be administered
topically or systemically by routes including, but not limited to,
intramuscular, intradermal, intravenous, subcutaneous,
intraperitoneal, intranasal, oral, mucosal, and external.
[0022] Depending upon the route of administration, the composition
can be formulated in various ways. For example, the composition can
be a liquid solution, a suspension, an emulsion, a syrup, a tablet,
a pill, a capsule, a sustained release formulation, a powder, a
granule, an ampoule, an injection, an infusion, a kit, an ointment,
a lotion, a liniment, a cream, or a combination thereof. The
composition can be sterilized or mixed with a pharmaceutically
acceptable carrier or excipient.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] The method for inhibiting xanthine oxidase described above
includes the step culturing Lactobacillus rhamnosus in a medium to
form a composition. In a preferred embodiment, the Lactobacillus
rhamnosus is Lactobacillus rhamnosus I21 deposited under Accession
No. DSM 28876.
[0028] The medium can be, but is not limited to, de
Man-Rogosa-Sharpe (MRS) broth, milk, and juice. In specific
embodiments, the medium is grape juice, mango juice, or orange
juice. In a particular embodiment, the method includes a step of
lyophilizing the composition to form a powder.
[0029] The method for inhibiting xanthine oxidase also includes a
step of contacting the xanthine oxidase with the composition
described above. 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 an
embodiment, the contacting step is performed by administering the
composition orally to a subject having xanthine oxidase.
[0030] Summarized above is a method for producing a composition for
reducing uric acid levels in a subject. The composition is produced
by first inoculating a medium with Lactobacillus rhamnosus. In a
specific embodiment, the Lactobacillus rhamnosus is Lactobacillus
rhamnosus I21 deposited under Accession No. DSM 28876.
[0031] The medium for inoculating the Lactobacillus rhamnosus can
be, but is not limited to, de Man-Rogosa-Sharpe (MRS) broth, milk,
and juice. In certain embodiments, the medium is grape juice, mango
juice, or orange juice.
[0032] After inoculating the medium with Lactobacillus rhamnosus,
the inoculated media is subjected to culturing, thereby forming the
composition for reducing uric acid levels in a subject. The
culturing step can be carried out at 37.degree. C. Additionally,
the culturing step can be carried out under facultative anaerobic
conditions. In an embodiment, the culturing is performed for 2
days.
[0033] The composition obtained by culturing Lactobacillus
rhamnosus 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 .mu.m filter. In a particularly preferred
embodiment, the sterilized liquid broth is first filtered or
centrifuged to remove the bacteria and then concentrated.
[0034] The method for producing a composition for reducing uric
acid levels in a subject can include a step of removing the
Lactobacillus rhamnosus from the composition. The Lactobacillus
rhamnosus can have a cell density of 1.times.10.sup.8 to
1.times.10.sup.9 cells/ml prior to the removing step. In a
preferred embodiment, the Lactobacillus rhamnosus cell density
prior to removing them is 1.times.10.sup.9 cells/ml.
[0035] In another embodiment, the method includes a step of
lyophilizing the composition to form a powder.
[0036] The composition described above for reducing uric acid
levels in a subject includes a metabolite of Lactobacillus
rhamnosus. The metabolite is an inhibitor of xanthine oxidase
activity. In a particular embodiment, the metabolite can be a
metabolite of Lactobacillus rhamnosus strain I21 deposited under
Accession No. DSM 28876.
[0037] In an embodiment, the composition can be a Lactobacillus
rhamnosus powder. In an alternative embodiment, the composition is
free of Lactobacillus rhamnosus.
[0038] In another embodiment, the composition can include, in
addition to the metabolite is of Lactobacillus rhamnosus, 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.
[0039] The composition 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.
[0040] In yet another embodiment, as mentioned above, the
composition includes a pharmaceutically acceptable excipient.
[0041] The composition can also be a food product. For example, the
composition can be a yogurt, a beverage, an ice cream, or a
cheese.
[0042] 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. 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
Lactic Acid Bacteria Produce a Xanthine Oxidase Inhibitory
Activity
[0043] Thirty four lactic acid bacteria strains were separately
inoculated onto De Man, Rogosa, and Sharpe (MRS) plates and
cultured at 37.degree. C. for 3 days. Bacteria strains were
isolated from healthy infant feces, bovine feces, milk solids,
bacon, fermented bean curd, botanical garden soil, pickles, and
sauerkraut. Bacteria were scraped from each plate using a 1 .mu.l
sterile inoculation loop, inoculated into 10 ml of MRS broth, and
incubated at 37.degree. C. under facultative anaerobic conditions
for 1 day to prepare an inoculum. The inoculum was then added to
MRS broth at 1% (v/v) and incubated for 1 day at 37.degree. C.
under facultative anaerobic conditions. The culture medium was
centrifuged and the supernatant collected for the analysis of
xanthine oxidase inhibitory activity.
[0044] Xanthine oxidase inhibitory activity was measured as
follows. First, 10 .mu.l of culture media from each strain was
added to a well in a 96-well plate. Then, 150 .mu.l of 50 mM
phosphate-buffered saline (PBS) and 80 .mu.l of 150 .mu.M xanthine
was added to each well. An initial absorbance value at 290 nm
(OD.sub.before) was determined before adding 10 .mu.l of xanthine
oxidase (0.1 U) into each well. After incubating the plate at
XOI ( % ) = 100 .times. [ 1 - ( OD after - OD before ) ] ( Blank OD
after - Blank OD before ) ##EQU00001##
25.degree. C. for 30 min., the absorbance value was measured again
at 290 nm (OD.sub.after). The xanthine oxidase inhibitory activity
(XOI) of each sample was calculated according to the following
formula:
[0045] The results are shown in Table 1 below. Among the 34 lactic
acid bacteria strains tested, two strains, namely, strains I21 and
F73 (shown in italics), inhibited xanthine oxidase activity more
than 40%.
TABLE-US-00001 TABLE 1 Xanthine oxidase inhibitory activity of
lactic acid bacteria strains strain E021 E027 E032 E100 E103 E106
E108 E109 E111 E112 % inh. .sup. 8.0 .sup.a 20.6 17.4 7.2 26.2 25.7
30.0 8.7 27.8 32.3 strain I01 I02 I03 I04 I07 I08 I10 I11 I15 I16 %
inh. 17.6 10.6 7.0 1.4 33.6 20.3 29.4 2.6 13.9 2.1 strain I18 I21
I28 I29 I30 I32 S10-V1 S16-6 S16-9 S16-10 % inh. 4.4 44.5 18.0 7.3
3.1 2.5 24.3 26.2 32.3 22.8 strain S17-2 F73 13-2 En3721 % inh.
29.7 68.1 24.7 18.6 .sup.a values are expressed as percentage
inhibition of xanthine oxidase activity
Example 2
HPLC Analysis of Xanthine Oxidase Activity Inhibition
[0046] Lactic acid bacteria strains F73 and I21 were inoculated
onto MRS plates and cultured at 37.degree. C. for 3 days. The
bacteria were scraped from the plate with a 1 .mu.l sterile
inoculation loop, inoculated into MRS broth, and incubated at
37.degree. C. for 1 day to prepare an inoculum. The inoculum was
then added to MRS broth and incubated at 37.degree. C. for up to 7
days. Samples were removed from the culture at day 1, day 2 and day
7, centrifuged, and the supernatant collected for the analysis of
xanthine oxidase inhibitory activity.
[0047] In a reaction tube, 880 .mu.l of xanthine (50 .mu.g/ml in
100 mM PBS) and 40 .mu.l of 50 mM PBS or 40 .mu.l of the culture
supernatants were premixed, and 80 .mu.l of xanthine oxidase (0.1
U) was added to initiate the reaction. The reaction was incubated
at 30.degree. 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 0.25 .mu.m membrane
filter and the content of xanthine was analyzed by HPLC. Xanthine
oxidase inhibitory activity of the samples was calculated as
follows:
XOI ( % ) = 100 .times. [ xanthine ] initial - [ xanthine ] after
sample [ xanthine ] initial - [ xanthine ] after control
##EQU00002##
[0048] The results are shown in Table 2 below.
TABLE-US-00002 TABLE 2 Inhibition of xanthine oxidase activity
strain 1 day .sup.a 2 days 7 days I21 26.74 .sup.b 27.39 24.63 F73
14.95 17.23 23.55 .sup.a number of days in culture at which sample
was removed .sup.b values expressed as percentage inhibition of
xanthine oxidase activity
[0049] The results demonstrated that xanthine oxidase inhibitory
activity of lactic acid bacteria strain I21 is higher than strain
F73. Notably, the xanthine oxidase inhibitory activity of strain
I21 reached a maximum after 1 day of fermentation. Prolonged
culturing of strain I21 for up to 7 days did not result in an
increase of xanthine oxidase inhibitory activity.
Example 3
Identification of Lactic Acid Bacteria Strain I21
[0050] Lactic acid bacteria strain I21 was isolated from the feces
of a healthy infant. An analysis of this strain revealed that it
was Gram-positive, catalase and oxidase negative, and non-motile.
Additionally, the strain did not produce endospores and did grow
under both aerobic and facultative anaerobic conditions.
[0051] The sequence of 16S rDNA from strain I21 (SEQ ID NO: 1) was
analyzed and determined to be most similar to Lactobacillus casei,
Lactobacillus paracasei subsp. paracasei, Lactobacillus paracasei
subsp. tolerans, Lactobacillus rhamnosus, and Lactobacillus zeae.
The 16s rDNA sequence similarity is as high as 98%.
[0052] An analysis of a partial sequence of the DnaK gene (SEQ ID
NO: 2) revealed that strain I21 shares 99% sequence identity to
Lactobacillus rhamnosus.
[0053] Strain I21 was also characterized with respect to the
ability to ferment certain carbohydrates using the analytical
profile index API.RTM. identification system. This test revealed
that strain I21 is a strain of Lactobacillus rhamnosus.
[0054] Applicants deposited Lactobacillus rhamnosus strain I21 on
Jun. 2, 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. The strain was assigned Accession No.
DSM 28876.
Example 4
Treatment of Experimental Uricemia
[0055] Lactobacillus rhamnosus I21 was inoculated on an MRS plate
and cultured at 37.degree. C. for 3 days. Bacteria were scraped
from the plate using a 1 .mu.l sterile inoculation loop, inoculated
into MRS broth, and grown at 37.degree. C. for 1 day to prepare an
inoculum. The inoculum (30 ml) was then added into 3 L MRS broth in
a 5 L fermenter and grown at 37.degree. C. for 2 days. The
fermentation broth was centrifuged at 3000 rpm for 15 min. The
supernatant was collected and lyophilized to produce the
Lactobacillus rhamnosus I21 fermentation product.
[0056] 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 a Lactobacillus rhamnosus I21
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 3 below.
TABLE-US-00003 TABLE 3 A fermentation product of Lactobacillus
rhamnosus I21 can reduce serum uric acid levels in experimental
animals. serum uric acid Experimental group .sup.a concentration
saline control 3.51 .+-. 0.02 mg/dL potassium oxonate (400 mg/kg)
4.91 .+-. 0.08 mg/dL potassium oxonate + allopurinol (10 mg/kg)
2.82 .+-. 0.28 mg/dL potassium oxonate + 150 mg fermentation
product 4.00 .+-. 0.49 mg/dL potassium oxonate + 200 mg
fermentation product 3.86 .+-. 0.13 mg/dL .sup.a mice (N = 10 per
condition) fed saline or the compounds indicated in a total volume
of 200 .mu.l
Other Embodiments
[0057] 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.
[0058] 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.
Sequence CWU 1
1
211537DNALactobacillus rhamnosusmisc_featurestrain I21 16s rDNA
1tcaggatgaa cgctggcggc gtgcctaata catgcaagtc gaacgagttc tgattattga
60aaggtgcttg catcttgatt taattttgaa cgagtggcgg acgggtgagt aacacgtggg
120taacctgccc ttaagtgggg gataacattt ggaaacagat gctaataccg
cataaatcca 180agaaccgcat ggttcttggc tgaaagatgg cgtaagctat
cgcttttgga tggacccgcg 240gcgtattagc tagttggtga ggtaacggct
caccaaggca atgatacgta gccgaactga 300gaggttgatc ggccacattg
ggactgagac acggcccaaa ctcctacggg aggcagcagt 360agggaatctt
ccacaatgga cgcaagtctg atggagcaac gccgcgtgag tgaagaaggc
420tttcgggtcg taaaactctg ttgttggaga agaatggtcg gcagagtaac
tgttgtcggc 480gtgacggtat ccaaccagaa agccacggct aactacgtgc
cagcagccgc ggtaatacgt 540aggtggcaag cgttatccgg atttattggg
cgtaaagcga gcgcaggcgg ttttttaagt 600ctgatgtgaa agccctcggc
ttaaccgagg aagtgcatcg gaaactggga aacttgagtg 660cagaagagga
cagtggaact ccatgtgtag cggtgaaatg cgtagatata tggaagaaca
720ccagtggcga aggcggctgt ctggtctgta actgacgctg aggctcgaaa
gcatgggtag 780cgaacaggat tagataccct ggtagtccat gccgtaaacg
atgaatgcta ggtgttggag 840ggtttccgcc cttcagtgcc gcagctaacg
cattaagcat tccgcctggg gagtacgacc 900gcaaggttga aactcaaagg
aattgacggg ggcccgcaca agcggtggag catgtggttt 960aattcgaagc
aacgcgaaga accttaccag gtcttgacat cttttgatca cctgagagat
1020caggtttccc cttcgggggc aaaatgacag gtggtgtatg gttgtcgtca
gctcgtgtcg 1080tgagatgttg ggttaagtcc cgcaacgagc gcaaccctta
tgactagttg ccagcattta 1140gttgggcact ctagtaagac tgccggtgac
aaaccggagg aaggtgggga tgacgtcaaa 1200tcatcatgcc ccttatgacc
tgggctacac acgtgctaca atggatggta caacgagttg 1260cgagaccgcg
aggtcaagct aatctcttaa agccattctc agttcggact gtaggctgca
1320actcgcctac acgaagtcgg aatcgctagt aatcgcggat cagcacgccg
cggtgaatac 1380gttcccgggc cttgtacaca ccgcccgtca caccatgaga
gtttgtaaca cccgaagccg 1440gtggcgtaac ccttttaggg agcgagccgt
ctaaggtggg acaaatgatt agggtgaagt 1500cgtaacaagg tagccgtagg
agaacctgcg gctggat 15372792DNALactobacillus
rhamnosusmisc_featurestrain I21 partial DNAK sequence 2agatgcggtt
atcacagttc cggcttactt taacgacagt cagcgtcagg caaccaagga 60tgccggtaag
atcgctggtt tgaatgttca acggattatc aacgaaccaa ccgcgtcagc
120cttggcttat ggtctggata aaggcgacaa agacgaaaag attttggttt
acgaccttgg 180cggcgggaca tttgatgttt ccatcctgca gttaggtgat
ggtgtcttcg aagtgctgtc 240aaccaatggc gatactcatt taggcgggga
tgattttgat aacaagatca tcgactggct 300tgtttccgaa ttcaaaaagg
ataacaacat tgacctgtct aaagacaaaa tggcaatgca 360acgcctgaag
gatgcagccg aaaaagctaa gaaggatctt tccggtgtga cccagacgca
420aatcagcttg ccatttattt ctgccggccc caacggccca ttgcacttgg
aacgcacttt 480aacccgtgca caatttgacg aaatgaccgc cgacttggtt
gctaagacca aggtgccagt 540tgaaaatgcg ctgaaagatg ctaaattgac
gaaagcagat attgacaaag taatcttaaa 600tggtggttca acacggatcc
ctgctgttca acaagcagtt aaaggaatgg actggcaaag 660atccggacca
cagcatcaac ccagacgaag cggttgcgct aggtgctgcc gttcagggtg
720gtgtcatttc cggtgacgtg aaggatgttg ttttgctgga tgttacgccg
ctgtcattag 780ggattgaaac ca 792
* * * * *