Bacillus subtilis takemi and compositions thereof

Details Bacillus subtilis takemi and compositions thereof Bacillus subtilis takemi and compositions thereof

2001-10-16

2004-11-02

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C424S093460, C426S061000, C426S052000, C426S053000

Reexamination Certificate

active

06812022

ABSTRACT:

TECHNICAL FIELD
The present invention relates to microorganisms of novel species and methods for using the same. More specifically, the present invention relates to microorganisms of novel species belonging to the genus Bacillus and methods for using the same.
TECHNICAL BACKGROUND
Mankind has utilized various microorganisms to date. For example, a wide variety of foods such as alcoholic drinks, fermented seasonings (e.g. soybean paste and soy sauce), fermented dairy products (e.g. cheese and yogurt) and bread have been produced using actions of microorganisms. In addition, microorganisms have also been utilized in the manufacturing of pharmaceuticals and agricultural chemicals, in the production of energy sources such as alcohol and methane, in the treatment of waste and wastewater, and so forth.
The technology of using functions of living things (especially microorganisms) in industrial processes, which is called biotechnology, is developing remarkably in these days. The discovery of novel, useful microorganisms contributes to the advance of biotechnology.
Under circumstances, it is an object of the present invention to provide novel, useful microorganisms.
It is another object of the present invention to provide methods of use of the microorganisms.
DISCLOSURE OF THE INVENTION
The present inventors have isolated from soil a microorganism of a novel species having a smell of coffee. Thus, the present invention has been achieved. The present invention provides microorganisms belonging to the genus Bacillus that are capable of reducing nitrates and contain chitin and/or chitosan in their cell walls. The microorganism of the invention may have a smell of coffee. The microorganism of the invention may be a microorganism belonging to
Bacillus subtilis
. As one example of the microorganism of the invention,
Bacillus subtilis takemi
(FERM BP-6589) may be given.
The present invention also provides a method for improving soil using the microorganism, and a composition for improving soil comprising the microorganism.
Further, the present invention provides a method for treating organic waste by fermentation using the microorganism, and a composition for treating organic waste by fermentation comprising the microorganism.
Further, the present invention provides a method for fermenting soybeans using the microorganism, and soybeans that have been fermented using the microorganism.
The present invention also provides a method of using the microorganism as a feed additive, and a feed additive comprising the microorganism.
The present invention also provides a method of using the microorganism for reducing bitterness, and a composition for reducing bitterness comprising the microorganism. The present invention also provides a method of using the microorganism as a food additive, and a food additive comprising the microorganism.
The present invention also provides a method for inhibiting bacterial growth using the microorganism, and an anti-microbial composition comprising the microorganism. Specific examples of target bacteria of which the growth can be inhibited by the microorganism of the invention include staphylococci, pathogenic
E. coli
O157 and O147, dermatophytes, and bacteria of the family micrococcaceae.
The microorganism of the invention is a bacterium belonging to the genus Bacillus that is capable of reducing nitrates and contains chitin and/or chitosan in its cell walls. The microorganism of the invention may have a smell of coffee. As a specific example of such a microorganism,
Bacillus subtilis takemi
(FERM BP-6589) may be given. This microorganism has been isolated as described below from a soil sample collected in Siberia.
Briefly, 500 &mgr;l of sterilized distilled water was added to 50 mg of a Siberian soil sample, which was then shaken for 30 min to prepare a suspension (presenting a brown color). Using this suspension as a stock solution, 10-2-10-7 dilutions were prepared by 10-fold serial dilution. Then, 100 &mgr;l of each dilution was plated on PDAYC medium (Potato Dextrose Agar medium containing Yeast Extract and Casion) and spread uniformly with a spreader, followed by stationary culture under the condition of all day light and at 20° C. As a result, formation of white colonies was observed in 10
−2
−10
−4
dilutions. It was found that these colonies were not filamentous fungi as a result of microscopic examination. Since all the colonies were white, it was presumed that a single species was growing there. Formation of no colonies was observed in dilutions below 10
−4
.
The characters of the isolated microorganism were as summarized below.
TABLE 1
Morphological Characters
Observation Item
Observation Results
Shape and size of cells
Broth agar medium
straight rod-shaped bacteria,
1.2-1.3 &mgr;m × 2.5-3.3 &mgr;m on
overnight culture at 30° C.
Broth liquid medium
straight rod-shaped bacteria,
0.9-1.2 &mgr;m × 4.6-7.2 &mgr;m
on overnight culture at 30° C.
Presence/absence
Polymorphism is not observed.
of polymorphism
Motility
Motility by peritrichous flagella*
is observed.
Spores
Formation of oval, subcentric
spores is observed.
Swelling of sporangia is not
observed.
The size of the spores is 0.9-1.1 &mgr;m ×
2.0-2.3 &mgr;m on 5 day culture on
broth agar medium at 30.
TABLE 2
Culture Characteristics in Individual
Media
Medium
State of Growth
Broth agar plate
Colonies present a rough
surface, and their periphery is
wave-like or notched. No luster is
observed.
Production of neither
characteristic colony pigments
nor diffusive pigments is
observed.
Broth liquid
Cells grow in the upper part of the
medium
medium; formation of a surface
membrane is observed; precipitation
is not observed.
Broth gelatin
Cell growth is observed in the upper
puncture
part of the medium. Liquefaction is
observed.
Litmus.milk
Cell growth in the upper part of
the medium and liquefaction throughout
the medium are observed, but no
solidification is observed
Production of acid is not observed.
TABLE 3
Physiological Properties: Part I
Test Item
Test Results
Denitrification reaction
−
Methyl red test
+
Production of indole
−
Production of hydrogen sulfide
1
TSI agar
−
Lead acetate liquid medium*
2
+
Assimilation of citrates
Koser medium
−
Christensen medium
+
Assimilation of inorganic nitrogen sources
Nitrates
+
Ammonium salts
+
Production of pigments
−
Urease
+
Oxidase
−
Range within which cells can grow*
3
pH
4.5-8.5
(good at 5.5-8.0)
Temperature
12-51° C.
(good at 25-45° C.)
OF (Hugh-Leifson) ∘
1
Test media were based on the media described in Takeharu Hasegawa (Ed), “Classification and Identification of Microorganisms (Vol. II)”, (1985), GAKKAI SHUPPAN CENTER.
*
2
Instead of adding lead acetate into the medium, a lead acetate test paper was suspended.
*
3
Test was performed using meat broth. pH was tested at intervals of 0.5, and temperature was tested at intervals of 1° C.
TABLE 4
Physiological Properties: Part II
Test Item
Test Result
Acid production*
1
L-Arabinose
+
D-Xylose
+
D-Glucose
+
D-Mannose
+
D-Fructose
+
D-Galactose
+
Maltose
+
Sucrose
+
Lactose
+
Trehalose
+
D-Sorbitol
+
D-Mannitol
+
Inositol
+
Glycerol
+
Starch
−
Gas production*
2
L-Arabinose
−
D-Xylose
−
D-Glucose
−
D-Mannose
−
D-Fructose
−
D-Galactose
−
Maltose
−
Sucrose
−
Lactose
−
Trehalose
−
D-Sorbitol
−
D-Mannitol
−
Inositol
−
Glycerol
−
Starch
−
*
1
As the test medium, the acid production medium disclosed in Gordon, R. E., Haynes, W. C. and Pang, C. H., “The Genus Bacillus” (1973), U.S. Department of Agriculture was used.
*
2
As the test medium, the gas production medium disclosed in Gordon, R. E., Haynes, W. C. and Pang, C. H., “The Genus Bacillus” (1973), U.S. Department of Agriculture was used.
TABLE 5
Chemotaxonomic Properties
Test Item
Test Result
Major quinone system
MK-7
It was also found that the GC content in the intracellular DNA

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