# US 11,898,139 B2 · Culturing method for genus Rhizobium bacteria

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- Number: US 11,898,139 B2
- Document ID: US11898139B2
- Status: Granted
- Kind: B2
- Application number: 16/758,950
- Filed: 2018-10-23
- Published: 2021-06-17
- Granted: 2024-02-13
- Earliest claimed: 2017-10-27
- Assignees: Kumiai Chemical Industry Co., Ltd.; K.I Chemical Industry Co., Ltd.; Okayama Prefectural Government
- Applicants: Kumiai Chemical Industry Co., Ltd.; K.I Chemical Industry Co., Ltd.; Okayama Prefectural Government
- Inventors: Toshinobu Yamazaki; Shingo Hattori; Tatsuya Horiuchi
- Web page: https://patentgrep.com/patent/US11898139B2

## CPC

- C12N 1/20: Bacteria; Culture media therefor
- C12N 1/04: Preserving or maintaining viable microorganisms (immobilised microorganisms C12N11/00)
- C12N 1/205: Bacterial isolates
- C12N 2500/34: Sugars
- C12N 2500/60: Buffer, e.g. pH regulation, osmotic pressure
- C12R 2001/41: Rhizobium
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS

## Abstract

Provided herein is a culturing method for bacteria of the genus Rhizobium whereby a low viscosity can be maintained in a culture solution during the culturing of and at the end of culturing of said bacteria, and that enables easy and efficient post-procedures, including concentration and separation of viable bacteria, among others. This is achieved by growing Rhizobium bacteria by liquid culture using a medium containing a disaccharide (for example, maltose, trehalose, lactose, sucrose) accounting for at least 25 mass % of the carbon source in the medium while maintaining a pH of 5.0 to 7.0 in the culture solution throughout the course of the culture, namely from the beginning of the culturing to the end of the culturing of said bacteria.

## Claims

### Claim 1

A method for inhibiting an increase in viscosity of a culture solution of Rhizobium bacteria, the method comprising culturing a Rhizobium bacterium by liquid culture using a medium comprising one or more carbon sources, wherein a disaccharide accounts for at least 25 mass % of the one or more carbon sources in the medium while maintaining a pH of 5.0 to 7.0 in a culture solution from a beginning of the culturing to an end of the culturing,
wherein the disaccharide is at least one selected from the group consisting of maltose, trehalose, and lactose, and
wherein the Rhizobium bacterium is a Rhizobium vitis ARK-1 strain with accession number FERM BP-11426, a Rhizobium vitis ARK-2 strain with accession number FERM BP-11427, a Rhizobium vitis ARK-3 strain with accession number FERM BP-11428, or an Agrobacterium radiobacter K84 strain.

### Claim 2 (depends on 1)

The method of claim 1, wherein the liquid culture is an at least 5-L mass culture.

### Claim 3

A method for inhibiting an increase in viscosity of a culture solution of Rhizobium bacteria and improving viable cell count of the Rhizobium bacterium in the culture solution, the method comprising:
culturing a Rhizobium bacterium by liquid culture using a medium comprising one or more carbon sources, wherein a disaccharide accounts for at least 25 mass % of the one or more carbon sources in the medium while maintaining a pH of 5.0 to 7.0 in a culture solution from a beginning of the culturing to an end of the culturing; and
concentrating and/or separating viable bacteria from the culture solution,
wherein the disaccharide is at least one selected from the group consisting of maltose, trehalose, and lactose, and
wherein the Rhizobium bacterium is a Rhizobium vitis ARK-1 strain with accession number FERM BP-11426, a Rhizobium vitis ARK-2 strain with accession number FERM BP-11427, a Rhizobium vitis ARK-3 strain with accession number FERM BP-11428, or an Agrobacterium radiobacter K84 strain.

### Claim 4 (depends on 3)

The method of claim 3, further comprising lyophilizing the viable bacteria.

### Claim 5 (depends on 3)

The method of claim 3, wherein the liquid culture is an at least 5-L mass culture.

### Claim 6 (depends on 1)

The method of claim 1, wherein the viscosity of culture solution is 17 to 44 mPa·s.

### Claim 7 (depends on 3)

The method of claim 3, wherein the viable cell count is 2×1010 to 4.4×1010 cfu/ml.

### Claim 8

A method for inhibiting an increase in viscosity of a culture solution of Rhizobium bacteria,
the method comprising culturing a Rhizobium bacterium by aeration-agitation culture using a medium comprising one or more carbon sources, wherein a disaccharide accounts for at least 25 mass % of the one or more carbon sources in the medium while maintaining a pH of 5.0 to 7.0 in a culture solution from a beginning of the culturing to an end of the culturing,
wherein the disaccharide is at least one selected from the group consisting of maltose, trehalose, and lactose, and
wherein the Rhizobium bacterium is a Rhzizobium vitis ARK-1 strain with accession number FERM BP-11426, a Rhizobium vitis ARK-2 strain with accession number FERM BP-11427, a Rhizobium vitis ARK-3 strain with accession number FERMI BP-11428, or an Agrobacterium radiobacter K84 strain,
whereby, at the time of culturing a Rhizobium bacterium, and, at the time of isolating and recovering a viable cell thereof, the viscosity of culture solution of Rhizobium bacteria is maintained by inhibiting the increase in viscosity.