# US 5,352,586 A · Method of determining the presence of an antibiotic produced by Lactobacillus reuteri

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- Number: US 5,352,586 A
- Document ID: US5352586A
- Status: Granted
- Kind: A
- Application number: 07/670,960
- Filed: 1991-03-18
- Granted: 1994-10-04
- Earliest claimed: 1987-05-01
- Assignees: Biogaia AB
- Inventors: Walter J. Dobrogosz; Sven E. Lindgren
- Web page: https://patentgrep.com/patent/US5352586A

## CPC

- C12N 1/205: Bacterial isolates
- C07D 319/06: not condensed with other rings
- C12P 17/06: containing a six-membered hetero ring, e.g. fluorescein
- C12R 2001/225: Lactobacillus
- Y10S 435/853: Lactobacillus
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

The antibiotic reuterin is obtained by cultivating strains of Lactobacillus reuteri under controlled conditions. Reuterin has inhibitory activity against Gram positive and Gram negative bacteria, against yeast, Saccharomyces cerevisiae, and against the protozoan, Trypanosoma cruzi. Reuterin producing strains are identified by grown inhibition of susceptible microorganisms in the presence of glycerol or glyceraldehyde.

## Claims

### Claim 1

A method for determining the presence of the antibiotic .beta.-hydroxypropionaldehyde as a detectable end-product in an aqueous solution of Lactobacillus reuteri cells comprising the steps of:
(a) placing Lactobacillus reuteri cells in an aqueous solution containing a compound selected from the group consisting of glycerol and glyceraldehyde as the carbon and energy source at a concentration of about 20-500 mM, and incubating said cells under anaerobic conditions;
(b) separating the Lactobacillus reuteri cells from a sample of the solution;
(c) analyzing the sample using high performance liquid chromatography;
(d) eluting the sample; and
(e) determining the presence of a peak intermediate in elution time to those of 1,3-propanediol and glycerol which is indicative of the presence of .beta.-hydroxypropionaldehyde in the sample.

### Claim 2

A method for identifying Lactobacillus reuteri isolates that produce the antibiotic .beta.-hydroxypropionaldehyde as a detectable end-product, which comprises the steps of:
(a) inoculating a suspension of microorganisms from an animal source on a solid Lactobacillus growth medium made to be highly selectively for Lactobacillus by addition of sodium acetate and adjustment of the medium pH to 5.5;
(b) incubating said inoculated growth medium under conditions that promote growth of Lactobacillus colonies;
(c) overlaying the inoculated growth medium and colonies with a liquified semisolid mixture containing a suspension of a living test microroganism and a compound selected from the group consisting of glycerol and glyceraldehyde as the carbon and energy source at a concentration of about 20-500 mM;
(d) incubating the overlaid inoculated medium under anaerobic conditions; and
(e) identifying in situ Lactobacillus reuteri colonies that produce .beta.-hydroxypropionaldehyde by detecting zones of growth inhibition in the test microorganism surrounding said colonies.

### Claim 3 (depends on 2)

The method for identifying Lactobacillus reuteri isolates of claim 2 wherein the carbon source is glycerol and the living test microroganism is Lactobacillus plantarum.

### Claim 4 (depends on 2)

The method for identifying Lactobacillus reuteri isolates of claim 2 wherein the conditions of incubation comprise an incubation temperature of 37 degrees C. for 48 hours.