# US 5,702,923 A · Method of making a lactococcal bacteriocin

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- Number: US 5,702,923 A
- Document ID: US5702923A
- Status: Granted
- Kind: A
- Application number: 07/840,503
- Filed: 1992-02-24
- Granted: 1997-12-30
- Earliest claimed: 1990-03-13
- Assignees: Quest International Flavors & Food Ingredients Company, division of Indopco, Inc.
- Inventors: Peter A. Vandenbergh; Shirley A. Walker; Blair S. Kunka
- Web page: https://patentgrep.com/patent/US5702923A

## CPC

- C07K 14/315: from Streptococcus (G), e.g. Enterococci
- A01N 63/50: Isolated enzymes; Isolated proteins (peptides A01N37/46)
- A23B 2/7295: {Antibiotics}
- A23B 2/783: Microorganisms; Enzymes
- Y10S 435/822: using bacteria or actinomycetales
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A23B: PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

A method of making a bacteriocin produced by Lactococcus lactis NRRL-B-18535 is described. The bacteriocin is useful in foods and other materials and has a wide spectrum of activity against Gram-positive bacteria in a pH range between 2 and 8.

## Claims

### Claim 1

A method for producing a bacteriocin in a growth medium which comprises:
(a) culturing live cells of Lactococcus lactis NRRL-B-18535 in a growth medium for the cells so as to produce the bacteriocin in the growth medium, and wherein the bacteriocin contains a protein having a molecular weight of about 6000 daltons, is inactivated by protease V and not inactivated by alpha-chymotrypsin, trypsin, lipase, pepsin and lysozyme, wherein the bacteriocin inhibits the growth of bacteria selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus carnosus, Pediococcus pentosaceus, Pediococcus acidilactici, Lactococcus cremoris, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus bulgaricus, Lactobacillus fermentum, Lactobacillus bifermentans, Lactobacillus plantarum and Listeria monocytogenes and has an pH for inhibition between about pH 2 and 8; and
(b) separating the bacteriocin in the growth medium from the cells.

### Claim 2

A method for producing a bacteriocin which comprises:
(a) culturing live cells of Lactococcus lactis NRRL-B-18535 in a growth medium for the cells to produce the bacteriocin;
(b) separating the bacteriocin from the cells; and
(c) isolating the bacteriocin from the growth medium, wherein the bacteriocin contains a protein having a molecular weight of about 6000 daltons, is inactivated by protease V and not inactivated by alpha-chymotrypsin, trypsin, lipase, pepsin and lysozyme, wherein the bacteriocin inhibits the growth of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus carnosus, Pediococcus pentosaceus, Pediococcus acidilactici, Lactococcus cremoris, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus bulgaricus, Lactobacillus fermentum, Lactobacillus bifermentans, Lactobacillus plantarum and Listeria monocytogenes and has an pH for inhibition between about pH 2 and 8.

### Claim 3 (depends on 2)

The method of claim 2 wherein the bacteriocin is lyophilized after isolation from the growth medium.

### Claim 4

A method for producing a bacteriocin which comprises:
(a) culturing live cells of Lactococcus lactis NRRL-B-18535 in a growth medium for the cells, so as to produce the bacteriocin in the growth medium;
(b) separating bacteriocin from the cells: and
(c) recovering the bacteriocin from the growth medium from the bacterocin by precipitating the bacteriocin in the growth medium and removing the precipitated bacteriocin to provide the bacteriocin, and wherein the bacteriocin contains a protein having a molecular weight of about 6000 daltons, is inactivated by protease V and not inactivated by alpha-chymotrypsin, trypsin, lipase, pepsin and lysozyme, inhibits the growth of bacteria selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus carnosus, Pediococcus pentosaceus, Pediococcus acidilactici, Lactococcus cremoris, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus bulgaricus, Lactobacillus fermentum, Lactobacillus bifermentans, Lactobacillus plantarum and Listeria monocytogenes and has an pH for inhibition between about pH 2 and 8.

### Claim 5 (depends on 4)

The method of claim 4 wherein the bacteriocin is lyophilized.

### Claim 6 (depends on 4)

The method of claim 4 wherein the bacteriocin partially purified from the growth medium by precipitation using ammonium sulfate and the precipitated bacteriocin is recovered from the growth medium.

### Claim 7

A method for producing a bacteriocin in the growth medium which comprises:
(a) culturing cells of Lactococcus lactis NRRL-B-18535 in a growth medium for the cells to produce, the bacteriocin in the growth medium;
(b) filtering the cultured growth medium in a microporous filter means so that the bacteriocin can pass through as a filtrate and which retains components of the growth medium and the cells on the filter means to produce the bacteriocin, and wherein the bacteriocin contains a protein having a molecular weight of about 6000 daltons, is inactivated by protease v and not inactivated by alpha-chymotrypsin, trypsin, lipase, pepsin and lysozyme, inhibits the growth of bacteria selected from the group consisting of Staphylococcus aureus, staphylococcus epidermidis, Staphylococcus carnosus, Pediococcus pentosaceus, Pediococcus acidilactici, Lactococcus cremoris, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus bulgaricus, Lactobacillus fermentum, Lactobacillus bifermentans, Lactobacillus plantarum and Listeria monocytogenes and has an pH for inhibition between about pH 2 and 8.

### Claim 8 (depends on 7)

The method of claim 7 further comprising lyophilizing the filtered bacteriocin.

### Claim 9 (depends on 7)

The method of claim 7 wherein the bacteriocin in the filtrate is precipitated with ammonium sulfate and then the precipitated bacteriocin is recovered from the growth medium is further purified by liquid chromatography.