# US 11,076,609 B2 · Use of lactic acid bacteria for preparing fermented food products with increased natural sweetness

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- Number: US 11,076,609 B2
- Document ID: US11076609B2
- Status: Granted
- Kind: B2
- Application number: 16/655,806
- Filed: 2019-10-17
- Published: 2020-03-26
- Granted: 2021-08-03
- Earliest claimed: 2012-04-25
- Assignees: Chr. Hansen A/S
- Applicants: Chr. Hansen A/S
- Inventors: Eric Johansen; Kim Ib Soerensen; Mirjana Curic-Bawden; Mette Pia Junge
- Web page: https://patentgrep.com/patent/US11076609B2

## CPC

- A23C 9/1238: {using specific L. bulgaricus or S. thermophilus microorganisms; using entrapped or encapsulated yoghurt bacteria; Physical or chemical treatment of L. bulgaricus or S. thermophilus cultures; Fermentation only with L. bulgaricus or only with S. thermophilus}
- A23C 19/0323: {using only lactic acid bacteria, e.g. Pediococcus and Leuconostoc species; Bifidobacteria; Microbial starters in general (using moulds A23C19/0682)}
- C12N 1/20: Bacteria; Culture media therefor
- C12N 9/1205: {Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases}
- C12N 15/01: Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
- C12N 15/1034: {Isolating an individual clone by screening libraries}
- C12N 15/746: {for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)}
- A23V 2400/123: Bulgaricus
- A23V 2400/249: Thermophilus
- A23C: DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF (preservation thereof A23B11/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A23V: INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION

## Abstract

Described are mutant Streptococcus thermophilus strains and mutant Lactobacillus delbrueckii subsp. bulgaricus strains that excrete glucose into milk when the milk is inoculated and fermented with such strains. Methods for obtaining such strains without genetic modification are described, including methods for obtaining such Streptococcus thermophilus strains by selecting and isolating from a pool of Streptococcus thermophilus strains derived from a galactose-fermenting Streptococcus thermophilus mother strain a pool of Streptococcus thermophilus strains which are resistant to 2-deoxyglucose, and selecting and isolating from the pool of Streptococcus thermophilus strains which are resistant to 2-deoxyglucose a Streptococcus thermophilus strain which exhibits a growth rate in M17 medium+2% galactose higher than its growth rate in M17 medium+2% glucose. The strains are useful for decreasing the lactose content of a fermented food product and for boosting growth of the probiotic BB-12®.

## Claims

### Claim 1

A method for obtaining a non-genetically modified Streptococcus thermophilus strain that carries a mutation in the DNA sequence of the glcK gene encoding a glucokinase protein, wherein the mutation inactivates the glucokinase protein or has a negative effect on expression of the gene, comprising:
(a) selecting and isolating from a pool of Streptococcus thermophilus strains derived from a galactose-fermenting Streptococcus thermophilus mother strain a pool of Streptococcus thermophilus strains which are resistant to 2-deoxyglucose; and
(b) selecting and isolating from the pool of Streptococcus thermophilus strains which are resistant to 2-deoxyglucose a Streptococcus thermophilus strain which exhibits a growth rate in M17 medium+2% galactose higher than its growth rate in M17 medium+2% glucose.

### Claim 2 (depends on 1)

A method according to claim 1, further comprising:
(c) selecting and isolating from a pool of 2-deoxyglucose resistant Streptococcus thermophilus strains derived from the Streptococcus thermophilus strain selected in step (b) a Streptococcus thermophilus strain that grows in M17 medium+2% sucrose but that has a growth rate in M17 medium+2% glucose of zero or at least 0-50% reduced compared to the growth rate of the mother strain in M17 medium+2% glucose.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the galactose-fermenting mother strain is selected from the group consisting of the Streptococcus thermophilus CHCC14994 strain that was deposited at Deutsche Sammlung von Mikroorganismen and Zellkulturen under accession no. DSM 25838, the Streptococcus thermophilus CHCC11976 strain that was deposited at Deutsche Sammlung von Mikroorganismen and Zellkulturen under accession no. DSM 22934, and strains derived therefrom.

### Claim 4 (depends on 1)

A method according to claim 1, wherein the obtained Streptococcus thermophilus strain increases the amount of glucose in 9.5% B-milk to at least 5 mg/ml when inoculated into the 9.5% B-milk at a concentration of 106-107 CFU/ml and grown at 40° C. for 20 hours.

### Claim 5 (depends on 1)

A method according to claim 1, wherein the obtained Streptococcus thermophilus strain increases the amount of glucose in 9.5% B-milk with 0.05% sucrose to at least 5 mg/ml when inoculated into the 9.5% B-milk with 0.05% sucrose at a concentration of 106-107 CFU/ml and grown at 40° C. for 20 hours.