# US 10,368,559 B2 · Streptococcus thermophilus strains > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,368,559 B2 - Document ID: US10368559B2 - Status: Granted - Kind: B2 - Application number: 14/905,125 - Filed: 2014-07-16 - Published: 2016-06-16 - Granted: 2019-08-06 - Earliest claimed: 2013-07-17 - Assignees: Dupont Nutrition Biosciences Aps - Applicants: Dupont Nutrition Biosciences Aps - Inventors: Philippe Horvath; Christophe Fremaux; Pascal Fourcassie - Web page: https://patentgrep.com/patent/US10368559B2 ## 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 9/127: using microorganisms of the genus lactobacteriaceae and other microorganisms or enzymes, e.g. kefir, koumiss (A23C9/13 takes precedence) - A23C 19/032: characterised by the use of specific microorganisms, or enzymes of microbial origin - A23C 21/02: containing, or treated with, microorganisms or enzymes - A23L 33/135: Bacteria or derivatives thereof, e.g. probiotics - C12N 1/20: Bacteria; Culture media therefor - C12N 1/205: Bacterial isolates - A23V 2002/00: Food compositions, function of food ingredients or processes for food or foodstuffs - A23V 2400/249: Thermophilus - C12R 2001/46: Streptococcus {; Enterococcus; Lactococcus} - A23C: DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF (preservation thereof A23B11/00) - A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B) - 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 - C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS ## Abstract The present invention relates to Streptococcus thermophilus strains, usable as starter cultures, able to provide both satisfactory rheological and organoleptic properties, and satisfactory shelf life to the media into which they are incorporated. In particular, these strains are also bacteriophage-resistant, thereby minimizing bacteriophage infection. The invention also provides a composition comprising one of these Streptococcus thermophilus strains, and feed or food products obtained with these strains. ## Claims ### Claim 1 A composition comprising a Streptococcus thermophilus strain and a food acceptable component, wherein: the food acceptable component is selected from cryoprotective agents and boosters; the Streptococcus thermophilus strain exhibits milk acidification kinetics characterized as follows: (1) an average speed of acidification between pH 6.00 and pH 5.30 which is at least 70×10−4 UpH/min, and (2) an average speed of acidification between pH 5.30 and pH 5.00 which is no greater than 22×10−4 UpH/min; and the average speeds of acidification of (1) and (2) are measured under the following conditions: ultra high-temperature pasteurized cow-milk milk having a fat content of fat 1.5% (w/w) is supplemented with 3% (w/w) skimmed milk powder to form a mixture at a temperature of from 20 to 25° C.; after the powder dissolves, the mixture is heated to 90° C. over a period of no greater than 35 minutes; the mixture is maintained at 90° C. for 10 minutes and then cooled to 35° C.-45° C. over a period of no greater than 45 minutes; 1 g/100 L of sodium formate is added to the mixture; the mixture is inoculated with the Streptococcus thermophilus strain, wherein: the Streptococcus thermophilus strain is in a preserved form at −80° C. in a milk-based medium, and the inoculation rate is 1×106 CFU/ml of the milk-base medium; and the inoculated mixture is incubated at a temperature of 43° C. (+/−1° C.), which is kept constant in a water bath, while measuring change in pH every 5 minutes for 24 hours. ### Claim 2 (depends on 1) The composition of claim 1, wherein the Streptococcus thermophilus strain is selected from the group consisting of: (1) the DSM 27029 strain, deposited under the Budapest Treaty on Mar. 21, 2013 in the name of Danisco Deutschland GmbH at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH; (2) the DSM 27030 strain, deposited under the Budapest Treaty on Mar. 21, 2013 in the name of Danisco Deutschland GmbH at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH; (3) the DSM 27031 strain, deposited under the Budapest Treaty on Mar. 21, 2013 in the name of Danisco Deutschland GmbH at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH; and (4) a mutant strain having all of the identifying characteristics of the DSM 27029 strain, of the DSM 27030 strain, or of the DSM 27031 strain. ### Claim 3 A composition comprising a Streptococcus thermophilus strain, wherein: the composition is in a liquid, frozen or dried-powder form; the Streptococcus thermophilus strain exhibits milk acidification kinetics characterized as follows: (1) an average speed of acidification between pH 6.00 and pH 5.30 which is at least 70×10−4 UpH/min, and (2) an average speed of acidification between pH 5.30 and pH 5.00 which is no greater than 22×10−4 UpH/min; and the average speeds of acidification of (1) and (2) are measured under the following conditions: ultra high-temperature pasteurized cow-milk milk having a fat content of fat 1.5% (w/w) is supplemented with 3% (w/w) skimmed milk powder to form a mixture at a temperature of from 20 to 25° C.; after the powder dissolves, the mixture is heated to 90° C. over a period of no greater than 35 minutes; the mixture is maintained at 90° C. for 10 minutes and then cooled to 35° C.-45° C. over a period of no greater than 45 minutes; 1 g/100 L of sodium formate is added to the mixture; the mixture is inoculated with the Streptococcus thermophilus strain, wherein: the Streptococcus thermophilus strain is in a preserved form at −80° C. in a milk-based medium, and the inoculation rate is 1×106 CFU/ml of the milk-base medium; and the inoculated mixture is incubated at a temperature of 43° C. (+/−1° C.), which is kept constant in a water bath, while measuring change in pH every 5 minutes for 24 hours. ### Claim 4 (depends on 3) The composition of claim 3, wherein the composition is in a frozen form. ### Claim 5 (depends on 3) The composition of claim 3, wherein the composition is in a freeze-dried powder form. ### Claim 6 (depends on 3) The composition of claim 3, wherein the Streptococcus thermophilus strain is selected from the group consisting of: (1) the DSM 27029 strain, deposited under the Budapest Treaty on Mar. 21, 2013 in the name of Danisco Deutschland GmbH at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH; (2) the DSM 27030 strain, deposited under the Budapest Treaty on Mar. 21, 2013 in the name of Danisco Deutschland GmbH at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH; (3) the DSM 27031 strain, deposited under the Budapest Treaty on Mar. 21, 2013 in the name of Danisco Deutschland GmbH at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH; and (4) a mutant strain having all of the identifying characteristics of the DSM 27029 strain, of the DSM 27030 strain, or of the DSM 27031 strain. ### Claim 7 A method for preparing a food or a feed product comprising: putting into contact a substrate with a culture of a Streptococcus thermophilus strain; and obtaining the product, wherein: the substrate comprises pasteurized milk; the Streptococcus thermophilus strain exhibits milk acidification kinetics characterized as follows: (1) an average speed of acidification between pH 6.00 and pH 5.30 which is at least 70×10−4 UpH/min, and (2) an average speed of acidification between pH 5.30 and pH 5.00 which is no greater than 22×10−4 UpH/min; and the average speeds of acidification of (1) and (2) are measured under the following conditions: ultra high-temperature pasteurized cow-milk milk having a fat content of fat 1.5% (w/w) is supplemented with 3% (w/w) skimmed milk powder to form a mixture at a temperature of from 20 to 25° C.; after the powder dissolves, the mixture is heated to 90° C. over a period of no greater than 35 minutes; the mixture is maintained at 90° C. for 10 minutes and then cooled to 35° C.-45° C. over a period of no greater than 45 minutes; 1 g/100 L of sodium formate is added to the mixture; the mixture is inoculated with the Streptococcus thermophilus strain, wherein: the Streptococcus thermophilus strain is in a preserved form at −80° C. in a milk-based medium, and the inoculation rate is 1×106 CFU/ml of the milk-base medium; and the inoculated mixture is incubated at a temperature of 43° C. (+/−1° C.), which is kept constant in a water bath, while measuring change in pH every 5 minutes for 24 hours. ### Claim 8 (depends on 7) The method according to claim 7, wherein the milk acidification kinetics of the strain are characterized by a ratio of the (1) average speed of acidification between pH 5.30 and pH 5.00 to the (2) average speed of acidification between pH 6.00 and pH 5.30 being less than or equals 25%. ### Claim 9 (depends on 8) The method according to claim 8, wherein the milk acidification kinetics of the Streptococcus thermophilus strain are characterized by a ratio of the (1) average speed of acidification between pH 5.30 and pH 5.00 to the (2) average speed of acidification between pH 6.00 and pH 5.30 being between 2 and 25%. ### Claim 10 (depends on 7) The method according to claim 7, wherein the genome of the Streptococcus thermophilus strain comprises a) a CRISPR4 locus comprising or consisting of the sequence as defined in SEQ ID NO:3 orb) a CRISPR4 locus comprising part(s) of SEQ ID NO:3. ### Claim 11 (depends on 7) The method according to claim 7, wherein the genome of the Streptococcus thermophilus strain comprises a) a CRISPR1 locus consisting of SEQ ID NO:19, orb) a CRISPR1 locus comprising SEQ ID NO:19 or comprising part(s) of SEQ ID NO:19, and optionally additional CRISPR1 [repeat-spacer] unit(s) of sequence R1-X1, wherein R1 is as defined in SEQ ID NO:20, and X1 is any sequence with a length of from 27 to 33 bp. ### Claim 12 (depends on 7) The method according to claim 7, wherein the genome of the Streptococcus thermophilus strain comprises a) a CRISPR3 locus consisting of SEQ ID NO:73, or b) a CRISPR3 locus comprising SEQ ID NO:73 or comprising part(s) of SEQ ID NO:73, and optionally additional CRISPR3 [repeat-spacer] unit(s) of sequence R3-X3, wherein R3 is as defined in SEQ ID NO:74, and X3 is any sequence with a length from 27 to 33 bp. ### Claim 13 (depends on 7) The method according to claim 7, wherein the Streptococcus thermophilus strain exhibits milk acidification kinetics characterized as follows: the (1) average speed of acidification between pH 6.00 and pH 5.30 is between 70×10−4 and 250×10−4; and the (2) average speed of acidification between pH 5.30 and pH 5.00 is between 1×10−4 and 20×10′. ### Claim 14 (depends on 7) The method according claim 7, wherein a composition comprising the Streptococcus thermophilus strain and at least one other microorganism is contacted with the substrate. ### Claim 15 (depends on 14) The method according to claim 14, wherein: the at least one other microorganism comprises a lactic acid bacterium; and the lactic acid bacterium is selected from: a different strain of the species Streptococcus thermophilus, a strain of the subspecies Lactobacillus delbrueckii subsp. bulgaricus, and a strain of the genus Bifidobacterium. ### Claim 16 (depends on 7) The method according to claim 7, wherein the Streptococcus thermophilus strain is selected from the group consisting of: (1) the DSM 27029 strain, deposited under the Budapest Treaty on Mar. 21, 2013 in the name of Danisco Deutschland GmbH at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH; (2) the DSM 27030 strain, deposited under the Budapest Treaty on Mar. 21, 2013 in the name of Danisco Deutschland GmbH at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH; (3) the DSM 27031 strain, deposited under the Budapest Treaty on Mar. 21, 2013 in the name of Danisco Deutschland GmbH at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH; and (4) a mutant strain having all of the identifying characteristics of the DSM 27029 strain, of the DSM 27030 strain, or of the DSM 27031 strain. ### Claim 17 (depends on 16) A food or a feed product prepared by the method of claim 16. ### Claim 18 (depends on 17) The food or feed product according to claim 17, wherein the product comprises a fermented dairy food. ### Claim 19 (depends on 17) The food or feed product according to claim 17, wherein the product is selected from the group consisting of a yogurt, cheese, buttermilk, quark, sour cream, kefir, fermented whey-based beverage, koumiss, milk beverage, yoghurt drink, fermented milk, matured cream, fromage frais, milk, dairy product retentate, processed cheese, cottage cheese, cream dessert, and infant milk. ### Claim 20 A method for preparing a food or a feed product comprising: putting into contact a substrate with composition comprising a culture of a Streptococcus thermophilus strain; and obtaining the product, wherein: the composition further comprises a food acceptable component; the food acceptable component is selected from cryoprotective agents, boosters, yeast extracts, sugars and vitamins; the Streptococcus thermophilus strain exhibits milk acidification kinetics characterized as follows: (1) an average speed of acidification between pH 6.00 and pH 5.30 which is at least 70×10−4 UpH/min, and (2) an average speed of acidification between pH 5.30 and pH 5.00 which is no greater than 22×10−4 UpH/min; and the average speeds of acidification of (1) and (2) are measured under the following conditions: ultra high-temperature pasteurized cow-milk milk having a fat content of fat 1.5% (w/w) is supplemented with 3% (w/w) skimmed milk powder to form a mixture at a temperature of from 20 to 25° C.; after the powder dissolves, the mixture is heated to 90° C. over a period of no greater than 35 minutes; the mixture is maintained at 90° C. for 10 minutes and then cooled to 35° C.-45° C. over a period of no greater than 45 minutes; 1 g/100 L of sodium formate is added to the mixture; the mixture is inoculated with the Streptococcus thermophilus strain, wherein: the Streptococcus thermophilus strain is in a preserved form at −80° C. in a milk-based medium, and the inoculation rate is 1×106 CFU/ml of the milk-base medium; and the inoculated mixture is incubated at a temperature of 43° C. (+/−1° C.), which is kept constant in a water bath, while measuring change in pH every 5 minutes for 24 hours. ### Claim 21 (depends on 20) The method according to claim 20, wherein the substrate comprises milk. ### Claim 22 (depends on 20) The method according to claim 20, wherein the substrate comprises pasteurized milk. ### Claim 23 (depends on 20) The method according to claim 20, wherein the composition is in a liquid, frozen, or freeze-dried powder form. ### Claim 24 (depends on 20) The method of claim 20, wherein the Streptococcus thermophilus strain is selected from the group consisting of: (1) the DSM 27029 strain, deposited under the Budapest Treaty on Mar. 21, 2013 in the name of Danisco Deutschland GmbH at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH; (2) the DSM 27030 strain, deposited under the Budapest Treaty on Mar. 21, 2013 in the name of Danisco Deutschland GmbH at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH; (3) the DSM 27031 strain, deposited under the Budapest Treaty on Mar. 21, 2013 in the name of Danisco Deutschland GmbH at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH; and (4) a mutant strain having all of the identifying characteristics of the DSM 27029 strain, of the DSM 27030 strain, or of the DSM 27031 strain. ### Claim 25 (depends on 24) A food or feed product prepared by the method of claim 24, wherein the food or feed product is a dairy food or feed product. ### Claim 26 (depends on 25) The food or feed product according to claim 25, wherein the product is selected from the group consisting of a yogurt, cheese, buttermilk, quark, sour cream, kefir, fermented whey-based beverage, koumiss, milk beverage, yoghurt drink, fermented milk, matured cream, fromage frais, milk, dairy product retentate, processed cheese, cottage cheese, cream dessert, and infant milk. ### Claim 27 A method for preparing a food or a feed product comprising: putting into contact a substrate with composition comprising a culture of a Streptococcus thermophilus strain; and obtaining the product, wherein: the composition is in a liquid, frozen or dried-powder form; the Streptococcus thermophilus strain exhibits milk acidification kinetics characterized as follows: (1) an average speed of acidification between pH 6.00 and pH 5.30 which is at least 70×10−4 UpH/min, and (2) an average speed of acidification between pH 5.30 and pH 5.00 which is no greater than 22×10−4 UpH/min; and the average speeds of acidification of (1) and (2) are measured under the following conditions: ultra high-temperature pasteurized cow-milk milk having a fat content of fat 1.5% (w/w) is supplemented with 3% (w/w) skimmed milk powder to form a mixture at a temperature of from 20 to 25° C.; after the powder dissolves, the mixture is heated to 90° C. over a period of no greater than 35 minutes; the mixture is maintained at 90° C. for 10 minutes and then cooled to 35° C.-45° C. over a period of no greater than 45 minutes; 1 g/100 L of sodium formate is added to the mixture; the mixture is inoculated with the Streptococcus thermophilus strain, wherein: the Streptococcus thermophilus strain is in a preserved form at −80° C. in a milk-based medium, and the inoculation rate is 1×106 CFU/ml of the milk-base medium; and the inoculated mixture is incubated at a temperature of 43° C. (+/−1° C.), which is kept constant in a water bath, while measuring change in pH every 5 minutes for 24 hours. ### Claim 28 (depends on 27) The method of claim 27, wherein the composition is in a frozen form. ### Claim 29 (depends on 27) The method of claim 27, wherein the composition is in a freeze-dried powder form. ### Claim 30 (depends on 27) The method according to claim 27, wherein the substrate comprises milk. ### Claim 31 (depends on 27) The method according to claim 27, wherein the substrate comprises pasteurized milk. ### Claim 32 (depends on 27) The method of claim 27, wherein the Streptococcus thermophilus strain is selected from the group consisting of: (1) the DSM 27029 strain, deposited under the Budapest Treaty on Mar. 21, 2013 in the name of Danisco Deutschland GmbH at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH; (2) the DSM 27030 strain, deposited under the Budapest Treaty on Mar. 21, 2013 in the name of Danisco Deutschland GmbH at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH; (3) the DSM 27031 strain, deposited under the Budapest Treaty on Mar. 21, 2013 in the name of Danisco Deutschland GmbH at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH; and (4) a mutant strain having all of the identifying characteristics of the DSM 27029 strain, of the DSM 27030 strain, or of the DSM 27031 strain. ### Claim 33 (depends on 32) A food or feed product prepared by the method of claim 32, wherein the food or feed product is a dairy food or feed product. ### Claim 34 (depends on 33) The food or feed product according to claim 33, wherein the product is selected from the group consisting of a yogurt, cheese, buttermilk, quark, sour cream, kefir, fermented whey-based beverage, koumiss, milk beverage, yoghurt drink, fermented milk, matured cream, fromage frais, milk, dairy product retentate, processed cheese, cottage cheese, cream dessert, and infant milk.