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EP-4735469-A1 - METHODS FOR AVOIDING MALTITOL-MEDIATED GROWTH INHIBITION

EP4735469A1EP 4735469 A1EP4735469 A1EP 4735469A1EP-4735469-A1

Abstract

The present invention relates to the use of specific strains of Streptococcus thermophilus to reduce the negative impact of the presence of disaccharide sugar alcohols during fermentation of a milk-based product, as well as methods for identifying specific strains of Streptococcus thermophilus , method for the preparation of a fermented milk-based product comprising disaccharide sugar alcohols, as well as methods for reducing the negative impact of the presence of disaccharide sugar alcohols during fermentation of a milk-based products.

Inventors

  • MAO, Yuejian
  • GUO, Rongkai

Assignees

  • International N&H Denmark ApS

Dates

Publication Date
20260506
Application Date
20240626

Claims (14)

  1. 1. Use of a sucrose-negative Streptococcus thermophilus strain to reduce the negative impact of the presence of a disaccharide sugar alcohol, such as maltitol, isomalt or lactitol during fermentation of a milk-based product with said strain.
  2. 2. Use according to claim 1, which sucrose-negative Streptococcus thermophilus strain is carrying one or more mutations selected from the group consisting of: 1) one or more mutation in one or more gene of the sucrose regulon; which mutation provides for a significant reduction in the ability to grow on and/or transport sucrose by said strain; and which mutation at least comprises 1) one or more of the scrA gene, and/or the scrB gene and/or the scrR gene, and ii) optionally a further mutation in the promoter region regulating the expression of the scrA, scrB and/or scrR genes, such as a mutation that protect the strain from reversing from the sucrose negative phenotype; and 2) optionally one or more further mutation affecting the glucose porter, such as affecting glcU and/or its expression, which mutation restores or improves glucose consumption of said strain when grown on glucose as the sole source of carbohydrates.
  3. 3. The use according to claim 2, which sucrose-negative, Streptococcus thermophilus strain has one or more mutation in the sucrose regulon in one or more of the scrA gene and the scrB gene.
  4. 4. The use according to claims 2 or 3, which mutation in one or more gene of the sucrose regulon is in the scrA gene effectively disrupting gene function, such as an insertion or deletion resulting in a frameshift of the open reading frame of the gene coding for the EIIABCsucrose protein leading to the translation of a truncated EIIABCsucrose protein encoded by the scrA gene, such as a mutation by insertion or deletion of a nucleotide, such as at or around a nucleotide corresponding to position 133 of SEQ ID NO:1, in the open-reading-frame (ORF) of the scrA gene.
  5. 5. The use according to any one of claims 2-4, which strain has a mutation in the promoter region regulating the expression of any one of the scrA, scrB and/or scrR genes.
  6. 6. The use according to claim 5, which mutation is a SNP in the promoter region of the scrA and/or scrB genes, such as a SNP in the promoter region of the scrB gene, such as a mutation near the -10 and -35 sequence of the promoter, such as a mutation of T to G corresponding to position 38 of SEQ ID NO:2 upstream of the scrB promoter, such as a mutation that will comprise the entire sequence identified by SEQ ID NO:2 or a variant sequence being at least about 90%, such as at least about 95% identical herewith pscrB829').
  7. 7. The use according to any one of claims 1-6, which sucrose-negative Streptococcus thermophilus strain is selected from: - a strain deposited under the Budapest Treaty in the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkul- turen, GmbH, on 18. January 2022 under number DSM34132; - a strain deposited under the Budapest Treaty in the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkul- turen, GmbH, on 18. January 2022 under number DSM34133; - a strain deposited under the Budapest Treaty in the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkul- turen, GmbH, on 18. January 2022 under number DSM34134; - a strain deposited under the Budapest Treaty in the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkul- turen, GmbH, on 18. January 2022 under number DSM34138; - a strain deposited under the Budapest Treaty in the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkul- turen, GmbH, on 18. January 2022 under number DSM34139; - a strain deposited under the Budapest Treaty in the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkul- turen, GmbH, on 18. January 2022 under number DSM34140; - a strain deposited under the Budapest Treaty in the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkul- turen, GmbH, on 18. January 2022 under number DSM34146; - a strain deposited under the Budapest Treaty in the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkul- turen, GmbH, on 18. January 2022 under number DSM34147; - a strain deposited under the Budapest Treaty in the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkul- turen, GmbH, on 16. February 2022 under number DSM34172; - a strain deposited under the Budapest Treaty in the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkul- turen, GmbH, on 6. February 2024 under number DSM34931; and - a strain deposited under the Budapest Treaty in the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkul- turen, GmbH, on 6. February 2024 under number DSM34932.
  8. 8. A method for identifying a sucrose-negative Streptococcus thermophilus strain, which method comprises the step of screening for and identifying Streptococcus thermophilus strains insensitive or with reduced sensitivity to a disaccharide sugar alcohol, such as malt- itol, isomalt or lactitol.
  9. 9. A method for the preparation of a fermented milk-based product comprising a disaccharide sugar alcohol, such as maltitol, isomalt or lactitol, the method comprising the fermentation of a milk-based substrate with a lactic acid bacteria starter culture in the presence of at least about 0.01% w/v of sucrose.
  10. 10. A method for reducing the negative impact of the presence of a disaccharide sugar alcohol, such as maltitol, isomalt or lactitol during fermentation of a milk-based product with a lactic acid bacteria starter culture, the method comprising the step of adding at least about 0.01% (w/v) of sucrose to the milk base before or during fermentation.
  11. 11. The method according to any one of claims 9 or 10, which lactic acid bacteria starter culture comprises or consists of at least one Streptococcus thermophilus strain as defined in claims 1-7, and optionally at least one Lactobacillus strain such as at a Lactobacillus delbrueckii subp bulgaricus strain, such as at a Lactobacillus acidophilus, such as at a Lactobacillus hel- veticus, optionally at least one Lactiplantibacillus strain such as at a Lactiplantibacillus plantarum, optionally at least one Lacticaseibacillus strain such as at a Lacticaseibacillus easel, such as at a Lacticaseibacillus paracasei, such as at a Lacticaseibacillus rhamnosus, optionally at least one Limosilactobacillus strain, such as at a Limosilactobacillus fermentum , optionally at least one Leuconostoc strain, such as at a Leuconostoc mesenteroides, and optionally at least one Bifidobacterium strain, such as at a Bifidobacterium animalis subsp. ani- malis, such as at a Bifidobacterium animalis subsp. lactis.
  12. 12. The use or method according to any one of claims 1-11, wherein said milk-based product is a dairy product, such as a yoghurt, a cheese, a buttermilk, quark, a sour cream, kefir, a fermented whey-based beverage, a koumiss, a milk beverage, a yoghurt drink, a fermented milk, a matured cream, a fromage frais, a milk, a dairy product retentate, a processed cheese, a cottage cheese, a cream dessert.
  13. 13. The use or method according to any one of claims 1-11, wherein said disaccharide sugar alcohol, such as maltitol, isomalt or lactitol is present in an amount of at least about 1% w/v, such as at least about 1.2% w/v, such as at least about 1.4% w/v, such as at least about 1.6% w/v, such as at least about 1.8% w/v, such as at least about 2.0% w/v, such as at least about 2.2% w/v, such as at least about 2.4% w/v, such as at least about 2.6% w/v, such as at least about 2.8% w/v, such as at least about 3.0% w/v, such as at least about 3.5% w/v, such as at least about 4.0% w/v, such as at least about 4.5% w/v, such as at least about 5.0% w/v, such as at least about 5.5% w/v, such as at least about 6.0% w/v.
  14. 14. A Streptococcus thermophilus strain, sucrose-negative Streptococcus thermophilus strain selected from the group consisting of: - a strain deposited under the Budapest Treaty in the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkul- turen, GmbH, on 18. January 2022 under number DSM34133; - a strain deposited under the Budapest Treaty in the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkul- turen, GmbH, on 6. February 2024 under number DSM34931; and - a strain deposited under the Budapest Treaty in the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkul- turen, GmbH, on 6. February 2024 under number DSM34932.

Description

METHODS FOR AVOIDING MALTITOL-MEDIATED GROWTH INHIBITION FIELD OF THE INVENTION The present invention relates to the use of specific strains of Streptococcus ther- mophilus to reduce the negative impact of the presence of disaccharide sugar alcohols during fermentation of a milk-based product, as well as methods for identifying specific strains of Streptococcus thermophilus, method for the preparation of a fermented milk- based product comprising disaccharide sugar alcohols, as well as methods for reducing the negative impact of the presence of disaccharide sugar alcohols during fermentation of a milk-based products. BACKGROUND OF THE INVENTION The food trend of natural-everything is seeping into the sugar avoidance trend as consumers desire natural solutions to less sugar. Sweetener blends deliver the sweet spot in reduced-sugar dairy applications and maltitol is one of natural sweeteners chosen by many customers. However, the presence of maltitol seems to significantly delay the fermentation time of commercial starter cultures, for example, YO-MIX 883 and YO-MIX PRIME 800. This delay mainly occurs at lag phase of the strains during milk acidification. After screening of commercial strains, this maltitol-caused growth inhibition seems to be quite common in Streptococcus thermophilus strains. SUMMARY OF THE INVENTION It is an object of embodiments of the invention to provide solutions for mitigating the negative impact of the presence of disaccharide sugar alcohols, such as maltitol, iso- malt or lactitol during the fermentation of a milk-based products. The present invention relates in a broad aspect to a new use of sucrose-negative Streptococcus thermophilus strains for reducing the negative impact of the presence of a disaccharide sugar alcohol, when using such strains. The present invention furthermore relates in a broad aspect to a method for iden- tifying new sucrose-negative Streptococcus thermophilus strains, methods for the prepa- ration of a fermented milk-based product comprising disaccharide sugar alcohols, such as maltitol, as well as methods for reducing the negative impact of the presence of disaccha- ride sugar alcohols, such as maltitol during fermentation. Accordingly, in a first aspect the present invention relates to the use of a sucrose- negative Streptococcus thermophilus strain to reduce the negative impact of the presence of a disaccharide sugar alcohol, such as maltitol, isomalt or lactitol during fermentation of a milk-based product with said strain. In a second aspect the present invention relates to a method for identifying a su- crose-negative Streptococcus thermophilus strain, which method comprises the step of screening for and identifying Streptococcus thermophilus strains insensitive or with re- duced sensitivity to a disaccharide sugar alcohol, such as maltitol, isomalt or lactitol. In a third aspect the present invention relates to a method for the preparation of a fermented milk-based product comprising a disaccharide sugar alcohol, such as malt- itol, isomalt or lactitol, the method comprising the fermentation of a milk-based substrate with a lactic acid bacteria starter culture in the presence of at least about 0.01% w/v of sucrose. In a further aspect the present invention relates to a method for reducing the negative impact of the presence of a disaccharide sugar alcohol, such as maltitol, isomalt or lactitol during fermentation of a milk-based product with a lactic acid bacteria starter culture, the method comprising the step of adding at least about 0.01% (w/v) of sucrose to the milk base before or during fermentation. DETAILED DESCRIPTION OF THE INVENTION The present inventors have found that sucrose uptake is associated with maltitol sensitivity of Streptococcus thermophilus strains. The present inventors therefore found that the maltitol-mediated growth inhibition could be annealed either by blocking genes of the sucrose regulon, such as the sucrose phosphotransferase system via scrA and scrB gene or by adding as low as 0.01% (w/v) of sucrose to block maltitol transportation. Accordingly, in a broad aspect the present invention relates to the use of a su- crose-negative Streptococcus thermophilus strain to reduce the negative impact of the presence of a disaccharide sugar alcohol, such as maltitol, isomalt or lactitol during fer- mentation of a milk-based product with said strain. In some embodiments the sucrose-negative Streptococcus thermophilus strain is carrying one or more mutations selected from the group consisting of: 1) one or more mutation in one or more gene of the sucrose regulon; which mutation provides for a significant reduction in the ability to grow on and/or transport sucrose by said strain; and which mutation at least comprises i) one or more of the scrA gene, and/or the scrB gene and/or the scrR gene, and ii) optionally a further mutation in the promoter region regulating the expression of the scrA, scrB and/or scrR genes, such as a mutation