Citrate-deficient lacticaseibacillus rhamnosus strains and uses thereof

Abstract

The aim of the present invention is to provide Lacticaseibacillus rhamnosus strains that can survive in an acidified milk product at ambient temperature without producing acetate or CO2. Such strains can be included as a probiotic in acidified milk products, such as yogurt. Acetate and CO2 are undesired products formed by the well-known LGG® strain.

Description

[0001] 17967-EP-EPA[2]

[0002] 1

[0003] CITRATE-DEFICIENT LACTICASEIBACILLUS RHAMNOSUS STRAINS AND USES THEREOF.

[0004] TECHNICAL FIELD

[0005] The present invention relates to novel citrate-deficient Lacticaseibacillus rhamnosus strains and uses thereof. The present invention also related to methods for obtaining citrate-deficient Lacticaseibacillus rhamnosus strains according to the present invention.

[0006] BACKGROUND ART

[0007] Flavor and gas formation are important attributes affecting customer acceptance of acidified milk products. Due to lack of cooling or temperature variation during transport and storage the flavor and / or gas formation of acidified milk products may change. In addition to change in the taste and mouth feel of the product, gas formation may also result in increased pressure, swelling and potentially blowing of the final product package.

[0008] Lacticaseibacillus rhamnosus (previously Lactobacillus rhamnosus) has been used for probiotic applications in Fresh Dairy, including its addition to pasteurized yoghurt which can be stored at room temperature. It is desirable for an acidified milk product to contain a high viable cell count of such probiotic strains. However, Lacticaseibacillus rhamnosus metabolizes citrate which results in the formation of carbon dioxide and acetate. Both acetate and carbon dioxide are undesirable in acidified milk products.

[0009] Thus, there is a need for improved methods for producing acidified milk products where the final product remains stable during storage, in particular when a cold chain is not possible or is at risk of being disrupted.

[0010] SUMMARY OF THE INVENTION

[0011] In a first aspect, the present invention provides a citrate-deficient Lacticaseibacillus rhamnosus strain. In a second aspect, the present invention relates to a composition comprising the citrate-deficient Lacticaseibacillus rhamnosus strain of the present invention.

[0012] In a third aspect, the present invention relates to a method for producing an acidified milk product containing Lacticaseibacillus rhamnosus, the method comprising adding (i) the citrate-deficient17967-EP-EPA[2]

[0013] 2

[0014] Lacticaseibacillus rhamnosus strain of the present invention or the composition of the present invention to (ii) a milk base or acidified milk product.

[0015] In a fourth aspect, the present invention relates to a kit comprising the Lacticaseibacillus rhamnosus strain of the present invention.

[0016] In a fifth aspect, the present invention relates to a method for obtaining a citrate-deficient Lacticaseibacillus rhamnosus strain, the method comprising:

[0017] (1) subjecting a citrate-positive Lacticaseibacillus rhamnosus mother strain to mutagenesis to obtain a pool of mutant strains;

[0018] (2) screening the pool of mutant strains of step (1) for citrate metabolism; and

[0019] (3) selecting a strain from the pool of mutants that is citrate-deficient.

[0020] FIGURES

[0021] re 1 Citrate utilization pathway. Citrate is converted to pyruvate via a two-enzyme process. Acetate and carbon dioxide are produced as side-products in this pathway.

[0022] DETAILED DESCRIPTION OF THE INVENTION

[0023] Definitions

[0024] All definitions of herein relevant terms are in accordance with what would be understood by the skilled person in relation to the herein relevant technical context.

[0025] In the context of the present invention in any of its embodiments, the expression "lactic acid bacteria" (sometimes abbreviated as "LAB") designates food-grade bacteria producing lactic acid as the major metabolic end-product of carbohydrate fermentation. These bacteria are related by their common metabolic and physiological characteristics and are usually Gram positive, low-GC, acid tolerant, non-sporulating, non-respiring, rod-shaped bacilli or cocci. Lacticaseibacillus rhamnosus is a species of LAB. "A-milk" refers to a solution that is prepared by reconstituting skimmed milk powder in distilled water and autoclaving the reconstituted skimmed milk at 115 °C for 15 minutes (see ISO 26323:2009). A-milk contains a dry matter content of 9.5% mass fraction.

[0026] "B-milk" refers to a solution that is prepared by reconstituting skimmed milk powder in distilled water and pasteurizing the reconstituted skimmed milk at 99 °C for 30 minutes (see ISO 26323:2009). B-milk contains a dry matter content of 9.5% mass fraction.17967-EP-EPA[2]

[0027] 3

[0028] The lactic acid bacterium strain of the invention may be an isolated strain, e.g., isolated from a naturally occurring source, or may be a non-naturally occurring strain, e.g., obtained recombinantly.

[0029] Recombinant strains will differ from naturally occurring strains by at least the presence of the nucleic acid construct(s) used to transform or transfect the mother strain.

[0030] The term "sequence identity" relates to the relatedness between two nucleotide sequences or between two amino acid sequences. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul etal. (1990) J. Mol. Biol. 215: 403-410 and Altschul etal. (1977) Nucleic Acids Res.

[0031] 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) website and for purposes of the present invention, the degree of sequence identity between two nucleotide sequences or two amino acid sequences is determined using the software made available by the NCBI. For nucleotide sequences, the default parameters of the megablast program are used (Expect threshold: 0.05; Word size: 28; Max matches in a query range: 0; Match / Mismatch Scores: 1,-2; Gap Costs: Linear; Filter: Low complexity regions; Mask: Mask for lookup table only). For amino acid sequences, the default parameters of the blastp program are used (Expect threshold: 0.05; Word size: 5; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence: 11 Extension: 1; Compositional adjustments: Conditional compositional score matrix adjustment; Filter: no options selected; Mask: no options selected). Sequence identity is calculated over the entire length of the reference sequence. For example, if a sequence is 100% identical to the reference sequence over just 70% of the entire length of the reference sequence, such a sequence has 70% identity with the reference sequence.

[0032] In the present context, the terms "strains derived from", "derived strain", "variant", "mutant" or "mutant derived from" should be understood as a strain derived from a strain of the invention or a mother strain by means of, e.g., genetic engineering, radiation and / or chemical treatment, and / or selection, adaptation, screening, etc. It is preferred that the derived strain is a functionally equivalent mutant, e.g., a strain that is citrate-deficient. Such a derived strain is a part of the present invention. Especially, the term "derived strain" or "mutant" refers to a strain obtained by subjecting a strain of the invention to any conventionally used mutagenesis treatment including treatment with a chemical mutagen such as ethyl methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV light or to a spontaneously occurring mutant. A mutant may have been subjected to several mutagenesis treatments (a single treatment should be understood one mutagenesis step followed by a screening / selection step), but it is presently preferred that no more than 20, no more than 10, or no more than 5, treatments are carried out. In a presently preferred derived strain, less than 1%, or less than 0.1%, less than 0.01%, less than 0.001% or even less than 0.0001% of the nucleotides in the17967-EP-EPA[2]

[0033] 4

[0034] bacterial genome have been changed (such as by replacement, insertion, deletion or a combination thereof) compared to the mother strain or strain of the invention.

[0035] The term "citrate-deficient" is used in the context of the present invention to characterize lactic acid bacteria which partly or completely have lost the ability to use citric acid as a source for maintaining cell viability or cell growth. In other words, the strain has partly or completely lost the ability to metabolize citric acid. A citrate-deficient strain of the present invention is less capable of metabolizing citric acid than a Lacticaseibacillus rhamnosus GG (LGG) strain. In some embodiments, a citrate-deficient strain of the present invention metabolizes less citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) xylitol than a LGG strain. In some embodiments, a citrate-deficient strain of the present invention metabolizes less citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) erythritol than a LGG strain. In some embodiments, a citrate-deficient strain of the present invention does not metabolize citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) xylitol. In some embodiments, a citrate-deficient strain of the present invention does not metabolize citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) erythritol. The citrate-deficient strain can be incubated in the acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) xylitol or erythritol for 30 days at 30 °C. The concentration of citric acid in the A-milk containing 0.1% (w / v) fructose and 2% (w / v) xylitol or erythritol can be measured using HPLC before adding the citrate-deficient strain and 30 days after adding the citrate-deficient strain. If the concentration of the citric acid is not reduced by 10% or more within 30 days, it can be concluded that the strain does not metabolize citric acid. The acidified A-milk of any one of the above embodiments is preferably obtained by adding hydrochloric acid to A-milk until the pH is reduced to 4.5.

[0036] The Lacticaseibacillus rhamnosus GG (commonly abbreviated as LGG) strain is a well-known publicly available probiotic strain. The probiotic strain is sold by Chr. Hansen A / S as LGG®. The genome sequence of the Lacticaseibacillus rhamnosus GG strain has been deposited in the database of the National Center for Biotechnology Information (NCBI) (see https: / / www.ncbi.nlm.nih.gov / ) and can be accessed under reference number GCF_028475085.1 as Lacticaseibacillus rhamnosus GG genome assembly ASM2847508vl. Reference is made to the database in the version accessible on 07 March 2024. The same genome sequence of the Lacticaseibacillus rhamnosus GG strain is provided in SEQ. ID NO: 1 of PCT / EP2025 / 056529.

[0037] The term "citrate-negative" is used in the context of the present invention to characterize lactic acid bacteria which completely have lost the ability to use citric acid as a source for maintaining cell viability or cell growth. In other words, the strain has completely lost the ability to metabolize citric acid. The term "acidified milk product" as used herein refers to a milk product wherein the preparation of the milk product involves acidification of a milk base. Acidification may be brought about by chemical17967-EP-EPA[2]

[0038] 5

[0039] acidification by adding an acid or compositions comprising acid. Examples of acids include but are not limited to adipic acid, citric acid (commonly found in lemon juice), fumaric acid, glucono-delta-lactone, hydrochloric acid, lactic acid, malic acid, phosphoric acid, succinic acid, and tartaric acid. Acidification may also be a result of fermentation by lactic acid bacteria(s) when added to a milk base or a preacidified milk base comprising one or more substrate(s) fermentable by the lactic acid bacteria(s). Acidification may also be a result of chemical acidification in combination with fermentation by lactic acid bacteria. Chemical acidification and fermentation by lactic acid bacteria may be conducted in the same step or in separate steps. The acidified milk product may be suitable for storage at ambient temperature. The acidified milk product is preferably pasteurized. In some embodiments the acidified milk product is a yogurt, buttermilk, kefir or quark. Preferably, the acidified milk product is a yogurt. More preferably, the acidified milk product is pasteurized yogurt. The yogurt may be set or stirred. The yogurt may be a strained yogurt (also referred to as Greek-style yogurt, chakka, labneh, suzma or yogurt cheese), a yogurt-based product such as fruit yogurt, or a yogurt-based beverage such as drinking yogurt.

[0040] The expression "ambient storage product" refers to a product, which is suitable for storage at ambient temperature for a period of time. Storage period may be between 1-12 month, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. The term "ambient temperature” in the present context refers to a temperature above 10°C, such as above 15°C, 20°C, 25°C, or 30°C.

[0041] The term "thermophilic" herein refers to microorganisms that thrive best at temperatures above 35°C. The industrially most useful thermophilic bacteria include Streptococcus spp. and Lactobacillus spp. The term "thermophilic fermentation" herein refers to fermentation at a temperature above about 35°C, such as between about 35°C to about 45°C. The term "thermophilic fermented milk product" refers to fermented milk products prepared by thermophilic fermentation of a thermophilic starter culture and include such fermented milk products as set-yoghurt, stirred-yoghurt and drinking yoghurt, e.g., Yakult.

[0042] The term "mesophilic" herein refers to microorganisms that thrive best at moderate temperatures (15°C-35°C). The industrially most useful mesophilic bacteria include Lactococcus spp. and Leuconostoc spp. The term "mesophilic fermentation" herein refers to fermentation at a temperature between about 22°C and about 35°C. The term "mesophilic fermented milk product" refers to fermented milk products prepared by mesophilic fermentation of a mesophilic starter culture and include such fermented milk products as buttermilk, sour milk, cultured milk, smetana, sour cream, Kefir and fresh cheese, such as quark, tvarog and cream cheese.

[0043] The term "thermophilic starter culture" herein refers to any starter cultures culture containing at least one thermophilic bacterium strain. Thermophilic starter cultures, such as combinations of17967-EP-EPA[2]

[0044] 6

[0045] Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus strains, are used to produce fermented milk products, such as yoghurt.

[0046] In the context of the present application, the term "milk" is broadly used in its common meaning to refer to liquids produced by the mammary glands of animals (e.g., cows, sheep, goats, buffaloes, camel, etc., "mammalian milk"). The term "milk" preferably refers to cow's milk.

[0047] The expression “milk base" may be any raw or processed milk ingredient or other material derived from milk that can be subjected to acidification as described herein. Thus, useful milk bases include, but are not limited to, whole milk, full fat milk, fat-free milk, low fat milk, skim milk, lactose-reduced milk, concentrated milk, reconstituted milk powder, condensed milk, dried milk, and cream. Obviously, the milk base may originate from any mammal, e.g., being substantially pure mammalian milk, or reconstituted milk powder.

[0048] "Homogenizing" as used herein means intensive mixing to obtain a soluble suspension or emulsion. If homogenization is performed prior to acidification, it may be performed so as to break up the milk fat into smaller sizes so that it no longer separates from the milk. This may be accomplished by forcing the milk at high pressure through small orifices.

[0049] "Pasteurizing" as used herein, means treatment of the milk, milk base or acidified milk product to reduce or eliminate the presence of live organisms, such as microorganisms. Preferably, pasteurization is attained by maintaining a specified temperature for a specified period of time. The specified temperature is usually attained by heating. The temperature and duration may be selected in order to kill or inactivate certain bacteria, such as harmful bacteria. A rapid cooling step may follow.

[0050] As used herein, the term "about" (or "around") means the indicated value ± 1% of its value, or the term "about" means the indicated value ± 2% of its value, or the term "about" means the indicated value ± 5% of its value, the term "about" means the indicated value ± 10% of its value, or the term "about" means the indicated value ± 20% of its value, or the term "about" means the indicated value ± 30% of its value; preferably the term "about" means exactly the indicated value (± 0%).

[0051] Throughout the description and claims the word "comprise" and variations of the word (e.g., "comprising", "having", "including", "containing") typically is not limiting and thus does not exclude other features, which may be for example technical features, additives, components, or steps. However, whenever the word "comprise" is used herein, this also includes a special embodiment in which this word is understood as limiting; in this particular embodiment the word "comprise" has the meaning of the term "consist of'.

[0052] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the17967-EP-EPA[2]

[0053] 7

[0054] singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0055] Lacticaseibacillus rhamnosus strains of the invention

[0056] It is an object of the present invention to provide Lacticaseibacillus rhamnosus strains that can survive in an acidified milk product at ambient temperature without producing acetate or CO2. This object has been achieved by the presently disclosed citrate-deficient Lacticaseibacillus rhamnosus strains of the invention.

[0057] In a first aspect, the present invention provides a citrate-deficient Lacticaseibacillus rhamnosus strain. In some embodiments, the citrate-deficient strain of the present invention metabolizes less citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) xylitol than a LGG strain. Preferably, the citrate-deficient strain of the present invention metabolizes less citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) xylitol than a LGG strain after 13 days (preferably, after 30 days) at 30°C. In some embodiments, the citrate-deficient strain of the present invention metabolizes less citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) erythritol than a LGG strain. Preferably, the citrate-deficient strain of the present invention metabolizes less citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) erythritol than a LGG strain after 13 days (preferably, after 30 days) at 30°C.

[0058] In some embodiments, the citrate-deficient strain of the present invention metabolizes less than 70% (e.g., less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, or less than 15%) of the available citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) xylitol after 13 days at 30°C. Preferably, the citrate-deficient strain of the present invention metabolizes less than 35% (e.g., less than 30%, less than 25%, less than 20%, or less than 15%) of the available citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) xylitol after 13 days at 30°C. In some embodiments, the citrate-deficient strain of the present invention metabolizes less than 70% (e.g., less than 65%, less than 60%,17967-EP-EPA[2]

[0059] 8

[0060] less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, or less than 15%) of the available citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) erythritol after 13 days at 30°C. Preferably, the citrate-deficient strain of the present invention metabolizes less than 35% (e.g., less than 30%, less than 25%, less than 20%, or less than 15%) of the available citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) erythritol after 13 days at 30°C.

[0061] In some embodiments, the citrate-deficient strain of the present invention metabolizes less than 85% (e.g., less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, or less than 15%) of the available citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) xylitol after 30 days at 30°C. Preferably, the citrate-deficient strain of the present invention metabolizes less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, or less than 15%) of the available citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) xylitol after 30 days at 30°C. In some embodiments, the citrate-deficient strain of the present invention metabolizes less than 85% (e.g., less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, or less than 15%) of the available citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) erythritol after 30 days at 30°C. Preferably, the citrate-deficient strain of the present invention metabolizes less than 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, or less than 15%) of the available citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) erythritol after 30 days at 30°C.

[0062] In some embodiments, the citrate-deficient strain of the present invention does not metabolize citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) xylitol. Preferably, the citric acid concentration in the A-milk containing 0.1% (w / v) fructose and 2% (w / v) xylitol is reduced by less than 10% after 13 days at 30°C. More preferably, the citric acid concentration in the A-milk containing 0.1% (w / v) fructose and 2% (w / v) xylitol is reduced by less than 10% after 30 days at 30°C. In some embodiments, the citrate-deficient strain of the present invention does not metabolize citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) erythritol. Preferably, the citric acid concentration in the A-milk containing 0.1% (w / v) fructose and 2% (w / v) erythritol is reduced by less than 10% after 13 days at 30°C. More preferably, the citric acid concentration in the A-milk containing 0.1% (w / v) fructose and 2% (w / v) erythritol is reduced by less than 10% after 30 days at 30°C.

[0063] The acidified A-milk of any one of the above embodiments is preferably obtained by adding hydrochloric acid to A-milk until the pH is reduced to 4.5. The citrate-deficient strain of the present17967-EP-EPA[2]

[0064] 9

[0065] invention may have at least partly lost its ability to utilize citric acid in accordance with any one of the above embodiments in both acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) erythritol and acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) xylitol.

[0066] All strains disclosed in Table 1 below do not metabolize citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) xylitol. All strains disclosed in Table 1 below also do not metabolize citric acid in acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) erythritol. As shown in the Examples of the present disclosure, citric acid concentrations did not decrease after 30 days at 30°C. In some embodiment, the citrate-deficient strain of the present invention is:

[0067] (i) the strain deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on April 3, 2025 under the accession no. DSM 35404;

[0068] (ii) the strain deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on June 25, 2024 under the accession no. DSM 35074;

[0069] (iii) the strain deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on June 25, 2024 under the accession no. DSM 35075; or

[0070] (iv) a citrate-deficient mutant derived from (i), (ii) or (iii).

[0071] In some embodiments, the Lacticaseibacillus rhamnosus strain of the invention shares at least 99.2% (e.g., at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, or at least 99.8%) average nucleotide identity (ANI) with DSM 35404. In some embodiments, the Lacticaseibacillus rhamnosus strain of the invention shares at least 99.9% average nucleotide identity (ANI) with DSM 35404. In a preferred embodiment, the Lacticaseibacillus rhamnosus strain of the invention shares at least 99.99% average nucleotide identity (ANI) with DSM 35404.

[0072] In some embodiments, the Lacticaseibacillus rhamnosus strain of the invention shares at least 99.2% (e.g., at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, or at least 99.8%) average nucleotide identity (ANI) with DSM 35074. In some embodiments, the Lacticaseibacillus rhamnosus strain of the invention shares at least 99.9% average nucleotide identity (ANI) with DSM 35074. In a preferred embodiment, the Lacticaseibacillus rhamnosus strain of the invention shares at least 99.99% average nucleotide identity (ANI) with DSM 35074.

[0073] In some embodiments, the Lacticaseibacillus rhamnosus strain of the invention shares at least 99.2% (e.g., at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, or at least 99.8%)17967-EP-EPA[2]

[0074] 10

[0075] average nucleotide identity (ANI) with DSM 35075. In some embodiments, the Lacticaseibacillus rhamnosus strain of the invention shares at least 99.9% average nucleotide identity (ANI) with DSM 35075. In a preferred embodiment, the Lacticaseibacillus rhamnosus strain of the invention shares at least 99.99% average nucleotide identity (ANI) with DSM 35075.

[0076] ANI can be calculated using FastANI version 1.32, "One to Many" mode with default settings (see, for example, Jain et al., 2018. Nat Commun. 9(1):5114 and Rodriguez-R et al., 2024. mBio.

[0077] 15(l):e0269623).

[0078] In some embodiments, the citrate-deficient strain of the present invention is citrate-negative. DSM 35074 and DSM 35075 are examples of citrate-negative strains.

[0079] In some embodiments, the Lacticaseibacillus rhamnosus strain of the present invention contains a loss-of-function mutation in the citN gene. A loss-of-function mutation refers to any mutation that reduces the expression of a gene or the activity of the gene product encoded by the gene. Without being bound by a particular theory, the citN gene encodes a citrate transporter and any loss-of-function mutation will result in citrate deficiency. The sequence of a native citN gene as well as the gene product encoded by the citN gene are provided below.

[0080] citN nucleotide sequence (SEQ ID NO: 1) - 1350 base pairs.

[0081] ATGTTACTCA CCGTCATTGC TTATGCCATG ATCATTGTCT TCATGTACGT CATCATGACT AAGAAACTTT CACCGTTTAC TTCCTTGGTT ATGATCCCGC TATTATTTGC GATTATTGCA ATGGTGGCTG GCGTGGCAAA GAAAGGAACG ATAGGCGATT TCGTGTTAAA AGGTCTGACG ACGACTGCTA ACACCGGGAT CATGCTCTTA TTCGCCATTC TTTATTTCTC CATTATGCTT GACGCTGGTT TGTTTGATCC GATTACGGCA CGCATGATCA AGATTGCAAA AGGCGACCCA ATGAAAGTGC TGATGGCAAC CGCAATTGTT GCGATGGCCG TTTCACTCAA CGGGGACGGC ACGACCACAA CTTTGATTTG TTGCTCGGCA TTTATTCCGA TTTATAAGAA GCTCAACATG AATATGATGA ACCTGGGTGT TTTGATTATT CTGCAAAATA CCATCATGAA CCTATTGCCT TGGGGTGGGC CAACTGCACG GGCAATGGCA GTTTTAAAAG TTGATGCGGA TATTTTGACC TACCTGTTGC CAGGGATGAT TTTGGCACTG GCTTATGTGA TCTTCTATGT GGCACCGCAC ATGGGTCGCG CAGAACGGAA ACGCTTGGGA GTTCGGGAGT TAACCGATGA AGAAATTGAC GAAATGACAT CGGTAGTGGA TCCGGAAGTT TCTGAAATTC GCCGCCCGAA TATGTTCCTG TTTAACGGTA TTTTGACGAT TGTTCTGATT GCCTGGTTGG TGGCAAGTTC CTTTATTAAA GCCATTGCAA TGCCGCCATT GTTGCTGTTC TTGGTCGGAA CCTGTATCGC ATTGATGGCA AACTATCCTA AGTTAGGCGA CCAAAGCAAA CGGATCGGCG CTAATGGTGG CGATGCCGTT CAAGTGGTTA TTCTGGTGTT TGCAGCCGGG GTCTTCATGG GTCTGTTCCA AGGAACCGGG ATGGCAGAAG CGTTGGCTAA GAGCTTTACC GCAATTATTC CAAATTCTAT GGCTGGCTTC TGGGGGCTCG TGATCGCATT GATTTCGGCG CCAGGGACAT TCTTCTTAAG TAATGATGGT TTTTACTTCG GCGTTATGCC GGTTTTGGCA ACTGCCGGCC GTGCCTATGG GTTTACTAAC17967-EP-EPA[2]

[0082] 11

[0083] ATGCAAATGG CATTGGCTTC ATTAATGGGT CAGGCATTCC ACTTACTGTC GCCATTGGTT GCTTTCATCT ACCTCTTACT GCGGCTAACC GGCTTGGATA TGGGTAAGTG GCAACGCGAA GCCGGAAAAT ATGCATTAGG CGTGTTTGCA ATTTTCGTGG TGACGGTTAT GCTCTTTGGT CACGTGCCGT TCTACCTGCC GCAAAAATAG

[0084] Protein sequence encoded by citN (SEQ ID NO: 2) - 450 amino acids.

[0085] MLLTVIAYAM IIVFMYVIMT KKLSPFTSLV MIPLLFAIIA MVAGVAKKGT IGDFVLKGLT TTANTGIMLL FAILYFSIML DAGLFDPITA RMIKIAKGDP MKVLMATAIV AMAVSLNGDG TTTTLICCSA FIPIYKKLNM NMMNLGVLII LQNTIMNLLP WGGPTARAMA VLKVDADILT YLLPGMILAL AYVIFYVAPH MGRAERKRLG VRELTDEEID EMTSVVDPEV SEIRRPNMFL FNGILTIVLI AWLVASSFIK AIAMPPLLLF LVGTCIALMA NYPKLGDQSK RIGANGGDAV QVVILVFAAG VFMGLFQGTG MAEALAKSFT AIIPNSMAGF WGLVIALISA PGTFFLSNDG FYFGVMPVLA TAGRAYGFTN MQMALASLMG QAFHLLSPLV AFIYLLLRLT GLDMGKWQRE AGKYALGVFA IFVVTVMLFG HVPFYLPQK

[0086] The mutant strains of the present invention having a mutation in the citrate transporter CitN listed in the table below all have a citrate-deficient phenotype.

[0087] Table 1: Position of CitN mutation in the strains (numbering is based on the corresponding position in the two seguences indicated). _

[0088] CitN mutation in: LGG® DSM 35074 DSM 35075 CM1 CM2 CM3 SEQ ID NO: 2 - T4I Q152* D359N C128* G162D SEQ ID NO: 1 -

[0089]

[0090] ACC- > ATC CAA- > TAA GAT- > AAT TGC->TGA GGT- > GAT

[0091] A loss-of-function mutation may, for example, result in a premature stop codon. DSM 35075 contains a mutation that results in the presence of a premature stop codon in the citN gene. The CAA codon encoding Q152 (numbering based on SEQ ID NO: 2) is mutated to a TAA codon in DSM 35075. The mutant strain CM2 has a mutation in the citN gene where the TGC codon encoding C128 is mutated to a TGA codon that results in a stop codon. In some embodiments, a loss-of-function mutation inserts a premature stop codon. In some embodiments the invention relates to a loss-of function mutation that inserts a premature stop codon resulting in a truncated version of the encoded protein, wherein the protein is truncated from a position corresponding to position 152 and / or 128 of SEQ ID NO: 2.

[0092] A loss-of-function mutation may also result in an amino acid substitution that reduces the expression or activity of the gene product. DSM 35074 has a mutation in the citN gene where the ACC codon encoding T4 is mutated to a ATC codon that results in a T4I substitution. The mutant strain CM1 has a mutation in the citN gene where the GAT codon encoding D359 is mutated to a AAT codon that results in a D359N substitution. The mutant strain CM3 has a mutation in the citN gene where the GGT codon encoding G162 is mutated to a GAT codon that results in a G162D substitution. In some embodiments,17967-EP-EPA[2]

[0093] 12

[0094] the invention relates to a loss-of-function mutation resulting in an amino acid substitution that reduces the expression or activity of the gene product. In some embodiments of the invention the loss-of-function mutation is an amino acid substitution at a position corresponding to position 4, position 162, and / or position 359 of SEQ ID NO:2. In some embodiments of the invention the loss-of-function mutation is a substitution selected from 41, 162D and / or 359N. In some embodiments of the invention the loss-of function mutation is T4I, G162D and / or D359.

[0095] In any of the above embodiments, the Lacticaseibacillus rhamnosus strain of the present invention may be a citrate-deficient mutant derived from an LGG strain. In some embodiments, the Lacticaseibacillus rhamnosus strain of the invention shares at least 99.2% (e.g., at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, or at least 99.8%) average nucleotide identity (ANI) with an LGG strain. In a preferred embodiment, the Lacticaseibacillus rhamnosus strain of the invention shares at least 99.9% (e.g., at least 99.91, 99.92, 99.93, 99.94, 99.95, 99.96, 99.97, 99.98 or 99.99%) average nucleotide identity (ANI) with an LGG strain. In some embodiments, the Lacticaseibacillus rhamnosus strain of the invention shares at least 99.99% average nucleotide identity (ANI) with an LGG strain. The genome sequence of a LGG strain is available under reference number GCF_028475085.1 (version accessible on 07 March 2024) or SEQ ID NO: 1 of PCT / EP2025 / 056529 as indicated above in the section entitled "Definitions".

[0096] In some embodiments, the Lacticaseibacillus rhamnosus strain of the present invention comprises a nucleotide sequence that is not identical to the genome sequence of the Lacticaseibacillus rhamnosus GG strain and has at least 99.00% identity to the genome sequence of the Lacticaseibacillus rhamnosus GG strain, preferably at least 99.10%, at least 99.20%, at least 99.30%, at least 99.40%, at least 99.50%, at least 99.60%, at least 99.70%, or at least 99.80% identity to the genome sequence of the Lacticaseibacillus rhamnosus GG strain, more preferably at least 99.90%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, or at least 99.98% identity to the genome sequence of the Lacticaseibacillus rhamnosus GG strain, even more preferably at least 99.99% identity to the genome sequence of the Lacticaseibacillus rhamnosus GG strain. The genome sequence of the Lacticaseibacillus rhamnosus GG strain is available under reference number GCF_028475085.1 (version accessible on 07 March 2024) or SEQ ID NO: 1 of PCT / EP2025 / 056529 as indicated above in the section entitled "Definitions".

[0097] In some embodiments, the Lacticaseibacillus rhamnosus strain of the present invention comprises a nucleotide sequence that differs by less than 100 nucleotides (e.g., less than 90, 80, 70, 60, 50, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27 , 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides) when compared to the genome sequence of the Lacticaseibacillus rhamnosus GG strain and is not identical to the genome sequence of the Lacticaseibacillus rhamnosus GG strain. In some17967-EP-EPA[2]

[0098] 13

[0099] embodiments, the Lacticaseibacillus rhamnosus strain of the present invention comprises a nucleotide sequence that differs by less than 35 nucleotides (e.g., less than 34, 33, 32, 31, 30, 29, 28, 27 , 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides) when compared to the genome sequence of the Lacticaseibacillus rhamnosus GG strain and is not identical to the genome sequence of the Lacticaseibacillus rhamnosus GG strain. In some embodiments, the Lacticaseibacillus rhamnosus strain of the present invention comprises a nucleotide sequence that differs by less than 31 nucleotides (e.g., less than 30, 29, 28, 27 , 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides) when compared to the genome sequence of the Lacticaseibacillus rhamnosus GG strain and is not identical to the genome sequence of the Lacticaseibacillus rhamnosus GG strain. In some embodiments, the Lacticaseibacillus rhamnosus strain of the present invention comprises a nucleotide sequence that differs by less than 16 nucleotides (e.g., less than 15, 14, 13, 12, 11 or 10 nucleotides) when compared to the genome sequence of the Lacticaseibacillus rhamnosus GG strain and is not identical to the genome sequence of the Lacticaseibacillus rhamnosus GG strain. The genome sequence of the Lacticaseibacillus rhamnosus GG strain is available under reference number GCF_028475085.1 (version accessible on 07 March 2024) or SEQ ID NO: 1 of PCT / EP2025 / 056529 as indicated above in the section entitled "Definitions".

[0100] In some embodiments, the Lacticaseibacillus rhamnosus strain of the present invention comprises a nucleotide sequence that differs in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides from the genome sequence of the Lacticaseibacillus rhamnosus GG strain. Preferably, the nucleotide sequence of the strain of the present invention differs in no more than 5000, 4000, 3000, 2750, 2500, 2250, 2000, 1750, 1500, 1250, 1000, 750, 500, 400300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15 nucleotides from the genome sequence of the Lacticaseibacillus rhamnosus GG strain. The genome sequence of the Lacticaseibacillus rhamnosus GG strain is available under reference number GCF_028475085.1 (version accessible on 07 March 2024) or SEQ ID NO: 1 of PCT / EP2025 / 056529 as indicated above in the section entitled "Definitions". In some embodiments, the Lacticaseibacillus rhamnosus strain of the present invention comprises a nucleotide sequence that differs by 1-100 nucleotides (e.g., 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-35 or 1-30 nucleotides) when compared to the genome sequence of the Lacticaseibacillus rhamnosus GG strain. In some embodiments, the Lacticaseibacillus rhamnosus strain of the present invention comprises a nucleotide sequence that differs by 2-100 nucleotides (e.g., 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-35 or 2-30 nucleotides) when compared to the genome sequence of the Lacticaseibacillus rhamnosus GG strain. In some embodiments, the Lacticaseibacillus rhamnosus strain of the present invention comprises a nucleotide sequence that differs by 3-100 nucleotides (e.g., 3-90, 3-80, 3-70, 3-60, 3-50, 3-40, 3-35 or 3-30 nucleotides) when compared to the genome sequence of the17967-EP-EPA[2]

[0101] 14

[0102] Lacticaseibacillus rhamnosus GG strain. In some embodiments, the Lacticaseibacillus rhamnosus strain of the present invention comprises a nucleotide sequence that differs by 4-100 nucleotides (e.g., 4-90, 4-80, 4-70, 4-60, 4-50, 4-40, 4-35 or 4-30 nucleotides) when compared to the genome sequence of the Lacticaseibacillus rhamnosus GG strain. In some embodiments, the Lacticaseibacillus rhamnosus strain of the present invention comprises a nucleotide sequence that differs by 5-100 nucleotides (e.g., 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-35 or 5-30 nucleotides) when compared to the genome sequence of the Lacticaseibacillus rhamnosus GG strain. In some embodiments, the Lacticaseibacillus rhamnosus strain of the present invention comprises a nucleotide sequence that differs by 10-100 nucleotides (e.g., 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-35 or 10-30 nucleotides) when compared to the genome sequence of the Lacticaseibacillus rhamnosus GG strain. The genome sequence of the Lacticaseibacillus rhamnosus GG strain is available under reference number GCF_028475085.1 (version accessible on 07 March 2024) or SEQ. ID NO: 1 of PCT / EP2025 / 056529 as indicated above in the section entitled "Definitions".

[0103] COMPOSITION OF THE INVENTION

[0104] In a second aspect, the present invention relates to a composition comprising the Lacticaseibacillus rhamnosus strain of the present invention. The Lacticaseibacillus rhamnosus strain may be in accordance with any of the embodiments disclosed herein (e.g., any embodiment described for the first aspect of the invention).

[0105] Preferably, the composition of the present invention in any of its embodiments comprises at least 1x10sCFU (colony-forming units) / ml or 1x10sCFU / g total LAB strains. It may be preferred that the composition comprises at least 1x10sCFU / ml or CFU / g total LAB strains.

[0106] Lactic acid bacteria, including bacteria of the species Lacticaseibacillus rhamnosus, are normally supplied to the dairy industry either as frozen (F-DVS) or freeze-dried (FD-DVS) cultures for bulk starter propagation or as so-called "Direct Vat Set" (DVS) cultures, intended for direct inoculation into a fermentation vessel or vat for the production of a dairy product. Accordingly, the composition of the present invention may be frozen or freeze-dried. Such frozen or freeze-dried composition generally comprise at least 10sCFU / g (e.g., at least 107, 108, 109, 1010or 1011CFU / g) of a LAB strain. In addition, the composition of the present invention may be provided in liquid form. Thus, in some embodiments, the composition is in frozen, dried, freeze-dried or liquid form.

[0107] In some embodiments, the composition:

[0108] (a) is freeze-dried or frozen; and / or17967-EP-EPA[2]

[0109] 15

[0110] (b) comprises at least 106CFU / g (e.g., at least 107, 108, 109, IO10or IO11CFU / g) of the Lacticaseibacillus rhamnosus strain of the present invention.

[0111] The composition of the present invention may additionally comprise cryoprotectants, lyoprotectants, antioxidants, nutrients, fillers, flavorants or mixtures thereof. The composition preferably comprises one or more of cryoprotectants, lyoprotectants, antioxidants and / or nutrients, more preferably cryoprotectants, lyoprotectants and / or antioxidants and most preferably cryoprotectants or lyoprotectants, or both. Use of protectants such as cryoprotectants and lyoprotectants are known to a skilled person in the art. Suitable cryoprotectants or lyoprotectants include mono-, di-, tri-and polysaccharides (such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch and gum arabic (acacia) and the like), polyols (such as erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol and the like), amino acids (such as proline, glutamic acid), complex substances (such as skim milk, peptones, gelatin, yeast extract) and inorganic compounds (such as sodium tripolyphosphate).

[0112] In some embodiments, the composition according to the present invention may comprise one or more cryoprotective agent(s) selected from the group consisting of inosine-5' -monophosphate (IMP), adenosine -5'-monophosphate (AMP), guanosine-5'-monophosphate (GMP), uranosine-5'-monophosphate (UMP), cytidine-5'-monophosphate (CMP), adenine, guanine, uracil, cytosine, adenosine, guanosine, uridine, cytidine, hypoxanthine, xanthine, hypoxanthine, orotidine, thymidine, inosine and a derivative of any such compounds. Suitable antioxidants include ascorbic acid, citric acid and salts thereof, gallates, cysteine, sorbitol, mannitol, maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, vitamins (such as vitamin B-family, vitamin C). The composition may optionally comprise further substances including fillers (such as lactose, maltodextrin) and / or flavorants.

[0113] In some embodiments of the invention the cryoprotective agent is an agent or mixture of agents, which in addition to its cryoprotectivity has a booster effect. The expression "booster effect" is used to describe the situation wherein the cryoprotective agent confers an increased metabolic activity (booster effect) on to the thawed or reconstituted culture when it is inoculated into the medium to be fermented or converted. Viability and metabolic activity are not synonymous concepts. Commercial frozen or freeze-dried cultures may retain their viability, although they may have lost a significant portion of their metabolic activity, e.g., cultures may lose their acid-producing (acidification) activity when kept stored even for shorter periods of time. Thus, viability and booster effect must be evaluated by different assays. Whereas viability is assessed by viability assays such as the determination of colony forming units, booster effect is assessed by quantifying the relevant metabolic activity of the thawed or reconstituted culture relative to the viability of the culture. The term "metabolic activity"17967-EP-EPA[2]

[0114] 16

[0115] refers to the oxygen removal activity of the cultures, its acid-producing activity, i. e. the production of, e. g., lactic acid, acetic acid, formic acid and / or propionic acid, or its metabolite producing activity such as the production of aroma compounds such as acetaldehyde, (a-acetolactate, acetoin, diacetyl and 2,3-butylene glycol (butanediol)).

[0116] In some embodiments, the composition of the invention comprises from 0.2% to 20% of the cryoprotective agent or mixture of agents measured as % w / w of the material. It is, however, preferable to add the cryoprotective agent or mixture of agents at an amount which is in the range from 0.2% to 15%, from 0.2% to 10%, from 0.5% to 7%, and from 1% to 6% by weight, including within the range from 2% to 5% of the cryoprotective agent or mixture of agents measured as % w / w of the frozen material by weight. In a preferred embodiment the culture comprises approximately 3% of the cryoprotective agent or mixture of agents measured as % w / w of the material by weight. The amount of approximately 3% of the cryoprotective agent corresponds to concentrations in the 100 mM range. It should be recognized that for each aspect of embodiment of the invention the ranges may be increments of the described ranges.

[0117] In some embodiments, the composition of the present invention is an acidified milk product. The acidified milk product may be suitable for storage at ambient temperature. The acidified milk product is preferably pasteurized. In some embodiments the acidified milk product is a yogurt, buttermilk, kefir or quark. Preferably, the acidified milk product is a yogurt. More preferably, the acidified milk product is pasteurized yogurt. The yogurt may be set or stirred. The yogurt may be a strained yogurt (also referred to as Greek-style yogurt, chakka, labneh, suzma or yogurt cheese), a yogurt-based product such as fruit yogurt, or a yogurt-based beverage such as drinking yogurt.

[0118] In some embodiments, the composition further comprises a sugar and / or a sugar alcohol. In some embodiments, the sugar is selected from fructose, galactose, glucose, and sucrose. Preferably, the sugar is fructose or sucrose. More preferably, the sugar is fructose. In some embodiments, the concentration of sugar is at least 0.001% (w / v), 0.005% (w / v), 0.01% (w / v), 0.05% (w / v), 0.10% (w / v), 0.15% (w / v), 0.20% (w / v), 0.25% (w / v), 0.30% (w / v), 0.35% (w / v), 0.40% (w / v), 0.45% (w / v), or 0.50% (w / v) or in the range of 0.05-0.50% (w / v) (e.g., 0.10-0.40% (w / v), or 0.15-0.30% (w / v)). In a preferred embodiment, the composition comprises at least 0.05% (w / v) fructose. More preferably, the composition comprises at least 0.1% (w / v) fructose.

[0119] In some embodiments, the sugar alcohol is a C4 sugar alcohol or a C5 sugar alcohol. The C4 sugar alcohol may be erythritol, D-threitol, or L-threitol. The C5 sugar alcohol may be xylitol, ribitol, D-arabitol, L-arabitol, D-lyxitol or L-lyxitol. Preferably, the sugar alcohol is xylitol or erythritol. More preferably, the sugar alcohol is erythritol. In some embodiments, the concentration of sugar alcohol is at least 0.001% (w / v), 0.005% (w / v), 0.01% (w / v), 0.05% (w / v), 0.10% (w / v), 0.15% (w / v), 0.20% (w / v),17967-EP-EPA[2]

[0120] 17

[0121] 0.25% (w / v), 0.5% (w / v), 1.0% (w / v), 1.5% (w / v), 2.0% (w / v), 2.5% (w / v), 3.0% (w / v), 3.5% (w / v), 4.0% (w / v), 4.5% (w / v), or 5.0% (w / v) or in the range of 0.5-5.0% (w / v), 1.0-4.5% (w / v), 1.5-4.0% (w / v), 2.0-3.5% (w / v), or 2.5-3.0% (w / v). The concentration is preferably at least 2% (w / v).

[0122] In some embodiments, the sugar alcohol is a C12 sugar alcohol. The C12 sugar alcohol may be a disaccharide. In some embodiments, the C12 sugar alcohol is maltitol. In some embodiments, the concentration of sugar alcohol is at least 0.001% (w / v), 0.005% (w / v), 0.01% (w / v), 0.05% (w / v), 0.10% (w / v), 0.15% (w / v), 0.20% (w / v), 0.25% (w / v), 0.5% (w / v), 1.0% (w / v), 1.5% (w / v), 2.0% (w / v), 2.5% (w / v), 3.0% (w / v), 3.5% (w / v), 4.0% (w / v), 4.5% (w / v), or 5.0% (w / v) or in the range of 0.5-5.0% (w / v), 1.0-4.5% (w / v), 1.5-4.0% (w / v), 2.0-3.5% (w / v), or 2.5-3.0% (w / v). The concentration is preferably at least 2% (w / v).

[0123] In a preferred embodiment, the composition comprises at least 1% (w / v) xylitol and / or erythritol. More preferably, the composition comprises at least 2% (w / v) xylitol and / or erythritol. In a preferred embodiment, the composition comprises at least 1% (w / v) erythritol. More preferably, the composition comprises at least 2% (w / v) erythritol.

[0124] In a preferred embodiment, the composition comprises at least 1% (e.g., at least 2%, 3% or 4%) (w / w) maltitol and / or erythritol. More preferably, the composition comprises at least 4% (w / w) maltitol and / or erythritol.

[0125] Sugar alcohol may be added as a single sugar alcohol or added as a mixture of two or more sugar alcohols. In some embodiments the disclosure relates to a combination of at least one C4 sugar alcohol and at least one C5 sugar alcohol. In some embodiments the at least one C4 sugar alcohol comprises erythritol. In some embodiments the at least one C5 sugar alcohol comprises xylitol. The ratio of C4:C5 sugar alcohols may vary. In some embodiments the C4:C5 ratio is selected from 1:99, 10:90, 20:80, 30:70, 40:60, 45:55, 50:50, 55:45, 60:40, 70:30, 80:20, 90:10, or 99:1. In some embodiments the erythritol to xylitol (Ery:Xyl) ratio is selected from 1:99, 10:90, 20:80, 30:70, 40:60, 45:55, 50:50, 55:45, 60:40, 70:30, 80:20, 90:10, or 99:1.

[0126] In some embodiments the disclosure relates to a combination of at least one C4 sugar alcohol and at least one C12 sugar alcohol. The C12 sugar alcohol may be a disaccharide. In some embodiments the at least one C4 sugar alcohol comprises erythritol. In some embodiments the at least one C12 sugar alcohol comprises maltitol. The ratio of C4:C12 sugar alcohols may vary. In some embodiments, the C4:C12 ratio is selected from 1:99, 10:90, 20:80, 1:3, 30:70, 40:60, 45:55, 50:50, 55:45, 60:40, 70:30, 80:20, 90:10, or 99:1. In some embodiments, the C4:C12 ratio is 20:80 to 30:60. In some embodiments the erythritol to maltitol (Ery:Mal) ratio is selected from 1:99, 10:90, 20:80, 1:3, 30:70, 40:60, 45:55,17967-EP-EPA[2]

[0127] 18

[0128] 50:50, 55:45, 60:40, 70:30, 80:20, 90:10, or 99:1. In some embodiments, the Ery:Mal ratio is 20:80 to 30:60 (e.g., 1:3).

[0129] In some embodiments, the composition (e.g., a pasteurized yogurt) further comprises:

[0130] (a) fructose; and

[0131] (b) xylitol and / or erythritol.

[0132] In some embodiments, the composition (e.g., a pasteurized yogurt) further comprises:

[0133] (a) at least 0.05% (w / v) fructose; and

[0134] (b) at least 1% (w / v) xylitol and / or erythritol.

[0135] In some embodiments, the composition (e.g., a pasteurized yogurt) further comprises:

[0136] (a) at least 0.05% (w / v) fructose; and

[0137] (b) at least 2% (w / v) xylitol and / or erythritol.

[0138] In some embodiments, the composition (e.g., a pasteurized yogurt) further comprises:

[0139] (a) at least 0.1% (w / v) fructose; and

[0140] (b) at least 2% (w / v) xylitol and / or erythritol.

[0141] In some embodiments, the composition (e.g., a pasteurized yogurt) further comprises:

[0142] (a) sucrose, fructose or galactose; and

[0143] (b) at least 2% (e.g., at least 3% or 4%) (w / w) maltitol and / or erythritol.

[0144] In some embodiments, the composition (e.g., a pasteurized yogurt) further comprises:

[0145] (a) sucrose, fructose or galactose; and

[0146] (b) at least 2% (e.g., at least 3% or 4%) (w / w) maltitol and erythritol. In some embodiments, the Ery:Mal ratio is 20:80 to 30:60 (e.g., 1:3).

[0147] USES OF THE STRAINS AND COMPOSITIONS OF THE PRESENT INVENTION

[0148] In a third aspect, the present invention relates to a method for producing an acidified milk product containing Lacticaseibacillus rhamnosus, the method comprising adding the Lacticaseibacillus rhamnosus strain of the present invention to a milk base or acidified milk product. The Lacticaseibacillus rhamnosus strain may be in accordance with any of the embodiments disclosed herein (e.g., any embodiment described for the first aspect of the invention). The Lacticaseibacillus rhamnosus may be added as part of a composition. The composition may be in accordance with any17967-EP-EPA[2]

[0149] 19

[0150] of the embodiments disclosed herein (e.g., any embodiment described for the second aspect of the invention).

[0151] In some embodiments, the composition or strain of the present invention is added to an acidified milk product. The acidified milk product may be suitable for storage at ambient temperature. The acidified milk product is preferably pasteurized prior to the addition of the strain or composition of the present invention. In some embodiments the acidified milk product is a yogurt, buttermilk, kefir or quark. Preferably, the acidified milk product is a yogurt. More preferably, the acidified milk product is pasteurized yogurt. The yogurt may be set or stirred. The yogurt may be a strained yogurt (also referred to as Greek-style yogurt, chakka, labneh, suzma or yogurt cheese), a yogurt-based product such as fruit yogurt, or a yogurt-based beverage such as drinking yogurt.

[0152] In some embodiments, the method further comprises adding a sugar and / or a sugar alcohol to the milk base or acidified milk product. In some embodiments, the sugar is selected from fructose, galactose, glucose, and sucrose. Preferably, the sugar is fructose or sucrose. More preferably, the sugar is fructose. In some embodiments, the concentration of sugar is at least 0.001% (w / v), 0.005% (w / v), 0.01% (w / v), 0.05% (w / v), 0.10% (w / v), 0.15% (w / v), 0.20% (w / v), 0.25% (w / v), 0.30% (w / v), 0.35% (w / v), 0.40% (w / v), 0.45% (w / v), or 0.50% (w / v) or in the range of 0.05-0.50% (w / v) (e.g., 0.10-0.40% (w / v), or 0.15-0.30% (w / v)). In a preferred embodiment, at least 0.05% (w / v) fructose is added. More preferably, at least 0.1% (w / v) fructose is added.

[0153] In some embodiments, the sugar alcohol is a C4 sugar alcohol or a C5 sugar alcohol. The C4 sugar alcohol may be erythritol, D-threitol, or L-threitol. The C5 sugar alcohol may be xylitol, ribitol, D-arabitol, L-arabitol, D-lyxitol or L-lyxitol. Preferably, the sugar alcohol is xylitol or erythritol. More preferably, the sugar alcohol is erythritol. In some embodiments, the concentration of sugar alcohol is at least 0.001% (w / v), 0.005% (w / v), 0.01% (w / v), 0.05% (w / v), 0.10% (w / v), 0.15% (w / v), 0.20% (w / v), 0.25% (w / v), 0.5% (w / v), 1.0% (w / v), 1.5% (w / v), 2.0% (w / v), 2.5% (w / v), 3.0% (w / v), 3.5% (w / v), 4.0% (w / v), 4.5% (w / v), or 5.0% (w / v) or in the range of 0.5-5.0% (w / v), 1.0-4.5% (w / v), 1.5-4.0% (w / v), 2.0-3.5% (w / v), or 2.5-3.0% (w / v). The concentration is preferably at least 2% (w / v).

[0154] In some embodiments, the sugar alcohol is a C12 sugar alcohol. The C12 sugar alcohol may be a disaccharide. In some embodiments, the C12 sugar alcohol is maltitol. In some embodiments, the concentration of sugar alcohol is at least 0.001% (w / v), 0.005% (w / v), 0.01% (w / v), 0.05% (w / v), 0.10% (w / v), 0.15% (w / v), 0.20% (w / v), 0.25% (w / v), 0.5% (w / v), 1.0% (w / v), 1.5% (w / v), 2.0% (w / v), 2.5% (w / v), 3.0% (w / v), 3.5% (w / v), 4.0% (w / v), 4.5% (w / v), or 5.0% (w / v) or in the range of 0.5-5.0% (w / v), 1.0-4.5% (w / v), 1.5-4.0% (w / v), 2.0-3.5% (w / v), or 2.5-3.0% (w / v). The concentration is preferably at least 2% (w / v).17967-EP-EPA[2]

[0155] 20

[0156] In a preferred embodiment, at least 1% (w / v) xylitol and / or erythritol is added. More preferably, at least 2% (w / v) xylitol and / or erythritol. In a preferred embodiment, at least 1% (w / v) erythritol is added. More preferably, at least 2% (w / v) erythritol is added.

[0157] In a preferred embodiment, the composition comprises at least 1% (e.g., at least 2%, 3% or 4%) (w / w) maltitol and / or erythritol. More preferably, the composition comprises at least 4% (w / w) maltitol and / or erythritol.

[0158] Sugar alcohol may be added as a single sugar alcohol or added as a mixture of two or more sugar alcohols. In some embodiments the disclosure relates to a combination of at least one C4 sugar alcohol and at least one C5 sugar alcohol. In some embodiments the at least one C4 sugar alcohol comprises Erythritol. In some embodiments, the at least one C5 sugar alcohol comprises Xylitol. The ratio of C4:C5 sugar alcohols may vary. In some embodiments the C4:C5 ratio is selected from 1:99, 10:90, 20:80, 30:70, 40:60, 45:55, 50:50, 55:45, 60:40, 70:30, 80:20, 90:10, or 99:1. In some embodiments the erythritol to xylitol (Ery:Xyl) ratio is selected from 1:99, 10:90, 20:80, 30:70, 40:60, 45:55, 50:50, 55:45, 60:40, 70:30, 80:20, 90:10, or 99:1.

[0159] In some embodiments the disclosure relates to a combination of at least one C4 sugar alcohol and at least one C12 sugar alcohol. The C12 sugar alcohol may be a disaccharide. In some embodiments the at least one C4 sugar alcohol comprises erythritol. In some embodiments the at least one C12 sugar alcohol comprises maltitol. The ratio of C4:C12 sugar alcohols may vary. In some embodiments, the C4:C12 ratio is selected from 1:99, 10:90, 20:80, 1:3, 30:70, 40:60, 45:55, 50:50, 55:45, 60:40, 70:30, 80:20, 90:10, or 99:1. In some embodiments, the C4:C12 ratio is 20:80 to 30:60. In some embodiments the erythritol to maltitol (Ery:Mal) ratio is selected from 1:99, 10:90, 20:80, 1:3, 30:70, 40:60, 45:55, 50:50, 55:45, 60:40, 70:30, 80:20, 90:10, or 99:1. In some embodiments, the Ery:Mal ratio is 20:80 to 30:60.

[0160] KIT OF THE INVENTION

[0161] In a fourth aspect, the present invention relates to a kit comprising the Lacticaseibacillus rhamnosus strain of the present invention. The Lacticaseibacillus rhamnosus strain may be in accordance with any of the embodiments disclosed herein (e.g., any embodiment described for the first aspect of the invention).

[0162] In some embodiments, the kit further comprises a sugar and / or a sugar alcohol. In some embodiments, the sugar is selected from fructose, galactose, glucose, and sucrose. Preferably, the sugar is fructose or sucrose. More preferably, the sugar is fructose.17967-EP-EPA[2]

[0163] 21

[0164] In some embodiments, the sugar alcohol is a C4 sugar alcohol or a C5 sugar alcohol. The C4 sugar alcohol may be erythritol, D-threitol, or L-threitol. The C5 sugar alcohol may be xylitol, ribitol, D-arabitol, L-arabitol, D-lyxitol or L-lyxitol. Preferably, the sugar alcohol is xylitol or erythritol. More preferably, the sugar alcohol is erythritol.

[0165] In some embodiments, the sugar alcohol is a C12 sugar alcohol. The C12 sugar alcohol may be a disaccharide. In some embodiments, the C12 sugar alcohol is maltitol.

[0166] In some embodiments, the kit comprises a C4 sugar alcohol, a C5 sugar alcohol and / or a C12 sugar alcohol. In some embodiments, the kit comprises a C4 sugar alcohol, and / or a C12 sugar alcohol. In some embodiments, the kit comprises a C4 sugar alcohol, and / or a C5 sugar alcohol

[0167] In some embodiments, the kit comprises:

[0168] (1) the Lacticaseibacillus rhamnosus strain of the present invention,

[0169] (2) fructose, sucrose or galactose; and

[0170] (3) xylitol, maltitol and / or erythritol.

[0171] In some embodiments, the kit comprises:

[0172] (1) the Lacticaseibacillus rhamnosus strain of the present invention,

[0173] (2) fructose; and

[0174] (3) xylitol and / or erythritol.

[0175] In some embodiments, the kit comprises:

[0176] (1) the Lacticaseibacillus rhamnosus strain of the present invention,

[0177] (2) sucrose, fructose or galactose; and

[0178] (3) maltitol and erythritol.

[0179] The sugar and / or sugar alcohol may be provided as a solid or a solution.

[0180] In some embodiments, the kit further comprises a starter culture. In some embodiments, the starter culture is a thermophilic starter culture. In some embodiments, the thermophilic starter culture comprises Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.

[0181] In some embodiments, the kit further comprises a Streptococcus thermophilus strain. In some embodiments, the kit further comprises a Streptococcus thermophilus strain and / or a Lactobacillus strain (e.g., Lactobacillus delbrueckii or Lactobacillus helveticus). In some embodiments, the kit further comprises a Streptococcus thermophilus strain and a Lactobacillus delbrueckii subsp. bulgaricus strain.17967-EP-EPA[2]

[0182] 22

[0183] The kit of the present invention may be used to make a composition in accordance with the second aspect of the present invention. The kit may be used in the method of the third aspect of the present invention.

[0184] METHODS FOR OBTAINING FURTHER CITRATE-DEFICIENT STRAINS

[0185] In a fifth aspect, the present invention relates to a method for obtaining a citrate-deficient Lacticaseibacillus rhamnosus strain (e.g., any one of the citate-deficient Lacticaseibacillus rhamnosus strains of the present invention according to the first aspect), the method comprising:

[0186] (1) subjecting a citrate-positive Lacticaseibacillus rhamnosus mother strain to mutagenesis to obtain a pool of mutant strains;

[0187] (2) screening the pool of mutant strains of step (1) for citrate metabolism; and

[0188] (3) selecting a strain from the pool of mutants that is citrate-deficient.

[0189] In some embodiments, the citrate-positive mother strain is a LGG strain or a strain that shares at least 99.9% ANI (e.g., at least 99.99% ANI) with a LGG strain. The genome sequence of a LGG strain is available under reference number GCF_028475085.1 (version accessible on 07 March 2024) or SEQ. ID NO: 1 of PCT / EP2025 / 056529 as indicated above in the section entitled "Definitions".

[0190] The mutagenesis can be performed using any conventionally used mutagenesis treatment including treatment with a chemical mutagen such as ethyl methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV light or to a spontaneously occurring mutant. In some embodiments, the mother strain is subjected to EMS mutagenesis.

[0191] Screening the pool of mutant strains of step (1) for citrate metabolism may involve growing one or more mutant strains on agar comprising an insoluble citrate salt (e.g., calcium citrate). The insoluble citrate salt makes the agar turbid. Strains that metabolize citrate exhibit clear zoning against the turbid background (see, for example, Galesloot et al., 1961. Neth Milk Dairy J. 15: 127-150; Nickels & Leesment, 1964. Milchwissenschaft. 19: 374-378; Reddy et al., 1973. Appl Microbiol. 24(6):947-952). Strains that do not create clear zones or produce clear zones that are more turbid than the citratepositive mother strain are selected.

[0192] In some embodiments, the method comprises:

[0193] (1) subjecting a citrate-positive Lacticaseibacillus rhamnosus mother strain to mutagenesis to obtain a pool of mutant strains;

[0194] (2) growing one or more mutant strain of the pool of mutants of step (1) on agar comprising an insoluble citrate salt (e.g., calcium citrate); and17967-EP-EPA[2]

[0195] 23

[0196] (3) selecting a strain from the pool of mutants that is citrate-deficient, wherein the citrate-deficient strain does not create clear zones on the agar or produces clear zones that are more turbid than the clear zones produced by the citrate-positive mother strain. In some embodiments, step (2) is performed by growing the one or more strain at 6-41°C (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41°C) for 1-14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days). In some embodiments, step (2) is performed by growing the one or more strain at 37°C for 3 days. The citrate-positive mother strain may be a LGG strain or a strain that shares at least 99.9% ANI (e.g., at least 99.99% ANI) with a LGG strain. The genome sequence of a LGG strain is available under reference number GCF_028475085.1 (version accessible on 07 March 2024) or SEQ. ID NO: 1 of PCT / EP2025 / 056529 as indicated above in the section entitled "Definitions".

[0197] DEPOSITS AND EXPERT SOLUTION

[0198] The applicant requests that a sample of the deposited microorganisms stated below may only be made available to an expert, subject to available provisions governed by Industrial Property Offices of States Party to the Budapest Treaty, until the date on which the patent is granted.

[0199] Table 2: Deposits were made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany.

[0200] Accession No. Deposit date Lacticaseibacillus rhamnosus DSM 35074 June 25, 2024 Lacticaseibacillus rhamnosus DSM 35075 June 25, 2024

[0201]

[0202] Lacticaseibacillus rhamnosus DSM 35404 April 3, 2025

[0203] EXAMPLES

[0204] Example 1: Generation and identification of citrate-deficient strains.

[0205] Ethyl methanesulfonate (EMS) mutagenesis was used to obtain a mutant pool from the mother strain L. rhamnosus LGG®. Pre-experiments were carried to establish efficacy and killing rate of EMS for this strain. A killing rate of > 95% was targeted. Based on this, 1 ml overnight culture (ODgzo = 3-4) in MRS-Difco was added with 20 pl EMS. The cultures were then incubated for 3 hours at 37°C and then diluted into series ranging from 10'2to 10'6. Subsequently, the diluted cultures are spread on MRS-IM + 2% (w / v) galactose agar plates to test for cell count. Mutant strains were selected from the mutant pool17967-EP-EPA[2]

[0206] 24

[0207] by high-throughput screening, which included mutant hit picking from agar, growth of mutants in broth, milk acidification and citrate utilization assays, selection of mutants with altered acidification or no citrate utilization were selected.

[0208] In more detail, MRS-IM + 2% (w / v) galactose agar pH 6.5 was poured into low profile square bioassay dish (Corning 431301). The mutant pools were spread on agar using sterile glass beads and incubated for 2 days at 37°C anaerobically. Approximately 5,000 colonies were hit-picked using QPix2 colony picker (Genetix) into 200-pl 96-well micro-titer plates containing MRS-IM broth with 2% (w / v) galactose. After an overnight incubation of the microtiter plates under anaerobic conditions at 37°C, 1% of the overnight inoculum was used for two different screening approaches:

[0209] Approach I) to inoculate milk supplemented with 0.1% (w / v) fructose + 0.3% (w / v) galactose with and without 2% (w / v) xylitol in 2-ml 96-well micro-titer plates. We followed acidification and selected strains (e.g., DSM 35404) that had a difference in acidification in the chosen conditions.

[0210] Approach II) To select strains that do not longer ferment citrate in a Leesment agar screening assay. Here the cells were spotted on Leesment agar with and without 0.3% galactose and incubated both anaerobically and aerobically. Cells that did not consume citrate were selected (e.g., DSM 35075, CM1, CM2, CM3).

[0211] Example 2: Citrate utilization in LGG® mutants.

[0212] We analyzed if the selected mutant strains did not show citrate consumption in milk in the right conditions. We used pre-acidified A-milk (pH 4.5) supplemented with different sugars and sugar alcohols to study the citrate utilization, as it would be the closest to application conditions. For acidification, we chose HCI to not interact with the lactate concentrations produced by LGG® during growth. We incubated LGG® and the mutant strains DSM 35075 and DSM 35404 in 2 ml of the different milks in 96 deep-well plates at 30 °C for 1 month. We analyzed citrate concentrations after 3, 13-14 and 27-30 days by HPLC.

[0213] When 0.1% fructose and 2% erythritol or xylitol were present in the milk both strains DSM 35075 and DSM 35404 did not ferment the citrate even after 1 month of storage at 30°C (see Tables 2 and 3). Table 3: Citric acid concentrations (g / L) measured in milk +0.1% (w / v) fructose +2% (w / v) xylitol after growth of LGG9or its mutant strains at 30°C and samples were taken at the indicated time. For LGG® and DSM 35075 triplicate measurements are shown.

[0214]

[0215] Time LGG® DSM 3507E DSM 35404 3 days 0.54 1.50 1.49 1.49 1.46 1.48 1.56

[0216]

[0217] ~l / 2 month <0.255 <0.255 <0.255 1.57 1.50 1.56 1.4717967-EP-EPA[2]

[0218] 25

[0219]

[0220] ~1 month <0.255 <0.255 <0.255 1.81 1.74 1.75 1.63

[0221] Table 4: Citric acid concentrations (g / L) measured in pre-acidified milk supplemented as indicated after growth of LGG® or its mutant strain at 30°C after 30 days.

[0222] Pre-acifidied milk supplemented with LGG® DSM 35404 +0.1% (w / v) fructose <0.288 0.45 +0.1% (w / v) fructose +2% (w / v) erythritol <0.288 1.56

[0223]

[0224] +0.1% (w / v) fructose +2% (w / v) xylitol <0.288 1.64

[0225] DSM 35075 has a mutation in the citrate transporter CitN (Q152*) and did not show citrate utilization in any of the tested conditions. DSM 35404 has several mutations, but none in the main citrate utilization pathway. This strain is able to utilize citrate under various conditions (see Table 4) but very surprisingly not in the application relevant condition with 0.1% (w / v) fructose + 2% xylitol or 2% erythritol.

[0226] Table 5: Citric acid concentrations (g / L) measured in pre-acidified milk supplemented with the indicated sugar and sugar alcohol (percentages refer to % w / v) after growth of DSM 35404 at 30°C for the indicated time period, fru = fructose; xyl = xylitol; gal = galactose.

[0227] +0.1% fru +0.2% gal

[0228] Days Milk pH 4.5 +0.1% fru +2%xyl +0.2% gal +2% xyl +2% xyl 3 1.15 1.53 1.56 1.05 1.26 1.40 13 <0.255 <0.255 1.47 <0.255 0.58 <0.255

[0229]

[0230] 27 <0.255 <0.255 1.63 <0.255 <0.255 <0.255

[0231] DSM 35074 behaved similarly to DSM 35075 and also did not show citrate utilization in any of the tested conditions. DSM 35074 has a mutation in the citrate transporter CitN (T4I).

[0232] Example 3: Survival of the mutant strains.

[0233] One of the major criteria is the survival of LGG® at ambient storage up to 6 months in post-pasteurized yogurt. We have seen earlier that LGG® did not survive as good in conditions, where it did not utilize the citrate, indicating that LGG® needs the carbon source consumed during shelf life for survival. Surprisingly, we found that the citrate-deficient mutants survived equally well in the tested conditions as LGG®, even without the citrate metabolism. Some exemplary data is provided in Table 5.17967-EP-EPA[2]

[0234] 26

[0235] We performed the same experiments as described in Example 2 and monitored the cell counts during the experiment.

[0236] Table 6: Cell count of LGG® and DSM 35075 in three different milk bases during storage at 30 °C for the indicated time (addition of fructose and xylitol are given in % w / v).

[0237] LGG® DSM 35075

[0238] prepreDays acidified +0.1% fru acidified +0.1% fru milk +0.1% fru +2% xyl milk +0.1% fru +2% xyl 12 2.03E+08 2.05E+08 1.98E+08 7.50E+07 7.75E+07 2.00E+07 19 3.50E+07 6.00E+07 3.75E+07 9.50E+07 1.03E+08 8.75E+06

[0239]

[0240] 43 2.60E+08 2.83E+08 1.00E+06 7.50E+07 1.35E+08 5.00E+05

[0241] Example 4: Producing an acidified milk product comprising the strains of the present invention. Two different milk bases were prepared with the following recipes.

[0242] Milk base 1 (MB1): Milk base 2 (MB2):

[0243] Full fat milk 92.1 % (w / w) 92.0 % (w / w)

[0244] Water 3.5 % (w / w) 1.6 % (w / w)

[0245] Sucrose 0.8 % (w / w) 0.8 % (w / w)

[0246] Modified starch 1.5 % (w / w) 1.5 % (w / w)

[0247] Pectin 0.1 % (w / w) 0.1 % (w / w)

[0248] Erythritol 0.7 % (w / w) 1.0 % (w / w)

[0249] Xylitol 1.3 % (w / w) - Maltitol - 3.0% (w / w)

[0250] MB1 and MB2 were heat-sterilized at 134°C for 4 seconds. The milk bases were then inoculated with starter cultures comprising Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (i.e., 100U / 1000L F-DVS YoFlex® Acidifix® 1.0 produced by Chr. Hansen A / S) and fermented at 43°C until a desired pH of 4.45 was reached. Then, the fermented milk product was pasteurized at 75°C for 25 seconds. The pasteurized yogurt was filled into sterile cups and stored at 6 °C until use.

[0251] The pasteurized yogurt was acclimated to 25°C and then aseptically inoculated with a Lacticaseibacillus rhamnosus strain. The viable cell count at inoculation was 9.90E+06 CFU / g (i.e., 9.90 x 106CFU / g). The LGG strain tested was F-DVS nu-trish® LGG® Al (Chr. Hansen A / S).17967-EP-EPA[2]

[0252] 27

[0253] Samples were shaken thoroughly to homogeneity without introducing air (a sterile spoon or mixer were used for bottles). The cups were then stored at 25°C for 72 hours. A pH of less than or equal to 4.3 was obtained. Then the cups were incubated at a pre-determined storage temperature. Viable cell count and CO2 generation was monitored during the incubation.

[0254] To measure CO2 generation, samples were placed in glass bottles with a rubber stopper lid. The lid was penetrated with a needle and %CC>2 in the head space of the bottle was measured using the portable Headspace Gaz Analyzer Dansensor® Checkpoints.

[0255] Table 7: Viable cell count of samples incubated at 25°C.

[0256] Strain 1 Milk base Day 0* Day 14 Day 28 Day 42 Day 56 LGG® MB1 1.20E+07 1.19E+07 9.55E+06 9.55E+06 7.30E+06 LGG® MB2 6.35E+07 7.95E+07 6.55E+07 6.55E+07 7.40E+07 DSM 35075 MB1 1.10E+07 8.80E+07 8.20E+06 8.20E+06 4.50E+06 DSM 35075 MB2 6.20E+07 4.00E+07 2.35E+07 2.35E+07 2.35E+07 DSM 35404 MB1 4.05E+07 1.10E+08 1.32E+07 1.32E+07 1.37E+07 | DSM 35404 MB2 4.25E+07 6.10E+07 4.90E+07 4.90E+07 2.70E+07 Blank ref MBl 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00

[0257]

[0258] Blank ref MB2 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 *72 hours after inoculation

[0259] Table 8: Viable cell count of samples incubated at 37°C.

[0260] Strain i i DayO* Day 7 Day 14 Day 21 Day 28 Day 42 Day 56 base

[0261] LGG® MBl 1.20E+07 1 1.33E+08 1 8.40E+06 1.12E+07 1.30E+07 1.78E+07 3.05E+07 LGG® 1 MB2 1 6.35E+07 1 1.64E+08 1 1.57E+08 4.60E+07 1.20E+07 4.25E+06 7.00E+06 DSM MBl i 1.10E+07 i 1.45E+08 i 9.25E+06 5.45E+06 3.55E+06 3.15E+06 1.50E+06 35075

[0262] DSM MB2 6.20E+07 1.33E+08 3.75E+07 1.37E+07 9.80E+06 2.20E+06 2.70E+06 35075

[0263] MBl 4.05E+07 ; 1.15E+08 1.90E+07 1.28E+07 ( 7.60E+06 ; 4.75E+06 3.80E+06 35404

[0264] DSM 1 MB2 4.25E+07 1 8.60E+07 1 4.05E+07 9.30E+06 1 1.46E+07 8.80E+06 6.70E+06 35404

[0265] Bla"kMBl 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 ref

[0266] Bla"kMB2 | 0.00E+00 j 0.00E+00 | 0.00E+00 0.00E+00 j 0.00E+00 j 0.00E+00 0.00E+00

[0267]

[0268] ref

[0269] *72 hours after inoculation17967-EP-EPA[2]

[0270] 28

[0271] Table 9: CO2 generation in samples incubated at 25°C.

[0272] Strain Milk base Day 0* Day 14 Day 28 Day 42 Day 56 LGG® MB1 4.05 5.50 5.30 5.81 5.59 LGG® MB2 4.10 5.85 8.15 12.75 17.60 DSM 35075 MB1 5.10 7.15 6.67 6.66 5.72 DSM 35075 MB2 4.20 6.25 5.94 5.68 5.57 DSM 35404 MB1 4.05 6.50 6.18 6.28 6.45 DSM 35404 MB2 4.15 5.75 5.75 5.78 5.16 Blank ref MB1 2.65 2.59 2.43 2.97

[0273]

[0274] Blank ref MB2 2.15 2.01 1.72 1.52 *72 hours after inoculation

[0275] Table 10: CO2 generation in samples incubated at 37°C.

[0276] Strain Milk base 1 DayO* Day 7 1 Day 14 1 Day 22 1 Day 28 1 Day 42 1 Day 56 LGG® MB1 4.05 7.15 9.00 8.34 10.76 17.57 26.39 LGG® MB2 4.10 8.45 22.75 14.29 14.39 10.04 7.26 DSM 35075 MB1 5.10 5.90 6.80 5.09 6.77 5.56 6.24 DSM 35075 MB2 4.20 10.25 10.95 8.78 7.14 6.45 8.01 DSM 35404 MB1 4.05 5.55 5.30 4.07 3.91 3.78 3.58 DSM 35404 MB2 4.15 4.70 6.40 5.59 6.47 6.47 4.39 Blank ref MB1 2.30 2.75 8.54 2.68 1.78 1.30

[0277]

[0278] Blank ref MB2 2.00 2.45 2.11 1.77 1.26 1.15 *72 hours after inoculation

[0279] T

[0280]

[0281] able 11: Concentration of citric acid (mg / g) in samples incubated at 25°C.

[0282] Strain Milk base Day 0* Day 14 Day 28 Day 42 Day 56 LGG® MB1 1.57 1.61 1.52 1.46 1.43 LGG® MB2 1.59 1.58 1.02 0.67 <0,288 DSM 35075 MB1 1.57 1.61 1.4 1.45 1.49 DSM 35075 MB2 1.63 1.46 1.47 1.46 1.45 DSM 35404 MB1 1.64 1.5 1.41 1.48 1.46 DSM 35404 MB2 1.59 1.5 1.38 1.44 1.45 Blank ref MB1 1.65 1.64 1.44 1.59 1.52

[0283]

[0284] Blank ref MB2 1.58 1.6 1.52 1.59 1.64 *72 hours after inoculation17967-EP-EPA[2]

[0285] 29

[0286] Table 12: Concentration of citric acid (g / L) in samples incubated at 37°C.

[0287] Strain Milk base Day 0* Day 7 Day 14 Day 21 Day 28 Day 42 Day 56 LGG® MB1 1.57 1.57 1.36 1.34 1.3 0.77 <0,288 LGG® MB2 1.59 1.21 <0,288 <0,288 <0,288 <0,288 <0,288 DSM 35075 MB1 1.57 1.53 1.47 1.44 1.45 1.42 1.5 DSM 35075 MB2 1.63 1.62 1.58 1.42 1.42 1.43 1.54 DSM 35404 MB1 1.64 1.61 1.45 1.4 1.37 1.34 1.45 DSM 35404 MB2 1.59 1.55 1.54 1.36 1.3 1.26 1.18 Blank ref MB1 1.65 1.62 1.5 1.51 1.59 1.56 1.49

[0288]

[0289] Blank ref MB2 1.58 1.53 1.57 1.5 1.6 1.52 1.52 *72 hours after inoculation

[0290] Table 13: Acetic acid generation in samples incubated at 25°C. Concentration of acetic acid is provided in mg / g.

[0291] Strain 1 Milk base Day 0* Day 14 Day 28 Day 42 Day 56 LGG® MB1 0.15 <0,150 0.23 0.29 0.31 LGG® MB2 0.15 0.2 0.51 0.83 1.26 DSM 35075 MB1 <0,150 0.17 0.19 0.21 0.24 DSM 35075 MB2 <0,150 0.16 0.21 0.22 0.24 DSM 35404 MB1 0.15 0.18 0.2 0.23 0.26 DSM 35404 MB2 0.15 0.19 0.21 0.25 0.31 Blank ref MB1 <0,150 <0,150 <0,150 <0,150 <0,150

[0292]

[0293] Blank ref MB2 <0,150 <0,150 <0,150 <0,150 <0,150 *72 hours after inoculation

[0294] Table 14: Acetic acid generation in samples incubated at 37°C. Concentration of acetic acid is provided in g / L.

[0295] Strain Milk base Day 0* Day 7 Day 14 Day 21 Day 28 Day 42 Day 56 LGG® MB1 0.15 0.2 0.32 0.32 0.45 0.77 0.96 LGG® MB2 0.15 0.46 1.23 1.26 1.26 1.33 1.32 DSM 35075 MB1 <0.150 0.17 0.19 0.21 0.23 0.25 0.25 DSM 35075 MB2 <0.150 0.17 0.23 0.22 0.25 0.26 0.27 DSM 35404 MB1 0.15 0.18 0.21 0.26 0.26 0.29 0.36 DSM 35404 MB2 0.15 0.19 0.25 0.25 0.29 0.35 0.46

[0296]

[0297]

[0298] Blank ref MB1 <0.150 <0.150 <0.150 <0.150 <0.150 <0.150 <0.15017967-EP-EPA[2]

[0299] 30

[0300]

[0301] Blank ref MB2 <0.150 <0.150 <0.150 <0.150 <0.150 0.15

[0302]

[0303] <0.150 *72 hours after inoculation

[0304] Tables 6-13 demonstrate that the strains of the present invention consume less citric acid thus generating less acetate and CO2 than the LGG® strain of the prior art. The strains of the present invention also remain viable to a similar degree as the LGG® strain of the prior art.

[0305] Example 5: Citrate utilization of LGG® citN mutant strains.

[0306] We analyzed citrate consumption of selected citN mutant strains as described in Example 2. We used pre-acidified A-milk (pH 4.5) supplemented with different sugars and sugar alcohols to study the citrate utilization, as it would be the closest to application conditions. For acidification, we chose HCI to not interact with the lactate concentrations produced by LGG® during growth. We incubated LGG® and the mutant strains in 2mL of the different milks in 96 deep-well plates at 30°C for 1.5 months. Citrate concentration was determined after 1 and 1.5 months by HPLC.

[0307] Table 15: Citrate concentration (g / L)

[0308] LGG® DSM 35075 CM1 CM2 CM3 1 month in 0.1% fru <0.288 1.74 1.66 1.62 1.67 1.5 months in 0.1% Fru + 3% Ery <0.288 1.47 1.48 1.55 - 1.5 months in 0.1% Fru + 2% Ery <0.288 1.52 1.625 1.61 -

[0309]

[0310] Example 6: Survival of citN mutant strains.

[0311] One of the major criteria is the survival of LGG® at ambient storage up to 6 months in post-pasteurized yogurt. We have seen earlier that LGG® did not survive as good in conditions, where it did not utilize the citrate, indicating that LGG® needs the carbon source consumed during shelf life for survival. Surprisingly, we found that the citrate-deficient mutants survived equally well in the tested conditions as LGG®, even without the citrate metabolism. The citN mutant strains were analyzed as described in Example 2 and cell counts were determined during the experiment. Data is shown in the table below. Table 16: Cell count of LGG* and its mutants in two different milk bases during storage at 30°Cfor 30 d

[0312]

[0313] ays (addition of fructose and xylitol are given in % w / v).

[0314] LGG® DSM 35075 CM1 CM2 CM3 0.1% Fru 5.01 E+06 3.02E+06 1.29E+07 5.01 E+06 2.51 E+06

[0315]

[0316] 0.1% Fru + 3% Ery 6.03E+06 4.47E+06 7.94E+06 1.51 E+06 1.51 E+0617967-EP-EPA[2]

[0317] 31

[0318] Example 7: Fermented milk products comprising Lacticaseibacillus rhamnosus strains CMl or CM2.

[0319] We have performed the experiment as described in Example 4. Milk base 2 (MB2) was heat-sterilized at 134°C for 4 seconds. The milk bases were then inoculated with starter cultures comprising Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (i.e., 100U / 1000L F-DVS YoFlex® Acidifix® 1.0 (Chr. Hansen A / S) and fermented at 43°C until a desired pH of 4.45 was reached. Then, the fermented milk product was pasteurized at 75°C for 25 seconds. The pasteurized yogurt was filled into sterile cups and stored at 6°C until use. The pasteurized yogurt was acclimated to 25°C and then aseptically inoculated with one of the Lacticaseibacillus rhamnosus strains CMl or CM2. The viable cell count at inoculation was 9.90E+06 CFU / g (i.e., 9.90 x 106CFU / g).

[0320] Samples were shaken thoroughly to homogeneity without introducing air (a sterile spoon or mixer were used for bottles). The cups were then stored at 25°C for 72 hours. A pH of less than or equal to 4.3 was obtained. Then the cups were incubated at a pre-determined storage temperature. Viable cells were counted, and CO2 generation was monitored during the incubation. To measure CO2 generation, samples were placed in glass bottles with a rubber stopper lid. The lid was penetrated with a needle and ° / oC02in the head space of the bottle was measured using the portable Headspace Gaz Analyzer Dansensor® CheckPoint3.

[0321] Table 17a: Viable cell count of samples incubated at 25°C.

[0322] STRAIN Day 0* Day 14 Day 28 CMl 2.0E+08 - 6.9E+07 - 5.3E+07 CM2 1.6E+08 - 5.8E+07 - 4.8E+07

[0323]

[0324] Blank 0.0E+00 - 0.0E+00 - 0.0E+00 * 72 hours after inoculation

[0325] Table 17b: Viable cell count of samples incubated at 37°C.

[0326] STRAIN Day 0* Day 7 Day 14 Day 21 Day 28 CMl 1.97E+08 7.10E+07 2.85E+07 2.30E+07 2.40E+07 CM2 1.56E+08 6.35E+07 4.40E+07 2.35E+07 2.15E+07

[0327]

[0328] Blank 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 * 72 hours after inoculation

[0329] Table 18a: CO2 generation in samples incubated at 25°C.

[0330] STRAIN Day 0* Day 14 Day 28 CMl 4.04 5.83 5.77 CM2 4.09 6.54 6.87

[0331]

[0332] Blank 2.75 3.45 2.79 * 72 hours after inoculation.

[0333] Table 18b: CO2 generation in samples incubated at 37° C.

[0334]

[0335] STRAIN Day 0* Day 7 Day 14 Day 22 Day 2817967-EP-EPA[2]

[0336] 32

[0337] CM1 4.04 6.76 7.60 7.32 7.03 CM2 4.09 6.63 7.84 7.39 7.78

[0338]

[0339] Blank 2.75 4.08 4.17 3.87 3.10 * 72 hours after inoculation.

[0340] Table 19a: Concentration of citric acid (mg / g) in samples incubated at 25°C.

[0341] STRAIN Day 0* Day 14 Day 28 CM1 1.27 1.23 1.20 CM2 1.26 1.27 1.32

[0342]

[0343] Blank 1.31 1.23 1.26 * 72 hours after inoculation.

[0344] Table 19b: Concentration of citric acid (mg / g) in samples incubated at 37°C.

[0345] STRAIN Day 0* Day 7 Day 14 Day 21 Day 28 CM1 1.27 1.26 1.31 1.21 1.24 CM2 1.26 1.32 1.23 1.20 1.22

[0346]

[0347] Blank 1.31 1.34 1.32 1.30 1.29 * 72 hours after inoculation.

[0348] Table 20a: Concentration of acetic acid (mg / g) in samples incubated at 25°C.

[0349] STRAIN Day 0* Day 14 Day 28 CM1 <0.150 <0.150 0.17 CM2 <0.150 <0.150 0.18

[0350]

[0351] Blank <0.150 <0.150 <0.150 * 72 hours after inoculation.

[0352] Table 20b: Concentration of acetic acid (mg / g) in samples incubated at 37°C.

[0353] STRAIN Day 0* Day 7 Day 14 Day 21 Day 28 CM1 <0.150 <0.150 0.19 0.19 0.23 CM2 <0.150 <0.150 0.17 0.21 0.25

[0354]

[0355] Blank <0.150 <0.150 <0.150 <0.150 <0.150 * 72 hours after inoculation.

[0356] The results demonstrate that the strains of the present invention consume less citric acid and generate less acetate and CO2 than the LGG® strain. The strains of the present invention also remain viable to a similar degree as the LGG® strain.(Original in Electronic Form)

[0357] (This sheet is not part of and does not count as a sheet of the international application) 0-1 Form PCT / RO / 134

[0358] Indications Relating to Deposited

[0359] Microorganism(s) or Other Biological

[0360] Material (PCT Rule 13bis)

[0361] 0-1-1 Prepared Using ePCT-Filing-Embedded

[0362] Version 4.17.008 MT / FOP 20260328 / 2.8

[0363] 0-2 International Application No.

[0364] 0-3 Applicant's or agent's file reference 17967-WO-PCT

[0365]

[0366] 1 The indications made below relate to

[0367] the deposited microorganism(s) or

[0368] other biological material referred to in

[0369] the description on:

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[0372] 1-3 Identification of deposit

[0373] 1-3-1 Name of depositary institution DSMZ Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures

[0374] 1-3-2 Address of depositary institution Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures

[0375] Inhof fenstr. 7B

[0376] D-38124 Braunschweig

[0377] Germany

[0378] 1-3-3 Date of deposit 25 June 2024 (25.06.2024)

[0379] 1-3-4 Accession Number DSMZ 35074

[0380] 1-4 Additional Indications Sample only to be issued to an Expert

[0381] 1-5 Designated States for Which All designations

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[0383] 1-6 Separate Furnishing of Indications

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[0386] International Bureau later(Original in Electronic Form)

[0387] (This sheet is not part of and does not count as a sheet of the international application) 2 The indications made below relate to

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[0389] other biological material referred to in

[0390] the description on:

[0391] 2-1 page 23

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[0393] 2-3 Identification of deposit

[0394] 2-3-1 Name of depositary institution DSMZ Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures

[0395] 2-3-2 Address of depositary institution Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures

[0396] Inhof fenstr. 7B

[0397] D-38124 Braunschweig

[0398] Germany

[0399] 2-3-3 Date of deposit 25 June 2024 (25.06.2024)

[0400] 2-3-4 Accession Number DSMZ 35075

[0401] 2-4 Additional Indications Sample only to be issued to an Expert

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[0404] 2-6 Separate Furnishing of Indications

[0405] These indications will be submitted to the

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[0409] other biological material referred to in

[0410] the description on:

[0411] 3-1 page 23

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[0413] 3-3 Identification of deposit

[0414] 3-3-1 Name of depositary institution DSMZ Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures

[0415] 3-3-2 Address of depositary institution Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures

[0416] Inhof fenstr. 7B

[0417] D-38124 Braunschweig

[0418] Germany

[0419] 3-3-3 Date of deposit 03 April 2025 (03.04.2025)

[0420] 3-3-4 Accession Number DSMZ 35404

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[0432]

[0433] 0-4-1 Authorized officer Kuiper-Cristina, Nathalie(Original in Electronic Form)

[0434] (This sheet is not part of and does not count as a sheet of the international application)

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Claims

17967-EP-EPA[2]33CLAIMS1. A citrate-deficient Lacticaseibacillus rhamnosus strain.

2. The Lacticaseibacillus rhamnosus strain of claim 1, wherein the strain does not metabolize citric acid in:(a) acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) xylitol; and / or(b) acidified A-milk containing 0.1% (w / v) fructose and 2% (w / v) erythritol.

3. The Lacticaseibacillus rhamnosus strain of claim 1 or 2, wherein the strain is a citrate-deficient mutant derived from a LGG strain.

4. The Lacticaseibacillus rhamnosus strain of any one of claims 1-3, wherein the strain is the strain deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr.7B, D-38124 Braunschweig, on April 3, 2025 under the accession no. DSM 35404, or a citrate-deficient mutant derived therefrom.

5. The Lacticaseibacillus rhamnosus strain of any one of claims 1-3, wherein the strain is citratenegative.

6. The Lacticaseibacillus rhamnosus strain of any one of claims 1-3 and 5, wherein the strain contains a loss-of-function mutation in the citN gene.

7. The Lacticaseibacillus rhamnosus strain of claim 6, wherein the mutation inserts a premature stop codon; or the mutation results in a substitution at a position corresponding to position 4, position 162 and / or position 359 in SEQ. ID NO:2, optionally a 41 substitution, a 359N substitution, and / or a 162D substitution.

8. The Lacticaseibacillus rhamnosus strain of any one of claims 1-3 and 5-7, wherein the strain is:(i) the strain deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on June 25, 2024 under the accession no. DSM 35074;17967-EP-EPA[2]34(ii) the strain deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on June 25, 2024 under the accession no. DSM 35075; or(iii) a citrate-deficient mutant derived from (i) or (ii).

9. A composition comprising the Lacticaseibacillus rhamnosus strain of any one of claims 1-8.

10. The composition of claim 9, wherein the composition:(a) is freeze-dried or frozen; and / or(b) comprises at least 106CFU / g of the Lacticaseibacillus rhamnosus strain.

11. The composition of claim 9, wherein the composition is an acidified milk product.

12. The composition of claim 11, wherein the acidified milk product is a pasteurized yogurt.

13. The composition of any one of claims 9-12, further comprising:(i) a sugar selected from fructose and sucrose; and / or(ii) a sugar alcohol.

14. The composition of claim 13, wherein the sugar is fructose, and the sugar alcohol is xylitol or erythritol.

15. A method for producing an acidified milk product containing Lacticaseibacillus rhamnosus, the method comprising adding (i) the Lacticaseibacillus rhamnosus strain of any one of claims 1-8 or the composition of 9 or 10 to (ii) a milk base or acidified milk product.

16. A method for obtaining a citrate-deficient Lacticaseibacillus rhamnosus strain, the method comprising:(1) subjecting a citrate-positive Lacticaseibacillus rhamnosus mother strain to mutagenesis to obtain a pool of mutant strains;(2) screening the pool of mutant strains of step (1) for citrate metabolism; and(3) selecting a strain from the pool of mutants that is citrate-deficient.

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