Method for producing a fermented product with bacteriophage-resistant s. thermophilus strains and corresponding strains

Abstract

The present disclosure is in the field of fermented food technology. It relates to new phage-resistant strains of the species Streptococcus thermophilus, compositions and fermented products comprising the strains, and methods for producing said fermented products.

Description

[0001] METHOD FOR PRODUCING A FERMENTED PRODUCT.

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to lactic acid bacteria strains of the species Streptococcus thermophilus with an improved resistance towards bacteriophages, i.e. improved phageresistance, compositions such as starter cultures comprising the strains, and fermented products comprising said strains.

[0004] BACKGROUND OF THE INVENTION

[0005] The food industry uses numerous bacteria, in particular lactic acid bacteria (LAB) in the production of fermented food. The choice of LAB impacts the characteristics and features of the fermented food product, such as e.g. taste, texture, and shelf life. In the dairy industry, LAB are used intensively in order to bring about the acidification of milk (by fermentation) but also in order to texturize the product into which they are incorporated.

[0006] Streptococcus thermophilus is one of the most common bacteria used worldwide as a starter in the production of fermented foods, such as cheese and yoghurt. This microorganism is a thermophilic, aerotolerant, Gram-positive coccus, and a member of a heterogeneous group of lactic acid bacteria. The extensive use of S. thermophilus in dairy plants, through cultivation in large vats, has resulted in increased susceptibility to bacteriophage infections. Indeed, phage outbreaks represent the major cause of slow or faulty fermentations, frequently leading to a lower quality of dairy products and suboptimal production costs. Bacteriophages, in short called phages, have been identified for many of the bacterial strains used in the industry of the species such as e.g. Lactococcus sp., Lactobacillus sp., Leuconostoc sp., Pediococcus sp. or Streptococcus sp.

[0007] The lytic development of bacteriophages involves adsorption of the phages to the host cell surface, injection of phage DNA into the cell, synthesis of phage proteins, replication of phage DNA, assembly of progeny phages and release of progeny from the host. Cell-mediated mechanisms of interference with any of these events can prevent a phage infection. The ability of bacterial cultures to resist bacteriophage infection during industrial use depends to a large extent on host strain characteristics affecting one or more of the above mechanisms. A factor, which may lead to frequent bacteriophage infections in lactic acid bacterial starter cultures, is the fact that fermentation conditions in the food industry including the dairy industry in general are non-sterile. Diverse treatments have been applied to minimize phage infections in the dairy environment. Predominant approaches include chemical and physical methods for equipment sanitation, as well as a culture replacement and strain rotation programs. The latter require strains with identical technological performance, but different phage sensitivities. Thus, isolation and characterization of bacteriophage insensitive mutants (BIMs) of strains used in dairy starter cultures has been widely performed. Several methods for generating BIMs of S. thermophilus have been proposed including insertional mutagenes, the secondary culture method, serial passaging in the presence of high phage titers, chemical mutagenesis, and transformation with an antisense mRNA-generating plasmid. Generally, the acquired resistance is due to the activation of intracellular resistance mechanisms, mainly clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems or restrictionmodification (R-M) systems. Additional phage-resistance systems, such as abortive infection (Abi) and superinfection exclusion (Sie), have also been detected in S. thermophilus. However, those mechanisms probably are not very widespread and therefore, they do not commonly mediate phage-resistance in BIMs of dairy strains.

[0008] It has not yet been possible to eliminate bacteriophage contamination under the current industrial conditions. In order to meet the requirements of the industry, it has become necessary to propose novel phage-resistant strains of Streptococcus thermophilus. Thus, development of new strains that are phage-resistant and which are suitable in methods that provide fermented food products having good texture are desirable.

[0009] SUMMARY OF THE INVENTION

[0010] An objective of the present invention is to provide strains of Streptococcus thermophilus with improved phage insensitivity and robustness. The present inventors have provided lactic acid bacteria of the species Streptococcus thermophilus, which are more resistant to phage attack than their (mother) strain from which they are derived and identified wildtype strains which as compared to reference strains. These strains also provide similar or improved properties such as reduced acidification time, and increased texture measured as shear stress and / or gel stiffness when used for fermenting milk as compared to fermented milk produced with their mother strain or a starter culture in the prior art.

[0011] The present disclosure provides in a first aspect a method for producing a fermented product comprising the steps:

[0012] (a) providing a strain of the species Streptococcus thermophilus, which strain is resistant towards the phage DSM 35227 and / or DSM 35228;

[0013] (b) adding the strain of (a) to a milk base; (c) fermenting the milk base at a temperature between 20°C and 50°C until a pH of 4.6 or less is reached; and

[0014] wherein the product has a post-acidification at or below 0.3 pH units measured at day 7 after fermentation at 43°C in a milk base comprising 3.5% protein, 3.0% fat, 0% added sugar and stored at 13°C.

[0015] In a second aspect the disclosure provides a phage-resistant strain of the species Streptococcus thermophilus, wherein said strain is resistant to the phage DSM 35227 and / or DSM 35228.

[0016] In a third aspect the disclosure provides a composition comprising the strain of the present disclosure.

[0017] In a fourth aspect the disclosure provides a fermented product comprising the strain of the present disclosure; or comprising the composition of the present disclosure.

[0018] In a fifth aspect the disclosure provides Use of the strain of the present disclosure; or the composition of the present disclosure for the manufacture of a fermented product.

[0019] BRIEF DESCRIPTION OF THE FIGURES FIGURE 1

[0020] Figure 1 shows acidification by the strain DSM 35226 of milk + / - phage DSM 35228.

[0021] FIGURE 2

[0022] Figure 2 shows acidification by the strain DSM 35231 og milk + / - the pool of 55 phages.

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] 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 the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. 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.

[0025] It will be appreciated that aspects and embodiments disclosed in any one of the sections "Method for producing fermented products", "Lactic acid bacteria strains", "Compositions", "Fermented Products", and Use" of the present disclosure may be equally relevant to other parts and sections of the specification. I is clear to the person skilled in the art that the various aspects and embodiments are not limited to the section(s) in which they are disclosed.

[0026] Method for producing fermented products

[0027] The disclosure provides methods for producing a fermented product a product obtainable by this method.

[0028] In the context of the present disclosure, the term "milk" is broadly used in its common meaning to refer to liquids produced by the mammary glands of animals or by plants. In accordance with the present invention the milk may have been processed and the term "milk" includes whole milk, skim milk, fat-free milk, low fat milk, full fat milk, lactose-reduced milk, or concentrated milk. Fat-free milk is non-fat or skim, milk product. Low-fat milk is typically defined as milk that contains from about 1% to about 2% fat. Full fat milk often contains 2% fat or more. The term "milk" is intended to encompass milks from different mammal and plant sources. Mammal sources of milk include, but are not limited to cow, sheep, goat, buffalo, camel, lama, mare and deer. Plant sources of milk include, but are not limited to, milk extracted from soy bean, pea, peanut, barley, rice, oat, quinoa, almond, cashew, coconut, hazelnut, hemp, sesame seed and sunflower seed. In the methods and products of the present invention, milk derived from cows is most preferably used as a starting material for the fermentation.

[0029] The term "milk" also includes fat-reduced and / or lactose-reduced milk products. Respective products can be prepared using methods well known in the art and are commercially available. Lactose-reduced milk can be produced according to any method known in the art, including hydrolyzing the lactose by lactase enzyme to glucose and galactose, or by nanofiltration, electrodialysis, ion exchange chromatograph and centrifugation.

[0030] The term "milk base" is broadly used in the present application to refer to a composition based on milk or milk components which can be used as a medium for growth and fermentation of LAB. The milk base comprises components derived from milk and any other component that can be used for the purpose of growing or fermenting LAB. Prior to fermentation, the milk base may be homogenized and pasteurized according to methods known in the art. "Homogenizing" as used herein means intensive mixing to obtain a soluble suspension or emulsion. If homogenization is performed prior to fermentation, 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. "Pasteurizing" as used herein means treatment of the milk substrate 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.

[0031] "Fermentation" in the methods of the present disclosure means the conversion of carbohydrates into alcohols or acids through the action of a microorganism. Preferably, fermentation in the methods of the invention comprises conversion of lactose to lactic acid. Fermentation processes to be used in production of fermented milk products are well known and the person of skill in the art will know how to select suitable process conditions, such as temperature, oxygen, amount and characteristics of microorganism(s) and process time. Obviously, fermentation conditions are selected to support the achievement of the present invention, i.e. to obtain a fermented milk product.

[0032] In one aspect the present disclosure relates to a method for producing a fermented product comprising the steps:

[0033] (a) providing a strain of the species Streptococcus thermophilus, which strain is resistant towards the phage DSM 35227 and / or DSM 35228;

[0034] (b) adding the strain of (a) to a milk base;

[0035] (c) fermenting the milk base at a temperature between 20°C and 50°C until a pH of 4.6 or less is reached; and

[0036] wherein the product has a post-acidification at or below 0.3 pH units measured at day 7 after fermentation at 43°C in a milk base comprising 3.5% protein, 3.0% fat, 0% added sugar and stored at 13°C.

[0037] The milk base may be any milk base suitable for producing a desired fermented product. In one embodiment the present disclosure relates to a milk base, wherein the milk base is of mammalian an / or vegetable origin. The amount of mammalian milk base may be in the range of 0-100%. The amount in percentage of mammalian to vegetable milk base may be 0:100; 1:99; 5:95; 10:90; 20:80; 30;70; 40:60; 50:50; 60:40; 70:30; 80:20; 90:10; 95:5; 99:1; or 100:0. A prophage is the genome of a bacteriophage, also called phage that is integrated into the genome of the bacteria or which exists as an extrachromosomal plasmid in the bacteria. Presence of prophages is important for the lysogenic cycle of phages. Prophages remains in the genome of the bacteria through cell divisions until activated by an external factor, such as UV light, leading to production of new phage particles that will lyse the cell and spread. In one embodiment the disclosure relates to the method, wherein the strain is prophage-negative.

[0038] In one embodiment the disclosure relates to the method, wherein the strain is resistant towards one or more phages selected from: DSM 23962, DSM 23994, DSM 35003, and DSM 35011.

[0039] In one embodiment the disclosure relates to the method, wherein the strain is present in a concentration of at least 107, 108, 109, 1010, 1011, or at least 1012CFU / g.

[0040] In one embodiment the disclosure relates to the method, wherein the fermented milk product has:

[0041] (a) a shorter acidification time to target pH;

[0042] (b) a reduced post-acidification at day 7; and / or

[0043] (c) an increased texture measured as gel firmness and / or shear stress;

[0044] as compared to (I) a fermented product made with a reference strain selected from DSM 26562 and / or DSM 17876; or (II) the commercial culture F-DVS eXact® Dahi 3 product no.

[0045] 717445 sold by Chr. Hansen A / S.

[0046] In one embodiment the present disclosure relates to the method, wherein the acidification time for producing the fermented product is reduced as compared to the acidification time for producing a fermented product made with the a reference strain selected from DSM 26562 and / or DSM 17876.

[0047] In one embodiment the present disclosure relates to the method, wherein the postacidification at day 7 of the fermented product is reduced as compared to the post-acidification at day 7 of a fermented product made with the a reference strain selected from DSM 26562 and / or DSM 17876.

[0048] In one embodiment the present disclosure relates to the method, wherein the texture of the fermented product is increased as compared to the texture of a fermented product made with a reference strain selected from DSM 26562 and / or DSM 17876. Texture may be measured as shear stress and / or as gel firmness as known by the skilled artisan. Rheological property may be measured with an HTR-3000 rheometer, model MCR302e, from Anton Paar. The method is using a rotational step, which is based on a rotational deformation of the sample from 0.273 s-1 to 300 s-1 and then back to 0.273 s-1 where the corresponding shear stress is measured. Preferably the shear stress is measured at the shear rate 30.2 s-1 (Pa) and / or 300 s-1 (Pa). In one embodiment the disclosure relates to the method, wherein the texture is measured as shear stress. In one embodiment the disclosure relates to the method, wherein the shear stress is measured at the shear rate 30.2 s-1 and / or 300 s-1. Gel firmness is a sensory descriptor of the fermented milk texture. It correlates with instrumental measurements such as positive compression area measured by texture analyzer and to complex modulus (G*) measured by rheometer. Gel firmness may be measured with a texture analyzer (TA. XT PlusC, Stable Micro Systems) or using a small-scale compression test (Hamilton robot & Mettler Toledo precision balance). Cohesiveness correlates with instrumental measurements such as negative compression area measured by texture analyzer. In one embodiment the disclosure relates to the method, wherein the texture is measured as any of gel firmness, shear stress, and / or cohesiveness.

[0049] In one embodiment the disclosure relates to the method, wherein the strain is selected from one or more of: DSM 35226; DSM 34681; DSM 35225; DSM 35230; DSM 35231; DSM 35232; and DSM 35233.

[0050] In one aspect the present disclosure relates to a fermented product obtainable by the method of the present disclosure.

[0051] Lactic acid bacteria strains

[0052] In the context of present disclosure, the term "lactic acid bacteria" or "LAB" is used to refer to 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. During the fermentation stage, the consumption of lactose by these bacteria causes the formation of lactic acid, reducing the pH and leading to the formation of a protein coagulum. These bacteria are thus responsible for the acidification of milk and for the texture of dairy product. As used herein, the term "lactic acid bacteria" encompasses, but is not limited to, bacteria belonging to the genus of Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., Lactococcus spp., such as Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus lactis, Bifidobacterium animalis, Lactococcus lactis, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus acidophilus, Bifidobacterium animalis and Leuconostoc spp. The term "mutant" should be understood as a strain derived from a strain of the present disclosure, for example by means of e.g. genetic engineering, radiation and / or chemical treatment. It is preferred that the mutant is a functionally equivalent mutant, e.g. a mutant that has substantially the same or improved properties in particular in relation to the effects on phage-resistance, texturizing properties, and fermentation or acidification time, as the deposited strain. Respective mutants represent embodiments of the present disclosure. The term "mutant" in particular refers to a strain obtained by subjecting a strain of the invention to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as ethane methane sulphonate (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 mutagenization treatments (a single treatment should be understood one mutagenization step followed by a screening / selection step), but it is presently preferred that no more than 20, or no more than 10, or no more than 5, treatments (or screening / selection steps) are carried out. In a presently preferred mutant, less than 5%, or less than 1% or even less than 0.1% of the nucleotides in the bacterial genome have been shifted with another nucleotide, or deleted, compared to the mother strain.

[0053] The term "variant" should be understood as a strain which is functionally equivalent to a strain of the present disclosure, having substantially the same or improved properties in particular in relation to the effects relating to phage-resistance, texturizing properties, and fermentation or acidification time, as the deposited strain. Such variants, which may be identified using appropriate screening techniques, are a part of the present invention.

[0054] As used herein, the term "bacteriophage" has its conventional meaning as understood in the art ie. a virus that selectively infects one or more bacteria. Many bacteriophages are specific to a particular genus or species or strain of bacteria. The term "bacteriophage" is synonymous with the term "phage". Bacteriophages may include, but are not limited to, bacteriophages that belong to any of the following virus families: Corticoviridae, Cystoviridae, Inoviridae, Leviviridae, Microviridae, Myoviridae, Podoviridae, Siphoviridae, or Tectiviridae. The bacteriophage may be a lytic bacteriophage or a lysogenic bacteriophage. A lytic bacteriophage is one that follows the lytic pathway through completion of the lytic cycle, rather than entering the lysogenic pathway. A lytic bacteriophage undergoes viral replication leading to lysis of the cell membrane, destruction of the cell, and release of progeny bacteriophage particles capable of infecting other cells. A lysogenic bacteriophage is one capable of entering the lysogenic pathway, in which the bacteriophage becomes a dormant, passive part of the cell’s genome through prior to completion of its lytic cycle.

[0055] In the present context, the term "phage-resistance" or "phage-resistant" refers to the ability of the lactic acid bacterium strain to propagate (at optimal growth temperature) in a milk base which contains 1000 phages per mL, i.e. the bacterium is able to reach a cell density above 10E8 cfu / mL after 48 hours when inoculated at a concentration of 10E5 cfu / mL. cfu is "cell forming units". In the present context, the term "phage-robust" is interchangeable with the term "phage-resistant".

[0056] The term "improved resistance to a bacteriophage" denotes that the bacteria strain when tested in e.g. a plaque assay, such as the assay described as "Determination of phageresistance by the agar overlay method" or the "Heap Lawrence assay" have an improved phage-resistance to at least one phage e.g. expressed as the difference in pfu / mL (plaque forming unit per mL) obtainable with said at least one bacteriophage on the given strain, compared to the pfu / mL obtainable with the same bacteriophage on the mother strain. A strain with improved resistance to a bacteriophage preferably show a reduction of pfu / mL of a factor at least 50, such as at least 100, e.g. 500, preferably at least 1000, more preferably at least a factor 10.000 or more. In one embodiment the present disclosure relates to the strain, wherein the strain is resistant to one or more bacteriophages or one or more sets of bacteriophages.

[0057] In one aspect the present disclosure relates to a phage-resistant strain of the species Streptococcus thermophilus, wherein said strain is resistant towards the phage DSM 35227 and / or DSM 35228.

[0058] In one embodiment the disclosure relates to the strain, wherein said strain is prophagenegative.

[0059] In one embodiment the disclosure relates to the strain, wherein the strain is resistant towards one or more phages selected from: DSM 23962, DSM 23994, DSM 35003, and DSM 35011. In one embodiment the disclosure relates to the strain, wherein the strain is not resistant to one or more of the antibiotics: ampicillin, chloramphenicol, clindamycin, erythromycin, tetracyclin and vancomycin.

[0060] In one embodiment the disclosure relates to the strain, wherein said strain is derived from DSM 35224.

[0061] In one embodiment the disclosure relates to the strain, wherein the strain is selected from DSM35226; DSM 34681; DSM 35225; DSM 35230; DSM 35231; DSM 35232; DSM 35233; or mutants or variants thereof.

[0062] In one embodiment the present disclosure relates to the strain, wherein the strain is able to acidify a milk base to pH 4.6 within 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 hours after start of acidification, wherein the one or more phages is selected from: DSM 35003, DSM 23962, DSM 23994, DSM 35011, DSM 35227, and DSM 35228. In one embodiment the present disclosure relates to the strain, wherein the strain provides a faster acidification of a milk base in the presence of one or more bacteriophages as compared to the reference strain DSM 26562 and / or DSM 17876.

[0063] pH may be measured by a pH electrode, or by using a pH indictor such as e.g. bromophenol purple or bromophenol green.

[0064] In one embodiment the present disclosure relates to the strain, wherein the acidification is conducted at a temperature in the range of 35-40°C, 36-38°C, or at a temperature of 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0065] Compositions

[0066] Lactic Acid Bacteria (LAB) are most commonly added to a milk base in the form of a composition, preferably as a starter culture. Alternatively, the LAB may be added to a milk base individually or as a kit-of part. The term "starter" or "starter culture" as used in the present context refers to compositions or cultures of one or more food-grade microorganisms, in particular to lactic acid bacteria, which are responsible for the acidification of milk base. Starter cultures may be available in various forms such as in fresh, frozen, spray-dried, freeze-dried, vacuum-dried, air-dried, tray-dried or liquid form, but are most frequently in frozen or freeze-dried form. The compositions may be available as "Direct Vat Set" (DVS) cultures and are intended for direct inoculation of a fermentation vessel or vat for the production of a fermented product, in particular a fermented food product such as a fermented milk product. Respective starter cultures are commercially available from numerous providers including from Chr. Hansen. One or more strains in the composition must be of the species of Streptococcus thermophilus. Strains from other genus such as Lactobacillus, Limosilactobacillus, Lacticaseibacillus, Ligilactobacillus, Lactiplantibacillus, Lentilactobacillus, Latilactobacillus, Companilactibacillus, Lactococcus, Leuconostoc, Pediococcus, and / or Bifidobacterium may also be comprised. Strains of the species such as Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus casei, Lacticaseibacillus casei, Lacticaseibacillus paracasei, and / or Bifidobacterium animalis are of particular interest. In one aspect the disclosure relates to a composition comprising the strain of the present disclosure.

[0067] In one embodiment the disclosure relates to the composition, comprising the strain DSM 35226 and one or more Streptococcus thermophilus strains selected from: DSM 33982; DSM 34679; and DSM 34681. In one embodiment the disclosure relates to the composition, comprising the strain DSM 35226 and DSM 33982. In one embodiment the disclosure relates to the composition, comprising the strain DSM 35226 and DSM 34679. In one embodiment the disclosure relates to the composition, comprising the strain DSM 35226 and DSM 34681. In one embodiment the disclosure relates to the composition, comprising the strain DSM 35225 and one or more Streptococcus thermophilus strains selected from: DSM 33982; DSM 34679; and DSM 34681. In one embodiment the disclosure relates to the composition, comprising the strain DSM 35225 and DSM33982. In one embodiment the disclosure relates to the composition, comprising the strain DSM 35225 and DSM 34679. In one embodiment the disclosure relates to the composition, comprising the strain DSM 35225 and DSM 34681. In one embodiment the disclosure relates to the composition, comprising one, two, three, or four of Streptococcus thermophilus strains selected from: DSM 35230; DSM 35231; DSM 35232; and DSM 35223.

[0068] In one embodiment the disclosure relates to the composition, further comprising DSM 19195 and / or DSM 21404.

[0069] The composition of the present disclosure may additionally comprise cryoprotectants, lyoprotectants, antioxidants, nutrients, fillers, flavorants or mixtures thereof. The composition may be in frozen or freeze-dried form. 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 cyroprotectants and lyoprotectantare 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). 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.

[0070] In one embodiment the present disclosure relates to the composition, wherein the composition further comprises cryoprotectants, lyoprotectants, antioxidants and / or nutrients. In one embodiment the present disclosure provides compositions in the form of a solid frozen or freeze-dried starter culture comprising lactic acid bacteria in a concentration of at least 107colony forming units (cfu) per g of starter culture or in a concentration of at least 108cfu / g, at least 109cfu / g, at least IO10cfu / g, at least IO11cfu / g of starter culture or in a concentration of at least 1012cfu / g of starter culture.

[0071] In one embodiment the disclosure relates to the composition, further comprising, either as a mixture or as a kit-of-part one or more lactic acid bacteria of a genus different from Streptococcus.

[0072] In one embodiment the disclosure relates to the composition, wherein the genus of the one or more lactic acid bacteria is Lactobacillus, Limosilactobacillus, Lacticaseibacillus, Ligilactobacillus, Lactiplantibacillus, Lentilactobacillus, Latilactobacillus, Companilactibacillus, Lactococcus, Leuconostoc, Pediococcus, and / or Bifidobacterium.

[0073] In one embodiment the disclosure relates to the composition, wherein the one or more lactic acid bacteria is Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus casei, Lacticaseibacillus casei, Lacticaseibacillus paracasei, Lactococcus lactis lactis, Lactococcus lactis cremoris, and / or Bifidobacterium animalis.

[0074] In one embodiment the disclosure relates to the composition, wherein the one or more lactic acid bacteria is Lactococcus lactis cremoris DSM 19195 and / or Lactococcus lactis lactis DSM 21404.

[0075] In one embodiment the disclosure relates to the composition, wherein the composition is in fresh, frozen, spray-dried, freeze-dried, vacuum-dried, air-dried, tray-dried or liquid form. Even though the fermented product and / or the food product itself comprise acid and flavor generated during fermentation it may be desired that fermented product and / or the dairy product comprises an ingredient selected from the group consisting of a fruit concentrate, a syrup, a probiotic bacterial strain or culture, a coloring agent, a thickening agent, a flavoring agent, a preserving agent and mixtures thereof.

[0076] Likewise, an enzyme may be added to the substrate e.g. the milk substrate before, during and / or after the fermenting, the enzyme being selected from the group consisting of an enzyme able to crosslink proteins, transglutaminase, an aspartic protease, lactase, chymosin, rennet and mixtures thereof.

[0077] Fermented Products.

[0078] Fermentation is carried out to produce fermented products. The term "fermented product", refers to products obtainable by the fermentation methods of the present disclosure and include cheese, yoghurt, fruit yoghurt, yoghurt beverage, strained yoghurt (Greek yoghurt, Labneh), quark, fromage frais and cream cheese. The term "cheese" is understood to encompass any cheese, including hard, semi-hard and soft cheeses, such as cheeses of the following types: Cottage, Feta, Cheddar, Parmesan, Mozzarella, Emmentaler, Danbo, Gouda, Edam, Feta-type, blue cheeses, brine cheeses, Camembert and Brie. The person skilled in the art knows how to convert the coagulum into cheese, methods can be found in the literature, see e.g. Kosikowski, F. V., and V. V. Mistry, "Cheese and Fermented Milk Foods", 1997, 3rd Ed. F. V. Kosikowski, L. L. C. Westport, CT. As used herein, a cheese which has a NaCI concentration below 1.7% (w / w) is referred to as a "low-salt cheese".

[0079] In the context of the present application, the term "yoghurt" refers to products comprising Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus and optionally other microorganisms such as Lactobacillus delbrueckii subsp. lactis, Bifidobacterium animalis subsp. lactis, Lactococcus lactis, Lactobacillus acidophilus and Lactobacillus paracasei, or any microorganism derived therefrom. The lactic acid strains other than Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, are included to give the finished product various properties, such as the property of promoting the equilibrium of the flora. As used herein, the term "yoghurt" encompasses set yoghurt, stirred yoghurt, drinking yoghurt, Petit Suisse, heat treated yoghurt, strained or Greek style yoghurt characterized by a high protein level and yoghurt-like products. In one embodiment the fermented product may be in the form of a stirred type product, a set type product or a drinkable product.

[0080] The term "stirred-type product" or "stirred product" specifically refers to a fermented milk product which sustains a mechanical treatment after fermentation, resulting in a destructuration and liquefaction of the coagulum formed under the fermentation stage. The mechanical treatment is typically but not exclusively obtained by stirring, pumping, filtrating or homogenizing the gel, or by mixing it with other ingredients. Stirred-type products typically but not exclusively have a milk solid non-fat content of 9 to 15%. In one embodiment the disclosure relates to a food product, wherein the food product is a stirred-type product, preferably wherein the food product is a stirred yogurt.

[0081] The term "set-type product" or "set product" includes a product based on milk which has been inoculated with a starter culture, e.g. a starter culture, and packaged next to the inoculating step and then fermented in the package. In one embodiment the disclosure relates to a food product, wherein the food product is a set-type product, preferably wherein the food product is a set yogurt.

[0082] The term "drinkable product" includes beverages such as "drinking yoghurt" and similar. The term "drinking yoghurt" typically covers a milk product produced by fermentation by the combination of Lactobacillus species and Streptococcus thermophilus. Drinking yoghurt typically has a milk solid non-fat content of 8% or more. Furthermore, the live culture count for drinking yoghurt drinks is typically at least 10E6 cell forming units (cfu) pr mL.

[0083] In particular, term "yoghurt" encompasses, but is not limited to, yoghurt as defined according to French and European regulations, e.g. coagulated dairy products obtained by lactic acid fermentation by means of specific thermophilic lactic acid bacteria only (i.e. Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus) which are cultured simultaneously and are found to be live in the final product in an amount of at least 10 million CFU (colony-forming unit) I g. Yoghurts may optionally contain added dairy raw materials (e.g. cream) or other ingredients such as sugar or sweetening agents, one or more flavoring(s), fruit, cereals, or nutritional substances, especially vitamins, minerals and fibers, as well as stabilizers and thickeners. Optionally the yoghurt meets the specifications for fermented milks and yoghurts of the AFNOR NF 04-600 standard and / or the codex StanA-IIa-1975 standard. In order to satisfy the AFNOR NF 04-600 standard, the product must not have been heated after fermentation and the dairy raw materials must represent a minimum of 70% (m / m) of the finished product.

[0084] In one embodiment the disclosure relates to a fermented product obtainable by the method of the present disclosure.

[0085] In one aspect the present disclosure relates to a fermented product comprising the strain of the present disclosure; or comprising the composition of the present disclosure.

[0086] In one embodiment the disclosure relates to the fermented product, wherein said product has: (a) a shorter acidification time to target pH;

[0087] (b) a reduced post-acidification at day 7; and / or

[0088] (c) an increased texture measured as gel firmness and / or shear stress;

[0089] as compared to (I) a fermented product made with a reference strain selected from DSM 26562 and / or DSM 17876 or (II) the commercial culture F-DVS eXact® Dahi 3 product no.

[0090] 717445 sold by Chr. Hansen A / S.

[0091] The strain may be present in a concentration of at least 107, 108, 109, 1010, 1011, or 1012CFU / g fermented product. The strain may be present in a concentration of about 107, 108, 109, 1010, 1011, or 1012cfu / g fermented product. In one embodiment the present disclosure relates to the fermented product, wherein the strain is present in a concentration of at least 107cfu / g.

[0092] In one embodiment the disclosure relates to the fermented product, wherein the fermented product is a Dairy product. In one embodiment the disclosure relates to the fermented product, wherein the dairy product is selected from a fresh dairy product such as yogurt, buttermilk, dahi, kefir; or a cheese such as fresh cheese or pasta filata.

[0093] Use

[0094] In one aspect the present disclosure relates to a Use of the strain of the present disclosure; or the composition of the present disclosure for the manufacture of a fermented product. In one embodiment the disclosure relates to the use, wherein said product has:

[0095] (a) a shorter acidification time to target pH;

[0096] (b) a reduced post-acidification at day 7; and / or

[0097] (c) an increased texture measured as gel firmness and / or shear stress;

[0098] as compared to (I) a fermented product made with a reference strain selected from DSM 26562 and / or DSM 17876 or (II) the commercial culture F-DVS eXact® Dahi 3 product no.

[0099] 717445 sold by Chr. Hansen A / S.

[0100] DEPOSIT AND EXPERT SOLUTION

[0101] 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.

[0102] Table 1: 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.

[0103]

[0104]

[0105] EXAMPLES MATERIAL & METHODS

[0106] F-DVS eXact® Dahi 3 (Chr. Hansen A / S product no. 717445) was used as Benchmark.

[0107] Table 2: Strains comprised in the Reference (R) and Experimental Compositions (C).

[0108]

[0109] The strains DSM 33982, DSM 14797, DSM 34679, DSM 19195, and DSM 21404 have all been described in W02023 / 094430, W02003 / 082019, PCT / EP2024 / 073295, WO2013 / 024178, and W02013 / 093049 respectively. The reference strains DSM 26562 and DSM 17876 have been described in W02017 / 005601 and W02007 / 095958 respectively.

[0110] EXAMPLE 1

[0111] Generation of a Phage-Resistant Mutant. DSM 35226 is a mutant derived from the mother strain DSM 35224. It was made in five rounds of phage hardening by selecting strains resistant towards phages including DSM 35227 and DSM 35228 attacking the mother strain.

[0112] In general, phage-resistant mutants were generated from a mother strain on M17-2% lactose agar plates with lOmM MgCh / CaCI? after plating O.lmL of an M17-2% lactose overnight culture of the host strain together with O.lmL of a phage lysate containing 1.0E09 phage particles per mL and incubation overnight at 37°C. For this, phages and host strain were mixed with 2.5mL of a top agar solution (molten agar with half (0.75%) of the standard agar concentration kept at 46-50 °C), and poured on a bottom M17-2% lactose agar. Both bottom and top agar were comprising lOmM MgCh / CaCI?. When the top agar was solidified incubation occurred with conditions described above.

[0113] Among a number of phage-resistant mutants which appeared on the agar plates after incubation, the putative phage-resistant mutants were three times colony purified and retested in plaque assay on M17-MgCl2 / CaCl2 lactose agar plates at 37°C test using the phage originally used for phage challenge. Phage-resistance towards the phage used originally for the phage challenge was confirmed by the absence of single plaques in the plaque test. Afterwards, additional phages towards the original wild type strains were tested against the phage-resistant mutants and, if a positive phage was identified, the mutant isolation procedure was repeated with that phage.

[0114] DSM 35226 was resistant to all phages attacking the mother strain DSM 35224 including the phages DSM 35227 and DSM 35228. Figure 1 shows the acidification of DSM 35226 of milk + / - the phage DSM 35228. The strain DSM 35224 is not sensitive i.e. resistant to the presence of the phage as the acidification profiles are similar irrespective of the presence or absence of the phage. Furthermore, the strain was also tested and shown resistant towards a panel of 55 phages.

[0115] The presence or absence of prophages in the chromosome of the S. thermophilus strains was conducted using the PHASTER (Phage Search Tool Enhance Release) program (https: / / phaster.ca). Analysis of the genome of DSM 35224 revealed the absence of prophages. This is an important feature since prophage free strains do not imply the risk of release of phage particles during the use in dairies which could attack other strains from the same species. Absence of prophages in a mother strain implies the absence of prophages in the strains derived from the mother strain.

[0116] EXAMPLE 2 Isolation of Phage-Resistant Strains

[0117] The phage-resistant Streptococcus thermophilus strains DSM 35225, DSM 35230, DSM35231, DSM 35232, DSM 35233, and DSM 35235 were selected from a pool of 1940 strains in three steps.

[0118] In the first step, strains suffering no phage hits from a phage pool testing, showing no antibiotic resistance toward Ampicillin, Chloramphenicol, Clindamycin, Erythromycin, Tetracycline nor Vancomycin, and having the fastest acidification rate were identified.

[0119] In the second step strains having no genetic relatedness and showing high gel firmness when tested at 43°C in 2mL of dahi milk (3.5% protein and 3.0% fat, made from low-heat skim milk powder, water and cream) supplemented with 0.003% Na-formate (0.6g Na-Formate: HCOONa, Mw 68.01 g mol-1 (Merck) dissolved in lOOmL sterile dH2O and sterile filtered), and 1% of a colored pH indicator (50mg bromocresol purple salt (Aldrich) and 50mg bromocresol green salt (Sigma) dissolved in a final volume of 40mL autoclaved dH2O. pH was adjusted to 7.0 with NaOH, dH2O was added to a final volume of 50mL, and 0.2pm sterile filtered).

[0120] In the third step, strains exhibiting low post-acidification defined as at or below 0.3 pH units at day 7 in lOOmL Dahi milk supplemented with 0.003% formate at 43°C were selected. The acidification rate and gel firmness were validated.

[0121] Phage-resistance of the strains was evaluated by phage-typing with a high number of phages from different lytic groups (phages with different host range and a high diversity concerning the phage sensitivity pattern). The phage-sensitivity I the phage-resistance of the Streptococcus thermophilus strains were tested in a colorimetric microtiter plate inhibition assay.

[0122] Test phages were distributed in microtiter plates containing sterilized 9.5% skim milk and a colorimetric pH indicator (indicator-milk). Each phage was added to a final concentration of 1.0E06 phages / mL. The control was indicator-milk without addition of phages.

[0123] The test strain was inoculated 1% in the microtiter wells from an overnight culture. The acidification was followed by periodical scanning of the microtiter plates on a flatbed scanner and translation of the color values into pH values by specific software resulting in pH curves for the strain infected with each single phage and the strain without added phages.

[0124] The pH of the phage containing well and the control well were compared when the control well without a phage reached pH 5.0. For each single phage-containing well the pH difference was measured at this control pH, and a sample was considered positive (phage sensitive) if the difference in pH was higher than 0.3 pH units. It was found that the new S. thermophilus strains DSM 35225, DSM 35230, DSM35231, DSM 35232, DSM 35233, and DSM 35235 were resistant to all of the 55 bacteriophages tested. Figure 2 shows the acidification by DSM 35231 of milk + / - the pool of 55 phages. The strain DSM 35231 is not sensitive i.e. resistant to the presence of the phages as the acidification profiles are similar irrespective of the presence or absence of the pool of 55 phages. The strains DSM 35225, DSM 35226, DSM 35230, DSM 35232, DSM 35233 all showed the same acidification profiles when tested I milk + / - the pool of 55 phages indicating phage-resistance. The strains were also tested towards the individual phages as shown in the table below. Table 3: Phage-resistance of strains towards selected phages.

[0125]

[0126] The phages DSM 23962 and DSM 23994 have been described in WO2011 / 092300. The phages DSM35003 and DSM 35011 have been described in a previous application filed by Chr. Hansen A / S.

[0127] The genomes of the strains DSM 35225, DSM 35230, DSM 35231, DSM 35232, DSM 35233, and DSM 35235 were furthermore analyzed for the presence of phage-resistance genes using the PADLOC program (PADLOC: a web server for the identification of antiviral defence systems in microbial genomes, Nucleic Acids Research, Volume 50, Issue Wl, 5 July 2022, Pages W541-W550). They showed a high number of phage-resistance genes. The analysis of these genomes also revealed the absence of prophages. This is an important feature since prophage free strains do not imply the risk of release of phage particles during the use in dairies which could attack other strains from the same species.

[0128] The presence or absence of prophages in the chromosome of the S. thermophilus strains was investigated with the PHASTER (Phage Search Tool Enhance Release) program (https: / / phaster.ca; ref: Arndt, D., Grant, J., Marcu, A., Sajed, T., Pon, A., Liang, Y., Wishart, D.S. (2016) PHASTER: a better, faster version of the PHAST phage search tool. Nucleic Acids Res., 2016 May 3). Texture Analyzer (TA. XT PlusC, Stable Micro Systems) records the force of resistance of the instrument probe as it penetrates the sample according to the predefined movement speed and amplitude. The measurement consists of forward and backward movement (back extrusion method) and the result is given as a positive compression area (gel firmness) and a negative compression area (cohesiveness). Before starting the experiment, a 2000g + / -0.1g weight is used to calibrate force of the Texture Analyzer and samples are placed at 13°C for at least 1 hour and not more than 2 hours. A 40mm Disk probe is fixed on the Texture Analyzer arm. Do not stir the sample and do not throw out / discard the whey if whey separation is observed in the samples. The compression method used for the analysis is then started with every sample. This method test speed is set to 5.00 mm / s with target distance of 15.00 mm and trigger force of 5.0 g.

[0129] Table 4: Acidification time and post-acidification of single strains.

[0130]

[0131] Table 5: Texture of single strains.

[0132]

[0133] EXAMPLE 3 Acidification time of the experimental compositions.

[0134] Set yoghurt were made with the four experimental compositions and the Benchmark.

[0135] A milk base (3.5% protein, 3.0% fat, 0% sugar) was made from fresh skim milk standardized with skim milk power and cream. The fermented products were produced by inoculating the milk base with the four experimental compositions and the benchmark respectively according to the following process.

[0136] Preparation of the milk base:

[0137] Mixing 55°C for Ihour

[0138] Homogenization 75°C (150 / 50 bar)

[0139] Pasteurization 95°C for 5 minutes

[0140] Fermentation:

[0141] Inoculation 500U / 5000L

[0142] Fermentation temperature 43°C

[0143] Target pH 4.6

[0144] Cooling a storage of the fermented product:

[0145] Cooling after fermentation Cooling from 43°C to 10°C over lOh, from 10°C to 5°C over lOh before keeping overnight at 5°C. The next day, store then at 13°C.

[0146] Storage Store at 13°C for 14 days

[0147] The acidification time was measured using the axone system (equipment initially developed for Chr. Hansen by Absciss, who is now part of ARDPI), which allows for continuous measurement of pH during fermentation.

[0148] Table 6: Acidification time to target pH.

[0149]

[0150] EXAMPLE 4

[0151] Texturizing properties measured by gel firmness Gel firmness was measured with a texture analyzer (TA. XT PlusC, Stable Micro Systems). The test mode was set to compression and the probe used was a 40mm disk. The pre-test, test and post-test speed were set at 5 mm / s, the distance to 15mm and the trigger force to 5grams. The data was measured at day +7, at 13°C, and the sample was stored at 13°C until the day of measurement.

[0152] Table 7: Gel firmness (g).

[0153]

[0154] EXAMPLE 5

[0155] Texturizing properties measured by shear stress

[0156] Rheological property was measured with an HTR-3000 rheometer, model MCR302e, from Anton Paar. The method is using a rotational step, which is based on a rotational deformation of the sample from 0.273 s-1 to 300 s-1 and then back to 0.273 s-1. The corresponding shear stress is measured.

[0157] The sample was stored at 13°C until the day of measurement and the shear stress at the shear rates of 30.2 s1and 300 s1was measured at day +7, at 13°C.

[0158] Table 8: Shear stress measured at two different shear rates.

[0159]

[0160] EXAMPLE 6

[0161] Acidification time of compositions comprising the new strains.

[0162] Set yogurt were made with the following blends: Rl, Cl, and C2.

[0163] F-DVS eXact® Dahi 3 (Chr. Hansen product no. 717445) was used as Benchmark.

[0164] A milk base (3.5% protein, 3.0% fat) was made from Low heat skim milk power and 38% cream. Four fermented products were produced by inoculating the milk base with the three experimental blends and the benchmark respectively according to the following process: Preparation of the milk base: Mixing 6°C for 2hours

[0165] Homogenization 65°C (200 / 50 bar)

[0166] Pasteurization 95°C for 5 minutes

[0167] Fermentation:

[0168] Inoculation 500U / 5000L

[0169] Fermentation temperature 43°C

[0170] Target pH 4.6

[0171] Cooling a storage of the fermented product:

[0172] Cooling after fermentation Cooling from 43°C to 26°C in 2h, then to 10°C over 8h, from 10°C to 5°C over lOh and keeping at 5°C. Next day, store at 13°C.

[0173] Storage Store at 13°C for 7 days

[0174] The acidification time was measured using Color of pH (a colorimetric method initially developed for Chr. Hansen), which allows for continuous measurement of pH during fermentation. A Hue (color) value of 135 corresponds to pH 4.6 which is the target value at which the fermentation was stopped.

[0175] Table 9: Acidification time to target Hue 135 (pH 4.6) in minutes.

[0176]

Claims

CLAIMS1. A method for producing a fermented milk product comprising the steps:(a) providing a strain of the species Streptococcus thermophilus, which strain is resistant towards the phage DSM 35227 and / or DSM 35228;(b) adding the strain of (a) to a milk base;(c) fermenting the milk base at a temperature between 20°C and 50°C until a pH of 4.6 or less is reached; andwherein the product has a post-acidification at or below 0.3 pH units measured at day 7 after fermentation at 43°C in a milk base comprising 3.5% protein, 3.0% fat, 0% added sugar and stored at 13°C.

2. The method according to claim 1, wherein the strain is prophage-negative.

3. The method according to any one of claims 1-2, wherein the strain is resistant towards one or more phages selected from: DSM 23962, DSM 23994, DSM 35003, and DSM 35011.

4. The method according to any one of claims 1-3, wherein the strain is not resistant to one or more of the antibiotics: ampicillin, chloramphenicol, clindamycin, erythromycin, tetracycline and vancomycin.

5. The method according to any one of claims 1-4, wherein the fermented milk product has:(a) a shorter acidification time to target pH;(b) a reduced post-acidification at day 7; and / or(c) an increased texture measured as gel firmness and / or shear stress;as compared to (I) a fermented product made with a reference strain selected from DSM 26562 and / or DSM 17876; or (II) the commercial culture F-DVS eXact® Dahi 3 product no. 717445 sold by Chr. Hansen A / S.

6. The method according to any one of claims 1-5, wherein the strain is selected from one or more of: A strain derived from DSM 35224; DSM35226; DSM 34681; DSM 35225; DSM 35230; DSM 35231; DSM 35232; DSM 35233; and mutants or variants thereof.

7. A fermented product obtainable by the method of any of claims 1-6.

8. A phage-resistant strain of the species Streptococcus thermophilus, wherein the strain is resistant towards the phage DSM 35227 and / or DSM 35228.

9. The strain according to claim 8, wherein said strain is prophage-negative.

10. The strain according to any one of claims 8-9, wherein the strain is resistant towards one or more phages selected from: DSM 23962, DSM 23994, DSM 35003, and DSM 35011.

11. The strain according to any one of claims 8-10, wherein the strain is not resistant to one or more of the antibiotics: ampicillin, chloramphenicol, clindamycin, erythromycin, tetracycline and vancomycin.

12. The strain according to any one of claims 8-11, wherein said strain is (I) derived from DSM 35224; or (II) selected from DSM35226; DSM 34681; DSM 35225; DSM 35230; DSM 35231; DSM 35232; DSM 35233; or mutants or variants thereof.

13. A composition comprising the strain according to any one of claims 8-12.

14. The composition according to claim 13, comprising the strain DSM 35226 and one or more Streptococcus thermophilus strains selected from: DSM 33982; DSM 34679; and DSM 34681, or the strain DSM 35225 and one or more Streptococcus thermophilus strains selected from: DSM 33982; DSM 34679; and DSM 34681.

15. The composition according to claim 14, further comprising DSM 19195 and / or DSM 21404.

16. The composition according to any one of claims 13-15, further comprising, either as a mixture or as a kit-of-part one or more lactic acid bacteria of a genus different from Streptococcus.

17. The composition according to claim 16, wherein the one or more lactic acid bacteria is Lactococcus lactis cremoris DSM 19195 and / or Lactococcus lactis lactis DSM 21404.

18. A fermented product comprising the strain according to any of claims 8-12; or comprising the composition according to any of claims 13-17.

19. The fermented product according to claim 18, wherein said product has:(a) a shorter acidification time to target pH;(b) a reduced post-acidification at day 7; and / or(c) an increased texture measured as gel firmness and / or shear stress;as compared to (I) a fermented product made with a reference strain selected from DSM 26562 and / or DSM 17876; or (II) the commercial culture F-DVS eXact® Dahi 3 product no. 717445 sold by Chr. Hansen A / S.

20. The fermented product according to any one of claims 7, 18 and 19, wherein the fermented product is a Dairy product.

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