Polysaccharide resulting in improved texture of milk-based products

Abstract

The present invention relates to novel exopolysaccharides that result in improved texture of milk-based products and LAB strains that produce such polysaccharides. The invention also relates to a method for preparing a product by using the polysaccharide and / or the strain, and a product comprising the polysaccharide and / or the strain. Finally, the invention relates to a method for producing the strain of the invention.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a novel exopolysaccharide resulting in an improved texture of a dairy product and a LAB strain producing such a polysaccharide. The present invention also relates to a method for manufacturing a food using the polysaccharide and / or the strain, and a food comprising the polysaccharide and / or the strain. Finally, the present invention relates to a method for manufacturing the strain of the present invention. Background Art

[0002] The texture of fermented milk is an important quality parameter that influences consumer acceptance. The texture of fermented milk depends on the bacteria used for fermentation and the process parameters. Lactic acid bacteria (LAB), such as strains of Streptococcus thermophilus, that provide good texture are beneficial for products such as yogurt. Exopolysaccharides produced by bacteria can have a positive impact on texture. The ability of LAB, such as Streptococcus thermophilus, to produce polysaccharides can lead to improved texture of fermented products.

[0003] Even if the potential to synthesize exopolysaccharides is encoded in the genome, polysaccharides are not always produced. Furthermore, the production of polysaccharides does not always increase the texture of products such as food. The amount, type and size of polysaccharides and their interaction with proteins (e.g. milk proteins) are decisive factors in the formation of texture in products such as dairy products (e.g. fermented milk products).

[0004] Some LAB strains contribute significantly to improved texture related to their ability to produce exopolysaccharides. Exopolysaccharides can be produced as unattached material secreted into the environment (commonly known as exopolysaccharides (EPS)) or as capsules attached to the surface of bacteria (known as capsular polysaccharides (CPS)). EPS consists of a single type of sugar (homopolysaccharides) or repeating units composed of different sugars (heteropolysaccharides). LABs that produce EPS are interesting because EPS acts as a natural viscosity enhancer and texture enhancer for fermented foods. In addition, EPS from food-grade LABs with clear rheological properties have the potential to be developed and utilized as food additives. EPS is known to improve the rheological properties (i.e. texture) of LAB fermented products by affecting viscosity, syneresis, hardness and sensory properties. The primary structural characteristics of polysaccharides (monosaccharide type and configuration, glycosidic bonds, non-sugar decoration, charge), conformation and molecular weight, the amount of polysaccharides, and the interaction of polysaccharides with other system components are all factors that can contribute to and influence the displayed technical functional properties (Zeidan et al., (2017), Polysaccharide production by lactic acid bacteria: from genes to industrial applications. FEMS Microbiology Reviews, 41 (1), S168-S200). The gene cluster encoding the enzyme responsible for the production of extracellular polysaccharides is usually called the eps gene cluster.

[0005] It has been shown that LAB produces EPS via two pathways encoded by different genomic elements: the sucrase-dependent pathway and the Wzy-dependent pathway. S. thermophilus has no sucrose-dependent pathway and can only synthesize exopolysaccharides via the Wzy-dependent pathway, which is encoded in a gene cluster referred to herein as the eps cluster.

[0006] There is a wide variety of possible polysaccharide structures (identified by their repeating units) that depend on their sugar building blocks, anomeric configuration, glycosidic bonds, non-sugar decorations and conformation of the monosaccharides. The selection of suitable polysaccharides and polysaccharide-producing cultures for use in fermented milk products depends on factors such as the functionality of the polysaccharide produced, the product type and the production process. For example, in the context of the present invention, suitable polysaccharides need to achieve the desired texture and must also meet other criteria in order to be commercially useful. Since texture is an important parameter for products, especially food and feed products (such as fermented milk products), texturing polysaccharides and polysaccharide-producing LAB strains (such as polysaccharide-producing Streptococcus thermophilus strains) are also needed. SUMMARY OF THE INVENTION

[0008] The inventors have discovered strains that produce extracellular polysaccharides, particularly EPS, that lead to improved texture of fermented milk. The inventors have also elucidated the structure of such polysaccharide repeating units. The structure of the extracellular polysaccharide of the present invention has not been reported. The present invention therefore relates to novel and creative extracellular polysaccharides, particularly EPS, and LAB strains that can produce these polysaccharides. The present invention also relates to compositions and starter cultures comprising such extracellular polysaccharides and / or strains, as well as methods for manufacturing products such as food or feed products using the extracellular polysaccharides and / or strains, and also to products such as food or feed products comprising the extracellular polysaccharides and / or strains of the present invention.

[0009] Thus, in a first aspect, the present invention relates to a polysaccharide, preferably an exopolysaccharide, even more preferably an EPS, having a repeating unit comprising one of the following backbones:

[0010] (i)→4)-β-D-Glcp-(1→4)-α-D-Glcp-(1→4)-β-D-Galp-(1→, or

[0011] (ii)

[0012]

[0013] The repeating unit comprising the main chain (i) further comprises at least one side chain connected to any sugar in the main chain, wherein the side chain comprises or consists of structure (a):

[0014]

[0015] Where (1->6) represents a linker attached to a monosaccharide in the backbone; and

[0016] The repeating unit comprising the main chain (ii) comprises at least one side chain attached to any sugar in the main chain, wherein the side chain comprises or consists of structure (b):

[0017]

[0018] Wherein, (1->2) represents the linker connected to the monosaccharide in the main chain.

[0019] In a second aspect, the present invention provides a lactic acid bacteria (LAB) strain capable of producing a polysaccharide according to the first aspect of the present invention or a polysaccharide comprising a repeating unit, the repeating unit comprising or consisting of one of the following structures: (02)

[0021] (03)

[0023]

[0024] In a third aspect, the present invention relates to a composition and / or a starter culture comprising the polysaccharide of the first aspect and / or the LAB strain of the second aspect of the present invention.

[0025] In a fourth aspect, the present invention relates to the use of the polysaccharide of the first aspect of the present invention or a polysaccharide comprising a repeating unit, the LAB strain of the second aspect and / or the composition / starter of the third aspect in improving product texture, such as increasing product viscosity, wherein the repeating unit comprises one of structures (2) and / or (3) or consists of one of structures (2) and / or (3).

[0026] In a fifth aspect, the present invention also relates to a method for producing a product, comprising the steps of using the polysaccharide of the first aspect of the present invention or a polysaccharide comprising a repeating unit, the LAB strain of the second aspect and / or the composition / starter of the third aspect, wherein the repeating unit comprises one of structures (2) and / or (3) or consists of one of structures (2) and / or (3).

[0027] In a sixth aspect, the present invention provides a product comprising the polysaccharide of the first aspect of the invention or a polysaccharide comprising a repeating unit, the LAB strain of the second aspect and / or the composition / starter of the third aspect, wherein the repeating unit comprises one of structures (2) and / or (3) or consists of one of structures (2) and / or (3).

[0028] In a seventh aspect, the present invention provides a method for producing the LAB strain defined in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0030] definition

[0031] All definitions of relevant terms in this document are consistent with the understanding of the relevant technical content of this document by those skilled in the art.

[0032] In the context of the present invention, the expression "lactic acid bacteria" ("LAB") refers in any of its embodiments to food-grade bacteria that produce lactic acid as the main metabolic end product of carbohydrate fermentation. These bacteria are related by their common metabolic and physiological characteristics, being Gram-positive, low GC, acid-tolerant, non-spore-forming, rod-shaped bacilli or cocci. During the fermentation phase, the consumption of carbohydrates by these bacteria results in the formation of lactic acid, which lowers the pH and leads to the formation of protein coagulum. Therefore, these bacteria are responsible for the acidification of milk and the texture of dairy products. The thermophilic Streptococcus strains of the present invention belong to lactic acid bacteria.

[0033] In the present specification and claims, "texturing strain" refers to a strain that preferably increases the viscosity of fermented milk, and more preferably produces fermented milk that, when heated for 300 s -1 When measured at a shear rate of 1.5 Pa, in the presence of 0.2% yeast extract, the shear stress of the fermented milk generated is higher than 17 Pa, for example, about 20 Pa, 25 Pa, 30 Pa, 35 Pa, 36 Pa, 36.5 Pa, 40 Pa, 41 Pa, 45 Pa, 50 Pa, 55 Pa, 60 Pa, 65 Pa, 66 Pa, 67 Pa, 70 Pa or more, and / or in the presence of 2% casein hydrolysate, the shear stress of the fermented milk generated is higher than 13.3 Pa, for example, 13.5 Pa, 15 Pa, 20 Pa, 25 Pa, 30 Pa, 35 Pa, 40 Pa, 41.2 Pa, 41.4 Pa, 41.5 Pa, 42 Pa, 45 Pa, 50 Pa, 55 Pa, 60 Pa, 63 Pa, 64 Pa, 64.5 Pa, 65 Pa, 70 Pa or more.

[0034] The textured LAB strains (e.g., thermophilic Streptococcus strains) of the present invention may be isolated strains, e.g., isolated from naturally occurring sources, or may be non-naturally occurring strains, e.g., recombinantly obtained strains. Recombinant strains differ from naturally occurring strains in that at least the nucleic acid construct(s) used to transform or transfect the parent strain are present.

[0035] "Texture" is also an important quality factor of the product, especially for fermented milk products. Consumer acceptance is often closely related to the textural properties. The texture of fermented milk depends on the exopolysaccharide structure, the bacteria used for fermentation, the process parameters and the milk composition. In the context of the present invention, the rheological properties (texture) of the fermented milk product, such as viscosity, can be measured as a function of the "shear stress" of the fermented milk product by the following method (see Example 1 for further details): When the pH of the fermented milk (e.g., mammalian-based milk or plant-based milk) reaches about pH 4.55 at the incubation temperature (e.g., 40°C), the fermented milk sample is cooled (e.g., cooled to 4°C) by transferring the container to ice water. The fermented milk sample is gently stirred manually with a rod equipped with a perforated disc until the sample is homogenous. In particular, the shear stress is measured as follows:

[0036] 200 mL skim milk (protein 38%, lactose 53%, fat <1.25) was heated to 99°C for 30 min, then cooled to the inoculation temperature, inoculated with 2 mL of overnight culture of lactic acid bacteria strain in the presence of 0.2% yeast extract or 2% casein hydrolysate, and allowed to stand at the inoculation temperature until pH 4.55, then cooled to 4°C, then gently stirred and incubated for 300 s. -1 The shear stress was measured at a shear rate of 2.5 Å, with the inoculation temperature being 40 °C.

[0037] The rheological properties of the sample, such as shear stress (i.e., texture), are preferably evaluated on the rheometer described in Example 1, i.e., an Anton Paar Physica rheometer (Anton The evaluation was performed using a bob-cup on a Flow Meter (Rheometer, TU Wien GmbH, Austria). During the measurement, the rheometer was set to a constant temperature of 13°C. The settings were as follows:

[0038] - Keep time (rebuild to somewhat original structure);

[0039] - 5 minutes without any physical stress (oscillation or rotation) on the sample;

[0040] - Oscillation step (measurement of elastic and viscous moduli, G′ and G″ respectively, and calculation of the complex modulus G*)

[0041] Constant strain = 0.3%, frequency (f) = [0.5…8] Hz

[0042] 6 measurement points within 60s (one every 10s);

[0043] - Rotation step (to measure shear stress at 300 1 / s);

[0044] -Two steps are designed:

[0045] - Shear rate = [0.3-300] 1 / s and 2) Shear rate = [275-0.3] 1 / s.

[0046] Each step contains 21 measurement points (every 10 seconds) within 210 seconds. Select 3001 / s (300 seconds -1 ) was used for further analysis as this is related to the thickness of the oral cavity when swallowing fermented dairy products.

[0047] In the present context, the terms "strains derived from", "derived strains" or "mutants" are to be understood as strains derived from the strains of the present invention by methods such as genetic engineering, irradiation and / or chemical treatment and / or selection, adaptation, screening, etc. Preferably, the derivative strains are functionally equivalent mutants, for example, strains having substantially the same or improved properties as the parent strain in terms of texturizing properties (e.g. viscosity enhancing properties). Such derivative strains are part of the present invention. In particular, the terms "derivative strains" or "mutants" refer to strains obtained by subjecting the strains of the present invention to any conventional mutagenic treatment, including treatment with chemical mutagens such as ethane methanesulphonate (EMS) or N-methyl-N′-nitro-N-nitroguanidine (NTG), UV light, or to spontaneously occurring mutants.

[0048] The mutant may have been subjected to several mutagenesis treatments (a single treatment is understood to be a mutagenesis step followed by a screening / selection step), but it is currently preferred that no more than 20, no more than 10 or no more than 5 treatments are performed. In preferred derivative strains of the present invention, 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 the bacterial genome have been altered (e.g. by substitution, insertion, deletion or a combination thereof) compared to the parent strain.

[0049] The terms "fermented milk" and "dairy" are used interchangeably herein. In the context of the present invention, the expression "fermented milk product" refers to a food or feed product in any of its embodiments, wherein the preparation of the food or feed product involves fermentation of a milk base with lactic acid bacteria. "Fermented milk product" as used herein includes, but is not limited to, products such as thermophilic fermented milk products or mesophilic fermented milk products. In this article, the term "thermophilic fermentation" refers to fermentation at a temperature above about 35°C, for example from about 35°C to about 45°C. The term "mesophilic fermentation" herein refers to fermentation at a temperature of about 22°C to about 35°C.

[0050] In the context of the present application, the term "milk" is widely used in its common meaning to refer to a liquid produced by the mammary glands of animals (e.g., cows, sheep, goats, buffaloes, camels, etc.) or produced using a plant base. The term "milkbase" or "milk substrate" can be any milk material fermented according to the present invention. In a preferred embodiment, the milk is cow's milk. According to the present invention, the milk may have been processed, so the term "milk" includes whole milk, skim milk, fat-free milk, low-fat milk, full fat milk, low-lactose milk or concentrated milk. Fat-free milk is a non-fat product or a skim milk product. Low-fat milk is generally defined as milk containing about 1% to about 2% fat. Whole milk generally contains 2% or more fat. Therefore, useful milk basis includes but is not limited to any milk or the solution / suspension of the milk sample product comprising protein, such as whole milk or low-fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, milk powder, whey, whey permeate, lactose, lactose crystal mother liquor, whey protein concentrate, cream or plant-based milk. Milk base can be derived from any mammal, such as substantially pure mammal milk or reconstituted milk powder. The plant source of milk includes but is not limited to the milk extracted from soybean, pea, peanut, barley, rice, oat, quinoa, almond, cashew, coconut, hazelnut, hemp, sesame seed, sunflower seed and broad bean. Preferably, plant-based milk is soy milk, which can be preferably supplemented with glucose, such as 0.5-5% glucose, 0.5-2% glucose or about 2% glucose.

[0051] The term "product" as used herein refers to any product that benefits from the addition of exopolysaccharides as described herein. As described herein, the product is preferably a food or feed product, more preferably a fermented food or feed product, and even more preferably a dairy product. The term "dairy product" as used herein refers to a food produced from milk.

[0052] Prior to fermentation, the milk base may be homogenized and pasteurized according to methods known in the art.

[0053] "Homogenization" as used in the context of the present invention in any of its embodiments refers to intensive mixing to obtain a soluble suspension or emulsion. If homogenization is performed before fermentation, homogenization can be performed to break down the milk fat into smaller particles so that it can no longer separate from the milk. This can be achieved by forcing the milk through small holes under high pressure.

[0054] "Pasteurization" as used in the context of the present invention refers to the treatment of a milk base in any of its embodiments to reduce or eliminate the presence of living organisms (e.g., microorganisms). Pasteurization is preferably achieved by maintaining a specified temperature for a specified period of time. The specified temperature is usually reached by heating. The temperature and time can be selected so as to kill or inactivate certain bacteria, such as harmful bacteria. A rapid cooling step may then be performed.

[0055] In the context of the present invention, "fermentation" in any of its embodiments refers to the conversion of carbohydrates into acids or alcohols or a mixture of the two by the action of microorganisms (LAB). Fermentation processes for the production of foods such as dairy products are well known, and those skilled in the art will know how to select appropriate process conditions, such as temperature, oxygen, microbial biomass and process time. The fermentation conditions are selected to support the implementation of the present invention, for example, to obtain in any of its embodiments a product, preferably a food or feed product, having improved textural properties, such as viscosity, compared to a product, preferably a feed or feed product, produced by the following method: the method does not involve the use of at least one EPS structure and / or a strain producing such a structure as described in the first aspect of the present invention, or does not involve the use of a strain or composition as described in the present invention.

[0056] As used herein, the term "about" (or "approximately (around)") refers to the indicated value ± 1% of its value, or the term "about" refers to the indicated value ± 2% of its value, or the term "about" refers to the indicated value ± 5% of its value, the term "about" refers to the indicated value ± 10% of its value, or the term "about" refers to the indicated value ± 20% of its value, or the term "about" refers to the indicated value ± 30% of its value; preferably, the term "about" refers to the indicated value exactly (±0%).

[0057] Throughout the specification and claims, the word "comprise" and variations of the word (e.g., "comprising", "having", "including", "containing") are generally not limiting and thus do not exclude other features that may be, for example, technical features, ingredients, substrates, components or steps. However, whenever the word "comprise" is used herein, this also includes specific embodiments in which the word is understood to be limiting; in this specific embodiment, the word "comprise" has the meaning of the term "consisting of".

[0058] Unless otherwise indicated herein or clearly contradicted by context, the use of the terms "a" and "an" and "the" and similar indicators in the context of describing the invention (especially in the context of the following claims) should be construed to cover both the singular and the plural. Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein, unless otherwise indicated herein or clearly contradicted by context. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context.

[0059] Unless otherwise stated, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the invention and does not limit the scope of the invention. No language in the specification should be construed as indicating any non-claimed element is essential to the practice of the invention.

[0060] Polysaccharides of the present invention

[0061] In a first aspect, the present invention provides a polysaccharide, preferably an exopolysaccharide (EPS), the repeating unit of which comprises or consists of one of the following backbones:

[0062] (i)

[0063]

[0064] or

[0065] (ii)

[0066]

[0067] The polysaccharide of the present invention preferably additionally comprises one or more side chains of one or more sugars connected to the main chain. Therefore, the polysaccharide of the present invention is preferably a branched polysaccharide. The polysaccharide of the present invention is a heteropolysaccharide (HePS) because two or more types of sugars are present in the repeating unit.

[0068] In one embodiment, the polysaccharide of the present invention, whose repeating unit comprises the main chain (i) defined above or consists of the main chain (i), includes at least one side chain connected to any sugar in the main chain, wherein the side chain comprises the following structure or consists of the following structure:

[0069] (a)

[0070]

[0071] Among them, (1->6) represents the linker connected to the monosaccharide in the main chain.

[0072] In one embodiment, the side chain defined in (a) is attached to the α-D glucose present in the main chain (i). Preferably, the side chain defined in (a) is attached to the second α-D glucose of the main chain (i), which corresponds to the monosaccharide marked with number 2 in the above main chain (i).

[0073] In one embodiment, the polysaccharide of the present invention, whose repeating unit comprises the main chain (ii) as defined above or consists of the main chain (ii), comprises at least one side chain connected to any sugar in the main chain, wherein the side chain comprises the following structure:

[0074] (b)

[0075]

[0076] Wherein, (1->2) represents the linker connected to the monosaccharide in the main chain.

[0077] In one embodiment, the side chain defined in (b) is attached to a D-glucose present in the main chain (ii). Preferably, the side chain defined in (b) is attached to an α-D glucose in the main chain (ii), which corresponds to the monosaccharide marked with the number 5 in the above main chain (ii). In another embodiment, the side chain defined in (b) is attached to a β-D-glucose and / or β-D-galactose of the main chain (ii).

[0078] The side chains connected to the main chain (i) or main chain (ii) of the polysaccharide of the present invention may contain one or more sugars connected to the terminal of the above-mentioned structure (a) or (b), that is, β-D-Galp and / or β-D-Glcp connected to the terminal of structure (a), or β-D-Glcp connected to the terminal of structure (b). In addition, at least one side chain connected to the main chain (i) of the polysaccharide of the present invention may contain one or more sugars connected to non-terminal monosaccharides, that is, β-D-Glcp monosaccharides marked with the number 5 in (a) above. Therefore, at least one side chain of the polysaccharide of the present invention may be a branched side chain. Such sugars connected to the side chains are preferably galactose and glucose, but may also be rhamnose, GalNAc, GlcNAc, ManNAc, fucose, ribose, nonionic sugars or mixtures thereof. In one embodiment, the side chain connected to the main chain of the polysaccharide of the present invention contains at least one additional sugar, preferably galactose or glucose, or a mixture thereof.

[0079] In one embodiment, the side chain of the polysaccharide of the present invention consists of the above structure (a) or (b), ie, the side chain attached to the polysaccharide main chain does not contain any additional sugar residues.

[0080] In one embodiment, the polysaccharide of the present invention comprises a repeating unit comprising or consisting of the following structure: (01)

[0082]

[0083] In some embodiments, the polysaccharide of the present invention comprises one or more phosphate groups in its repeating unit. In other embodiments, the polysaccharide of the present invention comprises one or more substituents in its repeating unit. These substitution modifications include sulfonation, phosphorylation, acetylation, selenization or a combination thereof.

[0084] LAB produces high molecular weight and low molecular weight polymers. The molecular weight of polysaccharides synthesized via the Wzy-dependent pathway can range from 8 kDa to more than 10,000 kDa. It is common for polysaccharides of different sizes to coexist, that is, LAB can simultaneously produce the same exopolysaccharide of different molecular weights, see Zeidan et al, 2017. In the present invention, the molecular weight (molecular weight, MW) of the polysaccharide of the present invention is not particularly limited, and preferably can be at least 1000 kDa, preferably in the range of 10,000 to more than 10,000 kDa. For example, the MW range of the polysaccharide of the present invention is 2000 to over 10000 kDa, such as 3000 to over 10000 kDa, or 4000 to over 10000 kDa, or 1000 to 8000 kDa, or 2000 to 8000 kDa, or 3000 to 8000 kDa, or 4000 to 8000 kDa, or 5000 to 8000 kDa, or 1500 to 7000 kDa, or 2000 to 7000 kDa, or 2500 to 7000 kDa, or 3000 to 7000 kDa, or 4000 to 7000 kDa, or 2500 to 6000 kDa, or 3000 to 6000 kDa, or 3000 to 5000 kDa.

[0085] Methods for determining the molecular weight of exopolysaccharides (eg EPS) are well known in the art. For example, polysaccharides can be separated (eg, as described in Example 2) and then the MW can be determined using size-exclusion chromatography (SEC).

[0086] Lactic acid bacteria strains of the present invention

[0087] In a second aspect, the present invention provides a lactic acid bacteria (LAB) strain capable of producing a polysaccharide according to the first aspect of the present invention or a polysaccharide comprising a repeating unit, the repeating unit comprising or consisting of one of the following structures: (02)

[0089] (03)

[0091]

[0092] Preferably, the LAB strain of the present invention produces at least one of said polysaccharides.Preferably, the LAB strain of the present invention is a Streptococcus thermophilus strain.

[0093] More preferably, the LAB strain of the present invention is strain (i) and / or strain (ii) and / or strain (iii):

[0094] Strain (i) produced fermented milk. In the presence of 0.2% yeast extract, the fermentation was carried out at 300 s. -1 When the shear rate is measured under the following conditions, the shear stress of the fermented milk is greater than 17 Pa, for example, about 20 Pa, 25 Pa, 30 Pa, 35 Pa, 36 Pa, 36.5 Pa, 40 Pa, 41 Pa, 45 Pa, 50 Pa, 55 Pa, 60 Pa, 65 Pa, 66 Pa, 67 Pa, 70 Pa or more:

[0095] 200 mL skim milk (protein 38%, lactose 53%, fat <1.25) was heated to 99°C for 30 min, then cooled to the inoculation temperature, inoculated with 2 mL of overnight culture of lactic acid bacteria strain in the presence of 0.2% yeast extract, and allowed to stand at the inoculation temperature until pH 4.55, then cooled to 4°C, then gently stirred and incubated for 300 s. -1 The shear stress is measured at a shear rate of 200° C., wherein the inoculation temperature is 40° C.; and / or

[0096] Strain (ii) produced fermented milk in the presence of 2% casein hydrolysate at 300 s. -1 When the shear rate is measured under the following conditions, the shear stress of the fermented milk is greater than 13.3 Pa, for example, 13.5 Pa, 15 Pa, 20 Pa, 25 Pa, 30 Pa, 35 Pa, 40 Pa, 41.2 Pa, 41.4 Pa, 41.5 Pa, 42 Pa, 45 Pa, 50 Pa, 55 Pa, 60 Pa, 63 Pa, 64 Pa, 64.5 Pa, 65 Pa, 70 Pa or more:

[0097] 200 mL skim milk (protein 38%, lactose 53%, fat <1.25) was heated to 99°C for 30 min, then cooled to the inoculation temperature, inoculated with 2 mL of overnight culture of lactic acid bacteria strain in the presence of 2% casein hydrolysate, and allowed to stand at the inoculation temperature until pH 4.55, then cooled to 4°C, then gently stirred and incubated for 300 s. -1 The shear stress is measured at a shear rate of 200° C., wherein the inoculation temperature is 40° C.; and / or

[0098] Strain (iii) produced EPS greater than 1 μg / mL / OD under the following conditions: 600 :

[0099] As described in Example 2, under anaerobic conditions, the strain was grown in a chemically defined medium supplemented with 2% lactose (w / v) at 40°C and a constant pH of 6.5 until it entered the growth stationary phase (5-6h); the cell culture was then centrifuged (13,700xg, 30min, 4°C), the supernatant was collected, and stored at -20°C to separate EPS.

[0100] Preferably, the LAB strain of the present invention is strain (i) and / or (ii):

[0101] Strain (i) produced fermented milk. In the presence of 0.2% yeast extract, the fermentation was carried out at 300 s. -1 When the shear rate is measured under the following conditions, the shear stress of the fermented milk is greater than 35 Pa, for example, about 36 Pa, 36.2 Pa, 36.5 Pa, 40 Pa, 41 Pa, 45 Pa, 50 Pa, 55 Pa, 60 Pa, 65 Pa, 66 Pa, 67 Pa, 70 Pa or more:

[0102] 200 mL skim milk (protein 38%, lactose 53%, fat <1.25) was heated to 99°C for 30 min, then cooled to the inoculation temperature, inoculated with 2 mL of overnight culture of lactic acid bacteria strain in the presence of 0.2% yeast extract, and allowed to stand at the inoculation temperature until pH 4.55, then cooled to 4°C, then gently stirred and incubated for 300 s. -1 The shear stress is measured at a shear rate of 200° C., wherein the inoculation temperature is 40° C.; and / or

[0103] Strain (ii) produced fermented milk in the presence of 2% casein hydrolysate at 300 s. -1 When the shear rate is measured under the following conditions, the shear stress of the fermented milk is greater than 40 Pa, for example, 41.2 Pa, 41.4 Pa, 41.5 Pa, 42 Pa, 45 Pa, 50 Pa, 55 Pa, 60 Pa, 63 Pa, 64 Pa, 64.5 Pa, 65 Pa, 70 Pa or more:

[0104] 200 mL skim milk (protein 38%, lactose 53%, fat <1.25) was heated to 99°C for 30 min, then cooled to the inoculation temperature, inoculated with 2 mL of overnight culture of lactic acid bacteria strain in the presence of 2% casein hydrolysate, and allowed to stand at the inoculation temperature until pH 4.55, then cooled to 4°C, then gently stirred and incubated for 300 s. -1 The shear stress was measured at a shear rate of 2.5 Å, with the inoculation temperature being 40 °C.

[0105] More preferably, the LAB strain of the present invention is strain (i) and / or (ii):

[0106] Strain (i) produced fermented milk. In the presence of 0.2% yeast extract, the fermentation was carried out at 300 s. -1 When the shear rate is measured under the following conditions, the shear stress of the fermented milk is greater than 40 Pa, for example, about 40.9 Pa, 41 Pa, 45 Pa, 50 Pa, 55 Pa, 60 Pa, 65 Pa, 66 Pa, 67 Pa, 70 Pa or more:

[0107] 200 mL skim milk (protein 38%, lactose 53%, fat <1.25) was heated to 99°C for 30 min, then cooled to the inoculation temperature, inoculated with 2 mL of overnight culture of lactic acid bacteria strain in the presence of 0.2% yeast extract, and allowed to stand at the inoculation temperature until pH 4.55, then cooled to 4°C, then gently stirred and incubated for 300 s. -1 The shear stress is measured at a shear rate of 200° C., wherein the inoculation temperature is 40° C.; and / or

[0108] Strain (ii) produced fermented milk in the presence of 2% casein hydrolysate at 300 s. -1 When the shear rate is measured under the following conditions, the shear stress of the fermented milk is greater than 41 Pa, for example, 41.2 Pa, 41.4 Pa, 41.5 Pa, 42 Pa, 45 Pa, 50 Pa, 55 Pa, 60 Pa, 63 Pa, 64 Pa, 64.5 Pa, 65 Pa, 70 Pa or more:

[0109] 200 mL skim milk (protein 38%, lactose 53%, fat <1.25) was heated to 99°C for 30 min, then cooled to the inoculation temperature, inoculated with 2 mL of overnight culture of lactic acid bacteria strain in the presence of 2% casein hydrolysate, and allowed to stand at the inoculation temperature until pH 4.55, then cooled to 4°C, then gently stirred and incubated for 300 s. -1 The shear stress was measured at a shear rate of 2.5 Å, with the inoculation temperature being 40 °C.

[0110] Even more preferably, the LAB strain of the present invention is strain (i) and / or (ii):

[0111] Strain (i) produced fermented milk. In the presence of 0.2% yeast extract, the fermentation was carried out at 300 s. -1 When the shear rate is measured under the following conditions, the shear stress of the fermented milk is greater than 65 Pa, for example, about 66 Pa, 66.9 Pa, 67 Pa, 70 Pa or more:

[0112] 200 mL skim milk (protein 38%, lactose 53%, fat <1.25) was heated to 99°C for 30 min, then cooled to the inoculation temperature, inoculated with 2 mL of overnight culture of lactic acid bacteria strain in the presence of 0.2% yeast extract, and allowed to stand at the inoculation temperature until pH 4.55, then cooled to 4°C, then gently stirred and incubated for 300 s. -1 The shear stress is measured at a shear rate of 200° C., wherein the inoculation temperature is 40° C.; and / or

[0113] Strain (ii) produced fermented milk in the presence of 2% casein hydrolysate at 300 s. -1 When the shear rate is measured under the following conditions, the shear stress of the fermented milk is greater than 64 Pa, for example, 64.5 Pa, 64.9 Pa, 65 Pa, 70 Pa or greater:

[0114] 200 mL skim milk (protein 38%, lactose 53%, fat <1.25) was heated to 99°C for 30 min, then cooled to the inoculation temperature, inoculated with 2 mL of overnight culture of lactic acid bacteria strain in the presence of 2% casein hydrolysate, and allowed to stand at the inoculation temperature until pH 4.55, then cooled to 4°C, then gently stirred and incubated for 300 s. -1 The shear stress was measured at a shear rate of 2.5 Å, with the inoculation temperature being 40 °C.

[0115] For further details, please refer to Examples 1 and 2.

[0116] Even more preferably, the LAB strain of the present invention is one of the following strains:

[0117] (i) strain DSM 34289;

[0118] (ii) strain DSM 34290;

[0119] (iii) strain DSM 34291; or

[0120] It comprises mutants or variants having maintained or further improved textural properties (eg having maintained or further improved shear stress properties).

[0121] As shown in the Examples, strain DSM 34289 is able to produce a polysaccharide comprising the following repeating units: (01)

[0123]

[0124] As shown in the Examples, strain DSM 34290 is able to produce a polysaccharide comprising the following repeating units: (02)

[0126]

[0127] As shown in the Examples, strain DSM 34291 is able to produce a polysaccharide comprising the following repeating units: (03)

[0129]

[0130] See Table 4.

[0131] The exopolysaccharide produced by the strain of the present invention, preferably EPS, is involved in the excellent texturizing ability required for producing fermented products (such as yogurt), as described in detail below. As shown in Example 1, strains producing such exopolysaccharides (such as DSM34289, DSM 34290 and / or DSM 34291) produce fermented milk with excellent texture.

[0132] Without being limited by theory, there is no good reason to believe that it is unlikely that another LAB strain (i.e. different from the specific strain of the present invention) capable of producing at least one of the novel exopolysaccharides discussed herein would not improve textural properties when used to ferment a milk base as described herein.

[0133] Compositions and / or starter cultures of the invention

[0134] In a third aspect, the present invention provides compositions and starter cultures comprising at least one polysaccharide and / or at least one LAB strain as defined in the first and second aspects of the present invention, respectively.

[0135] Preferably, the composition or starter culture of the present invention in any of its embodiments comprises at least 1×10 6 CFU (colony forming units) / ml of total LAB strains. It may be preferred that the composition comprises at least 1x10 8 CFU / ml of at least one (preferably one) lactic acid bacteria strain.

[0136] In one embodiment, the composition or starter culture of the present invention may further comprise at least one LAB strain of the present invention and yeast extract, preferably in an amount of about 0.2%. Yeast extract may be obtained from any source available to the skilled person, such as from Procelys (e.g., Yeast Extract For example, the composition or starter culture of the present invention may comprise one or more of strains DSM 34289, DSM 34290 and / or DSM 34291 and yeast extract, preferably in an amount of 0.2%, as described above.

[0137] In one embodiment, the composition of the present invention or starter culture can also include at least one LAB strain of the present invention and casein hydrolysate, preferably the amount of casein hydrolysate is about 2%. Casein hydrolysate can be obtained from any source available to the technician, for example, from Sigma-Aldrich (22090-500G). For example, as mentioned above, the composition of the present invention can include one or more of strain DSM 34289, DSM 34290 and / or DSM 34291 and casein hydrolysate, and the amount of casein hydrolysate is preferably 2%.

[0138] As described above in the context of the second aspect of the invention, the LAB strains of the invention are capable of producing fermented milk with improved texture, e.g., with high shear stress. Thus, the composition and / or starter culture of the invention are capable of generating at least the same shear stress as described for the LAB strains of the invention.

[0139] LAB, including bacteria of the species Streptococcus thermophilus, are typically supplied to the dairy industry as frozen (F-DVS) or freeze-dried (FD-DVS) cultures for bulk starter propagation or as so-called "Direct Vat Set" (DVS) cultures for direct inoculation into fermentation vessels or fermentation tanks to produce dairy products, such as fermented dairy products. Such lactic acid bacteria cultures are typically referred to as "starter cultures" or "starters." Therefore, the composition of the present invention may be frozen or freeze-dried. In addition, the composition or starter culture of the present invention may be provided in liquid form. Therefore, in one embodiment, the composition or starter culture is in frozen, dried, freeze-dried or liquid form.

[0140] Composition of the present invention or starter culture can also comprise cryoprotectant, lyoprotectant, antioxidant, nutrient, filler, flavoring or its mixture additionally.Said composition preferably comprises one or more in cryoprotectant, lyoprotectant, antioxidant and / or nutrient, more preferably cryoprotectant, lyoprotectant and / or antioxidant, most preferably cryoprotectant or lyoprotectant, or both.Protective agent such as the use of cryoprotectant and lyoprotectant is well known to those skilled in the art.Suitable cryoprotectant or lyoprotectant comprise monosaccharide, disaccharide, trisaccharide and polysaccharide (such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch and gum arabic (arabic gum) etc.), polyol (such as erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol etc.), amino acid (such as proline, glutamic acid), complex (such as skim milk, peptone, gelatin, yeast extract) and inorganic compound (such as sodium tripolyphosphate).

[0141] In one embodiment, the composition or starter culture of the present invention may include one or more cryoprotectants selected from the group consisting of inosine-5′-monophosphate (IMP), adenosine-5′-monophosphate (AMP), guanosine-5′-monophosphate (GMP), uridine-5′-monophosphate (UMP), cytidine-5′-monophosphate (CMP), adenine, guanine, uracil, cytosine, adenosine, guanosine, uridine, cytidine, hypoxanthine, xanthine, hypoxanthine, orotidine, thymidine, inosine and derivatives of any such compounds. Suitable antioxidants include ascorbic acid, citric acid and its salts, gallates, cysteine, sorbitol, mannitol, maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, vitamins (such as vitamin B group, vitamin C). The composition may also optionally include other substances, including fillers (e.g., lactose, maltodextrin) and / or flavoring agents.

[0142] In one embodiment of the invention, the cryoprotectant is an agent or mixture of agents which has a boosting effect in addition to cryoprotective properties.

[0143] The expression "booster effect" is used to describe the situation where a cryoprotectant confers an increased metabolic activity (boosting effect) to a thawed or reconstituted culture when it is inoculated into a medium to be fermented or converted. The concepts of viability and metabolic activity are not synonymous. Commercially frozen or freeze-dried cultures may retain their viability, although they may have lost a large part of their metabolic activity, for example, even if they are stored for a short period of time, the culture may lose its acidogenic (acidifying) activity. Therefore, viability and boosting effect must be evaluated by different assays. Viability is evaluated by viability assays, such as determining colony forming units, while the boosting effect is evaluated by quantifying the relevant metabolic activity of the thawed or reconstituted culture relative to the viability of the culture. The term "metabolic activity" refers to the deoxygenation activity of the culture, its acidogenic activity, i.e., the production of, for example, lactic acid, acetic acid, formic acid and / or propionic acid, or its activity to produce metabolites, such as the production of aromatic compounds such as acetaldehyde, α-acetolactate, acetoin, diacetyl and 2,3-butanediol (butylene glycol).

[0144] In one embodiment, when measuring with the %w / w of the material, the composition or starter culture of the present invention contains or comprises 0.2% to 20% cryoprotectant or reagent mixture. However, preferably, when measuring with the %w / w of the frozen material weight, the cryoprotectant or reagent mixture is added by weight in an amount of 0.2% to 15% range, 0.2% to 10% range, 0.5% to 7% range and 1% to 6% range, including cryoprotectant or reagent mixture in the range of 2% to 5%. In a preferred embodiment, when measuring with the %w / w of the material weight, the culture comprises about 3% cryoprotectant or reagent mixture. An amount of about 3% cryoprotectant corresponds to a concentration in the range of 100mM. It should be recognized that for each aspect of the embodiments of the present invention, the range can be an increment of the range.

[0145] In another aspect, the composition or starter culture of the invention contains or comprises an ammonium salt (e.g., an ammonium salt of an organic acid (e.g., ammonium formate and ammonium citrate) or an ammonium salt of an inorganic acid) as an enhancer (e.g., a growth enhancer or acidification enhancer) for bacterial cells, e.g., cells belonging to the species Streptococcus thermophilus, e.g., (substantially) urease-negative bacterial cells. The terms "ammonium salt," "ammonium formate," and the like are to be understood as a source of salts or a combination of ions. For example, the term "ammonium formate" or "source of ammonium salt" refers to a compound or mixture of compounds that, when added to a cell culture, provides ammonium formate or an ammonium salt. In some embodiments, the ammonium source releases ammonium into the growth medium, while in other embodiments, the ammonium source metabolizes to produce ammonium. In some preferred embodiments, the ammonium source is exogenous. In some particularly preferred embodiments, the ammonium is not provided by a milk substrate. Of course, it is to be understood that ammonia may be added instead of an ammonium salt. Thus, the term ammonium salt includes ammonia (NH3), NH4OH, NH4 + wait.

[0146] In one embodiment, the composition of the present invention may comprise a thickener and / or stabilizer, such as pectin (eg, HM pectin, LM pectin), gelatin, CMC, soy fiber / soy polymer, starch, modified starch, carrageenan, alginate and guar gum.

[0147] Use of polysaccharides, LAB strains and / or compositions / starters to improve product texture

[0148] In a fourth aspect, the present invention relates to the use of the polysaccharide or polysaccharide comprising a repeating unit according to the first aspect of the present invention, the LAB strain according to the second aspect and / or the composition / starter according to the third aspect of the present invention in improving the texture of a product, for example increasing the viscosity of the product, wherein the repeating unit comprises one of the structures (02) and / or (03) or consists of one of the structures (02) and / or (03). Preferably, the product is a fermented product or a dairy product, more preferably the product is a fermented dairy product.

[0149] The polysaccharide comprises a repeating unit, the repeating unit comprises or consists of the structure represented by (02 and / or 03) above, and the molecular weight (MW) of the polysaccharide is not particularly limited, and may preferably be at least 1000 kDa, preferably in the range of 1000 to more than 10000 kDa. For example, the polysaccharide comprises a repeating unit, the repeating unit comprises or consists of the structure represented by (02 and / or 03) above, and the MW of the polysaccharide is in the range of 2000 to more than 10000 kDa, such as 3000 to more than 10000 kDa, 4000 to more than 10000 kDa, or 1000 to 8000 kDa, or 2000 to 8000 kDa, or 3000 to 8000 kDa. kDa, or 4000 to 8000 kDa, or 5000 to 8000 kDa, or 1500 to 7000 kDa, or 2000 to 7000 kDa, or 2500 to 7000 kDa, or 3000 to 7000 kDa, or 4000 to 7000 kDa, or 2500 to 6000 kDa, or 3000 to 6000 kDa, or 3000 to 5000 kDa.

[0150] Therefore, in a fourth aspect, the present invention provides a method for improving the texture of a product (e.g., for increasing viscosity), the product preferably being a fermented product, preferably a dairy product, more preferably a fermented dairy product, wherein the method comprises using the polysaccharide, LAB strain, composition and / or starter culture of the present invention as described in the first to third aspects of the present invention, or using a polysaccharide comprising a repeating unit comprising or consisting of the structure represented by (02) and / or (03) above.

[0151] As described above, the LAB strain of the present invention according to the first aspect and the composition of the present invention according to the second aspect are capable of producing fermented milk, as measured in Example 1, in the presence of 0.2% yeast extract at 300 s. -1 When measured at a shear rate of 1.50 s, the shear stress of the fermented milk is greater than 17 Pa, for example, about 20 Pa, 25 Pa, 30 Pa, 35 Pa, 36 Pa, 36.5 Pa, 40 Pa, 41 Pa, 45 Pa, 50 Pa, 55 Pa, 60 Pa, 65 Pa, 66 Pa, 67 Pa, 70 Pa or more, and / or in the presence of 2% casein hydrolysate, at 300 s -1 When the shear rate is measured, the shear stress of the fermented milk is greater than 13.3 Pa, for example, 13.5 Pa, 15 Pa, 20 Pa, 25 Pa, 30 Pa, 35 Pa, 40 Pa, 41.2 Pa, 41.4 Pa, 41.5 Pa, 42 Pa, 45 Pa, 50 Pa, 55 Pa, 60 Pa, 63 Pa, 64 Pa, 64.5 Pa, 65 Pa, 70 Pa or more.

[0152] In a specific embodiment of the third aspect, the present invention provides the use of Streptococcus thermophilus strain DSM 34289, Streptococcus thermophilus strain DSM 34290 and / or Streptococcus thermophilus strain DSM 34291, or a mutant or variant thereof having maintained or further improved shear stress properties, in improving product texture (e.g., increasing viscosity), which product is preferably a fermented product, preferably a dairy product, more preferably a fermented dairy product.

[0153] Products and methods of producing them

[0154] In a fifth aspect, the present invention also relates to a method for producing a product such as a food or feed product, which comprises the step of using at least one exopolysaccharide of the present invention, a polysaccharide comprising a repeating unit defined in the above formula (02 and / or 03), a LAB strain of the present invention and / or a composition or a starter culture. The production of products such as food or feed products is carried out by methods known to those skilled in the art. In a sixth aspect, the present invention relates to a product such as a food or feed product, which comprises a polysaccharide of the first aspect of the present invention, a polysaccharide comprising a repeating unit defined in the above formula (02 and / or 03), a LAB strain of the second aspect and / or a composition / starter of the third aspect.

[0155] The present invention also relates to a method for producing a product such as a food or feed product, comprising at least one step of using at least one exopolysaccharide of the present invention and / or a polysaccharide comprising repeating units defined in the above formula (02 and / or 03). A fifth aspect of the present invention also relates to a method for producing a product such as a food or feed product, comprising at least one step of using at least one LAB strain of the present invention.

[0156] In another embodiment, the present invention relates to a method for producing a product, such as a food or feed product, comprising at least one step using the lactic acid bacteria strain Streptococcus thermophilus DSM 34289 or a mutant or variant thereof.

[0157] In another embodiment, the present invention relates to a method for producing a product, such as a food or feed product, comprising at least one step using the lactic acid bacteria strain Streptococcus thermophilus DSM 34290 or a mutant or variant thereof.

[0158] In another embodiment, the present invention relates to a method for producing a product, such as a food or feed product, comprising at least one step using the lactic acid bacteria strain Streptococcus thermophilus DSM 34291 or a mutant or variant thereof.

[0159] For example, the method of the present invention comprises using a 6 CFU / ml, preferably at least 1x10 8A composition of at least one LAB strain of the invention at a concentration of 100 CFU / ml ferments a milk substrate as described herein.

[0160] For example, the method of the present invention comprises using a 6 CFU / ml, preferably at least 1x10 8 CFU / ml of the strain Streptococcus thermophilus DSM 34289 or a mutant or variant thereof fermented milk substrate.

[0161] For example, the method of the present invention comprises using a 6 CFU / ml, preferably at least 1x10 8 CFU / ml of the strain Streptococcus thermophilus DSM 34290 or a mutant or variant thereof fermented milk substrate.

[0162] For example, the method of the present invention comprises using a 6 CFU / ml, preferably at least 1x10 8 CFU / ml of the strain Streptococcus thermophilus DSM 34291 or a mutant or variant thereof fermented milk substrate.

[0163] In another preferred embodiment, the method comprises fermenting a milk substrate as described herein (also referred to as "milk base" in the context of the present invention) with a composition and / or a starter as described herein.

[0164] In another preferred embodiment, the method further comprises the use of additional lactic acid bacteria, such as other strains of Streptococcus thermophilus or Lactobacillus bulgaricus, and / or the use of casein hydrolysate and / or yeast extract, preferably in the concentrations described herein (about 2% casein hydrolysate or about 0.2% yeast extract).

[0165] The present invention also provides products such as food and feed products, which contain the polysaccharide of the first aspect of the present invention, the polysaccharide containing the repeating unit defined in the above formula (02 and / or 03), the LAB strain of the second aspect and / or the composition / starter of the third aspect of the present invention. Preferably, the product is produced by the method of the fifth aspect of the present invention.

[0166] The product is preferably a fermented product. In another embodiment, the product is preferably a food or feed product, such as a dairy product. The product is preferably a fermented food or feed product, such as a fermented dairy product. In a preferred embodiment, the product comprises and / or is made from plant-based milk and / or mammalian milk.

[0167] Thus, the food may preferably be a dairy product, a meat product, a vegetable product, a fruit product or a cereal product. In a preferred embodiment, the food is a dairy product as defined above, such as yoghurt or In another preferred embodiment, the food is a plant-based (PB) food, such as a plant-based meat or dairy alternative, such as fermented soy milk or plant-based yogurt (also called "yogurt-like (YL) products" (PBYL)).

[0168] In a specific embodiment of the present invention, the fermented milk product is selected from the group consisting of: yogurt, buttermilk, sour milk, cultured milk, Smetana, sour cream, thick cream, cultured cream, ymer, fermented whey, Kefir, Yakult and fresh cheese, such as Quark, tvarog and cream cheese. In a preferred embodiment, the fermented milk product is yogurt. In another preferred embodiment, the fermented milk product contains another food selected from the group consisting of: fruit beverages, cereal products, fermented cereal products, chemically acidified cereal products, soy milk products, fermented soy milk products and mixtures thereof.

[0169] Fermented milk products typically contain protein levels of 1.0% to 12.0%, or 2.0% to 10.0%, by weight. In specific embodiments, sour cream contains protein levels of 1.0% to 5.0%, or 2.0% to 4.0%, by weight. In specific embodiments, quark contains protein levels of 4.0% to 12.0%, or 5.0% to 10.0%, by weight.

[0170] Preferably, the product is a food, more preferably a dairy product, and the method in any of its embodiments comprises fermenting a plant-based milk substrate (also referred to as "plant-based milk base" in the context of the present invention), such as soy milk, preferably soy milk supplemented with glucose, such as 0.5-5% glucose, preferably 0.5-2% glucose, more preferably about 2% glucose, with at least one LAB strain of the present invention and / or with a composition or starter culture of the present invention.

[0171] The food of the present invention may also advantageously contain "thickeners" and / or "stabilizers", such as pectin (eg HM pectin, LM pectin), gelatin, CMC, soy fiber / soy polymers, starch, modified starch, carrageenan, alginate and guar gum.

[0172] In another embodiment, the product is produced without substantially adding or without adding any thickener and / or stabilizer. By substantially free, it is understood that the product contains 0% to 20% (w / w) (e.g., 0% to 10%, 0% to 5%, or 0% to 2%, or 0% to 1%) of thickener and / or stabilizer.

[0173] Preferably, the product of the invention has an improved texture (e.g., as described in the specification and in Example 1, at 300 s) compared to a product produced by a comparable method not using at least one exopolysaccharide, LAB strain and / or composition or starter culture of the invention. -1 Improved viscosity when measuring shear stress).

[0174] Method for producing LAB strains

[0175] In a seventh aspect, the present invention provides a method for producing a LAB strain, preferably a LAB strain of the present invention.

[0176] For example, the present invention relates to a method for producing a LAB strain, wherein the method comprises the following steps:

[0177] (a) providing a LAB strain capable of producing the exopolysaccharide (EPS) defined in the first aspect of the present invention, preferably a thermophilic Streptococcus strain; and

[0178] (b) screening LAB strains that are capable of:

[0179] (i) To produce fermented milk, in the presence of 0.2% yeast extract, the fermented milk was heated for 300 s. -1 When the shear rate is measured under the following conditions, the shear stress of the fermented milk is greater than 17 Pa, for example, about 20 Pa, 25 Pa, 30 Pa, 35 Pa, 36 Pa, 36.5 Pa, 40 Pa, 41 Pa, 45 Pa, 50 Pa, 55 Pa, 60 Pa, 65 Pa, 66 Pa, 67 Pa, 70 Pa or more:

[0180] 200 mL skim milk (protein 38%, lactose 53%, fat <1.25) was heated to 99°C for 30 min, then cooled to the inoculation temperature, inoculated with 2 mL of overnight culture of lactic acid bacteria strain in the presence of 0.2% yeast extract, and allowed to stand at the inoculation temperature until pH 4.55, then cooled to 4°C, then gently stirred and incubated for 300 s. -1 The shear stress is measured at a shear rate of 200° C., wherein the inoculation temperature is 40° C.; and / or

[0181] (ii) Producing fermented milk, in the presence of 2% casein hydrolysate, when heated for 300 s -1When the shear rate is measured under the following conditions, the shear stress of the fermented milk is greater than 13.3 Pa, for example, 13.5 Pa, 15 Pa, 20 Pa, 25 Pa, 30 Pa, 35 Pa, 40 Pa, 41.2 Pa, 41.4 Pa, 41.5 Pa, 42 Pa, 45 Pa, 50 Pa, 55 Pa, 60 Pa, 63 Pa, 64 Pa, 64.5 Pa, 65 Pa, 70 Pa or more:

[0182] 200 mL skim milk (protein 38%, lactose 53%, fat <1.25) was heated to 99°C for 30 min, then cooled to the inoculation temperature, inoculated with 2 mL of overnight culture of lactic acid bacteria strain in the presence of 2% casein hydrolysate, and allowed to stand at the inoculation temperature until pH 4.55, then cooled to 4°C, then gently stirred and incubated for 300 s. -1 The shear stress is measured at a shear rate of 200° C., wherein the inoculation temperature is 40° C.; and / or

[0183] (iii) EPS produced under the following conditions is greater than 1 μg / ml / OD 600 :

[0184] As described in Example 2, under anaerobic conditions, the strain was grown in a chemically defined medium supplemented with 2% lactose (w / v) at 40°C and a constant pH of 6.5 until it entered the growth stationary phase (5-6h); the cell culture was then centrifuged (13,700xg, 30min, 4°C), the supernatant was collected, and stored at -20°C to separate EPS.

[0185] The invention includes any combination of the above-described elements, aspects, and embodiments in all possible variations thereof, unless otherwise indicated herein or clearly contradicted by context.The following describes embodiments of the invention by way of example only.

[0186] Preservation and Expert Solutions

[0187] The applicant requires that only samples of deposited microorganisms listed in the table below be provided to the expert before the patent grant date.

[0188] Table 1. Deposits made with a depository institution that has been granted the status of an international depository institution under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germany.

[0189] strains Accession number Collection date Streptococcus thermophilus DSM 34289 2022.06.14 Streptococcus thermophilus DSM 34290 <![CDATA[2022.06. 14 ]]> Streptococcus thermophilus DSM 34291 2022.06.14 Example

[0190] Example 1: Evaluation of the texture of milk fermented with a strain of Streptococcus thermophilus producing a desired exopolysaccharide

[0191] Strains that may be referred to as "texturizing" strains are measured as follows:

[0192] Strains grown overnight in M17 broth containing 2% lactose (or 2% galactose in the case of DSM 34289) were used to culture the culture medium in 200 mL supplemented with 2% casein hydrolysate (4 g, 200 mL; Sigma-Aldrich, 22090-500 G) or 0.2% yeast extract ( 545 MG, batch number 0005115910, batch AD 18A05030, Procelys) was lactated (1% inoculum). The strictly heat-treated skim milk was prepared by reconstituting skim milk powder (Arla Foods amba, Denmark) containing 38% protein, 53% lactose, less than 1.25% fat and 3.9% water to a dry matter content of 9.5%, then pasteurized at 99°C for 30 min and subsequently cooled to 40°C. The high temperature treatment ensures that the fermentation of this milk is not affected by inherent enzymes or bacteria. The inoculated milk samples were incubated at 40°C to about pH 4.55 and then cooled to 4°C. The samples were then gently stirred and used to measure shear stress at different shear rates. As described above, heat-treated skim milk supplemented with 0.2% yeast extract (medium A) or 2% casein hydrolysate (medium B) was heated at 300s due to fermentation. -1 The shear stress at the shear rate is higher than 17Pa or 13.3Pa, respectively, which is considered to have good texture.

[0193] On a rheometer (Anton Paar Physica rheometer with ASC, or Automatic Sample Changer, Anton The rheological properties of the samples were evaluated using a Bob cup at a Rheometer GmbH, Austria. During the measurement, the rheometer was set to a constant temperature of 13 °C. The settings were as follows:

[0194] - Keep time (rebuild to somewhat original structure);

[0195] - 5 minutes without any physical stress (oscillation or rotation) on the sample;

[0196] - Oscillation step (measurement of elastic and viscous moduli, G′ and G″ respectively, and calculation of the complex modulus G*)

[0197] Constant strain = 0.3%, frequency (f) = [0.5…8] Hz

[0198] 60s (in seconds) 6 measurement points (one every 10s);

[0199] - Rotation step (shear stress was measured at 300 1 / s);

[0200] -Two steps are designed:

[0201] - Shear rate = [0.3-300] 1 / s and 2) Shear rate = [275-0.3] 1 / s.

[0202] Each step contains 21 measurement points (every 10 seconds) within 210 seconds. Select 3001 / s (300 seconds -1 ) was used for further analysis as this is related to the thickness of the oral cavity when swallowing fermented dairy products.

[0203] The thermophilic Streptococcus strains of the present invention all contain an active eps gene cluster capable of producing the exopolysaccharide structure of the present invention, and show significantly higher texture when measured with enhanced shear stress compared to a non-textured thermophilic Streptococcus control strain.

[0204] Table 2. The results of fermentation of milk with the strain producing EPS of the present invention at a shear rate of 300 s -1 under shear stress.

[0205] strains Medium A Medium B DSM 34289 36.2 41.2 DSM 34290 40.9 41.4 DSM 34291 66.9 64.9 S. thermophilus control strain 17.0 13.3

[0206] Example 2: Evaluation of EPS production by strains of the present invention

[0207] Under anaerobic conditions, the strain was grown at 40°C and constant pH 6.5 in a chemically defined medium (CDMLac) supplemented with 2% lactose (w / v) until entering a growth stationary phase (5-6 hours). The culture was centrifuged (13700 x g, 30 min, 4°C), the supernatant was collected, and stored at -20°C to separate EPS. The culture supernatant was loaded into a 12kDa cut-off dialysis tube (Pur-A-Lyzer Mega 12000) and treated with Benzonase (25 kU) at 37°C during dialysis in 50mM NaNO3, 2mM MgSO4 overnight. Proteinase K was subsequently added, and the sample was dialyzed for 3h at 65°C in fresh 50mM NaNO3, 2mM MgSO4. Two volumes of ice-cold ethanol were added to the crude EPS and allowed to precipitate overnight at -20°C. The samples were centrifuged (3256 x g, 30 min, 4°C) and the ethanol was discarded. The pellets were dried at 37°C for 1 h to evaporate any remaining ethanol, suspended in MilliQ water, and rotated at 4°C for 30 min to dissolve the pellets. The samples were transferred to 1.5 mL test tubes and centrifuged at 20,000 x g for 15 min at room temperature. The purified EPS was transferred to a clean test tube and stored at -20°C until quantification. The polysaccharide fraction was quantified by determining the total sugar content by the phenol:sulfuric acid method (e.g., as described in DuBois et al. 1956. Colorimetric method for determination of sugars and related substances. Anal Chem 28: 350-356). The values ​​in Table 3 are the means ± standard deviations of at least 5 technical replicates.

[0208] Compared with the thermophilic Streptococcus control strain, the strain of the present invention mainly produces the secreted EPS of the present invention. It was previously believed that the more the amount of EPS, the higher the viscosity must be, i.e. the higher the shear stress. The EPS with the structure claimed can increase the viscosity, i.e. increase the shear stress (Example 1).

[0209] Table 3. EPS levels produced by strains of the present invention.

[0210]

[0211] Example 3: Determination of the structure of extracellular polysaccharides

[0212] The strain was grown in chemically defined medium (Otto medium) at 40°C for 24h under anaerobic conditions. The culture was then centrifuged (10000g, 1h, 4°C) and the supernatant was treated with 80% trichloroacetic acid (w / v) to a final concentration of 20%. The precipitation process was carried out at 4°C for 2h while stirring. The mixture was centrifuged (10000x g, 1h, 4°C) and the crude EPS was precipitated by adding an equal volume of acetone (4°C) to the supernatant. After precipitation overnight at 4°C with mild stirring, the sample was centrifuged (10,000g, 1h, 4°C). The particles were washed three times with 50% acetone (4°C), and the resulting particles were suspended in 50mL of distilled water and stirred vigorously. The suspension was subjected to 5min sonication to obtain a clear solution. Finally, the solution was dialyzed with distilled water (5L) at 4°C for 12h. The dialysis was repeated three times, each time with fresh distilled water. The contents of the dialysis bag were lyophilized, and the EPS residue was dissolved in 1 mL of D2O by vigorous stirring and sonication.

[0213] For structural elucidation by NMR, spectra were acquired on an AVANCE III 800 spectrometer (Bruker, Rheinstetten, Germany) operating at a proton frequency of 800.33 MHz and equipped with a triple-channel inverse detection probe (TXI) with pulsed field gradients. Two-dimensional spectra for structural elucidation were acquired at 60 °C using standard Bruker pulse programs. Proton-homonuclear shift correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), 1H- 13 C multiplication edited heteronuclear single quantum coherence spectroscopy (ed-HSQC) and 2D HSQC-TOCSY for complete assignment of all resonances in HSQC spectra. 1 H spectroscopy 3 J H1,H2 and measured from non-decoupled HSQC spectra 1 J C,H1 , which is used to determine the configuration of the sugar ring. The heteronuclear multiple bond connectivity spectrum (HMBC) is obtained to determine the position of the glycosidic bond. 1 H and 13 C chemical shift patterns and, where possible, by determining the different 3 J H，H coupling to determine the identity of the sugar monomer.

[0214] Table 4. Exopolysaccharides and strains producing them.

[0215]

Claims

1. A polysaccharide comprising repeating units comprising a backbone defined by one of the following structures: (i) →4)-β-D-Glcp-(1→4)-α(-D-Glcp-(1→4)-β-D-Galp-(1→ or (ii) The repeating unit comprising the main chain (i) further comprises at least one side chain connected to any sugar in the main chain, wherein the side chain comprises structure (a) or is composed of structure (a). composition: wherein (1->6) represents a linker attached to a monosaccharide in the backbone; and The repeating unit comprising the main chain (ii) further comprises at least one side chain connected to any sugar in the main chain, wherein the side chain comprises structure (b) or is composed of structure (b) composition: Wherein (1->2) represents the linker connected to the monosaccharide in the backbone.

2. The polysaccharide according to claim 1, wherein the polysaccharide comprises a repeating unit comprising or consisting of the following structure: (01) 3. The polysaccharide according to any one of claims 1-2, wherein the molecular weight of the polysaccharide is at least 1000 kDa.

4. A lactic acid bacteria strain which produces a polysaccharide as defined in any one of claims 1 to 3 or a polysaccharide comprising a repeating unit comprising or consisting of one of the following structures: (02) and / or (03) 5. The lactic acid bacteria strain according to claim 4, wherein the lactic acid bacteria strain is a strain that produces fermented milk, and in the presence of 0.2% yeast extract, the lactic acid bacteria strain is -1 When the shear rate is measured under the following conditions, the shear stress of the fermented milk is greater than 17 Pa: 200 mL skim milk (protein 38%, lactose 53%, fat <1.25) was heated to 99°C for 30 min, then cooled to the inoculation temperature, inoculated with 2 mL of the overnight culture of the lactic acid bacteria strain in the presence of 0.2% yeast extract, and allowed to stand at the inoculation temperature until the pH was 4.55, then cooled to 4°C, then gently stirred and incubated for 300 s. -1 The shear stress was measured at a shear rate of 1.5 Å, wherein the inoculation temperature was 40°C.

6. The lactic acid bacteria strain according to claim 4, wherein the lactic acid bacteria strain is a strain that produces fermented milk, and in the presence of 2% casein hydrolyzate, the lactic acid bacteria strain is -1 When the shear rate is measured under the following conditions, the shear stress of the fermented milk is greater than 13.3 Pa: 200 mL skim milk (protein 38%, lactose 53%, fat <1.25) was heated to 99°C for 30 min, then cooled to the inoculation temperature, inoculated with 2 mL of the overnight culture of the lactic acid bacteria strain in the presence of 2% casein hydrolysate, and allowed to stand at the inoculation temperature until the pH reached 4.55, then cooled to 4°C, then gently stirred and incubated for 300 s. -1 The shear stress was measured at a shear rate of 1.5 Å, wherein the inoculation temperature was 40°C.

7. The lactic acid strain according to any one of claims 4 to 6, wherein the lactic acid bacteria strain produces more than 1 μg / mL / OD under the following conditions 600 EPS strains: As described in Example 2, under anaerobic conditions, the strain was grown in a chemically defined medium supplemented with 2% lactose (w / v) at 40°C and a constant pH of 6.5 until it entered the growth stationary phase (5-6h); the cell culture was then centrifuged (13,700xg, 30min, 4°C), the supernatant was collected, and stored at -20°C to separate EPS.

8. The lactic acid bacterium according to any one of claims 4 to 7, wherein the bacterium is selected from the group consisting of strains DSM 34289, DSM 34290, DSM 34291 and mutants and variants thereof having maintained or further improved texturizing properties.

9. A composition comprising at least one polysaccharide as defined in any one of claims 1 to 3 or a polysaccharide comprising a repeating unit and / or a lactic acid bacteria strain as defined in any one of claims 4 to 8, wherein the repeating unit comprises one of the structures (02) or (03) or consists of one of the structures (02) or (03).

10. Starter culture comprising at least one lactic acid bacteria strain as defined in any one of claims 4 to 8 and / or a composition as defined in claim 9.

11. Use of at least one polysaccharide as defined in any one of claims 1 to 3 or a polysaccharide comprising repeating units comprising one of structures (02) or (03) or consisting of one of structures (02) or (03); a lactic acid bacteria strain as defined in any one of claims 4 to 8; a composition as defined in claim 9; and / or a starter culture as defined in claim 10 in improving product texture.

12. A method for producing a product, comprising the steps of using: at least one polysaccharide as defined in any one of claims 1 to 3 or a polysaccharide comprising a repeating unit, wherein the repeating unit comprises one of structures (02) or (03) or consists of one of structures (02) or (03); a lactic acid bacteria strain as defined in any one of claims 4 to 8; a composition as defined in claim 9; and / or a starter culture as defined in claim 10.

13. The method according to claim 12, wherein the product is a food or feed product.

14. The method according to any one of claims 12 or 13, further comprising the step of fermenting the substrate with the lactic acid bacteria strain defined in any one of claims 4 to 8, the composition defined in claim 9 and / or the starter culture defined in claim 10.

15. The method according to any one of claims 12 to 14, wherein the method comprises the use of other lactic acid bacteria, casein hydrolysate and / or yeast extract.

16. The method according to any one of claims 12-15, wherein the product is a dairy product or a plant-based product.

17. A product comprising at least one polysaccharide as defined in any one of claims 1 to 3 or a polysaccharide comprising repeating units, wherein the repeating units comprise one of structures (02) or (03) or consist of one of structures (02) or (03); a lactic acid bacteria strain as defined in any one of claims 4 to 8; a composition as defined in claim 9; and / or a starter culture as defined in claim 10.

18. The product according to claim 17, wherein the product comprises or consists of plant-based milk and / or mammalian milk.

19. A method for producing a lactic acid bacteria strain, wherein the method comprises the following steps: (a) providing a lactic acid bacteria strain capable of producing a polysaccharide as defined in any one of claims 1 to 3; and (b) screening for lactic acid bacteria strains capable of: (i) To produce fermented milk, in the presence of 0.2% yeast extract, -1 When the shear rate is measured under the following conditions, the shear stress of the fermented milk is greater than 17 Pa: 200 mL skim milk (protein 38%, lactose 53%, fat <1.25) was heated to 99°C for 30 min, then cooled to the inoculation temperature, inoculated with 2 mL of the overnight culture of the lactic acid bacteria strain in the presence of 0.2% yeast extract, and allowed to stand at the inoculation temperature until the pH was 4.55, then cooled to 4°C, then gently stirred and incubated for 300 s. -1 The shear stress is measured at a shear rate of 20° C., wherein the inoculation temperature is 40° C.; and / or (ii) Fermented milk was produced by heating the mixture for 300 s in the presence of 2% casein hydrolysate. -1 When the shear rate is measured under the following conditions, the shear stress of the fermented milk is greater than 13.3 Pa: 200 mL skim milk (protein 38%, lactose 53%, fat <1.25) was heated to 99°C for 30 min, then cooled to the inoculation temperature, inoculated with 2 mL of overnight culture of lactic acid bacteria strain in the presence of 2% casein hydrolysate, and allowed to stand at the inoculation temperature until pH 4.55, then cooled to 4°C, then gently stirred and incubated for 300 s. -1 The shear stress is measured at a shear rate of 200° C., wherein the inoculation temperature is 40° C.; and / or (iii) Produced more than 1 μg / ml / OD under the following conditions 600 EPS: As described in Example 2, under anaerobic conditions, the strain was grown in a chemically defined medium supplemented with 2% lactose (w / v) at 40°C and a constant pH of 6.5 until it entered the growth stationary phase (5-6h); the cell culture was then centrifuged (13,700xg, 30min, 4°C), the supernatant was collected, and stored at -20°C to separate EPS.

Images