streptococcus thermophilus strain

Abstract

The present invention relates to Streptococcus thermophilus strains that can be used as starter cultures, which, once incorporated into a culture medium, not only impart satisfactory rheological and organoleptic properties to the culture medium, but also allow the culture medium to have a satisfactory shelf life. In particular, these strains also have phage resistance, thus minimizing phage infection. The invention also provides compositions comprising one of these Streptococcus thermophilus strains, as well as feed or food products obtained with these strains.

Description

TECHNICAL FIELD

[0001] The present invention relates to Streptococcus thermophilus strains that can be used as starter cultures, which, when incorporated into a culture medium, not only impart satisfactory rheological and organoleptic properties to the culture medium, but also allow the culture medium to have a satisfactory shelf life. In particular, these strains also have phage resistance, thus minimizing phage infection. The present invention also provides compositions comprising one of these Streptococcus thermophilus strains, as well as feed or food products obtained with these strains. BACKGROUND

[0002] The food industry uses bacteria to improve the taste and texture of food or feed products. In the case of the dairy industry, lactic acid bacteria are often used, for example, to cause the acidification (via fermentation) of milk and to modify the texture of the product in which they are incorporated. Examples of lactic acid bacteria commonly used by the food industry include Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, and Bifidobacterium.

[0003] Lactic acid bacteria of the Streptococcus thermophilus species are widely used in the production of food or feed products, especially fermented products; during production, lactic acid bacteria are used alone or in combination with other bacteria. Lactic acid bacteria are particularly suitable for formulating starter cultures, which can be used to produce fermented milk (e.g., yogurt). Streptococcus thermophilus is widely used in the manufacture of yogurt and cheese, such as Emmental, Gouda, Cheddar, and Italian cheese. The high market value of these products makes Streptococcus thermophilus a species of great economic interest.

[0004] There is a need in the art for bacterial strains, especially Streptococcus thermophilus strains, that not only impart good or improved rheological or organoleptic properties (such as texture and flavor) to food or feed products, but also impart a satisfactory shelf life to food or feed products. SUMMARY

[0005] The present invention provides a Streptococcus thermophilus strain, the lactic acidification kinetics of which is characterized by an average acidification rate of at least 70.10 -4 UpH / min or equal to 70.10 -4UpH / min; the average acidification rate is less than 22.10 -4 UpH / min or equal to 22.10 -4 UpH / min; and / or the ratio of the average acidification rate (1) at a pH comprised between 5.00 and 5.30 and the average acidification rate (2) at a pH comprised between 5.30 and 6.00 is less than or equal to 25%. In a particular embodiment, the strain is the DSM 27029 strain, the DSM 27030 strain or the DSM 27031 strain, all strains being deposited at the Leibniz-Institut DSMZ on March 21, 2013.

[0006] The present application also provides a composition comprising or consisting of a culture of a Streptococcus thermophilus strain of the application, and optionally further comprising at least one other culture(s) of at least one other microorganism, in particular a lactic acid bacterium or a propionic acid bacterium.

[0007] The present application also relates to the use of a culture of a Streptococcus thermophilus strain of the application or of a composition of the application for the manufacture of a product, in particular a food or feed product, in particular a fermented product, in particular a fermented food or fermented feed product.

[0008] The present application also relates to a method for the manufacture of a product, in particular a fermented product, said method comprising: contacting a substrate, in particular a milk substrate, with a Streptococcus thermophilus strain or a composition of the application, or in the presence of a Streptococcus thermophilus strain or a composition of the application; optionally fermenting said substrate; and then obtaining said product.

[0009] The present application also provides a product, in particular a dairy product, in particular a fermented product, comprising a culture of a Streptococcus thermophilus strain of the application or a composition of the application. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 : Variation of the average acidification rate at a pH comprised between 5.00 and 5.30 (S2) as a function of the average acidification rate at a pH comprised between 5.30 and 6.00 (S1) for 69 Streptococcus thermophilus strains. Examples of strains of the application are represented with black squares, other Streptococcus thermophilus strains are represented with grey diamonds.

[0011] Figure 2(A) The relationship between the average acidification rate (S2) at pH values ​​between 5.00 and 5.30 and the average acidification rate (S1) at pH values ​​between 5.30 and 6.00 for 9 known Streptococcus thermophilus strains (gray rhombuses) and 3 strains of the present invention (black squares); (B) The S2 / S1 ratio (in %) of the 12 Streptococcus thermophilus strains in (A) above. Detailed Implementation

[0012] The inventors have identified thermophilic streptococcal strains with surprisingly atypical lactic acidification kinetics. Furthermore, the inventors have demonstrated that these strains can be used to produce or ferment feed or food products. Specifically, these strains impart satisfactory rheological and / or sensory properties, as well as satisfactory shelf life, to products at least similar to those obtained using existing thermophilic streptococcal strains. It is noteworthy that over 60 known thermophilic streptococcal strains have been disclosed in previous patent applications or literature, and it was during the study of these strains that we identified the thermophilic streptococcal strain with atypical lactic acidification kinetics.

[0013] This invention provides a thermophilic streptococcal strain, wherein the lactation kinetics of said strain are characterized as follows:

[0014] - When the pH value is between 5.30 and 6.00, the average acidification rate is at least 70.10. -4 UpH / min or equal to 70.10 -4 UpH / min,

[0015] - When the pH value is between 5.00 and 5.30, the average acidification rate is less than 22.10. -4 UpH / min or equal to 22.10 - 4 UpH / min.

[0016] The average acidification rate at a pH between 5.30 and 6.00 is referred to as S1 in this application. The average acidification rate at a pH between 5.00 and 5.30 is referred to as S2 in this application. Both the average acidification rates at pH between 5.30 and 6.00 and at pH between 5.00 and 5.30 are determined in milk substrates, particularly bovine milk (lactation kinetics), such as "Le Petit Vendéen". Both average acidification rates S1 and S2 are determined using any conventional method. Specifically, S1 and S2 are calculated using the Cinac system (CINAC, an automated system for controlling lactic acid fermentation agents; Corrieu G, Picque D, Perret B, Quemener P; Process Magazine; 1992: 1068; pp. 24-27). Automated systems for measuring acidification rates are well known to those skilled in the art. A reference system can be found, for example, in patent FR2629612. S1 and S2 are calculated from the same sample, particularly from the same lactic acidification curve. Example 1 discloses an example of data obtained using the CINAC system, from which the average acidification rates S1 and S2 can be calculated.

[0017] In one specific embodiment, S1 and S2 are calculated according to the assay method described in Assay I (described in detail below in Example 1), or S1 and S2 are calculated using Assay I. It is worth noting that S1 and S2 are calculated using only a single strain of Streptococcus thermophilus.

[0018] In one specific embodiment, the present invention provides a thermophilic streptococcal strain, wherein the lactation kinetics of said strain are characterized as follows:

[0019] - When the pH value is between 5.30 and 6.00, the average acidification rate is between 70.10. -4 With 250.10 -4 UpH / min is between 70.10 -4 With 200.10 -4 UpH / min is between 70.10 -4 With 180.10 -4 UpH / min, or between 70.10 -4 With 140.10 -4 Between UpH / min;

[0020] - When the pH value is between 5.00 and 5.30, the average acidification rate is between 1.10.-4 With 20.10 -4 UpH / min is between 2.10 and 2. -4 With 22.10 -4 UpH / min is between, or between 2.10 -4 With 20.10 -4 UpH / min is between.

[0021] In one specific embodiment, the present invention relates to a thermophilic streptococcal strain, wherein the lactation kinetics of said strain are characterized as follows:

[0022] - Based on the description in Method I, the average acidification rate is between 70.10 when the pH value is between 5.30 and 6.00. -4 With 250.10 -4 UpH / min is between 70.10 -4 With 200.10 -4 UpH / min is between 70.10 -4 With 180.10 -4 UpH / min, or between 70.10 -4 With 140.10 -4 Between UpH / min;

[0023] - Based on the description in Method I, the average acidification rate is between 1.10 when the pH value is between 5.00 and 5.30. -4 With 20.10 -4 UpH / min is between 2.10 and 2. -4 With 22.10 -4 UpH / min is between, or between 2.10 -4 With 20.10 - 4 UpH / min is between.

[0024] In a more specific embodiment, preferably calculated according to the description of determination method I, the average acidification rate (S1) when the pH value is between 5.30 and 6.00 is between 80.10. -4 With 120.10 -4 UpH / min is between 90.10 -4 With 110.10 -4 UpH / min, or between 95.10 -4 With 105.10 -4 UpH / min is between.

[0025] In a more specific embodiment, preferably calculated according to the description of determination method I, the average acidification rate (S2) when the pH value is between 5.00 and 5.30 is between 5.10. -4 With 20.10 -4 UpH / min, or between 10.10 -4 With 18.10 -4 UpH / min is between.

[0026] In one specific embodiment, the present invention relates to a thermophilic streptococcal strain, wherein the lactation kinetics of said strain are characterized as follows:

[0027] - Preferably, based on the calculations described in Measurement Method I, when the pH value is between 5.30 and 6.00, the average acidification rate is between 70.10%. -4 With 250.10 -4 UpH / min is between 70.10 -4 With 200.10 -4 UpH / min is between 70.10 -4 With 180.10 -4 UpH / min is between 70.10 -4 With 140.10 -4 UpH / min is between 80 and 10. -4 With 120.10 -4 UpH / min is between 90.10 -4 With 110.10 -4 UpH / min, or between 95.10 -4 With 105.10 -4 Between UpH / min;

[0028] - Preferably, based on the calculations described in Measurement Method I, when the pH value is between 5.00 and 5.30, the average acidification rate is between 1.10. -4 With 20.10 -4 UpH / min is between 2.10 and 2. -4 With 22.10 -4 UpH / min is between 2.10 and 2. -4 With 20.10 - 4 UpH / min is between 5.10 and 5.10. -4 With 20.10 -4 UpH / min, or between 10.10 -4 With 18.10 -4 UpH / min is between.

[0029] The present invention also provides a Streptococcus thermophilus strain, wherein the lactic acidification kinetics of the strain are characterized by the following: the ratio of the average acidification rate (1) (preferably calculated according to the description of determination method I) at pH values ​​between 5.00 and 5.30 to the average acidification rate (2) (preferably calculated according to the description of determination method I) at pH values ​​between 5.30 and 6.00 is less than or equal to 25%, less than or equal to 20%, or less than or equal to 18%. The average acidification rate at pH values ​​between 5.30 and 6.00 and the average acidification rate at pH values ​​between 5.00 and 5.30 are defined by the above embodiments and determined according to the above embodiments. The ratio (in %) is calculated as follows (the ratio of S2 to S1, or the S2 / S1 ratio):

[0030]

[0031] As described above, S1 and S2 are calculated from the same sample, and in particular from the same acidification curve, using the CINAC system.

[0032] In one specific embodiment, the S2 / S1 ratio is between 1% and 25%, between 2% and 25%, between 5% and 18%, between 8% and 18%, or between 10% and 18%.

[0033] In one specific embodiment, the present invention provides a thermophilic streptococcal strain, wherein the lactation kinetics of said strain are characterized as follows:

[0034] - When the pH value is between 5.30 and 6.00, the average acidification rate is at least 70.10. -4 UpH / min or equal to 70.10 -4 UpH / min;

[0035] - When the pH value is between 5.00 and 5.30, the average acidification rate is less than 22.10. -4 UpH / min or equal to 22.10 - 4 UpH / min; and

[0036] - The ratio of the average acidification rate (1) at pH values ​​between 5.00 and 5.30 to the average acidification rate (2) at pH values ​​between 5.30 and 6.00 is less than or equal to 25%, less than or equal to 20%, or less than or equal to 18%.

[0037] Preferably, the average acidification rate when the pH value is between 5.30 and 6.00 and the average acidification rate when the pH value is between 5.00 and 5.30 are calculated according to the content described in Measurement Method I.

[0038] The average acidification rate when the pH value is between 5.30 and 6.00 and the average acidification rate when the pH value is between 5.00 and 5.30 are defined by the above examples and measured according to the above examples.

[0039] In one specific embodiment, the present invention provides a thermophilic streptococcal strain, wherein the lactation kinetics of said strain are characterized as follows:

[0040] When the pH value is between 5.30 and 6.00, the average acidification rate is between 70.10. -4 With 250.10 -4 UpH / min is between 70.10 -4 With 200.10 -4 UpH / min is between 70.10 -4 With 180.10 -4 UpH / min is between 70.10 -4 With 140.10 -4 UpH / min is between 80 and 10. -4 With 120.10 -4 UpH / min is between 90.10 -4 With 110.10 -4 UpH / min, or between 95.10 -4 With 105.10 -4 Between UpH / min;

[0041] - When the pH value is between 5.00 and 5.30, the average acidification rate is between 1.10. -4 With 20.10 -4 UpH / min is between 2.10 and 2. -4 With 22.10 -4 UpH / min is between 2.10 and 2. -4 With 20.10 -4 UpH / min is between 5.10 and 5.10. -4 With 20.10 - 4 UpH / min, or between 10.10 -4 With 18.10 -4 Between UpH / min, and

[0042] - The ratio of the average acidification rate (1) between pH values ​​between 5.00 and 5.30 to the average acidification rate (2) between pH values ​​between 5.30 and 6.00 is between 1% and 25%, between 2% and 25%, between 5% and 18%, between 8% and 18%, or between 10% and 18%.

[0043] Preferably, the average acidification rate when the pH value is between 5.30 and 6.00 and the average acidification rate when the pH value is between 5.00 and 5.30 are calculated according to the content described in Measurement Method I.

[0044] The average acidification rate when the pH value is between 5.30 and 6.00 and the average acidification rate when the pH value is between 5.00 and 5.30 are defined by the above examples and measured according to the above examples.

[0045] The present invention relates to any thermophilic streptococcal strain defined by a combination of the following characteristics: having any of the S2 / S1 ratio ranges or maximum values ​​disclosed herein, having any of the S2 ranges or maximum values ​​disclosed herein, and having any of the S1 ranges or minimum values ​​disclosed herein.

[0046] As a specific embodiment, the present invention provides a Streptococcus thermophilus strain, wherein the lactation kinetics of the strain are characterized as follows:

[0047] - When the pH value is between 5.30 and 6.00, the average acidification rate is between 90.10%. -4 With 110.10 -4 UpH / min, or between 95.10 -4 With 105.10 -4 Between UpH / min;

[0048] - When the pH value is between 5.00 and 5.30, the average acidification rate is between 5.10. -4 With 20.10 -4 UpH / min, or between 10.10 -4 With 18.10 -4 Between UpH / min; and

[0049] - The ratio of the average acidification rate (1) when the pH value is between 5.00 and 5.30 to the average acidification rate (2) when the pH value is between 5.30 and 6.00 is between 8% and 18%, or between 10% and 18%.

[0050] Preferably, the average acidification rate when the pH value is between 5.30 and 6.00 and the average acidification rate when the pH value is between 5.00 and 5.30 are calculated according to the content described in Measurement Method I.

[0051] The methods described herein for calculating the average acidification rate (S2) between pH 5.00 and 5.30, the average acidification rate (S1) between pH 5.30 and 6.00, the S2 / S1 ratio, and / or the specific features of the CINAC system, are applicable to all embodiments of the thermophilic streptococcal strains of the present invention as defined in this application.

[0052] Given that all known Streptococcus thermophilus strains show a close correlation between the average acidification rate (S2) at pH values ​​between 5.00 and 5.30 and the average acidification rate (S1) at pH values ​​between 5.30 and 6.00 (i.e., the highest S1 value is accompanied by the highest S2 value), this invention proposes for the first time a Streptococcus thermophilus strain in which the average acidification rate (S2) at pH values ​​between 5.00 and 5.30 is not correlated with the average acidification rate (S1) at pH values ​​between 5.30 and 6.00.

[0053] The present invention also provides a Streptococcus thermophilus strain, which, in addition to having an average acidification rate (S1) at a pH value between 5.30 and 6.00 as defined herein, an average acidification rate (S2) at a pH value between 5.00 and 5.30 as defined herein, and / or b) an S2 / S1 ratio as defined herein, is characterized in that it contains at least one element selected from the following: CRISPR4 locus, CRISPR1 locus, and CRISPR3 locus, each element being defined below.

[0054] Jansen et al. (2002) OMICS J. Integ. Biol. 6: 23-33 described common structural features of the CRISPR-Cas system as: (i) the presence of multiple short direct repeat sequences (CRISPR repeat sequences), which are typically 24 to 40 bp partial palindromic sequences containing internal and terminal inverted repeat sequences of up to 11 bp, and showing little or no sequence variation within a given locus; (ii) the presence of non-repetitive spacer sequences of similar size (CRISPR spacer sequences) between the repeat sequences; (iii) the presence of a common leader sequence of a dozen to hundreds of base pairs in most species carrying multiple CRISPR loci; and (iv) the presence of one or more cas (CRISPR-associated) genes.

[0055] In this invention, the term "CRISPR locus" refers to a DNA fragment consisting of at least one [repetitive sequence-spacer sequence] unit and a terminal repeat sequence, the DNA fragment starting at the first nucleotide of the first CRISPR repeat sequence and ending at the last nucleotide of the terminal (last) CRISPR repeat sequence. Therefore, the composition of a CRISPR locus is as follows: first, at least one [repetitive sequence-spacer sequence] unit, particularly several [repetitive sequence-spacer sequence] units (all of which have the same or at least similar CRISPR repeat sequences), followed by a final terminal repeat sequence (whose sequence (especially its 5' portion) is the same as or similar to the CRISPR repeat sequence of the aforementioned [repetitive sequence-spacer sequence] unit). In the context of this invention, CRISPR loci (CRISPR1, CRISPR3, or CRISPR4 loci) are oriented as follows: the CRISPR leader sequence is a DNA fragment typically rich in A / T, which is adjacent to and upstream of the first CRISPR repeat sequence of the CRISPR locus. The CRISPR trailing sequence is a DNA segment that is immediately adjacent to and downstream of the terminal repeat sequence. Therefore, the CRISPR locus is located between the CRISPR leader sequence and the CRISPR trailing sequence.

[0056] In a first embodiment, the present invention provides a *Streptococcus thermophilus* strain as defined herein, the genome of which contains a CRISPR4 locus. In a specific embodiment, the present invention provides a *Streptococcus thermophilus* strain as defined herein, the genome of which contains a CRISPR4 locus defined by SEQ ID NO: 3 or a CRISPR4 locus containing one or more portions of SEQ ID NO: 3. SEQ ID NO: 3 comprises 12 CRISPR4 [repetitive sequence-spacer sequence] units and 1 terminal repeat sequence. The sequences of these 12 CRISPR4 [repetitive sequence-spacer sequence] units of SEQ ID NO: 3 are defined by SEQ ID NO: 4 to SEQ ID NO: 15, respectively. The CRISPR4 terminal repeat sequence is defined by SEQ ID NO: 16. The CRISPR4 leader sequence and CRISPR4 tail sequence located adjacent to the CRISPR4 locus in the specific embodiment of the *Streptococcus thermophilus* strain of the present invention are defined by SEQ ID NO: 2 and SEQ ID NO: 1, respectively.

[0057] In one specific embodiment, the present invention provides a Streptococcus thermophilus strain as defined herein, the genome of which includes a CRISPR4 locus comprising or consisting of a sequence defined by SEQ ID NO: 3.

[0058] In one specific embodiment, the present invention provides a Streptococcus thermophilus strain as defined herein, the genome of which includes a CRISPR4 locus comprising a portion of SEQ ID NO: 3 and a terminal repeat sequence defined by SEQ ID NO: 16 in the 5' to 3' direction.

[0059] In the context of the CRISPR4 locus, "a portion of SEQ ID NO:3" refers to a fragment of SEQ ID NO:3 containing at least three or exactly three consecutive CRISPR4 [repetitive sequence-spacer] units as contained in SEQ ID NO:3. Specifically, it contains at least 3, 4, 5, 6, 7, 8, 9, 10, or 11, or exactly 3, 4, 5, 6, 7, 8, 9, 10, or 11 consecutive [repetitive sequence-spacer] units as contained in SEQ ID NO:3. "Consecutive" means that the sequence in which the multiple CRISPR4 [repetitive sequence-spacer] units present in the portion of SEQ ID NO:3 are linked is the same as their order of appearance in SEQ ID NO:3 (e.g., SEQ ID NO:4-SEQ ID NO:5-SEQ ID NO:6, or SEQ ID NO:10-SEQ ID NO:11-SEQ ID NO:12). In one specific embodiment, a portion of SEQ ID NO:3 is a fragment of SEQ ID NO:3 containing at least three or exactly three consecutive CRISPR4 [repetitive sequence-spacer sequence] units selected from SEQ ID NO:4 to SEQ ID NO:15. In one specific embodiment, "a portion of SEQ ID NO:3" refers to 3, 4, 5, 6, 7, 8, 9, 10, or 11 consecutive terminal CRISPR4 [repetitive sequence-spacer sequence] units contained in SEQ ID NO:3. The term "terminal CRISPR4 [repetitive sequence-spacer sequence] unit contained in SEQ ID NO:3" refers to the CRISPR4 [repetitive sequence-spacer sequence] unit located at the 3' end (i.e., at the end of the trailing sequence) of the CRISPR4 locus in SEQ ID NO:3, that is, immediately preceding the terminal repeat sequence defined by SEQ ID NO:16. Therefore, the two consecutive terminal CRISPR4 [repetition sequence-spacer sequence] units of SEQ ID NO:3 refer to SEQ ID NO:14-SEQ ID NO:15, the three consecutive terminal CRISPR4 [repetition sequence-spacer sequence] units of SEQ ID NO:3 refer to SEQ ID NO:13-SEQ ID NO:14-SEQ ID NO:15, and so on.

[0060] In a second embodiment, either on its own or in conjunction with the first embodiment, the present invention provides a Streptococcus thermophilus strain as defined herein, the genome of which contains the CRISPR1 locus.

[0061] In one specific embodiment, the present invention provides a Streptococcus thermophilus strain as defined herein, the genome of which includes a CRISPR1 locus comprising a sequence defined by SEQ ID NO: 19, or consisting of a sequence defined by SEQ ID NO: 19, or the CRISPR1 locus comprising one or more portions of SEQ ID NO: 19.

[0062] SEQ ID NO: 19 comprises 32 CRISPR1 [repetitive sequence-spacer sequence] units and 1 terminal repeat sequence, which is similar to but different from the repeat sequences of these 32 CRISPR1 [repetitive sequence-spacer sequence] units. The sequences of these 32 CRISPR1 [repetitive sequence-spacer sequence] units of SEQ ID NO: 19 are defined by SEQ ID NO: 22 to SEQ ID NO: 53, respectively. The repeat sequences of all CRISPR1 [repetitive sequence-spacer sequence] units within the CRISPR1 locus defined herein are defined by SEQ ID NO: 20 (R1). The terminal repeat sequence is defined by SEQ ID NO: 21 (R'1). Notably, in one specific embodiment, the CRISPR1 locus defined by SEQ ID NO: 19, or a CRISPR1 locus containing one or more portions of SEQ ID NO: 19 as defined herein, is flanked by the CRISPR1 leader sequence and CRISPR1 tail sequence defined by SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

[0063] Following phage attack, one or more additional CRISPR1 [repetitive sequence-spacer sequence] units may be added within the CRISPR locus, particularly at the 5' portion (i.e., the end of the leader sequence) of the CRISPR1 locus as defined herein, that is, immediately after the last nucleotide of the CRISPR1 leader sequence. These additional CRISPR1 [repetitive sequence-spacer sequence] units have a sequence defined as R1-X1 in the 5' to 3' direction, where R1 is defined by SEQ ID NO: 20, and X1 is any sequence of length from 27 to 33 bp, specifically 28 to 32 bp, specifically 29 to 31 bp, specifically exactly 30 bp. Specifically, the sequences of any of these additional CRISPR1 [repetitive sequence-spacer sequence] units are selected from SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60. Non-limiting examples of additional CRISPR1 [repetitive sequence-spacer sequence] units that may be used according to the invention are defined by SEQ ID NO: 61 to SEQ ID NO: 70.

[0064] In one specific embodiment, the present invention provides a Streptococcus thermophilus strain as defined herein, the genome of which includes a CRISPR1 locus comprising or consisting of the sequence defined by SEQ ID NO: 19. In one specific embodiment, the CRISPR1 locus is composed from 5' to 3' as follows: at least one additional CRISPR1 [repetitive sequence-spacer] unit of sequence R1-X1, specifically at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more additional CRISPR1 [repetitive sequence-spacer] units of sequence R1-X1, followed by SEQ ID NO: 19.

[0065] In one specific embodiment, the present invention provides a Streptococcus thermophilus strain as defined herein, the genome of which includes a CRISPR1 locus comprising a portion of SEQ ID NO: 19 and a terminal repeat sequence defined by SEQ ID NO: 21 in the 5' to 3' direction.

[0066] In the context of the CRISPR1 locus, "a portion of SEQ ID NO: 19" refers to a fragment of SEQ ID NO: 19 that contains at least three or exactly three consecutive CRISPR1 [repetitive sequence-spacer sequence] units contained in SEQ ID NO: 19. Specifically, it contains at least three, four, five, six, seven, eight, nine, ten, eleven, thirteen, eleven, twelfth, thirteenth, eleventh, thirteen ... The term "continuous" means that the sequence in which multiple CRISPR1 [repetitive sequence-spacer sequence] units present in the portion of SEQ ID NO: 19 are connected is the same as the order in which they appear in SEQ ID NO: 19 (e.g., SEQ ID NO: 23-SEQ ID NO: 24-SEQ ID NO: 25, or SEQ ID NO: 42-SEQ ID NO: 43-SEQ ID NO: 44). In one specific embodiment, a portion of SEQ ID NO: 19 is a fragment of SEQ ID NO: 19 containing at least three or exactly three consecutive CRISPR1 [repetitive sequence-spacer sequence] units selected from SEQ ID NO: 22 to SEQ ID NO: 53. In one specific embodiment, "a portion of SEQ ID NO: 19" refers to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 consecutive terminal CRISPR1 [repetitive sequence-spacer sequence] units contained in SEQ ID NO: 19. The term "terminal CRISPR1 [repetitive sequence-spacer sequence] unit contained in SEQ ID NO: 19" refers to the CRISPR1 [repetitive sequence-spacer sequence] unit located at the 3' end (i.e., at the end of the trailing sequence) of the CRISPR1 locus in SEQ ID NO: 19, that is, immediately preceding the R1' terminal repetitive sequence defined by SEQ ID NO: 21. Therefore, the two consecutive terminal CRISPR1 [repetition sequence-spacer sequence] units of SEQ ID NO: 19 refer to SEQ ID NO: 52-SEQ ID NO: 53, the three consecutive terminal CRISPR1 [repetition sequence-spacer sequence] units of SEQ ID NO: 19 refer to SEQ ID NO: 51-SEQ ID NO: 52-SEQ ID NO: 53, and so on.

[0067] In one specific embodiment, the CRISPR1 locus, from 5' to 3', comprises: an integer number of [repetitive sequence-spacer sequence] units, including at least three consecutive CRISPR1 [repetitive sequence-spacer sequence] units as defined herein (as is defined herein as a portion of SEQ ID NO: 19), followed by the terminal repeat sequence of SEQ ID NO: 21. In another specific embodiment, the CRISPR1 locus, from 5' to 3', comprises: an integer number of [repetitive sequence-spacer sequence] units, including at least three consecutive CRISPR1 [repetitive sequence-spacer sequence] units selected from SEQ ID NO: 22 to SEQ ID NO: 53, followed by the terminal repeat sequence of SEQ ID NO: 21. In one specific embodiment, the CRISPR1 locus is composed from 5' to 3' as follows: at least one additional CRISPR1 [repetitive sequence-spacer sequence] unit of sequence R1-X1, specifically at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more additional CRISPR1 [repetitive sequence-spacer sequence] units of sequence R1-X1, at least three consecutive CRISPR1 [repetitive sequence-spacer sequence] units contained in SEQ ID NO: 19 (a portion of SEQ ID NO: 19), followed by the terminal repeat sequence of SEQ ID NO: 21.

[0068] In one specific embodiment, the CRISPR1 locus is composed from 5' to 3' as follows: at least one additional CRISPR1 [repetitive sequence-spacer sequence] unit of sequence R1-X1, specifically at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more additional CRISPR1 [repetitive sequence-spacer sequence] units of sequence R1-X1, as a portion of SEQ ID NO: 19 as defined above, followed by the terminal repeat sequence of SEQ ID NO: 21. In one specific embodiment, the CRISPR1 locus is composed from 5' to 3' as follows: at least one additional CRISPR1 [repetitive sequence-spacer sequence] unit of sequence R1-X1, specifically at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more additional CRISPR1 [repetitive sequence-spacer sequence] units of sequence R1-X1, 1 to 31 consecutive terminal CRISPR1 [repetitive sequence-spacer sequence] units contained in SEQ ID NO: 19, followed by the terminal repeat sequence of SEQ ID NO: 21.

[0069] In a third embodiment, whether on its own, in conjunction with the first embodiment, in conjunction with the second embodiment, or in conjunction with both the first and second embodiments, the present invention provides a Streptococcus thermophilus strain as defined herein, the genome of which contains the CRISPR3 locus.

[0070] In one specific embodiment, the present invention provides a Streptococcus thermophilus strain as defined herein, the genome of which includes a CRISPR3 locus comprising a sequence defined by SEQ ID NO: 73, or consisting of a sequence defined by SEQ ID NO: 73, or the CRISPR3 locus comprising one or more portions of SEQ ID NO: 73.

[0071] SEQ ID NO: 73 comprises 12 CRISPR3 [repetitive sequence-spacer sequence] units and 1 terminal repeat sequence, which is identical to the repeat sequences of the 12 CRISPR3 [repetitive sequence-spacer sequence] units. The sequences of the 12 CRISPR3 [repetitive sequence-spacer sequence] units of SEQ ID NO: 73 are defined by SEQ ID NO: 75 to SEQ ID NO: 86, respectively. The repeat sequences of all CRISPR3 [repetitive sequence-spacer sequence] units within the CRISPR3 locus defined herein are defined by SEQ ID NO: 74 (R3). The terminal repeat sequence is identical to R3 and is defined by SEQ ID NO: 74. In one specific embodiment, the CRISPR3 locus defined by SEQ ID NO: 73, or a CRISPR3 locus containing one or more portions of SEQ ID NO: 73 as defined herein, is side-connected with the CRISPR3 leader sequence and CRISPR3 tail sequence defined by SEQ ID NO: 71 and SEQ ID NO: 72, respectively.

[0072] Following phage attack, one or more additional CRISPR3 [repetitive sequence-spacer sequence] units may be added within the CRISPR3 locus, particularly at the 5' portion (i.e., the leader sequence terminus) of the CRISPR3 locus as defined herein, that is, immediately after the last nucleotide of the CRISPR3 leader sequence. These additional CRISPR3 [repetitive sequence-spacer sequence] units have a sequence defined as R3-X3 in the 5' to 3' direction, where R3 is defined by SEQ ID NO: 74, and X3 is any sequence of length from 27 to 33 bp, specifically 28 to 32 bp, specifically 29 to 31 bp, specifically exactly 30 bp, particularly any CRISPR spacer sequence. Specifically, the sequence of any of these additional CRISPR3 [repetitive sequence-spacer sequence] units is selected from SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, or SEQ ID NO: 93. Non-limiting examples of additional CRISPR3 [repetitive sequence-spacer sequence] units that may be used according to the present invention are defined by SEQ ID NO: 94 to SEQ ID NO: 103.

[0073] In one specific embodiment, the present invention provides a Streptococcus thermophilus strain as defined herein, the genome of which includes a CRISPR3 locus comprising or consisting of the sequence defined by SEQ ID NO: 73. In one specific embodiment, the CRISPR3 locus is composed of, from 5' to 3', at least one additional CRISPR3 [repetitive sequence-spacer] unit of sequence R3-X3, specifically at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more additional CRISPR3 [repetitive sequence-spacer] units of sequence R3-X3, followed by SEQ ID NO: 73.

[0074] In one specific embodiment, the present invention provides a Streptococcus thermophilus strain as defined herein, the genome of which includes a CRISPR3 locus comprising a portion of SEQ ID NO: 73 and a terminal repeat sequence defined by SEQ ID NO: 74 in the 5' to 3' direction.

[0075] In the context of the CRISPR3 locus, "a portion of SEQ ID NO: 73" refers to a fragment of SEQ ID NO: 73 containing at least three or exactly three consecutive CRISPR3 [repetitive sequence-spacer] units as contained in SEQ ID NO: 73. Specifically, it contains at least three, four, five, six, seven, eight, nine, ten, or eleven consecutive [repetitive sequence-spacer] units as contained in SEQ ID NO: 73. "Consecutive" means that the sequence in which the multiple CRISPR3 [repetitive sequence-spacer] units present in the portion of SEQ ID NO: 73 are linked together is the same as the order in which they appear in SEQ ID NO: 73 (e.g., SEQ ID NO: 76-SEQ ID NO: 77-SEQ ID NO: 78, or SEQ ID NO: 82-SEQ ID NO: 83-SEQ ID NO: 84). In one specific embodiment, a portion of SEQ ID NO: 73 is a fragment of SEQ ID NO: 73 containing at least three or exactly three consecutive CRISPR3 [repetitive sequence-spacer sequence] units selected from SEQ ID NO: 75 to SEQ ID NO: 86. In one specific embodiment, "a portion of SEQ ID NO: 73" refers to 3, 4, 5, 6, 7, 8, 9, 10, or 11 consecutive terminal CRISPR3 [repetitive sequence-spacer sequence] units contained in SEQ ID NO: 73. The term "terminal CRISPR3 [repetitive sequence-spacer sequence] unit contained in SEQ ID NO: 73" refers to the CRISPR3 [repetitive sequence-spacer sequence] unit located at the 3' end (i.e., at the end of the trailing sequence) of the CRISPR3 locus in SEQ ID NO: 73, that is, immediately preceding the terminal repeat sequence of SEQ ID NO: 74. Therefore, the two consecutive terminal CRISPR3 [repetition sequence-spacer sequence] units of SEQ ID NO: 73 refer to SEQ ID NO: 85-SEQ ID NO: 86, the three consecutive terminal CRISPR3 [repetition sequence-spacer sequence] units of SEQ ID NO: 73 refer to SEQ ID NO: 84-SEQ ID NO: 85-SEQ ID NO: 86, and so on.

[0076] In one specific embodiment, the CRISPR3 locus, from 5' to 3', comprises: an integer number of [repetitive sequence-spacer sequence] units, including at least three consecutive CRISPR3 [repetitive sequence-spacer sequence] units contained in SEQ ID NO: 73 (as defined herein as a portion of SEQ ID NO: 73), followed by the terminal repeat sequence of SEQ ID NO: 74. In another specific embodiment, the CRISPR3 locus, from 5' to 3', comprises: an integer number of [repetitive sequence-spacer sequence] units, including at least three consecutive CRISPR3 [repetitive sequence-spacer sequence] units selected from SEQ ID NO: 75 to SEQ ID NO: 86, followed by the terminal repeat sequence of SEQ ID NO: 74. In one specific embodiment, the CRISPR3 locus is composed from 5' to 3' as follows: at least one additional CRISPR3 [repetitive sequence-spacer sequence] unit of sequence R3-X3, specifically at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more additional CRISPR3 [repetitive sequence-spacer sequence] units of sequence R3-X3, at least three consecutive CRISPR3 [repetitive sequence-spacer sequence] units contained in SEQ ID NO: 73 (a portion of SEQ ID NO: 73), followed by the terminal repeat sequence of SEQ ID NO: 74.

[0077] In one specific embodiment, the CRISPR3 locus is composed from 5' to 3' as follows: at least one additional CRISPR3 [repetitive sequence-spacer sequence] unit of sequence R3-X3, specifically at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more additional CRISPR3 [repetitive sequence-spacer sequence] units of sequence R3-X3, as defined above in part of SEQ ID NO: 73, followed by the terminal repeat sequence of SEQ ID NO: 74. In one specific embodiment, the CRISPR3 locus is composed from 5' to 3' as follows: at least one additional CRISPR3 [repetitive sequence-spacer sequence] unit of sequence R3-X3, specifically at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more additional CRISPR3 [repetitive sequence-spacer sequence] units of sequence R3-X3, 1 to 11 consecutive terminal CRISPR3 [repetitive sequence-spacer sequence] units contained in SEQ ID NO: 73, followed by the terminal repeat sequence of SEQ ID NO: 74.

[0078] In one specific embodiment, the present invention provides a thermophilic streptococcal strain, wherein the lactation kinetics of said strain are characterized as follows:

[0079] a) When the pH value is between 5.30 and 6.00, the average acidification rate is at least 70.10. -4 UpH / min or equal to 70.10 -4 UpH / min, or between 70.10 -4 With 250.10 -4 UpH / min is between 70.10 -4 With 200.10 -4 UpH / min is between 70.10 -4 With 180.10 -4 UpH / min is between 70.10 -4 With 140.10 -4 UpH / min is between 80 and 10. -4 With 120.10 -4 UpH / min is between 90.10 -4 With 110.10 -4 UpH / min, or between 95.10 -4 With 105.10 - 4 When the upH / min is between 22.10 and 5.00, and the pH value is between 5.00 and 5.30, the average acidification rate is less than 22.10. -4 UpH / min or equal to 22.10 -4 UpH / min, or between 1.10 -4 With 20.10 -4 UpH / min is between 2.10 and 2. -4 With 22.10 -4 UpH / min is between 2.10 and 2. -4 With 20.10 -4 UpH / min is between 5.10 and 5.10. -4 With 20.10 -4 UpH / min, or between 10.10 -4 With 18.10 -4 Between UpH / min; and / or

[0080] b) The ratio of the average acidification rate (1) between pH values ​​between 5.00 and 5.30 to the average acidification rate (2) between pH values ​​between 5.30 and 6.00 is less than or equal to 25%, less than or equal to 20%, less than or equal to 18%, between 1% and 25%, between 2% and 25%, between 5% and 18%, between 8% and 18%, or between 10% and 18%.

[0081] Preferably, the average acidification rate at a pH value between 5.30 and 6.00 and the average acidification rate at a pH value between 5.00 and 5.30 are calculated according to the description of assay method I; and the genome of this strain contains at least one element selected from the CRISPR4, CRISPR1 and CRISPR3 loci as defined herein, preferably the genome of this strain contains one, two or three elements selected from the CRISPR4, CRISPR1 and CRISPR3 loci.

[0082] In one specific embodiment, the thermophilic streptococcal strain of the present invention is a strain that was deposited on March 21, 2013, under the name of Danisco Deutschland GmbH and with accession number DSM27029 [the DSM 27029 strain of this article] at the DSMZ-German Microbial Culture Collection Center of the Leibniz Institute (Inhoffenstr. 7B, D-38124 Braunschweig, Braunschweig) in accordance with the Budapest Treaty.

[0083] In another embodiment, the thermophilic streptococcal strain of the present invention is a strain that was deposited on March 21, 2013, in accordance with the Budapest Treaty, in the name of Danisco Deutschland GmbH and with accession number DSM27030 [the DSM 27030 strain of this document] at the Leibniz Institute DSMZ.

[0084] In another embodiment, the thermophilic streptococcal strain of the present invention is a strain that was deposited at the Leibniz Institute DSMZ on March 21, 2013, under the name of Danisco Deutschland GmbH and with accession number DSM27031 [the DSM 27031 strain of this document], in accordance with the Budapest Treaty.

[0085] We hereby confirm that the depositor, Danisco Deutschland GmbH (Busch-Johannsen-Strasse 1, D-25899 Niebüll, Germany), has authorized the applicant (DuPont Nutrition Biosciences ApS, Langebrogade 1, DK-1411 Copenhagen K, Denmark) to reference these deposited biological materials in this application, and unreservedly and irrevocably consents to the public access to its deposited biological materials.

[0086] With regard to those designations seeking European patent protection, a sample of the deposited microorganism may be obtained before the announcement of the grant of the European patent or before the date on which the application is rejected or withdrawn, or deemed to have been withdrawn, and the distribution of such sample shall be limited to an expert designated by the requester who requested the sample, and, where appropriate, with the consent of i) the applicant and / or ii) the European Patent Office (Article 32 of the Implementing Regulations of the European Patent Convention).

[0087] In one specific embodiment, the thermophilic streptococcal strain of the present invention is a mutant strain of DSM 27029, DSM 27030, or DSM 27031 disclosed herein, provided that the lactation kinetics of the mutant strain conform to the limitations given herein for any thermophilic streptococcal strain of the present invention, specifically, similar to the lactation kinetics of the DSM deposited strain from which the mutant strain is derived. Specifically, the lactation kinetics of the mutant strain are characterized as follows:

[0088] a) When the pH value is between 5.30 and 6.00, the average acidification rate is at least 70.10. -4 UpH / min or equal to 70.10 -4 UpH / min, or between 70.10 -4 With 250.10 -4 UpH / min is between 70.10 -4 With 200.10 -4 UpH / min is between 70.10 -4 With 180.10 -4 UpH / min is between 70.10 -4 With 140.10 -4 UpH / min is between 80 and 10. -4 With 120.10 -4UpH / min is between 90.10 -4 With 110.10 -4 UpH / min, or between 95.10 -4 With 105.10 - 4 When the upH / min is between 22.10 and 5.00, and the pH value is between 5.00 and 5.30, the average acidification rate is less than 22.10. -4 UpH / min or equal to 22.10 -4 UpH / min, or between 1.10 -4 With 20.10 -4 UpH / min is between 2.10 and 2. -4 With 22.10 -4 UpH / min is between 2.10 and 2. -4 With 20.10 -4 UpH / min is between 5.10 and 5.10. -4 With 20.10 -4 UpH / min, or between 10.10 -4 With 18.10 -4 Between UpH / min; and / or

[0089] b) The ratio of the average acidification rate (1) between pH values ​​between 5.00 and 5.30 to the average acidification rate (2) between pH values ​​between 5.30 and 6.00 is less than or equal to 25%, less than or equal to 20%, less than or equal to 18%, between 1% and 25%, between 2% and 25%, between 5% and 18%, between 8% and 18%, or between 10% and 18%.

[0090] Preferably, the average acidification rate when the pH value is between 5.30 and 6.00 and the average acidification rate when the pH value is between 5.00 and 5.30 are calculated according to the content described in Measurement Method I.

[0091] The term "mutant strain of DSM 27029, DSM 27030, or DSM 27031" refers to a thermophilic streptococcal strain whose genome is highly similar to that of DSM 27029, DSM 27030, or DSM 27031. In this application, the term "thermophilic streptococcal strain of the present invention" encompasses the aforementioned mutant strain. High genomic similarity includes:

[0092] - The genome of a Streptococcus thermophilus strain contains up to 150 mutation events compared to the genomes of DSM 27029, DSM 27030, or DSM 27031 strains, preferably up to 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, or 20 mutation events. Mutation events are defined as SNPs (single nucleotide polymorphisms) or INDELs (insertions, deletions, and combinations thereof). The number of mutation events is determined as follows: using the genomes of DSM 27029, DSM 27030, or DSM 27031 strains as controls, mutation events present in the mutant genome are identified, with each mutation event (SNP or INDEL) representing one mutation event (i.e., for example, an insertion of a sequence containing several nucleotides is considered only one mutation event). In this context, the genome sequence of the mutant strain of the present invention is defined by the number of mutation events contained in it compared to DSM27029, DSM 27030, or DSM 27031 strains. In addition to this definition, it may also be defined by the percentage of identity with the genome sequences of DSM 27029, DSM 27030, or DSM 27031 strains, wherein the percentage of identity herein refers to the percentage of sequences found in the genome of one strain that are present in the genome of another strain, specifically: a) the percentage of sequences found in the genome of DSM 27029, DSM 27030, or DSM27031 strains and present in the genome of the mutant strain, or b) the percentage of sequences found in the genome sequence of the mutant strain and present in the genome of DSM 27029, DSM 27030, or DSM 27031 strains. Therefore, mutant strains that differ from DSM 27029, DSM 27030, or DSM 27031 strains only in the presence of insertions (one or more) or deletions (one or more) have a genome that is 100% identical to that of DSM 27029, DSM 27030, or DSM 27031 strains, because the entire genome sequence of one strain is found in the genome of another strain.In one specific embodiment, the genome sequence of the mutant strain of the present invention, defined by the number of mutation events, is compared with that of DSM 27029 strain and DSM... The genomic sequence identity percentage of strain 27030 or strain DSM27031 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.92%, at least 99.94%, at least 99.96%, at least 99.98%, or at least 99.99%, wherein the identity percentage represents the percentage of sequences found in the genome of one strain and present in the genome of another strain; and / or.

[0093] - The genome sequence of a Streptococcus thermophilus strain shares at least 95% identity with the genome sequences of strains DSM 27029, DSM 27030, or DSM 27031, specifically with the genome sequences of strains DSM 27029, DSM 27030, or DSM 27031 deposited on March 21, 2013. The percentage of genomic sequence identity of strain 27031 is at least 90%, at least 91%, at least 95%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.92%, at least 99.94%, at least 99.96%, at least 99.98%, or at least 99.99%. Identity is described by comparing the two genomic sequences across their full length (global alignment) and can be calculated using any procedure based on the Needleman-Wunsch algorithm.

[0094] It is worth noting that, according to the definition given above, strains DSM 27029, DSM 27030, and DSM27031 are all mutant strains.

[0095] In one specific embodiment, the genome of the mutant strain contains at least one element selected from the CRISPR4, CRISPR1, and CRISPR3 loci as defined above, specifically, it contains one, two, or three elements selected from the CRISPR4, CRISPR1, and CRISPR3 loci.

[0096] In one specific embodiment, the present invention provides a thermophilic streptococcal mutant strain of DSM 27029, DSM 27030, or DSM 27031 as defined herein, wherein the genome of the mutant strain differs from that of the DSM 27029, DSM 27030, or DSM 27031 strain in that it has CRISPR4, CRISPR1, and / or CRISPR3 loci; specifically, the difference lies in its CRISPR4 locus, specifically in its CRISPR1 locus, specifically in its CRISPR3 locus, specifically in its CRISPR4 and CRISPR1 locus, specifically in its CRISPR1 and CRISPR3 locus, specifically in its CRISPR4 and CRISPR3 locus, specifically in its CRISPR4, CRISPR1, and CRISPR3 locus. The difference between DSM 27029, DSM 27030 or DSM 27031 strains is that the mutant strains of one or more CRISPR loci (CRISPR1 and / or CRISPR3 and / or CRISPR4) are, in this document, limited to CRISPR mutant strains of DSM 27029, DSM 27030 or DSM 27031 strains.

[0097] In one specific embodiment, the thermophilic streptococcal mutant strains of DSM 27029, DSM 27030, or DSM 27031, specifically the CRISPR mutant strains of DSM 27029, DSM 27030, or DSM 27031, are characterized as follows:

[0098] - Contains a CRISPR4 locus, which includes a sequence defined by SEQ ID NO: 3 or one or more portions of SEQ ID NO: 3, such as the CRISPR4 locus defined in any of the above embodiments; and / or

[0099] - Contains a CRISPR1 locus, which contains a sequence defined by SEQ ID NO: 19 or contains one or more portions of SEQ ID NO: 19, such as the CRISPR1 locus defined in any of the above embodiments. In one specific embodiment, the CRISPR1 locus of the mutant strain is composed from the 5' to 3' direction as follows: at least one additional CRISPR1 [repetitive sequence-spacer sequence] unit of sequence R1-X1, specifically at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more additional CRISPR1 [repetitive sequence-spacer sequence] units of sequence R1-X1, followed by SEQ ID NO: 19 (where R1 is defined by SEQ ID NO: 20, and X1 is any sequence of length 27 to 33 bp, specifically 28 to 32 bp, specifically 29 to 31 bp, specifically exactly 30 bp). In another specific embodiment, the CRISPR1 locus of the mutant strain is composed from the 5' to 3' direction as follows: at least one additional CRISPR1 [repetitive sequence-spacer sequence] unit of sequence R1-X1, specifically at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more additional CRISPR1 [repetitive sequence-spacer sequence] units of sequence R1-X1, at least three consecutive, specifically at least three consecutive terminal CRISPR1 [repetitive sequence-spacer sequence] units contained in SEQ ID NO: 19 (a portion of SEQ ID NO: 19), followed by the terminal repeat sequence of SEQ ID NO: 21; and / or

[0100] - Contains a CRISPR3 locus, which contains a sequence defined by SEQ ID NO: 73 or contains one or more portions of SEQ ID NO: 73, such as the CRISPR3 locus defined in any of the above embodiments. In one specific embodiment, the CRISPR3 locus of the mutant strain is composed from the 5' to 3' direction as follows: at least one additional CRISPR3 [repetitive sequence-spacer sequence] unit of sequence R3-X3, specifically at least or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more additional CRISPR3 [repetitive sequence-spacer sequence] units of sequence R3-X3, followed by SEQ ID NO: 73 (where R3 is defined by SEQ ID NO: 74, and X3 is any sequence of length 27 to 33 bp, specifically 28 to 32 bp, specifically 29 to 31 bp, specifically exactly 30 bp). In another specific embodiment, the CRISPR3 locus of the mutant strain is composed from 5' to 3' as follows: at least one additional CRISPR3 [repetitive sequence-spacer sequence] unit of sequence R3-X3, specifically at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more additional CRISPR3 [repetitive sequence-spacer sequence] units of sequence R3-X3, at least three consecutive, specifically at least three consecutive terminal CRISPR3 [repetitive sequence-spacer sequence] units contained in SEQ ID NO: 73 (a portion of SEQ ID NO: 73), followed by the terminal repeat sequence of SEQ ID NO: 74.

[0101] In one specific embodiment, the present invention provides a thermophilic streptococcal strain, wherein the lactation kinetics of said strain are characterized as follows:

[0102] a) When the pH value is between 5.30 and 6.00, the average acidification rate is at least 70.10. -4 UpH / min or equal to 70.10 -4 UpH / min, or between 70.10 -4 With 250.10 -4 UpH / min is between 70.10 -4 With 200.10 -4 UpH / min is between 70.10 -4 With 180.10 -4 UpH / min is between 70.10 -4 With 140.10 -4 UpH / min is between 80 and 10. -4 With 120.10 -4UpH / min is between 90.10 -4 With 110.10 -4 UpH / min, or between 95.10 -4 With 105.10 - 4 When the upH / min is between 22.10 and 5.00, and the pH value is between 5.00 and 5.30, the average acidification rate is less than 22.10. -4 UpH / min or equal to 22.10 -4 UpH / min, or between 1.10 -4 With 20.10 -4 UpH / min is between 2.10 and 2. -4 With 22.10 -4 UpH / min is between 2.10 and 2. -4 With 20.10 -4 UpH / min is between 5.10 and 5.10. -4 With 20.10 -4 UpH / min, or between 10.10 -4 With 18.10 -4 Between UpH / min; and / or

[0103] b) The ratio of the average acidification rate (1) between pH values ​​between 5.00 and 5.30 to the average acidification rate (2) between pH values ​​between 5.30 and 6.00 is less than or equal to 25%, less than or equal to 20%, less than or equal to 18%, between 1% and 25%, between 2% and 25%, between 5% and 18%, between 8% and 18%, or between 10% and 18%.

[0104] Preferably, the average acidification rate when the pH value is between 5.30 and 6.00 and the average acidification rate when the pH value is between 5.00 and 5.30 are calculated according to the content described in Measurement Method I, and wherein the Streptococcus thermophilus strain is not one or both of the following strains:

[0105] - DSM 27029 strain, deposited at the Leibniz Institute DSMZ on March 21, 2013.

[0106] - DSM 27030 strain, deposited at the Leibniz Institute DSMZ on March 21, 2013.

[0107] - DSM 27031 strain deposited at the Leibniz Institute DSMZ on March 21, 2013.

[0108] In any embodiment, a group of Streptococcus thermophilus strains whose genomes contain the CRISPR4 locus as defined above and / or contain the CRISPR1 locus as defined above and / or contain the CRISPR3 locus as defined above can be used as research subjects to identify the Streptococcus thermophilus strains of the present invention (including the Streptococcus thermophilus mutant strains as defined above).

[0109] In one specific embodiment, the thermophilic streptococcus strain of the present invention, specifically the mutant strain of DSM 27029, DSM 27030, or DSM 27031, is not the thermophilic streptococcus salivarius thermophilus strain deposited on May 6, 2011, with accession number CBS129457 at the Centraalbureau voor Schimmel-cultures (Fungal Biodiversity Centre, Utrecht, The Netherlands).

[0110] In one specific embodiment, the thermophilic streptococcal strain of the present invention, specifically the mutant strain of DSM 27029, DSM27030 or DSM 27031, is not the thermophilic streptococcal strain deposited at the China Fungal Culture Collection Center on May 6, 2011 with accession number CBS129458.

[0111] In one specific embodiment, the thermophilic streptococcal strain of the present invention, specifically, is a mutant strain of DSM 27029, DSM 27030, or DSM 27031 strain, which is neither the thermophilic streptococcal strain deposited at the Fungal Culture Collection Center on May 6, 2011 with accession number CBS129457 nor the thermophilic streptococcal strain deposited at the Fungal Culture Collection Center on May 6, 2011 with accession number CBS129458.

[0112] The present invention also provides a composition comprising or consisting of a culture of the thermophilic streptococcal strain of the present invention; specifically, comprising or consisting of a culture of thermophilic streptococcal strain DSM 27029, a culture of thermophilic streptococcal strain DSM 27030, or a culture of thermophilic streptococcal strain DSM 27031; particularly comprising or consisting of a culture of the thermophilic streptococcal mutant strain as defined above.

[0113] The compositions of the present invention (preferably, when used as a fermentation culture) can be pure cultures or mixed cultures. Therefore, we define a pure culture as one in which all or substantially all of the culture consists of the same thermophilic streptococcal strain of the present invention. In an alternative form, a mixed culture is defined as one that comprises several microorganisms, specifically several bacterial strains, including the thermophilic streptococcal strain of the present invention.

[0114] In one specific embodiment, the composition of the present invention is a pure culture of a Streptococcus thermophilus strain as defined herein, or is composed of such a pure culture.

[0115] In another embodiment, the composition of the present invention, in addition to comprising a culture of the thermophilic streptococcus of the present invention, also comprises at least one other microorganism. The term "microorganism" is defined herein as any organism that can be combined with the thermophilic streptococcus of the present invention, and is particularly useful for preparing products according to the present invention. The term "microorganism" encompasses yeasts, molds, and bacteria, such as lactic acid bacteria, Bifidobacterium, Brevibacterium, and / or Propionibacterium species.

[0116] In one specific embodiment of the mixed culture, the composition, in addition to containing a culture of the thermophilic streptococcus of the present invention, also contains at least one culture of lactic acid bacteria and / or at least one other culture of propionic acid bacteria. Suitable lactic acid bacteria include strains of *Lactococcus*, *Streptococcus*, *Lactobacillus* (including *Lactobacillus acidophilus*), *Enterococcus*, *Pediococcus*, *Leuconostoc*, and *Oenococcus*, or any combination of these strains. Lactococcus species include *Lactococcus lactis*, including *Lactococcus lactis subsp. lactis*, *Lactococcus lactis subsp. lactis biovar. diacetylactis*, and *Lactococcus lactis subsp. cremoris*. Other lactic acid bacteria species include *Leuconostoc* species, *Streptococcus thermophilus*, *Lactobacillus delbrueckii* subsp. bulgaricus, and *Lactobacillus helveticus*.

[0117] Therefore, as a specific embodiment, the present invention also relates to a composition as defined herein, which comprises or consists of the following substances: a culture of Streptococcus thermophilus of the present invention, at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 strains of Streptococcus thermophilus species that are different from the Streptococcus thermophilus strains of the present invention, and / or strains of Lactobacillus species, and / or any combination of the foregoing substances.

[0118] In one specific embodiment, the composition comprises or consists of the following substances: a culture of *Streptococcus thermophilus* of the present invention, at least one strain of *Streptococcus thermophilus* different from the *Streptococcus thermophilus* strain of the present invention, specifically at least or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 strains, and / or one or more strains of *Lactobacillus delbrueckii* subsp. bulgaricus, and / or one or more strains of *Lactobacillus helveticus*, and / or any combination of the foregoing substances. In another specific embodiment, the composition comprises or consists of the following substances: a culture of *Streptococcus thermophilus* of the present invention, one strain of *Streptococcus thermophilus* different from the *Streptococcus thermophilus* strain of the present invention, and one strain of *Lactobacillus delbrueckii* subsp. bulgaricus. In yet another specific embodiment, the composition comprises or consists of the following substances: a culture of *Streptococcus thermophilus* of the present invention, two strains of *Streptococcus thermophilus* different from the *Streptococcus thermophilus* strain of the present invention, and one strain of *Lactobacillus delbrueckii* subsp. bulgaricus.

[0119] In one specific embodiment, the composition comprises or consists of the following substances: a culture of Streptococcus thermophilus of the present invention, Lactococcus lactis subsp. lactis and / or Lactococcus lactis subsp. milk fat.

[0120] In one specific embodiment, the composition comprises or consists of the following substances: a culture of the thermophilic streptococcus of the present invention, and a complex mixed fermentation agent culture.

[0121] In any specific embodiment of the composition defined herein, whether the composition is a pure culture or a mixed culture, it further comprises at least one probiotic strain, such as Bifidobacterium animalis subsp. lactis, Lactobacillus acidophilus, Lactobacillus paracasei, or Lactobacillus casei.

[0122] In one specific embodiment, whether the composition defined herein is a pure culture or a mixed culture as defined above, it also particularly includes one or more components that can be added to food, such as, but not limited to, cryoprotectants (i.e., antifreeze agents), synergists, and / or common additives. The term "component" refers to any molecule or solution that is not a microorganism as defined above. For example, cryoprotectants include cyclodextrin, maltitol, trehalose, sucrose, maltodextrin, or combinations thereof. For example, synergists include nucleotides. For example, common additives include nutrients such as yeast extract, sugars, and vitamins.

[0123] In one specific embodiment, regardless of whether the composition defined herein is a pure culture or a mixed culture as defined above, or includes or does not contain additional components (one or more), it is either in liquid form, frozen form, or dry powder form (such as obtained after lyophilization).

[0124] In one specific embodiment, regardless of whether the composition of the present invention is a pure culture or a mixed culture as defined above, or whether it contains or does not contain additional components, it comprises a concentrated form of the thermophilic streptococcal strain of the present invention (and optionally at least one other microorganism) (concentrate), including freeze-dried or dried concentrates. Therefore, the concentration of the thermophilic streptococcal strain of the present invention in the composition is 10 ppm per gram of composition. 5 Up to 10 12 CFU (colony forming unit), preferably 10 7 Up to 10 12 Within the range of CFU, more preferably at least 10 per gram of composition. 7 At least 10 8 At least 10 9 At least 10 10 Or at least 10 11 CFU.

[0125] This invention also relates to the use of cultures of the thermophilic streptococcal strains of the present invention or the preparation of products, particularly food products or feed products, especially fermented products, specifically fermented food products or fermented feed products. Therefore, the present invention also provides a method for preparing a product, preferably a food or feed product, wherein the method comprises: a) contacting a substrate with a culture of the thermophilic streptococcal strain of the present invention or the composition defined herein, or contacting the substrate with the culture of the thermophilic streptococcal strain of the present invention or the composition defined herein in the presence of such a culture (or mixing the substrate with the culture of the thermophilic streptococcal strain of the present invention or the composition defined herein), b) optionally fermenting the substrate, and then c) obtaining the product. In one specific embodiment, the present invention also provides a method for preparing a fermented product, preferably a fermented food or feed product, wherein the method comprises: fermenting a substrate with a culture of the thermophilic streptococcal strain of the present invention or the composition defined herein, or fermenting the substrate in the presence of such a culture of the thermophilic streptococcal strain of the present invention or the composition defined herein, and then obtaining the fermented product.

[0126] This invention also relates to any product prepared from the thermophilic streptococcal strains of the present invention or the compositions defined herein, especially using the methods disclosed herein, or comprising or consisting of the thermophilic streptococcal strains of the present invention or the compositions defined herein. In one specific embodiment, the present invention provides a product obtained by or achievable through the methods described herein, particularly a food or feed product, especially a fermented product, specifically a fermented food or fermented feed product. The present invention also provides a product comprising a culture of the thermophilic streptococcal strain of the present invention or comprising the compositions defined herein, particularly a food or feed product, especially a fermented product, specifically a fermented food or fermented feed product.

[0127] Suitable products include, but are not limited to, food, food ingredients, food additives, food supplements, functional foods, feed, nutritional supplements, or probiotic supplements. According to the invention, "food" refers to a product intended for human consumption. According to the invention, "feed" refers to a product intended for feeding animals. As used herein, the term "food ingredient" includes formulations added to or potentially added to food, and includes formulations that can be used in small amounts in a wide variety of products requiring (e.g.) acidification. As used herein, the term "functional food" refers to a food that not only provides nutritional effects and / or taste satisfaction to the consumer, but also provides additional beneficial effects. Suitable products include, but are not limited to, fruits, vegetables, feed crops and feed vegetables (including derivatives), grains and grain derivatives, dairy products and dairy derivatives, meat, poultry, and seafood. The thermophilic streptococcal strains of the present invention or the compositions defined herein can be used to prepare food products, such as one or more of the following: confectionery products, dairy products, meat products, poultry products, fish products, and baked goods. For example, the thermophilic streptococcal strains of the present invention or the compositions defined herein can be used as ingredients in the following products: soft drinks, fruit juices or whey protein-containing beverages, health teas, cocoa beverages, milk beverages and lactic acid bacteria beverages, yogurt, drinking yogurt, and wine.

[0128] In one specific embodiment, the substrate to which the thermophilic streptococcal strain of the present invention or the composition defined herein (or mixed with the thermophilic streptococcal strain of the present invention or the composition defined herein) is a milk substrate. Therefore, in one specific embodiment, the present invention also relates to the use of the culture of the thermophilic streptococcal strain of the present invention or the composition as defined herein for the preparation of dairy products, particularly dairy food products or dairy feed products, especially fermented dairy products, specifically fermented dairy food products or fermented dairy feed products. Therefore, the present invention also provides a method for preparing dairy products, particularly dairy food products or dairy feed products, particularly fermented dairy products, specifically fermented dairy food products or fermented dairy feed products, wherein the method comprises: a) contacting the milk substrate with the culture of the thermophilic streptococcal strain of the present invention or the composition defined herein, or contacting the milk substrate with the culture of the thermophilic streptococcal strain of the present invention or the composition defined herein in the presence of the present invention; b) optionally fermenting the milk substrate; and then c) obtaining the product. In one specific embodiment, the present invention also provides a method for preparing fermented dairy products, preferably fermented dairy food or feed products, wherein the method comprises: fermenting a milk substrate with a culture of the thermophilic streptococcal strain of the present invention or a composition defined herein, or fermenting the milk substrate in the presence of a culture of the thermophilic streptococcal strain of the present invention or a composition defined herein, and then obtaining the fermented dairy product. In one specific embodiment, the milk substrate comprises solid articles, such as fruits, chocolate products, or cereal foods. In one specific embodiment, the present invention also relates to the use of the thermophilic streptococcal strain of the present invention or any composition (pure culture or mixed culture) as defined herein to achieve the effect that dairy products obtained or fermented with the thermophilic streptococcal strain or the composition, or dairy products obtained or fermented in the presence of the thermophilic streptococcal strain or the composition, exhibit reduced acidification compared to dairy products obtained or fermented not with the thermophilic streptococcal strain of the present invention, or dairy products obtained or fermented in the absence of the thermophilic streptococcal strain of the present invention; furthermore, the present invention also relates to the dairy product itself. In one specific embodiment, the invention also relates to the use of the thermophilic streptococcal strain of the invention or any composition (pure culture or mixed culture) as defined herein to obtain dairy products, specifically the use of yogurt, which maintains a stable pH of 4.4 ± 0.1 for 14 days when stored at a positive temperature below 10°C.In one specific embodiment, the invention also relates to the use of the thermophilic streptococcal strain of the invention or any composition (pure culture or mixed culture) as defined herein to obtain dairy products, specifically the use of yogurt, which, when stored at a positive temperature below 10°C, maintains a pH of 4.5 ± 0.1 or 4.4 ± 0.05 for 14 days, and optionally remains stable (i.e., within the same range) for 28 days.

[0129] The term "milk base" refers to a milky substance derived from animals and / or plants. In one specific embodiment, the milk base is derived from animals such as cows, goats, sheep, buffalo, zebras, horses, donkeys, camels, etc. The milky substance can be a natural milky substance, reconstituted milk, skim milk, or a milky substance supplemented with compounds (e.g., fats, yeast extract proteins, peptones, and / or surfactants) necessary for bacterial growth or subsequent processing of fermented milk. In one specific embodiment, the milk base is commercially available UHT milk (milky substance treated at ultra-high temperature (i.e., 130°C) for several seconds), specifically supplemented with 3% (w / w) semi-skimmed milk powder, and pasteurized by heating, specifically, at 90±0.2°C for 10±1 min. In another embodiment, the emulsion substrate is derived from plants, specifically from extracts of plant materials (plant milk) that have been processed or are derived, for example, from legumes (soybeans, chickpeas, lentils, etc.) or oilseeds (rapeseed, soybeans, sesame, cotton, etc.). The extract is a solution or colloidal suspension containing proteins that coagulate under chemical action, via acid fermentation, and / or heating. In another embodiment, the emulsion substrate is a mixture of animal milk (one or more) and plant milk (one or more) as defined above.

[0130] Therefore, the present invention provides a dairy product obtained using a milk base through or obtainable by the methods described herein, particularly a dairy food product or dairy feed product, especially a fermented dairy product, specifically a fermented dairy food product or fermented dairy feed product. The present invention also provides a dairy product comprising a culture of the thermophilic streptococcal strain of the present invention or comprising a composition defined herein, specifically a fermented dairy product. In one specific embodiment, the dairy product or fermented dairy product is or includes yogurt, cheese (e.g., sour curd cheese, hard cheese, semi-hard cheese, soft cheese), buttermilk, curd cheese, sour cream, kefir, fermented whey beverages, milk wine, milk beverages, yogurt beverages, fermented milk, ripened cream, light cheese, milk, dairy product residue, processed cheese, soft cheese, cream desserts, or infant formula, preferably these dairy products are based on a milk base derived from animals and / or plants.

[0131] Experiment

[0132] Example 1

[0133] The average acidification speed (SI) at a pH comprised between 5.30 and 6.00 was calculated, as well as the average acidification speed (S2) at a pH comprised between 5.00 and 5.30, and the ratio S2 / S1 Assay I

[0134] The assay was performed on strain DGCC7984

[0135] Add 3% (w / w) skim milk powder to commercially available semi-fat UHT milk (1.5% w / w fat content) [“Le Petit Vendéen”; GLAC, France]. After the milk powder dissolves, heat the mixture at 90°C for 10 minutes. The heating step involves increasing the temperature from 20-25°C to 90°C for no more than 35 minutes; the cooling step involves decreasing the temperature from 90°C to 35-45°C for no more than 45 minutes. Add 1 g / 100 L (w / v) sodium formate solution to the mixture, and then inoculate. Inoculate the bacterial strain stored at -80°C into milk-based medium. The inoculation rate is 1.10 g / ml of milk-based medium. 6 CFU. The incubation temperature was set at 43℃ ± 1℃, and a constant incubation temperature was maintained using a water bath during fermentation. pH changes were measured online using a Cinac system (CINAC, an automated system for controlling lactic acid starter cultures; Corrieu G, Picque D, Perret B, Quemener P; Process Magazine; 1992; no. 1068; pp. 24-27). pH values ​​were recorded every 5 minutes over 24 hours, and the results were summarized in tables or presented as CINAC curves.

[0136] The following three parameters were measured: time to pH = 6.00 (T) pH6.00 ), time (T) when pH = 5.30 pH5.30 ), time (T) when pH = 5.00 pH5.00 These parameters can be obtained directly from online records; if the time to reach the target pH value is not recorded in the table, linear interpolation can be performed between two records (see the example of strain DGCC7984 below).

[0137] Finally, the following parameters are defined to describe the lactation kinetics:

[0138] -S1=(6.00-5.30) / (T pH5.30 -T pH6.00 (UpH / min) [Average acidification rate when pH is between 5.30 and 6.00];

[0139] -S2=(5.30-5.00) / (T pH5.00 -T pH5.30 (UpH / min) [Average acidification rate when pH is between 5.00 and 5.30];

[0140] - The ratio of S2 to S1 = [S2 / S1 ratio] (in %).

[0141] Table 2

[0142] Assay I (as described above) was performed on strain DGCC7984. pH values ​​were recorded every 5 minutes over 24 hours, and the results are presented in Table 1.

[0143] Three parameters were measured: 1) time (T) for pH to reach 6.00. pH6.00 ), 2) Time (T) when pH = 5.30 pH5.30 ), 3) Time (T) when pH = 5.00 pH5.00 ).

[0144] Therefore, the time to pH = 5.30 was obtained directly from the online record (235 min). Since the time to pH = 6.00 is not included in the table (T... pH6.00 ) and time (T) at pH = 5.00 pH5.00 Therefore, the method described below (the example here is for estimating T) is used. pH6.00 The method involves linear interpolation between two records near pH = 6.00 and between two records near pH = 5.00. T is estimated using the same calculation model. pH5.00 .

[0145] -T pH6.00 =(6.00-pH1+T1*((pH1-pH2) / (T1-T2))) / ((pH1-pH2) / (T1-T2))

[0146] In this example, for pH1 = 6.04, T1 = 175 min; and for pH2 = 5.99, T2 = 180 min.

[0147] T pH6.00 =(6.00-6.04+175*((6.04-5.99) / (175-180))) / ((6.04-5.99) / (175-180))

[0148] T pH6.00 = (6.00 - 6.04 + 175 * ((0.05 / 5))) / (0.05 / 5)

[0149] T pH6.00=0.04+(175*0.01) / 0.01=179min

[0150]

[0151]

[0152] T pH6.00 (min)]]> 179 T pH5.30 (min)]]> 235 T pH5.00 (min)]]> 282.5 [S1 = (6.00 - 5.30) / (T pH5.30 - T pH6.00 ) (UpH / min)] 0.0125 [S2 = (5.30 - 5.00) / (T pH5.00 - T pH5.30 ) (UpH / min)] 0.0063 R(％) 50

[0153] Example 2

[0154] The SI, S2, and S2 / S1 ratio were determined for 69 S. thermophilus strains

[0155] Strains

[0156] The SI, S2, and S2 / S1 ratio were determined for these 69 S. thermophilus strains

[0157] Sixty-nine Streptococcus thermophilus strains deposited by DuPont were used. Of these 69 strains, seven had been previously disclosed in some patent applications and were deposited at the French National Center for the Collection of Microbial Cultures (CNCM); the genome sequences of two other Streptococcus thermophilus strains were available from the NCBI database.

[0158] Information on these 9 strains is summarized below:

[0159] (1) DGCC7809 strain: deposited in CNCM, accession number I-2425

[0160] (2) DGCC7710 strain: deposited in CNCM, accession number I-2423

[0161] (3) DGCC8014 strain: deposited in CNCM, accession number I-3617

[0162] (4) DGCC7984 strain: deposited in CNCM, accession number I-2980

[0163] (5) DGCC7666 strain: deposited in CNCM, accession number I-3782

[0164] (6) DGCC7681 strain, deposited in CNCM, accession number I-2432

[0165] (7) DGCC7891 strain: deposited in CNCM, accession number I-2429

[0166] (8) DGCC3198 strain: LMD-9, whose genome sequence can be obtained from the NCBI database, accession number NC_008532.1

[0167] (9) DGCC9742 strain: LMG 18311, whose genome sequence can be obtained from the NCBI database, accession number NC_006448.1

[0168] In this paper, the DGCC number is the internal reference number for DuPont's collection of microorganisms; the DSM and CNCM numbers are the numbers assigned by the Leibniz Institute DSMZ-German Center for the Preservation of Microbial Cultures and the French National Center for the Preservation of Microbial Cultures (Paris, France) respectively, in accordance with the Budapest Treaty.

[0169] Results and discussion

[0170] Method I (as described above) was applied to these 69 Streptococcus thermophilus strains, and the S1 value, S2 value, and S2 / S1 ratio were calculated as described above.

[0171] Figure 1

[0172] The average acidification rate (S1) of these 69 Streptococcus thermophilus strains was determined at pH values ​​between 5.30 and 6.00, and the average acidification rate (S2) at pH values ​​between 5.00 and 5.30. The S2 / S1 ratio was calculated. The 69 Streptococcus thermophilus strains were arranged in ascending order of S1 value (Table 3).

[0173] As can be seen from the table, the higher the S1 value of a Streptococcus thermophilus strain, the higher its S2 value. In short, a lower S1 value (below 70.10) indicates a higher S2 value. -4 Thermophilic Streptococcus strains with an upH / min (S2 value below 30.10) -4 UpH / min; while the S1 value is relatively high (at least 70.10). -4 Thermophilic Streptococcus strains with an upH / min (UH / min) and an S2 value of at least 35.10 -4 UpH / min. This is in Figure 1 It is obvious from the middle, Figure 1 The relationship between the S2 value and the S1 value of the strain is shown (gray diamond).

[0174] Unexpectedly, all three strains exhibited atypical lactation kinetics, meaning they had high S1 values ​​(at least 70.10). -4 UpH / min), while S2 value is below 22.10. -4UpH / min (even lower than the S2 value of some strains with lower S1 values). These strains are DSM 27029, DSM 27030, or DSM 27031 strains (deposited at the DSMZ-German Collection of Microbial Cultures at the Leibniz Institute on March 21, 2013, in accordance with the Budapest Treaty), showing that their S2 values ​​are not correlated with their S1 values, which can be seen from... Table 3 See this (black square).

[0175] This difference in acidification kinetics among the tested Streptococcus thermophilus strains can also be confirmed by calculating the ratio of S2 to S1 values ​​(in %). Therefore, generally speaking, a lower S1 value (below 70.10%) indicates a lower acidification kinetics among the tested Streptococcus thermophilus strains. -4 For *Streptococcus thermophilus* strains with an upH / min (UH / min), the S2 / S1 ratio is between 30% and 50%; while the S1 value is at least 70.10. -4 For thermophilic streptococcal strains with an UpH / min, the S2 / S1 ratio is at least 45% and at most 70%.

[0176] In comparison, strains DSM 27029, DSM 27030, and DSM 27031, although possessing higher S1 values ​​(at least 70.10), -4 The S2 / S1 ratio was below 25%, meaning it was the lowest among all the tested Streptococcus thermophilus strains. Even more surprisingly, the S2 / S1 ratios of these three strains ranged from an S1 value of at least 70.10. -4 The S2 / S1 ratio of thermophilic streptococcal strains with UpH / min is between 1 / 6 and 1 / 2.

[0177]

[0178]

[0179] Figure 2 Average acidification rate [S1(5.3-6.0), in units of 10⁻⁶] for 69 Streptococcus thermophilus strains at pH values ​​between 5.30 and 6.00. -4 UpH / min], the average acidification rate [S2(5.0-5.3), in units of 10⁻⁶] at pH values ​​between 5.00 and 5.30. -4 UpH / min], and the S2 / S1 ratio (in %); arranged in ascending order of S1 value; 1 to 9 This refers to strains (1) to (9) discussed above.

[0180] The average acidification rate (S1) of these three strains at pH values ​​between 5.30 and 6.00, the average acidification rate (S2) at pH values ​​between 5.00 and 5.30, and the S2 / S1 ratio are more distinctive compared to the nine thermophilic streptococcal strains labeled (1) to (9) mentioned above. Therefore, in Figure 2 A and Example 3 In B, it can be clearly seen that compared with the other 9 strains mentioned above, the S2 and S1 values ​​of these three strains are significantly uncorrelated, and the S2 / S1 ratio is very low.

[0181] Therefore, especially considering that the S1 value of these three strains with very low S2 / S1 ratios is at least 70.10... -4 Based on UpH / min, we believe that these three strains exhibit atypical lactation kinetics.

[0182] Genetic analysis of the DSM 27029, DSM 27030 and DSM 27031 strains, CRISPR mutant, and determination of the SI, S2, and S2 / S1 ratio

[0183] Genetic analysis CRISPR mutant

[0184] Determination of the SI, S2, and S2 / S1 ratio for the CRISPR mutant

[0185] Several regions of the genomes of DSM 27029, DSM 27030, and DSM 27031 strains were analyzed. Therefore, the sequences of the CRISPR4, CRISPR1, and CRISPR3 loci were determined.

[0186] In these three preserved DSM strains, the CRISPR4 locus consists of a sequence defined by SEQ ID NO: 3, containing 12 CRISPR4 [repetitive sequence-spacer sequence] units. It is flanked by a CRISPR4 leader sequence defined by SEQ ID NO: 2 and a CRISPR4 tail sequence defined by SEQ ID NO: 1.

[0187] In these three preserved DSM strains, the CRISPR1 locus consists of a sequence defined by SEQ ID NO: 19, containing 32 CRISPR1 [repetitive sequence-spacer sequence] units. It is flanked by a CRISPR1 leader sequence defined by SEQ ID NO: 17 and a CRISPR1 tail sequence defined by SEQ ID NO: 18.

[0188] In these three preserved DSM strains, the CRISPR3 locus consists of a sequence defined by SEQ ID NO: 73, containing 12 CRISPR3 [repetitive sequence-spacer sequence] units. It is flanked by a CRISPR3 leader sequence defined by SEQ ID NO: 71 and a CRISPR3 tail sequence defined by SEQ ID NO: 72.

[0189] Example 4

[0190] Several CRISPR mutant strains of the preserved strain have been obtained. The CRISPR mutant strains of this invention can be obtained using phage attack (described in detail in patent application WO2008 / 108989).

[0191] Preparation of a fermented milk using the DSM 27029 strain

[0192] The two CRISPR mutant strains of DSM 27029 are examples of several CRISPR mutant strains obtained, and their S1 value, S2 value, and S2 / S1 ratio have been characterized.

[0193] The S1 value of the first DSM 27029 CRISPR mutant strain (DSM 27029 M1) is 104.10. -4 UpH / min, S2 value is 18.10 -4 UpH / min, S2 / S1 ratio was 17%. The S1 value of the second DSM 27029 CRISPR mutant (DSM 27029 M2) was 112.10. -4 UpH / min, S2 value is 23.10 -4 UpH / min, S2 / S1 ratio is 20%.

[0194] These data confirm that the CRISPR mutant strain follows atypical lactation kinetics (i.e., an S1 value of at least 70.10). - 4 UpH / min, S2 value less than 22.10 -4 UpH / min, and / or the S2 / S1 ratio is less than 25%. These data also confirm that the different CRISPR loci, individually or in combination, as defined herein, guide those skilled in the art to identify the thermophilic streptococcal strains of the present invention.

[0195] Preparation of a fresh fermented milk

[0196] Preparation of a milk base

[0197] Inoculation

[0198] Fermented milk was prepared under the following experimental conditions:

[0199] Fermentation Add 3% skim milk powder to commercially available semi-fat UHT milk (1.5% w / w) and heat at 90°C for 10 minutes. Add 1 g / 100 L (w / v) sodium formate solution to the mixture and inoculate immediately.

[0200] Fresh fermented milk characteristic evaluation method The strains stored at -80℃ were inoculated into milk-based medium. A combination of strain DSM 27029, a second thermophilic streptococcal strain DGCC2057, and strain DGCC10697 belonging to the Lactobacillus delbrueckii subsp. bulgaricus were used. The inoculation rate was adjusted to 8.10 g / ml of milk-based medium. 5 CFU, 2.10 5 CFU, 1.10 4 CFU. Using the starter YOMIX. TM 465 LYO was used as a reference and inoculated at a commercial dose of 20 DCU / 100L.

[0201] Results and discussion The incubation temperature was set at 43℃ ± 1℃, and a constant incubation temperature was maintained using a water bath during fermentation. pH changes were measured online using a Cinac system (CINAC, an automated system for controlling lactic acid fermentation agents; Corrieu G, Picque D, Perret B, Quemener P; Process Magazine; 1992; no. 1068; pp. 24-27). At a pH of 4.60 ± 0.1, the fermentation product was cooled to 6℃ and then stored at 6℃. The time it took for the pH to reach 4.60 (at which point the product was cooled from 43℃ to 6℃) was calculated.

[0202] Viscosity (Pa.s) .

[0203] Fresh fermented milk can be described by its texture and flavor properties. Its characteristics were evaluated after storing fresh fermented milk at 6°C for 14 days. Rheological tools were used to evaluate the texture of the fermented milk. The viscosity of the fermented milk was measured using a Brookfield Engineering laboratories, Inc. viscometer equipped with a lifting stand. The following steps were then performed.

[0204] - Install a C-shaped T-shaped rotor on the viscometer.

[0205] Fill a 125mL glass yogurt jar with fermented milk.

[0206] Insert the viscometer into the yogurt container filled with fermented milk.

[0207] - Apply a rotation speed of 10 rpm to the viscometer, then

[0208] - After rotating for 30 seconds, read the viscosity value of the sample.

[0209] In addition, the extensibility of fresh fermented milk was assessed using a capillary fracture stretch rheometer (HAAKE CaBER 1, purchased from Thermo Electron Corp.). The protocol for evaluating fracture time is briefly described below.

[0210] - Heat the fermented milk sample to 20°C.

[0211] - Stir the fermented emulsion 20 times using a standard plastic spoon to break it up and homogenize it.

[0212] - Place 60 μL of homogenized fermented milk between the two plates of a capillary rupture tensile rheometer (HAAKE CaBER 1, purchased from Thermoelectric Corp.). The rheometer was set as follows: plate diameter: 6 mm; initial height: 2 mm; final height: 9.65 mm; impact speed: 0.24 mm / ms (impact time: 40 ms); laser micrometer: Class 1 infrared laser, 10 μm resolution.

[0213] - Measure the relative fracture time of the sample.

[0214] Three professional evaluators were invited to taste the samples. They were asked to evaluate the products under blind testing conditions (only the fermented milk was numbered). Six different sensory attributes were evaluated: "breakability," "thickness when scooped with a spoon," "stickiness," "thickness in the mouth," "viscosity in the mouth," and "acidity." Each attribute was scored on a scale of "0" to "4."

[0215] Relative break time (ms)

[0216] To achieve a pH of 4.60, the YOMIX starter culture requires 375 minutes, and the strain blend (containing DSM 27029) requires 437 minutes. Both times are standard technical times for preparing fermented milk at this temperature.

[0217] Blend YOMIX Table 4 65.5 99.3 Brittleness 56.8 46.0

[0218] Consistency with a spoonFreshly fermented milk made from a blend of DSM 27029 / DGCC2057 / DGCC10697 and commercially available culture YOMIX TM Comparative results of fresh fermented milk prepared by YOMIX 465.

[0219] Coagulability Consistency in the mouth Viscosity Acidity Blend YOMIX Table 5 1.0 4.0 3.0 4.0 3.0 0.0 Example 5 3.0 2.5 0.5 2.5 1.0 0.0

[0220] Preparation of a fermented milk using the DSM 27030 strain The sensory attribute scores of fresh fermented milk prepared from the aforementioned blend and YOMIX starter culture.

[0221] The yogurt made with the aforementioned blend exhibited significantly lower breakage than the yogurt made with YOMIX. This characteristic is likely of interest in stirred yogurt production. The yogurt made with the aforementioned blend also showed significantly higher consistency when scooped with a spoon and upon entering the mouth compared to the yogurt made with YOMIX. This result is valuable for stirred yogurt production technology. Our evaluation results clearly demonstrate that combining DSM 27029 with other strains helps yogurt producers create high-quality products that meet the needs of end consumers.

[0222] Therefore, we can conclude that fresh fermented milk producers can use DSM 27029 strain as part of the inoculum to produce yogurt with the characteristics of interest.

[0223] Preparation of a fresh fermented milk

[0224] Fresh fermented milk characteristic evaluation method

[0225] Results and discussion

[0226] Fermented milk was prepared according to the milk base preparation method and fermentation conditions described in Example 4. The bacterial strain stored at -80℃ was inoculated into the milk base medium to complete the inoculation. A combination of strains DSM 27030, the second thermophilic streptococcal strain DGCC2057, and strain DGCC10697 belonging to the Lactobacillus delbrueckii subsp. bulgaricus were used. The inoculation rate was adjusted to 8.10 g / ml of milk base. 5 CFU, 2.10 5 CFU, 1.10 4 CFU. Using the starter YOMIX. TM 465 LYO was used as a reference. Inoculation was performed at a commercial rate of 20 DCU / 100L. 110 mi ± 10 mi of the inoculated milk preparation was placed in a 125 ml yogurt container for fermentation.

[0227] To achieve a pH of 4.60, the YOMIX starter culture requires 375 minutes, and the combined strain requires 445 minutes. Both of these times are standard technical times for preparing fermented milk at this temperature.

[0228] Viscosity (Pa.s) .

[0229] As described in Example 4, determine and / or calculate the viscosity and sensory evaluation (texture and flavor attributes) of the milk.

[0230] Relative break time (ms)

[0231] Blend YOMIX Table 6 62.7 60.0 Brittleness 56.8 46.0

[0232] Consistency with a spoon Freshly fermented milk made from a blend of DSM 27030 / DGCC2057 / DGCC10697 and commercially available culture YOMIX TM Comparison of fresh fermented milk prepared by 465 (YOMIX).

[0233] Coagulability Consistency in the mouth Viscosity Acidity Blend YOMIX Table 7 2.0 3.0 1.5 3.5 1.0 0.0 Example 6 3.0 2.5 0.5 2.5 1.0 0.0

[0234] Preparation of a fermented milk using the DSM 27031 strain The scoring of several sensory properties of fresh fermented milk made from blends and YOMIX starter culture.

[0235] Yogurt made using the blend exhibited significantly lower breakage than yogurt made using YOMIX. This characteristic is noteworthy for stirred yogurt production. Yogurt made using the blend also showed significantly higher consistency in the mouth compared to yogurt made using YOMIX. This result is significant for stirred yogurt technology. The results clearly demonstrate that combining strain DSM 27030 with other strains can help yogurt producers deliver products with very high texture quality to end consumers.

[0236] Therefore, it can be concluded that using DSM 27030 as part of the inoculum provides fresh fermented milk producers with yogurts possessing the characteristics of interest.

[0237] Preparation of a fresh fermented milk

[0238] Fresh fermented milk characteristic evaluation method

[0239] Fresh fermented milk characteristics

[0240] Fermented milk was prepared according to the milk base preparation method and fermentation conditions described in Example 4. The bacterial strain stored at -80℃ was inoculated into the milk base medium to complete the inoculation. Strain DSM 27031 was combined with Streptococcus thermophilus strain DGCC2057 and Lactobacillus bulgaricus subspecies DGCC10697. The inoculation rate was adjusted to 7.10 g / ml milk base. 5 CFU, 3.10 5 CFU and 1.10 4 CFU. Using the starter YOMIX. TM 465 LYO was used as a reference. The starter culture was inoculated at a commercial dose of 20 DCU / 100L. Fermentation was carried out in a 125 mL yogurt container containing 110 mL + / - 10 mL of the inoculated milk preparation.

[0241] To reach a pH of 4.60, the YOMIX starter culture requires 375 minutes, and the bacterial composition requires 461 minutes. Both of these times are standard technical times for preparing fermented milk at this temperature.

[0242] Viscosity (Pa.s) .

[0243] As described in Example 4, determine and / or calculate the viscosity and sensory evaluation (texture and flavor attributes) of the milk.

[0244] Relative break time (ms)

[0245] Blend YOMIX Table 8 65.2 68.0 Brittleness 56.8 46.0

[0246] Consistency with a spoon Fresh fermented milk made from a blend of DSM 27031 / DGCC2057 / DGCC10697 and commercially available culture YOMIX TM Comparison of fresh fermented milk prepared by 465 (YOMIX).

[0247] Coagulability Consistency in the mouth Viscosity Acidity Blend YOMIX Table 9 3.0 3.5 1.5 3.5 1.0 0.0 Example 7 3.0 2.5 0.5 2.5 1.0 0.0

[0248] Preparation of a fermented milk using the DSM 27031 strain and comparison with fermented milks prepared with other S. thermophilus strains The scoring of several sensory properties of fresh fermented milk made from blends and YOMIX starter culture.

[0249] Yogurt made with the blend exhibited significantly higher consistency when scooped with a spoon and in the mouth compared to yogurt made with YOMIX. This result is significant for stirred yogurt technology. The results clearly demonstrate that combining strain DSM27031 with other strains can help yogurt producers deliver products with very high texture quality to end consumers.

[0250] Therefore, we can conclude that fresh fermented milk producers can use DSM 27031 strain as part of the inoculum to produce yogurt with the characteristics of interest.

[0251] Preparation of a fermented milk

[0252] Table 10 Fresh fermented milk characteristic evaluation method

[0253] pH variation during storage

[0254] Fermented milk was prepared according to the description of milk base preparation and fermentation conditions in Example 4. Two mixed cultures were prepared: Formula A contained *Streptococcus thermophilus* strains DGCC8897 and DSM 27031, and *Lactobacillus delbrueckii* subsp. bulgaricus strain DGCC10697; Formula B contained *Streptococcus thermophilus* strains DGCC8897 and DGCC7891, and *Lactobacillus delbrueckii* subsp. bulgaricus strain DGCC10697. Inoculation was performed using strains preserved in milk base medium at -80°C. The inoculation rates of mixed cultures A and B, and the S1 and S2 values ​​of each *Streptococcus thermophilus* strain used, are reported in Table 10. In Formula B, strain DGCC7891 was selected as a reference example because its S2 / S1 ratio (39%) was higher than that of strain DSM 27031 (16%).

[0255]

[0256] Results and discussion Inoculation rate (CFU / mL) of the mixed culture named after formulation A and formulation B, and the S1, S2, and S2 / S1 values ​​of the Streptococcus thermophilus strains used.

[0257] Fermentation was carried out in a 125 mL yogurt container containing 110 mL + / - 10 mL of inoculum milk preparation.

[0258] To achieve a pH of 4.60, Formula B requires 300 minutes, and Formula A requires 440 minutes. Both of these times are standard technical times for preparing fermented milk at this temperature.

[0259] Table 11 .

[0260] As described in Example 4, determine and / or calculate the viscosity and sensory evaluation (texture and flavor attributes) of the milk.

[0261] Brittleness

[0262] At pH 4.60 + / - 0.1, the fermentation product was cooled to 6°C and then stored at 6°C for 50 days. The pH values ​​of the fermented milk prepared from formula A or formula B and stored at 6°C were measured after 14 days, 28 days, and 50 days.

[0263] Consistency with a spoon

[0264] The characteristics of fresh fermented milk obtained by co-inoculation with different strains (formula A or B) are reported in Tables 11 and 12.

[0265]

[0266] Coagulability Comparison of fresh fermented milk prepared from formula A and formula B.

[0267] Consistency in the mouth Viscosity Acidity Formulation A Formulation B Table 12 Example 8 4 2 0 1 0 0 Preparation of a fermented milk using the DSM 27029 strain and comparison with fermented milks prepared with S. thermophilus strains 3 2 0.5 1 0.5 0

[0268] Preparation of a fermented milk using the DSM 27029 strain The sensory attributes of fresh fermented milk prepared from formula A or formula B are rated.

[0269] "Adhesion" and "thickness in the mouth" are less important for formulation A prepared by DSM 27031 than for formulation B. This result is significant for stirred yogurt technology.

[0270] The results clearly demonstrate that using strain DSM 27031 in combination with other strains can help yogurt producers provide end consumers with products that have high texture quality and a very mild taste.

[0271] Table 13

[0272] pH variation during storage Results and discussion

[0273] Table 14

[0274] Fermented milk was prepared according to the description of milk base preparation and fermentation conditions in Example 4. Two mixed cultures were prepared: Formula C contained *Streptococcus thermophilus* strains DGCC938 and DSM 27029, and *Lactobacillus delbrueckii* subsp. bulgaricus strain DGCC10697; Formula D contained *Streptococcus thermophilus* strains DGCC8897 and DGCC938, and *Lactobacillus delbrueckii* subsp. bulgaricus strain DGCC10697. Inoculation was performed using strains preserved in milk base medium at -80°C. The inoculation rates of mixed cultures C and D, and the S1 and S2 values ​​for each *Streptococcus thermophilus* strain used, are reported in Table 13.

[0275]

[0276] Brittleness Inoculation rate of the mixed cultures named after formulations C and D, and the S1, S2, and S2 / S1 values ​​of the Streptococcus thermophilus strains used.

[0277] Fermentation was carried out in a 125 mL yogurt container containing 110 mL + / - 10 mL of inoculum milk preparation.

[0278] To achieve a pH of 4.60 (at which point the product is cooled from 43°C to 6°C), the dairy product inoculated with Formula C requires 370 minutes of fermentation, while Formula D requires 389 minutes. Both of these times are standard technical times for preparing fermented milk at these temperatures.

[0279] Evaluation method for characteristics of fresh fermented milk.

[0280] As described in Example 4, determine and / or calculate the viscosity and sensory evaluation (texture and flavor attributes) of the milk.

[0281] Consistency with a spoon

[0282] The pH values ​​of the fermented milk obtained from formula C or formula D and stored at 6°C were measured after 14, 28, and 50 days.

[0283] Coagulability

[0284] The characteristics of fresh fermented milk obtained by co-inoculation with different strains (formula C or D) are reported in Tables 14 and 15.

[0285]

[0286] Consistency in the mouth Comparison of fresh fermented milk prepared from formula C and formula D.

[0287] As can be seen, the viscosity of fresh fermented milk C is significantly higher than that of fresh fermented milk D, which means that yogurt has the potential for a greater degree of overall texture variation.

[0288] Acidity Formulation C Formulation D Table 15 ​ ​ 2.5 4 1.5 4 0 ​ 4 1 0 1 0

[0289] ​ The scores of several sensory attributes of fresh fermented milk prepared from formula C or formula D.

[0290] The consistency when scooped with a spoon and the consistency in the mouth are more important for formula C prepared with DSM 27029 than for formula D. This result is significant for stirred yogurt technology. The results clearly demonstrate that using strain DSM 27029 in combination with other strains can help yogurt producers provide end consumers with products that have high texture quality and a very mild taste.

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

Claims

1. A Streptococcus thermophilus strain selected from the group consisting of: (1) the DSM 27029 strain, which was deposited on 21 March 2013 with the Leibniz-Institut DSMZ - German Collection of Microorganisms and Cell Cultures under the Budapest Treaty on behalf of Danisco A / S, Denmark; (2) the DSM 27030 strain, which was deposited on 21 March 2013 with the Leibniz-Institut DSMZ - German Collection of Microorganisms and Cell Cultures under the Budapest Treaty on behalf of Danisco A / S, Denmark; and (3) the DSM 27031 strain, which was deposited on 21 March 2013 with the Leibniz-Institut DSMZ - German Collection of Microorganisms and Cell Cultures under the Budapest Treaty on behalf of Danisco A / S, Denmark.

2. A composition comprising or consisting of a culture of a Streptococcus thermophilus strain according to claim 1.

3. The composition according to claim 2, further comprising at least one other microorganism.

4. The composition according to claim 3, wherein the at least one other microorganism is at least one lactic acid bacterium and / or at least one propionic acid bacterium.

5. The composition according to claim 4, wherein the at least one lactic acid bacterium is a different strain of the species Streptococcus thermophilus, and / or a strain of the subspecies Lactobacillus delbrueckii subsp. bulgaricus, and / or a strain of the genus Bifidobacterium, and / or any combination of these strains.

6. The composition according to any one of claims 2-5, further comprising one or more components that can be added to food.

7. The composition according to claim 6, wherein the one or more components that can be added to food are cryoprotectants, potentiators and / or common additives.

8. The composition according to any one of claims 2 to 5, which is in the form of a liquid, a frozen or a dry powder.

9. Use of a culture of a strain as defined in claim 1 or a composition as defined in any one of claims 2 to 8 for the preparation of a product.

10. The use according to claim 9, wherein the product is a food or feed product.

11. The use according to claim 9, wherein the product is a fermented product.

12. The use according to claim 11, wherein the product is a fermented food or a fermented feed product.

13. A method for preparing a product, wherein the method comprises contacting a substrate with a culture of a strain as defined in claim 1 or a composition as defined in any one of claims 2 to 8, or contacting a substrate in the presence of a culture of a strain as defined in claim 1 or a composition as defined in any one of claims 2 to 8; fermenting the substrate; and then obtaining the product.

14. The method of claim 13, wherein the product is a food or feed product.

15. The method of claim 13, wherein the product is a fermented product.

16. The method of claim 15, wherein the product is a fermented food or fermented feed product.

17. The method of claim 13, wherein the substrate is a milk substrate.

18. A product obtained using the method of claim 13.

19. The product of claim 18, wherein the product is a food or feed product.

20. The product of claim 18, wherein the product is a fermented product.

21. The product of claim 20, wherein the product is a fermented food or fermented feed product.

22. A product comprising a culture of a strain as defined in claim 1 or a composition as defined in any one of claims 2 to 8.

23. The product of claim 22, wherein the product is a food or feed product.

24. The product of claim 22, wherein the product is a fermented product.

25. The product of claim 24, wherein the product is a fermented food or fermented feed product.

26. The product of claim 18 or 22, which is a dairy product.

27. The product of claim 26, which is a cheese or milk.

28. The product of claim 26, which is a yogurt, a buttermilk, a curd cheese, a sour cream, a quark, a fermented whey beverage, a kefir, a milk beverage, a yogurt beverage, a fermented milk, a ripened cream, a cottage cheese, a ripened cheese, a processed cheese, a white soft cheese, a cream dessert, or an infant milk.

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