# Patent

## Title
Genetic cluster of strains of Streptococcus thermophilus having appropriate acidifying and texturizing properties for dairy fermentations

## Abstract

The invention relates to a genetic cluster of strains of Streptococcus thermophilus, which have a lysotype distinct from that of the strains currently used. Within this cluster novel acidifying and texturizing strains have been identified.

## Background
The present application is filed pursuant to 35 U.S.C. 371 as a U.S. National Phase application of International Patent Application No. pct/ep07/60463, which was filed Oct. 2, 2007, claiming the benefit of priority to French Patent Application No. 0608657, which was filed on Oct. 3, 2006. The entire text of the aforementioned applications is incorporated herein by reference in its entirety.
The invention relates to a genetic cluster of strains of Streptococcus thermophilus (S. thermophilus) having appropriate acidifying and texturizing properties for dairy fermentations.
Bacteriophages are viruses capable of attacking bacteria. During se viral attacks, the bacteriophages infect the bacterial culture, and multiply in order to finally destroy this culture. The technological impact on the milk processing industry (production of cheese, yoghurt, fermented milk) of these bacteriophages is significant since they can cause a complete cessation of fermentation and therefore prevent the production of these milk-derived products.
One way of combating the problems linked to the infection of the fermentations by bacteriophages is the use of strains having appropriate sensitivity spectra to the phages. In particular, producers of dairy ferments have developed strategies to combat the bacteriophages by constructing ferments constituted by several strains having distinct lysotypes, and using several of these ferments in rotation. It is clear that in order to be able to adopt this strategy it is important to possess a diversity of strains having the same functionalities (such as for example acidification, thickening power, flavouring etc.) but distinct lysotypes.
S. thermophilus are used extensively alone or in combination with other bacteria for the production of fermented food products. They are included in particular in the formulation of the ferments used for producing yoghurts. Strains of S. thermophilus are expected to participate in the formation of lactic curd by acidification of milk and in the development of the texture of the fermented product. A distinction generally drawn between 4 groups of S. thermophilus based on these functional properties: 1) non-texturizing and non-acidifying strains, 2) the non-texturizing and acidifying strains, 3) the texturizing and non-acidifying strains, and 4) the texturizing and acidify strains. A texturizing strain is a strain making, it possible to obtain fermented milks the gels of which can be described by their rheological properties.
Hitherto only four strains of S. thermophilus corresponding criteria of acidifying and texturizing strains have been described in the literature: Sfi39, CNCM I-2423, CNCM I-2426 and the strain CNCM I-2980 described in the Application WO2004/085607.
The rarity of such strains (rapid acidification and texturizing) makes it difficult to combat the bacteriophages during the fermentation of the milk. In fact, ideally, the fight against the bacteriophages would involve the combination in the same ferment of strains having similar technological properties but distinct lysotypes, then the use of ferments of this type in rotation. The ferments used in rotation should also have distinct lysotypes but similar technological properties. In the case of the texturizing and rapidly acidifying ferments, this approach is difficult in view of the small number of strains having these functional qualities.
Thus one of the problems which the invention proposes to resolve is to provide novel strains of S. thermophilus which have a lysotype distinct from the strains currently used, in particular strains which are acidifying and texturizing.
For this purpose, the invention relates to the strains of S. thermophilus in a genetic cluster which have a lysotype distinct from that of the acidifying and texturizing strains of S. thermophilus currently used. Within this cluster novel acidifying and texturizing strains have been identified.
The invention also describes a method making it possible to predict a strain's membership of a family of strains having identical or related lysotypes. This method analyzes the restriction polymorphism of the epsA-B-C-D region of the genome of S. thermophilus. 
By the epsA-B-C-D region is meant the region of the chromosome of S. thermophilus overlapping the epsA to epsD genes of the eps locus. The DNA fragment corresponding to this region, called the epsAD fragment, can be obtained by PCR reaction on the chromosomic DNA or S. thermophilus oligonucleotides of SEQ ID No1 and SEQ ID No2 as primers.
A subject of the present invention is a strain of Streptococcus thermophilus the epsAD fragment of which, after digestion by the restriction enzymes Mn/I, FokI and HindIII, has a restriction profile characterized by DNA fragments of 344±2 base pairs (bp), 341±2 bp, 305±2 bp, 299±2 bp, 277±2 bp, 210±2 bp, 160±2 bp, 142±2 bp, 100±2 bp, 79±2 bp, 75±2 bp, 66±2 bp, 42±2 bp, 23±2 bp and 9±2 bp.
The restriction profile of the epsAD fragment is determined by standard sequencing of the epsAD fragment followed by in silico determination of the restriction profile.
Typically the sequencing can be carried out with the CEQ8000 equipment (Beckman) and the in silico determination of the restriction profile can be carried out starting from the sequence of the epsAD fragment using the NEBcutter V2.0 tool accessible on the internet.
Typically a strain according to the invention comprises a nucleotide sequence having at least 80%, preferentially at least 90%, at least 95% or at least 97% and still more preferentially 100% identity with the nucleotide sequence of SEQ ID No4.
Typically a strain according to the invention comprises a nucleotide sequence having at least 80%, preferentially at least 90%, at least 95% or at least 97% and still more preferentially 100% identity with the nucleotide sequence of SEQ ID No5.
In order to calculate the percentage identity, a person skilled in the art will for example use the “BLAST 2 Sequences” tool (Tatusova & Madden, 1999) with the default parameters of the “blastn” program (for alignment of nucleotide sequences) or of the “blastp” program (for alignment of protein sequences). The percentage similarity between two protein sequences is calculated using the BLOSUM62 matrix.
Preferentially the strain according to the invention is texturizing. Preferentially the strain according to the invention acidifies rapidly. Still more preferentially the strain according to the invention is texturizing and acidifies rapidly.
By texturizing strain of Streptococcus thermophilus is meant a strain which produces fermented milks having, under the conditions described in the example, a viscosity greater than approximately 35 Pa·s, a thixotropic area of less than approximately 2000 Pa/s and/or a yield point of less than approximately 14 Pa.
By rapidly acidifying strain is meant a strain which under the conditions described in the example, a Vm of less than −0.0100 upH/min.
Preferentially, a strain according to the invention is the strain of Streptococcus thermophilus deposited on 14 Jun. 2006 at the Collection Nationale de Cultures de Microorganismes under no. CNCM I-3617 or a mutant strain which can be obtained from the latter.
Typically in order to obtain such mutant strains, a person skilled in the art can use the usual mutagenesis techniques such as UV irradiation or exposure to mutagenic chemical products (ethyl-methane-sulphonate, nitrosoguanidine, nitrous acid etc.).
Preferentially, a subject of the invention is the strain of Streptococcus thermophilus deposited on 14 Jun. 2006 in the name of Danisco France SAS, 20 rue de Brunel, 75017 Paris at the Collection Nationale de Cultures de Microorganismes under no. CNCM I-3617.
A person skilled in the art, starting from the restriction profiles described previously and/or from the sequences of SEQ ID No4 and/or No5, can identify the strains which belong to the same genetic cluster the strain CNCM I-3617. Typically in order to do this, he can use PCR and/or hybridization and/or DNA sequencing techniques.
A subject of the invention is also a bacterial composition comprising at least one strain according to the invention. By bacterial composition is meant a mixture of different strains, in particular a ferment, or a leaven.
The mixtures of preferred strains according to the invention are mixtures of Streptococcus thermophilus with other Streptococcus thermophilus, or mixtures of Streptococcus thermophilus with Lactobacillus delbrueckii subsp. bulgaricus, or mixtures of Streptococcus thermophilus with other Lactobacillus and/or with Bifidobacterium, or mixtures of Streptococcus thermophilus with Lactococcus, or mixtures of Streptococcus thermophilus with other strains of lactic bacteria and/or yeasts.
A subject of the invention is also a manufacturing process for a food product, a food complement, a dietary supplement or a product with probiotic properties, comprising a stage in which a strain according to the invention is used.
Typically the food product, the food complement, the dietary supplement or the product with probiotic properties is a dairy product, a meat product, a cereal product, a drink, a foam or a powder.
Preferentially the food product, the food complement, the dietary supplement or the product with probiotic properties is a dairy product. It is for example a fermented milk, a yoghurt, a matured cream, a cheese, a fromage frais, a milk drink, a dairy product retentate, a processed cheese, a cream dessert, a cottage cheese or an infant milk. Typically the dairy product comprises milk of animal and/or plant origin.
A subject of the invention is also a food product, a food complement, a dietary supplement or a product with probiotic properties comprising at least one strain according to the invention or the bacterial composition described previously.
The invention also describes a method for predicting the lysotype of a strain of S. thermophilus starting from analysis of the restriction polymorphism of the epsA-B-C-D region of its genome, comprising the following stages:
a) amplification of the epsAD fragment by PCR reaction on the chromosomic DNA of S. thermophilus using the oligonucleotides of SEQ ID No1 and SEQ ID No2 as primers;
b) sequencing of the epsAD fragment;
c) in silico determination of the restriction profile of the epsAD fragment after digestion by the restriction enzymes Mn/I, FokI and HindIII; and
d) comparison of the restriction profile obtained in Stage c) with the restriction profiles of the epsAD region of strains of S. thermophilus the lysotype which is known.
Examples of strains of Streptococcus thermophilus the lysotype of which is known are listed in Table 3.
The present invention is better illustrated below using the examples which follow. These examples are given only by way of illustration of the subject-matter of the invention, of which they in no way constitute a limitation.



BRIEF DESCRIPTIONS OF THE FIGURES
FIG. 1: Diagrammatic representation of the eps locus of various Streptococcus thermophilus having similarities in organization and sequence of the 5′ region including the epsA, epsB, epsC and epsD genes. The GENBANK access numbers of the nucleotide sequences are given in parentheses.
FIG. 2: Alignment of partial sequences of the epsA (A) and epsD (B)genes at the level f the regions targeted by the primers of SEQ ID NO:1 (in the epsA gene) and SEQ ID NO:2 (in the epsD gene). The sequences in the Figure are as follows SEQ ID NO:6 MTC310; SEQ ID NO: 7 Sfi39; SEQ ID NO:8 TypeI; SEQ ID NO:9 F19186; SEQ ID NO:10 TypeX; SEQ ID NO:11 TypeVI; SEQ ID NO:12 TypeV; SEQ ID NO:13 TypeIX; SEQ ID NO:14 NCDB2393; SEQ ID NO:15 LMG18311; SEQ ID NO:16 IP6757; SEQ ID NO:17 LMD-9; SEQ ID NO:18 TypeIII; SEQ ID NO:19 CNCM 12980; SEQ ID NO:20 TypeVII; SEQ ID NO:21 Type IV; SEQ ID NO:22 Type XI; SEQ ID NO:23 MR-1C; SEQ ID NO:24 CNRZ368; SEQ ID NO:25 CNRZ1065; SEQ ID NO:26 MTC360; SEQ ID NO:27 MTC330; SEQ ID NO:28 MR-2C; SEQ ID NO:29 Sfi6; SEQ ID NO:30 MTC310; SEQ ID NO: 31 Sfi39; SEQ ID NO:32 TypeI; SEQ ID NO:33 F19186; SEQ ID NO:34 TypeX; SEQ ID NO:35 TypeVI; SEQ ID NO:36 TypeV; SEQ ID NO:37 TypeIX; SEQ ID NO:38 NCDB2393; SEQ ID NO:39 LMG18311; SEQ ID NO:40 IP6757; SEQ ID NO:41 LMD-9; SEQ ID NO:42 TypeIII; SEQ ID NO:43 CNCM 12980; SEQ ID NO:44 TypeVII; SEQ ID NO:45 Type IV; SEQ ID NO:46 Type XI; SEQ ID NO:47 MR-1C; SEQ ID NO:48 CNRZ368; SEQ ID NO:49 CNRZ1065; SEQ ID NO:50 MTC360; SEQ ID NO:51 MTC330; SEQ ID NO:52 MR-2C; SEQ ID NO:53 Sfi6; SEQ ID NO:54 ST69 (AJ488593); SEQ ID NO:55 A2 (AJ488600); SEQ ID NO:56 D1 (AJ488599); SEQ ID NO:57 E1 (AJ488598); SEQ ID NO:58 I1 (AJ488597); SEQ ID NO:59 I1 (AJ488596); SEQ ID NO:60 N1 (AJ488595); and SEQ ID NO:61 Q1 (AJ488594).
FIG. 3: In silico determination of the profiles obtained according to the epsAD method of the various strains of S. thermophilic the sequence of the eps locus of which is described in the literature. The images of the restriction profiles were generated using the NEBcutter tool with the following parameters: Gel Type=2% agarose; Marker=100 bp DNA Ladder; DNA Type=Unmethylated; L=102 mm. The GENBANK access numbers of these strains are: AF448502 (MTC310), AF373595 (Sfi39), AF410175 (Type I), AJ289861 (IP6757), AF454496 (Type V), YI7900 (NCFB 2393), AJ272341 (FI9186), AF454497 (Type VI), NZ_AAGS01000017 (LMD-9), AF454501 (Type XI), AF454498 (Type VII), AF454495 (Type IV), AF454500 (Type X), AF454499 (Type IX), AY057915 (Type III), CP000023 (LMG 18311), CF000024 (CNRZ 1066), AF434993 (MTC360), AF430847 (MTC330), AY061649 (MR-2C), U40830 (Sfib). Z98171 (CNRZ 368), AF448249 (MR-TC). The eps sequence of the strain CNCM I-2980 is, described in the Application WO2004/085607.
FIG. 4: Diagrammatic representation of the organization of the eps locus of the strain CNCM I-3617, corresponding to the sequence SEQ ID No3. The open reading frames (ORF) represented by vertical hatching (region 1) have significant similarities with the epsA-epsB-epsC-epsD genes situated at the start of the eps locus in the great majority of the strains of S. thermophilus. The ORFs represented by horizontal hatching (region 2) have significant similarities with the eps11E, eps11F, eps11G; and eps11H genes of the type XI strain. The ORFs represented by a chequered pattern (region 4) have significant similarities with the eps4F gene of the type IV strain. The grey rectangle between regions 2 and 3 represents a non-coding region having sequence homologies with a region of the eps locus of the type IV strain. The ORFs represented by diagonal hatching (region 6) have significant similarities with the epsP, epsQ, epsS, epsR and epsT genes of the CNRZ368 strain. The unshaded ORFs (regions 3 and 5) have no significant similarities with the eps genes described for other S. thermophilus. The two black bands at the bottom of the figure represent the position of the sequences SEQ ID No4 and SEQ ID No5.



EXAMPLES
Biological Material
Table 1 and Table 3 show some of the strains used for the study. Some of these strains are obtained from the Danisco collection of strains and phages (DGCC: Danisco Global Culture Collection). The preparation of cultures of these strains was carried out according to the standard methods of microbiology.







TABLE 1







Description of some of the strains used for the study













Other
Texturizing
Acidifying

Bibliographical


Strain
name
propertya
propertya
Related strainsb
referencesc





CNCM I-3617

Yes
Yes
Unknown
None


CNCM I-2980

Yes
Yes
DGCC2056
WO2004/085607






DGCC8013,






DGCC8015


CNCM I-2423
MTC310
Yes
Yes
Sfi39, SY102
Lemoine et al. (1997);






FI9186, Type I
Germond et al. (2001);






DGCC945
Marshall et al. (2001)


CNCM I-2978
MTC360
Yes
No
Sfi6, CNCM I-
Lemoine et al. (1997);






733, CNCM I-
Doco et al. (1990);






734, CNCM I-
Marshall et al. (2001);






735, IMDO1, 2,
Stingele et al. (1996)






3, NCFB859,






EU21, MR-2C,






DGCC7773


CNCM I-2423
Type IV
Yes
No
Unknown
Rallu et al. (2002)


CNCM I-2429
Type VII
Yes
No
Unknown
Rallu et al. (2002)


CNCM I-2979

Yes
No
CNRZ368 MR-
Bourgoin et al. (1999);






1C
Low et al. (1998)


DGCC7966

Yes
No
Unknown
None


DGCC7919

No
Yes
Unknown
None


DGCC7766

No
No
Unknown
None





aon the basis of industrial use and the present study.


bon the basis of published sequence results of the eps locus and/or the structure of the polysaccharide and data internal to Danisco.


con the strain studied and/or on related strains.






The Strain CNC 617 Belongs to a Novel Genetic Cluster

The recent determination of the complete sequence of the chromosome of two strains of S. thermophilus CNRZ1066 and LMG18311 shows a high level of conservation of the genetic content and the organization of the genes in this species) Bolotin et al., 2004). One of the rare regions exhibiting major genetic differences between these two strains corresponds to the eps locus which codes for the genes involved in the biosynthesis of exopolysaccharides. Moreover, a great diversity has already been described at the level of this genetic locus (cf. FIG. 1) since several eps sequences had been determined for various strains of S. thermophilus (for a journal article, see Broadbent et al., 2003). Flanked by the deoD (coding for a purine-nucleoside phosphorylase) and pgm (coding for a phospho-glucomutase) genes, all the clusters of eps genes in S. thermophilus are composed of a conserved proximal region (from the epsA gene to the epsD gene) and of a highly variable distal region. In spite of a conserved organization of the epsA-B-C-D genes, a significant sequence polymorphism exists in this region. The polymorphism of this region has been used to develop a tool for genetic typing of the strains of S. thermophilus: the epsAD method.
epsAD method: The tool developed is based on the specific amplification (PCR) of the epsA-B-C-D region followed by the analysis of its restriction polymorphism (RFLP). For this purpose primers have been determined which allow the PCR amplification of this region for the great majority of the strains of S. thermophilus. They have been determined by the alignment of the sequences of the epsAD region (cf. FIG. 2) and allow the amplification of a DNA fragment of approximately 2480 base pairs (bp). The genomic DNA of S. thermophilus is purified using the “DNeasy Tissue Kit” (Qiagen), then the epsAD region is amplified by PCR according to the following parameters:
Composition of the reaction mixture (50 μL): buffer for the DNA polymerase ×1, MgCl2 2 mM, dNTP 200 μM each, genomic DNA 100 to 500 ng, primer EPSA632 (5′-AAATgAATTCAgAgCAAgCACTTg-3′ (SEQ ID No1)) 200 nM, primer EPSD1064 (5′-gTCATgTCAACTTTATTAAggACg-3′ (SEQ ID No2)) 200 nM, DNA polymerase 1.25 units, H2O qsf 50 μL.
Amplification Parameters:


    
    
        predenaturation at 94° C. for 1 min
        35 cycles alternating denaturation at 94° C. for 30 s, hybridization at 56° C. for 30 s, elongation at 72° C. for 3 min
        post-elongation at 72° C. for 6 min.
    
    


After amplification, the PCR product is checked by electrophoresis on 1.5% agarose gel. The size of the amplified product is approximately 2480 bp.
The sequence of the PCR fragment is determined (according to a method derived from Sanger et al., 1977) with CEQ8000 equipment (Beckman). The sequence is processed by the NEBcutter tool by selecting the restriction enzymes Mn/I, FokI and HindIII in order to establish its restriction profile in silico, and in particular in order to define the size of the restriction fragments.
For the strains of S. thermophilus the sequence of the eps locus of which is partially or completely available in the public databases (GENBANK for example), the in silico analysis of the theoretical restriction products of the PCR fragment of approximately 2480 bp with the restriction enzymes Mn/I, FokI and HindIII produces the restriction profiles shown in FIG. 3. These restriction profiles were established in silico on the basis of the sizes of the restriction fragments provided by the NEBcutter tool (cf. Table 2).







TABLE 2







Size of the DNA fragments determined by in silico digestion of the


epsAD fragment by the restriction enzymes MnlI, FokI and HindIII, for the strains


of S. thermophilus the sequence of the epsA-B-C-D genes of which is available.










GENBANK access




number of the eps
Size of the MnlI, FokI and HindIII digestion fragments


Strain of
locus, or other
of the epsAD region (size in base pairs determined by


S. thermophilus
reference
NEBcutter





MTC310
AF448502
778; 371; 344; 299; 175; 142; 100; 79; 75; 42; 35; 23; 9


Sfi39
AF373595
778; 371; 344; 299; 175; 142; 100; 79; 75; 42; 35; 23; 9


Type I
AF410175
778; 371; 344; 299; 175; 142; 100; 79; 75; 42; 35; 23; 9


IP6757
AJ289861
778; 371; 299; 247; 239; 175; 100; 76; 75; 42; 35; 23;




9; 3


Type V
AF454496
1264; 374; 305; 210; 123; 76; 66; 42; 9; 3


NCFB2393
Y17900
1264; 374; 305; 210; 123; 76; 66; 42; 9; 3


FI9186
AJ272341
1264; 374; 305; 210; 123; 76; 66; 42; 9; 3


Type VI
AF454497
1286; 305; 277; 210; 100; 76; 75; 66; 42; 23; 9; 3


LMD-9
NZ_AAGS01000017
848; 655; 371; 175; 111; 100; 75; 60; 42; 23; 9; 3


Type XI
AF454501
851; 356; 305; 299; 175; 111; 100; 75; 60; 46; 42; 23;




20; 9; 3


CNCM I-3617
SEQ ID No3
344; 341; 305; 299; 277; 210; 160; 142; 100; 79; 75;




66; 42; 23; 9


Type VII
AF454498
486; 341; 305; 299; 277; 210; 160; 100; 76; 75; 66;




42; 23; 9; 3


Type IV
AF454495
618; 551; 305; 299; 210; 100; 95; 79; 75; 46; 42; 23;




20; 9


Type X
AF454500
571; 356; 305; 299; 277; 210; 100; 79; 75; 66; 60; 42;




23; 9


Type IX
AF454499
987; 305; 299; 277; 210; 100; 79; 75; 66; 42; 23; 9


Type III
AY057915
341; 305; 299; 277; 247; 239; 210; 160; 100; 76; 75;




66; 42; 23; 9; 3


CNCM I-2980
WO2004/085607
778; 374; 305; 247; 239; 210; 123; 76; 66; 42; 9; 3


LMG18311
CP000023
778; 371; 299; 247; 239; 210; 100; 76; 75; 42; 23; 9; 3


CNRZ1066
CP000024
778; 371; 299; 247; 239; 175; 100; 76; 75; 42; 35; 23;




9; 3


MTC360
AF434993
778; 371; 299; 247; 239; 175; 100; 76; 75; 42; 35; 23;




9; 3


MTC330
AF430847
778; 371; 299; 247; 239; 175; 100; 76; 75; 42; 35; 23;




9; 3


MR-2C
AY061649
778; 371; 299; 247; 239; 175; 100; 76; 75; 42; 35; 23;




9; 3


Sfi6
U40830
778; 371; 299; 247; 239; 175; 100; 76; 75; 42; 35; 23;




9; 3


CNRZ368
Z98171
691; 371; 312; 305; 239; 112; 100; 76; 75; 63; 42; 35;




23; 22; 9; 3


MR-1C
AF448249
691; 371; 312; 305; 239; 112; 100; 76; 75; 63; 42; 35;




23; 22; 9; 3









Alternatively, the PCR product can be digested by the restriction enzymes Mn/I, FokI and HindIII under the following conditions: PCR product 15 to 30 μL, buffer 2 (New England Biolabs)×1, bovine serum albumin (New England Biolabs)×1, enzyme Mn/I (New England Biolabs) 1 unit, enzyme FokI (New England Biolabs) 1 unit, enzyme HindIII (New England Biolabs) 1 unit, H2O qsf 50 μL. Incubation at 37° C. for 1 hour.
The restriction fragments are then analyzed by electrophoresis. Electrophoresis on agarose gel can be used. However, in order to remedy the low resolution power of this type of electrophoresis (precision +/−10%) and the difficulty of visualizing fragments smaller than 100 bp, methods with a higher resolution (+/−0.1 to 1%) such as micro-fluidic electrophoresis (Agilent) or capillary electrophoresis may be preferred.
These methods (in silico analysis of the restriction profile or electrophoresis analysis of the restriction fragments) were applied to several hundreds of strains from the Danisco collection of S. thermophilus strains and the reference strains described in the literature.
The strains which have the same restriction profile have been grouped together genetic clusters (or genetic groups) denoted CL-1 to CL-12. Comparison of the restriction profiles was carried out by means of Bionumerics software version 3.5. Table 3 summarizes some of the results obtained.







TABLEAU 3







Summary of the results of genotyping and lysotyping











Strain
Genotype
Lysotype
Owner
Texturizing





CNCM I-3617
CL-1
Resistant
Danisco
YES


CNCM I-2423
CL-2
LT-2
Danisco
YES


CNCM I-2426


Danisco
YES


CNCM I-2424


Danisco
YES


Sfi39


Private
YES





collection



DGCC945


Danisco
YES


CNCM I-2432
CL-3
LT-4
Danisco
YES


CNCM I-2978
CL-4

Danisco
YES


DGCC7773


Danisco
YES


CNRZ1066


Public
YES





collection



DGCC7785


Danisco
YES


DGCC7788


Danisco
YES


DGCC7966


Danisco
YES


DGCC47


Danisco
YES


CNCM I-2980
CL-5
LT-5
Danisco
YES


DGCC2056


Danisco
YES


DGCC8013


Danisco
YES


DGCC8015


Danisco
YES


DGCC7790
CL-6
LT-6
Danisco
YES


DGCC7813


Danisco
YES


CNCM I-2979


Danisco
YES


CNRZ368


Public
YES





collection



MR-1C


University
YES


CNCM I-2429
CL-7
LT-7
Danisco
YES


ATCC BAA-491
CL-8
LT-8
Public
NO





collection



DGCC7689


Danisco
NO


DGCC1086


Danisco
NO


SMQ-301
CL-9

University
NO


DGCC7853


Danisco
NO


DGCC7919
CL-10
LT-10
Danisco
NO


DGCC7766
CL-11
LT-11
Danisco
NO


DGCC7809
CL-12
LT-12
Danisco
NO









Method for determining the sensitivity of a strain to a bacteriophage: The sensitivity of a strain to a bacteriophage is established by the lysis plaque method. 100 μl of a culture of the strain to be tested and 100 μl of an appropriate dilution of a serum containing the bacteriophage to be studied are used in order to seed 5 ml of an agar medium under suffusion (0.6% agar weight/volume) M17+glucose supplemented at 10 mM with CaCl2. The mixture is poured onto the surface of a solidified attar medium (1.5% agar weight/volume) M17+glucose supplemented at 10 mM with CaCl2. After incubation overnight at 42° C., the strain's sensitivity to the bacteriophage is evaluated by the presence of lysis plaques. The absence of lysis plaque signifies this strain's resistance to the bacteriophages tested. The spectrum of a strain's sensitivity to the bacteriophages, also called lysotype, is constituted by all of the sensitivities and resistances to the bacteriophages studied. A reference system with approximately sixty phages has been implemented in order to establish the lysotype of the strains of S. thermophilus in the Danisco collection. The strains which have the same lysotype have been grouped together in different groups denoted LT-n.
Some of the results are given in Table 3. It demonstrates in particular that the strains of the same genetic cluster virtually always have the same lysotype. The strain CNCM I-3617 belongs to a novel genetic group called CL-1 which has the very particular lysotype of being resistant to all the bacteriophages tested.
Sequence of the eps Locus
The genetic knowledge acquired with regard to S. thermophilus has shown that the eps locus is one of the major sites of heterogeneity between strains. This characteristic has already been exploited in part in order to develop the abovementioned genotyping method which uses the diversity in the epsA-B-C-D region which is the proximal region of the eps locus. An even greater diversity appears in the distal region of the eps locus (region encoding the glycosyl-transferases, see FIG. 1). This region gives the specificity of the exopolysaccharide and therefore induces at least in part the specificity of the strain's thickening power. This is therefore a region of the chromosome particularly indicated for unambiguously describing the strain CNCM I-3617 and the strains which are related to it. The nucleotide sequence of the eps locus of the strain CNCM I-3617 (starting from the epsA gene was obtained from a synthetic DNA fragment. This fragment was synthesized by PCR on a purified genomic DNA matrix of the strain CNCM I-3617 using two specific primers of conserved genes (deoD encoding a purine-nucleotide phosphorylase, and orf14.9 of unknown function) generally framing the eps locus in the S. thermophilus described in the literature. The sequence of 16037 bp originating from the strain CNCM I-3617 corresponds to the SEQ ID No3.
The sequences SEQ ID No4 and SEQ ID No5 (cf. FIG. 4) correspond respectively to the positions 6592 to 9391 and 10331 to 11373 of SEQ ID No3.
Genetic Organization of the eps Locus
FIG. 4 shows diagrammatically the genetic structure of the eps locus of the strain CNCM I-3617 established by analysis of its nucleotide sequence. The part upstream of the epsA open reading frame (ORF), the sequence of the start of the epsA ORF, and the part downstream of the epsT ORF are not known. Analysis of the sequence identifies 20 ORFs which are all oriented in the same direction. Due to their similarity of sequence with other known genes, and/or by the presence of specific protein units within products deduced from these ORFs, it is possible to attribute a putative function to them.
The structural analysis of the eps locus of the strain CNCM I-3617 shows that it possesses an overall organization similar to that of the eps loci already known (cf. FIG. 1).
The results of sequence comparison between the potential proteins deduced from the ORFs of the eps locus of CNCM I-3617 and those available in the public of databases (GENBANK) are summarized in Table 4. The eps locus of the strain CNCM I-3617 codes for proteins potentially involved in the synthesis of polysaccharide such as for example glycosyltransferases.
On the basis of these data, 6 regions can be distinguished (region 1 to region 6, from the 5′ end to the 3′ end of the eps locus; see FIG. 4):

    
    
        Region 1: this region is formed from 4 ORFs (epsA, epsB, epsC, epsD) for which the deduced proteins exhibit very great similarities (between 95.7 and 99.6%) with the proteins deduced from ORFs situated in the eps locus of strains of S. thermophilus. 
        Region 2: this region is formed from 5 ORFs (epsE, epsF, epsG, epsG′ and epsH) for which the deduced proteins exhibit very great similarities (between 96.6 and 99.3% proteins deduced from ORFs situated in the eps locus of the type XI strain (GENBANK access no. A17454501).
        Region 3: this region is formed from 3 ORFS (epsJ, epsK and epsL) for which the deduced proteins exhibit sufficient similarities (less than 81%) with proteins described in the literature to assign a probable function to them. However these ORFs are clearly distinct from ORFs already described in the literature.
        Region 4: this region is formed from one ORF (epsM) for which the deduced protein exhibits very great similarities (95.4%) with the protein deduced from the eps4F ORF situated in the eps locus of the type IV strain.
        Region 5: this region is formed from 2 ORFs (epsN and epsO) for which the deduced proteins exhibit sufficient similarities (less than 91%) with proteins of lactobacillae described in the literature to assign a probable function to them. However these ORFs are clearly distinct from ORFs already described in the literature in S. thermophilus. 
        Region 6: this region is formed from 5 ORFs (epsP, epsO, epsS, epsR and epsT) for which the deduced proteins exhibit very great similarities (between 98.9 and 100%) with the proteins deduced from ORFs situated in the eps locus of the strain CNRZ368 (GENBANK access no. Z98171).
    
    


Overall, the distal part of the eps locus resembles a hybrid assembly of genes certain of which had never previously been described.







TABLE 4







Analysis of the ORFs of the eps locus of the strain CNCM I-3617. The


position corresponds to the nucleotides at the start and end of the ORF in the


sequence SEQ ID No3. The probable function is that of the proteins deduced from


the ORF sequence. The best similarity is the protein or the deduced protein (the


GENBANK access number of the protein sequence is indicated in parentheses)


having the greatest similarity with the protein deduced from the ORF. The score is


the percentage similarity obtained for the best similarity using the “blastp” tool











ORF
Position
Probable function
Best Similarity
Score














epsA
 1 to 491
Transcriptional Regulator
Eps10A (AAN63761), S. thermophilus
99.4





Type X



epsB
 492 to 1223
Polymerization and/or
Eps5B (AAN63698), S. thermophilus
99.6




export of polysaccharides
Type V



epsC
1232 to 1924
Polymerization and/or
Eps1C (AAN63508), S. thermophilus
97.0




export of polysaccharides
Type I



epsD
1934 to 2674
Polymerization and/or
Eps5D (AAN63700), S. thermophilus
99.2




export of polysaccharides
Type V



epsE
2731 to 4098
Undecaprenyl-phosphate
Eps11E (AAN63787), S. thermophilus
98.9




glycosyl-1-phosphate
Type XI





transferase




epsF
4131 to 4574
Rhamnosyltransferase
Eps11F (AAN63788), S. thermophilus
99.3





Type XI



epsG
4546 to 5073
Epimerase
Eps11G (AAN63789), S. thermophilus
96.6





Type XI



epsG′
5089 to 5553
Epimerase
Eps11G (AAN63789), S. thermophilus
96.8





Type XI



epsH
5644 to 5940
UDP-glucose 6-
Eps11H (AAN63790), S. thermophilus
98.9




dehydrogenase
Type XI



epsJ
6661 to 7812
UDP-galactopyranose
Glf (ZP_00045853), Lactobacillus
81.1




mutase
gasseri ATCC 33323



epsK
7814 to 8896
Glycosyltransferase
EpsF (AAG44710), Lactobacillus
64.9





delbrueckii subsp. bulgaricus Lfi5



epsL
8896 to 9441
Glycosyltransferase
CpsI (CAC81257), S. thermophilus
65.7





FI9186



epsM
8896 to 9441
Glycosyltransferase
Eps4F (AAN63682), S. thermophilus
95.4





Type IV



epsN
 9540 to 10370
dTDP-4-dehydrohamnose
RfbD (YP_619620), Lactobacillus
81.4




reductase
delbrueckii subsp. bulgaricus ATCC






11842



epsO
10955 to 11563
dTDP-4-dehydrorhamnose
RfbC (NP_964906), Lactobacillus
90.6




3,5-epimerase
johnsonii NCC 533



epsP
11580 to 12422
Glycosyltransferase
EpsP (CAB52238), S. thermophilus
99.3





CNRZ368



epsQ
12519 to 13358
Glycosyltransferase
EpsQ (CAB52237), S. thermophilus
98.9





CNRZ368



epsS
13355 to 14641
unknown
EpsS (CAB52236), S. thermophilus
99.5





CNRZ368



epsR
14648 to 15790
Glycosyltransferase
EpsR (CAB52235), S. thermophilus
100





CNRZ368



epsT
15790 to 16037
unknown
EpsT (CAB52234), S. thermophilus
100





CNRZ368









Acidifying Property

The fermentation support is obtained by supplementing 100 ml of semi-skimmed UHT milk (Le Petit Vendéen®) with 3% (weight/volume) of skimmed milk powder (SUP'R TOP®, Eurial Poitouraine). The sterility of the solution is obtained by pasteurization for 10 min at 90° C. (at the core). The fermentation support thus obtained is inoculated with the strain to be tested at a rate of 106 cfu/ml, then incubated at 43° C. (in a water bath). The pH is continuously monitored using a CINAC apparatus (Ysebaert).
The acidifying properties of the strains of S. thermophilus can be described by the maximum rate of acidification, Vm (pH unit/min (pHu/min)), calculated by the maximum value of the first derivative of the pH curve as a function of time. Under these operating conditions, it is estimated that this variable is characteristic of the strain. Its value is constant whatever the physiological state of the micro-upon inoculation of the milk and the level of seeding. Two groups of strains are distinguished, the strains with so-called slow acidification, the Vm of which is greater than −0.0100 pHu/min, and the strains with so-called rapid acidification the Vm of which is less than −0.0100 pHu/min. The strain CNCM I-3617 clearly belongs to the group of the so-called rapid acidification strains (Table 5). This property is very often linked to the presence in the genome of the strains of a gene encoding the wall protease PrtS which could be detected in the genome of CNCM I-3617.







TABLE 5







Maximum rate of acidification of different strains of


Streptococcus thermophilus evaluated under the


operating conditions described.










Maximum rate




(×−1.E5 upH/min)















Standard




Strain
Average
deviation
prtS gene















CNCM I-2429
66
24
absent



DGCC7966
68
12
absent



CNCM I-2432
80
13
absent



DGCC7766
82
13
absent



CNCM I-2978
88
12
absent



DGCC7773
92
17
absent



CNCM I-2979
102
33
absent



CNCM I-2423
129
24
present



CNCM I-3617
144
7
present



CNCM I-2980
167
27
present



DGCC7919
190
22
present









Texturizing Property

The fermentation support obtained by supplementing 100 ml of semi-skimmed UHT milk (Le Petit Vendéen®) with 3% (weight/volume) of skimmed milk powder (SUP'R TOP®, Eurial Poitouraine). The sterility of the solution is obtained by pasteurization for 10 min at 90° C. (at the core). The fermentation support thus obtained is inoculated with the strain to be tested at a rate of 106 cfu/ml, then incubated at 43° C. (in a water bath) until a pH of 4.6 is obtained. The pH is continuously monitored using a CINAC apparatus (Ysebaert). The fermented milks thus obtained are placed in a ventilated oven at 6° C., until they are analyzed. Two types of rheological measurements are carried out: viscosity and flow. The viscosity measurements are carried out at a temperature of 2° C. on fermented milks after storage for 1, 7, 14 and 28 days at 6° C. The equipment used is a RVF-type Brookfield® viscosimeter (Brookfield Engineering Laboratories, Inc.) mounted on a Helipath stand Brookfield Engineering Laboratories, Inc.). The viscosimeter is equipped with a type C needle and the oscillation speed applied to the needle is 10 rpm. The flow measurements are carried out at a temperature of 8° C. on previously-stirred fermented milks, after storage for 14 days at 6° C. The equipment used is an AR1000-N rheometer (TA Instrument) equipped with co-axial cylinders (Radius 1=15 mm, Radius 2=13.83 mm, Height=32 mm, Air gap=2 mm). For the ascending segment, the stress applied in a continuous sweep varies from 0 to 60 Pa for a duration of 1 min according to a linear mode. For the descending segment, the stress applied in a continuous sweep varies from 60 to 0 Pa for a duration of 1 min according to a linear mode. The values taken into account are the thixotropic area and the yield point; the latter is calculated according to the Casson model.
The texturizing ability of a strain can be evaluated in a first phase by a viscosity measurement of the curd obtained under the operating conditions described above. The recognized non-texturizing strains provide viscosity values close to 30 Pa·s while the texturizing strains exceed 40 Pa·s. This texturizing ability can be more or less pronounced (Table 6). For example the strain CNCM I-2979 produces a curd the viscosity of which reaches 42 Pa·s, and the strain DGCC7966 makes it possible to obtain a clearly higher viscosity, of 70 Pa·s. The strain CNCM I-3611 provides curds the viscosity of which amounts to 54 Pa·s (Table 6). This value places this strain among the group of strains with a texturizing ability fully comparable to the industrial strains currently used to devise lactic ferments for the production of yoghurts and fermented milks.







TABLE 6







Viscosity of the fermented milks obtained with the


different strains tested, after storage at 6° C. for 14 days.










Viscosity in Pa · s














Standard



Strain
Average
deviation






DGCC7966
70.0
Nd



DGCC7773
55.0
3.3



CNCM I-3617
54.0
2.5



CNCM I-2980
53.0
2.9



CNCM I-2429
51.0
3.1



CNCM I-2978
49.6
4.2



CNCM I-2432
43.0
4.0



CNCM I-2979
42.2
3.0



CNCM I-2423
42.0
4.6



DGCC7919
28.0
Nd



DGCC7766
30.0
Nd





Nd: not determined






The rheological analyses using the AR1000-N rheometer made it possible to measure two rheological descriptors relevant for qualifying fermented milks: the yield point of the product (Pa) and the thixotropic area (Pa/s). These measurements are reported in Table 7 for each of the strains. For the fermented milk with the strain CNCM I-3617, the average values are 11.25 Pa and 352 Pa/s respectively. These values are significantly different from those measured on curds obtained with strains deemed non-texturizing (DGCC7766 or DGCC7919).







TABLEAU 7







Values of yield point and thixotropic area, Casson model,


measurements by the AR1000-N on fermented dairy products


with different strains after storage for 14 days at 6° C.












Yield point

Thixotropic




(Pa)

area (Pa/s)
















Standard

Standard



Strain
Average
deviation
Average
deviation
















CNCM I-2980
5.89
0.9
488
107



CNCM I-2423
8.86
0.9
1344
574



CNCM I-2978
10.51
0.4
728
153



CNCM I-3617
11.25
0.4
352
53



CNCM I-2432
12.27
1.3
1245
181



CNCM I-2429
13.32
1.2
1215
255



CNCM I-2979
13.56
Nd
1786
250



DGCC7773
14.00
Nd
60
Nd



DGCC7966
15.00
Nd
43
Nd



DGCC7919
15.91
0.2
33100
1415



DGCC7766
17.01
0.1
17083
1520





Nd: not determined






CONCLUSION
The strain CNCM I-3617 has several characteristics of interest for the construction of ferments and in particular for ferments used during the production of yoghurts or fermented milks. It exhibits a rare combination of functional properties (acidifying and thickening strain) and its lysotype is distinct from that of the other strains used in a standard manner for these applications.
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## Claims

1. A isolated strain of Streptococcus thermophilus, wherein said strain has an epsAD fragment which, after digestion by the restriction enzymes Mn/I, FokI and HindIII, has a restriction profile characterized by DNA fragments of 344±2 bp, 341±2 bp, 305±2 bp, 299±2 bp, 277±2 bp, 210±2 bp, 160±2 bp, 142±2 bp, 100±2 bp, 79±2 bp, 75±2 bp, 66±2 bp, 42±2 bp, 23±2 bp and 9±2 bp.

2. A isolated strain according to claim 1 comprising a nucleotide sequence having the nucleotide sequence of SEQ ID No4.

3. A isolated strain according to claim 1 comprising a nucleotide sequence having the nucleotide sequence of SEQ ID No5.

4. A isolated strain of Streptococcus thermophilus wherein said strain is texturizing and wherein said strain has an epsAD fragment which, after digestion by the restriction enzymes Mn/I, FokI and HindIII, has a restriction profile characterized by DNA fragments of 344±2 bp, 341±2 bp, 305±2 bp, 299±2 bp, 277±2 bp, 210±2 bp, 160±2 bp, 142±2 bp, 100±2 bp, 79±2 bp, 75±2 bp, 66±2 bp, 42±2 bp, 23±2 bp and 9±2 bp.

5. A isolated strain of Streptococcus thermophilus wherein said strain acidifies rapidly and wherein said strain has an epsAD fragment which, after digestion by the restriction enzymes Mn/I, FokI and HindIII, has a restriction profile characterized by DNA fragments of 344±2 bp, 341±2 bp, 305±2 bp, 299±2 bp, 277±2 bp, 210±2 bp, 160±2 bp, 142±2 bp, 100±2 bp, 79±2 bp, 75±2 bp, 66±2 bp, 42±2 bp, 23±2 bp and 9±2 bp.

6. The isolated strain of Streptococcus thermophiles deposited on 14 Jun. 2006 at the Collection Nationale of Culture of Microorganismes under no. CNCM 1-3617.

7. A bacterial composition comprising at least one isolated strain according to claim 1.

8. A process for producing a food product, a food complement, a dietary supplement or a product with probiotic properties comprising the step of adding or mixing at least one strain according to claim 1 in the formulation of said food product, food complement, dietary supplement or product with probiotic properties.

9. A process according to claim 8 in which the food product, the food complement, the dietary supplement or the product with probiotic properties is a dairy product, a meat product, a cereal product, a drink, a foam or a powder.

10. A food product, food complement, dietary supplement or product with probiotic properties comprising at least one strain according to claim 1 or the bacterial composition according to claim 7.

11. A dairy product comprising at least the strain according to claim 1 or the bacterial composition according to claim 7.

12. A dairy product according to claim 11 wherein said dairy product is a fermented milk, a yogurt, a matured cream, a cheese, a fromage frais, a milk drink, a dairy product retentate, a processed cheese, a cream dessert, a cottage cheese or an infant milk.

13. A dairy product according to claim 11 comprising milk of animal and/or plant origin.