Structuring fermenting milk bacteria

FIELD: food industry.

SUBSTANCE: strain Streptococcus thermophilus which produces lactic acid is described. Sequence of nucleic acids made of the strain producing polysaccharides are also described as well as food or pharmaceutical composition and milk product containing such strain.

EFFECT: strain has strong structural properties.

16 cl, 4 dwg, 6 tbl, 5 ex

 

The object of the present invention are strains of lactic acid bacteria containing at least one sequence selected from the group consisting of nucleotide sequences SEQ ID No. 1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No. 8, and the method of obtaining these strains. Finally, the present invention relates to food products containing and/or produced using the above-mentioned strains.

In the food industry is used by many bacteria, in particular in the form of enzymes, in particular, lactic acid bacteria to improve the taste and consistency of the products, as well as to increase the shelf life of these products. In the dairy industry, lactic acid bacteria are used intensively to ensure the souring of milk (fermentation), as well as for the structuring of the product in which they are administered.

Among the lactic acid bacteria used in food industry, you can specify delivery Streprococcus, Lactococcus, Lactobacillus, Leuconostoc, Pediococcus and Bifidobacterium.

Lactic acid bacteria of the species Streptococcus thermophilus are used extensively alone or in combination with other bacteria for food production, in particular fermented products. In particular, they are part of enzymes used for production of fermented milk such as yoghurt. Some the th of them play a predominant role in obtaining the necessary consistency of fermented product. This property is closely connected with the production of polysaccharides. Among strains of Streptococcus thermophilus distinguish structural strains and restrukturisasi strains.

Under the structural strain of Streptococcus thermophilus, you should understand the strain, forming a fermented milk having described in the examples, the terms of viscosity, about 35 PA·s, the area thixotropy below approximately 2000 PA/s and the threshold yield strength below approximately 14 PA. The strain Streptococcus thermophilus, you can determine how much structure, if it forms a fermented milk with the conditions described in the examples, the viscosity is above about 50 PA·s, the area thixotropy below approximately 1000 PA/s and the threshold yield approximately below 10 PA.

To address the challenges facing the food industry, there is a need to create new structures of strains of lactic acid bacteria, in particular Streptococcus thermophilus.

In this regard, the present invention is the creation of a strain of lactic acid bacteria possessing good properties of the structuring of food.

With this in mind, an object of the present invention is a strain of lactic acid bacteria containing at least one sequence selected from the group consisting of nucleotide sequences SEQ ID No. 1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No. 8.

This image is taneem also proposed a new strain of Streptococcus thermophilus, deposited on 26 February 2003 in the National collection of cultures of microorganisms under the number 1-2980.

The object of the present invention are the nucleotide sequence of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No. 8, and a nucleotide sequence containing at least one of these nucleotide sequences.

Another object of the present invention are plasmids used as vectors for cloning and/or expression containing at least one sequence selected from the group consisting of nucleotide sequences SEQ ID No. 1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No. 8, and a nucleotide sequence containing at least one of these nucleotide sequences.

The present invention also concerns of bacterial hosts, transformed with the above plasmids or vectors.

The object of the present invention is also a method of obtaining the above-described strains, namely, that these strains were obtained by transformation with plasmids or vectors containing at least one of the nucleotide sequences SEQ ID No. 1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No. 8, or with the vector containing the nucleotide sequence, sotiriadou is, at least one of these nucleotide sequences.

The present invention also available bacterial culture containing at least one of the above strain or containing at least one strain obtained by the method in accordance with the present invention.

Finally, the present invention relates to a food or pharmaceutical composition comprising at least one strain in accordance with the present invention or at least one strain obtained by the method in accordance with the present invention, or a bacterial culture in accordance with the present invention.

The present invention differs from the many advantages in terms of structuring of environments in which it is used. In particular, it allows to obtain gels, for example, based on fermented milk, which are grease, adhesive, coating, possessing fluidity and stability with respect to mixing and do not form grains.

Other advantages and distinctive features of the present invention will be more apparent from the following descriptions and examples are given as illustrations and not bearing a restrictive nature.

First of all, the present invention relates to a strain of lactic acid bacteria containing at least one sequence selected from the group consisting of nucleotide sequences SEQ ID No. 1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No. 8.

Lactic acid bacteria are gram-positive prokaryotes belonging to the group Firmicutes. They are heterotrophic and chemoorganotrophic; typically, anaerobic or aerotolerance, their metabolism may be Homo-fermentative or heterofermentative: lactic acid bacteria, mainly produce lactic acid by fermentation glutinosa substrate. Devoid of catalase lactic acid bacteria constitute a heterogeneous group of cocci, including the genera Aerococcus, Enterococcus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella or including Bacillus genera Lactobacillus and Carnobacterium. The name "lactic acid bacteria" is often extended to other related bacteria, such as Bifidobacterium.

Among strains of lactic acid bacteria, which can be used in the framework of the present invention, can be called the genera Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, Pediococcus and Bifidobacterium.

The preferred strain of lactic acid bacteria in accordance with the present invention is Streptococcus thermophilus.

Streptococcus thermophilus is a view that is present naturally in milk and is widely used in the food industry, in particular in the dairy industry, as what you can skachivat and structure of the milk. In this case we are talking about homofermentative thermophilic bacteria.

The present invention also concerns the strain Streptococcus thermophilus deposited on 26 February 2003 in the National collection of cultures of microorganisms under the number 1-2980.

The object of the present invention are the nucleotide sequence of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No. 8.

The nucleotide sequence of SEQ ID No. 2 to SEQ ID No. 8 are part of the operon about 14350 base pairs involved in the synthesis of polysaccharides (operon PS). This operon is included in the sequence SEQ ID No. 1. This means that in accordance with the present invention the sequence SEQ ID No. 1 comprising the sequence SEQ ID No. 2 to SEQ ID No. 8.

Primary structure of the operon, determined in accordance with the present invention, is shown in figure 1. Between deoD genes (before operon PS) and orfl4.9 (after operon PS) identified 17 ORFS (open reading frames), called eps13A, eps13B, eps13C, eps13D, eps13E, eps13F, eps13G, eps13H, eps13I, eps13J, eps13K, eps13L, eps13M, eps13N, eps13O, eps13P and IS1193. 16 the first ORF, located on the coding strands, potentially encode polypeptides involved in the synthesis assoclation or capsular polysaccharides; 17th ORF, located on the antiparallel strands, potentially encodes a functional transposon belonging to the family IS1193.

Can privo is th name and define the place of each ORF. Below is listed the name of each ORF, then the intended function selected protein and, finally, the position of the field containing this ORF (with respect to the sequence SEQ ID No. 1 specified in the list of sequences):

- eps13A: transcription regulator (342...1802)

- eps13B: polymerization (regulation of chain length) and/or transport of polysaccharides (1803...2534)

- eps13C: polymerization (regulation of chain length) and/or transport of polysaccharides (2543...3235)

- eps13D: polymerization (regulation of chain length) and/or transport of polysaccharides (3245...3985)

- eps13E: indicatinginformalpaymentsor (4042...5409)

- eps13F: indicatinginformalpaymentsor(5611...6195)

- eps13G: indicatinginformalpaymentsor (6251...6634)

- eps13H: beta-1,4-galactosyltransferase (6643...7092)

- eps13I:. beta-1,4-galactosyltransferase (7092...7607)

- eps13J: rhamnosyltransferase (7597...8493)

- eps13K: glycosyltransferase (8763...9797)

- eps13L: polymerase recurring units (9827...10969)

- eps13M: polymerase recurring units (10984...11793)

- eps13N: glycosyltransferase (11844...12578)

- eps13O: glycosyltransferase (to 12 633...13016)

- eps13P: transmembrane carrier (13049...14482)

- IS1193: transposon (addition (14614...15870).

The present invention also concerns a nucleotide sequence containing at least one sequence selected from the group consisting of the nucleotide who's sequences SEQ ID No. 1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8. Under a nucleotide sequence containing at least one of the above sequences in the framework of the present invention should be understood nucleotide sequence containing at least one ORF, product broadcast which has significant identity (percentage of identical residues is greater than or equal to 80% after matching sequences at the maximum correspondence between the positions of residues)at least one polypeptide sequence deduced from the ORF identified in the sequences SEQ ID No. 1 to SEQ ID No. 8.

The nucleotide sequence of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No. 8 can be embedded in a vector using genetic engineering, in particular using recombinant DNA technologies, well-known to experts.

The present invention also concerns vectors for cloning and/or expression containing at least one sequence selected from the group consisting of nucleotide sequences SEQ ID No. 1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No. 8, or defined above nucleotide sequences.

A preferred vector according to the present invention are plasmids. R is e can go on replicative or integrative plasmids.

These vectors and/or plasmids can be transformed bacteria to inclusion of these vectors and/or plasmids. The transformation can be performed using electroporation or conjugation, as is known to experts in this field of technology.

The present invention also concerns of bacterial hosts, transformed with the above plasmids or vectors.

Preferably the transformed bacteria are lactic acid bacteria. In particular, we are talking about lactic acid bacteria, which can be selected from the genera Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, Pediococcus and bifido bacteria bacterium.

The preferred strain of lactic acid bacteria in accordance with the present invention is Streptococcus thermophilus.

The present invention concerns also a method of obtaining strain or transformed bacteria-owners in accordance with the present invention, consists in the fact that they are obtained by transformation with a vector containing at least one sequence selected from the group consisting of nucleotide sequences SEQ ID No. 1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No. 8, or the nucleotide sequence containing at least one sequence selected from the group consisting of nucleotide sequences SEQ ID No. 1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6,SEQ ID No.7, SEQ ID No. 8.

According to the method in accordance with the present invention, the preferred vector is a plasmid.

Preferably according to the method in accordance with the present invention, the transformation is accompanied by integration into the genome of strain or bacteria-host transformed by at least one recombination event.

The present invention relates to a bacterial culture that contains at least one strain in accordance with the present invention contains at least one strain obtained by the method in accordance with the present invention, or containing at least one bacterium transformed in accordance with the present invention.

Under bacterial culture understand the mixture of different strains, in particular an enzyme or ferment.

Preferred mixtures of strains in accordance with the present invention are a mixture of Streptococcus thermophilus with other Streptococcus thermophilus or a mixture of Streptococcus thermophilus with Lactobacillus delbrueckii subspecies bulgaricus, or a mixture of Streptococcus thermophilus with other Lactobacillus and/or Bifidobacterium or of a mixture of Streptococcus thermophilus with Lactococcus, or a mixture of Streptococcus thermophilus with other strains of lactic acid bacteria and/or yeasts.

The present invention also concerns the use of strain in accordance with the present invention, or strain, the floor is built using the method in accordance with the present invention, or bacterial culture in accordance with the present invention for the production of a food product or food ingredient.

As a preferred food product or food ingredient in accordance with the present invention can be called a dairy product, meat product, grain product, beverage, mousse or powder.

The present invention also concerns a food or pharmaceutical composition comprising at least one strain in accordance with the present invention or at least one strain obtained by the method in accordance with the present invention, or a bacterial culture in accordance with the present invention.

The object of the present invention is also a dairy product containing at least one strain in accordance with the present invention or at least one strain obtained by the method in accordance with the present invention, or a bacterial culture in accordance with the present invention.

In the case of dairy product production by prominent in this area, in particular fermentation of dairy product by incorporating strain in accordance with the present invention.

As a dairy product in accordance with the present invention it is possible to specify square the Noe milk, yogurt, ripened cream, cheese, curd, milk drink, retentate dairy product, melted cheese, creamy dessert, rustic cheese or baby milk.

Preferably a dairy product in accordance with the present invention is the milk of the animal and/or vegetable origin.

As milk of animal origin, you can specify cow milk, sheep milk, goat's milk and Buffalo milk.

As milk plant origin can be any capable of ripening substance of plant origin, which can be used within the present invention, in particular derived from soy beans, rice or barley.

Figure 1 shows the structure of the operon PS strain in accordance with the present invention in comparison with the operon PS other known strains of Streptococcus thermophilus.

Figure 2 shows the factorial scheme 1-2 analysis by main components (AOC), obtained on the basis of sensory data, fermented dairy products, obtained using different the considered strains.

Figure 3 shows the average rating of the descriptive indicators of the STRUCTURE ON the SPOON for each of the examined strains. Interpretation of test results of Newman-Colza: the difference between the strains associated one letter is nesses the public.

Figure 4 shows the average rating of the descriptive indicators of the structure of the MOUTH for each of the examined strains. Interpretation of test results of Newman-Colza: the difference between the strains associated one letter is not significant.

What follows is a description of a specific, but not limiting examples.

EXAMPLES

1) Determining the sequence of the operon PS Streptococcus thermophilus CNCM I-2980

The DNA fragment containing the operon PS strain Streptococcus thermophilus CNCM I-2980, obtained by PCR amplification from genomic DNA isolated from this strain. Amplification was performed using thermal cycler Mastercycler (Eppendorf) using DNA polymerase, LA-Taq (BioWhittaker/Cambrex) and the following primers: 5'-GGGTGAACGTATCTCAGTAATGGGACTGG-3' and 5'-CCTGAGTTATGCGACGATTACTTGGCTG-3'. Amplification was performed under the following conditions: 14 cycles with alternation of denaturation at 98°C for 30 s and hybridization-synthesis at 68°C for 15 min 15 sec per cycle, then 1 additional cycle of synthesis at 72°C for 10 minutes Complete sequence of the PCR product was obtained by cloning fragments.

2) Molecular characterization of strain in accordance with the present invention in comparison with known strains of Streptococcus thermophilus

Sequence of the operon PS

The sequence of the operon PS strain Streptococcus thermophilus CNCM I-2980, consisting of approximately 17100 base pairs, sintesio the Ali by PCR using as a matrix of purified genomic DNA of strain and using two canned specific primers for genes (deoD, coding polynucleotides, and orf14.9 unknown function), usually framing operon PS from Streptococcus thermophilus that is described in the literature. The sequence contained between deoD genes and orf14.9 corresponding to the sequence SEQ ID No. l, is given together with a list of sequences.

Genetic organization of the operon PS

1 schematically shows the genetic structure of the operon PS strain CNCM I-2980 determined by analysis of the nucleotide sequence and structure of the operon PS 7 other strains of Streptococcus thermophilus. For the structures identified by the name of the strain and the access number in GENBANK (in parentheses), arrows indicate the location, size and direction of the gene from the initiation of the initiation codon to the termination codon. Color values and/or drawings is explained in figure 1.

- Comparison with literature

Structural analysis of operon PS strain CNCM I-2980 shows that his organization similar to the organization of known operons PS (see figure 1): the first ORF, potentially involved in the regulation of transcription of the operon PS, with subsequent 3 ORF, probably involved in the regulation of polymerization of the repeating units of PS and/or migration, with subsequent 11 ORF, encoding the glycosyltransferase gene and polymerase providing the Assembly of the repeating units, and with the subsequent 1 ORF potentially involved the military in the transfer of the repeating unit in the plasmid membrane.

Surrounding genetic environment of the operon PS strain CNCM I-2980 also similar with the natural genetic environment of other known operons CNCM 1-2980: before ORF deoD encoding polynucleotides, and once in the opposite direction ORF ISI193 and orf14.9, encoding respectively the transposon (belonging to the family of transposons IS1193, which is movable genetic elements) and a protein of unknown function.

However, comparison of the sequence between the proteins potentially encoded by ORF operon of strain CNCM I-2980, and proteins available in published databases (GENBANK), shows that the genetic content of the operon PS strain CNCM 1-2980 is new in its distal part.

- The proximal part of the operon PS strains of Streptococcus thermophilus and generally known today streptococcal contains 4 ORF, called epsA (or cpsA, or Sarah, or wzg), epsB (or cpsB, or SARV or wze), epsC (or cpsC, or SRS, or wzd) and epsD (or cpsD, or capD, or wze), the deduced polypeptide products for each of the 4 ORF have significant sequence similarity between strains. At this level of polypeptide products derived from ORF eps13A, eps13B, eps13C and eps13D operon PS strain CNCM I-2980, do not differ from the polypeptide products derived from other operons PS (the content of the same amino acids greater than or equal to 94%).

Immediately after ORF epsD in PL is known as operons PS from Streptococcus thermophilus ORF is present epsE (or cpsE, or Sarah, or wchA). In this case, the sequence similarity between homologous polypeptide products derived from different known sequences is significant; the product broadcast ORF eps13E strain CNCM I-2980 has significant similarity to homologues from other strains.

- Organization 4 the following ORF (eps13F, eps13G, eps13H and eps13J) operon PS strain CNCM I-2980, although it was previously described, but is less common among the various known structures of operons PS Streptococcus thermophilus. This organization was found, albeit in an incomplete form in the strains IP6757 (GENBANK access number AJ289861), type VII (GENBANK access number AF454498) and type III (GENBANK access number AY057915).

In the distal part of the operon 7 ORF eps13J, eps13L, eps13M, eps13N, eps13C) and eps13P are new and specific to the operon PS strain CNCM I-2980. Though poor, their sequence similarity on the protein level, or in some cases, the presence of specific protein drawings allows you to give the products of these ORFS intended function: potential glycosyltransferase or polymerase activity for product ORF eps13J-eps13C), transmembrane transport activity for PS product ORF eps13P.

3) Comparative rheological test strain Streptococcus thermophilus CNCM I-2980

Used strains of Streptococcus thermophilus CNCM I-2423, CNCM I-2429, CNCM I-2432, CNCM I-2978 and CNCM I-2979 are strains from the collection and organization "Rhodia Food". They are used mainly for industrial production of fermented milk products or yogurt because of their structural properties. They are typical representatives of the strains described in the prior art. In the future, they are treated in comparison with the strain CNCM 1-2980 and are tipichnii representatives structuring strains currently used in the agricultural sector.

Strains of Streptococcus thermophilus RD736 and RD676 are the strains produced by the company Rhodia Food, known for its weak organizational capacity. Polysaccharides that they can produce, and their operon PS is unknown. In the future, they are treated in comparison with the strain CNCM 1-2980 as typical representatives of restrukturisasi strains.

Fermented milk is received, adding 3% (wt./about.) skimmed milk powder in 100 ml of skimmed milk UHT 1/2 (Le Petit Vendeén®). The solution is pasteurized at 90°C for 10 min, with the temperature measured in the middle of the mass of milk. Thus obtained fermentation substrate inoculant test strain rate of 106CFU/ml (colony forming units/ml), then incubated at 43°C in a water bath to obtain a pH of 4.6. Registration pH carried out continuously. Thus obtained fermented milk product is placed in a ventilated Cabinet at 6°C on the analysis.

Spend two types of rheological measurements: determination of viscosity and fluidity. The viscosity measurement is carried out at 8°C in fermented milk after 1, 7, 14 and 28 days of storage at 6°C using a Brookfield viscometer® type RVF (Brookfield Engineering Laboratories, Inc.), mounted on the tripod Heilpath (Brookfield Engineering Laboratories, Inc.). The viscometer is equipped with a needle-type, and speed fluctuations, reported the needle is 10/min Measurement of flow is carried out at 8°C in fermented milk after 14 days of storage at 6°C after pre-mixing using rheometer AR 1000-N (TA Instrument)equipped with coaxial cylinders (radius 1=15 mm, radius 2=13,83 mm, height 32 mm, the intermediate space 2 mm). For a segment of the lifting force applied during continuous scanning, changing from 0 to 60 PA for 1 minute in a linear mode. For the segment of lowering the force applied during continuous scanning, varies from 60 to 0 PA for 1 minute in a linear mode. This takes into account the values of the field thixotropy and threshold yield; the latter is calculated by the model caisson.

The viscosity of the fermented milk obtained using a strain of Streptococcus thermophilus CNCM I-2980, was measured after 1,7, 14 and 28 days of storage at 6°C (table 1). The viscosity measured after one day of storage equal to 53.3 per PA·C. This value varies slightly is about time, that indicates the stability of the fermented milk obtained using a strain of Streptococcus thermophilus CNCM 1-2980. For comparison (table 1) viscosity values measured for the fermented milk obtained using strains RD736 and RD676, known for its low structural capacity, range from 26 to 30 PA·C. Other tested strains give fermented milk lower viscosity than the strain CNCM 1-2980. You can distinguish the first group of strains, giving a viscosity of 40 PA·s (CNCM I-2979, CNCM I-2423 and CNCM I-2432), and the second group, which includes CNCM 1-2980 providing a viscosity of 50 PA·S.

Table 1
The viscosity and pH of the fermented milk obtained by the use of different strains, after different periods of storage at 6°C
StrainsViscosity, PA·s/LV
1 day storage7 days storage14 days storage28 days storage
CNCM
I-2980
53,3/4,5053,0/of 4.4453,0/4,4253,5/4,40
CNCM
I-242
51,2/4,5551,9/4,4551,0/of 4.4451,2/of 4.44
CNCM
I-2978
50,4/4,5651,8/to 4.5249,6/4,4951,0/4,45
CNCM
I-2432
42,4/4,6042,2/4,5643,0/4,5543,0/4,45
CNCM
I-2423
42,0/4,6041,9/4.4042,0/4,3743,0/4,30
CNCM
I-2979
37,8/4,5440,7/4,4542,2/4,4242,2/4,33
RD67629,6/4,6030,0/4,5730,0/4,5730,0/to 4.52
RD73626,0/4,4526,0/4,3428,0/4,3427,0/4.26 deaths

The measurement of flow allowed us to identify two significant rheological factor (threshold yield and area thixotropy) for rheological description IC is asenovo milk (table 2). For fermented milk obtained from the strain CNCM 1-2980, average values are from 5.89 PA and 488 PA/s, respectively for a threshold yield for the region of thixotropy. These values differ significantly from the values obtained for the fermented milk prepared with strains that are considered restrukturisasi (RD676 and RD736). For example, fermented milk obtained from strain RD676, these average values are respectively 17,01 PA and PA 17083/sec. In the case of strains considered structure, the value of threshold yield and area thixotropy are much closer to the values obtained using strain CNCM 1-2980, however, significantly exceed them, which proves that higher structural capacity of the strain CNCM 1-2980.

Table 2
The value of threshold yield and area thixotropy (average values of three repetitions), measured with an instrument AR1000-N fermented milk obtained from different strains after 14 days of storage at 6°C
StrainsThe threshold yield stress (PA)Region thixotropy (PA/s)
CNCM I-2980of 5.8948
CNCM I-242913,321215 (2 values)
CNCM I-297810,51728
CNCM I-243212,271245
CNCM I-24238,861344
CNCM I-297913.56MHz1786
RD67617,0117083
RD73615,9133100

4) Organoleptic characteristics of the strain Streptococcus thermophilus CNCM 1-2980 in comparison with the control strains

Evaluation of fermented milk was carried out by sensory analysis after 14 days of storage at 6°C. Quantitative descriptive analysis fermented milk at the optimum temperature tasting of 12°C was produced by a panel of 9 experts on unstructured linear scale from 0 to 6 points. Analysis of the organoleptic profile was duplicated every few days. Pre-selected and trained experts evaluated the four descriptive indicators structure of the s on the spoon: brittle, the resistance to stirring, refluxing, grain and 4 descriptive indicators of the structure in the mouth: melting, adhesion, density, enveloping. Organoleptic differences were assessed by two-factor ANOVA analysis (called ANOVA) with fixed model with subsequent comparative test of the mean value Newman-Colza with alpha threshold of 5% on each of descriptive indicators. To visualize the space of product was applied to the analysis by main components (AOC) with the organoleptic opiatelike indicators with variable values and strains on individuals. Hierarchical ascending classification (CPI) allowed us to identify groups of strains on the basis of the AOC. Software used for these statistical analyses are Fizz® (Biosystems), Statgraphics® and Uniwin plus®.

Data on fermented milk obtained using a strain of Streptococcus thermophilus CNCM 1-2980 were compared with results from fermented milk obtained by using other control strains. The average values obtained for different strains on the certain parameters of the structure are shown in table 3, significant differences identified by ANOVA and comparative test on average values presented in table 4 and figure 3 and 4.

Table 3
The average rating assigned by the Commission organoleptic analysis for fermented milk obtained using various analysis strains, descriptive indices
StrainsIndicators on the spoonThe indicators in the mouth
BrittleResistance to mixingGrain sizeRunoffMeltingStickinessDensityEnveloping
CNCM
I-2980
0,89equal to 4.970,045,091,324,27of 5.06to 4.41
CNCM
I-2249
4,123,491,531,243,361,463,542.94
CNCM
I-2978
3,394,350,422,832,961,973,863,41
CNCM
I-2432
3,283,141,601,323,111,433,012,91
CNCM
I-2423
1,64as 4.020,163,442,252,483,613,30
CNCM
I-2979
3,982,792,360,743,710,722,392,22
RD676to 4.981,465,140,174,960,040,91RD7364,351,453,430,045,180,310,770,67

Table 4
Comparison of mean scores for each of the descriptive indicators of the patterns on the spoon and structure in the mouth with the help of test Newman-Colza with 5%. Interpretation of results: the difference between the strains associated with the same letter are not significant
StrainsIndicators on the spoonThe indicators in the mouth
BrittleResistance to mixingGrain sizeRunoffMeltingStickinessDensityEnveloping
CNCM
I-2980
EAndEAndD AndAndAnd
CNCM
I-2249
InDDInInSU
CNCM
I-2978
InEInSUInIn
CNCM
I-2432
CDDDInSU
CNCM
I-2423
DInEInininin
CNCM
I-2979
InDE InDD
RD676AndEAndFAndDED
RD736InEInFAndDED

Figure 3 and 4 shows the histogram of the results presented in table 4. All descriptive indicators considered restrukturisasi strains RD676 and RD736 significantly different from other strains. Among the structural strains of the strain CNCM I-2980 significantly and dramatically different in all indicators, except grain, on which he is not differentiated from strains CNCM I-2978 and CNCM I-2423.

AOC allows you to define the place of strains depending on their distance with respect to organoleptic indicators.

In scheme 1-2 AOC figure 2 presents 97,3% space product. Component 1 contrasts two groups of sensory variables. The first group includes variables: the us is ascioti to mixing, the density of the mouth, enveloping mouth, dripping on the spoon and stickiness on the spoon, characterizes the component 1 on the right. The second group includes the variables: melting in the mouth, brittle on the spoon and grain size on the spoon, characterizes the component 1 on the left. The first group anticorrelated with the second group. These variables allow us to analyze the place strains on this scheme. In addition, analysis of the CPI allows us to classify different groups of strains dashed in factorial scheme 1-2.

Factorial scheme contrasts several groups of strains that determine different properties of the structure. From these analyses it follows that the strains RD676 and RD736 give fermented milk brittle and granular structure on the spoon and melting in the mouth. However, they do not give a thick, sticky and enveloping structures in the mouth and resistant to stirring or flowing patterns on the spoon in comparison with other groups of strains. Consequently, as a result of their use receive unstructured fermented milk. In contrast, the strains CNCM I-2429, CNCM I-2432 and CNCM I-2979 produce srednemineralizovannaya fermented milk, the strains CNCM I-2423 and CNCM I-2978 produce structured fermented milk and strain CNCM I-2980 produces highly structured fermented milk. This analysis shows that the strain CNCM I-2980 will riday fermented milk special properties of the structure compared with all control strains.

5) Comparative test on the stability of the strain Streptococcus thermophilus CNCM I-2980 against phage

The strain sensitivity to bacteriophage determined by the method of the zones of lysis. 100 µl of the culture of the test strain and 100 μl of each dilution of serum containing considered the bacteriophage used for planting on the cooled agar medium (agar with 0.6% wt./about.) M17-glucose with the addition of 10 mm CaCl2. The mixture was poured onto the surface utverzhdenii agar medium (agar 1.5% wt./about.) M17-glucose with the addition of 10 mm l2. After incubation at 42°C for 16 hours assessment sensitivity to bacteriophage strain was performed according to the presence of zones of lysis. The absence of zones of lysis testifies to the stability of this strain with respect to the studied phages. Range of strain sensitivity to bacteriophages (also called lization) represents a set of values of sensitivity and resistance in relation to the studied bacteriophages.

Table 5 presents the bacteriophages used for this study, and their original strains/strains reproduction. We are talking about the strains and bacteriophages from the collection "Rhodia Food". Bacteriophages were selected according to their capability to infection control and structuring of strains.

Table is 5
Phage
Name of phageThe original strain
2972CNCM I-2423
4082RD729
4074CNCM I-2429
4154CNCM I-2429
1272CNCM I-2978
4128RD852
1255CNCM I-2432
1765RD728
4121RD862

For evaluation of industrial applicability of the strain CNCM I-2980 in connection with problems associated with bacteriophages, have been evaluated with lithotype strain CNCM I-2980 and its comparison with the control structuring strains. Table 6 shows the sensitivity of the strains to different phages (littp), determined by the method of the zones of lysis. It turned out that six of the investigated strains have different isotype. In particular, the strain CNCM I-2980 has littp that is different from lization other investigated structural strains. Indeed, the strain CNCM I-2980 was not infected with the tested phages.

Table 6
Littp tested strains
PhageStrains
CNCM I-2980CNCM I-2423CNCM I-2978CNCM I-2432CNCM I-2429CNCM I-2979
2972-+----
4082-+----
1272--+---
4128--+---
1255--- +--
1765---+--
4074----+-
4154----+-
4121-----+
+: sensitivity to the tested phage;
-: resistance to the tested phage.

1. The strain Streptococcus thermophilus, producing lactic acid, deposited on February 26, 2003 GV National collection of cultures of microorganisms under the number 1-2980.

2. The nucleotide sequence of SEQ ID No. 1, isolated from a strain according to claim 1, including PS operon for the production of polysaccharides.

3. The nucleotide sequence of SEQ ID No. 2, isolated from a strain according to claim 1, which causes the production of polysaccharides.

4. The nucleotide sequence of SEQ ID No. 3, isolated from a strain according to claim 1, which causes the production of polysaccharides.

5. The nucleotide sequence of SEQ ID No. 4, isolated from a strain according to claim 1, which causes the production of polysaccharides.

6. The nucleotide sequence of SEQ ID No. 5, selected from the strain according to claim 1, which causes the production of polysaccharides.

7. The nucleotide sequence of SEQ ID No. 6, selected from the strain according to claim 1, which causes the production of polysaccharides.

8. The nucleotide sequence of SEQ ID No. 7, selected from the strain according to claim 1, which causes the production of polysaccharides.

9. The nucleotide sequence of SEQ ID No. 8, selected from the strain according to claim 1, which causes the production of polysaccharides.

10. Nucleic acid isolated from a strain according to claim 1, which causes the production of polysaccharides containing at least one sequence selected the th group, consisting of nucleotide sequences SEQ ID No. 1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No. 8, or a nucleic acid that contains at least one ORF, product broadcast which has a percentage of identical residues over or equal to 80% with at least one polypeptide sequence deduced from the ORF identified in the sequences SEQ ID No. 1 to SEQ ID No. 8.

11. Application of the strain according to claim 1 for the production of a food product or food ingredient, or a pharmaceutical composition.

12. The application of claim 11, wherein the food product or food ingredient is a dairy product, meat product, grain product, beverage, mousse or powder.

13. Food or pharmaceutical composition comprising at least one strain according to claim 1.

14. Dairy product containing at least one strain according to claim 1.

15. Dairy product according to 14, characterized in that it comes to fermented milk, yogurt, ripened cream, cheese, cottage cheese, milk drink, ultracetultracet dairy product, melted cheese, creamy dessert, rustic cheese or milk for infants.

16. Dairy product 14 or 15, characterized in that it contains milk animal and/or vegetable origin.



 

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