MXPA98003427A - Production of l (+) - lact - Google Patents

Abstract

The present invention relates to a recombinant strain of L. johnsonii which has the ability to survive passage through the intestine by attaching to human intestinal cells and increasing phagocytosis of macrophages and having a sequence code of the D-lactate dehydrogenase gene which is a gene sequence code of L. johnsonii modified and that only produces L (+) - lactate

Description

PRODUCTION OF L (+) -LACTATE OBJECT OF THE INVENTION The present invention relates to genetically recombined bacterial strains, and to a method for producing these strains.

CURRENT STATE OF THE TECHNOLOGY Fermentation is a degradation of a carbon source during which the final hydrogen acceptor is an organic compound. Via the fermentation of lactic acid, certain bacterial strains produce a racemic mixture of the two isomeric forms of lactate, D (-) - lactate and L (+) - lactate, for the regeneration of NAD +, by the reduction of pyruvate by medium of two lactate des idrogenasas dependent on specific NAD. It is known that some individuals exhibit an intolerance to lactose reduction. This poor digestion of lactose is often due to the absence of a sufficient amount of β-galactosidase in the small intestine. Various studies (Kolars et al., N. Engl. J. Med., 310, 1-3, 1984; Marteau et al., Br. J. Nutr. 6_4_, 71-79, 1990; and Arrigoni et al., Am. J. Clin. Nutr., 60_, 926-929, 1994) have demonstrated the fact that these people digest and tolerate the lactose contained in yoghurts better than that contained in milk. This better digestion and better tolerance to lactose are due especially to the activity of β-galactosidase of the bacteria contained in yoghurts during intestinal transit. It is further known that D (-) - lactate can cause problems of acidosis in children. For these reasons, the World Health Organization (FAO / WHO, 1967, 1974) recommends that D (-) -lactate should not be added to children's foods, either independently or as a racemic mixture with the L (+) -lactate. Also, the daily intake limit of D (-) -lactate for adults preferably does not exceed 100 mg / kg of the human body. Bacterial strains that have been genetically recombined to produce only L (+) - lactate are now known. Thus, T. Bhowmik et al. (Appl. Microbiol. Biotechnol., 432-439, 1994) describes a technique for the isolation and inactivation, by directed mutagenesis, of the gene coding for the enzyme D-lactate dehydrogenase of the strain La ct oba ci llus hel ve ti cus CNRZ32, particularly the strain La ct oba ci ll us hel ve ti cus CNRZ32 (pSU 104), which produces only L (+) - lactate. This strain is obtained by electroporation of the integration vector pSU 104, which comprises the vector pSA3 and the internal fragment of Sal l-Sphl of 0.6 kb of the gene coding for the enzyme D-lactate dehydrogenase from Lact obaci ll us hel ve ti cus. However, bacterial strains with the ability to survive in the intestine, adhere to intestinal cells and effect immunomodulation, which have been genetically recombined to produce only L (+) - lactate, are not known at the present time. Now, it would be very valuable, for the preparation of food products, to have such bacterial strains with the ability to survive in the intestinal tract, to possess these beneficial properties on human health, and to produce only L (+) - lactate, to avoid Adverse effects due to D (-) - lactate. The object of the present invention is to satisfy these needs.

BRIEF DESCRIPTION OF THE INVENTION For this purpose, the present invention relates to a bacterial strain with the ability to survive in the intestine, to adhere to human intestinal cells, and to effect immunomodulation, which has been genetically recombined to produce only L (+) -lactate. The present invention relates especially to a bacterial strain in which the gene coding for the enzyme D-lactate dehydrogenase is inactivated. The present invention relates especially to a strain of Lactobacillus acidophilus, Lactobacillus j ohnsonii, Lactobacillus gasseri, Lactobacillus críspatus, Lactobacillus amylovorus or Lactobacillus gallinarum. The present invention is additionally related to strain CNCM 1-1851 and strain CNCM I-1852. A further object of the present invention is a method for producing a bacterial strain that has been genetically recombined to produce only L (+) - lactate.

Finally, the present invention relates to the use of a bacterial strain, obtained by carrying out the method according to the present invention, in the preparation of food products.

DETAILED DESCRIPTION OF THE INVENTION In the remainder of the description, the term "conjugative vector" will be used to denote a transferable DNA vector by conjugation between two strains of different species of lactic acid bacteria. Also, in the remainder of the description, the expression "strain with the ability to survive in the intestine" will be used to denote a bacterial strain of lactic acid which, after consumption, is found in the stool. Finally, in the remainder of the description, the expression "bacterial strain with the ability to effect immunomodulation" will be used to denote a bacterial strain of lactic acid that has a beneficial effect on the immune system, especially the property of increasing phagocytosis of the macrophages. The present invention therefore relates to a bacterial strain with the ability to survive in the intestine, to adhere to human intestinal cells and effect immunomodulation, which has been recombined genetically to produce only L (+) - lactate. The present invention relates especially to a bacterial strain with the ability to survive in the intestine, to adhere to human intestinal cells and effect immunomodulation, in which the gene encoding the enzyme D-lactate dehydrogenase has been inactivated. The strain according to the present invention may be a strain of Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus gasseri, Lactobacillus crispatus, Lactobacillus amylovorus or Lactobacillus gallinarum, for example. Two strains of Lactobacillus j ohnsonii which have been genetically recombined to produce only L (+) - lactate have been isolated in particular. These strains were deposited on 02/20/97, under the terms of the Budapest Treaty, in the Collection Nationale de Cultures de Microorganismes, INSTITUT PASTEUR, 25, rue du Docteur Roux, F-75724 PARIS CEDEX 15, where they were given the deposit number CNCM I-1851 and the deposit number CNCM 1-1852 respectively.

The present invention is further related to a method for preparing such a strain, wherein the sequence of the gene encoding the enzyme D-lactate dehydrogenase is isolated from a host bacterial strain with the ability to survive in the intestine, to adhere to cells human intestines and perform immunomodulation, a direct mutagenesis is carried out on this sequence to give a modified sequence, this modified sequence is integrated into a conjugative vector, the conjugative vector is transferred by conjugation into the host bacterial strain, and then the host bacteria in which the sequence coding for the enzyme D-lactate dehydrogenase has been replaced by homologous recombination with the modified sequence. In the method according to the present invention, the sequence of the gene encoding the enzyme D-lactate dehydrogenase can be isolated from the host bacterial strain by PCR, by cloning or by complementation, for example. A directed mutagenesis can be carried out on this sequence to give a sequence modified by the method of Extension of Superposition by Splice of Gene (Molecular Biotechnology, RM Horton, 1995, 3_, page 93-99), which consists in generating a sequence genetics in which one or more nucleotides, for example, are introduced or deleted. To integrate the modified sequence into a conjugative vector of a bacterial strain donor of the host bacterial strain, a donor bacterial strain containing a conjugative vector, which does not have the capacity to replicate in the host bacterial strain, can be selected. a construction by linking the modified sequence to the first vector, which is unable to multiply in the bacterial strain donor of the host bacterium, this construction can be introduced into the donor bacterial strain, and then the donor bacterium can be selected in which the first vector and the conjugative vector have recombined, for example. The conjugative vector containing the modified sequence is therefore transferred by conjugation to the host bacterial strain. Then, the host bacterium is selected, in which the sequence coding for the enzyme D-lactate dehydrogenase has been replaced by homologous recombination with the modified sequence. The host bacterium that has the conjugative vector integrated in its genome, for example, can be selected on a medium containing certain antibiotics. In fact, through the integration of the conjugative vector into their genome, these bacteria can express the antibiotic resistance genes contained in the vector sequence. Finally, the host bacterium is selected in which the DNA sequences of the conjugative vector have been removed from the genome, with the exception of the modified sequence. This can be done by carrying out a first selection on a medium containing antibiotics, to select the bacteria sensitive to these antibiotics, that is, the wild type bacteria and the genetically transformed bacteria, which now contain only the fragment of the modified sequence of the gene, for example. A color enzyme test can then be carried out in the presence of D-lactate dehydrogenase, tetrazolium salt and diaphorase, to differentiate the wild type bacteria from the genetically transformed bacteria according to the present invention, for example. This enzymatic test makes it possible to demonstrate the fact that bacteria that do not produce D (-) - lactate can not oxidize D (-) - lactate when the enzyme D-lactate dehydrogenase is added to the medium; consequently, the tetrazolium salt in the medium is not reduced by the enzyme diaphorase, in the absence of oxidized D-lactate, and these bacteria remain colorless. Then, PCR can be performed on the genomic sequences of the genetically recombined host bacteria according to the present invention, using primers specific for the host bacterial strain, the PCR product can then be digested in the presence of specific restriction enzymes, and the size of the fragments generated in this way can be compared with those obtained, after digestion with these same restriction enzymes, from the genome of the wild-type host bacterium, for example. Finally, the present invention relates to the use of a bacterial strain obtained by carrying out the method according to the present invention in the preparation of food products. The method of preparation of the bacterial strains according to the present invention, and those genetically recombined bacterial strains, are characterized in greater detail below by means of biochemical and molecular analysis, with reference to the appended drawings, in which: - the Figure 1 shows the vector pLL83, which is the product of the vector ligand pGEMT, sold by Promega, MADISON, Wl-USA, and the modified sequence of the gene coding for the enzyme D-lactate dehydrogenase, - Figure 2 shows the vector pMD14, which is constructed from the pBlueScript SK + vector of Escheri chi a col i (Stratagene, LA JOLLA, CA - USA) and which contains the chloramphenicol resistance gene (cat) of the pNZ12 vector (Gasson et al., J Bacteriol., 154, 1 9, 1983) and the 5 'region upstream of the erythromycin resistance gene sequence of the pAMßl vector (Clewell et al., J. Bacteriol., 3_3, 426-428, 1974), which was isolated from the pUC plasmid. -838 (Mollet et al., J. Bacteriol., 175, 4315-4344, 1993), Figure 3 shows the vector pLL88, which is the product of the ligand of the vector fragment pLL83 comprising the modified sequence of the gene coding for the enzyme D-lactate dehydrogenase, in the vector pMD14, and finally Figure 4 shows the vector pLL91, which comprises the vector sequence pLL88 and that of the vector pAMßl (Clewell et al., J. Bacteriol., 3! 3_, 426-428, 1974).

I. Isolation of the sequence of the gene that codes for the enzyme D-lactate dehydrogenase Lactobacillus j ohnsonii Lal: Lactobacillus jonhnsonii Lal was grown on an MRS medium overnight at 37 ° C. The culture was then transferred to a tube containing an MRS medium, and allowed to grow at a D06oo of about 1 The Lactobacillus j ohnsonii Lal genome was isolated by the method described in the article "DNA tested for Lactobacillus delbrueckii" (B. Mollet et al., Applied and Environmental Microbiology, June 1990, vol 56 (6), p. 1967 - 1970). The sequence of the gene coding for the enzyme D-lactate dehydrogenase from Lactobacillus jonhnson Lal was then isolated by PCR, using as sequence-specific primers the sequences SEQ ID NO: 1 and SEQ ID NO: 2 described below, which are sequences of regions of the gene encoding the enzyme D-lactate dehydrogenase from Lactobacillus helveticus (Eur. J. Biochem., Cloning and overexpression of Lactobacillus helveticus D-lactate dehydrogenase gene in Escherichia coli, Kochhar et al., 208, 799-805, 1992). ). This gives a fragment of 890 bp, which was cloned into the pGEMT vector sold by Promega, MADISON, Wl - USA, and then the sequence was determined. To isolate the complete sequence of the gene coding for the enzyme D-lactate dehydrogenase from Lactobacillus j ohnsonii Lal, a Southern blot was then effected with different restriction enzymes, and the probe used was the sequence previously obtained by PCR. A 3 kb nucleotide sequence comprising two open reading frames of opposite orientation was thus isolated. A high degree of homology was found between one of the open reading frames and the sequence of the gene encoding the enzyme D-lactate dehydrogenase from Lactobacillus helveticus. The sequence of this open reading frame has a length of 1014 nucleotides, and has a homology of 85% with the sequence of the gene coding for the enzyme D-lactate dehydrogenase of Lactobacillus helveticus, and a homology of 81% with that of Lactobacillus bulgaricus (FEBS Lett., Bernard et al., 1991, 290, 61-64). This sequence codes for a 338 amino acid polypeptide.

II. Directed mutagenesis of the sequence encoding the enzyme D-lactate dehydrogenase Lactobacillus jonhnsonii Lal: Direct mutagenesis was carried out on the sequence isolated by the Gene Splice Superposition Extension method (Molecular Biotechnology, R. M. Horton, 1995, 3_, 93-99). This sequence was subjected by PCR to a suppression of 11 nucleotides and to an insertion of 3 nucleotides in the center. These sequence modifications have the effect of creating a Dral restriction site and eliminating an EcoRV restriction site. These two modifications of restriction sites were used as a marker to demonstrate the presence of a modified sequence of the gene coding for the D-lactate dehydrogenase of Ct oba ci ll us j ohnsoni i Lal in the different vectors used in the remainder of the construction, and in the selection of the mutants that have integrated only the modified sequence of the gene that codes for the enzyme D-lactate dehydrogenase. The gene sequence modified in this manner encodes a polypeptide of 181 amino acids, instead of a 338 amino acid polypeptide.

III. Cloning of the modified sequence of the gene coding for the enzyme D-lactate dehydrogenase from La ct oba ci ll us j ohn soni i Lal in the vector pGEMT of Escheri chi a coli: The modified sequence of the gene coding for the enzyme D- Lactate dehydrogenase was cloned into the vector pGEMT of Es ch eri chi ac ol i. This was done by ligating the modified gene sequence in this pGEMT vector containing the ampicillin resistance gene. This ligand mixture was then introduced into Escheri chi to col XLl-Blue by electroporation, and positive clones were selected in the presence of X-gal and IPTG (Sambrook et al., Molecular cloning: A Laboratory Manual, 2nd edition, 1989 ). The resulting vector, as shown in Figure 1, was called pLL83. The pLL83 vector was then purified by the alkaline lysis method (Sambrook et al., Molecular cloning: A Laboratory Manual, 2nd edition, 1989). The fragment comprising the modified sequence of the gene coding for the enzyme D-lactate dehydrogenase was then isolated from the pLL83 vector purified in this manner. This was done by carrying out a digestion with the restriction enzyme Sph I at the restriction site of Sph I on the pLL83 vector. The T4 polymerase enzyme was then reacted at this breaking site, to add nucleotides and obtain a blunt cut. Finally, a digestion with the restriction enzyme Spel was carried out. In a parallel operation, a digestion was carried out on a vector that is unable to replicate in the donor bacterial strain, ct ococc u s s a c t i s, and in the host bacterial strain, La c t oba ci l l s s j ohns oni i. This digestion was carried out in a restriction site, after which the enzyme T4 polymerase was reacted in this break, to add nucleotides and obtain a blunt cut. Finally, a digestion with the restriction enzyme Spel was carried out. The vector pMD14, described in Figure 2, can be used in particular to produce this construct. It is possible to carry out a digestion on this vector pMD14 with the restriction enzyme EcoRl, then to react the enzyme T4 polymerase, and finally carry out a digestion with the restriction enzyme Spel. The vector fragment pLL83 comprising the modified sequence of the gene coding for the enzyme D-lactate dehydrogenase is then introduced into the vector pMD14.

The ligand mixture is then introduced into Escherichia coli XLl-Blue by electroporation. About one hundred colonies of Escherichia coli XL1-Blue constructed in this way were then placed on microfiltration plates, then transferred to a cellulose membrane, and then lysed in situ, and hybridized with an internal fragment of the cell. gene encoding the enzyme D-lactate dehydrogenase from Lactobacillus j ohnsonii Lal. Three positive clones containing the vector pLL88, described in Figure 3, were selected in this way. The determination of the vector sequence pLL88 demonstrates the fact that it comprises the modified sequence of the gene coding for the enzyme D-lactate dehydrogenase of Lactobacillus jonhnsii. Lal, the regions of pGEMT that flank this sequence, and the vector pMD1.

IV. Introduction of vector pLL88 in strain Lactococcus lactis MG1363 (pAMßl): Vector pLL88 was introduced into the strain Lactococcus lactis MG1363 (pAMßl) by electroporation. The transformants produced in this manner were then placed on a GM17 agar medium containing 12 μg / ml chloramphenicol, and incubated at 30 ° C overnight. The vector pLL88 can not be replicated in Gram-positive bacteria. All Lactococcus lactis MG1363 bacteria (pAMßl) having integrated, by homologous region, the pLL88 vector comprising the chloramphenicol resistance gene in the pAMßl conjugative vector, described in Figure 5, were thus selected on this medium containing chloramphenicol. This construct, comprising the sequence of vector pLL88 and that of vector pAMßl, will be named subsequently the vector pLL91, described in Figure 4.

V. Conjugation conditions: Lactococcus lactis MG1363 containing the vector pLL91 was grown on a GM17 medium containing 12 μg / ml of chloramphenicol, and the Lactobacillus johnsonii Lal was cultured on an MRS medium. 0.2% of the culture of Lactobacillus johnsonii Lal thus prepared was inoculated in a tube containing a freshly prepared MRS medium, and the mixture was incubated for 5 hours 00 minutes at 37 ° C. The cultures of Lactococcus lactis MG1363 and Lactobacillus j ohnsonii Lal were then subjected to centrifugation at 3000 rpm for 5 minutes, and each residue was transferred to 10 ml of LCMG medium (Efthymiou et al., An antigenic analysis of Lactobacillus acidophilus, J. Infect. Dis., 1962, 110, 258 - 267) containing 10 g of trypticase, 5 g of yeast extract, 3 g of triptose, 3 g of K2HP04, 3 g of KH2P04 2 g of ammonium citrate, 5 ml of solution enriched in mineral salts, 1 g of Tween 80, 1 g of sodium acetate, 20 g of glucose and 0.2 g of cysteine. 1 ml of culture of the donor strain The ct ococcus lac ti s MG1363 prepared in this way was then mixed with 10 ml of culture of the recipient strain Ct oba ci ll us j ohn soni i Lal prepared in this way, and the mixture It was subjected to centrifugation. The supernatant was discarded, and the concentrated residue was deposited on PEG agar medium plates (Takemoto et al., Agrie. Biol. Chem., 1989, 53_, 3333-3334) containing 5 g of PEG6000, 15 g of agar , 1000 ml of sugar-free LCMG solution, 100 ml of solution containing sugar and 10 ml of mineral salt solution. These plates were left at room temperature until the residue was dry, and then this was covered with 10 ml of LCMG medium containing 7% agar. These plates were then covered overnight at 37 ° C, then the agar containing the bacterial cells that had grown was cut, and this agar was placed in tubes containing 10 ml of LCMG medium. These tubes were then vigorously shaken, and the cultures prepared in this manner were diluted in TS medium containing 1 g / 1 tryptone and 8.5 g / 1 NaCl. The diluted cultures were then plated on MRS agar medium plates containing 100 μg / ml fosfomycin and 14 μg / ml chloramphenicol, and incubated at 37 ° C for 48 hours 00 minutes under anaerobic conditions.

SAW . Conjugation and integration of vector pLL91 in Lactobacillus j ohnsonii Lal: Lactococcus lactis MG1363 containing vector pLL91 was conjugated with Lactobacillus j ohnsonii Lal. This conjugation had a frequency of between 1 x 10 ~ 5 and 3 x 10 ~ 7 transforming cells / vessels. Six colonies of Lactobacillus j ohnsonii Lal resistant to chloramphenicol and phosphinomycin were then selected and cultured at 37 ° C on an MRS medium before being transferred for a few hours at 45 ° C, to select Lactobacillus jonhnsonii Lal bacteria. it had integrated the vector pLL91 within its genome, by region homologa, in the sequence that codes for the enzyme D-lactate dehydrogenase. In fact, the vector pAMßl containing the pLL91 vector is unable to replicate at a temperature above 42 ° C. Thus, all Lactobacillus jonhnsonii Lal bacteria that were resistant to chloramphenicol and could grow at 45 ° C had the vector integrated pLL91 within its genome, by homologous region, in the sequence that codes for the enzyme D-lactate dehydrogenase. Therefore, the integration of the vector pLL91 into the Lactobacillus j ohnsonii Lal genome is obtained by a simple crossing, either in the 5 'terminal region of the gene sequence coding for D-lactate dehydrogenase, or in region 3 'terminal of the sequence of this gene.

VII. Verification of the integration of the modified sequence of the gene coding for the D-lactate dehydrogenase enzyme into the chromosomal DNA of Lactobacillus johnsonii Lal: The integration of the vector pLL91 into the genome of Lactobacillus jonhnsonii Lal was verified by PCR. This was done using specific primers for the Lactobacillus j ohnsonii Lal genome, whose sequences are the sequences SEQ ID NO: 3 and SEQ ID NO: 4 described below, and primers specific for the vector pLL91, whose sequences are the sequences SEQ ID NO: 5 and SEQ ID NO: 6 described below. The fragments amplified in this way by PCR were then digested with the restriction enzyme EcoRV, whose restriction site is located in the original sequence of the gene coding for the enzyme D-lactate dehydrogenase, and with the restriction enzyme Dral, whose site of restriction is located in the modified sequence of the gene encoding the enzyme D-lactate dehydrogenase, to demonstrate the fact that the integration by a single crossing had taken place in the 5 'terminal region or in the 3' terminal region of the original sequence of the gene. Lactobacillus j ohnsonii Lal bacteria were then genetically modified by integration of the modified sequence of the gene within the 5 'terminal region of the original sequence of the gene, and a bacterium of Lactobacillus j ohnsonii Lal genetically modified by integration of the modified sequence of the gene. gene within the 3 'region of the original sequence of the gene. The integration of vector pLL91 into the genome of Lactobacillus j ohnsonii Lal was then verified by carrying out a Southern blot of the genome of these two genetically modified Lac t oba ci l l us j ohnsoni i Lal bacteria, selected above. This was done by digesting the genomic DNA of these two bacteria with different restriction enzymes, whose restriction sites were located in the genome, on the vector pLL91 and on the modified sequence of the gene coding for D-lactate dehydrogenase. The hybridization probe used is a fragment of the original sequence of the gene encoding the enzyme D-lactate dehydrogenase. This demonstrated the fact that the genome fragments of the two genetically engineered bacteria obtained after digestion with different restriction enzymes are of a different size from the fragments obtained after digestion of the genome of the bacterium La ct oba ci ll us j ohnsoni i Lal natural type with these same restriction enzymes.

VIII. Integration resolution: The integration was solved by releasing the pLL91 vector of the genome. The loss of the vector pLL91 was obtained either by a simple crossing between the 5 'terminal region of the original sequence of the gene coding for the enzyme D-lactate dehydrogenase and that of the modified sequence of the gene, or by a simple crossing between the terminal 3 'region of the original sequence of the gene encoding the enzyme D-lactate dehydrogenase and that of the modified sequence of the gene. The integration was resolved at 37 ° C, which is a permissive temperature for the vector, to favor the release of the vector pLL91 of the genome. An enzymatic color test was performed in the presence of D-lactate dehydrogenase, tetrazolium salt and diaphorase, to differentiate the wild-type bacteria from the genetically transformed bacteria according to the present invention. This enzymatic test makes it impossible to demonstrate the fact that bacteria that do not produce D (-) - lactate can not oxidize D (-) - lactate when the enzyme D-lactate dehydrogenase is added to the medium; consequently, the tetrazolium salt that is in the medium is not reduced by the enzyme diaphorase, in the absence of oxidized D-lactate, and these bacteria remain colorless. The Examples below are given to illustrate the use of a bacterial strain according to the present invention in the manufacture of food products.

The percentages are given by weight, unless indicated otherwise.

Example 1 Yoghurts were prepared from the strain The ct oba ci l l us j ohn soni i CNCM 1-1851 obtained by carrying out the method according to the present invention. This was done by preparing 500 ml of 9% reconstituted skimmed milk powder, adding 0.1% yeast extract, and sterilizing the mixture in an autoclave for 15 minutes at 121 ° C. This was then allowed to cool to 40 ° C before the incorporation of 10% by volume of an active culture of the strain La ct oba ci ll us j ohnsoni i CNCM 1-1851, which contained 5 x 108 microorganisms / cm3. This preparation was incubated for 4 hours 00 minutes at 40 ° C to produce a ferment containing about 2.5 x 108 microorganisms / cm 3. In a parallel operation, a ferment containing about 5 x 10 8 Strept bacteria ococcus thermophi l thickeners / cm 3 was prepared by the method described above. A mixture containing 1.5% fat and 3% skimmed milk powder was pasteurized at 90 ° C for 30 minutes. 1% of the Lactobacillus johnsonii ferment CNCM 1-1851 and 3% ferment of Streptococcus thermophilus were then added to this mixture. This preparation was then mixed, and incubated for 4 hours 20 minutes at 40 ° C to give a preparation of pH 4.6. This gives yoghurts of pleasant texture, in which the concentration of Lactobacillus j ohnsonii CNCM 1-1851 is 1 x 108 cells / cm3, and the concentration of Streptococcus thermophilus is 1 x 10a cells / cm3.

Example 2 The procedure is as described in Example 1, except that the yogurts produced were diluted 50% with sterile distilled water, so that this preparation could be used in parenteral nutrition in a hospital environment.

Example 3 A fermented slurry was prepared from the strain Lactobacillus johnsonii CNCM 1-1852 obtained by carrying out the method according to the present invention. This was done by heating 1 1 of milk at 120 ° C for 15 minutes, to denature it.

Then, it was cooled to 37 ° C, and inoculated with 5% v / v of Lactobacillus johnsonii CNCM 1-1852 obtained by carrying out the method according to the present invention. The preparation produced in this way was incubated at room temperature for 18 to 24 hours, until its acidity level reached a value of 1%. Finally, the fermented milk produced was bottled and stored in refrigeration.

Example 4 A fresh cheese was prepared from the strain Lactobacillus j ohnsonii CNCM 1-1852 obtained to carry out the method according to the present invention. This was done by heating 1 1 of milk at 72 ° C for 15 minutes, and then allowing it to cool to 19 ° C. This was then inoculated with 0.5% v / v of a mixture of bacteria containing a Lactococcus lactis cremoris, a Lactococcus lactis diacetylactis and the strain Lactobacillus johnsonii CNCM 1-1852. The resulting mixture was incubated at about 20 ° C, until the pH of the milk was 4.6. The milk coagulated in this way was then poured into nylon bags, to drain the excess water contained in the fresh cheeses produced.heese was then mixed with an antifungal agent, such as potassium sorbate, to prevent fungi. Finally, it was homogenized by slow mixing, to give a fresh cheese with a smooth texture. The fresh cheeses produced by this procedure were packed in small jars, which can be stored at 4-5 ° C for 4 to 5 weeks.

LIQUIDITY OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT (TO) NAME: SOCIETE DES PRODUITS NESTLE (B) ADDRESS: AVENUE NESTLE 55 (C) CITY: VEVEY (D) STATE: CANTON OF VAUD (E) COUNTRY: SWITZERLAND (F) POSTAL CODE: 1800 (G) TELEPHONE: 021 924 34 20 (H) TELEFAX: 021 924 28 80 (ii) TITLE OF THE INVENTION: PRODUCTION OF L- (+) LACTATE (iii) NUMBER OF SEQUENCES: 6 (iv) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAMMING ELEMENTS: PatentIn Relay # 1.0, Version # 1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) NUMBER OF HEBRAS: one (D) CONFIGURATION: linear ( ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 1: GCTTACGCTA TTCGAAAAGA CG 22 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) NUMBER OF FIBERS: one (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: GTAGTGTAGA AGGCGGTGTG TGG 23 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) NUMBER OF HEBRAS: one (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: TGGTTGCCAA GTATTAG_17_(2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) NUMBER OF HEBRAS: one (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 4: GCTAAGTCAT TAGTGCC 17 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) NUMBER OF HEBRA: a (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5: ACAAAAGCTG GAGCTCC 17 (2) INFORMATION FOR SEQ ID NO: 6: (i) FEATURES OF THE SEQUENCE: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) NUMBER OF HEBRAS: one (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: TTGACGTTGA GCCTCGG 17 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as antece.de, property is claimed as contained in the following:

Claims (9)

1. Bacterial strain with the ability to survive in the intestine, to adhere to human intestinal cells and to perform immunomodulation, characterized because it is genetically recombined to produce only L (+) - lactate.

2. Strain according to claim 1, characterized in that the gene coding for the enzyme D-lactate dehydrogenase is inactivated.

3. A strain according to claim 1, characterized in that it is a strain of Lac tobaci ll us acidophilus, Lactobacillus jonhnsonii, Lactobacillus gasseri, Lactobacillus cri spasus, Lactobacillus amyl ovorus or Lactobacillus gallinarum.

4. Strain according to claim 1, characterized in that it is deposited in the CNCM under the number 1-1851.

5. Strain d-e according to claim 1, characterized in that it is deposited in the CNCM under the number 1-1852.

6. Method for producing a strain according to claim 1, characterized in that: - the sequence of the gene coding for D-lactate dehydrogenase is isolated from a host bacterial strain with the ability to survive in the intestine, to adhere to intestinal cells human and to effect immunomodulation, a directed mutagenesis is carried out on this sequence, to give a modified sequence, - this modified sequence is integrated into a conjugative vector, the conjugative vector is transferred by conjugation within the host bacterial strain, - and then the host bacterium is selected in which the sequence coding for the enzyme D-lactate dehydrogenase has been replaced by homologous recombination with the modified sequence.

7. Method of compliance with the claim 6, characterized in that, to integrate the modified sequence into a conjugative vector, - a donor bacterial strain containing a conjugative vector that does not have the capacity to replicate in the host bacterial strain is selected, - a construction is produced by ligating the modified sequence within a first vector, which is unable to multiply in the donor strain, - this construct is introduced into the donor bacterial strain, - and then the donor bacterium is selected, in which the first vector and the conjugative vector are they have recombined.

8. Method of compliance with the claim 6, characterized in that the host bacterium is selected in which the DNA sequences of the conjugative vector have been removed from the genome, with the exception of the modified sequence.

9. The use of a bacterial strain obtained in carrying out the method according to any of claims 6 to 8, characterized in that it occurs in the preparation of a food product.