Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
2000-01-24
2001-07-10
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S252900
Reexamination Certificate
active
06258587
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to bacteria strains which produce lactate and to
L. johnsonii
strains and further to recombinant, i.e., genetically modified/bacteria strains.
Fermentation is a degradation of a carbon source during which the final hydrogen acceptor is an organic compound. By way of lactic acid fermentation, 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 means of two specific NAD-dependent lactate dehydrogenases.
Some individuals are known to exhibit an intolerance to the reduction of lactose. This poor digestion of lactose is often due to the absence of a sufficient amount of &bgr;-galactosidase in the small intestine. Various studies (Kolars et al., N. Engl. J. Med., 310, 1-3, 1984; Marteau et al., Br. J. Nutr., 64, 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 lactose tolerance are due especially to the activity of the &bgr;-galactosidase of the bacteria contained in yoghurts during intestinal transit.
It is further known that D(−)-lactate can give rise to acidosis problems in children. For these reasons, the World Health Organization (FAO/WHO, 1967; 1974) recommends that D(−)-lactate should not be added to children's food, either on its own or as a racemic mixture with L(+)-lactate. Also, the daily consumption limit of D(−)-lactate for adults preferably does not exceed 100 mg/kg of the human body.
Bacterial strains which have been genetically recombined so as to produce only L(+)-lactate are now known.
T. Bhowmik et al. (Appl. Microbiol. Biotechnol., 432-439, 1994) describe a technique for the isolation and inactivation, by directed mutagenesis, of the gene coding for the enzyme D-lactate dehydrogenase of the strain
Lactobacillus helveticus
CNRZ32, particularly the strain
Lactobacillus helveticus
CNRZ32(pSUW104), which produces only L(+)-lactate. This strain is obtained by the electroporation of integrating vector pSUW104, which comprises vector pSA3 and the 0.6 kb SalI-SphI internal fragment of the gene coding for the enzyme D-lactate dehydrogenase of
Lactobacillus helveticus.
However, bacterial strains with the capacity to survive in the intestine, adhere to intestinal cells and effect immunomodulation, which have been genetically recombined so as 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 capacity to survive in the intestinal tract, which possess these beneficial properties on human health and produce only L(+)-lactate, so as to avoid the adverse effects due to D(−)-lactate.
The object of the present invention is to meet these needs.
SUMMARY OF THE INVENTION
The present invention provides recombinant bacterial strains wherein the strains are modified genetically to produce only L(+)-lactate and which have the capacity to survive in the intestine, adhere to human intestinal cells and effect
The present invention relates especially to bacterial strains in which the gene coding for the enzyme D-lactate dehydrogenase is inactivated.
The present invention relates especially to strains of
Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus gasseri, Lactobacillus crispatus, Lactobacillus amylovorus
or
Lactobacillus gallinarum.
The present invention further relates to the strain CNCM I-1851 and the strain CNCM I-1852.
A further subject of the present invention is a method of producing a bacterial strain which has been genetically recombined so as to produce only L(+)-lactate.
Finally, the present invention relates to the use of bacterial strains, obtained by carrying out the method according to the present invention for the preparation of food products.
DETAILED DESCRIPTION OF THE INVENTION
In this description, the expression “conjugative vector” is used to denote a DNA vector transferable by conjugation between two strains of different species of lactic acid bacteria.
Also, in this description, the expression “strain with the capacity to survive in the intestine” is used to denote a lactic acid bacterial strain which, after consumption, is found in the stool.
Finally, in this description, the expression “bacterial strain with the capacity to effect immunomodulation” is used to denote a lactic acid bacterial strain which has a beneficial effect on the immune system, especially the property of increasing the phagocytosis of the macrophages.
The present invention therefore relates to bacterial strains with the capacity to survive in the intestine, adhere to human intestinal cells and effect immunomodulation, which has been genetically recombined so as to produce only L(+)-lactate.
The present invention relates especially to bacterial strains with the capacity to survive in the intestine, adhere to human intestinal cells and effect immunomodulation, in which the gene coding for the enzyme D-lactate dehydrogenase has been inactivated.
The strains according to the present invention can be a strain of
Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobaclllus gasseri, Lactobacillus crispatus, Lactobacillus amylovorus
or
Lactobacillus gallinarum
, for example.
Two strains of
Lactobacillus johnsonii
which have been genetically recombined so as to produce only L(+)-lactate have been isolated in particular. These strains were deposited on Feb. 20, 1997, 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 CIDEX 15, where they were aven the deposit number CNCM I-1851 and the deposit number CNCM I-1852 respectively.
The present invention further relates to a method of preparing such a strain, wherein the sequence of the gene coding for the enzyme D-lactate dehydrogenase is isolated from a host bacterial strain with the capacity to survive in the intestine, adhere to human intestinal cells and 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 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 are selected.
In the method according to the present invention, the sequence of the gene coding for 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 modified sequence by the Gene Splicing Overlap Extension method (Molecular Biotechnology, R. M. Horton, 1995, 3, 93-99), which consists in generating a gene sequence in which one or more nucleotides, for example, are introduced or deleted.
To integrate the modified sequence into a conjugative vector of a donor bacterial strain 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 can be produced by ligation of the modified sequence into a first vector which is incapable of multiplying in the donor bacterial strain of the host bacterium, this construction can be introduced into the donor bacterial strain, and then the donor bacteria in which the first vector and the conjugative vector have recombined, for example, can be selected.
The conjugative vector containing the modified sequence is therefore transferred by conjugation into the host bacterial strain.
The host bacteria in which the sequence coding for the enzyme D-lac
Delley Michele
Germond Jacques Edouard
Lapierre Luciane
Mollet Beat
Pridmore Raymond David
Achutamurthy Ponnathapu
Nestec S.A.
Tung Peter P.
Vogt & O'Donnell, LLP
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