MX2013011233A - Natural derivative of the lactobacillus johnsonii strain cncm i-1225 deficient in d-lactic acid production. - Google Patents
Natural derivative of the lactobacillus johnsonii strain cncm i-1225 deficient in d-lactic acid production.Info
- Publication number
- MX2013011233A MX2013011233A MX2013011233A MX2013011233A MX2013011233A MX 2013011233 A MX2013011233 A MX 2013011233A MX 2013011233 A MX2013011233 A MX 2013011233A MX 2013011233 A MX2013011233 A MX 2013011233A MX 2013011233 A MX2013011233 A MX 2013011233A
- Authority
- MX
- Mexico
- Prior art keywords
- cncm
- strain
- lactobacillus johnsonii
- composition
- natural derivative
- Prior art date
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Abstract
The present invention generally relates to the field of probiotic bacteria. In particular, the present invention relates to natural derivatives of the johnsoniistrain Lactobacillus CNCM I-1225 that are deficient in D-lactic acid production.
Description
NATURAL DERIVATIVE OF THE CEPA DE LACTOBACILLUS JOHNSONII CNCM 1-1225,
DEFICIENT IN THE PRODUCTION OF D-LACTIC ACID
The present invention generally relates to the field of probiotic bacteria. In particular, the present invention relates to natural derivatives of the strain of LactobacHIus johnsonii CNCM 1-1225 which are deficient in the production of D-lactic acid.
Strain Lactobacillus johnsonii CN CM 1 -1225, also known as Lactobacillus johnsonii NCC533, or Lactobacillus acidophilus La 1, or Lactobacillus johnsonii Lj1, a human isolate (Bernet-Camard, MF, et al., (1997) Appl. Environ Microbiol 63, 2747-2753), is a probiotic that is currently commercialized with great success under the trademark Lc1.
The strain LactobacHIus johnsonii CNCM 1-1225 has several well-documented health benefits, including, for example, activities for immunomodulation (Haller, D., et al., 2000, Infect. Immun. 68: 752-759; , D., et al., 2000, Gut 47: 79-87, or Ibnou-Zekri, N., et al., 2003, Infect.immun.71: 428-436), or inhibition of pathogens (Bernet, MF , et al., 1994, Gut, 35: 483-489), and a long history of safe use.
One aspect that has limited the application of LactobacHIus johnsonii CNCM 1-1225 in some product categories, for example, in products intended for young children and infants, is the predominant production of the D-lactic acid isomer from the fermentation of the sugars. For example, if LactobacHIus johnsonii CNCM 1-1225 is grown, in MRS medium, it ferments lactose to D- and L-lactic acid in a proportion of 60: 40%.
The CODEX Directive for Infant Formula (CODEX Infant Formula
Directive) discourages the use of D-lactic acid and bacteria that produce D-lactic acid by infants less than three years of age due to its limited elimination of D-lactic acid, which can result in D-lactic acidosis. lactate
The evidence to support this conclusion is limited and is based mainly on the presence of D-lactic acid in food and not on the administration of bacteria that produce D-lactic acid, which are natural inhabitants of the gastro-intestinal tract.
Accordingly, several publications have challenged this position (Connolly, E. et al., NTRAfoods 3 (3), 37-49, 2004; Haschke-Becher, E., et al., 2008, Ann. Nutr. Etab. : 240-244, Mack, DR, 2004, Can. J. Gastroenterol 18: 671-675) but until now the recommendation of the CODEX Directive for the Infant Formula remains unchanged.
CODEX has essentially excluded probiotics that produce D-lactic acid as a supplement in infant formulas but has inspired the genetic engineering of strains that produce only L-lactic acid. An example of this development is the generation of a genetically modified organism (GMO), in particular a genetically modified Lactobacillus johnsonii strain, where the gene (IdhD) of the D-lactate dehydrogenase (D-LDH) was isolated, and a cloned copy In vitro truncated IdhD gene was used to inactivate the genomic copy by gene replacement (Lapierre, L, et al., 1999, Appl. Environ Microbiol. 65: 4002-4007).
This genetically modified strain was produced only for laboratory purposes and has never been used in food products since its genetic material has been altered using recombinant DNA technologies and therefore the strain is considered a GMO.
However, it would be desirable to have available the possibility that
to make the numerous health benefits of Lactobacillus johnsonii CNC 1-1225 strain also available to individuals, which is the consumption of D-lactic acid or bacteria that produce D-lactic acid that is not currently recommended.
Therefore, there is a strong need in the state of the art of a natural probiotic strain, in particular a natural derivative of the strain Lactobacillus johnsonii CNCM 1-1225, which is deficient in the production of D-lactic acid and which nevertheless be viable
The inventors of the present invention have faced this need.
Accordingly, it was the aim of the present invention to provide the state of the art with a derivative of Lactobacillus johnsonii strain CNCM 1-1225, which can also be applied to products aimed at infants and young children while complying with the current regulations of the CODEX Directive for Infant Formula, and that is natural and that is not considered a GMO.
The inventors of the present invention were surprised to note that they were able to achieve the object of the present invention through the subject matter of the independent claims. The dependent claims further develop the concept of the present invention.
The inventors have investigated the possibility of isolating a variant of Lactobacillus johnsonii La1 natural (non-GMO), viable and genetically stable that produces only L-lactic acid.
Changes in the genome sequence exist naturally, for example, due to a wrong repair of damaged DNA or errors in DNA replication, with a relatively low frequency.
To find these natural derivatives of the Lactobacillus strain
johnsonü CNCM 1-1225 The inventors of the present invention have evaluated approximately 21,000 colonies of Lactobacillus johnsonü CNCM 1-1225 and in particular have identified 2 natural variants deficient in D-lactic acid which achieve the objective of the present invention.
The inventors of the present invention have further determined the concentrations of D- and L-lactic acid in the supernatants of the MRS cultures and have shown that the concentration of relative D-lactic acid compared to the total lactic acid concentration in the culture supernatant it is below 4%; in particular, in one case 3.7% and in the second case 0%.
An analysis of the DNA sequence, predominantly identified point mutations in the lactate dehydrogenase gene, which alter the amino acid sequence of the enzyme and therefore its catalytic properties.
Both natural derivatives showed to be viable and stable without examples of reversion towards the production of D-lactic acid.
Accordingly, one embodiment of the present invention is a natural derivative of the strain of Lactobacillus johnsonü CNCM 1-1225, where the derivative of the strain of Lactobacillus johnsonü CNCM 1-1225 is deficient in the production of D-lactic acid.
For the best knowledge of the inventors, this is the first time that a natural derivative of the strain Lactobacillus johnsonü CNCM 1-1225, which is deficient in the production of D-lactic acid, is provided.
The expression "deficient in the production of D-lactic acid" is understood for the purpose of the present invention that a strain produces less than 5%, preferably less than 4%, and ideally 0% D-lactic acid compared to the total lactic acid production. The concentrations of D- and L-lactic acid can be measured in the supernatant of cell-free culture.
The total amount of lactic acid produced by the natural derivatives of the Lactobacillus johnsonii strain CNCM 1-1225 is initially related to cell growth. When the cells enter the stationary phase and stop the division, they continue to metabolize sugars and produce more lactic acid. However, the proportion of D- and L-lactic acid produced was found constant.
A "natural" derivative of Lactobacillus johnsonii strain CNCM 1-1225 refers to a strain that is not considered a GMO. A natural derivative as such may for example be obtained by monitoring colonies that are subjected to changes in the genomic sequence that occurs naturally, for example, due to a wrong repair of damaged DNA or errors in DNA replication. This natural presence of errors can be increased by subjecting the colonies to stress conditions, for example by the application of ethyl methane sulfonate, MSE.
For the purpose of this application the term "GMO" must be defined in accordance with DIRECTIVE 2001/18 / EC of the PARLIAMENT AND OF THE EUROPEAN COUNCIL (OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL) of March 12, 2001 about the liberation deliberate towards the environment towards genetically modified organisms and the rejection of Directive Council Directive 90/220 / EEC. Accordingly, a "genetically modified organism (GMO)" refers to an organism, with the exception of humans, where the genetic material has been altered in such a way that it does not naturally exist by natural mating and / or recombination.
Within the terms of this definition, genetic modification occurs at least through the use of:
(1) recombinant nucleic acid techniques involving the formation of new combinations of genetic material by the insertion of nucleic acid molecules produced either by means that are outside the organism, towards any virus, bacterial plasmid or other vector system and its incorporation into a host organism where they do not naturally exist but where they are capable of continuous propagation;
(2) techniques that involve the direct introduction to an organism of heritable material prepared outside the organism including micro-injection, macroinjection and micro-encapsulation; or
(3) cell fusion (including protoplast fusion) or hybridization techniques where living cells with new combinations of heritable genetic material are formed through the fusion of two or more cells by means of methods that do not exist naturally.
A strain is considered a "derivative" of the strain Lactobacillus johnsonü CNCM 1-1225, if it has a nucleic acid identity of at least 99.95%, for example of at least 99.99%, preferably of at least 99.995%. For example, a strain is considered a derivative of Lactobacillus johnsonü strain CNCM 1-1225 if it has not more than 500, for example not more than 100, preferably not more than 50 nucleic acid changes compared to the nucleic acid sequence of Lactobacillus johnsonü CNCM 1-1225.
The inventors of the present invention have found that the natural derivatives of the Lactobacillus johnsonü CNCM 1-1225 strain of the present invention exhibit mutations in the d-ldh gene, which is responsible for the deficient phenotype of D-lactic acid.
Accordingly, the natural derivatives of Lactobacillus johnsonü strain CNCM 1-1225 can have an altered sequence for the D-lactate dehydrogenase enzyme. For example, the natural derivative of Lactobacillus johnsonü strain CNCM 1-1225 may comprise an amino acid change of a glycine per acid
aspartic at the position of amino acid 153 and / or an amino acid change of serine by leucine at amino acid position 204 of the enzyme sequence D-lactate dehydrogenase.
These changes apparently alter the function of the enzyme D-lactate dehydrogenase in an important way, so that there is extremely little or no D-lactic acid produced.
Changes in the protein sequence of the enzyme D-lactate dehydrogenase are based on changes in the nucleic acid sequence of the D-lactate dehydrogenase gene.
Any natural change in the nucleic acid sequence of the D-lactate dehydrogenase gene that inactivates the resulting enzyme can achieve the subject matter of the present invention. Also at least one deletion of one or more subsequent nucleotides within the wild-type sequence, or part or all of the D-lactate dehydrogenase gene, can achieve the subject matter of the present invention.
The inventors of the present invention have analyzed the nucleic acid sequence of the D-lactate dehydrogenase gene in the natural derivative of Lactobacillus johnsonii CNCM 1-1225 of the present invention.
Therefore, the present invention also relates to a natural derivative of the strain Lactobacillus johnsonii CNCM 1-1225, where the nucleic acid sequence of the D-lactate dehydrogenase gene in the natural derivative of the strain of Lactobacillus johnsonii CNCM 1 -1225 comprises a transition from G to A at nucleic acid position 498 and / or a transition from C to T at the 651 nucleic acid position.
While such changes that result in the inactivation of D-lactate dehydrogenase can occur spontaneously at low frequencies in nature, they are randomized, and can be repaired again to arrive at the parental sequence (wild type) on the same frequency.
This repair frequency may even be higher if the inactivated gene imparts a growth disadvantage to the variant. Given the large number of generations from the crop collection to the final product, it is important that these changes are stable, especially since they are single base pair changes, which are genetically less stable than deletions or deletions.
Advantageously, these changes were also found stable.
Two examples of natural derivatives of Lactobacillus johnsonii strain CNCM 1-1225 that achieve the objective of the present invention were isolated, purified and characterized in detail.
The present invention, therefore, relates to a natural derivative of the strain Lactobacillus johnsonii CNCM 1-1225, which can be selected from the group consisting of the strain of Lactobacillus johnsonii CNCM I-4434 and of the strain Lactobacillus johnsonii CNCM 1 -4441.
Both strains of Lactobacillus johnsonii CNCM I-4434 and Lactobacillus johnsonii CNCM 1-4441 were deposited on February 8, 201 1, with the National Collection of Microorganism Cultures (Collection Nationale de Cultures de Microorganismes, CNCM), Institui Pasteur, 25 Rue du Docteur Roux, F-75724 Paris Cedex 5, France, under the Budapest Treaty.
Both strains were completely sequenced.
The strain of Lactobacillus johnsonii CNCM 1-1225 was deposited on June 30, 1992, with the CNCM, under the Budapest Treaty.
The strain of Lactobacillus johnsonii CNCM I-4434 contains a total of 37 nucleic acid changes, including the elimination of a single base pair. This includes the expected change in the gene sequence of D-lactate dehydrogenase, which presumably is responsible for the deficiency in the production of D-lactic acid from this strain, plus two changes in genes in profane Lj965, which can be considered as "selfish" DNA with no probable impact on the physiology of the bacteria. The remaining predicted genes / proteins are mainly annotated only vaguely, but several changes affect the genes that are most important for bacterial growth. There are changes in six genes whose function is predicted, which is essential for growth, mainly LJ1 1 18 - DNA topoisomerase I, LJ 1 197 - penicillin binding protein 1 A, LJ1283 - orotate phosphoribosyltransferase, LJ1492 - alpha subunit of DNA polymerase III, LJ1493 - prolyl-tRNA synthetase and LJ1600 - peptide chain release factor 3. All these functions are essential for the growth of bacteria since they can not be supplemented through growth medium, the exception for this could be LJ1283. That the effects of individual, and cumulative, changes have no significant adverse effects on growth is confirmed by the growth of Lactobacillus johnsonii CNCM I-4434 at a similar rate as La1 in the MRS broth, and also achieves similar final UFCs in MRS and in industrial environment. A final final secondary change to be highlighted is LJ0854 - the small subunit of β-galactosidase, which has no consequences with respect to lactose fermentation.
The strain of Lactobacillus johnsonii CNCM 1-4441 contains a total of 27 nucleic acid changes, including the expected change in the D lactic acid dehydrogenase gene responsible for the deficiency in the production of D-lactic acid by this strain plus a change in each profane Lj965 and Lj928, plus a change in the transposase of the insertion sequence ISLjo5. These are changes in three genes whose function has been predicted to be important for growth, namely LJ1009 - Tu elongation factor, LJ1516 - particular protein recognition signal and LJ1600 - peptide chain release factor 3. Despite these individual changes and
Combined CNCM 1-4441 achieves similar UFC endings in MRS and in industrial environment compared to La1.
The present invention also extends to the natural derivatives of Lactobacillus johnsonii strain CNCM 1-1225 according to the present invention, where the natural derivative of Lactobacillus johnsonii strain CNCM 1-1225 is present as a biologically pure culture.
The natural derivatives of Lactobacillus johnsonii strain CNCM 1-1225 according to the present invention can be cultured according to any suitable method and can be prepared by addition to the compositions of the present invention, for example, by lyophilization or by spray drying.
The probiotic strain Lactobacillus johnsonii CNCM 1-1225 provides numerous well-documented health benefits some of which are detailed above.
The term "Probiotic" refers to microbial cell preparations or components of microbial cells that have a beneficial effect on the health or well-being of the host. (Salminen S, et al. "Probiotics: how should they be defined" Trends Food Sci. Technol. 1999: 10 107-10).
The natural derivatives of Lactobacillus johnsonii strain CNCM I-1225, according to the present invention, can essentially be considered as bioequivalent, in view of the benefits provided to health.
Scientific work has shown that also the supernatant of cell-free culture isolated from a biologically pure culture of Lactobacillus johnsonii strain CNCM 1-1225 has various health benefits (see for example, Bernet-Camard, M.- F., et al., 1997, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, pp. 2747-2753).
Therefore, the present invention further extends to the cell culture supernatant isolated from a biologically pure culture of a natural derivative of Lactobacillus johnsonü strain CNCM 1-1225 according to the present invention.
To the extent that the natural derivative of the Lactobacillus johnsonü strain CNCM 1-1225 and the cell-free culture supernatant of the present invention possesses numerous health benefits they can be used to treat or prevent disorders in the human or animal body. .
Accordingly, the present invention relates to a composition comprising the natural derivative of Lactobacillus johnsonü strain CNCM 1-1225 and / or the cell-free culture supernatant according to the present invention to be used in the preparation of a composition for use in a method intended for therapy treatment for the human or animal body.
The present invention also relates to the use of a composition comprising the natural derivative of the strain of Lactobacillus johnsonü CNCM 1-1225 and / or the supernatant of cell-free culture according to the present invention in the preparation of a pharmaceutical composition or of a medication.
The strain of Lactobacillus johnsonü CNCM 1-1225 has been extensively studied for its associated activities of probiotics, including immunornodulation (Haller, D., et al., 2000, Infect. Immun., 68, 752-759; Haller, D., et. al., 2000, Gut 47, 79-87, Ibnou-Zekri, N., et al., 2003, Infect.Immun. 71, 428-436), inhibition of pathogens (Bernet, MF, et al., 1994, Gut 35, 483-489), and binding to epithelial cells (Neeser, JR, et al., 2000, Glycobiology 10, 1 193-1 199; Granato, D., et al., 1999, Appl. Environ. Microbiol. 65, 1071-1077).
Due to bioequivalence, the natural derivatives of the strain of
Lactobacillus johnsonii CNCM 1-1225 and / or the cell-free culture supernatant thereof according to the present invention provides the same health benefits.
Accordingly, the present invention relates to a composition comprising the natural denucleated Lactobacillus johnsonii strain CNCM 1-1225 and / or the cell-free culture supernatant according to the present invention for use in the treatment or prevention of disorders related to a weakened immune system. The present invention also relates to the use of the natural derivative of the strain of Lactobacillus johnsonii CNCM 1-1225 and / or the supernatant of cell-free culture according to the present invention for the preparation of a composition for treating or preventing disorders related to a weakened immune system.
Disorders related to the weakened immune system are well known in the art and those skilled in the art will understand that the disorders are related to a weakened immune system.
Typical examples of disorders related to a weakened immune system may be selected from the group consisting of influenza, rhinitis, common cold, and combinations thereof.
The composition comprising the natural derivative of the strain of Lactobacillus johnsonii CNCM 1-1225 and / or the cell-free culture supernatant according to the present invention can also be used in the treatment or prevention of disorders related to cell attachment. cellular invasion by enterovirulent bacteria or viruses.
The present invention also extends to the use of the natural derivative of Lactobacillus johnsonii strain CNCM 1-1225 and / or the cell-free culture supernatant according to the present invention for the preparation of a composition for treating or preventing disorders related to cell attachment and cellular invasion by enterovirulent bacteria or viruses.
Enterovirulent bacteria and viruses are well known in the art. The European Food Safety Authority (EFSA) has published this list of pathogens in November 2010.
The enterovirulent bacterial or viral species may, for example, be selected from the group consisting of Salmonella strains; Campylobacter; Listeria; Escherichia coli, such as ETEC, EHEC, EPEC, or EIEC strains, for example; Yersinia; Shigella; Toxin-producing bacteria, such as Staphylococcus aureus, Clostridium botulinum, or Bacillus cereus; Vibrio vulnifucus / parahaemolyticus; rotavirus; norovirus; Verotoxigenic E. coli; Enterobacter sakazakii C. toxigenic perfringens (type A and B); parasites originating from food, such as Echinococcus, Toxoplasma, or Giardia; Helicobacter pylori; Clostridium difficile; Clostridium tetani; or combinations thereof.
It is evident to those skilled in the art, which disorders are related to which enterovirulent bacterial or viral species.
For example, the disorder related to cell attachment and cell invasion by enterovirulent bacterial or viral species can be selected from the group consisting of lower respiratory tract infections, gastrointestinal tract infections, otitis media, and combinations thereof.
The composition of the present invention can be any type of composition as long as it is suitable for administration to humans or animals.
In particular, the composition of the present invention may be for
administered orally, enterally, parenterally or topically. These can be provided in any galenic form normally available for the selected administration mode.
The composition of the present invention can be administered to any age group.
Preferably, the composition of the present invention is to be administered during the cold season, for example, from autumn to spring.
It can also be consumed at any time. It may be preferable to consume the composition of the present invention in the morning, for example, to stimulate the immune system for the day.
The composition, for example, can be selected from the group consisting of food compositions, pet food compositions, beverages, dairy products, nutritional formulas, infant formulas, food additives, nutraceuticals, pharmaceutical compositions, food ingredients and / or compositions. cosmetic
For example, the composition can be selected from the group consisting of acidified milk products, such as yogurt or yoghurt beverages; or powders based on milk.
In particular, powdered products may be preferable, if the composition is provided in the form of a stable powder during storage. To obtain storage stability and to ensure the viability of the probiotics, the composition can be provided with a water activity of less than 0.2, for example in the range of 0.19 to 0.05, preferably less than 0.5. The water activity or aw is a measure of the energy status of water in a system. It is defined as the vapor pressure of water that is derived from the powder / product divided by that of pure water at the same temperature; therefore, pure distilled water has a water activity of exactly one.
The compositions of the present invention can be cleaning, protective, treatment or care creams, skin care lotions, gels or foams, such as cleansing or disinfecting lotions, bath compositions or deodorant compositions.
In relation to this, the compositions for external topical administration can be, in particular, aqueous, idroalcoholic or oily solutions, solutions or dispersions of the lotion or serum type, emulsions of liquid or semi-liquid consistency, of the milk type, obtained by dispersion of a fatty phase in an aqueous phase (C7W) or vice versa (W / O), or suspensions or emulsions of soft, semi-solid or solid consistency, of the cream type, aqueous or anhydrous gels, microemulsions, microcapsules, microparticles, or vesicular dispersions of type ionic and / or nonionic.
A topical composition according to the invention can be advantageously formulated in any galenic form which is suitable for hair care, especially in the form of a hair lotion, a shampoo, especially an anti-dandruff shampoo, a hair conditioner, a detangler, a hair gel or cream, a styling lacquer, a hair setting lotion, a treatment lotion, a coloring composition (especially for oxidation staining) optionally in the form of a coloring shampoo, a lotion for hair restructuring, a permanent or curly composition, a lotion or gel to combat hair loss, an anti-parasitic shampoo or a medicated shampoo, especially an anti-seborrhea shampoo, a scalp care product, which It is especially anti-irritant, anti-aging or restructuring, or it activates blood circulation.
When the composition of the invention is an emulsion, the proportion of the fatty phase may be in the range of 5% to 80% by weight, and preferably 10% to 50% by weight, based on the total weight of the composition. The oils, emulsifiers and co-emulsifiers used in the composition in the form of an emulsion are chosen from those conventionally used in the field of cosmetics and / or dermatology. The emulsifier and the co-emulsifier can be present, in the composition, in a proportion ranging from 0.3% to 30% by weight, and preferably from 0.5% to 20% by weight, with respect to the total weight of the composition.
When the composition of the invention is a gel or oily solution, the fatty phase may represent more than 90% of the total weight of the composition.
Galenic forms for topical administration may also contain adjuvants which are customary in the field of cosmetics, pharmacy and / or dermatology, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, perfumes , binders, screens, odor absorbents and coloring agents. The amounts of these different adjuvants are those that are used conventionally in the field considered, and are, for example, from 0.01% to 20% of the total weight of the composition. Depending on their nature, these adjuvants can be introduced in the fatty phase and / or in the aqueous phase.
As fatty substances which can be used in the invention, mention may be made of mineral oils, such as, for example, hydrogenated polyisobutene and liquid petrolatum, vegetable oils such as, for example, a liquid fraction of shea butter, sunflower oil and apricot kernel oil, animal oils such as, for example, perhydrosqualene, synthetic oils, in particular Purcellin oil, isopropyl myristate and ethylhexyl palmitate, unsaturated fatty acids and fluorinated oils such as, for example, perfluoropolyethers. The use may also be composed of fatty alcohols, fatty acids, such as, for example, stearic acid and such as, for example, waxes, in particular, paraffin wax, carnauba wax and beeswax. The use may also comprise silicone compounds such as silicone oils and, for example, cyclomethicone and dimethicone, and silicone waxes, resins and gums.
As emulsifiers can be used in the invention, mention may be made, for example, of glyceryl stearate, polysorbate 60, the mixture of cetylstearyl alcohol / cetylstearyl alcohol oxyethylenated comprising 33 mol of ethylene oxide, marketed under the name Sinnowax AO® by the company Henkel, the mixture of PEG-6 / PEG-32 / glycol stearate marketed under the name of Tefose® 63 by the company Gattefosse, PPG-3 myristyl ether, silicone emulsifiers, such as cetyl dimethicone copolyol and monostearate or Sorbitan tristearate, PEG-40 stearate, or oxyethylenated sorbitan monostearate (20 EO).
As solvents that can be used in the invention, mention may be made of minor alcohols, especially ethanol and isopropanol, and propylene glycol.
The composition of the invention can also advantageously contain a thermal and / or mineral water, in particular selected from Vittel water, waters from the Vichy source, and water from the Roche Posay.
As hydrophilic gelling agents, mention may be made of carboxylic polymers such as carbomer, acrylic copolymers such as acrylate / alkyl acrylate copolymers, polyacrylamides, and in particular to the mixture of polyacrylamide, C13-14 isoparaffin and Laurato-7 marketed under the name of Sepigel 305® by the company SEPPIC, polysaccharides, for example derivatives such as
hydroxyalkylcelluloses, and in particular hydroxypropylcellulose and hydroxyethylcellulose, natural gums such as guar gum, locust bean gum, carob and xanthan gum, and clays.
As lipophilic gelling agents, mention may be made of modified clays such as bentones, metal salts of fatty acids, such as aluminum stearates and hydrophobic silica, or other ethyl celluloses and polyethylenes.
The compositions according to the invention can also be solid preparations which constitute soaps or cleaning sticks.
They can also be used for dandruff in the form of solutions, creams, gels, emulsions or foams, or alternatively in the form of aerosol compositions which also contain a propellant under pressure.
In the case of oral use according to the invention for oral administration, the use of an ingestible carrier or vehicle is preferred. The ingestible support or vehicle may be of a different nature depending on the type of composition under consideration.
Milk, yogurt, cheese, fermented milks, fermented products based on milk, ice cream, products based on cereal or products based on fermented cereal, breakfast cereals, cereal or granola bars, powders based on milk, formulas infant and baby foods, confectionery food products, of the chocolate or cereal type, animal feed, in particular for pets, tablets, capsules or gel pills, liquid bacterial suspensions, oral supplements in dry form and oral supplements in liquid form are especially suitable to be used as a support or ingestible vehicle.
The composition according to the invention to be administered orally can be formulated for example in the form of coated tablets, gel capsules, gels, emulsions, tablets, capsules, hydrogels, food bars, loose or compact powders, suspensions or solutions liquids, confectionery products, fermented milks, fermented cheeses, gum gum, toothpaste or spray solutions or food vehicles.
Tablets or pills, oral supplements in dry form and oral supplements in liquid form are suitable for use as dietary or pharmaceutical carriers or as food vehicles.
The composition can be, for example, a food supplement, which can be formulated by means of customary processes to produce in particular sugar-coated tablets, gel capsules, gels, emulsions, tablets, capsules and hydrogels that allow controlled release.
In particular, a microorganism according to the invention can be incorporated in all forms of food supplements or fortified foods, for example food sticks or compacted or non-compacted powders. The powders can be diluted in water, soda, dairy products or soy derivatives, or can be incorporated into food bars.
A microorganism of the invention, a fraction thereof and / or a metabolite thereof, can on the other hand be formulated with the usual excipients and components for such oral compositions or food supplements, i.e. in particular fatty and / or aqueous components, humectants , thickeners, preservatives, texture agents, flavor improvers and / or coating agents, antioxidants, preservatives and colorants that are common in the food industry.
Formulation agents and excipients for oral compositions, and in particular for food supplements, are known in the art and will not be subject to a detailed description in this application.
In therapeutic applications, the compositions are administered in a
enough to at least partially cure or stop the symptoms of a disease and its complications. An adequate amount to accomplish this is defined as "a therapeutically effective dose". The amounts effective for this purpose will depend on a number of factors known to those skilled in the art such as the severity of the diseases and the weight and general condition of the patient.
In prophylactic applications, as the compositions according to the invention are administered to a susceptible patient or otherwise at risk of suffering from a particular disease in an amount that is sufficient to at least partially reduce the risk of developing the disease. An amount as such is defined as "a prophylactically effective dose". Again, the precise amounts will depend on a number of patient-specific factors such as the patient's health and weight status.
The compositions of the present invention comprise at least one natural derivative of Lactobacillus johnsonii strain CNCM 1-1225 and / or the cell-free culture supernatant of the present invention in a therapeutically or prophylactically effective dose.
Those skilled in the art will be able to adjust the dosage accordingly.
For example, the composition may comprise the natural derivative of Lactobacillus johnsonii strain CNCM 1-1225 according to the present invention in an amount of 106 to 1012 ufe, for example 108 to 1010 ufe per daily dose.
Previous work has also shown that the strain of L. johnsonii CNCM 1-1225 non-replicating can be used in the treatment or prevention of disorders related to the immune system including infections, see application WO 2010/133475, fully incorporated herein by reference . It was found that strain L. L. otinsonii CNCM 1-1225 strongly induces the constitutive expression of hBD1, and that strain L. johnsonii CNCM 1-1225 thermally treated positively regulates hBD1, more intensely than its living counterpart.
Accordingly, the natural derivative of Lactobacillus johnsonii strain CNCM 1-1225 according to the present invention may also be present in a non-replicating form.
The "non-replicating" natural derivatives of the strain of Lactobacillus johnsonii CNCM 1-1225 include derivatives, which have been heat treated. This includes natural derivatives of Lactobacillus johnsonii strain CNCM 1-1225 that are inactivated, killed, are non-viable and / or are present as fragments such as DNA, metabolites, cytoplasmic compounds, and / or cell wall materials.
The term "non-replicating" means that there are no viable cells and / or colony forming units that can be detected by classical plating methods. Such classical plating methods are summarized in the microbiology book: James Monroe Jay, Martin J. Loessner, David A. Golden. 2005. Modern food microbiology. 7th edition, Springer Science, New York, N.Y. 790 p. In general, the absence of viable cells can be demonstrated as follows: there is no visible colony on agar plates or there is no increase in turbidity in the liquid growth medium after inoculation with different concentrations of bacterial preparations (samples "non-replicating") and with an incubation under appropriate conditions (aerobic and / or anaerobic atmosphere for at least 24 hours).
Obviously, microorganisms that do not replicate do not form colonies, therefore, this term should be understood as the amount of non-replicating microorganisms that are obtained from 106 and 1012 cfu / g of replicating bacteria.
The composition may also comprise the natural derivative of the strain
of Lactobacillus johnsonii CNCM 1-1225 according to the present invention in an amount of 0.005 mg - 5000 mg, for example 0.5 mg to 50 mg, per daily dose.
Those skilled in the art will understand that they can freely combine all the features of the present invention described herein, without departing from the spectrum of the invention described herein. In particular, the features described for the use of the present invention can be applied to the food product of the present invention and vice versa.
Advantages and additional features of the present invention are apparent from the following Examples and Figures.
Figure 1 shows the "survival curve" for Lactobacillus johnsonii CNCM 1-1225 treated with ethyl methane sulfonate.
Figure 2 shows the molecular process of the natural mutations originating from the oxidation of a G base to the segregation of DNA strands resulting in a mixture of parent and modified DNA types and a modified parental mixed colony.
Figure 3 shows the gene sequence of the D-lactate dehydrogenase gene from Lactobacillus johnsonii CNCM 1-1225 (SEQ ID NO: 1) and the changes identified in the corresponding genes in Lactobacillus johnsonii CNCM I-4434 and Lactobacillus johnsonii CNCM 1-4441 that they are locked in circles. The translated enzyme D-lactate dehydrogenase enzyme (SEQ ID NO: 2) is also shown with the corresponding changes for Lactobacillus johnsonii CNCM I-4434 and Lactobacillus johnsonii CNCM I-4441.
Figure 4A shows the sequence of the D-lactate dehydrogenase gene from Lactobacillus johnsonii CNCM I-4434 (SEQ ID NO: 6) with the base at position 61 1 enclosed in a square and altered compared to the Lactobacillus progenitor strain
johnsonii CNCM 1-1225 C).
Figure 4B shows the enzyme sequence of the D-lactate dehydrogenase of strain Lactobacillus johnsonii CNCM I-4434 (SEQ ID NO: 8) with the amino acid at position 204 enclosed in a square and altered compared to the progenitor strain Lactobacillus johnsonii CNCM 1-1225 (S).
Figure 5A shows the gene sequence of the D-lactate dehydrogenase gene from Lactobacillus johnsonii CNCM 1-4441 (SEQ ID NO: 7) with the base at position 458 enclosed in a square and altered compared to the parental strain Lactobacillus johnsonii CNCM 1- 1225 (G).
Figure 5B shows the protein sequence of the D-lactate dehydrogenase enzyme from Lactobacillus johnsonii CNCM 1-4441 (SEQ ID NO: 9) with the amino acid at position 153 enclosed in a square and altered compared to the parental strain Lactobacillus johnsonii CNCM 1-1225 (G).
Example 1
Treatment of cultures of Lactobacillus johnsonii CNCM 1-1225 with ethyl methane sulfonate. 100 nl_ samples, containing approximately 108 colony-forming units of Lactobacillus johnsonii CNCM I-1225 from a 16-hour culture, were washed 3 times with Dulbecco's phosphate-buffered saline. The cells were finally suspended in 1 ml of PBS and 0.10 was added. of ethyl methane sulfonate and incubated at 37 ° C without agitation. The treated cells were washed twice in PBS, the CFUs of the treated and untreated cultures were determined and plotted as the survivors to deliver the "survival curve" shown in Figure 1. The conditions that produce 1% of Survivors were initially targeted and enclosed in brackets with the before and after time points, cells diluted and plated as single colonies on MRS plates. The remaining treated cells were then used to inoculate 10 ml of MRS broth and incubated for 16 hours for growth at 37 ° C. The culture was then diluted and seeded on MRS plates to produce individual colonies for detection.
Mutations occur naturally in bacteria, primarily through the oxidation of guanosine residues (G) in double-stranded DNA and typically produce single-base pair changes when resolved by DNA replication as shown in Figure 2. This results in a natural mutation profile with changes of G by adenosine (A) and cytidine (C) by thymidine (T), depending on which strand of DNA was sequenced. Ethyl methane sulfonate acts by chemical oxidation of the G bases and results in the same mutation profile as natural mutations, mainly G by A and C by T, depending on which DNA strand was sequenced. This was subsequently confirmed by DNA sequencing of the D-lactate dehydrogenase gene and genome sequences, where the vast majority of the changes observed were either G by A or C by T.
Example 2
Selection of individual colonies for strains deficient in the production of D-lactic acid. Individual colonies treated with ethyl methane sulfonate were transferred to 96-well plates containing 200? of MRS broth and incubated at 37 ° C for 24 hours to form mini-cultures. Culture growth was estimated by absorbance at 620 nm using a Tecan sunrise microplate reader. Ten mixed? of the culture supernatant with 190 or a reaction mixture containing 100 mM Tris HCl pH 9, 2.5 mM EDTA pH 8.0, 20 U / ml D-lactate dehydrogenase (from Leuconostoc mesenteroides), NAD (? -nicotinamide) adenine dinucleotide) 1 mg / ml
plus 3% hydrazinium hydroxide and incubated at room temperature for 1 hour. The absorbance at 320 nm (formation of? -NADH) was measured using a Tecan sunrise microplate reader and the data was exported to Excel for analysis. Controls containing only MRS and culture supernatant of Lactobacillus johnsonii CNCM 1-1225 were included in each plate at concentrations of 5%, 25%, 50% or 100%, as standards for the normalization of the data.
This is a phenotypic analysis of the isolated colonies to determine the presence or absence of D-lactic acid in the culture medium after growth. When individual colonies are controlled for the presence of D-lactic acid it is also clear that "mixed" cultures containing 50% of a producer of D-lactic acid and 50% of a non-producer of D-lactic acid will result in a culture that is "positive" for the presence of D-lactic acid. The phenotype deficient in D-lactic acid to which we are directed, is then considered as "recessive" for the D-lactic acid production phenotype. This is important when considering the scheme of mutagenesis shown in Figure 2, as a single base of oxidized G that will result in a final colony that is a mixture of both parents and altered and therefore phenotypically positive individuals. for D-lactic acid. In this case the inclusion of growth in MRS broth after treatment with ethyl methane sulfonate was essential for the isolation of strains deficient in the production of D-lactic acid. After reviewing about 21,000 individual colonies treated with ethyl methane sulfonate, 15 colonies deficient in the production of D-lactic acid were isolated and subjected to further analysis.
Example 3
Determination of levels of D-lactic acid in culture medium. The
cultures were grown in MRS broth at 37 ° C for 16 hours and the bacteria were removed by centrifugation. To determine the concentrations of D-lactic acid, the cell-free culture supernatants were diluted in water, analyzed as described above and compared to a standard curve prepared with dilutions of sodium D-lactic acid. The concentrations of L-lactic acid were determined in the same way by the exchange of the enzyme D-lactate dehydrogenase with rabbit muscle L-lactic dehydrogenase and using sodium L-lactic acid as standard. The results of this analysis for CNCM 1 -4431 and CNCM I-4434 are shown in Table 1 and include the controls of Lactobacillus johnsonii CNCM 1 -1225, Lactobacillus johnsonii NCC9006 with an inactivated GMO gene of D-lactate dehydrogenase and Lactobacillus paracasei NCC2461 and Lactobacillus rhamnosus NCC4007, both considered as producing strains of L-lactic acid.
The strain of Lactobacillus paracasei NCC2461 (accession number: CNCM I-21 16) was deposited under the Budapest Treaty on January 12, 1999, with the CNCM (whose address has already been mentioned). The strain of Lactobacillus rhamnosus NCC4007 (accession number: CGMCC 1.3724) was deposited under the Budapest Treaty in October 2004, with the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinean Academy of Sciences, No 1, West Beichen Road, Chaoyang District, Beijing 100101, China. The strain of Lactobacillus johnsonii NCC9006 is the strain that is derived from Lactobacillus johnsonii La1, as described in the article by the author Lapierre et al. entitled "D-lactate dehydrogenase Gene (IdhD) Inactivation and Resulting Metabolic Effects in the Lactobacillus johnsonii strains La1 and N312" (Appl Environ Microbiol 1999, 65 (9): 4002).
Table 1 . Values of D- and L-lactic acid determined for Lactobacillus johnsonii CNCM 1-1225, Lactobacillus johnsonii CNCM 1-4431 and CNCM I-4434 from cell-free cultures. Included as controls are the strains NCC9006, a strain of LactobacHIus johnsonii with an inactivation of GMO in the gene of D-lactate dehydrogenase, LactobacHIus paracasei NCC2461 and LactobacHIus rhamnosus NCC4007, both considered as producing strains of L-lactic acid. Experiments were performed in triplicate and delivered as the mean value plus the standard deviation in parentheses.
Acid
% Acid D- Lactic acidic acid L-lactic acid D-lactic acid
lactic
Total*
CNCM 1-1225 14 4.8 (0.17) 9.2 (0.27) 65.7
CNCM I-4434 19 18.3 (0.3) 0.70 (0, 11) 3.7
CNCM 1-4441 20, 1 20, 1 (0.4) < 0.05 *** < 0.3 ****
NCC9006 21, 2 21, 2 (3.25) < 0.05 *** < 0.3
NCC2461 16.8 16.3 (0.97) 0.48 (0.02) 2.9
NCC4007 15.8 5.2 (0.49) 0.56 (0.03) 3.5
Lactic acid values in g / l.
only one of the samples delivered a value over the lower limit of quantification.
***, below the lower quantification limit.
"***,% calculated using the value of the lower quantification limit." The results show that the D-lactic acid production values for LactobacHIus johnsonii CNCM 1-1225 strain are approximately 65% and that the production values of D-lactic acid for the strain LactobacHIus johnsonii CNCM I-4434 and the strain LactobacHIus johnsonii CNCM 1-4441 are strongly reduced.For LactobacHIus johnsonii CNCM I-4434 there is a small biological variation in the production levels of acid D- lactic acid that vary between 3 and 4% of the total lactic acid, depending on the experiment.The production levels of D-lactic acid Lactobacillus johnsonü strain CNCM 1-4441 are very low and below the detection levels for this assay. This result is similar to the results obtained for the strain Lactobacillus johnsonü NCC9006 which contains an inactivated D-lactate dehydrogenase gene created using technology methods It is also noteworthy that the control strains Lactobacillus paracasei NCC2461 and Lactobacillus rhamnosus NCC4007, both considered as producers of L-lactic acid, and which produces 3 and 3.5% of D-lactic acid under these conditions, respectively. For these data the strain Lactobacillus johnsonü CNCM I-4434 and the strain Lactobacillus johnsonü CNCM 1-4441 can be considered as producers of L-lactic acid and phenotypically different from Lactobacillus johnsonü CNCM 1-1225.
Example 4
Identification of the changes in the genes of the D-lactate dehydrogenases. In order to identify mutations in the D-lactate dehydrogenase gene responsible for the deficient D-lactic acid phenotype, regions were amplified by PCR for DNA sequence analysis. The region was amplified from a μ? of bacterial culture using the primers P1 TCAGCACATAACCAGCAGCT (SEQ ID NO: 3) plus P2 GCAATAATACTGTCGCCGGT (SEQ ID NO: 4). The amplicons were purified and sequenced with the primers P1, P3 GTGTATAATAAAAGACGGTC (SEQ ID NO: 5) plus P2, and were compiled and analyzed in the suite of DNASTAR programs. The results are shown in Figure 3. As can be seen in Figure 3, the strain Lactobacillus johnsonü CNCM I-4434 contains a change of C by T in the base pair 651 of Figure 3 and Figure 4A, and that results in an amino acid change from serine to leucine at position 204 (S204L) in the signal domain 3 of the enzyme sequence D-lactate dehydrogenase (Figure 4B). Lactobacillus johnsonü strain CNCM I-4441 contains a change of G by A in base pair 498 of Figure 3 and Figure 5A, and which results in an amino acid change from glycine to aspartic acid in position 153 ( G153D) in the signal domain 1 of the sequence of the enzyme D-lactate dehydrogenase (Figure 5B). The gene sequencing data show that the phenotype deficient in the production of D-lactic acid in strains Lactobacillus johnsonii CNCM I-4434 and Lactobacillus johnsonii CNCM 1-4441 is accompanied by a corresponding change in the gene of D-lactate dehydrogenase and in the sequence of the enzyme.
Example 5
Determination of the phenotypic stability of the strains Lactobacillus johnsonii CNCM I-4434 and Lactobacillus johnsonii CNCM 1-4441. Given the large number of generations from the collection of crops to the final product, it is important that the phenotype deficient in D-lactic acid is stable and that the reversal towards the production of D-lactic acid is very rare. To investigate this, we cultivated strains of Lactobacillus johnsonii CNCM I-4434 and Lactobacillus johnsonii CNCM 1-4441 in MRS broth for a total of 100 generations and then tested 300 individual colonies for the production of D-lactic acid. The results are that none of the colonies tested showed levels of D-lactic acid above those determined for each strain. This analysis was also carried out after the pilot-scale production of spray-dried powders and Lc1 drinkable products, with the same results.
In lactic acid bacteria a single copy of D- or L-lactate dehydrogenase is essential for the production of lactic acid and for the regeneration of NADH, a co-factor in this reaction, for NAD. In lactic acid bacteria this is the only route to regenerate NAD under anaerobic conditions and is essential for its growth. In the case of deficient strains of D-lactic acid there is no selective pressure for the reversion to the production of D-lactic acid since the enzyme L-lactate dehydrogenase is sufficient to cover the lack of the activity of the D-enzyme. lactate dehydrogenase.
List of SEQ ID:
SEQ ID NO: SEQ ID NO: Short description
(DNA) (protein)
1 2 Lactobacillus johnsonii CNCM 1-1225 D-lactate dehydrogenase
3 n / a Particle P1 (see example 4)
4 n / a Splitter P2 (see example 4)
5 n / a Particle P3 (see example 4)
6 8 Lactobacillus johnsonii CNCM I-4434 D-lactate dehydrogenase (Figures 4A and 4B)
7 9 Lactobacillus johnsonii CNCM 1-4441 D-lactate dehydrogenase (Figures 5A and 5B)
Claims (15)
- CLAIMS 1 . Natural derivative of strain Lactobacillus johnsonii CNCM 1-1225, characterized in that the derivative of Lactobacillus johnsonii strain CNCM 1-1225 is deficient in the production of D-lactic acid. 2. Natural derivative of the strain of Lactobacillus johnsonii CNCM 1-1225 according to claim 1, characterized in that the sequence of the enzyme D-lactate dehydrogenase of the natural derivative of the strain of Lactobacillus johnsonii CNCM 1-1225 comprises an amino acid change of glycine by aspartic acid at amino acid position 153 and / or an amino acid change from serine to leucine at amino acid position 204. 3. Natural derivative of Lactobacillus johnsonii strain CNCM 1-1225 according to claim 1 or claim 2, characterized in that the nucleic acid sequence of the D-lactate dehydrogenase gene in the natural derivative of the strain of Lactobacillus johnsonii CNCM 1- 1225 comprises a transition from G to A at nucleic acid position 498 and / or a transition from C to T at the position of nucleic acid 651. 4. Natural derivative of the strain of Lactobacillus johnsonii CNCM 1-1225 according to any of claims 1 to 3, characterized in that it is selected from the group consisting of the strain of Lactobacillus johnsonii CNCM I-4434 and Lactobacillus johnsonii CNCM 1-4441. 5. Natural derivative of Lactobacillus johnsonii strain CNCM 1-1225 according to any of claims 1 to 4, characterized in that the natural derivative of Lactobacillus johnsonii strain CNCM 1-1225 is present as a biologically pure culture. . 6. Cell-free culture supernatant characterized in that it is isolated from a biologically pure culture of a natural derivative of Lactobacillus johnsonii strain CNCM 1-1225 according to any of claims 1 to 4. 7. Composition comprising the natural derivative of the strain of Lactobacillus johnsonii CNCM 1-1225 according to any of claims 1 to 5 and / or the culture supernatant, cell-free according to claim 6, characterized in that it is used in the preparing a composition for use in a method for the treatment of the human or animal body by therapy. 8. Composition characterized in that it comprises the natural derivative of Lactobacillus johnsonii strain CNCM 1-1225 according to any of claims 1 to 5 and / or the cell-free culture supernatant according to claim 6, for use in the treatment of disorders related to a weakened immune system. 9. Composition for use according to claim 8, characterized in that the disorder related to a weakened immune system is selected from the group consisting of influenza, rhinitis, common cold, and combinations thereof. 10. Composition characterized in that it comprises the natural derivative of Lactobacillus johnsonii strain CNCM 1-1225 according to any of claims 1 to 5 and / or the cell-free culture supernatant according to claim 6, for use in the treatment or prevention of disorders related to cell attachment and cellular invasion by enterovirulent bacteria or viruses. eleven . Composition for use according to claim 10, characterized in that the disorder related to cell attachment and cellular invasion by enterovirulent bacterial or viral species is selected from the group consisting of lower respiratory tract infections, gastrointestinal tract infections, otitis media, and combinations thereof, and wherein preferably the enterovirulent species are selected from the group consisting of Salmonella strains; Campylobacter; Listeria; Escherichia cotí, such as the strains ETEC, EHEC, EPEC, or EIEC, for example; Yersinia; Shigella; Toxin-producing bacteria, such as Staphylococcus aureus, Clostridium botulinum, or Bacillus cereus; Vibrio vulnifucus / parahaemolyticus; rotavirus; norovirus; Verotoxigenic E. coli; Enterobacter sakazakii; C. toxigenic perfringens (type A and B); parasites originating from foods, such as Equinococus, Toxoplasma, or Giardia; Helicobacter pylori; Clostridium difficile; Clostridium tetani; or combinations thereof. 12. Composition for use according to any of claims 7 to 11, characterized in that the composition is selected from the group consisting of food compositions, pet food compositions, beverages, dairy products, nutritional formulas, infant formulas, food additives , nutraceutical products, pharmaceutical compositions, food ingredients and / or cosmetic compositions. 13. Composition for use according to any of claims 7 to 12, characterized in that the composition is selected from the group consisting of acidified milk products, such as yogurt or yoghurt drinks; or powders based on milk. 14. Composition for use according to any of claims 7 to 13, characterized in that the composition comprises the natural derivative of the strain Lactobacillus johnsonii CNCM 1-1225 according to any of claims 1 to 5 in an amount of 106-1012 ufe per daily dose. 15. Composition for use according to any of claims 7 to 14, characterized in that it contains about 0.005 mg - 5000 mg of the natural derivative of Lactobacillus johnsonii strain CNCM 1-1225 according to any of claims 1 to 5 per daily dose.
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